Cloning And Activity Analysis Of Sheep KAP7 And KAP8 Gene Promoters

Wool is an important source of raw materials for the textile industry and animal husbandry economy income.The characterists such as fiber length,diameter and crimp of wool determine its ultimate economic value and utility.Fine wool sheep breeding has become an important study direction in the field of animal husbandry.During in recent years,the researchers had explored wool traits and their related genes with the aim to breed fine wool sheep with high yield and quality wool fibre through molecular marker assiated breeding.It has been proved that the the genetic diversity and express levels of high-glycine-tyrosine KAPs(HGTP)families of sheep are related to the thickness and length of wool.Promoter is an important element in regulating gene expression and its specific role in gene expression deserves further study.In view of this,the assays including the gene cloning,genetic polymorphism analysis,bioinformatics analysis,DNA methylation detection and forecast detection and core promoter activity analysis and fiber cell culture optimization were taken out to study HGTP family KAP7 and KAP8 gene promoters,and further to analyze the promoter activity and gene expression regulation mechanism.The results are summarized as follows:(1)We cloned the 5 ’promoter of KAP7 from Chinese Merino sheep and Suffolk sheep with a length of about 2290 bp.The wild type sequences of coarse and fine wool sheep were consistent.Genetic polymorphism analysis found 27 SNPs belonged to 25 haplotypes,of which 3 SNPs were unique to coarse wool sheep and 17 SNPS were unique to fine wool sheep.There were significant interspecific and intraspecific differences in KAP7 promoter.Bioinformatics analysis showed that-903 ～-853 is the core promoter region of KAP7.But there was no CpG island,and no DNA methylation occuring.The promoter sequence contains CREB,Hox C13,TST-1,C/EBP,AP-2,LEF-1,Sp1,POU2F1,Sp3 and other important transcription factor binding sites.(2)Sequence analysis revealed that there was a(CA)n repeat species-specific STR locus in the promoter region of KAP8 gene.When the repeat number n was17/18 the sheep breed is identified to fine wool sheep,and n was 23/24 is non-fine wool sheep,and n was 12+11/13+11 is a hybrid sheep.This STR locus can be used as a molecular marker for fine wool sheep breeding.The region located at-721 to-590 is the core promoter region,and there are several important transcription factor binding sites located in this region such as C/EBP,Sp1,Hoxc13,TST-1,CREB,NF-Kap PA-B,AP-2 and AP-1.GCGC repeats were found in the-500 ～-400 of representative sequences of coarse and fine wool.However,the degree of methylation modification was different.Three and one methylation modification sites had been detected in fine wool and coarse wool sheep,respectively.Furthermore,the methylation modification resulted in one transcription activator Sp1 binding site disappearing in fine wool sheep,which led to promoter activity discreasing.(3)A combination of trypsin and type I collagenase was used to digest the ear skin tissue of newborn lambs.By optimizing the culture process,fibroblasts were obtained successfuly.The results showed that after 2-3 days of cell culture,fibroblasts with 90% purity could be obtained.The time of this procedure was shortened to 7 days,which greatly shortened the culture cycle and improved the separation efficiency.(4)Seven and six gene deletion fragments of KAP7 and KAP8 promoters of Chinese Merino sheep were designed and cloned,respectively.The double luciferase reporter gene vector pGL3 was constructed and transfected into sheep fibroblasts to detect promoter activity.The results showed that the activities of PGL3-F7 and PGL3-F8 were significantly higher than those of pGL3 negative control.The promoters of KAP7 and KAP8 genes were typical eukaryotic promoters,which could drive the specific expression of exogenous reporter genes in sheep skin fibroblasts.And fragments of F7.1 and F8.1 were identified as the core promoter regions of KAP7 and KAP8 genes,respectively.There are strong activating transcription factor binding sequences such as C/EBPalp,AP-1,C/EBPbeta and Sp1 in both KAP7 and KAP8 gene promoters.Negative regulatory transcription factor binding sites such as OCT-1(POU),GATA-1 and NF-1 are specific in the promoter region of KAP7,and promoter activity is enhanced and gene expression is increased after deletion of this sequence.However,the specific mechanism of action and its application in sheep breeding need to further study.