本文選題：SNARE蛋白 + 動物毛色�。� 參考：《山西農(nóng)業(yè)大學(xué)》2015年博士論文

【摘要】：隨著科學(xué)的進步,人們生活水平的提高,新時代毛紡織業(yè)面臨的問題日益凸顯,單一的動物毛色不能滿足消費者的需求,化學(xué)染料對人體的健康有所危害,對環(huán)境的污染日趨嚴重,天然彩色動物毛纖維的生產(chǎn)是解決這一問題的理想途徑,從轉(zhuǎn)基因水平改變現(xiàn)有動物毛色有望實現(xiàn)天然彩色動物毛纖維的生產(chǎn),為此,毛色形成機理的探索顯得尤為緊迫。毛色形成包括黑色素生成,黑素小體運輸?shù)胶谒丶毎麡渫豁敳?黑素小體從黑素細胞向角化細胞的轉(zhuǎn)移,黑素顆粒在角化細胞中的重新分布降解。然而,現(xiàn)在大量的毛色形成機理的研究集中于色素生成和黑素小體運輸?shù)綐渫豁敹谁h(huán)節(jié),在色素顆粒由黑素細胞轉(zhuǎn)入角化細胞中的報道較少,鑒于黑素小體為膜性細胞器,本文將膜泡運輸、膜錨定與融合理論應(yīng)用到黑素小體的轉(zhuǎn)移過程,從一個新的領(lǐng)域探索毛色形成機理。黑素小體由黑素細胞轉(zhuǎn)移到角化細胞的過程有四種推測,其中一種是黑素小體由黑素細胞胞吐排出黑素細胞,通過胞吞進入角化細胞,胞吐胞吞過程會涉及到膜融合過程,SNRAE家族為膜融合相關(guān)蛋白,本文用生物信息學(xué)的方法對SNARE家族蛋白結(jié)構(gòu)功能域進行分析,分析其作用特點,并對部分SNARE家族蛋白在差異毛色的小鼠、綿羊和羊駝皮膚中的表達量進行普通PCR、RT-qPCR、免疫組化和Western blot檢測研究。1為了準確把握SNARE蛋白的功能,通過對SNARE蛋白的核酸和蛋白質(zhì)序列,進行蛋白質(zhì)三級結(jié)構(gòu)預(yù)測,跨膜區(qū)域預(yù)測分析,同源性分析和啟動子分析。結(jié)果顯示,所有預(yù)測的22種小鼠SNARE蛋白的三級結(jié)構(gòu)中均有α-螺旋,其中,12種Syntaxin的α-螺旋都在5個以上,7種VAMP蛋白所含α-螺旋結(jié)構(gòu)域較少,且較短,為3個左右。3種SNAP25蛋白含有較長α-螺旋,數(shù)量為2個以上。對22個小鼠SNARE蛋白進行跨膜區(qū)域的分析得出,Stx11、Snap23、Snap 25、Snap 29沒有跨膜區(qū)。10個物種Stx4 CDS區(qū)核酸序列的一致性是61.59%,氨基酸序列的一致性是68.74%,其羧基端的同源性較高。轉(zhuǎn)錄因子分析,Stx4的啟動子預(yù)測的轉(zhuǎn)錄因子中Foxd3、Usf-1和Pax6在黑色細胞中有表達,Stx17的預(yù)測的轉(zhuǎn)錄因子有SP1在黑素細胞中有表達。由此可知SNARE蛋白參與膜泡錨定融合,并可預(yù)測Stx4和Stx17可能在黑素細胞中表達。2 C57BL/6J小鼠背部皮膚是純黑色,腹部皮膚是灰色的。為了研究SNARE家族的基因是否與毛色差異有關(guān),我們通過實時定量PCR (RT-qPCR)檢測了這些基因在C57BL/6J小鼠背部和腹部皮膚的表達。結(jié)果表明,所有選定9個SNARE家族成員除了SNAP25和9個與它們相互作用的基因均在小鼠皮膚表達。SNAP23、STX17和STXBP1表達水平在腹部皮膚明顯高于背部皮膚,特別是STXBP1腹部皮膚表達比在背部皮膚高5倍,STX4、VAMP3、VAMP7、STXBP3A、DOC2B、EXOC3、SLC2A4和TXLNB表達在背部皮膚更高,與腹部皮膚相比。我們認為SNARE蛋白可能參與黑素小體轉(zhuǎn)移的過程,通過介導(dǎo)黑素小體錨定和融合將黑色素轉(zhuǎn)運到角化細胞。SNAP23、STX17和STXBP1抑制黑素小體轉(zhuǎn)移,而STX4、VAMP3、VAMP7、STXBP3A、DOC2B、EXOC3、 SLC2A4和TXLNB促進黑素體向角化細胞的轉(zhuǎn)移。3旨在探討突觸融合蛋白4(Syntaxin4;STX4)在皮膚組織細胞中的表達與定位,確定其是否與毛色形成存在相關(guān)性。選擇白、灰、黑3種毛色皮膚組織樣品(6只昆明小鼠白毛色背部皮膚、6只C57BL/6J小鼠灰毛色腹部皮膚和黑毛色背部皮膚)和體外培養(yǎng)小鼠黑色素細胞樣品,通過PCR擴增、Real-time PCR、免疫組化和Western blot技術(shù)對STX4的表達情況進行定性和定量分析。結(jié)果表明：在3種毛色皮膚樣品和黑色素細胞樣品中均擴增出897 bp CDS區(qū)序列片段;Real-time PCR檢測顯示,STX4在白灰黑3種毛色皮膚樣品中均有表達,黑色皮膚表達量最高,灰色皮膚表達量次之,分別是白色皮膚的3.44倍和1.92倍;免疫組化結(jié)果表明,在白色和黑色皮膚表達于整個毛囊,包括角化細胞;在體外培養(yǎng)黑色素細胞也顯示陽性表達；Western blot結(jié)果顯示,在白、灰、黑色皮膚和黑色素細胞樣品中均有STX4陽性條帶,表達量與熒光定量結(jié)果一致。綜上表明,STX4在小鼠皮膚組織、毛囊角化細胞、黑色素細胞有效表達,且表達量在白灰黑皮膚中呈遞增趨勢,推測STX4與小鼠毛色形成呈正相關(guān)性。4突觸融合蛋白4在小鼠毛色形成的相關(guān)性得到驗證,對綿羊毛色差異的形成是否有作用需進一步驗證。本實驗利用普通PCR、Western blot試驗、免疫組化染色對黑白毛色綿羊皮膚及體外培養(yǎng)黑素細胞中Stx4的表達情況進行檢測。PCR結(jié)果顯示,Stx4在黑白皮膚組織及體外培養(yǎng)黑素細胞RNA中均擴增出了間于750bp-1000bp長度的片段,條帶單一,大小正確；Western blot試驗顯示,突觸融合蛋白4在綿羊白色皮膚和黑色皮膚中均有表達,條帶大小為35 kDa,灰度結(jié)果分析,在黑色皮膚中顯著高于白色皮膚。免疫組化結(jié)果顯示,在白色皮膚和黑色皮膚毛囊均有STX4陽性表達。在白色皮膚毛囊上皮性根鞘,包括外根鞘和內(nèi)根鞘均能看到陽性結(jié)果,在毛囊上、中、下三部分都有陽性表達,在白色皮膚中鄰近毛乳頭的毛母質(zhì)細胞未見陽性著色,而黑色皮膚中有陽性著色。由此得出STX4表達量與綿羊毛色形成呈正相關(guān)關(guān)系。5突觸融合蛋白17與馬毛色形成有相關(guān)性,是否參與綿羊毛色形成。本試驗利用普通PCR、Western blot試驗、免疫組化染色對黑白毛色綿羊皮膚及體外培養(yǎng)黑素細胞中Stx17的表達情況進行檢測。普通PCR結(jié)果顯示,綿羊Stx17 CDS區(qū)序列成功在體外培養(yǎng)黑素細胞中擴增,條帶單一,大小間于1000bp與750bp之間。綿羊皮膚組織蛋白提取后進行免疫印跡顯示,綿羊組織中STX17蛋白分子量大小在33 kDa左右,條帶清晰整齊,灰度分析顯示,黑色皮膚中的表達量顯著高于灰色皮膚。6為了更進一步驗證Stx4和Stx17與毛色形成的相關(guān)性,選擇天然毛色豐富的羊駝作為研究對象,分析其STX4和STX17蛋白的三級結(jié)構(gòu)和跨膜區(qū)域,并與人類的該兩種蛋白進行比較,對羊駝皮膚組織進行這兩個蛋白的免疫組化染色,體外培養(yǎng)羊駝皮膚黑素細胞提取蛋白進行Western blot檢測。結(jié)果顯示,STX4羊駝比人類多兩個α-螺旋域,STX17羊駝的比人類的多一個α-螺旋域。羊駝和人類的這兩個蛋白的跨膜區(qū)域相類似。Western blot試驗顯示STX4和STX17理想大小的陽性條帶。推測STX4和STX17在黑素細胞中存在,并可能參與毛色的形成。結(jié)論：試驗結(jié)果發(fā)現(xiàn)不同毛色皮膚之間存在SNARE家族蛋白的差異表達。由此推斷,SNARE家族成員可能參與了小鼠、綿羊和羊駝的毛色形成,STX4與小鼠、綿羊和羊駝的毛色形成呈正相關(guān),STX17與毛色形成有相關(guān)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
【學(xué)位授予單位】：山西農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：S852.2

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本文編號：1956771