Soybean glyma.05g029800 gene enhancer regulatory element and sgRNA molecule thereof

Abstract

The application discloses a soybean Glyma.05g029800 gene enhancer regulatory element and an sgRNA molecule thereof. The enhancer regulatory element is an MHSbeta regulatory element, the nucleotide sequence of the MHSbeta 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:3-5, and has at least one of the nucleotide sequences shown as SEQ ID NO:2 and SEQ ID NO:6. The MHSbeta regulatory element can be used as an enhancer of the Glyma.05g029800 gene, the knockout of the MHSbeta regulatory element can reduce the expression amount of the Glyma.05g029800 gene and improve the nodule number of the soybean; the sgRNA molecule can cause large fragment deletion of the nucleotide sequence of the MHSbeta regulatory element, and has the advantages of low off-target rate and high editing efficiency.

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.05g029800 gene and its sgRNA molecule. More specifically, this invention relates to the use of the MHSβ regulatory element in regulating the expression of the Glyma.05g029800 gene, the use of the MHSβ regulatory element in regulating the number of soybean nodules, a method for regulating the number of soybean nodules, expression vectors, reagents, a CRISPR / Cas9 system, and a kit. Background Technology

[0002] Soybeans (Glycine max) are an important crop belonging to the legume family. Originating in East Asia, they are one of the world's most important food and oilseed crops. Rich in high-quality protein, vegetable oil, carbohydrates, and various nutrients, soybeans are widely used in food processing, animal feed, and bioenergy production.

[0003] During soybean growth, it establishes a mutually beneficial symbiotic relationship with a special type of bacteria—rhizobia. This symbiotic relationship is known as a symbiotic nitrogen-fixing system. Rhizobia are common soil bacteria that can form symbiotic nodules with soybean roots and fix nitrogen. Through interaction with soybean roots, rhizobia form nodule structures. These nodule structures are formed by rhizobia invading the root hair cells of soybean roots, establishing a close connection with the soybean root cells. In this symbiotic nitrogen-fixing system, soybeans provide a suitable environment and nutrients for rhizobia, while rhizobia provide the soybean with the nitrogen-fixing ability.

[0004] When external microorganisms infect plants, they can trigger a series of immune responses, including hypersensitive reactions (HR). Among these immune responses, hypersensitive-induced reaction (HIR) genes, belonging to the BAND7 protein family, play a crucial role. In soybeans, there is an HIR gene called Glyma.05g029800, which is highly expressed in soybean roots, especially in root nodules. However, when exposed to stimuli, this gene triggers a hypersensitive-induced reaction, thereby affecting the nodulation ability of soybeans.

[0005] Therefore, there is an urgent need for a method to increase the number of nodules in soybeans. Summary of the Invention

[0006] 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 discovers the use of the MHSβ regulatory element in the regulation of the Glyma.05g029800 gene.

[0007] This invention is based on the following discoveries of the inventors:

[0008] While researching the Glyma.05g029800 gene, the inventors unexpectedly discovered a novel enhancer targeting the Glyma.05g029800 gene. This enhancer, located downstream of the Glyma.05g029800 gene at Gm05:2556251..2556481(+strand), was named MHSβ. Furthermore, the inventors unexpectedly found that knocking out MHSβ in soybeans significantly reduced the expression level of the Glyma.05g029800 gene. In particular, compared to the wild-type group without MHSβ knockout, the MHSβ knockout mutant group showed a 26%–27% reduction in Glyma.05g029800 expression in leaves and an 81%–91% reduction in nodules. Additionally, the MHSβ knockout mutant group had a significantly higher number of nodules than the wild-type group without MHSβ knockout.

[0009] Based on this, in a first aspect, the present invention proposes the use of an MHSβ regulatory element (also referred to as MHSβ) in regulating the expression of the Glyma.05g029800 gene. According to an embodiment of the present invention, the nucleotide sequence of the MHSβ regulatory element is shown in SEQ ID NO:1. The present invention discovers that the MHSβ regulatory element can act as an enhancer of the Glyma.05g029800 gene to regulate its expression.

[0010] In two aspects, this invention proposes the use of MHSβ regulatory elements in regulating soybean nodulation. According to an embodiment of the invention, the nucleotide sequence of the MHSβ regulatory element is shown in SEQ ID NO:1. This invention discovers that MHSβ regulatory elements regulate soybean nodulation by modulating the expression of the Glyma.05g029800 gene.

[0011] In a third aspect, the present invention provides a method for regulating the number of nodules in soybeans. According to embodiments of the present invention, the nucleotide sequence of the MHSβ regulatory element in soybeans is modified or altered, the nucleotide sequence of the MHSβ regulatory element being shown in SEQ ID NO:1. This method can cause large fragment deletions in the nucleotide sequence of the MHSβ regulatory element, thereby completely or partially losing the activity of the MHSβ regulatory element, thus reducing the expression level of the Glyma.05g029800 gene and increasing the number of nodules in soybeans.

[0012] 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: 3-5, and optionally at least one of the nucleotide sequences shown in SEQ ID NO: 2 and SEQ ID NO: 6. This sgRNA molecule can cause large-fragment loss of soybean MHSβ regulatory elements, exhibiting advantages such as low off-target rate and high editing efficiency. Furthermore, by using this sgRNA molecule to cause large-fragment deletion of the nucleotide sequence of MHSβ regulatory elements, the activity of MHSβ regulatory elements is completely or partially lost, thereby reducing the expression level of the Glyma.05g029800 gene and increasing the number of nodules in soybeans.

[0013] 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 cause large-fragment deletions of the nucleotide sequence of the MHSβ regulatory element, thereby completely or partially losing the activity of the MHSβ regulatory element, thus reducing the expression level of the Glyma.05g029800 gene and increasing the number of nodules in soybeans.

[0014] 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 cause large fragment deletions of the nucleotide sequence of the MHSβ regulatory element, thereby completely or partially losing the activity of the MHSβ regulatory element, thus reducing the expression level of the Glyma.05g029800 gene and increasing the number of nodules in soybeans.

[0015] 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, a nucleic acid encoding a Cas9 molecule. This CRISPR / Cas9 system can cause large-fragment deletions of the nucleotide sequence of MHSβ regulatory elements, thereby completely or partially losing the activity of the MHSβ regulatory elements, thus reducing the expression level of the Glyma.05g029800 gene and increasing the number of nodules in soybeans.

[0016] 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, a nucleic acid encoding a Cas9 molecule. This kit can cause large-fragment deletions of the nucleotide sequence of the MHSβ regulatory element, thereby completely or partially losing the activity of the MHSβ regulatory element, thus reducing the expression level of the Glyma.05g029800 gene and increasing the number of nodules in soybeans. 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 shows the CRISPR / Cas9 knockout target site and its location in Glyma.05g029800-MHS in Example 1.

[0019] Figure 2 The construction process of the Glyma.05g029800-MHS-Cas9 vector in Example 2 is shown.

[0020] Figure 3 The mutation types are those of the two mutants in Example 3.

[0021] Figure 4 The expression levels of the Glyma.05g029800 gene in the leaves of wild-type and mutant plants in Example 4 are shown.

[0022] Figure 5 The expression levels of the Glyma.05g029800 gene in wild-type and mutant root nodules in Example 4 are shown.

[0023] Figure 6 The number of root nodules in wild-type and mutant in Example 4.

[0024] Figure 7 The nodulation status of wild-type and mutant in Example 4. 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, "regulatory elements" generally refer to DNA sequences produced by gene transcription regulation, mainly including promoters, enhancers, insulators, etc.

[0031] In this invention, "the nucleotide sequence of the MHSβ regulatory element is modified or altered" means that the nucleotide sequence of the MHSβ regulatory element is changed. These nucleotide sequence changes include sequence deletion, substitution, insertion, inversion, translocation, as well as sequence methylation and / or acetylation. After the nucleotide sequence of the MHSβ regulatory element is changed, it will affect the binding ability of the MHSβ regulatory element to transcription factors, thereby regulating the expression of the Glyma.05g029800 gene and regulating the nodulation ability of soybean.

[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, "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.

[0034] This invention proposes the use of MHSβ regulatory elements in regulating the expression of the Glyma.05g029800 gene, the use of MHSβ regulatory elements in regulating the number of soybean nodules, a method for regulating the number of soybean nodules, sgRNA molecules, expression vectors, reagents, a CRISPR / Cas9 system, and a kit.

[0035] The role of MHSβ regulatory elements in regulating Glyma.05g029800 gene expression

[0036] In a first aspect, the present invention proposes the use of an MHSβ regulatory element in regulating the Glyma.05g029800 gene. According to embodiments of the present invention, the nucleotide sequence of the MHSβ regulatory element is shown in SEQ ID NO:1. The present invention discovers that the MHSβ regulatory element can act as an enhancer of the Glyma.05g029800 gene to regulate its expression.

[0037] According to an embodiment of the present invention, the nucleotide sequence of the MHSβ regulatory element is modified or altered to reduce the expression level of the Glyma.05g029800 gene.

[0038] According to an embodiment of the present invention, the nucleotide sequence of the Glyma.05g029800 gene is shown in SEQ ID NO:17.

[0039] According to some optional embodiments of the present invention, the MHSβ sequence is located downstream of the Glyma.05g029800 gene at Gm05:2556251..2556481(+strand).

[0040] According to an embodiment of the present invention, the MHSβ regulatory element sequence is modified or altered in the following manner:

[0041] At least one of the following is performed on at least a portion of the sequence of the MHSβ 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 MHSβ regulatory element.

[0042] According to embodiments of the present invention, the MHSβ regulatory element sequence is modified or altered through gene editing systems and / or RNA interference.

[0043] According to an embodiment of the present invention, the gene editing system includes a CRISPR-Cas9 virus and a non-viral component.

[0044] 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 to 5, and optionally having at least one of the nucleotide sequences shown in SEQ ID NO:2 or SEQ ID NO:6.

[0045] According to an embodiment of the present invention, the gene editing system further includes the Cas9 protein.

[0046] Application of MHSβ regulatory elements in regulating soybean nodulation

[0047] In a second aspect, the present invention proposes the use of the MHSβ regulatory element in regulating the number of nodules in soybeans. According to an embodiment of the present invention, the nucleotide sequence of the MHSβ regulatory element is shown in SEQ ID NO:1. The present invention discovers that the MHSβ regulatory element can regulate the number of nodules in soybeans by regulating the expression of the Glyma.05g029800 gene.

[0048] According to an embodiment of the present invention, the nucleotide sequence of the MHSβ regulatory element is modified or altered to increase the number of nodules in the soybean.

[0049] According to an embodiment of the present invention, the MHSβ regulatory element sequence is modified or altered in the following manner:

[0050] At least one of the following is performed on at least a portion of the sequence of the MHSβ 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 MHSβ regulatory element.

[0051] According to embodiments of the present invention, the MHSβ regulatory element sequence is modified or altered through gene editing systems and / or RNA interference.

[0052] According to an embodiment of the present invention, the gene editing system includes a CRISPR-Cas9 virus and a non-viral component.

[0053] 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 to 5, and optionally having at least one of the nucleotide sequences shown in SEQ ID NO:2 or SEQ ID NO:6.

[0054] According to an embodiment of the present invention, the gene editing system further includes the Cas9 protein.

[0055] Methods for regulating the number of soybean nodules

[0056] In a third aspect, the present invention provides a method for regulating the number of nodules in soybeans. According to embodiments of the present invention, the nucleotide sequence of the MHSβ regulatory element in soybeans is modified or altered, the nucleotide sequence of the MHSβ regulatory element being shown in SEQ ID NO:1. This method can cause large fragment deletions in the nucleotide sequence of the MHSβ regulatory element, thereby completely or partially losing the activity of the MHSβ regulatory element, thus reducing the expression level of the Glyma.05g029800 gene and increasing the number of nodules in soybeans.

[0057] According to an embodiment of the present invention, the MHSβ regulatory element sequence is modified or altered in the following manner:

[0058] At least one of the following is performed on at least a portion of the sequence of the MHSβ 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 MHSβ regulatory element.

[0059] According to embodiments of the present invention, the MHSβ regulatory element sequence is modified or altered through gene editing systems and / or RNA interference.

[0060] According to an embodiment of the present invention, the gene editing system includes a CRISPR-Cas9 virus and a non-viral component.

[0061] According to embodiments of the present invention, the RNA interference includes at least one of siRNA, miRNA, and shRNA.

[0062] 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 to 5, and optionally having at least one of the nucleotide sequences shown in SEQ ID NO:2 or SEQ ID NO:6.

[0063] According to an embodiment of the present invention, the gene editing system further includes the Cas9 protein.

[0064] sgRNA molecules

[0065] 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: 3-5, and optionally at least one of the nucleotide sequences shown in SEQ ID NO: 2 and SEQ ID NO: 6. This sgRNA molecule can knock out the nucleotide sequence of soybean MHSβ regulatory elements, exhibiting advantages such as low off-target rate and high editing efficiency. Furthermore, this sgRNA molecule can cause large fragment deletions of the nucleotide sequence of MHSβ regulatory elements, thereby completely or partially losing the activity of the MHSβ regulatory elements, thus reducing the expression level of the Glyma.05g029800 gene and increasing the number of nodules in soybeans.

[0066] According to an embodiment of the present invention, the sgRNA molecule has a nucleotide sequence as shown in SEQ ID NO: 3.

[0067] According to an embodiment of the present invention, the sgRNA molecule has a nucleotide sequence as shown in SEQ ID NO: 4.

[0068] According to an embodiment of the present invention, the sgRNA molecule has a nucleotide sequence as shown in SEQ ID NO: 5.

[0069] According to embodiments of the present invention, the sgRNA molecule has nucleotide sequences as shown in SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5.

[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, SEQ ID NO: 5 and SEQ ID NO: 6.

[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 cause large-fragment deletions of the nucleotide sequence of the MHSβ regulatory element, thereby completely or partially losing the activity of the MHSβ regulatory element, thus reducing the expression level of the Glyma.05g029800 gene and increasing the number of nodules in soybeans.

[0073] According to embodiments 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 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 cause large fragment deletions of the nucleotide sequence of the MHSβ regulatory element, thereby completely or partially losing the activity of the MHSβ regulatory element, thus reducing the expression level of the Glyma.05g029800 gene and increasing the number of nodules in soybeans.

[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, a nucleic acid encoding a Cas9 molecule. This CRISPR / Cas9 system can cause large-fragment deletions of the nucleotide sequence of MHSβ regulatory elements, thereby completely or partially losing the activity of the MHSβ regulatory elements, thus reducing the expression level of the Glyma.05g029800 gene and increasing the number of nodules in 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, a nucleic acid encoding a Cas9 molecule. This kit can cause large-fragment deletions of the nucleotide sequence of the MHSβ regulatory element, thereby completely or partially losing the activity of the MHSβ regulatory element, thus reducing the expression level of the Glyma.05g029800 gene and increasing the number of nodules in soybeans.

[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 MHS region of the soybean Glyma.05G029800 gene

[0085] (1) Design of knockout target sites targeting the MHS region of the Glyma.05G029800 gene:

[0086] First, the inventors used MNase-seq sequencing technology to mine candidate enhancers across the entire genome of Williams82 soybean leaves. Using Bowtie software, they located reads from the MNase-seq results and backfilled them into the Williams82 genome sequence (https: / / phytozome.), adjusting the parameters allowing for base mismatches to retain reads that could only be backfilled into one region of the Williams82 genome. Second, the inventors divided the soybean genome into several 200bp non-overlapping regions. They counted the total number of backfilled reads generated from repeated experiments within each region, locating regions with an FDR < 0.01 and a length greater than 50bp as MHS sites, and further identifying these regions as candidate enhancer regions. Using this method, the inventors successfully located a candidate enhancer at Gm05:2556251..2556481(+strand) downstream of the Glyma.05g029800 gene, named MHSβ.

[0087] Subsequently, the inventors found the MHSβ sequence in the Williams82 genome sequence and designed sgRNAs on both sides and in the middle of the MHS using the online target website CRISPR-GE (http: / / skl.scau.edu.cn / ). The promoter used was the soybean endogenous promoter pM4. The following two principles were followed when designing the target sites: ① The target sequence of the sgRNA designed for the MHSβ sequence must be 5'-20nt-NGG; ② When designing the knockout site for the MHSβ sequence, the knockout site must be located on both sides and in the middle of the MHSβ. The five target sites were named MHSβ-sg1 (as shown in SEQ ID NO:2 in the sequence listing), MHSβ-sg2 (as shown in SEQ ID NO:3 in the sequence listing), MHSβ-sg3 (as shown in SEQ ID NO:4 in the sequence listing), MHSβ-sg4 (as shown in SEQ ID NO:5 in the sequence listing), and MHSβ-sg5 (as shown in SEQ ID NO:6 in the sequence listing). The CRISPR / Cas9 knockout target site and location of Glyma.05g029800-MHSβ are as follows: Figure 1 As shown.

[0088] (2) Predicting the off-target rate and editing efficiency of the knockout target site in the MHS region of the Glyma.05g029800 gene:

[0089] First, based on the predictions made on the CRISPR-GE website regarding the potential off-target rates of MHSβ-sg1, MHSβ-sg2, MHSβ-sg3, MHSβ-sg4, and MHSβ-sg5 designed for the MHS region of the Glyma.05g029800 gene in the above steps within the soybean genome exon region, the prediction results showed that the potential off-target rates of MHSβ-sg1, MHSβ-sg2, MHSβ-sg3, MHSβ-sg4, and MHSβ-sg5 within the soybean genome exon region were less than 40%.

[0090] Secondly, positive roots with root nodule structures were obtained through soybean hairy root transformation. Off-target effects and target editing efficiency of MHSβ-sg1, MHSβ-sg2, MHSβ-sg3, MHSβ-sg4, and MHSβ-sg5 were then examined. The editing efficiency results for the MHSβ target are shown in Table 1. The results showed no off-target effects for any of the five targets; furthermore, the editing efficiencies of MHSβ-sg1, MHSβ-sg2, MHSβ-sg3, MHSβ-sg4, and MHSβ-sg5 were all above 70%.

[0091] Table 1: Editing efficiency of MHSβ target

[0092]

[0093]

[0094] Example 2: Construction of Glyma.05g029800-MHSβ-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.05G029800-MHSβ with different targets:

[0097] 2.1 Construction of the single target Glyma.05G029800-MHSβ:

[0098] DNA fragments of MHSβ-sg2, MHSβ-sg3, and MHSβ-sg4 were amplified by PCR. The PCR products of these DNA fragments were then recovered by gel electrophoresis. The recovered PCR products were added to the ligation reaction system along with the vector pGES401. The resulting ligation products were transformed into competent E. coli DH5α cells. Positive clones were identified by colony PCR and sent to a sequencing company for sequencing. The correctly sequenced E. coli plasmid was extracted and transformed into Agrobacterium K599. The colony identification and sequencing results were correct, indicating that the single-target CRISPR / Cas9 knockout vector Glyma.05g029800-MHSβ-Cas9 was successfully constructed. This knockout vector contains the Bar selection marker gene (glufosinate resistance).

[0099] 2.2 Construction of the three-target Glyma.05G029800-MHSβ: Using the pGES401-three-target (MHSβ-sg2~4) empty vector as a template, two additional tRNA-sgRNA-sgRNA scaffold-tRNA structures were amplified by PCR, and a DNA fragment of three sgRNAs tandemly (i.e., the three-target Glyma.05G029800-MHSβ) was constructed using the goldgate method. The specific steps are as follows:

[0100] (1) PGES401-MHSβ-sg2, PGES401-MHSβ-sg3, and PGES401-MHSβ-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, MHSβ-sg2 1 μl, MHSβ-sg3 1 μl, MHSβ-sg4 1 μl, and ddH2O 5 μl.

[0101] (2) Two DNA fragments of 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.05G029800-MHSβ-F2 (protective base + BsaI recognition site + MHSβ-sg2 + sgRNA Scaffold, specific nucleotide sequence as shown in SEQ ID NO: 9), Glyma.05g029800-MHSβ-R2 (protective base + BsaI recognition site + BsaI cleavage site + MHSβ-sg3 + sgRNA Scaffold, specific nucleotide sequence as shown in SEQ ID NO: 10), Glyma.05G029800-MHSβ-F3 (protective base + BsaI recognition site + MHSβ-sg3 + sgRNA Scaffold, specific nucleotide sequence as shown in SEQ ID NO: 9). NO: 11), Glyma.05g029800-MHSβ-R3 (protective base + BsaI recognition site + BsaI cleavage site + MHSβ-sg4 + sgRNA Scaffold, the specific nucleotide sequence is shown in SEQ ID NO: 12).

[0102] (3) The two 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 three 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.

[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 DH5α competent E. 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: 15). The sequencing results showed that vector pGES401 contained the sequences of tRNA, MHS-sg2, sgRNAScaffold, tRNA, MHS-sg3, sgRNAScaffold, tRNA, MHS-sg4, sgRNAScaffold, 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 CRISPR / Cas9 knockout vector Glyma.05G029800-MHSβ-Cas9 targeting three targets has been successfully constructed. This knockout vector contains the Bar selection marker gene (glufosinate resistance).

[0104] 2.3 Construction of the five-target Glyma.05G029800-MHSβ: Using the pGES401-five-target (MHS-sg1~5) empty vector as a template, four additional tRNA-sgRNA-sgRNA scaffold-tRNA structures were amplified by PCR, and a DNA fragment of five sgRNAs tandemly (i.e., the five-target Glyma.05G029800-MHSβ) was constructed using the goldgate method. The specific steps are as follows:

[0105] (1) PGES401-MHSβ-sg1, PGES401-MHSβ-sg2, PGES401-MHSβ-sg3, PGES401-MHSβ-sg4, and PGES401-MHSβ-sg5 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, MHSβ-sg1 1 μl, MHSβ-sg2 1 μl, MHSβ-sg3 1 μl, MHSβ-sg4 1 μl, MHSβ-sg5 1 μl, and ddH2O 5 μl.

[0106] (2) Four DNA fragments of 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.05G029800-MHSβ-F1 (protective base + BsaI recognition site + BsaI cleavage site + MHSβ-sg1 + sgRNA Scaffold, specific nucleotide sequence as shown in SEQ ID NO: 7), Glyma.05g029800-MHSβ-R1 (protective base + BsaI recognition site + MHSβ-sg2 + sgRNA Scaffold, specific nucleotide sequence as shown in SEQ ID NO: 8), Glyma.05G029800-MHSβ-F2 (protective base + BsaI recognition site + MHSβ-sg2 + sgRNA Scaffold, specific nucleotide sequence as shown in SEQ ID NO: 8). Glyma.05g029800-MHSβ-R2 (protective base + BsaI recognition site + BsaI cleavage site + MHSβ-sg3 + sgRNA Scaffold, specific nucleotide sequence as shown in SEQ ID NO: 9), Glyma.05g029800-MHSβ-F3 (protective base + BsaI recognition site + MHSβ-sg3 + sgRNA Scaffold, specific nucleotide sequence as shown in SEQ ID NO: 11), Glyma.05g029800-MHSβ-R3 (protective base + BsaI recognition site + BsaI cleavage site + MHSβ-sg4 + sgRNA Scaffold, specific nucleotide sequence as shown in SEQ ID NO: 12), Glyma.05g029800-MHSβ-F4 (protective base + BsaI recognition site + MHSβ-sg4 + sgRNA Scaffold). Scaffold (specific nucleotide sequence shown in SEQ ID NO: 13), U Glyma.05g029800-MHSβ-R4 (protective base + BsaI recognition site + BsaI cleavage site + MHSβ-sg5 + sgRNA Scaffold (specific nucleotide sequence shown in SEQ ID NO: 14).

[0107] (3) The four 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 five 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-recovered.

[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: 15). The sequencing results showed that vector pGES401 contained the sequences of tRNA, MHSβ-sg1, sgRNAScaffold, tRNA, MHSβ-sg2, sgRNAScaffold, tRNA, MHSβ-sg3, sgRNAScaffold, tRNA, MHSβ-sg4, sgRNAScaffold, tRNA, MHSβ-sg5, sgRNAScaffold, 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 five-target Glyma.05G029800-MHSβ CRISPR / Cas9 knockout vector. This knockout vector contains the Bar selection marker gene (glufosinate resistance). The construction process of the five-target Glyma.05g029800-MHSβ-Cas9 vector is as follows: Figure 2 As shown. Furthermore, the inventors verified the target efficiency of the constructed vector Glyma.05g029800-MHSβ-Cas9 with single, three, and five targets through soybean hairy root transformation experiments. The results showed that the target efficiency of single, three, and five targets of Glyma.05g029800-MHSβ-Cas9 was high. This embodiment exemplarily demonstrates that the target efficiency of five targets of Glyma.05g029800-MHSβ-Cas9 was 90%, 88%, 80%, 75.5%, and 71.8%, respectively.

[0109] Example 3: Construction of the MHSβ regulatory element knockout phenotype of soybean Glyma.05g029800 gene

[0110] The single-target, three-target, and five-target vectors Glyma.05g029800-MHSβ-Cas9 constructed in Example 2 were transferred into the cotyledonary nodes of soybean through EHA105 Agrobacterium-mediated transformation into the wild-type Williams82 soybean variety, resulting in soybean single-target, three-target, and five-target Glyma.05g029800 enhancer CRISPR / Cas9 knockout mutant lines. In these soybean CRISPR / Cas9 knockout mutant lines with different target Glyma.05g029800 enhancers, positive seedlings with bar resistance were obtained by bar test strip detection (i.e., Q1, Q2, Q3, Q4, Q5, and Q30; the positive seedlings corresponding to the single-target Glyma.05g029800 enhancer CRISPR / Cas9 knockout mutant lines were Q1 and Q3; the positive seedlings corresponding to the three-target Glyma.05g029800 enhancer CRISPR / Cas9 knockout mutant lines were Q4 and Q5; and the positive seedlings corresponding to the five-target Glyma.05g029800 enhancer CRISPR / Cas9 knockout mutant lines were Q2 and Q30).

[0111] Using genomic DNA as a template, genomic fragments containing target sites were amplified using TOYOBO's high-fidelity enzyme KOD One™ PCR master Mix. The amplification primers were Glyma.05g029800-MHSβ-F (SEQ ID NO: 18) and Glyma.05g029800-MHSβ-R (SEQ ID NO: 19). 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 identified bar-resistant positive seedlings with large fragment knockouts (Q1, Q2, Q3, Q4, Q5, and Q30). Based on Sanger sequencing, the specific mutation types of these six large fragment knockout lines were determined by comparing the sequenced sequences with wild-type sequences using DNAman. Sequencing analysis revealed that all six homozygous knockout lines, Q1, Q2, Q3, Q4, Q5, and Q30, partially knocked out MHSβ.

[0112] This embodiment exemplifies the results of positive seedlings Q30 and Q2 in a CRISPR / Cas9 knockout mutant line with the five-target Glyma.05g029800 enhancer. For detailed results, please refer to [link to relevant documentation]. Figure 3 Among them, Q30 is a homozygous knockout line with a 625bp deletion in the MHSβ region, and Q2 is a homozygous knockout line with a 216bp deletion in the MHSβ region. Furthermore, both Q30 and Q2 homozygous knockout mutants were stably inherited by offspring after T0, T1, and T2 generations.

[0113] Example 4: Phenotypic analysis of the MHSβ regulatory element knockout mutant of soybean Glyma.05g029800 gene

[0114] Soybean mutants Q1, Q2, Q3, Q4, Q5, and Q30 (T2 generation homozygous mutants with large fragment deletions) prepared in Example 3 were sown in pots with wild-type control variety Williams82, with 30 plants in each line. The expression level of Glyma.05g029800 gene in Q1, Q2, Q3, Q4, Q5, Q30, and Williams82 was counted by RT-PCR, as were the root nodules in Q30, Q2, and Williams82. The expression level of Glyma.05g029800 gene, root nodule number, and nodulation status in Q1, Q2, Q3, Q4, Q5, Q30, and Williams82 were analyzed.

[0115] The results showed that, compared with wild-type Williams82, the expression level of Glyma.05g029800 in the leaves of MHSβ knockout mutants Q1, Q2, Q3, Q4, Q5 and Q30 was reduced, the expression level of Glyma.05g0298000 in the root nodules of MHSβ knockout mutants Q1, Q2, Q3, Q4, Q5 and Q30 was reduced, and the number of root nodules in MHSβ knockout mutants Q1, Q2, Q3, Q4, Q5 and Q30 was significantly higher than that in wild-type Williams82. Furthermore, the nodulation of MHSβ knockout mutants Q1, Q2, Q3, Q4, Q5 and Q30 was significantly better than that in wild-type Williams82.

[0116] This embodiment exemplarily demonstrates the results of positive seedlings Q30 and Q2 in a CRISPR / Cas9 knockout mutant line with the five-target Glyma.05g029800 enhancer. See details for further information. Figures 4-7 Compared with the wild-type Williams82, the expression level of Glyma.05g029800 in the leaves of the MHSβ knockout mutants Q30 and Q2 was reduced by 26%–27% (see details). Figure 4 In the root nodules of MHSβ knockout mutants Q30 and Q2, the expression level of Glyma.05g0298000 was reduced by 81%–91% (see details). Figure 5 Furthermore, the number of root nodules in the MHSβ knockout mutants Q30 and Q2 was significantly higher than that in the wild-type Williams82 (see details). Figure 6 Furthermore, the nodulation of MHSβ knockout mutants Q30 and Q2 was significantly better than that of wild-type Williams82 (see details). Figure 7Therefore, soybean MHSβ regulatory elements regulate soybean nodulation and affect nutrient accumulation by precisely controlling the expression of Glyma.05g0298000, and have important application prospects in soybean breeding.

[0117] 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.

[0118] 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. MHSβ regulatory elements in inhibiting Glyma.05g029800 Use in gene expression, the nucleotide sequence of the MHSβ regulatory element is shown in SEQ ID NO:1; Glyma.05g029800 The nucleotide sequence of the gene is shown in SEQ ID NO:

17. The nucleotide sequence of the MHS2 regulatory element is knocked out, reducing the... Glyma.05g029800 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:

3.

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

3.

3. A method for increasing the number of nodules in soybeans, characterized in that, include: The nucleotide sequence of the MHSβ regulatory element in soybean was knocked out, and the nucleotide sequence of the MHSβ 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:

3.

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

3.

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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