Molecular cloning and expression analysis of an Mnsuperoxide dismutase gene in sugarcane

Abstract

Superoxide dismutases (SODs) play an important role in stress-tolerance in plants. In this study, for the first time, a full-length cDNA sequence of MnSOD gene, termed as Sc-MnSOD (GenBank accession number: GQ246460), was obtained in sugarcane. Sequence analysis revealed that Sc-MnSOD gene was 919 bp long, including a 702 bp ORF, the 5’ UTR of 99 bp and 3’UTR of 118 bp. It encoded the 233 amino acid residues with a molecular weight of 25.3 KD and isoelectric point of 7.11. Protein domain prediction indicated that besides the conserved domain in MnSOD, Sc-MnSOD also had the signal peptides at the sites of 1 to 26 aa at the N-terminus. With SubLoc v1.0, Sc-MnSOD was localized in the mitochondria. In homology analysis, the MnSOD genes from different plant species were rather conservative. SDS-PAGE analysis and enzyme activity assay showed that the prokaryotic expression product was a fusion protein with SOD enzyme activity of 726 U·mg^-1. Real-time qPCR analysis demonstrated that the expression of Sc-MnSOD gene was greatly induced by the Ustilgao scitaminea, firstly induced and then inhibited by H₂O₂, and slightly influenced by SA, which suggested that it may play a role in the clearing of active oxygen and thus in disease resistance mechanism in sugarcane.

Key words: Reactive oxygen species, Saccharum officinarum, Mn-superoxide dismutase, prokaryotic expression, real-time quantitative PCR.