Hypoxia Adaption Study On HIF1B And HIF2A Genes From Plateau Schizothoracine Fishes

Hypoxia-inducible factor(HIF)gene played an important role in hypoxia adaption,which included two subtypes of α and β.Generally in mammals,α subtype can be divided into HIF1α,HIF2α,HIF3α,and in the bony fish,α subtype can be divided into HIF1A/1B,HIF2A/2B and HIF3.To analyze the characteristics of hypoxia-inducible factor(HIF)genes and their functional evolution to hypoxia,the complete open reading frame(ORF)of HIF1 B and HIF2 A gene were obtained and amino acid sequences were analyzed in Gymnocypris dobula and Schizothorax prenanti.The O RF of HIF1 B in G.dobula and S.prenanti were 2322 bp and 2313 bp,and encoded 773 and 770 amino acids residues,respectively;The ORF of HIF2 A gene are 2466 bp and 2502 bp,and encoded 821 and833 amino acids residues,respectively.The homology of the ODD domain(oxygen-dependent degradation domains)of HIF1 B between G.dobula and S.prenanti were 93.12%,and HIF2 A were 89.56%.A mutation(Lxx LAP to Pxx LAP)was found in the conserved CODD domain of HIF1 B in G.dobula(plateau schizothoracine fish).HIF1A/1B、HIF2A/2B、HIF3 of G.dobula were analyzed whether were positive selection by using bioinformatics method,and the results showed that Tibet plateau schizothoracine fish HIF1 B and HIF2 A were positive selection,relative to the schizothoracine fish of Yun Nan and S.prenanti.It may be a sign of functional evolution of HIF1 B and HIF2 A in plateau schizothoracine fish.Under the long-term hypoxia condition,the HIF1 B and HIF2 A mRNA expression levels of spleen,brain,skin,and gill were compared in G.dobula and S.prenanti,respectively.The qRT-PCR showed that the expression levels of HIF1 B and HIF2 A mRNA in teleost were significantly increased in acute hypoxia,whereas significantly decreased in long-term hypoxia condition.At the same time,the protein expression of HIF1 B in heart of G.dobula was fewer than S.prenanti by immunohistochemical.And Oreochromis niloticus was dealt with acute hypoxia(0.5h and 1 h),and HIF1 B and HIF2 A mRNA expression levels were analyzed by qRT-PCR.The results showed that HIF1 B and HIF2 A mRNA expression levels in skin and gill were significantly increased(***P<0.001),and HIF1 B mRNA in spleen and brain were also significantly increased(***P<0.001),but HIF2 A mRNA were essentially invariant.So there may be have two different mechanisms to adapt to the acute hypoxia and long-term hypoxia condition in teleost,respectively.HIF gene can activate nitric oxide synthase to synthetic nitric oxide.Because nitric oxide can increase the body’s ability to carry oxygen by diastoling blood vessels,so concentrations of NOX were measured using the nitrate reductase method assay.The result showed that serum NO metabolites of plateau schizothoracinae were both higher than S.prenanti(*** P<0.001).Plateau schizothoracinae included S.oconnori,S.macropogon and G.dobula.Explored the increasing reason of NO in the body,so NOS1(nitric oxide synthase 1)and NOS2B(nitric oxide synthase 2B)mRNA levels were measured by qRT-PCR.The results showed that both in long-term low oxygen and acute hypoxia,NOS1 and NOS2 B mRNA expressions in brain and heart of G.dobula were decreased.So the increasing reason of serum NO in G.dobula was not caused by the increasing of NOS1 and NOS2 B,then it may be caused by some other reasons.To sum up,there may be two kinds of mechanisms in bony fish to adapt to long-term low oxygen and acute hypoxia stress,respectively.These were foundation for further research of the biological functions of HIF1 B and HIF2 A in plateau schizothoracine fish plateau and its biological significance in hypoxia adaptation.