Deng Xian-Yu, Chen Xiao-Yan, Wang Zhi-Xue, Ou Pu, He Jian-Guo
School of Life Sciences, Hunan University of Science and Technology, Xiangtan, China.
Yi Chuan Xue Bao. 2006 Apr;33(4):365-72. doi: 10.1016/S0379-4172(06)60062-0.
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 polymorphic 16S-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.
根据16S rDNA 3'端和23S rDNA 5'端侧翼的保守序列设计PCR引物，通过PCR扩增两株创伤弧菌的16S - 23S rDNA基因间隔区（IGS），并克隆到pGEM - T载体中。选择不同的克隆进行测序，并用BLAST和DNAstar软件对序列进行分析。IGS序列分析表明，菌株ZSU006含有五种类型的多态性16S - 23S rDNA基因间隔区，即IGSGLAV、IGSGLV、IGSIA、IGSG和IGSA；而菌株CG021具有相同类型的IGS，但缺少IGSA。在这五种IGS类型中，IGSGLAV是最大的类型，包括tRNAGlu - tRNALys - tRNAAla - tRNAVal基因簇；IGSGLV包括tRNAGlu - tRNALys - tRNAVal基因簇；IGSAG包括tRNAAla - tRNAGlu；IGSIA包括tRNAIle - tRNAAla；IGSG包括tRNAGlu；IGSA包括tRNAAla。将这两株菌的所有IGS序列与GenBank中可获得的创伤弧菌ATCC27562的IGS序列进行种内多重比对，发现在所有菌株中tRNA基因侧翼的非编码区有几个高度保守的序列块，最显著的是前40个和后200个核苷酸，可用于设计种特异性PCR引物或检测探针。16S - 23S rDNA基因间隔区的结构变异为开发创伤弧菌诊断方法奠定了基础。
