基于iTRAQ技术对不同毛色绵羊皮肤相关蛋白组学分析和功能研究
发布时间：2018-11-26 17:55
【摘要】：哺乳动物的被毛颜色具有重要的经济学价值。绵羊的被毛是纺织业生产的重要原材料,因此,利用分子生物学手段调控绵羊被毛颜色具有重要的实践意义。动物皮肤和毛色的形成主要由黑色素细胞内产生黑色素的数量、种类和分布比例所决定的。目前已经发现的影响动物体内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技术是在蛋白组学上发展起来的一项新兴的生物技术,它可以同时标记8种不同样品,进行相对和绝对定量分析,此项技术可用于定性与定量同步进行,具有灵敏性高、高通量、分离能力强、结果可靠等特点,近年来,被应用在多种学科和领域。本研究以绵羊作为研究对象,应用iTRAQ技术对白色和黑色绵羊皮肤组织进行蛋白组学分析,并根据生物信息学分析方法和报告,筛选出有可能参与绵羊毛色形成的蛋白质,将筛选出的差异蛋白应用Western blot和QRT-PCR进行后续的定量分析验证,利用免疫组化、免疫荧光或激光共聚焦技术对差异蛋白进行表达定位分析。具体试验结果如下：1.G蛋白偶联信号通路中Gnαs和Gna11亚基在白色、黑色绵羊皮肤组织中的表达分析。研究显示,Gnas和Gna11均在绵羊皮肤组织中表达,且在黑色绵羊皮肤组织中的表达量显著高于白色绵羊皮肤中的表达(P0.05),转录和蛋白水平相一致;免疫组织化学和免疫荧光染色发现,Gnas和Gna11蛋白位于绵羊毛囊的毛乳头、外根鞘部位;此外,还发现Gnall mRNA和蛋白的相对表达量均高于Gnas的相对表达量。2.利用iTRAQ技术检测和分析白色和黑色绵羊皮肤组织中蛋白质的定量信息。本试验共获得不同肽段(unique peptide)9909个,鉴定到1704个蛋白质,其中有539个蛋白质具有5段及以上的不同肽段的覆盖。以白色绵羊皮肤组织为对照,对鉴定的1704个蛋白质数据进行整理分析,以差异表达上下调1.2倍(ratio士1.2)且P0.05为标准,共计筛选获得不同被毛颜色的差异蛋白的总数为136个,其中上调蛋白为101个,下调蛋白为35个。3.将LC-MS/MS检测到的蛋白质定量结果的原始数据进行GO注释、KEGG代谢通路分析和差异蛋白质互作网络生物信息学统计分析。GO注释结果显示：本试验中共134条蛋白序列被2022条GO功能条目注释;KEGG信号通路分析结果显示：共提取到与55个差异蛋白参与的121条信号通路。互作网络分析图发现在鉴定到的目的蛋白中有A2M、TTR、VIM、ALB、FGA、FGB、FGG、C1QC和APOA1等差异蛋白参与蛋白的直接相互作用。4.通过iTRAQ技术检测发现,Ang II蛋白存在于白色、黑色绵羊皮肤中,且为上调差异蛋白;利用Western blot和QRT-PCR进一步分析验证发现其与iTRAQ技术检测的结果相一致,即Ang Ⅱ蛋白在黑色绵羊皮肤的表达量显著高于白色绵羊皮肤(P0.05);免疫组织化学结果显示,Ang Ⅱ蛋白在白色、黑色绵羊皮肤毛囊中的毛乳头和外根鞘表达,且Ang Ⅱ蛋白在黑色绵羊毛囊中真皮乳头和外根鞘部的表达量均高于白色皮肤(P0.05);为研究Ang Ⅱ蛋白在绵羊被毛颜色的形成中的作用提供了新的思路。5. iTRAQ技术检测和鉴定发现,ACTB蛋白和FGA蛋白存在于白色、黑色绵羊皮肤中；ACTB蛋白在白色绵羊皮肤组织中的相对表达量显著高于黑色绵羊皮肤组织(P0.05);说明ACTB蛋白可能不适合作为毛色和色素沉积相关研究的内参基因或蛋白。FGA蛋白在黑色绵羊皮肤组织中的相对表达量显著高于白色绵羊皮肤组织(P0.05);利用激光共聚焦技术发现,ACTB蛋白主要存在于绵羊毛囊的外根鞘;FGA蛋白在毛囊的外根鞘、毛乳头和毛基质部都有表达;推测ACTB和FGA蛋白以及其所在的血小板信号通路可能参与绵羊被毛颜色的形成,但其调控绵羊被毛颜色形成的分子机制需要进一步深入研究和探讨。本研究应用iTRAQ技术对不同毛色绵羊皮肤组织中的蛋白质进行定量分析,通过生物信息学的分析和统计,我们发现了可能参与毛色形成的蛋白或信号通路,并对其进行后续的验证和分析。这一研究为将来蛋白质组学在调控绵羊被毛颜色的形成提供了试验基础,为探索和发现参与毛色形成机制提供了有力的证据。
[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，
本文编号：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，
本文编号：2359232
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