CN116970636A - Vaccine for preventing and treating potato virus Y and preparation method thereof - Google Patents

Vaccine for preventing and treating potato virus Y and preparation method thereof Download PDF

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Publication number
CN116970636A
CN116970636A CN202211358857.1A CN202211358857A CN116970636A CN 116970636 A CN116970636 A CN 116970636A CN 202211358857 A CN202211358857 A CN 202211358857A CN 116970636 A CN116970636 A CN 116970636A
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vaccine
pvy
seq
bacterial liquid
potato virus
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李俊营
杨铁钊
周方
周健飞
何冰
武兆云
徐世晓
孙聚涛
郭玉鸽
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Henan Tobacco Co ltd Pingdingshan Branch
Henan Agricultural University
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Henan Tobacco Co ltd Pingdingshan Branch
Henan Agricultural University
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8283—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for virus resistance
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Abstract

The invention belongs to the technical field of plant disease control, and in particular relates to a vaccine for controlling Potato Virus Y (PVY), which comprises pTRV1 and at least one recombinant expression vector pTRV 2-G-target gene, wherein the target gene is PVY-HC or PVY-NIb, and the sequences of the target gene are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2. 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 bacterial liquid can effectively silence exogenous invasion potato virus Y gene expression, the gene silencing efficiency is up to 54.7%, and the propagation of potato virus Y in plants is effectively inhibited, and the disease time is delayed.

Description

Vaccine for preventing and treating potato virus Y and preparation method thereof
Technical Field
The invention belongs to the technical field of plant disease control, and particularly relates to a vaccine for controlling potato virus Y and a preparation method thereof.
Background
The diseases caused by plant viruses are called "plant cancers" and cause very serious economic losses. Potato virus YPotato virus Y，PVY) has a wider host range, can infect various solanaceae plants, has serious harm especially on crops such as tobacco, potato, pepper and the like, and usually shows three types of symptoms of flowers, leaves, necrosis and leaves rolling in fields. The genome of potato virus Y has only one ORF, and is expressed and translated into a multimeric protein, and then encodes a series of functional mature proteins. Wherein, the main function of HC-pro is to inhibit gene silencing, participate in genome self-interaction, and influence the short-distance transportation of the virus between different cells or the long-distance transportation of the virus between different tissues; the NIb protease acts primarily during the amplification phase of the viral genome.
At present, chemical prevention and control are usually mainly used for preventing and controlling potato Y virus diseases 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 potato Y virus diseases, but breeding work is slow, and biological prevention and control technology has gradually become a new method for preventing and controlling diseases.
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 embrittlement 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 potato virus Y.
In order to solve the technical problems, the invention is realized by the following technical scheme:
a vaccine for preventing and treating potato virus Y comprises pTRV1 and at least one recombinant expression vector pTRV 2-G-target gene, wherein the target gene is PVY-HC or PVY-NIb, and the sequences are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2.
Preferably, the vaccine comprises pTRV1 and two recombinant expression vectors pTRV 2-G-target genes, wherein the target genes are PVY-HC and PVY-NIb respectively, and the sequences of the target genes are shown as SEQ ID NO.1 and SEQ ID NO.2 respectively.
Wherein, the recombinant expression vector pTRV 2-G-target gene is constructed by the following steps:
(1) Extracting total RNA of tobacco leaves infected with potato virus Y, 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-GFP empty plasmidBamHI/KpnI, double enzyme digestion, after recovering enzyme digestion products, obtaining a linearization carrier;
(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 PVY-HC, the primer pair is SEQ ID NO.3 and SEQ ID NO.4; when the target gene is PVY-NIb, the primer pair is SEQ ID NO.5 and SEQ ID NO.6.
The preparation method of the vaccine comprises the following steps:
(1) Transforming the recombinant expression vector pTRV 2-G-target gene into competent cells of agrobacterium GV3101 to obtain original bacterial liquid; 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 potato virus Y.
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 potato virus Y gene, the gene silencing efficiency is up to 54.7%, and the propagation of potato virus Y in plants is effectively inhibited, and the onset time is delayed.
Drawings
FIG. 1 pTRV2-G vector map;
FIG. 2 shows the gene amplification product of interest;
FIG. 3 PVY-HC gene expression levels after virus inoculation;
FIG. 4 PVY-NIb gene expression levels 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 PVY measured N The sequence of the gene (GenBank accession number: HQ 631374) was selected as a fragment suitable for constructing the VIGS vector, and the principle of selection was: 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.1-2 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
Total RNA of tobacco leaves infected with potato virus Y is extracted by TRIzol method, and the specific operation method is according to 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. ReactionThe conditions are that the pre-denaturation is carried out for 3min at 95 ℃; 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. BamHI/KpnI double digestion is carried out on pTRV2-G empty plasmid, and the digested products are recovered respectively to obtain the linearization vector. 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 a recombinant expression vector pTRV2-G-PVY-HC and pTRV2-G-PVY-NIb.
Transformation of recombinant expression vectors
Referring to the instruction of using Agrobacterium GV3101 competent cells of Shanghai Weidi biotechnology Co., ltd, the recombinant expression vectors pTRV2-G-PVY-HC and pTRV2-G-PVY-NIb were transformed into Agrobacterium GV3101 competent cells, respectively, to obtain an original bacterial solution. 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.
Preparation of the product
Inoculating TRV1 bacterial solution and original bacterial solution TRV2-G-PVY-HC and TRV2-G-PVY-NIb respectively to LB liquid culture medium, culturing at 28deg.C for 200r/min overnight, and adjusting concentration to OD 600 And (3) mixing the bacterial solutions of the TRV1 with the bacterial solutions of the TRV2-G-PVY-HC and the TRV2-G-PVY-NIb 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 penicillin bottle by using a freeze dryer to obtain the vaccine for preventing and treating potato virus Y.
The dosage of the product
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.
Time of use of the product
Leaf cutting is carried out at the seedling stage of the plants, and then vaccination is sprayed.
Gene silencing assay
The independent products (the last step is not mixed) of TRV2-G-PVY-HC and TRV2-G-PVY-NIb are respectively injected into six-leaf first-heart tobacco seedlings with uniform growth vigor, a 1 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, potato Y virus is respectively inoculated by friction 14 days after inoculation, 7 days after virus inoculation, RNA is extracted from new leaves, and the gene silencing effect corresponding to each product is measured.
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% >.
See FIGS. 3-4, with a PVY-HC gene silencing efficiency of 46.1% and PVY-NIb gene silencing efficiency of 54.7%.
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 morbidity conditions in a high-stage morbidity. 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 occurrence of tobacco potato Y, and the result shows that compared with the product without being inoculated by spraying, the disease occurrence rate is reduced, the control effect can reach 80.0%, 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 the control of potyvirus, characterized in that: comprises pTRV1 and at least one recombinant expression vector pTRV 2-G-target gene, wherein the target gene is PVY-HC or PVY-NIb, and the sequences are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2.
2. The vaccine for preventing and treating potato virus Y according to claim 1, characterized in that: comprises pTRV1 and two recombinant expression vectors pTRV 2-G-target genes, wherein the target genes are PVY-HC and PVY-NIb respectively.
3. The vaccine for preventing and treating potato virus Y according to claim 1, characterized in that: the recombinant expression vector pTRV 2-G-target gene is constructed by the following steps:
(1) Extracting total RNA of tobacco leaves infected with potato virus Y, 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-GFP empty plasmidBamHI/KpnI, double enzyme digestion, after recovering enzyme digestion products, obtaining a linearization carrier;
(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, will sequence positivelyThe exact monoclonal strain was propagated and plasmids were extracted.
4. A vaccine against potyvirus according to claim 3, wherein: regarding the corresponding primers in the PCR amplification of step (2): when the target gene is PVY-HC, the primer pair is SEQ ID NO.3 and SEQ ID NO.4; when the target gene is PVY-NIb, the primer pair is SEQ ID NO.5 and SEQ ID NO.6.
5. A method for preparing a vaccine for controlling potyvirus according to any one of claims 1 to 4, comprising the steps of:
firstly, transforming the recombinant expression vector pTRV 2-G-target gene into competent cells of agrobacterium GV3101 to obtain original bacterial liquid; 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;
respectively inoculating TRV1 bacterial liquid and each TRV2 initial bacterial liquid to LB liquid culture medium, culturing at 25-30deg.C for 150-250r/min overnight, and adjusting the concentration to OD 600 And (3) carrying out approximately equal to 0.8-1.0, mixing and shaking the cultured TRV1 bacterial liquid and the cultured single TRV2 bacterial liquid uniformly, mixing and freeze-drying the mixed materials, and thus obtaining the vaccine for preventing and treating potato virus Y.
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 a vaccine against potyvirus according to any one of claims 1 to 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.
CN202211358857.1A 2022-11-01 2022-11-01 Vaccine for preventing and treating potato virus Y and preparation method thereof Pending CN116970636A (en)

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