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

Abstract

The invention discloses a soybean Glyma. 05g029800 gene enhancer regulatory element and a sgRNA (small guide ribonucleic acid) molecule of the soybean Glyma. 05g029800 gene enhancer regulatory element. The enhancer regulatory element is an MHS beta regulatory element, and the nucleotide sequence of the MHS beta 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: 3-5, and at least one of nucleotide sequences as shown in SEQ ID NO: 2 and SEQ ID NO: 6. The MHS beta regulatory element can be used as an enhancer of the Glyma. 05g029800 gene, and after the MHS beta regulatory element is knocked out, the expression quantity of the Glyma. 05g029800 gene is reduced, and the nodulation quantity of soybeans is increased; the sgRNA molecule can enable a nucleotide sequence of an MHS beta regulation element to be deleted in a large fragment mode, and has the advantages of being low in off-target rate, high in 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.05g029800 gene enhancer regulatory element and its sgRNA molecule. More specifically, the present invention relates to the use of MHSβ regulatory elements in regulating the expression of 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, expression vectors, reagents, CRISPR / Cas9 system and kit. Background Art

[0002] Soybean (Glycine max) is an important crop belonging to the Leguminosae family. It originated in East Asia and is one of the most important food and oil crops in the world. Soybean is rich in high-quality protein, vegetable oil, carbohydrates and various nutrients, and is widely used in food processing, feed and bioenergy production.

[0003] During the growth process of soybean, it establishes a mutualistic relationship with a special type of bacteria, Rhizobium. This symbiotic relationship is called the symbiotic nitrogen fixation system. Rhizobia are a type of bacteria commonly found in soil that can form symbiotic nodules with soybean roots and fix nitrogen. Rhizobia form nodule structures through interaction with soybean roots. These nodule structures are formed by Rhizobia invading the root hair cells of soybean roots, and they establish a close connection with soybean root cells. In the symbiotic nitrogen fixation system, soybeans provide a suitable environment and nutrients for Rhizobia, while Rhizobia provide soybeans with nitrogen fixation capabilities.

[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 play an important role, and they belong to the BAND7 protein family. In soybeans, there is a HIR gene called Glyma.05g029800, which is highly expressed in soybean roots, especially in nodules. However, when faced with stimulation, 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 soybean nodules. Summary of the invention

[0006] 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 MHSβ regulatory element in regulating the Glyma.05g029800 gene.

[0007] The present invention is accomplished based on the following findings of the inventors:

[0008] When the inventor studied the Glyma.05g029800 gene, he unexpectedly discovered a new enhancer for the Glyma.05g029800 gene, which is located at Gm05:2556251..2556481 (+strand) downstream of the Glyma.05g029800 gene and named MHSβ. In addition, the inventor unexpectedly found that when MHSβ in soybean was knocked out, the expression level of the Glyma.05g029800 gene could be significantly reduced, especially compared with the wild-type group without MHSβ knockout, the expression level of Glyma.05g029800 in the leaves of the MHSβ knockout mutant group was reduced by 26% to 27%, the expression level of Glyma.05g029800 in the nodules of the MHSβ knockout mutant group was reduced by 81% to 91%, and the number of nodules in the MHSβ knockout mutant group was significantly greater than that in the wild-type group without MHSβ knockout.

[0009] Based on this, in the first aspect of the present invention, the present invention proposes the use of MHSβ regulatory element (also known as MHSβ) in regulating the expression of 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 finds that the MHSβ regulatory element can be used as an enhancer of the Glyma.05g029800 gene to regulate the expression of the Glyma.05g029800 gene.

[0010] In the second aspect of the present invention, the present invention proposes the use of MHSβ regulatory element in regulating the number of soybean nodules. 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 found that the MHSβ regulatory element regulates the number of soybean nodules by regulating the expression of the Glyma.05g029800 gene.

[0011] In the third aspect of the present invention, a method for regulating the number of soybean nodules is proposed. According to an embodiment of the present invention, the nucleotide sequence of the MHSβ regulatory element in the soybean is transformed or modified, and the nucleotide sequence of the MHSβ regulatory element is shown in SEQ ID NO: 1. This method can cause a large fragment of the nucleotide sequence of the MHSβ regulatory element to be deleted, thereby causing the activity of the MHSβ regulatory element to be completely or partially lost, thereby reducing the expression level of the Glyma.05g029800 gene and increasing the number of soybean nodules.

[0012] 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 NOs: 3 to 5, and optionally has at least one of the nucleotide sequences shown in SEQ ID NOs: 2 and 6. The sgRNA molecule can cause the loss of a large fragment of the soybean MHSβ regulatory element, and has the advantages of low off-target rate, high editing efficiency, etc. In addition, the sgRNA molecule can cause the deletion of a large fragment of the nucleotide sequence of the MHSβ regulatory element, thereby causing the complete or partial loss of the activity of the MHSβ regulatory element, thereby reducing the expression of the Glyma.05g029800 gene and increasing the number of soybean nodules.

[0013] 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 cause a large fragment of the nucleotide sequence of the MHSβ regulatory element to be deleted, thereby causing the activity of the MHSβ regulatory element to be completely or partially lost, thereby reducing the expression level of the Glyma.05g029800 gene and increasing the number of soybean nodules.

[0014] 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 cause the deletion of a large fragment of the nucleotide sequence of the MHSβ regulatory element, thereby causing the complete or partial loss of the activity of the MHSβ regulatory element, thereby reducing the expression level of the Glyma.05g029800 gene and increasing the number of soybean nodules.

[0015] 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 optionally encoding a Cas9 molecule. The CRISPR / Cas9 system can cause a large fragment of the nucleotide sequence of the MHSβ regulatory element to be deleted, thereby completely or partially losing the activity of the MHSβ regulatory element, thereby reducing the expression of the Glyma.05g029800 gene and increasing the number of soybean nodules.

[0016] 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 cause a large fragment of the nucleotide sequence of the MHSβ regulatory element to be deleted, thereby causing the activity of the MHSβ regulatory element to be completely or partially lost, thereby reducing the expression level of the Glyma.05g029800 gene and increasing the number of soybean nodules. 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 This is the CRISPR / Cas9 knockout target site and location of Glyma.05g029800-MHS in Example 1.

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

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

[0021] Figure 4 is the expression level of the Glyma.05g029800 gene in the leaves of the wild type and mutant in Example 4.

[0022] Figure 5 is the expression level of the Glyma.05g029800 gene in the wild type and mutant nodules in Example 4.

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

[0024] Figure 7 This is the nodulation situation of the wild type and mutant 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, "regulatory elements" generally refer to DNA sequences involved in gene transcription regulation, mainly including promoters, enhancers, insulators, and the like.

[0031] In the present invention, "the nucleotide sequence of the MHSβ regulatory element is transformed or modified" means that the nucleotide sequence of the MHSβ regulatory element is changed, and the forms of these nucleotide sequence changes include sequence deletion, substitution, insertion, inversion, ectopicity and 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 with the transcription factor, thereby regulating the expression of the Glyma.05g029800 gene and regulating the nodulation ability of soybean.

[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, "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.

[0034] The present invention proposes the use of MHSβ regulatory elements in regulating the expression of 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, CRISPR / Cas9 systems, and kits.

[0035] Use of MHSβ regulatory element in regulating Glyma.05g029800 gene expression

[0036] In the first aspect of the present invention, the present invention proposes the use of the MHSβ regulatory element in regulating 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 finds that the MHSβ regulatory element can be used as an enhancer of the Glyma.05g029800 gene to regulate the expression of the Glyma.05g029800 gene.

[0037] According to an embodiment of the present invention, the nucleotide sequence of the MHSβ regulatory element is transformed or modified 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 downstream of the Glyma.05g029800 gene Gm05:2556251..2556481 (+strand).

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

[0041] At least a portion of the sequence of the MHSβ regulatory element is deleted, replaced, inserted, inverted, and misplaced; or at least a portion of the sequence of the MHSβ regulatory element is methylated and / or acetylated.

[0042] According to an embodiment of the present invention, the MHSβ regulatory element sequence is transformed or modified by a gene editing system and / or RNA interference.

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

[0044] 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 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 a Cas9 protein.

[0046] Application of MHSβ regulatory element in regulating soybean nodulation number

[0047] In a second aspect of the present invention, the present invention proposes the use of the MHSβ regulatory element in regulating the number of soybean nodules. 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 found that the MHSβ regulatory element can regulate the number of soybean nodules 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 transformed or modified to increase the nodulation number of the soybean.

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

[0050] At least a portion of the sequence of the MHSβ regulatory element is deleted, replaced, inserted, inverted, and misplaced; or at least a portion of the sequence of the MHSβ regulatory element is methylated and / or acetylated.

[0051] According to an embodiment of the present invention, the MHSβ regulatory element sequence is transformed or modified by a gene editing system and / or RNA interference.

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

[0053] 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 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 a Cas9 protein.

[0055] Methods for regulating the number of soybean nodules

[0056] In the third aspect of the present invention, a method for regulating the number of soybean nodules is proposed. According to an embodiment of the present invention, the nucleotide sequence of the MHSβ regulatory element in the soybean is transformed or modified, and the nucleotide sequence of the MHSβ regulatory element is shown in SEQ ID NO: 1. This method can cause a large fragment of the nucleotide sequence of the MHSβ regulatory element to be deleted, thereby causing the activity of the MHSβ regulatory element to be completely or partially lost, thereby reducing the expression level of the Glyma.05g029800 gene and increasing the number of soybean nodules.

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

[0058] At least a portion of the sequence of the MHSβ regulatory element is deleted, replaced, inserted, inverted, and misplaced; or at least a portion of the sequence of the MHSβ regulatory element is methylated and / or acetylated.

[0059] According to an embodiment of the present invention, the MHSβ regulatory element sequence is transformed or modified by a gene editing system and / or RNA interference.

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

[0061] According to an embodiment 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 includes: sgRNA, the 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 a Cas9 protein.

[0064] sgRNA molecules

[0065] 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 NOs: 3 to 5, and optionally has at least one of the nucleotide sequences shown in SEQ ID NOs: 2 and 6. The sgRNA molecule can knock out the nucleotide sequence of the soybean MHSβ regulatory element, and has the advantages of low off-target rate and high editing efficiency. In addition, the sgRNA molecule can cause large fragments of the nucleotide sequence of the MHSβ regulatory element to be deleted, thereby completely or partially losing the activity of the MHSβ regulatory element, thereby reducing the expression of the Glyma.05g029800 gene and increasing the number of soybean nodules.

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

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

[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 cause a large fragment of the nucleotide sequence of the MHSβ regulatory element to be deleted, thereby causing the activity of the MHSβ regulatory element to be completely or partially lost, thereby reducing the expression level of the Glyma.05g029800 gene and increasing the number of soybean nodules.

[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 cause the deletion of a large fragment of the nucleotide sequence of the MHSβ regulatory element, thereby causing the complete or partial loss of the activity of the MHSβ regulatory element, thereby reducing the expression level of the Glyma.05g029800 gene and increasing the number of soybean nodules.

[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 optionally encoding a Cas9 molecule. The CRISPR / Cas9 system can cause large fragments of the nucleotide sequence of the MHSβ regulatory element to be deleted, thereby completely or partially losing the activity of the MHSβ regulatory element, thereby reducing the expression of the Glyma.05g029800 gene and increasing the number of soybean nodules.

[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 cause a large fragment of the nucleotide sequence of the MHSβ regulatory element to be deleted, thereby causing the activity of the MHSβ regulatory element to be completely or partially lost, thereby reducing the expression level of the Glyma.05g029800 gene and increasing the number of soybean nodules.

[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 in the MHS region of soybean Glyma.05G029800 gene

[0085] (1) Design of knockout targets for the MHS region of the Glyma.05G029800 gene:

[0086] First, the inventors used MNase-seq sequencing technology to mine candidate enhancers in the whole genome of Williams82 soybean leaves, and used Bowtie software to locate the reads of the MNase-seq sequencing results, backfilled the reads into the Williams82 genome sequence (https: / / phytozome.), and adjusted the parameters that allowed base mismatches accordingly, retaining the reads that could only be backfilled into one segment of the Williams82 genome. Secondly, the inventors divided the soybean genome into several 200bp non-overlapping regions, counted the total number of backfills generated by repeated experiments in the region, located the FDR<0.01 region with a length greater than 50bp as the MHS site, and located the FDR<0.01 region with a length greater than 50bp as the enhancer candidate region. Using the above 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 MHS through the target online website CRISPR-GE (http: / / skl.scau.edu.cn / ). The promoter was the soybean endogenous promoter pM4. The following two principles were followed when designing the target: ① When designing sgRNA for the MHSβ sequence, the target sequence format must be 5'-20nt-NGG; ② When designing knockout sites for the MHSβ sequence, the knockout sites must be located on both sides and in the middle of MHSβ. The five targets were named MHSβ-sg1 (as shown in SEQ ID NO: 2 in the sequence listing), MHSβ-sg2 (as shown in SEQID NO: 3 in the sequence listing), MHSβ-sg3 (as shown in SEQ ID NO: 4 in the sequence listing), MHSβ-sg4 (as shown in SEQID 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 shown.

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

[0089] First, according to the CRISPR-GE website, the possible 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 in the exon region of the soybean genome were predicted. The prediction results showed that the possible off-target rates of MHSβ-sg1, MHSβ-sg2, MHSβ-sg3, MHSβ-sg4 and MHSβ-sg5 in the exon region of the soybean genome were less than 40%.

[0090] Secondly, positive roots with nodule structures were obtained by soybean hairy root transformation method, and the off-target conditions and editing efficiency of targets of MHSβ-sg1, MHSβ-sg2, MHSβ-sg3, MHSβ-sg4 and MHSβ-sg5 were detected, and the results of the editing efficiency of the MHSβ target are shown in Table 1. The results showed that no off-target conditions were found for the five targets; and the editing efficiencies of MHSβ-sg1, MHSβ-sg2, MHSβ-sg3, MHSβ-sg4 and MHSβ-sg5 were all higher than 70%.

[0091] Table 1: Editing efficiency of MHSβ targets

[0092]

[0093]

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

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

[0098] DNA fragments of MHSβ-sg2, MHSβ-sg3, and MHSβ-sg4 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 the 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.05G029800-MHSβ single target Glyma.05g029800-MHSβ-Cas9 has been successfully constructed, and the knockout vector contains the Bar selection marker gene (phosphinothricin resistance).

[0099] 2.2 Construction of triple target Glyma.05G029800-MHSβ: Using pGES401-triple target (with MHSβ-sg2~4) empty vector as template, two additional tRNA-sgRNA-sgRNA Scaffold-tRNA structures were amplified by PCR, and a DNA fragment of three sgRNAs in series was constructed by the goldgate method (i.e., triple target Glyma.05G029800-MHSβ). 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: PGES401 plasmid 5 μl, T4 DNA Ligase 1 μl, T4 DNA LigaseBuffer 2 μl, BsaI 2 μl, MHSβ-sg2 1 μl, MHSβ-sg3 1 μl, MHSβ-sg4 1 μl, and ddH2O 5 μl;

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

[0103] (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: 15). The sequencing results showed that the vector pGES401 contained the sequences of tRNA, MHS-sg2, sgRNA Scaffold, tRNA, MHS-sg3, sgRNA Scaffold, tRNA, MHS-sg4, sgRNA Scaffold, and tRNA. The Escherichia coli plasmid with correct sequencing was extracted and transformed into Agrobacterium K599. The colony identification and sequencing results were correct, indicating that the CRISPR / Cas9 knockout vector of the triple-target Glyma.05G029800-MHSβ triple-target Glyma.05g029800-MHSβ-Cas9 had been successfully constructed, and the knockout vector contained the Bar selection marker gene (glufosinate resistance).

[0104] 2.3 Construction of five-target Glyma.05G029800-MHSβ: Using pGES401-five-target (MHS-sg1~5) empty vector as template, four additional tRNA-sgRNA-sgRNAScaffold-tRNA structures were amplified by PCR, and five sgRNA-concatenated DNA fragments (i.e., five-target Glyma.05G029800-MHSβ) were constructed by 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: 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 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.05G029800-MHSβ-F1 (protective base + BsaI recognition site + BsaI cleavage site + MHSβ-sg1 + sgRNA Scaffold, the specific nucleotide sequence is shown in SEQ ID NO: 7), Glyma.05g029800-MHSβ-R1 (protective base + BsaI recognition site + MHSβ-sg2 + sgRNA Scaffold, the specific nucleotide sequence is shown in SEQ ID NO: 8), Glyma.05G029800-MHSβ-F2 (protective base + BsaI recognition site + MHSβ-sg2 + sgRNA Scaffold, the specific nucleotide sequence is shown in SEQ ID NO: 9). NO: 9), Glyma.05g029800-MHSβ-R2 (protective base + BsaI recognition site + BsaI cleavage site + MHSβ-sg3 + sgRNA Scaffold, the specific nucleotide sequence is shown in SEQ ID NO: 10), Glyma.05G029800-MHSβ-F3 (protective base + BsaI recognition site + MHSβ-sg3 + sgRNA Scaffold, the specific nucleotide sequence is shown in SEQ ID 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), Glyma.05g029800-MHSβ-F4 (protective base + BsaI recognition site + MHSβ-sg4 + sgRNA Scaffold, the specific nucleotide sequence is shown in SEQ ID NO: 13), U Glyma.05g029800-MHSβ-R4 (protective base + BsaI recognition site + BsaI cleavage site + MHSβ-sg5 + sgRNA Scaffold, the specific nucleotide sequence is shown in SEQ ID NO: 14).

[0107] (3) The four 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 five sgRNA tandem DNA fragments. The specific PCR reaction conditions were: 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: 15). The sequencing results showed that the 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, 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 five-target Glyma.05G029800-MHSβ CRISPR / Cas9 knockout vector five-target Glyma.05g029800-MHSβ-Cas9 has been successfully constructed. The 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 Further, the inventors verified the target efficiency of the single-target, triple-target and five-target Glyma.05g029800-MHSβ-Cas9 of the above-constructed vectors through soybean hairy root transformation experiments. The results showed that the target efficiency of the single-target, triple-target and five-target Glyma.05g029800-MHSβ-Cas9 was high. This example exemplifies the target efficiency of the five-target Glyma.05g029800-MHSβ-Cas9, which 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, triple-target, and penta-target vectors Glyma.05g029800-MHSβ-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, triple-target, and penta-target Glyma.05g029800 enhancer CRISPR / Cas9 knockout mutant strains. Among these soybean different target Glyma.05g029800 enhancer CRISPR / Cas9 knockout mutant lines, positive seedlings with bar resistance were obtained after 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 triple-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, 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.05g029800-MHSβ-F (SEQ ID NO: 18) and Glyma.05g029800-MHSβ-R (SEQ ID NO: 19). 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, the positive seedlings Q1, Q2, Q3, Q4, Q5 and Q30 large fragment knockout strains with bar resistance were identified. Based on Sanger sequencing, the sequencing sequences were aligned with the wild-type sequences by DNAman, and the specific mutation types of the above six large fragment knockout strains Q1, Q2, Q3, Q4, Q5 and Q30 were determined. Sequencing analysis revealed that the six homozygous knockout strains, Q1, Q2, Q3, Q4, Q5 and Q30, all had partial knockout of MHSβ.

[0112] This example exemplifies the results of positive seedlings Q30 and Q2 in the five-target Glyma.05g029800 enhancer CRISPR / Cas9 knockout mutant strain. For specific results, see Figure 3 Among them, Q30 is a homozygous knockout strain with a 625bp deletion in the MHSβ part, and Q2 is a homozygous knockout strain with a 216bp deletion in the MHSβ part. Moreover, the two homozygous knockout mutants Q30 and Q2 can be stably inherited to the offspring through T0, T1, and T2 generation tests.

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

[0114] The T2 generation homozygous and large-fragment-deleted MHSβ knockout mutant soybeans Q1, Q2, Q3, Q4, Q5 and Q30 prepared in Example 3 and the wild-type control variety Williams82 were sown in pots, with 30 plants in each line. RT-PCR was used to count the expression levels of the Glyma.05g029800 gene in Q1, Q2, Q3, Q4, Q5, Q30 and Williams82, and the number of nodules in Q30, Q2 and Williams82 were counted. The expression levels of the Glyma.05g029800 gene, the number of nodules and the nodulation conditions in Q1, Q2, Q3, Q4, Q5, Q30 and Williams82 were analyzed.

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

[0116] Among them, this example exemplifies the results of positive seedlings Q30 and Q2 in the five-target Glyma.05g029800 enhancer CRISPR / Cas9 knockout mutant strains, see Figures 4 to 7 Compared with the wild-type Williams82, the expression of Glyma.05g029800 in the leaves of the MHSβ knockout mutants Q30 and Q2 was reduced by 26% to 27% (for specific results, see Figure 4 ), the expression of Glyma.05g0298000 in the nodules of the MHSβ knockout mutants Q30 and Q2 was reduced by 81% to 91% (for specific results, see Figure 5 ), and the number of nodules in the MHSβ knockout mutants Q30 and Q2 was significantly higher than that in the wild-type Williams82 (for specific results, see Figure 6 ), and the nodulation of MHSβ knockout mutants Q30 and Q2 was significantly better than that of wild-type Williams82 (for specific results, see Figure 7). Therefore, the soybean MHSβ regulatory element regulates soybean nodulation by precisely regulating the expression of Glyma.05g0298000, affecting nutrient accumulation, and has important application prospects in soybean breeding.

[0117] 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.

[0118] 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 MHSβ regulatory element in regulating the expression of the Glyma.05g029800 gene, the nucleotide sequence of the MHSβ regulatory element is shown in SEQ ID NO:

1.

2. The use according to claim 1, characterized in that The nucleotide sequence of the MHSβ regulatory element is transformed or modified to reduce the expression level of the Glyma.05g029800 gene; Optionally, the nucleotide sequence of the Glyma.05g029800 gene is as shown in SEQ ID NO:

17.

3. Use of the MHSβ regulatory element in regulating the number of soybean nodules, the nucleotide sequence of the MHSβ regulatory element is shown in SEQ ID NO:

1.

4. The use according to claim 3, characterized in that The nucleotide sequence of the MHSβ regulatory element is transformed or modified to increase the nodulation number of the soybean.

5. A method for regulating the number of soybean nodules, characterized in that: include: The nucleotide sequence of the MHSβ regulatory element in the soybean is transformed or modified, and the nucleotide sequence of the MHSβ 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 MHSβ regulatory element is transformed or modified in the following manner: Deleting, replacing, inserting, inverting and ectopic at least a portion of the sequence of the MHSβ regulatory element; or Methylating and / or acetylating at least a portion of the sequence of the MHSβ regulatory element; Optionally, the MHSβ regulatory element sequence 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 5, and optionally at least one of the nucleotide sequences shown in SEQ ID NOs: 2 and 6; 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 5, and optionally comprises at least one of the nucleotide sequences shown in SEQ ID NOs: 2 and 6.

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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