Title Page
Contents
LIST OF ABBREVIATIONS 9
Abstract 15
I. INTRODUCTION 17
1. Characterization of the genus Fabavirus 17
2. The species Petunia hybrida 20
3. Biological properties of BBWV2 23
4. Quantitative real time polymerase chain reaction (qRT-PCR) analysis 27
5. Purpose of this study 29
II. MATERIALS AND METHODS 30
1. Sources of viruses and hosts 30
2. Bacterial strain and recombinant plasmid vector 31
3. Purification of viral particles and viral RNA extraction from virions 33
3-1. Purification of virus 33
3-2. Morphology of virus particles 36
4. SDS-PAGE and western blot 36
4-1. SDS-PAGE analysis 36
4-2. Western blotting analysis 37
5. Viral RNA analysis 38
5-1. Viral RNA extraction 38
5-2. Viral RNA electrophoresis 41
6. Host range test 41
7. Primer design and RT-PCR analysis 42
7-1. Designing primers 42
7-2. RT-PCR analysis 44
8. Cloning and sequencing 46
9. Sequencing analysis 48
10. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis 49
10-1. Primer design 49
10-2. Standard curve method 51
10-3. qRT-PCR assay 52
III. RESULTS 53
1. Morphology of viral particles and biological properties of BBWV2-pet 53
1-1. Virus purification 53
1-2. SDS-PAGE and western blotting 55
1-3. Morphology of viral particles 57
2. Viral RNA extraction analysis 59
3. Host range and symptomatology of BBWV2-pet and BBWV2-RP5 61
3-1. Host range test 61
3-2. RT-PCR detection of plants infected with BBWV2-pet and BBWV RP5 63
4. qRT-PCR analysis of BBWV2-pet and BBWV2-RP5 75
4-1. Plasmid DNA standards after qRT-PCR 75
4-2. Validation of qRT-PCR 77
4-3. Absolute quantification of two BBWV2 isolates in host plants 84
IV. Discussion 87
V. References 90
VI. ABSTRACT IN KOREAN 98
Table 1. The list of hosts and symptoms infected with Broad bean wilt virus 2 24
Table 2. Primer pair for detection of BBWV2-pet and BBWV2-RP5 43
Table 3. Primer pairs for qRT-PCR of BBWV2-RP5, BBWV2-pet and 18S rRNA 50
Table 4. Experimental host range and symptoms of BBWV2-pet and BBWV2-RP5. 62
Figure 1. Genome structure of viral proteins of BBWV2-ME. 19
Figure 2. The sales quantities on different category types of petunia plants in USA... 22
Figure 3. The value of all sales at wholesale of petunia plants in USA in 2018. 22
Figure 4. Various symptoms of plants infected of BBWV2 isolated from Petunia... 26
Figure 5. pGEM-T Easy Vector (Promega, USA) was used to cloning. A: pGEM-T... 32
Figure 6. Purification of virus by performing sucrose density gradient... 35
Figure 7. Absorbance spectrum of purified virions of BBWV2-pet. Protein... 54
Figure 8. SDS-PAGE and western blotting analysis of CPs of BBWV2-pet. A:... 56
Figure 9. Electron micrographs of BBWV2-pet viral particles. The bar indicates... 58
Figure 10. Viral RNA extraction of purified virions of BBWV2-pet. M, RNA... 60
Figure 11. Symptoms of host plants inoculated with BBWV2-pet and BBWV2-RP5.... 64
Figure 12. Symptoms of host plants inoculated with BBWV2-pet and BBWV2-RP5.... 65
Figure 13. Electrophoretic patterns after detection of BBWV2-infected hosts using... 66
Figure 14. Symptoms of host plants inoculated with BBWV2-pet and BBWV2-RP5.... 67
Figure 15. Symptoms of host plants inoculated with BBWV2-pet and BBWV2-RP5.... 68
Figure 16. Electrophoretic patterns after detection of BBWV2-infected hosts using... 69
Figure 17. Symptoms of hosts inoculated with BBWV2-pet and BBWV2-RP5. A:... 70
Figure 18. Symptoms of host plants inoculated with BBWV2-pet and BBWV2-RP5.... 71
Figure 19. Electrophoretic patterns after detection of BBWV2-infected hosts using... 72
Figure 20. Symptoms of host plants inoculated with BBWV2-pet and BBWV2-RP5.... 73
Figure 21. Electrophoretic patterns after detection of BBWV2-infected hosts using... 74
Figure 22. Electrophoretic results of plasmid DNA standards after qRT-PCR. A:... 76
Figure 23. Amplification plots and standard curves of plasmid DNA of BBWV2-pet... 78
Figure 24. Melting curve plots of BBWV2-pet and BBWV2-RP5. 80
Figure 25. Electrophoretic patterns after qRT-PCR of BBWV2-infected hosts and... 82
Figure 26. Electrophoretic patterns after qRT-PCR of BBWV2-infected hosts and... 83
Figure 27. Expression quantity of BBWV2-pet in seven host plants. Inoculated and... 85
Figure 28. Expression quantity of BBWV2-RP5 in seven host plants. Inoculated... 86