一种用于植物基因沉默的新RNAi载体的构建

发布时间：2018-04-13 07:08

本文选题：基因沉默 + 遗传转化　；参考：《沈阳农业大学学报》2017年06期

【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。
[Abstract]:RNAi technique is a common method to study gene function.In order to solve the complex problem of plant RNAi vector construction at present, the pRNAi-E vector was obtained by inserting intron sequence from pKANNIBAL vector on the basis of plant overexpression vector pRI 101-AN.On this basis, the RNAi vector of the target gene can be constructed as long as the positive and negative fragments of the specific sequence of the target gene are respectively connected to the two sides of the intron.In order to verify the effect of pRNAi-E vector, the forward and reverse fragments of IAA29(Md IAA29 gene were inserted into pRNAi-E vector, and the RNAi vector pRNAi-IAA29 of Md IAA29 gene was obtained.The results of Agrobacterium tumefaciens mediated genetic transformation showed that the transcriptional level of IAA29 in transgenic apple was significantly lower than that in non-transgenic control.The results showed that the RNAi vector constructed based on pRNAi-E could effectively silence the expression of endogenous gene in apple.The pRNAi-E vector constructed in this study makes the construction of plant gene RNAi vector simple and efficient, and has certain significance for the research of plant gene function verification.
【作者单位】：沈阳农业大学园艺学院;
【基金】：国家自然科学基金项目(31470678)
【分类号】：Q943.2

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