Study On Identification And Physiological Characteristics Of Peptide Transporters In The Mammary Gland Of Lactating Dairy Cows

The thesis mainly detected the expression of the peptide transportors(Pep Ts) positioning and its potential functions of regulation in bovine mammary glands and mammary epithelial cells in middle lactation. The study aimed to determine the peptide transport exited in mammary gland of the lactating dairy cows and the role and mechanism of the peptide transport carriers in mammary epithelial cells. Thus, it provided theoretical basis for the further study of the molecular function and transport properties of the peptide transporters. Meanwhile it also provided theoretical basis and data accumulation for improving the intake and metabolic model of small peptides and the reasonable proportion of milk protein. Four parts of this research were shown as follow:1. Identification of mRNA gene levels of peptide transporters in the mammary gland of lactating dairy cowsMammary tissues randomly collected from three lactating Holstein dairy cows were used for extracting the total RNA and mammary epithelial cells culture. The expressions of Pep T1 and PepT2 in the mammary gland were detected using Real-time fluorescence quantitative and agarose gel technique.The results of the experiment showed that two kinds of peptide transporters had specific amplification and were highly expressed in both mammary gland of lactating dairy cows and in cultured mammary epithelial cells. The reliability of the results has been confirmed by sequencing. The homology of target sequence was 100% by analysis and comparison. The results showed that the PepTs gene was expressed in the mammary gland of lactating dairy cows.2. Immunological detection and localization of PepTs in the mammary gland of lactating dairy cowsThe total protein extracted respectively from mammary gland and mammary epithelial cells were used to detect the proteins expression of PepT1 and Pep T2 using Western Blot. The distribution of PepTs in mammary gland tissues and mammary epithelial cells were studied using immunohistochemistry and immunocytochemistry. The results of immunofluorescence showed that two kinds of peptide transporter proteins were highly expressed in the mammary gland of lactating dairy cows, Pep T1 was mainly found in the cell membrane of the cow mammary gland but PepT2 was mainly found in the cytoplasm of cow mammary glands.3. Physical、chemical characteristics and function regulation of Pep Ts in the mammary gland of dairy cowsEpithelial cells extracted from the mammary tissue were cultured in plate untill it grew up to 80% ~ 90% then vaccinated as the density of 1 x 105/well and extended until the fifth generation. The total RNA was extracted for RT-PCR and it run six times. We found that number of passage had significant effects on PepTs gene expression. Compared with the others, the significant effects were detected for either PepT1 or Pep T2(P﹤0.05) in the second generation. Therefore, the second generation cells were used for the following experiments. The treatments time were divided into six with six repeats in each group to detecte the influence of treatment time on gene expression of Pep Ts in the process of cell differentiation. The PepTs mRNA expression were increased then reduced and had a peak on 5th day(P﹤0.05). In order to study the regulation pattern of Pep Ts in mammary gland of lactating dairy cows, the different concentrations of lactation hormones(culture 24 h) and peptide substrates(48 h) were used for the next study. Test results showed that compared with addition of no peptides but free amino acids the gene expression of Pep Ts mRNA were increased(P﹤0.05) in the treatments of 0.0002, 0.002, 0.02,and 0.2 IU/m L Prolactin, 50, 500 and 5000 ng/m L Insulin as well as 100 and 1000 ng/m L hydrocortisone. The expression of Pep T1、Pep T2、 αs1 casein mRNA gene and the synthesis of milk protein were up-regulated(P ﹤ 0.05) by 10% Thr-Phe-Phe、5% Phe-Phe、10% Leu-Leu and 6 μg/m L Leu-Leu.4. Construction and expression of Eukaryotic Expression Vector of Bovine Mammary Gland Peptide Transporters GenePepT1 gene of lactating dairy cows was amplified and cloned by PCR. We constructed the recombinant plasmid named p IRES2-EGFP-Pep T1 by analyzing the specificity of PCR primers through digesting PCR products with restriction endonuclease and connections with T4 ligase.Pep T2 gene, pc DNA3.1-Pep T2, was provided by Sangon Biotech(Shanghai) limited company. p IRES2-EGFP-PepT1 and pcDNA3.1-Pep T2 were transfected in mammary epithelial cells then the changed the stability and expression quantity of PepTs and αs1 casein gene by RT-PCR after transfection. The results showed that peptide transporters(p IRES2-EGFP-Pep T1 and pcDNA3.1-PepT2) were constructed in the mammary gland of lactating dairy cows, and the p IRES2-EGFP-PepT1 and pcDNA3.1-PepT2 eukaryotic expression vector were successfully transfected in mammary epithelial cells. Compared with the control group, eukaryotic expression vector was highly expressed in the mammary gland of dairy cows and the αs1 casein mRNA expression was increased after Pep Ts were transfected by plasmid.In conclusion, the truth that the expression of Pep T2 and PepT1 both existed in the mammary gland of lactating dairy cows were confirmed in the Gene and protein levels in the cow mammary tissue and mammary epithelial cells. Transporters of Pep T2 and PepT1 were highly expressed, it is systematic to prove that period of cell differentiation process and lactation hormones and different substrates had significant effect on Pep Ts regulation present certain regularity. Peptide transporters(p IRES2-EGFP-PepT1 and pcDNA3.1-PepT2) in the mammary gland of lactating dairy cows were constructed. The stability of the p IRES2-EGFP-PepT1 and pcDNA3.1-Pep T2 expression was verified by RT-PCR. The p IRES2-EGFP-Pep T1 and pcDNA3.1-Pep T2 eukaryotic expression vectors were successfully transfected in mammary epithelial cells. Compared with the control group, transfection significantly increased the mRNA expression levels of PepTs and αs1 casein.