TAIL-PCR方法克隆约氏黄杆菌aroA基因序列

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基因序列,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1 230bp,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。

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

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 random primers 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.