An MTHFD2 protein and its applications

Abstract

This invention provides an MTHFD2 protein and its applications, belonging to the field of genetic engineering technology. The amino acid sequence of the protein is shown in SEQ ID NO.1, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.2. This invention also provides the application of the MTHFD2 protein in the preparation of products that inhibit insect growth and development, as well as a high-throughput screening method based on this protein. Experiments have confirmed that lufenuron can specifically bind to MTHFD2 and inhibit its enzymatic activity; knocking out the MTHFD2 gene in Drosophila using the CRISPR / Cas9 system leads to a decrease in egg production, a prolonged developmental period, and a significant reduction in pupation and emergence rates. This invention is the first to clearly identify MTHFD2 as a direct target of lufenuron, which can be used for screening novel insecticides, monitoring insect resistance, and creating green pesticides.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, and in particular relates to an MTHFD2 protein and its applications. Background Technology

[0002] Benzoylurea (BPUs) insecticides are a classic and important class of insect growth regulators (IGRs). With their advantages of high efficiency, low toxicity, and environmental friendliness, they have been widely used in the control of agricultural, forestry, and sanitary pests, especially showing excellent control efficacy against lepidopteran pests (such as diamondback moth, cotton bollworm, and pine caterpillar).

[0003] Currently, the types of insecticide mechanisms of action globally are relatively limited. Long-term use of single-category pesticides has led to increasingly prominent pest resistance problems, seriously threatening agricultural production safety and ecological sustainability. The traditional mechanism of action of lufenuron is to inhibit insect chitin synthesis, thereby blocking the formation of new epidermis, causing larval molting deformities and death, or inhibiting egg hatching, thus achieving pest control. However, its exact molecular target has long been controversial and remains unclear, and the differences in activity and resistance evolution mechanisms among different insects cannot be explained. This core bottleneck of unclear targets directly restricts the rational design of novel, highly efficient, and highly selective benzoylurea insecticides based on structure, and also hinders the precise development of resistance management strategies. Currently, the classification of insecticide mechanisms of action globally is limited, and long-term use of single-category pesticides has led to increasingly serious pest resistance problems. Developing green insecticides with novel targets and mechanisms of action has become an urgent need for green pest control in agriculture. Summary of the Invention

[0004] In view of this, one of the objectives of the present invention is to provide an MTHFD2 protein.

[0005] The second objective of this invention is to provide the application of the MTHFD2 protein in the preparation of products that inhibit insect growth and development.

[0006] A third objective of this invention is to provide the application of the MTHFD2 protein in monitoring insect resistance to pesticides.

[0007] The fourth objective of this invention is to provide the application of the MTHFD2 protein in pesticide identification.

[0008] The fifth objective of this invention is to provide the application of the MTHFD2 protein in regulating the sensitivity of insects to pesticides.

[0009] The sixth objective of this invention is to provide a method for identifying insecticides.

[0010] To achieve the above-mentioned objectives, the present invention provides the following technical solution: An MTHFD2 protein, the amino acid sequence of which is shown in SEQ ID NO.1.

[0011] Preferably, the nucleotide sequence of the gene encoding the MTHFD2 protein is shown in SEQ ID NO.2.

[0012] The present invention also provides the application of the MTHFD2 protein in the preparation of products that inhibit insect growth and development.

[0013] The present invention also provides the application of the MTHFD2 protein in monitoring insect resistance to pesticides.

[0014] The present invention also provides the application of the MTHFD2 protein in pesticide identification.

[0015] The present invention also provides the application of the MTHFD2 protein in regulating the sensitivity of insects to pesticides, and downregulating the expression of MTHFD2 protein reduces the sensitivity of insects to pesticides.

[0016] Preferably, the MTHFD2 protein serves as a target for the insecticide.

[0017] Preferably, the insecticide includes any one or more of the following: plant extracts, compounds with well-defined chemical structures, peptides, nucleic acids, polysaccharides, viral vectors, liposome vectors, or nanoparticle vectors.

[0018] Preferably, the insecticide targets lepidopteran or dipteran insects.

[0019] The present invention also provides a method for identifying insecticides, comprising the following steps: The analyte is added to the MTHFD2 protein, and the enzyme activity of the MTHFD2 protein in the presence of the analyte is measured. If the enzyme activity of the MTHFD2 protein decreases, the analyte is an insecticide.

[0020] Compared with the prior art, the present invention has the following beneficial effects: This invention provides an MTHFD2 protein associated with insecticide sensitivity, identifies and confirms that MTHFD2 is a direct target of lufenuron, and elucidates a novel mechanism of "targeting MTHFD2 → interfering with one-carbon metabolism → inhibiting chitin synthesis → insect death". This target is highly conserved and exhibits excellent selectivity. Insects and mammals have structural and metabolic differences, and lufenuron shows low toxicity and high safety to mammals.

[0021] This invention identifies the key binding sites of the MTHFD2 protein to lepidopteran insecticides, namely Leu143, Leu188, and Gln291, which can be directly used for rational drug design, significantly shortening the new drug development cycle and reducing costs.

[0022] This invention establishes an insecticide screening system based on the MTHFD2 protein, enabling the rapid discovery of novel MTHFD2 inhibitors and providing a stable technical platform for the development of green insecticides.

[0023] The MTHFD2 protein in this invention can be used for early monitoring of pest resistance, guiding the rotation of pesticides, and delaying the development of resistance. Attached Figure Description

[0024] Figure 1 The effect of folic acid on the symptoms of five lepidopteran insects after treatment with lufenuron.

[0025] Figure 2 The effect of folic acid on the epidermal structure of five lepidopteran insects after treatment with lufenuron was investigated; where Cu represents the epidermal layer, Ep represents the epithelial cell layer, and Bm represents the basal membrane.

[0026] Figure 3 The effects of lufenuron on folic acid content, trehalose, and chitin synthesis in cotton bollworm were investigated. Specifically, A represents the temporal variation of folic acid content in cotton bollworm; B represents the temporal variation of folic acid content in armyworm; C represents the temporal variation of folic acid content in beet armyworm; D represents the temporal variation of folic acid content in small armyworm; E represents the temporal variation of folic acid content in corn borer; F represents the expression level of MTHFD2 at different developmental stages of cotton bollworm; G represents the effect of lufenuron time on folic acid content in cotton bollworm; H represents the effect of lufenuron concentration on folic acid content in cotton bollworm; I represents the restorative effect of exogenous folic acid on the growth and development of cotton bollworm; J represents the effect of lufenuron on trehalose content in cotton bollworm; and K represents the effect of lufenuron on chitin content in cotton bollworm.

[0027] Figure 4 This is a sequencing map of the PCR amplification of the MTHFD2 gene.

[0028] Figure 5 The image shows the amino acid sequence alignment results of the cotton bollworm MTHFD2 protein with homologous proteins from other insects. Red indicates identical sequences, while blue and yellow indicate different sequences. The sequences compared are from the cotton bollworm (XP_049699294.2, H. armigera); the American cotton bollworm (XP_047032963, Helicoverpa zea); the fall armyworm (XP_035447993.2, Spodoptera frugiperda); and the beet armyworm (XP_022831585.1, Spodopteralitura).

[0029] Figure 6 Phylogenetic analysis of the MTHFD2 gene.

[0030] Figure 7 The images show the expression and purification results of the purified MTHFD21 protein (coomassie brilliant blue staining). The left image shows the expression of the purified MTHFD21 protein, and the right image shows the purification of the purified MTHFD21 protein.

[0031] Figure 8 This is a map of the MTHFD2 plasmid.

[0032] Figure 9 Subcellular localization of MTHFD2 protein in Sf9 cells, scale bar = 10 μm.

[0033] Figure 10 The figures show the relative expression levels of the MTHFD2 gene in different developmental stages and tissues of the cotton bollworm, as well as the expression changes after lufenuron treatment. Figure A shows the expression of the MTHFD2 gene at each developmental stage of the cotton bollworm; Figure B shows the expression of the MTHFD2 gene in different tissues of the 5th instar larvae of the cotton bollworm; Figure C shows the expression of MTHFD2 after lufenuron treatment. In the figures, Egg represents eggs, L1-L5 represent 1st to 5th instar larvae, FP represents female pupae, MP represents male pupae, FA represents female insects, MA represents male insects, HE represents head, EP represents epidermis, FG represents foregut, MG represents midgut, HG represents hindgut, MT represents Malpighian tubules, and FB represents fat body.

[0034] Figure 11 This section describes the construction and mutation detection of the MHFD2 mutant in Drosophila. Figure A shows a schematic diagram of the MHFD2 gene structure and sgRNA target site location in Drosophila. The green lines represent the five exons of the MHFD2 gene, the black lines represent the four introns of the MHFD2 gene, the red sequence is the target sequence of the sgRNA, and the blue sequence is the PAM sequence. Figure B shows the comparison analysis of different mutation types based on wild-type sequences. The dotted lines within the red boxes indicate missing bases. Figure C shows the agarose gel electrophoresis image of MHFD2 knockout Drosophila identified by PCR. Homozygous MHFD2ko Drosophila are sterile. MHFD2ko homozygous males and females were crossbred to preserve the strain. The offspring included both heterozygous and homozygous Drosophila, so homozygous and heterozygous detection was performed. Figure D shows the Western blotting detection of the MHFD2 mutant. Tubulin was used as an internal control gene.

[0035] Figure 12 The effect of MTHFD2 gene knockout on oviposition and developmental period in Drosophila is shown in the figure. In the figure, A represents the effect of MTHFD2 gene knockout on the number of eggs laid, and B represents the effect of MTHFD2 gene knockout on the developmental period in Drosophila.

[0036] Figure 13The figure shows the effect of MTHFD2 gene knockout on the growth and development of Drosophila; C in the figure represents the effect of MTHFD2 gene knockout on the hatching rate of Drosophila eggs, D represents the effect of MTHFD2 gene knockout on the pupation rate of Drosophila, and E represents the effect of MTHFD2 gene knockout on the emergence rate of Drosophila.

[0037] Figure 14 Changes in the sensitivity of MTHFD2 gene knockout fruit fly larvae to chlorfenapyr.

[0038] Figure 15 The results show the docking of lufenuron with the MTHFD2 molecule of cotton bollworm; Figure A shows the surface binding mode of lufenuron and MTHFD2 protein docking; Figure B shows the overall docking conformation of lufenuron and MTHFD2 protein; Figure C shows a two-dimensional schematic diagram of the key interaction between lufenuron and MTHFD2 protein; Figure D shows the three-dimensional interaction details of the binding pocket between lufenuron and MTHFD2 protein.

[0039] Figure 16 A shows the key amino acid residues of the interaction between lufenuron and cotton bollworm MTHFD2; A shows the changes in RMSD values ​​of the skeleton atoms of the MTHFD2-lufenuron complex (red), MTHFD2 (black), and lufenuron (blue); B shows the decomposition of the binding free energy of the MTHFD2-lufenuron complex; C shows the ASM analysis of MTHFD2 complexed with lufenuron.

[0040] Figure 17 This represents the activity of MTHFD2 dehydrogenase.

[0041] Figure 18 This is a kinetic analysis of the binding of lufenuron and MTHFD2 based on SPR; Figure A shows the specific binding response of lufenuron to MTHFD2; Figure B shows the specific binding response of methotrexate to MTHFD2.

[0042] Figure 19 Dixon kinetic analysis of the competitive inhibition of MTHFD2 by lufenuron.

[0043] Figure 20 The flowchart shows the high-throughput screening system for insecticides based on MTHFD2; Figure A shows the screening reaction system, Figure B shows the high-throughput operation flow of a 96-well plate, and Figure C shows the inhibitor determination criteria and IC50. 50 Fitting. Detailed Implementation

[0044] This invention provides an MTHFD2 protein, which is a bifunctional methylenetetrahydrofolate dehydrogenase cloned and expressed from the cotton bollworm (Helicoverpa armigera). To obtain this protein, total RNA was first extracted from 5th instar larvae of the cotton bollworm, and cDNA was obtained through reverse transcription. Specific primers were then designed based on predicted sequences from the cotton bollworm genome database, and the complete coding sequence was amplified using PCR technology. The PCR product was recovered by agarose gel electrophoresis and sequenced. The 909 bp coding region sequence was then assembled using DNAMAN software.That is, ATGGCCCGAATCCTCGACGGCAAAG CGCTGGCAGGAACAGTGAAGGATGAATTGAAGCAAGAAATTGCAAATTGGATTAACCTGGGTCACCGTGCACCGTCTATCCGATGCATCTTAGTAGGAGAAGACCCAGCTAGCCACACTTATGTTAACAACAAGATCATAGCCGCCAGATATGTAGGCATCAACGCTGAAGTGATTAGACGTGACAGCACAATAACAGAAGACCAACTTATTCAAGAAATACAAACACTAAACGCAGACAGCACTGTTGATGGCATCCTTGTACAACTACCAATTCCGGAGACTATGAGTGAGAGAAAAGTGTGCAATGCGGTCGCTCCTGAGAAGGACGTTGATGGTTTTCATATTGTGAACATCGGACAACTTTGTGTTGATATGCCAACGTTAGTTCCTGCGACCGCCTTGGCTGTGATAGAAATGCTGAAGAGATTCAACATCGAAACTTTTGGTCGCAACGCGGTCGTAGTAGGCCGATCGAAAAATGTTGGTCTGCCTATTGCAATGATGTTGCATAGTGACAAGAAACATGACAATGGTCTCGGAATGGATGCTACTGTTACTATCTGCCATCGTTACACACCTAAAGAACAGCTGGAATTCTTCTGCCAAAATGCCGATATTATTATTACGGCTACAGGTGTACCAAAACTTATCAAAGCAAACATGATCAAGCCCGGCGCTACCGTCATTGATGTTGGCATCACTAAAGTTACCGACGAAAATGGAAAATCTAGGCTAGTTGGCGATGTTGATTATGATGAGGTTAGCAAAGTAGCTGGTGCCGTGACGCCAGTCCCCGGTGGAGTGGGACCCATGACAGTCGCCATGTTGATGCACAACACATTCCAGGCTGCCAAACATCAGAGGGCAAAAGCACAGTTGCAG (SEQ ID NO.2), this sequence encodes 303 amino acids,Its amino acid sequence is specifically MARILDGKALAGTVKDELKQEIANWINLGHRAPSIRCI LVGEDPASHTYVNNKIIAARYVGINAEVIRRDSTITEDQLIQEIQTLNADSTVDGILVQLPIPETMSERKVCNAVAPEKDVDGFHIVNIGQLCVDMPTLVPATALAVIEMLKRFNIETFGRNAVVVGRSKNVGL PIAMMLHSDKKHDNGLGMDATVTICHRYTPKEQLEFFCQNADIIITATGVPKLIKANMIKPGATVIDVGITKVTDENGKSRLVGDVDYDEVSKVAGAVTPVPGGVGPMTVAMLMHNTFQAAKHQRAKAQLQ (SEQ ID NO.1). The coding sequence was cloned into a eukaryotic expression vector, transformed into DH10Bac competent cells, and recombinant rod cells were obtained through blue-white screening. These rod cells were then transfected into Sf9 insect cells, cultured for 72 hours, and the cells were collected. After sonication and centrifugation, the supernatant was purified using Ni-NTA affinity chromatography and molecular sieve chromatography to obtain recombinant MTHFD2 protein with a purity ≥90%. SDS-PAGE showed its molecular weight to be approximately 47.4 kDa, consistent with the theoretical value. Mass spectrometry identification revealed that this protein possesses typical bifunctional methylenetetrahydrofolate dehydrogenase activity, capable of catalyzing the conversion of 5,10-methylenetetrahydrofolate to 10-formyltetrahydrofolate, while simultaneously reducing NADP+ to NADPH. The protein expression level was highest in male pupae, with relatively high expression levels in eggs and first-instar larvae, and the highest expression level was observed in the larval epidermis. It was localized in the cytoplasm and nucleus, with higher expression levels in the nucleus than in the cytoplasm.

[0045] This invention also provides the application of the MTHFD2 protein in the preparation of products that inhibit insect growth and development. The products include reagents that inhibit the expression of the MTHFD2 gene or the activity of its encoded protein; the reagents are at least one of double-stranded RNA, antisense oligonucleotides, a CRISPR / Cas9 system, small molecule compounds, antibodies, or peptides; the CRISPR / Cas9 system contains sgRNA. This invention conducted MTHFD2 gene knockout experiments using Drosophila as a model organism. Specifically, two sgRNAs were designed targeting the second exon of the Drosophila MTHFD2 gene (CG18466), with targeting sequences GGTGCGCACCTCCTGGGCTA (SEQ ID NO.3) and TCATAAACCGGGAATGATAA (SEQ ID NO.4). After cloning the sgRNAs into an expression vector, they were co-injected with a Cas9 helper plasmid into Drosophila embryos to obtain MTHFD2 gene knockout strains. PCR and sequencing confirmed the existence of frameshift mutant lines MTHFD2-ko1 and MTHFD2-ko2, with deletions of 194bp and 218bp, respectively. Western blot analysis showed that the protein expression levels of MTHFD2-ko1 and MTHFD2-ko2 were 6.39% and 4.62% of the wild type, respectively. Phenotypic analysis revealed a significant decrease in egg production in the MTHFD2 knockout lines (approximately 55 eggs for MTHFD2-ko1 and approximately 63 eggs for MTHFD2-ko2), a reduction of 60–70% compared to the control group (P<0.001). The egg, larval, and pupal stages of MTHFD2-ko1 were shortened by 28%, 16.67%, and 8.11%, respectively; while those of MTHFD2-ko2 were shortened by 24%, 14.28%, and 12.16%, respectively. The average lifespan of the MTHFD2 knockout group was 9.75 ± 0.27 days, which was about 14.77% shorter than that of the wild type. The hatching rate (ko1: 30%, ko2: 16.25%) and pupation rate (ko1: 22.5%, ko2: 11.25%) of the MTHFD2 knockout eggs were significantly reduced, and the emergence rate was also affected, with MTHFD2-ko1 significantly reduced to 73.95%. This indicates that the suppression of the MTHFD2 gene significantly affects the reproductive capacity and growth and development process of Drosophila. Furthermore, this invention also confirms that lufenuron, as a potent inhibitor of MTHFD2, can directly bind to MTHFD2 (KD=0.305 nM) and inhibit its enzyme activity (Ki=4.305 μM). Treatment of third-instar larvae of cotton bollworm with 200 mg / L lufenuron significantly reduced folic acid content in the insects, inhibited trehalose and chitin synthesis, and resulted in typical molting disorders (double-headed sac symptoms). Exogenous folic acid supplementation significantly alleviated these poisoning symptoms, indicating that inhibiting MTHFD2 protein activity can also inhibit insect growth and development. In this invention, the product is preferably an insecticide or insect growth regulator.

[0046] In this invention, the insects are preferably lepidopteran or dipteran insects. The lepidopteran insects include, but are not limited to, cotton bollworms, beet armyworms, diamondback moths, armyworms, and corn borers; the dipteran insects include, but are not limited to, fruit flies and leaf miners.

[0047] The present invention also provides a method for screening compounds that inhibit the activity of the MTHFD2 enzyme, comprising the following steps: providing a recombinant MTHFD2 protein, the amino acid sequence of which is shown in SEQ ID NO.1; contacting the candidate compound with the MTHFD2 protein in a buffer containing the substrate 5,10-methylenetetrahydrofolate and the coenzyme NADP+, continuously monitoring the NADPH generation rate at a wavelength of 340 nm, and calculating the enzyme activity; comparing the enzyme activity measured above with the enzyme activity of a control group without the candidate compound, and calculating the enzyme activity inhibition rate; and screening for compounds that inhibit the activity of the MTHFD2 enzyme based on the enzyme activity inhibition rate.

[0048] In a specific embodiment of the present invention, the method is based on an in vitro enzyme activity detection system for recombinant MTHFD2 protein. First, recombinant MTHFD2 protein, whose amino acid sequence is shown in SEQ ID NO.1, is provided. It is expressed using a eukaryotic expression system (such as Sf9 insect cells) and purified by Ni-NTA affinity chromatography and molecular sieve chromatography to obtain an active protein with a purity ≥90%. A 100 μL reaction system is established in a 96-well plate, containing recombinant MTHFD2 protein at a final concentration of 20 μM, 50 mM Tris-HCl buffer (pH 7.8), 100 mM 2-mercaptoethanol, 100 μM NADP+, and candidate compounds at different concentrations. The concentrations of the candidate compounds are preferably set in a concentration gradient of 0.1–100 μM. A control group is set up: an equal volume of 0.1% DMSO or 0.01% Triton X-100 is used instead of the candidate compounds; a positive control group is set up: 50 μM lufenuron is added; a negative control group is set up: no enzyme is added, only buffer and substrate are present. The 96-well plate was pre-incubated at 25°C for 10–15 minutes to allow the candidate compounds to fully interact with the protein. Then, 5,10-methylenetetrahydrofolate (final concentration 50 μM) was added to each well to initiate the reaction. Immediately after initiation, absorbance was continuously monitored at 340 nm using a microplate reader, with readings taken every 30 seconds for a total monitoring time of 10 minutes. The linear range from 0 to 5 minutes after reaction initiation was used to calculate the NADPH production rate (ΔOD). 340The inhibition rate (%) is positively correlated with enzyme activity. The inhibition rate was calculated using the following formula: Inhibition rate (%) = (Control group rate - Treatment group rate) / Control group rate × 100%. Candidate compounds were graded based on their inhibition rate: compounds with an inhibition rate ≥ 70% exhibited strong inhibition of MTHFD2 enzyme activity; compounds with an inhibition rate between 40% and 70% exhibited moderate inhibitory activity; and compounds with an inhibition rate below 40% showed no significant inhibitory activity. For compounds with an inhibition rate ≥ 40%, 6–8 different concentrations (e.g., 0.1, 0.5, 1, 5, 10, 50, 100 μM) were further measured to determine the inhibition rate at each concentration. The dose-response curve was fitted using GraphPad Prism software, and the IC50 value was calculated. This method allows for rapid and high-throughput screening of MTHFD2 inhibitors from compound libraries, natural product extracts, or chemically synthesized products. The obtained inhibitors have the potential to be developed into novel insecticides.

[0049] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0050] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0051] Example 1 Effects of folic acid on the phenotype of cotton bollworm after lufenuron treatment.

[0052] 1. Effects of folic acid on five lepidopteran insects after lufenuron treatment.

[0053] Lufenuron technical grade was dissolved in N,N-dimethylformamide to prepare a stock solution, which was then diluted to 200 mg / L with 0.01% Triton X-100. Five species of 3rd instar lepidopteran larvae (cotton bollworm, beet armyworm, corn borer, armyworm, diamondback moth, and diamondback moth were treated with 200 mg / L lufenuron for 10 s immersion, and diamondback moth for 5 s immersion), with 0.01% Triton X-100 serving as a blank control. Twenty-four insects were used for each treatment. After treatment, each insect was fed 0.3 g / individual of artificial feed containing 0% v / v or 0.1% v / v folic acid, followed by a normal diet. Growth and symptoms of poisoning were observed and recorded using an Olympus microscopic imaging system.

[0054] The results are as follows Figure 1As shown, after treatment with lufenuron (200 mg / L), cotton bollworms, beet armyworms, and armyworms exhibited "double-headed sac" symptoms, meaning the old and new head shells overlapped, the thorax epidermis molted, revealing a light yellow new epidermis, and the old epidermis covered the new epidermis from below the thorax, completely enveloping the insect body, giving it a dark brown appearance. In contrast, diamondback moths and corn borers treated with lufenuron showed entirely black bodies, shrunken abdomens, and stiffened bodies. After folic acid supplementation, the poisoning symptoms of all tested pests were significantly reduced, and their molting ability was restored, indicating that folic acid has a therapeutic effect against lufenuron.

[0055] 2. Effects of folic acid on the epidermal structure of five lepidopteran insects after lufenuron treatment.

[0056] Five species of lepidopteran larvae were treated using the same method as described in "1. Effects of folic acid on five lepidopteran insects treated with lufenuron." After 72 h of treatment, the larvae were fixed in 4% paraformaldehyde. Samples were dehydrated using a gradient of ethanol (30%, 50%, 60%, 70%, 80%, 90%, 100%), cleared with xylene-ethanol mixture, embedded in paraffin, and then sectioned into 4 μm thick sections using a pathological microtome. Sections were dewaxed, rehydrated, stained with hematoxylin for 5 min, separated by hydrochloric acid-ethanol, stained with eosin for 3 min, dehydrated, cleared, and mounted. Images were acquired and analyzed using a fully automated slide scanning system.

[0057] The results are as follows Figure 2 As shown, histopathological observation revealed that, compared with the blank control group, lufenuron alone treatment caused significant thinning, loosening, and even dissolution of the epidermal layer (Cu) of five lepidopteran pests: cotton bollworm, diamondback moth, beet armyworm, and corn borer. This was accompanied by damage such as disordered arrangement of the epithelial cell layer (Ep), swelling and vacuolization, and blurred boundaries of the basal membrane (Bm). However, combined treatment with lufenuron and folic acid (Lufenuron + folate) significantly alleviated these destructive effects, restoring the epidermal thickness, epithelial cell morphology, and basal membrane structure of the tested pests to near-control levels. This suggests that folic acid can effectively antagonize the pathological damage to the epidermis of lepidopteran pests caused by lufenuron and maintain the integrity of the epidermal tissue structure.

[0058] 3. Effects of lufenuron on folic acid metabolism in cotton bollworm and the alleviating effect of folic acid supplementation.

[0059] Five lepidopteran 3rd instar larvae were treated using the same method as described in "1. Effect of folic acid on five lepidopteran insects after lufenuron treatment". Three lepidopteran larvae (3rd instar) were collected at different time points (1, 3, 6, 12, 24, 36, 48, 72, 96 h) after lufenuron (200 mg / L) treatment, as well as samples of different developmental stages of cotton bollworm: eggs (denoted as Egg), 1st to 6th instar larvae (denoted as L1 to L6), female pupae (denoted as FP), male pupae (denoted as MP), female adults (denoted as FA), and male adults (denoted as MA).

[0060] Third-instar larvae of cotton bollworm were immersed in different concentrations of lufenuron (0, 6.25, 12.5, 25, 50, 100 and 200 mg / L) for 10 seconds, and the immersed larvae were collected.

[0061] Among them, CK or Control means no treatment, Lufenuron or Treated means lufenuron-treated, CK+folate means no lufenuron-treated and fed 0.3 g / head of artificial feed containing 0.1% v / v folic acid, and Lufenuron+folate means lufenuron-treated and then fed 0.3 g / head of artificial feed containing 0.1% v / v folic acid.

[0062] The collected samples were ground in liquid nitrogen, and 1 g of each sample was added to 20 mg of porcine trypsin and phosphate-buffered saline (PBS, pH 7.4) to a final volume of 40 mL. The mixture was incubated at 37°C in the dark for 4 h, extracted at 95°C for 30 min, and centrifuged at 12,500 rpm for 8 min. The supernatant was filtered through a 0.2 μm filter. The folic acid content was calculated by measuring the absorbance using a microplate reader according to the instructions of the folic acid kit (P1001, VitaFast, Germany).

[0063] The results are as follows Figure 3 As shown, the effects of lufenuron on folic acid metabolism in five lepidopteran pests exhibited significant interspecific differences, with the cotton bollworm being the most sensitive to lufenuron. Under 200 mg / L lufenuron treatment, the folic acid content in the cotton bollworm rapidly decreased after 6 h, stabilizing at 23.5 μg / 100g after 24 h, only one-third of the control group. Figure 3 In the A section); folic acid levels in beet armyworms first increased and then decreased within 24 hours after treatment, dropping to half the level of the control at 72 hours. Figure 3 (B in the text); the diamondback moth and corn borer only began to decline 48 hours after treatment ( Figure 3 C and Figure 3 In the D); the number of armyworms was significantly lower than that of the control at all time points except 1-3 h. Figure 3 (E in the text).

[0064] Further analysis showed that the folic acid content of the cotton bollworm was specific to its developmental stage, with the highest levels observed in the second instar larvae. Figure 3 In the F group, the inhibitory effect of lufenuron showed a significant time- and concentration-dependent effect. Figure 3 G and Figure 3 (H in the original text). Exogenous folic acid supplementation can significantly alleviate the symptoms of lufenuron poisoning (H in the original text). Figure 3 In addition, lufenuron significantly inhibited trehalose and chitin synthesis. Treatment with 200 mg / L lufenuron reduced trehalose by 68.33%, and treatment with various concentrations of lufenuron reduced chitin by 22.92%–40.76%. This suggests that lufenuron affects trehalose accumulation and chitin deposition by interfering with folic acid metabolic homeostasis, ultimately leading to molting disorders and developmental abnormalities. Figure 3 J and Figure 3 (K in the middle).

[0065] 4. The alleviating effect of exogenous folic acid supplementation on the growth and development of cotton bollworm under lufenuron stress.

[0066] Cotton bollworm larvae were treated with 200 mg / L lufenuron (immersion for 10 s) and fed with feed containing 0% or 0.1% folic acid. At 3, 5, 7, and 9 days post-treatment, body weight was measured using an electronic balance, and body length was measured using Fiji software based on photographs, with the camera fixed 30 cm directly above the larvae and a ruler placed beside them. Developmental stages, number of pupae, and number of adults were recorded, and the pupation rate (number of pupae / number of 5th instar larvae × 100%) and emergence rate (number of adults / number of pupae × 100%) were calculated. After adult pairing, the number of eggs laid daily was recorded, and newly hatched larvae were individually reared and their development monitored. Results are shown in Tables 1-4.

[0067] Table 1. Effects of exogenous folic acid supplementation on body length of bollworm larvae under lufenuron stress.

[0068] Table 2. Effects of exogenous folic acid supplementation on the developmental stages of cotton bollworm under lufenuron stress.

[0069] Table 3 Effects of exogenous folic acid supplementation on pupal weight, emergence rate, and pupation rate of cotton bollworm under lufenuron stress.

[0070] Table 4. Effects of exogenous folic acid supplementation on the developmental stages of the first generation of cotton bollworm under lufenuron stress.

[0071] Compared with lufenuron alone, the lufenuron + folic acid combined treatment group showed that the larval body length recovered from 16.8 mm to 27.1 mm (Table 1), the 3rd instar development period shortened from 4.17 days to 2.50 days (Table 2), the pupation rate increased from 0% to 58.92%, the eclosion rate recovered to 87.78%, and the pupal weight reached 268.5 mg, which was not significantly different from the control group (Table 3). F1 generation follow-up showed that the detoxification effect of folic acid was persistent (Table 4).

[0072] Example 2 MTHFD2 gene cloning and sequence analysis.

[0073] Total RNA was extracted from 5th instar larvae of *Bollworm*. After integrity was assessed by agarose gel electrophoresis and concentration was determined by NanoDrop, cDNA was synthesized using 1 μg of total RNA as a template via the HiScript III RT SuperMix reverse transcription kit. Specific amplification primers were designed based on predicted sequences from the *Bollworm* genome database: upstream primer: 5'-AGGCATCAACGCTGAAGTCA-3' (SEQ ID NO.5); downstream primer: 5'-AACGTCACCAACTAGCCTGG-3' (SEQ ID NO.6). PCR amplification was performed using cDNA as a template. The reaction mixture consisted of: 25 μL 2×PCR Mix, 19 μL Nuclease-free water, 4 μL of upstream and downstream primer mixture (10 μM), and 2 μL cDNA. The program was: 95℃ pre-denaturation for 1 min; 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 1 min, 35 cycles; final extension at 72℃ for 10 min. The product was sequenced after detection by 1% agarose gel electrophoresis. Figure 4 The band was recovered and ligated into the pMD19-T vector, transformed into DH5α competent cells, and positive clones were screened and sequenced. The sequencing results were assembled and aligned using DNAMAN software, yielding a 909 bp coding region sequence, SEQ ID NO.2, named MTHFD2; encoding 303 amino acids. Figure 5), namely SEQ ID NO.1. Multiple alignments of the MTHFD2 amino acid sequences of *H. armigera* (XP_049702333.2), *Helicoverpa zea* (XP_047029291.1), *Spodoptera litura* (XP_022824655.1), and *Trichoplusia ni* (XP_026741196.1) were performed using DNAMAN software. The results showed that the amino acid sequence identity of MTHFD2 across different species was 86.75%, indicating that MTHFD2 has very high sequence similarity, is highly conserved, functionally conserved, and its domains are conserved, with no functional differentiation. Figure 6 ).

[0074] Example 3 Expression and purification of MTHFD2 protein.

[0075] The coding sequence shown in SEQ ID NO.2 was cloned into the pFastBac1 vector via the BamHI and XhoI sites, and transformed into DH10Bac competent cells to obtain a recombinant plasmid. The recombinant plasmid was transfected into Sf9 insect cells, and the cells were collected after culturing at 27°C for 72 hours. The cells were resuspended in lysis buffer (50 mM Tris-HCl pH 7.8, 300 mM NaCl, 10 mM imidazole, 1 mM MPMSF) and sonicated to obtain the supernatant. The supernatant was loaded onto a Ni-NTA affinity chromatography column and eluted with a gradient of buffers containing 20 mM and 250 mM imidazole, collecting the 250 mM imidazole elution peak. The affinity-purified protein was further purified by Superdex 200 molecular sieve chromatography, eluting with buffers containing 50 mM Tris-HCl pH 7.8 and 150 mM NaCl. SDS-PAGE electrophoresis results showed that the purified protein exhibited a single band at approximately 47.4 kDa, with a purity greater than 90%. Figure 7 ).

[0076] Example 4 Subcellular localization of MTHFD2 protein.

[0077] The constructed recombinant plasmid ( Figure 8Transformed into Top10 competent cells, plated on LB agar plates containing the corresponding antibody, and incubated overnight at 37°C; single colonies were picked and inoculated into 3 mL of LB liquid medium and incubated overnight at 37°C; 700 μL of bacterial culture was collected for preservation, and another 200 μL of bacterial culture was inoculated into 50 mL of LB liquid medium containing the antibiotic and cultured overnight at 37°C for expansion; the bacterial cells were collected for plasmid extraction. Cell transfection was then performed: Sf9 cells were passaged routinely, and the suspension cell density was adjusted to 1×10⁶ cells / mL before transfection. Cell smears were placed at the bottom of a 24-well plate, and 0.3×10⁶ cells were seeded in each well. 500 μL of Grace's Supplement medium containing 10% FBS without antibiotics was added, and the cells were incubated at 27°C for 30 min to allow cell adhesion. Transfection mixture was prepared: 100 μL of serum-free Grace's Unsupplement medium was added, along with 1 μg of plasmid DNA. After mixing, 2 μL of insect transfection reagent (transfection reagent: DNA = 2:1) was added, and the mixture was gently mixed. The plate was incubated at room temperature for 15-20 min. The original culture medium in the 24-well plate was aspirated, and 200 μL of transfection mixture was added to each well. The plate was sealed with sealing film and incubated at 27°C for 24 h. Then, 300 μL of Grace's Supplement medium containing 10% FBS without antibiotics was added, and the plate was incubated for another 48 h. Finally, immunofluorescence staining and confocal microscopy observation were performed: the transfected Sf9 cells were removed, the culture medium was discarded, and the cells were washed three times with pre-warmed 1×PBS; 500 μL of 4% paraformaldehyde was added to each well and the cells were fixed at room temperature in the dark for 30-40 min; the fixative was discarded, the cells were washed once with PBS, and an appropriate amount of glycerol was added to enhance the staining effect; DAPI dye was dropped onto the fixed cell samples on the slides and incubated at room temperature in the dark for 10 min; the dye was discarded, the slides were washed 2-3 times with PBS (3-5 min each time), and the slides were mounted with mounting adhesive and observed and photographed under a laser confocal microscope.

[0078] Further IF assays were performed to verify subcellular localization. For example... Figure 9 As shown, MTHFD2-GFP was successfully expressed in Sf9 cells. MTHFD2-GFP was located in the cytoplasm and nucleus, with higher expression levels in the nucleus than in the cytoplasm.

[0079] Example 5 Spatiotemporal expression pattern of MTHFD2 in cotton bollworm.

[0080] To investigate the expression pattern of the target gene at different developmental stages of *Bollworm*, samples were collected from each developmental stage (400 eggs; 100 first-instar larvae; 50 second-instar larvae; 20 third-instar larvae; 10 each of fourth, fifth, and sixth-instar larvae; 10 each of female pupae, male pupae, female adults, and male adults) as one biological replicate. Three independent replicates were established for each developmental stage. After sampling, samples were flash-frozen in liquid nitrogen and stored at -80°C for later use. To investigate the expression distribution of the target gene in different tissues, 2-day-old fifth-instar *Bollworm* larvae of uniform growth were selected, with 30 larvae per biological replicate (3 replicates). The larvae were rapidly dissected under a stereomicroscope in physiological saline, and seven tissues were collected: head, epidermis, fat body, foregut, midgut, hindgut, and Malpighian tubules. These tissues were cleaned with 0.7% NaCl solution, blotted dry with filter paper, and transferred to 1.5 mL centrifuge tubes, then stored at -80°C for later use. Total RNA was extracted from various developmental stages and tissues using the EasyPure RNA Kit. Quantification was performed using NanoDrop2000 to confirm that the A260 / A280 and A260 / A230 ratios were both between 1.8 and 2.2. Integrity was verified by 1% agarose gel electrophoresis. First-strand cDNA was synthesized according to the HiScript III RT SuperMix for qPCR (+gDNA wiper) instructions: 4 µL of purified RNA sample was added to 4×gDNA wiper Mix, followed by RNase-free ddH2O to a final volume of 16 µL. The mixture was heated at 42 °C for 2 min; then 4 µL of 5×HiScript III RT SuperMix was added, and the mixture was incubated at 37 °C for 15 min, 85 °C for 5 s, and stored at -20 °C. Using the obtained cDNA as a template, quantitative PCR primers were used, with actin-5C as an internal control, to quantitatively analyze the expression levels of MTHFD2 mRNA in different developmental stages and tissues of *Helicoverpa armigera*. The real-time quantitative PCR reaction system (20 µL) contained: 10.0 µL of 2×ChamQ Universal SYBR qPCR Master Mix, 1 µL of cDNA, 0.4 µL each of forward and reverse primers (10 µM), and ddH2O to a final volume of 20.0 µL. A two-step program was used: 95 °C pre-denaturation for 30 s; 95 °C denaturation for 5 s, followed by annealing and extension at 60 °C for 30 s, for a total of 40 cycles. The melting curve program was: 95 °C denaturation for 15 s, 60 °C annealing for 15 s, followed by a slow rise to 95 °C and a hold for 15 s. The relative expression level of the target gene was calculated using the 2-ΔΔCt method. Each sample had three technical replicates and three biological replicates. The oviparous stage was used as a reference for each developmental stage, and the head was used as a reference for each tissue.Further investigation was conducted on the effect of lufenuron treatment on MTHFD2 expression: Lufenuron stock solution was diluted to 200 mg / L with 0.01% Triton X-100. 5th instar cotton bollworm larvae of uniform size and vigorous activity were selected and immersed for 10 seconds. After the liquid was sucked off their bodies, they were fed fresh feed. The 0.01% Triton X-100 treatment was used as a control. Each treatment was repeated three times (24 test insects per replicate). Test insects were collected at 24, 48, 72, and 96 h after treatment, and the relative gene expression level was detected by qRT-PCR.

[0081] The MTHFD2 gene is expressed at all developmental stages of the cotton bollworm. The expression level of MTHFD2 is highest in male pupae, relatively high in eggs and first instar larvae, and lowest in female adults and third instar larvae. Figure 10 (A). The expression level of MTHFD2 in male pupae was 2.34 times that in the egg stage, 6.32 times that in the first instar, 8.26 times that in the second instar, 23.4 times that in the third instar, 9.36 times that in the fourth and fifth instars, 15.6 times that in the sixth instar, 26 times that in female pupae and male adults, and 78 times that in female adults. Overall, the expression level of MTHFD2 in cotton bollworms at different stages showed a trend of first decreasing and then increasing. MTHFD2 was expressed in different tissues of 5th instar larvae of cotton bollworms, and the expression level varied significantly among different tissues. MTHFD2 expression was low in the head (HE), foregut (FG), midgut (MG), and hindgut (HG) tissues, and the highest expression level was in the epidermis (EP), which was 40 times that in the foregut. The relative expression level of MTHFD2 in the epidermis (EP) was significantly different from that in the fat body (FB), with the former being about 1.3 times that of the latter. Figure 10 (B) After lufenuron treatment, the expression of MTHFD2 showed dynamic changes. MTHFD2 expression rapidly increased 48 h after treatment, and significantly decreased between 72 and 96 h. Figure 10 (C)

[0082] Example 6 Functional validation of the MTHFD2 gene based on CRISPR / Cas9.

[0083] Two sgRNAs targeting the second exon of the Drosophila MTHFD2 gene (CG18466) were designed with targeting sequences GGTGCGCACCTCCTGGGCTA (SEQ ID NO.3) and TCATAAACCGGGAATGATAA (SEQ ID NO.4). After cloning the sgRNAs into an expression vector, they were co-injected into Drosophila embryos along with a Cas9 helper plasmid to obtain MTHFD2 gene knockout lines. PCR and sequencing confirmed the existence of frameshift mutant lines MTHFD2-ko1 and MTHFD2-ko2, with deletions of 194 bp and 218 bp, respectively. Western blot analysis showed that the protein expression levels of MTHFD2-ko1 and MTHFD2-ko2 were 6.39% and 4.62% of the wild-type, respectively.

[0084] To construct a CRISPR / Cas9 gene-editing Drosophila strain targeting MTHFD2, the MTHFD2 gene sequence (Gene ID: CG18466) was first obtained from the Flybase database. The distribution of exons and introns was determined by alignment with NCBI, and coding exons longer than 200 bp were screened. sgRNAs (MTHFD2-ko1) with the sequence GGTGCGCACCTCCTGGGCTA (SEQ ID NO.3) and (MTHFD2-ko2) with the sequence TCATAAACCGGGAATGATAA (SEQ ID NO.4) were designed, adhering to the principles of 5'-N19-21-NGG-3' structure, GC content of 40%-60%, and avoidance of hairpin structures. Primers were synthesized after evaluation using the CRISPR Design online tool. The pEASY plasmid was digested with BbsI-HF, and the sgRNA was cloned into the digestion vector via inverse PCR. After transformation, single-clone screening, and sequencing verification, a high-purity sgRNA expression vector was extracted. The fruit fly strain BDSC78782 (y1sc) was selected. Using v1sev21;P{nanos-Cas9.R}attP2 as the genetic background, embryos were collected within 30 minutes. After sterilization, the sgRNA recombinant plasmid (300 ng / µL) was microinjected into each embryo (2 nL, 600 embryos in total). The injected embryos were cultured to obtain G0 generation adults, which were then crossed one-to-one with the balanced strain ywR13S (Sp / CyO; MKRS / TM2). Genotyping of the G1 generation was performed by PCR and gel electrophoresis (primers MTHFD2-F / R). Positive individuals were backcrossed to stabilize the mutant strain. In the G2 generation, MTHFD2-ko / TM2 individuals were selected for female-to-female crosses. Homozygous individuals were screened by PCR of the progeny, or heterozygous individuals were used for preservation. For propagation, 5 pairs of parents were inoculated into each tube, and the tubes were subcultured every 3-4 days for 3 consecutive times. After this process, the parents were removed, and the progeny adults were collected for later use. Fertility and developmental phenotype analysis of gene knockout strains: After culturing at 25℃ for 2 days, 20 fruit flies were placed in each bottle to lay eggs for 24 hours, and the number of eggs laid was counted. Newly laid eggs within 2 hours were collected, and single eggs were cultured in single tubes (80 replicates per genotype). Hatching, larval development, pupation, and emergence were recorded every 12 hours, and the pupation rate, emergence rate, and developmental duration of each stage were counted. The sensitivity of fruit fly larvae to lufenuron was determined using the feed-to-drug method: Lufenuron stock solution was mixed with culture medium to prepare eight concentration gradients of 0, 1, 5, 10, 20, 30, 40, and 50 mg / L. Second-instar larvae of wild-type and knockout strains were used as subjects, with 20 larvae per tube and 5 replicates per concentration. They were cultured at 25℃, and mortality was observed at 24, 48, 72, and 96 hours (death criteria included blackening of the body, elongation, and no response to stimulation). The corrected mortality rate was calculated, and the dose-mortality relationship was compared to analyze the effect of gene editing on lufenuron sensitivity.

[0085] The coding sequence (CDS, 3844bp) of the Drosophila MTHFD2 gene (located on chromosome 3, 8048 bp in length) was obtained from the Flybase database. This gene contains 5 exons and 4 introns. Two sgRNAs were designed targeting its 4th exon. Figure 11 (A). To verify the gene editing effect, PCR amplification and sequencing analysis were performed on the mutant genome. A fragment of approximately 800 bp was amplified in the control group (CK). Two independent homozygous mutant Drosophila strains were successfully obtained, named MTHFD2-ko1 and MTHFD2-ko2, respectively. The amplified fragments of MTHFD2-ko1 and MTHFD2-ko2 were approximately 600 bp and 500 bp, respectively. Sequencing analysis showed that MTHFD2-ko1 had a deletion between bases 31–226 of the CDS, a total of 194 bp was removed; MTHFD2-ko2 had a deletion of 218 bp in the corresponding region. The deletion lengths of both strains were not multiples of 3, resulting in frameshifts (…). Figure 11 (B)

[0086] Genotyping was performed by agarose gel electrophoresis. The homozygous MTHFD2-ko1 and MTHFD2-ko2 lines showed only about 600 bp and 500 bp mutant bands, respectively, while the wild-type control showed an 800 bp band, confirming the successful achievement of biallelic gene editing. Figure 11 (C). Western blotting was used to verify the knockout efficiency at the protein level. Tubulin was used as an internal control, and the loading amount was uniform. Quantitative analysis showed that the protein expression levels of MTHFD2-ko1 and MTHFD2-ko2 were 6.39±0.81% and 4.62±2.72% of the wild-type control group, respectively. Figure 1 (Middle D). The above results confirm that this study successfully constructed a homozygous mutant Drosophila strain of the MTHFD2 gene.

[0087] Phenotypic analysis of the obtained MTHFD2 homozygous mutant Drosophila strains showed that the deletion of MTHFD2 significantly affected the reproductive capacity and growth and development process of Drosophila. The egg production of the MTHFD2 knockout strains was significantly reduced (approximately 55 eggs for MTHFD2-ko1 and approximately 63 eggs for MTHFD2-ko2), a decrease of 60–70% compared to the wild type (P<0.001). This indicates that knockout of the MTHFD2 gene severely impairs the oviposition capacity of Drosophila. Figure 12 (A). MTHFD2 gene knockout significantly shortens the entire developmental process. Specifically, the egg, larval, and pupal stages of MTHFD2-ko1 were shortened by 28%, 16.67%, and 8.11%, respectively; while those of MTHFD2-ko2 were shortened by 24%, 14.28%, and 12.16%, respectively. Figure 12 (B) The average lifespan of the worms in the MTHFD2 knockout group was 9.75 ± 0.27 days, which was about 14.77% shorter than that in the CK group.

[0088] MTHFD2 knockout severely affected multiple developmental stages. Egg hatching rate (ko1: 30%, ko2: 16.25%) and pupation rate (ko1: 22.5%, ko2: 11.25%) were both significantly reduced. Emergence rate was also affected, with MTHFD2-ko1 significantly decreasing to 73.95% ( Figure 13 China A Figure 13 (Middle B and 13 Middle C).

[0089] The larval mortality rate of each knockout strain was determined 48 hours after treatment with lufenuron. Figure 14The results showed that gene editing significantly altered the Drosophila response to lufenuron compared to the wild-type (CK). While the mortality rates of MTHFD2-ko1 and MTHFD2-ko2 increased with increasing lufenuron concentration, exhibiting a concentration-dependent effect, they were significantly lower than the CK group at all concentrations. Overall, MTHFD2 gene knockout generally reduced the sensitivity of Drosophila to lufenuron, resulting in a lower mortality rate at the same concentration compared to the wild-type. This suggests that these genes may be involved in the toxicological pathway of lufenuron, and their editing may have weakened the lethal effect of lufenuron on Drosophila.

[0090] Example 7 Identification of key binding sites of lufenuron to MTHFD2.

[0091] To investigate the interaction mode between lufenuron and MTHFD2, molecular docking was first used for prediction: the 3D structure of lufenuron (CID: 71777) was downloaded from the PubChem database. The MTHFD2 protein obtained by ligand and homology modeling was pretreated by dehydration, hydrogenation, and charge addition using PyMOL. It was then converted to pdbqt format using AutoDock Tools. Semi-flexible docking was performed using the built-in Vina program in PyRx (the docking box covered the active pocket, with a grid spacing of 0.375 Å). The conformation with the lowest binding energy was selected as the initial complex, and visualization analysis was performed using Discovery Studio. Starting with the predicted complex structure, a 100 ns molecular dynamics simulation was performed using the Amber 24 software package. The system (protein ff14SB force field, small molecule GAFF force field, and charge calculation using RESP) was constructed using the LEaP tool and placed in a TIP3P water model, with Na+ / Cl- added to neutralize the charge. After energy minimization (first constraining the main chain, then unconstrained) and system equilibrium (NVT heating to 300 K, NPT equilibrium at 500 ps), a 100 ns unconstrained production simulation was performed under the NPT ensemble (temperature 300 K, pressure 1 bar, step size 2 fs, trajectory saved every 10 ps). RMSD, Rg, and RMSF were analyzed using CPPTRAJ. The binding free energy was estimated using the MM / GBSA method. 100 frames were uniformly extracted from the last 1 ns trajectory, and the contributions of van der Waals forces, electrostatics, polar solvation energy, and nonpolar solvation energy (unit: kJ / mol) were calculated. Key amino acids were identified by residue free energy decomposition. Finally, based on the optimized complex structure, computer simulation of alanine scanning was performed using the Residue Scanning module in Schrödinger Suite: residues within 5 Å of the ligand at the binding interface were selected and mutated one by one to alanine, and the change in binding free energy (ΔΔG) before and after the mutation was calculated. Residues with ΔΔG > 1 kcal / mol were identified as key sites that make important contributions to binding.

[0092] To identify the key amino acid residues involved in the interaction between lufenuron and the inter-grid spacing of cotton bollworm, molecular docking analysis first revealed that HIE 295, GLN 291, HIE 287, MET 284, ASN 288, LEU 137, PRO 135, THR 136, VAL 138, LEU 143, GLU 147, LYS 150, ILE 146, LEU 188, and ASN 186 are located within the binding pocket. Figure 15 Subsequently, molecular dynamics simulations were used to study the stability of the complex: the RMSD curve fluctuation range remained within 2 Å with no significant fluctuations, indicating high complex stability; the radius of gyration (Rg) curve remained stable throughout the simulation with minimal fluctuations, only showing slight fluctuations at the end of the simulation (around 40 ns), consistent with the SASA analysis results, further confirming the good overall stability of the complex; 100 ns equilibrium orbital simulations showed that the fluctuation values ​​of most amino acid residues in the binding pocket were within 30 Å, indicating that the binding pocket maintained a stable conformation during the simulation. Figure 16 (A). The binding free energy was calculated using the MM / PBSA method. The results showed that residues GLN291, LEU 137, THR 136, LEU 188, LEU 143, PRO 135, GLU 147, and GLU 116 contributed more than 1.0 kcal / mol to the binding free energy, playing an important role in ligand binding. Figure 16 (B) Alanine scanning mutagenesis (ASM) further showed that the binding free energy changed most significantly after mutations in LEU 188, LEU 143, and GLN 291, reaching 8.39, 8.12, and 5.30 kcal / mol, respectively. Mutations at these sites significantly weakened the binding of the protein to small molecules. Figure 16 (C)

[0093] Example 8 Determination of the inhibitory activity of lufenuron against MTHFD2 (Ki value).

[0094] The dehydrogenase activity of MTHFD2 was determined using an endpoint assay with 5,10-methylenetetrahydrofolate as the substrate. The reaction system (100 μL, 30℃, pH 7.8) contained: 0.05 M Tris / HCl buffer (pH 7.8), 100 mM 2-mercaptoethanol, 100 μM NADP+, different concentrations of 5,10-methylenetetrahydrofolate, and 20 μM MTHFD2 enzyme solution. The purified MTHFD2 was added to quartz 96-well plates to initiate the reaction. The absorbance at 340 nm was continuously monitored using a microplate reader. The NADPH generation rate, reflecting the substrate consumption rate, was used to characterize MTHFD2 activity. Calculate the reaction rate (μmol / L / min): v = (A sample - A blank control) / (εl t), where ε is the extinction coefficient of NADPH (0.00622μM-1·cm-1), l is the optical path length (the height of 100μL sample in a 96-well plate is approximately 0.276cm), and t is the enzyme reaction time (min).

[0095] The enzymatic kinetics of MTHFD2 were determined by ultraviolet spectrophotometry. Different concentrations of 5,10-methylenetetrahydrofolate were used as substrates to determine the corresponding reaction rates. Figure 17 As shown, the enzyme activity curve of MTHFD2 conforms to the typical Michaelis-Menten equation: in the low substrate concentration range, the reaction rate increases proportionally with the increase of substrate concentration; as the substrate concentration further increases, the rate of reaction slows down and gradually plateaus. Nonlinear fitting revealed that the Michaelis constant of MTHFD2 is 29.03 μmol / L, and Vmax is 42.50 μmol / L / min. These results indicate that the recombinant MTHFD2 protein possesses high catalytic activity and can effectively catalyze substrate conversion, laying the foundation for further research on the inhibitory effect of lufenuron on this enzyme.

[0096] Example 9 Sedimethomorph binding affinity (SPR) to MTHFD2.

[0097] The binding kinetics of lufenuron and MTHFD2 were analyzed using surface plasmon resonance (SPR) technology. First, ligand pre-enrichment was performed to determine the optimal coupling conditions: the coupling buffer was a 10-fold diluted PBS-P solution. The target coupling amount was calculated using the formula Rmax = (Analyte MW / Ligand MW) × Sm × RL (Rmax was set to 100 RU, Sm was defaulted to 1, and the actual coupling amount was 1.5 times the theoretical value). MTHFD2 was diluted to 20, 50, and 100 μg / mL with sodium acetate solutions at pH 4.0, 4.5, 5.0, and 5.5, respectively. A pre-enrichment program was run on the CM5 chip surface, and the pH condition with the highest coupling amount was selected for subsequent coupling. Subsequently, ligand coupling was performed: MTHFD2 was diluted to 20 μg / mL with sodium acetate at pH 4.5, and the protein was immobilized in channel 2 of the CM5 chip (channel 3 was a blank control) by amino coupling method (EDC / NHS activation, protein coupling, ethanolamine blocking). The coupling time was 900 s, the flow rate was 10 μL / min, and the temperature was 25℃. The actual coupling amount was then checked to see if it met the standard. Finally, a single-cycle kinetic mode was used to determine the binding parameters: lufenuron was dissolved in 5% DMSO, and the buffer was PBST (0.05% P2O) containing 5% DMSO. DMSO solutions were prepared in a gradient for solvent correction. Lufenuron was diluted with PBST containing 5% DMSO to 10, 5, 2.5, 1.25, and 0 nM, with methotrexate (1000, 500, 250, 125, and 0 nM) as positive controls. The Affinity module was selected, and the binding time was set to 60 s, the flow rate to 30 μL / min, and the dissociation time to 300 s. Each concentration was measured sequentially, and solvent correction was performed at the beginning and end (the pipeline was cleaned with 50% DMSO). The temperature was 25℃. The experimental data were imported into Biacore analysis software for fitting to obtain the binding constant (ka) and dissociation constant (kD).

[0098] Both lufenuron and methotrexate exhibit a specific binding response to MTHFD2. Figure 18 The binding response values ​​corresponding to a series of concentrations of lufenuron were kinetically fitted to obtain the affinity constant KD. The results showed that the KD value of lufenuron with MTHFD2 was 0.305 nM, indicating a high binding strength; while the KD value of methotrexate with MTHFD2 was 8.72 × 10⁴ nM, showing only a moderate binding strength. Compared with methotrexate, lufenuron showed a significantly higher binding affinity to cotton bollworm MTHFD2, and both binding models conformed to a steady-state binding mode. This result further confirms that lufenuron can directly act on the MTHFD2 protein, providing key evidence for revealing its molecular mechanism of targeting the folate metabolism pathway.

[0099] Example 10 Effects of lufenuron on MTHFD2 activity.

[0100] Using the same MTHFD2 enzyme inhibitory activity assay system as in Example 8, lufenuron technical was dissolved in DMF to prepare a 400 μM stock solution, which was then diluted with 0.1% Triton X-100 solution to create concentration gradients of 200, 100, 50, 25, 12.5, and 6.25 μM. For each sample group, 1 μL of lufenuron solution was added to a 96-well plate, followed by 84 μL of buffer and 5 μL of enzyme solution. The plate was incubated at room temperature for 15 min to allow for sufficient drug-enzyme reaction. Finally, 10 μL of substrate was added to initiate the reaction, and the plate was incubated at 30°C in the dark for 20 min. Positive and negative control groups were included. Absorbance at 340 nm was measured using a microplate reader, with six replicates for each treatment.

[0101] After clarifying the binding characteristics of lufenuron to MTHFD2, the inhibitory effect of lufenuron on it was further determined. Figure 19 As shown, under the same reaction conditions, lufenuron exhibited significant inhibitory activity against MTHFD2, with an inhibition constant Ki value of 4.305 ± 0.355 μmol / L, reaching the micromolar level, indicating that lufenuron is a potent inhibitor of MTHFD2. Combined with the SPR results, it can be clearly seen that lufenuron can not only efficiently recognize and bind to MTHFD2, but also effectively inhibit its catalytic function. The above studies, from the two dimensions of binding affinity and enzyme activity inhibition, jointly confirm the molecular mechanism by which lufenuron targets MTHFD2 in cotton bollworm.

[0102] Example 11 A high-throughput screening method for insecticides based on MTHFD2.

[0103] This embodiment establishes a high-throughput screening system based on MTHFD2 enzyme activity inhibition. The steps are as follows: A 96-well plate high-throughput screening system was established, with 100 μL of reaction solution per well containing: 50 mM Tris-HCl (pH 7.8), 100 μM NADP+, 100 mM 2-mercaptoethanol, 20 μM recombinant MTHFD2 protein (SEQ ID NO.1), and different concentrations of candidate compounds (final concentration 0.1~100 μM). After pre-incubation at 25℃ for 15 min, 5,10-methylenetetrahydrofolate oxygen (final concentration 50 μM) was added to start the reaction. Immediately, the reaction was continuously monitored at 340 nm wavelength for 10 min using a microplate reader, with readings every 30 seconds. The NADPH generation rate was calculated from the linear interval of 0-5 min. A blank control group (without candidate compounds, but with an equal volume of solvent), a negative control group (without enzyme), and a positive control group (50 μM lufenuron) were set up. The inhibition rate is calculated as follows: Inhibition rate (%) = (Control group rate - Treatment group rate) / Control group rate × 100%.

[0104] Based on inhibition rate, compounds are divided into three categories: those with an inhibition rate ≥70% are highly active compounds, those with an inhibition rate of 40%-70% are moderately active compounds, and those with an inhibition rate <40% have no significant inhibitory activity. Figure 20 For compounds with an initial screening inhibition rate ≥40%, further determination of IC50 was performed. 50 .

[0105] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An MTHFD2 protein, characterized in that, The amino acid sequence of the MTHFD2 protein is shown in SEQ ID NO.

1.

2. The MTHFD2 protein according to claim 1, characterized in that, The nucleotide sequence of the gene encoding the MTHFD2 protein is shown in SEQ ID NO.

2.

3. The use of the MTHFD2 protein of claim 1 in the preparation of products that inhibit insect growth and development.

4. The use of the MTHFD2 protein of claim 1 in monitoring insect resistance to pesticides.

5. The application of the MTHFD2 protein according to claim 1 in pesticide identification.

6. The application of the MTHFD2 protein according to claim 1 in regulating insect sensitivity to pesticides, characterized in that, Downregulating the expression of MTHFD2 protein reduces the sensitivity of insects to pesticides.

7. The application according to any one of claims 4 to 6, characterized in that, The MTHFD2 protein serves as a target for insecticides.

8. The application according to any one of claims 4 to 6, characterized in that, The insecticide includes any one or more of the following: plant extracts, compounds with well-defined chemical structures, peptides, nucleic acids, polysaccharides, viral vectors, liposome vectors, or nanoparticle vectors.

9. The application according to any one of claims 4 to 6, characterized in that, The insecticide targets lepidopteran or dipteran insects.

10. A method for identifying insecticides, characterized in that, Includes the following steps: Add the analyte to the MTHFD2 protein as described in claim 1, and measure the enzyme activity of the MTHFD2 protein in the presence of the analyte. If the enzyme activity of the MTHFD2 protein decreases, the analyte is an insecticide.

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