Application of PR-1B protein in tobacco plant breeding

Abstract

This application discloses the application of PR-1B protein in tobacco plant breeding, including the following steps: detecting the expression level of PR-1B protein in tobacco plants infected with black shank; when the expression level of PR-1B protein is overexpressed, the tobacco plant exhibits black shank resistance; the amino acid sequence of PR-1B protein is shown in SEQ ID No:1. The PR-1B protein disclosed in this application can be used reliably and sensitively to detect tobacco resistance to black shank, and provides a novel approach for screening black shank-resistant tobacco varieties and assisting traditional hybridization breeding, making it suitable for large-scale application.

Description

Technical Field

[0001] This application relates to the field of tobacco black shank detection technology, and in particular to the application of a PR-1B protein in tobacco plant breeding. Background Technology

[0002] Blackshank (BS) is a disease caused by the fungus *Phytophthora parasitica* Dast. var. *nicotianae* (Bredade Haan) Tucker. This fungus belongs to the *Phytophthora* genus, most notably associated with potato late blight. Pathogens of the *Phytophthora* genus can cause significant damage to their host plants. *Phytophthora parasitica* Dast. var. *nicotianae* (Bredade Haan) Tucker can infect 255 species of plants from 90 families, causing substantial harm. Blackshank attacks the roots, stems, and leaves of tobacco plants at different developmental stages, devastating tobacco production.

[0003] Black shank is currently widespread in major tobacco-growing countries such as China and the United States. In the early stages of infection, the black shank fungus absorbs nutrients from plant cells. As the plant cells gradually die, the fungus transitions to a necrotrophic lifestyle. Chemical control is one method to combat tobacco black shank, but it is costly and environmentally harmful. Therefore, utilizing tobacco resistance resources to improve main varieties is an important means of solving the tobacco black shank problem. There are four main physiological races of the black shank fungus: race 0, race 1, race 2, and race 3. Race 0 is the most widely distributed and therefore poses the greatest threat to tobacco cultivation in my country, while race 1 has poor adaptability. Races 2 and 3 are only distributed in parts of the United States, South Africa, and India, with even narrower distribution ranges.

[0004] Currently, there are two available tobacco resistance resources. The resistance of Florida301 is composed of multiple genes, providing different resistances against different black shank races. There are also two resistance resources controlled by single genes: the Php gene from Nicotiana lumbaginifolia, which has already been used in flue-cured tobacco, and the Phl gene from N. longiflora, currently only used for resistance to black shank in Burley tobacco. Both the Php and Phl genes confer vertical resistance to black shank race 0, but not resistance to race 1.

[0005] Flue-cured tobacco is currently the most widely cultivated tobacco variety in the world. Black shank disease (CS0) poses the greatest threat to flue-cured tobacco. However, it is difficult to select tobacco plants with significant resistance to CCS0 from among many tobacco plants, and there is a lack of existing technologies on how to improve the resistance of tobacco plants to CCS0. Summary of the Invention

[0006] This application provides an application of PR-1B protein in tobacco plant breeding to solve the technical problem in the prior art that it is difficult to quickly screen existing black shank resistant tobacco plants and obtain tobacco plants resistant to black shank.

[0007] This application provides an application of PR-1B protein in tobacco plant breeding, comprising the following steps: detecting the expression level of PR-1B protein in tobacco plants infected with black shank, and when the expression level of PR-1B protein is overexpressed, the tobacco plant has black shank resistance; the amino acid sequence of PR-1B protein is shown in SEQ ID No:1.

[0008] Preferably, the method includes the following steps: transcribing the gene coding sequence of the PR-1B protein into the gene sequence of the tobacco plant to be treated, as shown in Seq ID 2.

[0009] Using the above two methods, on the one hand, we can achieve rapid and effective screening of tobacco plants resistant to black shank based on this protein; on the other hand, we can also enable existing tobacco plants to overexpress this protein, thereby acquiring the characteristic of resistance to black shank.

[0010] By screening for differentially expressed proteins between resistant and susceptible tobacco plants after inoculation with black shank disease, a protein showing significant differential expression between inoculated and uninoculated plants was identified, and identified by mass spectrometry as pathogenesis-related protein 1B (PR-1B). Specifically, the differential protein sequence between yunyan300 and HD was determined using the ITRAQ method.

[0011] Using isobaric tags for relative and absolute quantification (iTRAQ) proteomics, peptides from the enzymatic digestion of leaf proteins of different resistant tobacco varieties were labeled with stable isotopes. The peptides were then assembled using a shotgun technique. The results showed that the pathogenesis-related protein 1B (PR-1B) was upregulated in resistant tobacco plants.

[0012] Based on the correlation between tobacco pathogenesis-related protein 1B (PR-1B) and tobacco resistance to black shank, the application of tobacco pathogenesis-related protein 1B (PR-1B) in tobacco disease resistance breeding and black shank resistance detection was proposed for the first time.

[0013] If the expression of the pathogenesis-related protein 1B (PR-1B) is upregulated in tobacco varieties, it suggests that the tested tobacco variety has high resistance to black shank disease.

[0014] The application of pathogenesis-related protein 1B (PR-1B) in tobacco can reliably and sensitively detect black shank disease in tobacco, and provide a new approach for screening black shank resistant tobacco varieties and assisting traditional hybridization breeding, which is suitable for large-scale promotion and application.

[0015] The specific operations used in the above two aspects are carried out according to the existing protein expression level detection methods and transgenic methods.

[0016] Preferably, the tobacco plant is obtained by hybridization of the disease-resistant flue-cured tobacco variety Coker371 and the Honghua Dajinyuan variety, followed by six generations of backcrossing and one generation of self-pollination using molecular marker technology.

[0017] The amino acid sequence of the pathogenesis-related protein 1B (PR-1B) described in this application is as shown in SEQ ID No: 1:

[0018] MGFFLFSQMPSFFLVSTLLLFLIISHSSHAQNSQQDYLDAHNTARADVGVEPLTWDNGVAAYAQNYVSQLAADCNLVHSHGQYGENLAQGSGDFMTAAKAVEMWVDEKQYYDHDSNTCAQGQVCGHYTQVVWRNSVRVGCARVQCNNGGYVVSCNYDPPGNVIGQSPY

[0019] The gene coding sequence of pathogenesis-related protein 1B (PR-1B) amino acid sequence is shown in Seq ID2:

[0020] ATGGAGAGAGTTAATAATTATAAGTTGTGCGTGGCATTGTTGATCATCAGCATGGTGATGGCAATGGCGGCGGCACAGAGCGCCACAAACGTGAGATCGACGTATCATTTATATAACCCACAGAACATTAACTGGGATTTGAGAGCAGCAAGTGCTTTCTGCGCTACTTGGGATGCCGACAAGCCTCTCGCATGGCGCCAGAAATATGGCTGGACTGCTTTC TGTGGTCCTGCTGGACCTCGAGGCCAAGATTCCTGTGGTAGATGCTTGAGGGTGACGAACACAGGAACAGGAACTCAAACAACAGTGAGAATAGTAGATCAATGCAGCAATGGAGGGCTTGATTTAGATGTAAACGTCTTTAACCAATTGGACACAAATGGAGTGGGCTATCAGCAAGGCCACCTTACTGTCAACTATGAATTTGTCAACTGCAATGACTAA

[0021] The beneficial effects that this application can produce include:

[0022] 1) The application of the PR-1B protein provided in this application in tobacco plant breeding enables rapid screening of tobacco plants resistant to black shank by measuring the expression level of the PR-1B protein, which serves as a marker for resistance to black shank. This eliminates the need for prolonged planting and observation of disease resistance, allowing for the selection of resistant plants from a large pool of tobacco plants. Detecting the gene sequence corresponding to this protein expression reliably and sensitively identifies black shank-resistant tobacco, providing a novel approach for screening black shank-resistant tobacco varieties and assisting traditional hybridization breeding, making it suitable for large-scale application.

[0023] 2) The application of the PR-1B protein provided in this application in tobacco plant breeding involves cloning the gene expression sequence of the PR-1B protein in the tobacco plant gene to cultivate tobacco plants with resistance to black shank disease. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the hybridization process of yunyan300 material in the embodiments of this application;

[0025] Figure 2 The phenotypes of the HD and yunyan300 tobacco varieties after inoculation with black shank fungus in the embodiments of this application;

[0026] Figure 3The pie charts and bar charts show the statistical results of differentially expressed proteins in resistant and non-resistant varieties in the embodiments of this application;

[0027] Figure 4 This is a heatmap showing the differential protein expression between resistant and non-resistant varieties in the embodiments of this application;

[0028] Figure 5 These are comparative photographs of OX-1 / OX-3 cells overexpressing the pathogenesis-related protein 1B gene and HD cells after inoculation with black shank fungus for 50 days, as shown in the embodiments of this application. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The components of the embodiments of the present invention described and shown in the accompanying drawings can generally be arranged and designed in various different configurations.

[0030] Therefore, the following detailed description of the embodiments of the invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the invention without inventive effort are within the scope of protection of the invention.

[0031] Technical means not detailed in this application and not used to solve the technical problems of this application are all set according to common general knowledge in the field, and multiple common general knowledge setting methods can be implemented. Example

[0032] Unless otherwise specified, all instruments, raw materials, and tobacco plants used in the following examples were obtained through commercial channels.

[0033] Unless otherwise specified, the experimental conditions in the following examples are generally performed according to conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer.

[0034] Example 1: Preparation of the tobacco black shank resistant variety yunyan300

[0035] The flue-cured tobacco variety Honghua Dajinyuan (HD) is extremely susceptible to black shank disease. To study the effect of the Php gene on the tobacco proteome, the disease-resistant flue-cured tobacco variety Coker371, which possesses the Php gene, was first crossed with the non-resistant variety Honghua Dajinyuan. Molecular marker technology was used for six generations of backcrossing and one generation of self-crossing to obtain a near-isogenic line of Honghua Dajinyuan containing the Php gene, namely the disease-resistant Hongda (yunyan300). The preparation process of this material is as follows: Figure 1 As shown, the specific hybridization operation here can be found in (CN200610110226.2 Paper tube method for tobacco hybridization).

[0036] The yunyan300 prepared according to the above method can minimize the influence of the Cocker371 genetic background on the proteome and improve the reliability of the test results.

[0037] Example 2: Inoculation procedure for tobacco black shank disease and results of infection in resistant tobacco cultivation

[0038] HD seeds were sown on 1 / 2 MS medium and cultured in an incubator at 25°C for about one month under a light:dark ratio of 16h:8h. Seedlings were then transplanted into trays.

[0039] Inoculation was performed 7 days after seedling transplanting: First, make a cut at the base of the plant stem with a knife. Then, add approximately 5g of P. nicotianae O-type infected rice (Oryza sativa L.) to the soil near the injured stem of each plant and gently cover with soil.

[0040] By culturing sterilized rice grains together with selected isolates of race 0 of Soot Black Shank, native to China, at 28 to 30°C for 10 to 14 days, Soot Black Shank can be colonized on the rice grains, resulting in infected rice grains.

[0041] After inoculation, the temperature was maintained at approximately 32°C. Soil moisture was kept uniform through irrigation. Plant growth for each genotype was recorded at 6 and 15 days post-inoculation. Photos of HD and yunyan300 plants after partial inoculation treatment are shown below. Figure 2 As shown, by Figure 2 As shown, the HD variety suffered severe lodging after cultivation, while yunyan300 remained completely unaffected by pathogens, suggesting that the resistant tobacco material yunyan300 exhibits significant disease resistance.

[0042] Example 3: Preparation of tobacco protein samples

[0043] The tobacco protein content of each sample listed in Table 1 was measured according to the following steps:

[0044] The sample was rapidly ground into powder using liquid nitrogen in a mortar. All the powdered sample was placed in liquid nitrogen and quickly transferred to pre-chilled 50 mL centrifuge tubes. 25 mL of pre-chilled 10% TCA / acetone (containing 65 mM DTT) was added to each tube, and the mixture was thoroughly mixed. The tubes were then stored at -20°C for 2 hours. The tubes were then centrifuged at 12000 g for 45 minutes at 4°C. The supernatant was discarded. Pre-chilled pure acetone was added, and the tubes were stored at -20°C for 2 hours. The tubes were then centrifuged at 12000 g for 45 minutes at 4°C. The supernatant was discarded. This step was repeated three times.

[0045] The obtained precipitate was freeze-dried under vacuum. Approximately 800 mg of the freeze-dried powder was placed in a 1.5 mL centrifuge tube and 400 μL SDT protein lysis buffer (4% SDS, 100 mM Tris-HCl, 100 mM DTT, pH 8.0) was added. The mixture was incubated in a boiling water bath at 100°C for 10 min, followed by sonication in an ice bath for 10 min (35 W for 2 s, with an 8 s interval), then incubated in a boiling water bath at 100°C for 5 min, and centrifuged at 14000 g for 30 min. The supernatant was collected.

[0046] The resulting supernatant was filtered using a 0.22 μm ultrafiltration tube.

[0047] 200 μg of each sample was used for FASP digestion.

[0048] Add 200 μL L U Abuffer (8 M Urea, 150 mM Tris-HCl pH 8.5) to the sample, mix well, centrifuge at 14000 g at room temperature for 30 min, discard the filtrate, and repeat 3 times. Add 100 μL L IAA (50 mM IAA in UA), vortex at 600 rpm for 1 min, incubate in the dark at room temperature at 300 rpm for 30 min, and centrifuge at 14000 g at room temperature for 30 min. Add 100 μL L U Abuffer, centrifuge at 14000 g at room temperature for 30 min, and repeat 3 times. Add 100 μL 100 mM / LDS buffer, centrifuge at 14000 g at room temperature for 30 min, and repeat 3 times. Finally, discard the filtrate and add 40 μL Trypsin buffer (1 μg Trypsinin 40 μL 100 mM / LDS buffer), and place on a constant temperature mixer (300 rpm, 18 h, 37 °C). Centrifuge at 14000g for 30 min at room temperature, collect the filtrate, replace with a new collection tube, add 40μL of 25mM / LDS buffer, centrifuge at 14000g for 30 min at room temperature, collect the filtrate, and quantify the peptides by OD280.

[0049] The concentration, volume, and total amount of each protein in the obtained samples are listed in Table 1.

[0050] The meanings of each sample in Table 1 are as follows: A represents yunyan300, A0-1 is the first treatment of yunyan300 inoculated with black shank pathogen 0 hours after inoculation, and this treatment is repeated twice to obtain A0-2 and A0-3;

[0051] A24-1 was the first treatment of yunyan300 24 hours after inoculation with black shank pathogen. This treatment was repeated twice to obtain A24-2 and A24-3.

[0052] A48-1 was the first treatment for yunyan300 48 hours after inoculation with black shank pathogen. This treatment was repeated twice to obtain A48-2 and A48-3.

[0053] HD is the flue-cured tobacco variety Honghua Dajinyuan. Similarly, HD0-1 is the first treatment of HD inoculated with black shank pathogen for 0 hours. This treatment was repeated twice to obtain HD0-2 and HD0-3.

[0054] HD24-1 was the first treatment of HD 24 hours after inoculation with black shank fungus. This treatment was repeated twice to obtain HD24-2 and HD24-3.

[0055] HD48-1 was the first treatment of HD 48 hours after inoculation with black shank bacteria. This treatment was repeated twice to obtain HD48-2 and HD48-3.

[0056] Table 1. Protein quantification results of the samples

[0057]

[0058] The data in Table 1 are the protein detection results for each treatment.

[0059] Example 4: LC-MS Analysis (Liquid Chromatography-Mass Array)

[0060] LC-MS analysis was performed on each sample in Table 1 according to the following procedures:

[0061] Solution A in the liquid chromatography was a 0.1% formic acid aqueous solution, and solution B was a 0.1% formic acid-acetonitrile solution (acetonitrile was 100%). The Thermo Scientific Analytical column (75 μm × 25 cm, 5 μm, 100 Å, C18) was equilibrated with 95% solution A. The sample was loaded into the Thermo Scientific Easytrap column (100 μm × 2 cm, 5 μm, 100 Å, C18) via an autosampler and then separated by the column.

[0062] The relevant liquid phase gradients are as follows: from 0 minutes to 40 minutes, the linear gradient of liquid B increases from 5% to 28%; from 40 minutes to 42 minutes, the linear gradient of liquid B increases from 28% to 90%; from 42 minutes to 60 minutes, liquid B remains at 90%.

[0063] The enzymatic hydrolysis products were desalted and separated by capillary high performance liquid chromatography and then analyzed by mass spectrometry using an Obitrap Fusion mass spectrometer (Thermo Finnigan, San Jose, CA).

[0064] Analysis duration: 60 min; Detection mode: positive ion; Precursor ion scan range: 375-1500 m / z; Primary mass spectrometry resolution: 120,000 atm / z200; Scan mode: Top-Speed; AGC target: 4e5; Primary maximum IT: 50 ms; Number of scan ranges: 1; Dynamic exclusion: 40.0 s; Data Dependent Mode: Cycle Time; Time between Master Scan: 3 s. The mass-charge ratio of peptides and peptide fragments was acquired using the following method: a secondary spectral scan (MS2 scan) was performed simultaneously with each primary scan (Master scan), with a scan cycle of 3 s; MS2 Activation Type: HCD; Secondary mass spectrometry resolution: 50,000 atm / z200; Microscans: 1; Secondary maximum IT: 105 ms; AGC target: 1e5.

[0065] Example 5 Data Analysis

[0066] In Example 4, the raw data from the LC-MS mass spectrometry analysis were RAW files. Database lookup and quantitative analysis were performed using the built-in software Mascot's ProteomeDiscoverer 2.1 (ThermoScientific). The NCBI database used was: P17032_NCBI_Nicotiana_tabacum_91636.fasta, containing 91636 sequences. Database lookup was performed using Mascot software.

[0067] The search parameters are as follows: single isotope mass, trypsin digestion, a maximum of 2 missed cleavage sites allowed, peptide charge number: 2+, 3+ and 4+, fixed modification is carbamidomethylation (C), dynamic modification is oxidation (M).

[0068] The maximum error for the precursor ion was 20 ppm, and the maximum error for the fragment ion was 0.1 Da. ProteomeDiscoverer 2.1 screened the peptides based on the identification results with an FDR ≤ 0.01 (High Confident) and output the results. A total of 31,106 peptides were obtained.

[0069] By comparing these peptides, a total of 4,512 unique proteins were obtained.

[0070] To determine the proteomic response of resistant and susceptible HD to black shank disease, the proteomes of yunnan300 and HD were compared at 0 hours, 24 hours, and 48 hours after vaccination. The quantitative comparison results are shown in [link to data]. Figure 3 .

[0071] Statistical results show that before inoculation with the pathogen, a total of 863 proteins were upregulated and 529 proteins were downregulated. 24 hours after inoculation, a total of 789 proteins were upregulated and 520 proteins were downregulated. 48 hours after inoculation, 864 proteins were upregulated and 593 proteins were downregulated.

[0072] A total of 518 proteins were upregulated and 347 proteins were downregulated across the three time periods. The protein expression patterns are as follows: Figure 3 As shown.

[0073] Proteins showing significant changes were selected for differential expression analysis using the pheatmap package in RStudio. A differential heatmap of the top 50 differentially expressed proteins was obtained, as shown below. Figure 4 As shown in the figure, the protein with significantly different expression in black shank resistant tobacco varieties is XP_016487756.1, which is shown in red in the figure. The gene corresponding to this protein is pathogenesis-related protein 1B (PR-1B). The amino acid sequence of this protein was obtained by blastP alignment on the NCBI website as shown in SEQ ID No:1, and its gene coding sequence on NCBI is shown in SEQ ID No:2.

[0074] Example 6: Overexpression of pathogenesis-related protein 1B (PR-1B) in susceptible varieties can enhance tobacco resistance to black shank.

[0075] The gene coding sequence of PR-1B protein was cloned from the disease-resistant yunyan300, and the PR-1B protein was overexpressed in the disease-susceptible HD to obtain the T0 generation transgenic tobacco, named OX-1 and OX-3.

[0076] Agrobacterium-mediated tobacco transformation and identification of transgenic plants

[0077] The recombinant plasmid, after being identified by PCR reaction and sequencing, was transformed into Agrobacterium LBA4404 using the freeze-thaw method. Positive Agrobacterium strains were identified by colony PCR, and the tobacco variety Yunyan 87 was transformed using the Agrobacterium-mediated leaf disc method.

[0078] The specific method is as follows:

[0079] (1) Under sterile conditions, put tobacco seeds into EP tubes and rinse them 2-3 times with sterile water;

[0080] (2) Soak in 75% alcohol for 30-60 seconds;

[0081] (3) Treat with 0.1% mercuric chloride for 5 minutes, and finally rinse with sterile water 5 times;

[0082] (4) Seeds were sown on MS medium and cultured in the tissue culture room of Yunnan Tobacco Agricultural Science Research Institute for 4 days in the dark. Then, cultured under light at 25℃ for 20-30 days.

[0083] (5) When the tobacco seedlings grow to 3-5cm (20-30 days), take the apical bud and place it on MS+BA0.2 mg / L (to strengthen the bud and promote rapid growth) medium for subculture;

[0084] (6) After subculture for 14 days (as long as there are small leaves), take leaves, 1cm x 1cm in size, cut off the petiole, scratch the surface and edge of the leaf, place them on MS + BA 1.0mg / L pH 6.0-6.5 pre-culture medium, place them face down and close to the medium, and pre-culture in the dark for 2-3 days.

[0085] (7) Then remove the pre-cultured leaves or stem segments and place them in the infection solution for infection. The night before infection, shake two bottles of Agrobacterium. Fill 2ml centrifuge tubes with bacterial solution, centrifuge at 4000rpm for 5min, and wash twice with bacterial suspension. Add 1.5ml of bacterial cells to the bacterial suspension at a ratio of 1:10 (10ml of bacterial suspension to 1 tube of 1.5ml of bacterial cells), add 25mg / L As (40ulAs in 40ml), and shake the infection solution continuously to ensure full contact with the cut surfaces of the leaves and stem segments. After 10min, remove the cells and place them on sterilized dry filter paper to absorb the bacterial solution.

[0086] (8) Place the leaves and stem segments back onto the pre-culture medium and co-culture them at 28°C in the dark for 2-3 days until micro-microbe spots form around the leaf cuts;

[0087] (9) Wash the bacteria. Take out the tobacco leaves and stem segments from the co-culture and rinse them 5 times with sterile water containing 500 mg / L eF. Place them on a shaker for 30 minutes the first time and shake for 5 minutes each time thereafter to wash away the Agrobacterium on the surface of the explants.

[0088] (10) After removing the sample, blot it dry with filter paper and transfer it to tobacco budding medium. The budding medium is MS + BA 1.0 mg / L + Hyg 25 mg / L + Cef 500 mg / L pH 5.8. Observe after 2 weeks. If no bacteria are found, reduce the Cef concentration. If bacteria are found, continue to maintain the Cef concentration.

[0089] (11) Change the culture medium every two weeks until adventitious buds appear (usually 2 weeks). Cut off the regenerated seedlings (about 1 cm long) and transfer them to subculture medium MS + BA 0.2-0.1 mg / L + Hyg 25 mg / L + Cef 500 mg / L pH 5.8;

[0090] (12) When the seedlings grow to 2cm in length (with small buds), they are transferred to rooting medium MS + NAA 0.2-0.1mg / L and cultured at 24±1℃, 12h light, and 1500lx for about three weeks to grow strong roots.

[0091] (13) When the roots grow to 2-3cm and the seedlings are about 7-10cm tall, remove them from the Erlenmeyer flasks, wash off the culture medium from the roots, and transplant them into flower pots for greenhouse cultivation.

[0092] Genomic DNA was extracted from transgenic tobacco seedlings using a Qiagen DNA extraction kit. Primers for the Basta resistance gene were designed for PCR amplification, and positive plants were screened. 25 positive plants were detected.

[0093] Total RNA was extracted from wild-type plants and 25 NtSAP5 gene-transformed T0 generation plants as described in Example 2, and Real-time PCR analysis was performed. The internal reference gene was 26s, and the expression of different lines was analyzed. The two plants with the highest expression levels ( Figure 4 ).

[0094] Fifty days after transplanting T0 generation transgenic tobacco, it was simultaneously inoculated with blackleg disease race 0 along with susceptible HD plants. Seven days after inoculation, the susceptible HD plants showed signs of wilting, while the PR-1B overexpressing plants OX-1 and OX-3 showed normal growth. (See details...) Figure 5 .

[0095] The *Acer rubrum* plant transgenic with the pathogenesis-related protein 1B gene was susceptible to infection by *Hypertida rubrum*, but exhibited significant tissue necrosis at the inoculation site, thus inhibiting further infection of tobacco plants by *Hypertida rubrum*. The overexpressing plants were named OX-1 and OX-3, while the control plant was HD. Figure 5This indicates that overexpression of pathogenesis-related protein 1B in tobacco can significantly improve tobacco's resistance to black shank disease.

[0096] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method of breeding tobacco plants, characterized by, The method comprises the following steps: cloning the gene coding sequence of the PR-1B protein in the disease-resistant Yunyan 300, wherein the amino acid sequence of the PR-1B protein is shown as SEQ ID No: 1, so that the PR-1B protein is overexpressed in the disease-susceptible flue-cured tobacco variety Honghuadajinyuan HD, and T0 generation transgenic tobacco is obtained; The transgenic tobacco has the characteristic of resisting black shank disease.

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