Application of slerf098 protein and its encoding gene in regulating steroidal alkaloid content to affect resistance of tomato to botrytis cinerea

Abstract

This invention belongs to the field of plant genetic engineering technology, specifically relating to the application of the SlERF098 protein and its encoding gene in regulating the content of steroidal alkaloids and influencing resistance to gray mold in tomatoes. The application involves the encoding gene enhancing tomato resistance to gray mold by regulating the metabolism of steroidal glycoside alkaloids. Genome-wide association analysis was used to locate the SlERF098 protein in tomato... SlERF098 Genes, and constructed SlERF098 Overexpression lines and RNA interference lines were observed. Experiments confirmed this. SlERF098 It can significantly activate the metabolic pathway of SGAs. GAMEs Gene expression. Furthermore, overexpression... SlERF098 Genes can increase the content of SGAs metabolites and resistance to gray mold in tomatoes, while RNA interference lines show the opposite trend. This invention provides a new target for molecular breeding of disease resistance and research on the synergistic regulation of plant immunity and metabolism, and has important agricultural application value.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology. Specifically, it relates to the application of the SlERF098 protein and its encoding gene in regulating the content of steroidal alkaloids and influencing resistance to gray mold in tomatoes. Background Technology

[0002] tomato( Solanum lycopersicum Tomatoes are an important economic crop widely cultivated globally. Tomato production is frequently affected by the necrotrophic vegetative fungus *Botrytis cinerea* (…). Botrytis cinerea The global disease caused by gray mold (Botrytis cinerea) is a necrotrophic fungus that can infect more than 200 crop species. Traditional chemical control methods easily lead to drug resistance and pollute the environment, making the discovery of endogenous disease-resistant gene resources an urgent need.

[0003] Metabolomics studies of Solanaceae plants have revealed a class of nitrogen-containing toxic secondary metabolites with significant phylogenetic distribution characteristics—steroidal glycoalkaloids (SGAs). These compounds are glycosylated products of steroidal alkaloids (SAs), and more than 100 structurally unique SGAs have been identified from Solanaceae plants. The biosynthetic pathway of SGAs involves GAME (… Glycoalkaloid Metabolism The gene cluster encodes a multi-enzyme cascade reaction system, including key steps such as hydroxylation, oxidation, and glycosylation modification. This is produced in tomatoes. α -tomatine, dehydrotomatine, and esculeoside A are the main characteristic SGAs. α Both β-tomatine and dehydrotomatine are widely found in immature fruits, leaves, and stems. α -Tomatine possesses significant antibacterial and insecticidal activity, helping plants resist pathogens and pests. Dehydrotomatine also exhibits antibacterial and insecticidal activity. α ESculeoside A is an intermediate product in the conversion of β-tomatine to esculeoside A, a conversion that helps reduce the toxicity and bitterness of fruits. Escureeoside A is found in relatively high concentrations in mature fruits, exhibiting not only lower toxicity but also retaining some antibacterial activity. Therefore, elucidating the transcriptional regulatory mechanisms of SGA metabolism and linking them to disease resistance phenotypes is a key scientific issue in overcoming the bottlenecks in disease-resistant breeding.

[0004] When plants are infected by gray mold, transcription factors within the plant receive signals from receptor-like protein kinases, regulating the expression of downstream genes and the generation of immune responses. Ma Chuangju et al. found that FvWRKY50 plays a key regulatory role in the strawberry fruit defense system. FvWRKY50 can positively regulate strawberry resistance to gray mold and the host immune response mechanism. Pin Yao Huang et al. also found that AtERF19 in Arabidopsis can negatively regulate resistance to gray mold. However, whether ERF family transcription factors mediate disease resistance through the regulation of secondary metabolites (such as SGAs), especially their effects on the tomato-gray mold interaction, has not been reported. Therefore, identifying its function in improving tomato gray mold resistance is of great significance for breeding new disease-resistant tomato varieties. Summary of the Invention

[0005] The technical problem to be solved by this invention is to provide an AP2 / ERF transcription factor SlERF098 and reveal its regulation of plant functions. GAMES The molecular mechanism by which the expression of [a substance] enhances plant resistance to gray mold.

[0006] To address the above problems, the present invention provides the following technical solution:

[0007] The first aspect of this invention provides an application for increasing protein content and / or activity, said application being any of the following:

[0008] A1) Application in improving resistance to gray mold in tomatoes;

[0009] A2) Application in cultivating tomatoes resistant to gray mold;

[0010] A3) Application in breeding tomatoes for resistance to gray mold or in improving tomato germplasm resources resistant to gray mold;

[0011] The protein, named SlERF098, meets the following conditions:

[0012] B1) The amino acid sequence of this protein is SEQ ID No. 2;

[0013] B2) A fusion protein with the same function is obtained by attaching a tag to the N-terminus and / or C-terminus of B1).

[0014] To facilitate the purification or detection of proteins in B1), a tag protein can be attached to the amino or carboxyl terminus of the protein, which consists of the amino acid sequence shown in SEQ ID No. 2 in the sequence listing.

[0015] The tagged proteins include, but are not limited to: GST (glutathione thiotransferase) tagged protein, His6 tagged protein (His-tag), MBP (maltose-binding protein) tagged protein, Flag tagged protein, SUMO tagged protein, HA tagged protein, Myc tagged protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomer red fluorescent protein), or AviTag tagged protein.

[0016] In this article, substances that increase the activity and / or content of the protein may be substances that promote or enhance gene expression, wherein the gene encodes the protein SlERF098.

[0017] The substance that promotes or enhances gene expression can be any of the biological materials described in E1)-E3) of this document.

[0018] A second aspect of the present invention provides the application of biomaterials related to the protein SlERF098, wherein the application may be any of the following:

[0019] D1) Application in improving resistance to gray mold in tomatoes;

[0020] D2) Application in the cultivation of tomatoes resistant to gray mold;

[0021] D3) Application in breeding tomatoes for resistance to gray mold or in improving tomato germplasm resources resistant to gray mold;

[0022] The biomaterial is any one of the following E1) to E7):

[0023] E1) Nucleic acid molecules that promote or enhance the expression of the gene encoding the protein;

[0024] E2) An expression cassette containing the nucleic acid molecules described in E1);

[0025] E3) A recombinant vector containing the nucleic acid molecule described in E1), or a recombinant vector containing the expression cassette described in E2);

[0026] E4) Recombinant microorganisms containing the nucleic acid molecules described in E1), or recombinant microorganisms containing the expression cassette described in E2), or recombinant microorganisms containing the recombinant vector described in E3);

[0027] E5) A transgenic plant cell line containing the nucleic acid molecule described in E1), or a transgenic plant cell line containing the expression cassette described in E2), or a transgenic plant cell line containing the recombinant vector described in E3;

[0028] E6) Transgenic plant tissue containing the nucleic acid molecules described in E1), or transgenic plant tissue containing the expression cassette described in E2);

[0029] E7) A transgenic plant organ containing the nucleic acid molecule described in E1) or a transgenic plant organ containing the expression cassette described in E2).

[0030] The nucleotide sequence shown in SEQ ID No. 1 is the nucleotide sequence of the gene encoding protein SlERF098 (CDS), with a length of 459 bp.

[0031] A third aspect of the present invention provides a method for cultivating plants resistant to gray mold, the method comprising increasing the content and / or activity of the protein SlERF098 in the target plant to obtain a disease-resistant plant with higher resistance to gray mold than the target plant.

[0032] In the above method, the increase in the content and / or activity of the protein SlERF098 in the target plant is achieved by increasing the expression level of the gene encoding the protein in the target plant.

[0033] In the above method, increasing the expression level of the gene encoding the protein SlERF098 in the target plant involves constructing a plant expression vector containing SEQ ID NO:1 using transgenic technology. This increases the expression level of the gene encoding the protein SlERF098 in the target plant genome, significantly increasing the content of the metabolites of steroidal glycoalkaloids, tomatidine, α-tomatine, and hydroxytomatine, thereby enhancing the plant's resistance to gray mold.

[0034] Any of the plants mentioned above can be plants of the genus Tomato.

[0035] The technical steps to achieve the present invention are as follows:

[0036] Using forward genetics and genome-wide association analysis (mGWAS), a significant locus (0969459154) was found on chromosome 9 in the metabolite Hydroxytomatidine. P =2.414421489E -20 Further analysis revealed that the location was tomato. SlERF098 Genes, and through construction SlERF098 overexpression lines ( SlERF098-OE ) and RNA interference strains ( SlERF098-kd Overexpression was found SlERF098 It can significantly activate SGAs metabolic pathways. GAMES Gene expression can be increased, and the production of SGA metabolites such as tomatidine and tomato can be enhanced.α β-tomatine and hydroxytomatine content and disease resistance-related genes SlAPX, SlCAT1, SlPI-II, SlPR1a and SlPR2 The expression of this gene enhances the tomato's resistance to gray mold, confirming the biological function and application pathway of this gene.

[0037] Compared with the prior art, the present invention has the following effects:

[0038] This invention demonstrates through transgenic experiments that the transcription factor SlERF098 can directly bind to key genes involved in the synthesis of tomato steroidal alkaloids (SGAs). GAMES The promoter region of the virus is activated, thereby significantly promoting its transcriptional expression. α - Tomatine ( α The biosynthesis of antimicrobial metabolites such as β-tomatine and tomatidine can effectively enhance the resistance of tomatoes to Botrytis cinerea (a fungus that causes bacterial growth). Botrytis cinerea ( ) disease resistance.

[0039] This invention not only provides a highly efficient target for molecular breeding of tomatoes to resist gray mold, but also opens up a new path for the synergistic improvement of crop disease resistance and fruit quality. The strategy proposed in this invention to enhance crop stress resistance by regulating the content of SGAs metabolites not only provides a theoretical basis for enhancing gray mold resistance using natural plant antimicrobial metabolites, but also provides an innovative approach for breeding high-quality, disease-resistant tomato varieties.

[0040] The present invention will be further described below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0041] Picture 1 To locate via mGWAS SlERF098 Gene: Picture 1 In the figure, A represents the association analysis between the metabolite content of SGAs as phenotypic data and the genotypic data of 329 tomato population materials. P= 2.414421489E -20 ); Picture 1 In the figure, B represents the transcriptional activation activity analysis of transcription factor SlERF098.

[0042] Picture 2 for SlERF098 Construction and identification of transgenic materials: Picture 2 A in the text is SlERF098-kd RNA interference (RNAi) target sites of the material; Picture 2 B in SlERF098 Gene overexpression lines ( SlERF098-OE ) and RNA interference strains ( SlERF098-kdExpression levels of ) were detected; results were based on three replicates, and error bars represent standard deviation (SD). ** indicates P A value less than 0.01 indicates that *** P The value is less than 0.001.

[0043] Picture 3 for SlERF098 Expression levels and main component contents of genes related to the SGAs synthesis pathway in transgenic materials: Picture 3 A in the diagram represents the biosynthetic pathway of SGAs. Picture 3 B in SlERF098 Expression levels of genes related to the SGAs synthesis pathway in transgenic plants; Picture 3 C in the text is SlERF098 Detection of the content of major SGA metabolites in transgenic plants; results were based on three replicates, error bars represent standard deviation (SD), * indicates P A value less than 0.05 indicates that ** P A value less than 0.01 indicates that *** P A value less than 0.001 indicates that **** P The value is less than 0.0001.

[0044] Picture 4 for SlERF098 It has been reported that genes can directly bind to the SGAs synthesis pathway. GAMES Gene promoters: Picture 4 In this context, A represents transcription factor SlERF098 and... GAMES Yeast monohybridization results of genes; Picture 4 B in the text represents the transcription factor SlERF098 binding. GAMES Emulsion migration arrest analysis (EMSA) of gene promoters. Picture 4 C in the text represents transcription factors SlERF098 and GAMES A schematic diagram of the gene luciferase complementation (LUC) system; Picture 4 In the diagram, D represents the experimental results of plot C; the results are based on three replicates, the error bars represent the standard deviation (SD), and * indicates... P A value less than 0.05 indicates that ** P A value less than 0.01 indicates that *** P A value less than 0.001 indicates that **** P The value is less than 0.0001.

[0045] Picture 5 for SlERF098 Genes positively regulate resistance to gray mold in tomatoes: Picture 5 A in the text represents the addition of Tomatidine and α -Inhibition diagram of Tomatine's PDA medium against Botrytis cinerea; Picture 5 B in the figure represents the WT and 5 days after inoculation with Botrytis cinerea. SlERF098 Phenotype of leaves of transgenic materials; Picture 5 C in the figure represents the internal reference gene of *Botrytis cinerea* 5 days after inoculation. BcActin and Botrytis cinerea cutinase gene BcCutA The amount of expression; Picture 5 In the figure, D represents the change in the expression level of resistance genes in WT, OE, and kd lines 5 days after inoculation with gray mold; the results are based on three replicates, and the error bars represent the standard deviation (SD). * indicates P A value less than 0.05 indicates that ** P A value less than 0.01 indicates that *** P A value less than 0.001 indicates that **** P The value is less than 0.0001.

[0046] Picture 6 As an introductory medium SlERF098 -pDonR207 plasmid map.

[0047] Picture 7 as an overexpression vector SlERF098 -pEAQ-HT-DEST2 plasmid map.

[0048] Picture 8 RNA interference vector SlERF098 -pK7GWIWG2R(II) plasmid map. Detailed Implementation

[0049] The following embodiments define the present invention and describe its application in isolating clones containing...

[0050] SlERF098

[0051] DNA fragments of the complete coding region of the gene, and verification

[0052] SlERF098

[0053] Methods for understanding gene function. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were all purchased from commercial sources.

[0054] Preparation of reagents and culture media:

[0055] (1) Abbreviations for reagents and solutions: The abbreviations of plant hormones used in the culture medium in this invention are as follows: KN (Kanamycin); SPM (Spectinomycin); KT (Kinetin); IAA (Indole-3-acetic acid); 2,4-D (2,4-Dichlorophenoxyacetic acid); AS (Acetosringone); DMSO (Dimethyl Sulfoxide); Tim (Timentin); ZR (trans-Zeatin-riboside); IBA (3-Indolebutyric acid); KH2PO4 (Potassium dihydrogen phosphate); MS (Murashige & Skoog).

[0056] (2) Main solution formulation:

[0057] 1) Preparation of MS liquid culture medium

[0058] MS Base salt 4.33 g;

[0059] MS Vitamin 0.1031 g;

[0060] 30 g of sucrose;

[0061] Dissolve at room temperature and bring the volume to 1000 mL with distilled water.

[0062] 2) ZR stock solution, IBA stock solution 2 g / L; IAA stock solution, KH2PO4, 2,4-D stock solution, KT stock solution 1 g / L; Tim stock solution 320 g / L; KN stock solution, SPM stock solution 50 g / L; AS stock solution 200 mM / L. All require filtration and sterilization.

[0063] (3) Culture medium formulation for tomato genetic transformation

[0064] 1) Pre- / co-culture medium

[0065] 1 L of MS liquid culture medium;

[0066] Agar powder 6 g;

[0067] ZR 0.5 mL;

[0068] IAA 0.1 mL;

[0069] Adjust the pH to 5.8, bring the volume to 1000 mL, and seal for sterilization.

[0070] 2) Screening culture medium

[0071] 1 L of MS liquid culture medium;

[0072] Agar powder 6 g;

[0073] ZR 0.5 mL;

[0074] IAA 0.1 mL;

[0075] Tim 0.6 mL;

[0076] KN / SPM 0.5 mL;

[0077] Adjust the pH to 5.8, bring the volume to 1000 mL, and seal for sterilization.

[0078] 3) Rooting medium

[0079] 1 L of MS liquid culture medium;

[0080] Plant-based gel (gum powder) 2.5 g;

[0081] IBA 0.5 mL;

[0082] Tim 0.5 mL;

[0083] KN / SPM 0.5 mL;

[0084] Adjust the pH to 5.8, bring the volume to 1000 mL, and seal for sterilization.

[0085] 4) YEB medium

[0086] Yeast extract 1 g;

[0087] Tryptone 5 g;

[0088] Beef extract 5 g;

[0089] 5 g of sucrose;

[0090] MgSO4•7H2O 0.5 g;

[0091] Agar powder 15 g;

[0092] Adjust the pH to 7.4, bring the volume to 1000 mL, and seal for sterilization.

[0093] Example 1: SlERF098 The discovery and localization of genes

[0094] The applicant conducted metabolite assays on 329 tomato germplasm resources from around the world, performed statistical analysis on the data, and selected varieties with significant differences in the biosynthesis of steroidal glycoside alkaloids (SGAs) for genome-wide association analysis. The results revealed a significant locus (0969459154) on chromosome 9 for the metabolite Hydroxytomatidine. P =2.414421489E -20 Further analysis revealed a transcription factor annotated as belonging to the AP2 / ERF family approximately 63 kb from this SNP; this gene was named [gene name missing]. SlERF098 Preliminary findings suggest that this gene may affect the biosynthesis of tomato SGAs. Picture 1 (A in the text). To detect whether the transcription factor SlERF098 has transcriptional activation activity, a [missing information - likely a transcriptional algorithm] was first constructed. SlERF098 The recombinant plasmid pGBKT7, along with the negative control pGBKT7 and the positive control pGBKT7-p53, was transformed into Y2HGold yeast strain. The results showed that on SD / -Trp-deficient medium, the transformed plasmid... SlERF098 Yeast cells containing pGBKT7, pGBKT7, and pGBKT7-p53 can all grow normally; in SD / -Trp-His+ α -gal-deficient culture medium, SlERF098 Yeast cells containing pGBKT7 and the positive control pGBKT7-p53 can grow normally, and the colonies are blue, while the negative control cannot grow. This indicates that the transcription factor SlERF098 has transcriptional activation activity. Picture 1 (B in the middle).

[0095] Example 2: SlERF098 Construction and transformation of gene overexpression vectors and RNA interference vectors

[0096] The construction methods for overexpression vectors and RNA interference vectors are as follows: The PCR products obtained by amplification are ligated into the pDonR207 entry vector using the BP reaction of GATEWAY cloning technology. Picture 6 ), screen for positive clones and confirm them by sequencing, and obtain SlERF098 The full-length cDNA of the gene was then ligated into the overexpression vector pEAQ-HT-DEST2 and the RNA interference vector pK7GWIWG2R(II) via the LR reaction of GATEWAY cloning technology. Picture 2 (A) The applicant will name the correctly sequenced clone... SlERF098-pEAQ-HT-DEST2 and SlERF098 -pK7GWIWG2R(II).

[0097] Will SlERF098 -pEAQ-HT-DEST2 ( Picture 7 )and SlERF098 -pK7GWIWG2R(II) Picture 8 It is used for transformation. That is, it is introduced into the tomato variety MicroTom through the Agrobacterium-mediated tomato genetic transformation system. After pre-culture, infection, co-culture, screening for resistant callus, differentiation, rooting, transplanting, and identification, transgenic plants are obtained.

[0098] Specific steps: (1) Soaking: Soak several seeds of the tomato variety MicroTom in water for 3-4 hours in advance, then pour out the water and add 75% alcohol for 45 seconds for disinfection, rinse five times with sterile water, add 20% NaClO for 18 minutes (in the dark), after disinfection, wash the seeds five times with sterile water, soak for about two minutes each time, pour out the sterile water, absorb the water slightly, and then transfer to 1 / 2 MS germination medium for dark culture; (2) Cutting seedlings for pre-culture: When the seeds germinate and grow to have two mature cotyledons, cut off the leaf tip and leaf base with scissors, place the cotyledons upside down with the back of the leaves facing up on the pre-culture medium, and pre-culture in the dark for about 1-2 days; (3) Agrobacterium culture: Add 20 μL of EHA105 competent cells (from Shanghai Weidi Biotechnology, commercial strain) to the constructed vector, place on ice and stand for 5 minutes, transfer to liquid nitrogen for 5 minutes, then water bath at 37℃ for 5 minutes, and finally place on ice for 5 minutes and add 100 μL YEB liquid medium, incubated on a shaker at 28°C for 1 h, the bacterial suspension was spread onto YEB solid plates with resistance, and incubated at 28°C for 1-2 days. After single colonies appeared, the colonies were transferred to YEB liquid medium and incubated on a shaker at 28°C for 12 h; (4) Agrobacterium infection and co-culture: the Agrobacterium suspension was adjusted to OD using liquid MS medium. 600 =0.2-0.3, gently shake the cotyledons in Agrobacterium suspension for 12-15 min, then transfer them to sterilized filter paper to dry, and place them on co-culture medium in the dark for 2 days; (5) Screening culture: transfer the cotyledons on the co-culture medium to the screening medium, change the medium every two weeks until adventitious buds grow from the callus tissue; (6) Rooting: cut off the adventitious buds, remove all the surrounding callus tissue, keep them as intact as possible, and transfer them to the rooting medium (insertion medium) for rooting culture; (9) Transplanting: select healthy plants that have grown roots and transplant them into nutrient soil. Avoid a large loss of water for 5 days after transplanting. After the plants grow normally, conduct identification.

[0099] The specific steps for identification are as follows:

[0100] (1) RNA extraction: RNA samples from tomato tissue were extracted using a Trizol kit (purchased from TransGen Biotech, Beijing). Fresh tomato leaf tissue was placed in a 2 mL centrifuge tube containing small steel balls and then frozen in liquid nitrogen. The centrifuge tubes containing the tomato samples were then ground into tissue powder at 30 Hz for 40 s in a pre-cooled grinder. The ground centrifuge tubes were then returned to liquid nitrogen. 1 mL of Trizol was added to each centrifuge tube, and the mixture was vortexed thoroughly. 200 μL of chloroform was added in a fume hood, followed by vigorous shaking for 15 s, and then the mixture was allowed to stand at 4°C for 10 min. 500 μL of isopropanol was added to a prepared 1.5 mL RNA-specific centrifuge tube. The sample, which had been allowed to stand at 4°C, was centrifuged at 12,000 rpm for 10 min at 4°C. 500 μL of the supernatant was aspirated and added to a 1.5 mL centrifuge tube containing 500 μL of isopropanol. Gently invert the container 4-5 times and incubate at 4°C for 10 min. Then centrifuge at 12000 rpm for 10 min at 4°C. Remove the supernatant, add 700 μL of 75% ethanol, vortex thoroughly until the precipitate floats, and centrifuge at 13000 rpm for 5 min at 4°C. Remove the supernatant. Incubate in a clean bench for approximately 10 min. Then add 20-40 μL of RNA lysis buffer and incubate at 60°C for 10 min for later use.

[0101] (2) RNA reverse transcription and real-time quantitative PCR (qRT-PCR): The RNA sample dissolved in water was subjected to one-step reverse transcription using the Tolo Harbour Script All-in-one RT EasyMix for PCR kit. The RNA sample concentration was first determined and then uniformly adjusted to 2500 ng. Then, the MIX was prepared as follows: 4 μL of 5×All-in-one RT buffer, 1 μL of All-in-one Enzyme Mix, and 5 μL of RNase-free ddH2O.

[0102] Gene expression levels were detected using quantitative real-time PCR (qRT-PCR). The reaction mixture consisted of 5 μL of 2xQ3 SYBR qPCR Master mix (Vazyme), 0.2 μL of qPCR F / R Primer, 2 μL of cDNA template, and 2.6 μL of ddH2O. SlactinAs an internal reference gene, the detection primer sequences were Slactin-qRT-F: 5'-GCCAAAGAAGATCAAGCACA-3' and Slactin-qRT-R: 5'-TCAGCATT AGGGCACTCCTT-3'. The relative expression levels were calculated using the 2-ΔΔCT method.

[0103] Use GraphPad Prism 8 software to process data and create graphs. Picture 2 B in the text is SlERF098 Gene expression levels, results showed SlERF098 exist SlERF098-OE The expression level was significantly increased in the plants, SlERF098-kd The expression level in the plant was significantly reduced.

[0104] Example 3: SlERF098 Expression levels and main substance content of genes related to SGAs synthesis pathway in transgenic materials

[0105] RNA and metabolic samples were collected from 6-week-old T2 generation transgenic materials. RNA extraction and reverse transcription were performed using the same methods as in Example 2. The study aimed to detect the published biosynthetic pathways of SGAs in the transgenic materials. GAMES The gene expression level is determined by the same procedure as in Example 2.

[0106] Picture 3 In the diagram, A represents the biosynthetic pathway of steroidal alkaloids (SGAs) in tomatoes: starting with cholesterol (the basic precursor for the synthesis of steroidal compounds), it is metabolized into dehydrotomatidine; dehydrotomatidine further reacts to produce tomatidine; tomatidine is further modified to synthesize... α - Tomatine ( α -tomatine) — This is the core SGA (sulfuric acid glycosides) of tomatoes in their immature stage. It has a bitter taste and some toxicity, and is used to defend plants against pests and diseases; as tomatoes ripen... α - Tomatine is enzymatically modified and eventually converted into esculeside A—the main SGAs in ripe tomatoes, which are non-bitter, low in toxicity, and make tomatoes edible. Picture 3 The B in the text is displayed SlERF098-OE and SlERF098-kd In genetically modified materials GAMES Significant differences exist in gene expression levels. Compared to wild-type WT, SlERF098-OE The main components involved in the biosynthesis of SGAs in plants GAMES Genes, including those involved in tomatidine biosynthesis GAME6, GAME11, GAME4, GAME12, GAME25 and participation α-tomatine biosynthesis GAME 1, Game 17, Game 18, Game 2 and promotes the biosynthesis of hydroxytomatine GAME31 And promotes the biosynthesis of esculeoside A Game 36, Game 40, Game 5 The expression levels were all significantly upregulated. SlERF098 The genes do not indiscriminately activate GAME family genes, but rather specifically regulate genes at "key nodes" in SGA synthesis, demonstrating their specific function. The main genes involved in SGA biosynthesis... GAMES Genes in SlERF098-kd The opposite trend was observed in the plants. Metabolic samples were freeze-dried for approximately one week, then ground into powder. 70% methanol was added at a ratio of sample:extract = 0.1 g:1 mL, and the mixture was vortexed to mix. The mixture was placed on ice and vortexed every 10 minutes for a total of three times. After overnight incubation, the mixture was vortexed again and centrifuged at 12000 rpm at 4°C. The supernatant was collected, filtered, and then analyzed by LC-MS. The results showed that ( Picture 3 (C) Compared with wild type, tomatidine, tomatidine, α The content of -tomatine and hydroxytomatine is in SlERF098-OE Significantly increased in plants, while SlERF098-kd tomatidine in the plant α The contents of β-tomatine and hydroxytomatine showed a significant decreasing trend.

[0107] Example 4: SlERF098 It has been reported that genes can directly bind to the SGAs synthesis pathway. GAMES gene promoters

[0108] SlERF098 Genes and GAMES Promoter binding was verified using yeast mono-hybridization, EMSA, and LUC.

[0109] Specific steps: Yeast hybridization: First, use the Gateway system to... GAME6, GAME11, GAME5, GAME1, GAME17 and GAME31 The promoter sequence was linked to the pHIS2 vector. SlERF098The full-length coding sequence was ligated into a pGADT7 vector. Different combinations of yeast were co-transformed into competent yeast cells Y187. After co-transformation, the cells were cultured in SD / -Leu-Trp deficient medium for 2 days. The resulting positive yeast clones were then added to SD / -Leu-Trp liquid medium and cultured with shaking for another day. After harvesting, the yeast culture was diluted with sterile water to a uniform OD600 of 10⁻⁶. 0 10 -1 10 -2 10 -3 Using a sterilized pipette tip, 2.5 μL of yeast culture was drawn up and different combinations of yeast mono- and heterozygous cultures were spotted onto SD / -Leu-Trp solid medium and SD / -Leu-Trp-His solid medium in a clean bench. The media were then sealed with sealing film and incubated in a 30℃ incubator for about 3 days to observe the results.

[0110] EMSA: SlERF098 The coding sequence was ligated into the PGEX-6P-1 protein expression vector via the Gateway system, transformed into BL21(DE3) intestinal competent cells, and expressed at low temperature under 1M IPTG induction. The expressed protein was purified using GST-tagged protein crosslinking purification resin. The protein was analyzed using the PlantTFDB website (http: / / planttfdb.gao-lab.org / ). GAMES Startup sub-region SlERF098 To detect potential binding sites, specific probes labeled with FAM fluorescent markers, competitive probes without markers, and mutant probes with base substitutions in the core conserved sequence were constructed. The purified protein and different probes were incubated in the dark for 30 min, followed by electrophoresis at 90 V for 50 min on a 3% non-denaturing polyacrylamide gel, and finally imaged using an infrared laser imaging system (ODYSSEY-FC, Gene Company Limited).

[0111] LUC: will GAME6, GAME11, GAME5, GAME1, GAME17 and GAME31 The promoter sequence 2 kb upstream of the start codon is ligated to the pH2GW7 vector. SlERF098The ligated vector was ligated into the pEAQ-HT-DEST2 vector. The ligated vector was transformed into Agrobacterium GV3101 competent cells and cultured at 30°C for 2-3 days. Single colonies were then transferred to liquid YEB medium for further amplification. The bacterial suspension was collected and diluted to OD600 of 1 using different combinations, and allowed to stand for 2-3 hours. Different combinations of Agrobacterium suspension were then injected into the dorsal cells of tobacco plants using a 1 mL syringe. The injected tobacco plants were placed in an incubator and cultured in the dark for 24 hours followed by 12 hours of light. Samples were then collected and ground into tissue powder using a pre-cooled grinder at 30 Hz for 40 seconds. The ground samples were promptly returned to liquid nitrogen. Extraction was performed according to the instructions of the Dual-Luciferase Reporter Assay System kit, and the results were measured using a microplate reader. The relative expression level of the reporter gene is expressed as the LUC / REN ratio.

[0112] The results of the yeast single-hybrid experiment showed that pGADT7- SlERF098 With pHIS2- proGAME6 pHIS2- proGAME11 pHIS2- proGAME5 pHIS2- proGAME1 pHIS2- proGAME17 pHIS2- proGAME31 The combination can be grown on SD / -Leu / -Trp / -His medium, i.e. SlERF098 Can be combined GAME6, GAME11, GAME5, GAME1, GAME17 and GAME31 promoter ( Picture 4 (A) EMSA results indicate that SlERF098 Can be combined with GAMES At the promoter, the band intensity decreased after adding a competing probe. However, after adding a mutant probe, the band intensity of the complex returned to its original state. Picture 4 (B in the text). LUC experimental results show that... SlERF098 Can be activated GAME6, GAME11, GAME5, GAME1, GAME17 and GAME31 promoter activity ( Picture 4 (C and D in the above). The above experiment proves SlERF098 By combining with the SGAs biosynthetic pathway GAMES Gene promoters regulate their expression levels, thereby affecting the content of related SGAs in tomatoes.

[0113] Example 5: SlERF098 Genes positively regulate resistance to gray mold in tomatoes

[0114] SlERF098 The regulation of resistance to tomato gray mold by genes was verified through metabolite inhibition experiments, gray mold spore inoculation treatment, and qRT-PCR.

[0115] Metabolite antibacterial experiment: Tomatidine and other metabolites were added to PDA medium at different concentration gradients of 0 ppm, 20 ppm, 50 ppm, 100 ppm and 200 ppm. α -Tomatine, and take pieces of gray mold of the same size and place them in solutions of different concentration gradients of tomatidine and α - The growth of Botrytis cinerea blocks was observed in PDA medium containing Tomatine.

[0116] Botrytis cinerea spore inoculation treatment: After culturing the strain on PDA medium for 7 days, 5 mL of sterile water was added to a culture dish covered with mycelia in a clean bench. The mycelia and spores were scraped off by bending a sterilized pipette tip and poured into a sterilized 50 mL centrifuge tube. The tube was thoroughly shaken to separate the spores, mycelia, and residual culture medium. The mixture was then poured into a new 50 mL centrifuge tube equipped with a filter head to filter out the mycelia and residual culture medium. Sterile water was added to bring the volume to 10 mL. The tube was centrifuged at 3500 rpm for 10 min, the supernatant was discarded, and 5 mL of a prepared spore resuspension (0.8 g maltose, 0.2 g peptone, and sterile water to a volume of 20 mL) was added. The mixture was thoroughly mixed by pipetting and observing the spore concentration under an optical microscope using a hemocytometer. The spore concentration was adjusted to 2 × 10⁻⁶ using the spore resuspension. 5 Prepare spores / mL for later use. Next, line a 25 cm × 25 cm petri dish with sterile, water-soaked tissue paper to maintain humidity. Select leaves of similar age and size, and add 5 μL of spore suspension to each side of the midrib on the underside of the leaf, ensuring the spore suspension is added at the same location on each leaf. Maintain humidity by spraying sterile water in a misting manner. Seal the dish with sealing film and incubate at 25°C. Observe for disease development after approximately 5 days, replenishing water as needed to keep the leaves moist.

[0117] Experimental results showed that in the initial stage, the bandwidth size caused by all concentration gradients of *Gnaphalium affine* blocks was approximately 6-7 mm. Observations after 48 h of treatment revealed that the SGA metabolites Tomatidine and... α Tomatine showed the most significant inhibitory effect on Botrytis cinerea at concentrations of 100 ppm and 200 ppm. The bandwidth of PDA media containing 20 ppm, 50 ppm, 100 ppm, and 200 ppm of tomatine reached 21 mm, 16 mm, 12.3 mm, and 9.7 mm, respectively. αThe bandwidths of the PDA medium for -Tomatine reached 22.3 mm, 19 mm, 16.7 mm, and 9.6 mm, respectively. This indicates that the SGA metabolites Tomatidine and α -Tomatine showed an inhibitory effect on gray mold with increasing concentration and time. Picture 5 (A) The results of Botrytis cinerea spore inoculation treatment showed that, phenotypic observation 5 days after inoculation with Botrytis cinerea spore suspension revealed that, compared with the control wild-type tomato (WT), SlERF098-OE The area of ​​tomato lesions on the plants was significantly reduced, and the diameter of the lesions also expanded less. SlERF098-kd The area of ​​lesions on the surface of the tomato plants increased significantly, and the water-soaked lesions were also larger. Picture 5 (B in the text). qRT-PCR results showed that compared with the control WT, SlERF098-OE In the plant BcCutA and BcActin The expression level of was significantly reduced, while SlERF098-kd In BcCutA and BcActin The expression level was significantly increased ( Picture 5 (C in the text). Compared to WT, disease resistance-related genes SlAPX, SlCAT1, SlPI-II, SlPR1a and SlPR2 exist SlERF098-OE Significant activation was observed in the plants. SlERF098-kd The plants, however, show the opposite trend. Picture 5 (D) in the above experiment proves SlERF098 It can positively regulate the resistance of tomatoes to gray mold.

Claims

1. The application of increasing protein expression levels in enhancing resistance to gray mold in tomatoes, characterized by: The amino acid sequence of the protein is shown in SEQ ID NO.

2.

2. A method for cultivating plants resistant to gray mold, characterized in that, The method includes increasing the expression level of the protein described in claim 1 in the target plant to obtain a disease-resistant plant with higher resistance to gray mold than the target plant, wherein the plant is tomato.

3. The method according to claim 2, characterized in that, The improvement in the expression level of the protein described in claim 1 in the target plant is achieved by increasing the expression level of the gene encoding the protein in the target plant.

4. The method according to claim 3, characterized in that, The method of increasing the expression level of the protein-coding gene in the target plant is to use transgenic technology to increase the expression level of the protein-coding gene.

5. The method according to claim 4, characterized in that, The method of using transgenic technology to increase the expression level of the protein-coding gene involves constructing a plant expression vector to increase the expression level of the protein-coding gene in the genome of the target plant. The plant expression vector contains nucleotides with sequences as shown in SEQ ID NO:

1.

6. The method according to claim 5, characterized in that, The increased expression of the gene encoding the protein significantly increased the content of tomato alkaloid, tomato alkaloid, α-tomato alkaloid, and hydroxytomato alkaloid, thereby enhancing the plant's resistance to gray mold.

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