Ishihara, Hirofumi: Analysis of two single trait loci affecting flavonol glycoside accumulations in Arabidopsis thaliana natural variations. 2007
Inhalt
- Table of Contents
- List of Abbreviations
- List of Figures
- List of Tables
- List of Appendices
- Abstract
- 1 Introduction
- 1.1 Flavonoids in A. thaliana
- 1.2 Arabidopsis thaliana natural variations as a tool for genome research
- 1.2.1 Molecular markers and Recombinant Inbred Line populations
- 1.2.2 Mapping of qualitative and quantitative traits loci
- 1.3 Identification of novel flavonoid derivatives based QTLs
- 1.4 Aims and Objectives
- 2 Materials and Methods
- 2.1 Enzymes and chemicals
- 2.2 Antibiotics
- 2.3 Bioinformatics tools and databases
- 2.4 Bacteria strains and DNA plasmid vectors
- 2.5 Standard molecular biology material and methods
- 2.5.1 Agarose gel electrophoresis
- 2.5.2 Primer design
- 2.5.3 Polymerase Chain Reaction
- 2.5.4 Purification of PCR product
- 2.5.5 DNA sequencing and assembling
- 2.6 Electro transformation of E. coli and A. tumefaciens
- 2.6.1 Preparation of electrocompetent E-coli strain XL1-Blue
- 2.6.2 Preparation of electrocompetent A. tumefaciens
- 2.6.3 Storage of bacterial cultures
- 2.7 Plant materials and methods
- 2.7.1 Plant materials
- 2.7.2 A. thaliana seed sterilization
- 2.7.3 Plant growth conditions in green house
- 2.7.4 Plant growth conditions in light room
- 2.7.5 A. tumefaciens mediated plant transformation
- 2.7.6 Histochemical analysis of β-glucuronidase (GUS) activity in plant tissues
- 2.7.7 Quick plant DNA extraction
- 2.7.8 Large scale plant DNA extraction
- 2.8 Metabolite analyses materials and methods
- 2.8.1 Metabolite analyses materials
- 2.8.2 Plant metabolite extraction
- 2.8.3 DPBA staining of the seedlings
- 2.8.4 High performance thin layer chromatography (HPTLC) analysis
- 2.8.5 High Performance Liquid Chromatography - photo diode array (HPLC PDA) and electro spray ionization/mass spectroscopy (ESI/MS) analyses
- 2.8.6 Gas chromatography - mass spectroscopy (GC-MS) analysis
- 2.9 QTL mapping and linkage analysis materials and methods
- 2.9.1 Mapping Materials
- 2.9.2 PCR based genotyping
- 2.9.3 Construction of the quercitrin standards curve
- 2.9.4 Normalization of quercitrin peaks for QTL analysis
- 2.9.5 QTL mapping using Windows QTL Cartographer
- 2.9.6 Calculating map positions of new markers (Linkage analysis)
- 2.10 Bacterial Artificial Chromosome (BAC) related materials and methods
- 2.10.1 BAC related materials
- 2.10.1.1 BAC screening filter
- 2.10.1.2 BIBAC custom sub-library
- 2.10.1.3 Ler BIBAC22K22 clone consensus sequence
- 2.10.1.4 Ler genome annotation
- 2.10.2 Screening of the Ler BiBAC library
- 2.10.3 Large scale low copy number plasmid extraction method
- 2.10.4 Large scale high copy number plasmid extraction method
- 2.11 RNA materials and methods
- 2.11.1 RNA materials
- 2.11.2 RNA Isolation for transcript profiling
- 2.11.3 cDNA synthesis
- 2.11.4 Semi quantitative reverse transcriptase –PCR (RT-PCR)
- 2.12 Transfection experiments materials and methods
- 2.12.1 Growth condition of A. thaliana-suspension culture At7
- 2.12.2 Protoplast isolation
- 2.12.3 Plasmid DNA extraction for transfection analysis
- 2.12.4 Transfection of A. thaliana-protoplasts
- 2.12.5 Protein extraction
- 2.12.6 Bradford assay
- 2.12.7 Measurement of luciferase activity
- 2.12.8 Measurement of β-glucuronidase activity
- 3 Results
- 3.1 Analysis of flavonoid derivatives in A. thaliana
- 3.1.1 Identification of recombinant inbred line (RIL) parental lines showing differences in flavonoid accumulation
- 3.1.2 Integration of HPTLC to HPLC chromatogram
- 3.1.3 Structure analysis of flavonol derivatives
- 3.1.4 Summary of metabolite analysis in A. thaliana seedlings
- 3.2 Identification of loci influencing quercetin 3-O-rhamnoside accumulation in Arabidopsis thaliana wildtype accessions
- 3.2.1 Phenotyping of the Lister and Dean Ler x Col RIL population
- 3.2.2 QTL mapping of the Qr4 trait
- 3.2.3 Identification of the candidate gene
- 3.2.4 TT10 sequence comparison
- 3.2.5 Association analysis of Qr4 accumulation with the TT10 alleles
- 3.2.6 In vivo TT10 promoter analysis by transfection experimets
- 3.2.7 Analysis of TT10 promoter activity in plant seedlings
- 3.2.8 Comparison of Qr4 accumulation between the seed coat and the seedling
- 3.2.9 Summary
- 3.3 Identification of qualitative loci influencing biosynthesis of flavonol glycosides
- 3.3.1 Flavonoid accumulation profile in distinct parts of A. thaliana seedlings
- 3.3.2 Scoring of the Km1 and Qr1 traits in the Ler x Col RIL population for a linkage analysis
- 3.3.3 Identification of the Km1/Qr1 locus in the A. thaliana genome by a linkage analysis
- 3.3.4 Fine mapping of the Km1/Qr1 locus
- 3.3.5 Identification of Ler BIBAC clones containing the target area
- 3.3.6 Integration of Ler BAC inserts to Col Chromosome 1 genomic sequence
- 3.3.7 Comparison of the targeted area between Col and Ler genomic sequences
- 3.3.8 Comparison of annotated sequences in Col and Ler
- 3.3.9 Summary of the Km1 and Qr1 locus
- 4 Discussion
- 4.1 Establishment of analytical method for identification of qualitative and quantitative differences in flavonoid accumulations in A. thaliana wildtype accessions.
- 4.2 Transparent Testa 10 involved in the quercitrin accumulation difference in A. thaliana wildtype accessions
- 4.3 The flavonoid derivatives accumulate in distinct parts of A. thaliana
- 4.4 Identification of candidate genes influencing qualitative traits
- 4.4.1 Complementation analysis of the Km1/Qr1 locus
- 4.4.2 Identification of the candidate gene involved in the Km1 and Qr1 trait
- 4.5 Outlook
- 4.6 Concluding remarks
- 4.7 Summary
- References
- Appendices
- List of Publication
- Acknowledgements