Lectins have long been established as potential agents for use in control of insect pest species. In this study the use of garlic bulb lectins against phloem-feeding hemipteran insect pests is investigated. There is no Bacillus thuringiensis (Et) toxin available which is effective against hemipteran insects. Plant lectins have been shown to be toxic to some insects, including aphids, in particular lectins from garlic. Subsequently heterodimeric (ASAI) and homodimeric (ASAlI) garlic bulb lectins were cloned and e!Cpressed in 31 recombinant yeast system, Pichia pastoris. Recombinant lectins were successfully purified and demonstrated to be functionally active in vitro by haemagglutination assays and toxic to a range of hemipteran insects: pea aphid (Acyrthosiphon pisum), peachpotato aphid (Myzus persicae) and rice brown planthopper (N. lugens) in ~ificial diet bioassays. To analyse the interactions between recombinant ASAII and the gut of A. pisum a pulldown assay was developed to identify specific interactions between ASAII and solubilized A. pisum gut proteins. Proteins which interacted with ASAll were subjected to matrix-assisted laser desoprtion ionization time of flight (MALDI-TOF) mass spectrometric analysis, this revealed that ASAll bound to alanyl aminopeptidase N (alanyl APN), a major constituent glycoprotein of the aphid gut which is rich in mannose oligosaccharides, and membrane-bound sucrase, an enzyme important in the maintenance of osmotic balance in the aphid gut. It was not possible to establish whether transport of ASAll into the haemolymph of A. pisum occurred using a Western blotting approach due to the lack of immunoreactivity of anti-ASA antibodies to the low levels of ASAII expected to be transported. A fusion protein of ASAll-Avidin was created to enable the conjugation of biotinylated peptides of potential insecticidal interest to lectin carrier proteins. One such peptide, leucomyosuppressin (LMS) from cockroach Diploptera punctata, was conjugated to ASAII-Avidin and toxicity to A. pisum was demonstrated in artificial diet bioassays. It was confirmed that avidin is transported into the A. pisum haemolymph, suggesting that if ASAII was not responsible for transport of LMS in to the haemolymph then it may be mediated by avidin. Using a bioinformatic approach a putative A. pisum LMS gene was assembled.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:486161 |
| Date | January 2008 |
| Creators | Wiles, Duncan Peter |
| Publisher | Durham University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://etheses.dur.ac.uk/1324/ |
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