非生物性逆境誘導阿拉伯芥熱休克轉錄因子調控低分子量熱休克蛋白質基因表現之研究

Abiotic Stresses Induce Heat Shock Factors to Regulate Small Heat Shock Protein Genes Expression in Arabidopsis thaliana

黃琮智(Tsung-Chih Huang)

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(2007年)

https://doi.org/10.6342/NTU.2007.03331

摘要

熱逆境為植物的環境逆境之一，隨著溫室效應造成全球暖化日趨嚴重，也因此成為重要的關切議題。當植物暴露於熱逆境時，會產生共同的生理反應來抵抗高溫逆境，此反應稱為熱休克反應(heat shock response)，會大量合成一群新的蛋白質來保護細胞，這些蛋白質稱為熱休克蛋白(heat shock proteins, HSPs)，其中以低分子量熱休克蛋白(small HSPs, sHSPs)的種類與數量為最多，而sHSPs的合成被認為是經由熱休克轉錄因子(heat shock factors, HSFs)的調控。有別於動物系統(只有4個HSFs)，阿拉伯芥具有21個HSFs，至於在非生物逆境下HSFs如何調控sHSPs的表現及其扮演的角色，則尚未有深入的研究。本實驗藉由逆遺傳(reverse genetics)方式，從T-DNA插入所造成HSFs knock-out的植株中，觀察在非生物逆境下sHSPs的表現，藉以了解阿拉伯芥21個HSFs所扮演的可能角色。由11個HSF T-DNA 插入植株中共篩選出八個HSF不表現(null mutant)株，包括AthsfA1e、AthsfA2、AthsfA4c、AthsfA5、AthsfA6a、AthsfA7a、AthsfA7b及AthsfB2b。藉由微陣列資料及反轉錄聚合酶鏈式反應(RT-PCR)的分析，得知AtHSFA2、AtHSFA7a、AtHSFA7b與AtHSFB2b在熱逆境下可能參與調控sHSPs基因的表現，而AtHSFA6a可能參與滲透壓與鹽逆境下之sHSPs基因的調控。重金屬與滲透壓的處理下，AthsfA7a及AthsfA6a植株與wild type相比，各有明顯不同的sHSPs差異性表現。在植物的耐熱性測試中，驗證了AtHSFA2對於延長植物後天耐熱性的重要性，而不同的HSF不表現株其耐熱性並不受熱逆境影響。總結，本實驗初步建立了阿拉伯芥熱逆境與其他非生物性逆境下HSFs和sHSPs的基因表現圖譜及上下游關係，並得知AtHSFA6a與AtHSFA7a在滲透壓逆境及重金屬中，可調控sHSPs的基因表現，但這些HSFs其真正的生理功能還需進一步的研究來證實。

關鍵字

熱休克轉錄因子 ； 低分子量熱休克蛋白 ； 熱逆境

並列摘要

Heat stress is one of the major environmental stresses. When plants are exposed in the elevated temperature, a general physiological process called heat shock (HS) response makes plants accumulate amounts of proteins called heat shock proteins (HSPs) to survive from heat stress. Among HSPs, small HSPs (sHSP) represent the unusual abundance and complexity in plants, and are mainly regulated by heat shock factors (HSFs). There are 21 HSFs in Arabidopsis in compared with 4 in mammalian, but the roles of each HSF in controlling sHSP gene expression and thermotolerance remain largely unaddressed. Hence, this study used reverse genetics analysis to clarify the roles of these HSFs in Arabidopsis. We screened 11 AtHSFs T-DNA insertion lines and 8 AtHSFs null mutant lines including AthsfA1e, AthsfA2, AthsfA4c, AthsfA5, AthsfA6a, AthsfA7a, AthsfA7b, and AthsfB2b were characterized and confirmed by RT-PCR. Through microarray database mining and RT-PCR analysis, some HSFs were induced by certain abiotic stresses. When HSF knock-out plants were treated with various abiotic stresses to examine sHSPs expression, the experiment results confirmed that under heavy metals treatments several sHSPs expression in AthsfA7a mutants was affected, and in AthsfA6a mutant sHSPs expression were also interfered during salt and osmotic stresses. Hence, AtHSFA7a and AtHSFA6a may regulate sHSPs expressions respectively in response to heavy metal and osmotic or salt stresses. When testing the thermotolerance in each HSF null mutant plants, results showed they didn’t lose the basal and acquired thermotolerance, except in AthsfA2. In summary, the expression profile of sHSPs and HSFs under HS and other abiotic stress as well as the regulation of sHSPs expression by several HSFs are established. In addition, AtHSFA7a and AtHSFA6a specifically functioning in regulation of sHSPs expressions in response to heavy metal and osmotic or salt stresses are suggested.

並列關鍵字

heat shock ； heat shock factors ； small heat shock proteins

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