Regulation Of Radioresistance-related RecBCD By Small RNAs In Kineococcus Radiotoleran Under Radiation Stress

Kineococcus radiotolerans is a gram-positive,radio-resistant bacterium,which was isolated from a radioactive waste and it has an extreme resistance to strong alkali,high salt,high concentration of metal ions,high chemical toxicity,high osmotic pressure and ?-ray radiation resistant,which because of its efficient and accurate DNA repair system of K.radiotolerans.RecBCD pathway is one of the most important pathways of homologous recombination repair,which acts on double strand DNA break repair.Double-strand DNA breaks(DSBs)repair is vitally significant for cell activity and homologous recombination.sRNAs(small non-coding RNAs)are widely used as an important regulator of gene expression in bacteria and those sRNAs are usually lack of protein coding function.Nonetheless,sRNAs can work on the translation or degradation of mRNA and sometimes also can regulate the function of some protein directly.Regulation of gene expression by sRNA is mainly based on the short and incomplete complementary base pairing between sRNA and mRNA.According to qRT-PCR results of K.radiotolerans with different irradiation dose(0,2,4,6,8,10,12 kGy)for 4 h,it's informed that the expression of RecBCD was increased with the increase of irradiation dose,which indicated that RecBCD was associated with the radioresistance characteristics of K.radiotolerans.The result of bioinformatics showed that there were 5 sRNAs regulating RecBCD and they were dentified in this paper.Thick cell wall and growing cluster led to the inability to transfer exogenous DNA into K.radiotolerans.Thus,mucin was used to treat K.radiotolerans and suppressed the aggregation.However,it failed as well after the aggregation was suppressed.Therefore,Escherichia coli was used as an exogenous host and the sRNA and 5'UTR::gfp fusion plasmids were co-maintained in the same cell.The regulatory protential of sRNA expression on a translational target-gfp fusion was detected by the analysis of flow cytometry and Western blotting.The results of flow cytometry and Western blotting showed that there were no significant differences in fluorescence intensity and GFP expression between control group and co-transformation groups,which indicating that sKRA059,sKRA214,sKRA155,sKRA051 and sKRA125 had no interaction with 5'UTR of RecBCD in E.coli.