Pheromone binding proteins of Epiphyas postvittana (Lepidoptera: Tortricidae) are encoded at a single locus
The light brown apple moth, Epiphyas postvittana (Tortricidae: Lepidoptera) uses a blend of (E)-11-tetradecenyl acetate and (E,E)-9,11-tetradecadienyl acetate as its sex pheromone. Odorant binding proteins, abundant in the antennae of male and female E. postvittana, were separated by native PAGE to reveal four major proteins with distinct mobilities. Microsequencing of their N-terminal residues showed that two were general odorant binding proteins (GOBPs) while two were pheromone binding proteins (PBPs). Full length cDNAs encoding these proteins were amplified using a combination of PCR and RACE-PCR. Sequence of the GOBPs revealed two genes (EposGOBP1, EposGOBP2), similar to orthologues in other species of Lepidoptera. Eleven cDNAs of the PBP gene were amplified, cloned and sequenced revealing two major phylogenetic clusters of PBP sequences differing by six amino acid substitutions. The position of the six amino acid differences on the protein was predicted by mapping onto the three-dimensional structure of PBP of Bombyx mori. All six substitutions were predicted to fall on the outside of the protein away from the inner pheromone binding pocket. One substitution does fall close to the putative dimerisation region of the protein (Ser63Thr). Expression of three of the cDNAs in a baculovirus expression system revealed that one class encodes an electrophoretically slow form (EposPBP1-12) while the other encodes a fast form (EposPBP1-2, EposPBP1-3). A native Western of these expressed proteins compared with antennal protein extracts demonstrated that PBP is also expressed in female antennae and that PBP may be present as a dimer as well as a monomer in E. postvittana. The fast and slow forms of EposPBP1 are allelic. Westerns on single antennal pair protein extracts and allele-specific PCR from genomic DNA both show a segregating pattern of inheritance in laboratory and wild populations. Radio labelled (E)-11-tetradecenyl acetate binds to both fast and slow PBP forms in gel assays. Taken together, the genetic and biochemical data do not support the hypothesis that these PBPs are specific for each component of the E. postvittana pheromone. However, duplication of this PBP locus in the future might allow such diversification to evolve, as observed in the other species.
Available from: Rubén Palma
- "The use of a degenerate primer designed by Newcomb et al (2002) for Epiphyas postvittana (Walker) in combination with a GCGT15 primer allowed a single-band cDNA (~550 bp) to be obtained (Fig 1b). With this partial sequence available, genespecific primers (GSPLB) were designed to determine the 5′- region and to obtain the full-length cDNA, which encode for a protein with 142 amino acid residues. "
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ABSTRACT: The European grapevine mothLobesia botrana(Denis & Schiffermüller) is an economically important insect in Europe. The species invaded vineyards in Chile, Argentina, and California during 2008–2010 causing severe problems. A major component of the sex pheromone, (E,Z)-7,9-dodecadienyl acetate (E7,Z9-12:Ac), is used in a mating disruption technique when grapevine moth populations are low or to monitor pest numbers. It is thought that these sexual pheromones are blends of volatiles that typically are specific to a species and are transported in the insect antenna by pheromone-binding proteins (PBPs) across the sensillar lymph to the olfactory receptors. Currently, an increasing number of Lepidopteran PBPs
are being identified and cloned. However, there are no studies of the olfactory system and of proteins involved in the olfactory perception of L. botrana at the molecular level. In the present study, we report, for the
first time, the sequence of a PBP from L. botrana(LbotPBP), which was determined using reverse transcription technology. Homology modeling was used to generate the three-dimensional protein structure. The model suggests that PBP consists of sixα-helices as follows: Lys2-Met23 (α1), Thr28-Phe36 (α2), Arg46-Leu59 (α3), His70-Asn80 (α4), Glu84-Asn100 (α5), and Cys108-Lys125 (α6), held together by three disulfide bridges, Cys19-Cys54, Cys50-Cys108, and Cys97-Cys117. Docking simulations based on this model suggested that Trp114 is a key residue in the recognition of acetate pheromones, such as E7,Z9-12:Ac. In silico results in this study are consistent with previous findings in whichE7,Z9-12:Ac acts as the most active compound in behavioral and electroantennographic assays.
Available from: summit.sfu.ca
Available from: Walter S Leal
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ABSTRACT: This chapter discusses the olfactory receptors and binding proteins that are useful in the understanding of odor sensitivity and discrimination. The odorant-binding proteins are essential for the dynamics of the olfactory system. The expression of a Drosophila odorant receptor in Xenopus oocytes provides direct evidence for its function, and its activation is orders of magnitude slower than normally observed in the in vivo function. Due to the lack of odorant binding proteins (OBPs) in the heterologous system, the response is extremely slow as compared to the dynamics of the insect olfactory system. Also, the kinetic studies demonstrate that the pH dependent conformational change in pheromone-binding protein from Bombyx mori (BmPBP) occurs in less than 4 msec, and structural biology suggests that the conformational change in BmPBP is an intramolecular mechanism for binding and release of pheromones by pheromone-binding proteins. The chapter also shows the possibility that each odor receptor (OR) can be stimulated by a small number of ligands with only one or a few of them reaching the dendrites.
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