多环芳烃生物降解功能新标记基因的筛选及其功能评估

The study focusing on the functional marker genes of polycyclic aromatic hydrocarbons (PAHs) biodegradation can provide technical supports for the detection of PAHs degrading microorganisms, the clarification of degradation pathways and mechanisms, and the evaluation of PAHs pollution bioremediation potential and efficiency. The pahAc gene coding for the large subunit of ring-hydroxylating dioxygenase of PAHs, has been widely used as PAHs degradation functional marker for decades. However, the previous studies found that pahAc had serious defects as a functional marker and pahE, which encode for the trans-o-hydrobenzylidenepyruvate hydratase aldolase, is likely to be a better functional marker of PAHs-degrading bacteria. In this project, firstly, we will establish a specific detection method for pahE and evaluate the potential of pahE gene as a functional marker for PAHs degrading bacteria by bioinformatics analysis and experiments. Secondly, we will analyze the diversity and relative abundance of pahE in a variety of PAHs polluted environments, such as soils, sediments, sludges, etc. to illustrate the distribution of PAHs degraders in these environments and discover novel PAHs degraders. Finally, we expect to establish the relationship between the microbial community structure, the diversity and abundance of pahE and PAHs degradation dynamics by microcosm experiments. These relationships are significant to evaluate the potential of pahE as a functional marker for assessment of PHAs degradation potential and efficiency in contaminated sites. The achievement of this study will provide a more accurate and powerful molecular tool for us to study the PAHs microbial degradation and PAHs function and provide a new theoretical and technical basis for the bioremediation of polluted environment PAHs.

研究多环芳烃（PAHs）生物降解功能标记基因可以为检测PAHs降解微生物、研究降解途径与机理、评价PAHs污染生物修复潜能和效率提供技术支撑。本课题组前期研究发现作为最常用的PAHs生物降解功能标记基因—PAHs起始双加氧酶大亚基编码基因pahAc，在指示PAHs降解功能时存在着严重缺陷，而醛缩酶基因pahE很可能是更好的PAHs降解功能标记基因。本课题拟通过生物信息学分析和实验研究，建立pahE的检测方法，比较pahAc与pahE对PAHs降解功能的指示作用，以充分评估pahE对PAHs降解功能的指示作用；其次，分析不同污染环境中pahE的多样性和丰度以表征PAHs降解微生物的多样性及分布特征，并发现更多未知的PAHs降解菌；最后，通过建立PAHs生物降解微宇宙模拟实验，分析微生物群落结构、pahE多样性和丰度等与PAHs降解效率的定量关系，评估pahE对污染环境中PAHs微生物降解潜能和效率的指示作用。新的PAHs降解功能指示基因pahE，将为我们研究PAHs降解微生物和功能提供更为准确的、有力的分子工具，为PAHs污染环境的微生物修复提供新的理论和技术基础。

近年来，随着高通量测序技术的发展，利用高通量测序直接研究环境中功能微生物的多样性及其生态功能成为一个新兴的领域。功能标记基因成为了解功能微生物多样性信息与其生态功能之间重要的分子工具，因而一个高特异高分辨率的功能标记基因对于研究功能微生物多样性是十分必要的。本项目针对已有研究中常用的PAHs好氧降解功能标记基因pahAc特异性差、分辨率低及其不能很好地表征PAHs降解活性等问题，通过生物信息学的方法，筛选出了更优的PAHs好氧降解新功能标记基因pahE；针对pahE设计了简并引物，建立了特异性的pahE检测方法。在此基础上，通过对纯菌和典型环境样品中PAHs好氧降解菌的pahE检测，评估pahE对PAHs好氧降解菌的指示作用及其适用性，结果表明pahE比pahAc更适合作为PAHs好氧降解菌的功能标记基因。通过微宇宙模拟实验监测PAHs降解过程中pahE多样性和丰度的变化，pahE丰度与PAHs降解率显著正相关（R2=0.908～0.922，P﹤0.01），表明pahE能较好地表征PAHs的降解潜能和活性。并采用靶向宏基因组技术探究了典型污染环境（油田土壤、红树林沉积物和城市路边土壤）中pahE的分布特征及其影响因子，结果表明pahE的组成和丰度主要受PAHs污染水平、营养水平、盐度和含水率等环境因子的影响。本项目首次以新的PAHs降解功能基因pahE为目的基因，探究了PAHs降解菌的分布规律及其影响因子，评估了污染场地的降解潜能，并掌握了不同污染场地中参与降解PAHs的降解菌属多样性以及与底物的对应性，本研究成果对于制定高效地PAHs污染生物修复策略有着重要的理论和指导意义。已发表SCI论文13篇（第一标注I论文11篇，第二标注2篇），EI论文1篇，申请发明专利1项。