Cloning, SNPs Detection And Expression PatternsAnalysis Of The Yak's EPAS1and VEGF-A Gene

In this study, four yak breeds (including Gannan,Datong,Tianzhu white and Pali)and ordinary cattle in high altitude were selected as experimental animals, two genesEPAS1and VEGF-A were cloned and sequenced using molecular biology techniques,the secondary and tertiary structure of the proteins encoded by these two genescombined some physical and chemical properties were predicted. The SNPs weredetected by using mixed DNApool sequencing technology combined with PCR-SSCPand PCR-RFLP technique, so as to test the effect of selection of the candidate genes inthe long-term evolutionary. At the same time, the relative mRNA expression levels ofEPAS1and VEGF-A in different tissues of all experimental animals were analyzed inorder to filter out the molecular beacons concerning the adaptation to high altitudehypoxia environment, so as to provide a theoretical basis for the further study of yakhigh altitude hypoxia adaptation mechanisms.The main results are as follows:The full-length of coding sequence (CDS) of yak's EPAS1and VEGF-A geneswere cloned.(1) EPAS1contains a complete ORF (2613bp) encoding870amino acids.The yak EPAS1protein has no signal peptide, is a hydrophilic protein which containscomplete domain of PAS superfamily protein.(2) VEGF-A gene contains a completeORF (573bp) encoding190amino acids. The VEGF-A protein has a signal peptide, isa hydrophilic protein which contains complete domain of Plate-derived growth factor(PDGF) family protein.3specific SNPs (g.83052C> T, g.83065G,> A, g.83067C> A) were detected inyak's EPAS1gene. The results of PCR-SSCP genotyping showed that5genotypeswere detected, named AA,AB,AC,BC and CD respectively. The Gannan yak andDatong yak living in the region in an altitude of3000m above had significantly higherCD genotype frequencies than that of Tianzhu White yak living in the2700m(P<0.05),while there were no significant differences of the other genotypes in the yakpopulations (P>0.05). By comparing the blood indicators of every genotype of Pali yaks, found that induviduals with CD genotype had significantly higher hemoglobinconcentration than that of the other genotype (P<0.05), suggesting that the CDgenotype may be more adapted to high altitude hypoxia environment.3SNP loci inthe three yak populations were in high linkage and Gannan yak and Datong yak hadsignificantly higher HapD haplotype frequencies than in Tianzhu white yak (P<0.05),therefore, EPAS1gene could be used as a molecular beacon of yak for adapting tohigh altitude hypoxia environment.2specific SNP (SNP g.8430T> C and the SNP g.14853G> A) were detected inyak's VEGF-A gene. The results of PCR-RFLP genotyping showed that the Gannanyak and Datong yak living in the region in an altitude of3000m above hadsignificantly higher GA, AA genotype frequencies, A allele frequency and haplotypeTAthan that of Tianzhu White yak living in the2700m (P<0.01), Suggested that G> Amutation may be conducive to yak to adapt to hypoxic environment. The neutralitytest results showed that these two SNP loci were subject to selection pressure, themain selective factor was plateau geographical environment, which was the yak livedin different geographical environment having a direct role in the choice.Relative expression levels of EPAS1and VEGF-A mRNA in various yak's tissueswere analyzed by real-time PCR. The results showed that EPAS1and VEGF-A wereexpressed in seven detected tissues of the yaks and highland cattle. The expression ofEPAS1mRNA levels was the highest in the lungs of the yaks and highland cattle, andthe highland cattle had very significantly higher expression level in lung and liverthan that of the yak (P<0.01). The expression levels of VEGF-A mRNA was mostabundant in pancreas and lungs of yaks and highland cattle, and the expression levelin spleen of the highland cattle was significantly higher than yaks (P<0.01). Thisresult showed that, when ordinary cattle entering into the plateau its adaptation to thephysiological regulation of the hypoxic environment occurred mainly in the lungs,liver and spleen.Correlation analysis of EPAS1and VEGF-A mRNA expression in differenttissues showed that the mRNA expression of EPAS1and VEGF-A gene had highly significant positive correlation in yak pancreas (P<0.01), and had a significantpositive correlation in liver and muscle (P<0.05), but only had highly significantpositive correlation in the lung of the highland ordinary cattle (P<0.01). This couldspeculate that in the process to adapt to the hypoxic environment by the highlandordinary cattle and yaks, the EPAS1gene regulation of VEGF-A gene may be differentin each organization. This result also reminded us that in the long-term evolution, yakhas developed a unique genetic characteristic to adapt to high altitude hypoxiaenvironment.