CN112410351A

CN112410351A - Duplex attenuated vaccine for resisting cucumber mosaic virus and potato virus X and application thereof

Info

Publication number: CN112410351A
Application number: CN202011259238.8A
Authority: CN
Inventors: 原雪峰; 刘珊珊; 葛玉倩; 于成明; 耿国伟
Original assignee: Shandong Agricultural University
Current assignee: Shandong Agricultural University
Priority date: 2020-11-12
Filing date: 2020-11-12
Publication date: 2021-02-26

Abstract

The invention discloses a duplex attenuated vaccine for resisting cucumber mosaic virus and potato virus X and application thereof, wherein the duplex attenuated vaccine is a cucumber mosaic virus RNA2 attenuated mutant plasmid vector pCC inserted with PVX gene segment_FR2‑2bPTⅡ‑PX_1967‑2166(ii) a The nucleotide sequence of the PVX gene segment is shown as SEQ ID NO.1, and the nucleotide sequence of the duplex attenuated vaccine is shown as SEQ ID NO. 2. The dual attenuated vaccine has cross protection effect on CMV and PVX; provides vaccine materials and effective control measures for field control of plant virus diseases caused by CMV and PVX.

Description

Duplex attenuated vaccine for resisting cucumber mosaic virus and potato virus X and application thereof

Technical Field

The invention relates to the technical field of plant virology and molecular biology, in particular to a dual attenuated vaccine for resisting cucumber mosaic virus and potato virus X and application thereof.

Background

The virus diseases are the second major plant diseases next to fungal diseases, cause different degrees of harm to most crops, and the prevention and control of the plant virus diseases are research hotspots and difficult problems. Cucumber Mosaic Virus (CMV) is one of the currently known plant viruses with the widest host range, and can infect more than 100 families and more than 1200 plants, so that various plants (crops, horticultural crops and the like) with important economic values have symptoms of yellowing, dwarfing, deformity and the like, and the yield and the quality of the plants are seriously influenced (Mochizuki et al, 2012). Potato Virus X (PVX) hosts are widespread and can infect 240 plants of 16 families, solanaceae being the main host, the first infected leaves usually show necrotic spots, followed by necrotic mottle, chlorosis, mosaic or vein chlorosis, resulting in severe yield reduction of plants (old corn pops, et al, 2016).

In nature, infection of the same host plant by different viruses sometimes exacerbates the symptoms of the viral disease and results in an increased concentration of at least one virus in the plant, a phenomenon known as synergism. The synergistic phenomenon of plant viruses exists widely in nature, and the damage caused by the compound infection of the cucumber mosaic virus and the potato virus X is larger, so that the design of a strategy capable of resisting various viruses at the same time is particularly important.

For the prevention and treatment of plant virus diseases, the most effective method is the screening and cultivation of disease-resistant varieties, but the method has the defects of long breeding period, easy loss of resistance of the disease-resistant varieties and the like. The other method is weak-toxicity cross protection, which is to use weak-toxicity mutant to infect and induce the immune system of the plant to prevent and control the later-stage infected strong virus strain of the same type; is an effective virus control strategy. The attenuated mutants may be naturally occurring or may be artificially prepared by molecular biological means.

For the double attenuated vaccine artificially prepared by a molecular biology means, because the fragment of the heterologous virus needs to be inserted during construction, the heterologous virus fragment with a certain length is inserted to ensure the prevention and treatment effect of potential gene silencing on the heterologous virus, and simultaneously the stability of the attenuated mutant (namely the stable existence after the heterologous virus fragment is inserted) is ensured, the construction difficulty of the double attenuated vaccine is higher compared with the attenuated vaccine resisting the single virus. However, a duplex attenuated vaccine which takes the cucumber mosaic virus as a basic vector and can resist the cucumber mosaic virus and the potato virus X at the same time is not reported at present.

Disclosure of Invention

Aiming at the prior art, the invention aims to provide a dual attenuated vaccine for resisting cucumber mosaic virus and potato virus X. The dual attenuated vaccine has cross protection effect on CMV and PVX; provides vaccine materials and effective control measures for field control of plant virus diseases caused by CMV and PVX.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect of the invention, a duplex attenuated vaccine for resisting cucumber mosaic virus and potato virus X is provided, wherein the duplex attenuated vaccine is a cucumber mosaic virus RNA2 attenuated mutant plasmid vector inserted with PVX gene segments; the nucleotide sequence of the PVX gene segment is shown as SEQ ID NO.1, and the nucleotide sequence of the duplex attenuated vaccine is shown as SEQ ID NO. 2.

In a second aspect of the present invention, a method for preparing the dual attenuated vaccine is provided, which comprises the following steps:

(1) with CMV_FnyRNA2 infectious clone plasmid pCB301-CMV_Fnyusing-R2 as template, obtaining intermediate carrier pCC by inverse PCR amplification technology_FR2-2bPT II; the intermediate vector pCC_FInserting TAATAG at the 2661 position of R2-2bPT II, and then sequentially introducing enzyme cutting sites of BamH I, SpeI and Sma I;

(2) the PVX gene fragment shown in SEQ ID NO.1 is inserted into an intermediate vector pCC by utilizing an enzyme digestion and connection method_FR2-2bPT II, namely constructing the dual attenuated vaccine pCC for resisting the cucumber mosaic virus and the potato virus X_FR2-2bPTⅡ-PX_1967-2166。

Preferably, in the step (1), the primer sequences for the reverse PCR amplification are shown as SEQ ID NO.3 and SEQ ID NO.4, respectively.

Preferably, in step (1), the conditions for the inverse PCR amplification are: denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 5s, extension at 72 ℃ for 8min, and 7 cycles; denaturation at 98 ℃ for 10s, extension at 68 ℃ for 8min, 25 cycles; storing at 4 ℃.

Preferably, in the step (2), the PVX gene segment shown in SEQ ID NO.1 is obtained by amplifying tobacco infected by PVX by using RT-PCR with the sequences shown in SEQ ID NO.5 and SEQ ID NO.6 as primers.

More preferably, in step (2), the reaction procedure of RT-PCR is: pre-denaturation at 94 ℃ for 2 min; denaturation at 94 ℃ for 30s, annealing at 53 ℃ for 30s, and extension at 72 ℃ for 10s for 30 cycles; extending for 2min at 72 ℃; storing at 4 ℃.

In a third aspect of the invention, the application of the dual attenuated vaccine in the prevention and treatment of plant virus diseases is provided; in particular, the plant virus disease is a plant virus disease caused by cucumber mosaic virus and potato virus X.

The invention has the beneficial effects that:

(1) the invention modifies the 2b protein coding sequence of cucumber mosaic virus, and obtains a cucumber mosaic virus attenuated mutant by inserting TAATAG at position 2661 and then sequentially introducing BamH I, SpeI and Sma I enzyme cutting sites; because the foreign fragment is inserted into the untranslated region, the modification mode of the invention can ensure that the inserted sequence only acts at the nucleotide level, and the controllability of the action of the vaccine is higher.

(2) For the obtained cucumber mosaic virus attenuated mutant, the invention further inserts the PVX gene segment after a new stop codon to obtain the cucumber mosaic virus attenuated mutant containing the PVX gene segment, prevents the infection of CMV and PVX virulent strains through cross protection, and can be used as a duplex attenuated vaccine for resisting the cucumber mosaic virus and the potato virus X.

(3) The inserted PVX gene fragment is a PVX gene conserved sequence with the length of 200bp, and has prevention and treatment potential on various PVX isolates.

Drawings

FIG. 1: basic vector pCB301-CMV_Fny-R2, intermediate vector pCC_FR2-2bPT II, and attenuated mutant pCC containing PVX_FR2-2bPTⅡ-PX_1967-2166A schematic diagram; in the figure, LB, T-DNA Left Border (LB); RB, T-DNA Right Border (RB); 35S, promoter (from gene 2x 35S); RZ, ribozyme (HDV-RZ); NOS，terminator(NOS terminator)。

FIG. 2: intermediate vector pCC_FThe electrophoresis schematic diagram of the enzyme digestion identification result of R2-2bPT II; wherein M is the D15000+2000 molecular weight standard of Tiangen company, and the fragment is about 7.6kb after enzyme digestion.

FIG. 3: plasmid vector pCC_FR2-2bPTⅡ-PX_1967-2166The enzyme digestion identification result electrophoresis diagram is shown; wherein M is D15000+2000 of Tiangen company, and an intermediate vector and a 200bp inserted PVX fragment are obtained after enzyme digestion.

FIG. 4: plasmid pCC_FR2-2bPTⅡ-PX_1967-2166Alignment of the PVX sequence contained in (1) with the full length of PVX (NCBI accession number EU 571480).

FIG. 5: pCC_FR2-2bPTⅡ-PX_1967-2166Weak pathogenic symptoms on medium smoke 100; mock, empty agrobacterium liquid; r1, CMV_Fny RNA1；R2，CMV_Fny RNA2；R3，CMV_Fny RNA3；pCC_FR2-2bPTⅡ-PX_1967-2166Containing CMV_FnyRNA2 full sequence and 200bp conserved sequence of PVX inserted after the 2b protein early stop codon.

FIG. 6: pCC_FR2-2bPTⅡ-PX_1967-2166Stability test of the PVX insert of (1) in plants; wherein M is a D2000 molecular weight standard from Tiangen corporation; mock, empty agrobacterium liquid; r2, CMV_Fny RNA2；R2-2bPTⅡ-PX_1967-2166Containing CMV_FnyThe complete sequence of RNA2, and a 200bp conserved fragment of PVX is inserted after the 2b protein early stop codon; dpi, days post inoculation.

FIG. 7: pCC_FR2-2bPTⅡ-PX_1967-2166Cross-protection effect on CMV and PVX; wherein mock is an empty agrobacterium liquid; post-infection by PVX + CMV, inoculating PVX and CMV; no pre-infection, No pre-injection inoculation of the attenuated mutant; pre-infection by R1/R2-2bPT II-PX_1967-2166/R3, preinjection inoculation containing CMV_Fny RNA1/R2-2bPTⅡ-PX_1967-2166/CMV_FnyR3.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

As introduced in the background art, for the duplex attenuated vaccine artificially prepared by molecular biology means, because the fragment of the heterologous virus needs to be inserted during construction, the insertion of the fragment with a certain length is ensured to effectively trigger the gene silencing effect on the heterologous virus, and the stability of the inserted fragment in the attenuated mutant is also ensured, so that the construction difficulty of the duplex attenuated vaccine is higher compared with the attenuated vaccine resisting the single virus.

The CMV genome consists of three positive single-stranded RNAs (RNA1, RNA2, and RNA3) that encode a total of 5 proteins. The 2b protein, produced from subgenomic RNA4A, is a gene silencing inhibitor that protects the viral genome to the greatest extent from degradation by the host plant (Csorba et al, 2015). As the CMV 2b protein is a virus-coded gene silencing inhibitor, the CMV attenuated mutant has strong inhibiting effect on the gene silencing effect of host plants on virus genomes, and the mutation and the modification of the coding gene of the CMV 2b protein are hot spots for the research of constructing the CMV attenuated mutant.

The inventor develops and designs a cucumber mosaic virus RNA2 attenuated mutant plasmid vector containing a tobacco PDS gene segment in earlier research (CN108486148), modifies a 2b protein coding sequence to obtain an attenuated mutant, and inserts the tobacco PDS gene segment in the modification process of the attenuated mutant to form naked eye visible induced gene silencing symptoms, thereby providing great convenience for the deep research of CMV-based attenuated vaccines. However, the plasmid vector is used for development and test of vaccine creation and cannot be used for field control because of the gene silencing effect on plant endogenous PDS genes.

Moreover, the modification strategy for the 2b protein coding gene in CN108486148 is as follows: with CMV_FnyRNA2 infectious clone plasmid pCB301-CMV_FnyThe intermediate vector pCB301-CMV of 2b protein early termination is obtained by taking-R2 as a template and utilizing an inverse PCR amplification method_Fny2Del2b, which is a multiple cloning site containing BamH I, SpeI and Sma I and a TAATA sequence inserted between position 2661 and position 2662 immediately after the stop codon of the 2a protein. In further subsequent studies, it was found that, by using this modification strategy, the newly inserted sequence in the intermediate vector can act simultaneously at the nucleotide and amino acid levels, wherein the action at the amino acid level may involve changes in the 2b elongase protein sequence and tertiary structure, resulting in low controllability of the vaccine effect.

Based on this, the modification strategy of cucumber mosaic virus 2b protein coding gene is improved, TAATAG is firstly inserted into 2661 of the intermediate vector, and then enzyme cutting sites of BamH I, SpeI and Sma I are sequentially introduced. The cloning sites BamH I, Spe I and SmaI introduced were mainly prepared for the insertion of different types of fragments into the vector. An exogenous insert can be introduced by using the combination of any two enzyme cutting sites, so that 3 enzyme cutting sites are necessary to be designed to facilitate the insertion of different exogenous sequences.

The PVX genome consists of a single positive single-stranded RNA of approximately 6.4kbp in length, encoding a total of 5 proteins. In order to construct a duplex attenuated vaccine capable of resisting both cucumber mosaic virus and potato virus X, the invention preferably selects a conserved fragment from 1967 to 2166 in a PVX genome and inserts the conserved fragment into a cucumber mosaic virus attenuated mutant on the basis of obtaining the cucumber mosaic virus attenuated mutant; the conservative fragment is obtained by comparing the whole genome sequences of a plurality of PVX isolates, so that the attenuated vaccine can be ensured to be finally obtained, and the strong toxicity of the plurality of PVX isolates can be prevented and treated.

In a preferred embodiment of the present invention, Cucumber Mosaic Virus (CMV) is given_Fny) RNA2 mutant plasmid vector pCC_FR2-2bPTⅡ-PX_1967-2166The construction method specifically comprises the following steps:

(1)pCC_Fconstruction of R2-2bPT II intermediate vector: according to CMV published in GenBank_FnySequence of RNA2 (accession number: D00355), primers Fny2-2bPT 3' -BSS-2662-F andfny2-2bPT 3' -BT-2661-R (see Table 1 for details). With pCB301-CMV_Fny-R2 is a template, PCR reverse amplification is carried out under the action of PrimeSTAR HS DNA Polymerase (TaKaRa), plasmid template degradation is carried out by DpnI (NEB), the PCR amplification fragment is self-ligated after BamH I enzyme digestion, the ligation product is transformed into escherichia coli DH5 alpha, and the intermediate vector pCC is obtained after colony PCR, plasmid digestion identification and DNA sequencing_FR2-2bPT II. The intermediate vector pCC_FR2-2bPT II was added after 2661 with TAATAG and BamH I, SpeI, SmaI cleavage sites to effect premature termination of the 2b protein while preserving as much as possible the integrity of the other nucleotide sequences (FIG. 1).

Table 1: primers used in the present invention

Note: the protected bases are in bold, the cleavage sites are underlined, and the non-viral sequences are in italics.

(2) Cloning of partial fragments of PVX: based on the complete sequence of 5 different strains of PVX published in GenBank (accession numbers: EU571480, EF423572, AB056718, KM659859, JF430080), the sequence was aligned using DNMAN software to find the conserved regions of the sequence and design primers PVX-BamHI-1967-F and PVX-SmaI-2166-R (see Table 1 for details). The total RNA of the plant infected by PVX tobacco is extracted, and a 200bp segment corresponding to 1967-2166 site of the PVX genome is amplified by RT-PCR.

(3)pCC_FR2-2bPTⅡ-PX_1967-2166The construction of (1): double digestion of pCC with BamH I and SmaI_FR2-2bPT II and PVX200bp fragments, then the fragments are connected after being recovered by glue, the connection product is transformed into escherichia coli DH5 alpha, and the vector pCC is obtained after colony PCR, plasmid restriction identification and DNA sequencing_FR2-2bPTⅡ-PX_1967-2166。

In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments.

The test materials used in the examples of the present invention, which were not specifically described, were all those conventional in the art and commercially available. In the examples of the present invention, the specific experimental conditions and methods are not specified, and the conventional conditions such as J. SummBruker et al, science publishers, 2002, molecular cloning guidelines (third edition); master catalog of speekt et al, scientific press, 2001, cell experimental guidelines; or according to conditions recommended by the manufacturer.

Example 1 intermediate vector pCC_FConstruction of R2-2bPT II:

with CMV_FnyInfectious clone plasmid pCB301-CMV_Fny-R2 is a template ((CMV)_FnyThe construction of infectious clone plasmid pCB301-Fny2 refers to "construction of Agrobacterium mediated CMV infectious clone and 2b deletion mutant", YaoMin et al, Chinese agricultural science, 2011,44 (14): 3060) and reverse PCR amplification is carried out under the action of PrimeSTAR HS DNA Polymerase (Takara), wherein the primer pairs are Fny2-2bPT 3'-BSS-2662-F and Fny2-2bPT 3' -BT-2661-R; PCR conditions were as follows: denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 5s, extension at 72 ℃ for 8min, and 7 cycles; denaturation at 98 ℃ for 10s, extension at 68 ℃ for 8min, 25 cycles; storing at 4 deg.C; the PCR product obtained by amplification is the linearized intermediate vector pCC_FR2-2bPTⅡ。

Plasmid template pCB301-CMV in PCR reaction is degraded by DpnI (NEB)_Fny-R2 under the reaction conditions: 1h at 37 ℃; 20min at 80 ℃; and (3) recovering the product by using an absolute ethyl alcohol precipitation method: the product was added to ddH₂Adding 50ul of sodium acetate with pH of 5.23.0 mM and 1ml of absolute ethyl alcohol into the mixture until the volume of O is 450 mu l, precipitating the mixture for 2h at minus 80 ℃, centrifuging the mixture at the normal temperature of 12000rpm for 10min, pouring the liquid out, adding 600 mu l of 70% ethyl alcohol into the mixture, centrifuging the mixture at the normal temperature of 12000rpm for 5min, pouring the liquid out, centrifuging the mixture at the normal temperature of 12000rpm for 1min, completely sucking the liquid in a centrifuge tube, adding ddH into the centrifuge tube₂O30. mu.l was redissolved.

Then, the product was recovered by digestion with BamHI (Takara), and the digested product was recovered by DNA recovery kit and then digested with BamHI (Takara), followed by isolation at T₄DNA ligase (Takara) was ligated overnight at 16 ℃ to transform the ligation into E.coli DH 5. alpha. and applied to LB plates with 100. mu.g/mL kanamycin for colony PCR, plasmid restriction and DNA sequencing,confirmation of the intermediate vector pCC_FR2-2bPTⅡ。

And (4) analyzing results: the PCR product is subjected to enzyme digestion by BamH I and then self-ligation, the transformed product is subjected to colony PCR primary screening, the quality-improved plasmid is subjected to enzyme digestion identification, the BamH I and the SmaI are selected for enzyme digestion identification respectively, the enzyme digestion result shows that the plasmid can be linearized by the two enzymes, the length of the linearized plasmid is about 7.6kb (figure 2), and the plasmid is basically confirmed to contain multiple cloning sites of BamH I, Spe I and SmaI; and finally confirmed by DNA sequencing of the plasmid.

Example 2 cloning of partial fragments of PVX:

extracting the total RNA of the Nicotiana benthamiana infected by PVX (EU571480) by using TransZol Up (TransGen), carrying out Reverse transcription reaction under the action of Reverse Transcriptase M-MLV (Takara), wherein Reverse primers are PVX-SmaI-2166-R, and the reaction conditions are as follows: denaturing at 80 deg.c for 3min without adding enzyme; after reverse transcriptase is added, the reaction is carried out for 1.5h at 42 ℃;

then taking the cDNA of the reverse transcription product as a template, and carrying out PCR reaction under the action of 2 XEs Taq MasterMix (Dye) (CWBIO), wherein the primer pair is PVX-BamHI-1967-F and PVX-SmaI-2166-R; PCR conditions were as follows: pre-denaturation at 94 ℃ for 2 min; denaturation at 94 ℃ for 30s, annealing at 53 ℃ for 30s, and extension at 72 ℃ for 10s for 30 cycles; extending for 2min at 72 ℃; storing at 4 deg.C; the PCR product is a 200bp conserved sequence fragment in PVX. And recovering the PCR product by a DNA recovery kit for later use.

And (4) analyzing results: the PVX fragment was amplified by PCR, and the electrophoresis result showed that the PCR product was about 200bp, which was consistent with the 200bp alignment of the PVX conserved region (FIG. 4).

Example 3 pCC_FR2-2bPTⅡ-PX_1967-2166Construction:

an intermediate vector pCC_FCarrying out double enzyme digestion on R2-2bPT II by BamH I and SmaI, and recovering the enzyme digestion product by a DNA recovery kit; the 200bp conserved sequence fragment in the PVXRdRp is subjected to double enzyme digestion by BamH I and SmaI, and the enzyme digestion product is recovered by a DNA recovery kit. The intermediate vector pCC digested in two times is used_FR2-2bPT II and PVX fragment at T₄Carrying out overnight ligation at 16 ℃ under the action of DNA ligase; the ligation product was transformed into competent cells of E.coli DH 5. alpha. and plated on LB plates with 100. mu.g/mL kanamycin, and the cells were screened by colony PCR and digested with enzymeIdentification and DNA sequencing confirmation to obtain positive clone pCC_FR2-2bPTⅡ-PX_1967-2166。

And (4) analyzing results: after the ligation product is transformed, the product is primarily screened by colony PCR, the quality-improved grains are subjected to enzyme digestion identification, double enzyme digestion identification of BamH I and SmaI is selected, the enzyme digestion result shows that an enzyme digestion strip of about 200bp is formed, the size of the rest enzyme digestion fragments is about 7.6kb (figure 3), and the plasmid is basically confirmed to contain a gene sequence of PVX200 bp; and finally identified by DNA sequencing of the plasmid.

Example 4 plasmid vector pCC_FR2-2bPTⅡ-PX_1967-2166Biological effects of

100 parts of agrobacterium infiltration infected medium tobacco: the wild-type CMV is first_FnyRNA1、pCC_FR2-2bPTⅡ-PX_1967-2166Wild type CMV_FnyThe plasmid of RNA3 was used to transform competent cells of Agrobacterium GV3101, plated on LB plates with 50. mu.g/ml kanamycin and 100. mu.g/ml rifampicin, incubated at 28 ℃ for 48h, and single plaques were picked for colony PCR verification, and then picked into 2ml LB medium (50. mu.g/ml kanamycin, 100. mu.g/ml rifampicin) and incubated at 28 ℃ for 24h at 200 rpm.

Induction: separately collecting CMV_FnyRNA1、pCC_FR2-2bPTⅡ-PX_1967-2166、CMV_FnyRNA3, 100. mu.L of the bacterial suspension was added to 5ml of LB medium (containing 50. mu.g/ml kanamycin, 100. mu.g/ml rifampicin), and cultured at 28 ℃ and 200rpm with shaking to OD₆₀₀Is 1.0-2.0.

Resuspending: centrifuging the induced bacterial liquid in a 10ml centrifuge tube at 6000rpm for 10min at room temperature; the cells were collected and resuspended in 1ml Agrobacterium cell resuspension solution (10mM MgCl)₂10mM MES, 0.1mM AS) and mixing, taking 50ul bacterial liquid to 1ml resuspension, measuring OD₆₀₀Value, adjusting the concentration to OD₆₀₀1.2, adjusting the OD of the residual bacteria liquid according to the ratio of the bacteria liquid to the resuspension liquid₆₀₀0.9-1.5, and making 3 kinds of bacterial liquid OD₆₀₀And (5) the consistency is achieved.

Adjusting the OD of the three types₆₀₀Bacterial solutions of value (each containing CMV_FnyRNA1、pCC_FR2-2bPTⅡ-PX_1967-2166、CMV_FnyRNA3) equal ratioThe mixture was mixed and allowed to stand at 28 ℃ for 3 hours.

Inoculation: taking a 1ml disposable injector, removing a needle head to absorb the agrobacterium tumefaciens bacterial liquid, selecting the 3-4-leaf stage medium tobacco 100, injecting the bacterial liquid in the injector between veins on the back of the leaves by using pressure, injecting two leaves for each plant, and at least covering 1/3 of the leaves by the injection amount of each leaf.

The inoculated plants are placed at 25 ℃ for culture, and the illumination time of 16 h/the darkness time of 8h are alternated.

And (4) analyzing results: 10d after inoculation, wild type CMV was inoculated_FnyThe leaves of the medium tobacco 100 of RNA1/RNA2/RNA3 are shrunk and the plants are dwarfed; inoculation of CMV_FnyRNA1/RNA2-2bPTⅡ-PX_1967-2166the/RNA 3 was essentially identical to the healthy controls, with no obvious symptoms of viral disease. Indicating pCC_FR2-2bPTⅡ-PX_1967-2166A weakly virulent mutant of CMV RNA2 (fig. 5).

To test the genetic stability of the mutants after tobacco inoculation, samples were taken from the leaves of the non-inoculated system 9 days after inoculation, and RT-PCR was used to test whether the heterologous viral insert was stably present. To determine whether the heterologous viral insert is stably present in CMV_FnyPrimers CM-Fny2-2510-F (SEQ ID NO.7) and CM-Fny2-2760-R (SEQ ID NO.8) are designed upstream and downstream of the insertion sequence of the heterologous fragment of RNA2, and RT-PCR detection is carried out.

And (4) analyzing results: pCC_FR2-2bPTⅡ-PX_1967-2166On day 9 after the inoculation of tobacco 100, virus replication signals were detected in the system leaves by RT-PCR, and the presence of the inserted PVX fragment was judged by the size of the amplified fragment (FIG. 6).

Example 5 plasmid vector pCC_FR2-2bPTⅡ-PX_1967-2166Cross protection effect test of

CMV inoculation Using the method of example 4_FnyRNA1/RNA2-2bPTⅡ-PX_1967-2166The cross-protection test was performed with/RNA 3. After inoculating the attenuated mutant for 10 days, infecting the plant which is inoculated with the attenuated mutant in advance and the plant which is not inoculated with the attenuated mutant in advance with the target virulent CMV and PVX respectively, and testing the cross protection effect.

And (4) analyzing results: non-inoculated mutation in early stage of CMV and PVX virulent direct inoculation100 middle-sized tobacco in the body is short and small as a whole, and shows dark and light green interphase floral leaf symptoms and typical rat tail-shaped morbidity characteristics caused by CMV infection; pre-inoculated CMV_FnyRNA1/RNA2-2bPTⅡ-PX_1967-2166The medium smoke 100 of RNA3 is inoculated with a target strong virus (CMV + PVX), the plant height is slightly dwarfed compared with a healthy control, the growth state is good, and only weak mosaic symptoms exist, which indicates that the CMV is pre-inoculated_FnyRNA1/RNA2-2bPTⅡ-PX_1967-2166The medium smoke 100 of the/RNA 3 has better cross-protection and prevention effects on CMV and PVX, and is a CMV attenuated vaccine for resisting the PVX (figure 7).

The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.

SEQUENCE LISTING

<110> Shandong university of agriculture

<120> dual attenuated vaccine for resisting cucumber mosaic virus and potato virus X and application thereof

<130> 2020

<160> 8

<170> PatentIn version 3.5

<210> 1

<211> 200

<212> DNA

1967-position and 2166-position fragment of <213> PVX (NCBI accession number EU571480)

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tggcacgaga attgctcgct atgaacagaa gtcctgccac catccctttg gacctactta 180

gagccagaga ctacggcagt 200

<210> 2

<211> 3268

<212> DNA

<213> Artificial sequence

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gtttatttac aagagcgtac ggttcaaccc ctgcctcccc tgtaaaactc cctagactta 60

aatcttttct ttctagtatc ttttctatgg ctttccctgc ccccgcattc tcactagcca 120

atcttttgaa cggcagttac ggtgtcgaca ctcccgagga tgtggaacgt ttgcgatctg 180

agcaacgcga agaggctgct gcggcctgtc gtaattacag gcccctaccc gctgtggatg 240

tcagcgagag tgtcacagag gacgcgcatt ccctccgaac tcctgacgga gctcccgctg 300

aagcggtgtc tgatgagttt gtaacttatg gtgctgaaga ttaccttgaa aaatctgatg 360

atgagctcct tgtcgctttt gagacgatgg tcaaacccat gcgtatcgga caactatggt 420

gccctgcgtt taataaatgt tcttttattt ccagcattgc tatggccaga gctttgttgt 480

tggcacctag aacatcccac cgaaccatga agtgttttga agacctggtc gcggctattt 540

acactaaatc tgatttctac tacagtgaag agtgtgaagc cgacgacgct cagatagata 600

tctcgtctcg cgatgtaccc ggttattctt tcgaaccgtg gtcccgaacg tctggatttg 660

aaccgccgcc catttgtgaa gcgtgcgaca tgatcatgta ccagtgcccg tgttttgatt 720

ttaatgcttt aaagaaatcg tgcgctgaga ggaccttcgc tgatgattat gttattgaag 780

gtttagatgg tgttgttgat aatgcgactc tgttgtcgaa tttgggtcca tttttggtac 840

ccgtgaagtg tcaatatgaa aaatgtccaa cgccaaccat cgcgattcct ccggatttaa 900

accgtgctac tgatcgtgtt gatatcaatt tagttcaatc catttgtgac tcgactctgc 960

ccactcatag taattacgac gactcttttc atcaagtgtt cgtcgaaagt gcagactatt 1020

ctatagatct ggatcatgtt agacttcgac agtctgatct tattgcaaaa attccagatt 1080

cagggcatat gataccggtt ctgaacaccg ggagcggtca caagagagta ggtacaacga 1140

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gtaactctct agcatccagt aactttgtca atgtcgttag taacttccac gattacatgg 1320

aaaaatggaa gtcctcaggt ctttcttatg atgatcttcc ggatcttcat gctgagaatt 1380

tgcagtttta tgaccacatg ataaaatccg atgtgaaacc tgtggtgagc gacacactca 1440

atatcgacag accggttcca gctactataa cgtatcataa gaagagtata acctcccagt 1500

tctcaccgtt attcacagcg ctattcgagc gcttccagag atgccttcga gaacgtatta 1560

ttcttcctgt tggtaagatt tcatcccttg agatggcagg atttgatgtc aagaacaagc 1620

actgcctcga gattgacctg tctaagtttg ataagtctca aggtgaattt cacttgctaa 1680

tccaggaaca cattttgaat ggtctaggat gtccagctcc gataactaag tggtggtgtg 1740

atttccatcg attctcttac attagagacc gtagagctgg tgttggtatg cctattagtt 1800

tccagagacg aactggcgat gcactcactt attttggcaa taccatcgtc accatggctg 1860

agtttgcctg gtgttatgac accgaccaat tcgaaaagct tttattctca ggcgatgatt 1920

ctctaggatt ttcactgctt ccccctgttg gtgacccgag taaattcaca actcttttca 1980

acatggaagc taaggtgatg gaacctgccg taccatatat ttgttcgaag ttcttactct 2040

ctgacgagtt cggtaacaca ttttccgttc cagatccatt gcgcgaggtt cagcggttag 2100

gaacaaagaa aattccctat tctgacaatg atgaattctt gtttgctcac ttcatgagct 2160

ttgttgatcg attgaagttt ttggaccgaa tgtctcagtc gtgtatcgat caactttcga 2220

ttttcttcga attgaaatac aagaagtctg gggaagaggc tgctttaatg ttaggcgcct 2280

ttaagaagta taccgctaat ttccagtcct acaaagaact ctattattca gatcgtcgtc 2340

agtgcgaatt gatcaattcg ttttgtagta cagagttcag ggttgagcgt gtaaattcca 2400

acaaacagcg aaagaattat ggaattgaac gtaggtgcaa tgacaaacgt cgaactccaa 2460

ctggctcgta tggtggaggc gaagaagcag agacgaaggt ctcacaaaca gaatcgacgg 2520

gaacgaggtc acaaaagtcc cagcgagaga gcgcgttcaa atctcagact attccgcttc 2580

ctaccgttct atcaagtgga tggttcggaa ctgacagggt catgccgcca tgtgaacgtg 2640

gcggagttac ccgagtctga gtaataggga tccctgttgg attcaaggcg ctagaaattc 2700

agagggatag gaatggaaca atgatcatgc ctatcacaga aatggtctcc gggttggaaa 2760

aagaggactt cccggaagga actccaaaag agttggcacg agaattgctc gctatgaaca 2820

gaagtcctgc caccatccct ttggacctac ttagagccag agactacggc agtcccgggg 2880

cctctcgttt agagttatcg gcggaagacc atgattttga cgatacagat tggttcgccg 2940

gtaacgaatg ggcggaaggt gctttctgaa acctcccctt ccgcatctcc ctccggtttt 3000

ctgtggcggg agctgagttg gcagtattgc tataaactgt ctgaagtcac taaacacatt 3060

gtggtgaacg ggttgtccat ccagcttacg gctaaaatgg tcagtcgtag aggaatctac 3120

gccagcagac ttacaagtct ctgaggcacc tttgaaacca tctcctaggt ttcttcggaa 3180

ggacttcggt ccgtgtactt ctagcacaac gtgctagttt cagggtacgg gtgcccccca 3240

cttttgtggg gcctccaaaa ggagacca 3268

<210> 3

<211> 44

<212> DNA

<213> Artificial sequence

<400> 3

tcggatccac tagtcccggg gcctctcgtt tagagttatc ggcg 44

<210> 4

<211> 36

<212> DNA

<213> Artificial sequence

<400> 4

atggatccct attactcaga ctcgggtaac tccgcc 36

<210> 5

<211> 33

<212> DNA

<213> Artificial sequence

<400> 5

atggatccct gttggattca aggcgctaga aat 33

<210> 6

<211> 28

<212> DNA

<213> Artificial sequence

<400> 6

atcccgggac tgccgtagtc tctggctc 28

<210> 7

<211> 20

<212> DNA

<213> Artificial sequence

<400> 7

agaatcgacg ggaacgaggt 20

<210> 8

<211> 21

<212> DNA

<213> Artificial sequence

<400> 8

ggggaggttt cagaaagcac c 21

Claims

1. A duplex attenuated vaccine for resisting cucumber mosaic virus and potato virus X simultaneously is characterized in that the duplex attenuated vaccine is a cucumber mosaic virus RNA2 attenuated mutant plasmid vector inserted with PVX gene segments; the nucleotide sequence of the PVX gene segment is shown as SEQ ID NO.1, and the nucleotide sequence of the duplex attenuated vaccine is shown as SEQ ID NO. 2.

2. The method for preparing the duplex attenuated vaccine of claim 1, which comprises the following steps:

3. The method according to claim 2, wherein in the step (1), the primer sequences for the reverse PCR amplification are shown as SEQ ID NO.3 and SEQ ID NO.4, respectively.

4. The method according to claim 2, wherein in the step (1), the conditions for the inverse PCR amplification are as follows: denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 5s, extension at 72 ℃ for 8min, and 7 cycles; denaturation at 98 ℃ for 10s, extension at 68 ℃ for 8min, 25 cycles; storing at 4 ℃.

5. The method according to claim 2, wherein in the step (2), the PVX gene segment shown in SEQ ID No.1 is obtained by using sequences shown in SEQ ID No.5 and SEQ ID No.6 as primers and amplifying the gene segment from tobacco infected with PVX by RT-PCR.

6. The method of claim 5, wherein in the step (2), the reaction procedure of RT-PCR is as follows: pre-denaturation at 94 ℃ for 2 min; denaturation at 94 ℃ for 30s, annealing at 53 ℃ for 30s, and extension at 72 ℃ for 10s for 30 cycles; extending for 2min at 72 ℃; storing at 4 ℃.

7. The use of the dual attenuated vaccine of claim 1 for the control of viral diseases in plants.

8. Use according to claim 7, wherein the plant viral disease is a plant viral disease caused by cucumber mosaic virus and potato virus X.