Trivalent attenuated vaccine resistant to PVY, PVX and CMV and application of trivalent attenuated vaccine resistant to PVY, PVX and CMV

Abstract

The invention provides a trivalent attenuated vaccine resistant to PVY, PVX and CMV and application, and belongs to the field of plant virology and molecular biology. The trivalent attenuated vaccine contains a mutant plasmid pCBFR2-2bPT-PVY8093-8292PVX85-284 of the cucumber mosaic virus (CMV) RNA2, and the sequence of the mutant plasmid is as shown in SEQ ID NO. 3. The invention also discloses a preparation method of the trivalent attenuated vaccine. According to the invention, the plasmid pCBFR1 containing the CMVFny isolate RNA1 and the aphid transmission site mutation plasmid pCBFR3-apno of the CMVFny isolate RNA3 are mixed and inoculated with the mutant plasmid in advance, so that the potato Y virus, the potato X virus and the cucumber mosaic virus can be prevented, meanwhile, the vaccine can prevent aphid transmission and avoid potential biological safety hazards, and vaccine materials and effective measures are provided for field prevention and treatment 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 capable of resisting PVY, PVX and CMV and its application, and in particular to a mutant plasmid containing potato virus Y (PVY), potato virus X (PVX), cucumber mosaic virus (CMV) and aphid-transmitted sites, and is used for preventing and controlling potato virus Y (PVY), potato virus X (PVX) and cucumber mosaic virus (CMV). Background Art

[0002] potato( Solanum tuberosum L.) is the fourth largest staple food crop in my country and occupies a core position in my country's agricultural economic system. Potato virus diseases have caused serious damage 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 caused is more than 80%. Potato virus X (PVX) has mild symptoms when it infects alone. Under natural conditions, it is usually co-infected with PVY. In severe cases, the yield loss can reach 50%. Although cucumber mosaic virus (CMV) has not been reported to significantly reduce potato yield, its detection rate in Gansu Province 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 infect together, they will inhibit the potato's defense mechanism and facilitate the infection of other viruses, causing serious losses in yield and quality.

[0003] The phenomenon of cross-protection is that after pre-vaccination with a weak strain, inoculation with a strong strain can significantly reduce the symptoms of the strong toxicity. The sources of the weak strains are partly obtained by natural selection and partly by artificial modification. CMV can infect more than 100 plant families such as Solanaceae and Cucurbitaceae, and has a wide host range. The 2b protein it encodes is one of the earliest discovered silencing suppressors. Early 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 on potatoes that can simultaneously resist potato virus Y, potato virus X and cucumber mosaic virus. Summary of the invention

[0004] In view of the above problems, the present invention provides a trivalent attenuated vaccine against PVY, PVX and CMV and its application, specifically a mutant plasmid pCB containing cucumber mosaic virus (CMV) RNA2 F R2-2bPT-PVY 8093-8292 PVX 85-284 , and containing CMVFny Plasmid pCB F R1, aphid-transmitted site mutation plasmid pCB F R3-△apno mixed inoculation is used to control potato virus Y (PVY), potato virus X (PVX) and cucumber mosaic virus (CMV). At the same time, the vaccine can prevent aphids from transmitting the virus, providing vaccine materials and effective prevention and 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 solution: In a first aspect of the present invention, a trivalent attenuated vaccine is provided that is resistant to PVY, PVX and CMV, wherein the trivalent attenuated vaccine contains a mutant plasmid pCB F R2-2bPT-PVY 8093-8292 PVX 85-284 ; The construction of the mutant plasmid of cucumber mosaic virus (CMV) RNA2 is as follows: In the presence of CMV Fny Based on the full-length sequence of RNA2 from the isolate, a double stop codon TAATAG was inserted between positions 2682 and 2683. Sma I restriction site, and 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 plasmid pCB F R2-2bPT-PVY 8093-8292 PVX 85-284 The nucleotide sequence is shown in SEQ ID NO.3.

[0006] In the second aspect of the present invention, the mutant plasmid pCB F R2-2bPT-PVY 8093-8292 PVX 85-284 Application in the control of PVY, PVX and CMV, and in the control of aphids; the application steps are as follows: pCB F R2-2bPT-PVY 8093-8292 PVX 85-284 , containing CMV Fny Plasmid pCB from isolate RNA1 F R1 and CMV Fny Isolate RNA3 aphid-transmitted site mutation plasmid pCB F R3-△apno was used to transform Agrobacterium to obtain Agrobacterium bacterial solution A, Agrobacterium bacterial solution B and Agrobacterium bacterial solution C. The three bacterial solutions were mixed and inoculated on potatoes. The mutant plasmid pCB F R3-△apno contains CMV Fny The full-length sequence of RNA3 of the isolate, with mutations at amino acids 129 and 162. The construction method is detailed in the patent application (patent number CN202411479723.4) Beneficial effects of the present invention: (1) Plasmid pCB containing the 2b protein premature termination mutant of cucumber mosaic virus RNA2 F R2-2bPT, the present invention inserts the conservative fragments of PVY and PVX after the termination codon to obtain the 2b protein premature termination mutant pCB of cucumber mosaic virus RNA2 containing PVY and PVX gene fragments F R2-2bPT-PVY 8093-8292 PVX 85-284 , this plasmid contains CMV Fny Plasmid pCB from isolate RNA1 F R1 and CMV Fny Isolate RNA3 aphid-transmitted site mutation plasmid pCB F The Agrobacterium of R3-△apno was mixed and inoculated to prevent infection by PVY, PVX and CMV, as a trivalent attenuated vaccine against potato virus Y, potato virus X and cucumber mosaic virus.

[0007] (2) The mutant plasmid pCB provided by the present invention F R2-2bPT-PVY 8093-8292 PVX 85-284 With CMV Fny Plasmid pCB from isolate RNA1 F R1 and CMV Fny Isolate RNA3 aphid-transmitted site mutation plasmid pCB F When Agrobacterium tumefaciens mixed with R3-△apno was inoculated into potatoes, the inserted virus fragment could still exist stably after three generations of subculture.

[0008] (3) The mutant plasmid pCB provided by the present invention F R3-△apno, mutates the amino acids at positions 129 and 162. The mutated vaccine can stably exist in the plant without affecting other functions, avoiding the biosafety risks caused by the spread of aphids during vaccination. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 Schematic diagram of constructing attenuated vaccine vector; from top to bottom are pCB F R1, pCB F R2-2bPT-PVY8093-8292 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).

[0010] Figure 2 This is a picture of the symptom effect of attenuated vaccine inoculated on potato 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.

[0011] 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.

[0012] 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 F The viral replication amount was plotted 5 days after inoculation of Agrobacterium R3-△apno and 12 days after inoculation of PVY+PVX+CMV.

[0013] Figure 5The stability test results of attenuated vaccines are shown in Figure 1; M is the D2000 molecular weight standard of GenStar; 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. DETAILED DESCRIPTION

[0014] The technical scheme of the present invention is described in detail below through specific implementation methods. It should be understood that the following specific implementation methods are only exemplary, and any changes or modifications, as long as they do not deviate from the technical scheme design of the present invention, should be within the scope of protection of the rights of the present invention. The present invention is described in detail below with reference to the examples. The experimental materials used in the examples of the present invention that are not specifically described are all conventional experimental materials in the field and can be purchased through commercial channels. If the specific experimental conditions and methods are not specified in the examples of the invention, conventional conditions are generally followed, such as the Molecular Cloning Experiment Guide (Fourth Edition), edited by J. Sambrook et al., Science Press, 2017; or the conditions recommended by the manufacturer. Schematic diagram of the construction of the attenuated vaccine vector is shown in Figure 1 shown.

[0015] Example 1. Plasmid pCB F R2-2bPT-PVY 8093-8292 PVX 85-284 Build Cloning of PVY fragment: 80 PVY strains were selected, as shown in Table 1-1 and Table 1-2. After sequence comparison analysis, the region with more conservative sequences 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. The PVY (NC001616) infectious cloning plasmid was used as a template, and the PVY fragment was cloned by PCR using 2×PhantaMax Master Mix (Dye Plus) (Vazyme). PCR conditions: 95 ℃ pre-denaturation for 3 min; 95 ℃ denaturation for 15 s, 55 ℃ annealing for 15 s, 72 ℃ extension for 15 s, 35 cycles; 72 ℃ extension for 5 min; 4 ℃ storage; PCR products were recovered by DNA gel recovery kit (Tiangen) and used for later 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.

[0016] Table 1-1 PVY selected strains

[0017] Table 1-2 PVY selected strains

[0018] Cloning of PVX fragment: 75 PVX strains were selected, as shown in Table 2-1 and Table 2-2. After sequence comparison analysis, the region with more conservative sequences was selected, which is RNA-dependent RNA replicase. Primers PVX-85-PVY-F and PVX-284-R were designed, and the PVX (EU571480) infectious cloning plasmid was used as a template. The PVX fragment was cloned by PCR using 2×Phanta Max Master Mix (DyePlus) (Vazyme). PCR conditions: 95 ℃ pre-denaturation for 3 min; 95 ℃ denaturation for 15s, 55 ℃ annealing for 15s, 72 ℃ extension for 15s, 35 cycles; 72 ℃ extension for 5 min; 4 ℃ storage; PCR products were recovered by DNA gel recovery kit (Tiangen) and used for later 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.

[0019] Splicing of PVY and PVX fragments: The PCR products of the above-mentioned recovered PVY and PVX were used as templates, the primers were PVY-8093-F and PVX-284-R, and the PCR reaction was carried out using 2×Phanta Max Master Mix (Dye Plus) (Vazyme). Fragment splicing conditions: 95 ℃ pre-denaturation for 3 min; 95 ℃ denaturation for 15 s, 55 ℃ annealing for 15 s, 72 ℃ extension for 15 s, 5 cycles; PCR conditions: 95 ℃ pre-denaturation for 3 min; 95 ℃ denaturation for 15 s, 55 ℃ annealing for 15 s, 72 ℃ extension for 20 s, 30 cycles. The PCR product is the fragment after splicing of PVY and PVX fragments. The PCR product was recovered by a DNA recovery kit (Tiangen) and set aside. 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.

[0020] Table 2-1 PVX selected strains

[0021] Table 2-2 PVX selected strains

[0022] pCB F R2-2bPT Sma I single enzyme digestion, the digestion product was recovered by DNA recovery kit (Tiangen), and the single enzyme digestion intermediate vector pCB F The products of R2-2bPT and PVY and PVX fragments were mixed at a molar ratio of 1:2 between vector and insert fragments and recombined using ClonExpress Ultra One Step Cloning Kit (Vazyme). The recombination primers were pCB F 2-PVYPVX-F, pCB F 2-PVYPVX-R, and the mixed product was reacted in a PCR instrument at 50 °C for 5 min. The recombinant product was transformed into competent cells of Escherichia coli DH5α, screened with LB solid medium containing 100 μg / mL kanamycin, and the positive clone plasmid pCB was confirmed by colony PCR and DNA sequencing. F R2-2bPT-PVY 8093-8292 PVX 85-284 , the plasmid pCB F R2-2bPT-PVY 8093-8292 PVX 85-284 The nucleotide sequence of is shown in SEQ ID NO.3. The primer pCB F2-PVYPVX-F, pCB F The nucleotide sequences of 2-PVYPVX-R are shown in SEQ ID NO.9 and SEQ ID NO.10.

[0023] Result analysis: After the ligation product was transformed, the product was initially 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 was confirmed. F R2-2bPT-PVY 8093- 8292 PVX 85-284 .

[0024] Example 2. Plasmid pCB F R2-2bPT-PVY 8093-8292 PVX 85-284 Biological effects Agrobacterium infiltration and infection of potato variety Nemesis No. 1: Screening: CMV Fny Plasmid pCB from isolate RNA1 F R1, pCB F R2-2bPT-PVY 8093-8292 PVX 85-284 、CMV Fny Isolate RNA3 aphid-transmitted site mutation plasmid pCB F R3-△apno were used to transform competent cells of Agrobacterium GV3101, respectively, and screened with LB solid medium containing 50 µg / ml kanamycin and 100 µg / ml rifampicin. Single colonies were picked and verified by PCR. The primers used were PVYPVX-F and PVYPVX-R, and the primer sequences are shown in SEQ ID NO.11 and SEQ ID NO.12.

[0025] Induction: Pick the verified positive Agrobacterium colonies separately, add them to 5 ml LB liquid medium (containing 50 µg / ml kanamycin and 100 µg / ml rifampicin), and culture overnight at 28°C with shaking at 200 rpm.

[0026] Resuspension: Centrifuge the induced bacterial solution at 6000rpm for 10min. Collect the bacterial cells and resuspend them in Agrobacterium resuspension solution (10mM MgCl2, 10mM MES, 0.1mM AS). Pipette and mix well to adjust the OD 600 is 1.5. Adjust the OD of the three 600 The bacterial suspensions with the same value were mixed in equal proportions and allowed to stand at 28°C for 3 h.

[0027] Inoculation: Take a 1 ml disposable syringe, remove the needle and absorb the mixed Agrobacterium solution, select Potato Killer No. 1 removed from the tissue culture bottle, and use pressure to inject the solution in the syringe between the veins on the back of the leaf. Inject two leaves per plant, and the injection amount of each leaf should cover at least 2 / 3 of the leaf.

[0028] The inoculated plants were cultured at 25°C with alternating 16 h light / 8 h dark periods.

[0029] Results: 7 days after inoculation, 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, with no obvious symptoms of viral disease. F R1 / pCB F R2 / pCB F R3 plants grew weakly. Figure 2 As shown; indicating that pCB F R2-2bPT-PVY 8093-8292 PVX 85-284 It is a weak mutant.

[0030] To detect the genetic stability of the mutant after inoculation of potatoes, after inoculation, three generations of subculture were performed, samples were taken from the top leaves of potato plants and RNA was extracted, and RT-PCR was used to detect whether the heterologous virus insertion fragment was stable. The primers used were 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.

[0031] Result analysis: pCB F R2-2bPT-PVY 8093-8292 PVX 85-284 After inoculation of potato and subculture, virus insertion fragments can be detected. According to the size of the fragments, it can be judged that the inserted PVY and PVX fragments are still present, such as Figure 5 shown.

[0032] After 4 h of starvation treatment, cotton aphids were inoculated into potato plants. The control group was inoculated with pCB vaccine at the aphid transmission site. F R1 / pCB F R2-2bPT-PVY 8093-8292 PVX 85-284 / pCB F R3; the aphid-transmitted mutant sequence is shown in SEQ ID NO. 4. The treatment group was the mutant aphid-transmitted site vaccine pCBF R1 / pCB F R2-2bPT-PVY 8093-8292 PVX 85-284 / pCB F R3-△apno, extract RNA from cotton aphid and detect the virus transmission efficiency through RT-PCR.

[0033] Result analysis: As shown in Table 3, the virus transmission rate of the control group was 92.0%, and the virus transmission rate of the treatment group was 4.0%, indicating that vaccination with mutant aphid transmission sites greatly reduced the virus transmission efficiency of aphids.

[0034] Table 3 Detection of aphid virus transmission efficiency

[0035] Example 3. Plasmid pCB F R2-2bPT-PVY 8093-8292 PVX 85-284 Cross-protection effect test Using the method in Example 2, pCB F R1 / pCB F R2-2bPT-PVY 8093-8292 PVX 85-284 / pCB F R3-△apno was used for cross protection test. Five days after inoculation with attenuated mutants, plants pre-inoculated with attenuated mutants and plants not pre-inoculated with attenuated mutants were infected with PVY, PVX, and CMV for 12 days to test their cross protection effect. The relative expression of the virus was detected by fluorescence quantitative method.

[0036] Results: When PVY, PVX and CMV were directly inoculated into potatoes, the plants were short, the leaves were mottled and wrinkled, and there were mosaic symptoms; F R1 / pCB F R2-2bPT-PVY 8093-8292 PVX 85-284 / pCB F After R3-△apno was inoculated with strong toxins (PVY, PVX and CMV), the plant height was slightly shorter than that of the control and the growth state was good, indicating that the pCB pre-inoculated potato F R1 / pCB F R2-2bPT-PVY 8093-8292 PVX 85-284 / pCB F R3-△apno has good cross-protection effect on PVY, PVX and CMV. Figure 3 The results of fluorescence quantitative analysis are shown in Figure 4As shown, 12 days after the injection of PVY+PVX+CMV by Agrobacterium, the control group (inoculated with PVY+PVX+CMV only) and the treatment group (inoculated with pCB F R1 / pCB F R2-2bPT-PVY 8093- 8292 PVX 85-284 / pCB F R3-△apno Agrobacterium tumefaciens was used for 5 days to detect the relative replication of PVY, PVX, and CMV viruses by fluorescence quantitative analysis. The relative replication of the viruses in the treatment group was significantly lower than that in the control group, indicating that pCB F R1 / pCB F R2-2bPT-PVY 8093-8292 PVX 85-284 / pCB F R3-△apno has a good cross-protection effect on PVY, PVX and CMV.

[0037] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A trivalent attenuated vaccine against PVY, PVX and CMV, characterized in that: The trivalent attenuated vaccine contains a mutant plasmid pCB1 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 The nucleotide sequence is shown in SEQ ID NO.

3.

2. The trivalent attenuated vaccine according to claim 1, characterized in that: The mutant plasmid pCB F R2-2bPT-PVY 8093-8292 PVX 85-284 The construction is as follows: In the presence of CMV Fny Based on the full-length sequence of RNA2 from the isolate, a double stop codon TAATAG was inserted between positions 2682 and 2683. Sma I restriction site, and 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, respectively.

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

4. The mutant plasmid pCB in the trivalent attenuated vaccine as claimed in claim 1 F R2-2bPT-PVY 8093-8292 PVX 85-284 Application in the control of aphids.

5. The use according to claim 3 or 4, characterized in that Here are the steps: pCB F R2-2bPT-PVY 8093-8292 PVX 85-284 , containing CMV Fny Plasmid pCB from isolate RNA1 F R1 and CMV Fny Isolate RNA3 aphid-transmitted site mutation plasmid pCB F R3-△apno was used to transform Agrobacterium to obtain Agrobacterium culture liquid A, Agrobacterium culture liquid B and Agrobacterium culture liquid C. The three culture liquids were mixed and inoculated onto potatoes.

6. The use according to claim 5, characterized in that The mutant plasmid pCB F R3-△apno contains CMV Fny The full-length sequence of RNA3 of the isolate was obtained, and the 129th and 162nd amino acids were mutated.

7. A medicine containing the trivalent attenuated vaccine as claimed in claim 1 or 2.

8. Use of the drug as claimed in claim 7 in preventing and treating PVY, PVX and CMV.

9. Use of the drug as claimed in claim 7 in controlling aphids.

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