Proteus mirabilis BCD3降解苄嘧磺隆的途径和分子机理研究

Bensulphuron-methyl, a sulfonylurea herbicide, is most widely used in rice paddy. Long-term use and abuse of bensulphuron-methyl has caused succession crop phytotoxicity and water pollution, which seriously damaged food safety and human health. Microbial degradation is generally considered to be an effective and safe way to remove pollution and residual. However, some key issues were unclear about biodegradation pathway, gene and enzyme involved in the regulation of degradation. An endophyte bacterium Proteus mirabilis BCD3 was isolated from barnyard grass, which could efficiently degrade bensulphuron-methyl and possessed broad degradation spectrum. In this study, a series of studies were carried out. (1) Degradation conditions were optimized by response surface method, and then metabolites of degradation were analyzed by mass spectroscopy and molecular biologic methods to deduce the metabolic pathway. (2) The transcriptomes of P. mirabilis were sequenced based on the high throughout next-generation sequencing technologies. The differentially expressed genes were analyzed using bioinformatics method. Candidate genes were screened and confirmed with RT-PCR. (3) Key genes were cloned and heterogenous expressed or removed for functional verification. Characteristics of enzyme were researched in this experiment. This study reveals the metabolism mechanism about the biodegradation of bensulphuron-methyl by P. mirabilis BCD3 from molecular level, which will provide a scientific basis for the development of herbicide phytotoxicity remediation and resistant crops.

苄嘧磺隆是水稻田用量最大的磺酰脲类除草剂，长期的盲目使用和滥用造成后茬作物药害和水体污染。微生物降解是一种安全有效的去除污染和残留的方式，但许多问题如微生物降解的代谢途径、参与调控降解的基因和酶等仍不清楚。申请者前期从稗草中筛选出能高效降解苄嘧磺隆的内生菌Proteus mirabilis BCD3，且降解谱广。本研究以该菌为对象开展以下工作：（1）用响应曲面法优化降解条件，借助质谱和分子生物学手段研究该菌对苄嘧磺隆的降解产物，揭示其降解途径；（2）采用高通量二代测序技术对该菌进行转录组测序，运用生物信息学对功能差异基因分析，筛选出候选基因，并使用RT-PCR进行验证；（3）克隆相关的降解基因，对获得的关键酶基因进行外源表达或基因敲除进行功能验证和酶学特性研究。本研究从分子水平上揭示P. mirabilis降解苄嘧磺隆的代谢机理，为该类除草剂药害污染修复提供科学依据。

苄嘧磺隆除草剂在水稻田的长期使用和滥用已造成后茬作物药害和水体污染，而微生物降解是一种安全有效的去除苄嘧磺隆污染和残留的方式。本研究利用植物内生菌Proteus mirabilis BCD3降解苄嘧磺隆，优化了P. mirabilis BCD3对苄嘧磺隆的降解条件（包括温度、pH、接菌量、初始浓度和碳源）；鉴定了苄嘧磺隆的2个降解产物并推断了菌株对苄嘧磺隆的降解途径（主要是通过裂解脲桥）；对P. mirabilis BCD3的降解酶进行定位并研究了胞内粗酶液的降解特性；对P. mirabilis BCD3进行基因组测序并分析功能基因；对8个降解相关基因进行表达量分析，筛选出3个候选基因；进行基因克隆、蛋白表达、蛋白纯化后，发现1个有效的降解基因GST并进行酶学特性研究。本研究的结果揭示P. mirabilis降解苄嘧磺隆的代谢途径，挖掘相关降解基因，为其在减少苄嘧磺隆的药害污染上的应用提供理论依据。