| Oxygen molecules are the material basis of life activities.When the body is deprived of oxygen,the homeostasis of internal environment is destroyed,and a series of adaptive responses will occur.After long-term adaptive evolution,Tibetan sheep have been genetically and physiologically and biochemically adapted to the plateau environment,and have good adaptive capacity to long-term hypoxic stress,but their hypoxic adaptation mechanisms are still not fully elucidated,and the functional genes regulating hypoxic adaptation in Tibetan sheep and their molecular mechanisms need further study.EPAS1,VHL,EPO and ZEB1 genes are thought to play an important role in the hypoxic signaling pathway.In this study,Tibetan sheep living in high altitude and low oxygen environment were selected as the research object,while lowland lake sheep were the control.Based on the oxygen concentration difference of sheep living environment,The m RNA expression levels of these four genes in the heart,lung,kidney,liver,spleen and longissimus dorsi muscle of sheep at different altitudes（100 m,2500 m,3500 m,4500 m）were determined by RT-q PCR,and the mode of action of gene expression characteristics in plateau hypoxia adaptation of Tibetan sheep was analyzed.The SNPs locus was detected by PCR-SSCP and KASP genotyping techniques,and the association between genetic polymorphism and blood gas was analyzed.The main research results are as follows:（1）Four alleles（A,B,C,D）were detected in exon 15 of EPAS1 gene,and their distribution was different between Tibetan sheep and Hu sheep populations.In Tibetan sheep population,B was the dominant allele,while in Hu sheep population,four alleles were common.Six single nucleotide polymorphisms（SNPs）were identified in the four alleles,one of which was a non-synonymous mutation（p.F606L）.A variant region of EPAS1 gene was associated with the elevation of p O2 and s O2.The expression levels of EPAS1 in the heart,liver,spleen and longissimus dorsi muscle of Tibetan sheep at three altitudes（2500 m,3500 m,4500 m）were significantly higher than those of Hu sheep（P<0.05）,and the expression levels were positively correlated with altitude,indicating that the expression of EPAS1 gene was related to oxygen concentration.Lower oxygen concentration leads to higher expression of this gene.（2）Three SNPs（g.3788 G>A,g.6400 G>C and g.6626 A>G）were detected in the second,third and 3’-UTR regions of VHL gene,which were named as VHL-SNP1,VHL-SNP2 and VHL-SNP3,and three genotypes were in all the three SNPs.Correlation analysis showed that VHL-SNP3 sites were significantly correlated with p CO2（P<0.05）,and 7 haplotype combinations were significantly correlated with p O2（P<0.05）.The expression level of VHL gene in lung of Tibetan sheep and Hu sheep was significantly higher than that of other 5 tissues（P<0.05）,while the expression level in lung and heart of Hu sheep was significantly higher than that of Tibetan sheep at 3 altitudes（P<0.05）,indicating that the expression level of VHL gene in lung of sheep had tissue and breed differences.（3）One SNPs（G.855 A>C）was detected in the first exon of EPO gene,and two SNPs（G.1985 T>G and G.2115 G>C）were detected in the fourth exon of EPO gene,which were named as EPO-SNP1,EPO-SNP2 and EPO-SNP3,and all three SNPs had three genotypes.Correlation analysis showed that EPO-SNP3 sites were significantly correlated with p O2（P<0.05）,and 7 haplotype combinations were significantly correlated with p O2（P<0.05）.The expression of EPO gene was the highest in the kidney and the lowest in the muscle of the two sheep breeds,indicating that the expression of EPO gene was different in tissues.The expression level of EPO gene in the heart,lung and liver of Tibetan sheep at 4500 m altitude was significantly higher than that of Hu sheep（P<0.05）,and the expression level of EPO gene in the heart of Tibetan sheep increased with the increase of altitude.（4）Four SNPs（g.35010273 A>G,g.35010343 A>G,g.35010347 G>C,g.35010376 A>G）were detected in exon 7 of ZEB1 gene,and one SNPs（g.35010458T>G）was detected in intron 7.Two SNPs（G.35035102 G>A and G.35036245）were detected in exon 12,which were named as ZEB1-SNP1,ZEB1-SNP2,ZEB1-SNP3,ZEB1-SNP4,ZEB1-SNP5,ZEB1-SNP6 and ZEB1-SNP7,respectively.All the 7SNPs had 3 genotypes.Correlation analysis showed that ZEB1-SNP3 and ZEB1-SNP6 sites were significantly correlated with p O2（P<0.05）,and ZEB1-SNP3sites were significantly correlated with s O2（P<0.05）.With the increase of altitude,the expression level of ZEB1 gene also increased.The expression level of ZEB1 in the lung of Tibetan sheep was the highest,and the expression level in the heart of Tibetan sheep at 4500 m altitude was significantly higher than that of Hu sheep（P<0.05）.The expression level of this gene in the lung of Hu sheep was significantly lower than that of Tibetan sheep at 3 altitudes（P<0.05）,indicating that the expression of this gene was different among different breeds.In summary,mutations and expression regulation of EPAS1,VHL,EPO and ZEB1 genes were associated with oxygen metabolism in sheep.The high level of expression of the four hypoxia-related genes in some tissues of the high-altitude Tibetan sheep population compared to the low-altitude population may be the molecular basis for the maintenance of normal biological functions of the organism under the hypoxic environment.Therefore,EPAS1,VHL,EPO and ZEB1 genes could be used as genetic markers for hypoxia adaptation studies. |
PDF Full Text Request