白背飞虱中肠内与SRBSDV P6蛋白互作的介体因子鉴定及功能分析

Southern rice black-streaked dwarf virus (SRBSDV) is transmitted by white-backed planthopper (WBPH; Sogatella furcifera Horváth) in a persistent-propagative manner. During SRBSDV infection in its insect vector, viroplasm, putative sites of viral replication, contained the nonstructural viral protein P5, P6, P9-1 and insect vector proteins. The P6 is innitially expressed, and interacts with P5 and P9-1 in insect vector, respectively. All these results demonstrated that P6 was essential for viroplasm formation and viral replication. In this study, yeast two hybrid experiments will be used to identify insect proteins interacting with SRBSDV P6 in the midgut. Gene clone, subcellular localization and RNA interference will be used to know about how insect proteins regulate SRBSDV form viroplasm in the midgut of insect vector. Results of this study will be useful in revealing the replication mechanisms of SRBSDV in its insect vector, which will facilitate the development of effective control measures against Southern rice black-streaked dwarf disease.

南方水稻黑条矮缩病毒（SRBSDV）由白背飞虱以持久增殖型方式进行传播。该病毒在介体昆虫内能够诱导形成病毒复制工厂—病毒原质，主要由病毒编码的P5、P6、P9-1蛋白及其介体因子参与形成。其中，P6蛋白在病毒侵染过程中较早表达，且分别和P5、P9-1蛋白进行特异性互作，在病毒原质形成过程中发挥重要作用。本项目拟以SRBSDV P6蛋白为主要诱饵蛋白，通过酵母双杂交钓取白背飞虱中肠内与病毒复制相关的介体因子。利用介体靶标蛋白的基因克隆，亚细胞定位和RNA干扰等技术弄清介体因子调控SRBSDV在白背飞虱中肠形成病毒原质的分子机制，为明确SRBSDV在昆虫体内的复制机理奠定基础。

本项目主要开展了南方水稻黑条矮缩病毒（SRBSDV）蛋白和介体白背飞虱蛋白的互作。计划书内选择SRBSDV P6蛋白为靶标，筛选鉴定白背飞虱体内和其互作的介体因子，但在项目实施过程中，未探寻到具有功能性的介体蛋白与SRBSDV P6蛋白互作。随后以SRBSDV P10外壳蛋白为靶标，筛选到昆虫体内一类伴侣蛋白PrefoldinⅡ能够和P10互作，并阐明了互作机制。本项目间接证明了SRBSDV在昆虫细胞内的复制主要由自身编码的几种蛋白参与，介体因子未参与病毒的复制过程，为田间早期防治该病毒病害提供指导思路（主要抑制病毒编码的复制蛋白表达）。本项目进一步明确了白背飞虱PrefoldinⅡ伴侣蛋白能够和P10蛋白进行互作，能够和P10 蛋白形成聚集包涵体运输到蛋白酶体，从而抵抗病毒的侵染。研究结果为南方水稻黑条矮缩病的科学防控提供了重要的理论基础。