Cloning And Functional Properties Of Rhodococcus Sp.NJ-530 DNA Photoremediation Enzyme

Photorehabilitation repair is an important DNA damage repair mechanism of organisms,which plays an important role in maintaining the continuity of genetic information and the integrity of genetic structure.Photorehabilitation repair refers to the use of light as a driving force to repair UV-damaged DNA under the action of DNA photolyase.Rhodococcus sp.NJ-530 is a bacterium that is isolated from Antarctic ice water and carries photolyase gene.Photolyase repair DNA damage caused by ultraviolet light has become one of the important ways to enhance its survival mechanism,due to long-term living in the Antarctic strong ultraviolet radiation,lower temperature environment.Therefore,it is meaningful to study the photorezyme repair activity of Rhodococcus sp.NJ-530 at the molecular level.In addition,the study can provide a basis for Antarctic bacteria to cope with the survival adaptability of strong ultraviolet radiation and other Antarctic species.In this study,the main research contents: the photolyase gene PHR of Rhodococcus sp.NJ-530 was obtained byPolymerase Chain Reaction(PCR)and codon optimized synthesis,and the bioinformatics analysis of PHR gene was carried out.The transcriptional regulation mechanism of Rhodococcus sp.NJ-530 photolyase gene PHR was studied by real-time quantitative PCR(qRT-PCR).To verify the function of PHR photolyase,the heterologous expression system was constructed to achieve heterologous expression inEscherichia coli BL21(DE3);Purification of the target protein by Ni-NTA affinity chromatography to obtain protein of higher purity;The repair activity of PHR photolyase in the expression strain and the in vitro repair activity were explored.For the first time,this paper reports the Rhodococcus sp.NJ-530 photolyasely genePHR.The full-lengthcDNA is 1146 bp,encoding 381 amino acids.The relative molecular mass of the predicted protein is 43.06 KDa,and the gene is uploaded to GenBank numbered MH844561;qRT-PCR results showed that PHR expression increased under UV light intensity of 90 ?W cm-2 and 5 °C,indicating that PHR gene is sensitive to UV and low temperature stress;The expressed protein was analyzed by SDS-PAGE and Western Blot.The results showed that the target protein was present in the cell disruption and the molecular weight was the same as the predicted molecular weight.The optimal expression conditions of PHR protein were pH 7.0,and IPTG concentration was 0.5mmol/l,and induction temperature was 15 °C.It was verified that the expression level of the target protein under the optimized conditions was basically consistent with the theoretical prediction value;after the inclusion body protein was revived and dissolved,the Ni-NTA chromatographic purification was carried out,and the results showed that the relative content of the target protein was increased and the content of the heteroprotein was decreased.Activity analysis experiments have shown that PHR photolyase have activity in vitro and in vivo,and can effectively repair UV-damaged DNA.