本文選題：青島文昌魚　切入點：過氧化氫酶　出處：《魯東大學(xué)》2016年碩士論文

【摘要】：H_2O_2是生物體內(nèi)經(jīng)常產(chǎn)生的活性氧之一。過量的H_2O_2幾乎對細胞的所有成分都是有害的,因此快速有效的去除H_2O_2對于需氧的原核生物和真核生物都是至關(guān)重要的。過氧化氫酶(catalase)是一種廣泛存在于各種生物內(nèi)的活性酶。早期對過氧化氫酶的研究主要集中在該酶的化學(xué)特性。隨著分子生物學(xué)及基因工程的迅速發(fā)展,越來越多的學(xué)者用分子手段來研究過氧化氫酶及其基因的結(jié)構(gòu)與功能。文昌魚隸屬于脊索動物門(Chordata)頭索動物亞門(Cephalochordata),是重要的研究脊椎動物起源與進化的海洋模式生物。文昌魚作為一種分類地位十分特殊的物種,近幾十年來隨著人類對其棲息地的破壞及海域環(huán)境的污染,其資源量急劇下降,成為瀕危物種。過氧化氫酶可以通過清除動物體內(nèi)氧自由基延緩衰老,并提高動物機體的免疫力。本文對文昌魚的過氧化氫酶基因進行了克隆,分析了其基因序列和蛋白質(zhì)序列,并與其他物種進行了同源性比對,構(gòu)建了系統(tǒng)發(fā)育樹,運用生物信息學(xué)方法推測了蛋白質(zhì)的一些性質(zhì),同時利用原位雜交技術(shù)和實時定量PCR技術(shù)對文昌魚過氧化氫酶的功能做了初步研究。這為深入研究文昌魚過氧化氫酶基因的結(jié)構(gòu)與功能奠定了基礎(chǔ),也為文昌魚的抗病及病害預(yù)防提供了一定的理論基礎(chǔ)。本文采用RACE技術(shù)首次克隆了文昌魚過氧化氫酶基因的全長cDNA序列,命名為AmphiCAT(GenBank登陸號:KU058636)。該基因全長為2640bp,其中3’非翻譯區(qū)(untranslated region,UTR)長度為981bp,5’非翻譯區(qū)為126bp,開放閱讀框(ORF)為1533bp,編碼510個氨基酸,預(yù)測分子量大約為57.85kDa。文昌魚過氧化氫酶基因cDNA推導(dǎo)的氨基酸序列包含一個長19個氨基酸的潛在活性位點序列HFNRERIPERVVHAKGHGA以及一個長9個氨基酸的血紅素配體信號序列RLFSYSDTH。生物軟件分析發(fā)現(xiàn)該蛋白質(zhì)序列無信號肽,推測其不屬于分泌蛋白;結(jié)構(gòu)預(yù)測表明文昌魚CAT蛋白為單功能的過氧化氫酶的clade3分支;系統(tǒng)進化分析表明文昌魚與軟體動物的親緣關(guān)系較近,且分類地位與傳統(tǒng)分類基本一致。以獲得的cDNA序列為模板設(shè)計引物,用文昌魚的基因組為模板進行了PCR反應(yīng),將獲得的序列拼接后獲得了一段5000bp左右的序列片段。將過氧化氫酶基因序列在NCBI中進行blast比對,推測其可能含有4個內(nèi)含子。原位雜交實驗表明,過氧化氫酶基因在文昌魚的肝盲囊、腸道、皮膚、性腺以及脊索中均有表達。在肝盲囊、腸道、皮膚等的高表達可能與這些器官直接接觸重金屬或致病菌等外界環(huán)境有關(guān)。實時定量PCR結(jié)果顯示過氧化氫酶的表達受重金屬的影響,當(dāng)鉻離子濃度為10mg/L時該酶的表達量會明顯升高,這可能與該酶中的巰基和重金屬發(fā)生作用有關(guān)。這也反應(yīng)了該酶與文昌魚的免疫有一定的關(guān)聯(lián)。雖本文對文昌魚的過氧化氫酶進行了研究,但是其在免疫方面的具體作用機制并不是很清楚,尚有待進一步的探索。
[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.
【學(xué)位授予單位】：魯東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S917.4;Q78

【參考文獻】

相關(guān)期刊論文 前10條

1 邢朝斌;劉巖;修樂山;龍月紅;吳鵬;;刺五加過氧化氫酶基因的克隆與表達分析[J];經(jīng)濟林研究;2014年02期

2 高杉;周遵春;董穎;楊愛馥;陳仲;王擺;關(guān)曉燕;蔣經(jīng)偉;姜北;;仿刺參過氧化氫酶基因全長cDNA的克隆及表達分析[J];中國農(nóng)業(yè)科技導(dǎo)報;2014年02期

3 劉慧春;朱開元;馬廣瑩;鄒清成;周江華;;紅掌抗寒相關(guān)AnCAT基因的克隆及表達分析[J];分子植物育種;2014年01期

4 黎東怡;杜青平;屈嘉儀;楊灃;;重鉻酸鉀對斑馬魚的急性毒性及組織過氧化氫酶活性的影響[J];廣東科技;2014年02期

5 林峻;林娟;葉秀云;;梅花鹿過氧化氫酶基因的克隆與表達[J];福州大學(xué)學(xué)報(自然科學(xué)版);2014年01期

6 唐斌;施佐X;郭紅雙;王u&;王世貴;張帆;;異色瓢蟲過氧化氫酶(Catalase)的基因克隆及序列分析[J];應(yīng)用昆蟲學(xué)報;2014年01期

7 蔡永智;祝建波;郝曉云;王愛英;徐登獻;;轉(zhuǎn)過氧化氫酶基因KatG棉花的抗旱性[J];西北農(nóng)業(yè)學(xué)報;2013年12期

8 彭丹;張霞;張富春;;鹽穗木過氧化氫酶基因的克隆與功能分析[J];西北植物學(xué)報;2013年10期

9 翁朝紅;謝仰杰;肖志群;李軍;黃良敏;張雅芝;;廈門海域文昌魚資源及其自然生態(tài)環(huán)境評價[J];集美大學(xué)學(xué)報(自然科學(xué)版);2012年04期

10 鄭清梅;韓春艷;溫茹淑;鐘艷梅;姚瓊鳳;侯雨文;;草魚過氧化氫酶全長cDNA的克隆、序列同源分析與組織表達[J];基因組學(xué)與應(yīng)用生物學(xué);2011年05期

相關(guān)碩士學(xué)位論文 前3條

1 張曉燕;青島文昌魚dax1基因的克隆與表達研究[D];魯東大學(xué);2015年

2 楊建威;青島文昌魚自然保護區(qū)生物資源與文昌魚資源調(diào)查研究[D];中國海洋大學(xué);2008年

3 魏然;鹽度對牙鲆非特異性免疫功能的影響和文昌魚抗氧化酶的活性[D];中國海洋大學(xué);2004年

，

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