苦荞转录因子FtMYB5和FtMYB6特异性调控黄酮醇合成的分子机制

MYB transcription factors (TFs) are the predominant TFs family acting as master regulator of plant flavonoids biosynthesis. The diversity and complexity of the mechanism on MYB regulating flavonoids metabolism results in different flavonoids accumulation in plant, including the types and contents. Tatary buckwheat (Fagopyrum tataricum Gaertn.) has been widely accepted as an ideal healthy coarse cereal based on the trait of containing abundance of flavonols, especially rutin. Unrevealing the regulation mechanisms of flavonol biosynthesis turn out to be an important way to improve rutin content. Based on the previous study of the two R2R3-MYB TFs classified into SG7 subfamily, FtMYB5 and FtMYB6, this project would perform the function deletion analyses of the promoters of FtMYB5 and FtMYB6 to identify the core elements of hormone-responsive and environment signal-responsive. Ectopic expression of FtMYB5 and FtMYB6 in tartary buckwheat and tobacco would be applied to assess the affection on the synthesis and metabolism of flavonol. Combining of RNA-seq and ChIP-seq with EMSA would be efficient to figure out the cis-element and DNA binding site of FtMYB5 and FtMYB6. This research would shed more lights on unrevealing the difference and diversity on the molecular mechanism of FtMYB5 and FtMYB6–mediated flavonol biosynthesis under different development stage, hormone stimulation and environment signal, which provides more theoretical bases on achieving the molecular breeding for tartary buckwheat with the more rutin or improving its rutin content through the artificial treatment with environment signal.

MYB转录因子是调控植物黄酮合成最主要的转录因子家族，其调控黄酮代谢机制的多样性和复杂性是引起植物黄酮种类和含量差异的重要原因。苦荞作为一种富含芦丁等黄酮醇的特色小杂粮，明晰其黄酮醇合成调控机制是一进步提高其芦丁含量的重要途径。针对本课题组获得的两个苦荞SG7亚家族R2R3-MYB转录因子FtMYB5和FtMYB6，本项目拟通过对其启动子功能分析鉴定其激素和环境因子应答核心元件；通过在苦荞毛状根和愈伤组织以及烟草中的超表达综合评价其对黄酮醇合成代谢的影响；结合转录组测序、CHIP-seq和EMSA等方法，明确其效应基因及其靶DNA结合位点特征。本研究可望揭示在生长发育、激素和环境信号的影响下，苦荞FtMYB5和FtMYB6参与调控黄酮醇生物合成的分子机制及其差异，为实现高芦丁含量苦荞的辅助分子育种和通过环境信号处理提高栽培苦荞芦丁含量供理论依据。

苦荞富含以芦丁为代表的黄酮类物质，是具有重要保健价值的特色杂粮。MYB转录因子在植物黄酮代谢的转录调控中具有关键作用，特别是SG7亚家族R2R3-MYB转录因子可特异性调控芦丁等黄酮醇的合成。前期研究发现，苦荞基因组中仅有两个SG7亚家族R2R3-MYB转录因子FtMYB5和FtMYB6。本项目的细胞学和生理学实验表明，FtMYB5和FtMYB6均具有转录激活活性且定位于细胞核，其基因表达可响应多种激素和环境信号的刺激，且在苦荞生长发育过程和光照处理下与苦荞芦丁含量积累呈正相关。FtMYB5和FtMYB6的启动子研究表明，它们具有不同的组织表达特异性，但其表达均强烈受到光处理的诱导。对转基因烟草分析发现，FtMYB5和FtMYB6可显著上调黄酮醇支路关键酶基因NtFLS的表达，不同程度下调花青素支路关键酶基因NtDFR的表达，从而显著提高了芦丁含量和降低花青素含量，导致花色变浅。转基因苦荞毛状根分析发现，FtMYB5和FtMYB6均可特异性上调FtFLS基因表达，促进芦丁、槲皮素和山奈酚等黄酮醇含量的显著增加。烟草瞬时转化实验显示，FtMYB5和FtMYB6均能激活FtFLS启动子，驱动LUC基因表达。转录组测序分析表明，FtMYB5和FtMYB6调控的差异基因在苯丙烷代谢途径均显著富集，且主要为上调基因。转基因苦荞毛状根代谢组检测分析发现，差异代谢物主要是苯丙烷类和聚酮类化合物，并富集到乙醛酸循环调控和苯丙烷类化合物中黄酮的合成调控。DAP-seq测序分析发现，富集到的FtMYB5和FtMYB6靶基因主要涉及转录调控和DNA结合过程，其中包括多个黄酮支路靶基因motif。尽管FtFLS是可能性最高的靶基因，但未能得到EMSA验证。本项目初步解析了苦荞中特异性黄酮醇正调控转录因子对芦丁合成的转录调控机制，丰富了苦荞芦丁品质形成调控网络，为高芦丁苦荞的辅助分子育种提供候选靶基因。

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