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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 | 王雅筠(Ya-Yun Wang) | |
dc.contributor.author | Feng-Chih Kang | en |
dc.contributor.author | 康峰誌 | zh_TW |
dc.date.accessioned | 2021-05-20T00:51:38Z | - |
dc.date.available | 2026-02-18 | |
dc.date.available | 2021-05-20T00:51:38Z | - |
dc.date.copyright | 2021-03-05 | |
dc.date.issued | 2021 | |
dc.date.submitted | 2021-02-08 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/8300 | - |
dc.description.abstract | 氮是植物生長發育必要的元素(如硝酸鹽或胜肽),在過去的研究中知道有機的氮源(如胜肽)吸收以及運動對植物的生理反應是非常重要的。AtPTR1是硝酸鹽轉運蛋白1 /胜肽轉運蛋白家族(NPF)的一員,可運輸多種胜肽但不能運輸硝酸鹽。在本研究中我們想藉由AtPTR1去了解,胜肽與硝酸鹽的受質專一性是如何決定的。我們挑選了多個點位並做出21種突變來研究其對於硝酸鹽以及雙肽運輸的影響。先用原生質體來檢視突變後的AtPTR1座落位置是否有表現在細胞膜上,發現H534L、H534Y、120-140、268-311座落位置由細胞膜改變為其他位置。使用酵母菌互補分析看雙肽的運輸,發現Y45I、Y346H、C31A、C31S、C318A、C318S、H534K之突變會降低對於雙肽的運輸;Y45IY46I、Y45IY46IY346H則喪失運輸能力。用非洲爪蟾卵母細胞表現系統看硝酸鹽的運輸,發現Y45I、Y46I、Y46A、Y45IY46I、Y346H、Y45IY46IY346H突變蛋白皆不能運輸硝酸鹽。在本研究中未發現可以影響受質決定的突變蛋白,然而這些結果讓我們了解到AtPTR1的座落位置會因特定點位的改變造成而受到影響,有的突變亦會造成其運輸能力的減弱或喪失,這些關於AtPTR1分子特徵的闡述,可作為模型去推論及研究NPF其他轉運蛋白的功能與特性。 | zh_TW |
dc.description.abstract | Nitrogen (such as nitrate and peptide) are essential for plant growth and development. The uptake and transport of organic N (such as peptides) is important for many physiological responses. In the past, AtPTR1 has been known as a plasma membrane-localized transporter and a member of the Nitrate transporter 1/Peptide transporter Family (NPF). AtPTR1 can transport several di-, tri-peptides, but not nitrate. In our study, we want to understand the difference in substrate specificity between AtPTR1 and other nitrate transporters in NPF. Therefore, we selected multiple sites to study the effect of nitrate and dipeptide transport. At first, we examined whether the localization of mutated AtPTR1 by the protoplast and found that H534L, H534Y, 120-140, and 268-311 has lost the plasma membrane-localized property. In yeast complementation assay, we analyzed the di-peptide uptake ability of mutated AtPTR1. We found Y45I, Y346H, C31A, C31, C318A, C318S, H534K reduced the di-peptide uptake ability, and Y45IY46I, Y45IY46IY346H loss of the di-peptide uptake ability. We also showed that no mutated AtPTR1 proteins can uptake nitrate by using the Xenopus oocyte expression system. In this study, the mutant protein that can affect substrate determination to nitrate haven’t been found. However, we understand some mutations of the specific sites can affect the localization of AtPTR1, and some mutations also can cause the reduction or loss of the di-peptide uptake ability. These findings of the substrate specificity of AtPTR1 can be used as a model to understand and study the functions and properties of other NPF members. | en |
dc.description.provenance | Made available in DSpace on 2021-05-20T00:51:38Z (GMT). No. of bitstreams: 1 U0001-0502202115561900.pdf: 7057150 bytes, checksum: 0be64eb00c718efff000defc722e63ab (MD5) Previous issue date: 2021 | en |
dc.description.tableofcontents | 口試委員會審定書 # 誌謝 ii LIST OF ABBREVIATIONS iii 摘要 v ABSTRACT vi CONTENTS vii LIST OF FIGURES x LIST OF TABLES xi LIST OF APPENDICES xii Chapter 1 Introduction 1 1.1 The importance of the organic nitrogen sources, and the three types of peptide transporters 1 1.2 PTR transporters are functional and widely necessary in diverse species 2 1.3 Nitrate transporter 1/peptide transporter family (NPF) evolves unique substrate specificity in plants 4 1.4 Peptide transporters of NPF share high sequence identity and similarity to other members but with different substrate specificity 6 Chapter 2 Materials and methods 8 2.1 Plasmid cloning 8 2.2 Plasmid subcloning 8 2.3 RNA extraction 9 2.4 Reverse transcription (RT) 10 2.5 Polymerase chain reaction (PCR) 10 2.6 PCR/gel purification 10 2.7 E. coli. Transformation 11 2.8 Plasmid extraction 11 2.9 Site-directed mutagenesis 12 2.10 Protoplast extraction 13 2.11 Transformation of protoplast and examination of protein localization 13 2.12 Yeast transformation 14 2.13 Yeast complementation assay 14 2.14 Analysis of growth curve in yeast 15 2.15 In vitro transcription 15 2.16 Functional analysis in Xenopus oocytes 16 2.17 Western blot 17 Chapter 3 Results 19 3.1 Sequence alignment of peptide and nitrate transporters 19 3.2 H534L, H534Y, 120-140 and 268-311 mutants failed to localize to the plasma membrane 20 3.3 Y45I, Y45IY46I, Y346H, Y45IY46IY346H, C31A, C31S, C318A, C318S mutants affect the di-peptide uptake ability of AtPTR1 21 3.4 Y45I, Y46I, Y46A, Y45IY46I, Y346H, Y45IY46IY346H mutants cannot uptake nitrate 22 Chapter 4 Discussions and conclusions 24 4.1 Protein structure modeling of native and mutated AtPTR1 proteins 24 4.2 The conformational change of Y45 may cause the reduction of the peptide biding ability 24 4.3 Y346 might complement the function of Y46 and lost function of Y45IY46I might result from increasing the cavity of the substrate binding site 25 4.4 The positive charge of Y346H may cause the reduction of the di-peptide uptake ability 26 4.5 The maintenance of polar and hydrophobic amino acid at residue 346 and 503, respectively, might be important for the substrate determination to nitrate 27 4.6 Sulfhydryl groups of C31 and C318 play an important role in the protein function of AtPTR1 27 4.7 Positive charge on H534 residue might be important for AtPTR1 to localize to the plasma membrane correctly 28 4.8 Conformation change of H534 may cause the reduction of peptide uptake ability 29 4.9 The loop between TM3 and TM4 (NPF-specific region) may affect the folding of protein and cause the change of localization 29 4.10 The central loop between TM6 and TM7 may be important for NPF to localized to plasma membrane correctly 30 Chapter 5 Figures 32 Chapter 6 Tables 68 Chapter 7 References 73 Chapter 8 Appendices 78 LIST OF FIGURES Figure 1. Sequence alignment of peptide and nitrate transporters. 37 Figure 2. Protein localization of mutated AtPTR1 in protoplasts. 42 Figure 3. Protein localization of AtPTR1, H534L, H534Y, 120-140, 268-311 in yeast cells. 44 Figure 4. Observation of protein expression in yeast cells. 48 Figure 5. Di-peptide uptake ability of mutated AtPTR1. 50 Figure 6. Growth curves of PTR1- and H534K-expressing yeast cells. 51 Figure 7. Nitrate uptake ability of mutated AtPTR1. 53 Figure 8. Summary of the important residues of AtPTR1. 54 Figure 9. Protein structure modeling of AtPTR1. 55 Figure 10. Comparison of important residues in the transporter pocket among AtPTR1, AtNPF6.3 and StPepT. 57 Figure 11. Protein structure modeling of mutated AtPTR1 in the transporter pocket. 60 Figure 12. Protein structure modeling of mutated AtPTR1 on the intracellular side. 63 Figure 13. Protein structure modeling of mutated AtPTR1 on the extracellular side. 67 LIST OF TABLES Table 1. The summary of aligned transporters. 68 Table 2. The summary of the candidate residues for analysis. 70 Table 3. Di-peptide uptake assay in different concentrations of His-Leu medium. 71 Table 4. Summary of important residues close to the substrate binding site. 72 LIST OF APPENDICES Appendix 1. Formula for A-tailing reaction 78 Appendix 2. Reverse-transcription mix 79 Appendix 3. PCR program of site-directed mutagenesis 79 Appendix 4. Enzyme solution 80 Appendix 5. W5 solution 80 Appendix 6. MMg solution 81 Appendix 7. PEG solution 81 Appendix 8. YPAD 81 Appendix 9. PLATE mixture 82 Appendix 10. Amino acid mixture 83 Appendix 11. Ura- 84 Appendix 12. Ura-His- 85 Appendix 13. Formula for in vitro transcription 86 Appendix 14. Ringer’s Buffer 86 Appendix 15. ND96 buffer without Ca2+ 87 Appendix 16. ND96 buffer with Ca2+ 87 Appendix 17. Program for micropipette puller 87 Appendix 18. Nitrate Uptake Buffer 88 Appendix 19. Oocyte Protein Extraction Buffer 88 Appendix 20. MOPS/SDS Running Buffer 89 Appendix 21. NuPAGE Gel Transfer Buffer 89 Appendix 22. Phosphate-Buffered Saline (PBS) buffer 89 Appendix 23. Primers for PTR1 cloning, mutagenesis, and subcloning. 90 Appendix 24. E. coli. strains 93 Appendix 25. Yeast strains 96 Appendix 26. Enzymes 97 | |
dc.language.iso | en | |
dc.title | 闡述AtNPF8.1/AtPTR1受質專一性的分子特徵 | zh_TW |
dc.title | Elucidating the molecular features of AtNPF8.1/AtPTR1 substrate specificity | en |
dc.type | Thesis | |
dc.date.schoolyear | 109-1 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 蔡宜芳(Yi-Fang Tsay),鄭貽生(Yi-Sheng Cheng),張英峯(Ing-Feng Chang) | |
dc.subject.keyword | 受質專一性,轉運蛋白,硝酸鹽,胜肽,NPF,AtPTR1, | zh_TW |
dc.subject.keyword | substrate specificity,transporter,nitrate,peptide,NPF,AtPTR1, | en |
dc.relation.page | 97 | |
dc.identifier.doi | 10.6342/NTU202100600 | |
dc.rights.note | 同意授權(全球公開) | |
dc.date.accepted | 2021-02-09 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 植物科學研究所 | zh_TW |
dc.date.embargo-lift | 2026-02-18 | - |
顯示於系所單位: | 植物科學研究所 |
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