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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
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
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
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
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
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
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
II . Enhanced Tolerance Against Osmotic Stresses of Escherichia coli Cells Expressing Soybean KS-Type Dehydrin 100
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
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
산화적인 환경에서 생명체는 활성산소종(ROS)으로 알려진 산화분자의 탐색 및 회복을 위한 효과적인 진화를 해왔다. 지금까지 산화적 스트레스를 포함한 여러 가지 ROS가 알려졌다. 과산화수소등과 같은 여러 종류의 ROS가 알려졌으며, 이러한 ROS는 SOD, GPx 및 Prx와 같은 항산화 효소를 포함한 항산화 시스템을 유발한다.
녹두에 저온스트레스를 처리 하였을 때 특이적으로 발현되는 2-Cys peroxiredoxin과 상동성을 나타내는 cDNA 클론을 확보하였고, VrPrx1이라 명명하였다. VrPrx1은 261개의 아미노산을 포함한 786bp로 구성되어 있으며, 계통분석도에서 VrPrx1은 식물종의 2-Cys Prx와 그룹을 이루었다. Sourthern blot 분석을 통해 녹두 게놈 상에 VrPrx1 유전자가 1개의 copy로 존재하는 것을 확인하였다. E. coli을 이용하여 분리한 재조합 VrPrx1 단백질은 산화 스트레스를 유도하는 MFO 시스템 내에서 DNA변성을 막아주었고, Dithiothreitol의 존재 하에서 H₂O₂와 TBHP의 제거 능력을 보였다.
VrPrx1 단백질은 환원제인 DTT와 Trx에 의해서 그 구조가 dimer 이상, oligomer 복합체에서 monomer의 형태로 변하는 것을 확인하였다. Western blot 분석 실험 결과, VrPrx1은 과산화수소와 Wounding 스트레스를 받은 녹두 식물체에서 강한 발현을 보였고, 반대로 건조 및 고염분 스트레스 하에서는 발현의 감소를 보였다. 담배 잎을 이용한 VrPrx1의 세포내 기관별 분포 양상 실험결과에서는 엽록체로 타겟팅 되는 것을 확인하였고, 더욱이 VrPrx1의 N-말단 부분의 70여개의 아미노산 그룹이 엽록체로 타겟팅 하는데 있어서 중요한 역할을 하는 것으로 나타났다. 애기장대를 이용하여 VrPrx1 유전자를 과다 발현시킨 형질 전환체 식물을 제작하였다. VrPrx1 형질전환체 식물은 산화유도제로 알려진 MV (Methyl viologen) 스트레스 처리시 대조구 식물체보다 높은 저항성을 보였다. 또한 VrPrx1 형질 전환체 애기장대는 MV 외에 건조, 저온 및 고염분 스트레스 처리 후 엽록소 함량 및 광합성 효율도 높았다.
Yeast two hybrid 실험을 통하여 VrPrx1와 상호작용을 하는 45종류의 partial 유전자들을 녹두 cDNA library로부터 확보하였다. 이들 유전자들 대부분 엽록체 내 존재한다고 알려져 있다. 이 partial 유전자 중 ONPG를 이용한 assay 분석 후 상호작용정도가 높은 것으로 나타난 Glutamine synthetase, Ubiqutin conjugating E2, ATP synthase를 선발하여 RACE를 통한 full length cDNA를 확보하였다. 이들 세 유전자와 VrPrx1l와 일대일 상호작용 여부를 확인하기 위하여 각각의 재조합 단백질을 제작하였다.
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