miR-let7a在辽宁绒山羊毛囊发育周期中的差异表达及其靶基因功能鉴定

发布时间：2018-04-26 11:10

本文选题：miRNA-let7a + 绒山羊　；参考：《吉林大学》2015年硕士论文

【摘要】：绒山羊是一类主要用于生产羊绒羊毛的山羊，目前世界上主要的品种有伊拉克绒山羊、澳大利亚绒山羊、巴基斯坦披肩山羊、河西绒山羊以及辽宁绒山羊等。在我国有10种绒山羊被列为遗传资源保护品种，其中包括青藏高原山羊、辽宁绒山羊、吕梁黑山羊和内蒙古绒山羊等。为了提高我国羊绒的产量和质量，，越来越多的具有优秀品质的绒山羊被选育出来，而毛囊是调控绒山羊被毛生长的重要组织，深入研究毛囊的周期发育调控机制对提高和改善羊绒品质有重要的作用。后基因组时代的到来，为研究毛囊周期发育开辟了新的道路。近年来，有关非编码小RNA的调控机制的研究逐渐成为热点问题。 microRNA（miRNA）是介导分子进行基因沉默的一类大家族非编码RNA，具有调节机体生长发育，细胞增殖分化，信号传导及免疫应答反应等多种功能。1993年首个miRNA（lin-4）被Victor Ambros和Rosalind Lee等在对秀丽线虫幼虫发育调控的研究中发现以后，越来越多的研究表明，miRNA在动物和植物中均有表达，并在动植物基础生命活动中发挥不同的作用。在动物中，let-7家族是研究最深入的miRNA之一，其主要编码let7a，let7b，let7c等12个基因，对细胞的增殖，分化及肿瘤的形成等方面起重要的调控作用。近年来已有研究表明let-7家族可以调控皮肤黑色素瘤的形成并在山羊和绵羊的皮肤组织中均有表达，但有关其如何调控毛囊的周期发育研究很少。因此，本研究在前期高通量测序的基础上，选择差异表达miRNA-let7a为研究对象，对绒山羊毛囊发育周期的调控机制进行了系统的研究。首先利用荧光定量PCR技术检测了miRNA-let7a在绒山羊毛囊发育生长期和退行期的表达差异，然后利用生物信息学软件miRGenV3.0对miRNA-let7a的靶基因进行预测，并进一步通过DAVID6.7基因注释工具中的GO和KEGG分析筛选出与毛囊发育周期相关的靶基因，随后通过荧光定量PCR和Western-blot技术分别在mRNA水平和蛋白水平对候选靶基因进行鉴定,并利用双荧光素酶报告基因检测法（Dual luciferase reporter gene assay）鉴定靶基因的作用靶点。由于细胞转染效率的高低直接影响到后续实验结果，因此我们利用MTT，荧光显微镜观察法及流式细胞法对转染效率进行了优化。通过对miRNA-let7a在绒山羊毛囊发育周期中的研究，阐明miRNA-let7a及其靶基因在绒山羊毛囊发育周期中的作用机理，为提高和改善绒山羊羊绒产量和品质提供了新的思路。具体研究结果如下： 1.通过荧光定量PCR技术检测miRNA-let7a的表达,发现其在绒山羊毛囊发育的不同时期均有表达，且在退行期中的表达量显著高于生长期。 2.利用在线生物学软件miRGenV3.0预测miRNA-let7a的靶基因，并通过DAVID6.7软件分析筛选出与毛囊发育周期相关的三个靶基因：Cmyc、FGF5和IGF-1R。利过生物信息学软件NCBI Blast对Cmyc、FGF5及IGF-1R进行同源性分析，结果显示三个靶基因与绵羊、牛、马等其他物种的同源性高达90%以上。 3.通过荧光定量PCR技术在mRNA水平上对候选靶基因Cmyc、FGF5及IGF-1R进行鉴定，结果显示三个候选靶基因在绒山羊生长期中表达量均高于退行期，进一步通过Western-blot技术在蛋白水平上对候选靶基因Cmyc和FGF5进行鉴定，结果表明候选靶基因Cmyc和FGF5在绒山羊生长期中表达量高于退行期，与荧光定量结果一致，并与miRNA-let7a表达趋势相反。这说明Cmyc和FGF5是miRNA-let7a的靶基因。 4.利用噻唑蓝染色法（MTT）、荧光显微镜观察法及流式细胞法对HEK-293T细胞转染效率进行检测。结果表明：每孔加入1μl脂质体，mimics与质粒载体共转染的最终浓度分别为100nM和0.8mg时，转染效率最高为53.13%，可以用于后续研究。 5.利用双荧光素酶报告基因检测法鉴定miRNA-let7a与其靶基因Cmyc和FGF5的作用靶点。结果显示，靶基因Cmyc及FGF5与miRNA-let7a“种子序列”结合的靶位点分别为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.

【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S827

【参考文献】