贵州石漠化区域细菌多样性研究
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摘要
从贵州石漠化重灾区安顺、毕节和黔西南等地采集土样,设计比较了TENS-乙酸铵直接提取法、SDS高盐直接提取法、间接提取法和试剂盒提取法对石漠化环境土壤DNA的提取效果。以试剂盒提取的DNA为模板,采取TA克隆策略,对6份土样的宏基因组DNA进行了16S rDNA扩增,胶回收PCR产物,和pMD-18T载体链接,转化宿主菌DH5α,通过蓝白斑试验筛选阳性克隆,提取质粒,随机挑取23个阳性克隆的质粒送生物公司测序,将测序结果登录NCBI,利用BLAST工具在GeneBank中查找相似性序列,找出与其相似性最高的菌种。利用ClusterW2进行在线多序列比对,构建出系统发育树。相似性比对结果以及系统发育分析揭示在贵州石漠化这一特殊生境中,微生物种类有限,除了三个在GeneBank中还没有的菌种外,几乎全为柠檬酸杆菌,优势菌为柠檬酸杆菌。
The soil samples were collected from rocky desertification areas, Anshun,Bijie in GuiZhou province and the southwest of GuiZhou .The extraction effects of the desertification environment soil DNA were designed and compared among TENS-ammonium acetate direct method, SDS high-salt direct method, indirect method and kit extraction method .The template of follow-up experiments was soil DNA extracted by kit extraction method .The follow-up experiments as follows: metagenomic DNA of soil samples extracted from 6 soil samples was amplified by 16SrDNA; PCR products were purified from agarose gel and linked into pMD-18T vector and transformed into host strain DH5α; positive clones were selected through blue and white blots test; plasmids were extracted; 23 positive clones plasmids were selected randomly and sequenced by biology company ,the sequenced results compared with the similar sequences searched by BLAST tool in the GeneBank on NCBI to get the highest similar bacteria .Finally, phylogenetic tree was constructed by using ClusterW2 on-line multiple sequence alignment. The similarity comparison results and phylogenetic analysis reveal that microorganisms are limited in the special desertification environment of Guizhou. They are nearly Citrobacter except three bacterias not to be found in the GeneBank .So the dominant bacteria is Citrobacter.
The soil samples were collected from rocky desertification areas, Anshun,Bijie in GuiZhou province and the southwest of GuiZhou .The extraction effects of the desertification environment soil DNA were designed and compared among TENS-ammonium acetate direct method, SDS high-salt direct method, indirect method and kit extraction method .The template of follow-up experiments was soil DNA extracted by kit extraction method .The follow-up experiments as follows: metagenomic DNA of soil samples extracted from 6 soil samples was amplified by 16SrDNA; PCR products were purified from agarose gel and linked into pMD-18T vector and transformed into host strain DH5α; positive clones were selected through blue and white blots test; plasmids were extracted; 23 positive clones plasmids were selected randomly and sequenced by biology company ,the sequenced results compared with the similar sequences searched by BLAST tool in the GeneBank on NCBI to get the highest similar bacteria .Finally, phylogenetic tree was constructed by using ClusterW2 on-line multiple sequence alignment. The similarity comparison results and phylogenetic analysis reveal that microorganisms are limited in the special desertification environment of Guizhou. They are nearly Citrobacter except three bacterias not to be found in the GeneBank .So the dominant bacteria is Citrobacter.
引文
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[5]Torsvik V , Goks? yr J , Daae F L. High diversity in DNA of soil bacteria,Appli. Environ Microbiol ,1990 ,56(3) :782-787.
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[8]杨官品,毛云翔.环境细菌宏基因组研究及海洋细菌生物活性物质BAC文库筛选[J].青岛海洋大学学报,2001,31(5):718-722
[9]叶姜瑜,罗固源.未培养微生物的研究与微生物分子生态学的发展[J].微生物学通报,2004,31 (5) :111-115
[10]Lawrence JR, Korber DR, Wolfaard GM, Caldwell DE: Behavioral strategies of surface colonizing bacteria[J]. Adv Microb Ecol 1995, 14:175.
[11] Schmidt TM, DeLong E F, Pace N R. Analysis of a marine picop lankton community by 16S rRNA gene cloning and sequencing[J]. Bacteriol, 1991, 173 (14) : 4371-4378
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[13] Stein J L, Marsh T L, Wu K Y, et al. Characterization of uncultivated prokaryotes: isolation and analysis of a 40 kilobase-pair genome fragment from a planktonic marine archaeon[J]. Bacteriol, 1996, 178 (3) : 591-599
[14]Handelsman J, Rondon M R, Brady S F, et al. Expression and Isolation of Antimicrobial SmallMolecules from Soil DNA[J]. Libraries Chem Biol, 1998, 5: 245-249
[15]张光亚,方柏山.宏基因组——生物催化剂的新来源[J].生命的化学, 2005,25(4) :278-281
[16]Jo Handelsman, RondonMR, Brady SF, et al. Molecular biological access to the chemistry of unknown soilmicrobes: a new frotier for natural p roducts[[J]. Chem2istry &Biology, 1998, 5: 245-249.
[17]Zhou.J.,et al. DNA recovery from soils of diverse composition[J]. Appl environ microbiol, 1996, 62 () :316-322.
[18] F. Martin-Laurent,et al. DNA extraction from soils: old bias for new microbial diversity analysis methods[J]. Appl environ microbial, 2001, 67 (5):2354-2359.
[19]Chritine Picard, at al. Detection and enumeration of bacteria in soil by direct DNA extraction and PCR[J]. Appl environ mirobiol,1992, 58 (9): 2717-2722.
[20]Yu-Li Tsai and Betty H. Olson. Rapid method for direct extraction of DNA from soil and sediments[J]. Appl environ microbiol, 1991, 57 (4) : 1070-1074.
[21]Carsten S. Jacobsen and Ole F. Rasmussen. Development and application of a new method to extract bacterial DNA from soil based on separation of bacteria from soil with cation-exchange resin[J]. Appl environ microbiol, 1992, 58 (8):2458-2462.
[22]Courtois Sophie, et al. Recombinant environmental libraries provide accss to microbial diversity for drug discovery from natural products[J]. Appl environ microbiol. 2003, 69 (1): 49-55.
[23]D.N.Miller,et al. Evaluation and optimization of DNA extraction and purification procedures for soil and sediments samples[J]. Appl Environ Microbiol,1999,65(11):4715-4724.
[24]Saleh2lakha S, MillerM, Campbell RG, et al. 2005.Microbial gene exp ression in soil: Methods, app licationsand challenges[J]. Journal of MicrobiologicalM ethods, 63:1–19
[25]冯美琴.宏基因组学的研究进展[J].安徽农业科学, 2008, 36(2) : 415- 416, 479
[26] Zhou J Z,Bruns M A,Tiedjie J M. DNA recovery from soil of diverse composition[J]. Appl Environ Microbiol,1996,62:316- 322.
[27]楼士林,杨盛昌,龙敏南,等.基因工程[M].北京:科学出版社,2002:85-257
[28]李海权,刁现民.基因组细菌人工染色体文库(BAC)的构建及应用[J].生物技术通报,2005 (1) :306- 308
[29] Rondon MR, August PR, Bettermann AD,et al. Cloning the soil metagenome: a strategy for accessing the genetic and functional diversity of uncultured microorganisms[J]. Appl Environ Microbiol, 2000 ,66(6):2541-2547.
[30]Courtois S, Cappellano C F, Ball M. Metagenomic profiling: microarray analysis of an environmental genomic library〔J〕. Appl. Environ Microbiol, 2003, 69(1):49-50
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