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

關媺媺(Mei-Mei Kuan)

指導教授 ： 李銘亮 博士

國立臺灣師範大學/理學院/生命科學研究所/博士(2007年)

摘要

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?(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?(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?體液中存在之誘生性抗菌蛋白質，而利用?翅目基因所設計之核酸引子對，進行反轉錄聚合?鏈鎖反應(RT-PCR)可檢測該誘生性抗菌蛋白質之訊息核?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?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?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

關鍵字

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

並列摘要

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.

並列關鍵字

ribosomal protein S40 ； ribosomal protein S60 ； phylogenetic ； Eriogyna cecropin ； phytoplasma

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