Tobacco polyphenol oxidase gene and application thereof in prodenia litura resistance

Abstract

The invention discloses a tobacco polyphenol oxidase gene and application thereof in resisting prodenia litura. The tobacco polyphenol oxidase gene is at least one of an NtPPO-1 gene, an NtPPO-2 gene and an NtPPO-3 gene; wherein the base sequence of the NtPPO-1 gene is as shown in SEQ ID No. 1; the base sequence of the NtPPO-2 gene is as shown in SEQ ID No. 2; the base sequence of the NtPPO-3 gene is as shown in SEQ ID No. 3; the regulation comprises the step of knocking out or reducing at least one of an NtPPO-1 gene, an NtPPO-2 gene and an NtPPO-3 gene, so that the resistance of the tobacco plant to the prodenia litura is reduced; through overexpression of at least one of the NtPPO-1 gene, the NtPPO-2 gene and the NtPPO-3 gene, the resistance of the tobacco plant to the prodenia litura is improved. The gene is subjected to single-gene or multi-gene overexpression in tobacco plants, the prodenia litura resistance of the plants can be greatly enhanced, and the gene has important significance and wide application prospects in the aspects of prodenia litura resistance mechanism research, molecular breeding and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant genes, in particular to a tobacco polyphenol oxidase gene and its application in Spodoptera litura resistance. BACKGROUND

[0002] Tobacco is susceptible to a variety of pests during growth, which seriously affects the yield and quality of tobacco. The larvae of Spodoptera litura can eat tobacco leaves, causing irregular damage or holes in tobacco leaves, and in severe cases, only leaf veins are left. At present, the prevention and control of Spodoptera litura in tobacco mainly relies on chemical pesticides, biological control, agricultural measures and other methods, but all have significant limitations, which is difficult to meet the needs of sustainable agriculture.

[0003] The abuse of chemical pesticides has caused serious resistance of Spodoptera litura, for example, Spodoptera litura has grown more than 10 times resistant to cypermethrin. Moreover, pesticide residues pollute the soil and water, endangering non-target organisms (such as bees and natural enemy insects); frequent application of chemical pesticides also increases planting costs; tobacco pesticide residues may affect consumer health, which is not in line with the trend of green agriculture. Using biological control, such as using natural enemy insects (such as trichogramma, trichogramma spp.) or microbial agents (such as Bacillus thuringiensis Bt, nuclear polyhedrosis virus NPV), is susceptible to the environment (temperature, humidity), and the field control effect is only 40%-60%. And it takes 3-7 days to take effect, which is slow and difficult to deal with explosive insect pests. The cost of natural enemy reproduction and release is high, which is difficult to cover large-scale planting areas. Using crop rotation, removing field residues or sex pheromones, and light traps can only reduce the insect population, but cannot completely control the damage. Moreover, it requires a large amount of labor, and the labor cost is high, which is not suitable for large-scale planting.

[0004] Current commercial tobacco varieties have weak resistance to Spodoptera litura, and need to rely on exogenous control. The use of tobacco endogenous polyphenol oxidase (NtPPO) gene mechanism to control Spodoptera litura has not been researched and developed. SUMMARY

[0005] The technical problem to be solved by the present application is to provide a tobacco polyphenol oxidase gene and its application in Spodoptera litura resistance, by identifying the tobacco NtPPO gene and constructing a high-efficiency expression vector, which is the first time to realize the endogenous gene-driven resistance to Spodoptera litura, filling the technical gap.

[0006] The technical problem to be solved by the present application is solved by the following technical scheme:

[0007] The application relates to an application of a tobacco polyphenol oxidase gene in regulating the resistance of tobacco plants to Spodoptera litura, wherein the tobacco polyphenol oxidase gene is at least one of NtPPO-1 gene, NtPPO-2 gene and NtPPO-3 gene; the base sequence of the NtPPO-1 gene is shown in SEQ ID No. 1; the base sequence of the NtPPO-2 gene is shown in SEQ ID No. 2; the base sequence of the NtPPO-3 gene is shown in SEQ ID No. 3; the regulation comprises reducing the resistance of the tobacco plants to Spodoptera litura by knocking out or reducing at least one of the NtPPO-1 gene, NtPPO-2 gene and NtPPO-3 gene; and increasing the resistance of the tobacco plants to Spodoptera litura by overexpressing at least one of the NtPPO-1 gene, NtPPO-2 gene and NtPPO-3 gene.

[0008] Preferably, in the above technical solution, the regulation of the resistance of the tobacco plants to Spodoptera litura is achieved by reducing the expression level of at least one of the NtPPO-1 gene, NtPPO-2 gene and NtPPO-3 gene in the tobacco plants or by knocking out at least one of the NtPPO-1 gene, NtPPO-2 gene and NtPPO-3 gene; wherein the knocking out is achieved by designing a specific target gene sequence sgRNA for the specific homologous segment of the NtPPO-1 gene, NtPPO-2 gene and NtPPO-3 gene, and the sequence is shown in SEQ ID No. 4.

[0009] Preferably, in the above technical solution, the expression level of at least one of the NtPPO-1 gene, NtPPO-2 gene and NtPPO-3 gene in the tobacco plants is reduced by CRISPR-Cas9 technology or RNAi gene silencing technology, wherein the sequence of the specific target gene sequence sgRNA of the CRISPR-Cas9 technology is shown in SEQ ID No. 4.

[0010] A recombinant vector for overexpressing a tobacco polyphenol oxidase gene, wherein the recombinant vector comprises a base sequence shown in SEQ ID No. 2 and / or SEQ ID No. 3.

[0011] Preferably, in the above technical solution, the recombinant expression vector is an NtPPO2-OX and / or NtPPO3-OX recombinant expression vector.

[0012] The base sequence of the primer NtPPO-2F for constructing the NtPPO2-OX is shown in SEQ ID No. 5, and the base sequence of the primer NtPPO-2R is shown in SEQ ID No. 6.

[0013] The base sequence of the primer NtPPO-3F for constructing the NtPPO3-OX is shown as SEQ ID NO. 7, and the base sequence of the primer NtPPO-3R is shown as SEQ ID NO. 8.

[0014] An expression vector comprises a polynucleotide targeting a target site shown as SEQ ID No. 4 in at least one of the NtPPO-1 gene, the NtPPO-2 gene and the NtPPO-3 gene, or a polynucleotide encoding an sgRNA of the sequence shown as SEQ ID NO. 4.

[0015] Preferably, in the above technical solution, the expression vector is a CRISPR gene editing vector.

[0016] A kit comprises an expression vector.

[0017] A cultivation method of a tobacco plant resistant to Heliothis assulta, comprising the following steps:

[0018] The constructed overexpression vector is transformed into Agrobacterium by using an electric shock transformation method;

[0019] The seeds of the common cultivated tobacco are treated with a 10% sodium hypochlorite solution for 10 min, then washed with sterile water, and sowed on 1 / 2MS medium;

[0020] After the seeds germinate and grow to the 4-leaf stage, tobacco transgenic infection is performed;

[0021] The tobacco seedlings with good growth are selected, the leaves are cut into 1cm*1cm in size, infected in the Agrobacterium infection solution for 15 min, then the excess bacterial solution on the leaves is absorbed with sterile filter paper, and the leaves are laid on tobacco differentiation medium and cultured at 28 DEG C in the dark for 2-3 days;

[0022] After the completion of the co-culture stage, the tobacco leaves are transferred to the differentiation medium for subsequent culture; when the new shoots start to germinate on the leaf surface, the new shoots are transferred to the screening rooting medium for further screening culture;

[0023] After the adventitious roots are observed to form and grow to 1cm in length, a hardening treatment is first performed for two days; then the roots are transplanted into nutrient soil for further cultivation to obtain the tobacco plant resistant to Heliothis assulta.

[0024] Preferably, in the above technical solution, the tobacco plant contains overexpressed at least one of the NtPPO-1 gene, the NtPPO-2 gene and the NtPPO-3 gene.

[0025] The above technical solution of the present application has the following beneficial effects:

[0026] The present application can greatly enhance the resistance of the plant to Spodoptera litura by overexpressing the NtPPO-1 gene and / or the NtPPO-2 gene and / or the NtPPO-3 gene of the polyphenol oxidase gene family in the tobacco plant. Compared with the control group, the overexpressed tobacco plant has a doubled resistance to Spodoptera litura. Meanwhile, by targeting the NtPPO-1 gene and / or the NtPPO-2 gene and / or the NtPPO-3 gene, the single gene or multi-gene reduced expression or knockout in the tobacco plant can greatly reduce the resistance of the tobacco to Spodoptera litura. BRIEF DESCRIPTION OF DRAWINGS

[0027] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the application and, together with the description, serve to explain the principles of the application.

[0028] Figure 1 The figure shows the induction of up-regulated expression of PPO genes in the tobacco leaf after the feeding treatment of the embodiment of the present application (Note: 0h is the control before the insect is fed, 5h is 5h after the insect is fed, and 9h is 9h after the insect is fed);

[0029] Figure 2 The figure shows the overexpression vector constructed in the embodiment of the present application;

[0030] Figure 3 The figure shows the gene knockout vector constructed in the embodiment of the present application;

[0031] Figure 4 The figure shows the mutation site analysis of the multi-gene knockout strain in the embodiment of the present application;

[0032] Figure 5 The figure shows the weight statistical analysis of the insects and the leaf damage display after the insects are inoculated into the multi-gene knockout mutant strain and the control plant in the embodiment of the present application;

[0033] Figure 6 The figure shows the weight statistical analysis of the insects and the leaf damage display after the insects are inoculated into the overexpression strain and the control plant in the embodiment of the present application;

[0034] Figure 7 The figure shows the PPO activity analysis of the multi-gene knockout mutant strain and the overexpression strain in the embodiment of the present application. DETAILED DESCRIPTION

[0035] Various exemplary embodiments of the present application will now be described in detail with reference to the accompanying drawings. Note that the relative arrangement, numerical expressions, and numerical values of the components and steps set forth in these embodiments do not limit the scope of the present application unless otherwise specifically stated.

[0036] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used are commercially available unless otherwise specified. The equipment used in the experiments is well known to those skilled in the art unless otherwise specified.

[0037] Example 1 Analysis of the insect-induced expression pattern of NtPPO gene

[0038] In order to analyze the expression of PPO gene in tobacco leaves after insect induction, the tobacco plants were treated with starved for 1 h Helicoverpa armigera larvae, and then the leaves before feeding, 5 h and 9 h after feeding were selected for quantitative PCR analysis.

[0039] Figure 1 For the expression analysis of PPO gene before and after insect feeding in the embodiments of the present application. As shown in Figure 1 After insect treatment, the expression of NtPPO-1 gene, NtPPO-2 gene and NtPPO-3 gene homologous genes in tobacco leaves was significantly up-regulated over time, especially the up-regulation of NtPPO-3 gene was higher than that of NtPPO-2 gene and NtPPO-1 gene, and the up-regulation of NtPPO-2 gene was the lowest, and the up-regulation of NtPPO-1 gene was between NtPPO-2 gene and NtPPO-3 gene. Therefore, we further analyzed the resistance of NtPPO-1 gene, NtPPO-2 gene and NtPPO-3 gene to Helicoverpa armigera.

[0040] Example 2 Construction of gene overexpression vector

[0041] NtPPO genes from Nicotiana tabacum were cloned, including three homologous genes NtPPO-1 gene (SEQ ID NO. 1), NtPPO-2 gene (SEQ ID NO. 2) and NtPPO-3 gene (SEQ ID NO. 3). According to the homology of the three NtPPO genes, NtPPO-2 gene and NtPPO-3 gene were selected to construct overexpression vectors, and were used for tobacco transgenic and identification for gene function analysis.

[0042] NtPPO-2 gene (SEQ ID NO. 2) and NtPPO-3 gene (SEQ ID NO. 3) were inserted into plant binary expression vectors by homologous recombination method to construct NtPPO2-OX and NtPPO3-OX recombinant expression vectors (as shown in Figure 2 ).

[0043] The specific process is as follows:

[0044] ① The NtPPO-2 gene is amplified using specific primers NtPPO-2F: 5'- ATGGCTTCTTCTTCTACTCTACC-3' (SEQ ID NO. 5); NtPPO-2R: 5'- TTAAAAACAATCCTTAAGTTCAATC-3' (SEQ ID NO. 6), and the NtPPO-3 gene is amplified using primers NtPPO-3F: 5'- ATGGCTTCTTCTTCTACTCTACC-3' (SEQ ID NO. 7); NtPPO-3R: 5'- TTAAAAACAATCCTTAAGTTCAATC-3' (SEQ ID NO. 8), and then the PCR products are recovered using a purification recovery kit; the expression vector is digested using BamHI, and the linearized expression vector is purified using a gel recovery kit. A certain amount of the target gene PCR recovery product and the linearized vector are mixed and incubated at 37°C for recombination.

[0045] ② 10 μL of the reaction solution is added to 50 μL of DH5α competent cells, mixed gently, and incubated on ice for 30 minutes. After 42°C heat shock for 45 seconds, the cells are quickly cooled on ice for 2 minutes. Then, 600 μL of SOD liquid medium is added, and the cells are incubated at 37°C for 1 h. The cells are collected by centrifugation at 4500 rpm for 2 minutes, and evenly spread on LB solid medium and cultured in a 37°C incubator.

[0046] ③ Multiple colonies on the transformation plate are picked and identified by colony PCR to obtain positive clones, and then the corresponding single colonies are picked and cultured in LB liquid medium for expansion, and used for plasmid extraction. The vector accuracy is further identified by plasmid PCR and sequencing.

[0047] Example 3 Construction of a multi-gene editing vector

[0048] According to the genomic sequence homologous segments of the NtPPO-1 gene (SEQ ID NO. 1), the NtPPO-2 gene (SEQ ID NO. 2), and the NtPPO-3 gene (SEQ ID NO. 3) cloned in Example 2, a specific target sequence for gene editing is designed, and a gRNA (guide RNA) sequence SEQ ID NO. 4 is synthesized:

[0049] 5'-TACAAGTACCATCTATGAAA-3'.

[0050] Figure 3The schematic diagram of the gene knockout vector constructed for the embodiment of the present application. The target site SEQ ID NO. 4 is designed for NtPPO-1 gene, NtPPO-2 gene and NtPPO-3 gene. Then the gRNA sequence fragment is recombined into the CRISPR / Cas9 gene editing vector containing a resistance marker as shown in Figure 3

[0051] Using the CRISPR / Cas9 gene editing vector system, the target sequence is edited to mutate the coding sequence of NtPPO-1 gene, NtPPO-2 gene and NtPPO-3 gene, so that it cannot be normally expressed or the amino acid sequence of the expression product is changed or prematurely terminated, so as to achieve the purpose of knocking out these genes.

[0052] Example 4 Genetic transformation of tobacco

[0053] Using the electroporation method, the plant overexpression vector and the gene editing vector constructed in the above Examples 2 and 3 are transformed into Agrobacterium EHA105, respectively.

[0054] The seeds of common cultivated tobacco are treated with 10% sodium hypochlorite solution for 10 minutes, then washed with sterile water several times, and sown on 1 / 2MS medium. After the seeds germinate and grow to 4-leaf stage, the leaf disc transformation method is used for tobacco transgenic infection. The tobacco seedlings with good growth are selected, the leaves are cut into about 1cm x 1cm in size, and then infected in the Agrobacterium infection solution for 15 minutes. The excess bacterial solution on the leaves is absorbed with sterile filter paper, and then placed on the tobacco differentiation medium at 28°C in the dark for 2-3 days of co-culture.

[0055] After the co-culture stage is completed, the tobacco leaves are transferred to the differentiation medium containing antibiotics for subsequent culture. When the new shoots start to sprout on the leaf surface, these newly sprouted shoots are transferred to the selection rooting medium for further selection culture. After observing the formation and growth of adventitious roots to about 1 centimeter in length, a two-day hardening treatment is first carried out. Subsequently, they are transplanted into nutrient soil for further cultivation.

[0056] Example 5 Screening and identification of gene editing lines

[0057] Screening of positive transgenic seedlings:

[0058] The genomic DNA of the T0 generation transgenic leaves is extracted using a kit, and PCR is used to detect whether the Cas9 gene sequence is contained in the genome, and whether the knockout vector has been inserted into the genomic sequence of the transgenic plants. The PCR product is detected by electrophoresis and sequencing to obtain positive transgenic seedlings. Then the gene editing site is detected and analyzed.

[0059] Analysis of editing mutation of target site​

[0060] The genomic DNA of the leaf of the positive transgenic seedling was used as a template to amplify the target gene sequence, and the primers were as follows:

[0061] NtPPO-1-F (SEQ ID No. 9): 5'-CCAACACCAACACCAACTCAT-3';

[0062] NtPPO-1-R (SEQ ID No. 10): 5'-TATCCTCACCGTGTTTTACAT-3';

[0063] NtPPO-2-F (SEQ ID No. 11): 5'-GGCTTCTTCTTCTTCTTCTACTCT-3';

[0064] NtPPO-2-R (SEQ ID No. 12): 5'-GACTTCTTGACCGAAATGACC-3';

[0065] NtPPO-3-F (SEQ ID No. 13): 5'-CCAGCATTTCCTCCATGGAAAA-3';

[0066] NtPPO-3-R (SEQ ID No. 14): 5'-GCAGTAAAAAACTGGATCTAAACCA-3'.

[0067] The target editing conditions of the planted T1 generation plants were identified, and gene editing events were detected at the target positions of the three genes. The editing efficiency of each target was determined according to whether the T1 generation had an editing event (Table 1). Overall, the editing efficiency of the target site was high, and the editing efficiency in the three genes was 56%, 72% and 48%, respectively.

[0068] Table 1. Statistics of each target editing condition of T1 generation plants

[0069]

[0070] Figure 4 Figure 1 is a diagram for analyzing the mutation sites of the multi-gene knockout strain of the embodiment of the present application. According to the analysis of the mutation sites of the multi-gene knockout strain of the embodiment of the present application, the following conclusions can be drawn. Figure 4 It can be seen that two base insertions occur in the coding sequence of the NtPPO-1 gene, and 4 base and 2 base deletions occur in the coding sequences of the NtPPO-2 gene and the NtPPO-3 gene, respectively, and the three Helicoverpa armigera related genes are effectively knocked out.

[0071] Example 6: Effect of NtPPO gene knockout plant on the body weight of Helicoverpa armigera

[0072] Select 3 transgenic tobacco plants and 3 control tobacco plants with basically consistent growth vigor, take the increased body weight of Helicoverpa armigera as the measuring index, detect the influence of overexpression lines, transgenic knockout tobacco and control plants on the body weight of Helicoverpa armigera. Select Helicoverpa armigera larvae with close body weight to treat each line, place 3 larvae on tobacco leaves. Continuously weigh the body weight of the larvae, compare and analyze the body weight of Helicoverpa armigera on transgenic tobacco and control plants, and the results are shown in Figure 5 The results of observing the damage of tobacco leaves after feeding larvae are shown in Figure 6 .

[0073] Figure 5 The figure of body weight statistical analysis of Helicoverpa armigera after inoculating the control plants and the multi-gene knockout mutant lines of the embodiment of the present application and the figure of tobacco leaf damage are shown. As shown in the A figure of Figure 5 , the body weight of the larvae fed by the multi-gene mutant plants is obviously increased, the body weight is increased by 0.12 g, and the increase is 240% at 3 days, and the body weight is increased by 0.17 g, and the increase is 340% at 5 days. It is proved that mutating these genes can reduce the resistance of the plants to Helicoverpa armigera.

[0074] As shown in the B figure of Figure 5 , the damage of tobacco leaves fed by the multi-gene mutant plants is obviously more serious than that of the control plants, which further proves that mutating these genes can reduce the resistance of the plants to Helicoverpa armigera.

[0075] Figure 6 The figure of body weight statistical analysis of Helicoverpa armigera after inoculating the control plants and the overexpression lines of the embodiment of the present application and the figure of tobacco leaf damage are shown. As shown in the A figure of Figure 6 , the body weight of the larvae fed by the overexpression plants is obviously reduced. Among them, the body weight of the control plants is increased by 0.1 g, and the increase is 200% at 3 days. The body weight of the NtPPO-2 gene overexpression line is increased by 0.07 g, and the increase is 140%. The body weight of the NtPPO-3 gene overexpression line is increased by 0.05 g, and the increase is 100%. Therefore, the NtPPO-2 gene overexpression line and the NtPPO-3 gene overexpression line have obvious resistance to Helicoverpa armigera. The body weight of the control plants is increased by 0.17 g, and the increase is 240% at 5 days. The body weight of the NtPPO-2 gene overexpression line is increased by 0.09 g, and the increase is 180%. The body weight of the NtPPO-3 gene overexpression line is increased by 0.06 g, and the increase is 120%. Therefore, the NtPPO-2 gene overexpression line and the NtPPO-3 gene overexpression line have obvious resistance to Helicoverpa armigera. As shown in the B figure of Figure 6 , the damage of tobacco leaves of the overexpression plants is obviously lighter than that of the control plants, which further proves that the NtPPO gene has the ability to enhance the resistance of the plants to Helicoverpa armigera.

[0076] Example 7 Detection of PPO activity of NtPPO gene knockout lines

[0077] The NtPPO gene encodes polyphenol oxidase protein, and the PPO activity of the overexpression and gene knockout lines is further detected.

[0078] Figure 7 Figure for detection and analysis of PPO activity in the multi-gene knockout mutant lines and overexpression lines of the embodiments of the present application. Figure 7 It can be seen that the PPO activity of the NtPPO-1 gene, NtPPO-2 gene and NtPPO-3 gene triple knockout tobacco plant is significantly decreased compared with the control plant, however, the PPO activity of the NtPPO-2 gene and NtPPO-3 gene overexpression lines is significantly increased, the trend is consistent with the resistance to Heliothis assulta, indicating that the increase of tobacco PPO activity is involved in the resistance of tobacco to Heliothis assulta.

[0079] Although the present application has been disclosed as above with examples, it is not intended to limit the present application, any person skilled in the art can make various selections and modifications without departing from the spirit and scope of the present application, therefore the protection scope of the present application is defined by the claims and equivalent forms thereof.

Claims

1. The use of a tobacco polyphenol oxidase gene in modulating the resistance of a tobacco plant to Heliothis virescens, characterized in that, The tobacco polyphenol oxidase gene is at least one of NtPPO-1 gene, NtPPO-2 gene and NtPPO-3 gene; wherein, the base sequence of the NtPPO-1 gene is shown as SEQ ID No. 1; the base sequence of the NtPPO-2 gene is shown as SEQ ID No. 2; and the base sequence of the NtPPO-3 gene is shown as SEQ ID No.

3. The regulation comprises reducing the resistance of the tobacco plant to Spodoptera litura by knocking out or reducing at least one of the NtPPO-1 gene, the NtPPO-2 gene and the NtPPO-3 gene; and increasing the resistance of the tobacco plant to Spodoptera litura by overexpressing at least one of the NtPPO-1 gene, the NtPPO-2 gene and the NtPPO-3 gene.

2. The use of a tobacco polyphenol oxidase gene according to claim 1 for modulating resistance of a tobacco plant to Heliothis virescens, characterized in that, The regulation of the ability of the tobacco plant to resist Spodoptera litura is achieved by reducing the expression level of at least one of the NtPPO-1 gene, the NtPPO-2 gene and the NtPPO-3 gene in the tobacco plant, or by knocking out at least one of the NtPPO-1 gene, the NtPPO-2 gene and the NtPPO-3 gene; wherein, the knocking out of the gene is achieved by designing a specific target gene sequence sgRNA for the specific homologous segment of the NtPPO-1 gene, the NtPPO-2 gene and the NtPPO-3 gene, and the sequence of the sgRNA is shown as SEQ ID No.

4.

3. The use of a tobacco polyphenol oxidase gene according to claim 1 for modulating resistance of a tobacco plant to Heliothis virescens, characterized in that, The expression level of at least one of the NtPPO-1 gene, the NtPPO-2 gene and the NtPPO-3 gene in the tobacco plant is reduced by using the CRISPR-Cas9 technology or the RNAi gene silencing technology, wherein, the sequence of the specific target gene sequence sgRNA of the CRISPR-Cas9 technology is shown as SEQ ID No.

4.

4. A recombinant vector overexpressing a tobacco polyphenol oxidase gene, characterized in that, The recombinant vector comprises the base sequence shown as SEQ ID No. 2 and / or SEQ ID No.

3.

5. The recombinant vector of claim 4, wherein the tobacco polyphenol oxidase gene is overexpressed. The recombinant expression vector is an NtPPO2-OX and / or NtPPO3-OX recombinant expression vector. The base sequence of the primer NtPPO-2F for constructing the NtPPO2-OX is shown as SEQ ID No. 5, and the base sequence of the primer NtPPO-2R is shown as SEQ ID No.

6. The base sequence of the primer NtPPO-3F for constructing the NtPPO3-OX is shown as SEQ ID No. 7, and the base sequence of the primer NtPPO-3R is shown as SEQ ID No.

8.

6. An expression vector, characterized by, The polynucleotide comprises a target site shown as SEQ ID No. 4 in at least one of the NtPPO-1 gene, the NtPPO-2 gene and the NtPPO-3 gene, or the sgRNA of the polynucleotide encoding the sequence shown as SEQ ID No.

4.

7. The expression vector of claim 6, wherein, The expression vector is a CRISPR gene editing vector.

8. A kit characterized in that, The expression vector comprises the expression vector of claim 7.

9. A method of breeding tobacco plants resistant to Heliothis virescens, characterized by, The method comprises the following steps: The constructed overexpression vector is transformed into Agrobacterium by using the electric shock transformation method; The constructed overexpression vector is transformed into Agrobacterium by using the electric shock transformation method; The seeds of common cultivated tobacco were treated with 10% sodium hypochlorite solution for 10 min, then washed with sterile water, and sowed on 1 / 2MS medium; After the seeds germinated and grew to 4-leaf stage, the tobacco transgenic infection was performed; The tobacco seedlings with good growth were selected, the leaves were cut into 1cm x 1cm in size, and then infected in the agrobacterium infection solution for 15 min, and the excess bacterial solution on the leaves was absorbed with sterile filter paper, and then laid on the tobacco differentiation medium and cultured at 28°C in dark for 2-3 days; After the completion of the co-culture stage, the tobacco leaves were transferred to the differentiation medium for subsequent culture; when the new shoots began to germinate on the leaf surface, the new shoots were transferred to the screening rooting medium for further screening culture; After the observation of the formation and growth of the adventitious roots to 1cm in length, the seedlings were first treated for two days, and then transplanted into the nutrient soil for further cultivation, and the tobacco plants resistant to heliothis subnota were obtained.

10. The breeding method according to claim 9, characterized by, The tobacco plants contain at least one of the overexpressed NtPPO-1 gene, NtPPO-2 gene and NtPPO-3 gene.

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