Study On Cloning And Expression Of Porcine CuZnSOD Gene And Its Relationship With Pork Quality And Anti-Oxidative Capacity

With the development of intensive modern husbandry, oxidations in the production of commercial pigs have appeared to be harmful of the immune function and meat quality. Antioxidant enzymes can alleviate or inhibit the negative effect of free radical, to improve the antioxidation ability, but also improve meat quality. Copper zinc superoxide dismutase (CuZnSOD) is a member of superoxide dismutase (SODs) family, is an important metal enzyme to defense oxidative damage. It can be ambulant to clear superoxide radical, and plays an important role in maintaining the balance of oxygen free radicals. In order to understand the structure and function of CuZnSOD gene, reveal the effect of the anti-oxidant in swine, and find the molecule marker correlated with meat traits, the cDNA and promoter of CuZnSOD gene were cloned and sequenced from muscles and ears of Laiwu black swine by RACE (rapid amplification of cDNA end) and Genome Walking techniques; and the eukaryotic expressing vector expressed in cells was constructed. The structure and function of CuZnSOD were analyzed by bioinformatics, and the gene expression profile in different tissues was examined by real-time PCR. Our research is mainly divided into 3 parts as follows:1. The results showed that the full sequence of CuZnSOD cDNA is 658 bp (GU944822), containing 76 bp sequence of 5′UTR and 120 bp sequence of 3′UTR, and coding region (CDS, 462 bp) encodes 153 amino acids. The isoelectric point (pI) of the protein is 6.03, and the molecular weight is 15.9 kDa. We use the total DNA of Laiwu ears as templates to amplify the promoter of CuZnSOD gene by the Genome Walking kit, and get the sequence of 1877bp. There were one O-glycosylation site at the third amino acid and one N-glycosylation site at the eighty-fourth amino acid. The percentage of alpha helix was 1.31%. The alignment similarities of the CDS sequence of swine CuZnSOD with those of cattle, human, rat, and mouse were 87.74%, 87.66%, 83.44%, and 83.23%, and the similarities of amino acid sequence were 90.26%, 94.12%, 92.21%, and 91.50%, respectively. CuZnSOD possesses the typical metal binding ligands (GFHVHQFGDNT). The phylogenic tree based on CuZnSOD protein sequence detected the closest relationship between swine and cattle.2. We use the total RNA of Laiwu muscle as template to amplify the whole length of cDNA fragment of CuZnSOD gene by PCR. TA cloning strate-gy was used to insert the target fragments into pUCm-T vec-tor. The recombinant plasmid was identified and noted as pUCm-T-CuZnSOD. Then, CuZnSOD was subcloned into pcDNA3.1(+), a eukaryotic expression vector. The plasmid of pcDNA3.1(+)/CuZnSOD was sequenced and was introduced into outer root sheath cell line by Lipofectamine TM 2000. The expression of CuZnSOD was detected by RT-PCR and QRT-PCR. Results: The eukaryotic expression plasmid pcDNA3.1(+)/CuZnSOD was successfully constructed. The recombinant eukaryotic expression vector of pcDNA3.1(+)/CuZnSOD has been constructed successfully which could express CuZnSOD, providing a tool for further gene therapy study. Results: we construct the pcDNA3.1(+)/CuZnSOD eukaryotic expressing vector. Moreover, we successfully express the goal gene. And it has laid a good experiment foundation for further research about the function of CuZnSOD.3. CuZnSOD mRNA is a broad-spectrum expression gene, which was detected in brain, heart, spleen, liver, kidney, lung, large intestine, small intestine, spinal cord, muscle, backfat, and stomach. In particular, high expression levels of CuZnSOD mRNA were detected in kidney, small intestine and lung, but low expressions were observed in heart and muscle tissues.