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

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题名:	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
作者:	黃乙統;Huang, Yi-Tung
贡献者:	生命科學系
关键词:	共免疫沉澱;桿狀病毒;Zelda
日期:	2022-09-26
上传时间:	2022-10-04 11:03:52 (UTC+8)
出版者:	國立中央大學
摘要:	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。 ;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.
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