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The roles of Epiphyas postvittana GOBP2 in odour detection 74 Table 3.1: A comparison of the EC50 values from dose response curves of EpOR1 obtained in Sf9 cell assays with DMSO and/or EpGOBP2 used as a solubilisation agent for the ligands methyl salicylate and geranyl acetate. Ligand components EC50[Methyl Salicylate] (M) EC50[Geranyl Acetate] (M) Saline only NR a NR DMSO only (1.8 ± 0.9) -12 (2.7 ± 0.4) -8 EpGOBP2 only (1.19 ± 0.82) -13 (1.9 ± 0.66) -10 DMSO + EpGOBP2 (1.33 ± 0.96) -13 (1.97 ± 2.5) -10 a. NR = no response 3.4 Discussion Insects can recognise a range of chemicals, most of which are hydrophobic in nature. The actual mechanism of odour recognition by insects is yet to be deduced; however theoretical models suggest solubilisation and targeting of the hydrophobic odours to the receptor neurons via soluble protein intermediates present in the sensillum lymph (Kaissling, 2009). We can test the roles of these soluble proteins in an in vitro setting by expressing the receptor in cell lines such as the Sf9 insect cell lines used in this study, and looking at the effect on receptor activation upon addition of these proteins. The ability of EpGOBP2 to solubilise plant volatiles of importance to E. postvittana was tested in a VOBA setting. The ten compounds tested were chosen based on their ability to activate EpOR1 expressed in the Sf9 cell assay system. EpGOBP2 was able to solubilise seven of these compounds; however the binding repertoire of EpGOBP2 may be more extensive if the range of tested compounds was to be expanded. For the scope of this study, only those compounds that elicited response to EpOR1 were chosen for testing their binding to EpGOBP2. The chemistries of these seven compounds differ considerably. Pentyl acetate is an ester that has a straight chain structure, while geranyl acetate and nerol are linear monoterpenes, eucalyptol is a cyclic monoterpene and α-farnesene is a sesquiterpene. Methyl salicylate is a benzoic ring phenol and octanol is a linear alcohol (O'Neil, 2006). The amount of each of these ligands bound by EpGOBP2 differs according to their chemistries, with EpGOBP2 having highest affinity for alcohol followed by the
The roles of Epiphyas postvittana GOBP2 in odour detection 75 phenol, then the terpenes and lastly the ester (Figure 3.5). However, the difference in the degree of binding of these ligands is small, with only a ten-fold difference between the highest octanol, and the lowest, pentyl acetate. Nevertheless, all these compounds are plant volatiles and may play a role in E. postvittana olfaction (as discussed in chapter 2). The moderate binding constants of EpGOBP2 for the range of plant volatiles tested reflect low affinity which supports the conclusion that GOBPs function to bind a range of odorants. These moderate binding constants of EpGOBP2 for a range of volatiles are indicative of a role in binding of a range of odorants by OBPs. Previous studies of GOBPs in other lepidopterans have shown their ability to recognise plant volatiles. M. sexta GOBP2 binds plant odorants such as (Z)-3-hexen-1-ol, geraniol, geranyl acetate and limonene (Feng and Prestwich, 1997) with moderate affinities (10 to 40 µM of these odorants were required in displacement assays to displace 50 % of the bound pheromone analog), comparable to those obtained for EpGOBP2 in this study. In the honeybee Apis mellifera, the OBP called ASP2 binds its ligands, mostly plant scents, with high binding affinities in the micromolar range (Briand et al., 2001). This shows that perhaps the Hymenoptera and Lepidoptera OBPs differ in their mechanisms of binding their ligands. Since EpGOBP2 binds a range of compounds with different shapes and chemical structures, it can be deduced that the binding pocket of EpGOBP2 must be spacious and flexible enough to harbor this varied range of compounds. The range of compounds tested in this study is limited; hence there might be other stronger binding ligand(s) that have not been tested yet. Increasing the test compound range will perhaps give insights into an even wider range of ligands for EpGOBP2, adding to the observation that while the PBP recognise species specific pheromones, the GOBPs are broadly tuned to recognise a range of compounds that includes (and might not be limited to) plant volatiles. When EpGOBP2 was pre-incubated with geranyl acetate or methyl salicylate, and added to Sf9 cells expressing EpOR1, a receptor activation response was observed (Figures 3.8 and 3.9), indicating that EpGOBP2 was able to solubilise the odorants enabling it to interact with the membrane bound EpOR1. This is the first documented role of a lepidopteran GOBP in odorant binding in an in vitro OR characterisation assay. Similar reconstitution assay studies have been done with lepidopteran PBPs,
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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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Page 45 and 46: General Introduction 29 Expression
Page 47 and 48: General Introduction 31 BmOr2 HvOr2
Page 49 and 50: General Introduction 33 for lepidop
Page 51 and 52: Functional characterisation of Epip
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Page 97 and 98: Identification of putative odorant
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5.1 Introduction 5 Concluding Discu
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Concluding Discussion 127 VOBA sett
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Concluding Discussion 129 redundant
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Concluding Discussion 131 against t
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Concluding Discussion 133 sequencin
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Appendix B 135 Buffered TB (Sambroo
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Appendix C 137 C. Appendix C - Clus
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Appendix D 139 D. Appendix D - Solu
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Appendix E 141 I II
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Appendix E 143 V
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Appendix E 145 VII continued Figure
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References 147 Ansebo, L., Ignell,
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References 149 Butenandt, A., Beckm
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References 151 Fedurco, M., Romieu,
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References 153 Große-Wilde, E., St
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References 155 Hrdy, I., F. Kuldova
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References 157 Kaissling, K. E. (19
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References 159 Krieger, J., Große-
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References 161 Lopez-Gomez, R. and
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References 163 Miura, N., Nakagawa,
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References 165 Pell, J. K., Macaula
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References 167 Røstelien, T., Stra
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References 169 Stowers, L. and Loga
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References 171 Turner, C. T., Davy,
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References 173 Vogt, R. G., Riddifo
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References 175 Widmayer, P., Heifet
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