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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 何孟樵(Meng-Chiao Ho) | |
| dc.contributor.author | Hao-Chung Jen | en |
| dc.contributor.author | 任浩中 | zh_TW |
| dc.date.accessioned | 2021-07-11T14:36:12Z | - |
| dc.date.available | 2022-08-31 | |
| dc.date.copyright | 2017-08-31 | |
| dc.date.issued | 2017 | |
| dc.date.submitted | 2017-08-18 | |
| dc.identifier.citation | [1] FAO (2015) The impact of disasters on agriculture and food security, FAO.
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[17] Voesenek, L. A. C. J., and Bailey-Serres, J. (2015) Flood adaptive traits and processes: an overview, New Phytol 206, 57-73. [18] Ella, E. S., Kawano, N., and Ito, O. (2003) Importance of active oxygen-scavenging system in the recovery of rice seedlings after submergence, Plant Sci 165, 85-93. [19] Parlanti, S., Kudahettige, N. P., Lombardi, L., Mensuali-Sodi, A., Alpi, A., Perata, P., and Pucciariello, C. (2011) Distinct mechanisms for aerenchyma formation in leaf sheaths of rice genotypes displaying a quiescence or escape strategy for flooding tolerance, Ann Bot-London 107, 1335-1343. [20] Voesenek, L. A. C. J., and Sasidharan, R. (2013) Ethylene - and oxygen signalling - drive plant survival during flooding, Plant Biology 15, 426-435. [21] Bailey-Serres, J., Fukao, T., Gibbs, D. J., Holdsworth, M. J., Lee, S. C., Licausi, F., Perata, P., Voesenek, L. A. C. J., and van Dongen, J. T. (2012) Making sense of low oxygen sensing, Trends Plant Sci 17, 129-138. [22] Bailey-Serres, J., and Chang, R. (2005) Sensing and signalling in response to oxygen deprivation in plants and other organisms, Ann Bot-London 96, 507-518. [23] Leung, S. K., and Ohh, M. (2002) Playing Tag with HIF: The VHL Story, J Biomed Biotechnol 2, 131-135. [24] Kaelin, W. G., and Ratcliffe, P. J. (2008) Oxygen sensing by metazoans: The central role of the HIF hydroxylase pathway, Mol Cell 30, 393-402. [25] Graciet, E., and Wellmer, F. (2010) The plant N-end rule pathway: structure and functions, Trends Plant Sci 15, 447-453. [26] Gibbs, D. J., Bacardit, J., Bachmair, A., and Holdsworth, M. J. (2014) The eukaryotic N-end rule pathway: conserved mechanisms and diverse functions, Trends Cell Biol 24, 603-611. [27] Hu, R. G., Sheng, J., Qi, X., Xu, Z. M., Takahashi, T. T., and Varshavsky, A. (2005) The N-end rule pathway as a nitric oxide sensor controlling the levels of multiple regulators, Nature 437, 981-986. [28] Gibbs, D. J., Lee, S. C., Isa, N. M., Gramuglia, S., Fukao, T., Bassel, G. W., Correia, C. S., Corbineau, F., Theodoulou, F. L., Bailey-Serres, J., and Holdsworth, M. J. (2011) Homeostatic response to hypoxia is regulated by the N-end rule pathway in plants, Nature 479, 415-U172. [29] Licausi, F., Kosmacz, M., Weits, D. A., Giuntoli, B., Giorgi, F. M., Voesenek, L. A. C. J., Perata, P., and van Dongen, J. T. (2011) Oxygen sensing in plants is mediated by an N-end rule pathway for protein destabilization, Nature 479, 419-U177. [30] Weits, D. A., Giuntoli, B., Kosmacz, M., Parlanti, S., Hubberten, H. M., Riegler, H., Hoefgen, R., Perata, P., van Dongen, J. T., and Licausi, F. (2014) Plant cysteine oxidases control the oxygen-dependent branch of the N-end-rule pathway, Nat Commun 5. [31] White, M. D., Klecker, M., Hopkinson, R. J., Weits, D. A., Mueller, C., Naumann, C., O'Neill, R., Wickens, J., Yang, J. Y., Brooks-Bartlett, J. C., Garman, E. F., Grossmann, T. N., Dissmeyer, N., and Flashman, E. (2017) Plant cysteine oxidases are dioxygenases that directly enable arginyl transferase-catalysed arginylation of N-end rule targets, Nat Commun 8. [32] Licausi, F., Pucciariello, C., and Perata, P. (2013) New Role for an Old Rule: N-end Rule-Mediated Degradation of Ethylene Responsive Factor Proteins Governs Low Oxygen Response in Plants, J Integr Plant Biol 55, 31-39. [33] Fukao, T., Xu, K. N., Ronald, P. C., and Bailey-Serres, J. (2006) A variable cluster of ethylene response factor-like genes regulates metabolic and developmental acclimation responses to submergence in rice(W), Plant Cell 18, 2021-2034. [34] Fukao, T., Harris, T., and Bailey-Serres, J. (2009) Evolutionary analysis of the Sub1 gene cluster that confers submergence tolerance to domesticated rice, Ann Bot-London 103, 143-150. [35] Tamang, B. G., and Fukao, T. (2015) Plant Adaptation to Multiple Stresses during Submergence and Following Desubmergence, Int J Mol Sci 16, 30164-30180. [36] Bailey-Serres, J., Fukao, T., Ronald, P., Ismail, A., Heuer, S., and Mackill, D. (2010) Submergence Tolerant Rice: SUB1's Journey from Landrace to Modern Cultivar, Rice 3, 138-147. [37] Fukao, T., Yeung, E., and Bailey-Serres, J. (2011) The Submergence Tolerance Regulator SUB1A Mediates Crosstalk between Submergence and Drought Tolerance in Rice, Plant Cell 23, 412-427. [38] Setter, T. L., Bhekasut, P., and Greenway, H. (2010) Desiccation of leaves after de-submergence is one cause for intolerance to complete submergence of the rice cultivar IR 42, Funct Plant Biol 37, 1096-1104. [39] Alpuerto, J. B., Hussain, R. M. F., and Fukao, T. (2016) The key regulator of submergence tolerance, SUB1A, promotes photosynthetic and metabolic recovery from submergence damage in rice leaves, Plant Cell Environ 39, 672-684. [40] Coleman, R. A., Taggart, A. K. P., Benjamin, L. R., and Pugh, B. F. (1995) Dimerization of the Tata-Binding Protein, J Biol Chem 270, 13842-13849. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/77861 | - |
| dc.description.abstract | 極端氣候會造成稻米產量下降，進而危及全球糧食安全，其中乾旱與洪水是兩大主因。依據氣候變遷模型預測，這兩種危害的發生頻率將會上升。帶有屬於第七群乙烯反應因子Sub1A-1基因的稻米品種，能夠於完全淹水的環境下，撐過超過兩週的時間。Sub1A-1除了能抵抗淹水逆境外，還能在稻米不缺氧的情況下，調控其抗旱、抗長期黑暗與抗水退的反應，這要歸功於Sub1A-1並不適用於N-end rule pathway的特性。N-end rule pathway屬於泛素-蛋白酶體系統(UPS)中的一部份，具有偵測植物體內平衡的功能，以及調控包含第七群乙烯反應因子在內，會受缺氧誘發的轉錄因子。有趣的是，Sub1A-1是唯一不會被N-end rule pathway降解得第七群乙烯反應因子。本論文的目的是藉由生物化學與生物物理實驗，來探討Sub1A-1是如何逃離N-end rule pathway。
對此目前有一種假設與兩種可能的原因；N-end rule pathway是受到空間障礙的干擾，導致相關酵素無法接近Sub1A-1位於N端的MCGG序列進行反應。第一種可能是，Sub1A-1具有穩固且特殊的結構能保護其N端，防止酵素接近。能藉由圓二色光譜儀與核磁共振光譜儀來判斷，其N端區域(domain)是否有此種特殊結構；又或者是Sub1A-1的二聚體型態具保護功能，所以我們以微量熱泳動儀(MST)與等溫滴定微量熱儀(iTC)測定不同蛋白區域間的結合親和力，以判定二聚體中間介面的位置。第二個可能是有其他植物細胞內的蛋白質參與，Sub1A-1與蛋白質形成能保護Sub1A-1的N端受質的複合體。為了瞭解是否有其他蛋白質參與，目前正在建立體外N-end rule實驗來驗證。 | zh_TW |
| dc.description.abstract | Rice losses as a result of extreme climate put a severe threat to global food security. Drought and flood are the most prevailing climate-related disasters hindering the growth of rice and climate models predict an increased frequency of these two abiotic stresses. Rice cultivars with the ERF-VII (group VII ethylene responsive factor transcription factors), Sub1A-1 can survive more than two weeks of submergence. Apart from inundation, Sub1A-1 also regulates the adaptive response to desubmergence, drought and prolonged darkness in normoxia owing to the ability to escape N-end rule pathway. N-end rule pathway, a part of the ubiquitin-proteasome system (UPS), that acts as a homeostatic sensor and regulates the key hypoxia-response transcription factors including ERF-VIIs in plants. Interestingly, Sub1A-1 is the only known rice ERF-VIIs that can escape N-end rule pathway. My aim is to study how Sub1A-1 escapes the N-end rule pathway by biophysical and biochemical approaches.
We have one hypothesis with two possible routes. We proposed that the N-end rule pathway may be disrupted by steric obstacles, so the enzymes cannot approach to the N-terminal MCGG sequence of Sub1A-1. One route is Sub1A-1 with a rigid and special structure to shield N-degron from enzymatic reaction. I studied the secondary structure of N domain by circular dichroism spectroscopy and nuclear magnetic resonance spectroscopy to examine if N-terminus possesses a special structure. The dimerization of Sub1A-1 may protect N-degron from the N-end rule, so we measured the binding affinity between different domains of Sub1A-1 by MST and iTC to determine the dimer interface. The second route is that another protein participates in the Sub1A-1 interaction. The Sub1A-1/protein complex may shield N-degron from N-end rule enzymes. In order to test if there is additionally protein involved, I am developing the in vitro reconstitution of N-end rule pathway assay. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-11T14:36:12Z (GMT). No. of bitstreams: 1 ntu-106-R04b46030-1.pdf: 3909373 bytes, checksum: ac6cd95699adf9cf9225d1ca7a53e3eb (MD5) Previous issue date: 2017 | en |
| dc.description.tableofcontents | Table of Contents
摘要 2 Abstract 3 Table of Contents 5 Chapter 1 12 Introduction 12 1.1 Climate-related Hazards 12 1.2 Biotic and Abiotic Stress Tolerance 13 1.3 Ethylene Transduction and ERF Transcription Factor 14 1.4 Adaptive Responses to Flooding 15 1.5 Oxygen Sensing 16 1.6 The N-end Rule Pathway 18 1.7 Sub1A-1 for Abiotic Stresses 21 1.8 Aim 23 Chapter 2 25 Materials and methods 25 2.1 Materials 25 2.1.1 Chemicals 25 2.1.2 Reagents 25 2.1.3 Resins and columns 26 2.1.4 Apparatus 26 2.2 Methods 27 2.2.1 Expression of recombinant Sub1A-1 N-alone, Sub1A-1 ΔC, Sub1A-1 AP2, Sub1A-1 ΔN, Sub1A-1 C-alone, ΔS Sub1A-1, AtMAP1A, OsPCO, OsATE protein 27 2.2.2 Expression of recombinant Sub1A-1 N-alone for NMR study 27 2.2.3 Purification of recombinant Sub1A-1 N-alone, Sub1A-1 ΔC, Sub1A-1 AP2, Sub1A-1 ΔN, Sub1A-1 C-alone and ΔS Sub1A-1, AtMAP1A, OsPCO1, OsATE, AtATE1 protein 28 2.2.3.1 Cell lysis by sonication 28 2.2.3.2 Immobilized metal ion affinity chromatography (IMAC) 28 2.2.3.3 SDS-PAGE Analysis 30 2.2.3.4 Heparin affinity chromatography (Sub1A-1 N-alone, Sub1A-1 ΔC and ΔS Sub1A-1) 30 2.2.3.5 Anion exchange chromatography (Sub1A-1 C-alone) 31 2.2.3.6 Size exclusion chromatography (SEC) (Sub1A-1 N-alone, Sub1A-1 C-alone and ΔS Sub1A-1) 32 2.2.4 Electrophoretic mobility shift assay (EMSA) 33 2.2.5 Size exclusion chromatography with multi-angle light scattering (SEC-MALS) 33 2.2.6 Circular dichroism (CD) spectroscopy 34 2.2.7 Nuclear magnetic resonance (NMR) spectroscopy 35 2.2.8 Microscale Thermophoresis 35 2.2.9 Isothermal titration calorimetry (iTC) 36 2.2.10 Crosslinking ofΔS Sub1A-1 37 Chapter 3 39 Result and Discussion 39 3.1 Expression and Purification of Various Sub1A-1 Constructs 39 3.2 Structural Studies of Sub1A-1 N-terminus alone 39 3.3 The Importance of C-terminal Domain of Sub1A-1 in N-end Rule Escaping 41 3.4 The Homo-dimerization Studies of Sub1A-1 C-alone and Sub1A-1 N-alone 41 3.5 Interaction Studies between Sub1A-1 Domains 42 3.6 In vitro Reconstitute of N-end Rule Pathway of Targeted Proteolysis 44 Chapter 4 45 Conclusion and Perspective 45 List of Figures 47 Figure 1. Strategies for submergence of rice. 47 Figure 2. Scheme of O2 deprivation sensing. 48 Figure 3. Scheme of N-end rule pathway in mammals and plants. 49 Figure 4. Functions of the N-end rule pathway in Arabidopsis. 50 Figure 5. Scheme of N-end rule pathway of ERF-VII. 51 Figure 6. The Sub1 locus encodes multiple ethylene-responsive factors, Sub1A, Sub1B and Sub1C. 52 Figure 7. Sub1 locus composition and submergence-induced mRNA accumulation in rice. 53 Figure 8. Various stresses induced throughout submergence and following desubmergence in plants 54 Figure 9. Scheme of Sub1A-mediated response to the progression of abiotic stresses during submergence in Rice. 55 Figure 10. Model of binding of target protein with 6-His tag and Ni-NTA 56 Figure 11. Far UV CD spectra associated with various types of secondary structure. 57 Figure 12. DSS (disuccinimidyl suberate) 58 Figure 13. Sulfo-SMPB (sulfosuccinimidyl 4-[p-maleimidophenyl]butyrate) 59 Figure 14. EMSA binding studies of Sub1A-1 and GCC probe. 60 Figure 15. Size exclusion chromatography result of Sub1A-1 (green) and Sub1A-1 with DNA (blue) 61 Figure 16. Purification result of recombination Sub1A-1 N-alone. 64 Figure 17. Purification result of recombinant Sub1A-1 C-alone. 67 Figure 18. The interaction between Sub1A-1 N-alone and Sub1A-1 C-alone experimented by size exclusion chromatography. 69 Figure 19. Purification result of recombinant ΔS Sub1A-1. 71 Figure 20. Purification result of recombinant Sub1A-1 ΔN by nickel-NTA column. 72 Figure 21. Purification result of recombinant Sub1A-1 AP2-alone by nickel-NTA column. 73 Figure 22. Purification results of the rAtMAP1A, OsPCO1, OsATE and AtATE1. 75 Figure 23. Purification result of recombinant Sub1A-1 ΔC. 76 Figure 24. The CD result. 77 Figure 25. The 15N-HSQC spectrum of Sub1A-1 N-alone. 78 Figure 26. Microscale thermophoresis study of protein–protein binding affinity. 79 Figure 27. The result of ex vivo experiment. 81 Figure 28. The SEC-MALS results of Sub1A-1. 82 Figure 29. The ITC result of Sub1A-1 N Domain and C Domain. 85 Figure 30. The SDS-PAGE analysis of crosslinking of ΔS Sub1A-1 by BM(PEG)3 and sulfo-SMPB. 86 Figure 31. The constructs of various recombinant Sub1A-1. 87 Figure 32. The constructs of various N-end rule enzymes.. 88 Figure 33. Purification of Sub1A-1 ΔC by size exclusion column. 89 List of tables 90 Table 1. Sub1 locus in rice varieties 90 Table 2. The amino acids sequence of recombinant ΔS Sub1A-1 91 Table 3. The amino acids sequence of recombinant Sub1A-1 NAP2 92 Table 4. The amino acids sequence of recombinant Sub1A-1 N-alone 93 Table 5. The amino acids sequence of recombinant Sub1A-1 AP2 94 Table 6. The amino acids sequence of recombinant Sub1A-1 AP2C 95 Table 7. The amino acids sequence of recombinant Sub1A-1 C-alone 96 Table 8. The amino acids sequence of recombinant ERF67 N+Sub1A-1 ΔN 97 Table 9. The amino acids sequence of recombinant ERF67 98 Table 10. Buffer ingredients 99 Reference 100 | |
| dc.language.iso | en | |
| dc.subject | Sub1A-1 | zh_TW |
| dc.subject | 對非生物壓力抗性 | zh_TW |
| dc.subject | N-end rule pathway | zh_TW |
| dc.subject | Sub1A-1 | en |
| dc.subject | Anti-abiotic stress | en |
| dc.subject | N-end rule pathway | en |
| dc.title | 稻米Sub1A-1如何脫逃N-end Rule Pathway之研究 | zh_TW |
| dc.title | Study on the Escape of Rice Sub1A-1 from N-end Rule Pathway | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 105-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 蕭超隆(Chiao-Long Hsiao),葉國楨(Kuo-Chen Yeh) | |
| dc.subject.keyword | Sub1A-1,N-end rule pathway,對非生物壓力抗性, | zh_TW |
| dc.subject.keyword | Sub1A-1,N-end rule pathway,Anti-abiotic stress, | en |
| dc.relation.page | 106 | |
| dc.identifier.doi | 10.6342/NTU201703709 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2017-08-18 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科學研究所 | zh_TW |
| 顯示於系所單位： | 生化科學研究所 | |
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