Chicken Manganese Superoxide Dismutase Cdna Cloning And Complete Sequence Analysis

As an essential trace element, mangnese (Mn) is of particular importance for chickens. Previous studies by our laboratory demonstrated that MnSOD in heart was affected by dietary Mn levels in practical corn-soybean meal basal diets for broilers and laying hens, and that Mn deficiency caused the reductions of MnSOD activity of heart and then resulted in the abnormalities of heart mitochondrial and endoplasmic reticulum ultrastructures in broilers. To further promote deep investigations on manganese nutrition of animals, this study identified and reported the entire nucleotide sequence of chicken MnSOD cDNA which has not been reported before. Firstly, a 1107 bp cDNA fragment encoding MnSOD from broiler chicken heart was amplified by 3?RACE ( rapid amplification of cDNA ends ) with degenerate primers designed on the N-terminal amino acid sequence of chicken MnSOD, and the amplified fragment was cloned and sequenced. The 3?RACE fragment from chicken MnSOD cDNA includes 597 bp of major coding region, and 411 bp of 3?untranslated region. Secondly, the 5?terminal region of the MnSOD transcript was determined by using 5?RACE procedure. The 5?RACE fragment is 521 bp in length, including 446 bp of coding region, 25 bp of 5?untranslated region, and 352 bp of overlapped region with 3?RACE fragment. Thirdly, the entire MnSOD cDNA sequence was obtained by combining the 3?RACE fragment with 5?RACE fragment. The final results showed that the MnSOD cDNA in chicken heart is 1108 bp in length, including 25 bp of 5?untranslated region, 675 bp of coding region and 408 bp of 3?untranslated region. The coding region encodes a peptide of 224 amino acid residues, in which there is a signal peptide of 26 amino acids and a mature peptide of 198 amino acids. The molecular weight of the mature peptide(subunit) is 22 kD, and its predicted isoelectric point is 6.88. The entire chicken MnSOD monomer chain forms two domains as predicted by PredictProtein i.e. the N-terminal domain dominated by 揳ll a -helix? and the C-terminal domain with an ?a + ~ structure. The predicted result is very similar to the known Tthermohilu.s MnSOD monomer second structure. Comparisons of the deduced amino acid sequence with those of human, rat, C.elegans, D.melanoga.ster and Tthermohilus show the amino acid homology of 82.4%, 84.7%, 62.4%, 59.3%, and 42%, respectively. Lastly, the chicken抯 entire MnSOD cDNA was amplified by End-to-end PCR with terminal primers designed on the entire nucleotide sequence of chicken MnSOD, and the entire cDNA copy was cloned into pUCm-T vector. The forward and reverse full-length cDNA clones named pCMSODF and pCMSODR were successfully obtained.