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The roles of Epiphyas postvittana GOBP2 in odour detection 60 antennae hence postulated to have a female oriented role, perhaps in the binding of some as yet unknown male specific pheromone. The fourth PBP, called PBP3 showed higher expression in male than female antennae, and has been postulated to be involved in binding of the pheromone component(s) of E. postvittana. The two GOBPs are members of the GOBP1 and GOBP2 family of GOBPs in moths and GOBP2 has a similar level of expression in male and female antennae (Newcomb et al., 2002). No data on sensillum localisation of EpGOBPs is available yet, however, the expression of A. polyphemus, B. mori and H. armigera GOBPs have been shown to be restricted to sensilla basiconica, hence a role of GOBPs in binding plant volatiles of importance to moths is assumed (Steinbrecht et al., 1995; Wang et al., 2003). Very limited functional data is as yet available for any moth GOBP, with the only study revealing that M. sexta GOBP2 binds the plant volatiles (Z)-3-hexen-1-ol, geraniol, geranyl acetate and limonene (Feng and Prestwich, 1997) and a recent study revealed B. mori GOBP2 binds bombykol and is able to discriminate it from the antagonist, bombykal (Zhou et al., 2009). Both M. sexta and B. mori GOBP1 have so far failed to bind any of the ligands they have been tested with (Vogt et al., 2002; Zhou et al., 2009) leading to the conclusion that the proteins tested might be inactive allelic variants. 3.1.1 Aim The aim of this research chapter is to test hypotheses of possible roles for GOBPs. GOBPs could be non-specific solubilising agents for a range of plant volatiles, or they could be ligand specific, like that observed for moth PBPs. GOBPs could form a complex with the ligand, and interaction of this complex confers increased sensitivity and selectivity onto the OR, as shown for H. virescens and B. mori PBPs in heterologous cell assay systems. With this knowledge of roles of PBPs in heterologous systems, a similar approach of using a reconstituted heterologous assay system can be used to test the hypothesised roles of GOBPs with E. postvittana GOBP2 (EpGOBP2) as a model: can EpGOBP2 act as a solubilising agent for plant volatiles? GOBP2 homologues in M. sexta and B. mori have been shown to bind plant volatiles and sex pheromone components hence we assume EpGOBP2 to have a similar ligand binding range. As part of this PhD research, E. postvittana OR1 was
The roles of Epiphyas postvittana GOBP2 in odour detection 61 characterised in an Sf9 cell assay system and shown to bind a range of plant volatiles. Some of the plant volatiles identified as ligands of EpOR1 have also been shown to be ligands for M. sexta GOBP2 (geraniol and geranyl acetate); hence leading us to hypothesis that EpGOBP2 might share ligands with EpOR1. The odorants used for testing EpGOBP2 hence is limited to the binding repertoire of EpOR1, as determined in chapter two. The Sf9 cell assay system heterologously expressing EpOR1 forms a good starting point for testing the role(s) of EpGOBP2. 3.2 Materials and methods 3.2.1 Recombinant Expression of EpGOBP2 A pET30a (Novagen) plasmid clone of EpGOBP2 (pET30a-EpGOBP2) was obtained from Duncan Stanley at Plant & Food Research. This clone encodes a protein containing an N-terminal His-tag and TEV protease recognition site upstream of the EpGOBP2 sequence (411 bp, GenBank accession no. AF411460), which is missing its N-terminal 20 amino acid signal peptide (Newcomb et al., 2002). The pET30a-EpGOBP2 plasmid was transformed into Rosetta-Gami2 cells (Novagen) and positive clones selected on kanamycin and chloramphenicol luria broth (LB) plates. pET30a-EpGOBP2 was over-expressed in buffered terrific broth (TB) with isopropyl-beta-D-1 thiogalactopyranoside (IPTG) induction, as stated in Hamiaux et al. (2009). Refer to appendix A for recipe of TB. Briefly, a colony of the transformed cells was used to inoculate a starter culture for protein expression consisting of 10 mL LB media, 15 µg/mL kanamycin, 34 µg/mL chloramphenicol and 0.01% glucose. This was incubated at 37°C overnight with shaking at 220 rpm after which 5 mL of the culture was used to inoculate 500 mL of buffered TB with 15 µg/mL kanamycin and 34 µg/mL chloramphenicol. The culture was left to grow at 37°C, 220 rpm until an optical density (OD600) of 0.6 to 0.8 was attained. IPTG was then added to the culture to a final concentration of 0.5 mM, and the culture let to grow overnight at 20°C shaking at 220 rpm. The cells were harvested by centrifugation at 13,000g at 4°C for 30 minutes, with the resulting pellet stored at -20°C until purification.
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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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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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