Method for increasing flavonoid content in soybean seeds and use thereof

Abstract

The present application relates to the technical field of soybean breeding, and specifically provides a method for increasing the content of flavonoids in soybean grains and application thereof.The method comprises the following steps: knocking out an E1 gene or an e1-as gene which is an allele of the E1 gene in soybean; the nucleotide sequence of the E1 gene is SEQ ID NO:1; and the nucleotide sequence of the e1-as gene is SEQ ID NO:5.Knocking out the above-mentioned genes in soybean can increase the content of flavonoids in soybean grains.The present application provides a brand-new breeding strategy for cultivating soybean strains with high flavonoid content, and has wide application prospect and economic value.

Description

Technical Field

[0001] This invention relates to the field of soybean breeding technology, and more specifically, to a method for increasing the content of flavonoids in soybean seeds and its application. Background Technology

[0002] Flavonoids in soybeans are important secondary metabolites formed during the soybean's growth process. Studies have shown that these compounds play important roles in cancer prevention, osteoporosis prevention, cardiovascular disease prevention, and menopausal syndrome relief. The flavonoids in soybean seeds mainly include isoflavones, such as genistein, genistein, and gentiopicrin. These compounds accumulate in the seeds, are primarily involved in plant reproduction, and promote human health by providing antioxidant protection and regulating hormones. Flavonoids play a crucial role in plant defense and soil microbial interactions. Therefore, increasing the flavonoid content in soybeans is of great significance. However, current research on flavonoids in soybean seeds is limited, and effective methods for increasing their content are lacking.

[0003] Studies on the soybean growth period gene E1 have shown that it plays a crucial role in regulating flowering and maturity, primarily by inhibiting flowering and prolonging the growth period. Mutants of the E1 gene exhibit early flowering characteristics. Current research mainly focuses on the molecular mechanisms by which the E1 gene affects flowering, but its functions in other aspects of soybean growth require further exploration. Summary of the Invention

[0004] The main objective of this invention is to provide a method for increasing the flavonoid content in soybean seeds and its application, so as to solve the problem of low flavonoid content in soybean seeds in the prior art.

[0005] To achieve the above objective, according to a first aspect of the present invention, a method for increasing the content of flavonoids in soybean seeds is provided, the method comprising: knocking out the E1 gene or the allele e1-as gene of the E1 gene in soybeans; wherein the nucleotide sequence of the E1 gene is SEQ ID NO: 1; and the nucleotide sequence of the e1-as gene is SEQ ID NO: 5.

[0006] Furthermore, the above knockout was performed using the CRISPR-Cas9 gene editing system.

[0007] Furthermore, the aforementioned CRISPR-Cas9 gene editing system includes sgRNA; the aforementioned sgRNA includes:

[0008] sgRNA1 or sgRNA2 targeting the E1 gene mentioned above;

[0009] sgRNA3 targeting the e1-as gene mentioned above;

[0010] The nucleotide sequence of sgRNA1 is SEQ ID NO: 2; the nucleotide sequence of sgRNA2 is SEQ ID NO: 3; and the nucleotide sequence of sgRNA3 is SEQ ID NO: 4.

[0011] Furthermore, the E1 gene was knocked out as follows: a deletion of bases at positions 55-301 of the nucleotide sequence of SEQ ID NO: 1.

[0012] Furthermore, the e1-as gene was knocked out as follows: a deletion of bases at positions 314-318 of the nucleotide sequence of SEQ ID NO: 5.

[0013] Furthermore, the flavonoids mentioned above are selected from isoflavones.

[0014] Furthermore, the aforementioned isoflavone compounds are selected from any one or more of the following: genistein, genistein, or gentiopicrin.

[0015] To achieve the above objectives, according to a second aspect of the present invention, the application of the above-described method in soybean breeding is provided.

[0016] Furthermore, the aforementioned soybean breeding includes increasing the content of flavonoids in soybean seeds.

[0017] By applying the technical solution of this invention, knocking out the E1 gene or its allele e1-as in soybeans can increase the content of flavonoids in soybean seeds. Therefore, the E1 gene and e1-as gene can be applied in soybean breeding. For example, compared to wild-type soybeans, mutant plants obtained by knocking out the E1 gene or e1-as gene show significantly higher levels of genistein, genistein, and gentiopicrin in their seeds. This invention provides a novel breeding strategy for cultivating soybean lines with high flavonoid content, possessing broad application prospects and economic value. Attached Figure Description

[0018] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0019] Figure 1 A schematic diagram of the E1 gene and e1-as gene knockout target sequences according to an embodiment of the present invention is shown.

[0020] Figure 2The diagram shows the detection of flavonoid content (daidzein, genistein, glycyrrhizin, daidzein, genistein and gentiopicrin) in the seeds of the e1-1 mutant according to an embodiment of the present invention.

[0021] Figure 3 The graph shows the detection of daidzein content in the seeds of the e1-1 mutant according to an embodiment of the present invention, where ** indicates p < 0.01 (t-test).

[0022] Figure 4 The graph showing the detection of genistein content in the seeds of the e1-1 mutant according to an embodiment of the present invention is shown, wherein **** indicates p < 0.0001 (t-test).

[0023] Figure 5 The diagram shows the detection of amygdalin content in the seeds of the e1-1 mutant according to an embodiment of the present invention, where **** indicates p < 0.0001 (t-test).

[0024] Figure 6 An edit type identification diagram of the e1-1 and e1-2 mutants according to an embodiment of the present invention is shown, where * indicates a region spanning 10 nucleotides.

[0025] Figure 7 The diagram shows the detection of flavonoid content (daidzein, genistein, glycyrrhizin, daidzein, genistein and gentiopicrin) in the seeds of the e1-2 mutant according to an embodiment of the present invention.

[0026] Figure 8 The graph shows the detection of daidzein content in the seeds of the e1-2 mutant according to an embodiment of the present invention, where ** indicates p < 0.01 (t-test).

[0027] Figure 9 The graph shows the detection of genistein content in seeds of the e1-2 mutant according to an embodiment of the present invention, where ** indicates p < 0.01 (t-test).

[0028] Figure 10 The diagram shows the detection of amygdalin content in the seeds of the e1-2 mutant according to an embodiment of the present invention, where * indicates p < 0.05 (t-test). Detailed Implementation

[0029] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0030] Terminology Explanation:

[0031] Flavonoids are a class of polyphenolic compounds widely found in plants, sharing a common chemical structure: two benzene rings (A and B rings) connected by a three-carbon bridge (C6-C3-C6). They are secondary metabolites in plants, participating in color formation, ultraviolet filtering, antioxidant protection, and pest and disease defense. Flavonoids include flavones, flavonols, isoflavones, flavanones, flavanols, and anthocyanins, among others.

[0032] Isoflavones: These compounds have a basic 15-carbon skeleton, typically a 3-phenylchromone structure. This differs from the 2-phenylchromone structure of other flavonoids. The structure of isoflavones gives them unique biological activities. Isoflavones exist in both glycosidic and non-glycosidic forms. Daidzein, genistein, and glycyrrhizin are glycosidic forms of isoflavones. These compounds contain a sugar molecule (usually glucose) in their structure, linked to the isoflavone portion via a glycosidic bond. Genistein, genistein, and gentiopicrin are non-glycosidic forms of isoflavones and do not contain a sugar molecule.

[0033] Daidzin: The 7-O-glucoside of daidzein. Daidzein typically has a hydroxyl group (-OH) at position 4 and is linked to glucose at position 7. Daidzin contains one glucose molecule, is highly water-soluble, and belongs to the glycosidic form of soy isoflavones.

[0034] Genistin: The 7-O-glucoside of genistein, which has hydroxyl groups at positions 5 and 7, with a glucose molecule attached at position 7. Genistin contains one glucose molecule, is highly water-soluble, and is found in soybeans. Similar to daidzein, but with a different aglycone: genistein.

[0035] Glycyrrhizin: Glycyrrhizin is the 7-O-glucoside of glycitein. Glycitein has a hydroxyl group at position 4, a methoxy group (-OCH3) at position 6, and a glucose molecule at position 7. It contains one glucose molecule and differs slightly in structure from other glycosides, affecting its biological activity.

[0036] Daidzein: An isoflavone compound. It is the aglycone form of daidzein, lacking a sugar motif and typically containing hydroxyl groups (-OH) at positions 4 and 7. Daidzein does not contain sugar molecules, only the basic isoflavone skeleton. It is highly lipid-soluble and is the non-glycosidic form of soy isoflavones.

[0037] Genistein: An isoflavone compound. It is the aglycone form of genistein, also lacking a glycosyl group, and has hydroxyl groups (-OH) at positions 5, 7, and 4. Due to the additional hydroxyl group at position 5, genistein exhibits outstanding antioxidant and estrogenic activities.

[0038] Formononetin: an isoflavone compound. Formononetin has a hydroxyl group (-OH) at position 4 and a methoxy group (-OCH3) at position 7. The methoxy group at position 7, instead of a hydroxyl group, causes it to differ in biological activity from the other two isoflavones.

[0039] Flavonoids possess a variety of biological activities and potential health benefits, including but not limited to: 1) Antioxidant effects: Flavonoids can scavenge free radicals in the body and reduce oxidative stress damage to cells. This helps prevent chronic diseases and delay aging; 2) Anti-inflammatory effects: They can inhibit the production of inflammatory mediators, thereby reducing inflammatory responses and combating various diseases caused by inflammation; 3) Cardiovascular protection: Flavonoids help improve vascular function, lower blood pressure and cholesterol levels, thereby reducing the risk of cardiovascular disease; 4) Anti-cancer effects: Through multiple mechanisms, including inducing apoptosis in cancer cells and inhibiting cancer cell proliferation and spread, flavonoids show potential in cancer prevention and treatment; 5) Antibacterial and antiviral effects: Some flavonoids have inhibitory effects on various bacteria and viruses, helping to enhance immune function; 6) Neuroprotective effects: They may have a protective effect on the nervous system through antioxidant and anti-inflammatory mechanisms, and may be used for the prevention and treatment of neurodegenerative diseases.

[0040] Soybean breeding: refers to the process of genetically improving soybeans through techniques such as selection, hybridization, and genetic engineering in order to cultivate new varieties with superior traits (such as high yield, disease resistance, stress resistance, and high quality).

[0041] As mentioned in the background section, the content of flavonoids in soybean seeds is low in existing technologies, which limits the application potential of soybeans. In this invention, the inventors attempted to increase the flavonoid content in soybean seeds by modifying soybean genes, and thus proposed the protection scheme of this invention.

[0042] In a first typical embodiment of the present invention, a method for increasing the flavonoid content in soybean seeds is provided. The method includes: knocking out the E1 gene or the e1-as allele of the E1 gene in soybeans; wherein the nucleotide sequence of the E1 gene is SEQ ID NO: 1; and the nucleotide sequence of the e1-as gene is SEQ ID NO: 5.

[0043] The E1 gene (gene number: Glyma.06G207800) in soybean is an important molecular site regulating flowering, but whether it has the function of increasing flavonoid content in seeds is unknown. The E1 gene in soybean is an intronless gene with three alleles: e1-as, e1-fs, and e1-nl. These four different genotypes are distributed in different varieties.

[0044] This study used two transgenic recipient materials. TL1 (Tianlong No. 1) contains the E1 genotype, with the nucleotide sequence SEQ ID NO: 1, and the E1 gene function is fully preserved. W82 (Williams 82) contains the e1-as genotype, with the nucleotide sequence SEQ ID NO: 5. Studies have shown that e1-as is an E1 percolation allele, which can be normally expressed in soybeans and retains some of the E1 gene function. The nucleotide sequence lengths of E1 and e1-as are identical, differing only by one base at nucleotide position 44.

[0045] The other two alleles, e1-fs and e1-nl, have significantly different nucleotide sequences from the E1 gene and have lost their function. Therefore, this invention did not investigate the mutation and function of these two genes (Z.Xia, et. Positional cloning and characterization reveal the molecular basis for soybean maturity locus E1 that regulates photoperiodic flowering, Proc. Natl. Acad. Sci. USA 109(32) E2155-E2164).

[0046] The sequence of SEQ ID NO: 1 is as follows:

[0047] atgagcaacccttcagatgaaagggagcagtgtcaaaagaagaggaaatccaccatatgcgaagcctctaactttaggacatcaaggagaagattctgcagcaacaacaaaaatgaagaggagatgaacaataagggagtttcaacaacactgaagctttacgatgatccttggaagatcaagaagacgctaaccgatagcgatttgggaatcctaagtagactcttgctggctgcagatttggtgaagaaacaaattttgcctatgttgggtgcatatcatgcaagagctgcagaaactgaagggaccccagttagagtttgggacatggacaccaaatccatgcaccaactcgttctaaagcgatggtcttcatccaagagctatgttcttattggaaagtggaaccaagatttcgtcagaagaagagatctcaggaaaggtgatgagatcggatttcattgggatccatataattgcgttttcaatttctgtgtccttaaacaagctatgccagagaattaa。

[0048] The sequence of SEQ ID NO: 5 is:

[0049] .

[0050] Knocking out the E1 gene or its allele e1-as gene in soybeans significantly increases the content of flavonoids in soybean seeds. For example, the knockout mutants of this invention (including the e1-1 mutant and the e1-2 mutant) show significantly increased contents of genistein, genistein, and gentiopicrin. In this invention, "increased" means that the content of flavonoids in the mutant seeds is increased by at least 10%, at least 8%, at least 6%, at least 4%, at least 2%, at least 1%, at least 0.1%, or at least 0.01% compared to the content of flavonoids in the seeds of the plant before the mutation.

[0051] It should be noted that gene knockout refers to the deletion or complete inactivation of a specific gene from an organism's genome using gene editing technology in order to study the gene's effects on the organism.

[0052] Methods for achieving gene knockout are well known to those skilled in the art, and include, but are not limited to: 1) using homologous recombination to introduce modified DNA fragments to replace the target gene with a non-functional or damaged version. When the exogenous DNA undergoes homologous recombination with a gene in the recipient cell genome that has the same or similar sequence, the target gene is replaced and inactivated; 2) using RNA interference to silence or degrade the mRNA of the target gene using small RNA molecules, preventing its translation into protein, thereby achieving gene knockout; or 3) using the CRISPR-Cas9 system to introduce targeted DNA breaks at specific locations in the genome using sgRNA and the Cas9 enzyme, leading to gene damage. CRISPR-Cas9 uses the non-homologous end joining repair pathway to generate frameshift mutations or fragment deletions at the break sites, silencing the gene and causing loss of function.

[0053] In other words, any method that can knock out the E1 gene or the e1-as gene is applicable to this invention. For example, RNA interference or the use of the CRISPR-Cas9 system to introduce targeted DNA breaks or homologous recombination at specific locations in the genome can all achieve this.

[0054] In a preferred embodiment of the present invention, the knockout is performed using a CRISPR-Cas9 gene editing system. In a more preferred embodiment of the present invention, the CRISPR-Cas9 gene editing system includes sgRNA; the sgRNA includes: sgRNA1 or sgRNA2 targeting the E1 gene; and sgRNA3 targeting the e1-as gene; wherein the nucleotide sequence of sgRNA1 is SEQ ID NO: 2; the nucleotide sequence of sgRNA2 is SEQ ID NO: 3; and the nucleotide sequence of sgRNA3 is SEQ ID NO: 4.

[0055] Designing sgRNA sequences based on the target gene sequence is a conventional method in the art. Any sgRNA capable of knocking out and inactivating the target gene is suitable for this invention, such as sgRNA1 or sgRNA2 targeting the E1 gene and sgRNA3 targeting the e1-as gene.

[0056] In a preferred embodiment of the present invention, the E1 gene is knocked out as follows: the bases at positions 55-301 of the nucleotide sequence of SEQ ID NO: 1 are deleted.

[0057] When the E1 gene in soybeans undergoes the aforementioned mutation, the resulting soybean mutants exhibit a large deletion in the amino acid sequence of the protein encoded by this gene. Amino acid sequence differences begin from amino acid position 19, leading to premature termination of the downstream sequence after frameshift. The sequence length decreases from 174 amino acids to 37 amino acids, resulting in loss of protein function. When the E1 gene is knocked out, the protein encoded by this gene loses its function, and the content of flavonoids in soybean seeds significantly increases.

[0058] In a preferred embodiment of the present invention, the above-mentioned e1-as gene is knocked out in the following manner: the bases at positions 314-318 of the nucleotide sequence of SEQ ID NO: 5 are deleted.

[0059] When the e1-as gene in soybean undergoes the aforementioned mutation, the amino acid sequence of the encoded protein in the resulting soybean mutant differs from amino acid position 105, leading to premature termination of the downstream sequence after a frameshift. The sequence length decreases from 174 amino acids to 146 amino acids, and the protein loses its function. When the e1-as gene is knocked out, the encoded protein loses its function, and the content of flavonoids in soybean seeds significantly increases.

[0060] In other words, knocking out the E1 gene or e1-as gene in soybeans can increase the content of flavonoids in soybean seeds, such as genistein, genistein, or gentiopicrin.

[0061] It should be noted that the specific mutation types described above are merely one manifestation of the loss of function of the E1 gene or the e1-as gene. Any other mutation type capable of causing the loss of function of these two genes is applicable to this invention. For example, the deletion of bases at positions 55-301 of the nucleotide sequence in SEQ ID NO: 1 in this invention knocks out the E1 gene in soybeans, thus inactivating its function. As another example, the deletion of bases at positions 314-318 of the nucleotide sequence in SEQ ID NO: 5 in this invention knocks out the e1-as gene, thus inactivating its function.

[0062] Furthermore, it should be noted that the nucleotide sequence lengths of the E1 gene and the e1-as gene in wild-type soybean are identical, differing only by one base at nucleotide position 44. Moreover, the mutation regions of the e1-1 and e1-2 mutants, which are derived from mutations of the two genes respectively, do not involve amino acid position 15.

[0063] This indicates that if the E1 gene undergoes a mutation of the same type as the e1-2 mutant (i.e., a deletion of bases 314-318 in the nucleotide sequence of SEQ ID NO: 1, resulting in an amino acid sequence difference starting from amino acid 105, leading to premature termination of the downstream sequence after a frameshift. The sequence length changes from 174 amino acids to 146 amino acids), the E1 gene will also be completely inactivated. Similarly, if the E1 allele e1-as gene undergoes a mutation of the same type as the e1-2 mutant (i.e., a deletion of bases 55-301 in the nucleotide sequence of SEQ ID NO: 5, resulting in an amino acid sequence difference starting from amino acid 19, leading to premature termination of the downstream sequence after a frameshift. The sequence length changes from 174 amino acids to 37 amino acids), the e1-as gene will also be completely inactivated.

[0064] In other words, this invention provides two mutation types that cause inactivation of the E1 gene or the e1-as gene for both the E1 gene and the e1-as gene.

[0065] In a preferred embodiment of the present invention, the flavonoid compound is selected from isoflavone compounds. In a more preferred embodiment of the present invention, the isoflavone compound is selected from any one or more of the following: genistein, genistein, or gentiopicrin.

[0066] Flavonoids, primarily isoflavones, are an important source of phytoestrogens and possess various biological activities. Therefore, increasing the flavonoid content in soybeans is of great significance. Genistein, genistein, and gentianin are all isoflavones. Genistein is a natural isoflavone and one of the most abundant isoflavones in soybeans, exhibiting weak estrogen-like effects and possessing various biological activities such as antioxidant, anti-inflammatory, anticancer, and cardiovascular protective properties. Genistein, another isoflavone mainly found in soybeans, has anticancer, anti-inflammatory, bone health-improving, and potential cardiovascular protective effects. Genistein has potential antioxidant, anti-inflammatory, and anticancer effects and may affect estrogen receptors; its content in soybeans is relatively low.

[0067] In a second typical embodiment of the present invention, the above-described method is provided for application in soybean breeding.

[0068] In a preferred embodiment of the present invention, the soybean breeding includes increasing the content of flavonoids in soybean seeds. In a more preferred embodiment of the present invention, the flavonoids are selected from isoflavones. In a further preferred embodiment of the present invention, the isoflavones are selected from any one or more of the following: genistein, genistein, or gentiopicrin.

[0069] The present invention will be further described in detail below with reference to specific embodiments, which should not be construed as limiting the scope of protection claimed by the present invention.

[0070] Example 1: Acquisition of e1 mutants (including e1-1 mutants and e1-2 mutants) and identification of gene editing type

[0071] (1) Obtaining the e1-1 mutant

[0072] The e1-1 mutant material used in this invention was donated by the Zhai Hong Laboratory of the Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences. This mutant was obtained by gene editing of the E1 gene of Tianlong No. 1 (code name TL1) using the WMC012 vector, with Tianlong No. 1 as the recipient material. The target sequences of the sgRNA used for gene editing are as follows: SgRNA1 (SEQ ID NO: 2): CAAGAGCTGCAGAAACTGAAGGG; SgRNA2 (SEQ ID NO: 3): CCACCATATGCGAAGCCTCTAAC. Detailed procedures are published in reference 1 (CRISPR / Cas9-mediated targeted mutation of the E1 decreases photoperiod sensitivity, alters stem growth habits, and decreases branch number in soybean, Front. Plant Sci., 14 December 2022 Sec. Crop and Product Physiology). The nucleotide sequence of the soybean wild-type E1 gene in this invention is SEQ ID NO: 1; this sequence is the genomic sequence of the E1 gene. The e1-1 mutant of the present invention refers to the e1-2 mutant in the above-mentioned reference 1.

[0073] (2) Obtaining the e1-2 mutant

[0074] The e1-2 mutant material used in this patent was provided by the Kong Fanjiang Laboratory at Guangzhou University. This mutant was obtained by gene editing the e1-as gene using the pYLCRISPRCas9P35-BS vector with W82 as the recipient material, and was named the e1-2 mutant. The sgRNA target sequence used for gene editing is as follows: sgRNA3 (SEQ ID NO: 4): AGGGACCCCAGTTAGAGTTTGGG. Detailed construction methods are published in Reference 2 (Diverse flowering responses subjecting to ambient high temperature in soybean under short-day conditions, Plant Biotechnology Journal (2023) 21, pp. 782–791). E1, E1L1a, and E1L1b mentioned in Reference 2 are homologous genes; therefore, the same CRISPR vector and target were used when designing the gene editing material. The target and editing information of the e1-2 mutant obtained by the above method is published in reference 3 (Noveland multifaceted regulations of photoperiodic flowering by phytochrome Ainsoybean. Proc Natl Acad Sci US A. 2022 Oct11; 119(41):e2208708119). The e1-2 mutant in this application refers to the mutants PHYA2 PHYA3 e1 E1la E1lb in reference 3. The nucleotide sequence of the e1-as gene in wild-type W82 soybean is SEQ ID NO: 5.

[0075] It should be noted that the methods for obtaining transgenic materials in this invention are all conventional methods in the field.

[0076] (3) Identification of gene editing types in e1-1 and e1-2 mutants

[0077] ① Extraction of mutant soybean genomic DNA using the CTAB method

[0078] Mutants e1-1 and e1-2 were planted in the soil. After the trifoliate compound leaves emerged, genomic DNA was extracted from the leaves using the CTAB method: Approximately 6 mM of fresh soybean leaves was placed in a 2.0 mL centrifuge tube, a clean steel ball with a diameter of 2 mm was added, and the tube was pre-cooled in liquid nitrogen. Then, the sample was sampled at 50 Hz for 1 min. 300 μL of CTAB extraction buffer was added, the tube was shaken thoroughly, and incubated at 65 °C for 30 min. After cooling to room temperature, 300 μL of phenol:chloroform:isoamyl alcohol was added. Mix (25:24:1); centrifuge at 12000 rpm for 10 min, transfer the supernatant to a new 1.5 mL centrifuge tube, add 0.6 times the volume of isopropanol, gently invert to mix, and let stand at -20℃ for 30 min; centrifuge at 12000 rpm for 10 min at room temperature, discard the supernatant, add 1 mL of 70% ethanol to rinse; centrifuge at 12000 rpm for 5 min at room temperature, blow under a clean bench for 5-10 min; add 50 μL of ddH2O, dissolve, and store at 4℃.

[0079] ② Identification of mutant gene editing types

[0080] PCR primers were designed upstream and downstream of the sgRNA target site in the soybean genome to amplify the genomic fragment containing sgRNA. The PCR products were sent to a sequencing company for sequencing to determine the gene editing status in this region. The amplification reaction system is shown in Table 1, and the amplification reaction procedure is shown in Table 2.

[0081] The nucleotide sequences of the amplification primers for both e1-1 and e1-2 mutants are as follows:

[0082] E1-test-F (SEQ ID NO: 14): AAGGGGAAATGTGCCTTCAC;

[0083] E1-Test-R (SEQ ID NO: 15): CGAGTTGGTGCATGGATTTGG.

[0084] Table 1:

[0085]

[0086]

[0087] Table 2:

[0088]

[0089] Sequencing results showed that the E1 gene in mutant e1-1 had a 247 bp deletion in the region near two sgRNAs. Specifically, the mutation type was a 247 bp deletion of nucleotides from position 55 to 301 in the nucleotide sequence of SEQ ID NO: 1 (see [link to SEQ ID NO: 1]). Figure 1In this mutant, a large segment of the amino acid sequence is deleted, with amino acid sequence differences starting from amino acid position 19, leading to premature termination of the downstream sequence after frameshift. The sequence length is reduced from 174 amino acids to 37 amino acids, and the protein loses its function (see...). Figure 6 ).

[0090] Figure 6 , SEQ ID NO: 6 is: atgagcaacccttcagatgaaagggagcagtgtcaaaagaagacgaaatccacc.

[0091] SEQ ID NO: 7 is:

[0092] atgagcaacccttcagatgaaagggagcagtgtcaaaagaagacgaaatccaccatatgcgaagcctctaactttaggacatcaaggagaagattctgcagcaacaacaaaaatgaagaggagatgaacaataagggagtttcaacaacactgaagc tttacgatgatccttggaagatcaagaagacgctaaccgatagcgatttgggaatcctaagtagactcttgctggctgcagatttggtgaagaaacaaattttgcctatgttgggtgcatatcatgcaagagctgcagaaactgaagggaccccag.

[0093] SEQ ID NO: 8 is:

[0094] aagggaccccagttagagtttgggacatggacaccaaatccatgcaccaactcgttctaaagcgatggtcttcatccaagagctatgttctt attggaaagtggaaccaagatttcgtcagaagaagagatctcaggaaaggtgatgagatcggatttcattgggatccatataattgcgttttcaattt ctgtgtccttaaacaagctatgccagagaattaa.

[0095] SEQ ID NO: 9 is:

[0096] gtttgggacatggacaccaaatccatgcaccaactcgttctaaagcgatggtcttcatccaagagctatgttcttattggaaagtggaaccaa gatttcgtcagaagaagagatctcaggaaaggtgatgagatcggatttcattgggatccatataattgcgttttcaatttctgtgtccttaaacaagcta tgccagagaattaa。[[ID=~1]] [[ID=~2]]

[0097] [[ID=~3]]SEQ ID NO: 10 is: [[ID=~4]] [[ID=~5]]

[0098] [[ID=~6]]MSNPSDEREQCQKKRKSTICEASNFRTSRRRFCSNNKNEEEMNNKGVSTTLKLYDDPW KIKKTLTDSDLGILSRLLLAADLVKKQILPMLGAYHARAAETEGTPVRVWDMDTKSMHQLV LKRWSSSKSYVLIGKWNQDFVRRRDLRKGDEIGFHWDPYNCVFNFCVLKQAMPEN。[[ID=~7]] [[ID=~8]]

[0099] [[ID=~9]]SEQ ID NO: 11 is: [[ID=~10]] [[ID=~11]]

[0100] [[ID=~12]]MSNPSDEREQCQKKTKSTICEASNFRTSRRRFCSNNKNEEEMNNKGVSTTLKLYDDPW KIKKTLTDSDLGILSRLLLAADLVKKQILPMLGAYHARAAETEGTPVRVWDMDTKSMHQLV LKRWSSSKSYVLIGKWNQDFVRRRDLRKGDEIGFHWDPYNCVFNFCVLKQAMPEN。[[ID=~13]] [[ID=~14]]

[0101] [[ID=~15]]SEQ ID NO: 12 is: [[ID=~16]] [[ID=~17]]

[0102] [[ID=~18]]MSNPSDEREQCQKKRKSTKGPQLEFGTWTPNPCTNSF。[[ID=~19]] [[ID=~20]]

[0103] [[ID=~21]]SEQ ID NO: 13 is: [[ID=~22]] [[ID=~23]]

[0104] MSNPSDEREQCQKKTKSTICEASNFRTSRRRFCSNNKNEEEMNNKGVSTTLKLYDDPW KIKKTLTDSDLGILSRLLLAADLVKKQILPMLGAYHARAAETEGTPGLGHGHQIHAPTRSKAMVFIQELCSYWKVEPRFRQKKRSQER.

[0105] The e1-as gene in mutant e1-2 has a 5 bp deletion at positions 314 to 318 of the nucleotide sequence of SEQ ID NO: 5 (see [link to SEQ ID NO: 5]). Figure 1 In this mutant, an amino acid sequence difference occurs starting from amino acid position 105, leading to premature termination of the downstream sequence after a frameshift. The sequence length changes from 174 amino acids to 146 amino acids, and the protein loses its function (see...). Figure 6 ).

[0106] Example 2: Determination of flavonoid content in the e1 mutant

[0107] (1) Extraction and detection of flavonoids in seeds

[0108] Seed sample preparation: After the soybean seeds mature, prepare seed samples and dry them thoroughly.

[0109] Detection: The dried seeds were ground using a grinder, and the ground sample was weighed. MeOH was added at a material-to-liquid ratio of 200 mg / mL, and the mixture was vortexed and then extracted using low-temperature ultrasonication for 30 min. After the sample had reacted completely, it was centrifuged at 4℃ and 12700 rpm for 10 min. The supernatant was collected and passed through a 0.22 μm organic membrane. The mixture was then centrifuged again at 4℃ and 12700 rpm for 10 min. The flavonoid content was determined by liquid chromatography using the supernatant.

[0110] (2) Measurement results:

[0111] like Figures 2-5 As shown, the results indicate that, compared to Tianlong No. 1, the e1-1 mutant showed a significant upregulation in the content of three important flavonoid compounds in the seeds, including daidzein, genistein, and formononetin.

[0112] like Figures 7-10 As shown, the results indicate that, compared to the W82, the e1-2 mutant has significantly higher contents of daidzein, genistein, and formononetin in its grains.

[0113] As can be seen from the above description, the above embodiments of the present invention achieve the following technical effects: compared with the wild type of Tianlong No. 1, the seeds of soybeans with E1 gene knockout (e.g., e1-1 mutant) and soybeans with E1 gene allele e1-as gene knockout (e.g., e1-2 mutant) show a significant upregulation of three important flavonoid compounds, namely daidzein, genistein and formononetin.

[0114] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for increasing the flavonoid content in soybean seeds, characterized in that, The method includes: knocking out [the pollutants] in soybeans. E1 Gene or the stated E1 Alleles of a gene e1-as Gene; Among them, the E1 The nucleotide sequence of the gene is SEQ ID NO: 1; e1-as The nucleotide sequence of the gene is SEQ ID NO: 5; The flavonoids are selected from any one or more of the following: genistein, genistein, or gentiopicrin.

2. The method according to claim 1, characterized in that, The knockout was performed using the CRISPR-Cas9 gene editing system.

3. The method according to claim 2, characterized in that, The CRISPR-Cas9 gene editing system includes sgRNA; the sgRNA includes: Targeted E1 The gene's sgRNA1 or sgRNA2; Targeted e1-as sgRNA3 of the gene; The nucleotide sequence of sgRNA1 is SEQ ID NO: 2; the nucleotide sequence of sgRNA2 is SEQ ID NO: 3; and the nucleotide sequence of sgRNA3 is SEQ ID NO:

4.

4. The method according to any one of claims 1-3, characterized in that, Knock out the following method E1 Gene: Deletion of bases at positions 55-301 of the nucleotide sequence of SEQ ID NO:

1.

5. The method according to any one of claims 1-3, characterized in that, Knock out the following method e1-as Gene: Deletion of bases at positions 314-318 of the nucleotide sequence of SEQ ID NO:

5.

6. The application of the method according to any one of claims 1-5 in soybean breeding.

7. The application according to claim 6, characterized in that, The soybean breeding program includes increasing the content of flavonoids in soybean seeds.

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