中国对虾来源哈维氏弧菌鉴定及生物学分析
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摘要
从患病中国对虾中分离到优势菌株,对其进行生理生化特性及分子生物学分析。TCBS培养基上菌落呈现绿色(以下称为TCBS-G),通过形态学、生理生化分析,菌株TCBS-G为革兰氏阴性菌,短杆状,两端钝圆,有鞭毛;Biolog GIII分析表明,该菌株生长代谢利用情况与弧菌吻合,与哈维氏弧菌特征相似。基于16S rRNA、gyrB、HSP60基因序列分析同源性,16S rRNA未将TCBS-G有效鉴定,gyrB、HSP60基因序列与哈维氏弧菌相聚类,聚合置信度可达99%。综合生物学及分子特征,判定TCBS-G为弧菌属的哈维氏弧菌(Vibrio harveyi)。底物平板试验结果显示TCBS-G菌株胞外产物具有淀粉酶、蛋白酶、脂肪酶和DNA酶活性;且能够扩增出哈氏弧菌溶血素基因的保守特异性片段。
In this study, the dominant strain was isolated from diseased Fenneropenaeus chinensis, the biochemical and molecular biological identification were conducted for this strain. The strain was green on TCBS medium(thus designated as TCBS-G). Based on physiological and biochemical analysis, TCBS-G was identified to be Gram-negative, in short rod shape, blunt ends, and with flagellum. Biolog GIII experiment showed that the growth and metabolism of TCBS-G were similar to that of Vibrio harveyi. Homology of TCBSG with Vibrio spp. was analyzed based on the sequences of 16 S rRNA gene, gyrB and HSP60. Based on the sequence of 16 S rRNA gene, TCBS-G was not identified. Phylogenetic tree based on gyrB and HSP60 genes showed that TCBS-G clustered together with V. harveyi, with a high bootstrap value of 99%. In summary, all above results confirmed that the isolated strain TCBS-G was V. harveyi. The substrate plate test showed that it could produce diastase, proteinase, lipase, and DNase. In addition, the conserved region in hemolysin gene of V. harveyi could be amplified by PCR with specific primers.
In this study, the dominant strain was isolated from diseased Fenneropenaeus chinensis, the biochemical and molecular biological identification were conducted for this strain. The strain was green on TCBS medium(thus designated as TCBS-G). Based on physiological and biochemical analysis, TCBS-G was identified to be Gram-negative, in short rod shape, blunt ends, and with flagellum. Biolog GIII experiment showed that the growth and metabolism of TCBS-G were similar to that of Vibrio harveyi. Homology of TCBSG with Vibrio spp. was analyzed based on the sequences of 16 S rRNA gene, gyrB and HSP60. Based on the sequence of 16 S rRNA gene, TCBS-G was not identified. Phylogenetic tree based on gyrB and HSP60 genes showed that TCBS-G clustered together with V. harveyi, with a high bootstrap value of 99%. In summary, all above results confirmed that the isolated strain TCBS-G was V. harveyi. The substrate plate test showed that it could produce diastase, proteinase, lipase, and DNase. In addition, the conserved region in hemolysin gene of V. harveyi could be amplified by PCR with specific primers.
引文
[1] Baumann P, Baumann L, Bang S S, et al. Reevaluation of the taxonomy of Vibrio, Beneckea, and Photobacteruim:abolish the genus Beneckea[J].Curr Microbiol,1980,4:127-132.
[2] Anonymous. Validation of the publication of new names and new combinations previously effectively published outside the IJBS[J].List no.6 Int J Syst Bacteriol,1981,31:215-218.
[3] Harris L J, Owens L. Production of exotoxins by two luminous Vibrio harveyi strains known to be primary pathogens of Penaeus monodon larvae[J].Dis Aquat Organ,1999,38:11-22.
[4] Teo J W P, Zhang L H, Poh C L. Cloning and characterization of a metalloprotease from Vibrio harveyi strain AP6[J].Gene,2003,303:147-156.
[5] Oakey H J, Cullen B R and Owen L. The complete nucleotide sequence of the Vibrio harveyi bacteriophage VHML[J].J Appl Microbiol,2002,93:1089-1098.
[6]郝云婕,韩素贞.gyrB基因在细菌系统发育分析中的应用[J].生物技术通报,2008(2):39-41.
[7]安然,易图永,肖启明,等.gyrB基因在细菌分类和检测中的应用[J].江西农业学报,2010,22(4):18-20.
[8]侯晓丽,陈智.分类及鉴别细菌的新靶标—gyrB基因[J].国外医学:流行病学传染病学分册,2005,32(1):38-41.
[9]温崇庆,薛明,何红,等.两株对虾幼体弧菌病病原的分离和鉴定[J].微生物学通报,2008,35(3):346-352.
[10]张文文,王庚申,施慧,等.一种引起脊尾白虾红体病病原菌的初步研究[J].浙江海洋学院学报:自然科学版,2014,33(1):65-71.
[11]余露军,蔡磊,陈小曲,等.诸氏鲻虾虎鱼致病性创伤弧菌的分离与鉴定[J].动物医学进展,2015,36(9):51-54.
[12]徐晓丽,邵蓬,李灏,等.豹纹鳃棘鲈致病性哈维氏弧菌的分离鉴定与系统发育分析[J].华中农业大学学报,2014,33(4):112-118.
[13] Liu P C, Lee K K. Cysteine proteinase is a major exotoxin of pathogenic luminous Vibrio harveyi in the tiger prawn, Penaeusmonodon[J].Lett Appl Microbiol,1999,28:428-430.
[14]张崇文,于涟,毛芝娟,等.哈维氏弧菌外膜蛋白OmpK基因的克隆及原核表达[J].水产学报,2006,30(1):9-14.
[15] Zhang X H, Meaden P G, Austin B. Duplication of hemolys in genesin a virulent isolate of Vibrio harveyi[J].Appl Environ Microbiol,2001,67:3161-3167.
[16] Pizzotto M, Hirst R G. Classification of isolates of Vibrio harveyivirulent to Penaeus monodon larvae by protein profile analysis and M13 DNA finger printing[J]. Dis Aquatic Organism,1995,21:61-68.
[17] Jiravanichpaisal P, Miyazaki T, Limsuwan C. Histopathology,biochemistry,and pathogenicity of Vibrio harveyi infecting black tiger prawn Penaeus monodon[J].J Aquatic Anim Health,1994(6):27-35.
[18] Liu P C, Lee K K, Yii K C, et al. Isolation of Vibrio harveyi from diseased kuruma prawn Penaeus japonicus[J].Current Microbiol,1996,33:129-132.
[19]李军,徐怀恕.中国对虾幼体致病菌哈维氏弧菌的分离及其生物学特性研究[J].海洋与湖沼,1998,29(4):353-361.
[20]苏杭.养殖凡纳滨对虾病原菌(哈维氏弧菌)的分离鉴定与防治[D].雅安:四川农业大学,2010.
[21]赵吉臣,王刚,邓中宁,等.不同条件下哈维氏弧菌(Vibrio harveyi)对墨吉明对虾(Fenneropenaeus merguiensis)致病性研究[J].海洋与湖沼,2015,6:1413-1419.
[22]白方方.哈维氏弧菌的密度感应系统及其信号干扰研究[D].青岛:中国海洋大学,2007.
[23]陈吉祥,杨慧,颜显辉,等.致病性哈氏弧菌溶血素基因克隆及其检测[J].中国水产科学,2005,12(5):580-586.
[24]张晓君,毕可然,阎斌伦,等.中国对虾糠虾幼体病原哈氏弧菌的鉴定及毒力基因检测[J].渔业科学进展,2014(3):105-111.
[2] Anonymous. Validation of the publication of new names and new combinations previously effectively published outside the IJBS[J].List no.6 Int J Syst Bacteriol,1981,31:215-218.
[3] Harris L J, Owens L. Production of exotoxins by two luminous Vibrio harveyi strains known to be primary pathogens of Penaeus monodon larvae[J].Dis Aquat Organ,1999,38:11-22.
[4] Teo J W P, Zhang L H, Poh C L. Cloning and characterization of a metalloprotease from Vibrio harveyi strain AP6[J].Gene,2003,303:147-156.
[5] Oakey H J, Cullen B R and Owen L. The complete nucleotide sequence of the Vibrio harveyi bacteriophage VHML[J].J Appl Microbiol,2002,93:1089-1098.
[6]郝云婕,韩素贞.gyrB基因在细菌系统发育分析中的应用[J].生物技术通报,2008(2):39-41.
[7]安然,易图永,肖启明,等.gyrB基因在细菌分类和检测中的应用[J].江西农业学报,2010,22(4):18-20.
[8]侯晓丽,陈智.分类及鉴别细菌的新靶标—gyrB基因[J].国外医学:流行病学传染病学分册,2005,32(1):38-41.
[9]温崇庆,薛明,何红,等.两株对虾幼体弧菌病病原的分离和鉴定[J].微生物学通报,2008,35(3):346-352.
[10]张文文,王庚申,施慧,等.一种引起脊尾白虾红体病病原菌的初步研究[J].浙江海洋学院学报:自然科学版,2014,33(1):65-71.
[11]余露军,蔡磊,陈小曲,等.诸氏鲻虾虎鱼致病性创伤弧菌的分离与鉴定[J].动物医学进展,2015,36(9):51-54.
[12]徐晓丽,邵蓬,李灏,等.豹纹鳃棘鲈致病性哈维氏弧菌的分离鉴定与系统发育分析[J].华中农业大学学报,2014,33(4):112-118.
[13] Liu P C, Lee K K. Cysteine proteinase is a major exotoxin of pathogenic luminous Vibrio harveyi in the tiger prawn, Penaeusmonodon[J].Lett Appl Microbiol,1999,28:428-430.
[14]张崇文,于涟,毛芝娟,等.哈维氏弧菌外膜蛋白OmpK基因的克隆及原核表达[J].水产学报,2006,30(1):9-14.
[15] Zhang X H, Meaden P G, Austin B. Duplication of hemolys in genesin a virulent isolate of Vibrio harveyi[J].Appl Environ Microbiol,2001,67:3161-3167.
[16] Pizzotto M, Hirst R G. Classification of isolates of Vibrio harveyivirulent to Penaeus monodon larvae by protein profile analysis and M13 DNA finger printing[J]. Dis Aquatic Organism,1995,21:61-68.
[17] Jiravanichpaisal P, Miyazaki T, Limsuwan C. Histopathology,biochemistry,and pathogenicity of Vibrio harveyi infecting black tiger prawn Penaeus monodon[J].J Aquatic Anim Health,1994(6):27-35.
[18] Liu P C, Lee K K, Yii K C, et al. Isolation of Vibrio harveyi from diseased kuruma prawn Penaeus japonicus[J].Current Microbiol,1996,33:129-132.
[19]李军,徐怀恕.中国对虾幼体致病菌哈维氏弧菌的分离及其生物学特性研究[J].海洋与湖沼,1998,29(4):353-361.
[20]苏杭.养殖凡纳滨对虾病原菌(哈维氏弧菌)的分离鉴定与防治[D].雅安:四川农业大学,2010.
[21]赵吉臣,王刚,邓中宁,等.不同条件下哈维氏弧菌(Vibrio harveyi)对墨吉明对虾(Fenneropenaeus merguiensis)致病性研究[J].海洋与湖沼,2015,6:1413-1419.
[22]白方方.哈维氏弧菌的密度感应系统及其信号干扰研究[D].青岛:中国海洋大学,2007.
[23]陈吉祥,杨慧,颜显辉,等.致病性哈氏弧菌溶血素基因克隆及其检测[J].中国水产科学,2005,12(5):580-586.
[24]张晓君,毕可然,阎斌伦,等.中国对虾糠虾幼体病原哈氏弧菌的鉴定及毒力基因检测[J].渔业科学进展,2014(3):105-111.
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