功能性分析甘藷之IbWIPK及IbMEK1參與生物性逆境抗性之研究

Abstract

SPORAMIN為甘藷中所特有的抗蟲基因，截至目前已發現IbNAC1轉錄因子可直接與SPORAMIN啟動子上的受傷反應元件專一結合，並在受傷訊息傳遞中，正向調控下游SPORAMIN基因表現，此訊息傳遞需要透過MAPK來放大受傷訊息以活化IbNAC1表現。本研究從甘藷cDNA基因庫中選殖出一個可經受傷、茉莉酸與過氧化氫誘導表現的MAPK基因，並命名為IbWIPK。以阿拉伯芥原生質體進行轉錄活性測試，發現相對於單獨表現proIbNAC1::GUS植株，共表現35S::IbWIPK與proIbNAC1::GUS之轉殖株具有較高的GUS 活性，由此得知，IbWIPK具有活化下游IbNAC1基因表現的能力。大量表現IbWIPK之阿拉伯芥轉殖株，相較於空載體轉殖之對照組植物，其對鹽逆境較敏感，並且具有茉莉酸、乙烯及生長素累積過多之植物形態特徵。茉莉酸、乙烯與生長素相關的指標性基因，在大量表現IbWIPK的阿拉伯芥具有較高的表現，因此可推測這些形態是由於過度累積茉莉酸、乙烯與生長素所造成。大量表現IbWIPK的阿拉伯芥轉殖株可藉由增加VSP2、CYSTEINE PROTEINASE與TERPENE SYNTHASE的表現量，來提高轉植株對斜紋夜盜蛾幼蟲的抗性。在大量表現IbWIPK甘藷轉殖株中，SPORAMIN活性也到大幅度的提升。此外，本研究中也釣取IbWIPK上游交互作用之蛋白分子IbMEK1(MAPKK)，並進行功能性分析。綜合這些結果，在甘藷抗蟲的機制上，IbWIPK可協同調控茉莉酸與乙烯的抗蟲訊息路徑，進而促使植物達到自我保護的防禦效果。

A NAC-domain transcription factor, IbNAC1, is known to positively regulate SPORAMIN expression, which has been identified as an unique insect-resistant gene in sweet potato, via binding to the wound-responsive cis-element of SPORAMIN promoter. This wound signal transduction requires MAPK cascades to amplify the signal to activate IbNAC1. In this study, we identified a wound-, MeJA- and H2O2-inducible MAPK gene named IbWIPK. Co-transformation of 35S::IbWIPK effector and proIbNAC1::GUS reporter in plant showed the increased GUS activity compared to reporter alone, suggesting that IbWIPK plays a positive and directive role on IbNAC1 activation. Notably, the Arabidposis plants overexpressing IbWIPK (IbWIPK-OE) exhibited sensitive to salt stress and showed JA/ET-response phenotype. The indicator genes of JA, ET and auxin were elevated in IbWIPK-OE plants, implying that those phenotypes were formed by accumulation of JA and auxin. These plants were increasingly resistant to Spodoptera litura due to high expression of VSP2, CYSTEINE PROTEINASE and TERPENE SYNTHASE. In transgenic sweet potato, the activity of SPORAMIN was enhanced by the overexpression of IbWIPK. Moreover, IbMEK1 (MAPKK), which was physically interacted with IbWIPK, was also identified and characterized in this study. Collectively, these results suggest that IbWIPK plays a critical role in coordinating herbivore signal in JA/ET pathway against herbivory in plants.