Application of soybean GmGASA1 in plant breeding and transgenic plant cultivation

Abstract

The invention discloses application of a soybean GmGASA1 gene, and relates to application of a soybean gibberellin regulation related protein GmGASA1 coding gene. The application of the soybean GmGASA1 gene is that the soybean GmGASA1 gene is applied to the improvement of the plant protein content, the seed amino acid content and the plant grain weight; the invention relates to an application of a soybean GmGASA1 gene in plant breeding. The invention relates to an application of a soybean GmGASA1 gene in cultivation of transgenic plants. Experimental results show that three transgenic lines are obtained through overexpression of the GmGASA1 gene in soybeans, and the protein content and the grain weight in transformed soybean seeds are remarkably improved compared with those in untransformed receptor plant seeds, so that the GmGASA1 gene can regulate and control the protein content, the amino acid content and the grain weight of the seeds. The GmGASA1 gene can be used for improving the soybean quality and increasing the soybean yield. The application of the GmGASA1 can effectively realize the breeding of new soybean varieties and the creation of new germplasm.

Description

Technical Field

[0001] The invention relates to an application of a soybean gibberellin regulation-related protein GmGASA1 encoding gene. Background Art

[0002] Soybean is one of the important sources of plant protein in the world, and the plant protein it provides accounts for about 68% of the total protein consumption in the world. The protein content of soybean accounts for about 40% of the weight of soybean grains, which is 4-5 times that of cereal foods. In terms of nutritional value, it contains all the essential amino acids needed by humans, especially rich in lysine, which is lacking in cereals. As a source of plant protein, soybeans are naturally cholesterol-free and rich in phytosterols that inhibit cholesterol absorption, which can assist in regulating hypercholesterolemia; at the same time, the arginine / lysine ratio of 1.2:1 not only improves protein utilization, but also synergistically maintains cardiovascular health through the vascular regulation function of arginine. At present, soybeans are important grain and oil crops and high-protein grain and feed crops in my country. The output of soybean protein is closely related to the edible protein industry, feed industry, and animal husbandry and aquaculture industry. Sulfur-containing amino acids (mainly methionine and cysteine) play a very important role in human health, and have the advantages of enhancing immune function, reducing inflammation, and improving cardiovascular health. However, the content of sulfur-containing amino acids in soybeans is very low, which cannot meet people's demand for sulfur-containing amino acids. It is necessary to significantly increase the content of sulfur-containing amino acids in soybeans, but we know very little about the genes that control sulfur-containing amino acids. More importantly, protein and grain weight are negatively correlated genetically, and it is not easy to increase soybean protein content and grain weight at the same time. At present, the protein content of new soybean varieties bred with yield improvement as the main breeding goal is generally not high, and the increase in yield cannot meet the corresponding increase in soybean protein content. Hundred-grain weight is an important factor affecting soybean yield and an important target trait in soybean breeding. Increasing soybean yield while increasing the protein content in seeds is an important direction of soybean breeding. However, there are few soybean genes that can simultaneously increase yield and protein content. Therefore, discovering and identifying genes that can synergistically increase yield and protein content as well as functional amino acid content (such as sulfur-containing amino acids) is of great significance for breeding high-yield and high-quality soybeans.

[0003] GmGASA1 belongs to the GASA / GAST gene family, which is common in plants and is characterized by genes encoding small proteins and a cysteine-rich short peptide at the C-terminus of the protein (Aubert et al., 1998). GAST1 was first identified in tomatoes, and preliminary analysis of family genes was subsequently carried out in plants including tomatoes, Arabidopsis, rice, wheat, grapes, 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 an important role in the biological regulation of plant development, including stem elongation, flowering, root development, photosynthesis, fruit ripening, and seed germination; some GASA also play a role in biotic and abiotic stress responses, such as heat stress, fungi, and nematodes. So far, there has been no report on the simultaneous improvement of seed protein, amino acids, and grain weight by GASA1 homologous genes in soybean. Summary of the invention

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

[0005] The soybean GmGASA1 gene of the present invention is used in plant breeding. The soybean GmGASA1 gene is used to increase seed protein content, seed amino acid content, seed grain weight and seed yield.

[0006] The soybean GmGASA1 gene of the present invention is used in cultivating transgenic plants. The soybean GmGASA1 gene is used for cultivating transgenic plants with increased seed weight and for cultivating transgenic plants with increased GmGASA1 gene expression level.

[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 after one or more amino acid residues are substituted and / or deleted and / or added;

[0009] (A2) a protein having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity with the amino acid sequence defined in (A1) and having the same function;

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

[0011] The present invention uses experiments to prove that the GmGASA1 gene is overexpressed in soybeans and three transgenic strains are obtained. The protein content in the transformed soybean seeds is significantly increased by 7.42%-8.86% compared with the untransformed soybean Dongnong 50 (DN50), the water-soluble protein content is significantly increased by 8.02%-9.00%, the content of sulfur-containing amino acids such as methionine and cysteine ​​is significantly increased by 4.57%-4.97% and 4.18%-5.99%, and the content of six essential amino acids including lysine, phenylalanine, threonine, isoleucine, leucine and valine is significantly increased by 5.53%-6.77%, 6.06%-8.74%, 6.14%-7.68%, 5.76%-8.90%, respectively. .40%-7.35%, 6.21%-8.00%, 6.70%-9.39%, alanine, glycine, glutamic acid, arginine, tyrosine, proline, serine, aspartic acid, 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%, 7.29%-9.28% respectively; meanwhile, grain weight increased significantly by 14%-34%, indicating that GmGASA1 gene can regulate seed protein, amino acid content and grain weight. GmGASA1 gene can be used to improve soybean protein and amino acid content while increasing soybean yield. Therefore, GmGASA1 has application potential in the breeding of new high-yield and high-quality soybean varieties and the creation of new germplasm.

[0012] The present invention can be seen from the experimental results that three transgenic lines were obtained by overexpressing the GmGASA1 gene in soybeans, and the protein content and grain weight in the transformed soybean seeds were significantly increased compared with the seeds of the untransformed recipient plants, indicating that the GmGASA1 gene can regulate the protein content and grain weight of the seeds. The GmGASA1 gene can be used to improve the quality of soybeans and increase soybean yield.

[0013] The application of the soybean GmGASA1 of the present invention in plant breeding and cultivation of transgenic plants can effectively realize the breeding of new soybean varieties and the creation of new germplasm. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The result of the PCR amplification product of GmGASA1 in Example 1 in agarose gel;

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

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

[0017] Figure 4 Schematic diagram of the overexpression vector of GmGASA1 in Example 2;

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

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

[0020] Figure 7 The results are the comparison of the protein (a) and water-soluble protein (b) contents in the GmGASA1 transgenic soybean material and the control seeds in Example 2;

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

[0022] Fig. 9 The comparison results of 100-grain weight (a) and grain appearance (b) of GmGASA1 transgenic soybeans in Example 2 are shown. DETAILED DESCRIPTION

[0023] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

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

[0026] Specific embodiment 2: This embodiment is different from specific embodiment 1 in that the soybean GmGASA1 gene is used to increase the protein content of seeds. Other aspects are the same as specific embodiment 1.

[0027] Specific implementation method 3: This implementation method is different from the specific implementation method 1 in that the soybean GmGASA1 gene is used to increase the amino acid content of seeds. The rest is the same as the specific implementation method 1.

[0028] Specific embodiment 4: This embodiment is different from the specific embodiment 1 in that the soybean GmGASA1 gene is used to increase seed weight. The rest is the same as the specific embodiment 1.

[0029] Specific embodiment 5: This embodiment is different from the specific embodiment 1 in that the soybean GmGASA1 gene is used to increase seed yield. The rest is the same as the specific embodiment 1.

[0030] Specific embodiment 6: This embodiment is different from the specific embodiment 1 in that: the soybean GmGASA1 gene is used in cultivating transgenic plants. The rest is the same as the specific embodiment 1.

[0031] Specific embodiment 7: This embodiment is different from specific embodiment 6 in that the soybean GmGASA1 gene is used to cultivate transgenic plants with increased seed protein content. Other aspects are the same as specific embodiment 6.

[0032] Specific embodiment eight: This embodiment differs from specific embodiment six in that the soybean GmGASA1 gene is used to cultivate transgenic plants with increased seed weight. Other aspects are the same as specific embodiment six.

[0033] Specific embodiment 9: This embodiment is different from specific embodiment 6 in that the soybean GmGASA1 gene is used to cultivate transgenic plants that can increase the expression level of the GmGASA1 gene. Other aspects are the same as specific embodiment 6.

[0034] Specific embodiment 10: This embodiment differs from specific embodiments 1 to 9 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 after one or more amino acid residues are substituted and / or deleted and / or added;

[0036] (A2) a protein having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity with the amino acid sequence defined in (A1) and having the same function;

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

[0038] Specific embodiment eleven: This embodiment differs from specific embodiments one to nine in that: the coding gene of the GmGASA1 protein comprises any of the following DNA molecules:

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

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

[0041] (B3) A DNA molecule encoding the GmGASA1 protein that has 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity with the DNA sequence defined in (B1) or (B2). Other aspects are the same as those of embodiments 1 to 9.

[0042] Specific embodiment 12: This embodiment is different from specific embodiments 1 to 9 in that the expression level of the GmGASA1 gene is introduced into the recipient plant and increased. The rest is the same as specific embodiments 1 to 9.

[0043] Specific implementation thirteen: This implementation differs from specific implementations one to nine in that the plant is a dicotyledonous or monocotyledonous plant. The rest is the same as specific implementations one to nine.

[0044] Specific embodiment 14: This embodiment differs from specific embodiments 1 to 9 in that the dicotyledonous plant is a leguminous plant. The rest is the same as specific embodiments 1 to 9.

[0045] Specific embodiment 15: This embodiment differs from specific embodiments 1 to 9 in that the leguminous plant is soybean. The rest is the same as specific embodiments 1 to 9.

[0046] Example 1

[0047] 1. Cloning of soybean GmGASA1 gene

[0048] The gene sequence information of GmGASA1 was searched according to the Phytozome database, and primers for amplifying GmGASA1 were designed according to 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] The soybean variety “Williams 82” (W82) was used as the material. Its developing seeds were taken and ground with liquid nitrogen. The powder was placed in a 1.5 ml EP tube, 1 ml of lysis buffer was added, vortexed to mix evenly, and then extracted according to the kit (Total RNA Kit, Tiangen, China).

[0050] The integrity of RNA was detected by 1% agarose gel electrophoresis. The total RNA obtained was used as a template, and the synthesis of cDNA was reverse transcribed according to the instructions of the reverse transcription kit (Vazyme HiScript 1st Strand cDNA Synthesis Kit, Nanjing, China) provided by Vazyme. PCR amplification was performed using cDNA as a template. The PCR reaction system was as follows: 2 μl template, 2 μl upstream and downstream primers, 25 μl 2×Phanta Max Master Mix, and finally ddH2O was added to make the volume 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, a total of 35 cycles; finally, 72℃ thorough extension for 5 min, followed by constant temperature at 4℃ for 30 min. Figure 1 The results of PCR amplification products of GmGASA1 in agarose gel.

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

[0052] 2. Analysis of GmGASA1 gene tissue expression

[0053] Seeds of soybean variety W82 were sown in pots filled with nutrient soil (peat soil: vermiculite, 2:1) and cultured in a greenhouse at 25°C, 16h / 8h (light / dark). The seed coat (SC) and cotyledon (COT) of W82 at three key developmental stages (early, middle and late) of nutrient accumulation of roots (V1 stage), stems (V1 stage), leaves (V1 stage), flowers, and seeds were collected and the samples were quick-frozen with liquid nitrogen and stored in a -80°C ultra-low temperature refrigerator for later use.

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

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

[0056] 3. Subcellular localization of GmGASA1

[0057] Specific primers were designed according to the known sequence of the GmGASA1 gene and the cloning site of the plant expression vector pBSK. The primers are as follows: the 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 the enzyme-cutting linker amplified by PCR is identified by gel electrophoresis, and the obtained product is recovered and purified by gel electrophoresis.

[0058] The recombinant vector was constructed by homologous recombination. The recombination reaction was carried out according to the instructions of Vazyme's ClonExpress IIOne StepCloning Kit (C112). The reaction system was as follows: 1 μl of vector, 2 μl of target fragment, 4 μl of 5×CE IIBuffer, 2 μl of Exnase II, and ddH2O was added to make the volume 20 μl. Reaction procedure: 37°C, 30 min for recombination reaction. The recombinant product was transferred into DH5α competent cells by freeze-thaw method, and the plasmid and empty plasmid were respectively transferred into the plasmid, plated, single cloned and verified by bacterial liquid PCR sequencing. The plasmid extracted from the bacterial liquid with correct sequencing was named pBSK-GmGASA1. Arabidopsis protoplasts were transiently transformed by PEG method. The transformed protoplasts were cultured in the dark at 28°C for 16 h, and then the fluorescence excitation signal intensity of the protoplasts was observed by LeicaTHUNDER system (Leica Microsystems, Wetzlar, Germany).

[0059] Figure 3 The results of subcellular localization of GmGASA1; the first row of the figure shows the imaging of the empty vector (carrying GFP) under a microscope, from left to right are bright field (Bright), chloroplast fluorescence channel (Chlorophyll), green fluorescence channel (GFP) and the overlay of the above three channels (Merged). The second row of the picture shows the imaging of GmGASA1-GFP protein under a microscope, and the arrangement of the pictures from left to right is the same as the first row.

[0060] Results Figure 3 , where the first row shows the results after empty plasmid transformation, from left to right they are bright field (Bright), chloroplast fluorescence channel (Chlorophyll), green fluorescence channel (GFP) and the overlay of the above three channels (Merged); the second row of the picture shows the imaging of pBSK-GmGASA1 vector transformation under a microscope, and the distribution of pictures from left to right is the same as the first row. The results show that the empty plasmid vector carrying GFP has green fluorescence signals in all tissues, while the GmGASA1 protein fluorescence signal is mainly located in the cytoplasm ( Figure 3 ).

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

[0062] 1. Construction of plant overexpression vector

[0063] Specific primers were designed according to the CDS sequence of the cloned GmGASA1 gene and the cloning site of the plant expression vector pTF101. The primers are as follows: the primer sequences are shown in SEQ ID NO.9 (5'-GACTCTAGAAACAGAGGATCCATGGCTCTCTCAAAGCTTCTAGT-3') and SEQ ID NO.10 (5'-TTCGAGCTCGCTGTTACTAGTAGGGCACTTGCG TCTGCCC-3'). The overexpression vector was constructed by homologous recombination, and the specific steps were the same as step 3 in the embodiment. The sequencing primer sequences are shown in SEQ ID NO.11 (5'-CATTTCATTTGGAGAGAACACG-3') and SEQ ID NO.12 (5'-AGCGGATAACAATTTCACACAG-3'). Finally, the pTF101-GmGASA1 plant overexpression expression plasmid was obtained and stored in a -20°C refrigerator for future use. Figure 4 Schematic diagram of the overexpression vector of GmGASA1; wherein the GmGASA1 gene is represented by an orange rectangle, and its 5' end is connected to the CaMV 35 promoter.

[0064] Schematic diagram of GmGASA1 overexpression vector is shown in Figure 4 , the CDS sequence of the target gene GmGASA1 is 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 example was transformed into Agrobacterium tumefaciens EHA105 by freeze-thaw method. The specific experimental operation process is as follows:

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

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

[0069] ③ Use a pipette to take 800 μl of YEP medium in the clean bench and revive it in a constant temperature shaker at 28°C and 150 rpm for 4 hours.

[0070] ④ Centrifuge at 5000×g for 2 min at room temperature, resuspend the bacteria with an appropriate amount of supernatant, spread 200 μl of the resuscitated bacterial solution evenly on the screening plate culture medium, and culture inverted at 28°C for 3 days.

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

[0072] Select good quality "Dongnong 50" soybean seeds and sterilize them in a fume hood using chlorine sterilization for 16 hours. Soak the sterile seeds in sterile water and culture them in a tissue culture room at 25°C for 24 hours. Cut the imbibed germinating soybeans longitudinally along the midline, remove the apical buds and axillary buds, and make slight scratches at the cotyledon nodes. Shake the transformed Agrobacterium solution to OD 600 = 0.5, centrifuge at 12000rpm, add the infection liquid to resuspend, and place the marked cotyledonary node explants in it, and let it stand at 28℃ for 30min. The infected cotyledonary node explants are dried with bacterial liquid, and the adaxial surface is placed on CCM solid culture medium, arranged neatly, and cultured in the dark in the tissue culture room for 3d. The cotyledonary node is placed in sterile water and washed 3 times, and the liquid is absorbed with a sterilized filter paper, and it is inserted into the recovery solid culture medium and cultured in the tissue culture room for 15d. The resistant buds that grow to 1-2cm are cut and inserted into the elongation culture medium, and cultured in the tissue culture room for 15d. When the elongated seedlings are strong, they are inserted into the rooting culture medium and cultured upright for 10d. When the elongated seedlings grow a proper amount of roots, they are removed and placed in humus mixed with vermiculite, and finally transferred to the greenhouse for 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 The results of bar test strip test (a) and PCR test (b) of GmGASA1 transgenic overexpression material; CK is the untransformed soybean recipient.

[0075] Figure 5 As shown in a, the untransformed soybean plant (CK) only showed one quality control band and no detection band, while the three transgenic overexpression lines all showed detection bands (red arrows); after the test, the leaves of the overexpression tissue culture seedlings were taken to extract DNA. PCR identification was performed using Novagen polymerase (2×Rapid Taq Master Mix), and the PCR system was as follows: 2×RapidTaq Master Mix 10μl, Primer 1 (10μM) 2μl, Primer 2 (10μM) 2μl, Template DNA 2μl, and finally ddH2O to 20μl. The reaction procedure is as follows: pre-denaturation at 95℃ for 3min; denaturation at 95℃ for 15sec, renaturation at 60℃ for 15sec, extension at 72℃ for 15sec, for a total of 35 cycles; complete extension at 72℃ for 5min. The PCR identification band that is consistent with the size of the target fragment is the overexpression positive seedling, and the results are shown in Figure 5 b. Next, the GmGASA1-OE transgenic soybean plants were further tested using qRT-PCR. Figure 6The results of the detection of GmGASA1 transcription level in transgenic soybean lines. The results showed that the expression level of GmGASA1 in 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 experiments all confirmed that the recombinant plasmid was successfully transferred into soybean and expressed successfully.

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

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

[0078] The results showed that compared with the control group, the protein content of seeds of the three strains overexpressing GmGASA1 was significantly increased by 7.42%-8.86% ( Figure 7 a), the water-soluble protein content increased significantly by 8.02%-9.00% ( Figure 7 b).

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

[0080] The amino acid content in the transgenic soybean T3 generation dry seeds was determined using a Perten DA7250 near-infrared analyzer (Perten, Sweden). Three independent transgenic lines were used in the experiment, and 5-8 plants were measured in each transgenic line. Figure 8 Comparison of amino acid content in GmGASA1 transgenic soybean and control seeds. Error bars represent mean ± SD; two-tailed T test was used for statistical analysis. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

[0081] The results show that ( Figure 8), the contents of 17 kinds of amino acids (alanine, methionine, glycine, glutamic acid, arginine, lysine, tyrosine, leucine, serine, threonine, aspartic acid, valine, isoleucine, histidine, cysteine, phenylalanine, proline) in the three strains overexpressing GmGASA1 were significantly increased compared with the untransformed recipient soybean DN50. Among them, the contents of sulfur-containing amino acids such as methionine and cysteine ​​were significantly increased by 4.57%-4.97% and 4.18%-5.99%, and the contents of 6 essential amino acids including lysine, phenylalanine, threonine, isoleucine, leucine and valine were 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%-9.90%, respectively. .39%, 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 100-grain weight

[0083] The harvested transgenic soybean seeds were dried in an oven at 30°C, and then the 100-grain weight of mature grains of the control material DN50 and the GmGASA1 overexpressing transgenic soybean plants was measured. Three independent transformation lines were selected for the experiment, and 5-8 plants were measured for each transformation line. Fig. 9 Comparison results of 100-grain weight (a) and grain appearance (b) of GmGASA1 transgenic soybeans; error bars represent 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-grain weight of the three strains overexpressing GmGASA1 increased significantly by 14%-34% ( Fig. 9 a), and the grain size was also significantly larger than that of the control ( Fig. 9 b).

Claims

1. Application of soybean GmGASA1 gene in plant breeding, 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 soybean GmGASA1 gene in plant breeding according to claim 1, characterized in that The soybean GmGASA1 gene is used to increase seed protein content.

3. The use of the soybean GmGASA1 gene in plant breeding according to claim 1, characterized in that The soybean GmGASA1 gene is used to increase the amino acid content of seeds.

4. The use of the soybean GmGASA1 gene in plant breeding according to claim 1, characterized in that The soybean GmGASA1 gene is used to increase seed weight.

5. The use of the soybean GmGASA1 gene in plant breeding according to claim 1, characterized in that The soybean GmGASA1 gene is used to improve seed yield.

6. The use of the soybean GmGASA1 gene in plant breeding according to claim 1, characterized in that Application of soybean GmGASA1 gene in breeding transgenic plants.

7. The use of the soybean GmGASA1 gene in cultivating transgenic plants according to claim 6, characterized in that The soybean GmGASA1 gene is used to breed transgenic plants with increased seed protein content.

8. The use of the soybean GmGASA1 gene in cultivating transgenic plants according to claim 6, characterized in that The soybean GmGASA1 gene is used to breed transgenic plants with increased seed weight.

9. The use of the soybean GmGASA1 gene in cultivating transgenic plants according to claim 6, characterized in that The soybean GmGASA1 gene is used to cultivate transgenic plants that can increase the expression level of the GmGASA1 gene.

10. The use according to any one of claims 1 to 9, characterized in that The protein encoded by the GmGASA1 gene is one of the following proteins: (A1) a protein having the same function as the amino acid sequence shown in SEQ ID NO.1 after one or more amino acid residues are substituted and / or deleted and / or added; (A2) a protein having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity with the amino acid sequence defined in (A1) and having the same function; (A3) A fusion protein obtained by linking a protein tag to the N-terminus and / or C-terminus of the protein defined in any one of (A1) to (A2).

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