miR2111在甘藷傷害逆境反應之探討

Abstract

植物的microRNA (miRNA)由20至24個核苷酸所組成的一段小分子，為非編碼RNA。近年來許多研究發現，miRNA不論是在植物的生長發育方面或是面對生物性逆境和非生物逆境，都扮演著重要的角色。在植物遭受的各種逆境中，傷害逆境對植物的影響最為嚴重，會妨害植物的生長與發育，並造成作物產量的損失。然而，現今的研究對於植物在遭受到傷害逆境下所產生的miRNA的調控反應之研究不是甚明瞭，因此在本篇研究中，利用次世代高通量小分子RNA定序的方式，結合生物資訊分析，經過篩選及比對miRNA資料庫(miRBase 21版)後，找到miR2111為一保守性的miRNA，透過機械性傷害處理後，其表現量受到抑制因而下降。根據甘藷的轉錄組資料庫進行其作用目標基因搜尋，miR2111的目標基因為F-box/kelch repeat protein (IbFBK)，當受到傷害性處理時，即時定量PCR分析其表現量則是受到傷害誘導而上升，且IbFBK的mRNA會被miR2111辨認，使IbFBK的mRNA在miR2111的第10和第11個核苷酸之間受到切割。在利用甘藷轉殖大量表現miR2111前驅物，傷害處理240分鐘後mature miR2111持續表現，因而抑制目標基因IbFBK，使其含量下降。此外，IbFBK被預測為E3 ligase SCF (SKP1-Cullin1-F-box) complex的其中一員，經酵母雙雜合系統分析，IbFBK運用其N端的F-box domain與IbSKP1結合形成SCF complex，而C端則是利用kelch-repeat domain與IbCNR8進行交互作用，使IbCNR8會被ubiquitin修飾。

Plant microRNAs (miRNAs), which are 20-24 nucleotides small non-coding RNAs, play crucial roles not only in the various developmental processes but also in the responses to biotic and abiotic stresses. One of the most severe stresses is wounding, which affects plant growth and development and causes food production lost. In this study, a wounding-reduced miRNA, miR2111, was identified in sweet potato (Impomoea batatas cv. Tainung 57). It was found that expression of miR2111 reduced after mechanical wounding. Furthermore, the target gene of miR2111 is IbFBK, which encodes the F-box/kelch repeat protein. Wounding can induce the expression of IbFBK, and IbFBK mRNA is cleaved in the region between 10th and 11th nucleotides of miR2111. In addition, wounding treatment in pre-miR2111 overexpressing transgenic sweet potato showed that IbFBK is downregulated. This results further indicated the existence of the relationship between miR2111 and IbFBK. IbFBK was predicted to be a component of E3 ligase SCF (SKP1-Cullin1-F-box) complex. Results of yeast two-hybried suggest that IbFBK, as a Kelch-type F-box protein, forms an SCF complex with IbSKP1, and binds to IbCNR8 for ubiquitin modification.