목차

CHAPTER I. General Introduction 1
1.1 Momordica charantia 2
1.2 Carotenoids 5
1.3 Carotenoid cleavage dioxygenases 5
1.4 Riboflavin 7
1.5 Multigene transfer to plants 7
CHAPTER II. Carotenoid Content and Expression of Phytoene Synthase and Phytoene Desaturase Genes 9
2.1 ABSTRACT 10
2.2 INTRODUCTION 11
2.3 MATERIALS AND METHODS 14
2.3.1 Plant materials 14
2.3.2 Real-time polymerase chain reaction analysis of M. charantia phytoene synthase and phytoene desaturase 16
2.3.3 Extraction and high performance liquid chromatography analysis of carotenoids from M. charantia 18
2.3.4 Principal component analysis 19
2.3.5 Statistical analysis 19
2.4 RESULTS 20
2.4.1 Gene expression of phytoene synthase (PSY) and phytoene desaturase (PDS) in different organs of M. charantia 20
2.4.2 Gene expression of PSY and PDS during fruit maturation in M. charantia 21
2.4.3 Principal components of carotenoid content during fruit maturation in M. charantia 23
2.4.4 HPLC analyses of carotenoids in M. charantia 26
2.5 DISCUSSION 32
CHAPTER III. Molecular Cloning and Characterization of cDNAs Encoding Carotenoid Cleavage Dioxygenase 34
3.1 ABSTRACT 35
3.2 INTRODUCTION 37
3.3 MATERIALS AND METHODS 40
3.3.1 Plant materials 40
3.3.2 Stress treatments 40
3.3.3 RNA isolation and cDNA synthesis 41
3.3.4 Cloning of the cDNAs encoding carotenoid cleavage dioxygenase 41
3.3.5 Sequence analysis 42
3.3.6 Real-time PCR 42
3.3.7 Statistical Analysis 43
3.4 RESULTS 44
3.4.1 Cloning of CCDs from M. charantia 44
3.4.2 Sequence analyses of McCCD1, McCCD4, and McNCED 44
3.4.3 Expression levels of McCCD1, McCCD4, and McNCED in different organs of M. charantia 51
3.4.4 Expression levels of McCCD1, McCCD4, and McNCED during fruit maturation in M. charantia 53
3.4.5 Transcriptional regulation of McNCED during seed germination and under abiotic stresses 53
3.5 DISCUSSION 58
CHAPTER IV. Riboflavin Accumulation and Characterization of cDNAs Encoding Lumazine Synthase and Riboflavin Synthase 61
4.1 ABSTRACT 62
4.2 INTRODUCTION 63
4.3 MATERIALS AND METHODS 66
4.3.1 Plant materials 66
4.3.2 RNA isolation and cDNA synthesis 66
4.3.3 Cloning of cDNA encoding lumazine synthase and riboflavin synthase 66
4.3.4 Sequence analysis 67
4.3.5 Real-time PCR 68
4.3.6 Riboflavin extraction and analysis 68
4.4 RESULTS 70
4.4.1 Cloning of LS and RS from M. charantia 70
4.4.2 Sequence analyses of McLS and McRS 71
4.4.3 Expression levels of McLS and McRS in different organs of M. charantia 75
4.4.4 Expression levels of McLS and McRS during fruit maturation in M. charantia 78
4.4.5 Analysis of riboflavin content in different organs of M. charantia 78
4.4.6 Analysis of riboflavin content during fruit maturation in M. charantia 80
4.5 DICUSSION 81
CHAPTER V. Co-ordination Overexpression of Momordica charantia’s Lumazine Synthase and Riboflavin Synthase by 2 Bicistronic Systems Involving IRES and 2A Sequences in Transgenic Tobacco 83
5.1 ABSTRACT 84
5.2 INTRODUCTION 85
5.3 MATERIALS AND METHODS 87
5.3.1 Plasmid construction 87
5.3.2 Tobacco transformation 90
5.3.3 Confirmation of the presence of transgenes in tobacco 90
5.3.4 RNA isolation and cDNA synthesis 91
5.3.5 Real-time PCR 91
5.4 RESULTS 93
5.4.1 Establishment of transgenic tobacco lines 93
5.4.2 Expression levels of McLS, McRS, LIR, and LAR in transgenic tobacco leaves 95
5.5 DISCUSSION 98
CHAPTER VI. Conclusion 99
REFERENCES 103
KOREAN ABSTRACT 114
ACKNOWLEDGMENTS 116