食用菌漆酶调控其体内残留农药代谢降解的分子机制研究

Pesticide residues are one of the most important influencing factors on edible fungi safety and their sustainable development. It has significant importance to environmental ecology and social economic by studying the molecular mechanisms of laccases in edible fungi regulating the pesticide residues, exploring the new environmental-friendly genes that can promote pesticide residue degradations and detoxications. This proposal plans to investigate the relationships between laccases metabolism, production of edible fungi and degradations of pesticide residues under the conditions of different edible fungi strains with degradation abilities to pesticide residues, and to analyze the correlations of the temporal and spatial changes of constituents and concentrations of laccases and pesticide residues in edible fungi and to obtain 2-3 edible fungi strains with higher content of laccases. Biotic and abiotic factors that can increase the expressions of key genes during the synthesis of laccases in edible fungi will be optimized. On this basis, this proposal is to identify and clone the genes with the higher degradation capabilities of pesticide residues of chlorpyrifos and chlorothalonil and to construct edible fungi strains with high expressions of key genes by using the technologies of the second generation high-throughput sequencings and gene chips. The degradation and detoxication abilities of strains with high expressions of key genes of laccases and common strains will be comparatively studied and their effects on pesticide residue degradations in edible fungi will be evaluated. Then this study will reveal molecular mechanisms of the laccases from edible fungi regulating pesticide residues degradation and detoxication and build a new approach to control the pesticide residues by enhancing the bioactivity of laccases in edible fungi.

研究食用菌中具有农药降解功能的漆酶对残留农药降解分子机制，发掘调控漆酶表达的环境友好型优良基因，对保障食品安全和农药污染环境修复都具有重要环境生态和社会经济意义。本项目通过研究不同品种食用菌菌株在农药处理后漆酶代谢-食用菌生产-农药降解之间的相互关系，分析漆酶组分、含量时空变化与残留农药降解相关性，并筛选2-3株具有高产漆酶能力食用菌菌株。对促进食用菌漆酶合成过程中关键基因表达的生物和非生物诱导因素进行优化，明确最佳选择性合成表达条件。采用第二代高通量测序和基因芯片技术鉴定、克隆食用菌漆酶中对常用农药毒死蜱和百菌清胁迫响应强烈的关键基因，并构建过表达漆酶关键基因食用菌菌株。比较过表达漆酶关键基因菌株与普通菌株对残留农药降解能力，验证食用菌漆酶关键基因过表达对残留农药降解作用,探明食用菌中漆酶对残留农药降解脱毒分子机制，建立一条基于强化食用菌体内生物活性物质漆酶的农药残留降解控制新途径。

农药的长期不合理使用会导致环境污染问题，在土壤和水体中农药残留问题较为严重时，作物在种植期间不施用或按规定使用农药，也会存在农药残留超标的问题。本实验是通过筛选出能高效产漆酶的食用菌菌株，并优化降解体系，研究食用菌漆酶对毒死蜱、百菌清、莠去津、嘧霉胺和异丙隆这五种农药的降解情况，以期用于农药污染的生物修复及其他作物的农药残留控制。主要研究结果如下：1.对漆酶（Trametes versicolor）的温度和pH稳定性进行研究，结果显示漆酶在pH=5，25℃时最稳定。研究毒死蜱、百菌清、嘧霉胺、异丙隆和莠去津的最适降解条件，结果显示毒死蜱的最适介体为香草醛，嘧霉胺和异丙隆为紫尿酸，莠去津为HBT，百菌清为乙酰丁香酮。异丙隆、嘧霉胺、莠去津的最适pH为4，毒死蜱、百菌清的为pH=5；异丙隆、嘧霉胺的最适降解温度为35℃，毒死蜱、百菌清和莠去津的最适降解温度为30℃。嘧霉胺和异丙隆的最适介体浓度为4 mmol/L,毒死蜱和百菌清的最适介体浓度为6 mmol/L，莠去津为8 mmol/L。2.从27种食用菌菌株中筛选出三株产漆酶较好的菌株（台秀、庆丰518、科杏），并分别对碳源、氮源、pH、诱导剂和金属离子这5个发酵条件进行研究，结果表明，台秀产酶的最适碳源为蔗糖，最适氮源为牛肉膏，最适pH为6，加入藜芦醇和Mn2+、Cu2+、Na+对其产漆酶有促进作用；科杏的最适碳源为可溶性淀粉，最适氮源为蛋白胨，最适pH为6，加入没食子酸、ABTS、亮蓝-R 和Ca2+、Na+离子对其产漆酶有促进作用；庆丰518的最适碳源为果糖，最适氮源为酵母粉，最适pH为6，加入黎芦醇诱和愈创木酚诱导剂和K+、Cu2+离子对其产漆酶有促进作用。3.对科杏、台秀、庆丰518这三种菌株所产漆酶的酶学性质进行研究，庆丰518漆酶最适温度20℃，在15℃时最稳定；科杏所产漆酶最适温度为30℃，在25和30℃时较稳定；台秀所产漆酶的最适酶活为35℃，在30和35℃较稳定。对不同来源的三种漆酶（科杏、台秀、庆丰518）降解同种农药的降解能力进行研究，结果表明，科杏-香草醛体系降解毒死蜱效果最好，台秀-乙酰丁香酮体系降解百菌清效果最好，科杏、台秀、庆丰518漆酶与HBT的降解体系对莠去津降解率都在94%以上，庆丰518-紫尿酸和科杏-紫尿酸体系降解嘧霉胺效果较好，台秀-紫尿酸和科杏-紫尿酸体系降解异丙隆效果较好。