Molecular cloning, tissue distribution and expression analysis of a manganese superoxide dismutase in blunt snout bream Megalobrama amblycephala

Fish Shellfish Immunol. 2014 Jun;38(2):340-7. doi: 10.1016/j.fsi.2014.03.036. Epub 2014 Apr 13.

Abstract

The full-length mitochondrial manganese superoxide dismutase cDNA of blunt snout bream Megalobrama amblycephala (denoted as MamMnSOD) was identified in liver using homology cloning and rapid amplification of cDNA ends. The full-length cDNA of MamMnSOD consisted of 986 bp, with an open reading frame encoding 224 amino acids, a 58-bp 5' untranslated region and a 256-bp 3' untranslated region. The deduced amino acid sequences of MamMnSOD showed high sequence homology to mitochondrial MnSODs from crustaceans. Several motifs, including three mitochondrial MnSOD signatures, amino acid residues responsible for coordinating the manganese, and the putative active center, were almost completely conserved in the deduced amino acid sequences of MamMnSOD. The mRNA expression of MamMnSOD in the tissues of heart, liver, spleen, kidney, muscle, intestine, and gill was examined by quantitative real-time PCR; the highest expression was in the liver. Transcription of MamMnSOD was kinetically modulated in response to nitrite stress in liver and gill tissues. The purified recombinant MamMnSOD showed potent antioxidant activity. Polyclonal antibodies generated from the recombinant product of MamMnSOD were used to specifically identify the native protein in liver of M. amblycephala. Collectively, the findings of this study strongly suggested that MamMnSOD combats oxidative stress and cellular damage induced by nitrite, by detoxifying harmful reactive oxygen species in M. amblycephala.

Keywords: Megalobrama amblycephala; Mitochondrial manganese superoxide dismutase; Nitrite; Transcriptional analysis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Blotting, Western / veterinary
  • Cloning, Molecular
  • Cyprinidae / genetics*
  • Cyprinidae / metabolism
  • DNA, Complementary / genetics
  • DNA, Complementary / metabolism
  • Fish Proteins / chemistry
  • Fish Proteins / genetics*
  • Fish Proteins / metabolism
  • Gene Expression Regulation, Enzymologic*
  • Molecular Sequence Data
  • Oxidative Stress
  • Phylogeny
  • Real-Time Polymerase Chain Reaction / veterinary
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Sequence Alignment / veterinary
  • Superoxide Dismutase / chemistry
  • Superoxide Dismutase / genetics*
  • Superoxide Dismutase / metabolism
  • Tissue Distribution

Substances

  • DNA, Complementary
  • Fish Proteins
  • Recombinant Proteins
  • Superoxide Dismutase