番茄果实耐贮性相关转录因子SlFSR分子调控网络研究

Tomato ripening mutants rin (ripening inhibitor) and Nr (Never-ripe) have high fruit storability, but the mechanism of molecular regulation is poorly understood. During study on tomato fruit ripening, we cloned a GRAS transcription factor family gene SlFSR, which expresses specificly in ripening fruit, and has a lower expression level in root, stem, leave, flower and immature green fruit, and its expression is strongly inhibited in the tomato ripening mutants rin and Nr. Silencing SlFSR improved significantly fruit storability and inhibited the expression of fruit storability-related genes, but no significant effects on the expression of the genes related to the metabolism of unsaturated fatty acid, lycopene biosynthesis and ethylene biosynthesis. The overexpression of SlFSR in the mutant rin decreased seriously fruit storability and significantly increased the expression of fruit storability-related genes, but no significant effects on the expression of the genes related to the metabolism of unsaturated fatty acid, lycopene biosynthesis and ethylene biosynthesis. We shall transform wild type tomato and mutant Nr with the SlFSR over-expression vector, get transgenic plants with over-expressed SlFSR. We shall analyse the phenotypes related to fruit ripening, quality and storability. By ChIP-Seq and RNA-Seq, we’ll analyse and clone the down-stream genes regulated by SlFSR. In addition, we'll clone the 5′ up-stream region of SlFSR promoter and construct a series of SlFSR promoter 5’ deletion mutant to analyse the cis-acting elements related to fruit ripening specific expression. With Yeast two-hybrid system、Co-Immunoprecipitation or Pull-down, the factors which can be co-operated with SlFSR will be confirmed. We’ll set up a gene regulatory network related to fruit ripening and storability. The research work will illuminate the function of SlFSR and shed more light on the molecular regulatory mechanism of fruit ripening and storability.

番茄突变体rin和 Nr果实极耐贮藏，但分子调控机制尚不清楚。我们克隆到一个果实成熟特异转录因子基因SlFSR，根茎叶及未成熟果实几乎不表达，在rin和 Nr中表达受到强烈抑制。沉默该基因，特异性地抑制细胞壁及果胶代谢有关基因的表达，果实表现出极强的耐贮性；在rin中超表达该基因，显著提高细胞壁及果胶代谢有关基因的表达，果实耐贮性降低；对乙烯、类胡萝卜素、不饱和脂肪酸、可溶性糖、有机酸等代谢无明显影响。表明SlFSR特异性地调控耐贮性有关基因的表达，是RIN和乙烯调控果实耐贮性途径的关键因子。本研究拟利用该基因的超表达和沉默株系，深入研究对果实成熟特性的影响；探索上游转录因子、SlFSR互作蛋白及顺式作用元件，挖掘下游靶标基因，建立该基因调控网络。本研究对明确SlFSR的功能，阐明rin和Nr果实极耐贮藏的分子调控机制具有非常重要的科学意义，在番茄等果实耐贮性遗传改良中具有广阔的应用前景。

番茄成熟突变体rin果实极耐贮藏，但其分子调控机制尚不清楚。本项目利用超表达技术，RNAi沉默技术和基因编辑技术培育了番茄果实成熟特异转录因子SlFSR基因超表达、沉默和敲除突变体，经表型观察、生理生化和分子水平检测，发现SlFSR正调控果实成熟，影响果实的耐贮性，果实色素的形成，可溶性糖、还原性谷胱甘肽等代谢过程，参与番茄果实品质调控。双荧光素酶报告系统和酵母单杂交系统证实，果实成熟转录因子RIN和SlERF6可以靶向结合SlFSR启动子，并且促进其转录，相反rin突变体中RIN-MC融合蛋白可以抑制SlFSR基因的转录。同时，SlFSR通过激活乙烯合成基因SlACO1和SlACO3的转录，抑制果实成熟负调控因子SlMADS1的转录，进而影响番茄果实成熟；SlFSR通过正向调控SlGALT7的转录，影响阿拉伯半乳糖蛋白糖基化修饰，调控果实细胞壁代谢，影响番茄果实耐贮性。本研究揭示了SlFSR的生物学功能，建立了SlFSR基因的调控网络，进一步阐明了番茄突变体rin果实耐贮性强的分子调控机理，在番茄等果实耐贮性遗传改良中具有广阔的应用前景。