Application of millet SiWRKY58 gene and encoded protein thereof in salt tolerance

Abstract

The invention discloses an application of a millet SiWRKY58 gene and an encoded protein thereof in salt tolerance, belongs to the technical field of gene engineering, and particularly relates to an application of the millet SiWRKY58 gene in regulation and control of the salt tolerance of crops, and the CDS sequence of the SiWRKY58 gene is as shown in SEQ ID NO.1. Setaria italica is used as a research object, transcriptome sequencing and qRT-PCR expression verification prove that the SiWRKY58 gene is subjected to salt stress induction up-regulation expression, heterologous expression experiments show that overexpression of the SiWRKY58 gene can obviously improve the salt tolerance of arabidopsis thaliana, and the SiWRKY58 gene has important significance on crop salt tolerance transformation, improvement of the crop salt tolerance and development of salt-tolerant crop breeding.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, and in particular relates to the application of the millet SiWRKY58 gene and its encoded protein in salt tolerance. Background Technology

[0002] Currently, irrigated farmland is affected by soil salinization, which has become one of the most pressing agricultural issues and an unavoidable problem affecting agricultural food security. Reports indicate that the gradual deterioration of the ecological environment, global warming, and inappropriate irrigation methods have exacerbated soil salinization. Salt stress induces ionic stress, osmotic stress, and secondary stress in plants, especially oxidative stress. Under salt stress, plants must adjust their physiological and biochemical processes, participating in the regulation of ionic and osmotic homeostasis and the repair of stress-induced damage to ensure the completion of their entire growth and development process.

[0003] Millet ( Setaria italic a L.) was anciently known as millet, foxtail millet, or sorghum, and belongs to the Poaceae family ( Gramineae ), genus *Setaria* ( Setaria Millet is a dual-purpose crop originating in my country, used for both food and forage. Furthermore, millet is characterized by its tolerance to poor soil and drought, small genome size, and short growth cycle, making it a model plant for studying the stress resistance mechanisms of C4 crops.

[0004] The WRKY transcription factor family consists of proteins containing one or two WRKY domains. A WRKY domain consists of a conserved WRKYGQK sequence motif and a zinc finger (C2H2 or C2HC) structure. WRKY transcription factors regulate the transcription of downstream key genes by specifically binding to the W-box [(T)(T)TGAC(C / T)] cis-elements in gene promoters. TGAC is the core conserved sequence of the W-box, determining the specific binding site between the WRKY transcription factor and its downstream promoter. Due to the varying number of WRKY domains and zinc finger motifs within the WRKY family, the WRKY transcription factor family is divided into three subfamilies: Group I typically contains two WRKY domains and a C2H2 zinc finger structure; Group II mostly contains one WRKY domain, and its zinc finger structure is also C2H2; members of Group III contain one WRKY domain, but the zinc finger structure in this subfamily is C2HC.

[0005] Abiotic stresses on plants in nature mainly include drought, salt damage, high temperature, cold, and mechanical damage. WRKY transcription factors play an important role in plant responses to these abiotic stresses. In Arabidopsis, heterologous expression of GmWRKY54 and GmWRKY49 can improve salt tolerance; GmWRKY123 can improve salt tolerance in soybean plants by binding to the GmCAX1 promoter; CmWRKY10 regulates drought tolerance in chrysanthemums through the ABA signaling pathway, and overexpression of CmWRKY10 can improve drought tolerance and the expression levels of related genes in chrysanthemums. Under salt stress, overexpression of cotton GhWRKY34 can maintain ion homeostasis in transgenic Arabidopsis, thereby improving its salt tolerance. In Arabidopsis, AtWRKY33 and AtbHLH122 jointly regulate the expression of the downstream potassium transporter gene AtKUP2, enhancing its salt tolerance. Overexpression of wheat TaWRKY17 in Arabidopsis and wheat significantly improves salt tolerance. Rice OsWRKY54 regulates salt tolerance by binding to the promoter of OsHKT1;5. In apple, MdWRKY75 enhances salt tolerance by regulating the expression of MdSOS3, an important calcium-binding protein in the SOS pathway.

[0006] However, there are currently few molecular mechanisms for millet's resistance to stress. Discovering its salt-alkali tolerance genes and elucidating its salt-alkali tolerance mechanism can provide a theoretical basis for breeding new salt-alkali tolerant varieties of millet. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention proposes the application of the millet SiWRKY58 gene and its encoded protein in salt tolerance. This invention discovers that overexpression of the SiWRKY58 gene can significantly improve the salt tolerance of Arabidopsis thaliana, and that the SiWRKY58 gene is of great significance for carrying out salt-tolerant crop breeding.

[0008] To achieve the above objectives, the present invention provides the application of the millet SiWRKY58 gene in regulating crop salt tolerance, the CDS sequence of which is shown in SEQ ID NO.1.

[0009] Preferably, the salt tolerance of crops is improved by overexpressing the SiWRKY58 gene.

[0010] More preferably, the crop is Arabidopsis thaliana.

[0011] The present invention also provides the protein encoded by the SiWRKY58 gene, the amino acid sequence of which is shown in SEQ ID NO.2.

[0012] The present invention also provides the application of the encoded protein in regulating crop salt tolerance.

[0013] Preferably, overexpressing the SiWRKY58 gene increases the expression level of the encoded protein, thereby enhancing the salt tolerance of crops.

[0014] More preferably, the crop is Arabidopsis thaliana.

[0015] The present invention also provides a recombinant vector comprising the SiWRKY58 gene or the encoded protein.

[0016] The present invention also provides a recombinant strain containing the SiWRKY58 gene or the encoded protein.

[0017] The present invention also provides the application of the recombinant vector or the recombinant strain in regulating crop salt tolerance.

[0018] Compared with the prior art, the present invention has the following advantages and technical effects: The present invention discloses the application of the millet SiWRKY58 gene and its encoded protein in salt tolerance. The present invention uses millet as the research object, and verifies the upregulation of SiWRKY58 gene under salt stress through transcriptome sequencing and qRT-PCR expression verification. Heterologous expression experiments show that overexpression of SiWRKY58 gene can significantly improve the salt tolerance of Arabidopsis thaliana. The SiWRKY58 gene is of great significance for modifying crop salt tolerance, improving crop salt tolerance, and carrying out salt-tolerant crop breeding. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 The results of salt tolerance tests for different Arabidopsis germplasms are shown. Among them, A is the control group of normal treatment of Black Soft Rice, B is the control group of normal treatment of Millet Bright Rice, C is the control group of normal treatment of Z-5, D is the Black Soft Rice 2.0% NaCl salt stress group, E is the Millet Bright Rice 2.0% NaCl salt stress group, and F is the Z-5 2.0% NaCl salt stress group. Figure 2 The results of relative expression levels of the SiWRKY58 gene in different Arabidopsis germplasms; Figure 3 The image shows the results of laser confocal microscopy localization of the SiWRKY58 gene-encoded protein. In the image, A represents the GFP group, B represents the mCherry group, C represents the bright-field group, and D represents the mixed bright-field and laser group. The scale bar is 250 μm. Figure 4The figure shows the leaf area statistics of wild-type Arabidopsis thaliana and Arabidopsis thaliana strains overexpressing the SiWRKY58 gene. In the figure, WT represents wild-type Arabidopsis thaliana, OE#6 and OE#9 represent strains overexpressing the SiWRKY58 gene, CK represents the normal treatment control group, NaCl represents the 150mM NaCl salt stress treatment group, and different lowercase letters represent significant differences. Figure 5 The figures show leaf area phenotypes of wild-type Arabidopsis thaliana and Arabidopsis thaliana strains overexpressing the SiWRKY58 gene. In the figures, 1 represents wild-type Arabidopsis thaliana in the normal treatment control group, 2 represents Arabidopsis thaliana in the normal treatment control group of strain OE#6 overexpressing the SiWRKY58 gene, 3 represents Arabidopsis thaliana in the normal treatment control group of strain OE#9 overexpressing the SiWRKY58 gene, 4 represents wild-type Arabidopsis thaliana in the 150mM NaCl salt stress treatment group, 5 represents Arabidopsis thaliana in the 150mM NaCl salt stress treatment group of strain OE#6 overexpressing the SiWRKY58 gene, and 6 represents Arabidopsis thaliana in the 150mM NaCl salt stress treatment group of strain OE#9 overexpressing the SiWRKY58 gene. Figure 6 The results show the sodium and potassium ion contents in the leaves of wild-type Arabidopsis thaliana and Arabidopsis thaliana strains overexpressing the SiWRKY58 gene. In the figure, A represents the sodium ion content and B represents the potassium ion content. WT represents wild-type Arabidopsis thaliana, OE#6 and OE#9 represent strains overexpressing the SiWRKY58 gene, CK represents the normal treatment control group, and NaCl represents the 150 mM NaCl salt stress treatment group. Different lowercase letters indicate significant differences. Figure 7 The results show the determination of malondialdehyde (MDA), catalase, peroxidase, and superoxide dismutase (SOD) contents in wild-type Arabidopsis thaliana and Arabidopsis thaliana overexpressing the SiWRKY58 gene. In the figure, A represents MDA content, B represents catalase content, C represents peroxidase content, and D represents SOD content. WT represents wild-type Arabidopsis thaliana, OE#6 and OE#9 represent lines overexpressing the SiWRKY58 gene, CK represents the normal treatment control group, and NaCl represents the 150 mM NaCl salt stress treatment group. Different lowercase letters indicate significant differences. Figure 8 The image shows the DAB staining results of Arabidopsis thaliana leaves under salt stress treatment. WT represents wild-type Arabidopsis thaliana, and OE#6 and OE#9 represent lines overexpressing the SiWRKY58 gene. Figure 9 The image shows the NBT staining results of Arabidopsis thaliana leaves under salt stress treatment. WT represents wild-type Arabidopsis thaliana, and OE#6 and OE#9 represent lines overexpressing the SiWRKY58 gene. Detailed Implementation

[0021] Various exemplary embodiments of the present invention are now described in detail. This detailed description should not be considered as a limitation of the invention, but rather as a more detailed description of certain aspects, features, and embodiments of the invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, for numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included within the scope of the invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range. Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art described herein. While only preferred methods and materials are described herein, any methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of the invention. All references to this specification are incorporated by reference to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail. Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary. The terms "comprising," "including," "having," "containing," etc., as used herein are open-ended, meaning they include but are not limited to.

[0022] The materials used in this invention were sourced as follows: Black Soft Valley, Millet Bright, and Z-5 varieties were preserved at the Key Laboratory of Resource Exploration and Biological Breeding in Coastal Saline-Alkali Land, Qingdao; RNA extraction kits were purchased from Nanjing Novizan Biotechnology Co., Ltd.; reverse transcription kits were purchased from Nanjing Novizan Biotechnology Co., Ltd.; 2×Phanta Max Master Mix (DyePlus) was purchased from Nanjing Novizan Biotechnology Co., Ltd.; NdeI and SalI restriction endonucleases were purchased from Thermo; Agrobacterium P19 bacterial culture, pRI101AN vector, and pcam35-egfp vector were obtained from the Key Laboratory of Resource Exploration and Biological Breeding in Coastal Saline-Alkali Land, Qingdao; ClonExpress® II. One-step cloning kit was purchased from Nanjing Novizan Biotechnology Co., Ltd.; KpnI and BamHI restriction endonucleases were purchased from Thermo; Agrobacterium GV3101 was purchased from Shanghai Weidi Biotechnology Co., Ltd.; YEP liquid medium was purchased from Qingdao Haibo Biotechnology Co., Ltd.; 1 / 2 MS medium was purchased from Qingdao Haibo Biotechnology Co., Ltd.; DAB (diaminobenzidine) powder and NBT (nitrotetrazole blue chloride) powder were purchased from Sigma.

[0023] Example 1 I. Gene Cloning: 1. Take leaves of 4-week-old millet variety "Mizi Liang" for gene cloning and isolation.

[0024] 2. RNA was extracted using an RNA extraction kit.

[0025] 3. The extracted RNA was reverse transcribed into cDNA using a reverse transcription kit.

[0026] 4. The SiWRKY58 gene was amplified by PCR using 2×Phanta Max Master Mix (Dye Plus) reagent. The CDS sequence of the SiWRKY58 gene was obtained from the NCBI website (https: / / www.ncbi.nlm.nih.gov / ), XP_004977390.1.

[0027] The CDS sequence of the SiWRKY58 gene is shown in SEQ ID NO.1, SEQ ID NO.1: ATGACGCTAGATCTGCAGGGTGACGAGCTCCTGCTGGCGCAGCTCCGTGAGCTGCTCTCGCCGTCGTCTCCGGCGGTTAAGGCAGTGGAGTCATGCGACGGGAGACGACGGCGCCGGCGGGGGAGCAAGAGAGCCCAGGACGACGACAACACCACCAATAACGGCAAAAGAAGGAGCAAGAAGCAGAAGAGCACATCGTCTTTTGTGACATCAGTGCCTGATTTCGATGGGTACCGGTGGAGGAAGTACGGCCAGAAGCAAATCGAAGGTGCCATGTACGCCAGGAGTTACTACAGGTGCACTCGCAGCGCAGAGCAAGGCTGCCCTGCCAAACGGACGGTGCAGCGCAACGACGACGGCGACAATGGCGGTGCCGCTCCAAAGTACACTGTGGTGTGCATGGGGGAGCACACCTGCACGGCCACCGACTCCCTGGAGGCGCCGGTCATCCTCGAGACCACCGCCGTCGTCGCCCCTGGTATTATTGGCACCAACAACAGACCTGATGAAGACGACAATGACGACACCTTTACATCAGCTGGTTCCACCACCACCACGGGTACCGGGGTTGAGTCTCCGGCGATCTCGGACATCACCTGGAGCAGCAGTAGTTGCGGCGGCGATTACGTGGTCGATGACTACGGGGCCGGGTTGTTCGATGTCCATGACAGCTGGGCTTCTTCGGCGTCGTTGCAGGAGATGGAGGACTTCACCGGACCGATCCGGTCGCCGGTCCACGTCCCCGCGGATGGTTGGACGATTGACCACTTCCTGCTGCAGCTAGCTAATAATGAGCCTGTTTCCCATTTCTCATCAGCTTGTTAA。

[0028] The amino acid sequence of the protein encoded by the SiWRKY58 gene is shown in SEQ ID NO.2. SEQ ID NO.2: MTLDLQGDELLLAQLRELLSPSSPAVKAVESCDGRRRRRRGSKRAQDDDNTTNNGKRRSKKQKSTSSFVTSVPDFDGYRWRKYGQKQIEGAMYARSYYRCTRSAEQGCPAKRTVQRNDDGDNGGAAPKYTVVCMGEHTCTATDSLEAPVILETTAVVAPGIIGTNNRPDEDDNDDTFTSAGSTTTTGTGVESPAISDITWSSSSCGGDYVVDDYGAGLFDVHDSWASSASLQEMEDFTGPIRSPVHVPADGWTIDHFLLQLANNEPVSHFSSAC.

[0029] II. Vector Construction and Agrobacterium Transformation: 1. The pRI101AN vector was digested with NdeI and SalI restriction endonucleases. Using homologous recombination, the SiWRKY58 amplified fragment was re-ligated with the NdeI, SalI and BamHI restriction sites of the target vector using the ClonExpress® II one-step cloning kit and primer pairs consisting of WRKY58-AN-F (nucleotide sequence as shown in SEQ ID NO.3) and WRKY58-AN-R (nucleotide sequence as shown in SEQ ID NO.4) to construct the overexpression vector.

[0030] 2. The pcam35-egfp vector was digested with KpnI and BamHI restriction endonucleases. Using homologous recombination, the SiWRKY58 amplified fragment was re-ligated to the KpnI and BamH restriction sites of the target vector pcam35-egfp using the ClonExpress® II one-step cloning kit and primers consisting of WRKY58-eGFP-F (nucleotide sequence as shown in SEQ ID NO. 5) and WRKY58-eGFP-R (nucleotide sequence as shown in SEQ ID NO. 6) to construct a subcellular localization vector.

[0031] 3. Agrobacterium GV3101 was transformed by ice bath and heat shock. After the bacterial culture was identified and sequenced correctly, Agrobacterium was mixed with 50% glycerol and stored at -80℃ for subsequent transformation in Arabidopsis and tobacco.

[0032] The nucleotide sequence of WRKY58-AN-F is shown in SEQ ID NO.3, SEQ ID NO.3: TCTTCACTGTTGATACATATGATGACGCTAGATCTGCAGGGTG.

[0033] The nucleotide sequence of WRKY58-AN-R is shown in SEQ ID NO.4, SEQ ID NO.4: TCCGGTACCCCCGGGGTCGACACAAGCTGATGAGAAATGGGAAA.

[0034] The nucleotide sequence of WRKY58-eGFP-F is shown in SEQ ID NO.5, SEQ ID NO.5: CGAACGATAGCCATGGTACCAATGACGCTAGATCTGCAGGGTG.

[0035] The nucleotide sequence of WRKY58-eGFP-R is shown in SEQ ID NO.6, SEQ ID NO.6: CATGCCTGCGGCCGCGCCGGATCCACAAGCTGATGAGAAATGGGAAA.

[0036] III. Arabidopsis transformation: Transformation of wild-type Arabidopsis thaliana Col-0: Before transformation, inoculate 500 μL of Agrobacterium into 50 mL of YEP liquid medium and incubate at 28°C until OD. 600 The bacterial cells were collected by centrifugation at a concentration of 1.0–1.2 and resuspended in infection medium (1×MS, 5% sucrose, 0.02% Silwet-L77, pH adjusted to 5.7 with KOH) to allow the OD to reach a certain level. 600 The value was 0.8; the Arabidopsis flower heads were immersed in the staining solution and gently shaken for 1 minute; the Arabidopsis inflorescence was covered with plastic wrap and cultured in the dark for 24 hours, then the plastic wrap was removed and the incubator was placed at 22℃ to obtain Arabidopsis seed.

[0037] IV. Screening and Identification of Transgenic Plants: 1. Seed disinfection: Dispense an appropriate amount of seeds into 1.5mL centrifuge tubes, add 1mL of 75% ethanol for 5min sterilization, and shake the centrifuge tubes continuously during this time. Then, use a pipette tip to remove the 75% ethanol, quickly add 1mL of anhydrous ethanol, and shake for 1min.

[0038] 2. Prepare sterile, dry filter paper in a clean bench. Quickly pour the seeds suspended in anhydrous ethanol onto the filter paper until the seeds are dried.

[0039] 3. Spread the sterilized seeds evenly on a 1 / 2 MS medium plate containing antibiotics, and seal the plate with sealing film.

[0040] 4. Cultivate the petri dishes in the incubator for 20 days. Transfer the resistant seedlings that have grown green true leaves into nutrient soil to continue growing, and harvest individual plants.

[0041] 5. The T2 generation single-plant seed screening is the same as the above steps. Select the lineage with a segregation ratio of 3:1, and transplant the resistant seedlings into the nutrient soil for single-plant seed collection.

[0042] 6. The same screening method was used for the T3 generation, and single plants of the strains without resistance segregation were selected for seed collection.

[0043] V. Instantaneous Transformation of Tobacco: 1. Small-scale culture: Use a sterile pipette tip to pick up a single clone and place it into 5 mL of YEP medium containing the corresponding antibiotic. Incubate overnight at 28°C and 180 rpm.

[0044] 2. Take 1.5 mL of the overnight culture and add it to 50 mL of YEP medium. Simultaneously add 6 μL of acetylsuccinone and incubate at 28°C and 180 rpm until the OD value is reached. 650 A value of 0.9 is sufficient.

[0045] 3. Collect the shaken bacterial culture into a 50mL centrifuge tube, centrifuge at 4000rpm for 10min, discard the supernatant, and resuspend the bacterial cells with the same volume of infection solution (100μM acetylsalicylic acid, 50mM MES, 10mM MgCl2).

[0046] 4. Mix the bacterial suspension containing the target carrier and the Agrobacterium P19 bacterial suspension at a ratio of 1:1 and let stand for 3 hours.

[0047] 5. Using a 1mL sterile syringe, draw up the prepared bacterial solution and slowly inject it into both sides of the main leaf vein. Mark the injection points and continue to cultivate the tobacco plants in the greenhouse for 3 days. Observe the plants using a laser confocal microscope.

[0048] VI. Determination of malondialdehyde (MDA) content: Take 0.1g of Arabidopsis thaliana leaves, place them in a pre-cooled mortar, add liquid nitrogen and grind into powder. Add 3mL of 10% trichloroacetic acid (TCA) and mix well. Centrifuge at 4000r / min for 10min, take 800μL of the supernatant and place it in a new 2mL centrifuge tube, add 800μL of 0.6% thiobarbituric acid (TBA). Heat the mixture at 100℃ for 15min, then immediately cool in an ice bath. Centrifuge the mixture at 10000r / min for 15min, take 200μL of the supernatant and place it in an ELISA plate. Measure the absorbance at 450nm, 532nm, and 600nm, respectively, and calculate the MDA content according to the formula. The calculation formula is: MDA concentration (nmol / g) = 6.45 × (A 532 -A 600 -0.56×A 450×60.

[0049] VII. Determination of the activity of oxidative scavenging related enzymes: Take 0.1g of Arabidopsis thaliana leaves and determine the contents of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT). Specific methods for activity assays are available from the Shanghai Sangon Biotech Co., Ltd. Superoxide Dismutase (SOD) Activity Assay Kit (D799593-0050), Peroxidase (POD) Activity Assay Kit (D799591-0050), and Catalase (CAT) Activity Assay Kit (D799597-0050).

[0050] 8. DAB staining: Weigh out DAB powder, dilute with distilled water to 1 mg / mL, add concentrated HCl to pH 3.8 to ensure complete dissolution, and adjust the pH to 5.8 with NaOH before use. Place Arabidopsis thaliana in the DAB solution and incubate at 28°C in the dark for at least 8 hours. Discard the staining solution, add 95% ethanol, and incubate in a boiling water bath for at least 10 minutes until the green color of the leaves is completely removed. Store in anhydrous ethanol.

[0051] IX. NBT staining: Weigh out NBT powder, dilute it with distilled water to 0.5 mg / mL, immerse Arabidopsis thaliana in NBT solution and stain at 28°C for 2 h, then bathe in boiling water with 80% ethanol for 15 min until the green color of the leaves is completely removed, and store in anhydrous ethanol.

[0052] 10. Experimental Results: like Figure 1 China A Figure 1 B, Figure 1 C, Figure 1 D, Figure 1 China E and Figure 1 As shown in Figure F, the collected millet germplasm was subjected to 2.0% NaCl salt stress treatment in the early stage, and the salt tolerance of different germplasm was analyzed. The phenotypic identification results showed that black soft millet was more sensitive to salt stress, while the varieties Mizi Liang and Z-5 showed higher salt tolerance.

[0053] like Figure 2 The differential gene analysis of the transcriptome showed that the expression level of the SiWRKY58 gene was induced in *Heiruangu*, *Miziliang*, and Z-5, with higher induced expression levels in the salt-tolerant varieties *Miziliang* and Z-5 than in the salt-intolerant variety *Heiruangu*. The qRT-PCR results were consistent with the trend of the transcriptome data, suggesting that SiWRKY58 may play a role in regulating plant salt tolerance.

[0054] like Figure 3 China A Figure 3 B, Figure 3 C and Figure 3As shown in Figure D, the p35s:SiWRKY58:GFP overexpression vector was constructed and infected with Tobacco Bengal leaves through an Agrobacterium transient transformation system. Laser confocal microscopy results showed that the SiWRKY58 protein was located in the nucleus.

[0055] like Figure 4 and Figure 5 As shown, the 35Spro:SiWRKY58 overexpression vector was constructed and used to infect Arabidopsis thaliana, and positive plants were obtained through screening. Wild-type WT, overexpression lines OE#6, and OE#9 Arabidopsis thaliana plants grown for 2 weeks were subjected to salt stress treatment with 150 mM NaCl, while the control group was watered with the same volume of water. Phenotypic observation after 10 days of treatment revealed no difference in leaf area between the wild-type and overexpression lines in the control group; however, under salt stress treatment, the leaf area of ​​the overexpression lines was higher than that of the wild-type.

[0056] like Figure 6 China A and Figure 6 As shown in Figure B, the sodium ion content in the leaves of wild-type and overexpression plants was measured. The results showed that under control conditions, there was no difference in sodium ion content between wild-type and overexpression plants. However, under salt stress treatment, the sodium ion content in both wild-type and overexpression plants increased significantly, with the sodium ion content in the wild-type being much higher than that in the overexpression line (e.g., ...). Figure 6 (A); Potassium ion content determination results showed that under salt stress, the potassium ion content of all lines decreased, with no difference between lines (e.g., A). Figure 6 (B)

[0057] like Figure 7 China A Figure 7 B, Figure 7 C and Figure 7 As shown in Figure D, under control conditions, there was no difference in MDA content between wild-type and overexpression lines. Under salt stress, the MDA content of all lines increased, with the MDA content in wild-type plants being higher than that in overexpression plants (e.g., ...). Figure 7 (A). Antioxidant enzyme activity assays showed that the activities of CAT, POD, and SOD enzymes were increased in all lines under salt stress. Under salt stress, the CAT and SOD enzyme activities of the overexpressing lines were higher than those of the wild-type control. There was no significant difference in POD enzyme activity among the lines under control and salt stress conditions (e.g., ...). Figure 7 B~ Figure 7 (D).

[0058] like Figure 8 The DAB staining results shown indicate that, under salt stress, the leaf color of overexpressing lines OE#6 and OE#9 was significantly lower than that of wild-type WT.

[0059] like Figure 9The NBT staining results shown indicate that, under salt stress, the leaf color of overexpression lines OE#6 and OE#9 was significantly lower than that of wild-type WT.

[0060] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of the millet SiWRKY58 gene in regulating crop salt tolerance, characterized in that, The CDS sequence of the SiWRKY58 gene is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, By overexpressing the SiWRKY58 gene, the salt tolerance of crops can be improved.

3. The application according to claim 1 or 2, characterized in that, The crop in question is Arabidopsis thaliana.

4. The protein encoded by the SiWRKY58 gene as described in claim 1, characterized in that, The amino acid sequence of the encoded protein is shown in SEQ ID NO.

2.

5. The application of the encoded protein as described in claim 4 in regulating crop salt tolerance.

6. The application according to claim 5, characterized in that, Overexpression of the SiWRKY58 gene increases the expression level of the encoded protein, thereby enhancing the salt tolerance of crops.

7. The application according to claim 5 or 6, characterized in that, The crop in question is Arabidopsis thaliana.

8. A recombinant vector comprising the SiWRKY58 gene of claim 1 or the protein encoded by claim 4.

9. A recombinant strain comprising the SiWRKY58 gene of claim 1 or the protein encoded by claim 4.

10. The application of the recombinant vector as described in claim 8 or the recombinant strain as described in claim 9 in regulating crop salt tolerance.

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