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

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