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研究生:林美秀
研究生(外文):MEI-HSIU LIN
論文名稱:在阿拉伯芥中一個鹽逆境及缺水逆境所誘導之AP2轉錄因子的功能分析
論文名稱(外文):Functional analysis of a salt-and water deficit-induced AP2 transcription factor in Arabidopsis
指導教授:詹明才
指導教授(外文):MING-TSAIR CHAN
學位類別:碩士
校院名稱:國立中央大學
系所名稱:生命科學研究所
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:2005
畢業學年度:93
語文別:中文
論文頁數:75
中文關鍵詞:AP2鹽逆境
外文關鍵詞:salt stressAP2
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中文摘要

植物在其生活史中常常會曝露在許多不同的非生物逆境,其中由乾旱、低溫及土壤中過高的含鹽量所導致的水份逆境是最常見的環境逆境。這些逆境會影響植物的代謝及基因表現,某些部份的基因表現會同時在不同的逆境下被誘導而改變植物的生理代謝,進一步的對抗或忍受逆境或者進而死亡。控制基因表現的主要機制在於轉錄層次的調節作用,藉由轉錄因子活化或抑制轉錄來調節基因的表現,而AP2/EREBP(APETALA2/Ethylene Responsive Element Binding Protein)這個轉錄因子家族便是其中之一,AP2/EREBP轉錄因子目前只在植物界才發現,且其AP2 binding domain具有高度的保留性。有許多的研究發現AP2/EREBP轉錄因子在植物發展及對抗環境及生物逆境具有重要的調節作用,例如CBF1、AP2、DREB……等。
利用微矩陣分析我們得到一個由乾旱及高鹽會共同誘導表現之基因:AF9(At4g34410) ,序列分析結果顯示其含有AP2 Donain,屬於AP2/EREBP轉錄因子。為了確認微矩陣分析的結果,我們先以生物資訊方式分析其基因中專一性片段,並利用PCR方法擴增此片段,以充當以後研究之探針。南方氏墨點法也確認此探針之專一性。利用北方式墨點法,我們發現這個基因會受高鹽逆境之誘導表現,亦受乾旱逆境誘導,但由於表現量不顯著,所以可能仍需進一步確認。而另外此基因也會受到過氧化氫、巴拉刈及重金屬銅、鎘的誘導,同時也發現植物荷爾蒙乙烯及茉莉酸甲酯也會誘導其表現,且在逆境早期便被誘導表現。AF9轉殖植物的篩選中,得到了四株T0代植株,並且進行T1種子的篩選,在篩選出的植株上發現其葉片多而小且其根系多。而以EMSA(Electrophoretic Mobility Shifting Assay)的方法也證明At4g34410會與GCC box鍵結,進而調節其對抗逆境之相關基因。並利用雙雜合系統找出與本基因有交互作用的蛋白質At1g65720、At5g18970,並藉由BiFC(Bimolecular fluorescence complementation)的方法證實了At1g65720與AF9的交互作用,希望藉由這些實驗可以進一步了解此AF9基因在植物逆境所扮演的角色。
Abstract
Plants are exposed to dynamic environmental stresses throughout their life cycle. Drought, low temperature and high salts in the soils are the most common stresses that alter metabolism and gene expression, which affects agricultural productivity. Although these stresses are quite different they do induce similar or different genes in order to cope with changes in the environment. The predominant mechanism for controlling gene expression in plants is regulated at transcriptional level and is mediated by transcription factors, which active or represses transcription. The AP2/EREBP is a gene family encodes transcription factors with highly conserved AP2-binding domain and is unique to plants. Some of AP2/EREBP family genes such as CBF1, AP2 and EREBP etc., have been characterized as key regulators in developmental processes and biotic or abiotic stresses.
Recently we have isolated a novel AP2/EREBP transcription factor induced by drought and salt stresses from Affymetrix microarray. The northern blot data revealed that expression of AF9 (At4g34410) was induced under salt stress and drought stress. However, expression under drought stress is not clear and this data need to be confirmed. In addition, AF9 was also induced by H2O2, MV, Cd, Cu, ethylene, and methyl jasmonate. Furthermore, it was induced in early phase of stresses. A binary vector consisting of AF9 gene was transferred to Arabidopsis via A. tumefaciens. Four T0 transgenic lines were obtained and collected T1 progeny. The leaves of T1 transgenic plants are more with many short hairy roots than wild-type. It was demonstrated that AF9 protein can bind to the GCC box (-AGCCGCCAC-) in promoter of responsive gene by EMSA. Moreover, two proteins, At1g65720 and At5g18970 were identified by bacterial two hybrid screening and confirmed by yeast two hybrid screening. These two genes were co-bombarded into onion epidermis to confirm the interaction with AF9. These results could help us to understand the function of AF9 gene in plants for further studies.
目 錄

中文摘要 ----------------------------------------------------------------------- Ⅰ
英文摘要 ----------------------------------------------------------------------- Ⅲ
目錄 ----------------------------------------------------------------------------- Ⅴ
圖表目錄 ----------------------------------------------------------------------- Ⅷ
縮寫與全名對照表 ----------------------------------------------------------- Ⅹ
壹、序論 ----------------------------------------------------------------------- 1
一、前言 -------------------------------------------------------------------- 1
二、植物對抗逆境的反應-------------------------------------------------- 1
三、在阿拉伯芥中AP2/EREBP轉錄因子介紹------------------------ 2
四、AP2/EREBP 轉錄因子的功能性角色------------------------------ 3
五、實驗起源與目的-------------------------------------------------------- 4
六、實驗策略----------------------------------------------------------------- 5
貳、材料與方法 -------------------------------------------------------------- 6
ㄧ、質體建構及雙雜合系統--------------------------------------------- 6
1、PCR 擴大基因片段----------------------------------------------- 6
2、純化PCR產物片------------------------------------------------- 6
3、載體與基因的建造------------------------------------------------ 7
4、製備大腸桿菌(E. coli)的勝任細胞----------------------------- 7
5、細菌的轉型作用--------------------------------------------------- 7
6、快速萃取質體DNA ----------------------------------------------- 8
7、從載體切除基因片段--------------------------------------------- 9
8、細菌的共同轉型作用-------------------------------------------- 10
9、酵母菌勝任細胞-------------------------------------------------- 11
10、酵母菌轉形作用------------------------------------------------ 11
11、Filter Lift Assay ------------------------------------------------ 12
二、阿拉伯芥基因植物的轉殖------------------------------------------ 12
12、製備農桿菌的勝任細胞--------------------------------------- 12
13、農桿菌的轉型作用 ------------------------------------------ 13
14、植物的轉型作用----------------------------------------------- 13
15、轉殖植株的篩選----------------------------------------------- 14
16、阿拉伯芥DNA之純化----------------------------------------- 15
17、阿拉伯芥total RNA 之純化-------------------------------- 15
18、RNA 電泳分析----------------------------------------------- 16
19、南方氏點墨法------------------------------------------------- 17
三、蛋白質的純化-------------------------------------------------------- 19
20、蛋白質的誘導表現及萃取----------------------------------- 19
21、蛋白質之電泳分析---------------------------------------- 20
22、蛋白質的純化--------------------------------------------- 21
23、西方點墨法------------------------------------------------- 22
24、蛋白質定量測定( Bradford assay ) --------------------- 23
25、EMSA (electromobility shifting assay) ------------------ 24
26、植物細胞轉殖作用----------------------------------------- 26
參、結果 --------------------------------------------------------------------- 28
一、以南方氏墨點法分析AF9 (At4g34410)基因探針的特異性- 28
二、以北方點墨法分析AF9基因在各種逆境的表現量--------- 28
三、阿拉伯芥轉殖植物的篩選與PCR確認轉植株-------------- 29
四、EMSA (Electrophoretic Mobility Shifting Assay) ------------- 30
五、細菌雙雜合系統的篩選------------------------------------------- 31
六、酵母菌雙雜合系統的確認--------------------------------------- 32
七、以Bimolecular fluorescence complementation (BiFC)在植物
細胞觀察蛋白質與蛋白質的交互作用----------------------- 33
肆、討論-------------------------------------------------------------------- 34
伍、圖表---------------------------------------------------------------------- 39
陸、參考文獻 -------------------------------------------------------------- 60
柒、附錄 -------------------------------------------------------------------- 63
圖 表 目 錄

圖一、南方氏墨點法分析AF9 (At4g34410)基因探針的特異性--- 39
圖二、北方點墨法分析AF9基因在高鹽分、乾旱及冷害處理下的表現情形----------------------------------------------------------------- 40
圖三、利用北方點墨法分析阿拉伯芥植物的AF9基因在重金屬硫酸銅及氯化鎘處理下的表現情形----------------------------------- 41
圖四、利用北方點墨法分析阿拉伯芥植物的AF9基因在過氧化氫及巴拉刈(Paraquat)處理下的表現情形---------------------------- 42
圖五、利用北方點墨法分析阿拉伯芥植物的AF9基因在乙烯及茉莉酸甲酯處理下的表現情形--------------------------------------- 43
圖六、 AF9基因轉殖植物的PCR確認----------------------------------- 44
圖七、 在不同的宿主BL21(DE3)及Rosetta(DE3)中誘導AF9蛋白質的表現情形-------------------------------------------------------- 45
圖八、 於16℃、20℃、30℃的溫度下誘導表現AF9蛋白質------ 46
圖九、 AF9蛋白質的純化及西方點墨法分析------------------------- 47
圖十、 利用EMSA的方法得知AF9可鍵結於GCC box,但無法鍵結於DRE element--------------------------------------------------- 48
圖十一、細菌雙雜合系統的第一次篩選--------------------------------- 49
圖十二、藉以抗鏈四環黴素作為第二次篩選確認陽性的方法---- 50
圖十三、將Target plasmids分群------------------------------------- 51
表一、第三次篩選確認其交互作用------------------------------------- 52
表二、所篩選確定與AF9有交互作用的基因定序後比對的資料-- 53
圖十四、酵母菌雙雜合系統的第一次篩選----------------------------- 54
圖十五、以β-galactosidase的活性測試以作為第二次的篩選-- 55
圖十六、以Bimolecular fluorescence complementation(BiFC)的方法在洋蔥表皮細胞中觀察AF9與96融合蛋白質的交互作用-------------------------------------------------------------------- 56
圖十七、在洋蔥表皮細胞觀察AF9、96、100的表現位置--------- 57
圖十八、觀察AF9、96、100共同轉殖於洋蔥表皮細胞的表現情形 58
陸、參考文獻

Bowman JL, Smyth DR, Meyerowitz EM (1989) Genes directing flower development in Arabidopsis. Plant Cell 1: 37-52.
Buttner M, Singh KB (1997) Arabidopsis thaliana ethylene-responsive element binding protein (AtEBP), an ethylene-inducible, GCC box DNA-binding protein interacts with an ocs element binding protein. Proc Natl Acad Sci U S A 94: 5961-6.
Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16: 735-43.
Elliott RC, Betzner AS, Huttner E, Oakes MP, Tucker WQ, Gerentes D, Perez P, Smyth DR (1996) AINTEGUMENTA, an APETALA2-like gene of Arabidopsis with pleiotropic roles in ovule development and floral organ growth. Plant Cell 8: 155-68.
Fujimoto SY, Ohta M, Usui A, Shinshi H, Ohme-Takagi M (2000) Arabidopsis ethylene-responsive element binding factors act as transcriptional activators or repressors of GCC box-mediated gene expression. Plant Cell 12: 393-404.
Gutterson N, Reuber TL (2004) Regulation of disease resistance pathways by AP2/ERF transcription factors. Curr Opin Plant Biol 7: 465-71.
Jofuku KD, den Boer BG, Van Montagu M, Okamuro JK (1994) Control of Arabidopsis flower and seed development by the homeotic gene APETALA2. Plant Cell 6: 1211-25.
Kizis D, Lumbreras V, Pages M (2001) Role of AP2/EREBP transcription factors in gene regulation during abiotic stress. FEBS Lett 498: 187-9.
Liu Q, Kasuga M, Sakuma Y, Abe H, Miura S, Yamaguchi-Shinozaki K, Shinozaki K (1998) Two transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought- and low-temperature-responsive gene expression, respectively, in Arabidopsis. Plant Cell 10: 1391-406.
Riechmann JL, Heard J, Martin G, Reuber L, Jiang C, Keddie J, Adam L, Pineda O, Ratcliffe OJ, Samaha RR, Creelman R, Pilgrim M, Broun P, Zhang JZ, Ghandehari D, Sherman BK, Yu G (2000) Arabidopsis transcription factors: genome-wide comparative analysis among eukaryotes. Science 290: 2105-10.
Riechmann JL, Meyerowitz EM (1998) The AP2/EREBP family of plant transcription factors. Biol Chem 379: 633-46.
Stockinger EJ, Gilmour SJ, Thomashow MF (1997) Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C-repeat/DRE, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit. Proc Natl Acad Sci U S A 94: 1035-40.
Walter M, Chaban C, Schutze K, Batistic O, Weckermann K, Nake C, Blazevic D, Grefen C, Schumacher K, Oecking C, Harter K, Kudla J (2004) Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation. Plant J 40: 428-38.
Wilson K, Long D, Swinburne J, Coupland G (1996) A Dissociation insertion causes a semidominant mutation that increases expression of TINY, an Arabidopsis gene related to APETALA2. Plant Cell 8: 659-71.
Erica, A. G. (2002). Protein-Protein Interactions: A Molecular Cloning Manual, Cold Spring Harbor Laboratory Press, New York.
Fields, S. and Song, O. (1989). A novel genetic system to detect protein-protein interactions.Nature 340, 245-246.
Bowman JL, Smyth DR, Meyerowitz EM (1989) Genes directing flower development in Arabidopsis. Plant Cell 1: 37-52.
Buttner M, Singh KB (1997) Arabidopsis thaliana ethylene-responsive element binding protein (AtEBP), an ethylene-inducible, GCC box DNA-binding protein interacts with an ocs element binding protein. Proc Natl Acad Sci U S A 94: 5961-6.
Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16: 735-43.
Elliott RC, Betzner AS, Huttner E, Oakes MP, Tucker WQ, Gerentes D, Perez P, Smyth DR (1996) AINTEGUMENTA, an APETALA2-like gene of Arabidopsis with pleiotropic roles in ovule development and floral organ growth. Plant Cell 8: 155-68.
Fujimoto SY, Ohta M, Usui A, Shinshi H, Ohme-Takagi M (2000) Arabidopsis ethylene-responsive element binding factors act as transcriptional activators or repressors of GCC box-mediated gene expression. Plant Cell 12: 393-404.
Gutterson N, Reuber TL (2004) Regulation of disease resistance pathways by AP2/ERF transcription factors. Curr Opin Plant Biol 7: 465-71.
Jofuku KD, den Boer BG, Van Montagu M, Okamuro JK (1994) Control of Arabidopsis flower and seed development by the homeotic gene APETALA2. Plant Cell 6: 1211-25.
Kizis D, Lumbreras V, Pages M (2001) Role of AP2/EREBP transcription factors in gene regulation during abiotic stress. FEBS Lett 498: 187-9.
Lee JH, Hong JP, Oh SK, Lee S, Choi D, Kim WT (2004) The ethylene-responsive factor like protein 1 (CaERFLP1) of hot pepper (Capsicum annuum L.) interacts in vitro with both GCC and DRE/CRT sequences with different binding affinities: possible biological roles of CaERFLP1 in response to pathogen infection and high salinity conditions in transgenic tobacco plants. Plant Mol Biol 55: 61-81.
Liu Q, Kasuga M, Sakuma Y, Abe H, Miura S, Yamaguchi-Shinozaki K, Shinozaki K (1998) Two transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought- and low-temperature-responsive gene expression, respectively, in Arabidopsis. Plant Cell 10: 1391-406.
Ohme-Takagi M, Shinshi H (1995) Ethylene-inducible DNA binding proteins that interact with an ethylene-responsive element. Plant Cell 7: 173-82.
Riechmann JL, Heard J, Martin G, Reuber L, Jiang C, Keddie J, Adam L, Pineda O, Ratcliffe OJ, Samaha RR, Creelman R, Pilgrim M, Broun P, Zhang JZ, Ghandehari D, Sherman BK, Yu G (2000) Arabidopsis transcription factors: genome-wide comparative analysis among eukaryotes. Science 290: 2105-10.
Riechmann JL, Meyerowitz EM (1998) The AP2/EREBP family of plant transcription factors. Biol Chem 379: 633-46.
Stockinger EJ, Gilmour SJ, Thomashow MF (1997) Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C-repeat/DRE, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit. Proc Natl Acad Sci U S A 94: 1035-40.
Sunkar R, Bartels D, Kirch HH (2003) Overexpression of a stress-inducible aldehyde dehydrogenase gene from Arabidopsis thaliana in transgenic plants improves stress tolerance. Plant J 35: 452-64.
Walter M, Chaban C, Schutze K, Batistic O, Weckermann K, Nake C, Blazevic D, Grefen C, Schumacher K, Oecking C, Harter K, Kudla J (2004) Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation. Plant J 40: 428-38.
Wilson K, Long D, Swinburne J, Coupland G (1996) A Dissociation insertion causes a semidominant mutation that increases expression of TINY, an Arabidopsis gene related to APETALA2. Plant Cell 8: 659-71.
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