巨大口蘑与双孢蘑菇褐变表现差异的发生机理

Browning is one of the most important factors affecting appearance quality and storability of fruitbody of mushroom. The fruitbody of Tricholoma giganteum exhibits a strong anti-browning property. The fruitbody shows any browning when it is damaged and stored for 40 days at 12 ℃ and 90% relative humidity. However, Agaricus bisporus browns rapidly after 3 days of storage under the same condition. To understand their differential browning,Tricholoma giganteum and Agaricus bisporus will be used as experimental materials. The cellular biology, biochemistry,enzymology and moleculal biology methods are adopted. The project will determine their physiological characteristics postharvest and investigate the location of browning substrates and enzymes in cells and their interreaction. The relationship between reactive oxygen species metabolism and cellular ultrastructure with browning will be analysed. The components, function, fluidity and permeability of cellular membrane are explored. Tyrosinase, a major browning enzyme of two mushrooms, are cloned and differential expression. The objective of the project will try to elucidate the major factors induceing the decompartmentation of the cellular membrane system and influencing membrane permeability, and the relationship with the compartmentation of browning substrate and polyphenol oxidase, then to understand the self-restoration function of membrane systems during storage and the key center leading to enzymatic browning, and finally to elucidate the difference in tyrosinase expression of two mushrooms. The project can understand better the different browning mechanism between Tricholoma giganteum and Agaricus bisporus based on physiological,biochemical, cellular and molecular points. The results obtained will demonstrate the mechanism of mushroom browning which can help to develop new technique during storage and transportation of mushroom.

褐变是食用菌采后商品品质降低甚至丧失的主要原因。巨大口蘑(Tricholoma giganteum)子实体具有明显抗褐变特性（即使损伤在12℃贮藏40天不变色），而双孢蘑菇3天就发生褐变。为了探明两者褐变差异发生机制，本项目以巨大口蘑、双孢蘑菇为研究对象，利用细胞生物学、生物化学、酶学和分子生物学等手段，明确两者采后生理特点，确定导致褐变差异关健酶和主要底物，定位酶-底物亚细胞分布，分析活性氧代谢及细胞超微结构变化与褐变的关系，确定膜脂主要组分及功能，阐明引起膜脂降解、影响膜流动性和导致酶-底物区域化丧失的关键因素，了解膜系统在采后过程中自我修复功能，确定引起酶促褐变的中心部位，克隆关键酶基因，并建立其高效表达系统，明确关键酶基因表达调控因子，从生理、生化、细胞、分子等多层次系统揭示两者褐变表现差异发生机理。研究结果将在理论上阐明食用菌褐变机理，在实践上将推动食用菌贮运保鲜新技术发展。

褐变是食用菌采后商品品质降低甚至丧失主要特征。巨大口蘑(Tricholoma giganteum)子实体具有明显抗褐变特性（即使损伤在12℃贮藏40天也不变色），而双孢蘑菇3天就发生褐变。我们利用细胞生物学、生物化学、酶学和分子生物学等手段，研究了两者褐变差异发生机制。.首先研究了它们与褐变相关酶活性、底物含量变化、膜系统变化、酶-底物亚细胞分布、活性氧代谢及细胞超微结构变化等与褐变间关系，获得了一系列研究成果，明确了巨大口蘑多酚氧化酶、酪氨酸酶表达量低，而双孢蘑菇表达量高是导致两者褐变表现差异主要原因。另外，双孢蘑菇磷脂酶A1和脂氧合酶活性高，自由基含量高，导致细胞膜快速降解，也是两者褐变表现差异原因。.通过分子生物学手段，克隆了4条双孢蘑菇的酪氨酸酶基因和5条巨大口蘑酪氨酸酶基因。由于两种蘑菇酪氨酸酶基因序列间最高同源率只有26.47%，差异太大，很难找到引起两者褐变差异的基因片段。.研究中，发现Fe2+对巨大口蘑酪氨酸酶有激活作用，能加速其褐变，于是通过RNA-seq对Fe2+处理和非Fe2+处理的巨大口蘑进行转录组测序，结果得到两个样本的差异表达基因共计2846条，其中有1796条上调基因，1050条下调基因。差异表达基因从GO分类上主要分布于代谢过程、细胞过程、催化活性、组装过程这4个过程。从KEGG途径上主要分布于代谢途径、次级代谢产物代谢途径；29条酪氨酸酶表达量显著上升，最高上升了2726倍；激活了酪氨酸和黑色素代谢通路；脂肪酸合成代谢被抑制，分解代谢被激活，细胞膜脂受到伤害，褐变加速。.综上所述，巨大口蘑、双孢蘑菇褐变差异表现主要原因：首先是巨大口蘑PPO、酪氨酸酶活性比双孢蘑菇低；其次，巨大口蘑与双孢蘑菇相比，其活性氧体系较稳定，破坏性较强的•OH含量较少，SOD、POD活力高；巨大口蘑底物含量低，而双孢蘑菇底物高，且底物合成代谢通路被激活；最后，巨大口蘑破坏膜脂结构的PLA1酶活力比双孢蘑菇低。这些结果从细胞水平、生理生化水平和分子水平上阐明了巨大口蘑不易褐变、而双孢蘑菇易褐变的机理，为今后食用菌的贮藏、运输和抗褐变育种提供了理论依据和实验材料。