博碩士論文 992204027 詳細資訊




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姓名 蔡孟諭(Meng-Yu Tsai)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 利用水稻HSP17.3啟動子探討阿拉伯芥熱休克因子在逆境下對細胞內蛋白質反應之角色分析
(Analysis of the roles of AtHSFs for cellular protein response under stresses by using OsHSP17.3 promoter)
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摘要(中) 除了熱逆境外,一種胺基酸衍生物azetidine-2-carboxylic acid (AZC) 能夠去誘導整個細胞內產生unfolded或misfolded蛋白質。因此,當大量的misfolded 蛋白質堆積在內質網 (endoplasmic reticulum, ER) 內時,即會誘使特定的基因與路徑進行調控蛋白質的修復,而這個反應過程即所謂的unfolded protein response (UPR)。且在UPR的過程中,此時或許在細胞質中也有另一個cytosolic protein response (CPR) 同時進行著。在CPR中,是有關於一群特定熱休克蛋白質的誘導表現。而在先前的研究中,我們發現到Oshsp17.3基因在阿拉伯芥系統中經過了AZC逆境處理後能被活化表現。所以在本篇研究中,我們想利用這套系統來探討究竟有哪些熱休克因子會參與在CPR中。根據RT-PCR分析結果顯示,AtHSFA2、AtHSFA4a、AtHSFA7a、AtHSFB2a 和AtHSFB2b在AZC逆境下的表現量有顯著的上升情況。因此,我們將建構好的Oshsp17.3基因啟動子結合GUS基因的質體送至阿拉伯芥熱休克因子突變株中,進而去比較這些轉殖株在AZC逆境下所呈現的GUS 活性。首先,根據RT-PCR分析結果顯示,在AZC逆境下無論在single或double熱休克因子突變株中,其它熱休克因子的表現並無顯著地受到影響。第二部分,在GUS 活性分析結果中顯示,含有Oshsp17.3pro::GUS轉殖株的GUS 活性,在athsfA2、athsfA4a、athsfA7a、athsfB2a和athsfB2b的突變株中是受到抑制的。第三部分,根據RT-PCR分析結果顯示,在Tunicamycin (Tm;為UPR的誘導物) 逆境下,阿拉伯芥熱休克因子並不會受到Tm活化表現。因此,推測上述這些熱休克因子在AZC逆境下會去參與調控Oshsp17.3基因的表現。且在這些熱休克因子中,AtHSFA2或許在經過AZC逆境所引起的CPR中扮演著主要的調節角色。
摘要(英) In addition to heat stress, an amino acid analog azetidine-2-carboxylic acid (AZC) is shown to induce production of unfolded or misfolded proteins in overall cells. Hence, the unfolded protein response (UPR), which is induced by the accumulation of misfolded proteins in the endoplasmic reticulum (ER), recruits specific genes and pathways to regulate protein repair in that compartment, and a parallel process, the cytosolic protein response (CPR), operates in the cytosol. The CPR is associated with the induction of a specific subset of HSP genes. In previous study, we found that Oshsp17.3 was expressed in Arabidopsis system under AZC treatment. In this study, we plan to use this system to detect which AtHSFs involved in CPR. RT-PCR analysis showed that the AtHSFA2, AtHSFA4a, AtHSFA7a, AtHSFB2a and AtHSFB2b were significantly induced by AZC. Thus we transfer the promoter of Oshsp17.3, which shows AZC responsiveness, fused with GUS reporter gene into these Arabidopsis hsf mutants and compare their GUS activity under AZC treatment. First, the RT-PCR analysis showed that the expression of other AtHSFs in the single or double athsf mutants were not significantly influenced by AZC treatment. Second, in the GUS activity analysis, we found that the GUS activity of Oshsp17.3pro::GUS was repressed in athsfA2, athsfA4a, athsfA7a, athsfB2a and athsfB2b mutants. Third, the RT-PCR analysis showed that AtHSFs were not activation under Tunicamycin treatment, which is an inducer of UPR. Hence, we suggest that these HSFs may involve in the regulation of the expression of Oshsp17.3 under AZC treatment. In these HSFs, AtHSFA2 may play a major role in the CPR of Arabidopsis in response to AZC treatment.
關鍵字(中) ★ 熱休克蛋白質
★ 熱休克因子
★ 細胞質蛋白質反應
★ 熱休克反應元素
關鍵字(英)
論文目次 摘要…………………………………………………………………………………………....…..i
Abstract………………………………………………………………………………………........ii
誌謝…………………………………………………………………………………………....…..iii
目錄………………………………………………………………………………………….....…..iv
圖表目錄……………………………………………………………………………………....…..vi
縮寫對照表…………………………………………………………………………………...….vii
壹、 緒論………………………………………………………………………………….....……..1
貳、 研究背景與目的………………………………………………………………………..…….6
參、 材料與方法………………………………………………………………………………..….8
一、水稻第一族小分子量熱休克蛋白質基因啟動子之轉殖……………………………..8
二、阿拉伯芥農桿菌轉殖……………………………………………………………….…10
三、阿拉伯芥轉殖株的分析…………………………………………………………….…13
四、阿拉伯芥基因表現分析………………………………………..…………….……….13
五、轉殖株之逆境處理與GUS活性 (GUS activity) 之分析………………………….…15
肆、 結果…………………………………………………………………………………..…..….18
一、以阿拉伯芥轉殖株表現系統進行Oshsp17.3啟動子分析……………..…………….18
二、AtHSFs在熱及AZC逆境下的表現情形…………….………………………………..19
三、AtHSFs突變株的鑑定…………………………………………………………….……19
四、Cross-link轉殖株之建立與AtHSFs在突變株中之表現……………………….……20
五、阿拉伯芥轉殖Oshsp17.3基因啟動子之轉殖株與cross-link轉殖株受熱逆境及AZC逆境處理的組織表現分析 (Histochemical GUS Staining)………………………………21
六、轉殖株受逆境處理誘導GUS活性表現之分析…………...…………………………..22
七、AtHSFs在Tunicamycin逆境下的表現情形…………………………………………..23
八、轉殖株受Tunicamycin逆境處理誘導GUS活性表現之分析…………………………24
伍、 討論………………………………………………………………………………………….25
一、在熱及AZC逆境下能夠誘導特定的熱休克因子進行表現………………………...25
二、AtHSFs在突變株中之表現…………………………………………...……………… 26
三、轉殖株受熱逆境及AZC逆境處理的組織表現分析 (Histochemical GUS Staining)..27
四、轉殖株受逆境處理誘導GUS活性表現之分析………………………….…………..27
五、AtHSFs在Tunicamycin逆境下的表現情形………………………………………….30
六、轉殖株受Tunicamycin逆境處理誘導GUS活性表現之分析…………………….…30
未來研究方向……………………………………………………………………………….31
陸、 參考文獻…………………………………………………………………………………….32
柒、 圖表……………………………………………………………………………..……….40
捌、 附錄………………………………………………………………………………………….49
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指導教授 葉靖輝(Ching-Hui Yeh) 審核日期 2013-11-1
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