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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 常玉強 | |
| dc.contributor.author | Hsiu-Chun Yang | en |
| dc.contributor.author | 楊琇淳 | zh_TW |
| dc.date.accessioned | 2021-06-15T16:16:47Z | - |
| dc.date.available | 2019-09-17 | |
| dc.date.copyright | 2015-09-17 | |
| dc.date.issued | 2015 | |
| dc.date.submitted | 2015-08-17 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/52505 | - |
| dc.description.abstract | 轉位子釣取系統 (transposon tagging) 是利用轉位子 (transposable elements) 創造插入型突變 (insertional mutation) 藉以研究基因功能的技術之一。此系統最大的潛力在於轉位子於生殖細胞轉位 (germinal transposition) 相較於體細胞轉位 (somatic transposition) 可產生較多獨立的插入型突變體。先前本實驗室使用來自玉米Ac / Ds (Activator-Dissociator) 轉位系統，利用水楊酸誘導啟動子PR-1a (Pathogenesis related-1a) 控制Ac轉位酶表現，並將轉位子構築於轉位酶內隱子 (intron)，建構了水楊酸誘導單次轉位系統 (COKC)。而本研究誘導COKC之轉殖水稻生殖細胞轉位，進而配合花藥培養，於單一世代生產同質結合突變體 (homozygous mutants)。該系統分別處理以下兩種水楊酸誘導方式：一、先誘導再分離花藥進行培養。二、同時誘導及花藥培養。前者轉位效率為5%，且皆為同質結合突變體；後者效率為20%，但之中90%為異質結合突變體 (heterozygous mutants)，推測轉位子於花藥培養誘導生殖細胞胚體化 (embryogenesis) 的同時，發生體細胞轉位。為了改善生殖細胞轉位效率，本研究利用水稻花粉不同發育時期之專一表現啟動子 (pollen-specific promoters) OsP1、OsP41和OsP128，建構生殖細胞轉位系統OsP1-0380PL、OsP41-0380PL和OsP128-0380PL，進行水稻及菸草轉殖而後同樣進行花藥培養。綜合上述研究成果，藉由水楊酸誘導系統COKC及生殖細胞轉位系統0380PL，可提升生殖細胞轉位效率，並於單一世代得到穩定的同質結合突變體。 | zh_TW |
| dc.description.abstract | Transposon tagging is an effective tool of insertional mutagenesis for studying gene function. A higher number of independent insertion mutants can be obtained from a few transgenic launch pads via germinal transposition. Our lab previously developed a one-time inducible Ac/Ds transposable system, COKC, where the Ds element in the intron of the Ac transposase is driven by the salicylic acid inducible promoter PR-1a (Pathogenesis related-1a). In order to increase germinal transposition and generate homozygous mutants, we treated floral tissues at uninucleate stages of microspore in COKC transgenic rice with salicylic acid in two salicilylic acid treatments. The first approach was pot induction, where rice panicle was induced in the pot prior to anther culture. The other approach was culture induction, where the rice anther was isolated first and then simultaneously induced and incubated. The transposition efficiency of anther-derived regeneration shoots from the two sets were 5 % and 20 %, respectively. For pot induction, all of the regeneration calli were homozygotes. In contrast, for culture induction, most of the regeneration calli were heterozygotes, which may result from somatic transposition after embryogenesis of the callus. In order to increase germinal transposition, the present study aims to construct three novel germinal transposon tagging systems OsP1-0380PL, OsP41-0380PL and OsP128-0380PL. In these systems, the Ac transposase is driven by each of the three pollen specific promoters identified in rice cultivar TNG67. The above tagging constructs were transformed into rice and tobacco through Agrobacterium. After that, the transformants were subjected to anther culture to observe the frequency of transposition in gametophytic process, where more homozygotes could be derived. In conclusion, the aim of this thesis is appling to improve germinal transposition to create stable homozygous mutants in a single generation in plants. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T16:16:47Z (GMT). No. of bitstreams: 1 ntu-104-D99621101-1.pdf: 3671324 bytes, checksum: c5587ccf83d20b8e67d36fdacbfd28ef (MD5) Previous issue date: 2015 | en |
| dc.description.tableofcontents | 口試委員會審定書 I
致謝 II 中文摘要 III 英文摘要 IV 第一章 前言 1 第二章 文獻回顧 2 2.1 植物轉位子 2 2.1.1 Ac/Ds 轉位子基因結構 3 2.1.2 Ac/Ds 轉位機制及插入序列特性 3 2.1.3 Ac/Ds轉位子插入型突變系統 4 2.2 建構轉位子插入型突變系統 5 2.2.1 雙構築及單一構築系統 6 2.2.2 可誘導單次轉位系統 7 2.2.3 生殖細胞專一轉位系統 8 2.2.4 轉位子之反劑量效應 10 2.3 花藥培養生產同質結合突變體 11 2.3.1 生產同質結合突變體 11 2.3.2 花藥培養產生單倍體或雙單倍體 12 2.3.3 應用花藥或花粉培養生產同質結合突變體 15 第三章 試驗架構 16 第四章 材料與方法 17 4.1 水楊酸誘導單次轉位系統 17 4.1.1 轉位子插入突變水稻起始系 17 4.1.2 水稻花藥培養 17 4.1.3 水楊酸誘導 18 4.1.4 花藥再生水稻突變系DNA萃取 18 4.1.5 Multiplex PCR評估花藥分化植株轉位 18 4.1.6 南方墨點法評估起始系為單一拷貝 18 4.1.7 Thermal asymmetric interlaced PCR分析插入突變系側翼序列 20 4.1.8. Multiplex PCR評估插入突變系為同質結合突變體 21 4.2 鑑別水稻生殖細胞專一啟動子 22 4.2.1 篩選水稻花粉專一表現基因 22 4.2.2 構築三基因啟動子片段於GUS表現系統 22 4.2.3 農桿菌轉型 23 4.2.4 水稻及菸草轉殖 23 4.2.5 PCR檢測轉殖株 25 4.2.6 花藥GUS染色及花粉發育時期評估 25 4.3 生殖細胞專一轉位系統 25 4.3.1 構築生殖細胞轉位系統 25 4.3.2 農桿菌轉型 26 4.3.3 水稻及菸草轉殖 26 4.3.4 PCR確定轉殖株及評估無自發轉位 26 4.3.5 南方墨點法判斷起始系為單一拷貝 26 4.3.6 水稻和菸草花藥培養 26 4.3.7 Multiplex PCR評估花藥分化植株轉位 27 第五章 結果 28 5.1 水楊酸誘導轉位系統 28 5.1.1 水楊酸誘導COKC轉位系統 28 5.1.2 COKC系統轉位評估 29 5.1.3 Multiplex PCR評估插入突變系為同質結合突變體 29 5.2 鑑別水稻生殖細胞專一性啟動子 31 5.2.1 候選基因具生殖細胞專一表現 31 5.2.2 轉殖水稻、菸草GUS蛋白表現評估 33 5.3 生殖細胞專一轉位系統 35 5.3.1 0380PL構築 35 5.3.2 生產轉位系統起始系 36 5.3.3 生殖細胞轉位系統進行花藥培養 37 第六章 討論 39 6.1 細胞專一性轉位 39 6.2 突變系特性 40 6.3 花藥培養生產同質結合突變體效率 41 6.4 水稻生殖細胞轉錄體 42 6.5 啟動子調控序列 43 第七章 引用文獻 46 圖目錄 圖一 水稻小孢子與花藥培養再分化植株 57 圖二 Multiplex PCR評估COKC轉殖株SA誘導花藥分化植株轉位 58 圖三 南方墨點法分析COKC轉殖株經水楊酸誘導花藥再分化植株轉位59 圖四 Multiplex PCR評估花藥分化植株插入突變體之同質性 60 圖五 RT-PCR 評估水稻三基因表現專一性 61 圖六 水稻花粉專一表現啟動子構築流程 62 圖七 OsP1啟動子序列及雄性生殖細胞專一性順式作用元件 63 圖八 OsP41啟動子序列及雄性生殖細胞專一性順式作用元件 64 圖九 OsP128啟動子序列及雄性生殖細胞專一性順式作用元件 65 圖十 三候選花粉專一性啟動子於轉殖水稻花藥GUS表現 66 圖十一 三候選花粉專一性啟動子於轉殖菸草花藥GUS表現 67 圖十二 三候選花粉專一性啟動子於水稻花粉不同核型發育期GUS表現 68 圖十三 三候選花粉專一性啟動子於菸草花粉不同核型發育期GUS表現 69 圖十四 OsP41啟動子系列刪除轉殖水稻花器GUS表現 70 圖十五 生殖細胞專一轉位系統0380PL構築流程 71 圖十六 PCR及multiplex PCR評估0380PL水稻轉殖株 72 圖十七 PCR及multiplex PCR篩選0380PL菸草轉殖株 73 圖十八 0380PL轉殖菸草花藥培養分化植株 74 圖十九 COKC轉殖水稻突變系定序結果 75 圖二十 三花粉專一啟動子相關順式作用元件分佈示意圖 76 表目錄 表一 轉殖水稻COKC插入突變系之側翼序列及專一性引子資訊 77 表二 花粉專一表現基因啟動子片段資訊 78 表三 花粉專一啟動子共同順式作用元件 79 表四 花粉專一啟動子雄性生殖細胞相關順式作用元件 80 表五 0380PL水稻及菸草轉殖系之單一拷貝起始系資訊 81 表六 系列刪除啟動子0380PL轉位系統轉殖系資訊 82 附錄目錄 附錄一 玉米Ac/Ds轉位子簡介 83 附錄二 Ac轉位子 轉位足跡形成機制 84 附錄三 雙構築及單一構築之轉位子插入型突變系統 85 附錄四 可誘導之轉位子插入型突變系統 86 附錄五 轉位子反劑量效應 87 附錄六 Ac/Ds單一構築轉位系統 88 附錄七 可誘導單次轉位系統COKC 89 附錄八 花粉專一表現基因於水稻花粉發育及各組織轉錄體分析結果90 附錄九 關鍵字對照及註釋 91 附錄十 本論文所使用引子一覽表 92 附錄十一 本論文所使用之培養基配方 94 附錄十二 本論文所使用之藥品來源 98 | |
| dc.language.iso | zh-TW | |
| dc.subject | 異質結合突變體 | zh_TW |
| dc.subject | 花粉專一表現啟動子 | zh_TW |
| dc.subject | Ac/Ds轉位子 | zh_TW |
| dc.subject | PR-1a啟動子 | zh_TW |
| dc.subject | 體細胞轉位 | zh_TW |
| dc.subject | 生殖細胞轉位 | zh_TW |
| dc.subject | 轉位子釣取系統 | zh_TW |
| dc.subject | 插入型突變 | zh_TW |
| dc.subject | 花藥培養 | zh_TW |
| dc.subject | 同質結合突變體 | zh_TW |
| dc.subject | germinal transposition | en |
| dc.subject | somatic transposition | en |
| dc.subject | pathogenesis related-1a | en |
| dc.subject | pollen-specific promoters | en |
| dc.subject | one-time inducible Ac/Ds transposable system | en |
| dc.subject | homozygous mutants | en |
| dc.subject | germinal transposon tagging systems | en |
| dc.subject | anther culture | en |
| dc.title | 構築生殖轉位系統生產同質結合突變體 | zh_TW |
| dc.title | Construction of transposon tagging system in germinal cells for creating homozygous mutants | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 103-2 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 杜鎮,洪傳揚,蔡育彰,黃永芬 | |
| dc.subject.keyword | 轉位子釣取系統,插入型突變,生殖細胞轉位,體細胞轉位,Ac/Ds轉位子,PR-1a啟動子,花藥培養,同質結合突變體,異質結合突變體,花粉專一表現啟動子, | zh_TW |
| dc.subject.keyword | anther culture,germinal transposition,germinal transposon tagging systems,homozygous mutants,one-time inducible Ac/Ds transposable system,pollen-specific promoters,pathogenesis related-1a,somatic transposition, | en |
| dc.relation.page | 98 | |
| 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 | 農藝學研究所 | zh_TW |
| 顯示於系所單位： | 農藝學系 | |
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