The Effect Of PprM And It’s DNA Binding Domain Gene Knockout On Radiation Resistance Of Deinococcus Radiodurans

PurposeDeinococcus radiodurans has a strong ability to resistant radiation and variousother extreme environments. Some researcher postulated that the pprM gene playedan important role for D. radiodurans in radiation-resistance.To analyze the functionof pprM and its DNA binding domain in D.radiodurans radiation resistance, weconstructed pprM gene and pprM DNA binding domain fragment knockout strains,DR△pprM and DR1pprM△CSD. The resistance ability under extreme environmentof mutant strains was compared with the wild type.Methods1.Physicochemical properties, advanced structure and biological function ofpprM, pprM DNA binding domain and PprM protein was analyzed by bioinformaticssoftwares.2. pprM and pprM DNA binding domain was deleted in D. radiodurans byoverlap PCR and homologous recombination in vivo. Survival ratio of mutant strainsand wild type was investigated after challenged with UV radiation, MMC andhydrogen peroxide (H2O2).Results1. Results of bioinformatic analysis showed that PprM is a DNA binding proteinwhich contains a Cold-Shock DNA-binding domain (DNA binding domain). Furtherresearch revealed that pprM is highly homologous with cspA. As a transcription factor,CspA protein regulates the DNA repair related gene expression by binding with thepromoter of these genes.2.Consider Deinococcus radiodurans Genomic DNA as a template, pprM gene3’end fragment and5’ end fragment were amplified by PCR, and then the PCR productwas used as a template to amplify a fragment with the pprM ORF deleted (△pprM) by overlap PCR. A fragment with pprM DNA binding domain deleted (pprM△CSD)by same method. The fragments of△pprM and pprM△CSD was connected with thesuicide plasmid pk18mobsacB, and the recombinant vector was transformed into E.coli JM109for proliferation, screened by sucrose. Transformation was identified byrestriction enzyme digestion and DNA sequencing. The recombinant vector was thentransfered into D. radiodurans, screened twice to obtain pprM and pprMDNA-binding domain gene knockout D. radiodurans strains. Survival ratio of mutantstrains and wild type was analyzed following MMC, Ultraviolet irradiation and H2O2treatment. Results showed that survival ratio of the mutant strains was much lowerthan that of the wild type.Conclusions1. We speculated that pprM function as a transcriptional factor in D. radioduransradioresistance.2. The△pprM and pprM△CSD mutant D. radiodurans strains wereconstructed successfully.3. The ability of mutant strains resisted for UV radiation and other extremeenvironments decreased, suggesting pprM and its DNA binding function played animportant role in radioresistance of D. radiodurans.