Annual report 2009

2009

Annual Report 2009 

(Apr. 2008 ~ Mar. 2009) 

(In the fiscal year 2008)

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, 

rape 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 

spectroscopy 

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 177 

Total number of employees 379 

Contract workers (Part time) 

493 

including 93 post-doc researchers 

Reemployment staff 6

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 

Resource Center 

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 2008 

◦Production of high quality silks having different fluorescent colors by transgenic 

silkworms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 

Toshiki TAMURA 1 , Tetsuya IIZUKA 1 , Hideki SEZUTSU 1 , Ken-ichiro TATEMATSU 1 , 

Isao KOBAYASHI 1 , Masayuki YONEMURA 1 , Keiro UCHINO 1 , Katsura KOJIMA 1 , 

Hiroaki MACHII 1 , Chihiro TAKABAYASHI 1 , Katsushige YAMADA 2 , Hiroyuki 

KURIHARA 2 , Tetsuro ASAKURA 3 , Yasumoto NAKAZAWA 3 , Junji YAMAGUCHI 4 , 

Nobuo KUWABARA 4 , Takashi NAKAMURA 5 , Kei YOSHII 5 , Atsushi MIYAWAKI 6 , 

Satoshi KARASAWA 7 , Hatsumi KOBAYASHI 7 ; 1 National institute of Agrobiological 

Sciences, 2 Toray Industries, Inc. 3 Tokyo University of Agriculture and Technology, 

4 

Gunma Sericultural Technology Center, 5 Textile Research Center of Gunma, 6 Riken, 

7 

Amalgaam, Ltd. 

◦Development of collagen vitrigel useful for tissue regeneration .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 

Toshiaki TAKEZAWA; Transgenic Animal Research Center 

◦Genome-wide analysis of Magnaporthe grisea gene structures with transcription 

evidence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 

Hisataka NUMA 1 , Marie NISHIMURA 2 , Tsuyoshi TANAKA 1 , Takashi MATSUMOTO 2 , 

Yoshiaki NAGAMURA 3 , Takeshi ITOH 1 ; 1 Bioinformatics Research Unit, 2 Plant-Microbe 

Interactions Research Unit, 3 Plant Genome Research Unit, 4 Genome Resource Center 

◦Functional analysis of Cyclops, a common symbiosis gene for root nodule and 

arbuscular mycorrhiza symbioses .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 

Koji YANO 1 , Mari BANBA 1 , Haruko IMAIZUMI-ANRAKU 1 , Makoto HAYASHI 2 ; 

1 

Plant-Microbe Interactions Research Unit, 2 Environmental Stress Research Unit 

◦RNA-directed DNA methylation induces transcriptional activation in plants . . . . . . . . . . . . . . . . 10 

Hiroshi TAKATSUJI; Plant Disease Resistance Research Unit 

◦Superior breadmaking quality of the rice flour of PDI mutant .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 

Yayoi ONDA, Yasushi KAWAGOE; Genetic Engineering Research Unit 

◦The genome of a lepidopteran model insect, the silkworm Bombyx mori . . . . . . . . . . . . . . . . . . . . . . . 13 

Kimiko YAMAMOTO, Keiko KADONO-OKUDA, Yoshitaka SUETSUGU, Kazuei MITA; 


◦Krüppel-homolog 1 (Kr-h1) mediates anti-metamorphic action of juvenile hormone . . . 15 

Chieka MINAKUCHI, Toshiki NAMIKI, Michiyo YOSHIYAMA, Tetsuro SHINODA; Insect 

Gene Function Research Unit 

◦Generation of Piglets from Cryopreserved Porcine In Vitro Matured and Fertilized 

Oocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 

Kazuhiro KIKUCHI 1 , Tamás SOMFAI 1 , Michiko NAKAI 1 , Naomi KASHIWAZAKI 2 , 

Takashi NAGAI 3 ; 1 Reproductive Biology Research Unit, 2 Azabu University, 3 National 

Institute of Livestock and Grassland Science

Research Activities 

Research Center 

・QTL Genomics Research Center . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 

・Transgenic Crop Research and Development Center . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 

・Transgenic Silkworm Research Center . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 

・Transgenic Animal Research Center . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 


・Plant Genome Research Unit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 

・Bioinformatics Research Unit .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 

・Genebank . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 

・Institute of Radiation Breeding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 

・Soybean Geome Research Team .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 


・Environmental Stress Research Unit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 

・Photobiology and Photosynthesis Research Unit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 

・Disease Resistance Research Unit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 

・Protein Research Unit .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66 

・Plant-Microbe Interactions Research Unit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 

・Plant Genetic Engineering Research Unit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72 


・Insect Genome Research Unit .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76 

・Invertebrate Gene Function Research Unit .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 

・Anhydrobiosis Research Unit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 

・Innate Immunity Research Unit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84 

・Insect Interaction Research Unit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87 

・Insect-Microbe Research Unit .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 

・Silk-Materials Research Unit .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94 

・Silk-Technology Unit .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98 


・Animal Genome Research Unit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 

・Reproductive Biology Research Unit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 

・Neurobiology Research Unit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114 

List of Publication 

・Original papers 

1) English . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116 

2) Japanese with English Summary .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140 

・Reviews and Monograph (In English) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142 

List of Organizations Exchanged MOU . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146 

Executive Members and Research Staff Members . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147 

・Members of NIAS Evaluation Committee . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156 

・Financial overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157 

・Location and How to access .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159

Topics of Research in This Year 

Production of high quality silks having different fluorescent 

colors by transgenic silkworms 

Toshiki TAMURA 1 , Tetsuya IIZUKA 1 , Hideki SEZUTSU 1 , Ken-ichiro TATEMATSU 1 , Isao 

KOBAYASHI 1 , Masayuki YONEMURA 1 , Keiro UCHINO 1 , Katsura KOJIMA 1 , Hiroaki 

MACHII 1 , Chihiro TAKABAYASHI 1 , Katsushige YAMADA 2 , Hiroyuki KURIHARA 2 , Tetsuro 

ASAKURA 3 , Yasumoto NAKAZAWA 3 , Junji YAMAGUCHI 4 , Nobuo KUWABARA 4 , Takashi 

NAKAMURA 5 , Kei YOSHII 5 , Atsushi MIYAWAKI 6 , Satoshi KARASAWA 7 , Hatsumi 

KOBAYASHI 7 

1 

National institute of Agrobiological Sciences, 2 Toray Industries, Inc. 3 Tokyo University of 

Agriculture and Technology, 4 Gunma Sericultural Technology Center, 5 Textile Research 

Center of Gunma, 6 Riken, 7 Amalgaam, Ltd. 

Sericulture in Japan has declined over the 

past 20 years with reductions in is successively 

reduce the amounts of annual cocoon production 

and the numbers of the farmers rearing the 

silkworms during 20 years. Thise reduction has 

been is caused by the low prices of the cocoons 

and raw silk. To promote the sericulture 

Iin Japan, it was thought to be necessary to 

develop new silks and cocoons that couldcan sell 

for much higher prices. than those of the ordinal 

one. In addition, the new silks and the cocoons 

would be distinguished can be discriminated 

from the cocoons and silks available from made 

by the foreign countries such as, like China, 

Brazil, and India etc. 

T h e R e c e n t l y , N a t i o n a l I n s t i t u t e o f 

Agrobiological Sciences have recently developed 

been established the technology for making a 

transgenic silkworms and for the systems for 

the expressing of the transgenic. By using this 

technology, the artificially synthesized gene can 

be introduced into the silkworm and expressed 

in the silk gland. We applied the method for the 

production of high quality of recombinant silks, 

and found that the characteristics of the silk can 

be modified by introducing introduction of an 

artificial silk gene. 

In this topic, we report the production of 

large amounts of recombinant silk and attempts 

to produce higher quality fabrics using 

recombinant silks. We first generated transgenic 

silkworms that produce silks having interesting 

green, red or orange fluorescence characteristics 

(Fig 1). To increase the production of 

fluorescent silk, these strains were back-crossed 

with the Japanese and Chinese parent races 

with selection in the successive generations. 

We generated the silkworms produced the 

high quality of the recombinant silks having 

different characters. The most interesting silk 

possesses the fluorescence of the green, red and 

orange color. We also developed a transgenic 

strain producing The very thin and strong silk 

by direct introduction of the artificial gene for 

thin silk into the thin silk race “Hachuring”. We 

then reared a large number of the silkworms 

and harvested a large amount of cocoons (Fig 1). 

This is also turned out to be produced by 

the transgenic silkworm. The Silk with a celladhesive 

character is also was produced by 

the same method. In this topic we report the 

production of large amounts of recombinant silk 

and attempted to produce the higher quality of 

fabrics using the recombinant silks. 

Annual Report 2009

We first generated the transgenic silkworms 

that produce the silks having the green, red 

and orange fluorescence or very thin fiber. The 

strains producing larger amount of the silks with 

the fluorescence were bred by back cross with 

Japanese and Chinese parent races and by the 

selection in the successive generations. The silkworm 

strain for the thin silk was performed by 

the direct introduction of the artificial gene for 

making the thin silk into the thin silk race. Then, 

we reared a large numbers of the silkworms and 

harvested large amounts of cocoons (Fig 1). 

It was known that the fluorescence color of 

the silk was lost is loosed when the silk was 

reeled by the usual hot steam and water an 

ordinal method. We also investigated different 

the reeling methods using lower temperature so 

that of the cocoon without loosing the modified 

characters could be retained and as a result, we 

found that the drying and boiling of the cocoons 

at a lower temperature and the use of, a vacuum 

treatment and use of specific reagents were are 

required applicable for the reeling. As a result 

of this innovative, we innovated the technology, 

to reel the recombinant silk could be reeled and 

successfully to produce large amounts of silks 

retaining remained the fluorescence color (Fig 1) 

as well as and the character of very thin fiber 

characteristic. Using these silks, we performed 

the production of the textile fabrics for (knit 

dresses, jackets and shawls) as shown in Fig 2 

and the interior accessories such as (lampshades 

and tapestries) as shown in Fig 2. 

From these studies, results, we concluded that 

the production of high quality silks is possible 

using the newly developed to use the technology 

developed and that the recombinant silks 

produced by the transgenic silkworms may open 

the new fieled in Japanese sericulture and contribute 

to the increase in of the silk production. 

White light Fluorescence White light Fluorescence 

Fig 1. The cocoons and silk made from the transgenic silkworms introduced green (upper) or red (lower 

fluorecence gene.) 

White light 

Fluorescence 

Fig 2. Woman’s wears made by the recombinant silk with the fluorescent colors. 


Development of collagen vitrigel useful for tissue 

regeneration 

Toshiaki TAKEZAWA 


The fibril density of traditional collagen gel 

(Fig 3) is quite low in comparison to that of connective 

tissue in vivo. Therefore, we aimed to 

develop a novel collagen scaffold with high fibril 

density. In addition, it was known that opaque 

egg white could be converted into a thin, 

transparent, rigid glass-like material by boiling 

and evaporating the moisture — a phenomenon 

known as vitrification of denatured proteins. 

We applied this vitrification technology to a 

traditional collagen gel (Fig 3). Collagen vitrigels 

with high fibril density were successfully prepared 

by a three-step process: a gelation step 

in which a type-I collagen sol forms an opaque 

and soft gel by incubation at 37C; a vitrification 

step in which the gel becomes a rigid material 

after sufficient drying; and a rehydration step 

with supplemental moisture that converts the 

vitrified material into a thin and transparent gel 

membrane with enhanced gel strength. In this 

report, we define a vitrigel as a gel in a stable 

state produced by rehydration following vitrification 

of a traditional hydrogel. The collagen 

vitrigel obtained possesses excellent protein 

permeability (Fig 3). 

Fig 3. Preparation methods of a traditional collagen gel and a novel collagen vitrigel and comparison 

of their properties. 


Furthermore, we also prepared frameworkembedded 

collagen vitrigel scaffolds by inserting 

a nylon membrane ring into the collagen sol 

prior to the gelation step (Fig 4). Anchoragedependent 

cells can be cultured on both 

surfaces of the scaffold by the manipulation of 

two-dimensional cultures, resulting in the reconstruction 

of a three-dimensional organoid (Fig 4). 

In studies of a crosstalk model between PC-12 

pheochromocytoma cells and L929 fibroblasts, 

we found that nerve growth factor secreted 

from L929 cells passed through the collagen vitrigel 

scaffold and induced the neurite outgrowth 

of PC-12 cells by its paracrine effect (Fig 5). 

We also found that a collagen vitrigel scaffold 

containing vascular endothelial growth factor 

(VEGF) showed a sustained release of VEGF in 

vitro and that its subcutaneous transplantation 

into a rat resulted in remarkable angiogenesis 

(Fig 6). These data suggest that the collagen 

vitrigel scaffold is useful for paracrine assays 

in vitro and drug delivery systems in vivo. 

Therefore, we expect that collagen vitrigels 

could contribute to studies in regenerative 

medicine and drug development and could serve 

as an alternative to animal experiments. 

Fig 4. Advantages of the culture technology utilizing a support material-embedded collagen vitrigel 

membrane scaffold. 


Fig 5. Paracrine effect between different types of cells vua a collagen vitrigel membrane scafford. 

Fig 6. Drug delivery system utilizing a collagen vitrigel membrane scafold. 


Genome-wide analysis of Magnaporthe grisea gene 

structures with transcription evidence 

Hisataka NUMA 1 , Marie NISHIMURA 2 , Tsuyoshi TANAKA 1 , Takashi MATSUMOTO 2 , 

Yoshiaki NAGAMURA 3 , Takeshi ITOH 1 

1 

Bioinformatics Research Unit, 2 Plant-Microbe Interactions Research Unit, 3 Plant 

Genome Research Unit, 4 Genome Resource Center 

Rice blast disease caused by Magnaporthe 

grisea is one of the most destructive diseases 

affecting rice production worldwide. In 2005, 

the draft genome sequence of M. grisea was 

released, and currently over 50,000 expressed 

sequence tags (ESTs) are available through the 

international sequence databases. However, the 

availability of 5’-end sequences of mRNAs from 

filamentous fungi is currently limited. For this 

reason, construction of a library enriched with 

long cDNAs provides useful information for 

accurate identification of M. grisea genes. 

In this study, we obtained both 5’- and 3’-end 

sequences of 16,368 cDNA clones, but could only 

sequence 5’-ends of 1,627 clones and only 3’-ends 

of 826 clones. As a result we produced 35,189 

ESTs from 18,821 clones in total. We aligned the 

ESTs to the genome supercontigs, and 14,791 

mate pairs (29,582 ESTs) were mapped within 

the same contigs. Of the 14,791 mate pairs, 2,704 

pairs could be concatenated to form a possible 

complete gene structure, whereas 12,087 pairs 

remained unconnected between 5’- and 3’-end 

sequences on the genome. To fill the gaps between 

mate pairs, we used our ESTs, previously 

published M. grisea ESTs and mRNAs from the 

public databases, and ab initio gene predictions. 

The concatenation of the ESTs, mRNAs, and 

predictions resulted in generation of 4,480 assemblies 

that include 12,499 mate pairs without gaps. 

We classified the assemblies into two groups; 

assemblies with complete coding sequences 

(CDSs), and assemblies with short hypothetical 

open reading frames (ORFs). The assemblies 

with complete CDSs were defined as those 

>100 amino acids in length, with coding regions 

containing both initiation and stop codons. All 

the other assemblies were defined as short ORF 

assemblies. As a result, 3,891 assemblies with 

complete CDSs and 589 assemblies with short 

ORFs were obtained (Table 1). Translated complete 

CDSs were subjected to BLASTP searches 

against UniProtKB and fungi proteins in RefSeq. 

Likewise, the nucleotides of the assemblies 

with short ORFs were subjected to BLASTX 

searches. We discarded M. grisea proteins from 

the BLAST hits because almost all of them were 

proteins predicted from the genome. As a result, 

3,432 (88.2%) of the complete CDSs and 120 (20.4%) 

of the short ORFs showed significant sequence 

similarity. In addition, we used InterProScan to 

find functional domains. 

An important point is the completeness of 

our cDNA clones. To validate that N-terminals 

of the clones were not truncated, we compared 

the M. grisea CDSs with non-fragment proteins 

in SwissProt of UniProtKB. We performed 

BLASTP searches with cutoff identity of 50%, 

and compared the N-terminals with those of 

the top hits. As a result, 1,125 of 3,891 complete 

CDSs showed similarity to database proteins, 

and the N-terminals of 981 (87.2%) CDSs were 

the same as or longer than those of database 

proteins. The results suggest that our cDNA 

clones substantially covered entire proteincoding 

regions. The clones containing complete 

protein sequences will be important resources 

for future experimental studies. 

All data of our EST sequences and predicted 

CDSs were integrated into a database, and are 

accessible through a widely used genome viewer 

at http://mg.dna.affrc.go.jp/ (Fig 7). Users can 

search the database with keywords about gene 

functions, domain names, Gene Ontology terms, 

and so on. Sequences entered by users can be 

aligned to our ESTs and assemblies through the 

BLAST search web service. 


Fig 7. M. grisea EST assemblies visualized on a web-based genome 

Table 1. Characteristics of assemblies with complete CDSs and assemblies with short ORFs. 

Complete CDS 

Short ORF 

Total number 3,891 589 

Average locus length (bp) 2,116 848 

Average transcript length (bp) 1,903 763 

Average number of exons 2.9 1.6 

Average exon length (bp) 651 475 

Average intron length (bp) 111 139 

GC content (%) 

Exon 54.0 49.6 

Intron 47.2 48.9 

Similarity to known proteins 3,432 120 

Including InterPro domains 2,827 160 

Note. - Untranslated regions are included in loci and exons. 


Functional analysis of Cyclops, a common symbiosis 

gene for root nodule and arbuscular mycorrhiza 

symbioses 

Koji YANO 1 , Mari BANBA 1 , Haruko IMAIZUMI-ANRAKU 1 , Makoto HAYASHI 2 

1 

Plant-Microbe Interactions Research Unit, 2 Environmental Stress Research Unit 

Plants require nitrogen, phosphorus, and 

potassium as major nutrients. Legume plants, e.g. 

pea, soybean, and alfalfa, establish intracellular 

symbiosis with soil bacteria, collectively called 

rhizobia, in root nodules to utilize atmospheric 

nitrogen. In addition, a majority of land plants 

establish intracellular symbiosis with arbuscular 

mycorrhizal fungi, which transport water and 

nutrients, especially phosphorus, from the soil 

beyond the area where plant roots can extend. 

Because nitrogen and phosphorus are two 

major nutrients that farmers have to supply 

for crop cultivation and the production of 

these fertilizers will be more and more difficult 

due to limited reserves, biological control and 

utilization of these symbioses may be factors 

contributing to sustainable agriculture in the 

future. To understand the mechanisms of plant 

intracellular symbiosis, we have been isolating 

and characterizing plant symbiotic genes that 

are necessary for successful symbiosis, using 

the model legume, Lotus japonicus. 

It has been pointed out that some of the mutants 

that are deficient in root nodule symbiosis 

also fail to establish symbiosis with arbuscular 

mycorrhiza. The genes responsible for both 

symbioses are called common symbiosis genes. 

There are seven common symbiosis genes predicted 

in L. japonicus, and Cyclops was the final 

gene remaining to be identified. Since common 

symbiosis genes play roles in fundamental aspects 

in this mutual relationship, we attempted 

to clone and characterize Cyclops. 

The root nodule phenotype of cyclops mutants 

is unique: while all other common symbiosis 

mutants show root hair deformation without 

curling, cyclops entraps bacteria inside the 

curled root hairs but nevertheless does not 

initiate infection threads (Fig 8). We identified 

the Cyclops gene with map-based cloning, and 

found that CYCLOPS has two nuclear localization 

signals and a coiled-coil domain, which is 

important for regulation of gene expression. We 

tested whether CYCLOPS is localized in the cell 

nucleus, with GFP fusion, and found CYCLOPS 

is indeed in the nucleus in planta. Another 

common symbiosis gene product, CCaMK is also 

localized in the nucleus. Therefore, we tested 

whether CYCLOPS interacts with CCaMK. With 

yeast two hybrid and BiFC assays, we could 

show the interaction. Moreover, CCaMK phosphorylates 

CYCLOPS as a substrate in vitro. We 

conclude that CYCLOPS functions with CCaMK, 

in common symbiosis signal transduction, by 

interaction and phosphorylation. Since both of 

the proteins are localized in the nucleus, the 

complex is important for regulation of symbiotic 

gene expression in order to establish successful 

infection (Fig 9). 


Fig 8. Root nodule infection phenotype of wild type Gifu (left) and a common symbiotic mutant 

cyclops (right). 

In the wild type root hair, bacteria (green) are incorporated into the infection thread to be 

delivered to the nodule primordium differentiated from cortex. In contrast, bacteria fail to invade 

host plant cells due to the lack of infection thread initiation in cyclops. Photographs were taken 

with confocal microscopy. The green fluorescence of bacteria is GFP expression. Plant cells were 

visualized with propidium iodide. 

Fig 9. Schematic presentation of CYCLOPS function. 

Upon recognition of symbiotic signals from symbionts (RN: root nodule, AM: arbuscularmycorrhiza), 

calcium signal is generated by a part of common symbiosis genes. The signal is 

then recognized by CCaMK and translated into phosphorylation signal. CCaMK interacts with 

and phosphorylates CYCLOPS to mediate symbiotic signals downstream, resulting in expression 

of symbiotic genes necessary for successful infection. 


RNA-directed DNA methylation induces transcriptional 

activation in plants 

Hiroshi TAKATSUJI 


A class-C floral homeotic gene of Petunia, 

pMADS3, is specifically expressed in the stamen 

and carpel of developing flowers. We have previously 

reported the ect-pMADS3 phenomenon 

in which endogenous pMADS3 is ectopically 

expressed in petal and sepal as a consequence of 

the introduction of a pMADS3 genomic sequence 

(intron 2) that contains pMADS3 transcriptional 

regulatory sequences. This phenomenon is 

similar to the transcriptional gene silencing in 

that they both are triggered by the introduction 

of transcriptional regulatory sequences; 

however, the former is unique in that the gene 

expression was “upregulated”. We investigated 

the molecular mechanism underlying the ectpMADS3 

phenomenon because it could lead to a 

new strategy to control gene expression. 

We reasoned that RNA-directed DNA 

methylation (RdDM) could be involved in the 

ect-pMADS3 phenomenon, so we examined 

whether pMADS3 ectopic expression could 

be reproduced when RdDM was induced in 

pMADS3 intron 2, which encompasses transcriptional 

regulatory sequence of pMADS3 for 

its floral-organ specific expression. To this end, 

we expressed in petunia the inverted-repeat 

(IR) sequences (RNA trigger) for sub-sequences 

of pMADS3 intron 2 and found that those for 

a particular intron-2 region induced pMADS3 

ectopic expression, resulting in petal-to-stamen 

homeotic conversion. We sequentially narrowed 

the region for IR expression, consequently specifying 

~100-bp region that is responsible for 

pMADS3 ectopic expression. DNA-methylation 

analysis revealed methylation at two CGs in a 

putative negative cis-element for pMADS3 floralorgan-specific 

transcription, which are highly 

conserved among various plant species. These 

results indicate that derepression of pMADS3 

transcription due to RdDM of a negative ciselement 

causes pMADS3 ectopic expression. 

The CG methylation was maintained over generations, 

along with pMADS3 ectopic expression, 

even in the absence of RNA triggers, suggesting 

that this mechanism could lead to the generation 

of a transcriptionally active epiallele, thereby 

contributing to plant evolution. Our results also 

reveal a putative negative cis-element for organspecific 

transcriptional regulation of class-C 

floral homeotic genes, which could be difficult to 

identify by other approaches. Furthermore, the 

strategy of expressing IR sequences to induce 

RdDM in particular cis-elements can be applied 

as a new technique to modify gene expression 

in various plant species without the permanent 

addition of transgenes. 

10 Annual Report 2009

Superior breadmaking quality of the rice flour of PDI 

(protein disulfide isomerase) mutant 

Yayoi ONDA, Yasushi KAWAGOE 

Genetic Engineering Research Unit 

Rice consumption has gradually declined to 

about 60kg per capita from more than 100kg 

40 years ago. In recent years, rice flour has 

received attention as an alternative to wheat 

flour owing to technological innovations for 

making fine flour from rice. However, there has 

been little effort aimed at breeding cultivars 

with superior milling or end-use qualities. Seed 

protein is a major determinant of end-use properties 

for processing in the food industries. The 

esp2 mutant [MNU-treated knockout mutant 

of a protein disulfide isomerase (PDIL1-1)], 

accumulates a large amount of proglutelins, the 

precursor of the major seed storage protein 

glutelin. PDIL1-1 is abundantly expressed in 

the developing endosperm and is involved in 

both oxidative protein folding and sorting of 

glutelins. Rice endosperm develops two types 

of protein bodies (PB-I containing prolamins 

and PB-II containing glutelins), whereas esp2 

forms small particles containing both prolamins 

and proglutelins (Fig 10). In a recent study, 

we demonstrated that the proglutelins in esp2 

accumulate as large protein complexes through 

inter-molecular disulfide bonds (Onda et al. 

2009). In addition, some starch granules purified 

from esp2 were much larger than those of wildtype 

(Fig 11). 

Having identified unique phenotypes of the 

esp2 seeds, we compared breadmaking quality 

of the rice flour of wild type and esp2. We 

consistently recognized three superior qualities 

in the esp2 flour: the first quality is that the 

esp2 flour was easier to knead because it was 

not as sticky as that of wild-type; the second is 

an improved extensibility of the dough during 

fermentation; and the third quality is higher 

plasticity of the dough. Baking tests showed that 

the bread of esp2 resulted in a larger volume 

than the control. The bread also was less prone 

to collapse during the cooling time after baking 

(Fig 12). We speculate that storage proteins of 

esp2 form gluten-like protein complexes in the 

ER, which probably improves the extensibility 

and plasticity of the dough. We plan to evaluate 

further the characteristics of esp2 flour on a 

commercial scale. 

Reference 

Onda Y, Kumamaru T, Kawagoe Y (2009) ER 

membrane-localized oxidoreductase Ero1 is 

required for disulfide bond formation in the 

rice endosperm Proc Natl Acad Sci USA 106: 

14156–14161 

Annual Report 2009 11

Fig 10. The esp2 endosperm develops small particles containing storage proteins. 

The wild-type endosperm develops two types of protein bodies: PB-I contains prolamins and 

PB-II stores glutelins and γ-globulin. In contrast, the esp2 endosperm develops small particles. 

Fig 11. Starch granules purified from wild-type and esp2. 

Wild type rice synthesizes compound granules consisting of up to several dozens of sharpedged 

granules. In contrast, some, but not all, starch granules purified from esp2 are much larger 

than those of wild type (arrows). 

Fig 12. Superior breadmaking quality of the esp2 flour. 

Rice flour (70%) of wild type or esp2 were mixed with wheat gluten (30%), and compared for 

breadmaking quality. The bread made from wild type flour (left) showed a prominent hollow 

region during the cooling time after baking, whereas the esp2 bread (right) did not collapse, 

indicating that the esp2 dough has a high plasticity. 


The genome of a lepidopteran model insect, the silkworm 

Bombyx mori 

Kimiko YAMAMOTO, Keiko KADONO-OKUDA, Yoshitaka SUETSUGU, Kazuei MITA 


Bombyx mori, the domesticated silkworm, is 

a major insect model for research, and the first 

lepidopteran for which draft genome sequences 

became available in 2004 by two whole genome 

shotgun sequencing projects of Japan and China. 

These independent data sets, however, were 

insufficient for building long scaffolds due to low 

sequencing coverage and/or lack of fosmid or 

BAC libraries. 

We have now merged and assembled the two 

datasets from whole-genome shotgun sequencing 

together with newly obtained fosmid- and 

BAC-end sequences (Fig 13). The remarkably 

improved new assembly is presented here. The 

8.5-fold sequence coverage of an estimated 432 

Mb genome was assembled into scaffolds with 

an N50 size of ~3.7 Mb; the largest scaffold was 

14.5 million base pairs (Table 2). With help of 

a high density SNP linkage map, we anchored 

87% of the scaffold sequences to all 28 chromosomes. 

A particular feature was the high repetitive 

sequence content estimated to be 43.6% and 

that consisted mainly of transposable elements. 

We predicted 14,623 gene models based on a 

GLEAN-based algorithm, a more accurate pre- 

diction than the previous gene models for this 

species. Over three thousand silkworm genes 

have no homologs in other insect or vertebrate 

genomes. 

Some insights into gene evolution and into 

characteristic biological processes are revealed 

by the fine genomic sequence. The massive 

silk production correlates with the existence of 

specific tRNA clusters, and of several sericin 

genes assembled in a cluster. The silkworm’s 

adaptation to feeding on mulberry leaves, which 

contain toxic alkaloids, is likely linked to the 

presence of new-type sucrase genes, apparently 

acquired from bacteria. The silkworm genome 

also revealed the cascade of genes involved in 

the juvenile hormone biosynthesis pathway, and 

a large number of cuticular protein genes. 

For efficient usage of genome information 

available for functional studies, genomic 

sequences, genetic maps and EST data were 

compiled into an integrated silkworm genome 

database KAIKObase, which is now public at 

the HP of NIAS at the URL http://sgp.dna.affrc. 

go.jp. 


Silkgland cells of male silkworm inbred strain p50T 

Fragmentation 

Fragments of 2kb and 7kb 

Both ends of 4 M 

fragments were sequenced 

Total 8 M reads 

Genomic DNA (Genome size: 4.75 Mb) 

Construct sequence contigs by assembling program, 

RAMEN ASSEMBLER 

Connected sequences 

(Contigs) by overlapping 

N50 contig = 15,500 b 

End sequences of bridge clones 

BAC (insert size: 150-200 kb): 150 K reads 

Fosmid (insert size: 40 kb): 300 K reads 

Connect contigs to 

scaffolds with bridge 

clones 

Scaffolds (connected contigs) 

N50 Scaffold = 3.7 Mb; Max scaffold = 14.5 Mb 

Fig 13. Overview of whole-genome shotgun sequencing by integration of Japan-China WGS data 

Table 2. Integrated WGS statistics 


Krüppel-homolog 1 (Kr-h1) mediates anti-metamorphic 

action of juvenile hormone 

Chieka MINAKUCHI*, Toshiki NAMIKI, Michiyo YOSHIYAMA, Tetsuro SHINODA 

Insect Gene Function Research Unit, * Present Address: Nagoya University 

Juvenile hormone (JH) prevents metamorphosis 

in insects, however the molecular mechanisms 

of JH action are still largely unknown. 

Recently, a transcription factor, Krüppelhomolog 

1 (Kr-h1), was found to be involved in 

the prevention of normal development of adult 

abdomen by artificial application of exogenous 

JH in the fruitfly, Drosophila melanogaster. 

However, it is not known whether Kr-h1 is 

involved in the anti-metamorphic action of 

endogenous JH in the larvae. To elucidate the 

JH signaling pathway in the larval stage, we 

have analyzed the function of Kr-h1 using the 

red flour beetle, Tribolium castaneum. 

First, the developmental and hormonal 

regulation of T. castaneum Kr-h1 expression 

was studied. The expression of Kr-h1 was high 

in the larval stage when the internal JH titer is 

supposed to be high, and was not detected in 

the pupal stage when JH titer is supposed to be 

very low. This developmental profile of Kr-h1 

correlates well with that of JHAMT (Juvenile 

hormone acid methyl transferase), a rate-limiting 

enzyme of JH biosynthesis, suggesting that the 

expression of Kr-h1 is regulated principally 

by endogenous JH. When JH biosynthesis was 

inhibited by the RNAi-mediated knockdown 

of JHAMT gene in the early larval stage, the 

expression of Kr-h1 was decreased. In addition, 

this decline was reversed by the application 

of exogenous JH mimic (JHM). These results 

indicate that the expression of Kr-h1 gene is 

maintained by JH in the larval stage. 

Next, we analyzed the function of Kr-h1 

in vivo using RNAi techniques. The RNAimediated 

knockdown of Kr-h1 in the early 

larval stage caused precocious metamorphosis 

(Fig 14A, B). Most of the RNAi animals died 

during pupation and only a few became 

miniature pupae. The treatment of JHM to the 

RNAi animals did not prevent the precocious 

metamorphosis indicating that Kr-h1 is not 

involved in JH biosynthesis, but in JH signaling. 

Furthermore, when the putative JH receptor 

gene, methoprene tolerant (Met), was knocked 

down by RNAi in the 4th instar larvae, the 

expression of Kr-h1, as well as Met, was suppressed. 

In contrast, Met was not suppressed 

by the knock down of Kr-h1. Kr-h1 thus works 

downstream of Met in the JH signaling pathway. 

Based on the results above, we proposed a 

novel model for JH signaling pathway (Fig 15). 

In this model, in the early larvae, high concentration 

of JH continues to induce Kr-h1, via the 

putative JH receptor Met, and Kr-h1 protein 

works as a larval specifier and prevents the 

larval-pupal metamorphosis. While in the last 

instar larvae, the expression of Kr-h1 declines 

because of the very low titer of JH, thus the 

anti-metamorphic action of Kr-h1 is canceled 

and the animals result in pupation. 


Fig 14. RNAi-mediated knockdown of Kr-h1 in the early larval stage. 

(A) Numbers of precocious animals induced by Kr-h1 RNAi (pink). (B) Typical phenotypes. Most of 

the animals were arrested during pupation and eventually died. 

Fig 15. Proposed model for JH signaling pathway in insect molting and metamorphosis in 

normal larvae. 


Generation of Piglets from Cryopreserved Porcine In Vitro 

Matured and Fertilized Oocytes 

Kazuhiro KIKUCHI 1 , Tamás SOMFAI 1 , Michiko NAKAI 1 , Naomi KASHIWAZAKI 2 , Takashi 

NAGAI 3 

1 

Reproductive Biology Research Unit, 2 Azabu University, 3 National Institute of Livestock 

and Grassland Science 

In domesticated animals, the cryopreservation 

of spermatozoa has been established and 

utilized as a reproductive technology; whereas, 

cryopreservation of oocytes of embryos has 

succeeded only in limited species and generally 

has not been used. In pigs, in vivo-derived 

embryos at the blastocyst stage can be cryopreserved 

by vitrification. Recently, embryos 

produced in vitro and vitrified at the 2- to 4-cell 

stages are reported to generate piglets after 

warming and transfer to recipients only when 

they were delipated (removed of cytoplasmic 

lipid) by micro-manipulation before vitrification 

(Nagashima et al, 2007). It is generally accepted 

that the cytoplasmic lipid in embryonic cells 

reduces the survival or developmental abilities 

after cryopreservation and warming. Until now, 

success has not been reported for unfertilized 

or fertilized porcine oocytes (=zygotes or 

1-cell stage embryos). Cryopreservation at the 

different fertilization or developmental stages 

is considered to have merit for preservation 

of genetic materials or utilization for breeding. 

Furthermore, easy procedures without micromanipulation 

would be welcomed for practical 

activities. In the present study, we vitrified in 

vitro-matured and fertilized oocytes after selecting 

oocytes with developmental ability by direct 

observation of pronuclei after centrifugation. 

We also confirm that the oocytes, after warming 

and transfer to recipients, have the ability to 

develop to the piglet stage. 

Porcine follicular oocytes were collected from 

prepubertal gilts and matured in vitro in a CO 2 

and O 2 gas-regulated incubator for about 44-46h. 

At 10h after in vitro fertilization with frozenthawed 

boar spermatozoa, the oocytes were 

centrifuged at 10,000 ×g for 20min to polarize 

cytoplasmic lipid droplets and to visualize 

pronuclei. Only oocytes forming 2 or 3 pronuclei 

(defined as monospermic and slightly polyspermic 

oocytes, respectively) and that have been 

reported to show high developmental ability 

(Somfai et al, 2008) were collected and used for 

the following experiments (Fig 16). Firstly, we 

examined the toxicity of the cryoprotectant, 

ethylene glycol. We evaluated two groups: 1) 

control [oocytes without any treatment were 

cultured in vitro as has been reported (Kikuchi 

et al, 2002)] and 2) toxicity control [oocytes 

treated with 35% (v/v) ethylene glycol, but not 

vitrified] and found no significant difference 

between the groups in both cleavage rates 

on Day 2 and blastocyst rates and their cell 

numbers on Day 6 (Table 3). Negative effects of 

the cryoprotectant on survival or developmental 

ability were not observed; therefore, we used 

this condition for the following cryopreservation 

experiment. The pronuclear oocytes were immersed 

in a solution containing ethylene glycol, 

vitrified by the solid surface vitrification method 

modified using aluminum foil, and stored in 

liquid nitrogen at –196C. After warming to 37C, 

the oocytes were cultured in vitro. The survival 

rates were not different between the two 

groups, but the cleavage and blastocysts rates 

of the vitrified oocytes were significantly lower 

(P < 0.05) than those of non-vitrified (control) 

oocytes. However, after the vitrified-warmed 

oocytes had developed to the blastocyst stage, 

their quality (cell number) was the same as that 

of the control (Table 4). These results suggest 

that vitrification reduces the rate of embryo 

development but does not affect their quality. 

Finally, vitrified-warmed oocytes (150 oocytes 

per recipient) were transferred to 5 recipients 


for which estrous was synchronized. A total of 

16 live piglets were born from 3 recipients (5, 2 

and 9 piglets, respectively) (Fig 17). They were 

grown normally and used for reproduction. 

We reported for the first time that porcine 

in vitro-matured and fertilized oocytes can be 

cryopreserved by vitrification (Somfai et al, 

2009). After some additional improvements, this 

method will be utilized for conservation of pig 

genetic resources and for bloodline activities 

resources in pigs. For the next step, unfertilized 

oocytes (immature or mature oocytes) also 

will be checked for cryopreservation ability. If 

this method proves to be successful, we will 

be able to generate embryos requested from 

cryopreserved sperm and oocytes. Furthermore, 

oocytes in ovarian tissue may be cryopreserved 

and utilized in combination for xenografting into 

immnodeficient animals (Kikuchi et al, 2008). 

References 

Kikuchi K, Onishi A, Kashiwazaki N, Iwamoto M, 

Noguchi J, Kaneko H, Akita T, Nagai T (2002) 

Successful piglet production after transfer of 

blastocysts produced by a modified in vitro 

system. Biol Reprod 66: 1033-1041 

Kikuchi K, Kashiwazaki N, Nagai T, Nakai M, 

Somfai T, Noguchi J, Kaneko H (2008) Selected 

aspects of advanced porcine reproductive 

technology. Reprod Dom Anim 43 (Suppl. 2): 

401–406 

Nagashima H, Hiruma K, Saito H, Tomii R, 

Ueno S, Nakayama N, Matsunari H, Kurome 

M (2007) Production of live piglets following 

cryopreservation of embryos derived from in 

vitro-matured oocytes Biol Reprod 76: 900 

–905 

Somfai T, Ozawa M, Noguchi J, Kaneko H, Karja 

NWK, Fahrudin M, Nakai M, Maedomari N, 

Dinnyés A, Nagai T, Kikuchi K (2008) In vitro 

development of polyspermic porcine oocytes: 

relationship between early fragmentation and 

excessive number of penetrating spermatozoa. 

Anim Reprod Sci 107: 131–147 

Somfai T, Ozawa M, Noguchi J, Kaneko H, 

Nakai M, Maedomari N, Ito J, Kashiwazaki N, 

Nagai T, Kikuchi K (2009) Live piglets derived 

from in vitro-produced zygotes vitrified at the 

pronuclear stage Biol of Reprod 80: 42–49 

Fig 16. In vitro matured and fertilized oocytes after centrifugation. 

Two and three pronuclei (arrows) are visible on Plates A and B, respectively. They are categorized 

as monospermic and slightly polyspermic oocytes , respectively. Asterisks (*) indicate paralyzed 

cytoplasmic lipid droplets. 


Fig 17. Piglets produced by the transfer of 

in vitro matured and fertilized oocytes after 

vitrification, storage in liquid nitrogen and 

warming. 

The photograph was taken 14 days after 

delivery. 

Table 3. Effect of cryoprotectant on in vitro developmental ability of in vitro matured and fertilized porcine oocytes 

Cryoprotectant 

No. oocyte 

examined 

Day 2 in culture 

No. (%) embryos 

cleaved 

No. (%) 

blastocysts 


Cell number / 

blastocyst 

– 

(Control) 

265 

+ 281 

235 

(88.1 ± 1.4) 

243 

(86.1 ± 2.4) 

65 

(23.2 ± 1.4) 

58 

(20.8 ± 1.4) 

38.0 ± 2.0 

41.2 ± 1.7 

Table 4. Effect of vitrification on in vitro developmental ability of in vitro matured and fertilized porcine oocytes 



Vitrification 

No. oocyte 

examined 

No. (%) 

oocyte survived 

No. (%) 

embryos 

cleaved 

No. (%) 

blastocysts 

Cell 

number / 

blastocyst 

– 

(Control) 

153 

+ 184 

153 

(100) 

172 

(93.4 ± 3.5) 

132 

37 

(86.3 ± 1.5) a (24.2 ± 2.0) a 41.6 ± 3.3 

131 

28 

(71.6 ± 7.5) b (15.2 ± 2.1) b 41.2 ± 3.4 

Mean ± SE are presented. 

a,b Values with different superscripts different significantly (P

Research Activities 


QTL Genomics Research Center 

Toward allele mining of the natural variations 

in Japanese rice germplasm 

There are diverse accessions in the germplasm 

of Asian cultivated rice, Oryza sativa, 

which has been cultivated under various 

environments. To find and analyze natural 

variations efficiently, core collection, a representative 

cultivar set is developed from diverse 

cultivars. Previously, we have selected World 

Rice Core-collection (WRC), which is consisted of 

69 accessions, from a wide range of geographical 

origins to cover the natural variation of Asian 

cultivated rice. This collection has been used to 

discover new locus or allele with agronomical 

and biological interests. On the other hand, sub 

core collection is also important to trace the 

diffusion of germplasm and find the primary 

factor of genetic differentiation among local 

populations. Japanese landrace is good target 

for developing local core collection, because 

Japan is located in the northernmost part of 

rice cultivation area in the world. In order to 

cover the variation in Japanese cultivars, core 

collection of Japanese landrace (JRC), which is 

consisted of 50 accessions, was developed from 

a original populations (236 Japanese landrace) 

based on genotype data of SSR markers, which 

are distributed on rice 12 chromosomes. 

Principal coordinate analysis of JRC showed 

that there are two cultivar groups, lowland 

type and upland type (Fig 1a). In the lowland 

type group, a few number of red rice cultivar 

were grouped with IR64, a type Indica. Most of 

the upland cultivars can be traced their origin 

by literatures. Relatively new introduction of 

upland rice is consistent with the rigid cluster 

of this cultivar group (Fig 1a). In the lowland 

type group, geographical cline was shown in 

the ordination of the accessions (Fig 1b). This 

cline suggested gradual diversification among 

lowland cultivars. A total of 50 accessions of JRC 

cover 88% of the allele diversity of SSR markers 

detected in the original population. 

Based on characteristics of morphological 

traits, JRC covers 50-80 % of the phenotypic 

variation in WRC. For example, in JRC, an 

equivalent level of variation was observed in 

heading date and culm length compared with 

that of WRC (Fig 2). Number and distribution 

of available DNA markers are not sufficient 

for the genome-wide genetic study of Japanese 

rice germplasm. To enhance potential of association 

study, we are currently discovering 

genome-wide SNPs from diverse accessions and 

developing a system of high throughput SNP 

genotyping. The seeds of WRC and JRC can be 

obtained from NIAS Genebank (http://www. 

gene.affrc.go.jp/databases-core_collections_en. 

php). 

A novel gene conferring durable blast resistance 

in rice 

Blast disease caused by Magnaporthe oryzae 

is the most devastating fungal disease of rice 

(Oryza sativa L). Race-specific resistance to 

this disease usually does not lead to durable 

resistance. In contrast, resistance controlled 

by quantitative trait loci (QTLs) is usually non- 


ace-specific, and considered to be associated 

with durability of resistance, although their 

molecular basis remains unknown. The previous 

genetic study has identified a major QTL, 

Pi21 on chromosome 4 and has revealed that 

a recessive allele (pi21) confers resistance. The 

pi21-mediated resistance lacks hypersensitive 

response, yet it restricts the development of lesions 

(Fig 3a). Some cultivars carrying pi21 have 

maintained resistance throughout a century 

of cultivation. However, the co-introduction of 

resistance and undesirable characteristics from 

donors has prevented the use of pi21 for the 

development of elite cultivars for the past 80 

years. 

High-resolution mapping delimited the Pi21 

locus to a 1705-bp region containing a heavy 

metal–transport/detoxification protein domain 

gene, Os04g0401000 where two deletions were 

detected in the resistant cultivar (Fig 3b). 

Complementation tests by transforming a genomic 

fragment containing the candidate gene 

from the resistant cultivar into the susceptible 

cultivar did not confer resistance, whereas 

transforming the susceptible Pi21 allele into 

resistant plant increased susceptibility to blast. 

Overexpression of susceptible Pi21 did not 

effect on R-gene mediated resistance. These 

results suggest that Pi21 is a negative regulator 

of basal defense, possibly due to defect in metal 

transporting. Analysis of natural variation of 

Pi21 gene suggested that the defect in pi21 

function might be attributed to both of the two 

deletions in proline-rich sequences and most rice 

cultivars carry susceptible Pi21 alleles. 

The cause of association between resistant 

Pi21 allele and undesirable grain characteristics 

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. The plants 

carrying the elite cultivar’s chromosomal 

sequence from a point less than 2.4 kb downstream 

of the Pi21 locus, that have selected 

from more than 6000 plants by marker assisted 

selection, showed equivalent level of eating 

quality to that of the elite cultivar, and the 

plants showed a high level of blast resistance 

(Fig 4). In contrast, plants carrying the donor 

chromosomal sequence up to 37 kb downstream 

of the Pi21 locus showed inferior eating quality. 

These results clearly show that the resistant 

pi21 allele does not penalize agronomic traits, 

and the cause of the association is tight linkage 

with genes causing undesirable effects. Mapbased 

cloning of Pi21, a quantitative trait locus 

(QTL) for broad-spectrum blast resistance 

identified a novel gene that negatively regulates 

defense response and provides a breakthrough 

to durably resistant cultivars without any penalties 

on agronomic traits, which has not been 

achieved by conventional rice breeding. 


a 

PC2 

Koshihikari 

IR64 

PC1 

b 

Number of 

accessions 

12 

10 

8 

6 

4 

2 

0 

0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18 0.2 0.22 0.24 

PC1 

Fig 1. Classification of Japanese landrace accessions. 

(a) Principal coordinate analysis of SSR genotypes of accessions showed that diversification of 

Japanese landrace is related to the cropping system, lowland and upland cultivation. Gray square 

and circle show lowland and upland accessions. Dark and white square shows control cultivar, IR64 

and Koshihikari. X- and Y-axis is Principal coordinate1 and 2. Part of lowland rice made a cluster with 

IR64, suggesting that they are Indica-like cultivars. (b) Histogram of PC1 analyzed for geographical 

accession group.white shows southern accessions, light grey shows central accessions, dark grey 

shows northeastern accessions, black shows north accessions. Geographical cline was found in the 

lowland type accessions. 

Heading date 

No. of accessions No. of accessions 

Culm length 

Days-to heading 

Culm length (cm) 

Fig 2. Phenotype variations for heading date and culm length in 69 World Rice Collections (WRC: 

purple) and 50 Japanese Rice Collections (JRC: yellow). 


a 

b 

C hromosome 4 

S 

L 

P i21 (1705bp) 

Os04g400900 

Os04g0401000 

Deletion (bp) 

21 48 

heavy-metal–transport/ 

detoxification protein 

domain 

proline-rich 

sequence 

AA 

AA-pi21 

Fig 3. Map-based cloning of pi21. 

(a) Diseased leaves of 30-day-old plants inoculated with blast fungus rase 007.0: AA, a susceptible 

culativar Aichiasahi; AA-pi21, a near-isogenic line for pi21 in Aichiasahi background. (b) Genetic and 

physical map around Pi21. Green boxes represent genes described in RAP-DB. Red arrows indicate 

the positions of two deletions in the resistant cultivar (Owarihatamochi) compared with the reference 

genome sequence (Nipponbare). 

a 

C omventional breeding 

b 

DNA 

marker 

a 

select plants 

carrying a and b 

a 

E ating 

quality 

pi21 

b 

b 

Blast 

resistance 

Molecular breeding 

pi21 

undesirable 

gene 

a 

c 

d 

b 

select plants 

carrying a, c and 

not carrying b, d 

a 

c 

d 

b 

E ating 

quality 

Blast 

resistance 

Fig4. Molecular breeding of pi21. 

(a) Removal of undesirable gene(s) tightly linked with the resistant pi21 gene based on information 

of physical gene location (Molecular breeding). (b) Field trial under sever blast infection condition. 

Recurrent blast-susceptible parent Koshihikari (left) and a line carrying resistant pi21-allele and 

improved eating quality (right) are indicated. (Figures are adopted from Science 325: 998-1001) 


Transgenic Crop Research and Development Center 

Characterization of a new rice glutelin gene 

GluD-1 expressed in the starchy endosperm 

Rice endosperm has been utilized as an excellent 

platform for producing foreign recombinant 

proteins because it is a natural storage organ 

for starch and proteins and can offer ample deposition 

space for foreign recombinant products. 

A number of biopharmaceuticals, nutriceuticals 

containing health-promoting peptides, and 

mineral-binding proteins have been produced 

using rice endosperm as a bioreactor. Because 

seed storage proteins (SSPs) are abundant in the 

endosperm, promoters of native SSP genes are 

often employed to achieve high accumulation of 

those compounds in the endosperm. 

Studies on the molecular mechanisms underlying 

endosperm-specific expression of cereal 

SSP genes have been carried out. It has been 

reported that several cis-elements, such as the 

prolamin box (P box: AAAG) and GCN4 (TGA(G/ 

C)TCA), ACGT and AACA motifs are conserved 

in many cereal SSP genes. Transcriptional 

factors that recognize these cis-elements have 

also been isolated, such as Opaque2-like bZIP, 

Dof and Myb proteins. Understanding these 

gene regulation mechanisms in greater detail 

would provide useful information for fine-tuning 

the expression of recombinant products in 

transgenic crops. 

We discovered a new glutelin by comparing 

the seed protein composition of various rice 

cultivars. The expression of this glutelin gene 

(GluD-1) has unique spatial and temporal 

features during seed development, although 

like the other glutelins, it is expressed in an 

endosperm-specific manner (Fig 1). GluD-1 was 

predominantly expressed in the inner starchy 

endosperm from about 5 DAF and steadily increased 

until maturity at 30 DAF (Fig 1). Since 

the 0.2kb upstream region is sufficient to confer 

GUS reporter expression, the essential regulatory 

elements controlling GluD-1 expression in 

the starchy endosperm are present within this 

region (Fig. 2). An in vitro gel-shift assay and 

an in vivo transient reporter assay indicated 

that such endosperm specificity is controlled by 

combinatorial interactions of a GCN4-like motif 

and a closely linked P box around position – 

200 from the ATG start site through binding 

by bZIP transcription factor RISBZ1 and DOF 

transcription factor RPBF. Its spatial specificity 

may be caused by deviations within the GCN4 

motif of the GluD-1 promoter. 

Development of rice-based oral vaccine 

against house dust mite allergy 

Transgenic plants are becoming common 

hosts for low cost production of valuable pharmaceuticals, 

vaccines and antibodies. Apart from 

extremely low risk of contamination with animal 

or human pathogens, transgenic plants can be 

used as a reliable delivery system for the oral 

administration of expressed valuable proteins. 

Among cereal crops, rice is especially an ideal 

potential oral delivery system of vaccines for 

clinical prophylactic and treatment of allergic 

disease as well as protecting against infection 

by pathogens. 

Conventional allergen-specific immunotherapy 

has been mainly performed by repeated subcutaneous 

injection of increasing concentrations 

of allergens throughout a period of 3-5 years 

with severe side effects including the risk of 

anaphylactic shock. A safe, easy and convenient 

treatment would benefit patients by taking advantage 

of the mucosal immune system via oral 

administration as an alternative to subcutaneous 

injection. 

House dust mite (HDM) of Dermatophagoides 

species is the most common source of indoor 

allergens associated with various allergic diseases 

such as asthma, rhinitis and atopic dermatitis. 

One of the most important properties of allergens 

is their ability to induce Th2 responses 

culminating in IgE antibody production. Der p 1, 

an allergen from feces of D. pteronyssinus, elicits 

IgE antibody responses in over 80% of patients 

who are sensitive to D. pteronyssinus and is 


considered to be the most immuno-dominant allergen 

involved in the expression of IgE-mediated 

hypersensitivity. Der p 1 has cysteine protease 

activity that is involved in the pathogenesis of allergies 

through cleavage of CD23 and CD25 from 

the surface of immune cells, IgE-independent 

mast cell activation, and increasing epithelial cell 

permeability. The mature Der p 1 has 222 amino 

acid residues possessing one N-glycosylation 

motif at 52nd Asn and the 34 th Cys residue also 

plays a crucial role in its enzyme activity. 

We developed transgenic rice seed expressing 

a major house dust mite allergen, Der p 1, as 

an oral vaccine. The C-terminal KDEL-tagged 

Der p 1 allergen specifically accumulated in 

seed endosperm tissue under the control of the 

endosperm-specific GluB-1 promoter. Der p 1 

reached a maximum concentration of 58µg/ 

grain and was deposited in the endoplasmic 

reticulum-derived protein body I (PB-I) (Fig 3). 

Plant-derived Der p 1 was posttranslationally 

modified with high-mannose-type glycan structure 

(Table 1). Glycosylated Der p 1 displayed 

reduced IgE binding capacity in comparison 

with its unglycosylated counterpart in vitro. 

Our results indicate that transgenic Der p 1 rice 

seeds are a safe, potential oral delivery vaccine 

for treatment of HDM allergy. 

Enhanced oral tolerogenicity of T cell epitopes 

coupled with cholera toxin B subunit 

Oral peptide immunotherapy using dominant 

T-cell epitopes provides a safe, easy and effective 

strategy against allergic and autoimmune 

diseases. We have recently reported that feeding 

mice with transgenic rice seed containing 

T-cell epitopes derived from major Japanese 

cedar pollen allergens Cry j 1 and Cry j 2 suppressed 

pollen-induced allergic responses and 

clinical symptoms. These results demonstrated 

the clinical potential of T-cell epitopes accumulated 

in rice seed for immunotherapy of allergic 

disorders. 

Cholera toxin B (CTB), a GM1 gangliosidebinding 

subunit of cholera toxin, has been 

shown to function as an effective carrier for 

mucosal antigens, as well as a non-toxic oral 

cholera vaccine antigen. Here, we applied this 

CTB molecule for the induction of oral tolerance 

against Japanese cedar pollen allergens. 

Allergen-derived tolerogenic T cell epitopes 

(3Crp) were expressed in transgenic rice seed 

as a fusion protein with either CTB or rice 

glutelin as a control (Fig 4). In an experimental 

mouse model of pollen allergy, a relatively high 

dose of CTB-free T cell epitopes (equivalent to 

15µg 3Crp) was required for the suppression of 

allergen-specific IgE antibody responses (Fig. 

4). In contrast, a significantly lower dose of the 

CTB-coupled T cell epitopes (equivalent to 0.3µg 

3Crp) was sufficient for IgE suppression (Fig 

4). Furthermore, the development of clinical 

symptoms such as pollen allergen-induced 

sneezing was suppressed by oral feeding of rice 

seed that accumulate T cell epitopes fused with 

CTB. Thus, our findings demonstrate that CTB 

enhances oral tolerogenicity of T cell epitope 

3Crp peptide, and support a promising potential 

role for CTB in peptide-based immunotherapy 

of Japanese cedar pollen allergy. 

Improvement of CoQ10-enriched rice 

We previously produced CoQ10-enriched 

transgenic rice plants by introducing the expression 

vector construct of S14:ddsA, in which 

the gene encoding DdsA, an enzyme involved 

in the CoQ10 biosynthesis pathway, was driven 

by the CaMV35S promoter. Here, in order to 

obtain a transgenic line accumulating higher 

concentration of CoQ10, we first expressed 

the S14:ddsA construct using the rice ubiquitin 

promoter as a much stronger promoter. In this 

approach, the highest expression line resulted in 

1.3-fold enhancement of CoQ10 in mature seed 

than the previous construct. We next produced 

another type of CoQ10-enriched rice plant by 

introducing two constructs simultaneously; one 

is DdsA driven by the rice ubiquitin promoter 

and the other is COQ2, the enzyme involved in 

CoQ biosynthesis pathway, driven by the CaMV 

35S promoter. Although the COQ2 gene was 

successfully expressed in the newly generated 

transgenic plants, CoQ10 level was very similar 

to that of Ubi:S14:ddsA plants. This result 

indicates that co-expression of DdsA and CoQ2 

does not result in an additive effect and that 

another approach is required to increase CoQ10 

production in rice seed. 


Fig 1. Temporal and spatial expression patterns of GluD-1. 

(A) Organ-specific expression pattern of GluD-1. R: root, S: shoot apex, L: leaf blade, F: flower, 

Se: seed at 15 days after flowering (DAF), and Ca: callus. (B) Temporal expression pattern during 

seed maturation of GluD-1. C–E) GUS expression driven by the 1.6kb GluD-1 promoter during 

seed maturation, at 7, 10, 15 DAF, respectively. al: aleurone and subaleurone layers, en: starchy 

endosperm, and em: embryo. 

Fig 2. Truncation analysis of the GluD-1 promoter. 

(A) Schematic representation of truncated promoter:GUS constructs for generating transgenic rice 

(left), and expression strength (right). (B)-(F) GUS expression at 7 DAF, driven by 1.6kb, 1.2kb, 0.6kb, 

0.4kb, and 0.2kb GluD-1 promoters, respectively. 


A 

HindIII Nco1 Ψ Sac1 EcoR 1 

LB pAg7 hpt 35S P GluB1 P SP Der p 1-KDEL 

GluB1T RB 

B 

Endo H 

 

 

C 

St 

kDa 

75 

50 

37 

PB-I 

PB-II 

PB-II 

25 

20 

15 

PB-I 

1 2 1 2 

Fig.3 

Fig 3. Characterization of transgenic rice expressing Der p 1. 

(A) Construct of binary vector used for expression of mite allergen Der p 1. (B) Analysis of Der p 

1 glycoprotein derived from rice seed after treatment with Endo H. (C) Analysis of intra-cellular 

localization of Der p 1 by immuno-electron microscopy. 

A 

CTB-free 3Crp 

3Crp 

LB 

pAg7 

HPT 

35S 

promoter 

2.3 kb GluB1 

promoter 

SP 

glutelin 

acidic subunit 

glutelin 

basic subunit 

GluB1 T 

RB 

CTB- coupled 3Crp 

LB 

pAg7 

HPT 

35S 

promoter 

2.3 kb GluB1 

promoter 

SP CTB 3Crp KDEL 

GluB1 T 

RB 

B 

3Crp 

(g) 

0 

1.5 

6 

15 

CTB-free 3Crp 

0.1 

0.3 

CTB-coupled 3Crp 

1 2 3 4 5 6 

Allergen-specific 

IgE Ab 

(reciprocal log 2 

titer) 

Fig 4. Efficacy of CTB-T cell epitope fusion protein expressed in rice seed. 

(A). Constructs for expression of CTB-free and CTB-coupled T cell epitope 3Crp peptide. (B) 

Suppression of allergen-specific IgE antibody responses by oral feeding of transgenic rice seed. 


Table Relative abundances of N-glycans detected in Der p 1 

Table 1. Relative abundances of N-glycans detected in Der p 1 

Fraction 

m/z (M + Na)+ 

MALDI -TOF -MS 

Calculated 

Glycoform 

Ratio 

(%) 

1 

1821.56 

1821.64 

Man 8 GlcNAc 2 

26.3 

2 

1983.39 

1983.78 


12.9 

4 

a 

c 

1659.36 

1983.03 

1659.50 

1983.78 


Glu 1 Man 8 GlcNAc 2 

1.6 

26.8 

5 

b 

2145.26 

2145.92 

Glu 1 Man 9 GlcNAc 2 

25.4 

6 

b 

e 

1143.90 

1335.40 

1143.05 

1335.22 

Man 3 XylGlcNAc 2 


4.9 

2.1 

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 studied the development of 

new methods for the generation of transgenic 

silkworm through construction of new vectors 

and development of new marker genes. We are 

also studying 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. 

Construction of a new injection system using 

the electric manipulator 

Efficient production of transgenic silkworms 

is essential for continuing the development and 

application of research in this area. The original 

method of using glass capillaries may not be 

appropriate for injection of DNA into silkworm 

eggs because the eggs possess thick and hard 

eggshells. Therefore, the injection of DNA into 

an egg was generally performed by the following 

processes: first, make a small hole in the 

eggshell with a tungsten needle; insert a glass 

capillary into the hole; and supply air pressure 

to the capillary to push the DNA solution into 

the egg. During this process, the penetration of 


Fig 1. The gene expression system of posterior part of silk glands of the 5th instar larva of the 

transgenic silkworm. Numbers indicate the stage of the 5th instar. 

the glass capillary into the small hole is a crucial 

step. To improve this step, we developed a 

method that uses an electric manipulator to automatically 

move the position of the hole in the 

eggshell. This method increased the speed of 

injection, the hatchability of eggs after injection, 

and the efficiency of production of transgenic 

silkworms. We concluded that the new system 

makes injection much easier compared to the 

original method for construction of transgenic 

silkworms. 

Development of a gene regulation system 

using the promoter of the fibroin gene of the 

domesticated silkworm Bombyx mori and the 

Japanese oak silkworm Antheraea yamamai. 

A system for expression of transgenes in the 

posterior silk gland had been developed using 

the GAL4 strain under the control of the promoter 

from the fibroin L chain gene of silkworm. 

This promoter, however, stimulated expression 

of EGFP driven by the GAL4 in young larvae 

which apparently also caused an abnormal shape 

of the posterior silk gland and reduced production 

of EGFP. To circumvent this problem, we 

made two constructs of the GAL4 gene — one 

under the control of the fibroin H chain gene 

promoter from domesticated silkworm, Bombyx 

mori, and the other under control of the fibroin 

H chain gene from oak silkworm, Antheraea 

yamamai. We generated transgenic silkworms 

with these fibroin H chain promoter constructs 

and investigated the expression pattern of 

EGFP after crossing with a homozygous UAS- 

EGFP strain. The expression of EGFP was only 

observed in the posterior silk gland after the 

2nd day of the 5th instar (Fig 1). In contrast to 

previous transgenic silkworms controlled by the 

fibroin L chain promoter, the abnormal shape of 

the silk gland was not observed in transgenic 

silkworms containing these H chain promoter 

constructs and production of silk proteins in 

the silk gland was no different from that of nontransgenic 

silkworms. The results indicate that 

the fibroin H chain promoter system is superior 

for the production of the recombinant proteins 

compared to the system using the fibroin L 

chain promoter. 

Production of modified GAL4 constructs to 

increase the expression level of the target 

gene 

To increase transgene expression level, we 

have shown that modification of the GAL4 construct 

is effective in experiments using cultured 

cells. In these experiments, we have shown that 

the level of expression of the best construct 

was more than 100 times higher than that of 

an unmodified GAL. Based on these results, 

we constructed a vector with the modified 

GAL4 construct under the control of the sericin 

gene promoter and then generated transgenic 

silkworm lines with the new construct. We 

confirmed that the lines expressed the target 

gene in the middle part of the silk gland of 

transgenic silkworms only during the middle 

and late stages of the final instar. In addition, we 

constructed new GAL4 strains that stimulate 


the expression of a target gene by heat-shock. 

These strains are expected to be very useful for 

achieving temporal expression of the transgene. 

Improvement of a production system for the 

recombinant proteins in middle part of silk 

gland 

To develop systems for production of useful 

recombinant proteins in the middle part of silk 

glands, we studied the effect of related factors 

on the production of recombinant proteins in 

transgenic silkworms. This year, we mainly 

tested the effects of the exon-intron of sericin 

1 and fibrohexamarin genes, tandem repeats 

and copy numbers. Addition of the exon and 

intron sequence of sericin 1 gene was not effective 

in increasing mRNA and protein of the 

target gene and the level was almost the same 

as that for the addition of the signal peptide 

sequence of sericin 1. In contrast, the addition 

of the exon-intron sequence of fibrohexamarin 

gene increased target gene mRNA levels and 

resulted in larger amounts of the target protein. 

Constructs with tandem repeats and increased 

copy numbers were effective in increasing the 

amount of protein produced in the middle silk 

gland of transgenic silkworms. We obtained 

transgenic silkworms that produce more than 1 

mg of the recombinant protein in a pair of the 

middle silk glands. From the results, we are 

constructing more efficient vectors compared to 

the old ones. 

Breeding of new silkworm strains for the 

production of recombinant protein 

To breed new silkworm strains that are 

adapted for the production of recombinant 

protein, we repeatedly performed selection 

and mating between transgenic strains and 

the strains that produce a large amount of silk 

protein. The amount of sericin protein produced 

by the newly bred strains reached more than 

75 mg, which is twice that of the original 

transgenic strains. The results suggest that the 

use of the new strain may drastically increase 

production. 

Production of the recombinant opioid receptor 

protein in transgenic silkworms 

In collaboration with Gunma University, we 

successfully produced recombinant protein of G 

protein opioid receptor in transgenic silkworms. 

The receptor produced by the silkworm possessed 

native activity of the receptor and the 

yield was comparable to that of the baculovirus 

expression system. 

Fig 2. Mass rearing of transgenic silkworms, larvae constructing cocoons, and spun cocoons 

expressing the orange fluorescent protein. 


Study of the development of recombinant silk 

produced by transgenic silkworms for practical 

use and industrialization 

Through collaborations with Toray Industries, 

Inc., Tokyo University of Agriculture and 

Technology, Gunma Sericultural Technology 

Center, Textile Research Center of Gunma, 

Riken, Amalgaam, Ltd., and Silk Technology unit 

of NIAS, we studied production of high quality 

recombinant silks. We successfully produced 

high quality silks having different fluorescent 

colors using transgenic silkworms. Furthermore, 

we bred strains for mass-rearing of silkworms 

and harvesting large amounts of the cocoons 

with the fluorescence color. Then, we performed 

a mass-rearing of the transgenic silkworms and 

successfully harvested large amounts of cocoons 

(Fig 2). We also developed the technology to 

spin large amounts of fluorescence-colored silks 

and produced large amounts of the recombinant 

silks (Fig 3). Tapestries, other textiles and lamp 

shades were produced from the recombinant 

silks and evaluated for practical use (Fig 4). In 

addition, we developed a means to produce thin 

silk by introducing a gene for an acidic peptide 

into silkworm. The silk produced by these 

transgenic silkworms is thinner than that of 

the original race. We showed the possibility of 

producing recombinant silk suitable for higher 

quality of clothes. We also demonstrated that 

artificial veins assembled from recombinant 

silks expressing a specific peptide related to the 

affinity of mammalian cells had better quality 

compared to those made from natural silk. 

Fig 3. Fluorescent-colored silks. 

Left: under white light. Right: illuminated by blue-LED light (viewed with a yellow filter). 

Fig 4. Tapestries (Jacquard) made with fluorescent-colored silks. 

Left: under white light. Right: illuminated by blue-LED light (viewed with a yellow filter); the characters 

and background pattern are fluorescing. 



Proto-oncogene MYCN transduced pigs produced 

by somatic cell nuclear transfer 

Neuroblastoma is a tumor of neural crest 

origin that comprises 8-10% of all childhood 

malignancies and causes 15% of cancer related 

death in children. Ninety percent of cases strike 

children before 10 years old. The transgenic 

mice that over-expressed proto-oncogene MYCN 

in neuroectodermal cells developed neuroblastoma. 

Although these mice are a model 

of neuroblastoma and are useful for study of 

pathogenesis and therapy, they are not optimal 

for research and development of surgery and 

chemotherapy treatments for humans. Pigs 

offer the advantage of having a similar size, diet, 

metabolism, lifespan and anatomy to humans. 

In the present study, we attempted to produce 

transgenic pigs expressing MYCN by nuclear 

transfer for use as a human disease model. 

The expression vector of human MYCN 

containing the rat tyrosine hydroxylase promoter 

and G418 resistant gene was transfected 

into cultured fibroblasts derived from a male 

porcine fetus. After selection by G418, the 

isolated clones were checked by RT-PCR for 

MYCN expression. The one colony expressing 

MYCN was used for nuclear transfer as donor 

cells. After nuclear transfer using 2863 oocytes 

matured in vitro, 454 embryos at the one-cell 

stage and 960 embryos at 2-8 cell stage were 

transferred into 7 synchronized recipient pigs. 

Four of 7 recipients were pregnant and 3 pigs 

delivered 14 piglets including 2 stillbirths and 

5 mummies (Fig 1). All 7 live piglets and the 2 

stillbirths showed introduction of MYCN gene 

by PCR analysis. Development of neuroblastoma 

in the cloned pigs should be examined later. 

Production of transgenic goats for 'Pharming' 

We have established a system to produce 

transgenic (Tg) goats by using somatic cell 

nuclear transfer (SCNT) technique. We have 

produced Tg goats expressing the human 

selenoprotein P (hSel P) gene under control of 

the bovine beta-casein core promoter for expression 

in the mammary gland. During this year, 

the female Tg offspring was mated with a wild 

type male. After parturition, the milk was collected 

regularly and content of the hSel P was 

evaluated by specific ELISA. In the analyses, 

hSel P production was highest at the 2nd day of 

parturition, and the concentration was 15ng/ml. 

After that it decreased to 2ng/ml and was 

secreted continuously for 8 weeks. Since the Tg 

Fig 1. Production of MYCN transgenic pigs by somatic cell nuclear transfer. 


goat carried only one copy of the hSel P gene, 

the concentration was lower than that found for 

the Tg mouse and rabbit which carried multiple 

copies of the same gene. The decrease in hSel P 

concentration may be related to the quantity of 

milk production during the lactation period. It is 

necessary to improve the expression level in the 

mammary gland; therefore, we have developed 

a new LA-SSP1 BAC vector with ~200kb DNA 

that includes the complete lactoalbumin gene 

from human BAC clone for higher control of 

gene expression in the mammary gland. 

GFP gene expression in gonads of chicken 

embryos 

The present study was carried out to develop 

techniques for introducing exogenous DNA into 

primordial germ cells (PGCs) and expressing 

the introduced DNA efficiently in the gonads of 

developing chicken embryos. PGCs circulating 

in the bloodstream were transfected with GFP 

gene in vitro or in vivo by lipofection or nucleofection. 

The manipulated PGCs successfully 

migrated to the germinal ridges and expressed 

GFP gene efficiently in the gonads of developing 

embryos (Fig 2). Intense GFP gene expression 

was observed in the gonads during the first 8 

days following the transfection, during which 

period the sexual differentiation of gonads and 

germ cells (GCs) takes place. The GFP gene 

expression then gradually declined during 

subsequent embryonic development until hatching. 

When PGCs were transfected in vivo by 

lipofection with linearised plasmid DNA, GFP 

gene was detected in the gonads of 4.3% (19/442) 

of embryos examined at 20.5 days of culture. 

In comparison, the frequency of detection was 

less than 1% in the gonads of embryos in which 

PGCs were transfected in vitro or with circular 

form plasmid DNA. In two of the embryos 

in which PGCs were transfected in vivo by 

lipofection with linearised plasmid DNA, GFP 

gene was expressed clearly in limited areas of 

the gonads of 20.5-day cultured embryos. The 

results obtained in this study suggest that the 

present in vitro and in vivo techniques for PGC 

manipulation provide a useful experimental 

system for studying gene functions in the 

sexual differentiation of gonads and GCs in early 

chicken embryos. 

Development of immortalized cell lines and 

novel tissue culture models 

Immortalized bovine brain cell lines provide 

ideal in vitro cellular infection models for bovine 

spongiform enthephalopathies (BSE) prion without 

enduring a species barrier. Here, we established 

an immortalized brain cell line (FBBC-1) 

Fig 2. Expression of GFP gene in the gonads of developing chicken embryos at (A) 6.5 days, (B) 8.5 

days, (C, D) 10.5 days, (E, F) 12.5 days, and (G, H) 20.5 days of culture. 


from primary cultures of cryopreserved fetal 

bovine brain tissues after transfection with 

SV40 large T antigen (Fig 3). FBBC-1 cells have 

spindle-like morphology and are stably passaged 

at a doubling time of about 24 hr up to at least 

100 population doublings after single cell cloning. 

After the treatment with dibutyryl-cyclic AMP, 

these cells ceased proliferation and extended 

neurite-like processes that were immunostained 

with the antibody against tubulin βIII, a 

marker of immature neurons. Up-regulation of 

tubulin βIII expression was also confirmed by 

immunoblotting. FBBC-1cells expressed cellular 

prion protein, so these bovine cells may provide 

the opportunity to develop an in vitro propagation 

model of cattle BSE prion. 

We previously succeeded in preparing a thin 

(thickness ca. 20µm), strong, transparent and 

protein-permeable membrane made of collagen 

fibrils in high density like corneal parenchyma 

in vivo. We named it collagen vitrigel membrane 

because its fabrication process utilizes a concept 

for the vitrification of heat-denatured proteins. 

As a first step to develop a corneal epithelial 

model useful for replacing the eye irritation test 

(Draize test), normal rabbit corneal epithelial 

(NRCE) cells were cultured on the collagen vitrigel 

membrane and their growth activity and 

morphology were analyzed. As a result, NRCE 

cells cultured on the collagen vitrigel membrane 

proliferated well and formed a monolayer of 

polygon-shaped cells the same as those on 

the control plastic dish did (Fig 4). These data 

suggest that the collagen vitrigel membrane 

can provide an appropriate scaffold for corneal 

epithelial cells and can contribute to an in vitro 

corneal epithelial reconstruction model. 

Fig 3. Establishment of an immortalized bovine brain cell line, FBBC-1 

(A) and its neuronal differentiation by the treatment with dibutyryl-cyclic AMP (B and C). FBBC-1 cells 

express cellular prion protein (D). 

Fig 4. Growth activity (A) and morphology (B) of NRCE cells cultured on collagen vitrigel or control 

plastic dish. 



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. 

Genome Resource Center 

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 

2008 are described as follows. 



The Unit has made major contributions to the 

construction and analysis of various genome resources 

from rice and other Gramineae species. 

Comparative analysis of rice centromeres 

Comparative analysis of orthologous genomic 

regions and gene sequences could help to reveal 

the mechanisms responsible for the molecular 

evolution of the rice genome and the functions 

of rice genes. We have established a system 

to conduct comparative genomics among the 

Oryza species or rice varieties with a focus 

especially on the genomic regions containing 

resistance gene clusters and centromeres, as 

well as functional genes related to speciation, 

domestication, and adaptation of cultivated rice. 

Oryza sativa contains two traditional subspecies: 

indica and japonica. To shed light on the nature, 

evolutionary timing, and evolutionary dynamics 

of rice centromeres, we have decoded a 2.25-Mb 

DNA sequence covering the centromeric region 

of chromosome 8 of an indica rice variety, 

‘Kasalath’(Kas-Cen8). Analysis of repetitive 

sequences in Kas-Cen8 led to the identification 

of 222 LTR-retrotransposon elements and 

584 CentO satellite monomers, which account 

for 59.2% of the region. A comparison of the 

Kas-Cen8 sequence with that of japonica 

rice ‘Nipponbare’ (Nip-Cen8) revealed that 

about 66.8% of the Kas-Cen8 sequence was 

collinear with that of Nip-Cen8. Although the 

27 putative genes are conserved between the 

two subspecies, only 55.4% of the total LTRretrotransposon 

elements in ‘Kasalath’ had 

orthologs in ‘Nipponbare’, thus reflecting recent 

proliferation of a considerable number of LTRretrotransposons 

following the divergence of 

subspecies indica and japonica within O.sativa. 

Comparative analysis of the subfamilies, time of 

insertion, and organization patterns of inserted 

LTR-retrotransposons between the two Cen8 regions 

revealed variations between ‘Kasalath’ and 

Fig 1. Genomic distribution of LTR-retrotransposon elements and CentO satellite repeats identified in 

the core domain (793 kb) of ‘Kasalath’ Cen8. 

The unshared and shared LTR-retrotransposons between ‘Kasalath’ and ‘Nipponbare’ Cen8 regions 

are given, respectively, above and below the sequence map. Asterisks indicate the intact or mostly 

intact LTR-retrotransposon elements, and numbers in parentheses represent the estimated insertion 

date (Mya). Solo LTRs are shown in red. 


‘Nipponbare’ in the preferential accumulation of 

CRR elements and expansion of CentO satellite 

repeats within the core domain of Cen8 (Fig 1). 

Together the results provide insights into the 

recent proliferation of LTR-retrotransposons and 

the rapid expansion of CentO satellite repeats 

underlying the dynamic variation and plasticity 

of plant centromeres. 

Gene expression analyses of rice plants 

infected with rice stripe virus (RSV) and rice 

tungro spherical virus (RTSV) 

Rice stripe disease is one of the major virus 

diseases in East Asia caused by rice stripe virus 

(RSV). Rice plants infected with RSV usually 

show symptoms such as chlorosis, weakness 

and necrosis in newly emerged leaves, and 

stunting. To reveal rice cellular systems 

influenced by RSV infection, temporal changes 

in the transcriptome of RSV-infected plants 

were monitored by a customized rice oligoarray 

system. The transcriptome changes in RSVinfected 

plants indicated that protein synthesis 

machineries and energy production in mitochondria 

were activated by RSV infection, while 

energy production in chloroplasts and synthesis 

of cell structure components were suppressed. 

Moreover, the transcription of plant hormoneregulated 

host genes, and those for gene silencing 

of invaders, were disarmed at the early 

infection stage of these viruses. Together with 

concurrent observations of virus concentration 

and symptom development, such transcriptome 

changes in RSV-infected plants implied that 

genes activated after RSV infection may have 

played roles to facilitate RSV propagation in 

host cells, while the genes suppressed may have 

been related to symptom development. 

Rice tungro disease is caused by rice tungro 

spherical virus (RTSV) and rice tungro bacilliform 

virus (RTBV). RTSV alone usually does not 

cause distinctive symptoms in indica rice plants. 

To elucidate the basis of asymptomatic response 

of rice to RTSV, gene responses in variety 

Taichung Native 1 (TN1) infected with RTSV 

were examined by a custom microarray. RTSV 

in TN1 increased rapidly at around 6 days after 

inoculation (DAI), and slightly increased again at 

around 15 DAI. Genes for translation machinery 

were especially activated at 6 and 15 DAI. More 

translation-related genes were activated at 6 

DAI than at 15 DAI, suggesting the association 

between activation of such genes and RTSV 

propagation. Some cell wall-, and chloroplastrelated 

genes, whose suppression is associated 

with symptom development, were indeed suppressed, 

but only temporarily by RTSV infection. 

Defense- and stress-related genes regulated 

by RTSV infection were largely divided into 

those regulated at 6 and 15 DAI and those at 

9 and 12 DAI. More defense- and stress-related 

genes were activated at 15 DAI than at 6 DAI, 

indicating a reverse association between the 

activated defense genes and RTSV propagation. 

Taken together, our results suggested 1) the 

lack of symptoms in TN1 by RTSV infection 

is ascribed to the observation that some genes 

associated with symptom development were indeed 

suppressed, but only temporarily, 2) RTSV 

multiplication is associated with activation of 

genes for translation machinery, and 3) defense 

and stress-responsive genes were selectively coactivated 

in response to RTSV propagation. 

Mapping of the eibi1 gene responsible for the 

drought hypersensitive cuticle in wild barley 

(Hordeum spontaneum) 

A waxy cuticle covers the aerial surfaces of 

the leaf and stem of many plants. The cuticle 

consists of two layers, the outer, translucent 

cuticle proper and an inner, opaque cuticular 

layer. One of the cuticle’s most important functions 

is to control water loss, a property which 

is especially important for plants growing in 

drought-prone environments. The spontaneous 

eibi1 mutant was identified in wild barley 

(Hordeum spontaneum Koch), and is hypersensitive 

to drought (Fig 2A, B). Its relative water 

loss rate is higher than that of other wilty 

mutants. A segregation analysis showed that 

eibi1, a drought hypersensitive cuticle wild 

barley mutant, was monogenic and recessive. 

This gene was mapped in two F2 populations, 

one made from a cross between the mutant and 

a cultivated barley (cv. Morex), and the other 

between the mutant and another wild barley. 

A microsatellite marker screen showed that 

the gene was located on barley chromosome 3H 


A 

B 

Fig 2. The drought sensitivity of the eibi1 mutant. 

(A) Five-leaf stage seedlings rehydrated after a drought stress applied by withholding water for 5d. Left 

hand pair of plants are eibi1, and the right hand pair are wild type. (B) Leaf segments cut from eibi1 (above) 

and wild type (below) plants after drying for 1h. 

and a set of markers already assigned to this 

chromosome, including both microsatellites and 

ESTs, were used to construct a genetic map. 

eibi1 co-segregated with barley EST AV918546 

and was located to bin 6. The synteny between 

barley and rice in this region is incomplete, with 

a large discrepancy in map distances, and the 

presence of multiple inversions. 

Flowering time genes in Triticeae 

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 flowering is completely 

opposite between rice (short-day plant) 

and wheat and barley (long-day plants) even 

Fig 3. Phylogenetic tree of the FT-like proteins from barley (Hv), rice (Os) and Arabidopsis (At). 

Sequences were aligned with ClustalX and the results are displayed graphically using Tree View. 

Bootstrap values out of 1000 bootstrap resamplings are shown at the nodes to assess the 

robustness of the tree. 


though all three are members of the Gramineae 

family. 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 (Fig 3). 

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, indicating that these FT-like 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 shortday 

(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 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 a 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 functional roles of 

FT-like genes in controlling flowering of barley 

might be differed from those of model plants, 

Arabidopsis and rice (Fig 3). 


Genome annotation based on ab initio predictions 

and protein homology 

With the recent advancement of next-generation 

sequencing technologies, a tremendous amount 

of nucleotide sequences are being produced. It is 

an urgent issue to efficiently conduct annotation 

that is a basis of further experimental genomics. 

We have developed an annotation pipeline that 

maps full-length cDNAs to genomes and identifies 

exon-intron structures. We expected that 

addition of ab initio predictions and structures 

based on protein homology would strengthen 

our genome annotation system. Therefore, first, 

for efficient ab initio predictions, the training 

process of computer programs for gene predictions 

was completely automated. In particular, 

the GRAPE software was employed to optimize 

parameters in the course of training because 

this is one of the most time-consuming steps. 

GeneZilla was used for our ab initio predictions. 

Second, protein sequences registered in the 

UniProt database were aligned to genomes with 

the Spaln program such that protein-coding 

regions can be inferred in the genomes. 

The ab initio prediction pipeline was applied 

to the rice and sorghum genomes. To validate 

the predictions, results were compared with the 

annotations of rice (RAP) and sorghum (JGI). 

Accuracy of our predictions was examined at 

the nucleotide, exon, and gene levels (Table 1). 

In all cases, annotations based on the automated 

training were almost equivalent or superior 

to those based on manual training. Therefore, 

our pipeline will be useful for novel genome sequences 

to be annotated in future. To evaluate 

gene predictions based on the protein-genome 

alignments, we used rice and wheat genomic 

DNA. At this moment, unlike ab initio predictions, 

the protein-based predictions displayed 

high specificity and low sensitivity (data not 

shown). We plan to test another program such 

as ProSplign. 


Table 1. Accuracy of manual and automated ab initio predictions 

Nucleotide level Exon level Gene level 

SN1 SP1 SN SP SN SP 

Rice genome 2 

Manual 0.86 0.83 0.63 0.62 0.29 0.22 

Automated 0.91 0.81 0.66 0.54 0.21 0.16 

Sorghum genome 3 

Manual 0.85 0.85 0.61 0.60 0.31 0.21 

Automated 0.92 0.84 0.67 0.57 0.23 0.16 

1 

SN, sensitivity; SP, specificity. 

2 

The rice genome annotation was retrieved from RAP: http://rapdb.dna.affrc.go.jp/. 

3 

The sorghum genome annotation was retrieved from JGI: http://genome.jgi-psf.org/Sorbi1/ 

Sorbi1.home.html. 

Comparative transcriptome analysis 

Comparison of transcriptomes adds to our 

knowledge about alternative usage of transcription 

start sites (TSSs), which is one of the key 

mechanisms to generate gene variation. We 

have examined diversified molecular evolution 

of TSSs in two representative flowering plants, 

rice and Arabidopsis. We conducted comprehensive 

analyses of large collections of full-length 

cDNAs and genome sequences, and determined 

45,917 representative TSSs within 23,445 loci 

of rice and 35,313 TSSs within 16,964 loci of 

Arabidopsis. As a result, about two TSSs per 

locus were defined in either species. 

The nucleotide features around TSSs displayed 

distinct patterns when the most upstream TSSs 

were compared to downstream TSSs. First, GCand 

AT-skews, which were indices of biased 

nucleotide compositions, were clearly different 

between upstream and downstream TSSs, 

and this difference was commonly observed in 

rice and Arabidopsis. Second, relative entropy 

analysis revealed that the most upstream 

TSSs retained canonical cis-elements, whereas 

downstream TSSs showed atypical nucleotide 

features. Third, rice and Arabidopsis seem to 

have acquired different downstream TSSs after 

their speciation. Finally, expression patterns 

inferred from clone library names were distinguishable 

between upstream and downstream 

TSSs (Fig 4). These results indicate that plant 

TSSs were generally diversified in downstream 

regions, resulting in the development of new 

gene expression patterns. We concluded that 

rice and Arabidopsis might have evolved novel 

TSSs in an independent manner, which led to 

diversification of these two species, although the 

patterns of their evolutionary processes were 

similar. 

Fig 4. An example of a relationship between TSS usage and libraries from which cDNA clones 

were collected. 

Gene structures of two different TSSs from a locus on chromosome 8 are illustrated. The 

numbers of clones collected are shown below. 


Genebank 

Genebank takes 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 “center-bank” in cooperation 

with “sub-banks” as follows: 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 

of GRs, 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 of GRs, quality control and 

improved multiplication and preservation methods; 

and 5) advanced information management 

and disclosure system to facilitate use of GRs 

to accomplish various objectives of the Project. 

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 

works on relevant GRs and biodiversity. 

The NIAS Genebank Project conserves 

241,507 accessions of plant GRs (PGRs), 24,898 

accessions of microorganism GRs (MGRs), and 

984 accessions of animal GRs (AGRs) (data on 30 

November 2008). A total of 15,714 accessions of 

PGRs, 1,342 accessions of MGRs and 186 accessions 

of AGRs were distributed upon request 

to domestic and/or foreign users for breeding, 

research and/or educational purposes. Up-todate 

passport and evaluation data of distributable 

accessions have been uploaded onto and 

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

go.jp/). 

Genetic Diversity of Sorghum Germplasm in 

Asia and Africa 

Sorghum bicolor (L.) Moench ssp. bicolor is 

a cereal cultivated for human consumption, 

for animal feed, and for producing bio-fuel (Fig 

5). The species was domesticated in northern 

Africa and has spread to other parts of Africa, 

then to the world such as Asia, Australia and 

the Americas. 

Genetic diversity in sorghum GRs from different 

regions of Asia and Africa were analyzed 

using PCR on rice EST and InDel marker 

information. Of the 48 sorghum landraces 

Fig 5. Sorghum accessions grown in a field at the NIAS, Tsukuba. 


studied, 36 accessions originating from different 

regions of Asia and Africa, were obtained 

from the NIAS Genebank, and the remaining 

12 accessions originating from Central Asian 

region were obtained from the N. I. Vavilov All- 

Russian Research Institute of Plant Industry, St. 

Petersburg, Russia. 

Multivariate analysis of polymorphisms demonstrated 

three geographical landrace groups 

(two African and one Asian) which evidently 

reflect the genetic differentiation of sorghum 

during its evolution and geographical distribution 

around the world. Our investigation showed 

that Asian sorghums form a separate geographical 

group that has a complex genetic structure. 

Three sub-groups were recognized in the Asian 

cluster: South Asian, Central Asian and East 

Asian. These sorghum germplasms will be used 

for further genetic studies and breeding. 

Comparative Genomics of Asian Legume 

Domestication: Azuki Bean and Rice Bean 

Both azuki bean (Vigna angularis) and rice 

bean (V. umbellata) were domesticated in Asia 

(Fig 6, 7) and might have experienced similar 

morphological and physiological changes such 

as increase in organ size, loss of seed dormancy, 

and loss of pod dehiscence. Diversification has 

occurred during crop evolution in some traits 

such as seed coat color. 

Fig 6. Crop Evolution of Azuki Bean. 

Fig 7. Crop Evolution of Rice Bean. 


QTL (Quantitative Trait locus) analysis of 

domestication-related traits for rice bean was 

conducted using a BC1F1 population (198 

individuals) derived from a cross between a wild 

rice bean accession and a cultivated rice bean. 

The rice bean linkage map, which consisted of 

326 molecular markers on 11 linkage groups 

covering 796.1 cM of the rice bean genome, 

showed a high level of synteny with the linkage 

map previously constructed in azuki bean 

(Han et al, 2005). In total, 73 QTLs and one 

morphological marker gene were detected for 

31 domestication-related traits of rice bean. 

The domestication-related QTLs of rice bean 

were considerably lower in number but with 

higher effects than those of azuki bean. Some 

QTLs were found both in crops and some were 

specific to either one. It is interesting to note 

a QTL specific to rice bean on linkage group 4 

shows a large contribution to seed size increase 

(Fig 8). 

Development of Method for Effective Depletion 

of Endogenous Primordial Germ Cell (PGC) using 

Busulfan Sustained-Release Emulsion in Chicken 

The production of avian germline chimeras 

by the transfer of foreign primordial germ 

cells (PGCs) into recipient embryos provides a 

useful tool for generating viable donor-derived 

offspring. This procedure would be a powerful 

tool for preservation of endangered and/or 

rare poultry GRs. However, efficiency of obtaining 

donor-derived offspring from germ lime 

chimeras remains very low. The proportion of 

donor-derived offspring may be determined by 

the ratio between donor- and recipient-derived 

PGCs in the gonads of recipient embryos. 

Elimination of endogenous PGCs in the gonads 

was regarded as one of the most effective methods 

to enhance the proportion of donor-derived 

PGCs in chimeric gonads. 

Busulfan, an alkylating agent, has been used 

extensively to treat myeloid leukemia. It induces 

sterility in mammals during germ cell migration, 

and has similar effects on developing embryos in 

birds. Therefore, we tried to develop a busulfan 

delivery system to improve the efficiency of 

endogenous PGCs depletion and to increase the 

ratio of donor PGCs in the gonads of recipient 

chicken embryos. Busulfan was delivered using 

a sustained-release emulsion, which was prepared 

by emulsifying equal amounts of Ca 2+ and 

Mg 2+ -free PBS containing 10% busulfan dissolved 

in N,N-dimethylformamide and sesame oil, using 

a filter. A busulfan sustained-release emulsion (75 

µg per 50µL) was injected into the yolk. The 

busulfan sustained-release emulsion significantly 

reduced the number of endogenous PGCs with 

Rice bean specific QTL 

Useful for Azuki bean 

seed size increase 

Contribution < 10% 

10% ≤ Contribution < 20% 

20% ≤ Contribution 

Azuki bean specific QTL 

Rice bean specific QTL 

Common QTL 

Fig 8. Comparative genome map showing seed size QTL. Left: Rice bean, Right: Azuki bean. 


A 

B 

Busulfan treated embryo (A) 

(Sterility = 97%) 

Busulfan treated embryo (B) 

(Sterility = 100%) 

Nontreated control 

Fig 9. PGC depletion in the gonads of 6 day embryo treated with busulfan (Red spots 

indicate PGCs). 

less variation compared with control (Fig 9). 

It is suggested that the sustained-release 

emulsion delivered a consistent amount of busulfan 

to the developing chick embryos. The PGCs 

transfer study showed that the proportion of 

donor PGCs in the gonads of busulfan sustainedrelease 

emulsion-treated embryos after six days 

incubation increased 28-fold compared with control. 

Exogenous PGCs are migrating and settling 

in gonads from which endogenous PGCs have 

been removed using a busulfan sustained-release 

emulsion. Injecting busulfan sustained-release 

emulsion into the yolk of recipient embryos was 

effective for depletion of endogenous PGCs and 

increasing the ratio of donor PGCs in the gonads 

of recipient chicken embryos. 

Re-identification of Fungal Strains Belonging 

to the Fusarium solani Species Complex 

Preserved at Genebank Based on Molecular 

Phylogenetic Analyses 

Fungal species belonging to the Fusarium 

solani species complex (FS complex) are very 

important plant pathogens for agriculture. 

Strains of the FS complex stored at the NIAS 

Genebank are often distributed as references 

of the species used for identification and 

pathogenicity tests made by the users. Correct 

identification and labeling (i.e. quality control) of 

the strains is, therefore, extremely important. 

We analyzed DNA sequences of the histone 

H3 gene region (ca. 450 nucleotides) of all 84 

strains of the FS complex preserved at the 

central bank of NIAS and re-evaluated their 

taxonomic and phylogenetic positions, as well 

as addition of molecular IDs (i.e., DNA sequence 

data) to them. The histone H3 gene region 

contains rich phylogenetic signals suitable for 

strain identification at the species level. Based 

on the phylogenetic analysis with data on typical 

strains of the FS complex species and its 

allies, 73 strains (87%) examined were found as 

belonging to the FS complex (Fig 10), consisting 

of many phylogenetic clades often corresponding 

to the formae speciales of Fusarium solani. 

Ten strains within the FS complex required 

their name change, and 11 strains (13%) were 

apparently non-FS complex species. Distribution 

of these erroneously-labeled strains to the users 

was stopped temporarily and will be restarted 

after their precise re-identification and correct 

labeling of their scientific names. 

In addition to the phylogenetic analyses of the 

Fusarium strains, molecular re-identification of 

Trichoderma, Colletotrichum and Agrobacterium 

strains in the NIAS Genebank is in progress. 

Reference 

Han OK, Kaga A, Isemura T, Wang XW, 

Tomooka N, Vaughan DA (2005) A genetic 

linkage map for azuki bean [Vigna angularis 

(Willd.) Ohwi & Ohashi] Theor Appl Genet 

111: 1278–1287 


Fig 10. Neighbor-joining (NJ) phylogenetic tree of 84 strains of the Fusarium solani species complex 

stored at the NIAS Genebank inferred from DNA sequences of the histone H3 gene region together 

with the data on reference strains. 

Eighty-seven percent of the Genebank strains (73 strains) were found as belonging to the FS 

complex, consisting of phylogenetic clades often corresponding to the formae speciales of F. solani. 

Eleven strains (13%) were revealed to be non-FS complex species, and 10 strains within the FS 

complex also require their name change. Fusarium matuoi was used for an outgroup taxon. 



Research activities of the Institute of 

Radiation Breeding are focused on the development 

of new strains of seed-propagated, vegetatively 

propagated species and woody crops 

through mutation by the application of various 

forms of irradiation. Mutations are induced by 

the following radiation sources: Gamma Field 

for γ-ray chronic irradiations to the growing 

plants, and Gamma Room for γ-ray acute 

irradiations to seed, bulbs, tubers, scions and in 

vitro materials. The Institute is also involved in 

the development of new technologies for plant 

breeding mainly utilizing γ-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. 

Molecular Characterization of Seed Protein 

Composition Mutants in Rice Plants 

Through the use of mutation breeding, we 

developed a rice cultivar LGC1, which has a low 

glutelin and high prolamine content, from cv. 

Nihonmasari. Because of the indigestibility of 

prolamine, LGC1 can be used as a low-protein 

rice in practice. It is thus useful for people 

such as patients with kidney disease who must 

restrict their protein intake. Furthermore, we 

developed two new rice cultivars, ‘LGC-Katsu’ 

and ‘LGC-Jun’, from a cross between LGC1 and a 

mutant line of Koshihikari (induced by gammaray 

irradiation) that was deficient in 26-kDa 

globulin (another easy-to-digest protein) (Fig 11). 

The glutelin content of these two new cultivars 

is reduced to about one-third that of regular 

cultivars, and 26-kDa globulin is completely 

absent. Consequently, the total amount of easyto-digest 

protein in the new cultivars is about 

half that of regular cultivars, and about 15% 

less than in LGC1. Because the parental mutant 

line was induced from cv. Koshihikari, which 

is one of the most widely cultivated cultivars 

and known as the best tasting rice in Japan, the 

taste of two new cultivars is very similar to cv. 

Koshihikari. 

This low glutelin trait is conferred by a 

dominant mutation Low glutelin content 1 

(Lgc1). The results of molecular characterization 

of the gene showed that Lgc1 suppresses the 

expression of the glutelin multigene family. 

Lgc1 associates with a 3.5-kb deletion between 

two highly similar and tandem repeated glutelin 

genes, GluB5 and GluB4, with a tail-to-tail 

inverted arrangement (Fig 12). As GluB5 lacks 

a transcription termination signal, transcription 

from the GluB5 promoter generates a readthough 

product containing a sense-truncated 

GluB5 and an antisense GluB4, which can 

form a hairpin RNA with the GluB4/5 dsRNA 

regions. Because dsRNA suppresses the expression 

of homologous genes, which is referred to 

as RNAi, this deletion is thought to be a trigger 

for the suppression of glutelin expression in 

LGC1. 

Interestingly, we found that the mutant glu1, 

which is missing a subunit of Glutelin B protein, 

had a gamma-irradiation-induced deletion of 

about 129.7 kb. This deletion involved both 

GluB5 and GluB4, suggesting that both genes 

were knocked out (Fig 13). Thus, the difference 

of deletion size and position between Lgc1 

and glu1 is thought to cause the differences 

of phenotypes, suggesting that the regulation 

of deletion size caused by radiation induced 

mutation will possibly play an important role in 

elucidation of the function of the genes. 

What steps should we take to decrease easyto-digest 

protein to levels even lower than 

those in LGC-Jun or LGC-Katsu Ohyama et 

al. reported the genetic transformation of rice 

plants with chimeric antisense cDNA for glutelin 

to achieve lower glutelin content than that of 

LGC1. Furthermore, Kuroda M. (personal communication) 

succeeded in reducing prolamine 


content using a transgenic technique. Therefore, 

the development of a rice plant with little or no 

glutelin, a deficiency of 26-kDa globulin, and low 

content of prolamine is plausible. 

To avoid the issue of public acceptance of 

genetically modified crops, in Japan we could 

use artificial mutants rather than gene transfer 

in order to decrease easy-to-digest protein to 

levels even lower than those in LGC-Jun or 

LGC-Katsu. In these two cultivars, the combined 

effects of two genes, Lgc1 and glb1, which 

causes 26-kDa globulin deficiency, resulted in 

a further decrease in the content of glutelin 

compared to LGC1. Lgc1 can suppress the 

26-kDa globulin deficient 

LGC1 

LGC-Jun, LGC-Katsu 

Koshihikari 

glutelin (acidic subunits) 

26-kDa globulin 

glutelin (basic subunits) 

13-kDa 16-k Da prolamine 

easy-to-digest 

proteins 

difficult-to-digest 

proteins 

Fig 11. SDS-PAGE analysis of total proteins in brown rice. 

Fig 12. Molecular characterization of Lgc1. 


glu1 

Large deletion (129.7 kb) 

Lgc1 

Small deletion (3.5 kb) 

GluB4 

GluB5 

GluB4 

GluB5 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Fig 13. Both glu1 and Lgc1 are the mutations involved in the GluB4 and GluB5 genes. 

production of GluB protein sufficiently, but there 

is not necessarily adequate suppression of GluA 

protein production. Consequently, there is still 

room for a further decrease in levels of easyto-digest 

protein. The whole genome sequence 

revealed by the genome projects has brought 

to light three genes that are responsible for the 

production of GluA protein. Two of them (glu2 

and glu3) have already been isolated by gammaray 

irradiation. To isolate this unknown fifth 

gene (i.e. in addition to the 4 genes Lgc1, glb1, 

glu2, and glu3), screening by a reverse genetic 

approach will be important. The accumulation 

of these five genes will produce a line that is 

almost completely free of glutelin and would 

be the ultimate rice line for minimizing the 

level of easy-to-digest protein. To accumulate 

the five genes, DNA marker-assisted selection 

is an indispensable tool, because each of the 

phenotypes expressed by the 5 genes is difficult 

to discriminate clearly by SDS-polyacrylamide 

gel electrophoresis. This research will not only 

include marker-assisted selection breeding, but 

also post-genome breeding that will follow the 

whole genome sequence mapping. 

Effects of dose and dose rate of gamma ray 

irradiation on mutation induction and nuclear 

DNA content in chrysanthemum 

Chrysanthemum is one of the most widely 

cultivated ornamental plants. As chrysanthemum 

is vegetatively propagated, mutation is 

an important tool for breeding new cultivars, 

and gamma rays and X-rays are widely used 

for mutation induction. However, in addition to 

mutations, radiation treatment induces radiation 

damages, such as chromosomal aberrations, in 

plants. A radiation method causing less radiation 

damage is desired for chrysanthemum. Dose 

rate is one important factor of radiation treatment 

and it is believed that the dose rate also 

influences radiation damage. However, data are 

not available regarding the effect of total irradiation 

dose and dose rate on mutation induction 

in chrysanthemum. Therefore, information on 

the effect of dose rate on radiation damage and 

mutation induction and the interaction between 

dose and dose rate is useful for radiation 

breeding. 

We investigated the effect of total irradiation 

dose and dose rate on flower color mutation and 


Total dose Dose rate 

Frequency of flower 

color mutation 

(Gy) (Gy/h) 

(%) 

0.5 4.6 (15/325) 

15 1 5.0 (18/363) 

2 4.3 ( 9/210) 

0.5 8.9 (22/248) 

30 1 12.1 (29/240) 

2 9.6 (26/272) 

0.5 12.2 (40/326) 

60 1 8.4 (22/262) 

2 12.0 (12/100) 

Table 2. Effects of total dose and dose rate of 

gamma ray irradiation on mutation induction. 

Numbers of flower color mutants/number of 

plants evaluated are shown in parentheses. The 

frequency of spontaneous mutation was 0.6%, 

as estimated using 937 plants that originated 

from non-irradiated leaf explants. 

Frequency (%) 

80 

60 

40 

20 

0 

80 

60 

40 

20 

0 

80 

60 

40 

20 

0 

15 Gy 30 Gy 60 Gy 

0.5 Gy/h 

Average = 99.4 

Variance = 1.6 

0.5 Gy/h 



0.5 Gy/h 



1 Gy/h 



2 Gy/h 



0.89-0.91 

0.91-0.93 

0.93-0.95 

0.95-0.97 

0.97-0.99 

0.99-1.01 

1.01-1.03 

80 

60 

40 

20 

0 

1 Gy/h 



2 Gy/h 



0.89-0.91 

0.91-0.93 

0.93-0.95 

Non-irradiation 



0.89-0.91 

0.91-0.93 

0.95-0.97 

0.97-0.99 

0.99-1.01 

1.01-1.03 

0.93-0.95 

0.95-0.97 

0.97-0.99 

0.99-1.01 

1.01-1.03 

Relative DNA content 

1 Gy/h 



2 Gy/h 



0.89-0.91 

0.91-0.93 

0.93-0.95 

0.95-0.97 

0.97-0.99 

0.99-1.01 

1.01-1.03 

Fig 14. Effects of total dose and dose rate of gamma ray irradiation on nuclear DNA content. 

Relative DNA content is expressed as the ratio of the nuclear DNA content of investigated plants 

divided by that of control plants maintained by vegetative propagation. Nuclear DNA content was 

measured in 50 regenerated plants per treatment. 

nuclear DNA content as an index of radiation 

damage in chrysanthemum. Chrysanthemum 

morifolium cv. ‘Taihei’ plants generated by in 

vitro culture were irradiated with a total dose of 

15, 30 and 60 Gy of gamma rays at a rate of 0.5, 1, 

2 and 5 Gy/h. Leaf explants cut from the irradiated 

plants were tissue-cultured, and frequency 

of flower color mutation was investigated. 

Nuclear DNA content was measured by flow 

cytometric analysis. Mutation frequency did not 

differ significantly among dose rates, indicating 

that mutation frequency was independent of 

dose rate and was dependent mainly on total 

dose (Table 2). Comparison of the average 

nuclear DNA content with each treatment 

revealed that it was influenced by both dose 


Mutation frequency (%) 

14 

12 

10 

8 

6 

4 

2 

r = -0.999 * 

2 Gy/h 

1 Gy/h 

0.5 Gy/h 

r = -0.898 

r = -0.679 

Fig 15. Relationship between reduction 

of nuclear DNA content and mutation 

induction. 

Relative DNA content is expressed as 

the ratio of the nuclear DNA content of 

investigated plants divided by that of 

control plants maintained by vegetative 

propagation. Nuclear DNA content was 

measured in 50 regenerated plants per 

treatment. 

0 

0.95 0.96 0.97 0.98 0.99 1 

Relative DNA content 

rate and total dose, and that the reduction in 

nuclear DNA content was less at low dose rates, 

even when total doses were high (Fig 14). It 

appears that similar mutation frequencies were 

obtained without a large reduction in nuclear 

DNA content by 0.5 Gy/h, when compared with 

2 Gy/h (Fig 15). Consequently, we conclude that 

gamma ray irradiations of high total doses at 

low dose rates efficiently induce mutations with 

less radiation damage in chrysanthemum. 

Distinction between two cytochimeras by flow 

cytometry 

In angiosperms, including most crops, both 

artificial and spontaneous mutations often 

generate and develop in a chimera state, that 

is, as a periclinal chimera. In practical mutation 

breeding for vegetatively propagated crops, stability 

of the chimera and the technique to isolate 

a complete mutant plant from the chimera are 

important. 

Cytochimeras (ploidy chimeras) are useful 

materials for the study of chimeras because 

the distribution of diploid and tetraploid cells in 

a target tissue or organ can be monitored by 

observation of the cellular and nuclear sizes by 

histological methods such as paraffin sectioning, 

unless polysomaty occurs in the target tissue or 

organ. Polysomaty is the co-existence of various 

ploidies of nuclei in a plant tissue and organ. 

Polysomaty may confound diploid-derived nuclei 

and tetraploid-derived nuclei in an observed 

tissue and organ. 

We have two types of mulberry periclinal 

cytochimeras with diploid and tetraploid cells, 

of which distributions of diploid and tetraploid 

cells differ from each other in the shoot apical 

meristem (SAM). One cytochimera has tetraploid 

cells in the L1 layer of the shoot apical 

meristem (4-2-2) and another has tetraploid cells 

in L2 and L3 layers (2-4-4) as demonstrated by 

paraffin sectioning of SAM. When we observed 

the SAM of seven cytochimeras by paraffin 

sectioning, all the cytochimeras were of these 

two types without exception indicating the two 

types of chimeras are likely the majority among 

mulberry cytochimeras. 

Flow cytometry has recently been used for 

analyzing the nuclear DNA contents and ploidy 

level of various plants. We investigated whether 

flow cytometry can be used to distinguish one 

type of periclinal cytochimera from another 

type. In a preliminary analysis, we determined 

whether polysomaty occurred in various 

organs of several mulberry species, which may 

cause misestimates of ploidy obtained by flow 

cytometric analysis. We did not find markedly 

multiple peaks that represent polysomaty in any 

flow cytometric histograms of the above-ground 

vegetative organs of mulberry. The histograms 

of the two cytochimeras indicated two peaks, 

one peak (2C peak) represents diploid nuclei and 

the other peak (4C peak) represents tetraploid 

nuclei. The two cytochimeras were identified 

by the percent of the area of the 4C peak to the 

total area of both 2C and 4C peaks. The percent- 


ages of the 2-4-4 cytochimeras examined were 

between 50.5 and 93.4%. In contrast, the percentages 

of 4-2-2 cytochimeras were 5.9-35.7%. The 

2-4-4 chimera also was easily distinguishable 

from diploid and tetraploid patterns obtained by 

flow cytometry (Fig 16). The combination of the 

cytochimeras and flow cytometry technique is a 

new method to analyze the state and change in 

chimeras. 

Fig 16. Histograms of diploid, 2-4-4 

cytochimera and tetraploid by flow 

cytometry. 


Soybean, Glycine max, is the most important 

legume and is the fourth crop next to rice, 

wheat and maize in terms of world crop production. 

Soybean contains abundant protein and 

oil, which make it a major source of nutritious 

food for humans and livestock. In addition, it 

provides industrial materials and biofuel. In 

Japan, soybean is an important source of traditional 

staple 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 methods for effective 

breeding and for the isolation of agronomically 

important genes. The most serious problems in 

soybean production in Japan are unstable and 

low yields. To overcome these problems, many 

traits including flood stress resistance, disease 

and pest resistance, low temperature tolerance, 

symbiotic ability and maturity depending on the 

regions where soybean is cultivated should be 

improved. Identification of the responsible genes 

or molecular markers linked to these traits is 

necessary. 

The estimated size of the soybean genome is 

1.1 Gb. To develop the basic resources for analyses 

of the whole genome, we have constructed 

two bacterial artificial chromosome (BAC) 

libraries from the Japanese soybean cultivar 

Enrei using the cloning restriction enzymes 


HindIII and MboI. We have sequenced Enrei 

BAC end sequences since last year to develop a 

BAC-based physical map and database by using 

Williams 82 genome assembly. We sequenced 

about 80,000 clones from a HindIII-library and 

about 20,000 clones from a MboI-library. To 

construct a BAC-based physical map of Enrei 

genome, BLASTn analysis between the BACend 

sequences of Enrei and genome assembly 

of Williams 82 (Glyma1 assembly, http://www. 

phytozome.org/soybean.php) was carried out. 

This physical map will be used for characterizing 

the genome structure of Japanese soybean 

cultivars in order to accelerate the isolation of 

agronomically important genes and to develop 

new breeding strategies for increasing the yield 

of Japanese cultivars. We are also constructing 

a soybean genome database, “DaizuBase”, to 

facilitate comparative genome analysis. Unified 

map of DaizuBase indicates the relationship 

between the linkage map and physical maps (Fig 

17). Gbrowse shows aligned Enrei BAC clones 

on Glyma1 assembly (Fig 18). At present, only 

the participants in the DD-project (Genomics for 

Agricultural Innovation of MAFF) can utilize 

DaizuBase, but consultation will be opened by 

the end of 2009. We introduced a next generation 

sequencer GS-FLX (Roche/454) to decode 

the Enrei genome for the characterization of 

the genome structure of Japanese (domestic) 

soybean. The huge sequencing ability will enable 

us to conduct a comparative analysis of the 

soybean genome structure. 

Since research on genome mapping of 

soybean at NIAS has been initiated recently, we 

currently focus on building up a genetic base of 

soybean and designing a marker development 

system to accelerate breeding in collaboration 

with soybean breeders and researchers in 

Japan. Though a genetic linkage map with 

about 1,800 markers has been developed in the 

U.S., the marker order on the map is sometimes 

Fig 17. Screen view of UnifiedMap on DaizuBase. 

This view offers information about DNA markers, BAC clones and the relationship between the 

linkage map and physical maps. 


Fig 18. Screen view of Gbrowse of DaizuBase. 

BAC contigs are shown on each chromosome of the Williams 82 genome assembly. 

obscure because the information is integrated 

from several linkage maps from different 

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 in order to obtain more 

precise marker information. We selected three 

parents, Enrei, Peking and Williams 82 for the 

construction of the genetic linkage map. Enrei 

is a very famous cultivar with high quality for 

food processing. On the other hand, Peking 

displays many useful characteristics such as 

cyst nematode resistance, soybean mosaic virus 

resistance, and Phytophthora rot resistance. In 

addition, Peking displays high stress resistance 

in germination under wet conditions, compared 

with modern cultivars. In Japan, the use of 

soybean fields converted from paddy fields has 

increased. Since seeds are sown before or at 

the beginning of the rainy season, excess water 

in soybean fields damages seeds and seedlings, 

leading to a reduction in yield. Good germination 

under wet conditions has become very important 

in Japan. We initiated the construction of a 

high density linkage map using these progenies. 

Initially, 294 genomic SSR markers developed 

by Cregan et al, (http://soybase.org/resources/ 

ssr.php), including markers that had been 

used for MAS and mapping by breeders and 

researchers, were anchored on the linkage map 

to cover the genome widely. Subsequently, 386 

newly developed EST-SSR markers by Hisano 

et al. (http://www.kazusa.or.jp/soymarker/) 

were integrated into the map. The genome 


Fig 19. A genetic linkage map based on F2 population derived from Enrei x Peking (left white 

bar). 

Physical map of Williams 82, Glyma 1.0, is aligned (right black bar) and marker locations are 

connected by black lines. For each chromosome, the physical map consists of a region of 

highly suppressed recombination between markers on the linkage map. These regions shown 

on the same scale on the right side are connected by gray lines and may correspond to the 

pericentrometric regions. 

sequence of a U.S. cultivar Williams 82, Glyma 0, 

has been available since January 2008 (Soybean 

Genome Project, DoE Joint Genome Institute, 

http://www.phytozome.net/soybean.php). New 

SSR markers were designed in large scaffolds 

(>100kb) and have been incorporated into the 

map in order to obtain a precise assembly of the 

genome sequences in Japanese soybean genetic 

background. Currently, 1117 SSR markers have 

been located on the map. This map spans a 

total length of 3044 cM with an average marker 

distance of 2.8 cM (Fig 19, left bar). There 

are several large gaps ( >10 cM) on the map. 

Severe segregation distortion (P=0.001) was 

observed on Gm06 (LG C2) and Gm11 (LG B1) 

(Fig 19 blue bar). The map length was longer 

(25%) than that of the soybean consensus map 

3.0 developed by Cregan et al. (http://soybase. 

org/LG2Xsome.php). The marker location on the 

linkage map (Fig 19, black dot beside left bar) 

was compared with that of the chromosomescale 

assembly of the soybean genome, Glyma 

1.0, updated in December 2008 (Fig 19, right 

black bar). In total, 1034 markers (93%) were 

anchored on the genome assembly. The 

other 75 markers were localized either on the 

unexpected chromosome (66) or unassigned 

scaffolds (9). Generally, the marker order on the 

linkage map revealed a close agreement with 

that of physical data. Although the relationship 

between genetic distance and physical 

distance appears to be ~360 kb/cM, assuming 

that the genome size is approximately 1.1 Gb, 

positional biases (23kb – 6.9Mb/cM) were found. 

In each chromosome, a genomic region that had 

considerably suppressed recombination between 

markers, possibly the pericentrometric region, 

was observed and the extent differed among 

the chromosomes. Currently, the cause of the 

large differences (Fig 19, red frame) on Gm05 

(LG A1) and Gm13 (LG F) and the other minor 

differences between Williams 82 and Enrei x 

Peking are being elucidated. This map will 

provide information to identify regions in the 

sequence assembly where additional information 

is required to resolve the marker order. 

The control of flowering time is an important 

objective in soybean because it is critical for 


Fig 20. Variation of gene structure of GmPhyA3 and photo-sensitivity. 

adapting cultivars to different cultivation areas 

and growing seasons. It had been reported that 

flowering time and maturity are controlled by 8 

alleles, E1/e1, E2/e2, E3/e3, E4/e4, E5/e5 E6/ 

e6, E7/e7 and J/j. By QTL analysis of the flowering 

time using a RIL population derived from 

Misuzudaizu and Moshidou Gong 503, three 

major QTLs, FT1, FT2 and FT3, were found to 

correspond to E1, E2 and E3, respectively. The 

strong candidate gene for E3, a phytochrome 

A gene (GmPhyA3), was isolated by map-based 

strategy using a residual heterozygous line 

(RHL) (Watanabe et al. in press). Allelism test 

and comparison of the gene structure showed 

that the gene function of Misuzudaizu and 

Harosoy was identical, though the gene from 

Harosoy harbored a large insertion at the third 

intron. One amino acid substitution or large 

deletion in the kinase domain might lead to a 

defect or loss of function of GmPhyA3 in the 

allele of Moshidou Gong 503 and Harosoy-e3, 

respectively. Moreover, a mutant line harboring 

non-functional GmPhyA3 due to a 40bp deletion 

at the first exon, displayed an early flowering 

phenotype compared with the original variety 

Bay under high R/FR conditions. These results 

strongly suggest that GmPhyA3 controls the 

flowering time and maturity of soybean. The 

phenotypes and gene structures are summarized 

in Fig 20. 



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. 


The 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 

The 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. 


The 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. 


The unit focuses on structures and functions 

of various proteins using NMR and X-ray 

diffraction to establish an important basis for 

utilizing genome information. 


The unit 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. 


The unit aims to develop new methods for 

further improvement of the safeness of GM 

crops and strategies leading to optimal regulation 

of the expression of introduced genes. 

The major research topics in fiscal 2008 are 

described in the following pages. 



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 2008 fiscal year. 

The voltage dependent chloride channels 

contribute to salt tolerance in rice 

Rice cells accumulate Na + ions in vacuoles 

to remove the ions from the cytoplasm and 

enhance the osmotic pressure under a high 

salt environment. Chloride ions accumulate 

simultaneously in vacuoles as counter ions. 

These ionic transport systems must contribute 

to salt tolerance in rice. Many papers report 

that Na + /H + antiporters localized in tonoplasts 

and plasma membranes transport Na + from the 

cytoplasm. On the other hand, we know little 

about intercellular behavior of chloride ions. 

The voltage-dependent chloride channel is 

an important candidate for a chloride transport 

system and five potential chloride channel genes 

were identified in the rice genome database. 

We isolated cDNA clones for these genes (Fig 

1). The cDNAs were transformed into a yeast 

mutant gef1, which is its only gene for the 

chloride channel, to test whether the cDNAs 

corrected the deficient mutant phenotype. The 

results indicated that the cDNAs encode chloride 

channel proteins. Two genes, OsCLC-1 and 

OsCLC-2, were analyzed in detail. Both genes 

have six introns, and the exon-intron structure 

was conserved in AtCLC-a, -b and -c from 

Arabidopsis thaliana. We showed immunochemically 

that proteins from both genes were localized 

in vacuolar membranes. Furthermore, we 

obtained rice Tos17-insertion mutants for each 

gene and analyzed the phenotypes to clarify the 

function of each gene. The mutant for OsCLC-2 

was more sensitive to salt stress than wild type, 

Nipponbare, and tolerance was recovered by 

transformation of OsCLC-2 or OsCLC-1 cDNA. 

However, Tos17 disruption of OsCLC-1 did not 

change salt tolerance. Furthermore, the disruption 

of OsCLC-2 increased the requirement for 

nitrate for optimal growth, but this was not the 

case in OsCLC-1. The nitrate requirement was 

diminished by the transformation of OsCLC-2 

or OsCLC-1 cDNA. In the field, growth was 

inhibited only in the OsCLC-2 mutant and yield 

and seed size also were decreased. 

Fig 1. The phylogenetic tree of CLC proteins (Red color indicates the rice CLC proteins). 


QTLs for deeper rooting in rice 

The global warming and desertification in 

recent years cause serious drought damage 

to the rice-growing 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 

(Fig 2). In an upland field, a new quantitative 

trait locus (QTL) controlling deeper rooting in 

rice was detected on chromosome 9 in a BC 2 F 2 

population derived from the cross between the 

lowland and the upland cultivars. Fine mapping 

of the target QTL was performed using the 

basket method to evaluate root growth (Fig. 

3). We compared the number of roots emerged 

from the bottom of the basket to the number of 

all roots emerged from the basket, and set the 

ratio as a parameter to evaluate deeper rooting. 

Using this method, a clear phenotypic difference 

was observed in root distribution between the 

parental lines. We selected eight BC 2 F 3 plants, 

in which recombination occurred in the region 

near the target QTL, and bred self-pollinated 

progeny (BC 2 F 4 ) out of these selected lines. We 

also selected two homozygous lines for lowland 

and upland cultivars without recombination in 

the region flanking the target QTL as genotype 

controls. The homozygous line having the allele 

of upland rice had a deeper rooting ratio of 

40.4%, compared with 2.6% for the homozygous 

line with the allele of lowland rice. This 

indicated that allelic differences governing root 

distribution in the target QTL were confirmed 

using the basket method (Fig 3). Accordingly, 

a deeper rooting QTL was mapped on chromosome 

9 as a single Mendelian factor. 

Fig 2. Deep root can absorb water deposited in the deep soil layers. 

Fig 3. Upland rice allele of deeper rooting QTL increased deep root in lowland rice. 


Functional analysis of seed dormancy regulator, 

Sdr4. 

A QTL termed Sdr4 is a pre-harvest sprouting 

resistance gene of ‘Kasalath’ indica rice. 

In an sdr4 mutant, the expression level of 

the DOG1 like gene, which is an ortholog of 

Arabidopsis seed dormancy regulator gene, 

namely Delay of Germination 1 (DOG1), was 

reduced, and the aquapolin gene, PIP1;3 which 

was reported to be a promoter of germination, 

was induced. These results were in agreement 

with the viviparous phenotype of sdr4 mutants. 

Furthermore, Sdr4 expression levels were 

reduced in Osvp1-1 and Osvp1-2 mutants, 

suggesting that the seed formation master regulator, 

OsVP1, regulates Sdr4 expression. This 

recent work of ours has revealed part of the 

seed dormancy regulation network; however, 

the downstream events in dormant and nondormant 

seeds are still unclear. 

To address downstream events, morphological 

and expression analyses of sdr4 mutant 

were performed. First, embryo size of the sdr4 

mutant was compared to that of wild type. At 

28 days after flowering (DAF), thin sections of 

seed were stained by toluidine blue (Fig 4A). 

It turned out that the size of embryo in sdr4 

mutant (M100) was larger than that in wild 

type (Npb). The weight of sdr4 mutant (M100) 

embryos was approximately 1.6-fold larger 

than wild type (Npb). Next, we determined the 

concentrations of phytohormones and found 

that IAA concentration of sdr4 mutant was 

reduced approximately 40% compared to that 

of wild type (data not shown). Consequently, 

we determined the expression levels of a key 

enzyme gene, Yucca1, for IAA synthesis. The 

expression levels were decreased in the sdr4 

mutant throughout seed maturation (Fig 4B) in 

agreement with the low IAA concentration. It 

has been reported that auxin or its precursor 

Trp inhibits germination of wheat, and secondary 

dormancy induction by high temperature 

is due to the accumulation of auxin in lettuce 

seeds. The result obtained here elucidated the 

contribution of auxin in seed dormancy regulation. 

GA and ethylene promote germination in 

general, so we also determined the expression 

levels of ACS genes, encoding a key enzyme 

in ethylene synthesis. Among the ACS genes, 

activity of ACS7 was highly induced in the 

sdr4 mutant. This result suggests that ethylene 

might also be involved in rice seed dormancy/ 

germination processes. Further analysis of the 

relationship between these phytohormones and 

seed dormancy will be required. 

Fig 4. Morphological and expression analysis of sdr4 mutant. 

(A) Comparison of embryos from sdr4 mutant (M100) and wild type (Npb). Seed sections 28 days 

after flowering (DAF) were stained with toluidine blue (left). The weight (n=50 with three repeats) 

of sdr4 mutant was approximately 1.6-fold larger than wild type (right). (B) The expression levels 

of the key enzyme gene in IAA synthesis, Yucca1 (left), and that in ethylene synthesis, ACS7 (right). 

Samples were collected from embryos 10 to 42 DAF. 


Photobiology and Photosynthesis Research Unit 

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. 

A Dof transcription factor gene RDD1 is 

regulated by the circadian clock and phytochromes 

and associated with grain size in rice 

Plants can adapt to changes in environmental 

conditions during the day, and mechanisms 

known as the circadian clock impose a persisting 

rhythm on plants over a period of approximately 

24 hours. The circadian clock regulates 

accumulation of transcripts for a variety of 

genes including transcription factor genes. We 

characterized a rice gene encoding a putative 

Dof transcription factor, named RDD1. The 

level of RDD1 transcripts showed daily oscillations 

irrespective of light conditions, indicating 

regulation by the circadian clock. Experiments 

of light responsiveness in etiolated coleoptiles 

revealed that the RDD1 transcripts accumulated 

in darkness and decreased after the onset 

of illumination. The expression analysis using 

phytochrome-deficient mutants indicated that 

phytochromes contributed to the regulation of 

RDD1 expression. 

Transgenic rice plants named RDD1-S and 

RDD1-AS which carried RDD1 in sense and 

antisense orientations, respectively, under 

the control of a constitutive promoter, were 

produced to examine the role of RDD1. 

Phenotypic analysis indicated that RDD1-AS 

plants were smaller than wild type and RDD1-S 

plants (Fig 5). Furthermore, RDD1-AS plants 

showed a decreased grain size (Fig 5A, inset), 

and the 1000-grain weight was reduced by 

24% compared to that of wild type plants. By 

contrast, RDD-S plants did not show any clear 

Fig 5. Phenotype of RDD1 transgenic rice plants. 

Wild type (cv. Nipponbare; WT), RDD1-S (S) and RDD1-AS (AS) plants were grown in a 

greenhouse to maturity. (A) Plants of the ripening stage and grains (brown rice). (B) Comparison 

of plant heights. Averages ±SE (n = 3–6) are shown. Asterisks indicate a significant difference 

from wild type plants using Student’s t-test (P < 0.05). 


phenotypes. Since expression of the transgene 

was very low in these plants, it is likely that 

there are some mechanism(s) controlling the 

transcript level of RDD1. RRD1 could be a target 

gene for improving rice productivity if the 

mechanism(s) controlling the RDD1 expression 

could be elucidated. 

Diversification of CK2 targets in circadian 

clocks and photoperiodic flowering in plants 

Photoperiodic control of flowering is regulated 

by both light signals and circadian clocks and 

varies among species, although molecular 

mechanisms conferring the distinct responses 

are largely unknown. Casein kinase II (CK2) 

is an evolutionarily conserved protein kinase 

known as a circadian clock component in the 

long-day plant Arabidopsis thaliana. Another 

circadian clock component CIRCADIAN CLOCK 

ASSOCIATED 1 (CCA1) is a CK2 target in A. 

thaliana, where it influences photoperiodic flowering. 

In rice, a short-day plant, Hd6 encodes a 

CK2α subunit and delays flowering under longday 

conditions. We found that the floweringtime 

control by the rice Hd6 CK2 α genetically 

requires a functional Hd1 gene, an A. thaliana 

CONSTANS orthologue (Fig 6). Unlike A. thaliana 

CK2, however, Hd6 does not affect circadianclock 

related gene expression in rice (Fig 7). The 

loss of control of circadian clocks by Hd6 CK2α 

could be due to an amino acid change from Ser 

to Glu in the corresponding CK2 target site of 

CCA1 in OsLHY (a CCA1 orthologue), although 

this site is critical for OsLHY function to control 

OsPRR1 gene, a rice orthologue of A. thaliana 

TOC1/PRR1 circadian clock gene. These observations 

disclose molecular mechanisms underlying 

how the evolutionarily-conserved CK2 has 

contributed to the biodiversity of flowering-time 

control in plants. 

Fig 6. Hd6 required the functional Hd1 to delay flowering under long-day conditions in rice. 

Rice plants of different Hd1 and Hd6 genotypes were grown under short-day (SD) or long-day (LD) 

conditions, and days to flowering after sowing were determined. ‘–’ and ‘+’ indicate non-functional 

and functional alleles, respectively, and ‘ox’, and ‘Tik’ for the Hd6 genotype indicate overexpressors 

and the dominant negative CK2α driven by the CaMV 35S promoter, respectively. Each bar 

represents means ± SD from 10 to 20 plants. 


Fig 7. Hd6 repressed FT-like genes and acted downstream of Hd1 under long-day conditions but did 

not control circadian clocks. 

(A-D) Bioluminescence analysis of cab1R::luc expression in constant darkness (DD; A) and constant 

light (LL; C) after light/dark entrainment for five days. Period lengths and relative amplitude errors in 

DD (B) and in LL (D) were calculated from luminescence data by an FFT-NLLS method. Hd6 and hd6 

lines were T3 (or T4) progeny lines from crosses with the cab1R::luc line of japonica cultivars with 

Hd6 and hd6 alleles, respectively. Hd6 overexpressing (ox) lines were from a cross between the Hd6 

ox line and the cab1R::luc line. (E-K) Expression levels of Hd6 (E), OsLHY (F), OsPRR1 (G), OsGI (H), 

Hd1 (I), Hd3a (J), and RFT1 (K) in Nipponbare (WT), NIL (Hd6) and two Hd6 ox lines measured by 

real-time quantitative RT-PCR. Data were normalized against ubiquitin expression levels. Means and 

SD were plotted from three biological replicates. Each plant was grown under long-day conditions 

(14.5L/9.5D) for 60 days. Young leaves from three plants were collected every 2h after dawn. 

Developing the high-throughput screening 

system for rice phenome analysis using digital 

image technology 

With the completion of the rice genome 

sequencing, the next major challenge is the 

systematic determination of gene function 

and searches for useful genes for breeding. 

Difficulties can then arise in the quantitative 

analysis of phenotypic traits. Comprehensive 

measurements of visible phenotypes throughout 

the growth process are especially important 

because it is difficult to predict when and where 

the growth pattern and/or rate varies to bring 

about phenotypic variation. To solve these 

problems, we have been developing a digital 

imaging system and constructing a large-scale 

image database for comprehensive analysis of 

visible phenotypes in rice seedlings. 


Fig 8A shows the imaging system. The 

seedling growth images and data are recorded 

every ten minutes for a week from germination 

until the second leaf becomes fully developed 

(Fig 8B). The image analysis software traces the 

shoot apex automatically and records its position 

based on orthographic projection data (Fig 

8C). From the traced data, various parameters 

that express dynamics and kinetics of seedling 

growth at high temporal resolution can be 

determined: these include timing of elongation 

start and stop, leaf appearance cycle, starting 

and ending position of leaf elongation, and velocity 

of leaf elongation (Fig 9). This system also 

enables us to analyze other kinetic parameters. 

Growing profiles of coleoptiles and seminal roots 

can be constructed from collected image data 

using an image processing algorithm. Combined 

with physiological and molecular biological 

methods, this systematic and comprehensive 

phenotypic profiling system may provide new 

insights into phenotypic trait measurements, 

advance our understanding of gene functions 

in plant growth and development, and provide 

new information useful for crop breeding and 

cultivation techniques. 

Rotation table 

Digital camera 

Rotation 

(B) Acquired growth images 

(A) Imaging system 

(C) Image analysis software software measuring measuring leaf tipleaf 

tip points and and leaf leaf length length automatically automatically 

Fig 8. The imaging system for rice seedling growth 

Detected leaf 

tip points 

Shoot apex height 

Horizontal distance of leaf tip 

Rice (Nipponbare) 

grown under dark 

conditions 

(A) Leaf elongation analysis 

(B) Coleoptile circumnutating analysis 

Fig 9. Data analyses for various growth parameters. 


Disease Resistance Research Unit 

Panicle blast resistance gene Pb1 encodes 

a CC-NB-LRR protein with unique molecular 

properties 

Rice blast, caused by Magnaporthe oryzae, is 

one of the most widespread and destructive 

diseases of rice. ‘Panicle blast’ is due to blast infection 

of panicles occurring after heading stage, 

and causes direct damage to rice yield and 

grain quality. Pb1 is a panicle blast resistance 

gene, which was identified as a major quantitative 

gene in rice cultivars derived from an 

indica rice cultivar, Modan. The blast resistance 

attributed to Pb1 is more effective during adult 

stages (adult resistance), especially for panicle 

blast, than during young stages (leaf blast), and 

has not experienced breakdown for more than 

20 years despite its cultivation in as much as 

100,000 ha a year (durable resistance). 

We have isolated the Pb1 gene by map-based 

cloning to investigate the mechanisms underlying 

its characteristic durable resistance. Pb1 

encoded a protein of CC–NB–LRR structure, 

which is conserved in many resistance (R) 

proteins that mediate gene-for-gene-based true 

resistance. Pb1 protein is unique in its NB 

domain as compared to those of R proteins; the 

NB domain of Pb1 lacks the P-loop and has a 

highly degenerated kinase-2 motif. Both the 

P-loop and kinase-2 motifs, which are highly 

conserved among R proteins, are implicated in 

the nucleotide binding that is required for the 

activation of R proteins; therefore, it is possible 

that the unique structure of NB domain is one 

of the determinants responsible for the durability 

of Pb1 resistance. In addition, Pb1 was found 

to physically interact with WRKY45, a central 

transcription factor involved in the salicylic 

acid signaling pathway in rice. This is another 

characteristic feature of Pb1 that may account 

for the durable resistance of Pb1 cultivars. 

Systematic screening of recessive rice-blast 

resistant mutant lines 

Use of host resistance genes will be the best 

solution to control crop diseases including rice 

blast. At present, dominant disease-resistance 

genes (R genes) are most commonly used for 

disease-resistance breeding. Because R-gene 

based resistance easily breaks down due to 

outbreaks of new fungal races, additional and 

more complicated systems may be required. 

Recently, a blast resistant gene that confers 

durable resistance was identified and found to 

be due to recessive mutation in a single gene 

(pi21). To effectively screen for new recessive 

resistance genes for fungal diseases, we have 

developed a new screening method that mimics 

natural infection conditions in greenhouses. We 

applied this method to screening for rice blast 

resistance lines from Tos17-mutant panel of cv. 

Nipponbare. As a result of screening ~20,000 

lines over three years, we have identified 11 candidates 

of mutant blast-resistant lines (CMrLs). 

Blast resistance tests under severe upland field 

conditions verified that 9 out of the 11 CMrLs 

were resistant against naturally occurring blast 

races, with the resistance being even stronger 

than those of reference field-resistance lines. 

The CMrLs were crossed with cv. Koshihikari 

and resulting F1 lines are now grown in the 

field to introgress the new resistance genes into 

the elite cultivar. 

Screening of rice-Arabidopsis FOX lines identified 

a multi-disease resistance gene 

By screening Arabidopsis lines that overexpress 

13,000 rice full-length cDNAs (FOX 

hunting lines), we identified 84 lines that are 

resistant to a bacterial pathogen, Pseudomonas 

syringae pv tomato DC3000 (Pst 3000) and 109 

lines that are resistant to a fungal pathogen, 

Colletotrichum higginsianum. Of these, 42 lines 

were resistant to both the pathogens and their 

causative genes were identified. When overexpressed 

in rice, one of those genes, OsPSR5, 

encoding a previously undescribed receptor-like 

cytoplasmic kinase, conferred resistance to both 

Xanthomonas oryzae and Magnaporthe oryzae, 


the causal pathogens of bacterial leaf blight 

and fungal blast diseases, respectively. These 

results confirm the utility of the rice-Arabidopsis 

FOX hunting lines in rapidly identifying genes 

that confer disease resistance to both monocot 

(rice) and dicot (Arabidopsis) plants. OsPSR5- 

overexpressing Arabidopsis plants showed only 

minor upregulation of PR1, a marker gene for 

the salicylic-acid (SA) pathway, after inoculation 

with Pst 3000, while the constitutively 

defense-activated cpr5-2 mutant showed high 

levels of PR1 expression with or without Pst 

3000 inoculation (Fig 10). By contrast, the 

OsPSR5-overexpressing plants showed greatly 

enhanced upregulation of PDF1.2, a marker 

gene for the ethylene/jasmonic acid (ET/JA) 

pathway, after Pst 3000 inoculation (Fig 10). 

These results indicate that ET/JA pathway is 

sensitized for pathogen infection in the OsPSR5- 

overexpressing plants. 

Components of ROS-mediated defense signaling 

in rice 

Production of reactive oxygen species (ROS) 

upon pathogen recognition is important for 

defense signaling in plants. However, molecular 

mechanisms involved in the signal transduction 

after ROS production is unclear. We identified a 

protein kinase, OsOxi1, as an interacting partner 

of OsPti1a, which negatively regulates defense 

induction in rice (Takahashi et al., 2007). OsOxi1 

belongs to the AGC kinase family and has high 

similarity to Arabidopsis Oxi1/AGC2-1 that 

regulates ROS-mediated defense signaling in 

Arabidopsis. Expression of OsOxi1 was tightly 

regulated temporally and spatially by ROS 

stimuli. Furthermore, OsOxi1 proteins were 

rapidly phosphorylated and activated after ROS 

treatment. These results suggest that OsOxi1 

functions downstream of ROS in activating 

defense responses in rice. A MAPK cascade 

is another candidate target of ROS-mediated 

signaling in rice. We revealed that OsMKK4- 

OsMPK3/6 cascade mediates defense signaling 

triggered by both fungal elicitor and ROS, and 

that this MAPK cascade is involved in the induction 

of defense-related genes, hypersensitive 

cell death, and phytoalexin production in rice. 

The analyses of these protein kinases, which 

function downstream of ROS, would help us 

understand the molecular mechanisms underlying 

defense signal transduction in rice. 

Genome-wide expression analysis reveals a 

role of OsNPR1 in rice defense program 

Salicylic-acid (SA) signaling pathway plays a 

pivotal role in disease resistance of dicot plants 

and NPR1 is a central positive regulator of the 

SA pathway in Arabidopsis. We have functionally 

characterized OsNPR1, a rice ortholog of 

NPR1, focusing on its role in blast disease resistance 

and identification of the genes regulated 

by OsNPR1. OsNPR1 knockdown compromised 

the blast resistance induced by benzothiadiazole 

(BTH, an SA analog) and OsNPR1 overexpression 

conferred strong blast resistance to rice, 

Fig 10. Expression of defense marker genes in OsPSR5-overexpressing Arabidopsis. 

PR1 and PDF1.2 are marker genes for the SA and the ET/JA pathways, respectively. Col-0, WT. 

OsPSR5:OX, OsPSR5 overexpressor. cpr5-2, disease resistant mutant constitutively expressing PR1. 


indicating the essential role of OsNPR1 in 

BTH-induced blast resistance. Genome-wide 

transcript profiling using OsNPR1-knockdown 

lines revealed that 358 genes out of 1,228 BTHupregulated 

genes and 724 genes out of 1,069 

BTH-downregulated genes were OsNPR1 dependent, 

indicating that OsNPR1 plays a major 

role in gene downregulation (Fig 11). Inspection 

of OsNPR1-dependent genes revealed that BTH 

downregulated many genes involved in photosynthesis 

(Fig 11) and chloroplast translation 

and transcription in an OsNPR1-dependent manner, 

indicating that OsNPR1 coordinately suppresses 

photosynthesis and chloroplast activities 

in response to the BTH-induced activation of the 

SA pathway. ABA-responsive genes were also 

downregulated OsNPR1-dependently, suggesting 

an antagonistic interaction of the SA signaling 

with the ABA signaling. None of the 11 BTHupregulated 

genes for WRKY transcription 

factors was OsNPR1 dependent, whereas most 

of those in Arabidopsis were NPR1 dependent, 

indicating that the role of OsNPR1 is distinct 

from that of NPR1 in Arabidopsis. These results 

are consistent with our proposed model for the 

rice SA-signaling pathway that branches to 

OsNPR1- and WRKY45-dependent subpathways. 

Fig 11. Percentage of OsNPR1- dependent rice genes among the BTH-upregulated and 

-downregulated genes in each functional category. 


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 a multitude of 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. 

Molecular basis for juvenile hormone perception 

by JHBP in hemolymph 

Juvenile hormones (JHs) are acyclic sesquiterpenoids 

and control an extraordinarily broad 

range of processes essential for the growth and 

development of insects. These processes include 

embryogenesis, molting and metamorphosis, 

reproduction, diapause, communication, migration/dispersal, 

caste differentiation, pigmentation, 

silk production, and phase transformation. 

Although the biological actions of JH are well 

documented, molecular mechanisms underlying 

JH actions are poorly understood due to a 

lack of structures of JH-protein complexes. JH 

is transported as a complex with its cognate 

binding protein (JHBP) present in hemolymphs 

from the site of its biosynthesis to the sites of 

its action in target organs. Formation of this 

complex provides protection of the chemically 

labile JH against nonspecific enzymatic degradation 

and/or sequestration. Therefore complex 

formation is critical for effective signaling to 

stabilize low amounts of the hormone. In the 

present study, we have determined threedimensional 

structures of Bombyx mori JHBP 

both in the presence and in the absence of JH. 

Our results established a molecular basis for 

JH perception by JHBP, thereby explaining the 

ligand specificity of the protein as well as the 

enhanced stability of the JHBP-bound hormone. 

The crystal structure of JHBP in the JHfree 

state was solved at 2.6nm resolution by 

X-ray crystallography. The overall structure of 

JHBP is an elongated β-barrel where a long 

spine helix α3 is wrapped by a meandering 

antiparallel β-sheet (Fig 12a). The central 

part of the β-sheet is five-stranded, whereas 

the same sheet is four-stranded at both ends. 

Shorter helices, α1 and α2, join the fourstranded 

parts of the β-sheet to form a wrap 

structure around the C- and N-terminal ends of 

the long spine α3-helix, respectively. There are 

two hydrophobic pockets in the JHBP structure; 

one is located near the C-terminus of α3 (first 

pocket) and the other is near the N-terminus of 

the same helix (second pocket). In the crystallized 

apoprotein, we clearly found two 2-methyl- 

2,4-pentanediol (MPD) molecules, one in the first 

pocket and the other in the second pocket (Fig 

12a). These MPD molecules were most likely 

incorporated from a precipitate solution during 

protein crystallization. 

The solution structure of JHBP complexed 

with JH III was determined by NMR spectroscopy. 

This is the first structure of a JH-protein 

complex that has been elucidated. Our complex 

structure established that only the first pocket 

of JHBP accommodates one molecule of JH III 

(Fig 12b). This observation is consistent with 

a 1:1 stoichiometry determined by Scatchard 

analysis of JH binding. The solution structure 

of the JH III-bound JHBP resembles the crystal 

structure of the MPD-bound JHBP. The middle 

portion of the spine α3-helix and the fourstranded 

part of the wrap structure covering 

the second hydrophobic pocket, superimpose 

well in alignment of these structures (Fig 

12c). However, pronounced displacements are 

observed at both ends of the wrap structure. In 

the JH III-bound JHBP, the C-terminus of α2 

moves towards the N-terminus of α3, resulting 

in a slightly narrower second pocket. At the 

opposite end of the protein, the wrap structure 

covering the first pocket, i.e., JH-binding pocket, 

is shifted further from the spine α3-helix in JHbound 

JHBP than in MPD-bound JHBP. The 

difference in this shift is 0.50–0.75nm for tips 

of the wrap. This shift leads to a significant 

difference in the size of the first pocket between 

the two structures (Fig 13), suggesting that the 

JH-binding pocket is radically flexible and could 

change its size in a ligand-dependent manner. 

The bound JH III molecule is completely 

buried inside the JHBP molecule (Fig 13b). 

This binding mode insulates the bound JH 

from the solvent region, and thereby prevents 

the hormone from nonspecific absorption and 

enzymatic degradation. The embedded JH 

III molecule is held in the binding pocket by 

several intermolecular hydrogen bonds. The 

Thr21 side chain forms a direct hydrogen bond 

with the ester oxygen of JH (Fig 13c). The 

side chain of Tyr128 is positioned such that its 


hydroxyl group can form hydrogen bond either 

with the carbonyl oxygen of the ester or with 

the epoxy oxygen (Fig 13c). It is also possible 

that the Tyr128 side chain forms two hydrogen 

bonds with both the ester C=O and the epoxy 

O atoms by associating with a bridging water 

molecule. Numerous non-polar interactions from 

24 residues of JHBP also contribute to keeping 

the JH molecule firmly held in the pocket 

(Fig 13c). Most of the JH-interacting residues 

identified in the structure of the complex are 

strictly conserved or conservatively substituted 

within the lepidopteran JHBP family. Results of 

our JH-binding assays with mutant JHBPs are 

consistent with the structural assignments of 

the JH III-JHBP complex. 

Fig 12. Overall structure of Bombyx mori JHBP. 

(a) Crystal structure in the JH-free state. The JHBP-bound 2-methyl-2,4-pentanediol (MPD) 

molecules are shown as space filling structures. (b) Solution structure of JHBP-JH III complex. JH III 

is shown as a space filling structure. (c) Three different views of the overlay of the solution structure 

of the JHBP-JH III complex (light green) and the crystal structure of the JH-free JHBP (pink). 

Fig 13. Ligand recognition by JHBP. 

Surface representations of JHBP around the JH-binding pocket of (a) the MPD-bound crystal 

structure and (b) the JH III-bound solution structure. The bound molecules are shown as ball-and 

stick models. The interior of the protein is shown in green whereas the exterior of the pocket is in 

purple. (c) A close-up view of the JHBP-bound JH III shown as a ball-and stick model. JHBP residues 

that contact to JH III are shown as stick models. Dotted lines depict possible direct hydrogen bonds 

between JH III and the protein. 



Higher plants interact with numerous kinds 

of microbes during their life cycle. These 

interactions are classified into symbiotic ones, 

such as mycorrhizal symbiosis where the fungus 

supplies inorganic nutrition, 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 to production and protection of crops. 

Evolutionary conservation of rice genes 

orthologus to a common symbiosis pathway 

in Lotus japonicus. 

Among legume genes involved in root nodule 

symbiosis, a distinct set of genes is called a 

common symbiosis pathway (CSP), because they 

are also essential to the arbuscular mycorrhiza 

(AM) symbiosis, another symbiotic interaction, 

which is widely distributed in the plant 

kingdom. Based on the concept that the RN 

symbiosis in legume plants has evolved from 

the ancient AM symbiosis, it is interesting to 

investigate whether the CSP is functionally 

Ljccamk 

ILjCCaMK 

Ljccamk 

IOsCCaMK 

Fig 14. Restoration of Symbiotic Defects in Ljccamk by OsCCaMK. 

OsCCaMK restores both mycorrhizal (B) and rhizobial endosymbioses (D) in Ljccamk mutants, as 

well as the mutant roots transformed with LjCCaMK (A and C). Scale bars; 0.1mm (A and B), 0.5mm (C 

and D). 


conserved in non-nodulating plants, such as 

rice. We identified three CSP gene orthologs, 

OsCASTOR, OsPOLLUX and OsCCaMK, in 

rice and demonstrated that OsPOLLUX and 

OsCCaMK are indispensable to AM symbiosis 

in rice. Interestingly, molecular transfection of 

either OsCASTOR or OsCCaMK fully complemented 

symbiosis defects in the corresponding 

mutant lines of Lotus japonicus for both the AM 

and RN symbioses (Fig 14). These results not 

only show a conserved genetic evidence for the 

AM symbiosis between rice and Lotus, but also 

indicate that the core of the CSP has been well 

conserved during the evolution of RN symbiosis. 

An inhibitory interaction between viral and 

cellular proteins underlies the resistance of 

tomato to non-adapted tobamoviruses 

Each virus species infects a particular, limited 

range of hosts. To a given virus, most plant species 

are ‘nonhosts’ to the virus, i.e., all members 

of the plant species are insusceptible to the 

virus. Resistance displayed by a nonhost plant 

species is rarely overcome, and therefore uncovering 

its mechanisms will help devise strategies 

to establish durable resistance. However, this 

type of resistance is difficult to study due to the 

inapplicability of classical genetics approaches, 

and factors that control the resistance to viruses 

remained unidentified. Previously, we identified 

Tm-1, a resistance gene of tomato to a tobamovirus 

Tomato mosaic virus (ToMV) and found 

that the Tm-1 protein binds to ToMV replication 

proteins to inhibit RNA replication. Tm-1 

is derived from a wild tomato species, Solanum 

habrochaites, and ToMV-susceptible tomato cultivars 

have an allelic gene tm-1. The tm-1 protein 

can neither bind to ToMV replication proteins 

nor inhibit ToMV multiplication. Tobacco 

mild green mosaic virus (TMGMV) and Pepper 

mild mottle virus (PMMoV) are tobamoviruses 

related to ToMV. TMGMV and PMMoV can 

efficiently multiply in tobacco plants, but not in 

any cultivars of tomato (Solanum lycopersicum); 

i.e., tomato is a nonhost species for TMGMV and 

PMMoV. This year, we asked if tm-1 plays a 

role in the resistance to these viruses shown by 

tomato. To test this possibility, we constructed 

transgenic tobacco plants that expressed tm-1, 

and inoculated these plants with TMGMV and 

PMMoV. We found that these tobacco plants 

showed resistance to TMGMV and PMMoV. 

The tm-1 protein bound to the replication 

proteins of TMGMV and PMMoV and inhibited 

their RNA replication also in vitro. In one of 

the tm-1-expressing tobacco plants, a tm-1- 

insensitive TMGMV mutant emerged. In tomato 

protoplasts, this mutant TMGMV multiplied as 

efficiently as ToMV, indicating that tm-1 is the 

only factor that restricts the multiplication of 

TMGMV in tomato protoplasts. In tomato plants, 

however, the mutant TMGMV multiplied with 

lower efficiency compared to ToMV, and caused 

systemic necrosis. These results suggest that an 

inhibitory interaction between the replication 

proteins and tm-1 underlies a multilayered 

resistance mechanism to TMGMV in tomato. 

Differential responses of rice to inoculation 

with wild-type and non-pathogenic mutants of 

Magnaporthe oryzae 

M. oryzae, causal agent of rice blast disease, 

germinates and forms appresoria, a differentiated 

organ specified to infection, before 

penetration. We analyzed the response of rice in 

each stage of development of the fungus before 

penetration using two mutant strains, mgb1 and 

mst12, which are deficient in the formation of 

appresoria and penetration pegs. These noninvasive 

mutants induced the much lower levels 

of phytoalexin accumulation compared to wild 

type, suggesting that fungal invasion is a key 

event to the massive production of phytoalexins 

in rice cells. Intense accumulation of H 2 O 2 also 

required fungal invasion. Microarray analysis 

of rice gene expression revealed mutantspecific 

gene expression (Fig 15), indicating that 

signal exchange between rice and M. oryzae 

occurs before fungal invasion into rice cells. 

In situ hybridization revealed that expression 

of a peroxidase and α-1,3-glucanase occurs 

after penetration as observed for phytoalexin 

production. 


Fig 15. Schematic diagram of rice genes responsive to inoculation with mgb1, mst12, or Wild Type 

(Guy11) in LTH (compatible) and IRBL 12-M (incompatible) rice at 24 and 48 hpi. 

Genes encoding PR proteins, plastid proteins, and ribosomal proteins are indicated in pink, green, 

and yellow, respectively, and up- and down-regulated genes are shown in red and blue, respectively. 

A and B indicate genes for ribosomal proteins, and C and D for plastid proteins, respectively. 

High efficiency of gene-knockout in a recombinant 

M. oryzae. 

The genome sequence of M. oryzae has been 

analyzed and annotated for more than 11000 

genes. In order to establish a durable strategy 

for the protection of rice from blast disease, it is 

indispensable to elucidate the function of genes 

involved in pathogenicity, and the knock-out of 

genes of interest is a powerful tool. Although 

we succeeded to enhance the efficiency of transformation 

by using Agrobacterium, it was still 

difficult to get targeted replacement of genes 

of interest because of high activity of the nonhomologous 

end-joining (NHEG) reaction. We 

constructed a recombinant M. oryzae in which 

MgLIG4 encoding DNA Ligase 4, an essential 

protein in NHEG, was destroyed. The mutant 

strain showed similar pathogenicity to the wild 

type. In this strain, the efficiency of targeted 

gene replacement was significantly higher 

compared to the wild type. 



Rapid and efficient Agrobacterium-mediated 

transformation in forage rice 

We previously reported the high-speed 

Agrobacterium-mediated transformation system 

in japonica and indica type of rice cultivars, 

Nipponbare and Kasalath, respectively. Using 

our transformation system, transgenic rice 

plantlets were also successfully obtained in rice 

cultivars for forage, Tachisugata and Tachiaoba 

(Fig 16). The transformation efficiency in these 

cultivars was lower than that in Nipponbare 

and Kasalath because of browning of growing 

calli grown on the regeneration medium. We 

have investigated the regeneration conditions, 

and it has been shown to be efficient to grow 

calli at low density on the regeneration medium. 

We are currently producing disease resistant 

transgenic forage rice. 

Toward the development of effective transgenic 

methods of sorghum 

Sorghum (Sorghum bicolor (L.) Moench) is 

one of the important annual crops of the world, 

because it can grow in arid soils and withstand 

drought. Recently, the usage of sorghum has 

been extended to an energy crop. Genetic modification 

via transformation of sorghum would 

be an indispensable tool for these purposes. We 

are now trying to develop effective transgenic 

methods for sorghum. To begin, we selected the 

most suitable cultivar, which has a high ability 

for regeneration, to employ for transgenic study. 

We furthermore constructed suitable culture 

and regeneration systems including the method 

for extracting immature embryos, components 

of culture medium, and temperature and light 

during culture. 

Production of a tryptophan fortified rice by 

gene targeting (GT) 

Rice is a poor source of tryptophan which is 

an essential amino acid for nonruminant animals. 

We previously reported the successful introduction 

of two point mutations corresponding to the 

S126F/L530D substitution into the endogenous 

OASA2 gene encoding anthranilate synthase 

— a key enzyme in tryptophan biosynthesis 

— via GT. In the leaves of these plants, free 

tryptophan was found to be accumulated over 

100-times compared to the original cultivar 

A 

B 

Fig 16. Genetic transformation in rice for forage. 

(A) Green fluorescence in 35S::GFP transformed calli under blue light. (B) Transgenic plants in T0 

generation. The transgene was stably inherited to the T1 generation. 


Nipponbare. In addition, we succeeded in obtaining 

plants in which Y367A or Y367A/L530D 

mutations were introduced into OASA2 enzyme 

by GT. We are currently analyzing these plants 

and their progenies. 

DNA mismatch repair system suppresses 

introduction of point mutations by T-DNAmediated 

GT in rice 

We previously reported a successful introduction 

of point mutations into the rice Acetolactate 

synthase (ALS) locus by T-DNA-mediated 

gene targeting (GT). ALS – a key enzyme in 

branched-chain amino acid synthesis – is the 

primary target for at least four structurally 

distinct classes of herbicides and several amino 

acid substitutions in ALS are known to confer 

herbicide tolerance. Our GT vector contained a 

partial rice ALS gene with mutations encoding 

the W548L/S627I substitution, which confer 

ALS-inhibiting herbicide, bispyribac-sodium (BS), 

tolerance and 2 silent mutations. In 3 out of 70 

GT plants, we found that point mutations on the 

targeting vector were incorporated into the rice 

ALS locus with a mosaic manner. We also found 

a mosaic manner modification of point mutations 

in the T-DNA which were integrated randomly 

into the rice genome. These results suggest 

the involvement of the DNA mismatch repair 

system in the course of T-DNA-mediated GT. 

Maintenance of silent states in plant chromatin 

RNA-directed modification of histones is essential 

for the maintenance of heterochromatin 

in higher eukaryotes. In fission yeast, RNAs 

transiently transcribed from heterochromatin 

upon DNA replication are processed into small 

interfering RNAs (siRNAs) and induce formation 

of repressive states of chromatin through 

histone modification, such as di-methylation of 

histone H3 lysine 9 (H3K9me2). In plants and 

mammals, cytosine methylation is an additional 

factor regulating inactive states of chromatin, 

but our knowledge about mechanisms explaining 

coexistence of cytosine methylation 

and repressive histone modification in plant 

heterochromatin is limited. In our current study, 

we showed data indicating that Morpheus' 

molecule 1 (MOM1), an epigenetic regulator for 

transcriptional gene silencing in Arabidopsis 

thaliana, silences a subset of heterochromatin 

loci at the downstream of RNA-directed DNA 

methylation (RdDM). Transcript profiling by 

a genome tiling array showed that a major 

fraction of the up-regulated loci in mom1 carry 

sequences related to transposable elements 

and homologous to 24 nt siRNAs accumulated 

in the wild type plant that are the hallmark of 

RdDM. Detailed analysis of a single-copy gene, 

Suppressor of drm1drm2cmt3 (SDC) that is 

regulated by RdDM at a tandem repeat in the 

Fig 17. Diagrams illustrating the division mechanisms of plastids in rice. 

(A) Chloroplasts in the leaf divide by binary fission. (B) Elongated amyloplasts containing small 

granules generate multiple constriction sites. This beads-on-a-string structure is seen when the 

endosperm cell is still small and rapidly expanding its cellular volume. At later stages of endosperm 

development, large amyloplasts divide by protrusion of small amyloplasts from the surface (Budding- 


promoter region, revealed that mom1 activates 

SDC with concomitant reduction of H3K9me2 

in the tandem repeat but without changes in 

siRNA accumulation and cytosine methylation. 

The results suggest that MOM1 transduces 

RdDM signals to repressive states of histone 

modification. 

Amyloplasts in rice endosperm divides at 

multiple sites 

The amyloplast in cereal endosperm is a terminally 

differentiated plastid where the carbon 

translocated from source tissues is converted 

to starch granules and stored until germination. 

The starch in the cereal endosperm 

provides not only a major source of dietary 

energy supply for human and farm animals 

but also is a valuable resource for non-food 

industries. Despite the enormous importance of 

amyloplasts as the organelle that synthesizes 

and stores starch, little is known about how the 

amyloplast proliferates in the cereal endosperm. 

The rice (Oryza sativa) endosperm provides an 

excellent model system for studying molecular 

mechanisms involved in amyloplast division 

and starch synthesis. We compared amyloplast 

division processes in the endosperm of wild 

type and a mutant of ARC5, a member of the 

dynamin superfamily. Plant growth and fertility 

of arc5 were not significantly different from the 

wild type. Unlike binary fission of chloroplast 

in the leaf, small amyloplasts in the endosperm 

of wild type divide simultaneously at multiple 

sites, generating a beads-on-a-string structure 

(Fig 17). In addition, large amyloplasts divide 

by budding-type division, giving rise to small 

amyloplasts attached to their surfaces (Fig 

17). ARC5 and FtsZ2-1 fused respectively to 

fluorescent proteins were targeted to the constriction 

sites in dividing amyloplasts. Both the 

loss of function of ARC5 and overexpression of 

ARC5 fusion proteins in the endosperm did not 

produce spherical amyloplasts with increased 

diameter, but produced either fused amyloplasts 

with thick connections or pleomorphic types, 

suggesting that proper stoichiometry between 

ARC5 and other components in the amyloplast 

division machinery is necessary for the completion 

of the late stage of amyloplast division. 

The size distribution of starch granules purified 

from arc5 was shifted to smaller granules and 

the starch gelatinization peak temperature was 

significantly higher than for wild-type starch, 

suggesting that amyloplast division processes 

have a significant effect on starch synthesis. 



The Division of Insect Sciences consists of 

eight research units and aims to develop up todate 

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. 


The unit is constructing the integrated 

genome database of silkworm genomic sequence 

information, EST information and map information, 

which is publicly available on the web. 


The unit will identify genes involved in the 

action of insect hormones, insect behaviors, 

invertebrate reproduction, embryonic development 

and regeneration. 


The unit is studying biochemical and 

molecular adaptations associated with anhydrobiosis 

in the sleeping chironomid, Polypedilum 

vanderplanki. 


The 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-Microbe Research Unit 

The 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. 


The 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 

The 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 

This laboratory is operated with funding 

provided by several supporting companies and 

backed by the Institute, and is conducting the 

insect symbiont genome project. 

Major topics in the Division in the fiscal year 

2008 are described in the follow reports. 


The unit aims to develop new technology in 

pest control based on the research for insectinsect 

and insect-plant interaction mechanisms. 



We merged the silkworm whole genome 

sequence data sets from Japan and China, which 

together cover 87% of silkworm genome, and 

have provided the high-accuracy silkworm nucleotide 

sequence information to the public via 

an integrated silkworm database, KAIKObase. 

For the practical use of the silkworm nucleotide 

sequence information, we continue to maintain 

and improve the database resources especially 

for the following three categories. 

Silkworm full-length cDNA analysis 

The complete gene structure information, fulllength 

cDNA information, and the annotation of 

silkworm genome sequences are indispensable 

for isolation and identification of important 

genes, for gene function analysis and for application 

of genetic engineering. To aid these applications, 

we have constructed a full-length cDNA 

library from previously unanalyzed tissues and 

selected candidates for the full-length cDNA 

clones from all ESTs with an aim of obtaining 

10,000 clones. 

In this year, we constructed and analyzed 

four full-length cDNA libraries: unfertilized egg; 

embryos 8 days post-fertilization; fat body of 

male fifth instar larvae; and malpighian tubules 

of fifth instar larvae. With the inclusion of 

previously constructed cDNA libraries, we now 

have 17 full-length cDNA libraries from different 

tissues (Table 1). Of the 187,286 obtained 5’ 

ESTs, 15,000 were selected for further analysis. 

Full nucleotide sequences were determined for 

the clones having 5’ cohesive ends using primerwalking. 

We have determined 10,143 full length 

cDNA sequences so far and have mapped them 

on the chromosomes using silkworm genome 

information. 

The number of genes on each chromosome 

showed good correlation with the lengths of the 

chromosome, indicating that the genes were 

randomly selected and distributed evenly. We 

are also trying the sequencing using Sorexa 

to accelerate complete sequencing of the fulllength 

cDNA clones. The sequence information 

is edited and stored in the full-length cDNA 

database, and available for researchers to use. 

Upgrade of silkworm genome information and 

its utilization 

To improve the regions with low-sequence quality 

caused by gaps and mis-assembly due to repeat 

sequences, and to further promote silkworm 

research, we performed the following studies: 

Table 1. List of full-length cDNA libraries 


・ Expansion of KAIKObase 

Bombyx Trap Database and the silkworm 

proteome database (KAIKO2DDB) were 

combined into KAIKObase (Fig 1). The EST databases 

for brown rice planthopper (Nilaparvata 

lugens) and sleeping chironomid (Polypedilum 

vanderpanki) were also expanded. The direction 

of five linkage groups in the SNP linkage map 

were aligned with the silkworm morphological 

marker linkage map. We also started to determine 

accurate positions for about 9,000 analyzed 

full-length cDNAs to construct a silkworm gene 

expression analysis system and an information 

infrastructure for silkworm genome annotation. 

EST clone mapping by linkage analysis was 

finished for linkage groups 4, 11, 13, and 15. 

In the BAC minimum tiling pass construction 

this year, we refined 959 tiling BACs in seven 

linkage groups, corresponding to 25% of the 

whole genome, and verified the assembly data 

by comparison of restriction enzyme maps. 

Moreover, we found 22 bridge BACs in 33 gaps 

in seven linkage groups which were confirmed 

by the FISH method. 

・ Efficient positional cloning of genes based on 

genome information 

Densovirus resistance gene (Nid-1): A target 

area was specified as a 34-kb region (Fig. 2) and 

expression analysis of the genes localized in this 

area was started. 

Pigment and non-diapausing egg genes (pnd, 

pnd-2): Candidate regions were narrowed to 158 

kb for pnd and 70 kb for pnd2. Expression of 

the genes localized within the candidate regions 

was compared in detail during embryogenesis 

Fig 1. Integrated silkworm database, KAIKObase. 

LG21 

LG17 

I 

II 

45.2 

28.6 

a 

b 

c 

24.5 

22.5 

14.3 

c 

16.4 

III 

IV 6.1 

V 5.1 

VI 

VII 

VIII 

20.8 

nsd-1 

1.0 

4.3 

e,f,g 

h,i 

nsd-2 

6.1 

d,e 

h,i 

4.9 

Nid-1 

14.8 

Fig 2. Investigation of silkworm densovirus resistance genes. 


in wild type and their expression in early 

embryogenesis confirmed by RT-PCR. 

Bacillus thuringiensis resistance gene: The 

candidate region was narrowed to 82 kb and 

one candidate gene was obtained. The function 

of the candidate gene is being confirmed by 

transgenic methods. 

Egg color related genes (w-2, re, pe): The candidate 

regions were narrowed to 215kb, 202kb, 

and 257kb, respectively, and some candidate 

genes were obtained. 

Comparative genome research among 

Lepidoptera by BAC-FISH method 

We isolated 159, 133 and 168 BAC clones 

from Manduca sexta, Ostrinia nubilalis and 

Heliothis virescens, respectively. The results 

of FISH analyses using the BACs as probes 

showed that these Lepidopteran species have 

similar chromosome structures. Molecular markers 

covering all 28 chromosomes of Manduca 

sexta were obtained. 

Construction of silkworm gene destruction 

mutation lines by Enhancer trap system 

Among the enhancer-trap strains created 

by BmA3-GAL4 mutator, expression analysis 

and insertion position analysis were performed 

for 105 strains. The mutator transposons were 

found to be randomly inserted into the silkworm 

genome (Fig 3). An enhancer trap database 

(Bombyx Trap DataBase) that combined 

expression positions, expression images and the 

genome insertion locations was constructed, 

integrated with the genomic database, and 

opened to the public (Fig 1). 

Reference 

Uchino K, Sezutsu H, Imamura M, Kobayashi I, 

Tatematsu K, Iizuka T, Yonemura N, Mita K, 

Tamura T (2008) Construction of a piggyBacbased 

enhancer trap system for the analysis 

of gene function in silkworm Bombyx mori 

Insect Biochemistry and Molecular Biology 

38(12): 1165-1173 

chr. 

0 

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 

10 

A 

U 

20 

Mb 

Transposed BmA3-GAL4 mutator 

Assigned: 89 

A BmA3-GAL4 original (193-2) 

Unassigned: 16 

U UAS-EGFP 

Fig 3. The distribution of insertions in each of the silkworm chromosomes. (Uchino et al, 2008) 



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 and amines to develop new 

pesticides. It also conducts molecular analyses 

of invertebrate reproduction, embryonic 

development and regeneration. The selected 

major research topics for 2008 from the present 

research unit are as follows: 

CA, but instead was predominantly transcribed 

in the corpora cardiaca (CC), an organ adjacent 

to the CA (Fig 4, 6). Indeed, by using a reversephysiological 

approach, we purified and 

characterized novel allatoregulatory peptides, 

short neuropeptide Fs (sNPFs) produced in AT 

receptor-expressing CC cells (Fig 5, 6), which 

may indirectly mediate AT activity on the CA 

(Fig 7). All of the above findings confirm the 

effectiveness of a systematic analysis of the receptor 

transcriptome, not only in characterizing 

orphan receptors, but also in identifying novel 

players and hidden mechanisms in important 

biological processes. 

Identification of allatotropin receptor and 

novel allatoregulatory neuropeptide receptors 

in Bombyx mori 

Neuropeptides are an important class of 

molecules involved in diverse aspects of insect 

physiology. The identification of their receptors 

has been studied extensively in the last decade; 

however, receptors for some key neuropeptides 

in insect development such as prothoracicotropic 

hormone, eclosion hormone and allatotropin (AT), 

remain undefined. Using the genome sequences 

of the silkworm, Bombyx mori, we performed 

the comprehensive cloning of neuropeptide G 

protein-coupled receptors from the insect and 

performed systematic analyses of their expression. 

Based on the expression patterns of orphan 

receptors, we identified the long-sought receptor 

for AT (BNGR A16), which is thought to 

stimulate juvenile hormone biosynthesis in the 

corpora allata (CA). Surprisingly, however, the 

AT receptor was not highly expressed in the 

Development of a method for in vivo RNA 

interference in the annelid Enchytraeus japonensis 

using glutamine synthetase gene as a 

positive control 

In the previous study, 165 regenerationupregulated 

genes (79 known and 86 unknown) 

were isolated from the oligochaete annelid 

Enchytraeus japonensis that can regenerate a 

complete individual from a small body fragment 

in several days (Myohara et al, 2006). To make 

functional analysis of these genes possible, we 

developed a method for in vivo RNA interference 

(RNAi) using glutamine synthetase gene 

(gs), one of most abundantly expressed genes 

during regeneration of E. japonensis, as a 

positive control. Formation of regeneration 

blastema was severely suppressed when 1-3µM 

gs-targeting small interfering RNA (siRNA) was 

administered by soaking E. japonensis body 

fragments at early stages of regeneration in a 

hypotonic medium (Fig 8A). The suppression 


Fig 4. Expression analysis of 6 Bombyx G-protein 

coupled neuropeptide receptors (BNGRs) within CC-CA 

complex on the fourth instar head capsule slippage. 

Vertical bars represent S.E. n=3. BNGR A16 represents 

AT receptor. 

Fig 5. Expression analysis of short neuropeptode F (sNPF) 

on the fourth instar head capsule slippage. 

Relative expression was shown as a percentage of the 

expression level in the CC. CC-CA, corpora cardiacacorpora 

allata complex; PG, prothoracic gland; BR, brain; 

FB, fat body; MG, midgut; EP, epidermis; MU, muscle; 

MP, Malpighian tubule; SiG, silk gland; SG, salivary gland; 

TE, testis; OV, ovary. Inset, in situ hybridization of sNPF. 

Scale bar, 100μm. 

Fig 6. Double staining of the CC-CA with immunohistochemistry 

for sNPF (upper) and in situ hybridization for 

BNGR-A16 (lower). 

Scale bar, 100μm. 

Fig 7. Schematic illustration of the regulatory pathways of 

JH biosynthesis identified in this study. 

Putative role of AT in the regulation of sNPF production in 

the CC remains to be elucidated. 

was induced by all four gs-targeting siRNAs 

tested, but not by non-targeting negative control 

siRNAs (Fig 8B), suggesting that the phenotype 

was induced by gene specific knockdown of 

gs expression. A real-time RT-PCR analysis of 

siRNA-treated E. japonensis intact worms also 

showed that the soaking method was effective 

for gene specific knockdown of targeted genes 

(Fig 8C). 

Reference 

Myohara M, Niva CC, Lee JM (2006) Molecular 

approach to annelid regeneration: cDNA subtraction 

cloning reveals various novel genes 

that are upregulated during the large-scale 

regeneration of the oligochaete, Enchytraeus 

japonensis. Developmemtal Dynamics 235: 

2051-2070 


Fig 8. In vivo RNAi for glutamine synthetase gene (gs) by siRNA soaking in Enchytraeus aponensis. 

A-B. Suppression of blastemal formation by gs-targeting siRNAs. Body fragments were treated with 

3µM gs-targeting siRNAs (gs121, gs680, gs801, gs1021) or non-targeting negative control siRNAs 

(NC#2, NC#5) for 3 days, fixed and stained with propidium iodide. Mean ± SEM of blastemal lengths 

(arrows in A) are shown as ratio to non-treated control in B. C. Suppression of gs gene expression 

in worms treated with a gs-targeting siRNA (gs801). Gs expression was measured by real-time RT- 

PCR analysis, normalized by β-actin gene expression and is presented as ratio to negative siRNA 

control (NC#5). 

Yamanaka N, Yamamoto S, Žitňan D, Watanabe 

K, Kawada T, Satake H, Kaneko Y, Hiruma 

K, Tanaka Y, Shinoda T, Kataoka H (2008) 

Neuropeptide Receptor Transcriptome Reveals 

Unidentified Neuroendocrine Pathways. 

PLoS ONE 3(8): e3048. doi: 10.1371/journal. 

pone.0003048 


Radiation Tolerance Linked to Anhydrobiosis 

in Polypedilum vanderplanki 

The larvae of the sleeping chironomid, 

Polypedilum vanderplanki, are able to withstand 

almost complete desiccation during which they 

enter a state of suspended animation (Hinton, 

1951); this process is known as anhydrobiosis 

(Keilin, 1959). The anhydrobiotic larva can 

tolerate various extreme conditions including 

high doses of radiation (Watanabe et al, 2006; 

Watanabe et al, 2006). It is of great interest to 

know how the desiccating larvae gain radiation 

tolerance during such drastic physiological 

changes upon anhydrobiosis (Kikawada et al, 

2006). 

We first examined effects of high-LET 

(linear energy transfer) radiation on 4 kinds of 

larvae (Fig 9): normal hydrated larva (W-low); 

intermediates between the anhydrobiotic 

and normal hydrated state (W-mid); almost 

completely dehydrated (anhydrobiotic) larvae 

(D-high); and desiccated larvae, taken after 

short rehydration, that are assumed to have a 

similar molecular profile to anhydrobiotic larvae 

(W-high) (Watanabe et al, 2007). These samples 

were exposed to 1 to 4500 Gy of 50 MeV 4He 

(LET ∞ =16.2 keV/µm) ion beam delivered 

from the azimuthally varying-field cyclotron at 

TIARA. Irradiated larvae were supplied distilled 

water and the diet. Larvae that moved their 

pharyngeal plunger were considered to be survivors. 

The results indicated that improvement 

of radiation tolerance in anhydrobiotic larvae, 

to some extent, can be due to the presence of 


an excess of the sugar trehalose, which could 

provide structural and biochemical protection 

to the cells in anhydrobiotic state (Sakurai et al, 

2008). 

Our recent results suggested that, however, 

there are more important factors. We focused on 

DNA-related aspects of the irradiation-response 

of the larvae, and analyzed DNA breaks and 

expression of genes involved in general stressresponse 

cascades and DNA reparation, after irradiation. 

We used the doses of ion beams, which 

were previously found to be the ID50 (median 

inhibitory dose) for adult emergence (70Gy) and 

pupation (160 Gy) of the chironomid (Hinton, 

1951; Keilin, 1959). The larvae were exposed 

to the 50 MeV 4 He (LET ∞ =16.2 keV/µm) ion 

beam delivered from the azimuthally varyingfield 

cyclotron at TIARA. DNA status and 

repair were analyzed with alkaline Comet Assay 

standard technique using the fat body cells as a 

model cell type. Estimation of relative percentage 

of DNA in comet “tails”, as a quantitative 

marker of DNA breaks, showed that the larvae 

experienced severe DNA damage after irradiation 

with both set of doses (70Gy and 160Gy) (Fig 

10, inset panel). However, we have not found any 

evidence of massive cell death and/or increasing 

level of apoptotic cells in irradiated larvae. The 

reparation of DNA to a level comparable to that 

of non-irradiated larvae took at least 96h (Fig 10). 

Moreover, non-irradiated dry larvae after revival 

also possessed cells with severely damaged 

DNA, which was also repaired within 96 h after 

rehydration (Fig 10). 

The DNA, damaged after 4 He irradiation 

and desiccation stress, should be mended by 

reparation enzymes and other stress-response 

associated proteins. Indeed, we cloned complete 

cDNAs of several stress-related genes from 

P. vanderplanki (ROS-response, chaperones, 

apoptosis-related and DNA-repair involved 

genes) and analyzed their expression after 

irradiation and desiccation stresses. Thus far, 

at least one gene (UV excision repair protein 

Rad23, family InterPro: IPR004806) significantly 

increased expression level in response to both 

radiation and desiccation stresses (Fig 11). 

On the basis of these results, we assume that 

Water 

(%) 

80 

40 

0 

Desiccation 

Rehydration 

100% RH 76% RH 5% RH In water 

D-high 

W-low W-mid W-high 

Fig 9. Preparation of larvae with different physiological 

conditions. 

Intact larvae (W-low) were incubated at 100% RH for 24h 

(W-mid), and further 76% RH, and 5% RH, each for a day 

(D-high). D-high larvae were rehydrated in distilled water 

for 1h (W-high). 

Trehalose 

(% dw) 

20 

10 

0 

0 

24 

48 

Time (h) 

72 

0 

24 

Damaged DNA (%) 

80 

60 

40 

20 

0 

a b 

12 24 48 96 168 

Time after irradiation (h) 

0 Gy 70 Gy 160 Gy Rehydration 

Fig10. Dynamics of DNA reparation in the larvae of P. 

vanderplanki exposed to 4He ion beam (0-160 Gy) and 

naturally reviving after anhydrobiosis (Rehydration). 

Inset panel shows a comet of cells from irradiated larva (70 

Gy, 24 h after). a: intact DNA. b: broken DNA. Damaged 

DNA (%) = b/(a+b)x100. 


Relative expression 

15 

10 

5 

0 

0 24 48 1 3 24 48 1 3 24 48 

Desi (h) Rehy (h) Irrad (h) 

Fig 11. Relative expression of Rad23 in the larvae of P. 

vanderplanki during desiccation (Desi), after rehydration 

(Rehy), and after irradiation with 70Gy of 4 He (Irrad). 

higher resistance of dry larvae to different types 

of radiation could be the result of the synthesis 

of stress-related proteins in the larvae prior to 

entering anhydrobiosis. The up-regulation of 

these genes could not prevent DNA-damage 

completely upon desiccation, but might give certain 

advantages for the recovery of the revived 

larvae after both desiccation and irradiation. 

References 

Hinton HE (1951) A new chironomid from 

Africa, the larvae of which can be dehydrated 

without injury Proc Zool Soc Lond 121: 371 

–380 

Keilin D (1959) The problem of anabiosis or 

latent life history and current concept Proc R 

Soc Lond 150B : 149–191 

Kikawada T, Nakahara Y, Kanamori Y, Iwata K, 

Watanabe M, McGee B, Tunnacliffe A, Okuda 

T (2006) Dehydration-induced expression of 

LEA proteins in an anhydrobiotic chironomid 

Biochem Biophys Res Commun 348: 56–61 

Sakurai M, Furuki T, Akao K, Tanaka D, 

Nakahara Y, Kikawada T, Watanabe M, 

Okuda T (2008) Vitrification is essential for 

anhydrobiosis in an African chironomid, 

Polypedilum vanderplanki Proc. Natl. Acad. 

Sci. USA 105: 5093–5098 

Watanabe M, Sakashita T, Fujita A, Kikawada 

T, Horikawa DD, Nakahara Y, Wada S, 

Funayama T, Hamada N, Kobayashi Y, Okuda 

T (2006) Biological effects of anhydrobiosis in 

an African chironomid, Polypedilum vanderplanki 

on radiation tolerance Int J Radiat 

Biol 82: 587–592 

Watanabe M, Sakashita T, Fujita A, Kikawada T, 

Nakahara Y, Hamada N, Horikawa DD, Wada 

S, Funayama T, Kobayashi Y, Okuda T (2006) 

Estimation of radiation tolerance to high 

LET heavy ions in an anhydrobiotic insect, 

Polypedilum vanderplanki Int J Radiat Biol 

82: 835–842 

Watanabe M, Nakahara Y, Sakashita T, 

Kikawada T, Fujita A, Hamada N, Horikawa 

DD, Wada S, Kobayashi Y, Okuda T (2007) 

Physiological changes leading to anhydrobiosis 

improve radiation tolerance in Polypedilum 

vanderplanki larvae J Insect Physiol 53: 573 

–579 



Selective cancer cell cytotoxicity of enantiomeric 

9-mer peptides derived from beetle 

defensins depends on negatively charged 

phosphatidylserine on the cell surface 

Four enantiomeric 9-mer peptides named 

D-peptide A, B, C and D were designed and 

synthesized on the basis of 43-mer insect defensins 

from two beetles. The D-9-mer peptides 

maintained bacterial membrane disruptive 

activity similar to the original peptides and also 

showed various extents of growth inhibitory 

activity against different cancer cell lines. Of 

these peptides, D-peptide B exhibited the highest 

selective cancer cell cytotoxicity against the 

mouse myeloma cell line, P3-X63-Ag8.653. Flow 

cytometric and scanning electron microscopic 

analysis revealed D-peptide B disrupts mouse 

myeloma membrane construction, whereas no 

cytotoxic effect on normal leukocytes was observed 

(Fig 12). Moreover, a strong correlation 

between negatively charged phosphatidylserine 

(PS) density in cancer cell membrane surface 

and sensitivity to D-9-mer peptides were 

observed in various cancer cell lines. These 

results suggest that D-9-mer peptides have 

negative charge-dependent selective cancer 

cell cytotoxicity targeting PS in the cancer 

cell membrane. In addition, synergic growth 

inhibitory activity against mouse myeloma was 

observed in combinations of D-peptide B and 

dexamethasone. These results suggest D-9-mer 

peptides are promising candidates for novel 

anticancer drugs. 

Mouse normal leukocyte 

Mouse myeloma P3-X63-Ag86.53 

Fig 12. Effect of D-peptide B on morphological changes and apoptosis induction in mouse myeloma. 

Effect of D-peptide B on the cell membrane in mouse normal leukocyte (bone marrow) and myeloma 

under scanning electron microscopy. Cells were incubated with or without 80μM D-peptide B for 24 

h at 37C before observation. 


Lipopolysaccharide elicits expression of immune-related 

genes in the silkworm, Bombyx 

mori. 

Lipopolysaccharide (LPS), a major cell wall 

component of gram-negative bacteria, was 

found to be unable to activate immune-related 

genes in Drosophila melanogaster. In contrast, 

highly purified LPS elicited immune-related 

gene expression in the fat body of Bombyx 

mori. However, the level of activation by highly 

purified LPS was lower than the activation by 

crude LPS and peptidoglycan (Fig 13A, B). 

Furthermore, synthetic lipid A also activated 

these genes, suggesting that B. mori possesses 

unknown signal pathways to activate immunerelated 

genes by LPS (Fig 13C). Up-regulation 

Fig 13. Quantification of Cecropin B1, Attacin and Lysozyme mRNA levels after stimulation by 

lipopolysaccharide (LPS) and lipid A in the Bombyx mori fat bodies. 

Total RNA was extracted from fat bodies excised from larvae 8 h after immunization and analysed by 

Northern blotting. Ribosomal protein 49 (RP49) mRNAs were analysed as an internal control. mRNA 

abundance was calculated based on the relative ratios of Cecropin B1, Attacin or Lysozyme mRNA to 

RP49 mRNA. Results of triplicate experiments are presented with their standard deviations. Asterisks 

(*) denote significant differences from saline- or dimethyl sulphoxide (DMSO)-injected samples (P < 

0.05). (A) Comparison of mRNA levels after stimulation by crude LPS (cLPS: 0.1 or 1.0 µg/larva) and 

TLRgrade TM LPS (pLPS: 0.1 or 1.0 µg/larva) from Salmonella abortus equi. (B) Comparison of mRNA 

levels after stimulation by cLPS: 1.0 µg/larva or pLPS: 1.0 µg/larva from S. abortus equi (Sa) and 

Escherichia coli (Ec). (C) Comparison of mRNA levels after stimulation by lipid A (1.8 µg/larva) and 

DMSO (control). 


of antimicrobial peptide genes by highly 

purified LPS was not confirmed in the immuneresponsive 

cell line, NIAS-Bm-aff3, suggesting 

that some factors necessary for signal transduction 

activated by LPS are deficient in this cell 

line. 

Anionic C-terminal proregion of nematode 

antimicrobial peptide cecropin P4 precursor 

inhibits bactericidal activity of mature peptide 

Nematode cecropins such as cecropin P1 are 

one of the best characterized antimicrobial peptides. 

Recently, an anionic proregion was found 

to be conserved at the C terminus of the nematode 

cecropin precursors. The proregion was 

chemically synthesized to evaluate its functions. 

The proregion inhibited the bactericidal activity 

of the mature peptide suggesting that the activity 

of the nematode cecropin is suppressed in its 

precursor form. Contrastingly, the putative freeform 

of the proregion, which could be generated 

by a convertase cleavage, was not inhibitory 

in trans, suggesting that the inhibitory effect 

of proregion was terminated after processing 

(Fig 14). The liposome dye-leakage caused by 

cecropin P4 was not affected by the presence of 

proregion, suggesting that the proregion did not 

directly inhibit membrane disruption. 

Fig 14. P4P inhibition of bactericidal activity of cecropin P4. P4P and mature cecropin P4 were mixed 

at various molar ratios and incubated with S. aureus for 2 h. 

The bactericidal activities were estimated by inoculation of the bacterial suspension after incubation. 

‘0:16’ is equal to ‘1:16’ without cecropin P4 to test the bactericidal activity of P4P only. ‘C’ represents 

negative controls with neither cecropin P4 or P4P. P4P-pf is a putative free-form of P4P. HRR is a 

peptide which is removed from P4P after a convertase cleavage. 



Transcriptome analysis of the salivary glands 

of the Brown planthopper Nilaparvata lugens 

The Brown planthopper (Nilaparvata lugens 

Stål, BPH) feeds on phloem sap of rice and is 

a serious insect pest. Saliva of BPH is thought 

to play very important roles in sucking the 

contents from sieve tubes. Therefore, study of 

the functions of salivary glands is important for 

understanding the mechanism of the feeding 

process of BPH, but few molecular biological 

studies have been done to date. Expressed- 

Sequence-Tags (EST) database from various 

tissues of BPH have been constructed by H. 

Noda et al. in our institute. An EST database for 

the salivary gland was also developed and it can 

be a powerful tool for research on the functions 

of salivary glands, especially for the feeding 

process. Here we analyzed the EST database of 

salivary gland of BPH. 

The database consists of 2,383 sequences and 

these sequences are divided into 723 clusters 

based on their sequence homology. A consensus 

sequence was derived for each cluster and used 

to search for other homologous sequences at 

the NCBI website using BLAST-P. An amino 

acid sequence was deduced from the consensus 

sequence and searches for signal sequences and 

other motifs were conducted. 

Proteins which play important roles in saliva 

are thought to have secretory functions and to 

be saliva specific. In the EST database of the 

salivary gland from BPH, there are fourteen 

clusters that are salivary gland specific and 

probably encode proteins with secretory signal 

peptides. Homology to any known functional 

proteins was only found for three clusters 

which are homologous to mucin-like protein 

of Laodelphax striatellus, phosphatidylinositolspecific 

phospholipase C of Homo sapiens and 

arginine vasopressin-like peptide of Nasonia 

vitripennis (Table 2). Several clusters are 

suggested to encode highly repetitive, low 

complexity proteins that may be components of 

the stylet sheath. 

The salivary gland of BPH was reported to 

have phenolase activity and alpha- and beta-glucosidase 

activities. Phenolase activity in the salivary 

gland of BPH is suggested to be involved 

in the formation of stylet sheath. However, the 

clusters in Table 1 were not associated with 

these enzymatic activities. In the EST database 

of the salivary gland from BPH, more than 

Table 2. Clusters related to secretory and salivary gland specific proteins from EST database of BPH 

salivary gland 


200 clusters have homological sequences with 

known enzymes and one cluster is likely related 

to alpha-glucosidase which digests sucrose, the 

most abundant carbohydrate in sieve tube sap. 

On the other hand, no cluster was found in the 

EST database of the salivary gland related with 

beta-glucosidase. This database also includes 

clusters linked to such carbohydrate enzymes 

as alpha-mannosidase, alpha-galactosidase, alphatrehalase 

and beta-mannosidase. Their actual 

substrates have not been researched yet. 

Further studies for functions of saliva 

are very important for understanding the 

mechanism of insect and plant interactions and 

analysis of the EST database of BPH salivary 

gland will contribute to this field. 

Regioselective formation of quercetin 

5-O-glucoside from orally administered quercetin 

in the silkworm, Bombyx mori 

The cocoons of some races of the silkworm, 

Bombyx mori, have been shown to contain 

5-O-glucosylated flavonoids, which do not occur 

naturally in the leaves of their host plant, 

mulberry (Morus alba). Thus, dietary flavonoids 

could be biotransformed in this insect. In this 

study, we found that after feeding silkworms 

a diet rich in the flavonol quercetin, quercetin 

5-O-glucoside was the predominant metabolite in 

the midgut tissue, while quercetin 5,4′-di-O-glucoside 

was the major constituent in the hemolymph 

and silk glands. UDP-glucosyltransferase 

(UGT) in the midgut could transfer glucose to 

each of the hydroxyl groups of quercetin, with a 

preference for formation of 5-O-glucoside, while 

quercetin 5,4′-di-O-glucoside was predominantly 

produced if the enzyme extracts of either the 

fat body or silk glands were incubated with 

quercetin 5-O-glucoside and UDP-glucose. These 

results suggest that dietary quercetin was 

glucosylated at the 5-O position in the midgut 

as the first-pass metabolite of quercetin after 

oral absorption, then glucosylated at the 4′ 

-O position in the fat body or silk glands. The 

5-O-glucosylated flavonoids retained biological 

activity in the insect, since the total free radical 

scavenging capacity of several tissues increased 

after oral administration of quercetin (Fig 15). 

Fig 15. Regioselective formation of quercetin 5-O-glucoside (right) from orally administered quercetin 

(left) by UDP-glucosyltransferase (UGT) in the midgut of the silkworm, Bombyx mori. 


Highly polymorphic microsatellite-like DNA 

detected from the mitochondrial genome of 

Orius predatory bugs. 

The flower bugs of the genus Orius are an 

important group of natural enemies preying 

on various kinds of herbivorous pests such as 

thrips, aphids, psylids, scales, and mites as well 

as immature stages of some lepidopteran and 

coleopteran insects. Despite the high potential of 

this group as biological control agents, ecological 

information is limited for only a small number of 

species. This situation is due to their small size 

Table 3. Properties of 37 DNA regions identified from the mitochondrial DNA of Orius 

minutus and O. tantillus. 

Length Polymorphic TV 

(bp) sites (%) (%) 

indel 

(no.) 

CDS 

ND2 972 20.2 51.5 0 

CO1 1539 13.5 42.3 0 

CO2 682 9.2 42.9 0 

ATP8 156 21.2 57.6 0 

ATP6 671 13.4 47.1 3 

CO3 786 14.9 41.9 0 

ND3 354 24.9 55.7 0 

ND5 1687 17.0 52.4 0 

ND4 1331 17.0 52.2 0 

ND4L 286 17.1 46.9 0 

ND6 486 22.4 52.4 6 

Cyt b 1137 16.1 36.1 0 

ND1 918 16.1 46.6 0 

rDNA 

16S 1187 10.2 73.0 10 

12S 770 11.6 60.0 9 

tRNA 

tRNA-Ile 65 10.8 71.4 0 

tRNA-Glu 65 1.5 0.0 0 

tRNA-Met 71 9.9 33.3 1 

tRNA-Trp 66 10.6 42.9 0 

tRNA-Cys 63 4.8 33.3 0 

tRNA-Tyr 66 10.6 50.0 3 

tRNA-Leu 70 10.0 57.1 0 

tRNA-Lys 70 2.9 50.0 0 

tRNA-Asp 65 9.2 0.0 4 

tRNA-Gly 63 1.6 100.0 0 

tRNA-Ala 62 4.8 66.7 0 

tRNA-Arg 61 6.6 0.0 2 

tRNA-Asn 65 12.3 20.0 3 

tRNA-Ser 67 4.5 0.0 1 

tRNA-Glu 64 18.8 28.6 5 

tRNA-Phe 65 20.0 45.5 2 

tRNA-His 64 7.8 0.0 2 

tRNA-Thr 65 10.8 60.0 2 

tRNA-Pro 67 9.0 40.0 1 

tRNA-Ser 70 10.0 20.0 1 

tRNA-Leu 66 0.0 0.0 0 

tRNA-Val 68 10.3 33.3 2 


and scarcity of genetic characters that can be 

used to detect variations among species, strains, 

and individuals of this group. To solve this 

problem, we have been analyzing the mitochondrial 

and nuclear DNA of this group. To date, 

we have determined the complete nucleotide 

sequences of the mitochondrial genomes of 

Orius minutus (16,149 bp) and O. tantillus (16,305 

bp). Comparison of the sequences revealed 

that relative evolutionary rates and strength 

of nucleotide substitution bias were different 

among different DNA regions (Table 3), suggesting 

that different regions can be used to detect 

genetic variations at different taxonomic levels. 

Among 38 DNA regions identified from the 

mitochondrial genome (two rRNA, 13 proteincoding, 

and 22 tRNA genes, and an A+T rich 

control region), the control region was revealed 

to be the most variable. The region was 

composed of three sub-regions; two of which 

contain directly repeated sequences similar to 

the nuclear mini-satellite DNA and another one 

contains A-C repetitive sequence similar to the 

nuclear microsatellite DNA (Fig 16). Preliminary 

analyses using PCR-primers designed to amplify 

the microsatellite-like sub-region indicated that 

the length of the sub-region was highly variable 

among individuals even within a population 

collected in the same locality. The mitochondrial 

microsatellite-like DNA can be used with the 

nuclear microsatellite DNA markers previously 

detected from Orius species to precisely analyze 

interrelationships among populations of Orius 

bugs. These markers could especially help gain 

insights into the relationship between introduced 

and endemic populations of the natural 

enemy. 

Identification of (R)-2-butanol as a sex 

attractant pheromone of the white grub 

beetle Dasylepida ishigakiensis (Coleoptera: 

Scarabaeidae), a serious sugarcane pest in 

the Miyako Islands of Japan 

The white grub beetle, Dasylepida ishigakiensis 

Niijima et Kinoshita, is a serious pest of 

sugarcane in the Miyako Islands of Okinawa, 

Japan. Two evident electroantennographic 

(EAG) responses were observed by solid-phase 

microextraction (SPME) sampling from female 

secretion and subsequent gas chromatographyelectroantennographic 

detection (GC-EAD) 

analyses. These were identified as 2-propanol 

and 2-butanol by GC-MS analyses. GC-EAD on a 

chiral column showed two evident EAG signals 

corresponding to 2-butanol enantiomers, but 

the ratio and amounts could not be determined 

because of the small amounts. (R)-2-Butanol 

elicited the strongest EAG response, followed by 

(S)-2-butanol and 2-propanol, which showed 1/10 

and 1/100 of the response of the first compound, 

Fig 16. Sequences of the mitochondrial microsatellite-like DNA region obtained from 19 individuals 

of Orius flower bug. 


espectively. In the field, only (R)-2-butanol was 

attractive to feral males when baited with 1 ml 

water solution of 1–100 µg of this compound, (S)- 

2-butanol, racemic mixture or 2-propanol on cotton 

ball lures (ca. 1.5 cm diameter) in vane-traps. 

When 2-propanol was blended with 10 µg/ml of 

(R)-2-butanol solution on cotton ball lures, male 

catches decreased as the amount of 2-propanol 

increased. From these results it was concluded 

that (R)-2-butanol was a component of the sex 

attractant pheromone of D. ishigakiensis while 

(S)-enantiomer and 2-propanol showed inhibitory 

effects when blended with the first compound 

(Fig 17). 

Fig 17. GC-EAD chromatogram of volatiles from D. ishigakiensis females by SPME and subsequent 

direct injection into GC-EAD system, and mass spectrograms of corresponding components. 

Insect-Microbe Research Unit 

Map-based cloning of Bacillus thuringiensis 

toxin resistance gene from Bombyx mori 

Map-based cloning for a resistance gene 

against Bacillus thuringiensis (Bt) toxin in 

the silkworm Bombyx mori (Lepidoptera: 

Bombycidae) selected a candidate gene that is 

expressed in the intestine. Bt toxin is widely 

used for controlling lepidopteran and dipteran 

insects as agricultural chemicals or geneticallyengineered 

plant products. Wide use of the 

toxin has caused appearance of resistant pest 

strains against the Bt toxin. To elucidate the 

mechanism of the Bt resistance and to have the 

knowledge to counteract the resistance, we have 

identified the resistance gene in the silkworm. 

Insecticidal effect of a Bt toxin, Cry1Ab, was 

examined in the resistant strain 401, susceptible 

strain 606, F1 generation of both strains, and 

BC1 backcrossed generations. A recessive gene 

exerted the resistance. Surviving resistant silkworms 

were examined for the resistance gene 

in the Bombyx chromosome using SNP (single 

nucleotide polymorphism) markers. One candidate 

gene was finally obtained after sequence 

comparisons and gene expression studies in 

the intestine among resistant and susceptible 

strains. The candidate gene from the susceptible 

strain was introduced into a resistant strain, 

expecting the transgenic silkworm to become 

Bt susceptible. The transgenic silkworm showed 


high mortality by the Bt toxin, proving that the 

candidate gene is related to the resistance in 

the silkworm. 

Comparison of the ultraviolet tolerance of 

Bacillus thuringiensis among drug formulas 

Bacillus thuringiensis (Bt), a bacterial pathogen 

of insects, has been used for insect pest 

control. Spores and insecticidal crystalline inclusions 

of Bt are inactivated by sunlight, when 

they were sprayed in the field. Ultraviolet (UV) 

degradation is a main factor of the inactivation 

of Bt. Little is known, however, about how UV 

affects Bt-insecticidal formulations. We have 

tested the stability of six commercial Bt products 

(Toarow-CT, Bacilex, Dipol, Delfin, Esmark 

and Xentari) under ultraviolet (UV) irradiation 

using a fluorescent lamp (ranges 270-370nm: 

peak 315nm). Solutions of 0.1% for each Bt 

product were exposed to the fluorescent lamps. 

The bioactivities of Bt solutions before and after 

irradiation were compared based on the growth 

inhibition of 2nd instar silkworm Bombyx mori 

using a diet incorporation method. Toarow-CT 

and Bacilex lost the bioactivity rapidly, Dipol 

and Delfin did moderately, and Esmark and 

Xentari did slowly. There was about six-fold 

maximum difference in half-life between BT 

formulas. Toarow-CT and Esmark were also 

tested in the field; each formulation was sprayed 

on leaves of mulberry trees and their residual 

insecticidal effect was tested by the silkworm 

assay. The effective period of Toarow-CT was 

shorter than that of Esmark. It is necessary to 

elucidate the factors affecting the UV-tolerance 

among BT formulas. 

Analyses of fusolin from an Entomopoxvirus 

for stimulating effect on virus infection 

The proteinaceous paracrystalline structures 

called spindles that are produced by Anomala 

cuprea entomopoxviruses (AcEPVs) enhance the 

infectivity of the EPV and some nucleopolyhedroviruses 

(NPVs). Fusolin, a major constituent 

protein of the spindles, is responsible for the 

viral enhancing activity. We have identified 

the molecular region of AcEPV fusolin that 

has the activity using a bioassay system 

including Bombyx mori NPV and B. mori 

larvae. Various AcEPV mutant fusolins were 

produced using baculovirus expression system 

and the expressed proteins were used for the 

bioassay. The N-terminal region (ca. 250 amino 

acid residues of a translated form) containing a 

signal peptide is essential for the activity and 

N-glycosylation is prerequisite; the rest of the 

fusolin is not essential. The N-terminal region 

had binding activity to chitin in vitro and the 

glycosylation worked for chitin binding. Chitin is 

a main component of the peritrophic membrane 

in the gut, and this binding should be associated 

with the disruption of this gut membrane. The 

non-essential region was easily eliminated by 

serine protease(s) in the digestive juice of B. 

mori midgut, whereas the essential region was 

more stable. In addition, AcEPV spindles were 

highly stable against various abiotic factors 

including high temperature and ultraviolet radiation. 

This nature of the spindles is desirable 

for field use with virus pathogen as a synergist. 

Antiviral function of the intestinal peritrophic 

membrane in Bombyx mori and Spodoptera 

litura 

In previous studies we showed that an oral 

administration of chitin synthesis inhibitors such 

as polyoxin, nikkomycin and captan to the silkworm, 

Bombyx mori (Lepidoptera: Bombycidae), 

drastically enhanced an infection of a nucleopolyhedrovirus 

(BmNPV) orally inoculated. The 

larvae that ingested the chemicals with a diet 

lost their peritrophic membrane (PM) in the gut. 

The PM is a chitinous tissue, which has been 

regarded as an intestinal barrier against pathogenic 

microbes such as a virus. Disablement of 

the antiviral mechanism of a pest insect would 

lead to an effective pest control method utilizing 

a viral insecticide. We presently examine the 

effect of the chitin synthesis inhibitors on the 

nucleopolyhedrovirus (SpltNPV) infection in the 

Spodoptera litura (Noctuidae) larva, which is a 

serious polyphagous pest worm. The S. litura 

larvae that ingested the above chitin synthesis 

inhibitors with a diet lost their PM in the gut, as 

was the case for B. mori. However, the infection 

rare of SpltNPV was scarcely affected by the 

chemical administration, which indicates that 

the PM in S. litura did not serve as a protec- 


tive barrier against the NPV. The degree of 

dependence on the PM for self-defense against 

pathogenic microbes would differ from insect 

species to species. 

Multiple coding sequences of genome-linked 

viral protein in dicistroviruses 

Picornaviruses have the genome-linked viral 

protein (VPg) that is covalently linked to the 

5' terminus of their genome. VPg functions as 

a primer for viral RNA replication. Previously 

characterized picornaviruses have a single copy 

of the VPg gene. The only known exception 

is the foot-and-mouth disease virus, which has 

three non-identical copies of the VPg coding 

sequences arranged in tandem. N-terminal 

Edman sequencing of the VPg of Plautia stali 

intestine virus (Dicistroviridae, Cripavirus) 

detected heterologus residues. The VPg 

sequence determined was triplicated in the 

nonstructural protein precursor. Multiple VPglike 

sequences were also found in 11 of the 14 

dicistroviruses with a maximum of 6 copies in 

Solenopsis invicta virus-1. The redundant VPg 

coding sequences suggest that they facilitate 

multiplication of dicistroviruses, because fewer 

cycles of translation of the nonstructural protein 

precursor would produce larger amounts of VPg 

proteins and allow production of larger amounts 

of coat protein precursors in the infected cell. 

Factors related to transmission of rice yellow 

dwarf phytoplasmas in the green rice 

leafhopper 

Phytoplasmas are important insect-transmitted 

pathogenic agents causing more then 700 

diseases in plants. They are passively acquired 

by phloem-feeding insects during feeding on infected 

plants. During the latent period (LP) that 

elapses from initial acquisition to the ability to 

transmit, the phytoplasmas move through and 

replicate in the competent vector’s body. Rice 

yellow dwarf phytoplasma (RYDP) replicates 

in the midgut, circulates in the hemolymph, 

and infects the salivary glands (SGs) during the 

LP. To be transmitted to plants, phytoplasmas 

must penetrate specific cells of the SGs and 

accumulate in the acinar cells of the salivary 

glands. Whole-mount in situ hybridization 

(WISH) using DIG-labeled phytoplasma-specific 

oligonucleotides revealed that RYDP are located 

in posterior lobes III of the SGs of the green rice 

leafhopper, Nephotettix cincticeps. 

To investigate factors associated with RYDP 

accumulation in SGs, the differential display 

method was performed. Differentially expressed 

genes between the SGs of RYDP-infected and 

uninfected vector insects were detected and the 

level of differential expression was evaluated 

by quantitative real-time PCR. A5SGR1 gene 

was up-regulated two-fold in the SGs of RYDPinfected 

leafhoppers. Additionally, WISH using 

DIG-labeled riboprobe of the A5SGR1 gene 

showed that A5SGR1 gene expression was 

restricted to the posterior lobes III. Future 

investigations will examine the relationship 

between A5SGR1 gene expression and RYDP 

accumulation in the acinar cells of the SGs. 

EST analyses of the brown planthopper 

Expressed sequence tag (EST) analyses 

of several important agricultural pests were 

performed for various aspects of studies 

including insect-microbe interaction. EST data 

of the brown planthopper, Nilaparvata lugens 

(Homoptera), a serious rice pest, has been 

released. More than 37,000 ESTs, excluding the 

mitochondrial genome, microbial genomes and 

ribosomal RNA sequences, have been produced 

from 18 libraries of various N. lugens tissues 

and stages. More than 10,000 clusters have been 

made from whole EST sequences, with average 

EST size of 627 bp. The actin gene was highly 

expressed in the planthopper, especially in the 

thorax. Several abundantly expressed genes 

are unique and show no homology in BLAST 

searches. Tissue-specifically expressed genes 

were found based on the expression frequency 

among the libraries. An EST database is available 

at http://bphest.dna.affrc.go.jp/. Sequences 

of clones from full-length cDNA libraries are 

going to be determined. 



The Silk-Materials Research Unit conducts research 

on 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 directed for use in medical areas such 

as cartilage regeneration scaffolds and wound 

dressings. We also develop fabrication technologies 

and approaches to improve the functional 

properties of silk proteins by use of transgenic 

and chemical processes. 

Influence of pore size on mechanical properties 

and chondrocytes culture of silk fibroin 

sponge 

We have reported that a silk fibroin porous 

3D structure (fibroin sponge) is applicable to 

be a cell scaffold for cartilage regeneration. We 

studied the influence of pore size on mechanical 

strength and cartilage regeneration of silk 

fibroin sponges in collaboration with Kyoto 

University. Within the experimental range of 

pore sizes, we did not detect significant differences 

in tensile strength or Young’s modulus 

for fibroin sponges. Furthermore, we found no 

significant changes in the mechanical properties 

between wet and dry state of fibroin sponges. 

Proliferation of chondrocytes and cartilage 

regeneration were improved for fibroin sponges 

with smaller pore size due to increased surface 

area compared to the larger pore size (Fig 18). 

The result suggests that an optimal pore size 

exists for cartilage regeneration in silk sponges. 

Characterization of Gel Film Prepared from B. 

mori Silk Sericin as a Wound Dressing 

Last year, a novel film material, named gel 

film, made of B. mori silk sericin was developed 

by drying a sericin hydrogel prepared from 

intact sericin that retained its native molecular 

weight. The gel film exhibits better water resistance 

than the conventional cast film due to the 

formation of intermolecular β-sheet networks 

during the gelation process. In this year, physical 

and biological properties of the gel film were 

investigated to verify its utility as a wound 

dressing. The dry gel film rapidly absorbed 

water and its water content equilibrated at 

about 80%. This value is comparable to those of 

other natural polymer-based wound dressings. 

The wet gel film exhibits elastic property with 

about 25% strain when it is uniaxially stretched. 

Fig 18. SEM photographs of fibroin sponges having different pore size. 


sericin 

gel film 

-serum 

+serum 

Cell number/cm -2 

1.5x10 4 

1.0x10 4 

5.0x10 3 

0.0 

cell cuture 

dish 

1.5x10 4 

1.0x10 4 

5.0x10 3 

0.0 

Fig 19. The number of mouse fibroblasts (Balb/3T3) adhered to the surface of the sericin gel film and 

the cell culture dish. 

Balb/3T3 were cultured for 2 h at 37°C in MEM with and without 10% fetal bovine serum. 

This elasticity reflects highly satisfactory handling 

property of the gel film in the wet state. 

Adhesion of mouse fibroblasts to the surface of 

the gel film was found to be low, especially under 

serum-free condition (Fig 19). This low cell 

adhesion was not due to the cytotoxicity of the 

gel film because the adhered cells propagated 

normally. These physical and biological properties 

of the sericin gel film indicate its usefulness 

as a dressing for moist wound healing. 

Gel Film Formation and Development of 

Morphology and Mechanical Properties upon 

Drawing of Silk of the Hornet Vespa 

The hydrogel of silk of the hornet Vespa 

simillima xanthoptera was pressed and dried 

to obtain a film. Wide angle X-ray diffraction 

(WAXD) and 13 C solid-state NMR experiments 

revealed that the hornet silk gel film (HSGF) 

possessed an α-helix with coiled-coil structure 

together with a β-sheet, which indicates that 

a structure similar to that of the native hornet 

cocoon is restored during the generation of 

HSGF. When HSGF was uniaxially drawn in the 

dry state, a necking phenomenon was observed 

(Fig 20), whereas, when in the wet state, the 

film became flexible and could be uniformly 

drawn to ≦ 2. The changes in the structures of 

gel film caused by drawing and heat-treatment 

were examined by WAXD, 13 C solid-state NMR, 

IR spectroscopy, scanning electron microscopy 

(SEM), and birefringence measurements. Even 

though the maximum draw ratio is only 2, 

the maximum tensile modulus and strength 

achieved in this study were higher than those 

of the films regenerated from silkworm silk and 

were comparable to those of native silk fiber 

spun from wild-type silkworm silk. 

Fig 20. SEM microphoto of the hornet silk gel film drawn uniaxially in the dry state. 

A necking phenomenon is observed. 


Spheroid formation of human hepatic cell line 

FLC-4 in lactose-modified silk fibroin sponges 

Since it is known that spherical multicellular 

aggregates (spheroids) of hepatocytes express 

higher liver-specific functions compared to 

monolayer hepatocytes, scaffold materials 

endowed with spheroid formation ability have 

been developed for hepatocyte culture. We 

designed three-dimensional porous sponges 

composed of lactose-modified silk fibroin (Lac- 

CY-SF) as a scaffold for spheroid formation of 

hepatocytes. Lac-CY-SF sponges were fabricated 

by freeze-drying of aqueous Lac-CY-SF solutions 

and insolubilized by the immersion into methanol 

aqueous solutions. FLC-4 cells, a functional 

human liver cancer cell line, was cultured in the 

sponges of Lac-CY-SF, silk fibroin, and collagen 

as a model of primary human hepatocytes. 

Morphologies of viable FLC-4 cells cultured in 

the sponges for 5 days were observed by phasecontrast 

microscopy after staining with neural 

red. Lac-CY-SF sponges induced the aggregation 

of the cells into spheroids with diameters 

enlarged to 80 µm in the inner pores (Fig 21) 

similarly to collagen sponges. In contrast, no 

cell aggregates could be seen in unmodified silk 

fibroin sponges. These results suggest that Lac- 

CY-SF sponges as well as conventionally-used 

collagen sponges were efficient for the spheroid 

formation of FLC-4 cells. 

materials. However, the molecular mechanism 

of the spinning process is not known yet. In 

order to analyze the molecular formation of 

native silk protein of Japanese spider Nephila 

clavta, circular dichroism (CD) spectroscopy was 

carried out. Significant structure changes were 

identified in the concentration dependence CD 

measurements (Fig 22). These data suggested 

that the spinning mechanism was involved in 

the aggregation of spider silk protein. Based on 

its hydrophobicity, the fiber forming region in 

silk protein was predicted from the amino acid 

sequence deduced from the cDNA sequence of 

the dragline protein. The candidate peptides 

were synthesized using the solid-phase method. 

Fiber formation experiments in several types of 

solvents are in progress. 

Production of pheromone receptors in insect 

cells for constructing a biosensor 

We cloned genes of pheromone receptors 

from the antennae of male silk moth (Bombyx 

mori) and synthesized their cDNAs. Among a 

number of pheromone receptors, BmOR1 and 

BmOR2 were selected. BmOR1 is reported 

as a sex pheromone receptor and BmOR2 is 

thought to be a signal amplifier. The precise 

role of BmOR2, however, is not yet apparent. 

We constructed the plasmid vector containing 

cloned genes and expression markers, such as 

Spinning mechanism of spider silk protein 

Spider silk is a particularly promising 

polymer molecule for protein-based functional 

Fig 21. Phase-contrast image of neutral red-stained 

FLC-4 cells cultured in Lac-CY-SF sponges for 5 

days. 

Fig 22. CD spectra of spider silk protein with decreasing 

concentration. 


EGFP and KusabiraOrange. They were transiently 

transfected by the lipofection method 

in insect cells, such as BmN and SF9. After 2 

days of culture, we observed expression of recombinant 

proteins, BmOR1-EGFP and BmOR2- 

KusabiraOrange, by the fluorescent microscope 

as shown in Fig 23. We confirmed the receptor 

expression by the immunoblotting method. 

Membrane fractions and cytoplasmic fractions 

were prepared from cultured cells using a commercial 

protein extraction kit. The fractionated 

proteins were blotted with antibodies to BmOR1 

and BmOR2. From the results, recombinant 

proteins were found in the membrane fractions 

specifically. Now we are examining the cell 

functions as a pheromone receptor by using 

electrophysiological methods, such as the whole 

cell clamp technique. 

Cloning of B. mori Phenylalanyl-tRNA 

Synthetase for Relaxing Its Amino Acid 

Recognition 

Aminoacyl-tRNA synthetases catalyze 

aminoacylation reactions of tRNAs with their 

cognate amino acids. The aminoacylation reaction 

is crucial for discriminating correct amino 

acids from others. Specificity of amino acid 

recognition can be relaxed by amino acid substitution 

in aminoacyl-tRNA synthetase active 

sites. The substrate specificity of phenylalanyltRNA 

synthetase (PheRS) has been extensively 

studied and several mutants which allow the 

incorporation of various Phe analogues have 

been developed. In this context, the genes 

encoding B. mori PheRS were cloned from the 

silk gland for the purpose of creating silk proteins 

containing phenylalanine analogues with 

unnatural functional groups. PheRS functions 

as a tetramer composed of two subunits, α and 

β, and its amino acid recognition site exists 

in the α subunit. The deduced amino acid 

sequence of B. mori PheRS α subunit exhibited 

similarity with PheRSs from other organisms 

such as S. cerevisiae and E. coli. In particular, 

the sequence around the amino acid recognition 

site is well conserved in B. mori PheRS (Fig 

24). From the sequence alignment, Thr407 and 

Ala450 of B. mori PheRS were expected to act 

as determinants of amino acid specificity. Amino 

acid substitution in these residues might relax 

the amino acid recognition as with PheRSs from 

S. cerevisiae and E. coli. 

Fig 23. Receptor-expressing Insect Cells (BmN). 

(Left: BmOR1-EGFP, Right: BmOR2-KusabiraOrange, The diameter of the cells is about 20µm.) 

Fig 24. Amino acid sequence alignment among B. mori, S. cerevisiae, and E. coli PheRS α subunits. 

Sequences around the amino acid recognition sites are shown. The presumed determinants of amino 

acid specificity (Thr407 and Ala450 for B. mori PheRS) are marked with boxes. 


Silk-Technology Unit 

Breeding of a strong cocoon filament race of 

the silkworm, Bombyx mori. 

Demand is rising for strong silk filaments for 

use in the fields of medicine and new materials. 

In response, we have been working to develop 

a new silkworm race which produces a strong 

cocoon filament. Three strong filament strains 

(N2, N4 and Y for J-parental strain; c, a and s 

for C-parental strain) were chosen from our 

breeding resources and genetic stocks in NIAS. 

We crossed these strains and individually 

selected the strong cocoon filament progeny. 

The filament strength of the population selected 

from the J-parental strain showed a wide deviation 

but has been gradually increased (Fig 25, 

left). The cocoon filament of C-parental strain 

has suddenly become stronger in the sixth 

generation after crossing, although it had been 

weaker than the parental strains until the fifth 

generation (Fig 25, right). Through this selection 

process, we have obtained both parental 

strains which produce strong cocoon filaments. 

Consequently, the cocoon filaments of their F1 

hybrid race, J x C, is also stronger than the 

conventional races (Table 4). 

We are also developing the polyphagous 

SERICIN HOPE for effective recovery of recombinant 

proteins from cocoon shells, and a chromosome 

substitution line for functional studies of 

practical traits using genome information. 

Fig 25. Changes of cocoon filament strength during the process of breeding high strength parental 

races J-(left) and C-(right). 

N2, N4, Y, C, A and S indicate the breeding resource strains. 

Table 4. Performances of cocoon filament of new silkworm race 


The luster/color of the silk thread and the 

silkworm races 

In our previous examinations, we determined 

that silk yarn was different in luster and color 

among silkworm races. We also observed that 

fine size of silk had higher luster than thick 

size of silk. To clarify whether luster depends 

on innate difference among races or on silk 

fineness, we used Triflumizole, which cause 

induction of three moltings (trimolter) on larva, 

to develop two types of silkworm to one of 

trimolter and the other of normal (four) moltings 

(tetramolter) in the same race. The trimolter 

make generally a finer thread than tetramolter. 

After cocooning, the luster and color of the raw 

silk were measured. In all races, the fiber size of 

the trimolter types became smaller than that of 

tetramolter types (Table 5), but the luster values 

did not consistently follow this pattern (Fig 23). 

In particular, the luster value of the “Hakugin” 

trimolter type was conspicuously lower than 

that of the tetramolter type. In addition, the trimolter 

types of “N137 × C146” and “C146” were 

smaller than the “Hakugin” tetramolter type, 

but their luster values were still low (Fig 26). It 

is considered that race has a bigger influence on 

luster than the fiber size. 

Table 5. The size (deniers) of cocoon filaments among silkworm races 

expressing trimolter or tetramolter types 

type 

silkworm race 

Hakugin Ariake N137×C146 C146 

trimolter 1.22 2.3 1.57 1.56 

tetramolter 1.76 3.71 2.91 2.65 

Fig 26. The luster value of raw silk among the silkworm races expressing trimolter (-3) or 

tetramolter (-4) types. 


The characteristics of silk thread, fabrics and 

floss produced by different silkworm races 

To investigate race-specific fabric characteristics, 

in addition to silk luster and color, we 

measured KES values for different fabrics made 

using several silkworm races for which cocoon 

filament sizes were different (Fig 27). We found 

the difference in the KES value for SHARI and 

“Shin-Akebono” was low and “Ariake” was a 

little high. We understood that these differences 

could be attributed to the silkworm races. 

To elucidate cocoon filament strength characteristics 

and assist development of high strength 

silkworm races, we investigated physical 

properties of 27 denier (d) raw silks from several 

high strength silkworm races. When tested with 

a tensile testing machine, a value of 5.35g/d for 

“SFC” had been the best until now. As shown 

in Table 6, “SFC2” was improved with a value 

of 5.60g/d and “SFC x SFN”, which is a hybrid 

silkworm showed the biggest value of 5.67g/d. 

We investigated the bulkiness of silk floss 

which is a product that could be developed 

directly from cocoons. Floss was obtained from 

cocoons of “Hakugin” which has thin filaments, 

from cocoons of “Ariake” which has thick 

filaments and from cocoons of “Oomi” (Fig 28). 

“Hakugin” and “Ariake” had almost the same 

bulkiness values and both were higher than 

“Oomi” silk floss. Also the bulkiness value of 

Fig 27. KES evaluation values of 

the fabrics. 

(KN-201-LDY); 1: Shin-Akebono 

(Spring), 2: Hakugin (Spring), 

3: Hakugin (Summer), 4: Ariake 

(Summer), 5: Hakugin (Autumn), 6: 

Ariake (Autumn), 7: N137×C146 

(Autumn), 8: C146×N137 (Autumn) 

Tenacity Elongation Young's modulus 

g/d kg/mm 2 

SFC2 5.60 20.0 1,326 

SFC3 5.17 22.7 1,225 

SFC4 5.30 20.0 1,219 

SFC×SFN 5.67 22.5 1,275 

C514×SFC 5.38 23.4 1,276 

SFC×C514 5.11 19.9 1,261 

Table 6. Physical properties 

comparison of raw silk from 

purebred and hybrid silkworms 

Bulkiness (cm 3 /g) 

30 

20 

10 

Fig 28. Bulkiness comparison of 

silk flosses. 

0 

Ariake 

Hakugin 

Oomi 

Materials 


“Hakugin” was a little superior to “Ariake”. 

Development of artificial blood vessels using 

cocoon filaments 

Artificial blood vessels of small inside 

diameter (1-5mm) have been developed from 

cocoon filaments using a combination of plaiting 

of silk threads and winding of cocoon filaments. 

In order to adapt artificial blood vessels to a 

living body, this year we produced vessels 

using cocoon filaments obtained from cocoons of 

transgenic silkworms, which contains a cell selfmultiplication 

protein (Fig 29). Graft operations 

with these vessels were tested with rats (Fig 

30). 

At various times after grafting, the artificial 

blood vessels were removed and sections of 

them were observed. Fig 31 shows sections 

after 2, 4 and 12 weeks. Fibroin fibers (white 

areas) decreased with time and collagen (red 

areas) increased with time. Thus, with time 

passing a part of collagen increased instead of 

fibroin fibers in a artificial blood vessel. 

Development of reeling methods for fluorescence 

transgenic cocoons and their products 

Large of quantities of transgenic silkworm 

cocoons expressing fluorescence protein genes 

were produced on a laboratory scale at the 

National Institute of Agrobiological Sciences. 

Methods for cocoon cooking and reeling were 

considered to adapt to the characteristics of 

those cocoons and materials such as knit suits 

and interior decorations were produced. 

The fluorescence color of cocoon filaments 

was decreased by treatment temperatures 

higher than 60C; therefore, new cocoon cooking 

methods were considered. We developed 

Fig 29. Artificial blood vessels; Left: 

1.5mm diam. Right: 1.0 diam 

Fig 30. Graft operation to rat produced 

from cocoon filaments 

Fig 31. Changes in areas of fibroin (white) and collagen (red) in sections of artificial blood vessels in 

accordance with time passing. Left: After 2 weeks, 

Center: After 4 weeks, Right: After 12 weeks blood vessels; Left: 1.5mm diam. Right: 1.0 diam 


a vacuum cooking method using an alkaline 

surface active agent so that cocoons could be 

reeled (Fig 32) without decreasing the fluorescence 

color of the silk. Fig 33shows pink silk 

thread due to introduced fluorescence protein 

genes originating from coral (DsRED (Discosoma 

sp Red)). Fig 34 shows silk skeins reeled from 

transgenic cocoons expressing the introduced 

Green Fuorescent Protein (GFP) and DsRED 

and the fluorescence resulting from illumination 

of silk skeins under blue LED. 

Fig 32. Vacuum type cocoon cooking machine (left) and the reeling state (right). 

Fig 33. Silk skeins of DsRED (left) and the fluorescence obtained by illumination with blue LED (right). 

Fig 34. Knit suits using GFP and DsRED (left) and the fluorescence occurring state by blue LED (right). 


The Laboratory of Special Projects 

The laboratory is operated with funding 

provided by several supporting companies 

and is backed by the National Institute of 

Agrobiological Sciences. The laboratory is 

conducting the insect symbiotic microorganism 

genome project in collaboration with the Insect- 

Microbe Research Unit of the Institute, and the 

College of Pharmacy, Nihon University. 

Construction of gene libraries expressible in E. 

coli from insect symbiont genomic DNA 

Symbiotic microorganisms (symbionts) exist 

in many species of insects, and are thought to 

produce substances that control insect growth, 

reproduction and sensitivity to infection by 

pathogenic bacteria among other effects. Some 

of the substances are expected to be useful for 

the development of novel medicine and agricultural 

chemicals. This special project aims to construct 

a system to search for novel biologically 

active substances that the symbionts produce. 

Brown-winged green bugs (Plautia crossota stali) 

were initially used as a model insect. After establishment 

of the experimental procedure and 

conditions, the P. crossota symbiont genomic 

DNA libraries, which are expressible in E. coli, 

have been constructed using a plasmid vector. 

About 100 clones were isolated from the abovementioned 

gene libraries (16,300 independent 

clones) and proteins expressed by each clone 

were analyzed by SDS-polyacrylamide gel 

electrophoresis (PAGE). Six discrete protein 

bands were detected on the gels. The structural 

analyses of each clone suggest that a specific 

protein was produced from the cloned specific 

gene of the P. crossota symbiont in the E. coli 

system. 

The expressible gene libraries were further 

constructed in a similar manner using genomic 

DNA prepared from the symbionts of brown 

marmorated stink bugs (Halyomorpha halys). 

In addition, the collaborator (Nihon University) 

is leading current research to determine the 

complete sequence of the total P. crossota 

symbiont genome and its annotation, and also 

has prepared the symbiont expressible gene 

library using a BAC vector. The extracts from 

the culture of each clone were found to show 

inhibitory activity in the Toll-Like Receptor-4 

assay system. 

Purification and characterization of the pigment 

extracted from H. halys gastric caeca 

Halyomorpha halys gastric caeca contains 

an interesting red pigment. Purification of the 

pigment was attempted in order to elucidate the 

structure of the pigment. Prior to purification, 

chemical and physical properties of the pigment 

were examined. Based on the findings obtained, 

the pigment was extracted from the gastric 

caeca of H. halys using SDS solution, and purified 

by SDS-PAGE followed by paper chromatography. 

Absorption spectrum of the pigment 

was found to show a maximum absorption at 

430 nm. The red pigment contained in gastric 

caeca of Halyomorpha brevis was identified 

by paper chromatography to be erythropterin 

which has an absorption maximum at 450 nm. 

From these findings, it seems unlikely that the H. 

halys pigment is erythropterin. It is necessary 

to obtain higher amounts of the purified pigment 

for further analyses of its structure. So far, 

bacteriocidal or growth-inhibitory activity of the 

pigment against E. coli, S. aureus and M. luteus 

has not been detected. 



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 

contribute significantly 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 

The 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. 


The 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 

The unit aims to elucidate the central mechanisms 

of learning, stress response, growth and 

reproduction in domestic animals to improve 

livestock management systems. The unit 

investigates neural functions of hypothalamicpituitary 

axis and limbic-cortex system and its 

control mechanisms by environmental factors. 

The major research topics in the fiscal year 

2008 are described as follows: 


The Animal Genome Research Unit 

Numbers of loci of swine leukocyte antigen 

classical class I genes differ depending on 

haplotypes 

The major histocompatibility complex (MHC) 

is one of the most complicated regions in the 

whole mammalian genome. It encodes many 

proteins that are closely involved in the immune 

response through the presentation of peptidic 

antigens to T lymphocytes. In the MHC region, 

many paralogous genes have been generated 

in the process of evolution from particular 

ancestral genes. The process of evolution of 

MHC class I genes in mammals is extremely 

complicated and there are many types of 

species-specific expansions of class I genes. 

Consequently, the structure of the gene family 

of MHC class I varies among species, and it is 

often difficult to find counterparts of a particular 

class I gene in closely related species. 

The porcine MHC class I genes are divided 

into two groups – classical and nonclassical – on 

the basis of their locations on the genome and 

their similarity to human or other mammalian 

class I genes. There are six classical class I 

genes: SLA-1, SLA-2, SLA-3, SLA-4, SLA-5, and 

SLA-9. Among the proteins encoded by these 

loci, SLA-1, SLA-2, and SLA-3 are functional and 

play a role in the presentation of intracellular 

peptidic antigens to cytotoxic T cells. SLA-4 

and SLA-9 are pseudogenes, and there is no 

evidence for the expression of SLA-5. An 

additional class I pseudogene, SLA-11, which 

has characteristics of both the classical and 

nonclassical class I genes, is located adjacent 

to the region containing the classical class I 

genes. The entire sequence of the porcine MHC 

(swine leukocyte antigen; SLA) region was 

completely sequenced recently by using a particular 

haplotype named Hp-1.1 found in a Large 

White pig. The sequencing of this region has 

demonstrated that the classical class I genes are 

arranged in a single contiguous region, and are 

ordered as SLA-1, SLA-5, SLA-9, SLA-3, SLA-2, 

SLA-4, and SLA-11 from the p-terminus of the 

chromosome. On the other hand, there are three 

nonclassical class I genes, SLA-6, SLA-7 and 

SLA-8, in a cluster. 

Studies to date have indicated that there is 

copy number variance of classical class I genes 

in the porcine MHC region. In particular, several 

studies have implied that the typical classical 

class I gene, named SLA-1, which is clearly 

defined in the Hp-1.0 haplotype (the portion of 

the region containing class I genes of the Hp-1.1 

haplotype), has duplications or deletions on the 

chromosome. On the other hand, another type 

of classical class I gene, named SLA-3, has deletions 

in particular haplotypes. However, genotyping 

of SLA-1 and SLA-3 in individuals whose 

haplotypes are not already known has often 

been performed by using cDNA, because the 

complexity and polymorphism of the sequences 

flanking the classical class I genes hinder the 

design of appropriate primers for polymerase 

chain reaction (PCR) amplification in genotyping. 

Therefore, the actual number of SLA-1 or 

SLA-3 loci on the genome has not been clearly 

demonstrated. We previously designed 40 microsatellite 

(MS) markers for the genotyping of 

haplotypes of SLA. The MS markers within the 

genomic region carrying MHC classical class I 

genes frequently produced more than two fragments 

from diploid genomic DNA. This implies 

that in these individuals there was duplication 

of the genomic region containing the classical 

class I genes, unlike in the Hp-1.0 haplotype. 

Previous studies have also implied that such 

copy number variation between haplotypes exists 

in MHC class I genes of other artiodactyls, 

i.e., cattle and sheep. In these animals, as in pigs, 

different haplotypes express varying numbers 

of class I genes. The variation in the number 

of class I genes expressed in cattle and sheep 

makes it difficult to designate locus names. 

We constructed two contigs using a bacterial 

artificial chromosome (BAC) library, which was 

generated with a Landrace breed pig, covering 

the region carrying MHC classical class I genes. 


The two contigs were derived from haplotypes 

different from Hp-1.0. We determined the whole 

sequences of the BAC clones contained in the 

two contigs. The contig corresponding to the 

first haplotype, which was designated Hp-28.0, 

was 541,313 bp long and ranged from the 

genomic region carrying TRIM26 and other 

members of the TRIM gene family (this region 

is located on the side of the p-terminal flanked 

by SLA-1 on swine chromosome [SSC] 7 of the 

Hp-1.0 sequence), to SLA-11, which is the class 

I gene most distal to the p-terminal of SSC7 in 

the region containing classical class I genes. The 

contig was extended to the middle of TRIM39, 

adjacent to SLA-11. The second contig, which 

was derived from another haplotype, which 

was designated Hp-62.0, was 254,346 bp long. It 

ranged from TRIM10 to SLA-4. Southern blotting 

with genomic DNA used for construction 

of the BAC library showed that one additional 

locus was inferred besides the loci contained 

in the contig; it might have corresponded to 

SLA-11 on the Hp-62.0 haplotype, although we 

could not isolate the BAC clones containing 

SLA-11 of Hp-62.0 from the library. Both of the 

contigs contained the genomic region containing 

loci corresponding to the typical SLA classical 

class I genes SLA-1, SLA-2, and SLA-3. 

The sequences derived from the Hp-28.0 and 

Hp-62.0 haplotypes contained 13 and 8 SLA 

classical class I genes, respectively (Fig 1). This 

was strikingly different from the case in the 

Hp-1.0 haplotype, which possesses seven SLA 

classical class I genes. We performed sequence 

comparisons of the observed loci with those 

on the Hp-1.0 haplotype. We detected five loci 

homologous to SLA-1/SLA-3 on the Hp-28.0 

haplotype (A01, A03, A05, A08, and A10). One 

of the loci corresponding to SLA-1/SLA-3 had 

a 2-bp deletion in exon 5 that generated a stop 

codon in exon 6 (A05). Another locus corresponding 

to SLA-1/SLA-3 had a 4-bp elongation 

in exon 6, resulting in a stop codon in exon 7 

(A03). On the other hand, the Hp-62.0 haplotype 

had three loci (B01, B04, and B06) homologous 

to SLA-1/SLA-3, one of which possessed an 

elongation similar to A03, resulting in a stop 

codon in exon 7 (B04). Phylogenetic analysis using 

entire coding sequences or using sequences 

of exons 2 and 3, as in a previous report of SLA 

class I nomenclature and the database for MHC 

polymorphisms, IPD-MHC (http://www.ebi. 

ac.uk/ipd/mhc/), demonstrated that A10 and 

B06 were included in the clade of known SLA-3 

sequences; we therefore classified these loci into 

SLA-3. Among the remaining loci similar to 

SLA-1/SLA-3, A01, A08 and B01 were grouped 

into a large clade containing many SLA-1 

alleles. In this study, we designated SLA-1 for 

these loci similar to authentic SLA-1. On the 

other hand, A03, A05, and B04 were grouped 

by themselves, away from the other SLA-1 

and SLA-3 loci, although these loci shared two 

characteristic bases – C at position 51 and G at 

position 118 in the coding sequence (CDS) – that 

clearly discriminate known SLA-1 alleles from 

SLA-3. These loci were designated SLA-12 

(Fig 1). 

We assigned the blocks to make the duplication 

and deletion process clear. The region 

encompassing two characteristic repetitive 

sequences, 5S rRNA and Tigger1a, was 

designated block A. The regions encompassing 

SLA-1-like (SLA-1 and SLA-12), SLA-5, SLA-9 

and SLA-3 loci and their flanking characteristic 

sequences were designated B, C, D and E, 

respectively. There were four repetitive units 

in the Hp-28.0 haplotype for the sequence corresponding 

to blocks B, C, D, and A in the Hp-1.0 

haplotype (Fig 2), although several regions were 

deleted in the repetitive units. The deletion of 

block D in the first repetitive unit (designated 

Hp-28.0-I) corresponded to the region carrying 

an SLA-9 locus and its upstream sequence. The 

second repetitive unit (Hp-28.0-II) lacked a large 

sequence, block C and the p-terminal side of 

block D, ranging from the downstream of A03 

(SLA-12) to the (GGGGA)n MS repeat located 

downstream of the absent SLA-5 locus. The 

second unit also lacked the SCRE upstream of 

A03 in block B. The most proximal unit to the 

centromere (Hp-28.0-IV) had a small deletion 

including a locus corresponding to SLA-9 (block 

D). In the Hp-62.0 haplotype, a large duplication 

of the sequence covering blocks B to A was 

observed; it also lacked block C, including a 

locus corresponding to SLA-5 and its upstream 

region in the unit proximal to the centromere 


(Hp-62.0-II). These observations show that active 

duplication and deletion processes have occurred 

in the region upstream of SLA-3 in the 

respective SLA haplotypes (Fig 2). 

The pig is considered a useful model animal 

for biomedical research – especially for transplantation 

research – because of the similarity 

of its organ size and cardiovascular system 

structure to those in humans. In such studies 

or transplantation applications using pigs, 

precise knowledge of the expression of antigenpresenting 

SLA molecules is indispensable. 

Determination of copy number variance of loci 

of MHC genes as presented in this study contributes 

to the analysis of how SLA haplotypes 

affect porcine immunological responses and 

disease resistance, and it will also help in the 

experimental design of transplantation research 

in pigs. 

Fig 1. BAC contigs derived from two haplotypes possessed by a Landrace individual. 

The haplotypes of the individual differed from haplotype Hp-1.0, which had been sequenced 

completely in previous studies. Genes on the genomic sequences are indicated by rectangles with 

arrows showing their orientation, according to a previous study. Loci with stop codon(s) in the 

region corresponding to CDSs or loci regarded as pseudogenes are asterisked. Detected genes 

corresponding to SLA classical class I genes are demonstrated as A01 to A13 on the Hp-28.0 

haplotype and B01 to B08 on the Hp-62.0 haplotype, with the corresponding class I genes named 

on the Hp-1.0 haplotype. SLA-1-like loci with different characteristic bases, newly found in this 

study, were designated as SLA-12. The duplicated loci were followed by sequential letters (a, b and 

c) from the most centromeric locus. MS markers (SLAMS034 and SLAMS035) from our previous 

reports are indicated by gray rectangles on the genomic sequences, along with the lengths of the 

PCR amplicons for the respective loci. BAC clones consisting of the contigs for the respective 

haplotypes are indicated by the large arrows with lengths in base pairs in parentheses. Locations of 

the fragments detected by Southern hybridization with the universal SLA classical class I probe are 

shown under the respective genomic sequences by hatched rectangles, with lengths in base pairs. 


Fig 2. Structural comparison around the duplicated regions containing loci corresponding to SLA-1, 

SLA-5, and SLA-9 among haplotypes. 

SLA classical class I loci, as well as characteristic repetitive sequences and MS repeats adjacent to 

the class I loci, are indicated in the three haplotypes. The regions encompassing two characteristic 

repetitive sequences, 5S rRNA and Tigger1a, were designated block A. The other regions 

encompassing loci and their characteristic flanking sequences were designated as follows: B (SLA-1/ 

SLA-12), C (SLA-5), D (SLA-9), and E (SLA-3). Loci that might have been deleted from the putative 

ancestral genomic sequence are asterisked. Repetitive units in the Hp-28.0 and Hp-62.0 haplotypes 

are designated Hp-28.0-I to -IV and Hp-62.0-I to -II, respectively, at the right side of the figure. 

Detection of QTL for average daily gain in a 

Large White population (a commercial pig 

breed) 

Growth rate is one of the important traits 

in pork production. Many researchers have 

investigated quantitative trait loci (QTL) affecting 

growth rates (or average daily gain), and 

some significant QTL effects were reported. 

Most QTLs were detected by using F2 populations 

between pigs from Asian local breeds and 

European commercial breeds. It is well known 

that European commercial breeds have been 

improved for many years and that they grow 

faster than Asian local breed pigs and wild 

boars. For improvement of commercial breed 

pigs currently, it is necessary to identify QTLs 

which have not been fixed in commercial breed 

pigs. 

We have investigated QTL affecting economical 

traits by using F2 populations and detected 

three QTL for growth rates on SSC (Sus scrofa 

chromosome) 4, 7 and 8 in an F2 population 

between a Japanese wild boar and Large White 

breed pigs (Fig 3). If diversity of these QTL 

were detected in commercial breed pigs, their 

application for pig breeding would be possible. 


In this study, we tried to detect the segregation 

of these QTL in a commercial Large White 

population bred in Tokushima prefecture. 

We used the data from 1,063 animals 

derived from five sires (Table 1, 2), which were 

produced in Livestock Research Institute of 

Tokushima prefecture from 1996 to 2004. Traits 

for growth rate are ADG1 (average daily gain 1): 

from weaning (28 day) to slaughter, and ADG2: 

from beginning of fattening (80.3 ± 9.0 day, 38.0 

± 8.5 kg) to slaughter. Among the groups classified 

by the sires, average weights at slaughter 

are almost equal; from 112.2 ± 6.0 kg to 113.5 

± 7.6 kg, but average rearing periods varied 

significantly from 177.7 ± 13.8 days to 187.2 ± 

10.4 days. For growth rates, ADG1 were from 

668.8 ± 59.1 g/day to 708.2 ± 71.1 g/day and 

ADG2 were from 712.2 ± 81.6g/day to 762.9 ± 

99.6 g/day. 

Five microsatellite markers, which were heterozygous 

in the sires, were arranged in each 

of the QTL regions on SSC4, 7 and 8, and were 

genotyped for meat animals. The QTL regions 

transmitted from the sires were determined 

by the haplotype of microsatellite markers, and 

half-sib analyses for ADG1 and ADG2 were carried 

out with the five sires. The result revealed 

that segregation of QTL effects for ADG2 

were detected on SSC4 with two sires, No.404 

and No.4407 (Table 3). The differences of allele 

effects detected for No.404 and No.4407 were 

37.6 g/day and 32.8 g/day (Fig 4), respectively, 

while the additive effect of the QTL on SSC4 in 

the F2 family between Japanese wild boar and 

Large White was 38.3 g/day. The proportion of 

phenotypic variance of ADG2 explained by the 

QTL on SSC4 was 6.4% and 5.2% in the cases of 

No.404 and No.4407, respectively. These effects 

of the QTL for ADG2 are corresponding to approximately 

four days difference of the fattening 

period. We think that this QTL will be useful to 

the breeding of Large White population and now 

the fine mapping of this QTL is in progress. 

Fig 3. QTL for average daily gain detected in F2 population between Japanese wild boar and Large 

White breed pigs. 

Table 1. Summary of the data for ADG1 

Weaning 

Slaughter 

Sire day weight day weight 

n ADG1 

404 28.0 ± 0.6 7.7 ± 1.6 185.4 ± 13.5 112.2 ± 6.0 205 668.8 ± 59.1 

503 28.1 ± 1.0 7.3 ± 1.8 187.2 ± 10.4 113.4 ± 6.9 212 668.8 ± 47.9 

2201 27.8 ± 0.8 7.9 ± 1.7 178.0 ± 14.9 112.7 ± 7.6 356 704.5 ± 71.3 

4407 28.0 ± 1.0 7.9 ± 1.6 177.7 ± 13.8 113.1 ± 7.1 189 708.2 ± 71.1 

8305 28.3 ± 0.7 8.0 ± 1.7 186.4 ± 14.1 113.5 ± 7.6 101 671.1 ± 62.2 

± SD 


Table 2. Summary of the data for ADG2 

Beginning of fattening Slaughter 

Sire day weight day weight 

n ADG2 

404 84.8 ± 6.9 38.2 ± 5.4 181.9 ± 12.8 111.8 ± 5.6 124 747.5 ± 92.9 

503 81.2 ± 9.6 36.6 ± 8.7 185.1 ± 10.6 112.4 ± 6.8 119 716.5 ± 67.2 

2201 80.9 ± 7.9 40.6 ± 8.6 177.4 ± 15.0 113.2 ± 7.0 298 750.3 ± 102.7 

4407 74.7 ± 8.8 35.6 ± 9.0 178.3 ± 14.1 113.0 ± 7.2 166 762.9 ± 99.6 

8305 78.5 ± 9.2 36.3 ± 8.3 186.9 ± 13.7 113.5 ± 7.8 94 712.2 ± 81.6 

± SD 

Table 3. Result of half-sib analysis of ADG1 and ADG2 

F-ratio / % variance explained 

Sire SSC4 SSC7 SSC8 

ADG1 404 5.77 0.62 0.61 

503 0.23 0.55 0.34 

2201 0.02 0.10 0.93 

4407 3.18 0.14 0.72 

8305 1.68 1.58 0.00 

ADG2 404 8.12* 6.4% 0.10 0.29 

503 0.04 0.01 0.00 

2201 0.14 0.63 0.18 

4407 7.10* 5.2% 0.05 1.05 

8305 0.28 6.38 0.12 

* p


Reproductive Biology Research Unit is seeking 

to clarify the molecular mechanisms regulating 

development/differentiation of germ and 

placental cells, and stem cells such as embryonic 

stem (ES) cells in mammals. The major research 

topics of this year are as follows: 

Production of blastocysts from reconstituted 

oocytes derived from porcine primordial 

oocytes grafted into nude mice 

Primordial follicles are a store for ovarian 

follicles and a potential resource of oocytes for 

medical, agricultural, and zoological purposes. 

We have been able to endow porcine oocytes 

in primordial follicles (primordial oocytes) with 

fertilizing ability by a combination of ovarian 

xenografting and subsequent in vitro culture 

of the collected oocytes (Kaneko et al. 2006). 

However, only a few of the fertilized oocytes 

(1%) attained the blastocyst stage probably due 

to a poor cytolasmic maturation of the porcine 

oocytes grown in nude mice. It is important to 

improve the developmental competence of the 

recovered oocytes in order to utilize the primordial 

oocytes for agricultural purposes. Here we 

introduce a new technology that is expected to 

enable us to rescue the recovered oocytes with 

low developmental ability. Ovarian tissues from 

20-day-old piglets, in which most of the follicles 

were primordial, were transplanted under the 

capsules of kidneys of ovariectomized nude 

mice. The mice then received continuous treatment 

of follicle stimulating hormone in order 

to enhance the growth of antral follicles in the 

xenografts. After in vitro maturation, porcine 

oocytes recovered from the xenografts were 

subjected to a serial centrifugation, and then, 

from the resulting cytoplasmic fragments, fragments 

containing metaphase II chromosomes 

(defined as karyoplasts) were selected. On the 

other hand, matured oocytes obtained from the 

prepubertal pig ovaries, which proved to have 

full developmental competence (Kikuchi et al. 

2002), were also subjected to the same centrifugation 

procedures and cytoplasmic fragments 

without metaphase II chromosomes (defined 

as cytoplasts) were selected. One karyoplast 

was electro-fused with 2 cytoplasts to produce 

reconstituted oocytes (Fig 5). These reconstituted 

oocytes were then transferred to in-vitro 

fertilization and cultured for 7 days. Out of the 

56 reconstituted oocytes, 4 oocytes developed to 

the blastocyst stage (Fig 6): the blastocyst rate 

was 7%. The number of cells of the blastocysts 

was 13.6 ± 1.8 (mean ±SE). These results 

demonstrate successful production of the reconstituted 

oocytes by fusion of one karyoplast, 

prepared from the xenogeneic oocytes, with 2 

cytoplasts from the normal in vitro matured 

oocytes. Reconstruction of oocytes enhanced 

the developmental ability of the porcine oocytes 

grown in nude mice. 

Establishment of pluripotent stem cells in 

domestic animals 

Pluripotent stem cells would be beneficial 

for generating precise genetically-modified 

animals based on genomic information. In 

mice, several types of pluripotent stem cells, 

such as embryonic stem (ES) cells, embryonic 

germ (EG) cells, and induced pluripotent stem 

(iPS) cells, that have the ability to generate 

germline chimera have been established, but 

not in domestic animals. We have attempted 

to establish pluripotent stem cells in rabbit, 

bovine and pig, and already have isolated some 

candidate cell lines. Rabbit ES cells formed 

slightly flattened colonies in contrast to mouse 

ES cells. They have alkaline phosphatase 

activity and express several undifferentiated 

cells markers, such as Pou5f1 (Oct3/4). Flow 

cytometry analysis also showed that SSEA-1 

positive cells were contained in the cells. Bovine 

ES-like cells showed a property almost the same 

as rabbit. In pig, we isolated cells that formed 

tight round-shape colonies like those of mouse 

ES cells. Although alkaline phosphatase activity 

and Pou5f1 (Oct3/4), NANOG expression were 


detected in the cells, they were not included the 

SSEA-1 positive cells. The differentiation ability 

of these cells to all three germ layers and germ 

cells is being estimated by in vitro and in vivo 

assays. We have also focused on the germ-line 

stem (GS) cells in bovine. Using flow cytometry, 

we established a useful system to enrich the 

cells expressing germ cell marker genes from 

whole calf testis (Fig 7). Studies are underway 

to establish GS cell lines from enriched spermatogonial 

cells. The knowledge gained from 

our studies will be useful for establishing protocols 

for multipotent stem cell lines of domestic 

animals, elucidation of the mechanisms of early 

development, and regenerative medicine using 

stem cell technology. 

Functions of prolactin-related proteins in 

ruminants 

The prolactin-related proteins (PRPs) are 

non-classical members of the prolactin/growth 

hormone family. Among ruminants, they are 

expressed in the cotyledonary villi of cattle and 

goat. We investigated placental PRP in sheep in 

order to gain a comprehensive understanding 

of the function and evolution of these molecules. 

We also examined the sequence properties, 

expression and lactogenic activation of the 

cloned genes. We cloned two novel ovine PRPs, 

named oPRP1 and oPRP2. oPRP2 had a typical 

PRP sequence similar to bovine PRP1 (bPRP1). 

oPRP1 had a short sequence identical with 

bovine or caprine type PRP but the reading 

frame was shifted. Both oPRPs were expressed 

in trophoblast giant binucleate cells (BNC) as 

in cattle and goat (Fig 8). oPRP1 expression 

declined from the early to the middle stage 

of gestation. In contrast, oPRP2 expression 

remained constant throughout the gestation 

period (Fig 9). oPRP2 was translated to form a 

mature protein in a mammalian cell expression 

system. Western blotting showed a molecular 

mass of 35 kDa for the FLAG-tag fusion oPRP2 

protein. This recombinant protein and bPRP1 

were bioassayed using Nb2 lymphoma cells; it 

was confirmed that neither ruminant PRP had 

lactogenic activity because the Nb2 lymphoma 

cells did not proliferate. We have identified 

two novel PRPs, oPRP1 and oPRP2, in ovine 

placenta. Both these ovine PRPs were localized 

and quantitatively expressed in BNC. Absence 

of lactogenic activity was confirmed for the 

oPRP2 molecule. The deduced molecular weight 

of oPRP1 was comparable with 16K fragment 

of pituitary prolactin. The 16K fragment of 

pituitary PRL is generated by post-translational 

enzymatic digestion and inhibits angiogenesis. 

It is anticipated that novel and known ruminant 

PRPs have common functions, except for lactogenic 

activity. 

Fig 5. Three cytoplasmic fragments 

just after electro-fusion. 

Fig 6. A blastocyst having 23 cells 

developed from a fusion oocyte. 


Fig 7. Enrichment of spermatogonial cells from calf testicular cells by flow cytometory. 

Fig 8. In situ hybridization of oPRP1 in ovine placentome on Day 45 of gestation. 

(A) anti-sense cRNA probe. (B) sense cRNA probe. Scale bars = 100 μm (main areas in A and B) and 

4 μm (right upper area in A). 

Fig 9. Real-time PCR analysis of (A) oPRP1 and (B) oPRP2 mRNA in ovine placenta. 

Expression of these mRNAs was normalized to the expression of GAPDH measured in the 

corresponding RNA preparation. Values are means ± SEM. Values with different letters (a, b, c and d) 

are significantly different (P< 0.05). 


The Neurobiology Research Unit 

The role of neuromedin U and neuromedin S 

in modulating stress responses in cattle 

The neuropeptides neuromedin U (NMU) 

and neuromedin S (NMS) were identified as 

endogenous ligands for two orphan G proteincoupled 

receptors, FM-3/GPR66 and FM-4/ 

TGR-1, currently identified as NMU type-1 

and type-2 (NMUR2) receptors, respectively. 

Since the NMUR2 is highly expressed in the 

central nervous system, the physiological roles 

of these neuropeptides in the central nervous 

system have been investigated mainly in 

rodents. Although NMU and NMS are reported 

to modulate stress responses mainly through 

corticotropin-releasing hormone (CRH) system 

in rodents, the in vivo effects of centrally 

administered NMU or NMS on stress regulation 

have not been fully elucidated in cattle. We 

examined adrenocorticotropic hormone levels, 

body temperature, and behavioral responses to 

intracerebroventricularly (ICV) administered rat 

NMU or rat NMS in steers. 

ICV NMU and NMS (0.2, 2, and 20 nmol/200µl) 

evoked a dose-related increase in plasma cortisol 

concentrations (CORT) (Fig 10). There was a significant 

time–treatment interaction for the time 

course of CORT (p

Fig 10. Effects of intracerebroventricular injections of 

200μl aCSF and 0.2, 2, and 20 nmol neuromedin U (NMU) 

or neuromedin S (NMS) on temporal changes in mean 

(+SE) plasma cortisol concentrations (CORT) in streets (A) 

and the mean (±SE) area under the CORT curve (AUC) 

from 0 to 90 min after the injection (B). 

Significant difference from the pre-injection value 

(-30 and 0 min) (p

List of Publications 

Original Papers 

1 ) English 

1 Abe H, Ohnishi J, Narusaka M, Seo S, Narusaka Y, Tsuda S, Kobayashi M (2008) Arabidopsis: Thrips 

system for analysis of plant response to insect feeding Plant Signaling & Behavior 3(7):446-447 

2 Abe T, Saburi J, Hasebe H, Nakagawa T, Kawamura T, Saito K, Nade T, Misumi S, Okumura T, Kuchida 

K, Hayashi T, Nakane S, Mitsuhasi T, Nirasawa K, Sugimoto Y, Kobayashi E (2008) Bovine quantitative 

trait loci analysis for growth, carcass, and meat quality traits in an F2 population from a cross between 

Japanese Black and Limousin Journal of Animal Science 86(11):2821-2832 

3 Akiyama K, Akimaru S, Asano Y, Khalaj M, Kiyosu C, Masoudi A.A, Takahashi S, Katayama K, Tsuji 

T, Noguchi J, Kunieda T (2008) A new ENU-induced mutant mouse with defective spermatogenesis 

caused by a nonsense mutation of the Syntaxin 2/Epimorphin (Stx2/Epim) gene Journal of 

Reproduction and Development 54(2):122-128 

4 Akiyama Y, Yamada-Akiyama H, Yamanouchi H, Takahara M, Ebina M, Takamizo T, Sugita S, Nakagawa 

H (2008) Estimation of genome size and physical mapping of ribosomal DNA in diploid and tetraploid 

guineagrass (Panicum maximum Jacq.) Grassland Science 54(2):89-97 

5 Ambrose W.M, Salahuddin A, So S, Ng S, Márquez S.P, Takezawa T, Schein O, Elisseeff J (2009) 

Collagen vitrigel membranes for the in vitro reconstruction of separate corneal epithelial, stromal, 

and endothelial cell layers Journal of Biomedical Materials Research Part B : Applied Biomaterials 

90B(2) : 818-831 

6 Ando A, Uenishi H, Kawata H, Tanaka-Matsuda M, Shigenari A, Flori L, Chardon P, Lunney J.K, 

Kulski J.K, Inoko H (2008) Microsatellite diversity and crossover regions within homozygous and 

heterozygous SLA haplotypes of different pig breeds Immunogenetics 60(7):399-407 

7 Ando S, Tsushima S, Kamachi S, Konagaya K, Tabei Y (2008) Alternative transcription initiation of the 

nitrilase gene (BrNIT2) caused by infection with Plasmodiophora brassicae Woron. in Chinese cabbage 

(Brassica rapa L.) Plant Molecular Biology 68(6):557-569 

8 Ando S, Tanabe S, Akimoto-Tomiyama C, Nishizawa Y, Minami E (2009) The supernatant of a conidial 

suspension of Magnaporthe oryzae contains a factor that promotes the infection of rice plants Journal 

of Phytopathology 157(7-8) : 420-426 

9 Aoto K, Shikata Y, Imai H, Matsumaru D, Tokunaga T, Shioda S, Yamada G, Motoyama J (2009) 

Mouse Shh is required for prechordal plate maintenance during brain and craniofacial morphogenesis 

Developmental Biology 327(1) : 106-120 

10 Arafat H.H, Tanaka K, Sawada H, Suzuki K (2009) Variation of lipopolysaccharide among the three 

major Agrobacterium species and the effect of environmental stress on the lipopolysaccharide profile 

Plant Pathology Journal 8(1) : 1-8 


11 Arakaki N, Nagayama A, Kobayashi A, Tarora K, Kishita M, Sadoyama Y, Mougi N, Kijima K, Suzuki 

Y, Akino T, Yasui H, Fukaya M, Yasuda T, Wakamura S, Yamamura K (2008) Estimation of abundance 

and dispersal distance of the sugarcane click beetle Melanotus sakishimensis Ohira (Coleoptera : 

Elateridae) on Kurima Island, Okinawa, by mark-recapture experiments Applied Entomology and 

Zoology 43(3) : 409-419 

12 Arakaki N, Nagayama A, Kobayashi A, Hokama Y, Sadoyama Y, Mogi N, Kishita M, Adaniya K, Ueda 

K, Higa M, Shinzato T, Kawamitsu H, Nakama S, Wakamura S, Yamamura K (2008) Mating disruption 

for control of Melanotus okinawensis (Coleoptera: Elateridae) with synthetic sex pheromone Journal of 

Economic Entomology 101(5) : 1568–1574 

13 Arakawa G, Watanabe H, Yamasaki H, Maekawa H, Tokuda G (2009) Purification and molecular 

cloning of xylanases from the wood-feeding termite, Coptotermes formosanus Shiraki Bioscience, 

Biotechnology and Biochemistry 73(3) : 710-718 

14 Arakawa T, Yukuhiro F, Noda H (2008) Insecticidal effect of a fungicide containing polyoxin B on the 

larvae of Bombyx mori (Lepidoptera : Bombycidae), Mamestra brassicae, Mythimna separata, and 

Spodoptera litura (Lepidoptera : Noctuidae) Applied Entomology and Zoology 43(2):173-181 

15 Arakawa T (2008) Synergistic effect of a fungicide containing polyoxin B with insect growth regulators 

(IGRs) in the killing of common cutworm, Spodoptera litura (Lepidoptera : Noctuidae) Applied 

Entomology and Zoology 43(2) : 167-171 

16 Arakawa T (2008) Epidemiological importance of silkworm body surface for persistence of viral 

contamination by nucleopolyhedrovirus of Bombyx mori L. in an artificial rearing environment 

Sericologia: Revue des vers à soie 48(1) : 23-30 

17 Arunkumar K.P, Mita K, Nagaraju J (2009) The silkworm Z chromosome is enriched in testis-specific 

genes Genetics 182(2) : 493-501 

18 Asadi M, Higaki T, Hinomoto N, Saboori A, Naghavi M.R (2009) Isolation and characterization of 

polymorphic microsatellite loci from the water mite Hygrobates fluviatilis (Acari : Hydrachnidia : 

Hygrobatidae) Molecular Ecology Resources 9(3):793-795 

19 Asamizu E, Shimoda Y, Kouchi H, Tabata S, Sato S (2008) A positive regulatory role for LjERF1 in the 

nodulation process is revealed by systematic analysis of nodule-associated transcription factors of 

Lotus japonicus Plant Physiology 147(4) : 2030-2040 

20 Ashikawa I, Hayashi N, Yamane H, Kanamori H, Wu J, Matsumoto T, Ono K, Yano M (2008) Two 

adjacent nucleotide-binding site–leucine-rich repeat class genes are required to confer Pikm-specific 

rice blast resistance Genetics 180(4) : 2267-2276 

21 Atsumi S, Inoue Y, Ishizaka T, Mizuno E, Yoshizawa Y, Kitami M, Sato R (2008) Location of the Bombyx 

mori 175 kDa cadherin-like protein-binding site on Bacillus thuringiensis Cry1Aa toxin FEBS Journal 

275(19):4913-4926 

22 Banba M, Gutjahr C, Miyao A, Hirochika H, Paszkowski U, Kouchi H, Imaizumi-Anraku H (2008) 

Divergence of evolutionary ways among common sym genes: CASTOR and CCaMK show functional 

conservation between two symbiosis systems and constitute the root of a common signaling pathway 

Plant and Cell Physiology 49(11) : 1659-1671 

23 Bang S.W, Ueno O, Wada Y, Hong S.K, Kaneko Y, Matsuzawa Y (2009) Production of Raphanus 

sativus (C 3 )-Moricandia arvensis (C 3 -C 4 intermediate) monosomic and disomic addition lines with each 

parental cytoplasmic background and their photorespiratory characteristics Plant Production Science 

12(1):70-79 

24 Bowolaksono A, Nishimura R, Hojo T, Sakumoto R, Acosta T.J, Okuda K (2008) Anti-apoptotic roles of 

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75 Inoue H, Kobayashi T, Nozoye T, Takahashi M, Kakei Y, Suzuki K, Nakazono M, Nakanishi H, Mori 

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82 Ishikawa M, Oda A (2008) Reevaluation of constant relative humidity chambers using phosphoric acid 

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105 Katsuma S, Kawaoka S, Mita K, Shimada T (2008) Genome-wide survey for baculoviral host homologs 

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107 Kawahigashi H, Hirose S, Ohkawa H, Ohkawa Y (2008) Transgenic rice plants expressing human P450 

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108 Kawakatsu T, Yamamoto M.P, Hirose S, Yano M, Takaiwa F (2008) Characterization of a new 

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112 Kawaoka S, Katsuma S, Meng Y, Hayashi K, Mita K, Shimada T (2009) Identification and 

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114 Khan N.A, Githiri S.M, Benitez E.R, Abe J, Kawasaki S, Hayashi T, Takahashi R (2008) QTL analysis of 

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137 Maedomari N, Kikuchi K, Nagai T, Fahrudin M, Kaneko H, Noguchi J, Nakai M, Ozawa M, Somfai T, 

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139 Maekawa-Yoshikawa M, Müller J, Takeda N, Maekawa T, Sato S, Tabata S, Brachmann A, Parniske M 

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147 Matsubara K, Kono I, Hori K, Nonoue Y, Ono N, Shomura A, Mizubayashi T, Yamamoto S, Yamanouchi 

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152 Meier S, Gehring C, MacPherson C.R, Kaur M, Maqungo M, Reuben S, Muyanga S, Shih M-D, Wei 

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154 Meng Y, Katsuma S, Daimon T, Banno Y, Uchino K, Sezutsu H, Tamura T, Mita K, Shimada T (2009) 

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155 Mihara M, Itoh T, Izawa T (2008) In silico identification of short nucleotide sequences associated with 

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157 Minakuchi C, Namiki T, Shinoda T (2009) Krüppel homolog 1, an early juvenile hormone-response 

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158 Minamino N, Osaki T, Yasue H, Katafuchi T (2009) Calcitonin receptor-stimulating peptide and 

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159 Minematsu T, Harumi T, Naito M (2008) Quantitative genotyping by amplifying the polymorphic 

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162 Miura K, Wu J, Sunohara H, Wu X, Matsumoto T, Matsuoka M, Ashikari M, Kitano H (2009) Highresolution 

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164 Mizuno H, Sasaki T, Matsumoto T (2008) Characterization of internal structure of the nucleolar 

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165 Mizuno N, Sugie A, Kobayashi F, Takumi S (2008) Mitochondrial alternative pathway is associated with 

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167 Morozumi T, Uenishi H (2009) Polymorphism distribution and structural conservation in RNAsensing 

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169 Muraji M, Arakaki N, Ohno S, Hirai Y (2008) Genetic variation of the green chafer, Anomala albopilosa 

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170 Muraji M, Nakahara S, Ishida T, Minoura K, Miyazaki I, Kohama T (2008) The Philippines is a possible 

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173 Murata M, Imamura T, Miyanoshita A (2008) Infestation and Development of Sitophilus spp. in pouchpackaged 

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174 Myronovych A, Murata S, Chiba M, Matsuo R, Ikeda O, Watanabe M, Hisakura K, Nakano Y, Kohno 

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175 Nagata M, Murakami E, Shimoda Y, Shimoda-Sasakura F, Kucho K, Suzuki A, Abe M, Higashi S, 

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176 Nakahara S, Muraji M (2008) Phylogenetic analysis of Bactrocera fruit flies (Diptera: Tephritidae) based 

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178 Nakahara Y, Watanabe M, Fujita A, Kanamori Y, Tanaka D, Iwata K, Furuki T, Sakurai M, Kikawada T, 

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179 Nakahara Y, Shimura S, Ueno C, Kanamori Y, Mita K, Kiuchi M, Kamimura M (2009) Purification and 

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180 Nakai M, Kaneko H, Somfai T, Maedomari N, Ozawa M, Noguchi J, Kashiwazaki N, Kikuchi K 

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181 Nakaminami K, Hill K, Perry S.E, Sentoku N, Long J.A, Karlson D.T (2009) Arabidopsis cold shock 

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182 Nakao H, Matsumoto T, Oba Y, Niimi T, Yaginuma T (2008) Germ cell specification and early 

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183 Nakashima N, Uchiumi T (2009) Functional analysis of structural motifs in dicistroviruses Virus 

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184 Nakashima N, Nakamura Y (2008) Cleavage sites of the “P3 region” in the nonstructural polyprotein 

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185 Nakatani K, Kobayashi Y, Haga A (2008) Release mechanism from a microsphere of a silkworm chitin 

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186 Nakaya Y, Kizaki K, Takahashi T, Patel O.V, Hashizume K (2009) The characterization of DNA 

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187 Namiki T, Niwa R, Higuchi A, Yoshiyama T, Mita K, Kataoka H (2009) A basic-HLH transcription factor, 

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188 Narita S, Shimajiri Y, Nomura M (2009) Strong cytoplasmic incompatibility and high vertical 

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189 Narusaka M, Kawai K, Izawa N, Seki M, Shinozaki K, Seo S, Kobayashi M, Shiraishi T, Narusaka Y (2008) 

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190 Nita M, Wang H-B, Zhong Y-S, Mita K, Iwanaga M, Kawasaki H (2009) Analysis of ecdysonepulse 

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191 Niwa R, Niimi T, Honda N, Yoshiyama M, Itoyama K, Kataoka H, Shinoda T (2008) Juvenile hormone 

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192 Noguchi J, Ozawa M, Nakai M, Somfai T, Kikuchi K, Kaneko H, Kunieda T (2008) Affected homologous 

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193 Numa H, Nishimura M, Tanaka T, Kanamori H, Yang C-c, Matsumoto T, Nagamura Y, Itoh T (2009) 

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194 Oda A, Amikura K, Kuchitsu K, Ishikawa M (2008) Phosphoric acid-assisted constant relative humidity 

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195 Ogasawara H, Aso H, Nagai Y, Matsumoto K, Okamura H, Tanaka S, Watanabe K, Ohwada S, 

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196 Ogawa T, Kawahigashi H, Toki S, Handa H (2008) Efficient transformation of wheat by using a mutated 

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197 Ohishi K, Ando A, Suzuki R, Takishita K, Kawato M, Katsumata E, Ohtsu D, Okutsu K, Tokutake K, 

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198 Ohta H, Tsuchihara K, Mitsumasu K, Yaginuma T, Ozoe Y, Asaoka K (2009) Comparative pharmacology 

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199 Okamoto S, Futahashi R, Kojima T, Mita K, Fujiwara H (2008) Catalogue of epidermal genes: Genes 

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200 Okuda-Shimazaki J, Kakehi N, Yamazaki T, Tomiyama M, Sode K (2008) Biofuel cell system employing 

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201 Okumura N, Matsumoto T, Hamasima N, Awata T (2008) Single nucleotide polymorphisms of the KIT 

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202 Osaka R, Nomura M, Watada M, Kageyama D (2008) Negative effects of low temperatures on the 

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203 Osakabe M (MH), Kotsubo Y, Tajima R, Hinomoto N (2008) Restriction fragment length polymorphism 

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204 Osanai-Futahashi M, Suetsugu Y, Mita K, Fujiwara H (2008) Genome-wide screening and 

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205 O’Donnell K, Ward T.J, Aberra D, Kistler H.C, Aoki T, Orwig N, Kimura M, Bjørnstad Å, Klemsdal S.S 

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206 Rezaeian A.H, Katafuchi T, Yoshizawa M, Hiraiwa N, Saito T, Nishibori M, Hamano K, Minamino 

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207 Riemann M, Riemann M, Takano M (2008) Rice JASMONATE RESISTANT 1 is involved in phytochrome 

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208 Roller L, Yamanaka N, Watanabe K, Daubnerová I, Žitňan D, Kataoka H, Tanaka Y (2008) The unique 

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209 Saika H, Toki S (2009) Visual selection allows immediate identification of transgenic rice calli efficiently 

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210 Saitoh K, Nishimura M, Kubo Y, Hayashi N, Minami E, Nishizawa Y (2008) Construction of a binary 

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211 Sakamoto H, Kageyama D, Hoshizaki S, Ishikawa Y (2008) Heat treatment of the adzuki bean borer, 

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212 Sakane I, Kamataki C, Takizawa Y, Nakashima M, Toki S, Ichikawa H, Ikawa S, Shibata T, Kurumizaka 

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213 Sakuraba Y, Kimura T, Masuya H, Noguchi H, Sezutsu H, Takahasi K.R, Toyoda A, Fukumura R, 

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214 Sakurai T, Sakamoto A, Muroi Y, Bai H, Nagaoka K, Tamura K, Takahashi T, Hashizume K, Sakatani M, 

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215 Salzberg S.L, Sommer D.D, Schatz M.C, Phillippy A.M, Rabinowicz P.D, Tsuge S, Furutani A, Ochiai 

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216 Sameri M, Nakamura S, Nair S.K, Takeda K, Komatsuda T (2009) A quantitative trait locus for reduced 

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217 Sano M, Tamada Y, Niwa K, Morita T, Yoshino G (2009) Sulfated sericin is a novel anticoagulant 

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218 Sasaki N, Ogata T, Deguchi M, Nagai S, Tamai A, Meshi T, Kawakami S, Watanabe Y, Matsushita Y, 

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219 Sato K, Matsuoka Matsunaga T, Futahashi R, Kojima T, Mita K, Banno Y, Fujiwara H (2008) Positional 

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221 Sato T, Okamoto J, Degawa Y, Matsunari S, Takahashi K, Tomioka K (2009) White rust of Ipomoea 

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222 Sato Y, Morita R, Katsuma S, Nishimura M, Tanaka A, Kusaba M (2009) Two short-chain 

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230 Sezutsu H, Tamura T, Yukuhiro K (2008) Uniform size of leucine-rich repeats in a wild silk moth Saturnia 

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231 Shibukawa T, Yazawa K, Kikuchi A, Kamada H (2009) Possible involvement of dna methylation on 

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232 Shibuya K, Fukushima S, Takatsuji H (2009) RNA-directed DNA methylation induces transcriptional 

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233 Shimada S, Ogawa T, Kitagawa S, Suzuki T, Ikari C, Shitsukawa N, Abe T, Kawahigashi H, Kikuchi 

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234 Shimizu H, Shimoda M, Yamaguchi T, Seong K-H, Okamura T, Ishii S (2008) Drosophila ATF-2 regulates 

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235 Shimizu T, Yoshii M, Wei T, Hirochika H, Omura T (2009) Silencing by RNAi of the gene for Pns12, a 

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236 Shimoda Y, Shimoda-Sasakura F, Kucho K, Kanamori N, Nagata M, Suzuki A, Abe M, Higashi S, 

Uchiumi T (2009) Overexpression of class 1 plant hemoglobin genes enhances symbiotic nitrogen 

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237 Shimogiri T, Nishibori M, Hiraiwa H, Hayashi T, Yasue H (2009) Assignment of 128 genes localized on 

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238 Shimura S, Kiguchi K, Kiuchi M (2008) Cell debris clearance in the integument of the Erisilkworm, 

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239 Shoji T, Inai K, Yazaki Y, Sato Y, Takase H, Shitan N, Yazaki K, Goto Y, Toyooka K, Matsuoka K, 

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240 Shomura A, Izawa T, Ebana K, Ebitani T, Kanegae H, Konishi S, Yano M (2008) Deletion in a gene 

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241 Soe O.K, Ohba Y, Imaeda N, Nishii N, Takasu M, Yoshioka G, Kawata H, Shigenari A, Uenishi H, Inoko 

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242 Somfai T, Ozawa M, Noguchi J, Kaneko H, Nakai M, Maedomari N, Ito J, Kashiwazaki N, Nagai T, 

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243 Somta C, Somta P, Tomooka N, Ooi P.A-C, Vaughan D.A, Srinives P (2008) Characterization of 

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244 Somta P, Kaga A, Tomooka N, Isemura T, Vaughan D.A, Srinives P (2008) Mapping of quantitative trait 

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245 Sone K, Suzuki A.A, Miyazawa S, Noguchi K, Terashima I (2009) Maintenance mechanisms of the pipe 

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246 Soyano T, Thitamadee S, Machida Y, Chua N-H (2008) ASYMMETRIC LEAVES2-LIKE19/LATERAL 

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247 Sugama S, Takenouchi T, Fujita M, Conti B, Hashimoto M (2009) Differential microglial activation 

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248 Sugama S, Takenouchi T, Kitani H, Fujita M, Hashimoto M (2009) Microglial activation is inhibited by 

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249 Sun G, Pourkheirandish M, Komatsuda T (2009) Molecular evolution and phylogeny of the RPB2 gene 

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250 Suto J (2008) Genetic dissection of testis weight in a mouse strain having an extremely large testis : 

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251 Suto J (2009) A novel growth retardation and abnormal gonad morphology locus on mouse 

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252 Suto J (2009) Identification of multiple quantitative trait loci affecting the size and shape of the 

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253 Suto J (2009) Metabolic consequence of congenital asplenia caused by the Dominant hemimelia 

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254 Suzuki K, Kaminuma O, Hiroi T, Kitamura F, Miyatake S, Takaiwa F, Tatsumi H, Nemoto S, Kitamura 

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255 Suzuki M, Tandon P, Ishikawa M, Toyomasu T (2008) Development of a new vitrification solution, 

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256 Suzuki M, Tsukamoto T, Inoue H, Watanabe S, Matsuhashi S, Takahashi M, Nakanishi H, Mori S, 

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257 Suzuki N, Yamazaki Y, Brown R.L, Fujimoto Z, Morita T, Mizuno H (2008) Structures of pseudechetoxin 

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258 Suzuki R, Shindo H, Tase A, Kikuchi Y, Shimizu M, Yamazaki T (2009) Solution structures and DNA 

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259 Suzuki R, Tase A, Fujimoto Z, Shiotsuki T, Yamazaki T (2009) NMR assignments of juvenile hormone 

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260 Suzuki R, Kuno A, Hasegawa T, Hirabayashi J, Kasai K, Momma M, Fujimoto Z (2009) Sugar-complex 

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261 Suzuki R, Fujimoto Z, Ito S, Kawahara S, Kaneko S, Taira K, Hasegawa T, Kuno A (2009) 

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262 Suzuki S, Sembon S, Iwamoto M, Fuchimoto D, Onishi A (2008) Identification of genes downregulated 

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263 Hashizume T, Onodera Y, Shida R, Isobe E, Suzuki S, Sawai K, Kasuya E, Nagy G.M (2009) 

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264 Tabata J, Yokosuka T, Hattori M, Ohashi M, Noguchi H, Sugie H (2008) Sex attractant pheromone 

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265 Tabunoki H, Shimada T, Banno Y, Sato R, Kajiwara H, Mita K, Satoh J (2008) Identification of Bombyx 

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266 Tada Y, Suzuki T, Takezawa T, Nomoto Y, Kobayashi K, Nakamura T, Omori K (2008) Regeneration 

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267 Taguchi K, Ogata N, Kubo T, Kawasaki S, Mikami T (2009) Quantitative trait locus responsible for 

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268 Taichi M, Yamazaki T, Kimura T, Nishiuchi Y (2009) Total synthesis of marinostatin, a serine protease 

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269 Takafuji A, Hinomoto N (2008) The distribution and geographic variation in diapause capacity among 

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270 Takagi H, Hiroi T, Yang L, Takamura K, Ishimitsu R, Kawauchi H, Takaiwa F (2008) Efficient induction of 

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271 Takahashi H, Nyamsamba D, Mandakh B, Zagdsuren Yo, Amano T, Nomura K, Yokohama M, Ito S, 

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272 Takai T, Ohsumi A, San-oh Y, Laza M.R.C, Kondo M, Yamamoto T, Yano M (2009) Detection of a 

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273 Takaiwa F, Sakuta C, Choi S-K, Tada H, Motoyama T, Utsumi S (2008) Co-expression of soybean 

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275 Takamiya T, Ohtake Y, Hosobuchi S, Noguchi T, Kawase M, Murakami Y, Okuizumi H (2008) Application 

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278 Takeda N, Sato S, Asamizu E, Tabata S, Parniske M (2009) Apoplastic plant subtilases support 

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279 Takemoto Y, Mitsuhashi W, Murakami R, Konishi H, Miyamoto K (2008) The N-terminal region of an 

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280 Takenaka Y, Masuda H, Yamaguchi A, Nishikawa S, Shigeri Y, Yoshida Y, Mizuno H (2008) Two forms 

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282 Takenouchi T, Iwamaru Y, Sato M, Yokoyama T, Kitani H (2009) Establishment of an SV40 large T 

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283 Takenouchi T, Nakai M, Iwamaru Y, Sugama S, Tsukimoto M, Fujita M, Wei J, Sekigawa A, Sato M, 

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284 Takeuchi Y, Hori K, Suzuki K, Nonoue Y, Takemoto-Kuno Y, Maeda H, Sato H, Hirabayashi H, Ohta H, 

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285 Takumi S, Shimamura C, Kobayashi F (2008) Increased freezing tolerance through up-regulation of 

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287 Tanaka D, Niino T, Tsuchiya Y, Shirata K, Uemura M (2008) Cryopreservation of shoot tips of 

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288 Tanaka H, Ishibashi J, Fujita K, Nakajima Y, Sagisaka A, Tomimoto K, Suzuki N, Yoshiyama M, Kaneko 

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290 Tanaka H, Sagisaka A, Fujita K, Kaneko Y, Imanishi S, Yamakawa M (2009) Lipopolysaccharide 

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291 Tanaka H, Sagisaka A, Nakajima Y, Fujita K, Imanishi S, Yamakawa M (2009) Correlation of differential 

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292 Tanaka K, Arafat H.H, Urbanczyk H, Yamamoto S, Moriguchi K, Sawada H, Suzuki K (2009) Ability of 

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293 Tanaka S, Zhu D-H (2008) Geographic variation in embryonic diapause, cold-hardiness and life cycles 

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294 Tanaka S, Yukuhiro F, Yasui H, Fukaya M, Akino T, Wakamura S (2008) Presence of larval and adult 

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295 Tanaka-Matsuda M, Ando A, Rogel-Gaillard C, Chardon P, Uenishi H (2009) Difference in number of 

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296 Tateno M, Toyooka M, Shikano Y, Takeda S, Kuwabara N, Sezutsu H, Tamura T (2009) Production and 

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297 Teramoto H, Kameda T, Tamada Y (2008) Preparation of gel film from Bombyx mori silk sericin and its 

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300 Tomioka K, Sawada H, Aoki T, Sato T (2008) Gray mold of pearl lupine caused by Botrytis cinerea 

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301 Tomioka K, Moriwaki J, Sato T (2008) Anthracnose of Polygonatum falcatum caused by Colletotrichum 

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302 Tomita R, Murai J, Miura Y, Ishihara H, Liu S, Kubotera Y, Honda A, Hatta R, Kuroda T, Hamada H, 

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303 Tomita S, Kikuchi A (2009) Abd-B suppresses lepidopteran proleg development in posterior abdomen 

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304 Tomoo K, Mukai Y, In Y, Miyagawa H, Kitamura K, Yamano A, Shindo H, Ishida T (2008) Crystal 

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305 Tribolium Genome Sequencing Consortium (2008) The genome of the model beetle and pest Tribolium 

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306 Tsubokura Y, Onda R, Sato S, Xia Z, Hayashi M, Fukushima Y, Tabata S, Harada K (2008) 

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309 Tanaka T, Koyanagi K.O, Itoh T (2009) Highly diversified molecular evolution of downstream 

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313 Uchiyama Y, Hosoe M, Sato T, Takahashi T (2009) Biological and immunological characteristics of 

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314 Ueda M, Nishikawa T, Fujimoto M, Takanashi H, Arimura S, Tsutsumi N, Kadowaki K (2008) Substitution 

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316 Uenishi H, Eguchi-Ogawa T, Toki D, Morozumi T, Tanaka-Matsuda M, Shinkai H, Yamamoto R, Takagaki 

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317 Ueno D, Kono I, Yokosho K, Ando T, Yano M, Ma J.F (2009) A major quantitative trait locus controlling 

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318 Ueno S, Kusaka K, Tamada Y, Minaba M, Zhang H, Wang P-C, Kato Y (2008) Anionic C-terminal 

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320 Umeda T, Katsuki J, Usami Y, Inoue K, Noguchi H, Fujimoto Z, Ashikawa Y, Yamane H, Nojiri H 

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323 Wang X, Sato S, Tabata S, Kawasaki S (2008) A high-density linkage map of Lotus japonicus based on 

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324 Watanabe M, Murata S, Hashimoto I, Nakano Y, Ikeda O, Aoyagi Y, Matsuo R, Fukunaga K, Yasue 

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325 Wicker T, Krattinger S, Lagudah E.S, Komatsuda T, Pourkheirandish M, Matsumoto T, Cloutier S, 

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326 Xia Z, Watanabe S, Chen Q, Sato S, Harada K (2009) A novel manual pooling system for preparing 

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327 Xu H.X, Jing T, Tomooka N, Kaga A, Isemura T, Vaughan D.A (2008) Genetic diversity of the azuki 

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328 Xu X, Hayashi N, Wang C.T, Kato H, Fujimura T, Kawasaki S (2008) Efficient authentic fine mapping of 

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330 Yamada T, Matsuda F, Kasai K, Fukuoka S, Kitamura K, Tozawa Y, Miyagawa H, Wakasa K (2008) 

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331 Yamada-Akiyama H, Akiyama Y, Ebina M, Xu Q, Tsuruta S, Yazaki J, Kishimoto N, Kikuchi S, Takahara 

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332 Yamagata T, Sakurai T, Uchino K, Sezutsu H, Tamura T, Kanzaki R (2008) GFP Labeling of 

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333 Yamagishi M, Tanaka S (2009) Overwintering biology and morphological characteristics of the 

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334 Yamaguchi H, Shimizu A, Hase Y, Degi K, Tanaka A, Morishita T (2009) Mutation induction with ion 

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335 Yamaguchi H, Shimizu A, Degi K, Morishita T (2008) Effects of dose and dose rate of gamma ray 

irradiation on mutation induction and nuclear DNA content in chrysanthemum Breeding Science 

58(3) : 331-335 

336 Yamamoto D.S, Tachibana K, Sumitani M, Lee J.M, Hatakeyama M (2008) Involvement of Mos-MEK- 

MAPK pathway in cytostatic factor (CSF) arrest in eggs of the parthenogenetic insect, Athalia rosae 

Mechanisms of Development 125(11-12):996-1008 

337 Yamamoto K, Takaya R, Tamada Y, Tomita N (2008) Effects of tribological loading history on the 

expression of tribological function of regenerated cartilage Tribology Online 3(2):148-152 

338 Yamanaka N, Yamamoto S, Žitňan D, Watanabe K, Kawada T, Satake H, Kaneko Y, Hiruma K, Tanaka Y, 

Shinoda T, Kataoka H (2008) Neuropeptide receptor transcriptome reveals unidentified neuroendocrine 

pathways PLoS ONE 3(8) : e3048 

339 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 

340 Yamanouchi H, Koyama A, Takyu T, Yoshioka T (2008) Flow cytometric analysis of various organs and 

cytochimeras of mulberry (Morus spp.) Journal of Insect Biotechnology and Sericology 77(2):95-108 

341 Yamanouchi H, Koyama A, Machii H, Takyu T, Muramatsu N (2009) Inheritance of a weeping character 

and the low frequency of rooting from cuttings of the mulberry variety ‘Shidareguwa’ Plant Breeding 

128(3) : 321-323 

342 Yano K, Yoshida S, Müller J, Singh S, Banba M, Vickers K, Markmann K, White C, Schuller B, Sato S, 

Asamizu E, Tabata S, Murooka Y, Perry J, Wang T.L, Kawaguchi M, Imaizumi-Anraku H, Hayashi M, 

Parniske M (2008) CYCLOPS, a mediator of symbiotic intracellular accommodation Proceedings of the 

National Academy of Sciences of the United States of America 105(51):20540-20545 

343 Yao S-G, Sonoda Y, Tsutsui T, Nakamura H, Ichikawa H, Ikeda A, Yamaguchi J (2008) Promoter 

analysis of OsAMT1;2 and OsAMT1;3 implies their distinct roles in nitrogen utilization in rice Breeding 

Science 58(3) : 201-207 


344 Yara A, Yaeno T, Hasegawa M, Seto H, Seo S, Kusumi K, Iba K (2008) Resistance to Magnaporthe 

grisea in transgenic rice with suppressed expression of genes encoding allene oxide cyclase and 

phytodienoic acid reductase Biochemical and Biophysical Research Communications 376(3):460-465 

345 Yasue H, Kitajima M, Tamada Y, Rezaeian A.H, Hiraiwa H, Hayashi T, Shimogiri T (2008) Assignment 

of 115 genes from HSA9 and HSA14 to SSC1q by RH mapping to generate a dense human-pig 

comparative map Animal Genetics 39(3) : 301-305 

346 Yasui H, Akino T, Fukaya M, Wakamura S, Ono H (2008) Sesquiterpene Hydrocarbons : Kairomones 

with a releaser effect in the sexual communication of the white-spotted longicorn beetle, Anoplophora 

malasiaca (Thomson) (Coleoptera : Cerambycidae) Chemoecology 18(4):233-242 

347 Yasuno N, Takamure I, Kidou S, Tokuji Y, Ureshi A, Funabiki A, Ashikaga K, Yamanouchi U, Yano 

M, Kato K (2009) Rice shoot branching requires an ATP-binding cassette subfamily G protein New 

Phytologist 182(1) : 91-101 

348 Yayou K, Ito S, Kasuya E, Sutoh M, Ohkura S, Okamura H (2008) Intracerebroventricularly administered 

oxytocin attenuated cortisol secretion, but not behavioral responses, during isolation in Holstein steers 

Journal of Veterinary Medical Science 70(7):665-671 

349 Yokota K, Fukai E, Madsen L.H, Jurkiewicz A, Rueda P, Radutoiu S, Held M, Hossain M.S, 

Szczyglowski K, Morieri G, Oldroyd G.E.D, Downie J.A, Nielsen M.W, Rusek A.M, Sato S, Tabata S, 

James E.K, Oyaizu H, Sandal N, Stougaard J (2009) Rearrangement of actin cytoskeleton mediates 

invasion of Lotus japonicus roots by Mesorhizobium loti The Plant Cell 21(1):267-284 

350 Yoshii M, Shimizu T, Yamazaki M, Higashi T, Miyao A, Hirochika H, Omura T (2009) Disruption of a 

novel gene for a NAC-domain protein in rice confers resistance to Rice dwarf virus The Plant Journal 

57(4):615-625 

351 Yoshioka K, Suzuki C, Onishi A (2008) Defined system for in vitro production of porcine embryos using 

a single basic medium Journal of Reproduction and Development 54(3):208-213 

352 Yukawa K, Kaku H, Tanaka H, Koga-Ban Y, Fukuda M (2008) Enhanced soybean infection by the 

legume “super-virulent” Agrobacterium tumefaciens strain KAT23 Bioscience, Biotechnology and 

Biochemistry 72(7) : 1809-1816 

353 Yun M-S, Chen W, Deng F, Yogo Y (2009) Selective growth suppression of five annual plant species by 

chalcone and naringenin correlates with the total amount of 4-coumarate : coenzyme A ligase Weed 

Biology and Management 9(1) : 27-37 

354 Zhang W, Ito H, Quint M, Huang H, Noël L.D, Gray W.M (2008) Genetic analysis of CAND1–CUL1 

interactions in Arabidopsis supports a role for CAND1-mediated cycling of the SCF TIR1 complex 

Proceedings of the National Academy of Sciences of the United States of America 105(24):8470-8475 

355 Zhao J, Shinkai M, Takezawa T, Ohba S, Chung U, Nagamune T (2009) Bone regeneration using 

collagen type I vitrigel with bone morphogenetic protein-2 Journal of Bioscience and Bioengineering 

107(3):318-323 

356 Zhou H-F, Zheng X-M, Wei R-X, Second G, Vaughan D.A, Ge S (2008) Contrasting population genetic 

structure and gene flow between Oryza rufipogon and Oryza nivara Theoretical and Applied Genetics 

117(7):1181-1189 

357 Zhu Z, Ohgo K, Watanabe R, Takezawa T, Asakura T (2008) Preparation and characterization of 

regenerated Bombyx mori silk fibroin fiber containing recombinant cell-adhesive proteins; Nonwoven 

fiber and monofilament Journal of Applied Polymer Science 109(5):2956-2963 

358 Zou Z, Picheng Z, Weng H, Mita K, Jiang H (2009) A comparative analysis of serpin genes in the 

silkworm genome Genomics 93(4):367-375 


2 ) Japanese with English Summary 

1 Ebitani T, Yamamoto Y, Yano M, Funane M (2008) Identification of quantitative trait loci for grain 

appearance using chromosome segment substitution lines in rice Breeding Research 10: 91- 99 

2 Fujino M, Nakanishi T, Ishihara J, Ono S, Doi Y, Sugiura M, Tomioka K (2008) New Yacon cultivars, 

“Andes no Yuki and “Salad Okame” Bulletin of the national agricultural research center for western 

region 7 : 131- 143 

3 Furuhata M, Yoshinaga S, Kaji R, Tamura K, Nabeshima H, Morita H, Yamashita H, Mizobuchi R, 

Okamoto M, Sakai M (2008) Characteristics of growth, yield and quality of rice in submerged direct 

seeding in late season in southern Japan Japanese Journal of the Crop Science 77: 273- 280 

4 Furuhata M, Yoshinaga S, Kaji R, Tamura K, Nabeshima H, Morita H, Yamashita H, Mizobuchi R, 

Okamoto M, Sakai M (2008) Growth, yield and quality characteristics of rice density seeded in the late 

season in southern Japan Japanese Journal of the Crop Science 77: 281- 287 

5 Goto Y, Niimi S (2008) Formation and maintenance of rat hepatocyte spheroids on lactose-silk fibroin 

conjugates in primary cultures Kobunshi Ronbunshu 65: 312- 316 

6 Kawaradani M, Nishimura A, Moriwaki J, Tomioka K, Sato T, Okada K, Nakasone W (2008) 

Anthracnose of perilla (Perilla ocymoides L.) caused by Colletotrichum destrctivum Japanese Journal 

of Phytopathology 74 : 335- 339 

7 Ikejima S, Tsuneyama I, Tanaka Y, Ninagi O, Hara W (2008) Identification of the attached chromosome 

in sex-limited normal marked silkworm, Bombyx mori Sanshi-konchu Biotec 77: 145- 151 

8 Irie K, Aye K, Nagamine T, Shirata K, Fujimaki H, Kikuchi F (2008) Analysis of Variation in days 

to heading of Myanmar rice landraces grown in Myanmar and Japan Tropical Agriculture and 

Development 1 : 1- 6 

9 Lee O, Hara W, Yokoyama T, Ninagi O (2008) Gene mapping of short lifespan ib imago by cDNA 

linkage in the silkworm Bombyx mori Sanshi-Konchu Biotec 77: 47- 52 

10 Kisaki G, Nakashima C, Araki I, Sato T (2008) Brown leaf spot of water convolvulus (Ipomoea aquatic 

Forssk.) caused by Cercospora apii sensu lato Proceedings of the Association for Plant Protection of 

Sikoku 43 : 23- 28 

11 Nakamura T, Nakatani H, Oda A, Ishikawa M (2008) No correlation between antifreeze activity and 

cold-inducible chitinase in bromegrass suspension cells Cryobiology and Cryotechnology 54 : 163- 

166 

12 Nakashima K, Miura M (2008) Development of a laser sensor-based size control unit to produce high 

quality thin silk Nippon silk Gakkaishi 17: 9- 13 

13 Nishishita N, Kimura H, Miyazawa M, Hirano Y (2008) Synthesis and evaluation of new type alginate 

hydrogel using self-assembly β-sheet peptides Kobunshi Ronbunshu 65: 745- 750 

14 Ohtaka N, Tomioka K, Sawada H, Aoki T, Sato T (2008) Description an mycelial compatibility 

of oncidium southern blight pathogen, Sclerotium rolfsii saccardo found in Kagawa Prefecture 

Proceedings of the Association for Plant Protection of Sikoku 43: 7- 12 

15 Sato T, Mori M, Moriwaki J, Tomioka K (2008) Anthracnose of Poinsettia (Euphorbia pulcherrima Willd.) 

caused by Colletotrichum capsici (Sydow) E. J. Butler & Bisby Proceedings of the Association for Plant 

Protection of Sikoku 43 : 1- 6 

16 Sekine T, Kanno H, Aoki, T. (2008) Occurrence of leaf and stem rot caused by Fusarium foetens on 

begonia elator hybrids (Begonia x hiemalis). Japanese Journal of Phytopathology 74(3): 164-166. 


17 Takeuchi Y, Kato H, Nemoto H, Ohta H, Sato H, Hirayama M, Hirabayashi H, Ideta O, Aoki N, Sakai 

M, Ebitani T, Taguchi-Shiobata F, Yamamoto T, Yano M, Imbe T, Ando I (2008) Breeding of “Koshihikari 

Kanto HD1” an isogenuic line of the rice cultivar Koshihikari with early heading Bulletin of the National 

Institute of Crop Science 9 : 1- 25 

18 Takashino K, Sakakibara M, Honda K, Noda T, Okada M (2008) Pheromone trap catch date of the 

diamondback moth in spring becomes earlier in Morioka City, North Japan Annual Report of Plant 

Protection of North Japan (59) : 145-148 

19 Tanabata T, Shimizu H, Shinomura, Takano, Miyamura (N)H, Saito T. (2008) An image measurement 

system for phenotype analysis of rice seedlings growth. IEEJ Trans EIS 128:962–969 


Reviews and Monograph (In English) 

I ) Reviews 

1 Couble P, Mita K, Xia Q (2008) Editorial: Silkworm genome Insect Biochemistry and Molecular Biology 

38(12) : 1035 

2 Fukui K (2008) Modeling mulberry shoot elongation growth Japan Agricultural Research Quarterly 

42(2) : 91-95 

3 Hashizume T, Kasuya E (2009) Methodology for the study of the hypothalamic-pituitary hormone 

secretion in cattle Animal Science Journal 80(1):1-11 

4 Itoh H, Ueguchi-Tanaka M, Matsuoka M (2008) Molecular biology of gibberellins signaling in higher 

plants International Review of Cell and Molecular Biology 268(6):191-221 

5 Izawa T (2008) The process of rice domestication : A new model based on recent data Rice 

1(2) : 127-134 

6 Izawa T, Konishi S, Shomura A, Yano M (2009) DNA changes tell us about rice domestication Current 

Opinion in Plant Biology 12(2) : 185-192 

7 Kawahigashi H (2009) Transgenic plants for phytoremediation of herbicides Current Opinion in 

Biotechnology 20(2) : 225-230 

8 Kikuchi K, Kashiwazaki N, Nagai T, Nakai M, Somfai T, Noguchi J, Kaneko H (2008) Selected aspects 

of advanced porcine reproductive technology Reproduction in Domestic Animals 43(Suppl. 2):401-406 

9 Krishnan A, Guiderdoni E, An G, Hsing Y-i.C, Han C-d, Lee M.C, Yu S-M, Upadhyaya N, 

Ramachandran S, Zhang Q, Sundaresan V, Hirochika H, Leung H, Pereira A (2009) Mutant resources in 

rice for functional genomics of the grasses Plant Physiology 149(1):165-170 

10 Matsumoto T, Wu J, Antonio B.A, Sasaki T (2008) Development in rice genome research based on 

accurate genome sequence International Journal of Plant Genomics 2008(348621):9 

11 Murata K, Wakabayashi Y, Kitago M, Ohara H, Watanabe H, Tamogami S, Warita Y, Yamagishi K, 

Ichikawa M, Takeuchi Y, Okamura H, Mori Y (2009) Modulation of gonadotrophin-releasing hormone 

pulse generator activity by the pheromone in small ruminants Journal of Neuroendocrinology 

21(4) : 346-350 

12 Nishimura M, Morita R, Kusaba M (2009) Utilization and molecular characterization of seed protein 

composition mutants in rice plants Japan Agricultural Research Quarterly 43(1):1-5 

13 Ohyama K, Takemura M, Oda K, Fukuzawa H, Kohchi T, Nakayama S, Ishizaki K, Fujisawa M, Yamato 

K (2009) Gene content, organization and molecular evolution of plant organellar genomes and sex 

chromosomes — Insights from the case of the liverwort Marchantia polymorpha Proceedings of the 

Japan Academy, Series B 85(3) : 108-124 

14 Sasaki T (2008) The rice genome structure as a trail from the past to beyond Plant Genomes, Genome 

Dynamics 4 : 131-142 

15 Sasaki T (2008) Breeding science in the genomics era Breeding Science 58(2):97 

16 Sasaki T, Antonio A.B (2008) The high-quality rice genome sequence and its impact on cereal 

genomics Rice Improvement in the Genomics Era:1-14 

17 Sasaki T (2008) From the editor’s desk Rice 1(1):1-2 


18 Sasaki T (2008) Making a difference in breeding science Breeding Science 58(3):199 

19 Sasaki T (2008) Good eating quality : Practically attainable but still genetically unsolved Breeding 

Science 58(4) : 345 

20 Sasaki T, Antonio B.A (2009) Sorghum in sequence Nature 457(29):547-548 

21 Sasaki T (2009) Bridging the gap between genomics and breeding Breeding Science 59(1):1 

22 Schulte D, Close T.J, Graner A, Langridge P, Matsumoto T, Muehlbauer G, Sato K, Schulman A.H, 

Waugh R, Wise R.P, Stein N (2009) The International barley sequencing consortium—At the threshold 

of efficient access to the barley genome Plant Physiology 149(1):142-147 

23 Sun Q-Y, Liu K, Kikuchi K (2008) Oocyte-specific knockout : a novel in vivo approach for studying 

gene functions during folliculogenesis, oocyte maturation, fertilization, and embryogenesis Biology of 

Reproduction 79(6) : 1014-1020 

24 Tanaka S (2008) Locust outbreaks and studies on phase polyphenism The International Conference on 

Sustainable Agriculture for Food, Energy and Industry 2008 :324-328 

25 Uenishi H, Shinkai H (2009) Porcine Toll-like receptors : The front line of pathogen monitoring 

and possible implications for disease resistance Developmental & Comparative Immunology 

33(3):353-361 

26 Vaughan D.A, Lu B-R, Tomooka N (2008) Was Asian rice (Oryza sativa) domesticated more than once 

Rice 1(1):16-24 

27 Wu J, Mizuno H, Sasaki T, Matsumoto T (2008) Comparative analysis of rice genome sequence to 

understand the molecular basis of genome evolution Rice 1(2):119-126 

28 Yang L, Wakasa Y, Takaiwa F (2008) Biopharming to increase bioactive peptides in rice seed Journal of 

AOAC International 91(4) : 957-964 

29 Yano M, Tuberosa R (2009) Genome studies and molecular genetics—from sequence to crops : 

genomics comes of age Current Opinion in Plant Biology 12(2):103-106 

30 Yano M (2009) Genomics-based germplasm enhancement and new paradigm in rice breeding 

Proceedings of the JSPS International Seminar 2008:51-59 

II ) Monograph 

1 Fukuta Y, Xu D, Yanoria M.J.T, Hairmansis A, Hayashi N, Kobayashi N (2009) Genetic characterization 

of universal differential variety sets developed under the IRRI-Japan Collaborative Research Project 

Advances in Genetics, Genomics and Control of Rice Blast Disease (IV) :325-335 

2 Hagio T (2009) Direct gene transfer into plant mature seeds via electroporation after vacuum treatment 

Electroporation and Sonoporation in Developmental Biology I (25):285-293 

3 Higuchi M, Matsui K, Ichikawa T, Kondou Y, Hasegawa Y, Kawashima M, Hirochika H, Matsui M (2008) 

Identification of photosynthesis-related genes in rice using FOX hunting system Photosynthesis. Energy 

from the Sun (11) : 597-600 

4 Ichikawa A, Ono H (2008) Stereochemical studies in insect chemistry : Synthesis and properties of 

chiral rsesolving agents Stereochemistry Research Trends 

5 Ichinose Y, Taguchi F, Takeuchi K, Suzuki T, Toyoda K, Shiraishi T (2008) Role of flagellin glycosylation 

in bacterial virulence Pseudomonas syringae Pathovars and Related Pathogens – Identification, 

Epidemiology and Genomics (III):167-174 


6 Kaga A, Vaughan D.A, Tomooka N (2008) Molecular markers in Vigna improvement : Understanding 

and using gene pools Molecular Marker Systems in Plant Breeding and Crop Improvement 

55(II.6) : 171-187 

7 Narita S, Kageyama D (2008) Wolbachia-induced sex reversal in Lepidoptera Insect Symbiosis, Volume 

3:295-319 

8 Sakai H, Itoh T, Gojobori T (2008) Processed pseudogenes and their functional resurrection in the 

human and mouse genomes Encyclopedia of Life Sciences (ELS): 

III ) Others 

1 Morishita T, Miyazawa Y, Saito H, Hayashi Y, Ryuto H, Fukunishi N, Abe T (2008) Semidwarf mutants of 

Tartary buckwheat induced by heavy-ion beam irradiation Riken Accelerator Progress Report 41:232 

2 Okuizumi H, Tamang A, Phuntsho U, Kawase M, Vaughan D.A, Tomooka N (2009) Plant genetic 

resources collaboration between Bhutan and the NIAS Genebank, Japan Journal of the Faculty of 

Agriculture Shinshu University 45(1-2):67-73 

3 Antonio B.A, Matsumoto T (2009) “What if” challenge of the rice biology in the “next-generation” 

sequencing era The Plant Genome 2(1):3-4 

4 Takeuchi K (2008) Studies on flagellin glycosylation and pathogenicity of Pseudomonas syringae 

Journal of General Plant Pathology 74(6):461-462 

5 Fukuoka S, Saka S, Koga H, Hayashi N, Takahashi A, Okuno K, Yano M (2008) Genetic and molecular 

dissection of quantitative resistance to blast pathogen in rice Crop Science Seminar in East Asia 

2008: Recent Progress in the Improvement of Biotic and Abiotic Stress Tolerance in Rice Proceedings: 

L-09 

6 Kouchi H (2009) Molecular genetics of symbiotic plant-microbe interactions using a model legume, 

Lotus japonicus Gamma Field Symposia 46():7-19 

7 Nakagawa H (2008) Project review of FNCA mutation breeding project FNCA 2008 Mutation Breeding 

Project, Sub-project Meeting on Disease Resistance in Banana 

8 Nakagawa H (2008) Mutation breeding and biological researches by the use of gamma ray irradiation 

inJapan FNCA 2008 Mutation Breeding Project Workshop 

9 Nishimura M (2008) Reseach on Rice Plant Mutations in Japan FNCA 2008 Mutation Breeding Project 

Workshop 

10 Nishimura M (2008) Creation of materials for breeding amylose library of primary rice plant varieties 

FNCA 2008 Mutation Breeding Project Workshop 

11 Nishio T, Takahashi Y, Nishimura M (2008) Application of TILLING to gamma-ray-irradiated rice and 

use of silent mutations for tracing farm products FAO/IAEA International Symposium on Induced 

Mutations in Plants 

12 Noda H (2008) Symbiote of insects Seminar at Nanjing Agricultural University 

13 Noda H (2008) Planthopper genocmics and gene functional studies Seminar at Nanjing Agricultural 

University 

14 Noda H (2008) Planthopper genocmics: How it can be useful planthopper management International 

Conference on Rice Planthoppers 


15 Saito T (2009) Neural activity of the temporal hippocampus by odor stimulation in the domestic pigs 

Seminar on Neurobiology 

16 Shiokai S, Hori Y, Nishimura M, Nishio T (2008) Fine mapping of the mutated gene of a genic-malesterile 

mutant in rice FAO/IAEA International Symposium on Induced Mutations in Plants :195 

17 Takezawa T (2008) Recent progress of tissue engineering-related studies utilizing collagen vitrigel and 

TOSHI-substrata The Elisseeff Lab Seminar, John Hopkins University 

18 Takezawa T (2008) Tissue regeneration engineering-related studies utilizing unique cell culture 

substrata External Seminar, Imperial College, London 

19 Yamamoto T (2008) The application of marker-assisted selection in rice breeding Agricultural 

Biotechnology Training-Workshop 


List of Organizations Exchanged 

MOU 

National Institute of Agrobiological Sciences (NIAS) is enhancing research collaboration with foreign 

Institutes and Universities. The Institute collaborative research would bring together the comparative 

advantages of both. For proceeding this, Memorandum of Understanding (MOU) between both 

was signed in each case. Table is shown the organization, term, and research theme, objectives and 

/or activities exchanged MOU. 

Organization 

Term 

Research theme, objectives and/or 

activities of MOU 

China 

Institute of Botany, Chinese 

Academy of Sciences 

2004. 08. 04 ~ 

2009. 08. 03 

Research cooperation on plant genomics 

in rice and other species 

U.S.A. 

Cornel University 

2005. 10. 10 ~ 

2010. 10. 09 

Inclusive agreement for research cooperation 

and exchange of researchers 

U.S.A. 

University of Minnesota 

2006. 02. 09 ~ 

2011. 02. 08 

Inclusive agreement for research cooperation 

and exchange of researchers 

China 

Southwest Agricultural 

University 

2006. 03. 24 ~ 

completion of 

analysis 

Genome sequencing and genome 

structure analysis in silkworm 

Thailand 

Kasetsart University 

2006. 05. 10 ~ 

2011. 05. 09 

Research cooperation on genetics and 

breeding in leguminous crops 

Switzerland 

Syngenta 

2006. 05. 24 ~ 

2011. 05. 23 

Contract for research cooperation to 

develop low-glutelin rice 

Czech 

Institute of Entomology, 

Biology Centre AVCR 

2006. 09. 25 ~ 

2009. 09. 24 

Research cooperation on insect silk 

fiber components and identification of 

their controlling genes 

IBSC 

The International Barley 

Genome Sequencing 

Consortium 

2006. 10. 06 ~ 

completion of 

analysis 

International research cooperation for 

barley genome analysis 

France 

French National Institute for 

Agricultural Research 

2006. 12. 26 ~ 

2010. 12. 25 

Research cooperation on genomics in 

insects, plants and animals 

Korea 

National Institute of 

Agricultural Biotechnology, 

Rural Development 

Administration 

2008. 5. 29 ~ 

2013. 5. 28 

Inclusive agreement for cooperation in 

the fields of agricultural science and 

biotechnology 

Australia 

The commonwealth scientific 

and industrial 

research organization 

2008. 7. 1 ~ 

2013. 6. 30 

Research cooperation and exchange of 

researchers 


Executive Members and Research 

Staff Members 

Executive Members 

President 

Vice-President 

Vice-President 

Auditor 

Auditor 

(as of March 31, 2008) 

Ishige Teruo 

Sasaki Takuji 

Shinbo Hiroshi 

Ichikawa Kunihiko 

Horio Yoshinori 

Research Staff 

Research Planning and Coordination 

Research 

Director-General 

Research Director 





Deputy Research Director 






Ohkawa Yasunobu 

Kawasaki Kenjiro 

Shirata Kazuto 

Machii Hiroaki 

Kawase Makoto 

Nakagawa Hitoshi 

(Asaoka Kiyoshi) 

(Onishi Akira) 

(Handa Hirokazu) 

(Nishizawa Yoko) 

(Kanno Masashi) 

(Kawamoto Natuo) 

Research Planning Section Head Noda Takashi 

Obo Masahiro 

(Yamamoto Shin-ichi) 

Evaluation Section Head Haga Atsunobu 

Information Management Section Head Mitsuhashi Hatsuhito 

Chief Researcher 

Kawada Masae 

Library Head Yoshida Fumio 

Public Relations Section Head Niino Takao 


Inoue Takashi 


GMO Research Promotion Section Head Tabei Yutaka 

(Domon Eiji) 

Safety Management Section Head Watanabe Shinichiro 

Technology Transfer and Research Cooperation Section Head Ohura Masanobu 


Kayano Toshiaki 


Hirogari Yasuhiro 

(Sakurai Michiharu) 

(Hagio Takashi) 

(Nakamura Masatoshi) 

Genetic Resources Management Section Head Kenmochi Fumikazu 

Technical Support Section Head Koyama Akio 

Head 

Kobayashi Toru 


QTL Genomic Research Center Director Yano Masahiro 


Sugimoto Kazuhiko 


Taguchi Fumio 


Yamamoto Toshio 


Ebana Kaworu 


Fukuoka Shuichi 


Yonemaru Jun-ichi 


Mizobuchi Ritsuko 


Yamanouchi Utako 


Ueda Tadamasa 

Researcher 

Yamamoto Shin-ichi 

Researcher 

Uga Yusaku 

Researcher 

Hori Kiyosumi 

Transgenic Crop Research and Development Center Director Takaiwa Fumio 


Domon Eiji 


Takahashi Sakiko 

Researcher 

Takagi Hidenori 

Researcher 

Kawakatsu Taiji 

Transgenic Silkworm Research Center Director Tamura Toshiki 


Yonemura Naoyuki 


Iizuka Tetsuya 



Researcher 

Researcher 

Tatematsu Kenichiro 

Sezutsu Hideki 

Kobayashi Isao 

Transgenic Animal Research Center Director Kitani Hiroshi 

Senior Researcher Naito Mitsuru 

Senior Researcher Sakurai Michiharu 

Senior Researcher Onishi Akira 


Matsubara Yuko 


Takezawa Toshiaki 


Takenouchi Takato 


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 

Plant Genome Research Unit Head Matsumoto Takashi 

Senior Researcher Kikuchi Shoshi 

Senior Researcher Komatsuda Takao 

Senior Researcher Handa Hirokazu 


Ogawa Taiichi 


Wu Jianzhong 


Kawahigashi Hiroyuki 


Mizuno Hiroshi 

(Izawa Takeshi) 

Bioinformatics Research Unit Head Ito Takeshi 


Maeda Miki 

Researcher 

Numa Hisataka 

Researcher 

Tanaka Tsuyoshi 

Researcher 

Sakai Hiroaki 

Genome Resource Center Head Nagamura Yoshiaki 


Baltazar Alcaraz Antonio 


Miyao Akio 



Researcher 

Koga Yasunori 

Sato Yutaka 

(Ichikawa Hiroaki) 

Genebank Director (Kawase Makoto) 

Senior Researcher Nagayasu Kenichi 

Senior Researcher Minezawa Mitsuru 

Senior Researcher Imanishi Shigeo 

Senior Researcher Sato Toyozo 

Senior Researcher Tomooka Norihiko 

Senior Researcher Aoki Takayuki 

Senior Researcher Sawada Hiroyuki 


Takeya Masaru 


Nagai Toshiro 


Kosegawa Eiichi 


Okuizumi Hisato 


Tomioka Keisuke 


Nishikawa Tomotaro 


Fukui Kuniaki 

Hayasaka Shoji 

(Kaga Akito) 

Institute of Radiation Breeding Director (Nakagawa Hitoshi) 

Senior Researcher Nishimura Minoru 


Takyu Toshio 


Morishita Toshikazu 


Muramatsu Noboru 


Shimizu Akemi 


Yamanouchi Hiroaki 

Researcher 

Morita Ryohei 

Soybean Genome Research Team Head Harada Kyuya 


Katayose Yuichi 


Kaga Akito 

Researcher 

Watanabe Satoshi 


Director 

Meshi Tetsuo 

Hagihara Kiyoshi 

Mochizuki Atsuko 

Environmental Stress Research Unit Head Hayashi Makoto 





Ogawa Masafumi 

Ishikawa Masaya 

Fukuda Atsunori 

(Sugimoto Kazuhiko) 

(Ueda Tadamasa) 

Photobiology and Photosynthesis Research Unit Head Takano Makoto 

Senior Researcher Miyao Mitsue 

Senior Researcher Ichikawa Hiroaki 

Senior Researcher Izawa Takeshi 


Takeichi Tetsuo 


Yazaki Yoshiaki 


Kiyota Seiichiro 


Inagaki Noritoshi 


Ishimaru Ken 


Baba Akiko 


Iwamoto Masao 


Sentoku Naoki 

Researcher 

Ito Hironori 

Plant Disease Resistance Research Unit Head Takatsuji Hiroshi 

Senior Researcher Kawasaki Shinji 

Senior Researcher Hayashi Nagao 


Mori Masaki 


Jiang Cian Je 


Sugano Shoji 


Yamazaki Muneo 

Researcher 

Takahashi Akira 

Researcher 

Inoue Haruhiko 

Protein Research Unit Head Yamazaki Toshimasa 

Senior Researcher Shimomura Shoji 

Senior Researcher Takase Kenji 

Senior Researcher Momma Mitsuru 


Kajiwara Hideyuki 


Fujimoto Zui 


Suzuki Rintaro 


Wako Toshiyuki 

Plant-Microbe Interactions Research Unit Head Minami Eiichi 

Senior Researcher Ishikawa Masayuki 


Nakayama Yasuji 











Researcher 

Researcher 

Researcher 

Researcher 

Nishizawa Yoko 

Kato Etsuko 

Ochiai Hirokazu 

Umehara Yosuke 

Mitsuhara Ichiro 

Nishimura Marie 

Seo Shigemi 

Yoshikawa Manabu 

Akimoto Chiharu 

Takeuchi Kasumi 

Otake Yuko 

Imaizumi Haruko 

Nakagawa Tomomi 

Plant Genetic Engineering Research Unit Head Toki Seiichi 


Hagio Takashi 


Kishimoto Naoki 


Miyahara Kenzo 


Habu Yoshiki 


Nakayama Shigeki 


Kawagoe Yasushi 

Researcher 

Saika Hiroaki 

(Tabei Yutaka) 

Division of Insect Science Director 

Director 

Takeda Satoshi 

Insect Genome Research Unit Head Mita Kazuei 


Kadono Keiko 


Yukuhiro Kenji 


Hirokawa Masahiko 


Tomita Shuichiro 


Yasukochi Yuji 


Komoto Natsuo 


Suetsugu Yoshitaka 

(Tamura Toshiki) 

(Tatematsu Kenichiro) 

Invertebrate Gene Function Research Unit Head Kiuchi Makoto 

Senior Researcher Myohara Maroko 

Senior Researcher Shinoda Tetsuro 

Senior Researcher Taniai Kiyoko 











Researcher 

Researcher 

Researcher 

Kotaki Toyomi 

Shiotsuki Takahiro 

Asaoka Kiyoshi 

Tanaka Yoshiaki 

Nakao Hajime 

Ichikawa Akio 

Shimoda Masami 

Hatakeyama Masatsugu 

Kamimura Manabu 

Shimura Sachiko 

Kihara (Yamamoto) Mami 

Kozaki Toshinori 

Anhydrobiosis Research Unit Head Okuda Takashi 


Researcher 

Kikawada Takahiro 

Cornette Richard Marcel 

Jacques 

Innate Immunity Research Unit Head Yamakawa Minoru 


Kato Yusuke 


Ishibashi Jun 


Tanaka Hiromitsu 

Insect Interaction Research Unit Head (Kawasaki Kenjiro) 

Senior Researcher Wakamura Sadao 

Senior Researcher Hattori Makoto 

Senior Researcher Tanaka Seiji 

Senior Researcher Inouchi Jun 

Senior Researcher Muraji Masahiko 


Nakamura Masatoshi 


Hirayama Chikara 


Konno Kotaro 


Tateishi Ken 


Hasegawa Tsuyoshi 


Hinomoto Norihide 


Yasui Hiroe 


Tamura Yasumori 


Maeda Taro 

Insect-Microbe Research Unit Head Noda Hiroaki 

Senior Researcher Hara Wajiro 

Senior Researcher Miyamoto Kazuhisa 









Researcher 

Researcher 

Mitsuhashi Wataru 

Watanabe Hirofumi 

Nakashima Nobuhiko 

Arakawa Toru 

Watanabe Kenji 

Wada Sanae 

Murakami Ritsuko 

Matsumoto Yukiko 

Kageyama Daisuke 

Silk-Materials Research Unit Head Tamada Yasushi 


Tomiyama Masamitsu 


Goto Yoko 


Toshima Yoshiyuki 


Miyazawa Mitsuhiro 


Hata Tamako 


Takasu Yoko 


Kameda Tsunenori 


Kuwana Yoshihiko 


Kojima Katsura 

Researcher 

Teramoto Hidetoshi 

Silk Technology Unit Head Takabayashi Chiyuki 


Nakajima Kenichi 


Mase Keisuke 


Okada Eiji 

Kinoshita Haruo 

Kawachi Ikuo 


Director 

Kurihara Mitsunori 

Animal Genome Research Unit Head Awata Takashi 

Senior Researcher Yasue Hiroshi 

Senior Researcher Hamajima Noriyuki 

Senior Researcher Kojima Misaki 


Hayashi Takeshi 


Harumi Takashi 


Mikawa Satoshi 


Watanabe Satoshi 


Uenishi Hirohide 

Researcher 

Ogawa Tomoko 


Reproductive Biology Research Unit Head Tokunaga Tomoyuki 

Senior Researcher Kaneko Hiroyuki 

Senior Researcher Noguchi Junko 


Ito Yoshiyasu 


Goto Hideo 


Kikuchi Kazuhiro 


Takahashi Toru 


Suto Junichi 


Hurusawa Tadashi 


Hosoe Misa 


Ohkoshi Katsuhiro 


Miyashita Norikazu 


Sakumoto Ryosuke 

Neurobiology Research Unit Head Okamura Hiroaki 


Saito Toshiyuki 


Yayo Kenichi 


Kasuya Etsuko 

Researcher 

Wakabayashi Yoshihiro 

General affairs 

Management Director-General 

Management Director 

Shimada Hiroaki 

Sato Shinetsu 

General Affairs Section Head Hoshi Motoo 

Accounting Section Head Oonuma Yoshinori 

Management and Supply Section Head Tateno Toshitake 

Audit and Compliance Section Head Saito Ryoichi 

( ) additional position 


Members of NIAS Evaluation 

Committee 

(as of March 31,2009) 

Kono Tomohiro 

Kubo Takeo 

Kobayashi Michihiro 

Nishimura Ikuko 

Shinozaki Kazuo 

Endo Takashi 

Gojobori Takashi 

Senoo Kenichiro 

Tokyo University of Agriculture 

School of Science, The University of Tokyo 

Nagoya University, Graduate School of Bioagricultural Sciences 

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 Nonprofit Organization 


FINANCIAL OVERVIEW 

Fiscal Year 2008 (April 2008 – March 2009) 

millions of yen 

TOTAL BUDGET 12,491 

OPERATING COSTS 4,647 

Personnel cost 3,928 

Personnel (387) 

President (1) 

Vice President (2) 

Auditor (2) 

Administrators (124) 

General administrators 

Field management and transportations 

Researchers (263) 

(Number of persons is shown in ( ) as of Mar 31, 2009) 

Administrative cost 440 

Facilities improvement expense 279 

RESEARCH PROMOTION COSTS 7,844 

Research Grant from MAFF 2,841 

Entrusted Research Expenses from MAFF 3,992 

Entrusted Research Expenses from MEXT 314 

Entrusted Research Expenses from others 697 

Entrusted Research Expenses 

from MEXT 

314 (3%) 

Entrusted Research Expenses from others 

697 (6%) 

Personnel 

3,928 (31%) 

Entrusted Research Expenses 

from MAFF 

3992 (32%) 

Research Grant from MAFF 

2,841 (23%) 

Administrative costs 

440 (4%) 

Facilities Improvement 

Expense 

279 (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 2009 

(Apr. 2008-Mar. 2009) 

No. 8 

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 2009

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