Use of a chilo suppressalis Csupobp4 gene

Abstract

The application belongs to the field of agricultural pest control and molecular biology technology, and provides application of Chilo suppressalis CsupOBP4 gene, which can be used for controlling Chilo suppressalis, detecting phoxim resistance of Chilo suppressalis or preparing phoxim synergist. The application provides a precise action target with clear structure for developing a new generation of insecticides, a pesticide synergist or a nucleic acid pesticide; and can be used for developing a resistance rapid detection technology based on the target, a new pesticide synergist and an RNAi biological pesticide and other green prevention and control products.

Description

Technical Field

[0001] This invention belongs to the field of agricultural pest control and molecular biology technology, specifically involving a rice stem borer odor-binding protein gene CsupOBP4 and its application in reducing the larval sensitivity to phoxim. Background Technology

[0002] The information disclosed in this background section is intended to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Borer borer ( Chilo suppressalis Rice stem borers (Chilodon grandiflorus) are among the most destructive borers in rice production, posing a serious threat to global food security. Currently, chemical control remains the primary method for controlling this pest, with phorate, a highly effective and broad-spectrum organophosphate insecticide, being widely used in the field. However, due to long-term, high-intensity use, rice stem borers have developed varying degrees of resistance to commonly used insecticides such as phorate, leading to decreased control effectiveness, increased pesticide usage, and consequently, a series of problems including environmental pollution, ecological damage, and pesticide residues.

[0004] Odor-binding proteins (OBPs) are a class of small, soluble proteins that play a crucial role in the chemoreceptive system of insects. Traditionally, they are thought to be responsible for recognizing, binding, and transporting odor molecules (such as sex pheromones and plant volatiles), thereby mediating insect behaviors such as foraging, courtship, oviposition, and predator avoidance. Recent studies have shown that OBPs, in addition to their olfactory recognition role, may also bind to exogenous harmful substances (including pesticides) and participate in detoxification processes. For example, in the peach aphid (… Myzus persicae In *Spodoptera litura*, overexpression of OBP3 and OBP7 was associated with chlorpyrifos resistance; in *Spodoptera litura* (… Spodoptera litura In the study of *Trichoderma esculenta*, GOBP2 exhibited high binding affinity for a variety of herbicides and insecticides; in the case of *Trichoderma esculenta* (…), GOBP2 showed high binding affinity for various ... Athetis lepigone In this study, AlepGOBP2 can bind to chlorpyrifos and phoxim. These findings suggest that OBPs may participate in resistance formation by reducing the effective concentration of insecticides reaching the target site through mechanisms such as "binding-masking" or "transport-metabolism".

[0005] Although the interaction between OBPs and insecticides has been preliminarily revealed in various pests, and several OBP members (such as GOBP1, GOBP2, and OBP8) have been reported to bind to host plant volatiles and sex pheromones in the rice stem borer, their role in insecticide resistance has not been systematically studied. In particular, the existence of specific binding OBPs in the rice stem borer against phoxim, their binding mechanisms, and physiological functions remain unknown, limiting the ability to understand resistance mechanisms at the molecular level and develop novel control strategies. Summary of the Invention

[0006] In view of the problems in the prior art, the present invention provides a rice stem borer odor-binding protein CsupOBP4, which can effectively increase the sensitivity of rice stem borer larvae to phorate by silencing this gene.

[0007] To achieve the above objectives, the present invention adopts the following technical solution.

[0008] A type of rice stem borer ( Chilo suppressalis Odor-binding protein, abbreviated as CsupOBP4, is used in the control of rice stem borer, the detection of rice stem borer resistance to phoxim, and the preparation of phoxim synergists.

[0009] Borer borer ( Chilo suppressalis The odor-binding protein has the amino acid sequence shown in SEQ ID NO:1. Preferably, the nucleotide sequence of the above protein is shown in SEQ ID NO:2.

[0010] The phoxim synergist is a CsupOBP4 protein inhibitor and an RNAi reagent.

[0011] The CsupOBP4 protein inhibitor can reversibly or irreversibly inhibit the function or activity of the CsupOBP4 protein. Preferably, the CsupOBP4 protein inhibitor is an antibody drug targeting the CsupOBP4 protein. Preferably, the CsupOBP4 protein inhibitor binds to at least one of Tyr26, Ile34, Leu53, Met118, Trp119, Leu122, His35, Ser49, and Tyr115. In some embodiments, the CsupOBP4 protein inhibitor binds to Ile34 and / or Met118 residues.

[0012] The RNAi reagent can target the mRNA of the CsupOBP4 gene to induce post-transcriptional silencing of the CsupOBP4 gene, wherein the mRNA is the mature mRNA of the CsupOBP4 gene or a precursor of the mRNA. Preferably, the RNAi reagent is selected from siRNA, shRNA, and gRNA.

[0013] A method for controlling rice stem borer includes the following steps: By targeting the CsupOBP4 protein, small molecule compounds, peptides, or nucleic acids that can inhibit its binding with phoxim were designed and screened for application to rice stem borer, thereby enhancing the insecticidal effect of phoxim.

[0014] A method for detecting resistance to phorate in rice stem borer includes the following steps: Rice stem borer larvae and susceptible rice stem borer larvae were collected, and the content of CsupOBP4 protein or its mRNA was detected. The resistance level of each field population to phorate was determined based on the expression level.

[0015] The present invention also provides a reagent and kit for detecting resistance to phorate, comprising a detection reagent containing CsupOBP4 protein or mRNA.

[0016] The present invention has the following advantages: The rice stem borer odor-binding protein CsupOBP4 provided in this invention plays a crucial role in mediating the low sensitivity of rice stem borer larvae to phorate, providing a structurally well-defined and precise target for the development of next-generation insecticides, pesticide synergists, or nucleic acid pesticides. This target originates from the pest's own resistance mechanism, exhibiting strong specificity and facilitating the design of green control products that are highly effective against the rice stem borer and safe for non-target organisms and the environment. Based on the positive correlation between CsupOBP4 expression levels and resistance levels, specific molecular detection methods and kits can also be established to achieve rapid and quantitative monitoring of resistant populations in the field. This provides a reliable technical tool for developing regional precision resistance management strategies and scientific pesticide application plans, helping to delay the development of resistance. The target protein provided by this invention has the potential to extend the lifespan of existing important insecticides such as phorate, reducing agricultural production costs and environmental pollution risks. This invention provides important theoretical basis and application prospects for the development of green control products such as rapid resistance detection technologies, novel pesticide synergists, and RNAi biopesticides based on this target. Attached Figure Description

[0017] Figure 1 The transcriptome differential analysis (A) and odor-binding protein expression levels (B) of rice stem borer larvae under phorate stress. Figure 2 The expression level of CsupOBP4 in rice stem borer larvae after different durations of phorate stress; Figure 3 The fluorescence binding curve (A) and Scatchard plot (B) of CsupOBP4 protein and probe 1-NPN are shown. Figure 4 The curves show the competitive binding of CsupOBP4 protein to three insecticides. Figure 5It is a CsupOBP4 three-dimensional structure prediction model based on AlphaFold2; Figure 6 This is a Laplace plot assessing the reliability of the CsupOBP4 protein model; Figure 7 Phoxim mainly works by molecularly docking with the CsupOBP4 protein; Figure 8 These are SDS-PAGE electrophoresis images of the CsupOBP4 Ile34 mutant (A) and the Met118 mutant (B); Figure 9 The binding curves (A), Scatchard plot (B), and binding affinity to phoxim (C) of the two mutant proteins, Ile34 and Met118. Figure 10 The gene silencing efficiency of CsupOBP4 after dsRNA feeding (A) and the lethality of phoxim to larvae under different treatments (B). Figure 11 This represents the mRNA expression level of CsupOBP4 in different populations of rice stem borer larvae. Detailed Implementation

[0018] The present invention will be further described below with reference to the embodiments and accompanying drawings, but the present invention is not limited to the following embodiments.

[0019] The kits and methods used in the following examples are merely examples of specific implementation methods. In actual implementation, other products or methods with equivalent functions may be used.

[0020] Example 1: Differences in CsupOBP4 expression in rice stem borer larvae under different treatments 1. Differential Gene Acquisition The toxicity of phoxim to third-instar larvae of the rice stem borer was assessed using an artificial feed-toxin mixture method. Phoxim was first dissolved in acetone to prepare a stock solution, which was then diluted to a series of concentrations with an aqueous solution containing 0.5% Triton X-100. This stock solution was then mixed with artificial feed at a 1:9 ratio. The control group received the same treatment with acetone and an aqueous solution containing 0.5% Triton X-100. Ten third-instar larvae were placed in each glass tube (1.5 cm in diameter, 7.5 cm in height) as a replicate, with five replicates per treatment. Larval mortality was recorded every 24 hours after treatment. Mortality was defined as the inability to turn over, lack of response, or ataxia within one minute of being gently touched with a soft brush. This experiment established a concentration-response relationship, and based on this, the sublethal concentration (LC50) of phoxim was determined. 30 ), obtained from phoxim LC 30The third instar rice stem borer larvae were treated, with untreated larvae serving as a control. TRIzol from ThermoFisher Scientific (USA) was used. TM Total RNA was extracted using reagents according to the manufacturer's instructions. First-strand cDNA was synthesized using the HiScript III first-strand cDNA synthesis kit (including gDNA removal), and a cDNA library was constructed. Paired-end sequencing was then performed on an Illumina Novaseq 2500 platform. Low-quality reads and adapter sequences were filtered from the raw sequencing data to obtain high-quality, high-throughput sequencing data. The data were aligned to the *Taxopoda dilatatus* reference genome using HISAT2 software, and transcript assembly and reconstruction were performed using StringTie software. The assembled single genes were compared with the NR, Swiss-Prot, COG, KOG, KEGG, and Pfam databases for functional annotation. Gene expression levels were standardized and quantified using FPKM values. Differentially expressed genes between the phorate-treated group and the control group were screened using a Fold Change ≥ 2 and an False Discovery Rate (FDR) < 0.01 as the threshold.

[0021] Experimental results are as follows Figure 1 As shown. Based on the above transcriptome data, volcano plot analysis ( Figure 1 Results (A) showed that, compared with the control group, the expression of 11,748 genes in the phorate-treated group did not change significantly; 50 significantly upregulated genes and 179 significantly downregulated genes were identified. Among the 19 odor-binding protein family genes identified, the expression difference of CsupOBP4 was particularly significant, with its expression level in the treated group being significantly higher than that in the control group. Figure 1 (B)

[0022] 2. Expression of CsupOBP4 at different time points after phoxim treatment RNA was extracted from the larvae of the treatment group and the control group 48 h and 72 h after phorate treatment and reverse transcribed into cDNA. The expression dynamics of CsupOBP4 at different time points after phorate treatment were detected by qRT-PCR.

[0023] Table 1 Primer pairs for specific qRT-PCR detection of the CsupOBP4 gene The results showed that, compared with the control group, CsupOBP4 was significantly upregulated at both 48 h and 72 h after treatment, indicating that the gene was continuously highly expressed under phorate stress. Figure 2 ).

[0024] Example 2: In vitro fluorescent competitive binding of CsupOBP4 protein to phoxim 1. Recombinant protein expression The sequence of CsupOBP4 containing a complete open reading frame was obtained from the transcriptome data of Example 1. The nucleotide sequence of the coding region of CsupOBP4 is shown in SEQ ID NO:2, and the amino acid sequence is shown in SEQ ID NO:1.

[0025] Table 2 Primer pairs for constructing the CsupOBP4 expression vector Using head cDNA from the rice stem borer larvae as a template, the coding region of the CsupOBP4 gene was amplified using specific primers CsOBP4-pet32a-F (SEQ ID NO:5) and CsOBP4-pet32a-R (SEQ ID NO:6). The PCR product was purified and processed using ClonExpress from Vazyme Biotech Co., Ltd. ® The One-Step Cloning Kit ligates the target fragment into the prokaryotic expression vector pET-32a to construct a recombinant expression plasmid. The correctly sequenced recombinant expression plasmid is transformed into BL21 (DE3) chemocompetent cells from Trangen Biotechologies. Single colonies are picked and inoculated for culture until the bacterial culture OD... 600 When the value reached 0.6, isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 1 mM, and the expression of the target protein was induced at 37℃.

[0026] The induced bacterial cells were collected by centrifugation and lysed by sonication to obtain a crude protein extract. The recombinant protein was purified by affinity chromatography using ABclonal's Ni-NTA agarose to obtain the histidine-tagged CsupOBP4 fusion protein. The molecular weight and purity of the purified protein were analyzed by SDS-PAGE. The resulting protein solution was dialyzed four times at 4°C in a urea-containing gradient buffer to finally obtain the biologically functional, soluble CsupOBP4 recombinant protein.

[0027] 2. CsupOBP4 protein competitively binds to phoxim via in vitro fluorescence. SpectraMax using Molecular Devices ® Fluorescence signal detection was performed using the M2 multi-functional microplate reader. N-phenyl-1-naphthylamine (1-NPN) was used as the fluorescent probe, with the excitation wavelength set at 337 nm and the emission wavelength scanning range of 400-415 nm.

[0028] The purified CsupOBP4 protein (2 μM) was dissolved in 20 mM Tris-HCl buffer (pH 7.4) and titrated with 1-NPN to achieve a final concentration range of 2–20 μM. Fluorescence intensity changes were measured, and the dissociation constant K between CsupOBP4 and 1-NPN was calculated by fitting a binding curve. d = 6.44 ± 0.32 μM ( Figure 3 (A). Scatchard plotting shows a single binding site between the protein and the probe. Figure 3 (B)

[0029] Phoxim, chlorantraniliprole, and chlorpyrifos were prepared into 1 mM stock solutions using methanol. In systems containing 2 μM SupOBP4 protein and 2 μM 1-NPN, phoxim, chlorantraniliprole, or chlorpyrifos were added to final concentrations ranging from 2 to 20 μM, and their competitive quenching effects on 1-NPN fluorescence were measured. The competitive dissociation constant (Ki) was calculated using the following formula: Ki = [IC 50 ] / (1 + [1-NPN] / K 1-NPN ); ; in, [IC 50 The concentration (μM) of the competing agent that reduces the initial fluorescence of 1-NPN by 50%; [1-NPN] is the concentration (μM) of free 1-NPN; K 1-NPN The value represents the binding constant between the protein and 1-NPN. The fluorescence values ​​of 1-NPN at different concentration gradients are calculated using the One sitebinding (hyperbola) equation. The results are as follows Figure 4 As shown, CsupOBP4 exhibits a significant binding affinity for phoxim, with a Ki of 4.95 ± 0.24 μM; while no significant binding was detected for chlorantraniliprole and chlorpyrifos, confirming that the CsupOBP4 protein has a high affinity and binding specificity for phoxim.

[0030] Example 3: In vitro fluorescence competitive binding of CsupOBP4 protein mutant to phoxim 1. Molecular docking (1) Three-dimensional structure prediction and model evaluation: The three-dimensional structure of the CsupOBP4 protein was predicted using AlphaFold 2, and the results are as follows: Figure 5As shown, the CsupOBP4 protein is composed of eight α-helices.

[0031] Table 3. Statistics of Ramachandran plots The model was evaluated using Ramachandran plots from Discovery Studio 4.5 software. The results are as follows: Figure 6 As shown in Table 3, 91.4% of the amino acid residues are located in the optimal allowed region, and no residues are located in the disallowed region, indicating that the obtained model has reliable stereochemical quality.

[0032] (2) Molecular docking and binding mode analysis: The three-dimensional structure of phoxim was obtained from the NCBI PubChem database. Using AutoDock Vina 1.1.2 software with default parameters, phoxim was docked to the predicted structure of CsupOBP4, and the optimal binding conformation was selected based on the lowest binding energy. The docking results were visualized and analyzed using PyMOL software.

[0033] The results are as follows Figure 7 As shown, phorate mainly interacts with nonpolar residues (Tyr26, Ile34, Leu53, Met118, Trp119, Leu122) and polar residues (His35, Ser49, Tyr115) in the CsupOBP4 binding pocket, forming hydrogen bonds with Ile34 and Met118 residues.

[0034] 2. Expression of mutant recombinant proteins Using Mut Express from Vazyme Biotech Co., Ltd. ® Using the Fast Mutagenesis Kit V2, mutant plasmids for Ile34 and Met118 were constructed according to the instructions. The mutant plasmids were then transformed into expression strains following the method described in Example 2, expression was induced, and the proteins were purified to obtain the corresponding mutant proteins: the Ile34 mutant and the Met118 mutant.

[0035] SDS-PAGE analysis showed that the molecular weight of the mutant protein obtained was consistent with the expected size. Figure 8 ).

[0036] 3. The CsupOBP4 protein mutant competitively binds to phoxim via in vitro fluorescence. The obtained mutant protein was subjected to in vitro fluorescence competitive binding detection according to the method in Example 2.

[0037] The Kd of the Ile34 mutant was 6.56 ± 0.35 μM, and the Kd of the Met118 mutant was 8.01 ± 0.47 μM; the binding curves of the mutants ( Figure 9 (A) and Scatchard diagram ( Figure 9 The mutant protein (Ile34) was similar to the wild-type protein, indicating that the mutation did not significantly affect the overall protein folding and probe binding; however, both the Ile34 and Met118 mutant proteins completely lost their ability to bind to phoxim. Figure 9 (C)

[0038] Example 4: In vitro fluorescence competitive binding of CsupOBP4 protein mutant to phoxim 1. Synthesis and Validation of dsRNA Primers CsOBP4-T7-F and CsOBP4-T7-R with a T7 promoter were designed and synthesized targeting the specific sequence of the CsupOBP4 gene.

[0039] Table 4 Primers for the T7 promoter Using the T7 RNAi Transcription Kit from Vazyme Biotech Co., Ltd., in vitro transcription was performed according to the instructions to synthesize double-stranded RNA (dsOBP4) targeting CsupOBP4. Simultaneously, control double-stranded RNA (dsEGFP) was synthesized using the enhanced green fluorescent protein (EGFP) gene as a template.

[0040] dsOBP4 was diluted with sterile water to approximately 600 ng / μL and evenly dropped onto the surface of the artificial feed. An equal volume of dsEGFP solution of the same concentration was used as a negative control. The treated feed was then provided to second-instar larvae for consumption. The experiment was conducted in 5 replicates, with 10 larvae per replicate.

[0041] Forty-eight hours after feeding with dsRNA, the larvae were fed a normal diet for another 48 hours. Larvae from both the treatment and control groups were then collected. The mRNA expression level of CsupOBP4 in the larvae was detected using the qRT-PCR method described in Example 1.

[0042] The results are as follows Figure 10 As shown in Figure A, compared with the dsEGFP control group, the expression level of CsupOBP4 in larvae fed with dsOBP4 decreased significantly to 44.3%.

[0043] 2. Effect of CsupOBP4 knockdown on phorate sensitivity Following the above method, 48 hours after feeding dsRNA, replace the feed with toxic feed (containing a sublethal concentration (LC50) of phorate).30 ), continue feeding for 48 h, and record and count the mortality rate of larvae in each group.

[0044] Under the treatment of phoxim LC 30 concentration, the larval mortality rate of the CsupOBP4 knockdown group was significantly higher than that of the control group (62% vs. 34%, P <0.01; Figure 10 B in).

[0045] Example 5 Application of CsupOBP4 in resistance monitoring According to NY / T 2058-2014 Technical Specification for Rice Stem Borer Resistance Monitoring, field populations of Chilo suppressalis were collected from three regions: Tangnan County of Nanchang (NC), Poyang County of Shangrao (PY), and Xiajiang County of Ji'an (XJ), and reared indoors to the 3rd instar larvae. The capillary droplet method was used to determine the toxicity of phoxim to the above populations and the long-term indoor reared strain (SS). The resistance ratio (RR) was calculated based on the LD of phoxim in Appendix B.1 of NY / T 2058-2014 50 value, and the resistance level was judged according to the criteria that 5.0<RR≤10.0 is low resistance level, 10.0<RR≤100.0 is medium resistance level, and RR>100.0 is high resistance level.

[0046] The method in Example 1 was used to detect the mRNA expression level of OBP4 in 3rd instar larvae of different populations. The results are shown in Figure 11 : There are significant differences in the OBP4 gene expression levels among the resistant Chilo suppressalis populations from the three regions. Among them, the OBP4 expression level of the Nanchang population is the highest, which is significantly higher than that of the other two regions and the susceptible population (SS), suggesting that its resistance level to phoxim is relatively the highest, reaching a medium resistance level; the expression level of the Xiajiang population is the second, with a low resistance level but a relatively high resistance risk; while the Poyang population has the lowest expression level, and compared with the susceptible population overall, the expression level has increased to a certain extent, with a low resistance level and a relatively low resistance risk. In general, all three field populations show a certain degree of resistance to phoxim, and are generally at a medium to low resistance level, among which the resistance development in Nanchang is the most obvious, followed by Xiajiang, and Poyang is relatively mild. The results suggest that the expression level of OBP4 can be used as a molecular marker for phoxim resistance assessment, to assist in evaluating the differences in resistance development or adaptation status of different field populations.

[0047] The above description is only the embodiments of the present application, and is not intended to limit the present application. For those skilled in the art, various modifications and variations can be made to the present application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present application shall be included in the scope of the claims of the present application.

Claims

1. The application of an RNAi reagent containing a rice stem borer odor-binding protein in the control of rice stem borers or in the preparation of phoxim synergists, characterized in that, The RNAi reagent targets the mRNA of the rice stem borer odor-binding protein, causing post-transcriptional silencing of the gene; The amino acid sequence of the rice stem borer odor-binding protein is shown in SEQ ID NO:1; The nucleotide sequence of the rice stem borer odor-binding protein is shown in SEQ ID NO:2; The RNAi reagent is dsRNA; The dsRNA was obtained by amplifying the nucleotides encoding the rice stem borer odor-binding protein shown in SEQ ID NO:2 using primers with nucleotide sequences as shown in SEQ ID NO:7 and SEQ ID NO:8, respectively. The amplified products were then transcribed using the T7 RNAiTranscription Kit.

2. The application according to claim 1, characterized in that, The mRNA is a mature mRNA or a precursor of mRNA.

3. A method for controlling the rice stem borer, characterized in that, Includes the following steps: Using the rice stem borer odor-binding protein as a target, nucleic acids that can inhibit its expression were designed and screened for application to the rice stem borer to enhance the insecticidal effect of phorate; the amino acid sequence of the rice stem borer odor-binding protein is shown in SEQ ID NO:1; The nucleic acid is dsRNA; The dsRNA was obtained by amplifying the nucleotides encoding the rice stem borer odor-binding protein shown in SEQ ID NO:2 using primers with nucleotide sequences as shown in SEQ ID NO:7 and SEQ ID NO:8, respectively. The amplified products were then transcribed using the T7 RNAiTranscription Kit.

4. A method for detecting resistance to phorate, characterized in that, Includes the following steps: Collect rice stem borer larvae and test the content of the protein or its mRNA as described in claim 1. Determine the resistance level of each field population to phorate based on the expression level.

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