Annual Report 2010

2010

Annual Report 2010 

(Apr. 2009-Mar. 2010) 

No. 9 

Published by National Institute of Agrobiological Sciences 

Kannondai, Tsukuba, Ibaraki, 305-8602, Japan 

TEL : 029 (838) 7406 

FAX : 029 (838) 7408 

URL : http://www.nias.affrc.go.jp/index_e.html

Annual Report 2010 

(Apr. 2009 ~ Mar. 2010) 

(In the fiscal year 2009)

Message from our president 

Teruo Ishige, President 

The National Institute of Agrobiological Sciences (NIAS), which is the largest agricultural 

research institute of basic life science in Japan, was established on 1 April 2001 as an independent 

administrative institution of the Ministry of Agriculture, Forestry, and Fisheries to be the center 

for basic studies to develop innovative agricultural biotechnologies and new bioindustries. Main 

research subjects of NIAS include genome research of plants, insects and animals, development 

of novel functional crops by gene-recombination technologies, functional analysis of genes for 

contribution to breeding applications, and development of new foundational materials for the 

creation of novel bioindustries. In 2004, the Institute had achieved the ultimate goal of decoding the 

entire rice genome sequence as a leading country of the 10 counties and regions that had organized 

the International Rice Genome Sequencing Project. The Institute also has decoded the silkworm 

genome sequence and developed technologies for the recombination of genes in crops, insects 

(silkworm), and animals. 

The Second Five-year Plan started on 1 April 2006 and the Institute has been advancing the 

technologies developed in the First Five-year Plan. In the Second Five-year Plan, the Institute 

focuses on: (1) improvement, diversity and utility of agrobiological resources - we utilize resources 

that we have built up in the course of research on the genes of rice, silkworms, and pigs and we 

have secured other genetic resources; (2) research on, and development of, innovative agricultural 

technologies using biological and genome information — we study organisms with a view to their 

ability to adapt to their environments, differentiate, and interact with other organisms; and (3) 

research and development aimed at creating new biotechnology-based industries — we develop 

biotechnologies to produce useful materials such as silk-based products that can be used in 

everyday items and in medical practice. There is an urgent need to develop technologies in these 

three subject areas, because each of them is important and is expected to contribute greatly to 

society. The Institute will make further efforts to maximize the benefits of biotechnology. 

Advanced biotechnologies are indispensable in helping to solve problems in the areas of global 

food supply, the environment, and medicine. Genetically modified (GM) crops such as maize, soybean,

ape seed and so on, have been adopted and grown in 23 countries (114.3 million ha) in 2007. The 

reason for this rapid spread is their capacity for high-yield potential. Also, due consideration 

has been given to securing safety in both the research and development of gene-recombination 

technology and its application; consequently, few concerns have arisen in regard to its influence on 

the environment and the safety of its use in food production. As the cultivated area of GM crops has 

increased sharply year after year, the public acceptance of GM crops in foods for humans and in 

animal feeds is increasing in many foreign countries. However, in Japan, GM crops developed using 

these new technologies are not yet practicable, because of insufficient public understanding and 

acceptance. 

The Institute has facilitated research on developing crops with high yields, crops with efficient 

biomass production, crops with ability to grow under harsh conditions, and crops with potential to 

help prevent diseases and keep people healthy. We have already isolated several useful genes and 

obtained intellectual property rights for their use. GM crops that can grow in adverse environments 

or that promote human health and facilitate prevention of common diseases have been developed. 

Novel basic technologies such as gene targeting and RNA interference techniques have been 

developed. 

We are facing problems associated with food shortages in the 21st century combined with 

ballooning grain prices as well as problems associated with the environment. Biotechnologies have 

great potential to address these problems and there is fierce global competition to lead in these 

fields. Gene-recombination technology and genome analysis are spreading widely because they 

provide opportunities to make use of scientific discoveries in many areas of biology. We believe that 

biotechnology research will contribute to the well-being of the human race. Thus, there is a need 

for the public to gain an understanding of the importance of the wise use of biotechnology for the 

future good of humankind. The Institute is willing and ready to lead in public education. 

At the midpoint of the Second Five-year Plan, all the staff at NIAS are determined to make a 

concerted effort to produce additional significant discoveries and results. We thank all of those 

involved for their cooperation, and we look forward to your continued support, understanding, and 

collaboration.

Summary of the Second Five-year Plan 

NIAS aims to create new industries that will dramatically increase agricultural productivity, 

create new demands for agricultural products, and open up new possibilities in the agricultural, 

forestry, and fisheries industries. Under the First Five-year Plan, we succeeded in obtaining worldleading 

results, such as the sequencing of the entire rice genome, the sequencing of the outline of 

the silkworm genome, the advancement of gene-recombination technology, and the production of 

genetically engineered pigs. 

During the five years beginning in 2006, the Institute aims to make a leap forward in both basic 

and pioneering research and technological development, mainly in the .eld of biotechnology. To this 

end, we are establishing four research centers that will place emphases on basis research, plant 

science, animal science, and entomological science. 

A Development, refinement and utilization of agro-bioresources 

(1) Enhancement and utilization of rice germplasm potential based on genomics approaches 

(2) Development and utilization of genome resources from Oryza and Gramineae crops 

(3) Development of insect genomic resources and its application 

(4) Development and utilization of swine genome resource 

(5) Development and utilization of soybean genome resource 

(6) Establishment of a bioinformatics research basis for comparative analysis between species 

(7) Collection, evaluation, multiplication, preservation and distribution of genetic resources 

(8) Development of mutant lines and varieties with new agronomically useful characteristics by 

radiation breeding 

B Research and development of innovative agricultural production 

technology based on genomics and physiology 

1) Research on environmental adaptation mechanisms in rice and their 

application 

(1) Research on environmental stress mechanisms in rice and their application 

(2) Research on responses to environmental light signals in rice and their application 

(3) Research on disease resistance mechanisms in rice and their application 

2) Analysis of adaptation mechanism to environmental condition in insect and 

development of insect control technique 

(1) Search and analysis of invertebrate gene function for development of pesticides 

(2) Elucidation of desiccation tolerance mechanism in insects and its application 

(3) Analysis of insect defense mechanism and its application 

3) Reproductive and neurobiological research in the livestock 

(1) Research of reproductive biology for gamete, placenta and stem cell 

(2) Neurobiological research in the control mechanism of instinctive behavior and endocrine 

system 

4) Research on the interactions between living organisms and development of 

their regulation techniques 

(1) Research on the plant-microbe interactions 

(2) Analysis of insect-insect and insect-plant interactions and its application 

(3) Functional analyses of insect-microbe interaction and their application 

5) Research on protein structures and functions by structural and analytical biology 

(1) Research on protein structures and functions by X-ray crystallography, NMR and MS 

spectrosco 

C Research for creation of new bio-industries by biotechnology 

1) Biotechnologies for production of safe and valuable products 

(1) Development of transgenic technology for ensuring agricultural and environmental safety 

and for producing valuable products 

(2) Development of health-promoting transgenic crops through research on high accumulation 

system of valuable products 

(3) Development of technology for the production of useful materials using transgenic insect 

(4) Development of transgenic animal models for protein production and medical application 

2) Development of materials for life and medical use by silk-technology 

(1) Development of medical materials by use of silk proteins 

(2) Development of the various silk materials for lives utilizing new silk functions 

Staff Number of Employees (As of April 2009) 

Reserchers 262 

Administrative staff 119 

Total number of employees 381 

Reemployment staff 6 

Contract workers (Part time) 

505 

including 76 post-doc researchers

Organization 

President 

Vice President (Plant Sciences) 

Vice President (Insect and Animal Sciences) 

Auditors 

Research Supporting Section 

Research Director-General 

Research Directors 

Deputy Research Directors 

Research Planning Section 

Evaluation Section 

Information Management Section 

Library 

Public Relations Section 

GMO Research Promotion Section 

Safety Management Section 

Technology Transfer and Research Cooperation Section 

Genetic Resources Management Section 

Technical Support Section 

Management Dierctor-General 

Management Director 

General Affairs Section 

Accounting Section 

Management and Supply Section 

Audit and Compliance Section 

Research Section 

Laboratory of Special Projects 

Insect Symbiotic Microorganism Genome Project 

Division of Genome and Biodiversity Research 

Director 

Plant Genome Research Unit 

Bioinformatics Research Unit 

Genome Resource Center 

Genebank 

Institute of Radiation Breeding 

Soybean Genome Research Team 

Division of Plant Sciences 

Director 

Environmental Stress Research Unit 

Photobiology and Photosynthesis Research Unit 

Plant Disease Resistance Research Unit 

Protein Research Unit 

Plant-Microbe Interactions Research Unit 

Plant Genetic Engineering Research Unit 

Division of Insect Sciences 

Director 

Insect Genome Research Unit 

Invertebrate Gene Function Research Unit 

Anhydrobiosis Research Unit 

Innate Immunity Research Unit 

Insect Interaction Research Unit 

Insect-Microbe Interaction Research Unit 

Silk-Materials Research Unit 

Silk Technology Unit 

Research Cente 

QTL Genomics Research Center 

Transgenic Crop Research and Development Center 

Transgenic Silkworm Research Center 

Transgenic Animal Research Center 

Division of Animal Sciences 

Director 

Animal Genome Research Unit 

Reproductive Biology Research Unit 

Neurobiology Research Unit

Contents 

Message from our President 

Summary of the Second Five-year Plan 

Organization 

Contents 

Research Highlights for 2009 

◦Map-based cloning and molecular breeding of pi21, a non-race-specific resistance 

gene to blast .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 

Shuichi Fukuoka, Norikuni Saka, Hironori Koga, Kazuko Ono, Takehiko Shimizu, 

Kawori Ebana, Nagao Hayashi, Akira Takahashi, Hirohiko Hirochika, Kazutoshi Okuno, 

Masahiro Yano 

◦Cleistogamous flowering in barley arises from the suppression of microRNA-guided 

HvAP2 mRNA cleavage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 

Sudha K. Nair, Ning Wang, Yerlan Turuspekov, Mohammad Pourkheirandish, 

Suphawat Sinsuwongwat, Guoxiong Chen, Mohammad Sameri, Akemi Tagiri, Ichiro Honda, 

Yoshiaki Watanabe, Hiroyuki Kanamori, Thomas Wicker, Nils Stein, Yoshiaki Nagamura, 

Takashi Matsumoto, Takao Komatsuda 

◦α-1,3-glucan functions as camouflage during infection in the rice blast fungus 

Magnaporthe oryzae .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 

Takashi Fujikawa, Marie Nishimura 

◦An inhibitory interaction between viral and cellular proteins underlies the resistance 

of tomato to a non-adapted tobamovirus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 

Kazuhiro Ishibashi, Masayuki Ishikawa Plant-Microbe Interactions Research Unit 

◦Host plant genome overcomes the lack of a bacterial gene for symbiotic nitrogen 

fixation .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 

Tsuneo Hakoyama, Koji Yano, Haruko Imaizumi-Anraku, Hiroshi Kouchi 

◦Transduction of RNA-directed DNA methylation signals to repressive histone marks 

in Arabidopsis thaliana .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 

Yoshiki Habu, Hisataka Numa, Manabu Yoshikawa 

◦Unique RNAi by RNA silencing inducible sequence in rice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 

Fumio Takaiwa, Hiroshi Yasuda, Yuhya Wakasa, Taiji Kawakatsu 

◦A new juvenile hormone isolated from a heteropteran insect .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 

Toyomi Kotaki 

◦Bombyx prothoracicostatic peptides activate the sex peptide receptor to regulate 

ecdysteroid biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 

Yoshiaki Tanaka 

◦Experimental production of wedding dress from high quality silk produced 

by transgenic silkworm through the collaboration of different industries .. . . . . . . . . . . . . . . . . . . . . 20 

Hiroaki Machii, Tetsuya Iizuka, Hideki Sezutsu, Naoyuki Yonemura, Ken-ichiro Tatematsu, 

Keiro Uchino, Isao Kobayashi, Chiyuki Takabayashi, Keisuke Mase1, Eiji Okada, 

Kenichi Nakajima, Toshiki Tamura 

◦Production of piglets from oocytes after injection of spermatozoa grown 

in immunodeficient mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 

Kazuhiro Kikuchi, Hiroyuki Kaneko, Michiko Nakai, Naomi Kashiwazaki 

◦Completion of draft sequencing of the pig genome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 

Hirohide Uenishi, Takeya Morozumi, Hiroyuki Kanamori, Tomoko Eguchi-Ogawa, 

Naoe Fujishima-Kanaya, Toshimi Matsumoto, Ayumi Mikawa, Naohiko Okumura, 

Hiroki Shinkai, Kohei Suzuki, Maiko Tanaka-Matsuda, Daisuke Toki, Takahito Bito, 

Nahoko Fujitsuka, Kozue Kamiya, Kanako Kurita, Ari Kikuta, Harumi Yamagata

Research Activities 

Research Center 

・QTL Genomics Research Center . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 

・Transgenic Crop Research and Development Center . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 

・Transgenic Silkworm Research Center . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 

・Transgenic Animal Research Center . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 


・Plant Genome Research Unit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 

・Bioinformatics Research Unit .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 

・Genome Resource Center .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 

・Genebank . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 

・Institute of Radiation Breeding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 

・Soybean Geome Research Team .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 


・Environmental Stress Research Unit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67 

・Photobiology and Photosynthesis Research Unit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 

・Plant Disease Resistance Research Unit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72 

・Protein Research Unit .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76 

・Plant-Microbe Interactions Research Unit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 

・Plant Genetic Engineering Research Unit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 


・Insect Genome Research Unit .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87 

・Invertebrate Gene Function Research Unit .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90 

・Anhydrobiosis Research Unit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 

・Innate Immunity Research Unit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 

・Insect Interaction Research Unit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99 

・Insect-Microbe Research Unit .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104 

・Silk-Materials Research Unit .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106 

・Silk-Technology Unit .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112 


・Animal Genome Research Unit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115 

・Reproductive Biology Research Unit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 

・Neurobiology Research Unit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122 

List of Publication 

・Original papers 

1) In English . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124 

2) In Japanese with English Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147 

・Reviews and Monograph (In English) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148 

List of Organizations Exchanged MOU . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150 

Executive Members and Research Staff Members . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151 

・Members of NIAS Evaluation Committee . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160 

・Financial overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161 

・Location and How to access .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162

Topics of Research in This Year 

Map-based cloning and molecular breeding of pi21, 

a non-race-specific resistance gene to blast 

Shuichi FUKUOKA 1 , Norikuni SAKA 2 , Hironori KOGA 3 , Kazuko ONO 1 , Takehiko SHIMIZU 4 , 

Kawori EBANA 1 , Nagao HAYASHI 1 , Akira TAKAHASHI 1 , Hirohiko HIROCHIKA 1 , Kazutoshi 

OKUNO 5 , Masahiro YANO 1 

1 

National Institute of Agrobiological Sciences, 2 Aichi Agricultural Research Center, 

3 

Ishikawa Prefectural University, 4 Institute of the Society for Techno-Innovation of 

Agriculture, Forestry and Fisheries, 5 Graduate School of Life and Environmental 

Sciences, University of Tsukuba 

Japanese upland rice (Oryza sativa L.) is a 

potential gene donor for durable resistance to 

blast disease caused by Magnaporthe oryzae. 

Pi21 is a major quantitative trait locus (QTL), 

for which an allele, pi21, from a Japanese upland 

rice, induces resistance by restricting development 

of lesions without involvement of a hypersensitive 

response. High-resolution mapping 

and complementation testing of pi21 identified 

a loss of function mutation of a heavy metal– 

transport/detoxification protein domain gene, 

Os04g0401000, encoding a protein structurally 

different from previously identified resistance 

proteins. 

Sequence analysis of the Pi21 gene from a 

number of worldwide cultivars identified 12 alleles. 

These alleles were discriminated based on 

insertion–deletion polymorphisms in the region 

containing proline-rich motifs that might be 

involved with the protein’s function. Evaluation 

of a series of chromosome segment substitution 

lines (CSSLs), each possessing one of the Pi21 alleles 

in the genetic background of a susceptible 

cultivar, clearly demonstrated that all except 

one were susceptible, and suggested that two 

deletions in the resistant allele are required to 

confer resistance (Fig 1A). The analysis also revealed 

that the resistance allele was present in 

some strains of japonica rice, suggesting that it 

could improve blast resistance of rice worldwide 

(Fig 1B). 

Japanese upland rice has been used as a 

source of blast resistance in conventional breeding 

program since the 1920’s, but the pi21 allele 

has not been used in irrigated rice cultivars, 

possibly due to co-introduction of resistance and 

undesirable grain characteristics. The cause 

of this troublesome association could be either 

tight linkage of genes controlling independent 

traits, known as “linkage drag,” or pleiotropic 

effects of the target gene on other traits; these 

two cases cannot be discriminated unless the 

linkage can be broken. Fine genetic analysis 

around Pi21 locus identified gene(s) associated 

with inferior eating quality within 40-kb of pi21. 

Desirable recombinants between pi21 and the 

genes conferring inferior eating quality were 

successfully selected from a breeding population 

over 6000 plants by using DNA markers for the 

region around Pi21. Field evaluation confirmed 

that the resistance allele does not penalize 

agronomic traits and that the cause of the 

association is tight linkage with genes causing 

undesirable effects. Map-based cloning of pi21 

Annual Report 2010 1

a 

Heavy metal 

binding 

Proline-rich 

ATG 82 83 567 

TAT 

b 

Lesion area % 

238 258 273 358 421 453 486 

In/del 

size (bp) 

0 10 20 30 

+45 

0 

-9 

-12 

-15 

-33 

-39 

-39 

-45 

-48 

-48 

-69 

b 

bc 

b 

b 

c 

bc 

bc 

b 

b 

bc 

b 

a 

Fig.1. Natural variations in Pi21 

(a) The insertion/deletion-size variation of Pi21 among Asian cultivated rice. In/del 

sizes of respective alleles from the Nipponbare-allele are indicated on right. (b) The average lesion 

areas caused by blast infection in backcrossed lines carrying the respective Pi21 alleles. The lesion 

area followed by different letters differ significantly according to Tukey’s HSD test at 5%. 

identified a novel gene that negatively regulates 

plant defense and provides a breakthrough to 

durably resistant cultivars without any penalties 

on agronomic traits, which has not been 

achieved by conventional rice breeding. 

Reference 

Fukuoka S, Saka N, Koga H, Ono K, Shimizu T, 

Ebana K, Hayashi N, Takahashi A, Hirochika 

H, Okuno K, Yano M (2009) Loss of function 

of a proline-containing protein confers 

durable disease resistance in rice. Science, 325: 

998-1001. 

2 Annual Report 2010

Cleistogamous flowering in barley arises from the 

suppression of microRNA-guided HvAP2 mRNA cleavage 

Sudha K NAIR 1 , Ning WANG 1 , Yerlan TURUSPEKOV 1 , Mohammad POURKHEIRANDISH 1 , 

Suphawat SINSUWONGWAT 1 , Guoxiong CHEN 1 , Mohammad SAMERI 1 , Akemi TAGIRI 1 , 

Ichiro HONDA 2 , Yoshiaki WATANABE 2 , Hiroyuki KANAMORI 3 , Thomas WICKER 4 , Nils 

STEIN 5 , Yoshiaki NAGAMURA 1 , Takashi MATSUMOTO 1 , and Takao KOMATSUDA 1 

1 

National Institute of Agrobiological Sciences, 2 National Institute of Crop Science, 

3 

Institute of the Society for Techno-innovation of Agriculture, Forestry and Fisheries, 

4 

Institute of Plant Biology, University of Zürich, 5 Department of Genebank, Leibniz 

Institute of Plant Genetics and Crop Plant Research 

Cleistogamous flowers shed pollen before 

opening, forcing plants with this habit to be 

almost entirely autogamous. Cleistogamy 

also provides a means of escape from cereal 

head blight infection, and minimizes pollenmediated 

gene flow (Fig 1A-C). Lodicule size 

differed markedly between cleistogamous and 

noncleistogamous cultivars. The lodicule in 

cleistogamous barley is atrophied. The first 

notable difference in their size became apparent 

at the white anther stage, where cell division 

was much more active in the noncleistogamous 

type (Fig 1D-G). We have isolated cleistogamy 

1 (Cly1) by positional cloning (Nair et al, 2010) 

and shown that it encodes a transcription factor 

containing two AP2 domains and a putative 

microRNA miR172 targeting site, which is an 

orthologue of Arabidopsis thaliana AP2 (Fig 2A). 

le 

pa 

st 

A 

lo 

B 

C 

D 

E 

cp 

st 

pa 

cp 

pa 

st 

F 

lo 

le 

G 

lo 

le 

Fig 1. The lodicules of cleistogamous and non-cleistogamous barley. 

(A) The lodicule [lo] located at the base of the stamen [st] opens the spikelet by pushing apart the 

lemma [le] and palea [pa]. (B) A non-cleistogamous and (C) a cleistogamous barley spike at anthesis. 

The lodicules of (D) a non-cleistogamous and (E) a cleistogamous barley. A section of the spikelet in (F) 

a non-cleistogamous and (G) a cleistogamous barley. The carpel [cp] is surrounded by the other floral 

organs. (Scale bars: D and E, 800µm; F and G, 200µm.) 


A 

(cM) 

B 

C 

Fig 2. Positional cloning of Cly1. 

(A) BACs contig harbour Cly1, which encodes AP2 transcription factor. The AZ and KNG Cly1 

sequences differ from one another by a SNP within the miR172 target site. (B) Sequence 

comparisons between alleles of the putative miR172 target site indicate that the alleles differ at 

the second and/or third variable positions (red asterisks). (C) Lodicule size at the yellow anther 

stage and at anthesis in a 274 accession germplasm set. 


Fig 3. Expression of Cly1. 

(A) The 3’ end of Cly1, including Exon 10 which carries the putative miR172 target site (red bar). The two regions 

targeted by RT-PCR are shown by arrows. The wavy line indicates the 5’ RACE RNA oligo-adapter ligated to 

cleaved mRNA. The probe used for in situ hybridization is also shown. (B) Cly1 expression in an immature spike 

at the awn primordium stage of the non-cleistogamous cultivar AZ, as detected by in situ hybridization with an 

anti-sense (left) and sense (right) Cly1 3’ UTR transcript. (C) A higher magnification of the presentation shown 

in (B). [lo] lodicule, [st] stamen. (D) Cly1 expression in the cleistogamous cultivar KNG. (E) Cly1 expression in 

an immature spike during development. The constitutively expressed actin gene was used as a control. Dark 

gray bars represent AZ and light gray bars KNG. The numbers below each bar refer to the developmental 

stage assayed (1- lemma primordium stage, 2- stamen primordium stage, 3- awn primordium stage, 4- white 

anther stage, 5- green anther stage, 6- yellow anther stage, 7- spike at anthesis). (F) Modified 5’ RACE ligations 

from (A) were amplified by nested PCR. (G) Cleavage at the miR172 site within Cly1. The 5’ termini of miR172- 

guided mRNA cleavage were determined by cloning and sequencing of the amplicon generated in (F). A rice 

miR172 sequence was aligned with the Cly1 mRNA sequences. Vertical arrows indicate the inferred 5’ termini of 

miR172-guided cleavage, and the number above each arrow indicates the proportion of clones showing these 

sites. (Scale bars: B, 500μm; C and D, 250μm). 

The length of the coding sequence is 1464 bp, 

and it is flanked by a 5’ UTR (480 bp) and 3’ 

UTR (346–368 bp). Cly1 encodes a predicted 

487 residue polypeptide with two AP2 domains. 

In situ RNA hybridization revealed that the 

expression of Cly1 was concentrated within the 

lodicule primordia (Fig 3 A-D). We established 

a perfect association between a synonymous 

nucleotide substitution at the miR172 targeting 

site and cleistogamy (Fig 2 B,C). Cleavage of 

mRNA directed by miR172 was detectable 

only in a non-cleistogamous background by 

a modified 5’ RACE experiment (Fig 3 E-G). 

We conclude that the miR172-derived downregulation 

of Cly1 promotes the development of 

the lodicules, thereby ensuring non-cleistogamy, 

while the single nucleotide change at the 

miR172 targeting site results in the failure of 

the lodicules to develop properly, producing the 

cleistogamous phenotype. 

Reference 

Nair SK, Wang N, Turuspekov Y, Pourkheirandish 

M, Sinsuwongwat S, Chen G, Sameri M, Tagiri 

A, Honda I, Watanabe Y, Kanamori H, Wicker T, 

Stein N, Nagamura Y, Matsumoto T, Komatsuda 

T (2010) Cleistogamous flowering in barley 

arises from the suppression of microRNA-guided 

HvAP2 mRNA cleavage. Proc. Natl. Acad. Sci. 

USA, 107: 490-495. 


α-1,3-glucan functions as camouflage during infection in 

the rice blast fungus Magnaporthe oryzae 

Takashi FUJIKAWA, Marie NISHIMURA 

Plant-Microbe Interaction Research Unit 

The fungal cell wall, the outermost layer of 

the cell, is a good target for recognition of fungal 

invaders by the host cells. Fungal cell walls are 

mainly composed of polysaccharides, e.g., β-glucans, 

α-glucans, chitin/chitosan, and mannans. In 

general,β-1,3-glucan and chitin form the skeletal 

core, while non-skeletal polysaccharides differ between 

fungal species and with growth conditions. 

Innate immune systems found in vertebrates, 

invertebrates and plants are activated upon 

receptor recognition of conserved structural patterns 

of cell-wall components of the fungal invaders 

and enables an immediate defence of the host 

cells against a broad range of fungi. As a result of 

activation of the innate immune systems, various 

defence responses are induced in the host cells. 

In plants, fungal cell wall chitin and β-glucans 

have been reported to evoke innate immune responses 

such as secretion of anti-fungal enzymes 

(β-glucanases and chitinases), production of antifungal 

agents and reinforcement of the plant cell 

wall. As such, recognition of the fungal cell-wall 

polysaccharide is very important in plant-microbe 

interactions. It was assumed that pathogens have 

mechanisms to protect themselves from host 

immune attacks during infection; however, the 

protection mechanisms in plant pathogens were 

poorly understood. 

To better understand fungal strategies circumventing 

the host plant immune responses, we 

used fluorescent labels to investigate localization 

of major cell wall polysaccharides of the rice blast 

fungus, Magnaporthe oryzae, during rice infection. 

Our cytological studies showed that α-1,3-glucan 

and chitosan were the major polysaccharides at 

accessible surface of infectious hyphae. However, 

β-1,3-glucan and chitin also became detectable 

after enzymatic digestion of α-1,3-glucan (Fig 1). 

Immunoelectron microscopic observation of 

infectious hyphae revealed that α-1,3-glucan 

was localized in the outer portion of the cell 

wall compared to β-1,3-glucan, supporting our 

microscopic observation that surface α-1,3-glucan 

masks β-1,3-glucan and chitin in the cell wall. 

Moreover, although no accumulation of α 

-1,3-glucan on germ tubes was observed on cover 

glass, it was clearly detected in the presence of 

exogenous plant wax or on plant surfaces (Fig 2). 

These results provide evidence that α-1,3-glucan 

accumulation is induced by a plant cue (wax). 

Using a genetic mutant related to signal transduction 

pathways in M. oryzae, we have shown that 

Mps1 MAP kinase, an ortholog of MAP kinase 

involved in cell-wall integrity in Saccharomyces 

cerevisiae, is required for the accumulation of α- 

1,3-glucan and is activated in the presence of the 

plant wax. We further demonstrated that the 

accumulation of α-1,3-glucan on the cell wall was 

correlated with resistance of the cell wall against 

chitinase digestion. Taken together, these results 

show that the fungus accumulates α-1,3-glucan 

on the surface of the fungal cell wall in response 

to plant wax in an Mps1 MAP-kinase-dependent 

manner to physically and functionally mask the 

cell wall. 

It is important to note that genes encoding α- 

1,3-glucanase are not found in the rice genome. 

Our results, together with the rice genome 

information, strongly suggest that the function 

of the surface α-1,3-glucan is to not only protect 

the fungal cell wall from digestive enzymes 

produced by plant cells but also to camouflage 

chitin and β-1,3-glucan in the cell wall from 

being recognized during infection by the innate 

immune system of rice (Fig 3). 


Fig 1. Detection of cell wall polysaccharides on infectious hyphae developed in rice sheath cells. 

After enzymatic digestion of α-1,3-glucan, β-1,3-glucan and chitin became detectable, indicating 

that α-1,3-glucan masks β-1,3-glucan and chitin in the fungal cell wall. Images were taken at 24h 

post inoculation. Ap: appressorium, Ih: infectious hypha. 

Fig 2. Induction of α-1,3-glucan accumulation on the fungal cell wall by plant cue (wax). 

α-1,3-glucan was only detected on appressorium developed on a cover glass (left panel). In 

contrast, α-1,3-glucan was detected in entire cells in the presence of plant wax (center panel) and 

on the surface of rice cells (right panel) . 

Fig 3. Stealth technology of the rice blast fungus. 

Rice innate immune system attacks fungal invaders by secretion of anti-fungal enzymes/agents 

upon recognition of chitin exposed on the fungal cell wall. However, rice failed to detect M. oryzae 

camouflaged with α-1,3-glucan mask that allows the fungus to successfully invade into rice cells. 

Reference 

Fujikawa T, Kuga Y, Yano S, Yoshimi A, Tachiki 

T, Abe K, Nishimura M (2009) Dynamics of cell 

wall components of Magnaporthe grisea during 

infectious structure development. Molecular 

Microbiology, 73: 553-570. 


An inhibitory interaction between viral and cellular proteins 

underlies the resistance of tomato to a non-adapted 

tobamovirus 

Kazuhiro ISHIBASHI, Masayuki ISHIKAWA 


Each virus infects only a limited range of 

hosts, and most plant species are ‘nonhosts’ to a 

given virus, i.e., all members of the species are 

insusceptible to the virus. Because mutant viruses 

that can multiply in nonhost plants rarely 

emerge, uncovering resistance mechanisms in 

nonhosts will help us devise strategies to confer 

durable virus resistance to crops. In nonhost 

plants, however, the factors that control virus 

resistance are not genetically tractable, and how 

the host range of a virus is determined remains 

to be revealed. 

Tm-1 is a semidominant resistance gene 

of tomato to a tobamovirus Tomato mosaic 

virus (ToMV). Previously, we found that Tm-1 

encodes a protein that binds to ToMV replication 

proteins and inhibits viral RNA replication. 

Tm-1 is derived from a wild tomato species, 

Solanum habrochaites, and ToMV-susceptible 

tomato (S. lycopersicum) cultivars have an allelic 

gene tm-1. Both Tm-1 and tm-1 encode proteins 

of 754 amino acids in which 25 residues are 

different. The tm-1 protein can neither bind to 

ToMV replication proteins nor inhibit ToMV 

RNA replication (Ishibashi et al, 2007). 

Tomato is a nonhost species for another 

tobamovirus Tobacco mild green mosaic virus 

(TMGMV). In this study (Ishibashi et al., 2009), 

we found that transgenic tobacco plants that 

express tm-1 exhibit resistance to TMGMV (Fig 

1). The tm-1 protein bound to the replication 

proteins of TMGMV and inhibited their RNA 

replication in vitro. In one of the tm-1-expressing 

tobacco plants, a tm-1-insensitive TMGMV mutant 

emerged. This mutant TMGMV multiplied 

in tomato protoplasts as efficiently as ToMV, 

suggesting that tm-1 is the only major factor 

that inhibits the intracellular multiplication of 

TMGMV in tomato. However, in tomato plants, 

the mutant TMGMV multiplied with lower efficiency 

compared to ToMV, and caused systemic 

necrosis (Fig 2), indicating that an inhibitory 

interaction between the replication proteins 

and tm-1 underlies a multilayered resistance 

mechanism to TMGMV in tomato. These results 

explain why viruses scarcely adapt to non-host 

organisms and could contribute to the development 

of crops that show durable resistance to 

viruses. 

References 

Ishibashi K, Masuda K, Naito S, Meshi T, 

Ishikawa M (2007) An inhibitor of viral RNA 

replication is encoded by a plant resistance 

gene. Proc. Natl. Acad. Sci. USA, 104: 13833– 

13838. 

Ishibashi K, Naito S, Meshi T, Ishikawa M (2009) 

An inhibitory interaction between viral and 

cellular proteins underlies the resistance of 

tomato to non-adapted tobamoviruses. Proc. 

Natl. Acad. Sci. USA, 106: 8778-8783. 


Fig 1. Transgenic tobacco plants that express tm-1 show resistance to TMGMV. 

Accumulation of the coat protein in TMGMV inoculated leaves of non-transgenic 

or tm-1-expressing tobacco was examined by SDS-PAGE and Coomassie blue 

staining. 

Fig 2. Tomato plants inoculated with the wild-type TMGMV or a TMGMV mutant that is 

insensitive to tm-1. 

Photograph of tomato plants (cv. Craigella GCR26: genotype tm-1/tm-1) inoculated with 

the wild-type (WT) TMGMV (right) or TMGMV mutant (TMGMV-T894M,F970Y) that is 

insensitive to tm-1 (left) at 16 days post inoculation. 


Host plant genome overcomes the lack of a bacterial gene 

for symbiotic nitrogen fixation 

Tsuneo HAKOYAMA 1 , Koji YANO 1,2 , Haruko IMAIZUMI-ANRAKU 1 , Hiroshi KOUCHI 1 

1 

Plant-Microbe Interaction Research Unit, 2 Present Address: National Institute for Basic 

Biology 

The major source of nitrogen for living 

organisms is atmospheric dinitrogen, which is 

fixed mainly by microorganisms that have an 

ability to reduce dinitrogen to ammonium ions 

by a nitrogenase system. In legume plants, soil 

bacteria of the family Rhizobiaceae are hosted 

within a symbiotic organ, the root nodule, in 

which the endosymbiotic bacteria are able to 

fix dinitrogen. This enables the host legumes 

to grow with very low nitrogen fertilization, 

thus being of critical importance in sustainable 

agriculture. Unlike many free-living diazotrophs, 

rhizobia are able to exhibit highly efficient 

nitrogen fixation only when they are in the 

host nodule cells as an endosymbiotic form, the 

bacteroid. This indicates that rhizobial nitrogen 

fixation is strictly controlled by the host plant. 

Fix − mutants of the host legumes, which 

form ineffective nodules with very low or no 

nitrogen fixation activity, are key tools in the 

identification of the host genes essential for the 

establishment of symbiotic nitrogen fixation. A 

Lotus japonicus Fix − mutant, fen1, forms morphologically 

normal but ineffective nodules with 

very low nitrogen fixing activity (Fig 1). The 

causal gene, FEN1, was isolated by map-based 

cloning and demonstrated to encode homocitrate 

synthase (HCS) by its ability to complement a 

HCS-defective mutant of Saccharomyces cerevisiae. 

FEN1 gene is expressed exclusively in root 

nodules. Homocitrate, a quite unusual compound 

in the plant kingdom, was present abundantly 

only in wild type nodules, but not in the fen1 

nodules. 

Homocitrate is a component of the iron– 

molybdenum cofactor (FeMo-co) in nitrogenase, 

where nitrogen fixation has been thought to occur. 

NIFV, which encodes HCS, has been identified 

from various diazotrophs but is not present 

in most rhizobial species. Therefore, we hypothesized 

that homocitrate supplied from the host 

plant cell cytosol is utilized for biosynthesis of 

FeMo-co and thus enables efficient nitrogen fixation 

by endosymbiotic rhizobia. This hypothesis 

could be confirmed by the facts that inoculation 

with Mesorhizobium loti, a Rhizobium species 

compatible to Lotus plants, carrying FEN1 or 

an authentic NIFV from a free-living nitrogenfixer, 

Azotobacter vinelandii, under the control 

of rhizobial NIFH promoter rescued perfectly 

the defect in nitrogen-fixing activity of the fen1 

nodules (Fig 2). An exogenous supply of homocitrate 

also recovered the nitrogen-fixing activity 

of the fen1 nodules through de novo nitrogenase 

synthesis in the rhizobial bacteroids. These 

results indicate that homocitrate derived from 

the host plant cells is essential for the efficient 

and continuing synthesis of the nitrogenase system 

in endosymbionts (Fig 3), and thus provide 

a molecular basis for the complementary and 

indispensable partnership between legumes and 

rhizobia in symbiotic nitrogen fixation. In addition, 

we suggest that the nodule-specific FEN1 

gene has been recruited from isopropyl-malate 

synthase (IPMS), which catalyzes condensation 

of 2-oxo acid and acetyl-CoA similarly to that of 

HCS and is present almost all living organisms 

as an essential component for leucine biosynthesis. 

Thus, our findings provide an important clue 

for understanding the co-evolution of metabolic 

pathways in two symbiotic partners. 


Fig 1. Symbiotic phenotype of the fen1 mutant. 

a) Growth in N-free medium after inoculation with M. loti. b), c) Nodules formed on fen1. d), e) 

Microphotographs of infected nodule cells. f) Nitrogenase (acetylene reduction) activity of fen1 

through the growth period. 

Fig 2. Complementation of the fen1 

mutant phenotype by inoculation 

of M. loti expressing Lotus FEN1 or 

Azotobacter NIFV. 

Fig 3. A schematic model for the function of FEN1 in symbiotic 

nitrogen fixation. 

Homocitrate produced by FEN1 in plant cell cytosol is supplied to 

bacteroids and utilized for biosynthesis of an active nitrogenase 

complex. 

Reference 

Hakoyama T, Niimi K, Watanabe H, Tabata R, 

Matsubara J, Sato S, Nakamura Y, Tabata S, 

Jichun L, Matsumoto T, Tatsumi K, Nomura 

M, Tajima S, Ishizaka M, Yano K, Imaizumi- 

Anraku H, Kawaguchi M, Kouchi H, Suganuma 

N (2009) Host plant genome overcomes the 

lack of a bacterial gene for symbiotic nitrogen 

fixation. Nature, 462: 514-517. 


Transduction of RNA-directed DNA methylation signals to 

repressive histone marks in Arabidopsis thaliana 

Yoshiki HABU 1 , Hisataka NUMA 2 , Manabu YOSHIKAWA 3 

1 

Genetic Engineering Research Unit, 2 Bioinformatics Research Unit, 3 Plant-Microbe 

Interactions Research Unit 

Heterochromatin is an inert structure in the 

nucleus composed of remnants of transposons 

and repetitive elements and is rich in repressive 

histone modifications. In fission yeast, repressive 

histone marks are recognized by a variety of 

protein complexes, including an RNA-induced 

transcriptional gene silencing complex and 

the Snf2/Hdac-containing repression complex 

that reinforces heterochromatin silencing. In 

mammals, the Mi-2/nucleosome remodeling and 

deacetylating complexes function to repress 

transcription through histone deacetylation and 

binding to methylated cytosines. In Arabidopsis 

thaliana accession Columbia, the major 

heterochromatin regions are located around 

centromeres, nucleolar organizer regions, and a 

region on the short arm of chromosome 4 called 

the heterochromatin knob. Plant heterochromatin 

is abundant in di-methylated histone H3 

lysine 9 (H3K9me2) and cytosine methylation, 

and maintenance of cytosine methylation at 

CG contexts is essential for the structural 

integrity of heterochromatin. Components of 

RNA-directed DNA methylation (RdDM) are 

also required for cytosine methylation, and 

transposons and repetitive sequences in heterochromatin 

are maintained in the inactive state 

through RdDM. Heterochromatin transcripts 

are processed into 24-nt siRNAs by the action 

of RNA processing machineries including 

RNA-DEPENDENT RNA POLYMERASE2 

and DICER-LIKE3, and the resulting siRNAs 

are incorporated into ARGONAUTE4, which 

directs DNA methylation in regions homologous 

to the siRNAs by DOMAINS REARRANGED 

METHYLTRANSFERASE2 (DRM2) in cooperation 

with DEFECTIVE IN RNA-DIRECTED 

DNA METHYLATION1 at all cytosine contexts 

(CG, CHG, CHH; H=A, T, or C). Methylated CG 

and CHG co-localize with H3K9me2, and CHG 

methylation especially is directly connected to 

H3K9me2 by CHROMOMETHYLASE3 (CMT3) 

and KRYPTONITE. However, the effect of 

drm2 and cmt3 mutations on symmetric and 

asymmetric cytosine methylation varies at 

different loci, and therefore complex, redundant, 

and locus-specific pathways have been proposed 

to silence various loci in the genome. 

MORPHEUS’ MOLECULE1 (MOM1) was 

identified in a screen for mutants that releases 

transcriptional gene silencing (TGS) of a cluster 

of transgenes. In addition to transgene TGS, 

mom1 mutants release silencing of remnants of 

transposons and silent copies of 5S rRNA genes. 

mom1 mutants have no effect on global cytosine 

methylation in the genome and in the target 

genes, and maintain morphologically normal 

chromocenters - nuclear foci consisting of 

centromeres and other heterochromatin regions. 

This contrasts sharply with effects observed in 

other mutants in which silent genes and transposons 

are activated with global loss of DNA 

methylation and alteration of nuclear morphology. 

The predicted MOM1 protein has a domain 

with limited similarity to the helicase domain 

of SWI2/SNF2 chromatin remodeling proteins, 

but this domain has recently been shown to be 

dispensable for silencing. Other than the nuclear 

localization signals, only a domain of unknown 

function has been shown to be essential for the 

silencing function of MOM1. 

To better understand the mechanism of TGS 

mediated by MOM1, we performed a global 

comparison of RNA accumulation between the 

wild type plant and mom1 using a genometiling 

array. The majority of up-regulated loci in 


transgenes and 

repetitive sequences 

small RNAs 

DNA methylation 

MOM1 

histone modification 

gene silencing 

Fig 1. The role of MOM1 in the RdDM pathway. 

In the RdDM pathway, aberrant RNAs derived from transgenes and endogenous repetitive 

sequences are processed into 24-nt small RNAs and direct methylation of DNA carrying sequences 

homologous to the small RNAs. DNA methylation and histone modification are intimately linked, and 

DNA methylation induces specific classes of histone modification such as di-methylation at lysine 9 

of histone H3; the detailed mechanism of this link is not known. Our current data have shown that 

MOM1 is involved in this link and transduces the signal of DNA methylation to histone modification 

for maintenance of gene silencing. 

mom1 carry remnants of transposons, mostly 

those of gypsy-like retrotransposons, and are 

clustered around the centromeric heterochromatin 

regions and the heterochromatin knob on 

chromosome 4. In addition, our data show that 

MOM1 is required for silencing of the singlecopy 

gene, SUPPRESSOR OF drm1 drm2 

cmt3, which is not related to transposons and is 

silenced through non-CG methylation at tandem 

repeats in the promoter. Furthermore, our data 

indicate that MOM1 transduces RdDM signals 

to repressive histone modification in genomic 

regions showing a characteristic dependency of 

CG methylation on non-CG methylation (Fig 1). 

References 

Habu Y (2010) Epigenetic silencing of endogenous 

repetitive sequences by MORPHEUS’ 

MOLECULE1 in Arabidopsis thaliana. 

Epigenetics, 5: 562-565. 

Habu Y, Yoshikawa M (2010) Locus-specific dependency 

of endogenous silent loci on MOM1 

and non-CG methylation in Arabidopsis thaliana. 

Plant Signaling & Behavior, 5: 724-726. 

Numa H, Kim J-M, Matsui A, Kurihara Y, 

Morosawa T, Ishida J, Mochizuki Y, Kimura H, 

Shinozaki K, Toyoda T, Seki M, Yoshikawa M, 

Habu Y (2010) Transduction of RNA-directed 

DNA methylation signals to repressive 

histone marks in Arabidopsis thaliana. The 

EMBO Journal, 29: 352-362. 


Unique RNAi by RNA silencing inducible sequence in rice. 

Fumio TAKAIWA, Hiroshi YASUDA, Yuhya WAKASA, Taiji KAWAKATSU 


RNA silencing is a powerful technology which 

has been widely used for reverse genetics and 

improving crop traits. RNA interference (RNAi) 

is generated through post-transcriptional gene 

silencing (PTGS) through which an artificial 

double-stranded RNA (dsRNA) of a target 

gene is processed into small interfering RNA 

molecules (siRNA), triggering target mRNA 

degradation. 

Many valuable crops have been developed 

by improving their agricultural traits through 

conventional breeding, such as utilization of 

natural variants as genetic resources or the 

use of induced mutations by treatment with 

chemical mutagens or γ-irradiation. However, 

conventional breeding approaches are hampered 

by limits in the natural gene pool, interspecies 

fertility barriers, gene duplication, restricted 

availability of mutants, and genetic variations. 

Therefore, genetic engineering technology, 

including RNAi, has been used to improve the 

agricultural traits and physiological quality of 

crop plants by directly manipulating target 

genes. 

Induction of artificial RNA silencing is becoming 

a more generally available and applicable 

technology and has great potential as an 

option for expanding breeding strategies. We 

previously reported that RNA silencing of the 

endogenous GluB subfamily, as well as modified 

glucagon-like peptide-1 (mGLP-1), was observed 

in endosperms of transgenic rice plants containing 

the mGLP-1 sequence under the control 

of the glutelin GluB-1 promoter containing its 

signal peptide (Fig 1). GLP-1 is a 30-amino acid 

serum peptide hormone produced by distal 

Fig 1. Suppression of seed storage proteins using RSIS. 

(left panel) Transformed constructs. (right panel) SDS-PAGE of seed proteins in wild type and 

transformants. In GluB less, GluB family (*) were suppressed. In GluB Glb less, GluB family and globulin 

were suppressed. In 13kD Proless, 13kD prolamins were suppressed. 


ileum L cells that stimulates insulin secretion 

from pancreatic β-cells, depending upon blood 

glucose concentrations. 

We showed that many genes expressed 

in various organs were easily and effectively 

suppressed by mGLP-1-mediated silencing in 

transgenic rice and that the mGLP-1 sequence 

acts as an RNA silencing inducible sequence 

(RSIS). Even multiple target genes with no 

homology to each other can be simultaneously 

suppressed by expressing RSIS linked to unique 

sequences derived from the target genes (Fig 1). 

Furthermore, by stacking several RSIS expression 

cassettes into a binary vector, more than 

3 genes could be suppressed through a single 

transformation procedure (Fig 2). 

Transgenic lines expressing target genederived 

siRNAs as well assiRNAs derived from 

RSIS were generated. These results suggest 

that RSIS-containing RNAs form dsRNAs that 

trigger siRNA biogenesis, followed by RNA 

silencing. 

Genetic engineering using RSIS-mediated 

RNAi may offer a useful technology for downregulating 

target genes in a dominant and 

tissue-specific manner. RSIS-mediated RNAi can 

be used as a convenient tool for the analysis of 

gene function, or for improving the physiological 

functions of crop plants (Fig 3). 

Fig 2. Multiple silencing by stacking RSIS expression cassettes. 

(left and middle panels) Transformed constructs. (right panel) SDS-PAGE of seed proteins in wild type 

and transformants. In 3-gluless, almost all glutelins were suppressed. In sspless, glutelins, globulin 

and 13kD prolamins were suppressed. 

Fig 3. Supposed model of RSIS-mediated RNA silencing and future perspectives. 

RNAs containing RSIS and partial sequences of target genes are transcribed from RSIS expression 

cassettes, and form dsRNAs, that trigger siRNA biogenesis, followed by RNA silencing. Using 

RSIS, multiple genes can be suppressed simultaneously. RSIS-mediated RNAi can be used as a 

convenient tool for the analysis of gene function, or for improving the physiological functions of crop 

plants, such as manipulating metabolic pathways and reducing multiple allergens. 


A new juvenile hormone isolated from a heteropteran 

insect 

Toyomi KOTAKI 

Invertebrate gene function research unit 

Juvenile hormone (JH) is a sesquiterpenoid insect 

hormone that controls metamorphosis and 

reproduction. It also plays roles in regulating 

various aspects of insect development, for example, 

diapause, polyphenisms, caste differentiation, 

mating behavior and pheromone production. 

Because JH is insect-specific, chemicals with JH 

activity can be used as insect control agents or 

insecticides with a low environmental impact. 

In fact, such insecticides are commercially available. 

Thus far, several forms of JH are known 

in different insect orders. The structure of JH in 

the suborder Heteroptera is, however, not identified 

yet, in spite of the fact that Heteroptera 

includes many economically important pests and 

a number of JH-related studies, including the 

first discovery of JH as a humoral factor, have 

been conducted in heteropteran insects. The 

purpose of the present study is to elucidate the 

structure of heteropteran JH using a stink bug, 

Plautia stali, a serious pest attacking fruit trees 

(Fig 1). 

To obtain structural information of putative 

heteropteran JH, corpora allata (CA), endocrine 

glands producing JH, were taken out of adults 

of P. stali and incubated in vitro. The product 

released into culture medium was extracted and 

subjected to mass spectrometry. As a result, 

compositional formula for the CA product was 

estimated to be C 16 H 26 O 4 . Based on this estimate 

and results of previous studies, the structure of 

heteropteran JH was deduced to be a bisepoxide 

of methyl farnesoate. A mixture containing 

32 bisepoxides of methyl E,E-farnesoate and 

methyl E, Z-farnesoate was prepared and then 

separated into >20 fractions using HPLC. Each 

fraction was assayed for JH activity (Fig 1), and 

two fractions were found to have JH activity. 

NMR analysis revealed that compounds in 

two fractions were a pair of stereoisomers 

expressed in the same structural formula (Fig 

2 a-d). These four stereoisomers were, however, 

not distinguishable from one another by NMR 

analysis. For this reason, each isomer was 

synthesized in a stereochemically pure form by 

asymmetric synthesis technique. HPLC analysis 

indicated that the elution time for compounds 1 

and 2 coincided with that for the two JH active 

fractions obtained from the bisepoxide mixture. 

To examine whether either compounds 1 or 2 

corresponded to the natural CA product, these 

samples were subjected to GC-MS analysis. 

The retention time and mass spectrum for 

natural CA product were identical to those 

for compound 2. Therefore, the structure of 

heteropteran JH was identified as compound 

2. Synthetic standard of compound 2 inhibited 

metamorphosis of P. stali nymphs in a dosedependent 

fashion (Fig 3) and was more potent 

than JH III (Fig 2 e). Based on the arrangement 

of two epoxy rings, this novel JH was named 

Juvenile Hormone III Skipped Bisepoxide 

(JHSB3). 

Because JHSB3 is novel and Heteropteraspecific, 

it can be a lead compound to develop 

insecticides selective to heteropterans with 

a low impact on non-target organisms. 

Likewise, at least a part of any physiological 

processes involved in JHSB3 is expected to be 

heteroptera-specific. Elucidating these processes 

may provide new insights into development of 

Heteroptera-specific control agents. 


Fig 1. A last instar nymph (A), an adult (B) and a nymph-adult intermediate (C) of P. stali. 

obtained by treatment of a test sample with JH activity. Scale bar: 5 mm. 

a 

c 

e 

b 

d 

Fig 2. Four stereoisomers of methyl 2,3;10,11-bisepoxyfarnesoate (a-d) as candidates of 

heteropteran JH and JH III (e). 

Compound b was found to be the natural JH in P. stali. 

Fig 3. Identification of JH in P. stali as compound b by GC-MS. 

A mixture of compounds a and b (black line), CA product (blue line) and a mixture of compounds a 

and b and the CA product (red line) were analyzed. Extracted ion chromatography for [M + NH 4 ] + ions 

at m/z 300 (CI, NH 3 ). 

Relative scutellum length 

0.7 

0.6 

0.5 

0.4 

0 

S 0.001 0.01 0.1 1 10 

Dose (µ g) 

Fig 4. Effect of JHSB 3 (open circle) and JH III (solid circle) on last instar nymphs of P. stali. 

Last instar nymphs were treated with JHSB 3 or JH III on the day of 4th ecdysis. After the following 

ecdysis, the relative length of scutellum was measured. “S” on the horizontal axis indicates solvent 

controls treated with hexane. 


Bombyx prothoracicostatic peptides activate the sex 

peptide receptor to regulate ecdysteroid biosynthesis 

Yoshiaki TANAKA 

Insect Gene Function Research Unit 

Insect molting and metamorphosis are 

induced by steroid hormones named ecdysteroids. 

Their biosynthesis had been thought 

to be regulated mainly by prothoracicotropic 

hormone (PTTH), which is known to stimulate 

ecdysteroid production in the prothoracic gland 

(PG) of insects. Recently, there is also growing 

evidence that the central neuroendocrine system 

exerts a prothoracicostatic effect in addition 

to PTTH, thus generating a timely fluctuation of 

the ecdysteroid titer in the hemolymph during 

insect development. Prothoracicostatic peptide 

(PTSP) is the first neuropeptide shown to suppress 

ecdysteroid production in the PG of the 

silkworm, Bombyx mori, but its physiological 

role in the ecdysteroid production has not been 

clearly explored yet. We have now characterized 

a Bombyx G protein-coupled receptor, 

which had previously been identified as an 

ortholog of the Drosophila sex peptide receptor 

(SPR), as a functional PTSP receptor. So we 

call this receptor Bombyx PTSPR/SPR. Bombyx 

PTSPR/SPR responded specifically to the 

PTSPs when examined using a heterologous 

expression system (Fig 1). PTSPR/SPR was 

highly expressed in the PG on the day 

before each larval and pupal ecdysis (Fig 2) 

when PTSPs are synthesized at a high level in 

the epiproctodeal glands - the peripheral neurosecretory 

cells that are located just anterior to 

the rectum. These results suggest that Bombyx 

PTSPR/SPR functions as a PTSP receptor 

in Bombyx and that the peripheral neurosecretory 

cells as well as the central neuroendocrine 

system play stage-specific roles in regulating ecdysteroid 

production in the PG, thus creating 

the finely-tuned fluctuation of ecdysteroid titer 

in hemolymph during insect development (Fig 3). 

Fig 1. Functional characterization of Bombyx PTSPR/SPR. 

Ca 2+ imaging analysis was performed using HEK293 cells expressing PTSPR/SPR. Doseresponse 

curves for PTSP-I, -III, -V and -VIII are shown. 


Fig 2. Tissue specificity of Bombyx PTSPR/SPR expression analyzed by quantitative RT-PCR. 

Transcript levels of PTSPR/SPR in the fourth instar head capsule slippage period are indicated in 

black, while those in the fifth instar day 2 are indicated in gray. PG: prothoracic gland; BR: brain; FB: 

fat body; MG: midgut; SG: silk gland; MT: Malpighian tubule; OV: ovary; TE, testis. 

Fig 3. Proposed model for the regulation of ecdysteroid production in the PG by various 

neuropeptides. 

PTTH and Bommo-myosupressin (BMS) are released from the brain and Bommo-FMRFamide (BRFa) 

is transported to PG by neural innervation. PTSP is released from peripheral neurosecretory cells 

(epiproctodeal gland) to suppress the ecdysteroid production. 


Experimental production of wedding dress from high 

quality silk produced by transgenic silkworm through the 

collaboration of different industries 

Hiroaki MACHII 1 , Tetsuya IIZUKA 1 , Hideki SEZUTSU 1 , Naoyuki YONEMURA 1 , Ken-ichiro 

TATEMATSU 1 , Keiro UCHINO 1 , Isao KOBAYASHI 1 , Chiyuki TAKABAYASHI 2 , Keisuke 

MASE 1,2 , Eiji OKADA 1,2 , Kenichi NAKAJIMA 2 , Toshiki TAMURA 1 

1 

Transgenic Silkworm Research Center, 2 Silk Technology Unit 

In order to restore the sericulture industry in 

Japan and create a new industry, it is vital to 

differentiate domestic silk from low-priced silk 

produced in China, Brazil and other countries 

and to develop a method to make silk of high 

added value. So far, our institute has developed 

a method to make high quality silk using 

transgenic silkworms, has developed a new 

method for reeling silk from transgenic cocoons, 

and has succeeded in making knit products 

from fluorescent color silk. However, remaining 

challenges include standardizing procedures 

for rearing transgenic silkworms on a largescale, 

demonstrating the weaving performance 

of fluorescent color silk as the warp thread, and 

instituting a process to obtain final products 

through the collaboration of different industries. 

Therefore, in order to stimulate the practical 

realization of transgenic silk, we developed a 

production manual to rear transgenic silkworms, 

wove a fabric with fluorescent color silks as 

warp threads, and produced high quality textiles 

with the collaboration of different industries. 

1. Improvement of transgenic silkworm 

strains producing high quality silks and making 

a manual to rear transgenic silkworms on 

a large-scale 

We improved transgenic silkworm varieties 

producing fluorescent silk and ultrafine silk 

and improved their quantitative traits such as 

cocoon weight, cocoon shell weight and cocoon 

shell percentage to equal or exceed the same 

traits in standard, non-transgenic varieties. 

We reared 40,000 silkworms of the transgenic 

varieties with green fluorescent color silk, 30,000 

silkworms of the transgenic varieties with red 

fluorescent color silk, and 140,000 silkworms of 

the transgenic varieties with ultrafine silk, which 

produced 8.1kg, 7.5kg and 24.8kg of raw silk, 

respectively. Moreover, we developed a rearing 

manual with standardized procedures suitable 

for the Cartagena Protocol on Biosafety based on 

the rearing data for these transgenic silkworms. 

2. Experimental production of wedding and 

reception dresses from high quality silks 

produced by transgenic silkworms through 

the collaboration of different industries 

A wedding dress and a colorful dress to be 

worn during the wedding reception were made 

as an experimental production using the high 

quality silks. Through the collaboration of public 

sectors and private companies such as Yumi 

Katsura International Co. Ltd, the wedding dress 

was made from silk satin “Mikado” woven with 

green fluorescent silk (Fig 1). The colorful dress 

for the wedding reception was made with the 

satin silk woven with the green fluorescent silk 

and red fluorescent silk as warp thread and 

woof thread, respectively, and a silk organdie 

was woven with red fluorescent silk (Fig 2). 

The interweaving resulted in the reception 

dress having a yellow-green fluorescent color. 

Moreover, through the collaboration of private 

companies and public sectors, we made a doll of 

the emperor and empress dressed in traditional 

costume with embroidery design using green 

fluorescent silk and red fluorescent silk (Fig 3). 

High quality silks, such as the fluorescent color 

silks, produced by transgenic silkworms can provide 

material for many industries such as textile, 

apparel and fashion industries. Therefore, it is 

an urgent issue to establish a large-scale rearing 

system for production of transgenic silkworms 


at the farmer’s level which also conforms to the 

Cartagena Protocol on Biosafety. 

References 

Tamura T, Iizuka T, Sezutsu H, Tatematsu 

K, Kobayashi I, Yonemura N, Uchino K, 

Kojima K, Machii H, Takabayashi C, Yamada 

K, Kurihara H, Asakura T, Nakazawa Y, 

Miyawaki A, Karasawa T, Kobayashi H, 

Yamaguchi J, Kuwabara N, Nakamura T, 

Yoshii K (2009) Production of high quality 

silks having different fluorescent colors using 

transgenic silkworms. AFF Research Journal, 

32(3): 7-10. 

Fig 1. Wedding dress showing green fluorescent color (Texture material: silk satin “Mikado”) 

(Left: white light, Center & Right: blue LED) 

 

S 

Fig 2. Colorful dress worn during the wedding reception was made with green fluorescent silk and red 

fluorescent silk (Texture material: silk satin, silk organdie) 

(Left: white light, Center & Right: blue LED) 

Fig.3 Doll of the emperor and empress dressed in traditional costume with embroidery design using 

green fluorescent silk and red fluorescent silk 

(Upper, left & right: white light, Center two: blue LED) 


Production of piglets from oocytes after injection of 

spermatozoa grown in immunodeficient mice 

Kazuhiro KIKUCHI 1 , Hiroyuki KANEKO 1 , Michiko NAKAI 1 , Naomi KASHIWAZAKI 2 

1 

Reproductive Biology Research Unit, 2 Azabu University 

Cryopreservation of male genetic resources 

is limited for ejaculated spermatozoa obtained 

from male animals. After thawing, preserved 

semen is widely used for artificial insemination 

in the animal industry. However, this method is 

acceptable only for mature male animals. The 

chances for the collection of spermatozoa and 

the quantity of spermatozoa per ejaculation are 

limited. Furthermore, we cannot collect any 

spermatozoa from juvenile animals. Freezing 

of spermatozoa - or semen banking - is not sufficient 

for conservation of male animal genetic 

resources. Recently, a new conservation method 

has been demonstrated in which spermatozoa 

can be grown and matured in testicular tissues 

that have been xenografted to immunodeficient 

mice. In the present study, we examine the 

competence of fertilization of porcine spermatozoa 

obtained from xenografts and the likelihood 

of development to embryos, fetuses and piglets. 

We performed several procedures; xenografting 

of testicular tissues to mice, collection of 

spermatozoa, in vitro maturation of porcine 

oocytes from slaughterhouse materials, intracytoplasmic 

sperm injection, in vitro culture 

of embryos and zygote transfer to recipient 

female pigs (Fig 1). Donor testes were obtained 

from 6- to 12-day old crossbred piglets. The 

seminiferous cords of donor testicular tissue at 

the time of grafting contained only gonocytes 

and spermatogonia. Immediately after removal 

of the testis, the cortex was cut into small 

pieces (1.5 mm cubes). Five- to 8-week-old male 

nude mice were anesthetized and received 

transverse linear incision in their back skin, and 

a subcutaneous space was created for grafting. 

Approximately 30 pieces of donor testicular tissue 

were then inserted (Day 0). On Day 118-280, 

we noticed the growth of grafted tissues (Fig 

2) and could collect spermatozoa after mincing 

of the tissues in 19 out of 27 euthanized mice 

(Fig 3). Some of the spermatozoa showed faint 

motility. Histological examination suggests the 

development of the seminiferous tubules in the 

grafts. Spermatozoa collected from the mice on 

Day 133-280 were injected into in vitro matured 

oocytes. They could be fertilized as confirmed 

by the formation of male and female pronuclei 

Fig 1. Procedures from xenografting to production of piglets 


Fig 2. Growth of grafted testicular tissues (arrows) on Day 125 after xenografting 

Fig 3. Sperm collected from minced tissues (Left, arrow) 

and its magnified typical one (Right) on Day 125 after 

xenografting 

Fig 4. A 50 days old-male piglet 

and development to the blastocyst stage after in 

vitro culture. Those embryos were examined by 

chromosomal analysis and found to have normal 

diploid sets suggesting normal competence for 

fertilization and embryo development by the 

spermatozoa obtained from the grafts (Nakai 

et al, 2009). Furthermore, we transferred the 

zygotes after intracytoplasmic sperm injection 

into the recipient females of which estrous was 

synchronized. Four out of 23 recipients became 

pregnant, and two went to term and delivered 

a total of 6 piglets (one female and five males) 

(Fig 4). The piglets were growing normally and 

showed sexual maturity. Until now, the only 

report that offspring can be obtained with the 

sperm from xenografts was found in rabbits 

(Shinohara et al, 2002). Our report is the first 

one for pigs - a large domesticated mammalian 

species (Nakai et al, 2010). 

We should confirm the reproductive abilities 

of the piglets in the next generation. This technology 

will be much more beneficial when the 

testicular tissues can be cryopreserved before 

xenografting. It is also acceptable not only for 

domesticated animals but also for genetically 

modified animals, juveniles and for high-value 

animals that die suddenly from accidents or 

diseases. Furthermore, xenografting or cryopreservation 

of ovarian tissues might also be 

possible for female genetic resources. 

References 

Nakai M, Kaneko H, Somfai T, Maedomari N, 

Ozawa M, Noguchi J, Kashiwazaki N, Kikuchi 

K (2009) Generation of porcine diploid blastocysts 

after injection of spermatozoa grown in 

nude mice. Theriogenology, 72: 2-9. 

Nakai M, Kaneko H, Somfai T, Maedomari N, 

Ozawa M, Noguchi J, Ito J, Kashiwazaki N, 

Kikuchi K (2010) Production of viable piglets 

for the first time using sperm derived from 

ectopic Testicular xenografts. Reproduction, 

139: 331-335. 

Shinohara T, Inoue K, Ogonuki N, Kanatsu- 

Shinohara M, Miki H, Nakata K, Kurome M, 

Nagashima H, Toyokuni S, Kogishi K, Honjo 

T, Ogura A (2002) Birth of offspring following 

transplantation of cryopreserved immature 

testicular pieces and in vitro microinsemination. 

Human Reproduction, 17: 3039–3045. 


Completion of draft sequencing of the pig genome 

Hirohide UENISHI 1 , Takeya MOROZUMI 2 , Hiroyuki KANAMORI 2 , Tomoko EGUCHI- 

OGAWA 1 , Naoe FUJISHIMA-KANAYA 2 , Toshimi MATSUMOTO 2 , Ayumi MIKAWA 2 , 

Naohiko OKUMURA 2 , Hiroki SHINKAI 1,2 , Kohei SUZUKI 2 , Maiko TANAKA-MATSUDA 2 , 

Daisuke TOKI 2 , Takahito BITO 2 , Nahoko FUJITSUKA 2 , Kozue KAMIYA 2 , Kanako KURITA 2 , 

Ari KIKUTA 2 , Harumi YAMAGATA 2 

1 

Animal Genome Research Unit, National Institute of Agrobiological Sciences, 2 Institute 

of Society for Techno-innovation of Agriculture, Forestry and Fisheries 

The pig genome consists of 18 pairs of autosomes 

and one pair of sex chromosomes. The 

size of the entire genomic sequence is about 2.7 

Gb, comparable with or slightly smaller than 

that of human. Genome information is essential 

for physiological analyses in pigs as well as 

other organisms. The international community 

for pig researchers has made efforts to develop 

components needed for sequencing of the entire 

pig genome, such as construction of a physical 

map of the pig genome by using radiation 

hybrid panels. Based on such analyses, sequencing 

of the entire pig genome was planned by 

the International Swine Genome Sequencing 

Consortium (SGSC), which was established in 

2003. SGSC consists of research institutes and 

universities in 12 countries and regions, including 

the National Institute of Agrobiological 

Sciences (NIAS) and the Institute of Society for 

Techno-innovation of Agriculture, Forestry and 

Fisheries (STAFF) from Japan. 

SGSC used a female Duroc pig, named T. J. 

Tabasco (Fig 1), for the genome sequencing. 

Sequencing was conducted by the “hybrid 

approach” composed of hierarchical and wholegenome 

shotgun sequencing. For hierarchical 

Fig 1. The female pig of Duroc breed subjected to 

draft genome sequencing, T. J. Tabasco. 

shotgun sequencing, a bacterial artificial 

chromosome (BAC) library was constructed 

with genomic DNA of T. J. Tabasco. The library 

was used for construction of a minimum-tiling 

path of the BAC clones covering the entire pig 

genome. The clones in the minimum-tiling path 

were subjected to shotgun sequencing at a 4 to 

8́ depth. The shotgun sequencing was mainly 

performed by the Wellcome Trust Sanger 

Institute, and the Japanese research team, 

Animal Genome Research Program (AGP) which 

consists of NIAS and STAFF, took charge of 

254 BAC clones located on chromosome 6 and 

7 (Fig 2). The 254 BAC clones corresponded to 

1.6% of the entire genome. AGP conducted the 

shotgun sequencing of each BAC clone at a 7.92 

× depth on average. Finally, SGSC conducted 

sequencing of 17,000 BAC clones and announced 

completion of draft sequencing of the pig 

genome in November 2009. In the announcement 

SGSC declared completion in sequencing 

of 98% of the physical map of the pig genome 

(Fig 3). The assembled pig genome sequence 

can be available in the Ensembl Database of the 

European Molecular Biology Laboratory (http:// 

www.ensembl.org/Sus_scrofa/Info/Index). On 

the other hand, whole genome shotgun sequencing 

of the same individual conducted by the 

Sanger Institute and groups of Korea and China 

will be integrated shortly in the next version of 

genome sequence assembly. 

The genome sequence in the Ensembl 

database is now being subjected to genome 

annotation by several groups that are interested 

in particular fields such as immunity or lipid 

metabolism. In the genome annotation, information 

of gene transcripts, particularly full-length 


cDNA sequences, is invaluable for correct 

mapping of exons of genes. In addition to the 

genome sequencing, we have also conducted 

full-length-enriched cDNA library construction 

for various tissues and cell populations of pigs, 

analyzed expressed sequence tags (ESTs), and 

sequenced the entire inserts of the cDNA clones. 

We have accumulated more than 280,000 ESTs 

mainly derived from full-length-enriched cDNA 

libraries, and completed sequencing of about 

25,000 cDNA clones extracted from the clones 

subjected to the EST analysis. Information of 

the cDNA and ESTs may be accessed through 

the Pig Expression Data Explorer (http://pede. 

dna.affrc.go.jp/) with results of BLAST similarity 

search, as well as through public nucleotide 

databases (DDBJ/EMBL/GenBank). This is 

a significant contribution to the annotation 

process of the pig genome. 

The draft sequence of the pig genome will 

generate much benefit in research of livestock 

and biomedical sciences. It enables us to localize 

many markers on the whole genome and to 

conduct analyses for detection of relationships 

between useful traits and genomic regions, 

which will contribute to pig breeding based on 

molecular information. Furthermore, information 

about the pig genome accelerates the use of pigs 

as biomedical model animals, because the body 

size of pig is comparable to human. Moreover, 

physiological systems such as cardiovascular 

system in pigs show high similarity to the 

human system. Pigs are regarded as large experimental 

animals for analyses that cannot be 

conducted with mouse or other small animals. 

To make the genome sequence more valuable, 

SGSC will continue the effort to improve the 

accuracy of the pig genome sequence. 

Chromosome 6 

(169.5 Mb) 

36.0 Mb 

Chromosome 7 

(129.2 Mb) 

6.3 Mb 

Contribution by AGP 

Fig 2. Contribution of the Animal Genome Research Program (AGP) in the International 

Swine Genome Sequencing Consortium (SGSC). 

AGP conducted sequencing of the genomic regions, which correspond to 42.3 Mb, on 

chromosome 6 and 7. 

Coverage of each chromosome (%) 

100 

90 

80 

70 

60 

50 

40 

30 

20 

10 

0 

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 X 

Chromosome 

Fig 3. Sequencing status of the pig genome at the announcement of completion of draft 

genome sequencing by SGSC. 

A Bar indicates the ratio of the region that is already sequenced in each chromosome. 


Research Activities 


QTL Genomics Research Center 

Genetic dissection of quantitative trait loci 

controlling stomatal conductance and chlorophyll 

content in rice 

Increasing stomatal conductance (gsc) to raise 

CO 2 supply to CO 2 fixation sites in leaves is 

suggested as one of the ways to improve yield 

potential in rice. Carbon isotope discrimination 

(Δ13C) and stomatal density may be indirect 

selection criteria to increase gsc provided 

that the traits are strongly related to gsc. We 

attempted to identify QTLs controlling Δ13C 

and stomatal density, and to elucidate their 

association with gsc and leaf photosynthesis. 

Substitution mapping by chromosome segment 

substitution lines (CSSLs) developed between 

a japonica cultivar Koshihikari and an indica 

cultivar Kasalath and subsequent advanced 

backcross QTL analysis detected a QTL for 

Δ13C at the vegetative and heading stages 

and for stomatal density at the heading stage 

within the same region on the long arm of 

chromosome 3. SL208, a backcross line in which 

most regions on chromosome 3 were substituted 

with the Kasalath segment, showed higher gsc 

than Koshihikari at vegetative stage, suggesting 

there may be a QTL pleiotropically controlling 

gsc and Δ13C at the vegetative stage on the 

long arm of chromosome 3. However, a QTL 

delaying heading stage, considered as Hd6, 

also was located within this region. Subsequent 

study using Kanto IL5, a Koshihikari-Hd6-near 

isogenic line, elucidated that Hd6 pleiotropically 

affected Δ13C and stomatal density by delaying 

the heading stage, but did not affect Δ13C and 

gsc during the vegetative stage. These results 

suggested that there should be a QTL controlling 

gsc and thus Δ13C at least during the 

vegetative stage located near Hd6 on the long 

arm of chromosome 3. 

Chlorophyll content is another important 

factor for leaf photosynthesis, a determinant of 

yield potential in rice. We searched for quantitative 

trait loci (QTLs) for Soil and Plant Analyzer 

Development (SPAD) value, an index of leaf 

chlorophyll content, and assessed their association 

with leaf photosynthesis. QTL analysis of 

lines derived from a cross between japonica cultivar 

Sasanishiki and high-yielding indica cultivar 

Habataki (Fig 1a) detected a QTL for SPAD 

value on chromosome 4. This QTL explained 

31% of the total phenotypic variance, and the 

Habataki allele increased the SPAD value. The 

CSSL with the corresponding segment from 

Habataki had a higher leaf photosynthetic rate 

and SPAD value than Sasanishiki, suggesting 

an association between SPAD value and leaf 

photosynthesis. The CSSL also had a lower specific 

leaf area (SLA) than Sasanishiki, reflecting 

its thicker leaves (Fig 1b and 1c). Substitution 

mapping in the Sasanishiki genetic background 

demonstrated that QTLs for SPAD value and 

SLA were co-localized in a 1798-kb interval. The 

results suggest that the phenotypes for SPAD 

value and SLA are controlled by a single locus 

or two tightly linked loci, and may play an important 

role in increasing leaf photosynthesis by 

increasing chlorophyll content or leaf thickness, 

or both. 


a) 

b) 

Habataki 

Dark green 

Sasanishiki 

Pale green 

 

 

 

c) 

SPAD value 

P n (μmol m -2 s -1 ) 

50 

40 

30 

20 

10 

0 

40 

30 

20 

10 

a 

a 

b 

b 

c 

c 

g s (mol m -2 s -1 ) 

SLA (cm 2 g -1 ) 

1.2 

0.9 

0.6 

0.3 

0.0 

210 

140 

70 

a 

a 

a 

b 

b 

ab 

Fig 1. (a) Difference of leaf color, which is an 

index of chlorophyll content, and association 

with leaf photosynthesis in Sasanishiki 

(japonica) and Habataki (indica). (b) Graphical 

genotype of SL414, a substitution line with 

the corresponding segment from Habataki 

in the Sasanishiki genetic background. (c) 

Comparisons of photosynthesis-related 

traits of flag leaves at heading stage among 

Sasanishiki, SL414, and Habataki. 

P n : leaf photosynthetic rate, g s : stomatal 

conductance, SLA, specific leaf area. 

0 

0 

Sasanishiki 

SL414 

Habataki 

Sasanishiki 

SL414 

Habataki 

Detection of QTLs for agronomically important 

traits in japonica rice cultivars 

While a considerable level of phenotypic 

variations could be observed among japonica 

rice cultivars, an extremely low frequency of 

DNA polymorphisms has prevented efficient 

genetic analysis for the agronomic traits among 

japonica rice cultivars. Recently, the genome 

sequence of japonica rice cultivar ‘Koshihikari’ 

was obtained by using next-generation sequencing 

technology. A large number of single 

nucleotide polymorphisms (SNPs) could be 

detected between Nipponbare and Koshihikari. 

These recent advances in molecular marker 

development have allowed effective large-scale 

screening for DNA polymorphism among japonica 

cultivars, and have greatly facilitated the 

study of quantitatively inherited traits among 

japonica cultivars. 

To identify quantitative trait loci (QTLs) 

associated with agronomically important traits 

in japonica rice cultivars, we developed a set of 

reciprocal backcrossed inbred lines (BILs) and 

chromosome segment substitution lines (CSSLs) 

derived from crosses between Nipponbare and 

Koshihikari. Using the Nipponbare/Koshihikari 

BILs and CSSLs, we tried to identify QTLs for 

pre-harvest sprouting resistance. Koshihikari 

showed strong resistance to pre-harvest sprouting, 

whereas Nipponbare showed moderate preharvest 

sprouting resistance (Fig 2a). In the 

BILs, we detected one major QTL on the short 

arm of chromosome 3 that accounted for 45% of 

the phenotypic variance and the genetic effect 

of this QTL has been confirmed using CSSLs. 

This QTL was further fine-mapped within a 474- 

kbp region by means of substitution mapping. 

Finally, this QTL was found to be co-localized 

with the low-temperature germinability gene 

qLTG3-1. The level of germinability under 

low temperature is strongly correlated with 

the level of pre-harvest sprouting resistance 

in the SLs. In addition, sequencing analysis 

revealed that Koshihikari qLTG3-1 was a loss 


of function allele with a 71-bp deletion, whereas 

Nipponbare qLTG3-1 was a functional allele. 

These results clearly suggested that the allelic 

difference in qLTG3-1 is likely to be associated 

with differences in both pre-harvest sprouting 

resistance and low-temperature germinability in 

Nipponbare and Koshihikari. 

Short-culm and dwarfism are desirable in rice 

breeding to improve lodging resistance and, as 

a result, yield. Koshihikari shows weak lodging 

resistance due to its slender and slightly long 

culm, whereas Nipponbare shows a shorter culm 

than Koshihikari and exhibits stronger lodging 

resistance (Fig 2b). A QTL, qCL1, which is 

involved in the culm-length difference between 

Nipponbare and Koshihikari, was detected on 

the short arm of chromosome 1 in the BILs 

in each of three consecutive growing seasons. 

The Nipponbare allele of qCL1 shortened culm 

length about 3.0 cm. qCL1 was mapped to a 2.6- 

Mbp region of the distal side of the long arm of 

chromosome 1, suggesting that qCL1 differed 

from the dwarf (d18) or semi-dwarf (sd1) genes 

that have been previously reported. We then 

conducted association analysis between culm 

length and SSR alleles near the qCL1 among 

Japanese japonica rice landraces and modern 

cultivars. Clear association was observed in the 

qCL1 region, suggesting that the novel allele 

for short culm length was originally distributed 

in Japanese rice landraces and has been used 

for introducing the semi-dwarf phenotype and 

improving plant architecture during practical 

breeding of modern cultivars. 

Clear phenotypic differences between 

Nipponbare and Koshihikari were observed 

in heading date, resistance to leaf blast, culm 

length, cool temperature tolerance at the 

booting stage, pre-harvest sprouting resistance, 

seed size and eating quality. We have already 

identified representative QTLs for respective 

agronomic traits by using Nipponbare/ 

Koshihikari BILs and CSSLs (Fig 3). Recently, 

whole genomic sequences have been decoded 

by the next-generation sequencing technology. 

Once we identified the QTL of those traits, it 

is easy to compare both genomic sequences 

to detect sequence polymorphisms. Extremely 

low level of sequence polymorphisms between 

the two cultivars may turn out to be strong 

advantages for identifying candidate functional 

nucleotide polymorphisms. Functional identification 

of QTLs for heading date, seed size and 

eating quality are now in progresses. 

a 

b 

Germination (%) 

120 (cm) 

100 

80 

Nipponbare 

100 

80 

Panicle 

60 

60 

1st internode 

40 

Koshihikari 

40 

2nd internode 

20 

0 

4 5 6 7 8 9 10 

Weeks after heading 

20 

0 

NipponbareKoshihikari 

** 3rd internode 

** 

* 

4th internode 

5th and 6th internode 

NipponbareKoshihikari 

Fig 2. Phenotypic differences in pre-harvest sprouting and culm length between Nipponbare and Koshihikari: 

(a) Changes in germination percentages from 4 to 10 weeks after heading. Red and blue lines indicate 

germination percentages for Nipponbare and Koshihikari, respectively. Panicles of Nipponbare (left) and 

Koshihikari (right) are at 7 weeks after heading. (b) Diagram of the panicle and each internode length of 

Nipponbare and Koshihikari. * and ** indicate significantly different lengths between Nipponbare and 

Koshihikari at P < 0.05 and P < 0.01, respectively. 


Eang quality 

Resistance to pre-harvest sproung 

Heading date 

 

Culm length 

Grain size 

Resistance to 

rice blast 

Heading 

date 

Cold tolerance at 

boong stage 

Fig 3. Chromosomal locations of major QTLs controlling several traits of agronomic interest. 

All QTLs were detected by using backcross inbred lines and chromosome segment substitution lines 

between Nipponbare and Koshihikari. 


26-Week oral safety study in macaques for 

transgenic rice containing major human T-cell 

epitope peptides from Japanese cedar pollen 

allergens. 

A study of repeated oral administration of 

transgenic rice containing a hybrid peptide 

of major human T-cell epitopes (7Crp) from 

Japanese cedar pollen allergens was carried 

out in cynomolgus macaques over 26 weeks. 

The monkeys were divided into three groups, 

each comprising three males and three females, 

administered a high dose of transgenic rice, a 

low dose of transgenic rice, or a high dose of 

the parental rice strain. The transgenic rice 

7crp#10 and the parental nontransgenic control 

were polished, steamed, mashed, and prepared 

in water at 40% (w/v). Monkeys were orally administered 

a high or low dose of transgenic rice 

or the nontransgenic control by gavage every 

day. No adverse effects on general behavior or 

body weight of animals were observed during 

the study. Analysis of blood from monkeys 

administered for 26 weeks showed that, with 

few exceptions, there were no significant differences 

in hematological or biochemical values 

between them. Additionally, neither pathological 

symptoms nor histopathological abnormalities 

were observed. Thus, it was concluded that 

oral administration of transgenic rice containing 

T-cell epitopes from Japanese cedar pollen allergens 

has no adverse effects. 


Compensation and interaction between 

RISBZ1 and RPBF during grain filling in rice. 

The rice RISBZ1 bZIP and RPBF DOF proteins 

are transcriptional activators of rice seed 

storage protein (SSP) genes in vivo. To ascertain 

the functions of these trans-activators in seed 

development, knock-down (KD) transgenic rice 

plants were generated in which the accumulation 

of RISBZ1 and RPBF was reduced in an 

endosperm-specific manner by co-suppression 

(KD-RISBZ1 and KD-RPBF). The accumulation 

of most SSPs changed little between individual 

KD mutants and wild type plants, whereas 

a double KD mutant (KD-RISBZ1/KD-RPBF) 

resulted in a significant reduction of most SSP 

gene expression and SSP accumulation (Fig 1). 

Reduction of both trans-activators also caused a 

greater reduction in seed starch accumulation 

than individual KD mutations. Storage lipids 

were accumulated at reduced levels in KD- 

RISBZ1 and KD-RISBZ1/KD-RPBF seeds. These 

phenotypes suggest combinatorial interactions 

between RISBZ1 and RPBF play an essential 

role during grain filling. The functional redundancy 

and compensation between RISBZ1 and 

RPBF possibly account for weak effects on the 

SSP levels in single KD mutants, and help maintain 

various processes during seed development 

in rice. Physical interaction between RISBZ1 

and RPBF may ensure that these processes are 

carried out properly. 

Expression of unprocessed glutelin precursor 

alters polymerization without affecting trafficking 

and accumulation. 

Rice glutelin is synthesized as a precursor in 

the endosperm endoplasmic reticulum and then 

deposited within the protein storage vacuole PB- 

II as an aggregate, with a high degree of polymerized 

higher-order structure comprising mature 

acidic and basic subunits after post-translation 

processing cleavage. In order to investigate the 

functional role of this processing and its effect 

on folding assembly, wild-type GluA2 and its 

un-processed form of cDNA (mGluA2) were 

expressed endosperm-specifically in the mutant 

rice a123 line lacking glutelin GluA1, GluA2, and 

GluB4. The mGluA2 precursor was synthesized 

and stably targeted to PB-II without processing 

in the transgenic rice seeds like the wildtype 

GluA2 (Fig 2). Notably, the saline-soluble 

mGluA2 precursor assembled with the other 

Fig 1. Generation of RISBZ1 and RPBF knock-down (KD) lines. 

Accumulation levels of RISBZ1, RPBF (upper) and seed storage proteins (lower) during grain filling in 

wild type, KD-RISBZ1, KD-RPBF and KD-RISBZ1/RPBF seeds. Protein samples were extracted from 

seeds at 7, 14 and 21 days after flowering (DAF), and from mature seed. Glutelins (precursor, acidic and 

basic subunits), α-globulin and 13-kDa prolamins are indicated. 


type of processed glutelin GluB as a trimer in 

PB-II, although such hetero-assembly with GluB 

was not detected in the transformant containing 

the processed GluA. Furthermore, the mGluA2 

precursor in the glutelin fraction was deposited 

in PB-II by forming a quite different complex 

from the processed mature GluA2 products. 

These results indicate that post-translational 

processing of glutelin is not necessary for trafficking 

and stable accumulation in PB-II, but is 

required for the formation of the higher-order 

structure required for stacking in PB-II. 

The 3’-untranslated region of rice glutelin 

GluB-1 affects accumulation of heterologous 

protein in transgenic rice. 

We compared the effect of the rice SSP gene 

GluB-1 terminator with the nopaline synthase 

(Nos) terminator on the accumulation of the 

modified house dust mite allergen mDer f 2 

driven by the maize ubiquitin promoter in 

transgenic rice. Accumulation of mDer f 2 in 

transgenic seed and leaf using the GluB-1 

terminator was greater than when using the 

Nos terminator construct (Fig 3). The mDer 

f 2 mRNA containing the GluB-1 3’UTR was 

processed and polyadenylated at the same sites 

as the native GluB-1 mRNA in the seeds but 

diverged in leaves of the transgenic plants. In 

contrast, the poly(A) sites of mDer f 2 containing 

Nos 3’UTR were more divergent in both seed 

and leaf. These results suggest that GluB-1 3’ 

UTR functions as a faithful terminator and that 

termination at the specific sites may play an 

important role in mRNA stability and/or translatability, 

resulting in higher levels of protein 

accumulation. 

Overexpression of BiP has inhibitory effects 

on the accumulation of seed storage proteins 

in endosperm cells of rice. 

SSPs are specifically and highly synthesized 

during seed maturation and are deposited 

into PBs via the ER lumen. The accumulation 

process is mediated by ER chaperones such 

as luminal binding protein (BiP) and protein 

disulfide isomerase (PDI). To examine the role of 

ER chaperones and the relationship between ER 

chaperones and levels of accumulation of seed 

storage proteins, we generated transgenic rice 

plants in which the rice BiP and PDI genes were 

overexpressed in an endosperm-specific manner. 

Fig 2. Immnolocalization of exogenous GluA2 and mGluA2 in rice endosperm cells. 

The 15 nm gold particles reveal that GluA2 or mGluA2 is localized in PB-II only in transgenic rice seed. Lower 

panels are enlargements of the areas outlined in white in the upper panels. PB-I, Protein body I; PB-II, protein 

body II. Scale bar=1 µm. 


Fig 3. Characterization of transgenic rice plants. 

(A) SDS-PAGE (upper panel) and Immunoblot of mDer f2 (lower panel). C: wild-type. 23 and 27: homozygous 

seeds of transgenic lines (pUbiDerGluB). 3 and 14: homozygous seeds of transgenic lines (pUbiDerNos). (B) 

Relative accumulation levels of mDer f2 in pUbiDerGluB and pUbiDerNos transgenic seeds. Horizontal bars: 

the average levels in each construct. Dot: an average of four transgenic seeds. (C) Comparison of mDer f 2 

accumulation in leaf (L), root (R) and seed (S) of pUbiDerGluB and pUbiDerNos transgenic plants 

The seed phenotype of the PDI-overexpressing 

transformant was almost identical to that of 

the wild type, whereas overexpression of BiP 

resulted in transgenic rice seed that displayed 

an opaque phenotype with floury and shrunken 

features. In the BiP-overexpressing line, the levels 

of accumulation of SSPs and starch contents 

were significantly lower compared with the wild 

type. Interestingly, overproduction of BiP in the 

endosperm of the transformant not only altered 

the morphological structure of ER-derived 

PB-I, but also generated unusual new PB-like 

structures with polysomes composed of a high 

electron density matrix containing glutelin and 

BiP and a low electron density matrix containing 

prolamins (Fig 4). These results suggested 

that the PB-like structure may be formed in the 

ER lumen, resulting in inhibition of translation, 

folding and transport of seed proteins. 

Analysis of ER stress in developing rice endosperm 

accumulating β-amyloid peptide. 

The common neurodegenerative disorder 

known as Alzheimer’s disease is characterized 

by cerebral neuritic plaques of amyloid β (Aβ) 

peptide. Plaque formation is related to the 

highly aggregative property of this peptide, 

because it polymerizes to form insoluble plaques 

or fibrils causing neurotoxicity. We expressed 

Aβ peptide endosperm-specifically as a new 

agent of endoplasmic reticulum (ER) stress 

to study ER stress occurring in plants. The 

dimer of Aβ(1-42) peptide was deposited at the 

periphery of distorted ER-derived PB-I protein 

bodies and severely inhibited the synthesis and 

deposition of seed storage proteins, resulting in 

the generation of many small and abnormally 

appearing PB bodies. This ultrastructural 

change was accounted for by ER stress leading 

to the accumulation of aggregated Aβ peptide 

in the ER lumen and a coordinated increase 

in ER-resident molecular chaperones such as 

BiPs and PDIs in Aβ-expressing plants. Aβ 

-expressing transgenic rice kernels exhibited an 

opaque and shrunken phenotype. When grain 

phenotype and expression levels were compared 

among transgenic rice grains expressing several 

other different recombinant peptides [Aβ, GLP- 

1, 7Crp peptide, house dust mite allergen Der 

p1], such detrimental effects on grain phenotype 

were correlated with the expressed peptide 

causing ER stress rather than expression levels 

of the other peptides. 


Transgenic Silkworm Research Center 

Introduction 

Systems for production of useful materials using 

transgenic organisms have been developed 

in plants and mammals and such systems are 

applicable to insects. The ability to produce 

protein in the domesticated silkworm, Bombyx 

mori, is very high since a large amount of silk 

protein is produced during the larval stage and 

extensive knowledge about silkworm science 

and technology has accumulated in Japan for 

more than 100 years. About 10 years ago, 

our institute developed innovative methods 

for construction of transgenic silkworms for 

efficient production of useful materials. Based on 

the fundamental science, transgenic silkworm 

research was thought to have very high 

potential for production of new materials as well 

as development of technology for a new field 

of agrobiological sciences. In the Transgenic 

Silkworm Research Center, we have been 

developing new methods for the generation of 

transgenic silkworm through construction of 

new vectors and development of new marker 

genes. We are also developing systems that 

control expression of the introduced foreign 

genes in silkworms and are breeding silkworm 

strains for the production of useful recombinant 

proteins and new silk fibers. 

Improvement of DNA injection method for 

producing transgenic silkworm 

Efficient production of transgenic silkworms 

is essential for both fundamental and applied 

studies in this field. We improved the efficiency 

of transgenesis in the silkworm using an in 

vitro synthesized source of transposase mRNA, 

in combination with a recently-developed new 

injection system using an electric manipulator. 

When a transposase mRNA was used as a 

helper in DNA injection for piggyBac-mediated 

germ line transformation, the efficiency was 

very high in comparison with injections where 

a plasmid DNA was used as a helper (Table 1). 

The percentage of emerging marker-positive 

individuals in the G1 generation was higher 

than all previous transformations that did not 

use the transposon helper mRNA. 

Gene function analysis using transgenic silkworm 

and RNAi method for developing new 

marker genes 

development of new marker genes is important 

for production of transgenic silkworms 

having multi-transgenes and for patent protection 

strategies. We have been studying the 

genes responsible for color mutations in eggs, 

larvae and cocoons for possible use as new 

marker genes. The functions of candidate genes 

were verified by germ line transformation and/ 

or RNAi methods. We identified the candidate 

gene for egg color mutation w-2 and studied the 

expression profiles. The function of candidate 

gene was verified by injection of dsRNA into 

the eggs. In the egg color mutant w-3, we rescued 

the mutant phenotype of translucent larval 

skin by forced-expression of Bmwh3 gene. 

In collaboration with National Institute of 

Infectious Diseases, we verified the function 

Table 1. Improved efficiency of transgenesis in the silkworm. 


of the candidate gene for Yellow cocoon (C) 

mutation. When the candidate gene Cameo2 

was expressed in the middle silk glands by 

GAL4/UAS system, the color of silk glands and 

cocoons became yellow (Fig 1). 

Through collaboration with Tokyo University, 

we identified sepiapterin reductase gene as the 

candidate gene for yellow body coloration lemon 

(lem) and lemon lethal (leml). We also verified 

the function of tyrosine hydoroxylase (BmTh) 

gene as the candidate gene for sex-linked chocolate 

(sch) and sch lethal (schl) mutations. The 

results indicated that these genes are usable as 

new maker genes. 

Construction of an efficient binary gene 

expression system for the production of 

recombinant protein in the silk gland 

Recently the demand for the mass-production 

of recombinant proteins of biomedical and 

pharmaceutical interest has been growing. The 

silkworm possesses the silk gland that has very 

high ability to produce and secrete proteins. 

Therefore, the silkworm is thought to be one 

of the most suitable organisms for the production 

of recombinant proteins. To construct an 

efficient production system for recombinant 

proteins, we investigated the promoter activity 

of sericin1, 2 and 3 genes (Ser1, Ser2 and Ser3) 

Fig 1. Coloration with carotenoid accumulation by forced-expression of Cameo2 gene 

using GAL4/UAS system. Silk glands (A) and cocoons (B) . 

Fig 2. Expression of EGFP in the middle silk gland of Ser1-GAL4/UAS-EGFP strain at the 5th instar larvae. 


of the silkworm using the GAL4/UAS binary 

gene expression system. The upstream region of 

Ser1 gene showed strong promoter activity giving 

a high level of expression of a transgene in 

the middle silk gland (MSG), and the expression 

was only observed in the middle and posterior 

region of MSG after the 2nd day of the 5th 

instar (Fig 2). The promoter region of Ser3 gene 

showed an intermediate activity in the anterior 

part of MSG. However, the upstream sequence 

of Ser2 did not show any activity. 

Since the strongest promoter activity is 

observed in Ser1, we attempted to construct 

a system for the production of recombinant 

proteins using the GAL4 construct with the 

Ser1 promoter (Ser1-GAL4). First we measured 

the amount of the EGFP protein in the original 

UAS-EGFP construct with the TATA region of 

Drosophila hsp70 gene and found that about 100 

µg of the protein was produced in a larva with 

both constructs Ser1-GAL4 and UAS-EGFP. 

Then we analyzed components of the UAS- 

EGFP construct (TATA region, signal peptide 

and intron sequences) for effects on production. 

In addition, the effects of Kozak sequence, gene 

copy number, enhancer and insulator were 

studied. We found that the optimization of these 

sequences was very effective for increasing 

the production of EGFP protein. We obtained 

transgenic lines that produced more than 500 

µg of EGFP protein per larva as an average (Fig 

3). We concluded from the results that an application 

of the binary system in the silkworm is 

useful as a tool for mass-production of recombinant 

proteins of biomedical and pharmaceutical 

interest. 

Recombinant protein production and breeding 

of silkworm strains 

We have been constructing a large-scale 

system for recombinant protein production. 

From 443 individuals of 5th instar silkworm, we 

extracted and purified about 200 mg of EGFP 

protein. Also we purified active interleukin-2 

protein from 320 individuals. This year again, 

breeding of silkworm strains adapted for the 

production of recombinant proteins have been 

continued and the amounts of sericin protein 

produced by these strains were sufficiently high 

and stable. 

Fig 3. Construction of various UAS-vectors (A) and the amount of EGFP protein produced using the vectors (B). 


Mass-rearing of recombinant silk strains 

We produced high quality recombinant silks 

such as fluorescent-colored silks and the finest 

silk in the world using transgenic silkworm last 

year. This year, the three strains of transgenic 

silkworms were reared on a large scale in collaboration 

with Gunma Sericultural Technology 

Center. Based on these tests, we compiled 

the methods and recommended processes for 

mass-rearing into a manual that is consistent 

with the provisions of the Cartagena Protocol 

on Biosafety. For the two strains producing 

the green and red fluorescent silks, 40,000 and 

30,000 individuals were reared, respectively 

(Fig 4). In addition, for the strain that spins 

extremely fine silk, 140,000 individuals were 

reared (Fig 4). From the harvested cocoons, 8.1 

kg, 7.5 kg and 24.8 kg of raw silks were derived, 

respectively (Fig 4). 

Manufacturing textile goods using recombinant 

silks 

To accelerate the practical use and industrialization 

of transgenic silks, we manufactured trial 

products using the raw silks derived by massrearing. 

In collaborations with Yumi Katsura 

International Co.,Ltd, Saiei Orimono Co.,Ltd. 

Tohoku Nenshi Co.,Ltd and Silk Technology 

Unit of NIAS, colorful and bridal dresses were 

manufactured as trial products using the green 

and red fluorescent-colored silks (Fig 5). In 

addition, through collaboration with Yoshihama- 

Ningyo Co.,Ltd and Ishikawa Co.,Ltd, the emperor 

and empress dolls for Girl’s Festival were 

made using the fluorescent-colored silks (Fig 5). 

The dolls are wearing the imitated costumes of 

Empress Michiko and Emperor Akihito in his 

enthronement ceremony and they were manufactured 

to celebrate the 20th anniversary. The 

green and red fluorescent-colored silks were 

used as embroidery threads (Fig 5). 

A 

Strain 

Number of 

silkworms 

reared (c.a.) 

Weight of total 

cocoons (kg) 

Weight of dried 

ocoons (kg) 

Drying rate (%) 

weight of law 

silks (kg) 

Green fluorescent 

silk strain 

40,000 63.3 27.4 43.3 8.1 

Red fluorescent 

silk strain 

30,000 48.8 24.9 50.9 7.5 

Finest silk strain 140,000 194.3 86.4 44.5 24.8 

B 

C 

Fig 4. Mass rearing of transgenic silkworm. 

Results of mass rearing (A), Larvae of transgenic silkworm (B), and 140,000 cocoons of the extremely 

fine silk strain (C). 


Fig 5. Trial products using transgenic silks. 

Colorful dress using the green and red fluorescent silks (A). Bridal dress using the green fluorescent silk (B). 

Emperor and empress dolls with the costumes using the green and red fluorescent silks (C). 

Transgenic Animal Research Center 

Successful cross-breeding of cloned pigs expressing 

endo-β-galactosidase C and human 

decay accelerating factor 

For successful organ xenotransplantation, 

genetically engineered pigs have been actively 

produced. As a first step, we produced two 

types of transgenic pigs by cell sorting and 

subsequent nuclear transfer. For inhibition of 

complement activity by expression of high levels 

of human decay accelerating factor (hDAF), 

transgenic cloned pigs expressing hDAF were 

produced. For reduction of αGal expression by 

its digestion enzyme, endo-β-galactosidase C 

(EndoGalC), transgenic cloned pigs expressing 

EndoGalC were produced as well. In the second 

step, we cross bred pigs expressing hDAF and 

EndoGalC and have now produced genetically 

engineered pigs effectively expressing both 

hDAF and EndoGalC genes. As a result, a total 

of 38 F1, 38 F2 and 27 F3 pigs were produced 

after crossbreeding (Yazaki et al, 2009). There 

was no obvious increase in stillbirths and no 

problem in growth rate or in sexual maturation. 

αGal expression levels in the cross-breeds were 

reduced up to 2 to 14% compared to that in 

the wild-type pigs. hDAF expression increased 

about 10- to 70-fold compared to that in human 

umbilical vein endothelial cells. In the third step, 


Recipient baboon 

Table 1. Pig to baboon kidney transplantation 

Donor transgenic pig 

IgM a IgG a Gal expression b hDAF 

expression c 

Graft survival 

35 8 2 45-fold 8 days 

66 12 3 11-fold 11 days 

74 2 14 25-fold 9 days 

51 16 8 27-fold 2 daysd 

a 

IgM and IgG binding level was expressed as a percentage of human pooled sera 

bound to wild-type pig cells. 

b 

Gal expression level was expressed as a percentage of wild-type pig fibroblasts. 

c 

hDAF expression level was expressed as a relative value of human positive control 

(HUVEC or HAEC). 

Baboon died due to the arterial line trouble with functioning graft. 

we used kidney transplantation to baboons to 

examine the usefulness of pig grafts with a high 

level of hDAF expression and EndoGalC-induced 

αGal reduction. Four trials of pig to baboon kidney 

transplantation clearly showed that hyper 

acute rejection could be avoided without any 

treatment for anti-pig antibody removal (Table 1). 

However, there was an immediate lapse into 

procoagulation after transplantation, resulting in 

acute vascular rejection. 

Culture of chicken primordial germ cells 

isolated from embryonic blood 

The generation of transgenic chickens is very 

useful for producing pharmaceutical materials 

in eggs and for the genetic manipulation of 

chickens. Primordial germ cells (PGCs) are the 

progenitor cells of ova or spermatozoa, and in 

chickens they circulate in the bloodstream before 

migrating to the germinal ridges, enabling 

PGC manipulation in vitro and their transfer 

between embryos. PGCs are, therefore, one 

of the most effective vehicles for introducing 

exogenous DNA into the germline of chickens. 

The method for generating germline chimaeric 

chickens by transferring PGCs has already 

been established, and has also achieved the 

generation of germline chimaeric chickens that 

produce donor-derived offspring efficiently. For 

the purpose of stable incorporation of exogenous 

DNA into PGCs, however, a method for an in vitro 

culture system for PGCs must be developed. 

The present study was carried out to develop 

a long-term in vitro culture system for chicken 

primordial germ cells (PGCs). PGCs were 

obtained from the blood of 2.5-day incubated 

embryos. GFP gene was transferred into the 

collected PGCs and then cultured on feeder cells 

derived from the gonads of 7-day incubated embryos. 

The GFP-positive cells attached loosely 

to the feeder cells, and their morphology varied 

from round to fibroblast-like in shape. They 

proliferated slowly and occasionally formed 

colonies (Fig 1). The PGCs cultured for 23-61 

days were transferred into the bloodstream 

of recipient embryos, and we examined their 

incorporation into the germline of chimaeric 

chickens. Test mating was carried out, and 

one germline chimaeric chicken was detected 

out of 28 putative chimaeric chickens. That 

one chicken was generated by transferring 58- 

day cultured PGCs and it produced one donorderived 

offspring out of 270 examined. Thus, a 

small percentage of the cultured PGCs retained 

the ability to migrate to the germinal ridges, 

thus giving rise to functional gametes, although 

most of the cultured PGCs differentiated during 

the culture period (Naito et al, 2010). In conclusion, 

a germline chimaeric chicken was generated 

by transferring PGCs cultured in vitro for 

about 2 months; the culture system for PGCs 

developed in the present study will contribute 

to the germline manipulation in chickens. 


Fig 1. Phase contrast micrographs of chicken primordial germ cells in culture (A, C, E, G) and 

expression of GFP gene (B, D, F, H). Scale Bar = 30mm 

P2X 7 R 

Fig 2. Schematic model for the regulatory mechanism of IL-1b maturation and release 

through the activation of P2X 7 R in microglial cells. 

Release and processing of interleukin-1β in 

microglial cells and development of a novel 

tissue culture model with collagen vitrigel 

membrane 

Interleukin (IL)-1β is one of the most potent 

pro-inflammatory cytokines. It is primarily 

released from activated microglia in the brain; 

and is also implicated in the induction and 

progression of pathogenesis in various neurodegenerative 

disorders. Therefore, clarification of 

the regulatory or modulatory mechanisms for 

maturation and release of IL-1β from microglia 

may provide therapeutic clues for neuroinflammatory/neurodegenerative 

diseases. Although 

the mechanisms for the release of mature IL-1β 

still remain controversial, emerging evidence 

suggests the pivotal roles of the P2X 7 receptor 

(P2X 7 R), one of the ionotropic P2X receptors for 

extracellular ATP, in the release of this cytokine 

(Takenouchi et al, 2009c). We demonstrated 

novel roles of lysophospholipids such as LPC 

and SPC in the regulatory pathway for the 


maturation and release of IL-1β mediated by 

P2X 7 

R activation in microglial cells (Fig 2). In 

addition, we demonstrated that P2X 7 R activation 

by ATP down-regulates the basal autophagic 

flux in microglial cells through the impairment 

of lysosomal functions leading to the stimulated 

release of lysosomal/autophagolysosomal components 

into the extracellular space (Takenouchi 

et al, 2009b). Because P2X 7 R plays a key role 

in the maturation and release of IL-11β, our 

study suggests basal autophagy may be another 

important regulatory pathway in the P2X 7 R- 

dependent maturation and release of IL-1β 

from microglial cells (Fig 2). Regulation of basal 

autophagy will provide a unique viewpoint to 

decipher the molecular mechanism of the ATPinduced 

maturation and release of IL-1βfrom 

microglial cells (Takenouchi et al, 2009a). 

We previously developed a collagen vitrigel 

membrane which is composed of high density 

collagen fibrils equivalent to connective tissues 

in vivo and is easily handled with tweezers. We 

have established a reconstruction method of 

rabbit corneal epithelium model by culturing 

normal rabbit corneal epithelial cells on the 

collagen vitrigel membrane substratum and 

inducing differentiation to form multilayers of 

the cells. However, to estimate eye irritation 

and permeability of chemicals towards humans, 

it is necessary to reconstruct a corneal model 

with barrier function utilizing human cells. In 

this study as a first step, we aimed for establishing 

a reconstruction method of human corneal 

epithelium model with barrier function utilizing 

both a human corneal epithelium-derived cell 

line (HCE-T) and the collagen vitrigel membrane 

substratum. Further, to confirm the utility of the 

human corneal epithelium model, the changes 

of its barrier function induced by exposing eyeirritant 

chemicals were measured. As a result, 

Fig 3. Human corneal epithelium model reconstructed on a collagen vitrigel membrane at 

one week-culture on the air-liquid interface. 

Observations by a phase contrast microscope (upper image) and an optical microscope 

after staining a cross-section with hematoxylin and eosin (lower image). 


HCE-T cells proliferated well on the collagen 

vitrigel substratum and gradually differentiated 

into multilayers on air-liquid interface culture, 

resulting in the time-dependent increase of 

transepithelial electrical resistance (TEER). The 

corneal epithelium model possessing five cell 

layers was well reconstructed after one weekculture 

on the air-liquid interface (Fig 3). The 

exposure of chemicals to the model induced the 

time-dependent changes of TEER in response 

to the characteristic of each chemical. These 

results suggest that eye irritant chemicals 

could be estimated by the barrier function of 

reconstructed human corneal epithelium model 

as an indicator. 

References 

Naito M, Harumi T, Kuwana T (2010) Long term 

in vitro culture of chicken primordial germ 

cells isolated from embryonic blood and incorporation 

into germline of recipient embryo. J. 

Poult, Sci., 47: 57-64. 

Takenouchi T, Fujita M, Sugama S, Kitani H., 

Hashimoto M (2009a) The role of the P2X 7 

receptor signaling pathway for the release of 

autolysosomes in microglial cells. Autophagy, 5: 

723-724. 

Takenouchi T, Nakai M, Iwamaru Y, Sugama 

S, Tsukimoto M, Fujita M, Wei J, Sekigawa 

A, Sato M, Kojima S, Kitani, H, Hashimoto M 

(2009b) The activation of P2X 7 receptor impairs 

lysosomal functions and stimulates the 

release of autophagolysosomes in microglial 

cells, J. Immunol., 182: 2051-2062. 

T a k e n o u c h i T , S u g a m a S , I w a m a r u Y , 

Hashimoto M, Kitani H (2009c) Modulation of 

the ATP-lnduced Release and Processing of 

IL-1β in Microglial Cells, Crit. Rev. Immunol., 

29: 335-345. 

Yazaki S, Iwamoto M, Onishi A, Miwa Y, Suzuki 

S, Fuchimoto D, Sembon S, Furusawa T, 

Hashimoto M, Oishi T, Liu D, Nagasaka T, 

Kuzuya T, Maruyama S, Ogawa H, Kadomatsu 

K, Uchida K, Nakao, A, Kobayashi T (2009) 

Successful cross-breeding of cloned pigs 

expressing endo-β-galactosidase C and human 

decay accelerating factor. Xenotransplantation, 

16: 511-521. 



The Division aims to develop and refine 

agro-bioresources for crop breeding and basic 

research based on the genome resources, 

genome information, genome analysis methods, 

and genetic resources developed or collected in 

NIAS. In addition, the Division aims to facilitate 

distribution of agro-bioresources to the research 

community and to efficiently discover and utilize 

genes with important functions by using those 

agro-bioresources. The Division also conducts 

comparative genomics studies by applying the 

results of rice genome research to other grass 

family crops and has initiated genome research 

on soybean. 

The Division consists of six research units 

and research aims of each unit are summarized 

as follows: 


The Unit aims to collect and sequence rice 

and barley full-length cDNAs which are useful 

resources for identification of important genes 

and analysis of their function. In addition, comparative 

genomics studies on wild rice species 

and grass family crops will be conducted with 

the aim of discovery and utilization of useful 

genes. 


The Unit aims to develop a new bioinformatics 

platform which enables researchers to 

analyze a large amount of biological information 

by comparing genome sequences and cDNA 

sequences of different species. By using this 

system, the Unit aims to identify genes with 

important functions. 


The Center aims to produce resources useful 

for functional analysis of rice genes, such as 

mutant rice lines and transgenic rice lines 

over-expressing a large collection of full-length 

cDNAs of rice, and to develop a database by 

integrating information obtained by the analysis 

of those resources. In addition, the Center aims 

to facilitate the maintenance and distribution of 

genome resources to the research community. 

Genebank 

Genebank aims to collect, preserve, evaluate 

and distribute plant, animal and microorganism 

genetic resources for breeding and research. 

To facilitate the efficient utilization of genetic 

resources, Genebank also aims to develop core 

collections of wild rice and Vigna species. 


The Institute aims to develop breeding 

materials with new traits such as disease 

resistance and high content of health-promoting 

ingredients. In addition, the Institute aims to 

develop radiation-induced mutant rice lines for 

identification and functional analysis of agronomically 

important genes. 


The Team aims to conduct genome research 

on soybean, a major source of protein and vegetable 

oil for animal and human nutrition, and 

to develop methods for isolation of useful genes 

and efficient breeding. The team started from 

June 2006. 

Major topics in the Division in the fiscal year 

2009 are described as follows. 



Gramineae plants, which include many major 

crops, are important both for us Japanese and 

people of all over the world. Plant Genome 

Research Unit has been devoted in construction 

and analysis of various genome resources form 

rice and other Gramineae species. 

Sequence analysis and elucidation of evolutionary 

and local adaptation of photoperiod 

sensitivity gene 

Heading date determines rice’s adaptation to 

its area and cropping season. Analysis of the 

molecular evolution of the Hd6 quantitative trait 

locus for photoperiod sensitivity in a total of 20 

cultivated varieties and wild rice species and 

revealed 74 polymorphic sites within its coding 

region. Moreover, natural mutations and 

modifications of the coding region of Hd6 within 

the genus Oryza have been suppressed during 

its evolution (Fig 1). Phylogenetic analysis and 

genome divergence using the entire Hd6 genomic 

region confirmed the current taxonomic 

sections of Oryza and supported the hypothesis 

of independent domestication of indica and 

japonica rice. 

SARAD on ARRAY: a new functions for the 

novel domain database 

We constructed a unique comparative genomics 

database termed the SALAD database 

(http://salad.dna.affrc.go.jp/salad/) from plantgenome-based 

proteome data sets by extracting 

evolutionarily conserved motifs by MEME 

software from 209,529 protein sequence annotation 

groups selected by BLASTP from the 

proteome data sets of 10 species: rice, sorghum, 

Arabidopsis thaliana, grape, a lycophyte, a moss, 

3 algae, and yeast. Similarity clustering of each 

protein group was performed by pairwise scoring 

of the motif patterns of the sequences (Fig 2). 

The SALAD database provides a user-friendly 

graphical viewer that displays a motif pattern 

diagram linked to the resulting bootstrapped 

dendrogram for each protein group. 

We also developed a viewer named ‘SALAD 

on ARRAYs’ to view arbitrary microarray data 

sets of paralogous genes linked to the same dendrogram 

in a window. The SALAD database is 

a powerful tool for comparing protein sequences 

and can provide valuable hints for biological 

analysis. 

Fig 1. Analysis of sequence similarity within the Hd6 region: conservation plot for nucleotides of the 

20 cultivated varieties and wild rice species within the Genus Oryza. 


Fig 2. SALAD database viewer. 

(A) Data of the GID1group. (A-1) Operation panel for manipulating the output, (A-2) Toolbox for 

constructing phylogenetic trees based on sequence alignments of selected multiple motifs. (A- 

3) Typical SALAD analysis: a dendrogram of sequences clustered according to the presence and 

similarity of extracted conserved motifs, and a diagram that displays positional information of the 

extracted motifs in each sequence. (B) Expansion section around GID1. Each motif is assigned to 

a sequence number and color in the ‘high percent similarity’ protein group, and the same color box 

indicates the same extracted motif. (C) Color key of species. 

Genome-wide analysis of NAC transcription 

factor family in rice 

We investigated 151 non-redundant NAC 

genes in rice and 117 in Arabidopsis. A complete 

overview of this gene family in rice is presented, 

including gene structures, phylogenies, 

genome localizations, and expression profiles. 

We also performed a comparative analysis of 

these genes in rice and Arabidopsis. Conserved 

amino acid residues and phylogeny construction 

using the NAC conserved domain sequence 

suggest that OsNAC gene family was classified 

broadly into two major groups (A and B) and 

sixteen subgroups in rice (Fig 3). We presented 

more specific phylogenetic analysis of OsNAC 

proteins based on the DNA-binding domain 

and known gene function, respectively. Loss of 

introns was observed in the segmental duplication. 

Homologous, paralogous, and orthologous 

searches of rice and Arabidopsis revealed that 

the major functional diversification within the 

NAC gene family predated the divergence of 

monocots and dicots. The chromosomal localizations 

of OsNAC genes indicated nine segmental 

duplication events involving 18 genes; nonredundant 

OsNAC genes were involved in 


Fig 3. Evolutionary relationship among the rice OsNAC domain sequences. 

The unrooted tree was generated by using the ClustalX program with the neighbor-joining 

method. OsNAC proteins were allocated to two distinct groups (A and B). 

tandem duplications (Fig 4). Expression levels 

of this gene family were checked under various 

abiotic stresses (cold, drought, submergence, 

laid-down submergence, osmotic, salinity and 

hormone) and biotic stresses [infection with 

rice viruses such as RSV (rice stripe virus) 

and RTSV (rice tungro spherical virus)]. Biotic 

stresses are novel work and increase the possibilities 

for finding the best candidate genes. 

A preliminary search based on our microarray 

(22K and 44K) data suggested that more than 

45 and 26 non-redundant genes in this family 

were upregulated in response to abiotic and 

biotic stresses, respectively. All of the genes 

were further investigated for their stress 

responsiveness by RT-PCR analysis. Six genes 

showed preferential expression under both 

biotic RSV and RTSV stress. Eleven genes were 

upregulated by at least three abiotic treatments. 

Our study provides a very useful reference for 

cloning and functional analysis of members of 

this gene family in rice. 

Transcriptome analysis based on rice transcript 

sequences 

The Rice Annotation Project has integrated 

the whole rice genomic sequence and full-length 

cDNA (FL-cDNA) sequences, however, 2,159 FLcDNAs 

have not been mapped to the genome 

sequence. Of the 2,159 sequences, 359 were 

mapped separately to multiple loci, either on 

different chromosomes or in distant regions, suggesting 

the generation of chimeric FL-cDNAs. 

A new methodology of whole mRNA sequencing 

(mRNA-seq) by using massive parallel 


Fig 4. Distribution of OsNAC genes on the 12 rice chromosomes (Mb scale). 

Genes with open reading frames in opposite orientations are marked on the chromosome. Straight 

lines connect the OsNAC genes presented on duplicated chromosomal segments, and tandemly 

duplicated gene clusters are marked by green bars. 

Fig 5. Thirty-six-base-pair reads mapped on the rice genome. The graph indicates the average 

depths of 36bp reads (blue). 

Gene models based on the full length-cDNA sequences are also shown (black boxes). 

sequencing technology was performed. We 

mapped 36bp reads from mRNA of rice tissues 

on the rice Nipponabare genome. The number 

of mapped reads was counted and graphically 

visualized on the rice genome (Fig 5). Gene models 

were constructed based on the piling-up of 

short reads on the rice genome for identification 

of unannotated transcripts. 

We started to study rice transcriptome upon 

phosphate stravation using the mRNA-seq 

technology described above. Phosphate (Pi) is a 

one of the essential macronutrients for viability. 

Plants have developed several methods of adapting 

to condition of Pi limitation morphologically 

and physiologically. Agricultural Pi deficiency 

is alleviated by the massive application of Pi 

fertilizers. However, the world’s reserves of 

rock Pi (mined for production of Pi fertilizers) 

are expected being depleted within the next 

century. The studies of the complex regulatory 

mechanisms in molecular level whereby plants 

acclimate to nutritional Pi deficiency are of 

great importance. This could lead to the development 

of manipulating the expression genes 

enabling growth in Pi deficiency environments, 

improve the P-use-efficiency of plants and 

reduce the P-fertilizer requirement of crops. So 

far, we identified many Pi deficiency responsible 

genes, including previously unannotated genes. 

Our study will provide a newly overview of the 

initial molecular events of Pi deficiency in rice. 


Cleistogamous flowering in barley: mechanism 

of suppression of microRNA-guided 

HvAP2 mRNA cleavage 

The cleistogamous flower sheds its pollen 

before opening, forcing plants with this habit to 

be almost entirely autogamous. Cleistogamy also 

provides a means of escape from cereal head 

blight infection, and minimizes pollen-mediated 

gene flow. We have isolated cleistogamy 1 (Cly1) 

by positional cloning, and show that it encodes a 

transcription factor containing two AP2 domains 

and a putative microRNA miR172 targeting 

site, which is an orthologue of Arabidopsis 

thaliana AP2. We conclude that the miR172- 

derived down-regulation of Cly1 promotes the 

development of the lodicules, thereby ensuring 

non-cleistogamy, while the single nucleotide 

change at the miR172 targeting site results in 

the failure of the lodicules to develop properly, 

producing the cleistogamous phenotype. 

Comparative genomics and expression analysis 

of flowering time genes 

Studies for the flowering time determination 

of wheat and barley and comparison of the 

results with those of rice are quite interesting 

because the light response for the flowering 

is completely opposite between rice (short-day 

plant) and wheat and barley (long-day plant) 

although wheat and barley belongs to Gramenae 

as well as rice. Last year we focused on the 

effects of barley FT-like genes on the flowering 

of barley. 

We performed expression and transgenic 

studies to clarify the functional roles of three 

FT-like genes and two other PEBP genes 

with regard to the flowering time of barley. 

Introduction of HvTFL1 and HvMFT1 into 

rice did not result in any changes in flowering, 

suggesting that these two genes have functions 

distinct from flowering. Overexpression of 

HvFT1, HvFT2, and HvFT3 in rice resulted in 

early heading (Fig 6), indicating that these FTlike 

genes can act as promoters of the floral 

transition. HvFT1 transgenic plants showed the 

most robust flowering initiation and HvFT1 was 

thought to be the key gene responsible for flowering 

in the barley FT-like gene family. HvFT2 

transgenic plants also showed robust flowering 

initiation, but HvFT2 was expressed only under 

short-day (SD) conditions, suggesting that its 

role is limited to specific photoperiodic conditions 

in barley. Flowering activity in HvFT3 

transgenic rice was not as strong as HvFT1 and 

Fig 6. Phenotype of transgenic rice plants overexpressing barley PEBP genes. 

Photographs show the transgenic plants at the heading stage under LD conditions as an example. 

The insets show enlarged photographs of panicles on the heads of transgenic rice plants. 


HvFT2 and was modulated by the photoperiod. 

These results suggest that HvFT3 functions in 

flowering promotion but that its effect is indirect. 

HvFT3 was mapped to chromosome 1HL, 

the chromosome that carries Ppd-H2, a major 

quantitative trait locus for flowering under 

SD conditions, and genomic sequence analyses 

revealed that there was structural difference of 

HvFT3 between Ppd-H2 and ppd-H2 cultivars. 

These data strongly suggest that HvFT3 may 

be identical to Ppd-H2. 

Our data revealed that the diversification of 

functional roles of FT-like genes in controlling 

flowering of barley, which are different from 

model plants, Arabidopsis and rice. 

References 

Yamane H, Ito T, Ishikubo H, Fujisawa M, 

Yamagata H, Kamiya K, Ito Y, Hamada M, 

Kanamori H, Ikawa H, Katayose Y, Wu J, 

Sasaki T, Matsumoto T (2009) Molecular and 

evolutionary analysis of the Hd6 photoperiod 

sensitivity gene within genus Oryza. Rice, 2(1): 

56-66. 

Mihara M, Itoh T, Izawa T (2010) SALAD 

database: a motif-based database of protein 

annotations for plant comparative genomics 

Nucleic Acids Research, Database issue. doi: 

10.1093/nar/gkp831 

Nuruzzaman M Manimekalai R, Sharoni AM, 

Satoh K, Kondoh H, Ooka H, Kikuchi S, Gene 

(2010) Gene, 465: 30-44. 

Mizuno H, Tanaka T, Sakai H, Kawahigashi 

H, Itoh T, Kikuchi S, Matsumoto T (2010) 

Characterization of 2159 Unmapped Fulllength 

cDNA Sequences of Oryza sativa L. 

ssp. japonica ‘Nipponbare’. Plant Molecular 

Biology Reporter, 28: 357-362. 

Nair SK, Wang N, Turuspekov Y, Pourkheirandish 

M, Sinsuwongwat S, Chen G, Sameri M, 

Tagiri A, Honda I, Watanabe Y, Kanamori H, 

Wicker T, Stein N, Nagamura Y, Matsumoto 

T, Komatsuda T (2010) Cleistogamous flowering 

in barley arises from the suppression of 

microRNA-guided HvAP2 mRNA cleavage. 

Proc. Natl. Acad. Sci. USA, 107: 490-495. 


Gene identification by cross-species fulllength 

cDNA mapping 

To cope with the deluge of emerging sequence 

information produced by new technologies, 

such as next-generation sequencing, the 

development of an efficient annotation system is 

needed. Exon-intron structures and the proteincoding 

sequences (CDSs) in genome sequences 

can be predicted either by ab initio predictions 

or by sequence similarity methods. While ab 

initio gene prediction programs may produce 

erroneous exon–intron structures, sequence 

similarity approaches generally show better 

results. Even though similarity methods based 

on full-length cDNAs (FLcDNAs) can be used 

to accurately determine exon-intron structures, 

sufficient numbers of FLcDNAs for the annotation 

are available in only a few plants, such 

as rice, maize and Arabidopsis. In this study, 

to fully utilize FLcDNA resources, algorithms 

have been developed for cross-species FLcDNA 

mapping and identification of complete CDSs 

by the extension of both ends of truncated 

CDSs. Cross-species mapping of 71,801 monocot 

FLcDNAs to the Oryza sativa genome resulted 

in the detection of 22,142 protein-coding regions. 

Moreover, in comparison with two cDNAmapping 

programs and three ab initio prediction 

programs, we found that our pipeline (see 

Fig. 1) was more capable of identifying complete 

CDSs (Table 1). We applied both cross-species 

and within-species mapping to ten monocot and 


dicot genomes and identified genes in 210,551 

loci. 

A web service of the pipeline for the FLcDNAs 

mapping to genomic DNA sequences is available 

(http://fpgp.dna.affrc.go.jp/index.html) (Fig 1). 

Users can submit a sequence of up to 1 Mb, 

and can specify dicot or monocot FLcDNAs to 

be mapped. After completion of the requested 

prediction, a URL that indicates the prediction 

results is supplied by e-mail. Results of the 

FLcDNA mapping are shown in a graph and a 

file of the gff3 format, in which predicted gene 

exon-intron are described, can be downloaded. 

Construction of the rice genome annotation 

based on the IRGSP build 5 

The rice genome sequence has been 

determined by the International Rice Genome 

Sequencing Project, and a novel genome assembly 

(build 5) was released in 2008. In accordance 

with this update, a new annotation data set, 

IRGSP/RAP build 5, was created. In addition 

to rice transcript evidence, 147,670 non-rice 

transcripts and protein sequences registered in 

UniProt/Swiss-Prot were utilized for the exonintron 

structure predictions. To find appropriate 

structures based on non-rice transcripts, the 

Table 1. Comparison of specificity (SP) and sensitivity (SN) in CDSs. 

Method 

Exon-intron Intronb All intronsb Entire CDS 

boundaryb 

SP SN SP SN SP SN SP SN 

This study 94.6 75.5 92.6 74.2 58.5 49.5 55.5 44.4 

GeneSeqer 87.9 77.9 83.7 75.5 42.3 38.9 3.3 2.8 

Sim4cca 90.8 74.3 87.9 73.0 40.5 47.2 -c -c 

GeneMark.hmm 87.6 87.6 76.2 80.7 22.1 25.6 24.0 25.1 

GeneZilla 87.4 77.0 76.4 68.3 29.9 30.7 31.8 35.1 

GlimmerHMM 91.3 75.5 81.7 69.1 36.3 35.8 39.4 38.7 

Note. - All values are expressed by percentages (%). 

Sim4cc does not report a representative transcript in a single locus, so that each mapping result was evaluated 

separately. 

CDS regions of the reference set were evaluated. 

Sim4cc does not predict CDS regions. 

Fig 1. A web service of the pipeline for the FLcDNAs mapping to genomic DNA sequences. 


aforementioned cross-species mapping system 

was used; for proteins, the ProSplign program 

developed by NCBI was employed. Clustering of 

the predicted structures on the basis of genomic 

positions resulted in 34,902 loci, 24,367 of which 

were identified by FLcDNAs of Oryza sativa 

(japonica group), while 6,815 were supported 

solely by non-rice transcripts (Table 2). Even 

though rice cDNAs are not cloned, these 6,815 

loci should contain convincing candidates of 

expressed rice genes. In addition to the loci in 

Table 2, another 9,975 genes were predicted 

computationally, but there was no evidence of 

their transcription and most may be pseudogenes 

or mis-predictions. 

In addition to the construction of the new 

annotation data, the Rice Annotation Project 

Database (RAP-DB) was reorganized. The rice 

annotation data can be searched for by gene 

symbols, functional descriptions, gene identifiers, 

and so on. Various types of non-rice information, 

such as non-rice transcript mapping and genome 

alignments, were integrated in the RAP- 

DB. A new inclusion is a data download system 

by which users can retrieve annotation data 

and sequences of a particular gene or genomic 

region. The positions of a genomic region can 

be specified either by names of markers or by 

gene identifiers. 

Table 2. Statistics of IRGSP/RAP build 5 annotation data. 

Evidence 

Number of loci 

O. sativa (japonica) FLcDNAs 24,367 

O. sativa (indica) and O. rufipogon FLcDNAs 1,275 

Other Oryza mRNAs 318 

Cross-species and paralog mapping 6,815 

Protein-mapping of Oryza sativa (japonica) 72 

Protein-mapping of Plants (with EST) 21 

Ab initio gene predictions (with EST) 2,034 

Total 34,902 


Developing a resource-based information 

infrastructure by gene expression profiling 

Understanding the function and regulation of 

all the rice genes essential for vegetative and 

reproductive growth is one of the major goals 

in rice genomics. This is particularly relevant 

at this point considering that 5 years after the 

completion of the rice genome sequence, more 

than 30% of approximately 32,000 annotated 

genes in rice are still uncharacterized in terms 

of function. In order to address this goal, we 

have embarked on a large-scale gene expression 

profiling of rice at different stages of growth 

and development under natural field conditions. 

Using the rice full-length cDNA sequences and 

the annotation of the rice genome sequence in 

RAP-DB (Rice Annotation Project Database, 

http://rapdb.dna.affrc.go.jp), we developed a 

4x44K oligomicroarray representing about 32,000 

genes predicted in the rice genome. This microarray 

platform was then used to characterize 

the transcriptome of rice tissues and organs by 

spatiotemporal gene expression profiling at the 

vegetative, reproductive and ripening stages. 

Continuous gene expression profiling was also 

performed for various tissues and organs to 

provide a detailed profile of the changes in rice 

transcriptome from transplanting to harvest- 


ing. As a result, we were able to establish an 

overview of the global transcriptional changes 

throughout the growth cycle as triggered by 

exogenous and endogenous signals under natural 

field conditions. In addition to tissue/organspecific 

gene expression, growth-stage specific 

signatures were also uncovered. Furthermore, 

the dynamic gene expression profile of leaves 

at regular intervals from transplanting until 

harvesting identified two drastic transcriptome 

changes associated with phase transition. 

The gene expression data derived from these 

analyses are now deposited in a database called 

RiceXPro (Rice Expresion Profile Database), 

which was designed to provide a resource-based 

infrastracture for expression profiling of every 

gene in rice under natural field conditions. 

A user-friendly interface facilitates 4 major 

functionalities: (1) an overview and search for 

the expression of each gene in various tissues 

and organs at vegetative, reproductive and 

ripening stages. Search can be initiated using 

a keyword (RAP locus ID, accession number 

or gene name) or based on the position of the 

gene in the chromosome; (2) a detailed view and 

search for the expression of each gene based 

on samples obtained at regular intervals during 

the entire growth cycle from transplanting to 

harvesting; (3) data mining function facilitated 

by analysis tools such as T-test and fold change 

(FC) to compare expression between two 

random samples; and (4) co-expression analysis 

tool that may be used to infer the function 

of uncharacterized genes. As a repository of 

expression data encompassing the entire growth 

in the field, RiceXPro can be used as a reference 

in elucidating the function of every gene in rice. 


Genebank 

Genebank has responsibility for conserving 

genetic resources (GRs) for food and agriculture 

in Japan and facilitating their use under the 

jurisdiction of the Ministry of Agriculture, 

Forestry and Fisheries. It implements the 

NIAS Genebank Project as the “central-bank” 

and coordinates several “sub-banks” that are 

research institutes in the National Agriculture 

and Food Research Organization (NARO), 

the Japan International Research Center for 

Agricultural Sciences (JIRCAS), the National 

Center for Seeds and Seedlings (NCSS), the 

National Livestock Breeding Center (NLBC), and 

the National Institute for Agro-Environmental 

Science (NIAES). The major activities of 

Genebank are 1) introduction, field survey 

and diversity studies, 2) characterization and 

evaluation of GRs toward increased active collection, 

3) development of genetic and breeding 

materials by using GRs, 4) durable preservation, 

quality control and improved multiplication 

and preservation methods, and 5) advanced 

information management and disclosure system 

to facilitate use of GRs in order to accomplish 

the Project purpose. Genebank cooperates with 

and is strongly supported by Genome Resource 

Center and Genetic Resources Management 

Section in NIAS for implementing the Project 

and carries out research projects on relevant 

genetic resources and biodiversity. 

NIAS Genebank and partner institutions 

conserve 242,960 accessions of plant GRs (PGRs), 

25,531 accessions of microorganism GRs (MGRs), 

and 989 accessions of animal GRs (AGRs) in the 

Project (data on 30 November 2009). In FY2009, 

9,484 accessions of PGRs, 1,520 accessions 

of MGRs and 748 accessions of AGRs were 

distributed to domestic and/or foreign users for 

breeding, research and/or educational purposes. 

Up-to-date passport and evaluation data of distributable 

accessions have been uploaded onto 

and disclosed at the website (http://www.gene. 

affrc.go.jp/). 

Field Survey in India (Tamil Nadu State) 

Under a Memorandum of Understanding 

with Tamil Nadu Agricultural University, a 

field survey of leguminous crops and their wild 

relatives was conducted in India. Central and 

northern Tamil Nadu State were surveyed and 

a total of 134 accessions (99 domesticated and 

35 wild) were collected and are now conserved 

at the Tamil Nadu Agricultural University. 

Landraces of pigeon pea (Cajanus cajan), horse 

gram (Macrotyloma uniflorum), hyacinth bean 

(Lablab purpureus), mungbean (Vigna radiata), 

Fig 1. A long tap root of Vigna trilobata (TN69) growing on sandy 

soil beside farmers house suggesting high drought tolerance. 


lack gram (Vigna mungo) and moth bean (Vigna 

aconitifolia) were collected. The wild species, 

Vigna aconitifolia, V. radiata, V. stipulacea 

and V. trilobata were found and collected. Two 

closely related wild species, V. stipulacea and 

V. trilobata were found to be common in Tamil 

Nadu. Although these two species showed 

quite similar morphological characters, they are 

adapted to wet heavy clay and dry sandy soil 

habitat, respectively. V. trilobata seems to be 

highly resistant to drought judging from its well 

developed deep tap roots that elongate in sandy 

soil (Fig 1). These two wild legumes are commonly 

used as food and forage in Tamil Nadu 

State. 

Trip reports (pdf file) are available from the 

NIAS genebank web site: 

http://www.gene.affrc.go.jp/pdf/report/ 

parts/2008_2-1.pdf 

Salt stress resistance of Vigna 

To search for the highly salt resistant Vigna 

accessions, 50 accessions of V. luteola, 50 of V. 

marina and 130 of V. vexillata were screened 

using hydroponic culture containing different 

concentrations of NaCl solution. Two V. luteola 

accessions (JP235857 Colombia, JP236231 Costa 

Rica) could survive 8 weeks under strong salt 

stress conditions (300mM NaCl solution for 4 

week followed by 400mM NaCl solution for 4 

weeks). V. marina accessions showed the highest 

salt resistance among three wild species. 

About 60% of the tested accessions could survive 

for 8 weeks under the strongest salt stress 

condition (350mM NaCl solution for 4 weeks 

followed by 500mM NaCl solution for 4 weeks). 

V. vexillata accessions showed the lowest salt 

resistance among three tested species. Only 4 

among 150 accessions tested could survive 8 

weeks under salt stress condition (250mM NaCl 

for 4 weeks followed by 350mM NaCl for 4 

weeks). In V. vexillata, a domesticated form was 

recently found cultivated in Indonesia. It was 

mainly cultivated for its tuber production. Three 

accessions of domesticated V. vexillata were 

tested and were found to be highly susceptible 

to salt (e.g. JP235864: Indonesia shown in Fig 2). 

Several wild V. vexillata accessions were much 

more resistant to salt stress (e.g. JP202330: 

Guyana, JP230747: Papua New Guinea, Fig 2), 

and these are considered a promising gene 

source for salt resistance breeding program. 

Molecular map development of Vigna radiata 

To facilitate the use of wild Vigna germplasm 

for breeding, a molecular linkage map 

of mungbean (V. radiata) was constructed 

this year. The map consists of 435 markers 

on 11 linkage groups, covering 727.1cM of the 

mungbean genome. In this map, markers from 

various related species were accumulated to enable 

comparative genomic studies. In particular, 

196 soybean EST markers developed by the 

Kazusa DNA Research Institute were placed on 

Fig 2. Response of Vigna vexillata plants under 250mM NaCl stress condition for 3 weeks. 

1) JP235864, Domesticated V. vexillata collected on Bali island, Indonesia 2) JP202330, Wild 

accession of V. vexillata collected in Guyana, 3) JP230747, Wild accession of V. vexillata collected in 

Papua New Guinea. 


Linkage 

Group 

Table 1. Markers from related species accumulated on the mungbean molecular linkage map 

No. of markers 

Azuki Mungbean Cowpea Common bean Soybean 

Total 

SSR SSR SSR SSR EST 

Length 

(cM) 

Average distance 

between markers 

(cM) 

1 23 3 10 0 31 67 90.2 1.4 

2 18 1 9 0 27 55 92.2 1.7 

3 8 2 8 1 23 42 74.7 1.8 

4 15 5 3 0 21 44 87.8 2.0 

5 15 3 3 1 17 39 56.5 1.5 

6 11 3 3 0 19 36 56.2 1.6 

7 8 5 6 1 13 33 47.9 1.5 

8 15 1 7 0 19 42 69.1 1.7 

9 10 5 2 3 8 28 57.2 2.1 

10 9 2 3 2 9 25 50.2 2.1 

11 9 0 5 1 9 24 45.1 2.0 

Total 141 30 59 9 196 435 727.1 1.8 

the mungbean map. The soybean EST markers 

placed on the mungbean map will be useful for 

comparative genomics between mungbean and 

soybean. In addition, NIAS genebank is currently 

developing azuki bean EST markers with 

Kazusa DNA Research Institute. 

Nucleotide polymorphisms of the bovine 

growth hormone secretagogue receptor 1a 

(GHSR1a) gene and their association with 

growth and carcass traits in Japanese black 

cattle 

Ghrelin growth hormone secretagogue receptor 

1a (GHSR1a) is involved in many important 

functions, including growth hormone secretion 

and food intake. To the best of our knowledge, 

there have been no reports to date on nucleotide 

polymorphisms from the 5’-flanking region 

to the 3’-UTR of the GHSR1a gene in cattle. 

Furthermore, the GHSR1a gene was reported 

as a potential candidate gene when we detected 

growth trait QTLs in Japanese Black cattle 

using microsatellite DNA markers and half-sib 

regression analysis. Here we have described (1) 

all possible nucleotide polymorphisms from the 

5’-flanking region to the 3’-UTR of the GHSR1a 

gene, and (2) an association between nucleotide 

polymorphisms of the gene and growth and 

carcass traits in Japanese Black cattle. 

The nucleotide sequencing of this gene 

revealed 47 single nucleotide polymorphisms 

(SNPs), 4 indels and 2 microsatellites [(TG)n, 5’ 

-UTR and (GTTT)n, Intron 1] (Table 1). The 19 

haplotypes were constructed from all nucleotide 

viability patterns and were divided into 3 major 

groups. Breed differences in allele frequencies 

of the 2 microsatellites, nt-7(C>A), L24V and 

DelR242 loci were found (P < 0.005). A DelR242 

SNP was found in the Japanese Shorthorn (frequency:~0.44) 

and Japanese Brown, but none 

was detected in the Japanese Black cattle. We 

carried out a statistical analysis of 5 nucleotide 

polymorphisms - [5’-UTR microsatellite (TG)n], 

nt-7(C>A), L24V, DelR242 and Intron 1-microsatellite 

[(GTTT)n] of the GHSR1a gene - and 

growth and carcass traits in Japanese Black 

cattle. Our analysis revealed that the 5’UTR 

microsatellite had a significant additive effect 

on carcass weight (CW) (P

Table 2. Summary of nucleotide polymorphisms of the bovine GHSR1a gene 

No. of polymorphism 

B. taurus 

Items Classification Subtotal B. taurus only and B. 

indicus 

Region 

5'-flanking + 

5'-UTR 

B. indicus 

only 

SNP 16 5 2 9 

1-bp deletion † 1 1 0 0 

Exon 1 SNP ‡ 4 4 0 0 

3-bp deletion 1 1 0 0 

Intron 1 SNP 23 6 7 10 

1-bp deletion § 1 0 1 0 

3-bp deletion ¦ 1 0 0 1 

Exon 2 SNP 1 0 0 1 

3'-UTR SNP 3 0 1 2 

Total 51 17 11 23 

5'-UTR Microsatellite (17) ¥ ( 3) ( 8) ( 5) 

(TG) n (10-33) £ (27-33) (19-26) (10-18) 

Intron 1 Microsatellite (4) ¥ ( 1) ( 2) ( 1) 

(GTTT) n (4,5,6,8) ¢ ( 8) (5, 6) ( 4) 

† 

nt-1117(A> - ). ‡ L24V(nt70 (C>G), nt456 (G>A), D191N (nt580 (G>A), nt667 (C>T). 

DelR242 (nt724-726 (AGG> - )). § nt2323 (T>-). ¦ nt1449-1451 (TTT>-). 

¥ 

Number of alleles. £ Number of (TG) repeats. ¢ Number of (GTTT) repeats. 

Re-identification of Fungal Strains Belonging to 

the Fusarium graminearum Species Complex 

and Related Species Preserved at Genebank 

Based on Molecular Phylogenetic Analyses 

Fungal species belonging to the Fusarium 

graminearum species complex (the FG complex) 

are important plant pathogens, especially 

for causing head blight of wheat and barley. 

Strains of the FG complex stored at the NIAS 

Genebank are often distributed as references for 

species identification and causal agents of their 

diseases. Correct identification of the strains is, 

therefore, essential. 

In the FY 2008, the central bank of the 

Genebank at NIAS received many strains of 

this fungal group transferred from the subbanks 

located in other agricultural institutes. 

We analyzed DNA sequences of the histone H3 

gene region (ca. 455 nucleotides) of 163 strains of 

the FG complex and related species to reevaluate 

their taxonomic and phylogenetic positions. 

Phylogenetic analysis of the histone H3 gene region 

revealed that 55 strains (ca. 34%) have been 

correctly identified, but names of 108 strains 

(ca. 66%) were based on the old classification 

systems requiring name changes. Distribution 

of these erroneously-labeled strains to the users 

was temporarily stopped and then restarted 

after the correction of their scientific names. 

Based on the results of molecular phylogenetic 

analyses and quality evaluation, 70 strains of 

Fusarium were selected as “Recommended 

strains for distribution” among Japanese strains 

of the genus. 

In addition to the phylogenetic analyses of the 

Fusarium strains, molecular re-identification of 

67 strains of Trichoderma, potential bio-control 

agents, stored at the NIAS Genebank was 

also completed. Similar molecular analyses on 

Colletotrichum and Agrobacterium strains in the 

NIAS Genebank are in progress. 


Fig 6. Neighbor-joining (NJ) phylogenetic tree of 163 strains of the Fusarium graminearumi species 

complex and related species stored at the NIAS Genebank inferred from DNA sequences of the 

histone H3 gene region. 

55 strains (34 %) were found as correctly identified, but 108 strains (66 % of the examined strains) 

have been labeled erroneously with old names based on previous classification systems and required 

name changes. Fusarium sp. MAFF 425244 was used for an out-group taxon. 



Research activities of the Institute of Radiation 

Breeding are focused on the development of 

new strains of seed-propagated species, vegetatively 

propagated species and woody crops 

through mutation by the application of various 

forms of irradiation. Mutation are induced by 

the following radiation sources: Gamma Field for 

gamma-ray chronic irradiations to the growing 

plants, and Gamma Room for gamma-ray acute 

irradiations to seeds, bulbs, tubers, scions and in 

vitro materials. The Institute is also involved in 

the development of new technologies for plant 

breeding mainly utilizing gamma-ray and ion 

beam irradiation and development of mutant 

resources for genomic analysis, including the 

elucidation of gene expression mechanisms 

in mutants. The Institute provides irradiation 

services and cooperative research at the request 

of universities, private industries, prefectural 

experiment stations, and national institutes of 

Ministry of Agriculture, Forestry and Fisheries. 

Mutagenic effects of ion beam irradiation on rice 

We had been investigating the nature of ion 

beams as a treatment for mutation breeding 

and compared it with gamma ray irradiation 

treatment. We already reported for the Annual 

Report on the usefulness of ion beams for 

mutation breeding in rice by comparing the 

chlorophyll mutation frequency per M 1 spike 

and by the relative frequency and type of 

chlorophyll mutants. In this report, we show 

additional data especially on mutation effectiveness 

and frequency, and on optimum irradiation 

dose, from studies of irradiation with 320MeV 

and 220MeV carbon ions (mean linear energy 

transfer = 86 and 122 keV/µm), 100 MeV helium 

ions (9 keV/µm), and gamma rays (0.2 keV/ 

µm). “Effectiveness” is defined as the number 

of mutations produced per unit dose, whereas 

“efficiency” is defined as the ratio of specific 

desirable mutagenic changes to plant damage in 

the M 1 generation, such as lethality and sterility 

To evaluate the “effectiveness”, the relationship 

between the irradiation dose and the 

mutation frequency per M 2 plant is shown 

(Fig 1). The frequency of chlorophyll mutation 

increased linearly with increasing irradiation 

dose. The doses required to obtain a 1% mutation 

frequency were: 4Gy with 220 MeV carbonion 

beam, 44Gy with 320 MeV carbon-ion beam, 

114Gy with 100 MeV helium-ion beam, and 

Mutation frequency 

per M 2 

plant (%) 

3 

2 

1 

0 

0 50 100 150 200 250 300 

Dose (Gy) 

Fig 1. Effect of ion beam and gamma-ray irradiation on mutation induction. 

The mutation frequency is determined as the number of chlorophyll mutants divided by the number 

of M 2 plants investigated, using the M 1 -plant progeny method. 

● : 220 MeV carbon-ion beam; ○ : 320 MeV carbon-ion beam; ▲ : 100 MeV helium-ion beam; △ : 

gamma rays. 


189Gy with gamma rays. Thus, the effectiveness 

increased with increasing linear energy transfer 

indicating that the “effectiveness” of ion beams 

was higher than that of gamma rays. 

To evaluate the “efficiency” on the basis of 

lethality, the relationship between the mutation 

frequency and the survival rate is shown in 

Fig 2. The mutation frequency per M 2 plant 

increased with decreasing survival rate caused 

by the irradiation treatment. The relationship 

between the mutation frequency per M 2 plant 

and survival rate was not linear; in the range 

where survival rate was 90–100%, the mutation 

frequencies increased markedly. In contrast, in 

the range where survival rate was 90% or less, 

the mutation frequency increased gradually. At 

a 70% survival rate, the 320 MeV carbon-ion 

beam showed the highest mutation frequency 

(2.0%), followed by the 100 MeV helium-ion 

beam (1.9%), 220 MeV carbon-ion beam (1.8%), 

and gamma rays (1.3%). Thus, on the basis of 

lethality, it was suggested that the “efficiency” 

of ion beams was higher than that of gamma 

rays. 

To evaluate the “efficiency” on the basis of 

fertility, the relationship between the mutation 

3 

2 

220 MeV 

carbon-ion 

1 

Mutation frequency per M 2 

plant (%) 

0 

0 20 40 60 80 100 

3 

2 

1 

3 

2 

1 

320 MeV 

carbon-ion 

0 

0 20 40 60 80 100 

100 MeV 

helium-ion 

0 

0 20 40 60 80 100 

3 

2 

1 

Survival rate (%) 

Gamma 

rays 

0 

0 20 40 60 80 100 

Fig 2. Relationship between survival rate and mutation frequency. 


of M 2 plants investigated, using the M 1 plant progeny method. Survival rate is expressed as the 

number of seedlings from irradiated seeds divided by the number of seedlings from the nonirradiated 

seeds. 


frequency and the fertility is shown in Fig 3. 

Fertility also was different in every irradiation 

treatment even if the same radiation type 

was applied at the same dose. Therefore, the 

mutation frequency and the fertility of each 

irradiation treatment were plotted, and their 

relationship is shown. Fertility and mutation 

frequency per M 2 plant showed a negative 

linear relationship for each type of radiation. 

The mutation frequency of ion beams increased 

significantly with decreasing fertility, and the 

same tendency was observed for gamma rays. 

The mutation frequencies at 60% fertility for the 

220 MeV carbon-ion beam treatment and the 

100 MeV helium-ion beam treatment were 1.4%, 

whereas those of 320 MeV carbon-ion beam 

treatment and gamma rays were 1.1%. 

Thus, on the basis of fertility, ion beams 

induced higher frequencies of mutation than 

gamma rays, suggesting that the “efficiency” of 

ion beams was higher than that of gamma rays. 

The optimum irradiation dose for obtaining 

the highest number of mutants from the irradiated 

seeds was clarified from the relationship 

between the irradiation dose and the number of 

mutated M 1 plants per sown M 1 seed (Fig 4). For 

both ion beams and gamma rays, the number of 

mutated M 1 plants per sown M 1 seed reached 

a maximum at a certain dose. At higher doses, 

the number of mutated M 1 plants per sown M 1 

3 

2 

220 MeV carbon-ion 

1 

Mutation frequency per M 2 

plant (%) 

0 

0 20 40 60 80 100 

3 

320 MeV carbon-ion 

2 

1 

0 

0 20 40 60 80 100 

3 

100 MeV helium-ion 

2 

1 

0 

0 20 40 60 80 100 

3 

2 

1 

0 

0 20 40 60 80 100 

Fertility (%) 

Gamma rays 

Fig 3. Relationship between fertility and mutation frequency. 


of M 2 plants investigated, using the M 1 plant progeny method. Fertility is based on seed set in 

panicles of the longest culm in 50 M 1 plants selected at random in each treatment. 


seed decreased with increasing dose. When quadratic 

curves were fitted to the plots for each 

radiation, the maximum numbers of mutated 

M 1 plants per sown M 1 seed was 8.5% at 22 Gy 

with the 220 MeV carbon-ion beam, 9.4% at 73 

Gy with the 320 MeV carbon-ion beam, 7.6% 

at 187 Gy with the 100 MeV helium-ion beam, 

and 5.8% at 209 Gy with gamma rays. Thus, the 

maximum numbers of mutated M 1 plants per 

sown M 1 seed of the 3 types of ion beams were 

higher than that of gamma rays. The dose at 

which the number of mutated M 1 plants per 

sown M 1 seed was highest almost corresponded 

to the shoulder appearing in the survival curves 

for both ion beams and gamma rays. 

0.12 

No. of mutated M 1 

plants 

per sown M 1 

seed 

0.10 

0.08 

0.06 

0.04 

0.02 

0.00 

0 50 100 150 200 250 300 

Dose (Gy) 

Fig 4. Relationship between irradiation dose and the number of mutated M 1 plants per 

sown M 1 seed. 

The number of mutated M 1 plants per sown M 1 seed is determined as the number of 

M 1 plants that produced chlorophyll mutants in their progeny (M 2 plant) divided by the 

number of M 1 seeds sown after irradiation. 

● : 220 MeV carbon-ion beam; ○ : 320 MeV carbon-ion beam; ▲ : 100 MeV helium-ion 

beam; △ : gamma rays. 


Soybean, Glycine max, is one of the most 

important legumes and is the fourth largest 

grain crop after rice, wheat and maize in terms 

of world crop production. Soybean is a staple 

source of nutritious vegetable protein and oil for 

humans and livestock. Additionally, it provides 

industrial materials and biofuel. In Japan, 

soybean is an important source of traditional 

foods such as tofu, natto, miso and soy sauce. In 

2007, we launched the soybean genome research 

program to characterize the genome structure 

of Japanese (domestic) soybean and to develop 

new strategies for effective breeding and for 

the isolation of agronomically important genes. 

The most serious problems in Japanese soybean 

production are unstable and low yield abilities. 

To overcome the problems, many agronomic 

traits including flooding tolerance, disease and 

pest resistance, low temperature tolerance, 

symbiotic ability and regional adaptability 

through maturity control should be improved. 

Information of molecular markers linked to 

these traits and identification of the responsible 

genes are essential for improvement. 

Soybean has an estimated genome size of 

1.1Gb. To develop the basic research resources 


Fig 1. Screen shot of Gbrowes on DaizuBase. 

This shot offers the information about BAC contigs, full-length cDNAs, DNA markers on Williams82 

genome assembly. 

for the whole genome analysis, we have constructed 

bacterial artificial chromosome (BAC) 

libraries from the Japanese soybean cultivar 

Enrei and determined the BAC-end sequences. 

We built up a BAC-based physical map of Enrei 

genome by the BLASTn analysis between the 

BAC-end sequences and the genome assembly 

of a U.S. soybean cultivar, Williams 82 (Glyma 1 

assembly, http://www.phytozome.org/soybean. 

php), and developed a database, “DaizuBase”, 

to visualize the entire Enrei genome (Fig 1). 

DaizuBase includes “Unified map”, which 

indicates the relationship between the linkage 

map and the physical map, and “Gbrowse”, 

which exhibits aligned Enrei BAC clones on the 

Glyma 1 assembly, and facilitates comparative 

genome analyses. We show here one example of 

such analyses for the differences between Enrei 

BAC end sequences and Williams 82 genome 

assembly (Fig 2). DaizuBase will accelerate the 

isolation of agronomically important genes and 

enable the development of new breeding strategies 

for improving productivity of local cultivars 

through the characterizations of the genome 

structure of Japanese soybean cultivars. At 

the present time, only participants in the DDproject 

(Genomics for Agricultural Innovation 

of MAFF) can utilize DaizuBase. In 2008, we 

introduced the next generation sequencer GS- 

FLX (Roche/454) to decode the entire Enrei 

genome, and upgraded from GS-FLX to GS-FLX 

Titanium in 2009. GS-FLX Titanium has a huge 

sequencing ability with 400 to 500 base pair 

read length and about one million high quality 

reads per run. Within 2010, we will assemble the 

whole genome shotgun sequence data of Enrei, 


Fig 2. Differences between Enrei BAC end sequences and the corresponding Williams 82 

genome sequences. 

This graph shows discrepancy rate for each chromosome. Green line shows gap rates, 

red line shows mismatch rates, and blue bar shows total difference rates. 

bacterial blight resistance, and Phytophthora rot 

resistance, and has provided such valuable traits 

to soybean breeding programs. 

The latest high density linkage map based on 

the F2 mapping population of Enrei × Peking 

consists of 2178 markers, including 282 USDA 

SSR markers, 383 EST-SSR markers, 510 SNP 

markers, and 1003 newly designed SSR markers. 

This map spans a total length of 2872 cM with 

an average marker distance of 1.3 cM, and provides 

a detailed genetic framework to achieve 

precise assembly of the genome sequence of 

Enrei. The marker location on the linkage map 

was compared with that on the chromosomescale 

assembly of the soybean genome, Glyma 

1 assembly. Generally, the marker order on the 

linkage map revealed close agreement with that 

of the assembly. Given the soybean genome size 

of approximately 1.1 Gb, the relationship between 

physical and genetic distance on average 

appears to be ~360 kb/cM with a range from 

50 kb/cM to ~7 Mb/cM were found for various 

positions. 

The length of genomic regions that have suppressed 

recombination between markers varied 

widely depending on each chromosome. As an 

example, the ratio of physical and genetic dissequenced 

with GS-FLX Titanium. 

An accurate and well-saturated genetic linkage 

map is fundamental to modern plant breeding 

because it allows both the identification 

of agronomic trait loci, including quantitative 

trait loci, as well as an understanding of genetic 

diversity and genome structure of genetic 

resources. Furthermore, such a linkage map 

is required for construction of a physical map. 

Though a genetic linkage map with about 1,800 

markers has been developed in USDA, the 

marker order on the map is sometimes obscure 

because the information is integrated from 

several linkage maps from different mapping 

populations. 

Limitations of precise marker location and 

resources on a soybean linkage map led us 

to construct a high density linkage map of 

Japanese soybean. We selected three parental 

varieties, Enrei, Peking and Williams 82 for the 

construction of the genetic linkage map. Enrei 

is one of the leading Japanese varieties with 

a high seed quality for food processing and is 

being sequenced as a representative Japanese 

soybean. On the other hand, Peking displays 

many useful characteristics such as cyst nematode 

resistance, soybean mosaic virus resistance, 


tance in the middle regions, 96-110 cM and 94- 

103 cM, of chromosomes 6 and 11, respectively, 

was more than 10 times higher than that in 

the other regions of these chromosomes (Fig 3). 

Such suppressed-recombination regions flanking 

centromeres are termed pericentromeres, 

but these regions are ill-defined in plants. Of 

the known loci controlling flowering time and 

maturity, E1 locus is important for soybean 

breeding in cool-temperature climates. However, 

this locus was located in close proximity to the 

region of low-recombination on chromosome 6 

(Fig 3, left), thus several tens of thousands of 

progenies have been examined to narrow down 

the candidate genes for E1 locus. Similar efforts 

would be required in conventional breeding in 

order to separate or recombine genes within 

the pericentromeric region. Surprisingly, such 

low-recombination regions were found to 

stretch more than halfway across the published 

soybean genome sequence (~555Mb, ca. 60%), 

and to be distributed in a patchy fashion in 

euchromatic regions (79 cM in right panel of 

Fig 3) as well as in the region surrounding 

centromere repeats (100 cM in cM in right panel 

of Fig 3). The distribution of low-recombination 

regions coincided with the abundance of LTRretrotransposons 

among transposable elements 

reported in soybean (Fig 3 red line). Those 

findings together with the highly duplicated 

genome imply that the structure of soybean 

genome is much more complex than the other 

crops, but nonetheless, marker assisted selection 

enables the new combinations of genes located 

in pericentromeric regions. 

Flowering time is one of the most important 

characters relating to soybean seed production 

through the adaptation to different cultivation 

areas and growing seasons. In addition, the 

growth habit such as plant shape is a basic factor 

to control yield components. In soybean, the 

stem growth habits have been divided into two 

types, indeterminate and determinate, according 

to the growth of shoot apical meristem (SAM). 

The indeterminate varieties develop leaves 

and flowers during most of their reproductive 

period. In contrast, the determinate varieties 

cease vegetative growth when the main stem 

Fig 3. The relationships between genetic distance, physical distance, and abundance of LTRretrotransposons 

in soybean chromosomes 6 and 11. 

Small black dots indicate marker locations. The middle regions of chromosome 6 (96-110cM) and 

chromosome 11 (94-103cM) are the main low-recombination regions. In both regions, physical/genetic 

distance ratio was as high as 2Mb/cM. Green arrows indicate the positions of centromeric repeats. The 

distribution of low-recombination regions coincided with the abundance of LTR-retrotransposons (red 

line). The length of sequence including intact elements and solo LTRs both of Gypsy-like and Copia-like 

families was used to calculate DNA contents of LTR-retrotransposon at the 100-kb window size. 


Fig 4. Structures, predicted amino acid sequences, and phylogenetic tree of soybean TFL1 

orthologues, GmTFL1a and GmTFL1b. 

A: Exon/intron structure of GmTFL1a and GmTFL1b. Exon (box) and intron sizes are indicated in 

base pairs. B: Multiple alignment of the predicted amino acid sequences of GmTFL1a, GmTFL1b, 

Lotus japonicus LjCEN/TFL1, pea (Pisum sativum) PsTFL1a, Arabidopsis TFL1, Arabidopsis ATC 

and Antirrhinum majus CEN. Highly conserved amino acids are in black, dark grey, or light grey 

depending on the level of identity (darker = higher level). Arrow indicates an amino acid substitution 

from arginine (R) in the Dt1 allele to tryptophan (W) in the dt1 allele. C: Phylogenetic relationships of 

TFL1/CEN proteins constructed using the Nj method with the program CLUSTAL_W. 


Fig 5. Plant morphology at the maturity stage in the near-isogenic line #6-22 for the Dt1 locus. 

The #6-22dt1 (dt1/dt1) plants were short with a few nodes because of stem termination after first 

flowering, whereas #6-22Dt1 (Dt1/Dt1) plants were tall and produced more nodes. Plants with 

intermediate phenotypes were heterozygous for the Dt1 locus. 

terminates in a terminal raceme. Two genes, 

Dt1 and Dt2, affect the stem growth habits. A 

recessive allele, dt1, and a dominant allele, Dt2, 

hasten the termination of apical stem growth, 

which decreases both plant height and number 

of nodes. The soybean genome research team 

and our collaborator identified sequences 

corresponding to the gene for Dt1. Positional 

information and expression analysis indicated 

that a homolog gene for TERMINAL FLOWER 

1 was considered to be a candidate gene for 

Dt1. Comparisons between genomic DNA and 

cDNA sequences revealed that soybean has 

two homolog genes, GmTFL1a and GmTFL1b. 

GmTFL1a was expressed highly in developing 

seeds and slightly in cotyledons and stem tips, 

whereas GmTFL1b was expressed only in root 

and stem tips. These genes are both composed 

of four exons as in the TFL1 genes of other 

plant species (Fig 4A), and are predicted to 

encode sequences with 173 amino acids (Fig 4B). 

A single base substitution in exon 4 resulted in 

the amino acid substitution at residue 166 from 

arginine in Dt1 allele to tryptophan in dt1 allele 

(Fig 4B). Gene structure including the arginine 

residue was highly conserved among plant species 

(Fig 4C). The association analysis with several 

soybean landraces showed that this amino 

acid substitution in exon 4 was probably a 

functional nucleotide polymorphism for soybean 

stem determination. Near isogenic lines for the 

Dt1 locus exhibited totally different plant shape 

at the maturity stage (Fig 5), and transformation 

with the genomic region of GmTFL1b from 

the indeterminate line complemented the stem 

growth habit in the determinate line. These 

results indicate that GmTFL1b controls the 

stem termination of soybean. Accumulation of 

the molecular knowledge relating to agronomic 

traits including flowering time and plant shape 

will give us the useful information about marker 

assisted selection and molecular breeding of 

soybean. 



Agricultural crops are important resources 

for human food and biomass energy. Food 

shortage in the near future is a serious concern, 

considering ongoing population growth and loss 

of potential cultivation areas in the world due to 

climatic instability and environmental destructions 

as a result of human activities causing 

unprecedented global warming. Plants grow in 

contact with various microorganisms, some of 

which act as pathogens and cause a drastic loss 

of yields. For a stable food supply, it is essential 

to maintain and improve crop productivity even 

under changing and stressful environments. For 

this purpose, profound understanding of plant 

development and functions is essential. 

The Division of Plant Sciences consists of six 

research units that aim to elucidate mechanisms 

of physiological phenomena such as plant growth, 

development, and responses to environments 

and pathogens. These units conduct diverse 

research programs that utilize various resources 

including the complete genomic sequence of rice. 

Development of basic technologies that should 

lead to improved crop productivity is also under 

way by understanding and utilizing diverse potentials 

of plants. Each research unit is engaged 

in the research described below. 

Environmental Stress Research Unit studies 

mechanisms of plants to withstand environmental 

stresses such as low temperatures, high salt, 

and drought to develop strategies for improving 

crop stress tolerance. 

Photobiology and Photosynthesis Research Unit 

studies mechanisms involved in photosynthesis 

efficiency and plant productivity and mechanisms 

of sensing and responding to light to 

develop strategies for improving light responses 

and utilization in crops. 

Plant Disease Resistance Research Unit analyzes 

various disease resistance genes, including 

those involved in rice-blast field resistance, and 

their functions to utilize them for improving 

disease resistance of rice. 


studies interactions between plants and 

microbes including symbionts and pathogens 

(fungi, bacteria, and viruses) for improving 

disease management and for efficient utilization 

of symbiotic microorganisms. 

Protein Research Unit focuses on structures 

and functions of various proteins using NMR 

and X-ray diffraction to establish an important 

basis for utilizing genome information. 


aims to develop new methods for further improvement 

of the safeness of the GM crops and 

strategies leading to optimal regulation of the 

expression of introduced genes. 

The major research topics in fiscal 2009 are 

described in the following pages. 


Environmental Stress Research Unit 

Abiotic stresses such as high salts, drought, 

high humidity, and low temperature are key 

factors for agricultural productivity. Plants potentially 

have various strategies to acclimate to 

environmental changes for optimal growth. To 

improve crop yields under different environmental 

stresses, we look for genes and substances 

that confer stress tolerance. Here we report a 

major achievement in the 2009 fiscal year. 

Selection of the genes related to salt tolerance 

using the rice FOX hunting system 

High levels of salinity in the soil cause serious 

problems for world food production. Rice has 

some mechanisms for protection against salt 

stress, but needs significant improvement. We 

are isolating genes participating in salt tolerance 

in rice and are trying to improve tolerance by 

reinforcing the function of these genes. The 

genes for salt tolerance in rice were selected 

by rice FOX (Full-length cDNA overexpressor 

gene) hunting system. We screened salt-tolerant 

lines from the FOX lines by short- and longterm 

treatments with 120 mM NaCl for 12-dayold 

plants and 100 mM NaCl for 1-month-old 

plants, respectively. Approximately 48.6% and 

48.3% of the FOX lines examined (3,347 lines) 

exceeded the mean growth of the control lines 

shoots and roots, respectively, after 3 days of 

the short-term treatment. In addition, 15.8% and 

16.0% of the FOX lines shoots and roots, respectively, 

exceeded the control mean by 1 standard 

deviation (σ), 3.1% and 3.6%: exceeded the mean 

by 2σ, and 0.7% and 0.8% exceeded the mean by 

3σ. The FOX lines exceeding the control mean 

by 3σ are candidates that may carry genes 

coding proteins related to stress tolerance, such 

as zinc finger family proteins, redox enzymes, 

protein kinases and heat shock proteins. On the 

other hand, approximately 13.1% of the FOX 

lines examined (4,038 lines) survived after 2 

months of the long-term treatment. In addition, 

9.2% of the surviving lines also showed better 

growth than the control lines in the short-term 

treatment, 2.8% exceeded the control mean by 

2σ and 0.2% exceeded the control mean by 3σ 

We are assessing reproducibility of the results 

and re-screening the selected lines. 

Dro1 involved in deep rooting under upland 

field conditions 

Global warming and desertification in recent 

years cause serious drought damage to the ricegrowing 

area in many developing countries 

in which farming relies on precipitation and 

not irrigation. Therefore, breeding of lowland 

rice with drought resistance is becoming a 

very important research topic. In an attempt 

to introduce drought resistance in lowland 

rice, we paid attention to the fact that upland 

rice shows deeper rooting than lowland rice. 

Deeper rooting is an important trait for drought 

avoidance because it enables the plant to absorb 

water deposited in deep soil layers. Mapping 

populations were derived from a cross between 

the lowland cultivar IR64 with shallow rooting 

and the upland cultivar Kinandang Patong (KP) 

with deep rooting. We mapped the deep rooting 

QTL, Dro1 (Deeper rooting 1), between the 

markers RM24393 and RM7424, which delimit 

a 608.4-kb interval in the reference cultivar 

Nipponbare. The Rice Annotation Project 

RAP2 database (http://rapdb.dna.affrc.go.jp/) 

predicts 54 genes in the candidate region for 

Dro1. We are currently in the process of mapbased 

cloning of Dro1. To clarify the influence 

of Dro1 on root distribution in an upland field, 

we quantified the root distribution in different 

soil layers by means of core sampling (Fig 1). A 

line homozygous for the KP allele of Dro1 (Dro1- 

KP) and IR64 did not differ in root dry weight 

in the shallow soil layers (0 to 25 cm), but root 

dry weight of Dro1-KP in deep soil layers (25 

to 50 cm) was significantly larger than that of 

IR64, suggesting that Dro1 plays a crucial role 

in increased deep rooting under upland field 

conditions (Table 1). To investigate effects of 

Dro1 on morphological and yield traits in upland 


field conditions, we harvested plants from Dro1- 

KP and IR64, and measured their morphological 

and yield traits. Although Dro1-KP did not differ 

significantly from IR64 in culm length, panicle 

length, panicle number and shoot dry weight, 

Dro1-KP increased panicle weight significantly 

compared to IR64. Drought stress occurred during 

the vegetative growth stage. Although leaf 

rolling occurred in IR64 at this time, it did not 

occur in Dro1-KP plants throughout the growing 

period (Fig 2). We cannot conclusively explain 

this difference between Dro1-KP and IR64, but it 

very likely resulted from the deeper rooting of 

Dro1-KP plants. The IR64 plants rooted mostly 

in the shallow soil and were, therefore, more 

susceptible to water stress; whereas, the Dro1- 

KP plants also rooted in the deep soil, where 

water would have been more abundant, allowing 

them to avoid or mitigate the water stress. This 

suggests that the KP allele of Dro1 significantly 

improved yield in an upland field under drought 

stress conditions. 

Table 1. Root dry weight in the shallow and deep soil layers in the IR64, Kinandang Patong and Dro1-KP plants. 

Lines 

Shallow soil 

Root dry weight (mg) 

Deep soil 

Beside 15-cm distance Beside 15-cm distance 

Mean SD Mean SD Mean SD Mean SD 

IR64 359.2 + 97.3 a 261.0 + 138.2 a 10.1 + 8.7 a 16.3 + 13.8 a 

Kinandang Patong 629.7 + 218.6 b 267.1 + 161.1 a 71.9 + 30.2 b 70.2 + 41.0 b 

Dro1-KP 370.2 + 95.8 a 245.0 + 89.9 a 50.8 + 19.1 b 59.6 + 16.0 b 

“Beside”, core sample taken from beside the hill; “15-cm distance”, core sample taken 15 cm from the hill. 

SD: standard deviation. Values labeled with different letters differ significantly among the four lines (P < 0.05, 

Tukey’s multiple-comparison test). 

Fig 1. Evaluation of the 

root distribution in the field 

as determined from core 

samples. 

Fig 2. Differences in leaf 

condition between IR64 

and Dro1-KP at 70 days 

after sowing. 


Photobiology and Photosynthesis Research Uni 

Plants utilize light as an energy source and 

also as external signals for monitoring changes 

in the environment. We are investigating regulation 

mechanisms of photosynthesis, metabolism 

and translocation, as well as molecular mechanisms 

of perception and transmission of light 

signals and the resultant responses of plants, 

aiming at developing innovative techniques 

for improved productivity, more efficient light 

utilization, and custom modification of growth 

and architecture of plants. 

Gene expression profiling of rice plants exposed 

to long-term CO 2 

enrichment 

Elevated CO 2 in the atmosphere enhances the 

growth and the productivity of most plant species. 

Although leaf photosynthesis is enhanced at 

an early stage of vegetative growth, prolonged 

exposure to elevated CO 2 leads to a decline of 

photosynthesis so that the final productivity and 

grain yield are lower than those expected in 

elevated CO 2 . To clarify mechanisms underlying 

these effects of elevated CO 2 , gene expression 

profiles of the leaves were compared between 

rice plants grown at elevated (68 Pa) and ambient 

(38 Pa) CO 2 concentrations in CO 2 -controlled 

chambers. To discriminate influences of the 

nitrogen availability from those of elevated CO 2 , 

rice plants were grown under three different 

soil nitrogen conditions (0, 0.6 and 1.2 g N per 

8-L pot). The 11th leaves of rice plants grown 

in this way showed typical symptoms observed 

in plants grown in elevated CO 2 , namely, 

reduced photosynthetic rate and decreased 

content of soluble protein on the leaf area basis. 

Comparison of gene expression profiles of the 

11th leaves by microarray analyses revealed 

considerable differences between ambient and 

elevated CO 2 . Forty six genes were upregulated 

(> 1.5-fold) and 35 genes were downregulated 

CO 2 fixation 

RbcS 

PGK 

GAPDH 

Rca 

Starch synthesis 

AGPaseS 

SS 

Calvin cycle 

FBPase SBPase 

PRK 

AGPaseL 

GB-SS 

Aldolase 

RuBP regeneration 

Fold over ambient CO 2 

0.6 1.7 

Fig 1. Effects of elevated CO 2 on the expression of genes related to the Calvin cycle and starch 

synthesis. 

Changes in the gene expression level in elevated CO 2 relative to ambient CO 2 are indicated by 

the color of dot. The three dots in each row represent results with low (left), medium (center), and 

high (right) nitrogen. For enzymes encoded by a gene family, data for genes highly expressed in 

the leaf blade are shown. RbcS: Rubisco small subunit; PGK: phosphoglycerate kinase; GAPDH: 

glyceraldehyde-3-phosphate dehydrogenase; Rca: Rubisco activase; FBPase: fructose bisphosphate 

phosphatase; aldolase, fructose bisphosphate aldolase; SBPase: sedoheptulose bisphosphate 

phosphatase; PRK: phosphoribulokinase; AGPaseS: ADP-glucose pyrophosphorylase small subunit; 

AGPaseL: ADP-glucose pyrophosphorylase large subunit; SS: starch synthase; GB-SS: granulebound 

starch synthase. 


(< 0.68-fold) in elevated CO 2 . These included 

many genes related to signal transduction and 

transcription regulation. By contrast, changes 

in expression levels of genes for photosynthesis 

and primary metabolism were rather small, 

ranging from 0.6- to 1.7-fold over those in ambient 

CO 2 . Even so, genes involved in different 

metabolic pathways showed different responses 

to elevated CO 2 . This was evident in genes 

encoding chloroplastic enzymes. Among genes 

for Calvin cycle enzymes, those for enzymes 

involved in CO 2 fixation were downregulated, 

whereas those for enzymes involved in regeneration 

of ribulose bisphosphate (RuBP) were 

upregulated under elevated CO 2 (Fig 1). Genes 

for enzymes involved in starch synthesis inside 

the chloroplast were also upregulated. These 

results suggest that each set of genes involved 

in a particular metabolic pathway/function are 

co-regulated so as to optimize metabolic balance 

in elevated CO 2 . 

Lodging resistance locus prl5 improves 

physical strength of rice plants irrespective of 

fertilization conditions 

Although high fertilizer application rates 

increase grain yield and biomass in rice, they 

reduce lodging resistance. Lodging resistance 

locus prl5 enhances resistance to the force pushing 

the plant down to the ground through delaying 

leaf senescence and increasing carbohydrate 

reaccumulation in culms. To verify whether 

prl5 is effective in enhancing lodging resistance 

at high fertilizer application rates, the effects 

of prl5 under different N-P-K basal application 

rates were investigated using a chromosome 

segment substitution line which contained 

prl5 from Kasalath in the genetic background 

of Koshihikari (CSSL prl5). High fertilizer 

application rates enhanced the pushing resistance 

of the lower part of a plant, but did not 

consistently affect that of the whole plant (Fig 

2A). prl5 enhanced pushing resistances of both 

the lower part and whole plants in all fertilizer 

application rates tested. Plant height, biomass 

and grain yield were higher with higher fertil- 

Fig 2. Pushing resistance, the chlorophyll content, and the content of non-structural carbohydrate 

(NSC) in culms of rice plants at the fully ripe stage. 

Koshihikari and CSSL prl5 were grown under designated fertilizer application rates. (A) Pushing 

resistances of the lower part (left panel) and the whole plant (right panel), which were measured, 

using a prostrate tester (Daiki Rika Kogyou Co., Tokyo, Japan), as the force required to bend stem 

irreversibly when plants were pushed down to an angle of 45° from the vertical. (B) Chlorophyll 

content of the second leaf blade. (C) NSC content in basal culms. Averages of data from five to six 

plants are shown. 


izer application rates, whereas stem diameter 

and dry weight of basal culms remained unaffected. 

At the fully ripe stage, chlorophyll and 

Rubisco contents in the leaves were unaffected 

by fertilizer application rates, but they were 

increased by prl5, an indication of delayed leaf 

senescence. Such effects were marked in the 

second leaf blades, which were senescing at the 

assay time (Fig 2B). The concentration of nonstructural 

carbohydrates (NSC) in basal culms 

was unaffected by high fertilizer application 

rates, whereas it was significantly increased 

by prl5 at the fully ripe stage (Fig 2C). These 

results suggest that high fertilizer application 

rates reduce lodging resistance by elevating the 

center of gravity of a plant and that prl5 can 

counteract this effect by enhancing the physical 

strength of the lower part through delaying leaf 

senescence and increasing NSC reaccumulation 

in culms irrespective of fertilizer application 

rates. 

Phytochromes are solely responsible for 

perception of red and far-red light in rice 

It is generally accepted that phytochromes 

are the sole photoreceptors for perceiving 

red and far-red light in plants. To confirm 

this hypothesis, a phytochrome triple mutant 

(phyAphyBphyC) was generated in rice and its 

responses to red and far-red light were monitored. 

Since rice only has three phytochrome 

genes (PHYA, PHYB and PHYC), this mutant 

has no phytochrome. Wild-type rice seedlings 

grown in darkness develop long coleoptiles 

while undergoing regular circumnutation. The 

triple mutants also show this characteristic 

skotomorphogenesis, even under continuous illumination 

with either red or far-red light (Fig 3). 

The morphology of the triple mutant seedlings 

grown under red or far-red light was completely 

the same as that of etiolated seedlings, and no 

expression of light-induced genes was detected 

in the mutant seedlings, an indication that 

phytochromes are the sole photoreceptors for 

perceiving red and far-red light, at least during 

rice seedling establishment. The shape of triple 

mutant adult plants was also dramatically 

altered in that the internodes were extended 

even during vegetative growth. Wild-type plants 

never elongated their internodes during vegetative 

growth. The triple mutants also flowered 

very early under long day conditions and set 

very few seeds due to incomplete male sterility. 

These observations indicate that phytochromes 

play an important role in maximizing photosynthetic 

abilities during vegetative growth in rice. 

Fig 3. The shoot-top movement of growing rice seedlings. 

Four lines of rice seedlings were grown in the monitoring system either in darkness (Dark) or under 

continuous red light illumination (Red) for a week. The top point pixels of the growing seedlings were 

extracted from each individual time-lapse image and overlaid onto the final picture. Orange lines, 

movement of coleoptile tops; green lines, movement of the first/second leaf tops. WT: Nipponbare; 

phyB: phyB single mutant; phyBC: phyBphyC double mutant; phyABC: phyAphyBphyC triple mutant. 


Plant Disease Resistance Research Unit 

Developmental expression pattern of Pb1 

originates from local genome duplication and 

may account for the durability of Pb1-based 

resistance 

Rice blast, caused by Magnaporthe oryzae, is 

one of the most widespread and destructive 

diseases of rice. The panicle blast occurring 

after heading stage causes yield loss and 

lowered quality of brown rice. We isolated the 

Pb1 (Panicle blast 1) gene by map-based cloning, 

which was identified as a major quantitative 

gene for panicle blast resistance, and investigated 

the mechanisms underlying its characteristic 

traits of resistance. 

In a Pb1+ cultivar, the level of blast resistance 

increased during plant growth compared 

with a Pb1- cultivar (Fig 1A). The resistance 

was weak during young stages, but became 

stronger in adult stages (adult resistance and 

panicle resistance). Real-time qRT-PCR analysis 

of Pb1 in the Pb1+ cultivar showed that the 

level of Pb1 transcripts was low during the 

early vegetative stages, but was upregulated 

in the 10-leaf and flag leaf stages, and further 

upregulated at the full heading stage (Fig 1A). 

Thus, the Pb1 expression pattern accounts for 

Fig 1. Developmental expression pattern and origin of Pb1 

(A) Developmental patterns of Pb1 expression and blast resistance. The developmental pattern of 

Pb1 expression in the leaves of Pb1+ cultivar, Koshihikari-Aichi-SBL, at the 2 (2L), 6 (6L), 10 (10L) 

and flag leaf (FL) stages, and in the panicle after heading, are shown by dotted lines. Spores of 

blast fungus (race 001.0), a race compatible with Nipponbare, were spray inoculated and disease 

symptoms were evaluated by the lesion numbers at the 2, 6, 10 and flag leaf stages, and the 

percentage of diseased grains relative to those in near isogenic Pb1- cultivar, Koshihikari (solid line). 

(B) Generation of Pb1 by local genome duplication. 


adult resistance and panicle resistance in Pb1+ 

cultivars. Plant resistance generally imposes 

fitness costs on fungal survival and, therefore, 

acts as adaptive selection pressure for fungal 

variants. The amount of fitness costs that plants 

impose on the blast fungus is the total of potential 

disease-resistance reactions over the plants’ 

lifetime, which is determined by the duration 

and strength of defense responses. Pb1 cultivars 

show resistance only during adult stages. In 

addition, Pb1-based resistance is of quantitative 

nature and not complete, even in adult stages 

and when overexpressed which likely accounts 

for the durability of Pb1-based resistance. 

Promoter:GUS analysis indicated that local 

genome duplication played a crucial role in 

the generation of Pb1 by placing a promoter 

sequence upstream of its coding sequence, 

thereby conferring a Pb1-characteristic expression 

pattern to a transcriptionally inactive 

‘sleeping’ resistance gene (Fig 1B). 

MAPK cascade regulates MAMPs-triggered 

defense metabolic flow in rice 

The earliest plant defense response against 

potential microbial pathogens is triggered 

by recognition of the pathogens through 

their microbial-associated molecular patterns 

(MAMPs). Mitogen-activated protein kinases 

(MAPKs) are known to play a pivotal role in 

mediating the MAMPs signals; however, the 

molecular mechanism governing the MAPKmediated 

signal transduction has been poorly 

understood. We identified two rice MAPKs 

(OsMPK3 and OsMPK6) and one MAPK kinase 

(OsMKK4), which were activated by chitin 

oligomer, a fungal MAMP, in cultured rice cells 

(Fig 2). OsMKK4 regulates kinase activities of 

both OsMPK3 and OsMPK6 through its phosphorylation 

activity. To characterize OsMKK4- 

OsMPK3/OsMPK6 cascade, we introduced a 

mutation into OsMKK4 to evoke constitutive 

activation of OsMKK4 (OsMKK4DD). Expression 

of OsMKK4DD in rice cultured cells induced 

various defense responses, such as cell death, 

biosynthesis of diterpenoid phytoalexins and 

lignin, but not generation of extracellular ROS 

(Fig 2). To identify the signal cascade lying 

downstream of OsMKK4-OsMPK3/OsMPK6, we 

performed a genome-wide transcript analysis. 

Conditional expression of OsMKK4 DD induced 

extensive alterations in gene expression, 

which implied dynamic changes of metabolic 

flow from glycolysis to secondary metabolite 

biosynthesis while suppressing basic cellular 

activities such as translation and cell division. 

Fig 2. A model for the role of the Os MKK4–Os MPK3/Os MPK6 

cascade in the MAMPs-triggered defense responses. 


Further, OsMKK4DD-induced cell death and 

expression of diterpenoid phytoalexin pathway 

genes were dependent on OsMPK6, but 

expression of phenylpropanoid pathway genes 

was not. Collectively, the data indicate that the 

OsMKK4–OsMPK6 cascade plays a crucial role 

in reprogramming plant metabolism during 

MAMP-triggered defense responses. 

Rice WRKY45 plays an important role in 

plant-activator induced blast and leaf blight 

resistance 

Plant ‘activators’, such as benzothiadiazole 

(BTH), protect plants from various diseases by 

priming the plant salicylic acid (SA) signaling 

pathway. We previously reported that a transcription 

factor identified in rice, WRKY45, plays 

a pivotal role in BTH-induced blast resistance by 

Fig 3. Microscopic analysis of the M. grisea infection process in WRKY45-ox plants. 

(A) Frequency of fungal invasion into rice cells. Nipponbare (NB) and WRKY45-ox rice plants (#15 and 

#21) were inoculated with M. grisea by intact leaf-sheath inoculation and examined for invasion into rice 

cells under a light microscope at 2 and 4 dpi. 

(B–D) Light micrographs of infection sites 48 h after inoculation. Leaf sheaths were treated with 0.8 M 

sucrose to cause plasmolysis. In Nipponbare, well-developed invading hyphae with their tips expanding 

into rice cells were observed; the cells were plasmolyzed (B). In WRKY45-ox leaves, most appressoria 

did not extend invading hyphae into rice cells (C). Fungus-invaded WRKY45-ox cells often exhibited 

cytoplasmic granulations (D). 

(E) Transmission electron micrographs of M. grisea attempted infection site in WRKY45-ox leaf sheath. 

No penetration was observed with H 2 O 2 accumulating between the cell wall and cytoplasm in the 

epidermal cell (fine black spots of Ce(OH) 2 OOH precipitates). 

Ap: appressorium; IH: invading hypha; Sp: spore. Arrowheads show plasma membranes of plasmolyzed 

epidermal cells. Bars = 10 µm. 

(collaboration with Ishikawa prefectural university ) 


mediating SA signaling. Here, we report further 

functional characterization of WRKY45. Different 

plant activators vary in action points, either 

downstream (benzothiadiazole and tiadinil) or 

upstream (probenazole) of SA. Rice blast resistance 

induced by both types of plant activators 

was markedly reduced in WRKY45-knockdown 

(WRKY45-kd) rice, indicating a universal role 

for WRKY45 in chemical-induced resistance. 

Light and transmission electron microscopic 

analysis showed that fungal invasion into rice 

cells was blocked at most attempted invasion 

sites in WRKY45-ox rice (Fig 3A-C, pre-invasive 

defense). Hydrogen peroxide accumulated 

within the cell wall just underneath invading 

fungus appressoria or between the cell wall and 

the cytoplasm, implying a possible role for H 2 O 2 

in pre-invasion defense (Fig 3E). In addition, a 

hypersensitive-reaction-like reaction was observed 

in rice cells, in which fungal growth was 

inhibited after invasion (Fig 3D, post-invasive 

defense). Thus, WRKY45-ox rice plants exhibit 

two layers of defense mechanism against blast 

fungus; a pre-invasion defense that blocks fungal 

invasion into cells, and a post-invasion defense 

accompanying HR-cell death that confines 

fungal growth to primarily invaded cells. The 

leaf blast resistance of WRKY45-overexpressed 

(WRKY45-ox) rice plants was much higher than 

other known blast-resistant varieties. WRKY45- 

ox plants also showed strong panicle blast 

resistance. BTH-induced leaf-blight resistance 

was compromised in WRKY45-kd rice, whereas 

WRKY45-ox plants were highly resistant to leaf 

blight. This indicates the broad-spectrum nature 

of WRKY45-mediated disease resistance and the 

versatility of its application. 

Abscisic acid interacts antagonistically with 

salicylic acid-signaling pathway in rice- 

Magnaporthe grisea interaction 

Plant hormones play important signaling roles 

in plant-pathogen interactions. Each hormone 

generates and transmits a distinct defense 

signal, while cooperative and/or antagonistic 

crosstalks between them are emerging as 

pivotal for outcomes of plant-pathogen interactions. 

We recently found that abscisic acid (ABA) 

antagonistically interacts with salicylic acid (SA) 

signaling pathway and thereby compromises 

blast resistance of rice plants. 

Exogenous application of ABA drastically 

compromised the rice resistance to both compatible 

and incompatible M. grisea strains, indicating 

that ABA negatively regulates both basal and 

R-gene-mediated blast resistance. ABA markedly 

suppressed the transcriptional upregulation of 

WRKY45 and OsNPR1, the two key components 

of the SA signaling pathway in rice, induced by 

SA/benzothiadiazole (BTH) or by blast infection. 

Overexpression of OsNPR1 or WRKY45 largely 

negated the enhancement of blast susceptibility 

by ABA, suggesting that ABA acts upstream of 

WRKY45 and OsNPR1 in the rice SA pathway. 

ABA-responsive genes were induced during 

blast infection in a pattern reciprocal to those 

of WRKY45 and OsPR1b in compatible riceblast 

interaction, but only marginally in the 

incompatible one. These results suggest that the 

balance of SA/ABA signaling is an important 

determinant for the outcome of rice-M. grisea 

interaction. ABA was detected in hyphae and 

conidia of M. grisea as well as in culture media, 

implying that M. grisea uses its own ABA to 

increase local ABA levels in host rice cells at 

infection sites, thereby suppressing the rice SA 

signaling pathway to avoid defense responses. 

It has often been observed that the incidence 

and severity of rice blast disease is enhanced 

by abiotic stresses, such as low temperature 

and drought. Previous studies and our current 

findings suggest possible linkage of ABA to this 

stress-associated rice susceptibility to blast disease. 

The crosstalk of SA/ABA signaling could 

also be important in balancing abiotic and biotic 

responses in rice to shift defense resources to 

the most life-threatening stress. 


Protein Research Unit 

Proteins are dynamic molecules that often undergo 

conformational changes while performing 

their specific functions. Hence, understanding of 

protein function requires a detailed knowledge 

of three-dimensional structure, dynamics, and 

interactions with other biomolecules. The 

Protein Research Unit utilizes X-ray crystallography 

and NMR spectroscopy to determine the 

protein structure and to monitor the dynamical 

behavior of a protein at many specific sites. In 

addition, structural and kinetic aspects of intermolecular 

interactions are studied by various 

experimental methods such as NMR, surface 

plasmon resonance, and analytical ultracentrifugation. 

Proteins involved in important biological 

phenomena are selected as targets. 

Crystal structure of β-L-arabinopyranosidase 

provides structural basis for substrate specificity 

and novel L-arabinopyranose binding 

property of the enzyme 

Arabinogalactan proteins (AGPs) are a family 

of plant cell surface proteoglycans and are 

considered to be involved in plant growth and 

development. Molecular and biological evidence 

indicates that AGPs have specific functions 

during root formation, promotion of somatic 

embryogenesis, and attraction of pollen tubes 

to the style. AGPs are characterized by the 

presence of large amounts of carbohydrate 

components rich in galactose (all the sugars in 

the present study are in the D-configuration 

unless otherwise specified) and L-arabinose and 

by protein components rich in hydroxyproline, 

serine, threonine, alanine, and glycine. Type 

II arabinogalactans and short oligosaccharides 

are the two types of carbohydrate attached to 

the AGP backbone. Type II arabinogalactans 

have β-1,3-linked galactosyl backbones in monoor 

oligo-β-1,6-galactosyl and/or L-arabinosyl 

side chains. L-Arabinose and lesser amounts of 

other auxiliary sugars, such as glucuronic acid, 

L-rhamnose and L-fucose, are attached to the 

side chains primarily at non-reducing termini. 

Since many putative protein cores exist and 

the structures of the carbohydrate moieties are 

complex, it has been difficult to differentiate one 

AGP species from another in plant tissues. This, 

in turn, has made it difficult to assign specific 

roles to individual AGPs. Glycoside hydrolases 

specific to particular sugar residues or to a type 

of glycosidic linkage would be useful tools in the 

structural analysis and assignment of AGPs. 

So far, there are many biochemical and 

structural reports on galactanases that 

hydrolyze β-1,3- or β-1,6-galactosyl linkages 

because the β-1,3-β-1,6-galactan backbone is 

the common structure of heterogeneous AGPs. 

In contrast, there are few reports on enzymes 

that liberate L-arabinopyranose. We have, for 

the first time, determined the crystal structures 

of β-L-arabinopyranosidase from Streptomyces 

avermitilis (SaArap27A) in the ligand-free 

state and in complexes with L-arabinose and 

galactose (Fujimoto et al, 2009; Ichinose et al, 

2009). The structure of SaArap27A consists of 

four domains (Fig 1A). The N-terminal catalytic 

domain (domain I, residues 45-339) has a (β/α) 8 

barrel, which is observed in glycoside hydrolase 

family 27 (GH27). The second domain (domain II, 

residues 340-430) is an eight-stranded antiparallel 

β-domain containing tandemly repeated 

Greek key motifs, but in imperfect shape. The 

relative arrangement of these two domains 

is the same as that of other GH27 enzymes. 

Domain II is located at the 7th and 8th α-helices 

of the catalytic domain. The third domain 

(domain III, residues 431-531) also contains eight 

antiparallel β-strands but comprises a β-jellyroll 

domain. This domain is located adjacent to 

domain II and also contacts the catalytic domain 

over the 5th α-helix and the loop after the 6th 

α-helix. The last domain, i.e. the C-terminal 

domain (domain IV, residues 532-658), is a ricintype 

lectin domain consisting of the β-trefoil 

fold and is of the carbohydrate-binding module 

belonging to family 13 (CBM13). This domain is 

in front of the catalytic domain covering the 6th 


α-helix and the loop before the 5th α-helix so 

that it contacts all three other domains forming 

a compact entity. 

Sugar complex crystals were prepared by 

soaking the SaArap27A crystals with L-arabinose 

or galactose solution, and the structures were 

determined. The relative positions of the 

domains did not change in comparison to the 

ligand-free state. In both the L-arabinose and 

galactose complexes, one sugar molecule was 

bound in the active site of the catalytic domain. 

The positions of the bound sugar molecules are 

almost the same in both the complexes, and the 

sugar-binding mechanism is also well conserved. 

The difference was only observed in recognition 

of the galactose (Gal)-O 6 atom, which is not 

present in L-arabinose. The Gal-O 6 atom has a 

unique hydrogen bond to Glu99 O ε1 atom of the 

enzyme in the galactose complex. 

The GH27 family includes α-galactosidase, 

α-N-acetylgalactosaminidase and isomaltodextranase. 

To date, five crystal structures have 

been determined for GH27-family members. 

Among these five, the amino-acid sequence of 

SaArap27A has the highest homology to that 

of rice α-galactosidase (41% identity and 56% 

similarity). The substrates of these enzymes 

are differentiated by hydroxymethylation of the 

fifth C atom (Figs. 1C and 1D). In comparison 

to the structure of rice α-galactosidase (Fig 1B; 

Fujimoto et al, 2003), there were no obvious 

differences in the sugar-binding manner, except 

for the recognition of the Gal-O 6 atom. In the 

structure of α-galactosidase (Fig 1F), the Gal-O 6 

atom makes a hydrogen bond with Asp52 

side chain while the corresponding residues of 

SaArap27A in the L-arabinose complex (Fig 1E), 

i.e. Glu99, are free from the hydrogen-bond 

network between the enzyme and the bound 

sugar. These observations suggest that Glu99 

plays a critical role in determining substrate 

specificity of the enzyme between galactose and 

L-arabinose. 

SaArap27A has almost the same K m values 

for PNP-β-L-Arap and PNP-α-Galp, 3.6 ± 0.4 mM 

and 5.1 ± 0.3 mM, respectively. However, the 

k cat values of the enzyme for PNP-α-Galp (2.3 ± 

0.1/min) was ~140 times lower than for PNP- β 

-L-Arap (317 ± 10/min), resulting in substrate 

specificity toward PNP-β-L-Arap (k cat /K m = 88 

/mM/min) over PNP-α-Galp (k cat /K m = 0.5 

/mM/min). To investigate the role of Glu99 for 

enzyme activity, a mutant enzyme (SaArap27A/ 

E99D) was constructed in which Glu99 was 

replaced by Asp. The K m and k cat values of the 

mutant for PNP-α-Galp and PNP-β-L-Arap were 

4.3 ± 0.1 mM and 29 ± 0.6/min, and 11.1 ± 0.9 

mM and 17 ± 1/min, respectively. The results 

demonstrate that substitution of Glu99 by Asp 

converts substrate specificity of SaArp27A from 

PNP-β-L-Arap (k cat /K m = 1.5/mM/min) to PNP-α 

-Galp (k cat /K m = 6.7/mM/min). 

The structure of the L-arabinose complex 

revealed that three L-arabinose molecules were 

bound in three subdomains (α, β, and γ) of the 

CBM13 of SaArap27A in the same manner. 

In contrast, only one galactose molecule was 

bound in one of these three subdomains in the 

galactose complex. Furthermore, all the sugar 

molecules bound in the three subdomains of 

CBM13 were found to be L-arabinose when the 

crystals were soaked in a mixture of L-arabinose 

and galactose. These results suggest a novel 

L-arabinose binding property for CBM13 of this 

enzyme. 

In summary, we have determined crystal 

structures of SaArap27A in the ligand-free 

state and in complexes with L-arabinose and 

galactose. The protein is classified as GH27 and 

consists of four modules. Although domains II 

and III themselves do not have sugar-binding 

activity, they may act as spacer domains to 

allow the C-terminal sugar-binding CBM13 access 

to the catalytic site in the N-terminal GH27 

domain. Based on these structures, together 

with the crystal structure of rice α-galactosidase 

previously determined by us, we investigated the 

substrate-recognition mechanism of SaArap27A. 

The enzymatic activity of SaArap27A differs 

from that of other α-galactosidases due to a 

single amino acid substitution. Furthermore, 

CBM13 of this enzyme has novel L-arabinose 

binding property. This is the first report of β 

-L-arabinopyranosidase as a new member of 

the GH27 family and CBM13 as an L-arabinosebinding 

module. 


Fig 1. Comparison of β-L-arabinopyranosidase from Strepromyces avermitilis (SaArap27A) and rice 

a-galactosidase. 

(A) Crystal structure of SaArap27A complexed with L-arabinose. Four bound L-arabinose molecules, 

one in the catalytic site of GH27 and three in the α-, β-, and g-subdomains of CBM13, and two 

catalytic residues are shown as ball-and stick models. (B) Crystal structure of rice a-galactosidase 

complexed with D-galactose. A D-galactose molecule bound in the catalytic site and two catalytic 

residues are shown as ball-and-stick models. Chemical structures of L-arabinose (C) and 

D-galactose (D). Close-up views of the catalytic pockets in SaArap27A complexed with L-arabinose 

(E) and rice a-galactosidase complexed with D-galactose (F). Side chains of the residues involved 

in substrate recognition are shown as ball-and-stick models. Hydrogen bonds between the enzyme 

and the bound sugar are shown as dotted lines. 

References 

Fujimoto Z, Ichinose H, Harazono K, Honda M, 

Uzura A, Kaneko S (2009) Crystallization and 

preliminary crystallographic analysis of β 

-L-arabinopyranosidase from Streptomyces 

avermitilis NBRC14893. Acta Crystlogr. F 

Struct. Biol. Cryst. Commun., 65: 632-634. 

Ichinose H, Fujimoto Z, Honda M, Harazono K, 

Nishimoto Y, Uzura A, Kaneko S (2009) A 

β-L-arabinopyranosidase from Streptomyces 

avermitilis is a novel member of glycoside 

hydrolase family 27. J. Biol. Chem., 284: 

25097-25106. 

Fujimoto Z, Kaneko S, Momma M, Kobayashi H, 

Mizuno H (2003) Crystal structure of rice α 

-galactosidase complexed with D-galactose. J. 

Biol. Chem., 278: 20313-20318. 



Higher plants interact with numerous kinds of 

microbes during their life cycle. These interactions 

are classified into symbiotic ones, such as 

mycorrhizal and rhizobial symbiosis where the 

fungi or bacteria supply inorganic nutrition and 

annmonia, and parasitic interactions which can 

result in lesion formation caused by infection 

with pathogenic viruses, bacteria and fungi that 

leads to damage in crop production. The final 

goal of this research unit is to elucidate the molecular 

mechanisms of plant-microbe interactions 

to establish novel strategies for production and 

protection of crops. In this section, our recent 

results on the rhizobia-legume, blast-rice and 

tobamovirus-tomato interactions are described. 

CERBERUS, a novel U-box protein containing 

WD-40 repeats, plays crucial roles in formation 

of the infection thread and development 

of nodules in the legume–Rhizobium symbiotic 

interactions. 

Soil-born N2-fixing bacteria, Rhizobium sp., 

infect roots of legume plants and form nodules 

where symbiotic N2 fixation occurs. After the 

early signal exchange between the host plants 

and bacteria, endosymbiotic infection of legume 

plants by the bacteria starts through infection 

threads (ITs), which are initiated within and 

penetrate from root hairs and deliver the endosymbionts 

into nodule cells. Despite recent progress 

in understanding the mutual recognition 

Fig 1. Root nodule phenotypes of wild type Gifu and cerberus. 

A, B: Nodulation phenotype of Gifu and cerberus at 14 dpi. Mature pink nodules were formed on 

wild type Gifu roots (A), whereas nodule organogenesis was arrested at premature stage on cerberus 

roots (B). Scale bar: 1 mm. 

C, D: Infection phenotype of Gifu and cerberus at 7 dpi. Infection process was visualized by 

inoculation of M.loti MAFF303099 carrying DsRed. In wild type Gifu, the bacteria infected root 

cells through an IT in a root hair (C). In cerberus, infection of bacteria was arrested at colonization, 

resulting in abortion of IT initiation (D). Scale bar: 20 µm. 

E, F: Nodule phenotype of Gifu (16 dpi) and cerberus (21 dpi). Sections were stained with toluidine 

blue. Wild type Gifu nodule developed infection cells containing bacteroids and uninfected cells in 

central zone (E). In cerberus, bacteria were not released into infection cells, resulted in premature 

nodule development (F). Scale bars: 100 µm. 


and early symbiotic signaling cascades in host 

legumes, the molecular mechanisms underlying 

bacterial infection processes and successive nodule 

organogenesis still remain to be understood. 

We isolated a novel symbiotic mutant of a model 

legume plant, Lotus japonicus, cerberus, deficient 

in IT formation and nodule organogenesis (Fig 

1A and B). In cerberus mutants, the bacteria 

colonized on curled root hair tips, but hardly 

penetrated into root hair cells (Fig 1C and D). 

ITs were occasionally formed inside the root 

hair cells, most of which were arrested within 

the epidermal cell layer. Nodule organogenesis 

was arrested prematurely, resulting in the 

formation of a large number of small bumps in 

which no endosymbiotic bacteria were found 

(Fig 1E and F). By map-based cloning, we 

isolated the causal gene, CERBERUS, coding for 

a novel protein harboring a U-box domain and 

WD-40 repeats. The expression of CERBERUS 

was detected in the roots and nodules, and was 

elevated after inoculation of Mesorhizobium 

loti. High levels of expression were observed 

in the primordia of developing nodules and 

the infected zone of mature nodules. These 

phenotypic and genetic analyses, together with 

comparisons with other legume mutants with 

defects in IT formation, strongly indicate that 

CERBERUS plays a key role in the very early 

steps of IT formation as well as in growth and 

differentiation of nodules. 

An inhibitory interaction between viral and 

cellular proteins underlies the resistance of 

tomato to a non-adapted tobamovirus. 

Each virus infects only a limited range of 

hosts, i.e., most living organisms are resistant to 

a particular virus. However, factors that restrict 

viral host range are difficult to analyze, and the 

mechanisms of the restriction had been poorly 

understood. Here, we found that the tm-1 protein, 

which binds to and inhibits the functioning 

of the replication proteins of Tobacco mild green 

mosaic virus (TMGMV), is one of the factors 

that underlie multi-layered resistance mechanisms 

in tomato to TMGMV. This result well 

explains why viruses rarely adapt to non-host 

organisms and could contribute to the development 

of crops that show durable resistance to 

viruses. For details, please refer to the Research 

Highlights section of this report. 

The supernatant of a conidia suspension of 

Magnaporthe oryzae promotes the infection 

of rice plants 

Magnaporthe oryzae, causal agent of rice blast 

disease, germinates and forms appresoria, a 

differentiated organ specific to infection, before 

penetration. During the infection process, the 

mode of signal exchange between rice and M. 

oryzae have been studied extensively and, in 

several reports, production of low molecular 

weight toxins that induces visible damages 

when applied to plants. However, the biological 

significance of these substances is not known. 

As reported last year, a drastic accumulation 

of H 2 O 2 is observed in the epidermal cells of 

rice leaf sheath. We investigated the roles of 

H 2 O 2 in the infection of rice by M. oryzae. The 

supernatant of conidia suspension (SCS) includes 

catalase activity, which is easily removed by 

washing conidia by centrifugation. In a leaf 

sheath assay with samples taken up to 48 hrs 

postinoculation, the washed conidia suspended 

in water showed lower infectivity than those 

suspended in SCS, and addition of catalase to 

the washed conidia restored the infectivity of 

incompatible and compatible isolates of M. oryzae 

(Fig 2). The absence or presence of catalase 

in the conidia suspension was well-correlated 

with the level of accumulated H 2 O 2 in inoculated 

leaf cells. These results strongly suggest that 

catalase activity in SCS promotes infection of 

M. oryzae by quenching H 2 O 2 . In the leaf blade, 

inoculation of compatible conidia in the presence 

of catalase produced more severe symptoms 

than that of conidia in the absence of catalase. 

These results suggest that the primary role of 

host-produced H 2 O 2 is toxicity that limits hyphal 

growth after penetration. 

We further found that SCS includes infectionpromoting 

activity after heat treatment of SCS 

(∆SCS) by which catalase is inactivated. The 

addition of ∆SCS stimulated the extent of invasion 

by infectious hyphae in the excised leaves 

of rice. The heat-stable activity was found in 

SCSs from all Japanese isolates of M. oryzae 

tested. Presence of SCS increased the ratio 


Fig 2. Effects of exogenous catalase on the infection of rice leaf sheaths by M. oryzae. 

Leaf sheath of Nipponbare was inoculated with washed conidia of M. oryzae P91-15B (incompatible) 

or Ina86-137 (compatible), and extent of infection was scored. (A) Ratio of “Infected”, “One cellinfected” 

and “Uninfected” appresoria at 48 hpi. (B) Schematic presentation of the extent of infection. 

Fig 3. Lesion formation in leaf blades of rice inoculated with M. oryzae in the presence of SCS. 

Excised leaves of Nipponbare were sprayed with the conidial suspension of a compatible isolate 

of M. oryzae after suspension in buffer (Buf) containing SCS or extract of medium (EM). At 6 

days postinoculation, lesions were counted according to the classification as shown in the upper 

picture. Vertical axis indicates the ratio of lesions. 

of susceptible lesion in the leaf blade (Fig 3). 

Interestingly, the effect was exclusively detected 

in compatible interactions, independent of 

the blast-resistance gene of rice and avirulence 

gene of M. oryzae. This is in contrast to catalase, 

which promoted the infection in the leaf sheath 

both in incompatible and compatible interactions. 

The non-host resistance of rice plants was 

not compromised by SCS, i.e., the presence of 

SCS did not stimulate the infection of Avena, 

Setaria, Panicum, Digitaria, and Eleusine isolate 

of M. oryzae in the leaf sheath of rice. These 

results suggest that SCS includes a heat-stable 

factor(s) that promotes M. oryzae infection 

during compatible interactions. Purification 

and further characterization of the infectionpromoting 

factor is in progress. 


Mstu1, an APSES transcription factor, is 

required for appressorium-mediated infection 

in Magnaporthe oryzae. 

The APSES protein family includes important 

transcriptional regulators of morphological 

processes in ascomycetes including Magnaoprthe 

oryzae. We isolated a deletion mutant of the 

Mstu1 gene that encodes an APSES protein 

Mstu1 in Magnaporthe oryzae. This mutant 

showed reduced conidiation and mycelial 

growth. Mstu1 formed a number of appressoria 

indistinguishable from the wild type, 

although appressorium formation was delayed. 

Rapid transfer of conidial glycogen and lipid 

droplets to incipient appressoria is required for 

appressorial turgor generation in M. oryzae, 

by which the fungus penetrates plant cuticles. 

Appressorial turgor was low in mstu1 and the 

mutant was deficient in appressorium-mediated 

invasion of rice leaves. The transport of conidial 

glycogen and lipid droplets to appressoria was 

remarkably delayed in mstu1, and a consequent 

delay in degradation of these conidial reserves 

was observed. These results indicate that Mstu1 

plays a key role in appressorium-mediated 

infection due to its involvement in the transfer 

of lipids and glycogen. 


Site-directed mutagenesis in higher plants 

Site-directed mutagenesis in higher plants 

remains a significant technical challenge for 

basic research and molecular breeding. Here, 

we demonstrate targeted-gene inactivation 

for an endogenous gene in Arabidopsis using 

zinc finger nucleases (ZFNs). Engineered ZFNs 

for a stress-response regulator, the ABA- 

INSENSITIVE4 (ABI4) gene, cleaved their 

recognition sequences specifically in vitro, and 

ZFN genes driven by a heat-shock promoter 

were introduced into the Arabidopsis genome. 

After heat-shock induction, gene mutations 

with deletion and substitution in the ABI4 gene 

generated via ZFN-mediated cleavage were 

observed in somatic cells at frequencies as high 

as 3%. The homozygote mutant line zfn_abi4- 

1–1 for ABI4 exhibited the expected mutant 

phenotypes, i.e., ABA and glucose insensitivity. 

In addition, ZFN-mediated mutagenesis was applied 

to the DNA repair-deficient mutant plant, 

atku80. We found that lack of AtKu80, which 

plays a role in end-protection of dsDNA breaks, 

increased error-prone rejoining frequency by 

2.6-fold, with increased end-degradation. These 

data demonstrate that an approach using ZFNs 

Fig 1. Free Trp contents in mature seeds of GT plants. 

Bar charts show Trp contents in the mature seeds of non-transformant (NT), and wildtype 

(WT) plants and plants homozygous (GT). Values are average±SD (n=3). 


Fig 2. Increased number of chromosomes is detected in polyploidy cell of OsCDKB2 

knockdown rice calli. 

OsCDKB2 knockdown mutants, which show increased populations of 4C, 8C and 

16C cells, contain nuclei with 24 chromosomes (2C, left panel), 48 chromosomes (4C, 

not shown) and 96 chromosomes (8C, right panel). 

can be used for the efficient production of 

mutant plants for precision reverse genetics. 

Designed mutation breeding via gene targeting 

—Tryptophan fortification in rice 

Tryptophan (Trp) fortification in rice is important 

for human food and animal feed. The activity 

of anthranilate synthase (AS), a key enzyme 

of Trp biosynthesis, is controlled by feedback 

regulation by Trp. Protein engineering of 

OASA2—an α subunit of AS in rice —showed 

that the S126F and L530D mutations conferred 

Trp insensitivity and enhanced catalytic activity, 

respectively. Molecular-designed breeding 

by site-directed mutagenesis via gene targeting 

(GT) is thought to be much more efficient than 

conventional breeding that depends on natural 

variation or random mutagenesis. We previously 

reported successful targeted mutagenesis of 

OASA2 via GT. 

OASA2 mRNA levels in plants homozygous 

or heterozygous for modified OASA2 were 

similar to that in wild-type plants. It has been 

demonstrated that gene modification via GT, 

unlike over-expression-based approaches, is much 

less at risk of gene silencing because of the use 

of an intrinsic promoter to express the modified 

OASA2, and due to the lack of any additional 

copies of OASA2. We confirmed that the free 

Trp contents in seedlings and mature seeds of 

both homozygous and heterozygous plants were 

higher than in the original cultivar. Surprisingly, 

the mature seeds of homozygous plants accumulated 

4256 ± 855 nmol/gDW (14.129 ± 0.907µg/ 

seed) Trp (Fig 1). This is estimated to be equal 

to half the daily Trp requirement of an adult human 

(4 mg/kg/day), assuming a person weighing 

60 kg eats a bowl of rice three times a day. On 

the other hand, the ratio of phenylalanine and 

tyrosine, which are synthesized in the branched 

pathway of Trp biosynthesis, to total amino acids 

was unchanged, suggesting that Trp can be 

accumulated specifically using this strategy. 

OsCDKB2 knockdown induces endomitosis in 

rice 

DNA damage occurring during S-phase 

induces two cellular responses needed for 

cell survival: activation of the DNA-repair 

machinery and delay, or even arrest, of cell 

cycle progression, providing cells sufficient time 

to repair the damaged DNA before proceeding 

into mitosis. DNA double strand break repair 

mechanism, homologous recombination (HR) 

is most proficient at S-G2 phase of mitotic cell 

cycle when sister chromatids are available 

as templates. We hypothesized that since HR 

repair mechanism is responsible for gene targeting, 

the enrichment of cells in S-G2 phase could 

increase gene targeting efficiency in plants. The 

analysis of Arabidopsis cell cycle mutants and 

wild-type plants treated with DNA damaging 

agents, however, showed that disturbance of 

G2/M progression enhanced transition from 

mitotic cycle to endocycle. Because endoreduplicated 

cells cannot proliferate, we thus 

concluded that disturbance of G2/M progression 

is not a good strategy for enhancement of gene 

targeting in Arabidopsis. On the other hand, in 

rice, endoreduplication is only reported in the 

endosperm and the DNA damage treatments do 

not induce endoreduplication. We hypothesized 


that inhibition of G2/M transition in rice would 

improve chances of gene targeting without 

causing endoreduplication. We generated RNAi 

knockdown of OsCDKB2, the gene that functions 

in G2/M progression and characterized 

cell cycle progression in the mutant. Flow 

cytometric analysis of calli showed that the 4C 

nuclei population had increased in several independent 

OsCDKB2 knockdown lines, suggesting 

that OsCDKB2 knockdown disturbs G2/M 

progression in rice mitotic cycle. Furthermore, 

we found nuclei with polyploid, such as 8C and 

16C, in several OsCDKB2 knockout lines (Fig 2). 

The endoreduplicating cells typically stop 

proliferation, but OsCDKB2 knockdown calli 

did not. Then, we expected that polyploidy 

detected in OsCDKB2 knockdown calli was due 

to endomitosis. To distinguish between endoreduplication 

and endomitosis, we an increased 

number of chromosomes in these polyploid cells 

and concluded that knockdown of OsCDKB2 

induces endomitosis in rice. 

Toward the development of effective transgenic 

methods of energy crops in Poaceae. 

Energy crops are plants, which grow with low 

cost but their large amount of harvested biomass 

can produce biofuels. In Poaceae, crops like 

sorghum and grasses like Miscanthus are categorized 

as energy crops. The biomass obtained 

from these plants, like sugar, starch and cellulose, 

is used to produce biofuels. Since last year, we 

started to develop effective transgenic methods 

for sorghum, Sorghum bicolor (L.) Moench, which 

can grow in arid soils and withstand drought. 

We chose one cultivar as material for transgenic 

experiments due to its high frequency of 

regeneration from calli and constructed suitable 

culture and regeneration systems. This year, we 

analyzed the frequency of regeneration among 

related varieties of this cultivar. We tried to 

develop DNA-bombardment and Agrobacteriummediated 

transformation procedures for use 

with sorghum. Furthermore, we applied the 

Agrobacterium-mediated transgenic method for 

sorghum to calli of one variety of wild sugarcane, 

Saccharum spontaneum L., which shows high 

frequency of regeneration. Although this species 

does not accumulate sugar in its stems, it could 

be very useful as an energy crop because it is 

not used for food. It may also be possible to apply 

genomic information of cultivated sugarcanes and 

other cereals in Poaceae by functional genomics 

for improvement of S. spontaneum. 

Generation of marker-free transformants by 

site-specific recombinase-gene under the 

control of male germline specific promoter 

The R recombinase gene of the site-specific 

recombination system R/RS was fused to the 

male germline specific promoter to remove a selectable 

marker after selection of transfomants. 

Total DNA was isolated from T2 plants, 

digested with restriction enzyme and analyzed 

by Southern hybridization. First, a GFP DNA 

inside of recognition sites of recombinase was 

used as a probe. The same membrane was 

used for a second hybridization in which a GUS 

DNA outside of the recognition site was used 

as a probe. Plants which have only a GUS DNA 

were obtained and marker gene removal was 

confirmed. 

Chromatin modification controls segregation 

distortion 

Segregation distortion is a phenomenon in 

which one of a pair of chromosomes or alleles 

in a heterozygote is preferentially transmitted 

to the next generation. Loci involved in 

segregation distortion have been found in 

various organisms and many of them have 

been mapped to chromosomal regions linked to 

heterochromatin associated with centromeres, 

suggesting possible roles of heterochromatin in 

segregation distortion. In this study, by using a 

rice inter-subspecific (japonica ×indica) cross, 

we found that global changes in chromatin 

modification altered the pattern of segregation 

distortion. Changes in the chromatin modification 

by either exogenous inhibition of histone 

deacetylaces or knockdown of the DDM1 gene 

that is required for the maintenance of global 

cytosine methylation resulted in similar but not 

identical distortion patterns. Knockdown of the 

DDM1 gene eliminated CENH3 in the centromeric 

regions and induced a steep increase of F 2 

heterozygotes at a chromosomal region linked to 

this centromere on chromosome 3 accompanied 


y induction of a meiotic recombination hot spot 

in a pericentromeric transposon-rich region. 

Septum formation in amyloplasts produces 

compound granules in the rice endosperm 

The size and shape of starch granules vary 

between species and the tissues from which 

they are purified. Storage tissues such as seed 

endosperm and tubers store starch in the form 

of granules in the amyloplast. In the rice (Oryza 

sativa) endosperm, each amyloplast produces 

compound granules consisting of several dozen 

polyhedral, sharp-edged, and easily separable 

granules; whereas in other cereals, including 

wheat (Triticum aestivum), barley (Hordeum 

vulgare), and maize (Zea mays), each amyloplast 

synthesizes one granule (Fig 3). Despite extensive 

studies on mutants of starch synthesis in cereals, 

the molecular mechanisms involved in compound 

granule synthesis in rice have remained elusive. 

We expressed green fluorescent protein fused to 

rice Brittle1 (BT1), an integral inner membrane 

protein that functions as an ADP-glucose 

transporter, to characterize dividing amyloplasts 

in rice endosperm. BT1-GFP was detected at the 

outer limit envelope of the amyloplast and the 

septa between granules (Fig 4). Serial Z-sections 

of the amyloplasts confirmed that BT1-GFP was 

localized in septa between granules and that the 

signal intensities in the septa were not uniform. 

These and other results suggested that the 

septum formation is primarily responsible for the 

synthesis of characteristic compound granules in 

the rice endosperm. 

Fig 3. Purified starch granules from wheat and rice. 

(Left) Wheat granules are simple-type, and are divided into the A-type (larger than 10 µm) or B-type 

(smaller than 10µm). (Right) Rice granules, separated during the purification, are compound-type, 

polyhedral, and sharp-edged. 

Fig 4. BT1-GFP is localized at the outer limit envelope and septa between granules in the amyloplast. 

The fluorescence signals of BT1-GFP (left panel) and stroma-targeted tpCherry (middle panel) are 

converted to green and magenta, respectively, and merged (right panel). Arrowheads point to septa 

between granules. Bars = 5 µm. 



The Division of Insect Sciences consists of 

eight research units and aims to develop up 

to-date agriculture techniques and to create 

new industries by analyzing insect-specific 

functions at the molecular level utilizing new 

biotechnology such as genomic information and 

transgenic silkworms. 

Insect Genome Research Unit is constructing 

the integrated genome database of silkworm 

genomic sequence information, EST information 

and map information, which is publicly available 

on the web. 


will identify genes involved in the action of 

insect hormones, insect behaviors, invertebrate 

reproduction, embryonic development and 

regeneration. 

Anhydrobiosis Research Unit is studying biochemical 

and molecular adaptations associated 

with anhydrobiosis in the sleeping chironomid, 

Polypedilum vanderplanki. 

Innate Immunity Research Unit will clarify 

mechanisms of insect antimicrobial proteins 

and synthetic peptides against bacteria and 

trypanosomes, and will utilize the results for 

agricultural and medical purposes. 

Insect Interaction Research Unit aims to 

develop new technology in pest control based 

on the research for insect-insect and insect-plant 

interaction mechanisms. 

Insect-Microbe Research Unit studies molecular 

mechanisms of the interaction between 

arthropods and their associated microorganisms, 

aiming to prevent arthropod-transmitted 

diseases of livestock and crops and to control 

agricultural pests with arthropod pathogens. 

Silk-Materials Research Unit studies physicochemical 

properties of silk proteins produced by 

silkworms, spiders and hornets and interactions 

with human cells to develop new materials for 

medical use. 

Silk Technology Unit will breed a new silkworm 

race having specific filament characters, 

such as high strength or lustrous appearance for 

applications in new medical materials or unique 

textiles, and will develop the silk materials for 

bedding and sanitary purposes. 

The laboratory of Special Project, which is 

operated with funding provided by several supporting 

companies and backed by the Institute, is 

conducting the insect symbiont genome project. 


Insect Genome Research Unit 

We developed an integrated silkworm genome 

database KAIKObase (Shimomura et al, 2009) to 

provide effective data mining and efficient utilization 

of the silkworm genome information for 

functional and applied genomics. The genomic 

sequences, map information and EST data are 

compiled into KAIKObase, which consists of 4 

map viewers, a gene viewer, sequence search, 

and keyword and position search. In addition, integration 

of the KAIKO2DDB for proteome data 

and Bombyx trap database for transgene and 

reporter data further enhance the functionality 

of KAIKObase (Figure 1). With this powerful 

database, we have succeeded in cloning the 

genes responsible for many Bombyx mutants. 

1) Data Sets 

Genomic sequence 

KAIKObase contains 43,462 assembled data 

configurations, including scaffolds and contigs 

not used in scaffolds, that correspond to a 

total genome size of 482 Mb (403 Mb without 

gaps). Among them, 192 scaffolds were mapped 

onto 28 chromosomes. In addition, a total of 

81,705 BAC-end sequences, 174,222 fosmid-end 

sequences, and 166,757 ESTs were included in 

KAIKObase. 

Map information 

The combined maps contain 16,209 gene 

models, 1,532 SNP markers, 770 trait markers, 

and 5,419 FPC contigs. The SNP markers 

b 

e 

g 

a 

c 

d 

f 

h 

i j k m l 

Fig 1. Flow chart for browsing KAIKObase. 

a) KAIKObase top page with links to PGmap and UnifiedMap; b) Keyword and position search 

function; c) Sequence search function using BLAST; d) Entry of fasta sequence and setting 

parameters; e) Result of keyword and position search; f) Result of sequence search; g) Bombyx trap 

database top page; h) Proteome database top page; i) PGmap showing an image of the genetic and 

physical maps; j) UnifiedMap showing the genetic map and various selectable physical map features; 

k) UTGB showing various selectable physical map functions; l) GBrowse showing various selectable 

physical map features; m) GeneViewer showing a sample gene profile. 


were identifed from BAC-end sequences. Trait 

markers represent the positions of transposon 

vectors (mutators) in transposon-insertion lines 

(enhancer-trap lines or gene-trap lines). The 

FPC contigs represent BAC contigs assembled 

by BAC fingerprinting. 

KAIKO2DDB (Proteome database) 

KAIKO2DDB provides information on the silkworm 

proteome. It contains 116 images of twodimensional 

polyacrylamide gel electrophoresis 

from different developmental stages and from 

various tissues. The spots provide information 

on products such as molecular weight and isoelectric 

point. Corresponding ESTs and selected 

gene models are shown on the gel images. 

Bombyx trap database 

The Bombyx trap database contains information 

of 288 transposon-insertion lines, e.g., 

enhancer-trap lines and gene-trap lines, the 

positions of insertions in the genomic sequence, 

expression profiles of genes for various developmental 

stages, organs and tissues including 

intensity of expression, and associated photos. 

2) Major Viewers and Tools 

PGmap 

The PGmap is an integration of the genetic 

map and physical map of the silkworm genome 

consisting of SNP markers, trait markers using 

the Bombyx trap database, bar charts of 

repeat sequences and gene sequences in the 

visible chromosomal range. A visual comparison 

between the genetic and physical lengths for 

entire chromosomes or a specified chromosomal 

region can be generated. The sequence of 

the selected region is linked to GBrowse as 

described below. 

GBrowse 

GBrowse provides a tracking function for restriction 

sites, FPC-contigs, 6-frame translation, 

DNA/GC content, contigs, ESTs, transcriptional 

profile, BAC, BAC-end, fosmid-end, gene models 

and genes, SNP-markers, and trait-markers. 

Pop-up balloons in the gene model track show 

links to: 1) sequence information displayed by 

GBrowse, 2) GeneViewer, and 3) the proteome 

database. Pop-up balloons associated with the 

trait-markers show links to sequence information 

displayed by the GBrowse function and 

the Bombyx trap database. A BLASTn search 

without a filter option was used to map ESTs 

onto the scaffold; the queries were ESTs using 

the scaffold as the database. Mapped ESTs with 

the top score for each BLAST result and an 

e-value less than 0.01 were chosen. 

GeneViewer 

The GeneViewer provides an overall profile of 

the gene models. Display items are: 1) image of 

nucleotide and spliced nucleotide and results of 

a domain search in the Pfam database; 2) links 

to KAIKO2DDB; 3) detailed information on the 

predicted gene including chromosome number, 

position of exons and GC content; 4) results 

of a homology search using BLASTn (top 3 

ESTs), BLASTp (top 10 proteins), HMMER and 

ProfileScan with the alignments of the sequence; 

5) results of amino acid analysis in PSORT, 

SOSUI, MOTIF and Gene ontology (InterProScan 

and mapping of InterPro entries to GO) with 

graphical representation of InterProScan; and 6) 

links to the nucleotide sequence, spliced nucleotide 

sequence and translated protein sequence 

of the predicted gene. 

Keyword and position search 

The mining function through a ke*yword and 

position search is indicated by the blue arrow 

in Figure 2. A direct search in KAIKObase for 

information such as scaffold, contig, FPC-contig, 

SNP-marker, trait-marker, gene model, EST/ 

cDNA, BAC, BAC-end, fosmid-end, and positions 

on scaffolds can be executed. A search for a 

specified region on a scaffold or chromosome is 

also available. Search results are shown as chromosomal 

images with the retrieved position, and 

listed with corresponding links to the browsers, 

one viewer and two independent databases. 

Sequence search (BLAST search) 

The entry to this class of data mining 

is shown by the yellow arrow in Figure 2. 

The query is a nucleic acid or an amino acid 

sequence; the tool allows finding the location of 

a scaffold or chromosome. The search result is 

listed with the data sorted in descending order 

of bit score with buttons linked to the four 

browsers. Manually entering the starting and 

ending nucleotide positions is possible at the top 

of the sequence search page. 

Although not shown here, KAIKObase also 


Fig 2. Links among browsers, viewer, and independent databases. 

The large red arrows represent mining from browsers and the pertinent browsers and database. The 

large blue arrows represent mining from a keyword search and the pertinent browsers and database. 

The large yellow arrows represent mining from a sequence search and the pertinent browsers and 

database. The dashed lines represent the flow of information from one database, browser, and 

viewer to another. 

has other map browsers, such as Unified map 

and UTBG. 

3) List of Identified Genes 

To date, genes involved in the following 

mutations have been identified using the 

silkworm genome information: lem (yellow body 

coloration), sepiapterin reductase gene (BmSpr) 

was identified by Meng et al. (2009); ow (waxy 

translucent), BmVarp, was identified by Ito et 

al. (2009); rb (red blood), kynureninase gene, 

BmKynu, was identified by Meng et al. (2009); 

C (Yellow cocoon), Cameo2 was identified by 

Sakudoh et al. (2010); Bts (brown head and tail 

spot), Bmyellow-e was identified by Ito et al. 

(2010); and Spli (soft and pliable), Bmacj6, was 

identified by Fujii et al. (2010). 

As mentioned above, KAIKObase is an integrated 

data resource with powerful and useful 

analysis tools, which substantially contributes 

not only to lepidopteran research in general, but 

also to facilitate sericulture improvement and 

development of new pest control strategies. 

Reference 

Shimomura M, Minami H, Suetsugu Y, Ohyanagi 

H, Satoh C, Antonio B, Nagamura Y, Kadono- 

Okuda K, Kajiwara H, Sezutsu H, Nagaraju J, 

Goldsmith MR, Xia Q, Yamamoto K, Mita K 

(2009) KAIKObase: An integrated silkworm 

genome database and data mining tool. BMC 

Genomics, 10: 486 

Meng Y, Katsuma S, Daimon T, Banno Y, 

Uchino K, Sezutsu H, Tamura T, Mita K, 

Shimada T (2009) The silkworm mutant 

lemon (lemon lethal) is a potential insect 

model for human sepiapterin reductase deficiency. 

Journal of Biological Chemistry, 284: 

11698-11705. 

Ito K, Katsuma S, Yamamoto K, Kadono-Okuda 

K, Mita K, Shimada T (2009) A 25 bp-long 

insertional mutation in the BmVarp gene 

causes the waxy translucent skin of the 

silkworm, Bombyx mori. Insect Biochemistry 

and Molecular Biology, 39: 287-293. 

Meng Y, Katsuma S, Mita K, Shimada T (2009) 

Abnormal red body coloration of the silkworm, 

Bombyx mori, is caused by a mutation 


in a novel kynureninase. Genes to Cells, 14: 

129-140. 

Sakudoh T, Iizuka T, Narukawa J, Sezutsu H, 

Kobayashi I, Kuwazaki S, Banno Y, Kitamura 

A, Sugiyama H, Takada N, Fujimoto H, 

Kadono-Okuda K, Mita K, Tamura T, 

Yamamoto K, Tsuchida K (2010) A CD36- 

related transmembrane protein is coordinated 

with an intracellular lipid-binding protein 

in selective carotenoid transport for cocoon 

coloration, Journal of Biological Chemistry, 

285: 7739-7751. 

Ito K, Katsuma S, Yamamoto K, Kadono-Okuda 

K, Mita K, Shimada T (2010) Yellow-E determines 

the color pattern of larval head and tail 

spots of the silkworm, Bombyx mori. Journal 

of Biological Chemistry, 285: 5624-5629. 

Fujii T, Kuwazaki S, Yamamoto K, Abe H, 

Ohnuma A, Katsuma S, Mita K, Shimada T 

(2010) Identification and molecular characterization 

of a sex chromosome rearrangement 

causing a soft and pliable (spli) larval body 

phenotype in the silkworm, Bombyx mori. 

Genome, 53: 45-54. 


The unit is conducting research to identify 

genes encoding the enzymes and neuropeptides 

involved in synthesis and degradation of the 

two major insect hormones (ecdysone and 

juvenile hormone) as well as those encoding 

amine receptors involved in controlling insect 

behaviors, and to develop screening methods 

for inhibitors of these enzymes and receptors of 

neuropeptides. It also conducts molecular analyses 

of invertebrate reproduction, embryonic 

development and regeneration. The selected 

major research topics of 2009 from the present 

research unit are as follows: 

Identification of a novel ecdysone biosynthesis 

gene nm-g/sro working in the “Black Box” 

Ecdysone is a principal steroid hormone that 

regulates molting and metamorphosis in insects. 

Ecdysone is synthesized from dietary cholesterol 

via a series of hydroxylation and oxidation 

steps in the prothoracic gland (PG) and ovaries. 

In the past decade, a number of genes encoding 

enzymes essential for ecdysone biosynthesis 

have been identified by molecular genetic 

studies using the fruit fly Drosophila melanogaster. 

The first step of ecdysone biosynthesis, 

conversion of cholesterol to 7-dehydrocholesterol 

(7dC), is mediated by the Rieske-domain protein 

Neverland (Nvd) (Fig 1). Meanwhile the terminal 

cholesterol 

7-dehydrocholesterol 

Nvd 

HO 

HO 

BLACK 

BOX 

ketodiol 

Nm-g/Sro 

CYP307A1/Spo 

ecdysone 

OH 

OH 

HO H H 

O 

OH 

CYP306A1/Phm 

CYP302A1/Dib 

CYP315A1/Shd 

HO 

HO H H 

O 

OH 

Fig 1. A schematic representation of the roles of Nm-g/Sro in ecdysteroid biosynthesis. 

Nm-g/Sro plays a crucial role in the conversion step(s) between 7-dehydrocholesterol and 

5β-ketodiol, the so-called Black Box. 


Fig 2. Phylogenetic tree of CCE proteins. 

MEGA 4.0 (Tamura et al, 2007) was used to construct the phylogenetic tree using the Minimum Evolution method. 

Asterisks in the cladogram indicate that bootstrap values were greater than 50%. The nomenclatures of the 

clades are according to Oakeshott et al. (2005) and Claudianos et al. (2006). B. mori JHE or CCE-1~5 proteins are 

colored purple. The GenBank (http://www.ncbi.nlm.nih.gov/Genbank/) accession number is indicated after the 

name of each CCE. Abbreviations: C. quinquefasciatus, Culex pipiens quinquefasciatus; C. tarsalis, Culex tarsalis; C. 

tritaeniorhynchus, Culex tritaeniorhynchus; L. cuprina, Lucilia cuprina; M. domestica, Musca domestica; H. irritans, 

Haematobia irritans; A. calandrae, Anisopteromalus calandrae; B. mori, Bombyx mori; B. tabaci, Bemisia tabaci; 

A. gossypii, Aphis gossypii; C. fumiferana, Choristoneura fumiferana; H. virescens, Heliothis virescens; M. sexta, 

Manduca sexta; D. melanogaster, Drosophila melanogaster; S. nonagrioides, Sesamia nonagrioides; M. brassicae, 

Mamestra brassicae; A. polyphemus ODE, Antheraea polyphemus odorant-degrading enzyme; S. littoralis, 

Spodoptera littoralis; P. hilaris, Psacothea hilaris; T. molitor, Tenebrio molitor; G. assimilis, Gryllus assimilis; A. 

aegypti, Aedes aegypti; A. polyphemus IE, Antheraea polyphemus integumental esterase; N. lugens, Nilaparvata 

lugens; M. persicae, Myzus persicae; A. polyphemus PDE, Antheraea polyphemus pheromone-degrading enzyme; 

P. japonica, Popillia japonica; A. mellifera, Apis mellifera; H. armigera; Helicoverpa armigera. 


steps, conversion of 5β-ketodiol to ecdysone, 

are catalyzed by three cytochrome P450 monooxygenases: 

Phantom (Phm; CYP306A1), 

Disembodied (Dib; CYP302A1) and Shadow 

(Sad; CYP315A1). These P450 genes have been 

identified from “Halloween” class mutant flies 

that are characterized by embryonic ecdysone 

deficiency. However, the middle biochemical 

reactions catalyzing the conversion from 7-dC 

to 5beta-ketodiol are poorly characterized and, 

therefore, are called ‘Black Box’ reactions. We 

have identified a novel ecdysteroidgenic gene, 

non-molting glossy (nm-g)/shroud (sro), which 

encodes a short-chain dehydrogenase/reductase 

working in the “Black Box” (Fig 1). This gene 

was first isolated by positional cloning of the 

nm-g mutant of the silkworm Bombyx mori, 

which exhibits a low ecdysteroid titer and consequently 

causes a larval arrest phenotype. In 

the fruit fly, Drosophila melanogaster, the closest 

gene to nm-g is encoded by the sro locus, one of 

the Halloween mutant members. The lethality 

of the sro mutant is rescued by the overexpression 

of either sro or nm-g genes, indicating that 

these two genes are orthologous. Both the nm-g 

and sro genes are predominantly expressed in 

tissues producing ecdysone, such as the prothoracic 

glands and the ovaries. Furthermore, the 

phenotypes caused by the loss of function of 

these genes are restored by the application of 

ecdysteroids and their precursor 5beta-ketodiol, 

but not by cholesterol or 7-dC. Altogether, we 

conclude that the Nm-g/Sro family protein is an 

essential enzyme for ecdysteroidogenesis active 

as part of the Black Box reactions. Because 

Nm-g/Sro enzyme is critical for insect development 

and is insect specific, we expect it to be 

an excellent target for developing novel insect 

growth regulators. 

Niwa R, Namiki T, Ito K, Shimada-Niwa Y, 

Kiuchi M, Kawaoka S, Kayukawa T, Banno 

Y, Fujimoto Y, Shigenobu S, Kobayashi S, 

Shimada T, Katsuma S, Shinoda T. (2010) Nonmolting 

glossy/shroud encodes a short-chain 

dehydrogenase/reductase that functions in 

the ‘Black Box’ of the ecdysteroid biosynthesis 

pathway. Development, 137: 1991-1999. 

Molecular characterization and functional 

analysis of novel carboxyl/cholinesterases 

with GQSAG motif in the silkworm Bombyx 

mori 

Juvenile hormone (JH)-selective esterases 

(JHEs) are one of the important JH-degradative 

enzymes in coordination with JH-synthetic 

enzymes that regulate JH titer. Multiple JHEs 

have not been reported for any insects to date. 

Carboxyl/cholinesterase (CCE) sequences are 

significantly more abundant in the silkworm 

Bombyx mori compared with other species according 

to genomic databases. Hence, functionally 

active JHEs were searched for in B. mori 

genomic database, KAIKOBLAST, in addition to 

the JHE gene (Bmjhe) we reported previously. 

A key for searching putative JHEs is the motif 

GQSAG that is highly conserved in almost all 

of the JHE genes sequenced. We identified 

five novel CCE genes (Bmcce-1~5) with the 

motif. Their cDNA sequences and intron-exon 

structures were determined. The developmental 

expression patterns of these CCE genes were 

compared by real-time quantitative PCR analysis 

and found that their expression patterns 

varied among developmental stages and organs. 

Those patterns were totally different from that 

of authentic Bmjhe. Of the proteins produced by 

the five genes, only BmCCE-5 had an hydrolytic 

activity with JH; however, it was concluded that 

this protein might not function as a JH-specific 

esterase in vivo because it had a high Km value 

for JH. On the other hand, BmCCE-5 hydrolyzed 

general esterase substrates efficiently. In 

addition, Bmcce-5 was strongly expressed in 

Malpighian tubules and the gut; therefore, its 

main contribution might be mainly for nutritional 

digestion or xenobiotic metabolism. Those 

results suggest that of the CCEs containing a 

GQSAG motif only BmJHE can function as a JHspecific 

degradation enzyme in the silkworm. 

Tsubota T, Shimomura M, Ogura T, Seino A, 

Nakakura T, Mita K, Shinoda T, Shiotsuki T 

(2010) Molecular characterization and functional 

analysis of novel carboxyl/cholinesterases 

with GQSAG motif in the silkworm Bombyx 

mori. Insect Biochem. Mol. Biol., 40: 100-112. 


Anhydrobiosis Research Unit 

Cells from an anhydrobiotic chironomid survive 

almost complete desiccation 

Dry-preservation of nucleated cells from 

multicellular animals represents a significant 

challenge in life science. As anhydrobionts can 

tolerate a desiccated state, their cells and organs 

are expected to show high desiccation tolerance 

in vitro. However, anhydrobiotic animals are 

generally of microscopic size, and thus not suitable 

for cytological analyses. On the other hand, 

larvae of the sleeping chironomid Polypedilum 

vanderplanki, which is the largest anhydrobiotic 

animal, could be used for isolation of cells or 

tissues. Indeed, we could successfully establish 

cell lines derived from embryonic tissues of 

the midge, designated as Pv11 and Pv210. 

Heterogeneous Pv11 exhibits varied forms, 

although a long spindle-shape is dominant (Fig 

1A, B). Cells tend to be unattached, and easily 

form a floating cell mass when cell densities are 

relatively high. Pv210 cells are unattached and 

typically spherical (Fig 1C, D). Both cell lines are 

the first lines established from an anhydrobiotic 

animal. Their growth is shown in Fig. 1E and F. 

Because P. vanderplanki larvae produce 

anhydrobiosis-related proteins induced by 

salinity stress, we examined whether Pv11 and 

Pv210 cells showed a response to salinity. In a 

preliminary study, however, we found that some 

anhydrobiosis-related genes were constitutively 

expressed in both cell lines cultured in the 

serum-containing medium used for routine 

culture, suggesting that cells were experiencing 

stress in the medium. Therefore, to minimize 

growth-related stress in subsequent experiments, 

the cells were incubated in a serum-free 

IPL-41 medium for a week prior to use. Then, 

medium was exchanged to NaCl-supplemented 

medium, and total RNAs were extracted after 

6 h. Quantitative PCR analysis showed that all 

anhydrobiosis-related genes tested were markedly 

induced by elevated NaCl levels in Pv11 

cells except for glucose-phosphatase which was 

unchanged (Fig 2), suggesting that individual 

cells can perceive stress and regulate gene 

expression compatible with entry into anhydrobiosis. 

For Pv210 cells, on the other hand, the 

response to high salinity was less clear: of the 

anhydrobiosis-related genes, only Tps, Tpp, and 

Treh were found to be salinity-inducible (Fig 2). 

As salinity stress induced the expression of a 

set of anhydrobiosis-related genes in both Pv11 

and Pv210 cells, each cell may autonomously 

control the physiological changes for the entry 

into anhydrobiosis. When desiccated with 

medium supplemented with 300 mM trehalose 

or sucrose and stored for 4 weeks in dry air 

(approximately 5% relative humidity), a small 

percentage of the cells was found to be viable 

upon rehydration, although surviving cells 

seemed not to be able to multiply. 

As the above result implicates anhydrobiotic 

ability in Pv11 and Pv210, we examined survival 

of these cells after almost complete desiccation. 

For both cell lines, 2x106 cells in 100 µl 

of culture medium used for routine passage 

(normal medium) were desiccated at 5% relative 

humidity (RH), and stored for one month under 

this condition. After rehydration, calcein-AM 

staining showed no survival, however (data not 

shown). 

Sugars are known to act as cryo- and 

desiccation-protectants for bacteria, yeast, and 

cells of multicellular organisms. Many anhydrobionts 

utilize disaccharides such as sucrose and 

trehalose as compatible solutes. IPL-41 medium 

contains 21.7 millimolar concentrations of several 

sugars (13.9 mM of glucose, 4.8 mM of sucrose, 

3 mM of maltose, but no trehalose), which might 

be not sufficient to exert protective effects. 

Therefore, we added 300 mM sugar (trehalose 

or sucrose) to IPL-41 medium. After four weeks 

incubation in a desiccator, water content of 

the samples was 11.3~13.9%. Upon rehydration, 

a small percentage of the cells desiccated in 

the presence of trehalose was found to be still 

alive (Fig 3A, C), as indicated by live-dead cell 

staining. When sucrose was used instead of 


trehalose, the survival rate of Pv11 decreased 

and surviving Pv210 cells were rare (Fig 3B, D). 

Prior to drying, Pv cell lines were conditioned 

in sugar-fortified medium for 24 h, in an attempt 

to improve desiccation tolerance. However, the 

results were similar to those observed without 

pre-conditioning (Fig 3E-G), except for Pv210 

with sucrose, in which a significant number of 

cells showed improved survival (Fig 3H). On the 

whole, trehalose seemed to be superior to sucrose 

in protection against desiccation, and Pv11 

seemed to be more tolerant than Pv210 (Fig 3K). 

Fig 1. Morphology and growth curves of Pv11 and Pv210. 

A-D: Photographs of Pv11 (A, B) and Pv210 (C, D) observed under a differential interference 

microscope. Bars are 50 µm. E,F: Growth curves of Pv11 and Pv210. Each cell line was seeded at 

1x10 5 (E) or 2x10 5 (F) per 500 µl in a well of 24-well multi-plate and their density was examined by 

counting viable cells using a hemocytometer every second day. 


Fig 2. Quantitative PCR of anhydrobiosis-related genes in Pv11 (left) and Pv210 (raight) cells 

stressed with NaCl. 

Each cell line was incubated in serum-free IPL-41 medium for a week, and then treated in a 

medium supplemented with NaCl (0.5% final concentration) for 6 h. Results were expressed as 

relative values based on a control, which was incubated in normal IPL-41 medium. Gp: glucosephosphatase, 

Tps: trehalose-phosphate synthase, Tpp: trehalose-phosphate phosphatase, Treh: 

trehalase, Tret1: trehalose transporter 1, Lea1: late-embryogenesis abundant protein 1, Lea2: lateembryogenesis 

abundant protein 2, Lea3: late-embryogenesis abundant protein 3. S: cells stressed 

by hypersalinity. C: cells incubated in serum-free medium. 

In all cases, surviving cells were spherical and 

slightly enlarged. Whether only a specific cell 

type in a heterogeneous population survived or 

whether surviving cells changed their morphology 

is unclear. The rehydrated cells were incubated 

in culture medium to see whether they 

could grow. Although a small proportion of the 

cells were still alive even 4 weeks after rehydration 

(Fig 3IJ), cells were apparently not able to 

multiply. A possible cause could be the very 

low density of living cells because their growth 

is strongly density-dependent. To improve this 

issue, conditioned medium that is expected to 

contain growth promotion factors was used for 


Fig 3. Desiccation tolerance of Pv11 and Pv210. 

A-D: Pv11 (A, B) and Pv210 (C, D) were directly desiccated with 300 mM trehalose (A, C) or sucrose 

(B, D). E-H: Pv11 (E, F) and Pv210 (G, H) were desiccated, following preconditioning with 300 mM 

trehalose (E, G) or sucrose (F, H). Viability was examined by double staining with calcein-AM (green: 

live cells) and PI (red: dead cells) immediately after rehydration. I-J: rehydrated cells were cultured for 

4 weeks and then survival was examined. Pv11 (I) and Pv210 (J) desiccated with trehalose, without 

preconditioning. K: survival rate of the desiccated cells. Bars indicate mean ± SD (n=3). NS means 

no significant difference between the groups (P>0.05, two-way ANOVA, Bonferroni post-tests). 

post-rehydration culture, but cells still failed to 

proliferate (data not shown). 

We also examined the anhydrobiotic ability of 

other cell lines, Sf9 (fall armyworm), S2 (fruit fly), 

CHO-K1 (Chinese hamster), NIH3T3-3-4 (mouse), 

and HuH-7 (human) by the same protocol, and 

found no survival at all. Therefore, at least with 

this drying method, survival after complete 

desiccation seemed to be specific to cells from 

anhydrobionts. 

To achieve our goal of a practical drypreservation 

method for nucleated cells, the 

survival rate after rehydration must first be 

increased. If stress is reduced to such a level 

that most cells would survive, damages may 

also be reduced to a repairable level. We have 

reported that P. vanderplanki larvae in which 

anhydrobiosis is successfully induced, cells 

could be stabilized by vitrification of intraand 

extracellular matrix (Sakurai et al., 2008). 

Therefore, whether such bioglasses can be 

formed inside the cell is one of the keys to 

resolving this problem. Furthermore, molecules 

involved in damage reduction must work well 

during the dehydrating phase. Although Pv11 

and 210 expressed anhydrobiosis-related genes 

constitutively in the culture medium, it is possible 

that their expression is not enhanced to 

levels comparable to those in desiccating larvae. 

We believe that if the innate ability for anhydrobiosis 

can be brought out fully, these cells 

will be successfully preserved by drying. To 

obtain this, further studies are needed to define 

adequate conditions, e.g., salt concentration for 

regulating gene expression, sugar concentration 

and medium amount for vitrification of the 

extracellular matrix and control of desiccation 

rate, and supplementation with other protective 

materials. 

This work was supported in part by 

Promotion of Basic Research Activities for 

Innovative Bioscience (PROBRAIN), and by a 

Grant-in Aid (Bio Design Program) from the 

Ministry of Agriculture, Forestry and Fisheries 

of Japan. 


Innate Immunity Research Unit 

Genome-wide analysis of host gene expression 

in the silkworm cells infected with 

Bombyx mori nucleopolyhedrovirus 

Nucleopolyhedrovirus (NPV) is a member of 

the family baculoviridae, enveloped DNA viruses, 

possessing a large circular double-stranded 

DNA (80-180 kb) genome. Although some lepidopteran 

host genes such as heat shock protein 

70 cognate have been shown to be up-regulated 

in the early stage of NPV infection, the genomewide 

host gene expression profile in response 

to NPV infection has not yet been analyzed. In 

this study, we analyzed the global expression 

profile of host genes in Bombyx mori (Bm) NPV 

infected silkworm cells by oligonucleotidebased 

DNA microarray and quantitive reverse 

transcriptase-polymerase chain reaction analysis. 

Our analysis showed that 35 genes were significantly 

up-regulated and 17 were significantly 

down-regulated. This is the first report of 

changes in the expression of these genes in response 

to NPV infection. We further quantified 

the levels of mRNA expression by quantitative 

reverse transcriptase-polymerase chain reaction 

and confirmed that the expression of 13 genes 

(such as BmEts and BmToll10-3) significantly 

increased (Fig 1) and 7 genes (such as Hsp20-1) 

significantly decreased after BmNPV infection. 

However, the expression levels of most genes 

were not dramatically changed except BmEts 

expression increased approximately 8 fold 12 h 

after BmNPV infection. 

Sagisaka A, Fujita K, Nakamura Y, Ishibashi J, 

Noda H, Imanishi S, Mita K, Yamakawa M, 

Tanaka H (2010) Genome-wide analysis of host 

gene expression in the silkworm cells infected 

with Bombyx mori nucleopolyhedrovirus. 

Virus Res., 147: 166-175. 

Fig 1. qRT-PCR analysis of genes up-regulated in response to BmNPV infection in NIAS-Bmoyanagi2. 

NIAS-Bm-oyanagi2 cells were infected with BmNPV. Total RNA was extracted 2, 6, 12, 24 h after 

BmNPV infection, and then qRT-PCR carried out with the specific primers for the 12 genes. The 

mRNA abundance unit was calculated as the number of mRNA molecules per 1,000 ribosomal 

protein RP49 mRNA molecules. Results of quadruplicate experiments are shown with the standard 

deviations. The asterisk denotes significant differences from non-infected (0 h) samples (p

Multiple functions of short synthetic enantiomeric 

peptides based on beetle defensins 

Four enantiomeric 9-mer peptides, D-peptide 

A (RLYLRIGRR-NH2), B (RLRLRIGRR-NH2), C 

(ALYLAIRRR-NH2) and D (RLLLRIGRR-NH2), 

were designed and synthesized on the basis of 

a beetle defensin antimicrobial peptide. These 

D-9-mer peptides have been reported to exhibit 

multiple functions including antimicrobial and 

antiprotozoan activity without cytotoxicity 

on normal fibroblasts and leukocyte cells. We 

revealed that the D-9-mer peptides inhibit telomerase 

activity (IC100 = 40 µM, Table 1). A new 

peptide, D-peptide C2 (ALYLAIRRRRRRRR- 

NH2), designed from D-peptide C to translocate 

into the cytoplasm by a penetrating sequence 

(octa-arginine,) showed extremely strong telomerase 

inhibitory activity (IC100 = 0.1 µM, Table 

Table 1. Telomerase inhibitory activity of enantiomeric peptides 

Sequence 

IC100 (µM) 

D-control peptide AKGFAANHS-NH2 >100 

D-peptide A RLYLRIGRR-NH2 40 

D-peptide B RLRLRIGRR-NH2 40 

D-peptide C ALYLAIRRR-NH2 40 

D-peptide D RLLLRIGRR-NH2 40 

D-peptide C2 ALYLAIRRRRRRRR-NH2 0.1 

Table 2. Comparison of inhibitory potency between D-peptide C and C2 

IC50 (µM)* 

Cos-1 Jurkat VA-13 

RERF- 

LC-AI 

U-251 MRC-5 

D-peptide C >640 574 >640 >640 >640 >640 

D-peptide C2 3.4 11.0 16.0 22.3 26.4 11.1 

* IC50 = 50% Inhibitory Concentration 

Fig 2. Effect of D-peptide C2 on mitochondrial potential in Jurkat cells 

Jurkat cells were incubated with no peptides (a, d) 10 µM of D-peptide C (b, e) or C2 (c, f) at 37°C 

for 24 h. After incubation, cells were stained by JC-1 dye for 30 min and effect of each peptide 

on mitochondria was determined by detecting change of JC-1 fluorescence. Red fluorescence 

indicates healthy mitochondria and green fluorescence indicates the loss of mitochondrial membrane 

potential. Scale bar indicates 100 µm. 


1). D-peptide C2 exhibited a great increase in 

cytotoxicity against various cancer cell lines 

(IC50 = 3.4~26.4 µM, Table 2). However, immediate 

death of cells suggested that the high 

cytotoxicity was not the effect of telomerase 

inhibitory activity. Mitochondrial swelling assay 

and microscopical observations of mitochondria 

revealed the major target of the D-peptide C2 is 

the mitochondrial membrane (Fig 2). 

Insect Interaction Research Unit 

Molecular characterization of laccase in the 

salivary glands of the green rice leafhopper, 

Nephotettix cincticeps 

The green rice leafhopper, Nephotettix 

cincticeps is a major insect pest of the rice 

plant in Japan. This insect has laccase (EC 

1.10.3.2) in its salivary glands and saliva, possibly 

playing an important role in detoxifying plant 

phenolics and in salivary sheath coagulation 

during feeding. We aimed to clarify the function 

of saliva-specific laccase in a vascularfeeding 

insect, N. cincticeps, for which we 

cloned 2 cDNAs (NcLac1S and NcLac1G) from 

the salivary glands and 1 cDNA (NcLac2) from 

the epidermis. The NcLac1S, NcLac1G, and 

NcLac2 transcripts encoded 701-, 792-, and 729- 

amino acid proteins, respectively. The putative 

proteins encoded by NcLac1S and NcLac2 were 

predicted to be soluble, whereas that encoded 

by NcLac1G was hydrophobic and predicted to 

have a C-terminal transmembrane domain. Realtime 

reverse transcriptase polymerase chain 

reaction analysis revealed that NcLac1S was 

expressed exclusively, and at a much higher 

level than NcLac1G and NcLac2 in the salivary 

glands. NcLac1G was also expressed in the epidermis, 

midgut, and Malpighian tubules. NcLac2 

expression was highest in the epidermis. In 

situ hybridization revealed NcLac1S expression 

in the V -cells of the salivary glands (Fig 1), 

in which laccase activity has been previously 

detected. Expression of NcLac1G and NcLac2 

were not detected clearly in all cells in the salivary 

glands. Therefore, NcLac1S is responsible 

Fig 1. Expression of NcLac1S in the posterior lobe of the salivary glands of the N. cincticeps adult female 

Left: anti-sense probe; Right: Sense probe. Arrow indicates the location of signals detected in the V-cells of 

the salivary glands. The bar represents 100 μm. 


for the laccase activity detected in the salivary 

glands and saliva of this insect. This is the first 

finding on gene cloning of salivary laccase from 

a vascular-feeding insect. 

A unique latex protein, MLX56, with chitinbinding 

and extensin domains defends 

mulberry trees from insects 

The mulberry (Morus spp.)-silkworm (Bombyx 

mori) relationship has been a well-known plantherbivore 

interaction for thousands of years. 

Previously, we found that mulberry leaves 

defend against insect herbivory by latex 

ingredients including sugar-mimic alkaloids and 

unknown defense protein (Konno et al, 2006). 

We recently succeeded to purify and identify a 

novel 56-kDa (394 amino acid) defense protein in 

mulberry latex designated mulatexin (MLX56) 

with an extensin domain, two hevein-like chitinbinding 

domains, and an inactive chitinaselike 

domain that provides mulberry trees with 

strong insect resistance (Wasano et al, 2009) 

(Fig 2 and 3). MLX56 is toxic to lepidopteran 

caterpillars, including the cabbage armyworm, 

Mamestra brassicae and the Eri silkworm, Samia 

ricini, at 0.01% concentration in a wet diet (Figs 

4A, B, D), suggesting that MLX56 is applicable 

for plant protection. MLX56 is highly resistant 

to protease digestion, and has a strong chitinbinding 

activity. Interestingly, MLX56 showed 

no toxicity to the silkworm, Bombyx mori 

(Fig 4C), suggesting that the mulberry-feeding 

species has developed adaptation to MLX56. 

Our results show that defensive proteins in 

plant latex play key roles in mulberry-insect 

interactions, and probably, also in other plantinsect 

interactions. Our results further suggest 

that plant latex, analogous to animal venom, is 

a treasury of applicable defense proteins and 

chemicals that has evolved through interspecific 

interactions (Agrawal and Konno, 2009). 

Establishment of continuous cell lines for 

investigations of polydnavirus 

Larval endoparasitoids can avoid the immune 

response of the host by function of polydnavirus 

(PDV) and venom. PDV infects hemocytes and 

affects the hemocyte function of the host. We 

established cell lines from the larvae of Cotesia 

kariyai, endoparasitoid, and larval hemocytes of 

Mythimna separata which is host of C. kariyai. 

The hemocytes of M. separata or whole tissue 

of C. kariyai larvae were pooled to set up cultures 

of each in 25-ml culture flasks. The culture 

medium used was MGM-464 supplemented with 

20% fetal bovine serum (FBS) and included 

Fig 2. Purification of the MLX56 protein from mulberry latex 

(A) Mulberry latex exuded from leaf laticifer (arrows). (B) SDS-PAGE profiles of the purified MLX56 

protein. Lane M, molecular marker; lane 1, total latex proteins; lane 2, purified MLX56 protein. 


1 µg/ml of methoprene. When cell number increased 

to cover 80% of the flask bottom, these 

cultures were subcultured by suspending the 

cells by pipetting. After the subculture, the cells 

were cultured in methoprene-free medium. The 

cell lines could be adapted to MGM-450 medium 

with 10% FBS after 10 passages. The cells were 

maintained over 100 passages by subculturing 

once a week. Continuous cell lines obtained from 

M. separata and C. kariyai were named, respectively, 

NIAS-Ms-23B and NIAS-Ck-1B (Figs 5 A 

and B). 

NIAS-Ms-23B was cultured in medium containing 

PDV, which was collected from ovaries 

of C. kariyai for confirmation of infectivity of 

PDV. The cells began to aggregate at 24 hours 

after culturing and were damaged at 48 hours 

(Fig 5C). 

NIAS-Ck-1B seems to maintain the PVD in 

the genome, because C. kariyai PVD are symbiotic 

viruses associated with C. kariyai. NIAS- 

Ms-23B and NIAS-Ck-1B constitute a valuable 

tool for investigations of PDV replication and 

infection. 

Fig 3. The gene encoding the MLX56 from the latex of mulberry trees 

(A) Nucleotide and deduced amino acid sequence of the mlx56 gene isolated from the cDNAs originated 

from mulberry latex. The putative hevein-like chitin-binding domains are underlined, and the putative 

extensin motif is double underlined. The potential polyadenylation signal is underlined by a broken line. The 

arrow indicates the cleavage site for the signal peptide. The GenBank accession number of the determined 

nucleotide sequence is EF535852. Another almost identical sequence identified from the latex had a single 

amino acid substitution (Leu234Pro) at the site indicated by the + mark. (B) Diagram showing the protein 

structure and deduced functional domains of the MLX56 protein. The start site of the mature protein is 

indicated by a black triangle. 


Fig 4. Insect toxicity tests of the MLX56 protein in the latex of mulberry trees against neonate larvae 

of three lepidopterous species 

(A)-(C) S. ricini and B. mori larval weights (mg) were measured on day 2 and day 5, and the M. 

brassicae larval weights were measured on day 6 and day 10. Bioassays were performed in triplicate 

or sexplicate, each time with five or ten larvae. Error bars indicate ± SD (n = 30). Values not followed 

by the same letters at the same point are significantly different (P < 0.01; Tukey’s test for multiple 

comparisons). Values in parenthesis indicate larval mortality (%). (A) S. ricini. (B) M. brassicae. (C) 

B. mori. (D) Neonate M. brassicae larvae fed an artificial diet containing 0.03% of purified MLX56 

protein for 6 days (right) or those fed a control artificial diet without MLX56 for 6 days (left). 

A two-step mechanism controls the timing of 

emerging behaviour for mating in adult males 

of the scarab beetle, Dasylepida ishigakiensis, 

a serious sugarcane pest in the Miyako Island 

of Japan 

Adults of the white grab beetle, Dasylepida 

ishigakiensis Niijima et Kinoshita (Kebukaakacha-kogane 

in Japanese), emerge from the 

soil around dusk for mating on subtropical 

islands. The present study examines the factors 

controlling the emergence of males. There 

are two steps involved. Several hours before 

emerging from the soil in the laboratory, adults 

are known to come to the soil surface where 

they expose their head to monitor environmental 

cues for mating activity. This “standby 

behaviour” is shown by adults in the field. The 

standby behaviour is facilitated by warm conditions, 

but the proportion of standby individuals 

is influenced not only by the temperature on 

that day but also by that on the previous day. 

Experiments in which beetles are exposed to a 

combination of photoperiod and thermoperiod, in 

and out of phase, have shown that temperature 

is more important in inducing standby and 

emerging behaviours than light alone. For the 

second step, factors such as temperature, light 

and the presence of the female sex pheromone 

determine whether males will leave the standby 

position and emerge onto the ground. The 

female sex pheromone ((R)-2-butanol) stimulates 

standby beetles to exhibit emerging and flight 

behaviours, but the effect depends on when it 

is presented to beetles (Fig 6). After the mating 

activity ends, beetles return to the soil; this 

behaviour is influenced by illumination and time 

of the day but not by temperature. The results 

suggest that D. ishigakiensis possesses a sophisticated 

mechanism controlling male emergence 

leading to mating behaviour. 

Virgin and mated males as well as virgin 

females, which are expected to emerge from the 

soil for mating on another evening, burrow into 

a relatively shallow layer of moist soil (< 3 cm), 


Fig 5. Cell morphology of NIAS-Ck-1B and NIAS-Ms-23B 

(A) NIAS-Ms-23B, (B) NIAS-Ck-1B and (C) NIAS-Ms-23B cultured in medium containing PDV at 48 

hours after culturing. 

% 

 

** 

* * 

** ** 

 

 

*** *** 

*** 

*** 

 

 

* 

With female sex pheromone 

Without female sex pheromone 

 

Time (min)1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 

Wm -2 

6.34 4.232.111.060.560.28 0.140.070.040.02 0.010.0050.0020.001 0 

Fig 6. Percentages of standby males that exhibited flight behaviours in the presence or absence of 

female sex pheromone under gradually darkening conditions 

Values are the means ± SE. Asterisks indicate significant differences between the treatment and 

control in each time (ANOVA, P < 0.05). n = 46 and 42 for with and without female sex pheromone, 

respectively. 

whereas mated females go down to a deeper 

layer (20–30 cm deep) and never come up to the 

surface again. 

A letter of appreciation with a memento was 

given to the Kebuka project of NIAS by the 

Miyako Branch of Technical Association for 

Sugarcane Production and the Miyako Branch of 

Insect Pest Management Office, because of our 

contribution and dedication to the development of 

methods for the control of the white grub beetle. 

References 

Agrawal AA, Konno K (2009) Latex: A model 

for understanding mechanisms, ecology, and 

evolution of plant defense against herbivory. 

Annual Review of Ecology, Evolution and 

Systematics, 40: 311-331. 

Hattori M, Tsuchihara K, Noda H, Konishi 

H, Tamura Y, Shinoda T, Nakamura M, 

Hasegawa T (2010) Molecular characterization 

and expression of laccase genes in the 


salivary glands of the green rice leafhopper, 

Nephotettix cincticeps (Hemiptera: Cicadellidae). 

Insect Biochemistry and Molecular Biology, 40: 

331-338. 

Konno K, Ono H, Nakamura M, Tateishi K, 

Hirayama H, Tamura Y, Hattori M, Koyama 

A, Kohno K (2006) Mulberry latex rich in 

anti-diabetic sugar-mimic alkaloids forces 

dieting on caterpillars. The Proceedings of 

the National Academy of Science USA, 103: 

1337-1341. 

Wasano N, Konno K, Nakamura M, Hirayama C, 

Hattori M, Tateishi K (2009) A unique latex 

protein, MLX56, defends mulberry trees from 

insects. Phytochemistry, 70: 880-888. 

Insect-Microbe Research Unit 

Bt toxin resistant gene was elucidated in the 

silkworm 

Toxins originated from insect pathogenic 

bacteria Bacillus thuringiensis (Bt toxins) are 

used for controlling lepidopteran and dipteran 

insects as agricultural chemicals or geneticallyengineered 

plant products. Wide use of the 

toxins has caused appearance of resistant pest 

strains against the Bt toxin. We have successfully 

cloned a resistant gene against a Bt toxin, 

Cry1Ab, from the silkworm, Bombyx mori, 

based on genome map and SNP markers of the 

silkworm. In order to confirm that the cloned 

gene is responsible for the resistance against 

the toxin, the gene was introduced into the 

silkworm by trasgenesis and gene expression 

was examined. 

The resistance gene showed recessive expression; 

therefore, the gene showing susceptibility 

was introduced into the resistant strain of the 

silkworm. The transgenic silkworm clearly 

showed susceptibility to the Bt toxin. The introduced 

gene was expressed in the silkworm as 

well as the homologous endogenous gene. It was 

confirmed that this gene is certainly responsible 

for the Bt toxin resistance in the silkworm. 

Biomass conversion systems of termites and 

their application 

We reported the first cellulase (endoglucanase) 

gene of insect origin from the termite 

Reticulitermes speratus (RsEG) in 1998. This has 

been followed by reports on homologues from 

various termites (termite EGs), cockroaches 

and some invertebrates. We constructed an EG 

mass-production system (up to 5 L scale of E. 

coli culture) using a mutant EG gene of a chimera 

of four termite EGs. In the meantime, we 

elucidated the detailed mechanism of an efficient 

biomass conversion that occurs in the gut of 

the termite (Coptotermes formosanus), which is 

one of the top-listed pest species. C. formosanus 

ingests wood and pulverizes it to less than 10 

micrometers in size by their mandibles and proventriculus. 

The small debris is then digested 

by the termite EG of extremely high concentration 

(over 1,000 units/ml) and beta-glucosidase in 

the midgut to produce glucose in a short period 

(assumed less than 1 hour). Then the undigested 

remainings are sent to hindgut to be further 

digested in symbiotic protozoan cells). Based 

on the current findings, we proposed a novel 

biomass conversion system using the mutant 

EG gene and created experimental models. 

Wolbachia, bacterial endosymbionts of invertebrates, 

interfere with sex-specific splicing 

of a sex-determining gene, doublesex 

In some strains of a butterfly Eurema 

mandarina, naturally occurring Wolbachia 

endosymbionts transform genetic male individuals 

into functional females. This feminization is 

one of the effects conferred by Wolbachia on its 

host arthropods. We focused on doublesex (dsx), 

i.e., one of the sex determining genes locating 

at the downstream of the sex determining 


gene cascade. The dsx gene is known to be 

conserved among insects and is subject to sexspecific 

alternative splicing. We identified a 

partial sequence of dsx in E. mandarina and 

found that the dsx was subject to sex-specific 

splicing in uninfected individuals but was subject 

to female-specific splicing in genetic male 

individuals that were feminized by Wolbachia. 

Many of the intersexual individuals generated 

by antibiotic treatments were subjected to both 

male-specific and female-specific splicing. The 

relative amounts of male-specific and femalespecific 

splicing products amplified from adult 

cDNA was dependent on the length of the 

antibiotic treatment during larval development. 

These results demonstrate that the feminizing 

Wolbachia endosymbionts continuously act on 

its hosts during larval development to maintain 

female-specific splicing of dsx under a male 

genotype. 

Next, we transfected Wolbachia into a cell line 

(M1) derived from male silkworm, Bombyx mori, 

wherein dsx gene is spliced in a male-specific 

manner. The splicing pattern of dsx in the 

Wolbachia-infected M1 cells changed from male 

type to female type. Following the antibiotic 

treatment to kill the bacteria, the splicing pattern 

of dsx reverted to male type. On the other 

hand, Wolbachia that can induce cytoplasmic incompatibility 

but not feminization did not affect 

the splicing pattern of dsx despite its high infection 

densities. This is the first representation of 

a Wolbachia-induced reproductive manipulation 

in a cell culture system and would allow us 

a further study to elucidate the underlying 

molecular mechanism of Wolbachia-induced 

manipulations of arthropod reproduction. 

An attempt to estimate migration routes of 

rice planthoppers in Asia based on their mitochondrial 

genes 

The brown planthopper (BPH, Nilaparvata 

lugens) and the white-backed planthoppers 

(WBPH, Sogatella furcifera) are serious pests 

for rice plants. Both pests annually migrate 

from tropical and subtropical regions into 

temperate regions in Asia, including Japan, 

Korea and northern China. To elucidate the 

geographical differences of the planthoppers 

and to estimate migration routes, we analyzed 

the variation of mitochondrial sequences in the 

region cox1–trnL2–cox2, in 579 BPH individuals 

(1928 bp) and 464 WBPH (1927 bp) from ten 

areas, Japan, China, Taiwan, Laos, Thailand, 

northern and southern Vietnam, northern and 

southern Philippines, and Papua New Guinea 

(PNG), collected in 1966-2009. The numbers of 

variation were 30 and 20 for BPH and WBPH, 

respectively. All BPH in PNG area showed PNGspecific 

variation. However, the samples from 

the other areas included many variations, and 

the variations were shared among many areas. 

BPH genetic distances among areas are very 

close to each other in Asia except for PNG and 

south Philippines. For WBPH, genetic distances 

were close among all areas. These data indicate 

that the two planthoppers might share their 

respective gene pool widely in Asia, suggesting 

that the migration routes would not be 

clearly elucidated based the mitochondrial gene 

sequences. 

EST analyses of important pests 

Important crops, vegetables and fruits are 

attacked by many pest species. However, the 

numbers of important pests whose genomic 

information are deposited in the databases are 

small, although a substantial amount of genomic 

or EST data of model insects are deposited 

in the public databases. Genomic information 

of pests is useful and important for molecular 

studies aiming at controlling these pests. We 

have been performing EST analysis of the 

brown planthopper (http://bphest.dna.affrc. 

go.jp/). The EST analyses were expanded to 

some other important pest species. The cotton 

aphid, Aphis fabae, and the common spider 

mites, Tetranychus urticae, are globally important 

pests for fruits and vegetable production, 

because they attack a wide range of plant hosts 

and show high reproductive rate. The green 

rice leafhopper, Nephotettix cincticeps, is a vector 

of virus and mycoplasma of rice plants. This 

species has specific beneficial symbionts for the 

hosts. The ESTs would be useful for the various 

studies of these important pests. 


Silk-Material Research Unit 

The research subject of Silk-Materials 

Research Unit is development of application 

technologies for silk proteins produced by 

silkworms, spiders, and hornets. We study 

physicochemical properties of these silk proteins 

and interactions with living body like cells. 

We develop new materials to use in medical 

areas such as cartilage regeneration scaffolds 

and wound dressings through development of 

fabrication and functioning technologies of the 

silk proteins by use of transgenic and chemical 

process. 

Gene expression of chondrocytes cultured in 

fibroin sponges having various pore sizes 

In collaboration with Kyoto University, we 

study the application of silk fibroin porous 3D 

structure (fibroin sponge) to be a cell scaffold 

for cartilage regeneration in tissue regeneration 

therapy. In order to design the fibroin sponge 

to be suitable for cartilage regeneration, gene 

expression by chondrocytes cultured on silk 

fibroin sponges with different pore size were 

studied. Collagen type I gene, which is a marker 

for dedifferentiation of chondrocytes, showed 

higher expression on larger pore size silk fibroin 

sponge. In contrast, higher gene expression of 

collagen type II and aggrecan which are cartilage 

component, as a differentiation marker, was 

observed on silk fibroin sponge with smaller 

pore size. We already reported good cartilage 

tissue regeneration around the surface region of 

silk fibroin sponge with smaller pore size. These 

results indicate highly differentiated cartilage 

tissue can be regenerated on silk sponge with 

smaller pore size. 

Model drug release from sericin gel film 

The highly hydrophilic nature of silk sericin 

makes it a promising candidate for developing 

novel medical and cosmetic materials. Sericin 

gel film, which possesses high water absorption 

and mechanical strength, has been developed 

by a novel film-forming process via gelation of 

sericin solution, and its physical and biological 

characteristics as a wound dressing have been 

Fig 1. Gene expression by chondrocytes cultured on silk fibroin sponge with 

various pore sizes. 7 days after incubation. 


Release (%) 

100 

90 

80 

70 

Rhodamine B (479 Da) 

60 

FITC-albumin (6.6 kDa) 

50 

40 

30 

20 

10 

0 

0 10 20 30 40 50 

Time (h) 

Fig 2. Release rate of the model drugs, Rhodamine B (479Da; circle) and FITC-albumin (6.6 

kDa; square), in PBS (pH 7.4) from sericin gel film at 37 °C. 

The release rate was calculated from the absorbance at 554 and 495 nm for Rhodamine 

B and FITC-albumin, respectively. 

investigated. In this year, the drug release from 

sericin gel film were investigated using two 

model drugs with different molecular weights, 

Rhodamine B (479 Da) and FITC-albumin (6.6 

kDa). Rhodamine B was loaded by dissolving it 

in ethanol used as a gelator and FITC-albumin 

was loaded by directly dissolving it in sericin 

solution before gelation. The release rate of 

these model drugs in PBS (pH 7.4) at 37℃ was 

measured form the absorbance at 554 and 495 

nm for Rhodamine B and FITC-albumin, respectively 

(Fig 2). As a result, the release of FITCalbumin 

was found to be much slower than that 

of Rhodamine B. This result indicates that the 

release rate of drugs from sericin gel film can 

be controlled by changing the pore size of threedimensional 

network in sericin gel film. 

Mechanical properties of a silk single fiber 

containing spider silk produced by transgenic 

silkworm technology 

We generated transgenic silkworm lines, 

which express a part of Spider (Araneus ventricosus) 

dragline silk (MaSp-2) protein as a part 

of fibroin fiber of silk by using a commercial 

silkworm strain “Chu-515”. To make clear the 

effect of spider silk protein on the physical 

properties of silk fiber, stress-strain analysis was 

performed using single fiber silk prepared from 

5th stage silkworm larvae. Fig. 3 shows the S-S 

curves of “chu-515” silk and “TKH29” (transgenic 

silkworm) silk of non-degummed (a), alkaline- 

degummed (b), and water immersed silk (c). The 

results shows that spider dragline silk protein 

effects on silk’s physical properties, especially on 

degummed silk and when the silk was wet. 

Drawing-induced Changes in Morphology 

and Mechanical Properties of Hornet Silk Gel 

Films 

Complete amino acid sequences of the four 

major proteins (Vssilk 1–4) of silk (hornet silk) 

obtained from yellow hornet (Vespa simillima, 

Vespinae, Vespidae) cocoons have been 

determined. The native structure of the hornet 

silk (HS), in which Vssilk 1–4 have an α-helix 

domain with coiled-coil a-helices and a β-sheet 

domain, is restored when hornet silk gel films 

(HSGFs) are formed by pressing and drying HS 

hydrogel. The HSGF can be stretched in the 

wet as well as dry state. Because the HSGF 

stretches uniformly in the wet state, we can 

obtain extended samples with accurate drawn 

ratio (DR) control. This makes it possible to 

carry out a detailed analysis of the changes 

in the structure and physical properties of 

the HSGF during wet drawing. Wet drawing 

induces orientation of the molecular chains with 

coiled-coil a-helix structures. The maximum DR 

achieved during wet drawing is only 2, and the 

molecular weight of HS is low; despite this, the 

maximum tensile strength and tensile modulus 

of the HSGF fabricated in this study are 170 

MPa and 5.5 GPa, respectively; these values are 


Fig 3. Stress-Strain analysis of silk fiber 

Single fibers were prepared from 5 th stage larvae of chu-515 and TKH29 (transgenic). The physical 

properties of the fibers were analyzed directly (a), after alkaline-degummed (b), or after immersion in 

water (c). 

Fig 4. Photographs of silk gel film obtained from yellow hornet 

(Vespa simillima xanthoptera). 


higher than those of the films prepared from 

silkworm silk. 

Effects of fibroin and sericin proteins on 

phospholipid bilayer membrane 

Since the phospholipid bilayer membrane 

(PBM) is a main structure of cell membranes, artificially 

reconstructed PBM is often substituted 

for cell membrane in in vitro experiments. We 

have studied the interaction between artificial 

PBM and B. mori silk proteins, in order to 

estimate the influence of those proteins on cells 

and assessed their safty/ Our study elucidated a 

strong interaction between silk fibroin and PBM; 

furthermore, microfibrils of native silk fibroin 

from the posterior silk gland were found to 

penetrate PBM (Fig 5). Such an interaction generally 

corresponds to cytotoxicity, for example 

as with bee venom melittin; however, the utilization 

of B. mori silk thread for surgical sutures 

has empirically demonstrated that B. mori silk 

fibroin is innocuous material. This inconsistency 

is to be elucidated in the future. Only a weak 

interaction was observed between sericin and 

PBM, which is negligible as compared with that 

of silk fibroin and PBM. 

Fig 5. Freeze-fracture electron micrographs of liposomes plus native silk fbroin from the posterior silk 

gland (PSGF). 

Scale bar = 200 nm in each panel. (a) PSGF alone as a reference, featuring a filament similar to a 

string of beads (arrows), nearly 15 nm in width. (b) Liposomes plus PSGF, showing a filament (arrow) 

and fractured liposomes (arrowheads). Two separated liposomes adhere to a filament. (c) Liposome 

skewered by a filament. The pore in the liposome (e) indicates that something pierced the membrane, 

leading to the conclusion that a filament pierced the membrane on the left side of the liposome (f), 

traversed the lumen while raising the membrane (g), and escaped from the lumen (e). (d) A liposome 

similarly skewered by a filament that pierced the membrane twice (h, i). 


Albumin secretion of human hepatic cell line 

FLC-4 cultured in lactose-modified silk fibroin 

(Lac-CY-SF) sponges 

We fabricated three-dimensional porous 

sponges composed of lactose-modified silk 

fibroin (Lac-CY-SF) bearing hepatocyte-specific 

β-galactose residues. Human hepatocellular 

carcinoma-derived FLC-4 cells were seeded 

in the sponges of Lac-CY-SF, silk fibroin (SF), 

and collagen, and cultured up to 3 weeks. Cell 

viability assay revealed that the viability of cells 

cultured in Lac-CY-SF sponges was still high at 

day 22 although the viability in collagen sponges 

at day 22 was remarkably lower than that in 

Lac-CY-SF and collagen sponges at day 8. The 

prolonged cell viability in Lac-CY-SF sponges 

means that Lac-CY-SF sponges have the 

capability to maintain the cells for a long period. 

Albumin secretion by FLC-4 cells cultured in 

sample sponges was quantified as one of the 

representative liver-specific functions by the 

ELISA assay (Fig 6). The amounts of albumin 

secreted by the cells cultured in Lac-CY-SF and 

collagen sponges were the same level at day 8 

and increased at day 15 compared to those at 

day 8. At day 22, the cells cultured in Lac-CY- 

SF sponges still showed high level of albumin 

while the cells cultured in collagen sponges 

showed very low level of albumin as well as the 

cells in SF sponges. These results suggest that 

the FLC-4 cells cultured in Lac-CY-SF sponges 

maintain hepatic functions throughout 3 weeks. 

Evaluation of raw spider silk single fiber by 

micro FTIR 

Spider dragline silk is a high performance biopolymer 

with exceptional mechanical properties. 

Artificial spider silk solution is prepared by a 

recombinant technique. However, the molecular 

process for fiber formation is still not completely 

understood. In this work, model peptides were 

chemically synthesized and examined for their 

ability to participate in fiber formation. A short 

model peptide derived from Japanese spider 

Nephila clavata was prepared by a solid phase 

peptide method, based on a prediction using 

the hydrophobic parameter of each individual 

amino acid residue. Significant fiber formation 

was observed in organic solvents, such as TFE. 

CD measurements indicated that the peptide is 

rich in β-sheet structure and that the formation 

of a β-sheet structure is required for the fiber 

formation. 

In addition, imaging technique with micro 

FT-IR spectroscopy was carried out in order 

to obtain an insight into the natural spider 

web of Nephila clavata. The results suggested 

that β-sheet structure is predominant in the 

radial and spiral threads. On the other hand, a 

different spectral feature which was due to the 

side chain from the amino acid was observed 

between the radial and spiral thread (Fig 7). 

Fig 6. Albumin secretion of FLC-4 cells cultured in sample sponges. 


Generation of a Bombyx mori phenylalanyltRNA 

synthetase mutant with relaxed substrate 

specificity 

To develop silk proteins containing unnatural 

amino acids by relaxing substrate specificity 

of aminoacyl-tRNA synthetases derived from 

the domesticated silkworm, Bombyx mori, 

phenylalanyl-tRNA synthetase (PheRS) genes 

have been cloned from silk glands of B. mori 

and the residues responsible for its substrate 

specificity have been predicted by sequence 

comparison with PheRSs from other organisms. 

In this year, a mutant B. mori PheRS gene 

was generated and its substrate specificity was 

assayed in vitro. Amino acid mutation to Gly 

was introduced at Ala 450 of B. mori PheRS 

α-subunit to enlarge its substrate recognition 

pocket. The mutant B. mori PheRS along with 

its wild-type counterpart were expressed in 

E. coli and purified. In vitro activity assay 

demonstrated that the mutant B. mori PheRS 

catalyzed aminoacylation of the synthesized 

B. mori tRNAPhe with p-chloro- and p-bromosubstituted 

L-phenylalanine analogs which were 

not recognized by the wild-type B. mori PheRS 

(Fig 8). 

Fig 7. FTIR spectrum of spider silks. 

Fig 8. Aminoacylation of the synthesized B. mori tRNA Phe with L-phenylalanine (Phe) or one of the 

Phe analogs by the wild-type B. mori PheRS (left panel) or the mutant B. mori PheRS (αA450G) (right 

panel). 

The aminoacylation reactions were carried out in the presence of Phe, p-fluoro-L-phenylalanine 

(F-Phe), p-chloro-L-phenylalanine (Cl-Phe), p-bromo-L-phenylalanine (Br-Phe), and p-iodo-Lphenylalanine 

(I-Phe). Unreacted tRNA Phe and its aminoacylated derivative (aa-tRNA Phe ) were 

separated on a 10% acid-urea polyacrylamide gel. 


Silk-Technology Unit 

The unit is conducting research to breed new 

silkworm races having specific filament characters 

such as high strength or luster to obtain new 

medical materials or a unique textile. It also develops 

useful silk materials of sanitary and bedding 

for babies, invalids and elderly that take advantage 

of the moisture absorption and insulation characteristics 

of silk. The selected research topics of 

2009 from the present research unit are as follows: 

Breeding of strong cocoon filament races of 

the silkworm, Bombyx mori 

In order to produce strong silk filaments 

required in medical and new material fields, we 

have been working to develop new silkworm 

races which produce strong cocoon filaments. 

Each three Japanese and Chinese strong filament 

strains maintained in our breeding resources 

and genetic stocks in NIAS Gene Bank were 

used as parents for cross-breeding. After several 

generations of individual and one-batch selection 

for cocoon filament strength, we obtained both 

Japanese and Chinese parental strains which produce 

strong cocoon filaments (Table 1). Then, the 

F1 hybrid strain between Japanese and Chinese 

strains were grown in the spring and autumn 

seasons, and economical raw silk performances 

were evaluated. The hybrid larvae were easy to 

rear compared to the parental strains, although 

heterosis was more evident for cocoon filament 

length and weight than for cocoon yield. Both the 

strength and Young’s module of the raw silk are 

higher than “Hakugin” which produces the thinnest 

and strongest fiber among the established 

strains so far. We are now preparing silk suture 

materials using the silk filament spun by this 

new hybrid race, SFN x SFC. 

Physical properties of twisted silk yarns 

In order to meet the demands for the twisted 

silk yarns with a variety of characteristics, we 

produced four sizes of raw silk threads ranging 

from very thin (10 denier) to extremely thick (100d). 

Their physical properties were such that, as the 

size increased, the tenacity decreased, elongation 

increased and Young’s modulus decreased. For the 

non-degummed twisted yarn, 1) tenacity increased 

slightly with the increase in the number of twists 

up to 500 T/m, but decreased at values greater 

than 500 T/m; 2) elongation becomes greater 

Table 1. Performances of the cocoon filament and raw silk of a new strongest filament hybrid race, SFNxSFC. 

2009 Spring 

Cocoon filament 

Raw silk 

Race 

 

 

Reelability Length Weight percentage Weight Size Strength Elongation Young's module 

) (g/d) (%) (kg/mm2) 

Ave 85.0 759 18.8 0.23 2.81 4.84 27.03 1030.00 

sd 12.77 46.78 0.90 0.01 0.07 0.09 0.90 2.65 

Ave 73.1 705 13.2 0.14 1.81 5.09 24.91 1097.14 

sd 8.95 106.58 0.95 0.02 0.09 0.11 0.71 22.60 

× 76.0 733 18.9 0.15 1.87 5.57 25.30 1117.00 

2009 Autumn 

 

SFC 

Ave 91.7 722 16.9 0.22 2.73 5.09 23.63 1027.67 

sd 1.53 45.88 0.82 0.03 0.52 0.43 0.49 72.53 

Ave 87.0 623 11.7 0.13 1.85 5.28 23.00 1140.33 

sd 2.00 44.02 0.97 0.01 0.05 0.05 0.56 105.98 

SFNxSFC 89.0 803 15.0 0.21 2.38 5.46 24.00 1149.00 

SFCxSFN 89.0 712 14.3 0.18 2.32 5.59 23.90 1194.00 

Reference 

Hakugin 76.0 1822 43.9 0.34 1.73 5.36 29.80 1011.00 


with the increase in the twist number except 

in 10d and 1000 T/m cases; 3) Young’s modulus 

decreased with the increase in the twist number; 

4) bending rigidity and hysteresis increased with 

the increase in the twist number, and it increased 

as the raw silk size increased for smaller numbers 

of twists but not as much for larger numbers of 

twists. The rate of increase related to 100d raw 

silk was smaller than those of other silk types. 

On the other hand, degummed yarns showed a 

little different result in which physical properties 

improved with the increase in thickness. Thus, we 

concluded that degummed twisted yarn made of 

extra-thick 100d silk yarn has good bending rigidity 

when it has many twists (Fig 1). 

Development of a new type of silk bed pad 

Several years ago, we developed “Silk-wave 

bed pad” in which cocoon filaments were reeled 

up in a separate state from a large number of 

cocoons (1,500–2,000 cocoons), dried perfectly and 

wound on a big wheel (Fig 2). In this type of silk 

bed pad, cocoon filaments are separated from 

each other and a sericin layer remains on the 

surface of cocoon filaments. 

The new type of silk bed pad is different from 

“Silk-wave bed pad”, that is, a large number of cocoons 

were reeled up and wound on a big wheel 

in wet state, then the silk thread was degummed 

to remove sericin from the cocoon filaments. 

Next, the silk threads, which were composed of 

fibroin filaments only, were dried and expanded 

by a draft force machine (Fig 2). The new type of 

silk bed pad manufactured in this way has higher 

bulkiness and pressure recovery properties compared 

with cotton and wool bed pads. This bed 

pad also is expected to be used for clothing pads 

such as outer wear, under wear, sports wear and 

so on. 

-4 

N m 2 /m) 

Bending rigidity (10 

0.2 

0.1 

0.0 

10d 

27d 

42d 

100d 

0 200 400 600 800 1000 

Twist number (T/m) 

Fig 1. Bending rigidity (left) and hysteresis (right) of degummed twisted yarn 

-2 N m/m) 

Hysteresis (10 

0.2 

0.1 

0.0 

10d 

27d 

42d 

100d 

0 200 400 600 800 1000 

Twist number (T/m) 

Mass reeling Winding in wet state Degumming 

Degumming Making bed pad Bed pads 

Fig 2. Manufacturing process of the new type of silk bed pad 



The Division of Animal Sciences consists of 

Animal Genome Research Unit, Reproductive 

Biology Research Unit, and Neurobiology 

Research Unit. 

Research Background 

Animal products, which are nutritionally 

better balanced and abundant in essential amino 

acids, are important for human diets. In Japan, 

low cost and high quality livestock production 

must be achieved, despite the severe limitation 

of land resources and high labour cost in comparison 

with countries as U.S.A. or Australia. In 

order to overcome these difficulties, it is important 

to improve livestock production technology, 

especially in animal breeding, reproduction, 

and management. In addition to the pursuit 

of economic efficiency, the concept of animal 

welfare has recently encouraged American and 

European countries to provide new standards 

for domestic animal management. Moreover, in 

new industries other than livestock production, 

there is a global competitive environment for 

research on transgenic domestic animals. On 

the other hand, gene sequencing in domestic 

animals is conducted in collaboration with 

international consortiums. The Animal Science 

Division accumulated genome information and 

has systematically used the information to 

improve domestic animals and create transgenic 

domestic animals for other fields such as 

medicine. To counteract the recent decrease 

in the conception rate in cattle and to improve 

somatic cell cloning technology, we conducted 

basic research to solve major problems in reproductive 

biology. Our research on the central 

regulatory mechanisms in instinctive behaviour 

would largely contribute to the development 

of animal production and husbandry. We have 

contributed internationally by participating in 

the international consortium on swine genome 

sequencing. 

Animal Genome Research Unit aims to improve 

the genome resources of domestic animals 

and to develop systems that promote the use of 

genome information to contribute to “the high 

quality and safe production of livestock” and “the 

new use of livestock as physiological models 

of humans.” The unit also identifies genome 

regions related to economically important traits 

and develops DNA markers that can be applied 

to improvement of livestock and to animal 

identification and traceability. 

Reproductive Biology Research Unit aims to 

improve the conception rate directly concerned 

with the animal industry and to establish new 

reproductive technology involving oogenesis 

and spermatogenesis. The unit investigates the 

proliferation and differentiation of germ cells 

and stem cells, the molecular mechanism of 

meiosis, and the process of implantation and 

placental formation. 

Neurobiology Research Unit aims to elucidate 

the central mechanisms of stress response, 

growth and reproduction in domestic animals 

to improve livestock management systems. The 

unit investigates neural functions of hypothalamic-pituitary 

axis and limbic-cortex system 

and its control mechanisms by environmental 

factors. 

The major research topics in the fiscal year 

2009 are described in the following pages. 


Animal Genome Research Unit 

QTL identification for inner muscle fat contents 

in Duroc breed pigs and development of 

a swine breeding population “Bono Brown” 

characterized as high intramuscular fat content 

(shimofuri). 

The standards of consumers in the choice of 

the pork are “safety”, “low cost”, and “high quality”. 

In Japan, many people say that domestic 

pork is “safe” and they have higher preference 

for it than imported pork. On the other hand, 

some consumers think that imported pork costs 

less and has the same quality as domestic pork 

and that it is not worth paying for domestic 

pork. In order for more consumers to choose 

domestic pork, improvement of the meat quality 

is important. Among traits of meat quality, 

intramuscular fat (IMF) content influences the 

tenderness, juiciness and flavor. High IMF 

contents are able to be judged by the appearance 

of sliced meats as “shimofuri”, so it is one 

of the most important traits affecting consumer 

acceptability of meat. 

In pigs, many studies for quantitative traits 

loci (QTL) have reported genetic regions affecting 

meat quality, but most of them have been 

detected between improved breeds and Asian 

local breeds or wild boar. In Japan, meat animals 

are generally produced by a three-way cross 

using Duroc sires and F1 (Landrace and Large 

White) dams, so genetic improvement of Duroc 

breed pigs would be effective. Therefore, we 

aimed to explore genetic regions affecting the 

IMF content in a Duroc population in collaboration 

with Gifu Prefectural Livestock Research 

Institute and STAFF-Institute. 

In Gifu prefecture, some meat animals showed 

extremely high IMF contents. We performed 

an analysis of consanguinity for them and 

found a common sire (D1). Then, we planned to 

construct an experimental family for analysis of 

IMF content by using the D1 sire. We thought 

that the D1 sire had the genetic ability to increase 

IMF content and that progeny produced 

by a cross of the D1 sire to Duroc dams in the 

other population would be heterozygous (Q/ 

q) at the loci for IMF content. In production of 

meat animals by using such heterozygous sires, 

segregation of IMF content would occur in the 

progeny population due to the allele types (Q 

or q) derived from the heterozygous sires and 

enable us to identify the genetic region affecting 

the IMF content. 

A heterozygous sire (named D2) was produced 

and mated to six dams of Large White 

breed. As a result of analysis with 191 progeny, 

the average IMF contents of barrows and gilts 

were 4.6 ± 1.5% and 3.5 ± 1.0%, respectively; 

4.1% on average of all the 191 individuals. These 

values were significantly higher than those of 

meat animals produced by a conventional line; 

3.3 ± 0.9% (73 barrows), 3.0 ± 0.9 % (78 gilts), 

and 3.2% on average. In this experimental family, 

there were some individuals presenting IMF 

contents higher than 10%. The IMF contents 

were therefore thought to be segregating in this 

population 

We performed genetic analysis of QTL for 

the IMF contents with the phenotype data and 

the genotype data of 120 microsatellite markers 

covering the whole genome for the 191 individuals 

produced with the D2 sire. As a result, two 

QTLs were detected on swine chromosomes 

(SSC) 7 and 14 (Fig 1). These two QTLs acted 

independently and QTL effects on SSC7 and 

SSC14 were 0.7% and 0.4% per single allele, 

respectively (Fig 2). The progeny heterozygous 

at both QTLs showed 1.1% higher IMF content 

on average. 

We then attempted to produce a breeding 

population, in which QTL types for both SSC7 

and SSC14 were fixed for the IMF-increasetypes. 

The D2 sire (Q/q) was mated to six 

independent Duroc dams (q/q). From the 

progeny, five heterozygous (Q/q) males and 

nine heterozygous (Q/q) females were selected 

by a marker assisted selection method with 

seven and six microsatellite markers for SSC7 

and SSC14, respectively. For the selection of 


the homozygous individuals (Q/Q) for the both 

of two QTLs on SSC7 and SSC14, 179 progeny 

were produced with the total of 23 matings 

between heterozygous animals. Until now, nine 

homozygous sires and 14 homozygous dams for 

both QTLs have been selected. 

For the analysis of meat quality of this population, 

60 individuals, which did not pass the 

aptitude test for sires and dams, were fattened 

and shipped. As a result of analysis of the meat 

quality, the average IMF contents were 6.3 

± 1.9% (21 barrows) and 5.8 ± 1.3% (39 gilts), 

which were approximately two times of those 

of meat animals produced from a conventional 

line; 3.2% on average described as above (Table 

1). This population was named “Bono Brown”, 

which was constructed from the Italian word 

of “bono” (delicious in Italian) and “brown” of 

Duroc hair color (Fig 3A). Now the pork production 

with “Bono Brown” is performed in Gifu 

prefecture (Fig 3B). 

Fig 1. Two QTLs for IMF contents in Duroc breed 

Solid and dotted lines indicate chromosome-wide significance level of 0.01% and 0.05%, respectively. 

Triangles indicate the positions of microsatellite markers used for an interval mapping analysis. 

Fig 2. QTL effects for IMF contents in F1 progeny of D2 sire and Large White dams 

Q means the IMF-increase-type alleles at the QTLs on SSC7 and SSC14, q means the non-increase type 

in Duroc pigs, and w means alleles of Large White, which were assumed to be the non-increase type. 

Fig 3. A: A sire of Bono Brown. B: Pork meat produced with Bono Brown 


Table 1. Summary for IMF contents in Bono Brown population 

Days of slaughter 

(115-120kg of live weight) 

Barrows (n=21) Gilt (n=39) 

174.6 ± 9.2 184.2 ± 15.7 

Back fat thickness (mm) 2.1 ± 0.6 1.8 ± 0.4 

IMF content (%) 6.3 ± 1.9 5.8 ± 1.3 

(3.1 ± 1.7) (3.0 ± 0.8) 

The values in parentheses indicate those in a conventional line 

Overexpression of NUDT7, a candidate quantitative 

trait locus for pork color, downregulates 

heme biosynthesis in L6 myoblasts 

Meat color determines initial acceptance or 

rejection in the marketplace. To strengthen consumer 

acceptance, improvement of meat color 

can be a top priority of the pork industry. Quantitative 

trait locus (QTL) analyses for pork color 

have revealed that variation in heme content, 

or Minolta measurement a* (redness), in skeletal 

muscle tissues is at least partly accounted for 

by a few relevant QTLs identified in the pig 

genome. In a previous study using F2 progeny 

from the mating of a Japanese wild boar 

(known as red meat) to Large White dams, we 

detected a QTL on swine chromosome 6 (SSC6) 

that explained 9% of phenotypic variance in 

heme content, and that the Japanese wild boar 

allele had a significant additive effect on the 

redness of pork. After fine-mapping the QTL 

on SSC6 and analyzing the genome structure, 

a nudix (nucleoside diphosphate linked moiety 

X)-type motif 7 (NUDT7) gene was located as 

a strong candidate of the QTL (Fig 4). As the 

substrate of NUDT7, oxidized CoA was listed 

from the study with yeast. Furthermore, the 

mouse NUDT7 showed substantial hydrolytic 

activity not only with oxidized CoA but also 

with acyl-CoA, including succinyl-CoA which is 

a substrate in the biosynthesis of heme. We also 

identified differential allelic expression of pig 

NUDT7 (Table 2), indicating that the transcription 

efficiency of the Japanese wild boar allele 

was less than that of the Large White allele. 

Thus, the lower NUDT7 expression in Japanese 

wild boar may contribute to the enrichment of 

meat redness. 

For the next step, we aimed to investigate 

the relationship between pig NUDT7 expression 

and heme content in muscle cells. Rat L6 

myoblast cell was selected as the in vitro model, 

because the cells differentiate into myotubes 

after confluence and synthesize endogenous 

heme and myoglobin. Rat L6 myoblasts were 

transfected with a mammalian expression vector 

for pig NUDT7 immediately after the induction 

of cell differentiation, and samples were harvested 

at 2, 4, 6, and 8 days (Fig 5). Before the 

induction of cell differentiation on day 0, the rat 

L6 myoblasts showed a fibroblast-like configuration. 

The myoblasts fused to form short linear 

myotubes on day 2, when the cells achieved 

full confluence. From days 4 to 6, the cells 

differentiated further and formed extensively 

longer myotubes. On day 8, circular cells were 

recruited to become centers of twitching movement. 

From the observation of cell cultures, 

cell differentiation difference was not observed 

between NUDT7- and control-transfected cells 

throughout the cell culture process. 

The abundance of pig NUDT7 mRNA in 

NUDT7-transfected cells increased (P < 0.05) 

from day 2 to day 4 by tenfold, but gradually 

declined (P < 0.05) from day 6 to day 8. 

Conversely, the amount of NUDT7 mRNA from 

control-transfected cells remained negligibly low. 

On day 0, when L6 cells were still undifferentiated 

myoblasts, heme content was 6.0 pmol/105 

cells. On days 2 to 8, the means of controltransfected 

cells ranged between 51.0 (day 8) 

and 64.0 pmol/105 cells (day 4), corresponding to 

increases of 8.5-fold (P < 0.01) and 10.7-fold (P < 

0.01). In contrast, the heme contents in NUDT7- 

transfected cells ranged between 14.2 (day 4) 


Fig 4. QTL analysis for the heme content and gene map in the candidate region 

Blue horizontal line indicates genome-wise 1 % significance level. Green and light blue horizontal 

lines indicate 1.0-LOD (7.8-cM) and 1.5-LOD (9.8-cM) support intervals. 

Fig 5. Heme content alteration by NUDT7 transfection during L6 myoblast differentiation 


Table 2 

Relative expression level of NUDT7 Wild boar and Large White alleles 

Heterozygous 

Individual 

Ratio of expression level 

Large White/Wild boar 

6801 1.434 ± 0.056

Fig 1. Incidence of hermaphrodism in B6-Chr Y AKR -Dh/+ N(2) males. Arrowheads indicate left testis 

and arrows indicate uterus and/or ovary formed on the right side. 

analysis revealed that at least one C57BL/6Jderived 

homozygous allele at a locus on chromosome 

13 was required for hermaphrodism and 

sex reversal. The genetic basis underlying sex 

determination is not yet completely understood, 

and one of the most powerful approaches to 

understanding the molecular basis of the sex determination 

pathway is to identify and analyze 

inherited sex-reversal conditions. The present 

condition was genetically distinct from known 

inherited sex-reversal conditions. It therefore 

offers a novel opportunity to investigate the 

genetic basis of sex determination in mammals. 

Identification and characterization of SOLD1, 

a novel member of the retrotransposonderived 

Ly-6 superfamily protein, in ruminant 

placenta. 

Ruminant placenta produces an array of 

proteins for the maintenance of gestation. 

Secreted protein of Ly-6 Domain 1 (SOLD1), 

a novel Ly-6 domain protein, was identified 

in ruminant placental trophoblast. The Ly-6 

domain is shared by Ly-6/uPAR superfamily 

protein. Initially, this gene was identified in 

the cow. The SOLD1 gene was an intronless 

structure containing the Alu retrotransposon, 

which was integrated via cytoplasmic reverse 

transcription (Fig 2). SOLD1 protein had distinct 

disulfide bonding pattern of cysteine residues 

and lacked a transmembrane domain. mRNA 

encoding SOLD1 was transcribed in trophoblast 

mononucleate cells and translated to the protein 

containing a secretory signal sequence (Fig 3. 

left). Following secretion, SOLD1 protein was 

localized in the extracellular matrices of the 

mesenchyme in placental villi (Fig 3. right). 

SOLD1 interacted with the telopeptide of type 

I collagen. The secretion of SOLD1 protein was 

basolaterally from mononuclear trophoblasts 

to villous mesenchyme and anchored by type I 

collagen. Consequently, SOLD1 gene was also 


Fig 2. Schematic structure of bSOLD1 gene. 

Fig 3. In situ hybridization (left) and immunohistochemistry (right) of SOLD1 in Day-60 bovine 

placenta BNC: trophoblast binucleate cell, MNC: trophoblast mononucleate cell, UE: uterine 

epithelium, US: uterine stroma, VM: villous mesenchyme. 

identified in sheep and goat; however, there was 

no similar sequence in other species examined 

(human, mouse, rat, pig and hen). Ovine and 

caprine SOLD1 amino acid sequences had high 

similarity with that of bovine. Bovine, ovine 

and caprine SOLD1 affected gene expression in 

mesenchymal fibroblast cell lines; nucleoredoxin 

expression was up-regulated and BCL1-like 13 

was down-regulated. In summary, our results 

suggest that SOLD1 acts as a modulator for 

cell proliferation and apoptosis. Furthermore, 

SOLD1 appears to be a ruminant-specific gene 

involved in implantation and placentation. 

Exploitation of stem cell technology in animal 

science 

Pluripotent stem cells would be beneficial for 

generating precise genetically-modified animals. 

In mice, several types of pluripotent stem cells, 

such as embryonic stem (ES) cells, embrynic 

germ (EG) cells, and induced pluripotent stem 

(iPS) cells have been established, but not in domestic 

animals. We established bovine ES cells 

from cloned blastocysts generated by somatic 

cell nuclear transfer (ntES cells) using combined 

conventional ES cell culture method and chemical 

inhibitor cocktail. The established ntES cells 

exhibited dome-shaped colonies quite similar to 

mouse ES cells (Fig 4), high alkline phosphatase 

activities (Fig 4), and expression of pluripotent 

cell markers. Bisulfite sequencing analysis 

showed that the methylation state of Oct3/4 

promoter was significantly decreased in these 

cell lines (Fig 5). These results indicate that 

somatic cells are initialized via nuclear transfer 

and gain the pluripotency under the ES cell 

culture condition. The ntES cells are expected 

to be available for generating both geneticallymodified 

chimeras and cloned cows. 


Fig 4. Phase contrast image and alkaline phosphatase staining of bovine ntES cells. 

Fig 5. DNA methylation state of Oct3/4 promoter in somatic cell and ntES cells. 

Neurobiology Research Unit 

Kisspeptin/neurokinin B/dynorphin A neurons 

in the arcuate nucleus generate GnRH pulse 

in goats 

Gonadotropin-releasing hormone (GnRH) 

neurons in the basal forebrain are the final 

common pathway through which the brain 

regulates reproduction. The pulsatile release of 

GnRH is a prerequisite for sustaining normal 

gonadotropin secretion in mammals; however, 

the mechanism that generates the rhythmic 

discharge of GnRH is unknown. Kisspeptin neurons 

in the hypothalamus play a key role in the 

regulation of GnRH neurons. Indirect evidence 

suggests the kisspeptin neurons in the arcuate 

nucleus (ARC) serve as the central pacemaker 

that drives pulsatile GnRH secretion. Moreover, 

it has been suggested that neurokinin B (NKB) 

and dynorphin (Dyn) co-expressed in ARC kisspeptin 

neurons are involved in the control of 

GnRH/luteinizing hormone (LH) secretion. In the 

present study, we sought to determine whether 

ARC kisspeptin neurons consist of the GnRH 

pulse generator, and NKB and Dyn play some 

roles in driving GnRH pulse generator activity 

in goats. First, using electrophysiological techniques 

to record multiple unit activity (MUA) in 

the close vicinity of ARC kisspeptin neurons, we 

found that bursts of MUA occurred at regular 


intervals and that these repetitive bursts (volleys) 

were invariably associated with discrete 

pulses of LH. Second, using double- and triplelabeling, 

we confirmed that kisspeptin, NKB and 

Dyn are co-expressed in the same population 

of neurons, and fibers containing kisspeptin/ 

NKB/Dyn surround ARC kisspeptin neurons. 

Finally, we observed that central administration 

of NKB induced MUA volleys and pulsatile LH 

secretion, whereas Dyn inhibited both. These 

results suggest that ARC kisspeptin neurons 

might represent the intrinsic source of rhythmic 

activity of the GnRH pulse generator, and kisspeptin/NKB/Dyn 

neurons form a network with 

each other by their collaterals and dendrites 

in the ARC. Moreover, electrophysiological 

studies suggest that NKB acts as an accelerator, 

whereas Dyn serves as a brake of the burst in 

kisspeptin/NKB/Dyn neurons. Taken together, 

we propose that stimulatory drive of NKB 

triggers simultaneous firings of the majority of 

kisspeptin/NKB/Dyn neurons, and inhibitory 

drive of Dyn immediately terminates the firings, 

thereby evoking episodic bursts in kisspeptin/ 

NKB/Dyn neurons, which in turn generate 

pulsatile GnRH release via kisspeptin. 

Figure legend 

The black and white background shows a 

photomicrograph of a dense network of kisspeptin 

neurons, fibers and varicosities in the 

arcuate nucleus and median eminence of the 

goat (stained by immunocytochemistry). Four 

panels of colored photomicrographs show the 

same 4 cells. These 4 cells were stained for 

kisspeptin (green, far left), neurokinin B (red, 

second from left), and dynorphin (blue, second 

from right); a merged image of all three cotransmitters 

is shown on the far right. The colored 

tracings below the photomicrographs show 

periodic bursts of multiple unit activity (MUA), 

with time on the x axis and electrical activity 

on the y axis. Three panels show MUA under 

normal conditions (green, left), after stimulation 

by NKB (red, middle), and after inhibition by 

dynorphin (blue, right). Each MUA burst was 

associated with a corresponding pulse of GnRH/ 

LH (as measured in the serum). These observations 

suggest that an ensemble of kisspeptin/ 

NKB/dynorphin neurons in the arcuate nucleus 

drives the ultradian release of GnRH and LH 

and is the proximate source of the GnRH “pulse 

generator” 


List of Publications 

Original Papers 

I. Original Papers in English 

1 Abe H, Shimoda T, Ohnishi J, Kugimiya S, Narusaka M, Seo S, Narusaka Y, Tsuda S, Kobayashi M (2009) 

Jasmonate-dependent plant defense restricts thrips performance and preference BMC Plant Biology 9 

( ):97 

2 Abe K, Osakabe K, Ishikawa Y, Tagiri A, Yamanouchi H, Takyuu T, Yoshioka T, Ito T, Kobayashi M, 

Shinozaki K, Ichikawa H, Toki S (2009) Inefficient double-strand DNA break repair is associated with 

increased fasciation in Arabidopsis BRCA2 mutants Journal of Experimental Botany 60(9):2751-2761 

3 Agung B, Piao Y, Fuchimoto D, Senbon S, Onishi A, Otoi T, Nagai T (2010) Effects of oxygen tension 

and follicle cells on maturation and fertilization of porcine oocytes during in vitro culture in follicular 

fluid Theriogenology 73(7) : 893-899 

4 Akama K, Kanetou J, Shimosaki S, Kawakami K, Tsuchikura S, Takaiwa F (2009) Seed-specific 

expression of truncated OsGAD2 produces GABA-enriched rice grains that influence a decrease in 

blood pressure in spontaneously hypertensive rats Transgenic Research 18(6):865-876 

5 Akasaka E, Watanabe S, Himaki T, Ohtsuka M, Yoshida M, Miyoshi K, Sato M (2010) Enrichment of 

xenograft-competent genetically modified pig cells using a targeted toxin, isolectin BS-I-B4 conjugate 

Xenotransplantation 17(1) : 81-89 

6 Akasaka S, Sasaki K, Harano K, Nagao T (2010) Dopamine enhances locomotor activity for mating in 

male honeybees (Apis mellifera L.) Journal of Insect Physiology 56(9):1160-1166 

7 Akiduki G (2010) Egg extract promotes cell migration and growth in primary culture of early embryos in 

the silkworm, Bombyx mori (Lepidoptera: Bombycidae) Applied Entomology and Zoology 45(1):153- 

161 

8 Albinsky D, Kusano M, Higuchi M, Hayashi N, Kobayashi M, Fukushima A, Mori M, Ichikawa T, Matsui 

K, Kuroda H, Horii Y, Tsumoto Y, Sakakibara H, Hirochika H, Matsui M, Saito K (2010) Metabolomic 

screening applied to rice FOX Arabidopsis lines leads to the identification of a gene-changing nitrogen 

metabolism Molecular Plant 3(1) : 125-142 

9 An C, Ishibashi J, Ragan E.J, Jiang H, Kanost M.R (2009) Functions of Manduca sexta hemolymph 

proteinases HP6 and HP8 in two innate immune pathways Journal of Biological Chemistry 

284(29) : 19716-19726 

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115 Kobayashi I, Kojima K, Sezutsu H, Uchino K, Tamura T (2009) Expression of the Japanese oak 

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116 Kobayashi K, Maekawa M, Miyao A, Hirochika H, Kyozuka J (2010) PANICLE PHYTOMER2 (PAP2), 

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117 Kobayashi T, Ogo Y, Aung M.S, Nozoye T, Itai R.N, Nakanishi H, Yamakawa T, Nishizawa N.K (2010) 

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118 Koch D, Sakurai M, Hummitzsch K, Hermsdorf T, Erdmann S, Schwalbe S, Stolzenburg J-U, Spanel- 

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119 Komatsuda T, Salomon B, von Bothmer R (2009) Evolutionary process of Hordeum brachyantherum 6x 

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120 Komori T, Miyata M, Yamamoto T, Nitta N, Hiei Y, Yano M, Ueki J, Komari T (2009) Isolation and 

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121 Konishi H, Noda H, Tamura Y, Hattori M (2009) Proteomic analysis of the salivary glands of the rice 

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122 Konno K, Hirayama C, Shinbo H, Nakamura M (2009) Glycine addition improves feeding performance 

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123 Kotaki T, Shinada T, Kaihara K, Ohfune Y, Numata H (2009) Structure determination of a new juvenile 

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124 Kozaki T, Bradyc S.G, Scott J.G (2009) Frequencies and evolution of organophosphate insensitive 

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125 Kuboyama T, Saito T, Matsumoto T, Wu J, Kanamori H, Taura S, Sato M, Marubashi W, Ichitani K (2009) 

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126 Kuroda Y, Tomooka N, Kaga A, Wanigadeva S.M.S.W, Vaughan D.A (2009) Genetic diversity of wild 

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127 Kurusu T, Hamada J, Nokajima H, Kitagawa Y, Kiyoduka M, Takahashi A, Hanamata S, Ohno R, 

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128 Kusaba M, Maoka T, Morita R, Takaichi S (2009) A novel carotenoid derivative, lutein 3-acetate, 

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129 Kuwana Y, Kojima K, Tamada Y (2009) Design and fabrication of biosensor device by use of receptor 

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130 Kuwano M, Takaiwa F, Yoshida K.T (2009) Differential effects of a transgene to confer low phytic acid in 

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131 Lee J-H, Muhsin M, Atienza G.A, Kwak D-Y, Kim S-M, De Leon T.B, Angeles E.R, Coloquio E, Kondoh 

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132 Liu B, Watanabe S, Uchiyama T, Kong F, Kanazawa A, Xia Z, Nagamatsu A, Arai M, Yamada T, 

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133 Lo N, Hayashi Y, Kitade O (2009) Should environmental caste determination be assumed for termites? 

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134 Lohmann G.V, Shimoda Y, Nielsen M.W, Jørgensen F.G, Grossmann C, Sandal N, Sørensen K, Thirup 

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135 Ma J.G, Yasue H, Eyer K.E, Hiraiwa H, Shimogiri T, Meyers S.N, Beever J.E, Schook L.B, Beattie C.W, 

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136 Madamba M.R.S, Sugiyama N, Bordeos A, Mauleon R, Satoh K, Baraoidan M, Kikuchi S, Shimamoto K, 

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137 Maekawa K, Ishitani K, Gotoh H, Cornette R, Miura T (2010) Juvenile Hormone titre and vitellogenin 

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138 Maeno K, Tanaka S (2009) Artificial miniaturization causes eggs laid by crowd-reared (gregarious) 

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139 Maeno K, Tanaka S (2009) The trans-generational phase accumulation in the desert locust : 

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140 Maeno K, Tanaka S (2009) Is juvenile hormone involved in the maternal regulation of egg size and 

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141 Marui J, Ohashi-Kunihiro S, Ando T, Nishimura M, Koike H, Machida M (2010) Penicillin biosynthesis 

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142 Masumoto C, Miyazawa S, Ohkawa H, Fukuda T, Taniguchi Y, Murayama S, Kusano M, Saito K, 

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143 Masuo Y, Imai T, Shibato J, Hirano M, Jones O.A.H, Maguire M.L, Satoh K, Kikuchi S, Rakwal R (2009) 

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144 Matsukura K, Matsumura M, Tokuda M (2009) Host manipulation by the orange leafhopper Cicadulina 

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145 Matsumoto Y, Yanase T, Tsuda T, Noda H (2009) Characterization of internal transcribed spacer 

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146 Mbéguié-A-Mbéguié D, Hubert O, Baurens F.C, Matsumoto T, Chillet M, Fils-Lycaon B, Sidibé-Bocs 

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147 Merkwitz C, Ricken A.M, Lösche A, Sakurai M, Spanel-Borowski K (2010) Progenitor cells harvested 

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148 Migheli Q, Balmas V, Harak H, Sanna S, Scherm B, Aoki T, O · Donnell K (2010) Molecular phylogenetic 

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149 Mihara M, Itoh T, Izawa T (2010) SALAD database: a motif-based database of protein annotations for 

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150 Minaba M, Ueno S, Pillai A, Kato Y (2009) Evolution of ASABF (Ascaris suum antibacterial factor)- 

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151 Mitsumasu K, Tanaka Y, Niimi T, Yamashita O, Yaginuma T (2009) Novel gene encoding precursor 

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152 Miwa Y, Yamamoto K, Onishi A, Iwamoto M, Yazaki S, Haneda M, Iwasaki K, Liu D, Ogawa H, 

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153 Miyamoto Y, Enosawa S, Takeuchi T, Takezawa T (2009) Cryopreservation in situ of cell Monolayers on 

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154 Mizuno H, Tanaka T, Sakai H, Kawahigashi H, Itoh T, Kikuchi S, Matsumoto T (2010) Characterization 

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155 Mochida K, Furuta T, Ebana K, Shinozaki K, Kikuchi J (2009) Correlation exploration of metabolic and 

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156 Morita R, Kusaba M, Iida S, Nishio T, Nishimura M (2009) Development of PCR markers to detect the 

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157 Morita R, Sato Y, Masuda Y, Nishimura M, Kusaba M (2009) Defect in non-yellow coloring 3, an α/β 

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158 Morita R, Kusaba M, Iida S, Yamaguchi H, Nishio T, Nishimura M (2009) Molecular characterization of 

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159 Moriwaki J, Sato T (2009) A new combination for the causal agent of tea anthracnose: Discula theaesinensis 

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160 Moriwaki K, Miyashita N, Mita A, Gotoh H, Tsuchiya K, Kato H, Mekada K, Noro C, Oota S, Yoshiki A, 

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161 Moriya K, Tsubota T, Ishibashi N, Yafune A, Suzuki T, Kobayashi J, Shiotsuki T, Utsumi T (2010) 

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162 Moriyama T, Terasawa K, Sekine K, Toyoshima M, Koike M, Fujiwara M, Sato N (2010) Characterization 

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163 Motoyama T, Maruyama N, Amari Y, Kobayashi K, Washida H, Higasa T, Takaiwa F, Utsumi S (2009) α' 

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164 Msalya G, Shimogiri T, Okamoto S, Kawabe K, Minezawa M, Namikawa T, Maeda Y (2009) Gene and 

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165 Msalya G, Shimogiri T, Nishitani K, Okamoto S, Kawabe K, Minesawa M, Maeda Y (2010) Indels within 

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166 Murakami R, Koyama A, Yasui H (2009) Fluctuation in the concentration of 1-deoxynojirimycin in 

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167 Nagai T, Yamasaki F (2009) Bacillus subtilis (natto) bacteriophages isolated in Japan Food Science and 

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168 Nagao H, Kurogi S, Kiyota E, Sasatomi K (2009) Kumanasamuha geaster sp. nov., an anamorph of 

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169 Nagaoka T, Doullah M.A.U, Matsumoto S, Kawasaki S, Ishikawa T, Hori H, Okazaki K (2010) 

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170 Nair S.K, Wang N, Turuspekov Y, Pourkheirandish M, Sinsuwongwat S, Chen G, Sameri M, Tagiri 

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171 Naito M, Harumi T, Kuwana T (2010) Long term in vitro culture of chicken primordial germ cells isolated 

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172 Naito M, Harumi T, Kuwana T (2010) Morphological characteristics of symmetrically developed ovaries 

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173 Nakahara Y, Imanishi S, Mitsumasu K, Kanamori Y, Iwata K, Watanabe M, Kikawada T, Okuda T (2010) 

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174 Nakai M, Kaneko H, Somfai T, Maedomari N, Ozawa M, Noguchi J, Ito J, Kashiwazaki N, Kikuchi 

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175 Nakamura H, Muramatsu M, Hakata M, Ueno O, Nagamura Y, Hirochika H, Takano M, Ichikawa H (2009) 

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176 Nakamura I, Rai B, Takahashi H, Kato K, Sato Y, Komatsuda T (2009) Aegilops section Sitopsis species 

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177 Nakamura Y, Kawai S, Yukuhiro F, Ito S, Gotoh T, Kisimoto R, Yanase T, Matsumoto Y, Kageyama D, 

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178 Nakao H (2010) Characterization of Bombyx embryo segmentation process : expression profiles of 

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179 Namba O, Nakamatsu Y, Tateishi K, Miura K, Tanaka T (2009) Cuticular encystment of Autographa 

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180 Navarro V.M, Gottsch M.L, Chavkin C, Okamura H, Clifton D.K, Steiner R.A (2009) Regulation of 

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181 Nielsen J.K, Nagao T, Okabe H, Shinoda T (2010) Resistance in the plant, Barbarea vulgaris, and 

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182 Nishimura M, Fukada J, Moriwaki A, Fujikawa T, Ohashi M, Hibi T, Hayashi N (2009) Mstu1, an APSES 

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183 Nochi T, Yuki Y, Katakai Y, Shibata H, Tokuhara D, Mejima M, Kurokawa S, Takahashi Y, Nakanishi 

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184 Noguchi M, Yoshioka K, Itoh S, Suzuki C, Arai S, Wada Y, Hasegawa Y, Kaneko H (2010) Peripheral 

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185 Nozoye T, Takaiwa F, Tsuji N, Yamakawa T, Arakawa T, Hayashi Y, Matsumoto Y (2009) Production of 

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186 Numa H, Kim J-M, Matsui A, Kurihara Y, Morosawa T, Ishida J, Mochizuki Y, Kimura H, Shinozaki K, 

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187 Ogiso E, Takahashi Y, Sasaki T, Yano M, Izawa T (2010) The role of casein kinase II in flowering time 

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188 Ogura T, Tan A, Tsubota T, Nakakura T, Shiotsuki T (2009) Identification and expression analysis of Ras 

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189 Ohara H, Nikaido M, Date-Ito A, Mogi K, Okamura H, Okada N, Takeuchi Y, Mori Y, Hagino-Yamagishi 

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190 Ohkura S, Takase K, Matsuyama S, Mogi K, Ichimaru T, Wakabayashi Y, Uenoyama Y, Mori Y, Steiner 

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191 Ohmori S, Kimizu M, Sugita M, Miyao A, Hirochika H, Uchida E, Nagato Y, Yoshida H (2009) MOSAIC 

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192 Ohnishi J, Hirai K, Kanda A, Usugi T, Meshi T, Tsuda S (2009) The coat protein of Tomato mosaic virus 

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193 Ohnuma T, Onaga S, Murata K, Fukamizo T, Taira T, Katoh E (2009) Structure and function of family 50 

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194 Ohshima Y, Tsukimoto M, Takenouchi T, Harada H, Suzuki A, Sato M, Kitani H, Kojima S (2010) γ 

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195 Ohtsuka M, Warita T, Sakurai T, Watanabe S, Inoko H, Sato M (2009) Development of CRTEIL 

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196 Okumura N, Hayashi T, Uenishi H, Fukudome N, Komatsuda A, Suzuki A, Shibata M, Nii M, Yamaguchi 

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197 Onda Y, Kumamaru T, Kawagoe Y (2009) ER membrane-localized oxidoreductase Ero1 is required for 

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198 Oono Y, Wakasa Y, Hirose S, Yang L, Sakuta C, Takaiwa F (2010) Analysis of ER stress in developing 

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199 Osakabe Y, Osakabe K, Chiang V.L (2009) Characterization of the tissue-specific expression of 

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200 Osakabe Y, Mizuno S, Tanaka H, Maruyama K, Osakabe K, Todaka D, Fujita Y, Kobayashi M, Shinozaki 

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201 Ozawa M, Nagai T, Somfai T, Nakai M, Maedomari N, Miyazaki H, Kaneko H, Noguchi J, Kikuchi K (2010) 

Cumulus cell-enclosed oocytes acquire a capacity to synthesize GSH by FSH stimulation during in 

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202 O’Donnell K, Sink S, Scandiani M.M, Luque A, Colletto A, Biasoli M, Lenzi L, Salas G, González V, 

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203 Pariasca-Tanaka J, Satoh K, Rose T, Mauleon R, Wissuwa M (2009) Stress response versus stress 

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204 Park Y-J, Nemoto K, Nishikawa T, Matsushima K, Minami M, Kawase M (2009) Molecular cloning 

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205 Park Y-J, Nemoto K, Nishikawa T, Matsushima K, Minami M, Kawase M (2010) Waxy strains of three 

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206 Penner G.B, Taniguchi M, Guan L.L, Beauchemin K.A, Oba M (2009) Effect of dietary forage to 

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207 Puleo C.M, Ambrose W.M, Takezawa T, Elisseeff J, Wang T-H (2009) Integration and application of 

vitrified collagen in multilayered microfluidic devices for corneal microtissue culture Lab on a Chip 

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208 Rezaeian A.H, Nishibori M, Hiraiwa N, Yoshizawa M, Yasue H (2009) Expression profile and localization 

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209 Rezaeian A.H, Isokane T, Nishibori M, Chiba M, Hiraiwa N, Yoshizawa M, Yasue H (2009) αCGRP 

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210 Riemann M, Bouyer D, Hisada A, Müller A, Yatou O, Weiler E.W, Takano M, Furuya M, Nick P (2009) 

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211 Roller L, Žitňanová I, Dai L, Šimo L, Park Y, Satake H, Tanaka Y, Adams M.E, Žitňan D (2010) Ecdysis 

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212 Sagisaka A, Fujita K, Nakamura Y, Ishibashi J, Noda H, Imanishi S, Mita K, Yamakawa M, Tanaka H 

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213 Saisho D, Pourkheirandish M, Kanamori H, Matsumoto T, Komatsuda T (2009) Allelic variation of row 

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214 Saito T, Fujiwara S, Sasaki Y, Niwa K, Nemoto T, Kasuya E, Sakumoto R, Yamaguchi T (2009) 

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215 Sakai H, Itoh T (2010) Massive gene losses in Asian cultivated rice unveiled by comparative genome 

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216 Sakata K, Ikawa H, Watanabe H, Ashikawa I, Shimizu Y, Horiuchi I, Antonio B.A, Numa H, Nagamura 

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218 Sakuma S, Pourkheirandish M, Matsumoto T, Koba T, Komatsuda T (2010) Duplication of a wellconserved 

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219 Sakumoto R, Vermehren M, Kenngott R.A-M, Okuda K, Sinowatz F (2010) Changes in the levels of 

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220 Sapsutthipas S, Urayama T, Yamate M, Tsujikawa M, Nishigaki H, Hagiwara K, Yunoki M, Yasue H, 

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221 Sasaki K, Abe T, Yoshida Y, Asaoka K (2009) A homeotic mutation influences the wing vibration 

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222 Sato K, Matsumoto T, Ooe N, Takeda K (2009) Genetic analysis of seed dormancy QTL in barley 

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223 Sato T, Maekawa S, Yasuda S, Sonoda Y, Katoh E, Ichikawa T, Nakazawa M, Seki M, Shinozaki K, 

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224 Sato T, Uzuhashi S, Hosoya T, Hosaka K (2010) A list of fungi found in the Bonin (Ogasawara) Islands 

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225 Sato Y, Yang P, An Y, Matsukawa K, Ito K, Imanishi S, Matsuda H, Uchiyama Y, Imai K, Ito S, Ishida Y, 

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226 Satoh K, Kondoh H, Sasaya T, Shimizu T, Choi I-R, Omura T, Kikuchi S (2010) Selective modification of 

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227 Sazuka T, Kamiya N, Nishimura T, Ohmae K, Sato Y, Imamura K, Nagato Y, Koshiba T, Nagamura Y, 

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228 Shahinnia F, Sayed-Tabatabaei B.E (2009) Conversion of barley SNPs into PCR-based markers using 

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229 Shahinnia F, Sayed-Tabatabaei B.E, Sato K, Pourkheirandish M, Komatsuda T (2009) Mapping of 

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230 Shehzad T, Okuizumi H, Kawase M, Okuno K (2009) Development of SSR-based sorghum (Sorghum 

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231 Shibata F, Sahara K, Naito Y, Yasukochi Y (2009) Reprobing multicolor FISH preparations in 

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232 Shibaya T, Sugawara Y (2009) Induction of multinucleation by β-glucosyl Yariv reagent in regenerated 

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233 Shimizu A, Kawasaki S (2009) Rapid construction of a high-density rice linkage map by high efficiency 

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234 Shimizu H, Tanabata T, Xie X, Inagaki N, Takano M, Shinomura T, Yamamoto K.T (2009) Phytochromemediated 

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237 Shimura S, Kiuchi M, Kiguchi K (2009) Spatial and temporal changes of mitotic activity in the epidermis 

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238 Shimura S, Kiuchi M, Kiguchi K (2009) Epidermal mitotic activity associated with knob formation in 

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239 Shiokai S, Shirasawa K, Sato Y, Nishio T (2010) Improvement of the dot-blot-SNP technique for 

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240 Somfai T, Noguchi J, Kaneko H, Nakai M, Ozawa M, Kashiwazaki N, Egerszegi I, Rátky J, Nagai T, 

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242 Sugama S, Takenouchi T, Cho B.P, Joh T.H, Hashimoto M, Kitani H (2009) Possible roles of microglial 

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243 Sugimoto K, Takeuchi Y, Ebana K, Miyao A, Hirochika H, Naho Hara N, Ishiyama K, Kobayashi M, 

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245 Suto J (2009) Hermaphrodism and sex reversal associated with the dominant hemimelia mutation in 

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246 Suto J (2010) Principal component and interacting QTL analyses identify novel gene loci associated 

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248 Suzuki K, Matsumoto T, Kobayashi E, Uenishi H, Churkina I, Plastow G, Yamashita H, Hamasima N, 

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249 Suzuki K, Kaminuma O, Yang L, Motoi Y, Takai T, Ichikawa S, Okumura K, Ogawa H, Mori A, Takaiwa 

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250 Suzuki M, Kusano M, Takahashi H, Nakamura Y, Hayashi N, Kobayashi M, Ichikawa T, Matsui M, 

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252 Takahara K, Kasajima I, Takahashi H, Hashida S, Itami T, Onodera H, Toki S, Yanagisawa S, Kawai- 

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253 Takahashi H, Yamamoto K, Ohtani T, Sugiyama S (2009) Cell-free cloning using multiply-primed rolling 

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254 Takahashi H, Kamakura H, Sato Y, Shiono K, Abiko T, Tsutsumi N, Nagamura Y, Nishizawa N.K, 

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257 Takai T, Yano M, Yamamoto T (2010) Canopy temperature on clear and cloudy days can be used to 

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265 Tamai A, Dohi K, Mori M, Meshi T, Ishikawa M (2010) Inducible viral inoculation system with cultured 

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274 The Nasonia Genome Working Group (2010) Functional and evolutionary insights from the genomes of 

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275 Tokuda G, Miyagi M, Makiya H, Watanabe H, Arakawa G (2009) Digestive β-glucosidases from 

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293 Ushizawa K, Takahashi T, Hosoe M, Kizaki K, Hashizume K (2010) Cleaved bovine prolactin-related 

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298 Wang A, Yamakake J, Kudo H, Wakasa Y, Hatsuyama Y, Igarashi M, Kasai A, Li T, Harada T (2009) Null 

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301 Wei J, Fujita M, Nakai M, Waragai M, Sekigawa A, Sugama S, Takenouchi T, Masliah E, Hashimoto 

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303 Wu J, Fujisawa M, Tian Z, Yamagata H, Kamiya K, Shibata M, Hosokawa S, Ito Y, Hamada M, Katagiri 

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Bombyx mori revealed by BAC-FISH mapping PLoS ONE 4(10):e7465 

319 Yayou K, Kitagawa S, Ito S, Kasuya E, Sutoh M (2009) Effects of intracerebroventricular administration 

of neuromedin U or neuromedin S in steers General and Comparative Endocrinology 163(3):324-328 

320 Yayou K, Nakamura M, Ito S (2009) Effects of AVP V1a and CRH receptor antagonist on psychological 

stress responses to frustrating condition in sheep Journal of Veterinary Medical Science 71(4) :431- 

439 

321 Yayou K, Ito S, Yamamoto N, Kitagawa S, Okamura H (2010) Relationships of stress responses with 

plasma oxytocin and prolactin in heifer calves Physiology & Behavior 99(3):362-369 

322 Yazaki S, Iwamoto M, Onishi A, Miwa Y, Suzuki S, Fuchimoto D, Sembon S, Furusawa T, Hashimoto 

M, Oishi T, Liu D, Nagasaka T, Kuzuya T, Maruyama S, Ogawa H, Kadomatsu K, Uchida K, Nakao A, 

Kobayashi T (2009) Successful cross-breeding of cloned pigs expressing endo-β-galactosidase C 

and human decay accelerating factor Xenotransplantation 16(6):511-521 

323 Yli-Mattila T, Gagkaeva T, Ward T, Aoki T, Kistler C, O’Donnell K (2009) A novel Asian clade within the 

Fusarium graminearum species complex includes a newly discovered cereal head blight pathogen from 

the Russian Far East Mycologia 101(6) : 841-852 

324 Yokosuka M, Hagiwara A, Saito T.R, Tsukahara N, Aoyama M, Wakabayashi Y, Sugota S, Ichikawa 

M (2009) Histological properties of the nasal cavity and olfactory bulb of the Japanese jungle crow 

Corvus macrorhynchos Chemical Senses 34(7):581-593 

325 Yokotani N, Ichikawa T, Kondou Y, Matsui M, Hirochika H, Iwabuchi M, Oda K (2009) Tolerance to 

various environmental stresses conferred by the salt-responsive rice gene ONAC063 in transgenic 

Arabidopsis Planta 229(5) : 1065-1075 

326 Yokotani N, Ichikawa T, Kondou Y, Maeda S, Iwabuchi M, Mori M, Hirochika H, Matsui M, Oda K (2009) 

Overexpression of a rice gene encoding a small C2 domain protein OsSMCP1 increases tolerance to 

abiotic and biotic stresses in transgenic Arabidopsis Plant Molecular Biology 71(4-5):391-402 

327 Yonemaru J, Ando T, Mizubayashi T, Kasuga S, Matsumoto T, Yano M (2009) Development of genomewide 

simple sequence repeat markers using whole-genome shotgun sequences of sorghum (Sorghum 

bicolor (L.) Moench) DNA Research 16(3) : 187-193 

328 Yonemura N, Mita K, Tamura T, Sehnal F (2009) Conservation of silk genes in Trichoptera and 

Lepidoptera Journal of Molecular Evolution 68(6):641-653 

329 Yoshii M, Yamazaki M, Rakwal R, Kishi-Kaboshi M, Miyao A, Hirochika H (2010) The NAC transcription 

factor RIM1 of rice is a new regulator of jasmonate signaling The Plant Journal 61(5):804-815 


330 Yoshiyama M, Yamakawa M (2009) Expression of a cysteine proteinase inhibitor gene in silkworm 

Bombyx mori following Trypanosoma brucei challenge Journal of Insect Biotechnology and Sericology 

78(3) : 149-153 

331 Yoshiyama M, Yamakawa M, Chigusa Y, Gibson W.C (2009) Characterization of trypsin-and 

chymotrypsin-like genes in the midgut of the tsetse fly Glossina morsitans morsitans (Diptera : 

Glossinidae) Medical Entomology & Zoology 60(1):13-22 

332 Yuki Y, Tokuhara D, Nochi T, Yasuda H, Mejima M, Kurokawa S, Takahashi Y, Kataoka N, Nakanishi U, 

Hagiwara Y, Fujihashi K, Takaiwa F, Kiyono H (2009) Oral MucoRice expressing double-mutant cholera 

toxin A and B subunits induces toxin-specific neutralising immunity Vaccine 27(43):5982-5988 

333 Yun M-S, Kawagoe Y (2009) Amyloplast division progresses simultaneously at multiple sites in the 

endosperm of rice Plant and Cell Physiology 50(9):1617-1626 

334 Zhu Z, Kikuchi Y, Kojima K, Tamura T, Kuwabara N, Nakamura T, Asakura T (2010) Mechanical 

properties of regenerated Bombyx mori silk fibers and recombinant silk fibers produced by transgenic 

silkworms Journal of Biomaterials Science, Polymer Edition 21(3):395-411 


II. Original Papers in Japanese with English Summary 

1 Hirayama C, Kosegawa E, Tamura Y, Nakamura M (2009) Analysis of flavonoids in the cocoon layer of 

the silkworm regionan races by LC-MS Sanshi-Konchu Biotec 78(1):57-63 

2 Kubota M, Tomioka K, Sato T (2009) Damping-off of broccoli caused by Rhizoctonia solani AG-1 IC 

Annual Report of The Kansai Plant Protection Society 51:27-28 

3 Kubota M, Tomioka K, Sato T (2009) Damping-off of Dahurian patrinia caused by Rhizoctonia solani 

AG-1 IB in Japan Japanese Journal of Phytopathology 75(2):116-118 

4 Morishita T, Yamaguchi H, Degi K, Shimizu A, Nakagawa H (2009) Comparison of growth and rhizome 

yield characters among turmeric species cultivated in Kanto area Japanese Journal of Crop Science 

78(4):509-514 

5 Nakayama K, Aoki T (2010) Foot rot of tomato, a new disease in Japan, caused by Fusarium solani f. 

sp. eumartii Japanese Journal of Phytopathology 76(1):7-16 

6 Okada Y, Kimura T, Ideta O, Domon E, Saito A (2010) Biodiversity risk evaluation of transgenic sweet 

potato ‘EP200’ and ‘EP220’ with coat protein (CP) gene of sweet potato feathery mottle virus server 

strain (SPFMV-S) in a closed and semi-closed greenhouse Breeding Research 12(1):16-21 

7 Okuizumi H, Nonaka E, Noguchi T, Sato T, Takamiya T, Iijima H, Murakami Y (2009) RLGS analysis of 

DNA methylation in plants DNA Polymorphism 17( ):80-84 

8 Sato T, Uzuhashi S (2010) Outbreak of white rust on Ipomoea spp. in Japan and host specificity of its 

pathogens Plant Protection 64(3) : 174-180 

9 Sugiyama S, Tsukamoto K, Yamauchi T, Yoshino T, Takahashi H, Kuwazaki S, Suetsugu Y, Narukawa 

J, Yamamoto K, Ohtani T (2009) Development of a novel genome analysis method based on scanning 

probe microscopy Hyomen Kagaku 30(8):427-432 

10 Takeya M, Kawada M, Hattori S, Yamasaki F, Kosegawa E, Nirasawa K, Minezawa M (2009) A system 

for managing characteristics/evaluation data of animal genetic resources Agricultural Information 

Research 18(4) : 168-176 

11 Takeya M, Yamasaki F, Tomooka N (2009) A web-based search and map display system for the 

integration of collection sites of plant genetic resources with geographic, climatic, and plant 

characteristic data Agricultural Information Research 18(2):82-90 

12 Usugi T, Hibino H, Tanaka M, Kodama K, Takesawa T, Chiba K, Iwadate Y (2010) Virus-like particles 

observed in plants with gentian tumorous symptoms Japanese Journal of Phytopathology 76(1):21-24 

13 Yokoyama T, Yamaguchi M, Tomooka N (2009) Analysis of genetic diversity of LysM domains in 

GmNFR5a genes : Perception of Nod factors produced by soybean bradyrhizobia and role of LysM 

domains in competitive root nodulation between B. japonicum and B. elkanii Soil Microorganisms 

63(1):9-17 


Reviews and Monograph (In English) 

III. Reviews and Monograph 

1 Agrawal A.A, Konno K (2009) Latex: A model for understanding mechanisms, ecology, and evolution 

of plant defense against herbivory Annual Review of Ecology, Evolution and Systematics 40( ):311-331 

2 Baranov V.M, Novikova N.D, Polikarpov N.A, Sychev V.N, Levinskikh M.A, Alekseev V.R, Okuda T, 

Sugimoto M, Gusev O.A, Grigor·ev A.I (2009) The Biorisk experiment : 13-month exposure of resting 

forms of organism on the outer side of the Russian Segment of the International Space Station : 

Preliminary results Doklady Biological Sciences 426(1):267-270 

3 Dang-Nguyen T.Q, Tich N.K, Nguyen B.X, Ozawa M, Kikuchi K, Manabe N, Ratky J, Kanai Y, Nagai 

T (2010) Introduction of various Vietnamese indigenous pig breeds and their conservation by using 

assisted reproductive techniques Journal of Reproduction and Development 56(1):31-35 

4 Hatakeyama M, Sumitani M, Yamamoto D.S, Lee J.M, Oka K (2009) The sawfly, Athalia rosae ruficornis 

(Hymenoptera) as a model insect for development and reproductive biology: what has been done and 

what should be done? Proceedings of Arthropodan Embryological Society of Japan (44):1-12 

5 Hayat S, Hasan S.A, Mori M, Fariduddin Q, Ahmad A (2009) Nitric oxide: Chemistry, biosynthesis and 

physiological role Nitric Oxide in Plant Physiology (1):1-16 

6 Henry R.J, Rice N, Waters D.L.E, Kasem S, Ishikawa R, Hao Y, Dillon S, Crayn D, Wing R, Vaughan D 

(2009) Australian Oryza : Utility and conservation Rice (Online First): 

7 Hinomoto N, Nagamori S, Kakimoto K, Shimizu T, Higaki T, Muraji M, Noda T, Kawasaki K (2009) 

Molecular identification and evaluation of Oris species (Heteroptera : Anthocoridae) as biological 

control agents Japan Agricultural Research Quarterly 43(4):281-288 

8 Ishikawa M, Ide H, Price W.S, Arata Y, Nakamura T, Kishimoto T (2009) Freezing behaviours in plant 

tissues : Visualization using NMR micro-imaging and biochemical regulatory factors involved Plant 

Cold Hardiness : From the Laboratory to the Field 1(3):19-28 

9 Izawa T (2010) Photoperiodic control of flowering in the short day plant Oryza sativa Photoperiodism: 

The Biological Calendar ( ) : 38-58 

10 Kadono-Okuda K (2009) Densovirus resistance in Bombyx mori Molecular Biology and Genetics of the 

Lepidoptera (18) : 337-348 

11 Katafuchi T, Yasue H, Osaki T, Minamino N (2009) Calcitonin receptor-stimulating peptide : Its 

evolutionary and functional relationship with calcitonin/calcitonin gene-related peptide based on gene 

structure Peptides 30(9) : 1753-1762 

12 Katoh E, Ando I (2010) High resolution solid state NMR, 13 C Encyclopedia of Spectroscopy and 

Spectrometry (Second Edition) ( ) : 894-904 

13 Kikuchi K, Somfai T, Nakai M, Nagai T (2009) Appearance, fate, and utilization of abnormal porcine 

embryos produced by in vitro maturation and fertilization Control of Pig Reproduction 8( ):135-147 

14 Kikuchi R, Handa H (2009) Photoperiodic control of flowering in barley Breeding Science 59(5):546-552 

15 Komatsuda T (2009) Breeding Science special issue: Triticeae Breeding Science 59(5):453-454 

16 Komatsuda T, Pourkheirandish M (2009) Mutational events in a homeobox gene Vrs1 that created a 

six-rowed spike in barley domestication Induced Plant Mutations in the Genomics Era ( ):71-73 

17 Miao Y-L, Kikuchi K, Sun Q-Y, Schatten H (2009) Oocyte aging : cellular and molecular changes, 

developmental potential and reversal possibility Human Reproduction Update 15(5):573-585 


18 Mitsuhashi W (2009) Insect virus proteins involved in the peroral infectivity of the viruses and their 

potential practical application in pest control Insect Viruses : Detection, Characterization and Roles 

(1):1-20 

19 Mitsuhashi W (2009) Recent advances in studies for the application of a protein produced by 

entomopoxviruses (Poxviridae) for insect-pest control Japan Agricultural Research Quarterly 

43(4):289-294 

20 Nakagawa H (2009) Induced mutations in plant breeding and biological researches in Japan Induced 

Plant Mutations in the Genomics Era ( ) : 48-54 

21 Noda H (2009) How can planthopper genomics be useful for planthopper management? Planthoppers: 

new threats to the sustainability of intensive rice production systems in Asia ( ):429-446 

22 Saika H, Toki S (2009) Towards a highly efficient gene targeting system in higher plants Japan 

Agricultural Research Quarterly 43(2) : 81-85 

23 Sasaki T (2009) Darwinism to breeding Breeding Science 59(2):107 

24 Sasaki T (2009) Breeding and biodiversity Breeding Science 59(3):207 

25 Sasaki T (2009) Legacy derived from Norin 10 Breeding Science 59(4):331 

26 Sasaki T (2010) On the 60 th anniversary of Breeding Science Breeding Science 60(1):1-2 

27 Takeda S (2009) Bombyx mori Encyclopedia of Insects (Second Edition) (31):117-119 

28 Takeda S (2009) Sericulture Encyclopedia of Insects (Second Edition) (232):912-914 

29 Takenouchi T, Sugama S, Iwamaru Y, Hashimoto M, Kitani H (2009) Modulation of the ATP-induced 

release and processing of IL-1β in microglial cells Critical Reviews in Immunology 29(4):335-345 

30 Takenouchi T, Sekiyama K, Sekigawa A, Fujita M, Waragai M, Sugama S, Iwamaru Y, Kitani H, 

Hashimoto M (2010) P2X7 receptor signaling pathway as a therapeutic target for neurodegenerative 

diseases Archivum Immunologiae et Therapiae Experimentalis 58(2):91-96 

31 Tomooka N (2009) The origins of rice bean (Vigna umbelata) and azuki bean (V. angularis): The evolution 

of two lesser-known Asian beans An Illustrated Eco-history of the Mekong River Basin ( ):33-35 

32 Wakasa Y, Yang L, Takaiwa F (2009) Production of bioactive peptide in transgenic rice seed 

Modification of Seed Composition to Promote Health and Nutrition ( ):101-120 

33 Watanabe H, Tokuda G (2010) Cellulolytic systems in insects Annual Review of Entomology 55( ):609- 

623 

34 Yamamoto T, Yonemaru J, Yano M (2009) Towards the understanding of complex traits in rice : 

Substantially or superficially? DNA Research 16(3):141-154 

35 Yamane H, Konno K, Sabelis M, Takabayashi J, Sassa T, Oikawa H (2010) Chemical defence and 

toxins of plants Comprehensive Natural Products II : Chemistry and Biology 4(8):339-385 

36 Yasui H (2009) Chemical communication in mate location and recognition in the white-spotted 

longicorn beetle, Anoplophora malasiaca (Coleoptera : Cerambycidae) Applied Entomology and 

Zoology 44(2) : 183-194 

37 Yoshioka H, Asai S, Yoshioka M, Kobayashi M (2009) Molecular mechanisms of generation for nitric 

oxide and reactive oxygen species, and role of the radical burst in plant immunity Molecules and Cells 

28(4):321-329 

38 Yuki Y, Takaiwa F, Kiyono H (2009) Transgenic rice for mucosal vaccine and immunotherapy Allergy 

Frontiers: Future Perspectives 6( ) : 149-166 


List of Organizations Exchanged 

MOU 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 


Executive Members and Research 

Staff Members 

Executive Members 

President 

Vice-President 

Vice-President 

Auditor 

Auditor 

(as of March 31, 2010) 

Ishige Teruo 

Sasaki Takuji 

Shinbo Hiroshi 

Hasegawa Mineo 

Ichikawa Kunihiko 

Research Staff 

Research Planning and Coordination 

Research Director-General 

Research Director 





Senior Researcher 

Deputy Research Director 







Kadowaki Koichi 

Kawasaki Kenjiro 

Shirata Kazuto 

Machii Hiroaki 

Nakagawa Hitoshi 

Kawase Makoto 

Haga Atsunobu 

(Takahashi Toru) 

(Miyazawa Mitsuhiro) 

(Seo Shigemi) 

(Nishizawa Yoko) 

(Sugano Shoji) 

(Sentoku Naoki) 

(Kuwana Yoshihiko) 

Research Planning Section Head Handa Hirokazu 

Chief Researcher Watanabe Kenji 

Chief Researcher Hagihara Kiyoshi 

Researcher 

Yamamoto Shinichi 


Kawasaki Shinji 

Evaluation Section Head Asaoka Kiyoshi 

Information Management Section Head Mitsuhashi Hatsuhito 

Chief Researcher 

Kawada Masae 

Library Head (Mitsuhashi Hatsuhito) 


Public Relations Section Head (Kawasaki Kenjiro) 

Chief Researcher Inoue Takashi A. 

GMO Research Promotion Section Head Tabei Yutaka 

(Domon Eiji) 

Safety Management Section Head Watanabe Shinichiro 


Tanaka Yoshiyuki 

Technology Transfer and Research Cooperation Section Head Hagio Takashi 

Senior Researcher Kayano Toshiaki 

Chief Researcher Hirogari Yasuhiro 

(Sakurai Michiharu) 

(Nakamura Masatoshi) 

(Kawagoe Yasushi) 

Genetic Resources Management Section Head Kenmochi Fumikazu 

(Tomioka Keisuke) 

Technical Support Section Head Koyama Akio 

Head 

Kobayashi Toru 


QTL Genomic Research Center Director Yano Masahiro 

Chief Researcher Fukuoka Shuichi 

Chief Researcher Sugimoto Kazuhiko 

Chief Researcher Taguchi Fumio 

Chief Researcher Ebana Kaworu 

Chief Researcher Yamamoto Toshio 

Chief Researcher Yonemaru Jun-ichi 

Chief Researcher Mizobuchi Ritsuko 

Chief Researcher Yamanouchi Utako 

Chief Researcher Uga Yusaku 

Researcher 

Hori Kiyosumi 

Transgenic Crop Research and Development Center Director Takaiwa Fumio 

Chief Researcher Ozawa Kenjiro 

Chief Researcher Domon Eiji 

Chief Researcher Takahashi Sakiko 

Researcher 

Takagi Hidenori 


Researcher 

Researcher 

Kawakatsu Taiji 

Ogo Yuko 

Transgenic Silkworm Research Center Director (Machii Hiroaki) 

Chief Researcher Yonemura Naoyuki 

Chief Researcher Sezutsu Hideki 

Chief Researcher Uchino Keiro 

Chief Researcher Iizuka Tetsuya 

Chief Researcher Tatematsu Kenichiro 

Researcher 

Kobayashi Isao 

(Mase Keisuke) 

(Okada Eiji) 

Transgenic Animal Research Center Director Kitani Hiroshi 

Senior Researcher Naito Mitsuru 

Senior Researcher Sakurai Michiharu 

Senior Researcher Onishi Akira 

Senior Researcher Takezawa Toshiaki 

Chief Researcher Matsubara Yuko 

Chief Researcher Takenouchi Takato 

Chief Researcher Sato Mitsuru 

Researcher 

Fuchimoto Daiichiro 

Researcher 

Akizuki Gaku 

Researcher 

Suzuki Syunichi 

Researcher 

Senbon Shoichiro 

(Tokunaga Tomoyuki) 

(Ohkoshi Katsuhiro) 


Director 

Hirochika Hirohiko 

Senior Researcher Duncan Alexander Vaughan 

Chief Researcher Ueda Tadamasa 

Plant Genome Research Unit Head Matsumoto Takashi 

Senior Researcher Kikuchi Shoshi 

Senior Researcher Komatsuda Takao 

Chief Researcher Wu Jianzhong 

Chief Researcher Ogawa Taiichi 

Chief Researcher Kawahigashi Hiroyuki 

Chief Researcher Mizuno Hiroshi 

Researcher 

Ono yoko 


(Izawa Takeshi) 

Bioinformatics Research Unit Head Ito Takeshi 

Chief Researcher Maeda Miki 

Researcher 

Numa Hisataka 

Researcher 

Tanaka Tsuyoshi 

Researcher 

Sakai Hiroaki 

Researcher 

Kawahara Yoshihiro 

Genome Resource Center Head Nagamura Yoshiaki 

Chief Researcher Baltazar Antonio 

Chief Researcher Miyao Akio 

Chief Researcher Koga Yasunori 

Researcher 

Sato Yutaka 


Nakayama Yasuji 

(Ichikawa Hiroaki) 

Genebank Director (Kawase Makoto) 

Senior Researcher Niino Takao 

Senior Researcher Nagayasu Kenichi 

Senior Researcher Minezawa Mitsuru 

Senior Researcher Sato Toyozo 

Senior Researcher Tomooka Norihiko 

Senior Researcher Aoki Takayuki 

Senior Researcher Sawada Hiroyuki 

Chief Researcher Nagai Toshiro 

Chief Researcher Takeya Masaru 

Chief Researcher Kosegawa Eiichi 

Chief Researcher Okuizumi Hisato 

Chief Researcher Tomioka Keisuke 

Chief Researcher Nishikawa Tomotaro 

Chief Researcher Fukui Kuniaki 

(Kaga Akito) 

(Tateishi Ken) 

Institute of Radiation Breeding Director (Nakagawa Hitoshi) 

Senior Researcher Nishimura Minoru 

Chief Researcher Muramatsu Noboru 

Chief Researcher Yamanouchi Hiroaki 

Chief Researcher Takyu Toshio 

Chief Researcher Shimizu Akemi 


Researcher 

Morita Ryohei 

Soybean Genome Research Team Head Harada Kyuya 

Chief Researcher Katayose Yuichi 

Chief Researcher Kaga Akito 

Researcher 

Watanabe Satoshi 


Director 


Researcher 

Meshi Tetsuo 

Yazaki Yoshiaki 

Mochizuki Atsuko 

Environmental Stress Research Unit Head Hayashi Makoto 

Senior Researcher Ishikawa Masaya 

Senior Researcher Ogawa Masafumi 

Chief Researcher Fukuda Atsunori 

(Sugimoto Kazuhiko) 

(Uga Yusaku) 

Photobiology and Photosynthesis Research Unit Head Miyao Mitsue 

Senior Researcher Ichikawa Hiroaki 

Senior Researcher Izawa Takeshi 

Chief Researcher Takeichi Tetsuo 

Chief Researcher Inagaki Noritoshi 

Chief Researcher Ishimaru Ken 

Chief Researcher Kiyota Seiichiro 

Chief Researcher Baba Akiko 

Chief Researcher Iwamoto Masao 

Chief Researcher Sentoku Naoki 

Researcher 

Ito Hironori 

Plant Disease Resistance Research Unit Head Takatsuji Hiroshi 

Senior Researcher Hayashi Nagao 

Chief Researcher Mori Masaki 

Chief Researcher Jiang Cian Je 

Chief Researcher Sugano Shoji 

Chief Researcher Yamazaki Muneo 

Chief Researcher Takahashi Akira 

Researcher 

Inoue Haruhiko 

Protein Research Unit Head Yamazaki Toshimasa 








Takase Kenji 

Momma Mitsuru 

Kajiwara Hideyuki 

Fujimoto Zui 

Suzuki Rintaro 

Wako Toshiyuki 

Plant-Microbe Interactions Research Unit Head Minami Eiichi 

Senior Researcher Ishikawa Masayuki 

Senior Researcher Nishizawa Yoko 

Senior Researcher Kato Etsuko 

Chief Researcher Ochiai Hirokazu 

Chief Researcher Mitsuhara Ichiro 

Chief Researcher Seo Shigemi 

Chief Researcher Umehara Yosuke 

Chief Researcher Nishimura Marie 

Chief Researcher Yoshikawa Manabu 

Chief Researcher Akimoto Chiharu 

Researcher 

Takeuchi Kasumi 

Researcher 

Otake Yuko 

Researcher 

Imaizumi Haruko 

Researcher 

Nakagawa Tomomi 

Plant Genetic Engineering Research Unit Head Toki Seiichi 

Chief Researcher Kishimoto Naoki 

Chief Researcher Habu Yoshiki 

Chief Researcher Kawagoe Yasushi 

Chief Researcher Miyahara Kenzo 

Chief Researcher Nakayama Shigeki 

Researcher 

Saika Hiroaki 

Researcher 

Endo Masaki 

(Tabei Yutaka) 

Division of Insect Science Director 

Director 

Kiuchi Makoto 

Insect Genome Research Unit Head Yamamoto Kimiko 

Senior Researcher Kadono Keiko 

Chief Researcher Yukuhiro Kenji 

Chief Researcher Hirokawa Masahiko 

Chief Researcher Tomita Shuichiro 





Yasukochi Yuji 

Komoto Natsuo 

Suetsugu Yoshitaka 

(Tatematsu Kenichiro) 

(Sezutsu Hideki) 

Invertebrate Gene Function Research Unit Head Shinoda Tetsuro 

Senior Researcher Myohara Maroko 

Senior Researcher Taniai Kiyoko 

Senior Researcher Shiotsuki Takahiro 

Chief Researcher Kotaki Toyomi 

Chief Researcher Tanaka Yoshiaki 

Chief Researcher Ichikawa Akio 

Chief Researcher Hatakeyama Masatsugu 

Chief Researcher Shimoda Masami 

Chief Researcher Kamimura Manabu 

Chief Researcher Nakao Hajime 

Chief Researcher Shimura Sachiko 

Researcher 

Kihara Mami 

Researcher 

Kozaki Toshinori 

Anhydrobiosis Research Unit Head Okuda Takashi 

Chief Researcher Kikawada Takahiro 

Researcher 

Cornette Richard Marcel Jacques 

Innate Immunity Research Unit Head Ishibashi Jun 

Senior Researcher Kato Yusuke 

Chief Researcher Tanaka Hiromitsu 

Insect Interaction Research Unit Head Noda Takashi 

Senior Researcher Wakamura Sadao 

Senior Researcher Hattori Makoto 

Senior Researcher Tanaka Seiji 

Senior Researcher Inouchi Jun 

Senior Researcher Muraji Masahiko 

Chief Researcher Nakamura Masatoshi 

Chief Researcher Konno Kotaro 

Chief Researcher Hirayama Chikara 

Chief Researcher Hinomoto Norihide 

Chief Researcher Yasui Hiroe 

Chief Researcher Tateishi Ken 





Researcher 

Hasegawa Tsuyoshi 

Tamura Yasumori 

Maeda Taro 

Tsujii Nao 

Insect-Microbe Research Unit Head Noda Hiroaki 

Senior Researcher Miyamoto Kazuhisa 

Senior Researcher Hara Wajiro 

Senior Researcher Mitsuhashi Wataru 

Chief Researcher Watanabe Hirofumi 

Chief Researcher Nakashima Nobuhiko 

Chief Researcher Arakawa Toru 

Chief Researcher Wada Sanae 

Chief Researcher Murakami Ritsuko 

Researcher 

Matsumoto Yukiko 

Researcher 

Kobayashi Tetsuya 

Researcher 

Kageyama Daisuke 

Silk-Materials Research Unit Head Tamada Yasushi 

Senior Researcher Goto Yoko 

Chief Researcher Tomiyama Masamitsu 

Chief Researcher Toshima Yoshiyuki 

Chief Researcher Miyazawa Mitsuhiro 

Chief Researcher Kameda Tsunenori 

Chief Researcher Hata Tamako 

Chief Researcher Kuwana Yoshihiko 

Chief Researcher Takasu Yoko 

Chief Researcher Kojima Katsura 

Chief Researcher Teramoto Hidetoshi 

Silk Technology Unit Head Takabayashi Chiyuki 

Chief Researcher Nakajima Kenichi 

Chief Researcher Mase Keisuke 

Chief Researcher Okada Eiji 


Kinoshita Haruo 


Director 

Kurihara Mitsunori 

Animal Genome Research Unit Head Awata Takashi 


Yasue Hiroshi 









Researcher 

Hamajima Noriyuki 

Kojima Misaki 

Hayashi Takeshi 

Mikawa Satoshi 

Harumi Takashi 

Watanabe Satoshi 

Uenishi Hirohide 

Ogawa Tomoko 

Reproductive Biology Research Unit Head Tokunaga Tomoyuki 

Senior Researcher Kaneko Hiroyuki 

Senior Researcher Noguchi Junko 

Senior Researcher Kikuchi Kazuhiro 

Chief Researcher Goto Hideo 

Chief Researcher Ito Yoshiyasu 

Chief Researcher Takahashi Toru 

Chief Researcher Suto Junichi 

Chief Researcher Hurusawa Tadashi 

Chief Researcher Hosoe Misa 

Chief Researcher Ohkoshi Katsuhiro 

Chief Researcher Sakumoto Ryosuke 

Chief Researcher Miyashita Norikazu 

Researcher 

Hayashi Kengo 

Neurobiology Research Unit Head Okamura Hiroaki 

Chief Researcher Saito Toshiyuki 

Chief Researcher Yayo Kenichi 

Chief Researcher Kasuya Etsuko 

Researcher 

Wakabayashi Yoshihiro 

General affairs 

Management Director-General 

Management Director 

Shimada Hiroaki 

( ) additional position 

Ota Hideo 

General Affairs Section Head Hoshi Motoo 

Accounting Section Head Oonuma Yoshinori 

Management and Supply Section Head Yokozeki Eiichi 

Audit and Compliance Section Head Saito Ryoichi 


Members of NIAS Evaluation 

Committee 

(as of March 31,2010) 

Kono Tomohiro 

Kubo Takeo 

Kobayashi Michihiro 

Nishimura Ikuko 

Shinozaki Kazuo 

Endo Takashi 

Gojobori Takashi 

Senoh Kenichiro 

Department of Bioscience, Tokyo University of Agriculture 

Graduate School of Science, The University of Tokyo 

Graduate School of Bioagricultural Sciences, Nagoya University 

Graduate School of Science, Kyoto University 

RIKEN, Plant Science Center 

Graduate School of Agriculture, Kyoto University 

National Institute of Genetics 

The Industry-Academia Collaboration Initiative 

(Non profit Organization) 


FINANCIAL OVERVIEW 

Fiscal Year 2009 (April 2009 – March 2010) 

millions of yen 

TOTAL BUDGET 12,319 

OPERATING COSTS 4,611 

Personnel cost 3,967 

Personnel (381) 

President (1) 

Vice President (2) 

Auditor (2) 

Administrators(119) 

General administrators 

Field management and transportations 

Researchers (262) 

(Number of persons is shown in ( ) as of Mar 31, 2010) 

Administrative cost 423 

Facilities improvement expense 221 

RESEARCH PROMOTION COSTS 7,708 

Research Grant from MAFF 2,820 

Entrusted Research Expenses from MAFF 3,957 

Entrusted Research Expenses from MEXT 278 

Entrusted Research Expenses from others 653 

Entrusted Research Expenses 

from MEXT 

278 (2%) 

Entrusted Research Expenses from others 

653 (5%) 

Personnel 

3,967 (32 %) 

Entrusted Research Expenses 

from MAFF 

3,957 (32%) 

Research Grant from MAFF 

2,820 (23%) 

Administrative costs 

423 (3%) 

Facilities Improvement 

Expense 

221 (2%) 

MAFF: Ministry of Agriculture, Forestry and Fishries, 

MEXT: Ministry of Education, Culture, Sports, Sciences and Technology 


Location: How to access to 

our National Institute of Agrobiological Sciences (NIAS) 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Transportation 

 

 

 

From Tokyo: Take the JR Joban Line from Ueno Station and get off at Ushiku Station. From the West Exit, take the 

Kantetsu Bus bound for Yatabe-shako, Tsukuba-Daigaku-Chuo, or Seibutsu-Ken-Ohwashi-Campus and get off at 

Norin-Danchi-Chuo. 

: Take the Tsukuba Express Line from Akihabara. Express train to Tsukuba (Station No. 20) leaves Akihabara 

every 15 minutes. Only taxi is available from Tsukuba Station to NIAS. Limited Express train leaves every 30 min and 

stops at Midorino (Station No. 17). Bus and taxi are available from Midorino Station to NIAS. 

From Tokyo International Airport (Narita): Take the Kanto-Tetsudo Bus bound for Tsuchiura via Tsukuba Center, and get off at 

Tsukuba Center and take a taxi. 

Headquarters Area 

2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602, Japan 

TEL: +81-29-838-7406 FAX: +81-29-838-7408 

http://www.nias.affrc.go.jp/ 

Ohwashi Area 

1-2 Ohwashi, Tsukuba, Ibaraki, 305-8634, Japan 

TEL: +81-29-838-6026 

NIAS Institutions outside Tsukuba 

●Hitachiohmiya (Inst. Radiation Breeding) 

2425 Kamimurata, Hitachiohmiya, Ibaraki, 319-2293, Japan 

TEL: +81-295-52-1138 

●Matsumoto (Sericultural Science Lab.) 

1-10-1 Agata, Matsumoto, Nagano, 390-0812, Japan 

TEL: +81-263-32-0549 

● Okaya (New Silk Materials Lab.) 

1-4-8 Gouda, Okaya, Nagano, 394-0021, Japan 

TEL: +81-266-22-3664 

● Hokuto (Insect Genetics Lab.) 

6585 Kobuchizawa, Hokuto, Yamanashi, 408-0044, Japan 

TEL: +81-551-36-2046

Annual Report 2010

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