構建並分析WRKY轉錄因子對阿拉伯芥Pathogenesis-related protein 1基因表現之轉錄調控網絡

Abstract

阿拉伯芥pathogenesis-related preoteins (PRs)在植物遭受病原菌入侵時會在植體內大量累積，其中PR1常被用作檢測是否發生系統性獲得抗性 (SAR)的指標基因，然而PR1轉錄層次上如何受到調控則尚未被研究清楚。分析發現PR1基因5’端上游1.5 kb序列中包含12個WRKY轉錄因子結合的W-box，推測WRKY轉錄因子及其基因調控網絡於PR1基因之表現具重要角色，因此本研究利用高通量篩選系統探討WRKY轉錄因子對PR1基因的轉錄調節。首先，試驗中建立一可快速構築WRKY轉錄因子大量表現的Gateway載體，共完成48個WRKY轉錄因子構築；其次，試驗中建立PR1 promoter / Luciferase轉殖阿拉伯芥；接著利用salicylic acid (SA)處理阿拉伯芥，釣取受SA誘導之WRKY基因；最後，利用polyethylene glycol (PEG)法分別將SA篩選所得WRKY蛋白質大量表現於PR1/Luciferase轉殖阿拉伯芥葉片原生質體，以分析各個WRKY基因對PR1啟動子活性的影響。結果顯示WRKY6, WRKY25, WRKY38, WRKY46, WRKY53轉錄因子大量表現可誘導luciferase活性達3∼8倍，顯示這些轉錄因子可能為正調控PR1表現的上游調控因子。本試驗結果可做為一新的研究方法，進一步確認調控PR1基因表現之重要因子及其可能參與的訊息傳遞路徑。

The Arabidopsis pathogenesis-related proteins (PRs) are induced by pathogen infection. The PR1 gene has been widely used as a marker gene for the onset of systemic acquired resistance (SAR); however, its upstream-transcriptional regulation remained unclear. In the 1.5 kb 5’-upstream region of PR1,12 WRKY transcription factors binding element (W-box) were observed. Thus, WRKY TF and its corresponding genes regulatory network are highly speculated to play important roles in regulation of PR1 gene expression. To understand how WRKY TFs participate in this process, a high-throughput system was set up. First, an overexpression gateway vector was created for efficient construction of 48 WRKY gene, and analysis of their transcriptional regulation on PR1. Second, transgenic Arabidopsis harboring PR1 promoter / Luciferase was generated. Third, induced expression of WRKYs were selected by salicylic acid treatment. Fourth, leaf protoplasts were used for studying the transcriptional regulation of WRKYs on the expression of PR1 / Luciferase. The result showed that overexpression of WRKY6, WRKY25, WRKY38, WRKY46, WRKY53 were significantly induced 3~8 folds of luciferase activity, indicating that these factors plays positive roles on PR1 expression. Our results represent a powerful approach to dissect key factors or components which involved either in gene regulation or signaling transduction pathway of PR1.