Effects of different concentrations of androgens on KAP24.1 gene expression in Hetian sheep and Karakul sheep.

The purpose of the experiment was to clone and eukaryotic expression of hair follicle keratin associated protein 24.1 (KAP24.1), study the effect of different concentrations of androgen on protein expression, and compare KAP24.1 gene in skin and hair follicles of different breeds of sheep expression, explore KAP24.1 Expression difference of gene among local sheep breeds in southern Xinjiang and its effect on wool quality. The body-side hair follicles of Plain-type Hetian sheep, Mountain-type Hetian sheep and Karakul sheep were used as experimental materials, and the KAP24.1 gene sequence of sheep in GenBank (accession number: JX112014.1) was used as the reference to design primers. The KAP24.1 gene was amplified by PCR, and the pMD19-T-KAP24.1 cloning plasmid was constructed. After double digestion and identification, the pEGFP-N1-KAP24.1 eukaryotic recombinant expression plasmid was constructed. After PCR and double digestion and identification, sequencing and sequence analysis were performed, and the expression was transfected into Hela cells. SDS-PAGE and Western blotting were used to detect the expression levels of androgen at different concentrations. The expression of KAP24.1 gene in different sheep skin follicles was detected by real-time fluorescent quantitative PCR. Three sheep KAP24.1 were cloned The CDS region sequence of gene is 759 bp, encoding 252 amino acids, all of which are unstable hydrophobic proteins.The results of similarity comparison showed that compared with the reference gene, the gene sequence similarity of Mountain-type Hetian sheep and Karakul Sheep was 99.47%, and that of Plain-type Hetian sheep was 99.34%. Phylogenetic tree analysis showed that the three sheep had the closest genetic relationship with Capra hircus and the furthest genetic relationship with Cervus canadensis.The secondary structure of KAP24.1 was mainly composed of random coil.PEGFP-N1-KAP24.1 was successfully constructed eukaryotic recombinant expression plasmid was successfully transfected into HeLa cells to obtain 58 kDa KAP24.1 recombinant protein. When the concentration of androgen is 10 mol / L, the protein expression is the highest. The expression of KAP24.1 gene in skin and hair follicles of Mountain-type Hetian sheep was significantly different from that of plain-type Hetian sheep (P < 0.05), and there was significant difference between Mountain-type Hetian sheep and Karakul Sheep (P < 0.05). The expression of Karakul Sheep was significantly higher than that of Plain-type Hetian sheep (P < 0.05). The 759 bp CDS sequence of KAP24.1 gene in sheep was cloned, and PEGFP-N1-KAP24.1 was constructed eukaryotic recombinant expression plasmid to obtain 58 kDa KAP24.1 recombinant protein. When the concentration of androgen was 10mol / L, the protein expression was the highest, and KAP24.1 gene was expressed in the skin and hair follicles of three sheep breeds, and the expression of Mountain-type Hetian sheep was the highest.