DsRNA, preparation method and application thereof, and insecticide
By synthesizing and injecting dsRNA of the V-ATPase D gene of the Eastern armyworm, RNA interference technology was used to fill the technological gap in the control of the Eastern armyworm, achieving a highly efficient and green pest control effect.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-31
- Publication Date
- 2026-03-31
AI Technical Summary
The lack of existing technologies for the design and control of dsRNA targeting the V-ATPase D subunit of the Eastern armyworm has led to severe damage from the Eastern armyworm and has impacted food security.
We designed and synthesized dsRNA of the V-ATPase D gene of the Eastern Armyworm. Using RNA interference technology, we injected the dsRNA into the Eastern Armyworm to inhibit the expression of the target gene, leading to the death of the pest.
It significantly increased the mortality rate of the Eastern armyworm and reduced the survival rate of the pest, thus achieving green control of the Eastern armyworm.
Smart Images
Figure CN121759458A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the fields of biotechnology and agricultural pest control, specifically to a dsRNA, its preparation method and application, and an insecticide. Background Technology
[0002] Eastern slime worm, Latin name Mythimna separata As a representative pest of the Noctuidae family in the Lepidoptera order, the insect is characterized by its strong migratory ability, high reproductive capacity, and voracious feeding behavior. It primarily damages gramineous crops such as rice, corn, and wheat. During the larval stage, it can devour crop leaves and bare stalks in a short period, leading to yield reductions of over 30%, and in severe cases, complete crop failure. Its periodic outbreaks have become a significant threat to my country's food security, necessitating the development of efficient and green new control technologies. Vacuole-type H⁺-ATPase is a transmembrane protein complex widely found in eukaryotes, composed of multiple subunits. It is responsible for catalyzing ATP hydrolysis and driving proton transmembrane transport, playing a central role in insect midgut digestion, ion homeostasis maintenance, and cell signal transduction. Vacuole-type H⁺-ATPase is abbreviated as V-ATPase.
[0003] RNA interference (RNAi) technology mediates the degradation of homologous mRNA through double-stranded RNA, achieving specific silencing of target genes. It boasts advantages such as target specificity, a clear mechanism of action, and good environmental compatibility, and is hailed as the "third revolution in pesticide development." RNA interference is abbreviated as RNAi, and double-stranded RNA is abbreviated as dsRNA. However, to date, there are no reports on the design and application of dsRNA targeting the V-ATPase D subunit of the Eastern armyworm. Summary of the Invention
[0004] To address the aforementioned technical problems, this invention provides dsRNA, its preparation method and application, and an insecticide.
[0005] A dsRNA for controlling the Eastern armyworm, wherein the nucleotide sequence of the sense strand of the dsRNA is shown in SEQ ID NO.7, and the nucleotide sequence of the antisense strand is the reverse complementary sequence of the sequence shown in SEQ ID NO.7.
[0006] This invention utilizes an RNA interference fragment from the V-ATPase D gene of *Armoria spp.* to provide dsRNA synthesized from this RNA interference fragment. Injecting the dsRNA provided by this invention into *Armoria spp.* significantly increases the mortality rate of the insects.
[0007] A method for preparing the dsRNA described above, using cDNA from *Armoria serratifolia* as a template, amplifying with primers shown in SEQ ID NO.3~SEQ ID NO.4 to obtain template 1, and amplifying with primers shown in SEQ ID NO.5~SEQ ID NO.6 to obtain template 2; The dsRNA was synthesized by in vitro transcription using template 1 and template 2.
[0008] The application of the dsRNA in the preparation of insecticides for controlling the Eastern armyworm.
[0009] Preferably, the insecticide is composed of the dsRNA and excipients.
[0010] Preferably, the excipients include a diluent.
[0011] Preferably, the diluent comprises water.
[0012] Preferably, the insecticide is used to reduce the survival rate of the Eastern Armyworm and inhibit its body length.
[0013] An insecticide for controlling the Eastern armyworm, comprising the dsRNA and water.
[0014] Preferably, the concentration of dsRNA in the insecticide is 500 ng / μL.
[0015] The present invention relates to the V-ATPase D gene of the Eastern Armyworm, the nucleotide sequence of which is shown in SEQ ID NO.1, with a nucleotide length of 741 bp; and the amino acid sequence of which is shown in SEQ ID NO.2, with a sequence length of 246 amino acids.
[0016] The application of the V-ATPase D gene of the Eastern Armyworm described in this invention in RNAi-mediated pest control.
[0017] The application of dsRNA in the control of the Eastern armyworm involves RNA interference in the larvae of the Eastern armyworm, leading to their death.
[0018] Preferably, the synthesized dsRNA is injected into the larvae of *Armoria spp.* at a dose of 500 ng / larva. After injection, it can significantly inhibit the expression of the MsV-ATPase D gene and significantly reduce the survival rate of *Armoria spp.*
[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides an RNA interference fragment of the V-ATPase D gene of the armyworm *Armoria spp.*, and the application of dsRNA synthesized from this RNA interference fragment in the control of *Armoria spp.*. RNA interference causes the death of *Armoria spp.* larvae, thus achieving green control of *Armoria spp.* Attached Figure Description
[0020] Figure 1The phenotypic characteristics of test insects 24h and 48h after injection of dsRNA of the V-ATPase D gene are shown. dsMsV-ATPase D represents the dsRNA of the injected V-ATPase D gene.
[0021] Figure 2 The figure shows the effect of injecting V-ATPase D gene dsRNA on the mortality rate of oriental armyworm larvae 24h and 48h after injection. In the figure, A represents the mortality rate 24h after injection, B represents the mortality rate 48h after injection, ds GFP is the control, ds MsV-ATPase D is the treatment group, and ds MsV-ATPase D represents the injection of V-ATPase D gene dsRNA. * represents P<0.05, ** represents P<0.01.
[0022] Figure 3 The effect of injecting dsRNA of the V-ATPase D gene on V-ATPase D gene mRNA expression was investigated at 24 h and 48 h. In this study, A represents the gene expression level at 24 h after injection, B represents the gene expression level at 48 h after injection, dsGFP represents the control group, dsMsV-ATPase D represents the treatment group, and dsMsV-ATPase D represents the injected V-ATPase D gene dsRNA. * represents P<0.05, and ** represents P<0.01.
[0023] Figure 4 Gel electrophoresis using dsRNA as a template.
[0024] Figure 5 Gel electrophoresis of purified dsRNA.
[0025] Figure 6 This is a diagram of dsRNA synthesis. Detailed Implementation
[0026] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods.
[0027] The nucleotide sequence of the Mythimna separata V-ATPase D gene of the present invention is: ATGTCTGGGAAAGATAGATTAGCGATTTTCCCTTCTCGGGGTGCTCAGATGTTGATAAAGGCACGTCTGGCTGGTGCACAGAAAGGCCATGGTCTCCTGAAGAAGAAGGCTGACGCCCTCCAAGTGAGGTTCCGTATGATTCTCAGCAAAATTATTGAGACAAAAACCCTTATGGGTGAGGTGATGAAGGAAGCAGCATTCTCTCTGGCTGAGGCCAAGTTCACAACTGGAGATTTCAACCAGGTGGTACTGCAAAACGTTACCAAGGCTCAGATCAAGATCCGCTCAAAGAAAGACAATGTTGCTGGTGTGACCCTCCCAATCTTCGAGTCTTACACAGACGGTACTGATACATACGAGCTAGCCGGTCTGGCCCGAGGAGGTCAGCAGCTCACCAAGCTGAAGAAGAACTTCCAGAGTGCTGTTAAACTGCTGGTTGAGTTGGCTTCCCTGCAGACGTCATTCGTGACTCTTGATGAGGTCATCAAGATCACCAACAGGCGTGTCAACGCTATTGAGCACGTAATCATTCCTCGATTGGAGCGTACTCTGGCGTACATCATCTCGGAGTTGGACGAGCTCGAGCGTGAGGAGTTCTACCGGCTGAAGAAGATCCAGGACAAGAAGAAGATCATCAAGGATAAGGCGGAAGCGCGCAAACAAGCGATGTTGCTAGCCGGTCAAGACCTGCAAGACTCCGCGAACTTGTTGGACGAGGGCGACGAAGACCTGCTGTTCTAA, denoted as SEQ ID NO.1.
[0028] The amino acid sequence of the V-ATPase D gene of the Eastern Armyworm of this invention is: MSGKDRLAIFPSRGAQMLIKARLAGAQKGHGLLKKKADALQVRFRMILSKIIETKTLMGEVMKEAAFSLAEAKFTTGDFNQVVLQNVTKAQIKIRSKKDNVAGVTLPIFESYTDGTDTYELAGLARGGQQLTKLKKNFQSAVKLLVELASLQTSFVTLDEVIKITNRRVNAIEHVIIPRLERTLAYIISELDELEREEFYRLKKIQDKKKIIKDKAEARKQAMLLAGQDLQDSANLLDEGDEDLLF, denoted as SEQ ID NO.2.
[0029] I. Extraction of total RNA from *Armoria spp.* Ten third-instar larvae of *Armoria serratifolia* stored at -80°C were selected as one sample group, with three replicates. Total RNA was extracted from the *Armoria serratifolia* samples using the Vazyme RNA isolater Total RNA Extraction Reagent kit. The entire experiment was performed on ice to prevent RNA degradation. Rubber gloves and masks were worn to prevent sample contamination, and the extraction process was strictly performed according to the kit instructions. The specific steps are as follows:
[0030] (1) Place the sample in a tissue homogenizer, add 1 mL of RNA isolater and homogenize quickly on ice until homogenized and there are no obvious large pieces of tissue. After thorough homogenization, pour the liquid into a sterile 1.5 mL centrifuge tube and centrifuge at 11200 rpm and 4℃ for 5 min in a high-speed refrigerated centrifuge. Transfer the supernatant to a new centrifuge tube.
[0031] (2) Add 1 / 5 volume of chloroform to the supernatant, shake vigorously for 15 seconds, and let stand on ice for 5 minutes. Centrifuge at 11200 rpm and 4℃ for 15 minutes, and carefully aspirate the upper aqueous phase into a new centrifuge tube.
[0032] (3) Add an equal volume of isopropanol, invert the tube to mix, and let stand on ice for 10 min. Centrifuge at 11200 rpm and 4℃ for 10 min. A white precipitate will appear at the bottom of the centrifuge tube. Discard the supernatant.
[0033] (4) Add 1 mL of 75% ethanol to the tube. The 75% ethanol is prepared with sterile water. Gently tap the bottom of the tube to suspend the precipitate. Invert the tube several times and let it stand on ice for 3 min. Centrifuge at 11200 rpm and 4℃ for 5 min and discard the supernatant.
[0034] (5) Repeat step (4) three times.
[0035] (6) Dry the precipitate in a clean environment at room temperature for 4 min, add 20 μL of RNase-free ddH2O, and shake thoroughly to dissolve the precipitate.
[0036] (7) Use a micro-ultraviolet spectrophotometer to detect the absorbance of the sample at 260 and 280 nm, and calculate the sample concentration and purity. Store the RNA sample in a -80℃ freezer until needed.
[0037] II. Reverse transcription to synthesize cDNA The total RNA extracted from *Armoria serratifolia* was used to remove genomic DNA from the sample, followed by reverse transcription to synthesize cDNA. The entire experiment was performed on ice, with rubber gloves and masks worn to prevent sample degradation and contamination. The procedure was strictly followed according to the kit instructions. Specific steps are as follows:
[0038] (1) Quantitative dilution of RNA: The RNA sample was diluted to 350 ng / μL with RNase-free ddH2O.
[0039] (2) Genomic DNA removal: Add 2.86 μL of template RNA, 4 μL of 4×gDNA wiper Mix, and 9.14 μL of RNase-free ddH2O to a 100 μL RNase-free centrifuge tube, vortex to mix, centrifuge, and incubate at 42℃ for 2 min in a constant temperature mixer.
[0040] (3) Reverse transcription reaction: Add 4 μL of 5×HiScript II qRT SuperMix II, vortex to mix, centrifuge, react at 50℃ for 15 min, increase the temperature to 85℃, react for 5 s. Store the cDNA sample at -20℃ for later use.
[0041] III. In vitro PCR amplification of dsRNA template F: taatacgactcactataggg GGCTCAGATCAAGATCCGCT, SEQ ID NO.3; R: TTGACCGGCTAGCAACATCG, SEQ ID NO.4; F: GGCTCAGATCAAGATCCGCT, SEQ ID NO.5; R: taatacgactcactataggg TTGACCGGCTAGCAACATCG, SEQ ID NO.6; Using cDNA as a template, template 1 was obtained by PCR amplification using SEQ ID NO.3~SEQ ID NO.4, and template 2 was obtained by PCR amplification using SEQ ID NO.5~SEQ ID NO.6.
[0042] The nucleotide sequences of template 1 and template 2 are inversely complementary. The nucleotide sequence of template 1 is: GGCTCAGATCAAGATCCGCTCAAAGAAAGACAATGTTGCTGGTGTGACCCTCCCAATCTTCGAGTCTTACACAGACGGTACTGATACATACGAGCTAGCCGGTCTGGCCCGAGGAGGTCAGCAGCTCACCAAGCTGAAGAAGAACTTCCAGAGTGCTGTTAAACTGCTGGTTGAGTTGGCTTCCCTGCAGACGTCATTCGTGACTCTTGATGAGGTCATCAAGATCACCAACAGGCGTGTCAACGCTATTGAGCACGTAATCATTCCTCGATTGGAGCGTACTCTGGCGTACATCATCTCGGAGTTGGACGAGCTCGAGCGTGAGGAGTTCTACCGGCTGAAGAAGATCCAGGACAAGAAGAAGATCATCAAGGATAAGGCGGAAGCGCGCAAACAAGCGATGTTGCTAGCCGGTCAA, denoted as SEQ ID NO.7.
[0043] PCR reaction system: On ice, add 25 μL of 2×E TaqMaster Mix (Dye), 19 μL of RNase-free ddH2O, 2 μL of cDNA template, 1 μL of forward primer, and 1 μL of reverse primer to a 100 μL RNase-free centrifuge tube, for a total volume of 50 μL. Vortex to mix, centrifuge, and place in a PCR instrument for reaction.
[0044] PCR reaction conditions: After a 2-minute pre-denaturation reaction at 94°C, the reaction was cycled 40 times. The cycle consisted of a 30-second denaturation reaction at 94°C, a 30-second annealing reaction, a 2-minute extension reaction at 72°C, and a final extension reaction at 72°C for 2 minutes. The reaction products were stored at 4°C.
[0045] IV. Gel Electrophoresis Preparation of TBE buffer: Weigh 0.375g of disodium ethylenediaminetetraacetate dihydrate, 5.4g of tris(hydroxymethyl)aminomethane and 2.75g of boric acid, dissolve them in deionized water and bring the volume to 1L.
[0046] 1. Gel electrophoresis (1) Gel preparation: Weigh 0.3g of agarose, add 30mL of TBE buffer to dissolve in an Erlenmeyer flask, and heat in a microwave oven for 1min. After cooling to room temperature, add 15μL of 10mg / mL EB staining agent. Gently shake to mix, assemble the gel plate, insert the comb, pour the mixed liquid into the gel plate, remove air bubbles, and let it air dry naturally at room temperature on the table. Remove the comb after it has set.
[0047] (2) Spotting: Place the sample in a TBE buffer electrophoresis tank, add TBE buffer until it covers the gel, add 5 μL of marker to the second well, and add 6 μL of PCR product to the remaining wells.
[0048] (3) Electrophoresis reaction conditions: current 80mA, voltage 120V, so that the sample moves from the negative electrode to the positive electrode. When the sample moves to 2 / 3 of the gel, the electrophoresis is stopped.
[0049] 2. Gel imaging The position and brightness of the PCR product bands were observed using an imaging system, and the results are as follows: Figure 4 As shown, the appearance of a single bright band with the band position consistent with the primer size indicates that the experiment was successful, and the product was sent to Shanghai Sangon Biotech Co., Ltd. for sequencing.
[0050] V. Purification of products from in vitro PCR amplification using dsRNA template The dsRNA PCR in vitro amplification products were purified using the Vazyme FastPure Gel DNA Extraction Mini Kit. 80 mL of anhydrous ethanol was added to Buffer GW and mixed thoroughly. Buffer GDP should be free of precipitate; if precipitate is present at the bottom of the vial, heat treatment at 50°C is performed. This experiment used the PCR reaction solution for purification and recovery; the specific operational steps are as follows:
[0051] (1) Transfer template 1 and template 2 to sterile 1.5 mL centrifuge tubes respectively, add RNase-free ddH2O to make up to 100 μL, add 500 μL of Buffer GDP and mix well.
[0052] (2) Place the adsorption column in the collection tube, transfer the above liquid into the adsorption column, centrifuge at 10,000 rpm and 4°C for 1 min in a high-speed refrigerated centrifuge, and discard the filtrate.
[0053] (3) Place the adsorption column back into the collection tube, add 700 μL of Buffer GW into the adsorption column, invert and mix well, centrifuge at 12000 rpm and 4℃ for 1 min, and discard the filtrate.
[0054] (4) Repeat step (3).
[0055] (5) Place the adsorption column back into the collection tube, centrifuge at 12000 rpm and 4℃ for 2 min, and discard the collection tube.
[0056] (6) Place the adsorption column in a sterile 1.5 mL centrifuge tube, add 20 μL of Elution Buffer to the center of the adsorption column, let stand for 2 min, centrifuge at 12000 rpm and 4 °C for 1 min, and discard the adsorption column.
[0057] (7) Use a micro-ultraviolet spectrophotometer to detect the absorbance of the sample at 260 and 280 nm, calculate the sample concentration, and store the purified products of template 1 and template 2 at -20℃.
[0058] VI. In vitro synthesis of dsRNA dsRNA was synthesized in vitro using the Vazyme T7 RNAi Transcription Kit, with 0.5 μg each of the purified products of template 1 and template 2.
[0059] dsRNA synthesis reaction system: On ice, add 4 μL of purified template 1, 4 μL of purified template 2, 8 μL of NTP Mix, 2 μL of 10×Transcription Buffer, and 2 μL of T7Enzyme Mix to a 100 μL RNase-free centrifuge tube. After adding the samples, mix well and centrifuge. Incubate at 37°C for 2 h in a PCR instrument to obtain the synthesized product.
[0060] Two-enzyme digestion system: Dilute 100 U / μL RNase T1 to 10 U / μL with RNase T1 Dilution Buffer, preparing fresh each time. On ice, add 20 μL of the synthesized product, 2 μL of diluted RNase T1, 1 μL of DNase I, and 17 μL of RNase-free ddH2O to a 100 μL RNase-free centrifuge tube. Mix well, centrifuge, and incubate at 37°C for 30 min to obtain dsRNA. Store the dsRNA at -20°C. The dsRNA synthesis diagram is shown below. Figure 6 As shown, the nucleotide sequence of the dsRNA sense strand is shown in SEQ ID NO.7, and the nucleotide sequence of the antisense strand is the reverse complementary sequence of the sequence shown in SEQ ID NO.7.
[0061] VII. dsRNA Purification (1) Transfer the synthesized dsRNA to a sterile 1.5 mL centrifuge tube, add an equal amount of phenol / chloroform 25:24:1 extraction buffer, centrifuge at 12000 rpm and 4℃ for 5 min in a high-speed refrigerated centrifuge, and aspirate the supernatant to a new centrifuge tube.
[0062] (2) Add an equal amount of isopropanol, invert the tube to mix, and let stand on ice for 5 minutes. Centrifuge at 12000 rpm and 4℃ for 5 minutes. A white precipitate will appear at the bottom of the centrifuge tube. Discard the supernatant.
[0063] (3) Add 1 mL of 75% ethanol to the tube. The 75% ethanol is prepared by using sterile water as the solvent. Gently tap the bottom of the tube to suspend the precipitate. Invert the tube several times and let it stand on ice for 3 min. Centrifuge at 12000 rpm and 4℃ for 5 min and discard the supernatant.
[0064] (4) Repeat step (3) three times.
[0065] (5) Dry the precipitate in a clean environment at room temperature for 4 min, add 20 μL of RNase-free ddH2O, and shake thoroughly to dissolve the precipitate.
[0066] (6) Use a micro-ultraviolet spectrophotometer to detect the absorbance of the sample at 260 and 280 nm, calculate the sample concentration and dilute it to 500 ng / μL to obtain purified dsRNA, which is stored at -20℃ for later use.
[0067] 8. Gel electrophoresis of purified dsRNA Mix 10 μL of purified dsRNA with 2 μL of 6× Loading Buffer, centrifuge, and load the sample into a TBE buffer electrophoresis tank. Add TBE buffer until the gel is submerged. Add 5 μL of marker to the second well and 6 μL of the mixture to the remaining wells.
[0068] The position and brightness of the PCR product bands were observed using an imaging system, and the results are as follows: Figure 5 As shown, the appearance of a single bright band with the band position consistent with the primer size indicates that the experiment was successful, and the product was sent to Shanghai Sangon Biotech Co., Ltd. for sequencing.
[0069] IX. In vitro injection and phenotypic observation of dsRNA 1 μL of purified dsRNA at a concentration of 500 ng / μL was drawn using a microsyringe and injected into the abdomen of the second segment of third-instar *Armoria spp.* using RNase-free ddH2O. Twenty larvae were used as a group, while the control group was injected with dsGFP. The experiment was repeated three times. Larvae were fed normally after injection. Mortality rates and phenotypic characteristics were recorded at 24 h and 48 h after injection.
[0070] The results are as follows Figure 1 , Figure 2 As shown in Table 1. Figure 2 In the middle A group, the mortality rate of *Oriental armyworm* 24 hours after injection was 36.67%. Figure 2 The mortality rate of *Oriental Armyworm* in the control group (B) was 43.33% 48 hours after injection, which was significantly different from the control group.
[0071] Table 1: Mortality, body length, and weight of third-instar larvae of *Armorium orientalis* 24 h and 48 h after gene silencing. Note: Different lowercase letters in the same column during the same time period indicate significant differences between treatments at the 0.05 level.
[0072] Meanwhile, the changes in pH values of the midgut and hemolymph of the test worms 24 hours after injection were statistically analyzed, and the results are shown in Table 2.
[0073] Table 2: Changes in pH values of the midgut and hemolymph after RNAi interference 10. RNAi efficiency detection After injecting dsRNA into third-instar larvae of *Armoria serratifolia* for 24 and 48 hours, 10 surviving larvae were collected from the experimental and control groups, respectively, for total RNA extraction and cDNA synthesis. Using β-actin as an internal reference gene, the relative expression level of the *Armoria serratifolia* V-ATPase gene was detected using RT-qPCR.
[0074] qRT-PCR assay: Using β-actin as an internal reference gene, qRT-PCR experiments were performed with three biological replicates and three technical replicates.
[0075] Prepare a 20 μL qRT-PCR reaction mixture in a 96-well plate: add 2 μL cDNA template, 0.4 μL forward primer, 0.4 μL reverse primer, 10 μL 2×ChamQ Universal SYBR qPCRMaster Mix, and 7.2 μL RNase-free ddH2O to each well. Centrifuge the entire plate and place it in a qRT-PCR instrument for reaction.
[0076] qRT-PCR reaction conditions: After a 30-s pre-denaturation reaction at 95℃, the reaction was cycled 40 times. The cycle consisted of 10 s denaturation at 95℃ followed by 30 s annealing at 60℃. The melting curve was set to the instrument's default program. After the reaction, the melting curve, amplification curve, and Ct value were observed. (Use 2−∆∆) Ct The relative expression levels were calculated using the method described above, and a bar chart was plotted using Prism 10.
[0077] The results are as follows Figure 3 As shown, compared with the control group, Figure 3 In sample A, the expression level of the MsV-ATPase D gene decreased by 62.93% 24 hours after injection. Figure 3 Gene expression levels decreased by 88.04% 48 hours after injection in B, demonstrating a significant silencing effect.
[0078] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.
[0079] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.
[0080] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.
Claims
1. A dsRNA for controlling the oriental armyworm, characterized by comprising a sequence represented by any one of SEQ ID NOs: 1 to 3. The nucleotide sequence of the sense strand of the dsRNA is shown as SEQ ID NO. 7, and the nucleotide sequence of the antisense strand is the reverse complement of the sequence shown as SEQ ID NO.
7.
2. A method of making the dsRNA of claim 1, characterized in that, The template 1 is obtained by amplification using the cDNA of the oriental armyworm as a template and the primers shown as SEQ ID NO. 3~SEQ ID NO. 4, and the template 2 is obtained by amplification using the cDNA of the oriental armyworm as a template and the primers shown as SEQ ID NO. 5~SEQ ID NO.
6. The dsRNA is synthesized by in vitro transcription using the template 1 and the template 2.
3. The use of the dsRNA of claim 1 in the preparation of an insecticide for controlling the oriental armyworm.
4. Use according to claim 3, characterized in that, The insecticide consists of the dsRNA and an auxiliary material.
5. Use according to claim 4, characterized in that, The auxiliary material includes a diluent.
6. Use according to claim 4, characterized in that, The diluent includes water.
7. Use according to claim 4, characterized in that, The insecticide is used to reduce the survival rate of the oriental armyworm and inhibit the body length of the oriental armyworm.
8. An insecticide for controlling the oriental armyworm, characterized by comprising the compound of claim 1. The insecticide is obtained by mixing the dsRNA of claim 1 and water.
9. The insecticide of claim 8, wherein, In the insecticide, the concentration of the dsRNA is 500 ng / μL.