Cloning and characterization of gene encoding a Mn-containing superoxide dismutase in Eutrema halophilum

Abstract

A gene encoding Mn-containing superoxide dismutase (Mn-SOD), designated as ThMSD, was cloned from salt cress (Eutrema halophilum) by reverse transcriptase — polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The full length of ThMSD (acc. No. EF413171) is 1 047 bp with an open reading frame (ORF) of 693 bp. The deduced 231-amino acid polypeptide had a predicted molecular mass of 25.5 kDa, an estimated pI of 9.08, and a putative Mn-binding site. Recombinant ThMSD protein was expressed in Escherichia coli and characterized. The SOD activity of ThMSD was inactivated by sodium azide but not by potassium cyanide or hydrogen peroxide confirming that ThMSD is a Mn-SOD. Real-time PCR revealed that ThMSD was expressed in roots, rosette leaves, stems, stem leaves, flowers, and siliques. ThMSD mRNA reached the highest content in roots and its content increased when plants were treated with NaCl (in a concentration dependent manner), ABA, and subjected to drought. ThMSD was transformed into Arabidopsis and the stress tolerance properties of transgenic lines were assayed. The seeds of transgenic lines exhibited significantly higher germination rate under 100 and 150 mM NaCl than the wild type. The root growth of transgenic lines was affected less obviously than the wild type under 100 mM NaCl. The above results indicate that ThMSD played an important role in E. halophilum tolerance to environmental stresses, especially NaCl stress.