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中国农学通报

中国农学通报 ›› 2011, Vol. 27 ›› Issue (4): 144-147.

• 林学 园艺 园林 • 上一篇    下一篇

Gateway技术快速构建百合双价抗病毒RNAi表达载体

徐品三 白建芳 刘纪文 李焕改   

  • 收稿日期:2010-09-03 修回日期:2010-11-02 出版日期:2011-02-10 发布日期:2011-02-10
  • 基金资助:

    国家自然科学基金

Construction of LSV and LMoV Binary Virus Resistant RNAi Vector Using Gateway Technology

  • Received:2010-09-03 Revised:2010-11-02 Online:2011-02-10 Published:2011-02-10

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摘要：

为获得适合转化百合的双价RNAi表达载体，以感病百合叶片的总RNA为模板，分别扩增400 bp左右的LSV和LMoV CP基因。通过重叠延伸PCR技术获得长约800 bp的LSV-LMoV融合基因。利用Gateway技术，通过BP反应及LR反应将LSV-LMoV融合基因克隆到双元载体pH7GWIWG2(Ⅱ)，成功构建了含两种病毒基因的RNAi表达载体pH7GWIWG2(Ⅱ)-LSV-LMoV。将构建好的双价表达载体导入农杆菌EHA105，PCR和测序表明所构建的载体及获得的工程菌与预期的完全一致。

关键词: 干旱监测评估, 干旱监测评估, 内蒙古地区, 服务系统

Abstract:

In order to gain the RNA interference (RNAi) transformation vector of Lily, two fragment genes about 400 bp of LSV and LMoV were obtained from the total RNA of Lily leaves infected. LSV-LMoV fusion gene was constructed by recombinant PCR technique. Using Gateway cloning technology, the RNAi transformation vector pH7GWIWG2 (Ⅱ) was constructed by BP and LR recombination reactions. PCR and sequencing analysis confirmed that the pH7GWIWG2 (Ⅱ)-LSV-LMoV vector, a binary vector containing two different virus genes, was obtained successfully. Virus susceptible Lily was transformed with pH7GWIWG2 (Ⅱ)-LSV-LMoV by Agrobacterium tumefaciens-mediated transformation.

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