Application of prodenia litura LOC111359120 gene in prevention and control of lepidoptera pests

Abstract

The invention belongs to the field of disease and insect pest prevention and control through biotechnology, and particularly discloses application of a prodenia litura LOC111359120 gene as a target spot in lepidoptera pest prevention and control, according to the application, lepidoptera pest prevention and control are achieved by inhibiting expression of the LOC111359120 gene, and the sequence of the LOC111359120 gene is shown as SEQ ID NO.1. The invention further discloses a preparation method of the prodenia litura LOC111359120 gene. According to the application, the LOC111359120 gene is knocked out at the genome level by utilizing a CRISPR / Cas9 gene editing technology, so that the deposition of exoskeleton melanin of pests can be obviously reduced, the body surface color becomes light, the resistance to nomuraea lerileyi is obviously weakened, and the natural mimicry ability and immune defense ability of prodenia litura larvae are changed, thereby effectively preventing and controlling the pests. The method is green and harmless, drug resistance can be avoided, and environmental and economic benefits can be brought to agricultural production.

Description

Technical Field

[0001] The invention belongs to the technical field of biotechnology for preventing and controlling pests and diseases, and specifically discloses the application of the Spodoptera litura LOC111359120 gene in the prevention and control of lepidopteran pests. Background Art

[0002] Spodoptera litura, also known as the lotus armyworm, is a polyphagous and voracious agricultural pest widely distributed in tropical and subtropical regions. Its larvae mainly feed on more than 300 crops from more than 100 families, including Cruciferae and Leguminosae, posing a major hidden danger to agricultural production and food security. Currently, the prevention and control of this insect mainly relies on chemical pesticides, but the long-term and large-scale use of chemical pesticides not only leads to an increase in pest resistance, but also seriously affects the ecological security of the environment and non-target organisms. Therefore, the development of new, safe, efficient and environmentally friendly alternative products is one of the key issues to be solved, which can promote the long-term sustainable development of agricultural ecosystems.

[0003] The larvae of the Spodoptera litura can simulate the surrounding environment through the color of their exoskeleton, cleverly blending into the natural background to avoid natural enemies. Melanin is one of the most important pigments in insect body color. In addition to changing the insect's body color, it can also act as a polymer to improve the physical resistance of the insect's exoskeleton. It can also combine with proteins such as proteases and chitinases to further prevent pathogens such as fungi, bacteria and even parasites from invading the exoskeleton. At the same time, the deposition of melanin in the insect's exoskeleton can reduce the damage of solar radiation to insects, reduce the risk of DNA damage and cell death, and regulate body temperature. These self-protection and survival defense strategies of the Spodoptera litura are the result of optimization under the action of natural selection, making it more adaptable to the environment. However, it also increases the difficulty of monitoring and prevention, making it a difficult problem in agricultural pest management.

[0004] There are 7 isoforms of the LOC111359120 gene, which belongs to the zinc finger (Zinc finger and BTB domain protein, ZBTB) transcription factor family. They all have a common BTB domain (Broad-Complex, Tramtrack, Bric-à-brac domain) at the N-terminus and two C2H2 zinc finger motifs (Zinc finger) at the C-terminus. The LOC111359120 protein has a high homology in insects such as Lepidoptera, Diptera, Coleoptera and Hymenoptera, and is relatively conservative in the evolutionary process. Moreover, as an important ZBTB transcription factor, it plays a key regulatory role in Drosophila embryogenesis, neuronal differentiation and gonadal development. However, the relationship between the LOC111359120 gene and Lepidoptera, especially the exoskeleton melanin and immune defense ability of Spodoptera litura, has not been reported. Summary of the invention

[0005] In view of the shortcomings of existing pest control technologies, the present application proposes a LOC111359120 gene that regulates the deposition of melanin on the exoskeleton of pests and its application in pest control. The nucleotide sequence of the LOC111359120 gene is shown in SEQ ID NO.1, and the pest is a Lepidoptera insect.

[0006] Taking the Spodoptera litura as an example, this application obtained a gene, LOC111359120, which significantly affects the deposition of melanin on the exoskeleton of the larvae of the Spodoptera litura, thereby reducing the natural mimicry and immunity capabilities, and developed a technology for knocking out the LOC111359120 gene at the genome level using CRISPR / Cas9 gene editing technology to prevent and control the Spodoptera litura.

[0007] In order to achieve the above objectives, this application provides the following technical solutions.

[0008] The first aspect of the present application is to provide an application of the LOC111359120 gene as a target in pest control. The pest control is achieved by inhibiting the expression of the LOC111359120 gene, the sequence of the LOC111359120 gene is shown in SEQ ID NO.1, and the pest is a Lepidoptera insect.

[0009] Preferably, the pest is Spodoptera litura; more preferably, Spodoptera litura.

[0010] In one embodiment of the present application, the inhibition of LOC111359120 gene expression is achieved by knocking out the LOC111359120 gene, comprising the following steps:

[0011] (1) Designing an sgRNA target for the Spodoptera litura LOC111359120 gene, the nucleotide sequence of which is shown in SEQ ID NO.2, and the nucleotide sequence of the nucleic acid molecule encoding the sgRNA is shown in SEQ ID NO.3;

[0012] (2) The sgRNA of the LOC111359120 gene was synthesized in vitro, mixed with Cas9 protein, and then injected into Spodoptera litura embryos. The mutants were hatched and screened.

[0013] In a specific embodiment of the present application, the sgRNA of the LOC111359120 gene was synthesized in vitro, mixed with the Cas9 protein and injected into the embryo of Spodoptera litura, and the changes in the melanin deposition of the exoskeleton of Spodoptera litura were observed and recorded to evaluate the effect of knocking out the LOC111359120 gene on the exoskeleton color of Spodoptera litura; the wild-type and LOC111359120 mutant larvae were immersed in the same concentration of Nomuraea rileyi spore suspension and Tween, and the death was counted to further evaluate the effect of knocking out the LOC111359120 gene on the immune defense ability of Spodoptera litura. The results showed that the mutation of the LOC111359120 gene of Spodoptera litura caused a significant reduction in the deposition of exoskeleton melanin, a lighter body color, and a significantly weaker resistance to Nomuraea rileyi (N. rileyi). Intervention of the LOC111359120 gene changed the natural mimicry and immune defense capabilities of the Spodoptera litura larvae, allowing for effective pest control.

[0014] The second aspect of the present application is to provide the use of a LOC111359120 gene expression inhibitor in the preparation of a product for controlling lepidopteran pests, wherein the LOC111359120 gene has a sequence as shown in SEQ ID NO.1, and the inhibitor includes a substance that reduces the activity of LOC111359120, or a substance that degrades LOC111359120, or a substance that reduces the expression level of LOC111359120.

[0015] Preferably, the inhibitor includes an sgRNA targeting the LOC111359120 gene, an expression cassette, a vector, a transgenic cell line, or a CRISPR / Cas9 system containing the sgRNA targeting the LOC111359120 gene.

[0016] More preferably, the target site sequence of the sgRNA is shown as SEQ ID NO.2, and the nucleotide sequence of the nucleic acid molecule encoding the sgRNA is shown as SEQ ID NO.3.

[0017] Preferably, the lepidopteran pest is Spodoptera litura; more preferably, it is Spodoptera litura.

[0018] Preferably, the product has one or more of the following applications:

[0019] (1) Used in biological pest control strategies;

[0020] (2) Used for the development of environmentally friendly pesticide formulations;

[0021] (3) Reduce melanin deposition on the exoskeleton of pests and improve the predation efficiency of natural enemies;

[0022] (4) Reduce the immune defense ability of pests and prepare products that are susceptible to infection by pathogens during the larval stage.

[0023] The third aspect of the present application is to provide a nucleic acid molecule for inhibiting the expression of the LOC111359120 gene, wherein the nucleic acid molecule is sgRNA, and the nucleotide sequence of the nucleic acid molecule encoding the sgRNA is shown in SEQ ID NO.3.

[0024] The fourth aspect of the present application is to provide an expression cassette, a vector, or a transgenic cell line containing the above-mentioned nucleic acid molecule for inhibiting the expression of the LOC111359120 gene.

[0025] The fifth aspect of the present application is to provide a drug for controlling lepidopteran pests, the active substance of the drug containing one or more of the above-mentioned nucleic acid molecules, expression cassettes, vectors, transgenic cell lines that inhibit the expression of the LOC111359120 gene, and pharmaceutically acceptable carriers or excipients.

[0026] The sixth aspect of the present application is to provide a method for controlling lepidopteran pests, wherein a CRISPR / Cas9 system targeting the LOC111359120 gene is introduced into the pest, wherein the CRISPR / Cas9 system contains an sgRNA targeting the LOC111359120 gene and a Cas9 protein, wherein the target site sequence of the sgRNA is shown in SEQ ID NO. 2, and the nucleotide sequence of the nucleic acid molecule encoding the sgRNA is shown in SEQ ID NO. 3. Furthermore, the introduction method is injection.

[0027] In one embodiment of the present application, the LOC111359120 gene can be efficiently knocked out by mixing the synthetic sgRNA and Cas9 protein into the embryos of Spodoptera litura, resulting in reduced melanin deposition in the exoskeleton of the larvae, lighter body color, and reduced disease resistance, thereby effectively controlling pests. This method has many advantages such as accuracy, repeatability, ease of implementation, and green and pollution-free, and has a good application prospect.

[0028] The seventh aspect of the present application is to provide a kit for use in screening drugs for controlling lepidopteran pests, the kit containing a reagent for detecting the expression level of the LOC111359120 gene, the LOC111359120 gene sequence being shown in SEQ ID NO. 1. The drug targets the LOC111359120 gene and inhibits its expression, and when low expression of the LOC111359120 gene is detected, the melanin deposition of the exoskeleton of the pest is reduced, the body surface color becomes lighter, and the disease resistance is reduced, then the drug to be screened can be used for controlling lepidopteran pests.

[0029] Preferably, the lepidopteran pest as described above is a Spodoptera moth; more preferably, it is a Spodoptera litura.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] (1) The present invention discloses for the first time the application of transcription factor LOC111359120 as a target in the prevention and control of Spodoptera litura, and develops a highly efficient knockout sgRNA target site, and develops a highly efficient control technology for Spodoptera litura, that is, by injecting sgRNA and Cas9 into Spodoptera litura embryos through CRISPR / Cas9 gene editing technology, knocking out the LOC111359120 gene of Spodoptera litura, and using LOC111359120 to change the color of the exoskeleton of Spodoptera litura, confirming that the loss of its protein function leads to a reduction in melanin deposition in the exoskeleton of Spodoptera litura larvae, making their body surface lighter and reducing resistance to pathogens. This genetic intervention changes the natural mimicry ability and immune defense ability of Spodoptera litura larvae. This not only significantly improves its visibility in the field environment, providing a more obvious predation target for natural enemies, but also reduces the disease resistance of pests, thereby further affecting their survival, so as to achieve the purpose of prevention and control.

[0032] (2) The present invention also proposes a strategy of using insect transcription factor LOC111359120 inhibitors as new environmentally friendly pest control agents. This method is easy to operate, has accurate target effectiveness and good sensitivity, can achieve long-term effective control of pest populations, and can significantly reduce the negative impact of chemical pesticide use on the environment. It is green and pollution-free; at the same time, it avoids the risk of pests developing resistance due to long-term exposure to chemical pesticides, provides an innovative solution for sustainable agriculture, and can bring long-term environmental and economic benefits to agricultural production. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Selection of target sites for knockout of the LOC111359120 gene by CRISPR / Cas9; capital letters represent exons, lowercase letters represent introns, and red letters represent PAM sequences.

[0034] Figure 2 This is a diagram of the mutant types screened after CRISPR / Cas9 knocked out the LOC111359120 gene; among them, capital letters represent exons, lowercase letters represent introns, red letters represent base substitutions, and "-" represents base deletions.

[0035] Figure 3 The phenotype of Spodoptera litura LOC111359120 gene knockout is reduced exoskeleton melanin deposition and lighter color.

[0036] Figure 4These are the survival curves of wild-type and LOC111359120 mutant larvae after infection with No. DETAILED DESCRIPTION

[0037] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0038] Unless otherwise specified, the experimental methods used in the examples of the present invention are all conventional methods; the materials, reagents, etc. used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0039] Example 1 Acquisition of the Spodoptera litura LOC111359120 gene fragment and synthesis of its sgRNA target site

[0040] 1. Obtaining the gene fragment of Spodoptera litura LOC111359120

[0041] Based on the genome and transcriptome database of Spodoptera litura, after sequence alignment analysis by BLAST, the nucleotide sequence of the Spodoptera litura LOC111359120 gene was obtained as shown in SEQ ID NO.1.

[0042] 2. Synthesis of sgRNA target sites for the Spodoptera litura LOC111359120 gene

[0043] (1) Target site selection

[0044] Based on the obtained LOC111359120 gene nucleotide sequence SEQ ID NO.1, according to the principle of 5'-GG-(N)18-NGG-3' target site design, a highly specific site was selected at the junction of the 5'-UTR and the first exon of LOC111359120. The target site sequence of sgRNA in this application is shown in SEQ ID NO.2, and the nucleic acid subsequence of sgRNA is shown in SEQ ID NO.3.

[0045] (2) Primer design

[0046] According to the obtained LOC111359120 gene sgRNA nucleic acid molecule sequence SEQ ID NO.3, the sgRNA primer P1 of the LOC111359120 gene was designed (Table 1). According to the sequence SEQ ID NO.1 of the LOC111359120 gene, the detection primer P2 for mutant screening was designed (Table 1). According to the pMD19-T vector sequence and the LOC111359120 gene sgRNA nucleic acid molecule sequence SEQ ID NO.3, the colony PCR detection primer P3 was designed (Table 1)

[0047] Table 1. Primers used in this example

[0048]

[0049] (3) Synthesis of sgRNA transcription template

[0050] 1) Extension of the short chain of sgRNA template for LOC111359120 gene:

[0051] The reaction system is shown in Table 2:

[0052] Table 2

[0053]

[0054] PCR reaction program: 98°C, 3 min; 98°C, 10 s, 55°C, 15 s, 72°C, 10 s, 30 cycles; 72°C, 10 min.

[0055] 2) Positive plasmid construction:

[0056] After the PCR amplification product was recovered and purified by agarose gel, the recovered product was connected to the pMD19-T vector for sequencing. The connection reaction system is shown in Table 3:

[0057] Table 3

[0058]

[0059] After the ligation product is placed in a 16°C ligation apparatus overnight, it is added to the competent cells, gently blown to mix, and placed on ice for 30 minutes. Then heat shock in a 42°C water bath for 90 seconds and cool in an ice bath for 10 minutes. Add 600μL of antibiotic-free LB liquid culture medium, culture in a shaker at 37°C, 180rpm for 1 hour, and centrifuge at 5000rpm for 5 minutes at room temperature. Concentrate the bacterial solution to 100μL, gently resuspend the bacterial solution with a pipette tip, and then evenly spread it on an ampicillin-resistant LB solid culture plate. Incubate overnight at 37°C. Screen positive clones, perform monoclonal colony PCR detection and sequencing verification, that is, the colonies with no nucleotide mutations in the sequencing results are the desired positive colonies.

[0060] 3) Amplification of sgRNA transcription template of LOC111359120 gene:

[0061] The positive clones screened above were expanded and cultured, and then plasmids were extracted and used as DNA templates to synthesize sgRNA templates transcribed in vitro by PCR. The reaction system is shown in Table 4:

[0062] Table 4

[0063]

[0064] PCR reaction program: 98°C, 3 min; 98°C, 10 s, 55°C, 15 s, 72°C, 10 s, 30 cycles; 72°C, 10 min.

[0065] The above reaction system was repeated for 6 tubes. After the reaction was completed, the PCR products were combined, and 200 μL of Nuclease-free water was added to make up to 500 μL. Then, an equal volume of Tris-saturated phenol: chloroform: isoamyl alcohol (25:24:1, pH = 8.0) was added for purification. After purification, the DNA concentration was determined using NanoDrop ND-2000 and verified by running on gel.

[0066] (4) In vitro transcription and purification of sgRNA

[0067] The method was further improved based on the instructions of the MEGAScript T7 kit to synthesize sgRNA in vitro and purify it.

[0068] 1) Add the reaction system shown in Table 5:

[0069] Table 5

[0070]

[0071] The above reaction solution was mixed and centrifuged, and then transcribed at 37°C for 8 h;

[0072] 2) After transcription is completed, add 1 μL of TURBO DNase, mix well and incubate at 37°C for 15 min;

[0073] 3) Add 390 μL of Nuclease-free water to make up the transcription product to 400 μL, then add 400 μL of Tris-saturated phenol: chloroform: isoamyl alcohol (25:24:1, pH>8.0), mix thoroughly, and centrifuge at 4°C, 12000 rpm for 10 min;

[0074] 4) Pipette the supernatant into a new centrifuge tube, add 1 mL of anhydrous ethanol and 50 μL of NaOAc (pH=5.2), mix well, and precipitate in a -80°C refrigerator for 30 min;

[0075] 5) After precipitation, centrifuge at 12000 rpm for 15 min at 4°C and discard the supernatant;

[0076] 6) Add 1 mL of 75% ethanol to the centrifuge tube to wash the precipitate, centrifuge at 4°C, 12,000 rpm for 5 min, discard the supernatant, and wash twice;

[0077] 7) Open the cap of the centrifuge tube and dry it on ice for 10 minutes to evaporate the residual ethanol;

[0078] 8) Add an appropriate amount of Nuclease-free water to dissolve RNA;

[0079] 9) Determine RNA concentration and run gel verification, and store in a -80℃ refrigerator for later use.

[0080] Example 2 Construction of mutant genotype of Spodoptera litura LOC111359120 (knockout using CRISPR / Cas9 system)

[0081] 1. Microinjection of Fertilized Eggs of Spodoptera litura

[0082] Disinfect or sterilize all supplies needed for the experiment before injection. Mix sgRNA and Cas9 protein to a final concentration of 300 ng / μL, then centrifuge at 4°C, 12000 rpm for 5 min.

[0083] After mating, collect fresh egg masses laid within 2 hours of Spodoptera litura. Use a brush to clean the surface hairs of the egg masses disinfected with formaldehyde. Transfer the eggs to a glass slide in a wet state and arrange the eggs with the front side facing up in a straight line with an ovulation needle. Use a microinjection instrument to inject about 9.6nL of the mixture into each egg. After the injection, place the eggs in an incubator at 25±1℃ for incubation.

[0084] 2. Detection and screening of mutants

[0085] (1) Extraction of mutant genomic DNA:

[0086] 1) Collect the epidermis of mutant pupae, add 600 μL digestion solution (EDTA, SDS, NaCl, proteinase K and Tris-HCl) and two grinding beads, grind thoroughly and digest in a 55°C water bath overnight;

[0087] 2) After digestion, add 600 μL of DNA extraction solution to the mixture, gently invert up and down to mix, and centrifuge at 12000 rpm for 10 minutes;

[0088] 3) Aspirate the upper aqueous phase and repeat step (2) until there is no obvious white layer at the boundary between the phenol phase and the aqueous phase;

[0089] 4) Add an equal volume of chloroform:isoamyl alcohol (24:1) to the supernatant, mix by inversion, centrifuge at 12000 rpm for 10 min, and aspirate the supernatant;

[0090] 5) Add 2 volumes of pre-cooled anhydrous ethanol to the supernatant, mix by inversion, precipitate at -20°C for 20 min, centrifuge at 12,000 rpm for 15 min, and discard the supernatant;

[0091] 6) Add 1 mL of pre-cooled 70% ethanol to wash the precipitate, centrifuge at 12000 rpm for 5 min, discard the supernatant, and repeat twice;

[0092] 7) Open the cap of the centrifuge tube and dry it in air for 10-15 minutes to allow the residual ethanol to evaporate until the precipitate becomes transparent, and add an appropriate amount of ddH2O to dissolve the DNA.

[0093] (2) Mutant screening:

[0094] The mutant genomic DNA was used as a template for PCR amplification and then base sequencing was performed. The sequencing results were compared and analyzed using SnapGene software, and different sequence base mutations were confirmed. After testing, the mutant genotype of Spodoptera litura LOC111359120 ( Figure 2 ). The PCR amplification reaction system is shown in Table 6:

[0095] Table 6

[0096]

[0097] The PCR reaction program was as follows: 98°C, 3 min; 98°C, 10 s, 55°C, 15 s, 72°C, 10 s, 30 cycles; 72°C, 10 min.

[0098] Example 3 Phenotype of Spodoptera litura LOC111359120 mutant

[0099] 1. Significant changes in exoskeleton melanin deposition

[0100] from Figure 3 It can be seen that after the CRISPR / Cas9 gene editing technology successfully knocked out the LOC111359120 gene, compared with the wild type, the melanin deposition in the exoskeleton of the LOC111359120 mutant larvae was significantly reduced, and the body color was significantly lighter ( Figure 3 ). The results indicate that knockout of the LOC111359120 gene can effectively inhibit the deposition of melanin in the exoskeleton of Spodoptera litura larvae, resulting in lighter color.

[0101] 2. Resistance to pathogenic fungi is significantly reduced

[0102] The 5th instar day 1 (L5D1) larvae of wild type and LOC111359120 mutant were immersed in 5×10 7 After 30 seconds in a suspension of N. rileyi spores at a concentration of spores / mL and 0.05% Tween (control), the number of deaths was counted every 12 hours. The results showed that the wild-type and LOC111359120 mutant larvae soaked in 0.05% Tween could pupate normally. The LOC111359120 mutant larvae infected by N. rileyi began to die at 120 hours and were all dead by 180 hours, while the wild-type larvae began to die at 144 hours and were not dead until 192 hours. The time when the LOC111359120 mutant larvae began to die and the time when all deaths occurred were significantly advanced ( Figure 4 ). The results showed that knockout of the LOC111359120 gene could effectively reduce the resistance of Spodoptera litura larvae to pathogenic fungi.

[0103] Obviously, the above embodiments of the present invention are only examples for clearly illustrating the technical solution of the present invention, and are not intended to limit the specific implementation methods of the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the claims of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

1. The use of LOC111359120 gene as a target in the control of lepidopteran pests, characterized in that: The application achieves the control of lepidopteran pests by inhibiting the expression of the LOC111359120 gene, and the sequence of the LOC111359120 gene is shown in SEQ ID NO.

1.

2. Use of a LOC111359120 gene expression inhibitor in the preparation of a lepidopteran pest control product, characterized in that: The inhibitor includes a substance that reduces the activity of LOC111359120, or a substance that degrades LOC111359120, or a substance that reduces the expression level of LOC111359120. The sequence of the LOC111359120 gene is shown in SEQ ID NO.

1.

3. The use according to claim 2, characterized in that: The inhibitor includes an sgRNA targeting the LOC111359120 gene, an expression cassette containing the sgRNA targeting the LOC111359120 gene, a recombinant vector, a transgenic cell line, or a CRISPR / Cas9 system.

4. The use according to claim 3, characterized in that: The target site sequence of the sgRNA is shown in SEQ ID NO.2, and the nucleotide sequence of the nucleic acid molecule encoding the sgRNA is shown in SEQ ID NO.

3.

5. The use according to claim 2, characterized in that: The lepidopteran pest is a noctuid armyworm.

6. The use according to claim 2, characterized in that: The lepidopteran pest is Spodoptera litura.

7. The use according to claim 2, characterized in that: The product has one or more of the following effects: a. Reduce the melanin deposition on the exoskeleton of pests; b. Reduce the immune defense ability of pests.

8. Use of a drug in the control of lepidopteran pests, characterized in that: The effective substance of the drug contains sgRNA targeting the LOC111359120 gene, one or more of an expression cassette, a vector, and a transgenic cell line containing the sgRNA targeting the LOC111359120 gene, and a pharmaceutically acceptable carrier or excipient. The target site sequence of the sgRNA is shown in SEQ ID NO.2, and the nucleotide sequence of the nucleic acid molecule encoding the sgRNA is shown in SEQ ID NO.

3.

9. A method for controlling lepidopteran pests, characterized in that: A CRISPR / Cas9 system targeting the LOC111359120 gene is introduced into the pest, wherein the CRISPR / Cas9 system contains an sgRNA targeting the LOC111359120 gene and a Cas9 protein, the target site sequence of the sgRNA is shown in SEQ ID NO.2, and the nucleotide sequence of the nucleic acid molecule encoding the sgRNA is shown in SEQ ID NO.

3.

10. Use of a kit for screening drugs for controlling lepidopteran pests, characterized in that: The drug targets the LOC111359120 gene and inhibits its expression. The kit contains a reagent for detecting the expression level of the LOC111359120 gene. The LOC111359120 gene sequence is shown in SEQ ID NO.1.

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