除草剂杀草丹的硫酯键水解代谢途径及其分子机制研究

Thiobencarb [S-(4-chlorobenzyl)-diethylthiocarbamate] is a highly effective herbicide that widely used in paddy field to control barnyard grass worldwide. Thiobencarb is very stable and has a long persistence in the paddy soil, and is highly toxic to aquatic organisms. Thus, it is important to elucidate the degradation mechanisms and ecotoxicology of thiobencarb. Metabolites identification results indicated that thiobencarb could be degraded through hydrolysis of thioester bond, N-deethylation, hydroxylation of the benzene ring, sulfoxidation, cleavage of the C-S bond and dechlorination, and hydrolysis of thioester bond is the main degradation pathway of thiobencarb in paddy soil. Microbial metabolism is the most important factor in the degradation of thiobencarb in soil. However, to date, only the pathway through cleavage of the C-S bond has been elucidated in Acidovorax sp. T1. In our preliminary study, a thiobencarb-degrading strain Bacillus sp. T2 was isolated from paddy soil. Metabolite identification results indicated that thiobencarb was initially degraded through hydrolysis of thioester bond. This project was designed to identify the downstream metabolites of thiobencarb degradation in strain Bacillus sp. T2, clone the genes involved in thiobencarb degradation and study the characteristics of the degrading enzymes. This project will reveal the process and mechanism of thiobencarb degradation through the pathway of hydrolysis of thioester bond, and the enzymes and genes involved in the process, and provide the theoretical basis for in-depth study on the degradation mechanism and ecotoxicology of thiobencarb in soil.

杀草丹是一种硫代氨基甲酸酯类除草剂，在世界范围内广泛用于稻田除稗。杀草丹性质稳定，残留期较长，对水生生物高毒，因此研究杀草丹的降解代谢过程和生态毒理具有非常重要的意义。从分解中间产物鉴定结果推测，土壤中杀草丹可以通过硫酯键水解、N-脱乙基、苯环羟基化、硫氧化、C-S键断裂和脱氯等途径降解。其中硫酯键水解是稻田中杀草丹好氧降解的主要途径，微生物代谢在其中起到关键作用，但迄今为止，仅有C-S键断裂途径在细胞和分子水平上得到确认。前期从土壤中分离到一株杀草丹降解菌株Bacillus sp. T2，代谢产物鉴定结果证明菌株T2通过硫酯键水解途径降解杀草丹。本项目将以菌株T2为材料，对其降解杀草丹下游代谢产物鉴定，结合对其基因组生物信息学分析，插入突变、降解酶酶学研究等，从遗传和酶学角度解析杀草丹通过硫酯键水解途径的降解机制，为深入研究杀草丹在土壤中的降解代谢过程及其生态毒理学研究提供理论依据。

杀草丹是一种特效防除稻田稗草的硫代氨基甲酸酯类除草剂，其化学性质稳定，水溶性差，在土壤及水体中残留期较长，严重威胁环境生态、动物及人类健康，研究杀草丹的降解代谢过程和生态毒理具有重要意义。本项目从土壤中分离到一株杀草丹降解菌株Bacillus sp. T2，GC-MS鉴定结果表明菌株T2通过硫酯键水解途径降解杀草丹，首先将其转化为对氯苄硫醇，对氯苄硫醇依次被氧化为对氯苯甲醛及对氯苯甲酸，最后进入三羧酸循环。利用转座子插入突变的方法克隆到杀草丹硫酯键水解酶基因thiA，该基因与低特异性II型硫酯酶YbtT具有较高序列相似度为58%。异源表达及突变株回补实验证明杀草丹硫酯酶ThiA能够将杀草丹水解并转化为对氯苄硫醇。此外，生物信息学分析发现菌株T2基因组中存在一整套编码对氯苯甲酸降解相关酶的基因簇，分别为对氯苯甲酸降解基因簇fcbB-A-T1-T2-T3-C、对羟基苯甲酸氧化酶基因pobA和原儿茶酸的3, 4-开环降解基因簇pcaHGBCD-IJF，通过荧光定量PCR表明这些基因簇参与杀草丹下游产物对氯苯甲酸的降解。同时利用来源于微生物的除草剂降解基因成功构建具有降解功能的CndA转基因拟南芥。本项目从细胞和遗传学角度揭示了杀草丹微生物降解代谢途径和机制，为深入研究土壤中杀草丹残留的分解转化过程及生物修复提供理论依据。