Use of hemocytin protein and its encoding gene in preparation of medicine for preventing and treating agricultural pests

Abstract

The present application relates to Hemocytin protein and its coding gene in the application of preparing the medicine for preventing and treating agricultural pests, and belongs to the technical field of pesticides.The present application provides the application of Hemocytin protein and its coding gene as a target protein of insecticidal medicine in preparing the medicine for preventing and treating agricultural pests, uses a natural secondary product of fungi, destruxin A, as a probe, identifies the target Hemocytin protein, provides the application method of the target as an RNAi target gene for preventing and treating pests, and discloses a way of developing a new type of insect immune inhibitor acting on the target.

Description

Technical Field

[0001] This invention relates to the application of Hemocytin protein and its encoding gene in the preparation of drugs for controlling agricultural pests, belonging to the field of pesticide technology. Background Technology

[0002] Pests seriously harm agricultural production, spread diseases, and threaten human health. Pesticides play a key role in pest control. There are many types of insecticides. According to pesticide registration information from the China Pesticide Information Network, there are currently 18,650 registered insecticide products in my country (http: / / www.chinapesticide.org.cn / hysj / index.jhtml, July 8, 2022). However, the structural types of insecticides are relatively few, mainly including about 30 molecular structural types such as organophosphates, carbamates, pyrethroids, neonicotinoids, nereistoxins, diamides, and benzoylurea. Correspondingly, the targets of insecticides are also few. According to data from the International Insecticide Resistance Action Committee (IRAC), apart from a very few insecticides with unknown mechanisms of action, there are only 28 targets of existing insecticides. Moreover, most existing insecticides act on a few target proteins in the nervous system, such as acetylcholinesterase, nicotinic acetylcholine receptors, voltage-gated sodium ion channels, ryanodine receptors, and γ-aminobutyric acid receptors. Insecticides with similar structures often have the same target, which accelerates the formation of cross-resistance in pests. Therefore, the development of insecticides with novel targets is of great significance in the management of insecticide resistance in pests.

[0003] Natural product models are one of the most effective ways to create new drugs. On one hand, new active lead compounds are discovered from natural products, and through molecular optimization and activity screening, novel insecticides are developed. For example, nereistoxin-based insecticides (such as carbaryl, chlorpyrifos, and chlorfenapyr) are derived from nereistoxin, an insecticidal lead compound found in nereis; pyrethroid insecticides are developed based on pyrethrin, a lead compound found in natural pyrethrum; and the highly effective insecticide and acaricide, chlorfenapyr (Ceftriaxone), is derived from Streptomyces. Streptomyces fumanus Understanding the secondary metabolite, dioxapyrrolomycin. On the other hand, using bioactive natural products as probes to discover new targets (proteins), and designing and synthesizing a series of candidate molecules based on the interaction patterns between probes and target molecules, thereby screening out novel drugs, has greatly improved the efficiency of new drug development and has become a hot topic in current pesticide development research.

[0004] Metarhizium anisopliae Metarhizium anisopliaeMetarhizium anisopliae is an important entomopathogenic fungus widely used in pest control. The non-ribosomal peptide compound it produces, destruxin A (DA), exhibits excellent insecticidal activity. Its molecular structure differs from existing insecticide structures. It acts on the insect's immune system, suppressing immune function, and its mechanism of action is unlike any existing insecticide. Identifying the target of DA allows for the development of novel insecticides targeting this target, playing a role in managing insecticide resistance.

[0005] Domestic and international research has shown that discovering novel insecticide targets within insects is of great significance for the development of new pesticides, the management of pesticide resistance in pests, and the development of RNAi control technology for pests. The content of this application differs from existing research findings, and its disclosure will promote the development of novel pesticide creation in my country. Summary of the Invention

[0006] In order to overcome the shortcomings of the prior art, the purpose of this invention is to provide the application of Hemocytin protein and its encoding gene in the preparation of drugs for controlling agricultural pests.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is: the application of Hemocytin protein as a target protein of insecticides in the preparation of drugs for controlling agricultural pests.

[0008] More specifically, an insecticide is designed targeting the insecticide target protein, using Metarhizium anisopliae A as a probe and Hemocytin protein as a target, with Hemocytin protein forming a docking model with the molecules of Metarhizium anisopliae A.

[0009] Furthermore, the amino acid sequence of the Hemocytin protein is shown in SEQ ID NO: 1.

[0010] In addition, the present invention also provides the application of the Hemocytin gene in the preparation of drugs for controlling agricultural pests, wherein the Hemocytin gene encodes the Hemocytin protein described in the present invention.

[0011] More specifically, insecticides designed to cause the loss or reduction of Hemocytin gene expression.

[0012] More specifically, the method of using the insecticide is as follows: dsRNA is designed based on the hemocytin gene sequence of the target agricultural pest, and then the dsRNA is released into the target agricultural pest. The hemocytin gene is knocked out using the RNAi principle, which kills the pest and achieves the purpose of controlling the damage caused by the pest.

[0013] Furthermore, the nucleotide sequence of the Hemocytin gene is shown in SEQ ID NO: 2.

[0014] Preferably, the agricultural pests include Lepidoptera, Hemiptera, Diptera, Coleoptera, or Orthoptera.

[0015] Preferably, the agricultural pest is the fall armyworm, diamondback moth, oriental fruit fly, cotton aphid, tobacco whitefly, or red flour beetle.

[0016] The present invention also provides an insecticide, the preparation method of which is as follows: by using metamizole A as a probe to form a molecular docking model between Hemocytin protein and metamizole A, the drug structure is designed to guide the chemical synthesis of small molecules, the biological activity is determined, and highly active molecules are screened out, thereby obtaining the insecticide.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] (1) This invention uses the natural secondary product of fungi - destruxin A as a probe to identify its target Hemocytin protein, provides a method for using this target as an RNAi target gene to control pests, and reveals the way to develop novel insect immunosuppressants that act on this target.

[0019] (2) This invention provides the application of Hemocytin protein as a target protein for insecticides in the preparation of drugs for the control of agricultural pests. The discovery of Hemocytin protein, a novel insecticide target in insects, is of great significance in the research and development of new pesticides, the management of pesticide resistance in pests, and the development of RNAi control technology for pests. The disclosure of the contents of this application has significant reference value for promoting the development of new pesticide creation in my country. Attached Figure Description

[0020] Figure 1 A represents the molecular structure of Metarhizium anisopliae A. Figure 1 B represents the electrophoretic bands of silkworm hemolymph treated with DARTS after treatment with Metarhizium anisopliae A;

[0021] Figure 2 A represents the peptide chain structure of hemocytin from the silkworm and its prokaryotic expression fragment. Figure 2 B represents the SPR analysis of the affinity of each fragment to DA;

[0022] Figure 3 For the coagulation test of 4th instar silkworm larvae; among them, Figure 3 Group A represents the group treated with Metarhizium anisopliae (A). Figure 3 B is the control group. Figure 3 C. RNAi-treated group;

[0023] Figure 4 Metarhizin A affects hemocytin-mediated hemocyte immunity in silkworm larvae;

[0024] Figure 5 The effect of morpholine A on the affinity of hemocytin for its three interacting proteins; among which... Figure 5 A. Hemocytin-interacting proteins isolated using the pull-down technique. Figure 5 B. To determine the effect of DA on the affinity of hemocytin for interacting proteins using the SPR method, the final concentration of DA was 1.0 μg / g body weight.

[0025] Figure 6 Analysis of the hemocytin peptide chain domains in several insect species;

[0026] Figure 7 The simulation results of amino acid fragments 1-600 of hemocytin in the silkworm and their docking with Metarhizium anisopliae A molecules on the Alphafold supercomputing system; among them, Figure 7 A is the overall structure diagram. The upper left corner shows the interaction surface between methiocarbazin A (purple-red) and the protein. Figure 7 B is a key residue in the interaction between metamizole A and hemocytin. Detailed Implementation

[0027] To better illustrate the purpose, technical solution, and advantages of this invention, the invention will be further described below with reference to specific drawings and embodiments. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0028] Firstly, the application of Hemocytin protein as a target protein for insecticides in the preparation of drugs for controlling agricultural pests.

[0029] More specifically, an insecticide is designed targeting the insecticide target protein, using Metarhizium anisopliae A as a probe and Hemocytin protein as a target, with Hemocytin protein forming a docking model with the molecules of Metarhizium anisopliae A.

[0030]

[0031]

[0032] Secondly, the present invention also provides the application of the Hemocytin gene in the preparation of drugs for controlling agricultural pests, wherein the Hemocytin gene encodes the Hemocytin protein described in the present invention.

[0033] More specifically, insecticides designed to cause the loss or reduction of Hemocytin gene expression.

[0034] More specifically, the method of using the insecticide is as follows: dsRNA is designed based on the hemocytin gene sequence of the target agricultural pest, and then the dsRNA is released into the target agricultural pest. The hemocytin gene is knocked out using the RNAi principle, which kills the pest and achieves the purpose of controlling the damage caused by the pest.

[0035] Furthermore, the nucleotide sequence of the Hemocytin gene is shown in SEQ ID NO: 2:

[0036]

[0037] Preferably, the agricultural pests include Lepidoptera, Hemiptera, Diptera, Coleoptera, or Orthoptera.

[0038] Preferably, the agricultural pest is the fall armyworm, diamondback moth, oriental fruit fly, cotton aphid, tobacco whitefly, or red flour beetle.

[0039] The present invention also provides an insecticide, the preparation method of which is as follows: by using metamizole A as a probe to form a molecular docking model between Hemocytin protein and metamizole A, the drug structure is designed to guide the chemical synthesis of small molecules, the biological activity is determined, and highly active molecules are screened out, thereby obtaining the insecticide.

[0040] To explain the technical solution of the present invention more clearly and in more detail, some embodiments are provided below for further illustration.

[0041] Example 1: Discovery and validation of a novel insecticide target – hemocytin

[0042] (1) Discovery of Hemocytin as a target of Metarhizium anisopliae A

[0043] Experimental Methods: The target molecule of Metarhizium anisopliae A was isolated and identified using the DARTS (drug affinity responsive target stability) method. DA was first prepared as a 10000 mg / L stock solution with dimethyl sulfoxide (DMSO). Before injection, the solution was diluted with an appropriate amount of Grace insect cell culture medium, and then 1 μL was injected into the hemocoel of 4th instar Bombxy mori larvae. The final concentrations of DA were set at 2.5 and 0.5 μg / g body weight. In the control group, each silkworm was injected with 1 μL of DMSO. The mixture was incubated at 25°C for 1 h, and then blood was collected to separate total hemolymph proteins. Subtilisin (S), streptoprotein (P), proteinase K (PK), thermolysin (T), and elastase (E) were used to treat total protein samples from hemolymphatic cells, with the treatment repeated four times. Samples treated with each protease were subjected to gel electrophoresis to separate specific bands. The specific bands were then cut and the proteins within them were isolated. The proteins were identified by mass spectrometry.

[0044] Experimental results: Electrophoresis revealed 39 specific bands ( Figure 1Mass spectrometry identified 58 proteins from these bands, among which hemocytin had the highest repetition rate, appearing in all 5 protease treatments and 15 times in a total of 39 samples, far exceeding other proteins (Table 1). This result preliminarily confirms that hemocytin is a target protein of Metarhizium anisopliae A.

[0045] Experimental Example 2: Validation of Hemocytin as a target of Metarhizium anisopliae A

[0046] The full-length peptide chain of the silkworm hemocytin protein is 3133aa. This protein is a multifunctional protein that is mainly involved in insect coagulation and hemolymphatic immune processes, but its specific functions and mechanisms have not yet been fully understood.

[0047] To further demonstrate that hemocytin is a target of DA, this invention conducted in vivo and in vitro experiments on silkworms and determined the interaction affinity between hemocytin homologs and DA in other important pests.

[0048] First, we verified the interaction between hemocytin and DA using in vitro experiments. We expressed, isolated, and purified all the proteins identified by the DARST method using a prokaryotic expression approach. Then, we determined the affinity coefficients K between these proteins and DA using surface plasmon resonance (SPR) technology. D (Table 1). Based on domain analysis, three fragments (F1, F2, and F3) of silkworm hemocytin were expressed and purified in prokaryotes. Figure 2 A), whose affinity coefficient K D The value is 2-8 μM ( Figure 2 (B) This value is much lower than the affinity coefficients of other proteins with DA (Table 1), indicating that hemocytin has the strongest affinity for DA.

[0049] Furthermore, we verified that hemocytin is a target protein of DA in silkworms by detecting coagulation. The experiment used fourth-instar silkworm larvae and included three groups: a DA treatment group, in which 1 μL of DA solution was injected into the hemocoel of fourth-instar larvae daily, with final concentrations of 0.01, 0.1, and 1.0 μg / g body weight; an RNAi group, in which in vitro synthesized dsRNA was injected into the hemocoel of fourth-instar larvae at a volume of 1 μL per larva; and a control group, in which each silkworm was injected with an equal volume of DMSO. The experiment was repeated three times. Twenty-four hours after injection, silkworm hemolymph was collected and placed on a glass slide for observation of coagulation under a stereomicroscope.

[0050] The results showed that the hemolymph in the control group immediately showed signs of clotting on the glass slide, and obvious flocculent aggregates were visible under the microscope. Figure 3), while DA processing group ( Figure 3 A) and RNAi group ( Figure 3 C) No coagulation phenomena were observed (Table 2, Figure 3 ).

[0051] Table 1. Proteins isolated and identified from silkworm hemolymph using the DARTS method and their affinity for DA.

[0052]

[0053]

[0054]

[0055] Table 2 Results of coagulation test on 4th instar silkworm larvae

[0056]

[0057] This invention also used immunofluorescence technology to verify that hemocytin is a target protein of DA in silkworms. First, it was demonstrated that hemocytin responds to the invasion of Metarhizium anisopliae. Using fourth-instar silkworm larvae as material, three treatment groups were established: Group A, injected only with 10... 7 Spores / mL Metarhizium anisopliae M. anisopliae One μL of conidial suspension was injected into the hemocoel of silkworms. Group B received the same treatment as Group A, but with the additional injection of hemocytin into the hemocoel, with a final concentration of 700 nM. Group C received the same treatment as Group B, but with the additional injection of DA into the hemocoel, with a final DA concentration of 1.0 μg / g body weight. The experiment was repeated three times. After a certain period following injection, silkworms treated with group A were sectioned and stained with fluorescent dye.

[0058] The results showed that hemocytin is involved in the response process of silkworms to Metarhizium anisopliae invasion. Figure 4 A), including mediating hemocyte recognition and cystation of *Metarhizium anisopliae* spores, forms a coagulation barrier on the epidermis to prevent the invasion of more spores. Simultaneously, blood smears were prepared from silkworms in each treatment group and observed using immunofluorescence staining. The results showed that in the *Metarhizium anisopliae* treatment group without DA and hemocytin (Group A) and in Group B with hemocytin and *Metarhizium anisopliae* injection, hemocyte cystation was significant, and hemocytin (green) formed a distinct fibrous structure. Figure 4 B); while in group C, cysts appeared only at the beginning (24 h after treatment), and later the cysts disappeared, blood cells dissolved, and hemocytin did not form fibrous structures. Figure 4 B).

[0059] This invention further isolated three interacting proteins of silkworm hemocytin using pull-down technology: ActinA3 (NP_001119726.1), Gelsolin (XP_021205043.2), and Attacin-like (NP_004926758.1), and used SPR technology to determine the effect of DA on their interaction with hemocytin. The results showed that DA significantly reduced the affinity between hemocytin and these three interacting proteins. Figure 5 ).

[0060] Table 3. SPR detection of hemocytin-DA interaction in different insects.

[0061]

[0062] This invention also determined the affinity of hemocytin to DA in other insects. Among Lepidoptera, Hemiptera, Diptera, Coleoptera, and Orthoptera insects and silkworms, the homology of hemocytin was 20%-60%, and all of them had structural domains such as TIL, VWD, C8, and FA58C, indicating that their functions were similar. Figure 6 SPR experimental results show that the fall armyworm... Spodoptera frugiperda Diamondback moth Plutella xylostella citrus fruit fly Bactrocera dorsalis cotton aphids Aphis gossypii whiteflies Bemisia tabaci and Red-eared Valley Beetle Tribolium castaneum Hemocytin in pests such as DA has a strong affinity for DA, and its K D The values ​​ranged from 9 to 35 μM (Table 3).

[0063] Experimental Example 3: Pest Control Technology and Application Based on Hemocytin Gene RNAi

[0064] The gene sequence of the target pest hemocytin was retrieved from the gene database. Primers for dsRNA of various pests were designed (Table 4). dsRNA was synthesized and purified. Quality-tested dsRNA products were used for bioassays. The synthesized dsRNA was injected into the target pests using a microsyringe, with 0.1–1 µL injected per insect. DMSO injection and no treatment were used as controls. Each treatment consisted of 30 insects, with three replicates. The mortality rate of the pests was checked after a certain period following treatment, and the corrected mortality rate was calculated.

[0065] Gene expression assay: 24 h after DA injection, total RNA was extracted using a rapid total RNA extraction kit. The tissue sample was ground with liquid nitrogen, 500 μL of lysis buffer was added and vortexed, followed by 200 μL of chloroform. The sample was centrifuged at 12000 rpm for 15 min at 4°C. The supernatant was collected and 70% anhydrous ethanol was added. All the liquid was then added to the RNA column in the collection tube and centrifuged at 10000 rpm for 1 min. The filtrate was discarded. The RNA column was washed with 500 μL RNA Wash Buffer 1 and 2, respectively, and finally eluted with 70 μL DEPC water. After passing quality checks, cDNA was synthesized using this as a template according to the reverse transcription kit instructions. The reaction mixture consisted of 10 μL RNA + 1 μL reverse transcriptase + 1 μL reverse transcription primer + 2 μL reaction buffer + 6 μL deionized water. Reverse transcription was performed at 55°C for 1 h. The relative expression level of the target gene was determined using quantitative real-time RT-qPCR. The insect GAPDH gene (glyceraldehyde-3-phosphate dehydrogenase) was used as an internal reference gene. The qPCR reaction system was as follows: 1 μL cDNA, 1 μL each of forward and reverse primers, 10 μL qPCR SuperMix, and 7 μL nucleic acid-free water. The reaction program was: 94 ℃ pre-denaturation for 30 s, 94 ℃ denaturation for 5 s, 60 ℃ annealing for 15 s, 72 ℃ extension for 10 s, 39 cycles followed by 95 ℃ for 10 s, and 65 ℃ to 95 ℃ for 5 s.

[0066] The results are shown in Table 5. After knocking out the hemocytin gene using RNA interference technology, the mortality rate of pests reached 65.47-85.64% at 24 hours and as high as 100% at 72 hours.

[0067] Table 4. RNAi primers for the hemocytin gene

[0068]

[0069] Table 5. Results of RNA interference experiments on the hemocytin gene in different insects.

[0070]

[0071] Experiment Example 4: Screening and Application of Novel Insecticides Based on the Interaction Mode of DA and Hemocytin

[0072] Protein structure analysis was performed using the Alphafold supercomputing system to simulate the 3D structure of hemocytin, and molecular docking was performed using molecular manipulation software (such as MOE (Molecular Operating Environment), Chemical Computing Group Inc., Montreal, QC, Canada). Figure 7 This study demonstrates the molecular docking model of hemocytin residues 1-600 in the silkworm and its interaction with DA. Using pharmacophore analysis, structure-based drug design was conducted based on the hemocytin-DA molecular docking model to guide the chemical synthesis of small molecules. Bioactivity assays were performed to screen for highly active molecules, ultimately leading to the development of novel insecticides targeting hemocytin.

Claims

1. Use of hemocytin gene in the preparation of a medicine for preventing and treating agricultural pests, characterized in that, The Hemocytin gene encodes the Hemocytin protein. Insecticides designed to cause the Hemocytin gene to be lost or reduced are used to target the Hemocytin gene. The method of using the insecticide is as follows: dsRNA is designed based on the hemocytin gene sequence of the target agricultural pest, and then the dsRNA is released into the target agricultural pest to knock out the hemocytin gene using the principle of RNAi. The nucleotide sequence of the Hemocytin gene is shown in SEQ ID NO: 2, and the target agricultural pest is the silkworm; or the Hemocytin gene ID is XM_035580296.1, and the target agricultural pest is the fall armyworm; or the Hemocytin gene ID is XM_048623765.1, and the target agricultural pest is the diamondback moth; or the Hemocytin gene ID is XM_011206389.3, and the target agricultural pest is the oriental fruit fly; or the Hemocytin gene ID is XM_027980777.1, and the target agricultural pest is the cotton aphid; or the Hemocytin gene ID is XM_019046539.1, and the target agricultural pest is the whitefly; or the Hemocytin gene ID is XM_015983564.1, and the target agricultural pest is the red flour beetle.

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