| Megalobrama amblycephala(Wuchang bream) has been cultured as an economically important freshwater fish in China. And it often suffers from hypoxic condition during pond culture, especially in hot season, resulting in the limitation of M. amblycephala production development. Further to understand the hypoxia regulatory mechanism of fish, we analyzed the effects of hypoxic stress on M. amblycephala physiological and biochemical characteristics(including antioxidant parameters, hematology parameters, and concentration of glucose and lactic acid). In addition, we cloned and characterized the M. amblycephala phd2 sequence and analyzed its expression under hypoxic condition. Moreover, we detected the expression of some genes involved in hypoxia signaling pathway under hypoxic stress. The main results are represented as follow: 1) The effects of hypoxia on M. amblycephala physiological and biochemical parametersThe analysis of the antioxidant enzyme(including T-AOC, SOD and GSH) activities showed that the T-AOC activity in serum increased in a time-dependent manner under hypoxia exposure, but was contrary in liver. In addition, with the decrease of the dissolved oxygen concentration, SOD activity in head kidney increased and then decreased, and GSH activity gradually reduced in intestine, liver and spleen. When the oxygen concentration reached 1.80 ± 0.55 mg/L, lactic acid and glucose accumulated in serum to supporting anaerobic metabolism. On the other hand, under hypoxic condition, the cell surface area of erythrocytes increased, and the proportion of thrombocytes and monocytes increased, but the proportion of lymphcoytes and eosinophils decreased. 2) The hypoxia effects on the expression of hypoxia regulated genes of M. amblycephalaThe results based on real-time PCR showed that under hypoxia condition, M. amblycephala glut2 m RNA level increased in head kidney, liver and gill, but decreased significantly in heart and peripheral blood, and the glut2 m RNA level increased then decreased in brain with continuous hypoxia treatment. The expression of ldh A in peripheral blood and muscle gradually decreased under hypoxia condition, but expressed contrarily in liver. In normal condition, the expression of both hb-α and hb-β in peripheral blood and heart exceeded in gill, muscle, liver, head kidney and brain. The expressions of hb-β in different tissues were far less than the expressions of hb-α. The hb(both hb-α and hb-β) m RNA levels in peripheral blood decreased and increased in liver and gill after hypoxia exposure. 3) Molecular cloning and sequence analysis of M. amblycephala phd2In this study, the phd2 c DNA sequence(1231 bp), including an open reading frame(ORF)(1077 bp) and encoding 358 amino acid residues was identified in M. amblycephala. The predicted Phd2 protein contained three conserved domains, MYND type zinc finger domain, Fe2 +-dependent 2OG-Fe(II) oxygenase superfamily domain, and P4Hc(prolyl 4-hydroxylase alpha subunit homologues) domain. Phylogenetic tree analysis showed that M. amblycephala Phd2 clustered together with other cyprinidae fish with high amino acid sequence similarity, especially with Crucian carp(89%). Phds generally fell into three distinct clades, and Phd2 and Phd3 were clustered together, suggesting that Phd2 probably shared the evolutionarily closer relationship with Phd3 than with Phd1. 4) M. amblycephala phd2 expression profiles under hypoxic conditionThe real-time PCR results showed that phd2 m RNA was ubiquitously expressed during embryonic development with highest level detected at 16 hpf(hours post-fertilization, for the mid-blastula stage). After hatching, the phd2 m RNA constantly expressed and gradually decreased. Additionally, M. amblycephala phd2 was widely expressed in heart, blood, muscle, head kidney, liver, brain and gill with higher levels detected in peripheral blood, heart and brain. And hif-1α showed lower m RNA levels than phd2 in all detected tissues under normoxia and hypoxia conditions.The hypoxia-reoxygenation analysis of M. amblycephala larvae revealed that the expression level of phd2 increased after 4 h hypoxia exposure and subsequently decreased after 12 h, while the opposite trend was observed for hif-1α. Noticeably, phd2 and hif-1α showed constant expression trend during reoxygenation, which is increasing during reoxygenation after 4 h hypoxia exposure and decreasing during reoxygenation after 12 h hypoxia exposure. In addition, in peripheral blood, muscle, head kidney, liver and brain, the phd2 m RNA increased under 4 h hypoxia(DO: 3.46 ± 0.59 mg/L) exposure, and dramatically reduced after another 10 h hypoxia exposure(DO: 1.8 ± 0.55 mg/L), while opposite trend was observed in heart and gill. The hif-1α expression was on the contrary with phd2 in peripheral blood, while it gradually decreased in heart, but gradually increased in liver along with continuous hypoxia treatment. |
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