Bacillus megaterium Q3降解二氯喹啉酸分子机理研究

Quinclorac (3,7-dichloro-8-quinoline-carboxylic) is an highly selective, widely used herbicide for controlling barnyard grass and certain dicot grasses in rice. However, the quinclorac residues are phytotoxic to many crops/vegetables, which become one of the major factors that affect rice and economic crop/vegetable ration in southern china. Degrading quinclorac using microbial is an effective way to reduce its phytotoxicity. In our preliminary study, we had isolated, identified and characterized an endophytic quinclorac degrading bacterium, Bacillus megaterium Q3. We assembled the whole genome of Q3 using de novo sequencing and predicted its gene annotations. However, the molecular mechanism of quinclorac degradation in Q3 is still unclear. In this project, we will apply systems biology methods to elucidate the enzymes for quinclorac degradation and transcription factors that control the quinclorac degradation. We will use RNA-seq to monitor the gene expressions of Q3 under quinclorac stress. We will identify the enzymes for quinclorac degradation by detecting differentially expressed genes. Furthermore, we will construct gene co-expression network to identify function modules and transcription factors that relate to quinclorac degradation. We will conduct biochemistry and genetic experiments to validate the functionality of enzymes and transcription factors. The findings of this proposal will significantly advance fundamental understanding of the molecular mechanism of quinclorac degradation in Q3. It will facilitate the modification and utilization of Q3 as a quinclorac degradation bacterium in the future.

二氯喹啉酸是一种广泛应用的稻田除草剂，由于对后茬作物易产生药害，已成为制约我国南方稻田轮作的主要因素之一。微生物降解是降低二氯喹啉酸药害的有效途径。Bacillus megaterium Q3是课题组分离得到的一株二氯喹啉酸高效降解内生细菌。前期，我们对Q3的生物学及降解特征进行了系统研究，已完成全基因组序列测定和注释，但其降解的分子机制尚不清楚。本项目拟运用系统生物学方法阐明Q3降解二氯喹啉酸的调控机制和分子机理。通过对Q3在二氯喹啉酸胁迫下的基因表达进行系列RNA-seq测序，比较不同胁迫条件下的基因表达差异，从而发掘Q3的二氯喹啉酸降解酶。同时，用基因表达数据构建基因共表达网络和转录调控网络，阐明Q3降解二氯喹啉酸的调控机制，并应用分子生物学方法验证降解酶和调控因子。本课题对于理解微生物降解二氯喹啉酸的分子机理具有重要的意义，也为进一步改造和利用Q3提供理论依据和技术基础。

二氯喹啉酸是南方稻田常用的除草剂之一，长期或过量施用该除草剂对后茬作物，尤其是双子叶作物产生严重的药害。项目组前期筛选到一株可高效降解二氯喹啉酸的菌株Bacillus megaterium Q3，后期发现该菌株降解二氯喹啉酸的效果不稳定。因此，项目组从长期施用二氯喹啉酸的土壤中重新筛选到一株可在7d内将50mg/L的二氯喹啉酸降解97%以上菌株F4，并将其鉴定为Mycobacterium sp.。盆栽试验结果表明，菌株F4的发酵液（含50mg/L二氯喹啉酸）对烟草无毒害效应。项目组基于HPLC分离纯化了菌株F4降解二氯喹啉酸的代谢产物，经串联质谱法和核磁波谱法测定，发现降解产物主要是二氯喹啉酸甲酯，其次是3-氯-7-羟基喹啉-8-羧酸，推测前者通过甲基化作用产生，而后者通过脱氯作用产生。综合降解途径及比较基因组学分析，推测有两类酶可能参与了二氯喹啉酸的降解：甲基转移酶和酰胺酶。F4基因组中共有77个甲基转移酶和13个酰胺酶，结合转录组测序及RT-PCR分析，我们选择了其中的4个基因作为敲除靶标（正在进行中）。后续研究中将以基因敲除及发酵谱分析为基础，并结合异源表达等手段进一步分析菌株F4降解二氯喹啉酸的分子机理。