| Milk is a valuable nutritious food,which contains proteins,fat,sugar,amino acids,minerals,vitamins,and other nutritional ingredients required by the human body.Mammary glands,as the sole organ of milk synthesis,secretion and storage,determine the yield and quality of milk.Due to the different dairy cow body condition,there are different levels of gene expression in Holstein dairy cow mammary gland during mid and late lactation,resulting different milk fat content.98%of milk fats are triglycerides(TAG).Triglyceride molecules are formed by the dehydration and condensation of one glycerol molecule and three fatty acid molecules.Long-chain acyl-CoA synthetase 4(ACSL4).a key enzyme in lipid synthesis,is a member of the ACSL family and long chain fatty acids C:12?C:22 are mainly catalyzed as long chain fatty acid CoA.However,this gene was little studied in bovine mammary epithelial cellsIn this study,the milk fat content of Holstein dairy cows in a lactation was detemined,and transcriptome sequencing was performed on the mammary tissues in the mid and late lactation,revealing the genetic changes in the mid and late lactation of Holstein dairy cows at the molecular level.The PPAR signaling pathway related to milk fat synthesis was significantly significant change in late lactation(P<0.05),and ACSL4 in this pathway was up-regulated in late lactation(P<0.05).Thus,according to the cow's ACSL4 gene sequence in NCBI,specific primers were designed in the conserved region,and the ACSL4 gene was cloned using bovine mammary epithelial cells as cDNA template.The sequence of the cloned genes was analyzed and the subcellular localization of the proteins was predicted using PSORT Prediction.pcDNA3.1+-ACSL4 eukaryotic expression vector was constructed and transfected into bovine mammary epithelial cells and the expression of ACSL4 in bovine mammary epithelial cells was inhibited by siRNA.Then the changes of triglyceride content and the expression of gene related lipid synthesis in the cells were measuredThe results show that:(1)Compared with the mid lactation,a total of 470 differentially expressed genes were found in the Holstein cow mammary tissues in late lactation,in which 266 were down-regulated and 204 were up-regulated.ACSL4 was significantly up-regulated in the late lactation(P<0.05);(2)The Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis results showed that the PPAR signaling pathway related to milk fat synthesis was significantly up-regulated in the late lactation;(3)The full length of bovine ACSL4 gene is 2136 bp,encoding 711 amino acids,including 89 positively charged residues and 77 negatively charged residues.The coding protein has a positive charge in the whole.the molecular weight is 79261.10,and the theoretical isoelectric point is 8.71.The coding region of ACSL4 is highly conserved in different varieties,and the protein encoded by ACSL4 mainly playes a role in the endoplasmic reticulum;(4)When ACSL4 was overexpressed in cow breast epithelial cells,the content of triglyceride increased significantly(P<0.05),and the expression levels of PABP3 and ELOVL6 were significantly up-regulated(P<0.05).while the expression levels of ACSL1.PPARD FASN.FADS2 and CPT1A were not significantly changed(P>0.05).(5)When ACSL4 was inhibited in bovine mammary epithelial cells,the content of triglyceride decreased significantly(P<0.05),and the expression levels of ACSL1,PABP3,CPT1A,ELOVL6 and FASN were significantly down-regulated(P<0.05).The expression levels of FADS2 and PPARD did not change significantly(P>0.05)The results revealed the hanges in gene expression levels during the mid and late lactation in Holstein dairy cows and the effects of ACSL4 on the changes of triglyceride expression levels in bovine mammary epithelial cells. |
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