羊布鲁氏菌非编码小RNA分子BSR-2的预测与鉴定

Prediction and Identification of Small Non-coding RNA BSR-2 in Brucella melitensis

利用RT-PCR和RACE等方法对生物信息学预测的羊布鲁氏菌非编码小RNA(smallnon-codingRNA,sRNA)BSR-2进行了实验鉴定,并通过分析BSR-2在胞内生存缺陷株中的转录情况以及预测BSR-2的靶标基因对BSR-2的功能进行初步探讨.RT-PCR结果表明,BSR-2在羊布鲁氏菌的总RNA中存在转录本,而且在不同的应激条件下转录水平不同.RACE结果表明,BSR-2长224nt,位于Ⅱ号染色体的BMEII0742和BMEII0743之间的基因间区.进一步的实验结果表明,BSR-2可能与布鲁氏菌的胞内生存能力相关.

The small non-coding RNA （sRNA） BSR-2 in the pathogenic bacterium B.melitensis was predicted by bioinformatics approach.The expression of BSR-2 was examined under different stress conditions （acid shock,limited nutrition,and oxidative stress） simulating the intracellular environment of macrophage by reverse transcription-PCR （RT-PCR）,and the precise start and endpoint of BSR-2 were determined by using the 5＇ and 3＇ RACE （rapid amplification of cDNA ends） experiments.The transcriptional profiles of BSR-2 in B.melitensis strain deficient for intracellular survival were analyzed.The mRNA targets of BSR-2 were predicted by TargetRNA.RT-PCR analysis showed that BSR-2-encodes transcripts were detected under TSB4.0 （acid shock ）,GEM7.0 （limited nutrition ）,and H2O2 （oxidative stress） in B.melitensis,verifying the presence of BSR-2.The 5＇ and 3＇ ends of BSR-2 were mapped by RACE experiments,showed that BSR-2 was 224 nt.Sequence analyses of BSR-2 indicated that BSR-2 was located on the intergenic regions between BMEII0742 and BMEII0743,transcribed independently of the flanking ORFs,and highly conserved in Brucella species.The transcriptional profiles of BSR-2 were significantly influenced by the mutation of virB and vjbR,which played important role in the Brucella intracellular survival.And BSR-2 was predicted to have three mRNA targets, including the transcriptional regulator AraC involved in virulence gene control in B.melitensis.These results indicated that the small non-coding RNA BSR-2 was located on the chromosome Ⅱ of B.melitensis and associated with the intracellular survival of B.melitensis.