阿拉伯芥熱休克因子結合蛋白在熱逆境反應及 生長發育時期之功能性研究

Functional Study of Heat Shock Factor Binding Protein (HSBP) in Heat Stress Response and Development Stages of Arabidopsis

李怡潔(Yi-Jie Li)

指導教授 ： 靳宗洛

國立臺灣大學/生命科學院/植物科學研究所/碩士(2017年)

https://doi.org/10.6342/NTU201704314

摘要

在前人研究中，阿拉伯芥熱休克因子結合蛋白(Heat Shock Factor Binding Protein, HSBP)已被證明透過利用自身的卷曲螺旋結構和熱休克因子(Heat Shock Facore,HSF)上的七胜肽重複區結合以達到在熱休克反應後期擔任負調控者的角色。在HSBP缺失的阿拉伯芥突變株中，我們觀察到了三個明顯的表現型缺陷，包括開花期的提早、果莢短小以及種子缺失三分之一的現象。為了瞭解這些表現型發生背後的分子機制，本篇研究中篩選並確認了HSBP的交互作用蛋白，包括光型態發育持續表現蛋白的交互作用蛋白一號(CIP1)、葉綠體移動類驅動蛋白一號及二號(KAC1/2)、種子儲存蛋白(MAG2)以及其三個共同作用蛋白(MIP1, 2 以及3)。HSBP和CIP1共同作用，參與光型態發育持續表現蛋白(COP1)影響開花時間以及光型態發育的調控。在HSBP突變株中偵測到不正常種子儲存蛋白的前驅物存在，暗示其在種子發育中可能扮演的角色。由於卷曲螺旋區域是HSBP的唯一功能性區域，推測其對結構的穩定性及功能是非常重要的。HSBP卷曲螺旋區域T17C以及R48K突變造成了聚合狀態的改變，而S35A突變則是會限制HSBP在細胞質而無法進入到細胞核中，I42K/M45K以及I49K/L52K突變株則是會對植株的耐熱性造成影響，結果確認卷曲螺旋對於HSBP結構以及生理功能具重要性。總體而言，本篇研究探究了HSBP在阿拉伯芥生長發育階段以及對抗熱逆境時所扮演的角色。

關鍵字

熱休克因子結合蛋白 ； 卷曲螺旋 ； 交互作用蛋白質 ； 光型態發育 ； 種子發育及儲存蛋白質

並列摘要

Arabidopsis heat shock factor binding protein (HSBP) has been reported as a negative regulator required for the attenuation of the heat shock response (HSR) by binding to the heptad repeat regions of HSF through its coiled-coil domain. In addition, three distinct phenotypes were observed in the HSBP-mutant plants, including early flowering, shorten silique length and one-third ratio of seed abortion. To decipher the molecular mechanisms behind these phenotypes and the functions of HSBP in developmental stages, I identified the interacting proteins of the HSBP through immunoprecipitation assay followed by the LC-MS/MS analysis. Cytoskeleton-associated proteins COP1 interactive protein1 (CIP1) and kinesin like protein for actin based chloroplast movement 1/2 (KAC1/2), as well as the ER-localized proteins, Maigo2 (MAG2) and MAG2-interactive protein1/2/3 (MIP1/2/3) were identified and confirmed as HSBP interacting proteins. HSBP along with CIP1 has effects on Constitutive photomorphogenic 1 (COP1) to participate the downstream flowering-time genes expression and photomorphogenesis control. The unprocessed seed storage protein precursors were detected in hsbp seeds with mis-shapen phenotype, indicating the possible role of HSBP in the intracellular protein trafficking and the seed maturation. The interaction between HSBP and KAC1/2 provides the insight into chloroplast cp-actin movement regulation. Since the coiled-coil domain is the only functional domain in HSBP, it is very important for its structure formation as well as the biological functions. The Arabidopsis HSBP-T17C and -R48K mutations cause the alteration of oligomerization status whereas the S35A variant restrains HSBP from entering into the nucleus. The I42K/M45K and I49K/L52K variants cause effects on heat stress tolerance ability. Together, we concluded that the mechanisms of HSBP versatile functions in response to heat stress and in the development stages in Arabidopsis thaliana.

並列關鍵字

HSBP ； interacting proteins ； photomorphogenesis ； seed storage proteins ； coiled-coil domain ； oligomerization status

參考文獻

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