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研究生:彭妍筑
研究生(外文):Yan-Jhu Peng
論文名稱:阿拉伯芥中控制雄蕊發育之AtRING3和AtRING4基因之選殖與功能性分析
論文名稱(外文):Characterization and functional analysis of AtRING3 and AtRING4 genes in regulating stamen development in Arabidopsis thaliana
指導教授:楊長賢楊長賢引用關係
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:2009
畢業學年度:97
語文別:中文
論文頁數:67
中文關鍵詞:泛素
外文關鍵詞:E3 ligase
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真核生物中E3蛋白(E3 ligase),藉由形成ubiquitin/26S proteasome(泛素-蛋白酶體)來調控蛋白質降解並在植物生長發育、荷爾蒙調節、抵抗逆境及防禦機制中扮演很重要的角色。而E3中的RING (Really Interesting New Gene) finger基因能專一辨識目標蛋白,藉由此RING finger domain與泛素-E2複合體結合,進而將目標蛋白泛素化(ubquitation),而使其進入26S protesome路徑將目標蛋白降解。為了研究E3 RING finger的功能,本研究由阿拉伯芥中選殖AtRING3和AtRING4來進行功能性分析。AtRING3及AtRING4均含有RING domain,分別轉譯成176及159個氨基酸。在mRNA轉錄層次的表現上,AtRING3在一周和兩周大的幼苗時期幾乎不表現,隨著生長發育在成熟植株中表現量逐漸提高,特別是在花苞表現量最高,而在營養葉表現量最低。而在AtRING4啟動子分析中,同樣發現在幼苗時期的頂芽分生組織及幼葉有明顯表現,而在花中之雌蕊、花萼和花瓣有高量的表現。在大量異位表現反向基因來抑制AtRING3或AtRING4基因的轉殖株中,都發現有花絲變短與花藥不開裂的雄不稔的性狀,與報導之defective in anther dehiscence1 (dad1) 突變株性狀相似。進一步的分析發現,抑制AtRING3突變株中,雄不稔之性狀與茉莉酸(JA)的生合成基因DEFECTIVE IN ANTHER DEHISCENCE1 (DAD1)的表現量顯著下降有關。而在大量異位表現反向AtRING3不孕轉殖株中外加MeJA處理,能促使轉殖株花藥開裂,並得到正常延長發育的果莢。綜合以上結果,推測AtRING3和AtRING4參與了阿拉伯芥的生長發育,尤其和調控茉莉酸之生合成及後續雄蕊的成熟發育及花藥開裂有關。
E3s play an important regulatory role in the control of protein degradation processes via the ubiquitin/26S proteasome pathway in eukaryotes. E3 RING (Really Interesting New Gene) finger is a particular class of zinc-finger domain that uses a particular disposition of cysteine and histidine residues to bind two zinc ions. To investigate the E3s function, AtRING3 and AtRING4 were isolated and characterized from Arabidopsis. AtRING3 encoded a 176 amino acid protein and AtRING4 encoded a 159 amino acid protein, both containing conserved RING domain. AtRING3 mRNA was absent in seeding and was detected in mature plants. AtRING3 mRNA was detected higher in flower than in the other organs in mature plants. AtRING4 mRNA was detected throughout the development. Further promoter assay by transforming constructs fusing the promoter of these genes with report GUS gene in Arabidopsis indicated GUS was expressed in stamen during early flower development and was detected in filament and anther- filament junction region of mature flower in AtRING3::GUS plants. GUS was detected in shoot apical meristem, young leaves, sepal, petal and carpel of flowers in AtRING4::GUS plants. Transgenic plants that ectopically expressed antisense or RNAi of AtRING3 resulted in male- sterility due to the indehiscence of the anther and production of short filament similar to that observed in defective in anther dehiscence1 (dad1) mutants. Similar male- sterility was also observed in transgenic plants ectopically expressed antisense of AtRING4. Further analysis indicated that the DAD1 expression was significantly down-regulated in AtRING3 RNAi mutants and external application of jasmonic acid (JA) rescued the un-dehiscence of the anthers in 35S::AtRING3 RNAi flowers. These results indicated that AtRING3/AtRING4 are likely the homologues with redundant function in controlling anther dehiscence during stamen development by regulating JA activity through activating the expression of DAD1.
中文摘要 iii
英文摘要 iv
前言 1
材料方法 8
結果 17
一、 AtRING3和AtRING4基因之特性分析 17
二、 AtRING3和AtRING4於阿拉伯芥野生型的表現情形 17
三、 AtRING3和AtRING4啟動子表現分析 18
(一) 啟動子片段的選殖與構築 18
(二)  AtRING3與AtRING4之啟動子活性分析(promoter assay) 18
四、 AtRING3、AtRING4轉殖植株之功能性分析 19
(一) 大量表現正向與反向基因之構築 19
(二)  AtRING3轉殖植株之性狀分析 19
(三)  AtRING4轉殖植株之性狀分析 20
(四) RNAi構築體(RNA interference) 21
(五)  RNAi轉殖植株性狀之分析 22
(六)  不孕性狀轉殖株內相關基因的表現量 22
五、35S::anti-AtRING3不孕植株荷爾蒙試驗 22
討論 24
參考文獻 28
圖表 32

圖表目次
表1、本研究所使用的引子(primer)序列 32
圖1、阿拉伯芥AtRING3 (At5g05280) mRNA序列與其相對氨基酸 34
圖2、阿拉伯芥AtRING4 (At5g01880) mRNA序列與其相對氨基酸 35
圖3、ATL蛋白結構與AtRING1、AtRING3和AtRING4氨基酸序列比對 36
圖4、RING-H2蛋白之演化樹分析 37
圖5、AtRING3於野生型阿拉伯芥之表現量分析 38
圖6、AtRING4於野生型阿拉伯芥之表現量分析 39
圖7、阿拉伯芥中AtRING3之啟動子的選殖 40
圖8、阿拉伯芥中AtRING4之啟動子的選殖 41
圖9、AtRING3之啟動子活性分析 42
圖10、AtRING4之啟動子活性分析 43
圖11、鑑定大量表現sense與anti-sense AtRING3和大量表現sense與anti-sense AtRING4之構築體 44
圖12、大量表現anti-AtRING3轉殖株之性狀分析 45
圖13、大量表現anti-AtRING3轉殖株表現量分析 46
圖14、利用共軛焦顯微鏡觀察大量表現anti-AtRING3轉殖株雄蕊 47
圖15、大量表現35S::AtRING3 sense轉殖株之分子鑑定轉殖株 48
圖16、大量表現anti-AtRING4轉殖株之性狀分析 49
圖17、大量表現anti-AtRING4轉基因植物表現量分析 50
圖18、AtRNG3 RNA interference之構築 51
圖19、AtRNG4 RNA interference之構築 52
圖20、AtRING3 RNA interference轉殖株之性狀分析 53
圖21、AtRING3、AtRING4 RNAi轉殖株鑑定分析 54
圖22、不孕轉殖株內 AtRING3、AtRING4與DAD1基因表現量 55
圖23、大量表現anti-AtRING3不孕植株茉莉酸互補實驗 56
附圖1、Ubiquitin (Ub) / 26S proteasome pathway示意圖 57
附圖2、不同形式之E3複合體示意圖 58
附圖3、花藥開裂的過程 59
附圖4、茉莉酸同步調控花粉成熟,花藥開裂與花器開啓的模示圖 60
附圖5、pGEM®-T Easy vector之載體圖譜(3015 bp) 61
附圖6、pEpyon 01K系列之載體圖譜 62
附圖7、pBI121之載體圖譜(13 kb) 63
附圖8、進行RNAi之轉接載體pBlueACTi 64
附圖9、pBI-mGFP1載體 65
附圖10、大量表現anti-NAC2轉殖株性狀 66
附圖11、AtRING3、AtRING4與其交互作用基因功能假說圖 67
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