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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 靳宗洛(Tsung-Luo Jinn) | |
dc.contributor.author | Chi-Min Liao | en |
dc.contributor.author | 廖期敏 | zh_TW |
dc.date.accessioned | 2021-06-08T03:29:49Z | - |
dc.date.copyright | 2019-08-18 | |
dc.date.issued | 2019 | |
dc.date.submitted | 2019-08-15 | |
dc.identifier.citation | Åkerfelt, M., Morimoto, R.I., and Sistonen, L. (2010). Heat shock factors: integrators of cell stress, development and lifespan. Nature Reviews Molecular Cell Biology 11, 545.
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/21264 | - |
dc.description.abstract | 熱休克因子結合蛋白(Heat-Shock-Factor Binding Protein, HSBP) 是一個小於10 kDa的小分子量蛋白質,它只有一個卷曲螺旋(Coiled Coil)作為它的功能區塊,透過卷曲螺旋與HSFs上的七肽區域結合藉此弱化熱休克反應,這個卷曲螺旋內的胺基酸在物種間具有高度保留性,在人類HSBP的研究中發現這個卷曲螺旋能夠透過疏水性作用力自我形成單體、三聚體及六聚體。阿拉伯芥中唯一基因HSBP也利用相同的方式來行使對熱休克反應之負向調控者的功能。在hsbp突變株中發現三種表型缺陷,包括提早開花、果莢短小和種子缺失的現象。前人研究中發現HSBP卷曲螺旋的點突變會影響HSBP行使其生理功能,包含改變不同聚體間的數量分布、熱逆境下HSBP在細胞內的位置及與HSF的結合能力及對阿拉伯芥的生長及發育。本研究中根據人類與玉米文獻中所提及的HSBP關鍵點突變位置,將阿拉伯芥HSBP之相對應胺基酸進行突變,以此改變HSBP不同的聚體間的比例或是降低跟HSF的交互作用,並將建構之HSBP卷曲螺旋突變版本用於hsbp突變株互補試驗,結果發現在HSBP M16D/F19D點突變轉殖株中,果莢短小與種子缺失的表型被回復,M16D/V20D和S35A轉殖株則只有果莢短小的表型被回復,而後天性耐熱力測試中,這三種轉殖株都沒有回復成野生型的熱耐受力。因此,HSBP的聚體在生長發育和熱反應中是有不同的功能的。另一方面,在前人研究中透過液相層析串聯式質譜儀(LC-MS/MS)與免疫共沉澱法(Co-IP)分析,找到COP1-interacting protein 1 (CIP1)、MAIGO2 (MAG2)和kinesin like protein for actin based chloroplast movement 1/2 (KAC1/2)為HSBP之 interacting proteins。hsbp在後天性耐熱力(acquired thermotolerance, AT)測試中有著較高的熱耐受力,而cip1突變株則跟野生型一樣。hsbp 和hsbp cip1 double mutant對藍光較不敏感,而cip1對紅光較敏感。因此,HSBP 可能參與了光訊號傳遞,生長發育與熱逆境反應中。 | zh_TW |
dc.description.abstract | Heat-shock-factor binding protein (HSBP) is a small 10-kDa protein and over 60% of protein consists of a highly conserved hydrophobic heptad repeat among species, and also self-associates leading to oligomerization. Changes in protein oligomerization status are known to be associated with regulation of various cellular processes. Arabidopsis HSBP, a single gene, is a negative regulator to attenuate the heat shock response (HSR) through interacting with the HSFs by the coiled-coil domain. HSBP participates in plant growth in addition to its well-known function in heat response. There are three major defective phenotypes in the hsbp mutant, e.g., early flowering, shorten silique, and seed abortion.
Our previous data showed that the HSBP coiled-coil domain point mutation caused the changing of oligomerization status, subcellular localization, and reducing the HSF interaction ability. By LC-MS/MS and Co-IP analysis, the COP1-interacting protein 1 (CIP1), kinesin like protein for actin-based chloroplast movement 1/2 (KAC1/2) and the ER-localized protein MAIGO2 (MAG2) were identified as HSBP interacting proteins. The hsbp cip1 double mutants showed higher thermotolerant phenotype, but cip1 was similar to the wild type (WT) plants. The HSBP-M16D/F19D mutation in order to break hexamer and favored the trimerization of HSBP recovered the hsbp short silique and seed abortion phenotypes to the WT. The HSBP-M16D/V20D and -S35A only recovered the hsbp shorten phenotype. However, the acquired thermotolerance on HSBP-M16D/F19D, -M16D/V20D and -S35A were not recovered. Thus, the oligomerization status of HSBP has different functions in response to heat stress and in the development stages Arabidopsis thaliana. | en |
dc.description.provenance | Made available in DSpace on 2021-06-08T03:29:49Z (GMT). No. of bitstreams: 1 ntu-108-R05b42020-1.pdf: 4697084 bytes, checksum: 7296341f624490aec5a44a2228a6619f (MD5) Previous issue date: 2019 | en |
dc.description.tableofcontents | ABSTRACT...............................................................................................................................6
摘要...........................................................................................................................................8 ABBREVIATIONS.....................................................................................................................9 INTRODUCTION....................................................................................................................10 Heat Shock Response............................................................................................................10 Heat Shock Factors................................................................................................................12 Heat Shock Factor Binding Protein........................................................................................13 The Coiled-Coil Domain.........................................................................................................16 Cytosolic Interacting Proteins of Arabidopsis HSBP...............................................................19 Motivation and Objectives......................................................................................................22 MATERIALS AND METHODS................................................................................................23 Plant Materials and Growth Conditions...................................................................................23 DNA/RNA Extraction, RT-PCR and qPCR (quantitative PCR)................................................24 Protein Extraction and Western Blot Analysis.........................................................................25 Transient Expression Assay in Arabidopsis Mesophyll Protoplasts........................................25 Acquired Thermotolerance Assay...........................................................................................26 β-Glucuronidase (GUS) Activity Assay and Cleared Tissue for Observation..........................27 Statistical Analysis..................................................................................................................28 Primers and Accession Numbers............................................................................................28 RESULTS................................................................................................................................29 The hsbp Seed Phenotype and GUS Staining Assay.............................................................29 Create the hsbp cip1 Double Mutant and Acquired Thermotolerance Assay.........................29 The Characterization of Mutated HSBP-3xFLAG Transgenic Plants.....................................31 Complementation of Transgenic Lines Phenotypes...............................................................31 The Acquired Thermotolerance of Transgenic Lines..............................................................32 The HSBP-Oligomeric Status in Response to Heat...............................................................32 The HSBP-Oligomeric Status of HSBP-mutant Lines............................................................33 Interaction of HSBP-mutants and HSFA2..............................................................................34 DISCUSSION.........................................................................................................................35 HSBP Is Involved in Photomorphogenesis and Heat Stress Response................................35 Heat Shock Factor Binding Protein (HSBP) and Coiled-Coil Domain....................................36 Conclusions............................................................................................................................39 Perspective and Future Work.................................................................................................40 TABLES..................................................................................................................................42 Table 1. Primer sequence for genotyping, RT-PCR and qPCR..............................................42 Table 2. The predicted results of mutated HSBP in human and maize, then corresponding the point mutation sites in Arabidopsis..........................................................................................44 Table 3. Phenotypic characterization of transgenic lines grow under normal condition..........45 FIGURES................................................................................................................................46 Figure 1. The seed phenotypes of hsbp mutant.....................................................................46 Figure 2. The expression pattern of HSBP in seed and seedling by GUS staining................47 Figure 3. Identification and characterization of cip1 and hsbp x cip1 double mutant..............48 Figure 4. Thermotolerance assay of the HSBP-interacting protein mutants...........................49 Figure 5. Measurement of hypocotyl length in hsbp mutant...................................................50 Figure 6. Sequence alignment of the HSBP homologs among different species and sites of mutations................................................................................................................................51 Figure 7. Expression of variant mutants of HSBP-3xFLAG in hsbp mutant background plants......................................................................................................................................52 Figure 8. Complementary of short silique phenotype in hsbp rescued by variant HSBP-mutation transgenes...............................................................................................................54 Figure 9. Complementary of early flowering phenotype in hsbp rescued by variant HSBP-mutation transgenes...............................................................................................................56 Figure 10. Complementary of seed abortion phenotype in hsbp rescued by variant HSBP-mutation transgenes...............................................................................................................57 Figure 11. Complementary of thermotolerance defect phenotype in hsbp rescued by variant HSBP-mutation transgenes....................................................................................................58 Figure 12. Complementary of thermotolerance defect phenotype in hsbp rescued by variant HSBP-mutation transgenes....................................................................................................60 Figure 13. Oligomerization status of HSBP in response to heat stress (HS)..........................61 Figure 14. Oligomerization status of HSBP in variant HSBP-mutation transgenic lines.........62 Figure 15. HSFA2 interacts with variant HSBP mutants analyzed by BiFC............................63 Supplementary data................................................................................................................64 APPENDICES..........................................................................................................................66 REFERENCES........................................................................................................................70 | |
dc.language.iso | en | |
dc.title | 阿拉伯芥熱休克因子結合蛋白的聚合狀態在熱逆境反應與生長發育之功能性研究 | zh_TW |
dc.title | Functional Study of the Oligomerization Status of Arabidopsis Heat Shock Factor Binding Protein (HSBP) in Development and Heat Response | en |
dc.type | Thesis | |
dc.date.schoolyear | 107-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 楊健志(Chien-Chih Yang),鄭秋萍(Chiu-Ping Cheng),張英?(Ing-Feng Chang),王雅筠(Ya-Yun Wang) | |
dc.subject.keyword | 熱休克因子結合蛋白,卷曲螺旋域,聚體, | zh_TW |
dc.subject.keyword | HSBP,coiled-coil domain,oligomerization status, | en |
dc.relation.page | 73 | |
dc.identifier.doi | 10.6342/NTU201903320 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2019-08-15 | |
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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