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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 林長平 | |
| dc.contributor.author | Yu-Yang Cheng | en |
| dc.contributor.author | 程諭揚 | zh_TW |
| dc.date.accessioned | 2021-06-15T04:15:22Z | - |
| dc.date.available | 2011-01-21 | |
| dc.date.copyright | 2010-01-21 | |
| dc.date.issued | 2010 | |
| dc.date.submitted | 2010-01-12 | |
| dc.identifier.citation | 1.林翠淳. 1996. 植物菌質體廣效型PCR引子之評估及疑似梨衰弱病病原菌質體之檢測. 國立台灣大學植物病理與微生物學研究所碩士論文。
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/45346 | - |
| dc.description.abstract | 在植物菌質體優勢免疫膜蛋白相關之研究中顯示，其為植物菌質體細胞膜之主要蛋白。優勢免疫膜蛋白基因已陸續由多種植物菌質體中被選殖出來，這些基因所編碼之膜蛋白依據其嵌入於細胞膜上之構型，計可區分為不同的三個類型，分別為第一型優勢免疫膜蛋白 Imp (immunodominant membrane protein, Imp)、第二型優勢免疫膜蛋白 IdpA (immunodominant membrane protein A, IdpA) 以及第三型優勢免疫膜蛋白 Amp (antigenic membrane protein, Amp)。此三型優勢免疫膜蛋白之間在胺基酸序列上不具相似性。此外，在植物菌質體中之優勢免疫膜蛋白基因之序列相同度，亦被證實是低於其上下游基因或是 non-coding regions。本研究利用聚合酵素連鎖反應並配合基因選殖技術，由本實驗室現有之植物菌質體中，選殖獲得花生簇葉病菌質體之第一型 imp 優勢免疫膜蛋白基因之全長序列及台灣梨衰弱病第二群菌質體之第一型 imp 優勢免疫膜蛋白基因之部分序列 、聖誕紅叢枝誘導性植物菌質體之第二型 idpA 及第一型 imp 優勢免疫膜蛋白基因之全長序列以及日日春葉片黃化病及台灣泡桐簇葉病菌質體之第三型 amp 及第一型 imp 優勢免疫膜蛋白基因之全長序列。依序列比對分析之結果，顯示同一型各優勢免疫膜蛋白基因間之序列相同度甚低，尤其在 hydrophilic domain 間之序列相同度更低於其他 domain 間之序列相同度。儘管為同一 16Sr group 中之植物菌質體，其彼此間之優勢免疫膜蛋白基因序列亦具有差異性之區域，因此可據以區分不同 16Sr group 或 subgroup 之植物菌質體。針對台灣目前僅有之同屬 16SrI group 之日日春葉片黃化病菌植體及台灣泡桐簇葉病菌質體，就其第三型優勢免疫膜蛋白基因 amp 序列間具差異性之區域，所設計出之專一性 PCR 引子對 ampf5/Amp1R 及 Amp1F/ampr4，以 PCR 反應可成功區分此二親緣關係相近之植物菌質體。此外，由反轉錄聚合酵素連鎖反應 (RT-PCR) 之結果顯示，日日春葉片黃化病菌質體第一型 imp 及第三型 amp 優勢免疫膜蛋白基因以及花生簇葉病菌質體第一型 imp 優勢免疫膜蛋白基因均可轉錄出 mRNA。 | zh_TW |
| dc.description.abstract | Previous studies have shown that an immunodominant membrane protein is a major portion of the total cellular membrane proteins in most phytoplasmas. Genes encoding immunodominant membrane proteins have been identified and sequenced from several taxonomic groups of phytoplasmas and have been classified into three distinct types: type 1 immunodominant membrane protein Imp, type 2 immunodominant membrane protein IdpA, and type 3 immunodominant membrane protein Amp. No amino acid similarity was revealed among different type of immunodominant membrane proteins. The sequence identity of immunodominant membrane protein genes between phytoplamas was also evidenced to be lower than those of their upstream or downstream genes or non-coding regions. In this study, cloning and sequencing of these three types immunodominant membrane protein genes in Taiwan were performed for three groups of phytoplasmas including the pear decline phytoplasma (PDTWII phytoplasma) and peanut witches'-broom (PnWB) phytoplasma in 16SrII group which have imp genes, poinsettia branch-inducing (PoiBI) phytoplasma in 16SrIII group which has idpA gene, and periwinkle leaf yellowing (PLY) phytoplasma and paulownia witches'-broom (PaWB-Taiwan) phytoplasma in 16SrI group which have amp genes. Sequence analysis of these cloned immunodominant membrane protein gene fragments revealed that the sequence identities were very low, especially among the sequences of hydrophilic domains. Each phytoplasmal immunodominant membrane protein gene has their own distinct sequence even the phytoplasmas belong to the same group. These distinct immunodominant membrane protein gene sequences can be used for the differentiation of groups or subgroups of various phytoplasmas. Specific PCR primer pairs ampf5/Amp1R and Amp1F/ampr4 were designed based on distinct regions of PLY phytoplasma and PaWB-Taiwan phytoplasma amp gene sequences for their identification and classification. RT-PCR analysis showed that the imp, amp genes of PLY phytoplasma and the imp gene of PnWB phytoplasma were transcribed in PLY phytoplasma and PnWB phytoplasma. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T04:15:22Z (GMT). No. of bitstreams: 1 ntu-99-R96633015-1.pdf: 2802211 bytes, checksum: bca8e9b458bd38bfe2239648dc56e80f (MD5) Previous issue date: 2010 | en |
| dc.description.tableofcontents | 論文口試委員審定書……………………………………………………II
誌謝……………………………………………………………………III 中文摘要…………………………………………………………………IV 英文摘要…………………………………………………………………VI 壹、前言…………………………………………………………………1 貳、前人研究……………………………………………………………4 一、植物菌質體之發現與其植物病理學 (plant pathology)………4 二、植物菌質體之分群…………………………………………………7 三、植物菌質體之生物特性與其分子生物學上之研究 ………………9 四、植物菌質體之優勢免疫膜蛋白之相關研究及其應用 …………11 參、材料與方法 ………………………………………………………18 一、研究材料來源與植物全 DNA (total DNA) 之純化 ……………18 (一) 試驗植物來源與繁殖……………………………………………18 (二) 健康及受植物菌質體感染之罹病植物全 DNA 之純化…………19 1. 大量抽取植物全 DNA ………………………………………………19 2. 微量抽取植物全DNA ………………………………………………20 二、植物菌質體之優勢免疫膜蛋白基因之選殖與分析 ……………21 (一) 第一型優勢免疫膜蛋白基因 imp (type 1 immunodominant membrane protein gene: imp) 之選殖與分析………………………21 1. 聚合酵素連鎖反應引子之設計……………………………………21 2. 聚合酵素連鎖反應 (polymerase chain reaction, PCR)………22 3. 聚合酵素連鎖反應產物之純化與選殖……………………………23 (1) 聚合酵素連鎖反應產物之純化……………………………………23 (2) 聚合酵素連鎖反應產物之選殖……………………………………23 (3) 嵌入片段菌落聚合酵素連鎖反應 (colony PCR) 分析…………24 (二) 第二型優勢免疫膜蛋白基因 idpA (type 2 immunodominant membrane protein gene: idpA) 之選殖與分析……………………26 1. 聚合酵素連鎖反應引子之設計……………………………………26 2. 聚合酵素連鎖反應 (PCR) …………………………………………26 3. 聚合酵素連鎖反應產物之純化與選殖……………………………27 (1) 聚合酵素連鎖反應產物之膠體萃取 (gel extraction)………27 (2) 聚合酵素連鎖反應產物之選殖……………………………………28 (3) 嵌入片段菌落聚合酵素連鎖反應 (colony PCR) 分析…………28 (三) 第三型優勢免疫膜蛋白基因 amp (type 3 immunodominant membrane protein gene: amp) 之選殖與分析………………………28 1. 聚合酵素連鎖反應引子之設計……………………………………28 2. 聚合酵素連鎖反應 (PCR) …………………………………………28 3. 聚合酵素連鎖反應產物之純化與選殖……………………………29 (1) 聚合酵素連鎖反應產物之純化……………………………………29 (2) 聚合酵素連鎖反應產物之選殖……………………………………30 (3) 嵌入片段菌落聚合酵素連鎖反應 (colony PCR) 分析…………30 三、由含第二型、第三型優勢免疫膜蛋白基因之植物菌質體中選殖之第一型優勢免疫膜蛋白基因 …………………………………………30 (一) 聚合酵素連鎖反應引子對之設計………………………………30 (二) 聚合酵素連鎖反應 ………………………………………………31 (三) 聚合酵素連鎖反應產物之純化與選殖 …………………………31 1. 聚合酵素連鎖反應產物之純化……………………………………32 2. 聚合酵素連鎖反應產物之選殖 ……………………………………32 3. 嵌入片段菌落聚合酵素連鎖反應 (colony PCR) 分析…………32 四、以 imp 基因序列及 16S rDNA 序列建構譜系樹 (phylogenetic tree)……………………………………………………………………32 五、花生簇葉病菌質體之第一型、日日春葉片黃化病菌質體之第一型及第三型優勢免疫膜蛋白基因 mRNA 之偵測…………………………33 (一) 健康與罹病植物全 RNA 之純化…………………………………33 (二) 反轉錄聚合酵素連鎖反應 (reverse transcription PCR, RT-PCR)…………………………………………………………………34 1. 反轉錄反應引子與聚合酵素連鎖反應引子對之設計……………34 2. 以反轉錄 (reverse transcription) 反應進行花生簇葉病菌質體第一型優勢免疫膜蛋白基因 imp 、日日春葉片黃化病菌質體第一型及第三型優勢免疫膜蛋白基因 imp 及 amp cDNA 之合成…………35 3. 聚合酵素連鎖反應…………………………………………………35 4. 聚合酵素連鎖反應產物之純化與選殖……………………………36 六、植物菌質體之優勢免疫膜蛋白基因推衍之胺基酸序列之特性分析…………………………………………………………………………37 七、以植物菌質體之第三型優勢免疫膜蛋白基因 amp 核酸序列設計專一性 PCR 引子對……………………………………………………37 (一) 16SrI group 植物菌質體專一性引子對之設計與應用………37 (二) 16SrI group之日日春葉片黃化病及台灣泡桐簇葉病菌質體專一性引子對之設計與應用…………………………………………………38 (三) 利用第三型優勢免疫膜蛋白基因 amp 核酸序列從事多重聚合酵素連鎖反應 (multiplex PCR)…………………………………………38 肆、結果…………………………………………………………………40 一、研究材料來源與全 DNA (total DNA) 之純化…………………40 二、植物菌質體優勢免疫膜蛋白基因之選殖與分析…………………40 (一) 第一型優勢免疫膜蛋白基因 imp 核酸序列、推衍之胺基酸序列、親疏水性及穿膜區域之分析………………………………………40 (二) 第二型優勢免疫膜蛋白基因 idpA 核酸序列、推衍之胺基酸序列、親疏水性及穿膜區域之分析………………………………………47 (三) 第三型優勢免疫膜蛋白基因 amp 核酸序列、推衍之胺基酸序列、親疏水性及穿膜區域之分析………………………………………48 三、以 imp 基因序列建構之譜系樹與 16S rDNA 序列建構之譜系樹之比較……………………………………………………………………52 四、花生簇葉病菌質體之第一型、日日春葉片黃化病菌質體之第一型及第三型優勢免疫膜蛋白基因mRNA 之偵測…………………………52 五、以植物菌質體之第三型優勢免疫膜蛋白基因 amp 核酸序列設計專一性 PCR 引子對……………………………………………………54 (一) 16SrI group 植物菌質體專一性引子對之測定………………54 (二) 16SrI group 之日日春葉片黃化病及台灣泡桐簇葉病菌質體專一性引子對之測定………………………………………………………55 (三) 利用第三型優勢免疫膜蛋白基因 amp 核酸序列從事之多重聚合酵素連鎖反應 …………………………………………………………55 伍、討論…………………………………………………………………56 陸、參考文獻……………………………………………………………64 柒、圖表…………………………………………………………………76 | |
| dc.language.iso | zh-TW | |
| dc.title | 植物菌質體之優勢免疫膜蛋白基因 imp, idpA 與 amp 之選殖與分析 | zh_TW |
| dc.title | Cloning and Analysis of Phytoplasmal Immunodominant Membrane Protein Genes imp, idpA and amp | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 98-1 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 徐源泰,詹富智,張碧芳,洪挺軒 | |
| dc.subject.keyword | 優勢免疫膜蛋白,聚合酵素連鎖反應,imp 基因,idpA 基因,amp 基因,反轉錄聚合酵素連鎖反應, | zh_TW |
| dc.subject.keyword | immunodominant membrane protein,polymerase chain reaction,imp gene,idpA gene,amp gene,RT-PCR, | en |
| dc.relation.page | 123 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2010-01-12 | |
| dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
| dc.contributor.author-dept | 植物病理與微生物學研究所 | zh_TW |
| 顯示於系所單位： | 植物病理與微生物學系 | |
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