2株创伤弧菌的16S-23S rDNA间区的克隆、测序及分析

根据细菌的16SrDNA3’端和23SrDNA5’端的高度保守区设计引物，PCR扩增了2株创伤弧菌（Vibrio vulnificus）的16S-23SrDNA间区（Intergenic spacer，IGS），克隆到pGEM-T载体上，测序。用BLAST和DNA star软件对16S-23SrDNA间区序列及其内的tRNA基因进行比较分析。结果表明，2株创伤弧菌共测出9条16S-23SrDNA间区序列，其中ZSU006测出5条，间区类型分别为：IGS^GLAV、IGS^GLV、IGS^LA、IGS^A和IGS^G.其中IGS^GLAv最大，包含tRNA^Glu、tRNA^Lys、tRNA^Ala。和tRNA^Val基因；IGS^GLV包含tRNA^Glu、tRNA^Lys。和tRNA^Val基因；IGS^LA，则包含tRNA^Ile和tRNA^Ala基因；IGS^G包含tRNA^Glu基因；而IGS^A仅包含tRNA^Ala基因。菌株CG021测出的16S-23SrDNA IGS序列有4条，除缺少IGS^A外，其余的IGS类型均与ZSU006的相同。与GenBank内的创伤弧菌ATCC27562的IGS序列比较，发现创伤弧菌所有类型的IGS的tRNA基因两端的非编码区具有较高的种内同源性。16S-23SrDNA间区结构的差异为建立一种新的创伤弧菌检测方法奠定了基础。

Cloning, Sequencing and Analysis of the 16S-23S rDNA Intergenic Spacers (IGSs) of Two Strains of Vibrio vulnificus

According to the conserved sequences flanking the 3' end of the 16S and the 5' end of the 23S rDNAs, PCR primers were designed, and the 16S-23S rDNA intergenic spacers (IGSs) of two strains of Vibrio vulnificus were amplified by PCR and cloned into pGEM-T vector. Different clones were selected to be sequenced and the sequences were analyzed with BLAST and the software DNAstar. Analyses of the IGS sequences suggested that the strain ZSU006 contains five types of polymorphic16S-23S rDNA intergenic spacers, namely, IGSGLAV, IGSGLv, IGSIA, IGSG and IGSA; while the strain CG021 has the same types of IGSs except lacking IGSA. Among these five IGS types, IGSGLAV is the biggest type, including the gene cluster of tRNAGlu - tRNALys - tRNAAla - tRNAVal; IGSGLV includes that of tRNAGlu-tRNALys-tRNAVal; IGSAG, tRNAAla-tRNAGlu; IGSIA, tRNAIle -tRNAAla; IGSG, tRNAGlu and IGSA, tRNAAla. Intraspecies multiple alignment of all the IGS sequences of these two strains with those of V. vulnificus ATCC27562 available at GenBank revealed several highly conserved sequence blocks in the non- coding regions flanking the tRNA genes within all of strains, most notably the first 40 and last 200 nucleotides, which can be targeted to design species-specific PCR primers or detection probes. The structural variations of the 16S-23S rDNA intergenic spacers lay a foundation for developing diagnostic methods for V. vulnificus.