Analysis of DNA and RNA by agarose gel electrophoresis

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Table of contents

1 Phenylpropanoids
1.1 Biosynthetic pathway of phenylpropanoids
1.2 Coumarins and furanocoumarins
1.2.1 Coumarins
1.2.2 Biosynthetic pathway of coumarins
1.3 Furanocoumarins
1.3.1 Distribution of furanocoumarins in the plant Kingdom
1.3.2 Role of furanocoumarins
1.3.3 Localization of furanocoumarins in plants
1.3.4 Storage of furanocoumarins in plant cell
1.3.5 Biosynthetic pathway of furanocoumarins
1.3.6 Biological properties of furanocoumarins
1.3.6.1 Photolysis and photo-oxydation of furanocoumarins
1.3.6.2 Photocylcloaddition and photo-dimerization
1.3.6.3 Reaction of furanocoumarins with nucleic acid
1.3.6.4 Reaction of furanocoumarins with lipids
1.3.6.5 Reaction of furanocoumarins with proteins
1.3.7 Furanocoumarins in the pharmaceutical industry
1.3.7.1 Photochemotherapy (PUVA)
1.3.7.2 Other implications of furanocoumarins as pharmaceutics
1.3.8 Toxicity caused by furanocoumarins
1.3.8.1 Phytophotodermatitis
1.3.8.2 Melanoma
1.3.8.3 Interaction of furanocoumarins with drugs
2 Prenyltransferases
2.1 Role of prenyltransferases in plants
2.1.1 Implication of prenyltransferases in the biosynthesis of primary metabolites
2.1.1.1 Biosynthesis of isoprenoids
2.1.1.1.1 Isopentenyl diphosphate isomerase (IPPI)
2.1.1.1.1.1 Reaction mechanism of GPP synthase
2.1.1.1.2 Subcellular Localization of the biosynthetic pathway of isoprenoids
2.1.1.1.3 Diversity of molecules derived from the isoprenoid biosynthetic pathway
2.1.1.2 Biosynthesis of isoprenoid quinones
2.1.1.2.1 DHNA phytyltransferases
2.1.1.2.2 HGA Prenyltransferases
2.1.1.2.3 p-hydroxybenzoic acid prenyltransferase
2.1.1.3 Prenyltransferases involved in the biosynthesis of other plant essential products
2.1.1.3.1 Cytokinins (Zeatin)
2.1.1.3.2 Biosynthesis of photosynthetic pigments
2.1.1.4 Post translational modification of proteins
2.1.2 Prenyltransferases in biosynthesis of secondary metabolites
2.1.2.1 General introduction
2.1.2.2 Biosynthesis of plant prenylated polyphenols
2.1.2.2.1 Polyphenols
2.1.2.2.2 Biological activities of prenylated polyphenols
2.1.2.2.3 Biochemical studies of polyphenol prenyltransferases of plants
2.1.2.2.3.1 Biochemical studies for soluble-type aromatic prenyltransferases
2.1.2.2.3.2 Biochemical studies for membrane bound aromatic prenyltransferases.
2.1.2.2.3.3 Isolation of aromatic prenyltransferases of plant polyphenols
2.1.2.2.3.3.1 Shikonin biosynthesis
2.1.2.2.3.3.2 Flavonoid and isoflavonoid biosynthesis
2.1.2.2.3.4 Structure activity relationship of Prenyltransferases
2.1.2.2.3.4.1 Three dimensional modeling of aromatic prenyltransferase
2.1.2.2.3.4.2 Reaction mechanism of aromatic prenyltransferases of plant polyphenols
2.1.2.2.3.5 Evolution of aromatic prenyltransferases of plants
2.1.2.2.3.6 Heterologous expression systems for the characterization of plants membrane-bound aromatic prenyltransferases
3 Objective of the project Material and Methods
1 Material
1.1 Plant material
1.1.1 P. crispum
1.1.2 N. benthamiana
1.1.3 R. graveolens
1.2 Bacterial strains
1.2.1 E. coli Top 10
1.2.2 E. coli GeneHogs® (Invitrogen)
1.2.3 E. coli M15
1.2.4 E. coli BL21 (DE3) (Novagen)
1.2.5 E. coli ccdb survival
1.2.6 E. coli HB101 (pRK2013)
1.2.7 E. coli MC1061
1.3 Agrobacterial strains
1.3.1 A. tumefaciens C58C1RifR (pGV2260)
1.3.2 A. tumefaciens LBA4404 (pAL4404)
1.4 Yeast strains
1.4.1 Saccharomyces cerevisiae WAT11 and WAT21
1.5 Recombinant plasmids and vectors
1.5.1 pYeDP60
1.5.2 pQE30 (Qiagen)
1.5.3 pGEX-KG (Amersham Biosciences)
1.5.4 pCR®8/GW/TOPO® (Invitrogen)
1.5.5 Binary vector systems
1.5.5.1 pBin-GW
1.5.5.2 pGWB2
1.6 Culture media
1.6.1 For bacteria
1.6.1.1 Liquid LB
1.6.1.2 Solid LB
1.6.1.3 YEB
1.6.2 Yeast medium
1.6.2.1 YPGA
1.6.2.2 SGI
1.6.2.3 YPGE
1.6.2.4 YPL
1.6.3 Culture media and conditions used for plants culture
1.6.3.1 Murashige and Skoog (MandS)
1.6.3.2 In vitro culture of R. graveolens
1.6.4 Growth of plants in soil
1.7 Antibiotics
2 Molecular biology
2.1 DNA Extraction from plant
2.2 RNAs Extraction from plant material
2.3 Amplification of DNA fragments by a Polymerase chain reaction (PCR) approach
2.3.1 Classic Polymerase chain reaction
2.3.2 High Fidelity PCR
2.3.3 Reverse transcription
2.3.4 Verification of the stably transformed plants using the Phire plant direct PCR k
2.3.4.1 Preparation of samples
2.3.4.2 Polymerase chain reaction for Phire plant direct PCR kit
2.4 Analysis of DNA and RNA by agarose gel electrophoresis
2.5 Extraction of DNA from agarose gel
2.6 Digestion of DNA fragment by restriction enzymes
2.7 Ligation
2.7.1 pCR8®/GW/ TOPO®
2.7.2 Ligation in other vectors
2.8 Construction of recombinant binary plasmid for genetic transformation of plant cells using the Gateway® Technology
2.8.1 Recombination reaction via Gateway® technology
2.9 Preparation of competent E. coli bacteria
2.9.1 Preparation of electro-competent bacteria
2.9.2 Preparation of chemically competent bacteria
2.10 Transformation of competent E. coli bacteria
2.10.1 Electroporation
2.10.2 Heat shock
2.11 Transformation of A. tumefaciens
2.11.1 Heat shock
2.11.1.1 Preparation of chemically-competent Agrobacteria
2.11.1.2 Heat shock transformation of Agrobacteria
2.11.1.3 Transformation of agrobacteria using the triparental mating method
2.11.1.3.1 Technical approach
2.12 Extraction of plasmidic DNA
2.13 Sequencing
2.14 Induction of the expression of genes by UV
3 Heterologous expression system
3.1 Prokaryote expression system
3.1.1 Expression
3.1.2 Purification of protein
3.2 Eukaryotes expression system
3.2.1 Yeast Expression system
3.2.1.1 Preparation of competent yeast
3.2.1.2 Transformation of yeast
3.2.1.3 Protein expression in yeast
3.2.1.3.1 Conditions of culture
3.2.1.3.2 Preparation of yeast culture
3.2.1.3.3 Microsomes preparation
3.3 Heterologous Expression in plants
3.3.1 Transient expression in N. benthamiana
3.3.2 Inoculum Preparation
3.3.3 Agro-infiltration of leaves
3.4 Stable transformation of R. graveolens
3.4.1 Germination of R. graveolens seeds
3.4.2 Preparation of bacterial inoculum
3.4.3 Transformation of hypocotyls of R. graveolens
4 Methods of biochemical analysis
4.1 Quantification of proteins
4.2 Quantification of P450 by CO spectrum
4.3 Measurement of enzymatic parameters
4.3.1 For Cytochrome P450
4.3.1.1 Determination of kinetic constants
4.3.2 For prenyltransferases
4.3.2.1 Determination of enzyme kinetics
4.3.2.2 Measurement of the inhibition of the Pt activity by furanocoumarins and determination of the inhibition constant
4.4 HPLC Analysis
4.4.1 Metabolisation of substrate
4.4.2 Analysis of extracts of phenylpropanoids
4.5 Analysis by mass spectrometry (MS)
4.5.1 Preparation of samples for analysis for quantification
4.5.1.1 Detection and quantification of hydroxylated products
4.5.1.2 Detection and quantification of prenylated products
4.6 Synthesis of substrats
4.6.1 Synthesis of CoA esters
4.6.1.1 Production of CoA esters
4.6.1.1.1 Kinetics of chemical reactions
4.6.1.2 Synthesis of shikimic and quinic acid esters
4.6.1.2.1 Synthesis of shikimic acid and quinic acid derivatives
5 Methods of analytical analysis
5.1 Preparation of phenylpropanoids extracts
5.2 Quantification of expression of gene by real time PCR
5.2.1 Preparation of material
5.2.2 Preparation of the reaction mix
6 Statistical analysis of data
Results and discussion
Chapter I: New putative aromatic prenyltransferases
1 Identification of candidate genes encoding for enzymes belonging to the aromatic prenyltransferases family
1.1 Identification and isolation of candidate genes
1.1.1 In silico data mining
1.1.1.1 A. gigas homogentisic acid phytyltransferases
1.1.1.2 Identification of a gene encoding for a putative Pt of P. sativa
1.1.2 PCR approach using degenerated primers
1.1 In silico analysis of the putative prenyltransferase sequences
1.1.1 Consensus sequences
1.1.2 Protein typology
1.1.3 Subcellular localization
1.1.4 A new group of prenyltransferases on the basis of phylogenetic analysis
1.2 Conclusion
Chapter II: Development of a heterologous transient expression system for membranous proteins using Nicotiana benthamiana
2 Development of heterologous expression system of N. benthamiana
2.1 Introduction
2.2 Validation of a transient expression system in N. benthamiana using the Green Fluorescent Protein (GFP)
2.2.1 Recombinant plasmid: pBIN-m-gfp5-ER
2.2.2 Preparation of Agrobacterium inoculum and inoculation of N. benthamiana leaves….
2.2.3 Determination of the best concentration of agrobacteria to be used for infiltrating N. benthamiana leaves
2.2.4 Improvement of the expression of GFP
2.2.4.1 Co-expression of GFP with p19
2.3 Development of a N. benthamiana -based transient expression system for the expression and the functional characterization of a membranous enzyme (CYP
2.4 Development of a N. benthamiana heterologous expression system in order to do the functional characterization of aromatic prenyltransferases
2.4.1 Presence and stability of potential substrates for Prenyltransferases involved in the biosynthesis of furanocoumarins
2.4.2 Validation of the N. benthamiana transient expression system for the expression of SfN8DT-1, a flavonoid prenyltransferase isolated from S. flavesc
2.4.2.1 Recombinant plasmid: pGWB5-SfN8DT-1
2.4.2.2 Preparation of inoculum and infiltration of plants
2.4.2.3 Development of N. benthamiana transient expression system for in vitro functional characterization
2.4.2.3.1 Preparation of plant microsomes and test for the enzyme activity
2.4.2.4 N. benthamiana transient expression system for in vivo characterization of SfN8DT-1
2.4.3 Discussion and conclusions
Chapter III: Functional characterization of the first aromatic prenyltransferase which catalyzes the prenylation of umbelliferone to produce demethylsube
3 Functional characterization of the umbelliferone prenyltransferase
3.1 Introduction
3.2 Cloning of the PcPt coding sequence into the pBIN-GW plasmid
3.3 Transient expression of PcPT in N. benthamiana
3.3.1 In vivo characterization of PcPt
3.3.2 Biochemical (in vitro) characterization of PcPt
3.3.2.1 Enzymatic characterization
3.3.2.1.1 Screening for other potential substrates
3.3.2.1.2 Biochemical and kinetic characterization of PcPt
3.3.2.1.2.1 Optimal incubation time
3.3.2.1.2.2 Optimal pH
3.3.2.1.2.3 Saturated concentration of DMAPP
3.3.2.1.2.4 Measurement of the kinetic constants of PcPt
3.3.2.1.2.5 Measurement of uncompetitive inhibition
3.4 Stable expression of PcPt
3.4.1 Construction of transgenic R. graveolens plants using PcPt as a transgene
3.4.2 Molecular characterization of transgenic plants
3.4.3 Relative Expression level of PcPt assessed by real time PCR
3.4.4 Quantification of coumarins and furanocoumarins
3.4.4.1 Is there a relationship between a tissue specific expression pattern of PcPt and the coumarin/furanocoumarin content in Parsley (P. crispum)?
3.4.4.1.1 Analysis of the coumarin and furanocoumarin composition in P. crispum plants
3.4.4.1.2 Quantification of the PcPt expression within various organs of P. crispum
3.4.5 Metabolic engineering of the biosynthetic pathway of furanocoumarin in N. benthamiana
3.4.5.1 Co-infiltration of tobacco leaves with Agro-pBIN-p19, Agro-pBIN-C2’H and Agro-pBIN-PcPt
3.4.5.2 Analyses of samples by mass spectrometry
3.4.6 Conclusion and Discussion
Chapter IV: Functional characterization of other putative aromatic prenyltransferases
4 Functional characterization of other putative aromatic prenyltransferases
4.1 Cloning of the six aromatic prenyltransferase coding sequence into a plant expression vector (either pBIN-GW or pGWB2)
4.2 Functional characterization of PsPt
4.2.1 General presentation
4.2.2 Umb, a substrate for PsPt ?
4.3 Functional characterization of CliPt
4.3.1 Transient expression of prenyltransferase of CliPt in N. benthamiana
4.3.1.1 In vivo characterization of CliPt
4.3.1.2 In vitro characterization of CliPt
4.3.2 Construction of transgenic R. graveolens plants overexpressing CliPt
4.3.2.1 Molecular characterization of CliPt transgenic plants
4.3.2.2 Overexpression of CliPt: detection through RT-qPCR
4.3.2.3 Metabolic profiles of transformed R. graveolens plants: Quantification of geranylated Umb and geranylated esculetin
4.3.3 Attempt for functional characterization for other aromatic prenyltransferases
4.3.3.1 Transient expression system of N. benthamiana
4.3.3.2 Stable expression in R. graveolens
4.4 Discussion and conclusions
5 General conclusion
6 Perspectives
7 Résumé en français pour validation par le conseil scientifique
7.1 Chapitre 1 : Identification et clonage de nouveaux gènes codants pour des Prenyltransferases
7.2 Chapitre 2 : Mise au point d’un système d’expression adapté pour l’expression de protéines membranaires
7.3 Chapitre 3: Caractérisation fonctionnelle de nouvelles prenyltransferases impliquées dans la synthèse de furocoumarines
Bibliography