Proteomics Analysis Of Dairy Cow Mammary Gland Epithelial Cells’Nuclear Phosphoprotein Associated With Lactation

Dairy cow is one of the most important animal in Chinese dairy industry, lactation ability of mammary gland will affect t the milk yield and quality. Phosphorylation is one of the most important post-translational modifications of proteins, which is related to many activities of life. Milk fat and milk protein synthesis are regulated by reversible protein phosphorylation, such as mTOR and STAT5. But, the mechanism by which the synthesis of milk proteins and milk fat is stimulated by nuclear phosphoproteins is less known.The dairy cow mammary gland epithelial cells’(DCMECs) nuclear phosphoprotein were investigated using mass spectrometry(MS) identification, with the analysis of protein fuction and phosphorylation site, we could get a further recognition the mechanism of how phosphoproteins effect milk synthesis. In the reseach we have known that the nuclear phosphoprotein could effect milk synthesis by identified some of the nuclear phosphoprotein with the biological method already used. And provide an important basis for the regulation of milk synthesis in proteomic analysis.Our work was divided into two parts:1. A dairy cow mammary epithelial cell line was established through culture method of tissue block and detection of cell biological characters. Extracted DCMECs’ nuclear protein was identification by MS. Using uniprot_Bos_taurus.fasta database, matching significant protein(Total score>65, P<0.05),664nuclear proteins and658nuclear phosphoproteins were identified by MS, at the meantime, phosphorylation site of the658nuclear phosphoproteins were identified by MS which are unknown in the database.2. The changes of DCMECs’ viability and proliferation treated with methionine were successfully established by RT-PCR, Western blotting and confocal scanning laser microscopy(CSLM). Methionine could enhance the expression of β-casein and STAT5in DCMECs (P<0.05), the number of lipid droplets were enhanced too. Determining the changes of mRNA expression between control group and methionine treated group of20proteins related to lactation in different functions from658nuclear phosphoproteins identified by MS. The result showed that: The expression level of RRP45, PK4, BAP18, CanX, TFIP11, ETIF3F and eEFIB were extremely higher than control (P<0.01); the expression level of DDX27, EIF4G2, TRIP12, Septin2and S6K1were higher than control (P<0.05); SF3A1, BAP1, PRP4, G3BP1and HSPB1had decreased expression than control, the difference was extremely significant (P<0.01); Meta and LDLRAP1had decreased expression than control, the difference was significant (P<0.05); the expression level of EGFR1shows no difference than control(P>0.05). Western blotting was used to detect the expression of eEFIB and S6K1which has already identified mRNA expression would higher than control, the result shows that methionine treated group were higher than control group(P<0.05). Infer to RRP45, PK4, BAP18, CanX, TFIP11, ETIF3F, eEFIB, DDX27, EIF4G2, TRIP12, Septin2and S6K1could take a role in positive regulation of lactation, BAP1, PRP4, G3BP1, HSPB1, SF3A1, MTDH and LDLRAP1could take a role in negative regulation of lactation.The results of this research have enriched the study content of nutritional proteomic of dairy cow, and have important basic theory and research methods.