Efficient Amplification and Sequence Analysis of aroA Gene Sequence of Flavobacterium johnsoniae through TAIL-PCR  

TAIL-PCR方法克隆约氏黄杆菌aroA基因序列(摘要)(英文)

作　　者：刘礼辉[1] 李宁求[1] 石存斌[1] 吴淑勤[1] 

机构地区：[1]中国水产科学研究院珠江水产研究所,广东广州510380

出　　处：《Agricultural Science & Technology》2010年第7期179-182,共4页农业科学与技术（英文版）

基　　金：Supported by The Natural Science Foundation of Guangdong Province(06024033);National Special Fund for the Agricultural Commonweal Industry(200803013);Earmarked Fund for Modern Agroindustry Technology Research System(nycytx-49-14)~~

摘　　要：[Objective] The aim was to study the sequence of aroA gene （synthetic gene in metabolic pathway of aromatic amino acids） of a pathogenic bacterium （Flavobacterium johnsoniae） causing gill-rote disease.[Method] Genomic DNA of strain M165 of F.johnsoniae was used as a template,three specific nested primers of 5＇end and 3＇end and arbitrary primer were used to amplify the aroA gene of strain M165 through Thermal asymmetric interlaced PCR （TAIL-PCR）.And the obtained sequence was analyzed.[Result] The electrophoresis determination result showed that the size of amplified product was consist with the expected product; sequencing analysis suggested that the full-length of aroA gene was 1 230 bp,enconding 410 amino acids.The amino acid sequence of aroA protein showed the highest level of similarity to amino acids sequence of aroA protein of F.johnsoniae.[Conclusion] TAIL-PCR provided a simple and efficient new method for the cloning of the gene sequence.Obtaining of full-length of aroA gene provided a foundation for further investigation on the effects of nutrition correlation factors such as aroA on the virulence of and virulence of F.johnsoniae.[目的]研究草鱼烂鳃病病原约氏黄杆菌芳香氨基酸代谢途径的合成基因aroA基因的全序列。[方法]以草鱼烂鳃病病原M165菌株基因组DNA为模板,分别利用5′和3′的3个特异性巢式引物与随机引物,通过热不对称交错PCR(TAIL-PCR)扩增菌株M165的aroA序列基因,并对其序列进行分析。[结果]电泳检测结果表明,扩增产物与预期扩增结果相符;测序结果分析表明,aroA基因序列全长1230bp,编码410个氨基酸。aroA蛋白序列与Flavobacterium johnsoniae的aroA蛋白质序列,具有高度同源性。[结论]TAIL-PCR方法为基因全序列的克隆提供了一种简便、高效的方法。aroA基因全序列的获得为进一步阐明aroA等营养相关因子对鱼类烂鳃病病原的致病力的影响奠定基础。

关 键 词：TAIL-PCR Gill-rote disease Flavobacterium johnsoniae aroA gene 

分 类 号：S941[农业科学—水产养殖]