番茄SlGRAS9调控果实品质及成熟衰老的机制研究

Fruit ripening was regulated by many transcription factors in plants. GRAS family are plant special transcription factors that play crucial roles on plant development and hormone signaling. However, there are few reports about GRAS regulating fruit quality and fruit ripening. Histone deacetylases (HDACs)-medicated acetylation are an integral component of the epigenetic network in eukaryotes. HDACs bind with transcription factors to regulate target genes that play key roles in plant growth and development. How HDACs and transcription factors cooperate with each other to regulate fruit ripening is unclear. In our previous study, overexpression of SlGRAS9 in tomato elevated quality and storability of fruits, and the interaction of SlGRAS9 and SlHDA3 has been identified by yeast two-hybrid. Besides, the transcription levels of SlGRAS9 and SlHDA3 were up-regulated significantly at breaker stage of the fruit, indicated that SlGRAS9 and SlHDA3 may be co-regulate fruit quality, ripening and senescence in tomato. In this project, the function of SlGRAS9 gene in tomato fruit quality and ripening will be investigated by transgenic methods, and the interaction of SlGRAS9 and SlHDA3 will be confirmed by yeast two-hybrid, BiFC, and CO-IP. The target genes and regulatory network will be identified by integrating the results of RNA-seq and ChIP-Seq. These results will illuminate the molecular mechanism of epigenetic regulation in quality, ripening and senescence of tomato fruit. Therefore, this project has theoretical and practical importance.

果实成熟受许多转录因子调控，GRAS家族作为植物特有的转录因子，在植物发育和激素信号转导方面发挥着重要作用，但是在果实品质及成熟衰老方面的调控功能目前尚不清楚。表观遗传重要调控因子组蛋白去乙酰化酶能够与特异转录因子结合调控下游基因表达，但对其协同调控果实成熟衰老的机制至今仍不清楚。我们前期研究发现，超表达SlGRAS9能够改善果实品质、提高番茄的耐贮性，酵母双杂交筛选发现它可与SlHDA3互作，因此推测它们可能协同调控果实的品质形成及成熟衰老过程。本项目通过转基因技术研究明确SlGRAS9在番茄果实品质形成和成熟衰老过程中的生理功能，并通过酵母双杂交、BiFC以及CO-IP技术鉴定SlGRAS9与SlHDA3的互作关系，并筛选共同调控的下游靶基因，明确其调控网络。研究结果对揭示表观遗传协同转录因子调控果实品质和成熟衰老的机理具有重要价值。

果实营养丰富，是膳食平衡的重要来源。果实成熟的调控网络是由许多转录因子单独或协同调控特定的下游基因来完成，解析调控果实成熟过程的核心转录因子对于果实品质改良十分必要。本项目利用生理生化和分子生物学等实验技术，系统的分析了在番茄果实中有着较高本底表达水平的SlGRAS9基因功能，主要获得以下结果：1. SlGRAS9通过直接调控叶绿素合成基因SlPOR-B促进叶绿素合成，同时通过调控叶绿体发育重要调整转录因子SlGLK1影响叶绿体发育。2. 敲除或下调SlGRAS9的番茄果实积累更多的叶绿素，进而提高果实光合作用速率，淀粉含量显著提高。3. SlGRAS9直接调控蔗糖转化酶基因SlLIN5和己糖激酶基因SlHK6影响果实的糖代谢。4. SlGRAS9通过调控角质单体合成基因SlHTH和角质多聚化重要调控因子SlGDSL1促进果实外果皮角质层增厚，从而提高果实硬度和采后货架期。5. SlGRAS9通过调控果实胶质液化重要调控因子SlMBP3影响细胞壁降解相关基因的表达，延迟果实软化过程。本项目的研究结果表明，转录因子SlGRAS9在番茄果实发育、成熟和采后整个过程中均发挥着重要作用，并且位于转录调控的上游位置，不仅能够直接调控结构基因，还可以直接调控多个功能十分强大的转录因子。研究结果为果实品质改良提供重要的理论基础和基因资源。