Use of induced transcription factor coWRKY3 in plant resistance to anthracnose

Abstract

The application belongs to the technical field of transcription factor application, and discloses application of transcription factor CoWRKY3 in resisting anthracnose of plants. The nucleotide sequence of the transcription factor CoWRKY3 is shown in SEQ ID NO. 1, and the application of the transcription factor CoWRKY3 in resisting anthracnose of plants includes application of the protein coded by the transcription factor CoWRKY3 in improving the anthracnose resistance of plants. The CoWRKY3 gene is of great significance to cultivation of transgenic crops of anthracnose-resistant varieties and improvement of the stress resistance of crops. The application firstly proposes and verifies the function of the CoWRKY3 gene in resisting anthracnose of tobacco, and enriches the gene resources for cultivation of crops with strong disease resistance.

Description

Technical Field

[0001] This invention belongs to the field of transcription factor application technology, specifically relating to the application of the inducible transcription factor CoWRKY3 in plant anthracnose resistance. Background Technology

[0002] Camellia oleifera Abel. is a woody oilseed plant endemic to my country, widely distributed in 14 provinces south of the Qinling Mountains and the Huai River. Along with olive, oil palm, and coconut, it is considered one of the world's four major woody edible oil source tree species. It is the raw material for various products such as tea oil, tea shells, tea seed cake, and tea roots. Tea oil, in particular, can effectively improve cardiovascular and cerebrovascular diseases, lower cholesterol, and inhibit the rise of triglycerides, making it a pure natural health-promoting edible oil with high nutritional and medicinal value. Anthracnose fungi are a class of globally distributed plant pathogens mainly found in tropical and subtropical regions. They can infect and cause anthracnose in more than 600 species of plants, including grasses, fruit trees, flowers, and vegetables, leading to significant economic losses. Anthracnose in Camellia oleifera, caused by Anthracnose fungi, is one of the major diseases affecting the camellia oleifera industry, severely restricting its development. With the rise of the camellia oil industry, frequent introductions of the plantation in various parts of the country, and the unrestrained overuse of chemical agents, the physiological races of this type of pathogen have changed rapidly, resulting in serious drug resistance. The original anthracnose-resistant camellia oil varieties have gradually lost their resistance, which has brought great difficulty to the prevention and control of anthracnose.

[0003] Transcription factors (TFs) are DNA-binding proteins that specifically interact with cis-acting elements, thereby influencing the transcription of a series of downstream genes. When plants encounter pathogenic stress, they trigger various defense mechanisms to resist adverse environments, generating a series of information transmissions that enable plant cells to respond to stress and enhance plant disease resistance. WRKY transcription factors participate in many signal transduction pathways in plants and play an important regulatory role in the response to biological stress.

[0004] Since their discovery, WRKY transcription factors have been a hot topic in plant defense response research. In model plants such as Arabidopsis thaliana, tobacco, and rice, WRKY members involved in disease resistance-related signal transduction pathways have been extensively studied. However, there are few reports on the cloning and functional studies of WRKY transcription factors related to disease resistance in Camellia oleifera, especially regarding the interaction between Camellia oleifera and anthracnose. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, this invention provides the application of inducible transcription factor CoWRKY3 in plant anthracnose resistance. The use of inducible transcription factor CoWRKY3 to cultivate anthracnose-resistant transgenic crops is of great significance for improving crop disease resistance.

[0006] The above-mentioned objective of the present invention is achieved through the following technical solution: the application of inducible transcription factor CoWRKY3 in plant anthracnose resistance, wherein the nucleotide sequence of inducible transcription factor CoWRKY3 is shown in SEQ ID NO.1.

[0007] Furthermore, the application of inducible transcription factor CoWRKY3 in plant anthracnose resistance includes the use of proteins encoded by CoWRKY3 to enhance plant resistance to anthracnose.

[0008] Furthermore, the amino acid sequence of the protein encoded by the aforementioned inducible transcription factor CoWRKY3 is shown in SEQ ID NO.2.

[0009] Furthermore, the nucleotide sequence of the forward primer for amplifying the aforementioned inducible transcription factor CoWRKY3 is shown in SEQ ID NO.3, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.4.

[0010] Furthermore, the nucleotide sequence of the forward primer for the overexpression vector of the aforementioned inducible transcription factor CoWRKY3 is shown in SEQ ID NO.5, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.6.

[0011] The beneficial effects of this invention compared to existing technologies are as follows: The CoWRKY3 gene is of great significance for breeding transgenic crops resistant to anthracnose and improving the stress resistance of crops; the phenotype of plants after anthracnose treatment shows that wild-type plants have more severe leaf disease and larger lesion areas than transgenic plants, while the transgenic plants show significantly enhanced disease resistance. This indicates that heterologous expression of the CoWRKY3 gene in tobacco significantly improves the plant's resistance to anthracnose. Therefore, this invention is the first to propose and verify the function of the CoWRKY3 gene in anthracnose resistance in tobacco, enriching the gene resources for breeding highly disease-resistant crops. Attached Figure Description

[0012] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0013] Figure 1 Schematic diagram of the conserved domains of the CoWRKY3 gene;

[0014] Figure 2 A phylogenetic tree diagram of the CoWRKY3 gene;

[0015] Figure 3 PCR detection diagram of genetically modified tobacco;

[0016] Figure 4 Figure showing the expression level analysis of CoWRKY3 in silent and control plants;

[0017] Figure 5 Phenotypic analysis of plant disease resistance;

[0018] Figure 6 Graph showing the peroxidase (POD) activity analysis of each plant after pathogen treatment;

[0019] Figure 7 Graph showing the superoxide dismutase (SOD) activity analysis of various plants after pathogen treatment;

[0020] Figure 8 Graph showing the analysis of phenylalanine ammonia-lyase (PAL) content in each plant after pathogen treatment. Detailed Implementation

[0021] The present invention is described in detail below through specific embodiments, but this does not limit the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the present invention are all conventional methods, and the experimental equipment, materials, reagents, etc. used can be obtained commercially. The following embodiments do not limit the present invention. For operation steps not described in detail in the embodiments, please refer to the corresponding section of the third edition of Molecular Cloning: A Laboratory Manual (J. Sambrook, E.F. Fritsch, et al., Science Press) or the instructions for the reagent kits used.

[0022] Example 1

[0023] Cloning of the CoWRKY3 gene in Camellia oleifera

[0024] Fresh leaves were collected from the Camellia oleifera cultivar Minyu 2 and preserved under liquid nitrogen for RNA extraction. The total RNA extraction steps are as follows:

[0025] S1. Grind the camellia oil sample in a mortar pre-cooled with liquid nitrogen, and continuously add liquid nitrogen to the mortar to prevent the sample from melting;

[0026] S2. Add the ground sample to a 1.5 mL sterile enzyme-free centrifuge tube containing 1 mL TRIZOL, weighing as you add. Take a sample of 50-60 mg, homogenize thoroughly, and let stand at room temperature for 5 min.

[0027] S3. Add 200 μL of chloroform, mix well, and centrifuge at 12000g for 15 min at 4℃;

[0028] S4. Take 500 μL of the supernatant and add it to a new centrifuge tube. Add 200 μL of chloroform, mix well, and centrifuge at 12000g for 15 min at 4℃.

[0029] S5. Take about 500 μL of the supernatant and add it to a new centrifuge tube (if there is still too much precipitate, you can extract it again), add 500 μL of isopropanol, gently mix the liquid in the tube, and let it stand at room temperature for 10 min.

[0030] Centrifuge at 6.4℃, 12000g for 10 min;

[0031] S7. Discard the supernatant, add 1 mL of 75% ethanol (prepared with DEPC in water) and gently wash the precipitate. Centrifuge at 7500g for 5 min at 4℃, and discard the supernatant;

[0032] S8. Swish the liquid from the tube wall to the bottom of the tube, remove the liquid completely with a pipette, and let it air dry in a fume hood for 10-20 minutes;

[0033] S9. Add an appropriate amount of DEPC H2O to dissolve the precipitate (promote dissolution at 65℃ for 10-15 min).

[0034] According to PrimeScript TM The cDNA was reverse transcribed into cDNA using the method described in the 1st Strand cDNA Synthesis Kit (Takara, Dalian, China). The primers listed in Table 1 below were designed and used as templates for amplification.

[0035] Table 1. PCR primer sequences for the CoWRKY3 gene (SEQ ID NO.3)

[0036]

[0037] Prepare the following mixtures in PCR tubes, and the reaction system is shown in Table 2 below:

[0038] Table 2 PCR reaction system

[0039]

[0040]

[0041] After gently mixing, centrifuge and place each tube on a PCR instrument. Set the reaction program as follows: 94℃ for 5 min, 1 cycle; 94℃ for 30 s, 55℃ for 30 s, 72℃ for 90 s, for a total of 35 cycles; 72℃ for 10 min, 1 cycle; store the product at -20℃.

[0042] Example 2

[0043] Construction of pMD 19T-CoWRKY3 cloning vector

[0044] CoWRKY3 gene PCR product recovery and purification

[0045] S1. The PCR product of the CoWRKY3 gene was recovered and purified using the TIANgel Maxi Purification Kit from Beijing Tiangen Biotech Co., Ltd. The purified target fragment was diluted to 50 ng / μL, and the ligation system described in Table 3 below was constructed according to the pMD 19T instructions:

[0046] Table 3. PCR Product Recovery, Purification, and Ligation System

[0047]

[0048] After gently mixing with a pipette, incubate at 16°C for 12 hours.

[0049] Sequence analysis of CoWRKY3

[0050] S2. Bioinformatics methods were used to analyze the sequence. Homologous sequences of CoWRKY3 were retrieved from the NCBI database. The CoWRKY3 gene contains a 1566 bp open reading frame encoding 522 amino acids, and conserved domains were analyzed. Figure 1 The molecular formula of CoWRKY3 is C3. 2478 H 3915 N 723 O 809 S 17 The protein has a molecular weight of 57.3247 kDa and a predicted isoelectric point of 6.53, indicating it is an acidic protein. Ser is the most abundant amino acid, accounting for 11.9% of the total. The total number of positively charged residues (Arg+Lys) is 55, and the total number of negatively charged residues (Asp+Glu) is 59. The instability coefficient is 57.87, indicating that CoWRKY3 is an unstable protein (unstable protein: coefficient > 40; stable protein: coefficient < 40). Sequences similar to the Camellia oleifera CoWRKY3 protein sequence were downloaded using BlastP. Multiple sequence alignment of Camellia oleifera CoWRKY3 with protein sequences from other plants was performed using BioEdit software. A phylogenetic tree was constructed and analyzed using MEGA7 software and Bootstrap. Figure 2 To assess its statistical reliability.

[0051] Example 3

[0052] Construction of overexpression vector pCAMBIA1300-CoWRKY3

[0053] (1) Extraction and enzyme digestion of pCAMBIA1300-mCherry plasmid

[0054] Escherichia coli DH5α containing the pCAMBIA1300-mCherry plasmid, stored at -80℃, was picked and plated onto LB agar containing 50 mg / L Kan, and incubated upside down at 37℃ for 12–16 h. A single colony of normal growth was picked and dissolved in a 200 μL tube containing 10 μL of sterile water to fully suspend the bacteria. 5 μL of the suspension was used for colony PCR detection. The remaining 5 mL of the suspension was added to liquid LB agar containing 50 mg / L Kan, and incubated at 37℃ with shaking at 200 rpm for 14–16 h.

[0055] (2) Ligation of the target fragment with the pCAMBIA1300-mCherry vector

[0056] The digested pCAMBIA1300-mCherry plasmid, along with the digested pMD 19T-CoWRKY3 gene fragment recovered from gel excision and stored at -20℃, and the pCAMBIA1300-mCherry vector framework, were used for amplification. Primers (listed in Table 4 below) were designed based on the restriction sites of the expression vector and the coding region sequence of the gene (the amplification primers contained SacⅠ and BamHI respectively, which were then ligated into pMD 19-T-Simple).

[0057] Table 4 Primer sequences for constructing CoWRKY3 overexpression vectors (SEQ ID NO.4)

[0058]

[0059]

[0060] Note: Bold text indicates protective bases; underlined text indicates Sac I and BamHI restriction sites, respectively.

[0061] The purified target fragment was diluted to 50 ng / μL and ligated into the overexpression vector pCAMBIA1300-mCherry. After digestion of the T-vector and expression vector with Sac I and BamHI, the target gene was ligated to the vector framework using T4 ligase, following the reaction system described in Table 5.

[0062] Table 5. Target gene-vector ligation reaction system

[0063]

[0064] After gently mixing with a pipette, the mixture was ligated at 16°C for 14 hours. The ligation product was then transformed into E. coli DH5α, and transformants were screened on resistant medium. Positive clones were selected, and plasmids were extracted for enzyme digestion identification.

[0065] Example 4

[0066] Transformation of Agrobacterium

[0067] (1) Preparation of Agrobacterium GV3101 competent cells

[0068] ① Pick Agrobacterium GV3101 stored at -80℃ and spread it on YEB solid medium containing 100mg / L Rif and 100mg / L Str, and incubate it upside down at 28℃ for 18-20h;

[0069] ② Pick a single colony with normal growth and inoculate it into 10 mL of liquid YEB medium containing 100 mg / L Rif, 100 mg / L Str and 50 mg / L Kan. Incubate at 28°C with shaking at 200 r / min for 18 h.

[0070] ③ Take 0.5 mL of the activated bacterial solution and inoculate it into a 500 mL Erlenmeyer flask containing 50 mL of YEB liquid medium. Incubate at 28℃ with full shaking at 200 r / min until the OD600 value of the bacterial solution is 0.5.

[0071] ④ Transfer the bacterial culture to a 50mL polypropylene plastic centrifuge tube, place it on ice for 10min, and centrifuge at 4000r / min for 5min at 4℃ to collect the bacterial precipitate.

[0072] ⑤ Discard the supernatant, gently resuspend the bacterial cells in 4 mL of freshly prepared 20 mmol / L CaCl2, and collect the bacterial cell precipitate by centrifugation at 4000 r / min for 5 min at 4℃.

[0073] ⑥ Discard the supernatant, gently resuspend the bacterial cells in 2 mL of 20 mmol / L CaCl2, and dispense 100 μL into each pre-chilled 1.5 mL tube on ice. The bacterial cells can be used directly for transformation.

[0074] (2) Transformation of Agrobacterium GV3101 and screening of positive strains

[0075] ① Take 1 μL of recombinant vector pCAMBIA1300-mCherry-CoWRKY3 and add it to GV3101 competent tube, and let it stand on ice for 30 min;

[0076] ② Place the tube on a float and freeze it in liquid nitrogen for 5 minutes, then immediately remove it and heat shock it in a water bath at 37°C for 5 minutes;

[0077] ③ Add 900 μL of YEB liquid culture medium preheated to 37℃ to the heat-shocked competent cells, and culture at 200 r / min for 2 h at 37℃, followed by centrifugation at 4000 r / min for 5 min.

[0078] ④ Discard 800 μL of supernatant, resuspend the remaining liquid and bacterial precipitate thoroughly, and spread them on YEB solid selection medium (100 mg / L Rif + 100 mg / L Str + 50 mg / L Kan). Incubate upside down at 28°C for 40-48 h. Pick single colonies that grow normally on YEB solid selection medium for colony PCR identification.

[0079] Example 5

[0080] Tobacco Conversion

[0081] S1. Virus-free tobacco seedlings

[0082] On a clean bench, tobacco seeds were first rinsed in 75% ethanol for 30 seconds, then transferred to a beaker containing 3% sodium hypochlorite solution and soaked for 10 minutes. After soaking, the solution was immediately discarded and the seeds were rinsed thoroughly with sterile water four times. Subsequently, the seeds were sown on sterile petri dishes containing MS solid medium, sealed with sealing film, and placed in an artificial climate chamber for 2 weeks of culture at 25±3℃ under 16h light / 8h darkness. After the tobacco seedlings emerged, each plant was transferred to a sterile tissue culture bottle containing MS solid medium and cultured for 3 weeks.

[0083] S2. Pre-culture of tobacco seedlings

[0084] On a clean bench, remove the petioles from tobacco leaves, scratch the edges and leaf surface, and cut them into 1cm×1cm pieces. Place them on sterile petri dishes containing MS pre-medium (MS + 0.5mg / L 6-BA + 0.1mg / L NAA, pH 6.0), seal them, and place them in an artificial climate chamber. Incubate for 2 days at 25±3℃ under 16h light / 8h darkness.

[0085] S3. Preparation of Agrobacterium infection solution

[0086] Agrobacterium GV3101 bacterial suspension containing the CoWRKY3 gene recombinant expression vector was added to YEB liquid selection medium and cultured at 28℃ with full shaking at 200 rpm for 18 h to activate the strain. Then, 1 mL of the activated Agrobacterium suspension was transferred to 50 mL of antibiotic-free YEB liquid medium and cultured at 28℃ with full shaking at 200 rpm until the OD600 value reached 0.5, at which point it was used to infect tobacco leaves.

[0087] S4. Co-cultivation

[0088] Place the pre-cultured leaves into the invasive staining solution and shake them on a shaker at 100 rpm for 5 minutes. Immediately remove the leaves and clean off any residual solution. Then place them on a sterile culture dish containing MS pre-medium, seal the dish, and place it in an artificial climate chamber. Co-culture in the dark at 25±3℃ for 2-3 days. The leaves are ready when tiny bacterial spots appear at the cut surfaces.

[0089] S5. Screening of Transformed Seedlings

[0090] The co-cultured tobacco leaves were gently rinsed in a beaker containing 500 mg / L Carb in sterile water until no flocculent hyphae appeared. Then, the residual liquid on the leaf surface was blotted dry with sterile filter paper and placed on a sterile petri dish containing screening medium (MS medium + 500 mg / L Carb + 50 mg / L Kan, pH 6.0). The dish was then sealed and placed in an artificial climate chamber and cultured at 25±3℃ for 16 h light / 8 h dark until differentiation and seedling emergence.

[0091] S6. Subculture and Rooting Culture

[0092] Select buds with intact growth points and good growth status on antibiotic medium, cut them completely, and transfer them to 1 / 2 MS medium (1 / 2 MS medium + 300 mg / L Carb + 50 mg / L Kan, pH 5.8) for rooting culture. After 3 weeks, cut the growth points again for subculture.

[0093] S7. Molecular detection of converted tobacco

[0094] Genomic DNA was extracted from leaves of untransformed wild-type tobacco (WT) and Kan-selected transformed tobacco using the genomic DNA extraction kit from Tiangen Biotech Co., Ltd. Using this genomic DNA as a template, amplification was performed using specific primers for the Camellia oleifera CoWRKY3 gene. The size of the target band was verified by 1% agarose gel electrophoresis. Three transgenic lines amplified bands of the same size as the target gene, while no amplified band was observed in WT plants. Figure 3 As shown, this indicates that the CoWRKY3 gene has been successfully transferred into tobacco. According to... Figure 4 Two lines with high expression levels were selected for resistance analysis.

[0095] Physiological analysis of the transgenic CoWRKY3 tobacco obtained in the above embodiments;

[0096] Disease resistance identification in genetically modified tobacco;

[0097] Select healthy, undamaged tobacco plants with similar growth conditions for spraying and inoculation, with an inoculation spore concentration of 10. 6 The concentration of 100 lesions / mL was sprayed and then transferred to an artificial climate chamber with a relative humidity of 100%. The cells were cultured in the dark at 28°C for 2 days, followed by 8 hours of light and 16 hours of darkness for 5 days, with water sprayed every 12 hours to maintain humidity. Seven days after inoculation, the leaves were cut off and the disease severity was assessed based on the total area of ​​lesions.

[0098] The activities of peroxidase (POD), superoxide dismutase (SOD), and phenylalanine ammonia-lyase (PAL) in wild-type and transgenic tobacco plants were measured before and after infection.

[0099] (1) POD activity assay:

[0100] ① Select tobacco leaves before and after pathogen infection, rinse the leaves with ddH2O; cut leaf tissue from the same part into 0.1g pieces and place them in a liquid nitrogen pre-cooled mortar, add 1mL of phosphate buffer (0.05mol / L, pH7.8), and grind them into a homogenate in an ice bath;

[0101] ② Transfer the entire homogenate to a 1.5 mL tube and centrifuge at 12000 r / min for 10 min at 4℃;

[0102] ③ Transfer 20 μL of supernatant enzyme solution to a new tube, and add 200 μL of 2% H2O2, 580 μL of phosphate buffer, and 200 μL of guaiacol in sequence. Measure the OD value at 470 nm on a microplate reader, with each measurement interval of 30 seconds. Calculate the ΔOD value per minute. 470 A value of 0.01 represents one enzyme activity unit. The formula is: POD activity (U / g FW) = (ΔOD) / (U / g FW ... 470 ×V) / (W×a×0.01×t), △OD 470 The change in OD value over the reaction time is represented by V, where V is the volume of supernatant enzyme solution, W is the sample weight, and t is the reaction time. WT before inoculation is taken as 1, and the relative POD activity of other groups is calculated as POD. 其它组 / POD WT .

[0103] (2) SOD activity assay:

[0104] ① Select tobacco leaves before and after infection with C. destructivum, rinse the leaves with ddH2O; cut leaf tissue from the same part into 0.1g pieces and place them in a liquid nitrogen pre-cooled mortar, add 1mL of phosphate buffer (0.05mol / L, pH7.8), and grind into a homogenate in an ice bath.

[0105] ② Transfer the entire homogenate to a 1.5 mL tube and centrifuge at 12000 r / min for 10 min at 4℃;

[0106] ③ Take 1.5 mL tubes as test tubes and control tubes respectively, and add 500 μL of 0.05 mol / L phosphate buffer solution, 100 μL of 130 mmol / L methionine solution, 100 μL of 750 μmol / L NBT solution, 100 μL of 100 μmol / L disodium EDTA solution, 100 μL of 20 μmol / L riboflavin solution, and 80 μL of ddH2O in sequence. Add 20 μL of enzyme extraction solution to the test tube, and replace the control tube with the buffer solution used for preparation.

[0107] ④ Let stand under a fluorescent lamp for 20 minutes, then immediately avoid light. Using the control tube as the zero value, measure the OD value at 560 nm using a microplate reader. One enzyme activity unit is defined as the inhibition of NBT photoreduction by 50%. The SOD activity is expressed as SOD activity (U / g FW) = (ΔOD). 470 CK-△OD 470 )×V / (△OD 470 Calculate CK×W×a×0.05; △OD 470 CK is the change value of the control tube, △OD 470 To measure the change in the measurement tube, V is the volume of the supernatant enzyme solution, a is the volume of enzyme used, and W is the sample weight. With WT before inoculation as 1, the relative SOD activity of other groups is equal to SOD. 其它组 / SOD WT .

[0108] (3) PAL activity assay:

[0109] ① Cut leaves from the same part of the tobacco plant before and after the tobacco anthracnose treatment, and wash the leaf surface with ddH2O; cut the leaf tissue from the same part into 0.1g pieces and place them in a liquid nitrogen pre-cooled mortar, add 1mL of mercaptoethanol (2mmol / L)-boric acid (0.1mol / L, pH8.8) buffer and a small amount of polyvinylpyrrolidone, and grind them into a homogenate in an ice bath;

[0110] ② Transfer the entire homogenate to a 1.5 mL tube and centrifuge at 12000 r / min for 10 min at 4℃;

[0111] ③ Transfer 250 μL of supernatant enzyme solution to a new tube, add 250 μL of 0.02 mol / L phenylalanine solution and 500 μL of distilled water sequentially, mix thoroughly, and incubate at 30℃ for 1 h. Measure the OD value at 290 nm. The ΔOD value is calculated as follows: ΔOD value is calculated per 1 h. 290 A value of 0.01 represents one enzyme activity unit. PAL activity (U / g FW) = (ΔOD) 290 ×V) / (W×a×0.01×t), △OD 290The change in OD value over the reaction time is represented by V, where V is the volume of the supernatant enzyme solution, W is the sample weight, and t is the reaction time. WT before inoculation is taken as 1, and the relative PAL activity of other groups is equal to the PAL value. 其它组 / PAL WT .

[0112] Seven days after live spraying inoculation, the lesion area results showed that WT tobacco leaves were severely affected, with mostly expanding lesions, obvious yellowing at the boundary between diseased and healthy tissue, and slight leaf wrinkling. The WT lesion area was relatively large. The CoWRKY3 transgenic strain was less affected, with mostly localized lesions and smaller lesion areas. Figure 5 ).

[0113] POD and SOD are important protective enzymes in the plant's antioxidant system, which can scavenge excess ROS and reduce cell damage. PAL is closely related to the synthesis of lignin, phytoalexins, and phenolic compounds, and plays an important role in plant disease resistance. Analysis results showed that before treatment, there were no significant differences in POD, SOD, and PAL levels between WT and transgenic lines. After treatment, the activities of all three defense enzymes increased to varying degrees, with significant differences. After treatment, the transgenic plants showed higher activities of all three defense enzymes than other lines. Figure 6 (7, 8). Based on the changes in physiological values ​​before and after pathogen treatment and in conjunction with the disease incidence, it can be seen that the activities of the three defense enzymes are closely related to the tobacco's disease resistance. CoWRKY3 may regulate the tobacco's resistance to anthrax by altering the activities of POD, SOD, and PAL.

[0114] The embodiments described above are merely preferred embodiments of the present invention, and not all feasible embodiments of the present invention. Any obvious modifications made by those skilled in the art without departing from the principles and spirit of the present invention should be considered to be included within the scope of protection of the claims of the present invention.

Claims

1. The application of inducible transcription factor CoWRKY3 in tobacco anthrax resistance, characterized in that, The amino acid sequence of the protein encoded by the inducible transcription factor CoWRKY3 is shown in SEQ ID NO.

2. The application is to overexpress the inducible transcription factor CoWRKY3 in tobacco to enhance the tobacco's resistance to anthrax.

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