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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 楊健志 | |
| dc.contributor.author | Shang-Hsuan Yeh | en |
| dc.contributor.author | 葉尚烜 | zh_TW |
| dc.date.accessioned | 2021-06-08T06:31:21Z | - |
| dc.date.copyright | 2006-07-28 | |
| dc.date.issued | 2003 | |
| dc.date.submitted | 2006-07-25 | |
| dc.identifier.citation | Altschul, S.F., Gish, W., Miller, W., Myers, E.W., and Lipman, D.J. (1990). Basic local alignment search tool. J Mol Biol 215, 403-410.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/25812 | - |
| dc.description.abstract | 我們在阿拉伯芥中發現四個與豬的 MAPR (membrane associated progesterone receptor conponent 1) 有 30~40% 相似度的同源性蛋白質，分別命名為 AtMAPR2、AtMAPR3、 AtMAPR4 與 AtMAPR5。在先前的研究指出 AtMAPR5 (putative membrane steroid binding protein, MSBP1) 可能為控制細胞延長之負調控因子，但對於其他 AtMAPRs 蛋白質的功能仍然缺乏全面性的了解。我們利用農桿菌基因轉殖的方式得到 AtMAPR4 基因靜默 (RNAi) 轉殖株及 AtMAPR4 基因過度表現 (overexoression) 轉殖株，由初步的性狀觀察發現 AtMAPR4 基因過度表現突變株在抽花序的時間有提早的現象；相反的 AtMAPR4 基因靜默突變株在抽花序的時間也有延遲的現象。由於先前我們也發現 AtMAPR4 基因在阿拉伯芥生長晚期，具有花的特異性表現，因此推測 AtMAPR4 可能與開花時間有關。另一方面，本實驗室先前的酵母菌雙雜合實驗結果顯示，AtMAPR5 可能與 RING-type 泛素接合酶 (At3g58030) 產生交互作用，且經序列比對後又發現另一個與其相似度高的 RING-type 泛素接合酶 (At2g42030)，因泛素接合酶具有辨識專一性目標蛋白質的功能，推測其可能與其他 AtMAPRs 蛋白質產生交互作用，因此我們將兩個 RING-type 泛素接合酶的基因選殖出來並在大腸桿菌中表現，作為將來進行與 AtMAPR 蛋白質交互作用實驗及 RING-type ubiquitin ligase 活性分析。 | zh_TW |
| dc.description.abstract | Four AtMAPRs (AtMAPR2, AtMAPR3, AtMPAR4, and AtMAPR5) in Arabidopsis sharing 30-40% similarity with porcine MAPR (membrane associated progesterone receptor component 1) have been identified. AtMAPR5 (putative membrane steroid binding protein, MSBP1) is proposed as a negative regulator of cell elongation (Yang et al., 2005). However, the functions of the other AtMAPRs are still unclear. By Agrobacterium-mediated transformation, we attempted to establish two transgenic plants where AtMAPR4 gene was either knocked-down by RNAi technology or overexpressed. Transgenic plants overexpressing AtMAPR4 showed that their bolting time was earlier than wild-type, whereas transgenic plants with knocked-down AtMAPR4 showed that they delayed flowering. We also found that AtMAPR4 was expressed higher in flowers by analyzing the tissue-specific expression in 35-day-old wild-type plants previously. It suggested that AtMAPR4 might be involved in the flowering. The second part of this research followed previous yeast two-hybrid assay experiment where AtMAPR5 might interact with RING-type ubiquitin ligase (At3g58030). Another RING-type ubiquitin ligase (At2g42030) shares high similarity with At3g58030. RING-type ubiquitin ligase could recognize target protein by specific domain. These two RING-type ubiquitin ligase were cloned and heterologously expressed in E.coli. They will be used in further pull-down experiment and ubiquitination assay. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T06:31:21Z (GMT). No. of bitstreams: 1 ntu-92-R93b47201-1.pdf: 2987063 bytes, checksum: 15e1df671364ae683f86fdfc6003211f (MD5) Previous issue date: 2003 | en |
| dc.description.tableofcontents | 目錄I
縮寫表IV 摘要VI AbstractVII 第一章 緒論 1.1 AtMAPRs 家族蛋白質之相關研究1 1.1.1 MAPRs 家族蛋白質1 1.1.2 AtMAPRs 家族蛋白質3 1.2 模式植物：阿拉伯芥 (Arabidopsis thaliana) 6 1.3 基因靜默 (RNA interference, RNAi) 簡介7 1.3.1 RNAi 的發展7 1.3.2 RNAi 的作用機制9 1.3.3 基因靜默載體介紹11 1.4 農桿菌 (Agrobacterium tumefaciens) 轉殖機制13 1.5 泛素-26S 蛋白酶體途徑 (Ub-26S proteasome pathway)15 1.5.1 泛素 (ubiquitin, Ub)15 1.5.2 泛素共價修飾酵素15 1.5.3 26S 蛋白酶18 1.5.4 去泛素化酶18 1.6 研究動機、目的與方向20 第二章 材料與方法 2.1 實驗材料21 2.1.1 植物材料21 2.1.2 載體21 2.1.3 大腸桿菌菌株22 2.1.4 農桿菌菌株23 2.2 實驗藥品23 2.2.1 一般化學試劑23 2.2.2 酵素23 2.2.3 培養基24 2.3 儀器設備26 2.4 實驗方法27 2.4.1 阿拉伯芥種植27 2.4.1.1 培養基之配製27 2.4.1.2 種子之表面消毒與低溫處理29 2.4.1.3 種子之無菌培養29 2.4.1.4 植株之土壤培養30 2.4.1.5 收集種子30 2.4.2 阿拉伯芥基因轉殖30 2.4.2.1 植物材料之準備31 2.4.2.2 農桿菌轉形31 2.4.2.3 農桿菌感染—花序浸潤法33 2.4.2.4 轉殖株的篩選34 2.4.2.5 染色體 DNA 之 PCR 檢測35 2.4.3 DNA之抽取與分析方法36 2.4.3.1 阿拉伯芥染色體 DNA 之抽取36 2.4.3.2 質體 DNA 之抽取與分析37 2.4.3.3 DNA 限制酶切割反應分析40 2.4.3.4 洋菜膠體電泳40 2.4.3.5 DNA 片段的分離及純化41 2.4.3.6 DNA 之磷酸化反應42 2.4.3.7 DNA 濃度估算42 2.4.4 RNA之抽取與表現量分析43 2.4.4.1 Total RNA 之抽取43 2.4.4.2 DNase 處理44 2.4.4.3 RNA 濃度估算44 2.4.4.4 RT-PCR46 2.4.5 RING-type ubiquitin ligase 基因序列選殖47 2.4.5.1 阿拉伯芥基因序列資料庫比對47 2.4.5.2 專一性引子的設計48 2.4.5.3 聚合酶鏈鎖反應（PCR）48 2.4.5.4 聚合酶鏈鎖反應產物純化 (PCR clean up)49 2.4.5.5 表現載體之建構49 2.4.5.6 大腸桿菌轉形50 2.4.6 重組蛋白質之表現與檢定53 2.4.6.1 重組蛋白質最佳 IPTG 誘導濃度、培養溫度之探討53 2.4.6.2 重組蛋白質之大量表現53 2.4.6.3 重組蛋白質之純化54 2.4.6.4 電泳檢定法54 2.4.7 蛋白質免疫轉印法57 2.4.7.1 蛋白質電泳轉印法57 2.4.7.2 酵素免疫染色法57 2.4.7.3 蛋白質 N 端定序…58 第三章 AtMAPRs 轉殖株之篩選 3.1 阿拉伯芥基因轉殖59 3.2 第一代轉殖株 (T1) 之篩選與培養60 3.2.1 染色體 DNA 之 PCR 檢測61 3.2.2 RNA表現量分析62 3.3 第二代轉殖株 (T2) 之篩選與培養65 3.4 第三代轉殖株 (T3) 之篩選與培養66 3.4.1 AtMAPR4 基因靜默與過度表現轉殖株之基因表現量分析67 3.4.2 AtMAPR4 基因靜默與過度表現轉殖株之培養與觀察68 3.4.3 光照對 AtMAPR4 的影響69 第四章 RING-type ubiquitin ligase 基因序列選殖 4.1 RING-type ubiquitin ligase 基因序列選殖71 4.1.1 阿拉伯芥基因序列資料庫比對71 4.1.2 RING2、RING3 基因序列選殖與表現載體的建構72 4.1.2.1 RING2、RING3 基因序列選殖72 4.1.2.2 表現載體的建構72 4.2 RING2 及 RING3 重組蛋白質之表現與檢定73 4.2.1 重組蛋白質之最佳表現條件之探討73 4.2.2 重組蛋白質之純化74 4.2.3 蛋白質定序75 第五章 結論與未來展望 5.1 結論76 5.2 未來展望76 參考文獻79 圖與表84 | |
| dc.language.iso | zh-TW | |
| dc.subject | 阿拉伯芥 | zh_TW |
| dc.subject | 農桿菌轉殖 | zh_TW |
| dc.subject | 基因靜默 | zh_TW |
| dc.subject | floral dip | en |
| dc.subject | RNAi | en |
| dc.subject | Arabidopsis | en |
| dc.subject | Agrobacterium | en |
| dc.title | 阿拉伯芥AtMAPRs基因靜默轉殖株之建構與性狀分析 | zh_TW |
| dc.title | Construction and analysis of transgenic Arabidopsis with AtMAPRs gene silencing vectors | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 94-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 蘇仲卿,常怡雍,邱子珍,王愛玉 | |
| dc.subject.keyword | 阿拉伯芥,基因靜默,農桿菌轉殖, | zh_TW |
| dc.subject.keyword | Arabidopsis,RNAi,Agrobacterium,floral dip, | en |
| dc.relation.page | 108 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2006-07-25 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 微生物與生化學研究所 | zh_TW |
| 顯示於系所單位： | 微生物學科所 | |
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