CN115843794B - Nanometer system of plant protein-based drug-encapsulated molecules and preparation method and application thereof - Google Patents
Nanometer system of plant protein-based drug-encapsulated molecules and preparation method and application thereof Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention relates to the technical field of plant disease resistance enhancement, in particular to a nano system of plant protein-based drug molecule encapsulation, a preparation method and application thereof. The specific technical scheme is as follows: the preparation process of nanometer system with medicine molecule coated onto vegetable protein base includes mixing medicine molecule and pyridine modified polyhydroxyethyl methacrylate in N, N-dimethylformamide, adding into vegetable protein solution, stirring, dialysis, centrifuging and freeze drying. The invention solves the problem that drug molecules are easy to degrade along with light in the field and have poor leaf surface wettability, so that the drug effect is reduced, and simultaneously provides a novel strategy for applying a nano system of coating drug molecules on a plant protein basis to enhance crop disease resistance.
Description
Technical Field
The invention relates to the technical field of plant disease resistance enhancement, in particular to a nano system of plant protein-based drug molecule encapsulation, a preparation method and application thereof.
Background
Food safety is critical to both human health and economic development. Many plant diseases not only reduce crop yield, but also are very susceptible to causing quality degradation of crops. For example, plant viral diseases (such as tobacco mosaic virus) are highly contagious, causing crop production losses each year, with economic values exceeding $1 million. Ningnanmycin and ribavirin are currently the most commonly used antiviral drugs. However, their wide application is hampered by the drawbacks of photosensitivity, high cost, poor therapeutic effect, etc. To achieve effective viral disease control, farmers are forced to overuse these agents or add adjuvants to pesticides, including organic solvents, surfactants and dispersants. Even under these conditions, the biological uptake of these compounds is less than 0.1%. Therefore, the problems of low pesticide effect, low pesticide utilization rate and the like of the traditional pesticide are increasingly remarkable, the environmental pollution is aggravated, and finally the ecological system and the human health are damaged. Thus, new methods and technical solutions are needed to increase global grain yield, minimize the impact of agriculture on the environment, and maintain the toughness of agricultural ecosystems.
The use of nanotechnology is expected to provide a new strategy for achieving sustainable "precision" agriculture. For example, researchers have shown that leaf surfaces are exposed to a composition containing ZnO or Fe 3 O 4 Can directly disable Tobacco Mosaic Virus (TMV), promote the growth of Nicotiana benthamiana and induce the defense response to plant viruses. The protective role of Nanomaterials (NMs) in inhibiting plant viruses has been reported to induce tobacco resistance by blocking the physical movement and replication of TMV. In the prior art, an alginate-based nanogel (CHI@ALGNP) loaded with Chloroindole Hydrazide (CHI) is prepared, has excellent foliar adhesion and promotes coatingAnd (3) slow release of the wrapping compound. The application of NMs in crop protection may bring about a paradigm shift in the formulation of pesticides, replace traditional agrochemicals, and increase the bioavailability of pesticides. Various core-shell nanoparticles (CS NPs) exhibit excellent properties including high encapsulation efficiency, stimuli-responsive pesticide release, and photochemical stability. Therefore, in order to achieve the goal of precise agriculture, it is essential to develop novel nanocarriers that provide an environmentally friendly preparation method and sustained pesticide release in response to stimulus.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a nano system for coating the drug molecules on the basis of plant proteins, a preparation method and application thereof, solves the problem that the drug effect is reduced due to the fact that the drug molecules are easy to degrade along with light and the wettability of leaf surfaces is poor in the field, and simultaneously provides a novel strategy for applying the nano system for coating the drug molecules on the basis of plant proteins to enhance the disease resistance of crops.
In order to achieve the above purpose, the invention is realized by the following technical scheme:
the invention discloses a preparation method of a nano system of a plant protein-based drug-loaded molecule, which comprises the steps of mixing the drug molecule with pyridine modified polyhydroxyethyl methacrylate in N, N-dimethylformamide, adding the mixture into a plant protein solution, uniformly stirring, dialyzing, centrifuging and freeze-drying to obtain drug-loaded nano particles.
Preferably, the drug molecule is at least one of a plant resistance inducer, an antiviral agent, a fungicide, a bactericide, an insecticide, a nematicide and an acaricide with low solubility, and the plant protein is a soluble soybean whey protein solution.
Preferably, the mass ratio of the pyridine modified polyhydroxyethyl methacrylate to the drug molecules is 2-8:1, the mass ratio of the vegetable protein to the drug molecules is 20:1, and the volume ratio of the N, N-dimethylformamide to the vegetable protein is 3:100.
Preferably, the concentration of the plant protein is 4-6 mg/mL.
Preferably, the pyridine modified poly (hydroxyethyl methacrylate) is prepared from poly (2-hydroxyethyl methacrylate) and nicotinyl chloride hydrochloride, and the relative molecular mass of the poly (2-hydroxyethyl methacrylate) is 20kDa.
Preferably, the dialysis condition is that the mixed solution after stirring is transferred into a dialysis bag, dialyzed for 24 hours in distilled water, centrifuged three times continuously, insoluble precipitate is removed, and the condition of each centrifugation is 2000rpm,5 min/time.
Correspondingly, the plant protein-based drug molecule-entrapped nano system prepared by the preparation method is provided.
Correspondingly, the nano system of the plant protein-based drug-encapsulated molecules prepared by the preparation method or the application of the nano system of the plant protein-based drug-encapsulated molecules in the preparation of the drug for enhancing the disease resistance of the agricultural pest hosts.
Preferably, the agricultural pest is tobacco mosaic virus, cucumber mosaic virus, potato virus X, rice bacterial leaf blight, citrus canker, tobacco bacterial wilt, kiwi fruit canker, rice bacterial leaf spot, cucumber gray mold, pepper wilt, rape sclerotinia, wheat scab, potato late blight, blueberry root rot, porter, dragon fruit anthracnose, rice sheath blight, green worms, caterpillars, aphids, scale insects, whiteflies, root knot nematodes, bursaphelenchus xylophilus, stem nematodes, cotton spider mites, citrus red mites.
The invention has the following beneficial effects:
1. the core-shell nano system of the plant protein-based drug-coated molecular disease cyanogen nitrate has the advantages of simple preparation method and high repeatability. The obtained core-shell spherical nano particles have stable and uniform diameter of about 130nm and encapsulation rate of 90 percent;
2. the core-shell nano system of the plant protein-based drug-encapsulated molecular disease nitro-compound has better photostability and wettability than the molecular disease nitro-compound, has long sustained-release and lasting period, releases active ingredients through in-vitro and in-vivo urea triggering and has better antiviral activity, the particle size of the obtained core-shell nano particles is small, and the problems of poor dispersibility, low effective utilization rate and the like of the traditional pesticide liquid can be solved.
3. The core-shell nano system of the plant protein-based drug-encapsulated molecular disease cyanogen nitrate prepared by the invention has the application prospect of promoting plant growth, improving the disease resistance of plants, cooperatively controlling plant virus diseases, reducing ecological environment pollution and grain safety problems and increasing grain yield.
Drawings
FIG. 1 is a nuclear magnetic resonance spectrum of a drug molecule, cyanogen nitrate (BQX);
FIG. 2 is a nuclear magnetic carbon spectrum of the drug molecule cyanogen nitrate (BQX);
FIG. 3 is a nuclear magnetic resonance spectrum of pyridine modified polyhydroxyethyl methacrylate (PyPHEMA);
FIG. 4 is a graph showing the results of a particle size analyzer measurement of the average particle size distribution, zeta potential and polydispersity index (PDI) of vegetable protein based nanoparticles (BQX@PP@S NPs);
FIG. 5 is a test result of encapsulation efficiency of vegetable protein-based nanoparticles (BQX@PP@S NPs) determined by HPLC;
FIG. 6 is a transmission electron microscopy image (100 nm,200nm,500 nm) of a vegetable protein based nanoparticle (BQX@PP@S NPs);
FIG. 7 is a Fourier infrared and Raman spectrum characterization result of vegetable protein-based nanoparticles (BQX@PP@S NPs), PP@S NPs, SWP, pyPHEMA, BQX;
FIG. 8 shows the results of surface element analysis of vegetable protein-based nanoparticles (BQX@PP@S NPs), PP@S NPs, SWP, BQX;
FIG. 9 shows the results of UV degradation and contact angle measurements of vegetable protein-based nanoparticles (BQX@PP@S NPs), BQX;
FIG. 10 is a graph of in vitro urea response release profile of vegetable protein based nanoparticles (BQX@PP@S NPs);
FIG. 11 shows the control effect of TMV in vivo with vegetable protein based nanoparticles (BQX@PP@S NPs);
FIG. 12 is a graph showing the control effect of urea response release on TMV in vivo with vegetable protein based nanoparticles (BQX@PP@S NPs);
FIG. 13 is a test result of plant protein based nanoparticles (BQX@PP@S NPs) induced disease resistance of tobacco plants;
FIG. 14 is the effect of plant protein based nanoparticles (BQX@PP@S NPs) on tobacco plant growth.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated.
The invention discloses a preparation method of a core-shell nano system of plant protein-based drug-encapsulated molecules for enhancing plant disease resistance, which comprises the following steps:
(1) Drug molecules and pyridine modified polyhydroxyethyl methacrylate (PyPHEMA) are simultaneously dissolved in N, N-dimethylformamide to obtain a mixed organic solution A; the medicinal molecule can be cyanogen nitrate (BQX) with purity not less than 98wt%, or at least one of insoluble plant attractant, antiviral agent, fungicide, bactericide, insecticide, nematicide and acaricide. Pyridine modified poly (hydroxyethyl methacrylate) (PyPHEMA) is prepared from poly-2-hydroxyethyl methacrylate (pHEMA) and nicotinyl chloride hydrochloride, and the relative molecular mass of the poly-2-hydroxyethyl methacrylate (pHEMA) is 20kDa. The mass ratio of the pyridine modified poly (hydroxyethyl methacrylate) (PyPHEMA) to the cyanogen nitrate (BQX) is 2-8:1.
(2) Slowly and dropwise adding the solution A into a prepared soluble soybean whey protein Solution (SWP) under the condition of continuous stirring to obtain a compound solution B; the mass concentration of Soy Whey Protein (SWP) is about 4 to 6mg/mL, preferably 5.15mg/mL, as determined by the Bradford method. The mass ratio of the soybean whey protein Solution (SWP) and the drug molecules (such as the cyanogen nitrate (BQX)) in the compound solution B is 20:1; the effective mass concentration of the drug molecule (such as the Benzonitrile (BQX)) is 250mg/L. The final volume ratio of N, N-dimethylformamide to soy whey protein Solution (SWP) was 3:100.
(3) And continuously stirring the composite solution B uniformly, dialyzing, continuously centrifuging for three times to remove insoluble precipitate, and freeze-drying the final drug-loaded nano particles (BQX@PP@S NPs). The dialysis conditions were transfer to dialysis bags (MWCO 3500 Da), dialysis in distilled water for 24 hours, and continuous centrifugation three times for removal of insoluble precipitate were 2000rpm,5 min/time.
The core-shell nano system of the plant protein-based drug-encapsulated molecules prepared by the invention can enhance the disease resistance of the host plant of agricultural diseases and insect pests, wherein the agricultural diseases and insect pests are virus diseases such as tobacco mosaic virus, cucumber mosaic virus, potato virus Y and the like; bacterial diseases such as bacterial leaf blight of rice, canker of citrus, bacterial wilt of tobacco, canker of kiwi fruit, bacterial leaf spot of rice and the like; fungal diseases such as gray mold of cucumber, pepper blight, sclerotinia rot of rape, wheat scab, potato late blight, blueberry root rot, grape vine cavity bacteria, anthracnose of dragon fruit, rice sheath blight and the like; insect diseases such as green insects, caterpillars, aphids, scale insects, whiteflies and the like; nematode diseases such as root-knot nematodes, bursaphelenchus xylophilus and stem nematodes; mite diseases such as spider mites and citrus red mites.
The invention is further illustrated below in conjunction with specific examples.
Example 1
Materials and methods of synthesis for use in the present invention
Materials: poly-2-hydroxyethyl methacrylate (pHEMA, MW=20 kDa), 8% aqueous Ningnanmycin, nicotinyl chloride hydrochloride (98%), anhydrous pyridine and all other chemicals and solvents were purchased from commercial sources. Defatted soy flakes (52.4% protein content (n×6.25, dry basis) with a Nitrogen Solubility Index (NSI) of 85%) were provided by the shandong Mo Defu group limited.
(1) The novel antiviral candidate drug of the designed and synthesized cyanogen nitrate (BQX): fig. 1,2 show: 1 HNMR(400MHz,CDCl 3 ):12.03(s,1H,NH),7.38-8.12(m,4H,Ar-H),4.28-4.34(q,J＝6.0Hz,2H,CH 2 ),2.22(s,3H,S-CH 3 ),1.33-1.37(t,J＝6.4Hz,3H,C-CH 3 ). 13 C NMR(400MHz,CDCl 3 ):168.11,168.08,141.91,134.15,132.43,126.59,126.16,125.88,116.63,82.89,61.59,17.02,14.17.
(2) Synthesis of pyridine modified polyhydroxyethyl methacrylate (PyPHEMA)
Poly-2-hydroxyethyl methacrylate (589.1 mg,4.54 mmol) was dispersed in anhydrous tetrahydrofuran (5.9 mL) and anhydrous pyridine (2.4 mL) and the mixture stirred at room temperature until the poly-2-hydroxyethyl methacrylate dissolved. To the mixed solution was added nicotinyl chloride hydrochloride (2.0003 g,11.24 mmol) to initiate the reaction, and the esterification reaction was carried out at room temperature for 72 hours, followed by distillation under reduced pressure to remove the solvent. By CH 2 Cl 2 Dissolving, washing with water for 6 times, anhydrous Na 2 SO 4 Drying and finally removing the solvent again. The resulting solid was dissolved in 6.0mL CH 2 Cl 2 In (2) was added dropwise to a mixed solvent of petroleum ether and ethyl acetate (100:1, v/v) under ice bath conditions until the white precipitate was completely precipitated. Finally, the mixed solvent is removed, and the precipitate is collected and dried. As shown in fig. 3: 1 H NMR(400MHz,DMSO-d 6 ,ppm):8.96(1H,pyridine-H),8.70(1H,pyridine-H),8.12(1H,pyridine-H),7.42(1H,pyridine-H),4.39(2H,OCH 2 ),4.15(2H,OCH 2 ),1.74(2H,CH 2 ),0.78-0.88(3H,CH 3 ).
(3) Preparation of Soy Whey Protein (SWP) solution
Extracting defatted soybean flakes and distilled water at a feed-to-liquid ratio of 1:8, and stirring and mixing at room temperature for 30 min. After insoluble precipitate is removed by centrifugation of the obtained suspension, the pH of the supernatant is adjusted to 4.5 by hydrochloric acid to obtain acid insoluble isolated soy protein as precipitate, and the isolated soy protein is removed by centrifugation. The supernatant was adjusted to ph=8.0 with 2.0M NaOH, centrifuged again and the solution was stored at 4 ℃. The prepared soy whey protein content was about 0.515% as analyzed by Bradford method.
EXAMPLE 2 preparation of drug-loaded nanoparticles
A certain amount of cyanogen nitrate (BQX) and pyridine modified polyhydroxyethyl methacrylate (PyPHEMA) are respectively weighed and dissolved in N, N-Dimethylformamide (DMF) to obtain organic solutions BQX and PyPHEMA, and 5 groups of organic solutions are respectively prepared in an EP tube of 1.5mL according to the mass ratio of PyPHEMA to BQX of 2:1, 3:1, 4:1, 6:1 and 8:1. Then, under the condition of continuous stirring, organic solutions with different proportions are slowly and dropwise added into a certain amount of Soybean Whey Protein (SWP) obtained by extracting defatted soybean flakes, and the mass ratio of the Soybean Whey Protein (SWP) to the cyanogen nitrate (BQX) is about 20:1. The final volume of the organic solvent in the mixed solution was maintained at 3% (v/v) of the aqueous solution, the mixed solution was stirred uniformly at room temperature, and transferred to a dialysis bag (MWCO 3500 Da) for dialysis in distilled water for 24 hours. Finally, removing insoluble sediment by continuous centrifugation for three times at 2000rpm and 5 min/time to obtain the nano particles.
PyPHEMA: BQX =2:1, 3:1, 4:1, 6:1, 8:1, (m/m); SWP BQX =20:1, (m/m) the prepared nanoparticles were named bqx@pp@s NPs, the above proteins: and (2) polymer: the nanoparticles with different mass ratios of 20:2:1, 20:3:1, 20:4:1, 20:6:1, 20:8:1 of the drug were measured by a particle sizer for nanoparticle size distribution, zeta potential, and polydispersity index (PDI). The results are shown in FIG. 4. The results show that the optimal mass ratio of SWP, pyPHEMA and BQX is 20:4:1.
The nanoparticles produced were characterized and Transmission Electron Microscopy (TEM) images were obtained on a FEI Talos F200C electron microscope (voltage: 200 kV)) (FEI, U.S. A.). Fourier Transform Infrared (FTIR) spectroscopy was performed using a Thermo Scientific Nicolet iS spectrometer (Thermo Scientific Nicolet iS, thermo Fisher Scientific, china). Chemical interactions were studied using a Horiba HR800 confocal Raman microscope spectroscopic system (Horiba HR800, horiba JobinYvon, france). X-ray photoelectron spectroscopy (XPS, thermo Fisher Scientific, u.s.a.) tests were performed after three washes and drying.
The result of the projection electron microscope is shown in fig. 6, and the result shows that the invention successfully prepares the nano-particle BQX@PP@S NPs with a core-shell structure by an environment-friendly non-covalent self-assembly method, and the average particle size is smaller than about 130nm. The fourier infrared characterization results are shown in fig. 7, and the surface element analysis results of the drug-loaded nanoparticles (bqx@pp@s NPs) and pp@s NPs, SWP, BQX are shown in fig. 8.
Example 3 drug loaded nanoparticle encapsulation efficiency test
Different concentrations (250, 200, 150, 100 and 50. Mu.g/mL) of the stock solution of cyanogen nitrate-N, N-dimethylformamide were prepared in 1mL of aqueous solution. And establishing a standard curve by adopting a high performance liquid chromatography. A viable regression equation is obtained. In the measurement process, the nano particles are subjected to self-assembly and then subjected to centrifugation for three times at 2000rpm for 5min to remove excessive cyanogen nitrate. Subsequently, the excess amount of sick cyanide was measured by High Performance Liquid Chromatography (HPLC). The result is shown in fig. 5, and the result shows that the prepared nano-particle BQX@PP@SNPs have smaller particle size, uniform and stable distribution and high encapsulation efficiency up to 90%.
Encapsulation efficiency was calculated according to the following formula:
the encapsulation Efficiency (ER) is determined by the relative ratio of the amount of mirabilite in the supernatant to the total amount of mirabilite initially added to the system. ER% = amount of encapsulated cyanogen/amount of total input cyanogen x 100%.
Example 4 test of drug-loaded nanoparticle photostability and blade surface wettability
To analyze the photostability of nanoparticles (bqx@pp@s NPs), bqx@pp@s NPs were suspended in distilled water, while an equal amount of BQX was suspended in 1.0% tween-80 distilled water. These test solutions were added to quartz tubes and placed under a 20cm distance of 6W germicidal ultraviolet lamp (254 nm), and the experiment was performed at room temperature. Samples containing BQX were collected from each quartz tube at time points 0, 1,2, 4, 8, 12, 24 and 48h, filtered through a 0.22 μm membrane filter, and analyzed for their intact BQX content by high performance liquid chromatography.
Wettability of the nanoparticles (bqx@pp@s NPs) was evaluated using a dynamic contact angle method. Tobacco leaves grown in a separate greenhouse were mounted on slides and droplets of different solutions were applied to the leaf surfaces using a micro-syringe. Dynamic contact angles were measured 10s later with a JC-2000D contact angle meter and each solution was tested three times on different areas of the leaf.
One of the main environmental factors limiting the efficacy of pesticides under field conditions is light, especially foliar spray. In order to compare the photosensitivity of BQX@PP@S NPs with that of the unpackaged compounds, the present invention examined the photostability of the samples exposed to ultraviolet light. Fig. 9A shows that the photodegradation of BQX is very rapid, with a loss of 45.0% of the active ingredient after 12h of irradiation. In contrast, NPs encapsulated BQX decomposed slower under uv light irradiation. For BQX@PP@S NPs, the cumulative residual rate of BQX in 48h is 85.3% which is 8.7 times the residual rate of unpackaged BQX. The existence of the core-shell structure of the BQX@PP@S NPs improves photochemical stability, which is a great impetus for the practical application of the NPs. The coated compound is shielded from decomposition under ultraviolet radiation by weakening the intensity of the ultraviolet light, thereby ensuring that the pesticide can be sprayed on the plant leaves, and the improved stability can reduce the pesticide dosage, the spraying times and prolong the antiviral efficiency.
The wettability of BQX@PP@S NPs on tobacco leaves was monitored by contact angle measurement. In these experiments, a small contact angle indicates a low probability of a droplet rolling off the blade surface. As shown in fig. 9B, the contact angle caused by bqx@pp@s NPs encapsulation is significantly reduced (44.5 °) compared to the contact angle of BQX dissolved in water (81.0 °), and therefore, the core-shell structure of bqx@pp@s NPs improves the wetting effect of hydrophobic drug molecules BQX on tobacco, potentially promoting drug deposition, leaf absorption, bioavailability, and biocompatibility.
Example 5 test of drug-loaded nanoparticle in vitro Urea response Release BQX
The effect of urea on bqx@pp@s NPs release BQX was examined by high performance liquid chromatography. In urea experiments urea (0, 0.1 and 0.2M) was added to a 20mL ethanol-water (50:50, v/v) mixture and tested for BQX release under different conditions. At various time points, 1.0mL of solution was collected and supplemented with an equal amount of urea-containing ethanol-water mixture (50:50, v/v). The release of BQX was calculated using a high performance liquid chromatography Agilent 1260 setting up a standard calibration curve at 380 nm. The cumulative release rate (Ri) of BQX was calculated as follows, where ρi (mg/L) is BQX, the mass concentration in the sample solution, and all experiments were repeated at least 3 times.
The results are shown in FIG. 10, and FIG. 10 clearly shows the effect of urea on the release of drug molecules BQX by BQX@PP@S NPs. In the control experiment (urea concentration 0M), BQX release after 12h was 61.7%. In contrast, bqx@pp@s NPs released 71.9% of BQX when the urea concentration was 0.2M. These results indicate that urea triggers bqx@pp@s NPs to release drug molecules BQX at a certain concentration.
Example 6 in vivo testing of Urea-triggered Release BQX in drug-loaded nanoparticles for antiviral Activity
In vivo antiviral activity assays were performed using TMV as the infectious agent using the half-leaf plaque method, and Ningnanmycin, a commercial antiviral compound, was used as a positive control under the same conditions, and each experiment was repeated three times. The in vivo urea reactivity BQX release test procedure was identical to the in vivo antiviral activity test procedure, and after the drug solution was spread evenly on the leaves for 12h, different concentrations (0, 0.1 and 0.2M) of urea were sprayed onto the leaves.
The results are shown in fig. 11, and fig. 11 shows the antiviral effect of NPs compared to uncoated BQX, with the commercial antiviral drug ningnanmycin as a positive control. These experiments show that foliar application of BQX@PP@S NPs can significantly reduce the number of spots caused by virus transmission, and the antiviral efficiency of 500mg/L is 20.7-49.9% after the tobacco plants are treated by the control sample BQX. When BQX@PP@S NPs are used, the curative effect of 500mg/L BQX is improved from 20.7-49.9% to 59.2-86.4%. Compared with unencapsulated BQX, the BQX@PP@S NPs with the concentration of 250mg/L significantly improves the antiviral efficiency, the therapeutic activity is 16.5%, the protective activity is 17.5%, and the inactivation rate is 42.4%. BQX@PP@S NPs exhibit optimal antiviral properties at 500mg/L, with 18.1%, 20.2% and 65.7% improvement in therapeutic, protective and inactivating activities over BQX, respectively. These results indicate that the antiviral activity of bqx@pp@s NPs is almost comparable to the currently most effective plant virus inhibitor, ningnanmycin. Based on these findings, BQX@PP@S NPs applications were used at a concentration of 500mg/L in the disease control performance and mechanism of action studies described below.
Since in vitro BQX@PP@S NPs release BQX faster at 0.2M urea concentration, plant leaves were uniformly sprayed with urea at different concentrations (0, 0.1 and 0.2M) 12h after BQX@PP@S NPs were applied. As shown in FIG. 12, the antiviral efficiency of urea with the concentration of 0.2M is improved by 3.2 to 6.5 percent, which is equivalent to that of Ningnanmycin. The results indicate that urea promotes the release of active ingredients in the nanoparticle body, and thus, urea-induced active ingredient release may be considered as one of the factors supporting the improvement of antiviral efficiency.
Example 7 drug loaded nanoparticle enhanced plant disease resistance test
Antioxidant enzyme activity assay: tobacco plants were the test material, and after treatment with BQX@PP@S NPs, BQX and Control (water), the leaves were smeared with TMV to infect the whole tobacco plant. In addition, the following experimental leaves were sampled at 1, 3, 5 and 7 days. Finally, SOD, POD, CAT and PAL enzyme activities were measured using a commercially available kit and analyzed using a microplate reader. To verify the effect of active oxygen scavenging, tobacco plant leaves were continuously sprayed with BQX@PP@S NPs, BQX and Control (water) for five days before being infected with recombinant TMV-GFP. 7 days after TMV-GFP inoculation, staining with 1mg/mL3, 3-diaminobenzidine was performed to monitor H in vivo 2 O 2 Top leaf of each sample was taken for the following RT-qPCR analysis). The leaves were immersed in a 3, 3-diaminobenzidine solution in the absence of light and gently infiltrated under vacuum for 30 minutes. To obtain a better dyeing effect, the leaves were shaken at 100 rpm for 4 hours, boiled in 90% ethanol for 20 minutes to completely decolorize the leaves, and finally photographed. RT-qPCR analysis: RNA was extracted from the tobacco leaves described above and was prepared according to PrimeScript TM Reverse transcription is carried out by the instruction of the RT kit, and then the expression quantity change of the disease-resistant related genes PR1 and PR2, the salicylic acid biosynthesis NbCIS gene and the abscisic acid biosynthesis NbNCED gene is measured by real-time fluorescence quantitative PCR.
As shown in FIG. 13, it can be seen from FIGS. 13A-D that BQX@PP@S NPs caused a significant change in the activity of defensive enzymes in tobacco leaves after treatment of tobacco. SOD levels increased rapidly, reaching a maximum 24 hours after contact. At this time, the SOD level of bqx@pp@s NPs treated plants was increased by 312% and 320% compared to that of BQX treated or control plants, respectively. Second, bqx@pp@s NPs also significantly increased CAT levels. CAT levels increased by 286% after 7 days of initial exposure compared to BQX treatment, indicating H caused by the innate resistance mechanism 2 O 2 Up-regulation of enzymes by increased levels. Fig. 13 also shows that bqx@pp@s NPs also resulted in an increase in POD level. After 5 days of initial contact, the content reached a maximum, which was 169% of the BQX treated plants. Finally, after a rapid rise, PAL levels peaked on the third day, increasing by 153% over BQX treatment. These observations indicate that bqx@pp@s NPs promote an increased variation in antioxidant enzymes in a range of tobacco plants. Diaminobenzidine (DAB) staining demonstrates ROS scavenging activity in plant tissues. FIG. 13E shows brown DAB staining of infected control leaves (left panel), indicating that the pathogen caused H 2 O 2 Excessive accumulation. After foliar application of BQX or bqx@pp@snps, the brown color of the leaves was significantly reduced. Compared to BQX treatment, bqx@pp@s NPs treatment resulted in fewer DAB staining areas and lower staining intensity, suggesting that NPs formulated plants reduced viral entry and less oxidative damage, thereby causing disease-resistant responses and other disease-resistant signal generation and transmission in plants.
The present invention detects the expression levels of two Pathogenic Related (PR) genes PR1 and PR2 regulated by SA. As shown in FIG. 13F, the relative expression levels of PR1 and PR2 treated with BQX@PP@S NPs were increased by 457% and 302%, respectively, compared to the control, while the expression level of the BQX treated plants was not significantly up-regulated. Upregulation of plant hormones such as SA and ABA plays a key role in stimulating the immunity of plants to viral infections and coordinating the activity of other hormones or hormone mimics. Since SA biosynthesis and accumulation in tobacco are controlled by the NbICS gene, the relative expression levels of NbICS were analyzed. NCED is a gene encoding a key enzyme for abscisic acid (ABA) -mediated reactions, and its expression has also been studied. BQX@PP@S NPs increased the expression of both groups of genes compared to BQX treatment. As shown in FIG. 13G, nbICS abundance increased by 193% and NCED expression increased by 189%.
Example 8 test of plant protein-based nanoparticles to promote plant growth
48 tobacco seedlings with consistent growth trend in 8 leaf periods are selected, and BQX@PP@S NPs, BQX or Control (water) are continuously sprayed on leaves of the tobacco plants for 5 days. Plants were grown in separate greenhouses for 7 days at 28 ℃/25 ℃ with 16h/8h light and dark cycles and 85% relative humidity. Firstly, the physiological form of the plant is recorded by using a digital camera, at this time, the plant height and the leaf width are measured, fresh plant tissues are collected, and the fresh weight is measured by washing and air-drying with distilled water. The tissue was then dried at 80 ℃ for 4 hours and the dry weight recorded. Finally, 1/3 of the plant leaves were used for the determination of the chlorophyll content, which were rinsed with tap water and then with distilled water. Chlorophyll content was measured with a commercial kit and the results were analyzed with an enzyme-labeled instrument.
The results are shown in fig. 13, and fig. 14A shows that the application of bqx@pp@s NPs significantly promoted the growth of tobacco plants, and leaf chlorophyll content was also significantly increased (113-126%) (fig. 14B). In addition, the fresh plant weight of BQX@PP@S NPs increased 133% compared to the initial BQX treatment, the dry weight increased 125%, the plant height increased 113%, and the leaf width increased 108% (FIG. 14C, D). Taken together, these data indicate that bqx@pp@s NPs improved plant growth index and chlorophyll content.
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (6)
1. The preparation method of the nano system of the plant protein-based drug-encapsulated molecules is characterized by comprising the following steps: mixing the drug molecules with pyridine modified polyhydroxyethyl methacrylate in N, N-dimethylformamide, adding the mixture into a vegetable protein solution, uniformly stirring, dialyzing, centrifuging, and freeze-drying to obtain drug-loaded nano particles;
the medicine molecule is cyanogen nitrate, and the plant protein solution is soluble soybean whey protein solution;
the mass ratio of the pyridine modified polyhydroxyethyl methacrylate to the drug molecules is 2:1, 3:1, 4:1, 6:1 and 8:1, the mass ratio of the vegetable protein solution to the drug molecules is 20:1, and the volume ratio of the N, N-dimethylformamide to the vegetable protein solution is 3:100;
the concentration of the vegetable protein solution is 4-6 mg/mL.
2. The method of manufacturing according to claim 1, characterized in that: the pyridine modified poly (hydroxyethyl methacrylate) is prepared from poly-2-hydroxyethyl methacrylate and nicotinyl chloride hydrochloride, and the relative molecular mass of the poly-2-hydroxyethyl methacrylate is 20kDa.
3. The method of manufacturing according to claim 1, characterized in that: the dialysis condition is that the mixed solution after stirring is transferred into a dialysis bag, dialyzed for 24 hours in distilled water, and centrifuged for three times continuously, insoluble precipitate is removed, and the condition of centrifugation each time is 2000rpm,5 min/time.
4. A nano-system of a plant protein-based drug-entrapped molecule prepared by the preparation method according to any one of claims 1 to 3.
5. The application of the nano system of the plant protein-based drug-encapsulated molecules prepared by the preparation method of any one of claims 1-3 or the nano system of the plant protein-based drug-encapsulated molecules of claim 4 in preparing the drug for enhancing the disease resistance of the agricultural pest hosts.
6. The use according to claim 5, characterized in that: the agricultural plant diseases and insect pests are tobacco mosaic virus, cucumber mosaic virus, potato virus X, rice bacterial leaf blight, citrus canker, tobacco bacterial wilt, kiwi fruit canker, rice bacterial leaf spot, cucumber gray mold, pepper wilt, rape sclerotinia, wheat scab, potato late blight, blueberry root rot, grape vine cavity bacteria, dragon fruit anthracnose, rice sheath blight, budworms, caterpillars, aphids, scale insects, whiteflies, root knot nematodes, bursaphelenchus xylophilus, stem nematodes, cotton leaf mites and citrus red mites.
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