R2R3-MYB转录因子PmEOBⅠ与PmMYB4引导梅花乙酸苯甲酯合成调控机制研究

Prunus mume can emit volatile organic compounds (VOCs) efficiently as major floral scent, benzenoid components represented by benzoyl acetate can account for more than 90% of total volatiles, for which P. mume is recognized as an excellent material to study the metabolism of benzenoid volatiles. The synthesis regulation and release of benzenoid compounds in flower are hotspots and difficult research topics at home and abroad. The rapid synthesis of volatile benzenoid is closely related to the pathway regulation of petal cells, but the research on the cooperation of driving genes on the benzenoid pathway of P. mume is still absent. Previous studies have supposed that PmEOBⅠ and PmMYB4 may be key genes in the regulation of volatile benzenoid metabolism. This study will combine data of petal cell RNA-seq and ATAC-seq throughout the process of floral volatile formation and emission to obtain credible chromatin open areas, and form the dynamic control on the metabolic network map of PmEOBⅠ and PmMYB4 in P. mume. With overexpression and RNA-interference of PmEOBⅠ and PmMYB4 in the protoplasts of petal and leaf cells, preliminary validation for the positive and negative roles on this metabolic pathway are peformed by the analysis of target gene expression patterns; the dual luciferase reporter system and EMSA assay are used to further verify their regulatory effects on target sites in the genome. This research trend to clarify the functions of PmEOBⅠ and PmMYB4 on their driving mode in the biosynthesis of benzoyl acetate, aiming at laying a benign foundation for molecular design breeding in the Prunus genus.

梅花能极速释放以乙酸苯甲酯为代表的花香成分，其中苯环类物质占总挥发性物质的90%以上。梅花花香的极速合成与花瓣细胞中调控基因的导向作用密切相关，但不同基因配合调控梅花乙酸苯甲酯合成代谢在国内外少有研究。前期研究发现，PmEOBⅠ与PmMYB4可能是梅花花香代谢调控的关键基因。本研究通过释香前后花瓣组织细胞的RNA-seq和ATAC-Seq联合数据分析，获得高置信度的染色体结构开放区域，并绘制出PmEOBⅠ与PmMYB4在梅花乙酸苯甲酯合成代谢网络上的动态调控图谱，获得其调控靶位点。借助原生质体分别过表达和抑制表达PmEOBⅠ与PmMYB4，通过靶基因的表达模式分析，初步验证其对代谢通路的激活/抑制作用；通过双荧光素酶报告体系与EMSA实验，进一步验证其对靶位点的调控作用。阐明PmEOBⅠ与PmMYB4相互配合调控乙酸苯甲酯合成的模式，旨在为李属植物香花的分子设计育种奠定基础。

梅花能极速释放以乙酸苯甲酯为代表的花香成分，花香的极速合成与花瓣细胞中调控基因的导向作用密切相关，关键基因的高表达促成了染色质开放区域结构变异，进而导致细胞命运的分化。本项目中，通过ATAC-seq的应用，我们发现开花过程中细胞在次级代谢前后，不同调控模式的增多，进一步的比较转录组研究表明这种差异可能是MYB对合成途径相关基因的差异表达进行调控。在绘制出两个核心PmMYB在梅花乙酸苯甲酯合成代谢网络上的动态表达规律后，通过获得高置信度的染色质开放区域分析，解析其调控靶位点，构建MYB基因转录调控的全息网络。初步阐明PmMYBs类转录因子间相互配合调控花香物质合成的模式。植物进化出这种性状可能是收到吸引传粉者提高传粉者拜访的频率的驱动，从而提高其授粉成功率。基因组水平上的深入研究对功能性基因的分子机制具有理论意义，为合成生物学提供参考的调控模式，同时相关应用基础研究将会对经济植物产业创新具有重要应用价值。