ARDRA分型测定刺参养殖环境中蛭弧菌多样性
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摘要
【目的】研究刺参养殖环境中蛭弧菌类微生物的多样性及其组成特征。【方法】对刺参养殖用水进行过滤,获得微生物并提取其总DNA,分别采用两对特异性引物Per和Bac扩增蛭弧菌类微生物相应的16S r RNA基因目的片段。通过核糖体DNA扩增片段限制性内切酶分析(ARDRA)方法,使用HaeⅢ和MspⅠ,双酶切各60个目的基因的单克隆,选不同条带克隆进行测序并对测序结果的多重序列分析比对。【结果】确定两对引物分别存在8和9种碱基差异序列,均属于噬菌弧菌属,分属于3个类群中。类群Ⅰ、Ⅱ为优势类群,且均为已知类群,类群Ⅲ为潜在的新类群;Per1(KP214541)和Bac44(KP214551)代表菌株分别为优势种。【结论】刺参养殖环境中蛭弧菌具有较高的多样性,包括已知类型和未知类型的蛭弧菌;可进一步进行蛭弧菌的分离、培养,用于刺参养殖中病害防治研究。
[Objective] To study the diversity and composition of Bdellovibrio-and-like organisms(BALOs) in mariculture environment of Apostichopus japonicus. [Methods] The aquaculture water of A. japonicus was filtered to obtain the microorganisms. Then the total DNA of microorganisms was extracted. The purpose fragments of 16 S r RNA gene of BALOs were amplified using two pairs of primers Per and Bac by PCR, respectively. Different patterns were determined with restriction enzymes Hae Ⅲ and Msp I by amplified ribosomal DNA restriction analysis(ARDRA). Positive clones were verified from 60 monoclonal strains. [Results] Eight and nine differences base sequences were obtained from two pairs of primers, respectively, and which were all belong to Bacteriovorax sp. and affiliated with three different clusters. Clusters Ⅰ and Ⅱ are dominant type which are known clusters; Cluster Ⅲ is a new one; The representative strains of Per1(KP214541) and Bac44(KP214551) are the dominant species. [Conclusion] The BALOs presented high diversity in mariculture environment of A. japonicus, including known and unknown, and the BALOs should be further isolated and cultured for the study of disease control in A. japonicus culture.
[Objective] To study the diversity and composition of Bdellovibrio-and-like organisms(BALOs) in mariculture environment of Apostichopus japonicus. [Methods] The aquaculture water of A. japonicus was filtered to obtain the microorganisms. Then the total DNA of microorganisms was extracted. The purpose fragments of 16 S r RNA gene of BALOs were amplified using two pairs of primers Per and Bac by PCR, respectively. Different patterns were determined with restriction enzymes Hae Ⅲ and Msp I by amplified ribosomal DNA restriction analysis(ARDRA). Positive clones were verified from 60 monoclonal strains. [Results] Eight and nine differences base sequences were obtained from two pairs of primers, respectively, and which were all belong to Bacteriovorax sp. and affiliated with three different clusters. Clusters Ⅰ and Ⅱ are dominant type which are known clusters; Cluster Ⅲ is a new one; The representative strains of Per1(KP214541) and Bac44(KP214551) are the dominant species. [Conclusion] The BALOs presented high diversity in mariculture environment of A. japonicus, including known and unknown, and the BALOs should be further isolated and cultured for the study of disease control in A. japonicus culture.
引文
[1]Snyder AR,Williams HN,Baer ML,et al.16S r DNA sequence analysis of environmental Bdellovibrio-and-like organisms(BALO)reveals extensive diversity[J].International Journal of Systematic and Evolutionary Microbiology,2002,52(6):2089-2094
[2]Huang HW,Zhan WB.Bdellovibrio bacteriovorus-A new tool in biological disease control in raising of sea cucumber[J].Hebei Fisheries,2007(1):37-38(in Chinese)黄华伟,战文斌.噬菌蛭弧菌——养殖刺参疾病生物防治的新工具[J].河北渔业,2007(1):37-38
[3]Huang L,Cai JP,Chen XH,et al.Elimination of potential pathogenic vibrios in oysters by Bdellovibrio sp.[J].Modern Food Science and Technology,2010,26(3):225-230(in Chinese)黄亮,蔡俊鹏,陈小红,等.应用蛭弧菌清除牡蛎中潜在致病弧菌的研究[J].现代食品科技,2010,26(3):225-230
[4]Zhang XN,Yang XL,Cao HP,et al.Optimization of preparation conditions of Bdellovibrio bacteriovorus granules[J].Journal of Shanghai Ocean University,2010,19(6):778-786(in Chinese)张小能,杨先乐,曹海鹏,等.噬菌蛭弧菌颗粒剂制备条件的优化[J].上海海洋大学学报,2010,19(6):778-786
[5]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
[6]Yang JX,Xu L,Cai JP.Prospects and problems of applying Bdellovibrio sp.in controlling pathogens in mariculture[J].Journal of Guangdong Ocean University,2004,24(3):79-82(in Chinese)杨吉霞,徐丽,蔡俊鹏.海水养殖中应用蛭弧菌控制病原菌的前景与问题[J].湛江海洋大学学报,2004,24(3):79-82
[7]Cai JP,Zhao J.The recent research progress on Bdellovibrio bacteriourus[J].Acta Microbiologica Sinica,2006,46(6):1028-1032(in Chinese)蔡俊鹏,赵俊.蛭弧菌的最新研究进展[J].微生物学报,2006,46(6):1028-1032
[8]Wen CQ.Diversity of Bdellovibrio-and-lile organisms in mariculture environments and biocontrol of bacteria by Bacteriovorax DA5[D].Guangzhou:Doctoral Dissertation of Sun Yat-sen University,2009(in Chinese)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:中山大学博士学位论文,2009
[9]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].Applied Microbiology and Biotechnology,2009,106(4):1154-1162
[10]Xue M,Guan ML,Wang FY,et al.Comparison of two media for isolation and diversity analysis of marine Bdellovibrio-and-like organisms in shrimp hatchery system[J].Microbiology China,2014,41(9):1723-1732(in Chinese)薛明,关敏丽,王飞燕,等.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
[11]Sambrook J,Russell DW.Molecular Cloning:a Laboratory Manual[M].Translated by Huang PT,Wang JX,Zhu HC,et al.3rd Edition.Beijing:Science Press,2002,8:87-96(in Chinese)萨姆布鲁克J,拉塞尔著DW.分子克隆实验指南[M].黄培堂,王嘉玺,朱厚础,等译.第3版.北京:科学出版社,2002,8:87-96
[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]Williams FN,Pi?eiro S.Ecology of the predatory Bdellovibrio-and like organisms[A]//Jurkevitch E.Predatory Prokaryotes[M].Berlin Heidelberg:Springer-Verlag,2007,4:213-248
[14]Baer ML,Ravel J,Pi?eiro SA,et al.Reclassification of salt-water Bdellovibrio sp.as Bacteriovorax marinus sp.nov.and Bacteriovorax litoralis sp.nov.[J].International Journal of Systematic and Evolutionary Microbiology,2004,54(4):1011-1016
[15]Li Y,Zheng W,Zheng TL.Advances in research of marine microbial diversity and molecular ecology[J].Microbiology China,2013,40(4):655-668(in Chinese)李祎,郑伟,郑天凌.海洋微生物多样性及其分子生态学研究进展[J].微生物学通报,2013,40(4):655-668
[16]Sockett RE,Lambert C.Bdellovibrio as therapeutic agents:a predatory renaissance[J].Nature Reviews Microbiology,2004,2(8):669-675
[2]Huang HW,Zhan WB.Bdellovibrio bacteriovorus-A new tool in biological disease control in raising of sea cucumber[J].Hebei Fisheries,2007(1):37-38(in Chinese)黄华伟,战文斌.噬菌蛭弧菌——养殖刺参疾病生物防治的新工具[J].河北渔业,2007(1):37-38
[3]Huang L,Cai JP,Chen XH,et al.Elimination of potential pathogenic vibrios in oysters by Bdellovibrio sp.[J].Modern Food Science and Technology,2010,26(3):225-230(in Chinese)黄亮,蔡俊鹏,陈小红,等.应用蛭弧菌清除牡蛎中潜在致病弧菌的研究[J].现代食品科技,2010,26(3):225-230
[4]Zhang XN,Yang XL,Cao HP,et al.Optimization of preparation conditions of Bdellovibrio bacteriovorus granules[J].Journal of Shanghai Ocean University,2010,19(6):778-786(in Chinese)张小能,杨先乐,曹海鹏,等.噬菌蛭弧菌颗粒剂制备条件的优化[J].上海海洋大学学报,2010,19(6):778-786
[5]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
[6]Yang JX,Xu L,Cai JP.Prospects and problems of applying Bdellovibrio sp.in controlling pathogens in mariculture[J].Journal of Guangdong Ocean University,2004,24(3):79-82(in Chinese)杨吉霞,徐丽,蔡俊鹏.海水养殖中应用蛭弧菌控制病原菌的前景与问题[J].湛江海洋大学学报,2004,24(3):79-82
[7]Cai JP,Zhao J.The recent research progress on Bdellovibrio bacteriourus[J].Acta Microbiologica Sinica,2006,46(6):1028-1032(in Chinese)蔡俊鹏,赵俊.蛭弧菌的最新研究进展[J].微生物学报,2006,46(6):1028-1032
[8]Wen CQ.Diversity of Bdellovibrio-and-lile organisms in mariculture environments and biocontrol of bacteria by Bacteriovorax DA5[D].Guangzhou:Doctoral Dissertation of Sun Yat-sen University,2009(in Chinese)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:中山大学博士学位论文,2009
[9]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].Applied Microbiology and Biotechnology,2009,106(4):1154-1162
[10]Xue M,Guan ML,Wang FY,et al.Comparison of two media for isolation and diversity analysis of marine Bdellovibrio-and-like organisms in shrimp hatchery system[J].Microbiology China,2014,41(9):1723-1732(in Chinese)薛明,关敏丽,王飞燕,等.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
[11]Sambrook J,Russell DW.Molecular Cloning:a Laboratory Manual[M].Translated by Huang PT,Wang JX,Zhu HC,et al.3rd Edition.Beijing:Science Press,2002,8:87-96(in Chinese)萨姆布鲁克J,拉塞尔著DW.分子克隆实验指南[M].黄培堂,王嘉玺,朱厚础,等译.第3版.北京:科学出版社,2002,8:87-96
[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]Williams FN,Pi?eiro S.Ecology of the predatory Bdellovibrio-and like organisms[A]//Jurkevitch E.Predatory Prokaryotes[M].Berlin Heidelberg:Springer-Verlag,2007,4:213-248
[14]Baer ML,Ravel J,Pi?eiro SA,et al.Reclassification of salt-water Bdellovibrio sp.as Bacteriovorax marinus sp.nov.and Bacteriovorax litoralis sp.nov.[J].International Journal of Systematic and Evolutionary Microbiology,2004,54(4):1011-1016
[15]Li Y,Zheng W,Zheng TL.Advances in research of marine microbial diversity and molecular ecology[J].Microbiology China,2013,40(4):655-668(in Chinese)李祎,郑伟,郑天凌.海洋微生物多样性及其分子生态学研究进展[J].微生物学通报,2013,40(4):655-668
[16]Sockett RE,Lambert C.Bdellovibrio as therapeutic agents:a predatory renaissance[J].Nature Reviews Microbiology,2004,2(8):669-675