抗黄瓜花叶病毒RNAi载体的构建及烟草的转化  

Construction of RNAi Vector which Resist Cucumber Mosaic Virus and Transformation of Tobacco

作　　者：张瑜[1,2] 郑银英[1,2] 赵峰玉[1,2] 乔亚红[1,2] 崔百明[1,2] 向本春[1,2] 

机构地区：[1]石河子大学农业生物技术重点实验室 [2]石河子大学生命科学院,新疆石河子832003

出　　处：《安徽农业科学》2010年第23期12337-12340,共4页Journal of Anhui Agricultural Sciences

基　　金：国际科技合作项目(2008DFA30560);973计划前期研究专项(2008CB117018);石河子大学高层次人才科研启动项目(RCZX200732)

摘　　要：[目的]构建抗黄瓜花叶病毒RNAi载体,并将载体转入烟草。[方法]采用RT-PCR方法,扩增黄瓜花叶病毒NS04加工番茄分离物的RNA2基因组的序列。选取CMVRAN2基因组中的复制酶片段作为靶序列,构建pBi35SCR2真核表达载体,并对表达载体进行鉴定;通过农杆菌介导的方法将表达载体转入烟草,用PCR的方法检测载体是否转入。[结果]系统进化树分析结果表明,RNA2中编码CMV-2a的序列与中国浙江的DQ412731分离物有较高核苷酸及氨基酸同源性,分别达到98.0%和96.5%;PCR结果表明,试验成功构建了pBi35SCR2真核表达载体,并成功将表达载体转入烟草。[结论]试验获得的转基因烟草可作为后期攻毒试验的材料,并为研究加工番茄抗黄瓜花叶病毒奠定了基础。[Objective] Aimed to construct RNAi vector of cucumber mosaic virus and transferred this vector into tobacco.[Method] RT-PCR method was used to amplify cucumber mosaic virus NS04 and process RNA2 gene sequen of tomato isolates. The analysis results of phylogenetic tree demonstrated that the sequence in RNA2 encoded CMV-2a had 98.0% and 96.5% homology with nucleotide and amino acid of DQ412731 isolate of Zhejiang,China. The replicase fragment in CMV RAN2 gene was taken as target sequence to construct pBi35SCR2 eukaryotic expression vector,then the expression vector was identified. Through agrobacterium-mediated method,the expression vector was transferred into tabacco and PCR method was used to check the transfer. The PCR results demonstrated that the experiment had successfully construct eukaryotic expression vector of pBi35SCR2 and the expression vector was successfully transferred into tabacco. [Conclusion] The obtained transgenic tobacco could be used as challenge test material in following experiment and provided foundation for studying processing tomato resist cucumber mosaic virus.

关 键 词：黄瓜花叶病毒 同源性 RNAI载体 烟草 转化 

分 类 号：R392.12[医药卫生—免疫学]