RNAi nanofomulation for prevention and control of cgmmv

Abstract

The present application provides RNAi nano-preparation, comprising QCS and dsRNA targeting CGMMV. The present application can effectively prevent and control CGMMV.

Description

Background Technology

[0001] Cucumber green mottle mosaic virus (CGMMV) is an important quarantine virus that harms cucurbitaceous crops. It can cause leaf mosaic, mottling, slow plant growth, and fruit rot, severely affecting yield and quality.

[0002] RNA interference (RNAi) pesticides are a potential new type of plant protection product that can specifically silence viral genes and achieve antiviral effects. However, naked dsRNA is easily degraded by nucleases, has poor stability and short duration of action, which limits its practical application.

[0003] Chitosan quaternary ammonium salt (QCS), as a natural cationic polysaccharide nanocarrier material, possesses advantages such as biodegradability, biocompatibility, and non-toxicity. After quaternization modification, the water solubility, positive charge density, and cell penetration of chitosan quaternary ammonium salt are significantly improved, making it an ideal nucleic acid delivery carrier. The applicant has conducted extensive research on CGMMV, especially its gene sequence and RNAi. However, there are no existing reports on using chitosan quaternary ammonium salt to encapsulate dsRNA targeting CGMMV for plant virus control. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this application provides an RNAi nanoformulation containing QCS and dsRNA targeting CGMMV, which can be used for plant virus control, especially for the control of CGMMV, a virus that can infect bottle gourd, melon, watermelon, zucchini, and / or cucumber; QCS is used to carry the dsRNA. The sequence of the dsRNA may be as shown in SEQ ID NO.1 / SEQ ID NO.2. The degree of substitution of the QCS may be 85%-95%, such as 90%; the QCS molecule may be hydroxypropyltrimethylammonium chloride chitosan.

[0005]

[0006] In vivo transcription can be performed using a prokaryotic expression system. The expression vector can be constructed through homologous recombination, and the backbone vector used can be the L4440 vector. For example, DNA containing the target fragment can be ligated to an L4440 vector that has been digested with EcoRI and transformed into HT115(DE3) strain to obtain prokaryotic expression strains containing the recombinant vectors L4440-CGIR and L4440-CGCP, respectively. Example of constructing a prokaryotic expression vector for the CGMMV encoding gene dsRNA (dsIR and dsCP sequences are SEQ ID NO.1 and SEQ ID NO.2, respectively): (1) Extract total RNA from the tissues (such as leaves) of cucurbit crops infected with CGMMV virus, including watermelon, cantaloupe, cucumber, zucchini or bottle gourd, and reverse transcribe it to synthesize cDNA. (2) Based on the viral genome sequence information, two pairs of primers were designed. The primer sequences are as follows: CGIR-F:5′-cgataagcttgatatcgaattcGAGTGGAACGTACCGGTTG-3′ CGIR-R:5′-gatctgatatcatcgatgaattcTATGTGAACCCCGGGGTC-3′ CGCP-F:5′-cgataagcttgatatcgaattcATGGCTTACAATCCGATCACA-3′ CGCP-R:5'-gatctgatatcatcgatgaattcCTAAGCTTTCGAGGTGGTAG-3' (3) Using cDNA as a template and CGIR-F / CGIR-R and CGCP-F / CGCP-R as primers, PCR amplification was performed to obtain two fragments with lengths of 1341bp and 531bp (containing the above template genes IR and CP). (4) Using the homologous recombination strategy, they were ligated to the L4440 vector and transformed into the HT115 (DE3) strain to obtain prokaryotic expression strains containing the recombinant vectors L4440-CGIR and L4440-CGCP, respectively. 2. Example of transcription of dsRNA of CGMMV encoding gene: (1) Each strain obtained in Example 1 above was pre-cultured in LB liquid medium (containing 100 ng / uL Amp and 10 ng / uL Tet), and then induced to culture by adding IPTG to obtain cells expressing dsRNA. (2) Use RNAiso plus or E. coli expression dsRNA extraction and purification kit (magnetic bead method) to extract dsRNA and obtain dsRNA extract containing dsIR or dsCP.

[0007] Optionally, the concentration of Na2SO4 in the chitosan quaternary ammonium salt (QCS)-mediated CGMMV dsRNA nanocarrier delivery system nanoformulation is 10-30 mM, such as 20 mM; the preparation of the nanoformulation may include establishing a mixture of QCS and dsRNA under the condition of 10-30 mM Na2SO4, preferably adding Na2SO4 to a final concentration of 20 mM.

[0008] Optionally, the nano-formulation is prepared by incubating a mixture at 50-60°C, followed by vigorous shaking and then allowing it to stand.

[0009] As an example, the preparation of nano-formulations includes: (1) Weigh 0.02g of QCS and dissolve it completely in 100 mL of H2O to obtain a 0.02% QCS working solution; (2) For the different dsRNA (dsIR, dsCP) extracts prepared in Example 2 above, take 10 mL of 0.02% QCS working solution, add 2 mg of dsRNA extract and 0.8 mL of 0.5 mol / L Na2SO4 in sequence, and make up to 20 mL with H2O.

[0010] (3) After mixing the systems, let them stand at room temperature for 2 min, then incubate them in a 55℃ water bath for 1 min, then quickly transfer them to a high-speed vortex apparatus and shake them violently for 30 s, and let them stand at room temperature for 10 min to form stable nanoparticles QCS-dsIR and QCS-dsCP.

[0011] This application also discloses the control of CGMMV by QCS-dsRNA, which can be formulated into a nanoparticle. The nanoparticle may contain the RNAi nanoparticle and a surfactant, such as polyether-modified heptamethyltrisiloxane, for example, the surfactant Silwet L-77.

[0012] The evaluation process may include: (1) Add Silwet L-77 surfactant (final concentration v / v of 0.02% in pesticide mixture) to QCS-dsIR and QCS-dsCP nanoformulation respectively, and mix thoroughly; (2) Spray them evenly on watermelon leaves, and rub them with CGMMV virus source 1 day later. Collect the inoculated leaves 3 days after inoculation to analyze the virus accumulation. Observe the disease status of plants 10-20 days after inoculation.

[0013] Beneficial effects

[0014] The QCS-dsRNA nanoparticle formulation and its nucleic acid pesticide of the present invention can significantly inhibit the replication and reproduction of CGMMV in the host, reduce its systemic infection capacity, and take effect by foliar spraying without the need for transgenics. At the same time, QCS is completely degradable and leaves no residue, making it safe for crops, the environment, and non-target organisms, and suitable for large-scale promotion. The present application also screened dsRNA and its ratio with QCS. The applicant found that when the ratio is too low, the effect of controlling CGMMV is not good. Attached Figure Description

[0015] Figure 1 Relative virus accumulation after spraying watermelons with QCS-dsIR and QCS-dsCP and inoculating them with CGMMV Figure 2 Symptoms of watermelons after being sprayed with QCS-dsIR and QCS-dsCP and inoculated with CGMMV. Detailed Implementation

[0016] Unless otherwise specified, all experimental and detection methods described below are conventional methods; all reagents and materials mentioned are commercially available unless otherwise specified. The following is an example of a continuous experiment: 1. Construction of a prokaryotic expression vector for the CGMMV encoding gene dsRNA To obtain the template genes IR and CP for transcription of dsIR and dsCP, total RNA was extracted from watermelon leaves infected with CGMMV virus and cDNA was synthesized. The specific operation was performed according to the instructions of RNAsimple Total RNA Extraction Kit (TIANGEN, DP419) and PrimeScript RT reagent Kit with gDNA Eraser (Perfect Real Time) (TAKARA, RR047A). Based on the viral genome sequence information, two pairs of primers were designed, and the primer sequences are as follows: CGIR-F:5′-cgataagcttgatatcgaattcGAGTGGAACGTACCGGTTG-3′ CGIR-R:5′-gatctgatatcatcgatgaattcTATGTGAACCCCGGGGTC-3′ CGCP-F:5′-cgataagcttgatatcgaattcATGGCTTACAATCCGATCACA-3′ CGCP-R:5'-gatctgatatcatcgatgaattcCTAAGCTTTCGAGGTGGTAG-3' PCR amplification was performed using cDNA as a template and CGIR-F / CGIR-R and CGCP-F / CGCP-R as primers to obtain two fragments with lengths of 1341 bp and 531 bp, respectively. The PCR amplification system consisted of: 10 μL 2×Q5 Hot Start Mix, 1 μL each of forward and reverse primers (10 mM), 1 μL cDNA, and H2O to a final volume of 20 μL. The reaction program was: 98℃ for 30 s; 98℃ for 10 s, 60℃ for 20 s, 72℃ for 1 min, 35 cycles; 72℃ for 5 min.

[0017] Using homologous recombination strategy, they were respectively combined with... Eco The L4440 vector, digested with RI, was ligated and transformed into HT115(DE3) strain to obtain prokaryotic expression strains containing recombinant vectors L4440-CGIR and L4440-CGCP, respectively. The homologous recombination system consisted of 2.5 μL NEBuilderHiFi DNA Assembly Master Mix (NEB), 1.5 μL L4440 digestion product, and 1 μL of recovered product, for a total volume of 5 μL. The mixture was thoroughly mixed and incubated at 50℃ for 15 min before transformation and screening.

[0018] 2. Transcription and purification of dsRNA of CGMMV encoding gene Each strain obtained in Experiment 1 was transferred to LB liquid medium (containing 100 ng / uL Amp and 10 ng / uL Tet) and cultured overnight at 37°C and 220 rpm. The next day, the bacterial culture was transferred to new LB liquid medium (containing 100 ng / uL Amp and 10 ng / uL Tet) at a ratio of 1:50 and cultured until the OD value was 0.4-0.6. IPTG solution was added to a final concentration of 0.4 mmol / L and induced at 37°C and 220 rpm for 5 h. The bacterial culture was centrifuged at 6000 rpm for 3 min, the supernatant was discarded, and the bacterial cells were collected. The bacterial cells collected from every 25 ml of bacterial culture were collected in one centrifuge tube for later use. Each 25ml of bacterial culture collected was considered a single sample, and RNA was extracted using RNAiso Plus. Add 1 mL of RNAiso Plus lysis buffer to each sample, vortex vigorously until the cells are fully dispersed and clear, then incubate at room temperature for 5 min. Add 200 μL of chloroform, vortex vigorously, and incubate at room temperature for 5 min. Centrifuge at 12,000 rpm for 15 min at 4 °C, and transfer the supernatant to a new centrifuge tube. Add 500 μL of isopropanol, mix by inverting, and incubate at room temperature for 10 min. Centrifuge at 12,000 rpm for 10 min at 4 °C, discard the supernatant, and add 1 mL of 75% ethanol to resuspend the RNA precipitate. Centrifuge at 12,000 rpm for 5 min at 4 °C, discard the supernatant. After the ethanol has completely evaporated, add 50 μL of RNase-free H2O and dissolve thoroughly to obtain dsRNA extracts of dsIR and dsCP. Determine the concentrations of dsIR and dsCP using a spectrophotometer for later use.

[0019] 3. Establishment of a chitosan quaternary ammonium salt (QCS) mediated CGMMV dsRNA nanocarrier delivery system Weigh 0.02 g of chitosan quaternary ammonium salt (hydroxypropyltrimethylammonium chloride chitosan QCS, Yuanye Biotechnology, catalog number: S26618, degree of substitution 90%) and dissolve it thoroughly in 100 mL of H2O to obtain a 0.02% QCS working solution. For different dsRNA extracts (dsIR, dsCP), take 10 mL of the 0.02% QCS working solution, add 2 mg of dsRNA and 0.8 mL of 0.5 mol / L Na2SO4, and bring the volume to 20 mL with H2O. After mixing the systems thoroughly, let them stand at room temperature for 2 min, then incubate them in a 55℃ water bath for 1 min, and then quickly transfer them to a high-speed vortex mixer and shake vigorously for 30 s. Let them stand at room temperature for 10 min to form stable nanoparticles QCS-dsIR and QCS-dsCP.

[0020] 4. Evaluation of the control effect of QCS-dsRNA on CGMMV Watermelon seeds were germinated and sown, and then raised at approximately 25°C. Once the cotyledons were fully expanded, dsRNA could be sprayed onto the watermelon leaves. 4 μL of Silwet L-77 surfactant (final concentration 0.02%) was added to 20 mL of QCS-dsIR and QCS-dsCP, and the mixture was thoroughly stirred. This solution was then sprayed evenly onto the watermelon leaves until the leaf surface was fully moistened. One day after spraying, the plants were inoculated with CGMMV virus via friction. After inoculation, the plants were cultured at approximately 25°C. Simultaneously, a positive control was established, comparing the CGMMV prevention effects of plants first sprayed with QCS (also using a surfactant) without dsRNA and then inoculated with the virus.

[0021] Leaves were collected on day 3 post-inoculation, and virus accumulation was analyzed using qRT-PCR. Disease incidence was observed and recorded 10-20 days after inoculation. Analysis showed that, compared with the positive control group, leaves treated with QCS-dsIR and QCS-dsCP on day 3 post-inoculation had significantly lower virus accumulation levels (75.1% and 69.3%, respectively). Figure 1 An investigation revealed that all plants in the positive control group developed symptoms on day 10 post-inoculation, while plants treated with QCS-dsIR and QCS-dsCP showed no symptoms until day 17 post-inoculation, at which point a few plants began to show symptoms. By then, the positive control group plants were already severely affected, exhibiting obvious mosaic patterns and stunted growth. Figure 2 Statistical analysis showed that the disease index of plants treated with QCS-dsIR and QCS-dsCP decreased, with the disease index of the QCS-dsIR group being 33.3, indicating a control effect of 57.9% on CGMMV. Exogenous spraying of QCS-dsIR and QCS-dsCP can inhibit virus replication, delay plant disease development, and reduce the severity of plant disease.

Claims

1. RNAi nanoformulations containing dsRNA as shown in SEQ ID NO. 1-2 supported by chitosan quaternary ammonium salt QCS.

2. The nano-formulation as described in claim 1, characterized in that, The QCS is hydroxypropyltrimethylammonium chloride chitosan.

3. Nanopharmaceutical formulation according to the preceding claim, characterized in that, The degree of substitution of the QCS is 85%-95%, such as 90%.

4. Nanoforumulation according to any of the preceding claims, characterized in that The mass ratio of dsRNA to the QCS used to carry it is 0.5-1.5:1, such as 1:

1.

5. Nanofomulation according to any of the preceding claims, characterized in that dsRNA is obtained through in vivo gene transcription. The mass ratio of the obtained dsRNA extract to the QCS used to carry it is 0.5-1.5:1, such as 1:

1.

6. Nanofomulation according to any of the preceding claims, characterized in that The concentration of Na2SO4 in the nano-formulation is 10-30 mM, such as 20 mM.

7. Nanofomulation according to any of the preceding claims, characterized in that The nano-formulation was prepared under conditions of 10-30 mM Na2SO4, preferably 20 mM Na2SO4.

8. A pharmaceutical preparation comprising any of the nanoformulations described in the prior claims.

9. The medicament according to the preceding claim, characterized in that it is a vaccine. The agent contains surfactants, such as polyether-modified heptamethyltrisiloxane.

10. The medicament of any preceding claim, characterised in that, The agent contains the surfactant Silwet L-77.

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