坚强芽孢杆菌(Bacillus firmus)16S—23S rDNA间区的克隆及多态性分析

CLONING AND ANALYSIS OF THE POLYMORPHISM OF THE 16S—23S rDNA INTERGENIC SPACER REGIONS OF BACILLUS FIRMUS

采用16S和23SrDNA保守区设计的引物对两株坚强芽孢杆菌(Bacillus firmus)16S—23SrDNA间区(Intergenic spacer region,ISR)进行了PCR扩增,并克隆到pMD18-T载体上进行测序;利用生物信息学进行了16S—23SrDNA间区序列及其内所含tRNA基因的分析。2株坚强芽孢杆菌共得到11条16S—23SrDNA间区特征区带(包括300、400、500和600bp等)序列,ISR类型包括ISR0、ISRG、ISRIA和ISRGLV四种,其中ISR0、ISRG和ISRIA可能在坚强芽孢杆菌中普遍存在。相同类型的ISR序列具有较高的相似性(达52.2%—100.0%),而不同类型的ISR序列间差异较大。此外,ISR序列在靠近16S和23S区域存在不同长度的保守区域,可能成为针对坚强芽孢杆菌特异性检测的引物和探针的靶区,这也为坚强芽孢杆菌新的检测方法的建立奠定了基础。坚强芽孢杆菌的16S—23SrDNA间区序列及其多态性分析均为首次报道。

Using the primers targeting the conserved regions flanking the 3＇ end of the 16S and the 5＇ end of the 23S rDNAs, the 16S--23S rDNA intergenic spacer regions （ISRs） of two strains of Bacillusfirmus were amplified with poly- merase chain reaction （PCR）. The amplified products were cloned into pMD18-T vector and sequenced, subsequently. The sequences and their contained tRNA genes were analyzed by bioinformatics methods. The sequences of 11 typical bands of ISRs （the length including 300bp, 400bp, 500bp, and 600bp, etc.） were obtained from the 2 strains of B. firmus. Analyses suggested that these sequences contain 4 types of polymorphic ISRs, namely, ISR°, ISRG, ISRIA and ISRGLV. The types ISR°, ISRG, and ISRIA may widely exist in B. firmus. Furthermore, the comparative analysis showed the sequences of the same type of ISRs have higher identity percentage （varying from 52.2% to 100.0%）, while the sequences of different ISR types differentiate greatly. Additionally, the hypothetical conserved domains different in length flanking the 16S and the 23S rDNAs may be candidates for the targets, according to which the species-specific PCR primer set and/or detection probes are possibly designed, and will lay a foundation for developing a novel diagnostic method for B. firmus. To date, the 16S-- 23S rDNA sequences and their polymorphism of B. firmus are first reported.