生物多样性 ›› 2013, Vol. 21 ›› Issue (1): 28-37.DOI: 10.3724/SP.J.1003.2013.10097

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东海表层沉积物纯培养与非培养细菌多样性

何建瑜, 刘雪珠, 赵荣涛, 吴方伟, 王健鑫*()   

  1. 浙江海洋学院海洋生物资源与分子工程实验室, 浙江舟山 316000
  • 收稿日期:2012-04-11 接受日期:2012-08-10 出版日期:2013-01-20 发布日期:2013-02-04
  • 通讯作者: 王健鑫
  • 基金资助:
    国家自然科学基金(31270160);浙江省自然科学基金(LY12C03003);浙江省科技厅公益性技术应用研究项目(2011C31017);舟山市科技局海洋类项目(10248);浙江省大学生科技创新项目(2012R411002)

Diversity of cultured and uncultured bacteria in surface layer sediment from the East China Sea

Jianyu He, Xuezhu Liu, Rongtao Zhao, Fangwei Wu, Jianxin Wang*()   

  1. Laboratory for Marine Living Resources and Molecular Engineering, Zhejiang Ocean University, Zhoushan, Zhejiang 316000
  • Received:2012-04-11 Accepted:2012-08-10 Online:2013-01-20 Published:2013-02-04
  • Contact: Wang Jianxin

摘要:

于2011年7月采集了浙江舟山沿岸海底沉积物样品(122°10′41″ E, 29°49′7″ N), 通过埋片原位观察、荧光显微计数、纯培养菌分离以及非培养细菌构建克隆文库的方法, 分析和研究了东海表层沉积物细菌群落结构及其多样性。埋片原位观察和荧光显微计数法的结果表明: 沉积物样品中细菌的细胞数量为(9.30±3.44) ×107 cells/g; 通过分离纯化共获得313株细菌, 分属于20个属, 4种培养基对细菌的分离效果依次为RO>M1>Zobell 2216>MR2A; 常规形态学与生理生化鉴定结果显示芽孢杆菌属(Bacillus)和海球菌属(Marinococcus)从数量和种类上皆为优势菌属, 分别占分离菌株总数的21.08%和17.25%; 对73株典型海洋细菌进行16S rDNA分子鉴定发现, 厚壁菌门(57.5%)、γ-变形杆菌纲(32.9%)、黄杆菌纲(4.1%)和放线菌纲(5.5%)等为主要类群。非培养细菌克隆文库序列分析发现: 细菌克隆子主要属于厚壁菌门和变形杆菌门, 而芽孢杆菌纲和γ-变形杆菌纲是上述两个门的优势类群。综合纯培养与非培养数据得出东海海域表层沉积物细菌多样性丰富, 具有进一步开发研究的价值。

关键词: 东海海域, 海洋细菌, 16S rDNA, 群落结构, 多样性

Abstract

Marine bacteria are an extremely important component of marine bioresources. We studied the community structure and diversity of cultured and uncultured bacteria in surface layer sediment sampled from the East China Sea (122°10′41″ E, 29°49′7″ N) by buried piece observation, fluorescence enumeration, pure culture techniques and 16S rDNA clone library analysis. The average abundance of bacteria was (9.30±3.44)×107 cells/g according to fluorescence microscopy enumeration and observation of buried slides. A total of 313 bacterial strains belonging to 20 genera was obtained from four kinds of culture medium and the separation efficiency of bacteria ranked as RO>M1>Zobell 2216>MR2A. In terms of morphology, physiology and biochemistry, Bacillus (21.08%) and Marinococcus (17.25%) were shown to be the most dominant and highly diverse bacteria. The analysis of 16S rDNA sequence from 73 representative bacteria showed most clones were affiliated with Firmicutes (57.5%): the other marine bacteria including gamma-Proteobacteria (32.9%), Flavobacteria (4.1%), and Actinobacteria (5.5%). Most clones were related to uncultivated bacterial clones retrieved from surface layer sediment and contained several highly divergent 16S rDNA gene sequences. After constructing a molecular library and phylogenetic analysis, the sequenced clones were shown to fall into two phyla of the domain Bacteria, Firmicutes and Proteobacteria, in which Bacillus and gamma-proteobacteria were the dominant groups, respectively. The results revealed that the species and community structure of bacteria in surface layer sediment from the East China Sea continental shelf were abundant and diverse, thus warranting further research and exploitation.

Key words: East China Sea, bacteria, 16S rDNA, community structure, diversity