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抑制南方水稻黑条矮缩病毒非结构蛋白P5-1的表达阻碍病毒在昆虫体内的增殖

引用本文：曹金强,王针针,陈红燕,毛倩卓,贾东升,魏太云.抑制南方水稻黑条矮缩病毒非结构蛋白P5-1的表达阻碍病毒在昆虫体内的增殖.植物保护学报,2015,42(3):418-424

DOI：10.13802/j.cnki.zwbhxb.2015.03.020

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作者	单位	E-mail
曹金强	福建农林大学植物病毒研究所, 福建省植物病毒学重点实验室, 福州 350002
王针针	福建农林大学植物病毒研究所, 福建省植物病毒学重点实验室, 福州 350002
陈红燕	福建农林大学植物病毒研究所, 福建省植物病毒学重点实验室, 福州 350002
毛倩卓	福建农林大学植物病毒研究所, 福建省植物病毒学重点实验室, 福州 350002
贾东升	福建农林大学植物病毒研究所, 福建省植物病毒学重点实验室, 福州 350002
魏太云	福建农林大学植物病毒研究所, 福建省植物病毒学重点实验室, 福州 350002	weitaiyun@fafu.edu.cn

中文摘要:为明确南方水稻黑条矮缩病毒(Southern rice black-streaked dwarf virus,SRBSDV)编码的非结构蛋白P5-1参于SRBSDV在介体白背飞虱体内侵染过程中的作用机制,通过原核表达蛋白制备SRBSDV编码的非结构蛋白P5-1的多克隆抗体,并应用Western blot和免疫荧光标记法检测抗体的特异性,以注射法将来源于P5-1基因的dsRNA(dsP5-1)注入获毒1 d的白背飞虱体内,5 d后通过免疫荧光标记法检测dsP5-1对SRBSDV在白背飞虱体内增殖的影响,同时以注射来源于GFP基因的dsRNA(dsGFP)为对照.结果显示,Western blot和免疫荧光标记分别检测到SRBSDV侵染水稻和白背飞虱表达的P5-1蛋白,表明所制备的P5-1抗体具有特异性.dsGFP处理的对照组白背飞虱带毒率高达81%,而dsP5-1处理的白背飞虱带毒率仅为21%,且P5-1蛋白的表达和SRBSDV在昆虫体内的增殖均受到抑制.表明P5-1蛋白是病毒在昆虫体内增殖的关键因子,可作为阻断病毒在昆虫体内增殖的理想靶标.

中文关键词:南方水稻黑条矮缩病毒 P5-1蛋白抗体制备 RNA干扰增殖

Knockdown of nonstructural protein P5-1 of Southern rice black-streaked dwarf virus prevents viral multiplication in vector insects

Author Name	Affiliation	E-mail
Cao Jinqiang	Fujian Province Key Laboratory of Plant Virology, Institute of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian Province, China
Wang Zhenzhen	Fujian Province Key Laboratory of Plant Virology, Institute of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian Province, China
Chen Hongyan	Fujian Province Key Laboratory of Plant Virology, Institute of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian Province, China
Mao Qianzhuo	Fujian Province Key Laboratory of Plant Virology, Institute of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian Province, China
Jia Dongsheng	Fujian Province Key Laboratory of Plant Virology, Institute of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian Province, China
Wei Taiyun	Fujian Province Key Laboratory of Plant Virology, Institute of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian Province, China	weitaiyun@fafu.edu.cn

Abstract:To investigate the function of the nonstructural protein P5-1 of Southern rice black streaked dwarf virus (SRBSDV) in the insect vector white-backed planthopper (WBPH), Sogatella furcifera (Horváth),here the prokaryotic expression of P5-1 was performed, followed by the preparation of polyclonal antibody against P5-1. Then the specificity of polyclonal antibody was tested by western blot and immunofluorescence. WBPH was microinjected with dsRNA targeting P5-1 gene (dsP5-1) of SRBSDV or dsRNA targeting GFP gene (dsGFP) at day one after virus acquisition. Five days later, the effect of dsP5-1 on viral mulitiplication in the insect vector was examined using immunofluorescence assay. The results showed that the protein P5-1 was detected in infected rice plants using western blot, and the specificity of polyclonal antibody against P5-1 was confirmed in viruliferous WBPH by using immunofluorescence. The rate of viruliferous insects in the control was 81%, while that of dsP5-1 microinjection was 21%. dsP5-1 significantly inhibited the expression of P5-1 and multiplication of SRBSDV in the body of WBPH vector. These results suggested that P5-1 was the key factor influencing viral multiplication in insect vector, which may serve as a target for blocking viral multiplication in WBPH.

keywords:Southern rice black streaked dwarf virus P5-1 protein antibody preparation RNA interference multiplication

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