Application of soybean gene Glyma. 13G252100 in regulation and control of disease resistance of soybeans to phytophthora

Abstract

The invention discloses application of a soybean gene Glyma. 13G252100 in regulation and control of the disease resistance of soybeans to phytophthora. The nucleotide sequence of the soybean gene Glyma. 13G252100 is shown as SEQ ID NO: 1. The invention also discloses application of the soybean gene Glyma. 13G252100 in regulation and control of the disease resistance of the soybeans to phytophthora. Wherein the expression quantity of the soybean gene Glyma. 13G252100 is reduced, so that the disease resistance of the soybean to phytophthora is improved; or the expression quantity of the soybean gene Glyma. 13G252100 is increased, so that the disease resistance of the soybeans to phytophthora is reduced. It is found for the first time that the disease resistance of soybeans to phytophthora can be regulated and controlled by regulating and controlling the expression of the Glyma. 13G252100 gene.

Description

Use of soybean gene Glyma.13G252100 in regulating soybean disease resistance to Phytophthora Technical Field

[0001] The present invention belongs to the field of plant genetic engineering technology and specifically relates to the use of the soybean gene Glyma.13G252100 in regulating soybean resistance to Phytophthora. More specifically, the present invention relates to an sgRNA molecule, expression vector, reagent, CRISPR / Cas9 system, kit, and a method for regulating soybean resistance to Phytophthora. Background Art

[0002] Soybean (Glycine max), originating in China, is a major crop worldwide. Widely cultivated, it is a crucial source of protein and vegetable fat in the human diet, a crucial feed protein for the livestock industry, and a raw material for the chemical industry.

[0003] However, soybeans are also facing various diseases, among which soybean phytophthora root rot is an important disease. Soybean phytophthora root rot is an oomycete disease caused by soybean phytophthora sojae (Phytophthora sojae). It is a devastating soil-borne disease that is fast-growing, has a wide range of damage and is difficult to prevent and control. Soybean phytophthora can occur at any stage of soybean growth. The spores of Phytophthora are most likely to germinate in an environment with high soil moisture and high temperature. First, the zoospores of Phytophthora are attracted by soybean root secretions (such as soy isoflavones and genistein), enter the plant cells through the germ tubes, and then spread to various parts of the soybean plant. This causes symptoms such as yellowing of leaves and waterlogging of stems. In severe cases, it may lead to a decrease in soybean yield and quality, or even cause complete failure of production. The main varieties of soybean cultivation are generally sensitive to this disease, so soybean phytophthora root rot has become an important factor limiting the increase in soybean yield.

[0004] Therefore, there is an urgent need to change the expression of disease-susceptibility genes in soybean to improve soybean's resistance to Phytophthora.

[0005] Summary of the Invention

[0006] The present invention aims to solve at least one of the technical problems existing in the prior art to at least a certain extent.

[0007] The inventors discovered that BIR1 in Arabidopsis is a negative regulator of cellular immunity and cell death. The loss of BIR1 can lead to spontaneous cell death, persistent activation of defense responses, and severe dwarfing of plants. Silencing the GmBIR1 (Glyma.18g246400) gene, which is homologous to Arabidopsis, can also lead to severe dwarfing of soybean plants and increased resistance to soybean mosaic disease and soybean spot pathogens, and can also lead to cell death and persistent activation of immune defense. Therefore, although the disease resistance of the Glyma.18g246400 gene-silenced strain in soybeans is enhanced, it will also seriously affect the normal growth of the plants. In order to overcome this problem, the inventors found the homologous gene Glyma.13G252100 of Glyma.18g246400 in soybeans and knocked out Glyma.13G252100 through gene editing technology. The results showed that Glyma.13G252100 only played a negative immune regulatory role when infected by soybean blight fungus, and the mutation of Glyma.13G252100 did not affect soybean yield.

[0008] Based on this, in a first aspect, the present invention proposes the use of the soybean gene Glyma.13G252100 in regulating soybean disease resistance to Phytophthora. The nucleotide sequence of the soybean gene Glyma.13G252100 is shown in SEQ ID NO: 1. The present invention is the first to discover that soybean disease resistance to Phytophthora can be regulated by regulating the expression of the Glyma.13G252100 gene.

[0009] In its second aspect, the present invention provides an sgRNA molecule. According to an embodiment of the present invention, the sgRNA molecule comprises at least one of the nucleotide sequences shown in SEQ ID NO: 2 and SEQ ID NO: 3. The sgRNA molecule of the present invention has a low off-target rate and high editing efficiency, and can partially or completely delete the sequence of the soybean gene Glyma.13G252100, thereby improving soybean resistance to Phytophthora.

[0010] In a third aspect, the present invention provides an expression vector. According to an embodiment of the present invention, the expression vector carries the sgRNA molecule described in the second aspect of the present invention and, optionally, a nucleic acid encoding a Cas9 molecule. The expression vector of the embodiment of the present invention can partially or completely delete the sequence of the soybean gene Glyma.13G252100, thereby partially or completely deleting the activity of the soybean gene Glyma.13G252100, thereby improving soybean resistance to Phytophthora.

[0011] In a fourth aspect, the present invention provides a reagent. According to an embodiment of the present invention, the reagent includes the sgRNA molecule described in the second aspect of the present invention or the expression vector described in the third aspect of the present invention. The reagent of the embodiment of the present invention can partially or completely delete the sequence of the Glyma.13G252100 gene, thereby partially or completely losing the activity of the Glyma.13G252100 gene, thereby improving soybean resistance to Phytophthora.

[0012] In a fifth aspect, the present invention provides a CRISPR / Cas9 system. According to an embodiment of the present invention, the CRISPR / Cas9 system comprises the sgRNA molecule described in the second aspect of the present invention and, optionally, a nucleic acid encoding a Cas9 molecule. The CRISPR / Cas9 system of the embodiment of the present invention can partially or completely delete the sequence of the Glyma.13G252100 gene, thereby partially or completely losing the activity of the Glyma.13G252100 gene, thereby improving soybean resistance to Phytophthora.

[0013] In its sixth aspect, the present invention provides a kit. According to an embodiment of the present invention, the kit comprises the sgRNA molecule described in the second aspect of the present invention or the expression vector described in the third aspect of the present invention; and optionally, a nucleic acid encoding a Cas9 molecule. The kit of the embodiment of the present invention can partially or completely delete the sequence of the Glyma.13G252100 gene, thereby partially or completely losing the activity of the Glyma.13G252100 gene, thereby improving soybean resistance to Phytophthora.

[0014] In a seventh aspect, the present invention provides a method for regulating soybean resistance to Phytophthora. According to an embodiment of the present invention, the method comprises: modifying the soybean gene Glyma.13G252100, the nucleotide sequence of which is shown in SEQ ID NO: 1. The method of the embodiment of the present invention can partially or completely delete the nucleotide sequence of the Glyma.13G252100 gene, partially or completely inactivate the Glyma.13G252100 gene activity, and thereby improve soybean resistance to Phytophthora. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments with reference to the following drawings, in which:

[0016] FIG1 is the qRT-PCR results of the Glyma.13G252100 gene in major tissues in Example 1.

[0017] Figure 2 is a schematic diagram of the CRISPR / Cas9 knockout target site and location of the Glyma.13G252100 gene in Example 1.

[0018] FIG3 shows the target editing status of the knockout mutant A-H1 in Example 2.

[0019] FIG4 is a diagram showing the differences in important agronomic traits between the wild-type receptor P3 and the knockout strain A-H1-T3 in Example 2.

[0020] Figure 5 is a diagram showing the results of identification of resistance to Phytophthora in Example 3 (Note: Figure A is before infection with the wild-type receptor P3; Figure B is after infection with the wild-type receptor P3; Figure C is before infection with A-H1; Figure D is after infection with A-H1). DETAILED DESCRIPTION

[0021] The following embodiments of the present invention are described in detail. The embodiments described below are exemplary and are only used to explain the present invention, and are not to be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, the techniques or conditions described in the literature in this field or the product specifications are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be obtained commercially.

[0022] In the present invention, the terms "contain", "include" or "include" are open expressions, that is, they include the contents specified in the present invention but do not exclude other aspects.

[0023] In the present invention, the terms "optionally", "optional" or "optionally" generally mean that the subsequently described event or circumstance can but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not occur.

[0024] In the present invention, "deleting, replacing, inserting, inverting, or translocating at least a portion of the Glyma.13G252100 gene sequence" refers to altering the nucleotide sequence of the Glyma.13G252100 gene, thereby affecting the transcription and translation processes of the Glyma.13G252100 gene. By altering the nucleotide sequence of the Glyma.13G252100 gene, the expression of the Glyma.13G252100 gene can be regulated. Examples of alterations in the nucleotide sequence of the Glyma.13G252100 gene include, but are not limited to, deletion, replacement, insertion, inversion, and translocation.

[0025] In the present invention, "chemically modifying at least a portion of the Glyma.13G252100 gene sequence" refers to altering the properties, structure, and function of the Glyma.13G252100 gene nucleotide sequence, thereby affecting the transcription and translation processes of the Glyma.13G252100 gene and regulating its expression. Modifications to the properties, structure, and function of the Glyma.13G252100 gene nucleotide sequence include, but are not limited to, methylation, phosphorylation, acetylation, oxidation, transactivation, and labeling.

[0026] In this context, "methylation" refers to the addition of a methyl group (CH3) to the Glyma.13G252100 gene, typically at the cytosine (C) base, forming 5-methylcytosine. Methylation is an important epigenetic modification that plays a key role in regulating gene expression and cell differentiation.

[0027] In the present invention, "phosphorylation" refers to the addition of a phosphate group (PO4 - ), typically occurring on deoxynucleotides in the Glyma.13G252100 gene. Phosphorylation can affect DNA structure and function and participate in processes such as DNA repair, recombination, and signal transduction.

[0028] In the present invention, "acetylation" refers to the addition of an acetyl group (CH3CO) to the Glyma.13G252100 gene, typically occurring on the histone protein of the Glyma.13G252100 gene. Acetylation can affect chromatin structure and gene expression regulation.

[0029] In the present invention, "stable inheritance" means that the mutant after gene editing can stably pass the mutant sequence to the offspring in the form of inheritance.

[0030] In the present invention, "gene editing", also known as genome editing or genome engineering, is an emerging and relatively precise genetic engineering technology that can modify specific target genes in the genome of an organism.

[0031] The present invention proposes the use of the soybean gene Glyma.13G252100 in regulating soybean disease resistance to Phytophthora, an sgRNA molecule, an expression vector, a reagent, a CRISPR / Cas9 system, a kit, and a method for regulating soybean disease resistance to Phytophthora.

[0032] Use of soybean gene Glyma.13G252100 in regulating soybean disease resistance to Phytophthora

[0033] In a first aspect, the present invention provides the use of the soybean gene Glyma.13G252100 in regulating soybean disease resistance to Phytophthora. The nucleotide sequence of the soybean gene Glyma.13G252100 is shown in SEQ ID NO: 1. The present invention is the first to discover that soybean disease resistance to Phytophthora can be regulated by regulating the expression of the Glyma.13G252100 gene.

[0034] According to an embodiment of the present invention, the expression level of the soybean gene Glyma.13G252100 is reduced to improve the disease resistance of the soybean to Phytophthora.

[0035] According to an embodiment of the present invention, the expression level of the soybean gene Glyma.13G252100 is increased to reduce the disease resistance of the soybean to Phytophthora.

[0036] According to an embodiment of the present invention, the reduction in the expression level of the soybean gene Glyma.13G252100 is achieved by:

[0037] At least one of a gene editing system and RNA interference.

[0038] According to an embodiment of the present invention, the gene editing system includes at least one of CRISPR-Cas9 system, CRISPR-Cas12a / Cpf1, Base Editors, TALEN and ZFN.

[0039] According to an embodiment of the present invention, the CRISPR-Cas9 system includes: sgRNA;

[0040] The sgRNA has at least one of the nucleotide sequences shown in SEQ ID NO: 2 and SEQ ID NO: 3.

[0041] According to an embodiment of the present invention, the increasing the expression level of the soybean gene Glyma.13G252100 is achieved by overexpressing the soybean gene Glyma.13G252100.

[0042] sgRNA molecules

[0043] In its second aspect, the present invention provides an sgRNA molecule. According to an embodiment of the present invention, the sgRNA molecule comprises at least one of the nucleotide sequences shown in SEQ ID NO: 2 and SEQ ID NO: 3. The sgRNA molecule of the present invention has a low off-target rate and high editing efficiency, and can partially or completely delete the sequence of the soybean gene Glyma.13G252100, thereby improving soybean resistance to Phytophthora.

[0044] According to an embodiment of the present invention, the sgRNA molecule includes: a nucleotide sequence as shown in SEQ ID NO: 2.

[0045] According to an embodiment of the present invention, the sgRNA molecule includes: a nucleotide sequence as shown in SEQ ID NO: 3.

[0046] According to an embodiment of the present invention, the sgRNA molecule includes: a nucleotide sequence as shown in SEQ ID NO: 2 and SEQ ID NO: 3.

[0047] expression vector

[0048] In a third aspect, the present invention provides an expression vector. According to an embodiment of the present invention, the expression vector carries the sgRNA molecule described in the second aspect of the present invention and, optionally, a nucleic acid encoding a Cas9 molecule. The expression vector of the embodiment of the present invention can partially or completely delete the sequence of the soybean gene Glyma.13G252100, thereby partially or completely deleting the activity of the soybean gene Glyma.13G252100, thereby improving soybean resistance to Phytophthora.

[0049] Reagents

[0050] In a fourth aspect, the present invention provides a reagent. According to an embodiment of the present invention, the reagent includes the sgRNA molecule described in the second aspect of the present invention or the expression vector described in the third aspect of the present invention. The reagent of the embodiment of the present invention can partially or completely delete the sequence of the Glyma.13G252100 gene, thereby partially or completely losing the activity of the Glyma.13G252100 gene, thereby improving soybean resistance to Phytophthora.

[0051] CRISPR / Cas9 system

[0052] In a fifth aspect, the present invention provides a CRISPR / Cas9 system. According to an embodiment of the present invention, the CRISPR / Cas9 system comprises the sgRNA molecule described in the second aspect of the present invention and, optionally, a nucleic acid encoding a Cas9 molecule. The CRISPR / Cas9 system of the embodiment of the present invention can partially or completely delete the sequence of the Glyma.13G252100 gene, thereby partially or completely losing the activity of the Glyma.13G252100 gene, thereby improving soybean resistance to Phytophthora.

[0053] Reagent test kit

[0054] In its sixth aspect, the present invention provides a kit. According to an embodiment of the present invention, the kit comprises the sgRNA molecule described in the second aspect of the present invention or the expression vector described in the third aspect of the present invention; and optionally, a nucleic acid encoding a Cas9 molecule. The kit of the embodiment of the present invention can partially or completely delete the sequence of the Glyma.13G252100 gene, thereby partially or completely losing the activity of the Glyma.13G252100 gene, thereby improving soybean resistance to Phytophthora.

[0055] Method for regulating soybean disease resistance to Phytophthora

[0056] In a seventh aspect, the present invention provides a method for regulating soybean resistance to Phytophthora. According to an embodiment of the present invention, the method comprises: modifying the soybean gene Glyma.13G252100, the nucleotide sequence of which is shown in SEQ ID NO: 1. The method of the embodiment of the present invention can partially or completely delete the nucleotide sequence of the Glyma.13G252100 gene, partially or completely inactivate the Glyma.13G252100 gene activity, and thereby improve soybean resistance to Phytophthora.

[0057] According to an embodiment of the present invention, the transformation process is achieved by:

[0058] At least one of deletion, substitution, insertion, inversion and misplacement of at least a portion of the sequence of the Glyma.13G252100 gene is performed; or

[0059] At least a portion of the sequence of the Glyma.13G252100 gene is chemically modified.

[0060] According to an embodiment of the present invention, the chemical modification includes at least one of methylation, phosphorylation and acetylation.

[0061] According to an embodiment of the present invention, the transformation treatment is to reduce the expression level of the soybean gene Glyma.13G252100, and the regulation is to improve the disease resistance of the soybean to Phytophthora.

[0062] According to an embodiment of the present invention, the modification treatment is to increase the expression level of the soybean gene Glyma.13G252100, and the regulation is to reduce the soybean's resistance to Phytophthora. According to an embodiment of the present invention, the method is used to construct a soybean Phytophthora disease model for screening drugs to treat soybean Phytophthora.

[0063] According to an embodiment of the present invention, the reduction of the expression level of the soybean gene Glyma.13G252100 is achieved by at least one of a gene editing system and RNA interference.

[0064] According to an embodiment of the present invention, the gene editing system includes at least one of CRISPR-Cas9 system, CRISPR-Cas12a / Cpf1, Base Editors, TALEN and ZFN.

[0065] According to an embodiment of the present invention, the gene editing system includes the sgRNA described in the second aspect of the present invention, the expression vector described in the third aspect of the present invention, or the reagent described in the fourth aspect of the present invention.

[0066] According to an embodiment of the present invention, the gene editing system is selected from the CRISPR / Cas9 system described in the fifth aspect of the present invention.

[0067] According to an embodiment of the present invention, the increasing the expression level of the soybean gene Glyma.13G252100 is achieved by overexpressing the soybean gene Glyma.13G252100.

[0068] According to an embodiment of the present invention, the overexpression of the soybean gene Glyma.13G252100 is achieved by introducing an expression vector containing the overexpression soybean gene Glyma.13G252100 into the soybean genome.

[0069] The sequence table of the present invention is as follows:

[0070] The scheme of the present disclosure will be explained below in conjunction with the examples. Those skilled in the art will understand 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 specified in the examples, they are carried out according to the techniques or conditions described in the literature in this area or according to the product instructions. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

[0071] Example 1

[0072] (1) Screening for homologous genes of GmBIR1 (Glyma.18g246400) in soybean

[0073] In this example, the GmBIR1 (Glyma.18g246400) gene sequence was searched through the Phytozome v13 website to screen gene sequences with more than 60% homology to GmBIR1 (Glyma.18g246400) as candidate sequences. The candidate homologous sequences were then cross-aligned with the Arabidopsis thaliana AtBIR1 genome sequence, and finally the homologous gene Glyma.13G252100 to soybean GmBIR1 (Glyma.18g246400) was determined as shown in Table 1.

[0074] Table 1: Homology alignment of GmBIR1 (Glyma.18g246400)

[0075] (2) Analysis of Glyma.13G252100 gene expression pattern

[0076] Using soybean transcriptome data from the Phytozome v13 website, we analyzed the expression levels of the Glyma.13G252100 gene in soybean roots, stems, leaves, and flowers. This analysis revealed that the Glyma.13G252100 gene is specifically expressed in soybean roots, but at very low levels. Further analysis of Glyma.13G252100 gene expression using RT-PCR, as shown in Figure 1, is largely similar to the data from the Phytozome website, further confirming that under normal growth conditions, Glyma.13G252100 is specifically expressed in soybean roots, but at very low levels.

[0077] Example 2

[0078] In this example, the benchmarking.com website was used to design gRNA target sites T1-1 (the primer for the T1-1 target site was Glyma.13G252100-sg1, and the sequence was shown in SEQ ID NO: 2) and T1-2 (the primer for the T1-2 target site was Glyma.13G252100-sg2, and the sequence was shown in SEQ ID NO: 3) on the exon of the Glyma.13G252100 gene, and the Glyma.13G252100-KO single target knockout vector Glyma.13G252100-KO-T1-1, Glyma.13G252100-KO-T1-2, and the double target knockout vector Glyma.13G252100-KO-T1-1-T1-2 were constructed, respectively.

[0079] 2.1 Construction process of single-target knockout vectors Glyma.13G252100-KO-T1-1 and Glyma.13G252100-KO-T1-2

[0080] tRNA-sgRNA Scaffold-tRNA structures were obtained by PCR amplification using pGES401-single target (Glyma.13G252100-sg1, Glyma.13G252100-sg2 empty) as templates, and the PCR products of the above DNA fragments were gel-recovered. The recovered PCR products were added to the ligation reaction system together with the vector pGES401, and the obtained ligation products were transformed into colorectal competent cells DH5α. After colony PCR identification of positive clones, the positive clones were sent to a sequencing company for sequencing, the correctly sequenced Escherichia coli plasmid was extracted and transformed into Agrobacterium K599. The colony identification was correct and the sequencing results were correct, indicating that the single-target Glyma.13G252100 CRISPR / Cas9 knockout vector single target Glyma.13G252100-KO-T1-1 and Glyma.13G252100-KO-T1-2 have been successfully constructed. The knockout vector contains the Bar selection marker gene (glufosinate resistance).

[0081] 2.2 Construction process of the dual-target knockout vector Glyma.13G252100-KO-T1-1-T1-2

[0082] Using pGES401-dual target (with Glyma.13G252100-sg1 and Glyma.13G252100-sg2) empty vector as template, amplify an additional tRNA-sgRNA-sgRNA Scaffold-tRNA structure by PCR, and construct a DNA fragment with two sgRNAs in series (i.e., dual target Glyma.13G252100-KO-T1-1-T1-2) using the goldgate method. The specific steps are as follows:

[0083] (1) PGES401-Glyma.13G252100-sg1 and PGES401-Glyma.13G252100-sg2 were obtained by ligation reaction. The specific ligation system was: PGES401 plasmid 5μL, T4 DNA Ligase 1μL, T4 DNA Ligase Buffer 2μL, BsaI 2μL, Glyma.13G252100-sg1 1μL, Glyma.13G252100-sg2 1μL, ddH2O 5μL;

[0084] (2) A tRNA-sgRNA-sgRNA Scaffold-tRNA DNA fragment was obtained by PCR amplification. The specific PCR reaction conditions were as follows: (37°C 5 min, 16°C 5 min) × 25 cycles, 37°C 15 min, 85°C 5 min; the specific amplification primers were: Glyma.13G252100-sg1-F (protective base + BsaI recognition site + Glyma.13G252100-sg1 + sgRNA Scaffold, the specific nucleotide sequence is shown in SEQ ID NO: 4), Glyma.13G252100-sg1-R (protective base + BsaI recognition site + BsaI cleavage site + Glyma.13G252100-sg2 + sgRNA Scaffold, the specific nucleotide sequence is shown in SEQ ID NO: 5).

[0085] (3) The tRNA-sgRNA-sgRNA Scaffold-tRNA DNA fragment obtained above was PCR amplified using the high-fidelity enzyme KOD One™ PCR master mix purchased from TOYOBO to obtain a PCR product of the two sgRNA-linked DNA fragments. The specific PCR reaction conditions were: 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, which was then gel-recovered.

[0086] (4) The recovered DNA fragments and vector pGES401 were added to the ligation reaction system at the same time, and the resulting ligation products were transformed into E. coli competent cells DH5α. Positive clones were identified by colony PCR and sent to a sequencing company for sequencing. The sequencing primer was STU-TEST-4R (nucleotide sequence as shown in SEQ ID NO: 6). The sequencing results showed that the vector pGES401 contained sequences of tRNA, Glyma.13G252100-sg1, sgRNA Scaffold, tRNA, Glyma.13G252100-sg2, 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 dual-target knockout vector Glyma.13G252100-KO-T1-1-T1-2 had been successfully constructed. The knockout vector contained the Bar selection marker gene (glufosinate resistance).

[0087] The constructed Glyma.13G252100-KO-T1-1, Glyma.13G252100-KO-T1-2, and Glyma.13G252100-KO-T1-1-T1-2 were transformed into Agrobacterium tumefaciens EHA101 and then transformed into recipient soybean P3 using the cotyledonary node method. Genetic large-fragment knockout mutants A-H1 (corresponding to the mutant constructed with the dual-target knockout vector Glyma.13G252100-KO-T1-1-T1-2), A-H2 (corresponding to the mutant constructed with the single-target knockout vector Glyma.13G252100-KO-T1-1), and A-H3 (corresponding to the mutant constructed with the single-target knockout vector Glyma.13G252100-KO-T1-2) were obtained. In this example, the results of mutant A-H1 (a mutant obtained by constructing the dual-target knockout vector Glyma.13G252100-KO-T1-1-T1-2) are exemplified. As shown in Figure 3, the T1-1 and T1-2 targets in mutant A-H1 are simultaneously broken and 189bp of the Glyma.13G252100 gene is knocked out. Subsequently, the agronomic traits such as plant height, number of nodes, and number of tillers of the wild-type receptor P3 and T3 generation homozygous A-H1 (also known as A-H1-T3) were investigated. As shown in Figure 4, the knockout strain A-H1-T3 had a slightly lower plant height and number of nodes than the wild-type receptor P3, but did not reach a significant level; the number of tillers and the number of pods per plant were not significantly different from those of the wild-type receptor P3. These results indicate that there is no significant difference between the Glyma.13G252100 mutant and the wild-type receptor P3 in important agronomic traits.

[0088] Example 3

[0089] In this example, the phytophthora-resistant varieties Heihe 32 and Hefeng 50 in Heilongjiang Province, the susceptible variety Heihe 38 were selected as controls, as well as the wild-type receptor P3 and the knockout mutant A-H1-T3 to be tested. 5 seeds were evenly sown in each pot, and repeated 4 times. After culturing under the same conditions for 7 days, the hypocotyl wound inoculation method was used for inoculation and then placed in a constant temperature and humidity incubator at a temperature of 35°C and a humidity of 80%. The disease incidence was counted after 7 days. The results showed that the mortality rate of the susceptible variety Heihe 38 identified in this identification was greater than 70%, so the identification result was valid. According to the identification results shown in Table 2 and Figure 5, the mortality rate of the wild-type receptor P3 strain was similar to that of Heihe 38, and it was identified as a susceptible variety; A-H1-T3 was identified as a resistant variety, and its disease resistance to phytophthora was significantly improved compared with the wild-type receptor P3, indicating that the Glyma.13G252100 gene negatively regulates the disease resistance response of soybean.

[0090] Table 2: Identification results of soybean Phytophthora root rot

[0091] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean 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 can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0092] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. Use of soybean gene Glyma.13G252100 in regulating the disease resistance of soybean to Phytophthora sojae, wherein the nucleotide sequence of the soybean gene Glyma.13G252100 is as shown in SEQ ID NO:

1.

2. The use according to claim 1, characterized in that Reducing the expression level of the soybean gene Glyma.13G252100 for enhancing the disease resistance of the soybean to Phytophthora sojae; or increasing the expression level of the soybean gene Glyma.13G252100 for reducing the disease resistance of the soybean to Phytophthora sojae.

3. The use according to claim 2, characterized in that, The reduction of the expression level of the soybean gene Glyma.13G252100 is achieved by the following means: At least one of gene editing systems and RNA interference.

4. The use according to claim 3, characterized in that, The gene editing system includes at least one of CRISPR-Cas9 system, CRISPR-Cas12a / Cpf1, Base Editors, TALEN and ZFN.

5. The use according to claim 4, wherein The CRISPR-Cas9 system includes: sgRNA; The sgRNA has at least one of the nucleotide sequences as shown in SEQ ID NO:2 and SEQ ID NO:

3.

6. The use according to claim 2, wherein The increase of the expression level of the soybean gene Glyma.13G252100 is achieved by overexpressing the soybean gene Glyma.13G252100.

7. A sgRNA molecule, characterized in that, Including: Having at least one of the nucleotide sequences as shown in SEQ ID NO:2 and SEQ ID NO:

3.

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

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

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

11. A kit, characterized in that, Including: The sgRNA molecule recited in claim 7 or the expression vector recited in claim 8; And Optionally a nucleic acid encoding a Cas9 molecule.

12. A method for regulating the disease resistance of soybeans to Phytophthora sojae, characterized in that, Including: Modifying the soybean gene Glyma.13G252100, wherein the nucleotide sequence of the soybean gene Glyma.13G252100 is as shown in SEQ ID NO:

1.

13. The method according to claim 12, wherein The modification is achieved by the following means: Performing at least one of deletion, substitution, insertion, inversion and translocation on at least a part of the sequence of the Glyma.13G252100 gene; or Or Performing chemical modification on at least a part of the sequence of the Glyma.13G252100 gene; Optionally, the chemical modification includes at least one of methylation, phosphorylation and acetylation.

14. The method according to claim 12, wherein The modification is to reduce the expression level of the soybean gene Glyma.13G252100, and the regulation is to enhance the disease resistance of the soybean to Phytophthora sojae; or The modification is to increase the expression level of the soybean gene Glyma.13G252100, and the regulation is to reduce the disease resistance of the soybean to Phytophthora sojae.

15. The method according to claim 14, wherein The reduction of the expression level of the soybean gene Glyma.13G252100 is achieved by at least one of gene editing systems and RNA interference.

16. The method according to claim 15, characterized in that The gene editing system includes at least one of CRISPR-Cas9 system, CRISPR-Cas12a / Cpf1, Base Editors, TALEN, and ZFN.

17. The method according to claim 16, wherein The gene editing system includes the sgRNA according to claim 7, the expression vector according to claim 8, or the reagent according to claim 9.

18. The method according to claim 16, characterized in that, The gene editing system is selected from the CRISPR / Cas9 system according to claim 10.

19. The method according to claim 14, characterized in that The increase in the expression level of the soybean gene Glyma.13G252100 is achieved by overexpressing the soybean gene Glyma.13G252100.

20. The method according to claim 19, wherein The overexpression of the soybean gene Glyma.13G252100 is achieved by introducing the expression vector containing the overexpressed soybean gene Glyma.13G252100 into the soybean genome.