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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 杜宜殷(Yi-Yin Do) | |
| dc.contributor.author | Yi-Ling Fan | en |
| dc.contributor.author | 范藝齡 | zh_TW |
| dc.date.accessioned | 2021-06-08T04:15:17Z | - |
| dc.date.copyright | 2010-08-18 | |
| dc.date.issued | 2010 | |
| dc.date.submitted | 2010-08-09 | |
| dc.identifier.citation | 李冠德. 2005. 可誘導性轉位子單次移除篩選標記基因之研究. 國立台灣大
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/22308 | - |
| dc.description.abstract | 基因轉殖作物中抗生素抗性基因與目標基因同時存在的情況下，可能造成人類對生態和食品安全的疑慮，可選擇非抗生素抗性基因作為篩選標誌，並於轉殖後去除轉殖植株內的篩選標誌來消除疑慮。突變的烯醇丙酮莽草酸磷酸合成酶 (5-enolpyruvylshikimate 3-phosphate synthase; EPSPS) 基因產物對殺草劑嘉磷塞 (Glyphosate) 具抗性，由於嘉磷塞對於環境不具影響且對動物是低毒性的，故本研究以聚合酶連鎖反應 (Polymerase chain reaction)，從轉殖水稻中選殖出突變之 EPSPS 基因，連接 CaMV 35S作為啟動子，構築為轉殖質體。以蝴蝶蘭癒傷組織作為材料進行轉殖，建立以 EPSPS 基因為篩選標誌的蝴蝶蘭轉殖系統。轉殖前測試蝴蝶蘭癒傷組織對嘉磷塞天然耐受性，結果顯示 1 mM 為合適篩選濃度。試驗中以嘉磷塞篩選蝴蝶蘭轉殖細胞，得到存活的細胞在 GUS組織化學活性分下，均具有活性表現，顯示利用嘉磷塞作為蝴蝶蘭癒傷組織的篩選試劑是可行的，且篩選效率較 G418 高約 4 倍。另外，本研究採取轉位子 (transpostion system) 系統去除篩選標誌之策略，轉殖細胞或植株經水楊酸誘導後，將可移除篩選標誌基因。經由聚合酶連鎖反應分析，已確認蝴蝶蘭轉殖細胞及菸草轉殖株。再以 5 mM 水楊酸進行轉位誘導後；觀察到轉位作用的發生。 | zh_TW |
| dc.description.abstract | The presence of antibiotic resistant genes in genetically engineered crops together with the target gene has generated a number of environmental and consumer concerns. use of the non-antibiotic selectable marker, or elimination of marker gene from transgenic plants are suitable strategies to reduce the concerns. Plants with mutated 5-enolpyruvyl shikimate 3-phosphate synthase (EPSPS) gene are resisted to the herbicide glyphosate, which has low toxicity and non-impact on environment and animals. In order to establish a non-antibiotic selection system for Phalaenopsis transformation in this research, the modified EPSPS gene was cloned by polymerase chain reaction (PCR) from transgenic rice, ligated with CaMV 35S promoter, constructed into vector. and transformed into Phalaenopsis calli. The nature tolerance of Phalaenopsis calli against glyphosate is at the concentration of 1 mM. After transformation, the Phalaenopsis calli were selected by glyphosate and the putative transgenic calli got positive results in GUS histochemical analysis. The selection efficiency was four folds higher than G418. It suggested that glyphosate is efficient to be a selective reagent for transformed Phalaenopsis calli. Meanwhile, transposon was used as a strategy to remove selection marker. Transgenesis of Phalaenops cells and tobacco plants were performed by PCR. Transposition was confirmed after transgenic calli treated with 5 mM salicylic acid for elimination of the transposon, via PCR with suitable primers. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T04:15:17Z (GMT). No. of bitstreams: 1 ntu-99-R96628142-1.pdf: 2197755 bytes, checksum: 489ca21ab2864c6b5d0e3ccc39d59bee (MD5) Previous issue date: 2010 | en |
| dc.description.tableofcontents | 內容目錄
論文口試委員審定書............................ii 謝誌.......................................................iii 中文摘要..................................................iv 英文摘要..................................................v 壹、前言..................................................1 貳、前人研究..............................................2 一、EPSPS 與嘉磷塞 (Glyphosate) 之特性...............2 二、EPSPS 對嘉磷塞之抗性機制.........................3 三、利用 EPSPS 基因作為篩選標誌基因..................4 四、去除篩選標誌基因之轉殖策略.......................5 參、材料與方法............................................9 一、試驗材料.........................................9 二、試驗方法........................................10 (一) 轉殖水稻之突變 EPSPS 基因選殖及轉殖載體之構築.10 (二) 蝴蝶蘭及菸草之基因轉殖........................16 (三) 蝴蝶蘭擬轉殖細胞及菸草轉殖株之分析............19 肆、結果.................................................33 一、蝴蝶蘭癒傷組織對嘉磷塞天然抗性試驗..............33 二、蝴蝶蘭擬轉殖細胞之 GUS 化學活性組織染色結果.....33 三、聚合酶連鎖反應分析擬轉殖細胞及轉殖株............34 四、比較 G418 及嘉磷塞對蝴蝶蘭癒傷組織之篩選效率....34 五、不同轉位子末端序列之辨識率......................34 六、處理水楊酸誘導轉位作用產生......................46 伍、討論.................................................54 一、以突變 EPSPS 基因作為蝴蝶蘭基因轉殖之篩選標誌基因...54 二、蝴蝶蘭擬轉殖細胞及菸草轉殖株之分析..............55 三、以水楊酸誘導轉位子轉位..........................55 陸、結語.................................................58 參考文獻.................................................59 圖目錄 圖一、中間質粒 pBIE1-3 構築流程圖........................23 圖二、轉殖質粒 pGES1-8 構築流程圖........................24 圖三、誘導性轉位子系統之構築示意圖.......................25 圖四、pGET 質體之構築....................................27 圖五、pGEnT 質體之構築...................................29 圖六、pBI-cES 質體之構築.................................30 圖七、pGcET 質體之構築...................................31 圖八、pGNUT 質體之構築...................................34 圖九、蝴蝶蘭癒傷組織於不同濃度嘉磷塞培養基處理一個月後生長情形..............37 圖十、蝴蝶蘭癒傷組織轉殖 pGET 之 GUS 活性分析............38 圖十一、蝴蝶蘭癒傷組織轉殖 pGEnT 之 GUS 活性分析.........39 圖十二、蝴蝶蘭癒傷組織轉殖 pGcET 之 GUS 活性分析.........40 圖十三、蝴蝶蘭癒傷組織轉殖 pGNUT 之 GUS 活性分析.........41 圖十四、轉殖 pGET 蝴蝶蘭癒傷組織之聚合酶連鎖反應分析.....42 圖十五、轉殖 pGEnT 蝴蝶蘭癒傷組織及菸草之聚合酶連鎖反應分析..................43 圖十六、轉殖 pGcET 蝴蝶蘭癒傷組織及菸草之聚合酶連鎖反應分析..................44 圖十七、轉殖 pGNUT 蝴蝶蘭癒傷組織之聚合酶連鎖反應分析....45 圖十八、轉殖 pGcET 蝴蝶蘭癒傷組織於 G418 與嘉磷塞經篩選六個月後之情形...............................................47 圖十九、跳躍子系統之轉位模式圖...........................50 圖二十、以 PCR 檢測轉殖蝴蝶蘭癒傷組織之轉位情形..........51 圖二十一、轉殖質體 pGcET 轉位後剩餘之 T-DNA 內序列定序結果..51 圖二十二、轉殖 pGcET 菸草植株之南方氏雜交分析……………..52 圖二十三、轉殖 pGcET 菸草植株之南方氏雜交分析……………..53 表目錄 表一、質體構築及分析所使用之引子………………………………..13 表二、比較 G418 與嘉磷塞對蝴蝶蘭轉殖細胞之篩選效率……….44 | |
| dc.language.iso | zh-TW | |
| dc.subject | 蝴蝶蘭 | zh_TW |
| dc.subject | 去除篩選標誌 | zh_TW |
| dc.subject | 嘉磷塞 | zh_TW |
| dc.subject | 烯醇丙酮莽草酸磷酸合成酶 | zh_TW |
| dc.subject | 轉位子 | zh_TW |
| dc.subject | 篩選標誌基因 | zh_TW |
| dc.subject | selectable marker | en |
| dc.subject | Phalaenopsis. | en |
| dc.subject | transposon | en |
| dc.subject | 5-enolpyruvyl- shikimate-3-phosphate synthase | en |
| dc.subject | glyphosate | en |
| dc.subject | selectable marker free | en |
| dc.title | 應用轉位子去除篩選標誌系統於蝴蝶蘭基因轉殖 | zh_TW |
| dc.title | Application of Transposon System for Selectable Marker Elimination in Transgenic Phalaenopsis | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 98-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.coadvisor | 黃鵬林(Pung-Ling Huang) | |
| dc.contributor.oralexamcommittee | 廖芳心(Fang-Shin Liao),常玉強(Yuh-Chyang Charng) | |
| dc.subject.keyword | 篩選標誌基因,去除篩選標誌,嘉磷塞,烯醇丙酮莽草酸磷酸合成酶,轉位子,蝴蝶蘭, | zh_TW |
| dc.subject.keyword | selectable marker,selectable marker free,glyphosate,5-enolpyruvyl- shikimate-3-phosphate synthase, transposon,Phalaenopsis., | en |
| dc.relation.page | 64 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2010-08-09 | |
| 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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