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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 salicomicum, 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. salicomicum, and it will be of great value to geneticists as well as evolutionary biologists. (C) 2017 The Authors. Production and hosting by Elsevier B.V. on behalf of King Saud University.