新穎miRNA-tag292在甘藷傷害反應之探討

The involvement of novel miRNA-tag292 in wounding response of sweet potato

謝若微(Jo-Wei Hsieh)

指導教授 ： 鄭石通

國立臺灣大學/生命科學院/植物科學研究所/碩士(2017年)

https://doi.org/10.6342/NTU201702625

摘要

由於植物無法移動，當面對逆境時無法藉由逃跑躲避，因此演化出許多應對的機制。在這些逆境中，傷害是最常見的逆境之一。近年研究發現microRNA (miRNAs) 參與受傷後的防禦機制。先前，實驗室前人利用次世代定序技術結合生物資訊分析找到許多在甘藷中受到傷害影響的新穎miRNA。本研究選定tag292，對其在甘藷體內的功能及目標基因IbRAP2.7進行探討。首先，tag292的前驅物可摺疊成miRNA特有的莖環結構，並於菸草短暫表現與甘藷大量表現tag292前驅物的植株中得知tag292可成功被剪切產生。於甘藷傷害反應下，tag292會受到抑制，其目標基因IbRAP2.7表現量會上升，在茉莉酸處理下也可獲得同樣的趨勢，顯示傷害對tag292與IbRAP2.7的調控可能是藉由茉莉酸來進行。tag292與目標基因IbRAP2.7的關係進一步藉由菸草短暫表現、甘藷轉殖系統以及剪切點分析證實在傷害處理下tag292確實對IbRAP2.7進行剪切並抑制其基因表現量。此外利用菸草原生質體證實IbRAP2.7主要會座落細胞核中。最後，藉由即時定量發現IbPR1a表現量在甘藷中大量表現IbRAP2.7會被誘導，反之大量表現tag292時被抑制，顯示IbPR1a在傷害下可能會被tag292和IbRAP2.7共同調控。總結以上的結論，甘藷遭受傷害會抑制tag292使其無法抑制目標基因IbRAP2.7，導致IbPR1a表現量上升。

關鍵字

甘藷 ； 傷害 ； 茉莉酸 ； 新穎miRNA ； tag292 ； RAP2.7 ； PR1a

並列摘要

Plants, anchored to the ground, are sessile organisms and thus have evolved complex survival mechanisms responding to stresses. Among these stresses, wounding is one of the most common ones. To date, it has been demonstrated that miRNAs participate in wounding defense mechanisms. Previously, we predicted several wound-responsible miRNA candidates based on the next generation sequencing and bioinformatics analysis. Here, we characterized the function of tag292 in planta and further identified its direct target Related to AP2.7 (IbRAP2.7). First, tag292 could be excised from putative precursor sequence (pre-tag292) proved by transient assay of tobacco and stable expression of transgenic sweet potato. Moreover, IbRAP2.7 mRNA cleavage induced by tag292 was confirmed by cleavage site mapping analysis, agro-infiltration assay and transgenic plant studies. The induction of IbRAP2.7 expression coincided with the reduction of tag292, demonstrating IbRAP2.7 was tag292 target. IbRAP2.7, a transcription factor, localized in the nucleus and endoplasmic reticulum of protoplasts. Finally, pathogenesis-related PR-1 family gene (IbPR1a) might be regulated via tag292/IbRAP2.7-mediated pathway in wounding response. These results concluded that IbRAP2.7 was induced following a decrease of tag292, leading to an increase of IbPR1a in sweet potato upon wounding.

並列關鍵字

sweet potato ； wounding ； jasmonate ； novel miRNA ； tag292 ； RAP2.7 ； PR1a

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