Identification of <i>GPD1</I> gene from yeast via fluorescence differential display-polymerase chain reaction (FDD-PCR)

Abstract

The main task of this work was to identify abiotic stress-induced gene(s) from yeast (Saccharomyces cerevisiae) and introduce it to a prokaryotic system to detect its effect on conferring tolerance to salt stress. Six isolates of yeast (S. cerevisiae) were evaluated under salt and osmotic stresses at concentrations of 2 M NaCl and 2 M sorbitol, respectively, in which one isolate was selected as the most tolerant against both stresses. Fluorescence differential display-polymerase chain reaction (FDD-PCR) was conducted for cDNAs after been exposed to 0 and 2 M NaCl for 0, 20, 40 and 60 min. After a number of DD-PCR runs, 350 fragments were observed, out of which 30 of them (9.14%) showed differential expression versus exposure times. They were classified into 12 patterns of gene expression. Three up-regulated DD fragments above 100 bp in size were chosen and cloned for subsequent molecular analysis and gene construction. DNA sequences were detected and subjected to homology searching via computer software. The results indicate that one DD fragment showed significant homology with a yeast DNA fragment on chromosome 4 expressing glycerol-3-phosphate dehydrogenase (GPD1); a NAD dependent key enzyme of glycerol synthesis essential for growth under osmotic stress. This fragment was chosen to recover full-length gene following the rapid amplification of cDNA ends (RACE) strategy, then gene was cloned and transformed into Escherichia coli. Expression of GPD1 gene was proven in transformed bacteria via northern blotting and glycerol-3-phosphate dehydrogenase enzyme activity. The overall results of stress tolerance for GPD1-transformed bacteria indicate the efficacy of utilizing the gene in conferring salt tolerance at the prokaryotic level.

Keywords: Salt stress, osmotic stress, cDNA, Northern blotting, rapid amplification of cDNA ends (RACE), GeneRacer