水稻毫糯扬抗白叶枯病新基因的克隆及作用机理分析

Rice bacterial blight is a very serious disease in rice production, breeding resistant varieties is one of the effective ways to control this disease. Rice variety Haonuoyang from Yunnan province was highly resistant to Xanthomonas oryzae pv.oryzae. R gene in Haonuoyang was preliminarily mapped within the 0.12Mb region of the long arm of chromosome 11,and was no allele with known Xa genes,temporarily named Xa42(t).This project will use hybrid cross near-isogenic lines of Haonuoyang and susceptible varieties Jingang30 were constructed as fine mapping group, map-based cloning of resistance genes in Haonuoyang will be completed using SSR,SNP,Indel molecular markers which distributed in location regions of 11 rice chromosome.Combining with deep genome sequencing and RNA-seq technology, structure and expression level of genes are analysis respectively , then get the candidate genes.Candidate genes will be constructed with GUS, GFP protien and overexpression vector respectively using the gateway technology, and site-directed gene mutagenesisusing CRISPR / cas9 system, transform rice to analysis function and subcellular localization of R gene product, to confirm Xa42 gene.The functional elements of Xa42 gene can be confirmed by analyzing its homology in different rice varieties,combining with functional domain.The resistance mechanism of Xa42 will be defined through comparing the hormone levels of the Haonuoyang, rice of overexpression and mutagenesisusing of Xa42 gene,Gene expression profile of Xa42 gene in rice tissues by being inoculated or non-inoculated by RT-PCR,and interaction network of Xa42 and other genes will be analysis.The project is to identify new R gene for resistance to rice bacterial blight, and further understand the mechanism of resistance to bacterial blight in rice.

水稻白叶枯病是水稻生产上重要的细菌病害，选育抗病品种是防治该病害的有效途径。云南地方稻毫糯扬高抗白叶枯病，其R基因初步定位在11号染色体长臂0.12Mb区段内，与已知抗病基因不等位，暂命名为Xa42（t）。本项目拟利用前期构建的毫糯扬抗性近等基因系与感病水稻金刚30杂交获得的F2群体，通过SSR、SNP分子标记、图位克隆，结合基因组深度重测序、RNA-seq技术，获得Xa42（t）候选基因；利用gateway技术分别构建候选基因过量表达、GUS 及GFP融合载体，利用CRISPR/Cas9系统进行基因敲除，转化水稻，进行功能分析和亚细胞定位，确定Xa42基因；分析Xa42功能域及在不同水稻上的同源性，确定其功能所需元件；分析病菌胁迫后Xa42抗性与激素水平的关系、基因表达谱，与其他基因的互作关系，明确Xa42的抗性机理。本研究对发掘利用抗水稻白叶枯病新R基因，揭示水稻抗性机理具有科学意义。

水稻白叶枯病是水稻生产上重要的细菌病害，选育抗病品种是防治该病害的有效途径。云南地方稻毫糯扬高抗白叶枯病，本项目前期将其携带的R基因初步定位在11号染色体长臂端，与已知抗病基因不等位，暂命名为Xa42（t）。本项目前期构建了毫糯扬近等基因系H197/金刚 30 杂交F2新群体，后应用其F3和F4群体，通过KASP标记进行重组单株的筛选及连锁分析将 Xa42(t)精细定位区域最终锁定在第11号染色体28863864-28905370bp，41.5Kb范围内，这个区段包含了5个预测的基因。结合基因组深度重测序、2个亲本间（CDNA及DNA）扩增测序比对和基因表达分析，确定G5这个基因为Xa42（t）的候选基因。经克隆该基因编码区全长为1738bp，翻译成氨基酸序列长度是579aa，与NCBI 网站上的 OsI_37091（EAY81912.1）的相似性达100%,预测编码一个scarecrow-like protein 9。通过转基因植株抗病表型分析表明该基因与水稻抗病相关，Xa42(t)的GFPs融合蛋白定位于细胞膜上，Xa42(t)介导的 HR-PCD 大部分为双层膜的膜泡和单层膜的膜泡结构等类自噬体结构为特征的细胞自噬。通过酵母双杂交系统筛选显示与Xa42(t)互作的蛋白有4个其功能分别是NAD（辅酶）绑定蛋白、热激蛋白等。通过对抗病基因Xa42(t)的过表达转基因植株具有一定的耐盐，耐寒及抗稻瘟病的广谱抗性。Xa42(t)受到JA的正向调控，同时引起病程相关蛋白表达，引发毫糯扬的抗病反应。研究结果丰富了抗水稻白叶枯病新R基因相关理论和技术基础，进一步揭示水稻抗白叶枯病的机理，为白叶枯病的防治提供了新的抗原和理论基础。项目研究发表论文4篇，其中SCI收录1篇；项目负责人入选2020 年云南省青年拔尖人才计划专项、项目组2人晋升研究员，1人晋升副研究员。