Osa_miRNAs在水稻-稻曲病菌互作中的调控机制研究

Based on the large-scale growing of high-yielding hybrid cultivars and the change of rice farming system, rice false smut (RFS) has occurred severely in major rice-growing regions and become one of the top three rice diseases. Up to date, no major resistant gene has been detected, and breeding resistant cultivars by traditional methods cannot be realized in the short term. Increasing evidences showed that miRNAs play important roles in plant immunity against pathogens. Thus, it is possible to obtain resistant cultivars through regulating the expression of genes associated with resistance by controlling the expression of certain Osa_miRNAs. Recently, we made transcriptome analysis and screened out 3 Osa_miRNAs that showed different expression patterns in resistant and susceptible rice cultivars after inoculated with Villosiclava virens. In current study, the regulation of Osa_miRNAs on expression of the predicted targets will be analyzed by using 5’-RLM-RACE method and further evaluated in Nicotiana benthimiana.

随着水稻栽培模式及品种布局的改变，我国稻曲病逐年加重，已成为新的水稻三大病害之一。水稻对稻曲病菌的抗性资源有限，至今未发现主效抗性基因，利用传统的育种方法培育抗病品种短期内难以实现。越来越多的证据表明，植物与病原菌互作过程中，miRNAS起着重要调控作用。利用现代分子技术控制水稻抗病相关Osa_miRNAs的表达，可调控水稻抗病基因的表达从而增强水稻对病原菌的抗性，有望短期内获得抗病品种。本项目在前期筛选到抗性相关Osa_miRNAs的基础上，将通过5’-RLM-RACE分析及烟草共表达系统就Osa_miRNAs对预测靶标基因的调控进行验证，鉴定出参与水稻抗稻曲病的Osa_miRNAs。

本项目选取致病力强的稻曲病菌YC11-28菌株接种于水稻抗性品种IR28和敏感品种晚籼98，分别收集接种0、3、5天的水稻穗部样品，挑选受侵染小花提取RNA，采用高通量测序技术完成了小RNA深度测序，总计检测出1856个sRNAs,其中，水稻中已知Osa_miRNAs 51个（miRBase中报道），预测得到新的Osa_miRNAs1778个，稻曲病菌中预测到27个miRNAs。经过表达量分析，在水稻的1778个预测Osa_miRNAs中，筛选出在抗/感水稻品种中差异表达的Osa_miRNAs共119个，其中51个Osa_miRNAs在敏感水稻晚籼98中上调表达，在抗性水稻中下调表达，其余68个Osa_miRNAs在敏感水稻晚籼98中下调表达，在抗性水稻中上调表达。目前，已挑选出4个接种后3天在抗/感水稻品种中表达量差异较大(差异5倍以上，与对照0天相比)的Osa_miRNAs 以及7个接种后5天在抗/感水稻品种中表达量差异较大(差异7倍以上，与3天相比)的Osa_miRNAs。根据以上Osa_miRNAs，从水稻中预测出了37个与抗病相关的靶标基因。此外，本课题组已经通过农杆菌注射接种在烟草中瞬时表达的方法，建立起验证miRNA调控靶标基因的成熟体系。