The odorant binding protein gene family from the genome of silkworm, Bombyx mori

Abstract

Background: Chemosensory systems play key roles in the survival and reproductive success of insects. Insect chemoreception is mediated by two large and diverse gene superfamilies, chemoreceptors and odorant binding proteins (OBPs). OBPs are believed to transport hydrophobic odorants from the environment to the olfactory receptors.

Results: We identified a family of OBP-like genes in the silkworm genome and characterized their expression using oligonucleotide microarrays. A total of forty-four OBP genes were annotated, a number comparable to the 57 OBPs known from Anopheles gambiae and 51 from Drosophila melanogaster. As seen in other fully sequenced insect genomes, most silkworm OBP genes are present in large clusters. We defined six subfamilies of OBPs, each of which shows lineage-specific expansion and diversification. EST data and OBP expression profiles from multiple larvae tissues of day three fifth instars demonstrated that many OBPs are expressed in chemosensory-specific tissues although some OBPs are expressed ubiquitously and others exclusively in non-chemosensory tissues. Some atypical OBPs are expressed throughout development. These results reveal that, although many OBPs are chemosensory-specific, others may have more general physiological roles.

Conclusion: Silkworms possess a number of OBPs genes similar to other insects. Their expression profiles suggest that many OBPs may be involved in olfaction and gustation as well as general carriers of hydrophobic molecules. The expansion of OBP gene subfamilies and sequence divergence indicate that the silkworm OBP family acquired functional diversity concurrently with functional constraints. Further investigation of the OBPs of the silkworm could give insights in the roles of OBPs in chemoreception.

Figure 1

Genomic locations of silkworm odorant-binding protein genes. 38 OBP genes are distributed across six chromosomes. Another four OBP genes (obp20, obp38, 39 and 44) are represented on chromosome 26, 16, 22 and 12, respectively. The four genes have been omitted for clarity in Figure 1. BmOBP9 and BmOBP28 are not mapped to chromosomes. The horizontal lines represent the locations of each OBP gene. Transcriptional orientations of OBP genes are indicated by (+) or (-).

Figure 2

Four representative OBP gene clusters present in silkworm. Four gene clusters are located on scaffold2529, scaffold2902, scaffold2943 and scaffold3052, respectively. Each gene is depicted by arrowheads presenting the orientation of transcription in the scaffold. Three non-olfactory genes between Obp1 and Obp2 in scaffold3052 have been omitted for clarity.

Figure 3

Alignment of the silkworm OBP-like family members. The signal peptides are boxed. Conserved residues are shown with a green or light green background. Highly conserved cysteine residues are marked by dark arrowheads. The rectangles under the alignment represent α-helices identified in BmorPBP1. Vertical bars indicate that the intron occurs between codons. Backward slanted separators and forward slanted separators point out the splice sites within codons after the first base and the second base, respectively. Six conservative splice sites are marked by hollow arrowheads.

Figure 4

Phylogenetic comparison of the OBP protein family members in the silkworm. An unrooted distance (neighbor-joining) tree was constructed using an alignment of the silkworm OBP-like family members after removing the highly divergent signal peptide sequences at the N-terminus. Bootstrap support (1000 replications) is indicated at the major nodes.

Figure 5

A Neighbor-joining unrooted tree of annotated OBP protein among B. mori, D. melanogaster, A. gambiae, T. castaneum and A. mellifera. Bootstrap support is based on 1000 resampled data sets. The tree is condensed to show only branches with 15% bootstrap support or higher. Color of gene names indicates species.

Figure 6

Expression patterns of silkworm OBPs in multiple tissues of larvae on day 3 of the fifth instar. The levels of expression are illustrated by a five grade color scale representing relative expression levels of < 500, 500–1000, 1000–2000, 2000–6000 and more than 6000.

Figure 7

The developmental expression patterns of silkworm OBPs. The columns represents fifteen different sample time points: day three of the fifth instar and fourteen different times after spinning: 0 hour, 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days and adult.

Figure 8

Four candidate PBP gene expression levels in the antennae of moths. Expression levels relative to the control gene BmActin3 are quantified by QRT-PCR. Bars on each column represent SD for three independent experiments.