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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 王愛玉 | |
| dc.contributor.author | Sheng-Hsiung Yeh | en |
| dc.contributor.author | 葉勝雄 | zh_TW |
| dc.date.accessioned | 2021-06-08T05:10:29Z | - |
| dc.date.copyright | 2011-07-26 | |
| dc.date.issued | 2011 | |
| dc.date.submitted | 2011-07-12 | |
| dc.identifier.citation | Adams MD, Celniker SE, Holt RA, Evans CA, Gocayne JD, et al. (2000) The genome sequence of Drosophila melanogaster. Science 287: 2185-2195
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/23814 | - |
| dc.description.abstract | 竹為生長速度最快之陸生植物。為了解綠竹於快速生長時期之基因調控方式，本研究使用差異性表現分析法 (differential display)，從八種不同生長時期綠竹之 cDNA 樣品中，篩選出306條具差異性表現之DNA 片段，並一一加以定序。經過 blast 比對後，將結果以 Gene ontology (GO) 資料庫進行分類比較。利用 DNA Microarray 分析，確認差異性表現分析法之結果，並透過逐步叢集分析 (k-means clustering analysis），將 306 條 DNA 片段區分為六個分群。在推測具有轉錄調節功能之 ESTs 中，挑選出一條BohLOL1。序列分析顯示其包含三個具有 DNA 結合功能的 zinc finger 保守區域，與阿拉伯芥 LSD1 及 LOL1 (LSD-One-Like 1) 具同源性。Reverse northern 分析顯示 BohLOL1 在綠竹快速生長時表現量上升。Real-time RT-PCR 分析顯示無菌竹苗中之 BohLOL1，會因為 auxins 及 cytokinins 之作用、病菌感染、2,6-dichloroisonicotinic acid (salicylic acid 之類似物) 及hydrogen peroxide 之添加而提高表現量。細胞定位與 Protein-DNA 交互作用分析結果指出，BohLOL1 可能在細胞核中，藉由與目標基因結合之方式，發揮其在綠竹快速生長與遭受逆境時之作用。本實驗提出並證實BohLOL1.與綠竹生長之關聯及其結合 DNA 之特性，對於後續針對綠竹快速生長時期基因調控之研究方向，極具參考價值。 | zh_TW |
| dc.description.abstract | Growth of woody bamboo is generally rapid and faster than other woody plants. To investigate what genes are involved in controlling the growth of bamboo, differential display RT-PCR was performed to analyze the gene expression profiles of bamboo (Bambusa oldhamii). 306 cDNAs were isolated and sequenced. The results of BLAST analysis were classified with Gene Ontology (GO) database. DNA microarray was carried out to confirm the results of the differential display. Real-time RT-PCR was applied to exam the results of microarray. Among the 306 ESTs, A cDNA, BohLOL1, encoding a protein containing three zf-LSD1 (zinc finger- Lesions Simulating Disease resistance 1) domains was choose for futher study. A phylogenetic analysis revealed that BohLOL1 is a homolog of Arabidopsis LSD1 and LOL1 (LSD-One-Like 1). The BohLOL1 gene was upregulated in shoots with higher rates of culm elongation. The expression level of this gene in multiple shoots of bamboo, was also upregulated by auxins, cytokinins, pathogen infection, 2,6-dichloroisonicotinic acid (a functional analog of salicylic acid) and hydrogen peroxide. The results suggest that BohLOL1 participates in bamboo growth and in the response to biotic stress. The DNA-binding assays and subcellular localization studies demonstrated that BohLOL1 is a nuclear protein.and affects the expression of target genes through protein-DNA interactions The growth-dependent up-regulation of BohLOL1 expression, which uniquely occurs in growing bamboo, might be one of the critical factors that contribute to the rapid growth of this remarkable plant. | en |
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| dc.description.tableofcontents | 摘要 i
Abstract ii 縮寫表 iii 第一章 研究背景 1 1.1 前言 1 1.2 功能性基因體之研究 2 1.3 植物生理與基因調節 2 1.4 竹之簡介 3 1.5 本研究之目的與策略 5 1.5.1 研究工具之選擇: 5 1.5.1.1 Differential Display 6 1.5.1.2 DD結果之確認 6 1.5.2 目標基因的選擇與研究 7 1.5.2.1 LSD1簡介 7 1.5.3 Microarray 簡介 8 1.5.4 Microarray 種類 9 1.5.4.1 DNA microarray 9 1.5.4.2 Protein microarray (Bertone and Snyder, 2005; Hober and Uhlen, 2008) 9 1.5.4.3 Tissue microarray: (Kononen et al., 1998) 9 1.5.5 Microarray 實驗架構 10 1.5.5.1 標靶 (target) 與探針 (probe) 之製備 10 1.5.5.2 雜交反應與訊號讀取 10 1.5.5.3 資料處理與分析 10 第一部份 綠竹不同生長階段基因表現差異分析 12 A.1 實驗材料 12 A.1.1 生長中之綠竹 12 A.1.1.1 綠竹樣品分類： 12 A.1.1.2 綠竹樣品前處理： 12 A.1.2 無菌竹苗 12 A.1.4 質體 13 A.1.5 核酸引子 13 A.1.5.1 Oligo(dT) 引子 13 A.1.5.2 H-AP 引子 14 A.1.5.3 其他引子 15 A.2 實驗藥品與儀器 15 A.2.1 實驗藥品 15 A.2.2 實驗儀器 15 A.3 實驗方法 17 A.3.1 RNA之抽取、檢定與分析 17 A.3.1.1 RNA 之抽取 (Chiu et al., 2006) 17 A.3.1.2 DNase I處理 17 A.3.1.3 RNA之定量 18 A.3.1.4甲醛瓊脂糖膠體電泳 (Lehrach et al., 1977) 18 A.3.2 差異表現分析法 18 A.3.2.1 反轉錄反應 (Reverse transcription; RT) 19 A.3.2.2 以聚合酶連鎖反應 (polymerase chain reaction; PCR) 增殖 19 A.3.2.3 具放射性之DNA 標準分子量製備 20 A.3.2.4 變性聚丙烯醯胺膠體電泳 20 A.3.2.5 差異性表現 DNA 片段的選取、回收與增殖放大 21 A.3.2.6以TA cloning建立重組質體 22 A.3.2.7 重組質體之轉形、篩選與鑑定 (Hanahan, 1983) 23 (1) Competent cells 之製備一 (氯化鈣法) 23 (2) Competent cells 之製備二 (電轉形法) 23 (3) 轉形 (heat shock法，用於氯化鈣法製備之 competent cells) 23 (4) 轉形 (電轉形法) 24 (5) 轉形株質體快速篩選法 24 (6) 質體 DNA 小量抽取 (Sambrook, 2001) 25 (7) 以限制酶分析確認 25 (8) 菌種保存 25 A.3.3 差異片段序列分析 (blastx) 26 A.3.4 Gene ontology 分析 27 A.3.5 Microarray之操作 27 A.3.5.1 標靶之製備 27 A.3.5.2 探針之製備 28 A.3.5.3 資料分析 28 A.3.6 real-time RT-PCR分析 29 A.3.6.1 RNA抽取 29 A.3.6.2 DNase I處理 29 A.3.6.3 反轉錄反應 (Reverse transcription; RT) 29 A.3.6.4 PCR反應 29 二、結果與討論 31 A.4.1差異表現分析法之結果探討 31 A.4.1.1 RNA抽取與分析結果 31 A.4.1.2 cDNA片段差異情形之探討與選取 31 A.4.1.3差異片段之回收，二次增殖及選殖 32 A.4.2 差異片段之序列分析結果 33 A.4.2.1 blast分析 33 A.4.2.2 Gene ontology分析 33 A.4.3 Microarray 分析結果探討 35 A.4.3.1 Microarray 資料分析 35 A.4.3.2 以 real-time RT PCR 驗證 microarray 之結果 36 A.4.3.3 以逐步叢集分析法 (k-means clustering) 進行分群之結果 37 A.4.3.4 未出土竹筍與 100 公分幼竹基因表現之差異探討 39 A.4.4 綠竹快速生長時期具差異表現基因之功能探討 40 A.4.4 1表現差異基因與植物生長發育之關聯 40 A.4.4.2表現差異基因與植物荷爾蒙之關聯 40 A.4.4.3表現差異基因與生理逆境之關聯 41 A.4.4 4其他功能之表現差異基因 41 三、結論 42 四、未來展望 43 第二部份 綠竹轉錄因子 BohLOL1 之研究 44 B.1 實驗材料 44 B.1.4 質體 44 B.1.5 核酸引子 45 B.2 實驗藥品與儀器 45 B.3 實驗方法 46 B.3.1 Reverse northern分析 46 B.3.1.1 標靶之製備 46 B.3.1.2 探針之製備 46 B.3.1.3 雜合反應 (Ausubel and Gitler, 1987) 46 B.3.2 BohLOL1 序列分析、表現與純化 47 B.3.2.1 BohLOL1 序列分析 47 B.3.2.2 表現質體之構築 47 B.3.2.3 BohLOL1 表現與純化 48 B.3.2.4 蛋白質定量 (Bradford, 1976) 48 B.3.2.5 蛋白質電泳 (Schagger and Vonjagow, 1987) 49 B.3.2.6 Western 轉印分析 (Towbin et al., 1979) 49 B.3.3 BohLOL1 抗體之製備 50 B.3.3.1 抗原之製備 50 B.3.3.2 對小鼠之免疫 50 B.3.4 BohLOL1細胞定位分析 51 B.3.4.1 洋蔥表皮細胞短暫表現分析 51 B.3.4.1.1 轉殖材料準備 51 B.3.4.1.2 表現質體構築 51 B.3.4.1.3 鎢粒子處理 51 B.3.4.1.4 DNA包覆 52 B.3.4.1.5 基因槍操作 52 B.3.4.1.6短暫表現之觀察 52 B.3.4.2 綠竹原生質體短暫表現分析 52 B.3.4.2.1 表現質體構築 52 B.3.4.2.2 綠竹苗原生質體分離、轉形及結果觀察 53 B.3.4.3 綠竹細胞核蛋白質分離 (Cox and Emili, 2006) 53 B.3.5.1 竹苗之培養與繼代 (Lin et al., 2007) 54 B.3.5.2 以不同條件處理竹苗 54 B.3.5.3 以竹病原菌感染竹苗 54 B.3.6 Real time RT-PCR分析 55 B.3.7 BohLOL1 之 DNA 結合活性分析 55 B.3.7.1 染色體 DNA 之抽取及 DNA 片段製備 55 B.3.7.2 非專一性探針之製備 55 B.3.7.3 濾紙結合分析 (Filter Binding Assay) (Papoulas, 2001) 56 B.3.7.4 Southwestern 分析 (Siu et al., 2008) 56 二、結果與討論 57 B.4.1 Reverse Northern分析 57 B.4 2 BohLOL1 之基因表現與功能探討 57 B.4.2.1 綠竹 BohLOL1 序列分析 57 B.4.2.2 BohLOL1 之基因表現分析 58 B.4.2.2.1 BohLOL1在不同生長階段之綠竹中的表現 58 B.4.2.2.2 SAR inducer 對綠竹苗中 BohLOL1 表現之影響 58 B.4.2.2.3 接種竹類病原菌對綠竹苗中 BohLOL1 表現之影響 59 B.4.2.2.4 氧化逆境對綠竹苗中 BohLOL1 表現之影響 59 B.4.2.2.5 外加植物荷爾蒙對綠竹苗中 BohLOL1 表現之影響 60 B.4.2.2.6 培養溫度對綠竹苗中BohLOL1表現之影響 60 B.4.3 重組 BohLOL1 之表現與純化 61 B.4.4 BohLOL1 多株抗體之製備 61 B.4.5 BoSD1細胞定位分析 61 B.4.5.1 綠竹細胞核蛋白質分離 61 B.4.5.2 BohLOL1在洋蔥表皮細胞中之表現 62 B.4.5.3 BohLOL1 在綠竹苗原生質體中之表現 62 B.4.6 BohLOL1 與 DNA 之交互作用 63 三、結論 64 四、未來展望 65 1. Protein-protein interactions 之研究 65 2. Protein-DNA interaction 之研究 65 Reference 65 | |
| dc.language.iso | zh-TW | |
| dc.title | 在綠竹生長過程中具差異性表現基因之檢定 | zh_TW |
| dc.title | Identification of genes differentially expressed during the growth of Bambusa oldhamii | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 99-2 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 宋賢一,曾志正,張珍田,楊健志,蔡孟勳 | |
| dc.subject.keyword | 竹生長發育,綠竹,抗病性,細胞核定位,植物轉錄因子,鋅指蛋白質, | zh_TW |
| dc.subject.keyword | LSD-One-Like 1,zf-LSD1 domain,bamboo growth,disease resistance,DNA-binding protein,nuclear localization, | en |
| dc.relation.page | 136 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2011-07-13 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科技學系 | zh_TW |
| 顯示於系所單位： | 生化科技學系 | |
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