负调控MYB转录因子在葡萄果实花色苷积累中的作用研究

The color of berry skin is an important economic trait for grapes. And it is closely related to the components and content of anthocyanins. Normally, the color of grape berry is stable for dark-colored grape, and it is not obviously affected by environmental conditions. However, the color of grape berry is easily influenced by environmental conditions for light-colored grape, such as shallow pink and red grape cultivars. The study of MybA transcription factors on biosynthesis of anthocyanin in grape berry has explained the formation mechanism of colored and colorless grape, but it is difficult to reveal the coloring mechanism of rich transitional color type. Therefore, we plan to use ‘Red Globe’ and ‘Benni Fuji’ that the color of berry is susceptible to environment and ‘Kyoho’ that the color of berry is stable as the experimental material. After the previous analysis of RNA-Seq, we select 5 candidate MYB transcription factors which may be related to negative regulation of anthocyanin biosynthesis. We will perform a series of molecular biology methods (such as gene sequence and structure analysis, gene expression analysis, analysis of protein interactions and genetically modified validation) to identify the key negative regulation MYB transcription factors, and to analyze their mechanism of action. Through this research, we expect to further explain the regulation mechanism of anthocyanin accumulation in grape berry skin. Then, we try to reveal the molecular mechanism of color variation in grape berry which can be provide theoretical basis for breeding work about the color of grape berry.

果实颜色是葡萄的重要经济性状之一，葡萄果实颜色由果皮中花色苷的组分和含量决定。果实颜色较深的葡萄品种着色稳定，不易受环境条件影响，果实颜色较浅的红色和粉红色葡萄品种的着色易受环境条件影响。MybA转录因子在花色苷合成调控方面的研究较为深入，解释了有色葡萄和无色葡萄的形成机理，但难以解释黄白色和紫黑色葡萄之间丰富过渡着色类型形成的原因。本项目拟以果实颜色受环境影响较大的红色葡萄‘红地球’‘红富士’以及果实着色较稳定的紫黑色品种‘巨峰’为试材，围绕前期研究筛选出的5个花色苷合成候选负调控MYB转录因子，通过基因序列及结构分析、基因表达分析、蛋白互作分析和转基因验证等分子生物学手段，确定葡萄果皮花色苷积累过程中的关键负调控MYB转录因子，并分析其作用机制。通过该研究，期望进一步阐释葡萄果皮花色苷积累的调控机理，揭示葡萄果实颜色变异的分子机制，为葡萄育种提供一定的理论依据。

果实颜色是葡萄的重要经济性状之一，由果皮中花色苷的组分和含量决定。果实颜色较深的葡萄品种着色稳定，不易受环境条件影响，果实颜色较浅的红色和粉红色葡萄品种的着色则易受环境条件影响。MYBA转录因子在花色苷合成调控方面的研究已较为深入，清楚地阐释了黄绿色系和红色系葡萄的变异机理，但难以解释红色系葡萄丰富着色类型的形成机理。本研究基于前期筛选的5个花色苷合成候选负调控MYB转录因子，通过转基因功能验证和作用因子挖掘，明确葡萄果实花色苷合成负调控MYB转录因子的功能及作用机制，并通过葡萄果实颜色QTL精细定位，进一步阐释葡萄果实颜色的分子调控机制。通过研究发现，前期筛选的5个MYB转录因子中，VdMYB14和VdMYB86在葡萄果实花色苷合成中发挥重要的负调控作用。VdMYB14和VdMYB86的表达模式与果皮中花色苷积累过程呈负相关关系。VdMYB14通过下调花色苷合成途径中UFGT基因的表达，上调原花色素途径中LAR和ANR基因的表达，导致类黄酮途径的中间产物由花色苷途径向原花青素途径通量转移，从而使花色苷的合成受到抑制。VdMYB86通过与花色苷合成调控中重要bHLH转录因子MYC1互作，抑制了MBW转录复合体对花色苷合成的调控作用，进而抑制了花色苷合成。基于高密度遗传图谱，结合群体果实颜色表型数据，定位到与葡萄果实颜色和花色苷合成调控相关的10个QTL位点，定位到的17号染色体的位点，正是VdMYB86基因所在的位点，进一步证实了其对葡萄果实颜色的调控作用。通过本研究，明确了VdMYB14和VdMYB86在葡萄果实花色苷合成中负调控作用，初步阐释了其分子调控机制，并定位到多个葡萄果实颜色调控位点，进一步深化了对葡萄果实颜色变异分子机制的认识，对在葡萄分子育种中的应用具有重要指导意义。