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[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。
[Objective] The research aimed to study the full-length sequence of aroA gene of the synthetic gene of aromatic amino acid in Flavobacterium yaudiii pathogen of grass carp. [Method] With genomic DNA from the pathogen M165 of the gill disease of grass carp as a template, three specific nested primers of 5 ’and 3’ and random primers were respectively used to amplify strain M165 by thermal asymmetric staggered PCR (TAIL-PCR) AroA sequence of the gene, and its sequence analysis. [Result] The results of electrophoresis showed that the amplified product was in good agreement with the expected amplification result. Sequence analysis showed that the aroA gene sequence was 1230 bp in length and encoded 410 amino acids. The aroA protein sequence shares a high degree of homology with the aroA protein sequence of Flavobacterium johnsoniae. [Conclusion] The TAIL-PCR method provided a simple and efficient method for cloning of the complete sequence of genes. The full-length aroA gene sequence provided a basis for elucidating the effects of aroA and other nutritional factors on pathogenicity of fish gill etiology.