Cloning And Modeling Of Yak Hemoglobin

The yaks originated from China with 95% of numbers all over the world. They were domesticated from the Bos Mutus before years of 5000-10000 by Jiang people. They mainly distribute in cold and hypoxic area with altitude of more than 3000m, where the oxygen content in the air is only 1/3 1/2 of that of sea level. They can not only reproduce normally, but also produce milk, meat, leather, hair, villus with the fame of "altitude vehicle". Their hemoglobin is both in high content, but also in high oxygen affinity. Lalthantluanga and Weber et al. thought that their high oxygen affinity mainly contributed to the β_II 135 Val residue from the primary structure other than high structure. Hemoglobin transfers O₂ and CO₂ in organism. We will clone the gene of globin and model the high structure of yak hemoglobin by insightII software for understanding its high oxygen affinity.We designed the primer for yak a-globin gene through the conservative region. The amplification results in a band of 754bp whose deduced residue is consistent with the known yak α1-globin chain. It has the most homology with cattle, then buffalo, the last for goat, sheep, horse and human, with the value of 99.71%, 98.58%, 96.03%, 95.57%, 68.55%, 51.13%, respectively. Each of two introns amplified only one band. The intergenic region of a-globin genes amplified a band of 2470bp. There were no genes in it after blasting. The upstream gene amplified a band of 1086bp, while the downstream is 1140bp. After cloning and sequencing, the upstream gene is agreement with α1-globin chain, while the downstream gene is agreement with known yak α2-globin chain. The studies above indicate that yak have two non-allele α-globin genes with the same length of 706bp. They locate tightly in the chromosome where α1-globin gene is in the front while a2-globin gene in the back.We used three and one primers designed from the cattle p-globin gene to amplify the β-globin gene of yak, respectively. Two alleles were obtained, β⁷3Asn and β¹35Asn. The deduced residues of allele β⁷3Asn are completely agreement with that of cattle while they aredifferent from the known yak βI-globin chain in one residue which is located in site 73. The result of experiment is Asn, while the known residue is Glu. The deduced residues of allele β¹35Asn are different from the known yak βII-globin chain in one residue which is located in site 135. The result of experiment is Asn, while the known residue is Val. The sample of 67 has these two alleles, while 1078 has the allele β73Asn, 64 has the allele β¹35Asn. Most importantly, the allele β¹35Asn is the characteristic of Chinese yak.Studies on the microsatellite polymorphism of β-globin locus in yak at four altitudes (4500m, 4000m, 3500m, 1700m) were undertaken. The H (heterozygosity) and PIC (polymorphic information contents) of yak were 0.8574 and 0.8426 respectively. 10 alleles were detected in yak including 7 unique ones. The remaining three were the common alleles of yak and cattle. The allele distribution for yaks of four altitudes was significantly different (P<0.01). There was a correlation between allele distribution and altitude with a correlation coefficient of 0.397. The genotype 121bp/103bp was found to be unique to the yak living at altitude of 4000m.The high polymorphic β-globin locus shows its possible natural selective value in the adaptation of yak to high altitude hypoxia and perhaps its possible regulatory role in the synthesis of hemoglobin.We were modeling the yak hemoglobin with known yak αI and βII globin chain using software insightII with the cattle carbonmonoxy hemoglobin as the reference protein. The model has the RMSD of 2.32 with X-ray structure after superimposing. There were nine hydrogen bonds in the surface of αIβI subunits, while four for cattle X-ray structure. Four and two hydrogen bonds in the surface of αIβII subunits for yak and cattle, respectively. At the environment of the different residues, the model had more hydrogen bonds than X-ray structure. Moreover, the βI135 Val of yak had mor...