Novel biopesticides targeting the neuromuscular system of the peach potato aphid Myzus persicae Aishah Mohammed Alatawi A thesis submitted for the Degree of Doctor of Philosophy School of Biology, Faculty of science, Agriculture and Engineering Newcastle University June/2016 Table of contents: List of figures: ............................................................................................................................. v List of Tables: ............................................................................................................................ xi List of Appendices: .................................................................................................................... xi Acknowledgements .................................................................................................................. xii Declaration: ............................................................................................................................. xiv Abbreviations ........................................................................................................................... xv Abstract: ................................................................................................................................ xviii 1 Chapter 1 Introduction ................................................................................................... 1 1.1 The global insect pest problem ........................................................................................ 1 1.1.1 Agricultural pests .................................................................................................. 1 1.2 Factors limiting the efficacy of conventional agrochemical pesticides ........................... 2 1.2.1 Insecticide resistance ............................................................................................ 3 Health consequences and environmental impacts ............................................................. 3 1.3 Development of bio- pesticides ....................................................................................... 4 1.3.1 Arthropod venoms as natural insect pest control agents ..................................... 5 Spider venoms: sources of novel bio-insecticides ............................................................ 6 Toxins purified from segestria florentina venom glands .................................. 7 Structure of spider venom peptides precursors and post-translational processing ............ 9 Structure and functional characteristics of Segestria. Florentina ...................... 9 Insecticidal targets of spider neurotoxins ........................................................ 11 Spider venom peptides targeting insect CaV Channels .................................. 12 Spider venom peptides targeting insect NaV channels ................................... 14 1.3.2 Marine cone snail from the Genus Conus: peptide nomenclature .................... 17 Structural motifs of cone snail venom peptides: ancestral variations ............................. 20 Potential insecticides targeting neurotransmitter receptors............................................. 21 Insecticides targeting nACh receptors ............................................................ 21 1.3.3 The venom peptide Acrorhagin-2a from the sea anemone Anthopleura maculate 23 Structural motifs of the sea anemone venom peptides .................................................... 23 Sodium ion channel as targets for neuropeptides as potential bioinsecticidal ................ 24 1.3.4 Marine cone snail from the Genus Conus: isolation ........................................... 24 Structure and function of the Genus Conus α -conotoxin E1 ......................................... 25 1.4 Neurotoxins and their use in fusion proteins ................................................................ 26 1.4.1 Spider venom toxins as insecticides.................................................................... 26 1.5 Expression of fusion proteins: Pichia pastoris as an expression host ............................ 27 1.5.1 pGAPZα as a plasmid vector for recombinant protein expression ..................... 28 1.6 Plant lectins .................................................................................................................... 28 i 1.6.1 Roles in plant defence ......................................................................................... 29 1.6.2 Rols of snowdrop lectin as a carrier molecule .................................................... 30 1.7 Synthetic fusion proteins ............................................................................................... 30 1.8 Using the peach potato aphid Myzus persicae both as a target and for other homopterans ........................................................................................................................... 31 1.8.1 Life cycle of Myzus persicae ................................................................................ 31 1.8.2 Control of Myzus persicae and need for alternative methods ........................... 32 1.9 Project aim ..................................................................................................................... 33 2 Chapter 2 Materials and Methods ................................................................................ 34 2.1 Microbial Expression systems and Insects ..................................................................... 34 2.2 Materials and Recombinant Techniques ....................................................................... 34 2.3 Standard molecular biology techniques ........................................................................ 35 2.3.1 Oligonuclueotide synthesis ................................................................................. 35 2.3.2 Polymerase Chain Reaction (PCR) ....................................................................... 35 2.3.3 Ligation and transformation of expression constructs into E. coli ..................... 37 2.3.4 Modification of plasmids for two copy insertion constructs .............................. 38 2.3.5 Transformation of expression constructs in Pichia Pastoris ............................... 38 2.3.6 Cloning and Expression of Cone Snail Toxins ...................................................... 39 2.3.7 Small-scale screening for fusion protein expression .......................................... 40 2.3.8 Protein Expression and Purification from P. pastoris ......................................... 40 Downstream processing of supernatant .......................................................................... 41 Ni-NTA Affinity Purification ......................................................................................... 41 2.4 Bioassays ........................................................................................................................ 42 2.4.1 Insects and Artificial Diet .................................................................................... 42 2.4.2 Insect Bioassays .................................................................................................. 43 Initial screening of recombinant fusion proteins from venom of the spider Segestria- florentina ......................................................................................................................... 43 Dose response effect of recombinant fusion proteins 2 X SFI1/GNA, SFI5/GNA, SFI8/GNA, and GNA alone ............................................................................................ 44 Initial screening of recombinant fusion protein from cone snails ................................... 44 Dose response effect of recombinant fusion proteins GNA/ alpha-Conotoxin EI .......... 44 2.5 Basic Analytical Techniques ........................................................................................... 45 2.5.1 Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) ......... 45 2.5.2 Immune assays by Western Blotting ................................................................... 45 2.5.3 Estimation of Protein concentration ................................................................... 46 2.6 DNA Sequencing ............................................................................................................ 47 2.7 Statistical Analysis .......................................................................................................... 47 ii 3 Chapter 3 Expression, purification and biological activity of fusion proteins incorporating the toxins from the venom of the spider Segestria florentina (SFIx) and snowdrop lectin (Galanthus nivalis agglutinin; GNA) ........................................................... 48 3.1 Introduction ................................................................................................................... 48 3.2 Results ............................................................................................................................ 49 3.2.1 Production and purification of fusion proteins incorporating SFI toxins and GNA 49 Assembly of the SFIx/GNA fusion protein construct ..................................................... 49 Assembly of the expression vector constructs containing two gene copies .................... 54 Expression of the recombinant protein SFIx/GNA ......................................................... 57 Yeast transformation and gene copy analysis ................................................................