Identification Of Insect Olfactory Related Genes Using Bioinformatics

Chemical sensing is critically important to insect survival. For insects, olfaction, which is the primary sensory perception modality, is used to detect odor molecules in the environment. These cues guide the insect towards food, mating partners, and oviposition sites and also facilitate detection of predators and toxic compounds. The antenna is a specialized organ for insect sensing, especially for olfaction. Several types of sensilla, which are specialized hair-like, multi-pore structures, cover the surface of the antennae. Olfactory receptor neurons(ORNs) and auxiliary structures are housed within the antennae, positioned at the sensilla root. For most of the olfactory sensilla, there are underlying 1-4 ORNs that extend their dendrites up into the sensilla and project their axons into the antennal lymph on towards the brain. The ORNs convert ecologically relevant volatile chemicals into an electrical impulse, which is transported to the primary olfactory center of the brain, the antennal lobe. Within the sensilla-ORN structure, a number of gene families have been identified as having active roles in olfaction. These include the odorant binding proteins(OBPs), and chemosensory proteins(CSPs), odorant receptors(ORs) and ionotropic receptors(IRs). Understanding of insect olfaction system may benefit to the development of insect behavior regulator, which regulate the population of insect in field by controlling insect’s behavior of reproduction and migration. The characters of insect olfaction related genes, which are highly divergent in sequence similarity and extremely low in expression, make it very difficult for traditional methods to discover, such as homology cloning and EST screening.Second generation high-throughput DNA sequencing platforms, such as Roche 454, SOLID and Illumina, are powerful tools for the genome-level study of pest insects. Development of de novo assembly strategies has expanded the applicability of second generation sequencing to non-model organisms without a proper reference genome. It can be easy to obtain transcriptome datasets with far higher throughput by using de Bruijn graphs and pair-end sequencing, which means that low abundance m RNAs can be detected at a cost of only 1% of traditional expressed sequence tag(EST) library technology. But the amount of data generated by NGS sequencing is in gigabase level, which requires new tools for insect scientists to mine in such large datasets. Our research presents an insect specified workflow to identify olfaction related genes from insect genome sequences or transcriptome. By apply this workflow, we analyzed data of five species to identify their olfaction related genes, and analyzed their evolutional relationship among some relative species.1. We identified a total of 45 candidate Ago OR genes in the genome of A. gossypii. Of these, 22 genes encode putative, complete functional proteins and 9 are apparent pseudogenes. The average identity percentage of all 45 Ago ORs versus Ap ORs is 54.76%, 26 Ago ORs have identities higher than 50%. 16 Ago OR/Ap OR orthologue cluster were found and their similarity among species are greater than 70%. 5 Ago OR species clades were also found, as their similarities is lower than 50%. By comparing their exon/intron structure, we identified 5 conserved intron splice sites among Ago ORs and Ap ORs. For IR genes, we identified 13 Ago IRs, in which 11 got full-length ORF. All the Ago IRs share conserved S1, S2, PORE domains but none of them retain the complete set of i Glu Rs characteristic residues. The expression profile showed that Ago ORs and Ago IRs mainly express in olfaction organisms but Ago IRs are also show expression in leg and body.2. Data mining of the C. suppressalis antenna transcriptome leads to the identification of 47 ORs, 20 IRs, 26 OBPs and 21 CSPs. Among the 47 candidate Csup ORs, 23 contain intact ORF and 6 belong to the pheromone receptor family. 13 out of 20 candidate Csup IRs contain full-length ORF; 13 Csup IRs have homolog genes found in other species and 5 Csup IRs are thought to be unique in C. suppressalis. 4 out of the 26 candidate Csup OBPs are thought to belong to the minus-C subgroup and the remaining 22 got all 6 conserved cysteine residues. Among the 21 candidate Csup CSPs, 18 contain intact ORF and signal peptide and all 21 CSPs contain 4 conserved cysteine residues.3. A total number of 110 olfaction related genes are found in the P. xylostella antenna transcriptome, including 53 candidate Pxyl ORs, 16 candidate Pxyl IRs, 24 candidate Pxyl OBPs and 15 candidate Pxyl CSPs. 23 out of 53 candidate Pxyl ORs contain full-length ORF and 7 are thought to be pheromone receptors. 9 out of 16 candidate Pxyl IRs contain the typical 3 transmembrane domains. 3 PBPs, 3 GOBPs and 19 OBPs make up the Pxyl OBP gene family and 20 of them have intact ORF and 23 have signal peptide.4. In antenna transcriptomes of two relative species H. zea and H. gelotopoeon, we respectively identified 62 Hzea ORs, 18 Hzea IRs, 38 Hzea OBPs and 60 Hgel ORs, 17 Hgel IRs, 38 Hgel OBPs. When analyze with olfaction genes from H. armigera, H. assulta and C. suppressalis, 17 OR ortholog groups are found in all 5 species. Meanwhile, ORs unique in Helicoverpa species are also found, which could be associated with Helicoverpa family specified chemical components.5. Phylogenomic analysis of H. zea, H. armigera, H. assulta and H. gelotopoeon reveals 1670 ortholog group(OG), which GO annotation are enriched in cellular process, metabolic process, single-organism process. Site-model Darwinish selection analysis shows that 379 OGs are under positive selection. These genes encode proteins making up cell, organelle and their function enriched in metabolic process, pigmentation, localization and response to stimulus.