利用叶蝉细胞瞬时表达系统研究水稻瘤矮病毒在介体细胞内的增殖机制

昆虫介体传播的动植物病毒严重威胁人畜健康和粮食生产安全。昆虫细胞系结合相应的瞬时表达载体系统，是研究无侵染性克隆病毒在细胞内增殖必不可少的技术手段。目前叶蝉细胞系广泛地应用于植物呼肠孤病毒的研究中，然而缺少叶蝉细胞特异的瞬时表达载体，导致无法更深入地探索水稻病毒在叶蝉细胞内的增殖机制。本项目拟通过定位和克隆黑尾叶蝉actin基因的promoter序列，开发能在叶蝉细胞中高效瞬时表达的载体系统。利用该系统验证RGDV非结构蛋白Pns7、Pns9和Pns12的细胞定位以及它们与主要的结构蛋白P3、P5、P8之间的互作情况。解析RGDV Viroplasm形成的分子机制以及其招募结构蛋白的机理。以期为植物呼肠孤病毒的研究提供新的技术手段，为探索病害的流行规律和制定有效的防控策略提供理论支撑

Animal and plant virus transmitted by Insect severely threaten animal health and crop production safety. In studying replication mechanism of virus without infectious clone, Insect culture cell and its specific transient expression system play an indispensable role. So far, Leafhopper cell line has been widely used in the research of phytoreovirus. However, it became difficult to investigate the molecular mechanism of rice virus replication in leafhopper cell, due to the absence of appropriate transient expression vector. In present proposal, we try to develop a highly efficient transient expression system for leafhopper cell by locating and cloning the promoter sequence of leafhopper action gene. This new system will be used to investigate the cellular location of RGBV non-structure protein Pns7, Pns9, Pns12 and their interaction with RGDV main structure protein P3, P5, P8. The result will lead us to identify the formation mechanism of RGDV Viroplasm and also explain how Viroplasm recruit structure protein, and which will provide a new system for the research of rice virus and theory basis for designing efficient control disease strategies in future.