Gene SlC3H73 for regulating drought resistance and disease resistance of tomato, application and prepared material

Abstract

The invention discloses a gene SlC3H73 for regulating and controlling drought resistance and disease resistance of tomatoes, application and a prepared material, relates to the technical field of biology, and particularly discloses an SlC3H73 gene with a nucleotide sequence shown as SEQ ID No. 1 and capability of coded protein of the gene in regulating and controlling the drought resistance and disease resistance of the tomatoes and application of the gene in improving the drought resistance and disease resistance of the tomatoes. The invention also discloses a tomato germplasm prepared by the application. Functional verification is carried out on the SlC3H73 gene by utilizing a transgenic technology, the drought resistance and disease resistance of tomatoes are remarkably improved by overexpressing the SlC3H73 gene, tomato germplasm with strong drought resistance and disease resistance is created, and the gene has important significance for further understanding a regulation mechanism of drought resistance and disease resistance of tomatoes and improving the quality of tomatoes.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a gene that regulates drought resistance and disease resistance in tomatoes. SlC3H73 Materials for application and preparation. Background Technology

[0002] tomato( Solanum lycopersicum Tomatoes, belonging to the genus *Solanum* of the Solanaceae family, are annual or perennial herbaceous plants. Their fruits are rich in nutrients and have a unique flavor, making them one of the three major globally traded vegetables widely cultivated worldwide. my country is not only a major consumer of tomatoes but also a producer and exporter of tomato products, holding a significant position in the global tomato product market. As an important warm-climate and water-loving vegetable crop, tomatoes are often affected by adverse conditions such as drought and pests during cultivation, resulting in slow seed germination, low germination rates, stunted plant growth, flower and fruit drop, and in severe cases, even plant death (Dong et al., 2024). When subjected to these stresses, plants can resist or adapt to adverse environments through signal transduction and corresponding molecular regulatory networks (Kim et al., 2024). Numerous studies have shown that transcription factors can transmit perceived adverse signals downstream by regulating the expression of certain target genes, ultimately enabling plants to respond to drought and pest stress (Baillo et al., 2019).

[0003] C3H is a member of the zinc finger protein (ZFP) family, containing a typical zinc finger motif (Znf) consisting of three carbon atoms and one hydrogen atom coordinated to zinc ions. Unlike other zinc finger proteins, C3H zinc finger proteins regulate gene expression by binding to mRNA (Bogamuwa et al., 2014). Previous studies have shown that C3H can regulate plant growth and development, such as flower development (Li, 2001), overwintering behavior (Schmitz et al., 2005), secondary cell wall biosynthesis (Chain et al., 2015), and leaf senescence (Kong et al., 2006). Simultaneously, C3H can also regulate plant responses to stresses, such as drought stress (Guo et al., 2022; Tian et al., 2024), salt stress (Jan et al., 2013; Zhang et al., 2023; Seok et al., 2024), and pathogen invasion (Guo et al., 2009). Deng Jie et al. used bioinformatics methods to identify 47 C3H members from the whole tomato genome and analyzed their sequence characteristics and tissue expression, but the biological functions of these C3H proteins are unclear.

[0004] Improving the adaptability of tomatoes has always been a pressing technical challenge that researchers have been eager to solve.

[0005] The relevant references are as follows.

[0006] 1. Dong Shuchao, Ling Jiayi, Zhao Liping, et al. Research progress on the regulation of drought resistance in tomato by transcription factors [J]. Jiangsu Agricultural Sciences, 2024, 51(09):9-16. 2. Kim, JS, Kidokoro, S., Yamaguchi-Shinozaki, K.&Shinozaki, K. Regulatory networks in plant responses to drought and cold stress. Plant Physiology 195, 170-189 (2024). 3. Baillo, EH, Kimotho, RN, Zhang, ZB&Xu, P. TranscriptionFactors Associated with Abiotic and Biotic Stress Tolerance and TheirPotential for Crops Improvement. Genes-Basel 10, (2019). 4. Bogamuwa, SP&Jang, JC Tandem CCCH Zinc Finger Proteins in Plant Growth, Development and Stress Response. Plant and Cell Physiology 55,1367-1375, (2014). 5. Li JJ, Jia XD, and Chen XM HUA1 a regulator of stamen andcarpel identities in arabidopsis, codes for a nuclear RNA binding protein. Plant Cell, 13(10): 2269-2281, (2001). 6. Schmitz R.J., Hong L., Michaels S., and Amasino R.M. FRIGIDA-ESSENTIAL1 interacts genetically with FRIGIDA and FRIGID-ALIKE1 to promotethewinter-annual habit of Arabidopsis thaliana. Development, 132(24): 5471-5478, (2005). 7. Kong Z.S., Li M.N., Yang W.Q., Xu W.Y., and Xue Y.B. A novelnuclear-localized CCCH-type zinc fnger protein OsDOS is involved indelayingleaf senescence in rice, Plant Physiol., 141(4): 1376-1388, (2006). 8. Jan A., Maruyama K., Todaka D., Kidokoro S., Abo M., Yoshimura E.,Shinozaki K., Nakashima K., and Yamaguchi-Shinozaki K.b. OsTZF1, a CCCH-tandemzinc finger protein, confers delayed senescence and stress tolerance inrice by regulating stress related genes, Plant Physiol., 161(3): 1202-1216(2013). 9. Guo Y.H., Yu Y.P., Wang D., Wu C.A., Yang G.D., Huang J.G., andZheng C.C. GhZFP1 a novel CCCH-type zinc finger protein from cotton, enhancessaltstress tolerance and fungal disease resistance in transgenic tobacco byinteracting with GZIRD21A and GZIPR5, New Phytol., 183(1): 62-75 (2009). 10. Chai, G. H. et al. Arabidopsis C3H14 and C3H15 have overlappingroles in the regulation of secondary wall thickening and anther development.Journalof Experimental Botany 66, 2595-2609, (2015). 11. Seok, H. Y. et al. AtC3H3, an Arabidopsis Non-TZF Gene, EnhancesSalt Tolerance by Increasing the Expression of Both ABA-Dependent and -IndependentStress-Responsive Genes. Int J Mol Sci 25, (2024). 12. Tian, Y. A. et al. The zinc-finger transcription factor ZFP8negatively regulates the drought stress response in Arabidopsis thalianabyinhibiting the transcriptional activity of ABF2. Journal of plantphysiology 303, (2024). 13. Zhang, Q. et al. The CCCH-Type Zinc-Finger Protein GhC3H20Enhances Salt Stress Tolerance in Arabidopsis thaliana and Cotton through ABASignalTransduction Pathway. Int J Mol Sci 24, (2023). 14. Guo, CM et al. RNA Binding Protein OsTZF7 Traffics Between theNucleus and Processing Bodies / Stress Granules and Positively RegulatesDroughtStress in Rice. Frontiers in Plant Science 13, (2022). Summary of the Invention

[0007] The objective of this invention is to address the technical problem of improving the adaptability of tomatoes by providing a gene that regulates the drought and disease resistance of tomatoes. SlC3H73 Materials for application and preparation. This invention utilizes transgenic technology to... SlC3H73 Gene function was validated through overexpression. SlC3H73 Genes significantly enhance the drought and disease resistance of tomatoes, and the creation of tomato germplasm with strong drought and disease resistance is of great significance for further understanding the regulatory mechanisms of drought and disease resistance in tomatoes and for improving tomato quality.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: A gene that regulates drought resistance and disease resistance in tomatoes SlC3H73 Its nucleotide sequence is shown in SEQ ID NO.1.

[0009]

[0010] The genes that regulate drought resistance and disease resistance in tomatoes SlC3H73 The amino acid sequence of the encoded protein is shown in SEQ ID NO.2.

[0011] SEQ ID No. 2: Amino acid sequence of C3H73 transcription factor: MVKLHFLSPLYIQTDHHRLNFKSPPLFQLAMIGERSRRIPTVDVPPWSYSDDQTANMQFMLSPSTNSISTPNNFNFSNFIVEDDYPLFC NDAEDIDEFELENREIELPVDVYACDNFRMYEFKVRRCGRGRSHDWTECPYVHPGEKARRRDPRKFHYSGTACPEFRKGNCNRGDSCEYAHGVFECWLHPGRYRTQ PCKDGGNCKRRVCFFAHSPEQLRVLGPGGSGSSPGPGSDSPRRYVKGLHFVSSPESSSPPSESPPMSPMTANSFTSLSRSVGSNSVSEVMASLRQLQLNRLNSMPSSSWNVQMGSPVFGSPRRPVIRPGFCSLPATPSGDPTRPGNRCFDLWENEEEPVMERVESGRDLRVKMFERLSKENPLDDPENPNPNINSGSGLNPDVGWVSDLIQ.

[0012] A method for regulating tomato performance, through SlC3H73 Gene overexpression to produce SlC3H73 Gene overexpression in tomatoes enhances their drought resistance and / or disease resistance; or through... SlC3H73 Gene mutations, producing SlC3H73 Mutant tomatoes reduce the drought resistance and / or disease resistance of tomatoes.

[0013] pass SlC3H73 Gene overexpression to produce SlC3H73 Gene overexpression in tomatoes increases the wax content in tomato leaves, thereby improving the drought resistance of tomatoes.

[0014] pass SlC3H73 Gene mutations, producing SlC3H73 Mutant tomatoes reduce drought resistance by decreasing the wax content in tomato leaves.

[0015] pass SlC3H73 Gene overexpression to produce SlC3H73 Gene overexpression in tomatoes increases the thickness of the waxy skin, thereby improving the disease resistance of tomatoes.

[0016] pass SlC3H73 Gene mutations, producing SlC3H73 Mutant tomatoes reduce disease resistance by decreasing the thickness of the waxy skin.

[0017] pass SlC3H73 Gene overexpression to produce SlC3H73 Gene overexpression in tomatoes enhances their storage resistance; or through... SlC3H73 Gene mutations, producing SlC3H73 Mutant tomatoes reduce the tomato's storage life.

[0018] The aforementioned methods are applied to improve tomato performance.

[0019] The aforementioned methods can be used to improve or reduce the drought and disease resistance of tomatoes.

[0020] The material prepared by the aforementioned method.

[0021] As mentioned earlier, improving the adaptability of tomatoes, such as drought resistance and / or disease resistance, has always been a pressing technical problem that researchers have been eager to solve. To this end, after long-term research, the inventors have identified a gene that regulates drought resistance and disease resistance in tomatoes. SlC3H73 Based on this discovery, the technical solution of this application was finally obtained. This application will... SlC3H73 Overexpression or mutation of genes in tomatoes has yielded tomato germplasm with strong or weak drought resistance and strong or weak disease resistance. This has important theoretical guiding significance and practical application value for the targeted improvement of tomato resistance to drought stress and pests and diseases, and for enriching tomato germplasm resources. Attached Figure Description

[0022] The present invention will be described by way of example and with reference to the accompanying drawings, wherein: Figure 1 Diagram of intermediate gene editing vectors; Figure 2 Diagram of gene editing expression vector; Figure 3 for SlC3H73 Genes in wild-type (WT) and SlC3H73 The relative expression level in tomatoes; Figure 4 Wild type (WT) and SlC3H73 Sequencing peak diagram of gene mutants; Figure 5 for SlC3H73 The mutation site of the gene mutant and the premature termination of the encoded polypeptide; Figure 6 For drought treatment SlC3H73 Gene mutant phenotype; Figure 7 For drought treatment SlC3H73 Gene overexpression tomato phenotype; Figure 8 for SlC3H73 Gene overexpression and relative water content in mutant tomato leaves; Figure 9 for SlC3H73 Gene overexpression and wax content per unit area of ​​mutant tomato leaves; Figure 10 for SlC3H73 Phenotypes of gene overexpression and mutant tomato leaves inoculated with gray mold; Figure 11 for SlC3H73 Infection area of ​​tomato leaves after inoculation with gray mold in gene overexpression and mutant tomatoes; Figure 12 for SlC3H73 Phenotypes of mutant tomato fruits after gene overexpression and inoculation with gray mold; Figure 13 for SlC3H73 The infection area of ​​gene-overexpressing and mutant tomato fruits after inoculation with gray mold; Figure 14 for SlC3H73 Gene overexpression and waxy skin in mutant tomatoes; Figure 15 for SlC3H73 Gene overexpression and the thickness of waxy skin in mutant tomatoes. Detailed Implementation

[0023] All features disclosed in this specification, or all steps in all disclosed methods or processes, may be combined in any way, except for mutually exclusive features and / or steps.

[0024] Any feature disclosed in this specification, unless otherwise stated, may be replaced by other equivalent or similar features. That is, unless otherwise stated, each feature is merely one example of a series of equivalent or similar features.

[0025] Example 1 Tomato C3H73 Cloning of genes As shown in the figure, based on the tomato whole genome sequence provided by SGN (http: / / solgenomics.wur.nl / ), Primer Premier 5 software was used to design... SlC3H73 The primers for gene cloning are as follows: SlC3H73-F: 5'-GCTCTAGAATGGTTAAGCTCCACTTTTTAT-3'; SlC3H73-R: 5'-CCGCTCGAGCTGGATCAGATCCGAAACC-3'.

[0026] Young leaves of Micro-Tom tomatoes were used as material. RNA was extracted using the RNAprep Pure plant total RNA extraction kit (purchased from Tiangen Biotech Co., Ltd.). First-strand cDNA was synthesized by reverse transcription using HiScript IV All-in-One Ultra RT SuperMix premix (purchased from Nanjing Novizan Technology Co., Ltd.) according to the product instructions. Using cDNA as a template, the above primers were used to perform PCR amplification with PrimeSTAR high-fidelity enzyme (purchased from Takara). The program was as follows: (1) 98℃, 10s; (2) 56℃, 10s; (3) 72℃, 10s [(1)-(3) 32 cycles]; (4) 72℃, 5min; (5) 4℃ incubation.

[0027] The amplification products were subjected to agarose gel electrophoresis and then gel recovery (gel recovery kit purchased from Omega Bio-Tek). The recovered products were constructed into the pEASY-Blunt Simple Cloning vector (purchased from TransGen). The recombinant plasmid pEASY-SlC3H73 was sent to BGI Genomics Co., Ltd. for sequencing confirmation.

[0028] The result SlC3H73 The nucleotide sequence of the gene is shown in SEQ ID No. 1, and the amino acid sequence of the protein encoded by the gene is shown in SEQ ID No. 2.

[0029] Example 2 Tomato SlC3H73 Construction of gene overexpression vectors and gene editing vectors 1. Tomato SlC3H73 Construction of gene overexpression vectors The recombinant plasmids pEASY-SlC3H73 and pBI121-Flag were digested with restriction endonucleases XbaI and XhoI, respectively. The digestion products were then recovered via agarose gel electrophoresis. SlC3H73 The fragment and the pBI121-Flag fragment were ligated at 22℃ and transformed into *E. coli*. The transformed *E. coli* were then incubated at 37℃ for 16-18 hours. Single colonies of *E. coli* were picked and inoculated into LB broth containing kanamycin sulfate, and cultured at 37℃ and 180 rpm for 14-16 hours. Plasmids were extracted and sent to BGI Genomics Co., Ltd. for sequencing analysis. SlC3H73 The gene overexpression vector was named pBI121-SlC3H73.

[0030] 2. Tomato SlC3H73 Construction of gene editing expression vectors (1) Tomato SlC3H73 Preparation of gene target adapters Based on the above... SlC3H73 Gene sequences were used to design two pairs of genes using Primer Premier 5 software. SlC3H73 The sgRNA primers for the gene are as follows: SlC3H75-CR-F1: 5'- ATTG AAGAAGTCGCCGGATTCCGA-3'; SlC3H75-CR-R1: 5'- AAAC TCGGAATCCGGCGACTTCTT-3'; SlC3H75-CR-F2: 5'- ATTG TCATCGGAGTACGACCACGG-3'; SlC3H75-CR-R2: 5'- AAAC CCGTGGTCGTACTCCGATGA-3'.

[0031] Primers SlC3H75-CR-F1 / SlC3H75-CR-R1 and SlC3H75-CR-F2 / C3H75-CR-R2 were used for forward and reverse pairing, respectively. The primers were then annealed to double-stranded DNA using a PCR instrument, yielding annealing products F1 / R1 and F2 / R2, respectively. The total volume of the annealing reaction was 100 μL, including 1 μL of forward primer (F1 or F2), 1 μL of reverse primer (R1 or R2), and 98 μL of ddH2O. The annealing program was: 94℃ for 3 min, 94℃ for 30 s (decreasing by 1℃ per cycle), for 88 cycles. Tomatoes were obtained using the above method. SlC3H73 Annealing products of gene targets, F1 / R1 and F2 / R2.

[0032] (2) Tomato SlC3H73 Connection between gene target and intermediate vector Using a cut-and-ligate method, the annealing products F1 / R1 and F2 / R2 were ligated into the gene editing intermediate vectors pYLgRNA-AtU3b and pYLgRNA-AtU6-1, respectively. Figure 1The reaction system is shown in Table 1. The ligation product was transformed into *E. coli* and cultured at 37°C for 16-18 hours. Single colonies of *E. coli* were picked and inoculated into LB broth containing ampicillin, and cultured at 37°C and 180 rpm for 14-16 hours. Plasmids were extracted and sent to BGI Genomics Co., Ltd. for sequencing analysis. The obtained recombinant vectors were named pYLgRNA-AtU3b-SlC3H73-1 and pYLgRNA-AtU6-1-SlC3H73-2, respectively.

[0033] Table 1 SlC3H73 Side-by-side ligation reaction system between gene target and intermediate vector

[0034] (3) Tomato SlC3H73 PCR amplification of gene sgRNA expression fragment Using pYLgRNA-AtU3b-SlC3H73-1 and pYLgRNA-AtU6-1-SlC3H73-2 plasmids as templates, two rounds of nested PCR were performed. The primers used are as follows: Primers for the first round of PCR amplification: UF: 5'-CTCCGTTTTACCTGTGGAATCG-3'; gRNA-R: 5'-CGGAGGAAAATTCCATCCAC-3'; Second round PCR amplification primers: Primers used for the pYLgRNA-AtU3b-SlC3H73-1 plasmid: Uctcg-B1': 5'-TTCAGAggtctcTctcgCACTGGAATCGGCAGCAAAGG-3'; gRctga-B2: 5'-AGCGTGggtctcGtcagGGTCCATCCACTCCAAGCTC-3'; Primers used for pYLgRNA-AtU6-1-SlC3H73-2 plasmid: Uctga-B2': 5'-TTCAGAggtctcTctgaCACTGGAATCGGCAGCAAAGG-3'; gRcggt-BL: 5'-AGCGTGggtctcGaccgGGTCCATCCACTCCAAGCTC-3'.

[0035] The amplification product was subjected to agarose gel electrophoresis and then recovered to obtain the desired product. SlC3H73The gene contains two sgRNA expression fragments, C3H73-sgRNA1 and C3H73-sgRNA2.

[0036] (4) Ligation of sgRNA expression fragment and gene editing expression vector The C3H73-sgRNA1 and C3H73-sgRNA2 fragments and the pYLCRISPR / Cas9-DN plasmid were combined ( Figure 2 After mixing, use restriction endonucleases. Bsa The I and T4 ligases were used for simultaneous digestion and ligation. The reaction system is shown in Table 2. The reaction conditions were 37℃ digestion for 5 min, 20℃ ligation for 5 min, and 5 cycles.

[0037] The ligation product was transformed into *E. coli* and cultured at 37°C for 16-18 hours. Single *E. coli* clones were picked and inoculated into LB broth containing kanamycin, and cultured at 37°C and 150 rpm for 16-18 hours. After plasmid extraction, clones identified by PCR were sent to BGI Genomics Co., Ltd. for sequencing analysis. SlC3H73 The gene editing expression vector was named pYLCRISPR / Cas9-DN-SlC3H73.

[0038] Table 2. Clone-to-ligation reaction system for sgRNA expression fragments and gene editing expression vectors

[0039] Example 3: Enhanced drought resistance ( SlC3H73 Gene overexpression in tomatoes) and reduced drought resistance ( SlC3H73 Creation of tomato materials (gene mutants) 1. SlC3H73 Creation of gene-overexpressing tomatoes (1) Agrobacterium-mediated genetic transformation of tomato The pBI121-SlC3H73 plasmid was transformed into Agrobacterium EHA105 using a freeze-thaw method. Tomatoes were then transformed using Agrobacterium-mediated genetic transformation, and the results were analyzed. SlC3H73 The specific steps for identifying gene-overexpressing tomatoes are as follows.

[0040] 1) Cultivation of sterile tomato seedlings This process must be completed in a clean bench. The specific steps are as follows: First, soak the seeds in sterile water in a centrifuge tube for 10 minutes, gently shaking to revive them. Pour out the sterile water from the tube and then use a micropipette to remove any remaining water. Next, add 75% alcohol for 30 seconds to disinfect, pour out the alcohol, and rinse once with sterile water. Pour out the rinse sterile water. Then, add a 15% sodium hypochlorite solution prepared with sterile water to the tube and soak the seeds for 20 minutes (gently shaking several times during this period). Pour out the sodium hypochlorite solution and rinse 3-5 times with sterile water. Finally, use sterile forceps to place an appropriate amount of seeds into half of MS medium and spread them evenly. Incubate in a light incubator.

[0041] 2) Pre-culture of tomato cotyledons Tomato seeds are cultured in an incubator for about 1-2 weeks. Once the cotyledons are fully open but the true leaves have not yet emerged, the cotyledons can be cut for pre-culture. The cotyledon cutting process must be completed in a clean bench. The specific procedure is as follows: Use sterile forceps to remove the tomato seedling. On a sterile culture dish, use sterile scissors or a scalpel to cut off the tomato cotyledons. Use sterile forceps to gently puncture the cotyledons 2-3 times. Place the cotyledons on the pre-culture medium. Seal the culture dish with sealing film and place it in an incubator in the dark for 2 days.

[0042] 3) Infection of tomato cotyledons One day in advance, expand the culture of Agrobacterium to infect the desired bacteria in 15 mL EP tubes using YEB liquid medium until the bacterial culture reaches OD. 600 Collect bacterial cells by centrifugation at 5000 rpm for 5 min at a depth of 0.6-0.8, and discard the supernatant. Tomato cotyledon infection must be performed in a clean bench. The specific procedure is as follows: Resuspend the above-mentioned Agrobacterium in the infection solution. Transfer cotyledons that have been cultured in the dark for 2 days on pre-medium to a sterile culture dish, add Agrobacterium, and gently agitate the dish to ensure that each cotyledon is in contact with the bacterial solution. After 10 minutes, remove the cotyledons and blot off excess bacterial solution with sterile filter paper. Transfer the cotyledons to a co-medium, seal the culture dish with sealing film, and place it in an incubator for 2 days in the dark.

[0043] 4) Differentiation culture Inside a clean bench, use sterile forceps to transfer the cotyledons from the previous co-culture step to the differentiation medium, arranging them evenly. Incubate in a light incubator until callus tissue develops from the cotyledons. Change the differentiation medium every 2-3 weeks until tomato seedlings differentiate.

[0044] 5) Rooting culture Use tweezers to transfer the callus tissue and differentiated seedlings onto a petri dish. Use a scalpel to make a 45-degree angled cut on the main stem of the seedling, and then use tweezers to insert the seedling 2-3 mm into the rooting medium along the direction of the main stem. Culture in a light incubator for about 15 days until roots develop.

[0045] 6) Transplanting of tissue culture seedlings Mix the soil with water, sterilize, and then divide it into small flowerpots. Make a hole in the center of the soil in each small flowerpot. Carefully remove the tissue culture seedling with tweezers and wash the roots under running water to remove the culture medium. Place the seedling in the pot, cover the roots with soil, and spray water on the surface of the seedling. Cover the seedling with a transparent plastic lid or plastic wrap. After about 3-5 days, open the ventilation hole or poke a few holes in the plastic wrap. After about 7-9 days, gradually remove the film to ensure light and temperature, water regularly, and fertilize every two weeks. The plant will grow for about 4 months until it ages.

[0046] The composition of the culture medium used for the above tomato tissue culture is shown in Table 3.

[0047] Table 3. Culture media used for tomato tissue culture

[0048] (2) SlC3H73 Identification of gene overexpression tomatoes Young leaves from Micro-Tom and transgenic plants were used as materials to synthesize first-strand cDNA using the method described in Example 1. Using tomato UBI as an internal reference gene, qRT-PCR amplification was performed using primers (shown in Table 4) and the above cDNA as a template. Two... -∆∆CT Legal analysis SlC3H73 Gene expression in different strains. Results are as follows: Figure 3 As shown, compared with Micro-Tom, strains 2, 8, and 9... SlC3H73 Gene expression levels increased significantly, specifically in Micro-Tom. SlC3H73 The 16.5, 22.85, and 9.27-fold increases in gene expression levels prove that these three lines are... SlC3H73 The overexpression (OE) strains were named SlC3H73-OE-2, SlC3H73-OE-8, and SlC3H73-OE-9, respectively.

[0049] Table 4. Detection using qRT-PCR SlC3H73 Primers and their sequences used to measure gene expression

[0050] 2. SlC3H73 Creation of gene mutants (1) Agrobacterium-mediated genetic transformation of tomato The pYLCRISPR / Cas9-DN-SlC3H73 plasmid was transformed into Agrobacterium EHA105 using a freeze-thaw method. Tomatoes were then transformed using the Agrobacterium-mediated genetic transformation method described in point 1 of this embodiment.

[0051] (2) SlC3H73 Identification of homozygous mutant lines without transgenic tags Genomic DNA was extracted from tomato tissue culture seedlings. Using this DNA as a template, sequences containing sgRNA were amplified using primers SlERF.F12-seqF and SlERF.F12-seqR with a high-fidelity enzyme. The amplified products were sent to BGI Genomics Co., Ltd. for sequencing analysis. Fragments showing bimodal peaks in the sequencing results were ligated into the pEASY-Blunt simple vector (purchased from TransGen Biotech) for multiple cloning sequencing to determine the mutated sequence. Tomato tissue culture seedlings screened using this method were identified as heterozygous mutant lines.

[0052] The primer sequences are as follows: SlERF.F12-seqF:CTTTTTATCACCCCTTTATATACAA; SlERF.F12-seqR: CTCTGTTACAATTTCCTTTACGAAA.

[0053] Heterozygous mutant lines were cultured until tomato fruits matured, and mature tomato seeds (T1 generation seeds) were harvested. After sterilization, T1 generation tomato seeds were sown on 1 / 2 MS medium and placed in a light incubator. Genomic DNA was extracted from tomato seedlings and PCR was used to identify the presence of the kanamycin sulfate resistance gene. The amplified products were observed after agarose gel electrophoresis. Using T1 generation seedlings that did not amplify the hygromycin resistance gene as material, high-fidelity enzymes were used to amplify the genomic sequence containing the sgRNA using SlERF.F12-seqF and SlERF.F12-seqR primers. The amplified products were sent to BGI Genomics Co., Ltd. for sequencing analysis. Lines showing a single peak and a mutation were considered homozygous mutant lines without a transgenic tag. SlC3H73 The gene mutants were named SlC3H73-CR-1, SlC3H73-CR-9, and SlC3H73-CR-15, respectively. Figure 4 and Figure 5 ). Here, CR is short for CRISPR / Cas9 gene editing technology.

[0054] 3. Drought resistance test (1) Phenotypic observation Will SlC3H73 Gene overexpression lines (SlC3H73-OE-2, SlC3H73-OE-8 and SlC3H73-OE-9) SlC3H73Mutant lines (SlC3H73-CR-1, SlC3H73-CR-9, and SlC3H73-CR-15) and Micro-Tom (wild-type) tomatoes were cultured in an artificial climate chamber. After 6 weeks of growth, the tomato seedlings were deprived of water, and their growth was observed. The results showed that after 4 days of water deprivation, some leaves of the wild-type tomatoes curled, but the three... SlC3H73 The seedlings of the gene mutant lines all exhibited severe wilting of leaves, indicating that compared to wild-type tomatoes, SlC3H73 Mutant tomatoes have reduced drought resistance. Figure 6 After five days of water deprivation, the leaves of wild-type tomatoes showed obvious wilting, but three... SlC3H73 Seedlings with overexpressed genes showed significantly less wilting, indicating that compared to wild-type tomatoes, SlC3H73 Tomatoes with overexpressed genes exhibit enhanced drought resistance. Figure 7 ).

[0055] (2) Determination of relative water content of leaves Cut wild-type plants that have undergone drought treatment. SlC3H73 Gene overexpression and SlC3H73 Leaves from the same leaf position of the gene mutant tomato were quickly weighed on a balance for fresh weight (FW). The leaves were then immersed in distilled water for 24 hours, removed, and wiped dry; the total weight (TW) was immediately measured. After weighing, the leaves were dried to constant weight and measured for dry weight (DW). The relative water content of the tomato leaves was calculated using the following formula: .

[0056] The results showed that, under drought conditions, compared with wild-type tomatoes, SlC3H73 The relative water content of the leaves of the gene mutant was significantly reduced, while SlC3H73 The relative water content of tomato leaves was significantly increased after gene overexpression. Figure 8 ).

[0057] (3) Determination of leaf wax content Take leaves from the same location on an 8-week-old tomato plant, flatten them with a glass plate, photograph them, and calculate the leaf area using ImageJ software. Wash the leaves thoroughly with water, dry them with filter paper, and weigh them on a balance (W1). Then, immerse the leaves in chloroform at 40-50℃ for 30 seconds and remove them quickly. Use a rotary evaporator at 80℃ to evaporate excess chloroform from the leaves for approximately 10 minutes. Remove the leaves and weigh them on a balance (W2). Calculate the wax content of the tomato leaves using the following formula: .

[0058] The results showed that, compared with wild-type tomatoes, SlC3H73The wax content in the leaves of the gene mutant was significantly reduced, while SlC3H73 Gene overexpression significantly increased the wax content in tomato leaves, suggesting... SlC3H73 Genes can influence the drought resistance of tomatoes by regulating the wax content of leaves. Figure 9 ).

[0059] 4. Disease resistance testing (1) Inoculation of gray mold Leaves infected with gray mold were located in the experimental field. Gray mold was scraped off using an inoculation loop and streaked onto PDA medium to obtain single colonies. Gray mold was cultured on PDA medium to expand its distribution. Experiments began when the gray mold had completely covered the PDA medium plate.

[0060] Leaves from 8-week-old tomato plants and mature tomato fruits were used as experimental materials. Using a punch approximately 5 mm in diameter, several small discs containing gray mold were prepared from PDA culture medium. A small hole was gently pricked on the back of each tomato leaf with a needle, and then the mold-covered side of the disc was placed over the hole. The procedure for the fruit was similar; a small hole was pricked in each fruit, and then the disc was placed over it.

[0061] (2) Phenotypic observation Take a styrofoam box, line the bottom with newspaper and soak it, place the leaves and fruit inside, then spray water into the box and close the lid. Place the styrofoam box in a cool place for 1 day, then open the lid, remove the PDA from the leaves and fruit, close the lid again and leave it for another day, then take photos to record the results, and use ImageJ to calculate the affected area.

[0062] The results showed that on the second day after inoculation with gray mold, SlC3H73 The infected area of ​​the mutant leaves was significantly larger than that of the wild-type tomato leaves, and SlC3H73 The infected area of ​​tomato leaves with overexpressed genes was significantly smaller than that of wild-type tomato leaves (e.g., Figures 10 - 13 (As shown).

[0063] (3) Observation of fruit peel wax Fresh tomato pericarp tissue was embedded in OCT and frozen at -20℃ to -30℃ before sectioning. Sections were dried at room temperature for 15-20 minutes, then fixed with 4% paraformaldehyde for 20-30 minutes. After rinsing with distilled water, the sections were transferred to 60% isopropanol for 5 minutes (to remove moisture and enhance staining). The sections were then immersed in Oil Red O working solution and stained in the dark for 8-15 minutes (at room temperature). Afterward, they were rinsed with 60% isopropanol for 3-5 seconds, and under a microscope, the staining was controlled until the background was colorless and the lipid droplets were bright red. After rinsing with running water, the nuclei were stained with hematoxylin for 1-2 minutes, followed by differentiation with 1% hydrochloric acid alcohol for 1-2 seconds. The sections were then rinsed with running water until the nuclei turned blue. Finally, glycerol-gelatin mounting medium was added (to avoid air bubbles). The prepared sections were observed and photographed under a microscope, and the thickness of the stained areas was measured using ImageJ software.

[0064] The results showed that, compared with wild-type tomatoes, SlC3H73 The thickness of the waxy coating on the pericarp of the gene mutant was significantly reduced, while SlC3H73 Gene overexpression significantly increases the waxy thickness of tomato peel. Figure 14 and Figure 15 ),hint SlC3H73 Genes can influence the disease resistance of tomatoes by regulating the thickness of the waxy coating on the fruit.

[0065] This invention is not limited to the specific embodiments described above. The invention extends to any new feature or combination disclosed in this specification, as well as any new method or process step or combination disclosed herein.

Claims

1. A gene that regulates drought resistance and disease resistance in tomatoes. SlC3H73 Its characteristics are, Its nucleotide sequence is shown in SEQ ID NO.

1.

2. The gene for regulating drought resistance and disease resistance in tomatoes according to claim 1. SlC3H73 Its characteristics are, The genes that regulate drought resistance and disease resistance in tomatoes SlC3H73 The amino acid sequence of the encoded protein is shown in SEQ ID NO.

2.

3. A method for regulating tomato performance, characterized in that, pass SlC3H73 Gene overexpression to produce SlC3H73 Gene overexpression in tomatoes enhances their drought resistance and / or disease resistance; or through... SlC3H73 Gene mutations, producing SlC3H73 Mutant tomatoes reduce the drought resistance and / or disease resistance of tomatoes.

4. The application of the method described in claim 3 or 4 in improving tomato performance.

5. The application according to claim 4, characterized in that, The aforementioned methods can be used to improve or reduce the drought and disease resistance of tomatoes.

6. The material prepared by the method described in claim 3 above.

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