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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 杜宜殷(Yi-Yin Do) | |
dc.contributor.author | Wan-Jyun Wei | en |
dc.contributor.author | 衛宛君 | zh_TW |
dc.date.accessioned | 2021-06-16T10:35:46Z | - |
dc.date.available | 2018-08-26 | |
dc.date.copyright | 2013-08-26 | |
dc.date.issued | 2013 | |
dc.date.submitted | 2013-08-14 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/60906 | - |
dc.description.abstract | 台灣蘭科植物病害主要以東亞蘭嵌紋病毒 (Cymbidium mosaic virus, CymMV) 及齒舌蘭輪點病毒 (Odontoglossum ringspot virus, ORSV) 感染為主,嚴重影響蘭花生長及降低其觀賞價值,又基因轉殖作物內含篩選標誌基因,易引起大眾對其安全性有所疑慮,本研究利用核糖核酸干擾技術 (RNA interference, RNAi),將同時含CymMV及ORSV鞘蛋白基因 250 bp 以反義-隱子-順義方式連接之默化載體,構築於轉位子剔除篩選標誌通用載體,轉殖至蝴蝶蘭及嘉德麗雅蘭中,期望獲得抗病且無篩選標誌之轉殖蘭花,並以模式植物邊沁菸草進行默化效力檢測。於邊沁菸草部分已完成轉殖株病毒接種試驗,證實轉殖株具有抗CymMV及ORSV之能力。蝴蝶蘭癒傷組織經轉殖後,以抗生素篩選,部分擬轉殖癒傷組織經GUS活性組織化學染色分析呈藍色反應,聚合酶連鎖反應分析結果合成出預期片段,確定轉殖事件之發生;轉殖癒傷組織經誘導再生為擬原球體, GUS染色分析亦呈藍色反應,目前持續於篩選培養基誘導抽芽中。為克服因蝴蝶蘭癒傷組織轉殖之篩選及再生時間過於冗長,本研究另以蝴蝶蘭葉片為培植體誘導擬原球體產生,約六個月即可完成再生,明顯快於癒傷組織之再生途徑,並已進行初步轉殖。於嘉德麗雅蘭轉殖部分,亦以再生速率較快之葉片為培植體進行轉殖,目前培植體持續於篩選培養基篩選中。 | zh_TW |
dc.description.abstract | Orchid diseases in Taiwan are caused mainly by infection of Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV). Both viruses affect normal growth of orchids seriously and reduce their marketable value. To achieve virus-resistant transgenic orchid without selectable marker genes, 25 bp DNA fragment based on RNA interference (RNAi) technology for both coat protein genes from CymMV and ORSV was constructed into vector combined with transposon as marker-free system and was transformed into Phalaenopsis and Cattleya. Nicotiana benthamiana was also transformed as a model to test silencing efficiency of RNAi plasmid. After inoculation with viruses, transgenic tobaccos have been proven resistant against CymMV and ORSV. Transformation events had been confirmed by GUS staining for Phalaenopsis survived calli and protocorm-like bodies after antibiotic selection. In order to shorten the time of antibiotic screening and regeneration for Phalaenopsis transformation, regeneration in six months was accomplished using leaf disc as explant. Both Phalaenopsis and Cattleya transformed leaf discs are on selective medium for shoot induction at this moment. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T10:35:46Z (GMT). No. of bitstreams: 1 ntu-102-R00628104-1.pdf: 2175434 bytes, checksum: 2a1664181bf1b38e7c9bd273692a1c70 (MD5) Previous issue date: 2013 | en |
dc.description.tableofcontents | 目錄
摘要 i Abstract ii 壹、 前言 1 貳、 前人研究 3 一、 蘭花 3 二、 東亞蘭嵌紋病毒 3 三、 齒舌蘭輪點病毒 4 四、 核糖核酸干擾技術 4 五、 轉錄後基因默化 6 (一) 轉殖反義股RNA模式之抗病機制 7 (二) 過度表現順義股RNA模式之抗病機制 7 (三) 雙股RNA模式之抗病機制 8 (四) 三種轉殖模式所造成之PTGS效率比較 8 (五) 轉錄後基因默化之應用於植物抗病的優點 9 六、 篩選標誌基因 9 (一) 剔除篩選標誌基因之轉殖策略 10 (二) 轉位子 (transposon) 系統 11 1. Ac / Ds系統剔除篩選標誌基因之質體構築 11 2. 誘導型啟動子剔除篩選標誌基因 12 (三) 正向篩選標誌基因 13 参、 材料與方法 14 一、 試驗材料 14 (一) 質體 14 (二) 試驗菌種 14 (三) 植物材料 14 二、 試驗方法 14 (一) RNAi質體pGcET-CyORi構築 15 1. 構築策略 15 2. DNA片段平端 (blunt end) 處理 15 3. 去磷酸化 (dephosphorylation) 15 4. DNA片段之回收 15 5. 接合反應 (ligation) 16 6. 大腸桿菌勝任細胞 (competent cell) 之製備 16 7. 大腸桿菌之轉型 (transformation) 16 8. 質體DNA少量製備 (minipreparation) 18 (二) 農桿菌之轉型 18 1. 農桿菌勝任細胞之製備 18 2. 農桿菌電穿孔法 (Electroporation) 轉型 19 3. 農桿菌質體小量製備 19 4. 農桿菌質體純化 20 (三) 基因轉殖方法 20 1. 邊沁菸草葉圓片轉殖 20 2. 蝴蝶蘭癒傷組織轉殖 21 3. 嘉德麗雅蘭PLB轉殖 21 4. 嘉德麗雅蘭及蝴蝶蘭葉片轉殖 22 5. 嘉德麗雅蘭及蝴蝶蘭葉片再生 22 (四) 擬轉殖株分析 22 1. GUS活性組織化學染色法 23 2. 植物基因組DNA之抽取 23 3. 聚合酶連鎖反應 (polymerase chain reaction, PCR) 分析 24 4. 核酸探針之製備和同位素標定 24 5. 南方氏雜交分析 25 (五) 病毒接種試驗 25 1. 接種CymMV及ORSV於轉殖邊沁菸草 25 2. 植物total RNA之抽取 26 3. 植物葉片蛋白質之抽取 26 4. 反轉錄聚合酶連鎖反應 (reverse transcription polymerase chain reaction, RT-PCR) 27 5. 酵素聯結免疫吸附法 (enzyme-linked immunosorbent assay, ELISA) 27 肆、 結果 29 一、 邊沁菸草 29 (一) 邊沁菸草擬轉殖葉片之GUS活性組織化學染色分析 29 (二) 邊沁菸草擬轉殖株之聚合酶連鎖反應分析 29 (三) 邊沁菸草擬轉殖株之南方氏雜交分析 29 (四) 接種CymMV及ORSV於邊沁菸草轉殖株之抗病能力分析 30 二、 蝴蝶蘭 39 (一) 蝴蝶蘭經癒傷組織之再生及轉殖 39 1. 蝴蝶蘭擬轉殖癒傷組織及擬原球體之GUS活性組織化學染色分析 39 2. 蝴蝶蘭擬轉殖癒傷組織之聚合酶連鎖反應分析 39 (二) 蝴蝶蘭經葉片之再生系統及轉殖系統的建立 39 1. 再生系統 39 2. 轉殖系統 40 三、 嘉德麗雅蘭 50 (一) 嘉德麗雅蘭pCRFG擬轉殖株之分子驗證 50 (二) 嘉德麗雅蘭葉片轉殖 50 伍、 討論 55 一、 蘭花基因轉殖 55 (一) 嘉德麗雅蘭 55 (二) 蝴蝶蘭 56 二、 無篩選標誌策略 57 三、 病毒鞘蛋白默化構築於轉殖邊沁菸草之抗病效果 58 (一) 病毒鞘蛋白基因默化構築 58 (二) 影響抗病效果之可能因素 59 陸、 結語 61 柒、 參考文獻 63 | |
dc.language.iso | zh-TW | |
dc.title | 應用轉位子去除篩選標誌於基因轉殖植物抗東亞蘭嵌紋病毒及齒舌蘭輪點病毒之研究 | zh_TW |
dc.title | Studies on Disease Resistance against Cymbidium Mosaic Virus and Odontoglossum Ringspot Virus with Transposon-directed Marker Elimination System in Transgenic Plants | en |
dc.type | Thesis | |
dc.date.schoolyear | 101-2 | |
dc.description.degree | 碩士 | |
dc.contributor.coadvisor | 黃鵬林(Pung-Ling Huang) | |
dc.contributor.oralexamcommittee | 廖芳心(Fang-Shin Liao),劉祖惠(Tsu-Hwie Liu) | |
dc.subject.keyword | 核糖核酸干擾,雙股RNA模式,無篩選標誌,蘭花基因轉殖,蘭花葉片再生, | zh_TW |
dc.subject.keyword | RNA interference,double-stranded RNA induced model,marker free strategy,orchid transformation,orchid leaf regeneration, | en |
dc.relation.page | 75 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2013-08-14 | |
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 |
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