Rhodococcus jialingiae djl-6-2降解多菌灵的途径及分子机制研究

Carbendazim is a kind of benzimidazole fungicide, which is used widely to control a broad range of fungal diseases in agriculture and forestry. Carbendazim is quite stable in soil and water, which in turn can lead to the contamination of foodstuffs. Carbendazim can hurt liver and endocrine system of animals even at low concentrations, which is harmful to the ecosystem and the health of humans. Microbial metabolism is the main mechanism responsible for the degradation of carbendazim in the environment, and the increasing attention has been put on the this method. However, the degradation mechanism of carbendazim has not been completely elucidated, and the complete degradation pathway and the related genes still need study. Rhodococcus jialingiae djl-6-2 was isolated by our lab; it could use carbendazim as the sole carbon and nitrogen source for its growth and completely degrade carbendazim. We have investigated several initial steps of its degradation pathway and cloned the mheI gene involved in the catalysis of the first step. The target of this program is to elucidate the complete degrading pathway of carbendazim by the method of substrates utilization and metabolites identification; to get the genomic sequence through the new sequencing technology, to clone all the genes involved in the pathway by the method of genome analysis combined with general molecular manipulation. The results of this study will elucidate the degradation pathway of carbendazim by Rhodococcus jialingiae djl-6-2 and its molecular mechanism, and provide the theoretical and technical foundation for the bioremediation of the carbendazim-contaminated environments.

多菌灵是一种应用广泛的苯并咪唑类杀菌剂。多菌灵残留破坏生态环境、危害人体健康。微生物降解是环境中多菌灵残留消除的主要因素。但目前对微生物降解多菌灵的机理研究还很不充分，完整的降解途径及参与的基因还不清楚。Rhodococcus jialingiae djl-6-2是本课题组分离的能以多菌灵为唯一碳、氮源生长且彻底降解多菌灵的菌株。我们已经对其降解途径进行了初步研究；克隆了催化该途径第一步反应的多菌灵水解酶的基因 mheI。本项目以该菌株为研究对象，通过底物利用和产物鉴定技术解析其完整的多菌灵降解途径；借助华大基因的DNA 高通量测序平台获得它的基因组序列；运用基因组学和常规分子生物学操作相结合的手段克隆和鉴定降解相关基因，从基因水平上阐明细菌降解多菌灵的途径。本研究的结果将解析微生物降解多菌灵的途径及分子机理，为多菌灵污染环境的生物修复提供理论和技术支撑。

多菌灵是一种应用广泛的苯并咪唑类杀菌剂。多菌灵残留破坏生态环境、危害人体健康。微生物降解是消除环境中多菌灵残留的主要因素。但目前对微生物降解多菌灵的机理研究还很不充分，完整的降解途径及参与的基因还不清楚。本项目以多菌灵降解菌Rhodococcus jialingiae djl-6-2为材料，从测定和解析djl-6-2的全基因组入手克隆菌株降解多菌灵的相关基因。此外还继续分离多菌灵降解菌株，为研究多菌灵降解菌的多样性提供材料。取了以下结果：1、解析了菌株djl-6-2的基因组，它包含一个大小为6,518,575 bp（CP025959）的染色体和3个环状质粒，大小分别为84,619 bp（CP025960）、 80,954 bp（CP025961）和15,865 bp（CP025962）。2、从菌株djl-6-2中克隆到一个新的多菌灵水解酶基因cmba，已申请专利（申请号为201810011521.5），该基因负责多菌灵降解的第一步。3、获得了菌株djl-6-2可能的负责多菌灵降解第二步的2-氨基苯并咪唑水解酶基因（orf 04172），正在进行功能方面的鉴定，近期就会有结果。4、分离到三株多菌灵降解菌株，分别为mbc-2T，djl-8T 和SD-4，将mbc-2T和djl-8T 鉴定为新种，分别命名为Nocardioides soli sp. nov. 和Nocardioides agrisoli sp. nov.。5、确定了菌株SD-4的多菌灵水解酶MheI中位于第16以及222位的半胱氨酸是其水解多菌灵的关键氨基酸。上述这些研究结果推动了微生物降解多菌灵分子机制的解析。.总体来说，本项目完成了预期研究目标。项目主持人已经以通讯作者发表标注项目号SCI录用文章11篇(第1标注6篇)；其中包括Appl. Environ. Microbiol 1篇和Journal of Hazardous Materials 2篇。培养研究生5名，其中博士2名，硕士3名。

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