Genes gmpmi1 and gmpmi1r for improving soybean resistance to soybean rust and application thereof

Abstract

The application discloses genes GmPMI1 and GmPMI1R for improving plant resistance to soybean blight and application thereof. The nucleotide sequences of the genes GmPMI1 and GmPMI1R are shown in SEQ ID NO. 1 or SEQ ID NO. 3 respectively. The protein encoded by the genes GmPMI1 and GmPMI1R in the application exerts an anti-disease effect by being secreted to the extracellular, thereby enhancing the plant disease resistance, especially the disease resistance to the Pythium, and the plant disease resistance to the Pythium can be significantly enhanced. The application can be applied to crop breeding and disease resistance improvement, and is expected to improve the plant disease resistance to the blight, and further promote the reduction of pesticide and fertilizer application and the increase of efficiency.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular biology and plant genetic engineering. Specifically, this invention relates to a gene GmPMI1 and GmPMI1R that enhances plant disease resistance and their applications. Background Technology

[0002] Phytophthora, belonging to the phylum Oomycetes, encompasses many pathogens capable of causing devastating plant diseases, posing a significant threat to global agricultural economies and ecosystems. While morphologically similar to fungi, oomycetes differ considerably in cell wall composition, biochemical metabolism, and reproductive methods. Systematically, they belong to different biological kingdoms. Therefore, fungicides and other methods used for fungal control are often ineffective against oomycete diseases. Furthermore, oomycetes are diploid organisms with complex and rapidly evolving genomes; the continuous emergence and development of new races of Phytophthora often leads to the loss of soybean disease resistance. Therefore, elucidating the broad-spectrum pathogenic mechanism of Phytophthora and using the core pathogenic factors as probes to discover broad-spectrum disease resistance genes in plants is of significant value for discovering new molecular breeding resources and improving the broad-spectrum and durable disease resistance of crops.

[0003] Plant cell walls serve as the first line of defense against pathogens. A multi-layered "arms race" has ensued between plants and pathogens around the plant cell wall, resulting in the evolution of a series of defensive and virulence factors. In the early stages of pathogen infection, pathogens secrete a series of cell wall hydrolytic enzymes that degrade the plant cell wall, promoting infection. For example, the apothecium effector PsXEG1 in soybean hydrolyzes hemicellulose in the plant cell wall in the early stages of infection, promoting *Phytophthora infestans* infection (Ma, et al. 2015; Ma, et al. 2017); the apothecium effector PiAA17s in pathogenic *Phytophthora infestans* can cleave pectin in the plant cell wall, thereby disrupting its integrity (Sabbadim, et al. 2021). Pectin, present in the middle lamella of adjacent plant cell walls, plays a role in binding cells together and is one of the main components of the plant cell wall. Plants regulate different growth and development processes by dynamically adjusting the degree of methyl esterification of cell wall pectin. For example, in Arabidopsis thaliana, the balance of methyl esterification of root cell wall pectin participates in regulating the normal function of the root clock and the production of lateral root primordia (Wachsman, et al. 2020). Demethylated pectin can interact with the LRX8-RALF4 complex in a charge-dependent manner, and the resulting complex plays an important role in maintaining the integrity and expansion of pollen tube cell walls (Moussu, et al. 2023). Recent studies have shown that the dynamic regulation of pectin methyl esterification is widely involved in plant-pathogen interactions. For instance, the effector PfAvr4-2 of *Phytophthora indicum* interacts with demethylated pectin in plant cell walls, thereby affecting the cross-linking of pectin with calcium ions, loosening the cell wall structure, and promoting infection (Chen, et al. 2021). The effectors of the AA17 family of *Phytophthora indicum* can only recognize and degrade demethylated pectin (Sabbadim, et al. 2021). These findings all indicate that in the "arms race" between pathogens and hosts, pectin demethylation has gradually become the main rate-limiting step in pectin degradation by pathogens. Therefore, identifying plant apoplastic pectin methylation repressor genes is of great significance for improving broad-spectrum resistance in crops. Summary of the Invention

[0004] The purpose of this invention is to provide a gene GmPMI1 and GmPMI1R for improving plant disease resistance, the protein they encode, and their applications.

[0005] The objective of this invention can be achieved through the following technical solutions:

[0006] In a first aspect, the present invention seeks protection for a DNA molecule as follows (a1) or (a2) or (a3):

[0007] (a1) A DNA molecule having a nucleotide sequence as shown in SEQ ID NO.1 or SEQ ID NO.3;

[0008] (a2) A DNA molecule that hybridizes with the DNA molecule described in (a1) under stringent conditions and encodes the same protein;

[0009] (a3) A DNA molecule that has 99%, 95%, 90%, 85% or more or 80% homology with the DNA sequence described in (a1) or (a2) and encodes the same protein.

[0010] Secondly, the present invention seeks protection for the protein encoded by the aforementioned DNA molecule.

[0011] Furthermore, the aforementioned proteins are (b1) or (b2) or (b3) or (b4) as follows:

[0012] (b1) A protein having an amino acid sequence as shown in SEQ ID NO.2 or SEQ ID NO.4;

[0013] (b2) A protein having the same function by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence described in (b1);

[0014] (b3) A protein that has 99%, 95%, 90%, 85%, or 80% homology with the amino acid sequence described in (b1) or (b2) and has the same function;

[0015] (b4) Fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of (b1) or (b2) or (b3).

[0016] This invention uses soybean genome as a template to design primers for amplification to obtain the full-length gene of GmPMI1 (as shown in SEQ ID NO.1), and GmPMIR (as shown in SEQ ID NO.2), which is a modified sequence of GmPMII, synthesized artificially. The amino acid sequence of the protein encoded by the GmPMI1 gene is shown in SEQ ID NO.2, and the amino acid sequence of the protein encoded by the GmPMIR gene is shown in SEQ ID NO.4.

[0017] Thirdly, the present invention seeks protection for biological materials containing the aforementioned DNA molecules, wherein the biological material is at least one of the following (c1)-(c4):

[0018] (c1) An expression cassette containing the above-mentioned DNA molecules;

[0019] (c2) A recombinant vector containing the above-mentioned DNA molecules, or a recombinant vector containing the expression cassette described in (c1);

[0020] (c3) A non-plant cell line containing the DNA molecule of claim 1, or a non-plant cell line containing the expression cassette of (c1), or a non-plant cell line containing the recombinant vector of (c2);

[0021] (c4) A recombinant microorganism containing the DNA molecule of claim 1, or a recombinant microorganism containing the expression cassette of (c1), or a recombinant microorganism containing the recombinant vector of (c2).

[0022] In a specific embodiment of the present invention, the recombinant vector is a recombinant expression vector, which is obtained by inserting the genes GmPMI1 and GmPMI1R or codon-optimized genes into the binary vector pBin-eGFP restriction site SmaI containing C-terminal eGFP, resulting in vectors pBin-GmPMI1-eGFP and pBin-GmPMI1R-eGFP.

[0023] When constructing a recombinant expression vector using the above-described gene, any type of enhancing promoter or constitutive promoter can be added before its transcription initiation nucleotide; in addition, when constructing a recombinant expression vector using the gene of the present invention, enhancers, including translational enhancers or transcriptional enhancers, can also be used.

[0024] The recombinant microorganisms are obtained by introducing the recombinant expression vector into a host bacterium, preferably *Escherichia coli* or *Agrobacterium*. To facilitate the identification and screening of transgenic plant cells or plants, the recombinant expression vector can be processed, such as by adding genes encoding enzymes or luminescent compounds that can be expressed in plants, antibiotic resistance markers, or chemical resistance markers.

[0025] Fourthly, the present invention claims protection for primer pairs used to amplify the full length or any fragment of the aforementioned DNA molecule, wherein the primer pairs are primer pairs shown in SEQ ID No. 5 and SEQ ID No. 6 (for amplifying the aforementioned gene GmPMI1), or primer pairs shown in SEQ ID No. 7 and SEQ ID No. 8 (for amplifying the aforementioned gene GmPMI1R).

[0026] Fourthly, the present invention seeks protection for the use of at least one of the above-described DNA molecules, proteins, or biological materials in (d1)-(d4).

[0027] (d1) Enhance plant immunity or disease resistance;

[0028] (d2) To cultivate new plant varieties with enhanced immunity or disease resistance;

[0029] (d3) Increase crop yield;

[0030] (d4) Develop new crop varieties with increased yields.

[0031] Furthermore, the above applications include expressing or overexpressing the aforementioned DNA molecules in target plants or increasing the activity or content of the protein described in claim 2 or 3 to enhance the immune resistance or disease resistance of target plants, or to cultivate new plant varieties with enhanced immune resistance or disease resistance, or to increase crop yield or cultivate new crop varieties with increased yield.

[0032] Fifthly, the present invention claims protection for a method for improving plant immune resistance or disease resistance, which involves expressing or overexpressing the aforementioned DNA molecules in a target plant or increasing the activity or content of the protein described in claim 2 or 3 to improve the immune resistance or disease resistance of the target plant, or cultivating new plant varieties with improved immune resistance or disease resistance, or increasing crop yield or cultivating new crop varieties with increased yield.

[0033] Furthermore, the improvement of plant immune resistance or disease resistance described in this invention refers to enhancing the plant's immune resistance to pathogens or its resistance to diseases caused by pathogens. The pathogens described in this invention are those capable of infecting major food and economic crops, and can be oomycetes, fungi, or bacteria. Even further, the pathogens are those that easily cause plant diseases, such as Phytophthora, Fusarium, or Rice blast fungus.

[0034] The target plants are soybeans, tobacco, tomatoes, or potatoes.

[0035] Specifically, introducing the aforementioned gene GmPMI1 or GmPMI1R, the protein encoded by the aforementioned gene GmPMI1 or GmPMI1R, the codon-optimized GmPMI1 or GmPMI1R gene, or the aforementioned biological material into crops can result in crop varieties with significant disease resistance and / or increased yield. Preferably, introducing the gene into soybean, tobacco, tomato, or potato can result in crop varieties with enhanced disease resistance and / or increased yield.

[0036] This study analyzed soybean extracellular secretions and found that the pectin methyl esterase repressor GmPMI1 is involved in soybean resistance to Phytophthora spp. infection. Using AI-assisted design, GmPMI1R was created. The modified GmPMI1R specifically targets and inhibits pectin methyl esterase in Phytophthora spp. without interfering with the normal function of the plant's pectin methyl esterase. Inoculation of soybeans overexpressing GmPMI1 or GmPMI1R genes with Phytophthora spp. resulted in a significant reduction in Phytophthora spp. biomass. The GmPMI1 and GmPMI1R genes play a crucial role in soybean disease resistance. Research on GmPMI1 and GmPMI1R in soybeans can drive research on extracellular resistance proteins in many other plants, such as tomatoes and potatoes, and can provide excellent disease resistance gene resources for disease resistance genetic engineering breeding.

[0037] The beneficial effects of this invention are:

[0038] The proteins encoded by the genes GmPMI1 and GmPMI1R described in this invention exert their disease-resistant effects by being secreted extracellularly, thereby enhancing plant disease resistance, especially against Phytophthora infestans. They have a broad spectrum of resistance and can significantly enhance plant resistance to Phytophthora infestans. This invention can be applied to the improvement of crop disease resistance in breeding, and is expected to improve plant resistance to Phytophthora infestans, thereby promoting the reduction and efficiency of pesticide and fertilizer application. Attached Figure Description

[0039] Figure 1 Symptoms of disease observed in the hairy roots of pFGC::GFP::GmPMI1 / GmPMIR and pFGC::GFP transgenic soybeans after inoculation with Phytophthora soybeanis.

[0040] Figure 2 Microscopic observation and counting of the number of oospores after inoculation of hairy roots of pFGC::GFP(EV) and pFGC::GFP::GmPMI1 / GmPMIR transgenic soybeans with Phytophthora soybeanis.

[0041] Figure 3 Real-time quantitative PCR was used to detect the biomass of Phytophthora soybean after inoculating the hairy roots of pFGC::GFP(EV) and pFGC::GFP::GmPMI1 / GmPMIR transgenic soybeans with Phytophthora soybean.

[0042] Figure 4 Western blot was used to detect the expression levels of GmPMI1 / GmPMIR in the hairy roots of transgenic soybean. Detailed Implementation

[0043] The following embodiments are provided to better understand the present invention, but do not limit the invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments were purchased from conventional biochemical reagent stores. The primers involved in the embodiments of the present invention were provided by Nanjing Genscript Biotech Co., Ltd.

[0044] Example 1

[0045] 1) Preservation and culture of the tested plants and strains:

[0046] (1) The soybean seeds tested were Hefeng 47.

[0047] (2) Escherichia coli strain JM109 and Agrobacterium tumefaciens strain K599 were preserved in our laboratory.

[0048] (3) The soybean Phytophthora strain P6497 was a gift from Professor Brett M. Tyler (Oregon State University, USA) (the existing technology has been disclosed) and is preserved in our laboratory.

[0049] 2) Cloning of the GmPMI1 / GmPMI1R gene:

[0050] (1) Using soybean genome as template, the full-length GmPMI1 / GmPMIR gene was amplified using the GmPMI1 / GmPMIR gene primers F / R;

[0051] pFGC::GFP::GmPMI-F

[0052] ttacatttacaattaccATGAAACCCACTTTCCTCCTTCTC(SEQ ID No.5)

[0053] pFGC::GFP::GmPMI-R

[0054] ctctagactcacctaggatccTAAGTTATTAGCGAAACCGTTAACC(SEQ ID No.6)

[0055] pFGC::GFP::GmPMIR-F

[0056] ttacatttacaattaccATGAAACCCACTTTCCTCCTTCTC(SEQ ID No.7)

[0057] pFGC::GFP::GmPMIR--R

[0058] ctctagactcacctaggatccTAAGTTATTAGCGAAACCAGCAACC(SEQ ID No.8)

[0059] (2) The 50 μL reaction system is: 10 μL of 5× buffer, 4 μL of 2.5 mM dNTPs, 0.5 μL of Takara PrimerSTARTaq enzyme, 1 μL of template cDNA, and water added to 50 μL. The final concentration of the primers before and after is 10 pmol.

[0060] (3) The PCR amplification program was: 98℃ pre-denaturation for 3 min, 98℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 1 kb / 1 min, 32 cycles, and 72℃ extension for 10 min.

[0061] (4) Nucleic acids were separated by electrophoresis on a 1% agarose gel. After ethidium bromide (EB) staining, the gel was photographed under UV light, the results were recorded, and the PCR products of genes such as GmPMI1 / GmPMIR were recovered by gel excision. The electrophoretic bands were recovered using an Agarose Gel DNA Purification Kit (TaKaRa). Sequencing revealed that the nucleotide sequence of the GmPMI1 gene is shown in SEQ ID No. 1, and its encoded protein is shown in SEQ ID No. 2. The nucleotide sequence of the GmPMIR gene is shown in SEQ ID No. 3, and its encoded protein is shown in SEQ ID No. 4.

[0062] 3) Construction of plant expression vectors:

[0063] (1) The PCR product of the recovered GmPMI1 / GmPMIR gene was ligated into the pFGC5941-GFP vector digested with NcoI and BamHI using homologous recombinase.

[0064] (2) Transform Escherichia coli competent cells JM109, spread them evenly on LB plates (containing 50 μg / mL kanamycin), and incubate at 37°C for 12 h;

[0065] (3) Colony PCR verification was performed using vector primers (pFGC5941-GFP-F and pFGC5941-GFP-R). The PCR products were subjected to nucleic acid electrophoresis on an agarose gel, stained, photographed, and positive clones were recorded.

[0066] pFGC5941-GFP-F: atttggagaggacacgctc (SEQ ID NO.9)

[0067] pFGC5941-GFP-R: tatcatgcgatcataggcgtc (SEQ ID NO. 10)

[0068] (4) Pick two positive single colonies and amplify them by shaking. Extract the plasmid according to the instructions of the plasmid extraction kit (Takara) and send it to Shanghai Sangon Biotech Co., Ltd. for sequencing.

[0069] (5) Transform the correctly sequenced plasmid into Agrobacterium K599 by Agrobacterium electroporation, add antibiotic-free LB, amplify at 28℃ for 1 h, and evenly spread on LB plates (containing 50 μg / mL kanamycin and 50 μg / mL streptomycin or containing 50 μg / mL kanamycin and 50 μg / mL rifampin) and incubate at 28℃ for 48 h.

[0070] (6) Colony PCR verification was performed using vector primers (pFGC5941-GFP-F and pFGC5941-GFP-R). Correct clones were selected and stored at -80℃ for subsequent experiments.

[0071] Example 2

[0072] (a) Overexpression of the GmPMI1 / GmPMI1R gene in soybean:

[0073] Referencing the soybean hairy root transformation method established by Kereszt et al. (A. Kereszt et al., Agrobacterium rhizogenes-mediated transformation of soybean to study rootbiology. Nature Protocols 2, 948-952 (2007)).

[0074] The specific steps are as follows:

[0075] 1) Sow soybean seeds (such as Hefeng 47, which are susceptible to disease) in moist vermiculite and place them in a greenhouse (25℃, 16h light / 8h darkness) for cultivation. After about 7 days, remove the soybeans and sterilize them for use in experiments. At this time, the cotyledon differentiation ability is strong.

[0076] 2) Six days after planting the beans, single colonies of Agrobacterium K599 were picked and placed in liquid LB containing antibiotics (50ug / mL kanamycin and 50ug / mL streptomycin) and cultured at 28°C for 24 hours.

[0077] 3) Cotyledon disinfection: Cut off the well-grown soybean cotyledons with a blade, treat the cotyledons with 75% alcohol for 1 minute, then treat with 10% sodium hypochlorite for 10 minutes, and then wash with sterile water 3 times.

[0078] 4) Preparation of bacterial suspension: In a clean bench, take 2 mL of bacterial suspension, centrifuge at 5000 rpm for 3 min, wash the bacterial cells twice with the prepared buffer (components: 10 mM 2-[N-morpholino]ethanesulfonic acid, 10 mM MgCl2, 200 μM Macetosyringone, pH 5.6), and finally adjust the OD600 value of the bacterial suspension to 0.6 for later use.

[0079] 5) Processing the soybean: Wear sterile gloves, cut the soybean off the petiole of the cotyledon, and make a wound in the middle of the lower epidermis of the cotyledon near the petiole end with a sterile scalpel blade.

[0080] 6) Inoculation: Place the treated soybeans with the skin facing down on MS medium, and drop 15-20 μL of bacterial solution into the wound. The bacterial solution should be sufficient to form slightly raised small blisters.

[0081] 7) Seal the petri dish with sealing film and place it in a greenhouse (25℃, 16h light / 8h darkness) for cultivation. Rhizomorphs will grow after about 3 weeks. Check for contamination daily and transfer contaminated dishes as needed.

[0082] 8) Hairy root growth: During the culture process, the wound at the inoculation site turns brown (2-3 days), and then callus tissue will grow in the middle or the entire wound. The callus tissue is granular or connected in sheets (it starts to grow in about 7 days). The callus tissue needs one to two weeks to grow roots.

[0083] 9) Preliminary identification of hairy roots can be achieved by fluorescence observation using a stereomicroscope (Leica MZ FLIII).

[0084] (II) Inoculation of soybean hairy roots overexpressing the GmPMI1 / GmPMI1R gene with Phytophthora soybeanis:

[0085] Soybean hairy roots exhibiting strong fluorescence were screened using a stereofluorescence microscope (Leica MZ FLIII). Hairy roots expressing the target gene and those with similar growth vigor to the control were selected and arranged side-by-side on sterile, water-moistened filter paper. Freshly cultured mycelial blocks of *Phytophthora sojae* strain labeled with red fluorescence were placed at the tips of both types of hairy roots. After incubation at 25°C for 36-48 hours, the roots were observed and photographed under a fluorescence microscope. Figure 1 Soybean hairy roots overexpressing GmPMI1 / GmPMIR had fewer soybean Phytophthora oospores compared to the control group overexpressing the empty vector EV. Figure 2 This indicates that GmPMI1 / GmPMIR can improve soybean resistance to Phytophthora sojae. After the observation period, samples were taken and frozen for subsequent determination of the biomass after Phytophthora sojae infection. Figure 3 ) and detection of protein expression levels in soybean hairy roots overexpressing GmPMI1 / GmPMIR ( Figure 4 ).

[0086] (III) Detection of Phytophthora biomass after Phytophthora infects soybean hairy roots:

[0087] Soybean hairy roots overexpressing the GmPMI1 / GmPMIR gene were collected 36 h after infection with Phytophthora sojae for Phytophthora biomass detection. Genome extraction was performed using a TIANGEN genome extraction kit according to the instructions, and the content and quality were determined using a spectrophotometer.

[0088] (iv) Real-time quantitative PCR reaction:

[0089] The PCR reaction system contained 5 μL gDNA, 10 μL SYBR Premix Ex Taq II (Tli RNase H Plus), 0.4 μL each of the pre- and post-primer primers, 0.4 μL ROX Reference Dye II, and 13.8 μL ddH2O. The reaction program was: I – 95℃ for 30 seconds, II – 95℃ for 5 seconds, 60℃ for 34 seconds, with 40 cycles in step II. The melting curve analysis program was: 95℃ for 15 seconds, 60℃ for 1 minute, and 95℃ for 15 seconds. Data analysis was performed using the 2-ΔΔCT method (KJLivak, TDSchmittgen, Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25, 402-408 (2001).).

[0090] The primers for the real-time quantitative PCR reaction are:

[0091] RT-Actin-GMCYP93A-F: CCAGAATGACGCTGAGTCAG (SEQ ID NO.11)

[0092] RT-Actin-GMCYP93A-R: GCAAATCGAAAGGCTTCAGG (SEQ ID NO. 12)

[0093] RT-Actin-P.sojae–F:ACTGCACCTTCCAGACCATC(SEQ ID NO.13)

[0094] RT-Actin-P.sojae–R:CCACCACCTTGATCTTCATG(SEQ ID NO.14)

[0095] (V) Detection of protein levels on soybean hairy roots overexpressing the GmPMI1 / GmPMI1R gene:

[0096] Soybean hairy roots overexpressing GmPMI1 / GmPMIR-GFP were collected 36 h after infection with *Phytophthora sojae* for the detection of GmPMI1 / GmPMIR protein expression levels. The collected soybean hairy roots were flash-frozen in liquid nitrogen, ground, and then mixed with protein extraction buffer (components: 150 mM NaCl, 50 mM Tris HCl, pH 7.5, 1.0% (v / v) NP-40, 1.0% (v / v) protease inhibitor cocktail). The mixture was incubated on ice for 30 min. The supernatant was collected by centrifugation at 18000 g and 80 μL was added to 20 μL of 5-fold protein loading buffer. The mixture was then boiled for 5 min. 10 μL of the sample was electrophoretically separated on an SDS-PAGE gel at 120 V for 1.5 h. After the reaction, the protein sample was transferred to a PVDF membrane and incubated with 5% PBST milk for sealing. After incubation with 1:5000 diluted GFP primary antibody (Abmart) for 2 hours, wash the membrane three times with PBST for 5 minutes each time; then incubate with 1:10000 diluted mouse antibody (LI-COR, irdye 800, 926-32210) for 30 minutes, wash the membrane three times with PBST for 5 minutes each time; scan the membrane and take pictures. Figure 4 Experimental results showed that Western blot revealed the GmPMI1 / GmPMIR-GFP fusion protein (~72 kDa), indicating that GmPMI1 / GmPMIR was successfully expressed in soybean hairy roots.

[0097] Experimental results showed that inoculating soybeans overexpressing the GmPMI1 / GmPMIR gene with *Phytophthora sojae* significantly reduced *Phytophthora sojae* biomass. The GmPMI1 / GmPMIR gene plays a crucial role in soybean disease resistance, and research on GmPMI1 / GmPMIR in soybeans can drive related research on extracellular resistance proteins in many other plants such as tomato and potato. These studies will better elucidate the disease resistance functions of plants against *Phytophthora sojae* and provide excellent disease resistance gene resources for disease resistance genetic engineering breeding.

[0098] Sequence List:

[0099] GmPMI1 gene sequence (SEQ ID NO.1)

[0100] ATGAAACCCACTTTCCTCCTTCTCTCTTATTCTTCACATTCACACTCTCCATCTCAC

[0101] CCCACCCGCCACCGCCGGTGACCGTTACGTTTCCGGCGAACAATTCCGGCGATGCCGA

[0102] CTTCATCCGCGCGAGCTGCAACGCGACCCTGTACCCCGACCTCTGCTTCTCCTCCCTC

[0103] TCCCGCTACGCCGCCGCCGTGCAGAGCAGCCACGCCGCCCTAGCACGTGTCGCCGTC

[0104] GCCGTGGCCCTCGCCAAGGCCCACGGCGCCGCGGCCTACCTCTCGCACCAGACCGC

[0105] GGCGGCCAGCGACGACGACTCCGGCGCCGGCTCCGCCCTGCACGACTGCTTCTCGA

[0106] ACCTCGAGGACGCTGTGGACGAGATCCGTGGCTCTCTTAAGCAGATGCGCCGGCTC

[0107] AAACCAGCCGGAGCCGGAAACTCTGACTCCAGCTCCGTCCGGTTTGGGCTCAGCAA

[0108] CGTGCTGACGTGGATGAGCGCCGCGCTCACTGACGAGGAAACGTGTACTGACGGAT

[0109] TCGAGGGTGTAGAGGAAGGTCCCGTGAAGACGAGCGTATGCGATCGCGTCACTAGG

[0110] GTCAAGAAGTTCACCAGCAATGCCTTGGCGCTGGTTAACGGTTTCGCTAATAACTTAG mPMI1 protein sequence (SEQ ID NO.2)

[0111] MKPTFLLSLLFFTFTLSHLTPPATAGDRYVSGDNSGDADFIRASCNATLYPDLCFSSLSRY

[0112] AAAVQSSHAALARVAVAVALAKAHGAAAYLSHQTAAASDDDSGAGSALHDCFSNLED

[0113] AVDEIRGSLKQMRRLKPAGAGNSDSSSVRFGLSNVLTWMSAALTDEETCTDGFEGVEE

[0114] GPVKTSVCDRVTRVKKFTSNALALVNGFANNLGmPMI1R gene sequence (SEQ ID NO.3)

[0115] ATGAAACCCACTTTCCTCCTTTCTCTCTTATTCTTCACATTCACACTCTCCCATCTCAC

[0116] CCCACCCGCCACCGCCGGTGACCGTTACGTTTCCGGCGACAATTCCGGCGATGCCGA

[0117] CTTCATCCGCGCGAGCTGCAACGCGACCCTGTACCCCGACCTCTGCTTCTCCTCCCTC

[0118] TCCCGCTACGCCGCCGCCGTGCAGAGCAGCCACGCCGCCCTAGCACGTGTCGCCGTC

[0119] GCCGTGGCCCTCGCCAAGGCCCACGGCGCCGCGGCCTACCTCTCGCACCAGACCGC

[0120] GGCGGCCAGCGACGACGCCTCCGCCGCCGGCTCCGCCCTGCACGCTTGCTTCTCGAA

[0121] CCTCGAGGACGCTGTGGACGAGATCCGTGGCTCTCTTAAGCAGATGCGCCGGCTCAA

[0122] ACCAGCCGGAGCCGGAAACTCTGACTCCAGCTCCGTCCGGTTTGGGCTCAAGTTCGT

[0123] GCTGGACGGAATGAGCGCCGCGCTCACTGACGAGGAAACGTGTACTGACGGATTCG

[0124] AGGGTGTAGAGGAAGGTCCCGTGAAGACGAGCGTATGCGATCGCGTCACTAGGGTC

[0125] AAGAAGTTCACCGCCAATGCCTTGGCGCTGGTTGCTGGTTTCGCTAATAACTTAG mPMI1 R protein sequence (SEQ ID NO.4)

[0126] MKPTFLLSLLFFTFTLSHLTPPATAGDRYVSGDNSGDADFIRASCNATLYPDLCFSSLSRYA

[0127] AAVQSSHAALARVAVAVALAKAHGAAAYLSHQTAAASDDASAAGSALHACFSNLEDAV

[0128] DEIRGSLKQMRRLKPAGAGNSDSSSVRFGLKFVLDGMSAALTDEETCTDGFEGVEEGPV

[0129] KTSVCDRVTRVKKFTANALALVAGFANNL

[0130] GmPMI1 / GmPMI1R gene primers:

[0131] pFGC::GFP::GmPMI - GFP - F ttacatttacaattaccATGAAACCCACTTTCCTCCTTTCTC(SEQID No.5)

[0132] pFGC::GFP::GmPMI - GFP - R ctctagactcacctaggatccTAAGTTATTAGCGAAACCGTTAACC(SEQ ID No.6)

[0133] pFGC::GFP::GmPMIR - GFP - F ttacatttacaattaccATGAAACCCACTTTCCTCCTTTCTC(SEQID No.7)

[0134] pFGC::GFP::GmPMIR - GFP - R ctctagactcacctaggatccTAAGTTATTAGCGAAACCAGCAACC(SEQ ID No.8)

[0135] Vector primers:

[0136] pFGC5941 - GFP - F: atttggagaggacacgctc(SEQ ID NO.9)

[0137] pFGC5941-GFP-R: tatcatgcgatcataggcgtc (SEQ ID NO. 10)

[0138] The primers for the real-time quantitative PCR reaction are:

[0139] qRT-GmActin-F: CCAGAATGACGCTGAGTCAG (SEQ ID NO. 11) qRT-GmActin-R: GCAAATCGAAAGGCTTCAGG (SEQ ID NO. 12) qRT-P.sojaeActin-F: ACTGCACCTTCCAGACCATC (SEQ ID NO. 13) qRT-P.sojaeActin-R: CCACCACCTTGATCTTCATG (SEQ ID NO. 12) NO.14).

Claims

1. Application of overexpressing DNA molecules with nucleotide sequences as shown in SEQ ID NO.1 or SEQ ID NO.3 in at least one of (d1)-(d2): (d1) Improve soybean resistance to Phytophthora sojae; (d2) Breed soybean varieties with improved resistance to Phytophthora indicum.

2. Application of increasing the content of proteins encoded by DNA molecules with nucleotide sequences such as SEQ ID NO.1 or SEQ ID NO.3 in at least one of (d1)-(d2): (d1) Improve soybean resistance to Phytophthora sojae; (d2) Breed soybean varieties with improved resistance to Phytophthora indicum.

3. The application according to claim 2, characterized in that, The protein in question is either (b1) or (b2): (b1) Proteins with amino acid sequences as shown in SEQ ID NO.2 or SEQ ID NO.4; (b2) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein described in (b1).

4. Application of biological materials containing DNA molecules with nucleotide sequences as shown in SEQ ID NO.1 or SEQ ID NO.3 in at least one of (d1)-(d2): (d1) Improve soybean resistance to Phytophthora sojae; (d2) Develop soybean varieties with improved resistance to Phytophthora indicum; The biomaterial is at least one of the following (c1)-(c4): (c1) An expression cassette containing a DNA molecule with a nucleotide sequence as shown in SEQ ID NO.1 or SEQ ID NO.3; (c2) A recombinant vector containing a DNA molecule with a nucleotide sequence as shown in SEQ ID NO.1 or SEQ ID NO.3; (c3) Non-plant cell lines containing DNA molecules with nucleotide sequences as shown in SEQ ID NO.1 or SEQ ID NO.3; (c4) Recombinant microorganisms containing DNA molecules with nucleotide sequences as shown in SEQ ID NO.1 or SEQ ID NO.3.

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