辽宁绒山羊IGF2和ZBED6基因分子特征、表达及甲基化研究

发布时间：2018-06-13 00:46

本文选题：辽宁绒山羊 + IGF2基因　；参考：《沈阳农业大学》2017年硕士论文

【摘要】：辽宁绒山羊是我国特有的绒肉兼用型山羊。本研究以辽宁绒山羊为研究对象,利用分子生物学技术,克隆IGF2基因和ZBED6基因进行序列测定,分析基因的5'调控区、编码区。借助生物信息学分析软件,将辽宁绒山羊IGF2基因和ZBED6基因序列与其他物种基因序列进行比对,并预测其CpG岛。本研究分析启动子的甲基化水平对基因表达的影响;推测编码蛋白质的二、三级结构和理化性质及在不同组织中的表达谱;为辽宁绒山羊的分子育种研究奠定理论基础。主要研究如下:1.IGF2和ZBED6的基因克隆与分子特征分析对辽宁绒山羊的IGF2和ZBED6基因的5'调控区和编码区进行特异性扩增,并进行克隆和测序,研究发现克隆出的IGF2基因序列总长度为2090bp,编码区长度为540bp,编码179个氨基酸,在5'调控区第263bp处发生A/T突变,第974bp处发生A碱基缺失突变,该蛋白中存在信号肽序列,为分泌性蛋白质,有较强的亲水性但不稳定。克隆出的ZBED6基因序列总长度为4851,编码区长度为2943bp,编码980个氨基酸,编码区中第838bp处发生A/G突变;第944bp处发生A/G突变;第1799bp处发生C/T突变;2276bp处发生A/G突变;2323bp处发生C/T突变;2333bp处发生G/T突变;2397bp处发生A/G突变;2480bp处发生A/G突变,该蛋白质中未发现明显的信号肽序列,亲水性较强且不稳定。2.IGF2和ZBED6的表达水平的研究对辽宁绒山羊的肌肉、心、肝、脾、肺、肾、睾丸七中不同的组织中的IGF2和ZBED6的相对表达量进行研究,发现在肌肉组织中ZBED6的表达量最高,IGF2的表达量相对较低;IGF2基因表达量结果是:肝脏心脏肾脏肌肉肺脏睾丸脾脏,ZBED6基因表达量结果是:肌肉心脏肝脏肾脏睾丸肺脏脾脏。3.IGF2和ZBED6启动子甲基化水平的研究对辽宁绒山羊肌肉组织中的启动子区进行预测,发现在IGF2基因启动子区有34个CpG位点,ZBED6基因启动子区有28个CpG位点,通过亚硫酸氢盐测序(BSP)检测发现,肌肉组织中的IGF2甲基化水平明显高于ZBED6的甲基化水平,结合IGF2和ZBED6的表达谱进行分析发现,在肌肉组织中IGF2的表达量不仅受到抑制因子ZBED6的调控,还受到启动子区的甲基化水平的影响,基因的甲基化水平越高其相对表达量越低。以上研究结果显示,在辽宁绒山羊的肌肉生长发育的过程中,IGF2和ZBED6基因对其有重要的调控作用,IGF2编码179个氨基酸,ZBED6编码980个氨基酸。IGF2和ZBED6在不同组织中存在差异性表达和DNA甲基化修饰,这些差异基因对肌肉细胞的生长分化以及代谢有重要的关系。本研究从分子水平对辽宁绒山羊肌肉生长发育进行研究,为辽宁绒山羊的育种工作提供理论依据。
[Abstract]:Liaoning cashmere goats are the unique cashmere goats in China. This study takes Liaoning cashmere goats as the research object, using molecular biology technology, cloning IGF2 gene and ZBED6 gene for sequencing, analyzing the 5'regulatory region and coding region of the gene. With the help of bioinformatics analysis software, the IGF2 and ZBED6 gene sequences of Liaoning cashmere goats are sequenced. The comparison of the gene sequences of other species and the prediction of its CpG island. This study analyzed the effect of the promoter methylation level on the gene expression, speculates the two, three level structure and physicochemical properties of the encoded protein and the expression profiles in different tissues; it lays a theoretical foundation for the molecular breeding of Liaoning cashmere goats. The main studies are as follows: 1.IGF2 and Z BED6 gene cloning and molecular characterization analysis of the IGF2 and ZBED6 gene of Liaoning cashmere goats was specifically amplified and cloned and sequenced. The total length of the IGF2 gene sequence was 2090bp, the length of the coding region was 540bp, 179 amino acids were encoded, and A/T mutations were occurring at 263bp at 5'regulation area. There is a deletion mutation of A base in 974bp, which contains a sequence of signal peptide, which is a secretory protein with strong hydrophilic but unstable. The total length of the ZBED6 gene sequence is 4851, the length of the coding region is 2943bp, the encoding of 980 amino acids, the A/G mutation at 838bp in the coding region, A/G mutation at 944bp, and 1799bp. C/T mutation, A/G mutation at 2276bp, C/T mutation at 2323bp, G/T mutation at 2333bp, A/G mutation at 2397bp, A/G mutation at 2397bp, and no apparent sequence of signal peptides in 2480bp, and the study on the muscle, heart, liver, spleen, lung of Liaoning Cashmere Goats The relative expression of IGF2 and ZBED6 in different tissues of kidney and testis seven was studied. It was found that the expression of ZBED6 in the muscle tissue was the highest and the expression of IGF2 was relatively low; the IGF2 gene expression results were: the liver and kidney, kidney, lung, lung, testis and spleen, and the result of the ZBED6 gene expression was the muscular heart liver kidney testis and the spleen and lung spleen. The study of.3.IGF2 and ZBED6 promoter methylation level was used to predict the promoter region in the muscle tissue of Liaoning cashmere goats. It was found that there were 34 CpG loci in the promoter region of the IGF2 gene, 28 CpG loci in the promoter region of the ZBED6 gene, and the occurrence of the occurrence by the sequencing of hydrogen sulphite (BSP), and the level of IGF2 methylation in the muscle tissue was significantly higher than that of ZBE. The methylation level of D6, combined with the expression profiles of IGF2 and ZBED6, showed that the expression of IGF2 in the muscle tissue was not only regulated by the inhibitory factor ZBED6, but also influenced by the level of methylation in the promoter region. The higher the level of the methylation of the gene, the lower the relative expression of the gene. The above results showed that the muscles of Liaoning cashmere goats were in the muscles. In the process of growth and development, IGF2 and ZBED6 genes play an important role in regulating it. IGF2 encodes 179 amino acids, ZBED6 encoding 980 amino acids.IGF2 and ZBED6 exist in different tissues, and there are differential expressions and DNA methylation modification. These differentially genes have important relationship with the growth and metabolism of muscle cells and metabolism. To study the muscle growth and development of Liaoning cashmere goat, and provide theoretical basis for breeding of Liaoning cashmere goat.
【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S827

【相似文献】