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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 杜宜殷(Yi-Yin Do) | |
| dc.contributor.author | Yu Wang | en |
| dc.contributor.author | 王瑜 | zh_TW |
| dc.date.accessioned | 2021-07-11T15:44:23Z | - |
| dc.date.available | 2025-08-20 | |
| dc.date.copyright | 2020-08-28 | |
| dc.date.issued | 2020 | |
| dc.date.submitted | 2020-08-19 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/79106 | - |
| dc.description.abstract | 蝴蝶蘭(Phalaenopsis spp.)是臺灣外銷最大宗花卉作物，受蕙蘭嵌紋病毒(Cymbidium mosaic virus, CymMV)及齒舌蘭輪斑病毒(Odontoglossum ringspot virus, ORSV)等病毒性病害嚴重影響品質。本論文期望運用基因編輯及核糖核酸干擾(RNA interference)技術，取得抗病毒之蝴蝶蘭。首先建立蝴蝶蘭之基因編輯平台，選取蝴蝶蘭八氫番茄紅素去飽和酶(phytoene desaturase, PDS)基因，以CRISPR/Cas9系統進行突變；此基因若喪失功能，會導致白化表觀。經分析P. amabilis PDS cDNA序列及選殖部分PDS基因片段，選擇位於第二個及第三個顯子的20 bp作為標靶序列，構築於表現載體，以農桿菌注射法及聚乙二醇法，分別轉入P. amabilis之葉片及原生質體。轉殖後抽取基因組DNA，進行高解析熔解曲線分析及次世代定序，結果顯示農桿菌注射法之葉片及聚乙二醇法之原生質體中，均造成標靶序列發生單一核苷酸之置換，表示標靶序列具有功能。穩定性轉殖採用花粉管導入法，分別將基因編輯表現質體及雙重病毒基因默化質體轉殖至蝴蝶蘭，得到之原球體於GUS活性組織化學染色後皆有藍點之表現；轉殖基因編輯表現質體得到之原球體具白化表觀，顯示PDS基因編輯成功。 | zh_TW |
| dc.description.abstract | Phalaenopsis orchids are the largest export floral crops in Taiwan. Infections by viruses such as Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) dramatically reduce the value of Phalaenopsis. In this study, genome editing and RNA interference (RNAi) technology were used to develop virus resistant Phalaenopsis. In order to establish a genome editing platform in Phalaenopsis, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) was used to mutate the phytoene desaturase (PDS) gene. Loss-of-function of PDS leading to the bleaching phenotype indicates the editing event happened. After the analysis of PDS cDNA sequence and cloning of partial PDS gene, two 20-nucleotide guide sequences located at the second and third exons, respectively, were selected to construct into the expression vector. The gene editing constructs were transformed into leaves and protoplasts of P. amabilis via agroinfiltration and polyethylene glycol (PEG)-mediated transformation, respectively. Genomic DNAs extracted from transformed explants were subjected to the high-resolution melting curve (HRM) analysis and next generation sequencing (NGS). Single nucleotide substitutions were detected in the guide sequence of edited cells. For stable transformation, gene editing expression plasmids and the virus dual gene silencing plasmid were transformed into Phalaenopsis via pollen tube pathway. Putative transgenic protocorms showed blue spots after the GUS assay. Several putative transgenic protocorms with albino phenotype indicated that PDS gene had been edited in these transformants successfully. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-11T15:44:23Z (GMT). No. of bitstreams: 1 U0001-1708202015492000.pdf: 16099838 bytes, checksum: 97bb8aace3ed80f72be0216c29b0b6bd (MD5) Previous issue date: 2020 | en |
| dc.description.tableofcontents | 摘要....................................................i Abstract...............................................ii 壹、前言.................................................1 貳、前人研究.............................................2 一、提升病毒抗性之植物基因轉殖.............................2 (一) 植物抗病毒相關基因之過量表現..........................2 1. 隱性抗性基因(recessive resistance gene)...............2 2. 顯性抗性基因(dominant resistance gene)................2 3. 抵禦媒介昆蟲之基因....................................3 4. 阻擋病毒於細胞間擴散之基因.............................3 (二) 植物抗病毒相關基因之抑制表現..........................4 1. 核糖核酸干擾之策略....................................4 2. CRISPR/Cas基因編輯系統於植物抗病毒之應用...............4 (1) CRISPR/Cas基因編輯系統...............................4 (2) 以PDS基因建立CRISPR/Cas9基因編輯平台..................5 (3) DNA病毒抗性之提升.....................................6 (4) RNA病毒抗性之提升.....................................6 二、常用的基因轉殖方法.....................................6 (一) 葉片農桿菌注射法(syringe agroinfiltration)............7 (二) 原生質體聚乙二醇法(polyethylene glycol, PEG)..........7 (三) 花粉管導入法(pollen tube pathway).....................8 (四) 基因槍法(gene gun)....................................9 (五) 農桿菌共感染轉殖法(Agrobacterium-mediated transformation)...10 參、材料與方法.............................................11 一、試驗材料...............................................11 (一) 菌種.................................................11 (二) 質體.................................................11 1. pBluescript II SK (-)..................................11 2. pGSfiI-gfpgus 2-8......................................11 3. pGKU-CyORg核糖核酸干擾質體...............................11 (三) 植物材料..............................................11 二、試驗方法...............................................12 (一) 基因結構分析..........................................12 1. 基因序列分析............................................12 2. 基因片段之選殖..........................................12 (1) 植物基因組DNA之抽取.....................................12 (2) 聚合酶連鎖反應..........................................13 (3) 聚合酶連鎖反應之產物回收.................................13 (4) 大腸桿菌之轉型..........................................13 (5) 小量質體DNA之製備.......................................13 (6) 定序分析...............................................14 (二) 基因編輯表現質體之構築..................................14 1. sgRNA標靶序列之挑選......................................14 2. 構築流程.................................................14 3. 大腸桿菌之轉型...........................................15 4. 大量質體DNA之製備........................................15 5. 農桿菌之轉型.............................................15 6. 農桿菌質體DNA之製備.......................................16 (三) 蝴蝶蘭之基因轉殖........................................16 1. 葉片農桿菌注射法..........................................16 2. 原生質體聚乙二醇法........................................17 3. 花粉管導入法..............................................17 (四) 基因轉殖結果分析.........................................18 1. GUS活性組織化學染色分析....................................18 2. 原生質體基因組DNA之抽取....................................18 3. 高解析度熔解曲線(high-resolution melting, HRM)............18 4. 次世代定序分析(next-generation sequencing, NGS)............19 肆、結果.....................................................20 一、蝴蝶蘭PDS基因之選殖.......................................20 二、sgRNA標靶序列之篩選及PDS基因編輯表現質體之構築..............20 三、PDS基因標靶序列編輯效果分析................................20 (一) 葉片農桿菌注射法.........................................20 (二) 原生質體聚乙二醇法.......................................21 四、蝴蝶蘭之穩定性基因編輯.....................................21 五、病毒雙抗質體之蝴蝶蘭穩定性轉殖..............................22 伍、討論.....................................................23 一、轉殖方法對基因編輯結果之影響................................23 (一) 基因編輯率...............................................23 (二) 基因編輯型態..............................................24 二、sgRNA構築對基因編輯效果之影響...............................25 三、核酸分解酶構築對基因編輯效果之影響...........................25 陸、結語......................................................27 柒、參考文獻..................................................47 捌、附錄......................................................56 | |
| dc.language.iso | zh-TW | |
| dc.subject | 農桿菌注射法 | zh_TW |
| dc.subject | 齒舌蘭輪斑病毒 | zh_TW |
| dc.subject | 蕙蘭嵌紋病毒 | zh_TW |
| dc.subject | 核糖核酸干擾技術 | zh_TW |
| dc.subject | 農桿菌共感染轉殖法 | zh_TW |
| dc.subject | 花粉管導入法 | zh_TW |
| dc.subject | 基因編輯 | zh_TW |
| dc.subject | 八氫番茄紅素去飽和酶 | zh_TW |
| dc.subject | 聚乙二醇法 | zh_TW |
| dc.subject | agroinfiltration | en |
| dc.subject | polyethylene glycol | en |
| dc.subject | Odontoglossum ringspot virus | en |
| dc.subject | pollen tube pathway | en |
| dc.subject | Agorbacterium-mediated transformation | en |
| dc.subject | phytoene desaturase | en |
| dc.subject | clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 | en |
| dc.subject | RNA interference technology | en |
| dc.subject | Cymbidium mosaic virus | en |
| dc.title | 蝴蝶蘭定點突變及抗病毒之基因轉殖 | zh_TW |
| dc.title | Genetic Transformation for Site-directed Mutagenesis and Virus Resistance in Phalaenopsis amabilis | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 108-2 | |
| 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-Hui Annie Liu),李昆達(Kung-Ta Lee) | |
| dc.subject.keyword | 基因編輯,八氫番茄紅素去飽和酶,農桿菌注射法,聚乙二醇法,花粉管導入法,農桿菌共感染轉殖法,核糖核酸干擾技術,蕙蘭嵌紋病毒,齒舌蘭輪斑病毒, | zh_TW |
| dc.subject.keyword | clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9,phytoene desaturase,agroinfiltration,polyethylene glycol,pollen tube pathway,Agorbacterium-mediated transformation,RNA interference technology,Cymbidium mosaic virus,Odontoglossum ringspot virus, | en |
| dc.relation.page | 56 | |
| dc.identifier.doi | 10.6342/NTU202003783 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2020-08-20 | |
| dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
| dc.contributor.author-dept | 園藝暨景觀學系 | zh_TW |
| dc.date.embargo-lift | 2025-08-20 | - |
| 顯示於系所單位： | 園藝暨景觀學系 | |
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