A two-stage synergistic osthole nano-preparation based on nano-delivery and RNA interference technology, and a preparation method and application thereof

Abstract

This invention provides a two-stage synergistic compound nanoformulation of osthol based on nanodelivery and RNA interference technology, its preparation method, and its application. The nanoformulation comprises: osthol, a hydrophilic-lipophilic amphiphilic block polymer nanocarrier, and at least one RNA interference substance. The RNA interference substance targets at least one of the following target genes: heat shock protein 70 gene, RNA-binding protein gene, and glucose dehydrogenase gene. This invention uses a nanocarrier to load osthol, and then uses transcriptome sequencing to screen for key RNAi target genes that can rapidly respond to osthol stress. An RNA interference substance targeting these genes is designed and synthesized, and combined with the osthol / carrier complex to achieve a two-stage synergistic effect on the insecticidal activity of osthol. This significantly enhances the sensitivity of peach aphids to osthol and promotes the rapid action of the pesticide, providing a new strategy for targeted synergistic effects of plant-derived pesticides.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to a two-stage synergistic compound nano-formulation of osthol based on nanodelivery and RNA interference technology, its preparation method and application. Background Technology

[0002] Osthole, a plant-derived pesticide, is characterized by low toxicity, safety, and environmental friendliness. It also possesses insecticidal and antibacterial biological activities and is widely used in agricultural pest and disease control, particularly in organic agriculture. However, its development is limited by drawbacks such as poor water solubility, slow onset of action, and low efficacy.

[0003] Current research on enhancing the efficacy of osthol mainly focuses on methods such as drug mixing or nanocarriers. Nanomaterials, as drug carriers, can achieve nanoscale loading and cell delivery, enabling faster and more effective delivery of various pesticides while reducing dosage. However, the poor water solubility of osthol necessitates the addition of large amounts of organic solvents or surfactants when combined with water-soluble nanocarriers. Therefore, the performance requirements for nanocarriers are very high, and the improvement in efficacy is limited.

[0004] RNA sequencing (RNA-seq) can systematically analyze the dynamic changes in gene expression profiles of pests under osthol stress, revealing their molecular response mechanisms. By utilizing key response genes and based on RNAi-induced gene silencing, the sensitivity of pests to osthol can be increased. Currently, there is a severe lack of RNAi candidate target genes targeting the mechanism of action of osthol. There is an urgent need to screen for precise and rapid RNAi candidate target genes that respond to osthol, and to verify the synergistic effect of RNAi, in order to further enhance the insecticidal efficacy of osthol. Summary of the Invention

[0005] In view of this, the present invention aims to propose a two-stage synergistic compound nano-formulation of osthol based on nanodelivery and RNA interference technology, its preparation method and application. The nano-formulation is formed by simultaneously loading osthol pesticide and RNAi drug on HLDP nanocarrier, which greatly improves the rapid action and insecticidal activity of osthol.

[0006] To achieve the above objectives, the technical solution of the present invention is implemented as follows: In a first aspect, the present invention provides a two-stage synergistic compound nanoformulation of osthol based on nanodelivery and RNA interference technology. The nanoformulation comprises: osthol, a hydrophilic and lipophilic amphiphilic block polymer nanocarrier, and at least one RNA interference substance. The RNA interference substance targets at least one of the following target genes: heat shock protein 70 gene, RNA binding protein gene, and glucose dehydrogenase gene.

[0007] Furthermore, the nanocarrier co-loads the osthol and the RNA interference substance to form the nano-formulation.

[0008] Furthermore, the nanocarrier is HLDP, and its structural formula is: ; Preferably, the mass ratio of osthol to the nanocarrier is 1:2 to 5, for example, it can be 1:2, 1:2.25, 1:2.5, 1:2.85, 1:3, 1:3.5, 1:4, 1:4.5, or 1:5. Preferably, the mass ratio of the RNA interference substance to the complex formed by osthol and the nanocarrier is 1:0.5 to 1.5, for example, it can be 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, or 1:1.5.

[0009] Furthermore, the RNA interference substance is dsRNA or hpRNA; And / or, the RNA interference substance includes dsRNA of the target gene Hsp70, comprising a sense strand with a nucleotide sequence as shown in SEQ ID NO:9 and an antisense strand with a nucleotide sequence as shown in SEQ ID NO:21; And / or, the RNA interference substance comprises hpRNA of the target gene Hsp70, comprising a sense strand with a nucleotide sequence as shown in SEQ ID NO:33 and an antisense strand with a nucleotide sequence as shown in SEQ ID NO:21; And / or, the RNA interference substance includes dsRNA of the target gene RBP, comprising a sense strand with a nucleotide sequence as shown in SEQ ID NO:10 and an antisense strand with a nucleotide sequence as shown in SEQ ID NO:22; And / or, the RNA interference substance includes dsRNA of the target gene GDH-1, comprising a sense strand with a nucleotide sequence as shown in SEQ ID NO:11 and an antisense strand with a nucleotide sequence as shown in SEQ ID NO:23; And / or, the RNA interference substance includes dsRNA of the target gene GDH-3, comprising a sense strand with a nucleotide sequence as shown in SEQ ID NO:12 and an antisense strand with a nucleotide sequence as shown in SEQ ID NO:24.

[0010] Furthermore, the method for preparing the dsRNA includes the following steps: (1) Using peach aphid cDNA as a template, PCR amplification was performed using primers to obtain the target gene fragment. The primer sequence is at least one of Hsp70-F (SEQ ID NO:1), Hsp70-R (SEQ ID NO:2), RBP-F (SEQ ID NO:3), RBP-R (SEQ ID NO:4), GDH-F1 (SEQ ID NO:5), GDH-R1 (SEQ ID NO:6), GDH-F3 (SEQ ID NO:7), and GDH-R3 (SEQ ID NO:8). (2) Using the target gene fragment as a template, PCR amplification is performed using primers with the T7 promoter sequence to obtain a DNA template for synthesizing dsRNA. The primer sequence is at least one of T7Hsp70-F (SEQ ID NO:13), T7Hsp70-R (SEQ ID NO:14), T7RBP-F (SEQ ID NO:15), T7RBP-R (SEQ ID NO:16), T7GDH-F1 (SEQ ID NO:17), T7GDH-R1 (SEQ ID NO:18), T7GDH-F3 (SEQ ID NO:19), and T7GDH-R3 (SEQ ID NO:20). (3) Using a DNA template as a template, T7 RNA polymerase is used for in vitro transcription. The transcription product is digested with DNase, precipitated and washed to obtain the dsRNA.

[0011] Furthermore, the method for preparing the hpRNA includes the following steps: (1) Using the Hsp70 gene fragment as a template, the forward fragment L and the reverse complementary fragment R are amplified using primers with restriction enzyme sites; the nucleotide sequence of the forward fragment L is shown in SEQ ID NO:33, the nucleotide sequence of the reverse fragment R is shown in SEQ ID NO:21, the forward fragment L and the reverse fragment R are connected by a stem-loop sequence, and the primer sequence with restriction enzyme sites is at least one of EcoRI-Hsp70L-F (SEQ ID NO:29), XbaI-Hsp70L-R (SEQ ID NO:30), XbaI-Hsp70R-F (SEQ ID NO:31), and XhoI-Hsp70R-R (SEQ ID NO:32); (2) The amplification product obtained in step (1) is digested with two enzymes and then ligated into an expression vector linearized with restriction endonuclease to obtain a recombinant expression vector; (3) The recombinant expression vector obtained in step (2) was transformed into HT115(DE3) RNase III-deficient Escherichia coli, IPTG was added to induce expression, the bacterial cells were collected, extracted and purified to obtain the hpRNA; Preferably, the stem-loop sequence is 50-200 bp in length, more preferably 100 bp, and even more preferably, the stem-loop sequence is the sequence shown in SEQ ID NO:34.

[0012] In a second aspect, the present invention provides a method for preparing nano-formulations as described in the first aspect, the method comprising the following steps: (1) Osthol was mixed and incubated with hydrophilic and lipophilic amphiphilic block polymer nanocarriers to obtain osthol / carrier complex; (2) Mix the RNA interference substance with the osthol / carrier complex, with or without the addition of an auxiliary binding agent, and allow the reaction to proceed until the nano-formulation is obtained; Preferably, in step (1), the incubation temperature is room temperature and the incubation time is 5~15 min; Preferably, in step (1), the mass ratio of osthol to the nanocarrier is 1:2 to 5, for example, it can be 1:2, 1:2.25, 1:2.5, 1:2.85, 1:3, 1:3.5, 1:4, 1:4.5, or 1:5; Preferably, in step (2), the auxiliary binding agent is an alkyl glycoside; Preferably, in step (2), the mass ratio of the RNA interference substance to the osthol / carrier complex is 1:0.5~1.5, for example, it can be 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, or 1:1.5.

[0013] Thirdly, the present invention provides a method for controlling peach aphids, the method comprising applying nano-preparations as described in the first aspect to crops infested by peach aphids.

[0014] Furthermore, the application method includes soaking or spraying.

[0015] Fourthly, the present invention provides a kit for controlling peach aphids, the kit comprising the nano-formulations as described in the first aspect.

[0016] Fifthly, the present invention provides the application of nano-formulations as described in the first aspect or kits as described in the fourth aspect in the control of agricultural pests.

[0017] Furthermore, the application includes at least one of the following: (1) Application in inhibiting the expression of Hsp70, RBP and GDH genes in peach aphid; (2) Application in the control of peach aphids; (3) Application in enhancing the insecticidal effect of osthol.

[0018] Furthermore, the application also includes at least one of the following: (4) Application in the preparation of products that inhibit the expression of Hsp70, RBP and GDH genes in peach aphids; (5) Application in the preparation of products for the control of peach aphids; (6) Application in the preparation of products that enhance the insecticidal effect of osthol.

[0019] In a sixth aspect, the present invention provides the application of the RNA interference substance in enhancing the insecticidal effect of osthol, wherein the RNA interference substance targets at least one of the following target genes: heat shock protein 70 gene, RNA binding protein gene, glucose dehydrogenase gene.

[0020] Compared with existing technologies, the osthol two-stage synergistic compound nano-formulation based on nanodelivery and RNA interference technology, its preparation method, and its application described in this invention have the following advantages: This invention uses a nanocarrier to load osthol, and then uses transcriptome sequencing to screen key RNAi target genes that can rapidly respond to osthol stress. RNA interference substances targeting these genes are designed and synthesized, and combined with the osthol / carrier complex to achieve a two-stage synergistic effect on the insecticidal activity of osthol. This significantly enhances the sensitivity of peach aphids to osthol and promotes the rapid effect of the pesticide, providing a new strategy for targeted synergistic effects of plant-derived pesticides. Attached Figure Description

[0021] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings: Figure 1 The particle size and morphology of the osthol / HLDP complex are shown. Figure 2 A schematic diagram of the 24-hour indoor bioassay results for the osthol / HLDP complex; Figure 3 24-hour in vitro bioassay and LC-assay of dsHsp70 in combination with osthol / HLDP complex 50 Calculation results diagram; Figure 4 A schematic diagram of the indoor bioassay results after 5 days of combined use of dsHsp70, dsRBP, dsGDH-1, dsGDH-3 and osthol / HLDP complex. Figure 5 A schematic diagram illustrating the silencing efficiency of Hsp70 under dsHsp70 processing; Figure 6 This is a schematic diagram showing the fusion ratio of dsRNA with the osthol / HLDP complex; Figure 7 A schematic diagram of the field simulation results of the hpHsp70 / osthol / HLDP compound nanoformulation; Figure 8 A schematic diagram illustrating the gene silencing effect of a compound nano-formulation of hpHsp70 / ostrichol / HLDP. Detailed Implementation

[0022] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0023] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0024] Example 1: Preparation and application of osthol / HLDP nanopesticides (1) Test insect materials The peach aphid is a strain that has been raised in our laboratory for multiple generations. The host plant is radish seedlings, and the rearing conditions are a constant temperature incubator with a temperature of 18℃ and a photoperiod of L:D=16:8.

[0025] (2) Test reagent materials 98% osthol technical grade (Shanghai Tengzhun Biotechnology Co., Ltd.), 1% osthol water emulsion (Inner Mongolia Qingyuanbao Biotechnology Co., Ltd.), HLDP nanocarrier (refer to the invention patent with publication number CN115124669B), ethanol (HPLC grade), Triton X-100 (Solepro, catalog number T8200), alkyl glycoside (Alkyl Polyglucoside, APG, Wanhua Supply Chain Service Co., Ltd., catalog number 0810).

[0026] (3) Preparation and characterization of nanocarrier / HLDP composite (3-1) Determination of drug loading rate 98% of osthol technical grade was dissolved in anhydrous ethanol to prepare a 30 g / L stock solution, which was then diluted with ultrapure water. A 500 mg / L osthol solution was mixed with an equal volume of HLDP nanocarrier, and incubated for 15 min. The complex solution was then transferred to a dialysis bag containing a regenerated cellulose membrane with a molecular weight cutoff of 1000 Da, and the dialysis fluid was changed every 3 h. The remaining liquid was freeze-dried using a vacuum centrifuge and weighed. The mass of osthol was recorded in detail, and the drug loading rate was calculated using the following formula: The formula for calculating drug loading rate is: Drug loading rate (%) = [(mass of osthol in the complex) ÷ (mass of the complex)] × 100%.

[0027] Calculations show that the average drug loading rate of osthol by the nanocarrier HLDP is 26.62%.

[0028] (3-2) Particle size determination Based on the drug loading rate, technical grade drug and commercial osthol / HLDP complex (final osthol concentration 50 mg / L) with a mass ratio of 1:2.85 were prepared. Particle size and polydispersity index (PDI) were measured using a dynamic light scattering (DLS) instrument and a Zetasizer Nano ZS. Samples were ultrasonically vibrated for 10 min before measurement to eliminate agglomeration at 25°C, and each sample was measured at least three times independently.

[0029] like Figure 1 As shown, the average particle size of osthol technical grade is as high as 3893.05 nm, while the average particle size of commercial osthol is 263 nm. However, after being combined with the nanocarrier HLDP, the average particle size of the technical grade complex is sharply reduced to 84.50 nm, and that of the commercial drug complex is reduced to 135.37 nm, a reduction of nearly half. This reduction in size facilitates the penetration of the drug into the insect epidermis, providing a physical basis for the enhanced insecticidal activity.

[0030] (3-3) Morphological observation The sample was treated with 2% phosphotungstic acid. After the sample was completely dry, an appropriate length of conductive adhesive was cut and pasted onto the sample stage. The sample was then placed on the conductive adhesive. Gold sputtering was performed to improve the conductivity of the sample. Before the test, a vacuum was drawn for about 10 minutes. The morphology of the sample under vacuum was observed using a field emission scanning electron microscope (SEM, Jsm7610fplus).

[0031] Scanning electron microscopy revealed that the monomers of osthol technical grade were regular short rods or near-ellipsoidal in shape, with relatively smooth particle surfaces, clear edge contours, and a certain tendency to aggregate. After binding with HLDP, the morphology of osthol changed significantly; the particles became nearly spherical, with good dispersibility and a relatively uniform distribution. Commercial osthol products, which are nearly spherical particles, showed almost no morphological change after binding with HLDP, but their size distribution became even more uniform.

[0032] (3-4) Indoor toxicity assay of osthol / HLDP complex Five concentration gradients of osthol technical (5 mg / L, 10 mg / L, 20 mg / L, 40 mg / L, 50 mg / L) and commercially available formulations (5 mg / L, 10 mg / L, 20 mg / L, 75 mg / L, 100 mg / L) were prepared. Water and HLDP treatments served as controls. The peach aphid was treated using an immersion method. Appropriately sized circular filter paper was placed in a 5 cm petri dish, moistened with a suitable amount of water, and fresh radish seedlings were placed inside. The radish leaves containing third-instar peach aphids, along with the aphids, were immersed in the solution for 15-20 seconds. After 15-20 seconds, the aphids were removed, placed on filter paper to absorb excess solution, and gently transferred to the fresh radish seedlings in the petri dish using a brush. A total of four treatments were performed, with each treatment repeated three times. After 24 hours, the survival status of the peach aphids was observed under a stereomicroscope, and the mortality rate was recorded.

[0033] Observations revealed that dead peach aphids turned brown, and dying individuals exhibited irregular shaking of their multiple legs. Figure 2 As shown, from the median lethal concentration (LC50) 50 From this perspective, after osthol technical grade and the nanocarrier HLDP form a complex, LC 50 The concentration of osthol decreased from 26.165 mg / L to 16.435 mg / L after being combined with the nanocarrier HLDP. 50 The concentration decreased from 33.181 mg / L to 9.128 mg / L. Regarding the increase in insecticidal activity, HLDP showed a synergistic effect with osthol at different concentrations. At a concentration of 20 mg / L, the insecticidal efficacy of HLDP against the technical grade pesticide increased from 27% to 45%, an increase of 18%; the insecticidal efficacy of HLDP against the commercial pesticide increased from 47% to 80%, an increase of 33%.

[0034] The results above indicate that the nanocarrier HLDP significantly enhances the insecticidal activity of osthol and exhibits a greater synergistic effect with commercially available pesticides.

[0035] Example 2: Co-use of dsRNA responding to osthol pesticide with osthol / HLDP nanopesticide (1) Synthesize dsRNA targeting hsp70, GDH, and RBP genes. Based on transcriptome sequencing results and the mechanism of action of osthol, four rapidly responding genes to osthol treatment and associated with metabolism, detoxification, and drug metabolism—heat shock protein 70 (Hsp70), glucose dehydrogenase (GDH-like), and RNA binding protein (RBP)—were screened from 423 differentially expressed genes (376 upregulated and 47 downregulated) as RNAi target genes to enhance the insecticidal efficacy of osthol / HLDP nanopesticides. For dsRNA design, fragments of appropriate size and high specificity were selected.

[0036] RNA was extracted from peach aphids using the Trizol method. Concentration was measured using a NanoDrop2000 ultra-micro spectrophotometer. RNA integrity was detected by agarose gel electrophoresis to obtain high-quality RNA. Using the extracted peach aphid RNA as a template, cDNA was obtained by reverse transcription using the HiScript® III All-in-one RT SuperMix Perfect for qPCR kit from Nanjing Novizan Biotechnology Co., Ltd. Upstream and downstream primers Hsp70-F (nucleotide sequence SEQ ID NO:1), Hsp70-R (nucleotide sequence SEQ ID NO:2), RBP-F (nucleotide sequence SEQ ID NO:3), RBP-R (nucleotide sequence SEQ ID NO:4), GDH-F1 (nucleotide sequence SEQ ID NO:5), GDH-R1 (nucleotide sequence SEQ ID NO:6), GDH-F3 (nucleotide sequence SEQ ID NO:7), and GDH-R3 (nucleotide sequence SEQ ID NO:8) were designed using the NCBI primer website. Using cDNA obtained from reverse transcription as a template, 1 μL of cDNA template, 10 μL of 2×Taq Plus Master Mix, 1 μL of upstream primer Hsp70-F, 1 μL of downstream primer Hsp70-R, and 7 μL of ddH2O were mixed to obtain a total volume of 20 μL. The mixture was then subjected to PCR under the following conditions: 94 ℃ for 3 min; 94 ℃ for 30 s, 60 ℃ for 30 s, 72 ℃ for 1 min, for 40 cycles; 72 ℃ for 5 min. The reaction amplified Hsp70 (nucleotide sequence SEQ ID NO:9), RBP (nucleotide sequence SEQ ID NO:10), GDH-1 (nucleotide sequence SEQ ID NO:11), and GDH-3 (nucleotide sequence SEQ ID NO:12) gene fragments.

[0037] CDS sequence of Hsp70 gene (SEQ ID NO: 9): 5’-TGTCCGTTGAACGAGGCATACATAATACTACTTGGCTACTATTTAACGCGACTTTCAGTGTTATTCACGAATTTTCCATTGCGGTTTTATACACAAAGTGATTTTAAATCGTTATCTAATTGCTGTACTACCTGCGTATACGTTTTAGTTTGAATTATCAAATTAATTTTCGTATTAATTGTGCGTGAATTATATCGACAATTATTGGCGAAATGGTTGGAAAAACAGCCATCGGTATCGACCTGGGTACCACCTATTCCTGCGTGGGTGTCTGGCAACACGGAAAAGTAGAGATCATCGCCAACGATCAAGGTAACAGGACCACCCC-3’ CDS sequence of RBP gene (SEQ ID NO: 10): 5’-AACCGTACCAGCAACACCAACAGTACAATTTCGGACCAAACCAACAGTACTACCCGGGTTCCGGATACTCCGCCGCCGGACAATACCCGACGCCGTACTCGGCTTACCCGTACTCCGCACAACCGTACTCTGCCCAACCGTACTCCGCCCAACCGTACTCCGCCCAACCGTACTCTGCCCAACCGTACTCAGCTCAACCGTACTCTGCCCAACAGTACAACGGCGGCTCTTACTCGCCATATCCGGGCGCCTTCCCCTCGTACTACGGTAATCAATTCGGCGTCCCGTCCGCCGCCGCCGCCACCGGTCAATACAACAGCCATTACTCGGCAGCCGCCGCCGCTCCGTACAAC-3’ CDS sequence of GDH-1 gene fragment (SEQ ID NO: 11): 5’-GACTTGTCGTGCTGGGGATTTTTCTACTCATTGCTAGCCCATGTTTTTCAATAAATATCTTTCGATATTTGGGTCAGGCTTACCGTAAAAATTTAATTAAATTCTACGAAGATCCTCAATTTGGAAATAAAAAAATACTAGATGAATACGATTTCATAGTGGTAGGAGCTGGTGCTGCAGGAGCAACAGTCGCTCGACGGTTAGCGGAAGTCTCAGGATGGAACATACTACTATTGGAAGCTGGAGGAGAAGAATCATTGATTACTTCTCTCCCAAGTATTGCTCACTATTTACAATTTACAAATTACAATTGGGCATACCACACGGAAGAAGAATTACATGCTTGTAAAGGACTTATAAACAAGACTTGCCCATGGCCAGCAGGAAAAGGATTGGGAGGAAGCACAATAATTAATAACAATATGTACACAAGAGGTAATGTAAGAGATTTTGATCGTTGGGCAGAAGCAGGAAATCGAGGGTGGTCTTTTAATGATGTACTACCATATTTCATAAAAAACGAAGACATAAACGTACCGGAACTAAAGAGGTCCCCTTATCACGGGGTCGGAGGACCATTGCCAATTAGCTATCCTGATTATAAGTCAAAACTGGTCGAAGCGTTCTTGGAGTCAGCACCTGAGGTCGGTATGTCTGTTGGTGACTATAACGCTCCGGG-3’ CDS sequence of GDH-3 gene fragment (SEQ ID NO: 12): 5’-TGGGAAGGAGATCGATGGGTGTACCCGACAGGCTGTACTACGAAACATATCACGGCGCTTTGGCACGTGACACGTGGTCGATCTGGGTTATGTTGATGTATCCGGAGAGCAGAGGACAGGTAAGGCTGCGCAGCGCTAATCCATTTGACAAACCGGTGATCAACGCGAACTTTTTCACCGACCCAATGGATTTGAAACGAATCGTGGCAGGCATAAAAATGACGATCGAGCTCAGCAAAACCAAGGCATTTCAAAAGTACGGTTCCCGTTTGCACAAAACACCCATGCTCGGGTGTAGGCATTTAGAATTTGGCACCGATCAATACTGGGAGTGTTGTGTGCAGACGATGACGATGCAAATGCACCACCAGAGTGGCACGTGCAAAATGGGACCGGAATGGGAC-3’ Forward primer Hsp70-F (SEQ ID NO: 1): 5’-TGTCCGTTGAACGAGGCATA-3’ Reverse primer Hsp70-R (SEQ ID NO: 2): 5’-GGGGTGGTCCTGTTACCTTG-3’ Forward primer RBP-F (SEQ ID NO: 3): 5’-AACCGTACCAGCAACACCAA-3’ Reverse primer RBP-R (SEQ ID NO: 4): 5’-GTTGTACGGAGCGGCGG-3’ Forward primer GDH-F1 (SEQ ID NO: 5): 5’-ACTTGTCGTGCTGGGGATTT-3’ Reverse primer GDH-R1 (SEQ ID NO: 6): 5’-CCCGGAGCGTTATAGTCACC-3’ Forward primer GDH-F3 (SEQ ID NO: 7): 5’-TGGGAAGGAGATCGATGGGT-3’ Reverse primer GDH-R3 (SEQ ID NO: 8): 5’-GTCCCATTCCGGTCCCATTT-3’ The band sizes of the products were checked using 1% agarose gel electrophoresis. Correct products were then extracted using the Novozymes FastPure Gel DNA Extraction Mini Kit to recover the target fragment. The recovered product was ligated into the pMD19T vector (Takara) and transformed into *E. coli* DH5α competent cells (Beijing Qingke). *E. coli* DH5α cells were plated on medium containing 100 mg / mL Amp antibiotics and cultured overnight. Positive single colonies were picked and sent to Beijing Sangon Biotech Co., Ltd. for sequencing. The sequencing results were compared with the NCBI database to identify strains containing the target sequence fragment. Plasmids were extracted using the PurePlasmid Mini Kit (Beijing Kangwei Century Biotechnology Co., Ltd.) as templates for the next reaction. Using the plasmid containing the target sequence fragment as a template, PCR amplification was performed using primers with the T7 promoter sequence. The primers used to amplify the target sequence fragment were named T7Hsp70-F (nucleotide sequence SEQ ID NO:13), T7Hsp70-R (nucleotide sequence SEQ ID NO:14), T7RBP-F (nucleotide sequence SEQ ID NO:15), T7RBP-R (nucleotide sequence SEQ ID NO:16), T7GDH-F1 (nucleotide sequence SEQ ID NO:17), T7GDH-R1 (nucleotide sequence SEQ ID NO:18), T7GDH-F3 (nucleotide sequence SEQ ID NO:19), and T7GDH-R3 (nucleotide sequence SEQ ID NO:20). The amplification system and conditions were the same as above. After the amplification reaction, the band size was detected by gel electrophoresis. Subsequently, the target product was recovered and purified as a DNA template for dsRNA synthesis.

[0038] T7Hsp70-F (SEQ ID NO:13):5'-TAATACGACTCACTATAGGGTGTCCGTTGAACGAGGCATA-3' T7Hsp70-R (SEQ ID NO:14): 5'-TAATACGACTCACTATAGGGGGGGTGGTCCTGTTACCTTG-3' T7RBP-F (SEQ ID NO:15): 5'-TAATACGACTCACTATAGGGAACCGTACCAGCAACACCA-3' T7RBP-R (SEQ ID NO:16): 5'-TAATACGACTCACTATAGGGGTTGTACGGAGCGGCGG-3' T7GDH-F1 (SEQ ID NO:17): 5'-TAATACGACTCACTATAGGGACTTGTCGTGCTGGGGATTT-3' T7GDH-R1 (SEQ ID NO:18): 5'-TAATACGACTCACTATAGGGCCCGGAGCGTTATAGTCACC-3' T7GDH-F3 (SEQ ID NO:19): 5'-TAATACGACTCACTATAGGGTGGGAAGGAGATCGATGGGT-3' T7GDH-R3 (SEQ ID NO:20): 5'-TAATACGACTCACTATAGGGGTCCCATTCCGGTCCCATTT-3' dsRNA was synthesized using the Promega T7 RiboMAX™ Express RNAi System kit. Specifically, 10 μL of RiboMAX™ Express T7 2X Buffer, 2 μL of Enzyme Mix, 1000 ng of DNA template, and RNase-free H2O were mixed to obtain a total volume of 20 μL. The mixture was incubated overnight at 37 °C. The overnight product was then incubated at 70 °C for 10 min and at room temperature for 20 min. Subsequently, 1 μL of RNase Solution 100-fold dilution (freshly prepared) and 1 μL of RQ1 RNase-free DNase were added to the mixture, and the mixture was incubated at 37 °C for 30 min. Then, 0.1 volume of 3M Sodium Acetate and 1 volume of isopropanol were added, and the mixture was incubated on ice for 5 min. Centrifuge at 13000 rpm for 10 min at 4 °C, aspirate the supernatant, add 500 μL of 70% ice-cold ethanol, gently pipette the precipitate, centrifuge at 13000 rpm for 5 min at 4 °C, discard the supernatant, dry in a clean bench for about 10 min, add 30 μL of enzyme-free water to dissolve the precipitate to obtain dsRNA products, named dsHsp70, dsRBP, dsGDH-1, and dsGDH-3. Dilute 10-fold and detect fragment concentration using a micro spectrophotometer, and detect fragment size using agarose gel electrophoresis. dsHsp70 consists of the sense strand with SEQ ID NO:9 and the antisense strand with SEQ ID NO:21; dsRBP consists of the sense strand with SEQ ID NO:10 and the antisense strand with SEQ ID NO:22; dsGDH-1 consists of the sense strand with SEQ ID NO:11 and the antisense strand with SEQ ID NO:23; and dsGDH-3 consists of the sense strand with SEQ ID NO:12 and the antisense strand with SEQ ID NO:24.

[0039] SEQ ID NO:21 is shown below: 5'-GGGGTGGTCCTGTTACCTTGATCGTTGGCGATGATCTCTACTTTTCCGTGTTGCCAGACACCCACGCAGGAATAGGTGGTACCCAGGTCGATACCGATGGCTGTTTTTCCAACCATTTCGCCAATAATTGTCGATATAATTCACGACAATTAATACGAAAATT AATTTGATAATTCAAACTAAAACGTATACGCAGGTAGTACAGCAATTAGATAACGATTTAAAATCACTTTGTGTATAAAACCGCAATGGAAAATTCGTGAATAACACTGAAAGTCGCGTTAAATAGTAGCCAAGTAGTATTATGTATGCCTCGTTCAACGGACA-3' SEQ ID NO:22 is shown below: 5’-GTTGTACGGAGCGGCGGCGGCTGCCGAGTAATGGCTGTTGTATTGACCGGTGGCGGCGGCGGCGGACGGGACGCCGAATTGATTACCGTAGTACGAGGGGAAGGCGCCCGGATATGGCGAGTAAGAGCCGCCGTTGTACTGTTGGGCAGAGTACGGTTGAGCTGAGTACGGTTGGGCAGAGTACGGTTGGGCGGAGTACGGTTGGGCGGAGTACGGTTGGGCAGAGTACGGTTGTGCGGAGTACGGGTAAGCCGAGTACGGCGTCGGGTATTGTCCGGCGGCGGAGTATCCGGAACCCGGGTAGTACTGTTGGTTTGGTCCGAAATTGTACTGTTGGTGTTGCTGGTACGGTT-3’ SEQ ID NO: 23 is specifically shown as follows: 5'-CCCGGAGCGTTATAGTCACCAACAGACATACCGACCTCAGGTGCTGACTCCAAGAACGCTTCGACCAGTTTTGACTTATAATCAGGATAGCTAATTGGCAATGGTCCTCCGACCCCGTGATAAGGGGACCTCTTTAGTTCCGGTACGTTTATGTCTTCGTTTTTTATGAAATATGGTAGTACATCATTAAAAGACCACCCTCGATTTCCTGCTTCTGCCCAACGATCAAAATCTCTTACATTACCTCTTGTGTACATATTGTTATTAATTATTGTGCTTCCTCCCAATCCTTTTCCTGCTGGCCATGGGCAAGTCTTGTTTATAAGTCCTTTACAAGCATGTAATTCTTCTTCCGTGTGGTATGCCCAATTGTAATTTGTAAATTGTAAATAGTGAGCAATACTTGGGAGAGAAGTAATCAATGATTCTTCTCCTCCAGCTTCCAATAGTAGTATGTTCCATCCTGAGACTTCCGCTAACCGTCGAGCGACTGTTGCTCCTGCAGCACCAGCTCCTACCACTATGAAATCGTATTCATCTAGTATTTTTTTATTTCCAAATTGAGGATCTTCGTAGAATTTAATTAAATTTTTACGGTAAGCCTGACCCAAATATCGAAAGATATTTATTGAAAAACATGGGCTAGCAATGAGTAGAAAAATCCCCAGCACGACAAGTC-3' SEQ ID NO: 24 is specifically shown as follows: 5'-GTCCCATTCCGGTCCCATTTTGCACGTGCCACTCTGGTGGTGCATTTGCATCGTCATCGTCTGCACACAACACTCCCAGTATTGATCGGTGCCAAATTCTAAATGCCTACACCCGAGCATGGGTGTTTTGTGCAAACGGGAACCGTACTTTTGAAATGCCTTGGTTTTGCTGAGCTCGATCGTCATTTTTATGCCTGCCACG ATTCGTTTCAAATCCATTGGGTCGGTGAAAAAGTTCGCGTTGATCACCGGTTTGTCAAATGGATTAGCGCTGCGCAGCCTTACCTGTCCTCTGCTCTCCGGATACATCAACATAACCCAGATCGACCACGTGTCACGTGCCAAAGCGCCGTGATATGTTTCGTAGTACAGCCTGTCGGGTACACCCATCGATCTCCTTCCCA-3' (2) Application of dsRNA and osthol / HLDP nano-formulation (2-1) Preparation of dsRNA / HLDP complex Take 4 μL of dsRNA solution with a concentration of 5000 ng / μL, mix it with HLDP complex at a mass ratio of 1:1, add 1 μL of LAPG (alkyl glycoside), and make up to 10 μL with deionized water. Let stand at room temperature for 5 min to allow the three to fully combine, and obtain a compound preparation with a final concentration of 2000 ng / μL of dsRNA.

[0040] (2-2) Indoor bioassay of dsHsp70, dsRBP, dsGDH-1, dsGDH-3 in combination with osthol / HLDP nanopesticides Using osthol commercial drug / HLDP complex LC 50 Four hours after treating peach aphids with the pesticide solution and the immersion method, third-instar peach aphids were gently transferred to a carbon dioxide analyzer using a brush. Once they were immobile and unconscious, 0.1 μL of the liquid was dripped onto the aphid's dorsal plate using a microsyringe under a stereomicroscope. The control group was dripped with dseGFP / HLDP and HLDP, while the treatment group was dripped with dsHsp70 / HLDP. After absorption, the aphids were placed in petri dishes containing moist filter paper and radish seedlings and observed in a light-controlled incubator at 18℃ and a light / D ratio of 16:8. Mortality rates were recorded, and the 24-hour toxicity equation and LC-1 were calculated. 50 .like Figure 3 As shown, the calculated LC of osthol / HLDP is... 50The LC-releasing capacity of dsHsp70 and osthol nanopesticide was 22 mg / L. 50 The concentration was 14 mg / L, demonstrating that silencing the Hsp70 gene significantly increased the peach aphid's sensitivity to osthol, making it lethal even at lower concentrations.

[0041] Using the same operating method, dsHsp70, dsRBP, dsGDH-1, and dsGDH-3 were treated in combination with osthol / HLDP nanopesticides. Patients were observed for 5 days, and mortality rates were recorded, with particular attention paid to rapid effectiveness and the rate of increase in mortality. Each group had 3 replicates, with 20 insects per replicate. Results are as follows: Figure 4 As shown, overall, the dsHsp70 combination group exhibited the fastest insecticidal speed and the highest cumulative mortality rate; while dsRBP, dsGDH-1 and dsGDH-3 also showed synergistic effects, but their effects were slightly inferior to Hsp70, verifying that all four selected genes were effective, with Hsp70 being the best target.

[0042] (2-3) Determination of interference effect First, use commercial osthol / HLDP complex LC. 50 Pretreatment of peach aphids with pesticide solution and immersion method. After the liquid was completely absorbed, the third instar peach aphids were gently transferred to the carbon dioxide meter with a brush. The dsRNA liquid was dripped onto the dorsal plate of the peach aphids using a microsyringe, 0.1 μL per aphid. The control group was dripped with dseGFP / HLDP, treatment group 1 was dripped with dsHsp70 / HLDP, and treatment groups 2 and 3 were treated with dsHsp70 / HLDP and water, respectively, using the peach aphids pretreated with the pesticide. Samples were taken 12 h and 24 h after treatment and transferred to 1.5 mL enzyme-free centrifuge tubes. RNA was extracted according to the Trizol method, then reverse transcribed into cDNA and diluted 4-fold. The diluted cDNA was used as a template. qRT-PCR was performed using EF-1 (elongation factor 1 alpha) as an internal control gene. The primers for the internal control gene were EF1-F (nucleotide sequence SEQ ID NO:25) and EF1-R (nucleotide sequence SEQ ID NO:26), and the primers for the Hsp70 gene were Q-Hsp70-F (nucleotide sequence SEQ ID NO:27) and Q-Hsp70-R (nucleotide sequence SEQ ID NO:28). Each treatment included three biological replicates, and each biological replicate included three technical replicates. Based on the CT value of each reaction well, 2... -ΔΔCT The relative expression levels of the target gene and internal reference gene in each group of samples were calculated using the method.

[0043] Quantitative primer sequences: EF1-F (SEQ ID NO:25):GTACTTCCCAGGCCGATTGT EF1-R (SEQ ID NO:26):AGGTGAAGGCCAATAGAGCG Q-Hsp70-F (SEQ ID NO:27):ACAATCAGTTGGCAGACAAGGAAG Q-Hsp70-R (SEQ ID NO:28): GTCCAGTACCGTGTATCTTCATCATC The qPCR reaction system was prepared using the TransStart® Top Green qPCR SuperMix kit: 10 μL of 2×TransStart® Top Green qPCR SuperMix, 0.4 μL of primer F, 0.4 μL of primer R, 1 μL of cDNA, and 8.2 μL of RNase-free ddH2O.

[0044] The reaction program is set as follows: (1) 94 ℃ 30 s (2) 94 ℃ 5 s (3) 60 ℃ for 15 s (4) 72 ℃ 10 s The program has 40 loops.

[0045] The results are as follows Figure 5 As shown, the expression level of Hsp70 gene in peach aphids was significantly upregulated under osthol / HLDP treatment. The expression level of Hsp70 gene in the dsHsp70 treatment group was significantly downregulated at both 12 h and 24 h compared to the control group. The expression level of Hsp70 gene in the osthol / dsHsp70 / HLDP group was still significantly downregulated at 12 h, indicating that dsHsp70 can effectively silence the transcriptional expression of the target gene Hsp70. This verifies the effectiveness and target specificity of RNAi delivery, inhibits the upregulation of early response gene expression caused by drug treatment, and proves that the increase in peach aphid mortality is indeed due to the increased sensitivity to osthol after the Hsp70 gene is specifically silenced, rather than the non-specific toxicity of dsRNA.

[0046] Example 3: Preparation and application of hpHsp70 / ostrichin / HLDP compound nano-formulation (1) Synthesis of hpHsp70 The Hsp70 fragment, which offers the greatest improvement in rapid efficacy, was selected to synthesize hairpin RNA. After RNA extraction, reverse transcription, PCR amplification of the target fragment, ligation and transformation into competent E. coli cells, and extraction of the target fragment plasmid, the plasmid was used as a template for subsequent hpRNA synthesis. The main methods included the construction of the recombinant vector and the induction of hpRNA expression.

[0047] Construction of recombinant vectors: The selected gene fragment Hsp70 was constructed into a recombinant vector. The target gene hpHsp70 was synthesized in large quantities using the HT115(DE3)RNase III expression system. EcoRI and XhoI were selected as restriction enzyme sites for the vector backbone, and XbaI was selected as the restriction enzyme site for the inserted gene. The secondary structure of the target fragment with stem-loop structure was predicted using the UNA Fold website (https: / / www.unafold.org / mfold / applications / rna-folding-form.php) to screen for stem-loops of suitable length. Then, using the target fragment plasmid as a template, a 428 bp forward fragment L (SEQ ID NO:33) with a 100 bp stem-loop (SEQ ID NO:34) and a 328 bp inverse complementary fragment R (SEQ ID NO:21) were amplified using primers with restriction sites EcoRI-Hsp70L-F (SEQ ID NO:29), XbaI-Hsp70L-R (SEQ ID NO:30), XbaI-Hsp70R-F (SEQ ID NO:31), and XhoI-Hsp70R-R (SEQ ID NO:32). The amplified products were then recovered by gel electrophoresis. Two fragments were sent to Qingke Biotechnology for sequencing. The correctly sequenced fragment was ligated into the PMD19-T vector and then transformed into E. coli DH5α competent cells. After screening for positive clones and verification by bacterial PCR, the correctly identified positive clones were mixed into 5 mL of ampicillin liquid medium, shaken, and the plasmid was extracted. Using this plasmid as a template, primers with restriction enzyme sites were used for large-scale amplification again. The amplified products were recovered by agarose gel electrophoresis.

[0048] Primer sequences with restriction enzyme sites: EcoRI-Hsp70L-F (SEQ ID NO:29): CGGAATTCCGTGTCCGTTGAACGAGGCATA XbaI-Hsp70L-R (SEQ ID NO:30): GCTCTAGAGCCCGTTTGGCGTCGAATACCG XbaI- Hsp70R-F (SEQ ID NO: 31): GCTCTAGAGCGGGGTGGTCCTGTTACCTTG XhoI- Hsp70R-R (SEQ ID NO: 32): CCTCGAGGTGTCCGTTGAACGAGGCATA 428 bp forward fragment L with a 100 bp stem-loop (SEQ ID NO: 33): 5'-TGTCCGTTGAACGAGGCATACATAATACTACTTGGCTACTATTTAACGCGACTTTCAGTGTTATTCACGAATTTTCCATTGCGGTTTTATACACAAAGTGATTTTAAATCGTTATCTAATTGCTGTACTACCTGCGTATACGTTTTAGTTTGAATTATCAAATTAATTTTCGTATTAATTGTGCGTGAATTATATCGACAATTATTGGCGAAATGGTTGGAAAAACAGCCATCGGTATCGACCTGGGTACCACCTATTCCTGCGTGGGTGTCTGGCAACACGGAAAAGTAGAGATCATCGCCAACGATCAAGGTAACAGGACCACCCCGAGTTATGTGGCATTCACGGACACTGAACGGTTGATCGGCGACGGTGCTAAGAACCAGGTGGCATTGAACCCCGTCAATACGGTATTCGACGCCAAACGG-3' (wherein, GAGTTATGTGGCATTCACGGACACTGAACGGTTGATCGGCGACGGTGCTAAGAACCAGGTGGCATTGAACCCCGTCAATACGGTATTCGACGCCAAACGG is the 100 bp stem-loop sequence: SEQ ID NO: 34) 328 bp reverse fragment R (SEQ ID NO: 21): 5'-GGGGTGGTCCTGTTACCTTGATCGTTGGCGATGATCTCTACTTTTCCGTGTTGCCAGACACCCACGCAGGAATAGGTGGTACCCAGGTCGATACCGATGGCTGTTTTTCCAACCATTTCGCCAATAATTGTCGATATAATTCACGCAC AATTAATACGAAAATTAATTTGATAATTCAAACTAAAACGTATACGCAGGTAGTACAGCAATTAGATAACGATTTAAAATCACTTTGTGTATAAAACCGCAATGGAAAATTCGTGAATAACACTGAAAGTCGCGTTAAATAGTAGCCAAGTA GTATTATGTATGCCTCGTTCAACGGACA-3' pET-28a(+) plasmid was extracted, and its concentration was determined. Linearized pET-28a(+) vector and target fragment were obtained using a double enzyme digestion method.

[0049] Double digestion system 1: 5 μL rCutSmart™ Buffer, 1 μL EcoRI, 1 μL XhoI, 1 μg pET-28a(+) plasmid, and enzyme-free water to a final volume of 50 μL. Double digestion system 2: 5 μL rCutSmart™ Buffer, 1 μL XbaI, 1 μL EcoRI, 1 μg pET-28a(+) plasmid, and enzyme-free water to a final volume of 50 μL. Double digestion system 3: 5 μL rCutSmart™ Buffer, 1 μL XbaI, 1 μL XhoI, 1 μg pET-28a(+) plasmid, and enzyme-free water to a final volume of 50 μL.

[0050] Double enzyme digestion reaction: react at 37 ℃ for 4 h, and incubate at 65 ℃ for 20 min.

[0051] The ligation reaction was performed using T4 DNA ligase: 0.5 μL of T4 DNA ligase, 1 μL of 10×Ligation Buffer, 10 ng of vector, 100 ng of fragment L, 100 ng of fragment R, and RNase-free ddH2O were added to a final volume of 10 μL. The mixture was incubated overnight at 4°C. The ligated products were then recovered by agarose gel electrophoresis.

[0052] The gel-recovered ligation product was transformed into DH5α competent cells, plated on Kan-resistant plates, and incubated overnight at 37°C inverted mode. The next day, single colonies were picked for sequencing. Strains with correct sequencing were mixed into 5 mL of Kan liquid medium for culture and plasmids were extracted. Double enzyme digestion was used to verify the correctness of the recombinant vector.

[0053] Transform 1 μL of the recombinant vector into 100 μL of HT115(DE3) RNase III competent cells, add 500 μL of antibiotic-free liquid medium, and revive at 37 ℃ and 220 rpm for 1 h. Spread the culture onto Kan antibiotic plates, incubate overnight at 37 ℃ upside down, pick single colonies, mix them into 5 mL of Kan liquid medium, and extract plasmids for preservation.

[0054] Induction of hpRNA expression: 500 μL of the verified bacterial culture was inoculated into 50 mL of 50 mg / L Kan liquid culture medium at a volume ratio of 1:100. The culture was amplified and shaken at 37 ℃ and 220 rpm until the OD600 was around 0.5. Then, 1 mM IPTG was added to the final concentration after bacterial filtration and the culture was continued for 4 h.

[0055] hpRNA was crudely extracted from the bacterial culture using the lysozyme method: Lysozyme was added to the induced bacterial culture to a final concentration of 1.3 mg / mL, and the reaction was stopped by incubating in a 37 ℃ water bath for 30 min, followed by a 75 ℃ metal bath for 5 min. The crudely extracted hpRNA was purified using Tiangen's RNA purification kit: RK solution was prepared fresh, and β-mercaptoethanol was added to RK to a final concentration of 1%. 100 μL of bacterial culture was transferred to an enzyme-free 1.5 mL centrifuge tube, and 350 μL of RK solution was added. The tube was inverted and thoroughly mixed. 250 μL of anhydrous ethanol was added, and the mixture was immediately transferred to an adsorption column. The column was centrifuged at 12000 rpm for 1 min, and the waste liquid was discarded. 500 μL of wash buffer RW (with anhydrous ethanol added) was added, and the mixture was allowed to stand at room temperature for 2 min, then centrifuged at 12000 rpm for 1 min, and the waste liquid was discarded. The previous step was repeated. The tube was centrifuged at 12000 rpm for 3 min. Transfer the adsorption column to a new 1.5 mL enzyme-free centrifuge tube, add 20 μL of enzyme-free water, let stand for 1 min, and then centrifuge. The purified product was subjected to agarose gel electrophoresis and concentration detection, and stored at -20 ℃.

[0056] (2) Screening of fusion ratio The osthol / HLDP complex was prepared at a mass ratio of 1:2.85. Separately, 100 μg of dseGFP solution was prepared and incubated at room temperature for 5 min. The osthol / HLDP complex and dseGFP were then mixed at mass ratios of 3:1, 2:1, 1:1, 1:2, and 1:3 (HLDP:dseGFP as the reference). The mixture was incubated at room temperature for another 5 min. The coating effect was detected by agarose gel electrophoresis, and the optimal ratio was selected based on the imaging results.

[0057] Electrophoresis results as follows Figure 6 As shown, complete encapsulation of dsRNA can be achieved when the osthol / HLDP complex binds to dsRNA at a 1:1 mass ratio.

[0058] (3) Preparation of hpHsp70 / ostrichin / HLDP compound nano-formulation First, a half-lethal concentration of osthol / HLDP nanoparticles was prepared. Then, the required volume of hpRNA was calculated based on the binding mass ratio of 1:1 and the concentration of hpRNA. After mixing the three for 5 minutes, the hpRNA / osthol / HLDP compound nanoparticles were obtained.

[0059] (4) Indoor bioassay of hpHsp70 / ostrichin / HLDP compound nano-formulation Indoor bioassay was conducted using a spray method: The osthol / HLDP / hpHsp70 compound nano-formulation and the osthol / HLDP complex were evenly sprayed onto radish leaves infested with third-instar peach aphids using a small spray bottle. HLDP and unloaded pET28-BL21 were used as controls. Each treatment was replicated three times, with 20 aphids per replicate. The mortality rate was recorded 5 days after treatment.

[0060] The results are as follows Figure 7 As shown, compared to using osthol / HLDP alone, the compound formulation with added hpHsp70 showed an insecticidal effect increased by more than 20% within 5 days after spraying. This demonstrates that the nano-formulation of this invention is equally efficient in simulated field spraying scenarios and has extremely high prospects for industrial application.

[0061] (5) Determination of interference effect Peach aphids were treated with a nano-formulation of osthol / HLDP / hpHsp70 and sprayed, with dseGFP and osthol nano-formulation as controls. Samples were collected after 24 h, and RNA was extracted, reverse transcribed into cDNA, and diluted 4-fold. The diluted cDNA was used as a template, with EF-1 as an internal control gene and Hsp70 as the target gene for qRT-PCR. Each treatment included three biological replicates, and each biological replicate included three technical replicates. Based on the CT value of each reaction well, 2... -ΔΔCTThe relative expression levels of the target gene and internal reference gene in each group of samples were calculated using the method.

[0062] Quantitative primer sequences: EF1-F (SEQ ID NO:25):GTACTTCCCAGGCCGATTGT EF1-R (SEQ ID NO:26):AGGTGAAGGCCAATAGAGCG Q-Hsp70-F (SEQ ID NO:27):ACAATCAGTTGGCAGACAAGGAAG Q-Hsp70-R (SEQ ID NO:28): GTCCAGTACCGTGTATCTTCATCATC The qPCR reaction system was prepared using the TransStart® Top Green qPCR SuperMix kit: 10 μL of 2×TransStart® Top Green qPCR SuperMix, 0.4 μL of primer F, 0.4 μL of primer R, 1 μL of cDNA, and 8.2 μL of RNase-free ddH2O.

[0063] The reaction program is set as follows: (1) 94 ℃ 30 s (2) 94 ℃ 5 s (3) 60 ℃ for 15 s (4) 72 ℃ 10 s The program has 40 loops.

[0064] The results are as follows Figure 8 As shown, compared with the control group, the expression level of Hsp70 gene in the osthol commercial drug / HLDP group was significantly increased, proving that Hsp70 is a key stress defense factor for peach aphids to resist osthol poisoning. In contrast, the expression level of Hsp70 gene in the osthol commercial drug / HLDP / hpHsp70 compound nano-formulation treatment group was significantly downregulated compared with the control group, proving that the hpHsp70 expressed by the engineered bacteria can play a good insecticidal synergistic role by significantly inhibiting the stress upregulation of Hsp70 induced by osthol.

[0065] In summary, this invention uses HLDP as a carrier to achieve simultaneous delivery of osthol and RNAi drugs. Through two-stage synergistic effects, it greatly improves the rapid action and insecticidal activity of osthol. It features a simple coating process, low cost, and the formulation can be applied by conventional spraying, making it convenient to operate. It has good prospects for industrial application and is suitable for organic agriculture and green pest control systems.

[0066] The embodiments described above are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

Claims

1. A two-stage synergistic compound nano-formulation of osthol based on nanodelivery and RNA interference technology, characterized in that, The nanoformulation comprises osthol, a hydrophilic and lipophilic amphiphilic block polymer nanocarrier, and at least one RNA interference substance; the RNA interference substance targets at least one of the following target genes: heat shock protein 70 gene, RNA binding protein gene, or glucose dehydrogenase gene.

2. The nano-formulation according to claim 1, characterized in that: The structural formula of the nanocarrier is: ; Preferably, the mass ratio of osthol to the nanocarrier is 1:2~5; Preferably, the mass ratio of the RNA interference substance to the complex formed by osthol and the nanocarrier is 1:0.5~1.

5.

3. The nano-formulation according to claim 1, characterized in that: The RNA interference substance is dsRNA or hpRNA; And / or, the RNA interference substance includes dsRNA of the target gene Hsp70, comprising a sense strand with a nucleotide sequence as shown in SEQ ID NO:9 and an antisense strand with a nucleotide sequence as shown in SEQ ID NO:21; And / or, the RNA interference substance comprises hpRNA of the target gene Hsp70, comprising a sense strand with a nucleotide sequence as shown in SEQ ID NO:33 and an antisense strand with a nucleotide sequence as shown in SEQ ID NO:21; And / or, the RNA interference substance includes dsRNA of the target gene RBP, comprising a sense strand with a nucleotide sequence as shown in SEQ ID NO:10 and an antisense strand with a nucleotide sequence as shown in SEQ ID NO:22; And / or, the RNA interference substance includes dsRNA of the target gene GDH-1, comprising a sense strand with a nucleotide sequence as shown in SEQ ID NO:11 and an antisense strand with a nucleotide sequence as shown in SEQ ID NO:23; And / or, the RNA interference substance includes dsRNA of the target gene GDH-3, comprising a sense strand with a nucleotide sequence as shown in SEQ ID NO:12 and an antisense strand with a nucleotide sequence as shown in SEQ ID NO:

24.

4. The nano-formulation according to claim 3, characterized in that, The method for preparing the dsRNA includes the following steps: (1) Using peach aphid cDNA as a template, PCR amplification was performed using primers to obtain the target gene fragment. The primer sequence is at least one of the sequences shown in SEQ ID NO:1-8. (2) Using the target gene fragment as a template, PCR amplification is performed using primers with the T7 promoter sequence to obtain a DNA template for synthesizing dsRNA, wherein the primer sequence is at least one of the sequences shown in SEQ ID NO:13-20; (3) Using a DNA template as a template, T7 RNA polymerase is used for in vitro transcription. The transcription product is digested with DNase, precipitated and washed to obtain the dsRNA.

5. The nano-formulation according to claim 3, characterized in that, The method for preparing the hpRNA includes the following steps: (1) Using the Hsp70 gene fragment as a template, the forward fragment L and the reverse complementary fragment R are amplified using primers with restriction enzyme sites; the nucleotide sequence of the forward fragment L is shown in SEQ ID NO:33, the nucleotide sequence of the reverse fragment R is shown in SEQ ID NO:21, the forward fragment L and the reverse fragment R are connected by a stem-loop sequence, and the primer sequence with restriction enzyme sites is at least one of the sequences shown in SEQ ID NO:29-32; (2) The amplification product obtained in step (1) is digested with two enzymes and then ligated into an expression vector linearized with restriction endonuclease to obtain a recombinant expression vector; (3) The recombinant expression vector obtained in step (2) was transformed into HT115(DE3) RNase III-deficient Escherichia coli, IPTG was added to induce expression, the bacterial cells were collected, extracted and purified to obtain the hpRNA; Preferably, the stem-loop sequence is 50-200 bp in length, more preferably 100 bp, and even more preferably, the stem-loop sequence is the sequence shown in SEQ ID NO:

34.

6. The method for preparing nano-formulations according to any one of claims 1-5, characterized in that, The preparation method includes the following steps: (1) Osthol was mixed and incubated with hydrophilic and lipophilic amphiphilic block polymer nanocarriers to obtain osthol / carrier complex; (2) Mix the RNA interference substance with the osthol / carrier complex, with or without the addition of an auxiliary binding agent, and allow the reaction to proceed until the nano-formulation is obtained; Preferably, in step (1), the incubation temperature is room temperature and the incubation time is 5~15 min; Preferably, in step (2), the auxiliary binding agent is an alkyl glycoside.

7. A kit for controlling peach aphids, characterized in that: The kit includes the nanoformulations as described in any one of claims 1-5.

8. The application of the nano-formulation as described in any one of claims 1-5 or the kit as described in claim 7 in the control of agricultural pests.

9. The application according to claim 8, characterized in that, The application includes at least one of the following: (1) Application in inhibiting the expression of Hsp70, RBP and GDH genes in peach aphid; (2) Application in the control of peach aphids; (3) Application in enhancing the insecticidal effect of osthol.

10. The application of RNA interference substances in enhancing the insecticidal effect of osthol, characterized in that, The RNA interference substance targets at least one of the following target genes: heat shock protein 70 gene, RNA binding protein gene, glucose dehydrogenase gene; Preferably, the RNA interference substance is dsRNA or hpRNA; And / or, the RNA interference substance includes dsRNA of the target gene Hsp70, comprising a sense strand with a nucleotide sequence as shown in SEQ ID NO:9 and an antisense strand with a nucleotide sequence as shown in SEQ ID NO:21; And / or, the RNA interference substance comprises hpRNA of the target gene Hsp70, comprising a sense strand with a nucleotide sequence as shown in SEQ ID NO:33 and an antisense strand with a nucleotide sequence as shown in SEQ ID NO:21; And / or, the RNA interference substance includes dsRNA of the target gene RBP, comprising a sense strand with a nucleotide sequence as shown in SEQ ID NO:10 and an antisense strand with a nucleotide sequence as shown in SEQ ID NO:22; And / or, the RNA interference substance includes dsRNA of the target gene GDH-1, comprising a sense strand with a nucleotide sequence as shown in SEQ ID NO:11 and an antisense strand with a nucleotide sequence as shown in SEQ ID NO:23; And / or, the RNA interference substance includes dsRNA of the target gene GDH-3, comprising a sense strand with a nucleotide sequence as shown in SEQ ID NO:12 and an antisense strand with a nucleotide sequence as shown in SEQ ID NO:24.

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