VvABF2介导葡萄果实白藜芦醇合成的转录调控机制解析

Due to the disease-resistance on plants and beneficial effects on human health of resveratrol, numerous studies have been focused on the regulation mechanism of its synthesis. Abscisic acid (ABA) could induce the accumulation of resveratrol in grape.VvABF2, an ABA-dependent transcription factor and associated with grape ripening, over-expressed in grape cell suspension could promote the increase of resveratrol concentration. However, the transcriptional regulatory mechanism of VvABF2 mediated resveratrol synthesis in grape berries is still unclear. In this research, we explore the relationship between the expression of VvABF2 and the promoter of VvMyb14 and VvSTS based on the analysis of bioinformatics. Then, studies on the interaction between VvABF2 and VvMyb14, VvSTS in vitro (yeast one-hybrid and electrophoretic mobility shift assay) and vivo (dual-luciferase reporter assay and chromatin immunoprecipitation) will be carried out. Finally, CRISPR/Cas9 technology combining with the grape cell suspension transformation system will be used to further study the role and regulatory mechanism of VvABF2 in the process when it mediates resveratrol synthesis in grape berries. This research will have important theoretical significance not only for studying the function of ABA signal transduction pathway in the process of resveratrol synthesis, but also for the improvement of grape varieties in the future.

白藜芦醇（Resveratrol, Res）对植物本身的抗病作用和对人体的保健作用，使其合成调控机制方面的研究成为近年来的热点。ABA能够诱导葡萄Res的合成，VvABF2是ABA-依赖的与葡萄果实成熟相关的转录因子，其过表达可促进葡萄细胞悬浮系中Res含量增加，但有关VvABF2介导葡萄果实Res合成的转录调控机制尚未阐明。本课题基于生物信息学分析，探究外源ABA处理葡萄果实中VvABF2的表达与VvMyb14及VvSTS启动子之间的关系；结合体外的酵母单杂交和EMSA实验及体内的双荧光素酶报告基因和ChIP-PCR实验，解析VvABF2与VvMyb14及VvSTS的相互作用；利用CRISPR/Cas9技术结合葡萄细胞悬浮转化体系进一步研究VvABF2在介导Res合成过程中的角色和调控机制。本课题对研究ABA信号转导途径在葡萄Res合成过程中的作用，及将来的品种改良都具有重要的理论意义。

基于白藜芦醇（Resveratrol，Res）对植物自身和人体保健都起着十分重要的作用，因而其在葡萄果实中的合成调控是近年来的研究热点。本研究在前期研究基础上，在‘品丽珠’（Cabernet Franc）葡萄的转色期前10天进行外源脱落酸（abscisic acid, ABA）及其合成抑制剂（氟啶酮，Fluridone）处理，研究葡萄果实Res合成与积累的转录调控机制。通过检测葡萄果实中Res含量及相关基因表达分析发现：外源ABA处理能够促进‘品丽珠’葡萄果实提前成熟，果实中Res含量增加, Fluridone处理延迟葡萄果实成熟，果实中Res含量降低；而外源ABA处理后葡萄果实发育过程中Res合成受转录水平的调控，ABA合成基因VvNCED1和VvNCED2表达升高，Res合成相关基因及其调控基因（VvSTS、VvMyb14及VvMyb15）表达升高，抑制剂处理后降低；此外，ABA信号转导途径中的转录因子VvABF2上调，酵母单杂交结果显示VvABF2与VvSTS和VvMyb14有相互作用，即ABA通过VvABF2调控葡萄果实Res合成。因此，本研究不仅可为人工提高葡萄果实Res含量奠定理论基础，也可极大降低Res在人体保健和疾病治疗方便的成本。