杀虫剂胁迫下白背飞虱与南方水稻黑条矮缩病毒互作的行为机制与分子机理

Southern rice black-streaked dwarf virus (SRBSDV) was a new kind of plant virus, which seriously damaged the growth and production of rice at Southeast asia. Some measures of "prevention virus disease by controlling media pests" were used to prevent and control plant virus disease. Nevertheless, the behavior mechanism was not clear that catching virus, transmitting virus, the incubation period about WPBH treated by insecticides, and molecular mechanism was not clear about interaction between WBPH and SRBSDV at the stress of insecticides. In this study, LD5, LD25 and LD50 of pymetrozine, imidacloprid, buprofezin and ethofenprox to WBPH at the stress of SRBSDV were determined, respectively. The biology of catching virus, transmitting virus, the incubation period about WPBH treated by insecticides was also studied. Based on this, dynamic distribution of 13 ORF from 10 genomes against SRBSDV from WBPH was studied using Real time PCR and the identification. The differential expression of proteome and protein identificaiton about WBPH was also studied using iTRAQ technology and 2-DE combined with bio-informatics. The interaction mechanism about WBPH and SRBSDV was studied using YSTH, BiFC, crystallology and Biacore in vivo and in vitro, respectivly.

南方水稻黑条矮缩病毒是当前严重为害东南亚稻区水稻种植的一种新型病毒，目前生产上主要采用"治虫防病"的综合措施来防治该病害，但仍然有两个方面的科学问题亟待解决。1）杀虫剂的胁迫下白背飞虱获毒、传毒和病毒循回期等行为机制是否发生改变？2）杀虫剂、白背飞虱和南矮病毒之间的互作的分子机理如何？本研究针对吡呀酮等候选杀虫剂，研究其对携带南矮病毒的白背飞虱的LD5、LD25和LD50，并研究杀虫剂胁迫作用下，白背飞虱对南矮病毒的获毒、传毒、病毒循回期等生物学数据。在此基础上，采用Real time PCR研究南矮病毒各个基因组在白背飞虱体内的含量动态分布，采用核素标记相对定量技术和2-ＤＥ结合生物信息学技术，研究杀虫剂、南矮病毒和白背飞虱的互作机制。采用YHTS和BiFC研究白背飞虱和南矮病毒在杀虫剂胁迫下的互作机制，采用复合物晶体培养和结构解析、Biacore等方法研究互作蛋白在体外的作用模式。

南方水稻黑条矮缩病是我国及越南等东南亚稻区水稻的重要病毒病。该病害潜隐性强、危害重、防控难度大，对水稻种植构成极大威胁。大量研究表明，杀虫剂的使用不仅是控制媒介害虫、减低害虫基数的有效措施；同时，它还能改变媒介昆虫生理代谢，影响媒介昆虫获毒、传毒、病毒在媒介昆虫体内的循回期等行为，改变媒介昆虫与病毒间的互作关系。本研究针对噻嗪酮等候选杀虫剂，研究其对携带SRBSDV的白背飞虱的杀虫活性。在此基础上，采用Real time PCR建立SRBSDV 13基因组的定量检测方法，并研究病毒在白背飞虱体内和水稻体内的含量动态分布。建立label free蛋白质组方法检测SRBSDV编码蛋白的方法，并研究SRBSDV在白背飞虱和水稻寄主体内的动态分布，发现了高丰度和低丰度表达的病毒蛋白。例如，P2、P5-1、P4、P8、P7-1 和P6 在水稻体内为高丰度，P5-2、P7-2、P9-2在水稻体内为低丰度。采用label free 蛋白质组学结合生物信息学技术，研究杀虫剂胁迫下SRBSDV和带毒的白背飞虱的互作机制。生物信息学分析发现WBPH肠道蛋白VTVPV是噻嗪酮胁迫下SRBSDV与WBPH互作的重要蛋白。在对VTVPV蛋白进行结构等相关生物信息学分析和预测的基础上，我们采用YHTS和BiFC证实了VTVPV和SRBSDV P10存在蛋白间的互作。ITC试验表明，VTVPV与P10存在分子间的互作，其解离常数（Kd）为7.24 μM。共结晶进一步证实了VTVPV和SRBSDV P10之间存在互作。