Abstract In the present study, we cloned a yolk protein gene, Bdyp1, of the oriental fruit fly Bactrocera dorsalis (Hendel) and its 2.7-kb 5’-flanking sequence (GenBank accession no. EU130922). The Bdyp1 gene comprises a 31-bp 5’- untranslational region (5’-UTR), three exons, and a 141-bp 3’-UTR. The 2.7-kb 5’-flanking region of Bdyp1 contains two putative TATA boxes close to the initiation site, 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 so forth. Transient transfection analysis of the Sf21 cells shows that the 2.4-kb frame of the 5’-flanking region of Bdyp1 exhibits the greatest activity in response to 10-2 nM of 20-hydroxyecdysone (20E). Promoter- deletion analysis reveals that 20E is most likely only interacting with the first two EcREs. In addition, the 20E-induced elevation of the promoter activity is suppressed by the addition of juvenile hormone III either before or after the 20E treatment. This is the first demonstration to show that JH is capable of suppressing the action of 20E on stimulating yolk protein gene expression.
摘要 本研究選殖得東方果實蠅 (Bactrocera dorsalis (Hendel)) 卵黃原蛋白基因 1 (yolk protein gene 1, Bdyp1) 及其 2.7 kb 之上游區域 (GenBank accession no. EU130922)。Bdyp1 基因由一段長 31 bp 之 5 端非轉譯區域 (5’-untranslational region, 5’-UTR)、三個外顯子 (exon) 及一段長 141-bp 之 3 端非轉譯區 (3’-UTR) 所構成。分析 Bdyp1 上游 2.7 kb 序列顯示，在靠近基因起始位 (initiation site) 處具有兩個類似TATA box的區域，6 個可能是蛻皮激素受器反應位 (ecdysone response element, EcRE) 分布在不同的位置，以及許多如雌性雙性基因 (female specific doublesex, dsxF)、GATA factor、E75、adult expression factor (AEF) 等等調控組織或性別表現專一性之轉錄因子 (transcription factor) 作用位置。細胞轉染 (cell transfection) 2.4 kb 上游 DNA 片段入 Sf21 細胞，激素測試結果顯示在 10-2 nM 蛻皮激素的刺激下啟動子的活性最高；進一步經啟動子刪除 (promoter deletion) 分析，結果顯示蛻皮激素作用主要的結合位 (binding site) 可能僅為調控區最靠近起始位之兩個 EcRE。另外，本研究首次發現蛻皮激素誘發啟動子活性的現象會因為處理青春激素 (無論在施用蛻皮激素之前或後) 而受到抑制。
| Key words: yolk protein gene promoter, juvenile hormone, 20-hydroxyecdysone, Bactrocera dorsalis, cis-regulation | | 關鍵詞: 卵黃原蛋白基因啟動子、青春激素、蛻皮激素、轉錄因子、東方果實蠅。 |
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