Amplification and Molecular Characterization of DREB1A Transcription Factor Fragment From Finger Millet [(Eleusine coracana (L.) Gaertn]

Abstract

Studies have shown that several plant species posses DREB1A and DREB2A (Dehydration-Responsive Element Binding Protein) orthologs. DREB transcription factors, also called C-repeat binding factors (CBFs), are the transacting elements/ transcription factors first identified in Arabidopsis which bind to low-temperature and dehydration responsive element (LTRE/DRE) found in several dehydrin (Dhn) promoters as well as in promoters of other cold and drought responsive genes and involved in dehydration-, cold-, and salinity-regulated gene expression. In this study, a fragment of DREB1A ortholog named EcDREB1A has been amplified from finger millet (Eleusine coracana), an important drought-tolerant grain crop with a rich genetic diversity grown in semi-arid tropics. In the current study, a systematic approach has been taken to predict a theoretical primer for EcDREB1A based on the cloned Arabidopsis thaliana AtDREB1A and other DREB genes for orthologous gene identification from finger millet. Sixteen different but related nucleotide sequences based on AtDREB1A gene were retrieved from different databases. A highly conserved region of 287 bp was detected on multiple sequence alignments through claustalw2 program and a set of primers (forward and reverse) was predicted using Primer3plus and Net software on the basis of this conserved region, assuming ideal conditions for primer length, GC content, formation of primer-dimers, hairpin-loops etc. The amplified genomic fragment of EcDREB1A was found to be 536 bp long, with possible introns as per translational analysis. Longest detected ORF in the amplified EcDREB1A fragment encodes a putative protein of 84 amino acids rich in serine (13.10%) with a predicted molecular mass of 9.29 kDa. Multiple sequence alignment of this EcDREB1A fragment with other DREB genes revealed presence of 9 highly conserved amino acids. Allele mining of EcDREB1A gene fragment across selected 5 finger millet cultivars revealed no variations on nucleotide, probably due to narrow genetic base in the test materials. Identification of novel regulatory genes involved in abiotic stress tolerance and allele mining in a manner similar to this presented herein might lead to a better and quicker solution for improving stress tolerance in crop plants.