水稻锯齿叶矮缩病毒介导DEAD-box RNA解旋酶调控microRNA表达的机制研究

MicroRNAs play a vital role in plant-virus interaction. Our previous study have been indicated that Rice ragged stunt virus (RRSV) highly induced miR319 expression to mediate the development of disease symptoms and facilitate virus infection. Small RNA sequencing has shown that other rice viruses also regulate rice microRNAome. However, the plant virus regulation mechanism of miRNA remains elusive. DEAD-box RNA helicase, a class of protein with highly conserved sequence in animals and plants, is involving in many RNA metabolism pathway and plays a role in regulating human miRNA expression. In this study, the effect of RRSV infection on expression profile of DEAD-box RNA helicases in rice will be analyzed; the mutant group of DEAD-box RNA helicases, which significantly affected by virus infection, will be obtained through Crispr/Cas9 system; finally, using de novo sequencing technologies, the role of DEAD-box RNA helicases in regulating miRNA expression will be determined. This project will further demonstrate the pathogenic mechanism of RRSV and lay the fundation for antiviral molecular breeding.

MicroRNA（miRNA）在植物与病毒互作中发挥重要作用。本实验室前期研究发现水稻锯齿叶矮缩病毒（RRSV）通过诱导miR319介导病害症状形成，促进病毒侵染。Small RNA测序结果显示其它水稻病毒侵染均影响水稻miRNA的表达。然而，病毒如何调控miRNA生成或加工的机制尚不清楚。DEAD-box RNA解旋酶是一类在动植物体内高度保守、参与各种RNA新陈代谢过程且对人内源miRNA具有调控功能的蛋白家族。本项目拟从DEAD-box RNA解旋酶角度出发，分析RRSV对水稻DEAD-box RNA解旋酶家族蛋白表达的调控，利用Crispr/Cas9系统获得受病毒显著影响的DEAD-box RNA解旋酶的突变体群体并通过深度测序技术明确DEAD-box RNA解旋酶对miRNA表达的调控及其调控miRNA表达的策略。该项目将进一步阐释RRSV致病机制，为抗病毒分子育种奠定理论基础。

MicroRNA（miRNA）是一类在植物与病毒互作中发挥重要作用的调控因子。然而，病毒如何调控miRNA生成或加工的机制尚不清楚。DEAD-box RNA解旋酶是一类在动植物体内高度保守、参与各种RNA新陈代谢过程且对人内源miRNA生成具有调控功能的蛋白家族。因此，本项目首先通过比较病毒侵染前后的DEAD-box RNA解旋酶基因的表达量变化筛选出OsRH37作为可能参与RRSV调控miRNA加工的基因。利用Crispr/Cas9系统和水稻遗传转化体系获得了OsRH37的突变体株系和转基因过表达株系，通过表型观察和生物统计发现OsRH37基因的突变体株系在苗期和成熟期均表现出一定的矮化表型，同时通过RRSV接种发现OsRH37基因的突变体株系对RRSV侵染更加敏感。利用qPCR实验进一步明确了OsRH37基因的突变体株系和转基因过表达株系中miRNA的差异表达，最终明确了OsRH37可能作为调控miRNA加工的上游因子参与RRSV的致病过程。本项目进一步阐明了miRNA介导的RRSV致病机制，也初步确定了DEAD-box RNA解旋酶基因参与调控miRNA加工的理论基础，为抗病毒分子育种提供了更多的潜在靶点。