两步法构建灰飞虱RNAi载体及水稻遗传转化  

Construction of RNA Interference Vector of Laodephax striatellus with A Two-step Method and Its Transformation in Rice

作　　者：王少岭[1] 杜强 尹鲜思 金如意 王志龙[1] 

机构地区：[1]湖南省作物种质创新与资源利用重点实验室/湖南农业大学农学院,长沙410128

出　　处：《作物研究》2015年第3期230-234,331,共5页Crop Research

基　　金：国家转基因生物新品种培育重大专项(2012ZX08009001);教育部"创新团队发展计划"项目(IRT1239);湖南省高校科技创新团队项目(2012)

摘　　要：灰飞虱不仅直接危害水稻,还是水稻条纹叶枯病和黑条矮缩病的传播媒介。为了探索防治灰飞虱及由灰飞虱引起的水稻病毒病的方法,本研究利用两步法快速高效构建灰飞虱RNAi载体,并应用于水稻遗传转化。针对灰飞虱的激活性蛋白激酶C受体(RACK)基因设计了一对特异性引物,通过PCR扩增一段200 bp的干扰片段,将干扰片段克隆到入门载体p ENTR,再经过LR反应使入门载体上的干扰片段整合入目的载体p ANDA,形成RNAi载体p ANDA-RACK,通过农杆菌EHA105介导的水稻转化法转入武陵粳1号,获得20株经鉴定为阳性的转基因植株,为防控灰飞虱及其传播的水稻病毒病提供了珍贵材料。Laodephax striatellus could not only damage rice directly, but also works as media to spread Rice Stripe Disease and Rice Black Streaked Dwarf Disease. To explore techniques for controlling Laodephax striatellus and rice virus diseases caused by it, RNAi vector was efficiently and rapidly constructed by a two - step method. One pair specific primer was de- signed for RACK gene from Laodephax striatellus to amplify 200 bp interference fragment, and then cloned it into entry vec- tor pENTR. Then the RNAi fragment was recombined into destination vector pANDA through LR reaction to form RNAi vector pANDA -RACK, which was transformed into rice cuhivar Wulingjing No. 1 mediated by Agrobacterium tumefaciens EHA105. Twenty transgenic plants were generated and identified by PCR, which plays a foundation against Laodephax stria- teUus and its transmitted virus diseases in rice.

关 键 词：水稻 灰飞虱 RNAI载体构建 转基因 

分 类 号：S511.03[农业科学—作物学] Q78[生物学—分子生物学]