深海沉积环境海洋放线菌纯培养策略研究

Marine actinobacteria are an important component of medicinal marine microbial resources, and have attracted worldwide concerns in recent years by biologists, chemists and ecologists. However, due to the lack of mature and effective isolation and cultivation methods for marine actinobacteria, more than 99 percent of marine actinobacteria are still unculturable in the lab conditions and thus are buried in the deep seabed, which largely hamper the utilization of these valuable resources. Therefore, development of efficient methods to culture marine actinobacteria becomes a bottleneck problem to be solved at present. .To better utilize the deep-sea sediment samples collected in recent years from South China Sea and Indian Ocean, we intend to establish effective isolation methods for marine actinobacteria. Firstly, the physiological properties of marine actinobacteria will be tested by considering the physical and chemical factors in the deep-sea sedimentary environments in conjunction with the bioinformatic analysis of actinobacterial community structure by pyrosequencing method (454 sequencing). Next, we will study the physiological properties of marine actinobacteria on the special nutritional requirements. With the guidance of the above background-information, we will focus on establishing the selective isolation methods and processes of marine actinobacteria from extreme environments like deep-sea. The following factors will be considered, (i) pretreatment process of samples; (ii) the design of the selective isolation media by optimizing exiting media for terrestrial actinobacteria; (iii) the design of selection media by simulating natural environment conditions; and (iv) the building of the high-throughput isolation method for cells in the sediment samples by combination of density gradient centrifugation separation and the single-cell sorting of flow cytometry..The implementation of this project to build the mature and efficient selective isolation methods for marine actinobacteria will lay a good ground for the rapid mining and evaluation of these untapped valuable resources in the world ocean, and the further research of its ecological distribution in the deep ocean, etc.

海洋放线菌是海洋药用微生物资源的重要组成，近年来引起国内外生物、化学、医药等领域专家的广泛关注。然而，由于缺乏成熟、有效的纯培养方法，致使99%以上的海洋放线菌资源仍然深埋海底，严重制约了对其的开发和利用，成为当前亟待解决的瓶颈问题。本项目拟以多年采集的南海及印度洋深海沉积样品为基础，以沉积环境理化因子及焦磷酸测序发现的深海沉积环境放线菌类群背景为指导，从海洋放线菌耐受物理、化学刺激和特殊的营养需求等生理学特性入手，深入探讨海洋放线菌选择性生理机制，建立适宜于深海放线菌挖掘的有效方法，包括建立样品预处理流程、高效选择性分离培养基、生态环境模拟培养等有效的分离方法与流程以及密度梯度离心与流式细胞仪相结合的高通量筛选手段等。本项目实施建立深海沉积环境放线菌高效的选择性分离方法将为海洋放线菌宝贵资源的快速挖掘和评价、海洋放线菌生态分布等研究打下良好的铺垫。

项目主要对深海环境放线菌纯培养分离方法进行研究，完成既定各项指标90%以上。. 本项目系统地对海洋放线菌盐度和温度适应性进行研究，发现添加5-10%盐浓度的培养基能提高海洋微生物的出菌率。深海低温沉积物样品，通过高温条件培养，能发现稀有放线菌类群和新物种资源。项目建立适宜于放线菌分离的深海沉积样品预处理流程1项，设计6种深海放线菌选择性分离培养基和3个产酶产酸微生物分离培养基，发明了一种微生物包埋微球连续制备装置；通过设计的选择性分离培养基和分离方法，共新增分离保藏海洋细菌3423株，新物种88个，完成鉴定并发表3个；新增海洋放线菌1060株，新增海洋放线菌新物种30个，在原有48个放线菌属级类群的基础上，本项目的实施新增海洋放线菌属级类群30个，总的海洋放线菌物菌株数量和物种类群应为最多。完成对80个沉积物样品的免培养微生物多样性测序及初步分析工作；通过对本组9株链霉菌和NCBI来源的22株链霉菌的基因组比较，分析海洋放线菌的海洋适应性特征，发现了20多个与海洋环境适应性关联性有很好的转运蛋白家族，以及海洋链霉菌具有较多规律间隔的短回文重复序列（CRISPR）元件，它能通过类似RNA沉默的机制抗噬菌体，较多的CRISPR元件也体现了海洋来源的链霉菌对海洋环境的适应性。比较基因组研究的结果，对海洋放线菌分离方法及培养基的设计有一定指导意义。. 项目系统地建立了海洋放线菌分离、培养的方法，积累了大量的海洋放线菌资源，为后期代谢产物、生物酶、新材料及海洋放线菌基因组研究打下基础。正式发表有项目标注的学术论文6篇，其中SCI论文4篇，中文核心2篇，微生物学通报审稿中的1篇。申请中国发明专利3项，授权2项，2013年5月申请美国专利2项并获得授权。获得2014年国家技术发明奖二等奖（第5名）、第十五届中国专利优秀奖（第5名）和2013年广东省专利金奖（第5名）各1项。