甘藷IbWIPK基因參與受傷防禦信息調控機制之探討

Abstract

Sporamin是甘藷塊根中的儲藏性蛋白質，具有胰蛋白酶抑制活性，平時在甘藷葉片中不會表達，但在受傷逆境下IbNAC1轉錄因子能正向調控sporamin大量表現於甘藷葉片，提高甘藷抗蟲能力。過去的研究已知，甘藷MAPK家族中的IbWIPK會被受傷誘導表現，並調控甘藷抗蟲基因IbNAC1及sporamin表現量，但受傷訊息調控IbWIPK基因表現的機制目前仍未明瞭。本研究首先選殖出884-bp的IbWIPK啟動子區域，經由啟動子活性分析實驗，進一步從IbWIPK啟動子中篩選出在TGACG-motif受傷活化IbWIPK啟動子的關鍵角色。更進一步研究發現，甘藷內生性bZIP轉錄因子—IbTGA1a可以直接的與TGACG-motif結合並提高報導基因表現的活性，且當甘藷葉片受傷時IbTGA1a之表達比IbWIPK更為迅速，這些結果顯示IbTGA1a為IbWIPK的上游調控因子。此外，本研究利用酵母菌雙雜合法篩選出可與IbWIPK蛋白質交互作用之轉錄因子—IbbHLH3及IbbHLH4及IbEIL1轉錄因子，我們推測IbWIPK可能透過磷酸化這些轉錄因子以調控茉莉酸與乙烯反應。綜合這些結果，本研究提出了一個受傷誘導IbWIPK表現的訊息傳遞機制，並透過IbWIPK蛋白質與下游目標蛋白質結合，以協同調控植物的荷爾蒙反應。

Sporamin is a storage protein of tuberous root with trypsin inhibitory activity in sweet potato. It has been reported that IbNAC1 transcription factor can upregulate sporamin expression in sweet potato leaves upon wounding, and enhance resistant ability against herbivory. Previously, a MAPK family protein, IbWIPK, has been identified as a wound-inducible activator for IbNAC1 and sporamin expression. However the wound-regulatory mechanism of IbWIPK expression is still unclear. In this study, the 884-bp of IbWIPK promoter region was cloned and analyzed. In the promoter activity analysis, we found that TGACG-motif is important in the activation of IbWIPK promoter in response to wounding. A combination of DNA/protein interaction resulted that a bZIP transcription factor, IbTGA1a, was screened to bind to and activate TGACG-motif. Furthermore, the expression of IbTGA1a was induced faster than IbWIPK after wounding. These results suggested that IbTGA1a is the upstream activator of IbWIPK. Additionally, two bHLH transcription factors, IbbHLH3 and IbbHLH4, and one IbEIL1 were found to interact with IbWIPK by yeast-two hybrid assays, suggesting that IbWIPK might modulate JA response and ethylene response by interaction and phosphorylation of these target proteins. Collectively, this study proposes a signal transduction pathway of IbWIPK expression during wounding stress, and it regulates the hormone responses by protein interaction and phosphorylation in sweet potato leaves.