东海表层沉积物纯培养与非培养细菌多样性

doi:10.3724/SP.J.1003.2013.10097

摘要/Abstract

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)×10⁷cells/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

何建瑜, 刘雪珠, 赵荣涛, 吴方伟, 王健鑫 (2013) 东海表层沉积物纯培养与非培养细菌多样性. 生物多样性, 21, 28-37. DOI: 10.3724/SP.J.1003.2013.10097.

Jianyu He,Xuezhu Liu,Rongtao Zhao,Fangwei Wu,Jianxin Wang (2013) Diversity of cultured and uncultured bacteria in surface layer sediment from the East China Sea. Biodiversity Science, 21, 28-37. DOI: 10.3724/SP.J.1003.2013.10097.

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链接本文: https://www.biodiversity-science.net/CN/10.3724/SP.J.1003.2013.10097

https://www.biodiversity-science.net/CN/Y2013/V21/I1/28

图/表 7

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培养基 Medium	稀释倍数 Dilution multiple	平板菌落数 No. of plate culture count	菌落单元数量 Numbers of colony- forming units(CFU/g)
M1	1,000	73	(7.30±.0.14)×10⁴
Zobell 2216	1,000	71	(7.10±0.08)×10⁴
MR2A	1,000	59	(5.90±0.09)×10⁴
RO	10,000	49	(4.90±0.17)×10⁵

培养基 Medium	稀释倍数 Dilution multiple	平板菌落数 No. of plate culture count	菌落单元数量 Numbers of colony- forming units(CFU/g)
M1	1,000	73	(7.30±.0.14)×10⁴
Zobell 2216	1,000	71	(7.10±0.08)×10⁴
MR2A	1,000	59	(5.90±0.09)×10⁴
RO	10,000	49	(4.90±0.17)×10⁵

代表菌株 Strain no.	登录号 Accession no.	相似菌株(登录号) Closest species (Accession no.)	相似性 Homology (%)
芽孢杆菌纲 Bacilli
DHC01	JQ904712	Bacillus pumilus strain PR13 (JQ435673)	99
DHC02	JQ904713	Bacterium YCG4 (JF775413)	97
DHC03	JQ904714	Bacillus tequilensis strain KM35 (JF411314)	99
DHC04	JQ904715	Bacillus sp. M71_S53 (FM992830)	100
DHC06	JQ904717	Bacillus cereus strain CB-10 (JF496530)	96
DHC07	JQ904718	Bacillus subtilis strain B7 (JQ086378)	100
DHC08	JQ904719	Bacillus horikoshii strain NBSL26 (FJ984529)	99
DHC10	JQ904720	Bacillus sp. RHS-str.320 (HE586887)	97
DHC11	JQ904721	Bacillus infantis strain AL10 (JF411305)	99
DHC12	JQ904722	Bacillus aryabhattai isolate PSB59 (HQ242772)	100
DHC13	JQ904723	Bacillus barbaricus strain P2 (GQ200827)	99
DHC14	JQ904724	Bacillus gibsonii strain S-2 (AY737309)	99
DHC15	JQ904726	Paenibacillus sp. GP26-03 (AM162316)	99
黄杆菌纲 Flavobacteria
DHC05	JQ904716	Cloacibacterium normanense strain tu33 (FJ544402)	100
放线菌纲 Actinobacteria
DHC09	JQ904725	Brevibacterium sp. 210_14 (GQ199716)	99
γ-变形菌纲 Gamma-proteobacteria
DHC16	JQ904727	Alteromonas sp. SCSWC10 (FJ461444)	99
DHC17	JQ904728	Pseudoalteromonas sp. B296 (FN295786)	98
DHC18	JQ904729	Pseudoalteromonas sp. VS-2 (FJ497709)	97
DHC19	JQ904730	Vibrio algoinfesta strain NBRC 14665 (AB680645)	99
DHC20	JQ904731	Vibrio harveyi EHP8 (FJ227114)	99
DHC21	JQ904732	Vibrio azureus strain CAIM 1457 (JN603238)	99
DHC22	JQ904733	Vibrio parahaemolyticus strain CM12 (EU660326)	99

代表菌株 Strain no.	登录号 Accession no.	相似菌株(登录号) Closest species (Accession no.)	相似性 Homology (%)
芽孢杆菌纲 Bacilli
DHC01	JQ904712	Bacillus pumilus strain PR13 (JQ435673)	99
DHC02	JQ904713	Bacterium YCG4 (JF775413)	97
DHC03	JQ904714	Bacillus tequilensis strain KM35 (JF411314)	99
DHC04	JQ904715	Bacillus sp. M71_S53 (FM992830)	100
DHC06	JQ904717	Bacillus cereus strain CB-10 (JF496530)	96
DHC07	JQ904718	Bacillus subtilis strain B7 (JQ086378)	100
DHC08	JQ904719	Bacillus horikoshii strain NBSL26 (FJ984529)	99
DHC10	JQ904720	Bacillus sp. RHS-str.320 (HE586887)	97
DHC11	JQ904721	Bacillus infantis strain AL10 (JF411305)	99
DHC12	JQ904722	Bacillus aryabhattai isolate PSB59 (HQ242772)	100
DHC13	JQ904723	Bacillus barbaricus strain P2 (GQ200827)	99
DHC14	JQ904724	Bacillus gibsonii strain S-2 (AY737309)	99
DHC15	JQ904726	Paenibacillus sp. GP26-03 (AM162316)	99
黄杆菌纲 Flavobacteria
DHC05	JQ904716	Cloacibacterium normanense strain tu33 (FJ544402)	100
放线菌纲 Actinobacteria
DHC09	JQ904725	Brevibacterium sp. 210_14 (GQ199716)	99
γ-变形菌纲 Gamma-proteobacteria
DHC16	JQ904727	Alteromonas sp. SCSWC10 (FJ461444)	99
DHC17	JQ904728	Pseudoalteromonas sp. B296 (FN295786)	98
DHC18	JQ904729	Pseudoalteromonas sp. VS-2 (FJ497709)	97
DHC19	JQ904730	Vibrio algoinfesta strain NBRC 14665 (AB680645)	99
DHC20	JQ904731	Vibrio harveyi EHP8 (FJ227114)	99
DHC21	JQ904732	Vibrio azureus strain CAIM 1457 (JN603238)	99
DHC22	JQ904733	Vibrio parahaemolyticus strain CM12 (EU660326)	99

代表克隆子 Clones no.	登录号 Accession no.	相似菌株(登录号) Closest species (Accession no.)	相似菌株分离源 Closest species origin	相似性 Homology (%)
芽孢杆菌纲 Bacilli
DHUC01	JQ904734	Bacillus cereus strain E22(GU826149 )	巴西土壤 Brazilian soils	99
DHUC02	JQ904735	Bacillus sp. C1(JN571045)	人造草坪土壤 Chinampa soils	98
DHUC03	JQ904736	Bacillus aquimaris strain T172 (HQ647277)	东乡野生水稻 Dongxiang wild rice	99
DHUC04	JQ904737	Bacillus subtilis strain MSI-9 (JN993708)	人参土壤 Ginseng soil	98
DHUC05	JQ904738	Bacillus megaterium strain XAS5-1 (JF496312)	生物土壤结皮 Biological soil crusts	100
DHUC06	JQ904739	Bacillus subterraneus strain DSM13966T (FR733689)	普通土壤 Normal soil	97
DHUC07	JQ904746	Bacillus barbaricus strain P2 (GQ200827)	盐田 Solar salterns	94
DHUC08	JQ904740	Uncultured Bacillus sp. (HQ397100)	普通土壤 Normal soil	100
DHUC09	JQ904741	Geobacillus stearothermophilus strain DoB50 (JQ359102)	铁皮石斛 Dendrobium officinale	99
DHUC10	JQ904747	Brevibacillus sp. 100-5 (FJ959373)	麦田土壤 Wheat field soil	94
α-变形菌纲 Alpha-proteobacteria
DHUC11	JQ904742	Uncultured alpha-proteobacterium (FJ205181)	不活动的热液区沉积物 Inactive hydrothermal field sediments	95
γ-变形菌纲 Gamma-proteobacteria
DHUC12	JQ904743	Uncultured Alteromonas sp. (HQ012278)	海洋 Marine	100
DHUC13	JQ904744	Vibrio harveyi strain EHP8(FJ227114)	枪乌贼发光器官 Loliginid squid light organs	100
DHUC14	JQ904745	Vibrio hepatarius (NR025491)	水生动物和海洋环境 Aquatic animals and the marine environment	98