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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 楊健志(Chien-Chih Yang) | |
| dc.contributor.author | Ching-Hsing Liang | en |
| dc.contributor.author | 梁景興 | zh_TW |
| dc.date.accessioned | 2021-06-15T16:14:06Z | - |
| dc.date.available | 2016-08-20 | |
| dc.date.copyright | 2015-08-20 | |
| dc.date.issued | 2015 | |
| dc.date.submitted | 2015-08-18 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/52412 | - |
| dc.description.abstract | 由酵母菌雙雜合篩選到INDUCER OF CBF EXPRESSION 1 (ICE1) 會與HEPTAHELICAL PROTEIN 1 (HHP1) 的N端產生交互作用。ICE1調控冷逆境訊息傳導被認為是一種離層酸非依賴性 (ABA-independent) 的路徑。本論文發現ICE1可能也會參與離層酸依賴性 (ABA-dependent) 的訊息傳導路徑。當ICE1的T-DNA插入突變株ice1-2播種在缺乏糖的1/2 MS培養基上時，幼苗長成率會明顯下降；但培養基中添加56 mM葡萄糖之後，幼苗長成率就可以回復到與野生型相同的程度。除了糖依賴性的性狀之外，ice1-2的發芽率和幼苗長成率對高濃度葡萄糖的感受性也比野生型高。另外ice1-2也觀察到對離層酸有過敏感 (hyper-sensitive) 性狀，推測ice1-2對葡萄糖的過敏感性狀可能是經由離層酸訊息傳導路徑產生的。葡萄糖和離層酸處理會使幼苗時期的ice1-2中兩個與離層酸訊息傳導有關基因ABA-INSENSITIVE 3 (ABI3) 和ABA-INSENSITIVE 4 (ABI4) 的表現量有過度誘導的現象。除了會參與冷逆境反應、氣孔發育和胚乳降解之外，本論文發現ICE1是離層酸依賴性路徑的一個負調控子。 | zh_TW |
| dc.description.abstract | INDUCER OF CBF EXPRESSION 1 (ICE1) was identified as an interaction partner of N-terminus of HEPTAHELICAL PROTEIN 1 (HHP1) by yeast two-hybrid. ICE1 mediates the cold stress signal via an abscisic acid (ABA)-independent pathway. A possible role of ICE1 in ABA-dependent pathways was identified in this study. Seedling growth was severely reduced in a T-DNA insertion mutant of ICE1, ice1-2, when grown on 1/2 MS medium lacking sugars, but was restored to wild-type (WT) levels by supplementation with 56 mM glucose. In addition to this sugar-dependent phenotype, germination and establishment of ice1-2 were more sensitive to high glucose concentrations than in the WT. Hypersensitivity to ABA was also observed in ice1-2, suggesting its sensitivity to glucose might be mediated through the ABA signaling pathway. Glucose and ABA induced much higher expression of two genes related to ABA signal transduction, ABA-INSENSITIVE 3 (ABI3) and ABA-INSENSITIVE 4 (ABI4), in ice1-2 than in the WT during establishment. In summary, in addition to its known roles in regulating cold responses, stomatal development, and endosperm breakdown, ICE1 is a negative regulator of ABA-dependent responses. | en |
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| dc.description.tableofcontents | 謝誌 1
目錄 3 摘要 7 Abstract 8 縮寫表 9 中英文對照表 11 第一章 前言 14 1.1. 糖訊息傳導 14 1.2. 滲透壓逆境的轉錄調控網路 15 1.3. 離層酸生合成 (biosynthesis) 17 1.4. 離層酸訊息傳導 18 1.5. Heptahelical Protein 1 (HHP1) 19 1.6. Inducer of CBF Expression 1 (ICE1) 20 1.7. 研究動機 21 第二章 材料與方法 22 2.1. 實驗材料 22 2.2. 實驗藥品 25 2.3. 儀器設備 27 2.4. 實驗方法 27 2.4.1. DNA限制酶切 27 2.4.2. 聚合酶鏈鎖反應 (polymerase chain reaction, PCR) 27 2.4.3. 洋菜糖膠體電泳 28 2.4.4. 甲醛洋菜糖膠體電泳 29 2.4.5. 分離與純化DNA片段 29 2.4.6. 純化PCR產物 30 2.4.7. 核酸定量 30 2.4.8. DNA接合反應 (Ligation) 30 2.4.9. T-A選殖 (T-A cloning) 30 2.4.10. 大腸桿菌熱衝擊法 (heat-shock) 轉形 31 2.4.11. 藍白篩選 31 2.4.12. 菌落PCR檢定法 31 2.4.13. 大腸桿菌質體DNA小量製備 32 2.4.14. 阿拉伯芥基因體DNA之抽取 32 2.4.15. 阿拉伯芥Total RNA之抽取 33 2.4.16. DNase處理 33 2.4.17. 反轉錄反應 (reverse transcription) 34 2.4.18. 定量式反轉錄聚合酶鏈鎖反應 (quantitative RT-PCR, qRT-PCR) 34 2.4.19. 定量式反轉錄聚合酶鏈鎖反應數據分析 35 2.4.20. 酵母菌雙雜合分析 35 2.4.21. 酵母菌勝任細胞製備 36 2.4.22. 酵母菌高效率轉形法 36 2.4.23. 計算酵母菌轉形效率 37 2.4.24. 挑選酵母菌雙雜合分析的正反應株 37 2.4.25. 純化酵母菌質體 37 2.4.26. 分析獵物 (prey) 質體中的cDNA序列 38 2.4.27. 建構HHP1的釣餌與獵物載體 38 2.4.28. 建構ICE1的釣餌與獵物載體 39 2.4.29. 阿拉伯芥種子表面消毒及低溫處理 39 2.4.30. 阿拉伯芥種子無菌培養 39 2.4.31. 阿拉伯芥種子土壤培養 39 2.4.32. 收集阿拉伯芥種子 40 2.4.33. 製備農桿菌勝任細胞與轉形 40 2.4.34. 農桿菌花序浸潤法 41 2.4.35. 篩選轉殖株的同型合子 (homozygote) 41 2.4.36. 鑑定T-DNA插入突變株 41 2.4.37. 篩選ICE1基因剔除突變株 43 2.4.38. 建構HHP1/ICE1雙重基因剔除突變株 43 2.4.39. 建構complementary ice1-2轉殖株 (c-ice1-2) 44 2.4.40. 分析種子與幼苗對葡萄糖、甘露醇、離層酸和fluridone的感受性 45 第三章 結果 46 3.1. 以酵母菌雙雜合分析篩選可能與HHP1有交互作用的蛋白質 46 3.2. 篩選ice1-2基因剔除突變株、建立hhp1-1/ice1-2雙重基因剔除突變株和建構ice1-2的互補轉殖株c-ice1-2 47 3.3. ice1-2的糖依賴性狀 (sugar-dependent) 47 3.4. ice1-2的葡萄糖過敏感性狀 48 3.5. 在不同濃度葡萄糖處理下ice1-2中基因表現量變化 49 3.6. ice1-2的離層酸過敏感性狀 51 3.7. 離層酸處理下ice1-2中基因表現量變化 51 第四章 討論 53 4.1. HHP1可能的交互作用蛋白質 53 4.2. 離層酸依賴性與離層酸非依賴性訊息傳導路徑 53 4.3. ICE1調控離層酸訊息傳導基因的表現 54 4.4. ice1-2中被表現的ICE1片段 55 4.5. 糖-離層酸-ICE1訊息傳導路徑 56 4.6. ICE1與HHP1的蛋白質交互作用 56 第五章 未來展望 58 第六章 參考文獻 59 圖與表 68 Fig. 1. Interaction candidates of pGBHHP1-N by yeast two-hybrid screening with cDNA library CD4-30. 68 Fig. 2. Interaction test of pGBHHP1-N and pGADICE1 and self-activation test of pGADICE1. 69 Fig. 3. Genotype and HHP1 and ICE1 expression analysis of the WT, the hhp1-1, the ice1-2 mutant, and the hhp1-1/ice1-2 mutant 70 Fig. 4. Genotype and ICE1 expression analysis of the WT, the ice1-2 mutant, and two c-ice1-2 mutants. 72 Fig. 5. Both ice1-2 and hhp1-1/ice1-2 display a sugar-dependent phenotype. 73 Fig. 6. ice1-2 displays a sugar-dependent phenotype. 75 Fig. 7. ice1-2 displays a sugar-hypersensitive phenotype. 78 Fig. 8. Higher induction of ABA signaling gene expression in ice1-2 in response to glucose. 80 Fig. 9. ice1-2 is also hypersensitive to ABA. 81 Fig. 10. Higher expression of ABA signaling genes in ice1-2 in response to ABA treatment. 82 Fig. 11. Expression of ABI3 and ABI4 in the WT, the ice1-2 mutant, and the two c-ice1-2 mutants in response to glucose and ABA. 83 Fig. 12 Putative partial ICE1 fragment expressed in ice1-2. 84 Fig. 13. A proposed model of the role of ICE1 in the cross-talk between ABA-dependent and ABA-independent pathways. 85 Table 1. Candidates of HHP1 interacting proteins obtained from CD4-30 library by yeast two-hybrid screening. 86 Table S1. Primer pairs used for the quantitative RT-PCR analysis. 87 Table S2. Primer pairs used for general PCR and cloning. 88 問答集 89 | |
| dc.language.iso | zh-TW | |
| dc.title | 阿拉伯芥ICE1在離層酸依賴性路徑之功能研究 | zh_TW |
| dc.title | Functional Studies of Arabidopsis ICE1 in Abscisic Acid Dependent Pathways | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 103-2 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 蘇仲卿,王愛玉,常怡雍,陳佩燁 | |
| dc.subject.keyword | HHP1,ICE1,離層酸,阿拉伯芥, | zh_TW |
| dc.subject.keyword | HEPTAHELICAL PROTEIN 1 (HHP1),INDUCER OF CBF EXPRESSION 1 (ICE1),abscisic acid (ABA),Arabidopsis, | en |
| dc.relation.page | 98 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2015-08-18 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科技學系 | zh_TW |
| 顯示於系所單位： | 生化科技學系 | |
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