Soybean immunoregulatory gene trehalase GmTRE, its encoded protein and application

Abstract

The application discloses a soybean immune negative regulation gene trehalase GmTRE, a coding protein thereof and application, and belongs to the technical field of crop genetic breeding. The technical problem to be solved by the application is how to improve the resistance of soybean to soybean downy mildew. To solve the above technical problem, the application provides application of a protein or a substance for regulating expression of a gene or a substance for regulating activity or content of the protein in regulation of the resistance of a plant to soybean downy mildew, and the GmTRE protein is a protein with an amino acid sequence shown in sequence 1 in the sequence listing. The application provides use of the GmTRE protein in genetic improvement breeding of soybean disease-resistant germplasm resources, and has a good application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of crop genetic breeding, and particularly relates to a soybean immune negative regulation gene trehalose GmTRE, an encoding protein thereof and application thereof. BACKGROUND

[0002] Soybean is one of the most important food and economic crops in the world, and plays an indispensable role in human food supply and energy security. As a perennial crop, soybean is continuously threatened by various diseases in production, including soybean Phytophthora root rot, soybean cyst nematode disease, virus disease, etc., which is one of the important factors restricting the improvement of soybean yield and quality in China. Among them, the soybean root rot caused by Phytophthora sojae is a devastating disease in soybean production. Soybean root rot is a soil-borne disease that can cause root rot, root stem rot, fruit rot, ulcer, wilting and spot, etc. Its transmission speed is fast, and it can cause plant absolute yield or plant extinction in severe cases. In agricultural production, the control of root rot mainly depends on the use of chemical fungicides containing metalaxyl and the planting of disease-resistant varieties. Compared with the traditional control of pesticides, breeding disease-resistant varieties is a more environmentally friendly, healthy and effective strategy to control plant diseases. However, due to the rapid variation of field Phytophthora sojae physiological races, the complex virulence structure and other reasons, the resistance of soybean varieties is often lost in production. These reasons make it particularly important to excavate new genes involved in the immune response process of soybean. Therefore, it is a challenging and practical hot field to carry out research on the mechanism of soybean resistance to Phytophthora and to develop new soybean resistance germplasm resources.

[0003] Trehalose is a non-reducing disaccharide, which is linked by 1,1 glycosidic bond between two pyranose glucose monomers, and its chemical name is α-D-glucopyranosyl-α-D-glucopyranoside. Trehalose is synthesized from uridine diphosphate glucose (UDP-G) and glucose-6-phosphate (G6P) into trehalose-6-phosphate (T6P) by trehalose-6-phosphate synthase (TPS), and then trehalose-6-phosphate (T6P) is catalyzed by trehalose-6-phosphate phosphatase (TPP) to synthesize trehalose. Trehalose can also be catalyzed by trehalase to generate two molecules of glucose. Trehalose is widely present in bacteria, yeast, animals and plants, but compared with other sugars involved in metabolic reactions in the body such as sucrose, fructose, glucose, etc., its content in the organism is extremely low, especially in higher plants. Trehalose is structurally stable in nature, has strong non-reducing property, can withstand heat or acid stimulation, and cannot be decomposed by general enzymes, but can be hydrolyzed by specific trehalase, which is a relatively stable sugar among natural disaccharides.

[0004] Trehalase is a trehalose hydrolase that can specifically and specifically decompose one molecule of trehalose into two molecules of glucose. In nature, trehalase is widely present in insects, mammals, microorganisms and plants and plays an important role. Trehalose in the body of insects cannot be hydrolyzed by general enzymes, and trehalase is the only enzyme in the body of insects that can decompose trehalose. Trehalase, also known as trehalose hydrolase, decomposes glucose to produce pyruvic acid through glycolysis pathway into tricarboxylic acid cycle, and finally oxidizes to form water and carbon dioxide. This most common degradation process can provide energy for the flight and growth and development of insects. During the special period of development, the trehalose in the body of insects will always remain at a high level, which plays an important role in the growth and development of insects by affecting the synthesis of chitin. Trehalase also exists widely in higher plants. Interestingly, only one trehalase gene is found in the genomes of rice and Arabidopsis. So far, there have been few studies on the function of trehalase. Studies have shown that the trehalase gene (OsTRE1; Os10g0521000) in rice may be involved in salt stress tolerance. In Arabidopsis, trehalose and trehalase may play a role in regulating the distribution of carbohydrates in plants. SUMMARY

[0005] The technical problem solved by the present application is how to improve the resistance of soybean to Phytophthora sojae.

[0006] To solve the above technical problem, in a first aspect, the present application provides the use of a protein or a substance for regulating the expression of a gene encoding the protein or a substance for regulating the activity or content of the protein in any one of A1-A7) below, wherein the protein can be a GmTRE protein;

[0007] A1), use in regulating the disease resistance of a plant;

[0008] A2), use in preparing a product for regulating the disease resistance of a plant;

[0009] A3), use in regulating the resistance of a plant to Phytophthora;

[0010] A4), use in preparing a product for regulating the resistance of a plant to Phytophthora;

[0011] A5), use in regulating the resistance of a plant to Phytophthora sojae;

[0012] A6), use in preparing a product for regulating the resistance of a plant to Phytophthora sojae;

[0013] A7), use in plant breeding or assisted plant breeding;

[0014] The GmTRE protein can be any one of a1)-a3) below:

[0015] a1), a protein with an amino acid sequence as shown in SEQ ID No. 1 in the sequence listing;

[0016] a2), a protein with an amino acid sequence having 80% or more identity to the amino acid sequence shown in a1) and being associated with the disease resistance of a plant, obtained by substitution and / or deletion and / or addition of amino acid residues of the amino acid sequence shown in a1);

[0017] a3), a fusion protein obtained by connecting a tag to the N terminus or / and C terminus of a1) or a2).

[0018] In the above use, the purpose of the plant breeding or assisted plant breeding is to prepare a plant resistant to Phytophthora sojae.

[0019] Further, in the above use, the GmTRE protein is derived from soybean.

[0020] SEQ ID No. 1 consists of 580 amino acid residues.

[0021] The above protein can be artificially synthesized, or a gene encoding the protein can be first synthesized and then expressed biologically to obtain the protein.

[0022] The protein tag refers to a polypeptide or protein fused and expressed with a target protein by DNA in vitro recombination technology, so as to facilitate the expression, detection, tracking and / or purification of the target protein. The protein tag can be a Flag protein tag, a His protein tag, an MBP protein tag, an HA protein tag, a myc protein tag, a GST protein tag and / or a SUMO protein tag, etc.

[0023] Further, in the above application, the substance for regulating the expression of the protein-coding gene or the substance for regulating the activity or content of the protein is a biological material, which can be any of the following:

[0024] B1), a nucleic acid molecule for inhibiting or reducing the expression of the gene in the above application or the activity of the GmTRE protein in the above application;

[0025] B2), an expression cassette containing the nucleic acid molecule of B1);

[0026] B3), a recombinant vector containing the nucleic acid molecule of B1) or a recombinant vector containing the expression cassette of B2);

[0027] B4), a recombinant microorganism containing the nucleic acid molecule of B1) or a recombinant microorganism containing the expression cassette of B2) or a recombinant microorganism containing the recombinant vector of B3);

[0028] B5), a transgenic plant cell line containing the nucleic acid molecule of B1) or a transgenic plant cell line containing the expression cassette of B2) or a transgenic plant cell line containing the recombinant vector of B3);

[0029] B6), a transgenic plant tissue containing the nucleic acid molecule of B1) or a transgenic plant tissue containing the expression cassette of B2) or a transgenic plant tissue containing the recombinant vector of B3);

[0030] B7), a transgenic plant organ containing the nucleic acid molecule of B1) or a transgenic plant organ containing the expression cassette of B2) or a transgenic plant organ containing the recombinant vector of B3);

[0031] B8), a nucleic acid molecule encoding the GmTRE protein in the above application;

[0032] B9), an expression cassette, a recombinant vector, a recombinant microorganism or a transgenic plant cell line containing the nucleic acid molecule of B8).

[0033] Further, in the above application, the nucleic acid molecule of B1) can be a DNA molecule expressing a gRNA targeting the gene in the above application or a gRNA targeting the GmTRE protein-coding gene in the above application.

[0034] Further, in the above-mentioned use, the nucleic acid molecule of B8) can be a DNA molecule according to any one of g1) to g3):

[0035] g1) a DNA molecule whose coding sequence of the coding strand is SEQ ID No. 2 in the sequence listing;

[0036] g2) a DNA molecule whose nucleotide sequence of the coding strand is SEQ ID No. 3 in the sequence listing;

[0037] g3) a DNA molecule having 80% or more identity to the DNA molecule according to g1) or g2), and which regulates plant disease resistance.

[0038] Further, in the above-mentioned use, the target sequence of the gRNA of B1) is selected from any one of T1) to T4):

[0039] T1) a DNA molecule whose nucleotide sequence is 5'-CAACCCCTCTTCTCTCCTT-3' (positions 341-359 of SEQ ID No. 3, located in the 1st exon, i.e., positions 104-122 of SEQ ID No. 2);

[0040] T2) a DNA molecule whose nucleotide sequence is 5'-TCGAAACCTTCGCCCATTC-3' (positions 386-404 of SEQ ID No. 3, located in the 1st exon, i.e., positions 149-167 of SEQ ID No. 2);

[0041] T3) a DNA molecule whose nucleotide sequence is 5'-GACTTACATACGCTGCTCC-3' (positions 718-736 of SEQ ID No. 3, located in the 1st exon, i.e., positions 481-499 of SEQ ID No. 2);

[0042] T4) a DNA molecule whose nucleotide sequence is 5'-TGGTTCGGTTGTCATTCCC-3' (positions 744-762 of SEQ ID No. 3, located in the 1st exon, i.e., positions 507-525 of SEQ ID No. 2).

[0043] Further, in the above-mentioned use, the plant is selected from any one of the following:

[0044] C1) a dicotyledonous plant;

[0045] C2) a plant of the family Fabaceae;

[0046] C3) a plant of the genus Glycine;

[0047] C4) Glycine max.

[0048] In the above-mentioned biological material, the expression cassette of B9) refers to a DNA capable of expressing the GmTRE protein in a host cell, which can include not only a promoter for initiating transcription of the GmTRE gene but also a terminator for terminating transcription of the GmTRE gene.

[0049] In the above-mentioned biological material, the recombinant microorganism of B4) can be a yeast, a bacterium, an alga, and a fungus.

[0050] In the above-mentioned biological material, the plant tissue of B6) can be derived from a root, a stem, a leaf, a flower, a fruit, a seed, pollen, an embryo, and an anther.

[0051] In the above-mentioned biological material, the transgenic plant organ of B7) can be a root, a stem, a leaf, a flower, a fruit, and a seed of a transgenic plant.

[0052] In the above-mentioned biological material, the transgenic plant cell line, the transgenic plant tissue, and the transgenic plant organ can or can not include propagation material.

[0053] To solve the above-mentioned technical problem, in a second aspect, the present application provides a method for regulating the resistance of soybean to P. sojae, which comprises M1) or M2):

[0054] M1), regulating the resistance of soybean to P. sojae by regulating the expression of the GmTRE protein in a recipient soybean or regulating the activity or content of the GmTRE protein;

[0055] M2), improving the resistance of a recipient soybean to P. sojae by applying validamycin to the recipient soybean.

[0056] Further, in the above-mentioned method, the method of M1) comprises M1-1) or M1-2),

[0057] M1-1), introducing the gene of the gRNA and the coding gene of the Cas protein in the above-mentioned application into a recipient soybean to inhibit or reduce the expression of the GmTRE protein coding gene in the recipient soybean or to inhibit or reduce the activity or content of the GmTRE protein in the recipient soybean, thereby obtaining a soybean with a higher resistance to P. sojae than the recipient soybean;

[0058] M1-2), introducing the nucleic acid molecule of B8) in the above-mentioned application into a recipient soybean to promote or improve the expression of the GmTRE protein coding gene in the recipient soybean or to promote or improve the activity or content of the GmTRE protein in the recipient soybean, thereby obtaining a soybean with a lower resistance to P. sojae than the recipient soybean.

[0059] To solve the above technical problems, in a third aspect, the present application provides a method for preparing a soybean with altered Phytophthora sojae resistance, the method comprising M1-1) or M1-2):

[0060] M1-1), introducing into a recipient soybean the gene encoding the GmTRE protein and the gene encoding the Cas protein to inhibit or reduce the expression of the GmTRE protein-encoding gene in the recipient soybean or to inhibit or reduce the activity or content of the GmTRE protein in the recipient soybean, to obtain a soybean with a higher resistance to Phytophthora sojae than the recipient soybean;

[0061] M1-2), introducing into a recipient soybean the nucleic acid molecule described in B8) of the above application to promote or increase the expression of the GmTRE protein-encoding gene in the recipient soybean or to promote or increase the activity or content of the GmTRE protein in the recipient soybean, to obtain a soybean with a lower resistance to Phytophthora sojae than the recipient soybean.

[0062] Further, in the above method, the recipient soybean can be a cell, tissue, organ or plant of soybean.

[0063] In an embodiment of the present application, the recipient soybean is a differentiated hairy root (root hair) of soybean.

[0064] To solve the above technical problems, in a fourth aspect, the present application provides the protein described in the above application and / or the biological material described in the above application.

[0065] In the present application, the Cas protein can be a Cas9 protein.

[0066] In the present application, the pathogenic bacteria of Phytophthora sojae can be Phytophthora sojae.

[0067] In an embodiment of the present application, the pathogenic bacteria of Phytophthora sojae can be Phytophthora sojae strain RFP-P6497.

[0068] In the present application, the identity refers to the identity of the amino acid sequence or the nucleotide sequence. The identity of the amino acid sequence can be determined using the homology search site on the Internet, such as the BLAST page of the NCBI homepage. For example, the identity (%) can be obtained by calculating the identity of the amino acid sequence in the advanced BLAST 2.1 using blastp as the program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as the Matrix, setting Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values), respectively, and then obtaining the value of the identity (%).

[0069] In the present application, the identity of 80% or more can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0070] In the present application, the regulation can be up-regulation or enhancement or increase, or can be down-regulation or inhibition or decrease.

[0071] In the present application, the substance that regulates the activity or the content of the protein can be a substance that knocks out the coding gene of the protein and / or a substance that regulates the expression of the coding gene of the protein.

[0072] In the present application, the substance that regulates the expression of the gene can be a substance that performs at least one of the following six regulations: 1) regulation at the transcription level of the gene; 2) regulation after the transcription of the gene (that is, regulation of the splicing or processing of the primary transcript of the gene); 3) regulation of the RNA transport of the gene (that is, regulation of the transport of the mRNA of the gene from the nucleus to the cytoplasm); 4) regulation of the translation of the gene; 5) regulation of the degradation of the mRNA of the gene; and 6) post-translational regulation of the gene (that is, regulation of the activity of the protein translated from the gene).

[0073] In the present application, the regulation of the expression of the gene can be inhibition or decrease of the expression of the gene, which can be achieved by gene knockout or by gene silencing.

[0074] The gene knockout refers to the phenomenon that a specific target gene is inactivated by homologous recombination. The gene knockout is the inactivation of a specific target gene by a change in the DNA sequence.

[0075] The gene silencing refers to a phenomenon that a gene is not expressed or lowly expressed without damaging original DNA. The gene silencing is premised on not changing the DNA sequence, so that the gene is not expressed or lowly expressed. The gene silencing can occur at two levels. One is the transcription level gene silencing caused by DNA methylation, heterochromatinization and position effect, and the other is the post-transcription gene silencing, that is, the gene is inactivated by specifically inhibiting the target RNA at the level after the gene transcription, including antisense RNA, co-suppression, gene quelling, RNA interference (RNAi) and micro RNA (miRNA) mediated translation inhibition.

[0076] In the present application, the substance for regulating gene expression can be an agent for inhibiting or reducing the expression of the gene. The agent for inhibiting or reducing the expression of the gene can be an agent for knocking out the gene, such as an agent for knocking out the gene by homologous recombination, or an agent for knocking out the gene by CRISPR-Cas9. The agent for inhibiting or reducing the expression of the gene can comprise a polynucleotide targeting the gene, such as siRNA, shRNA, sgRNA, miRNA or antisense RNA.

[0077] The present application has the following beneficial technical effects:

[0078] The soybean trehalase gene GmTRE can negatively regulate the immune response of plants by increasing the content of trehalose in plants. It is also found that the infection of soybean Phytophthora can be inhibited by applying Validamycin A, a TRE inhibitor, to soybean. The present application provides the use of GmTRE gene in the genetic improvement and breeding of soybean disease-resistant germplasm resources, which has good application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0079] Figure 1 Phenotype analysis of hairy roots of soybean after transient overexpression or knockout of GmTRE gene and inoculation of Phytophthora sojae. Among them Figure 1 A is the phenotype of the hairy roots overexpressing GmTRE after inoculation of Phytophthora sojae; Figure 1 B is the expression amount of GmTRE in the control and the hairy roots overexpressing GmTRE; Figure 1 C is the spore number and biomass analysis of the hairy roots overexpressing GmTRE after inoculation of Phytophthora sojae; Figure 1 D is the phenotype of the hairy roots with knockout of GmTRE after inoculation of Phytophthora sojae, and the figure shows four sgRNA sites; Figure 1 E is the spore number and biomass analysis of the hairy roots with knockout of GmTRE after inoculation of Phytophthora sojae; Figure 1 F is the Sanger sequencing analysis of the GmTRE gene editing type in the hairy roots.

[0080] Figure 2 TRE inhibitor validamycin A can inhibit the infection of soybean Phytophthora. Among them Figure 2 Table 1: Phenotype of soybean treated with water and validamycin A before inoculation of soybean Phytophthora Figure 2 Table 2: Phenotype of soybean leaf treated with water and validamycin A before inoculation of soybean Phytophthora Figure 2 Table 3: Disease length and biomass of soybean leaf treated with water and validamycin A before inoculation of soybean Phytophthora DETAILED DESCRIPTION

[0081] The application will be further described in detail below in conjunction with the specific embodiments. The examples given are only to illustrate the application, and are not intended to limit the scope of the application. The examples provided below can serve as a guide for further improvement by those of ordinary skill in the art, and do not in any way constitute a limitation on the application.

[0082] In the following examples, the experimental methods are conventional methods, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained commercially, unless otherwise specified.

[0083] In the following examples, the soybean is Huachun 6, which is a gift from the research group of Professor Nianhai He, South China Agricultural University, and is disclosed in the literature “Zhandong Cai. etc. CRISPR / Cas9-mediated gene editing of GmJAGGED1 increased yield in the low-latitude soybean variety Huachun 6. Plant Biotechnology Journal (2021) 19, pp. 1898-1900”. The biological material can be obtained from Shanghai Normal University by the public, and the obtained biological material can only be used for verification of the technical solutions in the present application, and cannot be used for other purposes.

[0084] In the following examples, the Phytophthora sojae is Phytophthora sojae strain RFP-P6497, which is conserved in our laboratory and disclosed in the literature “Peng Zhang. etc. The WY domain in the Phytophthora effector PSR1 is required for infection and RNA silencing suppression activity. New Phytologist (2019) 223: 839-852.” The biological material can be obtained from Shanghai Normal University, and the obtained biological material can only be used for verification of the technical solutions in the present application and cannot be used for other purposes.

[0085] In the following examples, the expression vector pFGC5941 is donated by Nanjing Agricultural University Wang Yuanchao laboratory and disclosed in the literature “Kaixuan Duan. etc. Large chromosomal segment deletions by CRISPR / LbCpf1-mediated multiplex gene editing in soybean. Journal of Integrative Plant Biology. September 2021 Volume 63 Issue 9 1620-1631.” The biological material can be obtained from Shanghai Normal University, and the obtained biological material can only be used for verification of the technical solutions in the present application and cannot be used for other purposes.

[0086] In the following examples, the gene editing vector pGES201 is donated by Fujian Agriculture and Forestry Guan Yuefeng laboratory and disclosed in the literature “Mengyan Bai. etc. Generation of a multiplex mutagenesis population via pooled CRISPR-Cas9 in soya bean. Plant Biotechnology Journal (2020) 18, pp. 721-731.” The biological material can be obtained from Shanghai Normal University, and the obtained biological material can only be used for verification of the technical solutions in the present application and cannot be used for other purposes.

[0087] In the following examples, the validamycin A is purchased from Yuanye Bio (item number: S17077).

[0088] The following examples use SPSS 25.0 statistical software for one-way analysis of variance, and Tukey multiple comparison method is used to test the difference between groups. Different letters represent significant differences (P<0.01).

[0089] Example 1, GmTRE and the protein encoded thereby

[0090] The genomic nucleotide sequence of the GmTRE gene is SEQ ID No. 3, wherein the positions 238-812, 902-1021, 1715-1830, 1956-2056, 2145-2254, 2489-2561, 2644-2805, 2897-2984, 3715-3814, 3905-4118, 4405-4488 are exons, the coding sequence of the GmTRE gene is a DNA molecule with the nucleotide sequence of SEQ ID No. 2, and the GmTRE protein (also called protein GmTRE) with the amino acid sequence of SEQ ID No. 1 is encoded.

[0091] Example 2, construction of soybean overexpression vector

[0092] 2.1, design of primers

[0093] The overexpression vector primers of the gene are designed according to the coding sequence of the GmTRE gene: pFGC5941-GmTRE-F: 5'-ATTACCATGGggcgcgccATGGCCGAGTACGCTCAAA-3' (the lowercase letter is the Asc I restriction site);

[0094] pFGC5941-GmTRE-R: 5'-TAGACTCACCTAggatccGCATTCTATGTTCCGATCTT-3' (the lowercase letter is the BamH I restriction site).

[0095] 2.2, amplification of target gene:

[0096] Total RNA was extracted from the leaves of two-week-old soybean seedlings using a kit, and cDNA was obtained by reverse transcription of the obtained total RNA using a reverse transcription kit. The cDNA was used as a template, pFGC5941-GmTRE-F / pFGC5941-GmTRE-R was used as primers, and high-fidelity DNA polymerase Kit of Nanjing Novozyme Company was used for PCR amplification to obtain the target gene fragment.

[0097] The PCR reaction system is as follows:

[0098]

[0099] The PCR reaction program is as follows:

[0100]

[0101] (Note: the extension time varies according to the sequence size, 1 kb / min)

[0102] After the PCR reaction, the agarose gel was loaded and electrophoresed for 18 min.

[0103] 2.3, Purification of target genes

[0104] The agarose gel electrophoresis gel containing the target gene was cut and purified using the EasyPure Quick Gel Extraction Kit from Beijing Transgene Co., Ltd. The steps are as follows:

[0105] 1) Under the irradiation of the ultraviolet light imager, the band containing the target fragment was cut and placed in a 2 mL centrifuge tube;

[0106] 2) Add 3 times the volume of GSB Buffer to the weight of the gel piece, and place it in a constant temperature metal bath or water bath at 55°C until the gel piece is completely melted;

[0107] 3) Let the gel solution temperature drop to room temperature, and after cooling, add the gel solution to the centrifugal column, centrifuge at 12000 rpm at room temperature for 1 min, and discard the effluent;

[0108] 4) Add 650 μL of Wash Buffer, centrifuge at 12000 rpm at room temperature for 1 min, and discard the effluent;

[0109] 5) Put the centrifugal column back into the centrifugal tube, repeat (4), and completely remove the residual WB;

[0110] 6) Move the centrifugal column to a new 1.5 mL centrifugal tube, let it stand for 2 min to volatilize the alcohol in the centrifugal column, and add 35-45 μL of Elution Buffer or ddH2O (EB or deionized water heated at 60-70°C before use can increase the elution rate) to the center of the centrifugal column;

[0111] 7) Centrifuge at 12000 rpm at room temperature for 1 min to obtain the recovered product, and use a Nano drop spectrophotometer to detect the concentration, OD260 / OD280 should be 1.7-1.9, i.e. the purified target gene is obtained.

[0112] 2.4, Vector digestion

[0113] The pFGC5941 vector was digested using FastDigest enzyme from Thermo Fisher Scientific, USA, and the enzyme digestion system was as follows:

[0114]

[0115] Add the sample to the PCR tube according to the above system, and mix the system slowly with a pipette gun. Place it at 37°C for 30-60 min. The enzyme digestion product is separated by 1% agarose gel in 1% TAE electrophoresis buffer at 160V for 18 min. Use EasyPure Quick Gel Extraction Kit kit to recover and purify.

[0116] 2.5, Connection of target gene and vector

[0117] The enzyme-digested vector obtained in 2.4 is connected with the target gene fragment obtained in 2.2 using ClonExpress II One Step Cloning Kit of Nanjing Novozyme Co., Ltd. The connection system is as follows:

[0118] Connection system:

[0119]

[0120] After adding the system, mix it gently with a pipette gun, and place it at 37°C for 30-60 min to obtain the connection product containing the recombinant expression vector pFGC5941-GmTRE.

[0121] 2.6, Transformation of connection product

[0122] 1) Take the prepared DH5a E. coli competent cells from the -80°C ultra-low temperature refrigerator, and thaw on ice for 5 min;

[0123] 2) After the competent cells are completely thawed, add the above connection product, and stand on ice for 20 min;

[0124] 3) After ice standing, heat shock at 42°C for 90 s, and cool on ice for 1 min;

[0125] 4) Add 700μL of sterilized and antibiotic-free LB liquid medium to the tube in the clean bench, mix it after mixing, and then place it in a 37°C constant temperature shaker at a speed of 250 rpm for 90 min;

[0126] 5) After the culture is completed, centrifuge at 12000 rpm at room temperature for 1 min to collect the bacterial body;

[0127] 6) Discard the supernatant, take 100μL of supernatant and precipitate, mix it thoroughly with a pipette gun, and add it to a screening plate containing the corresponding antibiotic, and evenly spread it with a spreader until the bacterial liquid is completely absorbed by the culture medium;

[0128] 7) Inverted culture in culture dish at 37℃ constant temperature incubator for 16-20h, single colony grows to visible to naked eye.

[0129] 2.7, Colony PCR

[0130] 1) Colony PCR amplification using Novizan 2xTaq Master Mix. Single colony was picked up with sterile gun head, streaked on LB plate containing corresponding resistance, and labeled. After streaking, the bacteria were put into prepared PCR reaction system, and the system was as follows:

[0131]

[0132]

[0133] 2) After streaking, the streaked plate was put into 37℃ constant temperature incubator, and the reaction system was subjected to PCR amplification, and the reaction program was the same as that of the target gene amplification.

[0134] 3) After amplification, the correctness of the PCR product was verified by 1% agarose gel. Single colony containing correct band size was picked into LB culture solution with corresponding resistance, and cultured at 37℃ with 250rpm for 12h or so.

[0135] The sequences of primer F and primer R are as follows:

[0136] Primer F: 5'-atttggagaggacacgctc-3';

[0137] Primer R: 5'-atcatgcgatcataggcgtc-3'.

[0138] 2.8, Plasmid extraction

[0139] Plasmid extraction was performed using EasyPure Plasmid MiniPrep Kit II kit of Beijing TRANSGENE company. The steps were as follows:

[0140] 1) 2.0mL centrifuge tube was used to collect the cultured bacteria solution, and centrifuged at 12000rpm for 1min at room temperature, and the supernatant was discarded after collecting twice;

[0141] 2) 250μL Resuspension Buffer (Rnase was added, and it needed to be stored at 4℃) was added, and the bacterial pellet was suspended by oscillation until the sterile pellet was precipitated;

[0142] 3) 250μL Lysis Buffer was added, and gently inverted 4-6 times;

[0143] 4) Add 350 μL Neutralization Buffer, gently invert 6-8 times, stand for 3 min, and let it fully lyse;

[0144] 5) 12000 rpm at room temperature for 10 min;

[0145] 6) Carefully transfer the supernatant to the centrifugal column, centrifuge at 12000 rpm at room temperature for 1 min, and discard the effluent;

[0146] 7) Add 650 μL Wash Buffer to the centrifugal column, centrifuge at 12000 rpm at room temperature for 1 min, and discard the effluent;

[0147] 8) Place the centrifugal column back into the centrifugal tube, centrifuge at 12000 rpm for 1 min, and completely remove the residual WB;

[0148] 9) Move the centrifugal column to a clean 1.5 mL centrifugal tube, open the cover and stand for 2 min, and let the alcohol in the tube evaporate completely;

[0149] 10) Add an appropriate amount (30 μL-50 μL) of Elution Buffer or ddH2O (EB or ddH2O is heated to 60-70 °C before use to increase the elution rate) to the center of the centrifugal column, and centrifuge at 12000 rpm for 1 min;

[0150] 11) The obtained plasmid is detected for concentration by spectrophotometer, 5 μL is taken for sequencing. The remaining plasmid is stored at -20 °C.

[0151] The sequencing results show that the recombinant expression vector pFGC5941-GmTRE is obtained by replacing the fragment between the Asc I and BamH I enzyme recognition sites of the vector pFGC5941 (small fragment between the Asc I and BamH I enzyme recognition sites) with a DNA molecule with the nucleotide sequence of SEQ ID No. 2, while keeping other nucleotide sequences of the vector pFGC5941 unchanged. The recombinant expression vector pFGC5941-GmTRE expresses the protein GmTRE with the amino acid sequence shown in SEQ ID No. 1.

[0152] Example 3, Construction of Soybean Gene Editing Vector

[0153] 3.1, Target selection

[0154] A target list is generated through an online target prediction website (http: / / skl.scau.edu.cn / home / ), and four target sites of the GmTRE gene are selected after analysis:

[0155] Target site of sgRNA1: 5'-CAACCCCTCTTCTCTCCTT-3' (positions 341-359 of SEQ ID No. 3, located in the 1st exon, i.e. positions 104-122 of SEQ ID No. 2);

[0156] Target site of sgRNA2: 5'-TCGAAACCTTCGCCCATTC-3' (positions 386-404 of SEQ ID No. 3, located in the 1st exon, i.e. positions 149-167 of SEQ ID No. 2);

[0157] Target site of sgRNA3: 5'-GACTTACATACGCTGCTCC-3' (positions 718-736 of SEQ ID No. 3, located in the 1st exon, i.e. positions 481-499 of SEQ ID No. 2);

[0158] Target site of sgRNA4: 5'-TGGTTCGGTTGTCATTCCC-3' (positions 744-762 of SEQ ID No. 3, located in the 1st exon, i.e. positions 507-525 of SEQ ID No. 2).

[0159] The primers for synthesizing the oligonucleotides were designed as follows:

[0160] pGES201-GmTRE-sgRNA1-F: 5'-GGATTGAAGGAGAGAAGAGGGGTTG-3';

[0161] pGES201-GmTRE-sgRNA1-R: 5'-AAACCAACCCCTCTTCTCTCCTTCCA-3';

[0162] pGES201-GmTRE-sgRNA2-F: 5'-GGATTGGAATGGGCGAAGGTTTCGA-3';

[0163] pGES201-GmTRE-sgRNA2-R: 5'-AAACTCGAAACCTTCGCCCATTCCA-3';

[0164] pGES201-GmTRE-sgRNA3-F: 5'-GGATTGGGAGCAGCGTATGTAAGTC-3';

[0165] pGES201-GmTRE-sgRNA3-R: 5'-AAACGACTTACATACGCTGCTCCCA-3';

[0166] pGES201-GmTRE-sgRNA4-F: 5'-GGATTGGGGAATGACAACCGAACCA-3';

[0167] pGES201-GmTRE-sgRNA4-R: 5'-AAACTGGTTCGGTTGTCATTCCCCA-3'.

[0168] 3.2, Annealing of oligos to double strands

[0169] The annealing reaction was prepared as follows:

[0170] 5μl of sense oligo (10μM), 5μl of antisense oligo (10μM), 100mM NaCl (final concentration), 50mM Tris-HCl pH 7.4 (final concentration), and water to 50μl.

[0171] The prepared annealing reaction buffer was mixed repeatedly, centrifuged briefly, and placed on the PCR machine, and the following program was run:

[0172] 95°C for 4min,

[0173] RAMP 0.1°C / s, 95°C to 16°C,

[0174] Keep 16°C,

[0175] After annealing, the double-stranded Oligos of the four targets were synthesized, which could be used immediately or stored at -20°C for long-term preservation.

[0176] 3.3, Linearization of pGES201 vector

[0177] Enzyme digestion system: Bsal 2μl, 10×cutsmart buffer 5μl, vector 20μl (≈250ng / μl), add H2O to 50ul, 37°C digestion for 2 hours.

[0178] After digestion, the linearized vector was recovered with agarose gel. The recovered linearized vector was quantified, and the working concentration of the linearized vector was usually 20-100ng / μl.

[0179] 3.4, Ligation

[0180] The four double-stranded Oligos synthesized by annealing in 3.2 were respectively connected with the linearized pGES201 vector obtained in 3.3. The connection system was as follows: 1μl of annealing product, 3μl of linearized vector, 1μl of 10×T4 buffer, 0.5μl of T4 ligase, and ddH2O to 10ul.

[0181] The ligation products containing recombinant vector pGES201-GmTRE-sgRNA1, pGES201-GmTRE-sgRNA2, pGES201-GmTRE-sgRNA3 or pGES201-GmTRE-sgRNA4 were obtained at 25°C for 2 hours or at 16°C overnight, respectively. The recombinant vector pGES201-GmTRE-sgRNA1 can express Cas9 protein and sgRNA1 targeting GmTRE gene; the recombinant vector pGES201-GmTRE-sgRNA2 can express Cas9 protein and sgRNA2 targeting GmTRE gene; the recombinant vector pGES201-GmTRE-sgRNA3 can express Cas9 protein and sgRNA3 targeting GmTRE gene; the recombinant vector pGES201-GmTRE-sgRNA4 can express Cas9 protein and sgRNA4 targeting GmTRE gene.

[0182] 3.5, Transformation

[0183] The ligation products were transformed into E. coli competent cells (DH5a competent), and the pGES201 vector was resistant to kanamycin (Kan) in prokaryotes.

[0184] 1) DH5a competent cells were taken out from -80°C and quickly inserted into ice. After 5 minutes, the bacterial mass was melted, the target DNA (plasmid or ligation product) was added and mixed, and the mixture was placed in ice for 30 minutes;

[0185] 2) Heat shock at 42°C for 60 seconds, quickly put back on ice and stand for 2 minutes, shaking will reduce the transformation efficiency;

[0186] 3) Add 300 μl of sterile medium (LB) without antibiotics to the centrifuge tube, mix well and place in a 37°C, 200 rpm shaker for 45-60 minutes;

[0187] 4) Take 300 μl of bacterial solution and directly spread on LB medium containing Kan antibiotic.

[0188] 5) Place the plate upside down in a 37°C incubator overnight.

[0189] 3.6, Positive clone identification

[0190] Pick a single colony and place it in 800 μL of kanamycin-resistant liquid LB medium at 37°C, 200 rpm shaker for 3-5 hours. Then perform colony PCR to obtain PCR products, and the PCR conditions are the same as in 2.7. The detection primers used are primers F / R, whose nucleotide sequences are as follows:

[0191] Primer F: 5'-TGTTTGTGAGATGAATATTGTTGGC-3';

[0192] Primer R: 5'-TATCATCATCCCCTAACCCATCTT-3'.

[0193] 3.7. Sanger sequencing

[0194] Quality plasmid for sequencing, confirmed by sequence alignment and sequencing peak map that the vector construction is correct.

[0195] Example 4. Soybean hairy root transformation

[0196] (1) Select undamaged soybean seeds and place them in a culture dish. Add 100 mL of pasteurized solution to a small beaker in a fume hood, then add 4 mL of concentrated hydrochloric acid. Place the small beaker in the middle of the large glass cover of the vacuum dryer, place the culture dish with soybeans in it, open the lid of the culture dish, close the lid of the dryer, and sterilize for 16-18 h;

[0197] (2) Open the glass cover, close the lid of the culture dish, and pour the liquid in the small beaker into the waste liquid tank. Place the culture dish in a clean bench, open the lid, and blow for 0.5-1 h using the air in the clean bench;

[0198] (3) Open the GM solid medium in the clean bench, and scatter 8-11 sterilized soybeans on each GM plate. After spotting, seal the culture dish with a breathable adhesive tape;

[0199] (4) Place the GM culture dish in the tissue culture room at 25°C with 16 h light and 8 h darkness for 4-5 d, until the radicles grow to about 2 cm in length;

[0200] (5) Take out Agrobacterium K599 carrying overexpression vector pFGC5941-GmTRE, Agrobacterium K599 carrying empty vector pFGC5941, or Agrobacterium K599 carrying empty vector pGES201, or Agrobacterium K599 carrying pGES201-GmTRE-sgRNA1-4, which are stored in a -80°C ultra-low temperature freezer, activate them, and then transfer them into new 5 mL LB liquid medium containing corresponding resistance, and add 100 μL of 0.5 M MES (2-morpholinoethanesulfonic acid) and 2 μL of 0.1 MAS (acetyl-syringone). Culture at 30°C, 250 rpm for 12-14 h on a shaker to obtain K599 bacterial solution.

[0201] (6) Add 400-500 μL of K599 bacterial solution to a 2 mL sterilized centrifuge tube in a clean bench, centrifuge at 12000 rpm / s for 1 min, add 2 mL of 10 mM MgCl2, shake to resuspend the bacterial cells, and wash twice to adjust the OD value to 0.6 as the bacterial solution for soybean infection.

[0202] (7) Take out the soybean grown on the GM culture dish, cut the cotyledon petiole with a sterilized scalpel, fix the soybean with a sterilized forceps, cut the cotyledon into two parts, and make a wound on the back of the soybean, and try not to make the wound too deep to avoid piercing the cotyledon.

[0203] (8) Put the cut soybean into the MS medium, and put 8-11 cotyledon leaves in each MS medium.

[0204] (9) Add an appropriate amount of washed bacterial liquid to the wound, inoculate 2 dishes with 7 types of Agrobacterium carrying different vectors respectively, close the lid, seal the medium with a breathable adhesive tape, and place it in a tissue culture room at 25°C with 16h light and 8h darkness for 3-4 weeks, until 1cm (or more than 1cm) hairy roots grow from the wound.

[0205] The hairy roots obtained by infecting soybean cotyledon with Agrobacterium carrying overexpression vector pFGC5941-GmTRE are GmTRE overexpression hairy roots (named PFGC-GmTRE or GmTRE). The hairy roots obtained by infecting soybean cotyledon with Agrobacterium carrying empty vector pFGC5941 are control group overexpression hairy roots (named PFGC-EV or EV). The hairy roots obtained by infecting soybean cotyledon with Agrobacterium carrying empty vector pGES201 are gene editing control group hairy roots (named Cas9-EV). The hairy roots obtained by infecting soybean cotyledon with Agrobacterium carrying gene editing vectors pGES201-GmTRE-sgRNA1, pGES201-GmTRE-sgRNA2, pGES201-GmTRE-sgRNA3, and pGES201-GmTRE-sgRNA4 are gene editing group hairy roots (named Cas9-GmTRE, and the four targets are represented by SG1, SG2, SG3, and SG4, respectively).

[0206] The preparation method of Agrobacterium K599 carrying overexpression vector pFGC5941-GmTRE is as follows:

[0207] (1) Take 100 μL K599 competent cell suspension from the -80°C refrigerator, and thaw at 37°C for 2 minutes;

[0208] (2) Add 1 μg of correctly sequenced plasmid to the competent cells, shake gently, and place on ice for 30 minutes;

[0209] (3) Place the ice-bathed sample in liquid nitrogen for quick freezing for 90 seconds;

[0210] (4) Thaw at 37°C for 2 minutes, and quickly place on ice for cooling for 2 minutes;

[0211] (4) In the clean bench, 1 mL of LB liquid medium (without antibiotics) was added to the tube, mixed and incubated at 30°C for 4 hours;

[0212] (5) 100 μL was taken in the clean bench and spread on the screening plate containing the corresponding antibiotic. The Petri dish was sealed with 3M sealing film and placed for 30 minutes, until the bacterial solution was completely absorbed by the culture medium;

[0213] (6) The Petri dish was inverted and incubated at 30°C for 2-3 days;

[0214] (7) After the single colonies grew, colony PCR was performed, and the experimental method was referred to 2.7. The single colony containing the target size band was shaken.

[0215] Agrobacterium K599 carrying empty vector pFGC5941 or Agrobacterium K599 carrying empty vector pGES201 and Agrobacterium K599 carrying pGES201-GmTRE-sgRNA1-4 refer to the preparation method of Agrobacterium K599 carrying overexpression vector pFGC5941-GmTRE, the only difference is that the overexpression vector pFGC5941-GmTRE is replaced by empty vector pFGC5941, empty vector pGES201, gene editing vector pGES201-GmTRE-sgRNA1-4.

[0216] Preparation of required reagents and culture medium:

[0217] GM medium:

[0218]

[0219] MS medium:

[0220]

[0221] Example 5, soybean hairy root inoculation

[0222] The experiment was divided into 4 groups, the first group used hairy roots named OE-GmTRE in Example 3, the second group used hairy roots named OE-EV in Example 3, the third group used hairy roots named Cas9-EV in Example 3, and the fourth group used hairy roots named Cas9-GmTRE (divided into SG1, SG2, SG3 and SG4) in Example 3.

[0223] The specific steps are: place a layer of filter paper in a culture dish and keep it moist with water; take out the soybean hairy roots transformed in Example 4, and arrange the root tips in one direction; place about 30 soybean hairy roots transformed by the same transformation method in each culture dish, and evenly place the RFP-P6497 mycelial block of P. sojae carrying RFP on top; wherein, according to the transformation method, a total of 7 groups of culture dishes are prepared: hairy roots overexpressing GmTRE (named PFGC-GmTRE or GmTRE), hairy roots overexpressing a control group (named PFGC-EV or EV), hairy roots of a gene editing control group (named Cas9-EV), hairy roots of a gene editing group (SG1), hairy roots of a gene editing group (SG2), hairy roots of a gene editing group (SG3), and hairy roots of a gene editing group (SG4), with three replicates for each group of culture dishes. Place the culture dishes at 25°C in the dark for 3 days, and then use them for subsequent experiments: observe the number of spores in the soybean hairy roots under a fluorescence microscope, and perform data statistics. Randomly sample the same mass of soybean hairy roots in each culture dish to extract DNA for biomass and editing type PCR sequencing analysis of P. sojae; randomly sample the hairy roots in each culture dish to extract RNA for determination of the relative expression amount of the GmTRE gene. Each group of experiments is repeated three times, and the average value is taken as the result.

[0224] The preparation method of the RFP-P6497 mycelial block of P. sojae carrying RFP is as follows: cut a small piece of P. sojae RFP-P6497 and transfer it to VA medium containing G418, and after 6 days of culture, cut a 1 cm wide and 2 cm long mycelial block and place it on the above root hairs. The composition of the VA medium containing G418 is as follows: 100 mL of V8 vegetable juice, 1 g of calcium carbonate, stir uniformly, centrifuge at 1500 rpm for 5 min, dilute the supernatant and distilled water at a ratio of 1:9 (i.e. 100 mL of supernatant + 900 mL of distilled water), and add 15 g of agar powder before sterilization; 121°C high temperature sterilization; add 25 μg / mL G418.

[0225] Among them, ChamQTM Universal Master Mix of Nanjing Novozyme Co., Ltd. is used to perform qPCR as follows: GmCYP2 and PsActin are used as biomass amplification internal standard genes for biomass analysis of P. sojae; and GmEF is used as an internal standard for determination of the relative expression amount of the GmTRE gene:

[0226] The following mixed liquid system (10 μL) is configured in a qPCR tube:

[0227]

[0228] The qPCR reaction is performed as follows:

[0229]

[0230] The primer sequences used for fluorescence quantitative PCR detection of biomass and relative expression amount are as follows:

[0231] GmCYP2-F: 5'-CGGGACCAGTGTGCTTCTTCA-3';

[0232] GmCYP2-R: 5'-CCCCTCCACTACAAAGGCTCG-3';

[0233] PsACTIN-F: 5'-ACTGCACCTTCCAGACCATC-3';

[0234] PsACTIN-R: 5'-CCACCACCTTGATCTTCATG-3';

[0235] GmEF1-F: 5'-TGCAAAGGAGGCTGCTAACT-3';

[0236] GmEF1-R: 5'-CAGCATCACCGTTCTTCAAA-3';

[0237] The results are as shown in Table 1. Figure 1 Table 1 A-E, Figure 1 Table 1 A is the phenotype of the hair roots overexpressing GmTRE inoculated with P. sojae compared with the control; Figure 1 Table 1 B is the expression amount of GmTRE in the control and the hair roots overexpressing GmTRE; Figure 1 Table 1 C is the analysis of spore number and biomass of the hair roots overexpressing GmTRE inoculated with P. sojae compared with the control; Figure 1 Table 1 D is the phenotype of the hair roots knocking out GmTRE inoculated with P. sojae compared with the control, and the figure shows four sgRNA sites; Figure 1 Table 1 E is the analysis of spore number and biomass of the hair roots knocking out GmTRE inoculated with P. sojae compared with the control; Figure 1 Table 1 F is the sequencing results of GmTRE edited at different target sites.

[0238] The results show that compared with the control hair roots, the hair roots overexpressing GmTRE are more susceptible to the disease, the relative biomass and spore number of P. sojae are significantly increased, and the relative expression amount of GmTRE gene is also significantly increased. Compared with the control hair roots, the hair roots knocking out GmTRE have enhanced resistance to P. sojae, and the relative biomass and spore number of P. sojae are significantly reduced.

[0239] Example 6, Soybean application of TRE inhibitor Validamycin A can inhibit P. sojae infection

[0240] Soybean seedlings and leaves were treated with 10 μM of validamycin A or H2O (control) and then inoculated with P. sojae strain RFP-P6497. Leaf disease symptoms were photographed three days after inoculation. The results are shown in Figure 1, wherein Figure 2 Figure 1 shows the results of the soybean phenotype three days after inoculation with P. sojae after treatment with water or validamycin A. Figure 2 Figure 1A shows the soybean phenotype three days after inoculation with P. sojae after treatment with water or validamycin A. Figure 2 Figure IB shows the soybean phenotype three days after inoculation with P. sojae after treatment with water or validamycin A. Figure 2 Figure 1C shows the soybean leaf disease length and P. sojae biomass three days after inoculation with P. sojae after treatment with water or validamycin A. Figure 2 The results shown in Figure 1 indicate that validamycin A reduces P. sojae infection by inhibiting the activity of GmTRE in soybean.

[0241] The soybean seedlings were treated with validamycin A by spraying, and the concentration of validamycin A was 10 μM. Each pot was sprayed with 20 mL, and 18 seedlings were planted in each pot. Three pots were set up in each group, and P. sojae strain RFP-P6497 was inoculated 24 hours after spraying validamycin A. The soybean leaves were treated with validamycin A by soaking, and the concentration of validamycin A was 10 μM. The leaves were soaked for 24 hours, and 30 leaves were planted in each pot. Three pots were set up in each group. P. sojae strain RFP-P6497 was inoculated 24 hours after soaking validamycin A. The P. sojae strain RFP-P6497 was inoculated by pouring 2000 spores / mL of the spore suspension into the roots of the soybean seedlings, and 1 piece of a 0.5 cm diameter mycelial block was inoculated on each leaf.

[0242] The application has been described in detail. For those skilled in the art, without departing from the purpose and scope of the application, and without unnecessary experiments, the application can be implemented in a wider range under the same parameters, concentrations and conditions. Although the application gives a special example, it should be understood that the application can be further improved. In general, according to the principle of the application, the present application intends to include any change, use or improvement of the application, including changes made by conventional techniques known in the art, which deviates from the scope disclosed in the present application.

Claims

1. Use of a protein or a substance that regulates expression of a gene encoding the protein or a substance that regulates activity or content of the protein in any one of the following: A1) modulating resistance of soybean to Phytophthora sojae; A2) preparing a product for modulating resistance of soybean to Phytophthora sojae; A3) breeding soybean with altered resistance to Phytophthora sojae; wherein the modulating resistance of soybean to Phytophthora sojae or the altered resistance to Phytophthora sojae is inhibiting or reducing expression of a gene or inhibiting or reducing activity or content of the protein, increasing resistance of soybean to Phytophthora sojae, promoting or increasing expression of a gene or promoting or increasing activity or content of the protein, decreasing resistance of soybean to Phytophthora sojae; and wherein the protein is a GmTRE protein that is a1) or a2): a1) a protein having an amino acid sequence represented by SEQ ID NO: 1 in the sequence listing; a2) a fusion protein obtained by linking a tag to the N-terminus or / and C-terminus of a1).

2. The substance that regulates expression of a gene encoding the protein or the substance that regulates activity or content of the protein is a biological material that is any one of the following: B1) a nucleic acid molecule that inhibits or reduces expression of the gene in the use of claim 1 or activity of the protein in the use of claim 1; B2) an expression cassette containing the nucleic acid molecule of B1); B3) a recombinant vector containing the nucleic acid molecule of B1) or containing the expression cassette of B2); B4) a recombinant microorganism containing the nucleic acid molecule of B1) or containing the expression cassette of B2) or containing the recombinant vector of B3); B5) a transgenic plant cell line containing the nucleic acid molecule of B1) or containing the expression cassette of B2) or containing the recombinant vector of B3); B6) a transgenic plant tissue containing the nucleic acid molecule of B1) or containing the expression cassette of B2) or containing the recombinant vector of B3); B7) a transgenic plant organ containing the nucleic acid molecule of B1) or containing the expression cassette of B2) or containing the recombinant vector of B3); B8) a nucleic acid molecule encoding the protein in the use of claim 1; B9) an expression cassette, a recombinant vector, a recombinant microorganism, or a transgenic plant cell line containing the nucleic acid molecule of B8).

3. The use according to claim 2, wherein: B1) the nucleic acid molecule is a DNA molecule that expresses a gRNA targeting the gene in the use of claim 1 or is a gRNA targeting the gene in the use of claim 1; and B8) the nucleic acid molecule is g1) or g2): g1) a DNA molecule having a coding sequence of SEQ ID NO: 2 in the sequence listing for a coding strand; g2) a DNA molecule having a nucleotide sequence of SEQ ID NO: 3 in the sequence listing for a coding strand.

4. The gRNA of B1) has a target sequence selected from any one of T1) to T4): T1) a sequence represented by SEQ ID NO: 4 in the sequence listing; T2) a sequence represented by SEQ ID NO: 5 in the sequence listing; T3) a sequence represented by SEQ ID NO: 6 in the sequence listing; and T4) a sequence represented by SEQ ID NO: 7 in the sequence listing. ​ ​ ​ ​ 2. Use according to claim 1, characterized in that: ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 4. Use according to claim 3, characterized in that: ​ T1) the nucleotide sequence is a DNA molecule of SEQ ID No. 3 from 341 to 359; T2) the nucleotide sequence is a DNA molecule of SEQ ID No. 3 from 386 to 404; T3) the nucleotide sequence is a DNA molecule of SEQ ID No. 3 from 718 to 736; T4) the nucleotide sequence is a DNA molecule of SEQ ID No. 3 from 744 to 762.

5. A method of modulating resistance of soybean to P. sojae comprising: The method comprises M1) or M2) or M3): M1) increasing the resistance of soybean to P. sojae by inhibiting or reducing the expression of the GmTRE protein or inhibiting or reducing the activity or content of the GmTRE protein in the recipient soybean; M2) increasing the resistance of the recipient soybean to P. sojae by applying validamycin to the recipient soybean; M3) reducing the resistance of soybean to P. sojae by promoting or increasing the expression of the GmTRE protein or promoting or increasing the activity or content of the GmTRE protein in the recipient soybean.

6. The method of claim 5, wherein: M1) the method is to introduce the gene of the gRNA and the coding gene of the Cas protein in the application of claim 3 or 4 into the recipient soybean to inhibit or reduce the expression of the GmTRE protein coding gene or inhibit or reduce the activity or content of the GmTRE protein in the recipient soybean, so as to obtain soybean with the purpose of higher resistance to P. sojae than the recipient soybean; M3) the method is to introduce the nucleic acid molecule of B8) in the application of claim 2 or 3 into the recipient soybean to promote or increase the expression of the GmTRE protein coding gene or promote or increase the activity or content of the GmTRE protein in the recipient soybean, so as to obtain soybean with the purpose of lower resistance to P. sojae than the recipient soybean.

7. A method of making a soybean having altered resistance to Phytophthora sojae comprising: The method comprises M1-1) or M1-2): M1-1) introducing the gene of the gRNA and the coding gene of the Cas protein in the application of claim 3 or 4 into the recipient soybean to inhibit or reduce the expression of the GmTRE protein coding gene or inhibit or reduce the activity or content of the GmTRE protein in the recipient soybean, so as to obtain soybean with the purpose of higher resistance to P. sojae than the recipient soybean; M1-2) introducing the nucleic acid molecule of B8) in the application of claim 2 or 3 into the recipient soybean to promote or increase the expression of the GmTRE protein coding gene or promote or increase the activity or content of the GmTRE protein in the recipient soybean, so as to obtain soybean with the purpose of lower resistance to P. sojae than the recipient soybean.

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