Effects Of BDH1 Gene On Lipid Metabolism Of Mammary Epithelial Cells In Dairy Goat

BDH1(β-hydroxybutyrate dehydrogenase 1)is a member of the gene family encoding short-chain dehydrogenase/reductase,and it is mainly involved in the metabolic processes of fatty acids,triglycerides and ketone bodies.The protein encoded by BDH1 catalyzes the interconversion of acetoacetate(ACAC)and(R)-3-hydroxybutyrate((R)-3-hydroxybutyrate,BHBA),which are produced during fatty acid catabolism Two major ketone bodies are important for lipid metabolism in the body.However,there is no report on the study of BDH1 gene on the lipid metabolism of dairy goat mammary gland.Therefore,it has important theoretical and practical significance to study the effect of BDH1 gene on dairy goat mammary lipid metabolism.In this study,dairy goat primary mammary epithelial cells were used as experimental materials,and the CDS region sequence of BDH1 gene was obtained by PCR cloning technology,and tissue expression profiling and bioinformatics analysis were performed on it.Eukaryotic overexpression vector and synthesis of effective si RNA,using overexpression and si RNA interference technology to explore the effect of BDH1 on lipid metabolism of milk goat mammary epithelial cells;combined with the study of BDH1 on lipid metabolism of BHBA-treated GMECs,to preliminarily explore the effect of BDH1 gene on milk Regulatory mechanisms of lipid metabolism in GMECs.The main results of this study are as follows:1.The CDS region of the goat BDH1 gene was successfully cloned,with a length of1035 bp,encoding a total of 344 amino acids.The goat BDH1 gene is closely related to sheep(XM＿004003025.4)and cattle(XM＿005201505.4),and is far from human(NM＿004051.5)and mouse(XM＿036160130.1).The BDH1 gene was expressed at a moderate level in the mammary gland of dairy goats.2.The milk goat pcDNA3.1-BDH1 eukaryotic over expression vector was successfully constructed.After over expression of BDH1 gene,the m RNA expression of SREBP1 gene was significantly decreased(P<0.05),the m RNA expression of ELOVL6,ACSL1 and GPAM genes was significantly decreased(P<0.01),and the m RNA expression of FABP3 gene was significantly increased(P<0.05);at the same time,it can significantly reduce the triglyceride content and lipid droplet accumulation in mammary epithelial cells(P<0.05);the intracellular fatty acid C10:0 content is extremely significantly increased(P<0.01),C14:0and C15 The content of :0 decreased significantly(P<0.05);the content of C17:1 decreased significantly(P<0.01).3.Synthesized effective si RNA-BDH1,and screened out si RNA-1032 with the best interference effect on BDH1 gene.Interfering with BDH1 gene can extremely significantly increase the m RNA expression of SREBP1,LXRα and LPL genes(P<0.01),significantly increase the m RNA expression of ELOVL6,CD36,GPAM and DGAT1 genes(P<0.05),and significantly reduce FABP3 and ATGL genes m RNA expression(P<0.05);at the same time,it can significantly increase the triglyceride content and lipid droplet accumulation in breast epithelial cells(P<0.05);the intracellular fatty acid C14:0,C20:1 and C20:4 content significantly increased(P<0.05),the content of C14:1 and C15:1 increased very significantly(P<0.01),and the content of C18:0 decreased extremely significantly(P<0.01).4.After BHBA treatment of dairy goat mammary epithelial cells,the m RNA expression of BDH1 gene was very increased(P<0.01),and the m RNA expression of FASN,ACCα,LPL,SREBP1,FABP3,ACSL1 and DGAT1 was extremely significantly decreased(P<0.01).GPAM gene m RNA expression decreased significantly(P<0.05);triglyceride content decreased significantly(P<0.05).After co-treatment of mammary epithelial cells with si RNA-1032 and BHBA,the m RNA expressions of FASN,LPL,SREBP1,ACSL1 and GPAM genes were extremely significantly increased(P<0.01),and the m RNA expression of FABP3 genes was extremely significantly decreased(P<0.01);glycerol Triester content increased very significantly(P<0.01).The results indicated that BDH1 could negatively regulate the inhibitory effect of BHBA on lipid synthesis in GMECs.In conclusion,BDH1 gene negatively regulates milk fat metabolism in dairy goats,and BDH1 gene can negatively regulate the inhibitory effect of BHBA on lipid synthesis in dairy goat mammary epithelial cells.This lays a theoretical foundation for in-depth exploration of the regulatory mechanism of BDH1 gene on lipid metabolism in GMECs.