Cloning of manganese superoxide dismutase gene and expression analysis in response to ammonia and nitrite challenge in Nibea albiflora

Manganese superoxide dismutase (MnSOD), a transition-metal-containing antioxidant enzyme that dismutates the superoxide radicals to either ordinary molecular oxygen (O₂) or hydrogen peroxide (H₂O₂), widely existed in many aerobic organisms. In this study, the cDNA of MnSOD, identified from Nibea albiflora, was 958 bp in length including 5′-untranslated region (UTR) of 47 bp, 3′-UTR of 233 bp and an open reading frame (ORF) of 678 bp encoding a polypeptide of 225 amino acids. The deduced amino acid sequence analysis showed that MnSOD contains a putative signal peptide in the N-terminus (1-27 aa), four Mn binding sites (His 53, 101, 190 and Asp 186) and a Mn/Fe SOD signature sequence (186-193 aa). Phylogenetic tree analysis indicated that N. albiflora has the closest relationship with Larimichthys crocea, and was classified into the same cluster with other teleosts (Epinephelus coioides, Takifugu fasciatus, Paralichthys olivaceus, Danio rerio and Anguilla japonica). Quantitative real-time qRT-PCR analysis showed that the mRNA transcripts of MnSOD were detected in all examined tissues and the predominant distribution was in heart, followed by brain, liver, gill, kidney, intestine, stomach, head-kidney, muscle and swim bladder, and the minimum level was displayed in spleen. Moreover, during the acute toxic experiment of ammonia nitrogen or nitrite nitrogen, N. albiflora showed more sensitive to ammonia nitrogen, and the 96 h median lethal concentration (LC₅₀) value was found to be 20.23 mg/L for ammonia nitrogen (or 0.57 mg/L for non-ionic ammonia) with safe concentration (SC) values of 2.02 mg/L (or 0.06 mg/L for non-ionic ammonia) and 99.08 mg/L with SC of 9.91 mg/L for nitrite nitrogen, successively. Furthermore, the temporal expression of MnSOD was significantly up-regulated in the liver, gill and head-kidney of N. albiflora. The expression features of the MnSOD suggested its important role in scavenging oxygen free radicals caused by ammonia nitrogen/nitrite nitrogen and could be used as an early biomarker for detection of environmental pollution.