Double-stranded RNA-nanomaterial composite carrier and application thereof in soybean rust prevention and treatment

Abstract

This invention discloses a double-stranded RNA-nanomaterial composite carrier and its application in the control of soybean rust. Using the PpAFF gene, a fungal extracellular membrane protein of soybean rust fungus, as a target, and its transcript as a template, double-stranded RNA is synthesized and sprayed onto the surface of soybean leaves. This silences the PpAFF gene, effectively alleviating soybean rust symptoms. The base sequence of this gene is shown in SEQ ID NO:1. The synthesized nanomaterial MNB can inhibit the germination of urediniospores of soybean rust fungus and stimulate immune responses in various plants such as soybean, wheat, rice, and pepper, improving crop resistance to the pathogen. The method of spraying the MNB-dsPpAFF complex onto the plant surface is green, safe, and highly effective. This invention provides a highly promising solution for the control of fungal diseases in important crops such as soybean rust and rice blast.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a double-stranded RNA-nanomaterial composite carrier and its application in the prevention and control of soybean rust. Background Technology

[0002] Soybean rust, the most devastating airborne fungal disease in the global soybean production system, is caused by Phakopsora pachyrhizi. It infects soybean leaves, hindering photosynthesis, causing leaf chlorosis and wilting, and shriveled grains, leading to a severe decline in soybean yield and quality. Since the first large-scale outbreak of soybean rust in Brazil in 2003, this disease has become a major threat to soybean production, causing over $2 billion in economic losses annually. In my country, the damage caused by soybean rust is equally significant, with yield reductions typically ranging from 10% to 30%, and losses exceeding 50% in severely affected fields, directly jeopardizing national food and oil security.

[0003] Currently, there is a lack of effective means to control soybean rust. Regarding disease-resistant breeding, the scarcity of resistant resources has hindered the traditional method of planting resistant varieties to control soybean rust. In terms of chemical control, the main reliance is on triazole and methoxyacrylate fungicides; however, the excessive and singular use of fungicides in the field has exacerbated the resistance problem, and the spread of resistant strains has reduced field efficacy by 40%–60%. Furthermore, the overuse of chemical pesticides has caused environmental pollution, pesticide residues, and other biosafety issues, raising public concerns about their potential harm to human health.

[0004] SIGS is a technology based on the exogenous application of artificially designed and synthesized double-stranded RNA or small interfering RNA (siRNA). This technology involves spraying RNA molecules targeting key genes of pathogens or insects onto the plant surface, allowing them to be absorbed by the harmful organisms and delivered into the cells, thereby specifically silencing the target genes and ultimately achieving effective suppression of pests and diseases. The field application of RNA insecticides based on this technology was listed as one of the top ten scientific breakthroughs in *Science* in 2024, and the first RNA pesticide has received registration approval from the U.S. Environmental Protection Agency.

[0005] Nanomaterials are particles with sizes ranging from 1 to 100 nm, existing in a variety of forms. Due to their unique physicochemical properties, nanomaterials exhibit multifunctional characteristics in the field of plant disease control. Photosensitive carbon dots (CDs) nanomaterials can disrupt fungal cell walls through photolysis by releasing reactive oxygen species (ROS), inhibiting the growth of mycelia and spore germination in various pathogenic fungi. Bioengineering of chitosan-iron nanocomposites (BNCs) synthesized by engineered bacteria can upregulate the transcription levels of multiple defense-related genes such as OsPRs, OsSOD, and OsAPX in rice plants. Binding to star polycations (SPc) can significantly increase the delivery efficiency of dsRNA in potato leaves, tobacco roots, and leaves, and promote the uptake of dsRNA by pathogenic *Phytophthora infestans* hyphae and spores.

[0006] In summary, utilizing nanomaterials as carriers for efficient and green control of plant diseases has become a research focus both domestically and internationally, and has demonstrated great application potential in actual production. Summary of the Invention

[0007] To address the technical problem of the lack of effective control methods for soybean rust in current production, the purpose of this invention is to provide a double-stranded RNA-nanomaterial composite carrier and its application in the control of soybean rust. This invention achieves green and efficient control of soybean rust by designing and synthesizing a double-stranded RNA targeting the PpAFF gene of the rust fungus, combining it with the nanomaterial MNB, and then spraying it onto the surface of soybean leaves.

[0008] The objective of this invention can be achieved through the following technical solutions:

[0009] In a first aspect, the present invention seeks to protect a nanomaterial MNB, which is a rhomboid nanoparticle formed by reacting ferrous sulfate with flavonoids as a backbone under alkaline conditions to generate FeO nanoparticles, and then combining them with chitosan and lysine.

[0010] Furthermore, the specific preparation process of the nanomaterial MNB includes the following steps:

[0011] (1) Preparation of FeO nanomaterials: A flavonoid compound solution was mixed with a ferrous sulfate solution, and an alkali was added to adjust the pH value to 9-12 to precipitate ferrous ions and generate FeO nanoparticles; after magnetic separation, water washing and drying, FeO nanomaterials were obtained; the flavonoid compound was rutin, luteolin or apigenin;

[0012] (2) Surface functionalization modification to prepare MNB: Chitosan and L-lysine were dissolved in dilute acetic acid aqueous solution to obtain a modification solution; FeO nanomaterials prepared in step (1) were added to the modification solution and stirred overnight; after magnetic separation, washing and drying, nanomaterials MNB were obtained.

[0013] Furthermore, the concentration of the flavonoid solution is 0.2-0.5 mM; the concentration of the ferrous sulfate solution is 0.5-1 M; and the molar ratio of the flavonoid compound to the ferrous sulfate is 1:2500-1:5000.

[0014] Furthermore, the final concentration of chitosan in the modification solution is 1-5 mg / mL; the final concentration of L-lysine in the modification solution is 1-5 mg / mL; the mass ratio of chitosan, L-lysine and FeO nanomaterials is 1:2-10:10-20; and the volume percentage concentration of the dilute acetic acid aqueous solution is 1%-10%.

[0015] Secondly, the present invention seeks to protect a double-stranded RNA-nanomaterial composite carrier, which is composed of double-stranded RNA molecule dsPpAFF and the aforementioned nanomaterial MNB in ​​a mass ratio of 1:1-20; wherein the double-stranded RNA molecule dsPpAFF is obtained by in vitro transcription or synthesis using the soybean rust fungus gene PpAFF, whose nucleotide sequence is shown in SEQ ID NO:1, as a template.

[0016] Thirdly, the present invention seeks protection for the use of the above-mentioned nanomaterial MNB in ​​the following (1) or (2) or (3):

[0017] (1) Application in inhibiting the germination of soybean rust fungus urediniospores or in the preparation of products for inhibiting the germination of soybean rust fungus urediniospores;

[0018] (2) Application in inducing plant immune responses or in the preparation of products for inducing plant immune responses;

[0019] (3) Application in the prevention and control of plant diseases or in the preparation of products for the prevention and control of plant diseases;

[0020] The plant is at least one of soybean, rice, wheat, chili pepper or alfalfa; the disease is soybean rust or rice blast.

[0021] Furthermore, in the above applications, the concentration of the nanomaterial MNB is 10-1000 ng / μL.

[0022] Fourthly, the present invention seeks protection for the use of the above-described double-stranded RNA-nanomaterial composite carrier in the following (1) or (2) or (3):

[0023] (1) Application in inhibiting the germination of soybean rust fungus urediniospores or in the preparation of products for inhibiting the germination of soybean rust fungus urediniospores;

[0024] (2) Application in inducing plant immune responses or in the preparation of products for inducing plant immune responses;

[0025] (3) Application in the prevention and control of plant diseases or in the preparation of products for the prevention and control of plant diseases.

[0026] Furthermore, in the above applications, the double-stranded RNA-nanomaterial composite carrier is prepared into a solution with a concentration of 10-1000 ng / μL of double-stranded RNA molecules dsPpAFF and 10-1000 ng / μL of nanomaterial MNB, and then applied to plant leaves by spraying.

[0027] Fifthly, the present invention seeks protection for the use of the soybean rust pathogen gene PpAFF, whose nucleotide sequence is shown in SEQ ID NO:1, as a nucleic acid pesticide target in the following (1) or (2) or (3):

[0028] (1) Application in inhibiting the germination of soybean rust fungus urediniospores or in the preparation of products for inhibiting the germination of soybean rust fungus urediniospores;

[0029] (2) Application in inducing plant immune responses or in the preparation of products for inducing plant immune responses;

[0030] (3) Application in the prevention and control of plant diseases or in the preparation of products for the prevention and control of plant diseases.

[0031] Sixthly, the present invention seeks protection for the use of the double-stranded RNA molecule dsPpAFF, obtained by in vitro transcription or synthesis using the soybean rust pathogen gene PpAFF (nucleotide sequence as shown in SEQ ID NO:1) as a template, in the following (1) or (2) or (3):

[0032] (1) Application in inhibiting the germination of soybean rust fungus urediniospores or in the preparation of products for inhibiting the germination of soybean rust fungus urediniospores;

[0033] (2) Application in inducing plant immune responses or in the preparation of products for inducing plant immune responses;

[0034] (3) Application in the prevention and control of plant diseases or in the preparation of products for the prevention and control of plant diseases.

[0035] Seventhly, the present invention claims protection for a method for inducing plant immune response or preventing plant diseases, the method being to spray the above-mentioned double-stranded RNA molecule dsPpAFF, double-stranded RNA-nanomaterial composite carrier or nanomaterial MNB onto the surface of plant leaves.

[0036] The double-stranded RNA molecule dsPpAFF is obtained by in vitro transcription or synthesis using the soybean rust fungus gene PpAFF, whose nucleotide sequence is shown in SEQ ID NO:1, as a template.

[0037] The nanomaterial MNB is a rhombic nanoparticle formed by reacting flavonoids with ferrous sulfate under alkaline conditions to generate FeO nanoparticles, and then combining them with chitosan and lysine.

[0038] The double-stranded RNA-nanomaterial composite carrier is composed of the double-stranded RNA molecule dsPpAFF and the nanomaterial MNB in ​​a mass ratio of 1:1-20.

[0039] In the technical solution of this invention, the plant is at least one of soybean, rice, wheat, chili pepper or alfalfa; the disease is soybean rust or rice blast.

[0040] This invention screens a soybean rust fungus nucleic acid pesticide target gene PpAFF, the nucleotide sequence of which is shown in SEQ ID NO:1 or the nucleotide sequence of SEQ ID NO:1 is modified by substitution and / or deletion and / or addition of one or more residues of a rust-related sequence derived from SEQ ID NO:1; the amino acid sequence of the protein synthesized by this gene PpAFF is shown in SEQ ID NO:2.

[0041] This invention designs a pair of primers for synthesizing the double-stranded RNA molecule dsPpAFF. Using the forward primer T7-dsPpAFF-F and the reverse primer T7-dsPpAFF-R, the PpAFF fragment is amplified from soybean rust fungus cDNA for use in double-stranded RNA synthesis. The nucleotide sequences of T7-dsPpAFF-F and T7-dsPpAFF-R are shown in SEQ ID NO:3 and SEQ ID NO:4, respectively.

[0042] This invention synthesizes a double-stranded RNA molecule, dsPpAFF, using the PpAFF gene fragment as a template. The dsPpAFF double-stranded RNA molecule is then applied to control soybean rust. By spraying the double-stranded RNA molecule, which targets PpAFF, onto the surface of soybean leaves, it effectively silences the PpAFF gene of the soybean rust fungus, reduces its expression level, and alleviates the incidence of soybean rust.

[0043] This invention provides a nanomaterial MNB and a double-stranded RNA-nanomaterial composite carrier, and applies the MNB nanomaterial and the double-stranded RNA-nanomaterial composite carrier to inhibit the germination of soybean rust fungus urediniospores and induce plant defense responses. The results show that co-incubating a solution of MNB nanomaterial with a suspension of soybean rust fungus urediniospores significantly inhibits spore germination rate. Spraying a solution of MNB nanomaterial onto the leaf surface of soybean, pepper, alfalfa, and wheat significantly upregulated the expression of plant defense genes. Combining the double-stranded RNA molecule dsPpAFF with MNB nanomaterial and spraying it onto the leaf surface has a significant control effect on soybean rust.

[0044] Compared with the prior art, the advantages and positive effects of this invention are as follows:

[0045] (1) Green and Safe: Traditional chemical control often involves environmental pollution and ecological risks, and the issues of pesticide residues and biosafety are of great concern. This invention loads environmentally friendly, safe, and non-toxic double-stranded RNA molecules onto the nanomaterial MNB and applies it through foliar spraying. Preliminary studies have confirmed that this MNB carrier has no significant adverse effects on soybean plant growth or on non-target microorganisms such as Escherichia coli and biocontrol fungus Trichoderma harzianum. Therefore, this technology provides a highly promising solution for the green control of soybean rust.

[0046] (2) Significant control effect: This invention loads double-stranded RNA molecules onto nanomaterial MNB for foliar spraying. Through the dual effects of inhibiting the growth and development of soybean rust fungus and stimulating the host's immune response, it significantly improves the control effect of soybean rust disease, with a control effect of up to 90%, showing broad application prospects. Attached Figure Description

[0047] Figure 1 This section describes the nanomaterial MNB and its combination with dsPpAFF. In the diagram, a represents the synthesis process and steps of MNB-dsPpAFF; b represents the potential difference characterization of the nanomaterial and MNB-dsPpAFF; and c represents the mass ratio of the MNB and dsPpAFF combination.

[0048] Figure 2 Both dsPpAFF and the nanomaterial MNB, as well as the complex formed by their combination, inhibited the germination of soybean rust urediniospores and appressorium formation. In the figures, a represents the germination status of soybean rust urediniospores after incubation with dsRNA or nanomaterials for 3 h and 12 h; b represents the statistical results of soybean rust urediniospore germination rate.

[0049] Figure 3The upregulation of defense genes induced by the nanomaterial MNB in ​​soybean, pepper, alfalfa, and wheat was investigated. In the figures, a represents the relative expression level of the soybean GmPR1 gene; b represents the relative expression level of the soybean GmPR2 gene; c represents the relative expression level of the soybean GmPR3 gene; d represents the relative expression levels of the pepper CaPR1, CaChilV3, and CaCAT genes; e represents the relative expression levels of the alfalfa MsPR1, MsNPR1, and MsSPL15 genes; and f represents the relative expression levels of the wheat TaPR1a, TaPR2, and TaPR5 genes.

[0050] Figure 4 Images show the effects of dsPpAFF, the nanomaterial MNB, and their combination on controlling soybean rust. Image a shows the control effect on soybean rust on soybean plants; image b shows the control effect on soybean leaves; and image c shows the average biomass of soybean rust on soybean leaves.

[0051] Figure 5 The image shows the effect of MNB-dsPpAFF solution on controlling rice blast. Image a shows the effect of rice blast control on rice leaves; image b shows the biomass of rice blast fungus on rice leaves. Detailed Implementation

[0052] To make the objectives and technical solutions of this invention clearer, specific examples are provided for further illustration, but the invention is not limited to these examples. The room temperature described in this invention is 25±10℃.

[0053] Example 1: Synthesis of double-stranded RNA molecule dsPpAFF based on PpAFF gene

[0054] (1) Total RNA extraction:

[0055] Using mixed samples of soybean rust fungus urediniospores at 1, 3, 5, 7 and 14 days after infection with the susceptible variety Williams 82, total RNA was extracted using the high-purity total RNA extraction kit from Shanghai Pudi Biotechnology Co., Ltd., following the instructions. The RNA content and quality were detected using a spectrophotometer.

[0056] (2) Reverse transcription to generate the first strand:

[0057] Use 0.8 μg of RNA as a template and synthesize cDNA according to the instructions of the third-generation full-length cDNA one-strand synthesis kit (genomic de-generated), bringing the volume to 20 μL. Use an appropriate amount of the reverse transcription product for subsequent gene cloning PCR.

[0058] (3) Using cDNA first strand as a template, perform PCR using standard methods to amplify the PpAFF gene fragment:

[0059] PCR primer amplification sequences:

[0060] Upstream primer T7dsPpAFF-F:

[0061] 5'- TAATACGACTCACTATAGGGGAATGCGCTGCCCATGGGG -3' (SEQ ID NO:3)

[0062] Downstream primer T7-dsPpAFF-R:

[0063] 5'- TAATACGACTCACTATAGGGCAGAGAGGTGAAAATAAAATG -3' (SEQ ID NO: 4),

[0064] The 50 μL reaction system consisted of: 25 μL of Novizan high-fidelity enzyme, 3 μL each of upstream and downstream primers, 1 μL of template cDNA, and water to a final volume of 50 μL.

[0065] The PCR amplification program was as follows: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 30 s, for 40 cycles, and a final extension at 72℃ for 10 min.

[0066] Electrophoresis was performed on an agarose gel, followed by ethidium bromide (EB) staining and photographing. The results were recorded, and the PCR product of the PpAFF gene fragment was recovered by gel excision. The electrophoretic bands were recovered using the Novizan Gel Extraction / DNA Purification Kit. The PCR product of the recovered PpAFF gene fragment was ligated into the T7 vector according to the instructions of the Novizan TA Compatible Introductory Cloning Kit to obtain the T7-PpAFF plasmid. The plasmid was transformed into E. coli competent cells JM109 and cultured in the dark at 37 °C for 16 h on LB agar plates containing 50 µg / mL kanamycin. After colony PCR verification, three positive clones were selected and sent to Sangon Biotech Co., Ltd. for sequencing. The sequence obtained should be consistent with the sequence of SEQ ID NO:1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO:2.

[0067] (4) Using the PpAFF gene fragment as a template, synthesize the double-stranded RNA molecule dsPpAFF:

[0068] Using 0.4 μg of the PpAFF gene fragment as a template, double-stranded RNA molecules dsPpAFF were synthesized according to the instructions for use of the in vitro dsRNA synthesis reagent from Novizan Pharmaceuticals, and the volume was adjusted to 20 μL. The content and quality of the double-stranded RNA were determined using a spectrophotometer.

[0069] Example 2: Synthesis of nanomaterial MNB

[0070] Add 20 mL of 0.2 mM rutin aqueous solution and 20 mL of 0.5 M ferrous sulfate aqueous solution to an Erlenmeyer flask. Stir the mixture with a mechanical stirrer for 15 min at room temperature. Adjust the pH of the reaction solution to approximately 12 with 0.2 M NaOH. Collect the precipitate using an external magnetic force, wash it three times with ultrapure water, and dry it at 60 °C for 12 h to obtain black solid FeO nanomaterials. Then, add 200 mg of FeO nanomaterials and 20 mL of 1% (v / v) acetic acid aqueous solution containing chitosan (20 mg) and L-lysine (40 mg) to a round-bottom flask. Stir the mixture overnight with a mechanical stirrer at room temperature. Separate the precipitate from the solution using an external magnetic force. Wash the product three times with ultrapure water and dry it at 60 °C for 12 h to obtain black solid MNBs nanomaterials (e.g., ...). Figure 1 (a) In this context, the MNBs carry a positive charge ( Figure 1 (b) has the ability to bind to negatively charged dsRNA. Figure 1 (b, c in the text).

[0071] Example 3: Nanomaterials MNB and double-stranded RNA molecules dsPpAFF inhibited the germination of soybean rust fungus urediniospores and appressorium formation, respectively.

[0072] Soybean rust fungus urediniospores stored at -80℃ were heat-shocked at 42℃ for 5 min, then dissolved and mixed with 0.1% (v / v) Tween-20 solution. After centrifugation at 12000 rpm for 5 min, the supernatant was removed, and the spores were prepared into a spore suspension with a final concentration of 100 spores / μL.

[0073] Weigh out MNB nanomaterial powder and add 2 mL of enzyme-free water. Place the mixture in an ultrasonic water bath at 55°C and sonicate for 1 h to fully dissolve the MNB. Dilute the dsPpAFF solution appropriately and mix it with the MNB solution at a 1:3 effective component mass ratio. Let the mixture stand at room temperature for 2 min, then incubate it in a 55°C water bath for 1 min, and finally let it stand at room temperature for 10 min to allow the two to fully combine.

[0074] The spore suspension was thoroughly mixed with dsPpAFF solution with a final concentration of 30 ng / μL, MNB solution with a final concentration of 90 ng / μL, and double-stranded RNA-nanomaterial composite carrier solution (containing 30 ng / μL dsPpAFF and 90 ng / μL MNB). Enzyme-free water (CK) and double-stranded YFP (dsYFP) were used as controls. After incubation in a dark incubator at 25℃ for 3 h and 12 h, the spore morphology and germination were observed using an optical microscope.

[0075] The results showed that the complex formed by MNB-dsPpAFF adsorbed onto the spore surface after co-incubation with soybean rust urediniospores for 3 h, significantly inhibiting spore germination with an inhibition rate as high as 100%. Although dsPpAFF solution had no significant effect on spores after 3 h of co-incubation, it inhibited the formation of its key infection structure, appressorium, at 12 h (e.g., ...). Figure 2 (As shown).

[0076] Example 4: Foliar application of MNB nanomaterial solution to induce immune responses in soybeans, peppers, alfalfa, and wheat plants.

[0077] The MNB nanomaterial solution (preparation method as described in Example 2) was diluted with water to 90 ng / μL. 0.1% (v / v) Tween-20 solution was added and mixed thoroughly, then sprayed evenly onto both sides of leaves from soybeans (2 weeks), peppers (4 weeks), alfalfa (4 weeks), and wheat (1 week) cultured for 2 weeks. Water spraying served as a negative control. After 12 or 24 hours, the treated leaves were flash-frozen in liquid nitrogen. Total RNA was extracted from the leaves according to the instructions of the high-purity total RNA extraction kit from Shanghai Pudi Biotechnology Co., Ltd., and the RNA content and quality were detected using a spectrophotometer. 0.8 μg of RNA was reverse transcribed into cDNA according to the instructions of the Novizan 3rd Generation Full-Length cDNA One-Strand Synthesis Kit (Genomic De-generated). The expression levels of various plant defense genes were detected using quantitative real-time PCR. GmCYP2 was selected as the soybean internal control gene, and GmPR1, GmPR2, and GmPR5 were selected as the defense genes to be detected in soybean; Caβ-tubulin was selected as the pepper internal control gene, and CaPR1, CaChilV3, and CaCAT were selected as the defense genes to be detected in pepper; MsActin was selected as the alfalfa internal control gene, and MsPR1, MsNPR1, and MsSPL15 were selected as the defense genes to be detected in alfalfa; TaActin was selected as the wheat internal control gene, and TaPR1a, TaPR2, and TaPR5 were selected as the defense genes to be detected in wheat. The primers used for the quantitative real-time PCR reaction are as follows:

[0078] GmCYP2 upstream primer:

[0079] 5'- CGGGACCAGTGTGCTTCTTCA-3' (SEQ ID NO: 5)

[0080] GmCYP2 downstream primer:

[0081] 5'-CCCCTCCACTACAAAGGCTCG-3' (SEQ ID NO:6)

[0082] GmPR1 upstream primer:

[0083] 5'-AACTATGCTCCCCCTGGCAACTATATTG-3' (SEQ ID NO:7)

[0084] GmPR1 downstream primer:

[0085] 5'-TCTGAAGTGGTAGCTTCTACATCGAAACAA-3' (SEQ ID NO:8)

[0086] GmPR2 upstream primer:

[0087] 5'-TGAAATAAGGGCCACGAGTCCAAATG-3' (SEQ ID NO:9)

[0088] GmPR2 downstream primer:

[0089] 5'-ATGGTACATGCAGACTTCAAGAATGCAGAT-3' (SEQ ID NO:10)

[0090] GmPR5 upstream primer:

[0091] 5'-GCGCTTGCTCCGCTTTCAACT-3' (SEQ ID NO:11)

[0092] GmPR5 downstream primer:

[0093] 5'-CTTGGAATAGACGGTGGGCTTGC-3' (SEQ ID NO:12)

[0094] Caβ-tubulin upstream primer:

[0095] 5'- TTAGGCGCAAGGCTTCTT-3' (SEQ ID NO: 13)

[0096] Caβ-tubulin downstream primer:

[0097] 5'-TGCTGTCGCATCCTGGTAT-3' (SEQ ID NO:14)

[0098] CaPR1 upstream primer:

[0099] 5'-CCTTACGGGGAAAACCTAGC-3' (SEQ ID NO:15)

[0100] CaPR1 downstream primer:

[0101] 5'-CGTACTGAGTTACGCCAGAC-3' (SEQ ID NO:16)

[0102] CaChilV3 upstream primer:

[0103] 5'- ACCAGAACAAGTGCTCAA-3' (SEQ ID NO:17)

[0104] CaChilV3 downstream primer:

[0105] 5'-CTCCACAGTATTCCCTAGTC-3' (SEQ ID NO:18)

[0106] CaCAT upstream primer:

[0107] 5'-GTCTACTATTCGGAGGATAAGC-3' (SEQ ID NO:19)

[0108] CaCAT downstream primer:

[0109] 5'- GTCCAATACGGTGTCTCTG-3' (SEQ ID NO: 20)

[0110] MsActin upstream primer:

[0111] 5'-ATTCACGAGACCACCTAC-3' (SEQ ID NO:21)

[0112] MsActin downstream primer:

[0113] 5'-GAGCCACAACCTTAATCTTC-3' (SEQ ID NO:22)

[0114] MsPR1 upstream primer:

[0115] 5'-TGACTCACCAGCACCATCT-3' (SEQ ID NO:23)

[0116] MsPR1 downstream primer:

[0117] 5'- GATATTGGGCCAACACCAAC-3' (SEQ ID NO: 24)

[0118] MsNPR1 upstream primer:

[0119] 5'-GGTTCTTTCCTCGTTGCTCA-3' (SEQ ID NO:25)

[0120] MsNPR1 downstream primer:

[0121] 5'-CGTCTGCGCTTCACTTGTCG-3' (SEQ ID NO:26)

[0122] MsSPL15 upstream primer:

[0123] 5'-GCGTTGATTCTCGTAACTGTTTGTC-3' (SEQ ID NO: 27)

[0124] MsSPL15 downstream primer:

[0125] 5'-TTCACAATGACAGCAAACAAGAGT-3' (SEQ ID NO: 28)

[0126] TaActin upstream primer:

[0127] 5'- GGAAAAGTGCAGAGAGACACG-3' (SEQ ID NO: 29)

[0128] TaActin downstream primer:

[0129] 5'-TACAGTGTCTGGATCGGTGGT-3' (SEQ ID NO:30)

[0130] TaPR1a upstream primer:

[0131] 5'-CGTCTTCATCACCTGCAACTA-3' (SEQ ID NO:31)

[0132] TaPR1a downstream primer:

[0133] 5'- CAAACATAAACACACGCACGTA-3' (SEQ ID NO:32)

[0134] TaPR2 upstream primer:

[0135] 5'-CCGCACAAGACACCTCAAGATA-3' (SEQ ID NO:33)

[0136] TaPR2 downstream primer:

[0137] 5'-CGATGCCCTTGGTTTTGGTAGA-3' (SEQ ID NO:34)

[0138] TaPR5 upstream primer:

[0139] 5'-ACAGCTACGCCAAGGACGAC-3' (SEQ ID NO:35)

[0140] TaPR5 downstream primer:

[0141] 5'-CGGCGTCCTAATCTAAGGGCAG-3' (SEQ ID NO:36)

[0142] Experimental results showed that spraying leaves with the nanomaterial MNB solution significantly induced the upregulation of defense genes GmPR1, CaPR1, MsPR1, and TaPR1a in soybeans, peppers, alfalfa, and wheat, thereby enhancing plant disease resistance (e.g., Figure 3 (As shown).

[0143] Example 5. Combining double-stranded RNA molecule dsPpAFF with nanomaterial MNB and spraying it on the leaf surface can effectively prevent soybean rust.

[0144] Soybean rust fungus was sprayed onto soybean leaves with dsPpAFF solution (final concentration 30 ng / μL), MNB solution (final concentration 90 ng / μL), and double-stranded RNA-nanomaterial composite carrier solution (containing 30 ng / μL dsPpAFF and 90 ng / μL MNB) as described in Example 3. Enzyme-free water (CK) and double-stranded YFP molecules (dsYFP) at the same concentration were used as controls. After the leaves were completely dried for 2 hours, a spore suspension (preparation method as described in Example 3) was sprayed onto the leaves for inoculation with soybean rust fungus. Infection was observed and photographed 13 days after inoculation, and samples were collected for RNA extraction. Soybean rust fungus biomass was detected using quantitative real-time PCR. Ubiquitin-3 was selected as the soybean internal reference gene, and α-tubulin was selected as the soybean rust fungus gene. The primers used for the quantitative real-time PCR reaction are as follows:

[0145] Ubiquitin-3 upstream primer:

[0146] 5'-GTGTAATGTTGGATGTGTTCCC-3' (SEQ ID NO:37)

[0147] Ubiquitin-3 downstream primer:

[0148] 5'-ACACAATTGAGTTCAACACAAACCG-3' (SEQ ID NO:38)

[0149] α-tubulin upstream primer:

[0150] 5'-CCAAGGCTTCTTCGTGTTTCA-3' (SEQ ID NO:39)

[0151] α-tubulin downstream primer:

[0152] 5'-CAAGAGAAGAGCGCCAAACC-3' (SEQ ID NO:40)

[0153] Results: Compared with water or double-stranded YFP molecule treatment, soybean rust symptoms on soybean leaves treated with dsPpAFF, MNB, and MNB-dsPpAFF were significantly reduced, with fewer spore masses. When dsPpAFF or MNB were used alone, the biomass of soybean rust infection was significantly reduced by approximately 35-45%. The control effect of the MNB-dsPpAFF complex was even more pronounced, reducing the biomass of soybean rust infection by approximately 80-90% compared to the control group. The MNB-dsPpAFF complex further enhanced the control effect compared to dsPpAFF or MNB alone. Figure 4 ).

[0154] Example 6. Double-stranded RNA molecule dsPpAFF combined with nanomaterial MNB and sprayed on the leaf surface can effectively prevent rice blast disease.

[0155] The MNB-dsPpAFF binding solution (dsPpAFF: 30 ng / μL, MNB: 90 ng / μL) from Example 3 was sprayed onto soybean leaves, with water (CK) and the same concentration of double-stranded YFP molecules (dsYFP) serving as controls. Twenty-four hours after treatment, rice leaves were sprayed with a suspension of Guy11 spores to inoculate against the fungus. Infection was observed and photographed six days after inoculation, and samples were collected for RNA extraction. The biomass of the fungus was detected using quantitative real-time PCR. OsRubq1 was selected as the rice internal reference gene, and 28S rDNA was selected as the gene for the fungus. The primers used for the quantitative real-time PCR reaction are as follows:

[0156] OsRubq1 upstream primer:

[0157] 5'-GTGGTGGCCAGTAAGTCCTC-3' (SEQ ID NO:41)

[0158] OsRubq1 downstream primer:

[0159] 5'-GGACACAATGATTAGGGATCA-3' (SEQ ID NO:42)

[0160] 28S rDNA upstream primer:

[0161] 5'-TACGAGAGGAACCGCTCATTCAGATAATTA-3' (SEQ ID NO:43)

[0162] 28S rDNA downstream primer:

[0163] 5'-TCAGCAGATCGTAACGATAAAGCTACTC-3' (SEQ ID NO:44)

[0164] Results: Compared with the control water and double-stranded YFP molecule treatment, the symptoms of rice blast on rice leaves treated with the MNB-dsPpAFF-bound solution were significantly weakened, the lesion area was reduced, and the biomass of rice blast fungus infection was significantly reduced, reaching 10%-15% of the control group (e.g., ...). Figure 5 (As shown).

[0165] sequence list

[0166] SEQ ID NO:1: Full-length nucleotide sequence of the PpAFF encoding gene, *Phakopsorapachyrhizi*, soybean rust fungus, 477 bp in length.

[0167] ATGGGGTTATACTCGGGTTTTGATTTTTTTGATCATCTTTCAAGTCTTTTATCTTGCCTTGGGAGATTCATTCGACAAAAATATCTATACTCGTCAAAACTCTCTCTCTACTTGCGCAAGAGACTGCTATACAAATGCAACTGCAAACACCGGTGCTTTAGGAACTTGCTCCCAGACGGATAACCTTTGCTTATGCAGGAGGGATGAGTTTGGCGATAGCGTAAAGGACTGTTGGGATA AATGTACGGACATTGAGGAAGCTGCTGCCAAAACTTGGTTCGAAACAGAATGCGCTGCCCATGGGGTTGCTGTGAGATTTAACAATGTTACAAGTGCCGTTTCAGAGACAGCATCTTCTGTGGCAAATACAATCAACAACGCACGAAATTCGCAGCCTAAGACTGTATCAATTTCAGTGGCATTAGCATTTTGTTCTCTTATGTTATACATTTTTATTTCACCTCTCTGTAGATCTTTCC

[0168] SEQ ID NO:2: Full-length protein amino acid sequence of PpAFF, soybean rust fungus (Phakopsorapachyrhizi), sequence length 159 aa

[0169] MGYTRVLIFLIIFQVFYLALGDSFDKNIYTRQNSLSTCARDCYTNATANTGALGTCSQTDNLCLCRRDEFGDSVKDCWDKCTDIEEAAAKTWFETECAAHGVAVRFNNVTSAVSETASSVANTINNARNSQPKTVSISVALAFCSLMLYILFHLSVDLS.

Claims

1. A nanomaterial MNB, characterized in that, The nanomaterial MNB is a rhombic nanoparticle formed by reacting flavonoids as a skeleton with ferrous sulfate under alkaline conditions to generate FeO nanoparticles, and then complexing with chitosan and lysine. The specific preparation process of the nanomaterial MNB includes the following steps: (1) Preparation of FeO nanomaterial: mix flavonoid solution with ferrous sulfate solution, add alkali to adjust the pH value to the range of 9-12, so that ferrous ions are precipitated to generate FeO nanoparticles; after magnetic separation, water washing and drying, FeO nanomaterial is obtained; the flavonoids are rutin; (2) Surface functionalization modification to prepare MNB: dissolve chitosan and L-lysine in dilute acetic acid aqueous solution to obtain a modification solution; add the FeO nanomaterial prepared in step (1) to the modification solution and stir overnight; after magnetic separation, washing and drying again, the nanomaterial MNB is obtained; The concentration of the flavonoid solution is 0.2-0.5 mM; the concentration of the ferrous sulfate solution is 0.5-1 M; the molar ratio of the flavonoids to ferrous sulfate is 1:2500-1:5000; The final concentration of chitosan in the modification solution is 1-5 mg / mL; the final concentration of L-lysine in the modification solution is 1-5 mg / mL; the mass ratio of chitosan, L-lysine and FeO nanomaterial is 1:2-10:10-20; the volume percentage concentration of the dilute acetic acid aqueous solution is 1%-10%.

2. A double-stranded RNA-nanomaterial complex carrier, characterized by, The composite carrier is composed of double-stranded RNA molecule dsPpAFF and nanomaterial MNB according to the mass ratio of 1:1-20; the double-stranded RNA molecule dsPpAFF is a soybean rust fungus gene with nucleotide sequence as shown in SEQ ID NO:1 PpAFF obtained by in vitro transcription or synthesis.

3. The nanomaterial MNB of claim 1 is used in (1) or (2) or (3) as follows: (1) for inhibiting the germination of soybean rust uredospores; (2) for inducing plant immune response, the plant being soybean, wheat, pepper or alfalfa; (3) for preventing and treating plant diseases, the plant disease being soybean rust.

4. Use according to claim 3, characterized in that, The concentration of the nanomaterial MNB used is 10-1000 ng / μL.

5. The double-stranded RNA-nanomaterial composite carrier of claim 2 is used in (1) or (2) or (3) as follows: (1) for inhibiting the germination of soybean rust uredospores; (2) for inducing plant immune response, the plant being soybean, wheat, pepper or alfalfa; (3) for preventing and treating plant diseases, the plant disease being soybean rust or rice blast.

6. Use according to claim 5, characterized in that, The double-stranded RNA-nanomaterial composite carrier is prepared into a solution with a concentration of 10-1000 ng / μL double-stranded RNA molecule dsPpAFF and 10-1000 ng / μL nanomaterial MNB, and is applied to the plant leaves by spraying.

7. A method of inducing an immune response in a plant, characterized in that, The method is to spray the double-stranded RNA-nanomaterial composite carrier of claim 2 or the nanomaterial MNB of claim 1 on the surface of plant leaves; the plant is soybean, wheat, pepper or alfalfa.

8. A method of controlling plant diseases, characterized by, The method is to spray the double-stranded RNA-nanometer material complex carrier of claim 2 on the surface of the plant leaves, and the plant disease is soybean rust or rice blast; or to spray the nanometer material MNB of claim 1 on the surface of the plant leaves, and the plant disease is soybean rust.

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