中国农业科学 ›› 2006, Vol. 39 ›› Issue (9): 1729-1735 .

• 作物遗传育种·种质资源 •    下一篇

水稻高效RNA干涉体系的建立及其在受体激酶基因功能研究中的应用

路铁刚 彭昊 张治国 宛淑艳 郭玉朋 翟英   

  1. 农科院生物技术研究所
  • 收稿日期:2005-12-12 修回日期:2006-01-09 出版日期:2006-09-10 发布日期:2006-09-10
  • 通讯作者: 路铁刚

Establishment of an efficient RNA interference system in rice and the application on the functional analysis of receptor-like kinase genes

  

  1. 农科院生物技术研究所
  • Received:2005-12-12 Revised:2006-01-09 Online:2006-09-10 Published:2006-09-10

摘要: 利用RNA干涉(RNAi)技术,可以特异性的抑制真核生物体中目标基因的表达。本研究构建了适合水稻转化的RNAi诱导载体pCADS1341。为检测其有效性,使用它构建了针对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。GUS染色分析结果表明该载体中RNAi诱导元件的瞬时表达可显著抑制GUS基因的表达。为探讨影响水稻中转基因诱导的RNAi效率的因素,对针对某个目标水稻基因的水稻RNAi植株进行了详细的分析。通过Southern和Northern杂交检测了T0代RNAi植株中T-DNA插入的拷贝数和目标基因的表达量,筛选出T-DNA为单拷贝插入,且目标基因的表达被高效抑制的株系。对来源于该株系的T1代植株进行了半定量RT-PCR检测,结果表明RNAi效应可以遗传给后代转基因水稻植株,但是不同个体中的RNAi效率存在差异。RNAi系统的表达量可能是决定RNAi效率的最关键因素。我们建立的高效RNAi技术体系对于水稻功能基因组学研究具有重要意义,利用这个系统我们已经构建了60多个水稻受体激酶基因的RNAi载体,系统的研究正在进行中。

关键词: RNA干涉, T-DNA, 水稻

Abstract: RNA interference (RNAi) technology can be used to specifically suppress the expression of target gene in eukaryotes. In this research, RNAi-inducing vector pCADS1341 was constructed for rice transformtion. In order to examine its effectiveness, RNAi vector targeting GUS gene was constructed using pCADS1341 and introduced into transgenic rice calli stably expressing GUS gene using gene-gun transformation method. GUS staining results indicated that transient expression of the RNAi apparatus could significantly suppress the expression of GUS gene. In order to investigate factors that affect RNAi efficiency, detailed analyses were carried out on rice RNAi plants targeting a given rice receptor-like kinase gene. T-DNA insertion copy number and expression level of target gene in each T0 RNAi plant were detected using Southern blot and Northern blot, respectively. A line with single-copy T-DNA insertion and well-suppressed target gene expression was selected out for semi-quantitative RT-PCR assay on its T1 offspring. The results indicated that RNAi effect could be inherited to the next generation of trangenic rice plants, but the RNAi efficiencies in different individuals were variable. Expression level of RNAi apparatus might determine RNAi efficiency. Our high efficiency RNAi system would play an important role in rice functional genomics research. We have successfully made more than six RNAi constructs for rice receptor-like kinase genes and systematic functional analysis of those genes are underway.

Key words: RNA interference, T-DNA, Rice