斜带石斑鱼3种致病性弧菌的分子生物学鉴定

从患病斜带石斑鱼分离到3株病原菌EcGS020802、EcGS021001、EcGS020801，经常规生理生化鉴定均属于弧菌属的种类。为了进一步确定其分类地位，测定了3株病原菌的16SrRNA和HSP60(heat shock protein，HsP60)基因部分序列。16SrRNA基因系统进化分析表明，3株病原菌与哈维氏弧菌、溶藻弧菌、副溶血弧菌亲缘关系较近，相互之间同源性均大于98．6％，差异不明显。HSP60基因序列分析表明，菌株EcGS020802、EcGS021001、EcGS020801 HSP60基因序列分别与哈维氏弧菌(AF230934)、溶藻弧菌(Ab230931)、副溶血弧菌(AF230951)HSP60基因的同源性最高，依次为95．7％、99．8％和99．8％，而与其它弧菌HSP60基因的同源性均低于90．6％，3株病原菌相互之间的同源性低于91．0％。HSP60基因构建的系统进化树表明，EcGS020802、EcGS021001、EcGS020801分别与Vibrio harveyi、Vibrio alginolyticus、Vibrio parahaemolyticus聚类。综合上述结果，菌株EcGS020802、EcGS021001、EcGS020801可分别鉴定为哈维氏弧菌、溶藻弧菌、副溶血弧菌。结果表明，HSP60基因比16SrRNA基因更适合用于海水鱼致病性弧菌种问的分类研究。

Molecular classification of three kinds of pathogenic Vibrios in orange-spotted grouper, Epinephelus coioides

Three strains of pathogenic bacteria, namely EcGS020802, EcGS021001 and EcGS020801 were isolated from diseased orange spotted grouper (Epinephelus coioides). By physiological and biochemical analyzing, they belonged to Vibrio. In order to further classify them, 16S rRNA and heat shock protein(HSP60) genes partial sequences were determined. Phylogenetic analysis based on 16S rRNA gene sequences indicated that three strains of pathogeny showed high levels of similarity to Vibrio harveyi, V. alginolyticus and V. parahaemolyticus. The identities among them, above 98.6%, were very high. Sequences analysis of HSP60 genes showed that they were very similar to V.harveyi (AF230934), V. alginolyticus (AF230931)and V.parahaemolyticus (AF230951), and their nucleotide identities were 95.7%, 99.8% and 99.8%, respectively. Moreover, the similarities with other Vibrio species were all less than 90.6%. The nucleotide similarities of HSP60 genes among three isolates were less than 91.0%. Phylogenetic tree of Vibrio based on HSP60 genes showed that strains EcGS020802,EcGS021001 and EcGS020801 clustered together with V. harveyi, V.alginolyticus and V. parahaemolyticus, respectively. Based on the results obtained, the strains EcGS020802, EcGS021001 and EcGS020801 were identified as V. harveyi, V. alginolyticus and V.parahaemolyticus, respectively. Results showed that HSP60 gene was fitter to classify marine Vibrios than 16S rRNA gene.