Gateway技术快速构建百合双价抗病毒RNAi表达载体  

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

作　　者：徐品三[1] 白建芳[1] 刘纪文[1] 李焕改[1] 

机构地区：[1]大连理工大学生命科学与技术学院,辽宁大连116023

出　　处：《中国农学通报》2011年第4期144-147,共4页Chinese Agricultural Science Bulletin

基　　金：国家自然科学基金"siRNA抑制百合无症病毒(LSV)表达的研究"(30771760)

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400bp左右的LSV和LMoVCP基因。通过重叠延伸PCR技术获得长约800bp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2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。In order to gain the RNA interference（RNAi） transformation vector of lily,two fragment genes about400 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.

关 键 词：百合无症病毒 百合斑驳病毒 RNAI载体 重叠延伸PCR GATEWAY技术 

分 类 号：Q782[生物学—分子生物学]