高效降解多环芳烃（PAHs）菌群的代谢网络及其相关基因研究

Polycyclic aromatic hydrocarbons (PAHs) can affect the function of hepar, lung, bladder, kidney of human, as well as inducing them to become cancerous, so the degrading of PAHs by microorganisms has become a hot spot of research. Microbial strains are the basis of the degrading PAHs. There have been a lot of reports of bacterial strains degrading PAHs but the efficiency of single strain degrading PAHs is low for the single strain only accomplish several steps of PAHs degraded pathway, and cause the degrading products accumulation (product feedback inhibition), and inhibit the PAHs degraded. Therefore, the microbial consortia screened of degrading PAHs from the different ecological environment is in the ascendant. The microorganisms from the soil that were polluted by PAHs had been cultured using the enrichment culture medium containing PAHs in this project. The microorganisms degrading PAHs could grow and enrich under PAHs stressing, and the microbial diversity and abundance had been analyzed using the high-throughput sequencing, and then had referred the degrading efficiency of PAHs in the enrichment culture medium to confirm the degrading PAHs capability of microbial consortium. The native consortia of high efficiency degrading PAHs had been screened. The metabolic network of native microbial consortia degrading PAHs will be constructed in this project through the transcriptome sequencing and gene annotation of native microbial consortia is going to be done. The bacterium strain will be isolated from the native microbial consortia. The transcriptome sequencing and gene annotation of bacterium strain is going to be done. The relative genes of bacterium strain degrading PAHs also will be cloned and expressed, and the function of expressed product of relative genes will be tested. The metabolic network of native microbial consortia degrading PAHs will be confirmed by the function of relative genes. This project will provide the theoretical support of native microbial consortia degrading PAHs process and metabolic regulation.

多环芳烃（PAHs）能诱发人体器官癌变，因此微生物降解PAHs已成为研究热点。菌种是PAHs微生物降解的关键，已有好多降解PAHs的菌株报道，然而单菌株降解PAHs只能完成降解途径的几步，故降解效果不明显，因此从各种生态环境中筛选能降解PAHs的菌群的研究正在方兴未艾。本项目已经用含PAHs的富集培养基对PAHs污染土壤微生物进行富集培养，在PAHs胁迫下能降解PAHs的微生物会富集生长，用高通量测序技术分析该系统微生物多样性和丰度，结合培养基中PAHs的降解率，确定该菌群降解PAHs的能力，已筛选出高效降解PAHs的土著菌群，进一步需要对该菌群的转录组进行测序和基因注释，构建菌群降解PAHs的代谢网络，同时从该菌群中分离能高效降解PAHs的细菌，进行转录组分析，并克隆和表达其相关基因，通过测定这些表达产物的功能来验证菌群降解PAHs的代谢网络，为菌群降解PAHs及其代谢调节提供理论支持。

目前石油污染土壤的问题越来越严重，造成了严重的环境污染和生态灾难。 石油污染不仅显著影响了土壤的正常代谢，还改变了土壤微生物菌群结构。了解石油污染土壤的微生物组成可以为土壤修复提供更好的生物学方法。采用16S rRNA和ITS技术对石油污染土壤中的细菌和真菌微生物进行了测定和分析，并对其理化性质(TOC、碱性水解氮、全磷、全钾、速效钾、Cu、Zn、Cd)进行了检测。结果表明，石油污染显著降低了土壤细菌和真菌的丰度和多样性，促进了Proteobacteria, Pseudomonas，Pseudoxanthomonas 和 Pseudoallescheria的相对丰度，改变了土壤原有细菌和真菌的优势菌群，也重塑了石油污染土壤细菌与真菌共现网络关系。石油污染土壤中TOC含量显著高于未污染土壤，碱性水解氮和速效钾含量显著低于未污染土壤，Cu含量在污染后显著升高。TOC是石油污染土壤微生物变化的关键驱动因素，对石油污染土壤微生物群落的重构和组成起着重要的调控作用。该研究为石油污染土壤的生物修复奠定了坚实的理论基础。. 目前对石油污染土壤中微生物基因组的基因功能特征、代谢产物及分布的研究有限，其调控机制尚不清楚。基于此，利用宏基因组学和GC-TOFMS代谢组学对石油污染土壤中的微生物和代谢物进行检测，并分析其功能基因代谢通路的变化。发现石油污染显著改变了土壤微生物和代谢产物的组成，促进了土壤中Pseudoxanthomonas和Mycobacterium的相对丰度，上调了油烃降解的关键酶脯氨酸羟化酶系统和β-葡萄糖苷酶，促进了外源性生物质降解和代谢途径，以及聚糖的生物合成和代谢、细胞运动、环境适应，进而促进苯类、脂类和类脂类、有机杂环化合物、有机氧化合物、苯丙烷和多酮类的代谢，并促进抗性基因的表达。同时石油污染土壤可促进石油降解菌的增殖，上调石油降解的相关酶脯氨酸羟化酶系统，进而促进苯、杂环化合物等石油污染物的代谢。本研究从分子调控机理方面为石油污染土壤的生物修复提供一定的参考，并为新型石油降解菌的筛选提供理论依据。