Preparation method of artificial defective interfering RNA and application thereof in anti-cucumber mosaic virus
By designing a specific artificial defective interfering RNA (R3-ADI), the problem of the inability to effectively inhibit CMV accumulation in existing technologies was solved, and an effective inhibition effect on CMV was achieved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- ZHEJIANG SCI-TECH UNIV
- Filing Date
- 2026-04-27
- Publication Date
- 2026-05-29
AI Technical Summary
Existing technologies cannot effectively suppress the accumulation of cucumber mosaic virus (CMV), and there is a lack of specific artificial defective interference RNA strategies targeting CMV.
An artificial defective interfering RNA (R3-ADI) was designed, whose structure includes the 5' UTR, IGR, CP open reading frame, and 3' UTR of CMV genomic RNA3 from the 5' end to the 3' end. An R3 mutant clone missing the MP open reading frame was constructed by site-directed mutagenesis PCR technology and named pCB301-R3-ADI. The cauliflower mosaic virus 35S promoter and NOS terminator were added to the recombinant expression vector.
It effectively inhibits the accumulation of CMV virus in plants and significantly reduces the expression of genomic RNA and protein of chimeric and wild-type CMV viruses, thus achieving an antiviral effect against CMV.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology, and in particular relates to a method for preparing artificial defective interfering RNA and its application in the fight against cucumber mosaic virus. Background Technology
[0002] Cucumber mosaic virus (CMV) is a typical member of the genus Cucumber mosaicvirus in the family Bromeliidae. As one of the plant RNA viruses with the widest range of infecting hosts, it can infect more than 1,200 species of monocotyledonous and dicotyledonous plants, covering vegetables, flowers, fruit trees and economic crops, seriously endangering agricultural production. Of particular note is that CMV is a dominant virus in vegetable crops. Artificial defective interfering RNA technology has been shown to effectively inhibit Tomato dwarf virus (J Virol. 1999;73(6):5070-5078). However, it is still unknown whether similar strategies can be used in the field of anti-CMV. Due to the significant differences in genome structure and replication mechanism among different viruses (especially those with distant evolutionary relationships), the required replication elements are also fundamentally different, and it is not possible to simply borrow or apply previous strategies. Instead, it is necessary to specifically design artificial interfering RNA structures based on the characteristics of different viruses, while retaining the necessary replication elements, so as to achieve the purpose of antiviral treatment. Summary of the Invention
[0003] The technical problem to be solved by the present invention is to provide an artificial defective interfering RNA that is resistant to CMV and its uses.
[0004] To address the aforementioned technical problems, this invention provides an artificial defective interfering RNA (artificial defective interfering RNA for interfering with CMV viral replication, R3-ADI), which includes the following elements (containing cis-acting elements required for CMV genome replication) sequentially from the 5' end to the 3' end: the 5' UTR (5'UTR) of CMV genomic RNA3, IGR, CP open reading frame, and the 3' UTR sequence (3'UTR).
[0005] That is, from the 5' end to the 3' end, the sequence includes the 5' untranslated region (UTR), the intergenic region (IGR), the CP open reading frame, and the 3' UTR sequence of CMV genomic RNA3.
[0006] The present invention also provides a recombinant DNA (DNA fragment) encoding artificial defective interfering RNA, comprising the sequence shown in SEQ ID NO:1.
[0007] The present invention also provides a recombinant expression vector containing the above-mentioned recombinant DNA.
[0008] As an improvement to the recombinant expression vector of the present invention, the recombinant expression vector further contains the cauliflower mosaic virus 35S promoter sequence, hammerhead ribozyme sequence, and NOS terminator sequence.
[0009] As a further improvement to the recombinant expression vector of the present invention, the pCB301-R3-ADI vector is provided, the sequence of which is shown in SEQ ID NO:2.
[0010] This invention utilizes the pCB301-Fny3 vector to construct an R3 mutant clone lacking the MP open reading frame via site-directed mutagenesis PCR technology, named pCB301-R3-ADI.
[0011] The present invention also provides the application of the above-mentioned artificial defective interfering RNA, recombinant DNA (DNA fragment), and recombinant expression vector in the fight against cucumber mosaic virus (anti-CMV infection).
[0012] It should be noted that although artificially designed defective interfering RNAs have been reported to be effective against viruses such as Tomato dwarf Virus (CMV), the specific defective interfering RNA structure that can inhibit the accumulation of a particular virus remains unknown due to the significant differences in the genomic structure and replication mechanisms of different viruses. CMV, as one of the most economically damaging viruses worldwide, currently lacks reported effective defective interfering RNAs. This invention, through analysis of the genomic structure and replication mechanism of CMV, develops an artificially designed defective interfering RNA vector that can effectively inhibit CMV accumulation in plants, thereby achieving antiviral effects.
[0013] In summary, this invention specifically designs artificially defective interfering RNA with anti-CMV effects by retaining essential replication elements based on the characteristics of the CMV virus's own genome structure. Attached Figure Description
[0014] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.
[0015] Figure 1 A schematic diagram of R3-ADI;
[0016] Figure 1 middle: (A) is a schematic diagram of the wild-type CMV RNA3 (R3) structure; (B) is a schematic diagram of the modified CMV R3-ADI structure, where the dashed line represents the missing wild-type R3 corresponding MP open reading frame in the R3-ADI.
[0017] Figure 2 This is a graph showing the detection of the activity of the pCB301-R3-ADI vector;
[0018] Figure 2 middle: (A) is a Northern Blot result for detecting CMV genome and C3-ADI accumulation.
[0019] (B) is a Western Blot result for detecting CP protein accumulation.
[0020] Figure 3 This is a detection graph of C3-ADI against chimeric CMV-eGFP virus in Example 3;
[0021] Figure 3 middle: (A) is a fluorescence image of a typical leaf under UV light three days after inoculation with CMV-eGFP+C3-ADI mixture; (B) Northern Blot results showing the accumulation of C3-ADI, C3-eGFP and subgenomic RNA4 in inoculated leaves three days after CMV-eGFP+C3-ADI mixed inoculation.
[0022] Figure 4 This is a Northern Blot result of the detection of CMV accumulation in the leaves of the wild-type CMV+C3-ADI mixed inoculation system three days after Example 4. Detailed Implementation
[0023] To facilitate understanding of the present invention, a more complete description is provided below, along with preferred embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to make the disclosure of the present invention more thorough and complete.
[0024] Artificial defective interfering RNA in embodiments of the present invention, such as Figure 1 As shown in (B), the following elements are sequentially included from the 5' end to the 3' end: the 5' untranslated region (5'UTR) of CMV genomic RNA3, the intergenic region (IGR), the CP open reading frame, and the 3' UTR sequence.
[0025] CMV belongs to the genus Cucumovirus of the family Bromoviridae. This virus is a triadic positive-sense RNA virus with three genomic RNAs, named RNA1, RNA2, and RNA3. RNA3 encodes the movement protein (MP) and the coat protein (CP), separated by the intergenic spacer region (IGR).
[0026] Based on the characteristics of CMV genome replication, this invention modifies its genome RNA3 while retaining essential replication elements, thereby obtaining artificial defective interfering RNA (R3-ADI) that interferes with CMV viral replication. By measuring the changes in the accumulation of CMV-derived RNA1, RNA2, and RNA3 in the R3-ADI treatment group and the control group, it is verified that it can effectively inhibit the accumulation of wild-type and chimeric CMV viruses, respectively, thus achieving the antiviral purpose.
[0027] The invention will now be described in further detail with reference to specific examples.
[0028] Example 1: Preparation of Artificial Defective Interference RNA Vector
[0029] 1. Constituent elements of ADI RNA
[0030] RNA3 in the CMV genome, such as Figure 1 As shown in (A) of the diagram. This invention modifies the CMV genome RNA3 to construct a sequence comprising the following elements from the 5' end to the 3' end: the 5' UTR (i.e., 5' UTR), IGR, CP open reading frame, and 3' UTR sequence (i.e., 3' UTR). All of these elements are derived from the CMV-Fny strain (Genbank accession no. D10538.1) and are CMV sequences themselves, as shown in (A). Figure 1 As shown in (B) in the diagram, it is named R3-ADI. The DNA sequence of R3-ADI is shown in SEQ ID NO:1.
[0031] 2. Construction of R3-ADI vector
[0032] Using the pCB301-Fny3 vector (Chinese Agricultural Science, 2011, 44(14): 3060-3068.), an R3 mutant clone lacking the MP open reading frame was constructed by site-directed mutagenesis PCR technology and named pCB301-R3-ADI.
[0033] The specific construction method is as follows:
[0034] 2.1) Using mutant PCR technology, plasmid pCB301-Fny3 was used as a template and mutant PCR amplification was performed using primers R3-ΔMP-F / R3-ΔMP-R to obtain the PCR product R3-ADI (DNA).
[0035] The primer sequences used for the mutant PCR are as follows:
[0036] R3-ΔMP-F: 5'-CGAGGCCACTTTGGTGCGTATTAGTATATAAGTATTTGT-3' (SEQ ID NO: 3),
[0037] R3-ΔMP-R: 5'-AAAGTGGCCTCGGGAAATCTAACACACTGTACC-3' (SEQ ID NO: 4);
[0038] The PCR amplification system was: 2×Canace Plus PCR buffer (containing Mg). 2+ 12.5 μL of dNTPs (Yisheng Biotechnology, Shanghai), 1 μL each of primers R3-ΔMP-F / R3-ΔMP-R (10 μmol / L), 1 μL of dimethyl sulfoxide, 1 μL of plasmid template pCB301-Fny309 (10 ng / μL), 0.5 μL of Hieff Canace Plus High-Fidelity DNA Polymerase (Yisheng Biotechnology, Shanghai), and ddH2O to a final volume of 25 μL; the PCR reaction program was: 98 ℃ for 3 min, 98 ℃ for 20 s, 60 ℃ for 10 s, 72 ℃ for 4 min, 12 cycles, 46 ℃ for 2 min, and 72 ℃ for 10 min.
[0039] Five μL of the amplified PCR product sample was subjected to agarose gel electrophoresis, and the target band was detected under ultraviolet light; the result was R3-ADI (DNA).
[0040] Add 2 μL of 10×Cutsmart buffer and 1 μL of DpnI enzyme (NEB) to the remaining 20 μL of PCR product, and incubate overnight at 37 °C to digest the plasmid template in the reaction system.
[0041] Transform 2 μL of the enzyme digestion product into Escherichia coli and spread it evenly on LB agar plates containing kanamycin (50 mg / mL) using a spreader. Incubate the plates at 37 °C with the plates inverted for 12 h.
[0042] Subsequently, single colonies were randomly selected from the cultured culture using a sterile pipette tip and cultured in LB liquid medium containing kanamycin (50 mg / mL) for 12 hours. The bacterial culture was then collected, and plasmids were extracted using the Axygen mini-prepare kit. The plasmids were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing verification. After confirmation by sequencing, the plasmid was named pCB301-R3-ADI.
[0043] 2.2) Take 30 μL of Agrobacterium GV3101 competent cells, add 2 μL of pCB301-R3-ADI plasmid, gently pipette to mix, stand on ice for 30 min, freeze in liquid nitrogen for 1 min, incubate in a 42 ℃ water bath for 1 min, then stand on ice for 2 min, add 500 μL of antibiotic-free LB liquid medium; and culture in a shaker at 28 ℃ and 220 r / min for 4 h.
[0044] After centrifugation at 6000 rpm for 3 min, the bacterial precipitate was collected and spread onto LB solid medium containing kanamycin, rifampin, and gentamicin antibiotics (all at a concentration of 50 mg / mL). The medium was then incubated at 28 °C for 2 days to obtain a single Agrobacterium colony containing pCB301-R3-ADI, which was named C3-ADI.
[0045] Example 2: Determination of replication activity of pCB301-R3-ADI vector
[0046] 1. The helper virus used to test the replication activity of the pCB301-R3-ADI vector was the CMV Fny strain, whose infectious clone consisted of three plasmids: pCB301-Fny1, pCB301-Fny2, and pCB301-Fny3. Agrobacterium GV3101 containing infectious clones of pCB301-Fny1, pCB301-Fny2, pCB301-Fny3, or pCB301-Fny3-eGFP were named C1, C2, C3, or C3-eGFP, respectively. pCB301-Fny1, pCB301-Fny2, and pCB301-Fny3 were donated by Professor Tao Xiaorong of Nanjing Agricultural University (Chinese Journal of Agricultural Science, 2011, 44(14): 3060-3068). pCB301-Fny3-eGFP is obtained by replacing the CP open reading frame in pCB301-Fny3 with an enhanced GFP (eGFP) open reading frame (Genbank accession no. AAB02572). Since the CP protein is essential for CMV systemic mobility, and pCB301-Fny3-eGFP has the CP replaced by the eGFP sequence, the chimeric virus (CMV-eGFP) obtained from this clone in subsequent embodiments loses its systemic mobility.
[0047] 2. The Agrobacterium single colony containing pCB301-R3-ADI obtained in Example 1 (hereinafter referred to as C3-ADI) and 500 μL of Agrobacterium C1, C2, C3, and C3-eGFP glycerol bacteria stored at -80 ℃ were cultured in 3 mL of LB liquid medium containing three antibiotics (kanamycin, rifampin, and gentamicin, all at a concentration of 50 μg / mL) at 28 ℃ for 12-16 h with shaking.
[0048] 300 μL of Agrobacterium tumefaciens culture for R3-ADI and C1, C2, C3, and C3-eGFP strains were added to 6 mL of LB liquid medium containing 50 μg / mL kanamycin and 50 μg / mL rifampin, respectively. Then, 1.5 μL of sterile 0.2 mol / L acetylsuccine (in dimethyl sulfoxide) and 300 μL of 0.5 mol / L 2-Morpholinoethanesulfonic acid (MES) buffer were added. The culture was incubated at 28°C for 16 h, and the cells were collected by centrifugation at 6000 rpm for 3 min. The absorbance of the bacterial culture was measured using a spectrophotometer. The concentration of Agrobacterium tumefaciens was adjusted with infiltration buffer to achieve the desired OD value. 600 = 0.15.
[0049] The specific formulation of the immersion buffer is as follows: 10 mL of 0.5 mol / L MES, 2.5 mL of 2 mol / L MgCl2, 500 μL of 0.2 mol / L acetylsylgenone, and deionized water to a final volume of 500 mL.
[0050] The specific formulation of 0.5 mol / L MES buffer is as follows: Dissolve 5.33 g of MES in 40 mL of deionized water, adjust the pH to 5.6 using 2 mol / L NaOH, and bring the volume to 50 mL.
[0051] Since RNA1 and RNA2 in the CMV genome encode replication-related proteins 1a and 2a, respectively, and these proteins interact to form a replication-active replicase complex, and RNA2 encodes protein 2b via a subgenomic strategy, this invention, in subsequent embodiments, uses Agrobacterium C2 lacking RNA2 expression as a negative control lacking replication function. This aims to demonstrate that the accumulation of specific R3-ADI derived from C3-ADI, CP protein expression, and subsequent disease resistance all depend on its ability to replicate.
[0052] 3. Group 1 was injected with infiltration buffer (blank control, i.e., mock) into each leaf of Nicotiana benthamiana. The injection volume was 1 mL of infiltration buffer per leaf.
[0053] Group 2 was injected with equal proportions of Benedictine smoke and mixed thoroughly to achieve a final concentration OD. 600 A C1+C3 mixed bacterial culture with a concentration of 0.15 was used as a negative control;
[0054] Group 3 was injected with equal proportions of Benedictine smoke and mixed thoroughly to achieve a final concentration OD. 600 A mixed bacterial culture of C1+C2+C3 with a concentration of 0.15 was used as a positive control;
[0055] Group 4: Inject Benedictine smoke into a mixture of equal proportions until the final concentration of OD is reached. 600 A C1+C3-ADI mixed bacterial culture with a concentration of 0.15 was used as the negative experimental group;
[0056] Group 5 was injected with equal proportions of Benedictine smoke to achieve a final concentration OD. 600 A C1+C2+C3-ADI mixed bacterial culture with a concentration of 0.15 was used as the positive experimental group.
[0057] The above-mentioned bacterial solution injection volume is 1 mL of activated bacterial solution per single leaf.
[0058] Select similar-sized Nicotiana benthamiana plants, puncture the fifth leaf with a needle, and inoculate the punctured leaf with 1 mL of the incubated mixed bacterial solution using a needleless syringe. The leaf position of the inoculated leaves in the experimental group and the control group should be consistent. All inoculated plants are placed in a plant growth chamber at 22-25 ℃ with a light duration of 16 h.
[0059] 4. Three days after inoculation, leaf samples were collected, and total RNA and total protein were extracted from the samples to analyze the expression of the genome in the plant. 0.1g of leaf sample was weighed, cut into small pieces, and placed in a 2 mL centrifuge tube containing two small steel balls. The tube was then placed in liquid nitrogen.
[0060] 4.1) Detection of ADI RNA vector RNA level accumulation:
[0061] ① Use a grinder to thoroughly grind the sample in the 2 mL centrifuge tube, and extract RNA according to the instructions of RNAisol (Newview Biotechnology, Hangzhou).
[0062] ② The concentration and purity of RNA were determined at 260 nm using a micro spectrophotometer.
[0063] ③ Adjust the RNA concentration of all groups to 1000 ng / μL according to the sample concentration to ensure that the loading amount is consistent between different samples.
[0064] ④ Add 10 μL of RNA loading buffer and 1 μL of RNA sample to the PCR tube, mix well, denature at 65 °C for 10 min, and place on ice for 3 min. Then perform formaldehyde agarose gel electrophoresis at 65 V for 50 min. Observe the RNA bands under UV light. If the bands are clear, intact and without degradation, continue electrophoresis at 65 V for 70 min.
[0065] Refer to the instructions for use of the Digoxigenin Label Detection Kit II (Roche, Switzerland) for the steps of RNA transfer and Northern hybridization.
[0066] ⑤ The probes for Northern blotting detection of the CMV genome target highly homologous nucleotide sequences at the 3' ends of CMV RNA1, RNA2, and RNA3. These probes were synthesized by Shanghai Bioengineering Co., Ltd. The probe sequences for the CMV genome are as follows (DIG represents digoxigenin):
[0067] 5'-DIG-GACTGACCATTTTAGCCGTAAGCTGGATGGACAACCCGTTC-3' (SEQ ID NO: 5);
[0068] Northern hybridization results are as follows Figure 2 As shown in (A):
[0069] Group 1 mock (blank control) showed no CMV source bands;
[0070] No viral genome bands were found in group 2 (C1+C3), indicating that the accumulation of transcriptional R3-ADI from C3-ADI could not be detected;
[0071] Group 3 (C1+C2+C3) served as a positive control, in which CMV genomic RNA (RNA1 / 2, RNA3, and subgenomic RNA4 derived from RNA3) could be detected. The above results indicate that in the presence of C1+C2, C3 can replicate normally to produce RNA3 and subgenomic RNA4.
[0072] No bands from the viral genome were detected in group 4 (C1+C3-ADI), indicating that the accumulation of transcriptional R3-ADI from C3-ADI could not be detected;
[0073] In group 5 (C1+C2+C3-ADI), RNA1 / 2 at the replication level, as well as RNA3-ADI derived from C3-ADI and subgenomic RNA4, were detected. These results indicate that in the presence of C1 and C2, C3-ADI can be replicated normally and produce subgenomic RNA4.
[0074] The above results indicate that the R3-ADI structure constructed in this invention can be normally replicated by CMV genomic RNA1 / 2 and generate subgenomic RNA4.
[0075] Note: The uniform brightness of the bands in the RNA Loading graph indicates that the amount of RNA loaded in each group was consistent during the experiment, eliminating the expression level deviation caused by differences in loading amount and ensuring the reliability of subsequent analysis.
[0076] 4.2) Detection of protein accumulation in the pCB301-R3-ADI vector:
[0077] Since pCB301-R3-ADI retains the CP gene, the accumulation of CP protein expressed by pCB301-R3-ADI was further examined.
[0078] Group 1 was injected with 1 mL of infiltration buffer (mock) into the smoker Benedictine.
[0079] Group 2 was injected with equal proportions of Benedictine smoke and mixed thoroughly to achieve a final concentration OD. 600 A C1+C3-ADI mixed bacterial culture with a concentration of 0.15 was used as a negative control group;
[0080] Group 3 was injected with equal proportions of Benedictine smoke and mixed thoroughly to achieve a final concentration OD. 600 The C1+C2+C3-ADI mixed bacterial culture with a concentration of 0.15 was used as the experimental group;
[0081] Group 4: Inject Benedictine smoke into a mixture of equal proportions until the final concentration of OD is reached.600 A mixed bacterial culture of C1+C2+C3 with a concentration of 0.15 was used as a positive control group.
[0082] The above-mentioned bacterial solution injection volume is 1 mL of activated bacterial solution per single leaf.
[0083] Select similar-sized Nicotiana benthamiana plants and puncture the fifth leaf with a needle. Inoculate the punctured leaf with the incubated mixed bacterial solution using a 1 mL needleless syringe. The leaf position of the inoculated leaves in the experimental group and the control group should be consistent. All inoculated plants are placed in a plant growth chamber at 22-25 ℃ with a light duration of 16 h.
[0084] Three days after inoculation, leaf samples were collected to extract protein for analysis of CP protein expression in the plant. 0.1 g of leaf sample was weighed, chopped, and placed in a 2 mL centrifuge tube containing two small steel balls, which was then placed in liquid nitrogen.
[0085] The sample in the 2 mL centrifuge tube was thoroughly pulverized using a grinder. The specific method for protein extraction is as follows:
[0086] ① Take 0.1 g of plant sample, freeze it with liquid nitrogen, and grind it in a grinder at 60 Hz for 20 s × 3 times;
[0087] ② Add 200 μL of protein extraction solution and 200 μL of 2× loading buffer, and vortex to mix.
[0088] ③ Denature at 95 ℃ for 10 min, centrifuge at 10000 r / min for 5 min, and collect the supernatant.
[0089] The specific formula for the protein extraction solution is: 500 μL 20×PBS, 1 mL 10% SDS, 200 μL β-mercaptoethanol, and deionized water to a final volume of 10 mL.
[0090] The specific formulation of the 2× loading buffer is as follows: 4 mL 10% SDS, 200 μL β-mercaptoethanol, 1 mL 1 M Tris-HCl (pH= 6.8), 2 mL glycerol, 300 μL saturated bromophenol blue, and deionized water to a final volume of 10 mL.
[0091] The denatured protein samples were subjected to SDS-PAGE gel electrophoresis. The specific method for protein hybridization is as follows:
[0092] ① Prepare SDS-PAGE gel (5% stacking gel concentration, 10% separating gel concentration).
[0093] ② Add 10 μL of protein sample to the sample well, perform electrophoresis at 80 V constant voltage for 30 min with 1×Running buffer, and then perform electrophoresis at 120 V constant voltage for 1 h.
[0094] ③ After electrophoresis, immerse the gel and nitrocellulose membrane (NC membrane) separately in 1× protein transfer buffer for 10 min. Assemble the transfer "sandwich" in the following order (from negative to positive): sponge pad, 3 layers of filter paper, gel, NC membrane, 3 layers of filter paper, sponge pad. Transfer at a constant voltage of 100 V and 4 ℃ for 1 h.
[0095] ④ After the transfer is complete, remove the membrane and immerse it in Ponceau S staining solution for 10 min. Rinse with deionized water until the background is clear, and take a picture to record the protein transfer.
[0096] ⑤ Immerse the NC membrane in 5% skim milk blocking solution and incubate at 4 ℃ for 4 h or overnight.
[0097] ⑥ Rinse with 1×TBS solution for 10 min.
[0098] ⑦ Incubate in the primary antibody dilution solution at 4°C for 4 hours or overnight.
[0099] ⑧ Rinse 3 times with 1×TBST solution, 10 min each time; rinse once with 1×TBS solution, 10 min each time.
[0100] 9. Incubate in the secondary antibody dilution solution at 4 ℃ for 1 h.
[0101] ⑩ Rinse 3 times with 1×TBST solution, 10 min each time; rinse once with 1×TBS solution, 10 min each time.
[0102] Next, after mixing equal volumes of chemiluminescent components A and B, the mixture is evenly applied to the front of the film. The film and X-ray film are then placed in a film press cassette and exposed in a darkroom for 10 minutes. The film is then rinsed in developing and fixing solutions for 20 seconds each.
[0103] The specific formula for 1×Running buffer is: 3.03 g Tris, 14.4 g glycine, 1 g SDS, and deionized water to a final volume of 1 L.
[0104] The specific formula for 10× protein transfer buffer is: 3.03 g Tris, 14.4 g glycine, and deionized water to a final volume of 1 L.
[0105] The specific formula for 10×TBS is: 121.1 g Tris, 87.66 g sodium chloride, pH adjusted to 7.8 with concentrated hydrochloric acid, and volume brought to 1 L with deionized water.
[0106] Western hybridization results are as follows Figure 2 As shown in (B), 3 days after vaccination, compared to the control group:
[0107] Group 1 mock (blank control) treatment did not detect the corresponding CP band;
[0108] Group 2 lacks C2, so C3-ADI cannot replicate to produce subgenomic RNA4, thus it cannot synthesize CP protein, and therefore no bands originating from CP can be detected;
[0109] Group 3 showed a detectable CP band. This result indicates that, in the presence of C1 and C2, C3-ADI can replicate to produce subgenomic RNA4, thereby synthesizing the CP protein;
[0110] Group 4, serving as a positive control, also showed a detectable CP band.
[0111] The above results indicate that, in the presence of C1 and C2, C3-ADI can generate subgenomic RNA4 via replication, thereby synthesizing the CP protein.
[0112] Among them, the results of NC membrane staining with virgin red after transfer showed that the bands in each group were uniform, indicating that the protein loading amount in each group was consistent during the experiment, eliminating the expression deviation caused by the difference in loading amount, and ensuring the reliability of subsequent analysis.
[0113] In summary, based on the above results, it can be concluded that in the presence of C1 and C2, C3-ADI can be replicated normally and produce subgenomic RNA4, and can express CP protein. These results indicate that the Agrobacterium tumefaciens lacking the R3-ADI structure constructed in this invention, and the C3-ADI Agrobacterium produced by subsequent transformation, possesses similar replication activity to wild-type RNA3 and the ability to produce CP protein via subgenomic expression in plants in the presence of C1 and C2.
[0114] Example 3: Application of C3-ADI against chimeric CMV-eGFP virus
[0115] To verify whether C3-ADI possesses anti-CMV functionality, this invention first tested its effect on inhibiting the accumulation of chimeric virus CMV-eGFP. In subsequent embodiments, following step 2 of Example 2, the pCB301 empty vector was transformed into Agrobacterium to obtain the corresponding Agrobacterium, named vector (empty vector), as a negative control. Following step 2 of Example 2, the C1, C2, C3-eGFP, vector, and C3-ADI Agrobacterium strains required in this embodiment were cultured, and the Agrobacterium concentration was adjusted using infiltration buffer. After being mixed in equal proportions, the mixed bacterial culture was OD... 600=0.15. In this embodiment, after mixing the three bacterial cultures C1, C2, and C3-eGFP (C1+C2+C3-eGFP), the infected plant can produce a chimeric virus CMV-eGFP, which is expressed by the subgenome. Therefore, the accumulation of this chimeric virus can be indirectly indicated by the eGFP protein expression level.
[0116] Group 1 was injected with a mixed bacterial culture of C1+C2+C3-eGFP+vector in equal proportions as a control in the form of Nicotiana benthamiana.
[0117] Group 2 was treated as the experimental group by injecting a mixed bacterial culture of C1+C2+C3-eGFP +C3-ADI in equal proportions into *Flavorum benthamianum*.
[0118] The injection volume of the above-mentioned bacterial solution was 1 mL of activated bacterial solution per single leaf. Selected Nicotiana benthamiana plants of similar size, and punctured the left and right sides of the 5th leaf with a needle. Using a 1 mL needleless syringe, the incubated bacterial solution was inoculated onto the punctured leaves, with the experimental and control groups inoculated onto the same leaf. All inoculated plants were cultured in a plant growth chamber at 22–25 ℃ with a light duration of 16 h.
[0119] Three days after inoculation, the fluorescence intensity of the experimental group and the control group was observed under ultraviolet light. Figure 3 As shown. Figure 3 (A) in the image shows a typical leaf infiltration spot under UV light. The results show that compared with the control group, the fluorescence signal generated by the C3-ADI experimental group derived from C3-eGFP expression of eGFP is weaker, indicating that R3-ADI can effectively inhibit the accumulation of chimeric CMV-eGFP virus.
[0120] Subsequently, inoculated leaves from four biological replicates of each treatment group were collected, and total RNA was extracted from the samples for further analysis of differences in chimeric viral RNA accumulation. The process of extracting total RNA from the samples and detecting CMV accumulation using Northern blotting was essentially the same as step 4 of Example 2. The only difference was the use of a CMV genomic RNA3-specific probe: 5'-CGGAGGGAGGATTCTGGGAACACGGAATCAGACTGG-DIG-3' (SEQ ID NO: 6, DIG represents digoxigenin group) for detection.
[0121] Northern hybridization results as follows Figure 3 As shown in (B), compared to the vector (empty vector) control group, the accumulation of RNA3 derived from the chimeric CMV genome (labeled as RNA3-eGFP in the figure) was significantly reduced in the C3-ADI experimental group. This result indicates that R3-ADI can effectively inhibit the accumulation of chimeric CMV-eGFP virus.
[0122] In summary, based on Example 3, it can be seen that R3-ADI can effectively inhibit the accumulation of chimeric CMV-eGFP virus at both the protein and genomic RNA levels.
[0123] Example 4: Application of C3-ADI against wild-type CMV virus
[0124] To further verify the effect of C3-ADI on resistance to wild-type CMV, the present invention further implements the following embodiments. According to step 2 of Embodiment 2, the C1, C2, C3, vector, and C3-ADI Agrobacterium species required in this embodiment are cultured, and the Agrobacterium concentration is adjusted using infiltration buffer. After being mixed in equal proportions, the OD of the bacterial culture is adjusted. 600 =0.15. In this embodiment, the mixture of three bacterial solutions, C1, C2, and C3 (C1+C2+C3), infects plants and produces wild-type CMV. Wild-type CMV has systemic mobility, so the difference in inhibitory effects can be indicated by detecting the amount of virus accumulated in the systemic leaves.
[0125] Group 1 was injected with infiltration buffer (mock) into the fumigation site of Benzodiazepam.
[0126] Group 2 was treated as a control group by injecting a proportionally mixed C1+C2+C3+vector bacterial suspension into *Nymphaea benthamiana*.
[0127] Group 3 was injected with a mixed bacterial suspension of C1+C2+C3+C3-ADI in equal proportions as the experimental group.
[0128] The above-mentioned bacterial solution injection volume is 1 mL of activated bacterial solution per single leaf.
[0129] Select similar-sized Nicotiana benthamiana plants and puncture the fifth leaf with a needle. Inoculate the punctured leaf with 1 mL of incubated bacterial solution using a needleless syringe. The leaf position of the inoculated leaves in the experimental group and the control group should be consistent. All inoculated plants are placed in a plant growth chamber at 22-25℃ with a light duration of 16 h.
[0130] Three days after inoculation, the 10th leaf of the system was collected, and total RNA was extracted from the sample to analyze viral RNA accumulation. 0.1g of leaf sample was weighed, cut into small pieces, and placed in a 2 mL centrifuge tube containing two small steel balls. The tube was then placed in liquid nitrogen.
[0131] The sample in the 2 mL centrifuge tube was thoroughly pulverized using a grinder, and total RNA was extracted according to the RNAisol (Simgen, China) reagent instructions.
[0132] The concentration and purity of RNA were determined at 260 nm using a micro-spectrophotometer.
[0133] Based on the sample concentration, the RNA concentration of all groups was adjusted to 1000 ng / μL to ensure consistent loading amounts across different samples.
[0134] Add 10 μL of RNA loading buffer and 1 μL of RNA sample to a PCR tube, mix well, denature at 65 °C for 10 min, and place on ice for 3 min. Then perform formaldehyde agarose gel electrophoresis at 65 V for 50 min. Observe the RNA bands under UV light. If the bands are clear, intact and without degradation, continue electrophoresis at 65 V for 70 min.
[0135] Refer to the instructions for use of the Digoxigenin Label Detection Kit II (Roche, Switzerland) for the steps of RNA transfer and Northern hybridization.
[0136] The probe for detecting the CMV genome using Northern hybridization is as described in step 4 of Example 2. Figure 4 As shown, compared to the control group, no viral genomic RNA was detected in the systemic leaves of the C3-ADI treatment group 3 days after inoculation. This result indicates that C3-ADI can effectively inhibit the accumulation of wild-type CMV virus in the systemic leaves of Nicotiana benthamiana at least 3 days after inoculation.
[0137] In the RNA Loading graph, the bands in each group have uniform brightness, indicating that the amount of RNA loaded in each group was consistent during the experiment. This eliminates the expression level deviation caused by differences in loading amount and ensures the reliability of subsequent analysis.
[0138] In summary, according to Example 4, it can be seen that 3 days after inoculation, C3-ADI can effectively inhibit the accumulation of wild-type CMV virus on the leaves of Nicotiana benthamiana compared with the control group.
[0139] Finally, it should be noted that the above examples are merely some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the disclosure of the present invention should be considered within the scope of protection of the present invention.
Claims
1. Artificial defective interfering RNA, characterized by: The sequence from the 5' end to the 3' end includes the following elements: the 5' UTR, IGR, CP open reading frame, and 3' UTR sequence of CMV genomic RNA3.
2. The recombinant DNA encoding the artificial defective interfering RNA as described in claim 1, characterized in that: It contains a sequence as shown in SEQ IDNO:
1.
3. A recombinant expression vector, characterized in that: The recombinant expression vector contains the recombinant DNA as described in claim 2.
4. The recombinant expression vector according to claim 3, characterized in that: The recombinant expression vector also contains the cauliflower mosaic virus 35S promoter sequence, hammerhead ribozyme sequence, and NOS terminator sequence.
5. The recombinant expression vector according to claim 3 or 4, characterized in that: The pCB301-R3-ADI vector has a sequence as shown in SEQ ID NO:
2.
6. The application of the artificial defective interfering RNA as described in claim 1 in the fight against cucumber mosaic virus.
7. The application of the recombinant expression vector as described in any one of claims 3 to 5 in the fight against cucumber mosaic virus.