水稻甲基转移酶基因OsMts1调控叶片早衰和抽穗期的分子机制研究

Rice premature leaf senescence is one of the important factors that affect rice yield and quality. It is very important to reveal the leaf-senescence mechanisms to high yield and quality of rice. In this project, a rice mutant with premature leaf senescence (pls1) was obtained from indica-type restorer line ZH8015 via 60Co irradiation treatment. Although normal development in early seedlings, the pls1 mutant displayed earlier leaf senescence with accumulated more reactive oxygen species, less chlorophyll content and photosynthesis rate compared with the wild type after tillering stage. Genetic analysis suggested that the mutant was controlled by a single recessive gene. Fine mapping and position cloning found that the 10th gene fragment was deleted, which encode the O-methltransferase (OsMts1). Although different kinds of leaf senescence-associated genes have been characterized, but methyltransferase regulates leaf senescence were poorly understood in plants. Functional complementation of OsMts1 in pls1 mutant completely restored to wild type. In this project, the biological functions of OsMts1 regulating rice leaf senescence were comprehensively characterized by using genetics, functional genomics, plant physiology, biochemistry and metabolomics tools to identify the key substrate molecules. Besides, the interaction protein of OsMts1 were screened by yeast two-hybrid and confirmed by Co-IP. These results could reveal the function and molecular mechanism of OsMts1 gene that regulates leaf senescence and heading date, which laid the foundation of hybrid rice germplasm improvement.

水稻叶片早衰与产量和品质密切相关，阐明叶片早衰的调控机制对水稻高产、优质十分重要。前期从钴-60辐射诱变籼型水稻恢复系“中恢8015” 得到叶片提前衰老突变体pls1，该突变体在分蘖盛期后叶绿素含量显著降低并提前抽穗；生理学指标测定发现突变体活性氧大量积累、光合速率显著降低。遗传分析表明该性状受隐性单基因控制；图位克隆发现pls1编码一个O-甲基转移酶OsMts1，功能互补pls1突变体能恢复正常表型，但其调控叶片衰老方面的功能和机制未知。本研究将综合遗传学、功能基因组学、植物生理学、生物化学和代谢组学等技术手段，重点研究OsMts1蛋白催化的关键底物分子；结合酵母双杂交及免疫共沉淀技术，筛选并鉴定OsMts1的互作蛋白及其可能的作用机制，旨在阐明该基因调控水稻叶片早衰的分子机制。研究结果可解析OsMts1基因在调控水稻叶片早衰和抽穗期的功能和分子机理，对改良杂交水稻早衰奠定理论基础。

本项目以水稻早衰突变体pls3为研究材料，该突变体表现为叶片过早衰老以及抽穗期提前，表型鉴定发现是由于活性氧大量积累导致。生理学分析发现该突变叶中叶绿素含量降低，而过氧化氢和丙二醛水平均升高；同时该突变体对外源过氧化氢高度敏感；其他衰老相关基因如Osh36和RCCR1在突变体中显著上调表达。图位克隆证明早衰表型是由于甲基转移酶OsMTS1发生基因缺失突变；OsMTS1功能互补该突变体能完全恢复至野生型表型。该基因编码褪黑色素生物合成途径中的O-甲基转移酶；生化酶活实验证明体外表达的重组OsMTS1蛋白能以N-乙酰羟色胺为底物合成褪黑色素，且外源施用褪黑色素能延缓该突变体的叶片衰老。研究结果表明OsMTS1编码的甲基转移酶能通过合成褪黑色素调控氧化还原平衡从而延缓叶片衰老。本研究为为培育耐早衰高产水稻品种以及富含褪黑色素的功能稻米提供理论基础。