甘藷中受一氧化碳調控之嶄新microRNA tag202調控Receptor-Like Kinase

Abstract

一氧化碳為二次訊息傳遞者，參與植物的生長發育。microRNA (miRNA)是一群20至24個核苷酸長的small RNA，具有降解目標基因mRNA等能力。然而，有關一氧化碳會透過miRNA調控植物生理反應的研究非常少。實驗室前人藉由small RNA定序找到受一氧化碳誘導的新穎miRNA—tag202。當甘藷受傷後，內生的一氧化碳含量受到抑制，tag202的表現量也同時下降。於菸草中短暫表現precursor form tag202 (pre-tag202)與其預測目標基因的互補序列片段，證實tag202可以抑制預測目標基因表現。且在大量與降低表現tag202的甘藷轉殖株中，目標基因的mRNA累積量與tag202的表現量呈相反趨勢。本研究釣取目標基因全長後於NCBI上分析，推測其應為receptor-like kinas (RLK)，且屬於CrRLK1L家族，因此改名為IbRLK。將tag202與IbRLK的互補序列進行點突變，證實IbRLK在相對於tag202的第10個核苷酸為tag202進行剪切的重要辨認點。將IbRLK接上GFP後觀察其於細胞中的存在位置，發現IbRLK位於菸草原生質體細胞膜上。並且在甘藷植株中， IbRLK與IbMEK1在葉片中表現量較高，兩蛋白質很可能共存於細胞中。藉由雙分子螢光互補作用(BiFC)，發現IbRLK會與IbMEK1在細胞膜上有交互作用，然而在酵母菌雜合 (Y2H)實驗中兩者沒有直接交互作用，推測IbRLK和IbMEK1之間可能有其他蛋白質幫助調控MAPK cascade。因此，受一氧化碳誘導的新穎miRNA tag202會調控IbRLK表現，而IbRLK可能藉由與IbMEK1發生交互作用影響下游基因的表現。

Carbon monoxide (CO), a secondary messenger, plays an important role in growth and immunity responses of plants and animals. MicroRNAs (miRNAs), the small noncoding RNAs with 21-24 nucleotides, direct messenger RNA cleavage or inhibit translation to regulate gene expression. However, little is known of the CO-responsive miRNAs and their regulation. According to previous studies, the expression of miRNA-tag202 increased after CO treatment in sweet potato (Ipomoea batatas cv Tainung 57). Furthermore, wounding treatment repressed the level of CO and suppressed the expression of tag202. Overexpressing tag202 precursor would reduce the expression level of target gene in both transient assay of tobacco and stable transformation of sweet potato. In this study, the target gene of tag202 encode a receptor-like kinase (RLK) on plasma membrane. IbRLK mRNAs were mostly cleaved at the 10th nucleotide from the 5′ end of the tag202-binding region by miRNA-tag202. Moreover, IbRLK interacted with IbMEK1, one of MAP2K of in the MAPK cascades. The interaction between IbRLK and IbMEK1 was confirmed by bimolecular fluorescence complementation (BiFC), but not by yeast-two hybrid assay (Y2H). There were other proteins between IbRLK and IbMEK1, and helped IbRLK interacted with IbMEK1. Therefore, the hypothesis is CO-induced tag202 expression to control IbRLK, which might interact with IbMEK1 to regulate downstream gene expression.