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

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.
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.