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研究生:劉耿全
研究生(外文):Keng-Chuan Liu
論文名稱:以酵母菌雙雜合系統篩選阿拉伯芥中與AtMAPRs有交互作用之蛋白質
論文名稱(外文):Searching Interacting Proteins of AtMAPRs in Arabidopsis Using Yeast Two-Hybrid Analysis
指導教授:楊健志
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:2005
畢業學年度:93
語文別:英文
論文頁數:74
中文關鍵詞:阿拉伯芥
外文關鍵詞:arabidopsis
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我們在阿拉伯芥中發現四個與豬的 MAPR (membrane associated progesterone
receptor conponent 1) 有同源性的蛋白質,並命名為 AtMAPR2、AtMAPR3、
AtMAPR4 與 AtMAPR5。雖然對此四個蛋白質的功能缺乏全面性的了解,但我們
推測 AtMAPRs 參與調控環境中訊息的接收,例如植物本身分泌的荷爾蒙、光照或
逆境。本實驗室先前的酵母菌雙雜合實驗結果顯示,AtMAPRs 可能與聚泛素
(polyubiquitin)、MYB3 轉錄因子、GTP 結合蛋白質 (AtTOC33) 以及丙胺酸-乙醛酸
轉胺? (alanine-glyoxylate aminotransferase,AGT1)。然而,AtMAPR3、AtMAPR4
與 AtMAPR5 所含有的推測性穿越膜區塊 (putative transmembrane domain) 可能影響
酵母菌雙雜合的結果。因此,我們使用除去一到四十個胺基酸的 AtMAPR5 當成釣
餌,透過酵母菌雙雜合系統來篩選主要以阿拉伯芥花製成的 cDNA 庫 (CD4-30)。
在二十六個分別可能與 AtMAPR5 有交互作用的蛋白質中,推測 C3HC4-type RING
蛋白質 (At3g58030) 與一種 FKBP-type peptidyl-prolyl cis-trans isomerase
(AtFKBP16-2; At4g39710) 可能分別直接與 AtMAPR5 在阿拉伯芥中一同發揮其效
能。根據這兩個可能與 AtMAPR5 產生交互作用的蛋白質,我們提出兩套模型來解
釋 AtMAPRs 可能的作用機制:(一) C3HC4-type RING 蛋白質可能具有 E3 ligase的
活性並且藉者調控 AtMAPRs 的降解而達到傳遞訊息的功能。(二) AtMAPRs 可能
於光合作用 (photosynthesis) 中與 AtFKBP16-2 一同扮演電子傳遞者的角色。許多工 作仍須繼續進行來證明此二假設的真實性。簡言之,酵母菌雙雜合系統的結果提供
許多資訊讓我們對 AtMAPRs 的功能做進一步的推測。
Four AtMAPRs (AtMAPR2, AtMAPR3, AtMPAR4, and AtMAPR5) sharing 30-
40% similarity with porcine MAPR (membrane associated progesterone receptor
component 1) have been identified in Arabidopsis. The function of the four AtMAPRs is
not clear, but we speculated that they could participate in regulating environmental
stimuli such as plant hormones, light, or stresses. Previous yeast two-hybrid assay
indicated that AtMAPRs might interact with polyubiquitin, MYB3 transcription factor,
AtTOC33 (GTP-binding protein), and AGT1 (alanine-glyoxylate aminotransferase).
However, the presence of putative transmembrane domain of AtMAPR3, AtMAPR4, and
AtMAPR5 might interfere with yeast two-hybrid results. In this work, we used truncated
AtMAPR5, lacking the 1-40 amino acid residues, as bait to screen flower two-hybrid
cDNA library, CD4-30. Among the 26 different interacting proteins obtained from the
screening, a C3HC4-type RING protein (At3g58030) and a FKBP-type peptidyl-prolyl
cis-trans isomerase, AtFKBP16-2 (At4g39710) was perhaps to function together with
AtMAPR5 in Arabidopsis. We proposed two models based on these two interacting
proteins that: (1) C3HC4-type RING protein might function as E3 ligase and promote the
degradation of AtMAPRs. (2) AtMAPRs might be involved in electron transferring in
photosynthesis with AtFKBP16-2. Further work has to be done to testify these two
assumptions. Briefly, these yeast two-hybrid results provided hints to the function of
AtMAPRs.
Abbreviations........................................................................................III
Abstract in Chinese..................................................................................V
Abstract...............................................................................................VI
Chapter 1. Introduction................................................................................................1
1.1 Genomic versus nongenomic steroid effects..........................................................1
1.2 MAPR and its homologues....................................................................................3
1.3 Ubiquitin-proteasome pathway in plant signaling................................................11
1.4 Yeast two-hybrid system.....................................................................................14
Chapter 2. Materials and Methods.............................................................................17
2.1 Materials.............................................................................................................17
2.1.1 Vectors.........................................................................................................17
2.1.2 cDNA library used in yeast two-hybrid system.............................................19
2.1.3 Microbiological materials.............................................................................20
2.1.4 Enzymes.......................................................................................................21
2.1.5 Mediums and Chemicals...............................................................................22
2.2 Methods..............................................................................................................25
2.2.1 Isolation and analysis of DNA......................................................................25
2.2.1.1 Purification of plasmid DNA from E.coli (Mini-preparation).................25
2.2.1.2 Purification of plasmid DNA from E.coli (Midi-preparation).................26
2.2.1.3 Restriction enzyme digestion.................................................................27
2.2.1.4 Agarose gel electrophoresis....................................................................27
2.2.1.5 DNA gel extraction................................................................................28
2.2.1.6 Detection of DNA concentration............................................................29
2.2.1.7 DNA Ligation........................................................................................29
2.2.2 E. coli transformation...................................................................................30
2.2.2.1 Heat-shock transformation.....................................................................30
2.2.2.2 Transformation by electroporation.........................................................31
2.2.3 Yeast two-hybrid system experiment............................................................33
2.2.3.1 Yeast transformation..............................................................................33
2.2.3.2 Screening an AD fusion cDNA library for putative interacting proteins.35
2.2.3.3 Isolation of plasmid DNA from yeast.....................................................36
2.2.4 Bioinformatics..............................................................................................37
Chapter 3. Results and Discussion.............................................................................39
3.1 Identification of AtMAPRs interacting proteins using yeast two-hybrid system...39
3.1.1 Verification of protein-protein interaction between AtMAPRs and their
putative interacting partners..................................................................................39
3.1.2 Preparation of yeast two-hybrid library, CD4-30...........................................40
3.1.3 Searching AtMAPRs interacting partners in CD4-30 cDNA library using
AtMAPR5(41-220) as bait.....................................................................................40
3.2 Putative interacting partners of AtMAPRs...........................................................42
3.2.1 C3HC4-type RING finger family protein......................................................43
3.2.2 FKBP-type peptidyl-prolyl cis-trans isomerase.............................................45
3.3 The possible role of AtMAPRs in vivo...................................................48
Chapter 4. References.............................................................................63
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