Mechanisms of Olfaction in Insects - ResearchSpace@Auckland ...

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Functional characterisation of Epiphyas postvittana odorant receptor 1 50 A C D E Figure 2.4: Dose response curves of the change in fluorescence exhibited by Sf9 cells transfected with pIB-EpOR1 to (A) methyl salicylate, (B) geranyl acetate, (C) citral (D) geraniol and (E) and geranial. The change in fluorescence of the pIB-EpOR1 transfected cells elicited by a range of ligand concentrations was calculated and the data represented by a sigmoidal curve for each ligand. Error bars represent the standard error of six to nine responding cells. B
Functional characterisation of Epiphyas postvittana odorant receptor 1 51 Table 2.2: EC50 (± standard error) and Hill slope estimates of the dose response curves of five best ligands for EpOR1. The recognition threshold is the lowest concentration to which EpOR1 transfected Sf9 cells elicited a measurable response to the test compound. Compound EC50 (M) Hill slope Recognition threshold (M) Methyl salicylate Geraniol Citral Geranial Geranyl acetate 2.4 Discussion (1.8 ± 0.95) x 10 -12 (5.8 ± 0.35) x 10 -11 (1.3 ± 2.5) x 10 -9 (5.3 ± 2.5) x 10 -8 (2.8 ± 0.4) x 10 -8 0.35 3.2 0.33 0.61 0.83 10 -15 10 -12 10 -10 10 -11 10 -10 Using an insect cell assay system, E. postvittana OR1 was functionally characterised to be a receptor for general plant odorants, including citral, geraniol, geranial, methyl salicylate, geranyl acetate, pentyl acetate, octanol, α-farnesene, nerol and 1,4-cineole. The presence of EpOR1 at the mRNA and protein levels in transfected Sf9 cells (detected by RT-PCR and western blot respectively) suggests that EpOR1 is being transcribed and translated in the Sf9 cells giving confidence that a functional assay is possible. Western blot of membrane fraction extracted from Sf9 cells transfected with pIB-EpOR1 showed three bands, one at approximately 25 kDa, the second at 50 kDa and the third at 60 kDa. The 50 kDa band corresponds closely to the theoretical molecular weight of myc-tagged EpOR1 at 49.6 kDa, while the higher 60 kDa band could be a glycosylated form of the protein, as has been postulated by Henningsen et al. (2002) for multiple bands on immunoblots of human histamine H2 receptor. The lowest band could be degraded protein or nonspecific binding as the pIB-V5/His transfected Sf9 cell membrane protein fraction (lane 3) also shows non-specific binding around 25 kDa. When the pIB-EpOR1 transfected Sf9 cells were stimulated with 10 -5 M citral, as shown in Figure 2.3, not all the cells showed an increase in fluorescence. This is due
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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
Page 15 and 16: List of Abbreviations gDNA Genomic
Page 17 and 18: 1.1 General overview 1 General Intr
Page 19 and 20: General Introduction 3 moth in loca
Page 21 and 22: General Introduction 5 Figure 1.1:
Page 23 and 24: General Introduction 7 1.5 Componen
Page 25 and 26: General Introduction 9 OBPs have al
Page 27 and 28: General Introduction 11 A model has
Page 31 and 32: General Introduction 15 Tanaka et a
Page 33 and 34: General Introduction 17 Table 1.1:
Page 37 and 38: General Introduction 21 et al., 200
Page 39 and 40: General Introduction 23 the attract
Page 41 and 42: General Introduction 25 leaves and
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
Page 65: Functional characterisation of Epip
Page 73 and 74: 3.1 Introduction 3 The roles of Epi
Page 75 and 76: The roles of Epiphyas postvittana G
Page 95 and 96: 4.1 Introduction 4 Identification o
Page 97 and 98: Identification of putative odorant
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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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