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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 楊健志 | zh_TW |
| dc.contributor.author | 吳柏璇 | zh_TW |
| dc.contributor.author | Po-Hsuan Wu | en |
| dc.date.accessioned | 2021-07-10T21:52:24Z | - |
| dc.date.available | 2024-07-31 | - |
| dc.date.copyright | 2019-08-28 | - |
| dc.date.issued | 2019 | - |
| dc.date.submitted | 2002-01-01 | - |
| dc.identifier.citation | Ashraf, M.P.J.C. and Harris, P.J.C. (2004). Potential biochemical indicators of salinity tolerance in plants, Plant Sci, 166: 3-16.
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Zipfel, C., Kunze, G., Chinchilla, D., Caniard, A., Jones, J.D., Boller, T. and Felix, G. (2006). Perception of the bacterial PAMP EF-Tu by the receptor EFR restricts Agrobacterium-mediated transformation, Cell, 125: 749-760. | - |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/77242 | - |
| dc.description.abstract | 近年來胜肽被廣泛的研究，其大部分的功能皆與參與植物訊息傳導和引發植物免疫系統防禦有關，而胜肽可作為植物生長或保護的調節劑並應用在農業上的特性也使胜肽的研究越來越受到重視。在本篇論文中，我們研究了做為訊息傳導胜肽的兩種胜肽，分別是植物激發胜肽 (Pep) 和快速鹼化因子 (RALF)，並希望從實驗結果中探討胜肽的潛在應用。在本研究中，我們首先建立異源表現重組胜肽 RALF14，RALF33 和 RALF34 的表現與純化系統，另外也利用化學合成的方法得到 Pep1、Pep3 和一些 RALF 部分保守序列的胜肽。本研究測試這些胜肽對阿拉伯芥面對鹽逆境，及對根長和發芽率的反應。在觀察根長的實驗中，發現在鹽逆境下用 Pep3 與 RALF34b 處理可使根長有加成性的抑制，但以 Pep1 與 RALF14 處理時，則沒有加成性的抑制現象。針對此結果，我們推測 RALF 和 Pep 在鹽逆境下是透過不同的路徑影響根長，且不同成員間的影響也不盡相同。此外，我們發現 RALF14 的較小片段 RALF14b 同樣可以引起根長的加成性抑制並使植物對鹽耐受性降低。最後，我們發現 Pep3 可以提高鹽逆境下老化種子的發芽率。這些發現揭示了這些訊息胜肽的功能比我們預期的更多，也提供了這些胜肽在農業應用中的不同面相的思考。 | zh_TW |
| dc.description.abstract | A diverse array of peptides involved in plant signaling and defense are gaining attention due to their possibility of being employed as a plant growth or protection regulator. In this study, known secreted peptides of plant elicitor peptides (Pep) and rapid alkalinization factors (RALF) families were explored for their potential applications in agriculture. Recombinant peptides of mature RALF14, RALF33 and RALF34 were constructed and purified in E. coli. Pep1, Pep3 and peptides containing the conserved motif of RALFs peptides were chemically synthesized. These peptides were applied on Arabidopsis thaliana to test their effects on salinity stress response, root lengths and seed germination. The results showed that under salinity stress, treatments with Pep3 and RALF34b, the smaller fragment of RALF34, could cause additive inhibition of roots length. However, the inhibition of root length caused by salinity was suppressed when treated with RALF14 and Pep1. RALF14b, the smaller fragment of RALF14, also caused additive inhibition of roots length and decreased salinity stress tolerance. These indicate that different members of RALF and Pep differentially affect root length under salinity stress. In addition, Pep3 could enhance the germination rate of aging seeds under salinity stress. These findings revealed that signaling peptides have more functions than we expected. Providing putative application of signaling peptides in agriculture. | en |
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| dc.description.tableofcontents | Abstract I
摘要 II Abbreviations III Chapter 1. Introduction 1 1.1 Signaling peptides in plants 1 1.2 Signaling peptides as plant growth regulators (PGRs) 3 1.3 RALF family and its function (Rapid ALkalinization Factor) 4 1.4 Receptors for RALF family 6 1.5 Pep family and its function (Plant Elicitor Peptide) 7 1.6 Receptors for PEP family 8 1.7 Salinity stress 9 1.8 Motivation and aim of the study 10 Chapter 2. Materials and methods 12 2.1 Experimental materials 12 2.1.1 Bacterial strains 12 2.1.2 Plant material 13 2.1.3 Media 13 2.1.3.1 E. coli media 13 2.1.3.2 Arabidopsis medium 14 2.1.4 Vectors 14 2.1.5 Peptides 15 2.2 Experimental methods 15 2.2.1 DNA extraction and analysis 15 2.2.1.1 Plasmid DNA extraction by Mini Plus 15 2.2.1.2 Plasmid DNA extraction by NucleoBond® Xtra midi 16 2.2.1.3 Plasmid DNA extraction by phenol-chloroform 18 2.2.1.4 Agarose gel electrophoresis 19 2.2.1.5 DNA quantification 20 2.2.2 Construction of expression plasmids 20 2.2.2.1 Polymerase chain reaction (PCR) 20 2.2.2.2 DNA purification 20 2.2.2.3 T-A cloning 21 2.2.2.4 Heat shock transformation 21 2.2.2.5 Colony PCR 22 2.2.2.6 DNA sequencing 22 2.2.2.7 Restriction enzyme digestion 23 2.2.2.8 DNA ligation 23 2.2.3 Protein analysis 23 2.2.3.1 Tris-Glycine SDS-PAGE 23 2.2.3.2 Tricine-SDS-PAGE 24 2.2.3.3 Coomassie Brilliant Blue R-250 staining 26 2.2.3.4 Western Blot 26 2.2.3.5 Stripping and reprobing in Western blotting 27 2.2.3.6 Liquid Chromatograph Tandem Spectrometer 28 2.2.4 Protein expression and purification 29 2.2.4.1 Small-scale expression test 29 2.2.4.2 Large-scale expression for production 30 2.2.4.3 Isolation of recombinant protein soluble fraction 31 2.2.4.4 HisTrap affinity chromatography 31 2.2.4.5 Dialysis 32 2.2.4.6 Bradford protein quantification assay 32 2.2.5 Arabidopsis planting and abiotic stress assays 33 2.2.5.1 Seed sterilization 33 2.2.5.2 Growth conditions 33 2.2.5.3 Peptide treatments 33 2.2.5.4 Salinity stress assay 34 2.2.5.5 Seed germination assay 34 2.2.6 Statistical analysis 34 2.2.6.1 Analysis of variance (ANOVA) 34 2.2.6.2 Student’s t-test 35 Chapter 3. Results 36 3.1 Preparation of recombinant peptide samples 36 3.1.1 Construction of expression system of RALF recombinant peptides in E. coli 36 3.1.2 Protein identification of RALF recombinant peptides by LC-MS/MS analysis 37 3.1.3 Purification of RALF recombinant peptides 38 3.2 Effects of peptides on Arabidopsis salinity stress responses 40 3.2.1 Defining the optimal sodium chloride (NaCl) concentration 40 3.2.2 Use of BP21 peptide and PBS as the controls 41 3.2.3 Effects of Pep family peptides on root growth under salinity stress 42 3.2.4 Effects of RALF family peptides on root growth under salinity stress 45 3.2.5 Effects of conserved motifs of RALF family peptides on root growth under salinity stress 47 3.2.6 Effects of Pep3 peptide on seed germination rate under salinity stress 50 Chapter 4. Discussion 52 4.1 Production yield of different recombinant peptides varied under the same purification method 52 4.2 Different members of RALF and Pep family can differentially affect root length under salinity stress 53 4.3 Pep3 can affect the germination rate of aging seeds under salinity stress 55 4.4 Future work 56 References 58 Figures 66 Figure 3.1 Expression of RALF14, RALF33 and RALF34 recombinant peptides 66 Figure 3.2 LC-MS/MS analysis of RALF14, RALF33 and RALF34 recombinant peptides 70 Figure 3.3 Purification of recombinant RALF14 by using HisTrap affinity column 71 Figure 3.4 Purification of recombinant RALF33 by using HisTrap affinity column 72 Figure 3.5 Purification of recombinant RALF34 by using HisTrap affinity column 73 Figure 3.6 Root growth of Arabidopsis thaliana wild type Col-0 under different NaCl concentrations 74 Figure 3.7 Root growth of Arabidopsis thaliana wild type Col-0 treated with chemically synthesized BP21 peptide and PBS under salinity stress 75 Figure 3.8 Root growth of Arabidopsis thaliana wild type Col-0 treated with chemically synthesized Pep3 peptide under salinity stress 76 Figure 3.9 Root growth of Arabidopsis thaliana wild type Col-0 treated with chemically synthesized Pep1 peptide under salinity stress 77 Figure 3.10 Root growth of Arabidopsis thaliana wild type Col-0 treated with recombinant RALF14 and chemically synthesized RALF14b peptides under salinity stress 79 Figure 3.11 Root growth of Arabidopsis thaliana wild type Col-0 treated with chemically synthesized RALF1a and RALF1b peptides under salinity stress 80 Figure 3.12 Root growth of Arabidopsis thaliana wild type Col-0 treated with chemically synthesized RALF34a and RALF34b peptides under salinity stress 81 Figure 3.13 Germination rate of aging Arabidopsis thaliana wild type Col-0 seeds treated with chemically synthesized Pep3 under salinity stress 82 Figure 3.14 Germination rate of healthy Arabidopsis thaliana wild type Col-0 seeds treated with chemically synthesized Pep3 under salinity stress 83 Figure 3.15 Comparison of primary structures and identity of RALF1, RALF14, RALF33 and RALF34 by using ClustalOmega and SIAS 84 Tables 85 Table 3.1 Expression conditions of RALF14, RALF33 and RALF34 85 Table 3.2 Different treatment of different peptide under salinity stress 86 Table 3.3 Different effects of different peptide treatment under salinity stress 87 Appendices 88 Appendix 2.1 Sequences of chemically synthesized peptide (Section 2.1.5) 88 Appendix 2.2 Sequences of recombinant peptide (Section 2.1.5) 88 Appendix 2.3 Primers for plasmid construction (Section 2.2.1) 88 Appendix 2.4 Tris-glycine SDS-PAGE solution configuration (Section 2.2.3.1) 89 Appendix 2.5 Tricine SDS-PAGE solution configuration (Section 2.2.3.2) 89 Appendix 2.6 Template of ANOVA in R programming (Section 2.2.6.1) 89 Supplementary data 91 Figure S1 Total root length of Arabidopsis thaliana wild type Col-0 under different NaCl concentrations 91 Figure S2 Total root length of Arabidopsis thaliana wild type Col-0 treated with chemically synthesized BP21 peptide and PBS under salinity stress 92 Figure S3 Total root length of Arabidopsis thaliana wild type Col-0 under different Pep3 concentrations 94 Figure S4 Total root length of Arabidopsis thaliana wild type Col-0 treated with chemically synthesized Pep3 peptide under salinity stress 95 Figure S5 Total root length of Arabidopsis thaliana wild type Col-0 treated with chemically synthesized Pep1 peptide under salinity stress 96 Figure S6 Total root length of Arabidopsis thaliana wild type Col-0 treated with recombinant RALF14 and chemically synthesized RALF14b peptides under salinity stress 97 Figure S7 Total root length of Arabidopsis thaliana wild type Col-0 treated with chemically synthesized RALF1a and RALF1b peptides under salinity stress 98 Figure S8 Total root length of Arabidopsis thaliana wild type Col-0 treated with chemically synthesized RALF34a and RALF34b peptides under salinity stress 99 Table S1 Elongation data (Figure 3.6 ~ Figure 3.12) in R programming 100 Table S2 Total length data (Figure S1 ~ Figure S8) in R programming 142 論文口試問答集及討論建議 210 | - |
| dc.language.iso | en | - |
| dc.subject | 鹽逆境 | zh_TW |
| dc.subject | 植物生長調節劑 | zh_TW |
| dc.subject | 訊息胜? | zh_TW |
| dc.subject | 植物激發胜? | zh_TW |
| dc.subject | 快速鹼化因子 | zh_TW |
| dc.subject | Rapid alkalinization factors | en |
| dc.subject | Salinity stress | en |
| dc.subject | Signaling peptides | en |
| dc.subject | Plant elicitor peptide | en |
| dc.subject | Plant growth regulator | en |
| dc.title | 訊息胜肽 AtPep 與 AtRALF 對阿拉伯芥在高鹽逆境反應與生長之影響 | zh_TW |
| dc.title | Effects of signaling peptides AtPep and AtRALF on the salinity stress response and growth of Arabidopsis thaliana | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 107-2 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 陳佩燁;鄭秋萍;李昆達;吳?承 | zh_TW |
| dc.contributor.oralexamcommittee | ;;; | en |
| dc.subject.keyword | 快速鹼化因子,植物激發胜?,訊息胜?,植物生長調節劑,鹽逆境, | zh_TW |
| dc.subject.keyword | Rapid alkalinization factors,Plant elicitor peptide,Signaling peptides,Plant growth regulator,Salinity stress, | en |
| dc.relation.page | 216 | - |
| dc.identifier.doi | 10.6342/NTU201902893 | - |
| dc.rights.note | 未授權 | - |
| dc.date.accepted | 2019-08-14 | - |
| dc.contributor.author-college | 生命科學院 | - |
| dc.contributor.author-dept | 生化科技學系 | - |
| 顯示於系所單位： | 生化科技學系 | |
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