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研究生:吳雨哲
研究生(外文):Yu-Che Wu
論文名稱:蝴蝶蘭乙烯不敏感基因(PeEIN3)之選殖與特性分析
論文名稱(外文):Molecular cloning and characterization of orchid ETHYLENE-INSENSITIVE3 (EIN3) gene
指導教授:吳文鑾
指導教授(外文):Wen-Luan Wu
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物學系碩博士班
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:78
中文關鍵詞:轉錄調控因子乙烯訊息傳遞乙烯不敏感基因蝴蝶蘭蘭花乙烯
外文關鍵詞:transcription factorPeEIN3EIN3ethylene insensitiveEILethyleneorchidphalaenopsisethylene pathway
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乙烯(C2H4)是一重要的植物氣體荷爾蒙,在植物生長發育過程,扮演著相當多樣的調節角色。過去研究大多著眼於乙烯如何在生物體內自然生成的生化反應機制,及植物在受乙烯刺激後會產生何種生理反應等等,現在則多從分子層級的角度切入,研究乙烯於高等植物中的訊息途徑。目前由阿拉伯芥的研究得知,植物對乙烯的感受性需由特殊的乙烯受器及一連串的訊息傳遞途徑來調控下游受乙烯調控的基因,以達成表型或生理機能的改變,而阿拉伯芥的轉錄調控因子ein3基因產生隱性突變,可以造成乙烯不敏感的性狀。為研究乙烯訊息傳遞的路徑及其相關分子在台灣原生種蝴蝶蘭中表現情形,本實驗以EIN3/EIL基因家族為研究對象,進行蝴蝶蘭PeEIN3 (Phalaenopsis equestris Ethylene-INsensitive3)基因的選殖,研究其構造,並探討其表現情形。首先以EST clone所得到的蝴蝶蘭之乙烯反應轉錄調控因子EIN3基因片段作為探針,進行姬蝴蝶蘭花苞cDNA基因庫篩選,分離出PeEIN3 cDNA共2411 bp (基因索引號AJ316086),其中開放譯架區為1866 bp可轉譯出621個氨基酸序列,轉譯氨基酸序列80殘基(residues)至300殘基處呈現高保留的特性。序列比對結果,與阿拉伯芥EIN3/EIL基因家族成員具有55-60%的相同度(identity)及68-70%相似度(similarity)。南方墨點分析結果顯示PeEIN3為單一拷貝基因,且其基因族系至少包含二個以上的成員。分子親緣關係之分析顯示PeEIN3在演化上與玉米的ZmEIN3、稻米的OsEIL位於同一個分類枝(clade),皆屬於單子葉基因群。北方墨點分析PeEIN3基因表現結果顯示,在蝴蝶蘭花苞、花、葉、根PeEIN3基因表現並無明顯的組織特異性。為進一步探討PeEIN3基因的功能是否與阿拉伯芥EIN3相似,故將姬蝴蝶蘭PeEIN3 cDNA利用農桿菌轉殖到阿拉伯芥野生型植株及乙烯不敏感突變植株(ein3-1),觀察轉殖阿拉伯芥T2世代的小苗發現無論是否有乙烯前驅物ACC的刺激,轉殖株均能持續表現triple response現象,顯示PeEIN3具有與EIN3/EIL基因家族相似的功能。
Ethylene (C2H4) is a gaseous hormone that affects many aspects on plant growth and development. Previous studies focused on the biosynthesis of ethylene and the ability of cells to perceive the hormone and respond in an appropriate manner. At present, the molecular events underlying the ethylene response pathway in higher plants have only begun to be understood. Ethylene perception requires specific receptors and a signal transduction pathway to coordinate down-stream responses. The ein3 gene of Arabidopsis encodes a transcription factor, mutation of ein3 confers recessive ethylene insensitivity. In this study, we cloned and characterized the EIN3 gene of Phalaenopsis equestris. The EIN3 gene fragment from EST clone was used as probe to screen a cDNA library constructed using poly(A)+ RNA from Phalaenopsis equestris flower buds. A cDNA encoding the EIN3 homolog, designated PeEIN3 (Accession No. AJ316086), was obtained and sequenced. The PeEIN3 cDNA is 2411 bp in length, encoding 621 amino acids. The deduced amino acid sequence shares 55-60% identity and 68-70% similarity with Arabidopsis EIN3 and EIN3-Like (EIL) genes. Southern blot analysis indicated that small gene family of EIN3 was present in Phalaenopsis equestris genomes. Molecular phylogenetic analysis of its evolutionary relationship revealed that PeEIN3 is linked to monocot genes, such as ZmEIN3 of maize and OsEIL of rice. Northern blot analysis of Phalaenopsis equestris leaf, root, flower and floral bud indicated that gene expression of PeEIN3 was not tissue-specific. To further explore if the similarity of sequence and expression is coupled to that in function of Arabidopsis EIN3, functional analysis of PeEIN3 through transgenic plants is performed. Sense cDNA of PeEIN3 was ectopically expressed in wild-type and ethylene-insensitive mutant Arabidopsis plants. Four-day-old T2 seedlings generated from both transgenic wild-type and mutant plants all exhibited triple response phenotype in either the presence or the absence of 1-aminocyclopropane- 1-carboxylic acid (ACC), a precursor of ethylene. These observations indicate that PeEIN3 is functionally similar to EIN3/EIL family.
目錄
中文摘要………………………………………...……………………….i
英文摘要..…………………………………………………………….….ii
致謝...........................................................................................................iii
第一章 緒言
一、乙烯對植物生長的調控…………………………..………………....1
二、乙烯訊息傳遞路徑…………………………………………….…….2
三、EIN3基因的特性分析……………………….………………………4
四、蝴蝶蘭簡介...…… …………………….……………………..……...6
五、蝴蝶蘭的乙烯相關研究………………………………….................7
六、研究目的..............................................................................................8
第二章 材料與方法
一、實驗材料..............................................................................................9
二、實驗步驟…...……………….……………………………………......9
(一) 蝴蝶蘭基因組DNA的萃取…..…… .……………………….9
(二) 聚合酵素連鎖反應擴增蝴蝶蘭PeEIN3基因與探針............10
1. 聚合酵素連鎖反應………. …...…………….…………..10
2. 由瓊脂凝膠中回收DNA片段…………………………..11
(三) 南方氏雜合實驗……………………….…………………….11 1. 隨機引子探針的製備.......................................................11
2. 蝴蝶蘭基因組DNA的限制酵素切割作用.....................12
3. 南方轉漬實驗...................................................................12
4. 雜合反應...........................................................................12
(四) 蝴蝶蘭Total RNA的抽取……...............…………………...13
1. 蝴蝶蘭Total RNA的抽取…….………………………...13
2. RNA電泳....................................………………………...14
(五) 北方雜合實驗.........................................................................14
(六) 蝴蝶蘭PeEIN3基因之篩選.....………………..…….……...15
1. 培養蝴蝶蘭cDNA基因庫…………..……...…………...15
2. 噬菌斑原位雜合反應..………....……………...………...15
3. pBluescript phagemid切割(excision)…………………….16
(七) 微量製備質體DNA........................……….………………...17
1. 培養細菌…...............................................………………17
2. 微量抽取…….......................................................………17
3. 限制酵素作用測試………………………………………17
(八) 核苷酸定序.............................................................................18
(九) 序列分析.................................................................................18
(十) 轉殖阿拉伯芥植株……......................................…..…….…18
1. 聚合酵素連鎖反應擴增PeEIN3譯碼區序列並回收....18
2. T-A cloning構築Teasy-PeEIN3質體..............................19
3. 大腸桿菌DH5a菌株熱休克(heat shock)轉型.................19
4. 微量抽取Teasy-PeEIN3質體DNA.................................19
5. 構築pBI121-PeEIN3質體................................................20
6. 大腸桿菌DH10B菌株熱休克轉型.................................20
7. 微量抽取pBI121-PeEIN3質體DNA..............................20
8. 農桿菌(Agrobacterium tumefaciens)電穿孔轉型............20
9. 阿拉伯芥轉基因實驗(transgenic experiment).................21
第三章 結果
一、蝴蝶蘭PeEIN3基因之篩選與序列分析........................................23
二、蝴蝶蘭PeEIN3基因族系組成........................................................24
三、蝴蝶蘭PeEIN3基因的演化分析....................................................25
四、蝴蝶蘭PeEIN3基因表現分析..........................................................25
五、蝴蝶蘭PeEIN3基因功能測試.........................................................26
第四章 討論
一、蝴蝶蘭PeEIN3基因結構與分子特性..............................................28
二、蝴蝶蘭PeEIN3基因族系組成與演化分析.....................................29
三、蝴蝶蘭PeEIN3基因表現.................................................................30
四、蝴蝶蘭PeEIN3基因轉殖阿拉伯芥分析..........................................31
參考文獻..................................................................................................33
附錄..........................................................................................................61
表目錄
表一、蝴蝶蘭PeEIN3基因序列分析與聚合酵素連鎖反應之引子序列
......................................................................................................39
表二、蝴蝶蘭PeEIN3基因與其他物種EIN3基因轉譯氨基酸序列相似度比對表..................................................................................40

圖目錄
圖一、PeEIN3篩選實驗之第四次噬菌斑原位雜合反應自動放射顯影圖
........................................................................................................41
圖二、pBluescript-PeEIN3 phagemid限制酵素作用檢測......................42
圖三、pBluescript-PeEIN3 phagemid圖譜及序列分析引子分佈位置示意....................................................................................................43
圖四、pBluescript-PeEIN3 phagemid插入片段之cDNA序列.............44
圖五、姬蝴蝶蘭之PeEIN3 cDNA序列及開放譯架區氨基酸序列......46
圖六、姬蝴蝶蘭PeEIN3與不同物種EIN3/EIL轉譯氨基酸序列保留性比較................................................................................................48
圖七、蝴蝶蘭PeEIN3基因族系組成分析..............................................51
圖八、以Neighbor-joining法構築蝴蝶蘭PeEIN3基因高保留譯碼區氨基酸序列演化樹...........................................................................52
圖九、姬蝴蝶蘭PeEIN3基因於不同器官中及乙炔處理後表現分析
.....................................................................................................53
圖十、姬蝴蝶蘭PeEIN3基因於植物不同生長時期表現分析...........54
圖十一、聚合酵素連鎖反應擴增PeEIN3完整譯碼區.......................55
圖十二、pGEM®-T Easy-PeEIN3質體DNA結構圖..........................56
圖十三、pBI121-PeEIN3質體DNA結構圖.........................................57
圖十四、pBI121-PeEIN3質體轉型農桿菌之限制酵素作用檢測........58
圖十五、PeEIN3基因功能測試結果.....................................................59
圖十六、聚合酵素連鎖反應擴增轉基因阿拉伯芥之PeEIN3近3’端非譯碼區序列..............................................................................60
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