抗烟草丛顶病转基因烟草的培育  

Constructing Transgenic Tobacco for Against Tobacco Bushy Top Disease

作　　者：张丽芳 赵兴能[4] 马美 施晓东 胡海林 陈海如[4] 莫笑晗[2] Zhang Lifang;Zhao Xingneng;Ma Mei;Shi Xiaodong;Hu Hailin;Chen Hairu;Mo Xiaohan(College of Bioresources and Food Engineering,Qujing Normal University,Qujing,655011;Yunnan Academy of Tobacco Agriculture Science,Kunming,650021;Qujing academy of agricultural sciences,Qujing,655011;College of Plant Protection,Yunnan Agricultural University,Kunming,650201)

机构地区：[1]曲靖师范学院生物资源与食品工程学院,曲靖655011 [2]云南省烟草农业科学研究院,昆明650021 [3]曲靖市农业科学院,曲靖655011 [4]云南农业大学植物保护学院,昆明650201

出　　处：《分子植物育种》2020年第5期1605-1610,共6页Molecular Plant Breeding

基　　金：云南省科技厅青年项目(2018FD080)资助。

摘　　要：烟草丛顶病由烟草丛顶病毒、烟草脉扭病毒、烟草脉扭病毒伴随RNA和烟草丛顶病卫星RNA复合侵染引起的毁灭性病害,为了构建这四种病毒的干扰载体,培育抗病毒烟苗,本研究首先利用PCR扩增、酶切、克隆、连接转化和胶纯化技术分别得到携带酶切序列约200 bp的四种病毒目的片段;其次,用目的片段与携带有对应酶切序列的pENTR?2B入门载体连接、检测和纯化得到携带有目的片段的入门重组载体pENTR?2B-Vims;最后,用入门重组载体与表达载体PK7GW1GW2进行LR反应,经PCR检测得到正确干扰重组载体后,再用叶盘法将目的基因转入烟草叶片中,进一步用组培技术培育组培苗。本研究为解决目前烟草丛顶病防治难题,提供烟草生产发展技术思路和理论依据。Tobacco bushy top disease is a devastating diseases,which are consist of tobacco bushy top virus,tobacco vein distorting virus,tobacco vein distorting virus accompany virus and tobacco bushy top disease satellite RN A.To obtain four RNAi vector and cultivate antiviral tobacco seedlings,this study would use PCR amplification,enzyme digestion,cloning,DNA connection and gel purification to obtain the four virus target fragments with about 200 bp respectively,which connect to pENTR?2B with the same enzyme loci and obtain recombinant pENTR?2B-Virus vector by PCR detection.Lastly,the above four RNAi with destination gene 200 bp vector were obtained by LR reaction with PK7GW1GW2 and pENTR?2B-Virus vector,which were transducted into Nicotiana tabacum K326 by leaf disc method at the same time.To lead to new ideas and theory basis on the control of diseases caused by tobacco virus disease and sustainable development of tobacco production.

关 键 词：烟草丛顶病 RNA干扰 Gateway重组技术 叶盘法 

分 类 号：S435.72[农业科学—农业昆虫与害虫防治]