PLRV 56KD蛋白基因植物转化载体的构建

Construction of plant transformation vector and transferring into potato of the plrv 56KD protein gene

为培育转基因抗马铃薯卷叶病毒的新材料 ,采用二次连接法将存在于克隆载体 p L R5 6中的马铃薯卷叶病毒 5 6 KD蛋白基因及 3 端非编码区构建到双元载体系统的中间表达载体质粒p ROK 中 ,采用三亲融合法和直接导入法转化根癌农杆菌 LBA4 40 4 ,PCR法检测。实验表明 ,二次连接法效果优于一次连接法 ,两种连接法对比 ,二次连接法更适用于大分子质粒的重组。直接导入法比三亲融合法更简便 ,二者转化效率没有显著差异。实验构建完成马铃薯卷叶病毒 5 6 KD蛋白基因及 3 端非编码植物转化载体 ,建立了高效。

The 56KD protein gene & 3 part non encoded region of the PLRV Chinese isolates which contained in plasmid pLR56 were constructed into binary vector plasmid pROK Ⅱ by Two-step link method, which was introduced into the agrobacterium tumefaciens LBA4404 by tri-parental conjugation and direct entering ways.Tests showed that Two-step link method was suitable for the recombinant of big plasmid,which was better than One-step method.Two transfermation method:tri-parental conjugation and direct entering ways were compared for PCR detection,tests showed that direct entering method was more sample and convenient.The plant transformation vector of the 56KD prodtein gene & 3 part non encoded region of the PLRV Chinese isolates was constructed successfully and a construction method was found to be an efficient and convenient method.$$$$