葡萄果实黄酮醇合成酶对UV-B的组织特异性响应机制研究

Flavonol synthase (FLS) is the key enzyme in controlling the synthesis of flavonols, which is a group of vital secondary metabolites in grape berry, study on its response mechanism to exogenous regulation is the key point to control the nutritional and sensory quality of grape berry and wine. Ultraviolet-B (UV-B) is an important regulation technology for plant secondary metabolite, which plays an important role in the regulation of flavonol accumulation, but its regulation mechanism has not been fully elucidated. In this study, by using the artificial simulation of UV-B technology and in vitro treatment technology, the external quality and inner quality of grape berries were studied during grape berry development. At the same time, the expression characteristics of FLS in different berry tissues during grape berry development were also studied before and after UV-B irradiation, the tissue-specific response of FLS to UV-B treatment during grape berry development were confirmed from different levels (enzyme activity level, translational level, transcriptional level, subcellular fraction level, and downstream product accumulation) by HPLC, Western blot, RT-PCR and immunogold localization methods, and the tissue-specific response mechanism of FLS to UV-B irradiation during grape berry development were revealed. The research achievements can provide a theoretical basis for further elucidation of both the regulation mechanism of UV-B on plant secondary metabolites and improvement mechanism of fruit quality.

黄酮醇合成酶（Flavonol synthase, FLS）是控制葡萄果实重要次生代谢产物黄酮醇合成的关键酶，研究其对外源调控的响应机制是解决葡萄及葡萄酒营养及感官品质难于控制的关键问题。UV-B是重要的植物次生代谢产物调控技术，对调控黄酮醇积累具有重要作用，但其机理尚未完全阐明。本项目通过人工模拟UV-B辐照及离体处理技术，跟踪葡萄果实发育进程，研究果实的外观及内在品质变化，同时以FLS为切入点，就辐照前后葡萄果实不同组织部位FLS的表达特性展开研究，利用HPLC、Western blot、RT-PCR、胶体金免疫电镜定位等方法从酶活性、翻译、转录、亚细胞分布和下游产物变化等层次证实果实发育过程中FLS表达对UV-B处理呈组织特异性响应，揭示葡萄果实发育过程中FLS对UV-B的组织特异性响应机制，从而为进一步阐明UV-B对果实次生代谢产物的调控机制和品质提升机制提供新的理论依据。

黄酮醇生物合成关键酶是控制葡萄果实重要次生代谢产物黄酮醇合成的关键酶，研究其对外源调控的响应机制是解决葡萄及葡萄酒营养及感官品质难于控制的关键问题。UV-B是重要的植物次生代谢产物调控技术，对调控黄酮醇积累具有重要作用，但其机理尚未完全阐明。本项目通过人工模拟UV-B辐照及离体处理技术，跟踪葡萄果实发育进程，研究果实的外观及内在品质变化，通过研究UV-B照射对葡萄果实黄酮醇生物合成途径关键酶（PAL,C4H,4CL,CHS，FLS）的影响，从酶活性、转录和下游产物变化等层次证实果实发育过程中葡萄果实黄酮醇生物合成关键酶对UV-B处理呈现组织特异性响应。实验结果表明采后适当剂量UV-B照射葡萄果实在不影响果实硬度及果梗褐变指数等外在品质的情况下可显著提高果实可滴定酸、总酚、总黄酮及总黄酮醇含量，减缓可溶性固形物及Vc降解，可较好改善酿酒葡萄采后品质。UV-B对葡萄果实采后品质的改善作用呈现明显的照射剂量依赖性和孵育时间依赖性。通过HPLC法对葡萄果实中的12种黄酮类化合物进行定性定量研究，发现适当剂量UV-B照射对黄酮醇种类无影响，但对黄酮醇含量具有重要影响，且各化合物的响应模式不同。葡萄果实中黄酮醇类化合物主要积累于葡萄果皮中，而在果肉及种籽中含量极低，说明UV-B对葡萄果实黄酮醇积累的诱导作用表现为组织特异性。适当剂量UV-B照射可显著促进生物合成途径上C4H,4CL,CHS和FLS基因的表达，表达水平分别为对照的5.01倍、1.89倍、3.55倍和28.66倍，且各关键基因的表达与UV-B之间呈现明显的照射剂量依赖性和照射时间依赖性。酶活性研究结果显示，UV-B对葡萄果实黄酮醇生物合成关键酶的调控可通过影响酶活性进行，且酶活性的变化呈现明显的发育阶段依赖性和组织特异性。本研究揭示了葡萄果实发育过程中黄酮醇生物合成关键酶对UV-B的组织特异性响应机制，从而为进一步阐明UV-B对果实次生代谢产物的调控机制和品质提升机制提供新的理论依据。