| This study successfully detected the expression of proton-dependent oligopeptide (POTs) in bovines’tissues. The bovine mammary epithelial cells (BMEC) were cultured with di-peptides to study the regulation of milk protein synthesis. The research could provide the basis for the functional research of POTs in dairy cows and the reference for the transport mechanism of small peptides in BMEC.The research consists of three experiments, as follows:Exp.1:This study was focused on analyzing the localization and expression of POTs in MG and BMEC. MG separated with less fat and rich in breast lobular under sterile conditions were cultured and purified. The immunofluorescence method was used to detect the localization and expression of PepT1 and PepT2 in MG and BMEC; qRT-PCR and Western blot methods were used to detect the specific expression of PepTl, PepT2, PHT1 and PHT2 mRNA in MG and BMEC. The results showed that PepT1 was mainly distributed in interlobular connective tissue, and PepT2 was mainly distributed in the mammary duct and acinus epithelial cells. In vitro culture of BMEC, PepT1 and PepT2 were mainly distributed in the membrane and cytoplasm. The expression of PepT1, PepT2 protein were observed in MG and BMEC by qRT-PCR and Western blot methods.Exp.2:This study was conducted to investigate the expression of POTs in bovines’ tissues. Four healthy 3-month-old Holstein cows were slaughtered. The specific expression of PepT1, PepT2, PHT1 and PHT2 mRNA in heart, liver, lung, bovine, kidney, spleen, thymus, breast (from eliminated late lactating dairy cows), muscle and the mucosa of rumen, reticulum, omasum, abomasum, duodenum, jejunum, ileum, cecum and colon was examined by qRT-PCR method. The results indicated that the expression of PepTl mRNA in jejunum and duodenum was dramatically higher than in Ileum, rumen, lung, colon, stomach valve, liver, cecum, reticulum, thymus, spleen, abomasum, kidney, breast, heart and muscle (P<0.01). The expression of PepT2 mRNA in breast was dramatically higher than in kidney, liver, lung, spleen, ileum, abomasum, thymus, duodenum, reticulum, cecum, heart, jejunum, colon, omasum, muscle and rumen (P<0.01). The expression of PHT1 mRNA in spleen, appendix and lungs was dramatically higher than in heart and muscle (P<0.01). The expression of PHT2 mRNA in Lungs, cecum and thymus was dramatically higher than in rumen, abomasum, reticulum, heart and muscle (P<0.01).Exp.3:This study was conducted to investigate the effect of milk protein synthesis on BMEC cultured with di-peptides. Adding different concentration of Met-Met, Val-Met and Leu-Met respectively to culture BMEC to screen out the best concentration. Based on the concentration, the experiment was divided into three groups which were di-peptide group (Met-Met, Val-Met, Leu-Met), and amino acid half instead of di-peptide group (Met-Met/Met, Met; Val-Met/Val, Met; Leu-Met/Leu, Met), amino acid equivalent replacement group (Met, Met; Val, Met; Leu, Met) to culture BMEC to evaluate the differences of functional regulation of protein translation. The results showed that the expression of mTOR, EIF-4E, S6K1 and SOCS-3 mRNA in group Met-Met/Met, Met was dramatically higher than in group Met-Met and group Met, Met (P<0.01). The expression of κ-casein and EIF-4E mRNA in group Val-Met/Val, Met was dramatically higher than in group Val-Met and group Val, Met (P<0.01). The expression of PepT2 mRNA in group Val-Met/Val, Met was very significantly higher than in group Val, Met and group Val-Met (P<0.05). The expression of PHT1 mRNA in group Leu-Met was significantly higher than in group Leu-Met/Leu, Met(P<0.05). The expression of κ-casein mRNA in group Leu-Met/Leu, Met was dramatically higher than in group Leu-Met and Leu, Met (P<0.01). The expression of EIF-4E mRNA in group Leu-Met was significantly higher than in group Leu, Met(P<0.05). The concentration difference of Met-Met, Val-Met and Leu-Met after the cell culture was measured by HPLC, and the disappearance of di-peptide in group Met-Met/Met, Met and group Leu-Met/Leu、Met was higher than in group Met-Met and group Leu-Met.Conclusion:(1) PepT1 was mainly distributed in interlobular connective tissueand PepT2 was mainly distributed in the mammary duct and acinus epithelial cells. (2) PepT1 and PepT2 was mainly distributed in cytoplasm of BMEC. (3) PepT1 mRNA was expressed in jejunum and duodenum abundantly; PepT2 mRNA was expressed in breast abundantly, PHT1mRNA was expressed in spleen, thymus and appendix abundantly and PHT2 mRNA was expressed inlung, appendix and thymus abundantly. (4) Di-peptide mixed with amino acid can promote the transcription of κ-casein and protein synthesis related genes. (5) Amino acids like Met, Val and Leu could promote the BMEC to transport di-peptide such as Met-Met, Val-Met and Leu-Met. |
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