基于16S rDNA和看家基因序列分析技术的鲍鱼养殖水体弧菌种类的鉴定

采用TCBS选择性培养基从厦门某鲍鱼养殖场的养殖水体中分离到19株细菌.经16S rDNA序列分析,所有菌株均属于弧菌属（Vibrio）,且与最近似的弧菌模式种的同源性都在98.99%以上.由于弧菌种间16S rDNA序列相似度极高,无法仅靠16S rDNA序列比对来鉴定这些菌株到种的水平.16S rDNA序列的系统发育分析也显示,大多数菌株与弧菌模式种无法归为一簇,表明16S rDNA基因在弧菌种的分类鉴定上分辨率不高.进一步采用基于4种看家基因（rpo A、pyr H、gap A和top A）的多位点序列分析技术（MLSA）对分离到的弧菌菌株进行分类鉴定,结果显示19株海洋弧菌归于溶藻弧菌（Vibrio alginalyticus）与魔鬼弧菌（Vibrio diabolicus）2个种,表明这两种弧菌是该鲍鱼养殖场水体环境中的优势弧菌种,在鲍鱼养殖弧菌病害防治方面需要重点关注.此外,看家基因与16S rDNA基因的多态性分析表明,4种看家基因的多态位点比率均高于16S rDNA.4种看家基因串联后的多态位点比率高达41.1%,远高于16S r DNA基因的13.4%,表明看家基因相较于16S rDNA基因有着更高的分辨率,更适合于海洋弧菌的分类鉴定.

Identification of Vibrio strains isolated from abalone aquaculture water based on 1 6S rDNA sequence analysis and multiliocus sequence analyses of housekeeping genes

A total of 19 bacterial strains were isolated from aquaculture water of an abalone farm in Xiamen.All these strains were identified as Vibrio spp.based on 16 S rDNA gene sequence analysis.Their 16 S rDNA sequencesshowed high degrees of similarity（≥98.99%） with closest matched reference sequences of Vibrio type species.However,due to extreme high similarity of 16 S rDNA sequences among Vibrio species,it was impossible to identify these Vibrio strains to species level only based on 16 S rDNA sequence analysis.Phylogenteic analysis also showed that most 16 S rDNA sequences of these isolates were not grouped with the reference sequences of Vibrio type species,suggesting that 16 S rDNA gene is not adequate for resolution of Vibrio species.Multilocus sequence analysis of 4 housekeeping genes（rpo A,pyr H,gap A,and top A） was further performed to identify these Vibrio isolates.The results showed that 19 isolates belong to two Vibrio species,Vibrio alginalyticus and Vibrio diabolicus,indicating that these two Vibrio species are dominant in aquaculture water of the abalone farm.Polymorphism analysis demonstrated that all of 4 housekeeping genes showed higher polymorphic sites than the 16 S rDNA gene.The concatenated sequences of 4 housekeeping genes exhibited 41.1% of polymorphic sites,much higher than that of 16 S rDNA（13.4%）.These results suggested that the housekeeping genes showed remarkable higher solution than 16 S rDNA gene for species identification of marine Vibrios.