组蛋白去乙酰化酶DoHDA7和DoHDA9基因调控铁皮石斛多糖合成的机制研究

Dendrobium officinale is a rare and precious traditional Chinese herbal medicine, which the main functional components are water-soluble polysaccharide. Epigenetic processes can regulate secondary metabolism, which is an important factor affecting the quality of Chinese herbal medicine. According to our previous study, during Dendrobium officinale plants were treated by low temperature, the content of Dendrobium officinale polysaccharide (DOP) increased obviously, at the same time, the expression of histone deacetylase DoHDA7 and DoHDA9 genes decreased significantly. However, the relationship between the content of DOP and the expression of histone deacetylase genes is not clear yet. This project will be performed to (1) investigate the content of DOP and the expression of histone deacetylase DoHDA7 and DoHDA9 genes of different growth and development period, different organs, under different stress conditions, such as high or low temperature, drought, etc. (2) investigate the level of histone acetylation and the expression of genes involved in DOP synthesis under different stress conditions, such as high or low temperature, drought, etc. (3) clone and analyze DoHDA7 and DoHDA9 genes and their promoters, investigate the molecular biochemical mechanisms of DoHDA7 and DoHDA9 gene action, and identify the target genes involved in DOP synthesis, which are regulated by DoHDA7 and DoHDA9 gene. Above research could help verify the molecular regulatory mechanism of DOP synthesis.

铁皮石斛是传统名贵珍稀中药材，水溶性多糖是其主要功效成份。表观遗传能够影响次生代谢且是影响中药材质量的重要因素。我们前期的研究发现，低温处理时，铁皮石斛多糖含量明显增加，组蛋白去乙酰化酶DoHDA7和DoHDA9基因的表达显著降低，但二者之间的关系尚不清楚。本项目将开展如下研究：（1）检测不同生长发育时期、不同器官、以及高低温和干旱等逆境条件下铁皮石中的多糖含量及DoHDA7和DoHDA9的表达模式，并进行关联分析；（2）检测高温、低温和干旱等逆境条件下铁皮石斛组蛋白乙酰化水平的变化，同时检测与多糖生物合成相关基因的表达模式，并进行相关性分析；（3）克隆分析DoHDA7和DoHDA9基因及其启动子，并通过转基因技术、VIGS技术和转录组测序等手段，明确DoHDA7和DoHDA9基因作用的分子生化机制和受其调控的与多糖合成代谢相关的靶基因。最终明确它们调控铁皮石斛多糖合成的机理。

铁皮石斛是我国传统名贵中药材，有效成分包括多糖、类黄酮等物质。组蛋白去乙酰化酶在调控植物次生代谢及非生物胁迫响应中起重要作用。本项目围绕铁皮石斛组蛋白去乙酰化酶基因的鉴定及其在调控植物次生代谢和响应非生物胁迫方面开展了一系列研究，得到如下结果：.1. 利用生物信息学方法从铁皮石斛基因组中共鉴定出14个组蛋白去乙酰化酶基因HDACs，系统进化树显示，DoHDA7与拟南芥AtHDA19具有较高的同源性，DoHDA9与AtHDA6具有较高的同源性。DoHDACs的启动子含有多种响应植物激素和非生物胁迫的顺式作用元件。.2. HDACs在不同组织和花发育不同时期的表达模式不同，并能够响应不同的激素处理（GA3、SA、ABA、MeJA）和非生物胁迫（盐、干旱、低温）；亚细胞定位结果显示DoHDA7，DoHDA9等均定位于细胞核中。.3. 不同器官中，茎秆中多糖含量最高；封顶期至开花期前，茎秆中多糖含量逐渐增加；非生物胁迫（干旱、低温）能够诱导多糖合成。.4. 转基因功能验证表明DoHDA7负调控拟南芥多糖和抗坏血酸合成基因的表达，DoHDA9能够提高拟南芥抗盐胁迫的能力。.5. 酵母单杂交文库筛选结果表明DoHDA7与DoCAT、DoSPIN及DoF3H等互作，DoHDA9与DoPOD、DoMYB及DoFLK等互作。.6. 利用组蛋白去乙酰化酶抑制剂TSA及SAHA处理铁皮石斛小苗，KEGG代谢通路富集结果表明组蛋白去乙酰化酶参与铁皮石斛多糖、类黄酮代谢和逆境胁迫响应过程。.本研究为铁皮石斛抗逆基因挖掘和优良品种选育提供了理论依据和基因资源。