抗CMV和ToMV RNAi载体的构建及加工番茄遗传转化  

Construction of RNAi vector with resistance to Cucumber mosaic virus and Tomato mosaic virus and genetic transformation of processing tomato

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

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

出　　处：《西北农林科技大学学报（自然科学版）》2014年第1期102-110,共9页Journal of Northwest A&F University(Natural Science Edition)

基　　金：国际科技合作与交流专项(20072072);国家自然科学基金项目(31260420)

摘　　要：【目的】利用农杆菌介导法将抗CMV和ToMV的RNAi表达载体pBi35STC12转入红番3号加工番茄,获得抗CMV和ToMV的加工番茄植株,为加工番茄病毒病的防治奠定基础。【方法】采用RT-PCR扩增,选取新疆加工番茄CMV分离物NS0-4 1a复制酶第1 051-1 350bp序列和ToMV分离物SCS-2 130/180ku复制酶第1 920-2 200bp序列,作为干扰片段CR12和To12。通过T克隆载体将CR12和To12连接成拼接片段TC12,构建成含反向重复结构拼接片段的RNAi表达载体pBi35STC12。通过农杆菌介导法,用其转化红番3号加工番茄,经过筛选、PCR检测得到转基因植株。【结果】成功构建了抗CMV和ToMV的RNAi表达载体pBi35STC12,并用其转化番茄植株,从转化的1 500个愈伤组织中获得33株再生植株,利用PCR对再生植株进行检测,显示从33株再生植株中获得了19株转基因植株,转化率为1.26%。【结论】构建了抗CMV和ToMV的RNAi表达载体pBi35STC12,用其转化红番3号加工番茄,获得了转基因植株。[Objective] The purpose of the study was to obtain transgenic processing tomato plant with the resistance to Cucumber mosaic virus （CMV） and Tomato mosaic virus （ToMV） by Agrobacterium-me diated transformation, and improve the prevention and control of virus disease. [Method] The 1 051--1 350 bp sequence of la replicase cloned from CMV isolate NS0-4 and the 1 920--2 200 bp sequence of 130/180 ku replicase cloned from ToMV isolate SCS-2 of Xinjiang processing tomato amplified by RT-PCR were used as interference fragments CR12 and To12. The two fragments CR12 and To12 were spliced into one fragment through pGEM-T easy. The recombinant plant expression vector pBi35STC12 containing inverted repeat fragments was constructed and introduced into processing tomato Hongfan 3 by Agrobacterium-mediated transformation. Then the transgenic plants were identified by PCR. [Result] The RNAi vector pBi35STC12 was successfully constructed and transformed to the processing tomato. 33 regenerated plants were obtained from ] 500 callus of processing tomato Hongfan 3.19 of them were identified to be transgen- ic plants by PCR and the transformational rate was 1.26%. [Conclusion] The RNAi vector pBi35STC12 was constructed and successfully transformed to the processing tomato Hongfan 3.

关 键 词：CMV TOMV RNAI载体 加工番茄 遗传转化 

分 类 号：S332.2[农业科学—作物遗传育种]