用多位点序列分析技术鉴定9株产高光学纯度D-乳酸野生菌株

A study on identification of wild strains produced high optical purity D-lactic acid via MLSA technology

目的对从某发酵食品中分离纯化出的9株产高光学纯度D-乳酸的野生菌株(SZD菌株)进行鉴定。方法先结合表型特征及16SrRNA基因序列分析将SZD菌株鉴定到种的水平,再通过综合分析hsp60、pheS、rpoA及tuf基因的多位点序列分析(MLSA)技术将其鉴定到亚种的水平。结果 SZD菌株的16SrRNA基因序列与棒状乳杆菌扭曲亚种及棒状亚种相应序列的相似率均达99.9%以上。SZD菌株的pheS基因序列与棒状乳杆菌棒状亚种及扭曲亚种的相应序列的相似率分别为100%和97.87%,而rpoA基因序列与棒状乳杆菌棒状亚种及扭曲亚种的相应序列的相似率分别为98.65%和100%;tuf基因序列与棒状乳杆菌两个已知亚种相应序列的相似率均为99.00%以上,而hsp60基因序列与棒状乳杆菌两个已知亚种相应序列的相似率均为99.00%以下。在用应用算术平均数的非加权成组配对法构建的基于16SrRNA、hsp60、tuf和pheS基因的进化树中,SZD菌株独立于棒状乳杆菌的两个已知亚种而自成一支。因此,将SZD菌株鉴定为棒状乳杆菌的一个新亚种。结论结合表型特征及16SrRNA基因序列分析将SZD菌株鉴定为棒状乳杆菌,最后通过基于hsp60、pheS、rpoA及tuf基因的MLSA技术将其鉴定为棒状乳杆菌一个新的亚种。

Objective To identify nine strains of wild type bacteria（SZD strains）which produced high optical purity D-lactic acid from a fermented food.Methods According to the phenotypic characteristics and analysis of 16S rRNA gene sequence,the SZD strains were identified to the species level,and then these isolates were identified to the subspecies level via the multilocus sequence analysis（MLSA）technology based on hsp60,pheS,rpoA and tuf genes.Results The similarity rates of the corresponding sequence of 16S rRNA gene among the SZD strains,Lactobacillus coryniformis subsp.coryniformis and Lactobacillus coryniformis subsp torquens were more than 99.9%.The similarity rates of the corresponding sequence of pheS gene among the SZD strains,Lactobacillus coryniformis subsp.coryniformis and Lactobacillus coryniformis subsp.torquens were 100% and 97.87%,respectively;while,the similarity rates of the corresponding sequence of rpoA gene between these strains were 98.65% and 100%,respectively.The similarity rates of the corresponding sequence of tuf gene among the SZD strains and two known subspecies of Lactobacillus coryniformis were more than 99.00%;in contrast,the similarity rates of the corresponding sequence of hsp60 gene among these strains were less than 99.00%.The phylogenetic trees constructed by unweighted pair-group method using arithmetic average based on 16S rRNA,hsp60,tuf and pheS genes showed that the SZD strains were independent of the two known subspecies of Lactobacillus coryniformis,and formed a unique branch.Therefore,the SZD strains were identified as a novel subspecies of Lactobacillus coryniformis.Conclusion According to the phenotypic characteristics and analysis of the l6S rRNA gene sequence,SZD strains are identified as Lactobacillus coryniformis.These isolates are identified as a novel subspecies of Lactobacillus coryniformis via the MLSA technology based on hsp60,pheS,rpoA and tuf genes finally.