刺参消化道中蛭弧菌类的生物多样性分析
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摘要
研究了刺参消化道中蛭弧菌类生物多样性。用刺参肠道内容物提取微生物总DNA,分别使用蛭弧菌类生物特异性引物Bd、Bac扩增获得16S r DNA目的片段,连接p MD19-T载体,转化到大肠埃希菌DH5α感受态细胞,经蓝白斑筛选及阳性克隆筛选,通过核糖体DNA扩增片段限制性内切酶分析(ARDAR)对阳性克隆进行分型,使用HaeⅢ和MspⅠ双酶切各60个阳性克隆,选取不同ARDAR型的阳性克隆测序,并进行生物学分析。结果显示:引物Bd、Bac的16S r DNA扩增产物分别分离获得3个和2个差异性序列,各属于一个类群,分属于蛭弧菌属(Bdellovibrio)和噬菌弧菌属(Bacteriovorax)。这些序列与数据库中相应菌株序列的最大相似性均为95.0%,这5个序列的菌株可能为潜在的新种。
The diversity of Bdellovibrio-like organisms inside the intestine of spiny sea cucumber( Apostichopus japonicas) was studied. Total microbial DNA was extracted using intestinal content from the sea cucumber. The purpose fragments of 16 S r DNA of BALOs were amplified using primers Bd and Bac respectively and connected to p MD 19-T vector,then transformed into E. coli DH5 α. The 60 positive clones were selected respectively by blue white screening and verified by PCR detection. The patterns of different clones were determined using amplified ribosomal DNA restriction analysis( ARDRA) with double restriction enzymes Hae Ⅲ and Msp I to cut 60 positive clones,respectively.Then,the positive clones sequence of different ARDAR patterns were selected and carried out biological analysis. The results showed that 3 and 2 different 16 S r DNA sequences of primers Bd and Bac amplified products were obtained respectively. They belong to two different clusters of Bdellovibrio and Bacteriovorax respectively. Comparison of these ssequences to those corresponding sequences in database all had a maximum similarity at 95%. These five sequences' strains possibly are potential new species.
The diversity of Bdellovibrio-like organisms inside the intestine of spiny sea cucumber( Apostichopus japonicas) was studied. Total microbial DNA was extracted using intestinal content from the sea cucumber. The purpose fragments of 16 S r DNA of BALOs were amplified using primers Bd and Bac respectively and connected to p MD 19-T vector,then transformed into E. coli DH5 α. The 60 positive clones were selected respectively by blue white screening and verified by PCR detection. The patterns of different clones were determined using amplified ribosomal DNA restriction analysis( ARDRA) with double restriction enzymes Hae Ⅲ and Msp I to cut 60 positive clones,respectively.Then,the positive clones sequence of different ARDAR patterns were selected and carried out biological analysis. The results showed that 3 and 2 different 16 S r DNA sequences of primers Bd and Bac amplified products were obtained respectively. They belong to two different clusters of Bdellovibrio and Bacteriovorax respectively. Comparison of these ssequences to those corresponding sequences in database all had a maximum similarity at 95%. These five sequences' strains possibly are potential new species.
引文
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[16]Pi珘neiro SA,Stine OC,Chauhan A,et al.Global survey of diversity among environmental saltwater Bacteriovoracaceae[J].Environmental Microbiology,2007,9(10):2441-2450.
[17]黄华伟,邓时铭,廖伏初.蛭弧菌的生物学特性及其在刺参养殖中的应用前景[J].河北渔业,2008,(5):1-4.
[2]蔡姝文.微生物制剂在水产养殖中的作用[J].现代农业科技,2014,18(1):251-270.
[3]王玉堂.蛭弧菌及其在水产养殖动物疾病防治中的应用(连载一)[J].中国水产,2014,(1):50-51.
[4]王玉堂.蛭弧菌及其在水产养殖动物疾病防治中的应用(连载二)[J].中国水产,2014,(2):46-47.
[5]王玉堂.蛭弧菌及其在水产养殖动物疾病防治中的应用(连载三)[J].中国水产,2014,(3):52-54.
[6]Dwidar M,Monnappa AK,Mitchell RJ.The dual probiotic and antibiotic nature of Bdellovibrio bacteriovorus[J].Biochemistry and Molecular Biology Reports,2012,45(2):71-78.
[7]温崇庆.海水养殖环境蛭弧菌类生物多样性及噬菌弧菌DA5对细菌的生物控制[D].广州:中山大学,2009.
[8]Wen CQ,Lai XT,Xue M,et al.Molecular typing and identification of Bdellovibrio-and-like organisms isolated from seawater shrimp ponds and adjacent coastal waters[J].Journal of Applied Microbiology,2009,106(4):1154-1162.
[9]薛明,关敏丽,王飞燕.两种培养基对对虾苗池海洋蛭弧菌的分离及其多样性分析[J].微生物学通报,2014,41(9):1723-1732.
[10]张志祥.鳗鲡肠道蛭弧菌的分离及其在预防水产养殖细菌性疾病的应用研究[D].厦门:集美大学,2009.
[11]李楠.鸡源蛭弧菌的分离、生物学特性研究及对鸡白痢的治疗试验[D].雅安:四川农业大学,2005.
[12]Davidov Y,Friedjung A,Jurkevitch E.Structure analysis of a soil community of predatory bacteria using culture-dependent and culture-independent methods reveals a hitherto undetected diversity of Bdellovibrio-and-like organisms[J].Environmental Microbiology,2006,8(9):1667-1673.
[13]Davidov Y,Huchon D,Koval SF,et al.A new alpha-proteobacterial clade of Bdellovibrio-like predators:implications for the mitochondrial endosymbiotic theory[J].Environmental Microbiology,2006,8(12):2179-2188.
[14]Zheng G,Wang C,Williams HN,et al.Development and evaluation of a quantitative real-time PCR assay for the detection of saltwater Bacteriovorax[J].Environmental Microbiology,2008,l0(10):2515-2526.
[15]Davidov Y,Jurkevitch E.Diversity and evolution of Bdellovibrio-and-like organisms(BALOs),reclassification of Bacteriovorax starrii as Peredibacter starrii gen.nov.,comb.nov.,and description of the Bacteriovorax-Peredibacter clade as Bacteriovoracaceae fam.nov.[J].International Journal of Systematic and Evolutionary Microbiology,2004,54(5):1439-1452.
[16]Pi珘neiro SA,Stine OC,Chauhan A,et al.Global survey of diversity among environmental saltwater Bacteriovoracaceae[J].Environmental Microbiology,2007,9(10):2441-2450.
[17]黄华伟,邓时铭,廖伏初.蛭弧菌的生物学特性及其在刺参养殖中的应用前景[J].河北渔业,2008,(5):1-4.