棉花VIGS病毒载体的构建及其在抗病基因功能鉴定的应用

doi:10.6048/j.issn.1001-4330.2018.07.004

新疆农业科学 ›› 2018, Vol. 55 ›› Issue (7): 1203-1208.DOI: 10.6048/j.issn.1001-4330.2018.07.004

棉花VIGS病毒载体的构建及其在抗病基因功能鉴定的应用

李建平, 郝晓燕, 李琴, 高升旗, 常晓春, 陈勋基, 足木热木·吐尔逊, 陈果, 黄全生

新疆农业科学院核技术生物技术研究所,乌鲁木齐 830091

收稿日期:2018-05-16 发布日期:2018-11-27
通信作者: 黄全生(1964-),男,新疆乌鲁木齐人,研究员,博士,研究方向为作物耐逆机制,(E-mail)hquansheng@126.com
作者简介:李建平(1977-),男,新疆乌鲁木齐人,副研究员,博士,研究方向为植物光信号转导,(E-mail)ljp7786@126.com
基金资助:
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)

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

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

Received:2018-05-16 Published:2018-11-27
Correspondence author: HUANG quan-sheng (1964-), male, Urumqi, Xinjiang, Professor, Ph.D, Ph.D. supervisor, the main research directions for crop adversity molecular biology, (E-mail)hquansheng@126.com
Supported by:
Basic Scientific R & D Program of Public Welfare Research Institutes of Xinjiang Uygur Autonomous Region of China "Expression Profiling of CDPK Family Genes Related to Verticillium wilt Resistance in Cotton and Cloning of Candidate Genes" (KYGY2016098)

摘要/Abstract

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GhCPK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GhCPK mRNA进行识别和切割作用,内源GhCPK mRNA降解而发生基因沉默。GhCPK基因沉默导致棉苗对黄萎病敏感性增加,证明GhCPK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GhCPKs基因在棉花抗病中的功能。

关键词: 棉花; 钙依赖蛋白基因; VIGS; 黄萎病

Abstract: 【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 identifyingGhCPKs gene in response to disease stress in cotton.

Key words: Cotton; CPK gene; VIGS; Verticillium dahlia

中图分类号:

S562
S188

李建平, 郝晓燕, 李琴, 高升旗, 常晓春, 陈勋基, 足木热木·吐尔逊, 陈果, 黄全生. 棉花VIGS病毒载体的构建及其在抗病基因功能鉴定的应用[J]. 新疆农业科学, 2018, 55(7): 1203-1208.

LI Jian-ping, HAO Xiao-yan, LI Qin, GAO Sheng-qi, CHANG Xiao-chun, CHEN Xun-ji, Zumuremu Tuerxun, CHEN Guo, HUANG Quan-sheng. The construction of VIGS virus vector and its application on identifying disease resistant gene in cotton[J]. Xinjiang Agricultural Sciences, 2018, 55(7): 1203-1208.

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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

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