Studies to influence the carbon metabolism of the C_1tn3 plant Nicotiana tabacum by expressing heterologous enzymes involved in C_1tn4-like CO_1tn2 fixation [Elektronische Ressource] / von Jun Li

Yang Wang

Sujets

• Biologie

Informations

• Publié par: rheinisch-westfalischen_technischen_hochschule_-rwth-_aachen
• Publié le: 01 janvier 2003
• Nombre de lectures: 2
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Studies to influence the carbon metabolism of the C plant 3
Nicotiana tabacum by expressing heterologous enzymes
involved in C -like CO fixation 4 2







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






Vorgelegt von

Master of Science
Jun Li

aus
Liaoning, V. R. China





Berichter: Universitä tsprofessor Dr. rer. nat. F. Kreuzaler
Universitätsprofessorin Dr. rer. nat. M. Frentzen


Tag der mündlichen Prüfung: 5. September 2003


Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfüg bar.













Dedicated to my dear parents, husband and son

























CONTENTS

1 1 Introduction

1.1 Photosynthesis and photorespiration in green plants 1
1.2 CO -concentrating mechanisms in plants 3 2
1.3 Approaches to diminishing photorespiration in C plants 5 3
1.4 Characteristics of C -pathway enzymes and their introduction into C plants 8 4 3
1.5 The task of the present study 18

21 2 Materials

2.1 Chemicals and enzymes 21
2.2 Bacterial strains 21
2.3 Plasmids 22
2.4 Plant material 26
2.5 Synthetic oligonucleotides 27
2.6 Antisera 29
2.7 Instruments and consumables 30

32 3 Methods

3.1 Molecular biological methods 32
3.1.1 Isolation of plasmid DNA from E. coli 32
3.1.2 Isolation of plant genomic DNA 33
3.1.3 Agarose gel electrophoresis 33
3.1.4 Isolation of DNA fragments from agarose gels 34
3.1.5 Polymerase chain reaction (PCR) 34
3.1.6 Sequencing of plasmid DNA 35
3.1.7 RNA analysis with RT-PCR and Northern hybridization 36
3.1.7.1 Isolation of total RNA from plant leaves 36
3.1.7.2 Reverse transcriptase-polymerase chain reaction (RT-PCR) 37
3.1.7.3 Northern blot analysis 38

3.2 Microbiological methods 39
3.2.1 Culture of bacteria 39
3.2.2 Transformation of E. coli 40
3.2.3 Complementation analysis 41
I

3.3 Culture and transformation of Nicotiana tabacum 43
3.3.1 Sterile culture 43
3.3.2 Sterilization of plant materials 43
3.3.3 Soil culture 44
3.3.4 Stable transformation of Nicotiana tabacum 44

3.4 Biochemical methods 47
3.4.1 Extraction of proteins from bacterial cells 47
3.4.2 Extraction of proteins from plants leaves 48
3.4.2.1 Preparation of crude leaf extracts 48
3.4.2.2 Preparation of chloroplast extracts 49
3.4.2.3 Purification of protein extracts 50
3.4.3 Determination of protein concentrations in leaf extracts 51
3.4.4 SDS-polyacrylamide gel electrophoresis (SDS-PAGE) 52
3.4.5 Western blot analysis 54
3.4.6 Determinations of enzyme activities (PC, ME, DK, PS and CK) 56
3.4.7 Analyses of metabolites 62
3.4.7.1 Determination of L-malate contents 62
3.4.7.2 Determination of UV protectants (flavonoids) contents 63
3.4.8 Determination of photosynthesis parameters 63
3.4.9 Analysis of plant growth 64
3.4.10 Statistical evaluation of experimental data 65

66 4 Results

4.1 Construction of a plant expression vector carrying the maize PC encoding
gene 66
4.1.1 Construction of a prokaryotic expression vector carrying ppc gene from Zea
mays 67
4.1.2 Phenotypic complementation test of the prokaryotic expression vector carrying
full-length coding region of maize ppc gene 70
4.1.3 Determination of PC activities in transgenic bacteria 72



II
4.2 Stable transformations of Nicotiana tabacum with “C -cycle” genes and 4
screening for transgenic plants 73
4.2.1 Transformation of tobacco with pVL amplicon expression constructs by means
of traditional leaf disc method and screening for transgenic plants by Western
blot analyses 74
4.2.2 Transformation of tobacco with pS expression constructs by means of optimized
transformation methods and screening for transgenic plants 76
4.2.2.1 Optimization of transformation methods during the transformation of tobacco
with pS expression constructs 77
4.2.2.2 Screening of regenerants for foreign proteins by Western blot analyses 80
4.2.3 Re-transformation of transgenic tobacco plants with a triple-“C -cycle” -genes 4
carrying vector and screening for transformants 86

4.3 Detections of foreign genes and determinations of “C -cycle” enzyme 4
activities and PT mRNA levels in transgenic tobacco plants 89
4.3.1 Detection of foreign genes in genomes of transgenic tobacco plants by PCR
analyses 90
4.3.2 Determinations of “C -cycle” enzymes activities and evaluations of ppt 4
transcription levels in transgenic tobacco plants 93
4.3.2.1 PC activity in cppc transformants and their progenies 94
4.3.2.2 ME activity in both crude leaf extracts and chloroplast extracts of the progenies
of Me2 transformants 96
4.3.2.3 PS activity in crude leaf extracts and in purified chloroplast extracts of ppsA
transformants and their progenies 97
4.3.2.4 CK activity in crude leaf extracts and chloroplast extracts of the progenies of
pckA transformants 101
4.3.2.5 PC activity in the progenies of Stppc or Stppc transformants 102 S9D-C4 -C4
4.3.2.6 DK activity in the progenies of pdk transformants 103
4.3.2.7 Evaluation of the ppt transcription in the progenies of transgenic tobacco plants 105
4.3.2.7.1 Isolation of leaf total RNA from ppt transgenic plants 105
4.3.2.7.2 Qualitative analysis of mRNA transcript of ppt gene by RT-PCR analysis 106
4.3.2.7.3 Quantitative assessment of mRNA transcript of the ppt gene by Northern blot 107
analysis


III
4.4 Establishment of C -like CO assimilation pathway(s) in tobacco by crossing 109 4 2
individual “C -cycle” gene overexpressors 4
4.4.1 Crosses between single PC and ME overexpressors 110 C
4.4.2 Crosses between single PC and CK overexpressors 112 C
4.4.3 Crosses between double PC /ME or PC /CK overexpressors and single PC , C C SD
PC , PC , DK or PS overexpressors 114 SF E
4.4.4 Crosses between different triple “C -cycle” enzymes overexpressors 116 4
4.4.5 Crosses between multiple “C -cycle” enzymes overexpressors and PT 4
overexpressors 118
4.4.6 Methods for efficient screening for desired hybrids of “C -cycle” genes 4
overexpressors 120
4.4.6.1 Simultaneous detection of multiple “C -cycle” genes in a single PCR reaction 121 4
4.4.6.2 Simultaneous detection of PC and PC or PC proteins on a single Western C SD SF
blot 123
4.4.6.3 Discrimination between enzyme activities of PC and PC on the basis of C SD
their differential responses to acetyl-CoA and L-aspartate 125

4.5 Effects of elevated activities of “C -cycle” enzymes on the biochemical and 4
physiological characteristics of transgenic tobacco plants 126
4.5.1 Effects of increased activities of “C -cycle” enzymes on the activities of 4
endogenous cytosolic and chloroplastic enzymes 127
4.5.2 Effects of increased PC activities on the leaf content of malate 130
4.5.3 Effects of increased PC activities on the leaf content of UV protectants
(flavonoids) 132
4.5.4 Effects of elevated activities of “C -cycle” enzymes on apparent CO 4 2
assimilation rate (A) and/or electron requirement for apparent CO assimilation 2
rate (e/A ratio) 133
4.5.4.1 Oxygen- and temperature-dependent inhibition of apparent CO assimilation 2
rate (A) as well as enhancement of electron requirement for apparent CO 2
assimilation (e/A ratio) in single and double transgenic tobacco plants 134
4.5.4.2 Apparent CO compensation point (G) and electron requirement for apparent 2
137 CO assimilation (e/A ratio) in multiple transgenic tobacco plants 2
4.5.4.3 Electron requirement for apparent CO assimilation (e/A ratio) in transgenic 2
plants with various C -like CO assimilation pathways 139 4 2

IV

4.6 Growth and formation of reproductive organs of transgenic tobacco plants 141
4.6.1 Growth of single PC or PC and double PC /ME overexpressors 141 C SD C
4.6.2 Growth of transgenic tobacco plants overexpressing “C -cycle” genes in 4
different combinations 144
4.6.3 Growth of the progenies of transgenic tobacco plants from various crosses 146
4.6.4 Growth of progenies of transgenic tobacco plants under limited CO 2
concentrations 156

158 5 Discussion

5.1 Establishment of C -like CO assimilation pathway(s) in Nicotiana tabacum 158 4 2
5.1.1 Agrobacterium-mediated transformation of with
heterologous “C -cycle” genes by means of optimized methods 158 4
5.1.2 Gene silencing in tobacco transformants carrying a viral expression system 160
5.1.3 Expression of “C -cycle” genes under the control of the CaMV 35S promoter 4
and crosses between transgenic tobacco plants harboring