Application of SlWRKY46 gene in improvement of tomato salt stress resistance

Abstract

The invention belongs to the technical field of modern agriculture, and particularly relates to application of an SlWRKY46 gene in improvement of tomato salt stress resistance. According to the invention, a tomato SlWRKY46 gene knockout and overexpression plant is constructed through a gene means, and the expression level of the SlWRKY46 protein is regulated to research the regulation mechanism of the SlWRKY46 protein on the salt resistance of tomatoes. Results show that salt stress can obviously induce transcription of SlWRKY46 and accumulation of SlWRKY46 protein, and overexpression of SlWRKY46 under salt stress can improve accumulation of tomato biomass, increase the content of green fluorescence in leaves and reduce the content of conductivity, so that salt stress tolerance of tomatoes is improved. In the modern agricultural industry, a gene resource is provided for cultivating a salt-tolerant tomato new variety, and the gene has a relatively good potential application value and lays a theoretical foundation for researching a mechanism of tomato plants responding to stress signals and a molecular mechanism of the tomato plants responding to complex and changeable environmental factors.

Description

Technical Field

[0001] This invention belongs to the field of modern agricultural technology, specifically involving the application of the SlWRKY46 gene in improving the salt stress resistance of tomatoes. Background Technology

[0002] tomato( Solanum lycopersicum Tomatoes (L.) belong to the genus *Solanum* of the Solanaceae family and are annual or perennial herbaceous plants, one of the most widely cultivated vegetable crops in the world. They are characterized by their preference for light and warm temperatures and their inability to tolerate salt. Salt stress is one of the major abiotic stresses affecting tomato growth and development, leading to yield reduction. Due to the long-term high temperature, high humidity, and lack of rainfall leaching in greenhouse soils, coupled with unreasonable practices such as continuous cropping, fertilization, planting, and irrigation, soil salinization in greenhouses is becoming increasingly serious, significantly reducing vegetable yield and quality. To alleviate the impact of salt stress on the greenhouse vegetable industry, various methods can be used to mitigate soil salinization during greenhouse cultivation. These include exogenous osmotic pressure regulators, exogenous growth regulators, rational fertilization, biological salt removal, engineering drainage, and changes in the cultivation environment. However, while exogenous material treatment and engineering drainage are quick to take effect, they are costly; biological improvement, although requiring less investment and without secondary pollution, is slow to show results. Therefore, exploring a scientific, effective, environmentally friendly, and low-cost approach to synergistically improve resistance and quality, in line with the modernization and sustainable development strategy of greenhouse agriculture, is particularly important.

[0003] Therefore, it is urgent to explore the response of tomatoes to salt stress and its molecular mechanism, to find key genes that can improve the salt stress resistance of tomatoes, and to enhance the resistance of tomatoes and reduce the harm of salt stress. Summary of the Invention

[0004] The first aspect of this invention aims to provide the application of overexpression of the SlWRKY46 gene in improving the salt stress resistance of tomatoes.

[0005] A second aspect of the present invention is to provide an application.

[0006] A third aspect of the present invention is to provide a method.

[0007] To achieve the above-mentioned objectives of this invention, the technical solution adopted by this invention is as follows: In a first aspect, the invention provides the application of overexpression of the SlWRKY46 gene in improving salt stress resistance in tomatoes.

[0008] In some embodiments of the present invention, the nucleotide sequence of the SlWRKY46 gene is shown in SEQ ID NO: 1.

[0009] A second aspect of the invention provides the application of a1) to a3) in at least one of b1) to b4): a1) Overexpression of SlWRKY46 protein; a2) Biomaterials associated with overexpression of SlWRKY46 protein; a3) Reagents that target and upregulate the expression level of SlWRKY46 protein and / or enhance the activity of SlWRKY46 protein; b1) Improve the salt stress resistance of tomatoes; b2) Develop tomato varieties; b3) Prepare products that improve the salt stress resistance of tomatoes; b4) Prepare products for breeding tomato varieties.

[0010] In some embodiments of the present invention, the biomaterial comprises at least one of c1) to c12): c1) Nucleic acid molecules encoding the SlWRKY46 protein; c2) An expression cassette containing the nucleic acid molecule described in c1); c3) A recombinant vector containing the nucleic acid molecule described in c1); c4) A recombinant vector containing the expression cassette described in c2); c5) Recombinant cells containing the nucleic acid molecules described in c1); c6) Recombinant cells containing the expression cassette described in c2); c7) Recombinant cells containing the recombinant vector described in c3); c8) Recombinant cells containing the recombinant vector described in c4); c9) Recombinant microorganisms containing the nucleic acid molecules described in c1); c10) Recombinant microorganisms containing the expression cassette described in c2); c11) Recombinant microorganisms containing the recombinant vector described in c3); c12) Recombinant microorganisms containing the recombinant vector described in c4).

[0011] In some embodiments of the present invention, the tomato variety includes the following feature: enhanced resistance to salt stress.

[0012] In some embodiments of the present invention, the nucleic acid molecule includes a nucleic acid molecule encoding the protein shown in SEQ ID NO: 2.

[0013] In some embodiments of the present invention, the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO: 1.

[0014] Those skilled in the art will understand that, due to the degeneracy of codons, other pairs SlWRKY46A technical solution that modifies the gene but still encodes a protein with an amino acid sequence as shown in SEQ ID NO: 2 can also achieve the same or similar technical effects as the present invention and is still within the scope of protection of the present invention.

[0015] In some embodiments of the present invention, the vector includes an overexpression vector.

[0016] In some embodiments of the present invention, the vector includes overexpression vectors, including but not limited to conventional overexpression vector tools in the art such as pFGC1008-HA.

[0017] In some embodiments of the present invention, the cells include at least one of *Escherichia coli* and *Agrobacterium*. *Escherichia coli* is a commonly used host cell in the art for constructing vectors and plasmids, while *Agrobacterium* is a common tool in the art for delivering DNA molecules to plants.

[0018] In some embodiments of the present invention, the biological material does not include reproductive material.

[0019] A third aspect of the invention provides a method comprising the step of increasing the expression level and / or activity of the SlWRKY46 protein in tomatoes.

[0020] In some embodiments of the present invention, the method is at least one of i1) to i2): i1) a method for improving the salt stress resistance of tomatoes; i2) a method for breeding tomato varieties.

[0021] In some embodiments of the present invention, the tomato variety includes the following feature: enhanced resistance to salt stress.

[0022] In some embodiments of the present invention, the tomato variety includes the following characteristics: increased salt stress resistance relative to a reference level; the reference level is the level of the wild type.

[0023] In some embodiments of the present invention, the step of increasing the expression level and / or activity of SlWRKY46 protein in tomatoes is to introduce a nucleic acid molecule encoding SlWRKY46 protein into tomato tissue or tomato cells.

[0024] In some embodiments of the present invention, the introduction method includes at least one of the following: using Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, and electroporation.

[0025] The beneficial effects of this invention are: This invention marks the first discovery of the application of tomato SlWRKY46 in tomato variety breeding. SlWRKY46 can regulate multiple aspects of tomatoes, including salt tolerance, biomass accumulation, photosynthetic levels, and plant height. This invention also marks the first construction of a tomato... SlWRKY46Transgenic plants with gene overexpression and gene knockout were studied for their functional characteristics. Through salt stress treatment experiments, it was found that... SlWRKY46 The gene plays a positive regulatory role in salt tolerance in tomatoes. This invention provides... SlWRKY46 Genes provide genetic resources for breeding new salt-tolerant tomato varieties and have good potential application value. In the field of modern agricultural industry, they lay a theoretical foundation for studying the regulatory network and signaling mechanism of tomato response to stress signals, and are of great significance and wide application value for breeding new salt-tolerant tomato varieties, promoting the commercialization of tomatoes, and modernizing the breeding process. Attached Figure Description

[0026] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 Evolutionary and structural analysis of the SlWRKY46 protein sequence.

[0027] Figure 2 In Embodiment 3 of the present invention SlWRKY46 Western blotting results of genes and proteins in tomato lines overexpressing the gene; where A represents tomato. SlWRKY46 Gene expression levels, where B represents the expression level of the tomato SlWRKY46 protein.

[0028] Figure 3 The mutant in Example 3 of this invention wrky46 Sequencing results of plant sgRNA sequences and mutant materials SlWRKY46 Gene expression levels; among which A mutant wrky46 A simplified diagram of the sequencing results of the sgRNA sequence of the plant, B represents tomato. SlWRKY46 Gene expression level.

[0029] Figure 4 Example 1, which illustrates the effects of this invention, shows tomatoes subjected to salt stress for different durations. SlWRKY46 Changes in gene expression.

[0030] Figure 5 The wild-type plants and overexpression plants in Example 1 of this invention SlWRKY46 -OE and mutants wrky46 The aboveground growth phenotype, fresh weight, and dry matter accumulation level of plants were measured 7 days after normal and salt stress treatments. Among them, A represents the aboveground growth of tomatoes, B represents the aboveground fresh weight of tomatoes, C represents the percentage decrease in aboveground fresh weight of tomatoes under salt stress for the three materials, D represents the aboveground dry weight of tomatoes, and E represents the percentage decrease in aboveground biomass of tomatoes under salt stress for the three materials.

[0031] Figure 6 The wild-type plants and overexpression plants in Example 1 of this invention are examples of the effects of this invention. SlWRKY46 -OE and mutants wrky46 The statistical results of plant height after 7 days of normal and salt stress treatment; where A is the statistical results of tomato plant height, and B is the percentage of tomato plant height reduction under salt stress in the three materials.

[0032] Figure 7 The wild-type plants and overexpression plants in the embodiments of this invention are examples of plants. SlWRKY46 -OE and mutants wrky46 The root growth phenotype, fresh weight, and dry matter accumulation levels of plants after 7 days of normal and salt stress treatments were measured. Among them, A represents the underground growth of tomatoes, B represents the fresh weight level of tomato roots, C represents the percentage decrease in fresh weight of tomato roots under salt stress for the three materials, D represents the dry weight level of tomato roots, and E represents the percentage decrease in biomass of tomato roots for the three materials under salt stress.

[0033] Figure 8 The wild-type plants and overexpression plants in Example 1 of this invention are examples of the effects of this invention. SlWRKY46 -OE and mutants wrky46 The relative electrical conductivity levels of leaves of plants after 7 days of normal and salt stress treatment.

[0034] Figure 9 The wild-type plants and overexpression plants in Example 1 of this invention are examples of the effects of this invention. SlWRKY46 -OE and mutants wrky46 Changes in the maximum photochemical efficiency (Fv / Fm) of PSII in leaves of plants after 7 days of normal and salt stress treatment.

[0035] Significance Explanation: Experimental data in this invention are expressed as mean ± standard deviation (n = 3). * and different letters indicate significant differences between treatments. P <0.05, Student's t test or Tukey's test). Detailed Implementation

[0036] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0037] Example 1 SlWRKY46 Phylogenetic analysis of transcription factors In order to build SlWRKY46 The transcription factor phylogenetic tree was constructed by first obtaining homologous sequences from closely related species such as tomato, Arabidopsis thaliana, and maize from the NCBI website, and then using MEGA11 software. For tomato (… Solanum lycopersicum L.) WRKY family, Arabidopsis thaliana ( Arabidopsis thaliana (L.) Heynh.) WRKY family, corn ( Zea mays Phylogenetic analysis was performed on the protein sequences of some members of the L. WRKY family. A phylogenetic tree was constructed using MEGA11, and the Maximum Likelihood (MJ) algorithm was employed for tree generation. The bootstrap parameter was validated by repeating the algorithm 1000 times. The analysis results are as follows: SlWRKY4 6 and SlWRKY45 It is most closely related to Arabidopsis thaliana AtWRKY46 and cotton GhWRKY46, but is more distantly related. Figure 1 ).

[0038] Previous studies have found that cotton WRKY46 can respond to salt stress, with its expression level increasing after 24 h of salt stress treatment, showing a certain degree of resistance to salt stress. For Arabidopsis thaliana, in open soil environments, plants overexpressing AtWRKY46 are more sensitive to salt stress; conversely, when cultured on closed agar plates, they show resistance to salt stress. SlWRKY46 with Arabidopsis AtWRKY46 ,cotton GhWRKY46 The homology is relatively distant, thus the identification and research of the function of tomato WRKY46 is more innovative.

[0039] SlWRKY46

[0040] The amino acid sequence of SlWRKY46 protein is: MGNKSFFIDLNTNPLLHNINRSPIPRETLDEELSRMREENKKLATTLTSLYEKYNSLQTHIIELQQKYSNHEEDNSKLLLSRKRKAEEDYCVNNSYINFEEASPKMPREITTNISTVCV KTNPSDQTSVVKDGYNWRKYGQKVTRDNPSPRAYFKCSFAPSCPVKKKVQRSVKDASILVATYEGEHNHPQPPQAEITVPLVNTTDPTFLNKFMKDINTNSVQQQYLVEQMASSLSKNPSFAATVATAISGLLF (SEQ ID NO: 2).

[0041] Example 2 SlWRKY46 Construction of gene overexpression vectors SlWRKY46 The gene originates from tomato and is identified as Solyc08g067340 in the tomato genome database (https: / / solgenomics.net / ). To investigate... SlWRKY46 The effect of overexpression on the salt stress resistance of tomatoes was first investigated by cloning a gene from the tomato genome. SlWRKY46 Gene. Based on coding region sequence analysis, specific primers were designed. SlWRKY46 -F and SlWRKY46 -R, and add restriction enzyme sites (AscI and KpnI) to the primers respectively. SlWRKY46 The sequence of -F is: ttacaattaccatggggcgcgccATGGGAAACAAGTCTTTTTTTATTGA (5'-3', SEQ ID NO: 3); SlWRKY46 The sequence of -R is: aacatcgtatgggtaggtaccAAAAAGTAATCCTGAGATGGCTGTAG (5'-3', SEQ ID NO: 4). PCR amplification was performed using KOD high-fidelity enzyme. SlWRKY46The PCR-amplified fragment and vector were digested with enzymes, and the SlWRKY46 fragment was ligated into pFGC1008-HA to obtain the plant overexpression vector pFGC1008::SlWRKY46-HA. The recombinant plasmid was sent to Youkang Company for sequencing confirmation. The nucleotide sequence of the obtained gene SlWRKY46 is shown in SEQ ID NO: 1; the amino acid sequence of the protein encoded by this gene is shown in SEQ ID NO: 2. The results showed that the cloned sequence was consistent with the sequence published in Solgenomics (Solyc08g067340). The positive plasmid was extracted for later use and named pFGC1008::SlWRKY46-HA.

[0042] Example 3 Construction of CRISPR / Cas9 gene knockout vector Designed using the CRISPR-P website (http: / / cbi.hzau.edu.cn / cgi-bin / CRISPR). SlWRKY46 The target sequences of the gene are sgRNA1: TTATTGCACAACATCAATAG (5'-3', SEQ ID NO: 5); sgRNA2: AGATTGAGTAGGATGAGAG (5'-3', SEQ ID NO: 6). Primers sgRNA-F1: CTGGTCTCTATTGaacaaagcaccagtggtctagtg (SEQ ID NO: 7) were designed based on the sgRNA sequences. WRKY46 -R1: CTGGTCTCTTTATTGCACAACATCAATAGtgcaccagccgggaa (SEQ ID NO: 8), sgRNA- WRKY46 -F2:GCTGGTCTCT ATAA gttttagagctagaaatagcaagtta (SEQ ID NO: 9), sgRNA- WRKY46 -R2: GCTGGTCTCTAAACCTCTCATCCTACTCAATTCTtgcaccagccgggaatcg (SEQ ID NO: 10). Then, using tRNA as a template, PCR amplification was performed with the designed primers and a high-fidelity enzyme. After verifying the correct bands with agarose gel electrophoresis, the positive plasmid was purified and extracted. It was then ligated into the pHEE401 vector using BsaI restriction enzyme and T4 ligase from the NEBGolden Assembly Kit. Subsequently, E. coli were transformed by heat shock. After the single-clone sequencing results matched the target sequence, the bacterial culture was preserved and the positive plasmid was extracted for later use, named pHEE401- SlWRKY46 .

[0043] Example 4 SlWRKY46 Obtaining transgenic plants The plant overexpression vector pFGC1008::SlWRKY46-HA and the gene editing vector pHEE401-SlWRKY46 were transformed into Agrobacterium GV3101 via electroporation. Wild-type (Ailsa Craig) tomato cotyledons were then infected. Callus induction, resistance-induced differentiation, and rooting culture were performed to obtain tissue culture seedlings, which were then used for single-plant verification and seed harvesting. The verification methods are as follows: quantitative real-time fluorescence assay and Western blotting were used for verification. SlWRKY46 In overexpression-positive transgenic plants, quantitative fluorescence experiments showed that, compared with wild-type, the overexpression lines... SlWRKY46 Gene expression is significantly upregulated (e.g.) Figure 2 (As shown in Figure A); Western blotting results showed that the wild-type did not have a SlWRKY46-HA protein band, while the overexpressing plants showed a clear SlWRKY46-HA band (as shown in Figure A). Figure 2 (As shown in Figure B). The primer sequences used for the quantitative fluorescence experiment are as follows: RT-SlWRKY46-F:CCCTCCGATCAAACTTCAGTGGTGA (SEQ ID NO: 11); RT-SlWRKY46-R: GGTGGTTGGGGATGATTGT (SEQ ID NO: 12); RT-SlACTIN2-F: TGTCCCTATTTACGAGGGTTATGC (SEQ ID NO: 13); RT-SlACTIN2-R: CAGTTAAAATCACGACCAGCAAGAT (SEQ ID NO: 14); RT-SlUBI3-F: GCCGACTACAACATCCAGAAGG (SEQ ID NO: 15); RT-SlUBI3-R:TGCAACACAGCGAGCTTAACC (SEQ ID NO: 16).

[0044] Positive results were verified using plant tissue DNA extraction, PCR, and sequencing techniques. SlWRKY46 The mutant transgenic plant (the plant transformed into the pHEE401-SlWRKY46 vector prepared in Example 2) was sequenced and found to be a mutant. wrky46 The plant (SlWRKY46 CRISPR / Cas9 knockout line) has a 1-base deletion at target site 121 and a 3-base deletion at target site 364. Figure 3(A), and due to the deletion of a base, a stop codon appears prematurely to stop translation, thereby achieving the effect of inactivating the WRKY46 protein; this example also tested wild-type and mutant proteins. wrky46 In the leaves WRKY46 Regarding gene expression levels, the study found that compared to the wild type, WRKY46 Gene expression showed a significant decreasing trend in mutants. wrky46 Almost undetectable in the material WRKY46 Gene expression ( Figure 3 (B)

[0045] Effect Example SlWRKY46 Salt tolerance evaluation of transgenic plants 1. Experimental materials The tomato varieties used in the experiment were wild-type Ailsa Craig and the variety obtained in Example 4. SlWRKY46 overexpression and SlWRKY46 CRISPR / Cas9 knockout lines.

[0046] 2. Experimental Methods This invention specifically uses a mixture of peat moss and vermiculite (2:1, v / v) to simulate real-world soil cultivation. Its advantages include simulating the physical support environment for roots, possessing a certain porous structure that allows roots to extend more naturally and interact with particles, thus more closely resembling the real soil rhizosphere environment. After salt stress drenching, the physiological responses of the entire plant under near-soil conditions can be measured, allowing for more precise measurement of changes in plant height and biomass after salt stress. In contrast, culture medium cultivation involves plant roots growing in a homogeneous, non-granular gel or liquid medium, which differs significantly from natural plant growth, resulting in lower ecological realism and potential discrepancies between experimental conclusions and production practices. Therefore, this invention employs a simulated soil cultivation method, which is more consistent with actual production practices.

[0047] Tomato seeds were disinfected by soaking in 55℃ warm water for 15 minutes, then transferred to a shaker at 28℃ and 200 rpm for 2 days to germinate. Once approximately 80% of the seeds showed signs of germination, they were sown in 72-cell trays containing a mixture of peat moss and vermiculite (2:1, v / v) for seedling cultivation. Seedlings were then cultured in a plant factory under the following conditions: room temperature 25℃ / 20℃, photosynthetic photon flux density 300 μmol·m⁻²·s⁻¹, and photoperiod 12 / 12 h (day / night). After emergence, the substrate was kept moist by watering according to its moisture content. Hogrange nutrient solution (PH1782, PHYGENE) was applied throughout the process. When the tomato seedlings reached the 3-leaf, 1-heart stage (approximately 15 days after sowing), they were transplanted into 10 cm diameter, 9 cm deep seedling pots and placed in the plant factory.

[0048] Salt stress treatment and control: Salt stress treatment was conducted when tomato seedlings reached the five-leaf stage. Treatment consisted of 200 mL of Hogrange nutrient solution (PH1782, PHYGENE) containing 250 mM NaCl per plant as the salt stress treatment, and an equal volume (200 mL) of Hogrange nutrient solution containing 0 mM NaCl as the control. Root irrigation was performed every three days. Phenotypic imaging was conducted 7 days after salt stress treatment, and dry weight, maximum photochemical efficiency of photosystem II, and relative conductivity were measured. For gene and protein experiments in response to salt stress, RNA samples were collected at 0 h, 6 h, 12 h, 24 h, 48 h, and 72 h after salt stress treatment.

[0049] Total RNA extraction, cDNA synthesis, and gene expression analysis in tomatoes: RNA was extracted from young tomato leaves subjected to salt stress for 0h, 6h, 12h, 24h, 48h, and 72h. After liquid nitrogen grinding of plant leaf tissues, total RNA was extracted using a plant total RNA extraction kit (Tiangen, Beijing) according to the manufacturer's instructions. After confirming the RNA sample concentration and quality with Nanodrop, the RNA was reverse transcribed into cDNA using the ReverTraAce qPCR RT Kit (Toyobo) (containing genomic DNA removal enzyme) according to the manufacturer's instructions. Real-time quantitative PCR was performed using the SYBR fluorescent dye kit (Takala) on a Roche Light Cyclist 480 PCR instrument. Tomato leaves were used as samples. ACTIN2 and UBI3 ( ubiquitin 3 The gene was used as an internal reference, and the relative expression level of the gene was calculated according to the method of (Livak and Schmittgen., 2001).

[0050] Protein extraction and Western blot analysis of tomato leaves: For the extraction of total protein from tomatoes, approximately 0.1 g of young, normally grown tomato leaves were ground into powder in liquid nitrogen. 0.2–0.3 mL of extraction buffer (100 mM HEPES, pH 7.5, 5 mM EDTA, 5 mM EGTA, 10 mM DTT, 10 mM NaVO3, 10 mM NaF, 50 mM glycerophosphate, 10% (v / v) glycerol, 1 mM PMSF and 5% (w / v) PVPP) was added. After vortexing, the mixture was centrifuged at 13000 rpm at 4 °C for 20 min. The supernatant contained the obtained protein. Protein content was quantified using Coomassie Brilliant Blue. The supernatant was then mixed with 2× loading buffer (250 mM Tris-HCl, pH 7.5). 6.8, 10% (w / v) SDS, 0.5% (w / v) bromophenol blue, 50% (v / v) glycerol, 10 mM DTT) in equal volumes, and denatured at 95 °C for 10 min. 80 or 100 μg of total protein was separated by 10% (w / v) SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The membrane was blocked at room temperature for 1 h with TBST (20 mM Tris, pH 7.5, 30 mM NaCl, 0.05% (v / v) Tween 20) containing 5% (w / v) BSA. The membrane was then washed 3-5 times with TBST for 5 min each time.

[0051] For WRKY46 protein: Incubation was performed for 1 h at room temperature with 0.1% HA polyclonal antibody (Abcam, ab18181, Cambridge, MA, USA). After washing, incubation was performed for 1 h at room temperature with anti-mouse-HRP conjugated antibody (Abcam, ab205719, Cambridge, MA, USA). The membrane was washed 5 times with TBST for 5 min each time. Finally, the signal on the blot was observed using a high-sensitivity chemiluminescence assay kit (Perkin Elmer, Massachusetts, USA) according to the manufacturer's instructions. Plant actin monoclonal antibody (Q30, Cat#YM3034) was used as a control for Western blot analysis.

[0052] The method for determining the relative conductivity of the plant is as follows: After treatment, tomato leaves are evenly cut into strips of appropriate length (avoiding the midrib). Three fresh samples, each weighing 0.2g, are quickly weighed and placed in graduated centrifuge tubes containing 20mL of deionized water. The tubes are capped and placed in a shaker at 28℃ for 1.5-2 hours for extraction. The conductivity R1 of the extract is measured using a conductivity meter. Then, the extract is heated in a boiling water bath for 15 minutes, cooled to room temperature, and shaken well. The conductivity R2 of the extract is measured again. Relative conductivity = R1 / R2 * 100%.

[0053] The specific method for determining the maximum photochemical efficiency of photosystem II is as follows: After the plant has been placed in a dark environment for 30 minutes to adapt, it is then illuminated with detection light (<0.5 μmol m-) using a chlorophyll fluorescence imaging spectrometer (IMAG-PAM; Heinz Walz, Germany). 2 s -1 The minimum fluorescence Fo was measured, and then saturated with a pulse of light (4000 μmol m). -2 s -1 The maximum fluorescence Fm was measured.

[0054] Fluorescence parameter calculation method: PSⅡ maximum photochemical efficiency (Fv / Fm) = (Fm-Fo) / Fm.

[0055] 3. Experimental Results 1) Tomato SlWRKY46 response to salt stress The tomato varieties used in the experiment were wild-type Ailsa Craig and the variety obtained in Example 3. SlWRKY46 overexpression and SlWRKY46 CRISPR / Cas9 knockout lines were sown in plastic pots filled with a 3:1 mixture of peat moss and vermiculite. After germination, the substrate was kept moist by watering according to its moisture content. Hoagland's solution was applied throughout the process. When the tomato seedlings reached the five-leaf stage, salt stress treatment was administered: 200 mL of Hoagland's solution (pH 1782, PHYGENE) containing 250 mM NaCl per plant was used as the salt stress treatment (NaCl), and an equal volume (200 mL) of Hoagland's solution containing 0 mM NaCl was used as the control. Treatments were performed every three days. After 7 days of salt stress treatment, phenotypic images were taken, and dry weight, maximum photochemical efficiency of photosystem II, and relative conductivity were measured. RNA samples were collected at 0h, 6h, 12h, 24h, 48h, and 72h of salt stress treatment.

[0056] First, wild-type Ailsa Craig tomato seedlings that had reached the five-leaf-one-heart stage were subjected to salt stress treatment by applying 200 mL of Hoagland nutrient solution containing 250 mM NaCl to each plant. RNA samples were collected at 0 h, 6 h, 12 h, 24 h, 48 h, and 72 h of salt stress treatment, and relevant tests were performed. SlWRKY46 The results of gene transcription are as follows Figure 4 As shown, salt stress can significantly induce SlWRKY46 Transcription, with increasing salt stress treatment time, SlWRKY46 Gene transcription initially showed a significant increase, followed by a gradual decrease. Salt stress treatment for 12 hours induced... SlWRKY46 The results, indicating the highest gene expression level, suggest that the tomato SlWRKY46 responds to salt stress.

[0057] 2) Effects of SlWRKY46 on tomato phenotype Next, the wild-type tomato seedlings with five leaves and one heart (WT) and the seedlings obtained in Example 3 were... SlWRKY46 Gene overexpression lines ( WRKY46 -OE) and mutant lines ( wrky46 Salt stress treatment was carried out: 200 mL of Hogland nutrient solution containing 250 mM NaCl per plant was used as salt stress treatment, and an equal volume (200 mL) of Hogland nutrient solution containing 0 mM NaCl was used as control. The plants were treated by root irrigation every three days. After one week, the growth phenotype and salt damage phenotype of wild-type, overexpression lines and mutant lines of tomato plants were observed.

[0058] Actual photographs showing the above-ground growth of tomatoes are shown below. Figure 4 As shown in Figure A, the results of the aboveground fresh weight weighing are as follows: Figure 5 As shown in Figure B, the decrease in fresh weight of the aboveground parts of tomatoes under salt stress was compared to that of the three materials. Figure 5 As shown in Figure C, the results of the aboveground dry weight weighing are as follows: Figure 5 As shown in Figure D, the aboveground biomass of tomatoes decreased by a certain percentage under salt stress compared to other materials. Figure 5 As shown in Figure E. The effects of salt stress on plant growth and development can be manifested in multiple ways. Among these, changes in plant biomass are a direct reflection of plant resistance to salt stress and a direct indicator of plant tolerance, reflecting to some extent the plant's growth status and ability to withstand salt stress. The results showed that after wild-type tomato plants were subjected to salt stress, the leaves turned yellow, and the aboveground fresh weight and biomass decreased significantly, with reductions of 50.69% and 43.62%, respectively. (Mutant) wrky46 The plants showed the worst tolerance to salt stress, and after salt stress... wrky46 The leaves at the base of the stem above ground turned yellow and fell off, causing the plant to die. Figure 5 (A). Moreover, after salt stress wrky46The mutant plants showed the greatest reduction in both fresh and dry weight of the aboveground parts, with decreases of 68.75% and 60.16%, respectively. However, WRKY46 -OE plants showed the strongest tolerance to salt stress, under salt stress WRKY46 -OE plants retain their vibrant green leaves, showing no signs of yellowing, and WRKY46 -OE plants showed the smallest reduction in aboveground fresh weight and biomass after salt stress, with decreases of 42.78% and 32.14%, respectively. Figure 5 (BE). Statistical results of tomato plant height phenotype are as follows: Figure 6 As shown. Studies can reveal that, under normal growth conditions, mutants... wrky46、 WT and WRKY46 -OE showed no significant difference in plant height. Crop salt tolerance can be assessed using indicators such as plant dry matter and plant height, with plant height being one of the more sensitive indicators of salt stress. The study also found that under salt stress treatment, WT, WRKY46 -OE and wrky46 The plant height of all three plants decreased significantly under salt stress. WRKY46 -OE plants showed the lowest percentage decrease in plant height, at 9.13%. wrky46 The mutant plants showed the highest reduction in plant height, at 31.91%, further proving... WRKY46 -OE plants are more tolerant of salt stress.

[0059] Actual photographs showing the underground growth of tomatoes are shown below. Figure 7 As shown in Figure A, the results of the fresh weight measurement of the underground part are as follows: Figure 7 As shown in Figure B, the decrease in fresh weight of tomato roots under salt stress was compared to that of the three materials. Figure 7 As shown in Figure C, the root dry weight weighing results are as follows: Figure 7 As shown in Figure D, the decrease in tomato root biomass under salt stress was compared to that of the three materials. Figure 7 As shown in Figure E, salt stress significantly inhibits tomato growth, resulting in stunted plants, yellowing leaves, and poor root development. The study found that under salt stress, the root system of WT tomato plants was significantly inhibited, with fewer roots and significantly reduced fresh and dry weight. Compared to the control group without salt stress, the reduction rates were 24.85% and 30.95%, respectively. wrky46 The plant's root system was most severely inhibited, with shorter and fewer roots, resulting in a significant reduction in both fresh and dry weight, decreasing by 38.22% and 55%, respectively. However, WRKY46 -OE plants showed the strongest tolerance to salt stress, under salt stress WRKY46 -OE plants retain their vibrant green leaves, showing no signs of yellowing. WRKY46-OE plants, initially with root systems comparable to WT plants, developed root systems nearly identical to WT plants after salt stress, and WRKY46 -OE plants showed the smallest reduction in root fresh weight and dry weight after salt stress, with decreases of 17.72% and 22.5%, respectively. Figure 7 (BE).

[0060] Overall, WRKY46 -OE plants showed the strongest tolerance to salt stress, with leaves remaining dark green without yellowing or falling off, and long, numerous lateral roots that were largely unaffected.

[0061] The results of the electrical conductivity measurement of tomato leaves are as follows: Figure 8 As shown. Under normal circumstances, plant cell membranes have selective permeability to substances. When plants are subjected to stress, the cell membranes are damaged, increasing membrane permeability and causing electrolytes to leak out of the cells. This leads to an increase in the conductivity of the cell extract. Therefore, the higher the relative conductivity, the greater the degree of damage to the plant tissue and the more severe the leakage of cellular molecules. The study found that under normal conditions, there was no significant difference in the relative conductivity among the three materials, which was around 22%. Overexpression of tomato under salt stress. WRKY46 -OE plants significantly improved the salt tolerance of tomatoes, with a leaf electrical conductivity content of 63.29%, significantly lower than that of wild type (WT) and... wrky46 Mutant strains, and mutants wrky46 The blade has the highest relative electrical conductivity of 91.2%.

[0062] In addition, the measurement results of the maximum photochemical efficiency of photosystem II of tomato plants are as follows: Figure 9 As shown. Fv / Fm is one of the most fundamental parameters of chlorophyll fluorescence, representing the chlorophyll fluorescence efficiency at which photosynthetic activity is at its peak. Studies have found that under normal conditions, there is no significant difference in Fv / Fm among the three materials, remaining around 0.8. Under salt stress... WRKY46 The -OE plants exhibited the highest maximum photochemical quantum yield (Fv / Fm) of PSII at 0.72, significantly higher than wild-type tomatoes (WT), while the mutant lines had the lowest at 0.35. This indicates that... WRKY46 -OE plants have a light absorption capacity closer to normal levels under salt stress.

[0063] In conclusion, in saline-alkali environments, due to the significant stress effect of high salt on plants, the normal development and growth status of tomatoes is more important than their total biomass. Overexpression... SlWRKY46The genetically modified tomato has a biomass that is basically the same as the wild type, but it has a leaf electrical conductivity and chlorophyll fluorescence that are closer to normal levels in saline-alkali environments. Its ability to resist salt stress is more suitable for survival and cultivation in saline-alkali environments, providing oily germplasm resources for tomato breeding on saline-alkali land.

[0064] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, unless otherwise specified, the embodiments of the present invention and the features thereof can be combined with each other.

Claims

1. overexpression SlWRKY46 application of genes in improving salt stress resistance in tomato; The SlWRKY46 The nucleotide sequence of the gene is shown as SEQ ID NO:

1.

2. Application of at least one of b1) to b4) from a1) to a3): a1) Overexpression of SlWRKY46 protein; a2) Biomaterials associated with overexpression of SlWRKY46 protein; a3) Reagents that target and upregulate the expression level of SlWRKY46 protein and / or enhance the activity of SlWRKY46 protein; b1) Improve the salt stress resistance of tomatoes; b2) Develop tomato varieties; b3) Prepare products that improve the salt stress resistance of tomatoes; b4) Prepare products for breeding tomato varieties.

3. The application according to claim 2, characterized in that: The biomaterial comprises at least one of c1) to c12): c1) Nucleic acid molecules encoding the SlWRKY46 protein; c2) An expression cassette containing the nucleic acid molecule described in c1); c3) A recombinant vector containing the nucleic acid molecule described in c1); c4) A recombinant vector containing the expression cassette described in c2); c5) Recombinant cells containing the nucleic acid molecules described in c1); c6) Recombinant cells containing the expression cassette described in c2); c7) Recombinant cells containing the recombinant vector described in c3); c8) Recombinant cells containing the recombinant vector described in c4); c9) Recombinant microorganisms containing the nucleic acid molecules described in c1); c10) Recombinant microorganisms containing the expression cassette described in c2); c11) Recombinant microorganisms containing the recombinant vector described in c3); c12) Recombinant microorganisms containing the recombinant vector described in c4).

4. The application according to claim 3, characterized in that: The tomato variety described above exhibits the following characteristics: enhanced resistance to salt stress.

5. The application according to claim 3, characterized in that: The nucleic acid molecule includes a nucleic acid molecule encoding the protein shown in SEQ ID NO:

2.

6. The application according to claim 5, characterized in that: The nucleic acid molecule includes the nucleotide sequence shown in SEQ ID NO:

1.

7. The application according to claim 3, characterized in that: The vector includes an overexpression vector.

8. The application according to claim 3, characterized in that: The cells include at least one of Escherichia coli and Agrobacterium.

9. A method comprising: the step of increasing the expression level and / or activity of SlWRKY46 protein in tomato; The method is at least one of i1) to i2): i1) a method for improving the salt stress resistance of tomatoes; i2) a method for breeding tomato varieties; The tomato variety described above has the following characteristics: improved resistance to salt stress; The tomato variety possesses the following characteristics: increased salt stress resistance relative to a reference level; the reference level is the level of the wild type. The step of increasing the expression level and / or activity of SlWRKY46 protein in tomatoes is to introduce the nucleic acid molecule encoding SlWRKY46 protein as described in any one of claims 3 to 6 into tomato tissue or tomato cells.

10. The method according to claim 9, characterized in that: The introduction method includes at least one of the following: using Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, and electroporation.

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