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Molecular Cloning And Difference Analyses Of HIF-1α Gene In Different Habitats Of Fish

Posted on:2013-01-06Degree:MasterType:Thesis
Country:ChinaCandidate:Y N TongFull Text:PDF
GTID:2210330374960743Subject:Aquatic biology
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The effect of anoxia is demonstrated at every level of an organism from molecule, cell tobehavior. The changes in oxygen over diel periods induce shifts in numerous metabolic, hormonaladaptations, hemoprotein adaptations, HIF-1gene transcripts levels, modulators of erythrocyte andmyoglobin and so on.. Hypoxia-inducible factor-1alpha (HIF-1α) is a key factor of transcript regulationin hypoxia environment, which can activate the transcription of more than60target genes to cope with thehypoxia. In this study, firstly, cDNA sequences of HIF-1α in Cassius auratus gibelio,qihesis, Silurns asotus,Paramisgurnus dabryanus were acquired by RT-PCR. HIF-1α cDNA sequence variation was analyzed infishes from different habitats, and gene expression was studied in different tissues or organs. We hope thatthese studies are helpful for understanding the correlation between HIF-1α sequence variation andhypoxia-tolerant ability in fish.Total RNA was extracted from heart, gill, kidney, muscle, and liver of fish, respectively. Specificprimers were desigened based on the conserved sequences of HIF-1α cDNA from different species inteleost, and used to amplify HIF-1α cDNA based on RT-PCR protocol in target fishes.The upstream anddownstream primers contain the restriction sites for Hind Ⅲ and XhoⅠ restriction endonucleases. TargetcDNA sequences of different fish species were respectively isolated and inserted into cloning vector pMD19-T, and then transferred in E.coli JM109through CaCl2method to produce recombinant choning vectors.Positive clones were identified by PCR and double restriction endonucleases digestion. Sequencing andBLAST homologous comprision indicated that HIF-1α cDNA sequences were obtained in target fishspecies.In C. auratus gibelio,the cDNA sequence consists of2325base pairs (bp) that encodes774amino acids.The deduced HIF-1α protein sequence of C. auratus gibelio shows a high conservation at the bHLH(basic helix-loop-helix domain), PAS(Per-ARNT-Sim), N-TAD(N-terminal transactivation domain), andC-TAD(C-terminal transactivation domain). C. auratus gibelio HIF-1α cDNA is97%sequence homologywith C. carassius qihesis, and shares95%sequence homology with Carassius carassius pengzesis,which is secondly high homology with Cyprinus carpio, Magalobrama amblycephala, Ctenopharyngodonidellus. With regard to the gene expression, HIF-1α is highly expressed in the heart, kidney.In S. asotus, cDNA sequence consists of2304bp that encodes767amino acids. The deduced HIF-1αprotein is high conservation, with90%homology, with Ictalurus punctatus. Four conserved domains arehigh similar to Myxocyprinus asiaticus,Hypophthalmichthys molitrix,C. cassius pengzesis.In P. dabryanus, the cDNA sequence consists of2316bp that encodes771amino acids, which shares85%sequence identify with H. molitrix, and is highly homologous to M. asiaticus, C. idellus, C. carassius.To further study of the HIF-1α function in C. auratus gibelio, we contructed a new recombinantvector of HIF-1α, in which ODD region of HIF-1α is removed by restriction endonucleases. The newdesigned recombinant vector will be helpful for understanding fuctions and regulations of HIF-1α gene andODD region.
Keywords/Search Tags:fish, Hypoxia-inducible factor-1alpha, cDNA sequence, tissue specific expression
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