Molecular characterization and expression studies of Eucalyptus globulus stress-responsive gene DHN-10

Abstract

The Eucalyptus globulus is one of the important forest tree species which reveals enhanced tolerance to multiple abiotic stresses. The enhanced tolerance of E. globulus against low temperature is linked to the presence of enhanced accumulation of lysine-rich dehydrin proteins. The accumulation of dehydrin protein of Mr 10 kDa in response to multiple abiotic stresses prevents cells from dehydration. It is encoded by a novel dehydrin-10 (DHN-10) gene. In the present study, we have used the DHN-10 gene of E. globulus of Pakistani origin, which was cloned in a bacterial expression vector pET30(a) and sequenced. We have found the 315 bp long coding sequence of this gene which has been enrolled as Eucalyptus GZJ-2018 DHN-10 gene under GenBank accession number MG948256.1. The in silico studies have identified several differences of this gene from the earlier enrolled DHN-10 genes: the studied gene possessed higher amphipathic character because of the presence of five additional electrically charged amino acids (two positively charged histidine, one negatively charged glutamate, and two negatively charged aspartate residues) and one extra lysine residue. The studied DHN-10 gene has been successfully expressed in the BL21-DE3 expression strain of Escherichia coli and 10 kDa protein has been detected on the nitrocellulose membrane. Our study is the first report of the sub-cloning of the DHN-10 gene and its expression outside the Eucalyptus cell.

Keywords

• escherichia coli expression strain
• eucalyptus dehydrin proteins
• in silico analysis
• sub-cloning