A combined preparation of a polypeptide toxin and a chemical pesticide, and its preparation method and application

Abstract

The present invention discloses a combined preparation of a polypeptide toxin and a chemical pesticide, its preparation method and application. The combined preparation includes the toxin PPTX-22 from Pardosa pseudoannulata and chlorantraniliprole. The amino acid sequence of the toxin PPTX-22 from Pardosa pseudoannulata is shown as SEQ ID NO.1. By analyzing the similarities and differences in the action mechanisms of the spider toxin PPTX-22 acting on the insect sodium channel and the pyrethroid insecticide chlorantraniliprole, and testing the combined action mechanism of the spider toxin and the pyrethroid insecticide, it is found that the action site of the toxin PPTX-22 is specific, and it synergistically regulates the insect sodium channel with chlorantraniliprole, forming synergism at the target level. Based on this, a toxin-insecticide combined preparation with enhanced efficacy against agricultural pests is obtained.

Description

Technical Field

[0001] The present invention relates to pest control, and in particular to a combined preparation of a polypeptide toxin and a chemical pesticide, and a preparation method and application thereof. Background Art

[0002] Spider toxins are excellent insecticides with good insecticidal activity and strong selective toxicity. Currently, no spider toxins that act on the ryanodine receptor in insects have been reported. The ryanodine receptor, named for its high affinity for ryanodine, is a receptor that controls intracellular Ca 2+ The released ligand-gated calcium channels are primarily located in the sarcoplasmic reticulum of muscle cells and the endoplasmic reticulum of nerve cells. Diamide insecticides, represented by chlorantraniliprole, are allosteric modulators of insect ryanodine receptors. They act by binding to the tetrameric ryanodine receptors in target insect muscle cells, causing the receptors to allosterically bind to them and open calcium channels. Intracellular calcium ions rapidly enter the cytoplasm through the sarcoplasmic / endoplasmic reticulum membranes, causing sustained muscle contractions in the insects, followed by paralysis and lethargy, immediate cessation of feeding, and eventual death. Since the launch of the first diamide insecticide, flubendiamide, in 1998, this class of insecticides has attracted widespread research interest due to its unique structure-activity relationship, and eight varieties have been marketed to date. Although chlorantraniliprole and flubendiamide in this class exhibit excellent insecticidal activity against lepidopteran pests, resistance is also becoming increasingly common. In 2008, reports in the Philippines and Thailand of a 775-fold resistance to chlorantraniliprole in diamondback moth were released.

[0003] At present, at least 9 species of Lepidoptera pests in different countries and regions around the world have developed different levels of resistance to flubendiamide or chlorantraniliprole. The emergence of resistance seriously threatens the service life of diamide insecticides such as chlorantraniliprole. Insecticide mixtures have the advantages of low investment and quick results in improving the control effect of pests and delaying the development of pest resistance. Insecticide mixtures usually involve mixing two or more insecticides of different types and modes of action in a certain proportion to achieve the purposes of expanding the control range, enhancing insecticidal activity, improving the control effect, and delaying and managing the resistance of pests. In the mixtures for controlling Lepidoptera pests, considering the differences and complementarities in the action mechanisms of single insecticides, they mainly include the mixtures between organophosphates and pyrethroids, between organophosphates and neonicotinoids, between diamides and macrolides, between diamides and neonicotinoids, etc. Only sporadic reports have been made on other types of mixtures. Regarding the research on bioactive macromolecular insecticidal components, it mainly focuses on aspects such as monomer insecticidal activity, insecticidal mechanism, and application demonstration. There is no report on the mixture test and application of polypeptide toxins and chemical insecticides. At the same time, due to the need to avoid the negative impact of two insecticides acting on the same target in the development of target mutation resistance, there is no reported mixture method of insecticides acting on different sites of the same target. Summary of the Invention

[0004] Object of the Invention: Aiming at the deficiencies in the prior art, the present invention provides a combined preparation of polypeptide toxin and chemical pesticide, the main components of which are the toxin PPTX-22 from Pardosa pseudoannulata and chlorantraniliprole. The mixture insecticide provided by the present invention is a combination of polypeptide toxin and chemical insecticide, which can be efficiently used for the control of major agricultural pests and sanitary pests, and solve problems in pest control such as the rapid development of pest resistance to chemical insecticides, the excessive use of chemical insecticides, and the low insecticidal activity of single active ingredients.

[0005] The present invention also provides a preparation method and application of the above combined preparation.

[0006] Technical Solution: To achieve the above object, a combined preparation according to the present invention, the toxin biological preparation includes the toxin PPTX-22 from Pardosa pseudoannulata and chlorantraniliprole, and the toxin PPTX-22 from Pardosa pseudoannulata is a mature peptide, and its amino acid sequence is as shown in SEQ ID NO.1.

[0007]

[0008] Among them, the molar ratio of the toxin PPTX-22 from Pardosa pseudoannulata to chlorantraniliprole is 1:5 - 5:1.

[0009] Preferably, the molar ratio of the Pardosa pseudoannulata toxin PPTX-22 to chlorantraniliprole is 1:5, 1:3 or 3:1.

[0010] The preparation method of the combined preparation of the present invention includes the following steps:

[0011] (1) Prepare the Pardosa pseudoannulata toxin PPTX-22;

[0012] (2) Dissolve chlorantraniliprole in dimethyl sulfoxide, and dissolve the Pardosa pseudoannulata toxin PPTX-22 in PBS buffer; mix the prepared mother liquors of chlorantraniliprole and PPTX-22 according to a ratio and dilute to prepare a series of concentration gradients.

[0013] Among them, in step (1), the Pardosa pseudoannulata toxin PPTX-22 is prepared by induced expression and purification or directly synthesized artificially according to the amino acid sequence.

[0014] Among them, in step (2), the technical scheme is to dissolve the technical chlorantraniliprole technical in dimethyl sulfoxide to prepare a mother liquor with a concentration of 100 mM; dissolve the toxin PPTX-22 in PBS buffer with pH = 7.4 to prepare a mother liquor with a concentration of 10 mM; mix the prepared mother liquors of chlorantraniliprole and PPTX-22 according to a series of ratios and dilute with PBS with pH = 7.4 to prepare a series of concentration gradients.

[0015] Application of the combined preparation of the present invention in the control of agricultural pests and sanitary pests.

[0016] Preferably, the agricultural pests and sanitary pests include Mythimna separata, Spodoptera frugiperda, Plutella xylostella, Chilo suppressalis, Spodoptera exigua, Cnaphalocrocis medinalis, Ectropis obliqua, Carposina sasakii, Ostrinia furnacalis, Agrotis ypsilon.

[0017] Among them, the combined preparation controls crop pests by spraying on plant leaves and / or the body surface of pests.

[0018] Application of the Pardosa pseudoannulata toxin PPTX-22 and chlorantraniliprole of the present invention in the preparation of a toxin-insecticide combined preparation having a significant synergistic effect on agricultural pests and sanitary pests.

[0019] The combined preparation provided by the present invention is used for the control of major agricultural pests and sanitary pests. Examples of pests include, but are not limited to: Mythimna separata, Spodoptera frugiperda, Plutella xylostella, Chilo suppressalis, Spodoptera exigua, Cnaphalocrocis medinalis, Ectropis obliqua, Carposina sasakii, Ostrinia furnacalis, Agrotis ypsilon, etc.

[0020] Mechanism of action: In this invention, the mechanism of the combined action of the toxin PPTX-22 from Pardosa pseudoannulata and chlorantraniliprole was studied through electrophysiological experiments. The results showed that PPTX-22 could induce an increase in the intracellular calcium ion concentration in Mythimna separata nerve cells, and blocking the calcium channels on the cell membrane with CdCl2 could not prevent the increasing trend of the intracellular calcium ion concentration, indicating that PPTX-22 does not act on the calcium channels on the cell membrane( Figure 1 ). Blocking the IP3R calcium channels on the endoplasmic reticulum in Mythimna separata nerve cells with 2-APB could not affect the increasing trend of the intracellular calcium ion concentration caused by PPTX-22, indicating that PPTX-22 does not act on the IP3R receptors on the endoplasmic reticulum( Figure 2 ). Blocking the ryanodine receptors on the endoplasmic reticulum in Mythimna separata nerve cells with a high concentration of ryanodine almost completely inhibited the increase in the intracellular calcium ion concentration caused by PPTX-22, indicating that PPTX-22 acts on the ryanodine receptors on the endoplasmic reticulum( Figure 3 ).

[0021] This invention further analyzed whether the toxin PPTX-22 and chlorantraniliprole act on the same binding sites on the insect ryanodine receptors. Figure 4 The results showed that when pretreated with PPTX-22 and then chlorantraniliprole was added, the previously added PPTX-22 had an impact on the increase in the calcium ion concentration in Mythimna separata nerve cells caused by the subsequently added chlorantraniliprole, but could not completely inhibit it. Vice versa, when pretreated with chlorantraniliprole and then PPTX-22 was added, the previously added chlorantraniliprole also had an impact on the increase in the calcium ion concentration in Mythimna separata nerve cells caused by the subsequently added PPTX-22, but could not completely inhibit it. This indicates that the binding sites of PPTX-22 and chlorantraniliprole on the insect ryanodine receptors overlap but are not identical, resulting in synergism at the target level.

[0022] This invention also studied the activity of different ratios of PPTX-22 and chlorantraniliprole on Mythimna separata nerve cells through electrophysiological experiments. Figure 5 The results showed that when the total concentration of the mixture was 6.5 μM, different ratios of PPTX-22 and chlorantraniliprole had different activities on the increase in the calcium ion concentration in Mythimna separata nerve cells, and some ratios had an obvious increasing effect (different lowercase letters in the figure indicate significant differences at the 0.05 level). Figure 6 The results showed that when the ratio of PPTX-22 and chlorantraniliprole in the mixture was 1:5, 1:3, and 3:1, the median effective concentration (EC 50 ) of the combination on the inhibition of the activity of Mythimna separata nerve cells was significantly lower than the EC 50 when using the toxin PPTX-22 alone (i.e., 1:0) and chlorantraniliprole alone (i.e., 0:1).(Different lowercase letters in the figure indicate significant differences at the 0.05 level). Based on the above results, when the combination of the toxin PPTX-22 and chlorantraniliprole proposed by the present invention is at the optimal ratio, its bioactivity against the target is significantly higher than that of the two single agents.

[0023] By analyzing the similarities and differences in the action mechanisms of the spider toxin PPTX-22 acting on the insect sodium ion channel and the pyrethroid insecticide chlorantraniliprole, and testing the combined action mechanism of the spider toxin and pyrethroid insecticides, the present invention found that the action site of the toxin PPTX-22 is specific, and it synergistically regulates the insect sodium ion channel with chlorantraniliprole, forming synergism at the target level. Based on this, a toxin-insecticide combined preparation with enhanced efficacy against agricultural pests was obtained. The present invention not only efficiently utilizes the toxin PPTX-22, but also discovers that PPTX-22 and chlorantraniliprole act on the same target but at different sites. For the first time, the same target insecticides are compounded. Generally, the compounding of two chemical insecticides needs to avoid acting on the same target. When two chemical insecticides or pesticides act on the same site and are used simultaneously, the two do not exert their effects simultaneously because there is no opportunity to attack the site, and the site is occupied by the highly toxic pesticide, so the low-toxicity one cannot show its toxicity. However, although the polypeptide toxin and the chemical pesticide provided by the present invention act on the same target, they act on different sites, so the two components can attack the site simultaneously without interfering with each other; and the simultaneous attack results in the superposition of effects, forming synergism.

[0024] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0025] (1) The present invention first discovered the spider toxin PPTX-22 acting on the insect ryanodine receptor, which synergistically regulates the insect ryanodine receptor with chlorantraniliprole, forming synergism at the target level. Based on this, a toxin-insecticide combined preparation with significant synergistic effect on pests is provided. The control effect of this combined preparation on pests is significantly better than that of the two single agents, reducing the usage amount of insecticides, lowering the control costs of various agricultural pests and sanitary pests, and playing an important role in pest resistance control.

[0026] (2) The combined preparation of the present invention is a compound of a polypeptide toxin and a chemical insecticide, and the compounding method of insecticides acting on the same target. Its median lethal concentration (LC 50 ) is significantly lower than that of the two single agents of PPTX-22 and chlorantraniliprole, and the co-toxicity coefficient of the compounding is 194-281 (more than 120 indicates synergism).

[0027] (3) The combined preparation of the present invention is simple to prepare, convenient to use, has little harm to plants, humans and the environment, and can be widely used in the field. Description of the Drawings

[0028] Figure 1 The activity of PPTX-22 on the calcium channels in the cell membrane of Mythimna separata nerve cells.

[0029] Figure 2 The activity of PPTX-22 on the IP3R calcium channels in the endoplasmic reticulum of Mythimna separata nerve cells.

[0030] Figure 3 The activity of PPTX-22 on the ryanodine receptors in the endoplasmic reticulum of Mythimna separata nerve cells.

[0031] Figure 4 The synergistic regulation mechanism of PPTX-22 and chlorantraniliprole on ryanodine receptors.

[0032] Figure 5 The activity of different ratios of PPTX-22 and chlorantraniliprole on Mythimna separata nerve cells.

[0033] Figure 6 The median inhibitory concentration EC50 value of different ratios of PPTX-22 and chlorantraniliprole on the activity of Mythimna separata nerve cells.

[0034] Figure 7 The synergistic coefficient of different ratios of PPTX-22 and chlorantraniliprole on the toxicity to Mythimna separata.

[0035] Figure 8 The synergistic coefficient of different ratios of PPTX-22 and chlorantraniliprole on the toxicity to Spodoptera frugiperda.

[0036] Figure 9 The synergistic coefficient of different ratios of PPTX-22 and chlorantraniliprole on the toxicity to Plutella xylostella.

[0037] Figure 10 The synergistic coefficient of different ratios of PPTX-22 and chlorantraniliprole on the toxicity to Chilo suppressalis.

[0038] Figure 11 The synergistic coefficient of different ratios of PPTX-22 and chlorantraniliprole on the toxicity to Spodoptera exigua.

[0039] Figure 12 The synergistic coefficient of different ratios of PPTX-22 and chlorantraniliprole on the toxicity to Cnaphalocrocis medinalis.

[0040] Figure 13 The synergistic coefficient of different ratios of PPTX-22 and chlorantraniliprole on the toxicity to Ectropis obliqua hypulina Wehrli.

[0041] Figure 14 The synergistic coefficient of different ratios of PPTX-22 and chlorantraniliprole on the toxicity to Carposina sasakii Matsumura.

[0042] Figure 15 The synergistic coefficient of the compound agents of PPTX-22 and chlorantraniliprole at different ratios against Ostrinia furnacalis

[0043] Figure 16 The synergistic coefficient of the compound agents of PPTX-22 and chlorantraniliprole at different ratios against Agrotis ypsilon Specific implementation manners

[0044] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0045] Example 1

[0046] 1. Test agents

[0047] The preparation method of the spider toxin PPTX-22 with Pardosa pseudoannulata reference is Example 1 in Patent 201810868357.X, or it can be directly synthesized artificially according to its amino acid sequence. The chlorantraniliprole technical was purchased from Sigma-Aldrich (St. Louis, MO, USA).

[0048] Specific preparation process:

[0049] Preparation of the toxin

[0050] Design and construction of the spider toxin gene

[0051] Based on the nucleotide sequence of the insecticidal gene PPTX-22 of Pardosa pseudoannulata shown as SEQ ID NO.2, and the amino acid sequence of the encoded protein shown as SEQ ID NO.3, its signal peptide, propeptide and mature peptide were predicted by SpiderP (http: / / www.arachnoserver.org / spiderP.html). The mature peptide sequence was codon-optimized according to the codon preference of Escherichia coli, and the PPTX-22 toxin gene sequence for expression in Escherichia coli was designed as shown in SEQ ID NO.4. The designed gene was synthesized by Invitrogen and sequenced by Nanjing Genscript. The synthesized gene was cloned into the Escherichia coli expression vector pET-32a(+), and the recombinant plasmid pET-32a(+)-PPTX-01 containing the target gene was constructed. Restriction enzyme cleavage sites of BanH I and EcoR I were introduced into this toxin gene.

[0052] Induced expression of the recombinant plasmid pET-32a(+)-PPTX-01

[0053] The recombinant plasmid pET-32a(+)-PPTX-02 was transformed into Escherichia coli BL21 strain. After growing on an LB plate containing carbenicillin for 16 h, different positive colonies were picked and added into LB liquid medium containing 100 mg / L carbenicillin respectively. They were cultured in a constant temperature shaker at 250 r / min and 37 °C for 12 h. The cultured bacterial liquid was inoculated into LB liquid medium containing 100 mg / L carbenicillin according to a volume ratio of 1:100, and cultured at 200 r / min and 37 °C for 4 h. At this time, the OD value of the bacterial liquid was 0.5 - 0.7; IPTG with a final concentration of 0.4 mmol / L was added into the pET-32a(+)-PPTX-01 recombinant, and it was induced for expression and cultured at 200 r / min and 37 °C for 5 h; 4 mL of the expressed bacterial liquid was taken, centrifuged at 12000 rpm for 5 min at 4 °C, the supernatant was discarded, 1.5 mL of 20 mM Tris-HCl, pH = 7.4 was added to the bacterial cell precipitate, resuspended and then sonicated to lyse the bacteria, with an intensity of 12%, for 10 min, running for 5 s and pausing for 5 s; after lysis, it was centrifuged at 18000 rpm for 10 min at 4 °C, the supernatant was taken and the precipitate was discarded, which was used for the detection of recombinant toxin.

[0054] Western Blot Detection

[0055] Take 50 μL of the lysate, add 17 μL of Loading Buffer and 2 μL of β-mercaptoethanol, boil at 100 °C for 5 min, add 20 μL of the mixture to the SDS-polyacrylamide gel, and add 10 μL of protein standard sample, run at 50 V for 50 min, then at 100 V until the loading buffer reaches the bottom of the gel; after electrophoresis, take out the gel and put it into the transfer buffer, soak the PVDF membrane in methanol for 1 min and then transfer it into the transfer buffer, and then perform transfer at 100 mA for 50 min; after transfer, take out the membrane, wash it 3 times with PBST, 5 min each time, block it with blocking solution for 2 h at 37 °C and 80 r / min; after blocking, take out the membrane, wash it 3 times with PBST, 10 min each time, transfer it into the blocking solution containing primary antibody (1:1000), incubate for 2 h at 37 °C and 80 r / min; after incubation with primary antibody, take out the membrane, wash it 3 times with PBST, 10 min each time, transfer it into the blocking solution containing secondary antibody (1:2000), incubate for 1.5 h at 37 °C and 80 r / min; after incubation with secondary antibody, wash it 3 times with PBST, 10 min each time, add luminescent solution, keep it in the dark for 3 min, dry the surface liquid with filter paper, and then perform chemiluminescence detection.

[0056] Large-scale Induced Expression of Recombinant Plasmid pET-32a(+)-PPTX-22

[0057] The cultured bacterial solution was inoculated into 100 mL of LB liquid medium containing 100 mg / L carbenicillin at a volume ratio of 1:100. The volume of the medium should not exceed 20% of the container volume. It was cultured at 200 r / min and 37 °C for 4 h. At this time, the OD value of the bacterial solution was 0.5 - 0.7. IPTG with a final concentration of 0.4 mmol / L was added to the pET-32a(+)-PPTX-22 recombinant, and it was induced and expressed at 200 r / min and 37 °C for 5 h. The expressed bacterial solution was taken, centrifuged at 12,000 rpm for 15 min at 4 °C, the supernatant was discarded, and 20 mM Tris-HCl with a volume of 20% of the medium volume (pH = 7.4) was added to the cell pellet. After resuspension, cell disruption was performed by ultrasonic treatment with an intensity of 12%, for 30 min, running for 5 s and pausing for 5 s. After cell disruption, it was centrifuged at 18,000 rpm for 30 min at 4 °C, the supernatant was taken, the precipitate was discarded, and the crude protein solution after disruption was used for the separation and purification of the recombinant toxin.

[0058] Purification of Recombinant Toxin PPTX-22

[0059] The crude protein solution after a large amount of induced expression and cell disruption was filtered through a 0.22 μm filter membrane, and purified using an AKTA avant automatic protein separation and purification system and a HisTrapTM HP nickel column (5 ml). First, the nickel column was equilibrated with 5 column volumes of binding buffer at a flow rate of 5 mL / min, the sample loading flow rate was 1 mL / min. After sample loading, the column was washed with 6 column volumes of binding buffer at a flow rate of 1 mL / min. Finally, the target protein was eluted with 3 column volumes of elution buffer at a flow rate of 5 mL / min. The yield of the purified protein was approximately 7.85 mg / L. The purified protein was the mature peptide, namely recombinant toxin PPTX-022, and its amino acid sequence was as shown in SEQ ID NO.1.

[0060] 2. Preparation Method of the Combined Preparation

[0061] Preparation of the combined preparation: The original chlorantraniliprole drug was dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution with a concentration of 100 mM; the toxin PPTX-22 was dissolved in PBS buffer with pH = 7.4 to prepare a stock solution with a concentration of 10 mM; the prepared stock solutions of chlorantraniliprole and PPTX-22 were mixed in a series of ratios such that the molar ratio of the toxin PPTX-22 of Pardosa pseudoannulata and chlorantraniliprole was 1:5 - 5:1, and diluted with PBS (pH = 7.4) to prepare a series of concentration gradients, and the total concentration range of the toxin and the pesticide was 0.5 - 2000 μM.

[0062] Example 2

[0063] 1. Test and Data Processing Method for the Activity of PPTX-22 and Chlorantraniliprole Acting on the Insect Ryanodine Receptor

[0064] After the 3rd instar larvae of Mythimna separata were anesthetized with 70% alcohol at room temperature, the nerve cords of the thorax and abdomen were dissected out, placed in physiological saline, and incubated at 28 °C for 6 minutes after adding 0.3% trypsin. The nerve cords were transferred to a petri dish, 1 mL of L-15 Leibovitz culture medium and 15% fetal bovine serum were added, and incubated at 28 °C for 2 h. Then, the nerve cells on the body wall in the petri dish were collected for subsequent tests. The nerve cells were incubated in physiological saline (NaCl 150 mM, KCl 4 mM, MgCl2 2 mM, CaCl2 2 mM, HEPES 10 mM, pH = 7.0) containing fluo-3AM (10 μM) at 28 °C for 30 min, then washed twice with physiological saline and transferred to the extracellular fluid for detection. The composition of the extracellular fluid was: NaCl 150 mM, KCl 4 mM, MgCl2 2 mM, EGTA 2 mM, Hepes 10 mM, pH = 7.0. The test insecticide or toxin was added to the extracellular fluid, and the fluorescence signal was recorded with an inverted fluorescence microscope Fluor 40×oil immersion objective (Olympus IX71) 3 min later. The excitation wavelength of the fluorescence signal was 488 nm, and the recorded signal was processed with a CCD (Image Pro-6.0). The fluorescence value F after adding the agent and the fluorescence value F0 without adding the agent were recorded, and F / F0 (%) was calculated as the activity index of the agent acting on the ryanodine receptor to trigger calcium ion release.

[0065] 2. Conclusion

[0066] According to the above experiments, the effects and activities of chlorantraniliprole, toxin PPTX-22, and the combination of toxin-chlorantraniliprole on the ryanodine receptor in the nerve cells of Mythimna separata were tested and analyzed, and the results are as Figures 1-6 shown.

[0067] From Figure 1 it can be seen that when adding 50 mg / L PPTX-22 and 1 mg / L CdCl2 at the final concentration, PPTX-22 can induce an increase in the intracellular calcium ion concentration in the nerve cells of Mythimna separata, and blocking the calcium channels on the cell membrane with CdCl2 cannot prevent the trend of the increase in the intracellular calcium ion concentration, indicating that PPTX-22 does not act on the calcium channels on the cell membrane.

[0068] From Figure 2 it can be seen that when adding 50 mg / L PPTX-22 and 1 mg / L 2-APB at the final concentration, blocking the IP3R calcium channels on the endoplasmic reticulum in the nerve cells of Mythimna separata with 2-APB cannot affect the trend of the increase in the intracellular calcium ion concentration caused by PPTX-22, indicating that PPTX-22 does not act on the IP3R receptor on the endoplasmic reticulum.

[0069] From Figure 3 It can be seen that when adding 50 mg / L PPTX-22 and 1 mg / L ryania at the final concentration, and using high-concentration ryania to block the putative receptor on the endoplasmic reticulum of Mythimna separata nerve cells, the increase in intracellular calcium ion concentration caused by PPTX-22 is almost completely inhibited, indicating that PPTX-22 acts on the ryanodine receptor on the endoplasmic reticulum.

[0070] From Figure 4 It can be seen that pretreatment with PPTX-22 and chlorantraniliprole both have an impact on the increase in calcium ion concentration in Mythimna separata nerve cells caused by subsequent addition of another substance, but cannot completely inhibit it, indicating that there is an interaction between the two substances in causing the increase in calcium ion concentration in nerve cells, suggesting that the sites where the two substances act on the ryanodine receptor overlap but do not completely coincide.

[0071] From Figure 5 It can be seen that under the condition of controlling the total concentration in the cell sap to be 6.5 μM, different molar ratios of PPTX-22 and chlorantraniliprole have different activities on the increase in calcium ion concentration in Mythimna separata nerve cells, and some ratios have an obvious increasing effect, such as the molar ratio of 1:5.

[0072] Set different concentrations for experiments and calculate the EC 50 value. From Figure 6 It can be seen that by comparing the EC 50 values of different molar ratios of PPTX-22 and chlorantraniliprole on the activity of Mythimna separata nerve cells, the EC 50 values of some ratios are significantly lower than those of PPTX-22 (1:0 is PPTX-22) and chlorantraniliprole (0:1 is chlorantraniliprole), indicating that these ratios have enhanced activity, such as the molar ratios of 1:5 and 1:2.

[0073] The above experiments indicate that the action sites of PPTX-22 and chlorantraniliprole on the insect ryanodine receptor overlap but do not completely coincide, resulting in synergism at the target level.

[0074] Example 3

[0075] 1. Bioassay method

[0076] Test on Lepidoptera insects: Mythimna separata, Spodoptera frugiperda, Plutella xylostella, Chilo suppressalis, Spodoptera exigua, Cnaphalocrocis medinalis, Ectropis obliqua, Carposina sasakii, Ostrinia furnacalis, Agrotis ypsilon.

[0077] The insecticidal activity of the combination preparation against Lepidoptera insects was determined by microinjection. The test insects were selected as 3rd instar larvae, with 3 replicates for each treatment and 20 test insects for each replicate. After the test insects were placed on ice for freezing paralysis, they were placed on a pre-prepared agarose gel with the ventral side facing up. 100nL of the combination preparation prepared in Example 1 was injected into the ventral epidermis between the last two pairs of abdominal legs of the test insects. The injected test insects were placed in a plastic culture dish filled with artificial feed and raised in a light incubator. The death of the test insects was checked after 24 hours. The experiment was divided into a control group and a treatment group. The control group was injected with an equal amount of PBS, pH = 7.4, and the treatment group was injected with different proportions of the compound agent.

[0078] 2. Data processing

[0079] The above bioassay results were processed using DPS v7.05 analysis software to calculate the toxicity regression curve equation, LC 50 , 95% confidence limits and correlation coefficients and other results.

[0080] The indoor screening of toxin biological agents adopts Sun Yunpei's co-toxicity coefficient method:

[0081] Actual Toxicity Index (ATI) of Toxin Biological Agent = LC of Agent A alone 50 / LC for mixing of reagents A and B 50 )×100

[0082] Theoretical Toxicity Index (TTI) of Toxin Biological Agents = ATI(A)×a+ATI(B)×b

[0083] a: represents the percentage of agent A in the mixture;

[0084] b: Indicates the percentage of agent B in the mixture.

[0085] Co-toxicity coefficient (CTC) = (actual toxicity index ATI of the compound agent / theoretical toxicity index TTI of the compound agent) × 100.

[0086] 3. Conclusion

[0087] According to the above experiments, the insecticidal activity and co-toxicity coefficient of chlorantraniliprole and toxin PPTX-22 when mixed in different ratios against various lepidopteran pests such as armyworm, fall armyworm, diamondback moth, striped stem borer, beet armyworm, rice leaf roller, tea looper, peach borer, corn borer, and black cutworm were tested and analyzed. Figures 7-16 shown.

[0088] from Figure 7 It can be seen that in the armyworm, the molar ratio of PPTX-22 to chlorantraniliprole is 1:5 and 1:3, and the co-toxicity coefficients are 234 and 137 respectively, with significant synergistic effect.

[0089] From Figure 8 It can be seen that in Spodoptera frugiperda, the co-toxicity coefficients of the molar ratio of PPTX-22 to chlorantraniliprole being 1:5, 1:3, and 5:1 are 248, 156, and 127 respectively, showing a significant synergistic effect.

[0090] From Figure 9 It can be seen that in Plutella xylostella, the co-toxicity coefficients of the molar ratio of PPTX-22 to chlorantraniliprole being 1:5, 1:3, and 9:1 are 281, 153, and 135 respectively, showing a significant synergistic effect.

[0091] From Figure 10 It can be seen that in Chilo suppressalis, the co-toxicity coefficient of the molar ratio of PPTX-22 to chlorantraniliprole being 1:5 is 261, showing a significant synergistic effect.

[0092] From Figure 11 It can be seen that in Spodoptera exigua, the co-toxicity coefficients of the molar ratio of PPTX-22 to chlorantraniliprole being 1:5, 1:3, and 9:1 are 221, 144, and 142 respectively, showing a significant synergistic effect.

[0093] From Figure 12 It can be seen that in Cnaphalocrocis medinalis, the co-toxicity coefficients of the molar ratio of PPTX-22 to chlorantraniliprole being 1:5, 1:3, and 5:1 are 233, 184, and 126 respectively, showing a significant synergistic effect.

[0094] From Figure 13 It can be seen that in Ectropis obliqua, the co-toxicity coefficients of the molar ratio of PPTX-22 to chlorantraniliprole being 1:5 and 1:3 are 194 and 159 respectively, showing a significant synergistic effect.

[0095] From Figure 14 It can be seen that in Carposina sasakii, the co-toxicity coefficients of the molar ratio of PPTX-22 to chlorantraniliprole being 1:5, 1:3, and 9:1 are 214, 144, and 122 respectively, showing a significant synergistic effect.

[0096] From Figure 15 It can be seen that in Ostrinia furnacalis, the co-toxicity coefficients of the molar ratio of PPTX-22 to chlorantraniliprole being 1:5, 1:3, and 3:1 are 242, 156, and 131 respectively, showing a significant synergistic effect.

[0097] From Figure 16 It can be seen that in Agrotis ypsilon, the co-toxicity coefficients of the molar ratio of PPTX-22 to chlorantraniliprole being 1:5 and 1:3 are 214 and 169 respectively, showing a significant synergistic effect.

[0098] TakingFigures 7-16 For example, when the toxin PPTX-22 and chlorantraniliprole are compounded in a certain ratio (such as 1:5, 1:3, 3:1), the compounding shows a synergistic effect on Mythimna separata, Spodoptera frugiperda, Plutella xylostella, Chilo suppressalis, Spodoptera exigua, Cnaphalocrocis medinalis, Ectropis obliqua, Carposina sasakii, Ostrinia furnacalis, Agrotis ypsilon, etc. And the activity is significantly higher than that of any one of the two used alone. A co-toxicity coefficient higher than 120 indicates a synergistic effect.

[0099] According to the above method, the LC 50 value (μM) and co-toxicity coefficient were determined when chlorantraniliprole, toxin PPTX-22, and their compound at a molar ratio of 1:5 were used. Taking the data shown in Table 1 as an example, when the toxin PPTX-22 and chlorantraniliprole are compounded at a ratio of 1:5, the median lethal concentration (LC 50 ) for various Lepidoptera pests such as Mythimna separata, Spodoptera frugiperda, Plutella xylostella, Chilo suppressalis, Spodoptera exigua, Cnaphalocrocis medinalis, Ectropis obliqua, Carposina sasakii, Ostrinia furnacalis, Agrotis ypsilon is significantly lower than that of the two single agents of PPTX-22 and chlorantraniliprole, and the co-toxicity coefficient of the compound is 194 - 281. According to the regulations of the Institute for the Control of Agrochemicals, Ministry of Agriculture, the judgment standard for the synergistic effect of the mixed formulation is: a co-toxicity coefficient greater than 120 indicates a synergistic effect.

[0100] Table 1. LC 50 values (μM) and co-toxicity coefficients of test insects for PPTX-22, chlorantraniliprole, and a 1:5 molar concentration ratio

[0101]

Claims

1. Application of a combined preparation of a polypeptide toxin and a chemical pesticide in the control of agricultural pests and hygienic pests. The combined preparation comprises Pardosa pseudoannulata toxin PPTX-22 and chlorantraniliprole. The amino acid sequence of the Pardosa pseudoannulata toxin PPTX-22 is as shown in SEQ ID NO.

1. The molar ratio of the Pardosa pseudoannulata toxin PPTX-22 to chlorantraniliprole is 1:

5.

2. The application according to claim 1, characterized in that, The preparation method of the combined preparation comprises the following steps: (1) Prepare Pardosa pseudoannulata toxin PPTX-22; (2) Dissolve chlorantraniliprole in dimethyl sulfoxide, and dissolve Pardosa pseudoannulata toxin PPTX-22 in PBS buffer. Mix the prepared mother liquors of chlorantraniliprole and PPTX-22 in proportion and dilute them to prepare a series of concentration gradients.

3. The application according to claim 2, wherein In step (1), the Pardosa pseudoannulata toxin PPTX-22 is prepared by induced expression and purification or directly synthesized artificially according to the amino acid sequence.

4. The application according to claim 2, characterized in that In step (2), dissolve the chlorantraniliprole technical in dimethyl sulfoxide to prepare a mother liquor with a concentration of 100-200 mM; dissolve the toxin PPTX-22 in PBS buffer with pH = 7.4 to prepare a mother liquor with a concentration of 10-20 mM; mix the prepared mother liquors of chlorantraniliprole and PPTX-22 according to a molar ratio of 1:5 and dilute with PBS with pH = 7.4 to prepare a series of concentration gradients.

5. The application according to claim 1, characterized in that, The agricultural pests and hygienic pests include Mythimna separata, Spodoptera frugiperda, Plutella xylostella, Chilo suppressalis, Spodoptera exigua, Cnaphalocrocis medinalis, Ectropis obliqua, Carposina sasakii, Ostrinia furnacalis, Agrotis ypsilon.

6. The application according to claim 1, wherein The combined preparation controls crops by spraying the plant leaves and / or the body surface of pests.

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