生物多样性 ›› 2013, Vol. 21 ›› Issue (1): 28-37. DOI: 10.3724/SP.J.1003.2013.10097
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:
Jianyu He, Xuezhu Liu, Rongtao Zhao, Fangwei Wu, Jianxin Wang*()
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。
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.
图1 采用埋片观察法得到的东海表层沉积物样品中微生物和有机质的原位分布图。红色箭头为微生物菌体, 绿色箭头为有机杂质。
Fig. 1 Direct observation of the sediment from East China Sea surface by the method of buried piece to find the distribution of microorganisms and organics in situ. Red arrows indicate microorganisms, and green arrows indicate organics.
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 |
表1 在4种培养基中利用稀释平板计数的方法统计纯培养的菌落单元总量
Table 1 Statistics of colony-forming units in four kinds of medias by the dilution method of plate counting
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 |
图2 不同菌属菌株数量分布图。Sb: 芽孢乳杆菌属; Hb: 盐芽孢杆菌属; Bb: 短芽孢杆菌属; Bi: 芽孢杆菌属; Pb: 类芽孢杆菌属; Plb: 脂肪杆菌属; Bbm: 短状杆菌属; Ab: 节杆菌属; Sl: 葡萄球菌属; Eo: 肠球菌属; Mo: 海球菌属; Pt: 巴斯德氏菌属; Vb: 弧菌属; Ao: 交替单胞菌属; Pd: 假单胞菌属; Ep: 丹毒丝菌属; Am: 壤霉菌属; Aib: 硫胺素芽孢杆菌属; St: 链球菌属; Aom: 放线菌属
Fig. 2 Distribution of the different genus. Sb, Sporolac- tobacillus; Hb,Halobacillus; Bb, Brevibacillus; Bi, Bacillus; Pb, Paenibacillus; Plb, Pimelobacter; Bbm, Brachybacterium; Ab, Arthrobactor; Sl, Staphylococcus; Eo, Enterococcus; Mo, Marinococcus; Pt, Pasteurella; Vb, Vibrio; Ao, Alteromonas; Pd, Pseudomonas; Ep, Erysipelothrix; Am, Agromyces; Aib, Aneurinibacillus; St, Streptococcus; Aom, Actinomyces.
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 |
表2 通过细菌16S rDNA基因序列对东海表层沉积物分离得到的纯培养细菌鉴定结果
Table 2 Identification of pure-cultured bacteria isolated from East China Sea surface sediment by bacterial 16S rDNA gene
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 |
图3 根据东海表层沉积物中纯培养得到的细菌菌株16S rDNA基因序列构建的系统发育树
Fig. 3 Phylogenetic tree based on 16S rDNA sequences of pure-cultured clones from East China Sea surface sediment
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 |
表3 东海表层沉积物非培养细菌的16S rDNA序列在数据库中的同源分类比较以及相似菌株的分离源分析
Table 3 Taxonomic affiliation of clones recovered in the bacterial clone libraries from East China Sea surface sediment and analysis of closest species origin
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 |
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