Cloning and expression analysis of MuNAC4 transcription factor protein from horsegram (Macrotyloma uniflorum (Lam.) Verdc.) conferred salt stress tolerance in Escherichia coli

Abstract

The NAC gene family encodes plant-specific transcription factors which play diverse roles in abiotic stress responses of plants. NAC TFs reported to be involved in the regulatory pathway of multiple abiotic stresses. In the present study, a salt stress-inducible NAC gene, named MuNAC4 (Macrotyloma uniflorum NAC4) from horsegram was isolated, cloned, characterized and studied its expression. Real-time PCR expression analysis showed up-regulation of MuNAC4 in horsegram across salinity, cold, drought and dehydration stress conditions. However, salt stress resulted a sixfold increase in MuNAC4 transcript levels. The involvement of MuNAC4 in abiotic stress tolerance was investigated by cloning MuNAC4 gene in expression vector pET28a and transformation in Escherichia coli BL21 (DH3). The apparent molecular weight of the recombinant protein was found to be 38.3 kDa as evident from SDS-PAGE analysis. The functional role of MuNAC4 in Escherichia coli confers the tolerance against salt (6 % NaCl), heavy metal (100 mM CuSO4) and water stresses (6 % PEG). The E. coli cells transformed with pET28a + MuNAC4 was showed better survival ratio and higher growth rates under NaCl stress than other abiotic stress conditions when compared to control cells (BL21/pET28a). This provides the experimental evidence that MuNAC4 protein enhance salt stress tolerance of E. coli cells suggesting their suitability as candidates for genetic manipulations for enhanced crop tolerance to salt stress and other abiotic stresses.