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; Functional analysis of a salt-and water deficit-induced AP2 transcription factor in Arabidopsis

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题名:	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;Functional analysis of a salt-and water deficit-induced AP2 transcription factor in Arabidopsis
作者:	林美秀;MEI-HSIU LIN
贡献者:	生命科學研究所
关键词:	AP2;鹽逆境;salt stress;AP2
日期:	2005-07-07
上传时间:	2009-09-22 10:19:05 (UTC+8)
出版者:	國立中央大學圖書館
摘要:	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.
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