除草剂二甲戊灵微生物降解途径及降解关键酶基因克隆

Pendimethalin is an important and promising dinitroaniline herbicide. It is the third largest herbicide in the world and widely used in farmlands of Jiangxi province and even across the country. Pendimethalin is very stable and persist in the environment, and threatens the safety of the ecosystem and human health. Besides, the degradation and transformation mechanisms of pendimethalin is not clear for now. Microbial metabolism is the most important factor in the degradation of pendimethalin in soil. Therefore, it is necessary to study the metabolic pathway, degradation mechanism, and microbial remediation progress of pendimethalin. Previously, we isolated and identified a pendimethalin-degrading bacterium Bacillus subtilis Y3 with high degradation efficiency, and cloned a nitroreductase gene pnr that was responsible for the initial degradation step of pendimethalin. Further studies aims at identifying the middle metabolites of pendimethalin by strain Y3 through the methods of analytical chemistry; cloning the genes involved in the downstream degradation process of pendimethalin through transposon insertion mutation, degrading protein(s) purification, and peptide fingerprint analysis; studying the transcription level of the degrading genes, and the enzymatic characteristics and kinetic constants of the degrading proteins. The coming results will reveal the entire metabolic pathway of pendimethalin by strain Y3 and the genes/enzymes involoved in the process. This study will provide the theoretial basis for the study on the transformation mechanism and ecological security of pendimethalin in soil, and for the microbial remediation of pendimethalin residual contamination in the environment.

二甲戊灵是一种广谱高效除草剂，用量占全球第3位，在江西省乃至全国广泛使用。但作为一种二硝基苯胺类化合物，其性质稳定，残留期长，对水生生物高毒并有潜在致癌性。大量研究表明微生物降解代谢是土壤中二甲戊灵消失的最主要因素，然而目前二甲戊灵微生物降解的完整过程和分子机制还不清楚，严重制约了对其环境行为和生态毒理的研究。Bacillus subtilis Y3是一株高效的二甲戊灵降解菌，前期工作鉴定了该菌降解二甲戊灵的部分途径，克隆到负责二甲戊灵第一步降解的硝基还原酶基因pnr。本项目拟通过分析化学的方法鉴定二甲戊灵下游代谢产物；通过随机插入突变、降解酶蛋白纯化及肽指纹图谱分析等方法克隆参与二甲戊灵下游降解过程的相关基因；研究降解基因的转录和降解关键酶的特性与动力学，揭示菌株Y3降解二甲戊灵的代谢途径及参与其中的基因和酶，为深入研究二甲戊灵在土壤中的转化规律、生态毒理及微生物降解修复提供理论依据。

二甲戊灵是一种广谱高效的二硝基苯胺类除草剂，在江西乃至全国范围被广泛使用，其化学性质稳定，残留期长。二甲戊灵在环境中残留对生态环境安全和人类健康都存在威胁，而微生物的代谢作用是消除环境中二甲戊灵残留的主要作用之一。但是目前二甲戊灵微生物降解的全过程和分子机制还没有得到阐明，这严重制约了对其环境行为和生态毒理的研究。.项目前期揭示了Bacillus subtilis Y3起始降解二甲戊灵将其转化为6-氨基二甲戊灵，并且克隆到了参与该过程的二甲戊灵硝基还原酶基因pnr。本研究以6-氨基二甲戊灵为降解底物，通过定时取样、UPLC-MS/MS等技术方法，从菌株代谢和分析化学水平上阐明菌株Y3可将6-氨基二甲戊灵脱N端烷基转化为3-硝基-4,5-二甲基-1,2-苯二胺。通过硫酸铵分级沉淀、native-PAGE凝胶电泳、蛋白质肽指纹图谱分析和基因组信息比对分析得到两个细胞色素P450加氧酶编码基因da1544和da2101、一个铁氧还蛋白NADP还原酶编码基因fr2829和一个铁氧还蛋白编码基因fd857，目前正通过基因表达和基因敲除对所得的几个基因进行功能验证。本项目研究丰富了二甲戊灵的微生物降解途径，后续研究也将进一步揭示微生物降解二甲戊灵的分子机制。.