Application of GmSAMS gene and protein in regulating soybean seed quality

Abstract

The application discloses application of a GmSAMS gene and protein in regulating soybean kernel quality, belongs to the technical field of genetic engineering, and particularly relates to application of the GmSAMS gene in regulating soybean kernel quality, wherein the nucleotide sequence of the GmSAMS gene is shown as SEQ ID NO. 1. The application can reduce the expression of a protein S-adenosylmethionine synthetase of the GmSAMS gene by silencing the GmSAMS gene, improve the protein content of soybean kernels, reduce the oil content of soybean kernels, reduce the total fatty acid content of soybean kernels, and make the soybean kernels smaller, so that the purpose of regulating the quality of soybean kernels is achieved.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, and in particular relates to the application of the GmSAMS gene and protein in regulating soybean seed quality. Background Technology

[0002] Soybeans Glycine max As a globally important food and oilseed crop, soybean is one of the main sources of plant protein and edible oil for humans. Its grain quality directly affects its nutritional value, processing performance, and market benefits. Grain quality mainly involves several complex traits, including protein and oil content and composition, amino acid balance, anti-nutritional factor content, and the efficiency of storage substance accumulation. Traditional breeding methods often face bottlenecks in improving these multi-gene-controlled quality traits, such as low selection efficiency, long breeding cycles, and difficulty in achieving synergistic improvement of multiple desirable traits. Therefore, analyzing the genetic regulatory network of soybean grain development and quality formation at the molecular level, identifying key functional genes, and elucidating their mechanisms of action have become important foundations for achieving high-efficiency molecular design breeding of soybean quality.

[0003] S-Adenosylmethionine synthase (SAMS) is a core enzyme in methionine metabolism in organisms, catalyzing the synthesis of S-adenosylmethionine (SAM) from ATP and methionine (Met). Studies have shown that SAM and its derivatives play a central role in plant growth and development, stress response, and secondary metabolism regulation. Currently, research on plant SAMS genes mainly focuses on the physiological function analysis of model plants (such as Arabidopsis and rice), confirming their involvement in embryonic development, stress response, and hormone synthesis. However, in soybean, although multiple SAMS gene families (such as the GmSAMS family) exist in the genome, systematic and in-depth research and clear understanding of the specific functions, mechanisms of action, and application potential of specific GmSAMS members in regulating soybean grain quality formation are still lacking.

[0004] Therefore, isolating and identifying the GmSAMS gene that is specifically or predominantly expressed in soybean seeds, and conducting in-depth research on the regulatory function and molecular mechanism of its encoded protein in the formation of seed quality traits, will not only enrich our basic understanding of soybean seed development biology, but more importantly, will provide new key gene targets and important technical means for the precise improvement of soybean nutritional quality using molecular biology methods (such as transgenic technology and gene editing technology), which has significant theoretical value and application prospects. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention proposes the application of the GmSAMS gene and protein in regulating soybean seed quality. By silencing the GmSAMS gene and reducing the expression of the GmSAMS gene protein S-adenosylmethionine synthase, this invention increases the protein content of soybean seeds, reduces the oil content and total fatty acid content of soybean seeds, and makes the soybean seeds smaller, thus successfully achieving the goal of regulating soybean seed quality.

[0006] To achieve the above objectives, this invention provides the application of the GmSAMS gene in regulating soybean seed quality. The nucleotide sequence of the GmSAMS gene is shown in SEQ ID NO.1. By silencing the GmSAMS gene, soybean seed quality is regulated, soybean seed protein content is increased, soybean seed oil content is decreased, soybean seed total fatty acid content is decreased, and soybean seeds are made smaller.

[0007] This invention also provides the application of the GmSAMS gene in regulating soybean seed protein content, thereby increasing soybean seed protein content by silencing the GmSAMS gene.

[0008] This invention also provides the application of the GmSAMS gene in regulating the oil content of soybean seeds, thereby reducing the oil content of soybean seeds by silencing the GmSAMS gene.

[0009] The present invention also provides the application of the GmSAMS gene in regulating the total fatty acid content of soybean seeds. By silencing the GmSAMS gene, the total fatty acid content of soybean seeds is reduced, thereby reducing the content of palmitic acid, stearic acid and linoleic acid in soybean seeds.

[0010] The present invention also provides the application of the GmSAMS gene in regulating soybean seed size, by silencing the GmSAMS gene to make soybean seeds smaller.

[0011] The present invention also provides the application of the protein of the GmSAMS gene in regulating soybean seed quality. The protein of the GmSAMS gene is S-adenosylmethionine synthase. The amino acid sequence of the protein of the GmSAMS gene is shown in SEQ ID NO.2. By reducing the expression of S-adenosylmethionine synthase, the quality of soybean seeds is regulated, the protein content of soybean seeds is increased, the oil content of soybean seeds is reduced, the total fatty acid content of soybean seeds is reduced, and the soybean seeds are made smaller.

[0012] The present invention also provides a recombinant plasmid for regulating soybean seed quality. The recombinant plasmid regulates soybean seed quality by silencing the GmSAMS gene, thereby increasing soybean seed protein content, decreasing soybean seed oil content, decreasing soybean seed total fatty acid content, and making soybean seeds smaller.

[0013] The present invention also provides a method for regulating soybean seed quality, including the step of silencing the GmSAMS gene using sgRNA.

[0014] Preferably, the sgRNA includes sgRNA1 and sgRNA2, the nucleotide sequence of sgRNA1 is shown in SEQ ID NO.3, and the nucleotide sequence of sgRNA2 is shown in SEQ ID NO.4.

[0015] Compared with the prior art, the present invention has the following advantages and technical effects: This invention provides the application of the GmSAMS gene and protein in regulating soybean seed quality. By using sgRNA to silence the GmSAMS gene, this invention discovered that silencing the GmSAMS gene and reducing the expression of the GmSAMS gene protein S-adenosylmethionine synthase can successfully increase soybean seed protein content, decrease soybean seed oil content, decrease soybean seed total fatty acid content, and make soybean seeds smaller. This invention demonstrates that the GmSAMS gene can regulate soybean seed quality, providing a novel key gene target for improving soybean nutritional quality, and has significant theoretical value and application prospects. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 The results of the in vitro enzyme activity assay of S-adenosylmethionine synthase in Example 1 are as follows: A represents the amplification product of pMAL-c4x-GmSAMS transformed into E. coli, where M represents DL2K plus marker, 1 represents positive control, 2 represents negative control, and 3-6 represent PCR amplification products of different single clones of E. coli transformed by pMAL-c4x-GmSAMS; B represents the SDS-PAGE detection results of SAMS purified protein, where M represents PageRuler Marker, 1-2 represent purified MBP-SAMS, and 3-4 represent purified empty MBP; C represents the Western Blot detection results of SAMS purified protein, where M represents PageRuler Marker, 1-2 represent purified MBP-SAMS, and 3-4 represent purified empty MBP; D is the SAMS standard curve; E represents the MBP-SAMS activity detection results. "" represents p<0.01; Figure 2 The results of the GmSAMS gene expression pattern analysis in Example 1; Figure 3 The images show the subcellular localization results of the GmSAMS protein in Example 1. A is the fluorescence field image of the empty GFP protein 35S::GFP in the GFP fluorescence field; B is the fluorescence field image of the empty GFP protein 35S::GFP in the RFP fluorescence field; C is the bright field image of the empty GFP protein 35S::GFP; D is the fluorescence fusion image of the empty GFP protein 35S::GFP; E is the fluorescence field image of the GmSAMS protein 35S::GmSAMS::GFP carrying the GFP protein in the GFP fluorescence field; F is the fluorescence field image of the GmSAMS protein 35S::GmSAMS::GFP carrying the GFP protein in the RFP fluorescence field; G is the bright field image of the GmSAMS protein 35S::GmSAMS::GFP carrying the GFP protein; and H is the fluorescence fusion image of the GmSAMS protein 35S::GmSAMS::GFP carrying the GFP protein. The scale bar is 25 μm. Figure 4 The images show the creation results of the GmSAMS gene mutant. Figure A shows the PCR amplification results of pGES201-GmSAMS sgRNA1, where M represents the DL5K plus DNA Marker, 1 represents the positive control, 2 represents the negative control, and 3-6 represent the PCR amplification products of different monoclonal E. coli transformed with pGES201-GmSAMS sgRNA1. Figure B shows the PCR amplification results of pGES201-GmSAMS sgRNA2, where M represents the DL5K plus DNA Marker, 1 represents the positive control, 2 represents the negative control, and 3-6 represent the PCR amplification products of different monoclonal E. coli transformed with pGES201-GmSAMS sgRNA2. Figure C shows the PCR amplification results of pGES201-sgRNA1 and pGES201-sgRNA2 plasmids transformed with EHA105, where M represents the Trans 2K Plus DNA Marker, 1 represents the positive control, 2 represents the negative control, and 3-6 represent the PCR amplification products of different monoclonal E. coli transformed with pGES201-GmSAMS sgRNA1. PCR amplification products of different monoclonal E. coli transformed by sgRNA1, 7~10 represent PCR amplification products of different monoclonal E. coli transformed by pGES201-GmSAMS sgRNA2; D represents the process of explant infection, recovery culture, shoot induction, shoot elongation and hardening after the expression vector was transformed into soybean DN50. Figure 5The results show the identification of GmSAMS gene mutants. A is a schematic diagram of GmSAMS gene mutant knockout; B is a comparison of off-target gene sequencing peaks; C is the PCR amplification results of gmsams-1 Csa9, where M represents DL5K Plus DNA marker, 1 represents water, 2 represents DN50, and 3-18 represent PCR amplification products of different gmsams-1 individual plants; D is the PCR amplification results of gmsams-2 Csa9, where M represents DL5K Plus DNA marker, 1 represents water, 2 represents DN50, and 3-16 represent PCR amplification products of different gmsams-2 individual plants; E is the PCR amplification results of gmsams-3 Csa9, where M represents DL5K Plus DNA marker, 1 represents water, 2 represents DN50, and 3-8 represent PCR amplification products of different gmsams-3 individual plants. Figure 6 The images show the results of total protein and fatty acid content determination in soybean seeds from GmSAMS gene mutants. In the images, A represents the total protein content determination result of the GmSAMS mutant, B represents the total fatty acid content determination result, C represents the fatty acid composition determination result, and D represents a transmission electron microscopy (TEM) image of soybean seeds from the GmSAMS mutant. The scale bar is 5 μm. In the images, WT and DN50 represent wild plants, gmsams-1, gmsams-2, and gmsams-3 represent GmSAMS gene mutants, MM represents maturity / material accumulation stage, LM represents late maturity, OBs represent oil bodies, and SSP represents protein bodies. " represents p<0.05", "" represents p<0.01; Figure 7 The results show the seed length and width measurements of soybeans with GmSAMS gene mutants. A is a phenotypic comparison of seed length in soybeans with GmSAMS gene mutants (scale bar: 1 cm); B is a phenotypic comparison of seed width in soybeans with GmSAMS gene mutants (scale bar: 1 cm); C is a statistical analysis chart of seed length in soybeans with GmSAMS gene mutants; and D is a statistical analysis chart of seed width in soybeans with GmSAMS gene mutants. In the figures, WT represents wild-type plants, and gmsams-1, gmsams-2, and gmsams-3 represent GmSAMS gene mutants. " represents p<0.05", " represents p<0.01", "This means p < 0.001. Detailed Implementation

[0018] Various exemplary embodiments of the present invention are now described in detail. This detailed description should not be considered as a limitation of the invention, but rather as a more detailed description of certain aspects, features, and embodiments of the invention. Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The specification and embodiments of this invention are merely exemplary.

[0019] Example 1 I. Enzyme activity assay of GmSAMS protein, i.e., S-adenosylmethionine synthase: Retrieved from the phytozome database GmSAMS A full-length CDS sequence of 1179 bp was obtained. CDS primers (excluding terminators) were designed at both ends of the CDS sequence of this gene using this sequence as a template. Cloning was performed using cDNA from DN50 leaves as a template with the CDS primers. GmSAMS The CDS sequence of the gene was obtained, and primers were designed to linearize the empty vector pMAL-c4x. GmSAMS -F: GAAGGATTTCAGAATTCATGGCAGAGACATTCCTATT, SEQ ID NO.6; GmSAMS -R: TCGACTCTAGAGGATCCTTAGGCCTTCTCCCACTTGAG, SEQ ID NO. 7; pMAL-c4x-F: GAATTCTGAAATCCTTCCCTCGATCCCGAGGTTG, SEQ ID NO. 8; pMAL-c4x-R: GGATCCTCTAGAGTCGACCTGCAGGCAAGCTTGG, SEQ ID NO. 9).

[0020] The products were subjected to agarose gel electrophoresis, and the gel was cut at the correct band position for purification. The purified target gene and the linearized vector fragment were subjected to homologous recombination using the Novizan ClonExpress II One Step Cloneing Kit. The homologous recombination system was prepared on ice as shown in Table 1.

[0021] Table 1 Homologous recombination system

[0022] After brief centrifugation, the cells were placed in a 37°C metal bath for 30 min and immediately transformed into *E. coli* DH5α competent cells (purchased from Shanghai Weidi Biotechnology Co., Ltd., DL1001). The cells were then evenly spread onto plates containing the corresponding antibiotics and incubated overnight at 37°C. Single colonies were picked and transferred to 1 mL of LB broth containing the corresponding antibiotics and incubated overnight at 37°C with a shaker until the culture became turbid. Positive single colonies were detected using Sanger sequencing. The sequencing results were compared with the gene CDS sequence on the Phytozome website to obtain the correctly sequenced recombinant plasmid pMAL-c4x-GmSAMS.

[0023] To test whether the GmSAMS protein possesses S-adenosylmethionine synthase activity, the expression vector pMAL-c4x-GmSAMS was constructed. The recombinant plasmid with correctly sequenced GmSAMS genes was transformed into *E. coli*. Rosetta-gami (DE3) (Purchased from Shanghai Weidi Biotechnology Co., Ltd., EC1012) After confirming the correct result by E. coli culture PCR (pMAL-c4x-GmSAMS: 1179bp), proceed to the next step of the experiment (such as...). Figure 1 (As shown in A).

[0024]

[0025] The amino acid sequence of the GmSAMS protein is shown in SEQ ID NO.2. NO.2: MAETFLFTSESVNEGHPDKLCDQISDAVLDACLEQDPDSKVACETCTKTNLVMVFGEITTKANVDYEKIVRDTCRNIGFVSNDVGLDADNCKVL VNIEQQSPDIAQGVHGHLTKKPEEIGAGDQGHMFGYATDETPELMPLSHVLATKLGARLTEVRKNGTCPWLRPDGKTQVTVEYYNDNGARVPIRVHTVLI STQHDETVTNDEIAADLKEHVIKPVIPEKYLDEKTIFHLNPSGRFVIGGPHGDAGLTGRKIIIDTYGGWGAHGGGAFSGKDPTKVDRSGAYIVRQAAKSIVASGLARRCIVQVSYAIGVPEPLSVFVDTYGTGKIHDKEILNIVKENFDFRPGMISINLDLKRGGNNRFLKTAAYGHFGREDPDFTWEVVKPLKWEKA.

[0026] Recombinant proteins were obtained through prokaryotic expression induction and purification. Empty MBP was used as a blank control. The purified proteins were subjected to Coomassie Brilliant Blue staining and Western Blot analysis. Results showed that the MBP-GmSAM fusion protein and the empty MBP protein exhibited single target bands at approximately 91 kDa and 44 kDa, respectively, consistent with the predicted protein sizes. This indicates successful protein purification and suitability for subsequent enzyme activity assays (such as enzyme activity determination). Figure 1 China B and Figure 1 (As shown in C).

[0027] Standard curves were plotted using SAMS protein standards at different concentrations (0 U / L, 25 U / L, 50 U / L, 100 U / L, 200 U / L, 300 U / L). S-adenosylmethionine synthase (SAMS) activity was measured using a plant S-adenosylmethionine synthase (SAMS) activity ELISA kit (catalog number EHJ-98664p, purchased from Xiamen Jia Hui Biotechnology Co., Ltd.). The specific experimental method was described in the manufacturer's instructions. Using empty vector MBP protein as a negative control, the MBP-SAMS enzyme activity was significantly higher than that of empty vector MBP, indicating that GmSAMS protein possesses S-adenosylmethionine synthase activity (e.g., ...). Figure 1 China D and Figure 1 (As shown in E).

[0028] II. Analysis of GmSAMS gene expression patterns: Samples were taken from various plant tissues (roots, stems, leaves, flowers, and pods) and seed development stages (cotyledon stage Cot, embryo morphogenesis stage EM, maturity / material accumulation stage MM, late maturity stage LM, and dry seed / dormancy stage DS) of soybean DN50 (from the Soybean Research Institute of Northeast Agricultural University). RNA was extracted from each tissue and reverse transcribed to obtain cDNA. GmActin2 was used as an internal reference gene, and the results were verified by qRT-PCR.

[0029] The results showed that the GmSAMS gene was expressed in all tissues, with the highest expression level in the stem, followed by the leaf, then the root and pod, and the lowest expression level in the flower. The GmSAMS gene was also expressed at all stages of seed development, with the highest expression level at the DS stage, followed by the MM stage, then the LM and EM stages, and the lowest expression level at the Cot stage. This indicates that the expression level of the GmSAMS gene gradually increases with seed development, reaching its highest level in the later stages of seed development, coinciding with seed maturation (e.g., ...). Figure 2 (As shown).

[0030] III. Subcellular localization analysis of GmSAMS protein: With restriction endonucleases ( HindIII and KpnI The empty Fu28 vector and the gel recovery product containing the target gene with restriction enzyme sites were cut and purified. The two were then ligated using Solution I ligase. The ligation product was transformed into competent *E. coli* DH5α cells and cultured in chloramphenicol-resistant plates until single colonies appeared. Single colonies were picked and transferred to 1 mL of LB broth containing the corresponding antibiotic and incubated overnight at 37°C with shaking until the culture became turbid. Positive single colonies were detected using Sanger sequencing. GmSAMS The CDS sequence is consistent, meaning the introductory carrier (Fu28-) has been completed. GmSAMS The construction of ).

[0031] Extracting Fu28- GmSAMS The pSOY1 vector plasmid was recombined using the LR reaction, and the product was transformed into competent *E. coli* DH5α cells. The cells were cultured in spectinomycin-resistant plates until single colonies appeared. Each single colony was picked and transferred to 1 mL of LB broth containing the corresponding antibiotic, and incubated overnight at 37 °C with a shaker until the culture became turbid. Positive single colonies were detected using Sanger sequencing. GmSAMS CDS sequence consistency means that the expression vector has been completed ( pSOY1-GmSAMS The construction of ) GmSAMS -GFP.

[0032] To investigate the subcellular localization of GmSAMS protein, a 35S:GmSAMS-GFP expression vector was constructed, and the recombinant plasmid was transformed into EHA105 bacteria (purchased from Shanghai Weidi Biotechnology Co., Ltd.). The plasmid was then co-transformed into tobacco leaves with a nuclear marker and a membrane marker, and the fluorescence signal was observed under a confocal microscope.

[0033] When empty GFP protein is expressed normally, GmSAMS protein carrying GFP protein can be observed to produce green fluorescent signals at the cell nucleus and cell membrane, and it fuses with the marker (e.g., Figure 3 China A~ Figure 3 As shown in Figure H), this indicates that the GmSAMS protein is located in the cell nucleus and cell membrane.

[0034] IV. Creation of GmSAMS gene mutants: Using the CRISPR-GE website (http: / / skl.scau.edu.cn / ), two superior sgRNAs with low off-target rates and located in the gene CDS region were selected from the GmSAMS genome sequence: sgRNA1 (SEQ ID NO.3: CGTCTCAAATGATGTGGGACTGG) and sgRNA2 (SEQ ID NO.4: GCTCATGGCATCAATTCAGGGG). Annealing reaction systems were prepared according to the system in Table 2.

[0035] Table 2 Annealing reaction system

[0036] Repeat the mixing of the prepared annealing reaction buffer, briefly centrifuge, and place on a PCR instrument. Run the following program: 95℃ for 4 min, temperature increase of 0.1℃ / s, 95℃~16℃, hold at 16℃. The annealed double-stranded nucleotides can be used immediately or stored long-term at -20℃.

[0037] Annealed double-stranded oligonucleotides were ligated with linearized pGES201 and transformed into E. coli DH5α competent cells. Positive colonies were picked and cultured to extract plasmids, and the sequences were confirmed by sequencing using CRISPR-F primers (5'-ggcgggaaacgacaatctgatc-3' (SEQ ID NO.5)).

[0038] The successfully constructed recombinant plasmid was transformed into Agrobacterium competent cells. Positive monoclonal identification showed a target band detected at 520 bp, the same size as the band in the positive control (recombinant plasmid), confirming successful transformation of sgRNA1 and sgRNA2 into the pGES201 vector, suitable for subsequent genetic transformation (e.g., ...). Figure 4 China A~ Figure 4(As shown in C). Expression vectors (pGES201-sgRNA1 and pGES201-sgRNA2) were transferred into recipient soybean DN50 cells via soybean genetic transformation. Following the soybean genetic transformation process (e.g., ... Figure 4 A total of 8 positive plants were obtained in the T0 generation (as shown in Figure D). Figure 4 (3~10 as shown in C).

[0039] V. Identification and Phenotypic Determination of GmSAMS Gene Mutants: 1. Identification of GmSAMS gene mutant materials: The eight T0 generation GmSAMS gene-positive plants obtained above were transplanted and harvested to obtain T1 generation mutant seeds, which were then propagated in a nursery for homozygous line isolation. Under the same conditions, T2 generation mutant plants and wild-type plants (WT) were planted, and leaf DNA was extracted. Specific detection primers were designed upstream and downstream of the gene editing site for PCR amplification, and the sequences were compared with the control group (WT).

[0040] like Figure 5 As shown in Figure A, three mutation types were detected in the GmSAMS gene mutants. The CDS sequence of gmsams-1 has a deletion of 8 bases (CTGAATTG) at target site 2, which causes premature termination of protein translation at amino acid 164. The CDS sequence of gmsams-2 has a deletion of 1 base (A) and an insertion of 1 base (G) at target site 2, which causes premature termination of protein translation at amino acid 137. The CDS sequence of gmsams-3 has an insertion of 1 base (A) at target site 2, which causes premature termination of protein translation at amino acid 167.

[0041] Genes with potential off-target sites were predicted using the CRISPR-GE website (http: / / skl.scau.edu.cn / ), and specific primers were designed for PCR amplification. No off-target effects were detected in any of the three types of GmSAMS gene mutants: gmsams-1, gmsams-2, and gmsams-3 (e.g., Figure 5 (As shown in B).

[0042] PCR identification of GmSAMS gene mutant materials using Cas9 gene-specific primers showed that some plants of gmsams-1, gmsams-2, and gmsams-3 did not show Cas9 gene segregation (e.g., Figure 5 C~ Figure 5 As shown in Figure E), this indicates that new editing types may emerge in the offspring. Therefore, lines homozygous for editing sites and without exogenous vector insertion were selected for subsequent phenotypic determination.

[0043] 2. Determination of oil and protein content in grains of GmSAMS gene mutant materials and observation by transmission electron microscopy: Under identical external conditions, GmSAMS gene mutant materials (gmsams-1, gmsams-2, and gmsams-3) and controls (wild WT plants) were planted. After the seeds matured, T2 generation transgenic seeds were collected according to different lines, and the protein content and total fatty acid content of the GmSAMS gene mutant materials were measured.

[0044] The crude protein and crude oil content of the harvested mutant plants were determined using a near-infrared analyzer (DA7250). Three plants of each mutant type were selected and planted independently. The total protein content was determined by Dumas nitrogen determination and the fatty acid content was determined by gas chromatography (as shown in Table 3).

[0045] Table 3 Total protein content and fatty acid content

[0046] The results showed that the average total protein content of mutant gmsams-1 plants was 44.78%, gmsams-2 plants 45.21%, gmsams-3 plants 45.12%, and WT plants 41.83%. The total protein content of the three lines (gmsams-1, gmsams-2, and gmsams-3) was significantly higher than that of the WT plants (e.g., ...). Figure 6 (As shown in A).

[0047] The fatty acid content of transgenic soybean seeds was determined. The results showed that the total fatty acid content of seeds from the gmsams-1, gmsams-2, and gmsams-3 mutant plants was significantly lower than that of the wild-type (WT) plant. Specifically, the average total fatty acid content of the gmsams-1 mutant plant was 19.08%, the gmsams-2 plant was 19.13%, the gmsams-3 plant was 19.06%, and the WT plant was 21.09% (e.g., WT ... Figure 6 (As shown in B).

[0048] Subsequently, the five major fatty acid components were analyzed. The palmitic acid content of the mutant plants gmsams-1 and gmsams-2 was significantly lower than that of the WT plants; the stearic acid content of gmsams-1 was significantly lower than that of WT, while the stearic acid content of gmsams-2 was significantly lower than that of WT; the oleic acid content of gmsams-1 was significantly higher than that of WT; the linoleic acid content of gmsams-1 and gmsams-3 was significantly lower than that of WT; and the linolenic acid content of gmsams-1 and gmsams-3 was significantly higher than that of WT (e.g., ...). Figure 6 (As shown in C).

[0049] Under the same planting conditions, samples were taken and fixed from soybean grains at the MM and LM stages of mutant materials and WT plants. After treatment, the morphology and number of oil bodies and protein bodies in cells at the same locations were observed using transmission electron microscopy. Black, round or nearly round structures in the field of view were protein bodies (SSPs). Small, densely packed transparent vacuoles near protein bodies within the cell wall were oil bodies (OBs). White, irregular, three-dimensional structures were starch granules. Compared with WT plants, the number of protein bodies in the gmsams-1, gmsams-2, and gmsams-3 mutant plants increased, while their volume decreased. There was no significant difference in the distribution of oil bodies and protein bodies, possibly because protein bodies and oil bodies were basically differentiated but still developing at the MM stage. At the LM stage, the mutant protein bodies increased in size and became more densely distributed, while the number of oil bodies decreased and their intracellular volume decreased (e.g., ...). Figure 6 (As shown in Figure D). In summary, the GmSAMS gene positively regulates the oil content of soybean seeds.

[0050] 3. Agronomic trait analysis of GmSAMS gene mutant materials: Under the same conditions, mutant plants gmsams-1, gmsams-2, and gmsams-3 and control wild plants (WT) were planted, and the changes in grain length and width were observed.

[0051] A direct comparison of soybean grains from WT and mutant plants clearly shows that the grain length and width of the gmsams-1, gmsams-2, and gmsams-3 mutants are significantly smaller than those of the WT wild-type control (e.g., ...). Figure 7 China A and Figure 7 (As shown in Figure B). Actual measurement results showed that the average grain length and width of mature seeds from the mutant plant were 5.03 mm and 4.98 mm, respectively, which were significantly different from the grain length and width of mature seeds from the wild plant WT (5.43 mm and 5.28 mm). Figure 7 C and Figure 7 (As shown in D). The above results indicate that the loss of function of the GmSAMS gene may reduce the grain length and width of soybeans, making the grains smaller. The GmSAMS gene may be involved in regulating the size of mature soybean grains.

[0052] 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 the GmSAMS gene in regulating soybean grain quality, characterized by, The nucleotide sequence of the GmSAMS gene is shown in SEQ ID NO.

1. The characteristic feature is that by silencing the GmSAMS gene, the quality of soybean seeds is regulated, the protein content of soybean seeds is increased, the oil content of soybean seeds is decreased, the total fatty acid content of soybean seeds is decreased, and the soybean seeds are made smaller.

2. The application of the GmSAMS gene as described in claim 1 in regulating soybean seed protein content, characterized in that, Increase soybean seed protein content by silencing the GmSAMS gene.

3. The application of the GmSAMS gene as described in claim 1 in regulating the oil content of soybean seeds, characterized in that, The oil content of soybean seeds can be reduced by silencing the GmSAMS gene.

4. The application of the GmSAMS gene as described in claim 1 in regulating the total fatty acid content of soybean seeds, characterized in that, Silencing the GmSAMS gene reduced the total fatty acid content of soybean seeds, thereby decreasing the content of palmitic acid, stearic acid, and linoleic acid in soybean seeds.

5. The application of the GmSAMS gene as described in claim 1 in regulating soybean seed size, characterized in that, By silencing the GmSAMS gene, soybean seeds can be made smaller.

6. The application of the GmSAMS gene protein as described in claim 1 in regulating soybean seed quality, characterized in that, The protein of the GmSAMS gene is S-adenosylmethionine synthase. The amino acid sequence of the GmSAMS gene protein is shown in SEQ ID NO.

2. By reducing the expression of S-adenosylmethionine synthase, the quality of soybean seeds is regulated, the protein content of soybean seeds is increased, the oil content of soybean seeds is reduced, the total fatty acid content of soybean seeds is reduced, and the soybean seeds are made smaller.

7. A recombinant plasmid for regulating soybean seed quality, characterized in that, The recombinant plasmid regulates soybean seed quality by silencing the GmSAMS gene described in claim 1, thereby increasing soybean seed protein content, decreasing soybean seed oil content, decreasing soybean seed total fatty acid content, and making soybean seeds smaller.

8. A method for regulating soybean seed quality, characterized in that, Includes the step of silencing the GmSAMS gene as described in claim 1 using sgRNA.

9. The method according to claim 8, characterized in that, The sgRNA includes sgRNA1 and sgRNA2, the nucleotide sequence of sgRNA1 is shown in SEQ ID NO.3, and the nucleotide sequence of sgRNA2 is shown in SEQ ID NO.4.

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