FtMYB2对荞麦类黄酮生物合成的代谢调控机制及抗逆功能研究

The MYB transcription factors play an important role in the flavonoids biosynthesis and abiotic stress response in plant. According to the previous study on MYB transcription factor of buckwheat, our results indicated that the expression levels of transcription factor FtMYB2 was obviously up-regulated by abiotic stress treatment in buckwheat, the expression level of the main genes in flavonoid biosynthetic pathway were regulated in buckwheat FtMYB2 overexpressed hairy roots, which indicated that FtMYB2 might play an important role in regulation of flavonoid biosynthetic pathway in buckwheat to response stresses, however, the regulation mechanism of FtMYB2 in buckwheat is still not clear. Based on the previous research, this proposal presents a study to identify and characterize the regulation mechanism and function of the transcription factor FtMYB2. Firstly, our aim is to obtain FtMYB2 over-expression and gene silencing transgenic hairy roots, combining the techniques of differential gene expression analysis to identify the main regulated target genes of FtMYB2 in flavonoid biosynthetic pathway in buckwheat; employ chromatin immunoprecipitation assay to clear the interacting between FtMYB2 and target genes. To analyze the targeted metabolites in each branches of flavonoid biosynthetic pathway by UPLC-MS-TOF-based metabonomics analysis, our research is trying to clear the regulated branches and target compounds of FtMYB2 in the regulation of flavonoids biosynthesis in buckwheat. Finally, the Agrobacterium-mediated transformation will be employed to transfer FtMYB2 to the model plant Arabidopsis, and the anti-abiotic stress ability and the network regulation way of FtMYB2 will be declared. The promoter of FtMYB2 will be cloned, and the cis-acting elements along with the upstream regulators of FtMYB2 will be identified by yeast one hybrid. Finally, to reveal the regulation mechanism of transcription factor FtMYB2 on the flavonoid biosynthetic pathway in buckwheat and the gene function of FtMYB2 when response to environmental stressors at the molecular and physiological metabolic level, our research will provide the scientific theory for improving the quality and anti-stress ability of buckwheat in the molecular breeding work.

MYB转录因子在植物类黄酮生物合成和非生物逆境应答中具有重要作用。前期研究表明荞麦转录因子FtMYB2特异响应非生物胁迫，并参与调控类黄酮合成途径结构基因的表达，但其调控机理并不清楚。本项目在前期研究基础上，拟重点研究FtMYB2调控荞麦类黄酮合成的机制与抗逆功能。首先，利用荞麦毛状根体系超量及抑制表达FtMYB2，结合表达谱测序筛选其类黄酮合成下游靶基因；采用染色质免疫共沉淀技术阐明其与候选靶基因的互作关系；其次，基于代谢组学分析比较超量及抑制表达FtMYB2荞麦毛状根的类黄酮代谢差异，明确FtMYB2调控的代谢支路和靶向产物；最后，经转基因拟南芥验证FtMYB2对非生物胁迫的抗性调控功能；通过启动子克隆和酵母单杂交明确其响应胁迫的顺式调控元件及上游调控基因。实现在分子和代谢水平上阐明FtMYB2对荞麦类黄酮合成的调控机制和抗逆功能，为提高荞麦品质和抗性的分子育种提供理论基础。

MYB转录因子在荞麦非生物逆境应答中具有重要作用。基于前期工作，本研究主要对荞麦转录因子FtMYB2在非生物胁迫中代谢调控的机制与抗逆功能进行了研究。本研究对荞麦转录组测序结果分析表明，其差异基因主要富集在苯丙烷代谢通路、类黄酮生物合成通路；从苦荞转录组数据中筛选得到35个MYB基因存在着显著差异，其中19个上调和16个下调，最终获得了荞麦响应胁迫相关的15个R2R3-MYB转录因子的基因信息；对初级代谢和次级代谢产物的代谢轮廓进行分析，荞麦中氨基酸类化合物和有机酸类化合物的含量多数呈上升趋势，其主要次级代谢物芦丁和槲皮素的合成显著上调。FtMYB2的基因表达与芦丁、对香豆素酸和槲皮素这三个类黄酮代谢物呈现出正相关。对转基因毛状根FtMYB2-OE和FtMYB2-RI进行转录组和靶向代谢组分分析，差异基因分析结果表明FtMYB2对荞麦代谢流的调控主要集中在苯丙烷类和类黄酮次生合成代谢途径。通过LC-QTOF-MS代谢组分检测分析，发现FtMYB2调控显著的次生代谢产物主要集中在黄酮烷类和黄酮醇类化合物，其次为异黄酮、黄酮类。酵母单杂交实验表明，FtMYB2可能与黄酮类化合物合成途径中的FtC4H、FtANS和FtRT的启动子部分结合，从而调控其基因表达水平，进而调控荞麦中类黄酮化合物合成途径的代谢。FtMYB2超表达株系一定程度上增加了拟南芥抗逆能力，并增加了拟南芥种子对ABA诱导信号的敏感性，提示该基因可能通过ABA信号调节通路参与非生物胁迫调控。本研究从分子和代谢水平上阐明FtMYB2对荞麦类黄酮合成的调控机制和抗逆功能，为提高荞麦品质和抗性的分子育种提供理论基础。