Locust reeler gene and its encoded protein in the prevention and treatment of locusts

Abstract

The present application relates to the technical field of genetic engineering, and provides application of locust Reeler gene and coded protein thereof in prevention and treatment of locusts, wherein the polynucleotide sequence of the locust Reeler gene comprises a sequence shown in SEQ ID NO:1, a complementary sequence, a degenerate sequence, a truncated sequence, or a homologous sequence or a complementary sequence having 90% or above identity with SEQ ID NO:1 and hybridizing under stringent conditions. The present application can design and synthesize interference molecules for specifically silencing the target gene by providing a molecular target of the Reeler gene, so as to specifically inhibit expression of the Reeler gene in the group-living locusts, thereby providing a key molecular basis for weakening the immune defense ability of the locusts to Metarhizium anisopliae, and realizing synergistic effect of immune sensitization and biological killing.

Description

Technical Field

[0001] This invention relates to the technical field of genetic engineering, specifically to the application of the locust Reeler gene and its encoded protein in locust control. Background Technology

[0002] Locusts (Locusta migratoria) are a major pest in agricultural and livestock production. This pest exhibits density-dependent phenotypic plasticity: they are solitary at low densities and gregarious at high densities. Swarms of locusts possess extremely strong migratory capabilities and destructive power, causing devastating losses to gramineous crops and other vegetation wherever they go.

[0003] Chemical control is the most direct and rapid means of combating locust plagues, boasting advantages such as high efficiency, rapid action, significant effects, ease of use, and wide application. However, the extensive use of chemical pesticides also brings numerous drawbacks, primarily including: causing severe environmental pollution, disrupting the balance of the ecosystem, harming non-target organisms (such as natural enemies and pollinating insects), and pesticide residues easily leading to food safety issues. Furthermore, long-term use of a single chemical agent can easily lead to locust resistance, further reducing the control effect and forcing farmers to increase pesticide dosages or switch pesticides, creating a vicious cycle. Summary of the Invention

[0004] The purpose of this invention is to overcome the above-mentioned problems in the prior art and provide the application of the locust Reeler gene and its encoded protein in locust control, so as to solve the technical problems of unsatisfactory control effect on gregarious locusts, slow action of biological pesticides or easy development of resistance in the prior art, and achieve efficient, precise and environmentally friendly locust control effect.

[0005] Based on this, the first aspect of the present invention provides a locust Reeler gene, wherein the polynucleotide sequence of the locust Reeler gene includes at least one of the following sequences: (a1) The polynucleotide sequence shown in SEQ ID NO:1; (a2) A complementary, degenerate, truncated, or homologous sequence of the polynucleotide sequence shown in SEQ ID NO:1, wherein the homologous sequence is a polynucleotide that has 90% or more identity with the nucleotide shown in SEQ ID NO:1; (a3) A polynucleotide or its complementary sequence that hybridizes with the polynucleotide sequence shown in (a1) or (a2) under strict conditions.

[0006] Furthermore, the amino acid sequence of the protein encoded by the locust Reeler gene includes at least one of the following sequences: (b1) The amino acid sequence shown in SEQ ID NO.2; (b2) A homologous sequence that has at least 90% or more identity with the amino acid sequence shown in SEQ ID NO.2 and has the same function; (b3) An amino acid sequence in which one or more amino acids are added, deleted, or replaced, and have the same function, in the amino acid sequence shown in SEQ ID NO.2; (b4) The amino acid sequence with the same function obtained by attaching a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO.2.

[0007] A second aspect of the present invention provides a biomaterial, said biomaterial being any one of the following (c1) to (c7): (c1) Gene silencing cassettes that silence the above polynucleotides; (c2) Gene silencing vectors that silence the above polynucleotides; (c3) Recombinant microorganisms that silence the above polynucleotides; (c4) Knockout cassette for the above polynucleotides; (c5) Knockout vectors for the above polynucleotides; (c6) Recombinant microorganisms with the above polynucleotides knocked out; (c7) introduced the recombinant insect cell lines of (c1)-(c6), which included locusts.

[0008] A third aspect of the present invention provides a dsRNA targeting the locust Reeler gene described in the first aspect of the present invention, said dsRNA comprising the nucleotide sequence shown in SEQ ID NO:3.

[0009] Preferably, the amplification primers for the dsRNA include the nucleotide sequences shown in SEQ ID NO:4 and 5.

[0010] A fourth aspect of the present invention provides a composition for controlling gregarious locusts, comprising: dsRNA targeting the locust Reeler gene; and a microbial agent.

[0011] Furthermore, the amplification primers for the dsRNA include the nucleotide sequences shown in SEQ ID NO:4 and 5; And / or, the microbial agent includes Metarhizium anisopliae; preferably, the microbial agent is Metarhizium locustii.

[0012] A fifth aspect of the present invention provides the application of the locust Reeler gene described in the first aspect, the biological material described in the second aspect, the dsRNA described in the third aspect, or the composition described in the fourth aspect in the control of gregarious locusts.

[0013] A sixth aspect of the present invention provides a method for controlling gregarious locusts, comprising: To determine whether a locust swarm is a high-density, gregarious type; Delivering dsRNA targeting the Reeler gene to locusts; and Microbial agents were applied to the locusts.

[0014] Furthermore, the microbial agent is Metarhizium anisopliae.

[0015] Furthermore, the dsRNA is delivered to locusts via injection, feeding, spraying, or delivery via nanocarriers.

[0016] The present invention, by adopting the above technical solution, has the following beneficial effects: This invention significantly weakens the immune defenses of locusts against Metarhizium anisopliae infection by specifically inhibiting the expression of the Reeler gene in gregarious locusts. Combined application of Metarhizium anisopliae on this basis produces a significant synergistic effect: even low doses of Metarhizium anisopliae can achieve highly effective lethality against locust populations. This dual strategy of "immunosensitization + biological killing" not only greatly improves the field control efficacy of Metarhizium anisopliae but also means that in production practice, the ideal control effect can be achieved by significantly reducing the application dosage of Metarhizium anisopliae, thereby reducing the selective pressure of Metarhizium anisopliae from the source of application and effectively delaying or even avoiding the risk of locusts developing resistance to Metarhizium anisopliae. Attached Figure Description

[0017] Figure 1 This figure shows a comparison of the relative expression levels of the Reeler gene mRNA and protein in locust populations at different densities.

[0018] Figure 2 Immunofluorescence staining images of the locust brain show the localization of Reeler protein in the brain: A. Reeler antibody labeling; B. Antisynaptic protein labeling of brain morphology; C. Merge image; D. Monolayer image.

[0019] Figure 3 This figure represents an evaluation of the silencing effect of the Reeler gene in locusts after injection of dsRNA.

[0020] Figure 4 A comparative diagram showing the effect of dsRNA injection on the developmental duration of locust nymphs.

[0021] Figure 5 Survival curves of locusts' resistance to Metarhizium anisopliae after injection of dsRNA targeting the Reeler gene. Detailed Implementation

[0022] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.

[0023] Unless otherwise defined, all scientific and technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art.

[0024] Locusts (Locusta migratoria) are agricultural and livestock pests, exhibiting density-dependent phenotypic plasticity. High-density gregarious individuals demonstrate significantly greater migratory ability and destructive power than solitary individuals. Biological control using insect pathogens such as Metarhizium anisopliae can reduce the use of chemical pesticides; however, the insecticidal speed of these pathogenic fungi is relatively slow, and their field efficacy is easily affected by environmental conditions such as temperature and humidity. Under the selective pressure of long-term use of Metarhizium anisopliae, locust populations also face the risk of gradually developing disease resistance through enhanced immune responses.

[0025] This application, through transcriptome analysis of locusts at different rearing densities, discovered that the transcriptional level of a gene containing the Reeler domain was significantly higher in the brains of high-density gregarious locusts than in low-density solitary individuals. Western blotting also confirmed that the expression level of its encoded protein increased with increasing density. Immunofluorescence localization showed that the protein encoded by the Reeler gene is distributed in the glial cell layer of the perineurium, located on the outer surface of the central nervous system, and participates in the formation of the blood-brain barrier structure. It is speculated that it plays a role in the locust's immune defense response, a function that has not yet been studied. Therefore, this invention further suggests that inhibiting the expression of this Reeler gene may weaken the immune defense capabilities of gregarious locusts.

[0026] The 19 kDa plasma protein in locusts is the first insect protein identified to contain the Reeler domain. This invention names it the Reeler protein. This protein is abundant in the hemolymph of adult locusts, and its synthesis is positively regulated by juvenile hormone. Currently, there are no functional studies on the locust Reeler gene, nor have there been any reports of dsRNA that can effectively silence the locust Reeler gene.

[0027] Based on this, embodiments of the present invention provide a locust Reeler gene, wherein the polynucleotide sequence of the locust Reeler gene includes at least one of the following sequences: (a1) The polynucleotide sequence shown in SEQ ID NO:1; (a2) A complementary, degenerate, truncated, or homologous sequence of the polynucleotide sequence shown in SEQ ID NO:1, wherein the homologous sequence is a polynucleotide that has 90% or more identity with the nucleotide shown in SEQ ID NO:1; (a3) A polynucleotide or its complementary sequence that hybridizes with the polynucleotide sequence shown in (a1) or (a2) under strict conditions.

[0028] Locusts exhibit enhanced migratory ability and destructive power in high-density swarms. Existing technologies often treat solitary and gregarious locusts indiscriminately, failing to precisely target key genes highly expressed in gregarious locusts, thus failing to weaken the intrinsic driving force behind their population outbreaks at the molecular level. The Reeler gene in locusts is highly expressed in gregarious locusts and plays an important role in locust development and immune defense. This invention utilizes dsRNA (double-stranded RNA) targeting this gene to specifically inhibit its expression in locusts. This inhibition significantly shortens the developmental duration of the locust nymph stage, allowing it to enter the molting stage more quickly, thereby reducing the time window for feeding and damage and disrupting the synchronicity of population outbreaks. Furthermore, it significantly weakens the locust's immune defense against Metarhizium anisopliae infection. When this dsRNA double-stranded RNA is used in combination with Metarhizium anisopliae, a significant synergistic effect is observed. Metarhizium anisopliae can more effectively infect and kill locusts with weakened immune systems, thus achieving highly effective control at lower doses. This synergistic strategy not only improves the control efficiency against gregarious locusts but also helps delay or prevent locusts from developing resistance by reducing the amount of Metarhizium anisopliae used, achieving precise, efficient, and environmentally friendly pest control.

[0029] In some embodiments, the polynucleotide sequence of the Reeler gene in (a1) is the polynucleotide sequence shown in SEQ ID NO:1. This polynucleotide sequence is derived from a laboratory locust transcriptome database and shows significantly upregulated expression in high-density locusts compared to low-density scattered locusts. Screening of annotation information for this polynucleotide sequence confirmed the presence of the Reeler domain, hence the name Reeler gene. Transcriptome data showed that the Reeler gene exhibited a density-dependent expression pattern in locust brains of different densities. Specifically, transcriptome analysis of the brains of locusts belonging to low, medium, and high density populations revealed that the FPKM value of this gene was highest in the medium-density population and relatively lower in the low and high-density populations, exhibiting a specific expression pattern that varied with density. To further verify this, Western blotting was performed on the Reeler protein levels in brains of different densities, and normalization analysis was performed using β-Tubulin as an internal reference protein. The results also showed that the higher the density, the higher the expression level of this protein, consistent with the trend of mRNA level changes.

[0030] In some embodiments, the sequence of the polynucleotide in (a2) is the complementary sequence of the sequence shown in SEQ ID NO:1. The complementary sequence is a complementary sequence formed according to the base pairing principle, and the complementary sequence can be an incompletely complementary sequence or a completely complementary sequence that has the same function as the polynucleotide sequence shown in SEQ ID NO:1.

[0031] In some embodiments, the sequence of the multiple nucleotides in (a2) is a degenerate sequence of the sequence shown in SEQ ID NO:1. A degenerate sequence means that after changing one or more of the polynucleotide sequences of SEQ ID NO:1, the types of amino acids encoded by the positions of the changed polynucleotide sequences remain unchanged, and the function and expression level of the gene are not affected.

[0032] In some embodiments, the polynucleotide sequence in (a2) is a truncated sequence of the polynucleotide sequence shown in SEQ ID NO:1. A truncated sequence refers to a shorter polynucleotide sequence extracted or cut from the nucleotide in SEQ ID NO:1, and the cDNA sequence or the type of amino acid encoded by the position of the polynucleotide sequence remains unchanged, so as not to affect the function and expression level of the gene.

[0033] In some embodiments, the sequence of the polynucleotide in (a2) is a homologous sequence to the sequence shown in SEQ ID NO:1. Homologous sequences include, but are not limited to, polynucleotides having approximately 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more identity with the nucleotide shown in SEQ ID NO:1. Examples include mutant genes, alleles, or derivatives.

[0034] For example, the "strict conditions" can be any of low-strict, medium-strict, and high-strict conditions, preferably high-strict conditions. For example, "low-strict conditions" can be 30°C, 5×SSC, 5×Denhardt solution, 0.5% SDS, and 52% formamide; "medium-strict conditions" can be 40°C, 5×SSC, 5×Denhardt solution, 0.5% SDS, and 52% formamide; and "high-strict conditions" can be 50°C, 5×SSC, 5×Denhardt solution, 0.5% SDS, and 52% formamide. Those skilled in the art should understand that higher temperatures yield polynucleotides with higher homology. Furthermore, those skilled in the art can choose a combination of factors affecting hybridization strictness, such as temperature, probe concentration, probe length, ionic strength, time, and salt concentration, to achieve the appropriate level of strictness.

[0035] In some embodiments, the amino acid sequence of the protein encoded by the locust Reeler gene includes at least one of the following sequences: (b1) The amino acid sequence shown in SEQ ID NO.2; (b2) A homologous sequence that has at least 90% or more identity with the amino acid sequence shown in SEQ ID NO.2 and has the same function; (b3) An amino acid sequence in which one or more amino acids are added, deleted, or replaced, and have the same function, in the amino acid sequence shown in SEQ ID NO.2; (b4) The amino acid sequence with the same function obtained by attaching a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO.2.

[0036] In some embodiments, the amino acid sequence in (b1) is the amino acid sequence shown in SEQ ID NO:2: MKLVVAAVLAMAASAVAAPLSAHGHVPSSTCADMLPVHGNAVPSTKPSPYTITVSPTSVNGGDTVRVHISGTEEFRGVYLQARRGQEPVGEFLLPAGENNKIALSDCPPGHNNAFSYISRTPLDTLDIDWKAPNTSDEIVFRATFVKSFSEFWVGVESPKIIIGTATSTDNAVAA.

[0037] In some embodiments, the amino acid sequence in (b2) is an amino acid sequence that has at least 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more identity with and has the same function as the amino acid sequence shown in (b1).

[0038] In some embodiments, the amino acid sequence in (b3) is the amino acid sequence shown in (b1) with the addition, deletion, or substitution of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) of amino acid residues, and having the same biological enzyme activity. The positions of the addition, deletion, or substitution of one or more residues are not within conserved structural domains and do not affect the activity.

[0039] In some embodiments, the amino acid sequence in (b4) is the amino acid sequence obtained by attaching a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO: 2. The tag is not specifically limited and can be added according to the intended use; for example, it can be, but is not limited to, HIS, GST, Flag, MBP, HA, c-Myc, eGFP, eYFP, or eCFP.

[0040] This invention also provides a biomaterial, which is any one of (c1) to (c7) below: (c1) Gene silencing cassettes that silence the above polynucleotides; (c2) Gene silencing vectors that silence the above polynucleotides; (c3) Recombinant microorganisms that silence the above polynucleotides; (c4) Knockout cassette for the above polynucleotides; (c5) Knockout vectors for the above polynucleotides; (c6) Recombinant microorganisms with the above polynucleotides knocked out; (c7) introduced the recombinant insect cell lines of (c1)-(c6), which included locusts.

[0041] In some embodiments, (c1) the gene silencing expression cassette refers to a DNA fragment capable of transcribing to produce dsRNA targeting the aforementioned polynucleotide, which typically includes a promoter (such as U6, H1, CMV, CAG promoters or Pol III, Pol II promoters), a sense strand and / or antisense strand sequence having more than 90% complementarity to the target polynucleotide, and a terminator (such as a Pol III terminator or a polyA signal), and optionally includes a circular coding sequence to form a hairpin dsRNA.

[0042] In some embodiments, (c2) the gene silencing vector refers to a DNA vector carrying the aforementioned gene silencing expression cassette. Specifically, it can be a plasmid vector (such as pUC series, pBR322, pET series, pGEX series, pBI series, pSilencer series, pSuper series), a viral vector (such as alfalfa silver-striped moth nucleopolyhedrovirus AcMNPV vector, silkworm nucleopolyhedrovirus BmNPV vector, lentiviral vector, adenovirus vector, adeno-associated virus vector), a bacteriophage vector (such as λ phage vector, M13 phage vector), or an artificial chromosome vector (such as bacterial artificial chromosome BAC, yeast artificial chromosome YAC), and may further include selective marker genes (such as antibiotic resistance genes, fluorescent protein marker genes, or auxotrophic markers) and / or reporter genes (such as lacZ, luciferase, GUS).

[0043] In some embodiments, in (c3), the recombinant microorganism that silences the aforementioned polynucleotides refers to a microorganism that has been transformed or transfected with the aforementioned expression cassette or vector, enabling it to express dsRNA or other silencing molecules targeting the polynucleotide. The microorganism may be selected from bacteria (such as Escherichia coli DH5α, BL21(DE3), HT115, Agrobacterium LBA4404, GV3101, EHA105, Bacillus, Pseudomonas, etc.), fungi (such as yeast, Aspergillus, Metarhizium anisopliae, Beauveria bassiana, etc.), or microalgae (such as Chlamydomonas). The recombinant microorganism may be used to prepare dsRNA or directly applied to the environment as a biocontrol agent.

[0044] In some embodiments, (c4) the knockout cassette for knocking out the aforementioned polynucleotide refers to a DNA fragment used to disrupt or delete the target polynucleotide through homologous recombination or site-directed mutagenesis. Taking homologous recombination as an example, it includes a 5' homologous arm, a 3' homologous arm, and a selection marker gene (such as an antibiotic resistance gene, a fluorescent protein gene, or a auxotrophic complement gene) located between the two. Optionally, it may also include a negative selection marker (such as an HSV-tk or sacB gene), as well as a promoter and a terminator. For the CRISPR / Cas9 system, the knockout cassette may be an sgRNA expression cassette or donor DNA.

[0045] In some embodiments, (c5) the knockout vector refers to a DNA vector carrying the knockout cassette, which may specifically be a plasmid vector (such as pKO series, pBluescript series derived vectors, pX330 series, pSpCas9(BB), lentiCRISPR series, etc.), a viral vector (such as lentiviral CRISPR vector, adenovirus vector, adeno-associated virus vector), or a suicide vector (such as pCVD442, pWM91), and may further contain the Cas protein coding sequence, the tracer RNA coding sequence, the origin of replication, and the selection marker.

[0046] In some embodiments, (c6) the recombinant microorganism that knocks out the above-mentioned polynucleotide refers to a microorganism that has been transformed or transfected with the above-mentioned knockout cassette or knockout vector, in which the target polynucleotide is completely or partially knocked out or inactivated. The microorganism can be selected from bacteria (such as Escherichia coli, Agrobacterium, Bacillus), fungi (such as yeast, Metarhizium acridum, Beauveria bassiana, Trichoderma), or microalgae. In particular, for locust control applications, the recombinant microorganism is preferably a knockout strain of Metarhizium acridum or Beauveria bassiana, which can yield strains with enhanced virulence or better environmental adaptability.

[0047] In some embodiments, in (c7), the recombinant insect cell line introduced with (c1)-(c6) includes locusts, specifically referring to insect cells or insect individuals transfected with any of the aforementioned biological materials. These cells can be selected from insect cell lines (such as the fall armyworm Sf9 and Sf21 cell lines, the white-spotted armyworm High Five™ cell line, the silkworm BmN and Bm5 cell lines, the fruit fly S2 and Schneider 2 cell lines, and primary or immortalized locust cells) or transgenic / gene knockout insect individuals (such as recombinant locust strains obtained through embryo microinjection, sperm-mediated transfection, or transposon systems, and orthoptera such as the desert locust). This recombinant insect cell line can be used to screen for highly efficient dsRNA sequences, conduct safety evaluations, study gene function, or serve as a live dsRNA production reactor for dsRNA preparation.

[0048] This invention also provides a dsRNA targeting the locust Reeler gene described above. The dsRNA comprises the nucleotide sequence shown in SEQ ID NO:3. The amplification primers for the dsRNA comprise the nucleotide sequences shown in SEQ ID NO:4 and 5. Using this pair of primers, a Reeler gene fragment of approximately 332 bp can be specifically amplified from locust cDNA. The design of this Reeler gene fragment sequence ensures that it is specific to the locust genome and will not mistakenly silence other unrelated genes. Because the primers SEQ ID NO:4 and SEQ ID NO:5 contain the T7 promoter, the amplified PCR product can be used as a template for in vitro transcription, resulting in the large-scale synthesis of dsRNA with a hairpin structure to interfere with the normal expression of the Reeler gene.

[0049] This invention also provides a composition for controlling gregarious locusts, the composition comprising: dsRNA targeting the locust Reeler gene; and a microbial agent.

[0050] In some embodiments, the microbial agent includes Metarhizium anisopliae.

[0051] This invention reveals that the Reeler gene plays a crucial role in the immune defense of locusts. When the expression of this gene is specifically suppressed by dsRNA, the locusts' natural immunity to Metarhizium anisopliae is significantly weakened. In this case, combined application of Metarhizium anisopliae produces a synergistic effect: locusts are more susceptible to infection and death by Metarhizium anisopliae, thus achieving ideal control effects with lower doses. This helps reduce drug costs and delay the development of drug resistance.

[0052] In some embodiments, the amplification primers for the dsRNA in the composition include the nucleotide sequences shown in SEQ ID NO:4 and 5. The dsRNA synthesized using this primer pair has a sequence that perfectly matches the target region of the locust Reeler gene, maximizing the efficiency of RNAi interference and ensuring the silencing effect on the Reeler gene, thereby laying the molecular basis for synergistic effects with Metarhizium anisopliae.

[0053] This invention also provides the application of the above-mentioned locust Reeler gene, the above-mentioned biological material, the above-mentioned dsRNA, or the above-mentioned composition in the control of gregarious locusts. Its applications include, but are not limited to: preparing locust control agents for spraying, feeding, or injection; establishing a sustainable control system based on engineered microorganisms or transgenic plants in locust-prone areas; and serving as a molecular tool for studying locust development and immune mechanisms.

[0054] This invention also provides a method for controlling gregarious locusts, the method comprising: To determine whether a locust swarm is a high-density, gregarious type; Delivering dsRNA targeting the Reeler gene to locusts; and Microbial agents were applied to the locusts.

[0055] First, it is necessary to identify whether the locust swarm is a high-density gregarious type. This can be achieved by observing the locusts' body color and behavior (whether they gather and are active), or by using molecular detection methods (such as detecting the expression level of the Reeler gene). Then, dsRNA targeting the Reeler gene is delivered to the locusts to suppress the expression of their immune-related genes. Finally, a microbial agent (Metarhizium anisopliae) is applied to the locusts to effectively kill them by utilizing their weakened immune state.

[0056] Specifically, identifying whether a locust swarm is high-density and gregarious can be done using methods known in the field. These include visually observing the locusts' body color (gregarious locusts typically have darker body color and more pronounced markings) and behavior (gregarious locusts exhibit stronger aggregation and migratory behavior), or detecting gregarious-specific molecular markers, such as the Reeler gene itself or its protein products, using molecular biology techniques. For example, a small number of locust samples can be collected from fields or habitats, and total RNA or total protein can be extracted from their hemolymph or head tissue. The expression level of the Reeler gene can then be rapidly detected using RT-qPCR or colloidal gold immunochromatographic assay strips. If the expression level is significantly higher than the threshold of the solitary control group (e.g., 3, 5, or 10 times higher than the average expression level in solitary locusts), the swarm can be identified as high-density and gregarious. This precise control method is highly targeted and can significantly reduce the impact on non-target organisms and low-density solitary locust populations, thus contributing to maintaining ecological balance and delaying pesticide resistance. In gregarious locusts, the Reeler gene is specifically highly expressed in the brain under high density, which allows the delivered dsRNA to silence the gene more efficiently and specifically, thereby weakening their immune defense to the greatest extent and achieving the goal of synergistic prevention and control with Metarhizium anisopliae.

[0057] In some embodiments, the *Metarhizium anisopliae* applied in the above method is preferably *Metarhizium anisopliae*. *Metarhizium anisopliae* exhibits high host specificity and pathogenicity against locusts and is one of the most commonly used biofungi in the field for locust control. Combining dsRNA targeting the Reeler gene with *Metarhizium anisopliae* can maximize the synergistic effect between the two, achieving optimal biocontrol results against the target pest.

[0058] In some embodiments, the dsRNA is delivered to locusts via injection, feeding, spraying, or delivery via nanocarriers. For example, in laboratory studies, the dsRNA solution can be directly injected into the locust nymph's body cavity using microinjection. In field applications, the dsRNA can be mixed with attractants to create poisoned bait for feeding, or the dsRNA can be encapsulated in nanomaterials (such as liposomes or chitosan nanoparticles) to create a spray for large-area application. Nanocarriers (such as liposome-based nanocarriers or inorganic nanocarriers) can protect the dsRNA from environmental degradation and improve its absorption efficiency across the insect's body wall.

[0059] The technical solutions 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.

[0060] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0061] The present invention will now be described in detail with reference to specific embodiments, which are intended to understand rather than limit the invention.

[0062] Example 1: Obtaining and analyzing the expression of the locust Reeler gene Based on the laboratory locust transcriptome database, compared with low-density scattered locusts, genes with significantly upregulated expression were obtained in high-density locusts. Gene annotation information was screened to obtain Reeler genes containing the Reeler domain.

[0063] 1. Total RNA extraction from locusts The entire process of RNA extraction from locust brain tissue was performed under RNase-free conditions using Trizol reagent (Invitrogen, USA) according to the manufacturer's instructions: (1) Take the fifth-instar locust nymphs raised at different densities (low-density solitary type and high-density gregarious type), take the brain tissue of 10 locust nymphs in each treatment group, put them into 1 mL of pre-cooled Trizol reagent, add grinding beads (two 2mm beads and one 3mm bead) to them, and put the centrifuge tube into the fully automatic sample rapid grinder. (2) Centrifuge at 4℃, 12000 r / min for 10 min, and then transfer the supernatant to a new 1.5 mL centrifuge tube; (3) Let the supernatant stand for 5 min, then add 0.2 mL of chloroform to the centrifuge tube, shake vigorously for 15 s, and let stand for 3 min; (4) Centrifuge at 12000 r / min for 15 min at 4℃. At this time, the solution in the tube will be divided into three layers. Use a pipette to transfer the uppermost layer to a new centrifuge tube, add 0.5 mL of 100% isopropanol, and let stand for 10 min. (5) Centrifuge at 12000 r / min for 10 min at 4℃, discard the supernatant, leave a precipitate at the bottom of the tube, add 1 mL of 75% anhydrous ethanol to it, and wash the precipitate. (6) Centrifuge at 7500 r / min for 5 min at 4℃, discard the supernatant, and place the centrifuge tube in a clean bench to dry for 5-10 min (do not dry completely). (7) Add 20-50 μL of Nuclease-free water according to the amount of precipitate, and use a pipette to blow and stir to fully dissolve the precipitate; (8) Incubate in a metal bath at 55℃ for 15 min, take 1 μL of RNA and dilute it appropriately. Use part of it for concentration determination and part of it for electrophoresis detection. (9) The remaining RNA samples can be used for subsequent experiments or stored at -80℃ for later use.

[0064] 2. Synthesis of first-strand cDNA The first-strand cDNA was synthesized using the GoScript™ Reverse Transcription System (Promega, USA). The specific steps included: (1) Add 1 μg RNA and 1 μL Oligo(dT)15 primer to a 200 μL enzyme-free PCR tube. The total reaction volume is 5 μL. Make up the volume with Nuclease-free water. (2) React at 70°C for 5 min in a PCR instrument, place on ice for 5 min, centrifuge at low speed, and centrifuge the liquid on the wall to the bottom of the tube; (3) Add 4 μl of GoScript™ 5X Reaction Buffer and 1 μL of Recombinant RNasin to the above reaction system in sequence. ® Ribonuclease Inhibitor, 1 μL PCR Nucleotide Mix, 1 μL LeoScript™ Reverse Transcriptase, 2 μL MgCl2, and then bring the total volume to 20 μL with Nuclease-free water; (4) 25℃, 5 min; 42℃, 1 h; finally 70℃, 15 min to terminate the reaction process; (5) The final reverse transcription product is stored at -20℃ for later use.

[0065] 3. Reeler gene cloning, expression, and purification: Using locust brain cDNA as a template, R gene-specific primers were used: Primer F (5'-3'):ATGAAGCTGGTGGTGGCTGC (SEQ ID NO:6) Primer R (5'-3'): CTAAGCAGCAACTGCGTTGT (SEQ ID NO:7) The full-length sequence of the R gene was amplified using the KOD One™ PCR Master Mix-Blue- (TOYOBO, Japan) kit. The reaction conditions were: 94℃ / 2 min; 94℃ / 10 ​​sec, 60℃ / 5 sec, 68℃ / 1 sec (35 cycles).

[0066] PCR product purification was performed using Wizard ® Perform the procedure using the SV Gel and PCR Clean-Up System (Promega, USA), following the instructions in the manual.

[0067] Using pEASY ® - The Blunt Cloning Kit (TranGen) cloned the purified product into the vector: 1 μL of PCR purified product, 1 μL of pEASY ® - Mix Blunt Cloning Vecto with 3 μL of nuclease-free water and incubate at 25°C for 10 min for ligation; add the ligation product to 50 μL of Trans1-T1 competent cells, gently tumble to mix, and incubate on ice for 30 min; heat shock at 42°C for 30 sec, then immediately place on ice for 2 min; add 250 μL of LB medium equilibrated to room temperature and incubate at 200 rpm / 37°C for 1 h; spread the cultured bacterial solution evenly on LB agar plates containing ampicillin and incubate overnight at 37°C; pick at least 10 clones and inoculate them into LB / Amp+ liquid medium, incubate at 200 rpm / 37°C for 5 h, amplify using M13F / R universal primers, detect bands by agarose gel electrophoresis, and send the bacterial solution containing the target band for sequencing verification.

[0068] Plasmid mini-prep: Plasmid mini-prep was performed using the TIANprep Mini Plasmid Kit (TIANGEN). The operating procedure was performed in accordance with the instruction manual.

[0069] Based on the target gene sequence, a pair of signal peptide-specific primers were designed, and restriction enzyme sites BamHI (GGATCC) and NotI (GCGGCCGC) were added. The specific operation is as follows: PCR amplification was performed using the plasmid as a template, and the product was purified. The purified product and the PET30a (+) vector were then double-digested with enzymes. The digestion system consisted of 1 μg of purified product, 1 μL each of BamHI and NotI, 5 μL of rCutSmart Buffer, and nuclease-free water to a final volume of 50 μL. The mixture was incubated at 37°C for 1 h. After purification, the product was ligated and transformed into Trans-T1 competent cells for bacterial amplification. After successful sequencing, plasmid mini-preparation was performed.

[0070] Plasmid R-PET30a (+) was transformed into BL21 (DE3) competent cells. Positive clones were selected and amplified in 1 mL LB liquid medium, followed by expansion culture in 500 mL medium. Then, 1 mmol / L IPTG was added, and the cells were induced at 18℃ / 220 rpm for 18 h. The cells were sonicated, and the supernatant was collected and purified by affinity chromatography using a nickel column to obtain the recombinant target protein. The protein was digested with recombinant enterokinase at 25℃ for 12 h, and then concentrated by ultrafiltration using a nickel column to obtain the His-tag-free R gene recombinant protein. The expression of the target protein at each stage was detected by 15% SDS-PAGE gel electrophoresis.

[0071] 4. Western blot detection of Reeler protein expression level Western blot was performed using a Reeler-specific polyclonal antibody synthesized by a biotechnology company. (1) Electrophoresis: 70V, 20min, then switch to 120V, 60min; (2) Soak the filter paper and PVDF membrane in the transfer buffer for 5 minutes, and place them in the clamping plate in the order of filter paper-glue block-PVDF membrane-filter paper, 300mA, 1h; (3) Place the PVDF membrane in TBST (5% skim milk powder) and seal it at room temperature for 1 hour; (4) Wash with TBST 3 times, 10 minutes each time; (5) The primary antibody against the target protein and the internal control protein was diluted 1:2000 in TBST (5% skim milk powder) and incubated overnight at 4°C; (6) Wash 3 times with TBST for 10 min each time. Dilute the target protein and internal control protein secondary antibody 1:10000 in TBST (5% skim milk powder) and incubate at room temperature for 1 h. (7) Wash with TBST 6 times, 10 minutes each time; (8) Using EasySee ®Western Blot Kit (TransGen) was used for color development.

[0072] The results are as follows Figure 1 As shown, the mRNA transcription level and protein level of the Reeler gene in the brain of high-density gregarious locusts were significantly higher than those in low-density solitary locusts, confirming that the gene has density-dependent expression characteristics.

[0073] Example 2: Brain localization of the Reeler protein in locusts To clarify the cellular localization of the Reeler protein in the locust brain, immunofluorescence staining and laser confocal microscopy were performed.

[0074] 1. Immunostaining Prepare the solution: Dissection buffer: 0.015% Triton X-100 dissolved in 1×PBS; 25µL sterile 30% Triton X-100 dissolved in 50 mL sterile 1×PBS, stored at 4°C; Wash buffer: 0.5% Triton X-100 dissolved in 1×PBS; 833µL sterile 30% Triton X-100 dissolved in 50mL sterile 1×PBS, stored at 4℃; Block buffer: Dissolve 1×NGS in wash buffer at 4°C; take 1.5 mL of 20×NGS from the -20°C freezer and add 30 mL of wash buffer. (1) Cut off the head of the fifth-instar locust nymph with medical scissors and place it in a dissection buffer. Dissect the brain under a stereoscope. (2) Transfer brain tissue to 1 mL of Fixation buffer (pre-cooled) and leave at room temperature for 2 h or overnight at 4°C; (3) The fixed brain was rinsed four times with 1 ml Wash buffer for 15 min each time, and then placed in 500 µL Block buffer and sealed at room temperature for 3 h. (4) Then add the primary antibody (diluted 1:200 in 500µL block buffer) and incubate at 4°C for 5 days; (5) Wash with Wash buffer 6 times for 20 min each time, then add secondary antibody (diluted 1:300 in 250µL Block buffer) and incubate at 4℃ for 3 days; (6) After incubation, rinse the tissue 6 times with 500µL Wash buffer for 20 min each time. Then dehydrate the tissue at room temperature with 500µL of graded alcohol at 50%, 70%, 90%, 96% and 100% (v / v) concentrations for 10 min at each concentration. Repeat the 100% concentration twice. (7) After dehydration, the tissue was placed in 250µL of methyl salicylate and cleared at 4°C for 30 minutes. Finally, the tissue was placed in the small hole of a special metal slide, neutral resin was added to the inside of the hole, and the slide was sealed with a coverslip and stored at 4°C in the dark.

[0075] 2. Laser confocal microscopy scanning Fluorescently labeled brain tissue was scanned layer by layer using a laser confocal microscope (FV1000, Olympus, Japan) at a resolution of 1024 × 1024 pixels, a scanning speed of 100 Hz, a pinhole size of 1, a line average of 4, and a layer spacing of 10 µm.

[0076] Brain nerve cells include two types: neurons and glial cells. To clarify the localization of Reeler proteins in the brain, anti-synaptic protein antibody (anti-SYNORF 1, DSHB) was used to label brain morphology and characterize brain anatomical structures, while Reeler protein-specific antibody (anti-R) was used to label Reeler proteins. Figure 2 The red area shows the brain structure of the 5th-instar locust nymph, and the green area shows the structure marked by the R protein antibody. The Merge image shows that the R protein antibody marker structure does not overlap with the brain morphology marked by the anti-synaptic protein antibody, but is wrapped in its outer layer. Therefore, it is speculated that the Reeler protein expression site may be located in the surface glial cell layer, which exists on the outer surface of the central nervous system and constitutes the blood-brain barrier.

[0077] Example 3: Validation of dsRNA synthesis and silencing effect targeting the Reeler gene 1. Synthesis of dsRNA The primer sequences used for RNA interference are shown below. Primers for amplifying the RNA interference fragment were designed using Primer 3 (https: / / primer3.ut.ee) based on Reeler's full-length sequence. The target dsRNA fragment size was 332 bp. The T7 promoter sequence is underlined.

[0078] Table 1 Primer Sequences The target fragment was amplified using Taq DNA polymerase (Tiangen, China). The reaction system was prepared according to the instructions, and the annealing temperature was 60℃.

[0079] Using Wizard ® The SV Gel and PCR Clean-Up System (Promega, USA) was used to purify the PCR products, following the instructions in the manual.

[0080] Using the purified product obtained in the above steps as a template, dsRNA was synthesized using the T7 RiboMAX™ Express RNAi System (Promega, USA) dsRNA in vitro synthesis kit. The system is shown in Table 2 below: Table 2 Reaction System The above reaction system was incubated at 37°C for 2 h, treated at 70°C for 10 min, and then naturally cooled to room temperature. (1) Add 1µL RQ1 and 1µL RNase (dilution ratio 1:200) to it and incubate at 37℃ for 30 min; (2) Add 0.1 volume of sodium acetate (pH = 5.2) and 1 volume of isopropanol, place on ice for 5 min, centrifuge at 15000 r / min for 10 min at 4℃; (3) Aspirate the supernatant, wash the precipitate with 500 μL of 70% pre-cooled ethanol, and air dry at room temperature for 10 min; (4) Add about 2 times the volume of Nuclease-Free Water to dissolve the precipitate, detect the dsRNA quality by 1.2% agarose gel electrophoresis, adjust the concentration to 1µg / µL, and store at -20℃ for later use.

[0081] 2. Evaluation of the silencing effect of dsRNA delivered by injection on the Reeler gene in locusts. Primers for the Reeler gene and primers for the internal control housekeeping gene rp49 are shown in Table 3 below: Table 3 Primers The RT-qPCR amplification reaction used SuperReal PreMix Plus (SYBR Green) (Tiangen, China), and the reaction system is shown in Table 4 below: Table 4 Reaction System Reaction program: 95℃ pre-denaturation for 15 min; 40 cycles (95℃, 10 s; 60℃, 30 s). The relative expression levels of the target gene were normalized using the housekeeping gene rp49, and a 23... -ΔΔCT The method is used for calculation.

[0082] like Figure 3 As shown, compared to the control group, injection of dsReeler can significantly reduce the Reeler gene transcription level.

[0083] Example 4: Effects of Reeler gene silencing on locust developmental behavior and resistance to pathogens 1. The effect of Reeler gene silencing on the developmental time of locust nymphs. Insects at similar developmental stages were selected. On the first day of the 3rd instar, 4 µL (1 µg / µL) of the candidate gene dsRNA was injected. On the first day of molting in the 4th instar, a second injection of the corresponding gene dsRNA was administered, 5 µL (1 µg / µL) per insect. Developmental status was recorded daily from the 3rd instar until the 5th instar. A 10 µL pointed microsyringe was used for injection. The control group consisted of a medium-density population injected with dsGFP, with the injection time and amount consistent with the experimental group.

[0084] Knockdown of the Reeler gene significantly affected the developmental course of the test insects from the 3rd to the 5th instar. Compared with the control group, the developmental time of the single instar to the 4th and 5th instars was significantly shortened in the Reeler gene knockdown group. Figure 4 ).

[0085] 2. Effects of Reeler gene silencing on locust resistance during the locust nymph stage. Metarhizium acridum (MAC): The strain was provided by the Institute of Zoology. A piece of fungal material containing conidia was taken from the edge of the culture medium using a punch and inoculated onto PDA solid medium, then incubated at 30°C. Once the conidia on the medium turned green, fresh green conidia were scraped off using a sterile pipette tip and placed in 1 mL of sterile water containing 0.1% Tween 80. The mixture was shaken to mix, and then counted using a hemocytometer.

[0086] Locust nymphs injected with dsGFP and dsReeler were raised to the 5th instar. On the first day of molting at the 5th instar, 5 µL of a diluted Metarhizium anisopliae solution with 10⁶ spores / mL was taken and injected into the area below the pronotum using a pipette. The control group was injected with 5 µL of sterile Tween water. The infected nymphs were placed in an incubator at 22-25℃ to continue growing, fed fresh wheat daily, and the number of dead individuals was recorded.

[0087] like Figure 5 As shown in the survival curves, the dsGFP-injected group exhibited stronger disease resistance than the dsReeler-injected group. Even at the end of the experiment, 50% of the individuals remained viable, compared to only 25% in the dsReeler group. This demonstrates that silencing the Reeler gene can serve as an immunosensitization strategy, weakening the locusts' immunity and enabling highly effective lethality with low doses of Metarhizium anisopliae, achieving a synergistic control effect of immunosensitization and biological killing.

[0088] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element. Furthermore, it should be noted that the scope of the methods and apparatuses in the embodiments of this application is not limited to performing functions in the order shown or discussed, but may also include performing functions substantially simultaneously or in the reverse order, depending on the functions involved. For example, the described methods may be performed in a different order than described, and various steps may be added, omitted, or combined. Additionally, features described with reference to certain examples may be combined in other examples.

[0089] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of a biological material in the control of gregarious locusts, characterized in that, The biomaterial is any one of the following (c1) to (c2): (c1) Silencing the gene silencing cassette of the locust Reeler gene shown in SEQ ID NO:1; (c2) Gene silencing vector for silencing the locust Reeler gene shown in SEQ ID NO:1; The gene silencing cassette or gene silencing vector generates dsRNA targeting the Reeler gene, the dsRNA sequence of the Reeler gene comprising the nucleotide sequence shown in SEQ ID NO:

3.

2. The application of a dsRNA targeting the locust Reeler gene shown in SEQ ID NO:1 in the control of gregarious locusts; said dsRNA comprising the nucleotide sequence shown in SEQ ID NO:3; The amplification primers for the dsRNA include the nucleotide sequences shown in SEQ ID NO:4 and 5.

3. A composition for controlling gregarious locusts, characterized in that, Includes: dsRNA targeting the locust Reeler gene; and microbial agents; The dsRNA comprises the nucleotide sequence shown in SEQ ID NO:3; The microbial agent is Metarhizium anisopliae; The polynucleotide sequence of the locust Reeler gene is the polynucleotide sequence shown in SEQ ID NO:

1.

4. The composition for controlling gregarious locusts according to claim 3, characterized in that, The amplification primers for the dsRNA include the nucleotide sequences shown in SEQ ID NO:4 and 5.

5. The use of the composition according to claim 3 or 4 in the control of gregarious locusts.

6. A method for controlling gregarious locusts, characterized in that, include: To determine whether a locust swarm is a high-density, gregarious type; Delivering dsRNA targeting the Reeler gene to locusts; as well as Apply microbial agents to the locusts; The microbial agent is Metarhizium anisopliae; The dsRNA includes the nucleotide sequence shown in SEQ ID NO:

3.

7. The method for controlling gregarious locusts according to claim 6, characterized in that, The dsRNA is delivered to locusts via injection, feeding, spraying, or delivery via nanocarriers.

Images