Application of soybean alkaline alpha-galactosidase gene GmAGA1 in regulating plant salt tolerance

Abstract

The application discloses a soybean alkaline alpha-galactosidase gene GmAGA1 The application belongs to the technical field of plant genetic engineering and crop breeding and relates to an application in regulating plant salt tolerance. GmAGA1 The gene is a gene for negatively regulating plant salt tolerance, and by inhibiting the expression of the gene or knocking out the gene, the peroxidase and superoxide dismutase activities of plants under salt stress can be improved, the chlorophyll content and the maximum photochemical quantum efficiency of photosystem II can be maintained, and thus the salt tolerance of plants can be significantly enhanced. GmAGA1 The application further provides a breeding method for salt-tolerant soybean strains, in which the CRISPR / Cas9 gene editing technology is used to knock out the gene, and a salt-tolerant soybean strain with stable heredity is screened.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering and crop breeding technology, specifically relating to the soybean alkaline α-galactosidase gene. GmAGA1 Application in regulating plant salt tolerance. Background Technology

[0002] Soybeans Glycine max (L.) Merr.) is an important oilseed and cash crop in my country. Its grains are rich in protein, fat, vitamins, and various bioactive substances, playing an irreplaceable role in ensuring food security and agricultural economic development. However, soybeans are quite sensitive to salt stress, and excessively high Na+ levels in the soil can exacerbate the problem. + Concentration can trigger osmotic stress and ion toxicity, disrupt intracellular ion homeostasis, induce oxidative stress, damage the photosynthetic system, and ultimately inhibit crop growth, leading to a significant decrease in yield.

[0003] In the regulatory network of plant responses to salt stress, sugar metabolism and its derived osmotic regulators play a central role. Raffinose family oligosaccharides (RFOs) are important compatible solutes in plants, acting as reactive oxygen species scavengers and membrane protectants, maintaining cell stability and mitigating stress damage during stress responses. Alkaline α-galactosidase is the rate-limiting enzyme in the RFO degradation pathway. By hydrolyzing the α-1,6-galactosidic bond at the terminal end of RFOs, it regulates the accumulation level of RFOs in plants, thereby affecting the plant's ability to adapt to stress.

[0004] Existing research indicates that salt stress can induce the expression of the raffinose synthase (GolS) gene in multiple species, promoting RFO synthesis and thus enhancing plant salt tolerance; α-galactosidase-mediated RFO hydrolysis also participates in plant salt stress responses, such as in grapes ( Vitis vinifera α-galactosidase in ) VvSIP Heterologous overexpression can enhance the salt tolerance of tobacco (Ben-Amar A, Daldoul S, Allel D, et al. Ectopic expression of a grapevine alkalinealpha-galactosidase seed imbibition protein VvSIP enhanced salinity tolerancein transgenic tobacco plants[J]. Funct Integr Genomics, 2022, 23(1): 12.). However, currently, there is little information available regarding the alkaline α-galactosidase gene ( AGAThe specific biological functions of [the plant] in the salt stress response of plant nutrient tissues still lack systematic research, especially in soybeans. AGA The link between the salt tolerance regulation mechanism of the gene and the metabolism and oxidative stress pathways of RFOs is not yet clear, and no soybean alkaline α-galactosidase gene has been found. GmAGA1 Reports on negative regulation of salt stress tolerance and its application in salt-tolerant breeding.

[0005] Therefore, it is necessary to explore the mechanisms by which soybeans regulate salt tolerance. AGA Defining the function and application value of genes and developing salt-tolerant soybean breeding technology have become urgent technical problems to be solved. Summary of the Invention

[0006] Technical problem solved: To address the above-mentioned technical problem, this invention provides a soybean alkaline α-galactosidase gene. GmAGA1 Its application in regulating plant salt tolerance, clearly defined GmAGA1 The gene's negative regulatory function can effectively improve plant salt tolerance by inhibiting or knocking out the gene; at the same time, it provides methods for breeding salt-tolerant soybean lines and provides new gene resources and technical solutions for salt-tolerant soybean breeding.

[0007] Technical solution: In a first aspect, the present invention provides a soybean alkaline α-galactosidase gene. GmAGA1 Application in regulating plant salt tolerance, the gene GmAGA1 It is a gene that negatively regulates plant salt tolerance, by repressing genes. GmAGA1 Gene expression or knockout GmAGA1 This improves the salt tolerance of plants.

[0008] Preferably, the gene GmAGA1 The nucleotide sequence is shown in SEQ ID NO.1: SEQ ID NO.1:

[0009] Preferably, the plant is soybean.

[0010] Secondly, the present invention provides a method for cultivating salt-tolerant soybean lines, comprising the following steps: S1. Using CRISPR / Cas9 gene editing technology, obtain the deletion... GmAGA1 Gene function gmaga1 Mutants; S2, to gmaga1 The mutants were screened and purified to obtain stably inherited mutants. gmaga1 Mutant strains; S3, for stable inheritance gmaga1 The mutant lines were tested for salt stress tolerance, and soybean lines with significantly higher salt tolerance than wild-type soybeans were selected as salt-tolerant soybean lines.

[0011] Preferably, the salt stress verification is performed by treating the plant with Hogland's nutrient solution containing 150 mmol / L NaCl for 3 to 7 days, and the salt tolerance is verified by observing the salt damage phenotype of the plant and measuring physiological indicators.

[0012] Preferably, the salt-tolerant soybean line includes gmaga1 The nucleotide sequence of the mutant is shown in SEQ ID NO.2.

[0013] Thirdly, the present invention provides a salt-tolerant soybean strain obtained by the cultivation method described in the second aspect, wherein the strain contains... GmAGA1 It has a gene function deficiency and its salt tolerance is significantly higher than that of wild-type soybeans.

[0014] Beneficial effects: 1) This invention is the first to clearly reveal the soybean alkaline α-galactosidase gene. GmAGA1 The study confirmed the gene's negative regulation of plant salt tolerance, filling a gap in the understanding of soybean salt tolerance. AGA The gap in research on salt tolerance function of genes has been filled, enriching the theoretical knowledge of plant salt tolerance regulatory networks and providing a new research direction for in-depth analysis of the molecular mechanisms of soybean salt stress response; 2) This invention inhibits or knocks out GmAGA1 The gene can selectively enhance the activity of peroxidase (POD) and superoxide dismutase (SOD) in plants, effectively remove reactive oxygen species produced under salt stress, and maintain high chlorophyll content and photosynthetic efficiency in plants, thereby significantly improving plant salt tolerance. Its regulatory mechanism is clear and its improvement effect is stable and reliable. 3) The salt-tolerant soybean breeding method provided by this invention adopts CRISPR / Cas9 gene editing technology, which has the advantages of precise targeting, high editing efficiency and simple operation. It can quickly breed stable genetic salt-tolerant soybean lines, which are highly practical and easy to promote. Attached Figure Description

[0015] Picture 1 This is an embodiment of the present invention. gmaga1 After being treated with salt stress for 0, 3, 12, and 24 h, mutant seeds were... GmAGA1 Image showing the results of gene expression level detection; Picture 2 These are different in the embodiments of the present invention. GmAGA1 A diagram showing the types of mutations and amino acid changes observed in gene knockout lines; Picture 3 In this embodiment of the invention, after 3 days of 150mM NaCl stress treatment, the wild type and... gmaga1 A schematic diagram of salt damage symptoms in mutant plants; Picture 4 This is an embodiment of the present invention. gmaga1 Figure 1 shows the results of chlorophyll-related index measurements between mutant and wild-type plants. Figure A shows the results of measurements using a handheld chlorophyll meter for wild-type (WT) and wild-type plants. gmaga1 Results of chlorophyll content in mutant plants; Figure B shows the results of measurements of wild-type (WT) and... gmaga1 Fv / Fm values ​​(reflecting photosynthetic efficiency) of mutant seedling leaves after 3 days of stress. Picture 5 This is an embodiment of the present invention. gmaga1 Graph showing the chlorophyll fluorescence intensity results of mutant and wild-type plants; Picture 6 This is an embodiment of the present invention. gmaga1 Figure showing the results of antioxidant enzyme activity assays in mutant and wild-type plants. Detailed Implementation

[0016] The present invention will be described in detail below with reference to specific embodiments: Example 1: GmAGA1 Gene knockout and salt tolerance verification The soybean variety tested was “Tianlong 1” (TL1), as a wild type (WT). gmaga1 The mutants were constructed using CRISPR / Cas9 gene editing technology, with T0 generation seeds as the starting material. Stable mutant lines were obtained through multiple generations of screening and purification.

[0017] 1.1 Salt stress treatment Wild-type seeds with plump, uniform size were selected and soaked overnight in water in a beaker to allow them to fully absorb water and swell. The soaked seeds were then densely planted in seedling trays for hydroponics. After germination and growth under darkness for 7 days, they were transferred to 1 / 2 MS nutrient solution and placed in an artificial climate incubator with a light-dark ratio of 16 h:8 h, a temperature of 25℃, and a relative humidity of 70%. When the plants developed their third trifoliate compound leaf, the seedlings were transferred to 1 / 2 MS nutrient solution containing 150 mmol / L NaCl for salt stress treatment. Plants without salt stress served as a blank control. The first trifoliate compound leaf was collected at 0, 3, 12, and 24 h of treatment. Each treatment was performed with 5 replicates and 3 biological replicates. Samples were flash-frozen in liquid nitrogen, rapidly ground, and stored at -80℃ for later use. GmAGA1 Gene expression level detection.

[0018] The results are as follows Picture 1 As shown: After salt stress treatment, wild-type soybeans... GmAGA1 Gene expression levels gradually increased with prolonged treatment time, reaching a peak after 24 h of NaCl treatment, which was 4.2 times higher than that of untreated (0 h) gene expression. GmAGA1 The gene expression was induced by salt stress, further suggesting its involvement in the soybean salt stress response process.

[0019] 1.2 gmaga1 Acquisition of mutants Using CRISPR / Cas9 gene editing technology, targeted editing GmAGA1 Gene (nucleotide sequence as shown in SEQ ID NO. 1), yielding three different deletion types. gmaga1 The mutants, namely those with deletions of 12bp, 9bp, and 6bp, have nucleotide sequences shown in SEQ ID NO.2 to SEQ ID NO.4: SEQ ID NO.2 ( gmaga1-12 ):

[0020] SEQ ID NO.3( gmaga1-9 ):

[0021] SEQ ID NO.4( gmaga1-6 ):

[0022] gmaga1-12 , gmaga1 -9 and gmaga1 The mutation types and amino acid changes observed in the -6 knockout line are as follows: Picture 2 As shown. Constructing a target GmAGA1 The CRISPR / Cas9 gene editing vector was used to genetically transform "Tianlong No. 1" soybean to obtain the T0 generation. gmaga1 Mutant seed.

[0023] 1.3 Screening and purification of mutants T0 generation gmaga1 The mutant seeds were sown and planted, and the mutants were screened out by PCR amplification combined with sequencing verification. GmAGA1 Homozygous gene knockout mutants were generated; these homozygous mutants were continuously seeded for three generations, with the stability of the mutation site verified by sequencing in each generation, ultimately yielding a stably inherited mutant. gmaga1 mutant lines gmaga1-12 .

[0024] 1.4 Salt Tolerance Verification 1.4.1 Seed surface disinfection (1) Spread the soybean seeds evenly in a 90mm×15mm sterile petri dish and open the lid for later use; (2) In the vacuum desiccator inside the fume hood, add 200 mL of 100% sodium hypochlorite solution to a 500 mL glass beaker; (3) Use a pipette to slowly add 5 mL of concentrated hydrochloric acid (HCl) drop by drop into the beaker. Vigorous bubbles will be generated when adding it, so handle with care. (4) Apply Vaseline to the interface of the vacuum dryer to seal it, and let it stand in the fume hood for 18-24 hours to fumigate and sterilize the seeds; (5) After sterilization, remove the petri dish under aseptic conditions, place it in a fume hood and blow for 2-3 hours to remove residual sterilization reagents, cover the dish and seal it with sealing film for later use.

[0025] 1.4.2 Plant Culture and Salt Stress Treatment Wild-type after surface disinfection and gmaga1 The mutant seeds were sown in nutrient pots containing a mixture of vermiculite and potting soil (3:1 volume ratio) and cultured for 14 days. The plants were then carefully removed, rinsed with deionized water, and transplanted into Hoagland solution for acclimatization for 2 days. They were then transferred to Hoagland solution containing 150 mmol / L NaCl for salt stress treatment. Plant growth was observed and recorded on days 0 and 3 of treatment. Each treatment was performed in triplicate, with 10 plants per replicate.

[0026] 1.4.3 Measurement of physiological indicators Chlorophyll content and chlorophyll fluorescence parameters were measured: The SPAD value of leaves was measured using a SPAD-502 chlorophyll meter (Konica Minolta, Japan), indirectly reflecting the relative chlorophyll content. A handheld plant chlorophyll fluorescence meter (GreenVision Technology (Wuxi) Co., Ltd.) was used. After leaves were placed in darkness for 30 minutes to acclimate, Fv (maximum photochemical efficiency) and Fm (maximum fluorescence value) were measured, and the Fv / Fm ratio was calculated. All measurements were performed between 14:40 and 17:30. Each biological replicate included five individual plants, for a total of three biological replicates.

[0027] Antioxidant enzyme activity assay: After 3 days of salt stress treatment, plant leaves (preferably young leaves) were collected, frozen in liquid nitrogen, and stored at -80℃. The activities of peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT) were measured according to the kit instructions of Beijing Solarbio Science & Technology Co., Ltd., with three biological replicates.

[0028] 1.4.4 Data Analysis Data were compiled using Microsoft Excel. Experimental results are expressed as mean ± standard deviation. The significance of differences was tested using t-test or one-way ANOVA, and P < 0.05 was considered significant.

[0029] Experimental results are as follows Picture 3-6 As shown: Under normal growth conditions, gmaga1 The mutant showed no significant difference in phenotype from the wild type, indicating that... GmAGA1 The gene deletion did not affect the normal growth and development of soybeans; after treatment with 150 mmol / L NaCl for 3 days, wild-type soybean plants showed obvious symptoms of salt damage, including yellowing and wilting of leaves. gmaga1 The mutants showed significantly milder salt damage symptoms and exhibited markedly better growth than the wild type.

[0030] Under salt stress, gmaga1 The SPAD and Fv / Fm values ​​of the mutant were significantly higher than those of the wild type, indicating that it had less chlorophyll degradation and less inhibition of photosynthesis; gmaga1 The mutant's POD activity was 2.84 times that of the wild type, and its SOD activity was 2.11 times that of the wild type, while its CAT activity showed no significant difference from the wild type, indicating that... gmaga1 Mutants can enhance the activity of POD and SOD, effectively scavenge reactive oxygen species generated under salt stress, reduce oxidative damage, and thus improve the salt tolerance of soybeans.

[0031] The above results confirm that GmAGA1 This gene negatively regulates soybean salt tolerance; knocking out this gene can significantly improve soybean salt tolerance.

[0032] Example 2: Cultivation of salt-tolerant soybean lines (1) Select plump, uniform, and disease-free fruits and vegetables. gmaga1 homozygous mutant ( gmaga1-12 Soak soybean seeds overnight, then spread them evenly on moist sterile filter paper and place them under short-day conditions to promote germination. (2) After the seeds germinate and grow two true leaves, transplant the seedlings into flower pots containing a mixture of vermiculite and nutrient soil (volume ratio 3:1). Sow an equal amount in each pot to ensure that the seedlings grow in a consistent environment. (3) Place the flowerpots in an artificial climate incubator and set the following conditions: temperature 25℃, photoperiod 14 h light / 10 h dark, relative humidity 60%~70%; (4) Water regularly during the growing season to keep the substrate moist and avoid drought or waterlogging; water with Hoagland nutrient solution every 7 days to ensure sufficient nutrients for plant growth; (5) Thin the seedlings in a timely manner according to the growth status of the plants, and keep 3 to 4 seedlings with the same growth in each pot to reduce competition between plants. (6) After the seedlings have grown in soil for 14 days, carefully remove the plants, rinse the roots with deionized water to remove any remaining substrate, and then transfer them into Hogland nutrient solution for 2 days to allow them to adapt to the hydroponic environment. (7) After the seedlings recovered from transplant shock, they were transferred to Hoagland's solution containing 150 mM NaCl for salt stress treatment. The plant growth was observed and recorded on day 0 and day 3 of the treatment. The lines with good growth and mild salt damage symptoms were selected as stable salt-tolerant soybean lines. Three biological replicates were set up for each treatment to ensure the reliability of the screening results.

[0033] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Soybean alkaline α-galactosidase gene GmAGA1 Its application in regulating plant salt tolerance is characterized by: The gene GmAGA1 It is a gene that negatively regulates plant salt tolerance, by repressing genes. GmAGA1 Gene expression or knockout GmAGA1 This improves the salt tolerance of plants.

2. The application according to claim 1, characterized in that: The gene GmAGA1 The nucleotide sequence is shown in SEQ ID NO.

1.

3. The application according to claim 1, characterized in that: The plant in question is soybean.

4. A method for cultivating a salt-tolerant soybean line, characterized in that, Includes the following steps: S1. Using CRISPR / Cas9 gene editing technology, obtain the deletion... GmAGA1 Gene function gmaga1 Mutants; S2, to gmaga1 The mutants were screened and purified to obtain stably inherited mutants. gmaga1 Mutant strains; S3, for stable inheritance gmaga1 The mutant lines were tested for salt stress tolerance, and soybean lines with significantly higher salt tolerance than wild-type soybeans were selected as salt-tolerant soybean lines.

5. The method for cultivating salt-tolerant soybean lines according to claim 4, characterized in that: The salt stress verification was performed using Hoagland nutrient solution with 150 mmol / L NaCl for 3–7 days.

6. The method for cultivating salt-tolerant soybean lines according to claim 4, characterized in that: The salt-tolerant soybean lines include gmaga1 The nucleotide sequence of the mutant is shown in SEQ ID NO.

2.

7. A salt-tolerant soybean strain, characterized in that: The strain was obtained by the breeding method according to any one of claims 4-6. GmAGA1 It has a gene function deficiency and its salt tolerance is significantly higher than that of wild-type soybeans.

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