VvBZR1与VvHY5互作调控酿酒葡萄花色苷合成的分子机制研究

Anthocyanins are predominant pigments of grape skins, which have important influence on grape berry quality and wine colour. Brassinosteroid (BR) and light signals play an essential role in the synthesis of anthocyanins. This project is based on the fact that exogenous BR could enhance the synthesis of anthocyanins and the expression of key genes in grape skins by signal transduction, and the transcription factors VvBZR1 and VvHY5 are related to the synthesis of grape anthocyanins. This project is proposed to further analyze the synergistic expression of VvBZR1 and VvHY5 and the synthetic genes of grape anthocyanins, and screen the target genes that VvBZR1 and VvHY5 regulate the synthesis of grape anthocyanins. The VvBZR1 and VvHY5 genes would be transformed into Arabidopsis thaliana. Accordingly, the relationship and effect between VvBZR1 and VvHY5 on anthocyanin synthesis would be analyzed through transgenic plants overexpressed by VvBZR1 and VvHY5 and down-regulated mutant plants. Additionally, the yeast two-hybrid, protein co-immunoprecipitation, GST pull-down and bimolecular fluorescence complementary techniques would be used to verify the interaction between VvBZR1 and VvHY5 proteins in vitro and in vivo. Moreover, the tobacco protoplast transient expression would be used to analyze the target genes of grape anthocyanin synthesis regulated by VvBZR1 and VvHY5. Reveal that the molecular mechanism of the VvBZR1 interacts with VvHY5 to regulation anthocyanins synthesis in red wine grape berry.

花色苷是赋予葡萄果实与葡萄酒颜色的主要物质，对其质量有重要影响。油菜素内酯（BR）与光信号对葡萄花色苷的合成起关键作用。外源BR可通过信号转导提高葡萄果皮花色苷积累与关键基因的表达，且转录因子VvBZR1和VvHY5与葡萄花色苷合成有一定关系，在此基础上，进一步分析VvBZR1和VvHY5与葡萄花色苷合成基因的协同表达关系，筛选VvBZR1与VvHY5调节葡萄花色苷合成的靶基因；克隆VvBZR1和VvHY5基因转化拟南芥，通过VvBZR1和VvHY5过表达的转基因植株及表达下调的突变体植株，分析VvBZR1和VvHY5调控花色苷合成的作用；通过酵母双杂交、蛋白质免疫共沉淀和双分子荧光互补技术等验证VvBZR1和VvHY5蛋白在体外和体内的互作；利用烟草原生质体瞬时表达分析VvBZR1与VvHY5共同调节花色苷合成的靶基因，揭示VvBZR1和VvHY5互作调控葡萄花色苷合成的分子机制。

花色苷作为赋予葡萄和葡萄酒颜色的主要物质，影响葡萄酒的口感和营养价值。油菜素内酯（BR）和光照对葡萄花色苷的合成起关键作用，BZR1和HY5分别是BR和光信号转导通路中关键的转录因子，与葡萄果实花色苷合成的关系密切，在此基础上，研究VvBZR1和VvHY5调控葡萄花色苷合成的分子机制。通过对田间生长和离体培养的葡萄果实进行油菜素内酯和光照处理，发现BR和光照能够促进果实成熟和花色苷、原花色素、类黄酮、总酚等酚类物质的合成，提高酿酒葡萄果实品质，同时提高花色苷相关基因及VvBZR1和VvHY5基因的表达量。并且光照是BR处理促进葡萄果实成熟和花色苷合成的必要条件。进一步分析发现VvMYB5b也与葡萄果实花色苷积累及VvBZR1的表达量趋势一致，说明VvBZR1分别与VvHY5和VvMYB5b相互作用调控葡萄果实花色苷的合成。因此，在前期生理实验的基础上，通过亚细胞定位分析发现VvBZR1、VvHY5、VvMYB5b均定位于细胞核；酵母双杂交、双分子荧光互补（BiFC）和免疫共沉淀（Co-IP）试验证明VvBZR1与VvHY5存在蛋白互作，同时酵母双杂交和BiFC试验证明VvBZR1也与VvMYB5b蛋白互作。将VvBZR1和VvHY5基因转入拟南芥，在光照条件下进行EBR处理能够促进植株茎中花色苷的积累，并且显著提高VvBZR1、VvHY5、AtBZR1基因和花色苷合成基因（AtCHS、AtCHI、AtPAP1、AtUFGT和AtDFR）的表达量，说明VvBZR1与VvHY5均有调控花色苷合成的功能。并对BZR-OE和HY5-OE转基因植株进行转录组测序分析，共筛选出与花色苷合成相关的8个上调表达基因，其中4个是参与花色苷合成的基因AtLDOX、AtTT4、AtCHS和AtGL2。酵母单杂交试验发现VvHY5与VvCHS1启动子区域互作，结合生理实验结果分析，VvCHS1是VvHY5在花色苷合成途径中的靶基因，VvHY5通过促进VvCHS1基因的表达促进赤霞珠葡萄果皮花色苷的合成。将VvBZR1和VvMYB5b稳定遗传转化红花烟草和瞬时转化赤霞珠葡萄果实，发现VvBZR1和VvMYB5b基因可以促进花色苷的合成。