The Regulation Of DGAT1 Gene On Lipid Metabolism In Mammary Gland Epithelial Cells Of Dairy Goat

The lipid content and composition in goat milk are important manifestations of its nutritional value.The lipids in goat milk are mainly composed of triglycerides(TAG).Diacylglycerol O-acyltransferase 1(DGAT1)is the only rate limiting enzyme in the synthesis of triglycerides,catalyzing the production of triglycerides from diacylglycerol(DAG)and acyl Co A(acyl Co A).Therefore,studying the function and transcriptional regulation of DGAT1 in lipid metabolism of dairy goat mammary gland epithelial cells has important theoretical and practical significance.In this study,we cloned the coding region sequence of DGAT1 from dairy goat and constructed overexpression vector of DGAT1;Screening out a DGAT1 specific interfering RNA with good effect;Using dairy goat mammary epithelial cells as raw materials,the effects of DGAT1 on lipid metabolism of GMECs were preliminarily revealed by using overexpression,interference,and lipomics techniques;The DGAT1 promoter sequence was cloned;The transcriptional active center of DGAT1 promoter was identified by deletion vector test;To screen key transcription factors and preliminarily explore the transcriptional regulation of transcription factors on DGAT1.The main results of this study are as follows:1.The CDS region sequence of DGAT1 1470 bp was successfully cloned,and the overexpression vector pc DNA3.1-DGAT1 was successfully constructed.Overexpression of DGAT1 in GMECs significantly increased the expression of PLIN3,and PLIN2 genes(P<0.05),and significantly increased the m RNA content of SREBP1 c,SCD1,FADS2,and FABP3(P<0.01);At the same time,it increased the triglyceride content(P<0.01)and promoted the accumulation of lipid droplets(P<0.05);The content of C20:3,C20:4,and C22:6was significantly increased(P<0.05).2.Screening out a DGAT1 interfering RNA with good effect.Inhibiting DGAT1 in GMECs significantly decreased the expression of CEBPA,FABP3,and ATGL(P<0.05),significantly increased the m RNA content of AGPAT6(P<0.01);Reduce the content of triglycerides(P<0.01)and lipid droplets(P<0.01);Significantly reduce C20:1 content.3.1630 lipid molecules were identified in GMECs.Overexpression of DGAT1 resulted in an upregulation of 24 lipid molecules,mainly triglycerides;Downregulated 448 lipid molecules,mainly glycerol phospholipids and sphingolipids.Differential lipids are mainly concentrated in glycerol phospholipid metabolism,c AMP signaling pathway,phospholipase D signaling pathway,phosphatidylinositol signaling system,α-linolenic acid metabolism,linoleic acid metabolism,and arachidonic acid metabolism.4.The DGAT1 promoter sequence 2071 bp was cloned.Bioinformatics analysis revealed binding sites such as RUNX1,CEBPA,SP1 and PPARG on the DGAT1 promoter;By constructing a deletion vector,it was found that the transcriptional active center of the DGAT1 promoter was located between-100 and-40 bp upstream of the transcriptional start site;After constructing single and double mutation vectors at the RUNX1 binding site,it was found that RUNX1 negatively regulates DGAT1 transcription through the RUNX1-1 site at the transcriptional active center;The expression of DGAT1 and RUNX1 in breast tissues at different lactation stages was detected and it was found that the expression of DGAT1 was the highest in early lactation and peak lactation,and the lowest in dry period.The expression level of RUNX1 was the lowest during peak lactation and the highest during dry period.In summary,DGAT1 plays an important role in lipid metabolism in dairy goats,and the transcription factor RUNX1 negatively regulates the expression level of DGAT1 gene.