A multi-component nanoformulation for improving the sensitivity of bean thrips to pesticides, and its preparation method and application

Abstract

The present invention discloses a multi-element nanoformulation for improving the sensitivity of bean thrips to drugs, and a preparation method and application thereof, and studies a hydrophilic-lipophilic diblock polymer (HLDP) and a targeted Nrf2 SUL / HLDP / ds Nrf2 Compound, for SUL / HLDP / ds Nrf2 The delivery efficiency of the complex in vivo and in vitro and the effect of SUL / HLDP / ds on the Nrf2 The insecticidal activity of the complex was tested and confirmed Nrf2 Regulation of pesticide detoxification and metabolism in BFTs, down-regulation Nrf2 It can inhibit the expression of most detoxification genes and reduce enzyme activity. SUL / HLDP / ds Nrf2 The complex showed high drug delivery efficiency and insecticidal activity both in vitro and in vivo, with a field efficacy of 89.89% against BFTs.

Description

Technical Field

[0001] The present invention relates to the technical field of pest control, and in particular to a multi-element nano preparation for improving the sensitivity of Thrips leucophylla to pesticides, and a preparation method and application thereof. Background Art

[0002] The frequent occurrence of "poisonous cowpea" incidents is mainly due to the high resistance of bean thrips (BFT, Meggalurothrips usitatus ) is caused by unscientific application of pesticides. Sand flies have developed a certain degree of resistance to different types of pesticides. The latest monitoring results show that the relative resistance ratio of sand flies in Hainan Province, China is as high as 65 times, resulting in a gradual decline in the efficacy of pesticides. At present, the main method of thrips management is to use different types of pesticides in combination or alternation, but this is not a long-term solution. Therefore, the development of new environmentally friendly agents has become an urgent need to solve the problem of high resistance of thrips.

[0003] The mechanisms of insecticide resistance mainly include metabolic resistance caused by increased detoxification enzyme activity and target resistance caused by reduced sensitivity of the target to insecticides. Metabolic resistance is a complex physiological process in which overexpression of detoxification genes in pests leads to changes in enzyme activity. Well-known detoxification enzymes include cytochrome P450 Enzyme ( P450 ), glutathione S-transferase (GST), peroxidase ( POD ), superoxide dismutase ( SOD ), UDP-glucuronosyltransferases (UGTs), and ATP-binding cassette transporters (ABC transporters). These enzymes help insects degrade or transform pesticides, thereby reducing their toxicity. However, resistance management against detoxification genes remains challenging due to their diversity.

[0004] RNA biopesticides are polynucleotide preparations that can interfere with or inhibit the transcription of specific genes in target organisms. Compared with traditional pesticides, RNA pesticides have many advantages, such as strong specificity, low development cost, and eco-friendliness. However, their field application faces technical bottlenecks, such as the easy degradation of double-stranded RNA (dsRNA) by RNases, the low delivery efficiency of dsRNA, and the high production cost, which limit the actual field application of RNA biopesticides. In recent years, the rapid development of nanotechnology in the field of agriculture has driven the continuous development of traditional agriculture in interdisciplinary fields. Nanocarrier delivery systems can protect dsRNA from degradation by ribonucleases and achieve effective delivery of dsRNA by activating clathrin-mediated endocytosis. In addition, nanocarriers can spontaneously load insecticides into insecticide / nanocarrier complexes, which have better characteristics such as nanoscale particle size, smaller contact angle, reduced surface tension, enlarged contact area, and enhanced plant absorption, thereby increasing insecticidal biological activity while reducing the application amount. Therefore, the use of targeted Nrf2 Nanocarriers co-delivered with dsRNA and insecticides provide a new approach to combat insect resistance. Summary of the invention

[0005] In view of the above-mentioned defects of the prior art, the present invention proposes a multi-component nanoformulation for improving the sensitivity of Macrothrips beanus to pesticides and a preparation method and application thereof, so as 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 nanoformulation for improving the sensitivity of bean thrips to pesticides, comprising the following components: sulfoxaflor, carrier HLDP and ds Nrf2 ,ds Nrf2 The nucleotide sequence of the gene is shown in SEQ ID NO: 1 (Genbank accession number: KAJ1521208.1); preferably, the carrier HLDP is a hydrophilic-lipophilic diblock polymer, and the structural formula of the carrier HLDP is as follows:

[0008] .

[0009] Preferably, the complex formed by sulfoxaflor and the carrier HLDP is Nrf2 The mass ratio is (1~6):1.

[0010] Preferably, the method for preparing the carrier HLDP comprises the following steps:

[0011] (1) 100 mg of n-butanol, 6.16 g of ε-CL, and 310 mg of Sn (Oct)2 were mixed and incubated in an oil bath at 90 °C for 9 h. After the reaction was complete, 30 mL of dichloromethane was added, followed by cold methanol to precipitate a white solid powder of linear polycaprolactone PCL.

[0012] (2) Add 3 g BIBB, 30 mL THF, and 4 g TEA to 3 g PCL solution, then add cold methanol to terminate the reaction. Filter and collect the precipitate, which is white powder PCL-Br.

[0013] (3) 100 mg PCL-Br was added to 0.87 g DMAEMA and dissolved in 2 mL THF. Then 15 mg CuBr and 37 mg PMDETA were added and reacted at 65 °C for 5 h. After the reaction, the mixture was dialyzed and freeze-dried to obtain a white solid product HLDP.

[0014] Preferably, the ds Nrf2 By chemical synthesis or in vivo synthesis; the chemical synthesis comprises the following steps: using primers containing T7 promoter sequence, using PCR amplification Nrf2 The gene fragment is transcribed into ds Nrf2 The in vivo synthesis comprises the following steps: using primers with restriction site specific primers, using PCR amplification to obtain Nrf2 The target sequence is double-digested and connected to the expression vector, and then transformed into the strain to obtain ds Nrf2 .

[0015] Preferably, the multi-component nanoformulation contains sulfoxaflor SUL, carrier HLDP and ds Nrf2 The concentration ratio is 0.177:0.876:0.219 mg / mL.

[0016] Preferably, a method for preparing a multi-component nanoformulation for improving the sensitivity of bean thrips to a drug is as follows: HLDP is titrated with sulfoxaflor SUL to form a SUL / HLDP complex; and ds Nrf2 , eventually forming SUL / HLDP / ds Nrf2 Complex, that is, multi-component nanoformulation.

[0017] Preferably, the multi-element nanoformulation involved in the present application is used in the preparation of a product for enhancing the sensitivity of Thrips macrothrips to pesticides.

[0018] Preferably, the multi-component nanoformulation involved in the present application is used in protecting cowpea leaves.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] (1) This application confirms that Nrf2 Regulation of pesticide detoxification and metabolism in BFTs, down-regulation Nrf2 It can inhibit the expression of most detoxification genes and reduce the activity of enzymes. The complex self-assembles through hydrogen bonds, van der Waals forces and electrostatic interactions, and its particle size is reduced to nanometers compared to SUL alone. SUL / HLDP / ds Nrf2 The composite has excellent leaf adhesion properties, with a small contact angle, reduced surface tension, increased contact area, improved retention and enhanced plant absorption. SUL / HLDP / ds Nrf2 The complex showed high drug delivery efficiency and insecticidal activity both in vitro and in vivo, with a field efficacy of 89.89% against BFTs.

[0021] (2) Compared with the pesticide SUL alone, the complex improved the contact angle, contact area, surface tension, leaf retention and plant absorption capacity. SUL / HLDP / ds Nrf2 The insecticidal activity of the complex was significantly enhanced, with a normalized synergistic ratio of 1.31-2.00, and it significantly inhibited Nrf2 and detoxification gene expression. In the wild, SUL / HLDP / ds Nrf2 The control effect of the complex on BFTs was 89.89%. Therefore, the multi-component nanoformulation developed in this study can effectively interfere with the expression of pest metabolic resistance genes, enhance the sensitivity of pests to pesticides, reduce pesticide dosage, and improve utilization efficiency. This is conducive to pest resistance management, effective use of pesticides, and sustainable agricultural development.

[0022] (3) The present invention provides a new RNAi target for sustainable management of improving the sensitivity of bean thrips to pesticides, and has great application prospects in environmentally friendly agriculture. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 for Nrf2 Key regulatory role in detoxification gene expression; Figure 1 a is ds Nrf2 Results of agarose gel retardation assay with HLDP; Figure 1 b is for ds Nrf2 Schematic diagram of treating adults of Macrothrips leucoderma with the / HLDP complex and conducting qRT-PCR detection; Figure 1 c is ds eGFP 、ds eGFP / HLDP,ds Nrf2 、ds Nrf2 / HLDP Nrf2 Interference effect; Figure 1 d is for adults of Thrips pea Nrf2 Diagram of gene changes after RNAi; Figure 1 e is Nrf2 KEGG enrichment analysis diagram after RNAi; Figure 1 f is Nrf2 Key detoxification genes after RNAi ( P450 , GSTs , POD and SOD ) heat map analysis; Figure 1 g is Nrf2 Key detoxification genes after RNAi ( P450 , GSTs , POD and SOD )’s FPKM value; Figure 1 h is Nrf2 Changes in expression of related detoxification and metabolism genes after RNAi; Figure 1 i is Nrf2 Key detoxification genes after RNAi ( P450 , GSTs , POD and SOD ) enzyme activity;

[0024] Figure 2 For SUL / HLDP / ds Nrf2 The self-assembly mechanism of the complex; Figure 2 a is highly efficient expression of ds Nrf2 and SUL / HLDP / ds Nrf2 Schematic diagram of the self-assembly mechanism of the complex; Figure 2 b ITC method for detecting HLDP and ds Nrf2 Gibbs free energy change of interaction (ΔG); Figure 2 c is ds Nrf2 Results of agarose gel blocking experiments with SUL / HLDP complex; Figure 2 d is SUL / HLDP / ds Nrf2 Changes in the zeta potential of the complex; Figure 2 e is SUL, SUL / HLDP, ds Nrf2 / HLDP、SUL / HLDP / ds Nrf2 Composite morphology and particle size;

[0025] Figure 3 For SUL / HLDP / ds Nrf2 Properties of the complex; Figure 3 a is HLDP, SUL, SUL / HLDP / ds Nrf2 Tyndall effect test of the complex; Figure 3b is ddH2O, HLDP, SUL, ds Nrf2 、SUL / HLDP / ds Nrf2 Contact angle of the composite; Figure 3 c is ddH2O, HLDP, SUL, ds Nrf2 、SUL / HLDP / ds Nrf2 Surface tension of the complex; Figure 3 d is ddH2O, HLDP, SUL, ds Nrf2 、SUL / HLDP / ds Nrf2 Surface tension contact area of ​​the composite; Figure 3 e is ddH2O, HLDP, SUL, ds Nrf2 、SUL / HLDP / ds Nrf2 The retention of the complex; Figure 3 f is ddH2O, HLDP, SUL, ds Nrf2 、SUL / HLDP / ds Nrf2 Plant uptake rate of the complex; Figure 3 g is SUL / HLDP, ds eGFP 、SUL / HLDP / ds eGFP The fluorescence intensity in the stem; Figure 3 h is SUL / HLDP, ds eGFP 、SUL / HLDP / ds eGFP The fluorescence intensity in the adult of Thrips leucophylla (in vivo); Figure 3 i is SUL / HLDP, ds eGFP 、SUL / HLDP / ds eGFP Fluorescence intensity in Drosophila S2 cells (in vitro); Figure 3 j is SUL / HLDP / ds Nrf2 Schematic diagram of the delivery ability of the complex in vivo and in vitro;

[0026] Figure 4 For SUL / HLDP / ds Nrf2 The insecticidal biological activity of the compound; wherein Figure 4 a is a schematic diagram of the treatment of Thrips leucoderma adults by immersion method; Figure 4 b is for immersion treatment using SUL, SUL / HLDP, SUL / HLDP / ds eGFP 、SUL / HLDP / ds Nrf2 Insecticidal biological activity of the complex; Figure 4 c is for immersion treatment using SUL, SUL / HLDP, SUL / HLDP / ds eGFP 、SUL / HLDP / ds Nrf2 Normalized synergistic ratio of the complex; Figure 4d is SUL / ds for immersion treatment eGFP 、SUL / ds Nrf2 、SUL / HLDP / ds eGFP 、SUL / HLDP / ds Nrf2 hour Nrf2 Interference efficiency; Figure 4 e is SUL / ds for immersion treatment eGFP 、SUL / ds Nrf2 、SUL / HLDP / ds eGFP 、SUL / HLDP / ds Nrf2 Time-related detoxification and metabolic gene interference efficiency; Figure 4 f is a schematic diagram of the root absorption method for adults of Thrips leucophylla; Figure 4 g is SUL, SUL / HLDP, SUL / HLDP / ds for root absorption treatment eGFP 、SUL / HLDP / ds Nrf2 Insecticidal biological activity of the complex; Figure 4 h is SUL, SUL / HLDP, SUL / HLDP / ds for root absorption treatment eGFP 、SUL / HLDP / ds Nrf2 Normalized synergistic ratio of the complex; Figure 4 i is SUL / ds for root absorption treatment eGFP 、SUL / ds Nrf2 、SUL / HLDP / ds eGFP 、SUL / HLDP / ds Nrf2 hour Nrf2 Interference efficiency; Figure 4 j is SUL / ds for immersion treatment eGFP 、SUL / ds Nrf2 、SUL / HLDP / ds eGFP 、SUL / HLDP / ds Nrf2 Time-related detoxification and metabolic gene interference efficiency; Figure 4 k is the field HLDP, ds Nrf2 / HLDP,SUL,SUL / HLDP,SUL / HLDP / ds Nrf2 The protective effect of spraying; Figure 4 l is HLDP for field use, ds Nrf2 / HLDP,SUL,SUL / HLDP,SUL / HLDP / ds Nrf2 Image of sprayed cowpea leaves. DETAILED DESCRIPTION

[0027] The technical solution of the present invention is further described in detail below in conjunction with specific implementation methods.

[0028] Reagents and manufacturers: SUL standard and commercial types were purchased from Shanghai McLean Biochemical Technology Co., Ltd. (China) and Corteva agriscience (USA), 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%) and Sn(Oct)2 purchased from Heowns BioChem Technologies (China), 2-(dimethylamino)ethyl methacrylate (DMAEMA, 99%) purchased from Energy Chemical (China), and tetrahydrofuran (THF), N,N,N',N',N',N"-pentamethyldiethyltriamine (PMDETA, 98%) and CuBr (99.999%) purchased from Sigma-Aldrich (USA). Other chemical reagents were provided by Beijing Chemical Plant (China).

[0029] Experimental example

[0030] 1 Methods

[0031] 1.1 Chemical synthesis of dsRNA

[0032] Thirty adult bean thrips were collected and ground thoroughly with a grinding rod. Total RNA was then extracted according to the instructions of the RNA Easy Fast Tissue / Cell Kit (TaKaRa, China). 1 μg of RNA was reverse transcribed using the PrimeScriptTM RTReagent Kit with gDNA Eraser (Perfect Real Time) (TaKaRa, Japan) to obtain cDNA. Nrf2 The target band was gel-recovered and then ligated into the cloning vector pMD19T-Vector (TAKARA) and transformed into the Escherichia coli DH5α strain (TSINGKE, China). The bacterial solution was verified by Sanger sequencing, and the plasmid was extracted from the bacterial solution with the correct sequencing. The plasmid was then used as a template and primers with a T7 promoter were used for amplification. The ds Nrf2 Enhanced green fluorescent protein ( eGFP ) gene as a control, eGFP The nucleotide sequence information is shown in SEQ ID NO: 2 (Genbank accession number: XM_013480425.1), and the primer information is shown in Table 1.

[0033] Table 1 Nrf2 , eGFP Primer information

[0034]

[0035] 1.2 Preparation and structure of hydrophilic-lipophilic diblock polymer (HLDP)

[0036] n-Butanol (100 mg, 1.35 mmol), ε-CL (6.16 g, 54 mmol) and Sn (Oct)2 (310 mg) were incubated in a 90℃ oil bath for 9 h. After the reaction was completed, dichloromethane (30 mL) was added, followed by cold methanol, and a white solid powder of linear polycaprolactone PCL was precipitated. 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 then cold methanol was added to terminate the reaction. The resulting precipitate was collected by filtration to obtain white powder PCL-Br. Finally, PCL-Br (100 mg, 0.04 mmol) was added to DMAEMA (0.87 g, 6.07 mmol), dissolved in 2 mL THF, and then CuBr (15 mg, 0.1 mmol) and PMDETA (37 mg, 0.21 mmol) were added. The reaction was carried out at 65°C for 5 h, and then dialyzed and freeze-dried to obtain a white solid product HLDP. The chemical structure of HLDP is:

[0037] .

[0038] 1.3 RNA-seq analysis Nrf2 Potential functions

[0039] Detection using RNAi technology and RNA-seq analysis Nrf2 Effects on detoxification and metabolism. In order to prepare ds Nrf2 / HLDP complex, the ds Nrf2 Best mass ratio with HLDP. Take 1 μgds Nrf2 The samples were incubated with HLDP at a mass ratio of 1:0, 1:1, 1:2, 1:3, 1:4, and 1:5 at room temperature for 15 min and analyzed by agarose gel electrophoresis. Nrf2 / HLDP complex. In RNAi experiments, cowpea was cut into 3 cm pieces and 10 μL of ds Nrf2 / HLDP complex, ds eGFP / HLDP complex, ds Nrf2 and ds eGFP The solution (600 ng dsRNA / worm) was applied to the small segments. These segments were fed with BFT adults in ventilated centrifuge tubes for 24 h, and BFT total RNA was extracted and transcribed into cDNA. Real-time fluorescence quantitative PCR (qRT-PCR) was used to detect Nrf2 The expression level of Nrf2 -F: AGTCCAACTTGAGAGGCAC;q Nrf2 -R: CCCAGAAGAAGCACCAAAT.

[0040] qRT-PCR was performed using the Step One Plus Real-Time PCR System (Applied Biosystems, USA) and SuperReal PreMix Plus (SYBR Green) (Tiangen Biotech, China). Nrf2 The relative mRNA levels were calculated using 2 −ΔΔCT Method assay, RPL (GenBank accession: XM_026436599) was used as the internal reference gene. Each treatment included three independent samples, each consisting of 30 adults of Macrothrips bean.

[0041] For RNA-seq analysis, we extracted ds Nrf2 / HLDP complex and ds eGFPTotal RNA of adults of Thrips leucophylla treated with HLDP / HLDP complex was then used for RNA-seq analysis by Novogene (China, https: / / cn.novogene.com / ) according to standard protocols. The above total RNA was used to construct RNA sequencing libraries using NEBNext® UltraTM RNA Library Prep Kitfor Illumina® (NEB, Beverly), and sequencing was performed on the Illumina Novaseq 6000 platform. The expression levels of various genes were determined by the number of fragments per million fragments transcribed (FPKM). Differentially expressed genes (DEGs) were identified with P≤0.05, fold change |log2 (foldchange)|≥1. The volcano plot visually displayed the distribution of DEGs for each comparison group. The enrichment of DEGs was analyzed using the Kyoto Encyclopedia of Genes and Genomes (KEGG). The heat map was based on the correlation coefficient calculated based on the FPKM value. The DEG set was created by taking the union of the differentially expressed genes from the control group, providing an intuitive visualization of the sample differences between groups and the sample duplications within the group. Three independent replicates were performed for each treatment.

[0042] 1.4 Nrf2 Evaluation of the effects of RNAi on detoxification gene expression and enzyme activity

[0043] RNA-seq results showed Nrf2 Regulate the resistance of BFTs to insecticides. Nrf2 Effects of RNAi on the expression of detoxification genes. According to the above method, we used ds Nrf2 / HLDP complex and ds eGFP / HLDP complex was used to treat adults of Macrothrips leucoderma. Total RNA was extracted 24 h later, and qRT-PCR was used to detect the expression of 14 detoxification genes. P450 s, GSTs , POD , SODThe expression levels of the 14 detoxification genes were Musi00116(Genbank accession number:KAJ1523289.1)、Musi007297(Genbank accession number:KAJ1528928.1)、Musi005799(Genbank accession number:KAJ1530963.1)、Musi005677(Genbank accession number:KAJ1530831.1)、Musi007941(Genbank accession number:KAJ1528017.1)、Musi010205(Genbank accession number:KAJ1525390.1)、Musi008167(Genbank accession number:KAJ1526581.1)、Musi003352(Genbank accession number:KAJ1526582.1)、 number:KAJ1521710.1), Musi004870 (Genbank accession number:KAJ1519595.1), Musi003877 (Genbank accession number:KAJ1520783.1), Musi009537 (Genbank accession number:KAJ1526399.1), Musi008322 (Genbank accession number:KAJ1526743.1), Musi002647 (Genbank accession number:KAJ1522355.1), RPL (Genbank accession number:XM_026436599), as shown in Table 2 for quantitative primers. Each treatment included three independent samples, each containing 30 BFT adults. Subsequently, the activity of detoxification enzymes in BFT adults of the above treatments was detected. 0.1 g of adult specimens were homogenized with 1 ml of extract to prepare crude enzyme solution (Suzhou Keming Biotechnology Co., Ltd., China). P450 , GST, POD and SOD The activity of the enzyme was determined by following the procedure of the kit (Suzhou Keming Biotechnology Co., Ltd., China). The total protein content of the enzyme source was quantified using the BCA protein assay kit (Beijing Biode Biotechnology Co., Ltd., China). Each experiment was repeated three times.

[0044] Table 2 Primer information of 14 detoxification genes

[0045]

[0046] 1.5ds Nrf2 Synthesis in vivo

[0047] The ds Nrf2 .according to Nrf2 Specific primers with restriction sites were designed and the target sequence was obtained by PCR cloning. The sequence was connected to the pET28a(+) expression vector by double restriction digestion and transformed into the BL21 (DE3) RNase III- strain to construct the pET28-BL21 (DE3) RNaseIII- expression system and incubated at 37°C overnight. A single colony was selected and cultured in 10 mL of LB liquid medium containing 100 mg / L kanamycin (Kan) at 37°C overnight. When OD 600 When the nm value reached 0.4, isopropyl β- d -1-thiogalactoside (IPTG) (1 mM) was added to induce expression. Lysozyme was then added to the culture medium to a final concentration of 1.3 mg / mL to destroy the cell wall and the bacteria were inactivated by storing at 75°C for 5 min. The resulting bacterial solution was ds Nrf2 .ds Nrf2 , eGFP The sequence information is shown in Table 3, and the underline indicates the restriction site.

[0048] Table 3 ds Nrf2 , eGFP Sequence information

[0049]

[0050] 1.6 SUL / HLDP / ds Nrf2 Analysis of complex assembly mechanism

[0051] As a widely used method, ITC was used to measure the main interaction force between SUL and HLDP. In Nano ITC (TA Instruments Waters, USA), 0.5 mM HLDP was titrated with 0.05 mM SUL. After completion, each titration peak was integrated using Origin7 software (OriginLab Co., USA). The experimental temperature was 25°C and the calculation formula was ΔG = ΔH - TΔS. In order to further clarify the interaction between SUL / HLDP complex and ds Nrf2 The assembly mechanism of 0.3 μM ds Nrf2The titration was performed with 0.0414 μM SUL / HLDP complex and the thermodynamic parameters were recorded. Nrf2 The cells were incubated with SUL / HLDP complexes at mass ratios of 1:0, 1:1, 1:2, 1:3, 1:4, 1:5, and 1:6 for 15 min and detected by agarose gel retardation method.

[0052] 1.7 SUL / HLDP / ds Nrf2 Study on particle size and morphology of composites

[0053] The particle size analyzer and zeta potential analyzer (Anton Paar, Austria) were used to measure the content of SUL (1 mg / mL), SUL / HLDP complex (SUL concentration: 1 mg / mL), ds Nrf2 / HLDP complex (HLDP: ds Nrf2 = 1:1) and SUL / HLDP / ds Nrf2 Particle size and zeta potential of the composite (SUL concentration: 1 mg / mL). Each treatment included three independent samples. 5 μL of each sample was placed on a copper grid plate for air drying, and its morphological characteristics were observed using a transmission electron microscope (TEM) (JEOL-F200, Japan).

[0054] The Tyndall effect is an optical scattering phenomenon of particles in a suspension or colloidal system. When a colloidal solution is irradiated with light of a certain wavelength, a bright light path can be formed in the liquid or gas system, reflecting whether the test liquid has undergone nanoscale aggregation. 40 mL of HLDP (based on PCL of SUL), SUL (0.1 mg / mL) and SUL / HLDP / ds were prepared respectively. Nrf2 The complex (SUL concentration: 0.1 mg / mL) solution was illuminated using a red laser pointer (wavelength 650 nm), and the images were taken in the dark using a Canon EOS R6Mark II camera (Canon, Japan).

[0055] 1.8 SUL / HLDP / ds Nrf2 Contact angle, contact area and surface tension analysis of composites

[0056] SUL / HLDP / ds Nrf2 Complex (SUL concentration was 0.1 mg / mL), ds Nrf2 (Based on HLDP and ds Nrf2The optimal binding mass ratio of ), SUL (0.1 mg / mL), HLDP (based on PCL of SUL), and ddH2O were dropped into cowpea leaves. After the droplets were stable for 20 seconds, the contact angle was observed and measured using a contact angle meter (datphysics OCA25A, Germany), and the contact angle of the droplet image was calculated using an ellipse fitting algorithm. Each solution was tested three times. At the same time, photos were taken using a micromanipulation system (Nikon, Japan), and the contact area was statistically analyzed using imagej1.8 (National Institutes of Health, USA). Each treatment included three independent samples.

[0057] Surface tension analysis was performed using a DCAT 21 surface tension meter based on the Wilhelmy plate method. ddH2O, HLDP (SUL-based PCL), SUL (0.1 mg / mL), ds Nrf2 (Based on HLDP and ds Nrf2 The best quality ratio) and SUL / HLDP / ds Nrf2 The complex (SUL concentration: 0.1 mg / mL) solution. The tensiometer is calibrated using the surface tension of water, ensuring the cleanliness of the plate and glassware. When measuring the sample, ensure that the surface tension value remains constant, indicating that equilibrium is achieved. Each sample is measured three times continuously, and the standard deviation does not exceed ±0.20 mN / m.

[0058] 1.9 SUL / HLDP / ds Nrf2 Determination of retention and plant uptake of the complex

[0059] The SUL / HLDP / ds was determined by gravimetric method. Nrf2 Retention of the composites on cowpea leaves. First, the initial weight (M1) of cowpea leaves (1.8 cm²) was measured. Then, the leaves were immersed in SUL (100 mg / L) and SUL / HLDP / ds Nrf2 The leaves were suspended in the air until no water droplets fell off naturally, and their weight (M2) was recorded. The retention rate was calculated as follows: Retention (mg / cm²) = (M2 -M1) ÷ leaf area. Each treatment included three independent samples.

[0060] UHPLC was used to detect the plant's resistance to SUL / HLDP / ds Nrf2Absorption of the complex. Rapeseed seedlings at the four-leaf stage of the same growth period were carefully washed with ddH2O and air-dried. Subsequently, the seedlings were immersed in SUL (0.2 mg / mL) and SUL / HLDP / ds Nrf2 The complex (SUL concentration: 0.2 mg / mL) solution was soaked for 1 min. After soaking for 6 h, the SUL remaining on the surface of the seedlings was thoroughly washed with ddH2O. 200 mg of seedlings were added to 20 mL of acetonitrile and 3 g of sodium chloride. The mixture was homogenized with an electric grinder, centrifuged at 10000 r / min for 10 min, and the supernatant was collected. Subsequently, 10 ml of the supernatant was evaporated to near dryness under nitrogen at 40°C. 6 ml of methanol-dichloromethane (5:95, v:v) was added and vortexed for 2 min. Purification was performed using an amino column (BKMAM, China), and the eluate was evaporated 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 membrane. The SUL concentration was determined by high-performance liquid chromatography. The specific conditions of UHPLC were as described above. Each treatment included three independent samples.

[0061] In order to facilitate the observation of plant absorption, the fluorescent ds eGFP , SUL / HLDP / ds eGFP Complex (ds eGFP : 1 μg). SUL / HLDP complex (0.808:4 μg) and ds eGFP (1 μg) was used as a control. The bean seedlings were cultured in the above solution for 6 h and the roots were washed with water. The fluorescence ds eGFP Root absorption.

[0062] 1.10 SUL / HLDP / ds Nrf2 Determination of the delivery efficiency of the complex in vitro and in vivo

[0063] Application of fluorescent ds eGFP Preparation of SUL / HLDP / ds eGFP The complex was evaluated for its delivery efficiency in adults of Thrips leucophylla (in vivo) and Drosophila melanogaster S2 cells (in vitro). 0.2 μL of SUL / HLDP complex (0.808:4 μg), ds eGFP (1 μg) and SUL / HLDP / ds eGFP Complex (ds eGFP: 1 μg) were applied to the back of BFT adults, and the adults were washed with ddH2O after 6 h of external application. Photos were taken using a fluorescence inverted microscope (LEICA, Germany), and the fluorescence intensity was quantified using ImageJ 1.8 (National Institutes of Health, USA). Each treatment included 3 biological replicates.

[0064] Drosophila S2 cells were cultured to a density of 5×105 / mL, and SUL / HLDP complex (0.808:4 μg), ds eGFP (1 μg) and SUL / HLDP / ds eGFP Complex (ds eGFP : 1 μg) was added to 0.5 mL of fresh cell culture medium. After 6 and 12 h of incubation, the cell culture medium was replaced with 0.4 mL of fresh culture medium. The mixture was gently pipetted and then dropped on a ConA-treated slide. The cells were fixed with 4% paraformaldehyde (PFA) for 20 min and 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 samples were included for each treatment.

[0065] 1.11 SUL / HLDP / ds Nrf2 Determination of the biological activity of the complexes against BFTs

[0066] Commercial SUL was used to determine the median lethal concentration (LC50) value for adults of Macrothrips bean. Cowpea segments (3 cm) were immersed in different commercial SUL solutions at concentrations of 0, 0.1, 0.2, 0.4, 0.8, 1.6, and 3.2 mg / mL for 1 min, and BFT adults were also immersed in the above solutions for 30 s. Cowpea segments were air-dried and placed in ventilated centrifuge tubes to feed 30 BFT adults, and the mortality rate was recorded 48 h after treatment. LC50 values ​​were calculated using SPSS software (version 23.0, USA), and the experiment was repeated 3 times.

[0067] According to the LC50 values ​​of commercial SUL, the following solutions were prepared: SUL (0.177 mg / mL), SUL / HLDP complex (0.177:0.876 mg / mL), SUL / HLDP / ds eGFP Complex (0.177:0.876:0.219 mg / mL), SUL / HLDP / ds Nrf2 Complex (0.177:0.876:0.219mg / mL), SUL / HLDP / ds Nrf2Complex (0.177:0.876:0.219mg / mL), ds eGFP / HLDP complex (0.219:0.876 mg / mL), ds Nrf2 / HLDP complex (0.219:0.876 mg / mL). Mortality was determined at 12, 24, 36 and 48 h after treatment using the same soaking method and root application method as above (BFT adults fed on cowpea seedlings treated with the above solution). The synergistic effect of insecticidal bioactivity was evaluated using the normalized synergy ratio = adult mortality of each formulation treatment ÷ average adult mortality of SUL treatment alone. The test was repeated 3 times, with 30 adults in each replicate. In addition, SUL / ds was detected at 12, 24, 36 and 48 h after treatment. eGFP Complex (0.177:0.219 mg / mL), SUL / ds Nrf2 Complex (0.177:0.219 mg / mL), SUL / HLDP / ds eGFP Complex (0.177:0.876:0.219 mg / mL) and SUL / HLDP / ds Nrf2 After treatment with complex (0.177:0.876:0.219 mg / mL) Nrf2 The expression levels of the detoxification genes ( P450 , GST, POD and SOD ) expression levels. Each treatment included three independent samples.

[0068] 1.12 SUL / HLDP / ds Nrf2 Field control effect determination of compound

[0069] In the Hongqi South Breeding Experimental Demonstration Base of Sanya Research Institute of China Agricultural University, SUL / HLDP / ds Nrf2 The control effect of the compound was evaluated in a cowpea field. The row spacing of cowpea seedlings was 0.5 m, and the spacing between plants in the row was 0.2 m. The density of adults on cowpea flowers was about 30 per flower. From 7:00 to 9:00 a.m. (when the bean petals were open), HLDP (0.876 mg / mL), ds Nrf2 / HLDP complex (0.219:0.876 mg / mL), SUL (0.177 mg / mL), SUL / HLDP complex (0.177:0.876 mg / mL), SUL / HLDP / ds Nrf2 Complex (0.177:0.876:0.219 mg / mL), SUL / HLDP / ds Nrf2The compound (0.177:0.876:0.219mg / mL) was sprayed at a dose of 100 mL / m². ddH2O was sprayed as a negative control. Each plot was about 20 square meters, with 40-60 plants planted. Ten plants were selected as 10 replicates in each plot, and the number of thrips on flowers was recorded at 0, 1, 3, 5 and 7 days after treatment. The insect population reduction rate and control effect were calculated using the following formula.

[0070] Pest population reduction rate (%) = (number of pests before application - number of pests after application) ÷ number of pests before application × 100;

[0071] Control effect (%) = (insect reduction rate in treatment area - insect reduction rate in control area) ÷ (100 - insect reduction rate in control area) × 100.

[0072] 2 Results

[0073] Statistical analysis: IBM SPSS statistical software (version 22) and GraphPad Prism 8 were used for graph generation and statistical analysis. Independent t-test or Duncan's multivariate range test was used to determine the significant difference between the two groups at the P < 0.05 significance level or multiple comparison analysis. Descriptive data are presented as mean and standard error of the mean.

[0074] 2.1 Nrf2 Key regulatory role in detoxification gene expression

[0075] Synthetic ds via nanocarrier-mediated RNAi Nrf2 Downward adjustment Nrf2 The expression of ds Nrf2 The best mass ratio with HLDP is 1:4. Nrf2 The band disappears, indicating that at this mass ratio, HLDP can be fully loaded with ds Nrf2 ( Figure 1 a). In subsequent experiments, ds Nrf2 / HLDP complex. Then, we used ds Nrf2 / HLDP complex treatment of adults of Macrothrips pea and detection by qRT-PCR Nrf2 interference, and Nrf2 RNA-seq analysis was performed on down-regulated samples ( Figure 1 b) The results showed that compared with the use of ds Nrf2 Compared with the control, ds Nrf2 / HLDP complex has better Nrf2 Disruption effect (reduced to 40.9%) Figure 1 c) Conduct tests on adults of Thrips leucophylla Nrf2After RNAi, volcano plot analysis identified 867 down-regulated genes and 1125 up-regulated genes ( Figure 1 d). KEGG analysis showed that DEGs were enriched in various metabolic pathways, such as starch and sucrose metabolism, carbon metabolism, fatty acid metabolism, pyrimidine metabolism, drug metabolism, and other enzymes ( Figure 1 e). Key detoxification genes ( P450 , GSTs , POD and SOD ) heat map analysis showed that Nrf2 After RNAi, the expression of these genes in BFT was significantly inhibited ( Figure 1 f), and the FPKM value also decreased significantly ( Figure 1 g). Further qRT-PCR results showed that Nrf2 After RNAi, the related detoxification and metabolism genes were significantly inhibited ( Figure 1 h). In addition, their enzyme activities were significantly reduced ( Figure 1 i).

[0076] 2.2 ds Nrf2 Biosynthesis and SUL / HLDP / ds Nrf2 Self-assembly mechanism of the complex

[0077] Will Nrf2 Constructed into pET28a(+) vector, ds was expressed in large quantities in the bacterial high-efficiency expression system pET28-BL21(DE3) RNaseIII- Nrf2 Then, HLDP self-assembled multifunctional nanoprotectors were applied to simultaneously load SUL and ds Nrf2 ( Figure 2 a). Then, ITC was used to detect the interaction between HLDP and SUL. According to the previous interpretation of ITC data, the high affinity constant (Ka) and low dissociation constant (Kd) indicated that HLDP and ds Nrf2 There is an effective and strong interaction between them, and this interaction is spontaneous, as the negative ΔG is -41.175 kJ / mol. The negative values ​​of ΔH (-119 kJ / mol) and ΔS (-311.3 kJ / mol) indicate that ds Nrf2 / HLDP complexes self-assemble through hydrogen bonding and van der Waals forces ( Figure 2 b) In order to further study ds Nrf2 The assembly mechanism of the SUL / HLDP complexes with different mass ratios of ds Nrf2 Agarose gel retardation experiment with SUL / HLDP complex. The results showed that as the mass ratio decreased, the ds Nrf2 The band intensity gradually disappears, indicating that electrostatic interaction exists in SUL / HLDP / ds Nrf2 The self-assembly process of the complex plays an important role ( Figure 2 c) In addition, ITC data showed that hydrogen bonding and van der Waals forces were involved in the interaction between the SUL / HLDP complex and ds Nrf2 It also plays an important role in the combination process ( Figure 2 d). Figure 2 As shown in d, complexation with HLDP increased the zeta potential of SUL from -5.31 mV to 28.38 mV, but the SUL / HLDP / ds Nrf2 The complex is negatively charged (-0.2 mV), which may be due to the negatively charged ds Nrf2 Due to complexation.

[0078] 2.3 SUL / HLDP / ds Nrf2 Characterization of the complex

[0079] Transmission electron microscopy (TEM) and dynamic light scattering (DLS) were used to detect the SUL / HLDP / ds Nrf2 Morphology and particle size of the composites. From several representative TEM images, it was observed that most of the SUL / HLDP / ds Nrf2 The composite is composed of nearly spherical particles. SUL, SUL / HLDP composite, ds Nrf2 / HLDP and SUL / HLDP / ds Nrf2 The particle sizes of the composites are 821.09, 140.30, 241.35 and 221.52 nm respectively. Figure 2 e). SUL / HLDP / ds Nrf2 The particle size of the composite is larger than that of the SUL / HLDP composite, which may be due to the ds Nrf2 The electrostatic adhesion on the surface of SUL / HLDP composite.

[0080] 2.4 SUL / HLDP / ds Nrf2 The complex exhibits the Tyndall effect

[0081] The Tyndall effect is a commonly used physical method to distinguish between colloids and solutions, reflecting whether the liquid has undergone nanoscale aggregation. Solutions that exhibit the Tyndall effect are actually colloidal solutions. When light passes through a colloidal solution, it is mainly scattered because the diameter of the dispersed particles is generally between 1-100 nm, which is smaller than the wavelength of the incident light. This scattering causes a bright path to be observed within the colloid, indicating the presence of the Tyndall effect. We use red lasers to detect HLDP, SUL, and SUL / HLDP / ds Nrf2 The results showed that, in addition to SUL, HLDP and SUL / HLDP / ds Nrf2The composites all showed the Tyndall effect, indicating that both solutions were at the nanoscale ( Figure 3 a).

[0082] 2.5 SUL / HLDP / ds Nrf2 Contact angle, surface tension and contact area of ​​composites

[0083] The contact angle, surface tension and contact area of ​​the droplets reflect the diffusion, deposition and adhesion of the droplets on the leaves. These factors are important factors affecting the efficacy of pesticides. Nrf2 The contact angle of the composite on cowpea leaves (33.31°) was significantly smaller than that of ddH2O (82.78°), HLDP (58.41°), SUL (68.17°) and ds Nrf2 (57.72°)( Figure 3 b) In addition, SUL / HLDP / ds Nrf2 The surface tension of the composite (44.18 mN / m) is also significantly lower than that of ddH2O (72.27 mN / m), HLDP (49.41 mN / m), SUL (63.74 ° mN / m) and ds Nrf2 (66.25mN / m)( Figure 3 c). Due to SUL / HLDP / ds Nrf2 The contact angle and surface tension of the composite were small, and its contact area with the cowpea leaves was the largest in the solution ( Figure 3 d).

[0084] 2.6 SUL / HLDP / ds Nrf2 Retention of the complex and improved plant uptake

[0085] We found that SUL / HLDP / ds Nrf2 The retention and plant absorption rate of the complex increased by 2.36 and 2.53 times, respectively ( Figure 3 e, f). The above results show that SUL / HLDP / ds Nrf2 The complex is easier to diffuse on the leaves and has a stronger affinity for the leaves, which promotes the absorption of pesticides and improves the utilization efficiency of pesticides. Nrf2 The absorption capacity of the complex was tested in cowpea roots. Nrf2 Absorption efficiency of the complex. Using SUL / HLDP complex, fluorescent ds eGFP and SUL / HLDP / fluorescent ds eGFP The complex cultured seedling roots and compared the fluorescence intensity of the seedling stems, and found that SUL / HLDP / fluorescent ds eGFPThe fluorescence intensity of the stems treated with the complex was the highest, significantly higher than that of the SUL / HLDP complex and the fluorescent ds eGFP The fluorescence intensity of the treated stems ( Figure 3 g). This indicates that HLDP can promote the absorption of dsRNA by cowpea roots.

[0086] We synthesized the fluorescent ds eGFP , and prepared SUL / HLDP / fluorescent ds eGFP The complex was then tested to evaluate its delivery efficiency in adults of Macrothrips leucophylla (in vivo) and Drosophila melanogaster S2 cells (in vitro). eGFP The fluorescence intensity of the adult worms treated with the complex was significantly higher than that of the adult worms treated with the SUL / HLDP complex and the fluorescent ds eGFP Processed BFT( Figure 3 h). Meanwhile, in vitro, SUL / HLDP / fluorescent ds eGFP The complex can effectively cross the cell membrane and enter Drosophila S2 cells ( Figure 3 i) In summary, the results show that SUL / HLDP / ds Nrf2 The complexes showed excellent delivery capabilities both in vitro and in vivo, and their advantages are summarized in Figure 3 j.

[0087] 2.7 SUL / HLDP / ds Nrf2 Insecticidal biological activity of the complex and its effect on detoxification metabolism

[0088] According to the regression equation of the toxicity of SUL to adults of Macrothrips bean, different preparations were prepared using the LC50 value of SUL and the PLC of HLDP, including SUL / HLDP complex, ds Nrf2 / HLDP compound, SUL / HLDP / ds Nrf2 The biological activities of these formulas were tested by immersion method and root absorption method ( Figure 4 a, f). The results showed that the immersion method and root absorption method ds Nrf2 / HLDP complex treatment had a slight lethal effect on BFT adults, with mortality rates of 28% and 24%, respectively ( Figure 4 b, g). After 48 h of treatment by immersion method and root absorption method, SUL / HLDP / ds Nrf2 The insecticidal biological activity of the complex was the highest, reaching 72% and 68%, respectively, which was significantly higher than 53% and 52% of SUL alone ( Figure 4 b, g). In addition, compared with using SUL alone, SUL / HLDP / ds Nrf2 The normalized synergistic ratios of the complexes were 1.35-1.43 and 1.31-2.00 ( Figure 4c, h). The doses required for the multi-component nanoformulation to achieve similar insecticidal activity after immersion and root application for 48 h were calculated. Nrf2 The above results show that under the action of HLDP, SUL / HLDP / ds Nrf2 The biological activity of the complex was significantly higher than that of SUL and ds Nrf2 Acting alone. This will greatly reduce the use of pesticides and is conducive to the sustainable development of green agriculture.

[0089] In addition, we use various solutions including SUL / ds by immersion and root absorption. eGFP Composite, SUL / ds Nrf2 Compound, SUL / HLDP / ds eGFP Compound and SUL / HLDP / ds Nrf2 Complex, explored Nrf2 and detoxification and metabolism genes ( P450 , GST, POD and SOD ) expression. The results showed that SUL / HLDP / ds Nrf2 The complex significantly inhibited BFT adult worms Nrf2 and detoxification and metabolism genes ( P450 , GST, POD and SOD ) expression. In addition, its interference efficiency is significantly higher than SUL / ds Nrf2 complex, indicating that HLDP enhances Nrf2 and RNAi interference efficiency of detoxification and metabolic genes ( Figure 4 d, e, i, j).

[0090] 2.8 SUL / HLDP / ds Nrf2 The compound has excellent control effect on BFTs in the field

[0091] Subsequently, the SUL / HLDP / ds Nrf2 The control effect of the compound on bean thrips. The density of adults on cowpea flowers was controlled at about 30 per flower. Then the field was sprayed with HLDP, ds Nrf2 / HLDP composite, SUL, SUL / HLDP composite and SUL / HLDP / ds Nrf2 The dosage of the compound was 100 mL / m². The number of BFT adults on cowpea flowers was recorded at 1, 3, 5, and 7 days after spraying, and the control effect was calculated. The evaluation results showed that 1 to 7 days after spraying, SUL / HLDP / ds Nrf2The efficacy of the composite agent gradually increased, reaching a maximum efficacy of 89.89% on the 7th day; this was significantly higher than 72.66% of SUL / HLDP composite, 62.88% of SUL, 24.56% of ds Nrf2 / HLDP complex and 3.98% HLDP ( Figure 4 k). In addition, the representative images clearly show the control effect of the above solutions on BFTs ( Figure 4 i) It is worth noting that HLDP helped SUL achieve nano-scale pesticides and improved Nrf2 and detoxification metabolism genes, thereby enhancing the insecticidal biological activity. Nrf2 The complex has a good control effect on BFTs.

[0092] 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 nanoformulation for improving the sensitivity of Thrips leucophylla to pesticides, characterized in that: Includes the following ingredients: sulfoxaflor, carrier HLDP and ds Nrf2 ,ds Nrf2 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 nanoformulation for improving the sensitivity of Thrips leucophylla to pesticides according to claim 1, characterized in that: The complex formed by sulfoxaflor and carrier HLDP reacts with ds Nrf2 The mass ratio is (1~6):

1.

3. The multi-component nanoformulation for improving the sensitivity of Thrips leucophylla to pesticides according to claim 1, characterized in that: The ds Nrf2 By chemical synthesis or in vivo synthesis; the chemical synthesis comprises the following steps: using primers containing T7 promoter sequence, using PCR amplification Nrf2 The gene fragment is transcribed into ds Nrf2 The in vivo synthesis comprises the following steps: using primers with restriction site specific primers, using PCR amplification to obtain Nrf2 The target sequence is double-digested and connected to the expression vector, and then transformed into the strain to obtain ds Nrf2 .

4. The multi-component nanoformulation for improving the sensitivity of Thrips leucophylla to pesticides according to claim 1, characterized in that: The multi-component nanoformulation contains sulfoxaflor, carrier HLDP and ds Nrf2 The concentration ratio is 0.177 mg / mL:0.876 mg / mL:0.219 mg / mL.

5. The method for preparing a multi-component nanoformulation for improving the sensitivity of Thrips leucophylla to a drug according to any one of claims 1 to 4, characterized in that: HLDP was titrated with sulfoxaflor to form a sulfoxaflor / HLDP complex; ds Nrf2 , and finally formed sulfoxaflor / HLDP / ds Nrf2 Complex, that is, multi-component nanoformulation.

6. Use of the multi-element nanoformulation according to any one of claims 1 to 4 in the preparation of a product for improving the sensitivity of Thrips leucophylla to drugs, characterized in that: The sensitivity to the drug is the sensitivity to sulfoxaflor.

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