RNA binding protein LOC111350322 and application thereof in pest control

Abstract

This invention belongs to the field of pest control technology and discloses an RNA-binding protein. LOC111350322 And its application in pest control. The inventors first discovered an RNA-binding protein that promotes Nrf2 protein expression in the agricultural pest *Spodoptera litura*, whose gene number is [gene number missing]. LOC111350322. Through cell and in vitro experiments and other techniques, it was confirmed that LOC111350322 It can bind to the 3'UTR of Nrf2, a key transcription factor for combating oxidative stress, to promote Nrf2 protein expression. In vivo experiments further confirmed that this is achieved through dsRNA interference. LOC111350322 The expression can downregulate the beet armyworm's ability to cope with plant secondary metabolites, increase the toxicity of plant secondary metabolites to the beet armyworm, and thus inhibit the growth of the beet armyworm, thereby achieving the purpose of pest control.

Description

Technical Field

[0001] This invention belongs to the field of pest control technology, specifically relating to an RNA-binding protein. LOC111350322 And its application in pest control. Background Technology

[0002] RNA-binding proteins (RBPs) are a class of functional proteins that can specifically bind to RNA molecules (such as mRNA, lncRNA, circRNA, etc.). They typically contain one or more conserved domains (such as RRM, KH, zinc finger structures, etc.), which recognize specific RNA sequences or secondary structures, thereby forming ribonucleoprotein complexes (RNPs). RNA-binding proteins are widely distributed in eukaryotes and prokaryotes, participating in almost all stages of the RNA life cycle and are key executors of gene expression regulation.

[0003] RNA-binding proteins play multiple functions within cells by regulating RNA metabolism. They can affect RNA splicing (e.g., SF2 / ASF regulates alternative splicing), editing, stability (e.g., HuR binding to mRNA delays degradation), and localization (e.g., ZBP1 mediates subcellular transport of mRNA). RNA-binding proteins can activate or inhibit translation by binding to the 5' or 3' untranslated region (UTR) of mRNA. They can also interact with miRNAs, lncRNAs, etc., to participate in gene silencing or chromatin modification.

[0004] Nrf2 Nuclear factor E2-associated factor 2 (NF-κB2) is a key transcription factor and a core regulator of cellular resistance to oxidative stress and electrophilic substance damage. Under normal circumstances, Nrf2 It binds to its inhibitory protein Keap1 in the cytoplasm and is rapidly degraded by proteases. When cells are subjected to oxidative stress, electrophilic agents, or chemical toxins, Nrf2 It dissociates from Keap1 and enters the cell nucleus, where it forms a heterodimer with a small Maf protein. It then binds to the antioxidant response element (ARE) in the promoter region of the target gene, initiating the expression of a series of antioxidant and detoxification genes. Nrf2 Its main function is to coordinate cellular antioxidant responses and phase II detoxification metabolism. The genes it regulates encode proteins including antioxidant proteins, detoxification enzymes, and drug transport proteins. In insects, Nrf2 Signaling pathways are a core defense mechanism for insects against external toxins such as plant secondary compounds and synthetic pesticides. When insects ingest toxins, Nrf2It will be activated and, together with its chaperone protein Maf, initiate the expression of numerous detoxification enzymes (such as GSTs, P450s, and carboxylesterases CarEs) and antioxidant genes. Overactivation of this system is a key reason for insect adaptation to plant secondary compounds and pesticides.

[0005] Spodoptera litura ( Spodoptera litura The beet armyworm (Spodoptera litura) is an omnivorous pest belonging to the Noctuidae family of Lepidoptera, capable of damaging nearly 300 plant species. Currently, pest control still relies primarily on chemical insecticides; however, long-term, unregulated, and excessive use of pesticides has led to pesticide resistance in pests. With the increasing resistance levels of the beet armyworm, the effectiveness of existing chemical control methods is declining, and control costs are rising significantly, posing a serious threat to agricultural production safety. Therefore, developing highly efficient, specific, and environmentally friendly new pest control technologies has become an urgent need for sustainable agricultural development. Summary of the Invention

[0006] The first aspect of the present invention is to provide LOC111350322 Application of inhibitors in the control of pests or in the preparation of products for the control of pests.

[0007] A second aspect of the present invention is to provide a dsRNA.

[0008] A third aspect of the present invention aims to provide biological materials related to dsRNA in relation to the second aspect of the present invention.

[0009] The fourth aspect of this invention is to provide a product.

[0010] The fifth aspect of this invention aims to provide applications of the dsRNA of the second aspect of this invention, the biomaterial of the third aspect of this invention, or the product of the fourth aspect of this invention.

[0011] The fifth aspect of this invention is to provide a method for controlling pests.

[0012] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of the present invention provides LOC111350322 The application of inhibitors in the control of pests or in the preparation of pest control products, the LOC111350322 The nucleotide sequence is shown in SEQ ID NO:1.

[0013] In some embodiments of the present invention, the LOC111350322 Inhibitors are inhibitors LOC111350322 Active substances, degradation LOC111350322 substances or reduction LOC111350322 At least one of the substances expressing the level.

[0014] In some embodiments of the present invention, the reduction LOC111350322 The substance expressing the reduced level is... LOC111350322 The substance expressing the level is at least one of a1)-a13): a1) Target LOC111350322 At least one of the following: siRNA, dsRNA, miRNA, ribozyme, shRNA, or CRISPR / Cas system; a2) encodes the nucleic acid molecule of a1); a3) An expression cassette containing the nucleic acid molecule described in a2); a4) A recombinant vector containing the nucleic acid molecule described in a2); a5) A recombinant vector containing the expression cassette described in a3); a6) Recombinant cells containing the nucleic acid molecules described in a2); a7) Recombinant cells containing the expression cassette described in a3); a8) Recombinant cells containing the recombinant vector described in a4); a9) Recombinant cells containing the recombinant vector described in a5); a10) Recombinant microorganisms containing the nucleic acid molecules described in a2); a11) Recombinant microorganisms containing the expression cassette described in a3); a12) Recombinant microorganisms containing the vector described in a4); a13) Recombinant microorganisms containing the vector described in a5).

[0015] In some embodiments of the present invention, the LOC111350322 The inhibitor is at least one of a1)-a13): a1) Target LOC111350322 a2) dsRNA; a3) nucleic acid molecule encoding a1); a4) expression cassette containing the nucleic acid molecule of a2); a5) recombinant vector containing the nucleic acid molecule of a2); a6) recombinant vector containing the expression cassette of a3); a7) recombinant cell containing the nucleic acid molecule of a2); a8) recombinant cell containing the recombinant vector of a4); a9) recombinant cell containing the recombinant vector of a5); a10) recombinant microorganism containing the nucleic acid molecule of a2); a11) recombinant microorganism containing the expression cassette of a3); a12) recombinant microorganism containing the vector of a4); a13) recombinant microorganism containing the vector of a5).

[0016] In some embodiments of the present invention, the cells do not include reproductive material.

[0017] In some embodiments of the present invention, the dsRNA comprises a double-stranded RNA consisting of the nucleotide sequence shown in SEQ ID NO:3 and the nucleotide sequence shown in its reverse complementary sequence.

[0018] In some embodiments of the present invention, the pests include lepidopteran insects.

[0019] In some embodiments of the present invention, the pest is one or more of the following: beet armyworm, tobacco hawk moth, cotton bollworm, diamondback moth, beet armyworm, and fall armyworm; preferably beet armyworm.

[0020] In some embodiments of the present invention, the LOC111350322 Inhibitors suppress Nrf2 protein expression, downregulate the pests' ability to cope with plant secondary metabolites, and inhibit the growth of pest larvae, thereby achieving the purpose of pest control.

[0021] In some embodiments of the present invention, the products include, but are not limited to, reagents, kits, drugs, and pesticides.

[0022] A second aspect of the present invention provides a dsRNA comprising a double-stranded RNA consisting of a nucleotide sequence shown in SEQ ID NO:3 and a nucleotide sequence shown in its reverse complementary sequence.

[0023] A third aspect of the invention provides biological materials related to dsRNA in the second aspect of the invention, said biological materials comprising any one of 1)-12): 1) A nucleic acid molecule encoding dsRNA of the second aspect of the present invention; 2) An expression cassette comprising the nucleic acid molecule of 1); 3) A vector comprising the nucleic acid molecule of 1); 4) A vector comprising the expression cassette of 2); 5) A recombinant cell comprising the nucleic acid molecule of 1); 6) A recombinant cell comprising the expression cassette of 2); 7) A recombinant cell comprising the vector of 3); 8) A recombinant cell comprising the vector of 4); 9) A recombinant microorganism comprising the nucleic acid molecule of 1); 10) A recombinant microorganism comprising the expression cassette of 2); 11) A recombinant microorganism comprising the vector of 3); 12) A recombinant microorganism comprising the vector of 4).

[0024] In some embodiments of the present invention, the cells do not include reproductive material.

[0025] In some embodiments of the present invention, the vector is a plasmid vector, a phage particle, a viral vector, a cell vector, a bacteriophage, a phage, an F phage, or an artificial chromosome. The plasmid vector may be an optional plasmid, the viral vector may be an optional virus, and the cell vector does not include propagation material.

[0026] A fourth aspect of the present invention provides a product comprising dsRNA of the second aspect of the present invention and / or biological material of the third aspect of the present invention.

[0027] In some embodiments of the present invention, the product is used to control pests or inhibit Nrf2 protein expression.

[0028] In some embodiments of the present invention, the products include, but are not limited to, reagents, kits, drugs, and pesticides.

[0029] In some embodiments of the present invention, the product further includes a pharmaceutically acceptable carrier, including but not limited to: diluents, buffers, suspensions, emulsions, granules, encapsulation agents, excipients, fillers, binders, sprays, transdermal absorbents, humectants, disintegrants, absorption enhancers, surfactants, colorants, flavoring agents, or adsorbents.

[0030] In some embodiments of the present invention, the pests include lepidopteran insects.

[0031] In some embodiments of the present invention, the pest is one or more of the following: beet armyworm, tobacco hawk moth, cotton bollworm, diamondback moth, beet armyworm, and fall armyworm; preferably beet armyworm.

[0032] A fifth aspect of the invention provides the use of the dsRNA of the second aspect of the invention, the biomaterial of the third aspect of the invention, and / or the product of the fourth aspect of the invention in any one of (b1)-(b4). (b1) Inhibit Nrf2 protein expression; (b2) Prepare a product that inhibits Nrf2 protein expression; (b3) Pest control; (b4) Prepare products for the prevention and control of pests.

[0033] In some embodiments of the present invention, the pests described in (b3)-(b4) include lepidopteran insects.

[0034] In some embodiments of the present invention, the pest is one or more of the following: beet armyworm, tobacco hawk moth, cotton bollworm, diamondback moth, beet armyworm, and fall armyworm; preferably beet armyworm.

[0035] In some embodiments of the present invention, the products include, but are not limited to, reagents, kits, drugs, and pesticides.

[0036] A sixth aspect of the present invention provides a method for controlling pests, comprising: reducing the amount of pesticides in the pests. LOC111350322 The steps of expression level and / or activity.

[0037] In some embodiments of the present invention, the LOC111350322 The nucleotide sequence is shown in SEQ ID NO:1.

[0038] In some embodiments of the present invention, the reduction of pests LOC111350322 The step of increasing the expression level and / or activity is to introduce the dsRNA of the second aspect of the present invention, the biological material of the third aspect of the present invention, or the product of the fourth aspect of the present invention into the insect body.

[0039] In some embodiments of the present invention, the method of introduction includes oral feeding or injection.

[0040] In some embodiments of the present invention, oral feeding includes pre-application, spraying, or misting to make food, pest habitats (soil, area, material, or environment where pests are growing or can grow, or materials, cultivated plants, plant propagation materials (such as seeds), soil, surface, or space) or water contain dsRNA of the second aspect of the present invention, biological materials of the third aspect of the present invention, and / or products of the fourth aspect of the present invention.

[0041] In some embodiments of the present invention, the pests include lepidopteran insects.

[0042] In some preferred embodiments of the present invention, the pest is one or more of the following: beet armyworm, tobacco hawk moth, cotton bollworm, diamondback moth, beet armyworm, and fall armyworm.

[0043] In some preferred embodiments of the present invention, the pest is the beet armyworm.

[0044] The beneficial effects of this invention are: The inventors have for the first time discovered an RNA-binding protein that promotes the expression of Nrf2 protein in the agricultural pest, the beet armyworm. Its gene number is [gene number missing]. LOC111350322 Through RNAi technology, LOC111350322 dsRNA was designed to interfere with the beet armyworm as a target. LOC111350322 Expression. This was confirmed through cell and in vitro experiments and other techniques. LOC111350322 It can bind to key transcription factors for anti-oxidative stress Nrf2 The 3' untranslated region (3'UTR) promotes Nrf2 protein expression. In vivo experiments further confirmed that this is achieved through interference with dsRNA. LOC111350322 The expression can downregulate the beet armyworm's ability to cope with plant secondary metabolites, increase the toxicity of plant secondary metabolites to the beet armyworm, and thus inhibit the growth of the beet armyworm, thereby achieving the purpose of pest control.

[0045] This invention provides new ideas and approaches for pest control, and has broad application prospects in the field of pest control. Attached Figure Description

[0046] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 To interfere in cells LOC111350322 Results of Nrf2 protein level detection after expression. (See figure.) P <0.01.

[0047] Figure 2 for LOC111350322 pass Nrf2 The results of the assay of luciferase activity regulated by the 3'UTR are shown in the figure. P <0.05.

[0048] Figure 3 To validate in vitro LOC111350322 Combination Nrf2 Experimental results at specific sites of the 3'UTR.

[0049] Figure 4 To feed at the same time LOC111350322 dsRNA and plant secondary metabolite I3C after the beet armyworm LOC111350322 mRNA level analysis. In the figure, P <0.01.

[0050] Figure 5 To feed at the same time LOC111350322 The statistical results of body weight in *Spodoptera litura* after dsRNA and plant secondary metabolite I3C were presented. In the figure, ns represents significant differences. P <0.01, P <0.001.

[0051] Figure 6 To feed at the same time LOC111350322 The mortality rate of Spodoptera litura larvae was determined by the addition of dsRNA and plant secondary metabolite I3C. (See figure.) P <0.001.

[0052] Figure 7 To feed at the same time LOC111350322 Characterization results of dsRNA and plant secondary metabolite I3C in Spodoptera litura. Detailed Implementation

[0053] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0054] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0055] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0056] Example 1 The inventors discovered a novel RNA-binding protein that promotes the expression of Nrf2 protein in the agricultural pest, the beet armyworm. Its gene number is [gene number missing]. LOC111350322 , LOC111350322 The nucleotide sequence of the gene is shown in SEQ ID NO:1, and the amino acid sequence is shown in SEQ ID NO:2.

[0057] 5'-ATGGCTGCTAACCACAGAAATGGTCAGGTTAATGGAGAAATCGCACAAAATGTAGACTGCCAGGAACCTGGCCCCAGTGGCGTGCACAGGGTCGGGAGACAGGCTGCAGCAGAAGGCGTCGCTCCACCCACTAGAAAACCACGTAAAACGCAAAAGTCGGTAGCAAACAAGATAAAAGAACTAAAGAAACAATTAGTACAACAGGAAAAACTAAAGATAGAAATAGAAGC TAAAATAAAAGAACTCAAGCAGGAATTGAAAGAGGCTGAGAAAGAATTGGAAGGAATGAAGAAAACTGCAAAAAAGAACAGAGAGCGTAGGAAGAAAGTTACAAAACGGAAGCAGAGTACACCACGGCAGACAAATAGAGTGTCACCACGGAGAAACGAAACTAATCCTAGCAGGACTGGGCAAGTCAAGTTTATTATCCGAGGTGAAGTACTTGAAGCAAATTAG-3' (SEQ DI NO:1), where the TAG at the end is the stop codon.

[0058] MAANHRNGQVNGEIAQNVDCQEPGPSGVHRVGRQAAAEGVAPPTRKPRKTQKSVANKIKELKKQLVQQEKLKIEIEAKIKELKQELKEAEKELEGMKKTAKKNRERRKKVTKRKQSTPRQTNRVSPRRNETNPSRTGQVKFIIRGEVLEAN (SEQ DI NO: 2).

[0059] Example 2 Interference LOC111350322 Effect of expression on Nrf2 protein synthesis This example was used to investigate the effect of interfering with LOC111350322 expression on Nrf2 protein synthesis. The specific experimental procedure is as follows: (1) In vitro synthesis LOC111350322 dsRNA Total RNA was extracted from Spodoptera litura cells Spli-221 using the Trizol method. 1 μg of total RNA was reverse transcribed into cDNA using Hifair® Ⅲ 1stStrand cDNA Synthesis SuperMix (Yisheng Biotechnology (Shanghai) Co., Ltd., catalog number 11141ES). Using the Spli-221 cell cDNA as a template, PCR was performed to amplify the DNA template for dsRNA synthesis. The dsRNA was synthesized according to the manufacturer's instructions for the T7 RiboMAX™ Express RNAi System (Promega, catalog number P1700).

[0060] Spodoptera litura LOC111350322 dsRNA (denoted as dsLOC111350322 The chain of justice is as follows: 5'-TGGCTGCTAACCACAGAAATGGTCAGGTTAATGGAGAAATCGCACAAAATGTAGACTGCCAGGAACCTGGCCCCAGTGGCGTGCACAGGGTCGGGAGACAGGCTGCAGCAGAAGGCGTCGCTCCACCCACTAGAAA ACCACGTAAAACGCAAAAGTCGGTAGCAAACAAGATAAAAGAACTAAAGAAACAATTAGTACAACAGGAAAAACTAAAGATAGAAATAGAAGCTAAAATAAAAGAACTCAAGCAGGAATTGAAAGAGGCTGA-3' (SEQ ID NO:3).

[0061] The sense strand of dsRNA in Dsgfp (control group) is: 5'-TACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCA ACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCAC-3' (SEQ ID NO:4).

[0062] Targeting the beet armyworm LOC111350322 The primer sequences used for the dsRNA are as follows: F: 5'-TAATACGACTCACTATAGGTGGCTGCTAACCACAGAAATG-3' (SEQ ID NO: 5); R: 5'-TAATACGACTCACTATAGGTCAGCCTCTTTCAATTCCTGCT-3' (SEQ ID NO: 6).

[0063] The primer sequences used above for the dsRNA of Dsgfp are as follows: Upstream primer F: 5'-TAATACGACTCACTATAGGTACGGCGTGCAGTGCTTC-3' (SEQ ID NO:7); Downstream primer R: 5'-TAATACGACTCACTATAGGGTGCTCAGGTAGTGGTTG-3' (SEQ ID NO:8).

[0064] (2) Transfer Spli-221 cells with good growth into 48-well plates and incubate overnight at 28°C.

[0065] (3) Use Fugene HD transfection reagent (Promega, catalog number E2311) to transfect the cells. LOC111350322 Transfect cells with dsRNA (according to the reagent instructions).

[0066] (4) Detection of protein content in Nrf2 Cellular proteins were extracted 48 h after transfection using RIPA Lysis Buffer (Beyotime Biotechnology). Protein samples were separated by 8% SDS-polyacrylamide gel electrophoresis and transferred to a membrane at 80 V on ice for 100 min. PVDF membranes were blocked with 5% BSA, and the primary antibody (i.e., Nrf2 antibody, as described in the article "Cen Y, Zou X, Zhong Q, et al. The TIAR-mediated Nrf2 response to oxidative stress is mediated through the Nrf2 The noncoding 3' untranslated region in Spodoptera litura. Free radicalbiology & medicine. 2022;184:17-29. doi:10.1016 / j.freeradbiomed.2022.03.016” was disclosed. The sample was incubated overnight at 4°C; washed 3 times with TBST; incubated with secondary antibody (i.e., horseradish peroxidase-labeled goat anti-mouse secondary antibody, purchased from Guangzhou Dingguo Biotechnology Co., Ltd., catalog number: IH-0031) at 37°C for 1 h; washed 3 times with TBST; and then chemiluminescence imaging was performed using Super ECL detection reagent (Yisheng Biotechnology (Shanghai) Co., Ltd.).

[0067] The results are as follows Figure 1 As shown, interference LOC111350322 Expression can significantly inhibit Nrf2 protein synthesis.

[0068] Example 3 Interference LOC111350322 Expressing Nrf2 Effects of 3'UTR activity This embodiment is used to examine the effect of interfering with the expression of LOC111350322 on... Nrf2 The effect of 3'UTR activity was investigated through the following experimental procedures: (1) Constructing the insertion Nrf23'UTR dual-luciferase vector: Using cDNA from Spodoptera litura Spli-221 cells as a template, Spodoptera litura was amplified using specific upstream primers (F: 5'-CTCGAGTTCCGCCTAATCAGTTCCCG-3', SEQ ID NO: 9) and downstream primers (R: 5'-GCGGCCGCGATGACCATACAATATAATCTAATGTTCCA-3', SEQ ID NO: 10). Nrf2 3'UTR. psiCHECK-2 was double-digested using Xho I and Not I restriction endonucleases (Takara). The 3'UTR fragment of *Spodoptera litura* Nrf2 was ligated into the psiCHECK-2 vector using T4 DNA ligase. The ligation product was transformed into DH5α competent cells. Positive clones were verified by colony PCR, and the plasmid was extracted from the positive clone bacteria; this plasmid serves as the reporter vector. Nrf2 3'UTR-psiCHECK-2.

[0069] (2) Transfer Spli-221 cells with good growth into 48-well plates and incubate overnight at 28°C.

[0070] (3) The reporter vector was transfected using the Fugene HD transfection reagent (Promega). Nrf2 3'UTR-psiCHECK-2 and LOC111350322 co-transfect cells with dsRNA; (4) Luciferase activity detection: 48 h after transfection, the luciferase activity in the cells was detected using the Dual-Luciferase® Reporter Assay System (Promega, catalog number E1910) according to its instructions.

[0071] The results are as follows Figure 2 As shown, the results indicate interference. LOC111350322 Expression can significantly inhibit the activity of Nrf2 3'UTR.

[0072] Example 4: In vitro detection of whether LOC111350322 binds Nrf2 3'UTR This embodiment is used for examination. LOC111350322 Can they be directly combined? Nrf2 3'UTR, the specific experimental procedure is as follows: The RNA probe was synthesized by Shanghai Jima Pharmaceutical Technology Co., Ltd., and its sequence is 5'-UUAUACACCUUCUUUUCAUUGCUACUUU-3' (SEQ ID NO:11). The 5' end of the binding probe is biotin-labeled, while the competing probe (Cold probe) is unlabeled. The purified LOC111350322-gst protein (amino acid sequence shown in SEQ ID NO:2) was obtained using a prokaryotic protein expression system, as follows: Using cDNA from Spodoptera litura Spli-221 cells as a template, the protein coding region sequence of Spodoptera litura LOC111350322 was amplified using specific upstream primers (F: 5'-GGTACCGCTGCTAACCACAGAAATGG-3', SEQ ID NO:12) and downstream primers (R: 5'-GCGGCCGCCTAATTTGCTTCAAGTACTTC-3', SEQ ID NO:13). pGEX-6p-1 was double-digested with BamHI and NotI restriction endonucleases (Takara). The LOC111350322 protein-coding region was ligated into the pGEX-6p-1 vector using T4 DNA ligase. The ligation product was transformed into BL21 competent cells, and positive clones were verified by colony PCR. Positive clones were inoculated at a 1:100 volume ratio into 100 mL of ampicillin-resistant LB medium and incubated at 37°C for 1.5 h. IPTG was added to a final concentration of 1 mM / L, and the culture was continued at 37°C for 4 h. Bacteria were collected by centrifugation, and the protein was purified using a GST-tagged protein purification kit (Shanghai Beyotime, catalog number: P2262) to obtain the purified LOC111350322-gst protein. RNA electrophoretic mobility shift assay (REMSA) was performed using the LightShift Chemiluminescent RNA EMSA kit (Thermo Scientific, catalog number 20158), strictly following the manufacturer's instructions.

[0073] The results are as follows Figure 3 As shown, the results indicate that the purified protein LOC111350322-gst can directly bind to... Nrf2 Specific sites of the 3'UTR.

[0074] Example 5 Interference LOC111350322 Expressing the impact on the beet armyworm This embodiment verifies interference. LOC111350322 The effect of [the substance] in the control of the beet armyworm was demonstrated through the following experimental procedure: (1) In vitro synthesis of prokaryotic systems LOC111350322 dsRNA Will LOC111350322The 268 bp PCR product was cloned into the L4440 vector. The recombinant L4440 vector was transformed into E. coli. HT115 (DE3) competent cells were used to verify positive clones using colony PCR. Positive clones were inoculated into 1 L of medium at a volume ratio of 1:100 and incubated at 37°C for 1.5 h. IPTG was added to a final concentration of 1 mM / L, and the cells were incubated at 37°C for 5 h. The bacteria were harvested by centrifugation at 4000 rpm for 5 min and resuspended in 5 mL of phosphate-buffered saline (PBS). The bacterial solution (OD) was... 600 ≈120) was added to 50 g of artificial feed containing 0.25 w / w% indole-3-carbinol (I3C), mixed, and then used to feed 1st instar larvae of the beet armyworm that had been pre-starved for 2 hours.

[0075] dsGFP was synthesized using the same method, and the bacterial solution expressing dsGFP (OD) was... 600 ≈120) was added to 50 g of artificial feed containing 0.25 w / w% I3C, and then used to feed 1st instar larvae of the beet armyworm that had been pre-starved for 2 hours as a control group.

[0076] The formula for artificial feed is 150 g wheat bran, 40 g yeast powder, 4 g vitamin C, 4 g sorbic acid, 4 g sucrose, 13 g agar powder, 4 g methylparaben, and 200 μL linoleic acid.

[0077] (2) Larvae LOC111350322 mRNA level detection Larvae were collected 48 h later, and total RNA was extracted from them using the Trizol method. 1 μg of total RNA was reverse transcribed into cDNA using the Hifair® Ⅲ 1st Strand cDNA Synthesis SuperMix (Yisheng Biotechnology (Shanghai) Co., Ltd., catalog number 11141ES). Larval RNA was then analyzed using the Hieff™ qPCR SYBR Green Master Mix Kit (Yisheng Biotechnology (Shanghai) Co., Ltd., catalog number 11201ES03). LOC111350322 The mRNA levels involved were measured. The detection primers included: F: 5'-TGTCACCACGGAGAAACGAA-3' (SEQ ID NO:14), R: 5'-CTTGACTTGCCCAGTCCTGC-3' (SEQ ID NO:15).

[0078] The results are as follows Figure 4 As shown, the results indicate that larvae fed with ds LOC111350322 E. coli 48 hours later LOC111350322The expression level of [the substance] was significantly downregulated, indicating that feeding it dsRNA could successfully interfere with [the process]. LOC111350322 Gene expression.

[0079] (3) Phenotypic detection of larvae after interfering with LOC111350322 expression Use containing LOC111350322 Spodoptera litura larvae were fed with artificial feed containing dsRNA and 0.25 w / w% I3C. Larval weight was recorded 48 h after feeding, and then every 24 h until 144 h. The number of larval deaths under different treatments was recorded, and the larval mortality rate at 144 h was calculated. Larval phenotypes were recorded using a stereomicroscope after 144 h of treatment.

[0080] The results are as follows Figures 5-7 As shown, interference within the body of the beet armyworm larvae LOC111350322 After expression, the larvae's ability to cope with plant secondary compounds is weakened, larval growth is inhibited, and larval mortality increases.

[0081] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. LOC111350322 The application of inhibitors in the control of pests or in the preparation of pest control products, the LOC111350322 The nucleotide sequence is shown in SEQ ID NO:

1.

2. The application according to claim 1, characterized in that, The LOC111350322 Inhibitors are inhibitors LOC111350322 Active substances, degradation LOC111350322 substances or reduction LOC111350322 At least one of the substances expressing the level.

3. The application according to claim 2, characterized in that, The reduction LOC111350322 The substance expressing the level is at least one of a1)-a13): a1) Target LOC111350322 At least one of the following: siRNA, dsRNA, miRNA, ribozyme, shRNA, or CRISPR / Cas system; a2) encodes the nucleic acid molecule of a1); a3) An expression cassette containing the nucleic acid molecule described in a2); a4) A recombinant vector containing the nucleic acid molecule described in a2); a5) A recombinant vector containing the expression cassette described in a3); a6) Recombinant cells containing the nucleic acid molecules described in a2); a7) Recombinant cells containing the expression cassette described in a3); a8) Recombinant cells containing the recombinant vector described in a4); a9) Recombinant cells containing the recombinant vector described in a5); a10) Recombinant microorganisms containing the nucleic acid molecules described in a2); a11) Recombinant microorganisms containing the expression cassette described in a3); a12) Recombinant microorganisms containing the vector described in a4); a13) Recombinant microorganisms containing the vector described in a5).

4. The application according to claim 3, characterized in that, The LOC111350322 The inhibitor is at least one of a1)-a13): a1) Target LOC111350322 dsRNA; a2) encodes the nucleic acid molecule of a1); a3) An expression cassette containing the nucleic acid molecule described in a2); a4) A recombinant vector containing the nucleic acid molecules described in a2); a5) A recombinant vector containing the expression cassette described in a3); a6) Recombinant cells containing the nucleic acid molecules described in a2); a7) Recombinant cells containing the expression cassette described in a3); a8) Recombinant cells containing the recombinant vector described in a4); a9) Recombinant cells containing the recombinant vector described in a5); a10) Recombinant microorganisms containing the nucleic acid molecules described in a2); a11) Recombinant microorganisms containing the expression cassette described in a3); a12) Recombinant microorganisms containing the vector described in a4); a13) Recombinant microorganisms containing the vector described in a5); Preferably, the dsRNA comprises a double-stranded RNA consisting of the nucleotide sequence shown in SEQ ID NO:3 and the nucleotide sequence shown in its reverse complementary sequence; Preferably, the pests include lepidopteran insects.

5. The application according to any one of claims 1-4, characterized in that, The LOC111350322 Inhibitors suppress Nrf2 protein expression, downregulate the pests' ability to cope with plant secondary metabolites, and inhibit the growth of pest larvae, thereby achieving the purpose of pest control.

6. A dsRNA comprising a double-stranded RNA consisting of a nucleotide sequence shown in SEQ ID NO:3 and a nucleotide sequence shown in its reverse complementary sequence.

7. A biomaterial related to the dsRNA of claim 6, wherein the biomaterial comprises any one of 1)-12): 1) A nucleic acid molecule encoding the dsRNA of claim 6; 2) An expression cassette containing the nucleic acid molecule described in 1); 3) A carrier containing the nucleic acid molecule described in 1); 4) A carrier containing the expression box described in 2); 5) Recombinant cells containing the nucleic acid molecules described in 1); 6) Recombinant cells containing the expression cassette described in 2); 7) Recombinant cells containing the vector described in 3); 8) Recombinant cells containing the vector described in 4); 9) Recombinant microorganisms containing the nucleic acid molecules described in 1); 10) Recombinant microorganisms containing the expression cassette described in 2); 11) Recombinant microorganisms containing the vector described in 3); 12) Recombinant microorganisms containing the vector described in 4).

8. A product characterized in that, The product comprises the dsRNA of claim 6 and / or the biomaterial of claim 7; Preferably, the product is used to control pests or inhibit Nrf2 protein expression.

9. The use of the dsRNA of claim 6, the biomaterial of claim 7, and / or the product of claim 8 in any one of (b1)-(b4); (b1) Inhibit Nrf2 protein expression; (b2) Prepare a product that inhibits Nrf2 protein expression; (b3) Pest control; (b4) Prepare products for the prevention and control of pests; Preferably, the pests mentioned in (b3)-(b4) include lepidopteran insects.

10. A method for controlling pests, comprising: reducing the amount of pesticides in the pests. LOC111350322 The steps of expression level and / or activity; Preferably, the LOC111350322 The nucleotide sequence is shown in SEQ ID NO:1; Preferably, the reduction of pests LOC111350322 The step of determining the expression level and / or activity is to introduce the dsRNA of claim 6, the biomaterial of claim 7, or the product of claim 8 into the insect body; Preferably, the pests include lepidopteran insects.

Images