Tomato SGT1 protein, application of tomato SGT1 protein in prevention and treatment of tomato gray mold and biological preparation for preventing and treating tomato gray
By purifying tomato SGT1 protein using an Escherichia coli recombinant system, the problem of plant-derived antimicrobial proteins being unable to inhibit Botrytis cinerea infection in existing technologies has been solved. This method enables early multi-target control of necrotrophic pathogens and improves the effectiveness of controlling tomato gray mold.
Patent Information
- Application Number
- CN202511488577.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-17
- Publication Date
- 2025-11-21
AI Technical Summary
Existing plant-derived antimicrobial proteins are difficult to effectively inhibit the infection of necrotrophic pathogens such as Botrytis cinerea, and their effects are delayed. The use of traditional chemical pesticides leads to resistance and environmental pollution, and cross-species screening of antimicrobial proteins is difficult to succeed.
The tomato SGT1 protein was purified using an E. coli recombinant protein expression system and used to inhibit the mycelial growth of Botrytis cinerea and disrupt the integrity of its conidia, achieving multi-target control in the early stage of infection.
It significantly inhibits the mycelial growth of Botrytis cinerea, disrupts the integrity of its conidia, blocks the infection process, improves control efficiency, overcomes the limitations of traditional antimicrobial proteins, and provides early and precise control.
Smart Images
Figure CN120988085A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of tomato disease control technology, and particularly relates to tomato SGT1 protein and its application in controlling tomato gray mold and biological agents for controlling tomato gray mold. Background Technology
[0002] Tomato (Solanum lycopersicum) is one of the world's most important economic crops, possessing extremely high agricultural and economic value. However, tomato production faces numerous disease threats, among which gray mold, caused by Botrytis cinerea, is one of the major diseases affecting tomato production. It has a wide host range and strong environmental adaptability, infecting flowers, fruits, leaves, and stems under various conditions, severely damaging tomato yield and quality. Annual yield losses due to tomato gray mold can reach 20%-30% of total yield, and in severe cases, even exceed 50%. Therefore, effectively controlling the field spread of tomato gray mold has become a crucial issue for ensuring the healthy development of the tomato industry.
[0003] Currently, the control of tomato gray mold mainly relies on chemical methods. However, the overuse of pesticides can easily lead to resistance in *Botrytis cinerea* and pose a risk of environmental pollution in the fields. In contrast, plant-derived antimicrobial peptides and proteins have significant disease control effects and, due to their natural origin, are environmentally friendly, thus attracting widespread attention. Therefore, the identification of plant-derived antimicrobial peptides and proteins is of great significance for the development of green and sustainable biopesticides or protein pesticides.
[0004] However, *Botrytis cinerea* is a typical necrotrophic pathogen, meaning it feeds on dead plant tissue. Unlike vivotrophic pathogens, which inhibit host cell death to obtain nutrients from living host cells, necrotrophic pathogens obtain nutrients by killing host cells, making their disease-treating strategy more "aggressive." Necrotrophic pathogens begin secreting toxins and degrading enzymes during spore germination and appressorium formation, which not only damage plant cell walls and promote self-infection but also degrade antimicrobial proteins, leading to their inactivation. Existing plant-derived antimicrobial proteins mostly target fungi in their hyphal expansion stage and are difficult to inhibit before pathogen colonization. Most discovered plant-derived antimicrobial proteins enhance resistance by activating PTIs (pattern-triggered immunity) or ETIs (effector-triggered immunity), but necrotrophic pathogens can directly induce plant cell death by secreting toxins and cell necrosis-inducing proteins, bypassing or inhibiting immune signaling pathways, thus weakening the immune-inducing effect of antimicrobial proteins. Existing screening of antimicrobial proteins is mostly based on model plants (such as Arabidopsis thaliana) or a few crops (such as rice and wheat). However, different crops (such as tomatoes and peppers) have different resistance mechanisms to necrotrophic pathogens, making cross-species screening of plant-derived antimicrobial proteins often difficult. Summary of the Invention
[0005] To address the problems of existing plant-derived antibacterial proteins being unable to effectively inhibit Botrytis cinerea infection and having a delayed effect, this invention provides tomato SGT1 protein and its application in controlling tomato gray mold, as well as biological agents for controlling tomato gray mold.
[0006] The technical solution of the present invention:
[0007] The tomato SGT1 protein has the amino acid sequence shown in SEQ ID No:1.
[0008] Furthermore, the tomato SGT1 protein is encoded by the SGT1 gene in the tomato variety Ailsa Craig, and the nucleotide sequence of the tomato SGT1 gene is shown in SEQ ID No:2.
[0009] An application of tomato SGT1 protein in the prevention and control of tomato gray mold.
[0010] Furthermore, the dosage of the tomato SGT1 protein used to control tomato gray mold is 10~80μM.
[0011] A biological agent for controlling gray mold in tomatoes, the biological agent containing tomato SGT1 protein, the tomato SGT1 protein having the amino acid sequence shown in SEQ ID No:1.
[0012] Furthermore, the tomato SGT1 protein is synthesized by a recombinant protein expression system containing the tomato SGT1 protein encoding gene, the nucleotide sequence of which is shown in SEQ ID No:2.
[0013] Furthermore, the recombinant protein expression system is a prokaryotic expression system or a eukaryotic expression system.
[0014] Furthermore, 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.
[0015] Furthermore, the Escherichia coli recombinant protein expression system contains the Escherichia coli pHAT2 expression vector.
[0016] The beneficial effects of this invention are:
[0017] This invention cloned the SGT1 gene from the cultivated tomato variety Alisa Craig and successfully purified the tomato SGT1 protein using an E. coli expression system. Experiments demonstrated that this protein effectively inhibits the mycelial growth of *Botrytis cinerea*, disrupts the integrity of its conidia, and significantly reduces the infectivity of *Botrytis cinerea* on tomato leaves. *Botrytis cinerea*, as a typical necrotrophic pathogen, relies heavily on the rapid germination of conidia and early toxin secretion for its pathogenicity. The tomato SGT1 protein of this invention disrupts the integrity of conidia, simultaneously blocking both physical infection and chemical toxicity pathways, directly inhibiting key biological processes in the early germination stage. This represents typical pre-infection inhibition and significantly improves the control efficiency of tomato gray mold. The tomato SGT1 protein provided by this invention overcomes the limitation of traditional antibacterial proteins that are only effective during the mycelial expansion stage, achieving precise control of necrotrophic pathogens through "early intervention and multiple targets," providing an important foundation for the development of novel biological agents for the control of tomato gray mold. Attached Figure Description
[0018] Figure 1 The images show the protein electrophoresis results of different samples in Example 1. M: marker, S: supernatant after ultrasonic disruption, P: precipitate after centrifugation, T: effluent after supernatant passes through His-Tag affinity chromatography packing, C: protein sample remaining on His-Tag affinity chromatography packing after elution, and 20, 60, 100, 200, and 500 are elution buffers containing different concentrations of imidazole.
[0019] Figure 2 The results of the plate antibacterial experiment in Example 2, showing the experimental group and the control group with different concentrations of tomato SGT1 protein added;
[0020] Figure 3 Microscopic images of Botrytis cinerea conidia in the treatment group and control group with different concentrations of tomato SGT1 protein added in Example 3;
[0021] Figure 4 This is a comparison chart showing the proportion of different forms of Botrytis cinerea conidia in the treatment group and the control group with different concentrations of tomato SGT1 protein added in Example 3.
[0022] Figure 5 Photographs of lesions obtained from tomato detached leaves treated with experimental group mixtures containing different concentrations of tomato SGT1 protein and control group mixtures in Example 4.
[0023] Figure 6 Example 4: Comparison of lesion area on detached tomato leaves treated with experimental mixtures containing different concentrations of tomato SGT1 protein and control mixtures. Detailed Implementation
[0024] The technical solution of the present invention will be further described below with reference to embodiments, but it is not limited thereto. Any modifications or equivalent substitutions to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention should be covered within the protection scope of the present invention. In the following embodiments, the process equipment or apparatus not specifically specified are all conventional equipment or apparatus in the art. Unless otherwise specified, the raw materials used in the embodiments of the present invention are all commercially available; unless otherwise specified, the technical means used in the embodiments of the present invention are all conventional means well known to those skilled in the art.
[0025] Example 1
[0026] This embodiment provides a method for expressing and purifying tomato SGT1 protein in an Escherichia coli recombinant protein expression system.
[0027] In this embodiment, cDNA of the cultivated tomato variety Ailsa Craig was used as a template. PCR amplification was performed using primers SGT1-AT2-F and SGT1-AT2-R shown in SEQ ID No:3 and SEQ ID No:4. The amplified product is the tomato SGT1 gene with a nucleotide sequence as shown in SEQ ID No:2, with a sequence length of 1113 bp. The last 3 bp are stop codons and are not translated into amino acids.
[0028] PCR products were recovered by agarose gel electrophoresis and ligated into the *E. coli* expression vector pHAT2 using conventional homologous recombination methods to obtain the pHAT2-SGT1 vector. The pHAT2-SGT1 vector and the empty pHAT2 vector were transformed into *E. coli* strain BL21(DE3) using a heat shock method. Positive plaques from BL21(DE3) strain were picked and cultured in LB liquid medium containing 50 mg / L ampicillin at 37°C and 220 rpm until OD600 = 0.6. 0.2 mM IPTG (isopropyl-β-D-thiogalactopyranoside) was added to the liquid medium, and the culture was carried out at 16°C and 160 rpm for 20 h. Bacterial cells were collected by centrifugation at 12000 rpm for 10 min and resuspended in 1×PBS, pH 7.0 buffer. The cells were then sonicated at 45% power for 15 min before affinity chromatography.
[0029] Subsequently, the protein was eluted with 1×PBS, 20mM imidazole, pH=7.0, 1×PBS, 60mM imidazole, pH=7.0, 1×PBS, 100mM imidazole, pH=7.0, 1×PBS, 200mM imidazole, pH=7.0, and 1×PBS, 500mM imidazole, pH=7.0, respectively. Impurities were eluted with 20-200mM imidazole and the target protein was eluted with 500mM imidazole, yielding the tomato SGT1 protein with the amino acid sequence shown in SEQ ID No:1, with a sequence length of 370aa.
[0030] Example 2
[0031] This embodiment investigated the antibacterial effect of the tomato SGT1 protein isolated and purified in Example 1 on Botrytis cinerea through a plate antibacterial test.
[0032] The plate inhibition test consisted of experimental and control groups. The tomato SGT1 protein isolated and purified in Example 1 was added to PDA solid medium at concentrations of 0 µM, 5 µM, 10 µM, and 20 µM as the experimental group. The purified protein from the *E. coli* strain transformed with the blank vector pHAT2 was added to the PDA solid medium at a concentration of 20 µM as the control group. After the plate medium solidified, mycelial blocks containing *Botrytis cinerea* strain B05.10 hyphae were inoculated using a 6 mm punch. The plates were then incubated at 25°C for 3 days. The radial diameter of the colonies on the medium was measured to verify the effect of SGT1 protein on the mycelial growth of *Botrytis cinerea*.
[0033] The results are as follows Figure 2 As shown, compared with the blank PDA plate without added tomato SGT1 protein, the low concentration of 5 μM tomato SGT1 protein inhibited the growth of Botrytis cinerea mycelium, and 20 μM tomato SGT1 protein significantly inhibited the growth of Botrytis cinerea mycelium.
[0034] Example 3
[0035] This embodiment investigated the effect of the tomato SGT1 protein isolated and purified in Example 1 on the development of Botrytis cinerea conidia through conidial morphology analysis.
[0036] Rinse the *Botrytis cinerea* strain B05.10, which has been cultured at 25°C for one week, with 2-3 mL of sterile water. After vigorous shaking, filter the solution, count the spores using a hemocytometer, and dilute the conidial suspension to a concentration of 1×10⁻⁶. 4The conidial suspension was mixed with SGT1 protein at concentrations of 10 µM, 20 µM, 40 µM, or 80 µM to form the tomato SGT1 protein treatment group. The conidial suspension was mixed with purified protein from E. coli strain transformed with pHAT2 empty vector at a concentration of 80 µM to form the control group (Mock). The conidial suspensions were inoculated onto hydrophobic slides, and the morphology of the conidia was observed and recorded using a stereomicroscope after 48 h.
[0037] The results are as follows Figure 3 and Figure 4 As shown, the conidial morphology analysis experiment showed that adding 10 μM low concentration of SGT1 protein to the same concentration of Botrytis cinerea conidial suspension affected the morphology and integrity of the conidia. Adding 80 μM concentration of SGT1 protein could significantly promote morphological changes and destroy their integrity.
[0038] Example 4
[0039] This embodiment examines the efficacy of the tomato SGT1 protein isolated and purified in Example 1 in controlling tomato gray mold.
[0040] Rinse the *Botrytis cinerea* strain B05.10, which has been cultured at 25°C for one week, with 2-3 mL of sterile water. After vigorous shaking, filter the solution, count the spores using a hemocytometer, and dilute the conidial suspension to a concentration of 1×10⁻⁶. 4 The conidial suspension was mixed with SGT1 protein at concentrations of 10 µM, 20 µM, 40 µM, and 80 µM as experimental groups. The conidial suspension was mixed with purified protein from E. coli strain transformed with pHAT2 empty vector at a concentration of 80 µM as the control group. 10 µL of the mixture was inoculated onto detached tomato leaves aged 3-6 weeks. The disease status of the inoculated leaves was observed 3-5 days after inoculation, and the area of lesions was recorded.
[0041] The results are as follows Figure 5 and Figure 6 As shown in the figure, the effect of SGT1 protein on controlling tomato gray mold indicates that inoculation of tomato leaves with a mixture of 20 μM tomato SGT1 protein and conidia of Botrytis cinerea strain B05.10 significantly inhibited Botrytis cinerea infection. 40 μM tomato SGT1 protein completely inhibited Botrytis cinerea infection of tomato leaves.
Claims
1. Tomato SGT1 protein, characterized in that, It has the amino acid sequence shown in SEQ ID No:
1.
2. The tomato SGT1 protein according to claim 1, characterized in that, The tomato SGT1 protein is encoded by the SGT1 gene in the tomato variety Ailsa Craig, and the nucleotide sequence of the tomato SGT1 gene is shown in SEQ ID No:
2.
3. The application of the tomato SGT1 protein as described in claim 1 or 2 in the prevention and control of tomato gray mold.
4. The application of the tomato SGT1 protein in controlling tomato gray mold according to claim 3, characterized in that, The dosage of the tomato SGT1 protein used to control tomato gray mold is 10-80 μM.
5. A biological agent for controlling gray mold in tomatoes, characterized in that, The biological agent contains tomato SGT1 protein, which has the amino acid sequence shown in SEQ ID No:
1.
6. The biological agent for controlling tomato gray mold according to claim 5, characterized in that, The tomato SGT1 protein was synthesized by a recombinant protein expression system containing the tomato SGT1 protein encoding gene, the nucleotide sequence of which is shown in SEQ ID No:
2.
7. The biological agent for controlling tomato gray mold according to claim 6, characterized in that, The recombinant protein expression system is either a prokaryotic expression system or a eukaryotic expression system.
8. The biological agent for controlling tomato gray mold according to claim 7, 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.
9. 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.