双抗CMV和ToMV RNAi载体的构建及烟草遗传转化  

Construction of RNAi Vector with Double Resistance to Cucumber Mosaic Virus and Tomato Mosaic Virus and Tobacco Genetic Transformation

作　　者：田桂英[1] 乔亚红[1] 向本春[1] 郑银英[1] 李诗林[1] 崔百明[1] 

机构地区：[1]石河子大学农业生物技术重点实验室/石河子大学农学院,石河子832003

出　　处：《石河子大学学报（自然科学版）》2012年第6期689-694,共6页Journal of Shihezi University(Natural Science)

基　　金：国际科技合作与交流专项(2008DFA30560)

摘　　要：为了获得抗CMV和ToMV的转基因烟草,本试验采用RT-PCR的方法从新疆加工番茄CMV分离物NS0-4克隆了1a复制酶第71～360bp和第1801～2050bp序列作为干扰片段CR9和CR14,以及从ToMV分离物SCS-4克隆了130/180kDa复制酶第658～940bp和第2551～2850bp序列作为干扰片段To9和To14。通过T克隆载体将CR9和To9及CR14和To14进行拼接,分别构建成含反向重复结构拼接片段的RNAi表达载体pBi35STC9和pBi35STC14;利用农杆菌介导法分别将表达载体侵染本氏烟(Nicotiana benthamiana),再生植株经分子检测,结果显示已成功获得了转pBi35STC9烟草9个单株及转pBi35STC14烟草8个单株,为进一步的攻毒试验奠定了基础。To obtain transgenic tobacco with double resistance to Cucumber mosaic virus（CMV）and Tomato mosaic virus（ToMV）,71～360 bp and 1801～2050 bp sequence of 1a replicase cloned from CMV isolate of Xinjiang processing tomato were used as interference fragments,CR9 and CR14.The 658 bp-940bp and 2551～2850 bp sequence of 130/180 kD replicase amplified from ToMV isolate were used as interference fragment To 9 and To14.The two fragments,CR9 and To9,were spliced into one fragment through pGEM-Teasy,CR14 and To14.Then the recombinant plant expression vector pBi35STC9 and pBi35STC14 were constructed in a way of inverted repeats.Through introducing vectors into Nicotiana benthamiana by Agrobacterium mediated transformation separately.The results showed that 9 positive transgenic plants of pBi35STC9 and 8of pBi35STC14 were obtained successfully by molecular detection,providing a foundation for further attack test.

关 键 词：CMV TOMV RNAI载体 转基因烟草 

分 类 号：S436.412.11[农业科学—农业昆虫与害虫防治]