Wang Huijuan, Huang Chunxiao, Chen Nan, Zhu Kecheng, Chen Boxiang, Wang Weimin, Wang Huanling
Key Laboratory of Freshwater Animal Breeding, Ministry of Agriculture, Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Ministry of Education, College of Fishery, Huazhong Agricultural University, 430070, Wuhan, PR China.
Key Laboratory of Freshwater Animal Breeding, Ministry of Agriculture, Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Ministry of Education, College of Fishery, Huazhong Agricultural University, 430070, Wuhan, PR China; Freshwater Aquaculture Collaborative Innovation Center of Hubei Province, 430070 Wuhan, PR China; Hubei BaiRong Improved Aquatic Seed Co., Ltd, 438800 Huanggang, PR China.
Comp Biochem Physiol B Biochem Mol Biol. 2015 Aug;186:28-35. doi: 10.1016/j.cbpb.2015.04.001. Epub 2015 Apr 11.
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.
缺氧诱导因子脯氨酰羟化酶2（Phd2）是铁（II）和2-氧代戊二酸依赖性双加氧酶家族的成员之一，是缺氧感应途径中的关键酶之一。在本研究中，在团头鲂（武昌鱼）中鉴定出phd2 cDNA序列（1231bp），包括一个开放阅读框（ORF），编码358个氨基酸残基。预测的Phd2蛋白包含三个保守结构域，具有关键调节活性的MYND型锌指结构域、具有脯氨酰羟化酶功能的Fe(2+)-依赖性2OG-Fe（II）加氧酶超家族结构域以及用于催化脯氨酸羟化的P4Hc（脯氨酰4-羟化酶α亚基同源物）结构域。实时PCR结果表明，phd2 mRNA在所有检测组织中均有广泛表达，在外周血、心脏和脑中表达水平较高，在所有胚胎发育阶段均有表达，尤其是在囊胚中期。在团头鲂幼鱼中，缺氧期间phd2和缺氧诱导因子1α（hif-1α）mRNA的表达趋势相反，phd2先升高（缺氧4小时）然后降低（缺氧12小时）。而在成鱼中，phd2 mRNA在缺氧4小时（溶解氧：3.46±0.59 mg/L）时出现短暂升高，在外周血、肌肉、头肾、肝脏和脑中随着进一步缺氧暴露至12小时而显著降低，但在心脏和鳃中表现出相反的表达趋势。外周血中hif-1α的表达与phd2相反，而在心脏中逐渐降低，但在肝脏中随着持续缺氧处理而升高。此外，在常氧和缺氧条件下，所有检测组织中hif-1α的mRNA水平也低于phd2。
