Tomato EAR transcription factor SlEAD1 and application thereof

Abstract

The invention relates to the field of modern agricultural industry, in particular to a tomato EAR transcription factor SIEAD1 and application thereof. According to the invention, a novel EAR motif transcription inhibition factor SlEAD1 of the tomato is identified, and a non-transgenic homozygous stead1 mutant is created by utilizing a CRISPR / Cas9 gene editing technology, so that the salt tolerance of the tomato is remarkably improved. The specific technical effects are shown as follows: under the salt stress condition, the salt tolerance of the stead1 mutant is obviously enhanced, and the physiological metabolism and antioxidant capacity of the stead1 mutant are obviously improved; molecular mechanism research shows that SlEAD1 participates in salt tolerance regulation and control by regulating and controlling expression of ABA signal channel key genes; through detection, the obtained mutant plant does not contain exogenous genes and meets non-transgenic requirements, and characters are stably inherited. The technology provides a new target gene and a salt-tolerant material with an application prospect for salt-tolerant breeding of tomatoes.

Description

Technical Field

[0001] The present invention relates to the field of modern agricultural industry, and in particular to a tomato EAR transcription factor SIEAD1 and applications thereof. Background Art

[0002] Tomatoes (Solanum lycopersicum L.), as an important cash crop in my country, play a key role in the vegetable industry. Their fruits are rich in nutrients, including the antioxidant lycopene, various vitamins (such as vitamin A, B vitamins, vitamins C and E), and essential minerals (including potassium, calcium, and iron). Data shows that my country's annual tomato production continues to climb, exceeding 8 million tons in 2023 for processing, a significant increase from the previous year. Over half of this production comes from greenhouse cultivation.

[0003] However, secondary soil salinization is an increasingly prominent problem during greenhouse cultivation due to continuous cropping and improper agricultural management practices (such as excessive fertilization and poor irrigation management). This phenomenon seriously affects the normal growth and development of tomato plants, leading to reduced fruit yield and deterioration in quality, resulting in significant economic losses to agricultural production. Currently, soil salinization has become a major obstacle to the development of the greenhouse tomato industry.

[0004] In this context, in-depth research on the molecular mechanisms of tomato's response to salt stress and the identification of key salt-resistance genes are crucial for breeding salt-tolerant varieties and mitigating the effects of salt damage. This research not only provides theoretical support for the sustainable development of protected agriculture but also has practical value in increasing farmers' incomes and promoting rural industrial revitalization. Summary of the Invention

[0005] The purpose of the present invention is to provide a tomato EAR transcription factor SIEAD1 and its application in response to the problems existing in the prior art.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] A tomato EAR transcription factor SlEAD1, whose nucleotide sequence is shown in SEQ ID NO.1.

[0008] The above-mentioned transcription factor SIEAD1 provides a key gene for regulating tomato salt tolerance and provides a target for salt-tolerant molecular breeding.

[0009] The present invention identifies a novel EAR motif transcriptional repressor SlEAD1 (Solanum lycopersicum EAR motif-containing ABAdown-regulated 1) in tomato and uses CRISPR / Cas9 gene editing technology to create a non-transgenic homozygous slead1 mutant, which significantly improves the salt tolerance of tomatoes. The specific technical effects are as follows: (1) Optimization of physiological indicators of salt stress: under 200mM NaCl treatment, the survival rate of the slead1 mutant is increased by more than 35% compared with the wild type, the malondialdehyde (MDA) content in the leaves is reduced by 42%, the catalase (CAT) activity is increased by 2.1 times, and the superoxide anion (O2 - ) and hydrogen peroxide (H2O2) accumulation decreased by 58% and 63%, respectively; (2) The molecular regulatory mechanism is clear: SlEAD1 negatively regulates salt tolerance by inhibiting the expression of key genes in the ABA signaling pathway (SlNCED2 and SlSnRK2s). qRT-PCR confirmed that the expression of SlEAD1 decreased by 60% after 4 hours of salt stress; (3) Breeding safety assurance: PCR detection of the mutant showed no exogenous Cas9 gene residues, meeting the requirements of non-transgenic, and the trait is stably inherited. This technology provides a new target gene with independent intellectual property rights and a salt-tolerant variety that can be commercialized for tomato cultivation in saline-alkali land.

[0010] A protein encoded by the tomato EAR transcription factor SlEAD1 gene, whose amino acid sequence is shown in SEQ ID NO.2.

[0011] The above-mentioned functional proteins that negatively regulate salt tolerance enhance salt tolerance through protein function loss.

[0012] A recombinant expression vector contains the tomato EAR transcription factor gene SlEAD1, wherein the vector is pCAMBIA1302 or a CRISPR / Cas9 gene editing vector.

[0013] Recombinant expression vectors provide molecular tools for gene manipulation to achieve overexpression or targeted editing of SlEAD1.

[0014] An expression cassette or transgenic cell line comprises the above-mentioned recombinant expression vector.

[0015] Expression cassettes or transgenic cell lines are application forms of expanded vectors, suitable for different genetic transformation systems.

[0016] Use of the aforementioned SlEAD1 gene, the aforementioned protein, the aforementioned recombinant expression vector, or the aforementioned expression cassette or transgenic cell line in any of the following:

[0017] (1) Regulate salt tolerance of tomatoes;

[0018] (2) Cultivate tomato plants with enhanced salt tolerance.

[0019] The application provided by the present invention clarifies the application scenarios of molecular tools and directly serves the goal of stress resistance breeding.

[0020] A non-transgenic slead1 mutant tomato plant was obtained by editing the SlEAD1 gene mentioned above through CRISPR / Cas9.

[0021] The non-transgenic slead1 mutant tomato plant provided by the present invention has significantly enhanced salt tolerance and is suitable for popularization and application.

[0022] A method for cultivating the above-mentioned non-transgenic slead1 mutant tomato plants comprises the following steps:

[0023] (1) Design sgRNA targeting the SlEAD1 gene and construct a CRISPR / Cas9 editing vector;

[0024] (2) Transform tomato callus and screen for positively edited plants;

[0025] (3) Obtain homozygous mutants without exogenous DNA through progeny separation.

[0026] The cultivation method provided by the present invention standardizes the breeding process and ensures the non-transgenic properties of the mutants.

[0027] A molecular marker for detecting salt tolerance of tomato, wherein the marker is based on the editing site of the tomato EAR transcription factor SlEAD1 as described above, and is used to identify slead1 mutants by PCR or sequencing.

[0028] The molecular marker for detecting salt tolerance of tomatoes provided by the present invention can accelerate the screening efficiency of salt-tolerant varieties.

[0029] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0030] The present invention identifies a novel EAR motif transcriptional repressor SlEAD1 (Solanum lycopersicum EAR motif-containing ABAdown-regulated 1) in tomato and uses CRISPR / Cas9 gene editing technology to create a non-transgenic homozygous slead1 mutant, which significantly improves the salt tolerance of tomatoes. The specific technical effects are as follows: (1) Optimization of physiological indicators of salt stress: under 200mM NaCl treatment, the survival rate of the slead1 mutant is increased by more than 35% compared with the wild type, the malondialdehyde (MDA) content in the leaves is reduced by 42%, the catalase (CAT) activity is increased by 2.1 times, and the superoxide anion (O2- ) and hydrogen peroxide (H2O2) accumulation decreased by 58% and 63%, respectively; (2) The molecular regulatory mechanism is clear: SlEAD1 negatively regulates salt tolerance by inhibiting the expression of key genes in the ABA signaling pathway (SlNCED2 and SlSnRK2s). qRT-PCR confirmed that the expression of SlEAD1 decreased by 60% after 4 hours of salt stress; (3) Breeding safety assurance: PCR detection of the mutant showed no exogenous Cas9 gene residues, meeting the requirements of non-transgenic, and the trait is stably inherited. This technology provides a new target gene with independent intellectual property rights and a salt-tolerant variety that can be commercialized for tomato cultivation in saline-alkali land. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The data show that SlEAD1 is localized in the cell nucleus and has transcriptional repressive activity.

[0032] Figure 2 The RT-PCR test results of SlEAD1 transgenic lines and Micro-Tom wild type.

[0033] Figure 3 This is a diagram showing the response of the SlEAD1 gene in tomatoes after 4 hours of salt treatment.

[0034] Figure 4 The salt tolerance data of slead1 mutant plants ( Figure 4 A in the figure shows the effect of NaCl treatment on the growth of wild-type, SlEAD1 overexpression and slead1 mutant plants. Figure 4 B is the staining image of hydrogen peroxide accumulation in leaves of wild type, SlEAD1 overexpression and slead1 mutant plants after NaCl treatment. Figure 4 C in the figure is the staining image of superoxide anion content in leaves of wild type, SlEAD1 overexpression and slead1 mutant plants after NaCl treatment. Figure 4 D in the figure is a bar graph showing the catalase activity in wild-type, SlEAD1 overexpressing and slead1 mutant plants after NaCl treatment. Figure 4 (E) is a bar graph showing the malondialdehyde content in wild-type, SlEAD1 overexpressing and slead1 mutant plants after NaCl treatment. DETAILED DESCRIPTION

[0035] The present invention will be described in detail below with reference to the accompanying drawings.

[0036] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0037] Example 1

[0038] During greenhouse cultivation, secondary soil salinization is an increasingly prominent problem due to continuous cropping and improper agricultural management practices (such as excessive fertilization and poor irrigation management). This phenomenon severely impacts the normal growth and development of tomato plants, leading to reduced yields and deteriorating fruit quality, resulting in significant economic losses to agricultural production. Currently, soil salinization has become a major obstacle to the development of the greenhouse tomato industry.

[0039] In this context, in-depth research on the molecular mechanisms of tomato's response to salt stress and identification of key salt-resistant genes are of great significance for breeding salt-tolerant varieties and alleviating the effects of salt damage.

[0040] As a key transcriptional repression domain in plants, the EAR motif has garnered significant attention for its molecular characteristics and functional mechanisms. The conserved EAR motif (L / F)DLN(L / F)xP was originally discovered in ERFs and some C2H2 zinc finger protein families, which exhibit transcriptional repression activity. Systematic sequence analysis further redefined it as two canonical motifs: LxLxL and DLNxxP (Kagale et al., 2010). Recent studies have demonstrated that EAR motif-mediated transcriptional repression is the primary form of transcriptional repression in plants. Transcriptional repressors containing the EAR motif can regulate plant responses to salt stress by inhibiting gene expression (Kagale et al., 2011; Yang et al., 2018). For example, GhZAT6 (zinc finger of Arabidopsis Thaliana), StERF3, MdZAT17, and GmERF5 regulate the expression of downstream target genes. GmERF5 has been shown to regulate plant salt tolerance through the ABA signaling pathway (Dong et al., 2015; Tian et al., 2015; Chen et al., 2021a; Wang et al., 2022a).

[0041] Based on genome-wide DLN××P and L×L×L sequence analysis, over 400 proteins containing the EAR motif have been identified in Arabidopsis thaliana, but the functional mechanisms of most members remain unelucidated (Yang et al., 2018). Notably, the short peptide nature of the EAR motif (consisting of only 6–7 amino acids) and the high sequence diversity of the proteins in which it reside suggest the existence of novel, yet-to-be-identified EAR motif proteins. During screening for ABA-responsive genes, the applicant's research group successfully discovered several novel EAR motif transcription factors, including SlEAD1 (Tian et al., 2017; Wang et al., 2020; Chen et al., 2021b; Wang et al., 2022b; Hussain et al., 2023; Li et al., 2023; Wang et al., 2024). SlEAD1, along with AtAITRs, AtASRs, and AtAUEs, all belong to the L×L×L EAR motif class. The applicant's previous studies found that SlEAD1 is an ABA-responsive gene. SlEAD1 negatively regulates the response of tomatoes to ABA by inhibiting the expression of ABA synthesis genes (SlNCED2) and signal transduction genes (SlSnRK2s), but whether it is involved in regulating the response of tomatoes to salt stress remains unknown (Wang et al., 2020).

[0042] Research has identified SlEAD1 as a novel EAR motif transcription factor (nucleotide and amino acid sequences are shown below), belonging to the L×L×L type EAR motif protein. Experimental validation confirmed that SlEAD1 is an ABA and salt stress-responsive gene and a nuclear-localized transcriptional repressor. Using gene editing technology, a slead1 mutant was generated. The salt stress response of wild-type and slead1 mutant tomatoes was compared, resulting in a new non-transgenic tomato variety with improved salt tolerance. The nucleotide and amino acid sequences of SlEAD1 are shown below:

[0043] The nucleotide sequence of the tomato EAR transcription factor SlEAD1 is shown in SEQ ID NO.1:

[0044] ATGAACATGATCGACGATCAACGTCGTGGTCCGCCACACGCGGCCTTATTAGCGTGGTGGTTATAGCAGTGATGATCGTTCCGTCATTAATCGGTGAAAATGGTGAAGCAATCACTGAATTCATCTCTGAACTTCTTACCCCAATTGGATTACTCCTTTTGCCTATAATTTTACTTCTC ACAATTCAATTTCTCTCATCTGATAGCGGCTCTTTCGTCTCGAGTATCTTTTCGAGTGGTGAGCCAAATTCAATTCATCGTGCTAGTGGTTCCCCTGTTGGTGTTGCTCTCGTTCTTCTCCTTGTTCTGTTTCTTCTGTATAATCCATTTTCGCTCTTTGGTGGTGATGATGAAGATTGA.

[0045] The amino acid sequence of the protein encoded by the tomato EAR transcription factor gene SlEAD1 is shown in SEQ ID NO.2:

[0046] MNMIDDQRRGPPHAALLAVVVIAVMIVPSLIGENGEAITEFISELLTPIGLLLLPIILLLTIQ FLSSSDSGSFVSSIFSGEPNSIHRASGSPVGVALVLLLVLFLLYNPFSLFGGDDED.

[0047] The specific steps are as follows:

[0048] Identification of SlEAD1 as a novel EAR motif transcriptional repressor: Transcriptome sequencing identified an ABA-responsive gene with unknown function. Amino acid sequence analysis revealed that the protein possesses a typical L×L×L EAR motif, hence the name SlEAD1 (Solanum lycopersicum EAR motif-containing ABA down-regulated 1). First, construct the PUC-GFP-SlEAD1 and PUC-GD-SlEAD1 plasmids:

[0049] a. Based on the CDs sequence, cloning primers F: 5'-CAAC ATATGAACATGATCGACGATCAACG-3' and R: 5'-CAAGAGCTCATCTTCATCATCACCACCA AAG-3' were designed with NdeI at the 5' end and SacI at the 3' end.

[0050] b. PCR cloning was performed using wild-type Micro-Tom cDNA as a template. The PCR reaction system and procedure are shown in Tables 1 and 2 below.

[0051] Table 1 Reaction system

[0052]

[0053] Table 2 Reaction procedure

[0054]

[0055] The cloned product was double-digested with NdeI and SacI, and the pUC19 vector was double-digested with the same enzymes. The enzyme digestion system is shown in Table 3.

[0056] Table 3 Enzyme digestion system

[0057]

[0058] The digested vector and fragment were ligated at room temperature, transformed into competent E. coli cells using heat stimulation, plated on LB solid medium containing Amp antibiotics, and cultured at 37°C overnight. The ligation system is shown in Table 4, and the transformation method is as follows:

[0059] Table 4 Connection system

[0060]

[0061] Transformation into competent E. coli

[0062] (1) Add 2 μL of the ligation product to 20 μL of DH5a competent E. coli cells and mix gently.

[0063] (2) On ice for 30 minutes.

[0064] (3) Heat shock in a 42°C dry box for 30 seconds and immediately place on ice for 2 minutes.

[0065] (4) Add 500 μL of liquid LB medium to a double-person single-sided horizontal clean bench. Place it in a 37°C constant temperature variable frequency shaker and shake for 1 hour.

[0066] (5) Under a double-person single-sided horizontal clean bench, evenly apply the culture medium on the surface of solid LB culture medium with 100 μg / ml Amp antibiotic. After the culture medium is completely dry, seal it with sealing film and place it upside down in a 37°C constant temperature incubator for 12-16 hours.

[0067] The colonies in the culture medium after overnight culture were selected and sent to Sangon for sequencing using cloning primer F. The bacterial solution with correct sequencing was amplified and cultured, and the plasmid was extracted for use in tomato protoplast transient transfection experiments.

[0068] Transient transfection experiments using tomato protoplasts demonstrated that SlEAD1 was localized in the cell nucleus and had transcriptional repression activity.

[0069] The steps for transient transfection of tomato protoplasts are as follows:

[0070] 1. Protoplast Preparation

[0071] (1) Select tomato leaves that have been growing well for about 28 days.

[0072] (2) Cut the leaves into small pieces and place them in a glass dish containing enzymatic hydrolysis solution. Perform enzymatic hydrolysis at room temperature in the dark for 5 hours to remove the cell walls of the leaves and release the protoplasts.

[0073] (3) Filter the enzymatic hydrolysate into a centrifuge tube using a filter membrane to remove undigested leaf fragments.

[0074] (4) Add half the volume of the enzyme solution to the centrifuge tube with 200 mM CaCl2 solution, and then centrifuge at low temperature (e.g., 4°C) to precipitate the protoplasts.

[0075] (5) After discarding the supernatant, add pre-cooled W5 solution equal to the volume of enzyme solution to wash the protoplasts. Remove dead cells and impurities in the supernatant by centrifugation. This step is usually repeated twice.

[0076] (6) Finally, the washed protoplasts were suspended in an appropriate amount of MMG solution for subsequent transfection experiments.

[0077] 2. Protoplast transfection

[0078] (1) Prepare the plasmid DNA prepared in the above steps.

[0079] (2) Add 100 ng of plasmid DNA to the prepared 200 μL protoplast suspension and mix gently.

[0080] (3) Add 210 μL of PEG solution and continue to mix and transfect for a total of 20 minutes to allow the plasmid DNA to enter the protoplasts.

[0081] (4) Add 800 μL of W5 solution to the mixture and remove PEG3350 and plasmid DNA that has not entered the protoplasts by centrifugation.

[0082] (5) Finally, the transfected protoplasts were resuspended in 1 mL of WI medium for subsequent culture and analysis.

[0083] SlEAD1 is localized in the cell nucleus and has transcriptional repression activity. Figure 1 shown.

[0084] Acquisition of SlEAD1 transgenic materials

[0085] The SlEAD1 CDs sequence was cloned and ligated into the PUC-HA vector using the same construction methods as described above for the PUC-GFP and PUC-GD vectors. The sequenced PUC-HA-SlEAD1 plasmid was then double-digested with PstI and SacI using the same digestion system and conditions as described above. The resulting product was then ligated with the pZP211 vector cut with the same restriction sites. After transformation into E. coli, the cells were screened in 100 mg / ml Spect resistance medium and sent to Sangon for sequencing.

[0086] Generate slead1 gene-edited mutants using CRISPR / Cas9 technology: Gene editing sites were identified based on the SlEAD1 genomic sequence. Visit http: / / www.crisprscan.org / ?page=sequence and copy the exon sequence of the target gene into the corresponding sequence frame to search for high-scoring targets. Visit http: / / www.rgenome.net / cas-offinder / and copy the 20 nt preceding the PAM of the scored target site into the corresponding sequence frame to assess off-target effects. Two target sequences were selected: a. GCCACACGCGGCCTTATTAG and b. GCGGCCTTATTAGCGGTGG. Two sets of primers were designed based on the target sequences for constructing the PHDE and PHEE vectors, respectively.

[0087] The primers used for the PHDE vector are as follows:

[0088] F:5'-CGCGGCCTTATTAGCGGTGGGTTTTAGAGCTAGAAATAGCAAGTTA-3'.

[0089] R:5'-CCACCGCTAATAAGGCCGGAATCACTACTTCGACTCTAGC-3'.

[0090] The primers used for the PHEE vector are as follows:

[0091] DT1-BsF,5'-ATATATGGTCTCGATTGGCGGCCTTATTAGCGGTGGGTT-3'.

[0092] DT1-F0,5'-TGGCGGCCTTATTAGCGGTGGGTTTTAGAGCTAGAAATAGC-3'.

[0093] DT2-R0,5'-AACCTAATAAGGCCGCGTGTGGCAATCTCTTAGTCGACTCTAC-3'.

[0094] DT2-BsR,5'-ATTATTGGTCTCGAAACCTAATAAGGCCGCGTGTGGC-3'.

[0095] After primer design, construct the CRISPR / Cas9 vector: For the PHDE vector, perform a four-primer PCR using the designed F and R primers with the common primers pER8-PgRNA-F: CCTGTCAAACACTGATAGTTTGTCCCAGGATTAGAATGATTAGG and TgRNA-pHDE-R: CAGTTAGGTCTAGACTAGTTTCCATCAGAGGTGTAACGGAATG. For the PHEE vector, perform a four-primer PCR using the four designed primers. The PCR system and reaction procedures are shown in Tables 5 and 6.

[0096] Table 5 PCR reaction system

[0097]

[0098] Table 6 Reaction procedure

[0099]

[0100] The PCR products were recovered by gel extraction, and the PHDE fragment was ligated to the pHDE 35S Cas9 vector after digestion with PmeI. The PHEE vector fragment was recovered and ligated to the pHEE401E vector. The ligation system is shown in Table 7:

[0101] Table 7 Connection system

[0102]

[0103] The ligation product was transformed into competent Escherichia coli using the same transformation steps as above. The product was plated on LB solid medium containing 50 μg / ml Kana antibiotics. After overnight culture at 37°C, a single clone was picked for amplification and culture, and the plasmid was extracted and sent to Sangon for sequencing. The sequencing primer sequence of the PHDE vector was: TermE9-U29-R: CAAGCTAGCTTACTCAGTTAGG, and the sequencing primer sequence of the PHEE vector was: U626-IDF: TGTCCCAGGATTAGAATGATTAGGC and U629-IDF: TTAATCCAAACTACTGCAGCCTGAC.

[0104] The sequenced PZP211-HA-SlEAD1, PHDE-SlEAD1, and PHEE-SlEAD1 plasmids were transformed into GV3101 Bacillus aeruginosa competent cells as follows:

[0105] (1) Take out GV3101 Agrobacterium competent cells from -80°C and place them on ice to thaw.

[0106] (2) After labeling, take 3 μL of the plasmid to be transferred and slowly add it to the melted Agrobacterium competent cells. Mix slowly with a pipette tip and place on ice for 30 minutes.

[0107] (3) After the ice bath, place the centrifuge tube from the ice back into liquid nitrogen and quickly cool for 2 minutes.

[0108] (4) Quickly remove the centrifuge tube from liquid nitrogen and place it in a 37°C constant temperature incubator for heat shock for 5 minutes.

[0109] (5) After the heat shock time is up, place the tube in ice for 2 minutes.

[0110] (6) Add 500 μL of LB liquid culture medium to the centrifuge tube, place it in a 30°C constant temperature variable frequency shaker, and shake the bacteria for 3-4 hours.

[0111] (7) Spread the bacterial suspension evenly on the surface of a solid LB medium containing 50 μg / ml Kana and 50 μg / ml Genta antibiotics. Allow to dry and seal with parafilm. Incubate in a 30°C incubator for 36-48 hours. The cultured colonies are then inoculated into a liquid medium containing the same antibiotics for expansion and use as a preparation for transforming tomato callus.

[0112] The callus tissues of wild-type tomato cotyledons and hypocotyls were genetically modified using tissue culture. Tissue culture seedlings were obtained through callus induction, resistance-induced differentiation, and rooting culture. RT-PCR, PCR, and sequencing techniques were used to screen and verify mutant plants with overexpression and gene editing of the SlEAD1 gene.

[0113] The primer sequences used for RT-PCR detection of overexpression were the same as those used at the time of cloning. The primer sequences used for sequencing were: F: CTTTTAACTTGAGCCTTACCCCCTC and R: CAAGAGCTCATCTTCATCATCACCA CCAAAG.

[0114] RT-PCR results showed that the expression levels of SlEAD1 gene in SlEAD1 transgenic lines SlEAD1#5 and SlEAD1#13 were significantly higher than that in Micro-Tom wild type, as shown below Figure 2 As shown: Figure 2 RT-PCR test results of SlEAD1 transgenic strain and Micro-Tom wild type)

[0115] The genomic sequence of SlEAD1 in CRISPR / Cas9-positive plants was amplified by PCR, and the amplified products were sent to Sangon for sequencing. The F primer used in PCR amplification was used, and the sequencing results were compared with the SlEAD1 genomic sequence in the wild type using DNAman. It was found that slead1-c1 had one additional base, slead1-c2 had five bases missing at the first target sequence editing site, and one additional base at the second target sequence position. Both mutants had premature termination codons to stop translation.

[0116] Non-transgenic, homozygous gene-edited slead1 mutant tomatoes were obtained through PCR and sequencing of subsequent generations. PCR screening for Cas9 protein in the vector was performed using primer sequences: F: GGACAACGAGGAGAATGAGG and R: TGTCTCGACCAGCTGCCTCTT. The PCR reaction system consisted of 20 μL of 1 μL E. coli, 1 μL of primers, 10 μL of 2× EsTaq Master Mix (Dye), and 8 μL of ddH2O.

[0117] Obtain varieties with increased salt tolerance: Wild-type and slead1 mutant plants were treated with 200 mM NaCl solution to simulate a salt stress environment. By evaluating the response of tomato plants to salt stress and measuring physiological indicators related to superoxide anion, hydrogen peroxide content, catalase activity, and malondialdehyde content, it was determined that the salt tolerance of non-transgenic and homozygous gene-edited slead1 mutant tomatoes was increased.

[0118] The specific operations are as follows:

[0119] First, Micro-Tom wild-type tomato seedlings were treated with water and 200 mM NaCl for 4 h, respectively. The expression of the SlEAD1 gene was detected by qRT-PCR (primer sequences: F-CATCGTGCTAGTGGTTCCCC and R-ATCATCACCACCAAAGAGCGA). The results are as follows Figure 3 As shown, salt treatment for 4 h could inhibit the expression of SlEAD1 gene, which indicated that the tomato SlEAD1 gene responded to salt stress.

[0120] Since SlEAD1 expression is affected by NaCl, and some EAR proteins are also involved in regulating plant salt stress responses, it is suggested that SlEAD1 may be involved in the salt stress response of tomato. Therefore, we examined the response of SlEAD1 overexpression and gene-edited mutant plants to salt stress and the changes in related physiological and biochemical indicators.

[0121] The specific steps of the tomato plant response to salt stress experiment are as follows: wild-type, overexpression and mutant tomatoes were sown in square pots filled with a mixture of nutrient soil and vermiculite, and grown normally for 2-3 weeks in a culture room at 25°C and 16h / 8h light. After taking pictures, they were irrigated with water and 200mM NaCl solution respectively until the tomato plants showed obvious differences. The survival rate was calculated after taking pictures.

[0122] After calculating the survival rate, samples were taken from the plants and their malondialdehyde (MDA) content was determined. 100 mg of leaves were placed in a 1.5 mL centrifuge tube and the extract from the malondialdehyde (MDA) kit (Nanjing Jiancheng Bioengineering Institute) was added. After grinding until homogenized, reagents 2 and 3 were added sequentially according to the instructions. The tubes were placed in a 95°C waterbath for 40 minutes. After the waterbath, they were immediately removed and cooled under running water. The supernatant was collected and the absorbance of each tube was measured in a spectrophotometer at 532 nm and a 1 cm optical path. The tubes were zeroed with distilled water.

[0123] After calculating the survival rate, samples were taken from the plants for catalase activity measurement. 100 mg of leaves were placed in a 1.5 mL centrifuge tube and the buffer provided in the Solebo Plant Catalase (CAT) Activity Kit was added. The sample was ground into a homogenate and centrifuged at 8000 g for 10 minutes. The supernatant was separated and used as the enzyme solution to be tested. According to the kit instructions, Reagent 1 and Reagent 2 were mixed in a specific ratio to prepare the CAT working solution. The sample and reagents were added to an ELISA plate, and the initial absorbance (A1) and the post-reaction absorbance (A2) were measured at 240 nm. Enzyme activity was then calculated.

[0124] NBT and DAB staining steps: Take leaves of normally grown wild type, overexpression and mutants and treat them with 200mM NaCl solution (water treatment as control) until wilting occurs. Use NBT and DAB buffer in the plant hydrogen peroxide staining solution (DAB method) and superoxide anion (NBT method) kit of Wuhan Sewell Biotechnology Co., Ltd. to stain for 4-6 hours (regular observation is required). Use 95% ethanol and heat at 80°C to decolorize until all chlorophyll is removed. Observe and take pictures.

[0125] The experimental results are as follows Figure 4 As shown, the results showed that the slead1 mutant plants had improved salt tolerance, reduced ROS accumulation, increased catalase activity, and reduced malondialdehyde content, while the overexpression plants had the opposite effect.

[0126] Preparation for obtaining varieties with increased salt tolerance using molecular breeding

[0127] To confirm that the SlEAD1 gene is a salt stress response gene: wild-type tomatoes were treated with 200 mM NaCl solution, and water treatment was used as a control. qRT-PCR was then used to detect the expression level of the SlEAD1 gene in the control and salt stress treated samples.

[0128] The CRISPR / Cas9 system was used to construct a gene editing vector, which was used to transform wild-type tomato callus tissue to obtain gene-edited positive plants. Non-transgenic tomatoes were then screened using PCR, and sequencing was used to detect the gene editing method of non-transgenic tomato plants, resulting in non-transgenic homozygous gene-edited slead1 mutant tomatoes.

[0129] Since SlEAD1 is a salt stress response gene, the obtained non-transgenic homozygous gene-edited slead1 mutant was used for salt stress response experiments.

[0130] The wild type and slead1 mutant were subjected to salt stress treatment using 200 mM NaCl solution for about 14 days. Normally watered plants served as controls. Each treatment and each line were replicated at least three times.

[0131] After treatment, the response of tomato plants to salt stress was observed.

[0132] The relevant physiological indicators of plants such as superoxide anion, hydrogen peroxide content, catalase activity and malondialdehyde content were measured.

[0133] The results showed that the salt tolerance of slead1 mutant plants was improved, the superoxide anion and hydrogen peroxide contents were reduced, the catalase activity (CAT) was increased, and the malondialdehyde (MDA) content was reduced.

[0134] The present invention identifies a novel EAR motif transcriptional repressor SlEAD1 (Solanum lycopersicum EAR motif-containing ABA down-regulated 1) in tomato and uses CRISPR / Cas9 gene editing technology to create a non-transgenic homozygous slead1 mutant, which significantly improves the salt tolerance of tomatoes. The specific technical effects are as follows: (1) Optimization of salt stress physiological indicators: Under 200mM NaCl treatment, the survival rate of the slead1 mutant is increased by more than 35% compared with the wild type, the malondialdehyde (MDA) content in the leaves is reduced by 42%, the catalase (CAT) activity is increased by 2.1 times, and the superoxide anion (O2 - ) and hydrogen peroxide (H2O2) accumulation decreased by 58% and 63%, respectively; (2) The molecular regulatory mechanism is clear: SlEAD1 negatively regulates salt tolerance by inhibiting the expression of key genes in the ABA signaling pathway (SlNCED2 and SlSnRK2s). qRT-PCR confirmed that the expression of SlEAD1 decreased by 60% after 4 hours of salt stress; (3) Breeding safety assurance: PCR detection of the mutant showed no exogenous Cas9 gene residues, meeting the requirements of non-transgenic, and the trait is stably inherited. This technology provides a new target gene with independent intellectual property rights and a salt-tolerant variety that can be commercialized for tomato cultivation in saline-alkali land.

[0135] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A tomato EAR transcription factor SlEAD1, characterized in that Its nucleotide sequence is shown in SEQ ID NO.

1.

2. A protein encoded by the tomato EAR transcription factor SlEAD1 gene according to claim 1, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

2.

3. A recombinant expression vector, characterized in that: The method contains the tomato EAR transcription factor gene SlEAD1 as claimed in claim 1, and the vector is pCAMBIA1302 or a CRISPR / Cas9 gene editing vector.

4. An expression cassette or transgenic cell line, characterized in that Comprising the recombinant expression vector according to claim 3.

5. Use of the SlEAD1 gene according to claim 1, the protein according to claim 2, the recombinant expression vector according to claim 3, or the expression cassette or transgenic cell line according to claim 4 in any of the following: (1) Regulate salt tolerance of tomatoes; (2) Cultivate tomato plants with enhanced salt tolerance.

6. A non-transgenic slead1 mutant tomato plant, characterized in that: The method is obtained by editing the SlEAD1 gene according to claim 1 through CRISPR / Cas9.

7. A method for cultivating non-transgenic slead1 mutant tomato plants according to claim 6, characterized in that: The following steps are involved: (1) Design sgRNA targeting the SlEAD1 gene and construct a CRISPR / Cas9 editing vector; (2) Transform tomato callus and screen for positively edited plants; (3) Obtain homozygous mutants without exogenous DNA through progeny separation.

8. A molecular marker for detecting salt tolerance of tomatoes, characterized in that: The marker is based on the editing site of the tomato EAR transcription factor SlEAD1 as claimed in claim 1, and the slead1 mutant is identified by PCR or sequencing.

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