CN110885796B - Attenuated vaccine for resisting potato virus X, preparation method and application thereof - Google Patents

Abstract

The invention relates to the field of plant antiviral genetic engineering, and discloses an attenuated vaccine against potato X virus, a preparation method and application thereof. The attenuated vaccine against potato X virus is based on a TVBMV attenuated mutant, and the TVBMV attenuated mutant is embedded with an effective gene fragment that can induce cross-protection to the potato X virus, and the effective gene fragment at least includes the RdRp gene of the potato X virus . The RdRp gene includes Rd1 gene, Rd2 gene and Rd3 gene, and one of Rd1 gene, Rd2 gene and Rd3 gene is embedded in the TVBMV attenuated mutant. The attenuated anti-potato virus vaccine of the present invention has stable action, can play an effective cross-protection effect, significantly reduces the damage of plants after being infected by a virulent strain of potato X virus, delays the onset of plant disease, and greatly reduces losses.

Description

Attenuated vaccine against potato X virus, preparation method and application thereof

technical field

The invention belongs to the field of plant antiviral genetic engineering, in particular to an attenuated vaccine against potato X virus, a preparation method and application thereof.

Background technique

Virus disease is an important disease on crops, causing huge losses to agricultural production. Due to the variety of crop virus diseases and the complicated transmission routes, there are no varieties with immunity or high resistance to virus diseases in production, and there are no drugs with specific effects on virus diseases on the market, so the prevention and control of virus diseases is very difficult.

Tobacco virus disease has always been an important factor restricting the production of tobacco leaves in my country. The main tobacco varieties currently in production are not ideal for resistance to virus diseases, and the control of tobacco virus diseases mainly relies on agricultural control and chemical control. However, there is no specific medicine for virus disease in production, and the effect of controlling tobacco virus disease by killing virus-transmitting insects is very poor, and the large-scale application of pesticides does not meet the requirements of sustainable development of tobacco leaf production. The use of attenuated strains to protect plants from the infection and damage of virulent strains (cross-protection) is a very effective means of preventing and controlling virus diseases, and has achieved success in the control of many crop virus diseases. Cross-protection has received increasing attention in recent years. Cross-protection refers to the phenomenon that plants are protected from subsequent virulent strains of viruses after being infected by attenuated strains. It has been successful in the control of many crop virus diseases. The key factors that limit the wide application of cross-protection are: first, there are not many types of attenuated strains currently available, which are inconvenient to use; is an amino acid site mutation. These attenuated strains or mutants run the risk of mutating into virulent strains when used to control viral diseases.

Potato virus X (PVX) belongs to the genus Potexvirus, which mainly infects Solanaceae crops such as tobacco, tomato, and potato, and is one of the main viruses on these crops. In agricultural production, PVX will cause serious losses to Solanaceae crops such as tobacco, pepper, and potato. Therefore, there is an urgent need for stable attenuated vaccines that can play an effective cross-protection role to prevent and treat viral diseases of plants.

The present invention has been made in view of this.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to overcome the deficiencies of the prior art, and to provide an attenuated vaccine against potato X virus, a preparation method and an application thereof. The attenuated anti-potato virus vaccine of the present invention has stable action, can play an effective cross-protection effect, significantly reduces the damage of plants after being infected by a virulent strain of potato X virus, delays the onset of plant disease, and greatly reduces losses.

In order to solve the above-mentioned technical problems, the basic conception of the technical scheme adopted in the present invention is:

The first object of the present invention is to provide an attenuated vaccine against potato X virus. The attenuated vaccine is based on a TVBMV attenuated mutant, and the TVBMV attenuated mutant is embedded with an effective gene fragment that can induce cross-protection to the potato X virus, so Said effective gene segment includes at least the RdRp gene of potato X virus.

The present invention is based on a self-constructed Tobacco vein banding mosaic virus (TVBMV) mutant, which does not cause visible symptoms after inoculating plants, is not transmitted by aphids, and is safe to use. The chimeric virus, ie attenuated vaccine, was obtained by ligating different gene fragments of Potato virus X (PVX) into TVBMV mutant by RT-PCR and enzyme ligation technology. Plants are then inoculated with the attenuated vaccine, and 15 days after inoculation the plants are infected with a virulent strain of Potato X virus (PVX). The results unexpectedly found that the RdRp gene segment of PVX could delay the onset of tobacco disease and reduce the loss of plants infected with viral diseases.

In a further scheme, the RdRp gene includes Rd1 gene, Rd2 gene and Rd3 gene, and one of Rd1 gene, Rd2 gene and Rd3 gene is embedded in the TVBMV attenuated mutant.

In a further scheme, the nucleotide sequence of the Rd1 gene is shown in Seq ID No.17.

In a further scheme, the nucleotide sequence of the Rd2 gene is shown in Seq ID No.18.

In a further scheme, the nucleotide sequence of the Rd3 gene is shown in Seq ID No.19.

Further scheme, described TVBMV attenuated mutant comprises the genome of tobacco vein mosaic virus HN39, and the 5' end of genome is connected with 35S promoter; And in the HC-Pro amino acid sequence of genome, contain mutation, at least the 52nd Arginine is mutated to glutamic acid.

Using reverse genetics technology, we identified the amino acids that HC-Pro in TVBMV regulates TVBMV pathogenicity, co-production and inhibition of RNA silencing. Attenuated mutants of TVBMV with multiple amino acid mutations, non-aphid transmission, and no synergistic effect with PVX were obtained by introducing mutations at these sites. These mutants are not prone to reverse mutation, are safe to use, and have good cross-protection effect on wild-type TVBMV.

In a further scheme, the mutations contained in the HC-Pro amino acid sequence of the genome also include: mutation of aspartic acid at position 198 to lysine.

In a further scheme, the mutations contained in the HC-Pro amino acid sequence of the genome also include: mutation of isoleucine at position 250 to aspartic acid, and mutation of glutamine at position 251 to glutamic acid.

The second object of the present invention is to provide a kind of preparation method of the attenuated vaccine against potato X virus as described in any of the above schemes, comprising the following steps:

(1) construct TVBMV attenuated mutant;

(2) obtaining the RdRp gene fragment that can induce cross-protection to potato X virus;

(3) Insert the obtained RdRp gene fragment into TVBMV attenuated mutant to obtain attenuated vaccine.

A further scheme, using the genome of Potato X virus as a template, utilizes PCR technology to amplify and obtains Rd1 gene, Rd2 gene and Rd3 gene, and then inserts into the multi-cloning site of TVBMV attenuated mutant respectively, and obtains attenuated virus containing each gene fragment. vaccine.

The third object of the present invention is to provide a kind of recombinant bacteria containing the attenuated vaccine of anti-potato X virus as described in any of the above schemes;

In a further scheme, the recombinant bacteria include Agrobacterium transformed into attenuated vaccines.

The 4th object of the present invention is to provide a kind of application of the attenuated vaccine of anti-potato X virus as described in any one of the above schemes in preventing and treating the application of the virulent strain of Potato X virus infecting plants;

Preferably, the plant is a dicotyledonous plant;

Preferably, the plant is tobacco.

The specific process of preparation and application of the attenuated vaccine of the present invention includes:

A. Obtainment of chimeric virus: The gene fragments of potato X virus were obtained by PCR technology, based on the self-construction of tobacco vein mosaic virus TVBMV attenuated mutants, and the multi-cloning sites were used to ligate each virus by enzyme ligation technology. The gene fragments are respectively connected to the attenuated mutants of TVBMV to obtain a chimeric virus, or a single-linked attenuated vaccine.

B. Screening of effective gene fragments: The attenuated vaccine obtained in A was inoculated into the host plant NC89 by the Agrobacterium infiltration method, the changes in symptoms caused by these attenuated vaccines were observed, and the gene fragments that could induce the cross-protection effect were screened.

C. Determination of cross-protection effect: Tobacco was inoculated with a single attenuated vaccine in advance, and 15 days later, the PVX virulent strain was respectively inoculated, and the protective effect of the attenuated vaccine on the virulent strain was observed.

After adopting the above-mentioned technical scheme, the present invention has the following beneficial effects compared with the prior art:

1. The attenuated vaccine of the present invention can play an effective cross-protection effect, significantly reduce the damage of plants after being infected by a virulent strain of potato X virus, delay the onset of plant disease, and greatly reduce losses.

2. The attenuated vaccine with RdRp gene fragment of the present invention can protect tobacco from the harm of PVX virulent strain, and the protection efficiency reaches 80%.

3, the attenuated vaccine of the present invention adopts the TVBMV attenuated mutant of self-construction, and on the basis of this TVBMV attenuated mutant, an effective gene fragment that can be induced to produce cross-protection to potato X virus is inserted, so the attenuated vaccine is not naturally present or artificially induced. It has a stable effect and is conducive to large-scale application.

The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

Description of drawings

The accompanying drawings, as a part of the present invention, are used to provide further understanding of the present invention, and the exemplary embodiments of the present invention and their descriptions are used to explain the present invention, but do not constitute an improper limitation of the present invention. Obviously, the drawings in the following description are only some embodiments, and for those of ordinary skill in the art, other drawings can also be obtained from these drawings without any creative effort. In the attached image:

Fig. 1 is a schematic diagram of TVBMV genome fragment amplification;

Figure 2 is a schematic diagram of the genome structure of pCamTVBMV;

Figure 3 shows the preventive and protective effects of attenuated vaccines containing different gene fragments on virulent PVX strains on the 15th day after inoculation.

It should be noted that these drawings and written descriptions are not intended to limit the scope of the present invention in any way, but to illustrate the concept of the present invention to those skilled in the art by referring to specific embodiments.

Detailed ways

In order to make the purposes, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and the following embodiments are used to illustrate the present invention , but are not intended to limit the scope of the present invention.

Example 1 Construction of multi-site TVBMV attenuated mutants

1. Construction of Infectious Clones of Tobacco Vein Mosaic Virus

Using the RNA of tobacco vein mosaic virus as a template, reverse transcription was performed with random primers. According to the restriction enzyme digestion map of the existing tobacco vein mosaic virus genome, it can be amplified in three parts, and assembled into a TVBMV full-length cDNA clone after enzyme digestion. First, the 35S promoter was fused to the upstream of the fragment from the 5′ untranslated region of TVBMV to the Nru I restriction site of the HC-pro gene by Overlap-PCR, and the inventor named the fragment p35S-HC; PCR amplification of HC- The fragment between the Nru I restriction site of the pro gene and the 6K2 Xho I restriction site was named pHC-6K2 by the inventor; the 6K2Xho I restriction site was amplified by PCR to the tail of the poly(A) A fragment, which the inventors named p6K2-polyA (as shown in Figure 1).

The reverse transcriptase used for cDNA synthesis was Moloney murine leukaemia virus reversetranscriptase (Promega); reverse transcription was performed with plant total RNA as a template and random primers as reverse transcription primers.

PCR amplification was performed using the obtained reverse transcription product as a template and corresponding primers. PCR products were subjected to 1% agarose gel electrophoresis. Three fragments of p35S-HC ₂₁₁₀ , pHC ₂₁₁₁ -6K2 ₆₀₇₅ and p6K2 ₆₀₇₆ -polyA were obtained after gel cutting and recovery. After ligating the three fragments, they were double digested with SbfI and Sma I, and then electrophoresed on a 0.8% agarose gel, recovered and ligated. To the Agrobacterium-mediated expression vector pCAMBIA0390, the inventors named the invasive clone constructed by this strategy as pCamTVBMV (as shown in Figure 2).

2. Construction of TVBMV attenuated mutants

After obtaining the invasive clone of Tobacco Vein Mosaic Virus, a TVBMV attenuated mutant was obtained by mutating key sites in the HC-Pro sequence. The specific method is as follows:

Mutation primers were designed. According to the method of Liu et al. (2008), site-directed mutation was performed on the conserved amino acid site of tobacco vein mosaic virus HC-Pro. The names and sequences of the mutated primers are shown in Table 1.

Table 1 Name and sequence of TVBMV HC-Pro mutation primers

Among them, primer 1 and primer 2 site-directed mutation of amino acid 52 of HC-Pro amino acid sequence from arginine to glutamic acid; primer 3 and primer 4 site-directed mutation of amino acid 198 of HC-Pro amino acid sequence, by aspartic acid Acid was mutated to lysine; primers 5 and 6 were site-directed to mutate amino acids 250 and 251 of the HC-Pro amino acid sequence, isoleucine at position 250 was mutated to aspartic acid, and glutamine at position 251 was mutated to Glutamate.

Using pCamTVBMV as template, PCR mutation system: 5×PCR Buffer 10 μL, dNTP (10 mM) 1 μL, mutation primer F (10 μM) 1 μL, mutation primer R (10 μM) 1 μL, template plasmid 10 ng, Phusion DNA polymerase 0.3 μL, ddH ₂ O make up to 50 μL.

PCR mutation program: 98°C/30sec; 98°C/10sec, Tmno+3°C/20sec, 72°C/5min, 20 cycles; 98°C/10sec; Tmpp/20sec; 72°C/15min; 4°C storage.

After mutation PCR, 1 μL of Dpn I was added to each reaction system, mixed well, and then digested at 37°C for 4 hours.

After the PCR reaction system was treated with Dpn I, 125 μL of absolute ethanol (2.5× volume) and 5 μL of 3M NaAc pH 8.0 were added, mixed and precipitated overnight; 12000 r/min, 10 min, discard the supernatant; 1 mL of 75% ethanol washed the precipitate, Discard the supernatant; dry the pellet and dissolve the pellet with 10 μL ddH2O.

The mutant precipitated product was transformed into Escherichia coli, and the transformed bacteria were evenly spread on LB plates containing X-gal and IPTG-Amp antibiotics, and a single colony was selected for cultivation, and the plasmid was extracted for sequencing. If the sequencing was correct, four site mutations were obtained. Attenuated mutants of TVBMV.

3. Study on pathogenicity of TVBMV attenuated mutants

The obtained mutant plasmid was transferred into Agrobacterium, and the recombinant bacteria were obtained after colony PCR verification. Then single spot was inoculated in liquid LB medium containing kanamycin (50 μg/mL), rifamycin (50 μg/mL) and tetracycline (50 μg/mL). Add 500 μL of bacterial liquid to 5 mL of LB medium containing 10 mmol/L 2-(N-morpholine)-ethylsulfonic acid (MES), 20 μmol/L acetosyringone (AS) and the above three antibiotics, at 28°C Shake culture to logarithmic growth phase.

The cells were collected by centrifugation and resuspended in 10 mmol/L MgCl ₂ , 10 mmol/LMES, 150 μmol/L AS, the concentration was adjusted so that the OD ₆₀₀ was about 0.5, and the cells were allowed to stand at room temperature for 3 hours. Take a 5mL disposable syringe, remove the needle and absorb the Agrobacterium solution, and infiltrate the back of the leaves of common tobacco (5-6 weeks old or 4-6 true leaves). Infiltrate 2 leaves per plant. The infiltrated plants were grown in a light incubator at 23°C (alternating 16 hours light/8 hours dark).

A number of common tobacco NC89 strains about 6 weeks old were selected and inoculated with TVBMV attenuated mutants. After 15 days of inoculation, it was found that the tobacco did not show symptoms. It shows that the TVBMV attenuated mutant will not cause visible symptoms after inoculating plants, will not be transmitted by aphids, and is safe to use.

Example 2 Amplification of Potato X Virus Related Gene Fragments and Construction of Attenuated Vaccine

1. Amplification of potato X virus-related gene fragments

Using the PVX-1985 genome as a template, RT-PCR was used to amplify each gene fragment. The embodiments provided by the present invention are all in accordance with conventional experimental conditions, and the primer sequences used are as follows:

Table 2 PVX gene fragment amplification primer sequences

Among them, primers 1 and 2 are used to amplify the Rd1 region of PVX, primers 3 and 4 are used to amplify the Rd2 region of PVX, primers 5 and 6 are used to amplify the Rd3 region of PVX, and primers 7 and 8 are used to amplify the PVX region. The CP region, primers 9 and 10 were used to amplify the TGB region of PVX. The nucleotide sequence of the amplified Rd1 gene is shown in Seq ID No.17, the nucleotide sequence of the Rd2 gene is shown in Seq ID No.18, and the nucleotide sequence of the Rd3 gene is shown in Seq ID No.19. The nucleotide sequence of CP gene is shown in Seq ID No.20, and the nucleotide sequence of TGB gene is shown in Seq ID No.21.

Using PVX-1985 as a template, RT-PCR was used to amplify its gene fragments, and the polymerase used was Phusion high-fidelity polymerase (Finnzymes).

The PCR reaction system is as follows:

2. Construction of attenuated vaccine

The recovered target fragments of each gene and TVBMV attenuated mutants were double digested with Xba I and Pac I, respectively. The enzyme digestion system and procedure are:

The volume of reaction components added is:

Add ddH2O to 20.0μL of solution and mix well, then digest with enzyme in a water bath at 37°C for 1.5h.

The ligation of the TVBMV attenuated mutant and the gene fragment, after the digestion product is recovered by gel, the ligation is carried out according to the ratio of the number of molecules of the vector and the fragment (1:3-1:10).

The connection system and method are as follows:

The components were mixed and ligated overnight at 16°C. Escherichia coli DH5α was transformed, and the ligated plasmid was verified by sequencing to obtain a chimeric virus.

Example 3: Chimeric virus inoculated plants

The chimeric virus obtained in Example 2 was transformed into Agrobacterium GV3101. After verification by colony PCR, recombinant bacteria were obtained. Then single spot was inoculated in liquid LB medium containing kanamycin (50 μg/mL), rifamycin (50 μg/mL) and tetracycline (50 μg/mL). Add 500 μL of bacterial liquid to 5 mL of LB medium containing 10 mmol/L 2-(N-morpholine)-ethylsulfonic acid (MES), 20 μmol/L acetosyringone (AS) and the above three antibiotics, at 28°C Shake culture to logarithmic growth phase.

The cells were collected by centrifugation and resuspended in 10 mmol/L MgCl ₂ , 10 mmol/LMES, 150 μmol/L AS, the concentration was adjusted so that the OD ₆₀₀ was about 0.5, and the cells were allowed to stand at room temperature for 3 hours. Take a 5mL disposable syringe, remove the needle and absorb the Agrobacterium solution, and infiltrate the back of the leaves of common tobacco (5-6 weeks old or 4-6 true leaves). Infiltrate 2 leaves per plant. The infiltrated plants were grown in a light incubator at 23°C (alternating 16 hours light/8 hours dark).

Test example Measurement of cross-protection effect

The attenuated vaccine was pre-inoculated with ordinary cigarettes for 15 days, and the diseased juice of PVX was rubbed to compare the cross-protective effects mediated by different fragments.

Specific methods include:

The 6-week-old ordinary cigarettes NC89 were selected and divided into 6 groups with 6 cigarettes in each group. The first group was the Mock group, only inoculated with TVBMV attenuated mutants without gene fragments. From the second group to the sixth group, they were inoculated with the single attenuated vaccine carrying the Rd1 gene, Rd2 gene, Rd3 gene, TGB gene and CP gene of potato X virus in sequence.

After 15 days of inoculation, 6 groups of common tobacco NC89 were inoculated with virulent strains of potato X virus.

Observation 15 days after challenge, the results are shown in Figure 3:

1. Plants (Mock group) that were pre-inoculated with TVBMV attenuated mutants not linked to other virus fragments, the upper leaves showed obvious mosaic symptoms, and all 20 inoculated plants were diseased.

2. Mosaic symptoms also appeared in tobacco pre-vaccinated with the attenuated vaccine with potato virus X (PVX) TGB and CP gene fragments, but the degree of mosaic was slightly lower than that of the Mock group.

3. The tobacco pre-vaccinated with the attenuated vaccine with the Rd1, Rd2 and Rd3 gene fragments of the potato virus (PVX) had no obvious or only weak mosaic symptoms.

Repeat the above test three times, observe and record the results respectively.

The tobacco morbidity results of three repeated experiments were summarized, and the results showed that:

Tobacco pre-vaccinated with attenuated vaccines with potato virus X (PVX) TGB and CP gene fragments showed a higher incidence of 85%-90%, and the protection efficiency was only 10%-15%.

Tobacco tobacco pre-vaccinated with attenuated vaccines with Rd1, Rd2 and Rd3 gene fragments of Potato Virus (PVX) has an incidence rate of 15%-20% and a protection efficiency of over 80%.

The above results show that the attenuated vaccine carrying the gene fragments Rd1, Rd2 and Rd3 of RdRp of PVX can delay the onset of tobacco, and has a good cross-protection effect on the virulent PVX strains.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention in any form. Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Within the scope of the technical solution of the present invention, personnel can make some changes or modifications to equivalent examples of equivalent changes by using the above-mentioned technical content, but any content that does not depart from the technical solution of the present invention is based on the technical solution of the present invention. Substantially any simple modifications, equivalent changes and modifications made to the above embodiments still fall within the scope of the solutions of the present invention.

Claims (8)

1. An anti-potexvirus attenuated vaccine is characterized in that the attenuated vaccine is based on TVBMV attenuated mutants, effective gene segments which can induce cross protection to potexvirus are embedded in the TVBMV attenuated mutants, and the effective gene segments at least comprise RdRp genes of the potexvirus; the RdRp gene comprises an Rd1 gene, an Rd2 gene and an Rd3 gene, and one of the Rd1 gene, the Rd2 gene or the Rd3 gene is embedded into the TVBMV attenuated mutant; the nucleotide sequence of the Rd1 gene is shown as Seq ID No.17, the nucleotide sequence of the Rd2 gene is shown as Seq ID No.18, and the nucleotide sequence of the Rd3 gene is shown as Seq ID No. 19;

the TVBMV attenuated mutant is obtained by four site mutations of pCamTVBMV in an HC-Pro sequence, wherein the site mutations are as follows: arginine at position 52 is mutated into glutamic acid, and aspartic acid at position 198 is mutated into lysine; isoleucine at position 250 is mutated to aspartic acid, and glutamine at position 251 is mutated to glutamic acid.

2. A method of preparing the attenuated vaccine of claim 1 against potexvirus, comprising the steps of:

(1) constructing a TVBMV attenuated mutant;

(2) obtaining RdRp gene segment capable of inducing cross protection to potato virus X;

(3) and inserting the obtained RdRp gene fragment into a TVBMV attenuated mutant to obtain the attenuated vaccine.

3. The preparation method of claim 2, wherein the gene group of potexvirus is used as a template, and the Rd1 gene, the Rd2 gene and the Rd3 gene are obtained by PCR amplification, and then are respectively inserted into the multiple cloning sites of the TVBMV attenuated mutant to obtain the attenuated vaccine containing each gene fragment.

4. A recombinant bacterium comprising the attenuated vaccine of claim 1 against potexvirus.

5. The recombinant strain of claim 4, wherein the recombinant strain comprises an Agrobacterium transformed with an attenuated vaccine.

6. Use of the attenuated vaccine against potexvirus according to claim 1 for controlling infection of plants by virulent strains of potexvirus.

7. The use of claim 6, wherein said plant is a dicotyledonous plant.

8. The use according to claim 6, wherein said plant is tobacco.

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