Application of siERF4 and siERF4-like genes in improving salt tolerance of foxtail millet

Abstract

The application discloses SiERF4 and SiERF4-like The application of the genes in improving the salt tolerance of millet belongs to the technical field of crop genetic breeding. SiERF4 The genes and SiERF4- like Single knockout or double knockout is performed on the genes to obtain a mutant strain of millet with significantly improved salt tolerance. SiERF4 The mRNA sequence coded by the gene is shown as SEQ ID NO. 1, and the amino acid sequence is shown as SEQ ID NO. 2; SiERF4-like The mRNA sequence coded by the gene is shown as SEQ ID NO. 3, and the amino acid sequence is shown as SEQ ID NO. 4. The application first finds that SiERF4 and SiERF4- like The gene is a negative regulation gene responding to salt stress, and knocking out the gene can significantly improve the salt tolerance of the millet, and has important practical significance for cultivating salt-tolerant millet varieties and developing and utilizing saline land.

Description

Technical Field

[0001] This invention relates to the field of molecular breeding technology, and in particular to... SiERF4 and SiERF4-like Application of genes in improving the salt tolerance of millet. Background Technology

[0002] Soil salinization has become one of the major environmental constraints affecting global food security and sustainable agricultural development. According to FAO statistics, the total area of ​​soil affected by salinization worldwide exceeds 833 million hectares, and more than 10% of farmland is salinized.

[0003] Saline soil typically contains high concentrations of sodium. + Cl - Ca 2+ SO4 2- and HCO3 - Its saturated hydraulic conductivity exceeds 4.0 dS·m -1 Salt stress (approximately 40 mM NaCl) significantly impacts plant growth and development. Due to factors such as climate change, inadequate agricultural irrigation, industrial pollution, and rising sea levels in coastal areas, soil salinization continues to worsen, making the development of salt-tolerant crop varieties an urgent priority. By deeply exploring the molecular mechanisms of plant responses to salt stress, revealing key biological processes and genetic regulatory networks, and based on this understanding, developing new crop varieties with superior salt tolerance will provide innovative solutions for the efficient utilization of saline-alkali soils, thereby promoting sustainable agricultural development and ensuring food security.

[0004] AP2 / ERF (APETALA2 / ETHYLENE RESPONSE FACTOR) is one of the largest plant-specific transcription factor families, defined by the AP2 / ERF domain, which consists of approximately 60-70 amino acids and participates in DNA binding. The AP2 / ERF transcription factor family is mainly divided into four subfamilies: DREB (dehydration response element binding), ERF (ethylene response element binding protein), AP2 (APETALA2), and RAV (associated with ABI3 / VP), as well as a few unclassified factors. Abiotic stress alters the production and distribution of plant hormones. Plant ERF transcription factors, as pivotal regulatory elements in hormone signaling networks, mediate multi-hormone signal transduction cascades such as ABA, ethylene, and jasmonic acid, achieving a dynamic balance between growth and development and stress response. ERF activates or inhibits downstream stress-response gene networks by directly binding to the GCC-box or DRE element of target gene promoters.

[0005] Under salt and PEG stress ERF38 Overexpression of this substance significantly increased the content of POD, SOD, soluble protein, and proline in poplar trees, indicating that... ERF38Genes can improve the salt tolerance and osmotic pressure resistance of transgenic poplar trees. Rice OsERF83 By directly binding to the GCC-box of the promoter region of drought stress response genes, the expression levels of OsLEA3 and OsP5CS were induced to be upregulated, thereby improving the drought resistance of rice. Overexpression of maize... ZmERF21 The plants subsequently showed a significant increase in chlorophyll content and activity of a series of antioxidant enzymes, including SOD, while also regulating... ZmAREB1 and ZmDREB2A By establishing a positive feedback regulatory pathway through key genes, the drought resistance of maize seedlings is enhanced. In apples, MdERF106 and MdMYB63 Interactions are mediated by regulating Na + / H + To improve the salt tolerance of plants. Corn ZmEREB57 Overexpression of strawberry can improve salt stress tolerance in maize and Arabidopsis by regulating the oxyphytic dermal acid (OPDA) and jasmonic acid pathways. Heterologous expression of strawberry in Arabidopsis... FaTINY2 It can improve the plant's tolerance to salt stress. Overexpression in pear CaERF2 Salt tolerance in plants can be improved by increasing the activity of oxidative scavenging enzymes.

[0006] Therefore, discovering genes related to salt tolerance in millet is of great significance for modifying the salt tolerance of millet, improving its salt tolerance capacity, and carrying out salt-tolerant crop breeding. Summary of the Invention

[0007] The purpose of this invention is to provide SiERF4 and SiERF4-like The application of genes in improving the salt tolerance of millet, through the manipulation of ERF family genes. SiERF4 and SiERF4-like Knockout can improve millet's tolerance to salt stress. Hybridizing mutants to obtain double knockout mutants can significantly improve the salt tolerance of millet.

[0008] To achieve the above objectives, the present invention provides SiERF4 and SiERF4-like Application of gene knockout in improving the salt tolerance of millet SiERF4 and / or SiERF4-like Genes that can improve the salt tolerance of millet; SiERF4 The mRNA sequence encoded by the gene is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2; SiERF4-like The mRNA sequence encoded by the gene is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4.

[0009] Preferably, methods to improve the salt tolerance of millet include: (1) Reduce malondialdehyde content; (2) Increase proline content; (3) Enhance the ability to scavenge oxidative stress; (4) Enhance the ability to maintain ion balance.

[0010] Furthermore, the present invention provides SiERF4 and SiERF4-like Application of gene knockout in the breeding of salt-tolerant millet varieties SiERF4 and / or SiERF4-like Genes that can improve the salt tolerance of millet; SiERF4 The mRNA sequence encoded by the gene is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2; SiERF4-like The mRNA sequence encoded by the gene is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4.

[0011] Therefore, the present invention SiERF4 and SiERF4-like The application of genes in improving the salt tolerance of millet has the following beneficial effects: (1) This invention utilizes CRISPR / Cas9 gene editing technology to target the genes in millet. SiERF4 Gene and SiERF4-like Single or double gene knockout yielded millet mutant lines with significantly enhanced salt tolerance. These mutants exhibited superior salt tolerance under salt stress, specifically: significantly reduced malondialdehyde (MDA) content, mitigating cell membrane damage; significantly increased proline content, enhancing osmotic pressure regulation; significantly increased POD and SOD activity, strengthening oxidative stress scavenging capacity; and significantly increased Na+ content. + Accumulation reduction, K + The content is stable and the ability to maintain ion balance is improved; the salt tolerance of the double knockout mutant is better than that of the single knockout mutant, and its growth under 2% NaCl salt stress is significantly better than that of the wild type.

[0012] (2) This invention is the first to discover that millet contains SiERF4 and SiERF4-like This gene is a negative regulator of salt stress response. Knocking out this gene can significantly improve the salt tolerance of millet, filling the research gap in the regulation of salt stress by millet ERF family genes and providing a new direction for the discovery of crop salt tolerance genes.

[0013] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0014] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention 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.

[0015] Figure 1 Subcellular localization results for SiERF4 and SiERF4-like proteins; Figure 2 A phenotypic comparison of different strains under salt stress; Figure 3 A comparison chart of MDA content in different strains under salt stress; Figure 4 For the proline standard curve; Figure 5 A comparison of proline content among different strains under salt stress; Figure 6 A comparison of POD activity among different strains under salt stress; Figure 7 A comparison of SOD activity among different strains under salt stress; Figure 8 Na+ levels in various strains under salt stress + Content comparison chart; Figure 9 K levels in various strains under salt stress + Content comparison chart; Figure 10 DAB staining results for each strain under salt stress; Figure 11 The results of NBT staining for each strain under salt stress are shown. Detailed Implementation

[0016] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0017] To make the objectives, technical solutions, and advantages of this application clearer, more thorough, and more complete, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. The following detailed descriptions are all descriptions of embodiments, intended to provide further detailed explanation of the present invention. Unless otherwise specified, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0018] The instruments, equipment, reagents, and materials used in the examples were all obtained commercially.

[0019] Example 1 S1. Leaves of 4-week-old millet variety "Mizi Liang" were used for gene cloning and isolation. RNA was extracted using an RNA extraction kit (Nanjing Novizan Biotechnology Co., Ltd.), and the extracted RNA was reverse transcribed into cDNA using a reverse transcription kit (Nanjing Novizan Biotechnology Co., Ltd.). The cDNA was then analyzed using 2×hanta Max Master Mix (Dye Plus) reagent (Nanjing Novizan Biotechnology Co., Ltd.) and PCR. SiERF4 Amplification of the SiERF4-like gene.

[0020] SiERF4 The mRNA sequence encoded by the gene is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2; SiERF4-like The mRNA sequence encoded by the gene is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4.

[0021] SEQ ID NO.1:

[0022] SEQ ID NO.2:MAPKNSALPPVSAATDGMVEPRFRGVRKRPWGRYAAEIRDPARKARVWLGTFDTAEAAARAYDAAALHFRGPKAKTNFPVAFAHPAPPPKMLAVSPSSSTVESSSRDSPAASPAAALPAPSLDLSLGMPPMVAAQPFLFLDPRLAVTVAVPAPVPCRPAVVAGANKATCREDEQSDTGSSSSVVDASPAVDVGFDLNMPPPAEVA。

[0023] SEQ ID NO.3:ATGGCGCCCCGGGCGGCGGACAAGTCGCCGGTGCCGCCGGCGACCGGCCTCGGTCTCGGCGTCGGCGGTGGAGTCGGAGGCGTTGGCATGGGCCCGCACTTCAGGGGCGTCCGGAAGCGCCCGTGGGGACGGTACGCCGCGGAGATCCGCGACCCTGCCAAGAAGAGCCGCGTGTGGCTGGGCACGTACGACACGGCTGAGGAGGCCGCCCGCGCCTACGACGCCGCCGCGCGCGAGTTCCGCGGCGCAAAGGCCAAGACCAACTTCCCGTTCGCGTCGCAGTGCCCCGTCGCCGCCGGCGCCGGCAGCCCCAGCAGCAACAGCACCGTGGACTCGAGCGGAGGCGGCAGCGCCTGCGGCGTCCAGGCGCCTATGCAGGCCATGCCGCTGCCCCCGGCCCTCGATCTCGATCTCTTCCACCGGGCGGCGGCCGTGACCGCGGTCTCCTCCGGCGGCATGCGGTTTCCGTTCAAGGGCTACCCTGTCGCGCGCCCGACGCCGCATCCGTACTTCTTCTACGAACAGGCGGCAGCGGCCGCCGCCGCGGCGGCCGGCTACCGGATGCTCAAGGTTGCCCCGCCGCCGGTCACCGTGGCCGCAGTCGCGCAGAGCGACTCCGACTCCTCGTCGGTGGTCGATCACACCCCTTCGCCGCCCCCGGTGACCACAAAGAAGGAGGTGTCCTTCGATCTGGATCTGAACTGGCCGCCGCCGGCGGAGAACTAG。

[0024] SEQ ID NO.4: MAPRAADKSPVPPATGLGLGVGGGVGGVGMGPHFRGVRKRPWGRYAAEIRDPAKKSRVWLGTYDTAEEAARAYDAAAREFRGAKAKTNFPFASQCPVAAGAGSPSSNSTVDSSGGGSA CGVQAPMQAMPLPPALDLDLFHRAAAVTAVTAVSSGGMRFPFKGYPVARPTPPHYFFYEQAAAAAAAAAGYRMLKVAPPPVTVAAVAQSDSDSSSVVDHTPSPPPVTTKKEVSFDLDLNWPPPAEN.

[0025] S2, Vector construction and Agrobacterium-mediated transformation: (1) The pcam35-egfp vector was digested with KpnI and BamHI restriction endonucleases (thermo); the vector was cloned using homologous recombination with the ClonExpress® II one-step cloning kit (Nanjing Novizan Biotechnology Co., Ltd.). SiERF4 and SiERF4-like The amplified fragment was re-ligated to the KpnI and BamH restriction sites of the target vector pcam35-egfp to construct a subcellular localization vector.

[0026] SiERF4 Forward primer for gene amplification: ERF4-eGFP-F: CGAACGATAGCCATGGTACCAATGGCGCCCAAGAACAGC, SEQ ID NO.5; Reverse primer: ERF4-eGFP-R: CATGCCTGCGGCCGCGCCGGATCCCGCCACCTCGGCCGGC, SEQ ID NO. 6; SiERF4-like Forward primers for gene amplification: ERF4l-eGFP-F: CGAACGATAGCCATGGTACCAATGGCGCGCCCCGGGC, SEQ ID NO.7; Reverse primer ERF4l-eGFP-R: CATGCCTGCGGCCGCGCCGGATCCGTTCTCCGCCGGCGGC, SEQ ID NO. 8.

[0027] (2) Design knockout targets using the Target Design website http: / / skl.scau.edu.cn / targetdesign / SiERF4 The target sequence of the gene is CAAGAACAGCGCTCTCCCCC CGG(SEQ ID NO.9, underlined indicates PAM site). SiERF4-like The target sequence of the gene is TTGGCATGGGCCCGCACTTC AGG (SEQ ID NO.10, underlined indicates PAM site). Construction of gene editing vector pHUE411- SiERF4 and pHUE411- SiERF4-like Gene editing was performed using CRSIPR / Cas9 technology.

[0028] SiERF4 The forward primer sequence for gene editing, SiERF4-F: GGCGCAAGAACAGCGCTCTCCCCC, SEQ ID NO. 11; Reverse primer sequence SiERF4-R: AAACGGGGGAGAGCGCTGTTCTTG, SEQ ID NO.12; SiERF4-like The forward primer sequence for gene editing, SiERF4l-F: GGCGTTGGCATGGGCCCGCACTTC, SEQ ID NO.13; Reverse primer sequence SiERF4-R: AAACGAAGTGCGGGCCCATGCCAA, SEQ ID NO.14.

[0029] (3) Agrobacterium rhizogenes EHA105 (Shanghai Weidi Biotechnology Co., Ltd.) was transformed by experimental methods such as 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 thaliana and tobacco.

[0030] S3, Genetic transformation of millet: (1) Select plump millet grains and polish them with sandpaper to remove the seed coat.

[0031] (2) Seed disinfection: Wash with 75% ethanol for 5 min, then disinfect with 25% NaClO for 20 min, inverting and mixing several times during the process to ensure thorough cleaning. Wash with sterile water 5-6 times in a laminar flow hood, and then dry the surface of the seeds on sterile filter paper.

[0032] (3) Seeds were sown in callus induction medium CIM, with 25 seeds placed in each dish.

[0033] (4) After incubation at 28℃ in the dark for 2 weeks, callus can be seen.

[0034] (5) In order to obtain high-quality regenerable callus, the primary callus was subcultured and transferred to a new CIM medium. After three subcultures, the callus turned pale yellow and could be used for transformation.

[0035] (6) Place the Agrobacterium strain in 3-5 mL of liquid LB medium (containing antibiotics) and shake gently in the dark for 1-2 days, then place it in 100 mL of liquid LB medium (containing antibiotics) and shake vigorously in the dark until OD is reached. 600nm Between 0.6 and 0.8.

[0036] (7) Infection: Centrifuge the cultured Agrobacterium tumefaciens in a 50 mL centrifuge tube at 6000 rpm for 10 min, collect the Agrobacterium tumefaciens, and then suspend it in IM liquid medium (resuspend twice, adding acetosyringone to the second IM medium).

[0037] (8) Dilute Agrobacterium with IM of infection solution (1:1, v / v, OD) 600nm =0.3-0.5), infecting active callus tissue for 15-30 minutes.

[0038] (9) After infection, the callus tissue was blotted dry on sterile filter paper, and then transferred to IM solid culture medium containing AS and co-cultured at 22°C in the dark for 3 days.

[0039] (10) After co-culture, the callus tissue was restored in CIM medium containing antibiotics (250 mg / L carbenicillin and 300 mg / L cephalosporin) and cultured in the dark at 28°C for 3 days.

[0040] (11) Cleaning the callus: Clean with sterile water 5 times. Add 250 mg / L carbenicillin and 300 mg / L cephalosporin to the sterile water during the last cleaning.

[0041] (12) Transfer the above callus to CIM selective medium containing selection markers (50 mg / L hygromycin, 250 mg / L carbenicillin and 300 mg / L cephalosporin) and culture in the dark for 2 weeks.

[0042] (13) Continue to subculture the yellow callus on selective medium until a fast-growing resistant callus is formed.

[0043] (14) Transfer the resistant callus to shoot induction medium (SIM) and culture for 4-5 weeks at a day / night temperature of 28 / 22℃ and a day / night duration of 16 / 10h.

[0044] (15) Transfer the regenerated shoot tips to root induction medium (RIM) and culture for 2-3 weeks.

[0045] (16) Transfer the rooted transgenic plants to the potting soil.

[0046] (17) Extract genomic DNA and perform PCR identification.

[0047] get SiERF4 Gene knockout lines erf4 and SiERF4-like Gene knockout lines erf41 And the double gene knockout strain produced by crossing two gene knockout strains. erf4erf41 .

[0048] CIM selective medium: 4 g / L N6 basal salt (containing vitamins) + 30 g / L sucrose + 2 mg / L 2,4-D + 0.3 g / L caseinate hydrolysate + 2.8 g / L proline + 0.1 g / L inositol + 0.1 mg / L 6-benzylaminopurine + 4 g / L plant gel, pH 5.8.

[0049] Infection solution IM: 0.44 g / L MS salt + 1× B5 vitamins + 68 g / L sucrose + 36 g / L glucose + 1 g / L aspartic acid + 1 g / L casein amino acids + 0.2 g / L cysteine ​​+ 2 mg / L 2,4-D + 200 μM AS, pH 5.2.

[0050] SIM bud induction medium: 4.43 g / L MS basal salts plus vitamins + 30 g / L sucrose + 1 g / L proline + 1 g / L aspartic acid + 0.5 g / L casein acid hydrolysate + 0.25 mg / L copper sulfate + 2 mg / L 6-BA + 0.2 mg / L NAA + 50 mg / L hygromycin + 8 g / L agar, pH 5.7.

[0051] RIM rooting medium: 2.2 g / L vitamin MS salt + 30 g / L sucrose + 0.1 g / L inositol + 2.6 g / L gelase, pH 5.6.

[0052] S4, Instantaneous Tobacco Transformation: (1) Small-scale culture: Use a sterile pipette tip to pick up a single clone and put it into 5 mL of YEP medium containing the corresponding antibiotic. Incubate overnight at 28°C and 180 rpm.

[0053] (2) Take 1.5 mL of the overnight culture and add it to 50 mL of YEP medium. At the same time, add 6 μL of acetylsuccinone and shake at 28°C and 180 rpm until the OD value is reached. 600nm Approximately 0.9 is sufficient.

[0054] (3) Collect the shaken bacterial solution 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 MMEs, 10mM MgCl2).

[0055] (4) Mix the bacterial solution containing the target carrier and P19 at a ratio of 1:1 and let stand for 3 hours.

[0056] (5) Use a 1mL sterile syringe to draw up the prepared bacterial solution and slowly inject the bacterial solution into both sides of the main leaf vein. Make a mark and continue to place the tobacco plant in the greenhouse for 2-3 days. Observe it with a laser confocal microscope.

[0057] Observation results as follows Figure 1 As shown, the results indicate that both SiERF4 and SiERF-like proteins are located in the nucleus.

[0058] S5, Salt stress treatment of millet seedlings: Wild-type and mutant lines were selected ( erf4 , erf41 , erf4erf41 Plump millet grains were sown in vermiculite. After 14 days of germination, seedlings with uniform growth were selected for salt stress treatment. The salt concentration was 2% NaCl. Phenotypic photos were taken after 10 days of salt stress treatment. Fifteen plants were treated for each line, and the experiment was repeated three times.

[0059] The results are as follows Figure 2 As shown, the results indicate that under salt stress treatment, the wild-type control showed the highest degree of wilting, while the mutant lines exhibited higher salt tolerance compared to the wild-type.

[0060] S6, Malondialdehyde (MDA) content determination: Wild-type and mutant strains were used. erf4 , erf41 , erf4erf41 0.1 g of millet leaves were placed in a pre-cooled mortar and ground into powder with liquid nitrogen. 3 mL of 10% trichloroacetic acid (TCA) was added and mixed thoroughly. The mixture was centrifuged at 4000 rpm for 10 min, and 800 μL of the supernatant was transferred to a new 2 mL centrifuge tube. 800 μL of 0.6% thiobarbituric acid (TBA) was added. The mixture was heated at 100 °C for 15 min, and then immediately cooled in an ice bath. The mixture was centrifuged at 10000 rpm for 15 min, and 200 μL of the supernatant was transferred to an ELISA plate. The absorbance was measured at 450 nm, 532 nm, and 600 nm, and the MDA content was calculated using the formula.

[0061] The calculation formula is: MDA concentration (nmol / g) = 6.45 × (A 532-A 600 -0.56×A 450 ×60.

[0062] The measurement results are as follows Figure 3 As shown, 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, and the MDA content in wild-type plants was higher than that in all mutant plants. erf4erf4l The double mutant MDA content is lower than erf4 and erf4l Mutant.

[0063] S7. Determination of proline content: Wild-type and mutant strains were used. erf4 , erf41 , erf4erf41 0.1g of millet leaves were placed in a pre-cooled mortar and ground into powder with liquid nitrogen. 3mL of 3% sulfosalicylic acid solution was added, and the mixture was extracted in boiling water for 15min. After centrifugation at 4000r / min for 10min, the supernatant was collected for analysis.

[0064] Preparation of the standard curve: Prepare 100 mL of 20 μg / mL proline standard solution. Divide the solution into 7 test tubes and add 0, 0.1, 0.2, 0.4, 0.6, 0.8, and 1 mL of proline standard solution, respectively. Add distilled water to make up any remaining volume if necessary. The proline standard curve is shown below. Figure 4 As shown.

[0065] Take 250 μL of the sample supernatant and standard solution, and add 250 μL of glacial acetic acid and 250 μL of colorimetric solution (acidic ninhydrin solution: 60 mL glacial acetic acid, 16.4 mL phosphoric acid, distilled water to a final volume of 100 mL, then add 2.5 g ninhydrin, dissolve and store in the dark). Mix well and incubate in a water bath at 100 °C for 30 min. Remove and cool to room temperature. Add 500 μL of toluene to each tube and shake repeatedly. After standing and allowing the layers to separate, aspirate 200 μL of the toluene layer into an ELISA plate and measure its absorbance at 520 nm to plot a standard curve.

[0066] The proline content was calculated based on the standard curve, and the results are as follows: Figure 5 As shown. Under control conditions, there was no difference in proline content between wild-type and overexpression lines. Under salt stress, the proline content of all lines increased, and the proline content in wild-type plants was lower than that in all mutant plants. erf4erf4l The double mutant has a higher proline content than erf4 and erf4l Mutant.

[0067] S8. Determination of the activity of oxidative scavenging related enzymes: Wild-type and mutant strains were used. erf4 , erf41 , erf4erf41 0.1g of millet leaves were used to determine the content of superoxide dismutase (SOD) and peroxidase (POD). Specific methods for activity determination can be found in the Superoxide Dismutase (SOD) Activity Assay Kit (D799593-0050) and Peroxidase (POD) Activity Assay Kit (D799591-0050) from Shanghai Sangon Biotech Co., Ltd.

[0068] The results of POD enzyme activity assay are as follows: Figure 6 As shown, under control conditions, there was no difference in POD enzyme activity among the different strains. Under salt stress treatment, the POD enzyme activity of all strains increased. erf4erf4l The POD enzyme activity in the double mutant lines was significantly higher than that in the wild-type control.

[0069] SOD enzyme activity assay results are as follows: Figure 7 As shown, under control conditions, there was no difference in SOD enzyme activity among the lines. Under salt stress treatment, the SOD enzyme activity in all lines increased, and the SOD enzyme activity in the mutant lines was significantly higher than that in the wild-type control.

[0070] S9, Na + K + Content detection: The sample was dried in an oven at 120°C for 30 minutes, then in an oven at 80°C overnight to constant weight. The dried sample was ground into powder and passed through a 0.5 mm sieve. A mixture of the powder and HNO3 (0.25 g) was digested with 5 mL of HNO3 and kept at 110°C until a colorless liquid was obtained (approximately 6 h). The liquid mixture was then cooled to room temperature and adjusted to a volume of 10 mL with deionized water. The Na+ content of the supernatant was determined using a Perkins-Elmer 360 atomic absorption spectrophotometer. + concentration.

[0071] Na + Content determination results are as follows Figure 8 As shown, under control conditions, the Na content in wild-type and mutant plants was significantly different. + There was no difference in Na content between wild-type and mutant plants under salt stress treatment. + The content of all three types was significantly increased, with the wild type showing the highest Na content. + The content was much higher than that of various mutant strains, and the mutant strains erf4 , erf4l and erf4erf4l ZhongNa + There was no difference in content.

[0072] K + Content detection results as follows Figure 9As shown, there was no difference in potassium ion content among the strains under control conditions, but under salt stress conditions, the potassium ion content of all strains decreased, with a significant decrease in potassium ion content in wild-type plants.

[0073] S10, DAB staining: Weigh DAB powder, dilute with distilled water to 1 mg / mL, add concentrated HCl to pH 3.8 to ensure complete dissolution, and adjust pH to 5.8 with NaOH before use. Place millet leaves in the DAB solution, vacuum for half an hour, and incubate overnight at 28°C in the dark. Discard the staining solution, wash several times with 95% ethanol until the green color of the leaves is completely removed, and store in anhydrous ethanol. DAB staining results are shown below. Figure 10 As shown, under salt stress treatment, the color of the leaves of each mutant line after staining was lower than that of the wild-type control.

[0074] S11, NBT staining: Weigh NBT powder, dilute with distilled water to 0.5 mg / mL, immerse Arabidopsis in the NBT solution under vacuum for half an hour, and stain overnight at 28°C. Discard the staining solution, wash several times with 95% ethanol until the green color of the leaves is completely removed, and store in anhydrous ethanol. The NBT staining results are shown below. Figure 11 As shown, under salt stress treatment, the color of the leaves of each mutant line after staining was lower than that of the wild-type control.

[0075] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. SiERF4 and SiERF4-like The application of genes in improving the salt tolerance of millet is characterized by: Knockout SiERF4 and / or SiERF4-like Genes that can improve the salt tolerance of millet; SiERF4 The mRNA sequence encoded by the gene is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2; SiERF4-like The mRNA sequence encoded by the gene is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.

4.

2. As described in claim 1 SiERF4 and SiERF4-like The application of genes in improving the salt tolerance of millet is characterized by, Improving the salt tolerance of millet includes: (1) Reduce malondialdehyde content; (2) Increase proline content; (3) Enhance the ability to scavenge oxidative stress; (4) Enhance the ability to maintain ion balance.

3. SiERF4 and SiERF4-like The application of genes in the breeding of salt-tolerant millet varieties is characterized by: Knockout SiERF4 and / or SiERF4-like Genes that can improve the salt tolerance of millet; SiERF4 The mRNA sequence encoded by the gene is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2; SiERF4-like The mRNA sequence encoded by the gene is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4.

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