DoMYB81调控铁皮石斛多糖生物合成的分子机制研究

Dendrobium officinale Kimura et Migo is the traditional rare medicinal herbs. Polysaccharides are regard as the main medicinal ingredients in D. officinale according to the Pharmacopoeia of the People's Republic of China. However, the molecular mechanism of the regulation of polysaccharide biosynthesis is not yet clear. Based on our previous studies, the DoMYB81 gene was identified as the candidate gene for regulating the polysaccharide biosynthesis in D. officinale. In order to study the regulation mechanism of polysaccharide biosynthesis in D. officinale, this project intends to carry out the following research: (1) Yeast One-Hybrid Library Screening System is used to analysis the combination of DoMYB81 and promoters of polysaccharide biosynthesis genes (DoPMM, DoGMPs and DoCSLAs), and then use EMSA technology to valid the combination. (2) Construction of expression vector and CRISPR/Cas9 vector of DoMYB81, then transform into D. officinale protoemm-like bodies (PLB), respectively. The polysaccharide content, monosaccharide composition and linkage will be analyzed and compared among transgenic lines and WT plants. The candidate target genes of DoMYB81 which related to the polysaccharide biosynthesis are identified by transcriptome sequencing analysis (3) The transcription factors which interact with DoMYB81 to regulating the transcription of polysaccharide biosynthesis genes are identified. And reveal the molecular mechanism of DoMYB81 regulating polysaccharide biosynthesis in D. officinale.

铁皮石斛是我国传统名贵中药材，依据《中华人民共和国药典》，铁皮石斛多糖是铁皮石斛的主要药效成份，但铁皮石斛多糖生物合成的调控机制尚不清楚。我们的前期工作发现，DoMYB81极有可能参与调控铁皮石斛多糖的生物合成。为了研究铁皮石斛多糖合成的调控机制，本项目拟开展以下研究：①酵母单杂交系统分析DoMYB81与多糖合成途径基因DoPMM、DoGMPs和DoCSLAs启动子的结合情况，EMSA实验对结合进行验证，明确DoMYB81可结合到铁皮石斛多糖合成途径基因的启动子区域；②在铁皮石斛中超表达和敲除DoMYB81，分析野生型、超表达株系和基因敲除株系的多糖含量、单糖组成和连接键型等，基于转录组测序分析鉴定下游与多糖合成相关的靶基因，明确DoMYB81调控铁皮石斛多糖合成。③鉴定与DoMYB81互作共同调控铁皮石斛多糖合成的转录因子。建立调控网络，阐明DoMYB81调控铁皮石斛多糖生物合成的机制。

铁皮石斛是传统名贵中药材，具有较大的药用价值和经济价值。本项目围绕MYB转录因子调控铁皮石斛多糖的合成开展研究工作，取得的主要结果如下：.1、DoMYB75转录因子是一个转录激活型转录因子，定位在细胞核内。表达模式分析表明DoMYB75极有可能参与铁皮石斛多糖合成。.2、克隆分析了DoMYB75的启动子，利用qPCR技术和DoMYB75pro::GUS转基因材料的GUS染色，分析了不同部位、不同处理下DoMYB75的表达模式。DoMMYB75在根、茎、叶和花均有表达，在茎的表达量最高。在盐和冷胁迫处理下，拟南芥的茎和叶染色明显比对照深；qPCR结果表明在上述胁迫下，DoMYB75在茎和叶中的表达量均显著上调，说明DoMYB75可能参与铁皮石斛的逆境胁迫响应。.3、在拟南芥中超表达DoMMYB75可显著提高多糖含量。构建Ubi::DoMYB75转录因子并转化铁皮石斛，多糖合成途径基因DoCSLA3、DoCSLA4、DoCSLA6、DoCSLA9和DoCSLA11在Ubi::DoMYB75转基因株系中的表达水平均显著高于野生型。双荧光素酶报告基因实验表明DoMYB75可以显著激活DoCSLA6、DoCSLA9和DoCSLA11的启动子活性。说明DoMYB75正调控DoCSLA6、DoCSLA9和DoCSLA11的转录。Ubi::DoMYB75转基因株系的抗盐能力显著高于野生型。.4、构建铁皮石斛酵母cDNA文库，以DoMYB75为诱饵，筛选DoMYB75的互作蛋白。一共筛选鉴定到20个互作候选蛋白。其中，有两个参与多糖合成的候选蛋白：phosphoglucomutase和UDP-glycosyltransferase 73E1-like。.本研究有助于人们深入认识铁皮石斛多糖生物合成的规律，而且为铁皮石斛的品质改良提供理论指导。