| Tibetan Plateau is the highest plateau in world, with an average altitude of 4000-5000 meters, located in southwest of China. The climate is dry, thin air, strong solar radiation and low temperatures. The ecosystem of Plateau is fragile and disrupt with external factors. Hypoxia-inducible factor is a transcription factor, a heterodimer of a and β subunits, a subunit is the key of its activity, and a variety of downstream genes can be regulated, so the cells adapt to hypoxia and maintain physiological homeostasis. Currently, the HIF family includes HIF-1, HIF-2 and HIF^3, the a subunit is HIF-1 a, HIF-2a and HIF-3a, which are sensitive to oxygen partial pressure within the cell, and adjust by the oxygen concentration. Research shows that recognition sites of hydroxyl sequence is PEMLAP (550-555) in HIF-1 a ODD domain in naked carp from Qinghai Lake, which is inconsistent with other vertebrates conserved sequences (LXXLAP, X represents any amino acid). This phenomenon imply that naked carp has different mechanism of hypoxia adaption with other teleost. Currently the study of HIF focuses on HIF-la, and little is known to HIF-2a’s molecular structure, expression and characteristic of hypoxia regulation. The aim of this study is to analyze hydroxylation of HIF-la ODD domain in naked carp, and clone HIF-2a genes with other three kinds of small mammals from Tibet Plateau, which will help to understand further the molecular mechanisms of hypoxia adaption of these animals.Firstly, total RNA was extracted from naked carp (Gymnocypris przewalskii) in liver tissue, and the full-length cDNA of naked carp HIF-1α was obtained by RT-PCR. An 207 bp fragment which includes the sequence between bases 1546-1752 (HI, coding the amino acid sequence 516-584, including PEMLAP). The mutated sequence (OE, LEMLAP) was produced by Overlap Extension PCR (OEPCR). Both fragments were cloned into expression vector pGEX-4T-2 GST-HI/GST-OE were expressed from pGEX-4T constructs in BL21(DE3) by induction with IPTG. GST fusion proteins were purified using GST Resin and their purity was determined by SDS/PAGE.Secondly, the coding sequence of zebrafish PHD2was cloned into expression vector pET-32. Trx-phd2 was expressed from pET-32-phd2 constructs in BL21(DE3) by induction with IPTG. Trx fusion proteins were purified using Ni Resin and dialyzed overnight at 4℃. PHD2 was found to specifically interact with both HI and OE, and OE interact with phd2 in a manner weaker than HI. Electrophoretic mobility of GST-OE is slightly faster than the non-hydroxylation of GST-OE strip, and there are no difference between the hydroxylation of GST-HI strip with mobility, it indicated that the recombinant protein of trx-PHD2 have hydroxylation activity. The hydroxylation of recombinant protein GST-OE has higher affinity with PHD2 than the recombinant protein GST-HI. Compared with other fish, it is more difficult to hydroxylate Pro residue within ODD domain of HIF-1α in Naked carp, it maybe the results of long-term adaptation to plateau salt lake.The total RNA was extracted from Naked carp (Gymnocypris przewalskii), Plateau zokor (Myospalax baileyi), Root Vole (Microtus oeconomus) and plateau pika (Ochotona curzoniae), and the genes of HIF-2α were cloned by RT-PCR and RACE. The full-length HIF-2α cDNA sequences from 4 kinds animals were 3013bp, 4431bp,3026 bp and 3196 bp respectively, containing open reading frames of 2502 bp,2607 bp,2625 bp and 2664 bp. The analysis of sequence showed that the HIF-2α sequences from 4 plateau animals share more than 50% amino acid identity with human. |
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