基于16S rRNA和gyrB基因串联DNA特征序列的气单胞菌鉴定方法

Identification of Aeromonas based on 16S rRNA and gyrB gene tandem DNA characteristic sequence

【目的】建立基于16S rRNA序列和gyrB基因串联DNA特征序列的属内菌种鉴定方法,为气单胞菌的种间区分提供技术支持。【方法】以2020—2021年在我国福建、江苏等地分离获得的水产源气单胞菌和NCBI已公布且完成全基因组测序的10种气单胞菌为研究对象,分别以16S rRNA序列、gyrB基因及2个基因序列串联后得到的新DNA特征序列作为构建系统发育进化树的依据,比较不同系统发育进化树的鉴定结果,并以运动性试验、溶血性试验、糖发酵试验进行辅助鉴定。【结果】在基于16S rRNA序列构建的系统发育进化树中,中间气单胞菌、嗜水气单胞菌、达卡气单胞菌、肠源气单胞菌等聚类在不同分支上,未能形成独立的种属进化分支;在基于gyrB基因构建的系统发育进化树中,达卡气单胞菌、豚鼠气单胞菌和嗜水气单胞菌聚类在同一分支上,而异嗜糖气单胞菌和维氏气单胞菌聚类在另一分支上。将16S rRNA序列和gyrB基因串联组成新DNA特征序列再构建系统发育进化树建树,能完全有效分离气单胞菌属的不同菌株,将不同种的气单胞菌独立聚为一支,较好地实现种间区分。【结论】将16S rRNA序列和gyrB基因串联组成新DNA特征序列再构建系统发育进化树,较基于16S rRNA序列或gyrB基因单独构建系统发育进化树具有更高的分辨力,是一种理想的保守序列相似性高的属内菌种鉴定方法。因此,在16S rRNA测序鉴定为气单胞菌的情况下可再次以gyrB基因为测序对象,获得序列信息后与16S rRNA序列串联构建系统发育进化树,能更加精确地将气单胞菌鉴定到种水平。

【Objective】This work was aimed to establish a identification method of Aeromonas based on 16S rRNA sequence and gyrB gene tandem DNA characteristic sequence,and to provide research technical support for the differentiation of Aeromonas at the species level.【Method】The aquatic bacteria,which were detected as Aeromonas later,isolated from Fujian,Jiangsu and other places in China from 2020 to 2021 and ten different species of Aeromonas that have completed genome sequencing on NCBI were taken as the experimental objects.The 16S rRNA sequence,gyrB gene and the new DNA characteristic sequences obtained from the tandem of the two sequences were used as the basis for the establishment of the evolutionary tree.The results were identified by comparing different evolutionary trees,meanwhile,exercise tests,hemolysis tests,and sugar fermentation tests were also be used for auxiliary identification.【Result】The results showed that the combined tree had better discrimination effect than the single gene tree.In the phylogenetic evolution tree constructed based on single 16S rRNA sequences,A.media,A.hydrophila,A.dhakensis,and A.enteropelogenes were dispersed on different branches,and did not form independent species and evolutionary branches.In the phylogenetic evolution tree constructed based on the single gyrB gene,A.dhakensis,A.caviae,and A.hydrophilawere clustered on the same branch,while A.allosaccharophila and A.veronii were also clustered on the same branch.By combining 16S rRNA sequence and gyrB gene in tandem to form a new DNA characteristic sequence,the phylogenetic evolutionary tree was constructed,which could completely and effectively isolate different strains of Aeromonas,and cluster different species of Aeromonas into an independent branch,so as to achieve better interspecific differentiation.【Conclusion】The phylogenetic tree constructed by combining 16S rRNA sequence and gyrB gene in tandem to form a new DNA sequence has better identification ability than the phylogenetic tree constructed solely based on 16S rRNA sequence or gyrB gene.It is an ideal method for genus species identification with high conserved sequence similarity.Therefore,in the case of the identification of Aeromonas by 16S rRNA sequencing,gyrB gene can be used as the sequencing object again,and after obtaining sequence information,the phylogenetic evolutionary tree can be constructed in tandem with 16S rRNA sequence to more accurately identify Aeromonas to the species level.