邻氨基苯甲酸合酶调控香菇耐热性的分子机制研究

Heat stress often infects seriously the mycelia growth, even causes mycelia apoptosis of Lentinula edodes. However, various strains generally show differences in the thermos-tolerance. In our previous research, the protein level of anthranilate synthase (TrpE) was significantly up-regulated in thermo-tolerant strain S606 after heat treatment at 40℃ for 24 h whereas there was a down-regulated transcriptional level. Moreover, the thermo-tolerance of mycelia would be significantly enhanced when the anthranilate is added into medium with strain S606. However, the mechanism of this protein TrpE involved in heat tolerance in L. edodes has not been determined until until now. In this proposal, the function of trpE would be identified via gene over-expression and gene silencing, and the target proteins that were interacted with the TrpE would be baited out by Co-Immunoprecipitation. Putative protein-interactions would be validated via in vitro and in vivo for explicating the interaction mechanism during the thermo-tolerance process. On the other hand, the candidate gene expression levels and metabolite dynamics engaged in tryptophan synthesis metabolic pathways would be evaluated, and the function of heat-tolerance related genes would be identified to reveal the heat-tolerance mechanism related to tryptophan biosynthesis metabolic pathways in L. edodes. This proposal have great significance in theoretical and applied prospect on the heat-tolerance molecular mechanism, genetic basis and breeding thermo-tolerant varieties of the edible mushroom in the future.

高温严重影响香菇菌丝生长，甚至导致菌丝凋亡，但香菇不同菌株的耐热能力明显不同。申请者在前期研究中发现，香菇耐热菌株S606在40℃胁迫24 h之后，邻氨基苯甲酸合酶蛋白表达水平显著上升，但转录水平却明显下降，尤其是在培养基中添加邻氨基苯甲酸能显著提高菌丝耐热能力，但邻氨基苯甲酸合酶影响香菇耐热性的机制却并不清楚。本项目拟一方面采用基因过表达和基因沉默方法鉴定trpE的基因功能，应用免疫共沉淀技术筛选与TrpE互作的蛋白，并开展蛋白间的体内体外互作验证，明确TrpE与互作蛋白共同影响香菇耐热性的机制；另一方面采用组学方法，分析与TrpE相关的色氨酸合成代谢途径中酶基因及代谢产物的表达水平，对与耐热性相关的酶基因进行功能分析，揭示色氨酸合成代谢途径影响香菇耐热性的机制。本项目对深刻揭示真菌耐热机制和遗传基础，培育食用菌耐热品种，具有重要的科学意义和实践价值。

高温常引起香菇菌丝体凋亡，造成重大的经济损失。申请人前期研究表明，高温胁迫后香菇耐热菌株S606邻氨基苯甲酸合成酶（TrpE）蛋白表达水平显著上调，而热敏感菌株Y3357蛋白表达水平极低，外源添加邻氨基苯甲酸能显著提高耐热香菇菌株S606菌丝的耐热能力。本项目通过基因过表达和基因沉默对香菇trpE基因功能进行研究，得到S606菌株LetrpE基因的RNAi转化子和超表达转化子；LetrpE基因RNAi转化子在40℃处理24h后，检测到从色氨酸到吲哚-3-乙酸合成途径中LetrpB、LeTam-1和LeYUCCA基因表达量均出现了下调，且热处理后能恢复生长，但野生型S606菌株可以恢复生长。LetrpE基因超表达转化子相较于出发菌株色氨酸代谢相关基因LetrpB和LeTam-1的表达水平均上调 2 倍以上，生长素合成基因LeYUCCA表达水平上调10倍以上；超表达转化子在41℃热处理24h后可以恢复生长，但出发菌株S606不能恢复生长。上述结果表明，香菇LetrpE基因是色氨酸代谢和吲哚乙酸代谢的关键基因，其表达水平变化能够影响香菇菌株的耐热性。通过对色氨酸代谢途径另两个酶基因LetrpB和LetrpD的功能分析发现：LetrpB和LetrpD基因也参与了香菇菌株S606的热胁迫应激反应。本项目通过酵母双杂交实验筛选到与香菇LetrpE互作的蛋白LeDnaJ07，并通过酵母双杂交点对点及BiFC实验验证了LetrpE与LeDnaJ07之间的互作；通过基因过表达和基因沉默对香菇LeDnaJ07基因功能研究表明，LeDnaJ07表达水平的变化可以影响香菇菌丝的耐热性、木霉抗性及胞内IAA含量，结合LeDnaJ07和LetrpE具有蛋白互作结果，说明LeDnaJ07可通过影响IAA的合成调节香菇菌丝的耐热性。本项目发现外源生长素类似物可通过调节细胞内氧化损伤提高热敏感菌株YS3357的耐热性。.综上所述，本项目对邻氨基苯甲酸合酶（EC:4.1.3.27）基因trpE的功能以及与TrpE互作的蛋白并对互作蛋白的功能进行了研究；明确与TrpE相关的色氨酸合成代谢途径与香菇耐热性相关的关键酶及其基因功能。项目资助发表SCI论文5篇，中文核心论文4篇。培养博士研究生2名，硕士研究生4名。项目投入经费60.0000万，支出57.9242万元，各项支出与预期相符，剩余2.0758万元。