The PHD2 Gene Cloning, Expression And Variantions In The Sheep

The subject in the study is totally six group, including Gansu Alpin Merino,Tibet sheep,Tan sheep,Tashikuergan sheep, Azak sheepand HU sheep.We clone the sequence of PHD2 gene in Tibet sheep and analyze its bioinformatics analysis,the relative expression of PHD2 gene m RNA in 13 tissues of sheep is detected by real-time fluorescence quantitative PCR technique.We analyze the genetic polymorphism of PHD2 gene by PCR-SSCP technique in the groups.In order to study the inherited character of PHD2 gene, and enrich the basic data of the study on the candidate genes of hypoxia adaptation in Tibetan sheep plateau.The main results and conclusions are as follows:(1)In the study we acquire the CDS Sequence of PHD2 gene in Tibetan sheep by clone technique. It contain 2609 bp and encode a putative 358 amino acids.Compared with the sheep sequence, there are two SNPs and all synonymous mutations in PHD2 gene sequence.Tibetan sheep PHD2 protein molecular formula is C1691H2674N496O514S12, molecular weight is 38561.5 and theoretical isoelectric point(PI) is 8.64.The protein is synthesized by the mitochondria, which is hydrophilic protein and stable. No signal peptide shear sites and transmembrane helical structures are found.There are two low complexity regions and three domains in the PHD2 protein of Tibetan sheep.Phosphorylation is the main method for the modification after the protein translation.The PHD2 protein in the cell play the role of translation, amino acid biosynthesis, the central intermediary metabolism, regulatory functions, replication, transcription and fatty acid metabolism.Biological action of enzyme is ligase.The protein secondary structureis mainly in random coil, and the tertiary structure is mainly composed of beta folding and random coil winding.(2) The PHD2 gene is expressed in all tissues of sheep, but there is a difference in each tissue expression. It is high expression level in the duodenum, middle expression level in the brain and spleen and low expression level in the other tissues.(3) PHD2 gene has rich polymorphism, high genetic variation in the sheep, which find 5 SNPs in intron 6-exon 7 region.PHD2 gene in intron 6-exon 7 region of 6 sheep populations detected 4 bands including AD, AB, BC, BD and regulated by 4 alleles including A, B, C, D. Sequence comparison show that, the A allele sequence and Gene Bank published sheep PHD2 gene sequence has no difference; B allele sequence compared to the original sequence has a A base insertion in 105; C allele sequence has a A base insertion in 105 compared to the original sequence and has a c.278 T>C base transversion; D allele sequence compared to the original sequence have base transversion c.188T>C, c.286 A>G and c.290C>A; There is no amino acid change because the target sequence in the intron.