Structural And Functional Studies Of Hypoxia Inducible Factors In Megalobrama Amblycephala

Blunt snout bream (Megalobrama amblycephala) is an important herbivorous economic fish, because of its easy to raise and delicious. The blunt-snout bream is an extremely hypoxia-intolerant fish. It will cause mass death and great economic losses under hypoxic conditions, which restricts the improvement of its production. HIF (Hypoxia-inducible factor, HIF) play a key role in the physiological response to hypoxia in animals, and its activity determined mainly by theαsubunit. HIF activates the transcription of oxygen-sensitive genes, such as EPO, IGFBP-1, VEGF and so on. To maintain survival under hypoxia condition. Present three types ofαsubunit have been found in fish, they are HIF-1α, HIF-2αand HIF-3α. But their molecular structure, expression patterns and regulatory mechanisms under hypoxia is still unclearly. So research on the structure and function of HIF-αs is important to explain molecular mechanism of bream response to hypoxia,In this study, we cloned the full-length cDNA of the bream HIF-1α, HIF-2αand HIF-3αby RT-PCR and RACE methods. The sequences of the three HIF-αs is 3815bp, 3121bp and 3059bp respectively, the open reading frame is 2325bp, 2508bp and 1932bp, encoding 774,835 and 643 amino acids. Sequence analysis showed that three bream HIF-αgenes belong to the bHLH family. HIF-1α, HIF-2αand HIF-3αN terminal contain bHLH and PAS (PAS-A and PAS-B) domain, which can bind target DNA andβ-subunit, and the three HIF-αs have high similarity in N terminal domain, reaching 56.4% 81.8%; HIF-1αand HIF-2αC terminus have N-TAD and C-TAD domains, which associated with activation of target gene transcription, but HIF-3αonly containing N-TAD domain, and the similarity of C terminal domain between three HIF-αs was only 39.3% low 57.1%. Analysis HIF-1α, HIF-2αand HIF-3αmRNA expression patterns of bream in different stages of embryonic development and tissues of adult fish by RT-PCR and whole embryo in situ hybridization. In the normoxic state, the expression pattern of bream HIF-1αand HIF-3αin different stages of embryonic development was relatively constant; but the HIF-2αexpression levels higher than the other phase in 8hpf, 28hpf, 48hpf and 52hpf. HIF- 1α, HIF-2αand HIF-3αexpression were detected in all tissues of bream adult fish. HIF-1αhas high expression level in the liver, gill and testis; HIF-2αin addition to other than the intestine and muscle tissues have higher expression; and HIF-3αexpression level in all tissues are stable. After hypoxic stress the HIF-2αmRNA in liver and kidney were significantly increased 910% and 320%, no significant changes in brain; HIF-3αmRNA expression were significantly increased 218%, 603% and 293% ,respectively in brain, liver and kidney; but HIF-1αmRNA levels in the corresponding tissues did not change significantly. The results implied that bream HIF-1α, HIF-2αand HIF-3αplay different physiological functions in the process of growth and development and hypoxic response.To investigate the functions of bream HIF-3α, we construct pCS2-HIF3α-eGFP expression vector and transfected to hela cells. The result of Bream HIF-3αprotein subcellular distribution showed that HIF-3αprotein was in the cytoplasm and nucleus, but concentrated in the nucleus. By site-directed mutagenesis technology two conserved proline in bream HIF-3αODD domain was mutated to alanine. The result of overexpression double-mutant HIF-3αmRNA in bream embryos were showed that overexpression of mutant HIF-3αmRNA don't effect embryonic development of bream. In this study we also constructed a transgenic vector pTgf2-zfβ-actin-HIF3α^Δ409/509system based on Tgf2 transposon, and do the transgenic experiment on bream. This study provides an experimental basis for exploring the molecular mechanisms of bream physiological adaptation to hypoxic environment, and laid a theoretical foundation for improving the capacity of bream tolerance to hypoxia.