Application of the LOC111359120 gene of the beet armyworm in the control of lepidopteran pests

Abstract

This invention belongs to the field of biotechnology for pest control, specifically disclosing the application of the LOC111359120 gene of the beet armyworm as a target in the control of lepidopteran pests. The application achieves pest control by inhibiting the expression of the LOC111359120 gene, the sequence of which is shown in SEQ ID NO.1. This application utilizes CRISPR / Cas9 gene editing technology to knock out the LOC111359120 gene at the genome level, resulting in a significant reduction in melanin deposition in the insect's exoskeleton, a lighter body color, and a significantly weakened resistance to *Nomura leishensis*. This alters the beet armyworm larvae's natural mimicry and immune defense capabilities, thereby achieving effective pest control. This method is green and harmless, avoids the development of pesticide resistance, and can bring both environmental and economic benefits to agricultural production.

Description

Technical Field

[0001] This invention belongs to the technical field of biotechnology for pest and disease control, and specifically discloses the application of the LOC111359120 gene of the beet armyworm in the control of lepidopteran pests. Background Technology

[0002] The beet armyworm (Spodoptera litura), also known as the lotus armyworm, is a polyphagous and voracious agricultural pest widely distributed in tropical and subtropical regions. Its larvae primarily feed on over 300 species of crops from more than 100 families, including cruciferous and leguminous plants, posing a significant threat to agricultural production and food security. Currently, pest control mainly relies on chemical pesticides. However, the long-term and excessive use of chemical pesticides has not only led to increased pesticide resistance in pests but also seriously impacted the environment and the ecological security of non-target organisms. Therefore, developing novel, safe, efficient, and environmentally friendly alternatives is one of the key issues that needs to be addressed to promote the long-term sustainable development of agricultural ecosystems.

[0003] The larvae of the beet armyworm can cleverly blend into the natural background by mimicking the color of their exoskeleton, thus avoiding predators. Melanin is one of the most important pigments in insect body color. Besides altering body color, it also acts as a polymer to enhance the physical resistance of the exoskeleton. Furthermore, it can bind to proteins such as proteases and chitinases, further preventing pathogens like fungi, bacteria, and even parasites from invading the exoskeleton. Simultaneously, melanin deposition in the exoskeleton reduces the damage from solar radiation, mitigating the risk of DNA damage and cell death, and also regulates body temperature. These self-protection and survival defense strategies of the beet armyworm are the result of natural selection, making it more adaptable to its environment. However, they also increase the difficulty of monitoring and control, making it a challenging issue in agricultural pest management.

[0004] The LOC111359120 gene has seven isoforms, belonging to the zinc finger and BTB domain protein (ZBTB) transcription factor family. They all share a common BTB domain (Broad-Complex, Tramtrack, Bric-à-brac domain) at the N-terminus and two C2H2 zinc finger motifs at the C-terminus. The LOC111359120 protein shows high homology in Lepidoptera, Diptera, Coleoptera, and Hymenoptera insects, indicating relatively high evolutionary conservation. Furthermore, as an important ZBTB transcription factor, it plays a crucial regulatory role in Drosophila embryogenesis, neuronal differentiation, and gonadal development. However, the relationship between the LOC111359120 gene and exoskeleton melanin and immune defense capabilities in Lepidoptera insects, particularly in the beet armyworm, has not been reported. Summary of the Invention

[0005] To address the shortcomings of existing pest and disease control technologies, this application proposes a LOC111359120 gene that regulates melanin deposition in 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 lepidopteran insect.

[0006] Taking the beet armyworm as an example, this application obtained a gene that significantly affects the deposition of melanin in the exoskeleton of the beet armyworm larvae, thereby reducing their natural mimicry ability and immunity—LOC111359120. Furthermore, it developed a technology to knock out the LOC111359120 gene at the genome level using CRISPR / Cas9 gene editing technology to control the beet armyworm.

[0007] To achieve the above objectives, this application provides the following technical solution.

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

[0009] Preferably, the pest is a noctuid moth; more preferably, it is a beet armyworm.

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

[0011] (1) Design the sgRNA target of the LOC111359120 gene of Spodoptera litura, 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 injected into the embryos of Spodoptera litura. Mutants were then selected after hatching.

[0013] In a specific embodiment of this application, the sgRNA of the LOC111359120 gene was synthesized in vitro, mixed with Cas9 protein, and injected into the embryos of *Spodoptera litura*. Changes in melanin deposition in 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*. Furthermore, the effect of knocking out the LOC111359120 gene on the immune defense ability of *Spodoptera litura* was evaluated by immersing wild-type and LOC111359120 mutant larvae, respectively, in the same concentration of *Nomuraea rileyi* spore suspension and Tween, and recording mortality. The results showed that knocking out the LOC111359120 gene in *Spodoptera litura* significantly reduced exoskeleton melanin deposition, resulting in a lighter body color, and significantly weakened resistance to *Nomuraea rileyi*. Intervention of the LOC111359120 gene alters the natural mimicry and immune defense capabilities of the beet armyworm larvae, enabling effective pest control.

[0014] The second aspect of this application provides the application of a LOC111359120 gene expression inhibitor in the preparation of products for controlling lepidopteran pests, wherein the LOC111359120 gene has the sequence shown in SEQ ID NO.1, and the inhibitor includes substances that reduce the activity of LOC111359120, or substances that degrade LOC111359120, or substances that reduce the expression level of LOC111359120.

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

[0016] More preferably, 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.

[0017] Preferably, the lepidopteran pest is a noctuid moth; more preferably, it is a beet armyworm.

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

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

[0020] (2) Used for developing environmentally friendly pesticide formulations;

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

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

[0023] A third aspect of this application is to provide a nucleic acid molecule that inhibits the expression of the LOC111359120 gene, said nucleic acid molecule being sgRNA, and the nucleotide sequence of the nucleic acid molecule encoding said sgRNA is shown in SEQ ID NO.3.

[0024] The fourth aspect of this application is to provide an expression cassette, vector, or transgenic cell line containing the nucleic acid molecule that inhibits the expression of the LOC111359120 gene as described above.

[0025] The fifth aspect of this application is to provide a drug for controlling lepidopteran pests, wherein the active ingredient of the drug contains one or more of the above-mentioned nucleic acid molecules, expression cassettes, vectors, and transgenic cell lines that inhibit the expression of the LOC111359120 gene, as well as a pharmaceutically acceptable carrier or excipient.

[0026] A sixth aspect of this application provides a method for controlling lepidopteran pests, comprising introducing a CRISPR / Cas9 system targeting the LOC111359120 gene into the pest's body, wherein the CRISPR / Cas9 system contains sgRNA targeting the LOC111359120 gene and a Cas9 protein, the target site sequence of the sgRNA being shown in SEQ ID NO.2, and the nucleotide sequence of the nucleic acid molecule encoding the sgRNA being shown in SEQ ID NO.3. Further, the introduction method is injection.

[0027] In one embodiment of this application, the LOC111359120 gene can be efficiently knocked out by mixing synthetic sgRNA with Cas9 protein and introducing it into the embryo of the beet armyworm. This results in reduced melanin deposition in the larval exoskeleton, lighter body color, and decreased disease resistance, thereby effectively controlling the pest. This method has many advantages, including precision, repeatability, ease of implementation, and being environmentally friendly, and has promising application prospects.

[0028] The seventh aspect of this application relates to the application of a kit in screening drugs for controlling lepidopteran pests. The kit contains reagents for detecting the expression level of the LOC111359120 gene, the sequence of which is shown in SEQ ID NO.1. The drug targets the LOC111359120 gene to inhibit its expression. When low expression of the LOC111359120 gene is detected, the pest's exoskeleton shows reduced melanin deposition, its body surface color becomes lighter, and its disease resistance decreases. Therefore, the drug to be screened can be used to control lepidopteran pests.

[0029] Preferably, the lepidopteran pest described above is a noctuid moth; more preferably, it is a beet armyworm.

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

[0031] (1) This invention discloses for the first time the application of transcription factor LOC111359120 as a target in the control of Spodoptera litura, and develops its highly efficient knockout sgRNA target site, and develops a highly efficient technology for the control of Spodoptera litura. Specifically, sgRNA and Cas9 are injected into Spodoptera litura embryos using CRISPR / Cas9 gene editing technology to knock out the LOC111359120 gene in Spodoptera litura. LOC111359120 is used to change the exoskeleton color 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 their resistance to pathogens. This gene-level intervention changes the natural mimicry ability and immune defense ability of Spodoptera litura larvae. This not only significantly improves their visibility in the field environment, providing natural enemies with more obvious predation targets, but also reduces the disease resistance of pests, thereby further affecting their survival and achieving the purpose of control.

[0032] (2) This invention also proposes a strategy of using an insect transcription factor LOC111359120 inhibitor as a novel environmentally friendly pest control agent. This method is easy to operate, has precise targeting, high effectiveness and sensitivity, and can achieve long-term effective control of pest populations. It can also significantly reduce the negative environmental impact of chemical pesticide use, making it green and pollution-free. At the same time, it avoids the risk of pests developing resistance due to long-term exposure to chemical pesticides, providing an innovative solution for sustainable agriculture and bringing long-term environmental and economic benefits to agricultural production. Attached Figure Description

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

[0034] Figure 2 A diagram showing the mutant types screened after knocking out the LOC111359120 gene in CRISPR / Cas9; where uppercase letters represent exons, lowercase letters represent introns, red letters represent base substitutions, and "-" represents base deletions.

[0035] Figure 3 This is a phenotype of reduced melanin deposition and lighter color in the exoskeleton of the beet armyworm LOC111359120 gene knockout.

[0036] Figure 4Survival curves of wild-type and LOC111359120 mutant larvae infected with Nomura leishi. Detailed Implementation

[0037] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. 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 of ordinary skill 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 the embodiments of this invention are conventional methods; unless otherwise specified, the materials and reagents used are commercially available.

[0039] Example 1: Acquisition of the LOC111359120 gene fragment from the beet armyworm and synthesis of its sgRNA target site.

[0040] 1. Obtaining the LOC111359120 gene fragment from the beet armyworm.

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

[0042] 2. Synthesis of sgRNA target site in the LOC111359120 gene of Spodoptera litura.

[0043] (1) Target site selection

[0044] Based on the obtained LOC111359120 gene nucleotide sequence SEQ ID NO.1, and 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 sequence of sgRNA is shown in SEQ ID NO.3.

[0045] (2) Primer design

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

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

[0052] Table 2

[0053]

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

[0055] 2) Construction of positive plasmids:

[0056] After PCR amplification products were purified by agarose gel electrophoresis, the purified products were ligated into pMD19-T vector for sequencing. The ligation reaction system is shown in Table 3.

[0057] Table 3

[0058]

[0059] After incubating the ligation product overnight in a ligator at 16°C, it was added to competent cells, gently mixed by pipetting, and incubated on ice for 30 min. Then, it was heat-shocked in a 42°C water bath for 90 s, followed by cooling on ice for 10 min. 600 μL of antibiotic-free LB broth was added, and the cells were incubated at 37°C and 180 rpm for 1 h on a shaker, followed by centrifugation at 5000 rpm for 5 min at room temperature. The bacterial culture was concentrated to 100 μL, gently resuspended with a pipette tip, and then evenly spread onto ampicillin-resistant LB agar plates. The plates were incubated inverted mode overnight at 37°C. Positive clones were screened, and single-clone colony PCR and sequencing verification were performed; colonies without nucleotide mutations in the sequencing results were considered the desired positive colonies.

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

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

[0062] Table 4

[0063]

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

[0065] The above reaction system was repeated 6 times. After the reaction, 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 by NanoDrop ND-2000 and verified by gel electrophoresis.

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

[0067] Based on the MEGAScript T7 kit instructions, further improvements were made to synthesize and purify sgRNA in vitro.

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

[0069] Table 5

[0070]

[0071] After mixing and centrifuging the above reaction solution, transcription was performed at 37°C for 8 hours.

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

[0073] 3) Add 390 μL of Nuclease-free water to bring 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 12000 rpm for 10 min at 4 °C.

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

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

[0076] 6) Add 1 mL of 75% ethanol to a centrifuge tube to wash the precipitate, centrifuge at 12000 rpm for 5 min at 4°C, discard the supernatant, and wash twice.

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

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

[0079] 9) Measure the RNA concentration and verify it by gel electrophoresis. Store in a -80℃ freezer for later use.

[0080] Example 2: Genotyping of the Spodoptera litura LOC111359120 mutant (using CRISPR / Cas9 knockout system)

[0081] 1. Microinjection of Spodoptera litura fertilized eggs

[0082] All supplies required for the experiment should be sterilized or disinfected before injection. Mix sgRNA and Cas9 protein to a final concentration of 300 ng / μL, then centrifuge at 12,000 rpm for 5 min at 4 °C.

[0083] After mating, fresh egg masses laid within 2 hours were collected. The egg masses, sterilized with formaldehyde, were brushed to remove surface hairs. While still moist, the eggs were transferred to a glass slide and arranged in straight rows with the face up using an oviposition needle. Approximately 9.6 nL of the mixture was injected into each egg using a microinjector. After injection, the eggs were placed 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 the mutant pupa, add 600 μL of digestion solution (EDTA, SDS, NaCl, proteinase K and Tris-HCl) and two grinding beads, grind thoroughly and digest overnight in a water bath at 55°C.

[0087] 2) After digestion, add 600 μL of DNA extraction buffer to the mixture, gently invert to mix, and centrifuge at 12,000 rpm for 10 min.

[0088] 3) Take off 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 inverting the container, centrifuge at 12000 rpm for 10 min, and collect the supernatant.

[0090] 5) Add 2 volumes of pre-cooled anhydrous ethanol to the supernatant, mix by inverting, precipitate at -20℃ for 20 min, centrifuge at 12000 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 let it air dry for 10-15 minutes to allow the residual ethanol to evaporate completely until the precipitate becomes transparent. Add an appropriate amount of ddH2O to dissolve the DNA.

[0093] (2) Mutant screening:

[0094] Following PCR amplification using mutant genomic DNA as a template, base sequencing was performed. The sequencing results were analyzed using SnapGene software for base sequence alignment to identify different sequence base mutations. After testing, the LOC111359120 mutant genotype of *Spodoptera litura* was identified. 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℃, 3 min; 98℃, 10 s; 55℃, 15 s; 72℃, 10 s; 30 cycles; 72℃, 10 min.

[0098] Example 3 Phenotypic of the Spodoptera litura LOC111359120 mutant

[0099] 1. Significant changes in melanin deposition on the exoskeleton

[0100] from Figure 3 It can be seen that after the LOC111359120 gene was successfully knocked out using CRISPR / Cas9 gene editing technology, compared with the wild type, the LOC111359120 mutant larvae showed significantly less melanin deposition in their exoskeletons and a significantly lighter body color. Figure 3 This result indicates that knocking out the LOC111359120 gene can effectively inhibit the deposition of melanin in the exoskeleton of the beet armyworm larvae, thus resulting in a lighter color.

[0101] 2. Significantly reduced resistance to pathogenic fungi.

[0102] Wild-type and LOC111359120 mutant larvae at the first day of the 5th instar (L5D1) were immersed separately in 5×10⁻⁶ water. 7 After 30 seconds in a suspension of *Nomura rileyi* spores at a concentration of 1 spore / mL and in 0.05% Tween (control), the number of deaths was counted every 12 hours. The results showed that both wild-type and LOC111359120 mutant larvae soaked in 0.05% Tween could pupate normally. LOC111359120 mutant larvae infected with *Nomura rileyi* began to die at 120 hours and were completely dead by 180 hours, while wild-type larvae began to die at 144 hours and were not completely dead until 192 hours. The onset and complete mortality time of LOC111359120 mutant larvae were significantly earlier. Figure 4 This result indicates that knocking out the LOC111359120 gene can effectively reduce the resistance of Spodoptera litura larvae to pathogenic fungi.

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

Claims

1. Knockout LOC111359120 The application of genes in the control of lepidopteran pests is characterized by... The LOC111359120 The gene sequence is shown in SEQ ID NO.1, and the lepidopteran pest is the beet armyworm.

2. The application according to claim 1, characterized in that, Using CRISPR / Cas9 gene editing technology to perform genome-wide editing LOC111359120 Gene knockout, the CRISPR / Cas9 system contains targeted... LOC111359120 The gene contains sgRNA and 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.

3. The application according to claim 1, characterized in that, The knockout LOC111359120 Genes have one or both of the following functions: a. Reduce melanin deposition in the exoskeleton of pests; b. Reduces the immune defense capabilities of pests.

4. A method for controlling lepidopteran pests, characterized in that, Target LOC111359120 The gene was introduced into the pest using the CRISPR / Cas9 system and knocked out. LOC111359120 The gene, the CRISPR / Cas9 system contains a targeted gene. LOC111359120 The sgRNA and Cas9 protein of the gene, LOC111359120 The gene sequence is shown in SEQ ID NO.1, the target site sequence of the sgRNA is shown in SEQ ID NO.2, the nucleotide sequence of the nucleic acid molecule encoding the sgRNA is shown in SEQ ID NO.3, and the lepidopteran pest is the beet armyworm.

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