组蛋白去甲基化酶SlJMJ6对番茄果实成熟相关基因甲基化修饰水平调控的研究

The control of tomato fruit ripening and senescence is important in fruit quality and postharvest preservation. It is the key point for fruit postharvest preservation technology control and an essential scientific issue for researchers. Fruit ripening and senescence is mainly controlled by gene expression, endogenous hormones and exogenous environmental signals. On the basis of previous work, this project focuses on a histone demethylase SlJMJ6, a quasi epigenetic factor, to explore its transcriptional regulation mechanism involved in fruit ripening and senescence. Firstly, the histone demethylation modification characteristics of SlJMJ6 protein are identified. Secondly, the tomato with overexpressed or knocked out of SlJMJ6 are established, the biosynthesis levels of carotenoids, chlorophyll, ethylene are detected, and the fruit storability is also tested. Then, the pathway of downstream gene regulated by SlJMJ6 in tomato fruit ripening and senescence is identified. Finally, the effect of SlJMJ6 on the histone methylation modification level is analyzed. These study results will provide an important theoretical basis for the regulation of fruit ripening and senescence, thereby improving of fruit quality and increasing commercial value of postharvest fruit.

番茄果实成熟衰老的过程控制对果实品质和采后保鲜起关键作用，是果实采后保鲜技术控制的关键点，也是研究者关注的重要科学问题。果实成熟衰老主要受基因表达、内源激素及外源环境信号的影响。本项目在前期工作的基础上，拟表观遗传因子——组蛋白去甲基化酶SlJMJ6为研究对象，探索其参与果实成熟衰老过程的转录调控机制。首先鉴定SlJMJ6蛋白的组蛋白去甲基化修饰特性；然后建立SlJMJ6的超表达和敲除的植株，检测这些植株中类胡萝卜素、叶绿素、乙烯变化、果实耐贮藏性等生理指标；鉴定SlJMJ6在番茄果实成熟衰老途径中调控的下游基因网络；最终分析SlJMJ6对下游基因的组蛋白甲基化修饰水平的影响。本研究将为调控果实成熟衰老、改良果实品质、提高果实采后的商品价值，提供重要的理论基础。

果实成熟是一个复杂而有序的过程，受内在（内源激素和遗传调控因子）和外部环境因素调节，它们在一个复杂的网络中调控成熟相关的基因的表达，最终导致果实成熟衰老。组蛋白甲基化是重要的表观遗传修饰方式之一，受组蛋白甲基转移酶和去甲基化酶动态调控，在植物的生长发育、花期调控、激素信号、非生物逆境胁迫等方面发挥重要作用。当前，组蛋白甲基化修饰在调节果实成熟衰老中的作用并不清楚。本项目在前期工作基础上，解析了番茄组蛋白去甲基化酶JMJ6在果实成熟中的作用及调控机制。主要研究结果如下：1、JMJ6在番茄果实中特异高表达，并且随着果实成熟进程逐渐上调；JMJ6过表达明显促进了番茄果实成熟，伴随着更高的乙烯合成速率、类胡萝卜素含量和可溶性果胶含量；RNA-seq分析表明，JMJ6调节一系列与成熟相关调节基因和功能基因的表达；2、JMJ6发挥去甲基化酶活性的结合Fe2+和α-酮戊二酸的关键氨基酸残基和识别甲基化赖氨酸的残基均不完全保守；烟草细胞核免疫荧光染色（in vitro）和组蛋白去甲基化酶体外活性（in vivo）分析表明，JMJ6是一个特异的组蛋白H3K27me3/2去甲基化酶；JMJ6过表达的番茄植株中H3K27me3/2组蛋白甲基化水平明显低于野生型植株，进一步证实了番茄JMJ6具有组蛋白H3K27me3/2去甲基化活性；3、通过RNA-seq、GFP-ChIP-Seq、H3K27me3-ChIP-seq联合分析鉴定出32个受JMJ6直接靶向、被JMJ6去H3K27me3和表达上调的基因，它们主要参与转录调控、乙烯生物合成、细胞壁降解、色素生物合成、激素信号转导等过程。T-qPCR和ChIP-qPCR进一步证实了11个与成熟相关的关键基因被JMJ6直接去H3K27me3甲基化而表达上调，表明JMJ6结合并降低了成熟相关基因位点的组蛋白H3K27me3修饰水平，从而激活其基因表达，促进番茄果实成熟。4、JMJ6能直接靶向DNA去甲基化酶DML2基因，并通过去除其基因位点H3K27me3激活DML2表达。综上所述，JMJ6是一个促进果实成熟的组蛋白H3K27me3去甲基化酶，通过降低成熟相关基因的H3K27me3修饰水平，激活基因表达，进而促进果实成熟。