Application of soybean gene GmACR39 in regulating and controlling saline-alkaline tolerance of plants

Abstract

The invention relates to the field of plant genetic engineering, in particular to novel application of a soybean ACR gene family member GmACR39 in improving saline-alkaline tolerance of plants. The invention provides application of a separated gene in preparation of a product for improving saline-alkaline tolerance of plants, and the gene encodes a nucleotide sequence shown in SEQ ID NO: 1 or encodes a protein which has at least 80% identity with SEQ ID NO: 2 and has a function of improving saline-alkaline tolerance of plants. The expression of the gene is up-regulated under saline-alkaline stress, and the saline-alkaline tolerance of a transgenic plant can be remarkably improved by over-expression of the gene. The invention also provides an expression vector containing the gene, an sgRNA molecule targeting a promoter of the gene, a CRISPR activation system, and related reagents, kits, a method for cultivating saline-alkaline tolerant plants and detection means. The invention provides a new gene resource and a molecular breeding tool for cultivating new varieties of saline-alkaline tolerant crops.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to soybean genes. GmACR39 Its applications in regulating plant salt and alkali tolerance, more specifically, involve expression vectors, sgRNA molecules, CRISPR activation systems, reagents, kits, methods for improving plant salt and alkali tolerance, and their related uses. Background Technology

[0002] Salt-alkali stress is one of the major abiotic stresses limiting crop growth and productivity. Large areas of saline-alkali land exist in my country and globally, seriously threatening food security. Soybeans ( Glycine max As an important food and oilseed crop, improving its salt and alkali tolerance is a crucial breeding objective. Utilizing molecular biology techniques to discover key salt and alkali tolerance genes and elucidate their functions is an effective approach to cultivating new salt and alkali tolerant varieties.

[0003] Amino acid metabolism plays a crucial role in plant stress responses. Proteins containing the ACT domain (Aromatic aminoacid, Chorismate mutase, and TyrA) (ACR proteins) are a family of proteins widely involved in amino acid metabolism and regulation. Members typically contain 2-4 repeating ACT domains and can function as amino acid sensors or metabolic regulators. Studies have shown that the ACR gene family has important functions in plant growth, development, and stress responses.

[0004] In the model plant Arabidopsis thaliana ( Arabidopsis thaliana In rice, 12 members of the ACR gene family have been identified. Among them, AtACR4 has been confirmed as a receptor kinase involved in regulating embryonic development; AtACR11, located in chloroplasts, has been shown to be associated with glutamine metabolism and nitrogen assimilation, and may participate in defense responses by regulating reactive oxygen species and salicylic acid accumulation. Oryza sativa In this study, nine ACR genes were identified. For example, OsACR7 and OsACR9 were confirmed to have glutamine sensing functions and were associated with the spatiotemporal expression of nitrogen assimilation genes. These studies demonstrate that the ACR gene family has conserved and diverse functions in plant basal metabolism and stress adaptation.

[0005] However, current research on the ACR gene family mainly focuses on model plants such as Arabidopsis thaliana and rice. In soybean, an important economic crop, the systematic identification, molecular characteristics, and specific functions of this gene family in responses to abiotic stresses (especially salt and alkali stress) remain unknown, and its potential application value has not yet been explored. Therefore, systematically identifying the soybean ACR gene family and screening and validating key salt-tolerant functional genes within it is of great significance for elucidating the molecular mechanisms of soybean salt-alkali tolerance and promoting molecular breeding practices. This is also a problem that urgently needs to be solved in this technological field. Summary of the Invention

[0006] This application aims to at least partially address one of the technical problems existing in the prior art. To this end, this application provides a soybean gene... GmACR39 Its use in regulating plant salt and alkali tolerance.

[0007] This application is based on the following discoveries of the inventors: During their systematic study of the soybean ACR gene family, the inventors discovered members... GmACR39 Expression levels were significantly upregulated under saline-alkali stress. Further functional validation experiments demonstrated that overexpression of the soybean gene in soybeans... GmACR39 This study significantly improved the survival rate, biomass (plant height, fresh weight, dry weight), and activity of key antioxidant enzymes (SOD, POD, CAT) of transgenic plants under salt-alkali stress, thereby effectively enhancing the plants' salt tolerance. This indicates that soybean genes... GmACR39 It plays a positive regulatory role in the response of soybean to salt and alkali stress.

[0008] Based on the above findings, in a first aspect, the present invention provides a soybean gene. GmACR39 The soybean gene's application in regulating plant salt and alkali tolerance. GmACR39 The gene contains any of the following nucleotide sequences: (1) a nucleotide sequence encoding a protein having the amino acid sequence shown in SEQ ID NO: 2; or a nucleotide sequence encoding a protein having at least 80% identity with SEQ ID NO: 2 and having the function of improving plant salt tolerance; (2) a nucleotide sequence shown in SEQ ID NO: 1. According to embodiments of the present invention, by increasing the expression level of this gene, the salt tolerance of plants can be effectively improved. This is the first time that the function and application potential of this gene in plant stress resistance (especially salt tolerance) has been revealed.

[0009] A second aspect of the present invention provides an sgRNA molecule. According to embodiments of the present invention, the sgRNA molecule targets soybean genes. GmACR39The promoter or enhancer region. The sgRNA molecule according to embodiments of the present invention can be used to construct a CRISPR activation (CRISPRa) system to enhance gene expression at the transcriptional level.

[0010] A third aspect of the present invention provides an expression vector. According to an embodiment of the present invention, the expression vector comprises the soybean gene described in the first aspect of the present invention. GmACR39 Or an expression cassette expressing the sgRNA molecule described in the second aspect of the present invention. Vectors according to embodiments of the present invention can be used for efficient transformation of plants to realize soybean gene expression. GmACR39 Overexpression.

[0011] A fourth aspect of the present invention provides a CRISPR activation system. According to embodiments of the present invention, the system comprises the sgRNA molecule described in the second aspect of the present invention and a fusion protein of an inactivated Cas9 protein and a transcription activator. The CRISPR activation system according to embodiments of the present invention can specifically upregulate endogenous soybean genes. GmACR39 The expression.

[0012] A fifth aspect of the present invention provides a reagent. According to embodiments of the present invention, the reagent comprises the sgRNA molecule described in the second aspect of the present invention, the expression vector described in the third aspect of the present invention, or the CRISPR activation system described in the fourth aspect of the present invention. The reagent according to embodiments of the present invention provides a material basis for implementing operations to improve the salt and alkali tolerance of plants.

[0013] A sixth aspect of the present invention provides a kit. According to embodiments of the present invention, the kit comprises the sgRNA molecule described in the second aspect of the present invention, the expression vector described in the third aspect of the present invention, or the CRISPR activation system described in the fourth aspect of the present invention. The kit according to embodiments of the present invention facilitates the implementation and standardization of related gene manipulations.

[0014] A seventh aspect of the present invention provides a method for improving the salt and alkali tolerance of plants. According to an embodiment of the present invention, the method includes enhancing soybean genes in plants. GmACR39 The expression level of the soybean gene GmACR39 It has the nucleotide sequence as defined in the first aspect of the present invention. Specifically, according to the method of the embodiments of the present invention, the endogenous gene can be transcribed and activated by introducing the above-described expression vector or by utilizing the above-described CRISPR activation system.

[0015] The eighth aspect of the present invention provides a soybean gene. GmACR39 The use of the encoded protein in regulating plant salt tolerance, the soybean gene GmACR39The encoded protein has the amino acid sequence shown in SEQ ID NO: 2; or has an amino acid sequence that is at least 80% identical to the sequence shown in SEQ ID NO: 2 and has the function of improving plant salt and alkali tolerance. According to embodiments of the present invention, the soybean gene... GmACR39 The encoded protein is a direct functional molecule that mediates salt and alkali tolerance. Its expression level is positively correlated with the salt and alkali tolerance of plants, providing a key target for elucidating the molecular mechanism of salt and alkali tolerance. It can also be used as an exogenous active ingredient to improve plant stress resistance.

[0016] A ninth aspect of the present invention provides a host cell. According to embodiments of the present invention, the host cell carries the expression vector described in the third aspect of the present invention, or has the soybean gene described in the first aspect of the present invention integrated into its genome and expressed therein. GmACR39 The host cell according to embodiments of the present invention is an effective tool for producing the protein, propagating the recombinant vector, or serving as an intermediate for plant genetic transformation. For example, Agrobacterium host cells can be used to efficiently mediate plant transformation, and plant host cells can be directly used to regenerate salt-tolerant transgenic plants.

[0017] A tenth aspect of the present invention provides a transgenic plant cell. According to an embodiment of the present invention, the transgenic plant cell comprises an exogenously introduced soybean gene as described in the first aspect of the present invention. GmACR39 Or containing the sgRNA molecule described in the second aspect of the present invention, and the soybean gene in the plant cell. GmACR39 The expression level of the [specific compound] was higher than that of the untransformed wild-type control cells. The transgenic plant cells according to embodiments of the present invention directly embody the application results of the present invention, exhibiting significantly enhanced salt and alkali tolerance, and can be directly used in agricultural production, or their seeds can be used for breeding and promotion as salt-tolerant varieties.

[0018] The eleventh aspect of the present invention provides the soybean gene described in the first aspect of the present invention. GmACR39 The protein described in the eighth aspect of the present invention, the sgRNA molecule described in the second aspect of the present invention, or the soybean gene specifically amplified in the first aspect of the present invention. GmACR39 The use of primer pairs in the preparation of reagents or kits for screening salt-tolerant plants.

[0019] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Attached Figure Description

[0020] The above and / or additional aspects and advantages of this application will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which: Figure 1This is a map showing the location and distribution of soybean GmACR gene family members on chromosomes in Example 1 of the present invention.

[0021] Figure 2 The phylogenetic tree constructed based on the ACR protein sequences of soybean and Arabidopsis thaliana in Example 1 of this invention shows that the GmACR gene is divided into three subgroups.

[0022] Figure 3 This is a diagram showing the distribution of conserved motifs, conserved domains, and gene structure (exons-introns) of the GmACR gene in Example 1 of this invention.

[0023] Figure 4 This is a collinearity analysis diagram of the GmACR genes within the soybean genome in Example 1 of the present invention, showing fragment replication events.

[0024] Figure 5 This is a genomic collinearity analysis diagram of the ACR genes of soybean and Arabidopsis thaliana in Example 1 of the present invention, showing the orthologous relationship.

[0025] Figure 6 This is a diagram showing the types and distribution of cis-acting elements in the promoter region of the GmACR gene in Example 1 of the present invention.

[0026] Figure 7 This is a heatmap showing the expression patterns of the GmACR gene in different soybean tissues (root, stem, leaf, flower, seed, root nodule) in Example 2 of the present invention.

[0027] Figure 8 This is a heatmap showing the expression pattern of the GmACR gene under different time points (0, 3, 6, 12, and 24 hours) of salt-alkali stress treatment in Example 2 of the present invention.

[0028] Figure 9 Wild-type soybean (WT) and in Example 4 of this invention GmACR39 Phenotypic comparison of overexpression (OE) hairy roots under normal conditions (Water) and salt-alkali stress (Stress).

[0029] Figure 10 The wild-type (WT) and salt-alkali stress strains in Example 4 of this invention . Biomass statistics of overexpression (OE) hairy roots, where A is fresh weight and B is dry weight.

[0030] GmACR39 The wild-type (WT) and salt-alkali stress strains in Example 4 of this invention Figure 11 Statistical graph of antioxidant enzyme activities in overexpressed (OE) hairy roots, including the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT). Detailed Implementation

[0031] The embodiments of this application are described in detail below. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0032] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this application, unless otherwise stated, "multiple" means two or more.

[0033] To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this invention, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0034] In this invention, the terms "comprising" or "including" are open-ended expressions, meaning they include the contents specified in this invention but do not exclude other aspects.

[0035] In this invention, the terms “optionally,” “optionally,” or “optionally” generally refer to events or conditions described subsequently that may but may not occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0036] In this article, the term "soybean gene" GmACR39 "Refers to soybeans ( GmACR39 A specific gene in the genome has the formal identifier of... Glycine max The naming convention is as follows: "Gm" represents soybean, "ACR" indicates that the protein encoded by this gene contains the ACR domain, and "39" is the specific member number in this family. Its nucleotide sequence is shown in SEQ ID NO: 1 of this patent.

[0037] In this paper, the term "ACT domain" refers to a conserved protein domain, initially discovered in enzymes involved in amino acid metabolism, and named after three enzymes: aspartate kinase, chorismate mutase, and prephenate dehydrogenase. This domain typically serves as a binding site for small molecules, such as amino acids, and participates in allosteric regulation of protein function.

[0038] In this paper, the term "ACR gene family" refers to the family of genes encoding proteins containing the ACT domain. Members of this family are involved in various physiological processes in plants, including amino acid metabolism and stress response.

[0039] In this paper, the term "overexpression" refers to the process by which a specific gene is expressed at a level significantly higher than its normal physiological level in a host organism or cell through genetic engineering techniques. In this invention, it refers to the enhancement of soybean gene expression levels by introducing recombinant vectors or gene editing techniques. Glyma.19G095400 Transcription and translation levels in plants.

[0040] In this paper, the term "salt-alkali stress" refers to the abiotic stress on plant growth caused by the simultaneous presence of excessively high concentrations of soluble salts (such as NaCl and Na2SO4) and alkaline salts (such as Na2CO3 and NaHCO3) in the soil. It is more complex than simple salt stress, involving multiple effects such as ion toxicity, osmotic stress, high pH, ​​and nutrient imbalance.

[0041] In this paper, the term "salt and alkali tolerance" refers to the ability of plants to maintain normal growth, development and reproduction under salt and alkali stress conditions, and is an important agronomic trait for evaluating plant stress resistance.

[0042] In this article, the terms "SOD (superoxide dismutase), POD (peroxidase), and CAT (catalase)" refer to key antioxidant enzymes in plants that constitute the reactive oxygen species (ROS) scavenging system. Changes in their activity under abiotic stress are often used to measure the oxidative stress level and resilience of plants. Higher activity generally indicates a stronger ability of the plant to scavenge harmful ROS.

[0043] In this document, the term "expression vector" refers to a DNA molecule constructed using DNA recombination technology that can carry a foreign gene in a host cell and drive its expression. In this invention, it specifically refers to a vector containing a promoter and a soybean gene. GmACR39 Plasmid vectors containing elements such as coding sequences and terminators.

[0044] In this paper, the term "CRISPR Activation System (CRISPRa)" refers to a gene regulation technology based on the CRISPR-Cas system. It specifically upregulates the transcriptional level of an endogenous gene by fusing a DNA-chopping Cas9 protein (dCas9) with a transcription activator and targeting the promoter region of a target gene under the guidance of a specific sgRNA.

[0045] In this paper, the term "enhancer" refers to a DNA sequence that significantly increases the transcription frequency of linked genes. Enhancers can be located upstream, downstream, or inside a gene, and their effects are generally independent of their distance or orientation relative to the promoter. The CRISPRa system can remotely activate gene expression by targeting enhancer regions.

[0046] In this paper, the term "primer" refers to a short single-stranded oligonucleotide that can anneal hybridize with a complementary DNA strand and serve as the starting point for DNA synthesis under the action of DNA polymerase.

[0047] In this article, the term "specific amplification" refers to the primary or exclusive amplification of the target sequence (i.e., specific amplification) by primer pairs in polymerase chain reaction (PCR). GmACR39 Under the same conditions, it does not amplify other non-target sequences (such as other ACR family members).

[0048] In this paper, the term "strict conditions" refers to conditions under which a probe or primer primarily hybridizes with its target sequence and substantially does not hybridize with other sequences. Strict conditions are sequence-dependent, for example: hybridization at 42°C in 50% formamide, 5x SSC, 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 μg / mL denatured and cleaved salmon sperm DNA, followed by washing at 65°C in 0.1x SSC.

[0049] In this paper, the term "conservative substitution" refers to the replacement of one amino acid residue with one or more other amino acid residues that have similar physicochemical properties (such as charge, size, hydrophilicity, and hydrophobicity). Examples include substitutions between leucine, isoleucine, and valine, or between lysine and arginine. Conservative substitutions are generally not expected to significantly alter the three-dimensional structure or function of a protein.

[0050] In this article, the term "nuclease-inactivated Cas protein" (also known as dCas protein, such as dCas9) refers to Cas proteins that have undergone mutations (e.g., the D10A and H840A mutations in Cas9). These proteins retain the ability to specifically bind DNA under the guidance of sgRNA, but lose the endonuclease activity that cuts the DNA double strand, and are therefore often used as DNA-binding scaffolds to carry transcription activators.

[0051] In this paper, the term "sgRNA (single guide RNA)" refers to a short synthetic RNA in the CRISPR-Cas system that contains a "guide" sequence complementary to the target DNA sequence and a "scaffold" structure that binds to the Cas protein. It is responsible for guiding the Cas protein to a specific location on the genome.

[0052] In this paper, the term "promoter" refers to a DNA regulatory region located upstream of the gene coding sequence, capable of binding RNA polymerase and transcription factors to initiate and regulate gene transcription. Promoters can be classified as constitutive (continuously active in various tissues), tissue-specific, or inducible (induced by specific signals) promoters.

[0053] In this paper, the term "transcriptome data" refers to the collection and abundance information of all RNA transcripts (mainly mRNA) in a cell or tissue under specific conditions or at a specific developmental stage. It is used to analyze gene expression patterns.

[0054] In this document, the term "host cell" refers to the cell used to receive and maintain exogenous DNA (such as a recombinant vector). In this invention, this includes microbial cells (such as *Escherichia coli*) used for vector amplification, intermediary cells (such as *Agrobacterium*) used for plant transformation, and the target plant cells themselves.

[0055] In this document, the term "transgenic plant cell" refers to a plant cell in which a foreign gene has been stably integrated into its genome and expressed through genetic engineering techniques. In this invention, it specifically refers to a plant cell in which a soybean gene has been integrated and overexpressed. GmACR39 Plant cells.

[0056] In this document, the term "biomarker" refers to a measurable biological indicator (such as the expression level or protein content of a specific gene) that can be used to indicate a physiological state, pathological process, or response to therapeutic intervention in an organism. In this invention, the soybean gene... GmACR39 The expression level of its proteins can serve as a predictive biomarker for the salt tolerance potential of plants.

[0057] In this paper, the terms “identity,” “homology,” or “similarity” are used to describe the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences relative to a reference sequence, determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN procedure (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Institute)). Foundation, Washington, DC). Numerous algorithms exist for aligning sequences and determining sequence identity, including: the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48: 443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2: 482; the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85: 2444; the Smith-Waterman algorithm (Meth. Mol. Biol. 70: 173-187 (1997); and the BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215: 403-410). Computer programs utilizing these algorithms are also available, including but not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul...). See, Meth.Enzym., 266:460-480 (1996); or GAP, BESTFIT, BLAST Altschul, etc., above, FASTA, and TFASTA, available in Genetics Computing Group (GCG) package, version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0058] In this paper, the term "at least 80% identity" means at least 80% identity with each reference sequence, which may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%.

[0059] In this paper, the term "Phytozome" refers to an integrated plant genome database that provides genome sequences, annotation information, and comparative genomics tools for a wide variety of plants.

[0060] In this paper, the term "PlantCARE" refers to a database used to analyze cis-regulatory elements and transcription factor binding sites in the promoter regions of plant genes.

[0061] In this paper, the term "Pfam database" refers to a widely used database of protein families and domains that identifies conserved functional regions in proteins using hidden Markov models.

[0062] The sequence list in this invention is as follows:

[0063] This application proposes a soybean gene. GmACR39 The following sections will describe in detail the uses of polynucleotide probes or primer pairs in regulating plant salt tolerance, expression vectors, sgRNA molecules, CRISPR activation systems, reagents, kits, methods to improve plant salt tolerance, host cells, transgenic plant cells, biomarkers, and related applications.

[0064] Soybean genes GmACR39 Applications in regulating plant salt and alkali tolerance In a first aspect, the present invention provides a soybean gene. GmACR39 The soybean gene's application in regulating plant salt and alkali tolerance. GmACR39 The gene contains any of the following nucleotide sequences: (1) a nucleotide sequence encoding a protein having the amino acid sequence shown in SEQ ID NO:2; or a nucleotide sequence encoding a protein having at least 80% identity with SEQ ID NO:2 and having the function of improving plant salt tolerance; (2) a nucleotide sequence shown in SEQ ID NO:1. According to embodiments of the present invention, by increasing the expression level of this gene, the salt tolerance of plants can be effectively improved. This is the first time that the function and application potential of this gene in plant stress resistance (especially salt tolerance) has been revealed.

[0065] According to an embodiment of the present invention, the soybean gene GmACR39 The expression level was increased to improve the salt and alkali tolerance of the plant.

[0066] According to an embodiment of the present invention, the soybean gene GmACR39 The increased expression level is achieved through overexpression of the gene.

[0067] According to an embodiment of the present invention, the overexpression is performed by expressing the soybean gene. GmACR39 This is achieved by introducing expression vectors into the plant genome.

[0068] sgRNA molecules In a second aspect, the present invention provides an sgRNA molecule. According to embodiments of the present invention, the sgRNA molecule targets soybean genes. GmACR39 The promoter or enhancer region. The sgRNA molecule according to embodiments of the present invention can be used to construct a CRISPR activation (CRISPRa) system to enhance gene expression at the transcriptional level.

[0069] According to embodiments of the present invention, the sgRNA molecule is suitable for enhancing soybean genes. GmACR39 transcriptional activity.

[0070] According to an embodiment of the present invention, the soybean gene GmACR39 It has a nucleotide sequence as shown in SEQ ID NO: 1.

[0071] According to an embodiment of the present invention, the sgRNA molecule has a nucleotide sequence as shown in SEQ ID NO: 3.

[0072] expression carrier In a third aspect, the present invention provides an expression vector. According to an embodiment of the present invention, the expression vector comprises the soybean gene described in the first aspect of the present invention. GmACR39 Or an expression cassette expressing the sgRNA molecule described in the second aspect of the present invention. Vectors according to embodiments of the present invention can be used for efficient plant transformation to realize soybean gene expression. GmACR39 Overexpression.

[0073] According to an embodiment of the present invention, the expression vector further comprises a nucleic acid sequence encoding a fusion protein of a nuclease-inactivating Cas protein and a transcription activator.

[0074] According to an embodiment of the present invention, the expression vector is an Agrobacterium-mediated transformation vector or a plant virus vector.

[0075] CRISPR activation system In a fourth aspect, the present invention provides a CRISPR activation system. According to embodiments of the present invention, the system comprises the sgRNA molecule described in the second aspect of the present invention and a fusion protein of an inactivated Cas9 protein and a transcription activator. The CRISPR activation system according to embodiments of the present invention can specifically upregulate endogenous soybean genes. GmACR39 The expression.

[0076] reagents In a fifth aspect, the present invention provides a reagent. According to embodiments of the present invention, the reagent comprises the sgRNA molecule described in the second aspect of the present invention, the expression vector described in the third aspect of the present invention, or the CRISPR activation system described in the fourth aspect of the present invention. The reagent according to embodiments of the present invention provides a material basis for implementing operations to improve the salt and alkali tolerance of plants.

[0077] Reagent test kit In a sixth aspect, the present invention provides a kit. According to embodiments of the present invention, the kit comprises the sgRNA molecule described in the second aspect of the present invention, the expression vector described in the third aspect of the present invention, or the CRISPR activation system described in the fourth aspect of the present invention. The kit according to embodiments of the present invention facilitates the implementation and standardization of related gene manipulations.

[0078] Methods to improve the salt and alkali tolerance of plants A seventh aspect of the present invention provides a method for improving the salt and alkali tolerance of plants. According to an embodiment of the present invention, the method includes enhancing soybean genes in plants. GmACR39 The expression level of the soybean gene GmACR39 It has the nucleotide sequence as defined in the first aspect of the present invention. Specifically, according to the method of the embodiments of the present invention, the endogenous gene can be transcribed and activated by introducing the above-described sgRNA molecule, expression vector, or using the above-described CRISPR activation system.

[0079] According to an embodiment of the present invention, the method of enhancing soybean genes in plants GmACR39 The expression level is achieved by: transforming the sgRNA molecule described in the second aspect of the present invention or the expression vector described in the third aspect of the present invention into plant cells or tissues; or using the CRISPR activation system described in the fourth aspect of the present invention to activate the endogenous soybean gene in plants. GmACR39 Transcriptional activation is performed.

[0080] According to an embodiment of the present invention, the plant is soybean.

[0081] use The eighth aspect of the present invention provides a soybean gene. GmACR39The use of the encoded protein in regulating plant salt tolerance, the soybean gene GmACR39 The encoded protein has the amino acid sequence shown in SEQ ID NO: 2; or has an amino acid sequence that is at least 80% identical to the sequence shown in SEQ ID NO: 2 and has the function of improving plant salt and alkali tolerance. According to embodiments of the present invention, the soybean gene... GmACR39 The encoded protein is a direct functional molecule that mediates salt and alkali tolerance. Its expression level is positively correlated with the salt and alkali tolerance of plants, providing a key target for elucidating the molecular mechanism of salt and alkali tolerance. It can also be used as an exogenous active ingredient to improve plant stress resistance.

[0082] According to an embodiment of the present invention, the soybean gene GmACR39 The expression level was increased to improve the salt and alkali tolerance of the plant.

[0083] According to an embodiment of the present invention, the soybean gene GmACR39 The increased expression level is achieved through overexpression of the gene.

[0084] According to an embodiment of the present invention, the overexpression is performed by expressing the soybean gene. GmACR39 This is achieved by introducing expression vectors into the plant genome.

[0085] host cells In a ninth aspect, the present invention provides a host cell. According to embodiments of the present invention, the host cell carries the expression vector described in the third aspect of the present invention, or has the soybean gene described in the first aspect of the present invention integrated into its genome and expressed therein. GmACR39 The host cell according to embodiments of the present invention is an effective tool for producing the protein, propagating the recombinant vector, or serving as an intermediate for plant genetic transformation. For example, Agrobacterium host cells can be used to efficiently mediate plant transformation, and plant host cells can be directly used to regenerate salt-tolerant transgenic plants.

[0086] According to an embodiment of the present invention, the host cell is a bacterial, fungal, or plant cell.

[0087] According to an embodiment of the present invention, the host cell is Agrobacterium, Agrobacterium tumefaciens, or yeast cell.

[0088] Transgenic plant cells In a tenth aspect, the present invention provides a transgenic plant cell. According to an embodiment of the present invention, the transgenic plant cell comprises an exogenously introduced soybean gene as described in the first aspect of the present invention. GmACR39 Or containing the sgRNA molecule described in the second aspect of the present invention, and the soybean gene in the plant cell. GmACR39The expression level of this compound was higher than that of untransformed wild-type control cells. According to embodiments of the present invention, the application results of the invention are directly demonstrated, exhibiting significantly enhanced salt and alkali tolerance, making it suitable for direct use in agricultural production, or its seeds can be used for breeding and promotion as salt-tolerant varieties.

[0089] use In an eleventh aspect, the present invention provides the soybean gene described in the first aspect of the present invention. GmACR39 The protein described in the eighth aspect of the present invention, the sgRNA molecule described in the second aspect of the present invention, or the soybean gene specifically amplified in the first aspect of the present invention. GmACR39 The use of primer pairs in the preparation of reagents or kits for screening salt-tolerant plants.

[0090] biomarkers In a twelfth aspect, the present invention provides a biomarker. According to an embodiment of the present invention, the biomarker is the soybean gene described in the first aspect of the present invention. GmACR39 transcription level or soybean gene as described in the eighth aspect of this invention GmACR39 The expression level of the encoded protein. The biomarkers according to embodiments of the present invention provide molecular-level quantitative indicators for early identification of crop salt-alkali tolerance, variety screening, and efficient tracking of the breeding process, which can accelerate the breeding process of salt-alkali tolerant varieties.

[0091] According to embodiments of the present invention, the biomarker is suitable for detecting the salt tolerance potential of plants.

[0092] Isolated polynucleotide probes or primer pairs In a thirteenth aspect, the present invention provides an isolated polynucleotide probe or primer pair. According to embodiments of the present invention, the isolated polynucleotide probe or primer pair is suitable for specifically detecting or amplifying the soybean gene described in the first aspect of the present invention. GmACR39 The probes or primer pairs according to embodiments of the present invention can be used efficiently and specifically for detecting soybean genes. GmACR39 The presence, copy number, and expression level of these biomarkers are the core tools supporting the application of these biomarkers, genotyping, and molecular detection.

[0093] The following will explain the solution of this application with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0094] Example Example 1: Genome-wide identification and bioinformatics analysis of the soybean GmACR gene family 1. Materials and Methods The soybean (Glycine max) reference genome sequence and gene annotation files (GFF3 format) were downloaded from the Phytozome website (version: Wm82.a4.v1). Based on the Hidden Markov Model (HMM) profile file of the Pfam database (PF01842, ACT domain), the Hummsearch program of HMMER software (version 3.3.2) was used for analysis. Multi-threaded computation (--cpu 8) was enabled during the analysis, with E-values ​​at both the whole sequence and domain levels less than 1 × 10⁻⁶. -5 The screening thresholds (corresponding to parameters -E 1e-5 and --domE 1e-5 respectively) were used to output the overall matching result table (--tblout) and the domain matching result table (--domtblout), respectively. All other parameters were set to default. This initial screening yielded candidate ACR proteins containing the ACT domain for subsequent analysis. Subsequently, the conserved domains of the candidate proteins were further validated using the SMART online tool and the NCBI CDD database (example: CDD validation showed that the GmACR1 protein sequence contained a typical ACT domain, with an E value <1e-10). Candidate proteins that did not contain any complete ACT domains (Pfam PF01842) were removed after CDD or SMART validation. Finally, 44 non-redundant soybean ACR genes were obtained and named... GmACR39 to GmACR1 The ExPASyProtParam tool was used to predict the amino acid number, molecular weight, and theoretical isoelectric point of the proteins encoded by each gene. TBtools software was used for chromosome localization visualization and collinearity analysis (MCScanX algorithm). Protein sequences were aligned using MAFFT (parameters: `mafft --auto input.fa>aligned.fa`). A phylogenetic tree was constructed using IQ-TREE (parameters: `iqtree -s aligned.fa -m MFP -bb 1000 -alrt 1000`).

[0095] 2. Results Forty-four GmACR genes were successfully identified from the soybean genome. Their basic information is shown in Table 1. The proteins range in length from 189 to 917 amino acids, with molecular weights ranging from 21.40 kDa to 100.42 kDa. Most proteins have a theoretical isoelectric point of acidity. Chromosomal localization analysis showed that these genes are unevenly distributed across 18 chromosomes (excluding chromosomes 9 and 12). See [link to relevant documentation] for details. GmACR44Based on their actual physical location on the chromosome, the genes are named sequentially. Figure 1 to GmACR1 .

[0096] Table 1: Information on GmACR gene family members

[0097] Phylogenetic analysis clearly divided all ACR proteins into three main subgroups (Groups I-III), see details below. GmACR44 Group III consists entirely of 16 soybean ACR genes, suggesting that this subgroup may have undergone specific expansion or functional differentiation during the evolution of soybean lineage.

[0098] Gene structure and conserved motif analysis showed that members within the same subgroup had similar structural and motif composition characteristics, see details below. Figure 2 For example, Group III has the simplest motif composition, which contrasts sharply with the other subgroups.

[0099] Collinearity analysis revealed 49 pairs of repetitive genes generated by fragment duplication, widely distributed across various chromosomes; see details below. Figure 3 This indicates that fragment duplication is the main mechanism driving the family expansion, and no tandem duplication events were found. Soybean-Arabidopa collinearity analysis identified 40 pairs of orthologous genes; see details below. Figure 4 This indicates that these genes are evolutionarily conserved.

[0100] Analysis of the promoter region revealed a variety of cis-regulatory elements, including photoresponsive elements (G-box, etc.), hormone-responsive elements (ABRE, etc.), and stress-responsive elements (MBS, etc.). See details in [link to relevant documentation]. Figure 5 This suggests that the GmACR gene may be widely involved in the regulation of a variety of physiological processes.

[0101] against Figure 6The promoter sequence 2000 bp upstream of the gene start codon (ATG) was first retrieved from the soybean genome database (Phytozome v13, Glycine max Wm82.a2.v1) in this embodiment. After converting the sequence to FASTA format, it was submitted to the PlantCARE online platform with default parameters and a confidence level of ≥95%. To establish screening criteria, cis-regulatory elements were predicted. After removing duplicates and low-confidence elements, the types, quantities, and precise locations of core elements such as light-responsive elements (G-box, GT1-motif), hormone-responsive elements (ABRE, TGA-element), and abiotic stress-responsive elements (MBS, LTR) were obtained. Examples of some prediction results are shown below: G-box (light response, start site 328, end site 333, strand orientation +, confidence 99%), ABRE (abscisic acid response, start site 756, end site 762, strand orientation -, confidence 98%), MBS (drought response, start site 1245, end site 1251, strand orientation +, confidence 96%). The element data output from PlantCARE was then compiled into an Excel spreadsheet containing four columns: "Feature Name," "Start," "End," and "Strand." This data was imported into the "Sequence Feature Plot" module of TBtools v1.120 software, with a sequence length set to 2000. The output is a PDF visualization of the graph, with a resolution of 300 dpi, a component rectangle height of 10, a font size of 12, and a sequence ruler interval of 200 bp. See details in the original text. GmACR The preview window shows that G-box elements are concentrated in the 300-500 bp region upstream of the promoter, while ABRE elements are dispersed in the 700-1500 bp range. The positive and negative chain elements exhibit an alternating distribution. These results suggest... Figure 6 Genes may participate extensively in the regulation of various physiological processes in soybean, such as light response, hormone signal transduction, and tolerance to abiotic stress, by binding to different transcription factors.

[0102] Example 2: Analysis of GmACR gene expression patterns under salt-alkali stress 1. Materials and Methods Plant materials and treatment: After germination, soybean cultivar 'Williams 82' seeds were transplanted into nutrient soil and cultured in an artificial climate chamber (temperature 25±2°C, photoperiod 16 hours light / 8 hours dark, light intensity 150-200 μmol m2). -2 s -1Plants were grown to the three-leaf stage. Salt-alkali stress groups and control groups were established. The salt-alkali stress group was hydroponically cultured with a 75 mM mixed solution of Na₂CO₃ and NaHCO₃ (molar ratio 5:1), while the control group was watered with an equal volume of distilled water. Root samples were collected at 0, 3, 6, 12, and 24 hours after treatment, flash-frozen in liquid nitrogen, and stored at -80℃.

[0103] RNA extraction and transcriptome sequencing: Total RNA was extracted from roots at each time point using a plant total RNA extraction kit. After quality control, strand-specific library construction and transcriptome sequencing on the Illumina platform were performed by a commercial sequencing company. Three biological replicates were set up for each time point.

[0104] Data Analysis: Hisat2 was used to align sequencing data to the soybean reference genome. StringTie was used for transcript assembly and gene expression estimation, with FPKM values ​​representing gene expression levels. Using this transcriptome dataset obtained in our laboratory, expression levels of 44 GmACR genes at different time points were extracted. TBtools software was used to create gene expression heatmaps.

[0105] 2. Results Transcriptome data analysis showed that the GmACR gene exhibited significantly different tissue expression patterns, see details below. GmACR For example, some genes are constitutively highly expressed (Group A), while... Figure 7 Genes such as [names] are specifically highly expressed in root nodules.

[0106] Time-series expression analysis under salt-alkali stress showed that most GmACR genes responded to stress, but in different patterns; see details below. GmACR11 .in, Figure 8 ( GmACR24 )and Glyma.11G046200 ( GmACR39 Genes such as ) were significantly upregulated 24 hours after stress and were classified as Group A, which are key candidate genes for stress response.

[0107] Example 3: Soybean Genes Glyma.19G095400 Construction of overexpression vectors 1. Materials and Methods Using soybean 'Williams 82' cDNA as a template, the soybean gene was amplified using high-fidelity DNA polymerase. GmACR39The complete open reading frame (ORF) sequence (SEQ ID NO: 1) was obtained. Specific restriction enzyme sites (such as BamHI and SacI) were introduced into the upstream and downstream primers, respectively; the nucleotide sequence of specific primer F is shown in SEQ ID NO: 8, and the nucleotide sequence of specific primer R is shown in SEQ ID NO: 9). The PCR product was purified, digested with enzymes, and ligated to the plant overexpression vector pCAMBIA1302.1 (or a similar vector containing a CaMV 35S promoter, a NOS terminator, and a herbicide resistance selection marker) that had undergone the same enzyme digestion treatment. The amplified target fragment and the target vector were then subjected to double digestion with BamHI and SacI to produce DNA fragments with complementary sticky ends. After the enzyme digestion reaction, the digestion products were purified to remove enzymes, buffer, and incompletely reacted nucleic acid components, obtaining the target fragment and linearized vector for the ligation reaction. The double-digested and purified target fragment was ligated to the vector using T4 DNA ligase. Specifically, the target fragment and vector were mixed at a predetermined molar ratio, and T4 DNA ligase and its matching reaction buffer were added. The ligation reaction was carried out under suitable conditions, allowing the target fragment to be directionally ligated to the vector backbone via its sticky ends. The ligation product was transformed into *E. coli* DH5α competent cells. Positive clones were screened by colony PCR and enzyme digestion identification, and the recombinant plasmid was sequenced for verification. The correctly sequenced recombinant plasmid was named p35S:: GmACR39 .

[0108] 2. Results The soybean genome has been successfully constructed, as confirmed by sequencing. GmACR39 plant overexpression vector p35S:: GmACR39 This ensured GmACR39 The correctness of the encoding sequence and its matching with the vector expression framework.

[0109] Example 4: Validation using soybean hairy root overexpression system GmACR39 Salt and alkali resistance 1. Materials and Methods Hairy root induction and transformation: The recombinant plasmid p35S:: constructed in Example 3 was used for hairy root induction and transformation. GmACR39 Electroporation to Agrobacterium tumefaciens ( GmACR39 In strain K599, sterile soybean 'Williams 82' seedlings were inoculated below the cotyledonary node with a bacterial suspension containing recombinant Agrobacterium (OD600=0.8, infection time 1h) and cultured in the dark on a co-culture medium for 3 days. They were then transferred to a rooting medium to induce hairy root growth.

[0110] Identification of transgenic hairy roots: Genomic DNA was extracted from hairy roots and analyzed using soybean genes. Agrobacterium rhizogenesPCR identification was performed using specific primers or promoter / gene-specific primers on the vector to screen for positive overexpression hairy root systems (OE). Hairy roots transformed into empty vectors were used as negative controls (VC).

[0111] Salt-alkali stress treatment and phenotypic observation: Hairy roots of OE and VC with consistent growth status were transferred to 1 / 2 MS liquid medium (MS524, 1 / 2 MS medium: manufacturer: Beyotime, catalog number: ST5008S, salt-alkali mixture directly dissolved in 1 / 2 MS medium without shaking) containing 75 mM salt-alkali mixed solution (same as in Example 2) or water for stress treatment. After 7-14 days of treatment, the growth status, color and other phenotypic characteristics of the hairy roots were observed and recorded.

[0112] Physiological parameters were measured: Hairy root samples were collected after stress treatment, and the following parameters were measured: ① Fresh weight and dry weight; ② Superoxide dismutase activity (WST-8 method, SOD kit: Beyotime S0101S); ③ Peroxidase activity (guaiacol method, POD kit: Biobox, catalog number: AKAO005C); ④ Catalase activity (ammonium molybdate method, CAT kit: Biobox, catalog number: AKAO003). All measurements were performed in at least three biological replicates.

[0113] 2. Results (1) Phenotypic analysis: Under normal conditions, there was no significant difference in hairy root growth between OE and VC. Under saline-alkali stress, hairy root growth of VC was significantly inhibited, and some roots turned brown; while OE- GmACR39 Hairy roots showed greater tolerance, with less growth inhibition and reduced browning. See details for further information. GmACR39 .

[0114] (2) Biomass analysis: Under salt and alkali stress, OE- Figure 9 Both the fresh weight and dry weight of the hairy roots were significantly higher than those of the VC hairy roots. See the detailed results below. GmACR39 .

[0115] (3) Antioxidant enzyme activity: Salt-alkali stress induced an increase in the activities of SOD, POD, and CAT in hairy roots. Compared with VC, OE- Figure 10 The activities of all three antioxidant enzymes in hairy roots increased to higher levels under stress conditions; see details below. GmACR39 .

[0116] Conclusion: Overexpression Figure 11 It can significantly enhance the tolerance of soybean hairy root system to salt and alkali stress, manifested in improved growth phenotype, maintenance of higher biomass, and activation of a stronger antioxidant protection system. This confirms the role of soybean genes. GmACR39It plays a positive regulatory role in the soybean's response to salt and alkali stress.

[0117] Example 5: Activation of endogenous soybean genes using the CRISPR activation system GmACR39 expression 1. Materials and Methods sgRNA design: targeting soybean genes GmACR39 Two to three specific sgRNAs were designed within 200 bp upstream of the transcription start site (TSS). This example demonstrates the sgRNA shown in SEQ ID NO: 3. Online tools were used to predict off-target effects, and sgRNAs with low off-target risk and high predicted activity were selected.

[0118] CRISPRa vector construction: The selected sgRNA sequence was cloned into a plant CRISPRa vector equipped with dCas9-VP64 (or dCas9-SunTag or other activation systems, vector source: AddGene catalog number #196291) to construct the recombinant vector pCRISPRa- GmACR39 .

[0119] Soybean hairy root transformation and verification: Following the method in Example 4, pCRISPRa- GmACR39 Soybeans were transformed with a vector to obtain transgenic hairy roots. Soybean genes were detected using qRT-PCR. GmACR39 The transcriptional level in CRISPRa hairy roots was used to verify whether its expression was specifically activated. The forward primer sequence of the internal reference gene used in qRT-PCR is shown in SEQ ID NO: 4, and the reverse primer sequence of the internal reference gene used in qRT-PCR is shown in SEQ ID NO: 5. The soybean gene used in qRT-PCR... GmACR39 The forward primer sequence is shown in SEQ ID NO: 6, and the soybean gene used for qRT-PCR is... GmACR39 The reverse primer sequence is shown in SEQ ID NO: 7.

[0120] 2. Results qRT-PCR analysis showed that, compared with the control hairy roots transformed with the empty CRISPRa vector, the roots transformed with pCRISPRa- GmACR39 In the hairy roots, soybean genes GmACR39 The mRNA level was significantly increased. Preliminary results showed that the salt and alkali tolerance of this hairy root was enhanced after undergoing salt and alkali stress treatment as described in Example 4. This indicates that the endogenous soybean gene was activated through CRISPRa technology. GmACR39 The expression of this is also an effective strategy to improve the salt and alkali tolerance of soybeans.

[0121] Example 6: Preliminary Analysis of Heterologous Expression and Activity of GmACR39 Protein 1. Materials and Methods Will GmACR39 The ORF was cloned into the prokaryotic expression vector pET-28a(+) to construct the recombinant plasmid pET-28a- GmACR39 GmACR39 The cells were transformed into E. coli BL21(DE3) competent cells. Recombinant protein expression was induced at 37°C for 4 hours with IPTG at a final concentration of 0.5 mmol / L, and soluble GmACR39 recombinant protein was obtained by nickel column affinity chromatography.

[0122] 2. Results SDS-PAGE electrophoresis showed a specific band at the expected molecular weight, which was confirmed by Western blotting to be the His-tagged GmACR39 protein. The purified protein can be used for subsequent biochemical analyses, such as investigating whether it has the amino acid-binding activity of known ACR family proteins, or its interaction with other stress-responsive proteins, thereby further elucidating its functional mechanism.

[0123] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0124] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application.

Claims

1. Soybean genes GmACR39 The soybean gene or the use of its encoded protein in regulating plant salt tolerance. GmACR39 Or the protein it encodes is selected from: (1) A nucleotide sequence encoding a protein having the amino acid sequence shown in SEQ ID NO: 2; or a nucleotide sequence encoding a protein having at least 80% identity with SEQ ID NO: 2 and having the function of improving the salt and alkali tolerance of plants; (2) The nucleotide sequence as shown in SEQ ID NO: 1; (3) A protein having the amino acid sequence shown in SEQ ID NO: 2; or a protein having at least 80% identity with SEQ ID NO: 2 and having the function of improving the salt and alkali tolerance of plants; Optionally, the soybean gene GmACR39 The expression level of [a substance] was increased to improve the salt and alkali tolerance of the plant. Optionally, the soybean gene GmACR39 The increased expression level is achieved through overexpression of the gene; Optionally, the overexpression is achieved by introducing the soybean gene... GmACR39 This is achieved by introducing expression vectors into the plant genome.

2. An sgRNA molecule, characterized in that, The sgRNA molecule targets soybean genes. GmACR39 promoter or enhancer regions; Optionally, the sgRNA molecule is adapted to enhance soybean genes. GmACR39 transcriptional activity; Optionally, the soybean gene GmACR39 It has the nucleotide sequence shown in SEQ ID NO: 1; Optionally, the sgRNA molecule has a nucleotide sequence as shown in SEQ ID NO:

3.

3. An expression carrier, characterized in that, Contains the soybean gene as described in claim 1 GmACR39 Or an expression cassette expressing the sgRNA molecule as described in claim 2; Optionally, the expression vector further comprises a nucleic acid sequence encoding a fusion protein of a nuclease-inactivating Cas protein and a transcription activator; Optionally, the expression vector is an Agrobacterium-mediated transformation vector or a plant virus vector.

4. A CRISPR activation system, characterized in that, include: The sgRNA molecule of claim 2, and the fusion protein of inactivated Cas9 protein and transcription activator.

5. A reagent, characterized in that, include: The sgRNA molecule of claim 2, the expression vector of claim 3, or the CRISPR activation system of claim 4.

6. A reagent kit, characterized in that, include: The sgRNA molecule of claim 2, the expression vector of claim 3, or the CRISPR activation system of claim 4.

7. A method for improving the salt and alkali tolerance of plants, characterized in that, include: Enhancing soybean genes in plants GmACR39 The expression level of the soybean gene GmACR39 It has the nucleotide sequence defined in claim 1; Optionally, the enhancement of soybean genes in plants GmACR39 The expression level is achieved in the following way: Transform the sgRNA molecule of claim 2 or the expression vector of claim 3 into plant cells or tissues; or Using the CRISPR activation system of claim 4 to activate endogenous soybean genes in plants GmACR39 Transcriptional activation occurs; Optionally, the plant is soybean.

8. A host cell, characterized in that, Carrying the expression vector of claim 3, or integrating and expressing the soybean gene of claim 1 into the genome. GmACR39 ; Optionally, the host cell is a bacterial, fungal, or plant cell; Optionally, the host cell is Agrobacterium, Agrobacterium tumefaciens, or yeast cell.

9. A transgenic plant cell, characterized in that, The transgenic plant cells contain the exogenously introduced soybean gene as described in claim 1. GmACR39 Or containing the sgRNA molecule as described in claim 2, and the soybean gene in the plant cell. GmACR39 The expression level was higher in untransformed wild-type control cells.

10. The soybean gene according to claim 1 GmACR39 Or the protein it encodes, the sgRNA molecule of claim 2, or the soybean gene of claim 1 specifically amplified. GmACR39 The use of primer pairs in the preparation of reagents or kits for screening salt-tolerant plants.

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