Enoki H, Izawa T, Kawahara M, Komatsu M, Koh S, Kyozuka J, Shimamoto K
Laboratory of Plant Molecular Genetics, Nara Institute of Science and Technology, Takayama, Ikoma, Japan.
Plant J. 1999 Sep;19(5):605-13. doi: 10.1046/j.1365-313x.1999.00549.x.
To examine whether the maize autonomous transposable element Ac can be used for the functional analysis of the rice genome, we used Southern blot analysis to analyze the behaviour of Ac in 559 rice plants of four transgenic families through three successive generations. All families showed highly active transposition of Ac, and 103 plants (18.4%) contained newly transposed Ac insertions. In nine of the 12 independent transpositions analyzed, their germinal transmission was detected. Partial sequencing of 99 Ac-flanking sequences revealed that 21 clones exhibited significant similarities with protein-coding genes in databases and four of them matched rice cDNA sequences. These results indicate preferential Ac transposition into protein-coding rice genes. To examine the feasibility of PCR-based screening of gene knockouts in rice Ac plants, we prepared bulked genomic DNA from the leaves of approximately 6000 rice Ac plants and pooled the DNA according to a three-dimensional matrix. Of 14 randomly selected genes, two gene knockouts were identified, and one encoding a rice cytochrome P450 (CYP86) gene was shown to be stably inherited to the progeny. Together, these results suggest that Ac can be efficiently used for the functional analysis of the rice genome.
为了研究玉米自主转座元件Ac是否可用于水稻基因组的功能分析，我们通过Southern杂交分析，对4个转基因家系的559株水稻植株连续三代中Ac的行为进行了分析。所有家系均显示Ac具有高度活跃的转座，103株植株（18.4%）含有新转座的Ac插入片段。在分析的12个独立转座事件中的9个中，检测到了它们在生殖细胞中的传递。对99个Ac侧翼序列进行部分测序，结果显示21个克隆与数据库中的蛋白质编码基因具有显著相似性，其中4个与水稻cDNA序列匹配。这些结果表明Ac优先转座到水稻蛋白质编码基因中。为了检验基于PCR筛选水稻Ac植株中基因敲除的可行性，我们从约6000株水稻Ac植株的叶片中制备了混合基因组DNA，并根据三维矩阵对DNA进行了汇集。在随机选择的14个基因中，鉴定出了2个基因敲除事件，其中一个编码水稻细胞色素P450（CYP86）基因的敲除事件被证明能稳定遗传给后代。综上所述，这些结果表明Ac可有效地用于水稻基因组的功能分析。
