罗非鱼海豚链球菌16S rRNA基因的序列测定和系统进化分析

为了从分子水平上对1株致病性罗非鱼链球菌进行分类学鉴定,利用原核生物16SrRNA基因通用引物对分离纯化的罗非鱼致病性链球菌进行16S rRNA基因的克隆及序列分析。结果扩增出长约1.5 kp目的片段,测序得到1条长度为1 447 bp核苷酸序列。核苷酸相似性分析表明,序列与NCB I公布的海豚链球菌(Streptococcus iniae,S.iniae)SCCF5L菌株16SrRNA基因核苷酸序列相似性最高(99.4%),暂称为中国广西株(S.iniae-CGX)。同时,亲源关系较近的S.iniae、S.difficilis和S.agalactiae代表菌株构建的系统发育进化树显示,所得菌株与S.iniae代表菌株组成同一进化分支,与S.agalactiae代表菌株组成的另一进化分支距离较近(95.5%),而与S.difficilis代表菌株组成的进化分支距离较远(92.3%)。上述研究证实,本试验从发病罗非鱼脑组织分离到的致病性链球菌为海豚链球菌。

Sequencing and phylogenetic analysis of the 16S rRNA gene of Streptococcus iniae

In order to identify the strain of Streptococcus iniae(S.iniae) from the infected tilapia at molecule level,the 16S rRNA gene was amplified by using the universal primers,and the PCR product was cloned and sequenced.A sequence of 1447 base pair(bp) was obtained from the bacteria.The sequence was compared to that derived from GenBank using BLASTn,and the closest sequence was the 16S rRNA gene of S.iniae SCCF5L strain,with a similarity of 99.4% and we name it S.iniae-CGX strain.The phylogenetic analysis was based on the 16S rRNA genes sequences of S.iniae-CGX strain and other related organisms,including the representative strains of S.iniae,S.difficilis and S.agalactiae species in GenBank database.The result showed that all strains of S.iniae specie formed a separate and distinct phylogenetic branch,the strains of S.difficilis and S.agalactiae species formed another two separate and distinct phylogenetic branches.The results above showed that the strain of Streptococcus from the tilapia brain was S.iniae species.