高效菌株SH14降解嘧菌酯的分子机制研究及应用

Azoxystrobin is one of the most frequently used fungicides worldwide. However, persistent use of the pesticide has resulted in serious environmental contamination problems and human healthy concerns. Microbial degradation has received increasing attentions as an efficient, cost-effective and safe approach to clean up contaminated environments. In our previous studies, one highly efficient azoxystrobin-degrading strain SH14 was isolated and identified as Ochrobactrum anthropi. To our knowledge, this is the first azoxystrobin-degrading microorganism. In addition, its degradation characteristics and the optimum degradation conditions were investigated. However, the degradation mechanism has not been investigated so far and remains unclear. In this study, we aim to screen for azoxystrobin-degrading genes from strain SH14 using mariner transposon random mutation. The transposon mutants without hydrolysis halo were screened on gentamycin-LB agar plates. The degrading gene sequences were amplified by Tail-PCR, then were cloned and expressed in Escherichia coli BL21. After point mutagenesis, bioinformatics analysis and GC-MS analysis of the degradation products, the conserved catalytic domain of the degrading enzymes was confirmed. The results may help to unveil the degradation mechanism of strain SH14. By using EP-PCR, the stable and efficient mutants were obtained to use for azoxystrobin degradation on cabbage and soils. These results will enrich the contents of pesticide toxicology and microbial degradation, and provide the key technical support for cleaning up azoxystrobin-contaminated environments.

嘧菌酯是一种应用极广泛的杀菌剂，其残留污染及危害问题日益凸显。微生物降解以其高效、廉价、安全等特点，成为处理农药残留的有效措施。在前期研究中，我们从环境中分离获得一株嘧菌酯高效降解菌SH14，经鉴定为苍白杆菌(Ochrobactrum anthropi)，并就其降解特性和降解条件进行深入研究，但该菌株的降解机理尚不清楚。本项目拟在前期研究基础上，运用转座子mariner随机插入突变的方法，经庆大霉素抗性筛选和降解过程中产生水解圈与否筛选降解功能丧失的突变体，并利用Tail-PCR技术扩增获得降解基因序列，同时进行诱导表达和蛋白纯化。通过生物信息学分析、定点突变及GC-MS分析酶催化降解产物，确定降解酶催化反应中心，阐明嘧菌酯降解机制。通过易错PCR随机突变，筛选降解稳定、高效突变菌应用于农药残留治理研究。研究结果将丰富农药毒理学及微生物降解内容，为嘧菌酯残留污染治理提供关键技术支撑。

嘧菌酯是一种应用极广泛的杀菌剂，其残留污染及危害问题日益凸显。微生物降解以其高效、廉价、安全等特点，成为处理农药残留的有效措施。在前期研究中，我们首次从环境中分离获得一株嘧菌酯高效降解菌SH14，经鉴定为苍白杆菌(Ochrobactrum anthropi)，并就其降解特性和降解条件进行深入研究，但该菌株的降解机理尚不清楚。本项目在前期研究基础上，运用转座子mariner随机插入突变的方法，经庆大霉素抗性筛选和降解过程中产生水解圈与否筛选降解功能丧失的突变体，并利用Tail-PCR技术扩增获得降解基因序列，同时进行诱导表达和蛋白纯化。通过生物信息学分析、定点突变及GC-MS分析酶催化降解产物，确定降解酶催化反应中心，阐明嘧菌酯降解机制。通过易错PCR随机突变，筛选降解稳定、高效突变菌应用于农药残留治理研究。研究结果将丰富农药毒理学及微生物降解内容，为嘧菌酯残留污染治理提供关键技术支撑。