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研究生: 洪彧丞
Hung, Yu-Cheng
論文名稱: 利用染色質免疫沉澱測序(ChIP-seq)實驗數據建立植物轉錄因子結合位置資料庫
Construction of a database for transcription factor binding sites identified by plant chromatin immunoprecipitation sequencing (ChIP-seq) experiments
指導教授: 張文綺
Chang, Wen-Chi
學位類別: 碩士
Master
系所名稱: 生物科學與科技學院 - 熱帶植物科學研究所
Institute of Tropical Plant Sciences
論文出版年: 2018
畢業學年度: 106
語文別: 中文
論文頁數: 74
中文關鍵詞: 轉錄因子轉錄因子結合位置基因調控染色質免疫沉澱測序PCBase
外文關鍵詞: TFs, TFBSs, gene regulatory mechanism, ChIP-seq, PCBase
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  • 植物在不同的生長發育階段或遭遇逆境時,都需要調節相關基因的表現量來幫助自身適應不同的環境。其中最重要的調控方式就是利用轉錄因子(transcription factors)結合到啟動子(promoter)上的近端調控因子(cis-elements)來控制下游基因的表現。因此,若想了解其中複雜的調控關係,研究轉錄因子和基因組(genome)上的結合位置 (transcription factor binding sites,簡稱TFBSs)顯然是一個相當重要的工作。近年來各種高通量技術蓬勃發展,其中染色質免疫沉澱測序(chromatin immunoprecipitation sequencing,簡稱ChIP-seq)被廣泛應用於分析整個基因組上可能的轉錄因子結合位置或尋找會被某個轉錄因子所調控的基因群。所以,若能將這些實驗資料進行全面且條理性的分析統整,勢必更能了解轉錄因子與基因之間的調控關係。到目前為止,只有3個公開資料庫有提供植物ChIP-seq實驗資料,分別為PCSD、ChIPBase v2.0和Expresso。但ChIPBase v2.0和Expresso分別只收錄來自阿拉伯芥(Arabidopsis thaliana)的26和20種轉錄因子。此外,PCSD也只提供關於阿拉伯芥、水稻(Oryza sativa)和玉米(Zea mays)三種植物的轉錄因子和其調控基因的基本資訊,使用者無法直接分析任意啟動子序列上是否存在實驗驗證的TFBSs。基於以上原因,本研究主要的目的為建立一個整合多種植物物種之ChIP-seq資料庫。我們由Gene Expression Omnibus (GEO)和Sequence Read Archive (SRA)中蒐集了阿拉伯芥、水稻、玉米、大豆(Glycine max)、番茄(Solanum lycopersicum)、棉花(Gossypium hirsutum)和深山南芥(Arabidopsis lyrata)共7種植物之ChIP-seq實驗資料,涵蓋662個samples和99種蛋白質(包含轉錄因子、組織蛋白和其他DNA結合蛋白)。以上所有資料經由系統性的流程處理,並由AQUAS及MACS2分析,最終獲得4,574,337筆蛋白質結合位置以及1,233,999筆蛋白質與基因之結合關係資訊。最後建立一個名為Plant ChIP-seq Database(簡稱PCBase)的資料庫以方便植物學家快速了解基因組DNA上多種蛋白質的結合位置。PCBase提供基因搜尋(Gene Search),蛋白質搜尋(Protein Search),啟動子分析(Promoter Analysis),基因組瀏覽(Genome Browser)及資料下載(Download)等五大功能來全面分析植物染色質狀態。使用者除了能輕易獲得轉錄因子和其轉錄因子結合位置座標及調控基因外,所有由ChIP-seq實驗資料得來的轉錄因子結合位置權重(TF binding position weight matrices)皆可用於分析任一段啟動子序列。我們希望PCBase可以幫助植物領域研究人員在研究植物的轉錄調控機制時更加便利。目前PCBase可由以下網址免費取得:http://pcbase.itps.ncku.edu.tw/

    Transcription factors (TFs) can bind to cis-elements on promoters and regulate gene expression during plant development and stresses responsiveness. Therefore, investigation of TFs and their binding sites (TFBSs) is important to understand the gene regulatory mechanisms. Recently, a high-throughput technology, chromatin immunoprecipitation sequencing (ChIP-seq), was applied to rapidly explore the binding sequences on a genome and regulatory genes of a TF in many studies. Consequently, the collection and analysis of ChIP-seq experimental data will be useful to further identify the relationships among TFs, TFBSs, and TF-targeted genes. Until now, only three databases provide ChIP-seq data from plants, those are PCSD (including Arabidopsis thaliana, Oryza sativa (rice), Zea mays (maize)), ChIPBase v2.0 and Expresso (including Arabidopsis thaliana), respectively. However, only 26 and 20 TFs can be accessed from ChIPBase v2.0 and Expresso, individually. Additionally, PCSD only provides the information of TFs and their target-genes; the experimental TF binding motifs can’t be applied in promoter analysis. Based on the above, the aim of this study is to construct a comprehensive plant ChIP-seq database. We collected ChIP-seq experimental data from seven plants including Arabidopsis thaliana, Oryza sativa (rice), Zea mays (maize), Glycine max (soybean), Solanum lycopersicum (tomato), Gossypium hirsutum (cotton) and Arabidopsis lyrata. The number of samples obtained from Gene Expression Omnibus (GEO) and Sequence Read Archive (SRA) are 662, which contain 99 proteins (TF/Histone/DNA binding protein). AQUAS and MACS2 programs systematically processed all of the raw data to identify the protein binding sites. Finally, we constructed a database named Plant ChIP-seq Database (PCBase) to comprehensively analyze plant chromatin status. There are five main functions in PCBase, such as Gene Search, Protein Search, Promoter Analysis, Genome Browser, and Download. The target genes and TFBS coordinates of a TF can be effectively retrieved. All experimental TF binding matrices can be utilized in any input promoter sequence. We hope this database can help scientists to study the transcriptional regulatory mechanisms in plants. This database is freely available at: http://pcbase.itps.ncku.edu.tw/

    目錄 考試合格證明 # 中英文摘要 I 誌謝 XII 目錄 XIII 表目錄 XV 圖目錄 XVI 一、 研究背景 1 1-1 植物研究的重要性 1 1-2 轉錄因子在調控基因表達中扮演重要角色 1 1-3 高通量技術廣泛應用於偵測轉錄因子結合位置 4 1-4 現存ChIP-seq實驗數據相關資料庫 5 1-5 研究目的 6 二、 資料蒐集與分析方法 8 2-1 彙整植物ChIP-seq實驗數據資料 8 2-2 ChIP-seq原始實驗數據處理及分析 9 2-3 整合基因註釋(ANNOTATION)資訊 11 2-4 蛋白質與調控基因分析 12 2-5 蛋白質與結合位置序列分析 13 2-6 啟動子分析 13 2-7 基因組瀏覽建立 15 三、 結果與討論 16 3-1 PCBase資料庫之建構策略及規劃 16 3-2 PCBase資料庫之五大主要分析功能 17 3-2-1 基因搜尋(Gene Search) 17 3-2-2 蛋白質搜尋(Protein Search) 18 3-2-3 啟動子分析(Promoter Analysis) 20 3-2-4 基因組瀏覽(Genome Browser) 21 3-2-5 資料下載(Download) 22 3-3 案例分析:以植物轉錄因子WRKY33驗證PCBase串聯分析功能 23 四、 結論 25 五、 參考文獻 27   表目錄 表一、目前ChIP-seq相關公開資料庫收錄物種比較 38 表二、PCBase所收錄之dataset與sample數量 39 表三、PCBase所收錄之轉錄因子、組織蛋白與其他DNA結合蛋白統計表 40 表四、PCBase所分析出所有蛋白質在基因組上之結合位點數量 41 表五、基因註釋來源資料庫版本與基因及轉錄體個數統計表 42 表六、蛋白質與基因之結合關係數量統計(已去除重複) 43 表七、由MEME及DREME分析所得之轉錄因子結合陣列及結合位置序列標誌數量 44 表八、PCBase與其他收錄植物ChIP-seq實驗資料之資料庫比較表 45   圖目錄 圖一、PCBase資料庫之系統架構圖 46 圖二、ChIP-seq實驗資料分析流程圖 47 圖三、PCBase網站首頁 48 圖四、PCBase物種選擇 49 圖五、基因搜尋(Gene Search)功能查詢 50 圖六、PSK5基因基礎資訊 51 圖七、PSK5基因功能分類(Gene Ontology) 52 圖八、PSK5調控關係資訊 53 圖九、PSK5基因受轉錄因子ERF115調控之實驗資訊 54 圖十、轉錄因子SEP3基本資訊 55 圖十一、轉錄因子SEP3調控資訊 56 圖十二、轉錄因子SEP3結合位置序列標誌 57 圖十三、轉錄因子SEP3與花朵發育重要基因SOC1之調控關係 58 圖十四、以視覺化圖像呈現轉錄因子SEP3與花朵發育重要基因SOC1之結合關係 59 圖十五、轉錄因子SEP3之偏好結合區域統計圖 60 圖十六、以Peak Browser呈現SEP3與AP1在SOC1 (AT2G45660.1)上的結合情形 61 圖十七、SEP2啟動子序列分析結果(一) 62 圖十八、SEP2啟動子序列分析結果(二) 63 圖十九、於Genome Browser檢視EDE1 (AT2G44190.1)基因 64 圖二十、資料下載(以阿拉伯芥轉錄因子為例) 65 圖二十一、PAD3基因基礎資訊 66 圖二十二、PAD3基因功能描述 67 圖二十三、PAD3基因之調控蛋白列表 68 圖二十四、PAD3基因與WRKY33轉錄因子之結合關係 69 圖二十五、WRKY33轉錄因子之調控基因群亦包含PAD3基因 70 圖二十六、WRKY33轉錄因子與PAD3基因結合關係 71 圖二十七、PAD3啟動子序列經Promoter Analysis分析結果(一) 72 圖二十八、PAD3啟動子序列經Promoter Analysis分析結果(二) 73 圖二十九、Genome Browser顯示不同實驗條件下WRKY33與PAD3的結合情形 74

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