新型农药氯虫苯甲酰胺降解菌筛选及其降解机理研究

Chlorantraniliprole is one of the most frequently used insecticides in the world with new mechanism of action. It long persists in the environment, which leads to the presence of residual cumulative risk. Biodegradation is a promising strategy in safety control of the pesticide for its safe, efficient and inexpensive application. In our previous studies, a microbial flora can degrade chlorantraniliprole has been accumulated from the environment. In this study, we aim at further isolation and identification of one highly efficient chlorantraniliprole -degrading strain. The gene libraries will be constructed and positive colony will be screened on ampicillin-LB agar plates. The degrading gene will be amplified and sequenced. The target gene function can be predicted according to the bioinformatics study. The induce target gene expression using Pet29(a) vector and purifying degrading protease will be studied. The protease biological properties will be detected and the chlorantraniliprole degradation products will be identified. The chlorantraniliprole enzyme catalyzed degradation pathway will be analyzed. The results may help to unveil the enzyme catalyzed degradation mechanism of chlorantraniliprole. This research will enrich biological resources for the microbial degradation of pesticides and provide the key technical support for cleaning up chlorantraniliprole -contaminated environments and food.

氯虫苯甲酰胺是一种使用量大、降解缓慢的新型杀虫剂，存在残留累积风险，而其环境消除行为研究鲜有报道。农药微生物降解具有安全、高效、低廉等优点，在农药残留安全控制方面具有广阔的应用前景。项目组成员从环境中筛选富集到氯虫苯甲酰胺降解菌群，拟进一步分离鉴定氯虫苯甲酰胺高效降解菌株并解析其生物降解机理；通过构建其基因组文库，筛选阳性菌落并克隆其关键降解基因；进而采用Pet29（a）表达载体诱导表达目的基因，分离纯化降解蛋白酶；同时测定降解酶酶活性质，鉴定降解产物，解析氯虫苯甲酰胺酶促降解机理。本项目研究旨在为农药微生物降解提供生物种质资源，为解决氯虫苯甲酰胺残留累积风险提供技术支持。

氯虫苯甲酰胺（CAP）是一种使用量大、降解缓慢的新型杀虫剂，在土壤中存在残留累积风险。为了获得CAP高效降解菌株，本项目通过在土壤中添加高剂量CAP，驯化培养CAP降解菌株，并采集加药土壤和其他耕作层土壤，筛选分离获得8株CAP降解菌株。筛选分离出的CAP降解菌株HN8，经全基因组测序、DNA-DNA杂交、生理生化测试、极性脂、G+C含量比较等鉴定试验，获得国际分类学细菌新种Mesorhizobium tritici sp. HN 8。研究接种量、温度和 pH 值对Mesorhizobium tritici sp.HN 8降解CAP速率的影响，筛选出最佳降解条件，并初步克隆表达了CAP降解基因，分离纯化获得了具有CAP高效降解特性的降解酶蛋白。此外，通过对加药土壤中细菌和真菌微生物核糖体转录间隔区（ITS）进行高通量测序，评价了CAP对土壤中细菌和真菌菌落结构的影响，发现CAP浓度与细菌和真菌菌落结构和多样性无显著相关性。