利用酵母同源重组系统快速构建Potyvirus病毒侵染性克隆方法  

A Simple and Rapid Method to Construct Potyvirus Infectious Clone by Yeast Homologous Recombination System

作　　者：赵光远[1,2] 庹德财[1,3] 沈文涛[1] 黎小瑛[1] 周鹏[1,2,3] 

机构地区：[1]中国热带农业科学院热带生物技术研究所,中国海南海口571101 [2]海南大学环境与植物保护学院,中国海南海口570228 [3]海南大学农学院,中国海南海口570228

出　　处：《生命科学研究》2015年第6期491-496,共6页Life Science Research

基　　金：国家自然科学基金资助项目(31171822;31371918)

摘　　要：前一个时期,虽然分子生物学技术有了相当大的进步,但在构建基因全长较大的RNA病毒时,例如马铃薯Y病毒属全长侵染性cDNA克隆或者是RNA体外转录物时,会遇到很大的困难,其cDNA转入大肠杆菌(E.coli)感受态细胞时,可能会产生对宿主菌有毒性的物质,从而导致非特异性重组。这些问题可以利用插入内含子等方法来解决,但这些方法大多耗时费力且成功率低。以番木瓜环斑病毒(Papaya ringspot virus,PRSV)为例,介绍了在不插入内含子的情况下利用酵母同源重组系统在两周之内构建完成其侵染性克隆的方法。Obtaining infectious clones often remains tedious, time-consuming and rather unpredictable, despite significant advances in molecular biology techniques during the last decades. In the case of Potyvirus＇ es, cDNA clones are frequently unstable due to the toxicity of some viral proteins for bacteria. The problem can be solved by inserting introns into the viral sequence, which requires additional steps in the cloning process and depends on the availability of suitable restriction sites in the viral sequence or adjudication of such sites by mutagenesis. For example with Papaya ringspot virus （PRSV）, it will be described how to construct the infectious clones or other viral eDNA vectors without insert introns by using yeast homologous recombination system in two weeks.

关 键 词：马铃薯Y病毒属 番木瓜环斑病毒 酵母同源重组 穿梭质粒 侵染性克隆 

分 类 号：Q782[生物学—分子生物学]