Function And Regulation Mechanism Of ?_S1-Casein Gene On Milk Protein Synthesis In Dairy Goats

Dairy products,such as cow milk and goat milk,are rich in protein that provides essential amino acids and bioactive peptides for human bodies.Milk protein can be divided into caseins and whey proteins,in which caseins are subdivided into?_S1-casein(CSN1S1),?_S2-casein(CSN1S2),?-casein(CSN2)and?-casein(CSN3).Despite the nutrition value of milk protein,food allergy caused by milk protein could not be ignored.Compared with other milk proteins,?_S1-casein is associated with higher rates of allergies in humans.The content of?_S1-casein in goat milk is much lower than that in cow milk,which is one of the main factor goat milk is not elicit allergic reactions.The content of?_S1-casein is closely related to the milk protein allergy,however,the function and regulation mechanism of CSN1S1 gene are still unclear.By studying the regulation of CSN1S1 gene on milk protein synthesis in goat mammary epithelial cells,as well as the transcriptional and post-transcriptional regulation mechanism of CSN1S1 gene,this study provides a theoretical basis for reducing the?_S1-casein content in milk and improving ruminant milk quality for human consumption.In this study,coding region of goat CSN1S1 gene was cloned and bioinformatics analysis was performed.By using RNA interference and adenovirus overexpression techniques,the regulatory effect of CSN1S1 gene on milk protein synthesis was investigated.The transcriptional regulation effect of STAT5 on CSN1S1 gene was studied by 5'-flanking region cloning and the activity of goat CSN1S1 promoter.The regulation mechanism of miR-204 family members on CSN1S1 expression was investigated by the3'-untranslated region(3'UTR)cloning and post-transcriptional regulation of CSN1S1 gene.The main results are as follows:1.Cloning and bioinformatics analysis of?S1-casein gene in dairy goats.The complete coding region 642bp of dairy goat CSN1S1 gene was cloned.By bioinformatics analysis of amino acid sequences of CSN1S1 genes in 10 mammals,goat,sheep and cattle had the close CSN1S1 gene affinities,while goat CSN1S1 gene is far related to mice and rats.Goat,sheep and cattle CSN1S1 gene functional motifs and domains are similar and they may have similar functions.Protein interaction analysis showed that?_S1-casein may interact with other milk protein components,such as?_S2-casein,?-casein,?-casein,?-lactoalbumin(LALBA),?-lactoglobulin(BLG)and so on.The expression of?_S1-casein and?-casein in goat milk and mammary tissue during lactation was determined.The trend of CSN1S1 and CSN2 gene expression in goat mammary gland during peak-lactation and middle-laction was opposite.The result revealed a negative correlation between the content of?_S1-casein and?-casein in goat milk.2.Regulation of goat?S1-casein gene on other milk protein synthesis.Construction of dairy goat CSN1S1 gene adenovirus vector,the overexpression of CSN1S1 gene in goat mammary epithelial cells(GMEC)significantly down-regulated the expression of CSN2 gene and the synthesis of?-casein.The expression of CSN2 gene and?-casein was significantly up-regulated by si RNA interference with CSN1S1 gene.Overexpression and interference of CSN1S1 gene did not affect the expression of milk protein genes such as CSN1S2,CSN3,LALBA and BLG.The activity of JAK2/STAT5 and m TOR pathways,which are closely related to milk protein synthesis,was also detected.We found that CSN1S1 gene significantly inhibited the phosphorylation of JAK2 and STAT5a,but did not affect the phosphorylation of m TOR.Rescue experiments showed that CSN1S1 gene inhibited CSN2 gene transcriptional activity and?-casein expression by down-regulating the phosphorylation level of STAT5a.The results showed that inhibition of CSN1S1 gene expression not only decreased the content of?_S1-casein,but also up-regulated the expression of?-casein in GMEC.3.Transcriptional activity analysis of?S1-casein gene promoter.The 5'-flanking promoter sequence 2201bp of dairy goat CSN1S1 gene was cloned,containing one TATA-box,located at-33?-26bp upstream of transcription start site.Transcription factor binding sites such as STAT5,C/EBP?,AP-1,GR and YY1,which are important for CSN1S1 promoter activity,were predicted by online software for example,JASPAR database.The results of promoter deletion showed that the active central region of CSN1S1 promoter was located at-110?-18bp upstream,which contained two STAT5binding sites(GAS sites).Overexpression of STAT5a gene significantly up-regulated the m RNA level and promoter activity of CSN1S1 gene.After treatment with STAT5 inhibitor STAT5-in-1,the phosphorylation level of STAT5 in cell nucleus was significantly decreased,and the promoter activity,m RNA and protein levels of CSN1S1 gene were significantly down-regulated.Through site-directed mutagenesis of three conserved GAS sites in the CSN1S1 promoter region,both GAS1 and GAS2 sites in the core region were binding sites of STAT5.Chromatin immunoprecipitation(Ch IP)assays confirmed the direct binding effect of STAT5 with these two sites.Therefore,STAT5 could directly regulate the transcription of CSN1S1 gene by GAS1 and GAS2 sites in GMEC,and mutation of these two sites can down-regulate the expression of CSN1S1 gene and the synthesis of?_S1-casein.4.Posttranscriptional regulation of the 3'-untranslated region of?_S1-casein gene.The 3'UTR sequence 429bp of dairy goat CSN1S1 gene was cloned,and miR-204family members,miR-204-5p and miR-211,were predicted to target the 275?281nt sites in the 3'UTR region of CSN1S1 gene by online software for example,Target Scan database.The expression of miR-204-5p and miR-211 in the goat mammary tissues during peak of lactation was significantly lower than that in the middle of lactation,which was contrary to the expression trend of CSN1S1 gene.The site mutation assays showed that miR-204-5p and miR-211 synergically inhibited the expression of CSN1S1 gene and?_S1-casein by targeting the 3'UTR specific sequence of CSN1S1 m RNA.In addition,miR-204-5p and miR-211 up-regulated the expression of CSN2 gene and the synthesis of?-casein,while up-regulated the activity of STAT5a.Rescue assays confirmed that miR-204-5p and miR-211 enhanced the expression of?-casein through the CSN1S1-STAT5a regulatory axis.The Ch IP assays showed that the transcriptional activity of CSN2 gene was up-regulated by promoting the STAT5a activity.miR-204-5p and miR-211 cooperativly down-regulated the content of?_S1-casein and up-regulated the content of?-casein by targeting CSN1S1 gene in GMEC.In conclusion,goat CSN1S1 gene negatively regulates?-casein expression,and?_S1-casein expression could be down-regulated through both transcriptional and post-transcriptional regulation ways in GMEC.This study described the function and expression regulation mechanism of CSN1S1 gene in dairy goats,and provided a theoretical basis for reducing?_S1-casein content in milk and decreaing ruminant milk protein allergy potential.