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| ???org.dspace.app.webui.jsptag.ItemTag.dcfield??? | Value | Language |
|---|---|---|
| dc.contributor.advisor | 張英峯(Ing-Feng Chang) | |
| dc.contributor.author | Pei-Yuan Chen | en |
| dc.contributor.author | 陳培元 | zh_TW |
| dc.date.accessioned | 2021-06-08T03:44:53Z | - |
| dc.date.copyright | 2021-01-20 | |
| dc.date.issued | 2020 | |
| dc.date.submitted | 2021-01-07 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/21743 | - |
| dc.description.abstract | WRKY轉錄因子在鹽逆境下扮演重要的角色，可活化或抑制一些逆境相關基因。此外，WRKY轉錄因子可藉由結合至其下游基因之啟動子上的順式作用元件，進而調控下游基因表達。然而，目前對於阿拉伯芥WRKY25轉錄因子與交互作用蛋白質，例如: 14-3-3和HDA6的交互作用功能尚未明朗；以及在鹽逆境下，WRKY25轉錄因子的下游調控基因仍未清楚。此篇研究中的基因表現結果顯示，DREB1A可能是WRKY25轉錄因子的下游目標基因，因此為了探討在鹽逆境下WRKY25轉錄因子和DREB1A之間的轉錄調控關係。本研究利用qPCR發現，在鹽處理下WRKY25大量表現株中的DREB1A基因表現與野生型相比有較高的趨勢。在染色質免疫沉澱實驗中，發現鹽處理下WRKY25轉錄因子會與DREB1A的啟動子結合。另外在鹽處理中，WRKY25大量表現株及dreb1a 功能獲得型突變株 (微過量表現DREB1A) 與野生型相比有較高的生存率，表示WRKY25及DREB1A具有抗鹽的功能。再者，在轉錄活性分析實驗中也顯示，鹽處理下WRKY25轉錄因子會活化DREB1A基因的轉錄。然而，當轉錄活性分析實驗同時加入WRKY25及14-3-3這兩種效應蛋白時，14-3-3似乎會去抑制DREB1A基因的轉錄。反之，實驗中發現HDA6似乎會去增強WRKY25活化DREB1A基因轉錄的效果。綜合上述結果，阿拉伯芥中會與WRKY25結合的蛋白，如14-3-3及HDA6，可能會影響WRKY25活化DREB1A基因轉錄的能力。 | zh_TW |
| dc.description.abstract | WRKY transcription factors play important roles in the regulation of salt stress response by activating or repressing several stress-inducible genes. In addition, WRKYs can bind to the cis-acting element W-box of downstream target genes to regulate their genes expression. However, the exact function of 14-3-3 and HDA6 proteins interacting with Arabidopsis WRKY25, and downstream genes targeted by WRKY25 under salt stress are still unclear. In this study, the gene expression analysis showed that DREB1A might be a target of WRKY25. Therefore, I aim to elucidate the transcriptional regulation relationship between WRKY25 and DREB1A in salt stress response. The expression of DREB1A in WRKY25 OE lines was higher than that in the wild type under the salt-treated condition. In chromatin immunoprecipitation (ChIP) assay, WRKY25 appeared to bind to the DREB1A promoter under salt stress. In addition, both WRKY25 OE lines and dreb1a gain-of-function line that slightly overexpressed DREB1A exhibited higher tolerance to salt stress in survival rate. Moreover, the transcriptional activity of DREB1A was also significantly activated by WRKY25 under the salt-treated condition in transactivation assays. However, 14-3-3 seemed to repress the transcriptional activity of DREB1A when WRKY25 and 14-3-3 effectors were both introduced. On the contrary, HDA6 appeared to enhance the transcriptional activity of DREB1A activated by WRKY25. Taken together, the WRKY25 binding proteins, 14-3-3 and HDA6, are suggested to affect the transcription activity of WRKY25 on DREB1A in Arabidopsis. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T03:44:53Z (GMT). No. of bitstreams: 1 U0001-0601202114594300.pdf: 3744642 bytes, checksum: cd2353497f850d309c0280e86daa8a7d (MD5) Previous issue date: 2020 | en |
| dc.description.tableofcontents | 誌謝 I 摘要 II Abstract IV Abbreviations VI List of figures XII Supplementary figures XIV Appendixes XVII 1. Introduction 1 1.1 Biotic and abiotic stresses 1 1.1.1 Responses of plants to salt stress 2 1.1.2 Signal transduction networks in abiotic stress responses 3 1.2 WRKY transcription factors 4 1.2.1 DNA binding motif of WRKY transcription factors 5 1.2.2 Classification of WRKY transcription factors 6 1.2.3 WRKY TFs in plant growth and development 7 1.2.4 WRKY Transcription factors in biotic stress responses 8 1.2.5 WRKY Transcription factors in abiotic stress responses 10 1.2.6 WRKY Transcription factors are potential 14-3-3 interacting proteins................................................................................................... 12 1.2.7 Phosphorylation of WRKY transcription factors 13 1.2.8 Chromatin remodeling proteins as WRKY interacting proteins 15 1.2.9 Downstream target genes of WRKY transcription factors 16 1.2.10 Dehydration-responsive element binding proteins (DREBs) 16 1.3 Project goals 18 2. Materials and Methods 20 2.1 Plant materials 20 2.2 Growth conditions 20 2.3 Generation of constructs for overexpression lines 21 2.4 Arabidopsis transformation by floral dipping 22 2.5 Isolation of T-DNA insertional mutants 23 2.6 RNA extraction and gene expression analysis 24 2.7 Semi-quantitative and quantitative real-time PCR analysis 25 2.8 Phenotyping of survival rates and seed germination rates 25 2.9 Chromatin immunoprecipitation quantitative real-time PCR (ChIP- qPCR) Assays 26 2.10 Isolation of Arabidopsis leaf protoplasts 27 2.11 Bimolecular Fluorescence Complementation (BiFC) 28 2.12 Subcellular localization of WRKY25 29 2.13 Transactivation assay 29 2.14 Purification of recombinant protein 30 2.15 In vitro kinase assay 31 2.16 Statistical analysis 32 3. Results 33 3.1 Isolation of wrky25 mutant and WRKY25 overexpression lines 33 3.2 Induction of WRKY25 in response to salt stress 34 3.3 Gene expression of DREB1A in WRKY25 overexpression lines under salt treatment 34 3.4 Isolation of T-DNA insertional line dreb1a 35 3.5 Comparison of survival rate under salt stress condition 36 3.6 Seed germination rate and vigor in ABA response 37 3.7 Seed germination rate and vigor in salt stress response 37 3.8 Gene expression of WRKY25 in aba2 mutant under salt treatment 38 3.9 WRKY25 can bind to the promoter of DREB1A in vivo in salt stress response 39 3.10 Protein–protein interactions between WRKY25, 14-3-3 and HDA6…………………………………………………………………………. 40 3.11 Mutation of Thr345 site of WRKY25 to Ala appeared to affect the interaction between WRKY25, 14-3-3 and HDA6 41 3.12 Subcellular localization of site-directed mutagenesis of WRKY25……………………………………………………………… …..42 3.13 Expression of DREB1A might be regulated by WRKY25, 14-3-3 and HDA6 in transactivation assay 42 3.14 HDA6 but not WRKY25 can be phosphorylated by CDPK16 in vitro in a kinase assay 44 4. Discussion 46 4.1 WRKY25 and DREB1A involve in salt stress response 46 4.2 The DREB1A promoter could be bound and regulated by WRKY25 in salt stress condition 50 4.3 Protein–protein interactions between WRKY25, 14-3-3 and HDA6…………………………………………………………………………. 52 5. References 57 Figures 70 Supplementary Figures 91 Appendixes……………………………………………………………………… 118 | |
| dc.language.iso | en | |
| dc.title | 探討鹽逆境下阿拉伯芥WRKY25調控DREB1A的表現 | zh_TW |
| dc.title | Regulation of DREB1A expression by WRKY25 in Arabidopsis thaliana under salt stress | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 109-1 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 吳克強(Ke-Qiang Wu),謝旭亮(Hsu-Liang Hsieh),鄭貽生(Yi-Sheng Cheng),張孟基(Men-Chi Chang) | |
| dc.subject.keyword | WRKY25,DREB1A,鹽逆境,生存率,離層酸,14-3-3,HDA6, | zh_TW |
| dc.subject.keyword | WRKY25,DREB1A,salt stress,survival rate,ABA,14-3-3,HDA6, | en |
| dc.relation.page | 131 | |
| dc.identifier.doi | 10.6342/NTU202100022 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2021-01-08 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 植物科學研究所 | zh_TW |
| Appears in Collections: | 植物科學研究所 | |
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