【摘要】 【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。更多还原

【Abstract】 【Objective】 A high efficiency RNA interference (RNAi) system was established in rice.【 Method】 RNAi-inducing vector pCADS1341 was constructed for rice transformation. In order to examine its effectiveness, RNAi vector targeting GUS gene was constructed using pCADS1341 and introduced into a transgenic rice calli stably expressing the GUS gene using a gene-gun transformation method. In order to investigate factors that affect RNAi efficiency, systematic RNAi analyses were carried out on rice gene AK121286. Using Southern and Northern blots, a line with single-copy T-DNA insertion and well-suppressed target gene expression was selected out from T0 RNAi plants for semiquantitative RT-PCR assay on its T1 offspring. 【Result】 GUS staining results indicated that a transient expression of the RNAi apparatus could significantly suppress the expression of the GUS gene. RT-PCR results indicated that the RNAi effect could be passed on to the next generation, but the RNAi efficiencies in different individuals were variable. 【Conclusion】 Expression level of the RNAi apparatus might determine RNAi efficiency. The high efficiency RNAi system would play an important role in rice functional genomics research.更多还原