青岛文昌鱼过氧化氢酶基因的克隆与表达研究

发布时间：2018-04-09 03:05

本文选题：青岛文昌鱼　切入点：过氧化氢酶　出处：《鲁东大学》2016年硕士论文

【摘要】：H_2O_2是生物体内经常产生的活性氧之一。过量的H_2O_2几乎对细胞的所有成分都是有害的,因此快速有效的去除H_2O_2对于需氧的原核生物和真核生物都是至关重要的。过氧化氢酶(catalase)是一种广泛存在于各种生物内的活性酶。早期对过氧化氢酶的研究主要集中在该酶的化学特性。随着分子生物学及基因工程的迅速发展,越来越多的学者用分子手段来研究过氧化氢酶及其基因的结构与功能。文昌鱼隶属于脊索动物门(Chordata)头索动物亚门(Cephalochordata),是重要的研究脊椎动物起源与进化的海洋模式生物。文昌鱼作为一种分类地位十分特殊的物种,近几十年来随着人类对其栖息地的破坏及海域环境的污染,其资源量急剧下降,成为濒危物种。过氧化氢酶可以通过清除动物体内氧自由基延缓衰老,并提高动物机体的免疫力。本文对文昌鱼的过氧化氢酶基因进行了克隆,分析了其基因序列和蛋白质序列,并与其他物种进行了同源性比对,构建了系统发育树,运用生物信息学方法推测了蛋白质的一些性质,同时利用原位杂交技术和实时定量PCR技术对文昌鱼过氧化氢酶的功能做了初步研究。这为深入研究文昌鱼过氧化氢酶基因的结构与功能奠定了基础,也为文昌鱼的抗病及病害预防提供了一定的理论基础。本文采用RACE技术首次克隆了文昌鱼过氧化氢酶基因的全长cDNA序列,命名为AmphiCAT(GenBank登陆号:KU058636)。该基因全长为2640bp,其中3’非翻译区(untranslated region,UTR)长度为981bp,5’非翻译区为126bp,开放阅读框(ORF)为1533bp,编码510个氨基酸,预测分子量大约为57.85kDa。文昌鱼过氧化氢酶基因cDNA推导的氨基酸序列包含一个长19个氨基酸的潜在活性位点序列HFNRERIPERVVHAKGHGA以及一个长9个氨基酸的血红素配体信号序列RLFSYSDTH。生物软件分析发现该蛋白质序列无信号肽,推测其不属于分泌蛋白;结构预测表明文昌鱼CAT蛋白为单功能的过氧化氢酶的clade3分支;系统进化分析表明文昌鱼与软体动物的亲缘关系较近,且分类地位与传统分类基本一致。以获得的cDNA序列为模板设计引物,用文昌鱼的基因组为模板进行了PCR反应,将获得的序列拼接后获得了一段5000bp左右的序列片段。将过氧化氢酶基因序列在NCBI中进行blast比对,推测其可能含有4个内含子。原位杂交实验表明,过氧化氢酶基因在文昌鱼的肝盲囊、肠道、皮肤、性腺以及脊索中均有表达。在肝盲囊、肠道、皮肤等的高表达可能与这些器官直接接触重金属或致病菌等外界环境有关。实时定量PCR结果显示过氧化氢酶的表达受重金属的影响,当铬离子浓度为10mg/L时该酶的表达量会明显升高,这可能与该酶中的巯基和重金属发生作用有关。这也反应了该酶与文昌鱼的免疫有一定的关联。虽本文对文昌鱼的过氧化氢酶进行了研究,但是其在免疫方面的具体作用机制并不是很清楚,尚有待进一步的探索。
[Abstract]:H_2O_2 is one of the active oxygen species often produced in organisms.Excessive H_2O_2 is harmful to almost all components of the cell, so the rapid and effective removal of H_2O_2 is essential for both aerobic prokaryotes and eukaryotes.Catalase (catalase) is an active enzyme widely found in various organisms.The early studies on catalase mainly focused on the chemical properties of catalase.With the rapid development of molecular biology and genetic engineering, more and more researchers use molecular methods to study the structure and function of catalase and its genes.Amphioxus belongs to Cephalochorta, a cephalocephala, which is an important marine model organism to study the origin and evolution of vertebrates.Amphioxus is a very special taxonomic species. In recent decades, with the destruction of human habitat and the pollution of marine environment, the resources of amphioxus have declined sharply and become endangered species.Catalase can delay aging and improve immunity by scavenging oxygen free radicals in animals.In this paper, the catalase gene of amphioxus was cloned, its gene sequence and protein sequence were analyzed, and compared with other species, the phylogenetic tree was constructed.Some properties of protein were speculated by bioinformatics, and the function of catalase in amphioxus was studied by in situ hybridization and real-time quantitative PCR.This provides a basis for further study on the structure and function of catalase gene in amphioxus, and also provides a theoretical basis for disease resistance and disease prevention of amphioxus.In this paper, the full-length cDNA sequence of catalase gene of amphioxus was first cloned by RACE technique and named as AmphiCAT(GenBank landing number: KU058636636.The total length of the gene is 2640 BP, of which the length of the 3'untranslated region UTRR is 981 BP 5'and the open reading frame ORF is 1533bp, encoding 510 amino acids, and the predicted molecular weight is about 57.85kDa.The amino acid sequence derived from catalase gene cDNA of amphioxus contains a 19 amino acid potential active site sequence HFNRERIPERVVHAKGHGA and a long 9 amino acid heme ligand signal sequence RLFSYSDTH.It was found that the protein sequence had no signal peptide and it was not a secretory protein, and the structure prediction showed that the amphioxus CAT protein was the clade3 branch of the monofunctional catalase.Phylogenetic analysis showed that the relationship between amphioxus and molluscs was close, and the taxonomic status of amphioxus was basically the same as that of traditional taxonomy.The obtained cDNA sequence was used as template to design primers and the amphioxus genome was used as template for PCR reaction. A sequence fragment about 5000bp was obtained by splicing the obtained sequence.The sequence of catalase gene was compared with blast in NCBI, and it was suggested that it might contain four introns.In situ hybridization experiments showed that catalase gene was expressed in the hepatic caecum, intestinal tract, skin, gonad and notochord of amphioxus.The high expression in the hepatic blind sac, intestine, skin and so on may be related to the exposure of these organs to the external environment such as heavy metals or pathogenic bacteria.The results of real-time quantitative PCR showed that the expression of catalase was affected by heavy metals, and the expression of catalase was significantly increased when the concentration of Cr ~ (2 +) was 10mg/L, which may be related to the production of mercapto and heavy metals in the enzyme.This also reflects a link between the enzyme and the immunity of amphioxus.Although the catalase of amphioxus was studied in this paper, the specific mechanism of catalase in the immunity of amphioxus is not very clear and needs further exploration.
【学位授予单位】：鲁东大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S917.4;Q78

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