簡易檢索 / 詳目顯示

研究生: 關媺媺
Mei-Mei Kuan
論文名稱: 研探野蠶親緣性與抗菌蛋白質誘生及昆蟲媒介擬菌質之分子檢測
The Study of Phylogeny, Inducible Antibacterial Substances in a Wild Silkworm and Detection of Phytoplasma in Leafhoppers by Molecular Approaches
指導教授: 李銘亮
Li, Ming-Liang
學位類別: 博士
Doctor
系所名稱: 生命科學系
Department of Life Science
論文出版年: 2009
畢業學年度: 96
語文別: 中文
論文頁數: 70
中文關鍵詞: 核醣核體S40蛋白質核糖核體S60蛋白質分子親緣性抗菌蛋白質擬菌質體
英文關鍵詞: ribosomal protein S40, ribosomal protein S60, phylogenetic, Eriogyna cecropin, phytoplasma
論文種類: 學術論文
相關次數: 點閱:172下載:0
分享至:
查詢本校圖書館目錄 查詢臺灣博碩士論文知識加值系統 勘誤回報
  • 本論文應用分子生物技術,以研探野生昆蟲之生物角色,針對以下議題進行討論:
    首先,自臺灣北部採集野生四黑目天蠺(Eriogyna pyretorum),經選殖該昆蟲40s核糖體蛋白質(RP L44及RP P40)與60s核糖體蛋白質(RP S23)之基因片段,進行其基因定序與詮釋,並與來自不同昆蟲與動物之對等基因片段進行比對,基於各種分子親緣演算法(Neighbor-Joint, Parsimony, and Maximum Likelihood method),建構並分析其分子性親緣系統樹,發現以該些核糖體蛋白質基因片段所重築之分子性親緣系統樹,與形態學為基礎之分類原則呈現平行性;因此,推論此些核糖體蛋白質基因,具有潛力能提供作為親源分析之分子標靶。
    其次,本文於試管中,進行野生四黑目天蠺(E. pyretorum)幼蟲之細菌免疫以誘生抗菌蛋白質實驗,發現利用酵素鏈結免疫吸附法(the enzyme-linked immunosorbent assay, ELISA)以家蠶抗體為探針,可定量檢測四黑目天蠺體液中存在之誘生性抗菌蛋白質,而利用鳞翅目基因所設計之核酸引子對,進行反轉錄聚合酶鏈鎖反應(RT-PCR)可檢測該誘生性抗菌蛋白質之訊息核苷酸(transient m-RNA)存在於脂肪體;經選殖該基因片段併定序分析,發現了2種抗菌蛋白質之存在,併以該野蠶學名分別命名:「Eriogyna cecropin A及Eriogyna cecropin B」;其中前者為抗菌蛋白質cecropin A之部份片段,另為抗菌蛋白質cecropin B之前身物(precursor),包含其前導蛋白質(leading protein)和其抗菌蛋白質cecropin B之全長。
    另為瞭解昆蟲傳播植物病原菌之潛在田間角色,採集絲瓜蔟葉病(Loofah witches’ broom)植株附近之幾種昆蟲,經分類、進行蟲體感染檢測,利用其可能病原體擬菌質體所設計之核酸引子對與探針,應用於聚合酶鏈鎖反應(PCR)併以分子探針雜交檢測,發現在葉蟬Hishimonus concavus測得存有擬菌質(Loofah witches’ broom phytoplasma)核苷酸陽性,推測其與該植病之擬菌質體感染傳播有關。
    關鍵字: 核糖核體S40蛋白質、核糖核體S60蛋白質、分子親緣性、抗菌蛋白質、擬菌質體

    To investigate the bio-functional roles of field trapped insects, there are 3 undetermined issues proposed in this thesis as follows:
    Firstly, a wild insect, Eriogyna pyretorum, which has been field trapped from northern Taiwan mountain, its taxa characteristics and phylogenetic role of this wild insect, are first time investigated. Following analyzing these partial cDNA sequences encoding ribosomal protein (RP) S23, L44 and P40, the molecule-based phylogenetic trees using these expressed sequence tags (ESTs) from E. pyretorum, other Lepidoptera and distant species were reconstructed. The sequences features and signatures of the corresponding Eriogyna ribosomal protein S23, L44 and P40 were annotated.
    Next, the induced antibacterial peptides on the wild insects, E. pyretorum, are explored. By the enzyme-linked immunosorbent assay (ELISA) and the polymerase chain reaction (PCR) based method using conserved primers derived from known Lepidoptera cecropins, the induced cecropin-like molecules in the hemolymph from bacteria infected larvae were detected. Further analysis the sequences of the cloned cDNA from the wild insects’ fat body using the blast analysis and phylogenetic method indicated there are putative molecules respectively encoding a partial Eriogyna cecropin A or encoding a precursor of Eriogyna cecropin B. Furthermore, the bio-functional and molecular properties of these putative antibacterial peptides were defined.
    Additionally, to understand the potential role of the pathogen transmitted by the vector insects, the candidate insects were field collected and processed by a PCR based assay to detect the status of pathogen’s infection. Since, the most abundant pathogens causing southern Taiwan Loofah witches’ broom disease in infected plants was the Loofah witches’ broom phytoplasma, field collected samples of suspect phytoplasma infected plants and their potential vector, leafhoppers, from southern Taiwan were processed to detect the presence of the putative phytoplasmas using strain-specific DNA hybridization and PCR assays. Based upon that Hishimonus concavus was detected positive for the Loofah witches’ broom infection, the potential role of this leafhopper associated to the putative phytoplasma transmission is then deduced.

    I.Abstract 3 II.Part 1: The Annotation of Cloned cDNA Encoding Ribosomal Protein Genes from a Wild Silkworm, Eriogyna pyretorum Introduction 7 Materials and Methods 9 Results 11 Discussion 15 References 19 Figures and Tables 21 III.Part 2: The Exploring of the Cecropin Like Antibacterial substances in a Wild Giant Silkworm, Eriogyna Pyretorum Introduction 31 Materials and Methods 33 Results 39 Discussion 43 References 46 Figures and Tables 50 IV.Part 3: Detection of the Incidence of Loofah Witches’ Broom Phytoplasma in Leafhoppers by a Molecular Approach Introduction 59 Materials and Methods 61 Results 63 Discussion 65 References 67 Figures and Tables 69

    Adoutte, A., Balavoine G., Lartillot N., Lespinet O., Prud´homme B., De Rosa R. 2000. The new animal phylogeny: reliability and implications, Proc. Natl. Acad. Sci. USA, 97: 4453-4456
    Castro, L. R. and Dowton, M. 2005. The position of the Hymenoptera within the Holometabola as inferred from the mitochondrial genome of Perga condei (Hymenoptera: Symphyta: Pergidae). Mol. Phylogenet. Evol. 34: 469-479.
    Doudna J. A. and Rath V. L., 2002. Structure and function of the eukaryotic ribosome: the next frontier. Cell, 109: 153-156.
    Hughes J., Longhorn S. J., Papadopoulou A., Theodorides K., De Riva A., Mejia-Chang M., Foster P.G., and Vogler A.P., 2006. Dense taxonomic EST sampling and its applications for molecular systematics of the Coleoptera (beetles). Mol. Biol. Evol. 23: 268-278.
    Liao D., Dennis P. P., 1994. Molecular phylogenies based on ribosomal protein L11, L1, L10, and L12 sequences. J. Mol. Evol., 38: 405-419.
    Muller E.C., Wittmann-Liebold B. 1997. Phylogenetic relationship of organisms obtained by ribosomal protein comparison. Cell. Mol. Life Sci., 53: 34-50
    Philippe H., 1993 MUST, a computer package for management utilitarians for sequences and trees, Nucleic Acids Res., 21: 5264-5272
    Philippe, H., Germot A., Moreira D. 2000, "The new phylogeny of eukaryotes", Curr. Opin. Genet. Develop.10: 596-601.
    Righi, M., Cibrelus P., Gabrion C. 1996. Réalisation expérimentale du cycle biologique de Taenia crassiceps (Cestoda, Taeniidae). Bull. Soc. Fr. Parasitol., 14: 71-78.
    Swofford D L (2003) PAUP*: Phylogenetic analysis using parsimony (*and other methods) version 4.0 (computer program). Sunderland (Massachusetts): Sinauer Associates.
    Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. 1997. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. Dec 15; 25: 4876-82
    Velculescu, V. E., Zhang, L., Zhou, W., Vogelstein, J., Basrai, M. A., Bassett, D. E. Jr., Hieter, P., Vogelstein, B. and Kinzler, K. W. (1997) Characterization of the yeast transcriptome. Cell, 88, 243–251.
    Whiting, M. F. 2002. Phylogeny of the holometabolous insect orders: Molecular evidence. Zool. Scr. 31: 3–15.
    Wittmann-Liebold, B., Köpke A. K. E., Arndt E., Krömer W., Hatakeyama T., Wittmann-Liebold, B., Köpke A. K. E., Arndt E., Krömer W., Hatakeyama T., Wittmann H.G. 1990. Sequence comparison and evolution of ribosomal proteins and their genes. Hill W.E., (Ed.) The Ribosome: Structure, Function, and Evolution, 598-616. American Society of Microbiology, Washington, DC.
    Wool, I. G., Endo Y., Chan Y. L., Gluck A., 1990. Structure, function, and evolution of mammalian ribosomes. Hill W.E., (Ed.) The Ribosome: Structure, Function, and Evolution, 203-214 American Society of Microbiology, Washington, DC.
    Abu Elzein EM, Crowther JR. 1978. Enzyme-labeled immunosorbent assay techniques in foot-and-mouth disease virus research. J Hyg (Lond.) 80(3): 391-9.
    Andra J., Berninghausen O. and Leippe M., 2001. Cecropins, antibacterial peptides from insects and mammals, are potently fungicidal against Candida albicans, Med. Microbiol. Immunol. (Berl) 189: 169-173.
    Andrea Giacometti, Oscar Cirioni, Wojciech Kamysz, Giuseppina D’Amato, Carmela Silvestri, Maria Simona Del Prete, Jerzy Lukasiak, Giorgio Scalise. 2004. In vitro activity and killing effect of the synthetic hybrid cecropin A–melittin peptide CA(1-7)M(2-9)NH2 on methicillin-resistant nosocomial isolates of Staphylococcus aureus and interactions with clinically used antibiotics. Diagnostic Microbiology and Infectious Disease 49: 197-200
    Andrea Giacometti, Oscar Cirioni. 1999. Antimicrobial activity of polycationic peptides. Peptides 20: 1265-1273
    Boman HG, Hultmark D. 1987. Cell-free immunity in insects. Annu. Rev. Microbiol. 41: 103–26
    Boman HG. 2003. Antibacterial peptides: basic facts and emerging concepts. J Intern Med. 254(3): 197-215.
    Chen HM, Leung KW, Thakur NN, Tan A, Jack RW. 2003. Distinguishing between different pathways of bilayer disruption by the related antimicrobial peptides cecropin B, B1 and B3. Eur J Bio chem. 270(5): 911-20.
    Chicharro C., Granata C., Lozano R., Andreu D. and Rivas L., 2001. N-terminal fatty acid substitution increases the leishmanicidal activity of CA(1–7)M(2–9), a cecropin-melittin hybrid peptide, Antimicrob. Agents Chemother. 45:2441-2449.
    Chung SC, In Hee Lee, Eungseok Kim, Seung Il Kim , HaK R. Kim. 2000. Antibacterial properties and partial cDNA sequences of cecropin-like antibacterial peptides from the common cutworm, Spodoptera litura. Comparative Biochemistry and Physiology (Part C) 125: 287-297.
    Devine D. A., Marsh P. D., Perciva R. S., Rangarajan M.. and Curtis M. A.. 1999. Modulation of antibacterial peptide activity by products of Porphyromonas gingivalis and Prevotella spp. Microbiology, 145: 965–971
    Fink J., Boman A., Boman H.G. and Merrifield R.B., 1989. Design, synthesis and antibacterial activity of cecropin-like model peptides, Int. J. Pept. Protein Res. 33: 412-421.
    Fink J., Merrifield R.B., Boman A. and Boman H.G., 1989. The chemical synthesis of cecropin D and an analog with enhanced antibacterial activity, J. Biol. Chem. 264, pp. 6260–6267.
    Giacometti A, and Cirioni O. 1999. Antimicrobial activity of polycationic peptides. Peptides. 20: 1265-1273
    Gudmundsson GH, Lidholm DA, A sling B, Gan R, Boman HG. 1991. The cecropin locus, cloning and expression of a gene cluster encoding three antibacterial peptides in Hyalophora cecropin. J. Biol Chem. 266: 11510–7.
    Hoffmann JA, Reichhart JM, Hetru C. 1996. Innate immunity in higher insects. Curr Opin Immunol. 8: 8-13.
    Holak TA, Engstrom A, Kraulis PJ, Lindeberg G, Bennich H, Jones TA, Gronenborn AM, Clore GM., 1988. The solution conformation of the antibacterial peptide cecropin A: a nuclear magnetic resonance and dynamical simulated annealing study. Biochemistry. 27: 7620-9
    Jin F, Xu X, Wang L, Zhang W, Gu D. 2006. Expression of recombinant hybrid peptide cecropinA(1-8)-magainin2(1-12) in Pichia pastoris: purification and characterization. Protein Expr Purif. 50: 147-56.
    Kato Y, Taniai K, Hirochika H, Yamakawa M. 1993 Mar. Expression and characterization of cDNAs for cecropin B, an antibacterial protein of the silkworm, Bombyx mori. Insect Biochem. Mol. Biol. 23(2): 285-90.
    Lehrer R. I, Rosenman M, Harwig S. S. S. L, Jackson R, Eisenhauer P. 1991. Ultrasensitive assays for endogenous antimicrobial polypeptides. J. Immunol. Methods 137: 167-173.24.
    Li M. L, Liao R. W, Qiu J. W, Wang Z. J, Wu T. M. 2001. Antimicrobial activity of synthetic all-D mastoparan M. Int J Antimicrobial Agents 13: 203-208.
    Lidholm, D. A, Gudmundsson G. H, Xanthopoulos K. G, Boman H. G. 1987. Insect immunity: cDNA clones coding for the precursor forms of cecropins A and D, antibacterial proteins from Hyalophora cecropia. FEBS Lett. 226, 8–12.
    Marchini, D., Manetti, A. G. O., Rosetto, M., Bernini, L. F., Telford, J. L., Baldari, C. T., Dallai, R. 1995. cDNA sequence and expression of the ceratotoxin gene encoding an antibacterial sex-specific peptide from the medfly Ceratitis captata (diptera). J. Bipl. Chem. 270, 6199-6204.
    Moore, A. J., Devine, D. A. and Bibby, M. 1994. Preliminary experi- mental anticancer activity of cecropins. Pept Res 7, 265-269.
    Sato H. and Feix J.B. 2006 Peptide-membrane interactions and mechanisms of membrane destruction by amphipathic α-helical antimicrobial peptides. Biochimica et Biophysica Acta (in press) http://www.elsevier.com/locate/bba or www. sciencedirect.com
    Shai, Y. 1998. Mechanism of the binding, insertion and destabilization of phospholipid bilayer membranes by a-helical antimicrobial and cell non-selective membrane-lytic peptides. Biochim. Biophys. Acta 1462: 55-70.
    Van Hofsten P, Faye I, Kockum K, Lee J Y, Xanthopoulos K G, Boman I A, Boman HG, Engstro¨m A, Andreu D, Merrifield RB. 1985. Molecular cloning, cDNA sequencing, and chemical synthesis of cecropin B from Hyalophora cecropin. Proc Natl Acad Sci USA. 82: 2240–3.
    Xu X, Jin F, Yu X, Ji S, Wang J, Cheng H, Wang C, Zhang W. 2007 Expression and purification of a recombinant antibacterial peptide, cecropin, from Escherichia coli. Protein Expr Purif.
    Yamano Y, Matsumoto M, Inoue K, Kawabata T, Morishima I. 1994. Cloning of cDNAs for cecropin A and B, and expression of the genes in the silkworm, Bombyx mori. Biosci Biotech Biochem. 58:1476–8
    Zasloff, M. 2002. Antimicrobial peptides of multicellular organisms. Nature. 415: 388-395

    無法下載圖示 本全文未授權公開
    QR CODE