拮抗木霉菌产生敌敌畏降解酶的诱导途径与作用机制研究

The large amounts of residues of organophosphate pesticides in agro-ecological system has became a great threat to the safety of environment and food and human as well as livestock, therefore, we use dichlorvos(DDV) as a model of organophosphate pesticide to study the mechanism of Trichoderma to produce DDV degrading enzymes under induction of DDV and to enzymatically degrade DDV ,the work if well completed would be great significant to globally understand molecular mechanism of enzymatic degradation of organophosphate pesticides by beneficial fungi. First of all, by using approaches of enzymology and immunochemistry, we identify the types of intracellular and extracellular degrading enzymes of diclorvos (DED)and DDV degraded intermediates as well as their releasing timing sequence. Secondly, the subcelluar localization of DED and identification of cell membrane molecular path allowing DDV and their degraded intermediates entrance into Trichoderma cell would be conducted in the aspect of immunochemistry ,membrance potential and gene function. Thirdly, based on previous screening at proteomic and transcriptomic level for finding differential proteins or genes related to sensing the induction of DDV and its degraded products,we furhter identify the crucial regulators involved in the induction of DED production and degradation process. Forthly the interrelation of those sensing regulators and their contribution to the induction of DED production are further investigated by yeast two hybrid or one hybrid method as wel as sited mutation to explore the molecular network and protein interaction domain involved in the mediation of DED production and activity in Trichoderma. Eventually, a comprehensive molecular model would be generated of DED induction and action mechansim. The study will fill a gap for investigating molecular and biochemical mechanism of fungi including Trichoderma to degrade organosphosphate pesticide residues in environments.

有机磷农药在生态系统中残留对农产品和人畜安全构成较大威胁，因此以敌敌畏为有机磷农药的模式分子，研究拮抗木霉菌诱导产生敌敌畏降解酶及作用机制具有重要意义。本研究通过酶学和分析化学技术，鉴定敌敌畏诱导下拮抗木霉菌胞内和胞外生产敌敌畏降解酶种类和时序，以及敌敌畏降解释放的中间产物种类和时序；通过免疫化学和基因gfp标记技术，明确降解酶的亚细胞定位；通过免疫化学技术、膜电位化学技术和基因功能分析技术，鉴定敌敌畏及降解中间产物穿透木霉细胞膜通道分子特性；在已筛选的木霉菌应答敌敌畏诱导作用和降解敌敌畏的相关蛋白或基因的基础上，鉴定调控木霉菌诱导产生敌敌畏降解酶和降解酶活性的关键因子（基因）；通过酵母单、双杂交和定点突变等技术鉴定调控因子与降解酶蛋白（基因）间相互关系和互作位点，明确调控网络结点基因。提出木霉菌诱导产生敌敌畏降解酶分子途径和酶促降解调控机制，填补真菌酶促降解有机磷农药分子机理研究空白。

通过蛋白质组学、转录组学和相关分子生物学方法对深绿木霉（Trichoderma atroviride）T23降解敌敌畏的作用机理进行了深入研究，主要包括五方面内容：⑴通过木霉菌应答敌敌畏胁迫的差异蛋白质组学分析，克隆T23的hex1基因，利用基因敲除及互补技术、电镜观察和生理生化分析等手段鉴定hex1基因的功能，发现hex1通过调控木霉菌细胞膜系统和抗氧化系统实现其耐受敌敌畏功能；⑵基于RNA-seq技术分析hex1调控木霉菌应答敌敌畏胁迫反应的基因表达情况，共获得5382个差异表达基因，鉴定了137个可能的hex1调控下敌敌畏胁迫应答基因，其中25个参与转运过程，分析不同突变株中7个候选ABC转运蛋白基因在敌敌畏胁迫下随时间的表达变化，发现PDR、MRP、MDR三个亚家族中各自基因具有类似的表达趋势，其中TaPdr2基因在6 h的表达量上调3000倍以上；⑶观察比较了野生株T23、敲除株细胞色素P450基因突变株（敲除突变株Δcyp11-19、过表达株OE-cyp11-19和回补株Δcyp11-19-C）对敌敌畏降解率的差异。通过每隔24h测定敌敌畏含量，发现菌株OE-cyp11-19敌敌畏降解能力最高，其次为Δcyp11-19-C且其同野生型T23之间的降解效率差异不显著，而敲除株Δcyp11-19降解敌敌畏的能力最低，但均高于敌敌畏的水解效率；⑷木霉菌基因组BLAST分析和分子对接，发现了与哺乳动物对氧磷酶PON1具有高度同源性的蛋白PON6可能与敌敌畏降解有关。通过同源克隆获得pon6基因，并证明其具有降解敌敌畏的功能，eGFP标记确定了PON6蛋白存在于细胞质中。最后通过RT-PCR和qPCR初步分析深绿木霉中pon6，cer 1、hex 、cyp11在降解敌敌畏过程中的相互关系；⑸明确了敌敌畏及其中间降解产物种类进入胞内主要转运通道的分子特征，通过简并引物同源克隆T23中的2个ABC转运蛋白基因TaPdr2和TaMrp1，利用ATMT技术和相关生理生化分析后发现敲除TaPdr2后，T23对敌敌畏的耐受能力显著降低，敌敌畏胁迫下菌丝麦角甾醇含量降低约50%,电解质外渗变化不显著；⑹在木霉菌胞外检测降解敌敌畏的产物，分别是2,2-二氯乙醇，二氯乙酸，乙酸二氯乙酯和磷酸二甲酯。由此，我们提出来木霉菌降解敌敌畏的主要分子机理。