Cloning Analysis Of EGLN1 And EPOR And Related MiRNAs Expression In Yaks

As the dominant animal species in qinghai-tibet plateau,yak has a strong adaptability to the plateau ecological environment such as low oxygen,cold and strong ultraviolet ray.It plays an important role in the development of animal husbandry on the qinghai-tibet plateau.In this study,TA cloning technology was used to clone the sequences of EGLN1 and EPOR gene CDS of yaks,to analyze the structure and function of their coding proteins,and to detect their expression levels in the brain,cerebrum,hypothalamus,heart,lungs,muscle,kidney and liver of Leiwuqi yaks and cattle(Tibetan cattle and Sanjiang cattle).Screening and identification of EPOR gene-related miRNAs and detection of their expression differences in tissues.The results are as follows:1.The total length of the CDS region of EGLN1 gene in Leiwuqi yak was 1263 bp,encoding 420 amino acids.Compared with the reference sequence of cattle,the AG conversion occurred at the 482 nd position of the base,and the encoding amino acid was mutated from asparagine to serine,which was a missense mutation.EGLN1 protein is a stable alkaline protein,showing hydrophilicity and mainly distributed in the nucleus(73.9%).EGLN1 protein has no transmembrane structure and is not membrane protein.After translation,the main modification method is phosphorylation,with a total of 30 phosphorylation sites.There are 3 low complexity regions and 1 P4 Hc functional domain.The secondary and tertiary structures are mainly irregular crimp.The total length of the EPOR gene CDS region in Leiwuqi yak was 1527 bp,encoding 508 amino acids.As high as 99.28%,compared with the cattle sequence,similarity of base mutations in A total of 11 of lead to amino acid mutations in six of them respectively.19th(G-C)alanine mutation for proline,119th(C-T)alanine mutation for phenylalanine,138th(G-T)glutamic acid mutation for valine,584th(G-A)arginine into lysine,659th(T-G)valine into glycine,1432th(G-A)glycine into arginine.Subcellular localization was mainly distributed in endoplasmic reticulum(44.4%),with secretory signal peptide(Sec/SPI),and transmembrane structure at position 251-273.The main method of post-translational modification is phosphorylation,with 50 phosphorylation sites.There are two low complexity regions,one fibrin type 3 domain and one transmembrane region.The secondary and tertiary structures were dominated by irregular curls(57.28%).2.EGLN1 gene was expressed in 8 tissues and organs of Leiwuqi yak,Tibetan cattle and Sanjiang cattle.In other tissues except the gluteus muscle,the expression level of EGLN1 was the highest in yaks,followed by that of Sanjiang cattle,and the lowest in Tibetan cattle.The expression level of EGLN1 was significantly higher in other tissues of yak than that of Tibetan cattle(P<0.01),and significantly higher in all tissues of Sanjiang cattle than that of Tibetan cattle(P<0.05).The expression levels of liver and kidney tissues of yak were significantly higher than other tissues.EPOR gene was expressed in 8 tissues and organs of three kinds of cattle,and the expression level in each tissue was the highest in Leiwuqi yak,followed by Tibetan cattle,and the lowest in Sanjiang cattle.The expression of EPOR in lung,kidney and gluteus muscle was significantly higher than that in other tissues(P<0.05).3.EPOR-related miRNAs(bta-let-7b,bta-mir-22-3p,bta-mir-30e-5p,bta-mir-125b)were screened by TargetScan and other databases,and were expressed in 8 tissues of three kinds of cattle.The expression of bta-let-7b in all tissues was the highest in Sanjiang cattle,followed by Tibetan cattle,and the lowest in Leiwuqi yaks.The expression of bta-mir-22-3p in all tissues(except lung and kidney)of Sanjiang cattle was significantly higher than that of Tibetan cattle and Leiwuqi yak(P<0.01).The expression of bta-mir-30e-5p in the tissues of both kinds of cattle was significantly higher than that of Leiwuqi yak(P<0.01).The expression of bta-mir-125 b in all tissues of Sanjiang cattle was significantly higher than that of Tibetan cattle(P<0.05),while the expression of bta-mir-125 b in all tissues of Tibetan cattle was significantly higher than that of Leiwuqi yak(P<0.01).The overall expression trend of the four miRNAs is opposite to that of EPOR gene,and these miRNAs may be negative regulators of EPOR gene.How they participate in the regulation of hypoxic adaptability of yaks needs further study in the future.In this study,for the first time,the EGLN1 and EPOR genes of Leiwuqi yak were cloned,analyzed by protein prediction and detected by RT-qpcr,and four miRNAs that were targeted by EPOR genes were detected by RT-qpcr.The results provide basic data and scientific basis for revealing the function of these two genes and the molecular mechanism of adaptation to the plateau,which is of great significance for the protection,development and utilization of yak genetic resources.