The Construction Of Deinococcus Radiodurans Ppri Gene Site-specific Integration Yeast And Research Of Its Extreme Resistance

Background and purpose: Deinococcus radiodurans is one of the most radioresistant organismsknown to date on the earth. Meanwhile, it is also extremely resistant to ultraviolet (UV) radiation,hydrogen peroxide, desiccation and a variety of DNA damaging agents. Studies showed that thesemultiple resistant phenotypes were attributed to its exceptional efficient DNA repair ability. Animportant repair gene pprI has been found in DR. It is a switch gene to start the expression of otherextreme resistance genes.Therefore, in this research, a yeast with DR-pprI gene transformed the thegenome was constructed; the change of extreme resistance of pprI gene site-specific integrationyeast was analyzed. This research will make further study of the DR pprI gene expression innon-radioresistant organisms.Methods:1. Based on the methods of polymerase chain reaction in vitro and homologous geneticrecombination in vivo, the DR-pprI gene was transformed into genome of yeast. Usewestern blot to detect expression of DR-pprI in yeast.2. Research the survival rate of the yeast dealt with UV and H2O2. Analyze the resistanceof radiation and oxidation effect of expression of DR-pprI in yeast; make further studyof the DR pprI gene expression in non-DR organisms.3. Use gas chromatography to detect the content of ethanol in pprI transformed yeastfermentation product, analyze the change of ethanol fermentation capacity of yeast withDR-pprI expression. Research the effect of DR-pprI expression to ethanol fermentationcapacity of yeast.Results:1. The connection product of three fragments-PGK1promoter, pprI gene and ADHIterminator was amplified successfully. Transformed the connection product into YIp5,single-enzyme digested the transformed plasmid and the pprI site-specific integrationyeast was successfully constructed. PprI protein was good expression by western blot.2. PprI expression yeast and wild type was analyzed after treated with UV irradiation andH2O2, the survival rate of PprI expression yeast were higher than the wild-type yeast(P<0.05).3. Result of gas chromatography showed ethanol fermentation product of DR-pprItransformed yeast was higher than wild type yeast.Conclusion:1.The DR-pprI site-specific integration yeast was constructed successfully.2.The expression of DR-pprI can enhance the resistance to UV and H2O2ofeukaryotic yeast.3. The expression of DR-pprI can enhance the ethanol fermentation capacity of yeast.