Soybean Glyma. 03G22970 gene enhancer regulatory element and sgRNA molecule thereof

Abstract

The invention discloses a soybean Glyma. 03G22970 gene enhancer regulatory element and a sgRNA (small guide ribonucleic acid) molecule of the soybean Glyma. 03G22970 gene enhancer regulatory element. The enhancer regulatory element is an MHS2 regulatory element, and the nucleotide sequence of the MHS2 regulatory element is as shown in SEQ ID NO: 1; the sgRNA molecule has at least one of nucleotide sequences as shown in SEQ ID NO: 3-4, and optionally has at least one of nucleotide sequences as shown in SEQ ID NO: 2 and SEQ ID NO: 5. The MHS2 regulatory element can be used as an enhancer of the Glyma. 03G22970 gene, so that the expression quantity of the Glyma. 03G22970 gene is reduced, and the oil content of soybeans is increased; the sgRNA molecule can partially or completely delete the sequence of the MHS2 regulatory element, and has the advantages of low miss rate, high editing efficiency and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic engineering, and specifically relates to a soybean Glyma.03G22970 gene enhancer regulatory element and its sgRNA molecule. More specifically, the present invention relates to the use of MHS2 regulatory elements in regulating Glyma.03G22970 gene expression, the use of MHS2 regulatory elements in regulating soybean oil content, a method for regulating soybean oil content, expression vectors, reagents, CRISPR / Cas9 systems and kits. Background Art

[0002] Soybean (Glycine max) is an important food and oil crop belonging to the Leguminosae family. It originated in northeastern Asia and is one of the world's main sources of protein and one of the most important vegetable oil crops.

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

[0004] However, nitrogen is one of the main limiting factors for soybean oil content. Soybean plants absorb nitrogen from the outside world and convert it into amino acids in the body, which then participate in the process of synthesizing proteins and other bioactive substances. Amino acids are one of the key precursors for the formation of soybean oil. Current research has found that the oil content of soybeans can be regulated by regulating proteins. For example, UMAMIT transporters are a common type of amino acid transporter in plants. They are responsible for the transport and distribution of amino acids in plants and can affect the oil content of soybeans. Summary of the invention

[0005] The present invention aims to solve one of the technical problems in the related art at least to a certain extent. To this end, the first aspect of the present invention finds the use of the MHS2 regulatory element in regulating the Glyma.03G22970 gene.

[0006] The present invention is mainly completed based on the following findings of the inventors:

[0007] The inventors found that the Glyma.03G22970 gene in soybeans is similar to UMAMIT25 and may have similar amino acid transport functions. Based on this, when the inventors studied the Glyma.03G22970 gene, they unexpectedly discovered an enhancer of the Glyma.03G22970 gene, which is located at Gm03:44336551..44336694 (+strand) upstream of the Glyma.03G22970 gene and was named MHS2. In addition, the inventors unexpectedly found that when the MHS2 gene in soybeans was knocked out, the expression level of the Glyma.03G22970 gene could be significantly reduced, especially compared with the wild-type group in which MHS2 was not knocked out, the expression level of the Glyma.03G22970 gene was reduced by 10% to 63%, the protein content in the seeds was reduced by 2%-6%, and the oil content in the seeds was increased by 1% to 5%.

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

[0009] In the second aspect of the present invention, the present invention proposes the use of MHS2 regulatory element in regulating the oil content of soybean, and the nucleotide sequence of the MHS2 regulatory element is shown in SEQ ID NO: 1. The present invention first discovered that the MHS2 regulatory element can regulate the oil content of soybean by regulating the expression of the Glyma.03G22970 gene.

[0010] In the third aspect of the present invention, a method for regulating the oil content of soybeans is proposed. According to an embodiment of the present invention, the method comprises: transforming and modifying the nucleotide sequence of the MHS2 regulatory element in the soybean, and the nucleotide sequence of the MHS2 regulatory element is as shown in SEQ ID NO: 1. The method of the present invention can partially or completely delete the nucleotide sequence of the MHS2 regulatory element, and partially or completely lose the 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 the fourth aspect of the present invention, the present invention proposes an sgRNA molecule. According to an embodiment of the present invention, the sgRNA molecule has at least one of the nucleotide sequences shown in SEQ ID NO: 3 to 4, and optionally has 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, partially or completely lose the activity of the MHS2 regulatory element, and the sgRNA molecule has the advantages of low off-target rate and high editing efficiency. In addition, the sgRNA molecule can partially or completely delete the sequence of the soybean MHS2 regulatory element, partially or completely lose the activity of the MHS2 regulatory element, reduce the expression of the Glyma.03G22970 gene, and increase the oil content of soybeans.

[0012] In a fifth aspect of the present invention, the present invention proposes an expression vector. According to an embodiment of the present invention, the expression vector carries the sgRNA molecule described in the fourth aspect of the present invention and optionally a nucleic acid encoding a Cas9 molecule. The expression vector can partially or completely delete the sequence of the MHS2 regulatory element, thereby partially or completely deleting the sequence of the MHS2 regulatory element activity, thereby reducing the expression level of the Glyma.03G22970 gene and increasing the oil content of soybeans.

[0013] In the sixth aspect of the present invention, the present invention proposes a reagent. According to an embodiment of the present invention, the reagent includes 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. The 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, thereby reducing the expression level of the Glyma.03G22970 gene and increasing the oil content of soybeans.

[0014] In the seventh aspect of the present invention, the present invention proposes a CRISPR / Cas9 system. According to an embodiment of the present invention, the CRISPR / Cas9 system comprises the sgRNA molecule described in the fourth aspect of the present invention and a nucleic acid that optionally encodes a Cas9 molecule. The 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, thereby reducing the expression of the Glyma.03G22970 gene and increasing the oil content of soybeans.

[0015] In the eighth aspect of the present invention, the present invention proposes a kit. According to an embodiment of the present invention, the kit 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; and optionally a nucleic acid encoding a Cas9 molecule. The 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, thereby reducing the expression of the Glyma.03G22970 gene and increasing the oil content of soybeans.

[0016] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0018] Figure 1 The Glyma.03G22970 gene in Example 1 is specifically and highly expressed in soybean seeds.

[0019] Figure 2 The position of the seed-specific MHS2 regulatory element in Example 1 on the genome.

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

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

[0022] Figure 5 is the expression level of the Glyma.03G22970 gene in the wild type and mutant in Example 4.

[0023] Figure 6 This is the protein content of wild type and mutant soybean seeds in Example 4.

[0024] Figure 7 This is the oil content of wild-type and mutant soybean seeds in Example 4. DETAILED DESCRIPTION

[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 understood 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 understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality" is two or more.

[0027] In order to make the present invention more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in the present invention, all other technical and scientific terms used in the present invention have the meanings commonly understood by those skilled in the art to which the present invention belongs.

[0028] In the present invention, the terms "comprise" or "include" are open expressions, that is, they include the contents specified in the present invention but do not exclude other contents.

[0029] In the present invention, the terms "optionally", "optional" or "optionally" generally mean that the subsequently described event or situation can but does not necessarily occur, and the description includes cases where the event or situation occurs and cases where it does not occur.

[0030] In the present invention, "the nucleotide sequence of the MHS2 regulatory element is transformed or modified" means that the nucleotide sequence of the MHS2 regulatory element is changed, thereby affecting the binding ability of the MHS2 regulatory element with transcription factors; as an enhancer of the Glyma.03G22970 gene, the expression of the Glyma.03G22970 gene can be regulated by changing the binding ability of the nucleotide sequence of the MHS2 regulatory element with transcription factors. Among them, the form of the nucleotide sequence change of the MHS2 regulatory element includes but is not limited to sequence deletion, substitution, insertion, inversion, ectopic, and sequence methylation and / or acetylation.

[0031] In the present invention, "regulatory elements" generally refer to DNA sequences involved in gene transcription regulation, mainly including promoters, enhancers, insulators, and the like.

[0032] In the present invention, "stable inheritance" means that the mutants after gene editing can stably pass on the mutant sequence to the offspring in the form of inheritance.

[0033] In the present 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 the present invention, "gene editing" is also called genome editing or genome engineering. It is an emerging and relatively accurate genetic engineering technology that can modify specific target genes in the genome of an organism.

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

[0036] Use of MHS2 regulatory element in regulating Glyma.03G22970 gene expression

[0037] In the first aspect of the present invention, the present invention proposes the use of the MHS2 regulatory element in regulating the Glyma.03G22970 gene, and the nucleotide sequence of the MHS2 regulatory element is shown in SEQ ID NO: 1. The present invention first discovered that the MHS2 regulatory element can be used as an enhancer of the Glyma.03G22970 gene to regulate the expression of the Glyma.03G22970 gene.

[0038] According to an embodiment of the present invention, the nucleotide sequence of the MHS2 regulatory element is transformed or modified 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 downstream of the Glyma.03G22970 gene Gm05:2556251..2556481 (+strand).

[0041] According to an embodiment of the present invention, the MHS2 regulatory element sequence is transformed or modified by the following methods:

[0042] At least a portion of the sequence of the MHS2 regulatory element is deleted, replaced, inserted, inverted, and misplaced; or at least a portion of the sequence of the MHS2 regulatory element is methylated and / or acetylated.

[0043] According to an embodiment of the present invention, the MHS2 regulatory element sequence is transformed or modified by a gene editing system and / or RNA interference.

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

[0045] According to an embodiment 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 includes: sgRNA, the sgRNA having at least one of the nucleotide sequences shown in SEQ ID NO:3 to 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 a Cas9 protein.

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

[0049] In the second aspect of the present invention, the present invention proposes the use of MHS2 regulatory element in regulating the oil content of soybean, and the nucleotide sequence of the MHS2 regulatory element is shown in SEQ ID NO: 1. The present invention first discovered that the MHS2 regulatory element can regulate the oil content of soybean by regulating 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 transformed or modified to increase the oil content of the soybean.

[0051] According to an embodiment of the present invention, the MHS2 regulatory element sequence is transformed or modified by the following methods:

[0052] At least a portion of the sequence of the MHS2 regulatory element is deleted, replaced, inserted, inverted, and misplaced; or at least a portion of the sequence of the MHS2 regulatory element is methylated and / or acetylated.

[0053] According to an embodiment of the present invention, the MHS2 regulatory element sequence is transformed or modified by a gene editing system and / or RNA interference.

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

[0055] According to an embodiment of the present invention, the gene editing system includes: sgRNA, the sgRNA having at least one of the nucleotide sequences shown in SEQ ID NO:3 to 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 a Cas9 protein.

[0057] Method for regulating soybean oil content

[0058] In the third aspect of the present invention, a method for regulating the oil content of soybeans is proposed. According to an embodiment of the present invention, the method comprises: transforming and modifying the nucleotide sequence of the MHS2 regulatory element in the soybean, and the nucleotide sequence of the MHS2 regulatory element is as shown in SEQ ID NO: 1. The method of the present invention can partially or completely delete the nucleotide sequence of the MHS2 regulatory element, and partially or completely lose the 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 regulatory element sequence is transformed or modified by the following methods:

[0060] At least a portion of the sequence of the MHS2 regulatory element is deleted, replaced, inserted, inverted, and misplaced; or at least a portion of the sequence of the MHS2 regulatory element is methylated and / or acetylated.

[0061] According to an embodiment of the present invention, the MHS2 regulatory element sequence is transformed or modified by a gene editing system and / or RNA interference.

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

[0063] According to an embodiment of the present invention, the gene editing system includes: sgRNA, the sgRNA having at least one of the nucleotide sequences shown in SEQ ID NO:3 to 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 a Cas9 protein.

[0065] sgRNA molecules

[0066] In the fourth aspect of the present invention, the present invention proposes an sgRNA molecule. According to an embodiment of the present invention, the sgRNA molecule has at least one of the nucleotide sequences shown in SEQ ID NO: 3 to 4, and optionally has at least one of the nucleotide sequences shown in SEQ ID NO: 2 and SEQ ID NO: 5. The sgRNA molecule can partially or completely delete the sequence of the soybean MHS2 regulatory element, and has the advantages of low off-target rate and high editing efficiency. In addition, the sequence of the MHS2 regulatory element can be partially or completely deleted by the sgRNA molecule, thereby partially or completely losing the activity of the MHS2 regulatory element, thereby reducing the expression of the Glyma.03G22970 gene and increasing the oil content of soybeans.

[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 an embodiment of the present invention, the sgRNA molecule has a nucleotide sequence as shown in SEQ ID NO: 3 and SEQ ID NO: 4.

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

[0071] Expression vector

[0072] In a fifth aspect of the present invention, the present invention proposes an expression vector. According to an embodiment of the present invention, the expression vector carries the sgRNA molecule described in the fourth aspect of the present invention and optionally a nucleic acid encoding a Cas9 molecule. The expression vector can partially or completely delete the sequence of the MHS2 regulatory element, thereby partially or completely deleting the sequence of the MHS2 regulatory element activity, thereby reducing the expression level of the Glyma.03G22970 gene and increasing the oil content of soybeans.

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

[0074] Reagents

[0075] In the sixth aspect of the present invention, the present invention proposes a reagent. According to an embodiment of the present invention, the reagent includes 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. The 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, thereby reducing the expression level of the Glyma.03G22970 gene and increasing the oil content of soybeans.

[0076] CRISPR / Cas9 system

[0077] In the seventh aspect of the present invention, the present invention proposes a CRISPR / Cas9 system. According to an embodiment of the present invention, the CRISPR / Cas9 system comprises the sgRNA molecule described in the fourth aspect of the present invention and a nucleic acid that optionally encodes a Cas9 molecule. The 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, thereby reducing the expression of the Glyma.03G22970 gene and increasing the oil content of soybeans.

[0078] Reagent test kit

[0079] In the eighth aspect of the present invention, the present invention proposes a kit. According to an embodiment of the present invention, the kit 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; and optionally a nucleic acid encoding a Cas9 molecule. The 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, thereby reducing the expression of the Glyma.03G22970 gene and increasing the oil content of soybeans.

[0080] The sequence table of the present invention is as follows:

[0081]

[0082]

[0083] The scheme of the present disclosure will be explained below in conjunction with the examples. Those skilled in the art will appreciate that the following examples are only used to illustrate the present disclosure and should not be considered to limit the scope of the present disclosure. Where specific techniques or conditions are not indicated in the examples, the techniques or conditions described in the literature in this area or the product instructions are used. Where the manufacturers of reagents or instruments are not indicated, they are all conventional products that can be obtained commercially.

[0084] Example 1 Design of CRISPR / Cas9 knockout target site in the seed-specific MHS2 region of the soybean seed highly abundantly expressed gene Glyma.03G22970

[0085] The Glyma.03G22970 gene is homologous to the Arabidopsis UMAMIT25 gene and is specifically expressed in soybean seeds, especially in the middle and late stages of soybean seed development. Figure 1 shown.

[0086] (1) Design of knockout targets for the MHS region of the Glyma.03G22970 gene:

[0087] First, this study used MNase-seq sequencing technology to mine candidate enhancers in the whole genome of Williams82 soybean leaves, flowers and grains. Bowtie software was used to locate the reads of MNase-seq sequencing results, and the reads were backfilled into the Williams82 genome sequence (https: / / phytozome.), and the parameters for allowing base mismatches were adjusted to retain reads that could only be backfilled into one segment of the Williams82 genome. The soybean genome was divided into several 200bp non-overlapping regions, and the total number of backfilled reads generated by multiple repeated experiments in this region was counted. The regions with FDR<0.01 and length greater than 50bp were located as MHS sites as enhancer candidate regions. Using this method, we successfully located a candidate enhancer for seed-specific enhancer at Gm03:44336551..44336694 (+strand) upstream of the Glyma.03G229700 gene, which is located in the seed-specific open chromatin region and named seed-specific-MHS2 (also known as MHS2). The specific location can be found in Figure 2 .

[0088] Subsequently, the seed-specific-MHS2 sequence was found in the Williams82 genome sequence, and sgRNA was designed on both sides and in the middle of MHS2 through the target online website CRISPR-GE (http: / / skl.scau.edu.cn / ). The promoter was the soybean endogenous promoter pM4. The following principles were followed when designing the target: (1) The target sequence was in the form of 5'-20nt-NGG; (2) The knockout site was located on both sides and in the middle of MHS2; this made it more likely that MHS2 would be completely knocked out, as well as partially knocked out. The four targets were named MHS2-sg1 (as shown in SEQ ID NO:2 in the sequence list), MHS2-sg2 (as shown in SEQ ID NO:3 in the sequence list), MHS2-sg3 (as shown in SEQ ID NO:4 in the sequence list), and MHS2-sg4 (as shown in SEQ ID NO:5 in the sequence list). The CRISPR / Cas9 knockout target site and location of seed-specific-MHS2 are shown in Figure 2. Figure 3shown.

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

[0090] First, according to the CRISPR-GE website, the possible 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 in the exon region of the soybean genome were predicted. The prediction results showed that the possible off-target rates of MHS2-sg1, MHS2-sg2, MHS2-sg3 and MHS2-sg4 in the exon region of the soybean genome were less than 33%.

[0091] Secondly, positive roots were obtained by soybean hairy root transformation method, and the off-target and target editing efficiency of MHS2-sg1, MHS2-sg2, MHS2-sg3 and MHS2-sg4 were detected. The results of the editing efficiency of the MHS2 target are shown in Table 1. The results showed that no off-target was found in the four targets; and the editing efficiency of MHS2-sg1, MHS2-sg2, MHS2-sg3 and MHS2-sg4 was higher than 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 example, PGES401 was used as the initial vector, and the PGES401 vector was driven by the pM4 promoter to express the Cas9 protein and sgRNA+sgRNA Scaffold.

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

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

[0098] DNA fragments of MHS-sg2 and MHS-sg3 were obtained by PCR amplification, and the PCR products of the above DNA fragments were gel-recovered. The above recovered PCR products were added to the ligation reaction system together with the vector pGES401, and the obtained ligation products were transformed into the competent cells of Escherichia coli DH5α. Positive clones were identified by colony PCR, and the positive clones were sent to a sequencing company for sequencing. The Escherichia coli plasmids with correct sequencing were extracted and transformed into Agrobacterium K599. The colony identification was correct and the sequencing results were correct, indicating that the CRISPR / Cas9 knockout vector single target Glyma.03G229705-MHS2-Cas9 with a single target Glyma.03G229705-MHS2 has been successfully constructed, and the knockout vector contains the Bar selection marker gene (phosphinothricin resistance).

[0099] 2.2 Construction of dual-target Glyma.03G229705-MHS2: Using pGES401-dual-target (MHS-sg2, MHS-sg3) empty vector as template, amplify an additional tRNA-sgRNA-sgRNA Scaffold-tRNA structure by PCR, and construct a DNA fragment of two sgRNAs in series by the goldgate method (i.e., 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: PGES401 plasmid 5μl, T4 DNA Ligase 1μl, T4 DNA Ligase Buffer 2μl, BsaI 2μl, MHS-sg2 1μl, MHS-sg3 1μl, ddH2O 5μl;

[0101] (2) A tRNA-sgRNA-sgRNA Scaffold-tRNA DNA fragment was obtained by PCR amplification. The specific PCR reaction conditions were as follows: (37°C 5min, 16°C 5min) × 25 cycles, 37°C 15min, 85°C 5min; 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 high-fidelity enzyme KOD One TM PCR master mix purchased from TOYOBO was used to perform PCR amplification on the DNA fragment of the tRNA-sgRNA-sgRNA Scaffold-tRNA obtained above to obtain a PCR product of the DNA fragment of two sgRNAs in series. The specific PCR reaction conditions are: 98°C pre-denaturation for 3 minutes, (98°C denaturation for 15 seconds, 58°C annealing for 15 seconds, 68°C extension for 20 seconds) × 35 cycles, and then 68°C extension for 5 minutes to obtain the PCR product, and the PCR product was gel-recovered.

[0103] (4) The recovered DNA fragments and vector pGES401 are added to the ligation reaction system at the same time, and the obtained ligation products are transformed into Escherichia coli competent cells DH5α. After colony PCR identification of positive clones, the positive clones are sent to a sequencing company for sequencing. The sequencing primer is STU-TEST-4R (nucleotide sequence as shown in SEQ ID NO: 12). The sequencing results show that the vector pGES401 contains the sequences of tRNA, MHS-sg2, sgRNA Scaffold, tRNA, MHS-sg3, sgRNA Scaffold, and tRNA. The Escherichia coli plasmid with correct sequencing is extracted and transformed into Agrobacterium K599. The colony identification is correct and the sequencing results are correct, indicating that the CRISPR / Cas9 knockout vector of the dual-target Glyma.03G229705-MHS2 dual-target Glyma.03G229705-MHS2-Cas9 has been successfully constructed, and the knockout vector contains the Bar selection marker gene (phosphinothricin resistance).

[0104] 2.3 Construction of four-target Glyma.03G229705-MHS2: Using pGES401-four-target (MHS-sg1~4) empty vector as template, three additional tRNA-sgRNA-sgRNA Scaffold-tRNA structures were amplified by PCR, and four sgRNA tandem DNA fragments (i.e., four-target Glyma.03G229705-MHS2) were constructed by 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: 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) Three tRNA-sgRNA-sgRNA Scaffold-tRNA DNA fragments were obtained by PCR amplification. The specific PCR reaction conditions are as follows: (37°C 5min, 16°C 5min) × 25 cycles, 37°C 15min, 85°C 5min; the specific amplification primers are: Glyma.03G229705-MHS2-F1 (protective base + BsaI recognition site + BsaI cleavage site + MHS2-sg1 + sgRNA Scaffold, the specific nucleotide sequence is shown in SEQ ID NO: 6), Glyma.03G229705-MHS2-R1 (protective base + BsaI recognition site + MHS2-sg2 + sgRNA Scaffold, the specific nucleotide sequence is shown in SEQ ID NO: 7), Glyma.03G229705-MHS2-F2 (protective base + BsaI recognition site + MHS2-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), Glyma.03G229705-MHS2-F3 (protective base + BsaI recognition site + MHS2-sg3 + sgRNA Scaffold, the specific nucleotide sequence is shown in SEQ ID NO: 10), Glyma.03G229705-MHS2-R3 (protective base + BsaI recognition site + BsaI cleavage site + MHS2-sg4 + sgRNA Scaffold, the specific nucleotide sequence is shown in SEQ ID NO: 11).

[0107] (3) The three tRNA-sgRNA-sgRNA Scaffold-tRNA DNA fragments obtained above were PCR amplified using the high-fidelity enzyme KOD One TM PCR master mix purchased from TOYOBO to obtain the PCR product of the four sgRNA tandem DNA fragments. The specific PCR reaction conditions are: 98°C pre-denaturation for 3 min, (98°C denaturation for 15 s, 58°C annealing for 15 s, 68°C extension for 20 s) × 35 cycles, and then 68°C extension for 5 min to obtain the PCR product, and the PCR product was gel recovered.

[0108] (4) The recovered DNA fragments and vector pGES401 were added to the ligation reaction system at the same time, and the obtained ligation products were transformed into E. coli competent cells DH5α. The positive clones were identified by colony PCR, and the positive clones were sent to a sequencing company for sequencing. The sequencing primer was STU-TEST-4R (nucleotide sequence as shown in SEQ ID NO: 12). The sequencing results showed that the 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 E. coli plasmid with correct sequencing was extracted and transformed into Agrobacterium K599. The colony identification was correct and the sequencing results were correct, indicating that the CRISPR / Cas9 knockout vector of the four-target Glyma.03G229705-MHS2 has been successfully constructed. The four-target Glyma.03G229705-MHS2-Cas9 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 shown.

[0109] Furthermore, the inventors verified the target efficiency of the single-target, dual-target and quad-target Glyma.03G229705-MHS2-Cas9 of the above-constructed vector through soybean hairy root transformation experiments. The results showed that the target efficiency of the single-target, dual-target and quad-target Glyma.03G229705-MHS2-Cas9 were all high. This example exemplifies the target efficiency of the quad-target Glyma.03G229705-MHS2-Cas9, which were 83.3%, 85.1%, 82.6% and 84.3%, respectively.

[0110] The constructed vector seed-specific MHS2-Cas9 was transferred into the Williams82 soybean variety through the EHA105 Agrobacterium-mediated soybean cotyledon node method to obtain the soybean Glyma.03G229705 enhancer CRISPR / Cas9 knockout mutant strain. The seed-specific MHS2-Cas9 vector construction process is as follows Figure 3 shown.

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

[0112] The single-target, dual-target, and quad-target vectors Glyma.03G229705-MHS2-Cas9 constructed in Example 2 were transferred into the wild-type Williams82 soybean variety through EHA105 Agrobacterium-mediated soybean cotyledon nodes to obtain soybean single-target, dual-target, and quad-target Glyma.03G229705-MHS2 enhancer CRISPR / Cas9 knockout mutant strains. Among these soybean different target point Glyma.03G229705-MHS2 enhancer CRISPR / Cas9 knockout mutant lines, 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 point Glyma.03G229705-MHS2 enhancer CRISPR / Cas9 knockout mutant line were T1 and T3, the positive seedlings corresponding to the double target point Glyma.03G229705-MHS2 enhancer CRISPR / Cas9 knockout mutant line were T4 and T5, and the positive seedlings corresponding to the quadruple target point Glyma.03G229705-MHS2 enhancer CRISPR / Cas9 knockout mutant line were T2, T22, and T27).

[0113] Using genomic DNA as a template, TOYOBO's high-fidelity enzyme KOD OneTM PCR master Mix was used to amplify the genomic fragment containing the target site. The amplification primers were Glyma.03G229705-MHS2-F (SEQ ID NO: 15) and Glyma.03G229705-MHS2-R (SEQ ID NO: 16). The PCR reaction conditions were: 98℃ pre-denaturation for 3min, (98℃ denaturation for 15s, 58℃ annealing for 15s, 68℃ extension for 30s) × 32 cycles, and then 72℃ extension for 5min. After PCR detection, 7 bar-resistant positive seedlings T1, T2, T3, T4, T5, T22 and T27 large fragment knockout strains were obtained. Based on Sanger sequencing, the sequencing sequences were aligned with the wild-type sequences by DNAman, and the specific mutation types of the above 7 large fragment knockout strains T1, T2, T3, T4, T5, T22 and T27 were determined. Sequencing analysis revealed that the seven large-fragment knockout strains, T1, T2, T3, T4, T5, T22 and T27, all partially or completely knocked out MHS2.

[0114] This example exemplifies the results of positive seedlings T2, T22 and T27 in the four-target Glyma.03G229705-MHS2 enhancer CRISPR / Cas9 knockout mutant strain. For specific results, see Figure 4 Among them, T2 is a large-fragment knockout strain with a partial deletion of 175bp in the MHS2 sequence, T22 is a homozygous complete knockout strain with a partial deletion of 216bp in the MHS2 sequence, and T27 is a large-fragment knockout strain with a partial deletion of 117bp in the MHS2 sequence. And the three mutants T2, T22 and T27 can be stably inherited to the offspring through T0, T1 and T2 generation testing.

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

[0116] The T2 generation homozygous and large-fragment deleted MHS2 knockout mutant soybeans T1, T2, T3, T4, T5, T22 and T27 prepared in Example 3 and the wild-type control variety Williams82 were sown in pots, with 30 plants in each line. The expression level of Glyma.03G229705 gene in T1, T2, T3, T4, T5, T22, T27 and Williams82 was counted by RT-PCR, and the protein and oil contents of T1, T2, T3, T4, T5, T22, T27 and Williams82 were analyzed by gas chromatography and elemental analyzer to detect nitrogen content. The expression level of Glyma.03G229705 gene, protein and oil contents in T1, T2, T3, T4, T5, T22, T27 and Williams82 were analyzed.

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

[0118] Among them, this example exemplifies the results of positive seedlings T2, T22 and T27 in the four-target Glyma.03G229705-MHS2 enhancer CRISPR / Cas9 knockout mutant strain, see Figures 5 to 7 Compared with the wild-type Williams82, the expression of Glyma.03G229705 in the MHS2 knockout mutants T2, T22, and T27 was reduced by 10% to 63% (for specific results, see Figure 5 ), and the protein content in the seeds of MHS2 knockout mutants T2, T22 and T27 decreased by 2%-6% (for specific results, see Figure 6 ), and the oil content in seeds of MHS2 knockout mutants T2, T22, and T27 increased by 1% to 5% (for specific results, see Figure 7 ). Therefore, soybean MHS2 regulatory elements can regulate protein accumulation in soybean grains by precisely regulating the expression of Glyma.03G229705, and have important application prospects in the breeding of high-protein and high-yield soybeans.

[0119] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0120] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. Use of the MHS2 regulatory element in regulating the expression of the Glyma.03G22970 gene, the nucleotide sequence of the MHS2 regulatory element is shown in SEQ ID NO:

1.

2. The use according to claim 1, characterized in that The nucleotide sequence of the MHS2 regulatory element is transformed or modified to reduce the expression level of the Glyma.03G22970 gene; Optionally, the nucleotide sequence of the Glyma.03G22970 gene is as shown in SEQ ID NO:

14.

3. Use of the MHS2 regulatory element in regulating the oil content of soybeans, the nucleotide sequence of the MHS2 regulatory element is shown in SEQ ID NO:

1.

4. The use according to claim 3, characterized in that: The nucleotide sequence of the MHS2 regulatory element is transformed or modified to increase the oil content of the soybean.

5. A method for regulating the oil content of soybeans, characterized in that: include: The nucleotide sequence of the MHS2 regulatory element in the soybean is transformed or modified, and the nucleotide sequence of the MHS2 regulatory element is shown in SEQ ID NO:

1.

6. The use according to claims 2 and 4 and the method according to claim 5, characterized in that The nucleotide sequence of the MHS2 regulatory element is transformed or modified by the following method: Deleting, replacing, inserting, inverting and ectopic at least a portion of the sequence of the MHS2 regulatory element; or Methylating and / or acetylating at least a portion of the sequence of the MHS2 regulatory element; Optionally, the nucleotide sequence of the MHS2 regulatory element is transformed or modified by a gene editing system and / or RNA interference; Optionally, the gene editing system includes CRISPR-Cas9 virus and non-virus; Optionally, the gene editing system comprises: An sgRNA having at least one of the nucleotide sequences shown in SEQ ID NOs: 3 to 4, and optionally having at least one of the nucleotide sequences shown in SEQ ID NOs: 2 and 5; Optionally, the gene editing system further comprises a Cas9 protein.

7. A sgRNA molecule, characterized in that include: The nucleic acid sequence of the present invention comprises at least one of the nucleotide sequences shown in SEQ ID NOs: 3 to 4, and optionally comprises at least one of the nucleotide sequences shown in SEQ ID NOs: 2 and 5.

8. An expression vector, characterized in that Carrying the sgRNA molecule of claim 7 and optionally a nucleic acid encoding a Cas9 molecule.

9. A reagent, characterized in that include: The sgRNA molecule of claim 7 or the expression vector of claim 8.

10. A CRISPR / Cas9 system, characterized in that: include: The sgRNA molecule of claim 7 and optionally a nucleic acid encoding a Cas9 molecule.

11. A kit, characterized in that: include: The sgRNA molecule of claim 7 or the expression vector of claim 8; as well as Optionally a nucleic acid encoding a Cas9 molecule.

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