SlGSTE8 in Spodoptera litura participated in the resistance to phoxim and chlorpyrifos
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Aug 05, 2023 version files 16.21 KB
Abstract
Glutathione S-transferases (GSTs) were reported to participate in insecticides resistance by metabolic and antioxidant activities. In our previous study, an ε class gene of GSTs, SlGSTe8 in Spodoptera litura, was screened out to be upregulated in a population resistant to pyrethroids and organophosphates. SlGSTe8 was highly expressed in the larvae stage, and the digestive tissue, foregut, midgut and hindgut. While the relative expression level was low in the pupae stage and other tissues. To further explore its role in the resistance to pyrethroids and organophosphates, the metabolic activity to insecticides by its recombinant protein was determined by Ultra Performance Liquid Chromatography, and its antioxidant enzyme activity was evaluated by disc diffusion assay. The recombinant protein showed significantly metabolic activity to phoxim and chlorpyrifos, but not to fenvalerate, cyhalothrin or β-cypermethrin. After incubation, the depletion rate of chlorpyrifos is 85.3%, higher than that of phoxim (17.5%). Also, the inhibition zone around filter discs decreased significantly after exposure to cumene hydroperoxide in recombinant plasmid than vector only, suggesting significant antioxidant activity of SlGSTE8. Further modeling and docking analysis indicated that the 3D structure of SlGSTE8 were well shaped for phoxim and chlorpyrifos, with the binding energy -5.58 and -5.15 kcal/mol, respectively. Our work provides evidence that SlGSTe8 in S. litura plays important roles in phoxim and chlorpyrifos resistance.
Methods
1. Insects and chemicals
Spodoptera litura populations used in this study were described in Li et al. (2021a). Briefly, GX population of S. litura, kindly provided by Guangxi Tianyuan Biochemistry Corp Ltd, served as a susceptible population. QJ population of S. litura, collected from the field in 2019, served as a resistant population to pyrethroids and organophosphates. Compared with GX population, QJ population showed 3659-, 295- and 207-fold resistance to β-cypermethrin, phoxim and chlorpyrifos, respectively. And the resistance level to fenvalerate and cyhalothrin were higher than 25000- fold (Li et al., 2021b). The insects were reared in the insectary at 26±1℃, 60-70% relative humidity, 14:10 h of light:dark photoperiod, without exposure to insecticides.
Fenvalerate (93.4%, Jiangsu Changlong Chemicals Co., Ltd, China), β-cypermethrin (95.0%, Beijing Huarong Biochemical Co., Ltd, China), cyhalothrin (98.4%, Beijing Huarong Biochemical Co., Ltd, China), phoxim (91.0%, Beijing Huarong Biochemical Co., Ltd, China) and chlorpyrifos (95.0%, Beijing Huarong Biochemical Co., Ltd, China) were used as the tested insecticides.
2. Characterization of the relative expression of SlGSTe8 in the QJ population
The spatiotemporal expression of SlGSTe8 in the QJ population and the comparison of its expression in the third instar larvae from QJ and GX populations were determined by quantitative RT-PCR (qRT-PCR). The first, second, third, fourth, fifth, sixth instar larvae and pupae, tissues including head, cuticle, foregut, midgut, hindgut, malpighian tubules, fat body from the fifth instar larvae were sampled for RNA extraction by TaKaRa MiniBEST Universal RNA Extraction Kit (Takara, Dalian, China). qRT-PCR reaction system was conducted using SuperReal PreMix Plus (SYBR Green, Tiangen, China) on ABI Prism 7500 Real-Time PCR System (Applied Biosystems by Life Technologies, Foster, CA, USA). Each reaction was carried out in triplicate with 3 independent mRNA samples. Primer pairs used in this study were listed in Table 1. EF1α and RPL10 were used as housekeeping gene (Lu et al. 2013). The relative expression was quantified according to the 2−ΔΔCt method.
3. Heterologous expression and the determination of enzyme activity of the recombinant protein SlGSTE8
The cDNA full length of SlGSTe8 was cloned and expressed in a prokaryotic expression system E. coli BL21 (DE3) (Tiangen). The heterologous expression of SlGSTE8 was conducted as described in Li et al. (2021a). Briefly, restriction enzyme sites Nde I and Xho I were introduced to the cDNA full length of SlGSTe8. Plasmid pET-26b(+) and the cDNA of SlGSTe8 were both digested by Nde I and Xho I (Takara), and the recombinant plasmid pET-26b(+)/SlGSTE8 was constructed using T4 DNA ligase (Tiangen). pET-26b(+)/SlGSTE8 was transformed in BL21 (DE3). Then, IPTG (Tiangen) at a final concentration of 1.0 mmol mL-1 was added in LB liquid medium to induce the expression following incubation at 37 °C, 160 rpm for 3 h. The recombinant protein was extracted using the following procedure: centrifuging at 4 °C, 10,000 g for 10 min; resuspending in potassium phosphate buffer (pH 7.0, 20 mmol L-1); lysing on ice by ultrasonic (Sonics and Materials, Inc., USA); final centrifuging at 4 °C, 20,000 g for 30 min. The supernatant was collected and purified by HisPurTM Ni-NTA Purification Kit (Thermo Fisher Scientific Inc., Shanghai, China) with a linear gradient of 0-300 mmol L-1 imidazole, desalted by Zeba™ Spin Desalting Columns (Thermo Fisher Scientific Inc.).
The extracts of pET-26b(+), crude recombinant protein and purified recombinant protein were detected by SDS-PAGE (12.5%). Briefly, a gel containing 12% seperation gel and 5% stacking gel was prepared. Samples were loaded in the gel and run in buffer at 80 v for 15 min and 150 v for 1 h. The gels were then transferred to Coomassie Blue R-250 (Tiangen) for staining. To detect the enzyme activity, Km and Vmax values of recombinant protein SlGSTE8 were determined using 2,4-dinitrochlorobenzene (CDNB) as the model substrate (Li et al., 2021a).
4 . in vitro metabolism activity of SlGSTE8 to pyrethroids and organophosphates
Mixture containing purified recombinant protein SlGSTE8 (180 μL, 1 mg L-1) and PBS (250 μL, 0.1 mol L−1, pH 6.8) was preheated at 37 °C and added with insecticides (20 μL, 500 mg L-1), freshly prepared GSH (50 μL, 100 μmol mL-1, pH 7.0) following incubation at 37 °C for 3 h. The reaction was stopped by adding acetonitrile at equal volume and the residual insecticides were extracted by adding saturated sodium chloride, shaking at 200 rpm for 2 h, centrifuging at 3,000 rpm for 2 min. The residual insecticides from the upper organic phase passed through a 0.22 μm membrane and quantified by Ultra Performance Liquid Chromatography using the detection method described in Li et al. (2021a). Briefly, the injection volume was 3 μL, and acetonitrile/water (80:20, v/v) was used as the mobile phase with flow rate of 0.4 mL min−1 using Acquity UPLC BEH C18 analytical column (2.1 mm × 100 mm, 1.7 μm, Waters Acquity UPLC system, USA). The residue of fenvalerate, β-cypermethrin, cyhalothrin, phoxim and chlorpyrifos were detected at 220, 220, 230, 280 and 290 nm, respectively using a PDA detector. The boiled recombinant protein SlGSTE8 was used as control.
5. The antioxidant activity of SlGSTE8 determined by disc diffusion assay
The antioxidant activity of SlGSTE8 was determined by disc diffusion assay using cumene hydroperoxide (CHP, J&K, Beijing, China) as the ROS inducer referred to Labade et al. (2018). A total of 0.2 mL of pET-26b(+)/SlGSTE8 expressed in E. coli with OD600=1 was spread evenly on the LB plate containing kanamycin (50 mg L-1) and IPTG (1 mmol L−1). Then the same volume of culture of empty vector pET-26b(+) in E. coli only was used as control. The plates were incubated at 37 ℃ for 1 h to make the bacterial resuscitate. Then, filter discs around 5 mm soaking with CHP at 0, 50, 100, 200, 300 mmol L−1 were laid on the surface of LB plate. After incubation at 37 ℃ for 48 h, the inhibition zone around each filter disc was measured twice by cross. Each treatment was repeated three times.
6. Homology modeling and molecular docking
The 3D protein structure of SlGSTE8 was modeled online by Swiss model server (Bienert et al., 2017; Waterhouse et al., 2018, https://swissmodel.expasy.org/) using GSTs isozyme E7 from Drosophila melanogaster (PDB ID: 4png.1.A) as the most suitable modeling template. Their sequence identity was 36.15%. The conserved domains of SlGSTE8 were analyzed by CD-search in NCBI (https://www.ncbi.nlm.nih.gov/cdd/?term=). The molecular structures of chlorpyrifos and phoxim were sketched in ChemDraw 20.0 (PerkinElmer, Shelton, CT, USA) and imported to Chem3D (PerkinElmer) to obtain its 3D structure. Molecular docking of SlGSTE8 with these insecticides were conducted using Autodock 4.2 (Morris et al., 2009; Autodock, San Diego, CA, USA) with the default parameters. The possible insecticide ligand-binding pockets and binding free energies were predicted. The interaction of optimal conformation of SlGSTE8 and ligand were further predicted with Protein-Ligand Interaction Profiler (PLIP, https://plip-tool.biotec.tu-dresden.de/plip-web/plip/index) online. To ensure a stable conformation of the complex, a Ramachandran plot of SlGSTE8 and insecticides were conducted using Procheck of SAVEs (Laskowski, et al., 1993; https://servicesn.mbi.ucla.edu/SAVES/). The results of modeling and docking were visualized by PyMOL (New York, NY, USA).
7. Statistical analysis
All the data were analyzed by SPSS 16.0 (SPSS, Chicago, IL, U.S.A.). The spatiotemporal expression of SlGSTe8 was analyzed by One-way analysis of variation (ANOVA) followed by Tukey’s HSD multiple comparisons test. Expression of SlGSTe8 in the QJ and GX populations, the in vitro metabolism activity of SlGSTE8 and the data from disc diffusion assay were evaluated by Students’ t-test. P < 0.05 was regarded to be statistically significant.
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