Cloning, expression and binding characteristic of chemosensory protein BminCSP3 gene in Bactrocera minax (Diptera: Tephritidae)

Abstract: 【Objective】Bactrocera minax is one of the most destructive pest insects of citrus crops. Like most insects, B. minax needs to rely on host plant volatiles to search and locate mates and food sources. Insect chemosensory proteins play an important role in discriminating and binding the odors to the ol-factory receptors. In order to provide a theoretical basis for revealing the molecular mechanism of olfac-tory recognition of B. minax, the binding characteristics of the chemosensory protein BminCSP3 highly expressed in the antennae of adults to odorant ligands were determined.【Methods】The gene-specific primers were designed to clone the cDNA that encodes mature BminCSP3 from the antennal total RNA.The PCR product was first cloned into pGEM-T easy vector. Positive clones were confirmed by se-quencing. The plasmids of positive clones were digested by EcoRⅠ and Hind Ⅲ QuickCut® restriction enzymes. The digested products were separated by agarose gel electrophoresis. The expected band was purified from the agarose gel and ligated into the empty expression vector pET-32a (+), which had been digested with the same restriction enzymes. The resulting recombinant plasmid was sequenced to con- firm that it encoded the mature protein. The recombinant vector was transformed into BL21 (DE3) com- petent cells. The positive clones were confirmed by sequencing. Protein expression was induced at 28 ℃ for 5 h with 0.5 mmol · L^-1 isopropyl-β-D-thiogalactopryranoside (IPTG) when the culture OD600 reached 0.6. The bacterial cells were harvested by centrifugation. The cellular pellets were resuspendedin the lysis buffer and then sonicated. After centrifugation, the recombinant proteins were present main-ly in the supernatant. Recombinant proteins in the supernatant were purified by Ni-NTA His·bind Resin column, according to the manufacturer’s protocol. The His-tag was removed by digestion with recombi-nant enterokinase, and the target proteins were purified again by the column mentioned above and ana-lyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The concentration of the purified proteins was measured using BCA Protein Assay Kit. The binding properties of BminC-SP3 with 14 odorant ligands were detected by fluorescence competitive binding assay. Emission fluores-cence spectra were performed on F-7000 fluorescence Spectrophotometer in 1 cm light path quartz cu-vette with 10 nm slit width for both excitation and emission. The excitation wavelength was set at 337 nm and emission spectra were recorded between 370 nm and 500 nm. The fluorescent probe N-phenyl-1-naphthylamine (1-NPN) and the ligands used in the binding assay were dissolved in spectrophotomet-ric-grade methanol to yield 1 mmol·L^-1 stock solution. To determine the dissociation constant between 1-NPN and BminCSP3, 2 μmol·L^-1 BminCSP3 dissolved in 20 mmol·L^-1 Tris-HCl (pH 7.4) was titrated with 1 mmol·L^-1 1-NPN to the final concentration of 2-22 μmol·L^-1. The fluorescence intensities at the maximum fluorescence emission between 370 nm and 500 nm were plotted against the concentration of added 1-NPN. The curve was linearized using Scatchard plots. The concentrations of bound 1-NPN were evaluated from the values of fluorescence intensity assuming that the protein was 100% active with a stoichiometry of 1∶1 (protein∶ligand) at saturation. The affinities of the selected ligands were measured by competitive binding assays by adding the ligands from 1 to 52 μmol·L ^-1 into the 1-NPN/ BminCSP3 solution (both at 2 μmol·L^-1). The emission spectra were recorded between 370 nm and 500nm and the maximum fluorescence intensities were plotted against the ligand concentration after nor-malization relative to the intensity at zero ligand. For these competitor ligands, the dissociation con-stants were computed from the corresponding IC50 values using the quation: Ki = [IC50]/(1+[1-NPN]/K1-NPN), where [1-NPN] was the free concentration of 1-NPN and K1-NPN was the dissociation constant of the protein complex/1- NPN. All values reported were collected in three independent measurements.【Results】The open reading frame (ORF) of BminCSP3 is 474 bp, encoding 157 amino acids and con-taining four conserved cysteine residues , and is a typical hallmark of the chemosensory protein family. BminCSP3 is a secretory protein with a signal peptide of 22 amino acids and isoelectric point of 5.41,and it is a hydrophilic protein with Grand average of hydropathicity of -0.397. It has no transmembrane domain. In order to study the binding properties of BminCSP3 with the tested compounds, we prepared the recombinant proteins. Then, a competitive binding assay was performed with 1-NPN as the fluores-cence probe. The probe was excited at 337 nm and emission spectra were recorded between 370 nm and 500 nm. The fluorescent intensity of the 1- NPN alone was fairly low, then a strong blue shift, and peaked at about 410 nm with the presence of BminCSP3. The intensity of this peak was used to deter-mine the dissociation constant of the BminCSP3/1-NPN complex. The fitted curve based on the intensi-ty of this peak upon 1-NPN titration is shown in Fig 4. The binding curve and Scatchard plot indicated a dissociation constant of 4.49 μmol · L^-1, which was used to calculate the dissociation constants (Ki) of different volatile compounds in the competitive binding assay. The results of fluorescence competitive binding experiment showed that the affinity of BminCSP3 with 14 odor ligands was weak, and their Kivalues were more than 20 μmol·L^-1. It is generally believed that the Ki value is more than 20 μmol·L^-1, indicating that the affinity between the protein and the ligand is weak.【Conclusion】In insects, CSPs  gene with high expression in antennae often has olfactory function. However, no odor ligand with high affinity for BminCSP3 was found in this experiment. It is speculated that although BminCSP3 is highly expressed in the antennae, the protein may not be involved in the olfactory recognition process of B.minax, and BminCSP3 has physiological functions other than olfaction. Alternatively, we have not found the most suitable odor ligand for BminCSP3 in the present study. The experimental results have laid a foundation for further study on the specific functions of BminCSP3.