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Title Page
Contents
LIST OF ABBREVIATIONS 8
Abstract 14
I. INTRODUCTION 15
1. Pepper mottle virus 15
2. GFP tagged PepMoV vector system 17
3. Serial passage experiment of plant virus 17
4. Purpose of this study 19
II. MATERIALS AND METHODS 20
1. Source of viruses 20
2. Source of plant 20
3. Bacterial strains and plasmids 20
4. Culture media 21
5. Passage experiment for population variation 21
6. Total RNA extraction 23
7. Reverse transcription-polymerase chain reaction(RT-PCR) 23
8. Cloning and screening of recombinant clones 25
9. Nucleotide sequencing and sequence analysis 26
10. Western blot analysis 27
11. Comparison of host ranges between PepMoV-Vb1/GFP and PepMoV 28
III. RESULTS 29
1. Analysis of pathological symptoms induced by PepMoV-Vb1/GFP 29
1-1. Symptomatology during serial passage 29
1-2. Host range study of PepMoV-Vb1/GFP 29
2. The initial viral population and passaging 34
3. Western blot analysis 34
4. Cloning and screening of recombinant clones 37
5. Analysis of sequencing data for PepMoV-Vb1 and PepMoV-Vb1/GFP population dynamics 37
5-1. Population variation of serially passaged viruses 37
5-2. Distribution and types of mutations 38
IV. DISCUSSION 79
V. REFERENCE 82
VI. ABSTRACT IN KOREAN 86
Table 1. The functions of potyviral proteins 16
Table 2. Primers for RT-PCR assay 24
Table 3. Host range and symptomatology induced by PepMoV-Vb1/GFP 33
Table 4. Nucleotide mutation frequency and type of amino acid alteration in the PepMoV-Vb1/GFP P1 region after serial passage 39
Table 5. Nucleotide mutation frequency and type of amino acid alteration in the PepMoV-Vb1/GFP CP region after serial passage 40
Table 6. Nucleotide mutation frequency and type of amino acid alteration in the PepMoV-Vb1/GFP GFP region after serial passage 41
Table 7. Locations and types of all mutations in the P1 coding region detected in the passage 1 population of PepMoV-Vb1/GFP 42
Table 8. Locations and types of all mutations in the P1 coding region detected in the passage 2 population of PepMoV-Vb1/GFP 43
Table 9. Locations and types of all mutations in the P1 coding region detected in the passage 3 population of PepMoV-Vb1/GFP 44
Table 10. Locations and types of all mutations in the P1 coding region detected in the passage 4 population of PepMoV-Vb1/GFP 45
Table 11. Locations and types of all mutations in the P1 coding region detected in the passage 5 population of PepMoV-Vb1/GFP 46
Table 12. Locations and types of all mutations in the P1 coding region detected in the passage 6 population of PepMoV-Vb1/GFP 47
Table 13. Locations and types of all mutations in the CP coding region detected in the passage 1 population of PepMoV-Vb1/GFP 48
Table 14. Locations and types of all mutations in the CP coding region detected in the passage 2 population of PepMoV-Vb1/GFP 49
Table 15. Locations and types of all mutations in the CP coding region detected in the passage 3 population of PepMoV-Vb1/GFP 50
Table 16. Locations and types of all mutations in the CP coding region detected in the passage 4 population of PepMoV-Vb1/GFP 51
Table 17. Locations and types of all mutations in the CP coding region detected in the passage 5 population of PepMoV-Vb1/GFP 52
Table 18. Locations and types of all mutations in the CP coding region detected in the passage 6 population of PepMoV-Vb1/GFP 53
Table 19. Locations and types of all mutations in the GFP coding region detected in the passage 1 population of PepMoV-Vb1/GFP 54
Table 20. Locations and types of all mutations in the GFP coding region detected in the passage 2 population of PepMoV-Vb1/GFP 55
Table 21. Locations and types of all mutations in the GFP coding region detected in the passage 3 population of PepMoV-Vb1/GFP 56
Table 22. Locations and types of all mutations in the GFP coding region detected in the passage 4 population of PepMoV-Vb1/GFP 57
Table 23. Locations and types of all mutations in the GFP coding region detected in the passage 5 population of PepMoV-Vb1/GFP 58
Table 24. Locations and types of all mutations in the GFP coding region detected in the passage 6 population of PepMoV-Vb1/GFP 59
Fig. 1. The PepMoV viral vector system and detection of expression in Nicotiana benthamiana at 14 days post-inoculation (dpi). 18
Fig. 2. Diagram of the serial passage procedure for generating virus populations. In the initial inoculation, in vitro transcripts of pSP6PepMoV-Vb1... 22
Fig. 3. Photographs of systemic symptoms produced by PepMoV-Vb1 and PepMoV-Vb1/GFP in Nicotiana benthamiana during six serial passages.... 31
Fig. 4. Infection of various host plant species by PepMoV-Vb1/GFP. Photographs were taken of Nicotiana debneyi (A, B) at 18 dpi; of... 32
Fig. 5. The RT-PCR analyses of PepMoV-Vb1/GFP during passage (PS) in systemically-infected leaves of N. benthamiana by RT-PCR of... 35
Fig. 6. Western blot analyses of PepMoV-Vb1/GFP coat protein (left) and turboGFP (right) in infected upper leaves of N. benthamiana during serial... 36
Fig. 7. In the P1 region, comparison of number of mutants and mutation type (synonymous, non-synonymous and silent) in the serial passages 1-6. 61
Fig. 8. In the CP region, comparison of number of mutants and mutation type (synonymous, non-synonymous and silent) in the serial passages 1-6. 62
Fig. 9. In the GFP region, comparison of number of mutants and mutation type (synonymous, non-synonymous and silent) in the serial passages 1-6. 63
Fig. 10. Nucleotide mutation frequency of transitions (Ts) and transversions (Tv) in the P1 region. 64
Fig. 11. Nucleotide mutation frequency of transitions (Ts) and transversions (Tv) in the CP region. 65
Fig. 12. Nucleotide mutation frequency of transitions (Ts) and transversions (Tv) in the GFP region. 66
Fig. 13. Distribution of changed amino acid in synonymous substitution mutations during serial passages. 67
Fig. 14. Distribution of changed amino acid in non-synonymous substitution mutations during serial passages. 68
Fig. 15. Distribution of changed amino acid in silent mutations during serial passages. 69
Fig. 16. Distribution of changed amino acid in all amino acid alteration during serial passages. 70
Fig. 17. In the P1 region, comparison of alternation between original and changed amino acid 71
Fig. 18. In the CP region, comparison of alternation between original and changed amino acid 72
Fig. 19. In the GFP region, comparison of alternation between original and changed amino acid 73
Fig. 20. Positions of mutations in PepMoV-Vb1 P1 (861nt) region based on the nucleotide sequence analysis of progeny viruses propagated during serial... 75
Fig. 21. Positions of mutations in PepMoV-Vb1 CP (819nt) region based on the nucleotide sequence analysis of progeny viruses propagated during serial... 76
Fig. 22. Positions of mutations in turboGFP (696nt) region based on the nucleotide sequence analysis of progeny viruses propagated during serial... 77
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