Molecular characterization and mRNA expression of HIF-prolyl hydroxylase-2 (phd2) in hypoxia-sensing pathways from Megalobrama amblycephala.

Abstract

HIF-prolyl-hydroxylase-2 (Phd2), amember of the iron (II) and 2-oxoglutarate-dependent dioxygenase family, is one of the key enzymes in hypoxia-sensing pathways. In this study, thephd2cDNA sequence (1231bp), including an open reading frame (ORF) and encoding 358 amino acid residues was identified inMegalobrama amblycephala(Wuchang bream). The predicted Phd2 protein contained three conserved domains, MYND type zinc finger domain with critical regulatory activity, Fe(2+)-dependent 2OG-Fe (II) oxygenase superfamilydomain with prolyl hydroxylase function, and P4Hc (prolyl 4-hydroxylase alphasubunit homologues) domain for catalyzing proline hydroxylation. The real-time PCR results showed thatphd2mRNA was ubiquitously expressed in all detected tissues with higher levels in the peripheral blood, heart and brain, and all embryogenesis stages, especially in mid-blastula stage. In larvaeM. amblycephala, the expression trend of thephd2and hypoxia-inducible factor 1 alpha (hif-1α) mRNA was opposite during hypoxia with an increase (hypoxia for 4h) and then decrease (hypoxia for 12h) forphd2. Whereas in adult fish, thephd2mRNA appeared a transient increase under hypoxia for 4h (DO: 3.46±0.59mg/L), and dramatically reduced with further hypoxia exposure to 12h in the peripheral blood, muscle, head kidney, liver and brain, but showed an opposite expression trend in the heart and gill. The hif-1αexpression was contrary withphd2in the peripheral blood, while it gradually decreased in the heart, but increased in the liver with continuous hypoxia treatment. Additionally,hif-1αalso showed lower mRNA levels thanphd2in all detected tissues under normoxia and hypoxia conditions.