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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 林讚標(Tsan-Piao Lin) | |
dc.contributor.author | Chen-Yin Weng | en |
dc.contributor.author | 翁禎吟 | zh_TW |
dc.date.accessioned | 2021-06-08T00:22:54Z | - |
dc.date.copyright | 2013-07-26 | |
dc.date.issued | 2013 | |
dc.date.submitted | 2013-07-18 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/17580 | - |
dc.description.abstract | 生物需要適時調控細胞內的代謝活動,藉此適應變動環境所引發的不同生理反應。位於葉綠體的thioredoxin m2 (Trxm2) 是廣泛存在各種生物體的氧化還原蛋白,可藉還原作用調控目標蛋白的活性。然而,以遺傳證據研究Trxm2是否參與在逆境耐受性的相關文獻仍是相當有限。為了瞭解Trxm2的生理功能,建構了過量表現Trxm2的阿拉伯芥轉殖株以進行研究。實驗發現35S::Trxm2轉植株會有熱耐受性下降,並伴隨著H2O2含量累積的現象。此外,也觀察到此轉植株在熱處理後,一些位於上游的HsfAs基因表現量有下降的趨勢。文獻報導指出HsfA1s是熱休克反應的主要調控者 (master regulators),可影響其他熱誘導基因的表現,當HsfA1s無法正常執行功能時會造成植物抵抗熱的能力下降 (Liu et al., 2011)。然而,對於Trxm2究竟是直接或間接影響HsfAs的表現尚待後續研究。有趣的是,觀察結果指出35S::Trxm2轉殖株相較於野生型會對不含蔗糖的培養基更加敏感。經由轉殖株體內葡萄糖的累積、NADPH含量降低並伴隨H2O2含量的提高,證明35S::Trxm2轉殖株的氧化五碳糖磷酸途徑 (OPP pathway) 受到影響。同時,透過氣相層析質譜儀(GC-MS) 分析可知,相較於野生型植物,35S::Trxm2轉殖株具有較高含量的果糖,但蔗糖含量卻是顯著下降。推測參與在蔗糖合成中的某個重要酵素,其活性會受到Trxm2調控。經由雙分子螢光互補系統 (Bimolecular fluorescence complementation) 證實,Trxm2會和參與在蔗糖合成中的fructose-1,6-bisphosphatase及UDP-glucose pyrophosphorylase交互作用,但是否具調控能力仍需更多實驗證明。綜合以上結果可知,Trxm2會藉由調控目標蛋白的活性以參與在熱逆境反應及蔗糖生合成路徑中。 | zh_TW |
dc.description.abstract | In a changing environment, cellular metabolism needs to be altered to enable an adapted physiological response. Chloroplast thioredoxin m2 (Trxm2) is an important oxidoreductase that can regulate the target protein activity by reduction. However, genetic evidence for its physiological importance to stress tolerance is largely lacking. To test the functional significance of Trxm2 in vivo, Arabidopsis transgenic plants overexpressing Trxm2 gene were studied in this research. We found that 35S::Trxm2 exhibited decreased thermotolerance accompanied by lower H2O2 contents. Furthermore, we also observed that the expressions of some HsfAs were decreased in response to heat stress in 35S::Trxm2 plants. It has been reported that HsfA1s were the master regulators that would regulate the expression of many heat-inducible genes which would affect thermotolerance (Liu et al., 2011). However, the connection between Trxm2 and HsfAs is unknown. Interestingly, we found that 35S::Trxm2 were more sensitive to non-sucrose medium than WT plants. We found that oxidative pentose phosphate pathway (OPPP) would be interrupted in 35S::Trxm2 plants because greater accumulation of glucose, less content of NADPH and higher content of H2O2. Meanwhile, analyses of neutral sugar by GC-MS showed that 35S::Trxm2 plants had higher fructose contents but lower sucrose contents compared with WT plants, indicating that sucrose synthesis was impaired. We assume that specific key enzyme involved in sucrose synthesis would be affected by Trxm2. By using BiFC, we found that Trxm2 would interact with fructose-1,6-bisphosphatase and UDP-glucose pyrophosphorylase which were involved in sucrose synthesis. More evidences are needed to identify whether Trxm2 would regulate the activity of these two enzymes. In conclusion, these results provide the evidence that Trxm2 might involve in heat stress response and sucrose synthesis by regulating the activities of target proteins. | en |
dc.description.provenance | Made available in DSpace on 2021-06-08T00:22:54Z (GMT). No. of bitstreams: 1 ntu-102-R98b42001-1.pdf: 3328301 bytes, checksum: f61a692b81d09931ba7e4a7156cb7732 (MD5) Previous issue date: 2013 | en |
dc.description.tableofcontents | 目錄
口試委員會審定書 誌謝 摘要……………………………………………………………………………………1 Abstract…………………………………………………………...…………………..2 縮寫對照表……………………………………………………………………………4 第一章 序論…………………………………...………………...…………………..6 1.0 前言……………...……………………………………………………6 1.1 硫氧化還原蛋白 (thioredoxin, Trx) 的介紹……………………..6 1.2 植物thioredoxin與環境逆境的相關研究……………...………….8 1.3 植物thioredoxin與OPP pathway的相關研究……………...……9 1.4 植物thioredoxin與蔗糖合成的相關研究……………...……….…10 1.5 實驗策略及研究目標……………...………………………………..11 第二章 材料與方法…………………...……...………………...………………….12 2.1 植物材料與生長條件……………...………………………………..12 2.2 Trxm2基因序列分析……………………………………………….12 2.3 Genomic DNA萃取………………………………..…...…………..12 2.4 聚合酶連鎖反應 (Polymerase Chain Reaction, PCR)………… 13 2.5 RNA萃取、RNase-free DNase I處理及cDNA合成…...….……14 2.6 即時定量聚合酶連鎖反應 (qRT-PCR)……………...……………15 2.7 構築及短暫表現的融合蛋白質之細胞位置分析………..………..16 2.8 GUS染色分析…………………………………………..………….16 2.9 植物轉形構築及轉殖基因植物的建立…………..…...……….…..16 2.10 熱逆境耐受程度測試……………………………………………... 17 2.11 鹽逆境和滲透壓逆境耐受程度測試…...….………………………18 2.12 NADPH萃取…………………………………………...……………18 2.13 H2O2萃取…………..……....……………………………….………..19 2.14 分析T-DNA homozygous lines…………………………………....19 2.15 中性糖萃取……………………………...….………………………20 2.16 雙分子螢光互補系統 (BiFC)……………..………...……………21 第三章 結果…………………………...……...………………...………………….24 3.1 Trxm2基因之序列分析……………..........………………………..24 3.2 Trxm2在植物細胞內表現之位置………………………………….24 3.3 Trxm2在植物組織內表現之位置……………..…..…...…………..24 3.4 35S::Trxm2轉殖株建構與基因表現檢測………………………… 25 3.5 35S::Trxm2轉殖株在熱逆境下之生長情形…....................….……25 3.6 35S::Trxm2轉殖株在鹽逆境和滲透壓逆境下之生長情形..…...…26 3.7 35S::Trxm2轉殖株在熱處理後體內H2O2含量檢測…….….……..27 3.8 35S::Trxm2轉殖株在熱處理後體內HsfAs 基因表現測量……….27 3.9 篩選trxm2 T-DNA插入品系…………..…...…………………..….28 3.10 trxm2 T-DNA插入品系在熱逆境下之生長情形……………..….....28 3.11 35S::Trxm2轉殖株體內NADPH含量檢測…...……….....………28 3.12 35S::Trxm2 轉殖株體內H2O2含量檢測…………..…..…….……..29 3.13 35S::Trxm2 轉殖株體內中性糖含量檢測….……………………..29 3.14 利用雙分子螢光互補系統 (BiFC) 驗證Trxm2與其他蛋白質的交互 作用…………………………………………..……………………....29 第四章 討論…………………………...……...………………...………………….31 4.1 Trxm2可能參與在熱逆境反應……….......…………………………..31 4.2 Trxm2可能參與在OPP pathway…………………………………….33 4.3 Trxm2可能影響蔗糖之生合成………….…..…..……......…………..34 4.4 總結………………………………………………………………… 36 參考文獻……………………………………………………………………………37 圖…………………………………………………………...…………………..45 Appendix……………………………………………………………………………61 | |
dc.language.iso | zh-TW | |
dc.title | 阿拉伯芥thioredoxin Trxm2過量表現會影響糖的含量及熱的耐受性 | zh_TW |
dc.title | Overexpression of thioredoxin Trxm2 leads to altered sugar content and thermotolerance in Arabidopsis | en |
dc.type | Thesis | |
dc.date.schoolyear | 101-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 鄭石通(Shih-Tong Jeng),靳宗洛(Jinn, Tsung-Luo),常怡雍(Yee-yung Charng) | |
dc.subject.keyword | 氧化還原蛋白,熱逆境,熱休克轉錄因子,氧化五碳糖磷酸途徑,蔗糖生合成, | zh_TW |
dc.subject.keyword | oxidoreductase,thermotolerance,heat shock factors,oxidative pentose phosphate pathway,sucrose synthesis, | en |
dc.relation.page | 71 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2013-07-18 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 植物科學研究所 | zh_TW |
顯示於系所單位: | 植物科學研究所 |
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