Characterization of the glyceraldehyde-3-phosphate dehydrogenase gene from the desert plant Haloxylon salicornicum using RT-PCR amplification and sequencing

Reverse transcriptase polymerase chain reaction (RT-PCR) is the most common molecular downstream application assay used to assess both quality and quantity of mRNA transcript, which heavily relies on using a sequence of an endogenous gene as a reference. In organisms with unknown genome sequence, designing of mRNA-specific PCR primers based on available sequence information of other species is an extremely useful approach to isolate homologous/orthologous genes. Here we report the isolation of a partial coding sequence of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene ortholog in Haloxylon salicornicum, a desert plant native to Kuwait, by designing a non-degenerate consensus primer pair based on conserved regions generated by a multiple alignment of orthologous GAPDH DNA sequences derived from closely-related species. Using both total RNA and genomic DNA extracted from leaf-stem segments, we proved that the designed primer pair is cDNA/mRNA specific, and there was no evidence of non-specific signals. Sequence and bioinformatics analyses of the obtained DNA fragment and its corresponding deduced amino acid sequence strongly suggested that it is a member of the cytosolic GAPDH family and that it is highly conserved. Based on the DNA sequence of the above-mentioned fragment, we also describe the RT-PCR amplification of a small-sized amplicon using a primer-template mismatch at the 3′-end region. The amplified products will be useful for PCR-based assays and gene expression studies of H. salicornicum, and it will be of great value to geneticists as well as evolutionary biologists.