干扰水稻瘤矮病毒(RGDV)非结构蛋白(Pns12)的表达抑制病毒在介体昆虫培养细胞内的复制  

Knockdown of Nonstructural Protein(Pns12) of Rice gall dwarf virus(RGDV) Inhibits Viral Replication in Insect Vector Cells

作　　者：郑立敏[1] 刘华敏[1] 陈红燕[1] 贾东升[1] 谢联辉[1] 魏太云[1] 

机构地区：[1]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

出　　处：《农业生物技术学报》2014年第11期1321-1328,共8页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金青年基金(No.31200118和No.31300136);福建省自然科学基金面上项目(No.2012J01087和No.2014J01085)

摘　　要：水稻瘤矮病毒(Rice gall dwarf virus,RGDV)属呼肠孤病毒科(Reoviridae)植物呼肠孤病毒属(Phytoreovirus),由介体电光叶蝉(Recilia dorsalis)以持久增殖型方式传播,其基因组第12条片段编码的非结构蛋白(nonstructural protein,Pns12)是提供病毒复制和子代病毒粒体装配场所——病毒原质(viroplasm)的组分之一。为了明确Pns12在RGDV侵染介体电光叶蝉培养细胞中的功能,本研究通过原核表达的Pns12蛋白免疫注射兔(Oryctolagus cuniculus),制备Pns12抗体,并应用免疫荧光标记和RNA干扰(RNA interference,RNAi)技术研究Pns12在介体培养细胞内的定位和参与病毒原质形成的过程。共聚焦显微镜观察到,病毒侵染的细胞中,与荧光素交联的Pns12抗体特异地标记在病毒原质上。干扰Pns12蛋白表达后,可有效地阻碍病毒原质的形成、子代病毒粒体的组装和病毒非结构蛋白Pns12和外壳蛋白P8蛋白的表达,表明Pns12作为病毒原质的组分参与了RGDV在介体培养细胞内的复制。也表明,Pns12可作为理想的靶标用于阻断电光叶蝉携带和传播RGDV。Rice gall dwarf virus（RGDV）, the genus Phytoreovirus in the family Reoviridae, is transmitted by the leafhopper vector（Recilia dorsalis） in a persistent-propagative manner. Nonstructural protein（Pns12）, encoded by segment 12 of RGDV, is one of the components of viroplasm which is the site for viral replication and assembly of progeny virons during viral infection in its insect vector cells. In this study, to investigate the functional role of Pns12 in the formation of viroplasm in its insect vector cells, the polyclonal antibody against Pns12 was prepared and purified, then conjugated to fluorescein isothiocyanate. The immunoglobulin G（IgG） of P8 was purified and conjugated to rhadamine. Immunofluorescence microscopy demonstrated that Pns12 antibodies specifically distributed in the viroplasm matrix during RGDV infection in the vector cells in monolayers （VCMs）, while outer capsid protein P8 were accumulated at the periphery of the viroplasm. The viroplasm increased in size over time. Knockdown of Pns12 by RNA interference （RNAi） induced by dsRNAs, targeting Pns12 gene of RGDV, significantly inhibited the formation of viroplasm compared with＆amp;nbsp;dsGFP, suggesting that RGDV replication was inhibited. Western blot showed that viral Pns12 and P8 expression reduced in the VCMs treated with dsPns12. Thus, the present study indicated that Pns12 of RGDV played an important role in viroplasm formation and viral replication in its insect vector cells. RNAi induced by dsRNAs derived from the viral genes of viroplasm matrix protein may be an ideal tool for inhibiting the infection and transmission of RGDV by leafhopper vectors.

关 键 词：水稻瘤矮病毒(RGDV) 介体昆虫培养细胞 非结构蛋白(Pns12) RNA干扰(RNAI) 

分 类 号：S435.111.4[农业科学—农业昆虫与害虫防治]