Application of overexpressing product of GmRHA1 gene in regulating soybean salt and alkali tolerance

Abstract

This invention relates to the field of genetic engineering technology, specifically to overexpression. GmRHA1 The application of gene-based products in regulating soybean salt and alkali tolerance, the aforementioned GmRHA1 The CDS sequence of the gene is shown in SEQ ID NO.1. This invention provides... GmRHA1 Genes are used to regulate salt and alkali tolerance in soybeans, and are expressed by overexpression in soybean plants. GmRHA1 Genes enhance the salt and alkali tolerance of soybeans.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to overexpression. GmRHA1 Application of gene-based products in regulating the salt and alkali tolerance of soybeans. Background Technology

[0002] Soybeans Glycinemax Soybeans have a long history of cultivation and are an important food crop, playing a crucial role in promoting agricultural development and ensuring food supply. Therefore, improving soybean yield and quality is particularly important. With the improvement of people's living standards, domestic market demand for soybeans continues to grow, but current production still cannot meet the demand. Salt-alkali stress severely inhibits the growth and development of soybeans and is a key factor restricting the improvement of their yield and quality.

[0003] Salt-alkali stress is a typical complex stress, combining osmotic stress and ion poisoning induced by high salt concentrations with alkaline stress caused by high pH. Stress induced by neutral salts such as NaCl and Na₂SO₄ is called salt stress; while stress induced by alkaline salts such as Na₂CO₃ and NaHCO₃, characterized primarily by high pH, ​​is called alkaline stress. In nature, salt and alkaline stresses often coexist; therefore, this complex stress is defined as salt-alkali mixed stress, and research on this complex stress is relatively limited.

[0004] Salinization significantly inhibits plant growth and development, and the annual reduction in economic crop yields due to salt stress is a serious problem. Salinization is mainly caused by excessive sodium in the environment. + Cl - This is due to accumulation, resulting in high concentrations of Na. + It competes with other ions for absorption and replaces calcium in the cell wall. 2+ Binding site, reducing extracellular Ca 2+ The activity of [the substance] triggers extracellular Ca [activity]. 2+ The concentration decreases. Under salt-alkali stress, the Na+ concentration in plant cytoplasm decreases. + Concentrations can reach over 100 mmol / L, thereby inhibiting plant photosynthesis, reducing the photosynthetic reaction rate, decreasing organic matter synthesis, and ultimately leading to reduced crop yields.

[0005] Excessive soil salinity can cause plant cells to lose water and increase intracellular osmotic pressure, which in turn can damage cell membranes and organelles. At the same time, harmful substances such as inorganic ions and reactive oxygen species (ROS) will accumulate in large quantities in the cells.

[0006] High salt concentrations can also trigger osmotic stress, oxidative stress, and ion stress in plants, leading to impaired water absorption, ion imbalance, and disruption of cellular homeostasis, severely inhibiting plant growth and development, and even causing plant death. Under alkaline stress, elements such as manganese, iron, and calcium are easily bound in the soil, resulting in nutrient deficiencies in plants; at the same time, high pH can also have a toxic effect on plant roots, causing disorders in the root organic acid metabolism system, thereby leading to corrosive damage to seedling roots and buds.

[0007] Therefore, breeding new salt-tolerant and high-quality soybean varieties has become an urgent task. Discovering novel salt-tolerant functional genes and deeply analyzing the soybean's response mechanism to salt-alkali stress are of significant theoretical and practical value for promoting salt-alkali tolerant soybean breeding and achieving stable and high soybean yields. Summary of the Invention

[0008] To develop a method for improving the salt and alkali tolerance of soybeans, this invention provides overexpression... GmRHA1 Application of gene products in regulating soybean salt and alkali tolerance. Overexpression in this invention... GmRHA1 The gene's product is used to regulate salt and alkali tolerance in soybeans and is overexpressed in soybean plants. GmRHA1 Genes can enhance the salt and alkali tolerance of soybeans.

[0009] This invention provides overexpression GmRHA1 The application of gene-based products in regulating soybean salt and alkali tolerance, the aforementioned GmRHA1 The CDS sequence of the gene is shown in SEQ ID NO.1.

[0010] This invention achieves this by overexpressing [the gene] in soybean plants. GmRHA1 Genes enhance the salt and alkali tolerance of soybeans.

[0011] Furthermore, overexpression GmRHA1 Gene's products are: GmRHA1 Gene overexpression vectors or overexpression GmRHA1 Recombinant bacteria.

[0012] Furthermore, the aforementioned GmRHA1 The steps for constructing a gene overexpression vector are as follows: Cloning soybeans GmRHA1 Genes, soybeans GmRHA1 Genes linked to the pCAMBIA1301 vector Captain I and Bam HI site, obtained GmRHA1 Gene overexpression vector.

[0013] Furthermore, the overexpression GmRHA1 The recombinant bacteria for gene preparation are as follows: GmRHA1 Gene overexpression vector was transferred into Agrobacterium rhizogenes competent cells to obtain overexpression. GmRHA1 Recombinant bacteria.

[0014] Furthermore, utilizing overexpression GmRHA1 Recombinant bacteria can enhance the salt and alkali tolerance of soybeans.

[0015] Furthermore, the transformation of soybeans using the Agrobacterium-mediated transformation method comprises: using the Agrobacterium-mediated transformation method to transform soybeans overexpressing Agrobacterium-mediated transformation. GmRHA1 Recombinant bacteria infected soybean explants to obtain transgenic soybean hairy root complex plants resistant to salt and alkali stress.

[0016] Furthermore, the Agrobacterium is K599.

[0017] Furthermore, the overexpression GmRHA1 Gene's products are used to alleviate the damage to soybean plants caused by salt and alkali stress.

[0018] Furthermore, the overexpression GmRHA1 Genes are used to increase chlorophyll content.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides overexpression GmRHA1 Application of gene products in regulating soybean salt and alkali tolerance, overexpression GmRHA1 Gene's products are: GmRHA1 Gene overexpression vectors or overexpression GmRHA1 A recombinant gene was cloned from soybean, yielding a 577bp gene. GmRHA1 The gene, whose full-length CDS is 459 bp and encodes 152 amino acids, was overexpressed by ligating into the pCAMBIA1301 vector. GmRHA1 Soybean hairy roots were transformed using Agrobacterium rhizogenes-mediated transformation, and the results were identified by qRT-PCR. GmRHA1 Soybean hairy root complex overexpression plants, i.e., overexpression with salt and alkali tolerance function GmRHA1 Soybean hairy root complex plant with genes.

[0020] The present invention provides GmRHA1 The gene is used to regulate the salt and alkali tolerance of soybeans and is overexpressed in soybean plants. GmRHA1 Genes can enhance the salt and alkali tolerance of soybeans. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 for GmRHA1 Gene cloning and identification; In the diagram, A is... GmRHA1 Electrophoresis diagram of PCR amplification products of the gene; where M: DL 2000 molecular weight standard; 0: blank control; 1-2: GmRHA1 PCR products of genes; B is GmRHA1 Electrophoresis diagram of positive colony PCR for gene identification; where M: DL 2000 molecular weight standard; 0: blank control; 1-3: colony PCR products.

[0023] Figure 2 pCAMBIA1301- GmRHA1 Double enzyme digestion identification; where M: DL 2000 molecular weight standard; 1: recombinant plasmid; 2-3: pCAMBIA1301- GmRHA1 Double enzyme digestion product.

[0024] Figure 3 for GmRHA1 PCR amplification of gene RNAi fragments; where M: DL 2000 molecular weight standard; 0: blank control; 1-4: GmRHA1 PCR products of gene RNAi fragments.

[0025] Figure 4 pTRV2- GmRHA1 Double enzyme digestion identification; where M: DL 2000 molecular weight standard; 1: recombinant plasmid; 2-3: pTRV2- GmRHA1 Double enzyme digestion product.

[0026] Figure 5 For containing pCAMBIA1301- GmRHA1 and pTRV2- GmRHA1 Agrobacterium colony identification by PCR; In the figure, A represents the component containing pCAMBIA1301- GmRHA1 Agrobacterium colony PCR identification; where M: DL 2000 molecular weight standard; 0: water control; 1: positive control (recombinant plasmid as template); 2-5: containing pCAMBIA1301- GmRHA1 PCR products of Agrobacterium K599 colonies; B contains pTRV2- GmRHA1 Agrobacterium colony PCR identification; where M: DL 2000 molecular weight standard; 0: water control; 1: positive control (recombinant plasmid as template); 2-6: containing pTRV2- GmRHA1 PCR products of Agrobacterium K599 colonies.

[0027] Figure 6 The text describes the culture process of a transgenic soybean hairy root complex plant; where A represents a soybean seedling with unexpanded cotyledons; BC represents infected soybean explants; and D represents a soybean hairy root complex plant.

[0028] Figure 7 For the transfer GmRHA1 qRT-PCR identification of the gene-derived soybean hairy roots.

[0029] Figure 8 For the transfer GmRHA1 GUS staining analysis of soybean hairy roots; In the figure, A represents the GUS staining results of normally cultured (overexpressing soybean hairy roots without stress); B represents overexpression after 24 hours of salt-alkali stress treatment. GmRHA1 GUS staining results of hairy roots of soybean hairy root complex plants.

[0030] Figure 9 Phenotype of transgenic soybean hairy root complex plants under salt-alkali stress; In the figure, A represents the phenotype of transgenic soybean hairy root complex plants subjected to 0h of salt-alkali stress; B represents the phenotype of transgenic soybean hairy root complex plants subjected to 12 hours of salt and alkali stress. C represents the phenotype of transgenic soybean hairy root complex plants subjected to 24 hours of salt and alkali stress.

[0031] Figure 10 The SOD and CAT activities of three soybean hairy root complex plants under salt-alkali stress were measured. In the figure, A represents the SOD activity of three soybean hairy root complex plants under salt-alkali stress; B represents the CAT activity of three soybean hairy root complex plants under salt-alkali stress.

[0032] Figure 11 The MDA content of hairy roots in three soybean hairy root complex plants under salt-alkali stress.

[0033] Figure 12 The content of proline in the hairy roots of three soybean hairy root complexes under salt-alkali stress.

[0034] Figure 13 The content of soluble sugars in the hairy roots of three soybean hairy root complexes under salt-alkali stress.

[0035] Figure 14 The chlorophyll content of leaves of three soybean hairy root complex plants under salt-alkali stress. Detailed Implementation

[0036] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0037] Example 1: GmRHA1 Gene cloning.

[0038] I. Experimental Materials and Methods 1. Experimental materials Plant material: Soybean variety Heinong 553.

[0039] Vector: Cloning vector pMD-19T Simple Vector (Takara).

[0040] Strains: Escherichia coli ( E. coli DH5α.

[0041] 2. Soybeans GmRHA1 Cloning of genes (1) Primer design Based on known sequence fragments, soybeans were retrieved and obtained from the soybean genome database (www.Phytozome.com / soybean). GmRHA1 Complete sequence of gene (Wm82.a4.v1|Glyma.13G009100.1); designed with primer 5.0. Bam HI and Psst I restriction site GmRHA1 Gene-specific primers, primer sequences are shown in Table 1.

[0042] Table 1 GmRHA1 Gene cloning primers Note: The underlined part is... Bam HI identification site, the double-underlined part is Psst I. Identification site.

[0043] (2) PCR amplification Total RNA was extracted from soybean leaves and cDNA was obtained by reverse transcription. Using the cDNA as a template, [the following was used]... GmRHA1 Gene-specific primers were used for PCR amplification. The reaction system is shown in Table 2, and the reaction conditions are shown in Table 3.

[0044] Table 2 Reaction System Table 3 Reaction conditions (3) Recovery, ligation and transformation of PCR amplification products After the PCR products were detected by 1% agarose gel electrophoresis, the target gene bands were recovered by gel cutting according to the EZNA® Gel Extraction Kit (OMEGA).

[0045] Will GmRHA1 The gene was ligated into the pMD19-Tsimple vector, and the recombinant vector constructed was named pMD19T- GmRHA1 The total volume of the connecting system is 10 μg, and its specific components are: Solution I 5 μg, GmRHA1 2 μg of gene recovery product, 1 μg of pMD19-Tsimple vector, and 2 μg of ddH2O were used for ligation under the following conditions: 16℃ water bath reaction for 16 h.

[0046] The recombinant vector pMD19T- was synthesized using a thermal shock method. GmRHA1 Transform into Escherichia coli DH5α competent cells, following the instructions for use of Escherichia coli DH5α competent cells. After transformation, spread the bacterial culture evenly on LB solid medium containing ampicillin resistance and incubate at 37°C upside down for 12 hours until single colonies grow on the medium.

[0047] (4) Identification and sequencing of positive recombinant clones Single colonies were selected for colony PCR identification to screen for positive clones. The PCR reaction system is shown in Table 2, and the PCR reaction procedure is shown in Table 3. The amplification products were verified by 1% agarose gel electrophoresis. Positive bacterial cultures were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing verification.

[0048] II. Experimental Results 1. GmRHA1 Gene cloning and sequencing Total RNA was extracted from soybeans and reverse transcribed into cDNA. Using cDNA as a template, a 577bp cDNA was amplified by PCR. GmRHA1 Target gene band ( Figure 1 (A). After ligating the target gene band into the pMD19-Tsimple vector, positive recombinant clones were screened by colony PCR. Figure 1 (B), and named the recombinant plasmid pMD19T- GmRHA1 .

[0049] Sequencing results showed that the amplified GmRHA1The CDS region is 459 bp in length (SEQ ID NO.1) and encodes 152 amino acids (SEQ ID NO.4).

[0050] SEQ ID NO.1: ATGGGCCTGTCAAGTCTCCCAGCACCATCTGAAGGAGTGTTATGTGTGCTTCTTGTAAACACTGCCTTGTCTATATCCATATTCAAAGGCATTGTTAGGACAATTCTACAAATTGTCGGTATCCGCGTTTCGTCGTTGTCTCCTTCACCAGACATCTCCCGAAACCCACCTGAGCCATTAGAATTCAACCTCAGCCCCTCGGAGGGTTTCATTGAAGAGTTCAGAAG CAGGACACCAACACTTAGGTTTGGCAGCATGTGTGGCAGTAAACAACCTCAACATGAATGTTGTTGTGTGTGTCTCACAAAGTTTGAACCAGAATCTGAGATAAACTGTTTATCATGTGGCCATATTTTTCACAAAGTGTGCATGGAGAAGTGGTTGGACTATTGGAACATTACATGCCCACTTTGCAGGACTTCCTTGATGCCTGAAGATGATGCATCTTGCTTTTGGTAA.

[0051] SEQ ID NO.4: MGLSSLPAPSEGVLCVLLVNTALSISIFKGIVRTILQIVGIRVSSLSPSPDISRNPPEPLEFNLSPSEGFIEEFRSRTPTLRFGSMCGSKQPQHECCCVCLTKFEPESEINCLSCGHIFHKVCMEKWLDYWNITCPLCRTSLMPEDDASCFW.

[0052] Example 2: Soybeans GmRHA1 Gene vector construction and genetic transformation of soybean hairy roots.

[0053] I. Experimental Materials and Methods 1. Experimental materials Plant material: Soybean variety Heinong 553.

[0054] 2. Vectors and strains Overexpression vector: pCAMBIA1301; RNAi vectors: pTRV2, pTRV1; Strains: Escherichia coli DH5α, Agrobacterium rhizogenes K599.

[0055] 3. Overexpression vector pCAMBIA1301- GmRHA1 Construction (1) Double digestion of vector and target gene Following the instructions of the EZNA® Plasmid Mini Kit I (OMEGA), the recombinant vector plasmid pMD19T- was extracted. GmRHA1 With the overexpression vector plasmid pCAMBIA1301. Bam HI and Psst I performed double digestion on the two plasmids mentioned above. The digestion system is shown in Table 4. The digestion conditions were 37℃ for 40 min. After the digestion products were detected by 1% agarose gel electrophoresis, the target fragment was recovered from the gel.

[0056] Table 4 Enzyme digestion system (2) Ligation of vector and target gene Enzyme digestion and recovery GmRHA1 The gene fragment was ligated with the pCAMBIA1301 vector fragment to generate the overexpression vector pCAMBIA1301- GmRHA1 The connection system is shown in Table 5. Connection conditions: 22℃, 1h; 65℃, 10min.

[0057] Table 5 Connection System (3) Identification of positive recombinant clones 10 μL of the ligation product was transformed into 100 μL of *E. coli* DH5α competent cells. The transformed bacterial culture was plated on LB agar containing 50 mg / L Kan resistance, and positive single colonies were screened. After picking a single colony and shaking it, primers were used to... GmRHA1 -S and GmRHA1 -A was used for PCR verification. Plasmids were extracted from the PCR-verified positive bacterial culture and used... Bam HI and Psst I paired the recombinant plasmid pCAMBIA1301- GmRHA1 Double enzyme digestion was performed for verification. The enzyme digestion system was the same as in Table 4, and the enzyme digestion conditions were 37℃ for 40 min.

[0058] 4. RNAi vector pTRV2- GmRHA1 Construction (1) Selection of RNAi fragments The gene sequences of RING-H2 type E3 ubiquitin ligases from multiple species were compared and analyzed using NCBI (https: / / www.ncbi.nlm.nih.gov / ) and combined with the target gene. GmRHA1 Based on domain characteristics, sequence fragments with high conservation and GC content preferably between 45% and 55% were selected. Candidate fragments were then compared with corresponding genome databases using BLAST to exclude fragments that could match non-target coding sequences, preventing interference with other homologous genes. Finally, sequence fragments with high specificity and interference efficiency were selected as the target sequence. GmRHA1 RNAi fragments of genes.

[0059] (2) GmRHA1 Cloning of gene RNAi fragments Designed using Primer 5.0 GmRHA1 Gene RNAi fragment-specific primers (Table 6), with corresponding restriction enzyme sites added to the 5' end of the primers and upstream of the primers. Xba I restriction site, downstream addition Bam HI restriction site. Using pMD19T- GmRHA1 Using plasmids as templates, RNAi- GmRHA1 -S, RNAi- GmRHA1 -A represents the primer pair. GmRHA1 The gene RNAi fragment was amplified by PCR. The PCR amplification system is shown in Table 7, and the amplification conditions are shown in Table 8. The amplification products were detected by 1% agarose gel electrophoresis and then recovered from the gel. The recovered gel products were ligated into a cloning vector to construct the recombinant vector pMD19T-RNAi- GmRHA1 The cells were transformed into E. coli DH5α competent cells, and positive recombinant clones were identified by PCR and sequenced for verification.

[0060] Table 6 GmRHA1 Gene RNAi fragment primers Note: The underlined part is... Xba I identifies the site; the double-underlined portion is... Bam HI recognition site.

[0061] Table 7 Reaction System Table 8 Reaction Conditions (3) Constructing the RNAi vector pTRV2- GmRHA1 pMD19T-RNAi- GmRHA1 With pTRV2 plasmid, via Xba I and BamAfter double digestion with HI and gel recovery of the target fragment, ligation was performed, followed by transformation into E. coli DH5α competent cells to construct the recombinant RNAi vector pTRV2- GmRHA1 The ligation product was plated on 50 mg / L Kan-resistant LB solid medium to screen for positive single colonies. After shaking, the colonies were verified by PCR and double enzyme digestion. The enzyme digestion system is shown in Table 9. The enzyme digestion conditions were 37℃ for 40 min.

[0062] Table 9 Enzyme digestion system 5. Genetic transformation of soybean hairy roots (1) Transformation and identification of Agrobacterium rhizogenes The overexpression vector pCAMBIA1301- was overexpressed using the freeze-thaw method. GmRHA1 and RNAi vector pTRV2- GmRHA1 The bacteria were transformed into Agrobacterium rhizogenes K599 competent cells. Single colonies on the Kan+Str double antibiotic medium were picked and inoculated into YEB liquid medium containing 50 mg / L Kan and 100 mg / L Str. After incubation at 28°C and 200 rpm with shaking, the bacterial culture was identified by PCR. The amplified products were detected by 1% agarose gel electrophoresis.

[0063] (2) Agrobacterium rhizogenes infects soybean explants Plump, disease-free soybean seeds of the Heinong 553 variety were selected and sown in vermiculite. Soybean seedlings that had been cultured for 7 days and whose cotyledons had not yet unfolded were selected, and explants were obtained through oblique root cutting. The explants were then treated as follows: soaked in a solution containing pCAMBIA1301- GmRHA1 Agrobacterium rhizogenes K599 bacterial culture (OD) 600 ≈0.7) 30 min, induction GmRHA1 Overexpression of hairy roots; mixing pTRV1- and pTRV2- GmRHA1 Agrobacterium rhizogenes K599 bacterial suspension (all OD) 600 ≈0.7) Mix at a volume ratio of 1:1, and immerse explants in the mixed bacterial solution for 30 min to induce induction. GmRHA1 RNAi hairy roots. Separately, explants were soaked in a vector-free Agrobacterium rhizogenes K599 bacterial solution for 30 min to induce hairy roots with empty bacterial cells, serving as a control (CK). All explants were cultured in the dark for 24 h after soaking, then transplanted into vermiculite, irrigated with 1 / 2 Hoagland nutrient solution, and cultured until hairy roots appeared.

[0064] (3) Identification of transgenic soybean hairy root complex plants qRT-PCR identification: Total RNA was extracted from the hairy roots of soybean hairy root complex plants overexpressing and containing RNAi, reverse transcribed into cDNA, and then detected by qRT-PCR. GmRHA1Gene expression levels. The internal reference gene for qRT-PCR is... GmActin11 (GenBank accession No. EMD46976). The qRT-PCR primer sequences are shown in Table 10, the qRT-PCR reaction system is shown in Table 11, and the qRT-PCR reaction procedure is shown in Table 12.

[0065] Table 10 qRT-PCR primer sequences Table 11 qRT-PCR reaction system Table 12 qRT-PCR reaction procedure GUS staining identification: Overexpression in normal culture and salt-alkali stress-treated cultures was identified separately. GmRHA1 Hairy roots of soybean complex plants were placed in GUS staining solution and stained overnight at 37°C in the dark, following the instructions of the GUS staining kit. After staining, the results were observed and recorded. Salt-alkali stress treatment involved overexpressing... GmbZIP71 Soybean hairy root complex plants were transplanted into a saline-alkali stress solution for 24 hours for stress treatment.

[0066] Salt-alkali stress solution: contains neutral and alkaline salts (the molar ratio of NaCl: Na₂SO₄: NaHCO₃: Na₂CO₃ is 9:1:9:1, Na… + 1 / 2 Hoagland nutrient solution (110 mmol / L, pH=8.42).

[0067] The concentrations of the following compounds are present: Ca(NO3)2·4H2O: 472.5 mg / L, KNO3: 303.5 mg / L, MgSO4·7H2O: 246.5 mg / L, KH2PO4: 68 mg / L, Fe-EDTA: 36.7 mg / L, H3BO3: 6.2 mg / L, MnSO4·H2O: 22.3 mg / L, ZnSO4·7H2O: 8.6 mg / L, CuSO4·5H2O: 0.025 mg / L, Na2MoO4·2H2O: 0.25 mg / L, KI: 0.83 mg / L, CoCl2·6H2O: 0.025 mg / L. The solvent is deionized water.

[0068] II. Experimental Results 1. Soybeans GmRHA1 Construction of gene vectors (1) Overexpression vector pCAMBIA1301- GmRHA1 Construction like Figure 2 As shown, using Bam HI and Psst The recombinant vector was identified by double enzyme digestion, and agarose gel electrophoresis showed two specific bands. The 577bp band was the target band, indicating successful vector construction and the acquisition of the overexpression vector pCAMBIA1301-. GmRHA1 .

[0069] (2) RNAi vector pTRV2- GmRHA1 Construction pMD19T- GmRHA1 Using the plasmid as a template, PCR amplification yielded a 206 bp sample. GmRHA1 Gene RNAi fragments ( Figure 3 ).use Xba I and Bam HI was used to identify the recombinant vector by double enzyme digestion. Figure 4 Electrophoresis showed two specific bands, with the 206 bp band being the target band, indicating successful vector construction and the acquisition of the RNAi vector pTRV2-. GmRHA1 .

[0070] 2. Identification of Agrobacterium rhizogenes K599 transformed by recombinant plant expression vector The overexpression vector pCAMBIA1301- was overexpressed using the freeze-thaw method. GmRHA1 With RNAi vector pTRV2- GmRHA1 The cells were transformed into *Agrobacterium rhizogenes* K599 competent cells and identified by PCR. Electrophoresis results are shown below. Figure 5 As shown, amplification was performed to obtain... GmRHA1 Gene-specific bands and GmRHA1 RNAi fragment bands were observed. The results indicate that both vectors were successfully transferred into Agrobacterium rhizogenes K599.

[0071] 3. Obtaining and identifying transgenic soybean hairy root complex plants (1) Obtaining transgenic soybean hairy root complex plants Using the overexpression vector pCAMBIA1301- GmRHA1 RNAi vector pTRV2- GmRHA1 Soybean explants were infected with Agrobacterium rhizogenes K599 culture without the carrier to obtain soybean hairy root complex plants. The culture process is as follows: Figure 6 As shown.

[0072] (2) Identification of transgenic soybean hairy root complex plants qRT-PCR identification: Results are as follows Figure 7 As shown, transfer pCAMBIA1301- GmRHA1Soybean complex plants overexpressing the vector ( GmRHA1 -OE1、 GmRHA1 -OE2、 GmRHA1 In -OE3), GmRHA1 The gene expression level was significantly higher than that of the K599 empty bacterial control (CK) (P<0.01), with an average expression level 8.3 times that of the control; the pTRV2- gene expression level was significantly higher than that of the control. GmRHA1 Plants with RNA interference vectors ( GmRHA1 -RNAi1, GmRHA1 -RNAi2, GmRHA1 In -RNAi3), GmRHA1 The gene expression level was significantly lower than that of the K599 empty bacterial control (CK) (P<0.01), with an average expression level of 1 / 5 of the control. This result indicates that the present invention has successfully obtained… GmRHA1 Soybean hairy root complex plants overexpressing and RNAi-silencing.

[0073] GUS staining: The transfected vector pCAMBIA1301- GmRHA1 soybean complex plants GmRHA1 -OE roots were cultured under normal conditions and subjected to salt-alkali stress, respectively. Hairy roots from different treatments were then stained with GUS. Results are as follows: Figure 8 As shown, a distinct blue signal appeared in the hairy roots under both culture conditions. These results indicate that positive transgenic soybean hairy root complex plants have been successfully obtained.

[0074] Example 3: Soybeans GmRHA1 Functional identification of genes.

[0075] I. Experimental Materials and Methods 1. Experimental materials change GmRHA1 Overexpression vector pCAMBIA1301- GmRHA1 Soybean hairy root complex plant ( GmRHA1 -OE), transRNA interference vector pTRV2- GmRHA1 Soybean hairy root complex plant ( GmRHA1 -RNAi) and soybean hairy root complex control plants CK obtained by infection with Agrobacterium rhizogenes K599 without vector (i.e. control plants CK transfected with empty K599 bacteria).

[0076] 2. Abiotic stress on transgenic soybean hairy root complex plants Will GmRHA1 overexpression of soybean complex plants ( GmRHA1 -OE), RNA interference GmRHA1 Genetic soybean complex plants ( GmRHA1The control plants (CK) and the RNAi-transformed K599 empty bacteria were cultured normally until the third pair of trifoliate leaves emerged. They were then transferred to 1 / 2 Hoagland nutrient solution and cultured for 2 days. The three plants were then subjected to salt-alkali stress solution for 24 hours, during which the phenotypic changes of the plants were observed and recorded.

[0077] Salt-alkali stress solution: contains neutral and alkaline salts (the molar ratio of NaCl: Na₂SO₄: NaHCO₃: Na₂CO₃ is 9:1:9:1, Na… + 1 / 2 Hoagland nutrient solution (110 mmol / L, pH=8.42).

[0078] 3. Determination of physiological indicators of transgenic soybean hairy root complex plants Collect samples after 12 hours and 24 hours of salt-alkali stress treatment GmRHA1 -OE、 GmRHA1 Leaves and hairy roots of soybean hairy root complex plants containing RNAi and CK were used to determine various physiological indicators.

[0079] Superoxide dismutase (SOD) activity was determined according to the method described in the following literature: Shen Xiangjuan. Soybean GmCBL7 Gene Cloning and Functional Analysis [D]. Harbin: Master's Thesis, Harbin Normal University. 2020. Catalase (CAT) activity, malondialdehyde (MDA) content, free proline content, and chlorophyll content were determined according to the methods described in the following literature: Zhai Jiayue. Soybeans GmWNK10 Cloning and Functional Study of Genes [D]. Harbin: Master's Thesis, Harbin Normal University. 2024. Soluble sugar content was determined according to the method described in the following literature: Chen Nan. Soybeans GmALMT33 Gene Cloning and Functional Analysis [D]. Harbin: Master's Thesis, Harbin Normal University. 2022. II. Experimental Results 1. Phenotypic analysis of salt and alkali tolerance in transgenic soybean hairy root complex plants soybean hairy root complex plants GmRHA1 -OE、 GmRHA1 -RNAi and K599 empty bacteria control plants (CK) were subjected to salt-alkali stress treatment, and the results were as follows: Figure 9 As shown. Before the stress (0h), all three plants grew well, with lush, green, and upright leaves. After 12h of salt-alkali stress, the leaves of the CK plant wilted and drooped slightly, with the wilting of the basal leaves being particularly noticeable, and the leaf margins showing wrinkling and drying. GmRHA1 RNAi plants exhibited significant wilting, drooping, and chlorosis, with particularly pronounced wrinkling and curling of the basal leaves; while GmRHA1-OE plants only had slightly drooping leaves, and the whole plant maintained good growth. After 24 hours of salt and alkali stress, the basal leaves of CK plants were completely shriveled and wrinkled, and the edges of the second to last pair of leaves also became wrinkled and curled. GmRHA1 -The RNAi plant was visibly dried out; GmRHA1 -OE plants only showed a slight lightening of leaf color, with some basal leaves becoming shriveled and wrinkled; overall, they maintained good growth. These results indicate that overexpression... GmRHA1 The gene can significantly improve the soybean's tolerance to salt and alkali stress.

[0080] 2. Determination of physiological indicators of transgenic soybean hairy root complex plants (1) Assay of antioxidant enzyme activity SOD activity assay: Under normal culture conditions, GmRHA1 The SOD activity of the hairy roots of the -OE soybean complex plants was significantly higher than that of the empty bacterial control plant CK (P<0.01), increasing by 4.99% compared to CK; GmRHA1 The SOD activity of the hairy roots of RNAi plants was significantly lower than that of CK (P<0.01), decreasing by 8.77% compared to CK.

[0081] The results are as follows Figure 10 As shown in A, after 12 hours and 24 hours of salt-alkali stress, GmRHA1 The SOD activity of the hairy roots of -OE plants was significantly higher than that of CK (P<0.01), increasing by 52.98% and 15.9% respectively compared to CK. GmRHA1 The SOD activity of the hairy roots of RNAi plants was significantly lower than that of CK (P<0.01), decreasing by 4.06% and 15% respectively compared to CK.

[0082] CAT activity assay: Under normal culture conditions, GmRHA1 -OE、 GmRHA1 There was no significant difference in CAT activity in the hairy roots of the three plants: RNAi, empty bacterial control (CK), and control group.

[0083] The results are as follows Figure 10 As shown in B, after 12 hours of salt-alkali stress, GmRHA1 The CAT activity in the hairy roots of the -OE soybean complex plants was significantly higher than that in the control plant CK (P<0.01), increasing by 80.41% compared to CK; GmRHA1 The CAT activity in the hairy roots of RNAi plants was lower than that of the control (CK) by 13.99%, but this was not statistically significant. After 24 hours of salt-alkali stress, GmRHA1 The CAT activity in the hairy roots of -OE plants was significantly higher than that of CK (P<0.01), increasing by 26.48% compared to CK; GmRHA1The CAT activity in the hairy roots of RNAi plants was significantly lower than that of CK (P<0.05), decreasing by 30.08% compared to CK.

[0084] Overall, after salt-alkali stress treatment, the activities of SOD and CAT, two antioxidant enzymes, in all three plants showed an increasing trend, among which... GmRHA1 The enzyme activities of -OE plants were all higher than those of the control CK. GmRHA1 The enzyme activities of RNAi plants were all lower than those of the control (CK). These results indicate that upregulation... GmRHA1 Gene expression can significantly improve the antioxidant capacity of soybean plants, thereby effectively reducing oxidative damage caused by stress.

[0085] (2) Determination of malondialdehyde (MDA) content Under normal culture conditions, GmRHA1 The MDA content in the hairy roots of the -OE soybean complex plants was significantly lower than that in the empty bacterial control plant CK (P<0.05), decreasing by 7.67% compared to CK; GmRHA1 The MDA content in the hairy roots of RNAi plants was significantly higher than that of CK (P<0.01), increasing by 11.18% compared to CK.

[0086] like Figure 11 As shown, after 12 hours and 24 hours of salt-alkali stress, GmRHA1 The MDA content in the hairy roots of the -OE soybean complex plants was significantly lower than that in the control (CK) (P<0.01), decreasing by 13.86% and 13.17% respectively compared to the CK. GmRHA1 The MDA content in the hairy roots of RNAi plants was significantly higher than that of CK (P<0.01), increasing by 33.37% and 20.78% respectively.

[0087] Overall, the MDA content of all three plants showed an increasing trend after salt-alkali stress treatment; among them GmRHA1 The MDA content of -OE plants was significantly lower than that of the control CK. GmRHA1 The MDA content in RNAi plants was significantly higher than that in the control (CK). These results indicate that upregulation... GmRHA1 Gene expression can significantly reduce the accumulation level of MDA in soybean plants, thereby mitigating oxidative stress damage caused by stress.

[0088] (3) Determination of proline content Under normal culture conditions, GmRHA1 -OE、 GmRHA1 There was no significant difference in proline content among the three plants: RNAi, empty bacterial control (CK), and control group.

[0089] The results are as follows Figure 12 As shown, after 12 hours of salt-alkali stress, GmRHA1The proline content in the hairy roots of -OE plants was significantly higher than that of the control plant CK (P<0.01), increasing by 45.11% compared to CK; GmRHA1 The proline content in the hairy roots of RNAi plants was lower than that of the control plant (CK), decreasing by 11.91%, but this was not statistically significant. After 24 hours of salt-alkali stress, GmRHA1 The proline content in the hairy roots of -OE plants was significantly higher than that of the control plant CK (P<0.01), increasing by 41.92% compared to CK; GmRHA1 The proline content in the hairy roots of RNAi plants was significantly lower than that in the control plant CK (P<0.05), decreasing by 13.75% compared to CK.

[0090] Overall, after salt-alkali stress treatment, the proline content of all three plants showed a continuous increasing trend; among them GmRHA1 -OE plants had higher proline content than the control CK. GmRHA1 - The proline content in RNAi plants was lower than that in the control (CK). These results indicate that upregulation... GmRHA1 Gene expression can increase proline accumulation in soybean plants, thereby enhancing the plant's osmotic regulation capacity and improving its tolerance to salt and alkali stress.

[0091] (4) Determination of soluble sugar content Under normal culture conditions, GmRHA1 -OE、 GmRHA1 There was no significant difference in the soluble sugar content among the three plants: RNAi, empty bacterial control (CK), and control group.

[0092] like Figure 13 As shown, after 12 hours and 24 hours of salt-alkali stress, GmRHA1 The soluble sugar content of the hairy roots of OE plants was significantly higher than that of the control plant CK (P<0.01), increasing by 59.04% and 60.26% respectively compared to CK. GmRHA1 The soluble sugar content of the hairy roots of RNAi plants was significantly lower than that of the control plant CK (P<0.01), decreasing by 12.43% and 20.23% respectively compared with CK.

[0093] Overall, the soluble sugar content of all three plants continued to increase after salt-alkali stress treatment; among them GmRHA1 The soluble sugar content of -OE plants was significantly higher than that of CK plants. GmRHA1 The soluble sugar content of RNAi plants was significantly lower than that of the control group (CK). These results indicate that upregulation... GmRHA1 Gene expression can significantly increase the accumulation of soluble sugars in soybean plants, thereby enhancing the plant's tolerance to saline-alkali stress.

[0094] (5) Determination of chlorophyll content Under normal culture conditions,GmRHA1 The chlorophyll content in the leaves of the -OE soybean complex plants was significantly lower than that in the empty bacterial control plant CK (P<0.01), decreasing by 9.67% compared to CK; GmRHA1 The chlorophyll content in the leaves of RNAi plants was significantly lower than that of the control (P<0.05), decreasing by 6.45% compared to the control.

[0095] like Figure 14 As shown, after 12 hours and 24 hours of salt-alkali stress, GmRHA1 The chlorophyll content of leaves in -OE plants was significantly higher than that in CK (P<0.01), increasing by 9.18% and 18.75% respectively compared to CK. GmRHA1 The chlorophyll content of the leaves of RNAi plants was significantly lower than that of CK (P<0.01), decreasing by 18.35% and 17.93% respectively.

[0096] Overall, after salt-alkali stress treatment, the chlorophyll content of all three plants showed a continuous decreasing trend, among which... GmRHA1 -OE plants had significantly higher chlorophyll content than the empty bacteria control (CK). GmRHA1 -The chlorophyll content of RNAi plants was significantly lower than that of the control (CK). These results indicate that upregulation... GmRHA1 Gene expression can effectively alleviate the damage to the photosynthetic system caused by abiotic stress, enabling plants to better maintain photosynthesis.

[0097] In conclusion, GmRHA1 The gene can positively regulate the tolerance of soybean to salt and alkali stress. This gene can alleviate the damage of stress to plants by increasing the activity of antioxidant enzymes, accumulating osmotic regulators, maintaining chlorophyll content, and reducing the level of membrane lipid peroxidation product MDA, thereby enhancing the stress resistance of soybeans.

[0098] Although preferred embodiments of the invention have been described, those skilled in the art, once they have learned the basic inventive concept, can make other changes and modifications to these embodiments.

[0099] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. Overexpression GmRHA1 The application of gene-based products in regulating soybean salt and alkali tolerance is characterized by, The GmRHA1 The CDS sequence of the gene is shown in SEQ ID NO.

1.

2. The overexpression according to claim 1 GmRHA1 The application of gene-based products in regulating the salt and alkali tolerance of soybeans is characterized by, overexpression GmRHA1 Gene's products are: GmRHA1 Gene overexpression vectors or overexpression GmRHA1 Recombinant bacteria.

3. The overexpression according to claim 2 GmRHA1 The application of gene-based products in regulating the salt and alkali tolerance of soybeans is characterized by, The GmRHA1 The steps for constructing a gene overexpression vector are as follows: Cloning soybeans GmRHA1 Genes, soybeans GmRHA1 Genes linked to the pCAMBIA1301 vector Kpn I and Bam HI site, obtained GmRHA1 Gene overexpression vector.

4. The overexpression according to claim 2 GmRHA1 The application of gene-based products in regulating the salt and alkali tolerance of soybeans is characterized by, The overexpression GmRHA1 The recombinant bacteria for gene preparation are as follows: GmRHA1 Gene overexpression vector was transferred into Agrobacterium rhizogenes competent cells to obtain overexpression. GmRHA1 Recombinant bacteria.

5. The overexpression according to claim 4 GmRHA1 The application of gene-based products in regulating the salt and alkali tolerance of soybeans is characterized by, Using overexpression GmRHA1 Recombinant bacteria can enhance the salt and alkali tolerance of soybeans.

6. The overexpression according to claim 5 GmRHA1 The application of gene-based products in regulating the salt and alkali tolerance of soybeans is characterized by, The transformation of soybeans using Agrobacterium infection involves: using Agrobacterium infection to transform soybeans overexpressing... GmRHA1 Recombinant bacteria infected soybean explants to obtain transgenic soybean hairy root complex plants resistant to salt and alkali stress.

7. The overexpression according to claim 6 GmRHA1 The application of gene-based products in regulating the salt and alkali tolerance of soybeans is characterized by, The Agrobacterium is K599.

8. The overexpression according to any one of claims 1 to 7 GmRHA1 The application of gene-based products in regulating soybean salt and alkali tolerance is characterized by, The overexpression GmRHA1 Gene's products are used to alleviate the damage to soybean plants caused by salt and alkali stress.

9. The overexpression according to claim 8 GmRHA1 The application of gene-based products in regulating the salt and alkali tolerance of soybeans is characterized by, The overexpression GmRHA1 Gene products are used to increase chlorophyll content.

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