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

HIF-prolyl-hydroxylase-2 (Phd2), a member of the iron (II) and 2-oxoglutarate-dependent dioxygenase family, is one of the key enzymes in hypoxia-sensing pathways. In this study, the phd2 cDNA sequence (1231bp), including an open reading frame (ORF) and encoding 358 amino acid residues was identified in Megalobrama 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 superfamily domain with prolyl hydroxylase function, and P4Hc (prolyl 4-hydroxylase alpha subunit homologues) domain for catalyzing proline hydroxylation. The real-time PCR results showed that phd2 mRNA 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 larvae M. amblycephala, the expression trend of the phd2 and hypoxia-inducible factor 1 alpha (hif-1α) mRNA was opposite during hypoxia with an increase (hypoxia for 4h) and then decrease (hypoxia for 12h) for phd2. Whereas in adult fish, the phd2 mRNA appeared a transient increase under hypoxia for 4h (DO: 3.46±0.59 mg/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-1α expression was contrary with phd2 in the peripheral blood, while it gradually decreased in the heart, but increased in the liver with continuous hypoxia treatment. Additionally, hif-1α also showed lower mRNA levels than phd2 in all detected tissues under normoxia and hypoxia conditions.