菌株Rhodococcus sp. YL-1降解杀虫剂噻嗪酮的途径及其分子机制

To control the rice planthoppers, large amount of buprofezin is used widely every year. The residues of buprofezin in soil, water and food (especially rice and drinking water) pose a threat to ecological balance and our health. Microorganisms play a very important role in the elimination of buprofezin residues in the natural conditions; microbial degradation is considered as a good method to eliminate pesticide residues. However, there are only several reports (all from our lab) about this process. Previously, in our lab, a buprofezin-degrading bacterial strain Rhodococcus sp. YL-1 was isolated from the rice field soils chronically exposed to buprofezin; a preliminary degradation pathway was identified and this strain also showed a potential in remediation of buprofezin residues. This project seeks to (1) indentify the entire metabolic pathway of buprofezin by strain YL-1; (2) clone the genes involved in the degradation of buprofezin and clarify the functions of these genes; (3) investigate the factors that affect the stability of buprofezin-degrading capability of strain YL-1. The results of this work would benefit agriculture production, food safety, and human and ecological health in buprofezin-contaminated sites.

作为对抗稻飞虱的主力农药之一，噻嗪酮每年被大量施用，其在水、土和食品（尤其是大米和饮用水）中的残留对生态平衡和人体健康构成威胁。在自然条件下，微生物是消除噻嗪酮残留的重要力量，微生物降解技术是解决其残留问题的理想选择，但目前相关的研究报道很少且都来自本实验室，在前期工作中我们从长期施用噻嗪酮的稻田土壤中分离到1株噻嗪酮高效降解菌Rhodococcus sp. YL-1，初步分析了其降解噻嗪酮的途径，并证明人为添加该菌株可以加快土壤中噻嗪酮残留的分解。为了更好地开发噻嗪酮残留微生物（酶）降解技术，本项目拟进一步阐明以下3个问题：①菌株YL-1降解噻嗪酮的完整途径；②关键降解酶基因；③降解酶基因的表达调控和遗传稳定性，如何通过遗传改造提高菌株降解性状的稳定性。

噻嗪酮是一种昆虫生长调节剂，能抑制昆虫几丁质的合成并干扰其新陈代谢，对飞虱、叶蝉和粉虱防治效果明显，广泛施用于水稻、茶叶、马铃薯、柑橘、蔬菜和棉花等植物。残留于环境和农产品中的噻嗪酮危害一些水生生物和某些非靶标昆虫，且对人体健康造成潜在威胁。在自然环境中，微生物是降解噻嗪酮的主要力量，微生物修复是清除噻嗪酮残留的理想手段。然而，微生物代谢噻嗪酮的分子机制还不清楚。此外，目前还缺乏降解性状稳定的菌株资源，限制了噻嗪酮残留污染的微生物修复。在前期的工作中，本实验室分离了一株能以噻嗪酮为唯一碳源生长的降解菌Rhodococcus qingshengii YL-1，但该菌株的降解性状不稳定。鉴于此，本项目的目标为：第一，以菌株YL-1为研究材料，阐明微生物代谢噻嗪酮的分子机制；第二，筛选噻嗪酮降解性状稳定的菌株，为微生物修复打下基础。我们首先通过UHPLC-MS分析，鉴定了菌株YL-1完整的上游代谢途径；接着获得噻嗪酮降解丢失的突变菌株，并通过比较基因组预测降解相关基因；然后通过遗传学和生化实验鉴定了噻嗪酮降解基因bfzBA3A4A1A2C，并发现bfzBA3A4A1A2C的编码产物还能降解联苯、联苯菊酯和黄酮等化合物；最后，通过富集培养法从长期施用噻嗪酮的稻田土中分离了噻嗪酮降解性状优良的菌株Rhodococcus sp. D-6。总体来看，研究结果加深了我们对微生物降解噻嗪酮的认识， 基因bfzBA3A4A1A2C和菌株D-6在微生物修复噻嗪酮残留方面有潜在应用价值。本项目圆满完成了预期目标。