Microsoft Word 黄媛_new_.doc

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1 46 5 Vol.46, No OCEANOLOGIA ET LIMNOLOGIA SINICA Sep., 2015 * ( ; ) Illumina MiSeq, 4 DNA, 16S rrna V3 V4, 67734, 2181 OTU, 441 bp, 400bp 99.74% 97% OTU,,, (Proteobacteria), 30.0% 64.7%, β- γ- δ-, N S,, 4 10% 15% (Unclassified), ; ; ; Illumina MiSeq Q938 doi: /hyhz , (, 2009) (, 2013), (Klammer et al, 2002),, (, 2013) Li (2011) (fluorescence in situ hybridization, FISH) (denaturing gradient gel electrophoresis, DGGE),, β- (β-proteobacteria) (AOB) ( ) 10 5 cells/g, (TOC), (2014) PCR (qpcr) (DGGE),, ;,,, ( ) 20,, 60km 2 12km 2,, (, 2003), 2003, 2008,, - - *, LY14C ;, 2014C33061,, @qq.com :,,, mchen63@163.com : , :

2 5 : 1203,,, MiSeq,,, ( E, N),, : (1) (, ); (2) (, ); (3) (, ); (4) (, ) 4 (0 10cm), 3, 2m, 1 4, 20 C 1.2 ph,,,, ph ph Analytik multi N/C 3100 ; EURO EA ; H 2 SO 4 -HClO 4 - (, 2015) MiSeq EZNA Soil DNA Kit (OMEGA ) DNA, 16S rrna V3 V4, : 338F (5 -ACTCCTACGGGAG GCAGCA-3 ); 806R (5 -GGACTACHVGGGTWTCTA AT-3 ) PCR (20μL): 5 FastPfu Buffer 4μL, 2.5mmol/L dntps 2μL, Forward Primer (5μmol/L) 0.4μL, Reverse Primer (5μmol/L) 0.4μL, FastPfu Polymerase 0.4μL, Template DNA 10ng : 94 C 120s, 94 C 30s, 55 C 30s, 72 C 45s; 28 3, PCR 2%, AxyPrep DNA PCR, Tris-HCl, 2%, PCR QuantiFluor -ST (Promega ),, Illumina MiSeq (, 2014) read 20, Trimmomatic FLASH overlap 0.2, barcode 0, OTU Usearch (vsesion 7.1 http: //qiime.org/), 97% (OUT) OTU, RDP (Wang et al, 2007)( 0.7) 97% OTU 16S : Silva (Release115 http: // OTUs Mothur (version v http: //www. mothur.org/wiki/schloss_sop#alpha diversity) Chao (http: // Chao) Shannon-Wiener (http: // , 4, 1, 4 ; 4, ph, 2 ph 5.17,

1204 46 表 1 西溪湿地沉积物样品理化性质 Tab.1 Physicochemical parameters in Xixi sediment ph (g/kg) (g/kg) (g/kg) 1 6.60±0.09 49.313±1.003 9.423±0.057 0.665±0.015 2 5.17±0.11 20.073±0.656 4.900±0.065 0.245±0.

3 表 1 西溪湿地沉积物样品理化性质 Tab.1 Physicochemical parameters in Xixi sediment ph (g/kg) (g/kg) (g/kg) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± RDP 97% OTU,, norank, (Proteobacteria) (Chloroflexi) (Firmicutes) (Bacteroidetes) (Nitrospirae) (Acidobacteria) (Chlorobi) (Spirochaetae) (Fusobacteria) (Actinobacteria) (Verrucomicrobia) (Gemmatimonadetes) 30 ( ) 252 ( 1, other ), (Ferribacterium) (Lactococcus) (Anaeromyxobacter) (Thiobacillus) (Pseudomonas) (Spirochaeta) (Thioalkalispira) Sulfuritalea Fig.1 1 Dominant bacterial community of each sample at the genus level

4 5 : 1205 (Crenothrix) (Syntrophus) Pseudorhodoferax 1 4, 8.29% 12.30%; 2 3, 13.52% 8.87%, 1 (Actibacter) (Beggiatoa) Ferruginibacter 6 ; 2 (Ferritrophicum) (Sorangium) (Neochlamydia) 10 ; 3 (Bdellovibrio); 4 (Methylocaldum) Propionivibrio, (Uncultured) (Unclassified), 30% 50%, 10% 20% 2.3 α-,, , barcode, 16S rdna Unique Reads, 97% OTU, OTU, OTU,, 441 bp, >400 bp 99.74% Reads OTU 2, ( 2) 2, ,,, Shannon- Wiener ( 3),,, ; 2000,, 4,, 3 PCR, ( 2) OTU Chao, 3 Shannon, 4823 Mothur α-, 2 Fig.2 Rarefaction curves of each sample 3 Shannon-Wiener Fig.3 Shannon-Wiener curves of each sample 表 2 不同样品细菌群落的多样性指数 Tab.2 The diversity index for bacterial community in different samples OTU OTU Chao Shannon

5 ,, 4 Chao Shannon, 2, 4, OTU, 1 454, 2 567, 3 472, ( 4), 4 OTU 224, 11.34% 1 4 OTU 264, 2 3 OTU 293, OTU, 1 4, 2 3, 4 ( 5),, 1 4, 2 3, 4 OTU Venn Fig.4 Venn diagram showing the unique and shared OTUs (3% distance level) 5 Fig.5 Multiple samples similarity tree 3 3.1,,,, PCR (DGGE) (FISH), 4,,, (Proteobaeteria), % 31.0% 31.5% 53.3%,, (Cifuentes et al, 2000; Madrid et al, 2001) Ravenschlag (2001), γ- (γ-proteobacteria) δ- (δ-proteobacteria) ; Li (2009) 16S rrna,,, β- γ- δ-,, C N S Fe, α β γ δ,, 4 (Nitrosococcus) (Nitrospira) (Nitrosomonadaceae_uncultured) Nitrospinaceae_ uncultured (Nitrospiraceae_uncultured),, ( ), β γ,, 4 (Thiobacillus); (Thioalkalispira) 2 3,

6 5 : 1207 (Beggiatoa) 1 ( ) (Desulfarculaceae_ uncultured), (Desulfobulbus) (Desulforhabdus) Desulfocapsa Desulfobacca (Desulfuromonadales_ unclassified) (Higashioka et al, 2013),, S -2 Zn 2+ Cu 2+ Hg 2+ Pb 2+ (, 1999) γ,,, 4 (Methylobacter) (Methylocystis) (Methylomicrobium) (Methylocaldum) Methylogaea Methylotenera (Methylococcaceae_ unclassified) (Methylophilaceae_uncultured), (Methylobacter), % 24.9% 43.0% 13.5%,, (Grégoire et al, 2011), (, 2012),, Björnsson (2002), ; (2013), COD ; Sorokin (2012), ; (2012), Levilinea,,, 4 10% 15% (Unclassified),, 30% 50%,, 3.2, Chao Shannon, 4 Chao Shannon,,,, (Córdova- Kreylos et al, 2006), 4, mg/g,, mg/g, mg/g,,, ;,,, (, 2012), ( 1),, 1 4, 2 3 ( 4 5) 1 4 (Ferribacterium), 8.29% 12.30%,, Fe +3, Ferribacterium Fe +3 Fe +2, (Cumming et al., 1999), 1 4, 2 3 (Lactococcus), 13.52% 8.87%,, 2 3,,,,,,,,,,,

7 ,, (, 2013), 40, DNA ( FISH DNA ),, (terminal restriction fragment length polymorphism, T-RFLP) (denaturing gradient gel electrophoresis, DGGE) DNA (single strand conformation polymorphism, SSCP),, (Singh et al, 2011; Ding et al, 2012),, SSCP ; 500bp DNA, DGGE 1998 Handelsman (Handelsman et al, 1998), (, 2013) Illumina Miseq, 4, OTU 2181,, Deng (2012) (Molecular Ecological Networks, MENs),,,,,, (, 2013),, , (3): 45 47,, , 40(1): 6 17,,, , 21(4): ,,, , 21(4): ,,, , 55(5): ,,, FISH MBR. ( ), 43(2): ,,, , 41(11): ,,, , 21(1): 28 37, , 19(5): , 402,,, , 32(2): ,,, , 26(2): ,, , 25(11): ,,, , 33(10): ,,, , 36(6): Björnsson L, Hugenholtz P, Tyson G W et al, Filamentous Chloroflexi (green non-sulfur bacteria) are abundant in wastewater treatment processes with biological nutrient removal. Microbiology, 148(8): Cifuentes A, Antón J, Benlloch S et al, Prokaryotic diversity in Zostera noltii-colonized marine sediments. Appl Environ Microbiol, 66(4): Córdova-Kreylos A L, Cao Y P, Green P G et al, Diversity, composition, and geographical distribution of microbial communities in California salt marsh sediments. Appl Environ Microbiol, 72(5): Cumming D E, Caccavo F Jr, Sping S et al, Ferribacterium limneticum, gen. nov., sp. nov., an Fe (III)-reducing microorganism isolated from mining-impacted freshwater lake sediments. Arch Microbiol, 171(3): Deng Y, Jiang Y H, Yang Y et al, Molecular ecological network analyses. BMC Bioinformatics, 13: 113 Ding X, Peng X J, Peng X T et al, Diversity of bacteria and archaea in the deep-sea low-temperature hydrothermal sulfide chimney of the Northeastern Pacific Ocean. Afr J Biotech, 11(2):

8 5 : 1209 Grégoire P, Fardeau M-L, Joseph M et al, Isolation and characterization of Thermanaerothrix daxensis gen. nov., sp. nov., a thermophilic anaerobic bacterium pertaining to the phylum Chloroflexi, isolated from a deep hot aquifer in the Aquitaine Basin. Syst Appl Microbiol, 34(7): Handelsman J, Rondon M R, Brady S F et al, Molecular biological access to the chemistry of unknown soil microbes: a new frontier for natural products. Chem Biol, 5(10): R245 R249 Higashioka Y, Kojima H, Watanabe M et al, Desulfatitalea tepidiphila gen. nov., sp. nov., a sulfate-reducing bacterium isolated from tidal flat sediment. Int J Syst Evol Microbiol, 63: Klammer S, Posch T, Sonntag B et al, Dynamics of bacterial abundance, biomass, activity, and community composition in the oligotrophic Traunsee and the Traun river (Austria). Water Air Soil Pollut: Focus, 2(4): Li H R, Yu Y, Luo W et al, Bacterial diversity in surface sediments from the Pacific Arctic Ocean. Extremophiles, 13(2): 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 Sinica, 30(3): Madrid V M, Aller J Y, Aller R C et al, High prokaryote diversity and analysis of community structure in mobile mud deposits off French Guiana: identification of two new bacterial candidate divisions. FEMS Microbiol Ecol, 37(3): Ravenschlag K, Sahm K, Amann R, Quantitative molecular analysis of the microbial community in marine Arctic sediments (Svaibard). Appl Environ Microbiol, 67(1): Singh R, Sheoran S, Sharma P et al, Analysis of simple sequence repeats (SSRs) dynamics in fungus Fusarium graminearum. Bioinformation, 5(10): Sorokin D Y, Lücker S, Vejmelkova D et al, Nitrification expanded: discovery, physiology and genomics of a nitrite-oxidizing bacterium from the phylum Chloroflexi. ISME J, 6(12): Wang Q, Garrity G M, Tiedje J M et al, Naïve Bayesian classifier for rapid assignment of rrna sequences into the new bacterial taxonomy. Appl Environ Microbiol, 73(16): BACTERIAL DIVERSITY AND COMMUNITY STRUCTURE IN SEDIMENTS OF XIXI WETLAND, HANGZHOU HUANG Yuan 1, FANG Xu 2, CHU Wen-Ke 1, CHEN Min 1 (1. College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou , China; 2. Zhejiang Institute of Microbiology, Hangzhou , China) Abstract The Illumina MiSeq sequencing-based analysis of the V3-V4 16S rrna gene region was used to detect bacterial diversity and community structure in Xixi wetland, Hangzhou. Overall, optimized reads with an average length of 441bp were obtained from four sediment samples. Bacterial diversity was very high in these samples, since a total of 2181 OTUs were identified which could be assigned into 30 different phyla and 252 genera. The dominant group belonged to Proteobacteria, accounting for 31.0% 64.7% of total sequences. Among Proteobacteria, the beta-, delta- and gamma-subdivisions were dominant classes. Chloroflexi, Firmicutes and Bacteroidetes were also very common, accounting for 7.0% 43.0%, 1.1% 14.7% and 1.0% 10.2% of total libraries, respectively. The ordination analysis showed that there were about 10% 15% of total sequences belonged to the unclassified group, which meant that there may be many undiscovered species in sediments. There were also large number of DNA sequences affiliated to the strains which related to N and S metabolism in beta-, gamma-, and delta-proteobacteria branches. This study will lay the foundation for future research on microbiology in Xixi wetland. Key words Xixi wetland; sediment; bacterial diversity; MiSeq high-throughput sequencing

Microsoft Word - 18 王健鑫.doc

Microsoft Word - 18 王健鑫.doc 43 4 Vol.43, No.4 2012 7 OCEANOLOGIA ET LIMNOLOGIA SINICA July, 2012 * ( 316000) DH-13 DNA, PCR TA 16S rdna, : (Proteobacteria, 41.5%), (Planctomycetes, 10.9%) (Actinobacteria, 8.9%) CFB group (7.9%),

Microsoft Word 黄 媛_new_.doc

Microsoft Word 黄媛_new_.doc