A multi-component nanocomposite for controlling pesticide resistance of peach aphid and its preparation method and application

Abstract

The present invention discloses a multi-element nanocomposite for controlling pesticide resistance of peach aphid, and its preparation method and application, clarifies the key regulatory role of the nuclear transcription factor erythroid 2-related factor 2 (nrf2)-small Maf protein (sMaf) pathway in broad-spectrum insecticide metabolic resistance, and proves nrf2 and sMaf Simultaneous downregulation can further inhibit the expression of detoxification genes, reduce the activity of detoxification enzymes, and provide a nearly spherical multi-component nanoprotectant IMI / HLDP / ds with a particle size of 256 nm. nrf2 ‑ sMaf The root absorption test showed that the insecticidal activity of the complex was through simultaneous inhibition of nrf2 and sMaf The normalized synergistic ratio was 1.65-2.29 times higher than that of nrf2-sMaf. This study elucidated the key regulatory role of the nrf2-sMaf pathway in the broad-spectrum metabolic resistance of peach aphid and developed a self-assembled multi-component nanoprotectant to achieve sustainable management of the broad-spectrum metabolic resistance of peach aphid.

Description

Technical Field

[0001] The invention relates to the technical field of pest control, and in particular to a multi-component nanocomposite for controlling pesticide resistance of peach aphid, and a preparation method and application thereof. Background Art

[0002] Myzus persicae Myzus persicae ) is one of the top ten pests in the world, capable of harming more than 100 species of plants from more than 40 families. It is a typical polyphagous insect and an important agricultural pest. Due to its short development period, strong reproductive ability and wide distribution, the control of peach aphid has long relied mainly on chemical / synthetic pesticides. However, the widespread and frequent use of pesticides has led to a high degree of resistance to almost all types of insecticides. Aphids are one of the 12 most resistant insects on record, with 7 different resistance mechanisms described, and they have developed resistance to 75 compounds. Studies have shown that aphids are also an excellent model for studying a range of basic ecological and evolutionary topics, including insect-plant interactions, virus transmission, phenotypic plasticity, symbiotic relationships and insecticide resistance.

[0003] Aphid resistance is mainly mediated by target and metabolic mechanisms. Therefore, controlling the metabolic resistance of pests and increasing their sensitivity to insecticides is one of the effective ways to deal with pest resistance. However, due to the diversity of detoxification genes, it is still challenging to address broad-spectrum metabolic resistance. Therefore, it is imperative to explore more effective and powerful target genes. In vertebrates, nuclear transcription factor erythroid 2-related factor 2 ( Nrf2 ) is a key transcription factor that regulates cellular antioxidant stress and plays an important role in inducing antioxidant responses and drug detoxification. It is involved in a variety of biological processes such as redox balance, drug metabolism and excretion, energy metabolism, cell apoptosis and aging. Studies have shown that Nrf2 It is closely related to the occurrence, development, and metastasis of major diseases such as tumors and liver diseases, and may also be closely related to the tumor resistance mechanism. Nrf2 is released and translocated to the nucleus, where it binds to downstream detoxification genes, including P450 , GSTs , CarEs , POD and ATP. Therefore, Nrf2 The gene may be a good target for improving broad-spectrum insecticide sensitivity by regulating these detoxification genes. Nrf2 It may play an indispensable regulatory role in pest resistance, but its signaling pathway involved in insecticide response is still unclear, and its effective application needs to be explored.

[0004] RNA interference (RNAi), as a technique to specifically knock out or shut down the expression of a specific gene, has been widely used in the identification of gene function and the treatment of human diseases. In insects, RNAi has been widely used to analyze gene function and develop RNA insecticides. However, double-stranded (dsRNA) is easily degraded by RNases, which reduces its stability, and its delivery into the cytoplasm is a challenge that limits the efficiency of RNAi. These technical bottlenecks have hindered the design and development of RNA insecticides. In recent years, the rapid development of nanotechnology has led to the development of precise drug delivery systems for specific sites. These nanocarrier-based delivery systems can increase the solubility of drugs and improve drug targeting. In the field of agriculture, nanocarrier-based delivery systems can load exogenous drugs for efficient delivery. These systems achieve efficient delivery of dsRNA while protecting it from ribonuclease degradation by activating the clathrin-mediated endocytosis pathway. Therefore, they significantly enhance the environmental stability of dsRNA and effectively stabilize the expression level of target genes through RNAi. At the same time, the nanocarrier delivery system can also be used to load insecticides and realize the nano-ization of insecticides, thereby improving the translocation of insecticides on the cuticle of insects, increasing insecticidal efficiency, reducing the use of insecticides, and providing a new method for ecological pest management. Therefore, the use of nanocarriers to co-load dsRNA and insecticides is expected to control insect metabolic resistance and increase sensitivity to insecticides. Summary of the invention

[0005] In view of the above-mentioned defects of the prior art, the present invention proposes a multi-component nanocomposite for controlling the pesticide resistance of peach aphid and its preparation method and application to solve the problems raised by the above-mentioned background technology.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] A multi-component nanocomposite for controlling the insecticide resistance of peach aphid, comprising the following components: imidacloprid, carrier HLDP and ds Nrf2 - sMaf ,ds Nrf2 - sMaf The nucleotide sequence of the gene is shown in SEQ ID NO:1.

[0008] Preferably, the complex formed by imidacloprid and the carrier HLDP is Nrf2 - sMaf The mass ratio is (1~5):1.

[0009] Preferably, the carrier HLDP is a hydrophilic-lipophilic diblock polymer, and its structural formula is as follows:

[0010] .

[0011] Preferably, the ds Nrf2 - sMaf It is obtained by chemical synthesis and efficient expression of engineering bacteria; the chemical synthesis and efficient expression of engineering bacteria include the following steps:

[0012] (1) Using primers containing the T7 promoter sequence, PCR was used to amplify Nrf2 and sMaf The gene fragment was reverse transcribed into ds Nrf2 and sMaf ;

[0013] (2) Utilization z / f-Nrf2 / sMaf Fusion primers were used to amplify Nrf2 and sMaf The forward complementary sequence and reverse complementary sequence of the sequence were then fused using fusion PCR. Nrf2 and sMaf forward complementary sequence and reverse complementary sequence;

[0014] (3) Use T4 DNA ligase to Nrf2 - sMaf -forward sequence sum Nrf2 - sMaf - antisense sequence was connected and introduced into the expression vector for culture; a single colony was picked and cultured in 5 mL LB liquid medium containing 50 mg / L kanamycin. 600 When the nm value was 0.4, isopropyl β- d -1-thiogalactoside (IPTG) was added for incubation, and then extracted with 75% ethanol to obtain ds Nrf2 - sMaf .

[0015] Preferably, the multi-component nanocomposite contains imidacloprid IMI, carrier HLDP, ds Nrf2 - sMaf The concentration ratio is 9.598:40.657:10.164 mg / L.

[0016] Preferably, the multi-component nanocomposite contains imidacloprid IMI, carrier HLDP, ds Nrf2 - sMaf The mass ratio is 15:68:17.

[0017] Preferably, a method for preparing a multi-component nanocomposite for controlling pesticide resistance of peach aphid comprises titrating imidacloprid with HLDP to form an imidacloprid / HLDP complex; Nrf2 - sMaf Titrate the imidacloprid / HLDP complex to form imidacloprid / HLDP / ds Nrf2 - sMafComplex, that is, multi-component nanocomposite.

[0018] Preferably, the multi-component nanocomposite of the present application is used in the preparation of a product for controlling pesticide-resistant green peach aphid.

[0019] Preferably, the multi-component nanocomposite of the present application is used in protecting rapeseed leaves.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] (1) The present application provides a new cationic polymer, called hydrophilic lipophilic diblock polymer (HLDP), which has hydrophobic and hydrophilic functional groups, allowing it to assemble with exogenous substances. The nanocarrier-based delivery system can load exogenous drugs for efficient delivery and significantly enhance the environmental stability of dsRNA.

[0022] (2) The present invention has developed a new self-assembled multi-component nanoprotective agent (IMI / HLDP / ds Nrf2 - sMaf Compound) can effectively control the pest of resistant peach aphid on rapeseed leaves. After external application and root absorption for 48 hours, IMI / HLDP / ds Nrf2 - sMaf The insecticidal activity of the complexes reached 77.33% and 74.67%, respectively, and the normalized synergistic ratios were 1.87-2.25 and 1.65-2.29, respectively, indicating that the use of nanocarriers to co-load dsRNA and insecticides can control insect metabolic resistance and increase sensitivity to insecticides. Nrf2 - sMaf Complex and IMI / HLDP / ds Nrf2 The topical dosage of the complex was 14.30% and 40.93% of that of IMI, respectively. The root absorption rates of the two solutions were 29.77% and 40.93% of that of IMI alone, respectively. The above results show that IMI / HLDP / ds Nrf2 - sMaf The biological activity of the complex is significantly higher than that of IMI alone, and can reduce the amount of pesticide used.

[0023] (3) The present invention provides a new RNAi target for the sustainable management of broad-spectrum metabolic resistance of peach aphid, reduces the use of chemical pesticides, and has great application prospects in environmentally friendly agriculture. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 for Nrf2 Expression changes of regulated detoxification genes, including Figure 1 a is Nrf2Schematic diagram of the operation of RNA-seq analysis to detect the expression level of detoxification genes in aphids after RNAi; Figure 1 b is ds Nrf2 / HLDP agarose gel retardation test results; Figure 1 c is ds eGFP 、ds eGFP / HLDP,ds Nrf2 / HLDP complex, ds Nrf2 RNAi efficiency; Figure 1 d is ds Nrf2 / HLDP complex treatment after aphid gene changes; Figure 1 e for ds Nrf2 KEGG analysis of aphids after treatment with / HLDP complex; Figure 1 f is Nrf2 Heat map analysis of key detoxification genes in aphids after RNAi; Figure 1 g is Nrf2 Aphid detoxification genes after RNAi P450s , GSTs , CarEs and PODs FPKM value;

[0025] Figure 2 The regulation of detoxification genes by the Nrf2-sMaf pathway; Figure 2 a is Nrf2 and sMaf The protein forms heterodimers and binds to ARE / EpRE to regulate the expression of detoxification genes; Figure 2 b is Nrf2 RNAi sMaf FPKM value; Figure 2 c After qRT-PCR test sMaf Received Nrf2 Positive regulation of Figure 2 dg is Nrf2 and sMaf After co-targeting P450 , GST , CarE and POD Expression level change diagram; Figure 2 h is Nrf2 and sMaf After co-targeting P450s , GSTs , CarEs and PODs Enzyme activity change diagram;

[0026] Figure 3 For IMI / HLDP / ds Nrf2 - sMaf Schematic diagram of the self-assembly mechanism of the complex; Figure 3 a Load IMI and ds for HLDP Nrf2 - sMaf Process diagram; Figure 3 b is the Gibbs free energy change value (ΔG) of the interaction between HLDP and IMI detected by ITC method; Figure 3 c is the potential change diagram after IMI and HLDP complexation, ds Nrf2 - sMaf Results of agarose gel retardation assay with IMI / HLDP complex; Figure 3 d is the IMI / HLDP complex and ds Nrf2 - sMaf Diagram of changes in hydrogen bonds and van der Waals forces during the binding process; Figure 3 e is IMI / HLDP / ds Nrf2 - sMaf The morphology and particle size of the composites;

[0027] Figure 4 For IMI / HLDP / ds Nrf2 - sMaf The adhesion performance and delivery efficiency of the composite are shown in FIG. Figure 4 a is ddH2O, HLDP, IMI, IMI / HLDP / ds Nrf2 - sMaf Contact angle diagram of the composite on rapeseed leaves; Figure 4 b is ddH2O, HLDP, IMI, IMI / HLDP / ds Nrf2 - sMaf Surface tension diagram of the complex on rapeseed leaves; Figure 4 c is ddH2O, HLDP, IMI, IMI / HLDP / ds Nrf2 - sMaf Contact area diagram of the composite on rapeseed leaves; Figure 4 d is IMI, IMI / HLDP / ds Nrf2 - sMaf Retention rate of the complex on rapeseed leaves; Figure 4 e is IMI, IMI / HLDP / ds Nrf2 - sMaf Plant uptake of the complex on rapeseed leaves; Figure 4 f is ddH2O, HLDP, IMI, IMI / HLDP / ds Nrf2 - sMaf Retention of the complex on rapeseed leaves; Figure 4 g is IMI / HLDP, ds eGFP 、ds eGFP / HLDP、IMI / HLDP / ds eGFP Fluorescence intensity of the complex on rapeseed leaves; Figure 4 h is IMI / HLDP, ds eGFP 、ds eGFP / HLDP、IMI / HLDP / ds eGFP Transport of the complex across the cell membrane in rapeseed leaves; Figure 4 i is IMI / HLDP / ds Nrf2 - sMaf Schematic diagram of the delivery ability of the complex in vivo and in vitro;

[0028] Figure 5 For IMI / HLDP / ds Nrf2 - sMaf The bioinsecticide activity of the compound against green peach aphid; wherein Figure 5 a is a process diagram of the topical preparation for green peach aphid; Figure 5 b is external treatment for peach aphid IMI / HLDP / ds Nrf2 - sMaf Insecticidal activity of the complex; Figure 5 c is external treatment for peach aphid IMI / HLDP / ds Nrf2 - sMaf Normalized synergistic ratio of the complex; Figure 5 d is IMI for external use against peach aphid / ds eGFP 、IMI / ds Nrf2 - sMaf 、IMI / HLDP / ds eGFP 、IMI / HLDP / ds Nrf2 - sMaf Processing Nrf2 Expression changes; Figure 5 e is IMI / ds for external use on peach aphid eGFP 、IMI / ds Nrf2 - sMaf 、IMI / HLDP / ds eGFP 、IMI / HLDP / ds Nrf2 - sMaf Processing sMaf Expression changes; Figure 5 f is IMI / ds for external use on peach aphid eGFP 、IMI / ds Nrf2 - sMaf 、IMI / HLDP / ds eGFP 、IMI / HLDP / ds Nrf2 - sMaf Detoxification and metabolism genes ( P450s , GSTs , CareE s and PODs ) expression changes; Figure 5 g is a process diagram of the green peach aphid root suction preparation; Figure 5h is the peach aphid root sucking treatment IMI / HLDP / ds Nrf2 - sMaf Insecticidal activity of the complex; Figure 5 i is peach aphid root sucking treatment IMI / HLDP / ds Nrf2 - sMaf Normalized synergistic ratio of the complex; Figure 5 j is the IMI / ds of the peach aphid root absorption eGFP 、IMI / ds Nrf2 - sMaf 、IMI / HLDP / ds eGFP 、IMI / HLDP / ds Nrf2 - sMaf Processing Nrf2 Expression changes; Figure 5 k is the IMI / ds of the peach aphid root absorption eGFP 、IMI / ds Nrf2 - sMaf 、IMI / HLDP / ds eGFP 、IMI / HLDP / ds Nrf2 - sMaf Processing sMaf Expression changes; Figure 5 l is the IMI / ds of the peach aphid root absorption eGFP 、IMI / ds Nrf2 - sMaf 、IMI / HLDP / ds eGFP 、IMI / HLDP / ds Nrf2 - sMaf Detoxification and metabolism genes ( P450 , GST , CareE and POD ) expression level changes. DETAILED DESCRIPTION

[0029] In order to enable those skilled in the art to better understand the technical content of the present invention, the technical solution of the present invention is further described in detail below in conjunction with specific implementation methods.

[0030] Reagents and manufacturers: IMI standard and commercial IMI were purchased from Shanghai McLean Biochemical Technology Co., Ltd., China and Bayer AG, Germany, respectively. Chemical reagents for the synthesis of HLDP included: ε-caprolactone (ε-CL), Sn(Oct)2, n-butanol, 2-bromo-2-methylpropyl bromide (BIBB), trimethylamine (TEA, 99%) purchased from Heowns BioChem Technologies (China); 2-(dimethylamino)ethyl methacrylate (DMAEMA, 99%) purchased from Energy Chemical (China); tetrahydrofuran (THF), N,N,N',N',N',N"-pentamethyldiethyltriamine (PMDETA, 98%) and CuBr (99.999%) purchased from Sigma-Aldrich (USA). Other chemical reagents were purchased from Beijing Chemical Plant (China).

[0031] Experimental example

[0032] 1 Methods

[0033] 1.1 Chemical synthesis of green peach aphid dsRNA

[0034] To synthesize dsRNA, we collected peach aphids from rapeseed leaves, extracted total RNA from peach aphids using the RNA Easy Fast Tissue / Cell Kit (TaKaRa, China), and used 1 μg of RNA as a template to reverse transcribe single-stranded cDNA using the PrimeScriptTM RT Reagent Kit with gDNA Eraser (Perfect Real Time) (TaKaRa, Japan) according to the kit instructions. The obtained single-stranded cDNA was used as a template to amplify the primers containing the T7 promoter sequence by polymerase chain reaction (PCR). Nrf2 and sMaf The gene fragment was cloned into pMD19T-Vector (TAKARA) and transformed into Escherichia coli DH5α strain (TSINGKE, China). The plasmid was extracted and verified by Sanger sequencing, and then dsRNA was synthesized using T7 RiboMAX Express RNAi System (Promega, USA). Nrf2 The nucleotide sequence of the gene is shown in SEQ ID NO:2 (Genbank accession number: XM_022304789.1). sMafThe nucleotide sequence of the gene is shown in SEQ ID NO:3 (Genbank accession number: XM_022317769.1), which lacks enhanced green fluorescent protein ( eGFP ) gene, the sequence information is shown in SEQ ID NO:4 (Genbank accession number: XM_013480425.1). This study synthesized ds eGFP As a control. Primer information is shown in Table 1:

[0035] Table 1 Nrf2 , sMaf and eGFP Primer information

[0036]

[0037] 1.2 Preparation and structure of hydrophilic-lipophilic diblock polymer (HLDP)

[0038] First, ε-CL (6.16 g, 54 mmol), n-butanol (100 mg, 1.35 mmol) and Sn (Oct)2 (310 mg) were added to the reaction tube, incubated in a 90℃ oil bath for 9 h, and cooled with liquid nitrogen to stop the reaction. Dichloromethane (30 ml) was added, and cold methanol was added for precipitation to obtain a white solid powder of linear polycaprolactone PCL. BIBB (3 g, 13.04 mmol), THF (30 mL), and TEA (4 g, 40 mmol) were added dropwise to the PCL (3 g, 0.58 mmol) solution, and ice-cold methanol was added to terminate the reaction. The precipitate was collected by filtration to obtain a white powder of PCL-Br. Finally, PCL-Br (100 mg, 0.04 mmol) and DMAEMA (0.87 g, 6.07 mmol) were dissolved in 2 mL of dry THF, and CuBr (15 mg, 0.1 mmol) and PMDETA (37 mg, 0.21 mmol) were added. The polymerization reaction was carried out at 65 °C for 5 h. After the reaction, the product was dialyzed and freeze-dried to obtain a white solid product of HLDP.

[0039] The chemical structure of HLDP is:

[0040] .

[0041] 1.3 Nrf2 RNAi and RNA-seq analysis, explore Nrf2 Potential functions

[0042] To determine ds Nrf2The best combined mass ratio with HLDP was 1 μg ds Nrf2 The samples were incubated with HLDP at a mass ratio of 1:0, 1:2, 1:3, 1:4, 1:5, and 1:6 at room temperature for 15 min, and ds Nrf2 In the following experiments, dsRNA / HLDP complexes were prepared at an optimal mass ratio of 1:4. Nrf2 / HLDP complex, ds eGFP / HLDP complex, ds Nrf2 and ds eGFP The solution (300 ng dsRNA per aphid) was applied to the back of the third-instar aphids. Total RNA was extracted 24 h later and reverse transcribed into cDNA. Real-time fluorescence quantitative PCR (qRT-PCR) was used to detect Nrf2 The expression levels of EF1 and EF2 were determined using the Step One Plus Real-time Fluorescence Quantitative PCR System (Applied Biosystems, USA) and SuperReal PreMix Plus (SYBR Green) (Tiangen Biotech, China). The relative mRNA level of each target gene was normalized to the abundance of the EF1 gene (GenBank accession: XM_022311019.1) using the 2-ΔΔCT method. Three independent samples were included in each treatment.

[0043] Also collect the above ds Nrf2 / HLDP complex and ds eGFP / HLDP complex-treated aphids, total RNA was extracted as above. RNA-seq was performed by Novogene (China, https: / / cn.novogene.com / ) according to standard protocols. RNA sequencing libraries were prepared using NEBNext® UltraTM RNA library Prep Kit for Illumina® (NEB, Beverly), and sequencing was performed on the Illumina Novaseq 6000 platform. The expression level of genes was estimated by the number of fragments per kilobase per million fragments transcribed (FPKM). P ≤ 0.05 and |log2 (fold change)| ≥ 1 were differentially expressed genes. The Kyoto Encyclopedia of Genes and Genomes (KEGG), a comprehensive database that integrates genomic, chemical, and system functional information (http: / / www.genome.jp / kegg / ), was used to analyze the enrichment of differentially expressed genes (DEGs). The heat map provides an intuitive visualization of sample differences between groups and sample repetitions within groups. The generation of the heat map was based on the correlation coefficient calculated based on the FPKM value. Each treatment was repeated three times independently.

[0044] 1.4 Highly efficient expression of dsRNA by engineered bacteria

[0045] The pET28-BL21 (DE3) RNase III-expression system was used to produce ds Nrf2 and ds Nrf2 - sMaf ,according to sMaf and Nrf2 gene sequence, design specific primers with restriction sites, and construct expression vectors. Nrf2 - sMaf The synthesis of Nrf2 / sMaf The fusion primers amplified Nrf2 and sMaf The forward complementary fragment and the reverse complementary fragment of the sequence. There is an overlap of about 20 bp between the forward and reverse complementary sequences. Fusion PCR was used to fuse Nrf2 and sMaf The forward complementary sequence and reverse complementary sequence of Nrf2 - sMaf - Forward and Nrf2 - sMaf - Antisense sequence was connected to the linearized expression vector pET28a(+). Incubate at 37℃ overnight. Select a single colony and culture it in 5 mL LB liquid medium supplemented with 50 mg / L kanamycin (Kan) at 37℃ overnight. 600 When the nm value reached 0.4, isopropyl β- d -1-thiogalactoside (IPTG) (1 mM) was added, incubated for 4 h, and extracted with 75% ethanol to obtain ds Nrf2 and ds Nrf2 - sMaf The primer information is shown in Table 2, and the underline indicates the restriction site.

[0046] Table 2 ds Nrf2 f and ds Nrf2 - sMaf Primer information

[0047]

[0048] 1.5 Analysis of the effects of the Nrf2-sMaf pathway on pesticide detoxification and metabolism

[0049] The prepared dsRNA was incubated with HLDP to form ds Nrf2 / HLDP complex, ds sMaf / HLDP complex and ds Nrf2 - sMaf / HLDP complex, 0.2 µL of each solution (300 ng dsRNA per aphid) was applied to the back of 3rd instar aphids, and total RNA was extracted 24 h after external application. sMaf and P450 , GST , CareE , POD The expression of detoxification and metabolic genes was measured, with EF1 gene (XM_022311019.1) as the internal reference gene. Each treatment included three independent samples.

[0050] This study determined the ds Nrf2 - sMaf / / HLDP complex, ds Nrf2 / HLDP complex and ds eGFP Effects of the HLDP / β-actin complex on the detoxification enzyme activities of the above aphids. 0.1 g of aphid sample was homogenized in 1 mL of extraction solution to prepare crude enzyme solution (Suzhou Keming Biotechnology Co., Ltd., China). P450 , GST , CareE and POD The activities were determined by microassay according to the operating procedures of the kits (Suzhou Keming Biotechnology Co., Ltd., China). The total protein content was quantified using the BCA protein assay kit (Beijing Biotech Biotechnology Co., Ltd., China). The activity of each detoxification enzyme was determined three times.

[0051] 1.6 IMI / HLDP / ds Nrf2-sMaf Self-assembly mechanism and morphology characteristics of the composite

[0052] Isothermal titration calorimetry (ITC) can accurately reflect the main interaction force between two substances. To determine the interaction between IMI and HLDP, 0.5 mM IMI was titrated with 0.05 mM HLDP in Nano ITC (TA Instruments Waters, USA). The interaction heat of each injection was calculated by integrating each titration peak using Origin7 software (OriginLab Co., USA). The experimental temperature was 25°C, and the ΔG calculation formula was ΔG = ΔH - TΔS. In order to further clarify the interaction between IMI / HLDP complex and ds Nrf2 - sMaf The assembly mechanism of Nrf2 - sMaf 0.0414 μM IMI / HLDP complex was titrated and the thermodynamic parameters were recorded.

[0053] To further verify the relationship between IMI / HLDP complex and ds Nrf2- sMaf The assembly property of 1 μg ds Nrf2 - sMaf Incubate with IMI / HLDP complex at a mass ratio of 1:0, 1:1, 1:2, 1:3, 1:4 and 1:5 for 15 min, and perform agarose gel retardation assay. Nrf2 - sMaf IMI in complex, IMI / HLDP complex with ds Nrf2 - sMaf With the best quality ratio (HLDP: ds Nrf2 - sMaf = 4:1) and dialyzed for 12 h. The concentration of IMI outside the dialysis bag was quantified using ACQUITYUPLC h-class (Waters, USA). The HPLC column was BEH C18 (2.1 × 100 mm, 1.7 μm), the column temperature was 35 °C, the mobile phase was acetonitrile and 0.1% formic acid aqueous solution, the injection volume was 5 μL, and the detection wavelength was 270 nm. A standard curve was constructed based on the chromatographic data of IMI dilutions (0, 4, 10, 20, 40, 100 μg / mL) to calculate IMI / HLDP / ds Nrf2 - sMaf The mass of IMI in the complex. Nrf2 - sMaf IMI / HLDP / ds was prepared by incubation at a mass ratio of 15:68:17. Nrf2 - sMaf Complex.

[0054] The concentrations of IMI (1 mg / mL), IMI / HLDP complex (IMI concentration of 1 mg / mL), and IMI / HLDP / ds were determined at 25 °C using a particle size analyzer and zeta potential analyzer (Anton Paar, Austria). Nrf2 - sMaf Particle size and zeta potential of the composite (IMI concentration was 1 mg / mL). Each treatment included three independent samples. The morphological characteristics of the above samples were further studied using a transmission electron microscope (JEOL-F200, Japan). 5 μL of each sample was dropped on a copper grid and observed after air drying.

[0055] 1.7 IMI / HLDP / ds Nrf2-sMaf Contact angle, surface tension, retention, systemic absorption and delivery efficiency of the composite

[0056] In this study, IMI / HLDP / ds Nrf2 - sMafThe contact angle, retention rate and surface tension of the composite (IMI concentration was 0.1 mg / mL) were measured. The contact angle analysis was performed using a contact angle meter (German datatorics OCA25A) to measure the IMI / HLDP / ds Nrf2 - sMaf The composite solution (IMI concentration of 0.1 mg / mL), IMI (0.1 mg / mL), HLDP (based on PCL of IMI), and ddH2O were dropped onto rapeseed leaves and glass slides, respectively. The droplets were observed and measured after stabilization for 20 s, and the contact angle of the droplet image was calculated using an ellipse fitting algorithm. Photos were taken using a micromanipulation system (Nikon, Japan), and the contact area was statistically analyzed using ImageJ 1.8 (National Institutes of Health, USA). Each solution was tested three times.

[0057] The surface tension was analyzed using a DCAT 21 surface tension meter based on the Wilhelmy plate method (Zheng et al., 2018) at 298 ± 0.1 K to measure ddH2O, HLDP (based on PCL of IMI), IMI (0.1 mg / mL), IMI / HLDP / ds Nrf2 - sMaf The complex (IMI concentration: 0.1 mg / mL) solution. Before each measurement, the platinum plate was cleaned with deionized water and ethanol and then sterilized in an alcohol flame. The tensiometer was calibrated using the surface tension of water to ensure the cleanliness of the plate and glassware. The sample was measured until its surface tension value remained constant, indicating that equilibrium had been reached. Each sample was measured three times in a row, and the standard deviation did not exceed ±0.20 mN / m.

[0058] The mass (M1) of rapeseed leaves (1.8 cm²) was measured at IMI (100 mg / L) and IMI / HLDP / ds. Nrf2 - sMaf The leaves were immersed in the solution of the complex (IMI concentration was 100 mg / L) for 1 min. The treated leaves were then suspended in the air until no droplets fell off naturally, and their weight (M2) was recorded. The retention of each sample was calculated as retention (mg / cm²) = (M2 - M1) ÷ leaf area. Each treatment included three independent samples.

[0059] Plant systemic capacity determination: Rapeseed seedlings at the same growth stage (four-leaf stage) were carefully washed with ddH2O and air-dried. Then the seedlings were immersed in IMI (0.2 mg / mL) and IMI / HLDP / ds Nrf2 - sMafThe seedlings were immersed in the complex (IMI concentration: 0.2 mg / mL) solution for 1 min, and then removed and placed on a clean surface. 6 h after immersion, the residual IMI on the surface of the seedlings was thoroughly washed with ddH2O. 200 mg of seedlings were added to 50 mL of acetonitrile, homogenized, centrifuged at 10000 r / min for 10 min, and the supernatant was taken and mixed with 5 g of sodium chloride. Then, 10 ml of the supernatant was evaporated to near dryness under nitrogen at 40°C. Subsequently, 2 ml of methanol-dichloromethane (1:99, v:v) was added and vortexed for 2 min. The solution was purified using an amino column (BKMAM, China), and the eluate was evaporated to near dryness under nitrogen at 40°C. Finally, 2 ml of methanol: water (1:1, v:v) was added, vortexed for 2 min, and filtered through a 0.22 μm organic filter membrane. The IMI concentration was determined by high-performance liquid chromatography. Each treatment included three independent samples.

[0060] IMI / HLDP / ds Nrf2 - sMaf Complex delivery efficiency determination: To detect IMI / HLDP / ds Nrf2 - sMaf To investigate the delivery efficiency of the complex, we synthesized the fluorescent ds eGFP , IMI / HLDP / ds eGFP The complex was used for fluorescence labeling. Fluorescent ds eGFP . IMI, HLDP and fluorescent ds eGFP (1 μg) were incubated at a mass ratio of 15:68:17 to prepare IMI / HLDP / ds eGFP A microsyringe (Hamilton, Switzerland) was used to inject 0.2 μL of IMI / HLDP / ds eGFP The complex solution was applied to the back of the 3rd instar aphid nymphs. The nymphs were washed with ddH2O 6 hours after external application. The fluorescence intensity was quantified using an inverted fluorescence microscope (LEICA, Germany) and imagej1.8 (National Institutes of Health, USA). eGFP (1 μg) and ds eGFP / HLDP complex (1:4 μg). Each treatment included 3 biological replicates.

[0061] IMI / HLDP / ds was detected using Drosophila S2 cells at a density of 5×105 / mL eGFP Cellular delivery of the complex. IMI / HLDP complex (0.944:4 μg), ds eGFP(1 μg) and ds eGFP / HLDP complex (1:4 μg), IMI / HLDP / ds eGFP The complex (0.944:4:1 μg). The cell culture medium was then replaced with 0.4 mL of fresh culture medium at 6 and 12 h after incubation. After gently pipetting to mix, the mixture was dropped onto a ConA-treated slide. The cells were fixed with 4% paraformaldehyde (PFA) for 20 min and then washed three times with 1 ml of PBS. The cells were sealed with DAPI sealing solution (Solarbio Life Sciences, China) and fluorescence was detected using a confocal microscope (Leica SP8, Germany). Fluorescence intensity was quantified using ImageJ1.8 (National Institutes of Health, USA). Three biological replicates were included for each treatment.

[0062] 1.8 IMI / HLDP / ds Nrf2-sMaf Determination of the bioinsecticide activity of the complex

[0063] The lethal concentration 50 (LC50) value of commercial IMI for 3rd instar aphid nymphs was determined. Rapeseed leaves were soaked for 1 min with commercial IMI formulations of different concentrations, 0, 1, 5, 20, 60 and 120 mg / L. 3rd instar nymphs fed on the treated leaves and their mortality was recorded after 48 h. LC50 values ​​were calculated using SPSS software (version 23.0, USA). Each treatment included 25 aphids and was repeated three times.

[0064] Based on the LC50 values ​​of commercial IMI, various solutions of IMI (9.598 mg / L), IMI / HLDP complex (9.598:40.657 mg / L), IMI / HLDP / ds eGFP Complex (9.598:40.657:10.164 mg / L), IMI / HLDP / ds Nrf2 Complex (9.598:40.657:10.164 mg / L), IMI / HLDP / ds Nrf2 - sMaf Complex (9.598:40.657:10.164 mg / L), ds eGFP (10.164 mg / L), ds eGFP / HLDP complex (10.164:40.657 mg L), ds Nrf2 (10.164 mg / L), ds Nrf2 / HLDP complex (10.164:40.657 mg / L), ds Nrf2 - sMaf (10.164 mg / L), dsNrf2 - sMaf / HLDP complex (40.657: 10.164 mg / L), 0.2 μL of each solution was applied to the back of 3rd instar aphids. In addition, rape roots were soaked in the corresponding solution, and the treated nymphs fed on the leaves. The mortality rate was recorded at 12, 24, 36, and 48 h after treatment. The synergistic effect of insecticidal activity was evaluated using the formula: Normalized synergistic ratio = mortality rate of nymphs treated with the new formula ÷ average mortality rate of nymphs treated with IMI alone. The experiment was repeated 3 times, with 25 nymphs in each repeat. In addition, the Nrf2 and sMaf The expression levels of the detoxification and metabolism genes were also detected. At the same time, the expression levels of the detoxification and metabolism genes in the aphids were detected after being treated with each solution for 48 h. Each treatment included three independent samples.

[0065] 2. Results and Discussion

[0066] 2.1 Nrf2 Regulates the expression of detoxification genes

[0067] For research Nrf2 Potential role in insecticide resistance, use Nrf2 RNA-seq analysis after RNAi detected the expression levels of detoxification genes in aphids ( Figure 1 a). Agarose gel retardation experiment showed that ds Nrf2 The optimal binding mass ratio with the nanocarrier HLDP is 1:4 ( Figure 1 b) Then, we prepared ds Nrf2 / HLDP complex, which can significantly reduce Nrf2 Expression in aphids, and RNAi efficiency was higher than that of ds alone Nrf2 ( Figure 1 c) Use ds Nrf2 / HLDP complex and ds eGFP Aphids were treated with ds / HLDP complexes, and total RNA was extracted for RNA-seq analysis. Nrf2 A total of 198 down-regulated genes and 167 up-regulated genes were identified in aphids treated with the HLDP / β complex. Figure 1 d) KEGG analysis showed that DEGs were enriched in multiple pathways, such as lysosome, peroxisome, fatty acid metabolism, fatty acid elongation, drug metabolism-other enzymes, drug metabolism-cytochrome P450 , cytochrome P450 Metabolism of exogenous drugs, etc. Figure 1 e).

[0068] Heat map analysis of key detoxification genes showed that Nrf2After RNAi, the expression of these genes in aphids was significantly suppressed ( Figure 1 f). Nrf2 After RNAi, P450s Seven genes were significantly down-regulated in the family ( Figure 1 g). Among them, 1 gene belonged to the CYP3, CYP5b and CYP6 subfamilies, and 4 genes belonged to the CYP4 subfamily. Studies have shown that the CYP3, CYP4 and CYP6 subfamilies in insects play a vital role in the formation of IMI resistance. It is worth noting that the FPKM values ​​of CYP3 and CYP4 subfamily genes decreased significantly from 64.64 and 91.33 to 7.19 and 39.36, respectively. At the same time, other detoxification genes, including GSTs , CarEs and PODs ,go through Nrf2 After RNAi, the FPKM value was also significantly reduced ( Figure 1 g). Therefore, our results show that Nrf2 It can serve as a good target for improving pest sensitivity to insecticides and managing metabolic resistance.

[0069] 2.2 Important regulatory role of the Nrf2-sMaf pathway in pesticide detoxification and metabolism

[0070] When organisms are exposed to reactive oxygen species (ROS), specific cysteine ​​residues in Keap1 are modified, leading to a conformational change in the Keap1-Cul3-E3 ubiquitin ligase. This interferes with Nrf2 ubiquitination Nrf2 Transferred to the cell nucleus. There, it interacts with sMaf The protein forms a heterodimer and binds to the antioxidant response element (ARE) / electrophilic response element (EpRE) of the target gene, inducing P450 , GST , POD , CarE and SOD The expression of various cell protection genes ( Figure 2 a) RNA-seq analysis showed that Nrf2 During RNAi, sMaf The FPKM value was significantly reduced ( Figure 2 b). This result was also verified by qRT-PCR, indicating that sMaf As Nrf2 Downstream targets of Nrf2 Positive regulation of Figure 2 c) It is worth noting that we found for the first time that Nrf2 / HLDP complex or ds sMaf Compared with the HLDP complex, topical application of dsNrf2 - sMaf / HLDP complex further inhibits sMaf In addition, in ds Nrf2 - sMaf In aphids treated with / HLDP complex, the detoxification gene ( P450 , GST , CarE and POD ) was further downregulated, indicating that coordinated RNAi of the two target genes is expected to be more effective in controlling insecticide resistance ( Figure 2 dg).

[0071] In this study, compared with targeting Nrf2 Compared with co-targeting Nrf2 and sMaf Significantly reduced P450s , GSTs , CarEs and POD The enzyme activities ranged from 0.15 to 0.10 nmol / min / mg prot, from 0.10 to 0.07 nmol / min / mg prot, from 0.46 to 0.38 U / mg prot, and from 7.21 to 5.76 U / mg prot, respectively. Figure 2 h). With ds Nrf2 Compared with the HLDP complex, ds Nrf2 - sMaf The / HLDP complex significantly inhibited the activities of four detoxification enzymes. The above results indicate that transcription factors Nrf2 Regulation of detoxification genes via the Nrf2-sMaf pathway ( P450 , GST , CarE and POD This study confirmed for the first time the regulatory role of the Nrf2-sMaf pathway in regulating the detoxification and metabolism of pesticides.

[0072] 2.3 IMI / HLDP / ds Nrf2-sMaf Self-assembly mechanism of the complex

[0073] The nanomaterial HLDP is a cationic polymer with hydrophobic and hydrophilic groups. The hydrophobic carbon chain can be assembled with hydrophobic active ingredients (AIs), and the hydrophilic carboxyl and hydroxyl groups make HLDP more stable in aqueous solution. In addition, HLDP has a positively charged surface, which allows it to load exogenous nucleic acid molecules in an aqueous environment. Therefore, HLDP is expected to combine with various types of groups and become a universal nanocarrier / adjuvant. This study used HLDP to self-assemble multifunctional nanoprotectants and simultaneously load IMI and ds Nrf2 - sMaf ( Figure 3a). The interaction between HLDP and IMI was detected by ITC. According to the previous interpretation of ITC data, the high affinity constant (Ka) and low dissociation constant (Kd) indicate that there is an effective and strong interaction between HLDP and IMI, and the negative ΔG value indicates that the interaction is spontaneous ( Figure 3 b). The negative values ​​of ΔH (−43.88 kJ / mol) and ΔS (−58.28 J / mol·K) indicate that the self-assembly of the IMI / HLDP complex is mainly driven by hydrogen bonding and van der Waals forces.

[0074] like Figure 3 As shown in c, complexation with HLDP can increase the zeta potential of IMI from -37.57 mV to 27.97 mV. As in previous studies, pesticide / nanocarrier complexes are usually positively charged, but IMI / HLDP / ds Nrf2 - sMaf The complex is negatively charged (-7.77 mV), which may be due to the negatively charged ds Nrf2 - sMaf Therefore, different mass ratios of ds Nrf2 - sMaf Agarose gel retardation experiments with IMI / HLDP complexes were performed to further investigate the ds Nrf2 - sMaf The results show that as the mass ratio decreases, the ds Nrf2 - sMaf The band intensity gradually disappears, indicating that the electrostatic interaction is Nrf2 - sMaf The self-assembly process of the complex plays an important role ( Figure 3 c) In addition, ITC data showed that in the IMI / HLDP complex with ds Nrf2 - sMaf In the binding process, hydrogen bonds and van der Waals forces also play an important role ( Figure 3 d) To further quantify IMI / HLDP / ds Nrf2 - sMaf IMI in complex, IMI / HLDP complex with ds Nrf2 - sMaf With a mass ratio of 4:1 (HLDP: ds Nrf2 - sMaf ) and dialyzed for 12 h. The IMI concentration outside the dialysis bag was quantified by HPLC. IMI: HLDP: ds was calculated based on the standard curve. Nrf2 - sMaf The mass ratio of IMI / HLDP and dsNrf2 - sMaf The effect of the combination on the IMI of HLDP load is negligible.

[0075] 2.4 IMI / HLDP / ds Nrf2-sMaf Characterization of the complex

[0076] Transmission electron microscopy observation of IMI / HLDP / ds Nrf2 - sMaf The morphology of the composites, representative transmission electron microscopy images show that IMI / HLDP / ds Nrf2 - sMaf The composites are mostly composed of nearly spherical particles of similar size ( Figure 3 e) IMI / HLDP / ds was determined by DLS method Nrf2 - sMaf Particle size of the composite. HLDP, IMI, IMI / HLDP composite and IMI / HLDP / ds Nrf2 - sMaf The particle sizes of the composites are 108.27 nm, 236.54 nm, 114.83 nm and 255.55 nm ( Figure 3 e). IMI / HLDP / ds Nrf2 - sMaf The particle size of the composite is larger than that of the IMI / HLDP composite, which may be due to the fact that ds Nrf2 - sMaf The results show that the HLDP complex is caused by electrostatic adhesion on the surface of IMI / HLDP complex.

[0077] 2.5 IMI / HLDP / ds Nrf2-sMaf The composite has better adhesion on plant leaves

[0078] Plant leaves are mainly composed of trichomes and wax layers, which have hydrophobic properties and often cause pesticide drift. The contact angle, surface tension, and contact area of ​​pesticide droplets are directly related to their deposition and adhesion, thus affecting the efficacy. In this study, among different formulations such as HLDP (58.67°), ddH2O (82.43°), and IMI (73.86°), IMI / HLDP / ds Nrf2 - sMaf The contact angle of the composite on rapeseed leaves is the smallest (43.44°) ( Figure 4 a). At the same time, IMI / HLDP / ds Nrf2 - sMaf The surface tension of the composite (55.08 mN / m) is also significantly lower than that of ddH2O (72.70 mN / m) and IMI (72.11 mN / m) ( Figure 4 b) Due to IMI / HLDP / dsNrf2 - sMaf The contact angle and surface tension of the composite are small, so its contact area is the largest among all the formulations ( Figure 4 c) As expected, IMI / HLDP / ds Nrf2 - sMaf The retention rate and plant uptake of the complex increased by 2.1 and 1.7 times, respectively, compared with IMI alone ( Figure 4 d,e). The above results show that IMI / HLDP / ds Nrf2 - sMaf The complex diffuses more easily on rapeseed leaves and has a stronger affinity for the leaves, which promotes the absorption of pesticides and improves the efficiency of pesticide utilization against piercing-sucking pests.

[0079] 2.6 IMI / HLDP / ds Nrf2-sMaf The complex has enhanced delivery efficiency in vitro and in vivo

[0080] Due to IMI / HLDP / ds Nrf2 - sMaf The surface tension of the composite was reduced, and its contact area in the axils of third-instar aphid nymphs was significantly increased compared with ddH2O, HLDP, and IMI, resulting in ds Nrf2 - sMaf and IMI retention in aphid cuticle increased significantly ( Figure 4 f) Subsequently, we synthesized the fluorescent ds eGFP , IMI / HLDP / ds eGFP The IMI / HLDP / ds complex was then expressed in aphids and Drosophila S2 cells. eGFP Complex and ds eGFP The fluorescence intensity of aphids treated with ds / HLDP complex was significantly higher than that of aphids treated with ds / HLDP alone. eGFP Aphids ( Figure 4 g). At the same time, in vitro, ds eGFP / HLDP complex or IMI / HLDP / ds eGFP The complex can be efficiently transported across the cell membrane into Drosophila S2 cells ( Figure 4 h). Our results show that IMI / HLDP / ds Nrf2 - sMaf The complex has excellent delivery capabilities both in vivo and in vitro, and its advantages are summarized in Figure 4 i.

[0081] 2.7 IMI / HLDP / ds Nrf2-sMaf The compound has good biological insecticide activity against green peach aphid

[0082] According to the toxicity regression equation, various formulations were prepared with the LC50 value of IMI, including IMI / HLDP complex, IMI / HLDP / ds Nrf2 Complex, IMI / HLDP / ds Nrf2 - sMaf The biological activity of the preparations was tested by topical application and root absorption. Figure 5 a, g). The results show that ds Nrf2 and ds Nrf2 - sMaf It has a slight lethal effect on the 3rd instar aphid nymphs. Under the action of HLDP, the insecticidal activity of IMI was significantly enhanced. After 48 hours of external application and root absorption treatment, IMI / HLDP / ds Nrf2 - sMaf The insecticidal activity of the complexes reached 77.33% and 74.67%, respectively, which was significantly higher than that of IMI / HLDP / ds Nrf2 Complex and IMI / HLDP complex ( Figure 5 b, h). Compared with IMI alone, IMI / HLDP / ds Nrf2 - sMaf The normalized synergistic ratios of the complexes were 1.87-2.25 and 1.65-2.29 ( Figure 5 c, i). Based on the toxicity regression equation, the dose of the multi-component nanoformulation required to achieve similar insecticidal activity after topical application and root absorption for 48 h was calculated. The results showed that IMI / HLDP / ds Nrf2 - sMaf Complex and IMI / HLDP / ds Nrf2 The topical dosage of the complex was 14.30% and 40.93% of that of IMI, respectively. The root absorption rates of the two solutions were 29.77% and 40.93% of that of IMI alone, respectively. The above results show that IMI / HLDP / ds Nrf2 - sMaf The biological activity of the complex is significantly higher than that of IMI alone and can reduce the amount of pesticide used. In addition, various solutions, including IMI / ds, can be applied topically and absorbed by the roots. eGFP Composite, IMI / ds Nrf2 - sMaf Complex, IMI / HLDP / ds eGFP Complex and IMI / HLDP / ds Nrf2 - sMaf The complex was studied Nrf2 , sMaf and detoxification and metabolism genes ( P450s , GSTs , CareE s and PODs ) expression. IMI / HLDP / dsNrf2 - sMaf Myzus persicae was successfully eliminated by external application and root absorption of the compound Nrf2 and sMaf Of note, similar to IMI / ds Nrf2 - sMaf Compared with the composite Nrf2 and sMaf The expression of HLDP was significantly reduced, indicating that HLDP increased the interference efficiency of RNAi, thereby enhancing the sensitivity of peach aphids to these agents ( Figure 5 d, e, j, k). In addition, detoxification metabolism genes ( P450s , GSTs , CareE s and PODs ) also showed the same interference trend after 48 h of treatment with the above solution ( Figure 5 f, l).

[0083] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A multi-component nanocomposite for controlling pesticide resistance of peach aphid, characterized in that: Includes the following ingredients: imidacloprid, carrier HLDP and ds Nrf2 - sMaf ,ds Nrf2 - sMaf The nucleotide sequence of the gene is shown in SEQ ID NO:

1. The carrier HLDP is a hydrophilic-lipophilic diblock polymer; the structural formula of the carrier HLDP is as follows: 。 2. The multi-component nanocomposite for controlling pesticide resistance of green peach aphid according to claim 1, characterized in that: The complex formed by imidacloprid and carrier HLDP reacts with ds Nrf2 - sMaf The mass ratio is (1~5):

1.

3. The multi-component nanocomposite for controlling pesticide resistance of green peach aphid according to claim 1, characterized in that: The ds Nrf2 - sMaf It is obtained by chemical synthesis and efficient expression of engineering bacteria; the chemical synthesis and efficient expression of engineering bacteria include the following steps: (1) Using primers containing the T7 promoter sequence, PCR was used to amplify Nrf2 and sMaf The gene fragment was reverse transcribed into ds Nrf2 and sMaf ; (2) Utilization z / f-Nrf2 / sMaf Fusion primers were used to amplify Nrf2 and sMaf The forward complementary sequence and reverse complementary sequence of the sequence were then fused using fusion PCR. Nrf2 and sMaf forward complementary sequence and reverse complementary sequence; (3) Use T4 DNA ligase to Nrf2 - sMaf -forward sequence sum Nrf2 - sMaf - Reverse sequence ligation, introduced into the expression vector for culture; pick a single colony and culture it in 5 mL LB liquid medium containing 50 mg / L kanamycin. 600 When the nm value was 0.4, isopropyl β- d -1-thiogalactoside was added for incubation, and then extracted with 75% ethanol to obtain ds Nrf2 - sMaf .

4. The multi-component nanocomposite for controlling pesticide resistance of green peach aphid according to claim 1, characterized in that: The multi-component nanocomposite contains imidacloprid, carrier HLDP, ds Nrf2 - sMaf The concentration ratio is 9.598:40.657:10.164 mg / L.

5. The multi-component nanocomposite for controlling pesticide resistance of green peach aphid according to claim 1, characterized in that: The multi-component nanocomposite contains imidacloprid, carrier HLDP, ds Nrf2 - sMaf The mass ratio is 15:68:

17.

6. A method for preparing a multi-component nanocomposite for controlling pesticide resistance of green peach aphid according to any one of claims 1 to 5, characterized in that: Imidacloprid was titrated with HLDP to form an imidacloprid / HLDP complex; Nrf2 - sMaf Titrate the imidacloprid / HLDP complex to form imidacloprid / HLDP / ds Nrf2 - sMaf Complex, that is, multi-component nanocomposite.

7. Use of the multi-component nanocomposite according to any one of claims 1 to 5 in preparing a product for controlling pesticide resistance of green peach aphid, characterized in that: The drug resistance is resistance to imidacloprid.

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