A trivalent attenuated vaccine against PVY, PVX and CMV and its application

Abstract

The present invention provides a trivalent attenuated vaccine against PVY, PVX and CMV and its application, and the present invention belongs to the field of plant virology and molecular biology. The trivalent attenuated vaccine contains a mutant plasmid pCB3 of cucumber mosaic virus (CMV) RNA2. F R2‑2bPT‑PVY 8093‑8292 PVX 85‑284 The sequence of the mutant plasmid is shown in SEQ ID NO.3. Fny Plasmid pCB from isolate RNA1 F R1 and CMV Fny Isolate RNA3 aphid-transmitted site mutation plasmid pCB F R3‑△apno is pre-mixed with mutant plasmids for inoculation to prevent potato virus Y, potato virus X and cucumber mosaic virus. At the same time, the vaccine can prevent aphids from transmitting the virus and avoid biosafety risks, providing vaccine materials and effective measures for field control of PVY, PVX and CMV.

Description

Technical Field

[0001] The present invention relates to the fields of plant virology and molecular biology, and relates to a trivalent attenuated vaccine that simultaneously resists PVY, PVX, and CMV and its application. Specifically, it relates to a mutant plasmid containing Potato virus Y (PVY), Potato virus X (PVX), Cucumber mosaic virus (CMV), and an aphid transmission site, which is used to control Potato virus Y (PVY), Potato virus X (PVX), and Cucumber mosaic virus (CMV). Background Art

[0002] Potato ( Solanum tuberosum L.) is the fourth major staple food crop in China and occupies a core position in China's agricultural economic system. Potato virus diseases have caused significant harm to the yield and quality of potatoes, and the yield loss caused by potato virus diseases is 20%-50%. Among them, Potato virus Y (PVY) is particularly harmful and is the main pathogenic factor. In severe cases, the yield loss can be more than 80%. When Potato virus X (PVX) infects alone, the symptoms are relatively mild. Under natural conditions, it usually co-infects with PVY, and the yield loss can reach 50% in severe cases. Although Cucumber mosaic virus (CMV) has not been reported to significantly reduce the potato yield, its detection rate in Gansu region of China has reached 50%. The 2b protein encoded by CMV and the HC-Pro protein encoded by PVY are both RNA silencing suppressors. If these two viruses co-infect, they will inhibit the defense mechanism of potatoes, which is beneficial to the infection of other viruses, thus causing serious losses in yield and quality.

[0003] The phenomenon of cross-protection is that after pre-inoculating with an attenuated strain and then inoculating with a virulent strain, the symptoms of the virulent strain can be significantly reduced. The sources of attenuated strains are partly obtained by natural selection and partly obtained by artificial modification. CMV can infect more than 100 families of plants such as Solanaceae and Cucurbitaceae, and has a wide host range. Its encoded 2b protein is one of the earliest discovered silencing suppressors. Premature termination of the 2b protein can significantly reduce the virulence of CMV without affecting its movement in plants. There has been no public report on a trivalent attenuated vaccine that can simultaneously resist Potato virus Y, Potato virus X, and Cucumber mosaic virus in potatoes. Summary of the Invention

[0004] In view of the above problems, the present invention provides a trivalent attenuated vaccine that simultaneously resists PVY, PVX, and CMV and its application. Specifically, it relates to a mutant plasmid pCB F R2-2bPT-PVY 8093-8292 PVX 85-284 , which contains CMVFny Plasmid pCB of RNA1 of the isolate F Plasmid pCB with R1 and mutation at the aphid transmission site F Inoculation with R3-△apno mixture is used to control Potato virus Y (PVY), Potato virus X (PVX) and Cucumber mosaic virus (CMV). At the same time, this vaccine can prevent aphid transmission of virus, providing vaccine materials and effective control measures for field control of plant virus diseases caused by PVY, PVX and CMV.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] In the first aspect of the present invention, a trivalent attenuated vaccine against PVY, PVX and CMV is provided. The trivalent attenuated vaccine contains a mutant plasmid pCB of Cucumber mosaic virus (CMV) RNA2 F R2-2bPT-PVY 8093-8292 PVX 85-284 ; The construction of the mutant plasmid of Cucumber mosaic virus (CMV) RNA2 is as follows:

[0007] Based on the full-length sequence of RNA2 of CMV Fny isolate, a double stop codon TAATAG is inserted between positions 2682 and 2683, Sma I restriction enzyme site, as well as the conserved sequences of Potato virus Y (PVY) and Potato virus X (PVX); the nucleotide sequences of the PVY and PVX fragments are shown in SEQ ID NO.1 and SEQ ID NO.2, and the nucleotide sequence of the plasmid pCB F R2-2bPT-PVY 8093-8292 PVX 85-284 is shown in SEQ ID NO.3.

[0008] In the second aspect of the present invention, the application of the mutant plasmid pCB F R2-2bPT-PVY 8093-8292 PVX 85-284 in the control of PVY, PVX and CMV, and in the control of aphids; the application steps are as follows:

[0009] The mutant plasmid pCB F R2-2bPT-PVY 8093-8292 PVX 85-284 and the plasmid pCB Fny containing RNA1 of CMV F isolate R1 and the plasmid pCB with mutation at the aphid transmission site of CMV Fny RNA3 FTransform Agrobacterium with R3-Δapno respectively to obtain Agrobacterium liquid A, Agrobacterium liquid B and Agrobacterium liquid C. Mix the three kinds of bacterial liquids and inoculate them onto potatoes;

[0010] The mutant plasmid pCB F R3-Δapno contains the full-length RNA3 sequence of CMV isolate and mutates the amino acids at positions 129 and 162. For the construction method, please refer to the applied patent (Patent No. CN202411479723.4) Fny and has mutations at amino acids 129 and 162. The construction method is detailed in the applied patent (Patent No. CN202411479723.4).

[0011] Advantages of the present invention:

[0012] (1) For the plasmid pCB F R2-2bPT of the premature termination mutant of the 2b protein of cucumber mosaic virus RNA2 obtained, the present invention inserts the conserved fragments of PVY and PVX after the stop codon to obtain the plasmid pCB F R2-2bPT-PVY 8093-8292 PVX 85-284 of the premature termination mutant of the 2b protein of cucumber mosaic virus RNA2 containing PVY and PVX gene fragments. This plasmid is mixed with the Agrobacterium containing the plasmid pCB Fny R1 of CMV isolate RNA1 and the plasmid pCB F R1 and CMV Fny of the plasmid of the aphid transmission site mutant of CMV isolate RNA3, pCB F R3-Δapno, and inoculated, which is used to prevent the infection of PVY, PVX and CMV, and serves as a trivalent attenuated vaccine against potato virus Y, potato virus X and cucumber mosaic virus.

[0013] (2) The mutant plasmid pCB F R2-2bPT-PVY 8093-8292 PVX 85-284 provided by the present invention is mixed with the Agrobacterium containing the plasmid pCB Fny R1 of CMV isolate RNA1 and the plasmid pCB F R1 and the plasmid pCB Fny of the aphid transmission site mutant of CMV isolate RNA3, pCB F R3-Δapno, and inoculated onto potatoes. After 3 subcultures, the inserted virus fragments can still stably exist.

[0014] (3) The mutant plasmid pCB F R3-Δapno provided by the present invention mutates the amino acids at positions 129 and 162. The mutated vaccine can stably exist in plants and does not affect other functions, avoiding the potential biosafety hazards caused by aphid transmission during vaccination. Brief Description of the Drawings

[0015] Figure 1 Schematic diagram of constructing attenuated vaccine vector; from top to bottom are pCB F R1, pCB F R2-2bPT-PVY 8093-8292 PVX 85-284 , pCB F Schematic diagram of R3-△apno; LB is the left border of the T-DNA region; RB is the right border of the T-DNA region; 35S is the promoter (2x35S); RZ is the ribozyme (HDV ribozyme); NOS is the terminator (NOS terminator).

[0016] Figure 2 This is a picture of the symptom effect of attenuated vaccine inoculated on potatoes 7 days later. The inoculated variety is Potato Killer No. 1. Among them, pCB F R1 contains CMV Fny Plasmid for isolate RNA1; pCB F R2-2bPT-PVY 8093-8292 PVX 85-284 For the constructed mutant plasmid; pCB F R3-△apno contains CMV Fny Isolate RNA3 aphid-borne site-mutation plasmid.

[0017] Figure 3 This is a diagram showing the cross-protection effect of attenuated vaccine inoculated on potatoes for 5 days and then inoculated with PVY+PVX+CMV for 12 days. The inoculated variety is Potato Killer No. 1; the blank is a healthy potato plant control; pCB F R1 / pCB F R2-2bPT-PVY 8093-8292 PVX 85-284 / pCB F R3-△apno is a mixed inoculation of Agrobacterium containing three plasmids; PVY+PVX+CMV is a mixed inoculation of Agrobacterium containing the corresponding infectious clone plasmids.

[0018] Figure 4 The results of the fluorescence quantitative PCR detection of virus replication are shown in Figure 1. Among them, a is the result of the detection of PVY replication; b is the result of the detection of PVX replication; c is the result of the detection of CMV replication; the control group was only inoculated with PVY+PVX+CMV virulent virus, and the treatment group was pre-inoculated with a mixture containing pCB F R1 / pCB F R2-2bPT-PVY 8093-8292 PVX 85-284 / pCB FViral replication amount graph 12 days after inoculating the strong virulence of PVY + PVX + CMV on Agrobacterium tumefaciens with R3-Δapno for 5 days.

[0019] Figure 5 It is the graph of the weak virus vaccine stability detection result; among them, M is the D2000 molecular weight standard of GenStar Company; pCB F R1 is a plasmid containing CMV Fny Isolate RNA1; pCB F R2-2bPT-PVY 8093-8292 PVX 85-284 Is the constructed mutant plasmid; pCB F R3-Δapno is a plasmid with a mutation at the aphid transmission site of CMV Fny Isolate RNA3. Detailed implementation manners

[0020] The technical solutions of the present invention are described in detail below through specific implementation manners. It should be understood that the following specific implementation manners are only exemplary, and any modification or change as long as it does not deviate from the technical solution design of the present invention should be within the scope of the claims of the present invention. The present invention is described in detail below in conjunction with embodiments. The test materials not specifically described in the embodiments of the present invention are all conventional test materials in the art and can be obtained through commercial channels. For the experiments in the embodiments of the invention where the specific experimental conditions and methods are not indicated, they are usually carried out under conventional conditions, such as Molecular Cloning: A Laboratory Manual (Fourth Edition) edited by J. Sambrook et al., Science Press, 2017; or according to the conditions recommended by the manufacturer. The schematic diagram of the construction of the weak virus vaccine vector is as Figure 1 Shown.

[0021] Example 1. Plasmid pCB F R2-2bPT-PVY 8093-8292 PVX 85-284 Construction

[0022] Cloning of PVY fragment: Eighty PVY strains were selected as shown in Table 1-1 and Table 1-2. After sequence alignment analysis, the region with relatively conserved sequence was selected. This region is located on the NIb gene, which is an RNA-dependent RNA replicase. Primers PVY-8093-F and PVY-8292-PVX-R were designed. Using the PVY (NC001616) infectious clone plasmid as a template and 2×PhantaMax Master Mix (Dye Plus) (Vazyme), this PVY fragment was cloned by PCR. PCR conditions: pre-denaturation at 95 °C for 3 min; denaturation at 95 °C for 15 s, annealing at 55 °C for 15 s, extension at 72 °C for 15 s, for 35 cycles; extension at 72 °C for 5 min; stored at 4 °C; after the PCR product was recovered using a DNA gel recovery kit (Tiangen), it was reserved for use. The nucleotide sequences of the primers PVY-8093-F and PVY-8292-PVX-R are shown in SEQ ID NO.5 and SEQ ID NO.6; the nucleotide sequence of the obtained PVY fragment is shown in SEQ ID NO.1.

[0023] Table 1-1 PVY Strains Selected

[0024]

[0025] Table 1-2 PVY Strains Selected

[0026]

[0027] Cloning of PVX fragment: Seventy-five PVX strains were selected as shown in Table 2-1 and Table 2-2. After sequence alignment analysis, the region with relatively conserved sequence was selected, which is an RNA-dependent RNA replicase. Primers PVX-85-PVY-F and PVX-284-R were designed. Using the PVX (EU571480) infectious clone plasmid as a template and 2×Phanta Max Master Mix (DyePlus) (Vazyme), this PVX fragment was cloned by PCR. PCR conditions: pre-denaturation at 95 °C for 3 min; denaturation at 95 °C for 15 s, annealing at 55 °C for 15 s, extension at 72 °C for 15 s, for 35 cycles; extension at 72 °C for 5 min; stored at 4 °C; after the PCR product was recovered using a DNA gel recovery kit (Tiangen), it was reserved for use. The nucleotide sequences of the primers PVX-85-PVY-F and PVX-284-R are shown in SEQ ID NO.7 and SEQ ID NO.8; the nucleotide sequence of the obtained PVX fragment is shown in SEQ ID NO.2.

[0028] Ligation of PVY and PVX fragments: Using the above-mentioned recovered PCR products of PVY and PVX as templates, with primers PVY-8093-F and PVX-284-R, perform PCR reaction using 2×Phanta Max Master Mix (Dye Plus) (Vazyme). Fragment ligation conditions: pre-denaturation at 95 °C for 3 min; denaturation at 95 °C for 15 s, annealing at 55 °C for 15 s, extension at 72 °C for 15 s, 5 cycles; PCR conditions: pre-denaturation at 95 °C for 3 min; denaturation at 95 °C for 15 s, annealing at 55 °C for 15 s, extension at 72 °C for 20 s, 30 cycles. The PCR product is the fragment after ligation of PVY and PVX fragments. After recovering the PCR product with a DNA recovery kit (Tiangen), it is reserved for use. The nucleotide sequences of the primers PVY-8093-F and PVX-284-R are shown in SEQ ID NO.5 and SEQ ID NO.8 respectively.

[0029] Table 2-1 PVX selected strains

[0030]

[0031] Table 2-2 PVX selected strains

[0032]

[0033] Digest the vector pCB F R2-2bPT with Sma I single enzyme, recover the enzyme digestion product with a DNA recovery kit (Tiangen). Mix the single enzyme digested intermediate vector pCB F R2-2bPT and the product of ligation of PVY and PVX fragments at a molar ratio of vector to inserted fragment of 1:2. Use the ClonExpress Ultra One Step Cloning Kit (Vazyme) recombination kit, and the recombination primers are pCB F 2-PVYPVX-F, pCB F 2-PVYPVX-R. React the mixed product in a PCR instrument at 50 °C for 5 min. Transform the recombinant product into competent cells of Escherichia coli DH5α, screen with LB solid medium containing 100 μg / mL kanamycin, and confirm the positive clone plasmid pCB F R2-2bPT-PVY 8093-8292 PVX 85-284 by colony PCR and DNA sequencing. The plasmid pCB F R2-2bPT-PVY 8093-8292 PVX 85-284The nucleotide sequence is as shown in SEQ ID NO.3. The primer pCB F 2-PVYPVX-F, pCB F The nucleotide sequences of 2-PVYPVX-R are as shown in SEQ ID NO.9 and SEQ ID NO.10.

[0034] Result analysis: After the ligation product was transformed, the product was preliminarily screened by colony PCR, and it was basically confirmed that the plasmid contained the gene sequences of PVY and PVX; the plasmid was extracted and sequenced, and finally the positive clone plasmid pCB F R2-2bPT-PVY 8093- 8292 PVX 85-284 .

[0035] Example 2. Biological effects of plasmid pCB F R2-2bPT-PVY 8093-8292 PVX 85-284

[0036] Agrobacterium infiltration to infect potato variety Kexing No.1:

[0037] Screening: The plasmids pCB Fny of CMV isolate RNA1, pCB F R1, pCB F R2-2bPT-PVY 8093-8292 PVX 85-284 , CMV Fny isolate RNA3 aphid transmission site mutant plasmid pCB F R3-△apno were respectively transformed into competent cells of Agrobacterium tumefaciens GV3101, and screened with LB solid medium containing 50 µg / ml kanamycin and 100 µg / ml rifampicin. After picking single colonies, PCR verification was carried out. The primers used were PVYPVX-F and PVYPVX-R, and the primer sequences are as shown in SEQ ID NO.11 and SEQ ID NO.12.

[0038] Induction: The verified positive Agrobacterium colonies were respectively picked and added to 5 ml of LB liquid medium (containing 50 µg / ml kanamycin and 100 µg / ml rifampicin), and cultured overnight at 28°C with shaking at 200 rpm.

[0039] Resuspension: The induced bacterial liquid was centrifuged at 6000 rpm for 10 min. The cells were collected and resuspended in Agrobacterium resuspension solution (10 mM MgCl2, 10 mM MES, 0.1 mM AS), and pipetted and mixed evenly. Adjust the OD 600 to 1.5. The OD of the three adjusted 600 ​Mix the bacterial liquid of the value in equal proportion and let it stand at 28°C for 3 h.

[0040] Inoculation: Take a 1 ml disposable syringe, remove the needle and suck the mixed Agrobacterium liquid. Select the first potato star removed from the tissue culture bottle, and use pressure to inject the liquid in the syringe between the veins on the back of the leaf. Inject two leaves per plant, and the injection volume per leaf should cover at least 2 / 3 of the leaf.

[0041] The inoculated plants were cultured at 25°C with an alternating cycle of 16 h light / 8 h dark.

[0042] Result analysis: 7 days after inoculation, inoculated with pCB F R1 / pCB F R2-2bPT-PVY 8093-8292 PVX 85-284 / pCB F R3-△apno was basically the same as the healthy control, without obvious virus disease symptoms. However, the growth of the plants inoculated with pCB F R1 / pCB F R2 / pCB F R3 plants was weak as Figure 2 shown; indicating that pCB F R2-2bPT-PVY 8093-8292 PVX 85-284 was a mild mutant.

[0043] To detect the genetic stability of the mutant after inoculating potatoes, after inoculation, subculture for three generations, sample the top leaves of potato plants and extract RNA, and use RT-PCR to detect whether the heterologous virus insertion fragment is stably present. The primers used are PVYPVX-F and PVYPVX-R, and the nucleotide sequences of the primers PVYPVX-F and PVYPVX-R are shown in SEQ ID NO.11 and SEQ ID NO.12.

[0044] Result analysis: After pCB F R2-2bPT-PVY 8093-8292 PVX 85-284 was inoculated on potatoes and subcultured, the virus insertion fragment could be detected. According to the size of the fragment, it was judged that the inserted PVY and PVX fragments still existed, as Figure 5 shown.

[0045] After starving the cotton aphids for 4 h, they were inoculated onto potato plants respectively. There were two groups of treatments. The control group was inoculated with the aphid transmission site vaccine pCB F R1 / pCB F R2-2bPT-PVY 8093-8292 PVX 85-284 / pCB FR3; The aphid-transmitted mutation sequence is shown in SEQ ID NO. 4. The treatment group was vaccinated with the mutant aphid-transmitted site vaccine pCB F R1 / pCB F R2-2bPT-PVY 8093-8292 PVX 85-284 / pCB F R3-△apno, cotton aphid RNA was extracted and the virus transmission efficiency was detected by RT-PCR.

[0046] Result analysis: As shown in Table 3, the virus transmission rate of the control group was 92.0%, and that of the treatment group was 4.0%, indicating that vaccination with the mutant aphid-transmitted site vaccine greatly reduced the aphid virus transmission efficiency.

[0047] Table 3 Detection of aphid virus transmission efficiency

[0048]

[0049] Example 3. Plasmid pCB F R2-2bPT-PVY 8093-8292 PVX 85-284 Cross-protection effect test

[0050] Using the method in Example 2, inoculate pCB F R1 / pCB F R2-2bPT-PVY 8093-8292 PVX 85-284 / pCB F R3-△apno for cross-protection test. 5 days after inoculating the attenuated mutant, 12 days after inoculating the plants pre-inoculated with the attenuated mutant and the plants not pre-inoculated with the attenuated mutant with PVY, PVX, and CMV, test their cross-protection effect. And detect the relative expression level of the virus by fluorescence quantitative method.

[0051] Result analysis: When PVY, PVX, and CMV were directly inoculated on potatoes, the plants were generally short, the leaves were mottled and wrinkled, and there were mosaic symptoms; while pre-inoculating pCB F R1 / pCB F R2-2bPT-PVY 8093-8292 PVX 85-284 / pCB F R3-△apno and then inoculating with strong virus (PVY, PVX, and CMV), the plant height was slightly shorter than that of the control, and the growth state was good, indicating that pre-inoculating pCB on potatoes F R1 / pCB F R2-2bPT-PVY 8093-8292 PVX 85-284 / pCB FR3-△apno has good cross-protection control effects against PVY, PVX, and CMV, as Figure 3 shown. The result analysis of fluorescence quantification is as Figure 4 shown. After 12 days of agroinfiltration inoculation with PVY + PVX + CMV, the relative replication amounts of PVY, PVX, and CMV viruses were detected by fluorescence quantification in the control group (only inoculated with PVY + PVX + CMV) and the treatment group (pre-mixed inoculation with agro-bacteria containing pCB F R1 / pCB F R2-2bPT-PVY 8093- 8292 PVX 85-284 / pCB F R3-△apno for 5 days). The relative replication amounts of viruses in the treatment group were significantly lower than those in the control group, indicating that pCB F R1 / pCB F R2-2bPT-PVY 8093-8292 PVX 85-284 / pCB F R3-△apno has good cross-protection control effects against PVY, PVX, and CMV.

[0052] The above are only the preferred embodiments of the present application and are not used to limit the present application. For those skilled in the art, various changes and modifications can be made to the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included within the protection scope of the present application.

Claims

1. A trivalent attenuated vaccine that is resistant to PVY, PVX, and CMV, characterized in that, The trivalent attenuated vaccine contains a mutant plasmid pCB of cucumber mosaic virus RNA2 F R2-2bPT-PVY 8093-8292 PVX 85-284 ; The plasmid pCB F R2-2bPT-PVY 8093-8292 PVX 85-284 has a nucleotide sequence as shown in SEQ ID NO.3 2. The trivalent attenuated vaccine according to claim 1, wherein The mutant plasmid pCB F R2-2bPT-PVY 8093-8292 PVX 85-284 was constructed as follows: Based on the full-length sequence of RNA2 containing a CMV Fny isolate, a double stop codon TAATAG is inserted between positions 2682 and 2683, Sma an I restriction site, and conserved sequences of potato virus Y (PVY) and potato virus X (PVX); the nucleotide sequences of the PVY and PVX fragments are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

3. Mutant plasmid pCB in the trivalent attenuated vaccine as claimed in claim 1 F R2-2bPT-PVY 8093-8292 PVX 85-284 Use in the prevention and treatment of PVY, PVX and CMV 4. Application of the mutant plasmid pCB in the trivalent attenuated vaccine as claimed in claim 1 F R2-2bPT-PVY 8093-8292 PVX 85-284 in the control of aphids.

5. A medicament comprising the trivalent attenuated vaccine as claimed in claim 1 or 2.

6. Use of the medicament as claimed in claim 5 for preventing and treating PVY, PVX and CMV.

7. Use of the medicament as claimed in claim 5 for preventing and treating aphids.

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