The Effect Of Gradient Deletion Leu,Ile,Met And Thr On Signal Pathway Of Milk Protein Synthesis In MAC-T

Amino acids are not only the substrate for protein synthesis,but also regulate the synthesis of proteins.Recently,in vitro experiments have verified that Met,Thr,Ile and Leu can affect the synthesis rate of casein,Ile and Leu can promote the phosphorylation of mTOR.In this study,Leu,Ile,Met and Thr of different concentrations were used to observe the effects the related pathways of mammary epithelial cells and protein signaling in cows.Experiment 1: Leu content is normal medium concentration of 0,25%,50%,75%,100%,there were no significant differences of cow mammary gland epithelial cell proliferation(P =0.95),but the performance of the rise in value,lack the same percentage of Met,Thr,is the same(P values were 0.89,0.89).However,as the content of Ile increased,there was a decrease in cell proliferation,but there was no statistically significant difference(P=0.97).Test 2: this experiment used the culture medium of the four amino acids as the control group to observe the effect of single amino acid on the expression of the mRNA in the signal pathway.When Leu was absent,the expression of 4eBP1 was significantly lower than that of the control group(P<0.01).There was no significant difference in mRNA expression of mTOR,S6K1,Akt and eEF2(P value was 0.51,0.28,0.62,0.16).When missing Ile,the expression of 4eBP1 amount compared with the control group showed a trend of decline after rising first(P=0.08),when adding Ile,mTOR,AKT,eEF2 mRNA expression quantity significantly reduced,with the increase of concentration of Ile after expressing quantity has a tendency to increase(P<0.01).When the Met was added,the expression of 4eBP1 decreased compared with the control group(P<0.05),while the other four mRNAs showed no significant difference.In the absence of Thr,there was no significant difference in the five mRNA.Test 3:The design was the same as the test 2.When the Leu concentration reached 25%,the phosphorylation degree of mTOR increased significantly(P<0.01),but there was nosignificant difference in the phosphorylation degree of eEF2,Akt and S6K1.The phosphorylation degree of Akt showed a trend of first decreasing and then increasing of Ile.The phosphorylation degree of mTOR increased significantly at 100% Ile concentration(P<0.01),but there was no significant difference in eEF2.The Akt phosphorylation in content have dropped 75%,100% Met(P = 0.37),while S6K1 in concentration of 50%,75% and100% when the degree of phosphorylation is significantly higher than the first three groups,and add the concentration of 0,25%,compared with the control group did not differ significantly(P < 0.01);Thr phosphorylation of mTOR in content is 0%,75%,significantly higher than other groups(P = 0.08),Akt phosphorylation degree as the amino acid added significantly reduced,and the addition amount of 100% rebound trend,there was no significant difference between other concentrations(P < 0.01),the phosphorylation of S6K1 level as add Thr gradually reduced,while 50% Thr is minimum,after a slow recovery(P <0.01).Above all,Leu,Ile,Met and Thr missing a single gradient has no effect on cell proliferation,and for some amount of mRNA expression and protein signaling pathways leading to the influence degree of protein phosphorylation,signal has confirmed that the four kinds of amino acids,and it also indicates that there may be antagonism between amino acids.