灰霉菌介导蓝莓采后角质层代谢产物诱导果实抗病性机理研究

Blueberries are considered as the most potential small berries with high nutrition content and antioxidant activity. However, the post-harvest fruits are vulnerable to decay, softening and senescence, resulting in significant deterioration following harvest. The cuticle layer (wax) of the blueberry surface is a natural barrier, and our previous study has confirmed that the waxy damage after harvest is closely related to fruit softening. The extracellular enzyme proteins released by plant pathogenic fungi can infect blueberry and degrade the cuticle, which leads to the change of fruit disease resistance. However, little information is available on the generated metabolic products, the influenced signal transduction and pathway, as well as the mechanism of disease resistance induction during the metabolic process of the cuticle. In this project, with techniques as electronic microscope, GC-MS and RNA-seq, we will investigate the changes in cell structure, physiological metabolism involved in disease resistance, hormone metabolism, and pathogenesis-related genes at molecular level in blueberries to elucidate the characteristics and mechanism of disease resistance induced by the cuticle metabolic products mediated with Botrytis cinerea infection, to clarify the relationship between metabolites and fruit resistance response, and to reveal the mechanism of postharvest blueberry cuticle against biological stress. Results from this project will provide direct evidence on the induced disease resistance by these products in blueberries infected with B. cinerea, enrich the knowledge on disease resistance of cuticles, and provide scientific basis for decreasing the loss due to microbial spoilage and prolonging shelf life.

蓝莓因营养丰富，美味可口，具有高抗氧化性等特点而成为最具潜力的小浆果之一。但蓝莓果实采后极易感染病原真菌而导致腐烂，失去食用和商品价值。果实表皮角质层是抵御病原真菌侵染的天然屏障，同时病原真菌通过胞外酶降解果实角质，产生角质代谢产物，改变果实抗病性，但其具体机制尚不清楚。项目拟选择蓝莓果实采后主要病原真菌灰霉菌为研究对象，从细胞结构变化、抗病生理代谢、激素代谢和抗病性相关基因等水平，通过电镜观察、GC-MS分析鉴定、RNA-seq等技术手段，研究灰霉菌侵染蓝莓果实所引起的角质代谢变化规律，进而探索灰霉菌侵染介导的角质代谢产物诱导的果实抗病性和抗病分子机制，阐明代谢产物与果实抗病应答的关系；揭示采后蓝莓角质层抵御生物胁迫作用机制。研究结果将为灰霉菌侵染介导的蓝莓果实角质层代谢产物参与诱导果实抗病性提供直接依据，丰富果实的角质抗病理论，为通过减少病原真菌侵染损失，延长贮藏保鲜期提供科学基础。

蓝莓因其高抗氧化性、保护视力的特点而被消费者喜爱。但蓝莓果实采后极易被病原真菌侵染而腐烂变质。蓝莓果实表皮角质层是抵御微生物侵染的重要屏障。本项目从蓝莓的主要病原真菌灰霉菌侵染果实角质层入手，研究灰霉菌突破蓝莓角质层的侵染机制，同时探究蓝莓果实蜡质组分对灰霉菌的抑菌机制及其在提高蓝莓果采后抗病性方面发挥的重要作用。本项目主要研究结果如下：.1.为了探究灰霉菌胞外角质酶在侵染蓝莓角质层时发挥的作用，从灰霉菌包胞外分泌物中分离纯化得到角质酶，分析了角质酶的酶学特性及其对蓝莓角质层的影响。结果发现，角质酶不仅可以使角质层厚度减少，同时还对蓝莓表皮蜡质产生作用，破坏蜡晶结构并改变其组分，这说明角质酶具有促进灰霉菌侵染的作用。.2.探究了灰霉菌孢子处理对蓝莓角质层的侵染机制。灰霉菌孢子首先附着在蓝莓果实角质层上，萌发分化形成菌丝，并降解菌丝周围的蜡质。GC-MS结果表明灰霉菌侵染引起蓝莓总蜡及蜡质三萜类组分的下降，提高了脂肪酸组分含量，同时引起蜡质合成及转运途径中的基因上调表达，这些结果表明灰霉菌侵染诱导了蓝莓蜡质采后合成与转运。.3.研究了蓝莓蜡质主要三萜物质熊果酸和齐墩果酸对灰霉菌的抑菌机制及其在蓝莓果实采后抗病性方面的作用。结果发现，熊果酸可能通过引起活性氧清除相关酶活性下降，导致灰霉菌细胞内活性氧爆发，改变细胞膜通透性、破坏细胞超微结构发挥抑菌效果；熊果酸、齐墩果酸均可以增强果实自身的抗病性，从而保持果实品质。