Tomato source antibacterial protein, application thereof in prevention and treatment of tomato gray mold and biological preparation for preventing and treating tomato gray mold
By cloning and preparing tomato antimicrobial proteins ABS258 and ABS121 from the tomato variety Ailsa Craig, and applying an Escherichia coli expression system, the problems of drug resistance and environmental pollution in the chemical control of tomato gray mold were solved, providing an efficient and safe biological pesticide solution.
Patent Information
- Application Number
- CN202511488582.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-17
- Publication Date
- 2025-11-25
AI Technical Summary
In the current technology, the control of tomato gray mold mainly relies on chemical pesticides, which leads to pesticide resistance and environmental pollution. In addition, there are few antimicrobial proteins derived from tomatoes, making screening and functional verification difficult, and there is a lack of efficient and safe natural antimicrobial protein resources.
Antimicrobial proteins ABS258 and ABS121 were cloned and identified from the cultivated tomato variety Ailsa Craig. They were prepared using an E. coli expression system and applied to biological agents for the control of tomato gray mold. This provides two antimicrobial proteins with different characteristics: ABS121 is a short-chain protein and ABS258 is a long-chain protein, which have the advantages of small molecular weight, low synthesis cost and strong anti-degradation ability, respectively.
It significantly inhibits the mycelial growth of Botrytis cinerea at low concentrations, preventing pathogen infection and overcoming the environmental pollution and resistance problems of chemical pesticides. It provides a candidate biopesticide with clear genetic origin and high compatibility with tomatoes.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the technical field of tomato disease prevention and control, and particularly relates to a tomato-derived antibacterial protein and application thereof in prevention and control of tomato gray mold and a biological agent for prevention and control of tomato gray mold. BACKGROUND
[0002] Tomato (Solanum lycopersicum) is one of the most important economic crops in the world, with extremely high agricultural and economic value. However, tomato production is threatened by various diseases, among which gray mold caused by Botrytis cinerea is one of the main diseases in tomato production, with a wide host range and strong environmental adaptability, and can infect flowers, fruits, leaves and stems of plants under various conditions. It seriously harms the yield and quality of tomatoes. The yield loss caused by tomato gray mold can reach 20%-30% of the total yield per year, and even more than 50% in severe cases. Therefore, how to effectively prevent and control the field prevalence of tomato gray mold has become a key problem to ensure the healthy development of the tomato industry.
[0003] At present, the prevention and control of tomato gray mold mainly relies on chemical means, but the overuse of pesticides can easily lead to the development of drug resistance in Botrytis cinerea and cause pollution risk to the field ecological environment. In contrast, plant-derived antibacterial peptides and proteins have significant disease control effect, and are friendly to the ecological environment due to their natural source characteristics, so they have attracted widespread attention. Therefore, identifying plant-derived antibacterial proteins and peptides is of great significance for the development of green and sustainable biological pesticides or protein pesticides.
[0004] The screening of plant-derived antibacterial proteins or peptides usually focuses on wild plants, economic crops and solanaceous vegetables with strong disease resistance, because they have formed a high-efficiency defense protein library during long-term evolution. Screening of antibacterial proteins from the crop's own gene library has the advantages of clear genetic resources, high compatibility for breeding, and natural expression products that are easier to pass safety assessment, which meets the needs of green agriculture. However, there are few tomato-derived antibacterial proteins, mainly because their disease resistance mechanism relies on the cooperation of multiple genes and induced expression, and most defense proteins are only transiently highly expressed after pathogen infection, with extremely low content in normal tissues. In addition, the functional redundancy of gene families is high, and the phenotype is difficult to show with single protein deletion, which makes screening and functional verification very difficult. At present, there is no clear report on tomato-derived antibacterial proteins for tomato gray mold, and the existing control means still face the bottleneck of drug resistance and environmental pollution, so it is urgent to mine efficient and safe natural antibacterial protein resources from the tomato's own gene library to develop green and sustainable disease control technology. SUMMARY
[0005] To solve the problem that the prior art lacks tomato-derived antibacterial proteins for preventing and controlling tomato gray mold, the present application provides a tomato-derived antibacterial protein and application thereof in prevention and control of tomato gray mold and a biological agent for prevention and control of tomato gray mold.
[0006] The technical scheme of the present application is as follows:
[0007] The tomato-derived antibacterial protein is as follows (1) or (2):
[0008] (1) Tomato ABS258 antibacterial protein, having an amino acid sequence as shown in SEQ ID No: 1;
[0009] (2) Tomato ABS121 antibacterial protein, the amino acid sequence of the tomato ABS121 antibacterial protein is 1 to 121 of the amino acid sequence of the tomato ABS258 antibacterial protein, specifically as shown in SEQ ID No: 2.
[0010] Further, the tomato-derived antibacterial protein is encoded by ABS258 gene in tomato variety Ailsa Craig, the nucleotide sequence of the tomato ABS258 antibacterial protein coding gene is as shown in SEQ ID No: 3, and the nucleotide sequence of the tomato ABS121 antibacterial protein coding gene is as shown in SEQ ID No: 4.
[0011] The application of a tomato-derived antibacterial protein in preventing and treating tomato gray mold.
[0012] Further, the tomato-derived antibacterial protein is used for preparing a biological preparation for preventing and treating tomato gray mold, and the use amount of the tomato ABS258 antibacterial protein and the tomato ABS121 antibacterial protein in preventing and treating tomato gray mold is 10-60 μM.
[0013] Further, the coding gene of the tomato-derived antibacterial protein is used for genetic improvement against tomato gray mold.
[0014] A biological preparation for preventing and treating tomato gray mold, the biological preparation contains a tomato-derived antibacterial protein, the tomato-derived antibacterial protein is tomato ABS258 antibacterial protein and / or tomato ABS121 antibacterial protein, the tomato ABS258 antibacterial protein has an amino acid sequence as shown in SEQ ID No: 1, and the amino acid sequence of the tomato ABS121 antibacterial protein is 1 to 121 of the amino acid sequence of the tomato ABS258 antibacterial protein, specifically as shown in SEQ ID No: 2.
[0015] Further, the tomato-derived antibacterial protein is synthesized by a recombinant protein expression system containing a coding gene of the tomato-derived antibacterial protein, the nucleotide sequence of the tomato ABS258 antibacterial protein coding gene is as shown in SEQ ID No: 3, and the nucleotide sequence of the tomato ABS121 antibacterial protein coding gene is as shown in SEQ ID No: 4.
[0016] Further, the recombinant protein expression system is a prokaryotic expression system or a eukaryotic expression system.
[0017] Further, the prokaryotic expression system is an Escherichia coli recombinant protein expression system; the eukaryotic expression system is a yeast cell recombinant protein expression system or a mammalian cell recombinant protein expression system.
[0018] Further, the Escherichia coli recombinant protein expression system contains an Escherichia coli pHAT2 expression vector.
[0019] The beneficial effects of the present application are as follows:
[0020] The present application successfully clones and identifies two novel antibacterial proteins ABS121 and ABS258 from the genome of cultivated tomato variety Ailsa Craig, which belong to different splice variants of the same protein and have the same 121-amino-acid core sequence. Both of the two proteins prepared by the Escherichia coli expression system exhibit significant Botrytis cinerea control effect: they can effectively inhibit the mycelial growth of Botrytis cinerea and prevent the pathogen from infecting tomato leaves at low concentrations. Among them, the tomato ABS121 antibacterial protein as a short-chain protein has the advantages of small molecular weight, low synthesis cost and high stability; while the tomato ABS258 antibacterial protein as a long-chain protein has stronger anti-degradation ability and more persistent antibacterial activity. The two antibacterial proteins can be produced on a large scale through various expression systems, providing important candidates for the development of new biological pesticides.
[0021] The present application obtains specific Botrytis cinerea proteins, which not only have the natural advantages of clear genetic source and high compatibility with tomatoes, but also provide flexible choices for pesticide development for different application scenarios with their differentiated protein structures, effectively overcoming the environmental pollution and drug resistance problems of traditional chemical pesticides. BRIEF DESCRIPTION OF DRAWINGS
[0022] Figure 1 The protein electrophoretogram obtained in the gel electrophoresis of different samples in the process of expressing and purifying the tomato ABS258 protein in the Escherichia coli recombinant protein expression system of Example 1, M: marker, S: supernatant after ultrasonic crushing, P: precipitate after centrifugation, T: eluate after the supernatant flows through the His-Tag affinity chromatography filler, C: protein sample remaining on the His-Tag affinity chromatography filler after elution, 20, 60, 100, 200 and 500 are eluents containing different concentrations of imidazole;
[0023] Figure 2The protein electrophoretogram of different samples in the process of expressing and purifying tomato ABS121 protein in the E. coli recombinant protein expression system in Example 1, M: marker, S: supernatant after ultrasonic disruption, P: precipitate after centrifugation, T: eluate after His-Tag affinity chromatography, C: protein sample remaining on the His-Tag affinity chromatography column after elution, 20, 60, 100, 200, 500: eluent containing different concentrations of imidazole;
[0024] Figure 3 The plate inhibition experiment results of the test group and the control group in Example 2 to which different concentrations of tomato ABS121 protein and ABS258 protein were added;
[0025] Figure 4 The photos of the lesion spots of tomato detached leaves treated with the mixed solution of the test group and the control group in Example 3 to which different concentrations of tomato ABS121 protein and ABS258 protein were added;
[0026] Figure 5 The comparison chart of the lesion spot area of tomato detached leaves treated with the mixed solution of the test group and the control group in Example 3 to which different concentrations of tomato ABS121 protein and ABS258 protein were added, A: tomato ABS121 protein, B: ABS258 protein. DETAILED DESCRIPTION
[0027] The technical solutions of the present application are further described below in combination with examples, but are not limited thereto. Any modification or equivalent replacement to the technical solutions of the present application without departing from the spirit and scope of the technical solutions shall be encompassed in the protection scope of the present application. The process equipment or device not specifically mentioned in the following examples is the conventional equipment or device in the art. If not specifically mentioned, the raw materials used in the examples of the present application are commercially available. If not specifically mentioned, the technical means used in the examples of the present application are the conventional means known to those skilled in the art.
[0028] Example 1
[0029] The present example provides a method for expressing and purifying tomato ABS258 antibacterial protein and tomato ABS121 antibacterial protein in an E. coli recombinant protein expression system.
[0030] The cDNA of cultivated tomato variety Ailsa Craig was used as a template, and the primer ABS258-AT2-F and ABS258-AT2-R shown in SEQ ID No: 5 and SEQ ID No: 6 were used for PCR amplification, and the obtained amplification product was the tomato 258 gene shown in the nucleotide sequence of SEQ ID No: 3, and the sequence length was 774 bp; the primer ABS258-AT2-F and ABS121-AT2-R shown in SEQ ID No: 5 and SEQ ID No: 7 were used for PCR amplification, and the obtained amplification product was the tomato 121 gene shown in the nucleotide sequence of SEQ ID No: 4, and the sequence length was 363 bp.
[0031] After the PCR product was recovered by agarose gel electrophoresis, it was connected to the E. coli expression vector pHAT2 by a conventional homologous recombination method to obtain the pHAT2-ABS258 vector and the pHAT2-ABS121 vector. The pHAT2-ABS258 vector, the pHAT2-ABS121 vector, and the pHAT2 empty vector were transformed into the E. coli BL21 (DE3) strain by heat shock method, respectively. The BL21 (DE3) strain transformation plate positive bacterial plaques were picked into liquid LB medium containing 50 mg / L ampicillin, and cultured at 37°C, 220 rpm for 20 h until the OD600=0.6. 0.2 mM IPTG (isopropyl-β-D-thiogalactoside) was added to the above liquid medium, and the culture was shaken at 16°C, 160 rpm for 20 h. The bacterial cells in the liquid medium were collected by centrifugation at 12000 rpm for 10 min, and the bacterial cells were resuspended with 1×PBS, pH=7.0 buffer. The bacterial cells were broken by ultrasonic crusher at 45% power for 15 min, and then affinity chromatography was performed.
[0032] Subsequently, elution was performed with 1×PBS, 20 mM imidazole, pH=7.0, 1×PBS, 60 mM imidazole, pH=7.0, 1×PBS, 100 mM imidazole, pH=7.0, 1×PBS, 200 mM imidazole, pH=7.0, and 1×PBS, 500 mM imidazole, pH=7.0 buffer, and the impurities were eluted with 20-200 mM imidazole, and the target protein was eluted with 500 mM imidazole, to obtain the tomato ABS258 protein shown in the amino acid sequence of SEQ ID No: 1, and the sequence length was 258 aa; and the tomato ABS121 protein, and the sequence length was 121 aa.
[0033] Example 2
[0034] In this example, the antibacterial effect of the tomato ABS121 protein and the ABS258 protein obtained by separation and purification in Example 1 on Botrytis cinerea was investigated by plate antibacterial test.
[0035] The tomato ABS121 protein and ABS258 protein obtained by separation and purification in Example 1 were added to PDA solid medium as test groups at concentrations of 0 µM, 5 µM, 10 µM and 20 µM, and the E. coli strain purified protein after transformation of the blank vector pHAT2 was added to PDA solid medium as a control group at a concentration of 20 µM. After the plate medium solidified, a 6mm puncher was used to access the mycelium block with Botrytis cinerea strain B05.10 mycelium, and after inoculation, the plate was incubated at 25°C for 3 days. The effect of tomato ABS121 protein and ABS258 protein on Botrytis cinerea mycelial growth was verified by measuring the radial diameter of the colonies on the medium.
[0036] The results are shown in Figure 3 Compared with the blank PDA plate without the addition of antibacterial protein, 10 μM of low-concentration tomato ABS121 protein or ABS258 protein had an inhibitory effect on Botrytis cinerea mycelial growth, and 20 μM of tomato ABS121 protein or ABS258 protein had a significant inhibitory effect on Botrytis cinerea mycelial growth.
[0037] Example 3
[0038] This example investigates the tomato gray mold disease control effect of the tomato ABS121 protein and ABS258 protein obtained by separation and purification in Example 1.
[0039] Botrytis cinerea strain B05.10 cultured at 25°C for one week was washed with 2-3 mL of sterile water, filtered after vigorous shaking, and the concentration of the conidial suspension was diluted to 1×10 4 The conidial suspension was mixed with tomato ABS121 protein or ABS258 protein at concentrations of 20 µM, 40 µM and 60 µM as test groups, and the conidial suspension was mixed with the E. coli strain purified protein after transformation of the blank vector pHAT2 at a concentration of 60 µM as a control group. 10 µL of the mixture was inoculated on 3-6 week old detached tomato leaves, and the disease condition of the inoculated leaves was observed 3-5 days after inoculation, and the lesion area was recorded.
[0040] The results are shown in Figure 4 and Figure 5As shown, the analysis of the effect of tomato ABS121 protein or ABS258 protein on the prevention and treatment of tomato gray mold showed that 20 μM of tomato ABS121 protein or ABS258 protein mixed with conidia of Botrytis cinerea B05.10 strain and inoculated on tomato leaves could significantly inhibit the infection of Botrytis cinerea on tomato leaves. 40 μM of tomato ABS121 protein or ABS258 protein could significantly inhibit the infection of Botrytis cinerea on tomato leaves.
Claims
1. A tomato-derived antibacterial protein, characterized in that, It is as follows (1) or (2): (1) Tomato ABS258 antimicrobial protein, having the amino acid sequence shown in SEQ ID No:1; (2) Tomato ABS121 antimicrobial protein, wherein the amino acid sequence of the tomato ABS121 antimicrobial protein is the first to the 121st position of the amino acid sequence of the tomato ABS258 antimicrobial protein, as shown in SEQ ID No:
2.
2. The tomato-derived antibacterial protein according to claim 1, characterized in that, The tomato-derived antimicrobial protein is encoded by the ABS258 gene in the tomato variety Ailsa Craig. The nucleotide sequence of the tomato ABS258 antimicrobial protein encoding gene is shown in SEQ ID No:3, and the nucleotide sequence of the tomato ABS121 antimicrobial protein encoding gene is shown in SEQ ID No:
4.
3. The application of the tomato-derived antibacterial protein as described in claim 1 or 2 in the prevention and control of tomato gray mold.
4. The application of the tomato-derived antibacterial protein according to claim 3 in controlling tomato gray mold, characterized in that, Tomato-derived antimicrobial proteins were used to prepare biological agents for the prevention and control of tomato gray mold. The dosage of both tomato ABS258 antimicrobial protein and tomato ABS121 antimicrobial protein for the prevention and control of tomato gray mold was 10-60 μM.
5. The application of the tomato-derived antibacterial protein according to claim 3 in controlling tomato gray mold, characterized in that, The gene encoding the antimicrobial protein derived from tomato was used for genetic modification to improve resistance to tomato gray mold.
6. A biological agent for controlling tomato gray mold, characterized in that, The biological agent contains tomato-derived antimicrobial proteins, namely tomato ABS258 antimicrobial protein and / or tomato ABS121 antimicrobial protein. The tomato ABS258 antimicrobial protein has the amino acid sequence shown in SEQ ID No:1, and the amino acid sequence of the tomato ABS121 antimicrobial protein is positions 1 to 121 of the amino acid sequence of the tomato ABS258 antimicrobial protein, as specifically shown in SEQ ID No:
2.
7. The biological agent for controlling tomato gray mold according to claim 6, characterized in that, The tomato-derived antimicrobial protein is synthesized by a recombinant protein expression system containing a tomato-derived antimicrobial protein encoding gene. The nucleotide sequence of the tomato ABS258 antimicrobial protein encoding gene is shown in SEQ ID No:3, and the nucleotide sequence of the tomato ABS121 antimicrobial protein encoding gene is shown in SEQ ID No:
4.
8. The biological agent for controlling tomato gray mold according to claim 7, characterized in that, The recombinant protein expression system is either a prokaryotic expression system or a eukaryotic expression system.
9. The biological agent for controlling tomato gray mold according to claim 8, characterized in that, The prokaryotic expression system is an Escherichia coli recombinant protein expression system; the eukaryotic expression system is a yeast cell recombinant protein expression system or a mammalian cell recombinant protein expression system.
10. The biological agent for controlling tomato gray mold according to claim 8, characterized in that, The E. coli recombinant protein expression system contains the E. coli pHAT2 expression vector.