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研究生:蔡佩璇
研究生(外文):Pei-Hsuan Tsai
論文名稱:阿拉伯芥中AtMAPRs基因靜默載體建構與表現分析
論文名稱(外文):Construction of the RNA gene silencing vector and RNA expression analysis of AtMAPRs in Arabidopsis
指導教授:楊健志
指導教授(外文):Chien-Chih Yang
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:微生物與生化學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:111
中文關鍵詞:基因靜默
外文關鍵詞:RNAi gene silencing
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固醇類荷爾蒙在大部分多細胞生物中, 可作為調節生長發育的訊息因子,其訊息傳導傳統上認為,是位在細胞質或細胞核的典型固醇類受體負責結合,稱為 “基因調控模式” (genomic action)。近年來則發現另一種 “非基因調控模式” (non-genomic action),其固醇類受體位在細胞膜上,惟仍所知有限。 MAPR(membrane associated progesterone receptor component1) 可能是參與 “非基因調控模式” 中的一種蛋白質。本實驗室先前已利用豬的MAPR胺基酸序列在阿拉伯芥基因體資料庫搜尋比對,找出四個相似性有30-40% 的同源蛋白質序列,並分別命名為AtMAPR2、3、4、5。過去已選殖出AtMAPR2、3、5基因,並表現蛋白質做出AtMAPR2的單株抗體,也利用酵母菌雙雜合系統篩選出與AtMAPRs有交互作用的蛋白質,但目前對於這四個AtMAPRs的生理弁鄐揖憚鴃C本篇論文將未選殖出來的AtMAPR4基因選殖出來,並完成AtMAPR2、4、5之基因靜默載體 pHG2AM2、pHG2AM4與pHG2AM5的建構供 RNAi 剔除轉殖株使用,並嘗試建立轉殖系統。另外本論文亦進行野生型阿拉伯芥中 AtMAPR基因在不同生長時期與不同器官中基因表現量的分析,將來得到RNAi 轉殖株可作為比較。在不同時期結果發現,AtMAPR2、AtMAPR4並未有明顯的RNA表現量差異,而AtMAPR3、AtMAPR5則在五天、七天及二十一天等叢生葉 (rosette leaf) 發育時期,其RNA表現量較高,AtMAPR5在二十一天亦可看到具有叢生葉的器官特異性,AtMAPR5或章鴭鬋O生葉的生長發育扮演重要角色。在二十一天大的器官,可觀察到AtMAPR2具有花、莖生葉 (cauline leaf) 與莖的器官特異性;AtMAPR3對花、根;AtMAPR4則表現量皆不高,更以莖的表現量最低;AtMAPR5對叢生葉、莖生葉及莖表現量較高。而三十五天的器官特異性分析,可觀察到AtMAPR2對花、叢生葉與長角果;AtMAPR3對花、根、叢生葉;AtMAPR4、AtMAPR5對花具有器官特異性。

Steroid hormones are the signaling factors for growth development. Two models of steroid signaling exist on animal, namely, “genomic effect” and “non-genomic effect”. The genomic effect, the conventional one, is mediated through nuclear receptors, while non-genomic effect is predicted to be perceived through membrane receptors. Our knowledge in non-genomic effect is far behind than we know about genomic effect. MAPR (membrane associated progesterone receptor component1) is one of a few proteins that may participate in the “non-genomic effect” in animals.
We are interested in the function of AtMAPRs, protein homologous to MAPR. As part of the effort to explore their physiological roles, we employ the RNAi technology to establish knock-out mutants. In this thesis, we described the construction of plasmids for RNAi experiment and a few transgenic attempts. The coding sequence of AtMAPR2, AtMAPR4 and AtMAPR5 were successfully subcloned as pHG2AM2, pHG2AM4, pHG2AM5, respectively, using BP cloning method. Although we did not obtain the transformants in Arabidopsis, the likely causes and improved protocols are discussed. The RNA levels for these genes in wild-type Arabidopsis were analyzed in different growth stages or tissues. These data are crucial for future comparison once the RNAi transformants are obtained. Similar RNA expression patterns for AtMARP2 and AtMAPR4 were observed. AtMAPR3 and AtMAPR5 expressed higher in 5 days, 7 days, and 21 days, implying that AtMAPR5 may play roles in rosette leaf development. In tissues from 21-day-old Arabidopsis, AtMAPR2 was expressed higher in flowers, cauline leaf, and stem; AtMARP3 showed higher expression in flowers, roots; AtMAPR4 expressed low in most organ; AtMARP5 was expressed in rosette leaf, cauline leaf, and stem. By analyzing the tissue-specific expression in 35-day-old plants, AtMAPR2 was expressed higher in flower, rosette leaf, and silique; while AtMAPR3 was expressed higher in flowers, roots, rosette leaf; AtMAPR4 and AtMAPR5 were expressed higher in flowers.


目錄
目錄………………………………………………………………………………….....I
縮寫表………………………………………………………………………………. IV
摘要…………………………………………………………………………………...V
abstract……………………………………………………………………………….VI

第一章 緒論
1.1 研究緣起………………………………………………………………………....1
1.2 MAPR的弁鉬P訊息傳導模式……….…………………………………………3
1.3 模式植物:阿拉伯芥 (Arabidopsis thaliana) ……………………………….....3
1.4 基因靜默 (RNA interference, RNAi)簡介
1.4.1基因靜默的發展…………………………………………………………..5
1.4.2原理介紹…………………………………………………………………..6
1.5 表現載體pHellsgate2的建構原理
1.5.1 gateway technology………………………………………………………16
1.5.2 λ噬菌體的整合與剪切作用 (λ phage integration and excision)……….17
1.5.3 基因靜默載體介紹……………………………………………………....20
1.6 農桿菌轉殖
1.6.1 基因轉形 (Transformation)……………………………………………...22
1.6.2農桿菌 (Agrobacterium tumefaciens) 轉殖機制………………………...23
1.7 本論文的研究動機、目的與方向………………………………………………25

第二章 材料與方法
2.1 實驗材料
2.1.1 植物材料…………………………………………………………………26
2.1.2 載體………………………………………………………………………26
2.1.3 大腸桿菌菌株……………………………………………………………27
2.1.4 農桿菌菌株………………………………………………………………28
2.2 實驗藥品
2.2.1 一般化學試劑…………………………………………………………....28
2.2.2 酵素………………………………………………………………………28
2.2.3 培養基…………………………………………………………………. ..28
2.3 重要儀器設備…………………………………………………………………...30
2.4 實驗方法
2.4.1 阿拉伯芥種植
2.4.1.1 培養基之配製……………………………………………….......31
2.4.1.2 種子之低溫處理………………………………………………...33
2.4.1.3 種子的無菌培養………………………………………………...33
2.4.1.4 植株的土壤培養………………………………………………...33
2.4.1.5 收種子…………………………………………………………...34
2.4.2 AtMAPR基因序列選殖
2.4.2.1 專一性引子設計………………………………………………...34
2.4.2.2 聚合酵素連鎖反應 (PCR) ……………………………………..35
2.4.2.3 聚合酵素連鎖反應產物純化 (PCR clean up) ………………...36
2.4.2.4 DNA濃度估算…………………………………………………..36
2.4.2.5 T-A cloning……………………………………………………….37
2.4.2.6 大腸桿菌轉形…………………………………………………...38
2.4.2.7 質體DNA製備法……………………………………………….40
2.4.2.8 DNA限制酶切割反應分析…………………………...…………43
2.4.2.9 洋菜膠體電泳…………………………………………………...43
2.4.3 表現載體建構
2.4.3.1 pHellsgate2質體增殖……………………………………………44
2.4.3.2 AttB-EZ 引子的聚合酵素連鎖反應 (AttB-EZ PCR)………….44
2.4.3.3 純化PCR反應………………………………………………….46
2.4.3.4 重組置換反應 …………………………………………………46
2.4.3.5 質體篩選………………………………………………………..47
2.4.3.6 PCR確認目標基因……………………………………………...47
2.4.3.7 定序……………………………………………………………..48
2.4.3.8 菌種保存………………………………………………………..48
2.4.4 阿拉伯芥基因轉殖
2.4.4.1 植物材料的準備………………………………………………..49
2.4.4.2 農桿菌轉形……………………………………………………..49
2.4.4.3 農桿菌感染……………………………………………………..52
2.4.4.4 轉植株的篩選…………………………………………………..54
2.4.5 基因型分析
2.4.5.1 RNA抽取與分析方法…………………………………………..54
2.4.5.2 RNA濃度估算…………………………………………………..56
2.4.5.3 RT-PCR ………………………………………………………….58

第三章 結果與討論
3.1 AtMAPR基因序列選殖…………………………………………………………61
3.2 表現載體建構
3.2.1 AtMAPRs部分序列選殖…………………………………………………61
3.2.2 次選殖目標基因到表現載體…………………………………………....62
3.3 阿拉伯芥基因轉殖……………………………………………………………...64
3.4 基因型分析
3.4.1 RNA的抽取…………………………………………………….………..65
3.4.2 RNA定量………………………………………………………………...67
3.4.3 RT-PCR…………………………………………………………………...67
3.4.4 RNA表現量分析………………………………………………………...68
3.4.5 RNA表現量分析的方法比較…………………………………………...72

第四章 結論與未來展望…………………………………………………………...74
參考文獻…………………………………………………………………………….76
圖與表……………………………………………………………………………….83
附錄…………………………………………………………………………………101
論文口試問答摘要…………………………………………………………………106


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