Soybean glyma.03g22970 gene enhancer regulatory element and sgRNA molecule thereof

Abstract

The application discloses a soybean Glyma.03G22970 gene enhancer regulatory element and an sgRNA molecule thereof. The enhancer regulatory element is an MHS2 regulatory element, and the nucleotide sequence of the MHS2 regulatory element is shown as SEQ ID NO:1. The sgRNA molecule has at least one of the nucleotide sequences shown as SEQ ID NO:3-4, and optionally has at least one of the nucleotide sequences shown as SEQ ID NO:2 and SEQ ID NO:5. The MHS2 regulatory element can be used as an enhancer of the Glyma.03G22970 gene, reduces the expression amount of the Glyma.03G22970 gene, and improves the oil content of soybean. The sgRNA molecule can make the sequence of the MHS2 regulatory element partially or completely missing, and has the advantages of low off-target rate, high editing efficiency and the like.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to an enhancer regulatory element of the soybean Glyma.03G22970 gene and its sgRNA molecule. More specifically, this invention relates to the use of the MHS2 regulatory element in regulating the expression of the Glyma.03G22970 gene, the use of the MHS2 regulatory element in regulating soybean oil content, a method for regulating soybean oil content, expression vectors, reagents, a CRISPR / Cas9 system, and a kit. Background Technology

[0002] Soybean (Glycine max) is an important food and oilseed crop belonging to the legume family. Originating in Northeast Asia, it is one of the world's most important sources of protein and one of the most important vegetable oilseed crops.

[0003] Soybean oil is a vegetable oil extracted from soybean seeds and is an important edible oil. It is rich in unsaturated fatty acids, linoleic acid, vitamin E, and other substances. Soybean oil has wide applications in food processing, cooking, and seasoning. It is used to make food products, vegetable oils, edible oils, and condiments.

[0004] However, nitrogen is one of the main limiting factors for soybean oil content. Soybean plants absorb nitrogen from the environment and convert it into amino acids within their bodies, which then participate in the synthesis of proteins and other bioactive substances. Amino acids are one of the key precursors for soybean oil formation. Current research has found that the oil content of soybeans can be regulated by controlling proteins. For example, UMAMIT transporters are a class of common amino acid transporters in plants, responsible for the transport and distribution of amino acids within the plant, and can affect the oil content of soybeans. Summary of the Invention

[0005] This invention aims to at least partially address one of the technical problems in the related art. To this end, a first aspect of this invention reveals the use of the MHS2 regulatory element in regulating the Glyma.03G22970 gene.

[0006] This invention is primarily based on the following discoveries of the inventors:

[0007] The inventors discovered that the Glyma.03G22970 gene in soybeans is similar to UMAMIT25, and may have similar amino acid transport functions. Based on this, while studying the Glyma.03G22970 gene, the inventors unexpectedly discovered an enhancer of the Glyma.03G22970 gene, located upstream of the Glyma.03G22970 gene at Gm03:44336551..44336694(+strand), named MHS2. Furthermore, the inventors unexpectedly found that knocking out the MHS2 gene in soybeans significantly reduced the expression level of the Glyma.03G22970 gene, especially compared to the wild-type group without MHS2 knockout, the expression level of the Glyma.03G22970 gene decreased by 10%–63%, the protein content in the seeds decreased by 2%–6%, and the oil content in the seeds increased by 1%–5%.

[0008] Based on this, in a first aspect, the present invention proposes the use of the MHS2 regulatory element (also known as MHS2) in regulating the expression of the Glyma.03G22970 gene, the nucleotide sequence of which is shown in SEQ ID NO:1. This invention is the first to discover that the MHS2 regulatory element can act as an enhancer of the Glyma.03G22970 gene to regulate its expression.

[0009] In two aspects, this invention proposes the use of the MHS2 regulatory element in regulating the oil content of soybeans, the nucleotide sequence of which is shown in SEQ ID NO:1. This invention is the first to discover that the MHS2 regulatory element can regulate the oil content of soybeans by controlling the expression of the Glyma.03G22970 gene.

[0010] A third aspect of this invention provides a method for regulating the oil content of soybeans. According to an embodiment of the invention, the method includes: modifying and altering the nucleotide sequence of an MHS2 regulatory element in soybeans, the nucleotide sequence of which is shown in SEQ ID NO:1. The method of this invention can partially or completely delete the nucleotide sequence of the MHS2 regulatory element, resulting in partial or complete loss of activity of the MHS2 regulatory element, thereby reducing the expression level of the Glyma.03G22970 gene and increasing the oil content of soybeans.

[0011] In a fourth aspect, the present invention provides an sgRNA molecule. According to embodiments of the present invention, the sgRNA molecule has at least one of the nucleotide sequences shown in SEQ ID NO: 3-4, and optionally at least one of the nucleotide sequences shown in SEQ ID NO: 2 and SEQ ID NO: 5. The sgRNA molecule of the present invention can partially or completely delete the sequence of the soybean MHS2 regulatory element, resulting in partial or complete loss of activity of the MHS2 regulatory element, and the sgRNA molecule has advantages such as low off-target rate and high editing efficiency. Furthermore, by using the sgRNA molecule to partially or completely delete the sequence of the soybean MHS2 regulatory element, the activity of the MHS2 regulatory element can be partially or completely lost, reducing the expression level of the Glyma.03G22970 gene and increasing the oil content of soybean.

[0012] In a fifth aspect, the present invention provides an expression vector. According to embodiments of the invention, the expression vector carries the sgRNA molecule described in the fourth aspect of the invention and, optionally, nucleic acid encoding a Cas9 molecule. This expression vector can partially or completely delete sequences of MHS2 regulatory elements, thereby partially or completely deleting sequences that activate MHS2 regulatory elements, thus reducing the expression level of the Glyma.03G22970 gene and increasing the oil content of soybeans.

[0013] In a sixth aspect, the present invention provides a reagent. According to embodiments of the present invention, the reagent comprises the sgRNA molecule described in the fourth aspect of the present invention or the expression vector described in the fifth aspect of the present invention. This reagent can partially or completely delete the sequence of the MHS2 regulatory element, thereby partially or completely losing the activity of the MHS2 regulatory element, thus reducing the expression level of the Glyma.03G22970 gene and increasing the oil content of soybean.

[0014] In a seventh aspect, the present invention provides a CRISPR / Cas9 system. According to embodiments of the invention, the CRISPR / Cas9 system comprises the sgRNA molecule described in the fourth aspect of the invention and, optionally, nucleic acid encoding a Cas9 molecule. This CRISPR / Cas9 system can partially or completely delete the sequence of the MHS2 regulatory element, thereby partially or completely losing the activity of the MHS2 regulatory element, thus reducing the expression level of the Glyma.03G22970 gene and increasing the oil content of soybeans.

[0015] In an eighth aspect, the present invention provides a kit. According to embodiments of the invention, the kit comprises the sgRNA molecule described in the fourth aspect of the invention or the expression vector described in the fifth aspect of the invention; and optionally, nucleic acid encoding a Cas9 molecule. This kit can partially or completely delete the sequence of the MHS2 regulatory element, thereby partially or completely losing the activity of the MHS2 regulatory element, thus reducing the expression level of the Glyma.03G22970 gene and increasing the oil content of soybean.

[0016] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0017] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0018] Figure 1 This demonstrates the specific high abundance expression of the Glyma.03G22970 gene in soybean seeds as shown in Example 1.

[0019] Figure 2 This shows the location of the seed-specific MHS2 regulatory element on the genome in Example 1.

[0020] Figure 3 This document describes the CRISPR / Cas9 knockout target site and its location for the Glyma.03G22970-MHS2 gene in Example 1, and the construction process of the Glyma.03G22970-MHS2-Cas9 vector in Example 2.

[0021] Figure 4 The mutation types are those of the three mutants in Example 3.

[0022] Figure 5 The expression levels of the Glyma.03G22970 gene in the wild type and mutant in Example 4 are shown.

[0023] Figure 6 The content of soybean seed protein in wild-type and mutant soybeans in Example 4 is shown.

[0024] Figure 7 The oil content of wild-type and mutant soybean seeds in Example 4 is shown. Detailed Implementation

[0025] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0026] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0027] To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this invention, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0028] In this invention, the terms "comprising" or "including" are open-ended expressions, meaning they include the contents specified in this invention but do not exclude other aspects.

[0029] In this invention, the terms “optionally,” “optionally,” or “optionally” generally refer to events or conditions described subsequently that may but may not occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0030] In this invention, "modification or alteration of the nucleotide sequence of the MHS2 regulatory element" refers to changes in the nucleotide sequence of the MHS2 regulatory element, thereby affecting its binding ability to transcription factors. As an enhancer of the Glyma.03G22970 gene, the MHS2 regulatory element can regulate the expression of the Glyma.03G22970 gene by altering its binding ability to transcription factors. These alterations can take various forms, including but not limited to sequence deletions, substitutions, insertions, inversions, translocations, and methylation and / or acetylation.

[0031] In this invention, "regulatory elements" generally refer to DNA sequences produced by gene transcription regulation, mainly including promoters, enhancers, insulators, etc.

[0032] In this invention, "stable inheritance" means that the mutant after gene editing can stably pass on the mutated sequence to its offspring in a genetic form.

[0033] In this invention, "transcription factor" refers to a group of protein molecules that can specifically bind to a specific sequence upstream of the 5' end of a gene, thereby ensuring that the target gene is expressed at a specific intensity at a specific time and space.

[0034] In this invention, "gene editing" is also known as genome editing or genome engineering. It is an emerging and relatively precise genetic engineering technology that can modify specific target genes in the genome of an organism.

[0035] This invention proposes the use of the MHS2 regulatory element in regulating the expression of the Glyma.03G22970 gene, the use of the MHS2 regulatory element in regulating soybean oil content, a method for regulating soybean oil content, sgRNA molecule, expression vector, reagents, CRISPR / Cas9 system, and kit.

[0036] The role of MHS2 regulatory elements in regulating Glyma.03G22970 gene expression

[0037] In a first aspect, this invention proposes the use of an MHS2 regulatory element in regulating the Glyma.03G22970 gene, the nucleotide sequence of which is shown in SEQ ID NO:1. This invention is the first to discover that the MHS2 regulatory element can act as an enhancer of the Glyma.03G22970 gene to regulate its expression.

[0038] According to an embodiment of the present invention, the nucleotide sequence of the MHS2 regulatory element is modified or altered to reduce the expression level of the Glyma.03G22970 gene.

[0039] According to an embodiment of the present invention, the nucleotide sequence of the Glyma.03G22970 gene is shown in SEQ ID NO:14.

[0040] According to some optional embodiments of the present invention, the MHS2 sequence is located downstream of the Glyma.03G22970 gene at Gm05:2556251..2556481(+strand).

[0041] According to an embodiment of the present invention, the MHS2 control element sequence is modified or altered in the following manner:

[0042] At least one of the following is performed on at least a portion of the sequence of the MHS2 regulatory element: deletion, substitution, insertion, inversion, and transposition; or methylation and / or acetylation is performed on at least a portion of the sequence of the MHS2 regulatory element.

[0043] According to embodiments of the present invention, the MHS2 regulatory element sequence is modified or altered through a gene editing system and / or RNA interference.

[0044] According to an embodiment of the present invention, the gene editing system includes a CRISPR-Cas9 virus and a non-viral component.

[0045] According to embodiments of the present invention, the RNA interference includes at least one of siRNA, miRNA, and shRNA.

[0046] According to an embodiment of the present invention, the gene editing system comprises: sgRNA having at least one of the nucleotide sequences shown in SEQ ID NO:3-4, and optionally having at least one of the nucleotide sequences shown in SEQ ID NO:2 or SEQ ID NO:5.

[0047] According to an embodiment of the present invention, the gene editing system further includes the Cas9 protein.

[0048] Application of MHS2 regulating element in regulating soybean oil content

[0049] In two aspects, this invention proposes the use of the MHS2 regulatory element in regulating the oil content of soybeans, the nucleotide sequence of which is shown in SEQ ID NO:1. This invention is the first to discover that the MHS2 regulatory element can regulate the oil content of soybeans by controlling the expression of the Glyma.03G22970 gene.

[0050] According to an embodiment of the present invention, the nucleotide sequence of the MHS2 regulatory element is modified or altered to increase the oil content of the soybean.

[0051] According to an embodiment of the present invention, the MHS2 control element sequence is modified or altered in the following manner:

[0052] At least one of the following is performed on at least a portion of the sequence of the MHS2 regulatory element: deletion, substitution, insertion, inversion, and transposition; or methylation and / or acetylation is performed on at least a portion of the sequence of the MHS2 regulatory element.

[0053] According to embodiments of the present invention, the MHS2 regulatory element sequence is modified or altered through a gene editing system and / or RNA interference.

[0054] According to an embodiment of the present invention, the gene editing system includes a CRISPR-Cas9 virus and a non-viral component.

[0055] According to an embodiment of the present invention, the gene editing system comprises: sgRNA having at least one of the nucleotide sequences shown in SEQ ID NO:3-4, and optionally having at least one of the nucleotide sequences shown in SEQ ID NO:2 or SEQ ID NO:5.

[0056] According to an embodiment of the present invention, the gene editing system further includes the Cas9 protein.

[0057] Methods for regulating soybean oil content

[0058] A third aspect of this invention provides a method for regulating the oil content of soybeans. According to an embodiment of the invention, the method includes: modifying and altering the nucleotide sequence of an MHS2 regulatory element in soybeans, the nucleotide sequence of which is shown in SEQ ID NO:1. The method of this invention can partially or completely delete the nucleotide sequence of the MHS2 regulatory element, resulting in partial or complete loss of activity of the MHS2 regulatory element, thereby reducing the expression level of the Glyma.03G22970 gene and increasing the oil content of soybeans.

[0059] According to an embodiment of the present invention, the MHS2 control element sequence is modified or altered in the following manner:

[0060] At least one of the following is performed on at least a portion of the sequence of the MHS2 regulatory element: deletion, substitution, insertion, inversion, and transposition; or methylation and / or acetylation is performed on at least a portion of the sequence of the MHS2 regulatory element.

[0061] According to embodiments of the present invention, the MHS2 regulatory element sequence is modified or altered through a gene editing system and / or RNA interference.

[0062] According to an embodiment of the present invention, the gene editing system includes a CRISPR-Cas9 virus and a non-viral component.

[0063] According to an embodiment of the present invention, the gene editing system comprises: sgRNA having at least one of the nucleotide sequences shown in SEQ ID NO:3-4, and optionally having at least one of the nucleotide sequences shown in SEQ ID NO:2 or SEQ ID NO:5.

[0064] According to an embodiment of the present invention, the gene editing system further includes the Cas9 protein.

[0065] sgRNA molecules

[0066] In a fourth aspect, the present invention provides an sgRNA molecule. According to embodiments of the invention, the sgRNA molecule has at least one of the nucleotide sequences shown in SEQ ID NO: 3-4, and optionally at least one of the nucleotide sequences shown in SEQ ID NO: 2 and SEQ ID NO: 5. This sgRNA molecule can partially or completely delete the sequence of the soybean MHS2 regulatory element, exhibiting advantages such as low off-target rate and high editing efficiency. Furthermore, by using this sgRNA molecule to partially or completely delete the sequence of the MHS2 regulatory element, the active portion or complete loss of the MHS2 regulatory element is achieved, thereby reducing the expression level of the Glyma.03G22970 gene and increasing the oil content of soybean.

[0067] According to an embodiment of the present invention, the sgRNA molecule has a nucleotide sequence as shown in SEQ ID NO: 3.

[0068] According to an embodiment of the present invention, the sgRNA molecule has a nucleotide sequence as shown in SEQ ID NO: 4.

[0069] According to embodiments of the present invention, the sgRNA molecule has nucleotide sequences as shown in SEQ ID NO: 3 and SEQ ID NO: 4.

[0070] According to embodiments of the present invention, the sgRNA molecule has nucleotide sequences as shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5.

[0071] expression carrier

[0072] In a fifth aspect, the present invention provides an expression vector. According to embodiments of the invention, the expression vector carries the sgRNA molecule described in the fourth aspect of the invention and, optionally, nucleic acid encoding a Cas9 molecule. This expression vector can partially or completely delete sequences of MHS2 regulatory elements, thereby partially or completely deleting sequences that activate MHS2 regulatory elements, thus reducing the expression level of the Glyma.03G22970 gene and increasing the oil content of soybeans.

[0073] According to embodiments of the present invention, the expression vector includes at least one of adenovirus expression vector, adeno-associated virus expression vector, retrovirus expression vector, and lentiviral vector.

[0074] reagents

[0075] In a sixth aspect, the present invention provides a reagent. According to embodiments of the present invention, the reagent comprises the sgRNA molecule described in the fourth aspect of the present invention or the expression vector described in the fifth aspect of the present invention. This reagent can partially or completely delete the sequence of the MHS2 regulatory element, thereby partially or completely losing the activity of the MHS2 regulatory element, thus reducing the expression level of the Glyma.03G22970 gene and increasing the oil content of soybean.

[0076] CRISPR / Cas9 system

[0077] In a seventh aspect, the present invention provides a CRISPR / Cas9 system. According to embodiments of the invention, the CRISPR / Cas9 system comprises the sgRNA molecule described in the fourth aspect of the invention and, optionally, nucleic acid encoding a Cas9 molecule. This CRISPR / Cas9 system can partially or completely delete the sequence of the MHS2 regulatory element, thereby partially or completely losing the activity of the MHS2 regulatory element, thus reducing the expression level of the Glyma.03G22970 gene and increasing the oil content of soybeans.

[0078] Reagent test kit

[0079] In an eighth aspect, the present invention provides a kit. According to embodiments of the invention, the kit comprises the sgRNA molecule described in the fourth aspect of the invention or the expression vector described in the fifth aspect of the invention; and optionally, nucleic acid encoding a Cas9 molecule. This kit can partially or completely delete the sequence of the MHS2 regulatory element, thereby partially or completely losing the activity of the MHS2 regulatory element, thus reducing the expression level of the Glyma.03G22970 gene and increasing the oil content of soybean.

[0080] The sequence list in this invention is as follows:

[0081]

[0082]

[0083] The present disclosure will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of the disclosure. Where specific techniques or conditions are not specified in the embodiments, they are performed in accordance with the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0084] Example 1: Design of CRISPR / Cas9 knockout target in the seed-specific MHS2 region of the high-expression gene Glyma.03G22970 in soybean seeds.

[0085] The Glyma.03G22970 gene is homologous to the Arabidopsis thaliana UMAMIT25 gene and is specifically expressed in soybean seeds, especially at high levels during the mid-to-late stages of soybean seed development. Figure 1 As shown.

[0086] (1) Design of knockout target sites targeting the MHS region of the Glyma.03G22970 gene:

[0087] First, this study utilized MNase-seq sequencing technology to mine candidate enhancers across the entire genome of Williams82 soybean leaves, flowers, and seeds. Bowtie software was used to locate reads from the MNase-seq results, and these reads were backfilled into the Williams82 genome sequence (https: / / phytozome). The parameters allowing for base mismatches were adjusted to retain reads that could only be backfilled into one region of the Williams82 genome. The soybean genome was divided into several 200bp non-overlapping regions. The total number of backfilled reads generated from multiple replicate experiments within each region was counted. Regions with an FDR < 0.01 and a length greater than 50bp were identified as MHS sites and used as candidate enhancer regions. Using this method, a seed-specific open chromatin region was successfully located at Gm03:44336551..44336694(+strand) upstream of the Glyma.03G229700 gene. This region was identified as a candidate enhancer for seed-specific enhancement and named seed-specific-MHS2 (also known as MHS2). For the specific location, please refer to [link to relevant documentation]. Figure 2 .

[0088] Subsequently, the seed-specific MHS2 sequence was found in the Williams82 genome sequence. sgRNAs were designed on both sides and in the middle of MHS2 using the online target website CRISPR-GE (http: / / skl.scau.edu.cn / ). The soybean endogenous promoter pM4 was selected as the promoter. The following principles were followed when designing the target sites: (1) the target sequence was in the form of 5'-20nt-NGG; (2) the knockout sites were located on both sides and in the middle of MHS2. This made it more likely to completely knock out MHS2, as well as partially knock out MHS2. The four target sites were named MHS2-sg1 (as shown in SEQ ID NO:2 in the sequence listing), MHS2-sg2 (as shown in SEQ ID NO:3 in the sequence listing), MHS2-sg3 (as shown in SEQ ID NO:4 in the sequence listing), and MHS2-sg4 (as shown in SEQ ID NO:5 in the sequence listing). The CRISPR / Cas9 knockout target sites and their locations for seed-specific MHS2 are shown below. Figure 3As shown.

[0089] (2) Predicting the off-target rate and editing efficiency of the knockout target site in the MHS region of the Glyma.03G22970 gene:

[0090] First, based on the predictions made on the CRISPR-GE website regarding the potential off-target rates of MHS2-sg1, MHS2-sg2, MHS2-sg3, and MHS2-sg4 designed for the MHS region of the Glyma.03G22970 gene in the above steps within the soybean genome exon region, the prediction results showed that the potential off-target rates of MHS2-sg1, MHS2-sg2, MHS2-sg3, and MHS2-sg4 within the soybean genome exon region were less than 33%.

[0091] Secondly, positive roots were obtained through soybean hairy root transformation. The off-target effects and editing efficiency of MHS2-sg1, MHS2-sg2, MHS2-sg3, and MHS2-sg4 were then examined. The editing efficiency results for the MHS2 target are shown in Table 1. The results showed no off-target effects for any of the four targets; furthermore, the editing efficiencies of MHS2-sg1, MHS2-sg2, MHS2-sg3, and MHS2-sg4 were all above 80%.

[0092] Table 1: Target Editing Efficiency

[0093] Number of hairy roots Number of positive roots Editing efficiency % MHS2-sg1 30 25 83.3％ MHS2-sg2 27 23 85.1％ MHS2-sg3 23 19 82.6％ MHS2-sg4 32 27 84.3％

[0094] Example 2: Construction of Glyma.03G229705-MHS2-Cas9 vector

[0095] In this embodiment, PGES401 is used as the initial vector. The PGES401 vector is driven by the pM4 promoter to express Cas9 protein and sgRNA+sgRNA Scaffold.

[0096] First, construct Glyma.03G229705-MHS2 with different target sites:

[0097] 2.1 Construction of single target Glyma.03G229705-MHS2:

[0098] DNA fragments of MHS-sg2 and MHS-sg3 were amplified by PCR, and the PCR products of these DNA fragments were recovered by gel electrophoresis. The recovered PCR products were then added to the ligation reaction system along with the vector pGES401. The resulting ligation products were transformed into competent DH5α E. coli cells. Positive clones were identified by colony PCR and sent to a sequencing company for sequencing. Plasmids from correctly sequenced *E. coli* were extracted and transformed into *Agrobacterium* K599. Correct colony identification and sequencing results indicated successful construction of the single-target CRISPR / Cas9 knockout vector Glyma.03G229705-MHS2-Cas9. This knockout vector contains the Bar selection marker gene (glufosinate resistance).

[0099] 2.2 Construction of the dual-target Glyma.03G229705-MHS2: Using the pGES401 dual-target vector (MHS-sg2, MHS-sg3) as a template, an additional tRNA-sgRNA-sgRNA scaffold-tRNA structure was amplified by PCR, and the DNA fragments of the two sgRNAs tandemly were constructed using the goldgate method (i.e., the dual-target Glyma.03G22970-MHS2). The specific steps are as follows:

[0100] (1) PGES401-MHS-sg2 and PGES401-MHS-sg3 were obtained by ligation reaction. The specific ligation system was as follows: PGES401 plasmid 5 μl, T4 DNA Ligase 1 μl, T4 DNA Ligase Buffer 2 μl, BsaI 2 μl, MHS-sg2 1 μl, MHS-sg3 1 μl, ddH2O 5 μl;

[0101] (2) A DNA fragment of tRNA-sgRNA-sgRNA Scaffold-tRNA was obtained by PCR amplification. The specific PCR reaction conditions were as follows: (37℃ for 5 min, 16℃ for 5 min) × 25 cycles, 37℃ for 15 min, 85℃ for 5 min; the specific amplification primers were: Glyma.03G229705-MHS2-F2 (protective base + BsaI recognition site + MHS-sg2 + sgRNA Scaffold, the specific nucleotide sequence is shown in SEQ ID NO: 8), Glyma.03G229705-MHS2-R2 (protective base + BsaI recognition site + BsaI cleavage site + MHS-sg3 + sgRNA Scaffold, the specific nucleotide sequence is shown in SEQ ID NO: 9).

[0102] (3) The DNA fragment of tRNA-sgRNA-sgRNA Scaffold-tRNA obtained above was amplified by PCR using the high-fidelity enzyme KOD One™ PCR master Mix purchased from TOYOBO, resulting in PCR products of two tandem DNA fragments of sgRNA. The specific PCR reaction conditions were: 98℃ pre-denaturation for 3 min, (98℃ denaturation for 15 s, 58℃ annealing for 15 s, 68℃ extension for 20 s) × 35 cycles, followed by a 68℃ extension for 5 min to obtain the PCR product, which was then gel-cleansed.

[0103] (4) The recovered DNA fragments and vector pGES401 were simultaneously added to the ligation reaction system, and the resulting ligation product was transformed into competent E. coli cells DH5α. Positive clones were identified by colony PCR and sent to a sequencing company for sequencing. The sequencing primers were STU-TEST-4R (nucleotide sequence as shown in SEQ ID NO: 12). The sequencing results showed that vector pGES401 contained the sequences of tRNA, MHS-sg2, sgRNA Scaffold, tRNA, MHS-sg3, sgRNA Scaffold, and tRNA. The correctly sequenced E. coli plasmid was extracted and transformed into Agrobacterium K599. The colony identification and sequencing results were correct, indicating that the dual-target Glyma.03G229705-MHS2 CRISPR / Cas9 knockout vector dual-target Glyma.03G229705-MHS2-Cas9 had been successfully constructed. This knockout vector contains the Bar selection marker gene (glufosinate resistance).

[0104] 2.3 Construction of the four-target Glyma.03G229705-MHS2: Using the pGES401-four-target (MHS-sg1~4) empty vector as a template, three additional tRNA-sgRNA-sgRNA scaffold-tRNA structures were amplified by PCR, and a DNA fragment of four sgRNAs tandemly (i.e., the four-target Glyma.03G229705-MHS2) was constructed using the goldgate method. The specific steps are as follows:

[0105] (1) PGES401-MHS2-sg1, PGES401-MHS2-sg2, PGES401-MHS2-sg3, and PGES401-MHS2-sg4 were obtained by ligation reaction. The specific ligation system was as follows: PGES401 plasmid 5 μl, T4 DNA Ligase 1 μl, T4 DNA Ligase Buffer 2 μl, BsaI 2 μl, MHS2-sg1 1 μl, MHS2-sg2 1 μl, MHS2-sg3 1 μl, MHS2-sg4 1 μl, and ddH2O 5 μl.

[0106] (2) DNA fragments of three tRNA-sgRNA-sgRNA Scaffold-tRNA were obtained by PCR amplification. The specific PCR reaction conditions are as follows: (37℃ for 5 min, 16℃ for 5 min) × 25 cycles, 37℃ for 15 min, 85℃ for 5 min; the specific amplification primers are: Glyma.03G229705-MHS2-F1 (protective base + BsaI recognition site + BsaI cleavage site + MHS2-sg1 + sgRNA Scaffold, specific nucleotide sequence as shown in SEQ ID NO: 6), Glyma.03G229705-MHS2-R1 (protective base + BsaI recognition site + MHS2-sg2 + sgRNA Scaffold, specific nucleotide sequence as shown in SEQ ID NO: 7), Glyma.03G229705-MHS2-F2 (protective base + BsaI recognition site + MHS2-sg2 + sgRNA Scaffold, specific nucleotide sequence as shown in SEQ ID NO: 7). Glyma.03G229705-MHS2-R2 (protective base + BsaI recognition site + BsaI cleavage site + MHS-sg3 + sgRNA Scaffold, specific nucleotide sequence as shown in SEQ ID NO: 8), Glyma.03G229705-MHS2-F3 (protective base + BsaI recognition site + MHS2-sg3 + sgRNA Scaffold, specific nucleotide sequence as shown in SEQ ID NO: 10), Glyma.03G229705-MHS2-R3 (protective base + BsaI recognition site + BsaI cleavage site + MHS2-sg4 + sgRNA Scaffold, specific nucleotide sequence as shown in SEQ ID NO: 11).

[0107] (3) The three tRNA-sgRNA-sgRNA scaffold-tRNA DNA fragments obtained above were amplified by PCR using the high-fidelity enzyme KOD One™ PCR master Mix purchased from TOYOBO, resulting in PCR products of four tandem sgRNA DNA fragments. The specific PCR reaction conditions were: 98℃ pre-denaturation for 3 min, (98℃ denaturation for 15 s, 58℃ annealing for 15 s, 68℃ extension for 20 s) × 35 cycles, followed by a 68℃ extension for 5 min to obtain the PCR product, which was then gel-cleansed.

[0108] (4) The recovered DNA fragments and vector pGES401 were simultaneously added to the ligation reaction system, and the resulting ligation product was transformed into competent DH5α coli cells. Positive clones were identified by colony PCR and sent to a sequencing company for sequencing. The sequencing primers were STU-TEST-4R (nucleotide sequence as shown in SEQ ID NO: 12). The sequencing results showed that vector pGES401 contained the sequences of tRNA, MHS-sg1, sgRNA Scaffold, tRNA, MHS-sg2, sgRNA Scaffold, tRNA, MHS-sg3, sgRNA Scaffold, tRNA, MHS-sg4, sgRNA Scaffold, and tRNA. The correctly sequenced *E. coli* plasmid was extracted and transformed into *Agrobacterium* K599. Correct colony identification and sequencing results indicate successful construction of the four-target Glyma.03G229705-MHS2 CRISPR / Cas9 knockout vector. This knockout vector contains the Bar selection marker gene (glufosinate resistance). The construction process of the four-target Glyma.03G229705-MHS2-Cas9 vector is as follows: Figure 3 As shown.

[0109] Furthermore, the inventors verified the target efficiency of the constructed vector Glyma.03G229705-MHS2-Cas9 with single, dual, and quadruple targets through soybean hairy root transformation experiments. The results showed that the target efficiency of single, dual, and quadruple targets Glyma.03G229705-MHS2-Cas9 was relatively high. This embodiment exemplifies that the target efficiency of quadruple targets Glyma.03G229705-MHS2-Cas9 was 83.3%, 85.1%, 82.6%, and 84.3%, respectively.

[0110] The constructed seed-specific MHS2-Cas9 vector was transferred into the Williams82 soybean variety via Agrobacterium tumefaciens-mediated transformation of soybean cotyledonary nodes using EHA105, resulting in a CRISPR / Cas9 knockout mutant line of the soybean Glyma.03G229705 enhancer. The seed-specific MHS2-Cas9 vector construction procedure is as follows: Figure 3 As shown.

[0111] Example 3: CRISPR / Cas9-mediated editing of the soybean Glyma.03G229705 gene MHS2 knockout mutant.

[0112] The single-target, dual-target, and quadruple-target vectors Glyma.03G229705-MHS2-Cas9 constructed in Example 2 were transferred into the cotyledonary nodes of soybean through EHA105 Agrobacterium tumefaciens, resulting in CRISPR / Cas9 knockout mutant lines of soybean with single-target, dual-target, and quadruple-target Glyma.03G229705-MHS2 enhancer. In these soybean CRISPR / Cas9 knockout mutant lines with different target Glyma.03G229705-MHS2 enhancers, positive seedlings with bar resistance were obtained after bar test strip detection (i.e., T1, T2, T3, T4, T5, T22, and T27; the positive seedlings corresponding to the single-target Glyma.03G229705-MHS2 enhancer CRISPR / Cas9 knockout mutant lines were T1 and T3, the positive seedlings corresponding to the double-target Glyma.03G229705-MHS2 enhancer CRISPR / Cas9 knockout mutant lines were T4 and T5, and the positive seedlings corresponding to the quadruple-target Glyma.03G229705-MHS2 enhancer CRISPR / Cas9 knockout mutant lines were T2, T22, and T27).

[0113] Using genomic DNA as a template, the genomic fragment containing the target site was amplified using TOYOBO's high-fidelity enzyme KOD One™ PCR master Mix. The amplification primers were Glyma.03G229705-MHS2-F (SEQ ID NO: 15) and Glyma.03G229705-MHS2-R (SEQ ID NO: 16). PCR reaction conditions were: 98℃ pre-denaturation for 3 min, followed by 32 cycles of (98℃ denaturation for 15 s, 58℃ annealing for 15 s, 68℃ extension for 30 s), and then a final extension at 72℃ for 5 min. PCR detection yielded seven bar-resistant positive seedling lines (T1, T2, T3, T4, T5, T22, and T27) with large fragment knockout. Based on Sanger sequencing, the specific mutation types of these seven large fragment knockout lines were determined by comparing the sequenced sequences with wild-type sequences using DNAman. Sequencing analysis revealed that the seven large fragment knockout lines—T1, T2, T3, T4, T5, T22, and T27—partially or completely knocked out MHS2.

[0114] This example demonstrates the results of positive seedlings T2, T22, and T27 in the CRISPR / Cas9 knockout mutant line with the four-target Glyma.03G229705-MHS2 enhancer. For detailed results, please refer to [link to relevant documentation]. Figure 4 Among them, T2 is a knockout line with a 175bp deletion in the MHS2 sequence, T22 is a homozygous complete knockout line with a 216bp deletion in the MHS2 sequence, and T27 is a knockout line with a 117bp deletion in the MHS2 sequence. Furthermore, these three mutants, T2, T22, and T27, can be stably inherited by offspring after T0, T1, and T2 generations.

[0115] Example 4: Phenotypic analysis of the MHS2 regulatory element knockout mutant of soybean Glyma.03G229705 gene

[0116] The T2 generation homozygous MHS2 knockout mutants T1, T2, T3, T4, T5, T22, and T27 prepared in Example 3, along with the wild-type control variety Williams82, were sown in pots with 30 plants per line. The expression level of the Glyma.03G229705 gene in T1, T2, T3, T4, T5, T22, T27, and Williams82 was counted by RT-PCR. The protein and oil content of T1, T2, T3, T4, T5, T22, T27, and Williams82 were analyzed by gas chromatography and elemental analysis. The expression level of the Glyma.03G229705 gene, protein, and oil content in T1, T2, T3, T4, T5, T22, T27, and Williams82 were analyzed.

[0117] The results showed that, compared with wild-type Williams82, the expression level of Glyma.03G229705 was reduced in MHS2 knockout mutants T1, T2, T3, T4, T5, T22 and T27. The protein content of MHS2 knockout mutants T1, T2, T3, T4, T5, T22 and T27 was higher than that of wild-type Williams82, and the oil content of MHS2 knockout mutants T1, T2, T3, T4, T5, T22 and T27 was higher than that of wild-type Williams82.

[0118] This embodiment exemplarily demonstrates the results of positive seedlings T2, T22, and T27 in the CRISPR / Cas9 knockout mutant line with the four-target Glyma.03G229705-MHS2 enhancer. See details for further information. Figures 5-7 Compared with wild-type Williams82, the expression level of Glyma.03G229705 in MHS2 knockout mutants T2, T22, and T27 was reduced by 10%–63% (see details). Figure 5 Furthermore, the protein content in the seeds of the MHS2 knockout mutants T2, T22, and T27 was reduced by 2%-6% (see details for specific results). Figure 6 Furthermore, the oil content in the seeds of the MHS2 knockout mutants T2, T22, and T27 increased by 1%–5% (see details). Figure 7 Therefore, soybean MHS2 regulatory elements have significant application potential in breeding high-protein, high-yield soybeans by precisely regulating the expression of Glyma.03G229705 to control protein accumulation in soybean seeds.

[0119] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0120] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. The MHS2 regulatory element in suppressing Glyma.03G229700 Use in gene expression, the nucleotide sequence of the MHS2 regulatory element is shown in SEQ ID NO:1; Glyma.03G229700 The nucleotide sequence of the gene is shown in SEQ ID NO:

14. The nucleotide sequence of the MHS2 regulatory element is knocked out, reducing the... Glyma.03G229700 The expression level of the gene; the knockout is achieved by targeted editing of the MHS2 regulatory element using sgRNA with a nucleotide sequence as shown in SEQ ID NO:

5.

2. The use of the MHS2 regulatory element in increasing the oil content of soybeans, wherein the nucleotide sequence of the MHS2 regulatory element is shown in SEQ ID NO:1, and the nucleotide sequence of the MHS2 regulatory element is knocked out to increase the oil content of soybeans; the knockout is achieved by targeted editing of the MHS2 regulatory element using sgRNA with the nucleotide sequence shown in SEQ ID NO:

5.

3. A method for increasing the oil content of soybeans, characterized in that, include: The nucleotide sequence of the MHS2 regulatory element in soybean was knocked out, and the nucleotide sequence of the MHS2 regulatory element is shown in SEQ ID NO:1; the knockout was achieved by targeted editing of the MHS2 regulatory element using sgRNA with a nucleotide sequence shown in SEQ ID NO:

5.

4. An sgRNA molecule, characterized in that, The nucleotide sequence of the sgRNA molecule is shown in SEQ ID NO:

5.

5. An expression carrier, characterized in that, It carries the sgRNA molecule as described in claim 4 and the nucleic acid encoding the Cas9 molecule.

6. A reagent, characterized in that, include: The sgRNA molecule of claim 4 or the expression vector of claim 5.

7. A CRISPR / Cas9 system, characterized in that, include: The sgRNA molecule and the nucleic acid encoding the Cas9 molecule as described in claim 4.

8. A reagent kit, characterized in that, include: The sgRNA molecule of claim 4 or the expression vector of claim 5; And the nucleic acid that encodes the Cas9 molecule.

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