博碩士論文 107821003 詳細資訊



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姓名 黃乙統(Yi-Tung Huang)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 以重組桿狀病毒表達系統建構果蠅Zelda基因 及其交互作用分子之篩選
(Construction of Drosophila Zelda using baculovirus system and screening for proteins interacting with Zelda)
檔案 [Endnote RIS 格式]    [Bibtex 格式]    [相關文章]   [文章引用]   [完整記錄]   [館藏目錄]   至系統瀏覽論文 (2027-9-16以後開放)
摘要(中) 在所有的動物中，初期的胚胎發育是由母源物質（maternal components）所支持與調控，此時期合子基因的表現是靜默的，直到特定的時間，合子基因表現被啟動（zygotic genome activation, ZGA），並接管了接下來的發育，這個轉換的過程叫做maternal to zygotic transition (MZT)。以黑腹果蠅（Drosophila melanogaster）為例，在MZT時期約有二波的ZGA，先後有五百多個及兩千多個基因被啟動，而轉錄因子Zelda則在此過程扮演了極重要的角色。目前已知Zelda的作用方式包括直接調控下游基因的轉錄（strong activator）及作為pioneer factor調控染色質的開放性，促進其他轉錄因子與基因體的結合，然而目前對於Zelda蛋白的功能性結構尚未完全釐清。此外，目前尚無直接證據顯示Zelda與轉錄起始複合體或表觀遺傳的調控子有直接作用，或藉由輔助因子（co-factors）交互作用。
為了近一步了解Zelda的分子機制，本研究包含了兩個部分：1. 純化全長Zelda蛋白：我們利用桿狀病毒表現系統（Bac-to-Bac），在昆蟲細胞異源表達6xHis tagged Zelda蛋白，並藉由Ni-NTA beads純化Zelda蛋白，目前已能穩定的生產與純化足量的Zelda蛋白，期在未來得以Cryo-EM分析Zelda的結構。2. 鑑識Zelda交互因子：我們萃取早期胚胎全蛋白與純化後的Zelda蛋白進行免疫共沉澱（Co-IP），再經質譜儀分析（mass spectrometry），最後得到134個候選蛋白，其中包括了polymerase subunits及histone deacetylase相關蛋白，未來會先針對與基因表現或發育相關之基因，進行生化與遺傳實驗，探討這些基因是否與Zelda作用機制有關。
摘要(英) In all animals, the early embryonic development is first controlled by the maternal components, while the zygotic gene expression is silent. Later, zygotic genes are activated and take over the control for the following development. This process is called maternal to zygotic transition (MZT). In Drosophila, during MZT, about 500 genes are activated initially follow by the activation of 2000 genes. In 2008, a maternal transcription factor, Zelda was reported as the key activator of these early zygotic genes (ZGA). It has been shown to be not only a strong activator but also an epigenetic regulator which opens chromatin during MZT. However, the structure and molecular function of Zelda is still not clear. Especially, there is no evidence showing that Zelda could directly interact with transcription machinery or histone regulators or through co-factors.
To better understand the mechanism of Zelda, this study included two parts. First, we ectopically expressed 6xHis-tagged Zelda in insect cells using Bac-to-Bac system and purified 6xHis-Zelda through Ni-NTA beads. So far, we are able to produce and purify sufficient and robust Zelda protein. We hope to further perform Cryo-EM analysis on Zelda to understand its structure and potential functional domains. Second, we carried co-immunoprecipitation of total protein extracts from early embryos with Zelda protein and identified 134 potential candidates interacting with Zelda. Among them includes proteins related to polymerase subunits and histone deacetylase. In the future, biochemical and genetic studies will be conducted to determine their interaction with Zelda, especially for those genes related to transcription and development.
關鍵字(中) ★ 共免疫沉澱
★ 桿狀病毒		 關鍵字(英) ★ Zelda
論文目次 中文摘要 i
Abstract iii
誌謝 iv
目錄 v
中英文對照表 ix
一、前言 1
1.1果蠅胚胎早期發育 1
1.2 Zelda是果蠅胚胎早期發育之重要因子 2
1.2.1 Zelda透過TAG sites啟動胚胎合子基因組 2
1.2.2 Zelda以多途徑調控基因表現 3
1.2.3 轉錄活化子（transcriptional activator）： 3
1.2.4 促進其他轉錄因子與基因體的結合： 4
1.2.5 降低核小體障礙： 4
1.2.6 Zelda的結構與功能性 5
1.3 Zelda的結構與功能性預測分析 7
1.3.1 Alpha Fold2 7
1.3.2 Cryo-EM（Cryo-electron microscopy） 8
1.4與Zelda交互作用的因子 9
1.4.1 與Zelda有genetic interaction的因子 9
1.4.2 與Zelda有physical interaction的因子 10
1.5桿狀病毒表現系統（Baculovirus Expression Vector System） 11
1.5.1桿狀病毒（Baculovirus） 11
1.5.2 桿狀病毒生活史 12
1.5.3 桿狀病毒表現系統 13
1.6研究動機及目的 15
二、實驗方法與材料 17
2.1 實驗材料 17
2.1.1 菌種與質體 17
2.1.2 聚合酶連鎖反應引子（Polymerase chain reaction primers） 17
2.1.3 細胞株 17
2.1.4果蠅品系 17
2.2 實驗方法 17
2.2.1大腸桿菌勝任細胞製作 17
2.2.2質體抽取（alkaline lysis） 18
2.2.3 Bacmid抽取 19
2.2.4 將zelda基因克隆至pFastBac-Ph-Lir-DsRed2載體 19
2.2.5 DNA electrophoresis 21
2.2.6 細胞培養及馴化 21
2.2.7 Bacmid與P0病毒製造 22
2.2.8收集P0-2病毒 23
2.2.9 半數細胞感染劑量 （50% tissue culture infective dose，TCID50） 23
2.2.10 桿狀病毒表現系統生產蛋白質（Bac-to-bac expression system） 24
2.2.11 Ni-NTA Agarose純化 24
2.2.12 西方墨點法 25
2.2.13免疫共沉澱（Co-Immunoprecipitation） 26
2.2.14蛋白濃縮 27
2.2.15 SDS-PAGE分析 27
三、實驗結果 29
3.1建構重組病毒載體pFB_HZldRed 29
3.2建構pFB_HZldRed bacmid 30
3.3轉染pFB_HZldRed bacmid至昆蟲細胞產生重組桿狀病毒 32
3.4測試以Ni-NTA純化6xHis-tagged Zelda之條件 33
3.5以純化Zelda與早期胚胎總蛋白萃取免疫共沉澱（Co-IP） 35
3.6蛋白質圖譜分析 38
四、實驗討論 40
4.1 培養重組桿狀病毒及生產Zelda蛋白 40
4.2 利用Ni-NTA純化6xZelda蛋白質 41
4.3 果蠅早期胚胎全蛋白萃取與Zelda免疫共沉澱 42
4.4 Zelda交互作用候選因子 42
4.5 未來應用 43
參考資料 45
附錄 49
附表1：Primer sequences 49
附表2：Bacmid篩選用PCR時間及溫度設定 49
附表3：西方墨點法相關試劑 50
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指導教授 粘仲毅 年中年(Chung-Yi Nien) 審核日期 2022-9-26
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