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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 林長平(Chan-Pin Lin) | |
| dc.contributor.author | Meng-Lu Tsai | en |
| dc.contributor.author | 蔡孟旅 | zh_TW |
| dc.date.accessioned | 2021-06-15T04:15:19Z | - |
| 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 | 陸、參考文獻
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/45345 | - |
| dc.description.abstract | 日日春葉片黃化病植物菌質體 (periwinkle leaf yellowing phytoplasma, PLY phytoplasma) 為本研究室於 2005 年於台灣桃園縣大園鄉發現並命名之新種植物菌質體，其 16S rDNA, 16S-23S rDNA intergenic spacer region (ISR) 及部分 23S rDNA 序列經初步比對分析後，顯示其親緣關係與第一群 (16SrI group) 植物菌質體最為接近。第一群植物菌質體所涵蓋之菌系種類繁多，可細分成十個亞群，為廣泛分佈於世界各地之重要病原，寄主範圍也相當廣泛。為進一步瞭解日日春葉片黃化病植物菌質體可能之植物寄主，及其在主要疫區之發病季節，本研究以 PCR 檢測方式，完成 2007 年 6 月至 2009 年 10月之主要疫區罹病植株月份監測，及可能之植物寄主調查。結果顯示日日春葉片黃化病植物菌質體之寄主植物除日日春外，尚有菊花、大波斯菊、夏堇、紫芳草、牛筋草及小黃瓜等六種，由上述植株中以 PCR 增幅出之植物菌質體 16S rRNA 基因片段，均經由選殖、定序與比對後確認為日日春葉片黃化病植物菌質體之序列。而月份監測結果顯示日日春葉片黃化病植物菌質體之發病季節於 2007 年為六月至十月；2008年為七月至十月，皆為夏、秋兩季；而2009 年則從四月開始即可在罹病植株中偵測到日日春葉片黃化病菌質體。為進一步釐清日日春葉片黃化病菌質體與第一群植物菌質體其他亞群間之親緣關係，本研究針對日日春葉片黃化病菌質體，及台灣故有且同屬第一群植物菌質體之泡桐簇葉病植物菌質體 (Paulownia witches’-broom phytoplasma PaWB-Taiwan) 選殖與定序體在亞群分類上常用之分子標誌 (molecular marker)：16S rRNA 基因，secY 基因，rplV-rpsC 基因及 tuf 基因，並加入第一群植物菌質體其他亞群 (subgroup) 之序列來建構譜系樹 (phylogenetic tree)。於本研究中共完成六棵譜系樹之建構，包括單獨以 16S rRNA 基因，secY 基因，rplV-rpsC 基因及 tuf 基因序列建構之譜系樹；與合併16S rRNA, rplV-rpsC, secY 基因序列，及合併rplV-rpsC, secY基因序列之譜系樹。譜系樹之結果顯示日日春葉片黃化病植物菌質體之親緣關係與 16SrI group 之 B 亞群 (16srI-B) 及 D 亞群 (16srI-D) 較接近；且在以 rplV-rpsC 序列建構之譜系樹中，PLY phytoplasma 自成一獨立支序群 (clade)，因此亦不排除其為一獨立之新亞群的可能性；而 PaWB-Taiwan phytoplasma 則與屬於 16SrI-D 之最早登錄於 NCBI 之泡桐簇葉病植物菌質體 (Paulownia witches’-broom phytoplasma PaWB) 較接近。另外，合併序列之譜系樹結果顯示其樹型與以單一基因序列獨立建構之譜系樹樹型具高度一致性，但其樹長 (tree length) 明顯大於以單一基因序列獨立建構之譜系樹，証實合併序列之譜系樹在譜系分析上具有較高的解析度 (resolution)。為釐清 PLY phytoplasma 在 16SrI group 中之亞群地位，及其與地緣相近的 PaWB-Taiwan phytoplasma 間之親緣關係，本研究亦利用植物菌質體不同菌系間，在群及亞群分類上所依據之電腦模擬 (in silico) RFLP (restriction fragment length polymorphism) 分析法，針對 PLY phytoplasma, PaWB-Taiwan phytoplasma 及在譜系分析中與其親緣關係相近菌系之 16S rRNA 基因序列進行分析並計算相似係數 (similarity coefficient)。in silico RFLP 之結果亦支持 PLY phytoplasma 成為 16SrI group 植物菌質體中一獨立之新亞群；而 PaWB-Taiwan phytoplasma 則被歸類到 16SrI-B 亞群，而非原登錄同為泡桐簇葉病病原 PaWB phytoplasma 所屬之 16SrI-D 亞群，顯示在本研究中針對 PaWB-Taiwan phytoplasma 選殖與定序之 16S rRNA 基因，可能為屬於 16SrI-B 之 rrnA，而非屬於 16SrI-D 之 rrnB。另外，本研究也針對上述親緣關係相似之菌系，輔以16S rRNA 基因序列之準限制酵素切位 (putative restriction site) 分析，結果也顯示 PLY phytoplasma 可和其他菌系有所區分。上述親緣分析結果顯示 PLY phytoplasma 應為 16srI group 中之新亞群。 | zh_TW |
| dc.description.abstract | A new disease named as periwinkle leaf yellowing (PLY) was first observed in a flower production farm in Dayuan Township (Taoyuan county, Taiwan) in August 2005. Sequence analysis of 16S rDNA, 16S-23S rDNA ISR, and partial 23S rDNA sequence revealed that the causative agent of PLY was closely related to the phytoplasmas of the aster yellows (AY) group (16SrI group) which cause diseases in many horticultural and vegetable crops worldwide, and can be delineated into 10 subgroups. Six cultivated plants including periwinkle plant, chrysanthemum, cosmos, torenia, Persian violet, goosegrass and cucumber, were determined to be the host plants of PLY phytolasma. Monthly PCR detection indicated that PLY phytoplasma was detected in host plants from June to October in 2007, and from July to October in 2007. However, it can be detected earlier since April in 2009. To further clarify the phylogenetic relationship of strain PLY among 16SrI phytoplasmas, six phylogenetic trees were constructed in this study. Beside the phylogenetic trees based on the independent analysis of 16S rRNA, rplV-rpsC, secY and tuf gene sequences, two other trees based on the analysis of the comprising gene sequence of 16S rRNA, rplV-rpsC and secY, and the comprising gene sequence of rplV-rpsC and secY were also constructed. The results indicate that the strain PLY was closely related to 16SrI-B and 16SrI-D subgroup, and could be a new subgroup based on the phylogenetic tree constructed by rplV-rpsC gene sequences. In addition, the phylogenetic analysis of the comprised gene sequences showed similar tree topology when compared with sequence analysis of the rplV/ rpsC gene or of the secY gene alone. The main difference is that the branch lengths were elongated in the comprised gene tree. To further confirm the subgroup affiliation of PLY phytoplasma, the 16S rRNA gene sequences of 10 closely related phytoplasma strains were digested in silico, and the similarity coefficients were then calculated. The results also support the conclusion that PLY phytoplasma might belongs to a new 16SrI subgroup. The putative restriction site analysis can also distinguish PLY phytoplasma from other close related phytoplasma strains in phylogenetic analysis. | en |
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| dc.description.tableofcontents | 目錄
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................................................................................................................3 一、植物菌質體之發現與其植物病理學....................................................................3 (一)、植物菌質體之發現與命名..............................................................................3 (二)、植物菌質體之病理學......................................................................................5 二、植物菌質體鑑定與分類相關技術之發展及其分群現況....................................6 (一)、傳統分類方法及其限制..................................................................................6 (二)、以核酸及血清學為基礎之分類方法..............................................................7 (三)、以軟體分析親緣關係之方法..........................................................................8 1. 譜系分析 (phylogenetic analyses) .....................................................................8 2. 電腦模擬限制酵素酵解片段長度多型性分析 (in silico RFLP analyses).....10 三、第一群 (16SrI group) 植物菌質體之相關研究................................................12 (一)、第一群植物菌質體之發現、分布與其植物病理學....................................12 (二)、分子標誌 (molecular marker) 在第一群植物菌質體亞群分類之應用.....13 (三)、日日春葉片黃化病植物菌質體在臺灣之研究............................................14 參、材料與方法..........................................................................................................17 一、日日春葉片黃化病植物菌質體可能之寄主植物調查......................................17 (一)、試驗植物來源與植物菌質體保存方式........................................................17 1. 可能之寄主植物採集方法................................................................................17 2. 帶菌日日春植株來源與植物菌質體保存方式................................................17 (二)、試驗植物全DNA之純化.............................................................................18 (三)、田間可能罹病植株中日日春葉片黃化病植物菌質體 ribosomal DNA 序列之 PCR 增幅 (amplification)、選殖 (cloning) 與分析.......................19 1. 聚合酵素連鎖反應 (PCR) ..............................................................................19 2. 聚合酵素連鎖反應產物之純化與選殖.........................................................21 3. 聚合酵素連鎖反應產物轉形株之特性分析...................................................23 (1) 以菌落聚合酵素連鎖反應 (colony PCR) 分析轉形株之選殖片段........23 (2) 轉形株選殖片段之核酸定序與序列分析..................................................24 二、田間可能罹病植株中日日春葉片黃化病植物菌質體之 PCR 月份監測......24 (一)、試驗植物來源................................................................................................24 (二)、田間可能罹病植株中日日春葉片黃化病植物菌質體之 PCR月份監測..25 三、日日春葉片黃化病植物菌質體之親緣分析......................................................25 (一)、以16S rRNA, rplV-rpsC, secY 及 tuf 基因建構譜系樹 (phylogenetic tree) .................................................................................................................25 1. 譜系分析序列來源............................................................................................25 2. 最大簡約法 (maximum parsimony, MP) 譜系樹建構...................................27 (二)、日日春葉片黃化病菌質體與臺灣泡桐簇葉病菌質體 16S rRNA基因序列之電腦模擬 (in silico) RFLP 分析及相似係數 (similarity coefficient) 之計算.............................................................................................................28 (三)、日日春葉片黃化病菌質體與臺灣泡桐簇葉病菌質體 16S rRNA 基因序列之準限制酵素切位 (putative restriction site) 分析...............................29 肆、結果.........................................................................................................................31 一、日日春葉片黃化病植物菌質體可能之寄主植物調查......................................31 (一)、田間疑似罹病植株之採集............................................................................31 (二)、試驗植物全 DNA 之純化............................................................................31 (三)、罹病植株中日日春葉片黃化病植物菌質體 ribosomal DNA 序列之 PCR 增幅 (amplification)、選殖 (cloning) 與分析...........................................31 1. 以聚合酵素連鎖反應 (PCR) 偵測疑似罹病株中之植物菌質體..............31 2. 聚合酵素連鎖反應產物之選殖與選殖株之特性分析................................32 3. 由不同罹病植株中選殖出之 rDNA 序列之比對分析.................................33 (四)、日日春葉片黃化病植物菌質體可能寄主植物之病徵觀察.......................34 二、田間日日春葉片黃化病植物菌質體之月份監測.............................................35 三、日日春葉片黃化病植物菌質體之親緣分析.....................................................35 (一)、最大簡約法譜系樹之建構...........................................................................35 1. 第一群植物菌質體 16S rRNA 基因之譜系樹建構......................................35 2. 第一群植物菌質體 rplV-rpsC 基因之譜系樹建構......................................36 3. 第一群植物菌質體 secY 基因之譜系樹建構...............................................37 4. 第一群植物菌質體 tuf 基因序列之譜系樹建構..........................................38 5. 合併 16S rRNA, rplV-rpsC, secY 基因序列之譜系樹建構..........................38 6. 合併 rplV-rpsC, secY 基因序列之譜系樹建構.............................................40 (二)、日日春葉片黃化病菌質體與臺灣泡桐簇葉病菌質體 16S rRNA基因序列之電腦模擬 (in silico) RFLP 分析及相似係數 (similarity coefficient) 之計算............................................................................................................40 (三)、日日春葉片黃化病菌質體與臺灣泡桐簇葉病菌質體 16S rRNA 基因序列之準限制酵素切位 (putative restriction site) 分析.............................42 伍、討論....................................................................................................................44 陸、參考文獻............................................................................................................51 柒、圖表....................................................................................................................59 | |
| dc.language.iso | zh-TW | |
| dc.title | 日日春葉片黃化病植物菌質體核醣體 RNA, rplV-rpsC, secY 與 tuf 基因之譜系分析 | zh_TW |
| dc.title | Phylogenetic Analyses of Periwinkle Leaf Yellowing Phytoplasma Based on Combined Analyses of rRNA, rplV-rpsC, secY and tuf gene sequences | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 98-1 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 詹富智,張碧芳,徐源泰,洪挺軒 | |
| dc.subject.keyword | in silico RFLP,日日春葉片黃化病植物菌質體,譜系分析,核醣體RNA基因,rplV-rpsC 基因,secY 基因,tuf 基因, | zh_TW |
| dc.subject.keyword | in silico RFLP,periwinkle leaf yellowing phytoplasma,phylogenetic analyses,rRNA gene,rplV-rpsC gene,secY gene,tuf gene, | en |
| dc.relation.page | 98 | |
| 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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