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大豆花叶病毒CP基因RNAi载体的构建及大豆遗传转化

, PP. 453-460

Keywords: 大豆花叶病毒,CP基因,RNAi,GATEWAY技术,农杆菌介导大豆转基因

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Abstract:

为获得含有大豆花叶病毒(Soybeanmosaicvirus,SMV)CP基因干扰片段的转基因大豆新材料,对11个SMV流行株系的CP基因进行了核苷酸序列比对,采用GATEWAY技术构建了RNA干扰(RNAinterference,RNAi)载体pB7GWIWG2(Ⅱ)-CPi,对重组载体测序比对,通过酶切以及PCR方法鉴定载体,并利用农杆菌介导的转基因体系进行大豆遗传转化。克隆出264bp高度保守的CPi干扰片段,通过测序证实其与目标序列的匹配度达到100%;重组质粒经过XbaⅠ单酶切后出现783bp和10818bp两条带,经过EcoRⅠ、HindⅢ双酶切后出现2256bp和9346bp两条带,说明2个ccdB结合位点均被CPi取代;PCR反应得到474bp和460bp两条带,说明CPi以反向重复形式与pB7GWIWG2(Ⅱ)组合。共获得10棵T0代转基因幼苗,经除草剂涂抹、PCR、PAT/bar转基因检测试纸检测后,6株为阳性,表明该方法可筛选出对大豆花叶病毒具有抗性的转基因大豆新种质,为SMV抗病育种工程提供良好的亲本材料。

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