Cloning And Characterization Of Superoxide Dismutase And Glutathione Reductase Gene From Rhodotorula Mucilaginosa AN5

The impact of human activities on the global environment is increasing,and the pollution of heavy metals in environment is becoming ever more serious.Antarctic organisms suffer multiple stresses from high salinity,strong radiation,low temperature,heavy metals and so on,leading to accumulation of reactive oxygen species(ROS).Antarctic organisms could synthesize cold-active antioxidant enzymes(SOD,GR,POD and CAT,etc.)to scavenge ROS damage,eventually achieving the accumulation of heavy metals or detoxification.In this study,we primaryly explored the role of yeast stress adaptability of SOD and GR gene or protein from Rhodotorula mucilaginosa AN5,including gene cloning,bioinformatics analysis,recombinant protein expression,purification,protein characterization and heavy metal tolerance test.(1)A novel SOD gene from Antarctic yeast Rhodotorula mucilaginosa AN5 was cloned,sequenced,and then expressed in Escherichia coli.The open reading frame(ORF)of R.mucilaginosa AN5 SOD(namely RmFeSOD)gene was 639 bp in length,which encoded a protein of 212 amino acids with a deduced molecular mass of 23.52 kDa and a pI of 7.89.The natural state RmFeSOD was presented in the form of homodimers after gel filtration chromatography and SDS-PAGE analysis.Recombinant RmFeSOD showed high stability in the pH 1.0-9.0 after incubation 1 h.Also,it was found to still keep relatively high thermostability,and maintained more than 80% activity at 50 °C for 1 h.By addition of 1 mmol/L metal ions,the results indicated that Zn2+,Cu2+,Mn2+ and Fe3+ promoted RmFeSOD enzyme activity,while Mg2+ exhibited inhibitory effect.RmFeSOD showed relatively low tolerance to some compounds,such as PMSF,SDS,Tween-80,Triton X-100,DMSO,?-ME and urea and a certain tolerance to the reductant DTT.In the experiment of copper stress,the Rm FeSOD recombinant E.coli exhibited better growth than non-recombinant bacteria,which revealed that RmFeSOD might play a significant role in the adaptability of heavy metals.(2)A glutathione reductase gene from Antarctic yeast Rhodotorula mucilaginosa AN5 was cloned,sequenced,and then expressed in Escherichia coli.The open reading frame of R.mucilaginosa AN5 GR(namely RmGR)gene was 1500 bp in length,which encoded a protein of 499 amino acids with a deduced molecular mass of 54.8 kDa and a pI of 6.07.The recombinant RmGR showed the maximum activity at p H 7.5 and 30 °C.Recombinant RmGR showed good pH stability in the p H range of 3.0-8.0 after 1 h incubation.It was found to still keep relatively high thermostability,and maintained more than 60% activity at 40 °C for 1 h.In the experiment of copper and cadmium stress,the RmGR recombinant E.coli exhibited better growth than non-recombinant bacteria,which revealed that RmGR might play an important role in the adaptability of heavy metals.