建立高通量篩選系統探討阿拉伯芥 WRKY 轉錄因子對 ascorbate peroxidase 1 基因之轉錄調節

Abstract

阿拉伯芥 ascorbate peroxidase (APX) 基因家族 9 個基因中，APX1 在植物抵抗強光逆境以及乾旱、高溫的雙重逆境抗性扮演重要角色，然而 APX1 如何受到轉錄調控則尚不清楚。由於 APX1 基因 5` 端上游 1 Kb 序列中含有 8 個 WRKY 轉錄因子結合之 W-box，因此本研究建構一套高通量系統以探討 WRKY 蛋白質對 APX1 的轉錄調節。首先，試驗中建構一個可快速構築 WRKY 轉錄因子大量表現的 Gateway 載體，並完成 48 個 WRKY 轉錄因子之構築；其次，試驗中建立 APX1 promoter / Luciferase 轉殖阿拉伯芥；最後，利用 Polyethylene Glycol (PEG) 法分別將各 WRKY 蛋白質大量表達於 PAPX1-0.5 K /Luciferase 轉殖阿拉伯芥葉片原生質體大量表達，以分析各個 WRKY 基因對 APX1 啟動子活性的影響。結果顯示 WRKY3, WRKY9, WRKY40, WRKY49, WRKY55, WRKY57, WRKY65 轉錄因子可大量表現誘導 APX1 基因表現 2 ~ 7倍，顯示這些因子可能為正調控 APX1 表現之上游調控因子。此高通量篩選系統可有效的篩選目標基因之上游轉錄因子，未來可望進一步利用此快速分析平台探討重要基因上游轉錄網絡。

The Arabidopsis ascorbate peroxidase (APX) gene family is composed of nine genes. The APX1 has been demonstrated to play a key role in tolerance to high light stress and drought / heat stress combination in plants; however, its upstream-transcriptional regulation remained unclear. In the 1 Kb 5`-upstream region of APX1, 8 WRKY transcription factor binding elements (W-box) were observed. Therefore, a high throughput system was established to study the transcriptional regulatory roles of WRKY on the expression of APX1. First, a gateway vector for overexpression was created for efficient construction of 48 WRKY genes, and analysis of their transcriptional regulation on APX1. Second, transgenic Arabidopsis harboring APX1 promoter / Luciferase was generated. Third, leaf protoplasts were used for studying the transcriptional regulation of WRKYs on the expression of APX1 / Luciferase. The results showed that overexpression of WRKY3, WRKY9, WRKY40, WRKY49, WRKY55, WRKY57, WRKY65 significantly induced 2 ~ 7 folds of luciferase activity, indicating that these factors play positive role on APX1 expression. The results suggested that the screening system is effective in screening upstream regulatory factors of APX1, and can be extended to studying the regulatory networks of target genes of interested.