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Thesis Final Version Nom/Logotip de la Universitat on s’ha llegit la tesi Towards the engineering of the monoterpene secoiridoid pathway in transgenic tobacco plants Bruna Miralpeix Anglada Dipòsit Legal: L.954-2013 http://hdl.handle.net/10803/123289 ADVERTIMENT. L'accés als continguts d'aquesta tesi doctoral i la seva utilització ha de respectar els drets de la persona autora. Pot ser utilitzada per a consulta o estudi personal, així com en activitats o materials d'investigació i docència en els termes establerts a l'art. 32 del Text Refós de la Llei de Propietat Intel·lectual (RDL 1/1996). Per altres utilitzacions es requereix l'autorització prèvia i expressa de la persona autora. En qualsevol cas, en la utilització dels seus continguts caldrà indicar de forma clara el nom i cognoms de la persona autora i el títol de la tesi doctoral. No s'autoritza la seva reproducció o altres formes d'explotació efectuades amb finalitats de lucre ni la seva comunicació pública des d'un lloc aliè al servei TDX. Tampoc s'autoritza la presentació del seu contingut en una finestra o marc aliè a TDX (framing). Aquesta reserva de drets afecta tant als continguts de la tesi com als seus resums i índexs. ADVERTENCIA. El acceso a los contenidos de esta tesis doctoral y su utilización debe respetar los derechos de la persona autora. Puede ser utilizada para consulta o estudio personal, así como en actividades o materiales de investigación y docencia en los términos establecidos en el art. 32 del Texto Refundido de la Ley de Propiedad Intelectual (RDL 1/1996). Para otros usos se requiere la autorización previa y expresa de la persona autora. En cualquier caso, en la utilización de sus contenidos se deberá indicar de forma clara el nombre y apellidos de la persona autora y el título de la tesis doctoral. No se autoriza su reproducción u otras formas de explotación efectuadas con fines lucrativos ni su comunicación pública desde un sitio ajeno al servicio TDR. Tampoco se autoriza la presentación de su contenido en una ventana o marco ajeno a TDR (framing). Esta reserva de derechos afecta tanto al contenido de la tesis como a sus resúmenes e índices. WARNING. Access to the contents of this doctoral thesis and its use must respect the rights of the author. It can be used for reference or private study, as well as research and learning activities or materials in the terms established by the 32nd article of the Spanish Consolidated Copyright Act (RDL 1/1996). Express and previous authorization of the author is required for any other uses. In any case, when using its content, full name of the author and title of the thesis must be clearly indicated. Reproduction or other forms of for profit use or public communication from outside TDX service is not allowed. Presentation of its content in a window or frame external to TDX (framing) is not authorized either. These rights affect both the content of the thesis and its abstracts and indexes. TOWARDS THE ENGINEERING OF THE MONOTERPENE SECOIRIDOID PATHWAY IN TRANSGENIC TOBACCO PLANTS by Bruna Miralpeix Anglada University of Lleida Departament of Plant Production and Forest Science Applied Plant Biotechnology Doctoral Dissertation 2013 To my parents, who love me above all else To my sister, wherever she is, she takes care of me Supervisor: Prof. Paul Christou Universitat de Lleida Institució Catalana de Recerca i Estudis Avançats Signature Date Co-supervisor: Prof. Teresa Capell Departement de Producció Vegetal i Ciència Forestal Universitat de Lleida Signature Date Table of contents Summary I Resum II Resumen III Acknowledgments IV Index of Figures VI Index of Tables X Abreviations XIII CHAPTER 1: GENERAL INTRODUCTION 1.1. Secondary metabolites: roles, uses and applications 1 1.2. Major classes of secondary metabolites 3 1.2.1. Alkaloids 3 1.2.2. Terpenoids 5 1.2.3. Phenols 6 1.3. Traditional sources of secondary metabolites 8 1.4. Catharanthus roseus 9 1.4.1. General information (plant, source of medicinal compounds) 9 1.4.2. Vincristina/vinblastine 13 1.5. References 16 CHAPTER 2: GENERATION OF A TRANSGENIC PLANT POPULATION ENGINEERED WITH EARLY MONOTERPENE SECOIRIDOID PATHWAY GENES 2.1. Abstract 21 2.2. Introduction 22 2.3. Aims and objectives 28 2.4. Material and methods 28 2.4.1. Donor plants and explant preparation 28 2.4.2. Plant transformation 29 2.4.3. Selection and regeneration of putative transgenic plants 32 2.4.4. DNA analysis 34 2.4.4.1. Genomic DNA extraction 34 2.4.4.2. Polymerase chain reaction (PCR) 34 2.4.5. mRNA analysis 36 2.4.5.1. RNA extraction 36 2.4.5.2. Reverse transcriptase polymerase chain reaction (RT-PCR) 36 2.4.5.3. mRNA (northern) blot analysis 37 2.5. Results 37 2.5.1. Recovery of transgenic plants 37 2.5.2. DNA analysis 38 2.5.3. mRNA analysis 42 2.6. Discussion 44 2.7. Conclusions 49 2.8 References 50 CHAPTER 3: METABOLIC ANALYSIS OF TOBACCO PLANTS EXPRESSING THE TWO EARLY GENES OF THE MONOTERPENE SECOIRIDOID BIOSYNTHETIC PATHWAY 3.1. Abstract 59 3.2. Introduction 59 3.2.1. Engineering of complex secondary metabolic pathways 60 3.3. Aims and Objectives 63 3.4. Material and methods 64 3.4.1. Transformation vectors 64 3.4.2. Transformation, selection and regeneration of transgenic plants 64 3.4.3. Quantitative real time PCR (qRT-PCR) 65 3.4.4. Nuclear magnetic resonance (NMR) spectroscopy 66 3.4.5. Multivariate data analysis 66 3.5. Results 67 3.5.1. Metabolic profiling 67 3.5.2. Quantitative real time PCR 69 3.6. Discussion 72 3.7. Conclusions 76 3.8. References 77 CHAPTER 4: PROTEOMIC ANALYSIS OF TRANSGENIC TOBACCO PLANTS EXPRESSING THE EARLY PART OF THE MONOTERPENE SECOIRIDOID ALKALOID PATHWAY GENES 4.1. Abstract 80 4.2. Introduction 80 4.2.1. Proteomic tools 80 4.2.2. Limitations of proteomic methods 83 4.2.3. Applications of proteomics in plants 86 4.2.4. Proteomic analysis of secondary metabolism 87 4.3. Aims and Objectives 89 4.4. Material and Methods 89 4.4.1. Plant material 89 4.4.2. Sample processing 89 4.4.3. Reduction, alkylation, digestion and iTRAQ-labeling 90 4.4.4. On-line MudPIT separation 90 4.4.5. MALDI-MS/MS and database search analysis 91 4.5. Results 92 4.5.1. Protein identification 92 4.5.2. Protein quantification 96 4.5.3. Geraniol synthase and geraniol 10-hydroxylase proteins 96 4.6. Discussion 96 4.7. Conclusions 101 4.8. References 102 CHAPTER 5: MODULATION OF THE MONOTERPENE SECOIRIDOID PATHWAY IN TRANSGENIC TOBACCO PLANTS BY THE TRANSIENT EXPRESSION OF DOWNSTREAM PATHWAY GENES 5.1. Abstract 105 5.2. Introduction 105 5.2.1. The shikimate and tryptophan pathways 107 5.2.2. The mevalonate (MVA) pathway 112 5.2.3. The 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway 113 5.2.4. Cross-talk between the MVA and MEP pathways 116 5.2.5. The monoterpene secoiridoid pathway 117 5.2.6. Multicellular organization of the monoterpenoid pathway 120 5.3. Aims and objectives 121 5.4. Materials and methods 122 5.4.1. Transformation constructs 122 5.4.2. Transformation, selection and regeneration of transgenic plants 123 5.4.3. Transient expression of monoterpene secoiridoid genes in transgenic tobacco leaves 124 5.4.4. Gas chromatography–mass spectrometry (GC-MS) 125 5.4.5. Liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) 126 5.5. Results 127 5.5.1. Transgenic plants expressing early genes in the monoterpene secoiridoid biosynthesis pathway do not accumulate geraniol following agroinfiltration with downstream pathway genes 127 5.5.2. Transgenic plants expressing early genes in the monoterpene secoiridoid biosynthesis pathway do not accumulate downstream metabolites following agroinfiltration with downstream pathway genes 129 5.6. Discussion 134 5.7. Conclusions 137 5.7. References 138 CHAPTER 6: ENGINEERING THE MONOTERPENE SECOIRIDOID PATHWAY BY COMBINATORIAL MULITGENE TRANSFER 6.1. Abstract 145 6.2. Introduction 145 6.2.1. Multigene engineering 145 6.2.2. Genetic engineering of the monoterpene secoiridoid pathway 148 6.3. Aims and objectives 153 6.4. Material and methods 153 6.4.1. Transformation constructs 153 6.4.2. Transformation, Selection and Regeneration of putative transgenic plants 155 6.4.3. mRNA Expression analysis 155 6.4.3.1. RNA extraction mRNA blot analysis 155 6.4.3.2. RT-PCR 156 6.5. Results 156 6.5.1. Selection of putatively transformed tissue and plant regeneration 156 6.5.2. Expression analysis 158 6.6. Discussion 161 6.7. Conclusion 166 6.8. References 167 CHAPTER 7: INTELLECTUAL PROPERTY DATABASE FOR THE ENGINEERING OF THE TERPENOID INDOLE ALKALOID PATHWAY IN PLANTS 7.1. Abstract 171 7.2. Introduction 172 7.2.1. Intellectual property 172 7.2.2. The prior art 173 7.3. Aims and objectives 175 7.4. Material and methods 175 7.4.1. Data sources 175 7.4.2. General search methodology 176 7.4.3. Actual searches – EPO, USPTO, JPO, CAMBIA 186 7.5. Results 181 7.5.1. Development of a focused search strategy 181 7.5.2. Development of an ideal search string 187 7.5.3. Quality control 188 7.5.4. Evaluation of the search results 190 7.6. Discussion 203 7.4. Conclusion 213 7.5. References 214 GENERAL CONCLUSION General conclusion 216 Abstract The reconstruction of biosynthetic pathways by genetic engineering in heterologous organisms is one of the current aims of metabolic engineering and synthetic biology.
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