Studies On HIF Signaling Pathway-related Genes From Haliotis Diversicolor Under Different External Stresses

In recent years,global climate change,particularly temperature increase,has already had observable effects on the natural and human environment.For marine ecosystems,increasing water temperature was the likely cause of some negative consequences,such as the blooming in microbial populations and the decrease in oxygen solubility.Aquatic hypoxia is a frequent event and a complex set of physiological and biochemical alterations are employed to cope with this environmental stress in aquatic organisms.Although many of these adjustments depend to a large extent on changes in the expression of genes were well understood in many vertebrates,little is known about the molecular regulation mechanism of hypoxic genes in mollusks.Hypoxia inducible factor 1(HIF-1)is a master transcription factor that regulates a variety of molecular responses to hypoxia.In this study,we report first on the molecular cloning and characterization of HIF-1α,HIF-1β and selenium-binding protein 1(SBP1)in Haliotis diversicolor.They were named HdHIF-1α,HdHIF-1β and HdSBP1.their signal peptides,domains and structures were predicted and analyzed by using bioinformatic methods.Real-time quantitative PCR(qRT-PCR)and RNAi were used to reveal the role of HIF signal pathway in hypoxia,thermal,hypoxia&thermal and Vibrio parahaemolyticus infections.The results were reported as follows:(1)The full length cDNA of HdHIF-1α,HdHIF-1β and HdSBP1 are 2833 bp,1919 bp and2269 bp respectively,encoding a protein of 711 aa,590 aa and 497 aa respectively.Similar to HIF-1of other species,HdHIF-1 has one basic helix–loop–helix(bHLH)domain and two Per-Arnt-Sim(PAS)domains,and HdHIF-1α has a oxygen-dependent degradation domain(ODDD)and a C-terminal transactivation domain(C-TAD).The synteny analysis of HIF-1α,HIF-1β and SBP1 were acquired by bioinformatics analysis.(2)HdHIF-1α transcripts were detected in the gills and hemocytes from small abalone under normoxia and environmental stress.Under hypoxia condition,the expression of HdHIF-1αm RNA in gills was significantly increased at 4 h,24 h and 96 h(P < 0.05);Under thermal stress,HdHIF-1α mRNA expression was significantly up-regulated in gills at 4 h(P < 0.05);Under hypoxia plus thermal stress,HdHIF-1α mRNA expression was significantly up-regulated in gills at 0 h、4 h and 24 h(P < 0.05);After Vibrio parahaemolyticus injection,HdHIF-1α mRNA expression was significantly up-regulated in gills at 3 h and 12 h(P < 0.05).Under hypoxia condition,The expression of HdHIF-1α was significantly increased in hemocytes at 24 h and 96h(P < 0.05);Under thermal stress,HdHIF-1α mRNA expression was significantly up-regulatedin hemocytes at 0 h and 4 h(P < 0.05);Under hypoxia plus thermal stress,HdHIF-1α mRNA expression was significantly up-regulated in hemocytes at 24 h and 96 h(P < 0.05);After V.parahaemolyticus injection,HdHIF-1α mRNA expression was significantly up-regulated in hemocytes at every phases(P < 0.05).(3)Under environmental stress,HdHIF-1β mRNA expression remained relatively constant in the gills and hemocytes.(4)HdSBP1 transcripts were detected in the gills and hemocytes from small abalone under normoxia and environmental stress.Under hypoxia condition,the expression of HdSBP1 mRNA in gills was significantly increased at 24 h(P < 0.05);Under thermal stress,HdSBP1 mRNA expression was significantly up-regulated in gills at-3 h and 4 h(P < 0.05);Under hypoxia&thermal stress,HdSBP1 mRNA expression was significantly up-regulated in gills at192 h(P < 0.05);After V.parahaemolyticus injection,HdSBP1 mRNA expression was significantly up-regulated in gills at 6 h and 24 h(P < 0.05).Under hypoxia condition,The expression of HdSBP1 was significantly increased in hemocytes at 192 h(P < 0.05);Under thermal stress,HdSBP1 mRNA expression was significantly up-regulated in hemocytes at 4 h(P< 0.05);Under hypoxia&thermal stress,HdSBP1 mRNA expression was significantly up-regulated in hemocytes at 0 h,4 h and 24 h(P < 0.05);After V.parahaemolyticus injection,HdSBP1 mRNA expression was significantly up-regulated in hemocytes at each phases(P <0.05).(5)In the gills and hemocytes from small abalone under normoxia and environmental stress,expression level of nitric oxide synthase(HdNOS)and tumor necrosis factor α(HdTNF-α)changed significantly.(6)When the HdHIF-1α was inhibited,the expression level of HdHIF-1α of the experimental group was lower than the blank control group and GFP group.Meanwhile,we tested the expression of the several HdHIF-1α target genes,such as HdTNFα,Caspase3(HdCasp3),Hexokinase(HdHK)and Glyceraldehyde 3-phosphate dehydrogenase(HdGAPDH).The qRT-PCR results showed that the expression level of these genes was lower in experimental group than in blank control group and GFP group.