Adaptation Mechanisms Analysis Of Tibetan Sheep To High-Altitude Hypoxia Based On Their Lung Histophysiology And Regulatory Genes

Tibetan sheep are distributed on the Tibetan plateau at an altitude of 2500-5000 m.Due to long-term breeding in the high-altitude hypoxic environment,a series of adaptive changes have occurred in multiple tissues and organs of Tibetan sheep,especially the lungs.The complex structure of the lungs enables gas exchange between inhaled air and circulating blood,and is one of the organs most responsive to hypoxia.In order to explore the adaptive changes of Tibetan sheep lungs at different altitudes on the Tibetan plateau,this study first measured the blood-gas indicators and observed lung histomorphology of Tibetan sheep distributed at altitude of approximately 2500 m（TL）,3500 m（TM）and 4500 m（TH）.Then mRNA-seq and Label-free proteome sequencing techniques were used to screen the key genes and proteins in their lungs related to high-altitude hypoxia adaptation.The SNPs detection and genotyping of key genes HIF-1α and HBA were then performed using Sanger sequencing and KASP genotyping techniques,and the association of genotype and haplotype combinations with blood-gas indicators was analyzed.The purpose of this study is to observe and analyze the histophysiology changes and the role of important regulatory genes in the lungs of Tibetan sheep at different altitudes,and to elucidate the hypoxia adaptation mechanism of Tibetan sheep.The research results will provide a reference for the development of plateau animal husbandry and promotion the conservation and exploitation of plateau animal genetic resources.The main research results are as follows:1.As altitude increased,PO₂ and SaO₂ decreased（P<0.05）,the air-blood barrier thinned,and the pulmonary artery vasodilatation and vasoconstriction capacity and volume increased（P<0.05）.Hct and Hb concentrations were higher at TM altitude than at TH altitude（P<0.05）,which beneficial to increased oxygen-carrying capacity of TM altitude Tibetan sheep.The total area of blood-oxygen exchange and P₅₀ were greater at TH altitude than at TM altitude（P<0.05）,which conduce to increased blood-oxygen exchange andO₂ transfer efficiency of TH altitude Tibetan sheep,while avoiding pulmonary hypertension.2.By mRNA-seq analysis of Tibetan sheep lungs,63,168 and 123 differentially expressed genes（DEGs）were screened out in the TL-vs-TM,TM-vs-TH and TL-vs-TH comparison groups,respectively.Gene function and pathway annotation identified 11 DEGs related to vascular development（FNDC1,HPSE,E2F8,SPARC,MFAP5,GJA4,FAP,COL1A1,COL1A2,COL3A1 and COL14A1）,4 related to gas transport（HBB,HBA1,APOLD1 and CHL1）and 2 related to oxidative stress response（GSTA1 and GSTA2）.Twelve DEGs were randomly selected and verified by RT-q PCR technology,and the results were consistent with the m RNA-seq data.3.In Tibetan sheep lungs Label-free proteome sequencing,149,71 and 137 differentially abundant proteins（DAPs）were screened out from the TL-vs-TM,TM-vs-TH and TL-vs-TH comparison groups,respectively.Protein function and pathway annotation identified 12DAPs related to vascular development（FLII,HRG,TAGLN,MPO,GSN,GNG2,CA2,COL14A1,LTBP4,MFAP5,EHD4 and SPARC）,6 related to gas transport（HBB,PRDX2,AHSP,HP,SPTB and DMTN）and 4 related to oxidative stress responses（GPX1,GPX3,GSTA1 and GSTA2）.Among them,COL14A1,MFAP5,SPARC,HBB,GSTA1 and GSTA2 were also identified by the m RNA-seq,which could be used as important candidate molecules to improve the high-altitude hypoxia adaptation of Tibetan sheep.The protein interaction network constructed for DAPs is also mainly related to vascular development,gas transport and oxidative stress response.The results of WB and immunofluorescence analysis of HBB protein were consistent with the proteome sequencing data.4.There was 1 SNP in each of exons 9,10 and 12 of the HIF-1αgene,the AA genotype individuals of SNP1 had high PO₂ and low Glu concentration（P<0.05）;the GG genotype individuals of SNP2 had high PO₂,Hct and Hb concentrations and low TCO₂（P<0.05）;and no significant association between the blood-gas indicators and each genotype of SNP3.There was 1 and 2 SNP in exon 1 and intron 1 of the HBB gene,respectively,the AA and AT genotype individuals of SNP4 had high SaO₂ and high P₅₀（P<0.05）,respectively.Lowering the frequency of the AA genotype at SNP1 and SNP4 and the GG genotype at SNP2 is beneficial to the dissociation ofO₂ from Hb and promotes blood circulation in TH altitude Tibetan sheep.This study showed that the blood-gas indicators and lung histomorphology of Tibetan sheep were significantly correlated with altitude.DEGs and DAPs screened at different altitudes by m RNA-seq and Label-free proteome sequencing were involved in influencing the vascular development,gas transport and oxidative stress responses of Tibetan sheep lungs,thereby Tibetan sheep can adapt to different degrees of hypoxic environment.The sequence variation of HIF-1α and HBA genes was associated with blood-gas indicators of Tibetan sheep,and also has a certain impact on the adaptation of Tibetan sheep to hypoxic environment.The results provide basic data for elucidate the molecular mechanism of Tibetan sheep adaptation to high altitude hypoxic environment.