皱纹盘鲍肠道菌群组成及 PCR-DGGE 指纹图谱分析 1, 2, 2, 2, 2, 1, 3 (1., 116045; 2., 712100; 3., 116023) 摘要 : 对养殖和野生皱纹盘鲍 (Haliotis discus hannai Ino ) 不同个体间肠道菌群数量和种类组成进行了 研究, 并采用 PCR-DGGE 指纹技术对比分析了二者肠道优势菌群的差异 研究结果表明, 养殖和野生 皱纹盘鲍肠道好氧及兼性厌氧菌总数分别为 (3.50±0.85) 10 6 个 /g, (3.03±1.10) 10 6 个 /g; 两组鲍鱼肠道 优势菌基本相同, 均为弧菌属 (Vibrio), 次优势菌均为玫瑰杆菌属 (Roseobacter) 和希瓦氏菌属 (Shewanella), 在养殖组中检测到芽孢杆菌属 (Bacillus), 表明皱纹盘鲍对肠道细菌具有特异选择性 此外, PCR-DGGE 指纹图谱结果表明, 从养殖和野生皱纹盘鲍肠道样品分别获得 14 条和 12 条扩增条带, 其中人工组中有 2 条特异性条带 ; 二者相似性系数 ( 戴斯系数 ) 为 92.31% 关键词 : 皱纹盘鲍 (Haliotis discus hannai Ino ); 肠道 ; 菌群 ; PCR-DGGE 中图分类号 : Q939.1 文献标识码 : A 文章编号 : 1000-3096(2013)05-0010-05,, [1],, [2-3] (Haliotis discus hannai Ino),,,, [4] [5],, Sawabe [6],, PCR-DGGE, 1 材料和方法 1.1 样品采集 10 126.72g±10.71 g 10.34cm±0.22cm 2010 10, ; 10 122.70g±15.50g 10.33cm±1.15cm, 5 1.2 方法 1.2.1 Sawabe [6] :, 70% 1min, : 2012-02-11; : 2012-07-17 : (2011203003) : (1979-),,,,, E-mail: xiaofeileng@126.com;,, E-mail: yqchang@dlou.edu.cn 10 / 2013 / 37 / 5
,, 10 4, 0.1 ml 2216E, 3, 22 5 d 80, 2216E 3, 80 1.2.2 (Bioteke) DNA, 16S rdna, 16S rdna GenBank GenBank 1.2.3 DNA (Bioteke) DNA, DNA (Nano drop 2000) DNA, 20 1.2.4 PCR-DGGE 16S V3 (F357-GC R518) 2 PCR -DGGE [7], 10% V3, 40% ~60% 10 Loading buffer 10 µl PCR 30 µl 40 V 1 h, 80V 10~12 h,, EB 20 min, Bio-Rad Quantity One(Bio-Rad Laboratories, CA, USA) 2 结果与分析 2.1 皱纹盘鲍肠道细菌数量和组成 (3.50±0.85) 10 6 /g, (3.03±1.10) 10 6 /g 2216E, 12 43 (GenBank : JQ083308- JQ083350) : 13 8, (Vibrio) (Roseobacter) (Shewanella) (Bacillus) (Salegentibacter) (Flammeovirga) (Tamlana) (Agarivorans), Vibrio halioticoli Vibrio cyclitrophicus (Vibrio alginolyticus) Vibrio Alcanivorax ; Shewanella colwelliana Shewanella pacifica ; 1 1,, Vibrio halioticoli, Vibrio halioticoli 76.47%±13.07% 9.74%±8.10% 6.54%±2.98%, 81.89%±10.70% 7.74%±4.27% 10.28%±6.71%,, (80%),, 2.2 16S rdna V3 可变区指纹图谱的建立及分析 16S rdna DGGE ( 1): 14 12,, 12, band2 band7 ; band4 band5 band14, 3 C s (Dice coefficient) [8], C s =2j/(a+b)(j A B a b ), 92.31%(24/26) 3 讨论,, Marine Sciences / Vol. 37, No. 5 / 2013 11
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1 16S rdna PCR-DGGE Fig.1 PCR-DGGE fingerprint and its pattern of the V3 region of 16S rdna of the bacteria from the gastrointestinal of Haliotis discus hannai 1~14. ; C. ; W. 1-14. no. of the band; C.the sample of cultured group; W. the sample of wild group [9] Tanaka [10] : 1, (Alginolytic non-motile fermenters) (Vibrio) (Cytophaga),, NMF(non-motile fermenters) 80%, V. halioticoli NMF 96%, V. halioticoli ( 76%) ( 7.74%) ( 6.54%), V. halioticoli,, ; [11-13], [14],,, [15-16],, Sawabe [6] 10 6 10 9 /g, 10 6 /g,, Ringø [17],, 16S rdna, (C s =92.31%), 12, 2 (band2 band7),,,, [18-19],,,,, : [1],,,. PCR-DGGE [J]., 2011, (3): 423-429. [2] Watson A K, Kaspar H, Lategan M J, et al. Probiotics in aquaculture: The need, principles and mechanisms of action and screening processes [J]. Aquaculture, 2008, 274: 1-14. [3] Iehata S, Inagaki T, Okunishi S, et al. Improved gut environment of abalone Haliotis gigantea through Pediococcus Marine Sciences / Vol. 37, No. 5 / 2013 13
sp. Ab1 treatment [J]. Aquaculture, 2010, 305: 59-65. [4],,,. [J]., 2006, 2: 117-124. [5],,,. [J]., 2010, (4): 64-69. [6] Sawabe T, Oda Y, Shiomi Y, et al. Alginate degradation by bacteria isolated from the gut of sea urchins and abalones[j]. Microbiol Ecol, 1995, 30: 193 202. [7],,. PCR-DGGE [J]., 2010, 3: 119-122. [8],,,. PCR-DGGE [J]., 2005, 25(4): 842-847. [9],,,. [J]., 2007, 5: 123-129. [10] Tanaka R, Sugimura I, Sawabe T, et al. Gut microflora of abalone Haliotis discus hannai in culture changes coincident with a change in diet [J]. Fish Sci, 2003, 9: 951-958. [11] Dang, H Y, Lovell C R. Bacterial primary colonization and early succession on surfaces in marine waters as determined by amplified rrna gene restriction analysis and sequence analysis of 16S rrna genes [J]. Appl Environ Microbiol, 2000, 66: 467-475. [12] Kim D, Baik K S, Kim M S, et al. Shewanella haliotis sp. nov., isolated from the gut microflora of abalone, Haliotis discus hannai [J]. Int J Syst Evol Microbiol, 2007, 57: 2926-2931. [13] Park S C, Baik K S, Kim M S,et al.shewanella marina sp. nov., isolated from seawater [J]. Int J Syst Evol Microbiol, 2009, 59: 1888-1894. [14] Spanggaard B, Huber I, Nielsen J, et al. The microflora of rainbow trout intestine: a comparis on of traditional and molecular identification [J]. Aquaculture, 2000, 182: 1-15. [15] Harrell L W, Etlinger H M, Hodgins H O. Humoral factors important in resistance of salmonid fish to bacterial disease. II. Anti-Vibrio anguillarum activity in mucus and observations on complement, [J]. Aquaculture, 1976, 4: 363-370. [16] Fletcher T C. Non-specific defence mechanisms in fish [J]. Develop Comp Immunol, 1982, 2(suppl.):123-132. [17] Ringø E, Strøm E, Tabachek J. Intestinal microflora of salmonids: a review[j]. Aquacult Res, 1995, 26: 773-789. [18] Tanaka R, Ootsubo M, Sawabe T, et al. Biodiversity and in situ abundance of gut microflora of abalone (Haliotis discus hannai) determined by culture-independent techniques [J]. Aquaculture, 2004, 241: 453-463. [19] Erasmus J H, Cook P A, Coyne V E. The role of bacteria in the digestion of seaweed by the abalone Haliotis midae [J]. Aquaculture, 1997, 155: 377-386. Microflora in the digestive tract of Haliotis discus hannai Ino and PCR- DGGE fingerprint analysis LENG Xiao-fei 1, JIANG Hai-feng 2, LIU Xiao-lin 2, WANG Gao-xue 2, ZHANG Yan 2, GAO Hong-tao 1, CHANG Ya-qing 3 (1. Pacific Fisheries Co.,LTD, Lü shun 116045, China; 2. College of Animal Science, Northwest A & F University, Yangling 712100, China; 3. Key Laboratory of Mariculture, Dalian Ocean University, Dalian 116023, China) Received: Feb.,11,2012 Key words: Haliotis discus hannai Ino; intestinal tract; microflora; PCR-DGGE Abstract: Microflora in the intestinal tract of cultured and wild abalones (Haliotis discus hannai Ino) individuals were studied in this paper. PCR-DGGE fingerprint was explored to analyze the differences between the dominant species in intestinal tract of these two groups. Results showed that the total aerobe and facultative anaerobe in the intestinal tract of cultured and wild abalone reached 3.50±0.85 10 6 and 3.03±1.10 106 cfu/g, respectively. The dominant species was similar. Vibrio was the dominant species, and Shewanella and Roseobacter were the secondary dominant species, Bacillus was detected in the cultured group only, suggesting that abalones have specific selectivity for microflora in the intestinal tract. Additionally, the result from PCR-DGGE fingerprint showed that 14 and 12 bands were observed from cultured and wild abalones, respectively. There were 2 special bands appeared in cultured group. The similarity between the two groups was 92.31%. 14 / 2013 / 37 / 5 ( 本文编辑 : 梁德海 )