本文關(guān)鍵詞： 鹿茸 Anxa-1 Anxa-2 cDNA克隆 生物信息學(xué)分析 熒光定量PCR　出處：《東北林業(yè)大學(xué)》2015年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：鹿茸角是雄性梅花鹿的第二性征,它是哺乳動(dòng)物中唯一可再生器官,在快速生長(zhǎng)期,其頂端增殖區(qū)的細(xì)胞分裂速度是腫瘤細(xì)胞分裂速度的近30倍,這樣的速度是其他哺乳動(dòng)物組織和器官所無(wú)法比擬的,鹿茸組織的細(xì)胞增殖、分化、凋亡受到特定基因的表達(dá)調(diào)控,但迄今仍無(wú)法從根本上闡明鹿茸生長(zhǎng)的生物學(xué)調(diào)控機(jī)制。本試驗(yàn)以東北梅花鹿(Cervus nippon hortulorum)鹿茸尖端間充質(zhì)組織為材料,以對(duì)多種惡性腫瘤細(xì)胞起著重要調(diào)節(jié)作用的膜聯(lián)蛋白基因?yàn)檠芯繉?duì)象,用RT-PCR技術(shù)克隆出包含膜聯(lián)蛋白A1 (Anxa-1)基因與膜聯(lián)蛋白A2 (Anxa-2)基因全部編碼區(qū)的cDNA序列,并運(yùn)用生物信息學(xué)方法及實(shí)時(shí)熒光定量技術(shù)對(duì)這兩種基因的氨基酸序列及不同生長(zhǎng)時(shí)期的表達(dá)情況進(jìn)行了分析。主要研究結(jié)果如下：1、東北梅花鹿鹿茸Anxa-1 和 Anxa-2基因cDNA克隆及生物信息學(xué)分析Anxa-1 與 Anxa-2基因序列測(cè)序長(zhǎng)度分別為1059bp和1180bp,分別編碼346個(gè)氨基酸和339個(gè)氨基酸。經(jīng)生物信息學(xué)分析,Anxa-1 和 Anxa-2基因編碼蛋白均為穩(wěn)定蛋白；相對(duì)分子質(zhì)量分別為38830.4和38612.0；理論等電點(diǎn)分別為6.17和6.92；Anxa-1蛋白一級(jí)結(jié)構(gòu)中亮氨酸占最大比例為10.4%, Anxa-2蛋白一級(jí)結(jié)構(gòu)中亮氨酸與賴(lài)氨酸占最大比例為10.0%；Anxa-1和Anxa-2蛋白均不具備信號(hào)肽,不屬于分泌蛋白；Anxa-1蛋白亞細(xì)胞定位于細(xì)胞質(zhì),Anxa-2蛋白亞細(xì)胞定位于細(xì)胞核；通過(guò)對(duì)Anxa-1和Anxa-2蛋白的二級(jí)結(jié)構(gòu)預(yù)測(cè),發(fā)現(xiàn)這兩種蛋白質(zhì)空間結(jié)構(gòu)均由a.螺旋、無(wú)規(guī)則卷曲、擴(kuò)展鏈三部分組成,并成功構(gòu)建了這兩種蛋白質(zhì)的三維結(jié)構(gòu)。分別對(duì)這兩種蛋白質(zhì)氨基酸序列進(jìn)行多重序列比對(duì),發(fā)現(xiàn)這兩種基因在不同物種間保守性較高,推測(cè)其在不同物種間功能相似,通過(guò)構(gòu)建系統(tǒng)進(jìn)化樹(shù),發(fā)現(xiàn)這兩種基因在進(jìn)化中分為兩支,一支為哺乳動(dòng)物類(lèi),一支為鳥(niǎo)類(lèi),說(shuō)明這兩種基因均由共同祖先基因進(jìn)化而來(lái),是重要的功能基因。2、鹿茸不同生長(zhǎng)時(shí)期Anxa-1和Anxa-2基因?qū)崟r(shí)熒光定量結(jié)果分析實(shí)時(shí)熒光定量PCR結(jié)果顯示：這兩種基因均在鹿茸不同生長(zhǎng)時(shí)期表達(dá)存在差異,其中Anxa-1基因在鹿茸的生長(zhǎng)前期表達(dá)量高于中期與后期；Anxa-2基因在鹿茸的生長(zhǎng)中期表達(dá)量高于前期與后期。
[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.
【學(xué)位授予單位】：東北林業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：S825;Q78

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1 曲昊淼;東北梅花鹿鹿茸組織Anxa-1、Anxa-2基因cDNA克隆及表達(dá)分析[D];東北林業(yè)大學(xué);2015年

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本文編號(hào)：1468545