Method for improving the killing effect of daphnis mori granulovirus on pests

Abstract

The present application belongs to the field of biological medicine, and relates to a method for improving the killing effect of Daphnis nigris cytoplasmic polyhedrosis virus on pests. Specifically, the present application takes Daphnis nigris and corresponding Daphnis nigris cytoplasmic polyhedrosis virus DnCPV as examples, provides application of JNK inhibitor in synergizing cytoplasmic polyhedrosis virus to killing ability of host pests, thereby protecting plants, and provides a plant protection method.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a method for improving the insecticidal effect of oleander hawk moth polyhedrosis virus on pests. Background Technology

[0002] The Apocynaceae family is a treasure trove of natural active ingredients, encompassing important medicinal species such as oleander, rauwort, yellow oleander, and periwinkle, rich in core medicinal substances such as alkaloids, cardiac glycosides, and terpenes. For example, the cardiac glycosides in oleander can be used to treat heart failure; the content of the classic antihypertensive drug reserpine in rauwort is twice that in common rauwort; and vincristine and vinblastine in periwinkle are broad-spectrum antitumor drugs used in the treatment of leukemia, lymphoma, and other diseases. The medicinal components of these plants have complex structures, unique activities, and are irreplaceable, with huge market demand.

[0003] The oleander hawk moth (Daphnis nerii) is a specialized and explosive pest of plants in the Apocynaceae family. The larvae voraciously devour leaves and tender shoots, and in severe cases, they can eat all the leaves off the entire plant, causing the photosynthesis of medicinal plants to collapse, growth to stop, the content of active ingredients to drop sharply, and even the entire crop to fail. This greatly reduces the yield of core medicinal substances (such as reserpine and cardiac glycosides) from the Apocynaceae family.

[0004] Currently, chemical pesticides are widely used to kill oleander hawk moths. However, with long-term use, pests have developed high resistance to pesticides, and conventional pesticides have relatively low efficacy. Moreover, pesticide residues seriously affect the safety of medicinal plants, restricting their use in medicine and export, forming a vicious cycle of "pests - pesticide residues - devaluation of plant medicinal value".

[0005] Oleander hawk moth polyhedrovirus (DnCPV) is an obligate parasitic pathogenic microorganism belonging to the Sphingidae family and is classified as a biological control resource. Its insecticidal mechanism is unique: the virus proliferates rapidly in the midgut epithelial cells of the oleander hawk moth, destroying intestinal tissue, causing the pest to stop feeding and eventually die. DnCPV is harmless to humans and animals, does not pollute the environment, does not harm natural enemies, and leaves no residue. It meets the "green, safe, and residue-free" production requirements for medicinal plants, making it an ideal alternative to chemical pesticides and a way to increase the yield of the core medicinal substances in oleander.

[0006] Although polyhedroviruses have the advantages of a broad host spectrum and high infectivity as insecticides, their limited infection and weak pathogenicity also restrict their application as biological insecticides.

[0007] Developing insecticides containing polyhedroviruses with stronger pathogenicity and shorter disease duration is urgently needed. Summary of the Invention

[0008] This invention provides the application of c-Jun N-terminal kinase (JNK) inhibitors in killing hawk moths and thus protecting their host plants, and provides a method for screening potential substances that kill hawk moths and thus protect their host plants.

[0009] In a first aspect of the invention, there is provided the use of a JNK inhibitor for enhancing the killing ability of a cytoplasmic polyhedrosis virus against a host pest.

[0010] In another preferred embodiment, a formulation is prepared for enhancing the killing ability of a polyhedrotic virus against a host pest.

[0011] In another preferred embodiment, the concentration of the JNK inhibitor in the formulation is 0.1-100 μM, more preferably 1-80 μM, even more preferably 10-50 μM, and most preferably 20-40 μM.

[0012] In another preferred embodiment, the cytoplasmic polyhedrosis virus kills the host pest.

[0013] In another preferred embodiment, the lethality refers to specific lethality and / or pathogenicity.

[0014] In another preferred embodiment, the plasmonic polyhedrovirus is a plasmonic polyhedrovirus derived from lepidopteran pests.

[0015] In another preferred embodiment, the plasmonic polyhedrovirus comprises: sphingomyeridae plasmonic polyhedrovirus, licheniformisridae plasmonic polyhedrovirus, or a combination thereof.

[0016] In another preferred embodiment, the sphingomyelida cytoplasmic polyhedrovirus comprises: oleander sphingomyelida cytoplasmic polyhedrovirus DnCPV, bean sphingomyelida cytoplasmic polyhedrovirus CbCPV, or a combination thereof.

[0017] In another preferred embodiment, the cytoplasmic polyhedrovirus of the Ficus pluvialis family comprises: DpCPV (Polyhedrovirus dpCPV).

[0018] In another preferred embodiment, the host pest of the cytoplasmic polyhedrosis virus is a lepidopteran pest.

[0019] In another preferred embodiment, the host pest of the cytoplasmic polyhedrosis virus is a sphingomyelidae pest.

[0020] In another preferred embodiment, the host pest of the polyhedrosis virus is a leaf moth (Dendrocalamus spp.)

[0021] In another preferred embodiment, the cytoplasmic polyhedrovirus is the oleander hawk moth cytoplasmic polyhedrovirus DnCPV.

[0022] In another preferred embodiment, the DnCPV includes DnCPV-23, DnCPV-NC, DnCPV-2, or a combination thereof.

[0023] In another preferred embodiment, the host pest of the morphological polyhedrosis virus includes: oleander hawk moth, bean hawk moth, pine caterpillar, camphor leech, or a combination thereof.

[0024] In another preferred embodiment, the JNK inhibitor significantly inhibits the JNK levels in cytoplasmic polyhedrosis virus host pests.

[0025] In another preferred embodiment, the JNK inhibitor comprises an inhibitor at the gene level, an inhibitor at the protein level, an inhibitor at the activity level, or a combination thereof.

[0026] In another preferred embodiment, the inhibitor of the activity level refers to an inhibitor of the JNK phosphorylation level.

[0027] In another preferred embodiment, the significant inhibition means that the JNK inhibitor inhibits the JNK level of the cytoplasmic polyhedrosis virus host pest by 10% or more, preferably 30% or more, more preferably 50% or more, even more preferably 70% or more, and most preferably 90% or more.

[0028] In another preferred embodiment, the JNK inhibitor is a JNK inhibitor that targets the JNK coding sequence of a plasmonic polyhedrosis virus host pest.

[0029] In another preferred embodiment, the host pest of the morphopolyhedrovirus is the oleander hawk moth, and the JNK coding sequence of the oleander hawk moth is shown in SEQ ID NO:9.

[0030] In another preferred embodiment, the JNK inhibitor comprises: a polynucleotide, a polypeptide, a small molecule compound, or a combination thereof.

[0031] In another preferred embodiment, the small molecule compound comprises: synthetic small molecule compounds, natural small molecule compounds, or combinations thereof.

[0032] In another preferred embodiment, the natural small molecule compound comprises: a traditional Chinese medicine monomer.

[0033] In another preferred embodiment, the herbal monomer comprises: agarwood sesquiterpene ZH-13, berberine, paeoniflorin, or a combination thereof.

[0034] In another preferred embodiment, the JNK inhibitor comprises: an antibody, a small molecule compound, microRNA, siRNA, shRNA, antisense oligonucleotide, nucleic acid aptamer, gene editor, or a combination thereof.

[0035] In another preferred embodiment, the small molecule compound comprises: SP600125, CC-401, AS601245, BI-78D3, TAT-JBD20, HRX215, or a combination thereof.

[0036] In another preferred embodiment, the siRNA comprises: SEQ ID NO:3, SEQ ID NO:4, or a combination thereof.

[0037] In another preferred embodiment, the siRNA consists of the sequence shown in SEQ ID NO:3 and the sequence shown in SEQ ID NO:4.

[0038] In another preferred embodiment, it is used for one or more of the following sub-uses: (Z1) Enhances the replication ability of plasmonic polyhedrosis viruses; (Z2) Improve the killing rate of polyhedrosis viruses against host pests; (Z3) Shorten the disease time of host pests in the presence of polyhedrosis viruses.

[0039] In another preferred embodiment, the improvement in the replication capacity of plasmonic polyhedrosis virus (PPV) means that, in the presence of a JNK inhibitor, the PPV level E1 is compared with the PPV level E0 under the same conditions in the absence of a JNK inhibitor, the ratio E1 / E0 is ≥1.3, preferably ≥1.5, more preferably ≥1.8, and most preferably ≥2.

[0040] In another preferred embodiment, the improvement of the kill rate of the cytoplasmic polyhedrosis virus (CPV) on the host pest means that, in the presence of a JNK inhibitor, the kill rate K1 of the CPV on the host pest is ≥1.1, preferably ≥1.2, more preferably ≥1.4, and most preferably ≥1.6.

[0041] In another preferred embodiment, the shortening of the disease onset time of host pests in the presence of plasmonic polyhedrosis virus (PPV) means that, compared with the disease onset time T1 of host pests in the presence of PPV under the presence of JNK inhibitor and the disease onset time T0 of host pests in the presence of PPV under the same conditions without JNK inhibitor, T1 / T0 ≤ 0.9, preferably ≤ 0.8, more preferably ≤ 0.7, and most preferably ≤ 0.6.

[0042] In a second aspect of the invention, a composite formulation is provided, the composite formulation comprising a first component and a second component; in, The first component includes a JNK inhibitor; The second component includes plasmonic polyhedrovirus.

[0043] In another preferred embodiment, the concentration of the JNK inhibitor in the compound formulation is 0.1-100 μM, more preferably 1-80 μM, even more preferably 10-50 μM, and most preferably 20-40 μM.

[0044] In another preferred embodiment, the concentration of the plasmonic polyhedrosis virus (CPV) is ≥10^5 PIB / mL, more preferably ≥10^6 PIB / mL, and most preferably ≥10^7 PIB / mL.

[0045] In a third aspect of the invention, the use of the compound formulation described in the second aspect of the invention is provided for killing host pests of cytoplasmic polyhedrosis viruses.

[0046] In another preferred embodiment, it is used to kill pests that are hosts of polyhedrosis viruses, thereby protecting plants.

[0047] In another preferred embodiment, the plant is a plant of the Apocynaceae family, a legume family, or a pine family.

[0048] In another preferred embodiment, the plants include: oleander, Rauvolfia pulidonis, yellow oleander and periwinkle, soybean, and Masson pine.

[0049] In another preferred embodiment, the plant is oleander.

[0050] In another preferred embodiment, the protection includes: preventing and / or mitigating plant morphological damage caused by pests, preventing and / or mitigating yield reduction caused by pests, preventing secondary spread of pests, interrupting the pest reproductive chain, or a combination thereof.

[0051] In another preferred embodiment, an agricultural reagent kit is prepared for killing host pests of cytoplasmic polyhedrosis viruses.

[0052] In a fourth aspect of the invention, an agricultural reagent kit is provided, the agricultural reagent kit comprising: (i) The compound formulation described in the second aspect of the present invention, (ii)Optionally, an agronomically acceptable carrier.

[0053] In another preferred embodiment, the agronomically acceptable carrier includes: an aqueous carrier, a mineral-inert carrier, an organically biodegradable carrier, a wetting agent, a dispersant, or a combination thereof.

[0054] In another preferred embodiment, the agricultural kit further comprises: nutrients, pesticides, or combinations thereof.

[0055] In a fifth aspect of the invention, a method for protecting plants is provided, the method comprising: applying to the plant the agricultural combination described in the second aspect of the invention, thereby killing cytoplasmic polyhedrosis virus host pests.

[0056] In another preferred embodiment, it is used to kill pests that are hosts of polyhedrosis viruses, thereby protecting plants.

[0057] In another preferred embodiment, the term "killing polyhedrosis virus host pests" refers to polyhedrosis virus host pests that kill the surface of the plant.

[0058] In another preferred embodiment, the application is selected from the group consisting of: spraying, soaking, sprinkling, injection, coating, or microcapsule.

[0059] In another preferred embodiment, the following sub-steps are included: (S1) Apply the JNK inhibitor to the plant, incubate, and (S2) Apply plasmonic polyhedrosis virus to the plants obtained in (S1) and incubate them; or (S1') Simultaneously apply JNK inhibitor and plasmopolyhedrovirus to the plant and incubate.

[0060] In another preferred embodiment, the following sub-steps are included: (S1) Apply the JNK inhibitor to the plant, incubate, and (S2) Apply plasmonic polyhedrosis virus to the plants obtained in (S1) and incubate them to protect the plants; or (S1') Simultaneously apply JNK inhibitor and plasmopolyhedrovirus to the plant, incubate, thereby protecting the plant.

[0061] In another preferred embodiment, the concentration of the JNK inhibitor is 0.1-100 μM, more preferably 1-80 μM, even more preferably 10-50 μM, and most preferably 20-40 μM.

[0062] In another preferred embodiment, step (S3) is further included after step (S2): applying a JNK inhibitor to the plant obtained in (S2) and incubating it.

[0063] In another preferred embodiment, the method further includes step (S4): removing residual additives and / or the remains of cytoplasmic polyhedrosis virus host pests from the plant surface.

[0064] In a sixth aspect of the invention, a method is provided for screening potential synergists to enhance the killing ability of cytoplasmic polyhedrosis viruses against their host pests, comprising the following steps: (a) In the experimental group, cytoplasmic polyhedrovirus host cells were cultured in the presence of the test substance; and in the control group, cytoplasmic polyhedrovirus host cells were cultured under the same conditions but in the absence of the test substance; and (b) The JNK levels in the cells of the experimental group and the control group were measured and compared. If the JNK level X1 in the experimental group was significantly lower than that in the control group, it would suggest that the test substance is a potential synergist for enhancing the killing ability of cytoplasmic polyhedrosis viruses on their host pests.

[0065] In another preferred embodiment, the JNK level includes the gene level, protein level, activity level, or a combination thereof of JNK.

[0066] In another preferred embodiment, the activity level is characterized by the level of JNK autophosphorylation.

[0067] In another preferred embodiment, the host cells derived from the plasmonic polyhedrosis virus are selected from the group consisting of: tobacco hawk moth cells, Sf9 cells, Sf21 cells, High Five cells, and primary midgut cells of pests.

[0068] In another preferred embodiment, the significant reduction means that X1 / X0 ≤ 0.9, more preferably ≤ 0.7, even more preferably ≤ 0.5, even more preferably ≤ 0.3, and most preferably ≤ 0.1.

[0069] In another preferred embodiment, the method further includes: (c) further conducting activity testing or functional testing on the potential synergists selected in the previous step.

[0070] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0071] Figure 1 The study showed that the JNK inhibitor SP600125 promotes DnCPV-23 replication.

[0072] Figure 2 This study showed that JNK gene expression inhibits DnCPV-23 replication in tobacco hawk moth cells. (A) siRNA knockdown effect; (B) Viral replication after JNK gene knockdown. *: Significant difference (P < 0.05); **: Extremely significant difference (P < 0.01); ***: Extremely significant difference (P < 0.001). Detailed Implementation

[0073] Extensive and in-depth research has led to the first discovery that inhibiting the JNK levels in host pests of plasmon polyhedrosis viruses (PPCVs) can promote PPCV replication and enhance the PPCV's ability to kill host pests. Taking the oleander hawk moth as an example, simultaneous application of PPCV and the JNK inhibitor SP600125 to the host pest resulted in a 90% kill rate on day 5 of infection, a 20% increase compared to the group treated with DnCPV alone; the onset of action was also significantly accelerated. This invention was developed based on these findings.

[0074] the term To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below. Other definitions are set forth throughout the application.

[0075] As used herein, the term “and / or” refers to and covers any and all possible combinations of one or more of the related listed items.

[0076] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0077] As used in this article, the term "or a combination thereof" means "or any combination thereof".

[0078] As used herein, the term "compound formulation" refers to an agricultural compound formulation. In a specific embodiment, the first component and the second component are mixed. In a specific embodiment, the first component and the second component are separate.

[0079] Insect virus pesticides Using insect viruses to control pests is a form of biological control. Compared to chemical control, insect virus control is environmentally friendly, less likely to induce drug resistance, and has advantages such as being green, harmless, safe, and stable.

[0080] Type polyhedrovirus Cypovirus (CPV) is a virus belonging to the family Reoviridae and the genus CPV. The virus particle is spherical (icosahedral), 50-70 nanometers in diameter, and possesses a segmented double-stranded RNA genome (molecular weight 0.3-2.7 × 10⁻⁶). 6The virus (Dalton) contains polygonal structures such as quadrilaterals and hexagons, ranging in size from 0.5 to 10 micrometers. Its host is insects, where it replicates primarily in the cytoplasm of midgut epithelial cells, leading to midgut necrosis and larval death. It is mainly transmitted vertically through feces and eggs, with a lethal cycle of 3-18 days, and can induce viral epidemics in insect populations.

[0081] CPV has its own complete intracapsular transcription / replicase (VP1 polymerase + VP3 capping enzyme), and the core replication machinery is encoded by the virus itself, without relying on replicase / core factors provided by the host.

[0082] This virus primarily infects the midgut cells of lepidopteran insect larvae and has been found in over 200 insect species, with more than 30 host insect species identified in my country. Spraying viral agents has proven effective in controlling pine caterpillars in Shandong, Guangdong, Yunnan, and other regions.

[0083] CPV has a wide host range, infecting more than 200 insect species, including pine caterpillars, silkworms, cotton bollworms, bean hawk moths, and oleander hawk moths. Different strains can cross-infect each other, but the degree of pathogenicity varies depending on the insect species. For example, the pine caterpillar polyhedrosis virus (DpCPV) can cross-infect lepidopteran larvae such as the beet armyworm and the cotton bollworm, and has a very high kill rate on beet armyworm larvae.

[0084] Oleander hawk moth polyhedrovirus ( Daphnis nerii Cypovirus (DnCPV), a novel cypovirus, is effective in killing the oleander hawk moth, thus protecting medicinal plants such as oleander, the medicinal plant Rauvolfia pulveratula, yellow oleander, and periwinkle, all belonging to the Apocynaceae family. Furthermore, this virus is also effective against the soybean hawk moth, one of the major pests of soybeans. Cypoviruses have the advantages of a broad host spectrum and high infectivity as insecticides; however, their limited infection range, weak pathogenicity leading to low killing efficiency, and long treatment cycles also limit their application as biological insecticides.

[0085] DnCPV is a double-stranded RNA virus belonging to the genus Cypovirus in the family Reoviridae. DnCPV originated from the naturally occurring oleander hawk moth (…). Daphnis nerii It was isolated from the larvae of the fall armyworm and is effective against the oleander hawk moth, and can also infect and kill various hawk moths. It can replicate in the Sf9 cells of the fall armyworm and is a commonly used model for insect virus research.

[0086] Cell lines are highly simplified, isolated in vitro systems, while complete hosts are complex systems with multiple organs, multiple immune systems, and multiple barriers. A compound that promotes viral replication in vitro may not necessarily enhance viral virulence in vivo, and could even have the opposite effect. The pathogenic effect of a virus on its host depends primarily on the following factors: (1) viral virulence, replication capacity, and ability to invade host cells; (2) viral tissue tropism (whether it attacks key organs); (3) viral immune evasion ability; (4) the intensity of the host's inflammatory response; and (5) the virus's ability to spread and proliferate in vivo. Therefore, obtaining potentiators of plasmonic polyhedrosis viruses that kill harmful parasites requires extensive screening and experimentation.

[0087] JNK inhibitors JNK inhibitors are molecules that specifically block JNK pathway signaling. Their core definition is: drugs or chemical tools that regulate cell fate (such as inflammation, apoptosis, regeneration, etc.) by interfering with the activity of JNK kinases, preventing them from transmitting "stress signals" downstream. In specific embodiments, JNK inhibitors include ATP-competitive inhibitors, substrate-competitive inhibitors, covalent inhibitors, JNK gene expression inhibitors, JNK upstream kinase inhibitors, or combinations thereof. In specific embodiments, the JNK inhibitor inhibits one or more JNK subtypes. In specific embodiments, the ATP-competitive inhibitors include SP600125, CC-401, AS601245, or combinations thereof. In specific embodiments, the substrate-competitive inhibitors include BI-78D3, TAT-JBD20, or combinations thereof. In specific embodiments, the covalent inhibitors achieve persistent inhibition by forming irreversible covalent bonds with specific amino acid residues (such as cysteine) on the JNK protein through a chemical reaction. In specific embodiments, the JNK upstream kinase inhibitors include MKK4 inhibitors (e.g., HRX215), MKK7 inhibitors, or combinations thereof. In specific embodiments, the inhibitors or antagonists include the following forms: polynucleotides, polypeptides, small molecule compounds, or combinations thereof. In specific embodiments, the small molecule compounds include: synthetic small molecule compounds, natural small molecule compounds, or combinations thereof. In specific embodiments, the natural small molecule compounds include: the traditional Chinese medicine monomer agarwood sesquiterpene ZH-13, berberine, paeoniflorin, or combinations thereof. In specific embodiments, the inhibitors include: antibodies, small molecule compounds, microRNA, siRNA, shRNA, antisense oligonucleotides, nucleic acid aptamers, gene editors, or combinations thereof.

[0088] SP600125 is an orally effective, reversible, ATP-competitive JNK inhibitor that inhibits the IC50 of JNK1, JNK2, and JNK3. 50The concentrations are 40, 40, and 90 nM, respectively. SP600125 is a potent inhibitor of ferroptosis. SP600125 can induce the transformation of autophagy into apoptosis in bladder cancer cells. Its structure is as follows: .

[0089] Uses of the present invention This invention provides a JNK inhibitor for enhancing the efficacy of oleander hawk moth polyhedrosis virus (OPV). Daphnis nerii The insecticidal effect of cypovirus (DnCPV).

[0090] Using cytoplasmic polyhedroviruses alone as biopesticides has drawbacks such as long incubation periods and insufficient lethality. Combining cytoplasmic polyhedroviruses with chemical pesticides pollutes the environment and may have side effects on humans and animals. Targeting specific host genes with inhibitors or siRNAs to promote viral replication can enhance viral lethality and is more environmentally friendly, showing promising application prospects.

[0091] This invention provides the JNK gene of the oleander hawk moth, an inhibitor targeting this gene, and siRNA to improve the killing effect on pests (increase killing efficiency and shorten the effective period) and protect landscape plants, medicinal plants, and legume crops.

[0092] In this invention, the DnCPV virus and the JNK inhibitor synergistically enhance the insecticidal rate against pests. In a specific embodiment, the DnCPV virus, at a certain concentration and in a certain manner (dosage form, administration method, etc.), synergistically enhances the insecticidal rate by targeting the host gene with an inhibitor.

[0093] In this invention, the DnCPV virus and JNK inhibitors synergistically enhance the speed of drug action. In a specific embodiment, the DnCPV virus, at a certain concentration and in a certain manner (dosage form, administration method, etc.), synergistically targets the host gene inhibitor to shorten the duration of drug efficacy, i.e., to exert its effect more quickly.

[0094] In this invention, the JNK gene sequence of the oleander hawk moth was cloned, providing a preliminary foundation for the subsequent development of drugs that target the JNK gene and synergistically promote the insecticidal effect of DnCPV.

[0095] This invention provides a method for protecting plants. The method includes the following sub-steps: (S1) Apply the JNK inhibitor to the plant, incubate, and (S2) Apply plasmonic polyhedrosis virus to the plants obtained in (S1) and incubate them to protect the plants; or (S1') Simultaneously apply JNK inhibitor and plasmopolyhedrovirus to the plant, incubate, thereby protecting the plant.

[0096] In specific implementation methods, those skilled in the art can select the appropriate plasmonic polyhedrosis virus based on the plant species and the type of pest that needs to be controlled.

[0097] The main advantages of this invention include: (a) This invention provides the application of JNK inhibitors in promoting DnCPV virus replication, enhancing the killing efficiency and onset rate of DnCPV virus, and provides agricultural compounds for the combined use of JNK inhibitors and DnCPV and their applications.

[0098] (b) The present invention provides a method for effectively killing CPV host pests, thereby protecting plants. This method is safe, efficient, fast-acting, long-lasting, green, low-cost, and simple to operate.

[0099] (c) The present invention provides a method for screening and killing CPV host pests to protect plants, which is of great significance for the cultivation of plants parasitized by CPV host pests.

[0100] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0101] Example 1: Experimental method.

[0102] (1) Materials and Methods 1) Cell lines, viruses, inhibitors, and antibodies Sf9 cell line (fall armyworm ovary cell line) was cultured at 28 °C on Grace insect medium (Gibco, USA) containing 10% fetal bovine serum (Gibco, USA). DnCPV-23 was initially isolated from and amplified in oleander hawk moth larvae. The DnCPV-23 polyhedral suspension used for cell infection was stored at 4 °C protected from light. The JNK inhibitor SP600125 (MCE, China) was used at a final concentration of 25 μM. In Western blotting experiments, rabbit polyclonal antibody against DnCPV-23 S1 protein (Yacoin, China) was used at a 1:500 dilution, and mouse monoclonal antibody against Tubulin (Yacoin, China) was used at a 1:10,000 dilution.

[0103] 2) Viral infection Treatment with 0.2M Na₂CO₃-NaHCO₃ (pH 10.8) resulted in a concentration of approximately 2 × 10⁻⁶. 7 Purified DnCPV-23 polyhedra were injected at a concentration of 1 mL / ml, and the pH was adjusted to 7.4 using 1 M Tris-HCl (pH 6.8) buffer. Free virus particles were stored at -80°C. Sf9 cells were seeded in 24-well plates and infected with free DnCPV-23 virus particles when the cells reached approximately 75% confluence. After 12 hours of incubation, the cell culture medium was replaced with Grace medium containing 10% (v / v) FBS, and cells were harvested at 24 and 48 hours post-infection. Uninfected cells served as controls.

[0104] 3) Reverse transcription quantitative PCR (RT-qPCR) This study analyzed the expression level of the DnCPV-23 S1 gene in transcriptome samples. Total RNA was isolated from the samples using TRIzol reagent (ThermoFisher, USA) for RT-qPCR analysis. Total RNA (500 ng per sample) was reverse transcribed into complementary DNA (cDNA) using the FastKing cDNA First-Strand Synthesis Kit (Tiangen, China). The cDNA obtained from reverse transcription was then analyzed for RT-qPCR at the target gene mRNA level using the SuperReal Quantitative Real-Time Premixed Reagent Enhanced Version (SYBR Green) kit (Tiangen, China). GAPDH was used as an internal control gene for experiments and analysis. Data were analyzed using the 2-ΔΔCt method. The reaction mixture consisted of 20 μL of SYBR Green, 0.4 μL of Rox, 0.3 μL each of 0.3 μmol / L forward and reverse primers, and 0.7 μL of cDNA; the reverse transcription mixture consisted of 20 μL of RNA and 500 ng; the RT-qPCR reaction conditions were: 95℃ for 5 min; 95℃ for 30 s; 60℃ for 30 s, for 40 cycles; fluorescence signals were collected at 60℃. The main instrument used was a QuantStudio™ 7Flex real-time quantitative PCR system (Applied Biosystems™, USA).

[0105] 4) Cell treatment Sf9 cells were used at a rate of 1 × 10⁶ cells per well. 5 Cells were seeded at a density of [number] cells / well in 24-well plates and cultured overnight. The following day, cells were pretreated for 2 hours with the JNK inhibitor SP600125 (MCE, China) at a final concentration of 25 μM. After 2 hours, 10 µL of free DnCPV-23 virus suspension without polyhedral particles was added to the cells, and the cells were cultured for another 6 hours. After incubation, the culture medium was discarded, and fresh culture medium containing the inhibitor was added, and the cells were cultured for another 24 or 48 hours.

[0106] 5) JNK siRNA transfection siRNA was transiently transfected into tobacco hawk moth cells using the riboFECT CP Transfection Kit (Borui Biotechnology) (sequence shown in Table 1). Tobacco hawk moth cells (1.2 × 10⁶ cells) were then transfected. 5Seeds were seeded onto 24-well plates. Transfection was performed the next day. For transfection, 1.25 μL of 20 μM siRNA was added to 30 μL of 1× riboFECT™ CP Buffer and gently mixed; then 3 μL of riboFECT™ CP Reagent was added, mixed, and incubated at room temperature for 10 min to prepare the transfection complex. After incubation, the transfection complex was added dropwise to the cells. The cells were then incubated at 28 °C for 3-4 days. Cells were then collected to assess gene knockdown.

[0107] Table 1. JNK gene siRNA sequence. 6) Western blotting Cells were collected at 300g for 5 min, the supernatant was discarded, and RIPA lysis buffer (Beyotime, China) was added. The cells were placed on ice for 20 min, then centrifuged (6000g, 6 min) to collect the supernatant. Reducing 5×SDS loading buffer was added to the supernatant, and the mixture was boiled at 100℃ for 7 min. The 5×SDS loading buffer formulation was: 10% (w / v) SDS, 250 mM Tris-HCl, 0.5% (w / v) BPB, 5% (v / v) β-mercaptoethanol, and 50% (v / v) glycerol. After separation by SDS-PAGE electrophoresis (80 V, approximately 2 h), the proteins on the separating gel were transferred to a PVDF (Millipore) membrane using a wet transfer method (100 V, approximately 1 h). After transfer, the PVDF membrane was blocked for 2 h at room temperature with TBST containing 5% skim milk (150 mM NaCl, 25 mM Tris-HCl, pH 7.4, 0.05% Tween-20), followed by overnight incubation with the corresponding primary antibody. The next day, the PVDF membrane was washed three times with TBST (10 min each time) and incubated for 2 h at room temperature with horseradish peroxidase-conjugated goat anti-mouse IgG or goat anti-rabbit IgG as secondary antibodies. Protein bands were imaged using a highly sensitive ECL luminescent reagent (Sangon Biotech, China) and analyzed using a ChemiScope 3000 mini system (Clinx, Shanghai, China). The primary antibodies were DnCPV-23 S1 protein rabbit polyclonal antibody (Icoin, 1:500 dilution) and Tubulin mouse monoclonal antibody (Icoin, 1:10,000 dilution). The secondary antibodies were goat anti-rabbit (CST, 1:10,000) and goat anti-mouse antibody (Subcoin, 1:10,000 dilution).

[0108] 7) Statistical methods Statistical analysis was performed using GraphPad Prism 5.0. Data are expressed as mean ± standard error (SEM). Unpaired two-tailed t-tests were used to compare values ​​between different groups. P < 0.05 was considered statistically significant (*); P < 0.01 was considered highly significant (**); P < 0.001 was considered extremely significant (***); and P > 0.05 was considered not significant.

[0109] Example 2: JNK CDS sequence cloning.

[0110] First, specific primers were designed based on the JNK genome sequences of the tobacco hawk moth and the silkworm: Primer F: ATGGTACAGTTTTACTCGG (SEQ ID NO:7); Primer R: CTATGTGGTGAGTGC (SEQ ID NO:8).

[0111] PCR procedures and gene cloning; Using midgut cDNA from the fourth instar of the oleander hawk moth as a template, PCR was amplified under the following conditions: 95 °C pre-denaturation for 5 min, followed by 32 cycles of denaturation at 95 °C for 30 s, annealing at 47 °C for 40 s, and extension at 72 °C for 1 min, with a final extension at 72 °C for 10 min. The PCR product was identified and recovered by agarose gel electrophoresis, and then ligated into the pMD19 T vector. The ligation reaction was carried out at room temperature for 1 h using ThermoScientific T4 DNA ligase. The ligation was then transformed into DH5α competent cells, and positive clones were obtained and sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. Sequencing results showed that we successfully cloned the full-length coding sequence (CDS) of the oleander hawk moth JNK gene, which is 1146 bp in length and is predicted to encode a 43.36 kDa protein. The CDS sequence of the DnJNK gene is as follows: >DnJNK

[0112] Example 3: The effect of JNK protein inhibitors on viral replication.

[0113] Before infection with DnCPV-23, Sf9 cells were pretreated for 2 h with JNK inhibitors (SP600125), p38 inhibitors, autophagy inhibitors, PI3K inhibitors, and JAK inhibitors, respectively. Western blotting was then performed to detect the expression of the viral protein VP1 (encoded by the viral S1 gene) to analyze the effect of these inhibitors on viral replication. Specifically, Sf9 cells were seeded in 24-well plates to achieve approximately 75% confluence and pretreated for 2 h with equimolar concentrations of JNK inhibitors (SP600125), p38 inhibitors, autophagy inhibitors, PI3K inhibitors, and JAK inhibitors, respectively, before infection with DnCPV-23. Two hours later, the cells were incubated with 10 μL of viral suspension containing free DnCPV particles without polyhedra for 6 h. After incubation, fresh cell culture medium containing the inhibitors was added to the cells. Samples were collected at 24 h and 48 h post-infection, and viral protein levels were determined by Western blotting using specific antibodies.

[0114] The results are as follows Figure 1 As shown in Table 2, the results indicate that 25 μM SP600125 treatment significantly promoted viral replication compared to the control group (DMSO). Treatment with other protein inhibitors failed to promote viral replication. These data suggest that the JNK pathway may inhibit DnCPV-23 replication.

[0115] Table 2. Relative expression levels of VP1 in different groups Example 4: The effect of siRNA knockdown of JNK gene expression on viral replication.

[0116] JNK siRNA was transfected into tobacco hawk moth cells using the riboFECT CP Transfection Kit. After 24 h of transfection, the cells were infected with DnCPV-23. Samples were collected at 24 h and 48 h post-infection, and viral replication was detected by RT-qPCR.

[0117] The results are as follows Figure 2 As shown in Figure A, the siRNA-2 group of siRNA sequences can significantly reduce the expression of JNK.

[0118] The effect of siRNA-2 on promoting DnCPV-23 replication is as follows: Figure 2 As shown in Figure B, knocking down the JNK gene significantly increased the viral replication level, as shown in Table 3, suggesting that the JNK gene can inhibit viral replication.

[0119] Table 3. Relative expression levels of DnCPV-23 VP1 in different groups Example 5: The effect of inhibitors on the virus killing effect.

[0120] After infection with the virus, the larvae experience reduced appetite and slow growth. Following infection, the larvae gradually turn grayish-brown, exhibit abnormal behavior, and progressively lose their appetite. Eventually, their bodies soften, but they do not liquefy upon death. Third-instar oleander hawk moth larvae were captured, reared for one day, and then starved for 24 hours. They were then fed oleander hawk moths with PBS or PBS containing inhibitors and / or the virus.

[0121] Twenty larvae from each of the following groups were tested: PBS group, JNK inhibitor SP600125 group (35 μM), DnCPV-23 virus infection group (10^7 PIB / mL), and DnCPV-23 virus fluid infection group containing inhibitor (inhibitor concentration 35 μM, virus concentration 10^7 PIB / mL). The kill rate was calculated over 5 days.

[0122] The results are shown in Table 4. After the addition of the inhibitor, the oleander hawk moth infected with DnCPV-23 had an earlier onset time and a significantly higher kill rate. This indicates that the addition of JNK inhibitor to DnCPV-23 virus preparation is expected to improve the insecticidal effect of the virus preparation. JNK inhibitor enhances the effect of DnCPV-23 virus preparation in killing the host of cytoplasmic polyhedrosis virus.

[0123] Table 4. Effects of inhibitors on viral killing efficacy All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. The use of a JNK inhibitor in enhancing the killing effect of synergistic polyhedroviruses on host pests. in, The JNK inhibitor promotes the replication of the polyhedrosis virus (PPV) by inhibiting the JNK level in the PPV host pest, thereby enhancing the PPV's ability to kill the host pest. The cytoplasmic polyhedrovirus is a cytoplasmic polyhedrovirus belonging to the family Sphingomycetes. The sphingolithoid polyhedroviruses are selected from the following group: oleander sphingolithoid polyhedrovirus DnCPV, bean sphingolithoid polyhedrovirus, or combinations thereof; The JNK inhibitor is selected from: small molecule compounds, siRNA, or combinations thereof; The small molecule compound refers to SP600125; The siRNA consists of the sequence shown in SEQ ID NO:3 and the sequence shown in SEQ ID NO:4; The JNK inhibitor suppressed the JNK level in the host pest of the plasmonic polyhedrosis virus by more than 30%.

2. The use as described in claim 1, characterized in that, The term "killing" refers to specific lethality and / or pathogenicity.

3. The use as described in claim 1, characterized in that, The host pests of the cytoplasmic polyhedrosis virus include: oleander hawk moth, bean hawk moth, or a combination thereof.

4. The use as described in claim 1, characterized in that, For one or more of the following purposes: (Z1) Enhances the replication ability of plasmonic polyhedrosis viruses; (Z2) Improve the killing rate of polyhedrosis viruses against host pests; (Z3) Shorten the disease time of host pests in the presence of polyhedrosis viruses.

5. A compound preparation, characterized in that, The compound formulation comprises a first component and a second component; The first component includes a JNK inhibitor, and the second component includes a plasmonic polyhedrosis virus. in, The cytoplasmic polyhedrovirus is a cytoplasmic polyhedrovirus belonging to the family Sphingomycetes. The sphingolithoid polyhedroviruses are selected from the following group: oleander sphingolithoid polyhedrovirus DnCPV, bean sphingolithoid polyhedrovirus, or combinations thereof; The JNK inhibitor is selected from: small molecule compounds, siRNA, or combinations thereof; The small molecule compound refers to SP600125; The siRNA consists of the sequence shown in SEQ ID NO:3 and the sequence shown in SEQ ID NO:4; The JNK inhibitor suppressed the JNK level in the host pest of the plasmonic polyhedrosis virus by more than 30%.

6. Use of the compound formulation according to claim 5 in killing pests that are hosts of polyhedrosis viruses; in, The cytoplasmic polyhedrovirus is a cytoplasmic polyhedrovirus belonging to the family Sphingomonas. The sphingomyelid polyhedroviruses are selected from the following group: oleander sphingomyelid polyhedrovirus DnCPV, bean sphingomyelid polyhedrovirus, or combinations thereof.

7. An agricultural reagent kit, characterized in that, The agricultural reagent kit contains: The compound formulation of claim 5, and an agronomically acceptable carrier.

8. A method for protecting plants, characterized in that, Applying the compound formulation of claim 5 to the plant thereby killing pests that are hosts of the polyhedrosis virus; The plant in question is a plant of the Apocynaceae family or a legume family; The cytoplasmic polyhedrovirus is a cytoplasmic polyhedrovirus belonging to the family Sphingomycetes. The sphingomyelid polyhedroviruses are selected from the following group: oleander sphingomyelid polyhedrovirus DnCPV, bean sphingomyelid polyhedrovirus, or combinations thereof.

9. The method for protecting plants as described in claim 8, characterized in that, Includes the following sub-steps: (S1) Apply the JNK inhibitor to the plant, incubate, and (S2) Apply plasmonic polyhedrosis virus to the plants obtained in (S1) and incubate them; or (S1') Simultaneously apply JNK inhibitor and plasmopolyhedrovirus to the plant and incubate.

10. A method for screening potential synergists to enhance the killing ability of cytoplasmic polyhedrosis viruses against their host pests, characterized in that, Includes the following steps: (a) In the experimental group, cytoplasmic polyhedrovirus host cells were cultured in the presence of the test substance; and in the control group, cytoplasmic polyhedrovirus host cells were cultured under the same conditions but in the absence of the test substance. and (b) The JNK levels in the cells of the experimental group and the control group were measured and compared. If the JNK level X1 in the experimental group was significantly lower than that in the control group, it would suggest that the test substance is a potential enhancer for enhancing the killing ability of cytoplasmic polyhedrosis virus on its host pests. in, The cytoplasmic polyhedrovirus is a cytoplasmic polyhedrovirus belonging to the family Sphingomycetes. The sphingomyelid polyhedroviruses are selected from the following group: oleander sphingomyelid polyhedrovirus DnCPV, bean sphingomyelid polyhedrovirus, or combinations thereof.

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