Application of NtWRKY6 gene in improving tobacco resistance to wild fire disease and tobacco germplasm improvement

Abstract

The application discloses NtWRKY6 The application of a gene in improving the resistance of tobacco to wild fire disease and tobacco germplasm resource improvement belongs to the technical field of plant genetic engineering. NtWRKY6 The application successfully constructs a tobacco plant with inhibited expression of the gene by using a virus-induced gene silencing (VIGS) technology. NtWRKY6 It is found that, compared with control plants, the tobacco plants with the gene silenced exhibit the characteristics of enhanced resistance, such as reduced plaque area and decreased accumulation of pathogenic bacteria. NtWRKY6 Further detection shows that the accumulation of active oxygen (H2O2 and O2 ‑ ) in the leaves of the tobacco plants with the gene silenced is significantly enhanced. NtWRKY6 NtWRKY6 The application proves that the gene has a negative regulation effect on the resistance of tobacco to wild fire disease, and provides a molecular target and data support for cultivating new tobacco disease-resistant varieties in the future.

Description

Technical Field

[0001] This invention relates to NtWRKY6 The application of genes in improving tobacco's resistance to wildfire and in the improvement of tobacco germplasm resources belongs to the field of plant genetic engineering technology. Background Technology

[0003] Tobacco wildfire is a bacterial disease caused by *Pseudomonas syringae* pv. tabaci, and is one of the major diseases affecting tobacco-growing regions worldwide. *Pseudomonas syringae* is a short rod-shaped bacterium with unipolar flagella and is Gram-negative. Its pathogenic mechanism hinges on the type III secretion system: the pathogen injects at least 22 type III effector proteins into tobacco cells through the needle-like structures of this system. These effector proteins suppress the plant's immune response, thereby establishing infection. Studies have shown that the loss of a single effector protein is insufficient to weaken the pathogen's virulence; the pathogen must cumulatively lose 14 to 22 effector proteins, especially core effector proteins such as AvrE1 and HopM1, to completely lose its infectivity, revealing the complexity of the pathogen's pathogenic mechanism. Furthermore, the pathogen produces a special amino acid called "wildfire toxin" during its metabolism, which damages the structure and function of chloroplasts, leading to the characteristic chlorotic halo on the leaves.

[0004] Tobacco wildfire disease primarily affects tobacco leaves, occurring mostly in the mid-to-late stages of growth. Its most typical symptoms are: small, dark brown, water-soaked, round spots initially appear on the leaves, which then enlarge, developing a reddish-brown necrotic center surrounded by a wide, distinct yellow halo. When moist, bacterial ooze oozes from the lesions; after drying, the lesions rupture and fall off. Besides tobacco leaves, stems, flowers, and capsules can also be affected, forming irregular small spots. Stem lesions are sunken, but the yellow halo is less pronounced.

[0005] Wildfire disease poses a significant threat to tobacco production, often breaking out and spreading after storms. In severe cases, leaves can rot and wither within days, leading to reduced yield and quality. The pathogen mainly overwinters in diseased plant debris, infected seeds, and the roots of weeds, spreading through rainwater or dew and entering through leaf stomata or wounds. High temperatures (24-30℃), high humidity (relative humidity above 81%), and stormy weather are highly conducive to disease outbreaks. Furthermore, improper field management, such as excessive nitrogen fertilization, overly dense planting, and premature topping, can cause young leaves to become waterlogged, exacerbating the disease.

[0006] Currently, methods for controlling tobacco wildfire disease in production include agricultural control, chemical control, and breeding disease-resistant varieties. Chemical control is particularly effective due to its broad-spectrum effectiveness, rapid and efficient application, ease of use, and lack of geographical or seasonal limitations, making it suitable for large-scale mechanized control. Chemical control, with its advantages such as effectiveness, has become a primary means of controlling wildfire disease. However, long-term use of chemical agents can easily cause poisoning in humans and livestock, environmental pollution, and lead to varying degrees of drug resistance in some pathogens. Breeding disease-resistant varieties is an economical and effective control method, but traditional breeding methods also suffer from problems such as long time cycles and high costs. With the establishment and development of recombinant DNA technology, the use of genetic engineering to ultimately breed tobacco varieties resistant to wildfire disease has opened up a new avenue for fundamentally solving the problem.

[0007] Plants rely on a complex series of signaling pathways to effectively defend against external threats while maintaining homeostasis. Transcription factors are key regulators of plant growth, development, and responses to external stimuli; their expression and activity are regulated at the transcriptional, post-transcriptional, and translational levels. Numerous families of transcription factors exist within plants, among which AP2, bHLH, bZIP, and WRKY have been found to participate in responses to biotic and abiotic stresses.

[0008] WRKY transcription factors are one of the largest families of transcription factors in plants. They are a class of DNA-binding proteins that play a role in plant stress responses by binding to the W-box (TGACC(A / T)) in the promoter of target genes, thereby activating or repressing the expression of downstream genes. Structurally, WRKY transcription factors contain one or two WRKY domains, including a highly conserved heptapeptide WRKYGQK at the N-terminus and a highly conserved zinc finger motif CX4-7-CX23-28-HX1-2-(H / C) at the C-terminus. Both motifs are essential for the interaction of WRKY proteins with the cis-elements of the W-box (TTGACT / C) (Cui Q, Yan X, Gao X, et al. Analysis of WRKY transcription factors and characterization of two Botrytis cinerea-responsive). LrWRKY genes from Lilium regalePlantPhysiolBiochem. 2018, 127: 525-536. Based on the number of WRKY domains and the structure of the zinc finger motif, WRKY proteins can be divided into three main groups. Groups I and II both contain a C2-H2 (C-X4-5-CX22-23-H-X1-H) zinc finger structure, and each contains two WRKY domains and one WRKY domain, respectively. Group III has a single WRKY domain, and the zinc finger structure is C2-HC (C-X7-C-X23-HXC). Group II can be further divided into five subgroups (IIa, IIb, IIc, IId, and IIe) (Ansar H, Xia L, Yahong W, et al. CaWRKY22 acts as a positive regulator in pepper response to Ralstonia solanacearum by constituting networks with CaWRKY6, CaWRKY27, CaWRKY40, and CaWRKY58. International Journal of Molecular Sciences, 2018, 19(5): E1426). The first cDNA encoding the WRKY protein was cloned from sweet potato (Ipomoea batatas) (Laudet V, Hänni C, Coll J, et al. Evolution of the nuclear receptor gene superfamily. EMBO J. 1992, 11(3):1003-13).

[0009] At the gene level, WRKY transcription factors can bind to the W-box TTGAC (C / T) in the promoters of target genes, regulating the expression of downstream genes through self-regulating forks to activate or inhibit their responses. At the protein level, WRKY transcription factors can interact with a variety of proteins, including MAP kinases, histone deacetylases, resistance R proteins, and various transcription factors, to regulate plant growth and development or various stress responses. In addition to regulating various stress responses, WRKY TFs are also one of the largest gene families among secondary metabolites such as phenols, flavonoids, lignins, and tannins. Studies have shown that in apples, MdWRKY11, MdWRKY40, and MdWRKY72 can regulate anthocyanin accumulation; and in Arabidopsis thaliana, heterologous expression of potato... WRKY6 The gene can enhance Arabidopsis thaliana's tolerance to cadmium; overexpression SlWRKY6 Genes can improve tomatoes' tolerance to low phosphorus levels; CmWRKY6-1 The gene plays a positive regulatory role in the chrysanthemum's response to stress from Fusarium oxysporum.

[0010] Although WRKY6 The gene is one of the WRKY family transcription factors, but currently there is little information about it. NtWRKY6 The mechanism of action and application value of genes in tobacco resistance to Pseudomonas syringae remain unclear. Therefore, identifying and utilizing core transcription factors such as NtWRKY6 as molecular modification targets is of great significance for breeding new tobacco lines resistant to wildfire. Summary of the Invention

[0011] The first objective of this invention is to provide NtWRKY6 The application of genes in improving tobacco resistance to wildfire disease, analysis NtWRKY6 The mechanism of gene action in tobacco's resistance to Pseudomonas syringae provides new gene loci for improving tobacco varieties using genetic engineering.

[0012] The second object of the present invention is to provide NtWRKY6 The application of genes in the improvement of tobacco germplasm resources provides a basis for utilizing... NtWRKY6 This lays the foundation for developing new tobacco varieties resistant to wildfire, using molecular modification targets.

[0013] To achieve the above objectives, in this invention NtWRKY6 The technical solution used in applying genes to improve tobacco's resistance to wildfire is: NtWRKY6 The application of genes in improving tobacco resistance to wildfire disease, inhibiting the growth of wildfire disease in tobacco. NtWRKY6 Gene expression enhances tobacco's resistance to wildfire; NtWRKY6 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0014] The beneficial effects of the above technical solution are as follows: This invention NtWRKY6 The application of genes in improving tobacco resistance to wildfire is a pioneering invention. This invention utilizes real-time PCR to discover… NtWRKY6 The transcription factor NtWRKY6, encoded by the gene, is expressed in the cell nucleus. Using virus-induced gene silencing (VIGS) technology, a novel gene silencing assay was successfully constructed. NtWRKY6 Tobacco plants with suppressed gene expression. It was then found that, compared to control plants, NtWRKY6 Gene-silenced tobacco plants, after inoculation with wildfire pathogens, exhibited enhanced resistance characteristics, including reduced lesion area and decreased pathogen accumulation. Further testing revealed... NtWRKY6 Reactive oxygen species (H2O2 and O2) in the leaves of gene-silenced tobacco plants - Accumulation is significantly enhanced. This invention proves... NtWRKY6The gene plays a negative regulatory role in tobacco resistance to wildfire, providing molecular targets and data support for the future breeding of new disease-resistant varieties.

[0015] As a further improvement, the enhancement of tobacco's resistance to wildfire disease involves reducing the area of ​​lesions on tobacco leaves, reducing the accumulation of pathogens, and increasing the accumulation of reactive oxygen species.

[0016] As a further improvement, the suppression is for constructing NtWRKY6 Gene silencing is achieved by using vectors to transform plants, followed by screening and identification.

[0017] As a further improvement, the aforementioned NtWRKY6 Gene silencing vectors NtWRKY6 The specific sequence in a gene is the guide sequence.

[0018] As a further improvement, the nucleotide sequence of the guide sequence is shown in SEQ ID NO.7.

[0019] As a further improvement, the transformation is to... NtWRKY6 Gene silencing was achieved by transforming Agrobacterium with a vector and using it as an infection solution to infect tobacco plants.

[0020] As a further improvement, the tobacco is Benzoic tobacco.

[0021] To achieve the above objectives, in this invention NtWRKY6 The technical solution adopted for the application of genes in the improvement of tobacco germplasm resources is as follows: NtWRKY6 The application of genes in the improvement of tobacco germplasm resources, the aforementioned NtWRKY6 The nucleotide sequence of the gene is shown in SEQ ID NO.1; the germplasm resource is improved to suppress... NtWRKY6 Gene expression was used to obtain tobacco varieties with enhanced resistance to wildfire.

[0022] The beneficial effects of the above technical solution are as follows: This invention has been experimentally proven to inhibit [the growth of tobacco plants]. NtWRKY6 The expression of this gene significantly enhances resistance to wildfire, indicating that this gene plays a negative regulatory role in tobacco's resistance to wildfire. This invention, through... NtWRKY6 In-depth research on gene function has provided new ideas for tobacco disease resistance breeding and genetic improvement.

[0023] As a further improvement, the tobacco variety with enhanced resistance to wildfire is obtained by the following method: NtWRKY6 Gene silencing was achieved by transforming Agrobacterium with a vector and using it as an infection solution to infect tobacco plants. Through screening and identification, tobacco varieties with enhanced resistance to wildfire disease were obtained.

[0024] As a further improvement, the tobacco is Benzoic tobacco. Attached Figure Description

[0025] Figure 1 This is a subcellular localization map of the tobacco NtWRKY6 protein in Example 1 of the present invention; Figure 2 The tobacco in Embodiment 2 of the present invention NtWRKY6 Electrophoresis diagram of gene-specific fragment amplification products; Figure 3 Tobacco VIGS in Embodiment 2 of the present invention NtWRKY6 plant NtWRKY6 Gene expression status (where the vertical axis represents relative expression level, *** represents...) P＜0.001 ); Figure 4 Tobacco VIGS in Embodiment 2 of the present invention NtWRKY6 Phenotype of plants after inoculation with wildfire disease; Figure 5 The tobacco in Embodiment 2 of the present invention NtWRKY6 Results of pathogen accumulation detection in silent plants and control plants (where * represents...) P＜0.05 ,***represent P＜0.001 ); Figure 6 The tobacco in Embodiment 2 of the present invention NtWRKY6 Accumulation of reactive oxygen species (DAB, NBT staining) in the leaves of silent plants. Detailed Implementation

[0026] Tobacco is an important model plant and economic crop. During its growth, it is often infected by various pathogens, among which wildfire disease caused by pathogenic species of *Pseudomonas syringae* is the most serious. This disease mainly affects the leaves, but can also infect the flowers, stems, and capsules. In the early stages, the disease mainly manifests as small, oily spots surrounded by a distinct yellow halo. In severe cases, multiple spots merge to form large, irregular patches, leading to extensive leaf death and cracking, ultimately resulting in decreased tobacco quality and yield loss. Current control measures mainly rely on chemical control, but the large-scale use of chemical agents can easily cause environmental pollution and pesticide residues, and can also increase the drug resistance of pathogens, making pathogen control more difficult. Therefore, using molecular breeding techniques to select disease-resistant varieties is particularly important.

[0027] Plant defense against pathogens mainly comprises two layers of defense systems: pathogen-associated molecular pattern-triggered immune responses (PTI) and effector-triggered immune responses (ETI), which are closely related to hormone signaling pathways such as salicylic acid (SA), jasmonic acid (JA), and ethylene (ETH). Among these, the interaction of the SA-JA signaling pathway is particularly crucial in tobacco's resistance to wildfire, and the WRKY transcription factor family is one of the largest transcription factor families in plants, playing an important role in plant stress responses and pathogen inactivation. NtWRKY6 is one of the WRKY family transcription factors; however... NtWRKY6 Whether genes are involved in the interaction with tobacco wildfire disease and regulate tobacco wildfire disease resistance has not been reported.

[0028] Based on this, the present invention provides, on the one hand, NtWRKY6 The application of genes in improving tobacco's resistance to wildfire disease, on the other hand, provides NtWRKY6 Application of genes in the improvement of tobacco germplasm resources.

[0029] The present invention will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the equipment and reagents used in the embodiments, experimental examples and comparative examples are all commercially available.

[0030] Unless otherwise specified, the following examples were conducted under conventional experimental conditions, such as those described in Sambrook et al.'s Molecular Cloning Laboratory Manual (Sambrook J & Russell DW, Molecular cloning: alaboratory manual, 2001), or as recommended by the manufacturer's instructions.

[0031] Biomaterials: Tobacco varieties: Seeds of both Benedictine and K326 were provided by the National Tobacco Gene Research Center of Zhengzhou Tobacco Research Institute.

[0032] Vectors: PBI121-GFP and PYY13 vectors were provided by the National Tobacco Gene Research Center.

[0033] Strains: DH5α chemocompetent cells, purchased from Takara Bio Inc.; GV3101 Agrobacterium tumefaciens competent cells, purchased from Shanghai Weidi Biotechnology Co., Ltd.

[0034] Pathogens Pseudomonas syringae pv. tabaci (Pst) Provided by Guizhou Provincial Tobacco Science Research Institute.

[0035] Primer synthesis and DNA sequencing were provided by Beijing BGI Genomics Co., Ltd.

[0036] Experimental reagents: RNA extraction kit, plasmid extraction kit, and DNA gel recovery kit were purchased from Beijing Codon Biotechnology Co., Ltd.; seamless cloning kit was purchased from Novizan Biotechnology Co., Ltd.; quantitative fluorescence kit and reverse transcription kit were purchased from Takara Bio Inc.; and DNA amplification enzyme was purchased from Kangrun Biotechnology Co., Ltd.

[0037] Experimental equipment: The PCR instrument was a T Professional Thermocycler manufactured by Biometra; the quantitative PCR instrument was a LightCycler 96 manufactured by Roche.

[0038] This invention NtWRKY6 Specific examples of the application of genes in improving tobacco resistance to wildfire and improving tobacco germplasm resources: The present invention provides the encoding gene (CDS sequence) of tobacco transcription factor NtWRKY6, which is 1647 bp in length and has the nucleotide sequence shown in SEQ ID NO.1. It encodes tobacco transcription factor NtWRKY6, which contains 548 amino acids and has the sequence shown in SEQ ID NO.2.

[0039] Example 1 To investigate the localization of the NtWRKY6 protein in tobacco cells, this embodiment constructs a subcellular localization vector, transiently transforms it into Nicotiana benthamiana, and then uses laser confocal microscopy to observe the specific expression location of the protein in tobacco cells. The specific implementation steps are as follows: 1. Construction of subcellular localization vectors Design primers NtWRKY6-PBI121-F and NtWRKY6-PBI121-R, to NtWRKY6 The gene was cloned into the PBI121 vector containing a GFP tag. The primer sequences are as follows: NtWRKY6-PBI121-F: 5'-GAACACGGGGGACTCTAGAATGGACAAAGGATGGG-3' (shown in SEQ IDNO.3); NtWRKY6-PBI121-R: 5'-GACTGACCACCCGGGgaGTTCCCTGAGA-3' (shown in SEQ ID NO. 4).

[0040] The PCR template was K326 leaf cDNA, the PCR amplification enzyme was 2×Taq PCR StarMix (Genestar), the PCR amplification system is shown in Table 1, and the PCR amplification program is shown in Table 2.

[0041] Table 1 PCR amplification system Table 2 PCR amplification program The amplified products were detected by 1.0% agarose gel electrophoresis, and then the target DNA fragment was recovered by gel excision and purification. The pBI121-GFP vector plasmid was double-digested with XbaI and SpeI, and the digestion system is shown in Table 3. The reaction conditions were 37℃ for 20 min.

[0042] Table 3 Enzyme digestion system The recovered vector fragment was then detected by 1.0% agarose gel electrophoresis after gel excision and purification. Using the ClonExpress II One Step Cloning Kit, the recovered target gene fragment and the linearized vector fragment were ligated using homologous recombination. The ligation system is shown in Table 4.

[0043] Table 4 Connection System After mixing, the mixture was incubated at 37°C for 30 min, and then cooled on ice after the reaction was complete. The ligation product was transformed into *E. coli* DH5α competent cells. Single colonies were picked and identified by PCR. Positive bacterial cultures were then sent for sequencing. The sequencing results and analysis showed that the cloned cells were... NtWRKY6 The gene sequence is shown in SEQ ID NO.1, and its encoded amino acid sequence is shown in SEQ ID NO.2. Plasmids were extracted from the correctly sequenced bacterial culture (the correctly sequenced vector was named pBI121-GFP::NtWRKY6 recombinant vector) and transformed into Agrobacterium tumefaciens competent cells GV3101. Positive single clones were picked and stored in sterile glycerol at a final concentration of 20% at -80°C for later use.

[0044] 2. Cultivation of Nicotiana benthamiana seedlings Nicotiana benthamiana seedlings were planted in plastic plant culture pots (nutrient soil: vermiculite = 3:1) and placed in the plant culture room of the National Tobacco Gene Research Center. The culture conditions were: temperature (24±2)℃, relative humidity 60%±2%, and 16 h light / 8 h dark culture. Once the seedlings reached the four-leaf stage, they were used for subcellular localization transient transformation experiments.

[0045] 3. Bacterial solution treatment and transformation Agrobacterium tumefaciens cultures containing the pBI121-GFP::NtWRKY6 recombinant vector, pBI121-GFP empty vector, and endoplasmic reticulum marker (pCAMBIA1300-35S-ER-mCherry), stored at -80℃, were cultured in a shaker at 28℃ until OD. 600The concentration reached 0.6-0.8. The bacterial cells were collected separately and then resuspended in a solution containing MgCl2, MES, and As. OD 600 Adjust to 0.8-1.0. Mix pBI121-GFP::NtWRKY6 and pBI121-GFP with endoplasmic reticulum marker resuspension at a 1:1 ratio and incubate in the dark for 3 h. Then, inoculate the resuspension into Nicotiana benthamiana from the underside of the leaf using a syringe. Incubate the inoculated plants in the dark for 8-10 h, and then place them in a culture room for 2-3 days under normal light.

[0046] 4. Subcellular localization observation Slides were prepared from tobacco leaves infected with Agrobacterium 2-3 days prior. Fluorescence was then observed and photographed using a laser confocal microscope (Olympus Spin SR10). The fluorescence parameters were: GFP excitation at 488 nm; RFP excitation at 541 nm. Subcellular localization results are shown below. Figure 1 As shown in the figure, the NtWRKY6 protein is located in the cell nucleus.

[0047] Example 2 In order to study NtWRKY6 The role of genes in tobacco resistance to wildfire disease: This embodiment utilizes a virus-induced gene silencing method. NtWRKY6 The gene expression level was reduced, and then the patient was inoculated with wildfire pathogens, thus suggesting... NtWRKY6 The function of the gene in tobacco wildfire resistance, and the specific implementation steps are as follows: 1. Construction of gene silencing vectors Using the SGN VIGS tool (https: / / vigs.solgenomics.net) from NtWRKY6 A 300bp silencing fragment was selected from the full-length gene, and specific primers were designed. The primer sequences are as follows: NtWRKY6-F: 5'-GACGACAAGACCCTGCAGGGTTTTTTCAAG-3' (shown in SEQ ID NO.5); NtWRKY6-R: 5'-GAGAAGAGCCCTGCAGTGATGAAATCCCATCATCTA-3' (shown in SEQ ID NO. 6).

[0048] amplification using K326 leaf cDNA as a template NtWRKY6 The gene silencing fragment and amplification products were analyzed by 1.0% agarose gel electrophoresis (see [link to analysis]). Figure 2 Then, the target DNA fragment is excised, purified, and recovered using a gel. The recovered target fragment is then ligated into an empty vector to construct VIGS- NtWRKY6The silencing vector and ligation product were transformed into *E. coli* DH5α competent cells. Single colonies were picked and identified by PCR. Positive bacterial cultures were then sent for sequencing. Sequencing results showed that the cloned cells... NtWRKY6 The gene silencing fragment sequence is shown in SEQ ID NO.7. Plasmids were extracted from the correctly sequenced bacterial culture and transformed into competent Agrobacterium tumefaciens cells GV3101. Positive monoclonal cultures were selected and stored at -80°C in sterile glycerol with a final concentration of 20%.

[0049] 2. Preparation of VIGS Injection The positive bacterial culture stored at -80℃ was expanded to OD on a shaker at 28℃. 600 The concentration reached 0.6-0.8. The bacterial cells were collected and then resuspended in a solution containing MgCl2, MES, and As. OD... 600 Adjust to 1. After 3 hours of dark incubation, add VIGS- NtWRKY6 pYY13 and PDS were mixed with pTRV1 in a 1:1 ratio, and then the resuspended bacterial solution was inoculated into Nicotiana benthamiana using a syringe (the cultivation of Nicotiana benthamiana seedlings was the same as in Example 1). The inoculated plants were placed in the dark and cultured overnight.

[0050] 3. System Silence Determination A PDS positive control was set up (complete leaf whitening was used as an indicator of systemic silencing). Plant type and leaf color changes were continuously observed for 14 days after inoculation. When leaf whitening appeared on the PDS plants, control plants at the same location were taken and treated with VIGS- NtWRKY6 RNA was extracted from the leaves of silent plants and reverse transcribed into cDNA for use in [further processing]. NtWRKY6 Gene silencing efficiency detection. NtActin As an internal control, VIGS- was analyzed using qRT-PCR. NtWRKY6 Silent plants NtWRKY6 Quantitative analysis of gene expression levels was performed.

[0051] The detection primers are shown below: qNtWRKY6-F: 5'-GTTTCTGTCCGTGCCCGA-3' (shown in SEQ ID NO.8); qNtWRKY6-R: 5'-ACAGCCATGGTGCAACGA-3' (shown in SEQ ID NO.9).

[0052] The primers for the internal reference gene are shown below: NtActin-F: 5'-TATTCCCTAGTATTGTTGGC-3' (shown in SEQ ID NO.10); NtActin-R: 5'-CTGGGGTATTAAAAGTCTCA-3' (shown in SEQ ID NO.11).

[0053] qRT-PCR reaction system (20 μL system): 2×TB Green Premix Ex Taq II, 10 μL; forward primer, 0.8 μL; reverse primer, 0.8 μL; cDNA template, 3 μL; sterile water, 5.4 μL.

[0054] The reaction procedure is as follows: Step 1: Pre-denaturation at 95℃ for 30s (1 cycle); Step 2: Denaturation at 95℃ for 5s, annealing at 60℃ for 30s (40 cycles).

[0055] Test results as follows Figure 3 As shown in the figure, compared with the control, the silent strains... NtWRKY6 Gene expression levels were significantly reduced.

[0056] 4. Inoculation with pathogens The pathogens used in the infection experiment were Pseudomonas syringae pv. tabaci (Pst) First, the strain was streaked onto antibiotic-free LB agar and incubated at 28°C for 24 hours to obtain single colonies. Then, a single colony was picked and inoculated into 50 mL of LB liquid medium, and cultured overnight at 28°C and 200 rpm with shaking until the cells reached the logarithmic growth phase (OD). 600 (≈0.8-1.0). After cultivation, the bacterial cells were collected by centrifugation at 5000 × g for 10 min and washed twice with sterile phosphate-buffered saline (PBS, 10 mM, pH 7.4) to remove residual culture medium. The bacterial cells were then resuspended in sterile PBS solution and the concentration was adjusted to OD0.05. 600 =0.001. The resulting bacterial suspension was used as the bacterial solution for subsequent leaf inoculation. Control plants with uniform growth and VIGS with clearly defined silencing efficiency were selected. NtWRKY6 The plants were inoculated with a suspension of tobacco wildfire pathogens by injecting the underside of the leaves.

[0057] 5. Resistance identification after inoculation (1) Observation and area statistics of lesions / symptoms Photographs were taken at 0 h, 12 h, 24 h, and 60 h post-inoculation, and the lesion area was quantified using ImageJ. Each group had at least three biological replicates, and each plant had at least three leaves injected.

[0058] The results showed that VIGS- NtWRKY6 The area of ​​lesions on the plants was significantly smaller than that of the control (see...) Figure 4 ).

[0059] (2) Absolute quantification of pathogen biomass Select the control group and VIGS- after vaccination NtWRKY6 Leaf samples from the same location in the silent strains were taken in equal areas using a perforator, and DNA was extracted. Absolute quantification was performed by qPCR using species-specific primers for wildfire pathogens. Ct values ​​were converted to DNA content (ng / μL) using a standard curve, and pathogen biomass was expressed in this unit (time points: 0, 12, 24, 60 hpi). Each group had at least three biological replicates.

[0060] The species-specific primer sequences for wildfire pathogens are as follows: tabB1F: 5'-ATTGAAGAAGCGTTCGAGCG-3' (shown in SEQ ID NO.12); tabB1R: 5'-GTTTCGGGTCGGCACGATTG-3' (shown in SEQ ID NO.13).

[0061] The results showed that VIGS- NtWRKY6 The pathogen DNA content (ng / μL) in the plants was significantly lower than that in the control material at both 24 hpi and 60 hpi. This indicates that... NtWRKY6 Gene silencing enhances the resistance of tobacco plants to wildfire (see...). Figure 5 ).

[0062] (3) Detection of reactive oxygen species (ROS) accumulation 48 h after inoculation, the isotope leaves were stained with DAB (H2O2, 10 mg / L, pH 3.8, 28℃ incubation for 6 h) and NBT (O2) staining. - (0.5 mg / mL, pH 7.0, under the same conditions); 95% ethanol decolorization imaging.

[0063] VIGS- NtWRKY6 The significantly enhanced DAB / NBT deposition in plants suggests an increased level of ROS accumulation (see [link to article]). Figure 6 ).

[0064] Based on the above results, NtWRKY6 The gene is closely related to tobacco's resistance to wildfire; inhibiting the expression of this gene significantly enhances tobacco's resistance to wildfire. In practical applications, genetic engineering techniques can be used to inhibit the expression of this gene in tobacco. NtWRKY6 Gene expression can reduce the area of ​​lesions on tobacco leaves, reduce the accumulation of pathogens, increase the accumulation of reactive oxygen species, and thus enhance the resistance of tobacco plants to wildfire disease. This can significantly accelerate the breeding process of wildfire-resistant tobacco varieties.

[0065] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. NtWRKY6 The application of genes in improving tobacco resistance to wildfire is characterized by: Inhibit tobacco NtWRKY6 Gene expression enhances tobacco's resistance to wildfire; NtWRKY6 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The method according to claim 1 NtWRKY6 The application of genes in improving tobacco resistance to wildfire is characterized by: The goal of improving tobacco's resistance to wildfire is to reduce the area of ​​lesions on tobacco leaves, reduce the accumulation of pathogens, and increase the accumulation of reactive oxygen species.

3. As described in claim 1 or 2 NtWRKY6 The application of genes in improving tobacco resistance to wildfire is characterized by: The suppression is for construction NtWRKY6 Gene silencing is achieved by using vectors to transform plants, followed by screening and identification.

4. The method according to claim 3 NtWRKY6 The application of genes in improving tobacco resistance to wildfire is characterized by: The NtWRKY6 Gene silencing vectors NtWRKY6 The specific sequence in a gene is the guide sequence.

5. The method according to claim 4 NtWRKY6 The application of genes in improving tobacco resistance to wildfire is characterized by: The nucleotide sequence of the guide sequence is shown in SEQ ID NO.

7.

6. The method according to claim 3 NtWRKY6 The application of genes in improving tobacco resistance to wildfire is characterized by: The transformation will NtWRKY6 Gene silencing was achieved by transforming Agrobacterium with a vector and using it as an infection solution to infect tobacco plants.

7. The method according to claim 1 or 2 NtWRKY6 The application of genes in improving tobacco resistance to wildfire is characterized by: The tobacco in question is Benzoic tobacco.

8. NtWRKY6 The application of genes in the improvement of tobacco germplasm resources is characterized by: The NtWRKY6 The nucleotide sequence of the gene is shown in SEQ ID NO.1; the germplasm resource is improved to suppress... NtWRKY6 Gene expression was used to obtain tobacco varieties with enhanced resistance to wildfire.

9. The method according to claim 8 NtWRKY6 The application of genes in the improvement of tobacco germplasm resources is characterized by: The tobacco varieties with enhanced resistance to wildfire were obtained through the following method: NtWRKY6 Gene silencing was achieved by transforming Agrobacterium with a vector and using it as an infection solution to infect tobacco plants. Through screening and identification, tobacco varieties with enhanced resistance to wildfire disease were obtained.

10. The method according to claim 8 or 9 NtWRKY6 The application of genes in the improvement of tobacco germplasm resources is characterized by: The tobacco in question is Benzoic tobacco.

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