MADS-RIN下游基因的鉴定及功能分析

Fruits are developmental structures that are unique to flowering plant and play a central role in seed maturation and dispersal. Ripening and senescence is a physiological feature of fruit. It is very important to explore the mechanisms regulating fruit ripening and senescence both in theoretical basis and practical implications. With many regulatory genes being identified in fruit, fruit-specific control of ripening and senescence by transcription factors has become an important research focus. Among them, transcription factor RIN has received more attention. In our previous study, using tomato as a model system, we have found five novel RIN-regulated direct target genes through the methods of proteomics-based chromatin immunoprecipitation, and firstly proved that aromatic metabolic pathway are directly regulated by RIN. Based on the previous work, this project is planed to use RNA-Seq technology to compare the gene expression of wild type and rin mutant for mining the downstream regulatory genes of RIN, especially transcription factors. Furthermore, we perform the methods of molecular biology, biochemistry, cell biology and plant physiology to explore the important roles of these transcription factors in fruit ripening and senescence, providing theory basis for engineering technology to delay fruit ripening and senescence.

果实是植物特有的发育器官，在种子成熟和传播过程中发挥重要作用。成熟衰老是果实发育的重要阶段，也是果实品质形成和保持的关键时期，研究果实成熟衰老的调控机制具有重要的理论意义和应用价值。近年来，转录因子研究为人们认识果实成熟衰老机制开辟了新视野，逐渐成为国际研究热点。其中，MADS-box转录因子家族成员RIN最受关注。申请人在前期工作中，以番茄果实为材料，通过蛋白质组学技术结合染色质免疫共沉淀等方法，鉴定出RIN调控的5个新靶标基因，并首次证实RIN直接调节芳香物质代谢途径。本项目拟在已有的研究基础上，利用RNA-Seq技术分析rin突变株和野生型番茄果实在成熟衰老过程中的基因表达差异，挖掘RIN调控的下游基因，特别是转录因子，并通过分子生物学、生物化学、细胞生物学和植物生理学等实验方法，深入分析RIN调控的下游转录因子在果实成熟衰老中的重要作用，为进一步利用基因工程技术来控制果实成熟衰老。

果实成熟受诸多因素的调控和影响，RIN转录因子是一个重要的节点。本研究从RIN调控的下游基因入手，挖掘了一个新的调控因子DOF，进一步通过分子和遗传学方法分析了DOF的功能机制。研究表明，DOF定位于细胞核，在BR期果实表达最强，能受外源乙烯诱导表达。相比于对照果实，DOF-RNAi转基因果实的乙烯峰值延后，番茄红素含量和硬度降低，果实成熟进程明显推迟。RNA-seq数据显示，DOF-RNAi果实中与成熟相关基因的表达下调。以上结果表明，DOF在果实成熟调控中起重要作用，但其分子调控机制仍需进一步研究。本研究对丰富果实成熟调控的网络研究具有重要意义。