阿拉伯芥熱休克轉錄因子結合蛋白之功能性研究

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。

關鍵字

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

並列摘要

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.

並列關鍵字

heat shock ； heat shock response ； heat shock protein ； heat shock facotr ； heat shock factor binding protein ； HSBP

參考文獻

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阿拉伯芥熱休克轉錄因子結合蛋白之功能性研究

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