SNARE蛋白在动物毛色形成中作用的研究

发布时间：2018-05-30 20:45

本文选题：SNARE蛋白 + 动物毛色　；参考：《山西农业大学》2015年博士论文

【摘要】：随着科学的进步,人们生活水平的提高,新时代毛纺织业面临的问题日益凸显,单一的动物毛色不能满足消费者的需求,化学染料对人体的健康有所危害,对环境的污染日趋严重,天然彩色动物毛纤维的生产是解决这一问题的理想途径,从转基因水平改变现有动物毛色有望实现天然彩色动物毛纤维的生产,为此,毛色形成机理的探索显得尤为紧迫。毛色形成包括黑色素生成,黑素小体运输到黑素细胞树突顶部,黑素小体从黑素细胞向角化细胞的转移,黑素颗粒在角化细胞中的重新分布降解。然而,现在大量的毛色形成机理的研究集中于色素生成和黑素小体运输到树突顶端环节,在色素颗粒由黑素细胞转入角化细胞中的报道较少,鉴于黑素小体为膜性细胞器,本文将膜泡运输、膜锚定与融合理论应用到黑素小体的转移过程,从一个新的领域探索毛色形成机理。黑素小体由黑素细胞转移到角化细胞的过程有四种推测,其中一种是黑素小体由黑素细胞胞吐排出黑素细胞,通过胞吞进入角化细胞,胞吐胞吞过程会涉及到膜融合过程,SNRAE家族为膜融合相关蛋白,本文用生物信息学的方法对SNARE家族蛋白结构功能域进行分析,分析其作用特点,并对部分SNARE家族蛋白在差异毛色的小鼠、绵羊和羊驼皮肤中的表达量进行普通PCR、RT-qPCR、免疫组化和Western blot检测研究。1为了准确把握SNARE蛋白的功能,通过对SNARE蛋白的核酸和蛋白质序列,进行蛋白质三级结构预测,跨膜区域预测分析,同源性分析和启动子分析。结果显示,所有预测的22种小鼠SNARE蛋白的三级结构中均有α-螺旋,其中,12种Syntaxin的α-螺旋都在5个以上,7种VAMP蛋白所含α-螺旋结构域较少,且较短,为3个左右。3种SNAP25蛋白含有较长α-螺旋,数量为2个以上。对22个小鼠SNARE蛋白进行跨膜区域的分析得出,Stx11、Snap23、Snap 25、Snap 29没有跨膜区。10个物种Stx4 CDS区核酸序列的一致性是61.59%,氨基酸序列的一致性是68.74%,其羧基端的同源性较高。转录因子分析,Stx4的启动子预测的转录因子中Foxd3、Usf-1和Pax6在黑色细胞中有表达,Stx17的预测的转录因子有SP1在黑素细胞中有表达。由此可知SNARE蛋白参与膜泡锚定融合,并可预测Stx4和Stx17可能在黑素细胞中表达。2 C57BL/6J小鼠背部皮肤是纯黑色,腹部皮肤是灰色的。为了研究SNARE家族的基因是否与毛色差异有关,我们通过实时定量PCR (RT-qPCR)检测了这些基因在C57BL/6J小鼠背部和腹部皮肤的表达。结果表明,所有选定9个SNARE家族成员除了SNAP25和9个与它们相互作用的基因均在小鼠皮肤表达。SNAP23、STX17和STXBP1表达水平在腹部皮肤明显高于背部皮肤,特别是STXBP1腹部皮肤表达比在背部皮肤高5倍,STX4、VAMP3、VAMP7、STXBP3A、DOC2B、EXOC3、SLC2A4和TXLNB表达在背部皮肤更高,与腹部皮肤相比。我们认为SNARE蛋白可能参与黑素小体转移的过程,通过介导黑素小体锚定和融合将黑色素转运到角化细胞。SNAP23、STX17和STXBP1抑制黑素小体转移,而STX4、VAMP3、VAMP7、STXBP3A、DOC2B、EXOC3、 SLC2A4和TXLNB促进黑素体向角化细胞的转移。3旨在探讨突触融合蛋白4(Syntaxin4;STX4)在皮肤组织细胞中的表达与定位,确定其是否与毛色形成存在相关性。选择白、灰、黑3种毛色皮肤组织样品(6只昆明小鼠白毛色背部皮肤、6只C57BL/6J小鼠灰毛色腹部皮肤和黑毛色背部皮肤)和体外培养小鼠黑色素细胞样品,通过PCR扩增、Real-time PCR、免疫组化和Western blot技术对STX4的表达情况进行定性和定量分析。结果表明：在3种毛色皮肤样品和黑色素细胞样品中均扩增出897 bp CDS区序列片段;Real-time PCR检测显示,STX4在白灰黑3种毛色皮肤样品中均有表达,黑色皮肤表达量最高,灰色皮肤表达量次之,分别是白色皮肤的3.44倍和1.92倍;免疫组化结果表明,在白色和黑色皮肤表达于整个毛囊,包括角化细胞;在体外培养黑色素细胞也显示阳性表达；Western blot结果显示,在白、灰、黑色皮肤和黑色素细胞样品中均有STX4阳性条带,表达量与荧光定量结果一致。综上表明,STX4在小鼠皮肤组织、毛囊角化细胞、黑色素细胞有效表达,且表达量在白灰黑皮肤中呈递增趋势,推测STX4与小鼠毛色形成呈正相关性。4突触融合蛋白4在小鼠毛色形成的相关性得到验证,对绵羊毛色差异的形成是否有作用需进一步验证。本实验利用普通PCR、Western blot试验、免疫组化染色对黑白毛色绵羊皮肤及体外培养黑素细胞中Stx4的表达情况进行检测。PCR结果显示,Stx4在黑白皮肤组织及体外培养黑素细胞RNA中均扩增出了间于750bp-1000bp长度的片段,条带单一,大小正确；Western blot试验显示,突触融合蛋白4在绵羊白色皮肤和黑色皮肤中均有表达,条带大小为35 kDa,灰度结果分析,在黑色皮肤中显著高于白色皮肤。免疫组化结果显示,在白色皮肤和黑色皮肤毛囊均有STX4阳性表达。在白色皮肤毛囊上皮性根鞘,包括外根鞘和内根鞘均能看到阳性结果,在毛囊上、中、下三部分都有阳性表达,在白色皮肤中邻近毛乳头的毛母质细胞未见阳性着色,而黑色皮肤中有阳性着色。由此得出STX4表达量与绵羊毛色形成呈正相关关系。5突触融合蛋白17与马毛色形成有相关性,是否参与绵羊毛色形成。本试验利用普通PCR、Western blot试验、免疫组化染色对黑白毛色绵羊皮肤及体外培养黑素细胞中Stx17的表达情况进行检测。普通PCR结果显示,绵羊Stx17 CDS区序列成功在体外培养黑素细胞中扩增,条带单一,大小间于1000bp与750bp之间。绵羊皮肤组织蛋白提取后进行免疫印迹显示,绵羊组织中STX17蛋白分子量大小在33 kDa左右,条带清晰整齐,灰度分析显示,黑色皮肤中的表达量显著高于灰色皮肤。6为了更进一步验证Stx4和Stx17与毛色形成的相关性,选择天然毛色丰富的羊驼作为研究对象,分析其STX4和STX17蛋白的三级结构和跨膜区域,并与人类的该两种蛋白进行比较,对羊驼皮肤组织进行这两个蛋白的免疫组化染色,体外培养羊驼皮肤黑素细胞提取蛋白进行Western blot检测。结果显示,STX4羊驼比人类多两个α-螺旋域,STX17羊驼的比人类的多一个α-螺旋域。羊驼和人类的这两个蛋白的跨膜区域相类似。Western blot试验显示STX4和STX17理想大小的阳性条带。推测STX4和STX17在黑素细胞中存在,并可能参与毛色的形成。结论：试验结果发现不同毛色皮肤之间存在SNARE家族蛋白的差异表达。由此推断,SNARE家族成员可能参与了小鼠、绵羊和羊驼的毛色形成,STX4与小鼠、绵羊和羊驼的毛色形成呈正相关,STX17与毛色形成有相关性。
[Abstract]:With the progress of science and the improvement of people's living standards, the problems faced by the wool textile industry in the new era are becoming increasingly prominent. The single animal hair color can not meet the needs of the consumers. The chemical dyes are harmful to the health of the human body, and the pollution to the environment is becoming more and more serious. The production of natural color animal wool fiber is an ideal way to solve this problem. It is expected to realize the production of natural color animal hair fibers from the change of the existing animal hair color from the transgenic level. Therefore, the exploration of the mechanism of hair color formation is particularly urgent. The formation of hair color includes melanin formation, melanosomes are transported to the top of the dendritic cells of melanocytes, the transfer of melanosomes from melanocytes to keratinocytes and the keratinization of melanin particles. However, a large number of hair color formation mechanisms are now focused on pigments and melanosomes transported to the apex of dendrites. There are few reports on the transfer of pigment particles into keratinocytes from melanocytes. In view of the fact that melanosomes are membranous organelles, the transport of membrane bubbles, the theory of membrane anchoring and fusion should be applied in this paper. Using the process of melanosomes transfer, we explore the formation mechanism of hair color from a new field. There are four kinds of speculations that melanin corpuscle is transferred from melanocyte to keratinocyte, one of which is melanin body exocytosis by melanocyte and enters diagonalization cell by endocytosis, and the process of endocytosis involves the membrane fusion process, SN The RAE family is a membrane fusion related protein. In this paper, the functional domain of SNARE family protein structure was analyzed by bioinformatics method, and its function characteristics were analyzed. The expression of some SNARE family proteins in the skin of sheep and alpaca in different hair color mice was carried out by ordinary PCR, RT-qPCR, immunohistochemistry and Western blot detection and study.1 for the purpose. Accurately grasp the function of SNARE protein, through the sequence of nucleic acid and protein of SNARE protein, the protein three level structure prediction, transmembrane region prediction analysis, homology analysis and promoter analysis. The results show that all the predicted 22 SNARE proteins of mice have alpha helix in the three structure, of which, 12 kinds of Syntaxin alpha helix are all 5 Above, the 7 VAMP proteins contain less alpha helix domain and shorter, and 3 SNAP25 proteins with 3.3 species contain longer alpha helix, and the number is more than 2. The analysis of the trans membrane region of 22 mice shows that Stx11, Snap23, Snap 25, Snap 29 without the.10 species in the Stx4 CDS region of the trans membrane region are 61.59%, ammonia, 61.59%, ammonia The consistency of the base acid sequence is 68.74%, its carboxyl terminal homology is high. Transcriptional factor analysis, Foxd3, Usf-1 and Pax6 are expressed in the black cells of the transcription factors predicted by the promoter of Stx4, and the predicted transcription factors of Stx17 are expressed in melanocytes by SP1. Thus, the SNARE protein is involved in the membrane foam anchorage fusion and can predict Stx4. And Stx17 may express the back skin of.2 C57BL/6J mice in melanocytes is pure black and the skin of the abdomen is gray. In order to study whether the SNARE family's gene is related to the hair color difference, we detected the expression of these genes in the back and abdomen skin of the C57BL/6J mice by real-time quantitative PCR (RT-qPCR). The results showed that all selected 9 were selected. The SNARE family members expressed.SNAP23 in the skin of mice except SNAP25 and 9 genes interacting with them in the skin of mice. The expression level of STX17 and STXBP1 was significantly higher in the skin of the abdomen than in the back skin, especially in the STXBP1 abdomen 5 times higher than in the back skin. STX4, VAMP3, VAMP7, STXBP3A, DOC2B, EXOC3, and expressions were expressed in the back skin. Higher, compared with the abdominal skin. We believe that SNARE protein may be involved in the process of melanosomes transfer by mediating melanosomes anchoring and fusion to transfer melanin to keratinocyte.SNAP23, STX17 and STXBP1 to inhibit melanosomes, while STX4, VAMP3, VAMP7, STXBP3A, DOC2B, EXOC3, SLC2A4, and TXLNB promote melanosomes to keratinocytes. The transfer of.3 was aimed at exploring the expression and localization of synaptic fusion protein 4 (Syntaxin4; STX4) in the skin tissue cells, determining whether it was associated with hair color formation. Select white, gray, and black 3 hair color skin tissue samples (6 Kunming mice, white hair color back skin, 6 C57BL/ 6J mice, gray hair color abdominal skin and black hair color back skin) and body. The samples of mouse melanocytes were cultured in vitro. The expression of STX4 was analyzed by PCR amplification, Real-time PCR, immunohistochemistry and Western blot. The results showed that 897 BP CDS region sequences were amplified in 3 hair color skin samples and melanocytes, and Real-time PCR detection showed that STX4 was 3 in grey black. The hair color skin samples were expressed, the black skin expression was the highest, the gray skin expression was 3.44 times and 1.92 times the white skin respectively. The immunohistochemical results showed that the white and black skin expressed in the whole hair follicle, including the keratinocyte, and the positive expression of the melanocytes in the culture in vitro; Western blot results. STX4 positive bands were found in white, gray, black skin and melanocyte samples, and the expression was in accordance with the fluorescence quantitative results. The results showed that the expression of STX4 in mouse skin tissue, hair follicle keratinocytes and melanocytes was effective, and the expression of STX4 was increasing in grey black skin. It was suggested that the formation of STX4 was positively correlated with the formation of hair color in mice. The correlation of 4 synaptic fusion protein 4 in the formation of hair color of mice is verified. Whether the formation of wool color difference in sheep needs further validation. This experiment uses common PCR, Western blot test, immunohistochemical staining to detect the expression of Stx4 in black and white sheep skin and in vitro cultured melanocytes,.PCR results, St X4 amplified the length of 750bp-1000bp between black and white skin tissue and in vitro cultured melanocyte RNA, with a single band and correct size. Western blot test showed that the synaptic fusion protein 4 expressed in the white skin and black skin of the sheep, the band size was 35 kDa, the gray result was analyzed, and the black skin was significantly higher in the black skin. In white skin and black skin follicles, the positive expression of STX4 was found in the white skin and black hair follicles. The positive results were found in the epithelial root sheath of the white skin follicle, including the outer root sheath and the inner root sheath. The positive expression was found in the hair follicles, in the three parts of the hair follicles, and the hair mother cells adjacent to the dermal papilla were not positive in white skin. The positive correlation between the black skin and the black skin was found. It was concluded that the expression of STX4 was positively related to the formation of sheep hair color..5 synapse 17 was associated with the formation of horse hair color, and whether it was involved in sheep hair color formation. This experiment used common PCR, Western blot test, immunohistochemical staining for black and white wool sheep skin and in vitro culture black. The expression of Stx17 in the vegetarian cells was detected. Common PCR results showed that the sequence of Stx17 CDS region of sheep was amplified successfully in cultured melanocytes in vitro, with a single band and between 1000bp and 750bp. The molecular weight of the STX17 protein in sheep was about 33 kDa in sheep skin tissue after the extraction of sheep skin tissue protein. With clear and tidy, gray analysis showed that the expression in black skin was significantly higher than that of grey skin.6. In order to further verify the correlation between Stx4 and Stx17 and hair color formation, the natural hair color rich alpaca was selected as the research object, and the three grade structure and transmembrane region of STX4 and STX17 protein were analyzed, and the two proteins of human were carried out. In comparison, the two proteins were immunohistochemical staining of Alpaca Skin Tissue and Western blot was detected in Alpaca Skin melanocyte extraction protein in vitro. The results showed that STX4 alpaca was more than human alpha helix domain, and STX17 alpaca was more than human in alpha helix domain. Alpaca and human two proteins in the transmembrane region. Similar.Western blot tests showed the positive bands of the ideal size of STX4 and STX17. Speculates that STX4 and STX17 exist in melanocytes and may be involved in the formation of hair color. Conclusion: the results showed that the differential expression of SNARE family proteins existed between different hair colored skins. Thus, it was concluded that members of the SNARE family might be involved in mice, sheep and sheep. The formation of hair color of camel is positively correlated with the hair color formation of mice, sheep and alpacas, and STX17 is related to the formation of hair color. STX4
【学位授予单位】：山西农业大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：S852.2

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