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【目的】在水稻中建立高效RNA干涉(RNAi)技术体系。【方法】构建适合水稻转化的RNAi诱导载体pCADS1341;为检测其有效性,使用它构建针对GUS基因的RNAi载体,并利用基因枪转化法导入GUS基因稳定表达的转基因水稻愈伤组织;为探讨影响水稻中转基因诱导的RNAi效率的因素,针对水稻基因AK121286进行系统的RNAi分析:通过Southern和Northern杂交从T0代RNAi植株中筛选出T-DNA为单拷贝插入,且基因的表达被高效抑制的株系,对来源于该株系的T1代植株进行半定量RT-PCR检测。【结果】GUS染色分析结果表明RNAi诱导元件的瞬时表达可显著抑制GUS基因的表达;RT-PCR结果表明RNAi效应可以遗传给后代,但是不同个体中的RNAi效率存在差异。【结论】RNAi系统的表达量可能是决定RNAi效率的最关键因素;本研究建立的高效RNAi技术体系对于水稻功能基因组学研究具有重要意义。
【Objective】 To establish high efficient RNA interference (RNAi) system in rice. 【Method】 RNA interference vector pCADS1341 was constructed for rice transformation. In order to test its effectiveness, RNAi vector targeting GUS gene was constructed and introduced into GUS gene stably expressing transgenic rice callus by gene gun transformation. In rice, the RNAi efficiency induced by transgene was systematically analyzed by RNAi analysis of the rice gene AK121286: T-DNA was selected from the T0 generation of RNAi plants by Southern and Northern blotting as a single copy insert and the gene expression was highly inhibited Line, T1 generation plants derived from this line were subjected to semi-quantitative RT-PCR. 【Result】 The results of GUS staining showed that the transient expression of RNAi inducing components could significantly inhibit the expression of GUS gene. The results of RT-PCR showed that the RNAi effect could be passed on to offspring, but the RNAi efficiency differed among different individuals. 【Conclusion】 The expression level of RNAi system may be the most crucial factor to determine the efficiency of RNAi. The high efficient RNAi system established in this study is of great significance for functional genomics research in rice.