Cloning and molecular characterization of vff1 gene encoding forisomes of Vicia faba [Elektronische Ressource] / vorgelegt von Maria Eugenia Fontanellaz

Maria Eugenia Fontanellaz

Sujets

• Biologie

Informations

• Publié par: rheinisch-westfalischen_technischen_hochschule_-rwth-_aachen
• Publié le: 01 janvier 2006
• Nombre de lectures: 435
• Langue: English
• Poids de l'ouvrage: 6 Mo

Extrait

Cloning and molecular characterization of vff1 gene encoding
Forisomes of Vicia faba





Von der Fakultät für Mathematik, Informatik and Naturwissenschaften der Rheinisch-
Westfälischen Technischen Hochschule Aachen zur Erlangung des akademischen
Grades einer Doktorin der Naturwissenschaften genehmigte Dissertation






vorgelegt von


M. Sc. Maria Eugenia Fontanellaz
aus Rosario, Argentinien










Berichter: Universitätsprofessor Dr. rer. nat. R. Fischer
Universitätsprofessor Dr. rer. nat. D. Prüfer

Tag der mündlichen Prüfung: 7. Dezember 2006

Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online
verfügbar.



Contents
___________________________________________________________________________

I INTRODUCTION
I.1 Phloem structure and function 1
I.1.1 Sieve element-companion cell complex 1
I.1.2 P-Proteins and regulation of phloem transport 2
I.1.3 Forisomes 5
2+ I.1.4 Ca -binding motifs and secondary protein structures 7
I.1.4.1 EF-hand 7
I.1.4.2 Coiled-coil domains 8
I.2 Promoter sequences and regulation of transcription 9
I.2.1 Phloem-specific promoters 9
I.3 Aim of this thesis 12

II MATERIALS AND METHODS 14
II.1 Materials 14
II.1.1 Chemicals and consumables 14
II.1.2 Enzymes and reaction kits 14
II.1.3 Buffers, media and solutions 15
II.1.4 Matrices and membranes 15
II.1.5 Primary antibodies, secondary antibodies and substrates 15
II.1.6 Biological material 15
II.1.6.1 Bacterial strains 15
II.1.6.2 Plants 16
II.1.6.3 Animals 16
II.1.7 Vectors 16
II.1.7.1 Bacterial expression vector 16
II.1.7.2 Plant expression vectors 17
II.1.8 Oligonucleotides 17
II.1.9 Equipment and applications 18
II.2 Methods 20
II.2.1 Plant material and isolation of Forisomes 20
II.2.2 Peptides sequencing 20
II.2.3 Isolation of genomic plant DNA 21
I Contents
___________________________________________________________________________
II.2.4 RNA isolation 21
II.2.5 Libraries construction and screening strategies 22
II.2.5.1 cDNA expression library 22
II.2.5.2 cDNA libraries 22
II.2.6 PCR technologies 24
II.2.6.1 Cloning of 5’ and 3’ ends of full-length cDNAs using
RACE-PCR 24
II.2.6.2 PCR amplification of cDNA 25
II.2.6.3 Identification of introns and genomic DNA cloning by
LD PCR 26
II.2.6.4 Genome Walking 26
II.2.7 Recombinant DNA technologies 27
II.2.7.1 Isolation of plasmid DNA from E.coli 27
II.2.7.2 Quantification of DNA 27
II.2.7.3 PCR amplification 27
II.2.7.4 Agarose gel electrophoresis of DNA 28
II.2.7.5 Preparative agarose gel electrophoresis 28
II.2.7.6 DNA sequence analysis 29
II.2.8 Preparation and transformation of E. coli 29
II.2.8.1 Preparation of electro-competent E. coli 29
II.2.8.2 Transformation of E. coli by electroporation 29
II.2.8.3 Culturing of E. coli and glycerol stock preparation 29
II.2.9 Preparation and transformation of Agrobacterium tumefaciens 30
II.2.9.1 Preparation of electro-competent Agrobacterium cells 30
II.2.9.2 Transformation of Agrobacterium by electroporation 30
II.2.9.3 Determination of the efficiency of recombinant bacteria
transformation 31
II.2.9.4 Growth of recombinant A. tumefaciens and preparation
of glycerol stocks 31
II.2.10 Generation and characterisation of transgenic plants 31
II.2.10.1 Transient transformation of tobacco leaves 31
II.2.10.2 Preparation of recombinant Agrobacteria 31
II.2.10.3 Vacuum infiltration of tobacco leaves 32
II.2.10.4 Stable transformation of tobacco plants 32
II Contents
___________________________________________________________________________
II.2.10.5 Growth of N. tabacum cv. Petite Havana SR1 33
II.2.10.6 Measurement of GUS activity and histochemical analysis 33
II.2.11 Expression and purification of recombinant proteins 34
II.2.11.1 Expression and purification of Forisome-GST fusion
proteins from E. coli 34
II.2.11.2 Fermentation at 4-liter scale 35
II.2.12 Protein analysis 36
II.2.12.1 Quantification of proteins 36
II.2.12.2 SDS-PAGE and Coomassie brillant blue staining 36
II.2.12.3 2D-PAGE analysis 37
II.2.12.4 Immunoblot analysis 38
II.2.13 Polyclonal antibodies production 38
II.2.13.1 Mouse immunization 38
II.2.13.2 Chicken immunization 39
II.2.13.3 Rabbit antisera 39
II.2.14 Determination of antisera titers 39
II.2.15 Confocal immunofluorescence microscopy 41

III RESULTS 41
III.1 Molecular characterization of Forisome genes 41
III.1.1 Analysis of isolated Vicia faba Forisomes by SDS-PAA
gel electrophoresis 41
III.1.2 Generation and characterization of Forisome-specific
polyclonal antibodies 41
III.1.2.1 Immunoblot analysis of isolated Vicia faba Forisomes 41
III.1.2.2 Immunoblot analysis of isolated Vicia faba Forisomes
on 2D-gel electrophoresis 42
III.1.3 Screening of cDNA Libraries 43
III.1.4 Immunoscreening of cDNA expression library 45
III.1.5 PCR screening of cDNA expression library 46
III.1.6 Cloning full-length Forisome cDNA 46
III.1.7 Isolation of total RNA from Vicia faba 51
III.1.8 Identification of the transcription start sites for vff1 52
III.1.9 Molecular characterisation of the vff1 genomic clone 53
III Contents
___________________________________________________________________________
III.2 Molecular cloning and characterization of the vff1 promoter 54
III.2.1 Construction of genome walking libraries 54
III.2.2 Identification and cloning of vff1 gene 5’-flanking region 54
III.2.3 Potential regulatory sequences in the vff1 promoter 56
III.2.4 Characterization of vff1 promoter in transgenic tobacco 58
III.2.4.1 Cloning of vff1 promoter into the pTRAk-GUS vector 58
III.2.4.2 Expression of vff1 Promoter-GUS fusions in tobacco
plants 60
III.2.4.2.1 Tissue specificity of vff1 promoter in transgenic
plants 60
III.2.4.2.2 Developmental regulation of vff1 promoter in
transgenic plants 63
III.2.4.3 Deletion analysis of vff1 promoter in transiently
transformed tobacco leaves 65
III.3 Bacterial expression and characterization of VFF1 66
III.3.1 Cloning of vff1 into the bacterial expression vector
pGEX-5X-3 66
III.3.2 Bacterial expression and purification of VFF1 66
III.3.3 Characterization of GST-VFF1 fusion protein by
immunoreativity toward Forisome-specific mouse and
chicken antisera 68
III.4 Immunological characterization of native and recombinant
Forisomes 70
III.4.1 Reactivity of polyclonal anti-GST-VFF1 antibodies to isolated
Forisomes 70
III.4.2 Characterization of native Forisomes by immunofluorescence 71
III.4.2.1 Reactivity of Forisome-specific polyclonal antibodies to
native Forisomes 71
III.4.2.2 Reactivity of VFF1-specific polyclonal antibodies to native
Forisomes 73

IV DISCUSSION 75
IV.1 Molecular characterization of Forisome genes 75
IV.1.1 Analysis of Vicia faba Forisome subunits 75
IV Contents
___________________________________________________________________________
IV.1.2 Cloning and characterization of vff1 gene 76
IV.1.2.1 Screening of cDNA libraries 76
IV.1.2.2 Database search and cloning of vff1 gene 78
IV.1.2.3 Sequence analysis of vff1 gene 78
IV.1.2.4 Motifs identified within the vff1 gene 79
IV.1.3 Immunological evidence of vff1 gene encoding a Forisome
protein 82
IV.1.3.1 Expression and purification of recombinant VFF1
protein 82
IV.1.3.2 Immunoreactivity of Vicia faba Forisome-specific
polyclonal antibodies to recombinant VFF1 proteins
and native Vicia faba Forisomes 83
IV.1.3.3 Immunoreactivity of VFF1-specific polyclonal antibodies
to recombinant VFF1 proteins and native Vicia faba
Forisomes 84
IV.2 Molecular characterizat