Influence Of Methionine On Subcellular Proteins Of Dairy Cow Mammary Gland Epithelial Cells

Low milk yield and milk protein content have been restricting the development of Chinese dairy industry, study of lactation regulation mechanism on dairy cows have both theoretical and practical significance. Methionine is an important precursor of milk protein synthesis, previous studies mainly focused on the impact of methionine on dairy production performance, while the effects of lactation regulation on dairy cow mammary gland epithelial cells (DCMECs) are poorly researched. It is needed to study the relationship between methionine and milk protein synthesis as well as lactation ability of mammary gland, understand the molecular mechanism at protein level and find the crucial proteins which correlate with lactating.In this study, in vitro cultured DCMECs model was successfully established. Effects of methionine on proliferation of DCMECs were evaluated by HPLC, RT-PCR, CASY and triglyceride reagent to determine the optimum concentration of methionine, two-dimensional electrophoresis was applied to analyze subcellular proteomics of healthy DCMECs and methionine-supplemented DCMECs cultured in vitro. The Image Master2-DE software was used to characterize different proteins and differentially expressed protein spots were identified by MALDI-TOF-MS.The main results of the present study were as follows:(1) Methionine was added to the DMEM/F12culture medium at concentrations of0,0.2,0.4,0.8,1.2and1.6mmol/L. Culture period was24h. The results showed that1.2mmol/L methionine significantly improved the proliferation of DCMECs (P<0.01), it’s the best concentration for DCMECs cultured in vitro.(2) We constructed the two-dimensional gel electrophoresis profiles for subcellular proteins of DCMECs. The2-DE profiles had both good resolution and reproducibility. Differentially expressed protein spots in DCMECs between control and methionine-supplemented were screened and identified by mass spectrum.5up-regulated protein spots in methionine-supplemented DCMECs group were screened out by Image Master6.0software. These spots were identified by MALDI-TOF-MS and searched in the NCBInr and EBI databases, including carnitine acetyltransferase, dihydropyrimidinase-related protein2, ornithine aminotransferase, NADH dehydrogenase [ubiquinone] flavoprotein2, apolipoprotein A-I-binding protein. The results showed these differentially expressed proteins were mainly involved in amino acid transport, energy metabolism and cytoskeleton composition, etc.(3) The2-DE results were verified on the mRNA level by RT-PCR. These differentially expressed proteins had the highest expression level in high lactation level dairy cows, the secondly expression level in low lactation level dairy cows, and the lowest expression level in dry period cows. The differentially displayed proteins between control and methionine-supplemented DCMECs are useful for lactating mechanism research. In summary, methionine can significantly promote milk protein synthesis-related genes expression and proteome changes. The results of protemics reveal the methionine play an important role in the lactation regulation of dairy cows, and this provide the experimental basis for further dairy cows lactation regulatory mechanism, and lay the foundation for subsequent research.