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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 | Po-Ya Wang | en |
dc.contributor.author | 王柏雅 | zh_TW |
dc.date.accessioned | 2021-06-15T16:18:11Z | - |
dc.date.available | 2020-08-28 | |
dc.date.copyright | 2015-08-28 | |
dc.date.issued | 2015 | |
dc.date.submitted | 2015-08-17 | |
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Ferguson, and K.-S. Chen. 2009. Ethylene-related genes show a differential response to low temperature during ‘Hayward’kiwifruit ripening. Postharvest Biol. Technol. 52:9-15. Yokotani, N., S. Tamura, R. Nakano, A. Inaba, and Y. Kubo. 2003. Characterization of a novel tomato EIN3‐like gene (LeEIL4). J. Exp. Bot. 54:2775-2776. Zhou, L., L. Dong, P.-Y. Jia, W.-R. Wang, and L.-Y. Wang. 2010. Expression of ethylene receptor and transcription factor genes, and ethylene response during flower opening in tree peony (Paeonia suffruticosa). Plant Growth Regul. 62:171-179. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/52547 | - |
dc.description.abstract | 文心蘭 (Oncidesa spp.) 為臺灣重要外銷切花,花朵及花藥蓋易脫落造成乙烯大量產生而迅速萎凋,為探討乙烯訊息傳導轉錄因子ETHYLENE INSENSITIVE 3 (EIN3)/EIN3-LIKEs (EILs) 基因表現特性,首先選殖文心蘭OgEIL1及OgEIL2,OgEIL1基因結構包含三個顯子及兩個位在5’非轉譯區之隱子,OgEIL2選殖得到轉譯區及轉譯起始點上游2.5 kb序列,兩基因轉譯起始點上游序列經資料庫比對顯示皆具有高溫、光、無氧逆境、激勃素及水楊酸等cis-acting element。進一步將OgEIL1啟動子、5’非轉譯區及隱子構築於含有β-glucuronidase的表達載體,轉殖至阿拉伯芥及菸草,觀察OgEIL1啟動子於各發育階段、生長調節劑及逆境誘導之表現情形。阿拉伯芥OgEIL1pro::GUS包含第一及第二隱子的轉殖株中啟動子表現於頂端分生組織、葉片、根尖、柱頭及花藥,OgEIL1pro::GUS只含第一隱子之轉殖株啟動子表現量較含兩隱子的轉殖株減少,OgEIL1pro::GUS只含第二隱子之轉殖株啟動子只於花瓣表現,未含隱子之OgEIL1pro::GUS轉殖株中啟動子只表現於頂端分生組織、葉片及柱頭,顯示第一隱子具有增強基因表現的功能,且第一及第二隱子皆具有調控啟動子表達部位的功能。IAA及SA可抑制而高溫則誘導OgEIL1pro::GUS同時包含兩個隱子及只含第一隱子的轉殖株之啟動子表現,顯示第一隱子具有對於IAA及SA之silencer且具有對於高溫的enhancer;創傷及乾旱可提升含有兩個隱子、只含第二隱子及無隱子之轉植株啟動子活性,顯示第二隱子具有對創傷及乾旱之enhancer;ABA可抑制含第一隱子轉殖株啟動子活性但提升含第二隱子轉殖株啟動子活性,顯示第一隱子具有對ABA的silencer且第二隱子具有enhancer;ACC、GA及鹽逆境可提升完全不含隱子及同時含二個隱子的轉殖株啟動子活性,但不影響只含單一隱子的轉殖株啟動子表現,顯示隱子中可能並非只有單一因子調控基因表現;MeJA及低溫處理可促進含兩隱子轉殖株的啟動子活性,顯示此兩隱子皆具有對MeJA及低溫的enhancer。四種不同表達載體之菸草轉殖株已經由PCR驗證轉殖株,並由南方氏雜交分析取得單一拷貝之轉殖株。 | zh_TW |
dc.description.abstract | The cut flower of Oncidesa Gower Ramsey is one of the most important ornamental flowers in Taiwan for exportation. However, the vase life tends to short resulting from the pollinia cap dislodging frequently and production of ethylene. In order to investigate the expression pattern of ETHYLENE INSENSITIVE 3 (EIN3) / EIN3-LIKEs (EILs), the OgEIL1 and OgEIL2 were cloned from Oncidesa. The gene structure of OgEIL1 contains three exon and two intron located in 5’-untranslated region. Several conserved cis-acting elements such as heat, light, anaerobic stress, GA, and MeJA. The OgEIL1 promoter, untranslated region and two introns were fused with β-glucuronidase gene to form different lengths of expression vectors and transformed into Arabidopsis and tobacco via Agrobacterium-mediated method to investigate promoter activity of OgEIL1. The promoter activity was observed by GUS histochemical staining at different development stages, plant growth regulators and stresses treatments. Histochemical staining analysis of transgenic Arabidopsis seedlings carrying OgEIL1pro::GUS containing intron 1 and intron 2 showed that GUS activity was detected in apical meristem, leaves, root tips, stigma and anther. There was low-level GUS activity in Arabidopsis seedlings carrying OgEIL1pro::GUS including intron 1. The GUS activity in Arabidopsis seedlings carrying OgEIL1pro::GUS containing intron 2 was detected only in petals. The GUS activity of Arabidopsis seedlings carrying OgEIL1pro::GUS containing intron 1 was detected in apical meristem, leaves, and stigma, therefore, intron 1 contained enhancer and tissue-specific expression probably is mediated by intron 1 and intron 2. There was lower GUS activity after IAA and SA treatments and higer activity after heat treatment in seedlings carrying OgEIL1pro::GUS including intron 1 or both introns, these results indicated there is a silencer response to IAA and SA and an enhancer response to heat in intron 1. There is a silencer in intron1 and an enhancer in intron2 response to ABA. ACC, GA, and salt increased the promoter expression in seedlings harboring OgEIL1pro::GUS or including both introns and decreased in seedlings harboring OgEIL1pro::GUS including intron 1 or intron 2. These results showed there are complex factors response to these treatments located in intron 1 and intron 2. There are enhancers in both introns response to MeJA and cold. The transgenic tobacco haboring four kinds of expression vectors were confirmed by polymerase chain reaction and Southern analysis for transgenic copy number. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T16:18:11Z (GMT). No. of bitstreams: 1 ntu-104-R02628112-1.pdf: 10106021 bytes, checksum: 7c4a7af4c2254cfca7e6f8f11c97d01a (MD5) Previous issue date: 2015 | en |
dc.description.tableofcontents | 壹、 前言 1 貳、 前人研究 2 一、 乙烯生合成路徑及訊息傳導 2 二、 EIN3/EILs結構與功能性分析 3 三、 EIN3/EILs基因表現之研究 3 四、 影響花瓣老化之重要轉錄因子 5 (一) 更年性花卉花瓣老化之轉錄因子 5 (二) 乙烯不敏感花卉花瓣老化之轉錄因子 6 五、 隱子對基因轉錄之調控 6 參、 材料與方法 8 一、 試驗材料 8 (一) 文心蘭基因組庫及EIN3同源cDNA選殖系 8 (二) 植物材料 8 1. 阿拉伯芥 (Arabidopsis thaliana ecotype Columbia) 8 2. 菸草 (Nicotiana tabacum L.cv. W38) 8 二、 試驗方法 8 (一) 文心蘭EIN3同源基因之篩選 8 1. 寄主細胞的製備 8 2. 文心蘭基因組庫之篩選 8 3. 核酸探針之製備 9 4. 噬菌體之複製 9 5. 噬菌體DNA之抽取 10 6. 噬菌體目標選殖系之限制酶圖譜分析 10 7. 文心蘭EIN3之次選殖 11 8. 質體DNA之小量製備 11 9. 植物基因組DNA之小量製備 11 10. Inverse Polymerase Chain Reation 12 11. 啟動子基因序列分析 12 (二) 啟動子暫時性及穩定性表現質體之構築 12 1. OgEIL1pro::GUS 表現質體pOEIL1pΔi1i2之構築 12 2. OgEIL1pro::GUS 包含第一隱子表現質體pOEIL1pΔi2之構築 13 3. OgEIL1pro::GUS 包含第二隱子表現質體pOEIL1pΔi1之構築 13 4. OgEIL1pro::GUS 包含第一隱子及第二隱子表現質體pOEIL1p之構築 13 5. OgEIL2pro::GUS (pOEIL2p) 表現質體之構築 14 (三) 基因槍暫時性表現分析 14 1. 金粒子製備及DNA包覆 14 2. 基因槍設定條件 14 (四) 農桿菌轉型與檢測 15 1. 農桿菌轉型 15 2. 農桿菌質體之小量製備 15 (五) 阿拉伯芥之基因轉殖 16 1. 阿拉伯芥植株培養 16 2. 阿拉伯芥之基因轉殖 16 (六) 菸草之基因轉殖 16 (七) GUS活性組織化學染色法 17 (八) 南方氏雜交分析 (Southern blot analysis) 17 (九) 轉殖株之啟動子活性誘導分析 17 1. 不同發育階段之啟動子活性分析 17 2. 不同非生物逆境誘導處理 17 3. 不同生長調節劑誘導處理 18 肆、 結果 19 一、 文心蘭EIN3同源基因OgEIL1及OgEIL2之選殖及分析 19 (一) 文心蘭EIN3同源基因限制酶圖譜分析 19 (二) 文心蘭OgEIL1及OgEIL2啟動子序列分析 19 二、 文心蘭OgEIL1啟動子暫時性表現分析 20 三、 文心蘭OgEIL1啟動子於阿拉伯芥穩定性轉殖分析 20 (一) 不同發育階段之阿拉伯芥OgEIL1pro::GUS轉殖株啟動子活性分析 20 (二) 不同生長調節劑處理阿拉伯芥之OgEIL1pro::GUS轉殖株啟動子活性分析 21 (三) 不同非生物逆境處理阿拉伯芥之OgEIL1pro::GUS轉殖株啟動子活性分析 22 四、 文心蘭OgEIL1啟動子轉殖菸草之分子驗證 23 (一) 菸草OgEIL1pro::GUS含第一及第二隱子 (pOEIL1p) 之轉殖株啟動子活性分析 23 (二) 菸草OgEIL1pro::GUS含第一隱子 (pOEIL1pΔi2) 之轉殖株啟動子活性分析 24 (三) 菸草OgEIL1pro::GUS含第二隱子 (pOEIL1pΔi1) 之轉殖株啟動子活性分析 24 (四) 菸草OgEIL1pro::GUS (pOEIL1pΔi1i2) 之轉殖株啟動子活性分析 24 伍、 討論 84 一、 OgEIL1啟動子於不同發育時期之組織活性分析 84 二、 乙烯對OgEIL1啟動子活性的影響 84 三、 激勃素對OgEIL1啟動子活性的影響 85 四、 水楊酸對OgEIL1啟動子活性的影響 85 五、 茉莉酸對OgEIL1啟動子活性的影響 85 六、 光照對OgEIL1啟動子活性的影響 86 七、 溫度對OgEIL1啟動子活性的影響 86 八、 創傷對OgEIL1啟動子活性的影響 87 九、 鹽逆境對OgEIL1啟動子活性的影響 87 陸、 結語 88 柒、 參考文獻 89 | |
dc.language.iso | zh-TW | |
dc.title | 文心蘭EIN3同源基因之選殖及啟動子活性分析 | zh_TW |
dc.title | Cloning and Promoter Activity Analysis of EIN3 Orthologs from Oncidesa Gower Ramsey | en |
dc.type | Thesis | |
dc.date.schoolyear | 103-2 | |
dc.description.degree | 碩士 | |
dc.contributor.coadvisor | 黃鵬林(Pung-Ling Huang) | |
dc.contributor.oralexamcommittee | 何錦玟(Chin-Wen Ho),李昆達(Kung-Ta Lee) | |
dc.subject.keyword | 文心蘭,乙烯,隱子,OgEILs,啟動子活性分析, | zh_TW |
dc.subject.keyword | Oncidesa spp.,ethylene,intron,OgEILs,promoter activity analysis, | en |
dc.relation.page | 93 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2015-08-17 | |
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 |
顯示於系所單位: | 園藝暨景觀學系 |
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ntu-104-1.pdf 目前未授權公開取用 | 9.87 MB | Adobe PDF |
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