烟草曲茎病毒来源的siRNA在病毒致病过程中的作用机理解析

目前对双生病毒致病机理的研究多集中于病毒蛋白与寄主蛋白互作，而有关病毒来源的小干扰RNA（vsiRNA）在其致病过程中的功能研究较少。本项目以烟草曲茎病毒（TbCSV）及卫星（TbCSB）为研究对象，拟通过small RNA测序分析来源于TbCSV的vsiRNA特征，以病毒载体介导过表达系统筛选与致病相关的vsiRNA；经生物信息学预测、降解组测序及生物学实验验证其寄主内源靶基因，利用病毒介导基因过表达和沉默系统及转基因技术明确靶基因在病毒侵染中的功能及调控致病性所依赖的生理或信号途径；对产生致病相关vsiRNA的病毒相应位点或寄主靶标切割位点进行突变及病毒挑战接种，最终解析vsiRNA与其靶标互作调控病毒致病性的分子机理。本研究基于vsiRNA角度解析TbCSV的致病机理，可拓展对双生病毒致病机理的认识和研究思路，以期为通过靶向病毒或寄主基因获得抗TbCSV的作物种质材料提供重要依据。

At present, the pathogenesis research of geminiviruses are mainly focus on the interaction betweenviral proteins and hosts, while the involvement of small interfering RNA derived from virus (vsiRNA) in the viral pathogenic process remains deep understanding. In this study, tobacco curly shoot virus (TbCSV) and its betasatellite (TbCSB) will be inoculated to Nicotiana benthamiana, and the characteristics of vsiRNA derived from TbCSV will be analyzed by small RNA high-throughput sequencing. The overexpression system mediated by virus vectors was used to screen the vsiRNA candidates involved in pathogenesis. The target genes will be predicted and confirmed by bioinformatics analysis, degradome sequencing and biological experiments, and function of target genes during virus infection and their roles in the physiological or signal pathways regulation will be clarified by using the virus-mediated gene over-expression and silencing system and transgenic technology. The molecular mechanism of vsiRNA interaction with the target genes in regulating viral pathogenicity will be further analyzed by site mutation of the corresponding viral sites produced vsiRNA or the cleavage sites of target genes and virus inoculation. In the study, the pathogenic mechanism of TbCSV will be uncovered from the perspective of vsiRNA. This will broaden our understanding about geminivirus pathogenesis and provide a novel perspective for producing tobacco or tomato germplasms resistant to TbCSV by targeting virus or host genes.