PROSiCAPE14 gene and application of protein encoded by PROSiCAPE14 gene in improving salt tolerance of plants

Abstract

The invention belongs to the technical field of plant genetic engineering, and particularly relates to application of a PROSiCAPE14 gene and a protein encoded by the PROSiCAPE14 gene to improvement of plant salt tolerance. According to the invention, it is found for the first time that salt stress can cause down-regulation of the expression of the millet PROSiCAPE14 gene. Under a salt treatment condition, the plant height and the root length of a PROSiCAPE14 gene coded small peptide externally applied to millet and a constructed PROSiCAPE14 gene overexpression strain are obviously worse than those of a control group, and the plant height and the root length of a PROSiCAPE14 gene silent plant are obviously better than those of the control group. It is indicated that the PROSiCAPE14 gene plays an important role in the salt stress response process of the millet, and a foundation is provided for further research on the effect of the CAPE14 gene in the adaptation of the millet to abiotic stress and other possible biological functions.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically involving PROSiCAPE14 Application of genes and their encoded proteins in improving plant salt tolerance. Background Technology

[0002] Millet is a small diploid C4 spike crop with advantages such as high water use efficiency, low nutrient requirements, and wide adaptability. In-depth analysis of the salt tolerance mechanism of millet is of great significance for utilizing marginal land and cultivating new salt-tolerant crop varieties.

[0003] In recent years, numerous researchers have employed various methods to elucidate the response mechanisms of millet under salt stress. For example, overexpressing the millet gene in Arabidopsis thaliana... SiMBR2 It can effectively reduce the accumulation of reactive oxygen species in the plant and improve drought tolerance, thereby enhancing plant stress resistance. Wang et al. discovered multiple [resources] in millet through transcriptome analysis and qRT-PCR technology. SiAAAP Genes may be involved in salt stress response. However, compared with model plants such as rice and Arabidopsis, research on the salt tolerance mechanism of millet is still in its early stages, with a limited number of identified genes and a complex regulatory network that needs further analysis. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the purpose of this invention is to address these shortcomings. PROSiCAPE14 Application of genes and their encoded proteins in improving plant salt tolerance.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: The first aspect of the present invention provides PROSiCAPE14 Application of genes and their encoded proteins in improving plant salt tolerance, wherein the encoded proteins are proteins as shown in (a1) or (a2) below: (a1) A protein consisting of the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing; (a2) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein defined in (a1).

[0006] A second aspect of the invention provides a product containing PROSiCAPE14 Application of genetic biomaterials in regulating the salt tolerance of millet.

[0007] A third aspect of the present invention provides a method for preparing transgenic plants, comprising inhibiting endogenous plant genetic material. PROSiCAPE14 Gene expression, silencing, or knockout of plant endogenous genes PROSiCAPE14 Genes; the stated PROSiCAPE14 The nucleotide sequence of the gene is shown in SEQ ID NO:2.

[0008] A fourth aspect of the present invention provides a method for improving the salt tolerance of plants, comprising inhibiting endogenous plant salt production. PROSiCAPE14 Gene expression, silencing, or knockout of plant endogenous genes PROSiCAPE14 Genes enhance plant salt tolerance; PROSiCAPE14 The nucleotide sequence of the gene is shown in SEQ ID NO:2.

[0009] The fifth aspect of the invention, PROSiCAPE14 Genes and their encoded proteins or containing them PROSiCAPE14 The application of the gene in biological materials or the methods described above in plant breeding, wherein the sequence of the PROSiCAPE14 gene is shown in SEQ ID NO:2.

[0010] The sixth aspect of the present invention, PROSiCAPE14 Genes and their encoded proteins or containing them PROSiCAPE14 The application of genetically modified biological materials or the methods described above in the genetic improvement of plant germplasm resources, wherein... PROSiCAPE14 The gene sequence is shown in SEQ ID NO:2.

[0011] Compared with the prior art, the technical solution of the present invention has the following beneficial effects: This invention is the first to discover that salt stress can cause millet to... PROSiCAPE14 Gene expression was downregulated. Under salt treatment conditions, exogenous application of salt to millet... PROSiCAPE14 Gene-encoded small peptides and their construction PROSiCAPE14 The plant height and root length of the gene-overexpressing lines were significantly worse than those of the control group. PROSiCAPE14 The gene-edited plants exhibited significantly greater plant height and root length than the control group. This indicates that... PROSiCAPE14 Genes play an important role in the response of millet to salt stress, providing a basis for further research. CAPE The role of genes in millet's adaptation to abiotic stresses and other possible biological functions provides a basis. Attached Figure Description

[0012] Figure 1 for PROSiCAPE Gene family expression pattern analysis diagram.

[0013] Figure 2 Millet under different salt stress conditions in embodiments of the present invention CAPE Gene family expression analysis; where A is PROSiCAPE2 Relative expression level, B is PROSiCAPE3 Relative expression level, C is PROSiCAPE5 Relative expression level, D is PROSiCAPE6 Relative expression level, E is PROSiCAPE7 Relative expression level, F is PROSiCAPE12 Relative expression level, G is PROSiCAPE14 Relative expression level, H is PROSiCAPE15 Relative expression level, I is PROSiCAPE18 Relative expression level.

[0014] Figure 3 This is an example of the subcellular localization of PROSiCAPE14 protein in tobacco in this invention; wherein, A is the subcellular localization of PROSiCAPE14 protein; and B is the yeast single-hybrid self-activation activity analysis.

[0015] Figure 4 This invention illustrates the effect of exogenous SiCAPE14 spraying on millet growth and salt tolerance in an embodiment of the invention. A represents the millet phenotypic diagram; B represents a comparison of millet root length bar graphs; C represents a comparison of millet plant height bar graphs; and D represents the DAB staining results of millet leaves. In the bar graphs, the same letter indicates no difference, and different letters indicate significant differences.

[0016] Figure 5 The figures above are phenotypic diagrams and biomass measurements of overexpressed millet under 0 and 150 mM NaCl treatments in this embodiment of the invention. In the figures, A represents the phenotypic diagram; B represents a comparison of plant height (bar chart); C represents a comparison of aboveground fresh weight (bar chart); D represents a comparison of aboveground dry weight (bar chart); E represents a comparison of root length (bar chart); F represents a comparison of root fresh weight (bar chart); and G represents a comparison of root dry weight (bar chart). In the bar charts, the same letter indicates no difference, and different letters indicate significant differences.

[0017] Figure 6 This invention relates to the determination of relevant physiological indicators of surface-treated plants under different salt concentrations in this embodiment; wherein, A represents DAB and NBT staining; B represents a comparison of sodium ion content measurement results in a bar chart; C represents a comparison of potassium ion content measurement results in a bar chart; and D represents Na... + / K + The results were compared using bar charts; in the bar charts, the same letter indicates no difference, while different letters indicate significant differences.

[0018] Figure 7 Wild-type millet and gene transiently silenced millet PROSiCAPE14 The results of gene expression level detection, where A represents the expression level of millet leaves after 12 h of treatment. PROSiCAPE14 Gene expression analysis, B represents the expression level of millet leaves after 48 h of treatment. PROSiCAPE14 Gene expression level analysis.

[0019] Figure 8 The figures show the root length and fresh weight analysis results of wild-type millet seedlings and millet seedlings with transient gene silencing; where A is the phenotypic diagram of different treatment groups, B is the root length analysis result of different treatment groups, and C is the fresh weight analysis result of different treatment groups; in the figures, ns indicates no significant difference, and * indicates p <0.05, ** indicates p <0.01.

[0020] Figure 9 As described in the embodiments of the present invention PROSiCAPE14 Promoter and transiently injected tobacco GUS staining analysis; where A is transiently injected tobacco GUS histochemical staining; B is yeast single-hybrid verification of SiMYBS1, SibHLH49 and PROSiCAPE14 Promoter interaction; C represents the carrier construction process.

[0021] Figure 10 Analysis of the SibHLH49 dual-luciferase reporter gene assay.

[0022] Figure 11 Analysis of the SiMYBS1 dual-luciferase reporter gene assay. Detailed Implementation

[0023] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0024] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.

[0025] In a typical embodiment of the present invention, PROSiCAPE14 Application of genes and their encoded proteins in improving plant salt tolerance, wherein the encoded proteins are proteins as shown in (a1) or (a2) below: (a1) A protein consisting of the amino acid sequence shown in SEQ ID NO:1 in the sequence listing; (a2) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein defined in (a1).

[0026] In some implementations, the PROSiCAPE14 The gene sequence is shown in SEQ ID NO:2.

[0027] MATTRTVAVAAFVLVFSTAISSMAQTTAQEQEFVALHNAARREVGVEDVVWNETVAAFARAYAARRAGDCKLEHSDQDERNKLGYGENIYMGPPGKDWTVAEAVQWWVDEKQFYDNVSGMCVVGKECGHYTQVVWGNTKAIGCARVKCDSGGIFITCNYTPAGNVIGKRPF (SEQ ID NO: 1).

[0028] ATGGCGACGACCAGGACGGTGGCAGTAGCAGCCTTTGTACTTGTGTTCTCCACGGCTATATCCTCAATGGCTCAGACCACGGCGCAAGAGCAGGAGTTCGTGGCGCTCCACAACGCCGCCCGTCGTGAGG TGGGTGTTGAGGATGTCGTCTGGAACGAGACCGTGGCGGCGTTCGCACGAGCCTACGCGGCAAGACGTGCTGGTGACTGCAAGCTCGAGCACTCGGACCAGGATGAGCGCAACAAGTTGGGCTACGGTGA AAACATTTACATGGGGCCTCCTGGTAAGGACTGGACGGTGGCGGAGGCCGTGCAATGGTGGGTGGACGAGAAGCAATTCTACGACAACGTCAGTGGTATGTGCGTGGTGGGGAAGGAGTGCGGGCACTAC ACCCAGGTGGTCTGGGGCAACACCAAGGCCATCGGCTGCGCGCGTGTCAAGTGTGACAGCGGTGGCATCTTCATCACCTGCAACTATACACCGGCGGGCAACGTCATCGGAAAACGTCCATTCTAG (SEQ IDNO:2).

[0029] In some implementations, the application is achieved by suppressing, silencing, or knocking out the PROSiCAPE14 gene.

[0030] In some embodiments, the suppression, silencing, or knockout of the PROSiCAPE14 gene expression is described. PROSiCAPE14 Genes include those obtained through gene knockdown, gene editing, and / or gene knockout technologies. PROSiCAPE14 Nucleic acid molecules with missing or inactivated genes.

[0031] Furthermore, the use of gene knockdown technologies (including RNA interference, Morpholino interference, antisense nucleic acid technology, and ribozyme technology), gene editing technologies (including zinc finger ribozyme gene editing, TALEN gene editing, and CRISPR gene editing), or gene knockout technologies (including complete gene knockout and conditional gene knockout) to suppress gene expression, silence, or knock out genes is well known to those skilled in the art. For example, targeting the protein can be used... PROSiCAPE14 Gene expression can be inactivated or silenced at the post-transcriptional or translational level by shRNA, siRNA, or miRNA encoding the gene. Target genes can also be knocked out using the CRISPR-Cas system, which contains sgRNA and Cas protein.

[0032] In some implementations, suppression PROSiCAPE14 Gene expression, silencing, or knockout PROSiCAPE14 The nucleic acid molecules of a gene include: (1) Double-stranded RNA (dsRNA), small interfering RNA (siRNA), microRNA (miRNA) and short hairpin RNA (shRNA) used in RNA interference technology; (2) Antisense RNA (asRNA) and antisense oligonucleotides (AON) used in antisense nucleic acid technology; (3) gRNA and sgRNA used in gene editing technology; (4) Aptamers and ribozymes, etc.

[0033] In some implementations, the silencing is achieved by using gene knockout technology to... PROSiCAPE14 Genes are silenced.

[0034] In some embodiments, the sequence of the antisense oligonucleotide is shown in SEQ ID NO:3.

[0035] T*T*G*C*AGGTGATGAAGA*T*G*C*C (SEQ ID NO: 3).

[0036] In some embodiments, the knockout is achieved using gene editing technology, through homologous recombination or non-homologous end joining repair, so that... PROSiCAPE14 Deletion, addition, truncation, or substitution of gene sequences.

[0037] In another typical embodiment of the present invention, the application of biomaterials containing the PROSiCAPE14 protein-coding gene in regulating the salt tolerance of millet is provided.

[0038] In some embodiments, the biological material is selected from at least one of recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, engineered bacteria, or non-renewable millet cells or tissues.

[0039] In some embodiments, the regulation is performed by adjusting the salt tolerance of millet by regulating the content or activity of the PROSiCAPE14 protein.

[0040] In some implementations, the content or activity of the PROSiCAPE14 protein is regulated using transgenic or gene-editing techniques.

[0041] In some implementations, the salt tolerance of millet is regulated by exogenous application of PROSiCAPE14-derived peptides.

[0042] In some embodiments, the salt tolerance of millet is improved by reducing the content or activity of the PROSiCAPE14 protein; or, the salt tolerance of millet is reduced by increasing the content or activity of the PROSiCAPE14 protein.

[0043] In another typical embodiment of the present invention, a method for preparing transgenic plants is provided, comprising inhibiting endogenous plant genetic material. PROSiCAPE14 Gene expression, silencing, or knockout of plant endogenous genes PROSiCAPE14 The sequence of the PROSiCAPE14 gene is shown in SEQ ID NO:2.

[0044] In another typical embodiment of the present invention, a method for improving plant salt tolerance is provided, comprising inhibiting endogenous plant salt production. PROSiCAPE14 Gene expression, silencing, or knockout of plant endogenous genes PROSiCAPE14 Genes that enhance plant salt tolerance, the aforementioned PROSiCAPE14 The gene sequence is shown in SEQ ID NO:2.

[0045] In another typical embodiment of the present invention, the application of the PROSiCAPE14 protein or its encoding gene or biological material containing its encoding gene or the above-described method in plant breeding is provided, wherein the amino acid sequence of the PROSiCAPE14 protein is shown in SEQ ID NO: 1; and the nucleotide sequence of the encoding gene is shown in SEQ ID NO: 2.

[0046] In some embodiments, the application in plant breeding is the application in cultivating highly salt-tolerant plants or in cultivating plants that grow in high-salt soil environments.

[0047] In another typical embodiment of the present invention, the application of PROSiCAPE14 protein or its encoding gene or biological material containing its encoding gene or the above-described method in the genetic improvement of plant germplasm resources is provided, wherein the amino acid sequence of the PROSiCAPE14 protein is shown in SEQ ID NO: 1; and the nucleotide sequence of the encoding gene is shown in SEQ ID NO: 2.

[0048] In this invention, the plant is selected from at least one of rice, corn, soybean, sunflower, sorghum, wheat, alfalfa, cotton, barley, millet, and foxtail millet. Foxtail millet is preferred.

[0049] The experimental materials and reagents used in the following examples were obtained from the following sources: The millet seed Ci846 was obtained from the Stress Plant and Molecular Biology Team of the College of Life Sciences, Shandong Normal University, in December 2021.

[0050] The bacterial strains used in the experiment included: Escherichia coli competent cells DH5α (Coolaber, CC501), yeast Y1H competent cells (Coolaber, CC308), Agrobacterium rhizogenes competent cells K599 (Coolaber, CC410), and Agrobacterium tumefaciens competent cells GV3101 (Coolaber, CC405).

[0051] The vectors used in the experiment included: Agrobacterium transient transformation expression vector pCAMBIA1300 (Coolaber, VT119), stable transformation expression vector pCAMBIA1300 (Coolaber, VT119), yeast experiment-related vector pAbAi / pGADT7 (Coolaber, VT009 / VT001), and dual-luciferase reporter gene experiment vector pGreenⅡ 0800-LUC (Coolaber, VT145).

[0052] The primers used for gene amplification and vector construction are shown in Table 1.

[0053] Table 1 Primer Sequences

[0054] The experimental methods involved in the following embodiments are as follows: Early treatment of millet: Select plump, brightly colored millet seeds and spread them evenly on petri dishes lined with tissue paper and filter paper for germination, keeping the petri dishes well-hydrated. After 1-2 weeks of cultivation, transfer to sand culture, watering daily with Hoagland nutrient solution (adjusted to pH 6.2), and allow to grow for approximately 2-3 weeks. Once the millet seedlings have grown to three leaves and one bud, treat them with 150 mM NaCl. NaCl treatment was performed at six time points: 0 h, 12 h, 24 h, 36 h, 48 h, and 72 h. Each treatment was replicated in triplicate. After treatment, rinse the leaves and other tissues thoroughly and dry them. Cut the roots, stems, and leaves into small pieces, wrap them in aluminum foil, flash-freeze in liquid nitrogen, and store at -80°C. The millet growth and treatment processes were conducted at 25°C–30°C.

[0055] Total RNA extraction from millet: Plant RNA was extracted using an RNA extraction kit (Novizan).

[0056] Millet cDNA synthesis: cDNA was synthesized using a reverse transcription kit (Novizan) and stored at -20°C.

[0057] PROSiCAPE14 Gene amplification: The CDS sequence of the gene was found on the Ensemble Plant website. Based on the information provided on the website, amplification primers were designed using DNAMAN. Note that primers should ideally be non-complementary and have similar annealing temperatures. The target sequence was then amplified using millet cDNA as a template. The amplification results were then detected by electrophoresis, and the target fragment was recovered using a gel extraction kit (Novozymes).

[0058] RT-qPCR Detection: cDNA Dilution: The reverse transcription product cDNA was removed, thawed on ice, and diluted with 2-5 volumes of RNase-free water. The mixture was inverted and briefly centrifuged. The quantitative enzyme preparation system from Qingke Biotechnology Co., Ltd. was used. RT-qPCR detection was performed using a Roche 96 real-time PCR instrument (Switzerland). Each group had three replicates, and the mean ± SD of the three replicates was used for data analysis. The RT-qPCR calculation method used a 2-1... -ΔΔCT Law.

[0059] Vector Construction: Ligation was performed using homologous recombination. First, primers with restriction enzyme sites were designed, and the target sequence was amplified using a template. The vector was then digested with enzymes. Agarose gel electrophoresis was used to verify the completeness of the digestion reaction. The digested products were purified using a DNA gel extraction kit (Novozymes) and ligated using TaKaRa Bio homologous recombination enzyme. The ligation products were placed in competent *E. coli* cells, followed by the addition of 900 µL of LB liquid medium and incubation at 37°C with shaking for 60 min. The cells were centrifuged at 5000 rpm for 3 min. Approximately 50 µL of supernatant was used to resuspend the cells. The cells were plated onto LB solid medium containing the corresponding antibiotic and incubated overnight at 37°C with shaking. Antibiotic resistance was determined based on the plasmid vector's resistance. All operations involving direct contact with the external environment were performed in a clean bench to prevent contamination. Colony PCR identification was then performed.

[0060] Plasmids were extracted using the Tiangen Rapid Plasmid Mini-Prep Kit, following the manufacturer's instructions. The plasmid was then transformed into GV3101 competent cells. GV3101-pSoup-P19 competent cells were removed from -80℃ and thawed on ice until a semi-transparent gel was formed. Under aseptic conditions, 5 µL of the plasmid to be transformed was added and gently pipetted 6–8 times. Transformation was then performed following the same procedure as for GV3101-pSoup-P19 competent cells. After transformation, 500 µL of LB liquid medium was added, and the cells were incubated at 28℃ on a shaker (200 rpm) for 3 h. The cells were centrifuged at 5000 rpm for 1 min, and 50 µL of supernatant was used to resuspend the cells. The resuspended cells were then evenly spread onto LB agar plates (rifampin + specific antibiotic) using a sterile spreader. All procedures were performed in a laminar flow hood to prevent contamination.

[0061] DAB staining: DAB staining is used for the qualitative detection of H2O2 accumulation levels in millet leaves under salt stress. Weigh 0.1 g of DAB dye and dissolve it in 100 mL of deionized water. Adjust the pH to 3.8 after dissolution. Then, select the middle part of the second or third leaf of the treated millet (the part should be consistent) and cut millet leaves of equal length. Adjust the pH of the above DAB staining solution to 5.8 using a pH meter. Immerse the cut millet leaves in the DAB staining solution and place them in the dark for at least 8 hours. Avoid placing them for too short or too long a time; too short a time will make staining difficult, while too long a time will easily lead to over-staining. Then, decolorize with 80% ethanol in a boiling water bath. Note: The staining solution should be prepared fresh before use.

[0062] NBT staining: NBT staining is used for superoxide anion staining in plant leaves. Dissolve PBS tablets in deionized water to prepare a PBS solution (10 mM). Weigh 0.05 g of NBT dye and dissolve it in 100 mL of PBS solution, then stir with a magnetic stirrer until dissolved. Next, select the middle section of the second or third leaf of the treated millet (any consistent section is acceptable) and cut millet leaves of equal length. Immerse these leaves in the NBT staining solution for 3-4 hours. Then decolorize with 80% ethanol in a boiling water bath.

[0063] Na + K + Content determination: Weigh 0.3 g of the plant tissue to be tested into a clean test tube, add 10 mL of Wahaha water, and boil for 2-3 hours. After cooling to room temperature, filter with filter paper and make up to volume, then remove impurities with a filter column, and finally determine the Na+ and K+ content using a flame spectrophotometer. Ion content (mmol / g FW) = [numerical value × volume (0.025 L) × dilution factor] / [atomic mass of the element (M) × sample mass (g)].

[0064] Subcellular localization analysis: The gene plasmid and empty vector plasmid were mixed 1:1 with the P19 helper plasmid and injected at multiple points using a 1 mL sterile syringe. After injection, the cells were cultured under light for 36-48 h, and then the subcellular localization was observed under a two-photon laser scanning confocal microscope (TCS SP8 MP, Leica, Germany).

[0065] The SiCAPE14 foliar spray method was used to identify the salt tolerance of millet: When the above-ground parts of millet reached approximately 7-8 cm in length, seedlings with uniform growth were selected for the experiment. Two groups were set up: a non-salt treatment and a 150 mM NaCl treatment. Millet leaves were sprayed with 0.5 μmol, 1 μmol, and 2 μmol of the small peptide SiCAPE14, respectively, while the control group was sprayed with an equal volume of sterile water. Each treatment was repeated in triplicate. The small peptide SiCAPE14 was synthesized by Qingdao Qingke Biotechnology Co., Ltd.

[0066] Y1HGold System Yeast Single-Heteromorphic Interaction Validation Experiment: The binding activity of the PROSiCAPE14 gene promoter to candidate transcription factors was verified using the Y1H system. The yeast single-heteromorphic interaction validation kit was used to perform the experiment.

[0067] GUS staining analysis for tissue localization: Using a perforator, obtain tissue samples and immerse the leaves to be stained in 5 mL of GUS staining working solution in an EP tube, ensuring the solution completely covers the leaf. Wrap the EP tube in aluminum foil and incubate overnight at 37°C. Observe and photograph using a fully automated stereofluorescence microscopy system (Leica).

[0068] Dual-luciferase reporter gene assay: The recombinant plasmid transformed into GV3101-pSoup-P19 and the empty vector plasmid were thawed on ice, then transferred to 50 mL LB liquid medium containing kanamycin, rifampin, and AS, respectively, and incubated overnight at 28°C with shaking. After the bacterial cells became turbid, they were shaken again, followed by incubation with shaking at 28°C. The OD of the bacterial culture was measured... 600 The OD value was approximately 0.6. The cells were centrifuged at 1700 g for 5 min, and the bacterial pellet was collected. The pellet was resuspended in MMA solution, and the OD value was adjusted. 600 The concentration was increased to 1.0, and the mixture was allowed to stand for 2-4 hours. The bacterial suspension was mixed 1:1 thoroughly. Healthy tobacco plants were selected, and the lower epidermis was injected using a 1 mL sterile syringe. To ensure consistency, different parts of the same leaf were used for both the experimental and control groups during injection. After incubation in the tissue culture room in the dark for 12 hours and under light for 24-36 hours, the lower epidermis was sprayed with a prepared D-fluorescein potassium salt solution. After standing for 5-10 minutes, the samples were observed and photographed using an animal in vivo three-dimensional imaging analysis system.

[0069] 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.

[0070] Example 1: Screening of Salt Stress-Related Genes Based on millet transcriptome data, 18 PROSiCAPE Gene expression patterns under 0, 24 h, and 7 d salt treatment conditions were analyzed. TPM values ​​were standardized and heatmaps were plotted. Results are as follows: Figure 1 As shown, it indicates PROSiCAPE2 , PROSiCAPE3 , PROSiCAPE5 , PROSiCAPE6 , PROSiCAPE7 , PROSiCAPE12 , PROSiCAPE14 , PROSiCAPE15 , PROSiCAPE18 These genes exhibit high levels of expression in the roots. Therefore, these nine genes corresponding to salt stress were selected for further experimental studies.

[0071] The expression of the above nine genes in millet was analyzed using RT-qPCR under salt treatments of 0, 12, 24, 36, 48, and 72 h. The results are as follows: Figure 2 As shown in Figures A to I, different genes exhibit differential expression under salt treatment conditions, such as PROSiCAPE2 Irregular expression levels PROSiCAPE5 Expression levels were upregulated, but not significantly increased after 24 hours. PROSiCAPE6 and PROSiCAPE14 The expression level is downregulated and follows a regular pattern. Based on the preceding analysis, we select... PROSiCAPE14 As a candidate gene for subsequent functional studies.

[0072] Example 2: Subcellular localization of PROSiCAPE14 protein in tobacco To investigate the subcellular localization of the PROSiCAPE14 protein in tobacco, this example constructs... PROSiCAPE14 Overexpression vector, and in PROSiCAPE14 A GFP (green fluorescent protein) tag was attached to the C-terminus of the gene to indicate its intracellular location. The PROSiCAPE14-GFP fusion gene was overexpressed in tobacco leaves using a transient expression system, followed by slide preparation and observation using a two-photon laser scanning confocal microscope. Results are as follows: Figure 3 As shown, PROSiCAPE14 Gene-encoded proteins are located on the cell membrane.

[0073] Example 3: Effect of exogenous SiCAPE14 on millet under salt treatment conditions In this embodiment, a small peptide (mature-derived peptide) of SiCAPE14 was artificially synthesized based on the amino acid sequence (SEQ ID NO:2) of the PROSiCAPE14 protein. The effect of SiCAPE14 on millet was detected by exogenously applying SiCAPE14 under salt treatment conditions. Control group, SiCAPE14 treatment group, 150 mM NaCl treatment group, and SiCAPE14 treatment group under 150 mM NaCl treatment were set up. Millet leaves were treated with exogenous spraying of different concentrations of SiCAPE14 (0.5 μmol, 1 μmol, 2 μmol), and the phenotypic diagrams are shown below. Figure 4 As shown in A. (As in...) Figure 4 As shown in B, without the addition of salt, exogenous application of SiCAPE14 had no effect on the growth of millet; Figure 4 As shown in C and D, under 150 mM NaCl treatment, the millet plant height of the group treated with exogenous SiCAPE14 was significantly lower than that of the 150 mM NaCl treatment group. Furthermore, physiological index measurements showed that the millet leaves treated with SiCAPE14 exhibited deeper DAB staining, consistent with previous findings. PROSiCAPE14 The gene expression in millet under salt treatment is consistent with that in millet, indicating that the gene plays a downregulation role.

[0074] Example 4 Overexpression PROSiCAPE14 The Influence of Genes on Salt Tolerance in Millet To explore in depth PROSiCAPE14 This study further obtained the influence of genes on the salt tolerance of millet. PROSiCAPE14 Stable transformation lines of genes.

[0075] After the millet plants have grown to the stage of three leaves and one bud, select plants with similar growth patterns and treat each line with 150 mM NaCl for 7 days. Then, photograph and record the phenotype. Figure 5 As shown in A, under conditions without salt stress, PROSiCAPE14 The growth of transgenic plants was not significantly different from that of the control group. However, after treatment with 150 mM NaCl, the growth of the overexpressing plants was worse than that of the control group. Biomass is an important indicator for assessing plant salt tolerance. Biomass was measured in overexpressing plants under both salt-free and salt-treated conditions, and the results are as follows: Figure 5 As shown in B, C, D, E, F, and G, under conditions without salt treatment, there were no significant differences in physiological indicators between the overexpression lines and the control group. However, under salt treatment conditions, the plant height and root length of the overexpression lines were significantly worse than those of the control group, and the aboveground fresh weight, aboveground dry weight, root fresh weight, and root dry weight were all slightly lower than those of the control group plants.

[0076] Under salt treatment conditions, various physiological indicators in plants, such as sodium ion concentration, potassium ion concentration, and relative conductivity, change. These changes can reflect the plant's growth status. The physiological indicators of overexpression lines under different salt concentrations were measured. Figure 6 As shown in B, C, and D, the plants maintain ion balance by excreting sodium ions and absorbing potassium ions. Under 150 mM NaCl treatment, the sodium ion content in each treated plant was significantly higher than that in the control group. + / K + The levels were also significantly higher than the control group. Leaves from the plant were removed and stained with DAB and NBT, such as... Figure 6 As shown in Figure A, the staining intensity of each strain under salt treatment was darker than that of the control group, indicating that the overexpression strains were more severely damaged under salt stress than the control group, and the reactive oxygen species content increased after salt treatment.

[0077] Example 5: Effect of PROSiCAPE14 gene knockout on millet salt tolerance The expression of the SiPROCAPE14 gene in millet seedlings of Ci846 was suppressed using the asODN (Antisense Oligonucleotide) method.

[0078] Artificial synthesis SiPROCAPE14The antisense oligonucleotide sequence was determined (T*T*G*C*AGGTGATGAAGA*T*G*C*C (SEQ ID NO:3); asterisks represent thiophosphorylation modification). Plump, uniformly sized millet seeds (Ci846) were selected and evenly spread on moist filter paper. After germination and growth for 2 weeks, they were transferred to a hydroponic container and cultured in nutrient solution for another 2 weeks. Then, the target gene was transiently silenced. Seedlings of uniform size were selected and divided into a control group and a treatment group. The seedlings were placed in transparent glass jars, with 20 mL of deionized water and deionized water containing the antisense oligonucleotide (concentration 0.2 OD / mL) added to each jar. After 12 h and 48 h of treatment, three seedlings were randomly selected from each control and treatment group, and RNA was extracted for gene expression level detection.

[0079] like Figure 7 As shown in A and B, compared with the control without antisense oligonucleotides, as-ODN treatment for 12 h significantly reduced leaf morphology. SiPROCAPE14 The expression of the target gene was effectively suppressed, and the gene silencing effect remained good even after 48 h of treatment, indicating that the technology has a good effect on transient silencing of the target gene in millet.

[0080] Salt stress treatment was applied to wild-type millet seedlings and millet seedlings with transiently silenced genes. The specific procedures were as follows: After transferring the millet seedlings to hydroponic containers, different millet lines were treated with Hogland nutrient solution or Hogland nutrient solution + 100 mM NaCl. Three days after treatment, root length and fresh weight of different treatment lines were measured. GraphPad Prism 10 software was used for plotting, and two-way ANOVA was used for data significance analysis.

[0081] The results are as follows Figure 8 As shown in A, B, and C, under 100 mM salt stress treatment, SiPROCAPE14 The gene-silenced lines exhibited superior growth compared to the wild-type lines. Measurements of root length and fresh weight, followed by significance analysis, revealed that both root length and fresh weight were significantly higher in the gene-silenced lines than in the wild-type lines. This indicates that the gene has a negative regulatory effect on the salt tolerance of millet, and reducing its expression can enhance the salt tolerance of millet.

[0082] Example 6 PROSiCAPE14 Promoter analysis The expression vector with the promoter tag was transformed into Agrobacterium tumefaciens EHA105, injected into tobacco, and treated with 100 mM NaCl for 24 h. Samples were then collected using a punch for GUS staining. Figure 9As shown in Figure A, when treated with 0 mM NaCl, the leaf with the promoter tag was stained blue by GUS staining solution. After treatment with 100 mM NaCl for 24 h, the staining became more intense, indicating that... PROSiCAPE14 The promoter of the gene contains response elements associated with salt stress.

[0083] Using the PlantRegMap website PROSiCAPE14 Analysis of cis-regulatory elements in the 2kb upstream promoter region of the gene was performed, and the results showed that... PROSiCAPE14 The promoter region contains numerous MYB and bHLH response elements. Extensive data indicate that the MYB and bHLH transcription factor families play crucial roles in plant responses to abiotic stress; therefore, subsequent yeast single-hybrid experiments were conducted for verification. Figure 9 As shown in Figure B, under treatment with abaminoside (AbA), SiMYBS1 and SibHLH49 significantly promoted yeast growth compared to the empty vector control. This indicates that SiMYBS1 and SibHLH49 may bind to... PROSiCAPE14 The promoter region activates its expression. To verify this interaction, a dual-luciferase reporter gene assay was performed. Figure 9 C in Figure 10 and Figure 11 As shown, MYBS1 and bHLH49 can specifically bind PROSiCAPE14 It stimulates the promoter region and significantly enhances the expression activity of downstream reporter genes.

[0084] The above research results indicate that PROSiCAPE14 The gene plays a downregulated role in the salt stress response of millet, and it may be activated by binding to upstream transcription factors, thereby participating in the salt stress response of millet.

[0085] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of them. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. PROSiCAPE14 The application of genes and their encoded proteins in improving plant salt tolerance is characterized by, The encoded protein is a protein as shown in (a1) or (a2) below: (a1) A protein consisting of the amino acid sequence shown in SEQ ID NO:1 in the sequence listing; (a2) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein defined in (a1).

2. The application as described in claim 1, characterized in that, The PROSiCAPE14 The gene sequence is shown in SEQ ID NO:

2.

3. The application as described in claim 1, characterized in that, The application suppresses PROSiCAPE14 Gene expression, silencing, or knockout PROSiCAPE14 Genetic implementation.

4. The application as described in claim 3, characterized in that, The inhibition PROSiCAPE14 Gene expression, silencing, or knockout PROSiCAPE14 Genes include those obtained through gene knockdown, gene editing, and / or gene knockout technologies. PROSiCAPE14 Nucleic acid molecules with missing or inactivated genes.

5. The application as described in claim 4, characterized in that, The nucleic acid molecules include: (1) Double-stranded RNA, small interfering RNA, microRNA and short hairpin RNA used in RNA interference technology; (2) Antisense RNA and antisense oligonucleotides used in antisense nucleic acid technology; (3) gRNA and sgRNA used in gene editing technology; (4) Aptamers and ribozymes.

6. The application as described in claim 5, characterized in that, The sequence of the antisense oligonucleotide is shown in SEQ ID NO:

3.

7. The application as described in claim 1, characterized in that, The plant is selected from at least one of rice, corn, soybean, sunflower, sorghum, wheat, alfalfa, cotton, barley, millet, and foxtail millet.

8. A method for improving the salt tolerance of plants, characterized in that, Including by inhibiting plant endogenous... PROSiCAPE14 Gene expression, silencing, or knockout of plant endogenous genes PROSiCAPE14 The gene enhances the salt tolerance of plants, and the sequence of the PROSiCAPE14 gene is shown in SEQ ID NO:

2.

9. The method as described in claim 8, characterized in that, The plant is selected from at least one of rice, corn, soybean, sunflower, sorghum, wheat, alfalfa, cotton, barley, millet, and foxtail millet.

10. The application of the method according to any one of claims 8 to 9 in plant breeding.

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