SbASR4 gene for regulating plant salt stress tolerance and application thereof

Abstract

The invention discloses a SbASR4 gene for regulating and controlling plant salt stress tolerance as well as an encoding protein and application thereof. The SbASR4 gene is derived from Sorghum baicola, and the encoded protein is simultaneously positioned in a cell membrane and a cell nucleus, does not have transcriptional activation activity, and is a negative regulation factor of plant salt stress response. The overexpression of the gene can significantly increase the sensitivity of the plant to salt and ABA, and the silence or knockout of the gene can greatly improve the salt tolerance of the plant. Mechanism research shows that the SbASR4 protein directly interacts with an SOS signal channel core kinase SOS2 in a plant body, and the SOS channel is negatively regulated through the interaction, so that the expression of a downstream ion transport gene is inhibited. The invention provides a method for improving the salt tolerance of plants by reducing the expression of SbASR4 or destroying the interaction of SbASR4 and SOS2, related genetic materials and products, and a brand new target and an effective strategy are provided for salt-resistant breeding of crops.

Description

A SbASR4 gene regulating plant salt stress tolerance and its application Technical Field

[0001] This invention belongs to the field of plant genetic engineering and molecular breeding technology, specifically involving an ASR family gene SbASR4 derived from sorghum (Sorghum bicolor) that negatively regulates plant salt stress tolerance and its encoded protein, as well as the method and application of using this gene to breed new salt-tolerant or salt-intolerant crop varieties. Background Technology

[0002] Soil salinization is one of the major abiotic stresses restricting global agricultural production. Statistics show that approximately 950 million hectares of land worldwide are affected by salinization, and this area continues to expand. Salt stress inhibits plant growth and development through multiple mechanisms, including ion toxicity, osmotic stress, and oxidative stress, leading to a significant decline in crop yields. Therefore, identifying key genes for salt tolerance in plants, elucidating their molecular regulatory mechanisms, and using genetic engineering techniques to cultivate new salt-tolerant crop varieties are of significant theoretical and practical importance for ensuring food security and improving arable land use efficiency.

[0003] The ASR (Abscisic Acid-Stress-Ripening) family of proteins is a class of plant-specific low-molecular-weight proteins, rich in histidine and possessing a typical ABA / WDS (Abscisic acid / Water Deficit Stress) domain. Previous studies have shown that ASR family proteins play important roles in various plant responses to drought, salt stress, and fruit ripening. However, the functions of ASR family members exhibit species specificity and functional differentiation; some members positively regulate plant stress tolerance, while others may exert negative regulatory effects. Therefore, conducting functional studies of ASR family members in different crops and clarifying the specific mechanisms of action of each member is crucial for precision molecular breeding.

[0004] Sorghum (Sorghum bicolor) is an important food and forage crop with high drought and salt tolerance, making it an ideal model plant for studying crop stress resistance mechanisms. However, the specific functions of the sorghum ASR family genes in salt stress response remain unclear, particularly regarding which members play a negative regulatory role and how to improve crop salt tolerance by regulating these negative regulatory factors. Systematic and in-depth research is lacking in this area.

[0005] This invention, through systematic expression analysis and functional verification of eight members of the sorghum ASR family, has for the first time discovered that SbASR4 is a key factor in the negative regulation of salt stress tolerance, providing a novel gene resource and breeding strategy for cultivating salt-tolerant crops using negative regulation. Summary of the Invention

[0006] This invention aims to provide a novel gene resource and its application method for improving plant salt stress tolerance. In the prior art, ASR family genes are mostly reported as positive regulators of stress response, but the existence of negative regulators in sorghum and how to utilize negative regulation strategies for salt-tolerant breeding have not been reported. This invention, through systematic expression analysis and functional verification, is the first to discover and confirm that the sorghum SbASR4 gene is a key negative regulator of plant salt stress tolerance. To solve the above technical problems, this invention provides: an SbASR4 protein with negative regulatory function on plant salt stress tolerance and its encoding gene; a method for significantly improving plant salt stress tolerance by reducing or inhibiting gene expression or protein activity; and a recombinant expression vector containing this gene, a gene editing system, and their application in salt-tolerant breeding.

[0007] Secondly, this invention provides a method for reducing plant salt stress tolerance or increasing plant sensitivity to abscisic acid (ABA), used to create sensitive plant materials. This invention further discovers that overexpression of the SbASR4 gene not only reduces plant salt tolerance but also significantly enhances plant sensitivity to ABA. This discovery provides new genetic materials and research tools for plant stress signal transduction research. To solve the above technical problems, this invention provides: a method for reducing plant salt stress tolerance or increasing plant sensitivity to ABA by increasing the expression level of the SbASR4 gene; and salt-sensitive or ABA-hypersensitive plant materials created using this method, and their applications in stress physiology research, compound screening, and other fields.

[0008] Furthermore, this invention aims to elucidate the molecular mechanism by which the SbASR4 protein negatively regulates salt tolerance in plants. Through protein-protein interaction analysis, this invention, for the first time, confirms that the SbASR4 protein interacts with the core kinase SOS2 protein of the SOS signaling pathway and affects the expression of downstream SOS pathway genes (such as SOS1 and NHX3). This discovery elucidates the molecular mechanism by which SbASR4 negatively regulates salt tolerance, namely, that SbASR4 interferes with the normal transduction of the SOS signaling pathway by interacting with SOS2, thereby weakening the plant's ability to regulate ion homeostasis.

[0009] Furthermore, this invention also provides specific application strategies for the SbASR4 gene in crop salt tolerance breeding. Based on the core finding that "low expression or loss of function of SbASR4 is positively correlated with enhanced salt tolerance in plants," this invention further provides technical solutions that can be directly applied to breeding practices, including: molecular marker-assisted breeding methods—using low expression or loss of function of the SbASR4 gene as a positive selection marker for salt tolerance, and rapidly screening salt-tolerant breeding materials by detecting the expression level or sequence variation of this gene in natural or mutagenic populations; and gene editing breeding methods—using gene editing technologies such as CRISPR / Cas9 to directionally knock out or mutate the SbASR4 gene, directly creating new crop germplasm with significantly improved salt tolerance.

[0010] To achieve the above objectives, the present invention adopts the following technical solution: (1) The present invention provides an isolated SbASR4 protein, the amino acid sequence of which is shown in SEQ ID NO:2, or a protein that has at least 90% sequence identity with SEQ ID NO:2 and has a negative regulatory function on plant salt stress tolerance. The protein is located in both the cell membrane and the cell nucleus and does not have transcriptional activation activity. The sequence of SEQ ID NO:2 is: MADEEKKHHHHLFHRHKQDGEEEASTGEVDYEKKEKHHKHLEQLGGLGAIAAGAYAIHEKHKAKKEPESAHGHKVKEEVAAVAALGAAGFAFHEHHQKKDAKKQGQS*.

[0011] (2) This invention provides an isolated gene encoding the above-mentioned SbASR4 protein, the nucleotide sequence of which is shown in SEQ ID NO:1, or has at least 90% sequence identity with SEQ ID NO:1 and encodes the protein. The gene is derived from sorghum (Sorghum bicolor), and its expression is significantly inhibited under salt stress and natural dehydration conditions, but its expression level is extremely high in the panicle and seeds, and it has no significant response to low nitrogen, low temperature and alkaline stress. The SEQ The sequence of IDNO:1 is: ATGGCTGATGAGGAGAAGAAGCACCACCACCTGTTCCACCGCCACAAGCAGGACGGCGAGGAGGAGGCGAGCACCGGCGAGGTGGACTACGAGAAGAAGGAGAAGCACCACAAGCACCTGGAGCAGCTCGGCGGGCTCCGGCGCCATCGCCGCCG GCGCATACGCTATTCACGAGAAGCACAAGGCGAAGAAGGAGCCCGAGAGCGCGCACGGGCACAAGGTGAAGGAGGAGGTGGCGGCGGTGGCGGCCCTGGGCGCGGCCGGGTTCGCGTTCCACGAGCACCACCAGAAGAAGGACGCCAAGAAGCAGGGCCAGAGCTGA.

[0012] (3) This invention provides a method for improving the salt stress tolerance of plants, including reducing the expression level of the SbASR4 gene or the activity of the SbASR4 protein in plants. Specific methods include, but are not limited to, gene editing technology, RNA interference technology, antisense RNA technology or artificial microRNA technology.

[0013] (4) The present invention provides a method for reducing the salt stress tolerance of plants or increasing the sensitivity of plants to abscisic acid, including increasing the expression level of the SbASR4 gene in plants.

[0014] In this embodiment of the invention, by overexpressing the SbASR4 gene in Arabidopsis thaliana, it was found that overexpression of SbASR4 significantly enhanced the sensitivity of Arabidopsis thaliana to salt stress. Furthermore, Arabidopsis thaliana overexpressing SbASR4 also showed significantly enhanced sensitivity to abscisic acid (ABA).

[0015] (5) This invention provides the application of the interaction between SbASR4 protein and SOS2 protein in regulating plant salt tolerance. The application includes: using the protein interaction between SbASR4 and SOS2 as targets, screening for compounds, peptides or nucleic acid molecules that can inhibit the interaction, for use in preparing products that improve plant salt tolerance; or screening for substances that enhance the interaction, for use in preparing products that reduce plant salt tolerance or enhance ABA sensitivity.

[0016] (6) This invention provides the application of the SbASR4 gene or SbASR4 protein in the breeding of salt-tolerant plant varieties. The application includes: using low expression or loss of function of the SbASR4 gene as a positive selection marker for salt tolerance to screen breeding materials with enhanced salt tolerance; or using gene editing technology to knock out / downgrade the SbASR4 gene to directly create transgenic or gene-edited plants with improved salt tolerance. Through molecular marker detection or sequencing identification, target gene-edited mutant lines can be screened, and homozygous stable offspring can be obtained through self-pollination or backcrossing. The plant is preferably sorghum or other crops.

[0017] Compared with existing technologies, this invention has the following significant advantages: First, it provides a novel resource of negative regulatory genes for salt tolerance, identifying and confirming for the first time the negative regulatory function of the SbASR4 gene in sorghum, providing a new breeding strategy for crop salt tolerance genetic improvement that shifts from the traditional approach of "enhancing positive regulatory factors" to "inhibiting negative regulatory factors." Second, the bidirectional regulatory strategy achieves comprehensive coverage of application scenarios. It can directly serve the core industrial demand for salt-tolerant crop breeding by reducing SbASR4 expression, and it can also create salt-sensitive or ABA-hypersensitive plant materials by increasing SbASR4 expression, serving research and development scenarios such as stress physiology studies and compound screening. Third, it reveals for the first time the molecular mechanism of SbASR4-SOS2 protein interaction, confirming that SbASR4 interferes with SOS signaling pathway transduction through direct interaction with SOS2. This mechanistic discovery not only deepens the understanding of the plant salt tolerance regulatory network but also provides an important theoretical basis for further elucidating the plant salt stress signal transduction mechanism. Fourth, it overcomes the inherent defects of overexpression strategies, resulting in higher breeding safety. The "inhibition of negative regulatory factors" strategy adopted in this invention does not introduce the overexpression of exogenous genes. Instead, it precisely knocks out or silences endogenous negative regulatory factors, which is closer to the breeding logic of natural variation. It has less impact on the normal growth and development of plants and has higher biosafety and industrial acceptance. Attached Figure Description

[0018] Figure 1. Expression patterns of SbASR family members in sorghum under different abiotic stresses and tissue expression profiles throughout the entire growth period. (A) Expression dynamics of SbASR family members under salt stress; (B) Relative expression levels of each gene under natural water loss conditions; (C) Time-dependent response of SbASR4 to low nitrogen stress; (D) Time-dependent response of SbASR4 to low temperature stress; (E) Response of SbASR4 to alkaline stress; (F) Expression patterns of SbASR4 in different tissues throughout the entire growth period of sorghum. Significance markers in the figure indicate differences from the 0-hour treatment: *P<0.05, **P<0.01, ***P<0.001, ns indicates no statistically significant difference; expression in root tissue is used as a control in Figure F.

[0019] Figure 2. Subcellular localization and transcriptional activation activity detection of SbASR4 in sorghum. (A) Co-localization analysis of SbASR4 and subcellular compartment marker proteins (Bars=30 μm); (B) Verification of SbASR4 transcriptional activation activity. Figure A shows the fluorescence signal of SbASR4-GFP in tobacco leaves 48 hours after expression. Nuclear markers (NLS-mCherry) and cell membrane markers (MCA-mCherry, mechanosensitive calcium channels) were used to indicate the corresponding subcellular structures, with the empty vector 35S::GFP serving as a control. In the yeast system, the empty vector pGBKT7 served as a negative control, while VP16 and WRKY47 served as positive controls for strong transcriptional activation.

[0020] Figure 3. Effects of salt stress on SbASR4 protein stability. (A) Transcriptional level of SbASR4 in Arabidopsis overexpression lines; (B) Fluorescence dynamics of SbASR4-GFP fusion protein in the roots of overexpressing plants under salt stress (bars=100 μm); (C) Quantitative analysis of the relative intensity of the corresponding fluorescence signals; (D) Fluorescence changes of transiently expressed SbASR4-GFP protein in leaves of Nicotiana benthamiana under salt stress (bars=50 μm); (E) Quantitative analysis of the relative intensity of fluorescence signals in the tobacco system. All fluorescence intensity data were analyzed using ImageJ software. Significance markers indicate differences relative to 0 h, ***P<0.001.

[0021] Figure 4. Effects of salt stress on early growth and development of SbASR4 overexpressing plants. (A) Early growth and development phenotypes of wild-type and SbASR4 overexpressing lines under normal conditions and salt stress (bars=1 mm); (B) Dynamics of early developmental progression of each genotype under normal conditions; (C) Dynamics of early developmental progression of each genotype under salt stress; (D) Delay ratio of time required for each genotype to reach the same developmental stage under salt stress. Developmental stage: 0.1, no germination; 0.5, roots exposed; 0.7, hypocotyl appearance; 1.0, cotyledon unfolding; and 1.02, two true leaves appearing. Significance markers indicate differences between overexpressing lines and wild-type (***P<0.001).

[0022] Figure 5. Effects of SbASR4 heterologous overexpression on the salt stress sensitivity of Arabidopsis thaliana. (A) Germination and seedling growth phenotypes of wild-type and SbASR4 overexpressing lines under different NaCl concentrations (bars=1 cm); (B) Germination rate statistics of each genotype under different salt concentrations; (C) Relative fresh weight of each genotype under different salt concentrations; (D) Root growth phenotypes of each genotype under salt stress (bars=1 cm); (E) Relative root length statistics of each genotype under salt stress. Significant markers indicate differences between overexpressing lines and wild-type (*P<0.05, **P<0.01, ***P<0.001).

[0023] Figure 6. Effects of SbASR4 heterologous overexpression on salt stress tolerance in Arabidopsis thaliana. (A) Phenotypes of wild-type and SbASR4 overexpressing lines after 7 and 10 days of treatment with 300 mmol / L NaCl (bars=4 cm); (BJ) Statistical analysis of multiple physiological indicators of each genotype under salt stress: (B) Survival rate; (C) Chlorophyll content; (D) Fresh weight; (E) Dry weight; (F) Relative water content; (G) Electrical conductivity; (H) Leaf Na+. + / K + Ratio; (I) Leaf Na + Content; (J) H2O2 accumulation. Significance markers indicate differences between overexpression lines and wild types (*P<0.05, **P<0.01, ***P<0.001).

[0024] Figure 7. Effects of SbASR4 silencing on salt stress tolerance in sorghum. (A) Phenotypes of control and SbASR4 gene-silenced plants after 7 and 10 days of treatment with 300 mmol / L NaCl (bars=7 cm); (B) Relative expression level of SbASR4 in gene-silenced lines; (CI) Comparison of multiple physiological indicators between control and SbASR4 gene-silenced plants under salt stress: (C) Survival rate; (D) Relative fresh weight; (E) Relative plant height; (F) Leaf NaCl concentration. + / K + (G) Electrical conductivity; (H) Na content of the blade + Content; (I) H2O2 accumulation. In the figure, pTRV:00 represents the empty vector control, and pTRV:SbASR4 represents the SbASR4 gene silencing line. Significance markers represent the differences between gene silencing lines and controls (*P<0.05, **P<0.01, ***P<0.001).

[0025] Figure 8. Effect of SbASR4 heterologous overexpression on Arabidopsis thaliana ABA sensitivity. (A) Time-dependent expression response of SbASR4 to 100 μmol / LABA treatment; (B) Root growth phenotypes of wild-type and SbASR4 overexpressing lines under different ABA concentrations (bars=1 cm); (C) Statistical analysis of relative root lengths of each genotype under ABA treatment. Significant markers in Figure A indicate differences from the 0-hour treatment; significant markers in Figure C indicate differences between overexpressing lines and wild-type (*P<0.05, **P<0.01, ***P<0.001).

[0026] Figure 9. SbASR4 participates in the regulation of Na⁺ homeostasis and ABA signaling pathways. (A) Expression analysis of key genes (SbSOS1, SbSOS2, SbNHX3), antioxidant gene (SbCAT), PP2C family genes (SbPP2C6, SbPP2C37, SbPP2C51), and energy metabolism gene (SbSnRK1.1) in control and SbASR4-silenced plants under normal growth and salt stress conditions; (B) Luciferase complementation assay to verify the protein interaction between SbASR4 and SbSOS2. SbSOS2-cLUC+SbASR4-nLUC was the experimental group, and nLUC+cLUC, SbASR4-nLUC+cLUC, and SbSOS2-nLUC+cLUC were negative controls. Significance markers in the figure indicate differences between the treatment and control materials under the same treatment (*P<0.05, **P<0.01, ***P<0.001). Detailed Implementation

[0027] The present invention will be described in detail below with reference to specific embodiments and accompanying drawings, but the scope of protection of the present invention is not limited to these embodiments. Unless otherwise specified, the reagents, materials and methods used in the present invention are conventional reagents, materials and methods in this technical field. pTRV1 and pTRV2 vectors are viral silencing vectors conventionally used in this field and can be obtained through public channels (ordered from the Arabidopsis thaliana Resource Center ABRC, pTRV1 (CD3-1039), pTRV2 (CD3-1040)). Example 1: Cloning, vector construction and genetic transformation of the SbASR4 gene 1.1 Experimental materials

[0028] Seeds of the sorghum (Sorghum bicolor L. Moench) variety BTx623 were preserved in our laboratory. The plant culture conditions were: photoperiod 14 h / 10 h (light / dark), temperature 28℃ / 25℃ (day / night), and light intensity 600 μmol·m⁻²·s⁻¹.

[0029] 1.2 SbASR4 Gene Cloning Based on the predicted sequence of SbASR4 (Sobic.004G171000) in the sorghum genome database (Phytozome v3.0), specific primers (without stop codons) were designed: Forward primer: 5'-ATGGCTGATGAGGAGAAGAAGC -3' Reverse primer: 5'-GCTCTGGCCCTGCTTCTTG -3' Sorghum seedling leaves were ground with liquid nitrogen and total RNA was extracted using TRIzol reagent (Invitrogen). Genomic DNA contamination was removed using DNase I (Vazyme). First-strand cDNA was synthesized using TransScript® One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen).

[0030] Using sorghum cDNA as a template, PCR amplification was performed using the high-fidelity DNA polymerase Phanta Max Super-Fidelity DNA Polymerase (Vazyme). The reaction mixture (50 μL) consisted of: 25 μL 2×Phanta Max Buffer, 1 μL dNTPs mix (10 mM), 2.5 μL forward primer (10 μM), 2.5 μL reverse primer (10 μM), 1 μL template cDNA (100 ng / μL), 0.5 μL Phanta Max Super-Fidelity DNA Polymerase, and ddH2O to a final volume of 50 μL. The PCR program was: 98℃ pre-denaturation for 30 s; 98℃ denaturation for 10 s, 58℃ annealing for 30 s, 72℃ extension for 30 s, for 35 cycles; and a final extension at 72℃ for 5 min.

[0031] Sequencing results showed that the SbASR4 coding region is 324 bp (SEQ ID NO:1) and encodes 107 amino acids (SEQ ID NO:2). Using the ExPASy ProtParam tool, the predicted molecular weight is 11.91 kDa, and the isoelectric point pI is 6.98.

[0032] 1.3 Construction of Overexpression Vector and Arabidopsis Transformation Vector: The SbASR4 coding sequence (excluding the stop codon) was amplified using primers containing homologous arms. The amplification product and the backbone vector were digested with hindIII and cloned into the pCAMBIA1300-35S-GFP vector using the ClonExpress II One Step Cloning Kit (Vazyme) via homologous recombination to construct the 35S::SbASR4-GFP fusion expression vector. After confirmation by sequencing, the vector was transformed into Agrobacterium GV3101.

[0033] Forward primer: 5'- caccaaatcgactctagaaagcttATGGCTGATGAGGAGAAGAAGC -3' Reverse primer: 5'- cttgctcaccatctgcagaagctt GCTCTGGCCCTGCTTCTTG -3' Arabidopsis transformation: Arabidopsis (Col-0) was transformed using the inflorescence immersion method. A single colony of Agrobacterium GV3101 containing the recombinant plasmid was inoculated into 5 mL of YEP liquid medium (containing 50 mg / L kanamycin and 50 mg / L rifampin) and cultured overnight at 28°C and 200 rpm. 1 mL of the bacterial culture was then transferred to 100 mL of YEP medium and cultured until OD500. 600=1.0~1.2. Collect bacterial cells by centrifugation and resuspend in 5% sucrose solution (containing 0.02% Silwet L-77) to OD0.05. 600 =0.8. Arabidopsis inflorescences were immersed in bacterial solution for 30 s, incubated in the dark for 24 h, and then cultured normally. T0 generation seeds were screened on 1 / 2 MS medium containing 50 mg / L hygromycin to obtain resistant plants. Two independent overexpression lines, SbASR4-OE2 and SbASR4-OE3, were selected for further research, with OE3 showing a significantly higher transcriptional level than OE2 (Figure 3A).

[0034] 1.4 Construction of gene silencing vector and sorghum transformation VIGS vector construction: The full-length SbASR4 coding region fragment (324 bp, sequence as shown in SEQ ID NO:1) was selected, digested with Kpn1 / XbaI, and cloned into the pTRV2 viral vector to construct pTRV2-SbASR4. After confirmation by sequencing, it was transformed into Agrobacterium GV3101.

[0035] Infection and silencing of sorghum plants: Single clones of Agrobacterium strain GV3101 containing pTRV1 and pTRV2-SbASR4 plasmids were cultured at 28°C in LB broth containing the corresponding antibiotics until OD200. 600 ≈ 1.0. Collect bacterial cells by centrifugation, resuspend in infection buffer (10 mmol / L MES, pH 5.6; 10 mmol / L MgCl2; 200 μmol / L acetylsylgenone) and adjust OD. 600 To 1.0. Mix bacterial suspensions containing pTRV1 and pTRV2-SbASR4 at a 1:1 volume ratio and incubate at room temperature in the dark for 3 hours. Select sorghum seeds whose radicles have just emerged from the seed coat (showing white hairs) after imbibition and immerse them in the above Agrobacterium mixture. Infect at 25°C with low-speed shaking (e.g., 50 rpm) or intermittent gentle shaking for 3-4 hours. After infection, gently rinse the seeds with sterile water, then sow them in sterile substrate and culture in a climate chamber (25°C, 16 hours light / 8 hours dark). Use plants carrying Agrobacterium carrying empty pTRV1 and pTRV2 vectors, treated in the same manner, as negative controls (the obtained control plants are designated pTRV:00). After approximately two weeks of culture, collect newly grown leaves and detect the expression level of the SbASR4 gene using RT-qPCR. Screen for silenced plants with significantly reduced SbASR4 expression (designated pTRV:SbASR4) for subsequent analysis.

[0036] Example 2: Expression pattern analysis of the SbASR4 gene

[0037] Salt stress treatment was applied to 2-week-old sorghum seedlings by irrigating them with a 150 mmol / L NaCl solution, and samples were taken at 0, 3, 6, 9, and 24 hours after the start of treatment. Separately, natural dehydration (drought) stress treatment was applied to sorghum seedlings grown in Hoagland nutrient solution at the same time: after removing the seedlings, excess moisture on the plant surface was blotted with filter paper, and samples were taken at 0, 1, 2, 6, and 12 hours at room temperature. All samples were immediately flash-frozen in liquid nitrogen and stored at -80℃ for later use.

[0038] Specific quantitative primers were designed using Primer Premier 5.0: SbASR1 forward primer: 5'-TGACGGAGGTGGTGTCCA-3'; SbASR1 reverse primer: 5'-GTGCTTCTCGTAGAGTGCGA-3'; SbASR2 forward primer: 5'-TGACCACCGGCGAGGA-3'; SbASR2 reverse primer: 5'-CTCGTGCTTCTCGTAAAGTGC-3'; SbASR3 forward primer: 5'-CCTTCGCCCTGTACGAGA-3'; SbASR3 reverse primer: 5'-TTGGAGTAGCGAAGAGCAGC-3'; SbASR4 forward primer: 5'-GGCGAGGTGGACTACGAGAA-3'. SR4 Reverse Primer: 5'-CCTTGTGCTTCTCGTGAATAGCG-3'SbASR5 Forward Primer: 5'-AAGCACTTGGAGCAGCTCG-3'SbASR5 Reverse Primer: 5'-ACCTCCTCCTTGATCCGGT-3'SbASR6 Forward Primer: 5'-GGTCGCACAGGATCAAGGAA-3'SbASR6 Reverse Primer: 5'-CGGCGATGGTGGCGAAAT-3'SbASR7 Forward Primer: 5'-CTTTGCCTTCCACGAACACC-3'SbASR7 Reverse Primer: 5'-CCCGCGCCTGGTAAATGAAA-3'SbASR8 Forward Primer: 5'- GAGAGGAGAGGGAGCACAAG-3'SbASR8 reverse primer: 5'- GCCCCTCGTACAGGGCAA-3' Quantitative PCR was performed using PerfectStart® Green qPCR SuperMix (TransGen) on a CFX Duet Real-Time PCR System (Bio-RAD). SbEIF4A (Sobic.004G039400) was used as an internal reference gene, and the relative expression level was calculated using the 2⁻ΔΔCt method.

[0039] As shown in Figure 1A, under 150 mmol / L NaCl treatment, the expression of SbASR1, SbASR2, and SbASR3 was significantly induced by salt stress, while the expression of SbASR4, SbASR5, SbASR6, SbASR7, and SbASR8 was significantly inhibited. Specifically, the expression levels of SbASR4 and SbASR8 decreased to half of their pre-stress levels after 3 hours of salt treatment (P<0.01), while the expression levels of SbASR5, SbASR6, and SbASR7 decreased significantly after 6 or 9 hours of treatment. Considering that salt stress is usually accompanied by cellular dehydration, the expression patterns of these genes under natural dehydration conditions were further examined, as shown in Figure 1B. Unlike salt stress, SbASR2 did not respond significantly to natural dehydration. Under natural dehydration conditions, except for SbASR4, which remained significantly inhibited (P<0.001), the expression of the other family members showed varying degrees of upregulation. As shown in Figures 1C-E, SbASR4 showed no significant response (ns) to low nitrogen, low temperature, and alkaline stress. Further expression profiling in different tissues throughout the entire growth period revealed that SbASR4 was expressed in roots, stems, leaves, leaf sheaths, panicles, flag leaves, flowers, and developing seeds, with the lowest expression level observed in roots. In stems, leaves, leaf sheaths, and flag leaves, its expression level was approximately 2-3 times that in roots, while it increased sharply in the panicle and during seed formation, reaching 600-fold and 200-fold respectively (P<0.001) (Figure 1F). These results suggest that SbASR4 likely plays an important regulatory role in the panicle development and grain formation stages of sorghum. Example 3: Subcellular localization and transcriptional activation activity analysis of SbASR4

[0040] The SbASR4 coding sequence (excluding the stop codon) was cloned into the pCAMBIA1300-GFP vector to construct the 35S::SbASR4-GFP fusion expression vector. The nuclear labeling vector was 35S::NLS-mCherry, and the cell membrane labeling vector was 35S::MCA-mCherry.

[0041] The recombinant vector was transformed into Agrobacterium GV3101 and injected into the abaxial surface of leaves of 4-5 week old Nicotiana benthamiana. The plants were cultured at 25°C for 48 h post-injection. Fluorescence signals were observed using a Zeiss LSM980 laser confocal microscope.

[0042] As shown in Figure 2A, the SbASR4-GFP fluorescence signal clearly overlaps with both NLS-mCherry and MCA-mCherry, indicating that SbASR4 is simultaneously localized in the cell membrane and nucleus (Bars=30 μm).

[0043] The SbASR4 coding sequence was cloned into the yeast expression vector pGBKT7 to construct pGBKT7-SbASR4. The vector was transformed into yeast strain AH109, ​​with VP16 and SbWRKY47 as positive controls and the empty pGBKT7 vector as a negative control.

[0044] As shown in Figure 2B, yeast clones carrying SbASR4 could only grow on SD-Trp medium, but could not grow on SD-Trp / -His, SD-Trp / -His / -Ade, or chromogenic media containing X-α-gal, indicating that SbASR4 does not possess transcriptional activation capabilities. Example 4: Effect of salt stress on the stability of SbASR4 protein

[0045] Given that salt stress significantly inhibits the transcriptional expression of SbASR4, we further investigated whether salt stress affects its protein stability. First, we constructed two independent stable transgenic lines in Arabidopsis thaliana overexpressing SbASR4, SbASR4-OE2 and SbASR4-OE3, with OE3 showing a significantly higher expression level than OE2 (Figure 3A). Seeds from the wild-type and homozygous transgenic lines were sown on 1 / 2 MS medium and grown vertically for 5 days. The seedlings were then transferred to 1 / 2 MS medium containing 150 mmol / L NaCl, and GFP signal changes were observed after 0, 3, 6, and 9 hours of treatment.

[0046] As shown in Figures 3B-C, the results show that the SbASR4 protein is highly sensitive to salt stress. After treatment with 150 mmol / L NaCl for 3 hours, the GFP fluorescence signal decreased significantly by about 80% (P<0.001), and the signal continued to weaken with the extension of treatment time, and almost disappeared after 9 hours (Bars=100 μm), indicating that salt stress can significantly reduce the protein stability of SbASR4.

[0047] To further validate these results, SbASR4-GFP was transiently expressed in *Nicotiana benthamiana* leaves, followed by sterile water (control) and salt treatments 36 hours after expression. As shown in Figures 3D-E, the GFP signal remained stable in the control group for 9 hours, while in the salt-treated group, the fluorescence signal dropped sharply to approximately 50% of its initial level after 3 hours, and was retained at only about 35% after 9 hours (Bars=50 μm). In conclusion, salt stress not only inhibits the transcriptional expression of SbASR4 but also significantly reduces its protein stability and promotes its degradation.

[0048] Example 5: Effect of SbASR4 overexpression on the salt stress sensitivity of Arabidopsis thaliana

[0049] 5.1 Early Growth and Development Analysis Seeds of wild-type and SbASR4 overexpression lines (OE2, OE3) were sown on 1 / 2 MS medium with or without 150 mmol / L NaCl, and their developmental process was observed (Figure 4A). The developmental time of root, hypocotyl elongation, and cotyledon unfolding stages was not significantly different from that of the wild type. Under normal conditions, OE2 and OE3 were slightly slower than the wild type in the root emergence stage, but their development in the subsequent hypocotyl elongation and cotyledon unfolding stages was basically consistent with that of the wild type. Notably, both overexpression lines were 0.5–1 day slower than the wild type in the formation of two true leaves (Figure 4B). Under salt stress, early development of all genotypes was inhibited, but the inhibitory effect of salt stress on the overexpression lines was significantly stronger than that on the wild type. For example, the formation of two true leaves was delayed to 15–16 days in the overexpression lines, while it only took about 12 days in the wild type (P<0.001) (Figures 4C, 4D). The above results indicate that salt stress has a more significant delaying effect on the early growth and development of SbASR4 overexpressing lines, further supporting the possibility that SbASR4 may act as a negative regulator in plant salt stress response.

[0050] To further investigate the function of SbASR4 in salt stress response, the effects of salt stress on the seed germination rate and root growth of its overexpression lines were examined. Seeds from the overexpression lines and wild-type were sown on 1 / 2 MS medium containing different concentrations of NaCl, and their germination and root phenotypes were observed (Fig. 5A, 5D). Under normal culture conditions, seeds of all genotypes germinated completely without significant differences. However, salt stress had a significantly stronger inhibitory effect on the germination and growth of the overexpression lines than on the wild type. With increasing salt concentration, the germination rate and seedling fresh weight of both overexpression lines were significantly lower than those of the wild type, with a more pronounced decrease (Fig. 5B, 5C). Particularly for the OE3 line with higher expression levels, its germination rate and fresh weight showed a gradual and maximum decreasing trend with increasing salt concentration. Regarding root elongation, the root length shortening of the overexpression lines under salt stress was also significantly greater than that of the wild type (Fig. 5D). Under 100 mmol / L NaCl treatment, the relative root length of the wild-type was 0.79 compared to the control, while that of OE2 and OE3 was 0.62 and 0.52, respectively. When the salt concentration increased to 150 mmol / L, the relative root length of the wild-type decreased to 0.48, while that of the overexpression lines further decreased to 0.32 and 0.06, respectively (P<0.001) (Figure 5E). These results indicate that overexpression of SbASR4 significantly enhances the sensitivity of Arabidopsis thaliana to salt stress.

[0051] 5.3 Evaluation of salt tolerance in Arabidopsis thaliana overexpressing SbASR4

[0052] Overexpression lines and wild-type plants, all 18 days old and of similar growth status, were subjected to 10 days of salt stress treatment (Figure 6A). Before the stress treatment, there were no significant differences in growth phenotypes among the different genotypes; however, after salt treatment, the overexpression lines showed poorer salt tolerance than the wild-type. After 10 days of treatment, all wild-type plants survived, while the survival rates of the overexpression lines decreased to 70% and 10%, respectively (P<0.001) (Figure 6B). In terms of physiological indicators, the chlorophyll content (Figure 6C), fresh weight (Figure 6D), dry weight (Figure 6E), and water content (Figure 6F) of the overexpression lines under salt stress were significantly lower than those of the wild-type (P<0.001), indicating that their overall physiological state was more severely inhibited. Simultaneously, the overexpression lines exhibited higher ion leakage rates (Figure 6G) and higher leaf Na+ levels. + The concentrations of Na⁺ and K⁺ (Fig. 6H), the Na⁺ / K⁺ ratio (Fig. 6I), and the H₂O₂ accumulation (P < 0.05 or P < 0.001) (Fig. 6J) indicate more severe cell membrane damage, weakened Na⁺ homeostasis regulation, and decreased reactive oxygen species scavenging capacity. In summary, these results consistently demonstrate that overexpression of SbASR4 significantly increases the sensitivity of Arabidopsis to salt stress, and that SbASR4 plays a negative regulatory role in plant salt stress response.

[0053] Example 6: SbASR4 Gene Silencing Enhances Sorghum Salt Tolerance. To verify the function of SbASR4 in the salt stress response of sorghum, we further constructed SbASR4-silenced plants using a virus-mediated gene silencing system and analyzed their salt tolerance (Figure 7A). RT-qPCR results showed that the expression level of SbASR4 in the silenced lines decreased by an average of approximately 46% (Figure 7B, P<0.01), meeting the requirements for subsequent functional verification. Phenotypic analysis under salt stress showed that silencing SbASR4 significantly improved the salt tolerance of sorghum. Under salt treatment, the survival rate of the silenced lines reached 75%, significantly higher than the 25% of the control group (Figure 7C, P<0.001). Before salt treatment, there were no significant differences in various physiological indicators among different genotypes; however, under salt stress, the trends of changes in each indicator were consistent with the phenotypic results. Compared to the control, the silent lines maintained better growth in terms of fresh weight (Fig. 7D) and plant height (Fig. 7E) on days 7 and 10 of salt treatment; simultaneously, their ion leakage rate (Fig. 7F) and leaf Na+ content were lower. + The salt content (Fig. 7G), Na⁺ / K⁺ ratio (Fig. 7H), and H₂O₂ accumulation (Fig. 7I) were all significantly lower than those in the control group (P<0.05 or P<0.001). These results consistently indicate that reducing SbASR4 expression can effectively improve the salt tolerance of sorghum, further supporting the negative regulatory role of SbASR4 in the salt stress response of sorghum.

[0054] Example 7: SbASR4 overexpression enhances Arabidopsis thaliana's sensitivity to ABA

[0055] Plants synthesize large amounts of abscisic acid (ABA) when subjected to drought or salt stress. To investigate whether SbASR4 is involved in ABA-mediated stress response, we first examined the transcriptional response of SbASR4 to ABA and analyzed the sensitivity of overexpressing lines to ABA. The results showed that, similar to most ASR family genes, the transcriptional expression of SbASR4 was significantly induced by ABA: it began to be upregulated 9 hours after ABA treatment, reaching approximately 40-fold and 120-fold induction levels at 12 and 24 hours, respectively (Figure 8A, P<0.001). Further analysis showed that under normal conditions, the taproot length of the overexpressing lines was slightly longer than that of the wild type; however, under ABA treatment, taproot elongation of the overexpressing lines was more significantly inhibited, and the degree of inhibition increased with increasing ABA concentration (Figure 8B, P<0.001). At 0.2 μmol / L ABA treatment, the primary root lengths of wild-type, OE2, and OE3 were 0.93, 0.70, and 0.61 times that of the control, respectively. When the ABA concentration increased to 0.5 μmol / L, the lengths of these three types decreased to 0.63, 0.42, and 0.30 times that of the control, respectively. Under 1.0 μmol / L ABA conditions, these lengths further decreased to 0.51, 0.15, and 0.16 times that of the control (Figure 8C, P<0.001). These results indicate that although SbASR4 transcription is strongly induced by ABA, its overexpression actually enhances the plant's sensitivity to ABA, suggesting that SbASR4 may participate in the ABA signaling pathway through post-translational regulation or protein stability regulation, and that there may be a complex regulatory relationship between its protein function and transcriptional response.

[0056] Example 8: SbASR4 affects the salt stress response of sorghum Na⁺ homeostasis genes and ABA signaling pathway.

[0057] Plants activate the SOS (Salt Overly Sensitive) signaling pathway to maintain ion homeostasis when subjected to salt stress. To investigate whether SbASR4 is involved in the SOS-mediated salt stress response, this study analyzed the expression changes of key genes in the SOS pathway in SbASR4-silenced lines.

[0058] The SbASR4 silencing lines and control lines were treated with 200 mmol / L NaCl, and RNA was extracted from each line. The expression levels of key genes in the SOS pathway and stress response genes were detected by RT-qPCR.

[0059] As shown in Figure 9A, under salt stress, the expression of SOS pathway genes SbSOS1, SbSOS2, and SbNHX3 was significantly upregulated in the control line, consistent with the classic salt stress response pattern. Notably, in the SbASR4 silencing line, the expression levels of these three genes were significantly higher than those in the control group: SbSOS1 expression was approximately 2.5 times that of the control group under salt stress (P<0.001), SbSOS2 expression was approximately 1.8 times that of the control group (P<0.001), and SbNHX3 expression was approximately 1.4 times that of the control group (P<0.05). Furthermore, the expression of the antioxidant enzyme gene SbCAT was also significantly higher in the silencing line than in the control group (P<0.05).

[0060] In contrast to the SOS gene, while the PP2C family genes SbPP2C6, SbPP2C37, and SbPP2C51 were induced to express under salt stress, their expression was significantly reduced in SbASR4-silenced lines (P<0.001). Specifically, under salt stress, the relative expression levels of SbPP2C6 in the control group and the silenced group were 6.2 and 2.8, respectively; for SbPP2C37, they were 5.1 and 2.1, respectively; and for SbPP2C51, they were 24.5 and 16.8, respectively. The expression of the energy metabolism-related gene SbSnRK1.1 in the silenced lines was also significantly higher than that in the control group (P<0.01). These results indicate that although SbASR4 transcription is induced by salt stress, its silencing actually enhances the expression of genes related to the SOS pathway, antioxidant defense, and energy metabolism. This suggests that SbASR4 may act as a negative regulator in the salt stress response, and there is a complex hierarchical relationship between its protein function and its regulatory effects on downstream genes. Example 9: Luciferase complementation imaging (LCI) confirmed the interaction between SbASR4 and SbSOS2.

[0061] To investigate the molecular mechanism by which SbASR4 regulates the SOS pathway, the protein-protein interaction between SbASR4 and SbSOS2 was analyzed using LCI technology. The SbASR4 coding sequence was cloned into the pCAMBIA-nLUC vector (N-terminal luciferase), and the SbSOS2 coding sequence was cloned into the pCAMBIA-cLUC vector (C-terminal luciferase). Both were transformed into Agrobacterium GV3101 and co-injected into leaves of Nicotiana benthamiana. Forty-eight hours later, a 1 mmol / L luciferin substrate was sprayed, and the fluorescence signal was detected using a plant in vivo imaging system.

[0062] As shown in Figure 9B, when SbASR4-nLUC and SbSOS2-cLUC are co-expressed in tobacco leaves, the reconstructed luciferase activity produces a strong fluorescent signal; while the negative control group (nLUC+cLUC, SbASR4-nLUC+cLUC, SbSOS2-cLUC+nLUC) shows no visible signal.

[0063] These results confirm the direct physical interaction between SbASR4 and SbSOS2 in plants, providing a molecular basis for explaining how SbASR4 negatively regulates the SOS pathway. Combined with gene expression analysis, it is speculated that SbASR4 may negatively regulate the expression of target genes such as SbSOS1 and SbNHX3 by interacting with SbSOS2, interfering with its normal function, or affecting downstream signal transduction. This interaction mechanism may have a dual effect: on the one hand, directly inhibiting SOS2 kinase activity, and on the other hand, indirectly inhibiting SnRK2 kinase by activating PP2C family genes, forming a multi-layered negative regulatory network on the SOS pathway.

[0064] In summary, SbASR4, as a negative regulator of sorghum's salt stress response, silencing it synergistically enhances ion homeostasis maintenance, reactive oxygen species scavenging, and energy metabolism adaptation. SbASR4 interacts directly with the core kinase SbSOS2, differentially regulating the expression of genes related to the SOS pathway, ABA signaling, and metabolic reprogramming, forming a sophisticated regulatory network for salt stress response. This discovery provides a new perspective for elucidating crop salt tolerance mechanisms and offers an important gene-editing target for molecular breeding of salt-tolerant sorghum.

[0065] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. 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.

[0066] In summary, this invention is the first to clone and identify an ASR family gene, SbASR4, that negatively regulates plant salt stress tolerance from sorghum, and systematically reveals its molecular mechanism and breeding application value. This invention found that SbASR4 encodes a DNA-binding protein located simultaneously in the cell membrane and nucleus, without transcriptional activation activity. The expression of this gene is significantly suppressed under salt stress and natural dehydration conditions, and it is specifically highly expressed in the panicle and developing seeds, suggesting that it may be involved in the trade-off regulation of reproductive development and stress adaptation. Functional studies show that SbASR4 is a key negative regulator of plant salt stress response: overexpression of SbASR4 significantly enhances plant sensitivity to salt and ABA, while silencing or knocking out SbASR4 significantly improves plant salt tolerance and ion homeostasis maintenance. Mechanism analysis confirmed that SbASR4 directly interacts with SOS2, the core kinase of the SOS signaling pathway, in plants, negatively regulating the expression of downstream ion transport genes such as SOS1 and NHX3, and synergistically regulating the expression of PP2C phosphatase and antioxidant genes, forming a multi-level negative feedback regulatory module for salt stress. Based on the above findings, this invention proposes a new molecular breeding strategy of "targeting negative regulatory factors and positively enhancing stress resistance". Compared with the traditional overexpression of positive regulatory factors, this strategy has the following significant advantages: (1) avoiding growth inhibition and metabolic burden that may be caused by constitutive overexpression; (2) using loss of function as a selection marker, making breeding screening simple and high-throughput; (3) being able to be superimposed with other positive regulatory traits without loss; and (4) being applicable to highly conserved homologous targets in a variety of gramineous crops. Sequence conservation analysis and pathway comparison studies showed that homologous genes of SbASR4 are widely present in the genomes of gramineous crops such as sorghum, rice, maize, wheat, and barley, and that SOS2 kinase and its interaction interface are highly conserved evolutionarily. Because the SOS signaling pathway is highly conserved in plants, and the SbASR4 homolog is widely found in gramineous crops, the technical solution of this invention is reasonably expected to be extended to crops such as rice, maize, wheat, and barley, providing important theoretical basis and technical reference for salt-tolerant breeding of these crops. This invention not only reveals a novel negative regulatory node for plant salt stress, enriching our understanding of the functional structure of the ASR family genes and the regulatory hierarchy of the SOS pathway, but also provides a plug-and-play gene editing target, a universal molecular breeding strategy, and a complete application verification system for the genetic improvement of crop salt tolerance. This invention has significant socio-economic benefits and broad commercial prospects.

Claims

1. An isolated SbASR4 protein, characterized in that, The amino acid sequence of the protein is shown in SEQ ID NO:2, or a protein that has at least 90% sequence identity with SEQ ID NO:2 and has a negative regulatory function on plant salt stress tolerance.

2. An isolated SbASR4 gene encoding the protein of claim 1, characterized in that, The nucleotide sequence of the gene is as shown in SEQ ID NO:1, or has at least 90% sequence identity with SEQ ID NO:1 and encodes the protein of claim 1.

3. A recombinant expression vector, characterized in that: (a) comprising the SbASR4 gene of claim 2, said gene being operatively linked to a constitutive or inducible promoter for overexpressing SbASR4 in plants; or (b) comprising a silencing sequence specifically targeting the SbASR4 gene of claim 2 for inhibiting SbASR4 expression in plants.

4. A method for improving the salt stress tolerance of plants, characterized in that, include: Reduce or inhibit the expression level of the SbASR4 gene as described in claim 2 in plants, or reduce or inhibit the activity of the SbASR4 protein as described in claim 1.

5. The method according to claim 4, characterized in that, The reduction or inhibition is achieved through gene editing technology, RNA interference technology, antisense RNA technology, or artificial microRNA technology.

6. The method according to claim 4, characterized in that, The plant is sorghum, or a variety of rice, corn, wheat, or barley containing the SbASR4 homologous gene.

7. A method for reducing plant salt stress tolerance or increasing plant sensitivity to abscisic acid, characterized in that, include: Increase the expression level of the SbASR4 gene as described in claim 2 in plants.

8. The method according to claim 7, characterized in that, The plant in question is Arabidopsis thaliana.

9. The SbASR4 protein according to claim 1, characterized in that, The protein can interact with the SbSOS2 protein in plants.

10. A method for cultivating salt-tolerant sorghum varieties, characterized in that, The breeding route includes any of the following: (a) a breeding route based on natural variation: (i) detecting the expression level or coding region mutation of the SbASR4 gene as described in claim 2 in sorghum breeding materials; (ii) selecting lines with reduced SbASR4 gene expression or loss of function; (iii) obtaining breeding materials or varieties with improved salt tolerance by fixing the homozygosity of this trait through self-pollination or backcrossing techniques; or (b) a breeding route based on gene editing: (i) using gene editing technology to edit the SbASR4 gene as described in claim 2 in sorghum to obtain mutant materials with reduced gene expression or loss of function; (ii) selecting mutant lines that have been identified and confirmed; (iii) obtaining homozygous fixed offspring through self-pollination or backcrossing techniques to cultivate sorghum varieties with improved salt tolerance.

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