| Insects possess sensitive chemosensory systems that recognize a variety of chemical compounds. Using these sensitive olfactory systems, insects detect their environment to identify enemies, to find food sources, to recognize mates and to locate oviposition sites. In insects, two main classes of polypeptides have been identified in the lymph of chemosensilla:odorant-binding proteins (OBPs) and chemosensory proteins (CSPs). CSPs may represent a new class of soluble carrier proteins involved in insect chemoreception. However, the physiological function of CSPs remains unclear. In this study, seven full-length cDNAs encoding chemosensory proteins were cloned from Aphis gossypii. In order to understand the role of CSPs in Aphis gossypii, quantitative real-time polymerase chain reaction (qPCR) was used to determine the expression patterns of the 7 CSPs genes. Finally, a varity of volatile chemicals were used to analyze the binding properties in competitive binding assays.1. Molecular cloning and sequence analysis of AgosCSP genesBased on the transcriptomic data and using degenerate PCR, seven full-length CSP cDNAs were identified from Aphis gossypii, which were respectively named as AgosCSPl (GenBank accession no. KC017750), AgosCSP2 (GenBank accession no. KC017751), AgosCSP3 (GenBank accession no. KJ451424), AgosCSP A (GenBank accession no. KJ451425), AgosCSP8 (GenBank accession no. KC017753), AgosCSP13 (GenBank accession no. FJ387488) and AgosCSP17(GenBank accession no.KC017754). Sequence analysis showed that all the 7 CSPs share typical characteristics in CSPs. Each member had four conserved cysteines.2. Expression profiles of AgosCSPsThe expression levels of AgosCSPs in different ecologically developmental stages, different forms, and various tissues in the apterous autumn adults were examined by RT-PCR. The results indicated that the CSPs were widely expressed in different stages and various tissues. Such expression profiles indicated important chemosensory and non-chemosensory functions. AgosCSPl had a higher expression level in the alatae autumn adults and in the legs, indicating its role in host recognition. The expression levels of AgosCSP1 in nymphs were significantly higher than those in adult. Moreover, it was highly expressed in the aphids from cotton than in the aphids from overwintering host hibiscus. These results suggest that the AgosCSP1 gene is most likely involed in the adaptation of nymphs to their hosts. Furthermore, AgosCSP3 was highly expressed in the alatae aphids from cotton, indicating its role in the behavior of alatae aphids on the cotton hosts.3. Prokaryotic expression and purification of AgosCSPsIn order to study the function of CSPs in the chemical communication system, the AgosCSP proteins were expressed using a prokaryotic expression system. The AgosCSP genes were individually introduced into the pGEX-4T-1 expression vector, and then were expressed in the BL21(DE) Escherichia coli after the induction of IPTG. SDS-PAGE analysis revealed that all the expressed AgosCSPs were soluble. These recombinant proteins were purified through affinity chromatography, and the GST-tags were removed by enzyme digestion. Finally, six AgosCSP proteins were obtained.4. Ligand-binding affinity analyses of AgosCSPThe binding affinities of the 6 AgosCSPs were tested by fluorescence competitive binding experiment with 85 odorant compounds including the alarm pheromones. AgosCSPl showed high binding affinities to 1-aminoanthracene (2.43 μmol·L-1) and P-ionone (22.67 pmol·L-1). Since 1-aminoanthracene is one of plant volatiles and β-ionone is a volatile widerly distributed in flowers and other tissues of many plant species, it is suggested that AgosCSP1 is involved in the host recognition. However, the remaining 5 AgosCSPs had low binding affinities to all the 85 odorant compounds. They may play other roles rather than olfaction in A. gossypii. |
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