Cloning And Expression Of HMOX1 And HMOX2 Genes And Their Association With Hypoxia Adaptation In Tibetan Sheep(Ovis Aries)

Heme oxygenase（HMOX）is a key rate-limiting enzyme in heme metabolism,which plays an important role in the regulation of hypoxia stress metabolism.Ovis aries have developed an unique hypoxic adaptation strategy to maintain their own oxygen balance during long-term evolution.In order to explore the role of heme oxygenase family genes（HMOX1 and HMOX2）in the hypoxia adaptation genetic mechanism of Tibetan sheep,this research selected Tibetan sheep（hypoxia group）and Hu sheep（matched control group）as the research object,determination of analysis the differences in blood physiological indexes between Tibetan sheep and Hu sheep,the mRNA sequences of HMOX1 and HMOX2 genes were cloned and analyzed in Tibetan sheep,and their structures and functions were analyzed by bioinformatics method,the heme oxygenase was used by RT-qPCR and PCR-SSCP molecular labeling techniques,the tissue expression of heme oxygenase family was analyzed,and the mutation sites were screened to analyze the effects of the mutation sites,which on the blood physiological indicators of Tibetan sheep.The main findings are as follows:1.Research on the blood physiology characteristics of hypoxic adaptation of Tibetan sheep12 blood indexes of Tibetan sheep（hypoxia group）and Hu sheep（control group）were analyzed,it was found that the four indicators of PO₂,SO₂,HGB and HCT are closely related to the metabolic transport of oxygen,which is hidden under hypoxic conditions,the Tibetan sheep were significantly lower than Hu sheep（P<0.01）in PO₂and SO₂,the Tibetan sheep were obviously higher than Hu sheep（P<0.01）in HGB and HCT,In the hypoxic state,the body’s hypoxia causes the blood to decrease in PO₂ and SO₂,while the Tibetan sheep’s HGB and HCT increase in order to increase the oxygen carrying capacity per unit volume of blood in a hypoxic environment.About the pH,PCO₂,HCO₃^-,TCO₂ and BE,the five indexes were related to balanced the blood acid and alkali.Tibetan sheep were visibly lower than Hu sheep（P<0.01）in PCO₂,HCO₃^-,TCO₂ and BE.There was no significant difference in pH index between the two groups（P>0.05）.Na⁺,K⁺and Ca²⁺were electrolyte indicators in blood.Tibetan sheep was visibly higher than Hu sheep（P<0.05）in Ca²⁺.There was no obvious difference between Na⁺and K⁺（P>0.05）.The reason for this difference may be related to the hypoxia of the Tibetan sheep in a hypoxic environment,which is related to the slowing of oxygen metabolism.2.Cloning and bioinformatics analysis of HMOX1 and HMOX2 genes in Tibetan sheep（1）The cDNA partial sequence of Tibetan sheep HMOX1 gene,the length is 1302 bp,including the 5’non-coding region（5’UTR）is 15 bp,and the 3’non-coding region（3’UTR）is 426 bp,In coding region（CDS）,the length of sequence is 867 bp and the encodes of amino acids is 288,Sequence comparison with sheep showed that there were 2（C337T and T522C）base mutation sites in CDS region of HMOX1 gene in Tibetan sheep,in which the C337T mutation caused the 133th cysteine to become arginine.Based on analysis,the physicochemical properties of Tibetan sheep HMOX1 protein showed that the Tibetan sheep HMOX1 protein belong to transmembrane hydrophilic protein.（2）The Tibetan sheep HMOX2 gene,the length of CDS region is 1005 bp and encodes of amino acids is 344.Sequence comparison with sheep showed that there were 3（G485C,T486G and A500T）base mutation sites in CDS region of HMOX2 gene in Tibetan sheep,in which the G485C and T486G mutations caused the 162th cysteine to become serine,and A500T caused the 167th glutamine to become leucine.Based on analysis,the physicochemical properties of Tibetan sheep HMOX2 protein showed that the Tibetan sheep HMOX2 protein belong to transmembrane hydrophilic protein.3.HMOX1 and HMOX2 genes were differentially expressed in different tissues of sheep（1）HMOX1 gene was expressed in all tissues of Tibetan sheep and Hu sheep.The gene was highly expressed in the spleen of Tibetan sheep（P<0.01）.The expression level of Tibetan sheep spleen was higher than that of Hu sheep（P<0.05）.The expression of HMOX1 gene was significantly different in lung and adipose tissue（P<0.01）;the expression level of HMOX1 gene was positively correlated with hemoglobin content in Tibetan sheep.（2）HMOX2 gene was expressed in all tissues of Tibetan sheep and Hu sheep.The gene was highly expressed in the lungs of Tibetan sheep.The expression level of Tibetan sheep heart and lung was higher than that of Hu sheep（P<0.01）.The lung and cardiac expression was positively correlated with its hemoglobin content of the sheep HMOX2 gene.4.Genetic variation of HMOX1 and HMOX2 genes in Tibetan sheep and evolution analysis of hypoxia tolerance（1）The HMOX1 gene detected two variant sequences and four SNPs from the first exon-the first intron,and the nucleotide variation was related to the indicators of PO₂,HGB and SO₂.Among them,the HGB value is significantly higher of Tibetan sheep that the allele B₁ was present.The second exon detected two variant sequences and two SNPs.The nucleotide variation was related to HCT,HGB and SO₂.Among them,the HCT,HGB and SO₂ values were positively improved of Tibetan sheep with allele B₂.The 3rd exon detected 3 variant sequences and 4 SNPs loci.The nucleotide variation was related to PO₂,HCT,HGB and SO₂.The A₃A₃ and B₃B₃ individuals were significantly higher than A₃B₃ type of HGB and HCT in Tibetan sheep,SO₂ value of A₃C₃ type is obviously higher than individuals type of A₃A₃ and B₃B₃;4th exon detects 2 mutation sequences and 1 SNPs locus,nucleotide variation is related to PO₂ and SO₂;Among the rest,there is an equipotential the lake sheep with the gene B₄ were higher of the PO₂ and SO₂ values,but Tibetan sheep with the allele B₄ had little effect of the PO₂ and SO₂ values.（2）The first exon-the first intron detected two variant sequences and two SNPs of the HMOX2 gene,and the nucleotide variation was related to the indicators of PO₂,HCT and SO₂,among which the allele B₅was absent,the HCT and HGB values were significantly increased of Tibetan sheep.The second exon-second intron detected two variant sequences and two SNPs.The nucleotide variation was related to the HCT index.Among them,the HCT value was significantly increased of Tibetan sheep with allele A₆.The second intron detected two variant sequences and two SNPs.The nucleotide variation was related to HCT and HGB markers.Among them,the HCT and HGB values were significantly increased of Tibetan sheep lacking allele B₇;the 5th exon-6th exon detected 2 mutation sequences and 1 SNPs locus.The genetic variation in this region was related to indicators of PO₂,HCT and SO₂,and the allele B₈ was PO₂（P=0.004）significantly correlated with HCT index（P<0.01）and positively correlated with SO₂（P=0.040）index（P<0.05）in Tibetan sheep.The variation of HMOX1 and HMOX2 gene sequences increases the HCT and HGB in the blood of Tibetan sheep,and promotes the oxygen-carrying capacity of red blood cells,which can effectively alleviate hypoxia in hypoxic environment.In conclusion,the hypoxia-specific expression of HMOX1 and HMOX2 genes can regulate the metabolism of downstream hemoglobin in the hypoxia metabolic pathway.The gene mutation has a certain correlation with blood parameters,and can be used as a candidate gene for hypoxia adaptation.For the molecular mechanism of hypoxia adaptation lays a foundation.