棉花VIGS病毒载体的构建及其在抗病基因功能鉴定的应用  

The construction of VIGS virus vector and its application on identifying disease resistant gene in cotton

作　　者：李建平[1] 郝晓燕[1] 李琴[1] 高升旗[1] 常晓春[1] 陈勋基[1] 足木热木.吐尔逊 陈果[1] 黄全生[1] LI Jian-ping;HAO Xiao-yan;LI Qin;GAO Sheng-qi;CHANG Xiao-chun;CHEN Xun-ji;Zumuremu Tuerxun;CHEN Guo;HUANG Quan-sheng(Research Institute of Nuclear and Biotechnologies,Xinjiang Academy of Agricultural Sciences, Urumqi 830091,China)

机构地区：[1]新疆农业科学院核技术生物技术研究所,乌鲁木齐830091

出　　处：《新疆农业科学》2018年第7期1203-1208,共6页Xinjiang Agricultural Sciences

基　　金：自治区公益性科研院所基本科研业务经费"棉花抗黄萎病相关CDPK家族基因的表达谱绘制及候选基因克隆"(KYGY2016098)~~

摘　　要：【目的】利用农杆菌介导的VIGS基因沉默技术,研究陆地棉抗病相关基因在棉花抗黄萎病中的功能。【方法】利用分子生物学技术构建VIGS病毒载体,建立VIGS病毒诱导沉默体系;利用此体系将棉花中的一个钙依赖蛋白(CDPK)基因,通过农杆菌介导在陆地棉中沉默,根据此基因沉默植株在病原菌环境下的发病情况研究该基因在棉花抗病中的功能。【结果】利用包含有Gh CPK抗病基因片段的VIGS病毒载体的农杆菌侵染棉花子叶,病毒载体上的目的基因片段由RNA聚合酶识别并合成双链RNA(double stranded RNA,dsRNA),dsRNA由Dicer酶识别并于其它RNA形成RNA诱导的沉默复合体(RNA induced silencing complex,RISC),RISC对内源Gh CPK mRNA进行识别和切割作用,内源Gh CPK mRNA降解而发生基因沉默。Gh CPK基因沉默导致棉苗对黄萎病敏感性增加,证明Gh CPK基因参与调控棉花抗病功能。【结论】病毒诱导的基因沉默技术(VIGS)能够快速有效的研究Gh CPKs基因在棉花抗病中的功能。【Objective】The purpose is to identify genes in response to Verticillium dahlia by the virus induced gene silencing(VIGS).【Method】With the construction of VIGS virus(virus induced gene silencing)vector by molecular way,we established an Agrobacterium-mediated virus-induced gene silencing(VIGS)assay in cotton.To study function of GhCPK gene family,a GhCPK gene was silenced in cotton using VIGS assay,then the disease index of the gene-silenced cotton seedlings were investigated when they were challenged with Verticillium dahlia.【Result】By infecting cotton cytoledons with VIGS virus vector containing GhCPK fragment,the GhCPK fragment is identified and catalyzed into dsRNAs.The combination of dsRNAs with other RNA produce RNA induced silencing comlex(RISC)which can target and degrade endogenous GhCPK mRNA and lead to the GhCPK gene silenced.As the result of silencing GhCPK gene in cotton,the GhCPK silenced cottons showed enhanced sensitivity to Verticillium dahlia,which suggested that GhCPK gene may play a role in regulating disease resitance in cotton.【Conclusion】The virus induced gene silencing technology is an efficient way for identifying GhCPKs gene in response to disease stress in cotton.

关 键 词：棉花 钙依赖蛋白基因 VIGS 黄萎病 

分 类 号：S562[农业科学—作物学] S188