cDNA cloning and tissue expression for l-gulonolactone oxidase gene in soft-shelled turtle Pelodiscus sinensis a species with the ability to synthesize ascorbic acid

The key enzyme required for ascorbic acid (vitamin C) biosynthesis in animals is l -gulonolactone oxidase (GLO). To understand molecular regulation of this enzyme in turtles, we cloned the complementary DNA (cDNA) for the GLO gene in soft-shelled turtle Pelodiscus sinensis . The P. sinensis GLO cDNA was 1639 bp long and consisted of a 29-bp 5′-untranslated region (UTR), a 287-bp 3′-UTR, and a 1323-bp open reading frame (ORF) encoding a 440-amino-acid protein with 50.6 kDa predicted molecular mass and 6.54 theoretical isoelectric point (p I ). Using MatGAT software, the amino acid sequence for P. sinensis GLO was analyzed against other known GLO sequences, and results revealed highest similarity (92.7%) and identity (81.6%) with red jungle fowl Gallus gallus . Phylogenetic analysis also revealed that the P. sinensis GLO amino acid sequence clustered together with red jungle fowl. Via both reverse-transcription polymerase chain reaction (RT-PCR) and enzyme activity analysis, we determined that GLO gene expression in soft-shelled turtle was kidney specific. The present study is the first to investigate the GLO gene and its tissue expression pattern in a reptile species.