辣椒脉斑驳病毒侵染性克隆构建的简易方法
Simple methods for constructing infectiouscDNA clone of ChiVMV
作者：刘 健(四川大学生命科学学院)；赵霏霏(四川大学生命科学学院)；陈应娟(四川大学生命科学学院)；吴阳晨(四川大学生命科学学院生物资源与生态环境教育部重点实验室)；朱 桐(四川大学生命科学学院, 能源植物生物燃油制备及利用国家地方联合工程实验室)；席德慧(四川大学生命科学学院, 能源植物生物燃油制备及利用国家地方联合工程实验室)；林宏辉(四川大学生命科学学院, 能源植物生物燃油制备及利用国家地方联合工程实验室)
Author：LIU Jian(College of Life Sciences, Sichuan University)；ZHAO Fei Fei(College of Life Sciences, Sichuan University)；CHEN Ying Juan(College of Life Sciences, Sichuan University)；WU Yang Chen(Key laboratory of Bio resources and Eco environment of Ministry of Education,College of Life Sciences, Sichuan University)；ZHU Tong(National Local Joint Engineering Laboratory for Energy Plant Bio oil Production and Application, College of Life Sciences, Sichuan University)；XI De Hui(National Local Joint Engineering Laboratory for Energy Plant Bio oil Production and Application, College of Life Sciences, Sichuan University)；LIN Hong Hui(National Local Joint Engineering Laboratory for Energy Plant Bio oil Production and Application, College of Life Sciences, Sichuan University)
收稿日期：2014-04-21 年卷（期）页码：2015,52(5):1151-1156
期刊名称：四川大学学报: 自然科学版
Journal Name：Journal of Sichuan University (Natural Science Edition)
关键字：辣椒脉斑驳病毒; 侵染性克隆; 体外转录; 重叠PCR
Key words：Chilli veinal mottle virus; Infectious clone; In vitro transcription; Overlapping PCR
基金项目：国家自然科学基金(31171835, 31270290); 四川省应用基础项目(2012JY0078); 中央高校基本科研业务费(2011SCU04B34)
中文摘要
本文介绍了一种构建辣椒脉斑驳病毒(Chilli veinal mottle virus , ChiVMV)侵染性克隆的简易方法. 基于辣椒脉斑驳病毒的全长确定序列, 根据基因内部的单一限制性酶切位点, 设计引物, 将ChiVMV分成KL1,KL2, KL3三个片段通过RT PCR和TA克隆的方法将3个片段构建到PMD19 T载体上. 通过双酶切将ChiVMV前面7000bp的片段构建到载体上, 从而将全部的ChiVMV cDNA分段构建到两个重组质粒中, 成功地避免了全长病毒序列在大肠杆菌中不稳定的现象. 进行体外转录时, 先通过PCR扩增出两个彼此有一定重叠的平末端片段, 再利用两个重叠片段为模板用重叠PCR的方法扩增出全长带T7启动子的ChiVMV cDNA, 并通过T7 RNA聚合酶成功转录出ChiVMV的病毒RNA.
英文摘要
In this study, a simple method for constructing Chilli veinal mottle virus (Chilli veinal mottle virus, ChiVMV) infectious clone was described. Primers were designed based on single restriction sites inside the gene of full length ChiVMV determined sequences, and divided ChiVMV into KL1, KL2, KL3 three fragments. These three fragments were built into the PMD19 T vector by RT PCR and TA cloning. Then the front 7000bp fragments of ChiVMV were construct to the vector by restriction enzyme digestion. So that the entire ChiVMV cDNA were segmented constructed to two recombinant plasmids and successfully avoided the phenomenon of full length viral sequence instability in E. coli. When in vitro transcription proceed, firstly two blunt end fragment overlap each other amplified by PCR . Then with two overlapping fragments used as template, full length ChiVMV cDNA with the T7 promoter was amplified by overlapping PCR. Finally the viral RNA of ChiVMV was successfully transcribed in vitro by T7 RNA polymerase.
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