以P型PRSV-VPg为模板多位点突变获得W型PRSV-VPg的研究  

Multi-Mutation of VPg Gene from PRSV-P to PRSV-W

作　　者：胡正龙[1,2] 肖旭倩[1,2] 沈文涛[2] 言普[2] 周鹏[1,2] 

机构地区：[1]海南大学,中国海南海口570228 [2]中国热带农业科学院热带作物生物技术研究所,中国海南海口571101

出　　处：《生命科学研究》2009年第2期104-108,共5页Life Science Research

基　　金：国家自然科学基金资助项目(30760134)

摘　　要：以P型PRSV-VPg(Papaya Ringspot Virus,VPggene)基因为模板利用高保真酶通过两对引物(突变4个位点),PCR扩增获得一条276bp和一条141bp的两个小片段.以此为基础,利用长引物延伸和套叠PCR的方法,定点突变剩余5个位点,最后拼接获得W型PRSV-VPg基因.结果表明通过上述方案可以成功改造P型PRSV-VPg基因,获得与W型PRSV-VPg同源的目的基因,成功率为100%.该技术可以绕开传统的通过寻找毒源进行RT-PCR方法和人工合成方法获得病毒基因,其技术操作简单,且节约时间和成本,并在人工合成基因、多位点基因定点突变等方面具有很好的借鉴作用和应用价值.According to the host range specificity, Papaya Ringspot Virus （PRSV） is classified into two types, called type W and P. The VPg gene of PRSV was involved in genome replication. To obtain the VPg gene of PRSV-W, the VPg gene of PRSV-P was modified into a VPg gene of PRSV-W by long primer extension and overlap extension PCR. The results showed that nine sites of VPg gene from PRSV-P were mutated and the VPg gene of PRSV-W was attained successfully. The research was benefit to further investigate both type of PRSV host specificity for infection of papaya.

关 键 词：艘Sy—yPg 基因改造 长引物延伸 套叠PCR. 

分 类 号：Q781[生物学—分子生物学]