油菜固醇調控的微型核糖核酸miR395a在阿拉伯芥中所扮演的角色

油菜固醇是一植物賀爾蒙，在植物體內扮演影響生長發育的角色。近年來已有研究以核糖核酸微陣列晶片以及蛋白質體學兩種高通量實驗，檢測油菜固醇對植物整體的影響，但對於微型核醣核酸所涉及的機制仍不明瞭。我們選用哥倫比亞型的阿拉伯芥，以微型核醣核酸微陣列晶片分析油菜固醇所調控的微型核醣核酸，並發現額外添加油菜固醇後，miR395a的表現量會顯著的上升，以即時定量聚合酶連鎖反應偵測其表現，也有相同的結果。為了釐清miR395a所涉及的機制，我們整合了生物資訊以及現有的核糖核酸微陣列晶片，預測其可能的目標基因，結果顯示GUN5極有可能是miR395a的目標基因之一。GUN5是一多功能的基因，涉及植物體內代謝，包含葉綠素合成，而近年的研究更指出其可能涉及離層酸的調控路徑。離層酸是一植物賀爾蒙，參與調適植物逆境，如藉由抑制種子萌發、延長根的長度以及側根的形成。我們利用即時定量聚合酶連鎖反應，進一步分析離層酸路徑裡的基因，結果顯示GUN5下游的基因表現量下降。為了證明miR395a與GUN5是否有直接的關聯，我們利用螢光分析檢測，結果顯示在含有miR395a以及融合蛋白GUN5-GFP的組別裡，螢光蛋白的表現量下降。另外，利用miR395a promoter-GUS轉植株則顯示miR395a表現在葉脈以及根的組織中。這些結果皆顯示油菜固醇所調控的生長路徑之一，或許是藉由miR395a來抑制GUN5的表現，以阻斷離層酸路徑，並減少葉脈周邊細胞葉綠素。

關鍵字

阿拉伯芥；油菜固醇；微型核糖核酸

並列摘要

Brassinosteroids (BRs) are essential plant hormones involved in growth and development. Many high-throughput researches of BRs have been provided by DNA microarray and proteomics; however, the microRNAs-involved mechanisms of BRs are still poorly understood. With the use of time-course microRNA array analysis, we identified BR-regulated microRNAs in Arabidopsis thaliana ecotype Columbia. We found that miR395a was significantly up-regulated after BR treatment, and validated the expression of miR395a and BR-regulated genes using quantitative PCR (q-PCR). In order to clarify the miR395a-involved mechanisms, we integrated bioinformatics methods and publicly available DNA microarray data to predict the potential targets of miR395a. According to these methods, GUN5 was identified as one of the possible targets to miR395a. GUN5 is a multifunctional protein involved in plant metabolism such as chlorophyll synthesis. Recent studies have shown that GUN5 might be involved in abscisic acid (ABA) pathway. ABA is a plant hormone that mediates the adaptation of plants to stress via inhibiting seed germination, root elongation and lateral root formation. We further investigated the gene expression of ABA pathway in BR-treated plants by performing q-PCR. As proposed, the downstream genes of GUN5 were down-regulated. We further used fluorescence assay to validate the relative expression ratio of miR395a and GUN5, and the result showed that the expression of GUN5-GFP fusion gene was down-regulated. Furthermore, the expression of miR395a was up-regulated in leaf veins and root tissues in BR-treated miR395a promoter-GUS plants. Our data revealed that miR395a may inhibit ABA responses by targeting GUN5, indicating that BR may affect the growth of Arabidopsis through miR395a-regulated ABA responses and causing less chlorophyll in the cell around leaf veins.

並列關鍵字

Arabidopsis thaliana ； Brassinosteroid ； miRNA

參考文獻

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油菜固醇調控的微型核糖核酸miR395a在阿拉伯芥中所扮演的角色

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