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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 張英? | |
dc.contributor.author | Chun-Wei Chun | en |
dc.contributor.author | 陳俊偉 | zh_TW |
dc.date.accessioned | 2021-06-17T00:32:47Z | - |
dc.date.available | 2017-03-19 | |
dc.date.copyright | 2012-03-19 | |
dc.date.issued | 2011 | |
dc.date.submitted | 2012-02-09 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/66376 | - |
dc.description.abstract | 植物在生長發育中,當環境劇烈改變時,像是乾旱、高鹽和極端溫度,植物會藉由不同的訊息傳遞路徑以適應不同的非生物性逆境。根據前人研究,阿拉伯芥乾旱誘導蛋白AtDi19可能會參與不同的逆境訊息傳遞路徑。目前在阿拉伯芥中共發現七個Di19基因,AtDi19-2 (At1G02750)是其中唯一被預測,不帶有核定位訊號(NLS)和核輸出訊號(NES)的蛋白,而其基因表現會受到高鹽處理所誘導,且不會受到離層酸(ABA)所調控,但目前對於AtDi19-2蛋白的功能還是未知的。本實驗室之前的研究,利用雙重性親合純化(tandem affinity purification)和pull-down分析,發現AtDi19-2會跟14-3-3蛋白和NUTCRACKER (NUC)轉錄因子分別有蛋白質和蛋白質的分子間交互作用。此外,利用雙分子互補螢光系統(bimolecular fluorescence complementation)和石英晶體微天平(quartz crystal microbalance)的方法來驗證AtDi19-2跟14-3-3 omega和NUC之間的交互作用。結果顯示AtDi19-2會在細胞質和細胞核中表現,除了跟14-3-3 omega在細胞質和細胞核內有交互作用外,也和NUC在細胞核內有交互作用。而且,di19-2 di19-5雙重突變株在高鹽處理下種子發芽率會比野生型高,有著對高鹽逆境比較不敏感的外表型。這些結果顯示AtDi19-2或AtDi19-5可能在高鹽逆境反應中扮演重要角色。 | zh_TW |
dc.description.abstract | Plants as sessile organisms must respond to changes in environmental conditions, such as various environmental stresses including too little water (drought), too much salt (salinity), and extreme temperature. The family of drought-inducible 19 (Di19) contain seven member with unknown functions in Arabidopsis. The AtDi19 family contains two atypical Cys/His zinc finger-like domains which may participate in different stress response and signaling pathways. AtDi19-2 (At1G02750) transcript level increased in response to high-salt stress, but its protein expression pattern and functions remain unknown. Previous study suggested that AtDi19-2 can interact with 14-3-3 proteins and NUC/AtIDD8 by use of tandem affinity purification and pull-down assay. Here, I use Bimolecular fluorescence complementation (BiFC) assays and quartz crystal microbalance (QCM), and confirmed that AtDi19-2 can interact with 14-3-3 proteins in nucleus and cytosol, and interact with NUC in the nucleus. In addition, I found that di19-2 di19-5 double mutant have higher germination rate than wild-type under condition of salt stress. This suggested that AtDi19-2 or AtDi19-5 may play important role in salt stress response. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T00:32:47Z (GMT). No. of bitstreams: 1 ntu-100-R98b42008-1.pdf: 34215437 bytes, checksum: 85a4d2221ed5af3952973cd2bba1e079 (MD5) Previous issue date: 2011 | en |
dc.description.tableofcontents | 誌謝 i
中文摘要 ii ABSTRACT iii Abbreviations iv CONTENTS vi LIST OF TABLES x LIST OF FIGURES xi 1.Introduction. 1 1.1 Abiotic stress 1 1.2 Osmotic stress 1 1.2.1 ABA-independent Pathways 2 1.2.2 ABA-dependent Pathways 3 1.2.3 Salt stress and ion homeostasis 3 1.3 Calcium-Dependent Protein Kinases (CDPKs) 4 1.4 Drought-inducible proteins Di19 5 1.4.1 Di19 in Arabidopsis thaliana and rice 5 1.4.2 Di19 in Triticum aestivum L. 6 1.4.3 Di19-2 in Gossypium hirsutum 7 1.4.4 AtDi19 interacts with Calcium-Dependent Protein Kinases (CDPKs) 7 1.5 14-3-3 protein 8 1.5.1 Structure of 14-3-3s 8 1.5.2 Functions of 14-3-3 8 1.5.3 14-3-3 Clients 9 1.5.4 Arabidopsis 14-3-3s 9 1.6 Nutcracker (NUC/AtIDD8) 10 1.7 Project goals 11 2.Materials and Methods 12 2.1 Plant materials and growth conditions 12 2.2 Preparation of E. coli DH5α competent cells 12 2.3 Transformation 13 2.4 Site-directed mutagenesis 13 2.5 Protoplast isolation 14 2.6 Transient expression 14 2.6.1 Plasmid Construction for Transient Expression 14 2.6.2 Transformation of Transient Expression plasmid 15 2.7 Bimolecular Fluorescence Complementation (BiFC) 15 2.7.1 Plasmid Construction of BiFC 15 2.7.2 Transformation of BiFC plasmids 16 2.8 Purification of recombinant protein 6His-Sumo-Di19-2 16 2.8.1 Plasmid Construction for generation of 6His-Sumo-Di19-2 16 2.8.2 Expression of 6His-Sumo-Di19-2 17 2.8.3 Purification of 6His-Sumo-Di19-2 17 2.9 Purification of recombinant protein MBP-NUC 18 2.9.1 Expression of MBP-NUC 18 2.9.2 Purification of MBP-NUC 18 2.10 Protein concentration measurement 19 2.11 Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) 19 2.12 Quartz Crystal Microbalance (QCM) 19 2.12.1 Sensor Washing 19 2.12.2 QCM analysis 20 2.13 Transactivation assay 20 2.14 In vitro kinase assay 21 2.15 Genomic DNA extraction 21 2.16 RNA extraction and RT-PCR analysis 22 2.17 Transgenic plant phenotyping 22 3.Results……. 24 3.1 Phylogenetic analysis of Di19-related proteins 24 3.2 Domain characterization of the AtDi19-2 sequence 24 3.3 Subcellular localization analysis of AtDi19-2 and 14-3-3 omega proteins 25 3.4 Protein–protein interactions between AtDi19-2 and 14-3-3s were confirmed by Bimolecular Fluorescence Complementation (BiFC) 25 3.5 Site-directed mutagenesis of AtDi19-2 26 3.6 Subcellular localization analysis of mutated AtDi19-2 27 3.7 The 14-3-3 binding sites of AtDi19-2 were not confirmed by BiFC 27 3.8 Dimerization of AtDi19-2 28 3.9 Protein−protein interaction between AtDi19-2 and transcription factor NUC was confirmed by BiFC 28 3.10 AtDi19-2 protein expression in E. coli 28 3.11 Analysis of protein–protein interaction between AtDi19-2 and NUC using quartz crystal microbalance 29 3.12 Transactivation of SUS4 was up-regulated by NUC and AtDi19-2 29 3.13 NUC was phosphorylated by AtCDPK1 and AtCDPK16 30 3.14 Isolation of a di19-2 di19-5 double mutant 31 3.15 The di19-2 di19-5 double mutant showed reduced sensitivity to NaCl 31 4.Discussion… 33 5.References… 37 Tables 48 Figures 51 Appendix 76 | |
dc.language.iso | zh-TW | |
dc.title | 阿拉伯芥Di19-2蛋白之功能性研究 | zh_TW |
dc.title | Functional study of Drought-Induced protein 19-2 in Arabidopsis thaliana | en |
dc.type | Thesis | |
dc.date.schoolyear | 100-1 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 鄭石通,林讚標,謝明勳,葉國楨 | |
dc.subject.keyword | 乾旱誘導蛋白,14-3-3,Nutcracker,雙分子互補螢光系統,石英晶體微天平,鹽分逆境, | zh_TW |
dc.subject.keyword | Drought-inducible 19,14-3-3,NUTCRACKER,bimolecular fluorescence complementation,quartz crystal microbalance,salt stress, | en |
dc.relation.page | 85 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2012-02-10 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 植物科學研究所 | zh_TW |
顯示於系所單位: | 植物科學研究所 |
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