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Biosynthesis and Regulation of Terpene Production in Accessions of Chamomile (Matricaria Recutita L.)

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Biosynthesis and Regulation of Terpene Production in Accessions of Chamomile (Matricaria Recutita L.) Biosynthesis and regulation of terpene production in accessions of chamomile (Matricaria recutita L.) Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) der Naturwissenschaftlichen Fakultät I – Biowissenschaften – der Martin-Luther-Universität Halle-Wittenberg vorgelegt von Prodyut Mondal geboren am 21.11.1983 in Kolkata, India Reviewers (Gutachter) 1. Prof. Dr. Jörg Degenhardt Institute of Pharmacy, Martin Luther University, Halle-Wittenberg Germany 2. Prof. Dr. Timo Niedermeyer Institute of Pharmacy, Martin Luther University, Halle-Wittenberg Germany 3. Prof. Dr. Timothy F Sharbel Global Institute for Food Security (GIFS), University of Saskatchewan, Saskatoon, Canada Tag der öffentlichen Verteidigung: 27.02.2020 Contents Table of Contents List of Figures ............................................................................................................................. i List of Tables ............................................................................................................................ iii Abbreviations ............................................................................................................................ iv 1. Introduction ................................................................................................................... 1 1.1 Essential oils in pharmaceutically used plants ............................................................ 1 1.1.1 Chamomile: An important medicinal herb .......................................................... 4 1.1.2 Pharmacological effects of chamomile essential oil ............................................ 5 1.1.3 The environment affects the composition of essential oil ................................... 6 1.1.3.1 Genetic efforts to improve the essential oil content ......................................... 7 1.1.3.2 Nitrogen and heavy metal affect the essential oil in chamomile ...................... 8 1.1.3.3 Drought effects on chamomile ......................................................................... 8 1.1.3.4 Biotic stress affects the quality of essential oils ............................................... 8 1.1.4 The content and composition of chamomile essential oil varies with extraction procedures and geographical origin. ................................................................................. 10 1.2 Biosynthesis of terpenes in plants ............................................................................. 11 1.3 Terpene synthases create a large structural diversity ................................................ 13 1.3.1 Structural and biochemical characteristics of terpene synthases ....................... 13 1.3.1.1 Specific structural motifs of terpene synthases .............................................. 14 1.3.1.2 The role of metal ion cofactors for TPSs activity .......................................... 15 1.3.1.3 Terpene synthases in chamomile.................................................................... 17 1.4 Biosynthesis of bisabolol and its oxides ................................................................... 17 1.5 Regulation of terpene synthases ................................................................................ 18 1.5.1 Gene transcription can regulate TPS activity..................................................... 18 1.5.1.1 Regulation of TPS activity during development ............................................ 19 1.5.1.2 Regulation of TPS activity in chamomile ...................................................... 20 Aims of the study .................................................................................................................... 21 2 Materials and methods ............................................................................................... 22 2.1 Chemicals .................................................................................................................. 22 2.2 Plant material............................................................................................................. 22 Contents 2.3 Nucleic acid techniques ............................................................................................. 24 2.3.1 Extraction of RNA ............................................................................................. 24 2.3.1.1 DNA (cDNA) synthesis ................................................................................. 24 2.3.1.2 DNA fragment amplification ......................................................................... 24 2.3.1.3 General conditions for PCR reactions ............................................................ 25 2.3.1.4 Agarose gel electrophoresis and isolation of DNA fragments ....................... 26 2.3.1.5 Quantification of DNA and RNA................................................................... 26 2.3.1.6 Quantitative real-time PCR ............................................................................ 26 2.3.1.7 Restriction digest of DNA .............................................................................. 27 2.3.1.8 Cloning Techniques (Ligation mixture) ......................................................... 28 2.3.1.9 DNA Plasmid transformation and purification .............................................. 28 2.3.1.10 Analysis of DNA and protein sequences .................................................... 28 2.3.1.11 Overexpression of α-bisabolol synthases in bacteria (E. coli) and protein extraction …………………………………………………………………………….29 2.3.1.12 Purification of α-bisabolol synthase ........................................................... 30 2.4 Microbiological method ............................................................................................ 30 2.4.1 Medium .............................................................................................................. 30 2.4.2 Antibiotics .......................................................................................................... 31 2.4.3 Bacteria .............................................................................................................. 31 2.4.4 Cultivation of Bacteria ....................................................................................... 31 2.4.5 Transformation of TOP 10 cells ........................................................................ 31 2.5 Extraction procedures ................................................................................................ 32 2.5.1 SPME for plant materials analysis ..................................................................... 32 2.5.2 n-hexane extraction ............................................................................................ 32 2.6 Chromatographic Method ......................................................................................... 32 2.6.1 SPME: Enzyme activity reaction ....................................................................... 33 2.7 Kinetic study of α-bisabolol synthase ....................................................................... 33 2.7.1 Determination the linear phase of the reactions ................................................. 33 2.7.2 Km values ........................................................................................................... 34 2.8 Determination of substrate concentration ................................................................. 34 2.8.1 Solvents used ..................................................................................................... 34 Contents 2.8.2 Extraction procedures ........................................................................................ 35 2.9 Statistical Analysis .................................................................................................... 36 3 Results .......................................................................................................................... 37 3.1 Analysis of volatile metabolite diversity in chamomile accessions .......................... 37 3.1.1 Metabolite concentrations during seedling development .................................. 37 3.1.2 Metabolite concentrations in stems and leaves of adult chamomile plants ....... 38 3.1.3 Metabolite concentrations in buds and flowers of adult chamomile plants ....... 42 3.2 α-bisabolol and its oxides are localized only in buds and flowers and their concentration varied with bisabolone oxide A ..................................................................... 48 3.3 Principal component analysis (PCA) of terpene metabolites in chamomile accessions ............................................................................................................................. 50 3.4 Identification of putative α-bisabolol synthases (MrTPS 7) from chamomile accessions ............................................................................................................................. 56 3.5 Dendrogram analysis of α-bisabolol synthase........................................................... 58 3.6 The transcript levels of α-bisabolol synthases of the chamomile accessions do not -bisabolol production ................................................................................. 60 3.7 Biochemical characterization of putative α-bisabolol synthases from chamomile accessions ............................................................................................................................. 62 3.8 Determination of substrate concentration in flowers
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