林讚標臺灣大學:植物科學研究所吳允哲Wu, Yun-ZheYun-ZheWu2010-05-112018-07-062010-05-112018-07-062009U0001-0808200921045900http://ntur.lib.ntu.edu.tw//handle/246246/181961植物生長過程中會受到各類非生物逆境所影響，諸如乾旱、高鹽、高溫、低溫等。其中乾旱會中止植物的水分供給，故干擾植物正常機能運作。植物會使用各種生理或生化機制以對抗乾旱。前人研究顯示植物會於轉錄層級進行基因調控，使下游基因受活化或抑制以因應乾旱逆境(Shinozaki and Yamaguchi-Shinozaki, 1997; Reddy et al., 2004)。我們從2002年發表的微矩陣序列分析資料(Seki et al., 2002)選出會受乾旱大量誘導的阿拉伯芥基因At4g28140進行研究，其於乾燥處理2小時後，表現量會提升至一般狀態的37.7倍。此轉錄調控因子屬於AP2/ERF superfamily，且含一個具高度保留性的AP2 DNA binding domain。根據北方式墨點實驗，我們發現At4g28140會受乾旱、高鹽及滲透壓逆境所誘導，但在低溫和高溫逆境下則無誘導表現。此外，At4g28140不受離層酸、乙烯、茉莉酸甲酯誘導表現，故其應屬於ABA-independent pathway。我們將At4g28140啟動子(1 kb)構築於載體後轉殖到阿拉伯芥中，經乾旱誘導報導基因GUS表現並加以染色後觀察組織專一性。而我們的研究結果顯示活性表現主要可見於根、莖、葉、花與果莢，特別是在維管束組織的表現最為顯著。我們建構了At4g28140與GFP的結合蛋白，透過基因槍將載體轉入洋蔥表皮細胞，在經由CaMV 35S啟動子調控下，可於細胞核中偵測到綠色螢光訊號。此基因的胺基酸序列分析顯示其具核定位訊號(NLS)。而電泳位移分析(Electrophoretic Mobility Shift Assay) 證實At4g28140蛋白會與GCC box (-AGCCGCCAC-) 以及DRE/CRT (-TACCGACAT-) element結合，而此兩者可見於逆境反應基因的啟動子序列中，故下游基因應可受At4g28140調控。在loss-of-function研究中，T-DNA insertion knockout突變株與野生型植株間的表現型並未有顯著差異，這可能是肇因於其他的逆境相關基因之功能性重覆。而在基因大量表現的研究中，35S::HA-At4g28140轉殖株無法產生可測得之蛋白質，推測可能原因為蛋白酶體的作用所致。綜上所述，本研究證實At4g28140為不需倚賴ABA，且可受到乾旱誘導表現的轉錄調控因子。而根據大量表現刪除C-terminal之At4g28140的實驗結果(資料由陳瑞宏提供)，此基因在乾旱訊息傳導路徑中可能扮演正向調控者的角色。Various abiotic stresses, including drought, high salinity, heat, and cold, affect plant growth. Drought deprived plants of water supply, thus interrupting the normal function. Plants use combination of different physiological and biochemical strategies against drought. Regulation of genes at transcriptional level has been described in response to drought stresses (Shinozaki and Yamaguchi- Shinozaki, 1997; Reddy et al., 2004). According to the microarray data (Seki et al., 2002), we selected At4g28140, which was highly induced to 37.7 fold after drought treatment for 2 hours. This putative transcription factor belongs to AP2/ERF superfamily and has a highly conserved AP2 domain. Our northern blot results revealed that At4g28140 was induced by drought stress, high salinity, and osmotic stress, but not by cold stress and heat stress. In addition, it was demonstrated that At4g28140 was not induced under ABA, ethylene, and methyl jasmonate treatments, and is involved in the ABA-independent pathway. We have found that At4g28140 was expressed in roots, stems, leaves, flowers and siliques, especially in vascular tissues by promoter-GUS assay. Using a fusion protein composed of the full length of At4g28140 coding region and GFP under the control of 35S promoter, subcellular localization study revealed that the GFP fluorescence was detected in the nuclei of onion epidermal cells. The amino acid sequence analysis predicted that this protein contains a nuclear localization signal. Electrophoretic mobility shift assay confirmed that the At4g28140 protein can bind to both GCC box (-AGCCGCCAC-) and DRE/CRT element (-TACCGACAT-) in the promoter regions of responsive genes. Loss-of-function study which used T-DNA knockout mutant did not show significant differences from wild-type plant. The finding suggested some functional redundancy of other stress-related genes. With respect to overexpression approach, 35S::HA-At4g28140 transgenic plants failed to produce detectable protein due to proteasome activity. In conclusion, in this study we provided evidence to show At428140 is an ABA-independent, dehydration-inducible transcription factor. Along with the overexpression approach deleting the C-terminal of At4g28140 (performed by Jui-Hung Chen), this gene may function a positive regulator in drought stress response pathway.口試委員審定書………………………………………………………………………………………………………………I謝……………………………………………………………………………………………………………………………II文摘要………………………………………………………………………………………………………………………IIIBSTRACT………………………………………………………………………………………………………………………VBBREVIATIONS…………………………………………………………………………………………………………………1NTRODUCTION…………………………………………………………………………………………………………………3 1.1 Drought signal transduction………………………………………………………………………………………3 1.2 The DRE/CRT element and the GCC box……………………………………………………………………………4 1.3 AP2/ERF transcription factors in Arabidopsis…………………………………………………………………6 1.4 Function of AP2/ERF transcription factors in the regulation of stress responses…………………7 1.5 Strategies and goals of the thesis………………………………………………………………………………9ATERIALS and METHODS………………………………………………………………………………………………………11 2.1 Plant Materials, Growth Conditions, and Drought, Chemical Treatment…………………………………11 2.2 Analysis of the At4g28140 Gene Sequence………………………………………………………………………12 2.3 DNA Extraction, PCR, and Sequencing……………………………………………………………………………12 2.4 Southern-Blot Analyses……………………………………………………………………………………………13 2.5 RNA Extraction and cDNA Synthesis………………………………………………………………………………13 2.6 Northern-Blot Analyses……………………………………………………………………………………………14 2.7 Generation of Plant Transformation Constructs and Transgenic Arabidopsis…………………………14 2.8 Histochemical Staining and Fixation……………………………………………………………………………15 2.9 Construction and Subcellular Localization Analysis of Transiently Expressed Fusion Proteins…16 2.10 Preparation of GST-Recombinant proteins……………………………………………………………………16 2.11 SDS-PAGE (SDS-Polyacrylamide Gel Electrophoresis) staining……………………………………………17 2.12 Western-blot Analyses……………………………………………………………………………………………18 2.13 Electrophoretic Mobility Shift Assay (EMSA) ………………………………………………………………18 2.14 Abiotic Stresses Tolerance Analysis of Loss-of-function Mutant………………………………………19 2.15 Real-Time PCR………………………………………………………………………………………………………19 2.16 Plant Protein Extraction and Quantification………………………………………………………………20ESULTS…………………………………………………………………………………………………………………………21 3.1 At4g28140 Gene in A. thaliana……………………………………………………………………………………21 3.2 Expression of At4g28140 Gene under Various Abiotic Stress Conditions and Hormone Treatments…21 3.3 Tissue-Specific Expression of At4g28140………………………………………………………………………22 3.4 Subcellular Localization of At4g28140 Protein………………………………………………………………23 3.5 At4g28140 Protein Binds to Both the GCC Box and DRE/CRT Element………………………………………24 3.6 Screening and Abiotic Stresses Tolerance Analysis of At4g28140 Loss-of-function Mutant………25 3.7 Expression Analysis of At4g28140 Overexpression Lines……………………………………………………26ISCUSSION……………………………………………………………………………………………………………………27 4.1 Expression of At4g28140 is induced by drought, high salinity and osmotic stress…………………27 4.2 At4g28140 is mainly expressed in vasculature and nucleus………………………………………………27 4.3 At4g28140 protein has DNA binding specificity………………………………………………………………29 4.4 Loss-of-function mutants of At4g28140 exhibit no significant phenotypes……………………………30 4.5 At4g28140 protein may be degraded rapidly by proteasome system………………………………………31IGURES…………………………………………………………………………………………………………………………33EFERENCES……………………………………………………………………………………………………………………56PPENDIXES……………………………………………………………………………………………………………………62application/pdf9963635 bytesapplication/pdfen-US阿拉伯芥乾旱轉錄因子AP2/ERFAt4g28140Arabidopsisdroughttranscription factorhttp://ntur.lib.ntu.edu.tw/bitstream/246246/181961/1/ntu-98-R96B42001-1.pdf