富半胱氨酸受体蛋白激酶OsCRK1调控水稻白叶枯病抗性的机理

Rice bacterial blight, caused by Xanthomonas oryzae pv.oryzae (XOO), is a worldwide rice disease, seriously affecting the yield of rice. Previous studies have proved that discovery and utilization of resistance genes in the host is the key to control this disease. Through the last NSFC project, we have developed a Near-isogenic line named as 5024 which shows a broad spectrum of resistance to bacterial blight. Recently, we conducted the transcriptome sequencing (RNA-seq) of 5024 and found a differentially expressed gene whose expression was highly induced by XOO. We designed this gene as OsCRK1, because it encodes a cysteine-rich receptor protein kinase(CRK). Further QRT-PCR analysis and RNAi silent experiment showed that OsCRK1 was associated with the bacterial blight resistance in 5024. So far, there is no report on a CRK gene involving in regulation of bacterial blight resistance in rice. Based on the above studies, this proposal aims to research on the gene expression patterns in terms of space and time; subcellular localization of the deduced protein OsCRK1. Further more, physiological and biochemical approaches including yeast two-hybrid, immune coprecipitation, protein spectrum technology will be used to search for the protein(s) interacting with OsCRK1 and the modification pattern of OsCRK1. Through these in-depth studies on OsCRK1, we expect to reveal the mechanism of OsCRK1-mediated disease resistance, to benefit rice molecular breeding for disease resistance, by providing anew gene and theoretical guidance for efficient use of OsCRK1.

由黄单胞杆菌引起的水稻白叶枯病是世界性水稻病害，严重制约水稻的高产稳产。实践证明，挖掘和利用寄主抗病基因是控制此病的关键。我们通过上一个基金项目获得广谱高抗白叶枯病的近等基因系5024，最近对其进行转录组测序(RNA-seq)，发现差异表达基因OsCRK1受白叶枯病菌诱导表达，编码一个富半胱氨酸受体蛋白激酶（CRK）。qRT-PCR分析、RNAi沉默等实验证明OsCRK1基因与5024的白叶枯病抗性相关。迄今还没有关于CRK基因调控水稻白叶枯病抗性的报道。本项目旨在前期研究的基础上，拟通过分子生物学手段包括基因的时空表达模式及亚细胞定位等解析该基因的调控途径；通过生理生化手段包括酵母双杂、免疫共沉淀、蛋白质谱技术等解析蛋白在体内体外的互作、修饰模式。通过对OsCRK1基因的深入研究，揭示其抗病机理，为水稻抗白叶枯病分子育种提供新的基因源和理论指导。

由稻黄单胞杆菌水稻致病变种引起的白叶枯病是世界性水稻病害，严重制约水稻的高产稳产。实践证明，挖掘和利用寄主抗病基因是控制此病的关键。我们从广谱高抗白叶枯病水稻材料5024中克隆出一个受病原菌诱导表达的抗病相关基因，将其命名为OsCRK1。通过生物信息学分析，该基因编码一个富半胱氨酸受体蛋白激酶。利用过表达、RNAi干涉及CRISPR/Cas9编辑等技术手段，验证了该基因的功能，明确了OsCRK1基因参与了抗病反应。通过亚细胞定位证明OsCRK1蛋白定位在细胞膜上。用抗病信号分子处理后发现，OsCRK1基因受SA、ABA诱导表达。利用酵母双杂交系统筛选到2个互作蛋白，并利用双分子荧光互补在体内进行了验证，结果表明OsCRK1蛋白与过氧化氢酶互作共同调控水稻抗病反应。我们还对3个水稻感病相关基因Pong2-1、Pong11-1和Xig1进行功能研究，并利用CRISPR/Cas9技术对感病基因进行编辑，获得了抗白叶枯病资源材料。本项目关于抗病、感病相关基因的解析对水稻抗白叶枯病分子育种提供了理论指导，创制的抗病材料为水稻抗病育种提供了新的资源，对水稻生产具有实践意义。