吉林克雷伯氏菌高效降解氯嘧磺隆作用机制的研究

Chlorimuron-ethyl (CHL) is an more effective, long residual Sulfonylurea herbicide applied in many kinds of crop plants all over the world now. It is well-known that microbes play an important role in the degradation of pesticides and herbicides. Numerous microbial groups that can degrade or inactivate these chemicals have been identified and the use of these detoxifying microbes has proven to be one of the important means of controlling environmental pollution. To reduce its toxic side effect on sensitive rotational crops and ensure food security, our team, for the first time, isolated an CHL-degrading bacterium strain named as Klebsiella jilinsis H. Zhang that with high efficient activity. For successful field applications, it is important to confirm its high CHL-degradation activity and investigate the molecular mechanism of degradation and soil remediation. The objectives and content of this study include the following:.1. Clone the functional and regulatory genes essential for CHL-degradation in K.jilinsis strain 2N3 with establishing DNA library, mutant library and genomic sequencing approaches. And study the functions of cloned genes by genetics methods and molecular operation such as gene knockout and complementation..2. Analyse temporal and spatial expression of the main functional genes to Chlorimuron ethyl and degradation level..3. Investigate the regulatory mechanisms of the CHL-degradation enzymes, further exploid a gene regulatory network with RNA sequencing or transcriptome Sequencing..4. Elucidate the metabolic mechanism of K. jilinsis mediated CHL-degradation using isotope labeling and HPLC-MS technology..5. Aim to edit and obtain high efficiency degradation engineering strains by using CRISPR technology. And It is much general scientific meaning in order to understand biodegradation and bioremediation of sulfonylurea herbicide.

氯嘧磺隆是一种高效长残留磺酰脲类除草剂，其对后茬敏感作物二次药害与残留污染严重。本课题组首次分离鉴定了一株氯嘧磺隆高效降解菌株－吉林克雷伯氏菌（Klebsiella jilinsis 2N3），并明确该菌株高效降解的生物学特性，亟待进一步深入研究其高效降解机制，为磺酰脲类除草剂生物修复提供科学依据。本项目拟研究：（1）筛选并克隆吉林克雷伯氏菌高效降解相关主要功能基因与调控基因，并验证相关基因的降解功能：（2）分析主要功能基因的表达动态与对氯嘧磺隆降解水平；（3）RNA-Seq转录组差异表达分析，明确其对氯嘧磺隆的降解表达谱与调控网络，揭示其高效降解作用机制；（4）利用HPLC-MS方法，解析该菌降解氯嘧磺隆的代谢途径；（5）CRISPR技术编辑获得高效降解工程菌株。对磺酰脲类除草剂高效降解与生物修复具有普遍科学意义。

氯嘧磺隆的残留药害严重。课题组首次鉴定氯嘧磺隆高效降解菌—吉林克雷伯氏菌（Klebsiella jilinsis）2N3菌株。进行了2N3菌株全基因组测序和高效降解的差异表达分析，挖掘了高效降解功能基因，揭示了2N3高效降解氯嘧磺隆的分子机制，并利用HPLC-MS明确了氯嘧磺隆生物降解途径。.首次分析2N3全基因组及其高效差异表达谱。其基因组大小为5.32 Mb，共5156个编码基因。验证突变体ΔORF0934和ΔORF 0492在36小时内对氯嘧磺隆的降解率显著下降。研究发现，2N3羧酸酯酶基因Kj-mhpC、细胞色素P450基因Kj-cysJ、谷胱甘肽-S-转移酶基因Kj-gst和水解酶基因等为高效降解的主要功能基因。转录分析表明，2N3降解氯嘧磺隆中共有839个差异表达基因；上调表达基因显著富集在“硫代谢”、“甲烷代谢”、“药物代谢-细胞色素P450”、“细胞色素P450对异生物质代谢”等代谢通路。.利用λ-Red同源重组系统，共获得了11个敲除突变体。突变体ΔKjcysJ，ΔKjmhpC1以及ΔKjgst分别显示出对氯嘧磺隆降解能力的缺失，其中ΔKjcysJ丧失了对氯嘧磺隆的降解能力。并优化降解酶的降解最适条件，明确体外对氯嘧磺隆的降解活性。证实其为2N3降解氯嘧磺隆的关键酶基因。同时发现，Kj-GST折叠结构域包含一个C端α螺旋折叠结构域和一个N端硫氧还原蛋白折叠结构域，其活性位点位于两个结构域之间，通过点突变实验发现由在降解氯嘧磺隆时的主要功能区域为GSH-H位点；HPLC-MS结果表明，氯嘧磺隆最终被2N3分解代谢成2-氨基-6-甲氧基嘧啶和邻磺酸苯甲酸亚胺。.本研究揭示了吉林克雷伯氏菌高效降解氯嘧磺隆机制，完成了研究内容与指标。研究结果在《Science of the Total Environment》等刊物上发表论文4篇。培养硕士研究生4人、博士研究生2人。