MQDs-PEI-coated miR43-CMCS / TA composite nanomaterial for preventing and treating plant viruses as well as preparation method and application of MQDs-PEI-coated miR43-CMCS / TA composite nanomaterial

Abstract

The invention discloses an MQDs-PEI-coated miR43-CMCS / TA composite nanomaterial for preventing and treating plant viruses as well as a preparation method and application of the MQDs-PEI-coated miR43-CMCS / TA composite nanomaterial, and belongs to the technical field of genetic engineering. The composite nano material is prepared from Mxene-QDs, polyetherimide, carboxymethyl chitosan, tannic acid and miR43 nano microspheres, the nano composite material is small in particle size and high in drug loading capacity, has an excellent protection effect and pH release capacity on miR43, can deliver miR43 into a plant body in a spraying mode and the like, and can be used for preparing the miR43 in the plant body by specifically shearing NbIAA29 in the plant. Therefore, replication and infection of the PVY virus and the TMV virus are inhibited, and important significance is achieved on prevention and treatment of the PVY virus and the TMV virus.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and in particular relates to a MQDs-PEI@miR43-CMCS / TA composite nanomaterial for preventing and controlling plant viruses, and a preparation method and application thereof. Background Art

[0002] After plants are infected with viruses, conventional chemical control agents are not effective. With the deepening of people's understanding of viruses and the extensive research on the mechanism of virus infection in hosts, many new methods for the prevention and control of viral diseases have emerged, mainly including physical and chemical control, transgenic breeding, RNAi-mediated and CRISPR-Cas and other RNA editing systems, and other new technologies for the prevention and control of viral diseases.

[0003] Transgenic breeding has a long cultivation cycle and the virus evolves quickly, so the prevention and control effect is not ideal. RNA editing based on the CRISPR-Cas system has a wide range of off-target phenomena. Among the RNAi mechanisms, dsRNA, siRNA, and miRNA can all regulate viral infection. As an innate antiviral mechanism, RNAi has a broader research prospect.

[0004] Direct delivery of nucleotide analogs (NA) in RNAi-mediated processes is difficult because they are susceptible to nuclease degradation and have poor physicochemical properties that limit the ease with which they can be transferred into cells. Therefore, safe and effective NA therapies require advanced delivery platform technologies.

[0005] Nanoparticles (NPs) have a huge chemical diversity and can exist in many forms: organic or biological products, carbon materials, polymers, semiconductors and inorganic materials, including metals and metal oxides. NPs have been studied for various biomedical applications such as delivery systems, biosensing, imaging and antimicrobial applications. Nanomaterials have desirable properties that allow them to be tuned into functional biological entities that can be targeted for delivery. Currently, there are many nanomaterials applied to the delivery of nucleic acid drugs. For example, multiplexers, dendrimers, hydrogels and artificial polymersomes and natural polymers such as cyclodextrin, chitosan, hyaluronic acid, dextran and gelatin can be used as delivery vehicles.

[0006] It can be seen that NA drug delivery has great potential and application prospects in the prevention and control of plant viruses. It works by activating or inhibiting important signaling pathways, or directly acting on the viral genome. After being effectively loaded by nanomaterials, nucleic acids can be successfully delivered to the cell nucleus to regulate immune responses, ultimately achieving effective prevention and control of plant viruses. Summary of the invention

[0007] The present invention provides a method for preparing a MQDs-PEI@miR43-CMCS / TA composite nanomaterial for preventing and controlling plant viruses, comprising the following steps:

[0008] (1) Synthesis of MQDs-PEI nanomaterials

[0009] The MXene-QDs solution and 5 mL of PEI solution were mixed and reacted; after the reaction was completed, the solution was centrifuged, and the product was washed and dried to obtain MQDs-PEI nanomaterials;

[0010] (2) Synthesis of MQDs-PEI@miR43 nanocomposites

[0011] The MQDs-PEI solution and the miR43 nanosphere solution were mixed and allowed to react. After the reaction was completed, the mixture was centrifuged, the supernatant was discarded, and the precipitate was washed to obtain the MQDs-PEI@miR43 nanocomposite material.

[0012] (3) Synthesis of MQDs-PEI@miR43-CMCS / TA nanocomposites

[0013] The MQDs-PEI@miR43 nanocomposite solution and the CMCS-TA mixed solution were mixed and reacted; after the reaction was completed, the solution was centrifuged, the supernatant was discarded, and the precipitate was washed to obtain the MQDs-PEI@miR43-CMCS / TA nanocomposite.

[0014] In the above preparation method, in the step (1), the volume ratio of the Mxene-QDs solution to the PEI solution is selected from 4 to 1:1 to 4, preferably 1:1.

[0015] In the above preparation method, in step (1), the concentration of the Mxene-QDs solution is selected from 1 to 10 mg / mL, preferably 2 mg / mL; the concentration of the PEI solution is selected from 5 to 20 mg / mL, preferably 10 mg / mL.

[0016] In the above preparation method, in step (1), the reaction conditions are selected from: vigorous stirring at 50-60°C for 12-24h; preferably: vigorous stirring at 60°C for 24h.

[0017] In the above preparation method, in step (2), the volume ratio of the MQDs-PEI solution to the miR43 nanosphere solution is selected from 1 to 8:1 to 10, preferably 1:1.

[0018] In the above preparation method, in step (2), the concentration of the MQDs-PEI solution is selected from 0.5 to 2 mg / mL; the concentration of the miR43 nanosphere solution is selected from 0.5 to 2 mg / mL.

[0019] In the above preparation method, in step (2), the static reaction conditions are selected from: static reaction at 25-35° C. for 1-4 hours; preferably: static reaction at room temperature for 2 hours.

[0020] In the above preparation method, in step (3), the volume ratio of the MQDs-PEI@miR43 nanocomposite solution and the CMCS-TA mixed solution is selected from 4 to 1:1 to 4, preferably 2:1.

[0021] In the above preparation method, in step (3), the concentration of the MQDs-PEI@miR43 nanocomposite solution is selected from 0.1 to 2 mg / mL, preferably 0.5 mg / mL; the concentration of the CMCS-TA mixed solution is selected from 0.5 to 2 mg / mL, preferably 1 mg / mL.

[0022] In the above preparation method, in step (3), the reaction conditions are selected from: stirring at 25-35°C for 4-12 hours; preferably: stirring at room temperature for 12 hours.

[0023] In the present invention, in the CMCS-TA mixed solution, the concentrations of carboxymethyl chitosan and tannic acid are the same.

[0024] In the above preparation method, the miR43 nanospheres can be prepared by conventional rolling circle amplification method, and the DNA nucleic acid sequence of the miR43 is as follows: 5'-CTGCCACCCCACTCAGATCTG-3'.

[0025] The present invention provides a MQDs-PEI@miR43-CMCS / TA composite nanomaterial prepared by the above method.

[0026] The present invention provides application of the above-mentioned MQDs-PEI@miR43-CMCS / TA composite nanomaterial in preventing and controlling plant viruses.

[0027] The present invention provides application of the above-mentioned MQDs-PEI@miR43-CMCS / TA composite nanomaterial in preparing medicines for preventing and treating plant viruses.

[0028] In the present invention, the plant virus is selected from one or both of PVY virus and TMV virus.

[0029] The beneficial effects of the present invention are:

[0030] The MQDs-PEI@miR43-CMCS / TA nanocomposite material prepared by the present invention has a small particle size, a high drug loading capacity, an excellent protective effect and pH release ability on miR43, can deliver miR43 into plants by spraying or the like, and specifically cleaves NbIAA29 in plants, thereby inhibiting the replication and infection of PVY virus and TMV virus, which is of great significance for the prevention and treatment of PVY and TMV viruses. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 SEM images of three materials: Mxene-QDs, MQDs-PEI@miR43, and MQDs-PEI@miR43-CMCS / TA;

[0032] Figure 2 Dynamic light scattering graphs; Figure A is miR43, Figure B is MQDs-PEI@miR43, and Figure C is MQDs-PEI@miR43-CMCS / TA;

[0033] Figure 3 Infrared absorption spectra; from top to bottom, they are Mxene-QDs, MQDs-PEI, MQDs-PEI@miR43 and MQDs-PEI@miR43-CMCS / TA;

[0034] Figure 4 The zate potentials of six materials: Mxene-QDs, miR43, CMCS-TA, MQDs-PEI, MQDs-PEI@miR43, and MQDs-PEI@miR43-CMCS / TA;

[0035] Figure 5 is the drug loading rate of MQDs-PEI@miR43 nanomaterials at different ratios;

[0036] Figure 6 The degradation of miR43 under different RNaseA concentrations;

[0037] Figure 7 is the contact angle of each material;

[0038] Figure 8 The miR43 release ability of MQDs-PEI@miR43-CMCS / TA composite nanomaterials at different pH;

[0039] Fig. 9 The qRT-PCR test results of PVY virus mRNA expression;

[0040] Fig.10This is the Western-blot test result of the expression of PVY virus PVY-CP protein;

[0041] Fig.11 Phenotypic observation of the control effect of each material on PVY virus;

[0042] Fig.12 The qRT-PCR test results of TMV virus mRNA expression;

[0043] Fig.13 The Western-blot test results of TMV virus TMV-CP protein expression;

[0044] Fig.14 This is the phenotypic observation of the control effect of each material on TMV virus;

[0045] Fig.15 Schematic diagram of the formation of miR43 nanospheres by rolling circle amplification;

[0046] Fig.16 This is a graph showing the change in the particle size of miR43 nanospheres with transcription time;

[0047] Fig.17 SEM images of miR43 nanospheres at different transcription times;

[0048] Fig.18 This is the zate potential of miR43 nanospheres formed after 4 hours of transcription. DETAILED DESCRIPTION

[0049] The materials used in the present invention are as follows:

[0050] Test plants: N. benthamiana were cultivated in a greenhouse with a photoperiod of 16h / 8h (light / dark) and a temperature of 25°C. Healthy N. benthamiana plants that had grown for 4 weeks were treated and simulated inoculated, and samples were taken seven days after inoculation. Three tobacco plants with the same growth conditions were treated and sampled for each treatment.

[0051] Test nanomaterials: Mxene-QDs (Mxene quantum dots, Beike Nano BKC3000166-1) were purchased from Beijing Beike New Materials Technology Co., Ltd.; carboxymethyl chitosan (CMCS) and tannic acid (TA) were purchased from Beijing Solebow Technology Co., Ltd.

[0052] In the following examples of the present invention, all solutions were prepared using ddH2O. The concentration of the Mxene-QDs solution was 2 mg / mL; the concentration of the carboxymethyl chitosan-tannic acid (CMCS-TA) mixed solution was 1 mg / mL, wherein the concentrations of carboxymethyl chitosan and tannic acid were the same; and the concentration of the polyetherimide (PEI) solution was 10 mg / mL.

[0053] In the present invention, the DNA nucleic acid sequence of miR43 is as follows:

[0054] 5'-CTGCCACCCCACTCAGATCTG-3'

[0055] Other materials used in the present invention, if not otherwise stated, can be obtained through commercial channels. Other terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art. The present invention is further described in detail below in conjunction with specific examples and with reference to data. The following examples are only for illustrating the present invention, and are not intended to limit the scope of the present invention in any way.

[0056] Example 1

[0057] The steps for preparing MQDs-PEI@miR43-CMCS / TA composite nanomaterials are as follows:

[0058] 1. Synthesis of MQDs-PEI nanomaterials

[0059] 5 mL of Mxene-QDs solution (2 mg / mL) and 5 mL of PEI solution (10 mg / mL) were transferred to a conical flask and mixed by ultrasound. The solution was vigorously stirred at 60 ° C for 24 h. The obtained solution was centrifuged at 8000 rpm for 3 min, and the precipitate was washed three times with deionized water to remove residual PEI. The product was dried in an oven at 40 ° C for 12 h to obtain PEI-modified Mxene-QDs, i.e., MQDs-PEI nanomaterials.

[0060] 2. Synthesis of MQDs-PEI@miR43 nanocomposites

[0061] Add 1 mL of miR43 nanosphere solution (1 mg / mL) to 1 mL of MQDs-PEI solution (0.5 mg / mL), vortex to mix, and stand at room temperature for 2 h. After ultrasound, centrifuge at 10,000 rpm for 5 min and discard the supernatant. Wash with ultrapure water three times to clean the precipitate and obtain the MQDs-PEI@miR43 nanocomposite.

[0062] 3. Synthesis of MQDs-PEI@miR43-CMCS / TA nanocomposites

[0063] 1 mL of MQDs-PEI@miR43 nanocomposite solution (0.5 mg / mL) was added to 0.5 mL of CMCS-TA mixed solution (1 mg / mL) and stirred at room temperature for 12 h. The solution was centrifuged at 10000 rpm for 10 min and the supernatant was discarded. The precipitate was washed three times with ultrapure water to obtain MQDs-PEI@miR43-CMCS / TA nanocomposite.

[0064] 1. Physical performance test

[0065] The physical properties of four materials, Mxene-QDs (MQDs), MQDs-PEI, MQDs-PEI@miR43, and MQDs-PEI@miR43-CMCS / TA, were tested, including SEM, DLS, infrared spectroscopy, and zeta potential. The test methods were all conventional methods.

[0066] 1. SEM image

[0067] Scanning electron microscopy (SEM) was used to observe the structure and morphology of MQDs-PEI@miR43-CMCS / TA composite nanomaterials, and Mxene-QDs and MQDs-PEI@miR43 were compared. Figure 1 As shown in the figure, Mxene-QDs are spherical with a particle size of less than 50nm. After adding miR43 nanospheres to MQDs-PEI, nucleic acids are bound around MQDs through electrostatic interactions, with a particle size between 50 and 100nm. When CMCS-TA is added, a protective film will be formed on the surface of the spheres, thereby protecting the nucleic acids and enhancing the adsorption capacity of the composite material to the leaves.

[0068] 2. DLS diagram

[0069] Dynamic light scattering (DLS) measurements such as Figure 2 As shown in the figure, the particle size of miR43 nanospheres is below 10nm, and the average hydrodynamic size after loading on MQDs-PEI increases to 10-30nm, which indicates that miR43 nanospheres are adsorbed on MQDs-PEI and form nanocomposites. After adding CMCS-TA, the average hydrodynamic size of MQDs-PEI@miR43-CMCS / TA composites increases to 100-300nm.

[0070] 3. Infrared spectroscopy

[0071] like Figure 3 As shown, the infrared absorption spectra of the nanocomposites have changed to varying degrees.

[0072] By Fourier transform infrared spectroscopy (FT-IR) ( Figure 3) investigated the structural molecules and chemical bonds of the four materials. Among them, 2956 peaks were attributed to CH peaks and 3326 peaks were attributed to NH peaks, indicating that PEI was successfully introduced into nanomaterials.

[0073] 4. Zeta potential

[0074] like Figure 4 As shown in the figure, after Mxene-QDs were modified with PEI, the potential changed from -10.2 mV to +14.4 mV. RNP-miR43 was added to MQDs-PEI to synthesize MQDs-PEI@miR43, and the detected zate potential was +7.8 mV. The charge was weakened because the negatively charged nucleic acid could combine with the positively charged MQDs-PEI through electrostatic interaction. CMCS-TA was further added to synthesize MQDs-PEI@miR43-CMCS / TA, and the detected zate potential was +20.6 mV. CMCS-TA was positively charged, which increased the charge of the solution.

[0075] 2. Drug loading performance test

[0076] 1. Drug loading rate

[0077] In order to study the effect of the mass ratio between MQDs-PEI and miR43 nanospheres on the drug loading rate, a nanomaterial drug loading experiment was conducted. The mass ratio of MQDs-PEI to miR43 nanospheres was set to 1:8, 1:4, 1:2, 1:1, 2:1, 4:1, 8:1, and 10:1 to prepare MQDs-PEI@miR43 nanocomposites, and the preparation method was the same as that described in Example 1. Then, gel electrophoresis was performed.

[0078] The test results are as follows Figure 5 As shown:

[0079] The results of the gel retardation experiment showed that when the ratio of MQDs-PEI to miR43 nanospheres was 1:8, the nucleic acid could not be completely loaded onto the nanomaterial, and most of the nucleic acid moved from the lane by electrophoresis. As the ratio of MQDs-PEI to miR43 nanospheres gradually increased, the amount of nucleic acid loading increased, until the ratio of MQDs-PEI to miR43 nanospheres was 4:1, the nucleic acid and MQDs-PEI material could be completely combined in the gel pores and no longer moved with electrophoresis. Then increasing the ratio of the material could also completely load the nucleic acid, indicating that the optimal ratio of MQDs-PEI to miR43 nanospheres is 4:1.

[0080] 2. RNA protection ability

[0081] Since RNA is very easy to degrade, we then studied whether MQDs-PEI can protect miR43 nanospheres from degradation in the presence of RNase. The miR43 nanospheres with a final concentration of 500 ng / mL or MQDs-PEI loaded with miR43 nanospheres were incubated with different concentrations of RNase A (final concentrations of 200 ng / mL, 150 ng / mL, 100 ng / mL, and 50 ng / mL) at 37°C for 30 min. The reaction products were then subjected to gel electrophoresis.

[0082] The test results are as follows Figure 6 As shown:

[0083] MiR43 without nanomaterial loading protection was degraded, while MQDs-PEI@miR43 was not degraded in the gel electrophoresis results. This shows that the MQDs-PEI composite material has a protective effect on nucleic acids and can protect them from RNase degradation.

[0084] 3. pH release ability

[0085] Phosphate buffer was used to change the pH of the MQDs-PEI@miR43-CMCS / TA nanocomposite solution to detect the pH release ability of the drug. The pH of the three-tube nanocomposite solution was adjusted to 6.5, 7.5, and 8.0 using phosphate buffer, and added to a 96-well plate. The absorbance of the solution was detected at a wavelength of 514nm with a time interval of 24h. The adsorption ability of the drug on the leaf was detected by measuring the contact angle between the drug and the leaf.

[0086] The test results are as follows Figure 7 and Figure 8 As shown:

[0087] It shows that as the pH decreases, the release rate of miR43 from the nanonucleic acid complex increases, and the release rate is fastest at pH 6.5. The contact angles of MQDs-PEI@miR43-CMCS / TA are 85.27° and 92.76°, the contact angles of miRNA are 105.50° and 105.37°, the contact angles of Mxene-QDs are 106.93° and 106.88°, and the contact angles of MQDs-PEI@miR43 are 100.80° and 96.06°. Therefore, the contact angle of MQDs-PEI@miR43-CMCS / TA with leaves is significantly reduced compared with the other three materials, showing that it has the strongest adsorption capacity with leaves.

[0088] 3. Antiviral effect verification

[0089] N. benthamiana was cultured in an artificial climate chamber at 25°C and a photoperiod of 16h / 8h (light / dark). N. benthamiana grown for about 4 weeks was used for antiviral effect verification. PVY and TMV source leaves were taken and ground with a mortar and added to PBS buffer. Quartz sand was used to rub the virus. The PVY source leaves were diluted 40 times with 0.01M PBS, and the TMV source leaves were diluted 80 times with 0.01M PBS to inoculate N. benthamiana leaves. After the quartz sand was evenly sprayed, the leaves were rubbed with a cotton swab to cause mechanical damage, and the virus could effectively infect the plant. The MQDs-PEI@miR43-CMCS / TA composite nanomaterial was sprayed on the tobacco leaves after the virus was inoculated using a high-pressure sprayer to fill the entire leaf. Photos were taken and samples were taken 3d and 5d after the virus was inoculated, and qRT-PCR, Western blot detection and PVY and TMV protein content were detected. Mxene-QDs (MQDs) group and blank control group (CK) were set up.

[0090] The results of the fluorescence quantitative detection of PVY virus mRNA content test are as follows Fig. 9 As shown:

[0091] The results showed that at 3dpi and 5dpi after virus treatment, the expression of the virus in Nicotiana benthamiana treated with MQDs-PEI@miR43-CMCS / TA was significantly lower than that in MQDs and CK, decreasing by more than 60% and 80%, respectively. Therefore, MQDs-PEI@miR43-CMCS / TA can play an anti-PVY virus infection effect.

[0092] Western-blot test results are as follows Fig.10 As shown:

[0093] The samples were taken for protein extraction 7 days after virus infection, and the expression of PVY-CP was semi-quantitatively detected by Actin protein concentration. The results showed that when MQDs-PEI@miR43-CMCS / TA was used to treat N. benthamiana, the protein expression of PVY-CP was also significantly inhibited, further demonstrating the antiviral effect of nanonucleic acid drugs.

[0094] The phenotypic observation results of Nicotiana benthamiana were as follows: Fig.11 As shown:

[0095] Among the three different treatments of MQDs-PEI@miR43-CMCS / TA, MQDs and CK, both MQDs and CK treatments showed obvious disease symptoms, with curling and yellowing of leaves. After treatment with MQDs-PEI@miR43-CMCS / TA, the growth status and disease symptoms of the plants were also improved, indicating that MQDs-PEI@miR43-CMCS / TA nanocomposites have good preventive and therapeutic effects on PVY virus.

[0096] The results of the fluorescence quantitative detection of TMV virus mRNA content test are as follows Fig.12 As shown:

[0097] At 3dpi and 5dpi after virus treatment, the virus expression levels in Nicotiana benthamiana treated with MQDs-PEI@miR43-CMCS / TA and MQDs-PEI-CMCS / TA were significantly lower than those in MQDs and CK. Therefore, MQDs-PEI@miR43-CMCS / TA can play an anti-TMV virus infection effect.

[0098] Western-blot test results are as follows Fig.13 As shown:

[0099] Samples taken 7 days after virus infection were used for protein extraction and the expression of TMV-CP was detected. The results showed that when MQDs-PEI@miR43-CMCS / TA was used to treat Nicotiana benthamiana, the protein expression of TMV-CP was also significantly inhibited, further demonstrating the antiviral effect of nanonucleic acid drugs.

[0100] The phenotypic observation results of Nicotiana benthamiana were as follows: Fig.14 As shown:

[0101] Among the four different treatments of MQDs-PEI@miR43-CMCS / TA, MQDs and CK, both MQDs and CK treatments showed obvious disease occurrence, while after treatment with MQDs-PEI@miR43-CMCS / TA, the growth status and disease occurrence of the plants were also improved, indicating that the MQDs-PEI@miR43-CMCS / TA nanocomposite material has a good preventive and therapeutic effect on TMV virus.

[0102] Example 2

[0103] In the present invention, a single miRNA is easily degraded. After the double-stranded DNA template is subjected to rolling circle amplification to form miR43 nanospheres, the protection ability of RNA can be enhanced, and a large amount of miR43 can be released to target target genes to prevent and treat viruses.

[0104] This example provides a method for preparing the miR43 nanospheres, the steps are as follows:

[0105] 1. Construction of circular DNA template

[0106] Template DNA is synthesized by designing four complementary DNA single strands. Through annealing, the four DNA single strands are combined to form a double strand based on the principle of base complementary pairing. T4 DNA ligase is added to connect the DNA, and finally a circular DNA with a stem-loop hairpin structure is formed.

[0107] Dissolve the lyophilized powder of single-stranded DNA (A-43, B-43, C-43 and D-43), centrifuge at 6000rpm for 60s, add appropriate amount of sterile water to dissolve, and vortex to mix. Prepare a mixed solution in an EP tube: 20μL single-stranded DNA, 8μL 1×PBS. Place the EP tube in a PCR instrument, react at 95℃ for 5min, and then slowly cool to room temperature within 2h (annealing). In the previous annealing system, add 10μL T4 DNA ligase buffer (NEB buffer) and 2μL T4 DNA ligase (NEB), connect overnight at 16℃, and then heat at 75℃ for 10min to inactivate T4 DNA ligase. Then purify the DNA, the specific steps are: remove enzymes and other impurities in the reaction product, draw the upper liquid into a new PCR tube, add 50 μL of sodium acetate (pH=5.3), 200 μL of anhydrous ethanol, vortex mix, stand at -20℃ for 2h, centrifuge at 4℃ for 15min, discard the supernatant, wash the precipitate three times with 75% anhydrous ethanol, discard the supernatant, dry and remove the residual ethanol, and obtain a circular DNA with a stem-loop hairpin structure. Store at -20℃.

[0108] The above single-stranded DNA sequence is as follows:

[0109] A-43: 5'- CTCACTATAG GGATGCAGGCACGA CTGCCACC -3'

[0110] B-43: 5'- CCACTCAGATCTG CTTGACAGATCTG-3'

[0111] C-43: 5'-GTTCTGGTGGCAGAGCACCGGACGATCCCTAT-3'

[0112] D-43:5'-AGTGAGTCGTATTACGAGGG TAATACGA -3'

[0113] In the above single-stranded DNA sequence, the solid line portion is the T7 promoter, and the dotted line portion is the miR43 sequence. There is a non-complementary sequence between the T7 promoter and miR43, and they cannot form a double strand through base complementary pairing, thereby forming a stem-loop structure.

[0114] 2. Synthesis of miR43 nanospheres by rolling circle amplification

[0115] The linear circular double-stranded DNA with T7 promoter sequence is used as a template and in vitro transcription is performed through T7 promoter, so that the DNA sequence downstream of T7 promoter can be transcribed to efficiently synthesize single-stranded RNA.

[0116] Prepare the following reaction solution: 2 μL 10× Transcription Buffer, 2 μL NTP Solution (containing ATP, GTP, CTP, UTP / Cy3-UTP), 0.5 μL RNase Inhibitor, 2 μL T7 RNA Polymerase, 2 μL DNA template, and add RNase free ddH2O to 20 μL.

[0117] The reaction solution (10×Transcription Buffer contains spermidine, the template DNA must be added last, and the reaction system can be scaled up) was centrifuged and then reacted at 42° C. for 2 h to form miR43 nanospheres in the system.

[0118] Add nuclease and react at 37°C for 2 h to remove T7 RNA Polymerase in the solution.

[0119] Then, RNA was purified by adding an equal volume of phenol / chloroform / isoamyl alcohol (25:24:1) to the reaction system, inverting to mix, and centrifuging at 12000rpm for 5min. The upper layer (water layer) was transferred to another clean centrifuge tube, and an equal amount of 5M ammonium acetate (pH=5.6) and 4 times the amount of 99.5% ethanol were added. After standing at room temperature for 5min, centrifuged at 15000rpm for 10min, and the supernatant was discarded. 100μL of 80% ethanol was added and centrifuged at 15000rpm for 5min at room temperature. The supernatant was discarded, and the residual ethanol was removed by drying to obtain miR43 nanospheres.

[0120] The schematic diagram of the above rolling circle amplification to form miR43 nanospheres is shown in Fig.15 shown.

[0121] 1. Characterization experiment of miR43 nanospheres

[0122] 1. Transcription time gradient experiment

[0123] In vitro transcription continuously produces miR43 chains, and their continuous self-assembly will also lead to the continuous enlargement of miR43 nanoparticles. However, tobacco leaves have cell walls and cell membrane structures, and nanomaterials with smaller particle sizes are easier to enter the plant body. Therefore, controlling the time of in vitro transcription is of great significance for the preparation of miR43 nanoparticles with smaller particle sizes.

[0124] In order to select the appropriate transcription time, the present invention designed six transcription times of 2h, 4h, 6h, 8h, 10h and 12h, and used scanning electron microscopy (SEM) to detect the change in the particle size of miR43 nanoparticles.

[0125] The test results are as follows Fig.16 and Fig.17 As shown:

[0126] At 4 hours of transcription, the particle size of miR43 nanoparticles was less than 10 nm. As the transcription time increased, the particle size of the nanoparticles continued to increase, and exceeded 100 nm at 12 hours. Therefore, the transcription time should be controlled within 4 hours.

[0127] 2. Zate potential

[0128] The miR43 nanospheres formed after 4 hours of transcription were tested for potential. Fig.18 As shown, miR43 nanospheres are negatively charged and can bind to positively charged nanomaterials through electrostatic interactions.

[0129] The above is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention without departing from the technical solution of the present invention still belongs to the protection scope of the technical solution of the present invention.

Claims

1. A method for preparing a MQDs-PEI@miR43-CMCS / TA composite nanomaterial for preventing and controlling plant viruses, characterized in that: The steps include: (1) Synthesis of MQDs-PEI nanomaterials The MXene-QDs solution and 5 mL of PEI solution were mixed and reacted; after the reaction was completed, the solution was centrifuged, and the product was washed and dried to obtain MQDs-PEI nanomaterials; (2) Synthesis of MQDs-PEI@miR43 nanocomposites The MQDs-PEI solution and the miR43 nanosphere solution were mixed and allowed to react. After the reaction was completed, the mixture was centrifuged, the supernatant was discarded, and the precipitate was washed to obtain the MQDs-PEI@miR43 nanocomposite material. (3) Synthesis of MQDs-PEI@miR43-CMCS / TA nanocomposites The MQDs-PEI@miR43 nanocomposite solution and the CMCS-TA mixed solution were mixed and reacted; after the reaction was completed, the solution was centrifuged, the supernatant was discarded, and the precipitate was washed to obtain the MQDs-PEI@miR43-CMCS / TA nanocomposite.

2. The preparation method according to claim 1, characterized in that: In the step (1): The volume ratio of the Mxene-QDs solution to the PEI solution is selected from 4 to 1: 1 to 4; and / or, The concentration of the Mxene-QDs solution is selected from 1 to 10 mg / mL; and / or, The concentration of the PEI solution is selected from 5 to 20 mg / mL; and / or, The reaction conditions are selected from: vigorous stirring at 50-60° C. for 12-24 hours.

3. The preparation method according to claim 1, characterized in that: In the step (2): The volume ratio of the MQDs-PEI solution to the miR43 nanosphere solution is selected from 1 to 8: 1 to 10; and / or, The concentration of the MQDs-PEI solution is selected from 0.5 to 2 mg / mL; and / or, The concentration of the miR43 nanosphere solution is selected from 0.5 to 2 mg / mL; and / or, The static reaction conditions are selected from: static reaction at 25-35° C. for 1-4 hours.

4. The preparation method according to claim 1, characterized in that: In the step (3): The volume ratio of the MQDs-PEI@miR43 nanocomposite solution to the CMCS-TA mixed solution is selected from 4 to 1:1 to 4; and / or, The concentration of the MQDs-PEI@miR43 nanocomposite solution is selected from 0.1 to 2 mg / mL; and / or, The concentration of the CMCS-TA mixed solution is selected from 0.5 to 2 mg / mL; and / or, The reaction conditions are selected from: stirring at 25-35° C. for 4-12 hours.

5. The preparation method according to claim 4, characterized in that: In the CMCS-TA mixed solution, the concentrations of carboxymethyl chitosan and tannic acid are the same.

6. The preparation method according to claim 1, characterized in that: The miR43 nanospheres are prepared by conventional rolling circle amplification method, and the DNA nucleic acid sequence of miR43 is as follows: 5'-CTGCCACCCCACTCAGATCTG-3'.

7. The MQDs-PEI@miR43-CMCS / TA composite nanomaterial for preventing and controlling plant viruses prepared by the method according to any one of claims 1 to 6.

8. Application of the MQDs-PEI@miR43-CMCS / TA composite nanomaterial according to claim 7 in preventing and controlling plant viruses.

9. Use of the MQDs-PEI@miR43-CMCS / TA composite nanomaterial according to claim 7 in the preparation of drugs for preventing and treating plant viruses.

10. The use according to claim 8 or 9, characterized in that: The plant virus is selected from one or both of PVY virus and TMV virus.

Images