新类病变基因LMM6调控水稻苗期抗稻瘟病的分子机制

It is one of hotspot of basis research on plant natural immune that lesion mimic mutants were used to study the mechanism of disease resistance in plants. We previously characterized a lesion mimic mutant, lmm6, which was obtained from an EMS- induced rice 02428 mutant library. The mutant lmm6 which was characterized with accumulation of ROS and increased expression of defense related gene showed enhanced resistance to blast fungus Magnaporthe grisea in seedling stage. With Map-based cloning, we successfully identified a novel single recessive nuclear gene LMM6. Bioinformatics analysis revealed that LMM6 encodes a lipase. In complementary test, overexpression of LMM6 in lmm6 mutant background complemented the lmm6 phenotype, confirming that the mutation identified in LMM6 is responsible for the lmm6 mutant phenotype. However, it is still unclear about the biochemical role of LMM6, and how LMM6 involved in rice innate immune activation to blast fungus. Therefore, in this project, genetics, molecular biology, biochemical studies will be conducted to uncover the molecular regulation mechanism of LMM6 gene mediated regulation on immunity by revealing the subcellular localization and transcription expression pattern of LMM6, characterizing the lipase activity, dissecting the function of resistance to rice blast with lmm6, LMM6 knock-out mutants and overexpression transgenic lines, and elucidating the molecular processes and metabolic regulations by using RNA-seq-mediated globally transcriptional analysis. These results will provide clues for our understanding of the resistance of plant against pathogens and exploring some new genes, and finally benefit the molecular breeding in agriculture.

利用类病变突变体研究植物抗病机理是植物天然免疫基础研究的热点之一。我们前期从粳稻02428的EMS诱变群体中鉴定了一份类病变突变体lmm6，该突变体中ROS明显富集，防卫相关基因表达量上升，在苗期接种稻瘟菌后表现出较强的抗病性。研究发现，lmm6类病变的表型由一个新的隐性基因LMM6控制，该基因编码一种脂肪水解酶。然而LMM6明确的生物学功能及其如何参与到lmm6抗稻瘟病的分子机制尚不清楚。本项目拟在前期研究基础上，综合运用遗传学、分子生物学、生物化学等方法，首先，分析LMM6基因表达模式、蛋白的亚细胞定位及LMM6的脂肪水解酶活性；然后，利用突变体lmm6、基因编辑技术创建的突变体和过表达转基因材料，剖析LMM6在水稻抗稻瘟病应答中的功能；最后，通过RNA-seq鉴定LMM6调控的分子网络，初步阐明其调控水稻苗期抗稻瘟病的分子机制。研究结果将为水稻抗病育种提供理论依据和新基因资源。

类病变突变体（LMM）是挖掘新抗病基因和研究植物抗病作用机理的重要材料。我们从粳稻02428的EMS诱变群体中鉴定了一份类病变突变体lmm6，前期发现该突变体中ROS明显富集，防卫相关基因表达量上升，在苗期接种稻瘟菌后表现出较强的抗病性。进一步发现，lmm6类病变的表型由一个新的隐性基因LMM6控制，该基因编码一种脂肪水解酶，该蛋白的第62位甘氨酸（G）变为谷氨酸（E）致了类病变坏死斑表型。本课题明确了水稻LMM6的定位、组织表达及诱导表达等特性；通过分析该基因过表达或突变后对稻瘟病抗性产生的变化，鉴定了LMM6基因在水稻抗稻瘟病反应中的调控功能以及其调控水稻抗病信号的特征性变化；通过体外检测LMM6蛋白脂肪酶活性，推测G62可能是LMM6活性中心的关键氨基酸残基之一；RNA-Seq分析，从中筛选差异表达基因：茉莉酸生物合成相关基因OsOPDA、参与调控细胞程序性死亡的天冬氨酸蛋白酶基因OsAP25、转录因子OsMADS26均在突变体中表达量显著上升。通过测定激素含量发现，与抗病应答相关茉莉酸在突变体中显著上升；酵母单杂交结果发现，OsAP25与OsMADS26互作，且目前已创建OsMADS26过表达及基因编辑材料。这些结果为解析LMM6调控水稻细胞程序性死亡与免疫应答的分子机制提供了重要证据，有助于深入剖析植物防卫反应的调控网络，为提高植物抗病性提供理论基础。