以桿狀病毒載體系統 建構與異源表達果蠅Zelda基因及其功能分析

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姓名

康致甄(Chih-Chen Kang) 查詢紙本館藏

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生命科學系

論文名稱

以桿狀病毒載體系統建構與異源表達果蠅Zelda基因及其功能分析
(Construction and ectopic expression of Drosophila Zelda using baculovirus system for functional analysis)

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至系統瀏覽論文 (2027-6-1以後開放)

摘要(中)

大多數動物早期胚胎發育是由母源基因產物來支持與調控，這些產物在卵子生成的過程中生成並寄存於卵子內。而合子基因組則維持不轉錄狀態，直到母源-子源轉換期 (maternal-to-zygotic transition, MZT) 才開始被激活表現，稱為zygotic genome activation (ZGA)，此時胚胎發育轉由合子基因組接管調控。在果蠅的MZT期間，初期約500-900個基因被激活，隨後約有2000-3000個基因激活[1, 2]。先前研究發現一母源轉錄因子Zelda在果蠅MZT時期扮演關鍵的角色，經由結合特定序列 (CAGGTAG及相關位點)，激活多數的早期基因。目前已知Zelda的作用方式包括: 1. 以轉錄激活因子活化目標基因，2. 增加染色質開放程度，以利其他轉錄因子與加強子 (enhancer) 結合並促進轉錄[2, 3]，3. 降低核小體的屏障[2, 4]，因此Zelda被視為先鋒因子(pioneer factor)。然而，其結構與分子功能機制仍未明朗，Zelda蛋白的表現與純化的困難更增加研究上的挑戰。本實驗主要目的是利用桿狀病毒表現系統為工具，於果蠅胚胎晚期的細胞株Schneider 2 (S2) cells異位表達6xHis-tagged Zelda 蛋白，以期未來得以Cryo-EM作結構分析、分子功能區 (functional domains) 分析、及交互因子的研究。
除了重組病毒的建構、病毒增殖及轉染、Zelda蛋白表現等各項條件的探討，本實驗並以RNA-seq及qRT-PCR檢視轉染異位表達Zelda對S2 cells基因組的影響。結果顯示大量基因 (5588) 表現量上升，僅有少部分下降，表現量上升的基因與文獻1到3小時受Zelda影響的基因相互比較[3]，有463個基因重疊 (50%)，特別是MZT初期表現基因。將表現量增加的基因按表現差異倍率排序，前300個基因進行基因功能分類分析，軸向發展相關基因佔多數，細胞化、細胞週期運轉、性別決定相關的基因亦包含在內，顯示異位表達Zelda可以重新激活這些早期胚胎發育基因，此結果呼應先前推測Zelda具有重塑染色質 (chromatin remodeling) 與細胞重新編程 (reprogramming) 的潛力。利用果蠅成蟲幹細胞標記初步探討異位表達Zelda是否足以使已分化細胞重新編程，結果發現其相關基因表現量皆有上升。綜合本研究及目前已知的斑馬魚MZT關鍵因子與誘導多能幹細胞 (induced pluripotent stem cells, iPSCs) 之核心誘導因子[5]，顯示以先驅因子重塑染色質，促進相關基因組表現，可能為細胞重新編程的策略之一。

摘要(英)

In all animals, the initial embryonic development is controlled by the maternal gene products, while zygotic gene expression remains silent until later time point at maternal to zygotic transition (MZT). During MZT, a major proportion of the maternal components is removed and the zygotic genome is activated (ZGA). During Drosophila MZT, about 500-900 genes are first activated (minor wave), followed by a major wave of gene expression (around 2000-3000 genes)[1, 2]. A maternal transcription factor, Zelda, was reported as the key activator of early ZGA[2, 4]. Zelda, is not only a strong activator but also acts as an epigenetic regulator which opens chromatin during MZT through a specific motif (CAGGTAG and its related sites), reduces nucleosome barriers and in turn facilitates chromatin accessibility[2, 3]. In that, Zelda is considered as a pioneer factor. However, the underlined molecular and structural functions of Zelda remain unclear. The difficulties to robustly express and purify Zelda protein make it even more challenging.
In this study，we successfully induced 6xHis-tagged full length Zelda proteins in Schneider 2 (S2) cells using the Bac-to-Bac expression system. S2 cells are derived from the late-stage Drosophila embryos and do not express Zelda. The conditions and timeline for virus production and transfection were optimized. Resulted Zelda protein will be subjected to future Cryo-EM analysis, functional domain dissection, interactor screening and global enhancer mapping by STARR-seq. We also applied RNA sequencing (RNA-seq) and qRT-PCR to characterize the effects of ectopically expressed Zelda on the gene expression of S2 cells. A large number of genes (5588) have been up-regulated. 463 up-regulated genes were overlapped with Zelda-dependent targets (50%) expressed in embryo during MZT[3]. Most of them are related to embryonic morphogenesis. Moreover, axis patterning genes were enriched in GO term analysis of top 300 up-regulated genes. Cellularization, cycle regulators, and sex determination related genes were also included. This result suggested that ectopically expressing Zelda can reactivate early embryonic development genes, and have the potential to reprogram differentiated cells. In fact, Drosophila adult stem cell markers were up-regulated by Zelda in S2 cells[5]. In all, Zelda, the key MZT factors of Zebrafish and iPSC (induced pluripotent stem cells) core reprogramming factors in mamalians share a similar feature as the pioneer factors which remodel the chromatin landscape and collectively and specifically activate subsets of genes, hinting a paradigm strategy to induce the pluripotency of cells.

關鍵字(中)

★ 果蠅早期發育

關鍵字(英)

★ MZT
★ Drosophila
★ ZGA
★ Zelda
★ pioneer factor

論文目次

目錄
中文摘要 i
Abstract iii
誌謝 v
目錄 vi
圖目錄 x
中英文對照表 xi
壹、前言 1
1.1 黑腹果蠅 (Drosophila melanogaster) 早期胚胎發育 1
1.2 母源-子源轉換期 (Maternal-to-zygotic transition, MZT) 1
1.3 轉錄因子Zelda 3
1.3.1 Zelda於果蠅MZT時期扮演關鍵角色 3
1.3.2 Zelda調控早期胚胎發育 3
1.3.3 Zelda促進早期TFs與DNA結合 5
1.3.4 Zelda克服核小體屏障 6
1.4 Pioneer factor與細胞重新編程之關係 6
1.5 重組桿狀病毒表達系統Bac-to-Bac expression system 8
1.6 研究動機及目的 8
貳. 實驗方法與材料 10
2.1 實驗材料 10
2.1.1 菌種與質體 10
2.1.2 聚合酶連鎖反應引子 (Polymerase chain reaction primers) 10
2.1.3 細胞株 10
2.2 實驗方法 10
2.2.1 質體之抽取 10
2.2.2 桿狀病毒質粒 (bacmid) 之抽取 11
2.2.3 大腸桿菌勝任細胞 (competent cell) 之製作 11
2.2.4 細菌轉形作用 (Transformation) 12
2.2.5 細胞轉染作用 (Transfection) 12
2.2.6 由電泳膠回收DNA 12
2.2.7 西方墨點法 (western blot) 13
2.2.8 限制酶切割 14
2.2.9 DNA接合反應 (DNA ligation) 14
2.2.10 病毒擴增 (Virus amplification) 14
2.2.11 病毒沉澱 14
2.2.12 半數細胞感染劑量 (50% tissue culture infective dose，TCID50) 15
2.2.13 Ectopic expression of Zelda in感染Schneider 2 細胞 15
2.2.14 萃取細胞RNA (RNA extraction) 16
2.2.15 目標RNA反轉錄至cDNA (Reverse-transcription of RNAs) 16
2.2.16 Quantitative reverse transcription PCR (RT-qPCR) 16
2.2.17 聚合酶連鎖反應擴增目標片段 (PCR amplification of target gene) 17
2.2.18 DNA 膠體電泳 (DNA gel electrophoresis) 17
2.2.19 轉錄組定序 (RNA Sequencing，RNA-Seq) 17
參、實驗結果 19
3.1 建立於S2 cells表達Zelda的重組病毒 (pFB_HZeldaRed_Actp) 19
3.2 構築pFB_HZeldaRed_actp bacmid 21
3.3 轉染pFB_HZeldaRed_actp至昆蟲細胞產生重組病毒 22
3.4 利用聚乙二醇沉澱法濃縮重組病毒 24
3.5 計算多重感染複數(multiplicity of infection，MOI) 24
3.6 評估重組病毒感染S2 cells後Zelda的表現量 25
3.7 利用螢光蛋白DsRed檢測感染率與DsRed表現的關係 27
3.8 S2 cells異位表達Zelda對基因表現之影響 28
3.8.1 與MZT時期Zelda targets比較 28
3.8.2 與MZT時期表現基因比較 29
3.8.3 GO term enrichment分析 31
3.8.4 Axis patterning 基因 32
3.8.5 Cellularization基因 33
3.8.6 Cycle regulator 基因 34
3.8.7 Sex determination基因 35
3.8.8 Cells apoptosis 基因 35
3.8.9 Stem cells marker 基因 36
3.9 利用qRT-PCR檢測早期zygotic基因表現 37
3.10 不同Zelda表現量對下游基因的影響 38
3.11 確認基因表現差異並非來自post-transcription modification 41
肆、實驗討論 43
4.1 培養擴大重組桿狀病毒 43
4.2 感染後zelda表現與天數之關係 44
4.3 在S2 cells中的異位表達Zelda 46
4.4 感染後細胞是否具有pluripotency 48
4.5 Ectopic expression of Zelda之應用 49
伍、結語 50
參考資料 51
附錄圖 55
附錄圖1. DNA定序確認構築的質體pFB_HZeldaRed_actp 55
附錄表 56
附錄表1. 實驗所使用之菌種與質體 56
附錄表2. 實驗coloning所使用之引子 57
附錄表3. 實驗qPCR所使用之引子 58
附錄表4. 西方墨點法相關試劑 60
附錄表5. Zelda and Zelda effects on zygotic gene expression 61

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指導教授

粘仲毅(Chung-Yi Nien)

審核日期

2022-6-27

推文