Use of glyma03g.201100 gene in improving salt tolerance of soybean or yeast
Patent Information
- Application Number
- CN202510370822.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-27
- Publication Date
- 2026-09-04
- Estimated Expiration
- 2045-03-27
AI Technical Summary
而芽期作为受胁迫的敏感时期，容易影响植株的后期生长，从而使得产量严重减产，造成很大的经济损失
Smart Images
Figure CN120230784B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology, specifically relating to the application of the Glyma03G.201100 gene in improving the salt tolerance of soybeans or yeast. Background Technology
[0002] Soybeans, as an oilseed and cash crop, have become one of the most widely cultivated and utilized economic and food crops in the world, accounting for approximately 25% of the global edible oil market and two-thirds of the world's protein demand. Soil salinization, however, is a global environmental stressor that seriously threatens the sustainable development of crop production. Statistics show that approximately 7% of the world's land area, or about 9.5 million square kilometers, is affected by soil salinization. Unlike drought stress, salt stress begins in the germination stage of plants and continues until they age. The bud stage is a sensitive period under stress, easily affecting the later growth of the plant, leading to severe yield reductions and significant economic losses.
[0003] Currently, the food problem facing humanity is becoming increasingly serious. It is necessary to make full use of saline-alkali land as a reserve resource and use modern biotechnology to explore the salt tolerance mechanism of soybeans and screen its salt tolerance genes. This can provide theoretical guidance and gene resources for the genetic improvement of crop salt tolerance, which is of great significance in addressing global soil salinization. Summary of the Invention
[0004] The purpose of this invention is to provide a method for improving the salt tolerance of soybeans.
[0005] This invention provides an application of the Glyma03G.201100 gene in improving the salt tolerance of soybeans or yeast.
[0006] This invention provides the application of Glyma03G.201100 protein in improving the salt tolerance of soybeans or yeast.
[0007] This invention provides the application of a recombinant vector containing the Glyma03G.201100 gene in improving the salt tolerance of soybeans or yeast.
[0008] Further specifying, the starting vector of the recombinant vector is pYES2.
[0009] This invention provides the application of recombinant microbial cells containing the Glyma03G.201100 gene in improving the salt tolerance of soybeans or yeast.
[0010] Further specifying, the microbial cells are eukaryotic microbial cells or prokaryotic microbial cells.
[0011] Further specifying, the salt concentration is 70-100 mg / mL NaCl.
[0012] This invention provides a breeding method for improving the salt tolerance of soybeans, the steps of which are as follows: Step 1: Ligate the gene shown in SEQ ID NO.1 with the vector pHair to obtain the recombinant vector; Step 2: Transform the recombinant vector described in Step 1 into Agrobacterium to obtain recombinant Agrobacterium; Step 3: The recombinant Agrobacterium described in Step 2 is transferred into soybeans to obtain transgenic soybean plants, and positive transgenic soybean plants are obtained after identification.
[0013] This invention provides a method for improving the salt tolerance of yeast, the steps of which are as follows: Step 1: Ligate the gene shown in SEQ ID NO.1 with the vector pYES2 to obtain the recombinant vector; Step 2: Transform the recombinant vector described in Step 1 into yeast to obtain recombinant yeast.
[0014] Further specifying, the primers for amplifying the sequence shown in SEQ ID NO.1 are SEQ ID NO.3 and SEQ ID NO.4.
[0015] Beneficial effects: Overexpression of the Glyma03G.201100 gene in soybean yielded transgenic soybean hairy roots. These roots were then subjected to salt stress treatment with 70 mm sodium chloride solution for 5 days. Root and leaf development were observed. Significant differences in MDA content were observed in the roots; under salt stress, MDA content significantly increased, while the MDA content in the transgenic overexpressing plants was significantly lower than that in the empty vector plants. Figure 7 Compared with the control plants, there were significant differences in the activities of POD, SOD, and CAT in the roots. Under salt stress, the activities of POD, SOD, and CAT were significantly increased, and the activities of POD, SOD, and CAT in transgenic overexpression plants were significantly higher than those in empty vector plants. Figure 8-10 This indicates that the hairy roots of genetically modified soybeans have also developed salt tolerance.
[0016] The Glyma03G.201100 gene was overexpressed in yeast. The transgenic yeast showed a significant difference in growth compared to yeast with the empty vector in YPDA medium under salt stress. However, no difference in yeast growth was observed in normal YPDA medium. This indicates that the transgenic yeast has developed salt tolerance. Attached Figure Description
[0017] Figure 1 Construct PCR amplification products for the soybean Glyma03G.201100 expression vector; Figure 2This is a PCR product of yeast transformed with a soybean expression vector; Figure 3 The results are from the validation experiment of transgenic yeast Glyma03G.201100; where CK is normal culture medium and S is salt stress culture medium. Figure 4 The identification results of the recombinant vector for Glyma03G.201100; Figure 5 The overexpression of the stress-induced gene in transgenic soybean hairy roots of Glyma03G.201100 was verified by qPCR. Figure 6 Results of PCR product construction for overexpression vector of Glyma03G.201100 transgenic soybean hairy root validation experiment.
[0018] Figure 7 Image showing the MDA detection results of hairy roots of transgenic soybean Glyma03G.201100; Figure 8 Figure 1 shows the SOD enzyme activity detection results of hairy roots of transgenic soybean Glyma03G.201100. Figure 9 Figure 1 shows the results of POD enzyme activity detection in the hairy roots of transgenic soybean Glyma03G.201100. Figure 10 Figure 1 shows the CAT enzyme activity detection results of hairy roots of transgenic soybean Glyma03G.201100. Figure 11 The successful transfection of soybean hairy roots of Glyma03G.201100 was verified by qPCR, and the gene overexpression results were confirmed. Detailed Implementation
[0019] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. These embodiments are intended to facilitate a better understanding of the invention, but are not intended to limit the invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional experimental methods in the art. Unless otherwise specified, the pharmaceuticals, reagents, and materials used in the following embodiments can be purchased commercially.
[0020] Example 1. Construction of recombinant vector The nucleotide sequence of the Glyma03G.201100 gene (SEQ ID NO.1): ATGGCCGATAAGCAACCCCACTTGAATGGTGCTTATTACGGTCCCGCCATTCCCCCGGCGGAGCAACCACACTACCGCCCTAGCCGCGAAAGAAGCTGCTGTTGCTGCCTCTTCGGAATCTTGTGGAAGATTCTGGTTGCACTCATTGTCCTCGTTGGCCTCGCGGTCCTCATCTTCTGGCTGGTGGTTCAACCCCGTTCCTTCAAGTTCCACGTCACGAAAGCCAACCTAACACAATTTGATTACTATACCAACAACAACACCCTTCACTACAACATGGTCCTCAACTTCACTGCACGCAACCCCAACAAAAAACTCAGCATATACTACGACAAAGTAGAGGCATTAGCATTCTACGAGGATGTCAGGTTCGCCAATTACAGTGTGATAACGCACATGAACTCCTTCCGCCAGTACAAGAAGACCACCAGCCACATGAGCGCCGTTTTCTCGGGGCAGCAAGTGTTGCCGCTCGACAACGACCTAGTCTCAGAGTTGAACCAAGACAAGAGTGGTGGGGTGTACGAGATCGATGTGAAGCTCTACTTCAGGATTAGGTTCAGGCTCGGGGATGTCAAAACCCGTCGCTTCAAGCCCGAGGTCAAATGTGATATCAGGGTTCCCTTGAGGACCAATGGCAGCGTAACTTTGTTTCAGACCACCAAGTGTGATGTCGATTACTAG; Protein sequence: (SEQ ID NO. 2) MADKQPHLNGAYYGPAIPPAEQPHYRPSRERSCCCCLFGILWKILVALIVLVGLAVLIFWLVVQPRSFKFHVTKANLTQFDYYTNNNTLHYNMVLNFTARNPNKKLSIYYDKVEALAFYEDVRFANYSVITHMNSFRQYKKTTSHMSAVFSGQQVLPLDNDLVSELNQDKSGGVYEIDVKLYFRIRFRLGDVKTRRFKPEVKCDIRVPLRTNGSVTLFQTTKCDVDY.
[0021] 1. Molecular cloning of the Glyma03G.201100 gene Synthesis of the first strand of cDNA: Calculate the volume based on the RNA sample concentration, take 1 µg of total RNA as a template, and perform RT-PCR reverse transcription into cDNA according to the reverse transcription kit instructions.
[0022] 2. Cloning of the Glyma03G.201100 gene and recovery and purification of PCR products. Primer design: First, the CDS sequence was identified using Phytozome, and primers were designed using Snapgene and sent to Shanghai Sangon Biotech for synthesis. The primer sequences are shown in Table 1. Table 1 Primer sequences
[0023] 3. Amplification of the Glyma03G.201100 gene fragment and recovery and purification of PCR products The PCR amplification system is shown in Table 2: Table 2 PCR system
[0024] The PCR reaction program was as follows: 94℃ pre-deformation for 2 min; 98℃ deformation for 10 s, 55℃ annealing for 45 s; 68℃ extension for 2 min, 35 cycles; 68℃ extension for 5 min. The PCR product length was verified by 1.0% agarose gel electrophoresis. Under UV light, the single target DNA band was removed as much as possible using a newly opened blade. The excised gel containing the DNA fragment was placed into a pre-weighed 1.5 mL centrifuge tube, weighed, and the product was recovered using a recovery kit. The experimental procedures were performed according to the manufacturer's instructions. The collected DNA solution was stored at -20℃.
[0025] 4. Obtaining the pYES2 plasmid The yeast expression vector used in this experiment was pYES2. A small amount of the purchased pYES2 plasmid was inoculated onto LB solid medium (100 mg / mL ampicillin) and cultured overnight. Then, single colonies were picked and cultured overnight in liquid medium. The pYES2 plasmid was extracted according to the instructions of the plasmid extraction kit.
[0026] To ligate the amplified target fragment into a vector, it is necessary to prepare a pYES2 plasmid and a gene fragment with the same sticky ends. First, the pYES2 plasmid was digested with EcoR I and Hind III, and the corresponding fragment was purified and recovered after agarose gel electrophoresis.
[0027] The enzyme digestion system is shown in Table 3: Table 3 Enzyme digestion system
[0028] Add the system components to a 200 μL centrifuge tube, gently mix, centrifuge briefly for 5 s, and then inactivate by heat at 37℃ for 30 min and 65℃ for 20 min.
[0029] Transgenic identification results: Recombinant Agrobacterium was obtained by transforming the recombinant vector into Escherichia coli. Single colonies were picked and pre-deformed at 95℃ for 3 min using the primers shown in Table 3; followed by deformation at 95℃ for 15 s, annealing at 55℃ for 15 s, and extension at 72℃ for 15 s, for a total of 30 cycles; and a final extension at 72℃ for 5 min. Colony PCR was then performed. The PCR product length was verified by 1.0% agarose gel electrophoresis. The results showed that the recombinant plasmid vector contained the target gene. Figure 1 ) Example 2. Construction of recombinant microbial cells 1. Ligation and transformation of the target fragment into a yeast expression vector The concentration of the purified fragment was measured, and the target fragment was ligated to the vector fragment using a homologous recombinase to construct the recombinant plasmid vector. The ligation system is shown in Table 4. Table 4 Connection System
[0030] Add the system components to a 200 μL centrifuge tube, gently mix, centrifuge briefly for 5 seconds, and then place at 50℃ for 30 minutes.
[0031] The recombinant plasmid vector was transformed into DH5α competent cells, and the culture medium contained 100 mg / mL ampicillin. After overnight incubation, colonies growing on the plates were found to be ampicillin-resistant. Single colonies were picked and inoculated into liquid medium containing ampicillin, and incubated overnight at 37°C and 200 rpm. Using the overnight culture as a template, PCR was performed. Recombinant plasmid vectors were extracted from PCR-positive colonies using a plasmid extraction kit.
[0032] 2. Yeast Conversion Freshly prepared competent yeast cells were used for transformation at room temperature, following the instructions in the transformation section. Finally, 50 μL of the transformation mixture was evenly spread onto SD-Ura (glucose) solid medium and incubated at 30°C for 3 days until white colonies appeared.
[0033] Select white single colonies that have grown and incubate them in SD-Ura liquid medium at 30°C and 200 rpm overnight. Take the bacterial solution and lyse the cells at 100°C for 5 min. Use 5 μL of the lysed bacterial solution as a template for PCR detection to determine whether the target gene has been accurately integrated into the yeast expression vector.
[0034] 3. Phenotypic identification of recombinant yeast The recombinant yeast was renamed INVScⅠ(pYES2-Glyma03G.201100), which is the Glyma03G.201100 gene introduced in Example 1. To verify the effect of salt stress on transgenic yeast, its growth was observed by applying 100 mmol NaCl salt stress, and the experiment was repeated three times.
[0035] (1) In a clean bench, select the successfully verified recombinant yeast and yeast with empty vector and place them in YPDA liquid medium (glucose) and culture overnight at 30°C and 200 rpm with shaking. (2) Measure the OD600 of the overnight bacterial culture and calculate the required amount of bacterial culture so that the OD value of 5 mL of induction medium with added galactose (SD-Ura) is 0.4; (3) Take the required volume of bacterial culture and centrifuge at 8000 rpm for 1 min; (4) First, resuspend the bacterial cells in 1 mL of SD-Ura, then add to a final volume of 5 mL; (5) Incubate overnight at 30℃ with shaking at 200 rpm; (6) Measure the OD600 of the overnight bacterial culture, and adjust the OD600 to 2.0 after calculation; (7) Dilute the bacterial solution by 10-3 times, 10-4 times, and 10-5 times respectively; (8) Using INVScⅠ(pYES2) transfected with empty vector yeast as a control, 5 μL of undiluted bacterial solution and diluted bacterial solution were inoculated onto normal YPDA solid medium and salt-stressed YPDA solid medium, respectively. (9) Incubate at 30℃ for 5 days; (10) Observe and record the growth of the plate, and analyze and compare the differences in yeast growth.
[0036] Salt stress treatment process: Diluted bacterial suspensions, with empty vector yeast as a control, were inoculated at 5 μL onto normal YPDA solid medium and YPDA solid medium under 100 mm sodium chloride stress, respectively. The cultures were incubated at 30℃ for 5 days. The growth of the transgenic yeast was observed and compared.
[0037] Positive results were confirmed by applying the primers shown in Table 3 to transgenic yeast samples. The samples were pre-deformed at 95℃ for 3 min; deformed at 95℃ for 15 s, annealed at 55℃ for 15 s; extended at 72℃ for 15 s, for 30 cycles; and finally extended at 72℃ for 5 min. Colony PCR was then performed. The PCR product length was verified by 1.0% agarose gel electrophoresis. Figure 2 ) This embodiment conducted a transgenic verification experiment in yeast to verify the function of Glyma03G.201100. The results showed that the transgenic yeast exhibited significant growth differences compared to the empty vector yeast in salt-stressed YPDA medium. However, no difference in yeast growth was observed in normal YPDA medium. This indicates that the transgenic yeast has developed salt tolerance. Figure 3 ).
[0038] Example 3. Genetic transformation of soybean hairy roots Obtaining pHair plasmid: The expression vector used in this experiment was pHair. A small amount of purchased pHair plasmid was inoculated onto LB solid medium (antibiotic: 50 mg / mL kanamycin) and cultured overnight. Then, single colonies were picked and cultured overnight in liquid medium. The pHair plasmid was extracted according to the instructions of the plasmid extraction kit.
[0039] To ligate the amplified target fragment into a vector, it is necessary to prepare a pHair plasmid and a gene fragment with the same sticky ends. First, the pHair plasmid is digested with asc I and xba I, and the corresponding fragment is purified and recovered after agarose gel electrophoresis.
[0040] The enzyme digestion system is shown in Table 5: Table 5 Enzyme digestion system
[0041] Add the system components to a 200 μL centrifuge tube, gently mix, centrifuge briefly for 5 s, and then inactivate by heat at 37℃ for 30 min and 65℃ for 20 min.
[0042] The concentration of the purified fragment was measured, and the target fragment was ligated to the vector fragment using a homologous recombinase to construct the recombinant plasmid vector pHair. Glyma03G.201100. The connection system is shown in Table 6: Table 6 Connection System
[0043] Add the system components to a 200 μL centrifuge tube, gently mix, centrifuge briefly for 5 seconds, and then place at 50℃ for 30 minutes.
[0044] The recombinant plasmid vector was transformed into DH5α competent cells, and the culture medium contained 50 mg / mL kanamycin. After overnight incubation, colonies growing on the plates were transformed kanamycin-resistant bacteria. Single colonies were picked and inoculated into liquid medium containing kanamycin and incubated overnight at 37°C and 200 rpm. Using the overnight culture as a template, bacterial PCR was performed. Recombinant plasmid vectors were extracted from PCR-positive colonies using a plasmid extraction kit.
[0045] The recombinant vector was successfully constructed and identified as follows: Single colonies of *E. coli* were picked and pre-deformed at 95℃ for 3 min using the primers shown in Table 3; followed by deformation at 95℃ for 15 s, annealing at 55℃ for 15 s, and extension at 72℃ for 15 s, for a total of 30 cycles; followed by a final extension at 72℃ for 5 min. Colony PCR was then performed. The PCR product was verified for length by 1.0% agarose gel electrophoresis. The results showed that the recombinant plasmid vector contained the target gene. Figure 4 ).
[0046] Transformation with Agrobacterium strain K599: Take 5 μL of pHair Add 100 μL of K599 competent cells to the Glyma03G.201100 vector plasmid, incubate on ice for 30 min, freeze in liquid nitrogen for 5 min, incubate at 37℃ for 5 min, add 800 μL of YT liquid medium, pre-culture at 28℃ and 200 rpm for 3 h, plate the bacterial culture on YT (50 μg / mL str + 50 μg / mL kana) solid medium, incubate in the dark at 28℃ for 2 days, select positive single clones, and shake the culture at 28℃ and 200 rpm for 16-18 h for soybean transformation.
[0047] Preparation of infection solution: Shake the bacteria 3-4 days in advance. Use Agrobacterium rhizogenes K599 empty vector culture and K599 overexpression culture. After culturing Agrobacterium on TY medium solid (containing KANA + STR antibiotic) for 2 days, pick single colonies and place them in a 2ml centrifuge tube containing 1ml of TY liquid medium. Incubate overnight at 28℃ and 200r / min. Take 500ul of the shaken bacterial solution and put it into 100ml of TY medium. Incubate overnight at 28℃ and 200r / min until turbid. Centrifuge the shaken bacterial solution at 28℃ and 4000r / min for 10min. After separating the bacterial slides, resuspend them in distilled water. The OD600 should be around 0.8. Centrifuge at 28 ℃ and 4000×g for 5 min, discard the supernatant, collect the bacterial cells, and resuspend the cells in an infection solution containing 45ul acetylsyl syringone (AS) at a final concentration of 100 µmol / L, 6ml MES at 0.5mol / L, 3ml MgCl2 at 1mol / L, and 500ml distilled water. Use this solution to infect soybeans.
[0048] Seed germination: Select plump soybean seeds and soak them in a diluted sodium hypochlorite solution for 3 minutes (stirring the solution continuously and sifting the seeds). Rinse the soaked seeds three times with distilled water. After treatment, sow them in vermiculite and germinate in a growth chamber.
[0049] Infection treatment: Hypocotyl seeds approximately 2 mm in diameter were wounded at the cotyledonary nodes to serve as explants for Agrobacterium-mediated root hair transformation. The prepared explants were immersed in a suspension of Aspergillus rhizogenes K599 containing the target construct. After infection, they were briefly air-dried on sterile filter paper. The explants were then placed on filter paper containing a small amount of liquid co-culture medium and incubated in the dark for 23 hours. Cultured for 1 day under C and 3 days under light.
[0050] Transgenic identification results: qrt-PCR was used to verify the successful transfection of soybeans and confirm gene overexpression. Figure 11 The expression levels of the Glyma03G.201100 gene in transfected soybean plants were compared with those in untransfected plants using qrt-PCR (primer F: TTTCTCGGGGCAGCAAGTG, SEQ ID NO.5); primer R: TGACATCCCCGAGCCTGA, SEQ ID NO.6). The expression level increased by approximately 14-fold.
[0051] Salt stress treatment process: Salt stress was applied using a 70 mg sodium chloride solution for 5 days, and the development of roots and leaves was observed. Figure 6 The following data were all obtained from the root systems of transgenic plants and unloaded plants treated with water for 5 days and 70 mm sodium chloride solution.
[0052] Results of transgenic identification: The transgenic material subjected to the above-mentioned stress was verified by qrt-PCR to confirm the successful transfection of soybean and to determine the gene overexpression results. Figure 5 The expression levels of the Glyma03G.201100 gene in transfected soybean plants were compared with those in untransfected plants using qrt-PCR (primer F: TTTCTCGGGGCAGCAAGTG, SEQ ID NO. 5); primer R: TGACATCCCCGAGCCTGA, SEQ ID NO. 6). The expression level increased by approximately 16-fold.
[0053] In this embodiment, physiological indicators of the function of Glyma03G.201100 were measured in a genetic transformation experiment of soybean hairy roots. The results showed that after 5 days of salt stress treatment, the MDA content in the roots of transgenic soybean plants was significantly different from that of control plants. Under salt stress, the MDA content was significantly increased, while the MDA content of transgenic overexpression plants was significantly lower than that of empty vector plants. Figure 7 Compared with the control plants, there were significant differences in the activities of POD, SOD, and CAT in the roots. Under salt stress, the activities of POD, SOD, and CAT were significantly increased, and the activities of POD, SOD, and CAT in transgenic overexpression plants were significantly higher than those in empty vector plants. Figure 8-10 This indicates that the hairy roots of genetically modified soybeans have also developed salt tolerance.
[0054] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.
Claims
1. The application of overexpression of the Glyma03G.201100 gene in improving the salt tolerance of soybean or yeast, characterized in that, The nucleotide sequence of the Glyma03G.201100 gene is shown in SEQ ID NO.
1.
2. The application of overexpression of Glyma03G.201100 protein in improving the salt tolerance of soybeans or yeast, characterized in that, The amino acid sequence of the Glyma03G.201100 protein is shown in SEQ ID NO.
2.
3. The application of a recombinant vector containing the Glyma03G.201100 gene in improving the salt tolerance of soybeans or yeast, characterized in that, The nucleotide sequence of the Glyma03G.201100 gene is shown in SEQ ID NO.
1.
4. The application according to claim 3, characterized in that, The starting vector for the recombinant vector is pYES2.
5. The application of recombinant microbial cells containing the Glyma03G.201100 gene in improving the salt tolerance of soybeans, characterized in that, The nucleotide sequence of the Glyma03G.201100 gene is shown in SEQ ID NO.
1.
6. The application according to any one of claims 1-5, characterized in that, The salt is NaCl, and its concentration is 70-100 mM.
7. A breeding method for improving the salt tolerance of soybeans, characterized in that, The steps of the method are as follows: Step 1: Ligate the gene shown in SEQ ID NO.1 with the vector pHair to obtain the recombinant vector; Step 2: Transform the recombinant vector described in Step 1 into Agrobacterium to obtain recombinant Agrobacterium; Step 3: The recombinant Agrobacterium described in Step 2 is transferred into soybeans to obtain transgenic soybean plants, and positive transgenic soybean plants are obtained after identification.
8. A method for improving the salt tolerance of yeast, characterized in that, The steps of the method are as follows: Step 1: Ligate the gene shown in SEQ ID NO.1 with the vector pYES2 to obtain the recombinant vector; Step 2: Transform the recombinant vector described in Step 1 into yeast to obtain recombinant yeast.
9. The method according to claim 7 or 8, characterized in that, The primers for amplifying the sequence shown in SEQ ID NO.1 are SEQ ID NO.3 and SEQ ID NO.4.