深海沉积环境uncultured放线菌多样性及其可培养研究

Recently, marine actinomycetes were become to the hot spots of marine microorganism research, because of its ability to produce the significant activity and novel structure of secondary metabolites. .While, since the new genus of Salinispora, the first obligate marine actinobacterial genus, was published at 2005, the huge 16S rRNA gene data of uncultured marine actinobacteria were deposited in the NCBI and GenBank. These data revealed that actinobacteria are widespread in the oceans and the dominant groups were Acidimicrobidae and Rubrobacteridae (up to 60%). Now, only more than 10 species are cultured in the two subclasses and a large number of them are still buried in the deep seabed. Strains in these two groups reveal the unique physiological characteristics, such as obligate acidophilic, thermophilic, radiation resistance, oxidize or reduce ferric iron, CO2 fixation and long-time generation, which were the valuable properties of experimental material. In this study, we firstly focus on the diversity and distribution of the uncultured Acidimicrobidae and Rubrobacteridae groups in the deep-sea sediment environments, with the Miseq sequencing method. This will provide the effective guidance to design the selective isolation media for these two special groups. At the same time, we study on these pure cultures’ physiological characteristics, especially on the nutrition requirements for growth, and explore the major limitations of their cultivability on the nutritional and environmental factors. Based on the above results, we will design the sub-groups special cultivable isolation methods, including the selective isolation media and enrichment procedures..This is the first time systematically to focus on culturing the uncultured Acidimicrobidae and Rubrobacteridae groups by using the isolation methods, and it will break the traditional bottleneck and establish the innovative pure culture procedures, simultaneously obtain prospective results on the rare bioresources, diversity, and populations of uncultured marine actinobacteria.

海洋放线菌因其能产生结构新颖活性显著的次生代谢产物，而成为海洋微生物研究的新热点。近10多年的研究发现，海洋环境存在大量uncultured放线菌类群，其中最优势种群为酸微菌亚纲和红色杆菌亚纲，约占60%以上。而两个亚纲目前仅发现10多个纯培养菌种，且具有耐酸、耐热、耐辐射、嗜铁、固定CO2、生长缓慢等非常独特的生理特性，是良好的实验材料。本项目拟利用通量测序方法对深海沉积环境中uncultured酸微菌和红色杆菌多样性进行分析，研究其种群结构、分布规律及其与环境间的关系，为可培养研究提供有效指导；同时对现有物种进行深入的生理特性研究，探索其对营养及环境因子的依赖性，结合免培养数据，分别从分离培养基和富集两个方面设计“种群特异”的培养方案。该项目首次对海洋环境中酸微菌和红色杆菌类群进行系统的研究，有望建立全新的分离培养方法，并在稀有生物资源、多样性、种群结构及分布等方面取得前瞻性的成果。

海洋放线菌因其能产生结构新颖活性显著的次生代谢产物，而成为海洋微生物研究的新热点。近10多年的研究发现，海洋环境存在大量uncultured放线菌类群，其中最优势种群为酸微菌亚纲和红色杆菌亚纲，约占60%以上。而两个亚纲菌株具有耐酸、耐热、耐辐射、嗜铁、固定CO2、生长缓慢等非常独特的生理特性，是良好的实验材料。对于海洋放线菌研究的现状，本项目主要开展了海洋沉积环境中uncultured 放线菌多样性研究、酸微菌和红色杆菌分离培养基的设计与优化、富集培养方法和可培养多样性研究。.通过对南海及印度洋深海沉积环境的样品进行了系统的研究，完成了31个样品的高通量测序，研究了其中免培养微生物的多样性情况，特别是本研究关注的放线菌类群，并精细分析了酸微菌纲和红色杆菌纲微生物的多样性和分布情况，探明红色杆菌和酸微菌在海洋沉积环境中普遍存在，且广泛分布于不同水深层中。基于已知酸微菌纲和红色杆菌纲微生物纯培养的生理学特征，设计出了40多个选择性分离培养基进行了系统的分离，建立了以光照培养为基础的红色杆菌菌株的选择性分离培养方案，以及酸微菌和嗜热油菌类群的选择性分离培养方案；三个最古老放线菌类群的选择性分离方法建立，为后期的深入挖掘该类群资源打下良好的基础。项目共分离保藏了5000多株海洋微生物，包括800多株海洋放线菌等宝贵的资源，红色杆菌202株，酸微菌和嗜热油菌22株，新科1个，新属6个，新物种20多个，为后期医药、农药、活性化合物、功能酶、新材料等积累丰富的、稀有的微生物资源。而且，深海沉积环境原始、古老，比较陆源生物的进化模式和深海的自然进化模式，有助于深入理解生命起源和物种进化等重大科学问题。.在本项目的资助下，参加6次国内会议并做了5次大会报告，参加3次国际会议，邀请微生物资源领域国际上著名的美国东北大学Slava Epstein教授等来南海所进行学术交流；已经发表SCI论文5篇，中文核心4篇，投稿待审SCI论文2篇，申请专利3项。