CDNA cloning, characterization and expression analysis of manganese superoxide dismutase in Ulva prolifera

Abstract

Superoxide dismutase (SOD) is an important antioxidant protein that protects organisms against various oxidative stresses by eliminating hydrogen peroxide. In the present study, a full-length cDNA sequence of manganese superoxide dismutase (MnSOD) from the Ulva prolifera (denoted as UpMnSOD) was cloned by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA end method. The UpMnSOD cDNA sequence contained 955 bp with an open reading frame of 699 bp encoding 232 amino acid residues. The cDNA contained a 5′-untranslated region (UTR) of 29 bp nucleotides and a long 3′-UTR of 227 bp nucleotides. The calculated molecular mass was 25.7 kDa, and the estimated isoelectronic point (pI) of this protein was 6.83. Histidine (His) 59, His103, His194, and aspartate (Asp) 190 were found to be activated sites in UpMnSOD. Multiple alignment analysis revealed that the deduced amino acid sequence of MnSOD shared high identity (86 %) with Ulva fasciata. A phylogenetic tree construct indicated that UpMnSOD clustered into one subgroup with the MnSOD from U. fasciata. Quantitative real-time analysis revealed that UpMnSOD expression was induced at both 35 and 5 °C. The result indicated that UpMnSOD expression levels reached a maximum point of 2.63-fold (P < 0.05) and 1.28-fold compared to the control at 25 °C after 6-h treatment at 35 and 5 °C, respectively, and that long-time heat shock and cold treatment decreased the mRNA expression levels of MnSOD. Furthermore, UpMnSOD expression increased after 15 °C treatment, a 2.4-fold increase after 12-h treatment. Treatment with 1 mmol·L−1 salicylic acid (SA) at 35 °C also increased the gene expression of UpMnSOD and reached a maximum of 1.48-fold of the control after 6-h treatment, followed by a gradual decrease. However, the gene expression level of UpMnSOD increased rapidly within 3 h then decreased quickly under SA 5 mmol·L−1 + 35 °C. These data indicate that MnSOD was perhaps involved in the acute response against temperature stress, and salicylic acid could alleviate the high temperature stress effect on U. prolifera.