广东星湖湿地放线菌多样性及活性初步研究

As an indispensable part in the wetland ecosystem, microorganisms play an important role in the material transformation and energy flow. It is becoming increasingly clear that un- and under-explored habitats, such as wetland ecosystem, is a rich source of novel actinomycetes which have the capacity to produce interesting new biologically active secondary metabolites, including some new antibiotics. These actinomycetes are relatively unstudied and are potential sources of novel natural products for exploitation in medicine, agriculture and industry. It is necessary to isolate, characterize and screen the reperesentatives of undiscovered actinomycete taxa from the wetland ecosystem. To better understand the diversity and antibacterial activity of actinomycetes associated with wetland, we set out to isolate, cultivate, characterize and screen actinomycetes from the soil materials collected from Xinghu wetland in Guangdong province, China. In order to improve the success of isolating individual members of the actinobacterial population as pure cultures as well as improving the ability to characterise the large numbers obtained in pure culture, our study will employe rational and holistic approaches including the use of selective isolation media, plating larger quantities of samples, incubating isolation plates for up to 8-12 weeks and excising colonies when they are visible. In addition, representative cultivable isolates will be examined to determine their phylogenetic diversity and to evaluate their ability to inhibit the growth of a panel of pathogenic microorganisms. To the best of our knowledge, this is the first study on the diversity of culturable actinomycetes associated with Xinghu wetland. It will contribute to our knowledge of wetland-associated actinomycetes and further increase the pool of microorganisms available for natural bioactive product screening. This work will serve as a prelude to developing the potential biotechnological applications of wetland-actinomycetes, and additional studies will be required to exploit available bioactive compounds from these resources.

微生物对湿地生态系统中的物质转化和能量流动起着重要作用，是湿地生态系统中不可缺少的重要成员。研究显示，湿地环境中蕴藏着丰富的放线菌资源，这些生存于湿地环境中的放线菌具有一些独特的次生代谢途径，能够产生一些结构特殊的活性次级代谢产物，是一类尚未充分发掘的重要菌种资源，因此，加强湿地环境放线菌资源的研究与开发具有重要的现实意义。本项目选择广东星湖湿地为研究对象，系统地开展该环境放线菌资源的选择性分离与培养、物种多样性分析及抑菌活性研究；研究中拟通过探索适合星湖湿地放线菌的选择性分离技术，提高放线菌菌株分离效率，获得更多的难培养或未培养放线菌资源，加大对该环境中未知放线菌的认识；同时，筛选具有拮抗耐药性细菌的活性放线菌菌株，为后续开展菌株次生代谢产物研究提供保障。本项目的实施对于进一步认知湿地环境放线菌分布特点，加强我国湿地放线菌资源的挖掘、保育、开发和利用具有重要的指导意义。

微生物对湿地生态系统物质转化和能量流动起着重要作用，是湿地生态系统中不可缺少的重要成员。研究显示，湿地环境中蕴藏着丰富的放线菌资源，这些生存于湿地环境中的放线菌具有一些独特的次生代谢途径，能够产生一些结构特殊的活性次级代谢产物，是一类尚未充分发掘的重要资源。.本项目选择广东肇庆星湖湿地底泥为研究对象，首先利用放线菌特异性引物PCR--DGGE分析星湖湿地样品放线菌类群组成特点，发现放线菌优势类群主要分布于类诺卡氏菌科、链霉菌科、小单孢菌科、微球菌科、纤维素单胞菌科 和原小单胞菌科；且群落结构受样品水溶性碳(WSOC)和速效磷(A-P)影响较大。然后，选取多样性较高的样品进行放线菌选择性分离培养，获得Actinocatenispora，Actinocorallia，Actinomadura， Actinopolymorpha等30余属1000多株放线菌菌株，其中包括3个微生物新物种，完成肇庆星湖湿地放线菌物种资源信息库建设。随后，联合采用基因筛选 和生物活性筛选方式对代表性菌株活性次级代谢潜力进行分析，发现具有高效稳定拮抗耐药性细菌的活性放线菌菌株3株，并完成菌株发酵条件的优化，为后续开展菌株次生代谢产物研究奠定基础。最后，采用Illumina测序技术开展肇庆星湖湿地底泥样品细菌多样性分析，发现星湖湿地底泥样品细菌主要分布于20个门，其中以Proteobacteria为主要类群，Acidobacteria，Actinobacteria，Bacteroidetes，Chloroflexi，Gemmatimonadetes等类群含量也较高，特别是Actinobacteria 类群含量在6.0% 到16.1%之间；同时，为了比较不同类型湿地环境放线菌分布多样性及探究湿地放线菌分布规律，我们特意增加了来自广州海珠湿地(城市淡水湿地)及南沙湿地(城市咸淡水交汇湿地)的湿地底泥样品与来自肇庆星湖湿地的底泥样品进行比较分析，发现来自南沙湿地(咸淡交汇)样品放线菌所占比例明显高于来自城市淡水湿地星湖湿地和海珠湿地样品。.我们的研究表明，在星湖湿地蕴藏着大量放线菌资源，但由于分离技术等限制，大量的放线菌还未能通过现有的选择性分离方法获得纯培养物。本项目的实施对于进一步认知湿地环境放线菌分布特点，加强我国湿地放线菌资源的挖掘、保育、开发和利用具有重要的指导意义。