The application of soybean GmGASA1 in plant breeding and cultivating transgenic plants

Abstract

The application of a soybean GmGASA1 gene, the application relates to the application of a gibberellin regulation related protein GmGASA1 coding gene of soybean.The application of the soybean GmGASA1 gene, namely the application of improving the protein content, the seed amino acid content and the grain weight of plants; the application of the soybean GmGASA1 gene in plant breeding; the application of the soybean GmGASA1 gene in cultivating transgenic plants.The experimental results show that three transgenic lines are obtained by overexpressing the GmGASA1 gene in soybean, the protein content and the grain weight of the transformed soybean seeds are significantly improved compared with the seeds of the untransformed receptor plants, which indicates that the GmGASA1 gene can regulate the protein content, the seed amino acid content and the grain weight of seeds.The GmGASA1 gene can be used for improving the quality of soybean and increasing the yield of soybean.The application of the GmGASA1 gene can effectively realize the breeding of new soybean varieties and the creation of new germplasm.

Description

Application of soybean GmGASA1 in plant breeding and the cultivation of transgenic plants Technical Field

[0001] This invention relates to the application of a gene encoding the soybean gibberellin regulatory-related protein GmGASA1. Background Technology

[0002] Soybeans are one of the world's most important sources of plant protein, providing approximately 68% of global protein consumption. Soybean protein accounts for about 40% of the soybean grain's weight, which is 4-5 times that of grains. Nutritionally, it contains all the essential amino acids required by humans, especially lysine, which is lacking in grains. As a plant-based protein source, soybeans are naturally cholesterol-free and rich in phytosterols, which inhibit cholesterol absorption and can help regulate hypercholesterolemia. Furthermore, the 1.2:1 arginine / lysine ratio not only improves protein utilization but also synergistically maintains cardiovascular health through arginine's vascular regulatory function. Currently, soybeans are an important grain and oil crop and a high-protein dual-purpose crop for both grain and feed in my country. Soybean protein production is closely related to the edible protein industry, feed industry, and livestock and aquaculture industries. Sulfur-containing amino acids (mainly methionine and cysteine) play a vital role in human health, offering benefits such as enhanced immune function, reduced inflammation, and improved cardiovascular health. However, soybeans have a very low content of sulfur-containing amino acids, which cannot meet people's needs for these amino acids. There is a need to significantly increase the sulfur-containing amino acid content in soybeans, but our understanding of the genes controlling sulfur-containing amino acids is limited. More importantly, there is a negative genetic correlation between protein content and grain weight, making it difficult to simultaneously increase both soybean protein content and grain weight. Currently, new soybean varieties bred primarily for yield enhancement generally have low protein content, and yield increases cannot meet the correspondingly increasing demand for soybean protein. 100-grain weight is a crucial factor affecting soybean yield and a key target trait in soybean breeding. Increasing both soybean yield and seed protein content is an important direction in soybean breeding. However, soybean genes capable of simultaneously increasing both yield and protein content are rare. Therefore, discovering and identifying genes that can synergistically enhance yield, protein content, and functional amino acid content (e.g., sulfur-containing amino acids) is of great significance for breeding high-yielding and high-quality soybeans.

[0003] GmGASA1 belongs to the GASA / GAST gene family, a family widely found in plants, characterized by genes encoding small proteins rich in cysteine ​​peptides at the C-terminus (Aubert et al., 1998). GAST1 was first identified in tomato, and subsequent preliminary analyses of the family genes were conducted in plants including tomato, Arabidopsis, rice, wheat, grape, and tobacco (Aubert et al., 1998; Roxrud et al., 2007; Muhammad et al., 2019; Lv et al., 2018; Ahmad et al., 2020). GASA proteins have been shown to play important roles in the biological regulation of plant development, including stem elongation, flowering, root development, photosynthesis, fruit ripening, and seed germination; some GASA proteins also function in responses to biotic and abiotic stresses, such as heat stress, fungi, and nematodes. To date, there are no reports in soybeans of GASA1 homologs simultaneously increasing seed protein, amino acid content, and grain weight. Summary of the Invention

[0004] This invention provides an application of soybean GmGASA1 in plant breeding and the cultivation of transgenic plants.

[0005] This invention relates to the application of the soybean GmGASA1 gene in plant breeding, which is used to improve seed protein content, seed amino acid content, seed weight, and seed yield.

[0006] This invention relates to the application of the soybean GmGASA1 gene in the cultivation of transgenic plants. The soybean GmGASA1 gene is used to cultivate transgenic plants with increased seed weight and to cultivate transgenic plants that can increase the expression level of the GmGASA1 gene.

[0007] Furthermore, the protein encoded by the GmGASA1 gene is one of the following proteins:

[0008] (A1) A protein having the same function as the amino acid sequence shown in SEQ ID NO.1, by substitution and / or deletion and / or addition of one or more amino acid residues;

[0009] (A2) is a protein that has 99% or more, 95% or more, 90% or more, 85% or more or more of the same properties and functions as the amino acid sequence defined in (A1);

[0010] (A3) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of any of the proteins defined in (A1)-(A2).

[0011] This invention demonstrates through experiments that overexpressing the GmGASA1 gene in soybean and obtaining three transgenic lines significantly increased the protein content of the transformed soybean seeds by 7.42%-8.86% compared to the untransformed soybean Dongnong 50 (DN50), significantly increased the water-soluble protein content by 8.02%-9.00%, and significantly increased the content of sulfur-containing amino acids such as methionine and cysteine ​​by 4.57%-4.97% and 4.18%-5.99%, respectively. The content of six essential amino acids, including lysine, phenylalanine, threonine, isoleucine, leucine, and valine, was also significantly increased by 5.53%-6.77%, 6.06%-8.74%, 6.14%-7.68%, and 5%, respectively. The protein and amino acid content of soybeans increased by 40%-7.35%, 6.21%-8.00%, and 6.70%-9.39%, respectively. The content of alanine, glycine, glutamic acid, arginine, tyrosine, proline, serine, aspartic acid, and histidine increased by 5.32%-6.63%, 6.38%-7.35%, 8.99%-10.94%, 10.26%-12.58%, 5.13%-5.98%, 5.85%-8.57%, 6.09%-6.82%, 7.06%-8.22%, and 7.29%-9.28%, respectively. Simultaneously, grain weight significantly increased by 14%-34%, indicating that the GmGASA1 gene can regulate seed protein, amino acid content, and grain weight. The GmGASA1 gene can be used to improve soybean protein and amino acid content while increasing soybean yield. Therefore, GmGASA1 has the potential for application in the breeding of high-yield and high-quality soybean varieties and the creation of new germplasm.

[0012] The experimental results of this invention show that overexpression of the GmGASA1 gene in soybean yielded three transgenic lines. The protein content and grain weight of the transformed soybean seeds were significantly higher than those of the untransformed recipient plants, indicating that the GmGASA1 gene can regulate seed protein content and grain weight. The GmGASA1 gene can be used to improve soybean quality and increase soybean yield.

[0013] The application of soybean GmGASA1 in plant breeding and the cultivation of transgenic plants can effectively realize the breeding of new soybean varieties and the creation of new germplasm. Attached Figure Description

[0014] Figure 1 shows the PCR amplification product of GmGASA1 in Example 1 on an agarose gel.

[0015] Figure 2 shows the tissue expression relative level analysis of GmGASA1 in Example 1;

[0016] Figure 3 shows the subcellular localization results of GmGASA1 in Example 1;

[0017] Figure 4 is a schematic diagram of the overexpression vector of GmGASA1 in Example 2;

[0018] Figure 5 shows the results of bar test strip detection (a) and PCR detection (b) of the GmGASA1 transgenic overexpression material in Example 2;

[0019] Figure 6 shows the results of GmGASA1 transcription level detection in transgenic soybean lines in Example 2;

[0020] Figure 7 shows the comparison results of protein (a) and water-soluble protein (b) content in soybeans of the GmGASA1 transgenic material and control seeds in Example 2;

[0021] Figure 8 shows the comparison results of amino acid content in GmGASA1 transgenic soybean and control seeds in Example 2;

[0022] Figure 9 shows the comparison results of 100-seed weight (a) and seed appearance (b) of GmGASA1 transgenic soybean in Example 2. Detailed Implementation

[0023] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all 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.

[0024] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0025] Specific Implementation Method 1: This implementation method is used in plant breeding of the soybean GmGASA1 gene, wherein the amino acid sequence of the protein encoded by the GmGASA1 gene is shown in SEQ ID NO.1.

[0026] Specific Implementation Method Two: This implementation method differs from Specific Implementation Method One in that the soybean GmGASA1 gene is used to increase seed protein content. Everything else is the same as in Specific Implementation Method One.

[0027] Specific Implementation Method Three: This implementation method differs from Specific Implementation Method One in that the soybean GmGASA1 gene is used to increase the amino acid content of seeds. Everything else is the same as in Specific Implementation Method One.

[0028] Specific Implementation Method Four: This implementation method differs from Specific Implementation Method One in that the soybean GmGASA1 gene is used to increase seed weight. Everything else is the same as in Specific Implementation Method One.

[0029] Specific Implementation Method Five: This implementation method differs from Specific Implementation Method One in that the soybean GmGASA1 gene is used to increase seed yield. Everything else is the same as in Specific Implementation Method One.

[0030] Specific Implementation Method Six: This implementation method differs from Specific Implementation Method One in that it involves the application of the soybean GmGASA1 gene in the cultivation of transgenic plants. Everything else is the same as in Specific Implementation Method One.

[0031] Specific Implementation Method Seven: This implementation method differs from Specific Implementation Method Six in that the soybean GmGASA1 gene is used to cultivate transgenic plants with increased seed protein content. Everything else is the same as in Specific Implementation Method Six.

[0032] Specific Implementation Method Eight: This implementation method differs from Specific Implementation Method Six in that the soybean GmGASA1 gene is used to cultivate transgenic plants with increased seed weight. Everything else is the same as in Specific Implementation Method Six.

[0033] Specific Implementation Method Nine: This implementation method differs from Specific Implementation Method Six in that the soybean GmGASA1 gene is used to cultivate transgenic plants that can increase the expression level of the GmGASA1 gene. Everything else is the same as in Specific Implementation Method Six.

[0034] Specific Implementation Method Ten: This implementation method differs from Specific Implementation Methods One through Nine in that the protein encoded by the GmGASA1 gene is one of the following proteins:

[0035] (A1) A protein having the same function as the amino acid sequence shown in SEQ ID NO.1, by substitution and / or deletion and / or addition of one or more amino acid residues;

[0036] (A2) is a protein that has 99% or more, 95% or more, 90% or more, 85% or more or more of the same properties and functions as the amino acid sequence defined in (A1);

[0037] (A3) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of any of the proteins defined in (A1)-(A2). The rest is the same as in Specific Embodiments 1 to 9.

[0038] Specific Implementation Method Eleven: This implementation method differs from Specific Implementation Methods One through Nine in that the gene encoding the GmGASA1 protein includes any of the following DNA molecules:

[0039] (B1) The DNA molecule shown in SEQ ID NO.2;

[0040] (B2) A DNA molecule that hybridizes under stringent conditions with the DNA molecule defined in (B1) and encodes the GmGASA1 protein;

[0041] (B3) A DNA molecule that has 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity with the DNA sequence defined by (B1) or (B2) and encodes the GmGASA1 protein. The rest is the same as in Specific Embodiments 1 to 9.

[0042] Specific Implementation Method Twelve: This implementation method differs from Specific Implementation Methods One through Nine in that it involves introducing and increasing the expression level of the GmGASA1 gene in the recipient plant. Everything else is the same as in Specific Implementation Methods One through Nine.

[0043] Specific Implementation Method Thirteen: This implementation method differs from Specific Implementation Methods One to Nine in that the plant described is a dicotyledonous or monocotyledonous plant. Everything else is the same as in Specific Implementation Methods One to Nine.

[0044] Specific Implementation Method Fourteen: This implementation method differs from Specific Implementation Methods One through Nine in that the dicotyledonous plant is a legume. Everything else is the same as Specific Implementation Methods One through Nine.

[0045] Specific Implementation Method Fifteen: This implementation method differs from Specific Implementation Methods One to Nine in that the legume plant mentioned is soybean. Everything else is the same as in Specific Implementation Methods One to Nine.

[0046] Example 1

[0047] 1. Cloning of the soybean GmGASA1 gene

[0048] The gene sequence information of GmGASA1 was found in the Phytozome database. Primers for amplifying GmGASA1 were designed based on the gene sequence. The primer sequences are shown in SEQ ID NO.3 (5'-TCTCTTTGCTATGGCTCTCTC-3') and SEQ ID NO.4 (5'-ATGGGTTGGAAGGTATCACA-3').

[0049] Using soybean variety “Williams 82” (W82) as the sample, its developed seeds were collected, crushed with liquid nitrogen, and the powder was placed in a 1.5 ml EP tube. 1 ml of lysis buffer was added, and the mixture was vortexed to make it homogeneous. Then, extraction was performed according to the kit (Total RNA Kit, Tiangen, China).

[0050] RNA integrity was assessed by 1% agarose gel electrophoresis. Using the obtained total RNA as a template, cDNA synthesis was performed according to the instructions of the Vazyme HiScript 1st Strand cDNASynthesis Kit (Nanjing, China). PCR amplification was then performed using the cDNA as a template. The PCR reaction mixture was as follows: 2 μl template, 2 μl each of forward and reverse primers, 25 μl 2×Phanta Max Master Mix, and finally, ddH2O was added to a final volume of 50 μl. The PCR program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 sec, 58℃ annealing for 15 sec, 72℃ extension for 60 sec, for a total of 35 cycles; final extension at 72℃ for 5 min, followed by isothermal treatment at 4℃ for 30 min. Figure 1 shows the PCR amplification product of GmGASA1 in agarose gel.

[0051] The PCR electrophoresis results of GmGASA1 are shown in Figure 1. After gel recovery, product purification, and T-vector ligation, the product was transformed into an E. coli enrichment plasmid. Sequencing yielded the complete coding sequence (CDS) of the soybean GmGASA1 gene, which is 303 bp in length. The CDS sequence is shown in SEQ ID NO.2.

[0052] 2. Tissue expression analysis of GmGASA1 gene

[0053] Soybean variety W82 seeds were sown in pots filled with nutrient soil (peat moss: vermiculite, 2:1) and cultured in a greenhouse at 25℃ for 16h / 8h (light / dark). Seed coats (SC) and cotyledons (COT) of W82 seeds were collected from the roots (V1 stage), stems (V1 stage), leaves (V1 stage), flowers, and seeds at three key developmental stages (early, middle, and late stages) for nutrient accumulation. The samples were flash-frozen with liquid nitrogen and stored in an ultra-low temperature freezer at -80℃ for later use.

[0054] Total RNA was extracted using a plant total RNA extraction kit, and RNA integrity was assessed by 1% agarose gel electrophoresis. cDNA synthesis was performed according to the instructions of the reverse transcription kit (Vazyme). The expression level of GmGASA1 in plant tissues was detected using quantitative real-time PCR (qRT-PCR). Primer sequences are shown in SEQ ID NO. 5 (5'-ATCAATCGGCATACGCACAG-3') and SEQ ID NO. 6 (5'-CAGCAAGTTCCACACGCTCT-3').

[0055] Figure 2 shows the relative tissue expression levels of GmGASA1. In the figure, Root: root tissue; Stem: stem tissue; Leaf: leaf tissue; Flower: flower tissue; SC1: seed coat tissue in early seed development; COT1: cotyledon tissue in early seed development; SC2: seed coat tissue in mid-seed development; COT2: cotyledon tissue in mid-seed development; SC3: seed coat tissue in late seed development; COT3: cotyledon tissue in late seed development. The results (Figure 2) show that GmGASA1 gene expression was not detected in roots, stems, leaves, and developing embryos, but it was expressed in the seed coats (SC1, SC2, SC3) at all three stages of flower and seed development, with the highest expression level in the mid-developing seed coat (SC2).

[0056] 3. Subcellular localization of GmGASA1

[0057] Based on the known sequence of the GmGASA1 gene and the cloning site of the plant expression vector pBSK, specific primers were designed as follows: primer sequences are shown in SEQ ID NO.7 (5'-GATAAGCTTGATATCGAATTCATGGCTCTCTCAAAGCTTCTAGT-3') and SEQ ID NO.8 (5'-CATTCTAGAACTAGTGGATCCAGGGCACTTGCG TCTGCCC-3'). The specific PCR process is the same as step 1 in this embodiment. The target gene fragment with enzyme digestion adapters amplified by PCR was identified by gel electrophoresis, and the obtained product was recovered and purified by gel electrophoresis.

[0058] Recombinant vectors were constructed using homologous recombination. The recombination reaction was performed according to the instructions of the Vazyme ClonExpress II One Step Cloning Kit (C112). The reaction mixture consisted of 1 μl of vector, 2 μl of target fragment, 4 μl of 5×CE II Buffer, 2 μl of Exnase II, and ddH2O to a final volume of 20 μl. The reaction program was 37℃ for 30 min. The recombinant product was then transformed into DH5α competent cells using a freeze-thaw method, along with the empty vector. The cells were plated, single clones were picked, and colony PCR sequencing was performed for verification. Plasmids from correctly sequenced colonies were extracted and named pBSK-GmGASA1. Arabidopsis protoplasts were transiently transformed using the PEG method. The transformed protoplasts were cultured in the dark at 28℃ for 16 h, and the fluorescence excitation signal intensity of the protoplasts was observed using the Leica THUNDER system (Leica Microsystems, Wetzlar, Germany).

[0059] Figure 3 shows the subcellular localization results of GmGASA1. The first row of the figure shows the microscopic images of the empty vector (carrying GFP), from left to right: bright field, chloroplast fluorescence channel (Chlorophyll), green fluorescence channel (GFP), and the superimposed image of the above three channels (Merged). The second row of the image shows the microscopic images of the GmGASA1-GFP protein, with the same left-to-right image arrangement as the first row.

[0060] The results are shown in Figure 3. The first row shows the results after transformation with the empty plasmid, from left to right: bright field, chloroplast fluorescence channel (Chlorophyll), green fluorescence channel (GFP), and a merged image of the three channels. The second row shows the images of the pBSK-GmGASA1 vector after transformation under a microscope, with the same distribution from left to right as the first row. The results indicate that the empty plasmid vector carrying GFP showed green fluorescence signals in all tissues, while the GmGASA1 protein fluorescence signal was mainly localized in the cytoplasm (Figure 3).

[0061] Example 2: Genetic Engineering Application of the Soybean GmGASA1 Gene

[0062] 1. Construction of plant overexpression vectors

[0063] Based on the CDS sequence of the cloned GmGASA1 gene and the cloning site of the plant expression vector pTF101, specific primers were designed as follows: primer sequences are shown in SEQ ID NO.9 (5'-GACTCTAGAAACAGAGGATCCATGGCTCTCTCAAAGCTTCTAGT-3') and SEQ ID NO.10 (5'-TTCGAGCTCGCTGTTACTAGTAGGGCACTTGCG TCTGCCC-3'). An overexpression vector was constructed using homologous recombination, following the same steps as in step 3 of the implementation method. Sequencing primer sequences are shown in SEQ ID NO.11 (5'-CATTTCATTTGGAGAGAACACG-3') and SEQ ID NO.12 (5'-AGCGGATAACAATTTCACACAG-3'). The pTF101-GmGASA1 plant overexpression plasmid was finally obtained and stored at -20℃ for later use. Figure 4 is a schematic diagram of the GmGASA1 overexpression vector; the GmGASA1 gene is represented by an orange rectangle, and its 5' end is connected to the CaMV 35 promoter.

[0064] Figure 4 shows a schematic diagram of the GmGASA1 overexpression vector. The CDS sequence of the target gene GmGASA1 is located between the restriction endonucleases BamH1 and Spe1.

[0065] 2. Transformation of Agrobacterium with overexpression vector

[0066] The recombinant plasmid obtained in step 1 of this embodiment was transformed into Agrobacterium tumefaciens EHA105 using the freeze-thaw method. The specific experimental procedure is as follows:

[0067] ① Use a pipette to aspirate 3 μl of the recombinant plasmid and transfer it into 100 μl of EHA105 competent cells, then gently mix.

[0068] ② Place the mixture on ice for 5 minutes, in liquid nitrogen for 5 minutes, incubate in a 37°C water bath for 5 minutes, and then immediately transfer it to an ice bath for 3 minutes.

[0069] ③ Take 800 μl of YEP culture medium with a pipette in a clean bench and revive it for 4 h in a constant temperature shaker at 28℃ and 150 rpm.

[0070] ④ Centrifuge at 5000×g for 2 min at room temperature, resuspend the bacterial cells with an appropriate amount of supernatant, take 200 μl of the revived bacterial solution and spread it evenly on the screening plate medium, and incubate at 28℃ upside down for 3 days.

[0071] 3. Transformation of soybean cotyledonary nodes infected with Agrobacterium

[0072] High-quality "Dongnong 50" soybean seeds were selected and sterilized in a fume hood using chlorine sterilization for 16 hours. The sterile seeds were then soaked in sterile water and cultured in a tissue culture room at 25℃ for 24 hours. After imbibition, the germinating soybeans were longitudinally split along the midline, and the apical and axillary buds were removed. Slight incisions were made at the cotyledonary nodes. The transformed Agrobacterium-mediated bacterial solution was shaken to OD... 600 Centrifuge at 0.5 μL, 12000 rpm, resuspend in the infection solution, and place the prepared cotyledonary explants in the solution. Let stand at 28°C for 30 min. Dry the infected cotyledonary explants by aspirating the bacterial solution, place them adaxially on CCM solid medium, arrange them neatly, and incubate in the dark in a tissue culture room for 3 days. Wash the cotyledonary nodes three times in sterile water, blot off the liquid with sterilized filter paper, and insert them into recovery solid medium. Incubate in a tissue culture room for 15 days. Cut off resistant shoots that have grown to 1-2 cm and insert them into elongation medium. Incubate in a tissue culture room for 15 days. Once the elongated seedlings are robust, insert them into rooting medium and culture upright for 10 days. When the elongated seedlings have developed sufficient roots, remove them and place them in humus mixed with vermiculite. Finally, transfer them to a greenhouse for further cultivation.

[0073] 4. Identification of positive transformation and gene expression analysis

[0074] The overexpression tissue culture seedlings transplanted into the soil were first tested for the bar gene (PAT / bar test strips). Figure 5 shows the results of bar test strip detection (a) and PCR detection (b) of GmGASA1 transgenic overexpression material; CK is untransformed soybean receptor.

[0075] Figure 5a shows that the untransformed soybean plants (CK) only showed one control band, with no detection band, while all three transgenic overexpression lines showed detection bands (red arrows). DNA was extracted from leaves of the overexpression tissue culture seedlings after the test. PCR identification was performed using Novizan polymerase (2×Rapid Taq Master Mix). The PCR system was as follows: 10 μl 2×RapidTaq Master Mix, 2 μl Primer 1 (10 μM), 2 μl Primer 2 (10 μM), 2 μl Template DNA, and finally adjusted to 20 μl with ddH2O. The reaction program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 sec, 60℃ annealing for 15 sec, 72℃ extension for 15 sec, for a total of 35 cycles; 72℃ complete extension for 5 min. Seedlings with PCR bands matching the target fragment size were considered positive for overexpression, as shown in Figure 5b. Next, qRT-PCR was used to further investigate the GmGASA1-OE transgenic soybean plants. Figure 6 shows the results of GmGASA1 transcriptional level detection in the transgenic soybean lines. The results showed that the expression level of GmGASA1 in the GmGASA1-OE overexpressing transgenic soybean plants was significantly higher than that in the control (**P<0.01) (Figure 6). The results of bar test strips, PCR molecular detection, and qRT-PCR all confirmed that the recombinant plasmid was successfully transformed into soybean and successfully expressed.

[0076] 5. Determination of protein content in genetically modified soybeans

[0077] The harvested transgenic soybean seeds were dried in a 30℃ oven. The protein and water-soluble protein content of mature seeds from the control material Dongnong 50 (DN50) and GmGASA1-OE transgenic soybean plants was then determined using a Perten DA7250 near-infrared analyzer (Sweden). Three independent transformation lines were selected for the experiment, and 5-8 individual plants from each line were analyzed. Figure 7 shows the comparison results of protein (a) and water-soluble protein (b) content in GmGASA1 transgenic soybean seeds and control seeds; error bars represent mean ± SD, and statistical analysis was performed using a two-tailed t-test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

[0078] The results showed that, compared with the control group, the seeds of the three lines that overexpressed GmGASA1 had significantly higher protein content by 7.42%-8.86% (Fig. 7a) and significantly higher water-soluble protein content by 8.02%-9.00% (Fig. 7b).

[0079] 6. Determination of amino acid content in genetically modified soybean seeds

[0080] The amino acid content in dried seeds of transgenic soybean generation T3 was determined using a Porton DA7250 near-infrared spectroscopy analyzer (Perten, Sweden). Three independent transgenic lines were used in the experiment, and 5-8 individual plants from each line were analyzed. Figure 8 shows the comparison of amino acid content between GmGASA1 transgenic soybean seeds and control seeds. Error bars represent mean ± SD; statistical analysis was performed using a two-tailed t-test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

[0081] The results (Figure 8) showed that the contents of 17 amino acids (alanine, methionine, glycine, glutamic acid, arginine, lysine, tyrosine, leucine, serine, threonine, aspartic acid, valine, isoleucine, histidine, cysteine, phenylalanine, and proline) in the three lines overexpressing GmGASA1 were significantly increased compared to the untransformed soybean DN50. Among these, sulfur-containing amino acids such as methionine and cysteine ​​significantly increased by 4.57%-4.97% and 4.18%-5.99%, respectively, while the contents of the six essential amino acids—lysine, phenylalanine, threonine, isoleucine, leucine, and valine—significantly increased by 5.53%-6.77%, 6.06%-8.74%, 6.14%-7.68%, 5.40%-7.35%, 6.21%-8.00%, 6.70%-9.99%, respectively. The percentages of alanine, glycine, glutamic acid, arginine, tyrosine, proline, serine, aspartic acid, and histidine increased by 5.32%-6.63%, 6.38%-7.35%, 8.99%-10.94%, 10.26%-12.58%, 5.13%-5.98%, 5.85%-8.57%, 6.09%-6.82%, 7.06%-8.22%, and 7.29%-9.28%, respectively.

[0082] 7. Genetically modified soybeans increase the weight of 100 grains.

[0083] The harvested transgenic soybean seeds were dried in an oven at 30℃. The 100-seed weight of mature soybeans from the control material DN50 and the GmGASA1 overexpressing transgenic soybean plants was then measured. Three independent transforming lines were used in the experiment, with 5-8 individual plants measured from each line. Figure 9 shows the comparison of the 100-seed weight (a) and seed appearance (b) of the GmGASA1 transgenic soybean material; error bars represent the mean ± SD. Statistical analysis was performed using a two-tailed t-test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

[0084] The results showed that, compared with the control DN50, the 100-seed weight of the three lines overexpressing GmGASA1 increased significantly by 14%-34% (Fig. 9a), and the seed size was also significantly larger than that of the control (Fig. 9b).

Claims

1. The application of the soybean GmGASA1 gene in increasing the protein content of soybean seeds, characterized in that... The amino acid sequence of the protein encoded by the GmGASA1 gene is shown in SEQ ID NO.

1.

2. The application of the soybean GmGASA1 gene as described in claim 1 in improving the amino acid content of soybean seeds.

3. The application of the soybean GmGASA1 gene as described in claim 1 in improving soybean seed weight.

4. The application of the soybean GmGASA1 gene as described in claim 1 in increasing the 100-seed weight of soybeans.

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