| Mammalian coat color has an important economic value. The wool is a major of raw material for textile production. Therefore, it is a great practical significance to regulate sheep wool color by molecular biology methods. Both hair and skin colors are dependent on the quantity, types and distribution of melanin produced by melanocytes. At present, it has been found to influence the color of the pigment formation genes include:MC1R, a-MSH, TYR, TYR-1, MITF, c-Kit, Agouti, Slc7?11, HGF, c-Met and so on. iTRAQ technology is a novel biological technique of proteomics, which can label 8 different samples and analyzed the relative and absolute quantification. The technology can be used for qualitative and quantitative simultaneously. The characteristics of iTRAQ technology are high sensitivity, high throughput, strong separatio efficiency, reliable results and so on. iTRAQ technology was applied to various academic fields in recent years. In the present project, the sheep were chose as experimental model to explore the expression of proteins by iTRAQ technology. The differential proteins that were involved in the coat color formation were screened by biological information analysis methods in sheep. After then, the expression levels of differential proteins were verified by Western blot and QRT-PCR. The expression and localization of differential protein were analyzed by immunohistochemistry, immunofluorescence and laser confocal microscopy. The results are as followed:1. We researched the expression of Gnas and Gn?11 of G protein coupled signaling pathway in white and black sheep skin tissues. The results showed that Gnas and Gnal 1 were expressed in both the white and black skins of sheep, and Gnas and Gnal 1 were significantly expressed at high levels in black sheep skin compared with that of white (P< 0.05), and transcripts and protein exhibited the same expression pattern in white and black sheep skins. Immunofluorescencence further demonstrated that signals of Gnas and Gn?11 were detected in hair papilla and outer root sheath. Furthermore, the expression of Gnall mRNA and protein was higher than that Gnas in two skin colors.2. The quantitative information of the proteins were detected and analyzed in white and black sheep skin tissues by iTRAQ technology. In our study, we obtained the 9909 unique peptides, and identified the 1704 proteins, including 539 proteins were covered by 5 different peptides or more peptides. The white sheep skin tissue as the control, and the 1704 protein datas were collected and analyzed by the ratio> ±1.2 and P< 0.05. A total of 136 differential proteins were obtained in different coat colors, including up-regulated were 101 proteins and down-regulated were 35 proteins.3. The protein quantitative results were obtained by LC MS/MS, the raw datas were statistical analysis by biological information analysis that including GO annotation, KEGG metabolic pathway analysis and differential protein interaction network. GO results showed that 134 proteins sequences were annotated by 2022 go functions; KEGG signal pathway analysis results showed that 121 KEGG pathways were extracted, which associated with 55 differential proteins. The differential protein interaction network results showed that the A2M?TTR?VIM?ALB?FGA?FGB?FGG?C1QC and APOA1 involved in direct interaction between differential protein and differential proteins.4. Ang ? protein was consisted in the white and black sheep skins, and was a up-regulated protein by iTRAQ technology. Our analysis validation results were consistented with the results of iTRAQ technology by Western blot and QRT-PCR, which was the expression of Ang ? was higher in black sheep skin than that in white skin (P< 0.05). Immunohistochemical analysis further demonstrated that Ang ? protein was localized in the hair papilla and outer root sheath of hair follicle in sheep. Furthermore, the expressions of Ang ? in the hair papilla and outer root sheath of black sheep were stronger than those in white sheep (P< 0.05; P< 0.01). These results offered a novel insight for further to investigate the role of Ang ? in the coat color formation in sheep.5. We found that ACTB and FGA proteins were consisted in the white and black sheep skins by iTRAQ technology. The expression level of ACTB protein was higher in white sheep skin than that of in black skin (P< 0.05), which suggested that ACTB protein was not suitable as reference gene or protein for coat color formation and pigmentation related research. The expression level of FGA protein was higher in black sheep skin than that of in white skin (P< 0.05). Laser confocal microscopy analysis further demonstrated that ACTB protein was localized in the outer root sheath of hair follicle in sheep. FGA protein was localized in the hair papilla, outer root sheath and hair matrix of hair follicle in sheep. We speculated that ACTB and FGA proteins were associated with platelet activation signal pathway may be involved in the formation of sheep coat color. However, the regulation of molecular mechanism in the formation of sheep coat color needs to research and discuss in further.In conclusion, we analyzed the protein quantitative analysis in different coat color sheep skin by iTRAQ technology and biological information, and detected that the differential proteins and signal pathways that may were involved the coat color formation in sheep. The differential proteins and signal pathways were validated and analyzed in the next experiment. The study provided an experimental model to explore proteomics in coat color regulation of sheep in future, and give a powerful experimental evidence on regulation mechanism in coat color formation. |
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