干酪乳杆菌的近缘种及亚种部分看家基因的系统发育分析

Phylogenetic analysis of Lactobacillus casei and closely related species/subspecies based on sequences of housekeeping genes

摘要:

【背景】16S rRNA基因序列分析已广泛应用于细菌的分类鉴定，但是存在一定局限性，而使用看家基因作为分子标记在近缘种及亚种间的系统发育分析中具有其独特的优势。【目的】研究16S rRNA、uvrC (核酸外切酶ABC，C亚基)和murE (UDP-N-乙酰胞壁酰三肽合酶)基因序列对干酪乳杆菌的近缘种及亚种的区分能力。【方法】采用分离自传统发酵乳中的6株干酪乳杆菌为研究对象，选取uvrC和murE基因片段，通过PCR扩增、测序，结合已公布的干酪乳杆菌的近缘种或亚种的相应序列计算遗传距离、构建系统发育树，并与16S rRNA基因序列分析技术进行比较。【结果】研究发现Lactobacillus casei及相近种间的uvrC、murE和联合基因(uvrC-murE)构建的系统发育树拓扑结构与16S rRNA基因结果基本一致，区别在于相似性的不同，其分别为79.00%?99.16%、89.08%?99.20%、76.56%?99.69%和99.58%?100%。基于16S rRNA基因不能区分干酪乳杆菌的近缘种及亚种，而看家基因uvrC和murE基因序列能够很好地区分干酪乳杆菌的近缘种及亚种，并且将uvrC和murE基因串联使用后，试验菌株与参考菌株的分类关系更加清晰。【结论】联合基因(uvrC-murE)可作为16S rRNA基因的辅助工具用于干酪乳杆菌的近缘种及亚种的快速准确鉴定。

Abstract:

[Background] 16S rRNA gene sequence is widely used in the classification and identification of bacteria, although still with some limitations. The housekeeping gene as a molecular marker has its unique advantages in phylogenetic analysis among related species and subspecies. [Objective] We used 16S rRNA, uvrC (excinuclease ABC subunit C) and murE (UDP-N-acetylmuramyl tripeptide synthase) gene sequences to identify the phylogenetic relationship among Lactobacillus casei and its related species/subspecies. [Methods] six L. casei strains isolated from traditional dairy products were selected. The uvrC and murE gene sequences of each isolates were obtained by polymerase chain reaction (PCR) methods and sequencing technology. Then, we estimated the genetic distance and constructed phylogenetic trees of 6 L. casei strains combined with 5 published related species/subspecies based on the sequences of 16S rRNA gene, uvrC and murE gene, respectively. [Results] Phylogenetic trees indicated that the homology of closely related L. casei species among uvrC, murE, the combined genes (uvrC-murE) and 16S rRNA gene were different, ranged from 79.00% to 99.16%, 89.08% to 99.20%, 76.56% to 99.69% and 99.58% to 100%. L. casei and its related species and subspecies cannot be distinguished by 16S rRNA gene sequence analysis. The phylogenetic tree based on the sequences of combined gene (uvrC-murE) had a distinct classification on genetic relationship of L. casei and its related species and subspecies. [Conclusion] Phylogenetic analysis based on the combined gene (uvrC-murE) can be used as a supplementary of 16S rRNA gene analysis for rapid classification and identification of L. casei and its related species and subspecies.