title: Identification, expression profiling and fluorescence-based binding assays of a chemosensory protein gene from the Western flower thrips, Frankliniella occidentalis.
abstract: Using RT-PCR and RACE-PCR strategies, we cloned and identified a new chemosensory protein (FoccCSP) from the Western flower thrips, Frankliniella occidentalis, a species for which no chemosensory protein (CSP) has yet been identified. The FoccCSP gene contains a 387 bp open-reading frame encoding a putative protein of 128 amino acids with a molecular weight of 14.51 kDa and an isoelectric point of 5.41. The deduced amino acid sequence contains a putative signal peptide of 19 amino acid residues at the N-terminus, as well as the typical four-cysteine signature found in other insect CSPs. As FoccCSP is from a different order of insect than other known CSPs, the GenBank FoccCSP homolog showed only 31-50% sequence identity with them. A neighbor-joining tree was constructed and revealed that FoccCSP is in a group with CSPs from Homopteran insects (e.g., AgosCSP4, AgosCSP10, ApisCSP, and NlugCSP9), suggesting that these genes likely developed from a common ancestral gene. The FoccCSP gene expression profile of different tissues and development stages was measured by quantitative real-time PCR. The results of this analysis revealed this gene is predominantly expressed in the antennae and also highly expressed in the first instar nymph, suggesting a function for FoccCSP in olfactory reception and in particular life activities during the first instar nymph stage. We expressed recombinant FoccCSP protein in a prokaryotic expression system and purified FoccCSP protein by affinity chromatography using a Ni-NTA-Sepharose column. Using N-phenyl-1-naphthylamine (1-NPN) as a fluorescent probe in fluorescence-based competitive binding assay, we determined the binding affinities of 19 volatile substances for FoccCSP protein. This analysis revealed that anisic aldehyde, geraniol and methyl salicylate have high binding affinities for FoccCSP, with KD values of 10.50, 15.35 and 35.24 μM, respectively. Thus, our study indicates that FoccCSP may play an important role in regulating the development of the first instar nymph and mediate F. occidentalis host recognition.
authors: State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China; Institute of Plant Protection, Ningxia Academy of Agriculture and Forestry Sciences, Yinchuan, China.
authors: Zhi-Ke
authors: ZK
authors: Zhang
authors: State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China.
authors: Zhong-Ren
authors: ZR
authors: Lei
mesh_terms: D015057:1-Naphthylamine / Q000031:analogs & derivatives / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D001681:Biological Assay / Q000379:methods*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D035264:Flowers / Q000469:parasitology*; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D017344:Genes, Insect; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008024:Ligands; D008297:Male; D008969:Molecular Sequence Data; D009928:Organ Specificity / Q000235:genetics; D010802:Phylogeny; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D015203:Reproducibility of Results; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013050:Spectrometry, Fluorescence; D061149:Thysanoptera / Q000235:genetics*
chemical_list: D018076:DNA, Complementary; D019476:Insect Proteins; D008024:Ligands; D011994:Recombinant Proteins; C005444:N-phenyl-1-naphthylamine; D015057:1-Naphthylamine
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