Porcine ZBED6 Gene And T?4 Gene Promoter Core Sequence Analysis

In this group,the ZBED6 gene and T?4 gene were screened out during the cold stress experiment of the early period swine pigs.There was a significant difference in the expression of both genes.The ZBED6 gene associated with muscle growth and development and the T?4 gene significantly related to hair follicle development and hair growth were The gene of interest was analyzed for promoter activity and selection of transcription factors to study the effects of transcriptional regulation on the growth traits of the boar.Mutation of zinc finger protein ZBED6(zinc finger bed domain containing 6,ZBED6)gene can promote significant upregulation of the transcription level of insulin-like growth factor 2(IGF2)in skeletal muscle,thereby promoting cell proliferation and muscle tube formation.The overexpression of ZBED6 not only inhibits the transcription of IGF2,but alsoregulates thousands of important genes that are closely related to developmental diseases,cancer and cardiovascular disease.However,the mechanism of transcriptional regulation of thegene itself is still unknown.The thymosin beta4(T?4)gene is a gene that is significantly associated with hair growth and hair follicle development.The outer hair root sheath cells of hair follicles showed significant proliferation effect with the intervention of thymosin ?4,indicating that thymosin ?4 stimulated hair follicle growth by stimulating the differentiation and proliferation of outer hair root sheath cells of hair follicles,but the specific mechanism was not yet clear.This study using molecular biology techniques to ZBED6 gene and T?4 gene cloning,the 5'end sequences by bioinformatics methods to predict two genetic potential of transcription factor binding sites,build ZBED6 gene and T?4 5' end sequences of different length of missing pieces of fluorescent reporter gene expression vector,using dual luciferase report system of the two analyses active gene promoter regions,missing mutation and expression technique is used to determine the two gene transcriptional regulatory elements,obtain the main results were as follows:(1)The "A" of the initial codon of the translation was "+1",and the cloned porcineZBED6 gene-2053~21 bp sequence was cloned,which was detected by the biluciferase reporter system and the promoter activity was activated.The smallest active region was found between-1808~1777 bp,and there was a positive regulating element.(2)Using on-line software to predict the-1808 to-1777 bp region of the ZBED6 gene,there are five transcription factor binding sites for CRETaul,NKx2-5(var.2),HINFP,Adf-1,and CREB3.Mutation of the recognition sites of Adf-1 and CREB3 could significantly reduce the promoter activity of ZBED6 gene(P<0.01).However,overexpression of CREB3 did not promote the expression of ZBED6,indicating that CREB3 is not a key transcription factor that affects ZBED6.Since the porcine's Adf-1 sequence is unknown,the ZBED6 gene's minimum activity region can only be determined within-1808 to-1777 bp.(3)With the "A" of the translation initiation codon as "+1",the porcine T?4 gene sequence of-1188 to +29 bp was cloned and detected by the dual luciferase reporter system,and it had promoter activity.In the segmented deletion,the smallest active region was located between-155 bp and-105 bp,with positive regulatory elements.(4)Using bioinformatics software to predict the-155~-105 bp region of T?4 gene,there were three transcription factor binding sites for ELK-1,MYBAS1 and E2F-1.Mutation of ELK-1 recognition site could significantly reduce the promoter activity of T?4 gene(P<0.05).Overexpression analysis of ELK-1 transcription element can promote the expression of T?4 gene,demonstrating that ELK-1 is a key factor in the transcription regulation of T?4 gene.