八种海洋经济动物肠道细菌群落的种群分子多样性分析
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摘要
本文以甲壳纲的中国明对虾(Fenneropenaeus chinensis)、凡纳滨对虾(Litopenaeus vannamei)、鹰爪虾(Trachypenaeus curvirostris)、口虾蛄(Oratosquilla oratoria)和辐鳍鱼纲的美国红鱼(Sciaenops ocellatus)、大泷六线鱼(Hexagrammos otakii)、鲈鱼(Lateolabrax japonicus)、石鲽(Kareius bicoloratus)、大菱鲆(Scophthatmus maximus)为研究对象,运用分子生物学方法对以上物种肠道细菌群落多样性进行了研究,并用细菌16S rDNA基因序列构建了分子系统发生树。本研究旨在利用细菌16S rDNA基因分析以上海洋生物肠道细菌群落种群多样性,为了解海洋生物肠道细菌构成做铺垫。主要内容分为以下三个部分:
1.直接提取中国明对虾、凡纳滨对虾、鹰爪虾、口虾蛄肠道微生物总DNA,并以其为模板扩增细菌16S rDNA,与T载体连接后建库。用限制性内切酶BsuRⅠ和Hin6Ⅰ对阳性克隆的PCR产物进行酶切分析,选取有代表性的克隆进行序列测定。序列及BLAST结果显示,中国明对虾、凡纳滨对虾、鹰爪虾肠道细菌70%以上属于γ-变形杆菌,其中以弧菌属占最大比重;口虾蛄肠道细菌种类比较少,且多为未培养的细菌。凡纳滨对虾肠道细菌种类最多,有弧菌属、气单胞菌属、发光杆菌属、假交替单胞菌属等。
2.通过分子生物学方法(RFLP)分析美国红鱼、鲈鱼、大泷六线鱼和石鲽肠道细菌多样性。测序结果显示四种鱼肠道细菌70%以上属于γ-变形杆菌,多样性指数分析结果表明鲈鱼肠道细菌多样性最为丰富。美国红鱼肠道细菌主要为弧菌属、发光杆菌属和螺原体属;鲈鱼肠道细菌主要为弧菌属和发光杆菌属;大泷六线鱼肠道细菌主要为弧菌属和Aliivibrio;石鲽肠道细菌主要为气单胞菌属和支原体属。
3.通过分子生物学方法分析大菱鲆肠道细菌多样性,从构建的16S rDNA文库中随机选取136个阳性克隆进行酶切(BsuRⅠ、Hin6Ⅰ和RsaⅠ)分析,一共得到46种酶切类型,其中仅有一个克隆的酶切类型有36种,表明大菱鲆肠道细菌多样性比较丰富。运用原位裂解法提取获得高质量的大菱鲆肠道微生物基因组DNA,用于宏基因组序列测定。
The intestinal bacterial diversity of Fenneropenaeus chinensis, Litopenaeus vannamei, Trachypenaeus curvirostris and Oratosquilla oratoria in Crustacea, and five fishes Sciaenops ocellatus, Hexagrammos otakii, Lateolabrax japonicus, Kareius bicoloratus and Scophthatmus maximus in Actinopterygii were analysed; phylogenetic trees were constructed based on the 16S rDNA gene of bacteria. The aim of this research is to analyze the intestinal bacterial diversity of these shrimps and fishes based on 16S rDNA gene, providing basic understanding of intestinal bacterial composition structure. The main work consists of the following three parts:
1. Culture-independent method was used, and 16S rDNA was amplified via PCR from total genomic DNA extracted from intestinal microorganisms of F. chinensis, L. vannamei, T. curvirostris and O. oratoria, with clone libraries constructed later. Positive clones were digested by restriction endonuclease BsuRⅠand Hin6Ⅰrespectively, and then representative clones were sequenced. DNA sequence analysis and BLAST analysis indicated that more than 70% of the intestinal bacteria of F. chinensis, L. vannamei and T. curvirostris belonged to the Gammaproteobacteria, with Vibrio possessed the highest proportion. The gut of Oratosquilla oratoria had very low bacterial diversity, and most of the bacteria were uncultured ones. The intestinal bacteria of L. vannamei had the highest bacterial diversity, including Vibrio, Aeromonas, Photobacterium, Pseudoalteromonas, etc.
2. Molecular method (RFLP analysis) was used to analyze the intestinal bacterial diversity of H. otakii, K. bicoloratus, S. ocellatus and L. japonicus. DNA sequence analysis indicated that more than 70% of the intestinal bacteria of these fishes belonged to the Gammaproteobacteria, and the statistical analysis revealed that the L. japonicus gut bacteria community had the highest diversity. The main bacterial composition of S. ocellatus was Vibrio, Photobacterium and Spiroplasma; the main bacterial composition of L. japonicus was Vibrio and Photobacterium; the main bacterial composition of H. otakii was Vibrio and Aliivibrio; the main bacterial composition of K. bicoloratus was Aeromonas and Mycoplasma.
3. Molecular method was used to analyze the intestinal bacterial diversity of Scophthatmus maximus. 136 positive clones were selected randomly from the constructed 16S rDNA library to be digested by restriction-endonuclease (BsuRⅠ、Hin6Ⅰ和RsaⅠ), and 46 RFLP Patterns were obtained, with 36 of them had only one clone, indicating that the intestinal bacterial of Scophthatmus maximus had relatively higher diversity. The total intestinal microorganism genomic DNA of Scophthatmus maximus was isolated with high quality using in situ degradation, which was used for metagenomic sequcing.
1.直接提取中国明对虾、凡纳滨对虾、鹰爪虾、口虾蛄肠道微生物总DNA,并以其为模板扩增细菌16S rDNA,与T载体连接后建库。用限制性内切酶BsuRⅠ和Hin6Ⅰ对阳性克隆的PCR产物进行酶切分析,选取有代表性的克隆进行序列测定。序列及BLAST结果显示,中国明对虾、凡纳滨对虾、鹰爪虾肠道细菌70%以上属于γ-变形杆菌,其中以弧菌属占最大比重;口虾蛄肠道细菌种类比较少,且多为未培养的细菌。凡纳滨对虾肠道细菌种类最多,有弧菌属、气单胞菌属、发光杆菌属、假交替单胞菌属等。
2.通过分子生物学方法(RFLP)分析美国红鱼、鲈鱼、大泷六线鱼和石鲽肠道细菌多样性。测序结果显示四种鱼肠道细菌70%以上属于γ-变形杆菌,多样性指数分析结果表明鲈鱼肠道细菌多样性最为丰富。美国红鱼肠道细菌主要为弧菌属、发光杆菌属和螺原体属;鲈鱼肠道细菌主要为弧菌属和发光杆菌属;大泷六线鱼肠道细菌主要为弧菌属和Aliivibrio;石鲽肠道细菌主要为气单胞菌属和支原体属。
3.通过分子生物学方法分析大菱鲆肠道细菌多样性,从构建的16S rDNA文库中随机选取136个阳性克隆进行酶切(BsuRⅠ、Hin6Ⅰ和RsaⅠ)分析,一共得到46种酶切类型,其中仅有一个克隆的酶切类型有36种,表明大菱鲆肠道细菌多样性比较丰富。运用原位裂解法提取获得高质量的大菱鲆肠道微生物基因组DNA,用于宏基因组序列测定。
The intestinal bacterial diversity of Fenneropenaeus chinensis, Litopenaeus vannamei, Trachypenaeus curvirostris and Oratosquilla oratoria in Crustacea, and five fishes Sciaenops ocellatus, Hexagrammos otakii, Lateolabrax japonicus, Kareius bicoloratus and Scophthatmus maximus in Actinopterygii were analysed; phylogenetic trees were constructed based on the 16S rDNA gene of bacteria. The aim of this research is to analyze the intestinal bacterial diversity of these shrimps and fishes based on 16S rDNA gene, providing basic understanding of intestinal bacterial composition structure. The main work consists of the following three parts:
1. Culture-independent method was used, and 16S rDNA was amplified via PCR from total genomic DNA extracted from intestinal microorganisms of F. chinensis, L. vannamei, T. curvirostris and O. oratoria, with clone libraries constructed later. Positive clones were digested by restriction endonuclease BsuRⅠand Hin6Ⅰrespectively, and then representative clones were sequenced. DNA sequence analysis and BLAST analysis indicated that more than 70% of the intestinal bacteria of F. chinensis, L. vannamei and T. curvirostris belonged to the Gammaproteobacteria, with Vibrio possessed the highest proportion. The gut of Oratosquilla oratoria had very low bacterial diversity, and most of the bacteria were uncultured ones. The intestinal bacteria of L. vannamei had the highest bacterial diversity, including Vibrio, Aeromonas, Photobacterium, Pseudoalteromonas, etc.
2. Molecular method (RFLP analysis) was used to analyze the intestinal bacterial diversity of H. otakii, K. bicoloratus, S. ocellatus and L. japonicus. DNA sequence analysis indicated that more than 70% of the intestinal bacteria of these fishes belonged to the Gammaproteobacteria, and the statistical analysis revealed that the L. japonicus gut bacteria community had the highest diversity. The main bacterial composition of S. ocellatus was Vibrio, Photobacterium and Spiroplasma; the main bacterial composition of L. japonicus was Vibrio and Photobacterium; the main bacterial composition of H. otakii was Vibrio and Aliivibrio; the main bacterial composition of K. bicoloratus was Aeromonas and Mycoplasma.
3. Molecular method was used to analyze the intestinal bacterial diversity of Scophthatmus maximus. 136 positive clones were selected randomly from the constructed 16S rDNA library to be digested by restriction-endonuclease (BsuRⅠ、Hin6Ⅰ和RsaⅠ), and 46 RFLP Patterns were obtained, with 36 of them had only one clone, indicating that the intestinal bacterial of Scophthatmus maximus had relatively higher diversity. The total intestinal microorganism genomic DNA of Scophthatmus maximus was isolated with high quality using in situ degradation, which was used for metagenomic sequcing.
引文
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2. Bowman JP, McCammon SA, Rea SM and McMeekin TA. The microbial composition of three limnologically disparate hypersaline Antarctic lakes. Fems Microbiology Letters, 2000(1):81-88.
3. Broderick NA, Raffa KF, Goodman RM and Handelsman J. Census of the Bacterial Community of the Gypsy Moth Larval Midgut by Using Culturing and Culture-Independent Methods. Appl Environ Microbiol, 2004(1):293-300.
4. Brown MV and Bowman JP. A molecular phylogenetic survey of sea-ice microbial communities (SIMCO). Fems Microbiol Ecol, 2001(3):267-275.
5. Boyd CE and Massaut L. Risks associated with the use of chemicals in pond aquaculture. Aquac Eng 1999(2)113-132.
6. Burns DG, Camakaris HM, Janssen PH and Dyall-Smith ML. Combined Use of Cultivation-Dependent and Cultivation-Independent Methods Indicates that Members of Most Haloarchaeal Groups in an Australian Crystallizer Pond Are Cultivable. Appl Environ Microbiol, 2004(9):5258-5265.
7. Castex M, Chim L, Pham D, Lemaire P, Wabete N, Nicolas JL, Schmidely P and Mariojouls C. Probiotic P. acidilactici application in shrimp Litopenaeus stylirostris culture subject to vibriosis in New Caledonia. Aquaculture, 2008(1):182-193.
8. Chen X, Li Q, Wang J, Guo X, Jiang X, Ren Z, Weng C, Sun G, Wang X, Liu Y, Ma L, Chen JY, Wang J, Zen K, Zhang J and Zhang CY. Identification and characterization of novel amphioxus microRNAs by Solexa sequencing. Genome Biol, 2009 (7):R78
9. Dempsey AC. Characteristics of bacteria isolated from penaeid shrimp. Crustaceana, 1987(1):89-94.
10. Dunbar J, Barns SM, Ticknor LO and Kuske CR. Empirical and theoretical bacterial diversity in four Arizona soils. Appl Environ Microbiol, 2002(68):3035-3045.
44. Qi W, K?ser M, R?ltgen K, Yeboah-Manu D and Pluschke G.Genomic diversity and evolution of Mycobacterium ulcerans revealed by next-generation sequencing. PLoS Pathog, 2009(9):e1000580.
45. Qi ZZ, Zhang XH, Boon N and Bossier P. Probiotics in aquaculture of China - Current state, problems and prospect. Aquaculture, 2009(1-2):15-21
46. Ranjan R, Grover A, Kapardar RK and Sharma R. Isolation of novel lipolytic genes from uncultured bacteria of pond water. Biochem Biophys Res Commun, 2005(1): 57-65.
47. Rattray RM, Perumbakkam S, Smith F and Craig AM. Microbiomic Comparison of the Intestine of the Earthworm Eisenia fetida Fed Ergovaline. Curr Microbiol, 2010(3):229-235
48. Rhee JK, Ahn DG, Kim YG and Oh JW. New thermophilic and thermostable esterase with sequence similarity to the hormone-sensitive lipase family, cloned from a metagenomic library. Appl Environ Microbiol, 2005(2): 817-825.
49. Sahu MK, Swarnakumar NS, Sivakumar K, Thangaradjou T, Kannan L. Probiotics in aquaculture: importance and future perspectives. Ind J Microbiol, 2008(3):1-10.
50. Saiton N and NeiM. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol, 1987(4):406-425.
51. Schloss PD and Handelsman J. Introducing DOTUR, a computer program for defining operational taxonomic units and estimating species richness. Appl Environ Microbiol, 2005(3):1501-1506.
52. Schloss PD and Handelsman J. Biotechnological prospects from metagenomics. Curr Opin Biotech, 2003(3):303-310.
53. Schmeisser C, Steele H and Streit WR. Metagenomics, biotechnology with non-culturable microbes. Appl Microbiol Biotechnol, 2007(5):955-962.
54. Sugita H, Oshima K and Tamura M. Bacterial flora in the gastrointestine of freshwater fishes in the river. Bull Japan Soc Sci Fish, 1983(9):1387-1395.
55. Sugita H and Ito Y. Identification of intestinal bacteria from Japanese flounder (Paralichthys olivaceus) and their ability to digest chitin. Letters in Applied Microbiology, 2006(3):336-342.
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