本文選題：miRNA-let7a + 絨山羊　； 參考：《吉林大學(xué)》2015年碩士論文

【摘要】：絨山羊是一類主要用于生產(chǎn)羊絨羊毛的山羊，目前世界上主要的品種有伊拉克絨山羊、澳大利亞絨山羊、巴基斯坦披肩山羊、河西絨山羊以及遼寧絨山羊等。在我國(guó)有10種絨山羊被列為遺傳資源保護(hù)品種，其中包括青藏高原山羊、遼寧絨山羊、呂梁黑山羊和內(nèi)蒙古絨山羊等。為了提高我國(guó)羊絨的產(chǎn)量和質(zhì)量，，越來(lái)越多的具有優(yōu)秀品質(zhì)的絨山羊被選育出來(lái)，而毛囊是調(diào)控絨山羊被毛生長(zhǎng)的重要組織，深入研究毛囊的周期發(fā)育調(diào)控機(jī)制對(duì)提高和改善羊絨品質(zhì)有重要的作用。后基因組時(shí)代的到來(lái)，為研究毛囊周期發(fā)育開辟了新的道路。近年來(lái)，有關(guān)非編碼小RNA的調(diào)控機(jī)制的研究逐漸成為熱點(diǎn)問(wèn)題。 microRNA（miRNA）是介導(dǎo)分子進(jìn)行基因沉默的一類大家族非編碼RNA，具有調(diào)節(jié)機(jī)體生長(zhǎng)發(fā)育，細(xì)胞增殖分化，信號(hào)傳導(dǎo)及免疫應(yīng)答反應(yīng)等多種功能。1993年首個(gè)miRNA（lin-4）被Victor Ambros和Rosalind Lee等在對(duì)秀麗線蟲幼蟲發(fā)育調(diào)控的研究中發(fā)現(xiàn)以后，越來(lái)越多的研究表明，miRNA在動(dòng)物和植物中均有表達(dá)，并在動(dòng)植物基礎(chǔ)生命活動(dòng)中發(fā)揮不同的作用。在動(dòng)物中，let-7家族是研究最深入的miRNA之一，其主要編碼let7a，let7b，let7c等12個(gè)基因，對(duì)細(xì)胞的增殖，分化及腫瘤的形成等方面起重要的調(diào)控作用。近年來(lái)已有研究表明let-7家族可以調(diào)控皮膚黑色素瘤的形成并在山羊和綿羊的皮膚組織中均有表達(dá)，但有關(guān)其如何調(diào)控毛囊的周期發(fā)育研究很少。 因此，本研究在前期高通量測(cè)序的基礎(chǔ)上，選擇差異表達(dá)miRNA-let7a為研究對(duì)象，對(duì)絨山羊毛囊發(fā)育周期的調(diào)控機(jī)制進(jìn)行了系統(tǒng)的研究。首先利用熒光定量PCR技術(shù)檢測(cè)了miRNA-let7a在絨山羊毛囊發(fā)育生長(zhǎng)期和退行期的表達(dá)差異，然后利用生物信息學(xué)軟件miRGenV3.0對(duì)miRNA-let7a的靶基因進(jìn)行預(yù)測(cè)，并進(jìn)一步通過(guò)DAVID6.7基因注釋工具中的GO和KEGG分析篩選出與毛囊發(fā)育周期相關(guān)的靶基因，隨后通過(guò)熒光定量PCR和Western-blot技術(shù)分別在mRNA水平和蛋白水平對(duì)候選靶基因進(jìn)行鑒定,并利用雙熒光素酶報(bào)告基因檢測(cè)法（Dual luciferase reporter gene assay）鑒定靶基因的作用靶點(diǎn)。由于細(xì)胞轉(zhuǎn)染效率的高低直接影響到后續(xù)實(shí)驗(yàn)結(jié)果，因此我們利用MTT，熒光顯微鏡觀察法及流式細(xì)胞法對(duì)轉(zhuǎn)染效率進(jìn)行了優(yōu)化。通過(guò)對(duì)miRNA-let7a在絨山羊毛囊發(fā)育周期中的研究，闡明miRNA-let7a及其靶基因在絨山羊毛囊發(fā)育周期中的作用機(jī)理，為提高和改善絨山羊羊絨產(chǎn)量和品質(zhì)提供了新的思路。具體研究結(jié)果如下： 1.通過(guò)熒光定量PCR技術(shù)檢測(cè)miRNA-let7a的表達(dá),發(fā)現(xiàn)其在絨山羊毛囊發(fā)育的不同時(shí)期均有表達(dá)，且在退行期中的表達(dá)量顯著高于生長(zhǎng)期。 2.利用在線生物學(xué)軟件miRGenV3.0預(yù)測(cè)miRNA-let7a的靶基因，并通過(guò)DAVID6.7軟件分析篩選出與毛囊發(fā)育周期相關(guān)的三個(gè)靶基因：Cmyc、FGF5和IGF-1R。利過(guò)生物信息學(xué)軟件NCBI Blast對(duì)Cmyc、FGF5及IGF-1R進(jìn)行同源性分析，結(jié)果顯示三個(gè)靶基因與綿羊、牛、馬等其他物種的同源性高達(dá)90%以上。 3.通過(guò)熒光定量PCR技術(shù)在mRNA水平上對(duì)候選靶基因Cmyc、FGF5及IGF-1R進(jìn)行鑒定，結(jié)果顯示三個(gè)候選靶基因在絨山羊生長(zhǎng)期中表達(dá)量均高于退行期，進(jìn)一步通過(guò)Western-blot技術(shù)在蛋白水平上對(duì)候選靶基因Cmyc和FGF5進(jìn)行鑒定，結(jié)果表明候選靶基因Cmyc和FGF5在絨山羊生長(zhǎng)期中表達(dá)量高于退行期，與熒光定量結(jié)果一致，并與miRNA-let7a表達(dá)趨勢(shì)相反。這說(shuō)明Cmyc和FGF5是miRNA-let7a的靶基因。 4.利用噻唑藍(lán)染色法（MTT）、熒光顯微鏡觀察法及流式細(xì)胞法對(duì)HEK-293T細(xì)胞轉(zhuǎn)染效率進(jìn)行檢測(cè)。結(jié)果表明：每孔加入1μl脂質(zhì)體，mimics與質(zhì)粒載體共轉(zhuǎn)染的最終濃度分別為100nM和0.8mg時(shí)，轉(zhuǎn)染效率最高為53.13%，可以用于后續(xù)研究。 5.利用雙熒光素酶報(bào)告基因檢測(cè)法鑒定miRNA-let7a與其靶基因Cmyc和FGF5的作用靶點(diǎn)。結(jié)果顯示，靶基因Cmyc及FGF5與miRNA-let7a“種子序列”結(jié)合的靶位點(diǎn)分別為CTGCCTC和ACTCCAT。
[Abstract]:Cashmere goat is a class of goats mainly used to produce cashmere wool. At present, the main varieties in the world are Iraqi cashmere goats, Australian cashmere goats, Pakistan shawl goats, Hexi cashmere goats and Liaoning cashmere goats. In our country, 10 kinds of cashmere goats are listed as protective varieties of genetic resources, including Tibetan plateau goats, Liaoning cashmere. Goats, Lvliang black goats and Inner Mongolia cashmere goats, in order to improve the production and quality of cashmere in China, more and more good quality cashmere goats are bred. The hair follicle is an important organization to regulate the growth of cashmere goats. The study of the regulation mechanism of the cycle development of the hair follicle has an important role in improving and improving the quality of cashmere. The arrival of the post genome era has opened a new way for the study of hair follicle cycle development. In recent years, the research on the regulation mechanism of the non coding small RNA has gradually become a hot issue.
MicroRNA (miRNA) is a class of large family non coded RNA mediated by molecular gene silencing. It has many functions such as regulating body growth, cell proliferation and differentiation, signal transduction and immune response. The first miRNA (Lin-4) of miRNA (Lin-4) was found in the study of the regulation of the development of the larvae of Caenorhabditis elegans by Victor Ambros and Rosalind Lee. More and more studies have shown that miRNA is expressed in both animals and plants and plays a different role in the basic life activities of animals and plants. In animals, the let-7 family is one of the most in-depth studies of miRNA, which mainly encodes 12 genes, such as let7a, let7b, let7c and so on. It plays an important role in cell proliferation, differentiation and tumor formation. Recent studies have shown that the let-7 family can regulate the formation of skin melanoma and can be expressed in the skin tissues of goats and sheep, but there are few studies on how to regulate the cycle development of hair follicles.
Therefore, on the basis of high throughput sequencing, the study selected differential expression miRNA-let7a as the research object, and systematically studied the regulation mechanism of the growth cycle of cashmere wool sac. First, the difference of miRNA-let7a expression in the long and degenerative period of the growth of wool sac was detected by the fluorescence quantitative PCR technique. The target gene of miRNA-let7a was predicted by the software miRGenV3.0, and the target genes related to the hair follicle development cycle were screened by GO and KEGG analysis in the DAVID6.7 gene annotation tool. Then the candidate target genes were identified by fluorescence quantitative PCR and Western-blot techniques at mRNA level and protein level respectively. Dual luciferase reporter gene assay (double luciferase reporter gene assay) was used to identify the target target of the target gene. As the cell transfection efficiency was directly affected by the results of the follow-up experiment, we used MTT, fluorescence microscope observation and flow cytometry to optimize the transfer efficiency. Through miRNA-let7a in cashmere. In the development cycle of goat hair follicle, the mechanism of miRNA-let7a and its target gene in the development cycle of cashmere wool sac were elucidated, and a new idea was provided to improve and improve the yield and quality of cashmere cashmere.
1. the expression of miRNA-let7a was detected by the fluorescence quantitative PCR technique. It was found that it was expressed in the different periods of the development of cashmere wool sac, and the expression in the degenerative period was significantly higher than that in the growth period.
2. the target genes of miRNA-let7a were predicted by the online biological software miRGenV3.0, and three target genes related to the hair follicle development cycle were screened by DAVID6.7 software. Cmyc, FGF5 and IGF-1R. bioinformatics software NCBI Blast had Homologous Analysis on Cmyc, FGF5 and IGF-1R, and the results showed that three target genes were with sheep, cattle and horses. The homology of other species is up to 90%.
3. the candidate target gene Cmyc, FGF5 and IGF-1R were identified by fluorescence quantitative PCR at mRNA level. The results showed that the expression of three candidate target genes in the growth period of cashmere goat was higher than that of the degenerative period. The candidate target gene Cmyc and FGF5 were identified by Western-blot technology at the protein level, and the candidate target gene was shown to be the candidate target gene. The expression of Cmyc and FGF5 in the growth period of cashmere goats was higher than that in the degenerative period, which was in accordance with the fluorescence quantitative results, and was contrary to the expression trend of miRNA-let7a. This shows that Cmyc and FGF5 are the target genes of miRNA-let7a.
4. the transfection efficiency of HEK-293T cells was detected by thiazolium staining (MTT), fluorescence microscope observation and flow cytometry. The results showed that the final concentration of mimics and plasmid carrier was 100nM and 0.8mg respectively, and the highest transfection efficiency was 53.13%, which could be used for follow-up study.
5. the target genes of miRNA-let7a and its target gene Cmyc and FGF5 were identified by the double luciferase reporter gene detection method. The results showed that the target loci of the target gene Cmyc, FGF5 and the miRNA-let7a "seed sequence" were CTGCCTC and ACTCCAT., respectively.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S827

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