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1 43 4 Vol.43, No OCEANOLOGIA ET LIMNOLOGIA SINICA July, 2012 * ( ) DH-13 DNA, PCR TA 16S rdna, : (Proteobacteria, 41.5%), (Planctomycetes, 10.9%) (Actinobacteria, 8.9%) CFB group (7.9%), (Acidobacteria) (Verrucomicrobia) (Nitrospira) (Firmicutes) ; (Crenarchaeota), Marine Crenarchaeotic Group I (MG I, 93.6%), Miscellaneous Crenarchaeotic Group (MCG) Marine Benthic Group C (MBGC) Marine Benthic Group A (MBGA),,, 16S rdna Q93, km 2, 48.6%,,, 1/2 5/6, 1/10 1/3 (, 2008),, (, 2001), (Marion et al, 2008),, (, 2000; Abreu et al, 2001; Delong, 1992), N, E, - - (, 2008) (>885km 3 /a),,,,,, (, 2010),,, (, 2006; Zeng et al, 2007), (, 2010; Park et al, 2008) 16S rrna, *, LY12C03003 ;, 2011C31017 ;, ;, 2010R411002,,, zswjx2575@163.com :,, shige @163.com : , :

2 DH-13, DH-13 ( N, E; 40m), 0 10cm 3cm 4,, 20, 80, 1.2 ( GB/T ; Heijs et al, 2008), 2ml 60 72h, ; (TOC) (TOC-V CSH, ) ; (TN) (SKALAR Primacs SN, ) 1.3 DNA FastPrep -24 (MP Biomedicals ) FastDNA spin kit for soil DNA, (Bio-Rad ) DNA, PCR 1.4 PCR PCR 27F (5 -AGAGTTT GATCMTGGCTCAG-3 ; M A C) 1492R (5 -TA CGGYTACCTTGTTACGACTT-3 ; Y C T), PCR Arch 21F (5 -TTCCGGTTGATCC YGCCGGA-3 ) Arch 958R (5 -YCCGGCGTTGAMT CCAATT-3 ) PCR : 95 4min; 94 30s, 50 30s, 72 60s, 35 ; 72 10min 1% PCR, DNA (QIAGEN) 1.5 PCR pmd-18t vector (TaKaRa) 16, (E. coli) DH5α,,,, 1.6 RDP, CHECK-CHIMERA ; BLASTN, ClustalX (Version 1.8), MEGA4 (Kumar et al, 2004); PHYLIP DNASIS, DOTUR (OTU)(Schloss et al, 2005) 16S rdna GenBank JN JN JN JN JN JN JN DH-13 1, (TOC) g/kg, (TN) g/kg, NO mg/kg, 11.3% 41.6%, (TOC/TN) S rdna 30 ( 1470bp), RDP II, 103, 1400bp, 0 3cm 3 5cm 5 8cm 8 10cm , DOTUR 97% OTU, 92 OTU 30 ( 900bp) 表 1 DH-13 站点表层沉积物环境化学指标 Tab.1 Chemical measurements of surface layer sediment in DH-13 station (g/kg) (g/kg) TOC/TN NO 3 (mg/kg) (%) DH-13 (0 3cm) DH-13 (3 5cm) DH-13 (5 8cm) DH-13 (8 10cm) :

3 4 : 807, 93, 800bp, 0 3cm 3 5cm 5 8cm 8 10cm , DOTUR 98% OTU, 10 OUT, 2 : DH- 13 Chao 1 ACE Shannon- Weaver, Simpson, DH-13 ; S rdna DH ( 1 2), (Proteobacteria, 41.5%) (Planctomycetes, 10.9%) (Actinobacteria, 8.9%) CFB group (7.9%); (Acidobacteria, 5.9%) (Verrucomicrobia, 3.9%) (Nitrospira, 1.9%) (Firmicutes, 1%) 20.1% (Proteobacteria),, 41.5%, (30.5%)(Li et al, 2008a), Alpha Beta Gamma Delta, Gamma-Proteobacteria ( 52%) Gamma-Proteobacteria DH132B23(5 ) DH133B05(3 ) (ELSC) Gamma-Proteobacteria (99%), DH133B08(2 ) DH132B24(2 ) DH132B31(2 ) Gamma-Proteobacteria ARTE12 (99%), DH132B01(2 ) Taketomi Gamma-Proteobacteria (95%), DH132B27(4 ) Gamma- Proteobacteria (97%), DH134B08 DH131B13 Gamma-Proteobacteria Delta-Proteobacteria, (Desulfovibrio Desulfobacter Desulfococcus Desulfonema ) (Desulfuromonas) Delta-Proteobacteria 35%, DH133B26 (3 ) Delta-Proteobacteria, DH132B04 (3 ) (, 2010) Delta-Proteobacteria, Delta-Proteobacteria, DH132B36 (3 ) ( ) (Myxococcales) 916-W4-24 (98%), DH134B07 (4 ) - Delta-Proteobacteria (98%), (SAB) (96%) Monterey (98%) Alpha Beta-Proteobacteria, Alpha-Proteobacteria 表 2 东海陆架表层沉积物细菌和古菌多样性指数一览表 Tab.2 Measures of bacterial and archaeal diversity from the surface layer sediment of the East China Sea DH13b DH13a Bias-corrected Chao 1 Schloss et al, Abundance-base Coverage Estimator (ACE) Schloss et al, S Shannon index (H ) H = Pi lnp i Simpson index (D) i= 1 S ni( ni 1) D = N( N 1) : S OTUs, N, n i OTU, P i i i= 1

4 Fig S rdna Phylogenetic tree of Proteobacteria according to 16S rdna gene sequences ), Beta-Proteobacteria (Planctomycetes), (Vergin et al, 1998),, DH134B06 (3 ) (SAB) (98%), DH132B30 (2 ) Salton (98%), DH132B11 (4 ) (ELSC) (95%), DH133B16 (2 ) (96%) CFB (Cytophaga-Flavobacterium-Bacterioides),,

5 4 : S rdna Fig.2 Phylogenetic tree of other bacteria according to 16S rdna gene sequences CFB 2 (Phakellia ventilabrum) (Bacteroidetes bacterium R8-Ret-T12-11d) 96%, (Monostroma oxyspermum) (Cytophaga sp.i-1858) 95%, CFB, 4 (ELSC) Cadiz (Montastraea faveolata) : G+C (Actinobacteria) G+C (Firmicutes), Bizerte ; (Siderastrea siderea) S rdna DH S rdna (Crenarchaeota) ( 3), Marine Crenarchaeotic Group I (MG I, 93.55%), Marine Benthic Group

6 S rdna Fig.3 Archaeal phylogenetic tree according to 16S rdna gene sequences C (MBGC, 3.22%) Miscellaneous Crenarchaeotic Group (MCG, 2.15%) Marine Benthic Group A (MBGA, 1.08%) Delong(1992),, MG I 13, 5, Cluster MG I ( 52%), Cadiz,, Cluster MG I ( 34%),, Napoli, MG I, MCG, Nankai (Sorensen et al, 2006) MCG (Singha et al, 2010) MBG, MBGA (Li et al, 2008b), MBGA (Costantino et al, 1999) MBGA MBGC, 3 (99%) (99%) MBGC 3 3.1,,

7 4 : 811,,,, TOC/TN( ), (Redfield ) 6.6,, 20 (Meyers, 1994),, DH13 ( ),, (5 10cm) (0 5cm),, (, 2008),,,,,,,,, TOC/TN : TOC/TN < 20, ; TOC/TN 20 30, ; TOC/TN > 30, DH13 3.2, (2005) 1) : ( cells/ml), Li (2011) FISH DGGE Beta- (β-aob) ( ) 10 5 cells/g, TOC, Feng (2009), ( 72.9%), (6.4%) (4.6%) (4.1%);, Gamma-Proteobacteria, Delta-Proteobacteria,, 50%, Gamma-Proteobacteria (Ravenschlag et al, 2001; Bowman et al, 2003; Inagaki et al, 2003), DH13 Feng (2009), 3.3, Karner (2001), 1/3,, (, 2001), Mandovi Zuari (Singha et al, 2010) 78%, Douro (Abreu et al, 2001) 85.7%, Dang (2008), Candidatus Nitrosopumilus maritimus SCM1,, ; (2010), 16S rdna 1), :, 85 97

8 812 43, Miscellaneous Crenarchaeotic Group (MCG), Marine Benthic Group B (MBG-B) South African Gold Mine Euryarchaeotic Group (SAGMEG) Anaerobic Methanot rophs 3 (ANME-3) Marine Crenarchaeotic Group I (MG I) Marine Benthic Group D (MBG-D), DH13, MG I 93.6%, Mandovi Zuari,, DH13,, 4 16S rrna DH-13, DOTUR,, 8,, 4, MG I,,,,,,, , 31(2): ,,, , 48(3): , :, 1 105,,, , 33 (1): 63 67, , 24(4): 22 26,,, ( ), 35(2): ,, , 25(2): 20 23,,, :, 40(6): Abreu C, Jurgens G, Marco P D et al, Crenarchaeota and Euryarchaeota in temperate estuarine sediments. Journal of Applied Microbiology, 90: Bowman J P, McCuaig R D, Biodiversity community structural shifts and biogeography of prokaryotes within Antarctic continental shelf sediment. Appl Environ Microb, 69: Costantino V, Jannasch H W, MacGregor B J et al, Population structure and phylogenetic characterization of marine benthic archaea in deep-sea sediments. Applied and Environmental Microbiology, 65(10): Dang H Y, Zhang X X, Sun J et al, Diversity and spatial distribution of sediment ammonia-oxidizing crenarchaeota in response to estuarine and environmental gradients in the Changjiang Estuary and East China Sea. Microbiology, 154: Delong E F, Archaea in coastal marine environment. Proc Natl Acad Sci, 89: Feng B W, Li X R, Wang J H et al, Bacterial diversity of water and sediment in the Changjiang estuary and coastal area of the East China Sea. FEMS Microbiol Ecol, 70: Inagaki F, Suzuki M, Takai K et al, Microbial communities associated with geological horizons in coastal subseafloor sediments from the Sea of Okhotsk. Appl Environ Microb, 69: Karner M B, DeLong E F, Karl D M, Archaeal dominance in the mesopelagic zone of the Pacific Ocean. Nature, 409: Kumar S, Tamura K, Nei M, MEGA3: Integrated software for molecular evolutionary genetics analysis and sequence alignment. Briefings in Bioinformatics, 5: Li J L, Bai J, Gao H W et al, Distribution of ammonia-oxidizing Betaproteobacteria community in surface sediment off the Changjiang River Estuary in summer. Acta Oceanol Sin, 30(3): Li T, Wang P, Wang P X, 2008a. Microbia diversity in surface sediments of the Xisha Trough, the South China Sea. Acta Ecologica Sinica, 28(3): Li Y, Li F, Zhang X et al, 2008b. Vertical distribution of bacterial and archaeal communities along discrete layers of a deep-sea cold sediment sample at the East Pacific Rise (approximately 13 degrees N). Extremophiles, 12(4): Marion K, Rainer W, Maja B, Microscale Investigations of microbial communities in coastal surficial sediments. Marine ecology, 29: Meyers P A, Preservation of elemental and isotopic source identification of sedimentary organic matter. Chemical Geology, 144: Park S J, Park B J, Pham V H et al, Microeukaryotic diversity in marine environments, an analysis of surface layer sediments from the East Sea. The Journal of Microbiology,

9 4 : (3): Ravenschlag K, Sahm K, Amann R, Quantitative molecular analysis of the microbial community in marine Arctic sediments (Svalbard). Appl Environ Microb, 67: Schloss P D, Handelsman J, Introducing DOTUR, a computer program for defining operational taxonomic units and estimating species richness. Applied and Environmental Microbiology, 71(3): Sorensen K B, Teske A, Stratified communities of active archaea in deep marine subsurface sediments. Appl Environ Microbiol, 72 (7): Vergin K L, Urbach E, Stein J L et al, Screening of a fosmid library of marine environmental genomic DNA fragments reveals four clones related to members of the order Planctomycetales. Appl Environ Microbiol, 64: Zeng Y H, Li H Y, Jiao N Z, Phylogenetic diversity of planktonic archaea in the estuarine region of East China Sea. Microbiological Research, 162: A PRELIMINARY STUDY OF MICROBIAL DIVERSITY OF THE SURFACE LAYER SEDIMENTS FROM THE EAST CHINA SEA WANG Jian-Xin, XU Xian-En, ZHOU Lian-Lian, TAO Shi, LIU Xue-Zhu, SHI Ge (Zhejiang Ocean University, Laboratory for Marine Living Resources and Molecular Engineering, Zhoushan, ) Abstract The environmental total DNAs were directly extracted from the surface layer sediment of the East China sea. The bacterial and archaeal 16S rdna gene libraries were generated by using PCR and TA cloning. Phylogenetic results show that there are eight phyla in the bacteria domain: Proteobacteria (41.5%), Planctomycete (10.9%), Actinobacteria (8.9%), CFB group (7.9%), Acidobacteria, Verrucomicrobia, Nitrospira and Firmicutes, the phylum Proteobacteria is predominant. In the archaea domain, all clones were belonged to Crenarchaeota and no one Euryarchaeota clone was detected. There are four divisions in the Crenarchaeota kingdom: Marine Crenarchaeotic Group I (MG I, 93.6%), Miscellaneous Crenarchaeotic Group (MCG), Marine Benthic Group C (MBGC), Marine Benthic Group A (MBGA), and MG I is the dominating group. DOTUR statistics of 16S rdna gene sequences from the libraries indicating a high richness in the sediment microbial communities. Key words Surface layer sediment, Microbial diversity, 16S rdna

Microsoft Word 黄媛_new_.doc

Microsoft Word 黄媛_new_.doc 46 5 Vol.46, No.5 2015 9 OCEANOLOGIA ET LIMNOLOGIA SINICA Sep., 2015 * 1 2 1 1 (1. 310036; 2. 310012) Illumina MiSeq, 4 DNA, 16S rrna V3 V4, 67734, 2181 OTU, 441 bp, 400bp 99.74% 97% OTU,,, 30 252 (Proteobacteria),