Cloning And Transcriptional Activity Of LF Gene Promoter Of Dairy Goat

Milk proteins are the main nutritional ingredient of goat milk,have the characteristics of high value,digestible and absorbable,also contain many kinds of active ingredients.Their types and concentrations greatly influence the quality of the milk.Lactoferrin(LF)is a multifunctional glycoprotein,as an important physiological active substance,it has extensive and unique biological activities and physicochemical characteristics,and has been used in the field of foodstuff,dairy industry,pharmacy,biochemistry,feed,cosmetic and other applications.Therefore,studies on transcriptional regulation mechanism of LF gene promoter of dairy goat have crucial value for breeding and the regulation of goat milk composition.The objective of this study was to clone the promoter region of LF of dairy goat and analyzed its structure and function.Eight promoter fragments with different length were obtained by progressive deletion.Then,the promoter fragments were cloned into pGL-3 luciferase reporter gene vectors.The constructs and the pRL-TK were transiently co-transfected into goat mammary epithelial cells.The core region of the promoter with basal transcriptional activity was determined with dual-luciferase reporter assays.We also investigated the transcription regulation mechanisms of goat LF promoter by transcriptional factors ER.The main results are as follows:1.Cloning of LF gene in goat mammary gland showed that the full-length of LF gene was 2 127 bp which encode 708 amino acids.After analyzed the secondary protein structure of LF,we found that this protein was mainly composed of alpha helix and irregular curl.In addition,LF tertiary structure 3D model diagram was also predicted.The concentrations of LF in colostrum and milk samples were determined using ELISA kits.Lactoferrin content in colostrum was 222.6±41.57 ?g/mL,then throughout the lactation period the mean concentrations remained stable,ranged between 34.61 and 51.94 ?g/mL.2.We obtained a 2 070 bp of the 5' flanking sequence,containing 1 935 bp upstream of transcription start site(+1),the first exon(+37 to +119),the first intron(+1 to +36)and part of the second intron.An intiation codon ATG was located in exon 1 and TATA-box was found in the promter of LF.Multiple alignment analysis indicated that the homology of LF promoter compared with bovine,human and mouse was 89.2%,55.25% and 39.85%,respectively.Morever,some consensus bingding sites for transcription factors such as PPAR-?,PPAR-?,LXR-?,C/EBP-?,Sp1,and NF-? were observed upon bioinformatics analysis.3.Detecting the promoting activities of deletion fragments by using the dual-lueiferase reporter assay system.The regulatory elements for goat LF transcription are located in forepart of LF promoter.The region from-84 to-46 is necessary and required for basal trsnscription activity of goat LF gene.The results of site-directed mutagenesis showed that mutation in ERE significantly decreased the promoter activity.These data suggested that three functional ERE elements on the LF promoter(-1 038 bp,-1 144 bp and-1 777 bp),which play an important role in maintaining the basal activity of promoter.In conclusion,the full-length of LF gene was 2 127 bp,the maximal concentrations of LF in milk were observed in colostral samples.The present study cloned the 5 'flanking sequence of goat LF promoter,and it has high homology with bovine LF promoter.The core region of goat LF promoter is from-84 to-46 bp by deletion analysis.The results of site-directed mutagenesis showed that EREs play an important role in transcriptional regulation of LF.