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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 林長平 | |
dc.contributor.author | Wuu-Yang Chen | en |
dc.contributor.author | 陳武揚 | zh_TW |
dc.date.accessioned | 2021-05-20T21:08:28Z | - |
dc.date.available | 2012-01-01 | |
dc.date.available | 2021-05-20T21:08:28Z | - |
dc.date.copyright | 2011-07-06 | |
dc.date.issued | 2011 | |
dc.date.submitted | 2011-05-17 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/10188 | - |
dc.description.abstract | 本研究利用差異性顯示法 (differential display, DD) 技術獲得日日春 (Catharanthus roseus) 為花生簇葉病 (peanut witches' broom, PnWB) 植物菌質體感染後表現具有差異性的 mRNA,並完成 64 個 cDNA 片段之選殖。以反向北方雜配反應 (reverse northern hybridization) 分析,篩選出 10 個選殖株,經核酸解序與比對分析,推測其基因功能。經同步聚合酵素連鎖反應 (real-time polymerase chain reaction, PCR) 分析上述 10 個基因於健康日日春及感染花生簇葉病植物菌質體之日日春體內的表現情形,顯示 7 個基因之表現具差異性。其中一個與 phi-1 相似基因之表現量上升 (up-regulated) 外,其餘基因之表現量則均下降 (down-regulated)。表現量下降的基因中,有 2 個為預測蛋白基因 (predicted protein gene),其餘則分別與 psaDa gene, ML (MD-2-related lipid recognition) domain protein gene, eukaryotic translation initiation factor SUI1 gene 及 plastidic aldolase NPALDP1 gene 具有高相似度。進而完成模式植物日日春 cDNA 基因庫之構築,並以菌落聚合酵素連鎖反應 (colony PCR) 篩選 cDNA 基因庫,取得 3 個基因之 cDNA 全長序列,並加以比對以確認基因名稱,分別為ML domain protein gene, eukaryotic translation initiation factor SUI1 gene 及 plastidic aldolase NPALDP1 gene,其功能分別與植物先天性免疫反應 (innate immune response)、逆境反應 (stress response) 與光合作用 (photosynthesis) 相關。在基因表現時序 (time course) 的研究中,發現在接種後第六週時,該三個基因的表現量呈現表現量下降的情形,與差異性顯示法所得之結果一致。此三個基因的功能及可能的交互作用機制經由文獻探討進行討論,以綜合評析日日春與花生簇葉病植物菌質體間交互作用之關係。 | zh_TW |
dc.description.abstract | The differential display (DD) strategy was applied to isolate periwinkle (Catharanthus roseus) cDNAs differentially expressed following infection with peanut witches' broom (PnWB) phytoplasma. Sixty four clones have been selected from differentially expressed cDNA fragments. After screening by reverse northern hybridization, 10 transcripts were selected and sequenced. The expression levels of them was quantified by real-time polymerase chain reaction (PCR), and 7 DD transcripts were identified as ones truly differentially expressed after PnWB phytoplasma infection. One showing homology with phi-1 gene was up-regulated, while the others were down-regulated. Except two of which were predicted protein genes, the other down-regulated genes share homology with psaDa gene, ML domain protein gene, eukaryotic translation initiation factor SUI1 gene, and plastidic aldolase NPALDP1 gene. Homologous genes were further confirmed for 3 DD transcripts by isolating full-length cDNA fragments from the cDNA library constructed in this study. Confirmed genes included ML domain protein gene, translation initiation factor SUI1 gene, and plastidic aldolase NPALDP1 gene, and were mainly involved in innate immune responses, stress responses, and photosynthesis. In the time course experiments, expression levels of these three genes were down-regulated at 6th week after inoculation, and the results agreed with those of differential display. The possible role of these genes in the periwinkle that was infected by PnWB phytoplasma is discussed. | en |
dc.description.provenance | Made available in DSpace on 2021-05-20T21:08:28Z (GMT). No. of bitstreams: 1 ntu-100-D91633001-1.pdf: 2708036 bytes, checksum: 02e1ae068016f0a236404c51f05411a4 (MD5) Previous issue date: 2011 | en |
dc.description.tableofcontents | 口試委員會審定書 I
致謝 II 中文摘要 IV 英文摘要 V 壹、前言 1 貳、前人研究 4 一、植物菌質體之發現及其植物病理學 4 (一)植物菌質體之發現 4 (二)植物菌質體之植物病理學 4 二、植物菌質體之分群 5 三、植物菌質體之生物特性及其分子生物學之研究 8 四、植物菌質體與寄主植物交互作用之研究 10 參、材料與方法 17 一、試驗植物來源與植物菌質體保存方式 17 二、試驗植物全 DNA 及全 RNA 之純化 18 (一)試驗植物全 DNA 之純化 18 (二)試驗植物全 RNA 之純化 19 1. 以 TRIzol® 溶液進行日日春全 RNA 之純化 19 2. 以 RNA 變性電泳膠確認全 RNA 之品質 20 三、以差異性顯示法 (differential display) 篩選日日春感染花生簇葉病植物菌質體後具差異性表現之基因 20 (一)差異性顯示法 (differential display) 之操作方法 20 1. 以 RNase-Free DNase 處理全 RNA 21 2. 反轉錄反應 (reverse transcription) 22 3. 以聚合酵素連鎖反應 (PCR) 增幅日日春 cDNA 22 4. 變性定序電泳膠片 (denaturing sequencing gel) 之製作及電泳分析 23 5. 以核酸銀染 (silver stain) 進行變性定序電泳膠片染色 24 6. 具差異性表現 cDNA 片段之回收 25 (二)差異性基因片段之選殖 (cloning) 與序列分析 26 1. 以 PCR 再增幅 (re-amplify) 差異性基因片段 26 2. 差異性基因片段 PCR 再增幅產物之純化 27 3. 黏結反應 (ligation) 27 4. 轉形反應 (transformation) 27 5. 以菌落聚合酵素連鎖反應 (colony PCR) 篩選差異性基因插入片段 28 (三)以反向北方雜配反應 (reverse northern hybridization) 快速篩選差異性基因 29 1. 以 PCR 增幅差異性基因之 cDNA 片段 29 2. 於尼龍膜點製矩陣 (nylon array) 30 3. 探針 (probe) 製作與雜配反應 (hybridization) 30 4. 差異性基因之核酸定序及序列分析 32 四、日日春 cDNA 基因庫之建構 32 (一)日日春 cDNA 合成 33 (二)將日日春 cDNA 基因庫建構於 pDNR-LIB 質體 36 1. 黏結反應 36 2. 轉形反應 36 (三)質體基因庫大小之測定 (titering) 37 五、以同步聚合酵素連鎖反應 (real-time PCR) 之相對定量測定日日春差異性基因之 mRNA 表現量 38 (一)日日春管家基因 (housekeeping gene) 之選殖 38 1. 日日春管家基因之評估 38 2. 以 PCR 增幅日日春管家基因 39 3. 日日春泛素 (ubiquitin) 基因 PCR 產物之純化、選殖與序列分析 40 4. 由篩選 cDNA 基因庫取得日日春 ubiquitin 基因序列全長 40 (1) 以 colony PCR 篩選 cDNA 基因庫取得日日春 ubiquitin cDNA 轉形株 40 (2) 轉形株選殖片段之核酸定序與序列分析 41 (二)差異性基因選殖株之篩選與分析 41 1. 差異性基因同步聚合酵素連鎖反應引子對之設計 41 2. 以相對定量方式測定差異性基因之 mRNA 表現量 42 (1) 樣本之收集 42 (2) 同步聚合酵素連鎖反應之操作方法 42 (3) 差異性基因表現量之計算 43 3. 日日春 cDNA 基因庫中差異性基因轉形株之篩選及特性分析 44 4. 差異性基因表現時序 (time course) 之測定 44 肆、結果 45 一、受花生簇葉病植物菌質體感染之日日春病徵表現 45 二、日日春感染花生簇葉病植物菌質體後具差異性表現基因之分析 45 (一)以差異性顯示法篩選差異性表現基因 45 (二)以反向北方雜配反應快速篩選差異性基因 46 (三)具差異性表現基因之序列分析 46 三、以同步聚合酵素連鎖反應之相對定量測定日日春差異性基因之 mRNA 表現量 47 (一)管家基因之選殖及分析 47 (二)同步聚合連鎖反應之引子對專一性測定 48 (三)以相對定量方式測定差異性基因之 mRNA 表現量之改變 49 四、日日春 cDNA 基因庫之差異性基因篩選與分析 49 (一)ML domain protein 基因之分析 50 (二)Eukaryotic translation initiation factor SUI1 基因之分析 51 (三)Plastidic aldolase 基因之分析 51 (四)差異性基因表現時序 (time course) 之測定 51 伍、討論 53 陸、參考文獻 64 柒、圖表 79 | |
dc.language.iso | zh-TW | |
dc.title | 日日春受花生簇葉病植物菌質體感染後之基因表現差異性研究 | zh_TW |
dc.title | Characterization of Catharanthus roseus Genes Regulated Differentially by Peanut Witches' Broom Phytoplasma Infection | en |
dc.type | Thesis | |
dc.date.schoolyear | 99-2 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 曾國欽,陳煜焜,張碧芳,徐源泰,林乃君,洪挺軒 | |
dc.subject.keyword | 花生簇葉病植物菌質體,日日春,交互作用,差異性顯示法, | zh_TW |
dc.subject.keyword | differential display,interactions,periwinkle, | en |
dc.relation.page | 118 | |
dc.rights.note | 同意授權(全球公開) | |
dc.date.accepted | 2011-05-17 | |
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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