利用反义基因沉默策略构建水稻突变体库及突变体筛选

Generation of a rice mutant library by shotgun antisense gene silencing and mutant screening

尝试利用鸟枪反义基因沉默策略构建水稻突变体库.利用水稻反义cDNA文库转化粳稻品种中花11,获得了来源于105块潮霉素(Hm)抗性愈伤组织的1486个转化植株.随机选择来源于11个独立转化系的335个T0植株进行Hm离体叶片筛选,结果表明所有植株均呈抗性,并且与PCR检测结果相吻合,说明T0植株中不存在假转化体.Southernblot分析结果表明T0植株大部分为单位点整合.T0及T1代转化植株均呈现了一些形态变异,如矮化、无分蘖或多分蘖、结实率降低、粒型或穗型改变、小穗结构改变等.利用Hm离体叶片筛选法对部分T1群体进行筛选,得到了1个变异性状与Hm抗性共分离的突变体,该突变体的变异表型为结实率比非转化对照下降约50%,每穗颖花总数比对照减少约40%,二者的总效应导致突变体单穗产量比对照约下降70%.

A strategy of shotgun antisense gene silencing was used to generate a rice mutant library. A total of (1 486) transgenic rice plants were obtained from 105 independent Hygromycin(Hm) resistant calli transformed with rice antisense cDNA library, using japonica cultivar Zhonghua 11 as transformation material. A total of 335 T_0 transformants from 11 independent calli were selected randomly to test Hm resistance by leaf segments screening. The results showed that all T_0 plants tested were Hm resistant, coincident with the results of PCR analysis, indicating that no escapee existed in T_0 transformants. The result of Southern blot analysis revealed that most of the transgenic plants contained single T-DNA insertion. Some morphological variations were observed in T_0 and T_1 transgenic plants such as dwarf, non-tiller or multi-tillers, decrease of seed-setting, changes of grain or panicle shape or spikelet structure. One mutant was characterized, the mutative traits of which cosegregated with Hm resistance. The mutant displayed about 50% decrease of seed-setting rate and 40% decrease of total number of spikelets per panicle, resulting in about 70% decrease of seed production per panicle as compared with non-transgenic control.