| With the progress of science and improvement of life level, wool industry faces the problem that is growing in the new era, a single color of wool cann't satisfy the needs of the consumers. Chemical dyes is harmful to people's health and environment, the natural colored animal wool production is an ideal way to solve these problems, the natural colored animal wool could be obtained by transgenic technologies, therefore, to explore the formation mechanism of the animal coat color is significant. There are many factors to influence animal coat colors, including melanin formation, melanosome transport to the tip of the melanocyte dendrite, melanin transfer from the melanocytes to the keratinocytes and melanin particles redistribution and degradation in keratinocytes. However, recent researches, have focused on the mechanism of melanogenesis and melanosome transport to the tip of the dendrite, the studies of the melanin particles transport from melanocytes to keratinocytes are less reported. This study focused on exploring the mechanism of coat color formation in a new research area by investigating melanin transfer using membrane vesicular transport, membrane anchoring and the fusion theories based on that the melanosome is the membranous organelle. There are four theories about melanin transfer process involved in membrane anchoring and fusion, and SNARE family are the related proteins. In this study, the structure domains and their functional characteristics of the SNARE family proteins were analyzed by bioinformatic methods, and the differential expression of the SNARE family proteins were detected in mice, sheep and alpaca with different coat color skins through PCR. RT-qPCR. Immunohistochemistry and Western Blot.1 In order to investigate the functions of SNAREs exactly, the SNAREs'nucleic acid and protein sequences, protein tertiary structure and transmembrane region were predicted and analyzed, and the homology and promoter were analyzed, the results showed that the tertiary structure of 22 kinds of mice SNAREs all had alpha helix, of which the number of alpha helixs of 12 kinds of syntaxins was more than five, but only some 3 and shorter for the alpha helix structure regain of 7 kinds of VAMPs, and more than 2 and longer for the alpha helix structure region of 3 kinds of SNAP25 proteins. However, there was no transmembrane region for Stx 11, Snap 23, Snap 25 and Snap 29 by analyzing the transmembrane region of the 22 SNAREs of mice. The identity of the nucleic acid sequence of the Stx4 CDS region in 10 species was 61.59%, and the identity of the amino acid sequence was 68.74%, the homology of the carboxyl terminal was higher. By analysis of the transcription factors, in the transcription factors predicted by Stx4 promoter, Foxd3, USF-1, pax6, were expressed in the melanocytes and SP1 was also expressed in the melanocytes predicted by Stx 17 promoter. So it was concluded that SNARE proteins involved in membrane anchoring and fusion, and Stx4, Stx 17 were probably expressed in melanocytes.2 In C57BL/6J mice, the back coat color is pure black, while it is gray on its abdomen. In order to investigate if SNAREs genes are related to the coat color difference, the expressions some genes were examined in the back and abdomen skins of C57BL/6J mice by quantitative real time PCR (RT-qPCR) analysis. The results showed that, in all selected 9 SNARE family members,9 interacting genes were expressed in the mice skin except for SNAP25. The expression abundances of SNAP23, STX17 and STXBP1 were significantly higher in abdomen skin compared with in back skin; in particular, STXBP1 expression was five times higher in abdomen skin than that in back skin; STX4, VAMP3, VAMP7, STXBP3A, DOC2B, EXOC3, SLC2A4, and TXLNB were expressed at significantly higher levels in back skin compared with in abdomen skin. It was recognized that the SNAREs could participate in the process of melanosome transfer by mediating the melanosome docking and fusion to transfer the melanin to the keratinocyte. SNAP23, STX 17 and STXBP1 inhibited melanosome transfer, while STX4, VAMP3, VAMP7, STXBP3A, DOC2B, EXOC3, SLC2A4, and TXLNB accelerated melanosome transfer to keratinocyte.3 The aim of the present study was to determine whether STX4 is associated with coat color formation in mice. The expression and localization of STX4 in mice skins and melanocytes were investigated. Three different coat color skins, white, grey and black, were collected from the back of 6 white Kunming mice, the abdomen of 6 C57BL/6 mice and the back of 6 C57BL/6 mice, respectively. Melanocytes were isolated from these skin samples and cultured in vitro. The expression of STX4 in these skin and cell samples were analyzed by standard RT-PCR, quantitative real-time PCR, immunohistochemical staining and Western Blot. The 897-bp sequence of STX4 CDS region was successfully amplified from three different coat color mice skins and melanocytes cultured in vitro by RT-PCR. Real -time PCR analysis revealed that STX4 is expressed in mice skins of all coat colors. STX4 showed highest expression in black skin, which is 3.44 times higher than white skin. The expression of STX4 in grey skin was 1.92 times higher than white skin. Immunohistochemical analysis showed that STX4 was expressed in whole hair follicles in white and black skins, included keratinocytes; it is expressed in melanocytes cultured in vitro. Western Blot results showed positive STX4 bands in white, grey and black skin samples and melanocytes cultured in vitro, These results are consistent with the real-time PCR results. In conclusion, STX4 is expressed in mice skins, hair follicles, keratinocytes, and melanocytes, and the expression of STX4 increases as the coat color deepens. Therefore, it is speculated that STX4 is positively correlated to coat color formation in mice.4 It was verified that STX4 was related with color formation in mice, but how about its function on the formation of sheep coat color difference is not clear. In this study, the STX4 expression in the skin tissues of black and white coat color sheep and in melanocytes cultured in vitro were detected by PCR, Western Blot and immunohistochemistry. The results showed that STX4 CDS region fragments were amplified from cDN A by PCR in skin tissues of black and white coat color sheep and melanocytes cultured in vitro, ranged from 750 bp to 1000 bp, band was single, size was correct; STX4 was expressed by Western Blot in the skin tissues of white and black sheep, band size was 35 kDa, it was significantly higher in the skin tissue of white sheep than in black sheep by gray scale analysis. STX4 was positively expressed in the hair follicles of white and black sheep by using immunohistochemistry. There was positive expression in white skin hair follicle epithelial root sheath including outer and inner root sheath, and also positively expressed in the three parts (upper, middle and lower) of the hair follicles; while there was no positive staining in the hair matrix cells of adjacent papilla region in the white skin tissue, but positively expressed in black skin. This elucidated STX4 expression was positively related with sheep coat color formation.5 STX17 is related with horse coat color formation, but it is not clear in the sheep coat color formation. In this study, the STX17 expression in the skin tissues of black and white coat color sheep and in melanocytes cultured in vitro were detected by general PCR, Western Blot and immunohistochemistry. The results showed that STX17 CDS region fragments were amplified successfully from cDNA by PCR in the melanocytes in vitro, ranged from 750 bp to 1000 bp, band was single, size was correct; The molecular weight of STX17 of sheep skin tissue was around 33 kDa, and band was clear and tidy by Western Blot. There was significantly higher STX17 expression in the black skin than in white skin by gray scale analysis.6 In order to further study the correlation of STX4 and STX17 with animal coat color formation, alpaca was selected as the research object, which has plentiful natural coat colors including 22 natural coat colors, STX4 and STX17 protein tertiary structures and transmembrane domains were analyzed and compared with the two proteins of human. The melanocytes extracted from skin and cultured in vitro were detected by Western Blot. Results showed there were two more alpha-helix domains of STX4 in alpaca than in human, and one more alpha-helix domain of STX17 in alpaca than in human. The two proteins' transmembrane regions were similar between alpaca and human. STX4 and STX17 had positive stripes with ideal sizes by Western Blot. This was speculated both STX4 and STX17 existed in the melanocytes and could participate in the formation of the coat color.Conclusions:There were differential expressions of SNARE proteins in the skin tissues with different coat colors, therefore SNARE proteins could participate in the coat color formation of mice, sheep and alpaca. STX4 are positively related with the coat color formation in mice, sheep and alpaca, and STX17 are related with the coat color formation. |
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