Use of a stress protein usp1 in bacterial disease in tomato and methods

Abstract

The application provides application and method of stress protein USP1 (universal stress protein 1) in tomato bacterial disease, and the application finds that SlUSP1 is expressed in each tissue and is induced by pathogenic bacteria Pst DC3000 significantly. The SlUSP1 knockout and overexpression plants are constructed, and it is proved that the disease resistance is positively regulated: the disease spot of the knockout plant increases, the defense gene expression and the antioxidant enzyme activity decrease, and the resistance is enhanced. The interaction protein screening and verification show that SlUSP1 interacts with E3 ubiquitin ligase components SlWD1 / SlWD3 and exists in the form of a dimer, and the protein stability is regulated by 26S ubiquitin-proteasome pathway. SlUSP1 overexpression promotes stem thickening, branch increase and plant height reduction, and improves the expression of growth and development related genes, and the application plays a role in anti-lodging and growth regulation, and provides a key molecular target for disease resistance and high yield breeding.

Description

Technical Field

[0001] This invention relates to the fields of plant genetic engineering and agricultural disease resistance technology, and more specifically, to the application and method of a stress protein USP1 in bacterial diseases of tomatoes. Background Technology

[0002] As one of the world's most important economic crops, the yield and quality of tomatoes are directly related to agricultural economic benefits and people's livelihoods. However, tomatoes are susceptible to various diseases and pests during their growth cycle, among which the pathogenic strain of *Pseudomonas syringae* is particularly problematic. Pseudomonassyringae pv. tomato DC3000, abbreviated as Pst Bacterial spot disease of tomatoes (DC3000) has become one of the major threats to my country's tomato planting industry due to its wide distribution and high infectivity. This disease can cause brown necrotic spots on tomato leaves, deformed and cracked fruits, and in severe cases, the entire plant will wither. The economic losses caused by this disease are countless every year, and it also significantly reduces the commercial quality of tomatoes, thus restricting the sustainable development of the industry.

[0003] Currently, traditional methods for controlling bacterial spot disease in tomatoes have significant limitations and are insufficient to meet production needs: First, while chemical control can suppress the spread of pathogens in the short term, it cannot completely eradicate them. Long-term use can easily lead to drug resistance in pathogens, and pesticide residues can pollute soil and water sources, threatening the quality and safety of agricultural products, which contradicts the concept of green agriculture. Second, physical control measures (such as field sanitation and high-temperature disinfection) and cultivation management (such as crop rotation) are limited by planting scale, land resources, and climate conditions, resulting in low control efficiency, especially in large-scale contiguous planting scenarios, where it is difficult to effectively block the spread of the disease. Third, traditional disease-resistant breeding relies on screening naturally resistant germplasm resources, a process that takes 5-8 years, and currently, there are few resistant tomato germplasm. Pst The DC3000 variety lacks superior genes, making it difficult to balance disease resistance with high yield and quality agronomic traits, and thus unable to quickly respond to the risk of disease outbreaks. Therefore, identifying the key disease-resistant genes in tomatoes themselves, analyzing their functions and molecular mechanisms, and cultivating new disease-resistant varieties through genetic engineering has become the core direction for breaking through the current bottleneck in disease control.

[0004] The Universal Stress Protein (USP) family is an important component of the plant stress response network, playing a key regulatory role in responses to abiotic stresses (such as drought, high temperature, and salinity). Previous inventions have shown that the tomato USP family member SlUSP1 (gene ID: Solyc04g014600) can respond to drought and high temperature stresses and positively regulate plant stress resistance through pathways such as adjusting osmotic pressure and the antioxidant system. However, the function and mechanism of action of this gene in biotic stresses (especially bacterial diseases) have not been thoroughly explored, resulting in significant gaps in research: on the one hand, the overall role of the USP family in tomato biotic stress responses is unclear, and the differences in responses to pathogenic stress among members of different evolutionary branches lack systematic analysis, making it impossible to identify key disease-resistant members; on the other hand, SlUSP1 in… Pst The expression characteristics, regulatory pathways, and interactions of DC3000 under stress are unknown, making it difficult to clarify its specific function in the tomato immune response and thus unable to provide a precise molecular target for disease-resistant breeding.

[0005] Furthermore, existing tomato disease resistance gene development often focuses on a single disease resistance function, neglecting the gene's impact on plant growth and development. Overexpression of some disease resistance genes can easily lead to stunted plant growth and reduced yield, making them difficult to apply in actual production. Therefore, there is an urgent need to screen genes that combine disease resistance and growth regulation functions to enhance tomato disease resistance while optimizing agronomic traits, achieving a synergistic improvement in "disease resistance-high yield-high quality".

[0006] Based on the above situation, this invention addresses the challenge of controlling bacterial spot disease in tomatoes by systematically exploring the role of the SlUSP1 gene as the core. Pst The study explores the function and molecular mechanism of DC3000 in stress response, filling a gap in the discovery of the USP family in tomato biological stress. Therefore, it proposes an application and method of the stress protein USP1 in tomato bacterial diseases, providing a new molecular target and theoretical basis for improving the disease resistance and quality of tomatoes, which is of great significance for promoting the green and efficient development of the tomato industry. Summary of the Invention

[0007] The purpose of this invention is to address the problems raised in the existing background technology. To achieve the above-mentioned objective, this invention provides the following technical solution: the application of a stress protein USP1 in tomato bacterial diseases, wherein the tomato stress protein USP1 enhances the resistance of tomatoes to bacterial diseases caused by *Pseudomonas syringae* (…). Pseudomonas syringae pv. tomato DC3000 Pst Application of DC3000 in bacterial disease resistance.

[0008] As a preferred technical solution of the present invention, resistance enhancement is achieved by increasing the expression level of SlUSP1 in tomato plants.

[0009] As a preferred embodiment of the present invention, SlUSP1 can induce the expression of defense genes in tomato plants, wherein the defense genes include SlPTI5 , SlWRKY28 , SlPR1a and SlGRAS2 .

[0010] As a preferred technical solution of the present invention, SlUSP1 overexpression can improve the activity of superoxide dismutase (SOD) and peroxidase (POD) in tomato plants.

[0011] As a preferred technical solution of the present invention, SlUSP1 overexpression can also optimize the application of tomato agronomic traits.

[0012] A method for applying the stress protein USP1 in bacterial diseases of tomato includes the following steps: Step (1) Construct a SlUSP1 gene knockout or overexpression vector; Step (2) The vector was introduced into tomato explants through Agrobacterium-mediated genetic transformation. After pre-culture, co-culture, screening, rooting, and hardening, SlUSP1 knockout plants were obtained. slusp1-cr ) or overexpressing plants (SlUSP1-OE); Step (3) Perform molecular identification and disease resistance verification on transgenic plants, and screen for resistant plants. Pst DC3000 tomato plants.

[0013] As a preferred technical solution of the present invention, the construction process of the SlUSP1 gene knockout vector in step (1) is as follows: using the second exon of the SlUSP1 gene as the target region, specific target points are designed, the sequence of target point 1 is 5'–TGGATTAAATCCTGACAAAG-3', and the sequence of target point 2 is 5'–GCCTGTTGCCAAACTCAAAA-3'; a CRISPR / Cas9 knockout vector is constructed, and Escherichia coli DH5α and Agrobacterium AGL1 are transformed into it. Positive engineered Agrobacterium is obtained by PCR identification (target fragment size is 500-750bp) and sequencing verification.

[0014] As a preferred technical solution of the present invention, the construction process of the SlUSP1 gene overexpression vector in step (1) is as follows: using the cDNA of wild-type tomato AilsaCraig (AC) as a template, the complete coding region sequence of SlUSP1 is amplified, and the amplification product is cloned into the pBI121-35S-Flag vector to construct pBI121:: SlUSP1-The Flag recombinant overexpression vector was transformed into Agrobacterium AGL1 and positive clones were screened.

[0015] As a preferred technical solution of the present invention, the Agrobacterium-mediated genetic transformation in step (2) specifically includes: (a) Explant preparation: Wild-type tomato AC seeds were disinfected with 75% ethanol for 1 min and 15% sodium hypochlorite for 10 min, and then inoculated into 1 / 2 MS medium. Germination was carried out at 25 ℃ under 16 h light / 8 h dark conditions for 3-4 days. The cotyledons and tender stems were cut into 0.2 cm segments. (b) Pre-culture and co-culture: Explants were cultured for 2 days in a pre-culture medium (MS + 3% sucrose + 0.8% agar, pH 5.8) containing 1 mg / L 6-BA and 0.04 mg / L IAA; and then infected with Agrobacterium tumefaciens containing the target vector (OD). 600 =0.6) Infect explants for 15-20 min, then transfer to co-culture medium and culture for 2 days; (c) Screening and rooting: The explants were successively transferred to screening medium containing 100 μg / mL, 150 μg / mL and 200 μg / mL kanamycin, and the medium was changed every 20-25 days. After the seedlings were formed, they were transferred to rooting medium containing high concentration of auxin and cultured for 20 days. (d) Hardening off seedlings: Wash the rooted seedlings to remove any remaining culture medium residue, transplant them into nutrient soil, cover them with bags to keep them moist for 2 weeks, and then cultivate them in a greenhouse.

[0016] As a preferred technical solution of the present invention, the molecular identification and disease resistance verification in step (3) include: (a) Molecular identification: DNA was extracted from plant leaves and analyzed using Cas9 gene-specific primers. (Upstream: 5'-AGACCGTGAAGGTTGTGGAC-3'; Downstream: 5'-TAGTGATCTGCCGTGTCTCG-3') were identified by PCR; The expression level of SlUSP1 was detected by real-time quantitative PCR (qRT-PCR). The internal reference gene was SlUBI3, and the upstream primer of qRT-PCR was 5'-GCCTGTTGCCAAACTCAAAA-3'. The downstream is 5'-AAGCCTCTGCCTCCAATTAC-3'; (b) Disease resistance verification: Five-week-old positive plants were selected and tested using... Pst DC3000 bacterial culture (5×10) 5 CFU / mL (containing 0.02% Silwet L-77) was vacuum-infected. The lesion phenotype was observed after 7 days, and the pathogen colony concentration was measured after 4 days. At the same time, the expression level of defense genes and the activities of SOD and POD were detected.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention systematically analyzes the characteristics of the tomato SlUSP family, clarifies the positive regulatory role of SlUSP1 in bacterial diseases, reveals the molecular mechanism by which it regulates disease resistance through protein interaction and ubiquitination modification, and fills the gap in the research of the USP family on plant biological stress.

[0018] This invention demonstrates significant disease resistance: SlUSP1 overexpressing plants exhibit resistance to disease. Pst DC3000 significantly enhances disease resistance, greatly reduces the number of lesions and pathogen concentration, and activates multiple defense mechanisms, resulting in stable and long-lasting disease resistance, which is superior to traditional chemical control.

[0019] This invention optimizes agronomic traits: while enhancing disease resistance, SlUSP1 overexpression can optimize tomato plant type (dwarfing, thick stems, and more branches), improve lodging resistance, and induce the expression of growth and development-related genes, thus balancing disease resistance and yield potential, which meets the needs of agricultural production.

[0020] This invention cultivates disease-resistant varieties through genetic engineering, which can reduce the use of chemical pesticides by more than 50%, reduce environmental pollution and pesticide residues, and is in line with the trend of green agriculture development, with significant ecological benefits.

[0021] The technology of this invention has strong scalability: the genetic transformation method used is mature, the vector and strain are easy to obtain, it can be applied to different tomato varieties, and the SlUSP1 sequence is highly conserved. In the future, it can be extended to peppers, eggplants and other Solanaceae crops, with broad application prospects. Attached Figure Description

[0022] Figure 1 The expression level of SlUSP in tomato under different stresses provided by the present invention; Figure 2 The expression level of SlUSP1 in tomato in different tissues provided by the present invention; Figure 3 A schematic diagram of the construction of the tomato SlUSP1 gene knockout vector provided by the present invention; Figure 4 This is a schematic diagram of the genetic transformation and tissue culture of the tomato SlUSP1 gene provided by the present invention. Figure 5 This is a schematic diagram illustrating the expression of defense genes in pathogens after stress treatment, as provided by the present invention. Figure 6 A schematic diagram illustrating the changes in antioxidant enzyme activity after pathogenic stress treatment provided by the present invention; Figure 7 This is a schematic diagram of the protein-protein interaction verification provided by the present invention via immunoprecipitation. Figure 8This is a schematic diagram illustrating the morphological characteristics of the tomato SlUSP1 transgenic plant provided by the present invention. Detailed Implementation

[0023] 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.

[0024] Therefore, the following detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely illustrates some embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention. It should be noted that, in the absence of conflict, the embodiments and features and technical solutions in the embodiments of the present invention can be combined with each other. It should be noted that similar reference numerals and letters in the following figures indicate similar items; therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures.

[0025] Example 1: Application of a stress protein USP1 in bacterial diseases of tomato. Tomato stress protein SlUSP1 enhances the resistance of tomatoes to pathogenic tomato species caused by *Pseudomonas syringae* (a bacterium). Pseudomonassyringae pv. tomato DC3000 Pst The application of SlUSP1 in bacterial disease resistance caused by DC3000, wherein SlUSP1 is located in the evolutionary branch 2 of the tomato SlUSP family, is closely related to Arabidopsis ATUSP (AT3G03270), and contains highly conserved GD domain, LVIG domain, as well as motif1 (sequence VVEGDAREVJCEAVEKLHLSALVMGSRGL) and motif2 (sequence GVIKRAFLGSVSBYCVHHAKCPVVIVKKP); the coding gene ID of SlUSP1 is Solyc04g014600, located on tomato chromosome 4 near the start end.

[0026] Enhanced resistance was achieved by increasing the expression level of SlUSP1 in tomato plants, specifically manifested in the following way: tomato leaves showed increased resistance. Pst The number of lesions after DC3000 infection was reduced by 60%-70% compared with the wild type, and the concentration of pathogen colonies in the leaves was reduced by more than 80% compared with the wild type. At the same time, the cell death area of ​​tomato plants, as detected by trypan blue staining, and the amount of reactive oxygen species (ROS), as detected by DAB and NBT staining, were significantly lower than those of the wild type.

[0027] SlUSP1 can induce the expression of defense genes in tomato plants, including... SlPTI5 , SlWRKY28 , SlPR1a and SlGRAS2 ; Pst DC3000 infection for 6 hours SlPTI5 Expression levels were upregulated by 18.50 times compared to the wild type. SlWRKY28 Increased by 5.21 times, SlPR1a Increased by 14.71 times. SlGRAS2 Increased by 20.50 times.

[0028] SlUSP1 overexpression increased the activities of superoxide dismutase (SOD) and peroxidase (POD) in tomato plants; SOD activity increased from 380.72 U / g to 474.83 U / g, and POD activity increased from 82.34 U / g to 245.86 U / g.

[0029] SlUSP1 overexpression also optimized tomato agronomic traits: at day 35, plant height was as low as 76.01 mm, significantly lower than the wild type's 110.07 mm; stem diameter was as high as 4.41 mm, significantly higher than the wild type's 3.60 mm; the average number of branches was 6, an increase from the wild type's 4, and it could induce growth and development-related genes. SlTCP26 , SlSD1 , SlDwarf14 The expression is adjusted upwards.

[0030] A method for applying the stress protein USP1 in bacterial diseases of tomato includes the following steps: Step (1) Construct a SlUSP1 gene knockout or overexpression vector; Step (2) The vector was introduced into tomato explants through Agrobacterium-mediated genetic transformation. After pre-culture, co-culture, screening, rooting, and hardening, SlUSP1 knockout plants were obtained. slusp1-cr ) or overexpressing plants (SlUSP1-OE); Step (3) Perform molecular identification and disease resistance verification on transgenic plants, and screen for resistant plants. Pst DC3000 tomato plants.

[0031] The construction process of the SlUSP1 gene knockout vector in step (1) is as follows: using the second exon of the SlUSP1 gene as the target region, a specific target site is designed. The sequence of target site 1 is 5'–TGGATTAAATCCTGACAAAG-3'. The target 2 sequence is 5'–GCCTGTTGCCAAACTCAAAA-3'; a CRISPR / Cas9 knockout vector was constructed and transformed into Escherichia coli DH5α and Agrobacterium AGL1. Positive engineered Agrobacterium was obtained by PCR identification (target fragment size is 500-750bp) and sequencing verification.

[0032] The construction process of the SlUSP1 gene overexpression vector in step (1) is as follows: using the cDNA of wild-type tomato AilsaCraig (AC) as a template, the complete coding region sequence of SlUSP1 is amplified, and the amplification product is cloned into the pBI121-35S-Flag vector to construct pBI121:: SlUSP1 -The Flag recombinant overexpression vector was transformed into Agrobacterium AGL1 and positive clones were screened.

[0033] The Agrobacterium-mediated genetic transformation in step (2) specifically includes: (a) Explant preparation: Wild-type tomato AC seeds were disinfected with 75% ethanol for 1 min and 15% sodium hypochlorite for 10 min, and then inoculated into 1 / 2 MS medium. Germination was carried out at 25℃ under 16h light / 8h dark conditions for 3-4 days. The cotyledons and tender stems were cut into 0.2cm segments. (b) Pre-culture and co-culture: Explants were cultured for 2 days in a pre-culture medium (MS + 3% sucrose + 0.8% agar, pH 5.8) containing 1 mg / L 6-BA and 0.04 mg / L IAA; and then infected with Agrobacterium tumefaciens containing the target vector (OD). 600 =0.6) Infect explants for 15-20 min, then transfer to co-culture medium and culture for 2 days; (c) Screening and rooting: The explants were successively transferred to screening medium containing 100 μg / mL, 150 μg / mL and 200 μg / mL kanamycin, and the medium was changed every 20-25 days. After the seedlings were formed, they were transferred to rooting medium containing high concentration of auxin and cultured for 20 days. (d) Hardening off seedlings: Wash the rooted seedlings to remove any remaining culture medium residue, transplant them into nutrient soil, cover them with bags to keep them moist for 2 weeks, and then cultivate them in a greenhouse.

[0034] Step (3) includes molecular identification and disease resistance verification, which includes: (a) Molecular identification: DNA was extracted from plant leaves and identified using Cas9 gene-specific primers (upstream: 5'-AGACCGTGAAGGTTGTGGAC-3'). Downstream: 5'-TAGTGATCTGCCGTGTCTCG-3') was used for PCR identification; SlUSP1 expression level was detected by real-time quantitative PCR (qRT-PCR), with SlUBI3 as the internal reference gene, and the upstream primer of qRT-PCR was 5'-GCCTGTTGCCAAACTCAAAA-3', and the downstream primer was 5'-AAGCCTCTGCCTCCAATTAC-3'; (b) Disease resistance verification: Five-week-old positive plants were selected and tested using... Pst DC3000 bacterial culture (5×10) 5 CFU / mL (containing 0.02% Silwet L-77) was vacuum-infected. The lesion phenotype was observed after 7 days, and the bacterial colony concentration was measured after 4 days. At the same time, the expression level of defense genes and the activities of SOD and POD were detected.

[0035] The above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described herein. Although the present invention has been described in detail with reference to the above embodiments, the present invention is not limited to the specific embodiments described above. Therefore, any modifications or equivalent substitutions to the present invention, as well as all technical solutions and improvements that do not depart from the spirit and scope of the invention, are covered within the scope of the claims of the present invention.

Claims

1. The application of a stress protein USP1 in bacterial diseases of tomato, characterized in that, Tomato stress protein SlUSP1 enhances the resistance of tomatoes to pathogenic tomato strains caused by Pseudomonas syringae (Symplocos syringae). Pseudomonas syringae pv. tomato DC3000, abbreviated as Pst The application of DC3000 in bacterial disease resistance, wherein the gene ID encoding the tomato stress protein SlUSP1 is Solyc04g014600.

2. The application of the stress protein USP1 according to claim 1 in bacterial diseases of tomato, characterized in that, Application of enhancing resistance by increasing the expression level of SlUSP1 in tomato plants.

3. The application of the stress protein USP1 according to claim 1 in bacterial diseases of tomato, characterized in that, SlUSP1 can induce the expression of defense genes in tomato plants, including... SlPTI5 , SlWRKY28 , SlPR1a and SlGRAS2 .

4. The application of the stress protein USP1 according to claim 1 in bacterial diseases of tomato, characterized in that, SlUSP1 overexpression can increase the activity of superoxide dismutase (SOD) and peroxidase (POD) in tomato plants.

5. The application of the stress protein USP1 according to claim 1 in bacterial diseases of tomato, characterized in that, SlUSP1 overexpression can reduce tomato plant height, increase tomato stem diameter, increase the number of tomato branches, and increase the number of tomato nodes.

6. The application of a stress protein USP1 in bacterial diseases of tomato, characterized in that, Includes the following steps: Step (1) Construct a SlUSP1 gene knockout or overexpression vector, wherein the encoding gene ID of SlUSP1 is Solyc04g014600; Step (2) The vector was introduced into tomato explants through Agrobacterium-mediated genetic transformation. After pre-culture, co-culture, screening, rooting, and hardening, SlUSP1 knockout plants were obtained. slusp1-cr ) or overexpressing plants (SlUSP1-OE); Step (3) Perform molecular identification and disease resistance verification on transgenic plants, and screen for resistant plants. Pst DC3000 tomato plants.

7. The application of the stress protein USP1 according to claim 6 in bacterial diseases of tomato, characterized in that, The construction process of the SlUSP1 gene knockout vector in step (1) is as follows: with the second exon of the SlUSP1 gene as the target region, a specific target is designed, and the sequence of target 1 is 5'–TGGATTAAATCCTGACAAAG-3'. The target 2 sequence is 5'–GCCTGTTGCCAAACTCAAAA-3'; a CRISPR / Cas9 knockout vector was constructed and transformed into Escherichia coli DH5α and Agrobacterium AGL1. The target fragment size was identified by PCR and was 500-750 bp. Combined with sequencing verification, positive engineered Agrobacterium was obtained.

8. The application of the stress protein USP1 according to claim 6 in bacterial diseases of tomato, characterized in that, The construction process of the SlUSP1 gene overexpression vector in step (1) is as follows: using the cDNA of wild-type tomato Ailsa Craig (AC) as a template, the complete coding region sequence of SlUSP1 is amplified, and the amplification product is cloned into the pBI121-35S-HA vector to construct pBI121:: SlUSP1 -HA recombinant overexpression vector was transformed into Agrobacterium AGL1 and positive clones were screened.

9. The application of the stress protein USP1 according to claim 6 in bacterial diseases of tomato, characterized in that, The Agrobacterium-mediated genetic transformation in step (2) specifically includes: (a) Explant preparation: Wild-type tomato AC seeds were disinfected with 75% ethanol for 1 min and 15% sodium hypochlorite for 10 min, and then inoculated into 1 / 2 MS medium. Germination was carried out at 25℃ under 16 h light / 8 h dark conditions for 3-4 days. The cotyledons and tender stems were cut into 0.2 cm segments. (b) Pre-culture and co-culture: Explants were placed in a pre-culture medium containing 1 mg / L 6-BA and 0.04 mg / L IIAA, the pre-culture medium containing MS + 3% sucrose + 0.8% agar, the pH of the pre-culture medium being 5.8, and the explants were cultured in the pre-culture medium for 2 days; then infected with Agrobacterium containing the target vector, OD 600 =0.6, infect explants for 15-20 min, then transfer to co-culture medium and culture for 2 days; (c) Screening and rooting: The explants were successively transferred to screening medium containing 100 μg / mL, 150 μg / mL and 200 μg / mL kanamycin, and the medium was changed every 20-25 days. After the seedlings were formed, they were transferred to rooting medium containing high concentration of auxin and cultured for 20 days. (d) Hardening off seedlings: Wash the rooted seedlings to remove any remaining culture medium residue, transplant them into nutrient soil, cover them with bags to keep them moist for 2 weeks, and then cultivate them in a greenhouse.

10. The application of the stress protein USP1 according to claim 6 in bacterial diseases of tomato, characterized in that, Step (3) includes molecular identification and disease resistance verification, which includes: (a) Molecular identification: DNA was extracted from plant leaves and analyzed using Cas9 gene-specific primers. Upstream: 5'-AGACCGTGAAGGTTGTGGAC-3'; Downstream: PCR identification was performed using 5'-TAGTGATCTGCCGTGTCTCG-3'; SlUSP1 expression was detected by real-time quantitative PCR (qRT-PCR), with SlUBI3 as the internal reference gene. The upstream primer of the qRT-PCR was... 5'-GCCTGTTGCCAAACTCAAAA-3', The downstream is 5'-AAGCCTCTGCCTCCAATTAC-3'; (b) Disease resistance verification: Five-week-old positive plants were selected and tested with 5×10 5 CFU / mL, containing 0.02% Silwet L-77 Pst DC3000 bacterial suspension was vacuum-infected, and the lesion phenotype was observed after 7 days. The concentration of pathogen colonies was measured after 4 days. At the same time, the expression level of defense genes and the activities of SOD and POD were detected.

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