基于核糖体图谱技术的果实成熟衰老翻译调控机制研究

Fruit ripening and senescence are complicated processes, which are regulated by multiple genes at various levels. In previous study, we found that fruit ripening and senescence are accompanied by the changes in abundance of a number of proteins, suggesting that translational regulation might play important roles in these processes, but the mechanisms are not clear. In this study, we plan to analyze, by using ribosome profiling, the changes in protein translation efficiency during fruit ripening and senescence in tomato. The genes whose expression is regulated at translational level will be identified and the function of translational regulatory elements will be analyzed. Ribosome profiling is a high-throughput and sensitive technique, which could be used for analysis of translational regulatory elements besides protein translation status. Therefore, ribosome profiling represents an effective method for the study of translational regulation. Through this technique, we will also study the changes in protein translation efficiency in tomato fruit after treatment with ethylene and ethylene inhibitor, and those under environmental conditions (light and temperature). The regulatory effects of ethylene and environment on fruit ripening and senescence at translational levels will be unraveled. Based on these studies, we hope to uncover the mechanisms of translational regulation in fruit ripening and senescence. It will contribute to the maintaining of fruit quality and the improvement of technology on fruit storage and preservation.

果实成熟衰老是复杂的生物学过程，受基因表达的多级调控。申请人前期的研究显示，成熟衰老过程伴随着多种蛋白质的显著变化，提示蛋白质翻译调控在此过程中发挥重要作用，但其内在机制不清。本项目拟通过核糖体图谱技术（ribosome profiling）分析番茄果实成熟衰老中蛋白质翻译效率的变化，鉴定在翻译水平可能受到调控的基因，并解析翻译调控序列对蛋白质翻译的调节作用。核糖体图谱技术具有高通量、高灵敏度的特点，在分析蛋白质翻译状况的同时，能够对翻译调控序列进行检测，因此是研究蛋白质翻译调控机制的有效方法。利用该技术，本项目也将分析果实经乙烯和乙烯受体抑制剂处理后，以及在不同环境条件下（光照和温度）蛋白质翻译效率的变化，诠释乙烯和环境条件在翻译水平调控果实成熟衰老的分子机理。通过以上研究，阐明果实成熟衰老的翻译调控机制，为果实采后品质保持和贮藏保鲜技术的改良提供理论依据。

果实成熟是复杂的生物学过程，受基因表达的多级调控。成熟过程伴随着色素合成、细胞壁代谢、香气产生等途径的多种蛋白质的显著变化，表明蛋白质翻译调控在此过程中发挥重要作用，但其内在机制不清。本项目以番茄为研究材料，对果实中核糖体图谱技术进行了条件摸索，包括核糖体提取和纯化方法、mRNA核酸酶种类选择及酶切条件、核糖体结合mRNA片段的富集和文库构建等；建立了适用于番茄、草莓等多种果实的蛋白质翻译效率检测技术。通过分析番茄果实成熟过程中m6A RNA甲基组变化，揭示RNA甲基化修饰在番茄果实中主要发生在终止密码子附近和3’非翻译区（3’UTR），并影响基因的mRNA稳定性和翻译效率；进一步分析发现RNA甲基化修饰可调节DNA去甲基化酶基因SlDML2的mRNA稳定性，从而调控番茄果实成熟；m6A去甲基化酶SlALKBH2在此过程中发挥核心作用。此外，阐明了光信号通路关键蛋白SlHY5影响重要核糖体蛋白的表达，进而调控果实成熟相关基因翻译效率的作用机制，从而诠释了光等环境信号在翻译水平调控果实成熟的分子机理。通过以上研究，在一定程度上阐明了果实成熟的翻译调控机制，为全面揭示果实成熟调控网络提供了参考，也为果实采后品质保持和贮藏保鲜技术的改良提供了理论依据。