一种用于植物基因沉默的新RNAi载体的构建  

Construction of a New RNAi Vector for Gene Silencing in Plants

作　　者：宋梦如 陈可钦 郭运娜 雷莹莹 代红艳[1] 

机构地区：[1]沈阳农业大学园艺学院,沈阳110161

出　　处：《沈阳农业大学学报》2017年第6期719-724,共6页Journal of Shenyang Agricultural University

基　　金：国家自然科学基金项目(31470678)

摘　　要：RNA干扰(RNA interference,RNAi)技术是研究基因功能的一种常用方法。为解决目前植物RNAi载体构建繁琐复杂的问题,本研究以常用的植物过量表达载体pRI 101-AN为基础,在其多克隆位点处插入源自pKANNIBAL载体的内含子序列,获得pRNAi-E载体。在此基础上,只要将目的基因特异序列的正反向片段分别连接到内含子两侧,即可构建目的基因的RNAi载体。为了验证pRNAi-E载体的效果,苹果IAA29(Md IAA29)基因的正向和反向片段被插入到pRNAi-E载体上,获得了Md IAA29基因的RNAi载体pRNAi-IAA29。通过农杆菌介导的遗传转化,获得了GL-3苹果的转基因株系。qRT-PCR数据显示转基因苹果植株中IAA29的转录水平比非转基因对照显著下调,表明基于pRNAi-E构建的RNAi载体能够有效地沉默苹果内源基因的表达。本研究构建的pRNAi-E载体使得植物基因RNAi载体构建变得简单高效,对于开展植物基因功能验证研究具有一定意义。RNA interference（RNAi） technique has been widely applied in gene function researches. To solve the problems that it is complicated and cost time to construct the RNAi vector for plants, in this study, a new RNAi empty vector, named pRNAi-E,was constructed by inserting the intron fragment from pKANNIBAL vector into the multiple cloning site of a plant overexpression vector-pRI 101-AN. RNAi vector for a target gene will be easily constructed by inserting the forward and reverse fragments into sides of intron of pRNAi-E vector. To validate the effect of pRNAi-E, a vector named pRNAi-IAA29 was constructed by inserting the forward and reverse fragments of apple IAA29 gene（Md IAA29） into pRNAi-E. Transgenic lines of GL-3 apple were obtained by Agrobacterium-mediated transformation, and the q RT-PCR data showed the transcription levels of Md IAA29 in transgenic lines were significantly down-regulated compared with that in non-transgenic control. It means that the RNAi vector based on pRNAi-E is effective to silence the expression level of genes in apple. It is simple and effective to construct RNAi vectors based on pRNAi-E, so it is meaningful for studies of gene functions.

关 键 词：基因沉默 遗传转化 载体 RNAI 

分 类 号：Q782[生物学—分子生物学]