利用GATEWAY^(TM)技术快速构建番木瓜环斑病毒HC-pro基因的RNAi表达载体  

Construction of a RNAi Expression Vector of PRSV-HC-pro Gene by GATEWAY^(TM) Technology

作　　者：高艳梅[1] 翟金玲[1] 黄惜[1] 

机构地区：[1]海南大学生物科学技术研究所,海南570228

出　　处：《热带作物学报》2009年第4期505-509,共5页Chinese Journal of Tropical Crops

基　　金：海南省教育厅高校科研项目(No.Hj2008-09);海南自然科学基金(No.808109)资助

摘　　要：通过RT-PCR扩增番木瓜环斑PRSV病毒HC-pro基因,序列比对结果表明,克隆到的该基因片段与已知的海南或华南地区已克隆的HC-pro基因只有约92%的同源性,说明PRSV病毒具有较大的变异性。为了培育番木瓜转基因抗病植株,利用pWATERGATE载体和GATEWAYTM技术,成功构建了番木瓜环斑病毒HC-pro基因的RNAi表达载体。具体步骤如下:①设计带特异性重组序列attB的HC-pro基因PCR的引物;②利用该引物进行RT-PCR扩增HC-pro基因;③通过BP反应,将扩增得到的HC-pro基因序列插入入门载体pDONR201中;④通过LR反应将HC-pro基因序列从pDONR201重组到目标载体pWATERGATE中,得到PRSVHC-pro基因的RNAi表达载体。RNA interference (RNAi) is one of the mechanisms, by which plants recognize and degrade exogenous dsRNA (such as viral RNA) . However, some viruses have evolved proteins (such as HC-pro of PRSV) that could efficiently inhibit RNAi of plants. In this context the HC-pro gene of PRSV was amplified through RT-PCR. Sequence alignment analysis showed that the cloned HC-pro gene was 92% homologous to other published HC -pro genes in Hainan or in South China, indicating that the PRSV genome varied. In order to obta...

关 键 词：番木瓜环斑病毒 GATEWAYTM技术 RNAI HC-PRO基因 

分 类 号：S436.67[农业科学—农业昆虫与害虫防治]