博碩士論文 100224019 詳細資訊




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姓名 張凱郁(Kai-yu Chang)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 阿拉伯芥中藉由核運輸接受器HIT2/XPO1A進行核質間運輸以促使植物耐受高溫逆境之專一分子的探索研究
(In search of Arabidopsis nuclear export receptor HIT2/XPO1A specific substrates that are essential for heat tolerance in plants)
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摘要(中) 受溫室效應的影響,地球氣候大幅改變,溫度顯著上升。因為植物無法自由行動,故其生長受全球暖化的影響尤其嚴重。先前本實驗室利用前向式遺傳學的方法 (forward genetics approach),篩選出一個對高溫逆境過度敏感之突變株,命名為heat-intolerant 2(hit2)。經基因定位之研究,確認導致此性狀的原因,是由於EXPORTIN1A (XPO1A)的基因發生突變,使該基因所編碼的核輸出接受器 (nuclear export receptors)失去功能(Wu et al., 2010)。經過不同加熱方式以及活性氧 (ROS)染劑的偵測,可知hit2是因無力對抗熱誘發氧化傷害,進而喪失先天性的耐熱能力 (basal thermotolerance),但仍保有後天性耐熱力 (acquired thermotolerance)。而在阿拉伯芥中,還有另一個與XPO1A相似的基因,XPO1B。兩者所編碼之胺基酸序列具有86%的相同度。但是xpo1b-1突變株仍保有對熱及氧化壓力之耐受能力。在加熱前後,野生型中HIT2及XPO1B轉錄程度並無差異。而相較於野生型,hit2植株中的XPO1B轉錄程度較高。酵母雙雜交和BiFC實驗中顯示,帶有核輸出序列的熱轉錄因子(HSF)與HIT2/XPO1A及XPO1B具有親合力上的差異。這些結果指出,儘管XPO1A及XPO1B之間可能有補償機制的存在,然而熱轉錄因子主要是由HIT2/XPO1A來負責其核質平衡,並啟動植物耐熱之必要反應。
摘要(英) The greenhouse effect has lead to serious global warming, and high temperature is a major limiting factor to plant growth and development.We have used a forward genetics approach to screen for heat-intolerant mutants of Arabidposis after ethyl-methane sulfonate (EMS)-mutagensis. hit2 was therefore isolated on the basis of its impaired ability to withstand moderate heat stress (37℃). The hit2mutant harbored a nonsense mutation of ArabidopsisEXPORTIN1A (XPO1A) gene, which encodes a nuclear export receptor that mediates translocation of NES containing proteins across nuclear envelope.Treatments under different heat stress regimes and tests with ROS-detecting dyes further revealed that hit2 was defective in basal but not in acquired thermotolerance due to the inability to protect plant against heat-induced oxidative stress. Arabidopsis contains two copies of XPO1 homologs, HIT2/XPO1A and XPO1B. Nevertheless, xpo1b knockout mutant was not sensitive to heat and oxidative stresses. The transcript levels of HIT2 and XPO1B in wilt type were similar regardless whether the plants were heat stressed or not. However, the transcript level of XPO1B in hit2 was higher than that in wilt type. Yeast two hybrid experiment and BiFC assay were then conducted and revealed that HIT2/XPO1A had a stronger affinity forNES-conraining heat stress transcription factors (HSFs) These resultscollectively indicated that, despite a possible compensatory mechanism between XPO1A and XPO1B, thenuclear-cytoplasmic balance of Arabidopsis HSFs is specifically mediated by HIT2/XPO1A, which is potentially necessary for heat tolerance in plants.
關鍵字(中) ★ 核輸出接受器
★ HIT2
★ 熱逆境
關鍵字(英)
論文目次 中文摘要 i
Abstract ii
致謝 iii
圖目錄 vi
表目錄 vii
縮寫名詞對照表 viii
一、緒論 1
1. 前言 1
2. 研究目的 3
二、 實驗材料與方法 5
1. 實驗材料 5
2. 實驗方法 12
三、結果 21
1. XPO1B突變不影響植物對任何形式高溫逆境的耐熱能力 21
2. hit2與xpo1b-1突變株並不會對強光逆境造成敏感性狀 21
3. XPO1B基因表現量會因hit2突變而上升 22
4. HIT2/XPO1A及XPO1B 與AtHSFA1s與AtHSFA2的結合有成員選擇與親合力強弱的差異 22
5. 阿拉伯芥中HSFA2從細胞核離開進入細胞質是由HIT2/XPO1A而非XPO1B所調節 23
6. XPO1抑制劑Leptomycin B會將AtHSFA2於細胞中的分布位置侷限在細胞核內 24
7. hit2與xpo1b-1突變株氧化逆境的耐受能力有差異性 25
8. HIT2/XPO1A突變會改變熱誘導抗氧化基因的表現模式 26
9. HIT2/XPO1A突變會改變熱逆境誘導活性氧在植物體內累積的變化模式 ..........................................................................................................................26
四、討論 28
1. hit2在不同逆境下的性狀探討 28
2. HIT2/XPO1A 與XPO1B具有功能性的差異 30
3. hit2對熱不具耐受性是由於AtHSFA2核質運輸失去平衡 33
4. hit2突變株清除ROS相關基因表現 34
五、參考文獻 37
六、附錄 45
1. hit2 dCAPS膠圖 45
2. 熱處理當下及復原期間植株的生長情況 46
3. 抗氧化基因縮寫名詞對照表 47
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指導教授 吳少傑(Shaw-jye Wu) 審核日期 2013-7-2
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