SbWRKY51 gene, promoter and application of SbWRKY51 gene and promoter in improvement of salt tolerance of sorghum

Abstract

The invention discloses a SbWRKY51 gene, a promoter and application of the SbWRKY51 gene and the promoter in improvement of salt tolerance of sorghum, and belongs to the technical field of plant genetic engineering. The SbWRKY51 gene is screened from sorghum, and the expression level of the SbWRKY51 gene is up-regulated after salt stress treatment. The protein coded by the gene belongs to a WRKY transcription factor family, has transcriptional activation activity, and can start the expression of a reporter gene. Sorghum overexpression strains and complementary strains of the gene are obtained, salt tolerance related physiological indexes are measured, overexpression of the gene can improve the seed germination rate, increase the seed root length, regulate ion balance and enhance the free radical scavenging capacity of plants, and the effect of the gene in the aspect of improving the salt tolerance is embodied. A further research finds that the SbWRKY51 gene can enhance the plant salt tolerance by regulating a lignin synthesis pathway. The technical scheme provides a basis for cultivating resistant plants, and has important guiding significance for enhancing the production potential of sorghum under a high-salt condition and promoting agricultural development.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically involving SbWRKY51 Genes and promoters and their application in improving the salt tolerance of sorghum. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Transcription factors play a central regulatory role in plant responses to stress. Among them, the WRKY transcription factor family has been widely confirmed to participate in the response to various biotic and abiotic stresses. However, current research mainly focuses on model plants and a few major crops. For sorghum, an important cereal crop, functional studies on its WRKY genes are relatively scarce and lagging behind, which greatly limits the exploration and application of genetic engineering techniques (such as regulating specific WRKY genes) to enhance the salt tolerance of sorghum.

[0004] Meanwhile, the translation of research on plant salt tolerance into practical applications faces significant challenges. Most existing findings originate from model plants or laboratory environments, resulting in low conversion efficiency for improving actual crop varieties such as sorghum. Accurately assessing the impact of salt stress on phenotypes, agronomic traits, and yield under complex field conditions is difficult, hindering the effective and predictable improvement of crops' salt tolerance capabilities. Therefore, it is urgent to deeply analyze the function of the WRKY gene unique to sorghum and develop effective translational application pathways to achieve breakthroughs in the breeding of salt-tolerant sorghum. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the purpose of this invention is to provide... SbWRKY51 This invention relates to genes and promoters and their application in improving the salt tolerance of sorghum. SbWRKY51 Gene studies have revealed that this gene enhances plant salt tolerance by regulating the lignin synthesis pathway.

[0006] Specifically, this invention screened sorghum from previous transcriptome data. SbWRKY51 Genes, combined with their relative expression levels in sorghum roots under different stresses, were used to infer... SbWRKY51 It is a transcription factor that positively regulates salt stress and plays a positive regulatory role in the salt tolerance of sorghum.

[0007] Through analysis SbWRKY51 Cis-acting elements on the promoter were found SbWRKY51The promoter region of the gene has two salt stress-related elements, eight methyl jasmonate response-related elements, seven light response-related elements, six abscisic acid response-related elements, and non-biostress-related elements. Therefore, it is speculated that SbWRKY51 The gene may play an important role in the growth and development of plants and various stress responses, and may also be involved in the response of plants to abiotic stress.

[0008] Cloning SbWRKY51 The full-length sequence of the promoter was cloned, and the expression vector with the promoter tag was injected into tobacco. After 24 h of spraying treatment with 100 mM NaCl, GUS staining was performed. The leaves with the promoter tag were stained blue by the GUS staining solution under 0 mM NaCl treatment, and the staining was found to be deeper after 24 h of 100 mM NaCl treatment, proving that SbWRKY51 The promoter of the gene has salt stress-related elements. After GUS staining of Arabidopsis thaliana at different growth stages, it was found that the gene was expressed in seeds, cotyledons, flowers, and pods.

[0009] Salt stress can cause ion toxicity in plants, interfere with K + and Na + homeostasis, and thus damage the selectivity of root cell membranes. Plants use the salt hypersensitivity (SOS) pathway for salt stress signal transduction and Na + tolerance. The ability to scavenge ROS is important for improving stress tolerance. In the Arabidopsis thaliana heterologous expression experiment, salt stress inhibited the growth of each strain of Arabidopsis thaliana. Under 0 mM NaCl treatment, the wild type and the complemented strain had basically the same growth, the overexpression strain had better growth, and the mutant had slightly poorer growth. Under 100 mM NaCl treatment, the growth of each strain was affected to some extent, the wild type and the complemented strain had similar growth, the mutant had the worst growth, and the overexpression strain was the least inhibited, indicating that the overexpression strain had acquired certain salt tolerance. The dry and fresh weights of each strain decreased to varying degrees under salt treatment, and the dry and fresh weights of the OE8 and OE15 strains were higher than those of the other strains, with the most obvious decrease in the mutant. The ion content of each strain of Arabidopsis thaliana was determined, and the Na + content and Na + / K + of the overexpression strain were significantly lower than those of the WT under salt stress. High levels of antioxidant enzyme activity in plant tissues can provide resistance to plants and reduce damage caused by oxidative stress. The CAT and SOD activities of the overexpression strain were higher than those of the control and mutant strains under salt stress, and the overexpression SbWRKY51 gene has enhanced free radical scavenging ability, which can reduce oxidative damage to the plant itself under salt stress.

[0010] In the induced hairy root transient transformation system of sorghum, transgenic sorghum was obtained by transforming K599 competent cells and invading plant bud tip explants. The relevant biomass of the transgenic plants and the controls was determined, and the results consistent with the physiological indicators of Arabidopsis were obtained. These results all show that overexpression of SbWRKY51 gene improves the salt tolerance of plants in the heterologous expression system of Arabidopsis or the transient transformation hairy root system of sorghum.

[0011] Through DAP-seq data analysis, the downstream target genes possibly regulated by the sorghum SbWRKY51 gene were found, and the qRT-PCR results show that among the genes related to the ROS signal pathway, SbAPX1 and SbGPX6 the expression amounts of the genes increase. Among the genes related to the ABA signal pathway, SbABF2 , SbSnRK2.4 the expression amounts of the genes significantly decrease, SbNCED3 the expression amounts of the genes significantly increase. Among the genes related to the synthesis of lignin, SbPAL4, SbC4H, SbCCR1, SbC3H, SbF5H, SbCOMT1 the expression amounts of the genes all significantly increase, and among them, SbCOMT1 the up-regulation fold of the gene is the most significant. In order to study the molecular mechanism of the biosynthesis of lignin in salt-adapted cells, the lignin content of the transgenic sorghum after salt stress was analyzed, and the results show that the lignin content of the transgenic plants after salt treatment increases and is higher than that of the control group, and it is speculated that SbWRKY51 the sorghum

[0012] In order to achieve the above purpose, the technical scheme of the present application is as follows: In the first aspect of the present application, the application provides SbWRKY51 gene or biological material containing the SbWRKY51 gene in any one or more of the following applications; (a1) improving the salt tolerance of plants; (a2) improving the germination rate of plant seeds in a salt stress environment; (a3) improving the root length of plants in a salt stress environment; (a4) improving the dry weight and / or fresh weight of plants in a salt stress environment; (a5) improving the oxidation tolerance of plants; (a6) improving the free radical scavenging ability of plants.

[0013] In some embodiments of the present application, the plants are selected from sorghum or Arabidopsis.

[0014] In some embodiments of the present application, the biological material containing the SbWRKY51 gene includes any one or more of a recombinant expression vector, an expression cassette, a transgenic cell line, a recombinant bacterium and a recombinant virus.

[0015] In a second aspect of the present application, there is provided SbWRKY51 a promoter of a gene or a biological material containing SbWRKY51 application of a biological material containing a promoter of a gene in any one or more of the following: (b1) improving salt tolerance of a plant; (b2) improving germination rate of a plant seed in a salt stress environment; (b3) improving root length of a plant in a salt stress environment; (b4) improving dry weight and / or fresh weight of a plant in a salt stress environment; (b5) improving oxidative tolerance of a plant; (b6) improving free radical scavenging ability of a plant.

[0016] In some embodiments of the present application, the plant is selected from Sorghum bicolor or Arabidopsis thaliana.

[0017] In a third aspect of the present application, there is provided a recombinant expression vector, expression cassette, transgenic cell line, recombinant bacteria or recombinant virus containing SbWRKY51 a gene and / or SbWRKY51 a promoter of a gene.

[0018] In a fourth aspect of the present application, there is provided a method for improving salt tolerance of a plant, comprising the steps of: constructing SbWRKY51 a gene and / or SbWRKY51 a promoter of a gene into an expression vector to form a recombinant expression vector, and then transforming the plant with the recombinant expression vector, so that the plant carries SbWRKY51 a gene and / or SbWRKY51 a promoter of a gene, and finally regulating salt tolerance of the plant through SbWRKY51 high expression of the gene. The recombinant expression vector described above can be constructed using existing plant expression vectors, which, in addition to containing the SbWRKY51 gene and / or SbWRKY51 promoter of a gene described in the present application, also contains the following cloning vectors, but is not limited to the following vectors: Agrobacterium vectors or vectors that can be used for plant microprojectile bombardment; for example, pCAMBIA1300-35S-GFP, pGBKT7, pCAMBIA2300-35S-GFP and pCAMBIA3301, etc., or other derived plant expression vectors.

[0019] The SbWRKY51 gene and / or SbWRKY51In constructing the recombinant expression vector, the promoter of the gene can be further added with any other enhancer, constitutive, tissue-specific or inducible promoter, such as the cauliflower mosaic virus (CAMV) 35S promoter, the maize ubiquitin gene Ubiquitin promoter (pUbi), the seed-specific expression promoter, the stress inducible promoter Rd29A, etc. In addition, the application of the application is used for SbWRKY51 the gene and / or SbWRKY51 In constructing the recombinant expression vector, the promoter of the gene can be further added with an enhancer, including a translation enhancer or a transcription enhancer, which can be an ATG start codon or a start codon adjacent region, etc., but should be the same as the reading frame of the coding sequence to ensure the correct translation of the entire sequence.

[0020] The source of the above-mentioned translation control signal and start codon is wide, which can be natural or synthetic; the translation initiation region can be from the transcription initiation region or the structural gene; in order to facilitate the identification and screening of the transgenic plant cells or plants, the plant expression vector used can be processed, such as adding a gene (selective marker gene) that can express an enzyme or a luminescent compound in plants to produce color change (GUS gene, luciferase gene, etc.), a marker gene of an antibiotic (such as nptII gene conferring resistance to kanamycin and related antibiotics, bar gene conferring resistance to herbicide phosphine, hph gene conferring resistance to antibiotic hygromycin, dhfr gene conferring resistance to methatrexate, and EPSPS gene conferring resistance to glyphosate), or a marker gene of a chemical reagent (such as herbicide-resistant gene), a mannose-6-phosphate isomerase gene providing the ability to metabolize mannose. If the safety of the transgenic plant is considered, no selective marker gene can be added, and the transformed plant can be directly screened by phenotypic traits. In some embodiments of the application, the plant is a dicotyledon, such as tobacco, Arabidopsis or sorghum. In some embodiments of the application, the transformation is: introducing the recombinant expression vector into the plant cell or tissue by a biological method; The biological method includes any one of Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation, Agrobacterium-mediated, gene gun, pollen tube introduction and liposome fusion.

[0021] The application has the following beneficial effects: The application first reports SbWRKY51 the gene and the promoter and the application thereof in improving the salt tolerance of sorghum. Specifically, the application screens the gene from sorghum SbWRKY51Genes whose expression levels are up-regulated after salt stress treatment. The encoded proteins belong to the WRKY class of transcription factors, have transcription activation activity, and can initiate reporter gene expression. Highland barley overexpression and backfill strains of the gene are obtained, and salt tolerance-related physiological indicators are measured, SbWRKY51 Overexpression of the gene can improve highland barley seed germination rate, increase seed root length, regulate ion balance, and enhance the plant's ability to remove free radicals, reflecting the role of the gene in improving salt tolerance. Further research has found SbWRKY51 The gene enhances plant salt tolerance by regulating the lignin synthesis pathway. The above technical solutions provide a basis for cultivating resistant plants and have important guiding significance for enhancing the production potential of highland barley under high salt conditions and promoting agricultural development, and have good practical application value. BRIEF DESCRIPTION OF DRAWINGS

[0022] The drawings accompanying the specification of this application serve to provide further understanding of the application, and the illustrative embodiments of the application and their descriptions serve to explain the application and do not constitute an improper limitation on the application.

[0023] Figure 1 Hydrophobicity analysis of SbWRKY51 protein.

[0024] Figure 2 Subcellular localization analysis of SbWRKY51 protein, wherein A is the control group pCAMBIA1300-35S-GFP, and B is the experimental group pCAMBIA1300-35S-GFP- SbWRKY51 .

[0025] Figure 3 Transcription activation activity analysis of SbWRKY51 , wherein A is the yeast competent cell AH109 cultured in SD / -Trp nutrient-deficient medium, B is the yeast competent cell AH109 cultured in SD / -Trp-His-Ade nutrient-deficient medium, and C is the yeast competent cell AH109 cultured in SD / -Trp-His-Ade + X-α-gal nutrient-deficient medium; and in the three nutrient-deficient media shown in A, B, and C, the left column is the yeast competent cell AH109 transformed with pGBKT7 empty vector, and the right column is the yeast competent cell AH109 transformed with pGBKT7- SbWRKY51 expression vector.

[0026] Figure 4 Seed germination period phenotype under different salt concentrations, wherein A is 0 mM NaCl (1 / 2 MS), B is 50 mM NaCl, C is 100 mM NaCl, and D is 150 mM NaCl.

[0027] Figure 5The values ​​represent the germination rates of seeds under different salt concentrations, where A is 0 mM NaCl (1 / 2 MS), B is 50 mM NaCl, C is 100 mM NaCl, and D is 150 mM NaCl.

[0028] Figure 6 The phenotypes of root length under different salt concentrations are shown, where A is 0 mM NaCl (1 / 2 MS), B is 50 mM NaCl, C is 100 mM NaCl, and D is 150 mM NaCl.

[0029] Figure 7 The root length was analyzed under different salt concentrations, where A was 0 mM NaCl (1 / 2 MS), B was 50 mM NaCl, C was 100 mM NaCl, and D was 150 mM NaCl.

[0030] Figure 8 Na content in leaves of various Arabidopsis thaliana strains under salt treatment + Content (A), K + Content (B) and Na + / K + (C).

[0031] Figure 9 The activities of catalase (A) and superoxide dismutase (B) in the leaves of various Arabidopsis thaliana strains under salt treatment.

[0032] Figure 10 GUS histochemical staining during transient injection of tobacco to the full length of the promoter.

[0033] Figure 11 The relative expression levels of genes related to the oxidative stress pathway (A), ABA signaling pathway (B), and lignin synthesis (C) under salt stress were analyzed.

[0034] Figure 12 The content of lignin in different sorghum lines under salt stress. Detailed Implementation

[0035] This invention analyzes sorghum SbWRKY5 The study elucidates the salt tolerance mechanism of gene 1 under salt stress, clarifies its position and role in the plant salt tolerance regulatory network, and reveals for the first time... SbWRKY51 Genes enhance plant salt tolerance by regulating the lignin synthesis pathway, providing a method based on SbWRKY51 A highly efficient salt-tolerant breeding technology scheme for sorghum was developed, establishing an efficient and stable sorghum genetic transformation system to achieve... SbWRKY51 Stable expression of the gene in sorghum provides technical support for salt-tolerant breeding of sorghum and is applicable to sorghum and other crops.

[0036] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0037] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0038] Example 1: Sorghum SbWRKY51 Bioinformatics analysis of genes SbWRKY51 Gene: SORBI_3006G206000 Amino acid sequence: MAVDLMGCYAPRRADDQLAIQEAAAESLRSLELLVSSLSTQAGAPHRAAHHLQQQQPFGEIADQAVSKFRKVISILDRTGHARFRRGPVESPPRAAAAPPVPAPAPALSLAPLAHVAPVSAAQPAPASQPPQSLTLDFTKPNLTMSGATSVTSTSF FSSVTAGEGSVSKGRSLMSSGKPPLSGHKRKPCAGAHSEATTNGGRCHCSKRRKNRVKRTIRVPAISSKIADIPPDEYSWRKYGQKPIKGSPYPRGYYKCSTVRGCPARKHVERATDDPAMLVVTYEGEHRHTPGAAGPSPLATASPVAAAVSAGNGHV (SEQ ID NO.1) Example 2: Sorghum SbWRKY51 Functional research of genes 1. Test materials The following seeds were collected: wild-type Arabidopsis thaliana (WT), atwrky11 (At4g31550), a homologous mutant of SbWRKY51 in Arabidopsis thaliana purchased from the Tair website, over-infected lines OE5, OE8, and OE15, as well as reintroduced lines R10, R12, and R16, sorghum inbred line M-81E, BTX623, and tobacco.

[0039] 2. Test methods 2.1 Construction of the expression carrier (1) The extraction of total RNA of sorghum: select the sorghum salt-tolerant inbred line M-81E seeds, after washing (24 h) and culturing, treat with different treatment liquids (Hoagland nutrient solution containing 150 mM NaCl, 150 mM mannitol, 150 μM ABA, respectively, for 0, 3, 6, 12, 24, 48 h) at the three-leaf-one-heart stage, and take 0.1 g of material to extract total RNA. The extraction of total RNA of sorghum is carried out according to the RNA extraction kit of Huayueyang.

[0040] (2) The synthesis of cDNA: according to the operation steps of the reverse transcription kit of Aikewei Biological Company, remove the genomic DNA, and then carry out the reverse transcription reaction to obtain cDNA and store at -20℃. Evo M - MLV

[0041] (3) SbWRKY51 The cloning of genes: use the primer to carry out PCR amplification with cDNA as the template, after the reaction, run agarose gel electrophoresis detection to detect whether the band of the target gene size is contained, if the target band is contained, use the gel recovery kit of Aikewei Biological Company to recover the target band.

[0042] Primer: SbWRKY51 -F: ATGGCCGTGGACCTGATGG (SEQ ID NO. 2); SbWRKY51 -R: GACATGGCCGTTGCCGGCGG (SEQ ID NO. 3).

[0043] (4) The construction of pMDTM18-T- SbWRKY51 Cloning vector: connect the target fragment with pMDTM18-T-Vector, transform the connection product into E. coli competent DH5α (the transformation steps are described in the product specification of DH5α Chemically Competent Cell of Shanghai Weidi Biological Technology Co., Ltd.), carry out colony PCR of the cloning vector, after the end of the PCR reaction program, prepare 1% agarose gel with 10xTAE, and carry out electrophoresis analysis to detect whether the band of the target gene size is contained, record the positive clones containing the target band, pick the corresponding streaked colonies on the culture medium, shake the bacteria to extract plasmid, and then send for sequencing. The extraction steps of the plasmid are described in the product specification of TIANperp Mini Plasmid Kit of Tiangen Biochemical Technology (Beijing) Biological Company.

[0044] Primer for colony PCR of the cloning vector: SbWRKY51 -F: ATGGCCGTGGACCTGATGG (SEQ ID NO. 2); SbWRKY51 ​-R: GACATGGCCGTTGCCGGCGG (SEQ ID NO. 3).

[0045] (5) Double enzyme digestion of the target gene and the vector: Double enzyme digestion of pCAMBIA1300-35S-GFP and pGBKT7 vector: according to the pCAMBIA1300-35S-GFP map, primers were designed, and the cloning vector was used as the template. SbWRKY51 The fragment with Kpnl and BamHI enzyme digestion sites was amplified. According to the map of pGBKT7 vector, the cloning vector was used as the template, and the fragment with EcoRI and BamHI enzyme digestion sites was amplified. After electrophoresis detection of the fragment consistent with the size of the target band, the target fragment was recovered by using the EcoRi biological gel recovery kit. The target fragment was connected to the expression vector to construct a recombinant plasmid, the connection product was transformed into E. coli competent DH5a, and the plasmid was sent for testing and the strain was preserved. SbWRKY51 SbWRKY51 SbWRKY51

[0046] Single enzyme digestion of pCAMBIA2300-35S-GFP vector: according to the map of pCAMBIA2300-35S-GFP vector, primers were designed, and the cloning vector was used as the template. SpeI was selected as the enzyme digestion site. Linear expression vector was obtained by single enzyme digestion, and the expression vector was homologously recombined with the gene fragment to construct pCAMBIA2300-35S-GFP- SbWRKY51 recombinant plasmid. SbWRKY51

[0047] (6) SbWRKY51 Cloning of gene promoter (i.e. Pro- SbWRKY51 Cloning vector): PCR reaction amplified [[ID= Gene promoter, after the reaction program, run agarose gel electrophoresis analysis, detect whether contains the size of the target gene band, if there is a target band, the next step is DNA fragment recovery, see the detailed steps of the EcoRi biological gel recovery kit.

[0048] PCR reaction amplified ​ Gene promoter primer L: ​ -Pro-F: TGATTAAAGAGTTAGACAAAATG (SEQ ID NO. 4); ​ -Pro-R: GGCCGGGCTTTATAGGTGG (SEQ ID NO. 5).

[0049] (7) Double enzyme digestion of pCAMBIA3301 vector and Pro- ​ ​​​​Cloning vector: according to the pCAMBIA3301 map design primer, to Pro- ​ Cloning vector as template, amplification of containing BamHI and NcoI enzyme cutting site fragment, construction of pCAMBIA3301-UBI-GUS-Pro- ​ Expression vector. Electrophoresis detected with the purpose of band size consistent with the fragment, with the EcoRi biological gel recovery kit reagent kit recovery purpose fragment. Purpose of fragment and expression vector connection, connection product transformation of E. coli competent DH5α, shake bacteria to improve plasmid sent test, save strain. With the single colony as a template colony PCR, while in the solid medium containing kanamycin line, overnight culture.

[0050] (8) expression vector transformation Agrobacterium competent GV3101, EHA105: the successful connection of expression vector plasmid 1-2 μL to Agrobacterium competent GV3101 or EHA105, transformation steps detailed in Shanghai Weidi Biotechnology Co., Ltd. product manual.

[0051] 2.2, gene expression pattern analysis: reverse transcription of cDNA as a template, using fluorescent quantitative PCR technology, with Sbactin-F, Sbactin-R as internal reference primer, qRT- ​ -F, qRT- ​ -R as gene quantitative primer, each treatment was done 3 technical replicates, analysis ​ gene expression pattern in sorghum M-81E.

[0052] Sbactin-F: CAACTTTGTCACCCGCGATGA (SEQ ID NO. 6) Sbactin-R: TCCAGAAACCTTAGCAGCCCA (SEQ ID NO. 7) qRT-S ​ -F: GGAAAAACCGCGTGAAGAGG (SEQ ID NO. 8) qRT- ​ -R: GTAAGGGGAGCCCTTGATGG (SEQ ID NO. 9) 2.3, subcellular localization: by Agrobacterium transient transformation of tobacco, the pCAMBIA1300-35S- ​ expression vector and empty load were transformed into Agrobacterium, and injected into tobacco leaves (tobacco before injection need to avoid light treatment, from the back of the tobacco injection), 36-72 h using two-photon laser scanning confocal microscope to observe fluorescence, determine ​ protein localization.

[0053] 2.4. Transcriptional activation activity analysis: The successfully constructed pGBKT7- ​ The expression vector and the empty pGBKT7 vector were transformed into AH109 competent cells (transformation steps are detailed in the AH109 Chemically Competent Cell product manual from Shanghai Weidi Biotechnology Co., Ltd.). The transformed yeast culture was diluted and plated onto different auxotrophic media (SD / -Trp, SD / -Trp-His-Ade, SD / -Trp-His-Ade + X-α-gal). Yeast growth was observed after culturing for 36-96 h to verify the results. ​ The transcriptional activation activity.

[0054] 2.5. Promoter cis-acting element analysis: The 2000 bp sequence of the gene promoter was downloaded from NCBI, its promoter function was predicted using the Plant CARE online website, and its cis-acting elements were analyzed using TBtools software.

[0055] 2.6 Transformation of Arabidopsis thaliana by inflorescence infection method (1) Seedling preparation: The day before infection, provide sufficient water to the wild-type Arabidopsis thaliana cultured in the artificial climate chamber, and cut off its blooming flowers and pods. (2) Preparation of bacterial culture: Take pCAMBIA1300-35S- ​ Agrobacterium strain (requires secondary activation) was inoculated into 50 mL LB liquid medium containing Kana and Rif, and incubated overnight at 28°C at 180 rpm / min until OD. 600 =Approximately 1. (3) Centrifugation: Centrifuge the re-shaken bacterial solution at 6000 rpm / min for 5 min, discard the supernatant, and collect the bacterial cells. (4) Preparation of resuspension: Resuspend the bacterial culture with 5% sucrose (at a ratio of 1.8 µL Swilt surfactant to 5 mL of deionized water). (5) Infecting inflorescences: Soak in bacterial solution for about 10-15 seconds, and infect inflorescences in lower positions as well. After infection, cover with a black plastic bag and culture in the dark for 24 hours. Remove the plastic bag and infect again after 5-7 days. Repeat this process about three times. Harvest seeds from a single seed (T1) and screen for about three generations until homozygous to obtain overexpression and replacement lines.

[0056] 2.7 GUS staining analysis of tissue localization: Immerse the seeds, leaves, flowers, rhizomes and other tissues to be stained in GUS staining working solution. Depending on the material conditions (based on gene promoter strength, the tenderness of the material and the thickness of the cuticle), incubate at 37℃ for 1 hour or overnight. Observe the GUS-positive blue spots with the naked eye or under a microscope to analyze the tissue localization of the gene.

[0057] 2.8, Determination of part of physiological indexes of Arabidopsis thaliana under salt stress (1) Measurement of dry and fresh weight: Arabidopsis thaliana was washed, dried and weighed to obtain fresh weight, and then dried to constant weight and weighed to obtain dry weight.

[0058] (2) DAB and NTB staining: DAB staining solution and NBT staining solution were prepared, and plant leaves were placed in the staining solution. After staining, fading and other steps, the color change of the leaves was observed, and the degree of oxidative stress of the plants was analyzed.

[0059] DAB staining solution: 0.1 g of DAB reagent was weighed into 100 mL of deionized water, first adjusted to pH 3.8 to dissolve the reagent, and then adjusted to pH 5.8. It is DAB staining solution.

[0060] PBS solution: One PBS tablet was dissolved in 100 mL of deionized water.

[0061] NBT staining solution: 0.05 g of NBT was weighed and dissolved in PBS solution, and the pH was adjusted to 7.4-7.6, which was NBT staining solution.

[0062] (3) Determination of Na + and K + : 0.3 g of sample was weighed, 10 mL of ultrapure water was added, and boiled for 2-3 h. After cooling, filtering and constant volume (25 mL), the Na + and K + content was determined by flame spectrophotometry.

[0063] (4) Determination of antioxidant enzyme activity: 0.2 g of roots and leaves under different treatments were taken respectively, 1.6 mL of 50 mM phosphate buffer (PBS pH=7.8) was added, and homogenate was prepared in a pre-cooled mortar. Transferred to a centrifuge tube, centrifuged at 4°C for 12000 rpm for 20 min, the supernatant was enzyme solution, and the activities of CAT and SOD were determined respectively.

[0064] (5) CTAB method for extracting DNA: CTAB method was used to extract Arabidopsis thaliana leaf DNA for subsequent gene identification.

[0065] 2.9, Establishment of Agrobacterium rhizogenes-mediated genetic transformation system (1) Sterilization and treatment of sorghum seeds: Sorghum BTX 623 seeds were selected, washed, sterilized (75% alcohol, 0.1%-0.2% Tween water, 2%-3% sodium hypochlorite, 0.1% Tween water, sterile water), and dried after a series of treatments.

[0066] (2) Sorghum planting: the sterilized seeds were planted on MS1 medium, and cultured in dark for 24 h. When the seeds showed 4-5 mm bud tips, the infection was performed.

[0067] (3) Transformation of the constructed vector into Agrobacterium rhizogenes competent K599: the plasmid was transformed into Agrobacterium competent K599, and after a series of treatments (in order: ice bath for 5 min, liquid nitrogen freezing for 5 min, 37°C water bath for 5 min, ice bath for 5 min), it was coated on TY solid medium containing kanamycin for culture, and colony PCR identification was performed and the strain was preserved.

[0068] (4) Activation of Agrobacterium rhizogenes K599 strain: pCAMBIA2300-35S-GFP- ​ and pCAMBIA2300-35S-GFP empty K599 strains were taken out from the -80°C ultra-low temperature storage box, inoculated into TY liquid medium containing antibiotics, and shaken for culture. The OD value was measured to make the concentrations of the two bacterial solutions consistent, and the bacterial bodies were collected for infection.

[0069] (5) Method for inducing sorghum hairy root system by Agrobacterium rhizogenes K599: the bacterial bodies were suspended with sterile water, and the bud tips of sorghum were infected. The infected bud tips were placed in MS2 medium for culture, and fluorescence was observed.

[0070] 2.10, Determination of lignin content: after the sample was dried, ground and sieved, acetylation treatment was performed using a lignin kit (Beijing Solabio Technology Co., Ltd.), and the absorbance value at 280 nm was measured using a spectrophotometer to calculate the lignin content.

[0071] 3, Test results and analysis 3.1, Analysis of hydrophilic and hydrophobic properties of SbWRKY51 protein: ​ The hydrophilic and hydrophobic properties of SbWRKY51 protein were analyzed. According to the analysis of the hydrophilic and hydrophobic properties of the amino acid sequence of SbWRKY51, it can be seen from the distribution of amino acids that the negative value region is greater than the positive value region, indicating that the protein is a hydrophilic protein. This property may affect the localization and function of the protein in the cell, and is of great significance for the study of the protein involved in the salt stress response mechanism.

[0072] 3.2, ​ Analysis of promoter elements Through online website analysis of the promoter of ​ the gene, it was found that ​The gene's promoter region contains two elements related to salt stress (GT1-motif), eight elements related to methyl jasmonic acid response (four TGACG-motifs and four CGTCA-motifs), seven elements related to light response (one C-box, two G-boxes, and four G-boxes), and six elements related to abscisic acid response (ABRE). This suggests... ​ Genes may play a role in plant growth and development and in responses to different stresses.

[0073] 3.3 ​ Analysis of gene expression patterns under different stresses M-81E cells cultured in hydroponic containers were treated with NaCl, mannitol, and ABA, and samples were taken at different treatment times for analysis. ​ The amount of expression. ​ Gene expression levels all showed a trend of first increasing and then decreasing. Under NaCl treatment, ​ Gene expression levels peaked at 3 hours. Similar to salt treatment, mannitol treatment... ​ Gene expression levels peaked at 24 hours. Under ABA treatment, ​ Gene expression levels peaked at 12 hours and then declined. Under different stress conditions... ​ Gene expression levels vary at different times, but ​ The gene showed an overall upregulation trend, and it is preliminarily speculated that the gene is a positively regulated transcription factor of salt stress, mannitol signaling pathway and ABA signaling pathway.

[0074] 3.4 Subcellular localization analysis of SbWRKY51 protein: The constructed pCAMBIA1300-35S-GFP- ​ Expression vectors were used to transiently transform tobacco using Agrobacterium, with tobacco injected with the pCAMBIA1300-35S-GFP empty vector as a control. The results were observed. ​ Location in tobacco. Results ( ​ The results showed that tobacco cells transfected with empty vectors exhibited GFP signals on both the cell membrane and nucleus, while those linked to empty vectors... ​ The gene vector showed a strong GFP signal only in the cell nucleus, confirming that the SbWRKY51 protein is located in the cell nucleus, providing a key basis for exploring its transcriptional regulatory function.

[0075] 3.5. Analysis of the transcriptional activation activity of SbWRKY51: Using pGBKT7 under no-load conditions as a comparison, pGBKT7 under no-load conditions and pGBKT7- ​ The expression vector was transformed into yeast competent cells AH109. (Example) ​As shown, after culture on different nutrient-deficient medium, both of them can grow normally on SD / Trp medium, only the yeast transformed with pGBKT7- ​ grew on SD / -Trp-His-Ade and SD / -Trp-His-Ade+X-α-gal medium, and the yeast turned blue on SD / -Trp-His-Ade+X-α-gal medium, which proved that SbWRKY51 had transcriptional activation activity and could initiate the expression of reporter gene, which was an important functional characteristic of its involvement in gene regulation.

[0076] 3.6, Germination of each line of Arabidopsis under salt treatment: ​ and ​ showed the seed germination phenotype (Fig. 3.6) and germination rate (Fig. 3.7) of each line of Arabidopsis (wild type, mutant, overexpression line, complementation line) under normal growth conditions and different salt concentrations (50 mM, 100 mM, 150 mM NaCl) treatment. ​ Under salt stress, the germination rate of each line decreased, the germination rate of the mutant was the lowest, the germination rate of the overexpression line was higher than that of the wild type, and the germination rate of the complementation line was similar to that of the wild type, indicating that the overexpression line had enhanced salt tolerance, which directly reflected the effect of SbWRKY51 on the salt tolerance of Arabidopsis seed germination. ​ ​

[0077] 3.7, Phenotype and root length analysis of each line of Arabidopsis under salt treatment: Each line of Arabidopsis was treated with 0, 50, 100 and 150 mM salt for 7 days, and the length of the main root was analyzed by ImageJ software. As shown in Fig. 3.7 and Fig. 3.8, with the increase of salt concentration, the root length growth of each line was inhibited, but the root length of the overexpression line was significantly longer than that of the wild type, mutant and complementation line, indicating that overexpression of SbWRKY51 could alleviate the inhibition of salt stress on seedling growth, and highlighted the positive effect of the gene on plant root growth under salt stress. ​ ​ ​

[0078] 3.8, Phenotype and dry and fresh weight determination of seedlings after salt treatment Under 0 mM NaCl treatment, the growth of each line was not much different. After 100 mM NaCl treatment, the growth of each line was affected to some extent, the wild type and the complementation line had similar growth, the mutant had the worst growth, and the overexpression line had the least inhibition, indicating that the overexpression line had acquired certain salt tolerance. Under 100 mM NaCl treatment, it was found that compared with the control, the dry and fresh weight of each line decreased to different extents, and the dry and fresh weight of the overexpression lines OE8 and OE15 was higher than that of other lines, and the decrease amplitude was lower than that of other lines, and the decrease amplitude of the mutant was the most obvious.​​​​​

[0079] 3.9 NBT and DAB staining of different Arabidopsis thaliana lines under salt treatment Plant leaves of similar size and shape from the same location were stained with NBT stain. Without salt treatment, there was no significant difference in staining intensity among the different strains. After salt treatment, the color of each strain deepened to varying degrees. The leaf staining intensity showed that the mutant strain had the deepest staining, indicating that it suffered the most severe oxidative damage.

[0080] DAB staining was performed on all lines, and the results were consistent with those of NBT staining. The OE line had the least accumulated H2O2 in its leaves and the lightest staining. This indicates that under salt stress conditions, ​ Overexpression of [a substance] can improve the oxidative tolerance of plants.

[0081] 3.10. Na+ content in leaves of different Arabidopsis thaliana strains under salt treatment + K + content: After treating Arabidopsis thaliana lines with 0 and 100 mM NaCl for 7 days, the Na content in the leaves was measured. + and K + Content. For example... ​ As shown, under non-salt treatment, the Na content of the WT and OEs lines and the Rs line... + Content and K + The content is almost the same; under salt treatment, the Na content of OEs is... + The content decreased significantly, and the Na content of wrky11 was significantly reduced. + The content increased significantly, with little difference between the WT and Rs lines, and the OEs line maintained a low Na content after salt treatment. + / K + The comparison revealed ​ Gene overexpression regulates ion balance in Arabidopsis thaliana, which is closely related to the plant's salt tolerance.

[0082] 3.11. Antioxidant enzyme activity in leaves of different Arabidopsis thaliana strains under salt treatment: The activities of antioxidant enzymes CAT and SOD in the leaves of various Arabidopsis strains were determined after seven days of treatment with 0 mM NaCl and 100 mM NaCl. The results are as follows: ​ As shown, the enzyme activities of the different strains were not significantly different under 0 mM NaCl treatment. After treatment with 100 mM NaCl, the antioxidant enzyme activities of all strains increased, and the CAT and SOD activities of the overexpression strains were higher than those of the control and mutant strains. The enzyme activities of the control and reinjection strains were not significantly different. This indicates that overexpression... ​ The enhanced ability of plants to scavenge free radicals due to this gene demonstrates its role in strengthening plant antioxidant capacity and improving salt tolerance.

[0083] 3.12, GUS histochemical staining of tobacco when the promoter is injected full-length transiently: The expression vector with promoter tag was transformed into EHA105 Agrobacterium and injected into tobacco, and GUS staining was performed after 24 h of spray treatment with 100 mM NaCl. As shown in Fig. 3.12, the leaves with promoter tag were stained blue under 0 mM NaCl treatment, and the staining was deepened after 24 h of 100 mM NaCl treatment, which proved that ​ the gene promoter had salt stress-related response elements, which was of great significance for studying the expression regulation mechanism of the gene under salt stress. ​

[0084] 3.13, GUS staining results of wild-type and transgenic Arabidopsis at different stages GUS tissue staining can be used to predict the organs of the gene and accurately observe the tissue localization of the gene. The full-length sequence of the cloned ​ gene promoter was connected to the vector containing the GUS tag, and after screening for homozygous plants in the T3 generation, GUS staining was performed on Arabidopsis at different growth stages. It was found that the gene was expressed in seeds, cotyledons, flowers, pods, and stems.

[0085] 3.14, DNA, RNA, and fluorescence level identification of transgenic plants Sterile and contamination-free hairy roots were selected and induced to culture for 14 d. Genomic DNA of the root system was extracted by CATB method for PCR detection. The detection of a band consistent with the size of the target band indicated that the transgenic plant was successfully induced and transformed. The RNA of each strain of hairy roots was extracted, and cDNA was obtained by reverse transcription. With cDNA as the template, Sbactin-S and Sbactin-A as the internal reference gene primers, qRT-SbWRKY51-F and qRT-SbWRKY51-R as the gene quantitative primers, and the hairy roots transformed with 2300 empty vector as the control, each transgenic strain was identified by qRT-PCR. After detection, OE2, OE3, OE4, OE5, OE8, OE9, and OE10 were all transgenic strains successfully transformed with the ​ gene.

[0086] The Agrobacterium K599 successfully transformed with the pCAMBIA2300-35S-GFP- ​ vector was used to infect the root tips of sorghum explants, with wild-type hairy roots as the negative control and hairy roots successfully transformed with the pCAMBIA2300-35S-GFP empty vector as the positive control. Fluorescence identification was performed using the full-automatic upright fluorescence microscopic imaging system of the experimental platform, and the related parameter settings of the instrument were unified. Fluorescence results showed that the successful transformation of ​ ​The fluorescence signal of the hairy roots induced by the gene was the strongest.

[0087] 3.15, Phenotype and biomass of each line of sorghum under salt treatment After 7 days of 150 mM NaCl treatment, the phenotype of each line was recorded. Under the condition of no salt stress, ​ The transgenic root length was not much different from the control. After NaCl treatment, the transgenic root system grew better than the control, the root length of the control group became shorter, the root system decreased, and the dry fresh weight decreased.

[0088] 3.16, Determination of Na + , K + content in the roots of each line of sorghum under salt treatment Each line of sorghum was treated with 0, 150 mM NaCl, and the Na + , K + content in the roots was determined after 7 days. Under non-salt treatment, the Na + content of the control and the overexpression line was not much different, and the K + content of the control was higher than that of the overexpression line. After salt treatment, the Na + content of the overexpression line increased less than the control, and the K + content decreased less than the control, so the overexpression line could maintain a lower Na + / K + ratio to resist salt stress.

[0089] 3.17, Determination of antioxidant enzyme activity of each line of sorghum under salt treatment The antioxidant enzyme SOD, CAT activity of each line of sorghum under salt treatment was determined. Under non-salt treatment, the antioxidant enzyme activity of the overexpression and control groups was not much different. After 7 days of 150 mM NaCl salt treatment, the antioxidant enzyme activity of all lines increased, and the SOD, CAT activity of the overexpression line was higher than that of the control group, indicating that after overexpression of the gene, the plant's ability to scavenge free radicals was enhanced, and under salt stress, the oxidative damage to the plant itself was reduced. ​

[0090] 3.18, Relative expression analysis of related genes under salt stress: The expression of genes related to oxidative stress, ABA signal pathway and lignin synthesis in transgenic sorghum was verified by qRT-PCR method. The results ( ​ ) showed that among the genes related to ROS signal pathway, SbAPX1 and SbGPX6 gene expression increased; among the genes related to ABA signal pathway, SbABF2 , SbSnRK2.4 expression decreased significantly, SbNCED3 ​The expression amount was significantly increased; in the genes related to lignin synthesis, SbPAL4 , SbC4H , SbCCR1 , SbC3H , SbF5H , SbCOMT1 The expression amount of all the genes was significantly increased, wherein SbCOMT1 The up-regulation fold of the gene (SORBI_3007G047300) was the most significant, which revealed SbWRKY51 The molecular mechanism of the gene regulating the response of sorghum to salt stress.

[0091] 3.10, the content of lignin of different sorghum strains under salt stress The sorghum strains were treated with 0, 150 mM NaCl respectively for 7 days, and then the lignin content in the roots was determined after drying the samples. As shown in Figure 12 Before the salt treatment, the lignin content of the sorghum strains was almost the same, and after the salt treatment, the lignin content of each strain was increased, and the increase of the lignin content of the transgenic plants was higher than that of the control group, which indicated that SbWRKY51 The gene might improve the salt resistance by regulating the genes related to lignin synthesis, and highlighted the correlation mechanism of the gene and the salt resistance of plants.

[0092] The above only describes the preferred embodiments of the present application and is not used to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. SbWRKY51 a gene or biological material containing SbWRKY51 application of a gene or biological material containing a gene in any one or more of: (a1) improving salt tolerance of a plant; (a2) improving seed germination rate of a plant under salt stress environment; (a3) improving root length of a plant under salt stress environment; (a4) improving dry weight and / or fresh weight of a plant under salt stress environment; (a5) improving oxidation tolerance of a plant; (a6) improving free radical scavenging ability of a plant.

2. Use according to claim 1, wherein The plant is selected from the group consisting of Sorghum bicolor and Arabidopsis thaliana.

3. The use according to claim 1, wherein containing SbWRKY51 Biological materials comprising the genes include any one or more of a recombinant expression vector, an expression cassette, a transgenic cell line, a recombinant bacteria, and a recombinant virus.

4. SbWRKY51 a promoter of a gene or a biological material comprising SbWRKY51 a biological material comprising a promoter of a gene for use in any one or more of the following; (b1) improving salt tolerance of a plant; (b2) improving seed germination rate of a plant under salt stress environment; (b3) improving root length of a plant under salt stress environment; (b4) improving dry weight and / or fresh weight of a plant under salt stress environment; (b5) improving oxidation tolerance of a plant; (b6) improving free radical scavenging ability of a plant.

5. The use according to claim 3, wherein the compound is ###0002### The plant is selected from the group consisting of Sorghum bicolor and Arabidopsis thaliana.

6. comprising SbWRKY51 a gene and / or SbWRKY51 a recombinant expression vector, expression cassette, transgenic cell line, recombinant bacteria or recombinant virus of a promoter of a gene.

7. A method for increasing salt tolerance in plants, characterized by, comprising the following steps: The SbWRKY51 gene and / or SbWRKY51 promoter of the gene is constructed into an expression vector to form a recombinant expression vector, and then the recombinant expression vector is transformed into a plant, so that the plant carries SbWRKY51 gene and / or SbWRKY51 promoter of the gene, and finally regulates the salt tolerance of the plant through SbWRKY51 high expression of the gene.

8. The method of claim 7, wherein, The expression vector is selected from the group consisting of Agrobacterium vector and vector applicable to plant microprojectile bombardment.

9. The method of claim 7, wherein, The plant is dicotyledon.

10. The method of claim 7, wherein, The transformation is introducing the recombinant expression vector into plant cells or tissues by biological method; The biological method includes any one of Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation, Agrobacterium mediation, gene gun, pollen tube introduction and liposome fusion.

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