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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 楊健志(Chien-Chih Yang) | |
| dc.contributor.author | Chia-Chia Yeh | en |
| dc.contributor.author | 葉佳佳 | zh_TW |
| dc.date.accessioned | 2021-06-08T05:58:41Z | - |
| dc.date.copyright | 2007-08-28 | |
| dc.date.issued | 2007 | |
| dc.date.submitted | 2007-08-10 | |
| dc.identifier.citation | 李平篤 (1970) 綠竹筍中可溶性 RNA 的細胞分裂素活性 國立臺灣大學農業化學研究所碩士論文.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/24936 | - |
| dc.description.abstract | 植物細胞分裂素的訊息經由具二元傳遞系統特徵的細胞分裂素受體傳導。此訊息傳遞方式首先於原核生物中發現。受到環境改變的刺激後，細胞分裂素受體啟動一連串的磷酸根轉移調控下游基因表現。阿拉伯芥中已找到三種細胞分裂素受體，AHK2、AHK3 和 CRE1/AHK4，三者序列高度相似，且功能上具重複性 (Redundancy)。
綠竹具有生長快速的特性。我們想知道此特性是否與細胞分裂素受體有關。本研究進行綠竹筍細胞分裂素受體 cDNA 之選殖與分析。利用非放射線標定細胞分裂素受體上具保守性的CHASE domain作為探針，對王愛玉教授實驗室提供之綠竹筍cDNA庫進行篩選。選殖出一段與阿拉伯芥細胞分裂素受體相似之 cDNA，命名為BCRE1。 BCRE1 全長 3479 鹼基對，可轉譯為 961 胺基酸，經序列比對後發現 BCRE1缺乏 5’ UTR及前端一百個胺基酸序列。利用5' RACE 進行 5' 端序列延長，但仍未得到完整的5' 端序列。BCRE1 所轉譯出的蛋白質包含三個疏水性穿膜區域、一個CHASE區塊、一個組胺酸激酶區塊及一個磷酸根接收區塊，其胺基酸序列與水稻及玉米已知的組胺酸激酶有80% 以上的同質性。將此基因的不同區塊分別以 pET16b 為表現載體轉殖入大腸桿菌 BL21 (DE3)表現重組蛋白質，找到誘導基因高表現量的最適條件，可進行後續結構與功能之研究。 | zh_TW |
| dc.description.abstract | The signal of cytokinins is mediated by cytokinin receptors which have two component system characteristics. This system was first discovered in prokaryotes.Sensing the environmental changes, cytokinin receptors initiate a serial phosphorelay which regulates downstream gene expression. Three cytokinin receptors, AHK2, AHK3 and CRE1/AHK4, have been found in Arabidopsis thaliana. There are high sequence homology and functional redundancy.
Bamboo is characterized by it fast growth. We were interested if this feature was related to cytokinin receptors. The purpose of this study was to isolate and characterize cytokinin receptor homologs from cDNA library of Bambusa oldhamii. The probe was designed and amplified based on the conserved CHASE domain. Five independent clones were detected by DIG-labeled probe from a sub cDNA library (5*104) provided by Dr. A. Y. Wang’s lab. One cDNA fragment homologous to cytokinin receptor of Arabidopsis thaliana was cloned and named as BCRE1. There are 3479 base pairs in BCRE1 gene which could be translated to 961 amino acids. By sequence alignment, BCRE1 lacked 5' UTR and the sequence encoding the first 100 amino acid compared to Arabidopsis thaliana. Elongation of BCRE1 by 5’ RACE was unsuccessful. The protein encoded by BCRE1 included one CHASE domain, flanked by two transmembrane domain and one transmembrane domain on its two ends, respectively. one histidine kinase domain, and one receiver domain. The primary structure of CRE1/ AHK4 from bamboo, rice and corn shared at least 80 % identity. To investigate the structure-function relationship of BCRE1, the histidine kinase (HK) and RR (response regulator) domains were cloned and expressed as recombinant proteins using E coli BL21(DE3). Overexpression were observed for both construction which can be used for further structural study. | en |
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| dc.description.tableofcontents | 目錄 …………………………………………………………………………………….I
摘要 ………………………………………………………………………………….IV Abstract ……………………………………………………………………………… V 縮寫表 ……………………………………………………………………………VI 第一章 緒論 1.1 細胞分裂素簡介………………………………………………………………. 1 1.1.1 細胞分裂素……………………………………………………………….1 1.1.2 細胞分裂素的代謝與合成……………………………………………….3 1.1.3 細胞分裂素對植物細胞產生的影響…………………………………….5 1.2 細胞分裂素受體 (cytokinin receptor)的簡介與訊息傳遞 …………………6 1.2.1 細胞分裂素受體簡介……………………………………………………7 1.2.2 二元傳遞系統…………………………………………………………10 1.3 綠竹 (Bambusa oldhamii)之簡介與特性 …………………………………14 1.4 研究動機與目的 ……………………………………………………………15 第二章 材料與方法 2.1 實驗材料………………………………………………………………………16 2.1.1 cDNA 庫的來源………………………………………………………16 2.1.2 載體 (vectors) …………………………………………………………16 2.1.3 大腸桿菌 (E coli) ………………………………………………………17 2.2 實驗藥品………………………………………………………………………17 2.3 實驗儀器………………………………………………………………………18 2.4 實驗方法………………………………………………………………………20 2.4.1 DNA之純化與分析 ……………………………………………………20 2.4.1.1目標基因放大 ………………………………………………………20 2.4.1.1.1 聚合脢鏈鎖反應 ………………………………………………20 2.4.1.1.2 A-tailing…………………………………………………………20 2.4.1.1.3 接合反應 ………………………………………………………21 2.4.1.1.4 質體 DNA 轉形……………………………………………… 21 2.4.1.1.4.1 勝任細胞 (competent cell) 之製備……………………21 2.4.1.1.4.2 質體轉形…………………………………………………22 2.4.1.1.4.3 藍白篩選法………………………………………………22 2.4.1.1.4.4 菌種保存…………………………………………………22 2.4.1.2 質體 DNA 之抽取 …………………………………………………23 2.4.1.2.1 小量純化法 ………………………………………………23 2.4.1.2.2 中量純化法 ………………………………………………23 2.4.1.3 DNA 片段之純化……………………………………………………24 2.4.1.4 DNA 洋菜膠體電泳分析 …………………………………………25 2.4.2 RNA 之純化與分析……………………………………………………25 2.4.2.1 綠竹之total RNA 抽取 ……………………………………………25 2.4.2.2 甲醛洋菜膠體電泳 …………………………………………………26 2.4.2.3 反轉錄脢-聚合脢鏈鎖反應(RT-PCR)………………………………27 2.4.3 cDNA庫篩選 ……………………………………………………………28 2.4.3.1 DIG 探針製備 ………………………………………………………28 2.4.3.2 價數 (Titer) 測定及cDNA庫之放大………………………………29 2.4.3.2.1噬菌體價數側定………………………………………………...29 2.4.3.2.2 cDNA庫之放大 ……………………………………………….29 2.4.3.3 cDNA library之轉印 ………………………………………………30 2.4.3.4 雜合反應 ……………………………………………………………30 2.4.3.5 正反應株 DNA 片段之選殖 ………………………………………31 2.4.3.5.1 挑選正反應噬菌體 ……………………………………………31 2.4.3.5.2 噬菌體胞內剪切 (in vivo excision) …………………………32 2.4.4 cDNA 的 5’ 序列之延長 ………………………………………………32 2.4.4.1 DNase 的前處理……………………………………………………32 2.4.4.2 5’ 序列的延長 ………………………………………………………32 2.4.5重組蛋白質之建構、抽取及電泳分析 …………………………………33 2.4.5.1重組蛋白質載體之建構 ……………………………………………33 2.4.5.2最佳蛋白質表現條件之探討 ………………………………………34 2.4.5.3 SDS 膠體電泳………………………………………………………35 2.4.5.4蛋白質染色 …………………………………………………………36 2.4.5.5蛋白質電泳轉印及酵素免疫染色法 ………………………………36 第三章 結果與討論 3.1 綠竹細胞分裂素受體 cDNA 之選殖………………………………………38 3.1.1 綠竹筍細胞分裂素受體 cDNA序列之選取 …………………………38 3.1.2以反轉錄聚合脢鏈鎖反應的產物設計探針……………………………40 3.1.3 BCRE1的cDNA 序列分析……………………………………………41 3.1.4 BCRE1 蛋白質一級結構分析 ………………………………………41 3.1.4.1 功能區塊預測 ……………………………………………………41 3.1.4.2 演化樹分析 ………………………………………………………42 3.2 BCRE1的重組蛋白質之表現 ………………………………………………43 3.2.1 BCRE1 表現載體之建構 ……………………………………………43 3.2.2 最適蛋白質誘導條件之測試 …………………………………………44 3.3 BCRE1 的 cDNA 5'序列的延長 ……………………………………………44 第四章 結論與展望 ………………………………………………………………47 參考文獻 …………………………………………………………………………50 圖與表 …………………………………………………………………………60 | |
| dc.language.iso | zh-TW | |
| dc.subject | 細胞分裂素 | zh_TW |
| dc.subject | 綠竹筍 | zh_TW |
| dc.subject | Bamboo | en |
| dc.subject | cytokinin | en |
| dc.title | 綠竹筍細胞分裂素受體cDNA之選殖與表現 | zh_TW |
| dc.title | Cloning and Expression of cDNA Encoding Cytokinin Receptor from Shoots of Bamboo (Bambusa oldhamii) | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 95-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 蘇仲卿,李平篤,陳佩燁 | |
| dc.subject.keyword | 綠竹筍,細胞分裂素, | zh_TW |
| dc.subject.keyword | Bamboo,cytokinin, | en |
| dc.relation.page | 82 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2007-08-10 | |
| 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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