温和气单胞菌TL97528株丝氨酸蛋白酶基因片段克隆与序列分析  

Cloning and sequence analysis of serine protease gene of Aeromonas sobria strain TL97528 from Trionyx sinensis

作　　者：沈锦玉[1] 李新华[1] 潘晓艺[1] 尹文林[1] 曹铮[1] 

机构地区：[1]浙江省淡水水产研究所,浙江湖州313001

出　　处：《上海海洋大学学报》2010年第2期151-156,共6页Journal of Shanghai Ocean University

基　　金：国家"十一五"科技支撑计划(2006BAD03B05)

摘　　要：中华鳖(Trionyx sinensis)细菌性疾病主要由气单胞菌感染引起的,通过脱脂奶平板检测及酶活性试验得出温和气单胞菌TL97528株分泌的胞外蛋白酶为丝氨酸蛋白酶,而丝氨酸蛋白酶是气单胞菌的主要毒力因子。根据已发表的嗜水气单胞菌的丝氨酸蛋白酶基因序列保守区域设计引物,PCR扩增出一长度为809 bp大小的特异片段,该序列在Genbank上的登录号为FJ357446。对该序列进行分析发现,已发表的温和气单胞菌(Aeromonas sobria)丝氨酸蛋白酶基因(GenBank登录号为AF253471)同源性为98%、与其他几株已发表的嗜水气单胞菌(Aeromonas hydrophila)丝氨酸蛋白酶基因(GenBank登录号分别为AF126213、AY841795、DQ127822、CP000462、CP000644、AF159142)同源性分别为98%、82%、82%、82%、81%、81%,与杀鲑气单胞菌(Aeromonas salmonicida)丝氨酸蛋白酶基因(GenBank登录号为X67043)同源性为80%。用DNAstar软件分析,与鳖源温和气单胞菌TK961010株的同源性为98%,与鱼源嗜水气单胞菌BSK-10、TPS-30和J-1株有较高的同源性,均为81%。同时分析了丝氨酸蛋白酶基因的进化关系,发现中华鳖分离的气单胞菌TL97528株与TK961010株关系较近;从鱼分离的气单胞菌BSK-10、TPS-30和J-1株聚为一簇,亲缘关系较近。Aeromonas sobria TL97528,isolated from soft-shelled turtle（Trionyx sinensis）,is the pathogen of Trionyx sinensis bacterial disease.And serine protease is the major virulence factor of aeromonas.A pair of primers was designed according to the nucleotide sequences of the serine protease gene of aeromonas species reported in GenBank.Desired fragment about 809 bp was amplified from TL97528 genome by the specific primers.The PCR product was cloned into PMD 18-T vector and sequenced.The sequence has been deposited in the GenBank database（accession number FJ357446） and was compared with the corresponding regions of aeromonas strains registered in GenBank and TL961010,BSK-10,TPS-30,J-1 by DNAstar.The nucleotide sequence of TL97528 showed 98% homology to AF253471,98% to AF126213,82% to AY841795,82% to DQ127822,82% to CP000462,81% to CP000644,81% to AF159142,80% homology to X67043 and 98% homology to TK961010,81% homology to BSK-10,TPS-30,J-1.Phylogenetic tree of serine protease gene sequence showed that strain TL97528 closed together with TK961010.BSK-10,TPS-30 and J-1 were clustered together.

关 键 词：中华鳖 气单胞菌 丝氨酸蛋白酶 基因 克隆 

分 类 号：S917[农业科学—水产科学]