灰飞虱微管蛋白及泛素融合蛋白在水稻条纹病毒克服唾液腺屏障中的功能解析

The small brown planthopper (SBPH) transmit rice stripe virus（RSV） in a persistent propagative manner. Salivary glands barrier is an important barrier in the transmission process. However, the mechanism that SBPH makes RSV overcome salivary glands barrier is not clear. In previous experiments, tubulin (LsTUB) and ubiquitin fusion (LsUBI) proteins were identified up-regulated in the viruliferous SBPH strains. After respectively RNA interference of the two proteins, the RSV transmission rates of SBPH were significantly decreased though the feeding behavior showed on difference. Based on the previous study, this project was carried out as following in order to explore the function of target proteins in RSV transmission. First, we will observe the subcellular localization of LsTUB and LsUBI. Then, we will detect the amount of RSV conquered salivary glands barrier after the target proteins interfered. Finally, we will further screen the interaction proteins of target proteins in RSV. The expected results of this project can clarify that LsTUB and LsUBI interacted with RSV and transfer it through salivary glands barrier via transmembrane transport. This project can both enrich the theoretical system of the interaction between persistent-propagatively transmitted virus and the vector, and also provide new ideas for the the prevention of virus disease by controlling the vectors.

灰飞虱（SBPH）以循回增殖的方式传递水稻条纹病毒（RSV），唾液腺屏障是传毒过程的重要屏障。然而，灰飞虱致使体内病毒穿过唾液腺屏障的机制尚不清晰。前期从带毒灰飞虱唾液腺中鉴定出两个上调表达的蛋白——微管蛋白和泛素融合蛋白。这两种蛋白的表达被干扰后，均不影响介体取食行为却显著降低介体传毒率。本项目在此基础上，为探索目的蛋白在病毒克服介体唾液腺屏障时的功能，着重开展以下研究：（1）完成目的蛋白的亚细胞定位；（2）目的蛋白表达被干扰后，检测穿过唾液腺的病毒量变化；（3）筛选目的蛋白在RSV中的互作蛋白。本项目预期结果能明确微管蛋白及泛素融合蛋白直接与病毒互作，并以跨膜运输的方式传递病毒使其克服介体唾液腺屏障。本项目既能丰富循回型病毒与介体互作的理论体系，亦能为介体传播病毒病的防治提供新思路。

水稻害虫灰飞虱以持久循回增殖的方式传播水稻条纹病毒病，严重危害了水稻生产。灰飞虱的唾液腺屏障是该传毒过程的重要屏障之一。然而，病毒穿过唾液腺屏障的机制尚不清晰。前期从带毒灰飞虱唾液腺中鉴定出两个上调表达的蛋白——微管蛋白和泛素融合蛋白。本研究通过RACE-PCR技术获得了微管蛋白（LsTUB）和泛素融合蛋白（LsUbi）的cDNA全长序列。利用荧光共聚焦显微镜观察到LsTUB与RSV在唾液腺、中肠和卵巢中共定位。随后Q-PCR实验表明，抑制LsTUB的表达会导致RSV载量降低和分布减少。进一步通过酵母双杂交和pull-down验证了LsTUB与RSV NS3的直接互作和两者的互作位点。BiFc实验检测烟草中RSV NS3沉默抑制活性，明确LsTUB的存在并不影响RSV NS3的沉默抑制活性。在RSV侵染的Sf9细胞实验中发现差异化表达LsTUB并不会影响RSV的复制。最终，证实抑制LsTUB显著阻碍RSV从最初感染的中肠细胞扩散到SBPHs的唾液腺中。对与LsUbi的研究，同样证实抑制LsUbi的表达会导致RSV载量降低和分布减少。然而酵母双杂交发现LsUbi与RSV的蛋白不存在直接互作关系。转而研究发现泛素系统关键蛋白泛素连接酶LsE3 Ring与RSV NS3有互作关系。因此，以上结果揭示灰飞虱LsTUB与RSV的非结构蛋白NS3互作促进其组织间扩散和水平传播；而推测灰飞虱LsUbi可能通过泛素结合酶LsE3 Ring结合RSV NS3促进病毒传播。本研究成果为丰富植物病毒和介体之间相互作用知识体系，并且为研发植物治虫防病的新策略提供理论指导。