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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 詹明才 | |
dc.contributor.author | Yi-Chiou Li | en |
dc.contributor.author | 李怡萩 | zh_TW |
dc.date.accessioned | 2021-06-08T05:03:50Z | - |
dc.date.copyright | 2011-02-20 | |
dc.date.issued | 2011 | |
dc.date.submitted | 2011-02-16 | |
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The E2 ubiquitin-conjugating enzyme, AtUBC1 and AtUBC2, plays redundant roles and are involved in activation of FLC expression and repression of flowering time in Arabidopsis thaliana. Plant J. 57: 297-288. 34. Yaeno, T., and Iba, K. (2008). BAH1/NLA, a RING-type ubiquitin E3 ligase, regulates the accumulation of salicylic acid and immune responses to Pseudomonas syringae. Plant Physiol. 148: 1032–1041. 35. Zhang, Y., Yang, C., Li, Y., Zheng, N., Chen, H., Zhao, Q., Gao, T., Guo, H., and Xie, Q. (2007). SDIR1 Is a RING Finger E3 ligase that positively regulates stress-responsive abscisic acid signaling in Arabidopsis. Plant Cell 19: 1912-1929. 36. Zhou, H., Wertz, I., O'Rourke, K., Ultsch, M., Seshagiri, S., Eby, M., Xiao, W., and Dixit, V.M. (2004). Bcl10 activates the NF-kappaB pathway through ubiquitination of NEMO. Nature 427: 167–171. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/23551 | - |
dc.description.abstract | 泛素化作用(ubiquitination)已經被證實參與在植物許多生長發育的調控路徑中,包括光訊息傳遞、植物胚胎發育,以及植物的防禦機制。泛素化作用分為三個步驟,主要由三個酵素參與進行一連串的分子活化作用,將泛素 (ubiquitin)接到受質上,而被泛素接合的受質蛋白則會被蛋白酶體所辨識,進而被蛋白酶體降解。這三個酵素分別為泛素激活酵素 (Ub-activating enzyme, UBA )(E1)、泛素接合酵素(Ub-conjugating enzyme, UBC) (E2)及泛素連接酶(Ub ligase) (E3)。在泛素化作用中,受質的專一性則是由接合酵素及連接酶一起決定。
在阿拉伯芥中,已經有37個蛋白質被鑑定為泛素接合酵素。然而大部分轉譯出這些蛋白的基因的生理功能都尚未被研究證實。本研究針對阿拉伯芥AtUBC8進行研究。在先前的研究中,已經知道AtUBC8參與在缺氮誘導的花青素累積、光形態發生以及植物荷爾蒙離層酸(ABA)的合成,但尚未有任何研究指出AtUBC8是否參與植物抵抗鹽逆境的反應中。本研究中發現阿拉伯芥AtUBC8在轉錄層次會被鹽處理誘導表現。我們發現UBC8-GFP主要表現在阿拉伯芥原生質體之細胞核及細胞質。此外,我們也建立了大量表現AtUBC8的阿拉伯芥轉殖株(UBC8 OX)以及無法正常表現AtUBC8的阿拉伯芥突變體(UBC8 KO)。我們將這兩種植株都以鹽處理,進而發現鹽處理兩天過後,UBC8 KO已經開始有白化的現象。鹽處理四天過後,50%的UBC8 OX仍存活著;UBC8 KO及wild-type plants只剩20%的存活率。我們也利用雙分子螢光復補法去確認UBC8確實會與PIP1B, LHCA 及TCTP互相作用。雖然阿拉伯芥UBC8如何調控抵抗鹽逆境的機制仍未知,但在此研究中,我們發現大量表現阿拉伯芥AtUBC8會使得植物對於鹽逆境較有耐受性,進而證實了阿拉伯芥AtUBC8會參與在植物抵抗鹽逆境的機制。未來我們可以利用所建立的阿拉伯芥UBC8 KO及UBC8 OX,以及已證實的會與UBC8有交互作用的蛋白質進一步去研究AtUBC8所參與的鹽逆境調控機制。 | zh_TW |
dc.description.abstract | Ubiquitylation plays important functions in many aspects of plant growth and development, including phytohormone and light signaling, embryogenesis, organogenesis, leaf senescence and plant defence. It is achieved in a multi-step reaction, sequentially involving an E1 (Ub-activating enzyme, UBA), an E2 (Ub-conjugating enzyme, UBC) and an E3 (Ub ligase). In the ubiquitination system, substrate specificity is mainly determined by E2 together with E3.
The Arabidopsis thaliana genome contains 37 genes encoding UBC homologs. However, the biological functions of these genes remain largely uncharacterized. The AtUBC8 has been reported to be involved in nitrogen limitation, photomorphogensis, and drought tolerance. However, the involvement of UBC8 in salt response hasn’t been reported. In this study, we tried to understand the UBC8 potential functions in salt responsive pathway. First of all, I found that the mRNA transcripts of AtUBC8 were induced by salt-stress in wild-type plants. In order to study the biological potential function of AtUBC8, I performed reverse genetic approach to investigate the role of UBC8 in salt responsive pathway. I’ve identified the UBC8 KO mutants and UBC8 OX transgenic plants. I validated that UBC8 are localized to nucleus and cytoplasm. I found that, in contrast to UBC8 KOs, the UBC8 OX seedling remained 50-55 % survivals after four days of salt treatment. On the other hand, I confirmed the PIP1B, LHCA and TCTP can interact with UBC8. Although the molecular mechanism how UBC8 regulates the salt responsive pathway in Arabidopsis remains unknown. In this study, we verified that AtUBC8 is involved in salt-responsive pathway. In the future, through studying the UBC8 interacting protein and utilizing the UBC8 KO UBC8 OX plants, we could further verify the role of AtUBC8 in salt signaling pathway. | en |
dc.description.provenance | Made available in DSpace on 2021-06-08T05:03:50Z (GMT). No. of bitstreams: 1 ntu-100-R97b42001-1.pdf: 1829724 bytes, checksum: 8f072d2e25ff262aa8906879826dc8b7 (MD5) Previous issue date: 2011 | en |
dc.description.tableofcontents | 口試委員會審定書.......................................................................................I
誌謝............................................................................................................II 中文摘要...................................................................................................III 英文摘要....................................................................................................V Introduction.................................................................................................1 The ubiquitin-26S proteasome system........................................................1 Ubiquitin-conjugating enzyme ..................................................................2 The study on AtUBC8...............................................................................3 Ubiquitination and salt stress in plant........................................................4 Objectives................................................................................................5 Materials and Methods..................................................................................7 Plant materials..........................................................................................7 Molecular Cloning of UBC8......................................................................7 A. Total RNA extraction........................................................................7 B. RNA electrophoresis.........................................................................8 C. Reverse transcription........................................................................9 D. Polymerase chain reaction.................................................................9 E. Purification of PCR reaction............................................................10 F. Construction of an entry clone..........................................................10 G. Preparation of XL1-Blue competent cells.........................................11 H. Transformation of E.coli.................................................................12 I. QIAprep miniprep kit.......................................................................12 J. Construction of the expression clone.................................................13 Establishment of UBC8 OX transgenic plants...........................................14 A. Preparation of electro-competent Agrobacterium..............................14 B. Transformation of Agrobacterium....................................................14 C. Floral dip transformation of Arabidopsis..........................................15 D. Screening of transgenic plants.........................................................16 E. DNA Extraction ..............................................................................16 F. Southern blot analysis......................................................................18 G. Preparation of probe........................................................................20 H. Hybridization.................................................................................20 I. Chemiluminescent Detection............................................................21 Identification of UBC8 KO......................................................................22 A. Selection scheme used for GABI-Kat T-DNA insertion strains..........22 B. Screening for the putative T-DNA insertion lines..............................23 C. Verification of UBC8 KO mutants....................................................23 Phenotypic characterization under salt stress............................................24 A. Salt stress treatment........................................................................24 B. quantitative RT-PCR.......................................................................24 Subcellular localization of UBC8.............................................................25 A. Construction of an expression clone.................................................25 B. Protoplast isolation.........................................................................25 C. Transient expression.......................................................................26 Identification of UBC8 interacting protein...............................................27 A. Bimolecular fluorescence complementation.....................................27 Results.......................................................................................................28 The mRNA transcripts of AtUBC8 are induced by salt-stress in wild type..28 UBC8 is localized to nucleus and cytoplasm.............................................29 The UBC8 T-DNA mutants are knock-out mutants....................................30 The UBC8 OX2 and OX8 are homozygous overexpression transgenic plants.32 Sustained expression of AtUBC8 confers salt tolerance.............................33 PIP1B, LHCA and TCTP can interact with UBC8......................................34 Discussion..................................................................................................36 Figures.......................................................................................................43 Tables........................................................................................................58 References.................................................................................................62 Appendix...................................................................................................69 | |
dc.language.iso | zh-TW | |
dc.title | 阿拉伯芥AtUBC8的功能性研究 | zh_TW |
dc.title | Characterization of Arabidopsis UBC8 potential function | en |
dc.type | Thesis | |
dc.date.schoolyear | 99-1 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 葉開溫,鄭秋萍 | |
dc.subject.keyword | 泛素化作用,鹽耐受性,泛素接合酵素,泛素,阿拉伯芥, | zh_TW |
dc.subject.keyword | ubiquitination,salt tolerance,AtUBC8,ubiquitin-conjugating enzyme,ubiquitin,Arabidopsis, | en |
dc.relation.page | 77 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2011-02-16 | |
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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