Isolation and Molecular Genetic Analysis of Brassinosteroid Signaling Mutants in Arabidopsis

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기리 라쥐 트라파씨
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활성 표지 애기장대|브라시노스테로이드|돌연변이체
Plant development is one of the most important aspects of plant’s life cycle that has extensively been studied at the morphological, genetic and molecular level. The generation of genetic variants, like mutants may increase genetic pool and gives the information about plant processes and their genetic control. Activation tagging is a new powerful tool to generate and identify new mutants, which emerged as an alternative for gene function analysis. This thesis reports the study on brassinosteroid hormone signaling mutants that controls the plant growth and development using mutants generated by an activation tagging-based approach in the model plant Arabidopsis thaliana.
Brassinosteroids (BRs) are polyhydroxylated steroid plant hormones that play essential role in growth and development. The mutant defective in BR signaling causes dwarfism, male sterility, abnormal vascular development and photomorphogenesis. Unlike animal steroid hormones, BRs are perceived at the plasma membrane by direct binding to the extracellular domain of the BRI1 receptor S/T kinase. BR pereception initiates a signaling cascade, acting through a GS3 kinase, BIN2, and the BSU1 phosphatase, which in turn modulates the phosphorylation state and stability of the nuclear transcription factors BES1 and BZR1.
An activation tagging library harboring 10,000 (F1) independent Arabidopsis transformants were generated and a pool system was established in order to screen brassinosteroid hormone signaling defective mutants. At the time of activation tagging library establishment a total of 150 mutants that are defective in normal phenotype were also screened in F1 generation. Brassinasteroid related mutants were screened using brassinazole a specific brassinosteroid biosynthesis inhibitor. In dark, brassinazole treated Arabidopsis develop as light grown plants and express light regulated genes, as do BR-deficient mutants. A total of 46 putative to brassinazole insensitive mutant were isolated that showed longer hypocotyls among the germinated seeds on brz at dark. All the mutants were identified T-DNA insertion by using TAIL-PCR, Adapter Ligation PCR Walking, plasmid rescue and IPCR. In this screening atbrzi1 and abz126 brassinszole insensitive mutants were taken for the study.
atbrzi1 has T-DNA insertion into the promoter region of gene At1g12380. In the brassinazole, hypocotyl length atbrzi1 is nearly doubled than wild-type. ATBRZI1 gene is located in the acrocentric region of the chromosome 1 and its homology gene At1g62870 is located the opposite acrocentric region of the same chromosome. Both genes have characteristic of Zinc finger BED-type motif located at the N-terminal region. In this study we predicted that the HLH (helix-loop-helix) type structure of BED-type zinc finger motif may bind the dephosphorylated BES1 and then involve in brassinosteroid hormone signaling in the respective way. Yet, research on role of zinc finger is not reported. However, our further ongoing experimental results may show the involvement of Znf BED-type motif in brassinosteroid hormone signaling by binding dephosphorylated BZR1 in the nucleus.
abz126 is a semi-insensitive to the brassinazole and a T-DNA has been inserted into the ninth exon of the GIGANTEA (GI) gene that is involved in control of flowering time. Loss of function in GI gene makes extremely delaying in flowering in Arabidopsis. Results of screen on brz, RT-PCR, different hormone dose response assay presumed that GI gene is involved in brassinosteroid hormone signaling and regulate the flowering time.
Alternative Title
활성 표지 애기장대에서 브라시노스테로이드 신호 돌연변이체의 분자유전학적 연구
Alternative Author(s)
Giri Raj Tripathi
조선대학교 대학원
일반대학원 유전자과학과
Awarded Date
Table Of Contents
Chapter I: ATBRZI1 mediates brassinosteroid regulated gene expression in A. thaliana = 1
Abstract = 2
Introduction = 4
A1.1 Activation tagging = 4
A1.2 Brassinosteroid = 6
A1.3 Zinc Finger = 9
Materials and Methods = 19
B1.1. Plant Material and Growth Condition = 19
B1.2. Mutant Screening = 20
B1.3. Extraction of plant genomic DNA = 21
B1.4. Determination of T-DNA insertion Site = 21
B1.4.1. Thermal Asymetric Interlaced (TAIL)-PCR = 21
B1.4.2. Plasmid rescue and Inverse PCR (IPCR) = 23
B1.4.3. Genotyping PCR = 27
B1.5. Seed germination Experiment = 27
B1.6. RNA isolation and cDNA Synthesis = 27
B1.7. RT-PCR Analysis = 28
B1.8. E. coli Preparations = 29
B1.8.1. Plasmid Preparation = 29
B1.8.2. Preparation of E. coli Competent Cells = 29
B1.9. Map-based Construct = 29
B1.9.1. cDNA Construct = 30
B1.9.2. gDNA Construct = 30
B1.9.3. ATBRZI1::GUS Construst = 31
B1.9.4. RNAi Construct = 31
B1.9.5. GFP fusion Construct = 31
B1.9.6. Protein expression Construct = 32
B1.9.7. Yeast-two hybrid Construst = 32
B1.9.8. Transformation of E. coli and Agrobacterium = 32
B1.10. Arabidopsis Transformation = 34
B1.11. Computer assisted Analysis and Presentation = 34
Results = 36
C1.1. Screening of activation tagging Arabidopsis = 36
C1.2. Isolation of atbrzi1 Mutant = 41
C1.3. Genotyping PCR and T-DNA Verification = 42
C1.4. Structure of ATBRZI Gene = 46
C1.4.1. Computer based analysis of Znf-BED-type motif = 46
C1.5. Constructs = 51
C1.6. Phenotypes of atbrzi1 Mutant = 58
Discussion = 62
D1.1 Mutant Screening = 62
D1.2 Possible involvement of ATBRZI1 in Brassinosteroid Signaling = 66
D1.3 Aplication and limitations of the Study = 74
References = 75
Chapter II:GIGANTEA is involved in brassinosteroid signaling in Arabidopsis = 86
Abstract = 87
Introduction = 88
Materials and Methods = 90
B2.1. Plant Material and Growth Condition = 90
B2.2. Isolation of abz126 Mutant = 91
B2.3. Hormone Assay = 91
B2.4. Determination of T-DNA Insertion Site = 91
B2.5. RNA Isolation and cDNA Synthesis = 92
B2.6. RT-PCR Expression Analysis = 93
Results = 94
C2.1. Screening of abz-126 Mutant in T-DNA Tagging Lines = 94
C2.2. Identification of T-DNA insertion Site = 96
C2.3. Expression of GI and Neighboring Genes = 96
C2.4. GI Gene Effects on Plant Size and Flowering Time = 98
C2.5. Response of Plant Hormones on Exogenous Application = 100
Discussion = 102
References = 105
Appendix I = 108
Appendix II = 116
Acknowledgments = 118
Dedication = 120
조선대학교 대학원
기리 라쥐 트라파씨. (2008). Isolation and Molecular Genetic Analysis of Brassinosteroid Signaling Mutants in Arabidopsis.
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General Graduate School > 4. Theses(Ph.D)
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