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研究生:張廉仕
研究生(外文):Lien-Shih Chang
論文名稱:阿拉伯芥中兩個E3 RING finger基因之功能性分析及文心蘭C與E MADS box基因RNAi載體之構築
論文名稱(外文):Functional analysis of two E3 RING finger genes in Arabidopsis thaliana and construction of RNAi constructs for C and E functional MADS box genes in Oncidium ‘Gower Ramsey’
指導教授:楊長賢楊長賢引用關係
指導教授(外文):Chang-Hsien Yang
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:2008
畢業學年度:96
語文別:中文
論文頁數:89
中文關鍵詞:阿拉伯芥文心蘭泛素開花基因RING9基因RING10基因
外文關鍵詞:Arabidopsisubiquitinflowering geneRING9RING10
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Ubiquitin (Ub) / 26S proteasome參與植物調節生長發育與荷爾蒙訊息等多方面生理功能的調控,而胺基酸的序列上有保守RING-finger domain的RING (Really Interesting New Gene) E3接合酶(E3 ligase)更是扮演重要的角色。本研究中從阿拉伯芥中選殖在演化樹上屬於同一個子群的AtRING9、10,進行功能性的分析。透過RT-PCR定量的方式發現AtRING9在14天幼年植株、腋葉、花苞及根部大量表現;而啟動子驅動GUS報導基因的表現,發現AtRING9主要表現在新生葉、小花苞、頂芽分生組織、葉脈組織及根部。AtRING10在RT-PCR定量中各部位都有表現,在幼年植株、新生花苞及根表現較多,而啟動子驅動GUS報導基因的表現,發現AtRING10主要表現在新生葉、小花苞、頂芽分生組織及根部。初步研究發現去除RING finger domain的AtRING9及全長之AtRING10,在接合GFP螢光分析結果中證實不會進核。透過異位大量表現AtRING10可以發現轉殖株的花序發育遭到抑制或造成終結花序,若異位大量表現反股AtRING10則發現轉殖植株失去頂芽優勢造成花序叢生及葉子捲曲。綜合上述,AtRING9、10對於新生組織特別是分生組織的發育應有一定的影響,而透過轉殖植物觀察進一步推論AtRING10對於分生組織及後續花序的發育扮演負調控的角色 (第一章)。
MADS box基因群對於植物花器生成佔重要的地位,而總共有五群MADS box基因依循一定模式相互協同調控一朵花的生成,所遵循的就是ABCDE model。在阿拉伯芥中,如果A功能基因失去功能會導致花器沒有花瓣與花萼,只有雄蕊跟雌蕊;若B功能基因失去功能會導致花器沒有雄蕊,只有花萼跟雌蕊;若C功能基因失去功能會導致花變成只具花萼及花瓣之重瓣花;E功能基因失去功能會導致所有花器被葉狀的萼片取代。本研究以阿拉伯芥MADS box基因的突變性狀為基準,透過RNAi技術抑制文心蘭C (OMADS4) 或E (OMADS6、7、11) 功能性MADS box基因表現,期望能在文心蘭當中看見重瓣花或是都是萼片的花,藉此創造新的文心蘭花型,也更進一步的確認實驗室所選殖到的OMADS box基因對文心蘭花器生成與調控所扮演真正的角色。本實驗因此對OMADS4設計其專一序列構築單一基因之RNAi構築體,而對E 功能之OMADS6、7、11則構築同時含三個基因專一序列之RNAi之構築體,以利後續文心蘭轉殖之實驗,期望能對多基因達到抑制效果 (第二章)。
E3s RING (Really Interesting New Gene) finger proteins contain a conserved RING motif and play key roles in ubiquitin-mediated proteolysis which has a central role in many processes of plant development. Two RING genes, AtRING9 and AtRING10, were isolated and characterized from Arabidopsis. AtRING9 mRNA is highly expressed in 14-d-old seedling, cauline leaf, flower buds and roots. Further promoter assay by transformed constructs containing AtRING9 promoter fused with report GUS gene indicated AtRING9 is expressed in young leaf, flower buds, SAM (shoot apical meristem), veins and roots. AtRING10 mRNA is expressed in all the organs tested with relatively high expression in seedling, flower buds and roots. Further promoter assay by transformed constructs fused the promoter of AtRING10 with report GUS gene indicated AtRING10 expressed in young leaf, flower buds, SAM and roots. Further analysis indicated that AtRING9 without RING finger domain and the full length of AtRING10 could not enter nucleus by fused with GFP. Ectopic expression of sense and anti-sense for AtRING10 was performed and phenotypic analyzed. The result indicated that sense transgenic plants delayed the flowering time and produced terminal flowers. The anti-sense transgenic plants lost apical dominance and produced curly leaves. These data indicated that AtRING9 and 10 may play roles in regulating SAM activity and controlling the inflorescence development (Chapter 1).
MADS box gene played a critical roles in floral organ development. ABCDE model predicts the formation of flower organ by the interaction of five classes of MADS box genes in plant. To generate transgenic flowers with novel phenotype of Oncidium ‘Gower Ramsey ’ with silence of the C (OMADS4) or E (OMADS6、7、11) function genes, RNAi strategy was performed. RNAi construct contained conserved region specific for OMADS4 was constructed. In addition, three conserved regions, specific for OMADS6, 7, 11 respectively, were constructed in the same RNAi construct. Further transformed these constructs into Oncidium in the future will expect to generate transgenic plants with novel flower phenotype (Chapter 2).
總摘要(中文)
總摘要(英文)

第一章 阿拉伯芥中兩個E3 RING finger基因之功能性分析
中文摘要 2
英文摘要 3
壹、前言 4
貳、材料方法 9
参、結果 18
一、 AtRING9、10之生物資訊 18
二、AtRING9、10於野生型阿拉伯芥之表現情形 18
三、AtRING9、10啟動子表現分析 19
(一)啟動子片段構築於含GUS報導基因載體 19
(二)AtRING9(P)、10 (P)之啟動子活性分析(promoter assay) 20
四、AtRING10轉殖植株之功能性分析 20
(一)大量表現正向與反向基因的構築體 20
(二)RNAi (RNA interference)構築體 21
(三)轉殖植株性狀之分析與鑑定 22
(四)轉基因植物的基因表現分析 22
(五)AtRING10在細胞層次的表現位置(cellular localization) 22
1. 阿拉伯芥轉殖植株 23
2. 農桿菌滲透法 23
五、AtRING9轉殖植株之建立 23
(一) RNAi (RNA interference)構築體 23
(二) AtRING9在細胞層次的表現位置(cellular localization) 24
1. 阿拉伯芥轉殖植株 24
2. 農桿菌滲透法 25
(三) 大量表現正向與反向基因的構築體 25
肆、討論 27
伍、參考文獻 30
陸、圖表
表1-1、本研究所使用之核酸引子(primer)序列 34
圖1-1、RING基因之演化樹圖譜 36
圖1-2、阿拉伯芥可轉譯出RING finger domain之AtRING9的cDNA序列 37
圖1-3、阿拉伯芥AtRING10的cDNA序列 38
圖1-4、AtRING9相關之生物資訊分析 39
圖1-5、AtRING10相關之生物資訊分析 40
圖1-6、AtRING9和AtRING10之胺基酸序列比對 41
圖1-7、阿拉伯芥轉譯出不具RING finger domain之AtRING9的cDNA序列 42
圖1-8、利用RT-PCR偵測AtRING9和AtRING10基因於野生型阿拉伯芥中的表現量 43
圖1-9、以PCR方法選殖AtRING9(P)基因啟動子(1.4 kb),並構築到pEpyon-01K載體中 44
圖1-10、以PCR方法選殖AtRING10(P)基因啟動子(1.3 kb),並構築到pEpyon-01K載體中 45
圖1-11、以GUS組織化學染色進行AtRING9基因啟動子之分析 46
圖1-12、以GUS組織化學染色進行AtRING10基因啟動子之分析 47
圖1-13、以PCR方法選殖AtRING10 基因 48
圖1-14 、利用colony PCR的方法確認AtRING10基因在pBI-mGFP載體中的方向性 49
圖1-15、以PCR方法選殖AtRING9、10 RNAi之sense與anti-sense片段 50
圖1-16、利用double digestion的方法確認AtRING9、10 RNAi之sense及anti-sense片段分別轉接入pBlueACTi載體 51
圖1-17、利用double digestion的方法確認AtRING9、10 RNAi片段轉接入pBI-mGFP1載體 52
圖1-18、大量表現正股AtRING10之轉基因植物 53
圖1-19、大量表現正股AtRING10轉基因植物之鑑定與表現量分析 54
圖1-20、大量表現反股AtRING10之轉基因植物 55
圖1-21、大量表現反股AtRING10轉基因植物之鑑定與表現量分析 56
圖1-22、以PCR方法選殖AtRING10(NS)基因片段,並構築到pBI-mGFP3載體中 57
圖1-23、以農桿菌滲透法觀察AtRING10在細胞層次的表現位置(cellular localization) 58
圖1-24、以PCR方法選殖不具有RING finger domain之AtRING9(NS)基因片段,並構築到pBI-mGFP3載體中 59
圖1-25、以農桿菌滲透法觀察不具RING finger domain之AtRING9在細胞層次的表現位置(cellular localization) 60
圖1-26、以PCR方法選殖不具有RING finger domain之AtRING9 基因 61

柒、附錄
附圖1-1、Ubiquitin (Ub) / 26S proteasome pathway示意圖 62
附圖1-2、不同型式之E3複合體示意圖 63
附圖1-3、pGEM®-T Easy vector之載體圖譜(3015 bp) 64
附圖1-4、pEpyon-01K之載體圖譜(10.9 kb) 65
附圖1-5、pBI-mGFP1之載體圖譜(13.7 kb) 66
附圖1-6、pBlueACTi之載體圖譜(3400 bp) 67
附圖1-7、pBI-mGFP3之載體圖譜(13.7 kb) 68
附圖1-8、不同的泛素連結方式與功能上之差異 69

第二章 文心蘭C與E MADS box基因RNAi載體之構築
中文摘要 71
英文摘要 72
壹、前言 73
貳、材料方法 77
参、結果 80
一、OMADS4 RNAi 載體構築 80
二、OMADS6,7,11 RNAi 載體構築 80
肆、討論 84
伍、參考文獻 85
陸、圖表
表2-1、本研究所使用之核酸引子(primer)序列 90
圖2-1、文心蘭中OMADS4之cDNA序列 91
圖2-2、以PCR方法選殖OMADS4 RNAi之sense與anti-sense片段 92
圖2-3、利用酵素雙截切的方法確認OMADS4 RNAi之sense及anti-sense片段分別轉接入pBlueACTi載體 93
圖2-4、利用酵素雙截切的方法確認OMADS4 RNAi片段轉接入pBI-mGFP1載體 94
圖2-5、文心蘭中OMADS6之cDNA序列 95
圖2-6、文心蘭中OMADS7之cDNA序列 96
圖2-7、文心蘭中OMADS11之cDNA序列 97
圖2-8、OMADS6, 7, 11 RNAi片段對實驗室local OMADS database BLAST結果 98
圖2-9、以PCR方法選殖OMADS6、7、11 RNAi之sense片段 99
圖2-10、利用二次PCR方式進行,選殖OMADS67 RNAi之序列片段 100
圖2-11、利用二次PCR方式進行,選殖OMADS6711 RNAi之序列片段 101
圖2-12、利用double digestion的方法確認OMADS6711 RNAi片段轉接入pBlueACTi載體 102
圖2-13、利用double digestion的方法確認OMADS6711 RNAi片段轉接入pBI-mGFP1載體 103
柒、附錄
附圖2-1、植物MADS box基因MIKC-type蛋白質結構圖 104
附圖2-2、阿拉伯芥中花器形成之“ABCDE”model 105
附圖2-3、利用二次PCR的方式,選殖OMADS6711 RNAi之序列片段 106
附圖2-4、pGEM®-T Easy vector之載體圖譜(3015 bp) 107
附圖2-5、pBlueACTi之載體圖譜(3400 bp) 108
附圖2-6、pBI-mGFP1之載體圖譜(13.7 kb) 109
(第一章)
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(第二章)
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邱怡芬(2007)文心蘭中調控開花時間相關基因之選殖與特性分析。國立中興大學生物科技研究所碩士論文。
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