副猪嗜血杆菌aroA基因鉴定及遗传进化分析

目的]细菌aroA基因参与芳香族氨基酸的生物合成,被成功应用于细菌分类和基因失活致弱突变菌株的构建.副猪嗜血杆菌(Hps)是感染猪出现多发性浆膜炎和关节炎的一种病原细菌,鉴定该菌aroA全基因序列将有助于鉴定遗传进化关系和突变分析.方法]利用PCR和细菌基因组步移技术鉴定Hps的aroA基因序列,进而对不同血清型菌株该基因序列进行鉴定,并与其它革兰氏阴性细菌进行比对和遗传进化分析.结果]自Hps血清5型基因组DNA中获得包含完整aroA基因的3.7 kb基因片段,其中aroA基因全长1314 bp,编码产物长度437 aa,分子量大小47.9 kDa,该基因上游为磷酸烯醇式丙酮酸羧化酶基因.自本试验选择的Hps不同血清型菌株中均可扩增出包含完整aroA基因的1476 bp片段,且这些不同血清型菌株间核酸序列同源性在97.7%以上.Hps血清5型aroA基因序列与巴氏杆菌科其它成员核酸序列同源性为70.6%-78.9%,与E.coli和S.typhi-murium的同源性分别为66.4%和67.2%.结论]本试验首次对Hps的15个血清型国际参考菌株及地方分离株aroA全基因序列进行了鉴定,序列比较结果显示aroA基因在革兰氏阴性细菌中具有较高的同源性.aroA基因鉴定对构建基因失活突变菌株以研究Hps生物学特性奠定了基础.

Identification and phylogenetic analysis on the aroA gene of Haemophilus parasuis

Objective] We clarified the genetic structure and phylogenetic relationship of the aroA gene in Haemophilus parasuis, a gene encoding 5'-enolpyruvylshikimate-3-phosphate synthetase. Methods] We used PCR and bacterial genome walking techniques to determine the aroA gene locus in the genome of H. parasuis serovar 5. Then we sequenced the aroA gene of H. parasuis reference strains and local isolates. All aroA gene sequences from H. parasuis, other members of the family Pasteurellaceae, Escherichia coli and Salmonella typhimurium were analyzed for phylogenetic relationship. Results] An approximately 3.7 kb fragment containing the whole aroA gene was obtained from the genomic DNA of H. parasuis serovar 5. Sequence analysis showed that the size of the whole aroA gene was 1314 bp, encoding a 437 aa product with molecular weight 47.9 kDa. The sequence immediately upstream of the aroA gene shared high similarity with phosphoenolpyruvate carboxylase gene. The whole aroA gene fragment could be amplified from all strains of H. parasuis serovars with a size of 1476 bp. The nucleotide sequence similarity was above 97.7% in H. parasuis serovars. The simi-larity was 70.6%-78.9% between H. parasuis serovar 5 and other members of Pasteurellaceae. The similarity between H. parasuis serovar 5 and E. coli or S. typhimurium was 66.4% and 67.2% respectively. Conclusion] No obvious difference was found in the aroA gene sequence of H. parasuis reference strains (15 serovars) and 3 local isolates. The nucleotide sequence of the aroA gene was well-conserved within Gram-negative organisms.