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姓名 翁慈珮(Chi-Pei Weng)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 阿拉伯芥熱誘導性狀突變株R45之基因定位及HSP40參與植物耐熱機轉之探究
(Gene mapping of Arabidopsis heat insensitive R45 mutant and the study of the role of HSP40 in heat-tolerance mechanism)
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摘要(中) 由於植物無法自由移動,是以必須演化出許多逆境抵禦機制,以因應瞬息萬變的生長環境。近年來溫室效應導致全球氣溫上升,而高溫對植物而言是環境中主要非生物逆境之一。高溫逆境會影響農作物的正常生長與發育、劣化品質。嚴重的話會導致植物的死亡,減損產量。是以了解植物抵抗高溫逆境的相關機轉,再運用這些知識來提升農作物對高溫的耐受力,以因應未來糧食供應之所需,有其必要性,也是本研究的出發點。在此,我們運用前向式遺傳學的策略,篩選出對熱敏感的突變植株,藉此找出植物耐受熱逆境的必要基因,並解析植物耐熱機制。利用此策略所篩選出的heat-intolerant 5(hit5),如篩選條件,對持續性溫和高溫敏感,然對短期熱休克則有更強的耐受力。在基因定位後,確認hit5的熱敏感性狀,是因ENHANCED RESPONSES TO ABA 1 (ERA1)基因發生突變所引起。從前人研究得知,此基因表現後所製造出的產物,為植物內蛋白質法尼酯轉移酶(Protein Farnesyltransferase, PFT)的β次單元。PFT由α及β兩個次單元所組成,催化反應為將一個含有15個碳鏈的脂質,接到特定的蛋白質的C端,屬於後轉錄修飾的一種形式。如同其名,ERA1基因突變會使得植株對於ABA敏感,而ABA為一已知能促使植物耐受逆境的重要賀爾蒙。為了釐清hit5的熱相關性狀與ABA調節是否有關,本研究進行了外加ABA及抑制ABA生合成影響hit5熱相關性狀的實驗,證實hit5熱相關性狀不受ABA調節。據此,推測另有其它目前未知的因子,作用在HIT5下游,分別促使植物因應不同形式的熱逆境。另根據文獻報導,若干植物分子量40 kD熱休克蛋白質家族成員,為PFT的受質。經實驗驗證,其中的一個成員的確有參與在HIT5所調節的熱逆境反應。與此同時,本研究再次以前向式遺傳學的策略,透過篩選能恢復hit5熱相關性狀回復成野生型之突變株,來找尋其他參與在HIT5調節熱逆境反應的遺傳因子,以助於更加釐清HIT5所調控的植物熱反應機制。
摘要(英) Plants are sessile organisms and thus vulnerable to the adverse environment in which they live. High temperature is one of the major environmental adversities that can decrease crop yields. To understand how plants resist high temperature stress, which is essential for developing heat-tolerant crops, we used a forward genetic approach to screen for EMS-mutagenized Arabidopsis mutants that are more thermosensitive than the WT. heat-intolerant 5 (hit5) was therefore isolated because prolonged heat incubation at 37°C for 4 d was lethal for hit5 but not for the WT seedlings. However, hit5 mutant has better ability to tolerance sudden heat shock treatment at 44°C for 40 min than WT. Map-based cloning revealed that HIT5 is ERA1 gene that encodes the β subunit of the protein farnesyltransferase (PFT). Protein farnesylation is a post-translational modification known to mediate abscisic acid (ABA)-regulated drought tolerance in plants. To verify the role of ABA in HIT5-mediated heat stress response (HSR), exogenous application of ABA or ABA synthesis inhibitor were proformed and the results indicated that these treatments did not affect the sensitivity of hit5 to either prolonged heat incubation or sudden heat shock treatment. These results suggest that other PFT substrates, play either a positive or a negative role in plant HSR, depending on the intensity and duration of high-temperature exposure, in an ABA-independent manner. According to published literature, some members of HSP40 family are substrates of PFT. There potential roles in the HIT5-mediated HSRs is being clarified. In addition, several revertants of hit5 were isolated by screening M2 individuals of EMS-mutagenized hit5 population. These revertants will be used to identify other genetic determinants participating in HIT5 medisted plant HSRs.
關鍵字(中) ★ 阿拉伯芥
★ 耐熱機轉
★ 基因定位
★ 蛋白質法尼酯化
關鍵字(英) ★ Heat-tolerance mechanism
★ Arabidospis
★ HEAT-INTOLERANT(HIT5)
★ Protein farnesylation
論文目次 中文摘要 i
英文摘要 ii
誌謝 iii
目錄 iv
圖表目錄 v
一、緒論 1
二、實驗材料與方法 6
1.實驗材料 6
2.耐熱回復突變株(Revertant 45)之位址定位 7
3.HSP40幼苗對抗離層酸(ABA)逆境測試 11
4.鹽分逆境對hit5及HSP40生長之測試 11
5.滲透壓逆境對於hit5及HSP40生長之測試 11
6.HSP40成株植物於開花週期之觀察 11
7.HSP40於高溫處理之耐熱性測試 12
8.HSP40 BiFC assay(bimolecular fluorescence complementation assay)質體建構 12
9.HSP40之雙分子螢光互補分析(BiFC assay)14
10.HSP40相關轉殖株於高溫處理之耐熱性測試 17
三、實驗結果 18
四、討論 23
五、參考文獻 30
六、附錄 30
1.hit5熱相關性狀不受ABA調節 55
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指導教授 吳少傑(Shaw-Jye Wu) 審核日期 2018-11-20
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