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標題: Regulation of yolk protein gene expression in the oriental fruit fly, Bactrocera dorsalis (Hendel), and establishment of its transgenic lines
作者: Chen, Shiu-Ling
出版社: 昆蟲學系所
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In current studies, a 2.7-kb upstream fragment of the putative promoter region of the oriental fruit fly, Bactrocera dorsalis yolk protein gene 1 (Bdyp1) was cloned, and the sequence comprises a series of potential regulatory elements responsible for the regulation of the gene expression, including six potential ecdysone response elements (EcREs), and numerous binding sites for various transcriptional factors, such as GATA, Doublesex, MAB-3, AEF-1, BBF-2, and others. Transient transfection analysis in Sf21 cells showed that the 2.4-kb frame of 5'-flanking region of Bdyp1 exhibits the greatest activity in response to the treatment with 10-2 nM of 20-hydroxyecdysone (20E). Moreover, promoter deletion and gel shift analysis further revealed that 20E is most likely interacting with the first two EcREs only. To address how transfactors regulate the Bdyp1 expression, RNA interference (RNAi) assays were used to demonstrate that female-specific Bddsx dsRNA not only specifically reduces its own transcript but also selectively inhibits the expression of Bdyp1 gene and delays the ovarian development. In addition, the properties of the Bdyp1 promoter were analyzed in vivo using germline transformation. The reporter gene, egfp, controlled by 2.4 and 0.4-kb of 5'-flank region of Bdyp1; and both exogenous genes were specifically expressed in fat body, indicating its tissue-specific expression of the yp1 promoter. However, the EGFP expression appears both in males and females, indicating loss of its sex-specific expression. It is thus speculated that loss of the sex-specificity in both transgenic lines may be caused by the position effect of transgenesis.

本研究選殖東方果實蠅(Bactrocera dorsalis (Hendel))(Tephritidae: Diptera)卵黃蛋白基因1(yolk protein gene 1, Bdyp1)之5’端調控、啟動子與基因本體部份核酸序列,經比對分析後得知調控區域具有蛻皮激素受器(ecdysone receptor, EcR)、雌性雙性基因(female specific doublesex, dsxF)、GATA factor、E75、adult expression factor (AEF)等激素、組織或性別表現專一性之轉錄因子(transcription factor)結合之反應位。於昆蟲細胞轉染(transfection)試驗中證實,卵黃蛋白基因啟動子會受激素誘發而活化,其中又以蛻皮激素(ecdysteroids)為主要調控激素,青春激素(juvenile hormone)僅具些微活化啟動子之效能,且蛻皮激素受器反應位(ecdysone response element, EcRE)之序列能與受蛻皮激素刺激之核蛋白結合,配合啟動子截切活性分析(promoter deletion assay)結果,顯示靠近轉錄起始位(transcription start code)之兩蛻皮激素受器反應位能受蛻皮激素刺激進而誘發卵黃蛋白基因表現,此部份結果與高等雙翅目昆蟲卵黃蛋白調控機制中所提之論點相符;再者,利用轉錄因子dsxf啟動RNA默化(RNA interference, RNAi)機制時,Bdyp1於基因及蛋白表現層次皆受到影響而有遲延表現之趨勢,卵巢發育亦明顯較注射雙股egfp之對照組緩慢。最後,藉由生殖細胞系的轉型作用(germline transformation)探討Bdyp1性別和組織表現之專一性,兩基因轉殖品系,pyp2.4與pyp0.4,之報導基因(綠色螢光蛋白)皆僅表現於脂肪體細胞而保留其組織專一性,但無法呈現預期之性別專一性,推測此現象乃由於基因轉殖外來片段時鑲嵌於染色體上位置效應(position effect)所導致。
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