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DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 杜宜殷(Yi-Yin Do) | |
dc.contributor.author | Chun-Lin Chen | en |
dc.contributor.author | 陳君琳 | zh_TW |
dc.date.accessioned | 2021-06-16T17:41:03Z | - |
dc.date.available | 2017-08-27 | |
dc.date.copyright | 2012-08-27 | |
dc.date.issued | 2012 | |
dc.date.submitted | 2012-08-14 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/64329 | - |
dc.description.abstract | 為瞭解苦瓜 (Momordica charantia L.) 質脂結合蛋白 (plastid lipid-associated protein) 之基因功能,本研究利用菸草 (Nicotiana tabacum L.) 作為轉殖材料,以分析苦瓜質脂結合蛋白基因 McPAP1 之啟動子活性及功能分析,結果顯示 McPAP1 啟動子活性於菸草轉殖株幼苗時期全株表現,而開花時期則專一性表現於生殖器官。誘導試驗顯示 McPAP1 啟動子於菸草轉殖株幼苗地上部與地下部具有顯著差異,於地上部可受到 BA、ABA、ACC 和 SA 以及創傷和 37℃ 黑暗誘導,受 GA 抑制活性表現;啟動子活性表現於地下部則受到 IAA、MeJA、高溫黑暗和乾旱誘導,受 BA 和 SA 抑制活性表現。過量表現 McPAP1 之菸草轉殖株具有植株高、花苞發育時間短、開花成熟期一致、平均節間長、葉基部大花朵數目多,且於乾旱和高光低溫環境耐受性較未轉殖株高等外表型,並且提高類胡蘿蔔素(carotenoid) 之貯存,但與類胡蘿蔔素生合成相關基因表現無關;綜合以上結果顯示,McPAP1 可能藉由穩定質粒結構,進而累積類胡蘿蔔素含量及穩定花器形成,由此推測 McPAP1 於營養生長階段與植株生長發育相關,而於生殖生長階段與花器發育具相關性。另外,本研究進一步建立胡瓜癒傷組織之再生及轉殖系統,以農桿菌媒介法將過量表現、默化表現以及蛋白質定位等構築轉殖至黃色鬆散之胡瓜癒傷組織,結果顯示以濃度 OD600 1.0 之農桿菌菌液、添加 100 μM AS、經超音波震盪 30 秒、進行感染 12 小時、共培養 48 小時之處理具較高轉殖率 94.80%。經由添加 0.1 mg•L-1 NAA、2 mg•L-1 Kinetin 以及 200 mg•L-1 kanamycin 之 MS 培養基同時進行篩選與再生,待轉殖細胞發生不定芽以獲得轉殖株。 | zh_TW |
dc.description.abstract | To understand the function of plastid lipid-associated protein gene McPAP1 from bitter gourd, promoter activity and overexpression in tobacco transformants were analyzed. McPAP1pro::GUS transgenic tobacco showed GUS activity in the whole plant during seedling development, and specifically expressed in flower. Results of plant growth regulator and stress treatments indicated that the differential expression of McPAP1 between upper-ground part and roots. BA, ABA, ACC, SA, wounding and 37℃/dark induced McPAP1 transcription in the upper-ground, but part of seedling GA reduced the gene expression . IAA, MeJA, drought and 37℃/dark induced McPAP1 transcription in the roots, but BA and SA reduced the gene expression. McPAP1-overexpressed tobacco showed longer stem, earlier flowering, shorter maturity period of flowers, longer average internode, wider leaf base, more flower number, and more tolerance under drought or 180 μmol•m-2/8℃. Transgenic tobacco contained higher content of carotenoids, but gene expression of carotenoid-related showed no difference with the untransformed plant. McPAP1 probably accumulates carotenoids and assists flower maturation by stabilizing plastid structure. It might be involved in plant vegetative growth and flower organ differentiation. Moreover, plasmids for overexpression, silencing and protein localization were introduced into cucumber callus by Agrobacterium-mediated transformation. Using Agrobacterium cells whose concentration at 1.0 OD600 and supplementing 100 μM AS. The highest transformation efficiencies 94.80 % were obtained when calli subjected to a combination of sonication for 30 seconds followed by 12 hours of infection, and 48 hrs of cocultivation. After transformation, cucumber calli were cultured on MS medium supplemented with 0.1 mg•L-1 NAA, 2 mg•L-1 Kinetin, and 200 mg•L-1 kanamycin for selection and regeneration via adventitious shoots to whole plant. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T17:41:03Z (GMT). No. of bitstreams: 1 ntu-101-R99628137-1.pdf: 2979161 bytes, checksum: a0502c853ba3467b7d33da859b5a5851 (MD5) Previous issue date: 2012 | en |
dc.description.tableofcontents | 壹、 前言………………………………………………………………………....1
貳、 前人研究…………………………………………………………………....3 一、 質粒之類型…………………………………………………………...3 二、 質粒小球之介紹……………………………………………………...3 (一) 質粒小球之脂質成分……………………………………………..4 (二) 質粒小球之蛋白成分……………………………………………..4 (三) 脂肪體與結合蛋白之共同特色…………………………………..4 (四) 質粒小球之功能…………………………………………………..5 三、 植物質脂結合蛋白之介紹…………………………………………..6 (一) 質脂結合蛋白之基本特性……………………………………….6 (二) 植物質脂結合蛋白基因之表現………………………………….7 (三) 植物質脂結合蛋白之功能分析………………………………….9 四、 苦瓜質脂結合蛋白之介紹………………………………………….12 (一) 苦瓜性別相關之蛋白質………………………………………….12 (二) 苦瓜質脂結合蛋白 cDNA 之選殖……………………………..12 (三) 苦瓜 McPAP1 與花性相關蛋白之比較………………………..12 (四) 苦瓜質脂結合蛋白之功能分析…………………………………13 五、 胡瓜之轉殖及再生系統…………………………………………….14 (一) 基因型和培植體來源……………………………………………14 (二) Vir 基因之誘導………………………………………………….14 (三) 篩選系統…………………………………………………………17 (四) 再生效率…………………………………………………………17 參、 材料與方法………………………………………………………………..18 一、 試驗材料…………………………………………………………….18 (一) 質體材料………………………………………………………...18 (二) 試驗用菌種………………………………………………………18 (三) 植物材料…………………………………………………………18 二、 試驗方法…………………………………………………………….20 (一) 轉殖株之啟動子活性分析………………………………………20 (二) 即時定量聚合酶連鎖反應 (Real-time quantitative polymerase chain reaction, real-time PCR)…………………………………….21 (三) 色素含量分析……………………………………………………22 (四) 逆境試驗…………………………………………………………22 (五) 胡瓜之基因轉殖…………………………………………………22 肆、 結果……………………………………………..…………………………26 一、 McPAP1pro:GUS 菸草轉殖株之啟動子活性分析………...……..26 (一) 不同發育階段與部位之 McPAP1 啟動子活性分析..…………26 (二) 不同植物生長調節劑與非生物逆境於McPAP1 啟動子活性分析……………..………………………………………………………26 二、 過量表現 McPAP1 之菸草轉殖株………………………………..35 (一) McPAP1基因表現……………………………………………….35 (二) 轉殖株之外表型態………………………………………………35 (三) 類胡蘿蔔素含量及生合成路徑基因分析………………………35 (四) 逆境試驗…………………………………………………………42 三、 建立胡瓜轉殖及再生系統………………………………………….42 (一) 癒傷組織誘導培養基試驗………………………………………42 (二) 芽體誘導培養基試驗……………………………………………47 (三) 芽體伸長及發根培養基試驗……………………………………47 (四) 癒傷組織之天然抗性測試………………………………………47 (五) 菌液濃度和感染時間對轉殖效率之影響………………………52 (六) 超音波震盪輔助農桿菌轉殖法…………………………………52 伍、 討論……………………………………………. …………………………57 一、 苦瓜質脂結合蛋白之啟動子活性……..…………………………..57 (一) 生長發育階段與部位……………………………………………57 (二) 植物生長調節劑之調控……………………………..…………..57 (三) 非生物逆境因子之調控…………………………………..……..59 二、 苦瓜質脂結合蛋白之基因功能…………………………………….59 (一) 過量表現 McPAP1 對菸草轉殖株之外表型態與發育之影響..59 (二) 類胡蘿蔔素含量及生合成路徑基因之表現……………………60 (三) 逆境試驗…………………………………………………………60 三、 胡瓜癒傷組織之再生及轉殖系統建立…………………………….61 (一) 胡瓜癒傷組織經多芽體途徑進行再生…………………………61 (二) 應用農桿菌媒介法於胡瓜癒傷組織轉殖………………………63 陸、 結語……………………………………………………………........…......66 柒、 參考文獻…………………………………………………………………..68 | |
dc.language.iso | zh-TW | |
dc.title | 苦瓜質脂結合蛋白基因 McPAP1 之功能分析及胡瓜基因轉殖系統之建立 | zh_TW |
dc.title | Functional Analysis of Plastid-lipid-associated Protein Gene McPAP1 from Bitter Gourd and Establishment of Cucumber Transformation System | en |
dc.type | Thesis | |
dc.date.schoolyear | 100-2 | |
dc.description.degree | 碩士 | |
dc.contributor.coadvisor | 黃鵬林(Pung-Ling Huang) | |
dc.contributor.oralexamcommittee | 何錦玟(Chin-Wen Ho),劉祖惠(Tsu-Hwie Liu) | |
dc.subject.keyword | 基因過量表現,類胡蘿蔔素,花器形成,基因轉殖,不定芽誘導, | zh_TW |
dc.subject.keyword | gene overexpression,carotenoids,flower development,transfotmation,adventitious shoots induction, | en |
dc.relation.page | 74 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2012-08-15 | |
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 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-101-1.pdf 目前未授權公開取用 | 2.91 MB | Adobe PDF |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。