五倍子抑制蓝莓采后灰霉病的物质组分及其作用机理研究

Blueberry are juicy and thin-skinned，susceptible to fungal infection and results in the decay of whole fruit, which restricts the development of blueberry industry. Because of its low toxicity and high efficiency, natural botanical preservative become one of the future directions for fresh fruit preservation. In our previous study, we found that ethanol extract of Galla chinensis has a strong inhibitory effect on pathogen (Botrytis cinerea) causing gray mold on postharvest blueberry. However the chemical constituents and mechanism of antifungal activity are still not clear. In this study, silica gel column chromatography and HPLC/MS/MS will be combined with bioactive tracer to isolate and identify the main chemical constituent from Galla chinensis, which responsible for the inhibitory effect on blueberry Botrytis cinerea. Moreover, in vitro, in vivo, fungal ultrastructure, membrane structure, and respiratory metabolism will be studied to identify the mechanism of the anti-Botrytis cinerea effect of Galla chinensis. Additionally, transcriptomics sequencing will be used to study the molecular mechanism. Results from this study are beneficial to reveal the antifungal substances from Galla chinensis and its possible mechanism of action to inhibit the blueberry fungus, providing technical and theoretical support for the development of new preservatives，effectively promoting the blueberry industrial development.

蓝莓果实皮薄多汁，采后易被微生物侵染而致使全果腐烂变质，是制约蓝莓产业发展的瓶颈。植物源抑菌剂以其天然低毒高效等优点成为今后水果保鲜的重要方向之一。前期研究发现五倍子醇提物对蓝莓采后灰霉病致病菌(灰葡萄孢霉)有较强抑制作用，但其抑菌的关键活性组分和作用机理尚不明确。本项目拟利用硅胶柱层析、HPLC/MS/MS并结合生物活性示踪，分离、纯化五倍子中对蓝莓灰葡萄孢霉起抑制作用的关键化学成分；拟分别从离体、活体、细胞超微结构、细胞膜结构、呼吸代谢角度研究五倍子抑制蓝莓灰葡萄孢霉的作用机理；并进一步利用转录组学手段解析五倍子抑制灰葡萄孢霉的分子生物学基础。研究结果有利于揭示五倍子抑制蓝莓致腐真菌的活性物质组分和作用机理，为研发安全的抑菌保鲜剂提供技术和理论支持，有效促进蓝莓产业健康发展。

蓝莓采后易被微生物侵染而引起腐烂变质，前期研究发现五倍子提取物可以有效抑制蓝莓采后主要致腐菌灰葡萄孢霉的生长。本研究通过筛选不同产地五倍子抑菌活性，获得湖南地区五倍子具有最高抑菌活性，经活性示踪结合分级萃取和HPLC分离制备，获得五倍子提取物中具有抑菌活性的单体，经质谱分析明确该成分为单宁类物质。研究灰葡萄孢霉在五倍子抑菌成分胁迫下，培养液电导率、可溶性蛋白质、膜脂质过氧化等指标变化，结合扫描电镜和透射电镜分析菌丝细胞表面形态及细胞超微结构变化规律，从超微观角度明确五倍子通过破坏细胞壁与细胞膜的完整性达到抑菌效果。此外，灰葡萄孢霉细胞内琥珀酸脱氢酶（SDH）、苹果酸脱氢酶（MDH）和ATP酶等关键酶活性经五倍子抑菌成分处理后显著降低，可见五倍子抑菌成分可影响灰葡萄孢霉呼吸应答代谢。通过转录组测序，筛选出1600余个差异表达基因，结合pathway分析，明确五倍子抑菌成分抑制灰葡萄孢霉的可能作用通路和靶点。本研究还以蓝莓果实的腐烂率、失重率等为指标，探究了五倍子抑菌成分对蓝莓采后果实品质和抗病性的影响，五倍子单宁处理一方面可以抑制灰葡萄孢霉的生长，另一方面可强化蓝莓果实的防御系统，有效减缓灰葡萄孢霉对蓝莓的侵染，蓝莓果实腐烂率降低72%以上，显著延长了保鲜期。