Title Page
Contents
LIST OF ABBREVIATIONS 9
Abstract 15
I. INTRODUCTION 17
1. Cucurbit-infecting Tobamovirus 17
2. Natural antiviral agents against plant virus 21
3. Catechin 24
4. Purpose of this study 28
II. MATERIALS AND METHODS 29
1. Sources of virus and plant 29
2. Sources of antiviral compounds 29
3. Purification of virus from host plant 30
4. Leaf disc assay for antiviral activity 31
5. Antiviral activity analysis with systemic host 33
5.1. Inactivation assay 34
5.2. Protection assay 35
6. Total RNA extraction 37
7. Total protein extraction 38
8. Reverse transcription-polymerase chain reaction (RT-PCR) 40
9. Western blot analysis 44
III. RESULTS 46
1. Leaf disc assays using Epigallocatechin gallate (EGCG) 46
2. Leaf disc assays using Epicatechin gallate (ECG) 51
3. Systemic host assays using Epigallocatechin gallate (EGCG) 56
3.1. Inactivation assay 56
3.2. Protection assay 64
4. Systemic host assays using Epicatechin gallate (ECG) 70
4.1. Inactivation assay 70
4.2. Protection assay 78
IV. Discussion 84
V. References 89
VI. ABSTRACT IN KOREAN 95
Table 1. List of Cucurbit-infecting Tobamovirus. 19
Table 2. List of antiviral agents against plant viruses. 23
Table 3. Previous studies of antiviral activity using catechin compounds. 26
Table 4. Coat protein primers sequences of zucchini green mottle mosaic virus and... 41
Table 5. 18S rRNA primer sequences and size (bp). 42
Table 6. Effect of antiviral two catechin compounds used in this study. 87
Figure 1. Schematic diagram of ZGMMV genome structure. RdRp, RNA depndent... 20
Figure 2. The structure of catechin compounds. Catechin compounds consist of ECG,... 25
Figure 3. Schematic diagram of leaf disc assay for antiviral activity analysis. 32
Figure 4. Schematic diagram of systemic host assay for antiviral activity analysis.... 36
Figure 5. Schematic diagram of total RNA and total protein extraction. 39
Figure 6. Schematic diagram of RT-PCR. 43
Figure 7. Schematic diagram of Western blot analysis. 45
Figure 8. Antiviral activity test for EGCG in ZGMMV-infected leaf discs. Data for... 48
Figure 9. Detection of ZGMMV viral RNA and 18S rRNA by RT-PCR analyses in... 49
Figure 10. Detection of ZGMMV by western blot analyses in the leaf disc antiviral... 50
Figure 11. Antiviral activity test for ECG in ZGMMV-infected leaf discs. Data for... 53
Figure 12. Detection of ZGMMV viral RNA and 18S rRNA by RT-PCR analyses in... 54
Figure 13. Detection of ZGMMV by western blot analyses in the leaf disc antiviral... 55
Figure 14. Inactivation assay of antiviral activity of EGCG in whole plant. The... 59
Figure 15. Inactivation assay of antiviral activity of EGCG in upper leaf. The... 60
Figure 16. Detection of ZGMMV viral RNA and 18S rRNA by RT-PCR analyses in... 61
Figure 17. Detection of ZGMMV by western blot analyses in the inactivation assay... 62
Figure 18. Electron microscopic observation of ZGMMV virion after treatment with... 63
Figure 19. Protection assay of antiviral activity of EGCG in whole plant. The... 66
Figure 20. Protection assay of antiviral activity of EGCG in upper leaf. The... 67
Figure 21. Detection of ZGMMV viral RNA and 18S rRNA by RTPCR analyses in... 68
Figure 22. Detection of ZGMMV by western blot analyses in the protection assay... 69
Figure 23. Inactivation assay of antiviral activity of ECG in whole plant. The... 73
Figure 24. Inactivation assay of antiviral activity of ECG in upper leaf. The... 74
Figure 25. Detection of ZGMMV viral RNA and 18S rRNA by RT-PCR analyses in... 75
Figure 26. Detection of ZGMMV by western blot analyses in the inactivation assay... 76
Figure 27. Electron microscopic observation of ZGMMV virion after treatment with... 77
Figure 28. Protection assay of antiviral activity of ECG in whole plant. The... 80
Figure 29. Protection assay of antiviral activity of ECG in upper leaf. The... 81
Figure 30. Detection of ZGMMV viral RNA and 18S rRNAby RTPCR analyses in... 82
Figure 31. Detection of ZGMMV by western blot analyses in the protection assay... 83
Figure 32. Chemical structure of the catechins ECG and EGCG. A: Basic catechin... 88