Agronomic performance and transcriptional analysis of genetically engineered zeaxanthin-rich potato (Solanum tuberosum L.) [Elektronische Ressource] / Xia Dong

Xia.Dong

Sujets

• Naturwissenschaften

Informations

• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2009
• Nombre de lectures: 22
• Langue: Deutsch
• Poids de l'ouvrage: 1 Mo

Extrait

TECHNISCHE UNIVERSITÄT MÜNCHEN
Lehrstuhl für Pflanzenzüchtung


Agronomic Performance and Transcriptional Analysis of Genetically
Engineered Zeaxanthin-rich Potato (Solanum tuberosum L.)

Xia Dong


Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für
Ernährung, Landnutzung und Umwelt der Technischen Universität München zur
Erlangung des akademischen Grades eines


Doktors der Naturwissenschaften


genehmigten Dissertation.



Vorsitzende: Univ.-Prof. Dr. Chr.-C. Schön
Prüfer der Dissertation:
1. Univ.-Prof. Dr. G. Wenzel
2. Priv.-Doz. Dr. V. Mohler




Die Dissertation wurde am 19.08.2009 bei der Technischen Universität München
eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für
Ernährung, Landnutzung und Umwelt am 16.11.2009 angenommen
TABLE OF CONTENTS

Table of Contents

Abstract.............................................................................................................................. iii
Zusammenfassung ..............................................................................................................v
List of Abbreviations ....................................................................................................... vii
List of Figures.................................................................................................................... ix
List of Tables .......................................................................................................................x
1. Introduction...........................................................................................................1
1.1 Zeaxanthin........................................................................................................1
1.1.1. General introduction.............................................................................1
1.1.2. Zeaxanthin and eye health ....................................................................3
1.1.3. Dietary gap of zeaxanthin .....................................................................4
1.2 Genetically engineered (GE) plants..................................................................5
1.2.1. GE plants with enhanced agronomic performance ...............................5
1.2.2. GE plants with enhanced nutritional value ...........................................6
1.2.3. Regulation of gene expression in GE plants.........................................8
1.3 Assessment of GE plants ..................................................................................8
1.3.1 Legal background.................................................................................8
1.3.2 Strategies.............................................................................................10
1.3.3 Methods..............................................................................................13
1.4 Aims of the work ............................................................................................17
2. Materials and Methods.......................................................................................18
2.1 Materials.........................................................................................................18
2.1.1 Plant materials.....................................................................................18
2.1.2 Chemicals and reagents ......................................................................19
2.2 Methods..........................................................................................................20
2.2.1 Growth conditions and sampling ........................................................20
2.2.2 Isolation and preparation of RNA.......................................................23
2.2.3 Qualification and quantification of RNA............................................26
2.2.4 PCR.....................................................................................................27
2.2.5 cDNA macroarrays.............................................................................29
2.2.6 cDNA microarrays..............................................................................34
2.2.7 Zeaxanthin and lutein measurement ...................................................40
i TABLE OF CONTENTS
3 Results..................................................................................................................42
3.1 Morphology and agronomic performance of GE potato clones .....................42
3.1.1 Under greenhouse conditions..............................................................42
3.1.2 Under open-field conditions ...............................................................43
3.2 Expression analysis of zep ..............................................................................48
3.2.1 Comparison between GE potato clones with Baltica..........................49
3.2.2 Comparison among conventional cultivars.........................................55
3.3 Expression profiling........................................................................................57
3.3.1 Analysis through cDNA macroarray ..................................................57
3.3.2 icroarray...................................................67
3.3.3 Comparison between macro- and microarray.....................................73
4 Discussion ............................................................................................................75
4.1 Morphology and agronomic performance of GE plants .................................75
4.1.1 Field testing of GE plants ...................................................................75
4.1.2 Transgene dispersal.............................................................................76
4.1.3 Zeaxanthin content under open-field conditions ................................78
4.2 Expression analysis of zep ..............................................................................79
4.2.1 zep expression in a tissue-specific manner .........................................79
4.2.2 zep expression kinetics in different tissues.........................................81
4.2.3 Post-transcriptional gene silencing .....................................................83
4.2.4 Natural and GE-derived variation in gene expression ........................84
4.3 Global expression comparison........................................................................85
4.3.1 Comparison through cDNA macroarray.............................................86
4.3.2 CompcDNA microarray .............................................89
4.3.3 Comparison between cDNA macro- and microarray .........................92
4.4 Conclusion and outlook..................................................................................93
Literature...........................................................................................................................95
Acknowledgements .........................................................................................................110
Appendix A: Chemicals..................................................................................................112
Appendix B: Kits, Enzymes and Oligonucleotides ......................................................113
Appendix C: Media and Buffers....................................................................................114
Appendix D: Planting Plan ............................................................................................115

ii ABSTRACT
Abstract

Age-related macular degeneration is the primary cause for blindness of aged people.
Recent studies have shown that enhanced carotenoid supply can lower the risk for this
disease. In order to provide a higher dietary intake, two genetically engineered (GE)
zeaxanthin-rich potato clones were derived from the potato (Solanum tuberosum L.)
cultivar Baltica. Both strategies rely on tuber-specific silencing of the zeaxanthin
epoxidase (zep) gene, either through co-suppression or anti-sense suppression. Primary
evaluation was carried out under greenhouse conditions in a previous study. However,
further evaluation including their agronomic performance, stability and tuber-specific
expression of the inserted zep gene and any potential unexpected changes at the
transcriptional level compared with their conventional counterpart cultivar Baltica needs
to be carried out under open-field conditions.
In this work, a three-year open-field trial was conducted for the two GE potato
clones, their conventional counterpart cultivar Baltica and the four other conventional
potato cultivars Désirée, Ditta, Selma and Sibu from 2005 to 2007 in two fields located in
Bavaria, Germany. Additionally, a two-year greenhouse experiment was also carried out
with the two GE potato clones and Baltica in 2005 and 2006 as control. The aims of this
work were to evaluate whether the inserted zep gene affects the general morphology,
agronomic performance and gene expression profiles of the two zeaxanthin-rich GE
potato clones in comparison to their conventional counterpart cultivar. In order to answer
these questions,