Application of tomato SlFMO1 gene in regulation and control of resistance of tomato fruits to gray mold

Abstract

The invention discloses an SlFMO1 gene and application of the SlFMO1 gene in regulation and control of resistance of tomato fruits to gray mold, and belongs to the technical field of gene engineering. A CRISPR / Cas9 gene editing technology is utilized, firstly, a CRISPR / Cas9-gRNA vector is constructed to accurately edit the SlFMO1 gene in tomatoes, GV3101 agrobacterium tumefaciens is utilized to conduct mediated transformation on the tomatoes, kanamycin serves as a resistance marker to conduct screening to obtain SlFMO1-deleted mutant tomatoes, it is found that when the SlFMO1 gene in the tomatoes is deleted, the resistance of tomato fruits to gray mold is remarkably reduced, and when the SlFMO1 gene in the tomatoes is deleted, the resistance of the tomato fruits to gray mold is remarkably reduced. The result shows that the SlFMO1 gene positively regulates and controls the resistance of the tomato to the gray mold. The discovery provides a new target for subsequent improvement of tomato varieties and creation of materials for resisting gray mold of fruits, and has important application prospects in molecular breeding for improving disease resistance of tomato fruits.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to tomatoes. SlFMO1 Application of genes in regulating the resistance of tomato fruits to gray mold. Background Technology

[0002] Tomatoes, as an important horticultural crop widely cultivated globally, are often severely threatened in terms of yield and quality by fungal diseases. Among the many fruit and vegetable diseases, those caused by the necrotrophic fungus Botrytis cinerea (Gastrodia elata) are particularly serious. Botrytis cinerea Gray mold, caused by *Botrytis cinerea*, is particularly prominent. *Botrytis cinerea* exhibits typical characteristics of thriving in low temperatures and high humidity, allowing it to rapidly infect and multiply plant tissues via conidia. When tomato stems, leaves, and fruits are infected, initial lesions are often water-soaked, followed by rapid softening and rotting of the tissue. In later stages, a typical gray mold layer forms on the surface of the affected area, leading to complete loss of commercial value. Furthermore, gray mold is highly susceptible to large-scale outbreaks and severe rot during post-harvest storage, cold chain logistics, and transportation of tomatoes. Statistics show that this disease causes post-harvest losses of 10% to 15% in tomatoes, resulting in significant economic losses to the fresh tomato supply chain and the tomato industry.

[0003] Currently, the control of tomato gray mold in production still heavily relies on chemical fungicides. Although chemical control is fast-acting and convenient, its long-term overuse has not only caused environmental pollution and food safety risks, but also accelerated the emergence of drug-resistant variants of gray mold, leading to a decline in the efficacy of existing chemical agents. Given the inherent limitations of current chemical control technologies, in-depth exploration of tomato's own disease-resistant gene resources, elucidating its molecular mechanisms of resistance to Botrytis cinerea infection, and thus creating green and safe new strategies for gray mold control, as well as cultivating new tomato varieties with excellent resistance, is not only an urgent need to ensure the sustainable development of the tomato industry, but also a key technological bottleneck that urgently needs to be overcome in the fields of plant functional genomics and bio-breeding. Summary of the Invention

[0004] The technical problem to be solved by this invention is: to provide tomatoes SlFMO1 The application of genes in regulating the resistance of tomato fruits to gray mold can help solve the problem of large losses caused by postharvest gray mold in tomatoes.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is: to provide a method for regulating the resistance of tomato fruits to gray mold. SlFMO1 Gene, SlFMO1 The nucleotide sequence of the gene is as follows: a. A nucleotide sequence as shown in SEQ ID NO: 1; or b. A nucleotide sequence that hybridizes under stringent conditions with the nucleotide sequence shown in SEQ ID NO: 1 and encodes a functional protein that regulates resistance to gray mold in tomato fruit; or c. A nucleotide sequence as shown in SEQ ID NO: 1 with one or more nucleotides deleted and / or added, encoding a sequence that encodes a functional protein that regulates the resistance of tomato fruit to gray mold.

[0006] The present invention also discloses a method comprising the above. SlFMO1 Recombinant gene expression vectors.

[0007] The present invention also discloses an engineered bacterium comprising the above-mentioned recombinant expression vector.

[0008] The present invention also discloses the above-mentioned SlFMO1 Application of genes, recombinant expression vectors, or engineered bacteria in regulating the resistance of tomato fruits to gray mold.

[0009] Based on the above technical solution, the present invention can be further improved as follows: Furthermore, by knocking out SlFMO1 Genes can be used to regulate the resistance of tomato fruits to gray mold.

[0010] Furthermore, by knocking out SlFMO1 Genes showed that the diameter of gray mold lesions in tomato fruits increased significantly, and the defensive genes in tomato fruits during storage... SlPR1 The expression of [a substance] was significantly reduced, and the activities of ascorbic acid peroxidase and peroxidase were both significantly reduced.

[0011] This invention also discloses a method for reducing the resistance of tomato fruits to gray mold, comprising the above-mentioned... SlFMO1 Gene knockout vectors were transferred into Agrobacterium and then used to infect tomato explants. After cultivation, the diameter of gray mold lesions in tomato fruits significantly increased, and the defensive gene in the tomato fruits increased during storage. SlPR1 The expression of [a specific enzyme] was significantly reduced, and the activities of ascorbic acid peroxidase and peroxidase were also significantly reduced, which decreased the resistance of tomato fruit to gray mold.

[0012] The beneficial effects of this invention are as follows: This invention utilizes CRISPR / Cas9 gene editing technology to first construct a CRISPR / Cas9-gRNA vector to target the gene editing process in tomatoes. SlFMO1 Genes were precisely edited, and tomatoes were transformed using Agrobacterium tumefaciens GV3101. Hygromycin was used as a resistance marker for selection. SlFMO1 Missing mutant tomatoes were found to contain... SlFMO1 When the gene is deleted, the resistance of tomato fruit to gray mold is significantly reduced. Phenotypic analysis revealed that the knockout gene... SlFMO1After gene modification, the diameter of botrytis cinerea lesions in tomato fruits increased significantly, and the defensive gene in tomato fruits increased during storage. SlPR1 The expression of [a substance] was significantly reduced, and the activities of ascorbic acid peroxidase and peroxidase were both significantly reduced, indicating [a deficiency]. SlFMO1 The gene positively regulates the resistance of tomatoes to gray mold. This discovery provides a new target for subsequent improvement of tomato varieties and the creation of materials with fruit resistant to gray mold, and has important application prospects in molecular breeding to improve the disease resistance of tomato fruits. Attached Figure Description

[0013] Figure 1 for SlFMO1 A schematic diagram of gene editing in gene knockout strains; Figure 2 Wild type and SlFMO1 Comparison of lesion diameters on green-ripe and red-ripe tomato fruits from gene knockout strains; Figure 3 Wild type and SlFMO1 Comparison of lesion diameter data for green-ripe tomato fruits from gene knockout strains; Figure 4 Wild type and SlFMO1 Comparison of lesion diameter data for red ripe tomato fruits from gene knockout strains; Figure 5 Wild type and SlFMO1 In the green ripe tomato fruits of gene knockout strains SlPR1 Gene expression comparison; Figure 6 Wild type and SlFMO1 In the ripe red tomato fruits of gene knockout strains SlPR1 Gene expression comparison; Figure 7 Wild type and SlFMO1 Comparison of peroxidase (POD) activity in green-ripe tomato fruits from gene knockout lines; Figure 8 Wild type and SlFMO1 Comparison of peroxidase (POD) activity in ripe red tomato fruits from gene knockout lines; Figure 9 Wild type and SlFMO1 Comparison of ascorbate peroxidase (APX) enzyme activities in green ripe tomato fruits of gene knockout lines; Figure 10 Wild type and SlFMO1 Comparison of ascorbate peroxidase (APX) enzyme activity in ripe red tomato fruits from gene knockout strains. Detailed Implementation

[0014] The specific embodiments of the present invention are described below to facilitate understanding of the invention by those skilled in the art. Unless otherwise specified, specific conditions are applied according to conventional conditions or the manufacturer's recommendations. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various modifications are obvious as long as they fall within the spirit and scope of the invention as defined and determined by the appended claims. All inventions utilizing the concept of this invention are protected.

[0015] SlFMO1 The nucleotide sequence of the gene is as follows:

[0016] SlFMO1 The amino acid sequence of the protein encoded by the gene is as follows: MAIIRSKIGIIGGGISGIAAAKQLAKYDPMVFEATECVGGVWKHCSYRSTKLQTPRCDYEFSDFPWTQRDNTNFPTYEEILDYLYSYAKHFDVLKFFNFNSKVVEIKFVGDREMNYFGEYGSLLTGNPV WEVAVRNNQSQTLQWYAFDFVVMCTGKYGDIPNIPNFPPKKGPKEFNGQVLHTLDYSKLDQEASTKLMKGKKVVVGYKKSAIDLAVECAEANQGPEGQPCTMVVRTLHWTVPHYSIWGLPFYLFYSTR SSQFLHERPNQGLFRTLLCHMLSPMRKAASKLIESYLEWKLPLEKYGLKPEHPFEEDYASCQMAILPENFFTEADKGKIMFKRTSKWWFWEGGVEFEDNTKLEADVVILATGFDGKKKIKAILPDPFRS LVEFPSGMIPLYRGTIHPLIPNMAFVGYLESVSNLHSAEIRCIWLSRLVDDLFKLPSVEKMLEQITQEMEIMKRTTRFYKRNCISTFSINHSDEICQEMGWQVWRKTNWLAEAFSPYNSQDYAEEK (SEQ ID NO: 2).

[0017] The primer sequences used in the following examples are shown in Table 1.

[0018] Table 1 Primer sequence listing

[0019] Example 1 Construction SlFMO1 CRISPR / Cas9 gene knockout system 1. Target sequence selection: The target site was selected using the CRISPR / Cas9 target online design website (https: / / chopchop.cbu.uib.no). The selected target site sequence was T1 (nucleotides 98-118 of SEQ ID NO: 1). SlFMO1 Location in genes such as Figure 1 As shown.

[0020] 2. Based on the target site sequence of gRNA, design gRNA amplification primers gRNA-F (SEQ ID NO: 3) and gRNA-R (SEQ ID NO: 4).

[0021] 3. Construction of gRNA expression vector: (1) Using plasmids as amplification templates, PCR amplification was performed using gRNA-F and gRNA-R, followed by purification by agarose gel electrophoresis to obtain gRNA fragments. The PCR amplification system is shown in Table 2.

[0022] Table 2 PCR amplification system

[0023] The PCR amplification program was as follows: 98℃ for 3 min pre-denaturation; 98℃ for 15 s denaturation, 55℃ for 10 s annealing, 72℃ for 1 min extension, 35 cycles; store at 4℃.

[0024] PCR products were recovered using the Omega Gel Extraction Kit from Beijing Yuntai Biotechnology Co., Ltd.

[0025] 4. The above PCR product was ligated to a CRISPR vector using a cut-and-ligate method to obtain a2-hyg-gfp-nCas9-tomato U6- SlFMO1 Expression carrier. The connection system is shown in Table 3.

[0026] Table 3 PCR Ligation System

[0027] The reaction conditions were: 37℃ for 5 min, 16℃ for 5 min, 50 cycles; 65℃ for 5 min; and stored at 16℃.

[0028] Example 2 Obtained SlFMO1 Gene knockout line plants 1. Screening of engineered bacteria (1) Transformation of Escherichia coli Place a2-hyg-gfp-nCas9-tomato U6- SlFMO1 The expression vector was transformed into E. coli DH5α and then plated on LB medium containing 50 mg / mL kanamycin. Single colonies were picked the next day for PCR amplification to check for successful ligation. The PCR primers were upstream primer F (SEQ ID NO: 5) and downstream primer R (SEQ ID NO: 6). The PCR amplification band was approximately 600 bp.

[0029] (2) Transformation of Agrobacterium Select a single colony that was correctly amplified by PCR, extract plasmids, and sequence them. The sequence of sequencing primer R is shown in SEQ ID NO: 7.

[0030] The plasmid with completely correct sequencing was transformed into Agrobacterium GV3101 and plated on LB solid medium containing 50 mg / mL rifampin and 50 mg / mL kanamycin. After a single colony grew, colony PCR was performed to verify whether it was a positive clone.

[0031] 2. Tomato conversion (1) Seed disinfection In a clean bench, prepare two 50mL test tubes, one with 75% alcohol and the other with 10-20% available chlorine sodium hypochlorite, to sterilize tomato seeds. First, soak the seeds in 75% alcohol for 2 minutes, then rinse three times with sterile water. Next, soak them in 10% (v / v) sodium hypochlorite solution for 10 minutes, then rinse six to seven times with sterile water. Inoculate the seeds into pre-prepared, sterilized 1 / 2 MS (Murashige and Skoog Medium) medium at room temperature, inoculating 25 seeds into each medium. Seal the medium and incubate in the dark. After 2-3 days of dark incubation at 25℃, transfer to light (16h light + 8h dark) for 5-7 days. Closely observe the cotyledon angle and true leaf growth on the 3rd-4th day under light.

[0032] (2) Cut cotyledons Add 0.2 mg / L of 2,4-D (2,4-Dichlorophenoxyacetic acid) and 0.1 mg / L of KT (Kinetin) to MS1 liquid medium (M519 medium). Remove seedlings with fully extended cotyledons, cut off the leaves, and then cut off a small cotyledon with a wound from each leaflet. Sow the cotyledons into MS1 medium, seal the seedlings, and culture them under light (16 h light + 8 h darkness) for 24-36 h.

[0033] (3) Infection Transformed Agrobacterium GV3101 was inoculated into LB medium containing 50 μg / mL rifampin (Rif) and 50 μg / mL kanamycin (Kan) and cultured at 28°C for 2 days. Single colonies were then picked and cultured in 2 mL of LB medium containing 50 μg / mL Rif and 50 μg / mL Kan in the dark at 28°C and 200 rpm for 1.5 days. Then, 100 μL of the bacterial culture was added to 10 mL of fresh LB medium containing 50 μg / mL Rif, 50 μg / mL Kan, and 100 μM acetylsyl syringone (AS) and cultured overnight at 28°C and 200 rpm. The cells were then collected by centrifugation at 4000 rpm for 10 min at room temperature, and centrifuged at 4000 rpm for 8 min in 2 mL of MS medium containing 1.0 mg / L indoleacetic acid (IAA) and 1.75 mg / L zeatin nucleoside (ZR). Finally, the bacterial cells were resuspended in 2 mL of MS salt medium, and the OD was adjusted. 600 Up to 0.3-0.4.

[0034] After suspending the bacterial culture, pour the culture into MS1 medium (MS liquid medium + 1.0 mg / L IAA + 1.75 mg / L ZR + 100 μM AS) containing 15 mL of infection solution, and adjust to OD200. 600 A suitable infection concentration is 0.3-0.4. Use tweezers to pick up explants from MS1 medium, place them in the infection solution with the wound side down, wrap the medium with aluminum foil, and incubate at 28°C and 80 rpm for 10 minutes. Use sterilized tweezers to pick up the infected explants, blot off excess bacterial solution on filter paper, and then place them on MS1 ​​medium. Incubate in the dark for 2 days.

[0035] (4) Transfer to MS2 callus culture After 48 hours of infection, the cotyledons were placed in MS2 medium (MS + 1.0 mg / L IAA + 1.75 mg / L ZR + 50 mg / L Kan + 200 mg / L Tim (Temperature)) and cultured at 26°C (16 hours light) / 18°C ​​(8 hours darkness) for 2 weeks.

[0036] (5) Transplantation of MS2 resistant buds Transfer the cotyledons that have grown a lot of callus to MS2 medium and culture for 1-2 weeks.

[0037] (6) Transplanting to MS3 for rooting culture The cotyledons of the resistant buds that have grown larger are transferred out, the callus is removed in a large dish, and then they are transferred to MS3 medium (MS + 0.2 mg / L IAA + 50 mg / L Kan + 200 mg / L Tim) and cultured for 2 weeks.

[0038] (7) Transplanting Transfer the mature and well-rooted seedlings out of the culture medium, add nutrient soil to sterilized pots, gently lift the plants from the MS3 medium, and wash the solid culture medium off the tomato roots in a slow stream of water to prevent fungal growth on the roots later. Dig a small hole in the center of the nutrient soil, gently place the tomato roots around the hole, press firmly, clearly label the variety type and insert it into the soil, cover each plant with a plastic cup, and place it in the culture room for cultivation.

[0039] 3. Identification of genetically modified plants Eight tomato seedling lines were harvested through tomato transformation. PCR was used to detect whether the target gene had been edited in these seedlings. Sequencing primers were designed based on the target site location, namely CR- SlFMO1 -F (SEQ ID NO: 8) and CR- SlFMO1 -R (SEQ ID NO: 9).

[0040] Genomic DNA was extracted from the leaves of eight transgenic lines and from the leaves of wild-type plants, and then PCR amplification was performed. The PCR reaction system is shown in Table 4.

[0041] Table 4. PCR reaction system for identifying transgenic edited plants

[0042] The PCR amplification program was as follows: 95℃ for 3 min pre-denaturation; 95℃ for 15 s denaturation, 55℃ for 10 s annealing, 72℃ for 1 min extension, 35 cycles; store at 4℃.

[0043] The PCR products were sequenced using CR- sequencing primers. SlFMO1 -F and CR- SlFMO1 -R. By comparing the sequencing results, plants that had undergone editing and those that had not were identified. The edited plants were determined to be either homozygous or heterozygous. If homozygous, phenotypic observation and recording were performed; if heterozygous, propagation was carried out to await the next generation of plants before sequencing. Seeds from the edited tomato T0 generation were collected, planted, and T1 generation seedlings were obtained for PCR testing. Further testing and screening of homozygous edited tomato plants followed.

[0044] like Figure 1 As shown, based on the sequencing results analysis, two homozygous lines were selected and denoted as CR- SlFMO1 -1 and CR- SlFMO1 -2, CR- SlFMO1 -1 represents the addition of a base G, CR- SlFMO1 -2 represents the addition of a base T.

[0045] Example 3 SlFMO1Gene regulation of tomato gray mold resistance (1) Select healthy wild-type (WT) calves with uniform color, size, and maturity at the green-ripe and red-ripe stages. SlFMO1 Gene knockout line (CR-) SlFMO1 -1 and CR- SlFMO1 -2) Soak the tomato fruit in a 1% NaClO solution for 2 minutes, then wash it 3 times with sterile water and let it air dry on filter paper.

[0046] (2) Remove the Botrytis cinerea culture dish from the 25℃ incubator. In a clean bench, use a pipette tip to draw liquid PDA and drop it onto PDB medium. Use a spreader to scrape spores and draw up the spore suspension. Filter the suspension through four layers of gauze. Take 10 μL and drop it onto a hemocytometer. Observe under a microscope and count the number of spores. Dilute according to the required concentration. During this process, add the surfactant Tween-80 at a ratio of 0.05% to finally prepare a 1×10⁻⁶ solution. 5 Spore concentration per mL.

[0047] (3) Place the dried fruit on a sterile workbench, prick the surface of the fruit with a sterilized needle (2 mm deep, 2 mm in diameter), and leave for 20 min; then add 5 μL of Botrytis cinerea spore suspension (1×10⁻⁶). 5 (Spores / mL) are applied to each wound.

[0048] (4) All treatments were cultured in an incubator at 25°C for 4 days. The diameter of the lesions was measured periodically, and samples were taken for the determination of relevant physiological indicators.

[0049] Each treatment group contained 15 fruits, with 3 replicates.

[0050] Depend on Figures 2 - 4 It can be seen that, compared to the wild type, SlFMO1 In gene knockout line plants, the diameter of gray mold lesions on tomato fruits at both the green-ripe and red-ripe stages was significantly increased, and the defense gene was also enhanced during storage. SlPR1 The expression of was significantly reduced ( Figure 5 and Figure 6 Its ascorbate peroxidase activity and peroxidase activity were both significantly reduced. Figures 7 - 10 ).illustrate SlFMO1 The reduced resistance of tomato fruits to gray mold due to gene deletion indicates that... SlFMO1 Genes positively regulate the resistance of tomatoes to gray mold.

Claims

1. Tomato SlFMO1 The application of genes in regulating resistance to gray mold in tomato fruits is characterized by, The SlFMO1 The nucleotide sequence of the gene is as follows: a. A nucleotide sequence as shown in SEQ ID NO: 1; b. A nucleotide sequence that, under stringent conditions, corresponds to the nucleotide sequence shown in SEQ ID NO: 1 and encodes a functional protein that regulates resistance of tomato fruit to gray mold; or c. The nucleotide sequence shown in SEQ ID NO: 1 is modified by substitution, deletion and / or addition of one or more nucleotides, and / or by missense mutation of one or more base pairs, and encodes a sequence that has a functional protein that regulates the resistance of tomato fruit to gray mold.

2. A recombinant expression vector, characterized in that, Includes the claims 1 SlFMO1 Gene.

3. A recombinant microorganism, characterized in that, It includes the recombinant expression vector as described in claim 2.

4. The claim 1 SlFMO1 The application of genes, the recombinant expression vector of claim 2, or the recombinant microorganism of claim 3 in regulating the resistance of tomato fruits to gray mold.

5. The application according to claim 4, characterized in that, By knocking SlFMO1 Genes can be used to regulate the resistance of tomato fruits to gray mold.

6. The application according to claim 5, characterized in that, By knocking SlFMO1 Genes showed that the diameter of gray mold lesions in tomato fruits increased significantly, and the defensive genes in tomato fruits during storage... SlPR1 The expression of [a substance] was significantly reduced, and the activities of ascorbic acid peroxidase and peroxidase were both significantly reduced.

7. A method for reducing the resistance of tomato fruit to gray mold, characterized in that, Will include the contents of claim 1 SlFMO1 Gene knockout expression vectors were transferred into Agrobacterium and then used to infect tomato explants. After cultivation, the diameter of botrytis cinerea lesions in tomato fruits significantly increased, and the defensive gene in the tomato fruits increased during storage. SlPR1 The expression of [a specific enzyme] was significantly reduced, and the activities of ascorbic acid peroxidase and peroxidase were also significantly reduced, resulting in a weakening of the resistance of tomato fruits to gray mold.

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