發(fā)布時間：2018-05-04 09:23

  本文選題：轉(zhuǎn)基因小鼠 + microRNA-137��； 參考：《山西農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：毛色生成調(diào)控機制的研究既可以幫助我們進一步了解哺乳動物生物學(xué)特征的發(fā)生機制,又可以促進天然毛色在毛用產(chǎn)品工業(yè)的應(yīng)用,避免由于染色對人們健康的危害,具有重要的研究意義。microRNA (miRNA)通過與其靶基因的3’UTR端的結(jié)合位點相結(jié)合,抑制或降解靶基因的表達,從而參與多種生物學(xué)過程的調(diào)控。在之前的研究中,我們成功地構(gòu)建了包括灰色和棕色毛色表型在內(nèi)的microRNA-137 (miR-137)轉(zhuǎn)基因小鼠,并發(fā)現(xiàn)在不同毛色轉(zhuǎn)基因小鼠的皮膚中,miR-137的表達量差異顯著。然而,miR-137導(dǎo)致轉(zhuǎn)基因小鼠發(fā)生毛色表型變化的毛色生成調(diào)控機制仍尚未研究。為了進一步研究miR-137的毛色生成調(diào)控機制,本文通過對灰色、棕色miR-137轉(zhuǎn)基因小鼠背部皮膚進行轉(zhuǎn)錄組測序,發(fā)現(xiàn)miR-137不同程度過表達導(dǎo)致小鼠毛色變化的過程中所調(diào)控的基因以及基因功能、影響的信號通路和生物學(xué)過程；利用對小鼠皮膚組織的HE染色和免疫熒光實驗驗證了上述信息學(xué)分析所提出的miR-137可以影響黑色素細(xì)胞內(nèi)黑色素合成過程這一假設(shè)；通過生物信息學(xué)預(yù)測和雙熒光報告實驗驗證,尋找miR-137導(dǎo)致小鼠毛色變化的過程中直接調(diào)控的毛色相關(guān)靶基因,同時在小鼠皮膚黑色素細(xì)胞中過表達miR-137,觀察其靶基因和靶基因毛色通路中下游基因的表達變化以及黑色素生成量的變化,揭示了miR-137調(diào)控毛色生成的作用機制。1.以灰色、棕色miR-137轉(zhuǎn)基因小鼠皮膚作為實驗材料,通過轉(zhuǎn)錄組測序技術(shù),檢測到兩種毛色小鼠皮膚中表達的基因約19,000個。利用這些基因在不同毛色小鼠皮膚中的表達量差異,我們篩選出93個表達量差異程度在2倍以上且符合統(tǒng)計學(xué)意義(0.8≤Diverse probability≤1)的表達量顯著差異的基因(差異基因)。為了驗證轉(zhuǎn)錄組測序結(jié)果,我們通過熒光定量實驗對差異基因在灰色、棕色轉(zhuǎn)基因小鼠皮膚中的相對表達量進行實驗驗證,結(jié)果顯示所選基因的表達量趨勢與測序結(jié)果一致,這證明了此次轉(zhuǎn)錄組測序數(shù)據(jù)的可靠性,同時為進一步對表達基因為基礎(chǔ)的后續(xù)分析奠定了重要的基礎(chǔ)。2.為了進一步了解miR-137調(diào)控兩種毛色小鼠表型產(chǎn)生的過程中所影響的基因功能和信號通路,我們首先利用Protein ANalysis THrough Evolutionary Relationships (PANTHER)軟件對所有差異基因的基因功能進行Go Ontology (GO)功能注釋并觀察差異基因在GO功能中的宏觀分布。繼而,通過Database for Annotation, Visualizationand Integrated Discovery (DAVID)軟件,對已經(jīng)注釋GO功能的差異基因進行GO功能富集,結(jié)果發(fā)現(xiàn)差異基因顯著富集于黑色素合成功能。然后,我們將差異基因富集到Kyoto Encyclopedia of Genes and Genomes (KEGG)數(shù)據(jù)庫中的信號通路中,發(fā)現(xiàn)差異基因顯著富集于黑色素合成這一通路。從而分析出,miR-137在黑色素細(xì)胞內(nèi)的黑色素合成過程中起到關(guān)鍵的調(diào)控作用,導(dǎo)致了不同毛色小鼠表型的產(chǎn)生。3.為了進一步探究miR-137在小鼠體內(nèi)過表達后從整體水平上調(diào)控小鼠體內(nèi)的生物學(xué)過程變化,我們通過GSEA分析手段,分析灰色、棕色小鼠皮膚轉(zhuǎn)錄組測序得到的全部表達基因所共同富集的生物學(xué)過程。為了保證分析的準(zhǔn)確性,我們選擇GSEA數(shù)據(jù)庫中收錄的已驗證基因功能的Hallmark基因集對轉(zhuǎn)錄組獲得的全部表達基因進行生物學(xué)過程富集分析。同時,為了進一步驗證上一章中我們對所選差異基因進行功能富集分析結(jié)果的準(zhǔn)確性,我們在選取Hallmark基因集的同時也選取GO gene sets基因集進行GSEA分析。從分析結(jié)果中我們發(fā)現(xiàn),miR-137在棕色小鼠體內(nèi)過表達確實引起了黑色素合成的變化,這與上一章所分析的結(jié)果一致,并發(fā)現(xiàn)miR-137不同程度的表達趨勢可以引起小鼠體內(nèi)不同生物學(xué)過程的變化：在灰色小鼠表型中,miR-137通過調(diào)控Myc下游基因和參與核糖體相關(guān)過程可以調(diào)控色素沉著更顯著的變化。4.在灰色、棕色小鼠體內(nèi)表達量差異的基因中對miR-137的靶基因進行生物學(xué)預(yù)測,并找出可能導(dǎo)致毛色直接變化的靶基因Kit和Myo7a 。結(jié)合第二、三章的分析結(jié)果中,miR-137在黑色素合成過程中影響了黑色素生成量的變化導(dǎo)致了棕色小鼠表型產(chǎn)生,從而得知miR-137可能通過影響SCF/Kit通路及下游黑色素合成相關(guān)基因而導(dǎo)致了棕色毛色小鼠表型的產(chǎn)生。5.利用HE染色和免疫熒光實驗,發(fā)現(xiàn)miR-137在轉(zhuǎn)基因小鼠過表達引起了黑色素細(xì)胞中黑色素生成量的改變。鑒于此,通過雙熒光報告實驗驗證了Kit是miR-137的靶基因,并發(fā)現(xiàn)miR-137在小鼠黑色素細(xì)胞中過表達可以引起Kit及其下游基因Tyr, Tyrp1和Dct (Tyrp2)的表達量發(fā)生變化,調(diào)控小鼠皮膚黑色素細(xì)胞中黑色素含量的生成。結(jié)論： (1)miR-137主要通過影響黑色素細(xì)胞中黑色素合成過程導(dǎo)致轉(zhuǎn)基因小鼠淺色表型產(chǎn)生。 (2)miR-137產(chǎn)生灰色、棕色兩種轉(zhuǎn)基因小鼠表型的作用機制是不同的。miR-137通過直接影響黑色素細(xì)胞中黑色素合成過程導(dǎo)致棕色小鼠表型產(chǎn)生,并且miR-137通過調(diào)控Myc下游基因及核糖體相關(guān)過程,導(dǎo)致更明顯的灰色小鼠表型產(chǎn)生。(3)Kit和Myo7a是miR-137導(dǎo)致轉(zhuǎn)基因小鼠毛色表型變化過程中直接調(diào)控的毛色相關(guān)靶基因。 (4)通過實驗驗證了上述分析提出的miR-137過表達影響黑色素細(xì)胞中黑色素合成過程這一作用機制的假設(shè)以及Kit與miR-137之間的靶向關(guān)系,并發(fā)現(xiàn)miR-137通過影響SCF/Kit通路中Kit及Kit下游參與黑色素合成過程的主效基因Tvr.Tyrp1和Dct (Tyrp2)的表達抑制黑色素細(xì)胞中黑色素的生成。
[Abstract]:The study of the regulation mechanism of hair color generation can help us not only understand the mechanism of mammalian biological characteristics, but also promote the application of natural hair color in wool products industry, and avoid the harm of dyeing to people's health. It has important research significance.MicroRNA (miRNA) through the 3 'UTR end of its target gene. In the previous study, we successfully constructed microRNA-137 (miR-137) transgenic mice, including gray and brown hair color phenotypes, and found the difference in miR-137 expression in the skin of different hair color transgenic mice. In order to further study the regulation mechanism of the hair color generation of miR-137, the transcriptional sequence of the back skin of the brown miR-137 transgenic mice was sequenced to find that the expression of miR-137 in different degrees led to the hair of mouse hair, in order to further study the regulation mechanism of hair color generation in transgenic mice. The signal pathways and biological processes regulated by genes and gene functions during the process of color change; the hypothesis that the miR-137 can affect melanin synthesis in melanocytes by the HE staining and immunofluorescence experiments on the skin of mice is used to predict the process of melanin synthesis in melanocytes; by Bioinformatics The test and double fluorescence report test proved that the hair color related target gene was directly regulated by miR-137 in the process of hair color change in mice, and miR-137 was overexpressed in the skin melanocytes of mice. The changes of the expression of the target gene and the target gene hair color pathway and the change of the melanin formation were observed, and miR-137 was revealed. The mechanism of regulation of hair color generation.1. uses grey, brown miR-137 transgenic mice skin as experimental material and about 19000 genes expressed in the skin of two hair color mice by transcriptional sequencing technology. The differences in the expression of these genes in the skin of different hair color mice were used to screen 93 differences in expression levels. In order to verify the sequencing results of the transcriptional group, we tested the expression of the differential genes in the gray and brown transgenic mice in order to verify the sequence of the transcriptional sequences, which were 2 times more than 2 times and conformed to the significant difference (0.8 < < Diverse probability < 1). The expression trend is consistent with the sequencing results, which proves the reliability of the transcriptional sequence data and lays an important foundation for further analysis of the gene based follow-up analysis,.2. in order to further understand the gene function and signal pathways affected by the miR-137 regulation of the phenotype of two hair color mice. The Protein ANalysis THrough Evolutionary Relationships (PANTHER) software was used to carry out Go Ontology (GO) functional annotations for all the gene functions of the differential genes and to observe the macro distribution of the differential genes in the GO function. The difference gene of GO function was enriched by GO function, and the difference gene was found to be enriched in the melanin synthesis function. Then, we enriched the differential genes into the signaling pathway in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, and found that the differential genes were enriched in the melanin synthesis pathway. MiR-137 plays a key regulatory role in melanin synthesis in melanocytes, leading to the production of.3. in different hair color mice in order to further explore the biological process changes in mice from the overall level of miR-137 after overexpression in mice. We analyzed the gray and brown mice by means of GSEA analysis. In order to ensure the accuracy of the analysis, we choose the Hallmark gene set in the GSEA database to carry out the biological process enrichment analysis of all the expressed genes obtained by the transcriptional group. In this chapter, the accuracy of the functional enrichment analysis of the selected differentially differentially expressed genes was obtained. We selected the Hallmark gene set at the same time as the GO gene sets gene set for GSEA analysis. From the analysis, we found that the overexpression of miR-137 in brown mice did cause changes in melanin synthesis, which was analyzed in the previous chapter. The results are consistent, and it is found that different degrees of expression of miR-137 can cause changes in different biological processes in mice: in the phenotype of gray mice, miR-137 can regulate the more significant changes in pigmentation by regulating the downstream genes of Myc and involved in ribosome related processes, and the genes of.4. in gray and brown mice are different in expression. The target gene of miR-137 is predicted by biology, and the target gene Kit and Myo7a that may lead to direct color changes are found. In the analysis of second, third chapters, miR-137 affects the change of melanin production in the melanin synthesis process, resulting in the phenotype production of brown mice, thus knowing that miR-137 may be affected by the influence of SCF/Kit. The pathway and the downstream melanin synthesis related genes led to the production of.5. in brown color mice by HE staining and immunofluorescence experiments. It was found that the overexpression of miR-137 in transgenic mice resulted in the change of melanin formation in melanocytes. In view of this, the target gene of miR-137 was verified by the double fluorescence Report. The overexpression of miR-137 in mouse melanocytes can cause changes in the expression of Kit and its downstream genes, Tyr, Tyrp1 and Dct (Tyrp2), and regulate the formation of melanin in murine skin melanocytes. Conclusion: (1) miR-137 mainly affects the light color phenotype of transgenic mice mainly by affecting melanin synthesis in melanocytes. (2) miR-137 produces grey and brown two transgenic mice, the mechanism of the action is that different.MiR-137 can lead to the phenotypic production of brown mice by directly affecting melanin synthesis in melanocytes, and miR-137 leads to more obvious phenotypes of grey mice by regulating the downstream genes and ribosome related processes of Myc. (3) K It and Myo7a are the hair color related target genes directly regulated by miR-137 in the hair color phenotype changes of transgenic mice. (4) the hypothesis of the effect of miR-137 overexpression on melanin synthesis process in melanocytes and the targeting relationship between Kit and miR-137, and the discovery of miR-137 through experiments were verified by experiments. The expression of major genes, Tvr.Tyrp1 and Dct (Tyrp2), involved in the process of melanin synthesis in the downstream of the SCF/Kit pathway, inhibits the formation of melanin in melanocytes.

【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】：2016
【分類號】：S852.2

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1 江珊;轉(zhuǎn)基因小鼠皮膚轉(zhuǎn)錄組測序揭示microRNA-137的毛色生成調(diào)控機制[D];山西農(nóng)業(yè)大學(xué);2016年

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本文編號：1842493