大豆花叶病毒CP基因RNAi载体的构建及大豆遗传转化

RNAi vector construction of Soybean mosaic virus CP gene and soybean transformation

为获得含有大豆花叶病毒(Soybean mosaic virus,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(RNA interference,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264 bp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783 bp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474 bp和460 bp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。

In order to obtain transgenic soybean plants carrying interference fragment of Soybean mosaic virus( SMV) CP gene,sequence alignment of CP genes of 11 popular SMV strains was conducted and the RNA interference( RNAi) vector pB7GWIWG2( Ⅱ)-CPi was constructed using GATEWAY technology. The recombinant vector was sequenced and identified by restriction enzyme digestion and PCR. Soybean transformation was conducted through Agrobacterium-mediated system. The most conserved interference fragment named CPi was cloned,which was 264 bp in length,and the sequencing results showed that the matching rate was 100%; the recombinant vector was digested into two bands of 783 bp and 10 818 bp with XbaⅠ,and 2 256 bp and 9 346 bp with EcoRⅠ mixed with Hind Ⅲ,indicating that the two ccdB sites were both replaced by CPi; two bands of 474 bp and 460 bp were obtained after PCRamplification,indicating that the interference fragment of CPi formed the inverted repeat structure in pB7GWIWG2( Ⅱ). Ten T0 transgenic seedlings were obtained through soybean genetic engineering and six plants were identified to be positive through herbicide painting,PCR and QuickStix Kit for Liberty Link( bar),demonstrating that the method could be applied for screening new germplasm of transgenic soybeans resistant to SMV,which provides the parent materials for SMV resistance breeding.