CN121058651A

CN121058651A - Double-stranded RNA-nano material composite carrier and application thereof in soybean rust prevention and treatment

Info

Publication number: CN121058651A
Application number: CN202511605003.2A
Authority: CN
Inventors: 王源超; 欧阳海兵; 施心成; 丁煜宾; 叶文武; 王燕; 段凯旋
Original assignee: Sanya Research Institute Of Nanjing Agricultural University; Nanjing Agricultural University
Current assignee: Sanya Research Institute Of Nanjing Agricultural University; Nanjing Agricultural University
Priority date: 2025-11-05
Filing date: 2025-11-05
Publication date: 2025-12-05

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

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

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a double-stranded RNA-nano material composite carrier and application thereof in soybean rust prevention and treatment.

Background

Soybean rust is the most destructive air-borne fungal disease in a global soybean production system, and pathogenic bacteria of soybean potato layer rust (Phakopsora pachyrhizi) infects soybean leaves to block photosynthesis, so that leaf green fading and grain collapse are caused, and the soybean yield and quality are seriously reduced. Since the first large-scale outbreak of soybean rust in brazil 2003, this disease has become a significant threat to soybean production, thus far causing economic losses of over 20 billion dollars each year. In China, the soybean rust disease is also not small, the yield reduction range in general year is 10% -30%, the loss of the heavy disaster field can exceed 50%, and the national grain and oil safety is directly endangered.

At present, effective means for preventing and treating soybean rust disease are lacking. In the aspect of disease-resistant breeding, the scarcity of resistance resources makes the traditional method for planting disease-resistant varieties for preventing and treating soybean rust disease blocked to a certain extent. In the aspect of chemical prevention and control, triazole and methoxy acrylic acid ester bactericides are mainly relied on for prevention and control, however, the problem of drug resistance is serious due to single and large-scale use of field bactericides, and the field prevention effect is reduced by 40% -60% due to diffusion of drug resistant strains. In addition, the abuse of chemical pesticides causes biosafety problems such as environmental pollution, pesticide residues, and the like, and the harm to human health also raises public concerns.

SIGS is a technique based on exogenous administration of artificially designed and synthesized double-stranded RNA or small interfering RNA (SMALL INTERFERING RNA, SIRNA). The technology is characterized in that RNA molecules of targeted pathogenic bacteria or insect key genes are sprayed on the surface of plants, so that the RNA molecules are absorbed by harmful organisms and transferred into cells, and the target genes are specifically silenced, so that the effective inhibition of plant diseases and insect pests is finally realized. The field application of RNA pesticides based on the technology is listed as one of ten major scientific breakthroughs in Science of 2024, and the first RNA pesticide has been approved by the registration of the United states environmental protection agency.

Nanomaterial is a particle with a size between 1-100 nm and has a rich form of existence. The nano material has unique physical and chemical properties, and shows multifunctional characteristics in the field of plant disease prevention and control. Photo-sensitive Carbon Dots (CDs) nanomaterials can release reactive oxygen species (Reactive oxygen species, ROS) through photolysis to destroy fungal cell walls and inhibit the growth of various pathogenic hyphae and spore germination. The chitosan-iron nanomaterial (Bioengineering of chitosan-iron nanocomposites, BNCs) synthesized by engineering bacteria can cause the up-regulation of the transcription level of various defense related genes such as OsPRs, osSOD and OsAPX in rice plants. Binding to the star polycation (Star polycation, SPc) can significantly increase the efficiency of dsRNA delivery in potato leaf, tobacco root and leaf and promote the uptake of dsRNA by phytophthora infestans filaments and spores.

In conclusion, the efficient green prevention and control of plant diseases are realized by using the nano material as a carrier, which becomes a focus of research at home and abroad and has great application potential in actual production.

Disclosure of Invention

Aiming at the technical problem that the soybean rust disease in the current practical production lacks an effective control means, the invention aims to provide a double-stranded RNA-nano material composite carrier and application thereof in the control of the soybean rust disease. The invention realizes green and efficient prevention and control of soybean rust by designing and synthesizing double-stranded RNA of targeted rust PpAFF genes and spraying the double-stranded RNA on the surface of soybean leaves after combining the double-stranded RNA with nano material MNB.

The aim of the invention can be achieved by the following technical scheme:

In a first aspect, the present invention claims a nanomaterial MNB, where the nanomaterial MNB is a diamond nanoparticle formed by reacting a flavonoid compound with ferrous sulfate under alkaline conditions to generate FeO nanoparticles, and then compositing the FeO nanoparticles with chitosan and lysine.

Further, the specific preparation process of the nano material MNB comprises the following steps:

(1) The preparation of the FeO nano material comprises the steps of mixing a flavonoid compound solution with a ferrous sulfate solution, adding alkali to adjust the pH value to 9-12, and precipitating ferrous ions to generate FeO nano particles, and carrying out magnetic separation, water washing and drying to obtain the FeO nano material, wherein the flavonoid compound is rutin, luteolin or apigenin;

(2) Preparing MNB through surface functionalization modification, namely dissolving chitosan and L-lysine in dilute acetic acid aqueous solution to obtain a modification solution, adding the FeO nanomaterial prepared in the step (1) into the modification solution, stirring overnight, and obtaining the nanomaterial MNB through magnetic separation, washing and drying again.

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

Further, the final concentration of the chitosan in the modification solution is 1-5 mg/mL, the final concentration of the L-lysine in the modification solution is 1-5 mg/mL, the mass ratio of the chitosan to the L-lysine to the FeO nano material is 1:2-10:10-20, and the volume percentage concentration of the dilute acetic acid aqueous solution is 1% -10%.

In a second aspect, the invention provides a double-stranded RNA-nanomaterial composite vector, which is formed by compositing double-stranded RNA molecules dsPpAFF and the nanomaterial MNB according to the mass ratio of 1:1-20, wherein the double-stranded RNA molecules dsPpAFF are obtained by in vitro transcription or synthesis by taking soybean rust germ gene PpAFF with a nucleotide sequence shown as SEQ ID NO. 1 as a template.

In a third aspect, the present invention claims the use of the nanomaterial MNB described above in (1) or (2) or (3) as follows:

(1) The application of inhibiting the germination of the soybean rust fungus summer spores or preparing a product for inhibiting the germination of the soybean rust fungus summer spores;

(2) Use of inducing a plant immune response or preparing a product for inducing a plant immune response;

(3) The application of the composition in preventing and controlling plant diseases or preparing products for preventing and controlling plant diseases;

The plant is at least one of soybean, rice, wheat, chilli or alfalfa, and the disease is soybean rust or rice blast.

Further, in the above application, the nanomaterial MNB is used at a concentration of 10-1000 ng/. Mu.L.

In a fourth aspect, the present invention claims the use of the double-stranded RNA-nanomaterial composite vector described above in (1) or (2) or (3) as follows:

(3) The application of the composition in preventing and controlling plant diseases or preparing products for preventing and controlling plant diseases.

Further, in the above application, the double-stranded RNA-nanomaterial composite vector is prepared into a solution with the concentration of 10-1000 ng/mu L of double-stranded RNA molecule dsPpAFF and 10-1000 ng/mu L of nanomaterial MNB, and the solution is applied to plant leaves by a spraying mode.

In a fifth aspect, the invention claims the use of soybean rust germ gene PpAFF with nucleotide sequence as shown in SEQ ID NO:1 as a nucleic acid pesticide target in (1) or (2) or (3) as follows:

In a sixth aspect, the present invention claims the use of double-stranded RNA molecule dsPpAFF obtained by in vitro transcription or synthesis using soybean rust germ gene PpAFF with nucleotide sequence as shown in SEQ ID NO:1 as a template in (1) or (2) or (3) as follows:

In a seventh aspect, the present invention claims a method for inducing a plant immune response or controlling plant diseases, wherein the method comprises spraying the double-stranded RNA molecule dsPpAFF, double-stranded RNA-nanomaterial composite carrier or nanomaterial MNB onto the surface of a plant leaf;

The double-stranded RNA molecule dsPpAFF is obtained by in vitro transcription or synthesis by taking a soybean rust germ gene PpAFF with a nucleotide sequence shown as SEQ ID NO. 1 as a template;

The nano material MNB is diamond-shaped nano particles formed by taking a flavonoid compound as a framework, reacting with ferrous sulfate under an alkaline condition to generate FeO nano particles, and then compositing with chitosan and lysine;

The double-stranded RNA-nanomaterial composite carrier is formed by compositing the double-stranded RNA molecules dsPpAFF and the nanomaterial MNB according to the mass ratio of 1:1-20.

In the technical scheme of the invention, the plant is at least one of soybean, rice, wheat, chilli or alfalfa, and the disease is soybean rust or rice blast.

The invention screens a soybean rust germ nucleic acid pesticide target gene PpAFF, the nucleotide sequence of which is shown as SEQ ID NO. 1 or the nucleotide sequence of SEQ ID NO. 1 is substituted and/or deleted and/or added with one or more residues to be related to rust, the sequence derived from SEQ ID NO. 1 is derived, and the amino acid sequence of the protein synthesized by the gene PpAFF is shown as SEQ ID NO. 2.

The present invention contemplates a pair of primers for synthesizing double stranded RNA molecules dsPpAFF, using forward primer T7-dsPpAFF-F and reverse primer T7-dsPpAFF-R to amplify PpAFF fragments from soybean rust cDNA for use in the synthesis of double stranded RNA. The nucleotide sequences of the T7-dsPpAFF-F and the T7-dsPpAFF-R are respectively shown as SEQ ID NO 3 and SEQ ID NO 4.

The invention synthesizes double-stranded RNA molecule dsPpAFF by using PpAFF gene fragment as a template. Double-stranded RNA molecule dsPpAFF is applied to prevention and treatment of soybean rust. The double-stranded RNA molecules synthesized by taking PpAFF as a target gene are sprayed on the surface of soybean leaves, so that the soybean rust PpAFF genes can be effectively silenced, the expression quantity of the soybean rust PpAFF genes can be reduced, and the occurrence of soybean rust is reduced.

The invention provides a nanomaterial MNB and double-stranded RNA-nanomaterial composite carrier, which are applied to inhibiting soybean rust fungus summer spore germination and inducing plant defense reaction. Research results show that the spore germination rate can be obviously inhibited by incubating the nano material MNB solution and the soybean rust fungus summer spore suspension. Spraying the nano material MNB solution on the surfaces of soybean, capsicum, alfalfa and wheat leaves can obviously cause up-regulation expression of plant defense genes. The double-stranded RNA molecule dsPpAFF is combined with the nano material MNB and sprayed on the surface of the leaf blade, so that the soybean rust disease can be prevented and treated obviously.

Compared with the prior art, the invention has the advantages and positive effects that:

(1) Green and safe, the traditional chemical control often accompanies environmental pollution and ecological risks, and the problems of pesticide residues and biological safety are concerned. The invention loads the environment-friendly, safe and nontoxic double-stranded RNA molecules on the nano material MNB, and applies the nano material MNB through foliar spraying. Preliminary researches prove that the MNB vector has no obvious adverse effect on non-target microorganisms such as soybean plant growth, escherichia coli, biocontrol bacteria trichoderma harzianum and the like. Therefore, the technology provides a solution with great potential for green prevention and control of soybean rust.

(2) The double-stranded RNA molecules are loaded on the nano material MNB for foliar spraying, and the double effects of inhibiting the growth and development of soybean rust and exciting host immune response are achieved, so that the prevention and control effects of soybean rust are remarkably improved, the prevention and control effects are up to 90%, and the broad application prospect is shown.

Drawings

Fig. 1 is a diagram of nanomaterial MNB and its binding to dsPpAFF. Wherein a is MNB-dsPpAFF synthesis flow and step, b is nano material and MNB-dsPpAFF potential difference characterization, c is mass ratio of MNB and dsPpAFF combination.

Figure 2 is dsPpAFF and nanomaterial MNB, both of which combine to inhibit soybean rust summer spore germination and attachment cell formation. Wherein a is germination condition after dsRNA or nano material is used for incubating with the soybean rust fungus summer spores for 3 h and 12 h, and b is germination rate statistics of the soybean rust fungus summer spores.

FIG. 3 shows that nanomaterial MNB induces up-regulated expression of soybean, capsicum, alfalfa, wheat defense genes. Wherein a is the relative expression of soybean GmPR gene, b is the relative expression of soybean GmPR gene, c is the relative expression of soybean GmPR gene, d is the relative expression of capsicum CaPR1, caChilV3 and CaCAT gene, e is the relative expression of alfalfa MsPR1, msNPR1 and MsSPL15 gene, and f is the relative expression of wheat TaPR a, taPR2 and TaPR5 gene.

Fig. 4 is a graph showing the effect of dsPpAFF and nano-material MNB on controlling soybean rust after combining the two. Wherein a is a soybean rust prevention and treatment effect diagram on soybean plants, b is a soybean rust prevention and treatment effect diagram on soybean leaves, and c is soybean rust average biomass statistics on the soybean leaves.

FIG. 5 shows the effect of MNB-dsPpAFF solution on controlling rice blast. Wherein a is a graph of the rice blast control effect on rice leaves, and b is the biological statistics of rice blast bacteria on the rice leaves.

Detailed Description

For the purpose of making the objects and technical solutions of the present invention more clear, the present invention is further described with specific examples, but is not limited to these examples. The room temperature of the invention is 25+/-10 ℃.

Example 1 Synthesis of double-stranded RNA molecule dsPpAFF based on PpAFF Gene

(1) Total RNA extraction:

The mixed sample of 1 d, 3d, 5 d, 7 d and 14 d after the soybean rust fungus summer spore infects the infected variety Williams 82 is used as a material, the total RNA is extracted by adopting a high-purity total RNA extraction kit of Shanghai Pu Di biological technology Co., ltd, and the RNA content and quality of the total RNA are detected by using a spectrophotometer according to the description operation.

(2) Reverse transcription generates the first strand:

0.8. Mu.g of RNA was used as a template for cDNA synthesis according to the instructions of the third generation full-length cDNA one-strand synthesis kit (genome removal), and the reaction was performed to a volume of 20. Mu.L. Appropriate amounts of the reverse transcription product were taken for subsequent gene cloning PCR.

(3) PCR was performed using the first strand of cDNA as a template, and PpAFF gene fragments were amplified by conventional methods:

PCR primer amplification sequence:

The upstream primer T7dsPpAFF-F:

5’- TAATACGACTCACTATAGGGGAATGCGCTGCCCATGGGG -3’(SEQ ID NO:3)

Downstream primer T7-dsPpAFF-R:

5’- TAATACGACTCACTATAGGGCAGAGAGGTGAAATAAAATG -3’(SEQ ID NO:4),

50. mu.L of the reaction system was 25. Mu.L of Hi-Fi enzyme from Novain, 3. Mu.L of each of the upstream and downstream primers, 1. Mu.L of template cDNA, and water was added to 50. Mu.L.

The PCR amplification procedure was 95℃pre-denaturation for 5min, 95℃denaturation for 30 s,56℃annealing for 30 s,72℃extension for 30 s, 40 cycles, and finally 72℃extension for 10min.

Electrophoresis separation was performed on agarose gel, ethidium Bromide (EB) staining was photographed, the results were recorded, and PCR products of PpAFF gene fragments were recovered by gel cutting. The electrophoresis bands were recovered using a Northey company gel recovery/DNA purification kit. The PCR product of PpAFF gene fragment recovered by cutting gel is connected to T7 carrier according to the description operation of TA compatibility entry cloning kit of Noruzan company to obtain T7-PpAFF plasmid, E.coli competent cell JM109 is transformed, colony PCR is verified to pick up three positive clones after 37 ℃ dark culture of LB solid plate containing kanamycin concentration of 50 mug/mL for 16h, and the sequence obtained by sequencing is consistent with SEQ ID NO:1 sequence, and the amino acid sequence of the coded protein is shown as SEQ ID NO: 2.

(4) Double-stranded RNA molecule dsPpAFF is synthesized by using PpAFF gene fragment as a template:

A0.4 mu g PpAFF gene fragment was used as a template for double-stranded RNA molecule dsPpAFF synthesis according to the instructions of the in vitro dsRNA synthesis reagent of Norpraise company, and the volume was fixed to 20. Mu.L. The content and quality of double-stranded RNA are detected by a spectrophotometer.

EXAMPLE 2 Synthesis of nanomaterial MNB

To the conical flask were added 20 mL of a 0.2 mM rutin aqueous solution and 20 mL of a 0.5M ferrous sulfate aqueous solution. The mixture 15 min was then stirred at room temperature with a mechanical stirrer. The pH of the reaction solution was adjusted to about 12 with 0.2M NaOH. The precipitate was collected by external magnetic force and washed three times with ultrapure water and dried at 60 ℃ for 12h to obtain a black solid FeO nanomaterial. Then, 200 mg FeO nm material and 20 a mL aqueous solution of 1% (v/v) acetic acid containing chitosan (20 a mg) and L-lysine (40 a mg) were added to the round bottom flask. The mixture was stirred at room temperature overnight with a mechanical stirrer. The precipitate was separated from the solution by external magnetic force. The product was washed 3 times with ultrapure water and dried at 60 ℃ for 12h to give a black solid MNBs nanomaterial (a in fig. 1), which MNBs is positively charged (b in fig. 1), and has the ability to bind to negatively charged dsRNA (b, C in fig. 1).

Example 3 nanomaterial MNB and double stranded RNA molecule dsPpAFF inhibit Leptosphaeria sojae germination and attachment cell formation, respectively

The spores of soybean rust were subjected to heat shock at 42℃of 5 min, then 0.1% (v/v) Tween-20 solution was added to dissolve and mix well, and after centrifugation at 12000 rpm of 5 min, the supernatant was removed to prepare spores as a spore suspension with a final concentration of 100 spores/. Mu.L.

Weighing nano material MNB powder, adding 2 mL of non-enzyme water, and placing in an ultrasonic water bath kettle at 55 ℃ and ultrasonic wave of 1h to fully dissolve MNB. Mixing dsPpAFF solution with MNB solution at a mass ratio of 1:3, standing at room temperature for 2 min, water-bathing at 55deg.C for 1min, and standing at room temperature for 10 min to combine the two.

Spore suspension was thoroughly mixed with dsPpAFF solution at a final concentration of 30 ng/. Mu.L, MNB solution at a final concentration of 90 ng/. Mu.L, and double-stranded RNA-nanomaterial composite carrier solution (containing dsPpAFF at 30 ng/. Mu.L and MNB at 90 ng/. Mu.L), and spore morphology and germination were observed by an optical microscope after incubation of 3h and 12h in a dark incubator at 25℃with non-enzymatic water (CK) and double-stranded YFP (dsYFP) as controls.

The result shows that the complex formed by MNB-dsPpAFF and the soybean rust fungus summer spores are incubated for 3h and then adsorbed on the surfaces of the spores, so that the germination of the spores is obviously inhibited, and the inhibition rate is as high as 100%. The dsPpAFF solution, after co-incubation with spores, while not significantly affecting the spores at 3h, was able to inhibit the formation of its critical invasive structure attachment cells at 12h (as shown in fig. 2).

Example 4 spraying of nanomaterial MNB solution onto leaf surfaces induces an immune response in soybean, capsicum, alfalfa, wheat plants.

The nano material MNB solution (the preparation mode is shown in example 2) is diluted to 90 ng/mu L by water, and 0.1% (v/v) Tween-20 solution is added for uniform mixing and then uniformly sprayed on the front and back sides of soybean cultured for 2 weeks, pepper cultured for 4 weeks, alfalfa cultured for 4 weeks and wheat leaves cultured for 1 week, and water is sprayed to serve as a negative control. 12 And h or 24h, quickly freezing the treated leaf with liquid nitrogen, extracting total RNA of the leaf according to the specification by using a high-purity total RNA extraction kit of Shanghai Pu Di biotechnology Co., ltd, and detecting the RNA content and quality by using a spectrophotometer. 0.8 μg of RNA was reverse transcribed into cDNA according to the instructions of the Novamat company third generation full-length cDNA one-strand synthesis kit (ungrouped). And detecting the expression quantity of each plant defense gene by utilizing fluorescent quantitative PCR. GmCYP2 is selected as a soybean reference gene, gmPR, gmPR, gmPR is selected as a soybean defending gene to be detected, ca beta-tubulin is selected as a capsicum reference gene, caPR, caChilV3, caCAT is selected as a capsicum defending gene to be detected, msActin is selected as a alfalfa reference gene, msPR1, msNPR, msSPL15 is selected as a alfalfa defending gene to be detected, taActin is selected as a wheat reference gene, taPR a, taPR2, taPR is selected as a wheat defending gene to be detected. The primers used for the fluorescence quantitative PCR reaction were chosen as follows:

GmCYP2 upstream primer:

5’- CGGGACCAGTGTGCTTCTTCA-3’(SEQ ID NO:5)

GmCYP2 downstream primer:

5’- CCCCTCCACTACAAAGGCTCG-3’(SEQ ID NO:6)

GmPR1 upstream primer:

5’- AACTATGCTCCCCCTGGCAACTATATTG-3’(SEQ ID NO:7)

GmPR1 downstream primer:

5’- TCTGAAGTGGTAGCTTCTACATCGAAACAA-3’(SEQ ID NO:8)

GmPR2 upstream primer:

5’- TGAAATAAGGGCCACGAGTCCAAATG-3’(SEQ ID NO:9)

GmPR2 downstream primer:

5’- ATGGTACATGCAGACTTCAAGAATGCAGAT-3’(SEQ ID NO:10)

GmPR5 upstream primer:

5’- GCGCTTGCTCCGCTTTCAACT-3’(SEQ ID NO:11)

GmPR5 downstream primer:

5’- CTTGGAATAGACGGTGGGCTTGC-3’(SEQ ID NO:12)

Ca beta-tubulin upstream primer:

5’- TTAGGCGCAAGGCTTTCTT -3’(SEQ ID NO:13)

ca beta-tubulin downstream primer:

5’- TGCTGTCGCATCCTGGTAT-3’(SEQ ID NO:14)

CaPR1 upstream primer:

5’- CCTTACGGGGAAAACCTAGC-3’(SEQ ID NO:15)

CaPR1 downstream primer:

5’- CGTACTGAGTTACGCCAGAC-3’(SEQ ID NO:16)

CaChilV3 upstream primer:

5’- ACCAGAACAAGTGCTCAA-3’(SEQ ID NO:17)

CaChilV3 downstream primer:

5’- CTCCACAGTATTCCCTAGTC-3’(SEQ ID NO:18)

CaCAT upstream primer:

5’- GTCTACTATTCGGAGGATAAGC-3’(SEQ ID NO:19)

CaCAT downstream primer:

5’- GTCCAATACGGTGTCTCTG-3’(SEQ ID NO:20)

MsActin upstream primer:

5’- ATTCACGAGACCACCTAC-3’(SEQ ID NO:21)

MsActin downstream primer:

5’- GAGCCACAACCTTAATCTTC-3’(SEQ ID NO:22)

MsPR1 upstream primer:

5’- TGACTCACCAAGCACCATCT-3’(SEQ ID NO:23)

MsPR1 downstream primer:

5’- GATATTGGGCCAACACCAAC-3’(SEQ ID NO:24)

MsNPR1 upstream primer:

5’- GGTTCTTTCCTCGTTGCTCA-3’(SEQ ID NO:25)

MsNPR1 downstream primer:

5’- CGTCTGCGCTTCACTTGTCG-3’(SEQ ID NO:26)

MsSPL15 upstream primer:

5’- GCGTTGATTCTCGTAACTGTTTGTC-3’(SEQ ID NO:27)

MsSPL15 downstream primer:

5’- TTCACAAATGACAGCAAACAAGAGT-3’(SEQ ID NO:28)

TaActin upstream primer:

5’- GGAAAAGTGCAGAGAGACACG-3’(SEQ ID NO:29)

TaActin downstream primer:

5’- TACAGTGTCTGGATCGGTGGT-3’(SEQ ID NO:30)

TaPR1a upstream primer:

5’- CGTCTTCATCACCTGCAACTA-3’(SEQ ID NO:31)

TaPR1a downstream primer:

5’- CAAACATAAACACACGCACGTA-3’(SEQ ID NO:32)

TaPR2 upstream primer:

5’- CCGCACAAGACACCTCAAGATA-3’(SEQ ID NO:33)

TaPR2 downstream primer:

5’- CGATGCCCTTGGTTTGGTAGA-3’(SEQ ID NO:34)

TaPR5 upstream primer:

5’- ACAGCTACGCCAAGGACGAC-3’(SEQ ID NO:35)

TaPR5 downstream primer:

5’- CGCGTCCTAATCTAAGGGCAG -3’(SEQ ID NO:36)

experimental results show that the nano material MNB solution can obviously cause up-regulated expression of soybean, capsicum, alfalfa, wheat defense genes GmPR a, caPR a, msPR a, taPR a and the like after being sprayed on the leaves, and plant disease resistance is enhanced (shown in figure 3).

Example 5. Double-stranded RNA molecules dsPpAFF are combined with nanomaterial MNB and sprayed on the surface of leaves to effectively prevent and treat soybean rust.

DsPpAFF solution with a final concentration of 30 ng/. Mu.L, MNB solution with a final concentration of 90 ng/. Mu.L and double stranded RNA-nanomaterial composite carrier solution (containing dsPpAFF at 30 ng/. Mu.L and MNB at 90 ng/. Mu.L) of example 3 were sprayed onto soybean leaves while using water without enzyme (CK) and double stranded YFP molecules (dsYFP) with the same concentration as controls. Spore suspension was sprayed on the leaves after treatment 2h were completely dried (preparation method see example 3) to inoculate soybean rust, and after inoculation 13 d, infection was observed, photographed, and samples were collected to extract RNA, and soybean rust biomass was detected by fluorescent quantitative PCR. The Ubiquitin-3 is selected as a soybean reference gene, and the alpha-tubulin is selected as a soybean rust gene. The primers used for the fluorescent quantitative PCR reaction were as follows:

Ubiquitin-3 upstream primer:

5’-GTGTAATGTTGGATGTGTTCCC-3’(SEQ ID NO:37)

a downstream primer of Ubiquitin-3:

5’-ACACAATTGAGTTCAACACAAACCG-3’(SEQ ID NO:38)

alpha-tubulin upstream primer:

5’-CCAAGGCTTCTTCGTGTTTCA-3’ (SEQ ID NO:39)

alpha-tubulin downstream primer:

5’-CAAGAGAAGAGCGCCAAACC-3’(SEQ ID NO:40)

As a result, compared with the water or double-chain YFP molecular treatment, the symptoms of soybean rust on soybean leaves treated by dsPpAFF, MNB and MNB-dsPpAFF are obviously reduced, the spore pile number is smaller, wherein when dsPpAFF or MNB is singly treated, the biomass of soybean rust infection is obviously reduced by about 35-45%, the prevention and treatment effect of soybean leaves treated by MNB-dsPpAFF conjugate is more obvious, the biomass of soybean rust infection is reduced by about 80-90% compared with that of a control group, and the MNB-dsPpAFF compound is further improved compared with dsPpAFF or MNB singly treated (figure 4).

Example 6 double stranded RNA molecules dsPpAFF are combined with nanomaterial MNB and sprayed on the leaf surface to effectively control rice blast.

The MNB-dsPpAFF binding solution of example 3 (dsPpAFF:30 ng/. Mu.L, MNB:90 ng/. Mu.L) was sprayed onto soybean leaves with water (CK) and the same concentration of double-stranded YFP molecules (dsYFP) as controls. Spraying a rice blast fungus Guy11 spore suspension on rice leaves after 24 h of treatment to inoculate the rice blast fungus, observing infection condition after 6 d of inoculation, photographing, collecting samples to extract RNA, and detecting the biomass of the rice blast fungus by utilizing fluorescent quantitative PCR. OsRubq 1A 1 was selected as a rice reference gene, and 28S rDNA was selected as a rice blast fungus gene. The primers used for the fluorescent quantitative PCR reaction were as follows:

OsRubq1 upstream primer:

5’-GTGGTGGCCAGTAAGTCCTC -3’(SEQ ID NO:41)

OsRubq1 downstream primer:

5’-GGACACAATGATTAGGGATCA -3’(SEQ ID NO:42)

28S rDNA upstream primer:

5’-TACGAGAGGAACCGCTCATTCAGATAATTA -3’ (SEQ ID NO:43)

28S rDNA downstream primer:

5’-TCAGCAGATCGTAACGATAAAGCTACTC -3’(SEQ ID NO:44)

As a result, compared with the control water and double-strand YFP molecular treatment, the symptoms of rice blast on the rice leaves treated by the solution after MNB-dsPpAFF binding are obviously reduced, the area of the disease spots is reduced, and the biomass infected by rice blast bacteria is obviously reduced to 10% -15% of that of the control group (shown in figure 5).

Sequence listing

SEQ ID NO. 1: full-length nucleotide sequence of PpAFF encoding gene, soybean rust germ (Phakopsora pachyrhizi), sequence length 477 bp

ATGGGTTATACTCGGGTTTTGATTTTTTTGATCATCTTTCAAGTCTTTTATCTTGCCTTGGGAGATTCATTCGACAAAAATATCTATACTCGTCAAAACTCTCTCTCTACTTGCGCAAGAGACTGCTATACAAATGCAACTGCAAACACCGGTGCTTTAGGAACTTGCTCCCAGACGGATAACCTTTGCTTATGCAGGAGGGATGAGTTTGGCGATAGCGTAAAGGACTGTTGGGATAAATGTACGGACATTGAGGAAGCTGCTGCCAAAACTTGGTTCGAAACAGAATGCGCTGCCCATGGGGTTGCTGTGAGATTTAACAATGTTACAAGTGCCGTTTCAGAGACAGCATCTTCTGTGGCAAATACAATCAACAACGCACGAAATTCGCAGCCTAAGACTGTATCAATTTCAGTGGCATTAGCATTTTGTTCTCTTATGTTATACATTTTATTTCACCTCTCTGTAGATCTTTCC

SEQ ID NO. 2: ppAFF full-length protein amino acid sequence, soybean rust germ (Phakopsora pachyrhizi), sequence length 159 aa

MGYTRVLIFLIIFQVFYLALGDSFDKNIYTRQNSLSTCARDCYTNATANTGALGTCSQTDNLCLCRRDEFGDSVKDCWDKCTDIEEAAAKTWFETECAAHGVAVRFNNVTSAVSETASSVANTINNARNSQPKTVSISVALAFCSLMLYILFHLSVDLS.

Claims

1. A nanomaterial MNB, characterized in that the nanomaterial MNB is a rhombic nanoparticle formed by reacting ferrous sulfate with flavonoids as a backbone to generate FeO nanoparticles under alkaline conditions, and then combining it with chitosan and lysine.

2. The nanomaterial MNB according to claim 1, characterized in that the specific preparation process of the nanomaterial MNB includes the following steps:

(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 a range of 9-12, so that ferrous ions were precipitated to form FeO nanoparticles; after magnetic separation, water washing and drying, FeO nanomaterials were obtained; the flavonoid compound was rutin, luteolin or apigenin;

(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.

3. The nanomaterial MNB according to claim 2, characterized in that the concentration of the flavonoid compound 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.

4. The nanomaterial MNB according to claim 2, characterized in that 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; and the volume percentage concentration of the dilute acetic acid aqueous solution is 1%-10%.

5. A double-stranded RNA-nanomaterial composite carrier, characterized in that the composite carrier is composed of a double-stranded RNA molecule dsPpAFF and the nanomaterial MNB described in any one of claims 1-4 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.

6. The use of the nanomaterial MNB according to any one of claims 1-4 in the following (1) or (2) or (3):

(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;

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

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

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

7. The application according to claim 6, wherein the concentration of the nanomaterial MNB is 10-1000 ng/μL.

8. The use of the double-stranded RNA-nanomaterial composite carrier according to claim 5 in the following (1) or (2) or (3):

9. The application according to claim 8, characterized in that the double-stranded RNA-nanomaterial composite carrier is prepared into a solution with a concentration of 10-1000 ng/μL of double-stranded RNA molecule dsPpAFF and 10-1000 ng/μL of nanomaterial MNB, and applied to plant leaves by spraying.

10. The application 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):

11. The application 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):

12. A method for inducing plant immune responses or preventing plant diseases, characterized in that the method involves spraying double-stranded RNA molecules dsPpAFF, double-stranded RNA-nanomaterial composite carriers, or nanomaterial MNB onto the surface of plant leaves; wherein the plant is at least one of soybean, rice, wheat, pepper, or alfalfa; and the disease is soybean rust or rice blast.

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.

The nanomaterial MNB is a rhomboid nanoparticle formed by reacting ferrous sulfate with flavonoids as the backbone to generate FeO nanoparticles under alkaline conditions, and then combining it with chitosan and lysine.

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.