芽胞杆菌ATC-3菌株降解乙草胺的分子机理研究

The widely use of the herbicide acetochlor could polluted farmland soil，caused serious security risks to animals, plants, human and ecological environment. The dynamic process of residual acetochlor in soil was closely related to the soil microorganisms, so it’s important to research the role of microorganisms in the bioremediation of soil acetochlor residue elimination. In previous research, a acetochlor-degrading strain named ATC-3 was isolated from contaminated soil. When acetochlor was provided as the sole carbon source, the degradation efficiency in liquid medium was more than 85% after 6 days of inoculation with strain ATC-3. In order to find out the process of acetochlor degradation, the ATC-3 strain was used as the object to analyze the utilization of different substrates, study . The stydy of degrading- metabolite will be carried out by GC-MS to analyze the metabolic pathway of acetochlor degradation by strain ATC-3. We also want to obtain the degrading- genes by shotgun and verify its function. The degrading-enzymes will be purified by ammonium sulfate fractional precipitation, anion exchange and Sephadex gel chromatography,and then the enzymatic properties will be analyzed. Through the project, we could understand the molecular mechanism of acetochlor -degradation, which would provide new resources and ideas for the bioremediation of acetochlor contaminated soil.

除草剂乙草胺的大量使用会污染农田土壤，并对动植物、人类和生态环境造成严重的安全隐患。乙草胺在土壤中的降解与土壤微生物密切相关，探索微生物在土壤乙草胺消除作用对于污染土壤的微生物修复具有重要意义。Bacillus sp. ATC-3是本项目组从环境中自行分离的乙草胺降解菌株，在以乙草胺为唯一碳源的基础盐培养基中37℃培养6天可以将100 mg•L-1 的乙草胺降解85%以上。为探索该菌株降解乙草胺的过程，本项目以ATC-3菌株为研究对象，分析其对不同相关底物的利用情况，并通过气相色谱-质谱联用等技术研究降解过程中的各种中间产物，解析菌株降解乙草胺的代谢途径；利用鸟枪法克隆乙草胺降解基因，验证其功能，并通过硫酸铵分级沉淀、阴离子交换和葡聚糖凝胶层析纯化其降解酶，研究其酶学特性。本项目拟在代谢途径以及基因和酶水平上阐明ATC-3菌株降解乙草胺的分子机制，为乙草胺污染的微生物修复提供理论依据。

乙草胺是我国生产量和使用量最多的三大除草剂之一，由于环境中残留的乙草胺会对环境产生不利影响，因此因而寻求环境友好、生态安全的乙草胺残留消除技术显得十分重要。本研究筛选到一株乙草胺降解细菌ATC-3。在唯一碳源培养基中，7d内可将100 mg/L的乙草胺降解80%以上。经16S rRNA 序列分析，将该菌株鉴定为芽孢杆菌属(Bacillus sp.)。菌株ATC-3降解乙草胺的最适温度为 30°C, 最适pH值为7。利用GC-MS分析研究菌株降解乙草胺的代谢过程，并且检测到两种主要的代谢产物，推测ATC-3菌株降解乙草胺的代谢途径。建立ATC-3基因文库，从大约20000个转化子中获得1个阳性克隆，成功克隆到ATC-3菌株降解乙草胺的水解酶基因，命名为atcH。该基因含903个碱基，编码301个氨基酸，分子量32kDa，atcH与CMEPA水解酶基因damH同源性为78%。以pET29a为载体，构建表达载体菌株E.coli BL21(DE3)-(pET29a-atcH)，经诱导培养后，实现了重组酶AtcH在E.coli BL21(DE3)中的异源表达。表达载体经Ni2+-NTA亲和层析纯化后，研究了重组酶的降解特性。atcH等电点为4.6、最适反应温度30℃，最适pH为7，金属离子Mg2+能促进乙草胺降解酶atcH的酶活，而Zn2+，K+对atcH的酶活有抑制作用本项目目前在投中文核心论文1篇，申请发明专利1项，顺利完成了本项目研究内容。项目实施有利于对环境中的乙草胺降解提供理论依据。