Rice Genetics IV - IRRI books - International Rice Research Institute

Generation of T-DNA insertionaltagging lines in riceGynheung An, Jong-Seong Jeon, Sichul Lee, Ki-Hong Jung, Sung-Hoon Jun, Dong-Hoon Jeong,Jinwon Lee, Seonghoe Jang, Shin-Young Lee, Kiyoung Yang, Byoungho Lee, Sinok Moon,Kyungsook An, Min-Jung Han, Jung-Hwa Yu, Namok Lee, Su-Young An, Sun-Hee Park,Eun-Sik Song, In-Soon Park, and Hyun-Sook LeeT-DNA insertions have produced approximately 30,000 transgenic lines ofrice. Polymerase chain reaction and genomic DNA gel-blot analyses have shownthat approximately 65% of the population contains more than one copy of theinserted T-DNA. Hygromycin resistance tests determined the number of T-DNA inserts to be an average of 1.4 genetic loci per plant. From this, it canbe estimated that at least 40,000 taggings have been generated.The binaryT-DNA vector used in the insertion contained the promoterless ß-glucuronidase(gus) reporter gene with an intron as well as multiple splicing donors andacceptors immediately next to the right border. This gene trap vector is designedto detect a gene fusion between gus and the endogenous gene that istagged by the T-DNA. The leaves, roots, mature flowers, and developing seedsof the transgenic rice plants were subjected to histochemical GUS assays.The results showed that 1.6–2.1% were GUS-positive in the tested organsand that their gus expression patterns were organ- or tissue-specific or ubiquitousin all parts of the plant. This large population of T-DNA tagged lineswill be useful for identifying insertional mutants in various genes and fordiscovering new genes in rice.Strategies to discover the functions of plant genes have recently been developingrapidly. These strategies have been largely based on genetic approaches such as mutantidentification and map-based gene isolation (reviewed in Martin 1998). The useof a transposon insertion to inactivate genes has been employed for functional studiesin several plant species. Similarly, mutagenesis through the use of transfer DNA (T-DNA) has also been developed for tagging genes in Arabidopsis (Babiychuk et al1997, Feldmann 1991, Krysan et al 1999). T-DNA insertion is believed to be a randomevent and the inserted genes are stable through multiple generations (reviewedin Azpiroz-Leehan and Feldmann 1997).The development of several strategies for screening T-DNA or transposon insertionsin a known gene and recovering sequences flanking the insertions have madeGeneration of T-DNA insertional tagging lines in rice 253