16S rrna ARDRA Q938 A (2008) : (2006BAD07B03, 2006BAD08A08, 2006BAD17B08); (NYHYZX07-050)

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1 Acta Microbiologica Sinica 48(10) 1344~1350; 4 October 2008 ISSN ; CN /Q 16S rrna * ( ) 16S rrna DNA PCR 16S rrna Hinf Hae 16S rrna ARDRA(Amplified Ribosomal DNA Rstriction Analysis) OTU γ β α 124 OTUs 92 OTUs 16S rrna ARDRA Q938 A (2008) [1] [2] 16S rrna 16S rrna 16S rrna, : (2006BAD07B03, 2006BAD08A08, 2006BAD17B08); (NYHYZX07-050) * Tel/Fax: ; xiebingyan2003@yahoo.com.cn : (1981 ),,,, liuweiqi10124@126.com : ; :

2 16S rrna./ (2008) 48(10) 1345 DNA 16Sr RNA ~15cm 10 2 mm 20 ph g/kg N g/kg 25.63% ph g/kg N g/kg 28.71% PCR pgem-t DNA Promage PCR PTC-200 3K DNA DNA [3] 3 DNA K/SDS [4] DNA DNA 1.3 PCR 16S r RNA PCR 27f 5 -AGAGTTTGATCCT- GGCTCAG r 5 -TACGGCTACCTTGTTACG- ACTT-3 PCR 10 Buffer5 μl Mg 2+ (25 mmol/l)4 μl dntp(5 mmol/l)2 μl 10 pmol/l 3 μl DNA 10 ng Taq DNA (5U)1 μl ddh2o 50 μl PCR 94 5 min 94 1 min min min 55 1 min 72 1 min min SrRNA PCR 1% 1500 bp DNA pgem-t (Promega, USA) TOP10 IPTG X-Gal 37 T7 SP6 PCR T7: 5 -TAATACGACTCACTATAGGG-3 SP6: 5 -ACGATTTAGGTGACACTATAG ARDRA PCR rdna ARDRA Amplified Ribodomal DNA Restriction Analysis Hinf Hae (37 ) 1.2% OTU [5] OTU 1~3 1.6 NCBI BLAST 2 Sequence [6] 97% [7] CHECK-CHIMERA [8] [9] GenBank Blast RDPII CLUSTAL-X 1.83 [10] MEGA 3.1 Kimura 2 [11]

1346 Weiqi Liu et al. /Acta Microbiologica Sinica (2008) 48(10) 2 2.1 DNA DNA DNA DNA DNA PVPP EDTA DNA DNA DNA DNA 2.2 PCR 16S rrna 16S rrna 1500 bp 2.

3 1346 Weiqi Liu et al. /Acta Microbiologica Sinica (2008) 48(10) DNA DNA DNA DNA DNA PVPP EDTA DNA DNA DNA DNA 2.2 PCR 16S rrna 16S rrna 1500 bp S rrna 10 DNA PCR PCR 3 3 PCR PCR 10 DNA PCR PGEM-T 16S rrna 16S rrna PCR 16S rrna T7 SP6 PCR EcoR ARDRA Hinf Hae OTU 45 OTU 79 OTU RFLP 26 1 coveragec [12] 90.6% coveragec 16S rdna OTU a C=1 n1/n N 16S rdna n1 16S rdna OTU CoverageC Fig. 3 1 Hinf (A) Hae (B) Restriction fragment patterns of Hinf and Hae

4 16S rrna./ (2008) 48(10) % RFLP 1 3 GenBank 16S rrna 88%~99.7% 16S rrna ARDRA OTU 38 OTU 54 OTU OTU 18 2 coveragec 91.4%,, GenBank/ EMBL/DDBJ 16S rrna EU EU EU EU EU EU S rrna EU EU Sr RNA Fig. 2 Rarefaction curves generated for 16S rrna genes in the clone library from samples collected in the mangrove soil. Clones were grouped into phylotypes based on sequence similarity of 97% S rdna S rrna 500 bp GenBank Blastn CLUSTAL-X % % 16SrDNA 97% 40% 98% 10% 16S rrna 85% 16S rrna 3-A 259 OTUs Proteobacteria 53.02% 90-99% 97% -Proteobacteria β-proteobacteria α-proteobacteri 22.42% 16.7% 13.9% BJ-156 EU Proteobacteria BJ-156 Bdellovibrio bacteriovorus HD100 (BX842648) 93% (Proteobacteria) Bacteroidetes 17.6% Actinobacteria 6.7% (Acidobacteria) 6.7% (Gemmatimonadetes) 2.4% (Firmicutes) 2.4% (Chloroflexi) 1.0% (Planctomycetes) 0.7% 10 16S rrna 3-B Proteobacteria 62.7% α-proteobacteri γ-proteobacteria β-proteobacteria 43.4% 14.1% 5.2% Bacteroidetes 12.5% (Acidobacteria) 3.7% (Firmicutes) 3.8% (Planctomycetes) 2.0% 3 ARDRA ARDRA 16S rrna (species) ARDRA OTU operational taxonmic unit OTUs

5 1348 Weiqi Liu et al. /Acta Microbiologica Sinica (2008) 48(10) 3 16S rrna Fig. 3 Phylogenetic tree based on the 16S rrna sequences of the clones obtained from Beijing vegetable soil(a) and from Shandong vegetable soil(b). The tree was constructed via the neighbor-joining method. Bootstrap values above 1000 are shown as percentage. Bar indicates 5% nucleotide changes per 16S rrna position. UC represents uncultured bacterium.

6 16S rrna./ (2008) 48(10) 1349 coveragec 16S rrna 124 OTUs 92 OTUs 16S rrna γ β α 16S rrna Handelsman [13] 16S rrna Proteobacteria (48.6%) Acidobacteria (15.3%) Borneman [14] Wisconsin 16S rrna 124 (16.1%) - - (21.8%) G+C (21.8%) 53.02% 62.7%. Handelsman [13] Borneman [14] 48.6% 16.1% [15] [16] 16S rrna 16S rrna 16S rrna [1],.. (Pratacultural Science), 2002, 19(9): [2] Kennedy AC. Bacterial diversity in agroecosystems. Agriculture, Ecosystems and Environment, 1999, 74: [3],,,. DNA. (Journal of Agro-environment Science), 2005, 24(5): [4] Zhou JZ, Bruns MA, Tiedje JM. DNA Recovery from Soils of Diverse Composition. Appl Environ Microbiol, 1996, 62(2): [5] Vaneechoutte MR, Rossau R, Devos P, et al. Rapid identification of bacteria of the Comamonadaceae with amplified ribosomal DNA restriction analysis (ARDRA ). Fems Microbiology Letters, 1992, 93: [6] Tatiana AT, Thomas LM. Blast 2 sequences a new tool for comparing protein and nucleotide sequences. Fems Microbiology Letters, 1999, 174: [7] McCaig AE, Glover LA, Prosser JI. Molecular analysis of bacterial community structure and diversity in unimproved and improved upland grass pastures. Applied and Environmental Microbiology, 1999, 65: [8] Cole JR, Chai B, Marsh TL, Farris RJ, et al. The Ribosomal DatabaseProject (RDP-II): previewing a new autoaliger that allows regular updates and the new prokaryotic taxonomy. Nucleic Acids Research, 2003, 31: [9] Schloss PD, Handelsman J Toward a census of bacteria in soil. Plos Computational Biology, 2(7): [10] Thompson JD, Gibson TJ, Plewniak F, et al. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research, 1997, 25: [11] Kumar S, Tamura K, Nei M. MEGA3: Integrated software for Molecular Evolutionary Genetics Analysis and sequence alignment. Briefings in Bioinformatics, 2004, 5: [12] Good IL. The population frequencies of species and the estima-

7 1350 Weiqi Liu et al. /Acta Microbiologica Sinica (2008) 48(10) tion of population parameters. Biometrika, 1953, 40: [13] Schloss PD, Handelsman J. Status of the Microbial Census. Microbiology and Molecular Biology Reviews, 2004, 68(4): [14] Borneman J, Skroch PW, Palus JA, et al. Molecular microbial diversity of anagricultural soil in Wisconsin. Applied and Environmental Microbiology, 1996, 62: [15] Ekundayo EO Effect of common pesticides used in the Niger delta basin of southern Nigeria on soil microbial populations. Environmental Monitoring and Assessment, 2003, 89: [16],,,.. (Chinese Journal of Soil Science), 2006, 37: Analysis of soil bacterial diversity by Using the 16S rrna gene Library Weiqi Liu, Zhenchuan Mao, Yuhong Yang, Bingyan Xie * (Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing , China) Abstract: [Objective] Soil microorganisms play an important role in the vegetable soil ecosystem. Through 16S rrna gene cloning library technology to analysis the composition of bacteria community structure in the typical vegetable soil. To revealed the microbial diversity in the typical vegetable soil, and laid the foundation for the relationship between land-use changed and the ecological environment. [Methods] Total microbial DNA was directly extracted from a typical vegetable soil of Beijing and Shandong province. The clone library of 16S ribosomal RNA genes was amplified using PCR with universal bacterial primer sets. The PCR products were then subcloned into pgem-t vector. Each unique restriction fragment polymorphism pattern, created by using two restriction endonucleases (Hinf and Hae ), was designated as an operational taxonomic unit (OTU). Amplified DNA was used for diversity analysis. [Results] Constructed bacterial phylogenetic trees of the samples revealed the γ-proteobacteria, β-proteobacteria and α-proteobacteria groups were dominant in both clone libraries. Bacterial species composition from Beijing and Shandong province included 124 OTUs and 92 OTUs, respectively. [Conclusion] the dominant species of bacteria populations are proteobacteria in the typical vegetable soil of Beijing and Shandong areas. But bacterial diversity in the two typical vegetable soil samples was reduced. This phenomenon may be directly related to Continuous cultivation for many years and plant a single vegetable species. At the same time, this phenomenon may also be an important reason to cause vegetable soil diseases widespread occurred and soil degradation. Keywords: bacterial diversity; 16S rrna gene clone library; amplified ribosomal DNA restriction analysis; phylogenetic analysis Supported by the Subject of National Key Technology Research and Development Program of the Eleventh Five-year Plan (2006BAD07B03, 2006BAD08A08, 2006BAD17B08) and the Fund Scientific Research for Agricultural Public Welfare (NYHYZX07-050) * Corresponding author. Tel/Fax: ; xiebingyan2003@yahoo.com.cn Received: 4 March 2008/ Revised: 31 July 2008 ˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇ

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