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研究生:陳秋伶
研究生(外文):Chiu-Ling Chen
論文名稱:阿拉伯芥中與調控花藥開裂之DAF作用蛋白質之功能性分析
論文名稱(外文):Characterization of the DAF interacting proteins in regulating anther dehiscence in Arabidopsis thaliana
指導教授:楊長賢楊長賢引用關係
口試委員:楊俊逸林彩雲
口試日期:2017-07-20
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生物科技學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2017
畢業學年度:105
語文別:中文
論文頁數:78
中文關鍵詞:花藥不開裂DAF花器發育
外文關鍵詞:anther dehiscenceDAFflower development
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DAF 【DEFECTIVE IN ANTHER DEHISCENCE1 (DAD1)-Activating Factor】 是一個阿拉伯芥中透過活化茉莉酸 (JA) 生合成途徑中DAD1基因之表現以調控花藥開裂的RING-finger E3 ligase蛋白。本實驗室利用酵母雙雜合系統尋找可能被DAF降解之候選目標受質蛋白質,選定CSD190、CSD760和CSD220進行研究。本研究藉由分析候選目標受質蛋白質的功能性,推測其與DAF之間的關聯性。實驗結果顯示,在阿拉伯芥中,異位大量表現CSD190、CSD760及CSD220皆會出現植株在花朵發育過程中花藥無法正常開裂而導致雄不稔之外表型。且在這些轉殖株中也發現DAD1的表現量受到抑制而下降,此性狀與前人發表之35S:DAF antisense/RNAi結果相似。而以測定花粉活性的亞歷山大染色法針對花藥進行染色,結果顯示35S::CSD190、35S::CSD760與35S::CSD220轉殖株的花粉與野生型的花粉一樣具有活性。再者,將野生型植株的花粉經人工方式授粉至其柱頭上,可觀察到正常延長的果莢,表示轉殖株雌蕊的功能正常無虞。特別的是,35S::CSD190轉殖株中花粉管有提早萌發的現象,且啟動子表現位置分析結果顯示其在花粉發育中晚期也有所表現,文獻指出CSD190為一Ubiquitin-like蛋白,推測CSD190可能在花粉發育中晚期與其他E3 ligase蛋白作用以調控花粉發育,因此在35S::CSD190轉殖株中,花粉早期表現CSD190導致不稔性狀產生。而CSD190在花朵發育階段12後,其表現與DAF在雄蕊上的位置重疊,但蛋白表現並未受到降解,因此推測CSD190與DAF為共同作用關係,而並非受DAF降解之受質蛋白。而CSD760與CSD220因表現位置與DAF類似,且異位大量表現轉殖株性狀與35S:DAF antisense/RNAi相似,故推測CSD760和CSD220仍具受DAF降解之潛力。再者,關於雌蕊的部分,DAFL1是一個與DAF相似的蛋白,作用同為E3 ligase。且DAFL1單獨表現於雌蕊上,依據表現位置推測CSD190在雌蕊上也是扮演協助DAFL1的角色。CSD760於雌蕊的表現也是和DAF有相同的情況,推測可能是屬於DAFL1的受質。這些實驗結果皆有助於了解DAF與其作用蛋白質在植物中調控花藥開裂的機制,但仍需更多的實驗加以闡述其相互關係。
DAF 【DEFECTIVE IN ANTHER DEHISCENCE1 (DAD1)-Activating Factor】, a RING-finger E3 ligase protein, regulates anther dehiscence by activating the expression of DAD1 in the jasmonic acid (JA) biosynthesis pathway. To identify the putative targets which will be degraded by DAF, yeast two-hybrid analysis was performed and several candidate target proteins such as CSD190, CSD760 and CSD220 were identified. In this study, we further investigated the function of these candidate proteins. The ectopic expression of either CSD190, CSD760 or CSD220 in Arabidopsis showed similar phenotype of anther indehiscence and male sterility throughout flower development. The expression of DAD1 was also down-regulated in the over-expressing transgenic plants. This result are similar to that of 35S::DAF antisense/RNAi flowers. The result of Alexander’s staining indicated that pollen viability of the 35S::CSD190, 35S::CSD760 and 35S::CSD220 transgenic plants is similar to the wild-type pollen. When manually pollinating the transgenic pollen to its stigma, normal elongation of the siliques was observed. This indicates that the function of the pistil for the transgenic plants is normal. Especially, 35S::CSD190 plants showed earlier germination of pollen tube. In pCSD190::GUS plants, the GUS activity was detected in late stages of pollen development but the GUS activity of pDAF::GUS was not detected in this stage. We suggested that CSD190 may play a role in pollen development with other E3 ligase proteins to regulate pollen development since CSD190 is an ubiquitin-like protein and 35S::CSD190 plants showed male-sterility. The expression pattern of CSD190 overlapped with the expression of DAF in stamens after the flower development stage 12. However, CSD190 seemed not to be degrade by DAF, thus CSD190 and DAF may has auxiliary relationship. CSD760 and CSD220 still have the potential to be the substrate of DAF since the expression pattern of CSD760 and CSD220 is similar to the pattern of DAF. 35S::CSD760 and 35S::CSD220 plants also showed similar phenotype to 35S::DAF antisense/RNAi plants. Furthermore, on the part of the pistil, DAFL1 is a protein similar to DAF, acting as E3 ligase. DAFL1 is shown on the pistil alone. According to the performance location, inference CSD190 in the pistil is also playing to help DAFL1 role.
CSD760 is located in the pistil position and DAF is the same, suggesting that may belong to the DAFL1 substrate. These results provided new information for understanding the mechanism of DAF and its interacting proteins in regulating anther dehiscence in plants. Further experiments will be performed for investigating the correlation.
摘要 i
ABSTRACT ii
前言 1
一、 泛素/26S蛋白酶體路徑 (Ubiquitin/26S proteasome pathway) 1
二、 Ubiquitin/26S proteasome pathway參與之蛋白 1
三、 RING-finger domain 4
四、 雄蕊發育與雄不稔 (Stamen development and male-sterile) 5
五、 茉莉酸生合成 (Biosynthesis of jasmonic acid) 5
六、 實驗室前人之相關研究 6
七、 研究動機與目的 7
材料與方法 10
結果 18
一、 DAF候選作用蛋白質特性分析 18
二、 DAF候選作用蛋白質 (CSD) 於阿拉伯芥之表現分析 18
三、 大量表現DAF候選作用蛋白質轉殖株之功能性分析 21
四、 雄不稔性狀之轉殖株茉莉酸處理試驗 22
五、 雄不稔性狀之轉殖株雜交試驗 23
討論 24
參考文獻 28
圖表 33
表1、本論文研究所使用之引子 (primer) 序列 33
圖1、CSD190之編碼序列 (coding sequence) 與胺基酸序列 35
圖2、CSD760之編碼序列與胺基酸序列 36
圖3、CSD220之編碼序列與胺基酸序列 37
圖4、CSD190於野生型阿拉伯芥之各部位表現量 38
圖5、CSD760於野生型阿拉伯芥之各部位表現量 39
圖6、CSD220於野生型阿拉伯芥之各部位表現量 40
圖7、CSD基因於阿拉伯芥雄蕊中各時期表現量 41
圖8、pCSD190::GUS的載體構築與分子選殖 42
圖9、pCSD760::GUS的載體構築與分子選殖 43
圖10、pCSDs:: CSDs-GUS的載體構築與分子選殖 44
圖11、pCSD190::GUS啟動子活性分析 46
圖12、pCSD190:: CSD190-GUS啟動子活性分析 47
圖13、pCSD760::GUS啟動子活性分析 48
圖14、大量表現CSD基因的分子選殖 49
圖15、大量表現CSD基因之轉殖株性狀分析 50
圖16、大量表現CSD190轉殖株表現量分析 52
圖17、大量表現CSD760轉殖株表現量分析 53
圖18、大量表現CSD220轉殖株表現量分析 54
圖19、大量表現CSD基因轉殖株之果莢透化 55
圖20、大量表現CSD基因轉殖株之亞歷山大染色 56
圖21、大量表現CSD190轉殖株之花藥內部螢光圖 57
圖22、大量表現CSD基因轉殖株內CSD與DAD1基因表現量 58
圖23、大量表現CSD基因轉殖株與野生型阿拉伯芥雄蕊授粉 59
圖24、大量表現CSD基因轉殖株自花授粉 60
圖25、CSD190於花器中作用之假設模型圖 61
圖26、CSD與DAF作用假設模型圖 62
附圖 63
附圖1、Ubiquitin (Ub) / 26S proteasome pathway示意圖-1 63
附圖2、Ubiquitin (Ub) / 26S proteasome pathway示意圖-2 64
附圖3、各種不同形式之E3複合體示意圖 65
附圖4、RING finger domain示意圖 66
附圖5、阿拉伯芥花藥開裂示意圖 67
附圖6、茉莉酸生合成途徑 68
附圖7、花粉成熟、花藥開裂和開花同步調控模型 69
附圖8、DAF在阿拉伯芥中調控花藥開裂的功能模型 70
附圖9、酵母菌雙雜交原理 71
附圖10、過氧化物之膜蛋白複合體模型 72
附圖11、pGEM®-T Easy vector之圖譜 73
附圖12、pEpyon-01K之載體圖譜 74
附圖13、pEpyon-32K之圖譜 75
附圖14、花發育階段示意圖 76
附圖15、DAF:GUS 啟動子表現模式圖 77
附圖16、DAFL1:GUS 啟動子表現模式圖 78
許巍瀚 (2012)。阿拉伯芥中調控細胞分裂與配子體發育相關基因之功能性分析。
國立中興大學生物科技學研究所。博士論文。

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