Transcriptome Sequencing And Expression Of KAP2.12 Gene In Hair Follicles Of Hetian And Karakul Sheep

Hetian sheep belongs to the characteristic breed of sheep in Hetian area of southern Xinjiang.Due to the difference of producing areas,Hetian sheep can be divided into plain area(HP)and mountainous area(HS).This study aims to obtain transcriptome information of sheep skin tissue by transcriptome sequencing to explore the molecular mechanism of signaling pathways related to hair follicle development.Then,on the basis of transcriptome sequencing,a gene of sheep hair follicle keratin was cloned and expressed,which provided a theoretical basis for the verification of functional genes of keratin.The main research contents and results are as follows:(1)Transcriptome study of sheep skin tissueThrough the study of skin tissue of three different breeds of sheep,the differences among the samples were analyzed.With KL as control,there were 5269 up-regulated genes for HS,9051 down-regulated genes for HS,3698 up-regulated genes for HP,and 5214 down-regulated genes for HP.Through GO enrichment and KEGG enrichment analysis,178 DEGs were found to be related to 33 pathways according to the screening conditions,which provided a theoretical basis for further study on the molecular mechanisms regulating the growth and development of hair follicles.(2)Cloning and prokaryotic expression of KAP2.12 geneThe whole length of KAP 2.12 gene CDS was obtained from the skin hair follicles of HS,HP and KL by TA cloning technique,and bioinformatics analysis was performed on the obtained genes.KAP2.12 gene was constructed into p ET28a(+)prokaryotic expression vector to obtain p ET28a(+)-KAP2.12 prokaryotic expression vector,and then transformed into strain BL21(DE3)for prokaryotic expression of the gene.SDS-PAGE and Western Blot were used to detect the protein,and the target protein with a size of 15 k D was finally obtained.(3)Eukaryotic expression of KAP2.12 geneThe eukaryotic expression primers of KAP2.12 gene were designed and constructed into the eukaryotic expression vector p EGFP-N1 to obtain p EGFP-N1-KAP2.12.The recombinant expression vector p EGFP-N1-KAP2.12 was then transferred into COS-7 cells for fluorescence detection and Western Blot analysis.The size of the target protein was about 43 k D.