Molecular Characterization Of Two Glutathione Peroxidase Genes In Oryza Sativa

Glutathione peroxidases (GPX, EC1.11.1.9) are a group of enzymes that regulate the levels of reactive oxygen species in cells and tissues and widely distributed in animal, plant, fungi and bacteria. GPX is considered to play an important role in resistance to oxidative stress. In this paper, two GPX genes (OsGPX3andOsGPX4) were cloned from Oryza sativa. Studies of gene structure, phylogenetic analysis, expression pattern, protein structure and enzymatic activity were conducted and the main results were as followings:1. OsGPX3and OsGPX4genes were cloned from rice cDNA The length of OsGPX3and OsGPX4open reading frame were717and705bp, respectively.OsGPX3and OsGPX4genes encode two proteins with238and234amino acid residues, with a calculated molecular mass of25.84kDa and25.07kDa, respectively. Gene structure analysis showed OsGPX3and OsGPX4both contain six exons, and the length of the corresponding exons were similar, but the length of the corresponding introns were different. Protein sequence analysis revealed high similarity among all plant GPX, and three conserved Cys were found in GPX. Phylogenetic analysis showed OsGPX3and OsGPX4and other plant GPX grouped together, with high bootstrap. Protein structure modeling revealed OsGPX3and OsGPX4have typical TRX structure, including six (3sheets and four a helices.2. We investigated the expression pattern of rice OsGPX3and OsGPX4in different tissues (including root, stem, mature leaf, immature leaf and leaf sheath) under normal growth condition. Reverse transcription PCR revealed that the OsGPX3and OsGPX4were expressed in various tissues including root, stem, mature leaf, immature leaf and leaf sheath. This suggests that OsGPX3and OsGPX4were constitutive expression genes in O. sativa.3. The OsGPX3. and OsGPX4proteins were overexpressed in E. coli, and were purified by Ni-affinity chromatography. The OsGPX3and OsGPX4proteins showed enzymatic activities towards the substrates H2O2, tBOOH and COOH, with2.80±0.03,1.19±0.04,2.99±0.05μmol/min per mg for OsGPX3, and1.26±0.01,0.70±0.01、1.31±0.02μmol/min per mg for OsGPX4. OsGPX3showed higher enzymatic activities to three substrates than did OsGPX4, suggesting functional divergence between the OsGPX3and OsGPX4genes.By integrating sequence analysis, gene expression, enzymatic characterization and protein structure modeling, we identified two constitutively expressed genes named OsGPX3and OsGPX4from O. sativa, with broad substrate spectrum to peroxide. Our study suggested that GPX may play a unique role in plant oxidative stress resistance, and GPX may be good candidate for genetic improvement to enhance the plant oxidative stress resistance ability.