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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 黃鵬林(Pung-Ling Huang) | |
| dc.contributor.author | Fu-Yu Hung | en |
| dc.contributor.author | 洪福佑 | zh_TW |
| dc.date.accessioned | 2021-06-08T04:58:52Z | - |
| dc.date.copyright | 2010-08-20 | |
| dc.date.issued | 2010 | |
| dc.date.submitted | 2010-08-19 | |
| dc.identifier.citation | 王清玲 安寶貞 翁振宇 張清安 張翠瑛 陳淑佩 黃晉興 謝廷芳. 2005. 蝴蝶蘭
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/23294 | - |
| dc.description.abstract | 摘要
為了分析默化蘭花病毒鞘蛋白基因的效果，本研究選用穩定轉殖喜姆 比蘭嵌紋病毒(Cymbidium mosaic virus, CymMV)或齒舌蘭輪斑病毒 (Odontoglossum ringspot virus, ORSV)全長與短片段鞘蛋白所構築之各默化 載體的邊沁菸草(Nicotiana benthamiana)，並以感病病葉萃取汁液直接接 種、Potato virus X (PVX)重組病毒與infectious clone三種方法分別進行接 種，記錄外觀病徵並以反轉錄聚合酶連鎖反應檢測植株中病毒鞘蛋白RNA 產物累積，檢測現有默化轉殖株的默化能力。試驗結果顯示三種病毒接種 方法皆可以將CymMV或ORSV順利的感染未轉殖之邊沁菸草，產生可辨識 的病徵，且會隨著時間增加趨於明顯。同時也可以在接種約一到兩週後的 葉片組織中，測得CymMV或ORSV鞘蛋白RNA的累積。CymMV或ORSV鞘 蛋白默化效果分析結果顯示：全長鞘蛋白RNAi及鞘蛋白短片段RNAi構築 之轉殖株，經接種後大部份轉殖株都表現出較輕微的病徵，同時也有較少 的病毒鞘蛋白mRNA產物累積。 | zh_TW |
| dc.description.abstract | To evaluate the silencing effect on Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) coat protein gene silenced construction, infected leaf sap, recombinant-Potato virus X (PVX) and virus infectious clone were used to inoculate Nicotiana benthamiana transformants containing different hairpin RNA (hpRNA) construct of RNA interference. Observation of virusderived symptoms and analysis by reverse transcription polymerase chain reaction (RT-PCR) were performed for investigation of silencing effect in transgenic lines. Untransformed N. benthamiana could be inoculated successfully with CymMV and ORSV by all three inoculation methods based on virusinduced symptoms appearance and accumulation of coat protein mRNA. After inoculation, transgenic lines containing hpRNA construct for coding region or 25 bp of coat protein cDNA form CymMV or ORSV showed milder virus-induceed symptoms and accumulated lesser coat protein RNA than untransformed N. benthamiana. These results confirmed the silencing effect of coat protein genes by hpRNA construct. | en |
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| dc.description.tableofcontents | 內容目次
論文口試委員審定書................................................................................................................................i 誌謝................................................................................................................................................................ii 摘要...............................................................................................................................................................iii Abstract.......................................................................................................................................................iv 壹、前言......................................................................................................................................................1 貳、前人研究............................................................................................................................................2 一、蘭花病毒......................................................................................................................................2 二、基因默化及其作用機制..........................................................................................................5 三、植物防禦病毒之機制...............................................................................................................6 四、影響植物病毒接種成功率之因子.......................................................................................8 參、材料與方法.....................................................................................................................................11 一、試驗材料....................................................................................................................................11 (一) 基因默化轉殖質體............................................................................................................11 1.全長CymMV外鞘蛋白基因之RNAi構築質體 .......................................................11 2.全長ORSV外鞘蛋白基因之RNAi構築質體..............................................................11 (二) 植物材料...............................................................................................................................11 (三) 植物病毒來源.....................................................................................................................12 1. 感病病葉..............................................................................................................................12 2. 重組病毒..............................................................................................................................12 3. 全長病毒 infectious in vitro RNA transcripts..........................................................12 二、試驗方法....................................................................................................................................13 (一)菸草擬轉殖植株之篩選與馴化.....................................................................................13 (二) 轉殖株病毒接種...............................................................................................................13 1. 酶切及純化 template DNA............................................................................................13 2. T7 promoter啟動之 in vitro transcription reaction................................................13 3. T3 promoter啟動之 in vitro transcription reaction................................................16 4. 病毒機械接種的操作......................................................................................................16 (三) Total RNA之抽取 .............................................................................................................17 (四) 轉殖株分子檢測................................................................................................................18 1. 反轉錄聚合酶連鎖反應(reverse transcription-PCR, RT-PCR)..........................18 2. siRNA 檢測分析................................................................................................................18 (1) DNA片段回收...............................................................................................................18 (2) 探針的製備與同位素標定........................................................................................19 (3) siRNA enrichment.........................................................................................................19 (4) siRNA 雜交分析...........................................................................................................19 肆、結果....................................................................................................................................................21 一、 病毒接種方式之致病效果比較分析................................................................................21 (一) 重組PVX病毒接種邊沁菸草之致病效果分析.......................................................21 (二) CymMV及ORSV infectious transcripts接種邊沁菸草之致病效果分析........24 (三)三種不同病毒來源進行接種之比較...........................................................................24 二、 默化構築轉殖菸草以病葉直接接種CymMV以及ORSV之分析...........................33 (一) CymMV外鞘蛋白默化載體轉殖株之抗病效果分析...........................................33 (二) ORSV外鞘蛋白默化載體轉殖株之抗病效果分析...............................................33 三、默化構築轉殖菸草接種PVX-CymMVgp5及PVX-ORSVgp5之分析.....................37 (一) CymMV外鞘蛋白默化載體轉殖株之抗病效果分析...........................................37 (二) ORSV外鞘蛋白默化載體轉殖株之抗病效果分析...............................................41 四、 CymMV及ORSV infectious transcripts 接種轉殖菸草效果之分析.....................41 (一) CymMV外鞘蛋白默化載體轉殖株之抗病效果分析...........................................41 (二) ORSV外鞘蛋白默化載體轉殖株之抗病效果分析...............................................45 伍、討論....................................................................................................................................................51 一、外鞘蛋白默化構築轉殖菸草抗病效果分析.................................................................51 二、利用不同接種方法接種於邊沁菸草的效果.................................................................54 三、不同外鞘蛋白不同保守序列默化構築的效果............................................................58 陸、參考資料..........................................................................................................................................60 | |
| dc.language.iso | zh-TW | |
| dc.subject | 基因默化 | zh_TW |
| dc.subject | 喜姆比蘭嵌紋病毒 | zh_TW |
| dc.subject | 齒舌蘭輪斑病毒 | zh_TW |
| dc.subject | CymMV | en |
| dc.subject | infectious clone | en |
| dc.subject | recombinant virus | en |
| dc.subject | PTGS | en |
| dc.subject | ORSV | en |
| dc.title | 抗喜姆比蘭嵌紋病毒與齒舌蘭輪斑病毒之基因默化轉殖菸草抗病效力分析 | zh_TW |
| dc.title | Studies on RNAi Mediated Virus Immunity against Cymbidium mosaic virus or Odontoglossum ringspot virus in Nicotiana benthamiana by Silencing Coat Protein Gene | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 98-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 張清安(Chin-An Chang),葉信宏(Hsin-Hung Yeh),杜宜殷(Yi-Yin Do) | |
| dc.subject.keyword | 喜姆比蘭嵌紋病毒,齒舌蘭輪斑病毒,基因默化, | zh_TW |
| dc.subject.keyword | CymMV,ORSV,PTGS,recombinant virus,infectious clone, | en |
| dc.relation.page | 69 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2010-08-19 | |
| dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
| dc.contributor.author-dept | 園藝學研究所 | zh_TW |
| 顯示於系所單位： | 園藝暨景觀學系 | |
文件中的檔案：
| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| ntu-99-1.pdf 未授權公開取用 | 69.37 MB | Adobe PDF |
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