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Study on Cloning and Expression of Porcine CuZnSOD Gene and Its Relationship with Pork Quality and Anti-Oxidative Capacity

Author: DuJinFang
Tutor: ZengYongQing
School: Shandong Agricultural University
Course: Animal Genetic Breeding and Reproduction
Keywords: Pig CuZnSOD Clone Expression Analysis QRT-PCR Antioxidant Meat quality characteristics
CLC: S828
Type: Master's thesis
Year: 2011
Downloads: 42
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Abstract

With the the modern husbandry intensification of the continuous improvement of the dangers of oxidation in commercial pig production has a large number of apparent negative impact of live animals immune function and post-mortem meat characteristics. The antioxidant enzymes alleviate or suppress the negative effects of free radicals, improve the antioxidant capacity of commercial pigs, thereby improving the commercial pig meat. CuZnSOD (Copper Zinc Superoxide Dismutase), superoxide dismutase (SODs) one of the members of the family, is an important oxidative damage of the collective defense of metalloenzymes metastatic superoxide anion radical in maintaining oxygen free The yl balance plays an important role. In order to further knowledge and understanding of the the CuZnSOD gene structure and function, reveals CuZnSOD pig anti-oxidative function, look for meat quality traits associated molecular markers. In this study, using RACE (Rapid Amplification of cDNA End) and Genome Walking the method, cloned and sequenced the of Laiwu pigs CuZnSOD gene cDNA and the promoter region, the analysis of gene structure and function, and constructed the eukaryotic expression vector that can be expressed in cells. QRT-PCR to detect the CuZnSOD varieties and differences in the organization of the gene expression, to study the expression of the meat quality traits and meat quality antioxidant. This study is mainly divided into the following three parts: 1 of CuZnSOD gene cDNA sequence of full-length of 658 bp (GenBank accession number: GU944822) contains 76 bp 5'UTR and 120 bp of 3'UTR sequence. All CDS sequence of 462 bp, encoding 153 amino acids, a molecular weight of 15.9 kDa and an isoelectric point PI was 6.03. Genome Walking kit for amplification of the genome of a promoter sequence, obtained 1876bp fragment was amplified. The CuZnSOD gene encoding the amino acid sequence, the third amino acid residue is present at one O-glycosylation sites, and the first 86 amino acid residues present at an N-glycosylation sites. Α-helical secondary structure accounted for only 1.31%. Highly conserved during evolution, the coding region of homology with human, bovine, mouse and rat were 87.74%, 87.66%, 83.44% and 83.23%; amino acid sequence homology to 90.26% and 94.12%, respectively, 92.21% and 91.50%, respectively. The phylogenetic tree constructed based on protein sequence, indicating that the pig and cattle closest genetic relatives. 2 by RT-PCR method, Laiwu pig muscle total RNA as template to amplify the full-length cDNA coding sequence of the CuZnSOD gene using TA cloning technologies, inserted into the target fragments were identified in pUCm-T vector, recombinant plasmid was named pUCm-T-CuZnSOD. Then CuZnSOD further cloned into the eukaryotic expression vector pcDNA3.1 (). With liposomes after the sequencing, verify restructuring pcDNA3.1 () / of CuZnSOD plasmid transfection to goat hair follicle outer root sheath cells the CuZnSOD gene mRNA by QRT-PCR detection of the expression. Results: successfully constructed pcDNA3.1 () / of CuZnSOD eukaryotic expression plasmids, transfection goat outer root sheath cells, QRT-PCR test results show that the transfection CuZnSOD successfully expressed in the outer root sheath cells of the goat hair follicles. The eukaryotic expression plasmid pcDNA3.1 () / of CuZnSOD laid a foundation for further study CuZnSOD succulent antioxidant role. 3 at the mRNA level, CuZnSOD is a broad-spectrum expressed genes can be detected in the brain, heart, spleen, liver, kidneys, lungs, large intestine, small intestine, spinal cord, muscle, backfat and stomach, with a variety within CuZnSOD mRNA expression there are significant differences in the organization (P lt; 0.05), a higher amount of expression in the kidney, small intestine and lung, lower expression in heart and muscle tissue. The the CuZnSOD gene mRNA expression in muscle, backfat and liver three organizations there are obvious differences between the varieties (P lt; 0.05), and the CuZnSOD gene mRNA expression always Laiwu pigs gt; Roule black pig gt; Yorkshire . CuZnSOD mRNA expression higher the higher the SOD activity, MDA content is lower the higher the water holding capacity of the muscle, the more vivid flesh-colored, and the meat is more tender.

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