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核农学报  2014 

TMV、PVM和CMV干扰载体构建及对人参果遗传转化

DOI: 10:11869/j.issn.100-8551.2014.01.0044, PP. 44-51

Keywords: 烟草花叶病毒,马铃薯M病毒,黄瓜花叶病毒,ihpRNA,转基因人参果

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Abstract:

人参果是以营养器官繁殖为主的多年生草本植物,连年种植易造成多种病毒病积累。为了寻求一种能同时抵御多种病毒病的方法,本试验以危害人参果的3种主要病毒为研究对象,根据GenBank中烟草花叶病毒(TMV)P126基因、马铃薯M病毒(PVM)CP基因和黄瓜花叶病毒(CMV)2b蛋白基因序列,利用E-RNAi网站提供的分析软件,筛选出含有较多有效小干扰RNA[siRNA,19nt]的长dsRNA(LongdsRNA)片段作为RNAi的靶序列。用重叠延伸PCR方法将三个靶标片段融合在一起,构建了一种具有内含子(intro)的反向重复结构和嵌合基因的植物RNAi表达载体pCEIHFR。将pCEIHFR通过冻融法导入农杆菌LBA4404并转化人参果,经过草甘膦筛选和PCR检测,确认其中的24株为转基因阳性植株。

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