烟草抗马铃薯Y病毒相关miRNA的筛选与功能分析

Potato virus Y (PVY) is one of the most important viral pathogens that infects various commercial crops including potato, tomato and tobacco and causes severe quality and yield losses. Lacking of the molecular mechanism of plant resistance to PVY is now a serious impediment to the disease control and plant resistant breeding. RNA silencing, a conserved gene regulatory mechanism, is also an important antiviral or antibacterial mechanism. We have obtained some local tobacco varieties in Guizhou province that exhibit resistance against PVY, part of them exhibits strong resistance to PVY, which are ideal materials for studying the molecular mechanism of plant-virus interaction. In this project, we will employ the deep sequencing technique to compare the miRNA expression patterns of both resistant and susceptible varieties infected by PVY virus to screen the key miRNAs that may participate in regulation of tobacco resistance to PVY. The miRNA overexpression or deletion vector were constructed and will be reintroduced into tobacco to clarify their function in anti-PVY by measuring the PVY infection timing, symptoms, replication and other indicators. In this research, we will analysis the differences of miRNA expression patterns between resistant and susceptible varieties infected by PVY virus for the first time. This research will lay the foundation for PVY disease control and breeding resistant varies.

马铃薯Y病毒（Potato Virus Y，PVY）是生产上严重影响马铃薯、番茄、烟草等多种作物产量和品质的病毒之一,目前尚无有效的防治方法。RNA 沉默是植物固有的机制，可在转录和转录后水平调节基因的表达，也是天然的抗病毒防御机制。本项目首先通过深度测序对抗病烟草和感病烟草感染PVY后miRNA的表达差异进行系统分析，建立上述2种烟草感染PVY miRNA表达模式，进一步比较分析上述2种材料在PVY感染后miRNA表达模式的差异，筛选差异表达miRNA基因，对筛选的miRNA基因进行靶基因的预测与验证。构建筛选miRNA的超量表达或者敲除载体并转化烟草，通过转基因烟草与对照在接种PVY后发病时间、病毒症状、复制量等指标鉴定候选miRNA基因抗病毒。 本研究首先分析PVY侵染对不同抗性水平烟草miRNA表达模式的差异，在此基础上筛选miRNA并鉴定其功能，为该病害的防治和抗病育种奠定基础。

近年来，PVY（Potato Virus Y，PVY）来在全国的发生及其造成的损失正呈急剧上升的趋势，PVY侵染烟草后可引起烟草黄化、矮化、叶脉坏死等症状，使烟叶失去烘烤价值，造成严重的降质、减产，乃至绝产。RNA 沉默是基因在转录和转录后水平的一种调控手段，也是天然的抗病毒防御机制。因此，利用RNA沉默来进行抗PVY育种是分子育种的新方向。本项目通过深度测序对烟草得抗病品种和感病品种在感染PVY后miRNA的表达差异进行系统分析，建立上述2种烟草感染PVY miRNA表达模式，进一步比较分析了表达模式的差异，筛选差异表达miRNA基因，对筛选的miRNA 基因进行靶基因的预测与验证。为进一步分析差异表达得miRNA的功能，构建筛选miRNA的超量表达以及CRISPR/Cas9敲除载体并转化烟草，通过转基因烟草与对照在接种PVY后发病时间、病毒症状、复制量等指标鉴定了候选miRNA抗病毒功能。本项目共对10个miRNA进行了超量表达，5个miRNA进行了基因敲除，并分别研究了它们对PVY抗性的作用。. 此外，对感病材料感染PVY后的转录组和降解组进行了测序，通过转录组和降解组数据的联合分析，发现烟草翻译控制肿瘤蛋白(translational controlled tumor protein, TCTP)是PVY产生的小RNA的靶基因，并证明该基因是一个新的抗PVY基因。该基因的发现不仅打破了国外对PVY抗性基因知识产权的封锁，也为马铃薯、辣椒等其他茄科植物的抗病育种提供新的思路。. 本项目分析了PVY 侵染对不同抗性水平烟草miRNA表达模式的差异，在此基础上筛选miRNA 并鉴定其功能。利用多组测序的数据的联合分析，鉴定了烟草中的新的抗病基因。因此，本研究加深了烟草与PVY互作的分子机制，为该病害的防治和抗病育种奠定基础。