一个GRAS基因在水稻与白叶枯病菌互作中的功能研究

The GRAS gene family is an important plant-specific gene family that has diverse functions in plant growth, development, and responses to environment. The rice genome has 57 GRAS genes. However, the knowledge of GRAS genes in rice-pathogen interactions is largely unknown. Previously a rice mutant which developed spontaneous lesions (lesion mimic) on the leaves and showed enhanced resistance to Xanthomonas oryzae pv. oryzae (Xoo) was treated with ethyl methanesulfonate (EMS) in our lab. A mutant with no lesion mimic and reduced resistance to Xoo phenotypes was further analyzed in this project. GRASx was then identified as a putative mutated gene using the MutMap method. In a rice database based on the long read CHIA-PET (Chromatin interaction analysis by paired-end tag sequencing) technic, the GRASx locus was found to form a chromatin loop with the G3Pa locus coding for an enzyme which regulates the metabolism of glycerol-3-phosphate. In this research, the function of GRASx and G3Pa in rice-Xoo interaction will be analyzed, and the details of how the GRASx- and G3Pa-formed chromatin loop influence their expression will be elucidated. Further, whether the two genes were involved in the regulation of glycolysis pathway and the metabolism of reactive oxygen species will be studied. This project will improve the understanding of functional mechanism of GRAS genes, and facilitate the elucidation of the molecular mechanism of plant defence system in the view of three-dimensional genome architecture, and also provide supports for rice resistance breeding.

GRAS基因家族是植物特有的一类大家族，广泛参与调控植物的生长发育和各种胁迫响应。水稻中共有57个GRAS类基因，但是人们对其在水稻-病原互作中的功能了解甚少。申请人所在的实验室对一个叶片自发产生类病斑且抗白叶枯病的水稻突变体进行了EMS诱变。本项目对其中一个类病斑和抗病性消失的突变体展开研究，利用MutMap方法鉴定得到了GRASx基因。通过查找数据库，发现GRASx基因同一个编码产物参与3-磷酸甘油代谢的基因G3Pa在三维空间结构上形成一个染色质环。本项目将研究这两个基因在水稻抗病反应中的功能，通过分析二者形成的染色质环对其表达量的影响，以及二者在糖酵解和活性氧代谢中的功能，解析GRASx调控水稻抗白叶枯病的分子机理。本研究结果将拓宽人们对GRAS基因功能及作用机制多样性的理解，有助于人们从三维基因组学的角度理解植物免疫反应的分子机理，并为育种工作提供基因资源和理论支撑。

水稻白叶枯病是危害全球范围内水稻生产的重要细菌性病害，克隆抗病基因、解析水稻的抗病机制是培育抗病品种的重要步骤。我们前期获得了一个对白叶枯病表现出广谱抗性的水稻类病斑突变体osedr1，但是该基因介导的抗病信号通路未知。通过对该突变体进行EMS诱变，我们利用MutMap方法获得了OsEDR1信号通路中的重要基因GRASx。但是GRASx与OsEDR1并不存在直接互作，以GRASx为基点，我们进一步通过酵母双杂交、生物信息学分析等技术手段，鉴定到GRASx的互作蛋白OsMPK6以及OsMPK6的互作蛋白OsMPKK10.2，以及这一信号通路中的另一蛋白OsVQ1。GRASx和OsVQ1位于OsMPKK10.2-OsMPK6信号级联的下游，通过不同的机制调控活性氧信号路径进而调控水稻的抗病性；但这一信号路径被OsEDR1负调控。我们的结果揭示了水稻白叶枯病抗性的新的分子机理，也为相应的育种工作提供了基因资源。