CARDIFF UNIVERSITY PhD Thesis Topic: Towards Optimisation of L-DOPA synthesis in Mucuna pruriens. By Kibedi Bilal Cabral (Cardiff School of Biosciences) This thesis is submitted to Cardiff University in partial fulfilment of the requirements for the degree of DOCTOR OF PHILOSOPHY (BIOSCIENCES). AUGUST 2014. I APPENDIX I. DECLARATION. This work has not been submitted in substance for any other degree or award at this or any other university or place of learning, nor is being submitted concurrently in candidature for any degree or other award. II DEDICATION. I dedicate this work to my parents, my academic mentors and all those who have supported me to complete my PhD program. III ABSTRACT. This study examines the potential for increasing natural L-DOPA drug biosynthesis in Mucuna pruriens by silencing or “knocking down” expression of putative DOPA/tyrosine decarboxylase (Mp-ty/ddc) in situ. Mp-ty/ddc codes for DOPA/tyrosine decarboxylase (Mp-TY/DDC) which converts L-DOPA to dopamine in plants. The hypothesis of the work was that silencing the Mp-ty/ddc gene would result in accumulation of L-DOPA in the plant tissues. This work involved isolation and characterisation of 1.73 kb putative full-length ORF of Mp-ty/ddc. The gene showed 74% homology with TY/DDC protein alignments of other plants in the same taxa, although no enzyme activity was detected when the gene product was heterologously expressed. In addition, a protocol was developed for Agrobacterium mediated transformation of M. pruriens so as to be able to manipulate expression of the DOPA genes in situ. The cotyledonary nodal and hypocotyl tip explants regenerated shoots on M.S media supplemented with 50 μM BA, 0.5 μM NAA and 50 mg l-1 kanamycin selection also the nptII transgene was detected by PCR. The Agrobacteria strains GV3101 harbouring a pGREEN vector and carrying an Mp- ty/ddc antisense were used for the plant transformation experiments. Further work showed that the Mp-ty/ddc gene copy number was 1, the gene expression was highest in roots and stems, followed by seeds and was very low in leaves. On the other hand, L-DOPA-content in seeds was 17-fold higher relative to leaves and 15 fold relative to stems and roots. IV LIST OF ABBREVIATIONS AND ACRONYMS. Amp: Amperes. BA: 6-Benzylaminopurine. BCIP: 5-bromo-4-chloro-3-indolyl phosphate. Bp: Base pairs of Nucleotides. ddc: DOPA decarboxylase gene. DDC: DOPA decarboxylase enzyme DEPC: Diethylpyrocarbonate. DNA: Deoxyribonucleic acid. DOPA genes: Genes in the L-DOPA synthesis and metabolic pathway. E. coli: Escherichia coli. EDTA: Ethylenediaminetetraacetic acid eGFP: Enhanced Green Florescent Protein.. ESTs: Expressed sequence tags. Epi: Epinephrine HPLC: High performance liquid chromatography IPTG: Isopropyl-ß-D-1-thiogalactoside. LB: Luria Bertani medium. L-DOPA: L-3, 4-dihydroxyphenylalanine. mM: Millimoles. mRNA: Messenger ribonucleic acid. V Mp-TY/DDC: Putative DOPA/tyrosine decarboxylase enzyme extracted from Mucuna pruriens. Mp-ty/ddc: Putative DOPA/tyrosine decarboxylase gene isolated from Mucuna pruriens. Mp-TYOH: Putative tyrosine hydroxylase enzyme from Mucuna pruriens. Mp-tyoh: Putative tyrosine hydroxylase gene from Mucuna pruriens. MS: Skoog and Murashige basal nutrient media. MSD4X2: MSO, NAA, 6-BAP (ref above). MSO: Skooge and Murashige basal salts x1, Sucrose (3% w/v) (and for solid, Agar (1% w/v)). NAA: Naphthalene acetic acid. NBT: Nitro blue tetrazolium. NCBI: National Council for Biotechnological Information. nM: Nanomolar PCR: Polymerase Chain Reaction PD: Parkinson’s disease. RCF: Relative centrifugal force. RH: Relative humidity. RNA: Ribonucleic acid. RPM: Revolutions per minute. qRT-PCR: Quantitative Real-time Polymerase Chain Reaction. SDS: Sodium dodecylsulfate. SSC: Tri-sodium citrate. VI TPL: Tyrosine phenol-lyase. TY/DDC: DOPA/tyrosine decarboxylase enzyme ty/ddc: DOPA/tyrosine decarboxylase gene Tyr: Tyrosine hydroxylase. X-gal: 5-bromo-4-chloro-3-indolyl1-ß-D-galactopyranoside VII ACKNOWLEDGEMENTS. I extend my sincere gratitude to my academic mentors/Principal investigators; Dr Hillary Rogers and Dr Carsten Muller for their intellectual generosity, excellent supervision and support which has enabled me to complete my PhD study program. In the same vein I extend special to my advisor/mentor Prof James Murray for his distinguished technical advice and support. In addition, I thank Dr Barend de Graaf, Dr Dafydd Jones, staff at Bristol University School of Biological Sciences and colleagues in my PhD study research group at Cardiff University School of Biosciences for the support and advice. I am grateful to the Director Post graduate studies, Prof Hellen White-Cooper, Prof Paul Kemp and the school administration for the enormous support all through my PhD study program. Last but not least, I thank my sponsor The Islamic Development Bank Merit Scholarship office, my parents Dr and Mrs Kirunda Kivejinja, my family and friends for the financial and moral support that has enabled me to pursue and complete my PhD study Program. VIII CONTENTS Page CHAPTER 1: GENERAL INTRODUCTION 19 1.1: Introduction 19 1.2: Sources of L-DOPA 21 1.2.1: Industrial synthesis of L-DOPA 21 1.2.2: Natural sources of L-DOPA 25 1.2.3: A brief account of Mucuna pruriens – a potential bio-factory for L-DOPA 28 1.2.4: Biosynthesis and biochemistry of L-DOPA in M. pruriens 30 1.3: Molecular pharming and Advances in Applied Biotechnology 34 1:4: Aim and objectives of the PhD work 38 CHAPTER 2: GENERAL MATERIALS AND METHODS 39 2.1: Plant material growth conditions and harvesting of material 39 2.1.1: Plant material 39 2.1.2: Growth conditions 39 2.1.3: Harvesting and storage of plant material 40 2.2: Molecular Biology procedures 40 IX 2.2.1: Genomic DNA extraction 40 2.2.1.1: Large Scale Extraction 40 2.2.1.2: Small scale crude genomic DNA quick extraction protocol for PCR amplification 42 2.2.2: RNA extraction 42 2.2.2.1: Previous methods of RNA extraction from leaves, stems and roots of M. pruriens 42 2.2.2.2: Extraction of RNA from proteinaceous M.pruriens seeds 44 2.2.3: DNAse treatment 45 2.2.4: cDNA synthesis 46 2.2.5: Designing primers 46 2.2.5.1: Gene specific primers 46 2.2.5.2: Degenerate primers 47 2.2.6: Polymerase Chain Reaction (PCR) 47 2.2.7: Agarose gel electrophoresis 48 2.2.8: Purification of PCR products using a QIAquick PCR Purification Kit 49 2.2.9: Extraction of a DNA band from agarose gels using a QIAquick Gel Extraction Kit 49 2.2.10: Preparation of pZERO-2-T for cloning PCR products 50 2.2.11: Dephosphorylation of DNA 5’-termini 52 2.2.12: DNA ligation in pZERO-2-T or pGEM-T easy vector 53 2.2.13: Preparation of bacterial competent cells 53 2.2.14: Transformation of competent cells 54 2.2.15: Colony PCR 55 X 2.2.16: Plasmid DNA purification using QIAprep Spin Miniprep Kit 56 2.2.17: Purification of Nucleic Acids using Phenol-Chloroform 57 2.2.18: Purification and concentration of nucleic acids by Isopropanol precipitation 57 2.2.19: Restriction enzyme digestion of plasmid DNA 58 2.2.20: Analysis of sequence data 58 2.2.21: SDS-PAGE 59 CHAPTER 3: CHEMICAL PROFILE OF L-DOPA IN MUCUNA PRURIENS 61 3.1: Introduction 61 3.1.1: Aims, objectives and approaches used in Chapter 3 67 3.2: Materials and Methods 68 3.2.1: Plant material 68 3.2.2: Extraction of L-DOPA from M. pruriens 68 3.3.3: HPLC analysis of M. pruriens extracts 69 3.3.3.1: Preparation of standards 69 3.3.3.2: Measurement of L-DOPA and dopamine-content by HPLC 70 3.4: Results 71 3.4.1: Preliminary range finder trial 71 3.4.2: L-DOPA concentrations 73 XI 3.5: Discussion 75 CHAPTER 4: ISOLATION, CHARACTERISATION AND EXPRESSION OF A MUCUNA PRURIENS DOPA DECARBOXYLASE LIKE-GENE (Mp-ddc) 77 4.1: Introduction 77 4.1.1: Aims, objectives and approaches used in Chapter 4 81 4.2: Methods and Materials 82 4.2.1: Plant Materials 82 4.2.2: Extraction of nucleic acids and cDNA synthesis 82 4.2.3: Rapid amplification of cDNA ends 82 4.2.3.1: 3’ RACE and Primer design 82 4.2.3.2: 3’ RACE cDNA synthesis and PCR amplification 84 4.2.3.3: 5’ RACE and Primer design 85 4.2.3.3.1: Principles of 5’ RACE 85 4.2.3.4: 5’ RACE and PCR amplification 87 4.2.4: Southern analysis 88 4.2.4.1: Principle of Southern blot analysis 88 4.2.4.2: Restriction digestion and Gel electrophoresis 89 4.2.4.3: Southern blot 92 4.2.4.4: Pre-hybridisation and Probe generation 95 4.2.4.5: Hybridisation 97 4.2.4.6: Washing and development 97 XII 4.2.5: Real PCR (qRT- PCR) 98 4.2.5.1: Principle of Real time qRT-PCR 98 4.2.5.2: qRT-PCR Primer Design 100 4.2.5.2.1: Designing Degenerate primers for endogenous genes in M. pruriens 100 4.2.5.2.2: Designing Gene-specific qRT-PCR primers 101 4.2.5.3: Normalisation of endogenous gene expression 101 4.2.5.4: Real-time PCR Amplification 102 4.2.5.5: Analysis of Real-time PCR results 103 4.3: Results 105 4.3.1: Isolation of a portion of a putative M. Pruriens DOPA/tyrosine decarboxylase gene DC1 105 4.3.2: Sequence results for the cloned 0.4 kb Mp-ty/ddc genomic product 107 4.3.3: Obtaining the full 3’ end of the Mp-ty/ddc mRNA using 3’ RACE 111 4.3.4: Towards obtaining the full end of the Mp-ty/ddc mRNA using 5’ RACE 117 4.3.5: Isolation of a further 5’ fragment of the Mp-ty/ddc gene based on degenerate PCR primers 120 4.3.6: Analysis of the Mp-ty/ddc assembled sequence 126 4.3.7: Southern analysis and Mp-ty/dc gene copy number in M.