Soybean GmMPK4, GmICE2 and GmPR1 genes, recombinant expression vectors and transformants thereof in the prevention and treatment of soybean phytophthora root rot

Abstract

The application provides soybean GmMPK4, GmICE2 and GmPR1 genes, a recombinant expression vector and a transformant thereof in the application to the prevention and treatment of soybean phytophthora root rot, and belongs to the technical field of plant genetic engineering. The soybean GmMPK4 gene can enhance the sensitivity of soybean to soybean phytophthora root rot, mainly through phosphorylation of GmICE2 by GmMPK4, promotion of protein degradation of GmICE2 and inhibition of transcription regulation of GmPR1, so as to enhance the sensitivity of soybean to soybean phytophthora root rot. Overexpression of the soybean GmICE2 can enhance the resistance of soybean to soybean phytophthora root rot, and GmICE2 can enhance the defense ability of soybean to soybean phytophthora root rot by promoting the expression of the disease-resistant gene GmPR1. The application provides valuable gene resources for promoting the research and application of a plant defense system and the cultivation of new soybean varieties with high disease resistance, and has important application value in the genetic engineering breeding of soybean disease resistance.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant genetic engineering, and particularly relates to a soybean GmMPK4, GmICE2 and GmPR1 gene, a recombinant expression vector and a transformant thereof in the application of preventing and treating soybean Phytophthora root rot. BACKGROUND

[0002] Soybean Phytophthora root rot is caused by Phytophthora sojae Kaufmann & Gerdemann, which leads to root and stem rot of soybean, and is one of the most devastating diseases in the world, which has caused a serious threat to soybean production. The variation of Phytophthora sojae is high, and with the increase of the number of new populations and the differentiation of virulence, the resistant varieties are easy to lose resistance. Therefore, disease-resistant genetic breeding is one of the good ways to solve this problem.

[0003] Mitogen-activated protein kinase (MAPK) transmits the external stimulus perceived by the receptor to the cell through the MAPKKK-MAPKK-MAPK cascade phosphorylation, so that the cell produces specific physiological responses to the exogenous signal to adapt to the biological and non-biological stress brought by the outside world. MAPK kinases can respond to various non-biological stresses, thereby being activated, including ozone, drought, high temperature, heavy metals, oxidative stress, salt, cold, contact and water stress, ultraviolet light and nutrient absorption. In addition, in terms of biological stress, MAPKs resist the invasion of pathogenic bacteria by regulating multiple defense responses, including defense gene activation, plant innate immunity, stomatal closure, active oxygen production, hypersensitive response and overall disease resistance. MAPK cascade plays an important role in plant immunity, and understanding its cascade network system is of great significance for us to further understand plants.

[0004] MAPK cascade phosphorylation is a key component of plant immune response. In Arabidopsis, rice, wheat, tomato, tobacco and other plants, the MAPK cascade signaling pathway has been identified as an important phosphorylation signaling pathway. MAPK genes have been identified in a variety of plants, with 20 MAPK genes in Arabidopsis genome, 17 in rice, 21 in poplar, 15 in wheat, 16 in tomato, 21 in maize and 17 in tobacco. The MAPK cascade pathway phosphorylates substrates and induces the expression of resistance genes. Many transcription factors have been identified as substrates of the MAPK cascade in the process of biological and non-biological stress response, including bHLH transcription factors. GmICE2 belongs to bHLH transcription factors, which play an important role in plant response to cold stress and stress resistance.

[0005] PR (Pathogenesis-related genes) genes are one of the most abundant genes produced during plant defense responses and play a key role in the defense response of plants to pathogenic fungi. Overexpression of PR1, PR2 or PR3 in various plants increases resistance to a variety of pathogenic fungi. PR genes as downstream target genes of transcription factors can enhance the resistance of soybean to P. sojae in the defense response of P. sojae.

[0006] The regulation of plant defense responses occurs at various levels from the accumulation and regulation of transcription to the translation and modification of proteins, involving the participation and coordination of various regulatory mechanisms such as immune response proteins, transcriptional regulatory factors and defense-related genes. So far, the molecular defense mechanism of soybean to P. sojae is not clear. SUMMARY

[0007] In view of this, the purpose of the present application is to provide a soybean GmMPK4, GmICE2 and GmPR1 gene, a recombinant expression vector and a transformant thereof for use in the prevention and treatment of soybean Phytophthora root rot.

[0008] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions:

[0009]

[0010] The application further provides a protein encoded by the GmMPK4 gene, and an amino acid sequence of the protein is shown as SEQ ID NO. 2, and specifically, MSVVESGEHNNIRGVPTHGGRYVQYNIYGNLFEVSRKYVPPIRPVGRGAYGIVCAAVNAETGEEVAIKKIGNAFDNRIDAKRTLREIKLLRHMDHANIMSIKDIIRPPQKENFNDVYLVSELMDTDLHQIIRSNQQLTDDHCRYFLYQLLRGLKYVHSANVLHRDLKPSNLLLNANCDLKIADFGLARTTSETDFMTEYVVTRWYRAPELLLNCSEYTAAIDIWSVGCILGEIITRQPLFPGKDYVHQLRLITELIGSPNDASLGFLRSDNARRYVKQLPQYPKQNFSARFPDMSPGAVDLLEKMLIFDPNRRITVDEALSHPYMAPLHDINEEPVCTRPFSFDFEQPSFTEEDIKELIWRESVKFNPVPPVY.

[0011] The application further provides an application of overexpressing the soybean GmMPK4 gene in enhancing the sensitivity of crops to the Phytophthora root rot fungus.

[0012] The application further provides an application of overexpressing the soybean GmMPK4 gene in degrading the disease-resistant protein GmICE2 in crops.

[0013]

[0014] The application further provides a protein encoded by the GmICE2 gene, and an amino acid sequence of the protein is shown as SEQ ID NO. 4, and specifically MNMERYTLPAPPNIIPEEEENIAIAMGAPLIPSFKSMLQQHPQQLDSYFNSSISSTTIPFVPNMDSFLSLDPFSPSFFNNSSSSSNMALDPGFDLGLDTGLHSSAPAPHLFSQAQMMGFEFSELEALAGPGNVNVPFADGAKAAALLRSPHFEPPQQQPTLYRKRRGTAAEIPGLEMVRRKGRKWQEGGGEGEEGSSADVGGSGLNYESDEQNESNGLKLSENGGDNKGKKKGLPAKNLMAERRRRKKLNDRLYMLRSVVPKISKMDRASILGDAIDYLKELLQRINDLHHELESTPPGSSLTPSSSTSFQPLTPTLPTLPCRVKEELYPGTLPSPKNQAAKVEVRVREGRTVNIHMFCTRRPGLLLSTMKALDNLGLDVQQAVISCFNGFALDVFKAEQCREGQDVLPEQIKAVLSDSAGFHGMM.

[0015] The application further provides an application of overexpressing the soybean GmICE2 gene in improving the resistance of crops to Phytophthora root rot.

[0016] The application further provides an application of overexpressing the soybean GmICE2 gene in improving the transcription of the disease resistance pathway related gene GmPR1 in crops.

[0017] The application further provides a recombinant expression vector comprising a target gene and a pCAMBIA3301 overexpression vector, wherein the target gene is a soybean GmICE2 or GmPR1 gene; the nucleotide sequence of the GmPR1 gene is shown as SEQ ID NO. 5, in particular, ATGGGGTACATGTGCATTAAGATTTCGTTTTGTGTGATGTGTGTGTTGGGGTTGGTGATCGTGGGTGATGTTGCCTACGCTCAAGATTCAGCAGAAGACTACGTGAATGCACACAATGCAGCACGAGCAGAGGTGGGTTCTCAATCACCAAGACAAACAGTGATTGTTCCAAGTTTGGCTTGGGATGATACGGTTGCTGCTTATGCAGAGAGCTATGCTAATCAACGCAAAGGTGACTGCCAACTGATCCACTCTGGTGGTGAATACGGAGAGAATATTGCAATGAGCACTGGTGAACTAAGTGGCACAGATGCAGTGAAAATGTGGGTTGATGAGAAATCCAACTATGACTATGATTCTAACTCTTGTGTTGGAGGAGAGTGCCTGCACTACACACAGGTCGTTTGGGCTAACTCGGTGCGTCTTGGATGTGCCAAAGTGACATGTGATAACGGAGGCACTTTCATCACTTGCAACTATGATCCCCCTGGCAACTTTGTTGGTGAAAGACCCTACAAACTGTAG; and the amino acid sequence of the protein encoded by the soybean GmPR1 gene is shown as SEQ ID NO. 6. The amino acid sequence is: MGYMCIKISFCVMCVLGLVIVGDVAYAQDSAEDYVNAHNAARAEVGSQSPRQTVIVPSLAWDDTV AAYAESYANQRKGDCQLIHSGGEYGENIAMSTGELSGTDAVKMWVDEKSNYDYDSNSCVGGECLH YTQVVWANSVRLGCAKVTCDNGGTFITCNYDPPGNFVGERPYKL.

[0018] The method for constructing the recombinant expression vector comprises the following steps:

[0019] 1) linearizing the pCAMBIA3301 overexpression vector to obtain a linearized expression vector pCAMBIA3301;

[0020] 2) The soybean GmICE2 or GmPR1 gene is connected to the pEasy-Blunt vector respectively, and then transformed into the E. coli competent cells for propagation to obtain pEasy-Blunt-GmICE2 or pEasy-Blunt-GmPR1;

[0021] 3) The specific primers with vector end sequences at both ends are designed by taking the obtained pEasy-Blunt-GmICE2 or pEasy-Blunt-GmPR1 as a template, and the target fragments of the GmICE2 or GmPR1 gene are amplified by PCR;

[0022] 4) The target fragments are inserted into the linearized expression vector pCAMBIA3301 to obtain the recombinant expression vector for overexpressing the soybean GmICE2 or GmPR1 gene.

[0023] The application further provides a transformant for overexpressing the soybean GmICE2 or GmPR1 gene, and the soybean GmICE2 or GmPR1 gene is introduced into E. coli; and the E. coli is a Trans1-T1 competent cell.

[0024] Compared with the prior art, the application has the following beneficial effects:

[0025] The application finds through experimental research that the soybean GmMPK4 gene can enhance the sensitivity of soybean to soybean Phytophthora root rot, mainly by phosphorylating GmICE2 through GmMPK4 to promote the protein degradation of GmICE2 and inhibit the transcription regulation of GmPR1, thereby enhancing the sensitivity of soybean to soybean Phytophthora root rot. Overexpression of the soybean GmICE2 can enhance the resistance of soybean to soybean Phytophthora root rot, and GmICE2 enhances the defense ability of soybean to soybean Phytophthora root rot by promoting the expression of the disease-resistant gene GmPR1. The finding provides important genetic basis and theoretical support for the research on the related mechanism of resistance to soybean Phytophthora root rot, provides valuable genetic resources for promoting the research and application of the plant defense system and cultivating new soybean varieties with high disease resistance, and has important application value in the soybean disease-resistant genetic engineering breeding. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 It is the domain analysis of the GmMPK4 gene;

[0027] Figure 2 It is the expression quantity analysis result of the GmMPK4 gene induced by the soybean Phytophthora fungus in different soybean varieties;

[0028] Figure 3 It is the subcellular localization result of the GmMPK4 gene;

[0029] Figure 4Yeast two-hybrid assay to verify the self-activation activity of GmMPK4;

[0030] Figure 5 Yeast two-hybrid assay to screen the interacting proteins of GmMPK4;

[0031] Figure 6 Yeast two-hybrid assay to verify the interaction between GmMPK4 and candidate proteins;

[0032] Figure 7 Pull-down assay to verify the interaction between GmMPK4 and GmICE2;

[0033] Figure 8 Luciferase complementation imaging assay to verify the interaction between GmMPK4 and GmICE2;

[0034] Figure 9 Bimolecular fluorescence complementation assay to verify the interaction between GmMPK4 and GmICE2;

[0035] Figure 10 Co-immunoprecipitation assay to verify the interaction between GmMPK4 and GmICE2;

[0036] Figure 11 In vitro phosphorylation assay to verify the phosphorylation modification of GmMPK4 on GmICE2;

[0037] Figure 12 In vivo phosphorylation assay to verify the phosphorylation modification of GmMPK4 on GmICE2;

[0038] Figure 13 Protein degradation assay to verify that phosphorylation of GmICE2 by GmMPK4 reduces the protein stability of GmICE2 (wherein A is the result of Western blot, and B is the gray value of Western blot protein band);

[0039] Figure 14 Ubiquitination degradation assay to verify that phosphorylation of GmICE2 by GmMPK4 promotes the ubiquitination degradation of GmICE2;

[0040] Figure 15 Ubiquitination degradation assay to verify that phosphorylated GmICE2 promotes its degradation through the 26S proteasome pathway;

[0041] Figure 16 Ubiquitination degradation assay to verify that phosphorylation promotes the in vivo degradation of GmICE2 in soybean;

[0042] Figure 17 The expression analysis results of GmICE2 gene induced by P. sojae in different soybean varieties;

[0043] Figure 18 Domain analysis of GmICE2 gene;

[0044] Figure 19 Subcellular localization results of GmICE2;

[0045] Figure 20 Transcriptome sequencing results of GmICE2;

[0046] Figure 21 Regulatory pathways in which GmICE2 might be involved;

[0047] Figure 22 Luciferase assay to verify that GmICE2 activates the expression of downstream target gene GmPR1 promoter (wherein A is the imaging chart of chemiluminescence imaging system, and B is the relative activity of LUC);

[0048] Figure 23 ChIP assay to verify that GmICE2 directly binds to the promoter region of GmPR1 (wherein A is the analysis result chart of E-box element in the promoter, and B is the detection result chart of ChIP-qPCR);

[0049] Figure 24 qRT-PCR assay to verify the transcriptome results of GmICE2 (wherein A is the relative expression level of stress-related genes in GmICE2-OE, and B is the relative expression level of stress-related genes in GmICE2-RNAi);

[0050] Figure 25 Luciferase assay to verify that GmMPK4 phosphorylates GmICE2 to regulate the transcription of GmPR1 (wherein A is the imaging chart of chemiluminescence imaging system, and B is the relative activity of LUC);

[0051] Figure 26 Luciferase assay to verify that the simulated phosphorylation of GmICE2 inhibits the transcriptional regulation of GmPR1 (wherein A is the imaging chart of chemiluminescence imaging system, and B is the relative activity of LUC);

[0052] Figure 27 Luciferase assay to verify that the deactivation of phosphorylation of GmICE2 promotes the transcriptional regulation of GmPR1 (wherein A is the imaging chart of chemiluminescence imaging system, and B is the relative activity of LUC);

[0053] Figure 28 Nikon D7000 camera photographing results of the cotyledon of GmMPK4 transgenic plants;

[0054] Figure 29The results of the analysis of the lesion area of cotyledon and the accumulation of P. sojae of GmMPK4 transgenic plants and controls (wherein A is the lesion area of cotyledon, B is the biomass accumulation of P. sojae) ;

[0055] Figure 30 The results of the photograph of the root of GmMPK4 transgenic plants by Nikon D7000 camera;

[0056] Figure 31 The results of the analysis of the lesion area of root and the accumulation of P. sojae of GmMPK4 transgenic plants and controls (wherein A is the lesion area of cotyledon, B is the biomass accumulation of P. sojae) ;

[0057] Figure 32 The results of the photograph of GmMPK4 transgenic soybean hairy roots by Nikon D7000 camera;

[0058] Figure 33 The results of the analysis of the lesion area and the accumulation of P. sojae of GmMPK4 transgenic soybean hairy roots (wherein A is the biomass accumulation of P. sojae in GmMPK4-OE roots, B is the biomass accumulation of P. sojae in GmMPK4-RNAi roots) ;

[0059] Figure 34 The results of the photograph of GmICE2 transgenic plants cotyledon by Nikon D7000 camera;

[0060] Figure 35 The results of the analysis of the lesion area of cotyledon and the accumulation of P. sojae of GmICE2 transgenic plants and controls (wherein A is the lesion area of cotyledon, B is the biomass accumulation of P. sojae) ;

[0061] Figure 36 The results of the photograph of the root of GmICE2 transgenic plants by Nikon D7000 camera;

[0062] Figure 37 The results of the analysis of the lesion area of root and the accumulation of P. sojae of GmICE2 transgenic plants and controls;

[0063] Figure 38 The results of the photograph of GmICE2 and GmICE2 phosphorylation mutant transgenic soybean hairy roots by Nikon D7000 camera;

[0064] Figure 39 The results of the analysis of the lesion area and the accumulation of P. sojae of GmICE2 and GmICE2 phosphorylation mutant transgenic soybean hairy roots (wherein A is the biomass accumulation of P. sojae in GmICE2-OE, GmICE2-OE and GmICE2-PhoA roots, B is the biomass accumulation of P. sojae in GmICE2-RNAi, GmICE2-RNAi and GmICE2-PhoA roots) ; 6A -OE and GmICE2 6DBiomass accumulation of P. meadii in GmMPK4-OE roots, C is the lesion area in GmMPK4-RNAi plants, D is the biomass accumulation of P. meadii in GmMPK4-RNAi plants; 6A GmMPK4-OE and GmMPK4-RNAi 6D Biomass accumulation of P. meadii in GmMPK4-OE roots, C is the lesion area in GmMPK4-RNAi plants, D is the biomass accumulation of P. meadii in GmMPK4-RNAi plants;

[0065] Figure 40 Nikon D7000 camera photographing results of GmPR1 transgenic soybean hairy roots;

[0066] Figure 41 Lesion area analysis results of GmPR1 transgenic soybean hairy roots and controls (wherein, A is the lesion area in GmPR1-OE roots, B is the lesion area in GmPR1-RNAi roots) ;

[0067] Figure 42 Biomass accumulation analysis results of P. meadii in GmPR1 transgenic soybean hairy roots and controls (wherein, A is the biomass accumulation of P. meadii in GmPR1-OE roots, B is the biomass accumulation of P. meadii in GmPR1-RNAi roots).

[0068] Note: "OE" in the above figures is the abbreviation of GmMPK4-OE or GmICE2-OE, and "RNAi" is the abbreviation of GmMPK4-RNAi or GmICE2-RNAi. DETAILED DESCRIPTION

[0069] The technical solutions provided by the present application will be described in detail below in combination with examples, but they should not be understood as limiting the scope of protection of the present application.

[0070] The transcriptome sequencing and related derivative pictures in the present application are completed by Wuhan Maitever Biological Technology Co., Ltd.

[0071] Example 1 Cloning of soybean GmMPK4, GmICE2 and GmPR1 genes

[0072] 1.1 Primer design

[0073] According to the coding region sequence of GmMPK4, GmICE2 or GmPR1, the primer is designed by Primer Premier 5, and the CDS full-length sequence of soybean GmMPK4, GmICE2 or GmPR1 gene is obtained by PCR amplification. The sequences of the primers are shown in Table 1, Table 2 and Table 3.

[0074] Table 1 Primer sequence for amplifying soybean GmMPK4 gene

[0075] Name Sequence (5'→ 3') GmMPK4F GTCAATAAGTTCACGGGTTC (SEQ ID NO. 7) GmMPK4R GACTGGTGGAACAGGATTG (SEQ ID NO. 8)

[0076] Table 2 Primer sequences for amplifying the soybean GmICE2 gene

[0077] Name Sequence (5'→ 3') GmICE2F CACAGCAATATAGCATCGCA (SEQ ID NO. 9) GmICE2R CACAGCAATATAGCATCGCA (SEQ ID NO. 10)

[0078] Table 3 Primer sequences for amplifying the soybean GmPR1 gene

[0079] Name Sequence (5'→ 3') GmPR1F CAAAAACCTTTCCTTTTAG (SEQ ID NO. 11) GmPR1R CACAGGGAACATCCTTTAT (SEQ ID NO. 12)

[0080] The GmMPK4 and GmICE2 sequences were analyzed using the ORF Finder in NCBI.

[0081] Experimental results: such as Figure 1 As shown. The full-length cDNA sequence of GmMPK4 is 1122 bp, encoding 373 amino acids, including a kinase domain of 287 amino acids, an ATP binding site, an activation-loop, and a conserved TEY motif. The C-terminus has a conserved ED domain.

[0082] like Figure 18 As shown. The full-length cDNA sequence of the GmICE2 gene is 1281 bp, encoding 426 amino acids, including a 56-amino acid bHLH domain, a 42-amino acid ACT domain, 6 MAPK phosphorylation sites (green background), 9 DNA binding sites (underlined), and 20 amino acids that can form dimers (red text).

[0083] 1.2 RNA Extraction

[0084] Leaves of highly resistant soybean variety "Suinong 10" and highly susceptible soybean variety "Dongnong 50" were ground into 100 mg powder in liquid nitrogen. 60 mg was placed in a 1.5 mL centrifuge tube, 1 mL of Trizol reagent was added, and the mixture was immediately shaken for 20 s to allow for complete lysis. The mixture was then placed on ice for 5 min, 200 μL of chloroform was added, and the mixture was thoroughly mixed and allowed to stand for 8 min. The mixture was then centrifuged at 12,000 × g at 4 °C for 15 min. After centrifugation, 500 μL of the supernatant was transferred to a new AXgen enzyme-free EP tube, and 500 μL of isopropanol was added. The mixture was thoroughly mixed and allowed to stand on ice for 8 min. The mixture was then centrifuged at 12,000 × g at 4 °C for 10 min, and the supernatant was discarded. The precipitate was washed with 75% ethanol prepared with DEPC water, and the supernatant was discarded. After the precipitate dried, an appropriate amount of DEPC water was added to dissolve the RNA, thus obtaining RNA.

[0085] 1.3 Reverse transcription of RNA

[0086] The reverse transcription was performed according to the instructions of ReverTraAce qPCR RT Kit (purchased from TOYOBO Co., Ltd., Japan), specifically: the extracted RNA was denatured at 65°C for 5 min, and then immediately cooled on ice, and the reverse transcription system (0.5 μg of RNA, 0.5 μL of Primer Mix, 2.0 μL of 5×RT Buffer, 0.5 μL of RT Enzyme Mix, to 10 μL of Nucease-free Water) and the reverse transcription program of 37°C for 15 min and 98°C for 5 min were set to perform reverse transcription to obtain cDNA; after the reaction, it was diluted and stored at -20°C.

[0087] 1.4 PCR reaction

[0088] The reaction system (1.5 μL of GmMPK4 / GmICE2 / GmPR1 F, 1.5 μL of GmMPK4 / GmICE2 / GmPR1 R, 25 μL of KOD Master Mix, 4 μL of cDNA template, 18 μL of deionized water) and the reaction program of 98°C for 3 min pre-denaturation; (98°C for 10 s, 60°C for 5 s, 68°C for 5 s) x 35; 68°C for 10 min extension were set to obtain the CDS sequence (1122 bp) of the soybean GmMPK4 gene using the amplification primer sequence in Table 1 as the template; the CDS sequence (1281 bp) of the soybean GmICE2 gene was cloned using the amplification primer sequence in Table 2; and the CDS sequence (525 bp) of the soybean GmPR1 gene was cloned using the amplification primer sequence in Table 3.

[0089] The PCR amplified product was detected by 1% agarose gel electrophoresis, and then the target fragment was recovered by cutting the gel and connected to the pEasy-blunt vector (purchased from Shanghai Novagene Bio-technology Co., Ltd.) to obtain pEasy-blunt-GmMPK4, pEasy-blunt-GmICE2 and pEasy-blunt-GmPR1, which were then transformed into E. coli DH5α competent cells (purchased from Shanghai Novagene Bio-technology Co., Ltd.) for sequencing alignment.

[0090] Example 2 Construction of recombinant expression vector

[0091] 1.1 Linearization of vector

[0092] The vector plasmid was extracted according to the AXgen plasmid extraction instructions, specifically: the subcellular localization vector liquid of pCAMBIA1302 (stored in the Key Laboratory of Soybean Biology of the Ministry of Education of Northeast Agricultural University), the overexpression vector liquid of pCAMBIA3301 (stored in the Key Laboratory of Soybean Biology of the Ministry of Education of Northeast Agricultural University), the pGreenII 0800 liquid (purchased from Shanghai Novizen Biotech Co., Ltd.), the Pcambia1300 liquid (purchased from Shanghai Novizen Biotech Co., Ltd.) and the pFGC5941 interference vector liquid (stored in the Key Laboratory of Soybean Biology of the Ministry of Education of Northeast Agricultural University) were taken into 2 mL centrifuge tubes, and centrifuged at 12,000 x g for 1 min at room temperature, and the supernatant in the EP tube was discarded; 250 μL of Solution I was added to resuspend the bacteria in the EP tube, 250 μL of Solution II was added, and gently mixed, 350 μL of Solution III was added, and gently mixed; centrifuged at 12,000 x g for 10 min at room temperature; 700 μL of supernatant was taken and added to the preparation tube provided in the kit, centrifuged at 12,000 x g for 2 min at room temperature, and the supernatant filtered in the preparation tube was discarded, 500 μL of Wash Buffer I was added to the preparation tube, and centrifuged at 12,000 x g for 2 min at room temperature; the supernatant filtered in the preparation tube was discarded, 700 μL of Wash Buffer II was added to the preparation tube, and centrifuged at 12,000 x g for 2 min at room temperature; the supernatant filtered in the preparation tube was discarded, and the preparation tube containing the vector plasmid solution was transferred to the EP tube provided in the kit, 40 μL of Elution Buffer was added to the EP tube, and centrifuged at 12,000 x g for 2 min at room temperature, and the elution was repeated once, and the double enzyme digestion system (0.5 μL of enzyme 1, 0.5 μL of enzyme 2, 1 μL of 10 x buffer, 8 μL of vector plasmid, 10 μL of system) was set to obtain linearized vector.

[0093] 1.2 Gel recovery

[0094] The vector after enzyme digestion was recovered according to the instructions of the OMEGA gel recovery kit. The specific steps were as follows: the agarose gel was placed on the blue light gel cutter, and the separated target fragment was cut and placed in a 1.5 mL EP tube; 200 μL of Binding Buffer was added to the EP tube, and the gel was completely dissolved in a 60°C metal bath for 5 min; the dissolved gel was transferred to the adsorption column provided in the kit, centrifuged at room temperature at 12,000 x g for 30 s, and the supernatant in the EP tube was discarded; 300 μL of Binding Buffer was added to the adsorption column, centrifuged at room temperature at 12,000 x g for 30 s, and the supernatant in the EP tube was discarded; 700 μL of Wash Buffer was added to the adsorption column, centrifuged at room temperature at 12,000 x g for 30 s, and the supernatant in the EP tube was discarded, and the washing was repeated once; the adsorption column was centrifuged at room temperature at 12,000 x g for 1 min, then transferred to a new 1.5 mL EP tube, 20 μL of Elution Buffer was added, centrifuged at room temperature at 12,000 x g for 1 min, and the elution was repeated once to obtain the gel recovery product.

[0095] The concentration of the gel recovery product was detected by a micro-nucleic acid protein analyzer, and then diluted to 100 ng / μL-400 ng / μL to ensure that the sample added in the recombination reaction was in the optimal amount of linearized vector and inserted fragment.

[0096] 1.3 Amplification of GmMPK4, GmICE2 or GmPR1 target fragment

[0097] According to the instructions of ClonExpress II One Step Cloning Kit (Vazyme, China), specific primers with vector end sequences at both ends (see Table 4 for specific sequences) were designed using the Vazyme online primer design software CE Design V1.04, and pEasy-Blunt-GmMPK4, pEasy-Blunt-GmICE2 and pEasy-Blunt-GmPR1 were used as templates to amplify the GmMPK4, GmICE2 and GmPR1 target fragments.

[0098] Table 4 Specific primers with vector end sequences at both ends

[0099]

[0100]

[0101] Note: The last letter "F" in the name represents a forward primer, and "R" represents a reverse primer.

[0102] 1.4 Recombination reaction

[0103] The recombination reaction system was configured on ice (1 μL linearized vector, 2 μL gene fragment, 4 μL 5×CE buffer, 2 μL Exnase, to 20 μL deionized water, 20 μL system). The target fragment obtained in 1.3 was inserted into the linearized pCAMBIA3301, pFGC5941, pCAMBIA1302, pGBKT7, pCAMBIA1300, pSAT6, pGreenII0800 vector, respectively, to obtain the recombination vectors GmMPK4-bar, GmMPK4-Flag, GmMPK4-RNAi, GmMPK4-GFP, GmMPK4-BD, GmMPK4-cLuc, GmICE2-nLuc, GmMPK4-cYFP, GmICE2-nYFP, GmMPK4-Flag, GmICE2-GFP, GmICE2-Myc, GmICE2-RNAi, GmICE2-GFP, pGmPR1-LUC, GmPR1-Myc, GmPR1-RNAi, respectively.

[0104] Transformation of E. coli Trans1-T1 competent cells

[0105] The recombination reaction liquid after the recombination reaction in the above-mentioned Example 2 was transferred to 100 μL Trans1-T1 competent cells with a pipette, gently mixed, placed on ice for 25 min, heat shocked in a 42°C water bath for 45 s, and then immediately placed in an ice bath for 2 min; 500 μL of LB liquid medium was taken in the clean bench with a pipette and added to the competent cells in the ice bath, and the recovery was performed at 37°C, 150 rpm constant temperature shaker for 1 h; centrifuged at room temperature, 5,000×g for 2 min, and the supernatant was taken out with a pipette in the clean bench and the bacterial cells were resuspended with the remaining supernatant; 200 μL of the resuspended bacterial liquid was taken and plated on LB plate medium (containing 50 mg / mL Kana), evenly coated, and placed in a 37°C incubator for constant temperature culture overnight; several single colonies were picked in a suitable amount of LB screening medium (containing 50 mg / mL Kana) and cultured at 37°C, 220 rpm constant temperature shaker overnight; 2 μL of the cultured bacterial liquid was subjected to PCR amplification. The reaction program of the PCR was: 94°C for 3 min pre-denaturation; (94°C for 30 s, 60°C for 30 s, 72°C for 1 min) × 30; 72°C for 10 min extension. The PCR reaction system was: 1 μL Prime F (10 μM), 1 μL Prime R (10 μM), 12.5 μL 2×Taq Master Mix, 2 μL bacterial liquid, 8.5 μL deionized water, 25 μL system.

[0106] After the PCR reaction was completed, 20 μL of the reaction solution was taken from the PCR reaction tube for electrophoresis; the bacterial culture corresponding to the sample with the successful amplification of the target band was selected and stored as glycerol bacteria, and the plasmid was extracted and transformed into Agrobacterium 4404 competent cells.

[0107] The method for transforming plasmids into Agrobacterium 4404 competent cells is as follows: Agrobacterium 4404 competent cells (purchased from Shanghai Novizan Biotechnology Co., Ltd.) are placed on ice and allowed to thaw. Then, GmMPK4-bar, GmMPK4-RNAi, GmICE2-bar, and GmICE2-RNAi plasmids are added. After mixing, the cells are incubated on ice for 5 min, in liquid nitrogen for 5 min, in a water bath at 37°C for 5 min, and then incubated on ice for another 5 min. Subsequently, 700 μL of LYEP liquid medium (purchased from Shanghai Novizan Biotechnology Co., Ltd.) is added and the cells are incubated on a shaker at 28°C for 2 h. The cells are then plated and cultured to obtain Escherichia coli Trans1-T1 competent cells.

[0108] Example 4: Analysis of expression patterns of soybean GmMPK4 and GmICE2 genes

[0109] Once the seedlings of the highly resistant soybean variety "Suinong 10" and the highly susceptible soybean variety "Dongnong 50" reached the V1 stage, Phytophthora soybean physiological race No. 1 (a dominant race in Heilongjiang Province, preserved in the Key Laboratory of Soybean Biology of the Ministry of Education, Northeast Agricultural University) was inoculated into the hypocotyls of "Dongnong 50" and "Suinong 10". After inoculation, samples were taken from the treatment group and the control group (without Phytophthora soybean inoculation) at 0h, 3h, 6h, 9h, 12h, 24h, 36h, 48h, and 72h, respectively. The obtained samples were quick-frozen with liquid nitrogen and stored at -80℃ for later use.

[0110] Experimental results: such as Figure 2 As shown in the figure, the transcriptional level of GmMPK4 in the resistant variety "Suinong 10" did not change significantly. However, in the susceptible soybean variety "Dongnong 50", the transcriptional level of GmMPK4 increased tenfold 9 hours after inoculation with Phytophthora soybeanae, compared with 0 h after inoculation, indicating that GmMPK4 is significantly induced by Phytophthora soybeanae in susceptible varieties.

[0111] like Figure 17 As shown in the figure. The results indicated that the transcriptional level of GmICE2 did not change significantly in susceptible soybean variety "Dongnong 50". However, in the resistant variety "Suinong 10", compared with 0 h after inoculation with Phytophthora soybeanis, it gradually increased after 9 h after inoculation, reaching a peak (7-fold) at 24 h after inoculation with Phytophthora soybeanis, and then gradually decreased, indicating that GmICE2 is significantly induced by Phytophthora soybeanis in resistant varieties.

[0112] Example 5: Subcellular localization of GmMPK4 and GmICE2 proteins

[0113] The plasmids pCAMBIA1302-GmMPK4 and pCAMBIA1302-GmICE2 were extracted using a high-throughput method and transformed into Arabidopsis protoplast cells (preserved in the Key Laboratory of Soybean Biology, Northeast Agricultural University) via PEG-mediated transformation. The subcellular localization of GmMPK4-GFP and GmICE2-GFP was observed under a confocal fluorescence microscope. The empty vector pCAMBIA1302-GFP (purchased from Shanghai Novizan Biotechnology Co., Ltd.) was used as a blank control.

[0114] Experimental results: such as Figure 3 and Figure 19 As shown in the figure, the results indicated that Arabidopsis protoplasts transformed with the pCAMBIA1302-GmMPK4 plasmid exhibited green fluorescence in both the cytoplasm and nucleus, indicating that GmMPK4 is localized in both the cytoplasm and nucleus. In contrast, Arabidopsis protoplasts transformed with the pCAMBIA1302-GmICE2 plasmid showed green fluorescence only in the nucleus, consistent with the nuclear localization control, indicating that GmICE2 is localized in the nucleus and is a nuclear localization protein.

[0115] Example 6: Interaction between GmMPK4 and GmICE2

[0116] 1.1 Screening of GmMPK4 interacting proteins

[0117] 1.1.1 Yeast self-activation verification of GmMPK4

[0118] The full-length CDS of GmMPK4 was cloned into the pGBKT7 yeast expression vector to form the recombinant plasmid GmMPK4-BD. Y2H competent cells were prepared in the competent cells, and the self-activation activity of the GmMPK4 protein was studied by co-transformation with different combinations of plasmids. The plasmid combinations were designed as follows: pGBKT7-GmMPK4 and pGADT7 empty vector, pGBKT7-53 and pGADT7-SV40 as positive controls, and pGBKT7 and pGADT7 empty vector as negative controls. Successfully transformed bacterial cultures were analyzed by spot plasmid analysis on SD / -Trp-Leu (DDO), SD / -Trp-Leu-His-Ade (QDO), and SD / -Trp-Leu-His-Ade / X-α-Gal (QDO+X-α-Gal) plates.

[0119] Experimental results: Yeast strains containing the pGBKT7-GmMPK4 co-transformation plasmid grew normally on SD / -Trp-Leu selection plates, but could not grow on SD / -Trp-Leu-His-Ade and SD / -Trp-Leu-His-Ade / X-α-Gal selection plates. The results are as follows: Figure 4The results showed that GmMPK4 protein did not have self-activation activity, and the complete amino acid sequence could be used for yeast library screening and yeast two-hybrid identification.

[0120] 1.1.2 GmMPK4 interacting protein screening

[0121] In order to analyze whether there are other proteins that form dimers with GmMPK4, the yeast liquid containing the cDNA library and the Y2H yeast liquid containing the bait vector GmMPK4-BD were reacted, and the interaction proteins were screened by plating. The grown yeast spots were picked and placed in four-lack SD / -Trp / -Leu / -His / -Ade liquid medium, and incubated at 30°C overnight. The yeast liquid was spotted on four-lack SD / -Trp / -Leu / -His / -Ade+X-α-gal solid medium, and incubated at 30°C in an incubator for 3 days. Positive yeast colonies were selected for PCR amplification to obtain the full-length cDNA sequence of the candidate interacting proteins. Figure 5

[0122] After systematic screening of the yeast two-hybrid library, a total of 268 candidate proteins were identified, which are expected to reveal the potential interaction mechanism of GmMPK4 in subsequent studies. In this study, five transcription factors GmMYB48, GmILR3, GmICE2, GmAP2, and GmNAC018 related to biotic and abiotic stress, and an E3 ubiquitin ligase GmSDIR1 were selected as candidate proteins interacting with GmMPK4.

[0123] 1.1.3 GmMPK4 and candidate protein yeast two-hybrid interaction analysis

[0124] To further explore whether the candidate proteins GmMYB48, GmILR3, GmICE2, GmAP2, GmNAC018, or GmSDIR1 interact with GmMPK4, six genes were first cloned from the soybean disease-resistant variety “Suinong 10” using RT-PCR technology. They were constructed into PGADT7 vectors, respectively.

[0125] The recombinant vectors GmMYB48-PGADT7, GmILR3-PGADT7, GmICE2-PGADT7, GmAP2-PGADT7, GmNAC018-PGADT7, and GmSDIR1-PGADT7 were co-transformed with GmMPK4-PGBKT7 into Y2H yeast competent cells, and the positive control combination and negative control combination were co-transformed into Y2H Gold yeast cells. They were plated on two-lack DDO, four-lack QDO, and four-lack+X-α-gal deficient medium, respectively, and incubated at 30°C in an incubator for 3 days.​

[0126] The experimental results are shown in Table 1. Figure 6 Table 1: The results of yeast two-hybrid assay. The results show that yeast grows well on the double-deficient medium, indicating that the yeast transformation is not a problem; on the quadruple-deficient and quadruple-deficient + X-a-gal deletion medium, the AD-SV40+BD-P53 positive, the experimental groups GmMPK4-BD+GmICE2-AD and GmMPK4-BD+GmAP2-AD grow well on the quadruple-deficient medium and exhibit a-gal activity, while the experimental groups GmMPK4-BD+GmMYB48-AD, GmMPK4-BD+GmILR3-AD, GmMPK4-BD+GmNAC018-AD, GmMPK4-BD+GmSDIR1-AD and the negative control (BD+AD) cannot grow normally on the quadruple-deficient medium, indicating that GmICE2 or GmAP2 interacts with GmMPK4, while GmMYB48, GmILR3, GmNAC018 or GmSDIR1 does not interact with GmMPK4. GmICE2 was selected as the interacting protein of GmMPK4 for further study in this study.

[0127] Example 7: Verification of the interaction between GmMPK4 and GmICE2

[0128] 1.1 Pull-down interaction analysis of GmMPK4 and GmICE2

[0129] To explore whether GmMPK4 and GmICE2 proteins interact in vitro, pull-down assay was used for verification. The CDS sequence of GmMPK4 was constructed into the pGEX4T-1 vector to obtain the GmMPK4-pGEX4T-1 recombinant vector, which was sequenced and identified correctly. The recombinant plasmid GmMPK4-pGEX4T-1 was transformed into the prokaryotic expression strain ShuffleT7 strain, and a single colony was picked and inoculated in LB medium containing 50 μg / mL kanamycin and cultured at 37°C, 220 rpm for overnight to obtain activated bacterial liquid. The next day, 1 mL of bacterial liquid was added to LB medium without antibiotics at 37°C for about 2 h, and the OD600 was 0.5. 1 mL of bacterial liquid was taken in a 1.5 mL centrifuge tube, centrifuged, and the supernatant was discarded and stored at -20°C for later use. 0.1 mM IPTG was added to induce at 28°C for 4 h, and 1 mL of sample was taken, and the whole bacterial liquid was centrifuged and frozen for later use. The sampled bacterial liquid was subjected to SDS-PAGE electrophoresis, and the gel was stained with Coomassie brilliant blue staining solution to detect the expression of the target protein and carry out subsequent purification work.

[0130] After resuspending the bacteria with lysis buffer added with lysozyme and PMSF, the bacteria were ultrasonically lysed and centrifuged, and the supernatant was incubated with Ni-NTA gel for 1 h, and then the gel was washed with washing buffer, and washed for 3-5 times, and then the purified recombinant protein was eluted with washing buffer, and the elution product was detected by SDS-PAGE electrophoresis, and the purified GmICE2-His protein was obtained. Similarly, the recovered GmMPK4-GST protein was detected by western blot. The above results show that the concentration and purity of GmMPK4-GST and GmICE2-His recombinant proteins meet the requirements of pull-down experiment, and subsequent research can be carried out. The obtained GmMPK4-GST, GmICE2-His and control GST proteins were subjected to in vitro pull-down analysis.

[0131] Experimental results are shown in Table 1. Figure 7 As shown in Table 1. The results show that after GmMPK4-GST and GmICE2-His are co-incubated and subjected to pull-down treatment, the specific band of GmICE2-His can be clearly seen; while when GST control and GmICE2-His are co-incubated and subjected to the same pull-down operation, GmICE2-His band cannot be detected. Therefore, it can be concluded that GmMPK4 and GmICE2 interact in vitro.

[0132] 1.2 Verification of tobacco SLCA interaction of GmMPK4 and GmICE2

[0133] To verify whether GmMPK4 and GmICE2 can interact in plants, the full-length CDS of GmMPK4 and GmICE2 without stop codon was constructed into pCAMBIA1300-cLUC and pCAMBIA1300-nLUC plant expression vectors, respectively, to obtain recombinant vectors GmMPK4-cLUC and GmICE2-nLUC. After the constructed recombinant vectors were introduced into GV3101 (carrying pSoup-p19 helper plasmid) Agrobacterium strain, Agrobacterium containing both recombinant plasmids GmMPK4-cLuc and GmICE2-nLuc was used to co-transfect tobacco leaves to achieve transient gene expression. To ensure the accuracy of the experiment, two different negative control experiments were set up: one group of (GmICE2-nLuc + cLuc) co-transfected tobacco leaves, and another group of (GmMPK4-cLuc + nLuc) co-transfected leaves; at the same time, a positive control experiment was set up, i.e. (Fls2-nLuc + Gβ-cLuc) co-transfected tobacco leaves. After the transfection of the tobacco leaves, 1 mM D-Luciferin solution was applied, and then Tian Neng 5200 type chemiluminescence imaging system was used for imaging.

[0134] Experimental results: as shown in Figure 8 The results show that GmMPK4-cLuc and GmICE2-nLuc or positive control transfection area all present significant chemiluminescence signals, while no chemiluminescence is observed in the negative control transfection area. The above results show that GmMPK4 and GmICE2 can interact in vivo in plants.

[0135] 1.3 BiFC interaction analysis of GmMPK4 and GmICE2

[0136] To further study the interaction of GmMPK4 and GmICE2 in vivo in plants, the BiFC technology was used to verify the interaction of GmMPK4 and GmICE2 in vivo in plants. GmMPK4 and GmICE2 were respectively fused with the yellow fluorescence protein tag (YFP, Yellow fluorescence protein) divided into N-terminal and C-terminal two parts for expression, and the plant expression vectors of GmMPK4 and YFP N-terminal fusion expression and GmICE2 and YFP C-terminal fusion expression were constructed. The obtained plant expression vectors GmMPK4-nYFP and GmICE2-cYFP were transformed into Top10 E. coli strain and a large amount of plasmid was extracted, and the recombinant vector plasmids GmMPK4-nYFP and GmICE2-cYFP were co-transformed into Arabidopsis protoplasts by PEG-mediated method. GmMPK4-nYFP, cYFP empty vector, and GmICE2-cYFP, nYFP empty vector co-transformed Arabidopsis protoplasts were used as negative control. Histone H2B fused with mCherry was used as a nuclear control, and co-transformed Arabidopsis protoplast cells. Laser confocal microscope was used to image Arabidopsis protoplast cells.

[0137] Experimental results: as shown in Figure 9 The results show that yellow fluorescence is observed in the nucleus of GmMPK4-nYFP and GmICE2-cYFP co-transformed Arabidopsis protoplast cells, while no yellow fluorescence is observed in the control, indicating that GmMPK4 and GmICE2 proteins interact in the plant nucleus.

[0138] 1.3 Co-IP interaction analysis of GmMPK4 and GmICE2

[0139] Further, the interaction between GmMPK4 and GmICE2 in plants was verified by co-IP, and the specific operation was as follows: first, GmMPK4-Flag and GFP-GmICE2 two recombinant plasmids were transformed into GV3101 Agrobacterium (carrying pSoup-p19 helper plasmid) respectively; then, the Agrobacterium carrying pCAMBIA1300-GmMPK4-Flag and pBin-GFP-GmICE2 recombinant plasmids were mixed, and co-transfected into tobacco leaves by injection for co-IP analysis.

[0140] The experimental results are shown in Table 1. Figure 10 As shown in Table 1, when GmMPK4-Flag and GmICE2-GFP proteins coexist, obvious positive bands can be seen, and when GmMPK4-Flag and GFP proteins coexist, no band is detected. This result shows that GmMPK4 interacts with GmICE2 in plants.

[0141] Example 8 In vitro and in vivo phosphorylation analysis of GmMPK4 and GmICE2

[0142] 1.1 In vitro phosphorylation of GmICE2 by GmMPK4

[0143] GmMPK4 directly interacts with GmICE2, and to further study whether GmICE2 is a direct phosphorylation substrate of GmMPK4, in vitro phosphorylation analysis was performed. First, it was found by PPSP system analysis that GmICE2 has 6 MAPK phosphorylation sites. The protein phosphorylation site was mutated to alanine (A), and the protein will lose the function of being phosphorylated by MAPK kinase. In this study, the 6 conserved Thr and Ser of GmICE2 6A (T72A / T149A / 296A / 303A / 314A / 335A) were mutated to GmICE2 6A mutant was constructed into the PET29b prokaryotic expression vector, and the results showed that the 6 conserved threonine and serine of GmICE2 were successfully mutated to alanine.

[0144] Studies have shown that GmMKK2 can phosphorylate GmMPK4 in vitro, and in this study, GmMKK2DD with continuous phosphorylation was constructed into a PGEX4T-1 prokaryotic expression vector containing a GST tag, which activated GmMPK4 as an upstream kinase of GmMPK4. Further, GmICE2 6A -His and GmMKK2 DD -GST were transformed into T7 Express lysY competent cells, and the proteins were induced with 0.5mM IPTG. The induced GmICE2 6A-His with GmMKK2 DD -GST protein was used for subsequent experiments. Then, an in vitro phosphorylation assay was performed using Phos-tag PAGE.

[0145] Experimental results: such as Figure 11 As shown. By Figure 11 It can be seen that only GST empty carrier (lane 1) or GmMKK2 DD When GST (lane 9) is present, GmICE2-His shows no phosphorylation signal, indicating that the empty GST vector or GmMKK2 is present. DD -GST cannot directly phosphorylate GmICE2-His; although GmMPK4 can directly phosphorylate GmICE2 (lane 4) (the gray value of the phosphorylated protein during migration is 0.43), GmMKK2... DD The addition of GST significantly enhanced the phosphorylation signal of GmMPK4 (lane 6) on GmICE2 (the gray value of the migrating phosphorylated protein was 0.75), indicating that GmMKK2... DD Activation of GmMPK4 can enhance the phosphorylation signaling of GmICE2; however, GmMKK2 DD -GmMPK4 cascade can weakly phosphorylate GmICE2 6A (The grayscale value of the migrated phosphorylated protein is 0.21), indicating that GmMKK2 is activated. DD -GmMPK4 cascades can still phosphorylate other sites besides these 6 phosphorylation sites (lane 7).

[0146] Based on these results, GmICE2 is a phosphorylation substrate downstream of the GmMKK2-GmMPK4 cascade, and six conserved threonine and serine residues in GmICE2 (Ser72, Ser149, Thr296, Thr303, Thr314, and Ser335) are key phosphorylation sites.

[0147] 1.2 GmMPK4 phosphorylation of GmICE2 in vivo

[0148] To verify whether GmMPK4 phosphorylates GmICE2 in vivo, GmICE2-Myc was expressed alone or co-expressed with GmMPK4-Flag in the hairy roots of uninfected and Phytophthora soybean. 6A -Myc and GmMPK4 K68,69R -Flag (kinase inactivation mutant of GmMPK4: 68th and 69th lysine of GmMPK4 are mutated to arginine) is used as a negative control.

[0149] Western blot results are as followsFigure 12 As shown, the results indicated that when the transgenic hairy roots were not inoculated with *Phytophthora sojae*, phosphorylation of GmICE2 was detected when GmICE2 and GmMPK4 were co-expressed (gray value of migrated phosphorylated protein was 0.47). After inoculation with *Phytophthora sojae*, both expression of GmICE2 alone and co-expression of GmICE2 and GmMPK4 showed phosphorylation signals, but the phosphorylation signal when GmICE2 and GmMPK4 were co-expressed (gray value of migrated phosphorylated protein was 0.73) was significantly stronger than that when GmICE2 was expressed alone (gray value of migrated phosphorylated protein was 0.49). The phosphorylation signal of GmICE2 and GmMPK4 co-expression after inoculation with *Phytophthora sojae* was significantly stronger than that in the uninoculated roots. These results indicate that GmMPK4 can phosphorylate GmICE2 in vivo, and that *Phytophthora sojae* infection can enhance the in vivo phosphorylation of GmICE2 by GmMPK4.

[0150] Example 9: GmMPK4 phosphorylation of GmICE2 promotes the ubiquitination and degradation of GmICE2.

[0151] 1.1 GmMPK4 phosphorylation of GmICE2 reduces the protein stability of GmICE2.

[0152] To investigate whether phosphorylation affects the stability of the GmICE2 protein, GmICE2-Myc or GmICE26A-Myc were expressed alone in Arabidopsis protoplasts with or without the proteasome inhibitor MG132; or GmMPK4-Flag and GmICE2-Myc were co-expressed, or GmMPK4 was co-expressed. K68,69R -Flag and GmICE2-Myc, co-expression of GmMPK4-Flag and GmICE2 6A -Myc, co-expression of GmMPK4 K68,69R -Flag and GmICE2 6A -Myc. After incubation for 4 hours, total protein was extracted.

[0153] Western blot results are as follows Figure 13 As shown, the results indicate that compared to GmICE2-Myc, GmICE2... 6A -Myc stability was significantly enhanced; when co-expressed with GmMPK4, the stability of GmICE2-Myc was significantly reduced compared with other combinations. The addition of MG132 increased the protein abundance of GmICE2, but did not increase the abundance of the internal control protein. These results indicate that phosphorylation of GmMPK4 can significantly reduce the stability of GmICE2 protein.

[0154] 1.2 Phosphorylated GmICE2 promotes its degradation via the 26S proteasome pathway.

[0155] Total protein was extracted from wild type soybean, and the extracted total protein was incubated with prokaryotic induced expression of GmICE2-His or GmICE2 6A -His protein under 22℃ water bath condition, and samples were taken at different incubation time points (0, 0.5, 1 and 3h), and the sampled protein was subjected to western blot analysis, with Actin as a control. 26S proteasome inhibitor MG132 was added, and the ubiquitination degradation of GmICE2-His and GmICE2 6A -His was analyzed at the determined degradation time point of 3h.

[0156] The experimental results are shown in Table 1. Figure 14 Table 1: The experimental results are shown in Table 1. The results show that GmICE2-His or GmICE2 6A -His recombinant protein was significantly degraded at 3h of incubation, however, when 26S proteasome inhibitor MG132 was added to the incubation system, and both were incubated for 3h, the degradation was significantly inhibited. The above experimental results reveal that GmICE2 protein is degraded by ubiquitination modification, and this ubiquitination degradation is carried out through the 26S proteasome pathway. It can be seen from the protein gray value analysis that the degradation degree of GmICE2 6A at different time points is lower than that of GmICE2, indicating that phosphorylation can enhance the protein degradation of GmICE2.

[0157] 1.3 Phosphorylation promotes the in vivo degradation of GmICE2 in soybean

[0158] GmICE2 6A -Myc and GmICE2-Myc recombinant vectors were transformed into K599 Agrobacterium by ice melting method and transformed into soybean hairy roots, and after treatment with 26S proteasome inhibitor MG132 and protein synthesis inhibitor CHX at room temperature for 1h, further analysis of GmICE2 6A -Myc (non-phosphorylated GmICE2) and GmICE2-Myc transgenic soybean hairy roots.

[0159] The experimental results are shown in Table 1. Figure 15 In the presence of protein synthesis inhibitor (CHX), the protein level of GmICE2-Myc in GmICE2-Myc transgenic soybean hairy roots was greatly reduced, while the protein level of GmICE2 6A -Myc in GmICE2-Myc transgenic soybean hairy roots changed little. These results show that phosphorylation promotes the in vivo degradation of GmICE2.

[0160] 1.4 GmMPK4 promotes the in vitro ubiquitination degradation of GmICE2

[0161] Total proteins were extracted from WT, GmMPK4-OE and GmMPK4-RNAi transgenic soybean hairy roots, and total proteins of WT, GmMPK4-OE and GmMPK4-RNAi transgenic soybean hairy roots were treated with 120 μM MG132 or DMSO for 1 h. The treated WT, GmMPK4-OE or GmMPK4-RNAi proteins were incubated with GmICE2-His proteins at 22 °C water bath condition, and samples were taken at 0 h, 0.5 h, 1 h and 3 h, and the sampled proteins were subjected to western blot analysis. Actin was used as a control, Anti-His was used to detect the protein expression of GmICE2, and the immunoblot images were quantified and analyzed for protein relative abundance using Image J software.

[0162] The experimental results are shown in Table 1. Figure 16 The results show that overexpression of GmMPK4 (GmMPK4-OE) significantly enhances the degradation of GmICE2 protein compared with control EV transgenic soybean hairy roots, and interference of GmMPK4 gene (GmMPK4-RNAi) significantly inhibits the degradation process of GmICE2 protein. Further experiments show that when 26S proteasome inhibitor MG132 is added to the co-incubation system of GmMPK4-OE, EV or GmMPK4-RNAi and GmICE2-His protein, the degradation rate of GmICE2-His protein is significantly reduced. The above results collectively reveal that GmMPK4 can promote the ubiquitination degradation of GmICE2, and this promotion is achieved through a 26S proteasome-dependent pathway.

[0163] Example 10 Screening of GmICE2 downstream target genes

[0164] 1.1 RNA-Seq analysis of GmICE2

[0165] To further explore the mechanism of GmICE2 in soybean disease resistance, the present study performed RNA-Seq analysis on transgenic soybean hairy roots overexpressing GmICE2 (GmICE2-OE) and control soybean hairy roots containing only empty vector (EV) to identify potential target genes regulated by GmICE2.

[0166] The experimental results are shown in Table 1. Figure 20 Based on the RNA-Seq data, the present study identified a total of 2377 differentially expressed genes (fold change > 2, false discovery rate < 0.01), including 1613 up-regulated genes and 764 down-regulated genes.

[0167] As shown in Table 2. Figure 21The KEGG classification annotation results showed that these genes were widely involved in important biological processes such as plant-pathogen interaction, plant hormone signal transduction, phenylpropanoid metabolism, MAPK cascade, isoflavone and flavonoid biosynthesis. The above results showed that the expression level of GmICE2 in soybean could significantly affect the expression of genes related to stress response, and it might play a key role in these physiological processes.

[0168] 1.2GmICE2 directly binds to the promoter region of GmPR1

[0169] The reporter vector 35S:GmICE2 and EV empty vector (purchased from Shanghai Novozyme Biotechnology Co., Ltd.) and the effector vector pGmPR1-LUC, pGmPR1-LUC(Mut) (CAGTTG (pGmPR1) motif at position-473bp was mutated to CCGTCG) were transformed into N. benthamiana leaves (stored in the Key Laboratory of Soybean Biology and Education, Northeast Agricultural University), and the specific transformation steps were as follows:

[0170] In the clean bench, 50 μL of recombinant reaction liquid containing pGmPR1-LUC, pGmPR1-LUC(Mut) obtained in Example 2 was added to 50 mL YEP medium (purchased from Shanghai Novozyme Biotechnology Co., Ltd.), and cultured overnight at 28°C, 220 rpm constant temperature shaker, and the OD 600 = 0.6 of the secondary expansion bacterial liquid was obtained. The secondary expansion bacterial liquid was transferred to a 50 mL EP tube, centrifuged at 8,000 x g for 10 min, and the supernatant was discarded; 10 mL of Agrobacterium infection solution (10 mM MES, 150 mM acetosyringone, 10 mM MgCl2) was used to resuspend the bacterial body and adjust the OD 600 = 0.8, and equilibrated at 25°C in the dark for 4 h. Tobacco plants growing to 6-7 leaves were selected, and Agrobacterium containing the corresponding vector was co-injected into the tobacco leaves to obtain leaves containing 35S:GmICE2+pGmPR1-LUC, 35S:GmICE2+pGmPR1-LUC(Mut) and EV+pGmPR1-LUC, respectively. The tobacco plants were placed in the dark at room temperature for 3 days and then imaged in the chemiluminescence imaging system.

[0171] The experimental results are shown in Table 1. Figure 22 (quantitative PCR determination) showed that GmICE2 could activate the expression of GmPR1 gene by binding to the E-box on the GmPR1 promoter.

[0172] Example 11 Application Test

[0173] 1.1 Preparation of GmMPK4 and GmICE2 transgenic plants

[0174] Formulation germination medium (pH 5.8): 1.0 mg of 6BA, 3.21 g of B5 mixed salt, 20 g of sucrose and 8 g of agar, co-culture medium (pH 5.4): 0.31 g of B5 mixed salt, 3.9 g of MES, 30 g of sucrose and 8 g of agar, recovery medium (pH 5.8): 3.1 g of B5 mixed salt, 0.6 g of MES, 30 g of sucrose and 8 g of agar, elongation medium (pH 5.8): 4.43 g of MS mixed salt, 0.6 g of MES, 30 g of sucrose and 8 g of agar, rooting medium (pH 5.8): 4.43 g of MS mixed salt, 0.6 g of MES, 20 g of sucrose and 8 g of agar.

[0175] Select the growth of healthy, full, uniform size "Dongnong 50" soybean seeds, chlorine disinfection overnight. In the clean bench with high temperature sterilization of the tweezers after disinfection of soybean seeds in the germination medium, and placed in the light culture rack under room temperature light culture 5d.

[0176] Meanwhile, 100 μL of p35S:GmMPK4 containing Agrobacterium 4404 competent cells and p35S:GmICE2, GmICE2-RNAi recombinant plasmid containing Agrobacterium 4404 competent cells were taken into 10 mL of YEP medium (containing 50 mg / L Kana, purchased from Shanghai Novozyme Biotechnology Co., Ltd.), 28℃, 220 rpm constant temperature culture overnight. In the clean bench, 800 μL of p35S:GmMPK4 and p35S:GmICE2, GmICE2-RNAi recombinant plasmid containing 4404 Agrobacterium was taken for bacterial liquid expansion, twice inoculation activation to OD 600 = 0.6, 8,000 × g centrifugation for 15 min, discard the supernatant, and resuspend the activated p35S:GmMPK4 and p35S:GmICE2, GmICE2-RNAi recombinant plasmid containing 4404 Agrobacterium with Agrobacterium infection solution.

[0177] In the clean bench, the lower hypocotyl of the soybean cultured for 5d was removed with a high temperature sterilized surgical knife, and the 4404 Agrobacterium liquid containing p35S:GmMPK4 and p35S:GmICE2, GmICE2-RNAi recombinant plasmid was drawn from the center of the cotyledon to the growth point, and the cut soybean was immersed in the suspended 4404 Agrobacterium liquid containing p35S:GmMPK4 and p35S:GmICE2, GmICE2-RNAi recombinant plasmid for 30 min, and then the cotyledon was evenly arranged on the prepared co-culture medium. After 4d of light culture, the soybean was transferred to the recovery medium, and then sequentially transferred to the elongation and rooting medium for subsequent culture after the callus grew. GmMPK4-OE transgenic plants, GmICE2-OE transgenic plants and GmICE2-RNAi transgenic plants were obtained.

[0178] 1.2 Preparation of GmMPK4, GmICE2, GmICE2 6A , GmICE2 6D , GmPR1 transgenic soybean hairy roots

[0179] To construct the transgenic hairy roots carrying GmMPK4, GmICE2, GmICE2 6A , GmICE2 6D , GmPR1 genes, the soybean hairy root-specific optimized medium formula is prepared as follows:

[0180] (1) Preparation of the medium

[0181] Soybean growth medium (1L): Accurately weigh 20g sucrose, 3.21g B5 salt and 0.6g MES, and adjust the pH value of the mixture to 5.8. Then, add 8g agar for sealing and sterilization treatment. In a sterile operation table, add an appropriate amount of 6-BA plant hormone to the prepared medium, mix thoroughly, and then dispense for use.

[0182] Root induction medium (1L): Accurately measure 30g sucrose, 2.215g 1 / 2MS salt and 0.6g MES, mix and adjust the pH value to 5.8. Then, seal and sterilize 8g of agar. In a sterile operation table, add 1000mg cefotaxime and 500mg carbenicillin disodium to the treated medium, mix thoroughly, and then dispense for use.

[0183] (2) Culture of transgenic soybean hairy roots

[0184] Select healthy and full soybean seeds without disease traces, and perform 16h chloroform disinfection treatment. The disinfected seeds are placed in the previously prepared soybean growth medium and cultured for one week under natural environmental conditions. When the soybean seedlings develop to a fresh green color and the seed coat is tightly covered, the seedlings are removed and the embryo of the cotyledon part is accurately cut off. Then, an appropriate wound for bacteria inoculation is created on the back of the cotyledon.

[0185] The recombinant vector construction and transformed bacteria liquid need to be expanded and cultured until the OD 600 value reaches 0.8. At this time, the bacteria liquid is centrifuged using a high-speed centrifugal device to collect the bacteria. Then, the bacteria precipitate is resuspended using an AS-added MgCl2 solution. The resuspended p35S:GmMPK4, GmMPK4-RNAi, p35S:GmICE2, p35S:GmICE2 6A , p35S:GmICE2 6D, GmICE2-RNAi, p35S:GmPR1-Myc, GmPR1-RNAi bacterial solution is accurately applied to the previously created cotyledon wound site, then sealed, and finally transferred to a special soybean light culture room for culture for about three weeks to obtain GmMPK4-OE transgenic soybean hairy roots, GmMPK4-RNAi transgenic soybean hairy roots, GmICE2-OE transgenic soybean hairy roots, GmICE2 6A -OE transgenic soybean hairy roots, GmICE2 6D -OE transgenic soybean hairy roots, GmICE2-RNAi transgenic soybean hairy roots, GmPR1-OE transgenic soybean hairy roots, GmPR1-RNAi transgenic soybean hairy roots.

[0186] 1.3 Detection of GmMPK4, GmICE2, GmPR1 transgenic soybean and soybean hairy roots

[0187] 1.3.1 The ability of GmICE2 to directly bind to the GmPR1 promoter is analyzed by ChIP experiments. Specifically, 1 g of fresh GmICE2-OE transgenic plants and control Dongnong 50 plants (Dongnong 50 seeds are stored in the Key Laboratory of Soybean Biology of the Ministry of Education, Northeast Agricultural University) are collected, cut into small pieces of about 5 mm with scissors, and then placed in 50 mL centrifuge tubes. Add 37 mL MC buffer containing 1% formaldehyde, place in ice, vacuum pump to vacuum infiltration fixation of samples for 30 min, add 0.41662 g glycine powder, terminate the reaction, place the centrifuge tube in the drum, rotate at 4°C for 20 min, discard the MC buffer, add new MC buffer for washing, and obtain the pretreated sample.

[0188] M1 buffer, M2 buffer and M3 buffer were prepared. The pretreated sample was drained into a mortar and ground in liquid nitrogen for 30 min, pre-cooled M1 buffer was added and the sample was transferred to a 2 mL centrifuge tube, centrifuged at 14000 rpm for 5 min at 4°C, the precipitate was resuspended with pre-cooled M2 buffer, centrifuged at 14000 rpm for 5 min at 4°C, the supernatant was removed, and the above steps were repeated 5-10 times; an appropriate amount of M3 buffer was added to repeatedly pipette the precipitate, and the centrifuge tube was placed in a low-temperature refrigerated centrifuge and centrifuged for 6 min, and the above steps were repeated 6 times. An appropriate amount of ultrasonic buffer was added to repeatedly pipette the precipitate, and the precipitate was placed in an ultrasonic crusher and ultrasonically treated for 20 min, and then transferred to a low-temperature refrigerated centrifuge and centrifuged at 14000 rpm for 10 min, and after centrifugation, the supernatant was transferred to a new centrifuge tube, 75 μL of the supernatant was taken as an Input sample and placed in a centrifuge tube, 30 μL of Anti-Myc agarose beads was added, the centrifuge tube was placed in a rotating drum and rotated overnight at 4°C, centrifuged at 2500 rpm for 5 min at 4°C, and the sample was collected, the agarose beads were gently resuspended with 1 mL of high-salt buffer, the centrifuge tube was placed in a rotating drum and rotated for 8 min at 4°C, and the above steps were repeated 4 times; the agarose beads were gently resuspended with 1 mL of IP buffer, the centrifuge tube was placed in a rotating drum and rotated for 8 min at 4°C, and the above steps were repeated 4 times, centrifuged at 2500 rpm for 5 min at 4°C, and the supernatant was discarded, 200 μL of elution buffer was taken with a pipette and added to the agarose beads and mixed well to elute, the centrifuge tube was placed in a metal bath and incubated at 65°C for 15 min, centrifuged at 13000 rpm for 5 min, and the supernatant was transferred to a new 1.5 mL centrifuge tube. The elution was repeated twice. The collected elution sample was centrifuged at 13000 rpm for 5 min, and the supernatant was used for subsequent DNA analysis.

[0189] An appropriate amount of 5M sodium chloride was taken with a pipette and added to the supernatant, the centrifuge tube was placed in a metal bath and incubated at 65°C overnight, an appropriate amount of Rnase A was added, the centrifuge tube was placed in a water bath and incubated at 37°C for 25 min, an appropriate amount of Proteinase K was added, the centrifuge tube was placed in a water bath and incubated at 42°C for 2 h, an appropriate amount of isopropyl alcohol and chloroform was added, the centrifuge tube was placed in a centrifuge and centrifuged at 14000 rpm at room temperature for 8 min. The isopropyl alcohol was pre-cooled, the supernatant was taken with a pipette and added to a new centrifuge tube, an equal volume of pre-cooled isopropyl alcohol was added, mixed well, and placed at -20°C for 2 h, centrifuged at 14000 rpm at room temperature for 15 min, the supernatant was discarded, and 40 μL of water was used to elute the DNA for subsequent qRT-PCR detection.

[0190] The experimental results are shown in Table 1. Figure 23 The results show that GmICE2 can directly bind to GmPR1.

[0191] The transcription levels of the down-regulated differentially expressed genes related to stress response in the GmICE2-OE and GmICE2-RNAi transgenic plants compared with the WT plants (the Dongnong 50 plants without GmICE2) were detected by qRT-PCR technology. The specific primer sequences are shown in Table 5.

[0192] Table 5 Primer sequences of stress-related genes in the GmICE2 transcriptome

[0193]

[0194]

[0195] The experimental results are shown in Table 6. Figure 24 The results show that the expression amount of GmPR1 in the GmICE2-OE transgenic plants is the highest compared with the rest of the genes, and the down-regulation multiple in the GmICE2-RNAi transgenic plants is the lowest.

[0196] 1.3.1 The ability of GmMPK4 phosphorylation to affect the binding of GmICE2 to the GmPR1 promoter was analyzed by LUC test, and the specific steps were as follows: the reporter p35S:GmICE2 6A -Myc, p35S:GmICE2 6D -Myc, p35S:Flag-GmMPK4-p35S:GmICE2-Myc (double original expression vector), p35S:GmMPK4-p35S:GmICE2 6A -Myc and the EV empty vector (purchased from Shanghai Novozyme Biotechnology Co., Ltd.) and the effector vector pGmPR1-LUC were respectively transformed into N. benthamiana leaves (stored in the Key Laboratory of Soybean Biology and Education, Northeast Agricultural University), and the specific transformation steps were as follows:

[0197] In the super-clean workbench, 50 μL of the recombinant reaction liquid containing pGmPR1-LUC obtained in Example 2 was added to 50 mL of YEP medium (purchased from Shanghai Novozyme Biotechnology Co., Ltd.), and cultured overnight at 28°C, 220 rpm constant temperature shaker, to obtain secondary expansion bacterial liquid with OD 600 = 0.6. The secondary expansion bacterial liquid was transferred to a 50 mL EP tube, centrifuged at 8,000 x g for 10 min, and the supernatant was discarded; 10 mL of Agrobacterium infection liquid (10 mM MES, 150 mM acetosyringone, 10 mM MgCl2) was used to resuspend the bacterial body and adjust the OD 600= 0.8, 25℃, light avoidance, 4h. Select tobacco plants growing to 6-7 leaves, co-inject Agrobacterium of the corresponding vector into tobacco leaves, and obtain leaves containing pGmPR1-LUC reporter plasmid and EV empty effect plasmid, pGmPR1-LUC reporter plasmid and p35S:GmICE2-Myc effect plasmid, pGmPR1-LUC reporter plasmid and p35S:Flag-GmMPK4-p35S:GmICE2-Myc effect plasmid, pGmPR1(Mut)-LUC reporter plasmid and p35S:Flag-GmICE2, respectively; obtain leaves containing reporter plasmid pGmPR1-LUC and empty effect plasmid EV, reporter plasmid pGmPR1-LUC and effect plasmid p35S:GmICE2-Myc, reporter plasmid pGmPR1-LUC and effect plasmid p35S:GmICE2 6D -Myc, reporter plasmid pGmPR1-LUC and effect plasmid Flag-GmICE2, respectively; obtain leaves containing reporter plasmid pGmPR1-LUC and empty effect plasmid EV, reporter plasmid pGmPR1-LUC and effect plasmid GmICE2 6A -Myc, reporter plasmid pGmPR1-LUC and effect plasmid p35S:Flag-GmMPK4-p35S:GmICE2 6A -Myc (double original expression vector), reporter plasmid pGmPR1(Mut)-LUC and effect plasmid p35S:Flag-GmICE2 6A Place the tobacco plants in dark at room temperature for 3d, and then place them in a chemiluminescence imaging system for imaging.

[0198] Experimental results: as shown in Figure 25 to Figure 27 The results show that GmICE2 can directly bind to pGmPR1, thereby activating its expression.

[0199] 1.3.2 Detection of GmMPK4, GmICE2, GmPR1 overexpressing transgenic soybean plants and soybean hairy roots

[0200] Extract the proteins of GmMPK4-OE, GmICE2-OE, GmPR1-OE plants obtained in step 1.1, and perform western blot detection with Anti-bar as the antibody (purchased from Shanghai Novicure Biotechnology Co., Ltd.). Use qRT-PCR to detect the expression amount of GmMPK4 in GmMPK4-OE plants, the expression amount of GmICE2 in GmICE2-OE plants, and the expression amount of GmPR1 in GmPR1-OE roots.

[0201] 1.3.3 Detection of GmMPK4, GmICE2, GmPR1 interfering transgenic soybean plants and soybean hairy roots

[0202] DNA was extracted from GmMPK4-RNAi, GmICE2-RNAi, GmPR1-RNAi soybean plants obtained in step 1.1, and the exogenous bar gene was detected in the GmICE2-RNAi soybean plants. The expression levels of GmMPK4 in GmMPK4-RNAi plants, GmICE2 in GmICE2-RNAi plants, and GmPR1 in GmPR1-RNAi plants were detected by qRT-PCR.

[0203] 1.3.4 Resistance analysis of GmMPK4, GmICE2, and GmPR1 transgenic soybean plants and soybean hairy roots

[0204] Soybean seedlings with consistent growth status and length were selected, and the cotyledon and the root were inoculated with fungus, respectively. After 48 h of inoculation, the plants were observed and photographed by a Nikon D7000 camera. Then the samples were quickly frozen in liquid nitrogen, and the RNA of the transgenic plants was extracted. The biomass accumulation of the fungus was detected by qRT-PCR, and the lesion area was calculated by ImageJ software (https: / / imagej.nih.gov / ij / index.html).

[0205] The experimental results are shown in Table 1. Figure 28 to Figure 42 As shown in Table 1, GmMPK4 is a negative regulatory factor in response to P. sojae infection, GmICE2 is a positive regulatory factor in response to P. sojae infection, GmMPK4 phosphorylation of GmICE2 can weaken the resistance of GmICE2 to P. sojae, and GmPR1 is a positive regulatory factor in response to P. sojae infection.

[0206] Based on the above experimental results, the protein GmICE2 that interacts with GmMPK4 was identified by screening the interacting proteins of the negative regulatory factor GmMPK4 of P. sojae. The phosphorylation test results further showed that GmMPK4 can phosphorylate and degrade GmICE2. Overexpression of the GmMPK4 gene can improve the sensitivity of soybean to P. sojae, mainly in the form of GmMPK4 disease resistance phenotype identification, phosphorylation regulation of GmICE2, and other corresponding characteristics. The downstream target gene GmPR1 directly regulated by GmICE2 was screened by transcriptome sequencing of the positive regulatory factor GmICE2 of P. sojae. It was found that overexpression of the GmICE2 gene can improve the resistance of soybean to P. sojae, mainly in the form of GmICE2 disease resistance phenotype identification, regulation of the disease resistance target gene GmPR1, and other corresponding characteristics. It is indicated that the soybean GmMPK4 gene may play an important role in stress through post-translational modification of proteins.

[0207] The above merely describes the preferred embodiments of the present application, and it should be pointed out that, for those skilled in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as falling within the protection scope of the present application.

Claims

1. Overexpression of soybean GmICE2 The application of genes in improving crop resistance to soybean Phytophthora root rot is characterized by, The GmICE2 The nucleotide sequence of the gene is shown in SEQ ID NO.

3. The soybean root rot caused by *Phytophthora sojae* is a fungus. Phytophthora sojae Caused by Kaufmann & Gerdemann.

2. The application according to claim 1, characterized in that, The soybean GmICE2 Genes enhance disease resistance pathways in crops GmPR1 The transcription, the GmPR1 The nucleotide sequence of the gene is shown in SEQ ID NO.

5.

3. The application according to claim 1 or 2, characterized in that, Includes the following steps: 1) The pCAMBIA3301 overexpression vector was linearized to obtain the linearized expression vector pCAMBIA3301; 2) The soybeans GmICE2 The gene was ligated into the pEasy-Blunt vector, then transformed into competent E. coli cells for propagation, yielding pEasy-Blunt- GmICE2 ; 3) Obtain pEasy-Blunt- GmICE2 Using soybean as a template, specific primers with vector terminal sequences at both ends were designed, and the soybean was amplified by PCR. GmICE2 The target segment of the gene; 4) The target fragment is inserted into the linearized expression vector pCAMBIA3301 to obtain soybean overexpression. GmICE2 Recombinant gene expression vectors.

4. The application according to claim 3, characterized in that, It also includes overexpressing the soybean GmICE2 The recombinant expression vector of the gene was introduced into Escherichia coli; the Escherichia coli was Trans1-T1 competent cells.

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