SLC7A5 Gene Regulating The Synthesis Of Milk Protein And Milk Fat Through MTOR In Dairy Goat Mammary Epithelial Cells

LAT1(Large neutral amino acids transporter small subunit 1)is a Na+ and p H-independent transporter protein encoded by the SLC7A5(Solute carrier family 7 member5)gene,which has amino acid(AA)transporter activity such as leucine and tryptophan.It is involved in carboxylic acid and thyroid hormone transport as well as exogenous transport.It forms a complex with the disulfide bond of glycoprotein 4F2hc(4F2 cell-surface antigen heavy chain)to transport essential amino acids such as leucine and regulates cell growth by regulating the uptake of AA.The transcriptome sequencing of the SLC7A5 gene in Saanen dairy goats with extremely high and low milk protein rate and milk fat rate revealed that the SLC7A5 gene is a key candidate gene for regulating milk protein and milk fat synthesis,so the Goat mammary epithelial cells(GMECs),the SLC7A5 gene(CDS),were used as the test material.The effect of the SLC7A5 gene on the synthesis of milk protein and milk fat in GMECs was investigated by constructing an overexpression vector and interfering RNA and transfecting GMECs.The mechanism of the SLC7A5 gene regulating milk protein and milk fat synthesis was investigated by rescue assay.The following main findings were obtained:1.Effect of SLC7A5 gene on the expression of key genes for milk protein and milk fat synthesis in GMECsThe SLC7A5 overexpression plasmid was successfully constructed and the recombinant eukaryotic expression plasmid p EGFPC1-SLC7A5 was obtained;meanwhile,based on the sequence of the SLC7A5 gene,si RNA was designed and synthesized,and the best silencing effect of si RNA-1063 on SLC7A5 gene was screened.Overexpression the of SLC7A5 gene in GMECs resulted in significant up-regulation(P < 0.05)of m RNA and protein expression of CSN1S1,a gene related to milk protein synthesis;significant up-regulation(P < 0.05)of m RNA expression levels of SCD1,C/EBPβ and ACACA,a gene related to milk fat synthesis,and increased accumulation of lipid droplets.Interfering with the SLC7A5 gene,the m RNA and protein expression of CSN1S1,a gene related to milk protein synthesis,was significantly down-regulated(P < 0.05);the m RNA expression levels of SCD1,FASN and C/EBPβ,a gene related to milk fat synthesis,were significantly down-regulated(P < 0.05),and the accumulation of lipid droplets was reduced.In conclusion,the SLC7A5 gene had significant regulatory effects on milk protein and milk fat synthesis in GMECs.2,Effect of interfering with SLC7A5 gene on AA/Leu-stimulated m TOR signaling pathwayAfter interfering with the SLC7A5 gene,the activity of m TOR was significantly inhibited,and the phosphorylation levels of S6 K and S6 were significantly reduced(P <0.05);under AA or Leu stimulation,the phosphorylation levels of S6 K and S6 were gradually enhanced in both the interfered and control groups with the extension of stimulation time,but the enhancement of phosphorylation levels of both proteins in the interfered group was smaller than that in the control group.It can be seen that the SLC7A5 gene regulates the m TOR signaling pathway by regulating the uptake of AA,especially Leu.Inhibition of SLC7A5 gene expression attenuated the uptake of AA or Leu by GMECs to a certain extent,which in turn attenuated the role of the m TOR signaling pathway.These results suggest that SLC7A5 gene can activate m TOR signaling pathway under the stimulation of AA,especially Leu,and then exert regulatory effects on biological processes.3.SLC7A5 gene regulates milk protein and milk fat synthesis in GMECs through the m TOR signaling pathwayThe addition of the m TOR inhibitor Trion1 while overexpressing the SLC7A5 gene in GMECs showed that the CSN1S1 m RNA expression level was down-regulated compared to the SLC7A5 overexpression alone group,and the protein results were consistent with the fluorescence quantification results.However,the addition of m TOR agonist MHY1485 while interfering with SLC7A5 did not regress the CSN1S1 m RNA expression level compared with the SLC7A5 alone group;the regression effect of protein results was not significant.This may be due to the reduced availability of AA after the reduction of cellular SLC7A5 resulting in the inability to sustain the translation process.The results of milk lipid-related gene assay showed that the m RNA expression levels of SCD1 and FASN were reduced when SLC7A5 gene was simultaneously overexpressed and Torin1 was added,and the m RNA results of SCD1 and FASN were consistent when SLC7A5 gene was interfered with while MHY1485 was added.This reveals that in GMECs SLC7A5 gene may promoteαS1-casein synthesis and affect milk protein synthesis through positive regulation of m TOR signaling pathway;SLC7A5 gene may regulate the expression of SCD1 and FASN genes through m TOR signaling pathway and affect the synthesis of milk lipids.This study revealed that the SLC7A5 gene regulated the synthesis of milk protein and milk fat in GMECs through the m TOR signaling pathway under the stimulation of AA,especially Leu,by interference and overexpression techniques.The results provide a theoretical basis to investigate the molecular mechanism of milk protein and milk fat synthesis in GMECs,improve the molecular regulatory network of milk component synthesis and metabolism in dairy goats,and thus improve the quality of goat milk.