ISOLATION, IDENTIFICATION AND CHARACTERIZATION OF OsRGLP2 PROMOTER BINDING PROTEIN(S)

Abstract

Germin and germin-like proteins (GLPs) belong to cell wall glycoproteins that have shown asignificant resistance to detergent action, denaturation by heating, and degradation by proteases. GLPs expression is reportedly modulated during exposure to pathogens and abiotic stresses. Yet, little is identified about the regulatory mechanism of the GLP genes. The promoter of OsRGLP2 gene was isolated and cloned by Mahmood et al. (2007). This promoter showed strong expression ofthe GUS gene in transgenic tobacco during salinity, dehydration and wounding stresses. In this study, the regulatory cis-elements and their binding proteins for OsRGLP2 promoter are characterized. Various putative stress responsive cis-regulatory motifs and their specific binding proteins were identified by in silico analysis. DNA binding domains of OsWRKY71, OsDOF18 and OsMYB1 were cloned, overexpressed in E. coli and then purified by affinity chromatography using Glutathione Sepharose resin followed by cationic exchange chromatography. Electrophoretic mobility shift assays (EMSAs) have shown that recombinant OsWRKY71, OsDOF18 and OsMYB1 proteins were capable to interact with DIG labeled fragments of OsRGLP2 promoter containing W-box, AAAG and WAACCA motifs respectively. Binding was further confirmed by competitor EMSA and EMSA with mutant oligonucleotides. These regulatory elements were also active in binding with nuclear factors from rice nuclear proteins extract in vitro as confirmed by competitive EMSA. Expression analysis was performed to check the level of OsWRKY71, OsDOF18 and OsMYB1 against salt, cold, heat, wounding and drought stresses. Expression of OsWRKY71 was found to be induced in case of salt and cold stresses while OsDOF1 was expressed at relatively high level in response to salinity and drought. OsMYB1 expression was 23 fold higher in response to wounding which demonstrates the valueofOsMYB1 up-regulation in response to wounding stress inrice. In order to investigatethe functionof OsWRKY71, OsMYB1 and OsDOF18againstdiverse abiotic stresses, recombinant plasmids were subjected to transformationin E. coli and their effect on E. coli growth was analyzed. The E. colicells containing pGEX-OsWRKY71, pGEX-OsDOF18 and pGEX-OsMYB1 has shown different levels of tolerance against salt, drought, cold and heat stresses as compared to empty pGEX-4T1 vector. In silico characterization suggested the involvement of these proteins in protein-protein interaction. In conclusion, the positive response of OsWRKY71, OsDOF18 and OsMYB1 in abiotic stresses suggests their association with OsRGLP2 promoter and the importance of these proteins in providing protection to plants under abiotic stresses. Overexpression of OsWRKY71, OsDOF18 and OsMYB1 genes in crop plants may help in obtaining stress tolerant lines.