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研究生:吳上豪
研究生(外文):Shang-Hao Wu
論文名稱:阿拉伯芥HSA32調控長期後天耐熱性與HSP101降解途徑之研究
論文名稱(外文):Studies on the mechanism of HSA32-mediated long-term acquired thermotolerance and HSP101 degradation in Arabidopsis
指導教授:常怡雍
口試委員:葉國禎楊健志
口試日期:2016-07-22
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:生化科技學系
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2016
畢業學年度:104
語文別:英文
論文頁數:51
中文關鍵詞:阿拉伯芥HSA32HSP101長期後天耐熱性細胞自噬26S 蛋白酶體
外文關鍵詞:Arabidopsis thalianaHSA32HSP101Long-term acquired thermotoleranceAutophagy26S proteasome
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熱鍛鍊增強植物對劇烈熱逆境的耐受力,這個現象又稱為後天耐熱性。先前研究發現缺乏HSA32的阿拉伯芥及水稻幼苗有長期後天耐熱性缺陷,但是保有短期後天耐熱性。結果顯示HSA32係藉由抑制HSP101的降解,以延遲後天耐熱性之衰退,可是HSP101降解與HSA32調節HSP101穩定性的機制仍然不清楚。已知蛋白質降解主要經由26S蛋白酶體與細胞自噬作用兩種途徑,欲了解HSP101經何種機制分解,本實驗利用強效之26S蛋白酶體抑制劑bortezomib與細胞自噬突變株atg5分別阻礙這兩種途徑。結果顯示阻礙任一途徑皆只能部分抑制HSP101降解,然而將hsa32 atg5雙突變株施以bortezomib同時阻礙兩條蛋白質降解途徑時,HSP101的降解速率回復至野生型水平。根據實驗結果總結,受熱誘導產生的HSP101在植物回到室溫後,會經由26S蛋白酶體與細胞自噬逐漸分解,而HSA32的作用在於抑制HSP101透過這兩個途徑降解。

Heat acclimation enhances plant thermotolerance against severe heat stress, a phenomenon known as acquired thermotolerance. Previous research demonstrated that
Arabidopsis and rice seedlings of HSA32 knockout (hsa32) mutant have defect in long-term acquired thermotolerance (LAT), but retain normal short-term acquired thermotolerance (SAT). It was shown that HSA32 regulates LAT by specifically preventing HSP101 from degradation. However, the mechanisms of HSP101 degradation and HSA32-mediated HSP101 stability remain unclear. Proteolysis can be categorized into 26S proteasome and autophagy pathways. The strong 26S proteasome inhibitor bortezomib and autophagy related mutant atg5 were used to specifically block proteolysis pathways to investigate which route is responsible for the degradation of HSP101. The results demonstrated that blocking either pathway in the absence of HSA32 resulted in partial suppression of HSP101 degradation. However, when hsa32 atg5 double mutant was treated with bortezomib, the HSP101 level was
restored to that of the wild type. Taken together, the results show that HSP101 undergoes post-stress degradation through both 26S proteasome and macroautophagy pathways, which is suppressed by HSA32.

Table of Contents
口試委員會審定書......…………....…………………………………………………....i
致謝……..……………………………………………………………………………...ii
摘要.................................................................................................................................v
Abstract...........................................................................................................................vi
Abbreviation..................................................................................................................vii
Chapter 1 Introduction......................................................................................................................1
1.1 Heat shock response in plants..........................................................................1
1.2 Long-term acquired thermotolerance in plants................................................3
1.3 Interplay between HSP101 and HSA32...........................................................4
1.4 Macroautophagy proteolytic system in plants.................................................7
1.5 Specific aims..................................................................................................10
Chapter 2 Materials and Methods..................................................................................11
2.1 Plant materials and growth condition.............................................................11
2.2 Genomic DNA extraction and genotyping PCR............................................12
2.3 Total RNA extraction and RT-PCR................................................................13
2.4 Carbon starvation phenotypic analysis..........................................................14
2.5 Immunoblot....................................................................................................15
2.6 Thermotolerance assay...................................................................................16
2.7 Bortezomib treatment on heat acclimated seedlings......................................18
Chapter 3 Results...........................................................................................................19
3.1 Characterization of a loss-of-function mutant of ATG5.................................19
3.2 Arabidopsis atg5 mutant is more tolerant to acute heat stress but less tolerant to chronic heat stress than wild type..............................................................19
3.3 Disruption of macroautophagy alters degradation of some HSPs.................21
3.4 Disruption of macroautophagy in hsa32 partially rescues the defective LAT phenotype.......................................................................................................21
3.5 Both macroautophagy and 26S proteasome proteolytic pathways contribute to HSP101 degradation in hsa32....................................................................22
Chapter 4 Discussion.....................................................................................................25
4.1 Thermotolerance of atg5..................................................................................25
4.2 Heat-induced HSP101 degradation pathway...................................................27
Chapter 5 Future Works.................................................................................................29
Tables and Figures.........................................................................................................31
References......................................................................................................................45

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