外源一氧化氮对采后树莓果实鞣花酸生物合成代谢的调控作用

Ellagic acid is an important natural phenolic antioxidant that has positive effects on human health. Raspberry fruit is rich in ellagic acid, but the content of ellagic acid in raspberry fruit significantly decreased during the period of frozen storage. Therefore, improving the content of ellagic acid is helpful to improve the economic benefit of raspberry fruit. As a bioactive molecule, nitric oxide (NO) can be induced by exogenous ellagic acid, and can also improve the ellagic acid content in raspberry fruit. However, there are few studies on the regulation mechanism of ellagic acid biosynthesis of raspberries during storage. This project intends to study the effects of NO on the regulation of reactive oxygen balance from both enzymatic and non-enzymatic pathways and the effects on the fruit quality of raspberries. The effects of NO on intermediate metabolites of ellagic acid biosynthesis are studied by means of chemical biology and metabolomics. The regulations by NO on the activity and gene expression of key enzymes in the biosynthesis of ellagic acid are studied using biochemistry, molecular biology and proteomics methods. The modifications of NO on the key enzymes are simulated and kinetic analysis are performed to study the regulation of NO on the functions of the key enzymes. The project studies the regulation by NO on ellagic acid biosynthesis of post-harvest raspberry using the methods of chemical biology, physical chemistry, metabolomics to provide theoretical basis for improving the content of ellagic acid and storage quality of post-harvest raspberry fruit.

鞣花酸是重要的天然酚类抗氧化剂，对人类健康具有积极作用。树莓果实富含鞣花酸，但在冻藏期间鞣花酸含量显著降低。因此，提高采后树莓中鞣花酸含量有助于提升树莓的经济效益。作为生物活性分子，一氧化氮（NO）能被外源鞣花酸诱导产生，也能提高树莓果实中鞣花酸含量。但NO对树莓果实贮藏期间鞣花酸生物合成的调控机理仍少有研究。本项目拟从酶促和非酶促途径研究NO对树莓果实活性氧平衡的调控作用和对果实品质的影响；采用化学生物学和代谢组学手段研究NO对鞣花酸生物合成途径中间代谢产物的影响；采用生物化学、分子生物学、蛋白质组学等方法研究NO对鞣花酸生物合成关键酶的活性和基因表达的调控作用；模拟计算NO对关键酶的修饰作用并进行动力学分析以研究NO对关键酶功能的调控作用。本项目从化学生物学、物理化学、代谢组学等方面研究NO对树莓鞣花酸生物合成途径的调控作用，为提高采后树莓果实鞣花酸含量及贮藏品质提供理论依据。

鞣花酸是存在于众多水果中的酚类化合物，作为天然抗氧化剂能够增强人体抗氧化能力并提高免疫力。树莓果实中富含鞣花酸，但树莓果实极其不耐贮藏，而长期冷藏会显著降低果实中鞣花酸的含量。因此，改良保鲜方法，维持采后树莓果实的贮藏品质，提高树莓中鞣花酸含量，能够增加树莓的经济效益。一氧化氮（NO）作为生物活性小分子能够有效延缓果实的采后衰老，保持果实的贮藏品质。前期研究发现，鞣花酸处理能提高果实内源NO含量，而NO处理也能提高果实内源鞣花酸的含量，由此推测鞣花酸代谢和NO代谢之间可能存在相互调控作用，但NO对贮藏期间树莓果实鞣花酸生物合成的调控机理仍少有研究。本项目用外源NO（气体/溶液）、cPTIO（NO 清除剂）和氮气/清水（对照）处理树莓果实，0°C贮藏，从化学生物学、结构化学、组学等方面对贮藏期间树莓果实抗氧化能力和鞣花酸生物合成代谢进行研究，结果发现：NO处理能够通过直接提高树莓果实中抗氧化酶的活性，促进细胞内活性氧的清除，降低活性氧的含量，提高抗氧化剂（抗坏血酸、类黄酮、总酚、花青素等）的含量，同时提高AsA-GSH循环、苯丙烷类代谢、花青素代谢相关酶的活性来增强细胞的抗氧化能力；NO处理能够提高树莓果实中单宁酰基水解酶（RuTAN）、漆酶型酚氧化酶（RuLAC）、葡糖基转移酶的活性，提高RuTAN、RuLAC 基因表达水平，提高树莓果实中五没食子酰葡萄糖、没食子单宁、鞣花酸的含量，降低没食子酸的含量；结合转录组测序数据获得RuTAN、RuLAC的序列，理论模拟计算发现NO与RuTAN、RuLAC的相互作用主要是通过对半胱氨酸残基进行亚硝基化修饰。此外，项目还探究了NO对树莓果实中氨基酸及糖代谢的影响。通过研究明确了NO对树莓果实抗氧化能力的调控作用，初步阐明了NO对鞣花酸生物合成途径的调控机制，为提高采后树莓果实鞣花酸含量及贮藏品质提供理论依据。