CN116814681A - Vaccine for preventing and treating cucumber mosaic virus and preparation method thereof - Google Patents
Vaccine for preventing and treating cucumber mosaic virus and preparation method thereof Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
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- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention belongs to the technical field of plant disease control, and particularly relates to a vaccine for controlling Cucumber Mosaic Virus (CMV), which comprises pTRV1 and four recombinant expression vectors pTRV 2-target genes, wherein the target genes are CMV-1a, CMV-2a, CMV-MP and CMV-CP, and the sequences of the target genes are respectively shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO. 4. When the vaccine is used on tobacco, buffer solution is added into the vaccine to activate the vaccine, and the vaccine is uniformly mixed; and (3) shading and storing at room temperature, adding water into the obtained mixed solution, and uniformly spraying the mixed solution onto tobacco seedlings subjected to leaf cutting, wherein the spraying amount is that water drops visible to the naked eyes exist on leaf surfaces. The invention can effectively silence the expression of exogenous invasion cucumber mosaic virus genes, the gene silencing efficiency is up to 60.4%, the propagation of the cucumber mosaic virus in plants is effectively inhibited, and the onset time is delayed.
Description
Technical Field
The invention belongs to the technical field of plant disease control, and particularly relates to a vaccine for controlling cucumber mosaic virus and a preparation method thereof.
Background
The diseases caused by plant viruses are called "plant cancers" and cause very serious economic losses. Cucumber mosaic virusCucumber mosaic virusCMV) is a very serious viral disease, and the virus can reach any site other than the growth point. Cucumber mosaic virus is one of the most economically important plant viruses with the largest host range and the largest distribution. Cucumber mosaic virus is a trisomy plus sense single stranded RNA virus, RNA1 contains 1 ORF encoding 1a replicase protein; RNA2 contains 2 ORFs, the 5 'end encodes a 2a replicase protein and the 3' end encodes a 2b protein. Previous studies found that the symptom of plants infected by cucumber mosaic virus is determined by 1a and 2a replicase proteins, and the two proteins have synergistic effect; RNA3 also has 2 ORFs, with the 5 'end encoding 31kDa of the intercellular Motor Protein (MP) and the 3' end encoding a 24-26kDa of the Coat Protein (CP).
At present, chemical control is mainly used for controlling cucumber mosaic virus in production, but the problems of medicament residue, environmental pollution and the like are easily caused. The cultivation of disease-resistant varieties is also one of effective ways for preventing and controlling cucumber mosaic disease, but breeding work is slow, and biological prevention and control technology has gradually become a new method for disease prevention and control.
Virus-induced gene silencing (VIGS) is a technique for reverse genetic manipulation of plants and is a manifestation of plant defense mechanisms. The agrobacterium-mediated VIGS technique transfers a viral vector carrying a gene fragment of interest into plant cells via agrobacterium, mediates mRNA degradation of the targeted homologous gene, causing silencing of the gene of interest.
Disclosure of Invention
In order to solve the problems, the invention constructs a tobacco brittle fracture virus TRV vector targeting the related gene fragment and converts agrobacterium competent cells, thereby laying a foundation for the subsequent establishment of a TRV-mediated VIGS system for preventing and treating cucumber mosaic virus.
In order to solve the technical problems, the invention is realized by the following technical scheme:
a vaccine for preventing and treating cucumber mosaic virus comprises pTRV1 and at least one recombinant expression vector pTRV 2-target gene, wherein the target gene is CMV-1a, CMV-2a, CMV-MP or CMV-CP, and the sequences are respectively shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO. 4.
Preferably, the vaccine comprises pTRV1 and four recombinant expression vectors pTRV 2-target genes, wherein the target genes are CMV-1a, CMV-2a, CMV-MP and CMV-CP, and the sequences of the target genes are respectively shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO. 4.
Wherein, the recombinant expression vector pTRV 2-target gene is constructed by the following steps:
(1) Extracting total RNA of tobacco leaves infected with cucumber mosaic virus, and reversing the total RNA into cDNA;
(2) Then carrying out PCR amplification by taking the cDNA as a template, then carrying out agarose gel electrophoresis detection, and recovering a target fragment;
(3) pTRV2 empty vector plasmid was subjected toEcoRI/BamHI double enzyme digestion, recovering enzyme digestion products, obtaining linearityCarrying out carrier formation;
(4) Ligating the target fragment of the amplified product obtained in the step (2) with the linearization vector obtained in the step (3), and then converting the target fragment into escherichia coliDH5αCompetent cells, (monoclonal strains with correct sequencing) were propagated and plasmids were extracted.
Preferably, with respect to the corresponding primers in the PCR amplification of step (2): when the target gene is CMV-1a, the primer pair is SEQ ID NO.5 and SEQ ID NO.6; when the target gene is CMV-2a, the primer pair is SEQ ID NO.7 and SEQ ID NO.8; when the target gene is CMV-MP, the primer pair is SEQ ID NO.9 and SEQ ID NO.10; when the target gene is CMV-CP, the primer pair is SEQ ID NO.11 and SEQ ID NO.12.
The preparation method of the vaccine comprises the following steps:
(1) Transforming the recombinant expression vector pTRV 2-target gene into competent cells of agrobacterium GV3101 to obtain an original bacterial solution; uniformly smearing original bacterial liquid on LB (containing rifampicin with the concentration of 20-30mg/mL and kanamycin with the concentration of 40-60 mg/mL) solid culture medium, inversely culturing for 40-50h at the temperature of 25-30 ℃, picking single bacterial colony, performing PCR amplification, detecting and verifying by agarose gel electrophoresis, and storing the original bacterial liquid with correct verification in an ultralow temperature refrigerator for standby;
(2) Inoculating TRV1 bacterial liquid and each TRV2 initial bacterial liquid onto LB liquid culture medium, respectively, culturing at 25-30deg.C for 150-250r/min overnight, and adjusting concentration to OD 600 And (3) carrying out mixing and shaking on the cultured TRV1 bacterial liquid and the cultured single TRV2 bacterial liquid respectively, wherein the volume ratio is 1: (0.8-1.2), and then mixing and freeze-drying the mixture to obtain the vaccine for preventing and treating cucumber mosaic virus.
The use method of the vaccine comprises the following steps: adding buffer solution into the vaccine to activate the vaccine, and uniformly mixing the vaccine; the volume ratio of the buffer solution to the vaccine is 1: (0.8-1.2); the mixture was stored under shade at room temperature, and the ratio of the obtained mixture to the solution was 1: (8-12) adding water, and then uniformly spraying the water onto tobacco seedlings subjected to leaf cutting, wherein the spraying amount is that water drops visible to the naked eyes are on the leaf surfaces.
The invention has the following positive and beneficial effects:
the invention can effectively silence the expression of exogenous invasion cucumber mosaic virus genes, the gene silencing efficiency is up to 60.4%, the propagation of the cucumber mosaic virus in plants is effectively inhibited, and the onset time is delayed.
Drawings
FIG. 1 pTRV2 vector map;
FIG. 2 shows the gene amplification product of interest;
FIG. 3 CMV-1a gene expression levels after virus inoculation;
FIG. 4 CMV-2a gene expression level after virus inoculation;
FIG. 5 CMV-MP gene expression levels after virus inoculation;
FIG. 6 CMV-CP gene expression level after virus inoculation.
Detailed Description
The following detailed description of specific embodiments of the invention is, but it should be understood that the invention is not limited to specific embodiments. The experimental methods in the embodiment of the invention are conventional methods unless otherwise specified.
1. Primer design
Based on the CMV determined (GenBank accession number: GCA_ 000864745.1), a fragment suitable for constructing the VIGS vector was selected on the principle: the sequence length is 200-800 bp; GC content is 45% -55%; the sequence specificity is high, the sequence is not overlapped with other sequences of the virus, the used vector sequence and the tobacco genome sequence, and the off-target effect is prevented; and the inverted repetition is avoided, so that the hairpin structure is prevented from being formed. Finally, the fragment sequences SEQ ID NO.1-4 used to construct the VIGS vector were selected. After the selection of fragments was completed, oligo 7 software was used to design specific primers, the sequences of which are shown in Table 1.
TABLE 1 primer sequences designed according to the invention
。
2. Construction of VIGS vector
Extracting total RNA of tobacco leaves infected with cucumber mosaic virus by TRIzol method,the specific operation method is based on the specification of TRIzol product of Tiangen Biochemical technology (Beijing) limited company. The cDNA was inverted by referring to the Beijing full gold biotechnology Co., ltd. TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix specification. PCR amplification was performed using the obtained cDNA as a template and the corresponding primers in Table 1 using P505 high fidelity enzyme from Norpran Biotechnology Co., ltd, respectively, and the amplification system was as follows: 2X Phanta Max Buffer. Mu.L, dNTP Mix (10 mmol/L) 1. Mu.L, phanta Max Super-Fidelity DNA Polymerase. Mu.L, 10. Mu. Mol/L upstream and downstream primers each 2. Mu.L, 20. Mu. Mol/L cDNA template 1. Mu.L, add ddH 2 O was added to 50. Mu.L. The reaction condition is pre-denaturation at 95 ℃ for 3min; denaturation at 95℃for 15s, annealing at 68℃for 15s, elongation at 72℃for 30s,35 cycles; and at 72℃for 5min. Agarose gel electrophoresis was performed and the target fragment was recovered using a gel purification kit from Beijing full gold Biotechnology Co., ltd., see FIG. 2. pTRV2 empty vector plasmid was subjected toEcoRI/BamAnd (3) HI double enzyme digestion, and obtaining a linearization vector after respectively recovering enzyme digestion products. ClonExpress using Norpran Biotechnology Co., ltd ® II, respectively connecting the amplified products with a linearization vector by using a recombinant cloning kit, and then converting the amplified products into escherichia coliDH5αCompetent cells. And (3) propagating and extracting plasmids for preservation by using the monoclonal strains with correct sequencing to obtain recombinant expression vectors pTRV2-CMV-1a, pTRV2-CMV-2a, pTRV2-CMV-MP and pTRV2-CMV-CP.
3. Recombinant expression vector transformation
The recombinant expression vectors pTRV2-CMV-1a, pTRV2-CMV-2a, pTRV2-CMV-MP and pTRV2-CMV-CP were transformed into competent cells of Agrobacterium GV3101, respectively, to obtain an original bacterial solution, referring to the instructions for using competent cells of Agrobacterium GV3101 of Shanghai Weidi Biotechnology Co. Uniformly smearing the obtained original bacterial liquid on LB (containing rifampicin with the concentration of 25mg/mL and kanamycin with the concentration of 50 mg/mL) solid culture medium, and inversely culturing for 48 hours at the temperature of 28 ℃; single colonies were picked, amplified by PCR, and verified by agarose gel electrophoresis detection. And (5) respectively propagating the bacterial solutions with correct verification, and then storing the bacterial solutions to an ultralow temperature refrigerator at the temperature of-80 ℃ for standby.
4. Product preparation
TRV1 bacterial liquid and original bacterial liquid TRV2-CMV-1a, TRV2-CMV-2a, TRV2-CMV-MP, TRV2-CMV-CP are respectively inoculated on LB liquid medium, cultured at 28deg.C for 200r/min overnight, and the concentration is adjusted to OD 600 And (2) mixing the bacterial solutions of the TRV1 with the bacterial solutions of the TRV2-CMV-1a, the TRV2-CMV-2a, the TRV2-CMV-MP and the TRV2-CMV-CP which are cultured respectively, shaking uniformly (mixing in equal proportion), mixing the four mixed shaking solutions together in equal proportion, and then filling the mixed shaking solutions into a penicillin bottle according to 15 ml/bottle, and freeze-drying the mixed shaking solutions by using a freeze dryer to obtain the vaccine for preventing and treating the cucumber mosaic virus. .
5. Product dosage
Before the product is used, the buffer solution is added to activate the product, the buffer solution is injected into penicillin bottles (15 milliliters of buffer solution is injected into each bottle) by a syringe, and the mixture is uniformly shaken. The product is stored for 12-24 hours under shade at room temperature, and the time is not longer than 24 hours. And adding water into the mixed solution according to a ratio of 1:10, and uniformly spraying the mixed solution onto tobacco seedlings subjected to leaf cutting by using a sprayer, wherein the spraying amount is that water drops visible to the naked eyes exist on leaf surfaces.
6. Product use time
Leaf cutting is carried out at the seedling stage of the plants, and then vaccination is sprayed.
7. Gene silencing assay
The single products (the last step is not mixed) of TRV2-CMV-1a, TRV2-CMV-2a, TRV2-CMV-MP and TRV2-CMV-CP are respectively injected into six-leaf first-heart tobacco seedlings with uniform growth vigor, a1 mL injector is used for injecting vaccine on the back of tender leaves of tobacco plants, 2 leaves are injected into each tobacco plant, 20 tobacco plants are injected into each product, cucumber mosaic virus is respectively rubbed and inoculated 14 days after inoculation, 7 days after virus inoculation, new leaf extraction RNA is taken for measuring the gene silencing effect corresponding to each product.
The calculation formula of the gene silencing efficiency is as follows: [ (control CMV gene expression level-treatment CMV gene expression level)/control CMV gene expression level ] ×100% >.
Referring to FIGS. 3-6, CMV-1a did not achieve the desired silencing effect, CMV-2a gene silencing efficiency was 60.4%, CMV-MP gene silencing efficiency was 32.9%, and CMV-CP gene silencing efficiency was 58.9%.
8. Tobacco field test
And (3) carrying out a large-area test on the flat-topped mountain in the Henan tobacco area, cutting leaves and spraying the product before transplanting tobacco seedlings, and investigating different treatment disease conditions in the disease peak period. According to the method for classifying and investigating tobacco diseases and insect pests (GB/T23222-2008), the disease grade is identified by taking plant as a unit, and the morbidity, disease index and prevention and treatment effect are calculated according to the following formula.
After transplanting, 60d, 80d and 100d are used for investigating the disease condition of tobacco cucumber mosaic, and the result shows that compared with the case of spraying and inoculating the product and not inoculating the product, the disease rate is reduced, the control effect can reach 78.3%, and the control effect is good as a whole.
Morbidity = (number of diseased plants/total number of investigation) x 100%;
disease index= (Σ number of disease plants at each stage×disease level)/(total number of investigation×highest disease level) ×100;
control effect= (control disease refers to-treatment disease refers to)/control disease refers to x 100%.
The foregoing examples are provided as part of the presently preferred embodiments of the invention, and the embodiments of the invention are not limited to the foregoing examples, but are intended to be equivalent alternatives, modifications, substitutions, combinations, and simplifications that may be made without departing from the spirit and principles of the invention.
Claims (10)
1. A vaccine for controlling cucumber mosaic virus, characterized in that: comprises pTRV1 and at least one recombinant expression vector pTRV 2-target gene, wherein the target gene is CMV-1a, CMV-2a, CMV-MP or CMV-CP, and the sequences are respectively shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO. 4.
2. The vaccine of claim 1, wherein: the vaccine comprises pTRV1 and four recombinant expression vectors pTRV 2-target genes, wherein the target genes are CMV-1a, CMV-2a, CMV-MP and CMV-CP, and the sequences of the target genes are respectively shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO. 4.
3. The vaccine of claim 1, wherein: the recombinant expression vector pTRV 2-target gene is constructed by the following steps:
(1) Extracting total RNA of tobacco leaves infected with cucumber mosaic virus, and reversing the total RNA into cDNA;
(2) Then carrying out PCR amplification by taking the cDNA as a template, then carrying out agarose gel electrophoresis detection, and recovering a target fragment;
(3) pTRV2 empty vector plasmid was subjected toEcoRI/BamHI double enzyme digestion, after recycling enzyme digestion products, obtaining a linearization vector;
(4) Ligating the target fragment of the amplified product obtained in the step (2) with the linearization vector obtained in the step (3), and then converting the target fragment into escherichia coliDH5αCompetent cells, expanding and extracting plasmids.
4. The vaccine of claim 1, wherein: regarding the corresponding primers in the PCR amplification of step (2): when the target gene is CMV-1a, the primer pair is SEQ ID NO.5 and SEQ ID NO.6; when the target gene is CMV-2a, the primer pair is SEQ ID NO.7 and SEQ ID NO.8; when the target gene is CMV-MP, the primer pair is SEQ ID NO.9 and SEQ ID NO.10; when the target gene is CMV-CP, the primer pair is SEQ ID NO.11 and SEQ ID NO.12.
5. A method of preparing a vaccine according to any one of claims 1 to 4, characterized in that: the method comprises the following steps:
(1) Transforming the recombinant expression vector pTRV 2-target gene into competent cells of agrobacterium GV3101 to obtain an original bacterial solution; uniformly smearing original bacterial liquid on an LB solid culture medium, inversely culturing for 40-50h at 25-30 ℃, picking single bacterial colonies, performing PCR amplification, detecting and verifying by using agarose gel electrophoresis, and storing the original bacterial liquid with correct verification after propagation in an ultralow temperature refrigerator for standby;
(2) Separating TRV1 bacterial liquid and each TRV2 initial bacterial liquidInoculating to LB liquid culture medium, culturing at 25-30deg.C for 150-250r/min overnight, and adjusting concentration to OD 600 And (3) carrying out mixing and shaking on the cultured TRV1 bacterial liquid and the cultured single TRV2 bacterial liquid respectively to obtain about 0.8-1.0, mixing the mixed shaking liquids together, and freeze-drying to obtain the vaccine for preventing and treating cucumber mosaic virus.
6. The method of manufacturing according to claim 5, wherein: the LB solid medium contains rifampicin at a concentration of 20-30mg/mL and kanamycin at a concentration of 40-60 mg/mL.
7. The method of manufacturing according to claim 5, wherein: mixing and shaking uniformly, wherein the volume ratio of the cultured TRV1 bacterial liquid to the single original TRV2 bacterial liquid is 1: (0.8-1.2).
8. A method of using the vaccine of any one of claims 1-4, comprising the steps of: when the vaccine is used on tobacco, a buffer solution is added into the vaccine to activate the vaccine, and the mixture is uniformly mixed; and (3) shading and storing at room temperature, adding water into the obtained mixed solution, and uniformly spraying the mixed solution onto tobacco seedlings subjected to leaf cutting, wherein the spraying amount is that water drops visible to the naked eyes exist on leaf surfaces.
9. The method of use according to claim 8, wherein: the volume ratio of the buffer solution to the vaccine is 1: (0.8-1.2).
10. The method of use according to claim 8, wherein: the volume ratio of the mixed solution is 1: (8-12) adding water.
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| CN202211358914.6A CN116814681A (en) | 2022-11-01 | 2022-11-01 | Vaccine for preventing and treating cucumber mosaic virus and preparation method thereof |
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