Application of PeBL1 gene and its protein in tomato disease control

Abstract

This invention provides the application of the PeBL1 gene and its protein in the control of tomato diseases. Specifically, this invention provides the application of the PeBL1 protein in improving the resistance of tomatoes to pathogens and / or in the preparation of products that enhance the resistance of tomatoes to pathogens, wherein the pathogens include Fusarium oxysporum tomato neck and root rot specific type (FORL) and tomato brown fruit wrinkle virus (ToBRFV), and the amino acid sequence of the PeBL1 protein is shown in SEQ ID NO:1. The PeBL1 protein can enhance the resistance of tomatoes to Fusarium oxysporum tomato neck and root rot specific type, significantly reduce the accumulation of Tomato brown fruit wrinkle virus on tomatoes, and reduce the impact of the virus on the growth of tomato plants.

Description

Technical Field

[0001] This invention relates to the field of plant disease control technology, specifically to the application of the PeBL1 gene and its protein in the control of tomato diseases. Background Technology

[0002] Any discussion of prior art throughout the specification should not be construed as an admission that such prior art is well-known or constitutes part of common general knowledge in the art.

[0003] Tomatoes are a crop of significant economic importance worldwide and one of the most widely cultivated vegetables globally. With the adjustment of the agricultural industrial structure, China, as the world's largest tomato producer, has seen its tomato planting area continuously expand, with an annual output of approximately 675 million tons. Large-scale greenhouse cultivation meets the ever-increasing market demand. However, continuous planting has led to an increase in soil-borne diseases, seriously threatening the safe production of tomatoes. Tomatoes face threats from various fungal and viral diseases during their growth, including tomato gray mold, tomato neck rot and root rot, tomato brown wrinkled fruit virus, and tomato yellow leaf curl virus.

[0004] Tomato crown and root rot has become one of the most serious soil-borne diseases in greenhouse and field tomato cultivation in recent years. Tomato crown and root rot (FCRR) is a highly destructive soil-borne disease caused by *Fusarium oxysporum* f.sp. radicis-lycopersici (FORL), first discovered and identified in Japan in 1974. FORL was discovered in South America in 1976 and first found in China in 2007. To date, FORL has caused severe losses in China, Israel, Japan, the United States, Mexico, South Korea, Canada, and parts of Europe, and is one of the major soil-borne diseases of tomatoes after root-knot nematode disease and tomato yellow leafroll virus. In my country, tomato crown and root rot has occurred in many regions, such as Shandong, Liaoning, and Ningxia, posing a serious threat to greenhouse tomato production. The incidence of this disease is particularly high in Northeast and North China, with the situation being especially severe in Shouguang, Shandong Province. Reports indicate that in Shouguang's greenhouses, the incidence of tomato neck and root rot exceeds 80%, with a mortality rate exceeding 30%, leading to a significant drop in tomato yield. When tomato plants are infected with this disease, distinct dark brown lesions form at the contact point between the soil and the stem base. The stem base constricts, and in severe cases, seedlings break off at the lesions and quickly wilt and die, severely impacting tomato yield. Therefore, effective control measures must be implemented to curb the spread and impact of tomato neck and root rot.

[0005] Tomato brown rugose fruit virus (ToBRFV) was first characterized in 2016. ToBRFV possesses a positive single-stranded RNA genome encapsulated in rod-shaped particles approximately 300 nm in length and 18 nm in diameter. The virus is highly contagious and can be transmitted through contact, propagation material, seeds, and bumblebees. Both infected tomato plants and fruits exhibit obvious symptoms, including: brown or yellow rimming or spots on leaves, leaf deformation (such as curling or twisting), stunted plant growth, and overall poor growth; and brown sunken spots or streaks on fruits, irregular fruit shape, possible wrinkling, and abnormal fruit color development, such as discoloration or uneven ripening. ToBRFV infection leads to severe reductions in yield, marketability, and fruit quality, posing a persistent and destructive threat to tomato production. Three resistance genes have been identified: Tm-1, Tm-2, and Tm-2. 2 These methods can protect tomato plants from various tomato viruses, such as Tomato Mosaic Virus (ToMV) and Tobacco Mosaic Virus (TMV), but they do not confer resistance to ToBRFV. Currently, the main strategy for managing ToBRFV is to implement good hygiene practices, including the use of disinfection tools and equipment, and the timely removal of infected plants to reduce the risk of virus transmission.

[0006] PeBL1 originates from *Brevibacillus laterosporus*, a soil bacterium known to produce a variety of secondary metabolites with biocontrol potential. PeBL1 has been shown to enhance the resistance of tomatoes and cucumbers to aphids (*Myzus persicae*) by increasing the accumulation of jasmonic acid (JA), salicylic acid (SA), and ethylene (ET). SA can promote the production of certain insect-harmful secondary metabolites, such as phenylpropanoids, which can reduce plant palatability or directly poison insects. JA is one of the main hormones regulating plant responses to insect attacks; its accumulation can stimulate the production of insect-resistant compounds, such as protease inhibitors. These inhibitors can interfere with aphids' digestive system, reducing their food intake and survival rate. JA can also promote the release of volatile organic compounds (VOCs), which can attract aphid predators such as ladybugs and parasitic wasps, thus mitigating aphid damage through biocontrol. ET participates in the tissue repair process after plant injury. After aphids feed on the plant, ET can help the plant repair damage and restore normal physiological functions more quickly. Summary of the Invention

[0007] This invention provides the application of PeBL1 protein in enhancing the resistance of tomatoes to specific pathogens (Fusarium oxysporum f.sp. radical-lycopersici (FORL) and Tomato Brown Fruit Virus (ToBRFV)) and / or in the preparation of products that enhance tomato resistance to pathogens. PeBL1 protein can enhance the resistance of tomatoes to the FORL fungus and ToBRFV virus, significantly reducing their accumulation on tomato plants and their impact on tomato plant growth, thus helping to reduce the incidence and severity of diseases. Furthermore, the application method of PeBL1 protein is simple; an effective amount can be applied directly to the tomato plant for the prevention and control of FORL and ToBRFV, thereby protecting tomato plants and improving tomato quality and yield.

[0008] Specifically, the present invention provides the following technical solutions.

[0009] In a first aspect of the invention, the use of PeBL1 protein in improving the resistance of tomatoes to pathogens and / or in preparing products that improve the resistance of tomatoes to pathogens is provided, wherein the pathogens include Fusarium oxysporum tomato neck rot and root rot specific type (FORL) and tomato brown wrinkled fruit virus (ToBRFV), hereinafter referred to as FORL and ToBRFV, respectively, and the amino acid sequence of the PeBL1 protein is shown in SEQ ID NO:1.

[0010] In embodiments of the present invention, the improvement of tomato resistance to pathogens includes at least one of the following:

[0011] 1) Enhance the defense and / or control of Fusarium oxysporum tomato neck rot and root rot specific variants in tomatoes;

[0012] 2) Enhance tomato defense and / or control of tomato brown wrinkled fruit virus infection;

[0013] 3) Reduce or eliminate symptoms caused by infection with Fusarium oxysporum tomato neck rot and root rot specific type;

[0014] 4) Reduce or eliminate symptoms caused by infection with tomato brown wrinkled fruit virus;

[0015] 5) Prevent the occurrence of tomato neck rot and root rot; and

[0016] 6) Reduce or eliminate the symptoms of tomato neck rot and root rot.

[0017] By enhancing the resistance described above, it is possible to resist and control the infection of tomatoes by FORL and / or ToBRFV, effectively reduce or eliminate the symptoms caused by FORL and / or ToBRFV infection, prevent the occurrence of tomato neck rot and root rot and / or reduce or eliminate the symptoms of tomato neck rot and root rot, thereby helping to reduce the probability and severity of disease occurrence, thus protecting tomato plants and improving the quality and yield of crops.

[0018] The resistance to FORL includes resisting and controlling FORL infection in tomatoes, enhancing resistance levels, and preventing or mitigating the impact of FORL infection on tomato growth. Preventing or mitigating the impact of FORL on tomato growth includes, but is not limited to, preventing or mitigating stem lesions, such as stem thinning, lignification, and lodging; relieving or alleviating growth retardation and overall poor growth, such as stunted growth and wilting; and preventing or mitigating root system changes, such as sparse fibrous roots and underdeveloped root systems. For example, in one embodiment of the invention, applying PeBL1 protein to FORL-infected tomato plants can enhance the tomato's resistance to FORL, reduce the impact of FORL infection on tomato growth, relieve or alleviate growth retardation, significantly improve the control effect after FORL infection, and can be used for the treatment of FORL infection in tomato plants, effectively limiting the further spread of the disease.

[0019] The ToBRFV resistance includes resisting ToBRFV infection in tomatoes and preventing or mitigating the effects of ToBRFV on tomato growth. Preventing or mitigating the effects of ToBRFV on tomato growth includes, but is not limited to, preventing or mitigating leaf lesions such as curling, yellowing, and wrinkling; relieving or alleviating stunted growth and poor overall growth, such as dwarfism; and preventing or mitigating brown sunken spots or streaks on fruits, irregular fruit shape, wrinkling, and abnormal color development, such as discoloration or uneven ripening. For example, in some embodiments of the present invention, tomato plants sprayed with PeBL1 protein, when exposed to ToBRFV infection, can significantly improve the resistance level of tomatoes to ToBRFV, reduce the accumulation of ToBRFV on tomatoes, reduce the impact of the virus on tomato plant growth, and effectively improve the prevention and control of ToBRFV infection.

[0020] In a second aspect of the invention, the use of a nucleic acid molecule encoding the PeBL1 protein in enhancing the resistance of tomatoes to pathogens and / or in preparing products that enhance the resistance of tomatoes to pathogens is provided, wherein the pathogens include FORL and ToBRFV, and the amino acid sequence of the PeBL1 protein is shown in SEQ ID NO:1.

[0021] In an embodiment of the present invention, the improvement of the tomato's resistance to pathogens is as defined in the first aspect above.

[0022] In a third aspect of the invention, the use of a vector, expression cassette, or transgenic cell line comprising a nucleic acid molecule encoding a PeBL1 protein in enhancing the resistance of tomatoes to pathogens and / or in preparing products that enhance the resistance of tomatoes to pathogens, wherein the pathogens include FORL and ToBRFV, and the amino acid sequence of the PeBL1 protein is shown in SEQ ID NO:1.

[0023] In one embodiment of the present invention, the vector containing the nucleic acid molecule encoding the PeBL1 protein can be constructed by inserting the target gene into an expression vector. In one embodiment, the expression vector is a plasmid, preferably pET30a(+).

[0024] In an embodiment of the present invention, the improvement of the tomato's resistance to pathogens is as defined in the first aspect above.

[0025] In a fourth aspect of the invention, the use of recombinant bacteria comprising a nucleic acid molecule encoding PeBL1 protein in enhancing the resistance of tomatoes to pathogens and / or in preparing products that enhance the resistance of tomatoes to pathogens is provided, wherein the pathogens include FORL and ToBRFV, and the amino acid sequence of the PeBL1 protein is shown in SEQ ID NO:1.

[0026] The recombinant bacteria containing the nucleic acid molecule encoding the PeBL1 protein can be obtained by transferring a recombinant expression vector containing the target gene into a host cell. In some embodiments, the host cell is a prokaryotic cell, preferably *Escherichia coli*. For example, in some embodiments, the recombinant expression vector pET30a(+)-PeBL1 is synthesized using the pET30a(+) plasmid as the expression vector, and then an engineered *E. coli* strain containing the PeBL1 gene, BL21-pET30a(+)-PeBL1, is constructed using *E. coli* as the host strain. This recombinant strain is capable of expressing and producing the PeBL1 protein.

[0027] In an embodiment of the present invention, the improvement of the tomato's resistance to pathogens is as defined in the first aspect above.

[0028] In a fifth aspect of the invention, a product for improving the resistance of tomatoes to pathogens is provided, comprising any one of the substances described in 1) to 3) below:

[0029] 1) Protein molecules, peptides, or variants thereof with amino acid sequences as shown in SEQ ID NO:1;

[0030] 2) Nucleic acid molecules, nucleotide fragments, or variants thereof encoding the amino acid sequence shown in SEQ ID NO:1; and

[0031] 3) Vectors, expression cassettes, transgenic cell lines or recombinant bacteria containing a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:1.

[0032] In embodiments of the present invention, the improvement of tomato resistance to pathogens includes at least one of the following:

[0033] 1) Enhance the defense and / or control of Fusarium oxysporum tomato neck rot and root rot specific variants in tomatoes;

[0034] 2) Enhance tomato defense and / or control of tomato brown wrinkled fruit virus infection;

[0035] 3) Reduce or eliminate symptoms caused by infection with Fusarium oxysporum tomato neck rot and root rot specific type;

[0036] 4) Reduce or eliminate symptoms caused by infection with tomato brown wrinkled fruit virus;

[0037] 5) Prevent the occurrence of tomato neck rot and root rot; and

[0038] 6) Reduce or eliminate the symptoms of tomato neck rot and root rot.

[0039] By enhancing the resistance described above, it is possible to resist and control the infection of tomatoes by FORL and / or ToBRFV, effectively reduce or eliminate the symptoms caused by FORL and / or ToBRFV infection, prevent the occurrence of tomato neck rot and root rot and / or reduce or eliminate the symptoms of tomato neck rot and root rot, thereby helping to reduce the probability and severity of disease occurrence, thus protecting tomato plants and improving the quality and yield of crops.

[0040] The resistance to FORL includes resisting and controlling FORL infection in tomatoes, enhancing resistance levels, and preventing or mitigating the impact of FORL infection on tomato growth. Preventing or mitigating the impact of FORL on tomato growth includes, but is not limited to, preventing or mitigating stem lesions, such as stem thinning, lignification, and lodging; relieving or alleviating growth retardation and overall poor growth, such as stunted growth and wilting; and preventing or mitigating root system changes, such as sparse fibrous roots and underdeveloped root systems. For example, in one embodiment of the invention, applying PeBL1 protein to FORL-infected tomato plants can enhance the tomato's resistance to FORL, reduce the impact of FORL infection on tomato growth, relieve or alleviate growth retardation, significantly improve the control effect after FORL infection, and can be used for the treatment of FORL infection in tomato plants, effectively limiting the further spread of the disease.

[0041] The ToBRFV resistance includes resisting ToBRFV infection in tomatoes and preventing or mitigating the effects of ToBRFV on tomato growth. Preventing or mitigating the effects of ToBRFV on tomato growth includes, but is not limited to, preventing or mitigating leaf lesions such as curling, yellowing, and wrinkling; relieving or alleviating stunted growth and poor overall growth, such as dwarfism; and preventing or mitigating brown sunken spots or streaks on fruits, irregular fruit shape, wrinkling, and abnormal color development, such as discoloration or uneven ripening. For example, in some embodiments of the present invention, tomato plants sprayed with PeBL1 protein, when exposed to ToBRFV infection, can significantly improve the resistance level of tomatoes to ToBRFV, reduce the accumulation of ToBRFV on tomatoes, reduce the impact of the virus on tomato plant growth, and effectively improve the prevention and control of ToBRFV infection.

[0042] In some embodiments of the present invention, the product includes reagents, pharmaceuticals, and pesticides. The product can be used to prevent and control infection from the fungus FORL and / or the virus ToBRFV.

[0043] In one embodiment of the present invention, the product may be a protein with an amino acid sequence as shown in SEQ ID NO:1, namely PeBL1 protein, and the product may be in solid or liquid form, such as powder or solution. Considering the convenience of storage and transportation, the product is preferably in powder form, which can be dissolved in a solvent (such as water) or form a suspension when used, and exerts its effect by spraying it onto tomato plants (such as leaves, fruits, stems, and roots).

[0044] In one embodiment of the present invention, the product may be a recombinant bacterium containing a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:1, which can express and produce a protein with the amino acid sequence shown in SEQ ID NO:1, namely the PeBL1 protein. The recombinant bacterium strain is, for example, a strain that can survive in soil or can live symbiotically on the roots, stems, and leaves of plants, and is harmless to the environment and humans, preferably an endophytic bacterium of tomatoes. The product may be solid or liquid, such as freeze-dried bacterial powder or liquid inoculum, and can be mixed or sprayed into tomato potting soil or culture medium during use.

[0045] In a sixth aspect of the invention, a method for improving the resistance of tomatoes to pathogens is provided, comprising: applying to tomatoes any of the products described in the fifth aspect above. The pathogens particularly refer to the fungus FORL and / or the virus ToBRFV.

[0046] In one embodiment of the present invention, the applied product is a solution containing PeBL1 protein.

[0047] In one embodiment of the invention, the application is to spray a solution containing PeBL1 protein onto tomato plants, such as leaves, fruits, stems and roots, preferably leaves.

[0048] In one embodiment of the present invention, the application concentration is not less than 2 μM, preferably not less than 4 μM.

[0049] For example, in one implementation, when used to improve the resistance of tomatoes to FORL or to prevent and control tomato neck rot and root rot, the application concentration is not less than 2 μM, for example, it can be 2-4 μM.

[0050] For example, in one embodiment, when used to improve the resistance of tomatoes to ToBRFV or to prevent ToBRFV infection, the application concentration is not less than 2 μM, preferably not less than 4 μM, more preferably not less than 8 μM, such as 2-8 μM.

[0051] Compared to existing technologies, the advantages of this invention include:

[0052] This invention provides the application of the PeBL1 gene and its protein in the control of tomato diseases. Specifically, this invention provides the application of the PeBL1 protein in improving tomato resistance to pathogens and / or in the preparation of products that enhance tomato resistance to pathogens. The improvement in tomato resistance to pathogens includes enhancing tomato resistance to FORL, tomato resistance to tomato neck rot and root rot, and tomato resistance to ToBRFV. The PeBL1 protein can enhance tomato resistance to FORL and ToBRFV, significantly reducing the accumulation of FORL and ToBRFV on tomatoes and reducing the impact of FORL and ToBRFV on tomato plant growth. Furthermore, the application method of the PeBL1 protein is simple; an effective amount can be applied to the leaves, fruits, and / or stems of the tomato plant, enabling its use for the prevention and control of FORL and ToBRFV in tomato plants. Attached Figure Description

[0053] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. Hereinafter, embodiments of the invention will be described in detail with reference to the accompanying drawings, wherein:

[0054] Figure 1 : Structural diagram of the recombinant expression vector pET30a(+)-PeBL1.

[0055] Figure 2 Electrophoresis results of PCR products of positive clones of Escherichia coli BL21 after transformation with pET30a(+)-PeBL1 recombinant expression vector.

[0056] Figure 3 Protein standard curve.

[0057] Figure 4 PeBL1 protein nickel column purification results, where M: protein molecular weight standard, FT: loading flow-through buffer, W: washing buffer, E: elution buffer.

[0058] Figure 5 Microscopic examination results of Fusarium oxysporum, a specialized strain of tomato neck and root rot.

[0059] Figure 6 Standards for classifying the severity of tomato neck and root rot.

[0060] Figure 7 Tomato plant height in each treatment group one month after FORL infection.

[0061] Figure 8 Tomato growth status in each treatment group one month after FORL infection.

[0062] Figure 9 The incidence of neck rot and root rot in tomatoes in different treatment groups one month after FORL infection.

[0063] Figure 10 Changes in the roots and stems of tomatoes in different treatment groups one month after FORL infection.

[0064] Figure 11 Tomato plant height in each treatment group on day 35 after ToBRFV inoculation.

[0065] Figure 12 The viral accumulation levels in each treatment group on day 35 after ToBRFV vaccination were as follows: 1: PBS treatment group; 2: 8 μM hypersensitive protein treatment group; 3: 2 μM PeBL1 protein treatment group; 4: 8 μM PeBL1 protein treatment group. Detailed Implementation

[0066] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer.

[0067] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art. The reagents and raw materials used in this invention are readily available through conventional means, and unless otherwise specified, they shall be used in accordance with conventional methods or product instructions. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0068] Example 1 Construction and protein expression of the recombinant E. coli expression vector for the PeBL1 gene

[0069] 1 Experimental Methods

[0070] 1.1 Construction of PeBL1 gene recombinant expression vector

[0071] Using the PeBL1 gene as the target gene, the nucleotide sequence of which is shown in SEQ ID NO:2, the recombinant expression vector pET30a(+)-PeBL1 was synthesized using the pET30a(+) plasmid as the expression vector. The gene sequence was synthesized by Shanghai Sangon Biotech Co., Ltd., and the structure of pET30a(+)-PeBL1 is shown in Figure 2. Figure 1 As shown.

[0072] 1.2 Obtaining engineered Escherichia coli containing the PeBL1 gene

[0073] The pET30a(+)-PeBL1 recombinant expression vector was transformed into Escherichia coli BL21. Single clones were picked and cultured in LB medium containing a final concentration of 50 μg / mL kanamycin overnight at 37°C using a shaker. Colony PCR was then performed, and the electrophoresis results are shown below. Figure 2 As shown, bright bands were observed at their respective sizes. The corresponding bacterial cultures were submitted to Shanghai Sangon Biotech Co., Ltd. for sequencing and sequence alignment. The homology with the target gene was 100%, proving the successful construction of the engineered Escherichia coli containing the PeBL1 gene—BL21-pET30a(+)-PeBL1.

[0074] 1.3 Expression of PeBL1 protein

[0075] (1) Activation of strain: Take 10 μL of Escherichia coli glycerol bacteria containing the PeBL1 gene constructed in Example 1.2 and inoculate it into 10 mL of LB medium containing a final concentration of 50 μg / mL kanamycin. Incubate overnight at 37°C in a shaker.

[0076] (2) Expanded culture: Take the above-mentioned activated strain and inoculate it into 100 mL of LB medium containing a final concentration of 50 μg / mL kanamycin at an inoculation rate of 1%. Incubate at 37°C and 180 rpm with shaking until OD. 600nm It is around 0.6;

[0077] (3) Induction of expression: IPTG was added to the bacterial culture to a final concentration of 0.1 mM, and cultured at 37°C and 180 rpm for 6 h with shaking. The bacterial cells were collected by centrifugation (5000 rpm, 10 min, 4°C) and stored at -20°C.

[0078] 1.4 Purification of PeBL1 protein

[0079] Add 2-5 mL of non-denaturing lysis buffer to each gram of bacterial precipitate wet weight, thoroughly resuspend the bacterial cells, and sonicate on ice to lyse the cells. Use an ultrasonic power of 200-300 W, sonicating for 2 seconds each time, with a 4-second interval, for a total of 99 times. The sonicated liquid should be clear. Centrifuge at 12000 rpm for 10 min, collect the supernatant, and purify the PeBL1 protein according to the operating procedures of the His-tagged protein purification kit (purchased from Shanghai Beyotime Biotechnology Co., Ltd.).

[0080] 1.5 Dialysis for desalination

[0081] The purified target protein was dialyzed using a regenerated cellulose dialysis bag (purchased from Shanghai Beyotime Biotechnology Co., Ltd.) with 1×PBS solution as the dialysate. The protein sample was placed in the dialysis bag, clamped at both ends, and the container containing the dialysis bag was placed at 4°C. The dialysate was changed every 3-4 hours, and the product was harvested after dialysis for 24 hours.

[0082] 1.6 Protein Concentration Determination

[0083] Protein concentration was detected using the Bradford Protein Assay Kit (Shanghai Beyotime Biotechnology Co., Ltd.). A protein concentration standard curve was constructed by setting concentration gradients of 0 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 0.75 mg / mL, 1.125 mg / mL, and 1.5 mg / mL. Figure 3 The content of the target protein was determined using Coomassie Brilliant Blue staining solution.

[0084] 1.7 SDS-PAGE gel electrophoresis verification

[0085] (1) Preparation of separating gel and stacking gel

[0086] The separating gel and stacking gel were prepared according to the instructions of the SDS-PAGE rapid gel preparation kit (purchased from Shanghai Beyotime Biotechnology Co., Ltd.).

[0087] (2) Sample preparation and electrophoresis

[0088] Add 5×SDS gel loading buffer to the sample, heat at 100℃ for 10 min, and load 10 μL of sample. Electrophore at 70V for 30 min until bromophenol blue enters the separating gel, then increase the voltage to 100V and electrophore until bromophenol blue reaches the bottom, ending the electrophoresis.

[0089] (3) Staining

[0090] The gel was stained with the staining solution from the Coomassie Brilliant Blue staining kit (Beijing Solarbio Science & Technology Co., Ltd.) for about 150 minutes, and then destained overnight with the destaining solution. After that, it could be observed and photographed.

[0091] 2 Experimental Results

[0092] After induction and expression of pET30a(+)-PeBL1 recombinant E. coli, the cells were lysed, purified by nickel column chromatography, and subjected to SDS-PAGE followed by Coomassie brilliant blue staining. Figure 4 As can be seen, a clear target band was observed near 18.4 kDa, with few impurity bands, indicating successful purification of PeBL1 protein. The concentration of the target protein PeBL1 was determined to be 1.29 mg / mL using the Bradford method. Fermentation of 1 L of BL21-pET30a(+)-PeBL1 recombinant E. coli culture yielded 93.0 mg of purified PeBL1 protein.

[0093] Example 2 PeBL1 protein resistance to tomato neck rot and root rot test

[0094] 1. Experimental Materials and Methods

[0095] 1.1 Test Materials

[0096] The *Fusarium oxysporum* strain specifically for tomato neck and root rot was preserved in the laboratory of Shandong Bilan Biotechnology Co., Ltd. The hypersensitive protein (also known as Harpin protein, trade name MessengerR, Kangzhuangsu) was purchased from Hunan Agricultural University Science and Technology Development Co., Ltd.

[0097] 1.2 Culture of FORL, the pathogen causing tomato neck and root rot

[0098] (1) Wipe the ultra-clean workbench clean and sterilize it for 30 minutes.

[0099] (2) Sterilize the scalpel by burning it over an alcohol lamp flame. After the scalpel cools down, cut a 1cm×1cm piece of bacteria from a PDA plate covered with Fusarium oxysporum FORL.

[0100] (3) Inoculate the bacterial block onto a new PDA (Qingdao Haibo Biotechnology Co., Ltd.) plate and incubate at 28℃ for 7 days; or inoculate the bacterial block into 200mL YPDA medium (Qingdao Haibo Biotechnology Co., Ltd.) and incubate at 25℃ and 160rpm / min for 7 days.

[0101] (4) Take 10 μL of FORL bacterial solution and drop it into the center of the slide. Use tweezers to pick up the coverslip and cover it from one side of the bacterial solution, being careful not to create air bubbles. Place it on the microscope stage and examine and photograph it.

[0102] 1.3 Tomato Breeding

[0103] After sterilizing the collected soil at 121℃ for 30 minutes, let it sit overnight. Mix the sterilized soil with the substrate at a 2:1 ratio (i.e., potting soil), keep it moist by sprinkling water, and evenly spread it in the seedling foam box. Use tweezers to pick up tomato seeds (variety: Zhongshu No. 4) and place them evenly in the seedling foam box, then cover with soil. Keep the soil moist by spraying water and place it in a 25℃ environment to wait for germination.

[0104] 1.4 Tomato root soaking and inoculation with Fusarium oxysporum

[0105] After tomato seedlings have been cultivated for about 3 weeks, gently remove the seedlings with 2-3 true leaves from the seedling foam box. Wash the soil off the roots with water, trim the tip of the taproot by about 0.5cm, and then place the seedlings in a 10cm thick container. 7 Soak in a CFU / mL FORL spore suspension for 30 min, then transplant into sterile culture soil.

[0106] 1.5 Experimental Grouping

[0107] (1) Two days after the above 1.4 infection with FORL, 60 tomato seedlings with uniform growth were selected and randomly divided into 4 treatment groups (i.e., PBS control group, 4μM hypersensitive protein treatment group, 2μM PeBL1 protein treatment group, and 4μM PeBL1 protein treatment group). Each treatment group was replicated with 15 seedlings. The 2μM and 4μM PeBL1 protein treatment groups were diluted with sterile 1×PBS to a concentration of 2μM and 4μM, respectively. The 4μM hypersensitive protein treatment group was diluted with sterile 1×PBS to a concentration of 4μM. The PBS control group was treated with sterile 1×PBS.

[0108] (2) The prepared protein solution was sprayed onto the tomato leaves of the corresponding groups using a spray bottle (the control group was sprayed with PBS), ensuring the solution was suspended but not dripping. Approximately 15 days later, the protein solution was sprayed again (the control group was sprayed with PBS). One month after inoculation with Fusarium oxysporum FORL, the disease incidence was assessed, the disease index was calculated, and the phenotype of each strain was observed and recorded.

[0109] 1.6 Evaluation Methods

[0110] Based on the disease incidence of tomatoes in different treatment groups one month after FORL infection, the disease was graded according to the grading criteria shown in Table 1. (Refer to DB15 / T 3323-2024 Inner Mongolia Autonomous Region Local Standard: Technical Specification for Indoor Identification of Tomato Resistance to Neck and Root Rot)

[0111] Table 1 Disease Grading Standards

[0112]

[0113] The host resistance levels are shown in Table 2.

[0114] Table 2 Host Resistance Levels

[0115]

[0116]

[0117] The disease index and relative control effect are calculated based on the disease grading results and plant height. The disease index is counted according to the formula as follows:

[0118]

[0119] In the formula:

[0120] DI: Disease Index.

[0121] DV: A numerical value representing the severity of the illness, with values ​​of 0, 1, 2, 3, and 4.

[0122] n: the number of plants at each disease level;

[0123] DVx: The representative value for the highest disease level, which is 4;

[0124] N: The total number of plants surveyed using the same material.

[0125] Relative prevention and control effect = [(disease index of control group - disease index of treatment group) / (disease index of control group)] × 100%.

[0126] 2. Experimental Results

[0127] 2.1 FORL microscopic examination results

[0128] After culturing in YPDA liquid medium at 25°C for 7 days, microscopic examination was performed. 10 μL of FORL bacterial suspension was dropped into the center of a glass slide, covered with a coverslip, and placed on the microscope stage for examination. The microscopic results are as follows: Figure 5 As shown, the results indicate that the hyphae are slender, septate, colorless, and transparent; the conidiophores are short, lateral to the hyphae, initially existing singly, and later arranged in clusters; it is a typical Fusarium oxysporum morphology, with no contamination.

[0129] 2.2 Effects on growth performance

[0130] One month after infection with FORL, the tomato plants in each group were graded for disease severity, and the plant height of different treatment groups was measured. Figure 6 The diagram illustrates the plant classification, and the plant height of each group is shown in Table 3. Figure 7 As shown in the figure. The results indicated that the tomato plant height in the 4 μM PeBL1 protein treatment group was significantly higher than that in the PBS control group, increasing by 10.78% compared to the PBS control group and by 3.72% compared to the 4 μM hypersensitive protein treatment group.

[0131] Table 3. Tomato plant height in each group one month after FORL infection.

[0132]

[0133]

[0134] Note: The same lowercase letter in the same column indicates no significant difference (p≥0.05), and different lowercase letters in the same column indicate significant difference (p<0.05);

[0135] 2.3 Disease resistance

[0136] One month after FORL infection of tomato seedlings, the incidence of tomato neck rot and root rot in different treatment groups was statistically analyzed, and the disease index and relative control effect were calculated. The results are shown in Table 4. Figure 8 , Figure 9 and Figure 10 As shown in the figure. The results indicated that PeBL1 protein could enhance the resistance of tomatoes to Fusarium oxysporum FORL. The disease index of the 2 μM and 4 μM PeBL1 protein treatment groups was lower than that of the PBS control group and the 4 μM hypersensitive protein treatment group. The relative control effect was increased by 29.16% and 45.01% respectively compared with the PBS control group, and by 19.04% and 37.15% respectively compared with the 4 μM hypersensitive protein treatment group. 2 μM PeBL1 protein treatment could improve the resistance of tomatoes to Fusarium oxysporum FORL, and 4 μM PeBL1 protein treatment was more effective.

[0137] Table 4. Disease incidence of tomato neck rot and root rot

[0138]

[0139] Example 3 PeBL1 protein resistance to Tomato Brown Curly Fruit Virus (ToBRFV) test

[0140] 1. Experimental Methods

[0141] The tomato brown wrinkled fruit virus was preserved in the laboratory of Shandong Bilan Biotechnology Co., Ltd. The hypersensitive protein (also known as Harpin protein, trade name MessengerR, Kangzhuangsu) was purchased from Hunan Agricultural University Science and Technology Development Co., Ltd.

[0142] 1.1 Tomatoes were sprayed with different concentrations of PeBL1 protein and inoculated with the virus.

[0143] Two weeks after transplanting, 40 tomato plants of similar growth were selected and randomly divided into 4 groups, treated according to Table 5. Protein was sprayed until the droplets were suspended but not dripping (the control group was sprayed with PBS). 48 hours after spraying, ToBRFV virus was diluted 1×PBS at a ratio of 1:30 and inoculated into each treatment group by friction, with 100 μL of virus sap injected into each tomato plant.

[0144] Table 5 Grouping and Processing Methods

[0145]

[0146]

[0147] 1.2 Measure the plant height of tomatoes in each group on day 35 after ToBRFV inoculation.

[0148] On the 35th day after tomatoes were inoculated with the virus, the distance from the base of the rootstock to the top of the main stem in each group of tomatoes was measured with a ruler, and the average value and error were calculated.

[0149] 1.3 The disease index of tomatoes in each group was measured on day 35 after ToBRFV inoculation.

[0150] Tomatoes inoculated with ToBRFV were photographed on day 35, with 3 plants in each group. The symptoms of tomatoes infected with ToBRFV were graded according to the 9-level grading method shown in Table 6 ("Methods of Plant Disease Research (3rd Edition)", by Fang Zhongda, December 1998). The severity of the disease on each plant in each group was counted, and the disease index and relative control effect were calculated. The host resistance grading standards are shown in Table 6.

[0151] Table 6 Scores of Different Grades of Diseases

[0152]

[0153] Disease Index (DI) = [Σ(number of diseased plants at each level × disease level value) / (total number of plants surveyed × highest disease level value)] × 100.

[0154] Relative prevention and control effect = [(disease index of control group - disease index of treatment group) / (disease index of control group)] × 100%.

[0155] 1.4 SDS-PAGE detection of virus accumulation in tomatoes

[0156] (1) Sample treatment: Leaves from the same leaf position of tomatoes in different treatment groups were collected, ground with liquid nitrogen, and the ground samples were weighed in 1.5 mL centrifuge tubes. Reagents were added at a ratio of sample:1×PBS = 1:2 (mass-volume ratio), and the mixture was shaken thoroughly. The mixture was centrifuged at 12000 rpm for 10 min at 4℃. 200 μL of the supernatant was taken into a 1.5 mL centrifuge tube, 50 μL of 5×SDS loading buffer was added, and the mixture was boiled in water for 10 min (800 W) and placed on ice.

[0157] (2) Prepare protein gel: Use the SDS-PAGE gel rapid preparation kit (purchased from Shanghai Beyotime Biotechnology Co., Ltd.) to prepare separating gel. After mixing each component in sequence, add deionized water to cover the liquid surface and let it solidify at room temperature for about 20 minutes. Drain the liquid on the gel and pour the stacking gel in the same way. Insert a comb to avoid generating air bubbles.

[0158] (3) Protein electrophoresis: Separate proteins by electrophoresis. Load 10 μL of each sample and 8 μL of the marker. Electrophore at 70 V for 30 min until the bromophenol blue enters the separating gel. Increase the voltage to 100 V and electrophore until the bromophenol blue reaches the bottom, then stop the electrophoresis.

[0159] (4) Staining

[0160] The gel was stained with Coomassie Brilliant Blue for about 150 minutes, then destained overnight with a destaining solution, and finally observed and photographed.

[0161] (5) Results Analysis

[0162] ImageJ software was used to perform grayscale analysis on the protein bands.

[0163] 2. Experimental Results

[0164] 2.1 Plant height of tomatoes in different treatment groups on day 35 after ToBRFV inoculation

[0165] The results are shown in Table 7 and Figure 11 As shown in the figure. The results showed that the average plant height of tomatoes in the 8 μM PeBL1 protein treatment group was significantly higher than that in the PBS group and the 8 μM hypersensitive protein treatment group by 16.34% and 12.54%, respectively (p < 0.05). This indicates that spraying tomatoes with 8 μM PeBL1 protein can reduce the impact of virus inoculation on plant growth.

[0166] Table 7. Tomato plant height on day 35 after ToBRFV inoculation in different treatment groups.

[0167]

[0168] Note: The same lowercase letter in the same column indicates no significant difference (p≥0.05), and different lowercase letters in the same column indicate significant difference (p<0.05);

[0169] 2.2 Disease index of tomatoes in different treatment groups after ToBRFV inoculation

[0170] On day 35 after ToBRFV inoculation, the leaves of plants in the PBS treatment group showed severe discoloration, curling, narrowing and deformity; the tomato plants treated with 8μM PeBL1 protein grew normally compared with the plants in other treatment groups, and the leaves did not show obvious discoloration and curling.

[0171] As shown in Table 8, the disease index of plants treated with 8 μM PeBL1 protein was the lowest at 50.56, while the disease index of plants treated with PBS was the highest at 80.25. The 8 μM PeBL1 protein treatment group showed the best control effect, with a control efficacy of 37%.

[0172] Table 8 Disease index and control effect of TobRFV rubbing inoculation on tomatoes on day 35

[0173]

[0174] 2.3 Detection of virus accumulation in tomatoes on day 35 after inoculation with Tomato Brown Fruit Curling Virus (ToBRFV)

[0175] The SDS-PAGE results are shown below. Figure 12 The results of the analysis using ImageJ software are shown in Table 9.

[0176] Table 9. Protein gray values ​​of different treatment groups

[0177]

[0178] Among them, the IntDen value of the target protein is the gray value of the target protein (viral coat protein), which reflects the relative content of the target protein.

[0179] Internal reference protein IntDen value: The gray value of the internal reference protein (β-Actin), used to correct for variability between samples.

[0180] Relative expression level: The relative expression level of the target protein, with the expression level of the PBS group as the baseline (100%), and the expression level of other treatment groups relative to the PBS group.

[0181] Internal control calibration: The expression level of the target protein is corrected using the IntDen value of the internal control protein to reduce the variation and error in the experiment.

[0182] Relative expression level calculation: The relative expression level is the target protein expression level after correction based on the internal reference protein. The usual formula is: Relative expression level = [(Target protein IntDen value / Internal reference protein IntDen value)] 处理组 ÷ (Target protein IntDen value / Internal reference protein IntDen value)PBS组 ×100%.

[0183] The results showed that, compared with the PBS group, the relative expression level of the target protein (viral capsid protein) was significantly decreased in all treatment groups (including the 8 μM hypersensitive protein treatment group, the 2 μM PeBL1 protein treatment group, and the 8 μM PeBL1 protein treatment group). Specifically, the highest virus accumulation was observed in plants in the PBS treatment group on day 35 after ToBRFV inoculation. The accumulation level of viral CP (viral capsid protein) in plants treated with 8 μM hypersensitive protein was 55.46% of that in the PBS treatment group. The lowest virus accumulation was observed in the 8 μM PeBL1 protein treatment group, approximately 46.59% of the viral load in the PBS control group; the accumulation level of viral CP in the 2 μM PeBL1 protein treatment group was approximately 54.27% of that in the PBS control group. This indicates that treatment with hypersensitive protein and PeBL1 protein reduced the accumulation of the target protein. PeBL1 protein at different concentrations (2 μM and 8 μM) significantly reduced the relative expression level of the target protein (viral capsid protein), with the lowest relative expression level observed in the 8 μM PeBL1 protein group, suggesting that PeBL1 protein may have a dose-dependent effect. Viral capsid proteins are major structural components of viral particles, and their synthesis and accumulation are closely related to viral replication. Reduced capsid protein content typically reflects a decrease in viral replication and assembly processes. Treatment with PeBL1 protein significantly reduced the accumulation of viral capsid proteins, further demonstrating the effectiveness of PeBL1 protein in inhibiting viral replication and accumulation.

[0184] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Use of a PeBL1 protein for increasing the resistance of a tomato to a pathogen and / or for the manufacture of a product for increasing the resistance of a tomato to a pathogen, wherein, The pathogen is selected from the group consisting of Fusarium oxysporum f. sp. radicis-lycopersici and Tomato brown rugose fruit virus, the amino acid sequence of the PeBL1 protein is shown as SEQ ID NO: 1, the application is achieved by contacting the PeBL1 protein with the tomato plant in an effective amount, the effective amount refers to the concentration of the PeBL1 protein is not less than 2 μM; The improved resistance of the tomato to the pathogen is selected from at least one of the following: 1) improving the defense of the tomato and / or controlling the infection of Fusarium oxysporum f. sp. radicis-lycopersici; 2) improving the defense of the tomato and / or controlling the infection of Tomato brown rugose fruit virus; 3) reducing or eliminating the symptoms caused by the infection of Fusarium oxysporum f. sp. radicis-lycopersici; 4) reducing or eliminating the symptoms caused by the infection of Tomato brown rugose fruit virus; 5) preventing the occurrence of tomato collar and root rot; and 6) reducing or eliminating the symptoms of tomato collar and root rot.

2. Use of a nucleic acid molecule encoding a PeBLl protein for increasing the resistance of a tomato plant to a pathogen and / or for the manufacture of a product for increasing the resistance of a tomato plant to a pathogen, wherein, The pathogen is selected from the group consisting of Fusarium oxysporum f. sp. radicis-lycopersici and Tomato brown rugose fruit virus, the amino acid sequence of the PeBL1 protein is shown as SEQ ID NO: 1, the application is achieved by contacting the PeBL1 protein with the tomato plant in an effective amount, the effective amount refers to the concentration of the PeBL1 protein is not less than 2 μM; The improved resistance of the tomato to the pathogen is selected from at least one of the following: 1) improving the defense of the tomato and / or controlling the infection of Fusarium oxysporum f. sp. radicis-lycopersici; 2) improving the defense of the tomato and / or controlling the infection of Tomato brown rugose fruit virus; 3) reducing or eliminating the symptoms caused by the infection of Fusarium oxysporum f. sp. radicis-lycopersici; 4) reducing or eliminating the symptoms caused by the infection of Tomato brown rugose fruit virus; 5) preventing the occurrence of tomato collar and root rot; and 6) reducing or eliminating the symptoms of tomato collar and root rot.

3. Use of a vector, an expression cassette, a transgenic cell line comprising a nucleic acid molecule encoding a PeBLl protein in increasing the resistance of a tomato to a pathogen and / or in the manufacture of a product for increasing the resistance of a tomato to a pathogen, wherein, The pathogen is selected from the group consisting of Fusarium oxysporum f. sp. radicis-lycopersici and Tomato brown rugose fruit virus, the amino acid sequence of the PeBL1 protein is shown as SEQ ID NO: 1, the application is achieved by contacting the PeBL1 protein with the tomato plant in an effective amount, the effective amount refers to the concentration of the PeBL1 protein is not less than 2 μM; The improved resistance of the tomato to the pathogen is selected from at least one of the following: 1) improving the defense of the tomato and / or controlling the infection of Fusarium oxysporum f. sp. radicis-lycopersici; 2) improving the defense of the tomato and / or controlling the infection of Tomato brown rugose fruit virus; 3) reducing or eliminating the symptoms caused by the infection of Fusarium oxysporum f. sp. radicis-lycopersici; 4) reducing or eliminating the symptoms caused by the infection of Tomato brown rugose fruit virus; 5) preventing the occurrence of tomato collar and root rot; and 6) reducing or eliminating the symptoms of tomato collar and root rot.

4. A product for increasing the resistance of a tomato to a pathogen, characterized in that, The pathogen is selected from the group consisting of Fusarium oxysporum f. sp. radicis-lycopersici and Tomato brown rugose fruit virus, the amino acid sequence of the PeBL1 protein is shown as SEQ ID NO: 1, the application is achieved by contacting the PeBL1 protein with the tomato plant in an effective amount, the effective amount refers to the concentration of the PeBL1 protein is not less than 2 μM; The improved resistance of the tomato to the pathogen is selected from at least one of the following: 1) improving the defense of the tomato and / or controlling the infection of Fusarium oxysporum f. sp. radicis-lycopersici; 2) improving the defense of the tomato and / or controlling the infection of Tomato brown rugose fruit virus; 5. The product of claim 4, wherein, 3) reducing or eliminating the symptoms caused by the infection of Fusarium oxysporum f. sp. radicis-lycopersici; 4) reducing or eliminating the symptoms caused by the infection of Tomato brown rugose fruit virus; 5) preventing the occurrence of tomato collar and root rot; and 6) reducing or eliminating the symptoms of tomato collar and root rot. The product includes reagents, drugs, pesticides. The product includes reagents, drugs, pesticides.

6. A method of increasing the resistance of a tomato to a pathogen comprising: A product as described in claim 4 or 5 is applied to a tomato plant such that it contacts the tomato plant in an effective amount, the pathogen is Fusarium oxysporum f. sp. radicis-lycopersici and Tomato brown rugose fruit virus, the effective amount is a concentration of PeBL1 protein in the applied product of not less than 2 μM.

7. The method of claim 6, wherein, The product is a solution comprising PeBL1 protein.

8. The method of claim 7, wherein, The application is spraying a solution comprising PeBL1 protein to a tomato plant.

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