| 摘要: | 高溫逆境因子(Heat stress factors, Hsfs)是啟動熱休克蛋白質及抗氧化基因表現，以幫助植物細胞抵禦高溫傷害的主要調節因子。阿拉伯芥 Class A Hsfs 共有15個成員，均帶有nuclear localization signal (NLS) 及 nuclear export signal (NES)，是以Hsfs可經由核內、外的空間分布來進行其調節者的角色。生物分子要進入或離開細胞核，皆須透過核運輸受體 (nuclear transport receptor) 的媒介，再經由核孔(nuclear port complex) 通過核膜。然而，不同的核運輸受體有不同的受質(substrates)，同一受質也有可能經由不同的核運輸受體轉運，是以細胞內是否存在著對耐受高溫逆境具有專一性之核運輸受體，一直未有被報導。運用前向性遺傳學 (forward genetics) 的方法，我們篩選出一個對高溫逆境失去耐受性之阿拉伯芥突變株，命名為hit2。該突變株對熱休克(heat shock)及持續性高溫逆境過度敏感。經基因定位，確認hit2 的突變點位在可辨識NES 之核運輸受體EXPORTIN1A (XPO1A)基因上。阿拉伯芥有兩個XPO1 同源基因，分別為XPO1A 及 XPO1B 。而hit2 的研究，除了證明植物存在著對耐受高溫逆境具有專一性之核運輸受體，也揭露出一個全新的熱逆境訊號傳遞調節點，更提出幾個瞭解植物耐熱機轉亟待解答的重要問題。其一，XPO1A 與XPO1B於植物熱逆境耐受能力上，顯然有不同的分子反應與生理角色，不同之處為何？其二、是否有XPO1A專一辨認的class A Hsfs？如果有，該成員為何？其三，受XPO1A專一辨識之Hsfs，其細胞核/質之分佈在高溫逆境下是否會經由HIT2而改變？本專題研究計劃，即針對如上問題，設計了包含酵母菌雜合 (yeast two-hybrid) 系統，螢光蛋白標定(YFP-fusion)分析，雙分子螢光互補(Bimolecular fluorescence complementation, BiFC)測試，以及相關生理遺傳等實驗，來做一完整的分析。此外，本研究計畫也將繼續對本實驗室所篩選出之其它hit突變植株，進行基因定位及生理研究。藉由對更多熱逆境敏感突變株的發現與研究，我們將可獲得更多新資訊，以演繹出更全面的植物耐熱機轉網路。 Many studies have shown that plants can convey signals from and trigger appropriate responses to environmental stimuli by changing the nucleo-cytoplasmic partitioning of certain regulatory proteins. Meanwhile, the efficient and directed translocation of regulatory proteins across nuclear envelop requires the mediation of nuclear transport receptors. This implies that nucleo-cytoplasmic trafficking mediated by nuclear transport receptors is vital for plant tolerance to stress. However, owing to the fact that a receptor may have a broad range of cargo substrates, or one species of macromolecule may use several different receptors for nuclear translocation, the existence of a nuclear transport receptor with a specific role in plant stress tolerance had not been demonstrated. Recently, we isolated an Arabidopsis hit2 mutant that is hypersensitive to sustained high temperature and sudden heat shock. The mutated locus was later mapped to the EXPORTIN1A (XPO1A) gene. XPO1 is a nuclear export receptor that interacts with leucine-rich nuclear export signals (NESs), which are found in all 15 members of the Arabidopsis class A heat shock factors (Hsfs). Meanwhile, in Arabidopsis, two XPO1 genes (XPO1A, At5g17020 and XPO1B, At3g03110) have been identified. These findings not only demonstrate that a nuclear transport receptor with a specific role for tolerance to environmental stress dose exist in plants, but also raise many crucial questions as (i) what is the difference between XPO1A and XPO1B in plant heat tolerance; (ii) is there any specific Hsf that is recognized by only one of the XPO1; (iii) the identity of the Hsf whose pattern of nuclear-cytoplasmic partitioning through XPO1A must change in order for plants to survive under heat stress. To answer these critical questions and to provide new insights into the importance of regulatory control at the level of nucleo-cytoplasmic trafficking for plant stress responses, an array of experiments, including yeast two-hybrid analysis, fluorescent protein fusion, bimolecular fluorescent complementation assay, and various physiological and genetic analysis are thus proposed in this study. Meanwhile, the endeavors to map other heat-hypersensitive mutants that have been isolated in this laboratory will be carried on. As with more essential genes in plant heat tolerance identified, we can better clarify the complex mechanisms by which normal plants tolerate environmental stress. 研究期間：10008 ~ 10107 |
