CN119410639A - A dsRNA and its application in preparing nano preparation for preventing and controlling pepper blight - Google Patents

Abstract

The invention discloses a dsRNA and its application in preparing a nano preparation for preventing and treating pepper blight, and relates to the technical field of pepper blight prevention and treatment. The dsRNA sequence is shown in SEQ ID NO.2; the dsRNA sequence is used to inhibit the HK2 gene, and the nucleotide sequence is shown in SEQ ID NO.1. The dsRNA is applied to the preparation of the nano preparation MSNs-dsRNA for antagonizing pepper phytophthora and preventing and treating pepper blight, and the expression of the HK2 gene can be targeted and inhibited. The MSNs-dsRNA nano preparation is highly effective in preventing and treating the blight caused by pepper phytophthora infection, and is expected to be developed into a new green fungicide.

Description

DsRNA and application thereof in preparation of nano preparation for preventing and treating pepper epidemic disease

Technical Field

The invention relates to the technical field of pepper epidemic disease prevention and control, in particular to dsRNA and application thereof in preparation of a nanometer preparation for pepper epidemic disease prevention and control.

Background

The Phytophthora (Phytophthora) pathogenic bacteria have more than 120 species, can harm almost all dicotyledonous plants, are called as plant killers, and have the characteristics of high spreading speed, easy explosion and disaster formation, difficult control and the like, and are called as crop epidemic disease in production. Phytophthora capsici (P.capsici) is the most extensive host pathogen in Phytophthora, and can harm more than 500 plants of 26 families such as Solanaceae, leguminosae, brassicaceae, cucurbitaceae, rosaceae, etc., and the economic loss caused by each year reaches billions of dollars, thus seriously affecting the quality safety and ecological environment safety of vegetables in the world. The use of bactericides is still one of the main means of controlling epidemic diseases at present. However, currently available oomycete inhibitors are relatively few in variety, and long-term use of single-acting site agents is likely to cause the problem of drug resistance of plant pathogenic oomycetes, so that the creation of a green, safe and efficient synergistic new crop epidemic prevention and control strategy is urgently needed in production.

The nucleic acid pesticide has the advantages of strong specificity, short effect period, no residue, small influence on non-target organisms and the like, is praised as the 3 rd revolution in pesticide history, and is a hot spot in the field of novel green pesticide creation. Spray-induced gene silencing (SIGS) is a novel plant protection method based on RNA interference (RNAi), which controls the expression of target genes of endogenous genes or pathogens of plants not by means of recombinant viruses or transgenic plants, but by means of exogenous application of double-stranded RNAs (double-STRANDED RNA, DSRNA) or small interfering RNAs (siRNA). In recent years, it has been reported that it has a good control effect on part of plant pathogenic fungi. The study subjects mainly include fusarium causing fusarium wilt, botrytis causing gray mold of crops, fruits, vegetables, flowers after harvest, sclerotium bacteria causing stem rot, and some other important pathogenic fungi. For example, koch et al show that spraying dsRNA (CYP 3-dsRNA) targeting 3 Fusarium graminearum Fusarium graminearum ergosterol biosynthesis genes (CYP 51A, CYP51B, CYP C) on the surface of barley Hordeum vulgare leaves can effectively inhibit Fusarium graminearum growth. However, in vitro application of dsRNA is unstable in the environment and is easily degraded by RNA degrading enzyme, ultraviolet light and high temperature, so that the technical problems of difficult effective introduction into target organisms and the like are solved, and the practical application effect of the sprayable nucleic acid pesticide is severely limited. Thus, how to enhance the stability of dsRNA is an urgent problem in the art to be solved.

The nano material has the characteristics of small size effect, interface effect, good biocompatibility and the like under the nano scale, can be used as a dsRNA carrier to enhance the stability of dsRNA in the environment and the capability of entering target biological tissues or cells, further improves the targeted delivery efficiency, and is an effective means for solving the problem of difficult exogenous application of nucleic acid pesticides. Mesoporous Silica Nanoparticles (MSNs) are materials with regular nanoscale pore structures, have pore diameters of 2-50 nm, and have good adsorption and cation exchange capacities. The medical uses of MSNs in anti-cancer, antimicrobial and therapeutic applications are particularly prominent because of their excellent properties of delivering many different small molecules and more recently biologicals (mRNA, siRNA, antigens, antibodies, proteins and peptides) at the target site. At the same time, it has a key advantage in improving the bioavailability of various cargo, including biological agents. Therefore, MSNs have great application potential as nucleic acid pesticide vectors.

Because the target genes which can effectively inhibit the growth and development of pathogenic bacteria and infect the pathogenic bacteria through gene silencing in phytophthora in vivo are not clear, the application of SIGS in crop epidemic disease prevention and control is greatly restricted. Therefore, the development and identification of more RNA molecules for different target genes is a precondition and focus for the research of RNAi pesticides and targeted prevention and control technologies. Histidine kinase (HK HISTIDINE KINASE) is a key component responsible for signal collection and transmission in a two-component signal system, which can sense and transmit various environmental and intracellular stimulation signals, and the component system is commonly existing in bacteria, fungi, colistin and higher plants and widely participates in physiological and biochemical processes of cells. Notably, no homologous genes have been identified to date in mammals and humans. Thus, histidine kinase has the potential to be a target for phytophthora capsici. The early research of the team shows that the histidine kinase gene HK2 is a key factor for regulating the growth and development of phytophthora capsici and the pathogenic process. At present, no report on the aspect of preventing and treating pepper epidemic disease by using the gene related dsRNA exists.

Therefore, how to use the gene as a target, design a dsRNA fragment with high efficiency and apply the dsRNA fragment to the control of pepper epidemic disease by combining with mesoporous silica nano materials is a problem which needs to be solved by the technicians in the field.

Disclosure of Invention

In view of the above, the invention provides dsRNA and application thereof in preparation of nano-preparation for preventing and treating pepper epidemic disease.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

In one aspect, the embodiment of the invention provides dsRNA, the dsRNA sequence is shown as SEQ ID NO.2, the dsRNA sequence is used for inhibiting an HK2 gene, and the nucleotide sequence of the HK2 gene is shown as SEQ ID NO. 1.

The second aspect of the embodiment of the invention also provides application of the dsRNA in preparation of nano preparations for antagonizing phytophthora capsici and preventing and treating phytophthora capsici.

The third aspect of the embodiment of the invention provides a nano preparation for preventing and treating pepper epidemic disease, which comprises dsRNA, mesoporous silica and polyethyleneimine, wherein the mass ratio of the dsRNA to the mesoporous silica to the polyethyleneimine is 1:80:12.

In a preferred embodiment, the dsRNA is at a concentration of 1.0mg/mL, the mesoporous silica is at a concentration of 1.0mg/mL, and the polyethylenimine is at a concentration of 0.5mg/mL.

The fourth aspect of the embodiment of the invention provides application of the nano preparation in preparing chemical preparations for preventing and treating pepper epidemic diseases.

The fifth aspect of the embodiment of the invention provides a method for preventing and treating pepper epidemic disease, wherein the nano preparation is sprayed on pepper leaves.

Compared with the prior art, the technical scheme has the following technical effects:

1) The invention clones to brand new double-stranded ribonucleic acid dsRNA for the first time, targets phytophthora capsici histidine kinase gene HK2 efficiently, induces the silencing of target genes through RNAi, has silencing efficiency reaching more than 55%, has no influence on the expression of non-target genes, and has high specificity. Meanwhile, the target gene HK2 is an important functional gene in the growth and development and pathogenic process of phytophthora, and a homologous gene thereof has not been identified in mammals and humans, so that the target gene HK2 has high safety to non-target organisms.

2) The nano mesoporous silica used in the invention has the excellent characteristics of high specific surface area, good biocompatibility, adjustable aperture, high loading capacity and the like, and can remarkably improve the transfection efficiency, the interference efficiency and the stability of dsRNA.

3) Experiments of inoculating phytophthora capsici after spraying the MSNs-dsRNA nano preparation on tobacco leaves show that the nano preparation can obviously inhibit the infection of phytophthora capsici, compared with a control group, the disease spot area of the treatment group is reduced by 72.6%, and experiments of pot plants of tomatoes show that after spraying the MSNs-dsRNA nano preparation, the incidence of tomatoes plants is reduced by 83.3%.

In conclusion, the dsRNA provided by the invention can inhibit the expression of HK2 genes in a targeted manner, and the MSNs-dsRNA nano preparation has high efficiency on preventing and treating epidemic diseases caused by phytophthora capsici infection, and is hopeful to be developed into a novel green bactericide.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.

FIG. 1 is a diagram showing the result of detecting a transcript by electrophoresis in example 1, wherein lane M is DL 2000DNA marker and lane 1 is the dsRNA band of the present invention.

FIG. 2 is a diagram showing the electrophoresis detection of dsRNA and MSNs. Wherein, lane M is DL 2000DNA marker, lane 1 is dsGFP, lane 2 is dsGFP combined with MSNs, lane 3 is dsRNA of the invention, and lane 4 is the combination of dsRNA of the invention and MSNs.

FIG. 3 shows the expression of histidine kinase gene HK2 after treatment of Phytophthora capsici with the MSNs-dsRNA nanoformulation of example 3.

FIG. 4 is a chart showing leaf spot phenotype of S.capsici on leaf in vitro of Nicotiana benthamiana after treatment with the MSNs-dsRNA nano-preparation of example 4, and B is a chart showing spot area analysis.

FIG. 5 is a graph showing the occurrence of pepper epidemic disease on tomato seedlings after treatment with the MSNs-dsRNA nano-preparation of example 5, wherein A is a graph showing the occurrence of tomato seedling plants, and B is a histogram of statistical analysis of disease index.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The sequence in the dsRNA is derived from a cDNA coding sequence of phytophthora capsici histidine kinase gene HK2, and the sequence is shown as SEQ ID NO. 1. A base sequence of 247bp in HK2 (shown as SEQ ID NO. 2) is selected as a transcription template of the target dsRNA.

ATGGTTGGCATCGGTCAGGATATCACGGAGCGTATTGCGCAAGAGCAAGAGTATAGCCGTCTCATTGATACGGCTAATGCTCCGATCTTTGGTGTAGACATCAACGGCCGCGTGAATATTTGGAACCGCAAGGCCGCCGATATCATGCAGTACACGAACGAGGATGTGCTGGGCAAGGATCTGGTAGCGGAATTCATCTCGGAGGAGTACAAGGTTCCAGTGCGAAGAGTTCTCGAGAAGGCGTTCGAGGGTGTGGAGACGGCGAACTTTGAGTTCCCATTGATCACAAAGGCGGGTCGCCGTGTGGAGATTTTGTTGAATGCAACGCCGAGATATAACGAGCATGGTGAAGTGAAGGGTATGGTTGGAATCGGCCAGGACATTACGGAGCGTATCGCTCAAGAGCAGGAGCATAGTAGAATGATTGATACTGCTAATGCTCCAATCTTCGGTGTTGACACGGATGGGCGTGTTAATATTTGGAACAGAAAGGCTGCTGATATTATGCAGTACACGAACGAGGATGTTCTTGGTAAGAACTTGGTGGAGCAATTTATTTCTGAGGAGTACCGTGTCGCAGTTCGAAGTGTTCTAGAGAAAGCTTTCAAGGGTGTTGAAACTGCGAACTTCGAGTTTCCGTTGATAACAAAAGCTGGCCGTCGCGTAGAAATCTTGCTGAATGCTACACCACGCTACAATGAGCGGGGGGAGCATGGGCAGATTGTTGGCGTCGTGGGTATCGGCCAGGATATCACGGATCGTATCGCCCAAGAGCAAGAGTACACGAGATTGATCGACTCAGCCAACGCCCCAATCTTCGGCGTGGACGTGAACGGGTGTGTGAATATCTGGAACAAAAAGGCTGCCGAAATCACGCAATACACGCCAAACGACGTGATGGGAGAGAATTTGGTGGAGAAGTTCATCACGGAAGACTATCGCGAAGCCGTTGGTCTCGTTCTATCGAAGGCGTGCGAAGGAACGGAGACCGCTAACTTTGAGTTCCCACTGATGACTAAGGCTGGCCGCCGCGTGGAGATCTTGCTGAATGCTACGTCTCGTTTTAATGAGATCGGTGAGGTTATGGGAGTGGTGGGAATCGGCCAGGACATCACTGAGCGTATTGCCCAAGAACAGGAGTACACTCGTCTGATCGACACGGCCAACGCCCCGATTTTCGGTGTCGACATCAACGGCCACGTAAACATTTGGAACCGAAAGGCAGCTGAGACGACCCAATACACAAATGCAGAGGTCCTTGGGAAGGACTTGGTGGCCGAGTTCATTTCGAAGGAGTATAAGGTTCCCGTCCGCAGTGTATTGGAGAAAGCTTTTGAAGGTGTGGAGACAGCCAATTTCGAGTTCCCGTTGATCACGAAGGCCGGTCGCCGAGTGGAAATCCTGTTAAATGCCACGCCGCGATACAACGAACACGGCGAAGTAATGGGGATGGTAGGTATTGGCCAGGATATCACCGAGCGCATTGCTCAGGAGCAGGAGTACACTCGCTTGATCGATACAGCTAATGCGCCGATCTTCGGTGTGGATATTAACGGACGCGTGAACATCTGGAACCGGAAAGCTGCAGATATTATGCAGTACACGAATGAAGACGTGCTGGGCAAGGATTTGGTTGCAGAGTTCATCTCGAAGGAGTACAAGGTGCCTGTGCGCAGTGTGTTAGAGAAAGCATTCGAAGGTGTCGAAACTGCGAACTTCGAGTTCCCGTTGATCACGAAGGCCGGTCGCCGCGTGGAGATCCTGTTGAACGCGACGCCGAGATACAATGAGCGCGGCGAGGTGATGGGTATGGTTGGCATCGGCCAGGATATCACGGAGCGTATTGCGCAAGAGCAAGAGTATAGCCGTCTCATTGATACGGCTAATGCTCCGATCTTTGGTGTAGACATCAACGGCCGCGTGAATATTTGGAACCGCAAGGCCGCCGATATCATGCAGTACACGAACGAGGATGTGCTGGGCAAGGATCTGGTAGCGGAGTTCATCTCGGAGGAGTACAAGGTTCCAGTGCGAAGAGTTCTCGAGAAGGCGTTCGAGGGTGTGGAGACGGCGAACTTCGAGTTCCCGTTGATCACAAAGGCGGGTCGCCGTGTGGAGATTTTGTTGAATGCAACGCCGAGATATAACGAGCATGGTGAAATTGTTGGCGTCGTGGGTATCGGCCAGGATATCACGGATCGTATCGCCCAAGAGCAAGAGTACACGAGATTGATCGACTCAGCCAACGCCCCAATCTTCGGCGTGGACGTGAACGGGTGTGTGAATATCTGGAACAAAAAGGCTGCCGAAATCACGCAATACACGCCAAACGACGTGATGGGAGAGAATTTGGTGGAGAAGTTCATCACGGAAGACTATCGCGAAGCCGTTGGTCTCGTTCTATCGAAGGCTGTGTTAGAGAAAGCATTCGAAGGTGTCGAAACTGCGAACTTCGAGTTCCCGTTGATCACGAAGGCCGGTCGCCGCGTGGAGATCCTGTTGAACGCGACGCCGAGATACAATGAGCGCGGCGAGGTGATGGGTATGGTTGGCATCGGCCAGGATATCACGGAGCGTATTGCGCAAGAGCAAGAGTATAGCCGTCTCATTGATACGGCTAATGCTCCGATCTTTGGTGTAGACATCAACGGCCGCGTGAATATTTGGAACCGCAAGGCCGCCGATATCATGCAGTACACGAACGAGGATGTGCTGGGCAAGGATCTGGTAGCGGAGTTCATCTCGGAGGAGTACAAGGTTCCAGTGCGAAGAGTTCTCGAGAAGGCGTTCGAGGGTGTGGAGACGGCGAACTTCGAGTTCCCGTTGATCACAAAGGCGGGTCGCCGTGTGGAGATTTTGTTGAATGCAACACCGAGATATAACGAGCATGGTGAAGTGAAGGGTATGGTTGGAATCGGCCAGGACATTACGGAGCGTATCGCTCAAGAACAGGAGTACAGTCGCCTGATTGACACGGCCAATGCTCCTATCTTCGGAGTGGATGCCAATATGTGTGTCAATATTTGGAACAGGAAAGCTGCTCAGATCACGAACTATTCGATTGGGGAAGTTATGGGTGAAAATCTTGTGGAGACGTTTATTTCGCCTGAATTCCGACCGATTGTGGCTGAAGTGCTGTCCCAAGCTCTAACAGGAGTGGAGACCGCGAATTTTGAATTTCCTCTAATTACTCGTCCAGGCACAAGAATTGAGATTTTGTTGAACGCAACCCCGCGCTATGACTTAACTGGTAACATTGTTGGTGTCGTGGGTATCGGGCAGGATATCACGGATCGGATTGCCCAAGAACACGAGTACTTCCGACTGATTGATACAGCGAATGCCCCGATCTTCGGTATTGATACTAATGGTCGCATCAATGAATGGAATCAGAAGATCGAGGCGATCACTGGCTACCACAAATCGAGTGTTCTGGGCTTGTCGTTGGTGCACACTTTCATCACCCCAGAAAGTAGACAACAAGTTCGGCAGTTGCTAAACCAAGCTCTGATCGGCATTGATGTTGGTGAGATGGAACTTCCGATGACAACCAAGCGTGGTGTGTTCCTGCTTTTGCTTGTGAATGCATCCAGTAAGAAGGACATGCATGGAAACATTCGTGGTGTAATCGGTGTGGGACAAGATTACACTGCCAGAAAGCATATGGAGGCTGCCAAGGTGAACTTCCTGGCCTCGTTTAGTCATGAGCTGCGAACGCCGTTGAATGGTGTGCTGGGTATGCTGGAATTGCTGAAAGAGCAGCCACTTGATAAATCCATCGAGAGATACGTGCACATGGCATACGTGTCCGGATCTCTTCTTCTAAATTTGATCAACGACATTTTGGACTTGTCGAAAATCGAGGCTGGGCACCTCGAGATCTCAACAGCTCCGTTCCAGATGCATGACCTGCTGGATTACTCGATTGAGATCTTTAAGTTCAAGGCTCGAGAGCGTGGTCTGAAGCTTGAGCTGAAATGTGGTGATAACGTGCCAAAGGCTGTCATCGGTGATGTTGTGCGTCTACGCCAAGTGCTGTTGAACTTGCTTTCAAACGCAATTAAGTTTACGAACACGGGCTCAATTACGGTTGCTTGCAGCGTCGTGCATTCCCCTGAATTGCCTCCACAGTTCAAAAAGCTGTTGTTCCAAGTCATTGATACGGGCATTGGCATGGATGCAGAGGAGAAGATGCGCCTGTTTTCACTGTTTACGAAGTTGGAGCGCACTCGCCAGAACAATCCTACTGGTTCTGGCCTGGGTCTAGCTATTTGCAAGCAGCTTGCGGAACTTATGGATGGCTCAATTGATGTCGACAGTGAGTTGGGGGTTGGCAGCAACTTTTTTTTTACAGTGGTCGTGAGATTAATTGACGAGGTCGATCCCAAGCATGCTTACTACTCCTCAGAGGACTTCCTTGATCCATCGACAGCATTGCTTTCACCTGATGGAGCTGTACGTACGGTCGAAAATGGAGAGAACGCATCGGTGCGAGTGGAGGTTCCTAAGCAGGCGCGAATCTTGGTCGTTGAGGATAACGAGTTTAATTGGGAAGTGGTGAAGTGCTTCCTGCAGCAAGACGACCACCTACTTCAATGGGAGGTGAATGGCCGTGATGCTGTTAAGGCCTACAAGGAAAATCACACGGAATTTGACCTGATCTTCATGGATTGCGAAATGCCTATTATGGATGGATATACTGCTACGAACGCAATTCGCGAGTTTGAGCAGCAGCAGAACCTTCCTCGTATTCCAATTCTCGGCTTGACAGCGTATGCGATGAGCGGAGACCGACAGAAGTGTCTTGACTGTGGCATGGATGAGTTCATGGTGAAACCCATCTCGAAGCTGAGTCTTCGTAAAGCCATCCGGCAGTGGATGCGTATCCGTTACCTTGGCCAACAGAACGCTGCACTGGGAGCTGTGGATGTTACACTCATGGACGCAGTCTCGACTGCCAGACTTGCACCCGCCTCACGCCACATGCAGCAGTTGGATCTTGCTCAGGCTATCTCGAACCTGGAGCTGGATGATCCGATGTCTATTGGCCTCCAAAGTGGCCGAGCGGTTCGGACTGCCACACCGACGACTTCGATCTTCAGCTTGGGCCCCTCAGTGAACAACGTCTCACGAGCGACAAGCCAGAATGATTTGCCTGATCTGCTAAGGCTATCTCGAAACACGAGCGGGGCTTCAGTTTCGAGCCCGCCGGCGCCTGAGAAGCAAAACTCCGAGACGTCTACAACGCTGCCTTATGTCAAGACCCCGTTTACTGGAGTCACAACTAGCCCAGCCTTAAGTGGATCACGTTCTCCTGTGATGAATCCAACGCTTTGGAGCCATCCACCGTTCAACAAAAAGGATCTACCGACGGGTCGACCGCAGTCCTGGTCTAGCCTTCACATGGAAACTGGCAGTTCTGTCGAAGATGATACACCGATGGAGCCTGCTGCAACTCATGAAGAGACTACTGTATCACACTCGATTGACCCGATGAGCATCGAGATCCCAGAGGGAGACCCCGTCAACTACACGTTGGGTGTGGATCAATGTGGAGGCCACGAAGAGCTCTTCTTGACTCTTCTGGAGAAGTTCGCGACAACCTCGGAATCTATAACCACTAGGGTGATCGAGGCGCACGAGCACAATGACTTCGTAACTGCTCGTCGTGAAGCTCACTCGCTTAAAGGATCCTCTGCCTACGTGGCTGCTCTTCGTTTGTCGAAATGCGCCTTCCGTGTTCAAGTCGCGTACGAGCATCTGATAGCGCAACAGGCGAACGGCGATGGCTCCGACACGTTGGCTGCGAAGGAAATTGTGGATAAATCTGTAAAGCTGCTGACCAAGGAGCAGAAACTTCTCCGTGGCTACCTCCGACGCAACTTCGAGTTCAAGAGTCAAGCGGTGTCATCCCAAGCGACTTCAGATCAGCAGCCTGACAACCAAGACAAACAATCAACCGGCCCATGCTGCATAATGTGA, As shown in SEQ ID NO. 1.

CTCGGCTTGACAGCGTATGCGATGAGCGGAGACCGACAGAAGTGTCTTGACTGTGGCATGGATGAGTTCATGGTGAAACCCATCTCGAAGCTGAGTCTTCGTAAAGCCATCCGGCAGTGGATGCGTATCCGTTACCTTGGCCAACAGAACGCTGCACTGGGAGCTGTGGATGTTACACTCATGGACGCAGTCTCGACTGCCAGACTTGCACCCGCCTCACGCCACATGCAGCAGTTGGATCTTGCTC, As shown in SEQ ID NO. 2.

The main materials are Promega T7 RiboMAX ^TM Express RNAI SYSTEM kit from Promega, DNA MARKER from Takara Bio-engineering Co., ltd., 2X Phanta Max Ma ster Mix from Nanjinouzan Biotechnology Co., ltd., and the rest of the reagents from Bio (Shanghai) technologies Co. Primers were synthesized by the technology company of biological engineering (Shanghai). The tomato material used was AC variety, phytophthora capsici LT1534 was given away by the university of agriculture plant protection college of south kyo and was stored in the present laboratory.

Example 1 in vitro synthesis of dsRNA.

Step one, preparing a dsRNA transcription template.

① Culturing phytophthora capsici leonian for 3d by using a liquid V8 culture medium, collecting mycelium, and extracting genome DNA of the phytophthora capsici leonian by adopting a CTAB method.

② PCR amplification is carried out by taking the extracted phytophthora capsici genome DNA as a template and using dsRNA specific primers (SEQ ID NO.3 and SEQ ID NO. 4). When the fragment is amplified, a T7 promoter sequence is added to the 5' end of two primers of PCR, and the amplified fragment automatically contains the T7 promoter sequence.

GGATCCTAATACGACTCACTATAGGCTCGGCTTGACAGCGTAT,SE Q ID NO.3。

GGATCCTAATACGACTCACTATAGGGAGCAAGATCCAACTGCT,SE Q ID NO.4。

PCR reaction system (50. Mu.L) used:

The PCR reaction procedure was:

After the reaction, 5. Mu.L of the reaction product was detected by 1.2% agarose gel electrophoresis, 100V pressure-stabilizing electrophoresis was performed for 25min, and the bands were observed by a gel imager. Gel imaging showed a single band at around 247bp, consistent with the expected sequence size, and fragment recovery was performed by DNA purification kit from american biotechnology limited, guangzhou.

And step two, dsRNA synthesis, namely using electrophoresis products recovered by glue as templates, and using a T7RiboMAX ^TM Express RNAI SYSTEM transcription kit of Promega to synthesize dsRNA.

Reaction system (20 μl):

Reaction conditions were incubation for 30min at 37 ℃, annealing for 10min at 70 ℃, and then cooling to room temperature. 100U/. Mu.L of RNase T1 was diluted to 10U/. Mu.L with RNase T1 Dilution Buffer. For every 20. Mu.L of reaction product, 1. Mu.L of freshly diluted RNase solution and 1. Mu.L of DNase I were added and incubated at 37℃for 30min.

And step three, detecting the transcription product by electrophoresis, wherein the result is shown in figure 1. Wherein, lane M is DNA DL2000 marker, lane 1 is HK2-dsRNA.

And step four, dsRNA purification, namely adding 55 mu L of 95% ethanol into the transcription product, uniformly mixing and placing the mixture on ice for 5min, and centrifuging the mixture for 10min at 12000 rmp. The supernatant was carefully aspirated and the pellet was washed with 0.5ml of 70% cold ethanol and after washing all ethanol was removed. The pellet was left to air dry at room temperature for 15 minutes and the transcript dsRNA was resuspended by adding 50. Mu.L nuclease-free water. The absorbance of the product A ₂₆₀ was measured, its concentration was determined and stored at-20 ℃.

Example 2 preparation of MSNs-dsRNA nanoformulations.

Step one, preparing Mesoporous Silica (MSNs) working solution, namely dissolving nano material MSNs in DEPC water, and uniformly mixing by ultrasonic vibration, wherein the final concentration is 1mg/mL.

Step two, combining MSNs and dsRNA, namely uniformly mixing 1 mu g dsRN A targeting GFP gene and 1 mu g dsRNA targeting HK2 gene with 1.2 mu L polyethylenimine PEI (0.5 mg/mL) respectively, and shaking for 10min to fully mix. 4. Mu.L of mesoporous silica (1.0 mg/mL) was added, mixed and stabilized at room temperature for 10min. Thereafter, the solution was mixed with 4. Mu.L of sodium citrate buffer (0.1M, pH 4.0), and then 9.8. Mu.L of nuclease-free water was added to give a final volume of 20. Mu.L, to obtain MSNs-dsRNA nanoformulation targeting GFP gene (abbreviated as MSNs-dsGFP) and MSNs-dsRNA nanoformulation targeting HK2 gene (abbreviated as MSNs-dsHK 2).

And step three, electrophoresis detection, namely detecting MSNs-dsRNA nano particles by using 1.2% agarose gel electrophoresis and performing 110V stabilized voltage electrophoresis for 25min. The results are shown in FIG. 2, wherein lane M is DNA DL2000marker, lane 1 is dsGFP, lane 2 is MSNs-dsGFP, lane 3 is dsHK2, and lane 4 is MSNs-dsHK2. As can be seen from fig. 2, lanes 2 and 4 have no distinct dsRNA bands, indicating that Mesoporous Silica (MSNs) can completely load dsRNA, forming stable MSNs-dsRNA nanoformulations.

Example 3 detection of expression of the phytophthora capsici target gene HK2 by MSNs-dsRNA nanoformulations.

Step one, treating phytophthora capsici by using the MSNs-dsRNA nano preparation, namely inoculating phytophthora capsici strain LT1534 into a V8 solid culture medium, and culturing for 2 days in a light-proof incubator at 28 ℃. Fresh mycelia at the edge of the colonies were inoculated into 10mL of V8 liquid medium, and then 10. Mu.L of the MSNs-dsGF P, MSNs-dsHK nm preparation prepared in example 2 was added to the medium, while the same volume of MSNs and water was used as a control. The above treatments were placed in a 28℃incubator for cultivation. After co-incubation for 3d, the mycelia were removed and the water was blotted dry, and the mycelia were collected in liquid nitrogen and rapidly frozen and stored at-80 ℃ for later use.

And step two, RNA extraction and detection, namely performing RNA extraction of phytophthora capsici mycelium by using FastPure Universal Plant Total RNA Isolation Kit kit (Nanjinouzan biotechnology Co., ltd.). The method comprises the following specific steps:

1) After the mycelium pellet was ground in liquid nitrogen, 600. Mu.L of Buffer EL was added, and the mixture was centrifuged at 12000rpm for 5 minutes with vigorous shaking for 30 sec.

2) The supernatant was centrifuged at 12000rp m for 30sec in the range of about 500. Mu.L to FastPure gDNA-Filter Columns III, and FastPure gDNA-Filter Columns III was discarded to collect the filtrate.

3) 250 Mu L of absolute ethyl alcohol is added into a collecting pipe, and the mixture is stirred and mixed for 15s.

4) The mixture was transferred to FastPure RNA Columns V, centrifuged at 12000rpm for 30sec, and the filtrate was discarded.

5) 700. Mu.L Buffer RWA was added to FastPure RNA Columns V and centrifuged at 12000rp m for 30sec, and the filtrate was discarded.

6) To FastPure RNA Columns V was added 500. Mu.L of Buffer RWB, and the mixture was centrifuged at 12000rp m for 30sec, and the filtrate was discarded.

7) Repeating step 6).

8) FastPure RNA Columns V was placed back in the collection tube and centrifuged at 12000rpm for 2min.

9) FastPure RNA Columns V was transferred to a new RNase-free Collection Tube s 1.5.5 mL centrifuge tube, and 100. Mu.L of RNase-free ddH ₂ O preheated at 65℃in advance was suspended and added dropwise to the center of the adsorption column membrane, and the mixture was left standing at room temperature for 5min and centrifuged at 12000rpm for 1min.

Step three, detecting reverse transcription of a sample:

1) gDNA was removed and the reaction system was as shown in Table 1 below

TABLE 1

4×gDNAwiperMix	4μL
		Template RNA	800ng
Nuclease-free water	0-11μL
		Total volume of	16μL

2) Gently beating and mixing by a pipette. 42 ℃ for 2min.

3) Reverse transcription to cDNA, reaction system is shown in Table 2;

TABLE 2

5×HiScriptIIIqRTSuperMix	4μL
		Reaction liquid in the last step	16μL
Total volume of	20μL

4) Gently beating and mixing by a pipette. The reaction conditions were 37℃for 15min and 85℃for 15sec.

And fourthly, qRT-PCR detection, namely adopting a Real-time PCR method to detect the relative expression quantity of the target gene HK2 and the housekeeping gene action respectively, and calculating the silencing efficiency of the target gene HK2 and the housekeeping gene action. The results showed that the expression level of HK2 in the MSNs-dsHK2 treated group was significantly reduced by more than 55% compared to the control group (fig. 3).

Example 4 in vitro leaf blade inoculation method of Nicotiana benthamiana to detect the inhibitory Effect of MSNs-dsRNA nanoformulations on Phytophthora capsici

Phytophthora capsici strain LT1534 is inoculated in a V8 solid culture medium and cultivated for 2d in a constant temperature incubator at 28 ℃ in a dark place. 10. Mu.L of the MSNs-dsRNA (200 ng/. Mu.L) nano-preparation prepared in example 2 was dropped on the leaf of Nicotiana benthamiana, then a freshly cultured Phytophthora capsici mycelium pellet (diameter: 5 mm) was inoculated at the drop, placed in a 28 ℃ incubator for humidity culture, and after 48 hours, the experimental result was observed. There are 5 groups of treatments, (1) treatment with clear water as control, (2) treatment with a suspension of Mesoporous Silica (MSNs) alone, (3) treatment with a mixture of PEI-MSNs, (4) treatment with MSNs-dsGFP, and (5) treatment with MSNs-dsH K2. 5 leaves were treated for each experiment and repeated 3 times.

As a result, as shown in FIG. 4, the lesion area of treatment group 5 (MSNs-dsHK 2) was significantly smaller than that of control group 1 (Mock), and the lesion area was reduced by 72.6%, while there was no significant difference between treatment groups 2,3, and 4 and control group 1. The result shows that the MSNs-dsRNA nano preparation has remarkable inhibition effect on phytophthora capsici infection.

Example 5 application of MSNs-dsRNA nanoformulations in the prevention and treatment of pepper epidemic disease

Step one, preparing zoospores of phytophthora capsici, namely inoculating phytophthora capsici strain LT1534 into a V8 solid culture medium, and culturing for 2 days in a 28 ℃ constant temperature incubator in a dark place. Fresh mycelia at the edge of the colonies were inoculated into 10mL of V8 liquid medium and cultured at 28℃for 3d. Taking out mycelium blocks, continuously washing 3 times with sterile water until the mycelium turns white, spreading the mycelium in a culture dish, adding 10mL of sterile water, carrying out 24-hour strong light treatment, placing the culture dish under a microscope, observing a large number of sporangia, placing in a4 ℃ refrigerator for 30min, taking out, placing at room temperature for 30min, and filtering to remove the mycelium to obtain phytophthora capsici zoospore. The zoospore suspension was adjusted to a concentration of 10 ⁴ per mL using a hemocytometer for further use.

Step two, tomato seedling treatment, namely sowing tomato seeds after accelerating germination, and taking two true leaves for inoculation experiments after the tomato seeds grow. Clear water, MSNs and MSN-dsGFP are respectively arranged as a control group, MSN-dsHK2 is a treatment group, and 6 pot plants in each group are repeated for 3 times. Spraying a nano preparation containing 10 mug dsRNA on each plant of leaves, moisturizing and shading for 12 hours at 25 ℃, vertically scratching the roots with a surgical knife at a position 0.5cm away from the stem of a tomato seedling to perform root injury treatment, sucking 5mL of zoospore suspension (10 ⁴/mL) with a pipetting gun to perform root irrigation inoculation, continuously performing dark and moisturizing treatment for 24 hours, moisturizing for 2 days at 25 ℃ under normal light (10 hours of light and 14 hours of dark), counting diseased plants, and photographing.

As shown in FIG. 5, the incidence rate of the plants in the control group is more than 85%, the incidence rate of the MSN-dsHK2 in the treatment group is 16.7%, and the prevention and control efficiency is 83.3%. The MSN-dsRNA nano preparation provided by the invention can effectively reduce the occurrence of pepper epidemic disease, and has a wide market application prospect.

In the present specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, and identical and similar parts between the embodiments are all enough to refer to each other.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims (6)

1. The dsRNA is characterized by having a sequence shown in SEQ ID NO.2, wherein the dsRNA sequence is used for inhibiting an HK2 gene, and the nucleotide sequence of the HK2 gene is shown in SEQ ID NO. 1.

2. Use of the dsRNA of claim 1 for the preparation of a nano-formulation for antagonizing phytophthora capsici and controlling phytophthora capsici.

3. A nano preparation for preventing and treating pepper epidemic disease, which is characterized by comprising the dsRNA, mesoporous silica and polyethyleneimine according to the mass ratio of 1:80:12.

4. The nano-preparation for preventing and treating pepper epidemic disease according to claim 3, wherein the concentration of the dsRNA is 1.0mg/mL, the concentration of the mesoporous silica is 1.0mg/mL, and the concentration of the polyethyleneimine is 0.5mg/mL.

5. The use of the nano-preparation according to claim 4 for preparing a chemical preparation for controlling pepper epidemic disease.

6. A method for controlling pepper epidemic disease, characterized in that the nano-preparation of claim 3 is sprayed on pepper leaves.