Locked nucleic acid molecules targeting tyrosyl hydroxylase g-quadruplexes and uses thereof

Abstract

The application provides a locked nucleic acid molecule targeting a G-quadruplex of tyrosine hydroxylase and application thereof, and belongs to the field of biotechnology and pesticide development. TH The sequence of PQS3-2 is a target sequence, and a locked nucleic acid molecule sequence is designed by using a bioinformatics analysis tool, and the locked nucleic acid molecule is synthesized and purified by using a chemical synthesis technology, the molecule can specifically target TH The PQS3-2 G-quadruplex is combined to interfere with normal functions, so that the expression of the tyrosine hydroxylase gene is inhibited.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and pesticide development, specifically to a locked nucleic acid molecule that targets the G-quadruplex of the tyrosine hydroxylase gene promoter and its application in the development of nucleic acid pesticides. Background Technology

[0002] Beet armyworm ( Spodoptera exigua ) belongs to the order Lepidoptera ( Lepidoptera Noctuidae ( Noctuidae The beet armyworm (Spodoptera exigua) is an insect belonging to the genus Spodoptera exigua. It has a wide host range and can cause serious damage to various economic crops, including vegetables (such as cabbage, bok choy, and spinach), cotton, tobacco, sugar beets, corn, and soybeans, leading to reduced yields or even total crop failure. The beet armyworm larvae are voracious eaters, capable of devouring crop leaves in a short time, causing the crop to lose its photosynthetic capacity and, in severe cases, resulting in reduced yields or even total crop failure. Therefore, timely and effective control measures are crucial for ensuring agricultural production.

[0003] In the control of beet armyworm, traditional chemical pesticides (such as organophosphates, pyrethroids, and carbamates) have long been the main control method. While applying pesticides during the larval stage can suppress adult populations and reduce the number of the next generation of larvae, it still presents several problems: First, resistance is a significant issue: the beet armyworm has a short generation cycle and high reproductive capacity, and long-term, large-scale use of chemical pesticides leads to rapid development of resistance. Currently, the beet armyworm has developed high levels of resistance to many commonly used insecticides, leading to increasing control difficulties and requiring ever-increasing dosages, creating a vicious cycle. Second, chemical pesticides have long residual periods in soil, water, and the atmosphere, exerting toxic effects on non-target organisms (such as natural enemies, pollinators, and soil microorganisms), disrupting the balance of the farmland ecosystem. Furthermore, excessive pesticide residues directly affect the quality of agricultural products and food safety, and the use of chemical pesticides leads to a decline in biodiversity, impacting sustainable agricultural development. Therefore, there is an urgent need for new green control technologies to replace or supplement traditional chemical pesticides.

[0004] With the rapid development of agricultural biotechnology, RNA interference (RNAi) technology has been increasingly applied in the pesticide field due to its characteristics such as easy degradation, no residue, high specificity, and environmental friendliness. Nucleic acid pesticides work by designing nucleic acid molecules complementary to the key gene sequences of pests, specifically inhibiting the expression of target genes, thereby preventing pest growth and development and achieving pest control. Compared with traditional chemical pesticides, nucleic acid pesticides are highly specific, acting only on specific genes of target pests and are safe for non-target organisms; they are easily degraded in the environment and leave no persistent residues; furthermore, nucleic acid pesticides act on essential genes of pests, making it less likely to induce resistance, and based on genomic information, nucleic acid molecules targeting new targets can be rapidly designed, offering high precision and controllability.

[0005] G-quadruplexes (G4) are four-stranded higher-order nucleic acid structures formed by Hoogsteen hydrogen bonds in guanine (G)-rich regions of nucleic acids under specific conditions (such as the presence of cations). G-quadruplexes are widely distributed in key regulatory regions of eukaryotic genomes, such as promoter regions, telomere regions, and the 5' untranslated region of mRNA, and are closely related to various biological processes, including gene transcription, translation, and telomere maintenance. In gene promoter regions, the formation of G-quadruplexes can affect the binding of transcription factors to DNA, the recruitment of RNA polymerases, and chromatin conformation, thereby regulating gene transcriptional activity. Studies have shown that nucleic acid molecules targeting specific G-quadruplexes can stabilize or disrupt their structure by binding to the G-quadruplex, thereby regulating the expression of proto-oncogenes (such as c-MYC, KRAS, and BCL-2). However, in the field of plant protection, pest control methods targeting G-quadruplex structures have not yet been developed.

[0006] Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the dopamine biosynthesis pathway in insects. It catalyzes the hydroxylation of L-tyrosine to L-DOPA, a crucial first step in the synthesis of catecholamine neurotransmitters such as dopamine, norepinephrine, and epinephrine. L-DOPA produced by TH is a precursor to melanin synthesis, and melanin plays a vital role in insect epidermal hardening, body color formation, and immune defense. Inhibition of TH... TH Gene expression can hinder the formation of the new cuticle in the beet armyworm, affecting its normal molting and development process, leading to larval failure, impaired pupation, and even death. Therefore, TH Genes are ideal targets for developing nucleic acid pesticides; however, there are currently no nucleic acid pesticide products developed that target tyrosine hydroxylases. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention designs and synthesizes a specific locked nucleic acid molecule, named TH-G4-LNA, targeting the G-quadruplex TH PQS3-2 in the promoter region of the beet armyworm tyrosine hydroxylase gene. This molecule specifically targets the TH PQS3-2 G-quadruplex, binding to it and interfering with its normal function, thereby inhibiting its activity. TH Gene expression.

[0008] To achieve the above objectives, the present invention provides the following technical solution to address the technical problem:

[0009] In a first aspect, the present invention provides a locked nucleic acid molecule that targets the beet armyworm tyrosine hydroxylase promoter G-quadruplex THPQS3-2, wherein the sequence of the beet armyworm tyrosine hydroxylase promoter G-quadruplex THPQS3-2 has a nucleotide sequence as shown in SEQ ID NO.1.

[0010] Secondly, the present invention provides a locked nucleic acid molecule that targets the G-quadruplex THPQS3-2 of the promoter of the beet armyworm tyrosine hydroxylase gene, wherein the locked nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO.2, or has a nucleotide sequence that has more than 90% homology with the nucleotide sequence shown in SEQ ID NO.2.

[0011] In some embodiments, the locked nucleic acid molecule contains a locked nucleic acid-modified nucleotide, the site of which is targeted at the G-tetrad plane of the G-quadruplex TH PQS3-2.

[0012] In some embodiments, the locked nucleic acid molecule is chemically modified with a sugar ring, introducing a methylene bridge between the 2' oxygen atom and the 4' carbon atom of the ribose to "lock" the sugar ring in a specific RNA conformation.

[0013] In some embodiments, the preparation of the locked nucleic acid molecule includes the following steps:

[0014] First, the sequence of the G-quadruplex TH PQS3-2 in the promoter region of the beet armyworm tyrosine hydroxylase gene was used as the target sequence, and the LNA sequence was designed using bioinformatics analysis tools. Then, the LNA molecule was synthesized and purified using chemical synthesis techniques.

[0015] Thirdly, the present invention provides a nucleic acid pesticide for controlling agricultural pests, comprising the aforementioned locked nucleic acid molecule as an active ingredient, and a pesticide-acceptable adjuvant or carrier.

[0016] In some embodiments, the adjuvant includes at least one of a nucleic acid protectant, a penetration enhancer, a spreading agent, or a surfactant; preferably, the nucleic acid protectant includes liposomes, nanomaterials, chitosan, or hydrogels.

[0017] In some embodiments, the working concentration of the locked nucleic acid molecules in the nucleic acid pesticide is 5 μM to 10 μM.

[0018] In some embodiments, the agricultural pest is a Lepidoptera noctuid moth.

[0019] In some embodiments, the agricultural pest is the beet armyworm.

[0020] Fourthly, this invention provides the application of nucleic acid pesticides in the preparation of formulations for controlling lepidopteran agricultural pests.

[0021] Fifthly, the present invention provides a method for controlling agricultural pests, wherein the aforementioned nucleic acid pesticide is applied to the agricultural pest or the crop it damages.

[0022] In some embodiments, the nucleic acid pesticide is applied to crop plants, the pest's living environment, or directly to the pest.

[0023] In some embodiments, the nucleic acid pesticide application method includes injection, micro-injection, feeding, spraying, soaking, or root irrigation;

[0024] In some embodiments, nucleic acid pesticides are injected directly into the insects, suitable for laboratory research and precision application; or mixed into feed and ingested by the insects, suitable for larval control; or formulated into aqueous solutions or nano-preparations for foliar spraying, suitable for field production.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0026] This invention provides a locked nucleic acid molecule that targets the G-quadruplex of tyrosine hydroxylase, exhibiting high stability and specificity. The locked nucleic acid molecule (TH-G4-LNA) provided by this invention incorporates LNA modification, possessing extremely high nuclease resistance and thermostability, enabling it to exert a sustained effect in pests, with a significant and long-lasting gene silencing effect.

[0027] The pesticide molecule of this invention is based on the beet armyworm. TH The gene promoter-specific G-quadruplex sequence was designed and found to be homologous to the human genome after BLAST alignment. It is highly safe for non-target organisms and environmentally friendly, opening up a new approach for the development of green nucleic acid pesticides by regulating pest genes at the DNA structure level.

[0028] In this invention, in vivo biological experiments show that this molecule can significantly prolong the development time of beet armyworm larvae, greatly reduce their pupation rate and pupal weight, and has a significant lethal effect, thus providing an effective method for controlling agricultural pests. Attached Figure Description

[0029] Figure 1 TH PQS3-2 G-quadruplex structure diagram.

[0030] Figure 2Promoter activity diagrams of wild-type and mutant TH PQS3-2 sequences under different concentrations of LNA treatment. pGL3-basic is the blank plasmid, pGL3-F203 is the plasmid containing the TH PQS3-2 sequence, and pGL3-F203mut is the plasmid containing the THPQS3-2 mut sequence.

[0031] Figure 3 The effect of injecting enzyme-free water into the control group on the growth and development of 5th instar larvae of the beet armyworm.

[0032] Figure 4 The effect of injecting 5 μM TH-G4-LNA on the growth and development of 5th instar larvae of the beet armyworm.

[0033] Figure 5 The effect of injecting 10 μM TH-G4-LNA on the growth and development of 5th instar larvae of the beet armyworm.

[0034] Figure 6 The effect of TH-G4-LNA injection on the pupation rate of beet armyworm.

[0035] Figure 7 The effect of TH-G4-LNA injection on the pupal weight of beet armyworm.

[0036] Figure 8 Injection of TH-G4-LNA against beet armyworm TH A diagram illustrating the impact of gene expression levels. Detailed Implementation

[0037] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to specific examples. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0038] Unless otherwise specified, the experimental methods used in this invention are conventional methods; unless otherwise specified, the materials and reagents used are commercially available.

[0039] Example 1: Design and Synthesis of TH-G4-LNA Molecule

[0040] First, targeting the G-quadruplex TH PQS3-2 in the promoter region of the beet armyworm tyrosine hydroxylase (TH) gene, a specific locked nucleic acid (LNA) molecule was designed and synthesized, named TH-G4-LNA. The specific operation is as follows:

[0041] 1. Identification and analysis of target sequences:

[0042] The promoter sequence of the beet armyworm tyrosine hydroxylase gene was obtained from the NCBI database (GenBank: KR058777.1). Circular dichroism spectroscopy (CD), non-denaturing polyacrylamide gel electrophoresis (Native-PAGE), and one-dimensional nuclear magnetic resonance (1H-NMR) were used to identify the G-quadruplex formation sequence TH PQS3-2 located in this promoter region, which was then used as the target molecule for locking nucleic acid. The nucleic acid sequence of the target molecule TH PQS3-2 is: 5'-GGACCGGGGGATGCCGG-3' (SEQ ID NO. 1).

[0043] Single-base mutant sequences were designed (as shown in Table 1), and single-stranded DNA was synthesized. The DNA sequence was diluted to 5 μM with 10 mM KH2PO4 / K2HPO4 (phosphate buffer, pH 7.0) and 100 mM KCl solution. The solution was heated at 95°C for 5 min in a constant-temperature metal bath, then naturally cooled to room temperature and incubated at 4°C for 2 h. A 15% non-denaturing polyacrylamide gel was prepared: 7.5 mL 40% (19:1) acrylamide, 4 mL 5 × TBE, 0.5 mL KCl (2 mol / L), 8 mL ddH2O, 120 μL 10% APS, and 20 μL TEMED. Pre-electrophoresis was performed at 80 V for 20 min, followed by adding the DNA to the gel wells and electrophoresis at 100 V for 2 h (4°C). 4 µL of SYBR Gold was dissolved in 40 mL of 1 × TBE (containing 50 mmol / L KCl). The gel was placed in a staining cassette containing the staining agent and stained for 15 min. The bands were detected using a Bio-Rid gel imaging system, and analyzed using ImageLab software. The conformation of the G-quadruplex structure formed by the sequence was analyzed, and the structural model diagram is shown below. Figure 1 As shown.

[0044] Table 1. Mutated TH PQS3-2 DNA sequence

[0045]

[0046] 2. Design and modification of LNA molecules:

[0047] Based on the principle of complementary base pairing, locked nucleic acid fragments were designed and coupled with human (… Homo sapiensBLAST homology analysis of the genome confirmed that no 100% homologous sequence exists in the human genome. The LNA sequence (named TH-G4-LNA), highly complementary to the target sequence, was synthesized, modified, and purified by Shanghai Bioengineering Co., Ltd., with a fragment length of 17 nt.

[0048] The nucleotide sequence of TH-G4-LNA is: 5'-C C GGCATC C C C CGGT C C-3' (SEQ ID NO.2); where the bold underlined part refers to the locked nucleic acid modified nucleotide, and the modification site targets the G-tetrad plane of TH PQS3-2.

[0049] 3. Purification and Quality Control:

[0050] TH-G4-LNA molecules were synthesized using chemical synthesis techniques and purified by high-performance liquid chromatography (HPLC) to ensure purity. The accuracy of its chemical structure was confirmed using nuclear magnetic resonance (NMR) technology, and its molecular weight and purity were determined by mass spectrometry (MS) analysis, ensuring the quality of each batch of TH-G4-LNA molecules.

[0051] Example 2: Cellular Experiment Verification of the Inhibition of Promoter Activity by TH-G4-LNA

[0052] A reporter gene plasmid containing the TH PQS3-2 sequence in its promoter region was co-transfected with TH-G4-LNA molecules into Sf9 cells. Untransfected Sf9 cells were used as a blank control group. Oligo(dT) was used to treat the cells. 18 The treatment served as a negative control. After 24 hours, the activity ratio of the two luciferases was measured using a dual-luciferase reporter gene system to assess the TH-G4-LNA molecule's effectiveness. TH The effect of gene promoter activity was investigated, and the specific experimental steps are as follows:

[0053] (1) Cells were cultured in sterile cell culture flasks using complete culture medium and allowed to adhere to the cell walls.

[0054] (2) When the cell density reaches 90%, gently blow the cells away from the bottle wall to suspend them, and count the cells using a hemocytometer;

[0055] (3) Dilute the cells with complete culture medium to a concentration of 1×10⁻⁶. 5 Add 1 mL of cells to each well of a 24-well adherent cell culture plate until the cell concentration reaches 1 × 10⁻⁶ cells / mL. 5 Cells / wells are placed in a cell culture incubator for 24 hours to allow the cells to adhere to the incubator.

[0056] (4) The amount of transfection solution used per well is prepared according to the following system, as shown in Table 2:

[0057] Table 2. Preparation of Transfection Reagents

[0058]

[0059] Prepare super mixes of transfection solutions A and B in Eppendorf tubes, then slowly add solution B dropwise to solution A, gently invert the tube 3-5 times, mix well, and let stand at room temperature for 20 minutes.

[0060] (5) Remove the cell plate from the incubator, remove the complete culture medium, wash the cells in each well with incomplete culture medium, and remove the washing medium.

[0061] (6) Add 800 μL of incomplete culture medium to each well, and then slowly add 200 μL of the incubated A+B mixture to each well. Then return the cells to the cell culture incubator and continue culturing for 5.5 h;

[0062] (7) Remove the cell plate from the incubator, remove the transfection solution from the wells, add 1 mL of complete culture medium to each well, and then incubate in the cell culture incubator for 24 h. Then collect the cells for luciferase activity assay, and perform the operation according to the kit instructions (Dual-Luciferase® Reporter Assay, SystemDual Luciferase® dual luciferase reporter gene detection system (firefly / Kidney luciferase detection reagent), purchased from Promega); set up 3 replicates for each group.

[0063] Experimental results are as follows Figure 2 As shown, compared with the blank control group and the negative control group, cells treated with TH-G4-LNA (10 μM and 30 μM) showed that... TH Gene promoter activity was significantly reduced. Treatment with 10 μM TH-G4-LNA reduced luciferase activity by about 14%, and when the concentration was increased to 30 μM, luciferase activity decreased by about 40%, indicating that TH-G4-LNA can effectively and specifically inhibit the transcriptional regulatory activity of this promoter.

[0064] Example 3: Indoor Control and Gene Silencing Effect Experiment of Beetroot Cutworm

[0065] To verify the effects of TH-G4-LNA molecules on the growth and development of beet armyworm at the in vivo level and to evaluate its application potential as a candidate molecule for nucleic acid pesticides.

[0066] 1. Test insects and environment:

[0067] Healthy, uniformly developed 5th instar beet armyworm larvae were selected. They were reared in the laboratory at 27±1℃, photoperiod L14:D10, and relative humidity (RH) of 75%. This ensured that the influence of environmental factors on the experimental results was minimized.

[0068] 2. Experimental grouping and treatment:

[0069] Four groups were set up: a blank control group, a negative control group, and treatment groups with different concentrations of TH-G4-LNA (see Table 3). Each group had three replicates. LNA molecules were precisely injected into the larvae using a microinjector at a dose of 0.2 μL, corresponding to final concentrations of 5 μM / larva and 10 μM / larva, respectively. Each larva was injected once.

[0070] Table 3 Experimental Groups

[0071]

[0072] 3. Measurement of observation indicators:

[0073] During the experiment, the growth and development of the larvae were observed and recorded daily, including indicators such as survival rate, developmental stage transition time, pupation rate, and pupal weight. Simultaneously, larval and pupal samples at different developmental stages were collected for subsequent molecular biological analysis to determine the molecular pairing of TH-G4-LNA. TH The inhibitory effect on gene expression was detected by quantitative real-time PCR (qPCR). TH Gene expression levels were used to verify the effects of TH-G4-LNA molecules at the molecular level.

[0074] (1) Survival rate: The number of surviving larvae in each group was counted daily, and the survival rate was calculated. The effect of TH-G4-LNA treatment on larval survival was observed, and its lethal effect and inhibitory effect on larval growth were analyzed.

[0075] Depend on Figure 3-5 It was found that the cumulative mortality rate of larvae increased with increasing TH-G4-LNA concentration. The cumulative mortality rate of the control group was 14.4% ± 1.2%, the cumulative mortality rate of the 5 μM TH-G4-LNA group was 21.7% ± 2.1%, and the cumulative mortality rate of the 10 μM TH-G4-LNA group was 30.0% ± 3.0%, indicating that TH-G4-LNA treatment significantly increased the mortality rate of larvae, and the lethal effect was stronger in the high concentration group.

[0076] (2) Developmental stage transition time: Record the time when the larvae enter the prepupa (PP) stage from the fifth instar larva (L5) stage and the time when they enter the pupal stage (P) stage from the PP stage.

[0077] Depend on Figure 3-5 The results showed that the development time of the TH-G4-LNA-treated group was significantly prolonged compared with the control group. From the L5 to PP stage, the development time was 1.3±0.5 days in the control group, 2.5±0.8 days in the 5 μM group, and 3.2±1.1 days in the 10 μM group; from the PP to P stage, the development time was 1.2±0.3 days in the control group, 2.0±0.7 days in the 5 μM group, and 1.8±0.6 days in the 10 μM group. This indicates that TH-G4-LNA can significantly delay the development process of larvae, and this delaying effect is positively correlated with concentration.

[0078] (3) Pupation rate and pupal weight: After development is complete, the pupation rate of each group is counted and the pupal weight is measured.

[0079] like Figure 6-7 As shown, the pupation rate in the control group was 85.6% ± 2.1%, and the pupal weight was 0.131 g ± 0.002 g; the pupation rate in the 5 μM group decreased to 53.3% ± 5.3%, and the pupal weight slightly decreased to 0.128 g ± 0.003 g; the pupation rate in the 10 μM group further decreased to 43.3% ± 6.7%, and the pupal weight significantly decreased to 0.121 g ± 0.005 g (a decrease of 7.6%). These results indicate that TH-G4-LNA not only prolongs the development time but also significantly reduces the pupation rate, thus inhibiting the development of the beet armyworm.

[0080] (4) TH gene expression level: The expression level of TH gene in larvae injected with TH-G4-LNA was determined by real-time quantitative PCR (qRT-PCR). TH Gene expression level.

[0081] The PCR program is as follows: pre-denaturation at 95 ℃ for 30 s; the cycling steps include: denaturation at 95 ℃ for 5 s, annealing at 60 ℃ for 10 s, extension at 72 ℃ for 30 s, for 40 cycles, with fluorescence signal acquisition during the extension step; the melting curve program is as follows: the temperature starts at 60 ℃ and gradually increases to 95 ℃ at a rate of 0.3 ℃ / s, with real-time monitoring of fluorescence signal during the heating process.

[0082] In qRT-PCR experiments, the ΔΔCt method was used to calculate the relative mRNA expression level: assuming a doubling of product quantity per cycle, the Ct value obtained during the exponential phase of the PCR reaction was used to infer the initial template quantity; a difference of one cycle (Ct=1) corresponds to a difference of two-fold in the initial template quantity. The relative mRNA expression level of the target gene in each sample was normalized by the expression level of the housekeeping gene β-actin in the sample. The relative mRNA expression level of the target gene in each sample can be calculated using the following formula: Relative mRNA expression level = 2 ^(-△△Ct)Graphpad Prism 10.1 software was used for plotting and differential significance analysis. Primer information is shown in Table 4.

[0083] Table 4 qRT-PCR primer information

[0084]

[0085] like Figure 8 As shown, compared with the control group, 5 μM TH-G4-LNA reduced... TH Gene expression decreased by 93.4%, while the 10 μM treatment group decreased by 94.1%, exhibiting concentration saturation inhibition characteristics.

[0086] 4. Analysis of experimental results:

[0087] In-depth analysis of the recorded data was conducted, and statistical methods were used to verify the significant differences between the TH-G4-LNA molecule treatment group and the control group. The results showed that TH-G4-LNA molecules significantly inhibited the growth and development of the beet armyworm in multiple ways, including reducing survival rate, prolonging development time, reducing pupation rate, and decreasing pupal weight. From a molecular mechanism perspective, this inhibitory effect is mainly attributed to the highly efficient suppression of TH gene expression by TH-G4-LNA molecules. Further research indicated that the specific binding of TH-G4-LNA molecules to the TH PQS3-2 G-quadruplex interfered with its normal function, thereby affecting growth and development. TH Gene transcriptional activity. This gene silencing effect can be achieved through various pathways, such as inhibiting the binding of transcription factors to promoter regions, affecting DNA topology, or recruiting chromatin remodeling complexes, ultimately leading to... TH Gene expression is downregulated. Furthermore, experiments in this invention have revealed that the TH-G4-LNA molecule possesses high stability and targeting ability, enabling it to exert a sustained effect within pests, with a long duration of action and sustained efficacy. These characteristics make the TH-G4-LNA molecule a promising candidate for pest control, providing a strong foundation for developing novel, efficient, and environmentally friendly nucleic acid pesticides.

[0088] In summary, this invention successfully developed the TH-G4-LNA molecule, which can specifically target the beet armyworm. TH The G-quadruplex TH PQS3-2 in the gene promoter region effectively inhibits TH Gene expression is disrupted, interfering with the growth and development of the beet armyworm. TH-G4-LNA treatment significantly reduced the survival rate, prolonged development time, and decreased pupation rate and pupal weight of the beet armyworm, with the effect being concentration-dependent. This invention provides a highly efficient and environmentally friendly nucleic acid pesticide candidate molecule for pest control, and is expected to promote the development of pest control technology and effectively solve the problems caused by traditional chemical pesticides.

[0089] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A locked nucleic acid molecule targeting the G-quadruplex TH PQS3-2 promoter of beet armyworm tyrosine hydroxylase, characterized in that, The sequence of the beet armyworm tyrosine hydroxylase promoter G-quadruplex TH PQS3-2 has the nucleotide sequence shown in SEQ ID NO.

1.

2. A locked nucleic acid molecule targeting the G-quadruplex TH PQS3-2 promoter of the beet armyworm tyrosine hydroxylase gene, characterized in that, The locked nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO.2, or has a nucleotide sequence that is more than 90% homologous to the nucleotide sequence shown in SEQ ID NO.

2.

3. The locked nucleic acid molecule as described in claim 1 or 2, characterized in that, The locked nucleic acid molecule contains a locked nucleic acid-modified nucleotide, the site of which is targeted at the G-tetrad plane of the G-quadruplex TH PQS3-2.

4. The locked nucleic acid molecule as described in claim 1, characterized in that, The preparation of the locked nucleic acid molecule includes the following steps: First, the sequence of the G-quadruplex TH PQS3-2 in the promoter region of the beet armyworm tyrosine hydroxylase gene was used as the target sequence, and the LNA sequence was designed using bioinformatics analysis tools. Then, the LNA molecule was synthesized and purified using chemical synthesis techniques.

5. A nucleic acid pesticide for controlling agricultural pests, characterized in that, It contains the locked nucleic acid molecule as described in any one of claims 1 to 4 as the active ingredient, and a pesticide-acceptable adjuvant or carrier.

6. The nucleic acid pesticide as described in claim 5, characterized in that, The working concentration of the locked nucleic acid molecules in the nucleic acid pesticide is 5 μM to 10 μM.

7. The nucleic acid pesticide as described in claim 5, characterized in that, The agricultural pests mentioned are noctuid moths belonging to the order Lepidoptera.

8. The nucleic acid pesticide as described in claim 7, characterized in that, The agricultural pest in question is the beet armyworm.

9. The use of the locked nucleic acid molecule as described in any one of claims 1 to 4, or the nucleic acid pesticide as described in any one of claims 5 to 8, in the preparation of formulations for controlling lepidopteran agricultural pests.

10. A method for controlling agricultural pests, characterized in that, The nucleic acid pesticide according to any one of claims 5 to 8 is applied to the agricultural pest or the crop it damages, wherein the application method includes injection, feeding or spraying.

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