虎杖中一个新的MYB转录因子在白藜芦醇合成中的功能研究

Resveratrol have been implicated in many beneficial effects on human health. Although stilbene synthases, the key enzymes responsible for resveratrol biosynthesis, have been isolated and characterized from several plant species, the transcriptional regulation underlying resveratrol biosynthesis is unknown. MYB have been well characterized in many plant species, however it is still not clear about the role of MYB involved in resveratrol biosynthetic pathway in Polygonum cuspidatum. In our previous study, transcriptional responses were investigated in Polygonum cuspidatum leaves before and after exposure to UV-C irradiation using comparative RNA-seq based transcriptomic analysis and digital gene expression profiling analysis. Our study found that a new MYB transcription factor, PcMYB1, could regulate resveratrol biosynthetic pathway in Polygonum cuspidatum. To clarify tissue-specific expression profiles of target gene, some molecular biology technologies will be applied in this work, including expression analyses of PcMYB1 in different tissues by real time fluorescence quantitative-PCR, the construction of the promoter-GUS fusion vector and GUS histochemical analysis. The subcellular localization of PcMYB1 will be analyzed using onion epidermal strips transformation system, and its transcriptional activity will be determined by yeast hybrid system. To assess the biological functions of target gene, overexpression and RNA interference of PcMYB1 in hairy roots will be applied. All together, these results will illustrate the function of PcMYB1 in resveratrol biosynthetic pathway of Polygonum cuspidatum, and provide new important information for the research of regulatory mechanism and the genetic improvement of Polygonum cuspidatum .

白藜芦醇具有重要的药用和商业价值。转录因子对白藜芦醇合成的调控已经成为新的热点研究问题。MYB转录因子功能具有多样性，但目前药用植物虎杖中尚未有关于MYB转录因子参与调控白藜芦醇合成的报道。项目组成员曾以紫外胁迫处理的虎杖叶片为材料，利用转录组测序、数字基因表达谱等技术，在虎杖中发掘了一个新的与白藜芦醇合成相关的MYB转录因子PcMYB1。该项目拟进一步解析其功能，采用现代分子生物学、生物信息学、细胞生物学、转录组学等方法，对转录因子PcMYB1的时空表达特点、亚细胞定位、转录活性、过量表达、RNA干涉等进行系列分析研究，从而进一步明晰转录因子PcMYB1的功能，项目研究的实施不仅可丰富虎杖白藜芦醇合成的转录调控理论，同时还可为虎杖品质遗传改良奠定基础。

药用植物虎杖中尚未有关于MYB转录因子参与调控白藜芦醇合成的报道。本项目成功克隆了与白藜芦醇合成相关的新基因PcMYB1及其启动子。PcMYB1在虎杖根、茎、叶中均有表达，其中根的基因表达水平最低，但根的白藜芦醇含量最高，PcMYB1基因的表达模式与白藜芦醇含量呈负相关。构建PcMYB1启动子驱动的GUS融合表达载体，GUS染色分析表明该启动子具有强的启动子活性并且无组织特异性。亚细胞定位和酵母单杂交实验表明PcMYB1定位于细胞核，具有转录抑制活性。构建PcMYB1基因过量表达载体，利用优化后的发根农杆菌介导的虎杖转基因体系获得转基因毛状根。与对照组CK相比，过表达毛状根中白藜芦醇含量显著降低42%，黄酮含量显著降低28%；过表达毛状根中白藜芦醇合成相关基因表达显著下调(包括PcRS等)；同时黄酮合成相关基因表达均下调(包括PcCHS等)。表明PcMYB1基因过量表达抑制虎杖中白藜芦醇和黄酮的积累。另一方面，构建RNA干涉载体，利用相同方法获得转基因毛状根。与对照组CK相比，PcMYB1-RNAi毛状根中白藜芦醇和黄酮合成相关基因表达上调并且提高了白藜芦醇和黄酮的含量。表明抑制PcMYB1转录促进虎杖中白藜芦醇和黄酮的积累。前人报道的VvMYB14等都属于R2R3-MYB的S2亚家族，在葡萄白藜芦醇生物合成中起正调控作用。然而，本项目研究的PcMYB1属于R2R3-MYB的S4亚家族，是一个转录抑制子，PcMYB1在虎杖白藜芦醇生物合成中起负调控作用。本项目研究结果可以丰富白藜芦醇合成的转录调控理论。