谷子抗锈基因Rusi1介导的抗病反应相关元件的筛选、鉴定与功能研究

Rust is an important fungal disease in foxtail millet, and there were plant lodging and no harvest when the disease is epidemic. Planting resistant varieties is the most economical and effective strategy to reduce damage caused by rust. But resistance loss occurs easily in the application of rust resistant varieties. Therefore, it is of great significance to clone rust resistance genes and reveal the resistance mechanism for rapid breeding of rust-resistant varieties and prolonging the service life of the varieties. In the project, based on the cloning of Rusi1, the rust resistant candidate gene in foxtail millet, Shilixiang (SLX), we will do research on the Rusi1-mediated early immune mechanism at RNA omics level. The transcriptome sequencing (RNA-seq) and small RNA sequencing of the leaves of SLX and Yugu1 (susceptible) collected from different time points (0h, 8h, 12h, 16h, 24h and 36h) after rust inoculation were separately carried out. The aim is to screen the differentially expressed components (genes and sRNAs) involved in Rusi1-mediated disease resistance response at the early stage to the greatest degree, and further to verify the functions of the identified important components with the agrobacterium-mediated transformation of foxtail millet. The study will lay the foundation for further understanding the rust resistance mechanism, and provide candidate genes for the molecular breeding of rust resistant varieties and the sustainable utilization of the rust resistance gene in foxtail millet.

锈病是谷子上重要病害，流行年份植株倒伏、颗粒无收。生产中利用抗锈品种是最经济有效的防治方法。但抗锈品种应用中易出现抗性丧失现象，因此，克隆抗锈基因、揭示抗锈机理对快速培育抗锈品种，延长品种使用寿命具有重要意义。本项目是在谷子十里香抗锈候选基因Rusi1已克隆的基础上，拟在RNA组学层面进一步对该基因介导的早期抗锈病机理展开研究。对十里香与豫谷1号（感病）接种谷锈菌后不同时间点（0h、8h、12h、16h、24h和36h）的叶片材料进行转录组测序（RNA-seq）和小RNA测序，高通量的筛选鉴定Rusi1介导的早期抗锈病反应的差异表达元件（基因和sRNAs），并对重要的抗病相关元件进行谷子转基因功能验证。一方面，从分子水平上揭示谷子抗锈的分子调控途径，另一方面，为开展谷子抗锈分子育种及抗锈基因可持续利用提供候选基因。

谷锈病（Uromyces setariae-italicae Yoshino）是谷子上重要的气传流行性病害，利用抗病品种是最经济、有效的防治方法，谷锈菌群体毒性的改变是导致谷子抗锈品种丧失抗性的主要原因，因此，克隆谷子抗锈病基因，揭示抗锈病的分子调控途径，挖掘并利用起关键调控作用的抗病相关基因，对延长品种使用寿命具有重要意义。因此，本项目以谷子高抗锈病资源十里香为研究对象，在RNA组学层面对谷子早期抗锈病机理展开研究，取得了以下主要进展：（1）利用转录组测序和sRNAs测序，系统鉴定到谷子抗锈病相关的基因149个、miRNAs 78个、miRNA与靶基因表达呈负相关的“miRNAs-靶基因”对49个，为谷子抗锈病分子机理研究提供了候选元件；（2）转录组分析和抗病相关基因的表达研究表明，谷子早期抗锈病反应是抗病、感病反应共有的基础免疫反应信号通路和抗病反应特异的信号通路共同作用的结果；较感病反应相比，上调表达的免疫相关元件在抗病反应中大部分表现出更早的、更剧烈的、更持久的诱导表达；real-time PCR鉴定到SiMYB041、SiMYB074、SiMYB100、SiMYB177、SiMYB202、SiMPK3、SiMPK4-1、SiMPK17-1的表达与谷子抗锈病相关，其中SiMYB074、SiMYB100和SiMYB202可能通过SA和JA信号途径协同参与谷子的早期抗病反应，SiMPK4-1可能通过JA信号途径正调控谷子对谷锈菌的抗性；首次提出了谷子早期抗锈病反应的分子路径模式图，初步解析了谷子抗锈病分子机理。（3）通过转基因功能验证，获得2个具有抗锈病功能的抗病相关基因SiTPS27和SiPK6，为谷子抗锈病分子育种提供了候选基因。