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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/58708完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 黃鵬林(Pung-Ling Huang) | |
| dc.contributor.author | Tzu-Fan Hsiang | en |
| dc.contributor.author | 向子帆 | zh_TW |
| dc.date.accessioned | 2021-06-16T08:26:48Z | - |
| dc.date.available | 2019-03-18 | |
| dc.date.copyright | 2014-03-18 | |
| dc.date.issued | 2014 | |
| dc.date.submitted | 2014-01-20 | |
| dc.identifier.citation | PartI
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/58708 | - |
| dc.description.abstract | 第一部分 香蕉果實後熟相關基因MhDnaJ
香蕉 (Musa spp.) 為世界重要經濟果樹之一，了解其果實後熟生理有助改善櫥架壽命。MhDnaJ基因自香蕉第三級後熟果實之cDNA庫篩選而得，其胺基酸序列屬type III之J-protein，利用即時定量反轉錄聚合酶連鎖反應 (real-time reverse transcription polymerase chain reaction) 檢測北蕉 (Musa `Pei Chiao') 不同組織內屬type III J-protein之MhDnaJ基因表現，結果顯示後熟果實基因表現量最高，推測此基因與香蕉果實後熟相關。基因過量表現及默化MhDnaJ基因之轉殖阿拉伯芥及菸草經聚合酶連鎖反應分析及南方氏雜交分析，證明目標序列已插入植物之基因組中。分析過量表現MhDnaJ之轉殖菸草對於鹽類逆境之抗性，結果顯示CaMV 35Spro:MhDnaJ-1轉殖系於高鹽環境下具有較佳的耐受性。藉分析前人研究有關香蕉後熟相關轉錄體資料，選取10個後熟相關之J-protein基因，進行未轉殖北蕉、及默化ACC oxidase (MhACO1和MhACO2) 之轉殖株果實基因表現量分析，以進一步探討J-protein與香蕉果實後熟之相關性。 第二部分、苦瓜鈣離子依賴型核酸分解酶基因McCAN1 McCAN1基因選殖自苦瓜開花後16天果實所製備之cDNA庫，其胺基酸序列與鈣離子依賴型核酸分解酶 (Ca2+-dependent nuclease) 具同源性，本研究利用大腸桿菌表達McCAN1重組蛋白，並利用His•Bind Resin (Novagen Co.)進行純化，經檢測酵素活性結果顯示，McCAN1於37℃分解基因組DNA之活性最佳，而70℃高溫下仍有活性，且在pH5~7環境下具有最佳活性。為了分析McCAN1之基因功能，將過量表現及基因默化質體轉殖至阿拉伯芥 (Arabidopsis thaliana ecotype Columbia) 及菸草 (Nicotiana tacacum L. cv. W38)，且已獲得轉殖植物。同時本研究亦利用花藥特異性之文心蘭金魚草素合成酶基因 (Oncidium ‘Gower Ramsey’ aureusidin synthase, OgAS1) 啟動子於菸草表達McCAN1 之cDNA，以期獲得雄不稔 (male-sterile) 性狀，經聚合酶連鎖反應 (polymerase chain reaction) 分析及南方氏雜交分析 (Southern hybridization analysis)，證明目標片段已整合入植物基因組中，且完成拷貝數檢測。 | zh_TW |
| dc.description.abstract | PartI
Banana (Musa spp.) is one of the most important economic fruits, and the elucidation of fruit ripening has been an important issue for improving the shelf life. The MhDnaJ gene was isolated from a cDNA library constructed from the ripening fruit at stage 3. Gene expression of MhDnaJ, classified to type III J-protein, in different tissues of banana was analyzed by real-time reverse transcription PCR. MhDnaJ is proposed to be involved in the regulation of fruit ripening because its mRNA was most abundant in ripening fruit. Both overexpressing- and silencing- MhDnaJ transgenic Arabidopsis and tobacco plants have been identified. To analyze the resistant of salt tolerance for overexpression MhDnaJ transgenic tobacco, the results show CaMV 35Spro: MhDnaJ-1 transgenic line at high salt environment with better tolerability. To explore the relationship of J-protein with banana fruit ripening, MhDnaJ and ten more J-protein genes were chosen for gene expression analysis in wild type and ACC oxidase gene silenced transgenic fruits based on previous transcriptome analysis. PartII McCAN1 gene was cloned from the cDNA libray of the bitter gourd fruit of 16 days after flowering and genomic library. In our study, McCAN1 recombinant protein was expressed in E. coli to examine the enzyme acitivity. The results showed that McCAN1 best activity at 37 ℃, still active at 70 ℃ high temperature, and in the pH 5 ~ 7 environment with optimal activity. Both overexpression and silencing plasmids of McCAN1 were transformed separately to Arabidopsis thaliana ecotype Columbia and Nicotiana tacacum L. cv. W38 for analysis of gene function, and have obtained transgenic plants. Furthermore, McCAN1 cDNA driven by the anther-specific promoter of Oncidium `Gower Ramsey' aureusidin synthase gene (OgAS1) was transformed to generate the posible male-sterile tobacco plants. Polymerase chain reaction and Southern analysis have been demonstrated the target fragments were integrated into the plant genome and copy numbers of transformants have been identified. | en |
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| dc.description.tableofcontents | 第一部分 香蕉果實後熟相關基因MhDnaJ 1
中文摘要 1 Abstract 2 壹、前人研究 3 一、 熱休克蛋白 (Heat shock proteins, Hsps) 3 二、 J-protein家族之分類 3 三、 J-protein之結構 4 (一) J-domain 4 (二) glycine/phenylalanine rich region (G/F region) 5 (三) Zinc-finger domain 5 (四) C-terminal domain 6 四、Hsp70s:J-proteins之作用機制 6 五、J-protein之相關功能性研究 7 (一) 逆境之耐受性 7 (二) 開花之調控 7 (三) 熱敏感配子型之雄不稔性 8 (四) 與病毒移動蛋白之交互作用 8 (五) 細胞死亡及抗病性 9 (六) 植物之光感知 9 六、香蕉轉錄體之後熟相關J-protein 基因表現 10 貳、材料與方法 11 一、試驗材料 11 (一)菌種材料 11 (二)植物材料 11 (三)質體材料 11 二、試驗方法 13 (一) 質體之構築 13 (二) 阿拉伯芥之穩定性轉殖及轉殖株之篩選 15 (三) 菸草葉圓片轉殖 22 (四) GUS活性組織化學染色分析 23 (五) 總RNA之抽取 23 (七) 即時定量聚合酶鏈鎖反應 (Real-time quantitative polymerase chain reaction, real-time PCR) 25 參、結果 27 一、MhDnaJ過量表現之轉殖系分析 27 (一) 阿拉伯芥轉殖株之分析 27 (二) MhDnaJ過量表現轉殖菸草之鹽類逆境試驗 27 二、MhDnaJ基因默化之轉殖系分析 28 (一) 阿拉伯芥轉殖株之分離率計算 28 (二) 阿拉伯芥擬轉殖株之鑑定 28 三、香蕉各組織部位MhDnaJ之基因表現分析 28 四、MhDnaJ及10種香蕉後熟相關J-protein之胺基酸保守性區域分析 29 肆、討論 39 一、MhDnaJ基因之表現特性 39 二、MhDnaJ對於逆境之效應 39 三、比較分析香蕉10種後熟相關J-protein之基因表現 40 伍、結語 43 陸、附錄 44 柒、參考文獻 47 第二部分、苦瓜鈣離子依賴型核酸分解酶基因McCAN1 52 中文摘要 52 Abstract 53 壹、前人研究 54 一、Ca2+ dependent nuclease (CAN)之相關研究 54 (一) 胡瓜 (Cucumis sativus L.) 雌花雄蕊之發育 54 (二) 影響植物之老化 55 (三) 木本植物次生木質部 (secondary xylem)之發育 56 二、雄不稔性狀之策略 56 (一) 傳統細胞核雄不稔品種之選育 57 (二) 利用GMS相關分子標誌輔助作物細胞核雄不稔性之育種 57 (三) 利用花器特異性啟動子驅動核酸分解酶相關基因 57 三、花器特異性啟動子之相關應用 58 (一)利用絨氈層特異性啟動子表達核酸分解酶基因以導致雄不稔性 58 (二) 利用胚珠特異性啟動子驅動iaaM基因以導致單為結果 58 貳、材料與方法 59 一、試驗材料 59 (一)菌種材料 59 (二)植物材料 59 (三)質體材料 59 二、試驗方法 68 參、結果 82 一、McCAN1胺基酸親緣性比對及特徵性區域分析 82 二、利用大腸桿菌表達及純化McCAN1重組蛋白及酵素活性試驗 82 (一)誘導條件測試 82 (二)McCAN1重組蛋白之純化 83 (三)McCAN1酵素活性檢測 83 三、轉殖McCAN1蛋白質定位質體於阿拉伯芥 84 四、McCAN1過量表現之轉殖株分析 84 (一)阿拉伯芥轉殖系之分析 84 (二)菸草轉殖系之分析 84 五、McCAN1基因默化之轉殖株分析 85 (一)阿拉伯芥轉殖系之分析 85 (二)菸草轉殖株之分析 85 六、花藥特異性啟動子驅動McCAN1基因 (OgAS1pro:McCAN1) 之轉植株分析 85 (一)菸草轉殖株之鑑定 85 (二)菸草轉殖系之基因表現分析 86 (三) FDA花粉活力檢測 86 (四)阿拉伯芥之穩定性轉殖 86 肆、討論 109 一、McCAN1之胺基酸序列分析 109 二、McCAN1與CsCaN之基因表現及啟動子相關特徵性區域分析 109 三、McCAN1之酵素活性 110 四、McCAN1過量表現之轉殖系分析 110 五、花藥特異性啟動子驅動McCAN1 (OgAS1:McCAN1) 之穩定性基因轉殖 111 伍、結語 117 陸、附錄 118 柒、參考文獻 119 | |
| dc.language.iso | zh-TW | |
| dc.subject | 香蕉 | zh_TW |
| dc.subject | 基因過量表現 | zh_TW |
| dc.subject | 基因默化 | zh_TW |
| dc.subject | 轉錄體分析 | zh_TW |
| dc.subject | 苦瓜 | zh_TW |
| dc.subject | 花藥特異性啟動子 | zh_TW |
| dc.subject | 雄不稔性 | zh_TW |
| dc.subject | male-sterility | en |
| dc.subject | Banana | en |
| dc.subject | gene overexpression | en |
| dc.subject | gene silencing | en |
| dc.subject | transcriptome analysis | en |
| dc.subject | bitter gourd | en |
| dc.title | 香蕉果實後熟相關基因MhDnaJ及苦瓜鈣離子依賴型核酸分解酶基因McCAN1之研究 | zh_TW |
| dc.title | Studies on the Fruit Ripening-Associated Gene MhDnaJ from Banana and Calcium Dependent Nuclease Gene McCAN1 from Bitter Gourd | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 102-1 | |
| dc.description.degree | 碩士 | |
| dc.contributor.coadvisor | 杜宜殷(Yi-Yin Do) | |
| dc.contributor.oralexamcommittee | 何國傑(Kuo-Chieh Ho),洪傳揚(Chwan-Yang Hong) | |
| dc.subject.keyword | 香蕉,基因過量表現,基因默化,轉錄體分析,苦瓜,花藥特異性啟動子,雄不稔性, | zh_TW |
| dc.subject.keyword | Banana,gene overexpression,gene silencing,transcriptome analysis,bitter gourd,male-sterility, | en |
| dc.relation.page | 123 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2014-01-20 | |
| 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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