东北梅花鹿鹿茸组织Anxa-1、Anxa-2基因cDNA克隆及表达分析

发布时间：2018-01-27 13:10

本文关键词： 鹿茸 Anxa-1 Anxa-2 cDNA克隆生物信息学分析荧光定量PCR　出处：《东北林业大学》2015年硕士论文　论文类型：学位论文

【摘要】：鹿茸角是雄性梅花鹿的第二性征,它是哺乳动物中唯一可再生器官,在快速生长期,其顶端增殖区的细胞分裂速度是肿瘤细胞分裂速度的近30倍,这样的速度是其他哺乳动物组织和器官所无法比拟的,鹿茸组织的细胞增殖、分化、凋亡受到特定基因的表达调控,但迄今仍无法从根本上阐明鹿茸生长的生物学调控机制。本试验以东北梅花鹿(Cervus nippon hortulorum)鹿茸尖端间充质组织为材料,以对多种恶性肿瘤细胞起着重要调节作用的膜联蛋白基因为研究对象,用RT-PCR技术克隆出包含膜联蛋白A1 (Anxa-1)基因与膜联蛋白A2 (Anxa-2)基因全部编码区的cDNA序列,并运用生物信息学方法及实时荧光定量技术对这两种基因的氨基酸序列及不同生长时期的表达情况进行了分析。主要研究结果如下：1、东北梅花鹿鹿茸Anxa-1 和 Anxa-2基因cDNA克隆及生物信息学分析Anxa-1 与 Anxa-2基因序列测序长度分别为1059bp和1180bp,分别编码346个氨基酸和339个氨基酸。经生物信息学分析,Anxa-1 和 Anxa-2基因编码蛋白均为稳定蛋白；相对分子质量分别为38830.4和38612.0；理论等电点分别为6.17和6.92；Anxa-1蛋白一级结构中亮氨酸占最大比例为10.4%, Anxa-2蛋白一级结构中亮氨酸与赖氨酸占最大比例为10.0%；Anxa-1和Anxa-2蛋白均不具备信号肽,不属于分泌蛋白；Anxa-1蛋白亚细胞定位于细胞质,Anxa-2蛋白亚细胞定位于细胞核；通过对Anxa-1和Anxa-2蛋白的二级结构预测,发现这两种蛋白质空间结构均由a.螺旋、无规则卷曲、扩展链三部分组成,并成功构建了这两种蛋白质的三维结构。分别对这两种蛋白质氨基酸序列进行多重序列比对,发现这两种基因在不同物种间保守性较高,推测其在不同物种间功能相似,通过构建系统进化树,发现这两种基因在进化中分为两支,一支为哺乳动物类,一支为鸟类,说明这两种基因均由共同祖先基因进化而来,是重要的功能基因。2、鹿茸不同生长时期Anxa-1和Anxa-2基因实时荧光定量结果分析实时荧光定量PCR结果显示：这两种基因均在鹿茸不同生长时期表达存在差异,其中Anxa-1基因在鹿茸的生长前期表达量高于中期与后期；Anxa-2基因在鹿茸的生长中期表达量高于前期与后期。
[Abstract]:Antler antler is the second sexual characteristic of male sika deer. It is the only renewable organ in mammals. In the rapid growth period, the cell division rate in the apical proliferative region is nearly 30 times faster than that in the tumor cell. This speed is incomparable to other mammalian tissues and organs. The cell proliferation differentiation and apoptosis of velvet antler tissue are regulated by specific gene expression. However, the biological regulation mechanism of velvet antler growth has not been elucidated at all. The present study was carried out on Cervus nippon hortulorum. The mesenchymal tissue at the tip of velvet antler is the material. The study focused on the integrin gene, which plays an important role in the regulation of various malignant tumor cells. RT-PCR technique was used to clone the cDNA sequence containing all the coding regions of the membrane binding protein A1 (Anxa-1) gene and the membrane protein A2 (Anxa-2) gene. The amino acid sequences of these two genes and their expression at different growth stages were analyzed by bioinformatics and real-time fluorescence quantitative technique. The main results are as follows: 1. CDNA cloning and bioinformatics Analysis of Anxa-1 and Anxa-2 genes of Deer Antler in Northeast Sika Deer. The length of Anxa-2 gene was 1059bp and 1180bp, respectively. It encodes 346 amino acids and 339 amino acids respectively. The proteins encoded by Anxa-1 and Anxa-2 genes are stable proteins by bioinformatics analysis. The relative molecular weight was 38830.4 and 38612.0, respectively. The theoretical isoelectric points are 6.17 and 6.92, respectively. The proportion of leucine in the primary structure of Anxa-1 protein was 10.4 and that of lysine in the primary structure of Anxa-2 protein was 10.0. Neither Anxa-1 nor Anxa-2 protein had signal peptide and was not a secretory protein. The Anxa-1 protein subcells were located in the nucleus of the cytoplasmic Anxa-2 protein subcells. Based on the prediction of the secondary structures of Anxa-1 and Anxa-2 proteins, it is found that the spatial structures of the two proteins are composed of a. Helix, irregular curls and extended chains. The three dimensional structure of the two proteins was successfully constructed. The amino acid sequences of the two proteins were compared with each other, and it was found that the two genes were highly conserved among different species. By constructing phylogenetic tree, the two genes are divided into two branches, one is mammal and the other is bird. These two genes were evolved from the common ancestor gene, which is an important functional gene .2. Real-time fluorescence quantitative analysis of Anxa-1 and Anxa-2 genes in different growth stages of pilose antler the results of real-time fluorescence quantitative PCR showed that the expression of these two genes were different in different growth stages of velvet antler. The expression of Anxa-1 gene in the early stage of growth was higher than that in the middle and late stages of antler growth. The expression of Anxa-2 gene in antler was higher than that in early stage and late stage.
【学位授予单位】：东北林业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S825;Q78

【相似文献】