Cloning Expression And UVC Resistant Function Of RecO And RecR Gene From Kineococcus Radiotolerans

Kineococcus radiotolerans is a strongest ionizing radiation and UV tolerance,Gram-positive bacteria,and it can be survived in strong alkali,high salt,high metal ion concentration,high osmotic pressure and other extreme the environment.It is considered to be that the extreme resistance of K.radiotolerans is due to the efficient repair of genes damage.RecFOR pathway is one of the major homologous recombination repair of bacteria,which acts on DNA damage repair and mainly composed of RecF,RecO and RecR.So far,no data are available on the K.radiodurans RecFOR molecular regulation mechanism.In present study we constructed prokaryotic expression system for recO and rec R as the agents of RecFOR from K.radiodurans and tested the protective effect of transfection of recO and recR gene on oxidation.The purpose of this study is to experiment data,and observe the protective effect of the target gene when the host was exposed to ultraviolet radiation,and to provide experimental evidence for the study of subsequent UV biological protection.Physico-chemical properties of RecOR were analyzed by series of bioinformatics tools,and the results showed that RecO and RecR were stable.The secondary structure of RecOR contains high-proportioned irregular curl and?-helix,and?-helix less.3D model of protein tertiary structure of RecO and RecR was built by SWISS-MODEL with the method of homology modeling.This provided some help for further study of Rec FOR in K.radiodurans.PCR primers were designed from the GenBank database index.K.radiodurans gene sequence of recO and recR and the K.radiodurans genome DNA was used as the template for PCR amplification.The results are cloned into the pGEX-2T plasmid.Recombinant plasmids were transformed into the E.coli BL21(DE3).Optimization expression of induction conditions of RecO and RecR:Research the effects of protein expression with different concentration of IPTG,different induction time and temperature in order to determine the optimization expression of induction conditions of RecO and RecR.The expression of RecO and RecR protein had the highest expression level at 28?,0.1 mmol/L IPTG for 5 h and 30?,0.5 mmol/L IPTG for 2~3 h respectively.We analyzed anti-UV ability of RecO and RecR recombinant bacteria by different doses of UV irradiation.The survival ability of E.coli pGEX-2T-RecO,E.coli pGEX-2T-RecR,E.coli BL21(DE3)and E.coli pGEX-2T were inhibited after UVC irradiation.When the UV radiation dose was more than 8 J/m~2,the recombinant strains RecO and RecR started to show obvious repairing ability,and the survival rates were above the two groups of blank control,indicating that RecO and RecR proteins from K.radiotolerans can enhance UV resistance of E.coli BL21(DE3).The expression of target genes was detected by q RT-PCR at the gene level.The results showed that the expression of K.radiotolerans recR was higher 15 times than that of the K.radiotolerans recO.Transformation of recO from K.radiotolerans increased the expression of RecFOR related genes in E.coli BL21(DE3).In contrast,transformation of recR from K.radiotolerans had no significant influence on the expression of RecFOR related genes in E.coli BL21(DE3).The results indicated that recO and recR from K.radiotolerans increased the UV-resistance of E.coli BL21(DE3)through different regulatory pathways.The results of this study provide a scientific basis for the study of radiation-resistant mechanism of K.radiotolerans.