A medicine for preventing and treating plant diseases, a preparation method and application thereof

Abstract

The application discloses a medicine for preventing and treating plant diseases, a preparation method and application thereof, and belongs to the technical field of biological pesticides. The medicine for preventing and treating plant diseases comprises a hydrogel and nbe-miR159a; and the nucleic acid sequence of the nbe-miR159a is shown as SEQ ID NO:1. The isoGPBG pH-responsive hydrogel is used to coat the nbe-miR159a; not only the transmembrane transport obstacle of plant miRNA is broken through, efficient delivery and controllable release of the antiviral nucleic acid medicine are realized, but also the miRNA can be protected from degradation. The medicine has good inhibiting effect on potato Y virus and Pseudomonas solanacearum, and has important application prospects in the prevention and treatment of plant bacterial diseases and viral diseases.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological pesticides, and particularly relates to a medicine for preventing and treating plant diseases, a preparation method thereof and application. BACKGROUND

[0002] Plant virus disease is a major biological disaster threatening global food security, which spreads rapidly and is difficult to control, causing hundreds of billions of dollars in economic losses each year. Traditional chemical pesticides have problems such as environmental residues and drug resistance, and the RNAi-based biological control technology is difficult to popularize and apply due to poor nucleic acid stability and low delivery efficiency.

[0003] Developing new biological pesticides with good stability and high delivery efficiency can provide a new idea for the prevention and treatment of plant virus diseases, and has important significance for promoting sustainable agricultural development and ensuring global food security. SUMMARY

[0004] In view of the problems in the prior art, the purpose of the present application is to provide a medicine for preventing and treating plant diseases, a preparation method thereof and application.

[0005] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0006] A medicine for preventing and treating plant diseases, comprising a hydrogel and nbe-miR159a; the nucleic acid sequence of the nbe-miR159a is shown in SEQ ID NO: 1.

[0007] On the basis of the above scheme, the hydrogel is isoGPBG hydrogel, and the preparation method is as follows:

[0008] (1) Mix a 0.5 M potassium hydroxide solution and a phosphate buffer solution according to a volume ratio of 1:19 to obtain a mixed solution;

[0009] (2) Take 0.025 mmol of isoguanosine and 0.025 mmol of phenylboronic acid and add them to 1 mL of the mixed solution prepared in step (1); heat in a water bath at 100℃ until the mixture turns into a clear liquid;

[0010] (3) Add 0.025 mmol of guanosine to the mixture prepared in step (2) and continue to heat in a water bath at 100℃ until the solution is clear, and then gradually cool at room temperature to obtain the isoGPBG hydrogel.

[0011] On the basis of the above scheme, the 5' end of the nbe-miR159a is labeled with a fluorescent group, and the full chain is modified with methoxy.

[0012] On the basis of the above scheme, the 5' end of the nbe-miR159a is labeled with a Cy3 fluorescent group.

[0013] On the basis of the above scheme, the concentration of the nbe-miR159a is 10 µM.

[0014] The preparation method of the plant disease control drug is as follows:

[0015] (1) Mix a 0.5 M potassium hydroxide solution and a phosphate buffer solution according to a volume ratio of 1:19 to obtain a mixed solution;

[0016] (2) Add 0.025 mmol of isoguanosine and 0.025 mmol of phenylboronic acid to 1 mL of the mixed solution prepared in step (1); heat in a water bath at 100 DEG C until the mixture turns into a clear liquid;

[0017] (3) Add 0.025 mmol of guanosine to the mixture prepared in step (2) and continue to heat in a water bath at 100 DEG C until the solution is clear;

[0018] (4) Add nbe-miR159a to the product prepared in step (3) and mix thoroughly, and gradually cool at room temperature to obtain the product.

[0019] The application of the above plant disease control drug is for preventing and treating bacterial diseases of plants or for preventing and treating viral diseases of plants.

[0020] On the basis of the above scheme, the bacterial disease of the plant is a disease caused by Pseudomonas solanacearum, and the viral disease of the plant is a disease caused by Potato virus Y.

[0021] On the basis of the above scheme, the prevention method is to dilute the plant disease control drug and then perform root irrigation treatment on the plant.

[0022] On the basis of the above scheme, the final concentration of the diluted plant disease control drug is 25 µM.

[0023] Advantages of the technical scheme of the present application:

[0024] The present application discloses a plant disease control drug, which specifically uses a pH-responsive isoGPBG hydrogel to coat nbe-miR159a; not only breaks through the membrane transport barrier of plant miRNA, realizes efficient delivery and controllable release of antiviral nucleic acid drugs, but also protects miRNA from degradation. The drug has good inhibitory effect on Potato virus Y and Pseudomonas solanacearum, and has important application prospects in the prevention and treatment of bacterial diseases and viral diseases of plants. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 It is an isoGPBG hydrogel synthesis reaction process schematic diagram;

[0026] Figure 2 isoGPBG and isoGPBG nbe-miR159a Hydrogel pictures;

[0027] Figure 3 isoG, PBA, G, dried isoGPBG and isoGPBG nbe-miR159a Fourier transform infrared spectra (wherein, A is the Fourier transform infrared spectrum at the wavelength of 1200-4000, B is the Fourier transform infrared spectrum at the wavelength of 600-2000);

[0028] Figure 4 isoG, PBA, G, dried isoGPBG and isoGPBG nbe-miR159a Powder X-ray diffraction pattern;

[0029] Figure 5 isoGPBG nbe-miR159a Hydrogel rheology assay (wherein, A is isoGPBG nbe-miR159a Hydrogel viscosity test; B is isoGPBG nbe-miR159a Hydrogel frequency sweep test);

[0030] Figure 6 isoGPBG nbe-miR159a Hydrogel characterization graph (wherein, A is isoGPBG nbe-miR159a Scanning electron microscope results of lyophilized hydrogel, scale bar is 50 µm; B is isoGPBG nbe-miR159a Atomic force microscope results of lyophilized hydrogel in tapping mode, scale bar is 30 µm);

[0031] Figure 7 isoGPBG nbe-miR159a pH responsiveness of hydrogel (wherein, A is the remaining isoGPBG nbe-miR159a amount of hydrogel; B is the cumulative release curve of nbe-miR159a from isoGPBG nbe-miR159a hydrogel under different pH conditions);

[0032] Figure 8 isoGPBG nbe-miR159a Effect of concentration of isoGPBG on tobacco growth (scale bar = 5 cm);

[0033] Figure 9 Localization of nbe-miR159a in N. benthamiana tissues (scale bar for root-irrigated leaves is 100 µm, scale bar for root-irrigated roots and infiltrated leaves is 50 µm);

[0034] Figure 10qRT-PCR detection of the relative level of nbe-miR159a in N. benthamiana tissues;

[0035] Figure 11 isoGPBG nbe-miR159a Hydrogel slowly releases nbe-miR159a (A is the time-dependent release of nbe-miR159a from isoGPBG nbe-miR159a B is the isoGPBG nbe-miR159a effectively protects nbe-miR159a at room temperature);

[0036] Figure 12 isoGPBG nbe-miR159a Phenotype of isoGPBG

[0037] Figure 13 isoGPBG nbe-miR159a treated plants after qRT-PCR detection of the relative PVY RNA accumulation;

[0038] Figure 14 isoGPBG nbe-miR159a treated plants after WB detection of PVY coat protein expression;

[0039] Figure 15 isoGPBG nbe-miR159a Antibacterial rate of the hydrogel against P. solanacearum;

[0040] Figure 16 isoGPBG nbe-miR159a Antibacterial plate experiment of the hydrogel against P. solanacearum;

[0041] Figure 17 Disease incidence of K236 tobacco after inoculation with P. solanacearum;

[0042] Figure 18 Disease index statistics of K236 tobacco after inoculation with P. solanacearum;

[0043] Figure 19 isoGPBG nbe-miR159a Cell viability detection of BY2 cells treated with the hydrogel for 72 h (scale = 100 µm);

[0044] Figure 20 isoGPBG nbe-miR159a Seed germination after treatment with the hydrogel;

[0045] Figure 21 isoGPBG nbe-miR159a Seed germination rate after treatment with the hydrogel.

[0046] In the above figures, "*" indicates p < 0.05; "**" indicates p < 0.01; "***" indicates p < 0.001; "****" indicates p < 0.0001; and "ns" indicates no significant difference. DETAILED DESCRIPTION

[0047] The terms used in the present application have the meanings generally understood by those of ordinary skill in the art, unless otherwise specified. The present application is described in further detail below in conjunction with specific examples and with reference to the data. The following examples are merely intended to illustrate the present application and in no way limit the scope of the present application.

[0048] The experimental methods in the following examples are all conventional methods, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The experimental materials, reagents, and drugs used in the following examples can be purchased through general channels, unless otherwise specified.

[0049] The materials such as N. benthamiana and plant virus sources were preserved and cultured by the Plant Protection Research Group of Tobacco. N. benthamiana was cultured in a greenhouse under the conditions of humidity 55% ± 5%, temperature 25°C ± 1°C, and light cycle 16 h\8 h.

[0050] Example 1

[0051] A medicine for preventing and treating plant diseases, comprising a hydrogel and nbe-miR159a;

[0052] The hydrogel is an isoGPBG hydrogel, and the preparation method is as follows:

[0053] (1) A 0.5 M potassium hydroxide solution and a phosphate buffer solution (PBS, pH = 7.4) were mixed according to a volume ratio of 1:19 to obtain a mixed solution;

[0054] (2) 0.025 mmol of isoguanosine (isoG) and 0.025 mmol of phenylboronic acid (PBA) were added to 1 mL of the mixed solution prepared in step (1); and the mixture was heated in a water bath at 100°C until the mixture turned into a clear liquid;

[0055] (3) To the mixture prepared in step (2), 0.025 mmol of guanosine (G) was added, and heating at 100°C in a water bath was continued until the solution was clear. The pH-responsive isoGPBG hydrogel (hereinafter referred to as isoGPBG) was obtained after gradually cooling at room temperature. The concentration of the hydrogel was 25 mM (the concentration represents the initial molar concentration of the key components isoG, PBA or G. When the hydrogel needs to be diluted in the following method, it is based on this concentration).

[0056] The reaction principle (Fig. 1) is as follows: Figure 1 Potassium hydroxide provides an alkaline environment, which promotes the deprotonation of the hydroxyl group (-OH) of isoG and G to form a more nucleophilic oxygen anion (-O⁻). Under alkaline conditions, the boronic acid group (-B(OH)2) of PBA reversibly condenses with the cis-diol structure (2', 3'-OH of ribose) of isoG or G to form a benzene boronic acid ester bond. This bond is stable under alkaline conditions, but will hydrolyze under neutral or acidic conditions (pH 7.4 or below), giving the gel pH responsiveness. Guanosine molecules form G-quartets through Hoogsteen hydrogen bonds under alkaline conditions, and further stack into G-nanowire structures as physical cross-linking points of the hydrogel. IsoG can be incorporated into G-quartets due to structural similarity, enhancing the stability of the gel and adjusting the mechanical properties.

[0057] The nucleic acid sequence of the nbe-miR159a is shown in SEQ ID NO: 1, the 5' end is labeled with a Cy3 fluorescent group, and the whole chain is modified by methoxy.

[0058] nbe-miR159a: 5'-AAGCUGCCGACCUAUGGAUUCC-3' (SEQ ID NO: 1).

[0059] Example 2

[0060] A method for preparing a medicine for preventing and treating plant diseases, comprising the following steps:

[0061] (1) Mix 0.5 M potassium hydroxide solution and phosphate buffered saline (PBS, pH=7.4) according to a volume ratio of 1:19 to obtain a mixed solution;

[0062] (2) Take 0.025 mmol of isoG and 0.025 mmol of PBA and add them to 1 mL of the mixed solution prepared in step (1); heat in a water bath at 100°C until the mixture turns into a clear liquid;

[0063] (3) To the mixture prepared in step (2), 0.025 mmol of guanosine (Guanosine, G) was added, and the water bath was continued to heat at 100°C until the solution was clear;

[0064] (4) To the product prepared in step (3), nbe-miR159a was added and mixed thoroughly, and the final concentration of nbe-miR159a was 10 µM; gradually cooled at room temperature, and isoGPBG was obtained nbe-miR159a Hydrogel, the concentration of which was 25 mM (the concentration represents the initial molar concentration of the key components isoG, PBA or G, and when the hydrogel needs to be diluted in the following method, it is based on this concentration).

[0065] The nucleic acid sequence of the nbe-miR159a is shown in SEQ ID NO: 1, which is labeled with a Cy3 fluorescent group at the 5' end, and the whole chain is modified by methoxy.

[0066] nbe-miR159a: 5'-AAGCUGCCGACCUAUGGAUUCC-3' (SEQ ID NO: 1).

[0067] The isoGPBG prepared by the above method and isoGPBG nbe-miR159a The appearance of the hydrogel is shown in Figure 2 Since the nbe-miR159a is labeled with a Cy3 fluorescent group, its solution presents a characteristic pink color, and after adding the nbe-miR159a, the hydrogel also presents the same pink characteristic, which preliminarily confirms the successful loading of the nbe-miR159a.

[0068] The isoG, PBA, G, and dried isoGPBG and isoGPBG nbe-miR159a were analyzed by Fourier transform infrared spectroscopy. Specifically, by potassium bromide tabletting method, using Fourier transform infrared spectrometer, the isoG, PBA, G, isoGPBG, isoGPBG -1 were recorded in the range of 4000-600 cm nbe-miR159a Fourier transform infrared (FT-IR). The results are shown in Figure 3 FT-IR analysis showed that the phenylboronic acid ester bond played a key role in the formation of isoGPBG nbe-miR159a hydrogel, and realized the pH responsiveness of the hydrogel through dynamic covalent crosslinking. Specifically, the FT-IR spectrum of PBA (phenyldiboronic acid) at 3290 cm -1 and 1340 cm -1The characteristic peaks of stretching and bending vibration of B-OH were observed at 1095 cm -1 and 1250 cm-1, respectively. It is worth noting that a new absorption peak appeared at 1095 cm nbe-miR159a for both dried isoGPBG hydrogel and isoGPBGnbe-miR159a hydrogel, which corresponds to the stretching vibration frequency of B-O-C bond. Meanwhile, the addition of nbe-miR159a did not cause significant shift or disappearance of the characteristic peaks in FT-IR spectrum of isoGPBG hydrogel, indicating that the encapsulation of miRNA did not affect the basic chemical structure of the hydrogel.

[0069] X-ray powder diffraction (PXRD) was used to investigate the structural characteristics of isoGPBG and isoGPBG nbe-miR159a hydrogels in lyophilized state. The results are shown in Fig. 6. PXRD test further confirmed the structural characteristics of isoGPBG and isoGPBG nbe hydrogels. A strong diffraction peak was observed at 2Q ~ 27.4° (d = 3.31 A), which is consistent with the distance of π-π stacking between two G / isoG-quartet. Importantly, the incorporation of nbe-miR159a did not cause significant changes in the crystal structure of isoGPBG hydrogel, with no obvious changes in the position and intensity of the diffraction peak. Figure 4

[0070] Rheometer was used to investigate the rheological behavior of the hydrogels. To determine the viscoelastic properties of the hydrogels, the storage modulus (G') and loss modulus (G'') were measured. Oscillatory measurements were performed using a parallel plate with a diameter of 25 mm. isoGPBG -miR159a hydrogel was heated to clarity and then quickly transferred to a preheated parallel plate at 80°C. This can prevent the sample from re-solidifying into a gel during the transfer process and before the experiment begins. After the transfer was completed, silicone oil was carefully added along the outer edge of the parallel plate to seal it and prevent the sample from volatilizing. When the temperature dropped to 37°C, the actual test began. Frequency sweep test was performed with a strain of 0.1% and a frequency range of 0.1 to 100 Hz. At the same time, strain sweep test was performed at a fixed frequency of 1 Hz, with a strain range of 0.01% to 100%. In addition, viscosity test was performed at a shear rate range of 0.1 to 100 s -1 Figure 5 The results are shown in Fig. 7. Rheological test results showed that the viscosity of the hydrogel decreased with the increase of shear rate from 0.1 s to 10 s, with good shear thinning behavior (G' < G''). Figure 5 ​In A), within the strain range of 0.01% to 4.50%, the storage modulus (G′) is higher than the loss modulus (G′′), confirming that the hydrogel has good viscoelasticity and structural stability ( Figure 5 In B), it can effectively prevent the premature release of nbe-miR159a.

[0071] With the help of scanning electron microscopy (SEM) and tapping-mode atomic force microscope (TM-AFM), observe the internal morphology and structure of the freeze-dried isoGPBG nbe-miR159a hydrogel. The results are as Figure 6 shown. The SEM image shows a unique sheet-like structure ( Figure 6 In A), while the TM-AFM image reveals its honeycomb structure ( Figure 6 In B). This complex structural arrangement may endow the hydrogel with unique physical and chemical properties, opening up new avenues for its application in RNA delivery.

[0072] isoGPBG nbe-miR159a pH responsiveness test of isoGPBG hydrogel:

[0073] The isoGPBG hydrogel loaded with nbe-miR159a was immersed in PBS solutions with different pH values respectively. After 5 days, observe the remaining amount of isoGPBG nbe-miR159a hydrogel, and determine the cumulative release curve of nbe-miR159a from the isoGPBG nbe-miR159a hydrogel under different pH conditions. The results are as Figure 7 shown. Under acidic conditions, the size of the remaining isoGPBG nbe-miR159a hydrogel is significantly smaller than that under alkaline conditions, specifically showing pH = 5.5 < pH = 6.8 < pH = 7.4 < pH = 8.4, indicating that the degradation rate of the hydrogel increases with the increase in acidity ( Figure 7 In A). Cy3 has specific optical properties, with an excitation wavelength of 530 nm and an emission wavelength of 570 nm. When measuring the release curve of the isoGPBG nbe-miR159a hydrogel, utilize the fluorescence properties of Cy3 to indirectly and quantitatively analyze the release amount of nbe-miR159a by detecting the Cy3 fluorescence intensity at different time points. Figure 7 In B shows the release of nbe-miR159a from isoGPBG nbe-miR159aRelease curve in hydrogel. The experimental results show that the release amount of nbe-miR159a under acidic conditions (pH=5.5 and 6.8) is significantly higher than that under neutral (pH=7.4) and alkaline (pH=8.4) conditions (p<0.01). This is because the phenyldiboronic acid ester bond has reversibility and is relatively stable under neutral / alkaline conditions, so that the hydrogel remains solid. Under acidic conditions, the phenyldiboronic acid ester bond will break due to protonation, causing the disintegration of the cross-linked network, so that the hydrogel changes from a solid gel to a liquid solution. This result further confirms that isoGPBG nbe-miR159a The hydrogel can release nbe-miR159a under acidic conditions.

[0074] Example 3

[0075] isoGPBG nbe-miR159a Effect of concentration on the growth and development of N. benthamiana

[0076] Different volumes of isoGPBG nbe-miR159a The hydrogel (concentration of 25 mM) was added to the Hoagland nutrient solution at pH 6.8, so that the isoGPBG nbe-miR159a The final concentration of the hydrogel in the Hoagland nutrient solution was 2 µM~1125 µM; the roots of N. benthamiana were immersed in the isoGPBG nbe-miR159a The hydrogel was cultured in the Hoagland nutrient solution; the growth status of N. benthamiana was observed, and the results are shown in Figure 8 The isoGPBG nbe-miR159a The hydrogel had the smallest effect on the phenotype of the plant and the largest drug loading capacity when the final concentration of the hydrogel in the Hoagland nutrient solution was 25 µM.

[0077] Effect of administration method on the delivery effect of nbe-miR159a

[0078] isoGPBG nbe-miR159a The hydrogel was used to treat N. benthamiana plants by methods such as infiltration (leaves) and root irrigation, and the transportation of nbe-miR159a in the plant body was observed by laser confocal microscopy after 24 h, wherein the infiltration (leaves) was that the isoGPBG nbe-miR159a The hydrogel was mixed with PBS in equal volume and injected into the leaves of N. benthamiana; the root irrigation was that the isoGPBG nbe-miR159a The hydrogel was mixed with PBS in equal volume and injected into the leaves of N. benthamiana; the root irrigation was that the isoGPBG nbe-miR159a The amount of the hydrogel was the same. The results are shown in Figure 9 The nbe-miR159a-Cy3 was observed in the leaves of the infiltration group, the roots and leaves of the root irrigation group, indicating that the isoGPBG nbe-miR159aThe nbe-miR159a in the hydrogel can be released and transported into the plant body. At the same time, the root system of the root irrigation group absorbs the isoGPBG nbe-miR159a The nbe-miR159a released in the hydrogel and delivered to the leaves by the roots indicates that the root irrigation method can effectively enable the long-distance transportation of nbe-miR159a in the plant body. Thus, the root irrigation method is the most convenient and effective method.

[0079] The isoGPBG nbe-miR159a The hydrogel and the isoGPBG hydrogel are used to treat N. benthamiana by root soaking, specifically, isoGPBG nbe-miR159a The hydrogel and the isoGPBG hydrogel are added to the Hoagland nutrient solution (the final concentration of the hydrogel is 25 µM), and after 24 h of root soaking, qRT-PCR is used to detect the relative level of nbe-miR159a in the leaves and roots of N. benthamiana, and the Hoagland nutrient solution without the addition of the hydrogel is used as a control (NC). The results are shown in Figure 10 , and the relative level of nbe-miR159a in the leaves and roots of the hydrogel treatment group is significantly higher than that in the isoGPBG hydrogel treatment group and the control group. nbe -miR159a The hydrogel can effectively deliver nbe-miR159a.

[0080] The nbe-miR159a labeled with a Cy3 fluorescent group is added to an aqueous solution to prepare an nbe-miR159a solution with a concentration of 10 µM; the prepared nbe-miR159a solution is added to a 96-well plate and evenly divided into two groups, and isoGPBG nbe-miR159a hydrogel is added to the nbe-miR159a solution in one group, mixed, and cooled to obtain isoGPBG nbe-miR159a hydrogel; the other group is not added with the hydrogel. The above nbe-miR159a solution and isoGPBG nbe-miR159a hydrogel solution are placed at room temperature for 9 d, and the release of nbe-miR159a in the solution over time is determined by detecting the Cy3 fluorescence intensity at different time points based on the fluorescence characteristics of Cy3, and the results are shown in Figure 11 , and the release amount of nbe-miR159a in the isoGPBG nbe-miR159a hydrogel solution gradually increases over time, and after 48 h, the fluorescence intensity of the isoGPBG nbe-miR159a hydrogel solution is significantly higher than that of the nbe-miR159a solution. Agarose gel electrophoresis is used to detect nbe-miR159a in the nbe-miR159a solution and isoGPBG nbe-miR159a hydrogel solution, and the results are shown in Figure 11As shown in Figure B, after 9 days at room temperature, almost no band was detected in the nbe-miR159a solution, while isoGPBG... nbe-miR159a Clear bands were still present in the hydrogel solution, indicating that the hydrogel can effectively protect nbe-miR159a from degradation to a certain extent at room temperature.

[0081] Example 4

[0082] isoGPBG nbe-miR159a Application of hydrogels in the prevention and control of potato virus Y (PVY)

[0083] isoGPBG nbe-miR159a The hydrogel slowly releases nbe-miR159a and isoGPBG when placed in Hogrange nutrient solution (pH=6.8). nbe-miR159a The final concentration of the hydrogel in Hoagland nutrient solution was 25 µM. The roots of *Nicotiana benthamiana* were immersed in the solution containing isoGPBG. nbe-miR159a In the Hoagland hydrogel, the released nbe-miR159a fully penetrates into the plant through the root system and acts on plant cells. After 24 h of treatment, *Tobacco Benzovia* leaves were inoculated with PVY. The inoculation method was as follows: PVY-infected tobacco leaves (systemic infection stage) were homogenized in pre-cooled 0.03 M potassium phosphate buffer (pH 7.2) or PBS buffer containing 1% (w / v) sodium sulfite (1:30, w / v), centrifuged at 12,000×g for 10 min at 4°C to remove tissue fragments. The supernatant was the crude virus extract. The OD of the crude virus extract was measured using a UV spectrophotometer. 260 Adjust the final concentration of the inoculum to 0.8-1.2 OD. 260 / mL. The dilution ratio is usually 1:30 (weight of infected leaf / volume of buffer solution) to ensure a unit area (cm²) 2 The inoculation volume was approximately 10 μL. PVY virus solution was inoculated onto *Tobacco Bengal* leaves using a mechanical friction inoculation method, with each experiment repeated three times; disease development was observed. isoGPBG hydrogel prepared according to the method in Example 1 and Hoagland nutrient solution (NC) without any added hydrogel were used as controls. Seven days after PVY inoculation, the phenotypes of each treatment group were observed as follows: Figure 12 As shown, compared with the control group, isoGPBG nbe-miR159a This study increased the resistance of Nicotiana benthamiana to PVY. RNA was extracted from the upper leaves of each treatment group, and the relative PVY RNA accumulation in the leaves of each treatment group was detected by qRT-PCR. The results were compared with those of the uncoated nbe-miR159a treatment group. Figure 13 As shown, isoGPBG nbe-miR159aThe relative expression of PVY in the hydrogel treatment group was significantly lower than that in the control group. The nbe-miR159a treatment group alone was not effective, and nbe-miR159a was easily degraded. WB analysis was further used to analyze the expression of PVY viral coat protein in each treatment group, and β-Actin was used as the internal reference protein. The results are shown in Figure 14 The PVY / β-Actin ratio was used to normalize the data, and the results are shown in Table 1. The isoGPBG nbe-miR159a After hydrogel and isoGPBG gel treatment, the accumulation of PVY viral coat protein was inhibited and was significantly lower than that in the control group. Therefore, the isoGPBG nbe-miR159a Hydrogel root irrigation treatment can effectively improve the resistance of N. benthamiana to PVY virus.

[0084] Table 1. PVY / β-Actin ratio data normalization

[0085]

[0086] Example 5

[0087] isoGPBG nbe-miR159a Inhibition of P. solanacearum by hydrogel

[0088] The minimum inhibitory concentration test was carried out by standard LB dilution method: isoGPBG nbe-miR159a The final concentration of hydrogel in LB liquid medium was 10 mM, 5 mM, 2.5 mM, 1.25 mM and 0 mM, respectively. Then, 100 μL of 5.4×10 4 CFU / mL P. solanacearum suspension was accurately added to each 100 μL of hydrogel sample with different concentrations. The mixture was incubated in a 37°C incubator at 300 rpm for 6 h, and then the inhibition rate was detected by a microplate reader (OD=600). The results are shown in Figure 15 The isoGPBG nbe-miR159a Hydrogel can effectively inhibit the activity of P. solanacearum, and the inhibition rate can reach 80%. 100 μL of the mixture of the above P. solanacearum and hydrogel after co-culture was evenly spread on NA medium for overnight culture, and finally the colonies were counted and imaged. The results are shown in Figure 16 With the increase of isoGPBG nbe-miR159a hydrogel concentration, the inhibition effect was significantly improved, and when the concentration was 10 mM, the inhibition rate could reach more than 80%, which was consistent with the results of the microplate reader.

[0089] Example 6

[0090] isoGPBGnbe-miR159a Application of hydrogels in the control of Ralstonia solanacearum

[0091] Select healthy K326 tobacco seedlings at the 4-6 leaf stage, carefully pull them out, gently shake to remove soil from the roots, and use sterile scissors to trim a small number of fibrous roots 1-2 cm from the root tip (creating micro-wounds to promote infection). Place the seedlings back into planting pots. Use isoGPBG prepared according to the method in Example 2. nbe-miR159a The hydrogel was prepared into a 10 mM solution using Hoagland's nutrient solution and slowly poured around the roots of K326 tobacco seedlings. After 24 hours, the seedlings were then irrigated with the prepared Ralstonia solanacearum inoculum solution (1×10⁻⁶). 8 CFU / mL), with a dosage of 10-20 mL per plant. The normal treatment group (NC) was not irrigated with isoGPBG. nbe-miR159a Hydrogel solution and Ralstonia solanacearum inoculum; control group (CK) without isoGPBG irrigation nbe-miR159a Hydrogel solution was used, but Ralstonia solanacearum inoculum was applied as irrigation. After inoculation, the soil was kept moist (relative humidity 70%-80%), and cultured at a constant temperature of 28-30℃. Disease incidence was observed regularly. Results were as follows... Figure 17 As shown, without isoGPBG nbe-miR159a The normal treatment group (NC) treated with Ralstonia solanacearum grew well in Hogrange nutrient solution, and after isoGPBG... nbe-miR159a The experimental group treated with Ralstonia solanacearum showed better growth than the control group (CK). Disease index analysis showed that the disease index in the treatment group was significantly lower than that in the control group (CK). Figure 18 ), indicating isoGPBG nbe-miR159a Hydrogels not only have significant effects in antiviral applications, but also show great potential in the field of antibacterial applications, providing new ideas and methods for the integrated prevention and control of plant diseases.

[0092] Example 7

[0093] isoGPBG nbe-miR159a Toxicity testing

[0094] At the cellular level, tobacco BY2 cells (Nicotiana tabacum L.cv Bright Yellow2, BY2) were used as the test subject. BY2 cells were placed in a solution containing isoGPBG. nbe-miR159a The hydrogel was cultured in an environment free of isoGPBG (final hydrogel concentration 25 μM). nbe-miR159a BY2 cells cultured in hydrogel medium served as a control. After a period of culture, cell viability and structure were measured. Figure 19 As shown, compared with the control group, isoGPBG nbe-miR159aThe cell morphology of the hydrogel culture group was not obviously destroyed, and the cell viability was maintained at about 90%. This result clearly shows that the hydrogel does not interfere with the normal physiological functions of BY2 cells and has no significant negative impact on their activity.

[0095] At the level of plant seeds, seed germination experiments were carried out using N. benthamiana seeds as a representative. The seeds were soaked in isoGPBG nbe-miR159a In the aqueous solution of the hydrogel (the final concentration of the hydrogel was 25 μM), the BY2 cells were cultured without isoGPBG nbe-miR159a The aqueous solution of the hydrogel was used as a control, and the seeds were cultured under strictly controlled suitable environmental conditions, and the germination dynamics and germination rate of the seeds were closely observed. The results are shown in Table 2. Figure 20 and Figure 21 As shown in Table 2, the isoGPBG nbe-miR159a The germination rate and germination speed of the seeds in the hydrogel treatment group were basically consistent with those of the control group, and no abnormal growth morphology was observed during the germination process. This fully shows that the hydrogel does not hinder seed germination and can ensure the normal opening of the growth and development process of the seeds. Combining the experimental results at the cell and seed levels, it is strongly proved that the isoGPBG nbe-miR159a hydrogel has good biocompatibility with target crops.

[0096] The above description is only a preferred embodiment of the present application, and is not intended to limit the other forms of the present application. Any person skilled in the art can use the disclosed technical content to make changes or modifications to equivalent embodiments. However, any simple modification, equivalent change and modification made according to the technical essence of the present application to the above embodiments, without departing from the technical solution of the present application, still falls within the protection scope of the present application.

Claims

1. A drug for preventing and treating plant diseases, characterized in that, The plant disease control medicine comprises a hydrogel and nbe-miR159a; the nucleic acid sequence of the nbe-miR159a is shown as SEQ ID NO:1; The hydrogel is an isoGPBG hydrogel, and the preparation method is as follows: (1) 0.5 M potassium hydroxide solution and phosphate buffer solution are mixed according to a volume ratio of 1:19 to obtain a mixed solution; (2) 0.025 mmol of isoguanosine and 0.025 mmol of phenylboronic acid are added to 1 mL of the mixed solution prepared in step (1); heating is performed in a water bath at 100 DEG C until the mixture is converted into a clear liquid; (3) 0.025 mmol of guanosine is added to the mixture prepared in step (2), and heating is continued in a water bath at 100 DEG C until the solution is clear; cooling is performed at room temperature, and the isoGPBG hydrogel is obtained; When the plant disease control medicine is used for controlling plant diseases, it is diluted to a final concentration of 25 µM.

2. The agent for controlling plant diseases according to claim 1, wherein The 5' end of the nbe-miR159a is labeled with a fluorescent group, and the whole chain is modified by methoxy.

3. The agent for controlling plant diseases according to claim 2, wherein The 5' end of the nbe-miR159a is labeled with a Cy3 fluorescent group.

4. The agent for controlling plant diseases according to claim 1, wherein The concentration of the nbe-miR159a is 10 µM.

5. A process for the preparation of a pharmaceutical for controlling plant diseases as set forth in any one of claims 1 to 4, characterized in that, The steps are as follows: (1) 0.5 M potassium hydroxide solution and phosphate buffer solution are mixed according to a volume ratio of 1:19 to obtain a mixed solution; (2) 0.025 mmol of isoguanosine and 0.025 mmol of phenylboronic acid are added to 1 mL of the mixed solution prepared in step (1); heating is performed in a water bath at 100 DEG C until the mixture is converted into a clear liquid; (3) 0.025 mmol of guanosine is added to the mixture prepared in step (2), and heating is continued in a water bath at 100 DEG C until the solution is clear; (4) nbe-miR159a is added to the product prepared in step (3) and mixed thoroughly, and cooling is performed gradually at room temperature, and the isoGPBG hydrogel is obtained.

6. The use of a plant disease control agent as claimed in any one of claims 1 to 4, characterized in that The plant disease control medicine is used for controlling plant bacterial diseases or for controlling plant viral diseases; The plant bacterial disease is a disease caused by Ralstonia solanacearum, and the plant viral disease is a disease caused by Potato virus Y; The Ralstonia solanacearum is Ralstonia solanacearum that causes tobacco diseases.

7. The use of a drug for controlling plant diseases according to claim 6, characterized in that, The control method is to dilute the plant disease control medicine and then perform root irrigation treatment on plants.

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