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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 鄭梅君(Mei-Chun Cheng) | |
| dc.contributor.author | Zi-Bin Huang | en |
| dc.contributor.author | 黃梓斌 | zh_TW |
| dc.date.accessioned | 2023-03-19T23:35:45Z | - |
| dc.date.copyright | 2022-10-08 | |
| dc.date.issued | 2022 | |
| dc.date.submitted | 2022-09-12 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/86077 | - |
| dc.description.abstract | 因為氣候變遷的影響，極端的氣候變化正逐漸影響全球多個農業地區，其中對植物所造成了包含高鹽、乾旱、淹水等非生物逆境，使得農作物生長面臨挑戰，人類的糧食危機也正在醞釀。植物會因應不同逆境於轉錄層次進行基因調控，其中ETHYLENE RESPONSE FACTOR 1 (ERF1) 作為植物激素—乙烯，的下游基因，可調節生物和非生物逆境反應基因的表達，並在逆境耐受中發揮重要作用。根據前人的報導，ERF1 的蛋白在黑暗中很不穩定。在這項研究中，我們透過體外和體內的免疫沉澱分析證實了ERF1蛋白可以與 CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) 蛋白進行交互作用。COP1 是一種在光訊息傳遞中起負面調控作用的 E3 連接酶；我們利用雙分子螢光系統 (BiFC) 顯示了ERF1只能在黑暗下與 COP1 結合。為了瞭解COP1是否也參與在逆境反應下，我們進行了鹽逆境的測試。結果顯示，cop1 突變體相較於野生型有較長的主根以及較高的萌芽率，而且在cop1突變體中過度表達 ERF1 會提高它們的存活率；在黑暗條件下，ERF1下游基因，如P5CS1和PDF1.2，在cop1 突變體中的表達皆高於野生型，並在 ERF1 / cop1 植物中甚至更高，代表COP1 通過負調控ERF1表現來負調節部分的逆境反應。另一方面我們也好奇ERF1是否也參與了光訊號的傳遞；在下胚軸測試中，我們發現與野生型相比ERF1過表達植物的下胚軸較野生型的短，而ERF1 RNAi植株的下胚軸較長；在黑暗進入光照的條件下，與野生型相比ERF1過表達植株中的光反應相關基因，如Fd2、HEMA1、CAB3，都有顯著較高、葉綠素含量也明顯較高表現，代表ERF1在光型態發生中扮演正向調控者的角色。此外，COP1蛋白在黑暗條件下和ACC（乙烯前驅物）處理下會在細胞核中累積，代表除了光照以外尚有其他調節COP1的機制存在。我們的研究證實了一項調控光照與逆境交互影響下的分子機制。 | zh_TW |
| dc.description.abstract | ETHYLENE RESPONSE FACTOR 1 (ERF1) regulates biotic and abiotic stress-responsive gene expression and plays important role in stress tolerance. Previous report showed that ERF1 is unstable in the dark. Here, using in vitro and in vivo immunoprecipitation assays, we showed that ERF1 could interact with CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), an E3 ligase that plays a negative role in light signaling. Moreover, bimolecular fluorescence complementation assay showed that ERF1 interacts with COP1 only in the dark condition. In our salt stress tests, cop1 mutants showed longer primary root elongation, and overexpressing ERF1 in cop1 mutants enhanced their survival rate. The expressions of ERF1 downstream genes, including P5CS1 and PDF1.2, were higher in cop1 mutants under darkness, and were even higher in ERF1/cop1 plants compared to WT, suggesting that COP1 negatively regulates stress responses through destabilizing ERF1. On the other hand, ERF1 overexpression plants showed shorter hypocotyl elongation whereas ERF1 RNAi knockdown lines showed longer hypocotyl elongation compared to WT. The light-responsive genes such as Fd2, HEMA1, CAB3 were significantly increased in ERF1 overexpression lines, suggesting that ERF1 plays a positive role in photomorphogenesis. In addition, COP1 protein accumulates in the nucleus in dark condition and under ethylene precursor treatment, indicating that there might be other mechanisms regulating COP1. Our results reveal a novel mechanism underlying the light-stress signaling crosstalk. | en |
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| dc.description.tableofcontents | 目錄 致謝 I 摘要 II 目錄 IV 圖表目錄 VII 附錄目錄 VIII 第一章 序論 1 引言 1 第一節 高鹽逆境的訊息傳導 1 第二節 阿拉伯芥中的AP2/ERF轉錄因子 2 第三節 ERF1的背景 3 第二節 COP1與植物的光暗型態發生 4 第五節 光接收體對COP1/SPA1的活性調控機制 5 第六節 實驗策略與目標 7 第二章 材料與方法 8 第一節 植物材料 8 (一) 阿拉伯芥野生型 8 (二) ERF1大量表現轉殖株的建構與篩選 8 (三) ERF1 RNAi轉殖株 與COP1-HA大量表現轉殖株 8 第二節 植物生長條件 9 (一) 種子消毒與播種 9 (二) 白化苗生長條件 9 第三節 以雙分子螢光互補系統進行蛋白質交互作用實驗 9 (一) 質體建構與製備 9 (二) 阿拉伯芥原生質體製備 9 (三) 原生質體之質體轉殖 10 第四節 免疫共沉澱分析 10 (一) 半體內免疫共沉澱 10 (二) 體外免疫共沉澱 11 第五節 西方墨點法分析 11 第六節 突變株的鹽逆境耐受性分析 12 (一) 根長測試分析 12 (二) 發芽率測試分析 12 第七節 突變株的光照敏感度分析 12 (一) 下胚軸延長測試 12 (二) 短暫紅光誘導之種子發芽率測試 13 第八節 葉綠素含量分析 13 第九節 基因表現量分析 13 (一) RNA萃取 13 (二) cDNA 合成 14 (三) Real-time qPCR 14 第十節 核蛋白分離實驗 14 第三章 結果 16 第一節 透過BiFC分析證實ERF1和COP1在光照與黑暗中的交互作用 16 第二節 透過pull-down與共免疫沉澱分析證實ERF1和COP1的交互作用 16 第三節 鹽逆境下COP1缺失有利於根的生長 17 第四節 COP1缺失提升ERF1下游的逆境反應基因表現 17 第五節 ERF1大量表現抑制黑暗中下胚軸生長 18 第六節 ERF1大量表現促進光照誘導的種子發芽 18 第七節 ERF1大量表促進葉綠素的生合成 19 第八節 ERF1大量表現提升光反應相關基因表現量 19 第九節 乙烯訊號使核內COP1蛋白不穩定 20 第四章 討論 21 第一節 ERF1在黑暗下與COP1交互作用並可能受到其泛素化降解 21 第二節 乙烯處理會使COP1在核內不穩定 21 第三節 COP1會透過ERF1影響其下游目標基因來部分負調控逆境反應 22 第四節 ERF1促進了植物的光型態發生並被COP1負向調控 22 第五節 未來研究方向 23 第六節 結論 23 參考資料 25 圖表 32 附錄 44 圖表目錄 Figure 1. ERF1和COP1的BiFC實驗分析ERF1與COP1的交互作用 36 Figure 2. ERF1與COP1的體外免疫共沉澱分析ERF1與COP1的交互作用 37 Figure 3. ERF1與COP1的半體內免疫共沉澱分析ERF1與COP1的交互作用 38 Figure 4. cop1突變株在鹽逆境下的根生長測試 39 Figure 5. cop1突變株在鹽逆境下的發芽率測試 40 Figure 6. cop1突變株在鹽逆境下的ERF1下游逆境反應基因mRNA表現 41 Figure 7. ERF1 大量表現與RNAi轉殖株的下胚軸生長測試 42 Figure 8. ERF1大量表現與RNAi轉殖株的發芽率測試 43 Figure 9. ERF1 大量表現與RNAi轉殖株的葉綠素含量測試 44 Figure 10. ERF1 大量表現與RNAi轉殖株的光反應相關基因表現調控測試 45 Figure 11. 黑暗與乙烯處裡下的COP1蛋白累積測試 46 Table 1. qPCR所用的引子列表 47 附錄目錄 附錄1、植物體內的鹽訊號傳遞模型 44 附錄2、AP2/ERF1在激素路徑中的角色 45 附錄4、以Y2H 驗證ERF1與COP1之蛋白質間的交互作用 47 附錄5、各轉殖株的ERF1基因表現量 48 附錄6、COP1將ERF1泛素化 49 附錄7、ERF1下游所調控的各種逆境反應相關基因 50 | |
| dc.language.iso | zh-TW | |
| dc.title | 探討阿拉伯芥中COP1與ERF1的交互作用以及光照與逆境交互影響下的生理功能 | zh_TW |
| dc.title | Investigating the interaction between ERF1 and COP1 and their functions in light-stress signaling crosstalk in Arabidopsis | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 110-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 常怡雍(Yee-Yung Charng),楊健志(Chien-Chih Yang),洪傳揚(Chwan-Yang Hong),許富鈞(Fu-Chiun Hsu) | |
| dc.subject.keyword | 鹽逆境,光型態發生,COP1,ERF1,阿拉伯芥, | zh_TW |
| dc.subject.keyword | Arabidopsis,ERF1,COP1,photomorphogenesis,salinity stress, | en |
| dc.relation.page | 52 | |
| dc.identifier.doi | 10.6342/NTU202203297 | |
| dc.rights.note | 同意授權(全球公開) | |
| dc.date.accepted | 2022-09-13 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科技學系 | zh_TW |
| dc.date.embargo-lift | 2023-08-10 | - |
| 顯示於系所單位： | 生化科技學系 | |
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