Identification Of Noncoding Small RNA And Its Influence On Growth In Mycobacterium

ObjectiveMicroRNA are18-30nt, non-coding single-stranded RNA, were generated by miRNA precursor (70-80nt), which has stem-loop structure. They can target at the3’UTR of target gene, and induce translation inhibition or gene degradation. MicroRNAs (miRNAs) have mufti-functions. They are widely found in plant, mammalian, drosophila melanogaster, C. elegans, and viruses, and involved in growth, development, tumor origin, metastasis, invasion and programmed cell death.Here we identified noncoding small RNA in mycobacterium by bioinformatics and molecular biology, and explore their function on growth.MethodsFirst, the total RNA was isolated from mycobacterium, then small RNA was isolated from the total RNA, small RNA (18-30nt) was isolated by denatured RNA page,5’terminate was dephosphorylated, and3’terminate was ligased to adaptor, then5’terminate was phosphorylated, and also ligased to adaptor, RT-PCR was carried out, and PCR product was harvested, then the PCR product was ligased to TA vector, and analyzing was done. Next, we blasted the sequences with the genome of mycobacterium, those totally match with genome are predicted by mfold for the RNA secondary structure, and then, Northern boltting was carried out for confirmation in the total RNA of BCG. Finally, we blocked the noncoding small RNA and investigated the influence on growth.Results1. We obtained20small RNA range from18-30nt by cloning and sequencing.2. We identified these small RNA in the genome, and predicted the second structures by bioinformatic methods.3. We transformated antisence oligo into the mycobacterium, and found it could enhance the growth of mycobacterium.ConclusionOur results firstly explored noncoding small RNA (similar with miRNA) in the mycobacterium, and found some small RNAs had influence on the growth of mycobacterium. Studying noncoding small RNA in the mycobacterium can board the studies of miRNA in the nature, and explore its function, and provided a promising view of treating tuberculosis by gene therapy.