Title Page
국문초록
Contents
Introduction 12
CHAPTER I. Molecular characterization of 2-Cys peroxiredoxin isolated from mungbean (Vigna radiata) 16
Materials and methods 17
1. Plant Materials, Growth Conditions and Treatments 17
2. Cloning of VrPrx1 Gene Using 5' Rapid Amplification cDNA Ends (5' RACE) 17
3. Phylogenetic Analysis 18
4. Genomic DNA Blot Hybridizations 18
5. Production of His-tagged VrPrx1 Recombinant Protein in E. coli 19
6. DNA Protection and Peroxidase Activity of VrPrx1 Recombinant Protein 20
7. Antibody Production, SDS-PAGE and Western Blot Analysis 21
8. Construction and Subcellular Localization of Series of VrPrx1-GFP Fusion Proteins 22
9. Generation of VrPrx1 Overexpressing Arabidopsis 24
10. Measurements of Chlorophyll Content and Fv/Fm of Transgenic Lines Treated with Methyl Viologen (MV), Drought, Low temperature and NaCl Stress 24
Results 26
1. Cloning of a VrPrx1 and Analysis of Its Amino Acid Sequence 26
2. Genomic Organization and RNA Expression of VrPrx1 32
3. Peroxidase Activity and Redox State of Recombinant VrPrx1 Protein 34
4. Subcellular Localization of VrPrx1 42
5. Improved Tolerance of Plants Overexpressing VrPrx1 to Photooxidative Stress 45
Discussion 50
CHAPTER II. Novel proteins interactiong with VrPrx1 isolated from mungbean (Vigna radiata) 54
Materials and methods 55
1. Yeast Two-Hybrid Screening 55
2. Liquid Culture Assay Using ONPG as Substrate 56
3. Cloning of Genes Using 5' Rapid Amplification cDNA Ends (5' RACE) 56
4. Construction and Subcellular Localization of GFP-VrGS, UBC and ATP synthase Fusion Proteins 57
5. Production of His-tagged and c-Myc tagged VrGS, 85, 137, 695 Recombinant Protein in E. coli 58
Results 60
1. Analysis of Target Proteins of VrPrx1 by Y2H Screening 60
2. Liquid Culture Assay Using ONPG as Substrate 61
3. Subcellular Localization of VrGS, UBC, ATP synthase 68
4. Production of His-tagged and c-Myc-tagged VrGS Recombinant Protein in E. coli 71
5. Interactions Between the VrPrx1 protein and 85, 137 and 695 73
Discussion 75
CHAPTER III. Molecular cloning and characterization of the soybean genes induced by environmental stress 77
I. Molecular cloning and characterization of the soybean DEAD-box RNA helicase gene induced by low temperature and high salinity stress 78
Introduction 79
Materials and method 81
1. Plant materials and treatments 81
2. Cloning of GmRH gene using 5' and 3' rapid amplification cDNA ends (5' RACE) 81
3. Phylogenetic analysis 82
4. Northern blot analyses 82
5. Construction and subcellular localization of GmRH-GFP fusion protein 83
Results 85
1. Isolation and phylogenetic analysis of the soybean DEAD-box RNA helicase gene. 85
2. Determination of copy number and RNA expression of GmRH 93
3. Nuclear localization of GmRH-GFP protein in protoplasts 95
Discussion 97
II . Enhanced Tolerance Against Osmotic Stresses of Escherichia coli Cells Expressing Soybean KS-Type Dehydrin 100
Introduction 101
Materials and methods 102
1. Plant materials and treatments 102
2. Expreesion of the His-tag SLTI66 and SLTI629 fusion protein in E. coli 102
3. SDS-PAGE and western blot analysis 103
4. Freeze and thaw experiment 104
5. Salt stress test in E. coli 104
Results and discussion 106
1. Molecular cloning of KS-type dehydrin genes 106
2. Production of SLTI66 and SLTI629 recombinant protein in E. coli 110
3. Enhanced tolerance to freezing and salt stress by SLTI66 or SLTI629 in E. coli 112
References 117
Abstract 130
CHAPTER II. 7
Table 1. Sequence comparison of clones isolated from Y2H screening. 63
Table 2. Continued 64
CHAPTER III. 7
Table 1. List of RNA helicases homologous to GmRH 92
CHAPTER I. Molecular characterization of 2-Cys peroxiredoxin isolated from mungbean (Vigna radiata) 8
Figure 1. Amino acid sequence analysis of VrPrx1. Sequence alignment of mungbean VrPrx1 with 2-cys peroxiredoxins. Protein alignment was generated using CLUSTALW at the T-coffee website (Notredame et al.,... 28
Figure 2. Phylogenetic tree of VrPrx1 and 2-Cys Prxs. ClustalX (ver. 1.18) and Treeview (ver. 1.6.6) programs were used to analyze phylogenetic relationships. The phylogenetic relationships showed that... 30
Figure 3. Southern blot and northern blot analyses of VrPrx1. Twenty microgram of genomic DNA was digested with EcoRI, HindIII, or XbaI and loaded on each lane. 33
Figure 4. SDS-PAGE and western blot analyses of His-VrPrx1 protein. (A) Vector and VrPrx1 cells were induced by IPTG and proteins were resolved on a 12 % SDS-PAGE and the gel was coomassie-stained.... 36
Figure 5. Protection assay of DNA by the VrPrx1 protein in MFO system and peroxidase activity of VrPrx1. A total of 500 ng plasmid DNA wsa incubated with DTT and FeCl₃.... 37
Figure 6. Peroxidase activity of VrPrx1. (A-B) the level of H₂O₂by VrPrx1. (1 mg/ml) was measured differing incubation time and concentration in the presence and absence of DTT (C-D).... 38
Figure 7. Kinetic activity of VrPrx1 peroxidase activity. The level of H₂O₂by VrPrx1 protein (1 mg/ml) was measured in the presence of DTT.... 39
Figure 8. Bandshift of recombinant VrPrx1 protein in western blot analyses with or without DTT (A) and in the presence of H₂O₂ (B). (A) VrPrx1 protein (3 μg) was boiled in the sample buffer supplemented with... 40
Figure 9. Western blot analyses of VrPrx1 protein. Mungbean plants were treated with non-stress, low temperature (4 ℃), drought, wounding, hydrogen peroxide, or high salinity (0.1 M NaCl) stress.... 41
Figure 10. Subcellular localization of GFP and VrPrx1-GFP in Arabidopsis transgenic plant. GFP and GFP fusion protein were observed in leaf and root tissues using a confocal microscopy.... 43
Figure 11. Subcellular localization of GFP and VrPrx1-GFP in tobacco protoplasts. Localization of each GFP fusion protein was analyzed in the protoplasts isolated from the tabacco leaves after 36 h of... 44
Figure 12. Overexpression of VrPrx1 improves antioxidant activities in Arabidopsis. (A) Western blot analysis was carried out using VrPrx1 polyclonal antibodies with WT and the 35S-VrPrx1 plants.... 47
Figure 13. Maximal PSII photochemical efficiency and chlorophyll retention in leaves from WT, VrPrx1-overexpressing Arabidopsis plants subjected to photooxidative treatments.... 48
Figure 14. The maximal PSII photochemical efficiency in leaves of Arabidopsis plants subjected to different abiotic stress treatment. Stress treatments were applied to Arabidopsis plants grown for 24 to 28 days... 49
CHAPTER II. Novel proteins interactiong with VrPrx1 isolated from mungbean (Vigna radiata) 9
Figure 1. Construction of pBD-VrPrx1 as a bait and pAD-library as a prey for yeast two hybrid. (A) We N-termial region of VrPrx1 was deleted. The cDNA library was constucted from stressed mungbean... 62
Figure 2. Liquid β-galactosidase assay using 2-nitrophenyl β-D-galactosidase ONPG as a substrate. β-galactosidase activity is expressed in U (=nmol min-1). The value was displayed as the average β-galactosidase activity for three... 65
Figure 3. Amino acid sequence analysis of VrGS. Sequence alignment of mungbean VrGS with other GS. Protein alignment was generated using CLUSTALW at the T-coffee website (Notredame et al., 2000), and... 67
Figure 4. Subcellular localization of GFP and VrGS-GFP in tobacco protoplasts. Localization of each GFP fusion protein was analyzed in the protoplasts isolated from the tabacco leaves after 36 h of... 69
Figure 5. Subcellular localization of GFP and ATP synthase-GFP, UBC-GFP in tobacco protoplasts. Localization of each GFP fusion protein was analyzed in the protoplasts isolated from the tabacco leaves after 36... 70
Figure 6. SDS-PAGE and western blot analyses of His-VrGS-c-Myc protein. (A) The proteins were induced by IPTG and the proteins were resolved on a 12% SDS-PAGE and the gel was coomassie-stained.... 72
Figure 7. The interactions were assayed in the GAL4 (a regulator of galactose - induced genes) Y2H system to retest the interactions of pBD-VrPrx1 with pAD-85, pAD-137, pAD-695.... 74
CHAPTER. III 10
I. Molecular cloning and characterization of the soybean DEAD-box RNA helicase gene induced by low temperature and high salinity stress 10
Figure 1. Structural map of GmRH protein. (A) Comparison of the conseved motifs in GmRH with consensus from other DEAD-box RNA helicase proteins.... 88
Figure 2. Phylogenetic relationship of GmRH with paralogous and orthologous proteins. (A) Two protein sequences predicted as paralogs, HsRH (Homo sapiens) and XlRH (Xenopus laevis), and 15 Arabidopsis... 90
Figure 3. Northern blot analysis of RNA expression of GmRH gene during low temperature (LT), drought, ABA, and salt stresses. (A) For low temperature stress, soybean plants were placed in the refrigerator... 94
Figure 4. Diagrammatic representation of GmRH(1 - 335)- and GmRH(131 - 355)-GFP fusion constructs (A) and confocal images of GFP, GmRH (1 - 335)- and GmRH(131 - 355)-GFP protein in N. benthamiana protoplasts (B, C)....(이미지참조) 96
II . Enhanced Tolerance Against Osmotic Stresses of Escherichia coli Cells Expressing Soybean KS-Type Dehydrin 11
Figure 1. Alignment of deduced amino acid sequences of SLTI66, SLTI629 and other similar cold-stress proteins. The alignment was generated using CLUSTALW (Notredame et al., 2000) at the T-coffee... 108
Figure 2. Phylogenetic tree of deduced amino acid sequences of SLTI66, SLTI629 and other similar coldstress proteins. ClustalX (ver. 1.18) and Tree top programs (http://www.genebee.msu.su/service/phtree_reduced.ht... 109
Figure 3. SDS-PAGE analysis (A) and western blot analysis of His-SLTI66 and -SLTI629 produced in E. coli. M; molecular weight marker, V; soluble proteins of the cells containing pET100 vector, 66;... 111
Figure 4. Freezing-stress tolerance test of E. coli cells carrying pET100 vector, soybean SLTI66 or, SLTI629 gene. The E. coli cells producing His-SLTI66 (black), His-SLTI629 (diagonal) and with pET100 vector... 114
Figure 5. NaCl salt-stress tolerance test of E. coli cells with vector and cold-regulated protein genes (SLTI66, SLTI629). The E. coli cells with pET100 vector (A), with SLTI66 gene (B), and SLTI629 (C) were cultured... 115