Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/90094
標題: Characterization and functional analysis of an E3 ubiquitin ligase gene in the glucose-insensitive mutant in Arabidopsis thaliana
阿拉伯芥中對葡萄糖不敏感突變株之E3泛素化連結酶基因的特性及功能分析
作者: 謝禮臣
Li-Chen Hsieh
關鍵字: 離層酸
葡萄糖
非生物性逆境
泛素化
乾旱

滲透壓
ABA
glucose
abiotic stress
ubiquitination
drought
salt
osmosis
E3 ubiquitin ligase
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摘要: 當植物遭遇非生物性逆境(abiotic stress)時,會啟動一系列複雜的泛素化 (ubiquitination)過程,以因應外來環境壓力,其中E3 ubiquitin ligases (UL)能專一性的降解標的物。本實驗室從高濃度葡萄糖生長培養基篩選,並確定對葡萄糖不敏感性 (insensitivity)的阿拉伯芥T-DNA插入的ul突變株。AtUL基因編譯出一條含411個胺基酸的E3 ubiquitin ligase,分子量為45.4 kDa。此蛋白質含C4HC3的RING motif以及在C端的四個穿膜區 (transmembrane domain)。利用阿拉伯芥原生質體做細胞定位分析,將融合EGFP的蛋白質定位在細胞質膜上。此基因明顯受滲透壓、鹽、離層酸 (abscisic acid, ABA)及乾旱誘導。種子在不同濃度的離層酸、鹽及滲透壓下,ul突變株216及480的萌發與子葉綠化程度比野生株高,顯示兩突變株較野生株不敏感。從植株耐鹽試驗得知ul突變株較具耐鹽特性。葉片蒸散之測試,顯示兩突變株失水程度高於野生株,顯示突變株較不耐旱。三周大的突變株經乾旱12天或利用低濃度離層酸處理,其葉片氣孔張開比率明顯高於野生株。乾旱15天後之回水測試,兩突變株存活率明顯低於野生株。檢測旱境下的離層酸含量發現,ul突變株的離層酸含量雖有增加,但明顯低於野生株上升幅度。這些結果同樣指出ul突變株的不敏感性。利用即時定量聚合酶鏈鎖反應 (quantitative real-time polymerase chain reaction)分析,在離層酸處理下,兩突變株的離層酸生合成基因、訊息傳遞基因與多數受逆境誘導基因的mRNA均明顯低於野生株。利用上下位鑑定發現,AtUL基因位於HXK1與ABI4基因上游,並扮演正調控子角色,參與葡萄糖介導產生的離層酸訊息傳導過程。
Upon abiotic stresses, plants may initiate a series of complicate processes of ubiquitination in which E3 ubiquitin ligase can specifically degrade its target proteins. We have screened to identify an Arabidopsis T-DNA insertion mutant, ul, that exhibited glucose-insensitivity in the presence of high glucose conditions. AtUL gene encodes an E3 ubiquitin ligase that has 411 amino acids with a calculated molecular mass of 45.4 kDa. The protein contains a C4HC3 RING-variant domain and a region of four transmembrane domains at the C-terminus. Subcellular localization analysis demonstrated that protein fused with EGFP was located to the plasma membrane. The gene can be significantly induced by glucose, salt, abscisic acid (ABA) and dehydration. The two mutant lines 216 and 480 displayed germination and cotyledon greening percentages higher than wild-type (WT) under various concentrations of ABA, salt and mannitol, suggesting that they are ABA-insensitive. Salt resistance has also demonstrated that ul mutants exhibited significantly resistance to salt. Transpiration analysis of the two mutant leaves displayed higher water loss than WT, suggesting of the insensitivity of the mutant. The stomatal openness in the two mutant lines was markedly higher than that in WT when leaves of 3-week-old plants were either treated with low concentrations of ABA or dehydrated for 12 days. The survival rate of the two mutant lines was lower than that of WT when plants were recovered after 15 days of dehydration. The ABA content in the ul seedling increased 1.3 fold which is lower than that in the WT after 4 hour dehydration also suggesting of the insensitive nature of two mutant lines. At the molecular level, quantitative real-time polymerase chain reaction analysis revealed that most transcripts of ABA/stress responsive genes were repressed in the two mutant lines compared to WT. We have demonstrated that AtUL acts in a signaling network upstream of HXK1 and ABI4 and plays a positive role in the regulation of glucose mediated ABA signaling pathway.
URI: http://hdl.handle.net/11455/90094
文章公開時間: 2018-08-03
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