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Concluding Discussion 126 The aims of this thesis were to decode the binding partners of EpOR1, a member of a group of receptors shown to be essential in odorant perception in insects; to show the role, if any, of E. postvittana GOBP2 in odorant binding by EpOR1 in a heterologous expression system; and to expand the known OR repertoire of E. postvittana and identify potential candidates for the PR(s) amongst these. 5.2 Summary of results and discussion 5.2.1 EpOR1 characterisation EpOR1 was previously identified from a male antennal EST library of E. postvittana by Jordan et al. (2009). EpOR1 forms a clade with ORs from other moths, including PRs from B. mori, H. virescens, P. xylostella, D. indica and M. separate (Krieger et al., 2002; Krieger et al., 2004; Krieger et al., 2005; Nakagawa et al., 2005; Mitsuno et al., 2008). However qRT-PCR analysis did not show male-biased expression for EpOR1 so a functional analysis of EpOR1 was conducted to identify its binding repertoire. Transient expression of EpOR1 in insect Sf9 cells revealed that EpOR1 binds a range of plant volatiles in vitro (Table 2.1). Some of these volatiles have implications in plant based insect repellents (geraniol), act as oviposition deterrents (citral), are released by plants under herbivore attack (methyl salicylate) and are natural constituents of plant aroma (geranyl acetate). This characterisation of EpOR1 reveals it to be a receptor for general odorants and shows that at least some moth ORs, like the Drosophila ORs are broadly tuned, recognising a range of compounds. The decoding of binding partners of ORs has implications in dealing with insect pests by targeting their chemosensory systems. This will aid to the development of sustained and environment friendly insecticides. 5.2.2 EpGOBP2 reconstitution of the Sf9 cell assay E. postvittana GOBP2, identified by Newcomb et al. (2002) was successfully used for reconstituting the Sf9 cell assay system for functional characterisation of EpOR1. Recombinant EpGOBP2 was able to bind seven of the ten compounds tested in a
Concluding Discussion 127 VOBA setting (Figure 3.5). These results indicate that EpGOBP2 is broadly tuned recognising a range of compounds. EpGOBP2 was further tested for its ability to solubilise two ligands of EpOR1, methyl salicylate and geranyl acetate in the Sf9 cell assay. These experiments showed that EpGOBP2 could be used to replace DMSO as a solubilisation agent for both methyl salicylate and geranyl acetate (Figures 3.7 and 3.8). EpGOBP2 also enhanced the sensitivity with which EpOR1 recognise these compounds in vitro as determined by comparing EC50 values (Table 3.1). The increased sensitivity observed for EpOR1 in the presence of EpGOBP2 could be a combination of a role as a solubilising agent and/or as an activated ligand and suggest that species-specific OBPs can be used to reconstitute the Sf9 cell assay system for improving the sensitivity of the system. This is the first documented role of a lepidopteran GOBP in odorant binding in an in vitro OR characterisation assay. 5.2.3 Identification of putative ORs from E. postvittana The initial development of an antennal EST library at Plant and Food Research Ltd enabled the identification of three ORs from E. postvittana (Jordan et al., 2009). A further 49 ORs have been identified as part of this project using 454 transcriptome and Illumina genome sequencing (Table 4.6). These ORs share some common features with B. mori ORs identified so far. Twenty-four E. postvittana ORs have one- to-one orthologs with B. mori ORs. Expansions seem to have occurred in 13 of the B. mori ORs, with E. postvittana having between 2 to 4 ORs for each of the 13 B. mori ORs. EpOR1 and EpOR6 fall into the moth PR clade. However, EpOR1 has been shown to be the receptor for plant volatiles, with similar expression levels in male and female antennae. For EpOR6 tissue expression analysis by qRT-PCR has yet to be performed. The other possibility is that some of the phylogenetics is not correct as not all ORs are full length and members of the PR clade may be placed elsewhere currently. Only the full lengths of all the identified E. postvittana ORs will determine their true phylogeny. It could also be that the PRs are missing from the list of identified ORs, as the E. postvittana OR repertoire is approximately twenty ORs short compared to B. mori OR repertoire, thereby indicating that not all E. postvittana ORs have been identified. Since the PRs from six other moth species fall into the moth PR clade, we assume the E. postvittana PR(s) to be part of this clade also. On the other hand, not all members of this clade are PRs (for example BmOR4, 5, 7 and 9, as
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Abstract Abstract Olfaction or the
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Acknowledgements Acknowledgements I
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Contents v Contents Abstract……
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Contents 4.2.5 Degenerate PCR .....
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List of Figures List of Figures Fig
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List of Tables List of Tables Table
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List of Abbreviations gDNA Genomic
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1.1 General overview 1 General Intr
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General Introduction 3 moth in loca
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General Introduction 5 Figure 1.1:
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General Introduction 7 1.5 Componen
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General Introduction 9 OBPs have al
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General Introduction 11 A model has
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General Introduction 15 Tanaka et a
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General Introduction 17 Table 1.1:
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General Introduction 21 et al., 200
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General Introduction 23 the attract
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General Introduction 25 leaves and
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General Introduction 29 Expression
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General Introduction 31 BmOr2 HvOr2
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General Introduction 33 for lepidop
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Functional characterisation of Epip
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3.1 Introduction 3 The roles of Epi
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Page 163 and 164: References 147 Ansebo, L., Ignell,
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Page 171 and 172: References 155 Hrdy, I., F. Kuldova
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Page 177 and 178: References 161 Lopez-Gomez, R. and
Page 179 and 180: References 163 Miura, N., Nakagawa,
Page 181 and 182: References 165 Pell, J. K., Macaula
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Page 187 and 188: References 171 Turner, C. T., Davy,
Page 189 and 190: References 173 Vogt, R. G., Riddifo
Page 191 and 192: References 175 Widmayer, P., Heifet
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