Directional Transformatin And Function Identification Of High Activity Soybean MnSOD Gene

Superoxide dismutase (sod) is the most important of scavenging free radicals andantioxidant enzyme, is the cornerstone of the free radicals of life science research.This study aimed to modified the activity of superoxide dismutase in soybean by lowtemperature PCR. The superoxide dismutase were induced expressed inE.coli.Determining the enzyme activity and screening of high activity strains.Themain research contents and results are as follows.1、The target gene inserted into the cloning vector successfully: pREP5N-MnSOD as a template with MnSOD specific primers,the six amplified superoxidedismutase gene at low annealing temperatures were inserted into PMD18-T Vector*1builting a cloning vecter successfully.2、The expression vector pPM1-pPM6had been constructed.:Digesting thepurpose amplification at low temperature by PCR and the vector of pREP5N,connection and connecting the product into E.coli DH5α PCR and enzyme digestion,ultimately construction of expression vector pPM1-pPM6successfully.3、The analysis results Purpose gene sequences and protein structure:The purposeof the gene sequence compared with known gene sequence of soybean MnSOD, theanalysis results show that the genetic sequence consistency of86.57%on average,amino acid sequence homology of82.58%on average.Using protein structure analysissoftware to analyze its structure, and the results showed that the protein belongs toSOD-Mn superoxide dismutase (SOD) enzyme protein.4、Get the engineering strains of E.coli DH5α（pPM1）-E.coliDH5α（pPM6）with our fabrication: pPM1-pPM6plasmid will be expression vector into competentescherichia. The expressed product was identified by SDS-PAGE,we detected that theresults of the purpose Protein of relative molecular mass which was consistent withthe expected molecular mass. Using NBT to test the enzyme activity, aim proteaseenzyme activity is1.95times higher than ontrol bacteria on average. when annealingtemperature is28.7℃highest enzyme activity was2.32times of the original.PCR technique in low temperature is kind of effective method, the results forapplication will provide more technical support toproduct uperoxide dismutase,E.coli DH5α fermentation to product SOD has certain guidance for industrialization,as well.