Application of SlERF098 protein and coding gene thereof in regulating steroidal alkaloid content to affect botrytis cinerea resistance

Abstract

The invention belongs to the technical field of plant genetic engineering, and particularly relates to an application of SlERF098 protein and a coding gene thereof in regulating and controlling steroid alkaloid content to influence botrytis cinerea resistance. According to the application, the resistance of tomatoes to gray mold is enhanced by regulating and controlling the metabolism of steroid glycoalkaloid through the coding gene. The tomato SlERF098 gene is positioned through whole genome association analysis, and an overexpression strain and an RNA interference strain of the tomato SlERF098 gene are constructed. Experiments prove that the SlERF098 can obviously activate the expression of the GAMEs gene in the SGAs metabolic pathway. In addition, overexpression of the SlERF098 gene can improve the content of SGAs metabolites in tomatoes and the resistance to gray mold, while RNA interference strains present an opposite trend. The invention provides a new target for research on disease-resistant molecular breeding and plant immunity-metabolism coordinated regulation and control, 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-resistant phenotypes is a key scientific issue in overcoming the bottlenecks in disease-resistant breeding.

[0004] When the plant is infected by Botrytis cinerea, the transcription factor in the plant body will receive the signal of the receptor-like protein kinase, which will regulate the expression of downstream genes and the production of immune response. Ma Chuangju et al. found that FvWRKY50 has a key regulatory role in the defense system of strawberry fruit. FvWRKY50 can positively regulate the resistance of strawberry to Botrytis cinerea and the host immune response mechanism. Pin Yao Huang et al. also found that AtERF19 in Arabidopsis can negatively regulate the resistance to Botrytis cinerea. However, whether the ERF family transcription factor mediates disease resistance by regulating secondary metabolites (such as SGAs), especially the effect on tomato-Botrytis cinerea interaction, has not been reported. Therefore, identifying its function in improving tomato Botrytis cinerea resistance will have very important significance for breeding new disease-resistant tomato varieties. SUMMARY

[0005] The technical problem to be solved by the present application is to provide an AP2 / ERF transcription factor SlERF098 and to reveal the molecular mechanism of its regulation of the expression of GAMEs to enhance the resistance of plants to Botrytis cinerea.

[0006] To solve the above problems, the present application provides the following technical solutions: The present application provides the following technical solutions: A1) application in improving the resistance of tomato to Botrytis cinerea; A2) application in cultivating tomato resistant to Botrytis cinerea; A3) application in tomato Botrytis cinerea resistance breeding or tomato Botrytis cinerea resistance germplasm improvement; The protein is named SlERF098 and meets the following conditions: B1) the amino acid sequence is the protein of SEQ ID No. 2; B2) a fusion protein with the same function is obtained by connecting a tag to the N terminus and / or C terminus of B1).

[0007] In order to facilitate the purification or detection of the protein in B1), a tag protein can be connected to the amino terminal or carboxyl terminal of the protein consisting of the amino acid sequence shown in SEQ ID No. 2 in the sequence listing.

[0008] The tag proteins include, but are not limited to, a GST (glutathione S-transferase) tag protein, a His6 tag protein (His-tag), a MBP (maltose binding protein) tag protein, a Flag tag protein, a SUMO tag protein, a HA tag protein, a Myc tag protein, an eGFP (enhanced green fluorescent protein), an eCFP (enhanced cyan fluorescent protein), an eYFP (enhanced yellow green fluorescent protein), an mCherry (monomeric red fluorescent protein), or an AviTag tag protein.

[0009] Herein, the substance for improving the activity and / or content of the protein can be a substance for promoting or improving the expression of a gene encoding the protein SlERF098.

[0010] The substance for promoting or improving the expression of a gene can be specifically a biological material according to any one of E1) to E3) herein.

[0011] The second aspect of the present application provides an application of a biological material related to the protein SlERF098, which can be any one of the following: D1) an application in improving the resistance to gray mold disease of tomato; D2) an application in breeding tomato resistant to gray mold disease; D3) an application in breeding or improving the germplasm resource of tomato resistant to gray mold disease; The biological material can be any one of the following E1) to E7): E1) a nucleic acid molecule for promoting or improving the expression of a gene encoding the protein; E2) an expression cassette containing the nucleic acid molecule according to E1); E3) a recombinant vector containing the nucleic acid molecule according to E1), or a recombinant vector containing the expression cassette according to E2); E4) a recombinant microorganism containing the nucleic acid molecule according to E1), or a recombinant microorganism containing the expression cassette according to E2), or a recombinant microorganism containing the recombinant vector according to E3); E5) a transgenic plant cell line containing the nucleic acid molecule according to E1), or a transgenic plant cell line containing the expression cassette according to E2), or a transgenic plant cell line containing the recombinant vector according to E3); E6) a transgenic plant tissue containing the nucleic acid molecule according to E1), or a transgenic plant tissue containing the expression cassette according to E2); E7) a transgenic plant organ containing the nucleic acid molecule according to E1), or a transgenic plant organ containing the expression cassette according to E2).

[0012] 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.

[0013] 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.

[0014] 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.

[0015] 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.

[0016] Any of the plants mentioned above can be plants of the genus Tomato.

[0017] The technical steps to achieve the present invention are as follows: 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 ), found overexpression 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.

[0018] Compared with the prior art, the present application has the following effects: The present application proves by transgenic experiment that the transcription factor SlERF098 can directly bind to the promoter region of the key gene of tomato steroidal alkaloid (SGAs) synthesis GAMEs , activates its transcription expression, thereby significantly promoting α - the biosynthesis of tomato base (tomatine), tomatidine and other antibacterial metabolites, and the accumulation of these antibacterial substances can effectively enhance the disease resistance of tomato to Botrytis cinerea. α Botrytis cinerea

[0019] The present application not only provides an efficient target for tomato gray mold disease resistance molecular breeding, but also opens up a new path for the research of crop disease resistance and fruit quality improvement. The strategy of improving crop stress resistance by regulating the content of SGAs metabolites proposed in the present application not only provides a theoretical basis for enhancing gray mold resistance by using plant natural antibacterial metabolites, but also provides an innovative idea for breeding new varieties of high-quality disease-resistant tomatoes.

[0020] The present application will be further described below in conjunction with the accompanying drawings and examples. DETAILED DESCRIPTION

[0021] Figure 1 To locate the SlERF098 gene by mGWAS: Figure 1 A in the above formula is to use the content of SGAs metabolites as phenotype data for association analysis with the genotype data of 329 tomato population materials P= 2.414421489E -20 ); Figure 1 B in the above formula is the transcription activation activity analysis of the transcription factor SlERF098.

[0022] Figure 2 To construct and identify the transgenic materials: SlERF098 A in the above formula is the RNA interference (RNAi) target site of the material; Figure 2 B in the above formula is the expression amount detection of the overexpression strain SlERF098-kd ) and the RNA interference strain Figure 2 ) of the gene; the results are based on three repetitions, and the error line represents the standard deviation (SD), ** indicates that the SlERF098 value is less than 0.01, and *** indicates that the SlERF098-OE value is less than 0.001. SlERF098-kd P P

[0023] Figure 3 ​​​​​​SlERF098 Expression of genes related to SGAs synthesis pathway in transgenic materials and contents of main substances: Figure 3 A in FIG. 1 is a schematic diagram of the biosynthetic pathway of SGAs; Figure 3 B in FIG. 1 is SlERF098 Expression of genes related to SGAs synthesis pathway in transgenic plants; Figure 3 C in FIG. 1 is SlERF098 Detection of contents of main SGAs metabolites in transgenic plants; results are based on three replicates, error bars represent standard deviation (SD), * indicates P value less than 0.05, ** indicates P value less than 0.01, *** indicates P value less than 0.001, **** indicates P value less than 0.0001.

[0024] Figure 4 A in FIG. 2 is SlERF098 The promoter of the gene can directly bind to the SGAs synthesis pathway reported GAMEs gene: Figure 4 A in FIG. 3 is the transcription factor SlERF098 binding to GAMEs gene of yeast one-hybrid results; Figure 4 B in FIG. 3 is the transcription factor SlERF098 binding to GAMEs Gel shift retardation assay (EMSA) of the promoter of the gene; Figure 4 C in FIG. 3 is the transcription factor SlERF098 binding to GAMEs Luciferase assay (LUC) system schematic diagram of the gene; Figure 4 D in FIG. 3 is the experimental results of C figure; results are based on three replicates, error bars represent standard deviation (SD), * indicates P value less than 0.05, ** indicates P value less than 0.01, *** indicates P value less than 0.001, **** indicates P value less than 0.0001.

[0025] Figure 5 A in FIG. 4 is SlERF098 The gene positively regulates the resistance of tomato to gray mold: Figure 5 A in FIG. 5 is the inhibition diagram of PDA medium added with Tomatidine and α -Tomatine on gray mold; Figure 5 B in FIG. 5 is the phenotype of WT and SlERF098 transgenic material leaves after inoculation of gray mold for 5 days; Figure 5C in the figure is the expression level of the internal reference gene of Botrytis cinerea after 5 days of inoculation BcActin and the expression level of Botrytis cutinase gene BcCutA . Figure 5 D in the figure is the change of the expression level of the disease resistance gene of WT and OE and kd strains after 5 days of inoculation; the results are based on three repetitions, and the error line represents the standard deviation (SD), * represents P value less than 0.05, ** represents P value less than 0.01, *** represents P value less than 0.001, **** represents P value less than 0.0001.

[0026] Figure 6 is an entry vector SlERF098 -pDonR207 plasmid map.

[0027] Figure 7 is an overexpression vector SlERF098 -pEAQ-HT-DEST2 plasmid map.

[0028] Figure 8 is an RNA interference vector SlERF098 -pK7GWIWG2R (II) plasmid map. DETAILED DESCRIPTION

[0029] The following examples define the present application and describe the application in isolation and cloning of DNA fragments containing SlERF098 the complete coding segment of the gene, and the method for verifying SlERF098 the function of the gene. The experimental methods in the following examples are all conventional methods unless otherwise specified. The experimental materials used in the following examples are all purchased from commercial channels unless otherwise specified.

[0030] Preparation of reagents and culture medium used: (1) Abbreviations of reagents and solutions: The abbreviations of plant hormones used in the medium of the present application 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).

[0031] (2) Main solution formula: 1) Preparation of MS liquid medium MS Base salt 4.33 g; MS Vitamin 0.1031 g; Sucrose 30 g; Dissolve at room temperature and dilute with distilled water to 1000 mL.

[0032] 2) ZR stock solution, IBA stock solution is 2 g / L; IAA stock solution, KH2PO4, 2,4-D stock solution, KT stock solution is 1 g / L; Tim stock solution is 320 g / L; KN stock solution, SPM stock solution is 50 g / L; AS stock solution is 200 mM / L. All need to be sterilized by filtration.

[0033] (3) Medium formula for genetic transformation of tomato 1) Pre / co-culture medium MS liquid medium 1 L; Agar powder 6 g; ZR 0.5 mL; IAA 0.1 mL; Adjust pH to 5.8, dilute to 1000 mL, and sterilize by sealing.

[0034] 2) Selection medium MS liquid medium 1 L; Agar powder 6 g; ZR 0.5 mL; IAA 0.1 mL; Tim 0.6 mL; KN / SPM 0.5 mL; Adjust pH to 5.8, add water to 1000 mL, and sterilize.

[0035] 3) Rooting medium MS liquid medium 1 L; Plant gum 2.5 g; IBA 0.5 mL; Tim 0.5 mL; KN / SPM 0.5 mL; Adjust pH to 5.8, add water to 1000 mL, and sterilize.

[0036] 4) YEB medium Yeast extract 1 g; Tryptone 5 g; Beef extract 5 g; Sucrose 5 g; MgSO4•7H2O 0.5 g; Agar 15 g; Adjust pH to 7.4, add water to 1000 mL, and sterilize.

[0037] Example 1: SlERF098 Discovery and localization of genes Applicants conducted statistical analysis on the measurement data of 329 tomato germplasm resources from all over the world, selected varieties with significant differences in the biosynthesis of steroidal glycoside alkaloids SGAs, and performed whole genome association analysis. It was found that there was a significant locus of metabolite Hydroxytomatidine on chromosome 9 (0969459154, P =2.414421489E -20 ). Further analysis found that there was a transcription factor annotated as AP2 / ERF family about 63 Kb away from the SNP, and the gene was named SlERF098 . It is preliminarily considered that the gene may affect the biosynthesis process of tomato SGAs (A in Figure 1 ). In order to detect whether the transcription factor SlERF098 has transcription activation activity, first, the SlERF098The 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. Figure 1 (B in the middle).

[0038] Example 2: SlERF098 Construction and transformation of gene overexpression vectors and RNA interference vectors 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. Figure 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. Figure 2 (A) The applicant will name the correctly sequenced clone... SlERF098 -pEAQ-HT-DEST2 and SlERF098 -pK7GWIWG2R(II).

[0039] Will SlERF098 -pEAQ-HT-DEST2 ( Figure 7 )and SlERF098 -pK7GWIWG2R(II) Figure 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.

[0040] Specific steps: (1) Point seeding: the seeds of tomato variety MicroTom are counted and put in water for 3-4 h in advance, then the water is poured out and 75% alcohol is added for disinfection for 45 s, the seeds are washed with sterile water for five times, 20% NaClO is added for disinfection for 18 min (avoiding light), after disinfection, the seeds are washed with sterile water for five times, each time for about two minutes, then the sterile water is poured out, the seeds are slightly absorbed, and then transferred to the germination medium 1 / 2 MS for dark culture; (2) Cutting seedlings for pre-culture: when the seeds germinate and grow to two mature cotyledons, the leaf tip and leaf base are cut off with scissors, and the cotyledons are laid on the pre-culture medium with the back of the leaf facing up, and pre-cultured in the dark for about 1-2 days; (3) Agrobacterium culture: the constructed vector is added to 20 μL EHA105 competent cells (from Shanghai Weidi Biology, commercial strain), placed on ice for 5 min, transferred to liquid nitrogen for 5 min, then 37°C water bath for 5 min, finally placed on ice for 5 min, then added with 100 μL YEB liquid medium, 28°C shaking culture for 1 h, the bacterial liquid is coated on the resistant YEB solid plate, and cultured at 28°C for 1-2 days. After single colonies appear, the colonies are transferred to YEB liquid medium, and cultured at 28°C on a shaking table for 12 h; (4) Agrobacterium infection and co-culture: the suspension of Agrobacterium is adjusted to OD 600 =0.2-0.3 using liquid MS medium, the cotyledons are gently shaken in the Agrobacterium suspension for 12-15 min, then transferred to sterile filter paper to absorb, and placed in the dark for 2 days of co-culture medium; (5) Screening culture: the cotyledons on the co-culture medium are transferred to the screening culture medium, the medium is replaced every two weeks until callus grows adventitious buds; (6) Rooting: the grown adventitious buds are cut off, all the surrounding callus is removed, and the intact ones are transferred to the rooting medium (inserted into the medium) for rooting culture; (9) Transplanting: the healthy plants with roots are transplanted into nutrient soil, 5 days after transplanting, the water loss is avoided, and the plants are identified after normal growth.

[0041] The specific identification steps are as follows: (1) RNA extraction: Tomato tissue RNA samples were extracted using the Trizol kit (purchased from Beijing Zison Company). Fresh tomato leaf tissue was placed in a 2 mL centrifuge tube containing small steel beads, then placed in liquid nitrogen for freezing. The centrifuge tube containing the tomato sample was then placed in a pre-cooled grinder for 40 S treatment at 30 HZ to pulverize the tissue powder. The ground centrifuge tube was placed back in liquid nitrogen. Then 1 mL of Trizol was added to each centrifuge tube, mixed thoroughly, and then 200 μL of chloroform was added in a fume hood and shaken vigorously for 15 S. The sample was then placed at 4°C for 10 min. 500 μL of isopropanol was added to the prepared 1.5 mL RNA centrifuge tube. The sample was centrifuged at 4°C for 10 min at 12000 rpm. The supernatant was removed and 500 μL of isopropanol was added to the 1.5 mL centrifuge tube. Gently invert 4-5 times, and then place at 4°C for 10 min. Then centrifuge at 4°C for 10 min at 12000 rpm. Remove the supernatant and add 700 μL of 75% ethanol. Mix thoroughly until the precipitate floats, then centrifuge at 4°C for 5 min at 13000 rpm. Remove the supernatant and blow for about 10 min in a clean bench. Then add 20-40 μL of RNA dissolving solution, then 60°C water bath for 10 min and store for later use.

[0042] (2) RNA reverse transcription and real-time fluorescent quantitative PCR (qRT-PCR): The RNA sample dissolved in the water bath was subjected to one-step reverse transcription using the Tolo Harbor Script All-in-one RT EasyMix for PCR kit. The RNA sample concentration was determined and adjusted to 2500 ng. Then the MIX was configured as follows: 4 μL of 5x All-in-one RT buffer, 1 μL of All-in-one Enzyme Mix, and 5 μL of RNase-free ddH2O.

[0043] The expression level of the gene was detected by real-time fluorescent quantitative PCR (qRT-PCR) method, and the reaction system was as follows: 5 μL of 2x Q3 SYBR qPCR Master mix (purchased from Vazyme Company), 0.2 μL of qPCR F / R Primer, 2 μL of cDNA template, and 2.6 μL of ddH2O. The SlactinThe primer sequence for detecting the internal reference gene is Slactin-qRT-F: 5'-GCCAAAGAAGATCAAGCACA-3', Slactin-qRT-R: 5'-TCAGCATT AGGGCACTCCTT-3', and the experimental data is calculated by 2-ΔΔCT method to calculate the relative expression amount.

[0044] The data is processed and plotted using GraphPad Prism 8 software. Figure 2 B in the above formula (I) is SlERF098 The expression level of the gene, and the results show that SlERF098 In the SlERF098-OE plant, the expression amount is significantly increased, and in the SlERF098-kd plant, the expression amount is significantly decreased.

[0045] Example 3: SlERF098 Expression amount of SGAs synthesis pathway related genes and main substance content in transgenic materials The 6-week-old T2 generation transgenic materials are taken as samples, and RNA samples and metabolic samples are taken. The RNA sample extraction and reverse transcription are the same as in Example 2. The expression amount of the GAMEs gene in the published SGAs biosynthesis pathway in the transgenic material is detected, and the specific operation is the same as in Example 2.

[0046] Figure 3 A in the above formula (I) is the steroidal alkaloid SGAs biosynthesis pathway in tomato: taking cholesterol as the initial precursor (which is the basis for the synthesis of steroidal compounds), it is converted into dehydrotomatidine through metabolic reaction; dehydrotomatidine continues to react to generate tomatidine; tomatidine is further modified to synthesize α tomatidine α -tomatine—this is the core SGAs in the immature stage of tomato, which has bitterness, certain toxicity, and is used for plant defense against pests and diseases; with the maturation of tomato, α tomatidine is enzymatically modified and finally converted into esculeside A—this is the main SGAs in mature tomatoes, which has no bitterness, low toxicity, and makes tomatoes edible. Figure 3 B in the above formula (I) shows that SlERF098-OE and SlERF098-kd The expression level of the GAMEs gene in the transgenic material has a significant difference. Compared with the wild type WT, SlERF098-OE the main GAMEs gene involved in the biosynthesis of SGAs in the GAME6, GAME11, GAME4, GAME12, GAME25 plant, including α tomatidine biosynthesis-related α andGAME1, GAME17, GAME18, GAME2 and promotes the biosynthesis of hydroxytomatine GAME31 And promotes the biosynthesis of esculeoside A GAME36, GAME40, GAME5 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 ( Figure 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 plants α The contents of β-tomatine and hydroxytomatine showed a significant decreasing trend.

[0047] Example 4: SlERF098 It has been reported that genes can directly bind to the SGAs synthesis pathway. GAMEs gene promoters SlERF098 Genes and GAMEs Promoter binding was verified using yeast mono-hybridization, EMSA, and LUC.

[0048] 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. SlERF098 The 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.

[0049] 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).

[0050] 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. SlERF098 The 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.

[0051] The yeast one-hybrid experiment results show that pGADT7- SlERF098 , pHIS2- proGAME6 , pHIS2- proGAME11 , pHIS2- proGAME5 , pHIS2- proGAME1 , pHIS2- proGAME17 , pHIS2- proGAME31 can grow on the SD / -Leu / -Trp / -His medium, that is SlERF098 can bind to the promoters (A in GAME6, GAME11, GAME5, GAME1, GAME17 and GAME31 ). The EMSA results show that Figure 4 can bind to the promoters of SlERF098 , and the band is weakened after adding the competitive probe. However, the band intensity of the complex is restored to the original state (B in GAMEs ) after adding the mutant probe. The LUC experiment results show that Figure 4 can activate the activities of the promoters of SlERF098 and GAME6, GAME11, GAME5, GAME1, GAME17 (C and D in GAME31 ). The above experiments prove that Figure 4 regulates the expression amount of the SlERF098 gene by binding to the promoter of the GAMEs gene in the SGA biosynthetic pathway, thereby affecting the content of the related SGAs in tomatoes.

[0052] Example 5 SlERF098 Gene positively regulates the resistance of tomatoes to gray mold SlERF098 The regulation of the gene on the resistance of tomatoes to gray mold is verified through the metabolite antibacterial experiment, the spore inoculation treatment of gray mold, and the qRT-PCR.

[0053] Metabolite antibacterial experiment: 0 ppm, 20 ppm, 50 ppm, 100 ppm, and 200 ppm different concentration gradients of Tomatidine and Tomatine are added to the PDA medium, and the same size of the gray mold block is placed in the PDA medium added with different concentration gradients of Tomatidine and Tomatine to observe the growth of the gray mold block. α α

[0054] ​​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.

[0055] 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. α The 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. Figure 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-kdThe area of ​​lesions on the surface of the tomato plants increased significantly, and the water-soaked lesions were also larger. Figure 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 ( Figure 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. Figure 5 (D) in the above experiment proves SlERF098 It can positively regulate the resistance of tomatoes to gray mold.

Claims

1. Use for increasing the protein content and / or activity, characterized in that, The application is any one of the following: A1) application in improving tomato resistance to Botrytis cinerea; A2) application in cultivating tomato resistant to Botrytis cinerea; A3) application in breeding or improving tomato germplasm resources resistant to Botrytis cinerea; The protein meets the following conditions: B1) the amino acid sequence is the protein of SEQ ID No. 2; B2) a fusion protein with the same function obtained by connecting a tag to the N-terminus and / or C-terminus of B1).

2. Use of a biological material associated with the protein as claimed in claim 1, characterised in that, The application is any one of the following: D1) application in improving tomato resistance to Botrytis cinerea; D2) application in cultivating tomato resistant to Botrytis cinerea; D3) application in breeding or improving tomato germplasm resources resistant to Botrytis cinerea; The biological material is any one of the following E1) to E7): E1) a nucleic acid molecule promoting or improving the expression of the gene encoding the protein in claim 1; E2) an expression cassette containing the nucleic acid molecule of E1); E3) a recombinant vector containing the nucleic acid molecule of E1), or a recombinant vector containing the expression cassette of E2); E4) a recombinant microorganism containing the nucleic acid molecule of E1), or a recombinant microorganism containing the expression cassette of E2), or a recombinant microorganism containing the recombinant vector of E3); E5) a transgenic plant cell line containing the nucleic acid molecule of E1), or a transgenic plant cell line containing the expression cassette of E2), or a transgenic plant cell line containing the recombinant vector of E3); E6) a transgenic plant tissue containing the nucleic acid molecule of E1), or a transgenic plant tissue containing the expression cassette of E2); E7) a transgenic plant organ containing the nucleic acid molecule of E1), or a transgenic plant organ containing the expression cassette of E2).

3. Use according to claim 2, characterized in that, The nucleotide sequence encoding the protein is shown in SEQ ID No.

1.

4. A method of breeding a plant resistant to gray mold disease, characterized by, The method comprises increasing the content and / or activity of the protein in claim 1 in the target plant, to obtain a plant with higher resistance to Botrytis cinerea than the target plant, and the plant is tomato.

5. The method of claim 4, wherein, The increase in the content and / or activity of the protein in claim 1 in the target plant is achieved by increasing the expression amount of the gene encoding the protein in the target plant.

6. The method of claim 5, wherein, The increase in the expression amount of the gene encoding the protein in the target plant is achieved by using transgenic technology to increase the expression amount of the gene encoding the protein in claim 1 in the genome of the target plant.

7. The method of claim 6, wherein, The increase in the expression amount of the gene encoding the protein in the genome of the target plant is achieved by using a plant expression vector comprising SEQ ID NO:

1.

8. The method of claim 6, wherein, The increase in the expression amount of the gene encoding the protein significantly increases the content of steroidal glycoside alkaloid metabolites such as solanidol, solanidane, alpha-solanine and hydroxysolanine, thereby enhancing the resistance of the plant to Botrytis cinerea.

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