16SrRNA和secA1基因构建临床诺卡菌的系统发育树比较

Comparison of Phylogeny of Clinical Nocardia Species Based on 16S rRNA and secA1 Gene Sequences

目的:比较16S rRNA和secA1基因构建诺卡菌分属Nocardia wallacei、Nocardia farcinica、Nocardia cyriacigeorgica及Nocardia otitidiscaviarum4个种系统发育树的多样性。方法:将8株临床分离的诺卡菌菌株分别接种于脑心浸出液琼脂培养基分离培养,观察其生长情况;提取细菌DNA,采用聚合酶链式反应扩增其16S rRNA和secA1基因序列并测序,所获序列与NCBI数据库进行BLAST比对,鉴定其菌种名;选取本研究鉴定的4个属共有的40个有效发表种及本研究分离的8株诺卡菌菌株的种,通过邻接法、最大似然法分别构建16S rRNA和secA1基因序列的系统发育树并比较。结果:本研究所收集的诺卡菌分属Nocardia wallacei、Nocardia farcinica、Nocardia cyriacigeorgica及Nocardia otitidiscaviarum 4个种,且Nocardia wallacei、Nocardia farcinica及Nocardia cyriacigeorgica基于secA1基因构建的系统发育树较16S rRNA在种内菌株亲缘关系的分化差异程度高及分类多样性多(P<0.05),而Nocardia otitidiscaviarum的分化程度和分类多样性在16S rRNA和secA1基因构建的系统发育树间差异无统计学意义(P>0.05)。结论:secA1基因用于诺卡菌种群的系统发育分析和分子进化的研究,较16S rRNA具有高度分辨率的优势。

Objective： To compare and analyze the diversity of 16S rRNA and secA1 gene in phylogeny of four Nocardia species ： Nocardia wallacei, Nocardia farcinica, Nocardia cyriacigeorgica and Nocardia otitidiscaviarum. Method： 8 clinical strains were inoculated in Brain Heart Infusion Agar medium separately, and growth condition was observed; DNA was extracted and sequenced of the 16S rRNA and secA1 gene by PCR, tested sequence was compared with NCBI database by BLAST and identifing its species name; 40 strains including four species and 8 strains of Nocardia isolated in this study were selected for the evaluation in this research; 16S rRNA and secA1 gene were compared, the phylogenetic tree was constructed by neighbor -joining analysis and maximum parsimony. Results： The collected clinical strains belong to four species. Comparing to the phylogenetic tree based on 16S rRNA and secA1 gene, the species of Nocardia wallacei,Nocardia farcinica and Nocardia cyriacigeorgica shows a high degree of differentiation in related strains and an obvious taxonomic diversity （P 〈 0.05 ）, but this feature in this specie of Nocardia otitidiscaviarum shows no significant difference （ P 〉 0.05 ） . Conclusion ： secA1 gene displays the advantage of high resolution in phylogenetic and molecular evolution analyses comparing with 16s rRna.