【作者】 刘秀明；

【导师】 张金霞；

【作者基本信息】 中国农业科学院 ， 微生物学， 2013， 博士

【摘要】 海藻糖在微生物中不仅是一种储存性物质，也是应激保护性物质。海藻糖可以稳定细胞膜和蛋白质天然结构，提高抗逆性。海藻糖在非生物胁迫抗逆响应中作用，正受到越来越广泛的重视。我国食用菌仍以农业设施生产为主，常受到气候变化的温度波动影响。突发的高温气候常导致菌丝生长缓慢，子实体产量和品质下降。因此，深入研究食用菌的高温胁迫响应机制，将为食用菌丰产优质技术研发奠定理论基础。本研究以侧耳属主要栽培种糙皮侧耳（Pleurotus ostreatus）和肺形侧耳（Pleurotuspulmonarius）的不同热敏感型菌株为材料，研究了外源海藻糖对高温胁迫下菌丝体内活性氧产生和抗氧化酶类活性变化的影响，以及不同热敏感型肺形侧耳高温胁迫及恢复培养条件下胞内海藻糖代谢途径在酶活性和基因水平的响应。主要研究结果如下：高温胁迫（最适生长温度+12℃）下，外源海藻糖处理对糙皮侧耳和肺形侧耳菌丝体内与抗氧化损伤有关生理指标的影响趋势相同。外源海藻糖处理可以降低高温下TBARS的显著积累，缓解膜的氧化伤害，表现为O-2和H2O2的积累减少，诱导脂氧合酶（LOX）活性降低。外源海藻糖对抗氧化酶活性产生不同的影响：保护超氧化物歧化酶（SOD），抑制过氧化氢酶（CAT）、抗坏血酸过氧化物酶（APX）和谷胱甘肽还原酶（GR），不影响过氧化物酶（POD）。外源海藻糖提高抗热性与其保护抗氧化酶SOD活性和直接清除O-2和H2O2的作用有关。海藻糖提高生物的抗热性与浓度有关，对热敏感型菌株的保护效应高于其对耐热型强菌株的保护效应。正常生理条件下，肺形侧耳不同热敏感型菌株的胞内海藻糖本底值无显著差异。高温胁迫前期海藻糖大量积累，热敏感型菌株显著高于耐热型菌株，海藻糖-6-磷酸合成酶（TPS）和合成海藻糖反应方向海藻糖磷酸化酶（TP）活性较耐热型菌株的增幅更显著。TreP途径和OtsA-OtsB途径都是高温胁迫初期海藻糖合成的主要途径。胁迫中后期，不同热敏感型菌株的中性海藻糖酶（NTH）和降解海藻糖反应方向TP都参与海藻糖的降解。热敏感型菌株TPS活性保持持续升高，而耐热型菌株TPS活性呈下降趋势。高温胁迫后恢复培养，肺形侧耳不同热敏感型菌株的海藻糖含量迅速降至对照水平。热敏感型菌株的下降速率高于耐热型菌株。但是，TPS活性仍持续稳定升高，海藻糖-6-磷酸酯酶（TPP）是热敏感型菌株OtsA-OtsB途径合成海藻糖的限制酶。恢复期NTH和降解海藻糖反应方向TP活性增加。对高温胁迫及恢复培养期间海藻糖代谢相关基因表达量分析表明，TPS、TPP、TP活性变化与其相应基因的表达量变化一致，而NTH的活性变化与nth基因表达量的持续增加变化不一致。推测海藻糖对高温胁迫的应激响应是通过转录和翻译共同调控。高温胁迫及其后的恢复条件下，不同热敏感型菌株的海藻糖应激响应不完全相同，这主要来自tps基因表达的差异。更多还原

【Abstract】 In microorganisms, trehalose functions both as a reserve carbohydrate and as a stress metabolite.Trehalose accumulation is a crucial defense mechanism that stabilizes proteins and biologicalmembranes during periods of stress. The research focus on the role of trehalose in the abiotic stressresistance responses has attracted more and more attentions.The productions of mushrooms in China are always conducted in the traditional mushroom tents,which makes the mushroom vulnerable to climate fluctuations. Sudden change of high temperatureclimate would inhibit the mycelium growth, impair fruiting, and finally affect the quality of mushroom.Oxidative stress triggered by heat stress is the main reason that leads to the damage. Therefore, in-depthstudy of the thermal response mechanisms would provide a theoretical basis for high yield and highquality of mushroom.We investigated the alleviative effects of exogenous trehalose on heat-induced oxidative stress invarious sensitive strains of Pleurotus ostreatus and Pleurotus pulmonarius, which are the maincultivated species of Pleurotus. The responses of trehalose metabolism at the level of enzyme activityand gene expression during heat stress and the recovery period in the two different sensitive type strainsof P. pulmonarius were also studied. Our conclusions were as follows:Under heat stress (at12℃above optimum growth temperature), exogenous trehalose effectedsimilarly on physiological indexes of resistance to oxidative damage between P. ostreatus and P.pulmonarius. Exogenous trehalose could alleviate the oxidative damage induced by heat stress byreducing the generation rate of O-2and H2O2, the activity of LOX and the content of TBARS.Additionally, exogenous trehalose had much different effects on the activities of enzymetic antioxidants,including the positive effect on the activity of antioxidants SOD, the negative effect on enzymeticantioxidants including CAT, APX and GR, and no effect on enzymetic antioxidants POD under the heatstress. The reason for improved heat resistance was that the exogenous trehalose protected SOD activityand scavenged H2O2and O-2. The protective effect was related to trehalose concentration, and the effecton the heat sensitive strains was higher than that on heat resistant strains.Under normal physiological conditions, the in vivo trehalose content in the heat sensitive strain andthe heat tolerant strain did not show a significant difference. An interesting phenomenon is that thetrehalose content increased first and then decreased finally. The increased extent of activities oftrehalose-6-phosphate synthase (TPS) and trehalose phosphorylase (TP) in the direction of trehalosesynthesis in heat sensitive strain was higher than that in heat tolerant strain during the early stress period.TreP pathway and OtsA-OtsB pathway was the main pathway at the early heat stress. In later stressperiod, neutral trehalase and TP were involved in trehalose degradation in two strains. Meanwhile, theTPS activity increased continuously in heat sensitive strain, while reduced in heat tolerant strain.The trehalose content of P. pulmonarius strains rapidly decreased to the control level under heatstress recovery conditions and the decreasing rate in heat sensitive strain were faster than that in heat tolerant strain. But, the TPS activity increased continuously in heat sensitive strain, and TPP was arestriction enzyme in the OtsA-OtsB pathway. Neutral trehalase and TP were involved in trehalosedegradation in two P. pulmonarius strains under heat stress recovery conditions.Analysis of trehalose metabolism-related gene expression during the heat stress and recoverycondition suggests that, in contrast with the case of TPS, TPP and TP, NTH activity was depressedduring heat stress, even though the mRNA of the nth gene increased. The trehalose stress response toheat was presumed to be regulated at the transcription and the translation levels. The response ofmetabolic pathway of trehalose in different sensitive P. pulmonarius strains to heat stress and recoverywere not exactly the same, and that was mainly caused by the mRNA expression of the tps gene.更多还原