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研究生:許時逢
研究生(外文):Shih-Feng Hsu
論文名稱:阿拉伯芥熱休克轉錄因子結合蛋白之功能性研究
論文名稱(外文):Functional Study of Heat Shock Factor Binding Protein in Arabidopsis thaliana
指導教授:靳宗洛靳宗洛引用關係
指導教授(外文):Tsung-Luo Jinn
學位類別:博士
校院名稱:國立臺灣大學
系所名稱:植物科學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:英文
論文頁數:58
中文關鍵詞:熱逆境熱休克反應熱休克蛋白熱休克轉錄因子熱休克轉錄因子結合蛋白
外文關鍵詞:heat shockheat shock responseheat shock proteinheat shock facotrheat shock factor binding proteinHSBP
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熱休克反應為所有生物體共通的現象。此現象受到熱休克轉錄因子與熱休克蛋白的嚴格調控,可避免細胞受到熱逆境的傷害。人類HSP70和熱休克轉錄因子結合蛋白(Heat shock factor binding protein; HSBP)可與活化的熱休克轉錄因子結合,促使其喪失轉錄活性,因而終結熱休克反應。然而在植物系統中,HSBP對於熱逆境的調控機制不甚清楚。本論文藉由遺傳學及分子生物技術,探討這個普遍表現於各個組織與會受熱誘導表現的阿拉伯芥AtHSBP的功能。經由觀察AtHSBP缺陷突變株的表現型,得知AtHSBP可調控胚胎發育,導致種子的發育不良。檢測其熱耐受性,確定突變株具有較高的誘導耐熱性,但是對於先天耐熱性並沒有顯著的影響,推測AtHSBP在熱逆境中對於耐熱性的獲得扮演負向調控者的角色。在阿拉伯芥原生質中表現AtHSBP-GFP,得知在正常生長情況以及37°C加熱1個小時後,AtHSBP只表現於細胞質中;在回復到正常生長情況1個小時後,AtHSBP會由細胞質轉移到細胞核內;而回復到正常生長情況2個小時後,細胞核內已偵測不到AtHSBP的表現。原生質體雙雜合系統分析中,確認AtHSBP除了自身外,也可與熱休克轉錄因子(AtHSFA1a,AtHSFA1b與AtHSFA2) 進行交互作用。藉由點突變分析AtHSBP 的C端疏水性功能區,發現於正常生長情況下此保守區可侷限AtHSBP表現於細胞質中,且與熱休克轉錄因子的交互作用扮演重要的角色。另外EMSA分析得知AtHSBP可以降低AtHSFA1b與熱休克元素結合的能力,因而降低AtHSFA1b的轉錄活性。利用即時定量聚合酶鍊鎖反應與西方墨點轉印分析,得知AtHSBP缺陷突變株在遭受熱逆境時,熱休克蛋白基因的表現量及蛋白累積量比野生型多;而AtHSBP的過度表現株,其熱休克蛋白基因的表現量及蛋白累積量下降,導致其誘導耐熱性能力降低。這些發現對於植物熱逆境的調控機制提供新的視野,並揭露AtHSBP在HSR扮演一個負向調控者與對於種子發育的重要性。

Heat shock response (HSR) is a universal mechanism in all organisms. It is under tight regulation by heat shock factors (HSFs) and heat shock proteins (HSPs) after heat shock (HS) to prevent stress damage. On the attenuation of HSR, HSP70 and HSF binding protein 1 (HSBP1) interact with HSF1 and thus dissociate trimeric HSF1 into an inert monomeric form in humans. However, little is known about the effect of HSBP with thermal stress in plants. This report describes our investigation of the role of AtHSBP in Arabidopsis by genetic and molecular approaches. AtHSBP was heat-inducible and ubiquitously expressed in all tissues; AtHSBP was also crucial for seed development after fertilization and during embryogenesis, as demonstrated by AtHSBP-knockout lines showing seed abortion. Thermotolerance results showed that AtHSBP participates in acquired thermotolerance but not basal thermotolerance and is a negative regulator of HSR. Subcellular localization revealed that the cytosolic-localized AtHSBP translocated to the nucleus in response to HS. Protoplast two-hybrid assay results confirmed that AtHSBP interacts with itself and with the HSFs, AtHSFA1a, AtHSFA1b, and AtHSFA2. The mutagenesis assay shows that the conserved residues of the hydrophobic heptad repeat domains in AtHSBP are important for retaining AtHSBP in the cytoplasm under normal growth conditions and for interacting with AtHSFs. AtHSBP also negatively affected AtHSFA1b DNA-binding capacity in vitro. Quantitative PCR and western blot analysis demonstrated that altered levels of AtHSBP lead to differential HSP expression mainly during the recovery from HS. These studies provide a new insight into HSBP in plants and reveal that AtHSBP is a negative regulator of HSR and required for seed development.

Abstract in Chinese ---------------------------------------------------------------------------------- 4
Abstract in English ----------------------------------------------------------------------------------- 5
Abbreviations ------------------------------------------------------------------------------------------ 6

Introduction
1. Heat Shock Response and Thermotolerance ------------------------------------------------- 7
2. Heat Shock Proteins -------------------------------------------------------------------------------- 7
3. The Regulation of HSR by Heat Shock Transcription Factors --------------------------- 9
4. Heat Shock Factor Binding Proteins ---------------------------------------------------------- 11
5. Progresses and Goals ----------------------------------------------------------------------------- 13

Materials and Methods
1. Plant Material and Growth Condition -------------------------------------------------------- 14
2. Overexpression of AtHSBP, Complementation test and Transformation ------------ 14
3. Semi-Thin Sections and Light microscopy -------------------------------------------------- 15
4. Cleared Tissue for Observation of Siliques ------------------------------------------------- 15
5. Thermotolerance Tests --------------------------------------------------------------------------- 15
6. Transient Expression in Arabidopsis Mesophyll Protoplasts --------------------------- 16
7. Protoplast Two-Hybrid (P2H) and GUS Activity Assay --------------------------------- 17
8. Recombinant Protein Purification and Electrophoretic Mobility Shift Assay (EMSA) ---------------------------------------------------------------------------------------------------------- 17
9. Real-Time Quantitative PCR ------------------------------------------------------------------- 18
10. Western Blot Analysis -------------------------------------------------------------------------- 19
11. Primers and Oligomers ------------------------------------------------------------------------- 19
12. Accession Numbers ----------------------------------------------------------------------------- 19

Results
1. AtHSBP is a Functional Homolog among Plants and Animals ------------------------- 20
2. Heat-Inducible AtHSBP are Ubiquitously Expressed in All Tissues of Arabidopsis ---------------------------------------------------------------------------------------------------------- 20
3. The Phenotypic Characterization of AtHSBP-Knockout and AtHSBP- Overexpressed Lines ------------------------------------------------------------------------------ 21
4. The Defective AtHSBP May not Affect Ovule Development but May Influence Embryogenesis after Fertilization to Result in Seed Abortion ------------------------- 22
5. Altered Levels of AtHSBP Regulate Thermotolerance ----------------------------------- 22
6. Cytosolic-Localized AtHSBP Translocates to the Nucleus in Response to Thermal Stress ------------------------------------------------------------------------------------------------- 23
7. AtHSBP Interacts with AtHSFs and Itself in vivo ----------------------------------------- 24
8. Characterization of the AtHSBP Motif for Subcellular Localization and Interaction with AtHSFs -------------------------------------------------------------------------------- 24
9. AtHSBP Negatively Affects AtHSFA1b HSE-Binding Capacity in vitro ----------- 25
10. AtHSBP Functions as a Negative Regulator of HSR ------------------------------------ 26

Discussion
1. Structural Analysis of AtHSBP ---------------------------------------------------------------- 28
2. Heat-Inducible AtHSBP Plays an Important Role in Seed Development and May Confers Multiple Functions than Monocots ------------------------------------------------ 28
3. The Translocation of Cytosolic-Localized AtHSBP to the Nucleus during the Recovery from HS is Associated with the Attenuation of HSR ---------------------- 29
4. The Residue Ser35 and the HR Domains on C Terminus of AtHSBP are Important for AtHSBP Translocation or for Interacting with AtHSFs ---------------------------- 30
5. AtHSBP May Coordinate with HSP70 to Attenuate the HSR -------------------------- 31
6. Conclusions ----------------------------------------------------------------------------------------- 32
7. Perspectives ----------------------------------------------------------------------------------------- 33

Tables --------------------------------------------------------------------------------------------------- 35
Figures -------------------------------------------------------------------------------------------------- 40
References --------------------------------------------------------------------------------------------- 54


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