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研究生(外文):Chang-Lung Lee
論文名稱(外文):Functional Analyses of Arabidopsis TUBBY-Like Proteins
指導教授(外文):Jei-Fu Shaw
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在先前的研究中,我們定義了阿拉伯芥 (Arabidopsis)中的類肥胖蛋白質 (AtTLP) 基因家族是屬於F-box 蛋白質家族中的一個獨特類型。 此基因家族中的一員 (AtTLP9),已被證實參與了植物荷爾蒙離層酸 (ABA) 的訊息傳遞過程 (Lai et al., 2004)。 在此,我們利用酵母菌雙雜合系統 (yeast two-hybrid system),證明了此家族中的一員 AtTLP2 和 ASK1 具有專一性的作用。 而利用一系列的剔除分析方式 (serial deletion analysis),則進一步的闡明 F-box功能性區塊 (domain) 及 tubby 功能性區塊均可能參與了 AtTLP2 和 ASK1 間的交互作用。 進一步研究指出在此家族中AtTLP5、AtTLP7 及 AtTLP8 這三個類肥胖蛋白質基因家族的成員並無法和 ASK1 產生交互作用。 功能性區塊置換的實驗指出,將AtTLP7和AtTLP2的F-box功能性區塊做交換會造成AtTLP2失去了和ASK1的作用能力。 利用生物資訊學的分析結果,我們推論 AtTLP2 中相對應於人類 SKP2 蛋白質中脯胺酸 (proline) 113的脯胺酸52,可能參與了 AtTLP2和ASK1 間的交互作用。 將 AtTLP2 蛋白質中的脯胺酸52定位點突變為白胺酸 (leucine) 後,驗證了AtTLP2 和 ASK1 的之間的作用力會有大幅的減弱。 在阿拉伯芥葉肉細胞原生質體 (mesophyll protoplasts)短暫表達 AtTLP2的結果顯示 AtTLP2會作用於細胞核中。 這些研究結果指出 AtTLP2可能會參與辨識細胞核內的某些蛋白質,並藉由遍存素標定的方式將其分解 (ubiquitin-mediated proteolysis)。
The Arabidopsis TUBBY-like protein (AtTLP) gene family has been recently characterized as a distinct class of F-box proteins in Arabidopsis. AtTLP9, one member in this family, has been shown to be involved in phytohormone abscisic acid (ABA) signaling (Lai et al., 2004). In the present work, AtTLP2, a representative member of AtTLPs, was shown to specifically interact with Arabidopsis SKP1-like 1 (ASK1) using the yeast two-hybrid system. Serial deletion analyses demonstrated that both F-box domain and tubby domain were required for AtTLP2-ASK1 interaction. Three AtTLPs (AtTLP5, AtTLP7 and AtTLP8) were shown to be unable to interact with ASK1. Domain swapping indicated the replacement of F-box domain of AtTLP2 by that of AtTLP7 resulted in the loss of interacting activity with ASK1. Bioinformatics analysis suggested that residue P52 in AtTLP2 corresponding to P113 in human SKP2 might be involved in AtTLPs-ASK1 interaction. Site directed mutagenesis of P52L in AtTLP2 was confirmed to greatly reduce the interacting activity with ASK1. Transient expression of AtTLP2 in Arabidopsis mesophyll protoplasts indicated that AtTLP2 is localized in the nucleus. These results strongly suggest that AtTLP2 probably acts through targeting some regulatory protein(s) in nucleus for ubiquitin-mediated proteolysis.
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