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標題: | 阿拉伯芥中一個受乾旱誘導的轉錄因子At2g20880的功能性分析 Functional study of a drought-induced transcription factor, At2g20880, in Arabidopsis |
作者: | Mei-Chun Cheng 鄭梅君 |
指導教授: | 林讚標(Tsan-Piao Lin) |
關鍵字: | 阿拉伯芥,乾旱誘導,轉錄因子,At2g20880,AP2/ERF, Arabidopsis,drought-induced,transcription factor,At2g20880,AP2/ERF, |
出版年 : | 2008 |
學位: | 碩士 |
摘要: | 植物在其生活史中常常會暴露在許多不同的非生物逆境,包括乾旱、低溫及高鹽等。這些逆境會誘導許多基因的表現,進而調控植物的生理代謝來對抗逆境。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下游基因的表現。我們從Shinozaki在2002年發表的微矩陣序列分析資料中選出被乾旱大量誘導的At2g20880基因。At2g20880轉錄因子的表現量在乾旱逆境下比正常生長高出24.5倍,其屬於AP2/ERF superfamily的一員並具有高度保留性的AP2 DNA binding domain。利用北方式墨點法,我們發現At2g20880會受乾旱、滲透壓以及高鹽逆境的誘導,但並不會受到熱逆境和冷逆境的誘導。此外,此基因也不會受到離層酸、乙烯以及茉莉酸甲酯的誘導。我們以EMSA (Electro- phoretic Mobility Shifting Assay)的方法也證明At2g20880會與GCC box (-AGCC- GCCAC-)以及DRE/CRT (-TACCGACAT-) element鍵結,進而調節其對抗逆境之相關基因。將At2g20880啟動子(1.3 kb)構築於pKGWFS7載體後轉殖到阿拉伯芥中,利用報導基因GUS表現加以染色後觀察組織專一性的表現,發現活性表現主要分布於根部、莖部和葉部,並且在葉脈及維管束組織表現特別顯著;另外在花及果莢的活性表現則較微弱。利用CaMV 35S啟動子表現At2g20880與GFP之結合蛋白,以基因槍將載體打入洋蔥表皮細胞,發現At2g20880在細胞質中有表現;但將載體暫時表現在利用PEG轉型的阿拉伯芥原生質體中,發現At2g20880與GFP結合蛋白卻表現在細胞核中;另外將大量表現此結合蛋白的載體轉殖到阿拉伯芥中,觀察根部細胞內的表現,發現綠色螢光蛋白在轉殖株被乾旱處理後,會從細胞質轉移到細胞核中。由此結果我們推測At2g20880需要與其它乾旱逆境誘導之蛋白質結合後,才能轉移至細胞核內作用。Pull-down assay以及免疫沉澱(co-immunoprecipitation)也被利用來進一步研究與At2g20880結合的蛋白,但目前並沒有找到任何與之結合的蛋白。結合本實驗室前人對At2g20880 gain-of-function的研究,可以得知At2g20880只有在乾旱逆境中才能發揮轉錄因子的功能,屬於ABA-independent pathway,在乾旱訊息傳導路徑中扮演ㄧ個正向調控者的角色。 Plant growth and productivity are affected by various abiotic stresses such as heat, cold, drought, and high salinity. Exposure to these stresses induces various biochemical and physiological changes in the process of acquiring stress tolerance. Assortments of genes with diverse functions have been described to respond to these stresses at the transcriptional level. According to the microarray data (Kamiya et al., 2002), we have selected a drought-induced gene, At2g20880, which was induced by 24.5 folds after 2h drought treatment, for studying more details in this thesis. This putative transcription factor belongs to an AP2/ERF superfamily and has a highly conserved AP2 domain. Our Northern blot data revealed that At2g20880 was induced by drought stress, salt stress, and osmotic stress, but not by heat stress and cold stress. In addition, At2g20880 was not induced by ABA, ethylene, and methyl jasmonate treatments. It was demonstrated that the At2g20880 protein can bind to both GCC box (-AGCCGCCAC-) and DRE/CRT element (-TACCGACAT-) in the promoter regions of responsive genes by EMSA. Using promoter-GUS assay, we have found that At2g20880 was expressed in roots, stems, and leaves, especially in vascular tissues, but had lower expression levels in flowers and siliques. Subcellular localization study using a fusion protein consisting of the full length of At2g20880 coding region and GFP fusion under the control of 35S promoter revealed that the GFP fluorescence was detected in the cytosol in onion epidermal cells. However, it was also detected in the nuclei of Arabidopsis protoplasts. Stable transgenic lines were also generated to observe its subcellular localization. Our study indicated that At2g20880-GFP was detected in the nuclei only in PEG-treated protoplasts and drought-treated plants. The amino acid sequence analysis by PredictNLS server showed this protein lacks a typical nuclear localization signal, implying that protein-protein interaction is required for nuclear import during drought treatment. Pull-down assay and co-immuno- precipitation assay were also applied to study the interacting proteins with At2g20880. However, no interacting protein was detected so far. Together with our previous gain-of- function study of At2g20880, At2g20880 may have a function only under drought condition and is a positive regulator in ABA-independent dehydration stress response. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/37693 |
全文授權: | 有償授權 |
顯示於系所單位: | 植物科學研究所 |
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