Overexpression Of Btn Gene Promotes Lipid Droplet Synthesis In Dairy Cows

Milk fat is one of the main components of milk,which can provide energy and improve milk flavor.It has a high nutritional value,and milk fat content is an important economic indicator of the dairy industry,affecting the quality and price of milk.The mammary gland is the main organ for the synthesis of milk fat,and blactofornin BTN1A1(BTN)plays an important role in the synthesis of lipid droplets in mammary epithelial cells.Therefore,this study used the over-expressed BTN protein to study its role in the formation of breast cell lipid droplets,laying the foundation for elucidating the mechanism of fat synthesis in breast epithelial cells.In this experiment,BTN-PEGFP-N1 recombinant vector was firstly obtained and transfected into the MAC-T of breast epithelial cells in dairy cows.The subcellular localization of green fluorescent BTN protein was observed by fluorescence microscopy.The Lenti CRISPR V2 plasmid was used as the backbone.Btn recombinant lentiviral vector was packaged into virus to infect MAC-T cells,and Btn-overexpressing MAC-T cell line was constructed and its genotype was identified.Overexpression of Btn m RNA was detected by real-time fluorescence quantitative PCR(QPCR).Nile red staining and microplate reader were used to observe the effect of Btn overexpression on lipid droplet morphology.The results showed that the Btn gene was 1578 bp in the CDS region,and Btn could be located on the top of the cytoplasmic membrane by fluorescence microscopy.The recombinant lentiviral vector with Btn was successfully constructed,and the use of lentivirus to mediate Btn in MAC-T cells was achieved.In the overexpression,the Btn1a1 overexpressed 52961-MAC-T cell line was screened by puromycin,and the Btn m RNA expression level of 52961-MAC-T cells was increased 400-fold compared to the control cells(p<0.0001);Detection of lipid droplet fluorescence values revealed that Btn overexpression significantly increased lipid droplet content(p<0.0001).This study shows that overexpression of Btn in mammary epithelial cells in dairy cows can affect the expression of fat synthesis genes in cells and promote the synthesis of lipid droplets in mammary epithelial cells.