Rice Genetics IV - IRRI books - International Rice Research Institute

Retrotransposons of riceas a tool for the functional analysisof genesH. Hirochika, A. Miyao, M. Yamazaki, S. Takeda, K. Abe, R. Hirochika, G.K. Agrawal,T. Watanabe, K. Sugimoto, T. Sasaki, K. Murata, K. Tanaka, K. Onosato, A. Miyazaki,Y. Yamashita, and N. KojimaFive endogenous active retrotransposons have been found in rice. Amongthem, the most active one, called Tos17, was characterized in detail. Tos17is silent under normal conditions and becomes active only under tissue cultureconditions. Five to 30 transposed Tos17 copies were found in each plantregenerated from culture. Tos17 was shown to transpose preferentially intolow-copy-number, gene-rich regions, indicating that Tos17 can be used as anefficient insertional mutagen. A collection of 32,000 regenerated rice linescarrying about 256,000 insertions was generated, and these lines are beingused for forward and reverse genetic analyses. By using a transposon-taggingstrategy, causative genes for viviparous, dwarf, semidwarf, brittle culm,pale green, and narrow leaf mutations, among others, have been cloned. Forreverse genetic studies, two strategies are being employed. One is the polymerasechain reaction (PCR) screening of mutants of the gene of interest.We screened 12,000 lines and found mutants of 15 genes, including MAPK,MADS-box, and P450 genes, among the 47 genes analyzed. This suggeststhat at least 37,000 lines are required for saturation mutagenesis. Anotherimportant strategy is the random sequencing of mutated genes by isolatingthe sequences flanking transposed Tos17. The flanking sequences are amplifiedby TAIL (thermal asymmetric interlaced)- and suppression-PCR anddirectly sequenced. Until now, 7,376 independent flanking sequences from2,134 lines have been determined and mutants of different classes of geneshave been identified.Because of an explosion in the number of sequenced genes of rice with unknownfunction revealed by large-scale analysis of expressed sequence tags (ESTs) (Sasakiet al 1994, Yamamoto and Sasaki 1997) and genome sequencing (Sasaki and Burr2000), development of a systematic method suitable for discovering the biologicalfunctions of these genes becomes extremely important. To determine gene function,gene inactivation is a powerful tool. Among the gene inactivation strategies, insertionalmutagenesis seems most suitable for a systematic functional analysis of a largenumber of genes. In Arabidopsis, whose entire genomic sequencing will be completedsoon, several insertional mutagens have been used for efficient mutagenesis.Retrotransposons of rice as a tool . . . 279