基于iTRAQ技術(shù)對(duì)不同毛色綿羊皮膚相關(guān)蛋白組學(xué)分析和功能研究
發(fā)布時(shí)間：2018-11-26 17:55
【摘要】：哺乳動(dòng)物的被毛顏色具有重要的經(jīng)濟(jì)學(xué)價(jià)值。綿羊的被毛是紡織業(yè)生產(chǎn)的重要原材料,因此,利用分子生物學(xué)手段調(diào)控綿羊被毛顏色具有重要的實(shí)踐意義。動(dòng)物皮膚和毛色的形成主要由黑色素細(xì)胞內(nèi)產(chǎn)生黑色素的數(shù)量、種類和分布比例所決定的。目前已經(jīng)發(fā)現(xiàn)的影響動(dòng)物體內(nèi)黑色素形成的基因包括：MC1R、α-MSH、TYR、TYR-1、LMITF, c-Kit、 Agouti、Slc7al1、HGF、c-Met等基因和蛋白。iTRAQ技術(shù)是在蛋白組學(xué)上發(fā)展起來(lái)的一項(xiàng)新興的生物技術(shù),它可以同時(shí)標(biāo)記8種不同樣品,進(jìn)行相對(duì)和絕對(duì)定量分析,此項(xiàng)技術(shù)可用于定性與定量同步進(jìn)行,具有靈敏性高、高通量、分離能力強(qiáng)、結(jié)果可靠等特點(diǎn),近年來(lái),被應(yīng)用在多種學(xué)科和領(lǐng)域。本研究以綿羊作為研究對(duì)象,應(yīng)用iTRAQ技術(shù)對(duì)白色和黑色綿羊皮膚組織進(jìn)行蛋白組學(xué)分析,并根據(jù)生物信息學(xué)分析方法和報(bào)告,篩選出有可能參與綿羊毛色形成的蛋白質(zhì),將篩選出的差異蛋白應(yīng)用Western blot和QRT-PCR進(jìn)行后續(xù)的定量分析驗(yàn)證,利用免疫組化、免疫熒光或激光共聚焦技術(shù)對(duì)差異蛋白進(jìn)行表達(dá)定位分析。具體試驗(yàn)結(jié)果如下：1.G蛋白偶聯(lián)信號(hào)通路中Gnαs和Gna11亞基在白色、黑色綿羊皮膚組織中的表達(dá)分析。研究顯示,Gnas和Gna11均在綿羊皮膚組織中表達(dá),且在黑色綿羊皮膚組織中的表達(dá)量顯著高于白色綿羊皮膚中的表達(dá)(P0.05),轉(zhuǎn)錄和蛋白水平相一致;免疫組織化學(xué)和免疫熒光染色發(fā)現(xiàn),Gnas和Gna11蛋白位于綿羊毛囊的毛乳頭、外根鞘部位;此外,還發(fā)現(xiàn)Gnall mRNA和蛋白的相對(duì)表達(dá)量均高于Gnas的相對(duì)表達(dá)量。2.利用iTRAQ技術(shù)檢測(cè)和分析白色和黑色綿羊皮膚組織中蛋白質(zhì)的定量信息。本試驗(yàn)共獲得不同肽段(unique peptide)9909個(gè),鑒定到1704個(gè)蛋白質(zhì),其中有539個(gè)蛋白質(zhì)具有5段及以上的不同肽段的覆蓋。以白色綿羊皮膚組織為對(duì)照,對(duì)鑒定的1704個(gè)蛋白質(zhì)數(shù)據(jù)進(jìn)行整理分析,以差異表達(dá)上下調(diào)1.2倍(ratio士1.2)且P0.05為標(biāo)準(zhǔn),共計(jì)篩選獲得不同被毛顏色的差異蛋白的總數(shù)為136個(gè),其中上調(diào)蛋白為101個(gè),下調(diào)蛋白為35個(gè)。3.將LC-MS/MS檢測(cè)到的蛋白質(zhì)定量結(jié)果的原始數(shù)據(jù)進(jìn)行GO注釋、KEGG代謝通路分析和差異蛋白質(zhì)互作網(wǎng)絡(luò)生物信息學(xué)統(tǒng)計(jì)分析。GO注釋結(jié)果顯示：本試驗(yàn)中共134條蛋白序列被2022條GO功能條目注釋;KEGG信號(hào)通路分析結(jié)果顯示：共提取到與55個(gè)差異蛋白參與的121條信號(hào)通路�；プ骶W(wǎng)絡(luò)分析圖發(fā)現(xiàn)在鑒定到的目的蛋白中有A2M、TTR、VIM、ALB、FGA、FGB、FGG、C1QC和APOA1等差異蛋白參與蛋白的直接相互作用。4.通過(guò)iTRAQ技術(shù)檢測(cè)發(fā)現(xiàn),Ang II蛋白存在于白色、黑色綿羊皮膚中,且為上調(diào)差異蛋白;利用Western blot和QRT-PCR進(jìn)一步分析驗(yàn)證發(fā)現(xiàn)其與iTRAQ技術(shù)檢測(cè)的結(jié)果相一致,即Ang Ⅱ蛋白在黑色綿羊皮膚的表達(dá)量顯著高于白色綿羊皮膚(P0.05);免疫組織化學(xué)結(jié)果顯示,Ang Ⅱ蛋白在白色、黑色綿羊皮膚毛囊中的毛乳頭和外根鞘表達(dá),且Ang Ⅱ蛋白在黑色綿羊毛囊中真皮乳頭和外根鞘部的表達(dá)量均高于白色皮膚(P0.05);為研究Ang Ⅱ蛋白在綿羊被毛顏色的形成中的作用提供了新的思路。5. iTRAQ技術(shù)檢測(cè)和鑒定發(fā)現(xiàn),ACTB蛋白和FGA蛋白存在于白色、黑色綿羊皮膚中；ACTB蛋白在白色綿羊皮膚組織中的相對(duì)表達(dá)量顯著高于黑色綿羊皮膚組織(P0.05);說(shuō)明ACTB蛋白可能不適合作為毛色和色素沉積相關(guān)研究的內(nèi)參基因或蛋白。FGA蛋白在黑色綿羊皮膚組織中的相對(duì)表達(dá)量顯著高于白色綿羊皮膚組織(P0.05);利用激光共聚焦技術(shù)發(fā)現(xiàn),ACTB蛋白主要存在于綿羊毛囊的外根鞘;FGA蛋白在毛囊的外根鞘、毛乳頭和毛基質(zhì)部都有表達(dá);推測(cè)ACTB和FGA蛋白以及其所在的血小板信號(hào)通路可能參與綿羊被毛顏色的形成,但其調(diào)控綿羊被毛顏色形成的分子機(jī)制需要進(jìn)一步深入研究和探討。本研究應(yīng)用iTRAQ技術(shù)對(duì)不同毛色綿羊皮膚組織中的蛋白質(zhì)進(jìn)行定量分析,通過(guò)生物信息學(xué)的分析和統(tǒng)計(jì),我們發(fā)現(xiàn)了可能參與毛色形成的蛋白或信號(hào)通路,并對(duì)其進(jìn)行后續(xù)的驗(yàn)證和分析。這一研究為將來(lái)蛋白質(zhì)組學(xué)在調(diào)控綿羊被毛顏色的形成提供了試驗(yàn)基礎(chǔ),為探索和發(fā)現(xiàn)參與毛色形成機(jī)制提供了有力的證據(jù)。
[Abstract]:The hair color of the mammal is of great economic value. The wool of sheep is an important raw material for textile production, so it is of great practical significance to control sheep's hair color by means of molecular biology. The formation of animal skin and hair color is mainly determined by the amount, type and distribution ratio of melanin in the melanin cells. The genes that have been found to affect the formation of melanin in the animal body include the genes and proteins such as MC1R, C-MSH, TYR, TYR-1, LMITF, c-Kit, Agouti, Slc7al1, HGF, c-Met, and the like. iTRAQ technology is a new biological technology developed on proteomics, which can mark eight different samples at the same time, carry out relative and absolute quantitative analysis, and the technology can be used for qualitative and quantitative synchronization, and has high sensitivity, high flux and strong separation capability, The result is reliable and so on. In recent years, it has been applied in many subjects and fields. In this study, sheep were used as the research object, and the proteomic analysis of the white and black sheep skin tissues was carried out by using the iTRAQ technique, and the protein that could be involved in the color formation of the sheep was screened according to the bioinformatics analysis method and the report. Western blot and QRT-PCR were used to carry out the subsequent quantitative analysis and validation, and the differential protein was expressed and analyzed by immunohistochemistry, immunofluorescence or laser confocal technique. The results of the specific test are as follows: 1. The expression of Gn and GGA11 subunits in the G protein coupled signal pathway in white and black sheep skin tissue. The results showed that the expression of Gnas and Gna11 in the skin tissue of sheep was significantly higher than that in the white sheep skin (P0.05), and the transcription and protein levels were in line with that of the white sheep skin. Gnuas and Gna11 proteins were located in the hair papilla and the outer root of sheep hair follicle. In addition, it was found that the relative expression of Gnall mRNA and protein was higher than that of Gnas. The quantitative information of the protein in the white and black sheep skin tissues was detected and analyzed using the iTRAQ technique. In this study, a total of 9909 different peptide fragments were obtained, and 1704 proteins were identified, of which 539 were covered with 5 or more different peptide segments. Based on the white sheep skin tissue, the identified 1704 protein data were analyzed and the difference was reduced by 1. 2 times (ratio 1. 2) and P 0.05. The total number of the differentially expressed proteins was 136, and the up-regulated protein was 101. The down-regulation protein was 35. The raw data of the quantitative results of the protein detected by LC-MS/ MS were analyzed by GO, KEGG metabolic pathway analysis and differential protein cross-network bioinformatics. The results of GO annotation show that the total of 134 protein sequences in the test are annotated by the function of 2022 GO; the results of the analysis of the KEGG signal pathway show that a total of 121 signal paths with the participation of 55 differential proteins are extracted. The results showed that A2M, TTR, VIM, ALB, FGA, FGB, FGG, C1QC and APOA1 were involved in the direct interaction of protein in the identified target protein. in that detection of iTRAQ technique, the Ang II protein is present in the white and black sheep skin, and is an up-regulation difference protein; and the result of the verification is further analyzed by Western blot and QRT-PCR, and the result is consistent with the result of the iTRAQ technique detection, The results showed that the expression of Ang鈪，
本文編號(hào)：2359232
[Abstract]:The hair color of the mammal is of great economic value. The wool of sheep is an important raw material for textile production, so it is of great practical significance to control sheep's hair color by means of molecular biology. The formation of animal skin and hair color is mainly determined by the amount, type and distribution ratio of melanin in the melanin cells. The genes that have been found to affect the formation of melanin in the animal body include the genes and proteins such as MC1R, C-MSH, TYR, TYR-1, LMITF, c-Kit, Agouti, Slc7al1, HGF, c-Met, and the like. iTRAQ technology is a new biological technology developed on proteomics, which can mark eight different samples at the same time, carry out relative and absolute quantitative analysis, and the technology can be used for qualitative and quantitative synchronization, and has high sensitivity, high flux and strong separation capability, The result is reliable and so on. In recent years, it has been applied in many subjects and fields. In this study, sheep were used as the research object, and the proteomic analysis of the white and black sheep skin tissues was carried out by using the iTRAQ technique, and the protein that could be involved in the color formation of the sheep was screened according to the bioinformatics analysis method and the report. Western blot and QRT-PCR were used to carry out the subsequent quantitative analysis and validation, and the differential protein was expressed and analyzed by immunohistochemistry, immunofluorescence or laser confocal technique. The results of the specific test are as follows: 1. The expression of Gn and GGA11 subunits in the G protein coupled signal pathway in white and black sheep skin tissue. The results showed that the expression of Gnas and Gna11 in the skin tissue of sheep was significantly higher than that in the white sheep skin (P0.05), and the transcription and protein levels were in line with that of the white sheep skin. Gnuas and Gna11 proteins were located in the hair papilla and the outer root of sheep hair follicle. In addition, it was found that the relative expression of Gnall mRNA and protein was higher than that of Gnas. The quantitative information of the protein in the white and black sheep skin tissues was detected and analyzed using the iTRAQ technique. In this study, a total of 9909 different peptide fragments were obtained, and 1704 proteins were identified, of which 539 were covered with 5 or more different peptide segments. Based on the white sheep skin tissue, the identified 1704 protein data were analyzed and the difference was reduced by 1. 2 times (ratio 1. 2) and P 0.05. The total number of the differentially expressed proteins was 136, and the up-regulated protein was 101. The down-regulation protein was 35. The raw data of the quantitative results of the protein detected by LC-MS/ MS were analyzed by GO, KEGG metabolic pathway analysis and differential protein cross-network bioinformatics. The results of GO annotation show that the total of 134 protein sequences in the test are annotated by the function of 2022 GO; the results of the analysis of the KEGG signal pathway show that a total of 121 signal paths with the participation of 55 differential proteins are extracted. The results showed that A2M, TTR, VIM, ALB, FGA, FGB, FGG, C1QC and APOA1 were involved in the direct interaction of protein in the identified target protein. in that detection of iTRAQ technique, the Ang II protein is present in the white and black sheep skin, and is an up-regulation difference protein; and the result of the verification is further analyzed by Western blot and QRT-PCR, and the result is consistent with the result of the iTRAQ technique detection, The results showed that the expression of Ang鈪，
本文編號(hào)：2359232
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