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研究生:康峰誌
研究生(外文):Feng-Chih Kang
論文名稱:闡述AtNPF8.1/AtPTR1受質專一性的分子特徵
論文名稱(外文):Elucidating the molecular features of AtNPF8.1/AtPTR1 substrate specificity
指導教授:王雅筠
指導教授(外文):Ya-Yun Wang
口試委員:蔡宜芳鄭貽生張英峯
口試委員(外文):Yi-Fang TsayYi-Sheng ChengIng-Feng Chang
口試日期:2021-01-27
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:植物科學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2021
畢業學年度:109
語文別:英文
論文頁數:97
中文關鍵詞:受質專一性轉運蛋白硝酸鹽胜肽NPFAtPTR1
外文關鍵詞:substrate specificitytransporternitratepeptideNPFAtPTR1
DOI:10.6342/NTU202100600
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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
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