Application of GmEXO70 gene in enhancing soybean resistance to nematode

Abstract

This invention discloses GmEXO70 Application of genes in enhancing soybean resistance to nematodes. This invention is the first to discover and validate a gene encoding the EXORDIUM protein through single-cell transcriptome analysis. GmEXO70 It is specifically and highly expressed in the syncytia of disease-resistant soybean varieties, and its expression is strongly induced by nematode infection. Overexpression was confirmed by constructing an overexpression vector and transforming soybean hairy roots with Agrobacterium rhizogenes. GmEXO70 It can significantly increase the expression level of this gene in roots. Further resistance evaluation showed that, compared with the empty vector control, overexpression significantly increased the expression level of this gene. GmEXO70 In the root system, the proportion of nematodes developing to the J3 / J4 stage was significantly reduced, with most nematodes arresting at the J2 stage. Furthermore, immunofluorescence in situ hybridization directly confirmed at the transcriptional level that... GmEXO70 The gene was specifically enriched in nematode-induced syncytia. This invention provides a new key gene for soybean nematode resistance breeding. GmEXO70 .

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, specifically to GmEXO70 Application of genes in enhancing soybean's resistance to nematodes. Background Technology

[0002] Soybean Cyst Nematode (SCN) is one of the most serious pathogens affecting soybean production worldwide. It infects soybean roots and induces vascular bundle cells to reprogram into syncytiums as feeding sites, disrupting the root system's transport and absorption of substances, leading to stunted plant growth, yellowing, dwarfing, and even death, often causing yield losses of 10% to 30%.

[0003] In studies on soybean nematode resistance mechanisms, reported resistance genes are mostly enriched at the transcriptional regulatory level, such as... WRKY , MYB , NAC The transcription factor family. These genes mainly activate the plant's immune response and enhance its resistance to nematode infection by regulating the expression of downstream defense-related genes. However, the plant's resistance to nematodes is a complex spatiotemporal dynamic process. In addition to transcriptional remodeling in the nucleus, the targeted transport of substances in the cytoplasm, cell wall strengthening, and precise delivery of immune-active substances are equally important. The exocyst complex is a multi-protein complex composed of eight subunits (SEC3, SEC5, SEC6, SEC8, SEC10, SEC15, EXO70, EXO84), which plays a core mediating role in regulating the targeted fusion of secretory vesicles with the plasma membrane (exocytosis). Among them, the EXO70 subunit, as a key component in the connection between the complex and the plasma membrane, determines the targeting specificity of vesicle transport.

[0004] Although existing research has shown that the exosome-related gene family, to which EXO belongs, is involved in plant cell polar growth and defense responses against certain fungi and bacteria, its functional role in the specific interaction system of soybean nematode resistance remains almost entirely unexplored. This is especially true regarding… GmEXO How gene-like structures regulate the transport of vesicles containing resistance substances, participate in cell wall modification during syncytium formation, and their specific molecular mechanisms in coordinating host spatial immune defense have not yet been systematically elucidated.

[0005] Therefore, we need to delve deeper and clarify the following: Glyma.04G100400 As the representative 70The biological functions of family genes in the process of nematode resistance can not only break the current limitation of soybean resistance research to transcriptional regulators, but also reveal the soybean resistance mechanism from the new dimension of intracellular vesicle transport and precision defense, providing core gene resources with independent intellectual property rights for the breeding of high-yield, broad-spectrum nematode-resistant soybean varieties. Summary of the Invention

[0006] In view of the above-mentioned technical problems, the present invention proposes a... GmEXO70 The specific technical solution for applying genes to enhance soybean's resistance to nematodes is as follows: The present invention provides in a first aspect GmEXO70 The use of genes or related biological materials in at least one of the following: A1) Regulate the ability of soybeans to resist nematodes or prepare products that regulate the ability of soybeans to resist nematodes. A2) Cultivate soybeans with improved nematode resistance or prepare products that enhance soybean nematode resistance; A3) Preparation of genetically modified soybeans; The GmEXO70 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0007] Furthermore, the biomaterial includes one or more of the following: The GmEXO70 The gene-encoded protein and / or containing the above GmEXO70 Gene recombinant vectors, recombinant microorganisms, or transgenic soybean cell lines.

[0008] Furthermore, the aforementioned GmEXO70 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2.

[0009] The containing GmEXO70 Gene recombination vectors include basic vectors and GmEXO70 The underlying vector includes the pAGM4673 vector.

[0010] Furthermore, the containing GmEXO70 Recombinant microorganisms include Agrobacterium rhizogenes ARqua1.

[0011] Furthermore, the regulation of soybean resistance to nematodes includes: regulating the development of soybean cyst nematodes within soybean roots.

[0012] Furthermore, the regulation of soybean's resistance to nematodes includes: by upregulating... GmEXO70 Gene expression is used to enhance soybean's resistance to nematodes.

[0013] In a second aspect, the present invention provides a capability to increase GmEXO70The application of gene-expressing biological materials in regulating soybean nematode resistance, breeding nematode-resistant soybeans, or preparing transgenic soybeans; GmEXO70 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0014] Further, the biomaterial includes: GmEXO70 The gene encodes a protein and / or contains upregulated proteins. GmEXO70 Recombinant vectors for gene expression, recombinant microorganisms, or transgenic soybean cell lines.

[0015] Furthermore, the enhanced nematode resistance includes inhibiting the development of soybean cyst nematodes within soybean roots.

[0016] Furthermore, the present invention utilizes soybeans ( Glycine max Overexpression in ) GmEXO70 Genes can increase the level of EXORDIUM protein in plant root syncytia, delaying or inhibiting the development of nematodes in the roots, thereby reducing the parasitism rate of nematodes.

[0017] Furthermore, the soybean varieties include, but are not limited to, Williams 82, Forrest, or PI 88788.

[0018] Furthermore, the nematode development process refers to reducing the proportion of soybean root nematodes that have developed to the J3 and J4 stages in order to enhance soybean's resistance to nematodes.

[0019] In a third aspect, the present invention provides a method for improving the nematode resistance of soybeans, comprising: Will be able to increase GmEXO70 Gene-expressing biological materials are transferred into soybeans.

[0020] In a fourth aspect, the present invention provides a method for cultivating soybeans with enhanced nematode resistance, comprising: Will be able to increase GmEXO70 Gene-expressing biological materials are transferred into soybeans.

[0021] In a fifth aspect, the present invention provides a soybean with enhanced nematode resistance, the soybean comprising upregulating... GmEXO70 Biological materials for gene expression; The GmEXO70 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0022] Compared with the prior art, the present invention has the following beneficial effects: This invention screens gene expression characteristics of the syncytium, a feeding structure of nematodes, and the results show that... GmEXO70The gene expression level in the syncytia of resistant varieties was significantly higher than that in susceptible varieties, and it exhibited an induced expression pattern during infection. Based on this, to achieve cross-variety transfer of resistance traits, this invention further verifies transgenic function, demonstrating the ability to transfer highly induced genes from resistant resources. GmEXO70 The gene was translocated into the susceptible cultivar Williams 82 for expression. Results showed that the introduction of this gene significantly enhanced the resistance level of the susceptible cultivar to cyst nematodes, manifested by an effective delay in the developmental process of the nematodes in the roots. This was achieved through gene introduction and regulation. GmEXO70 Genes can rapidly enhance the nematode resistance of soybean varieties without relying on complex hybridization breeding, and have significant value for promotion and application and industrialization prospects. Attached Figure Description

[0023] Figure 1 This is a single-cell atlas of soybean roots and nematode infection (UMAP cluster diagram).

[0024] Figure 2 for GmEXO70 ( Glyma.04G100400 Dot plot of gene expression in different cell types and varieties.

[0025] Figure 3 A photomicrograph of the hairy roots of a positive transgenic soybean with red fluorescence (RFP / tdTomato).

[0026] Figure 4 for GmEXO70 Results of gene overexpression detection in the hairy roots of transgenic soybean.

[0027] Figure 5 For overexpression GmEXO70 The inhibitory effect of genes on the development of soybean cyst nematodes; among which, Figure 5 A shows the root phenotype results 14 days after inoculation, stained with acid fuchsin. Scale bar: 500µm. Figure 5 B represents the corresponding statistical data.

[0028] Figure 6 for GmEXO70 Figure showing the results of gene-induced reduction of the final sporangium number on soybean root surface; among which, Figure 6 A shows the root phenotypes of the control and overexpressing plants 21 days after inoculation with soybean cyst nematodes. Scale bar: 2 mm. Figure 6 B is a bar chart showing the number of cysts per root.

[0029] Figure 7 To detect using digoxigenin-labeled probe fluorescence in situ hybridization (FISH) technology GmEXO70 Expression localization of the gene in infected soybean roots; scale bar: 50µm.

[0030] Figure 8 This is a model constructed based on WGCNA analysis, consisting of 13 key genes. GmEXO70 A co-expression regulatory network for nematode resistance; among which, Figure 8 A represents the module division results of the weighted gene co-expression network analysis (WGCNA); Figure 8 B is GmEXO70 Results of Gene Ontology (GO) enrichment analysis. Detailed Implementation

[0031] To enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. It should be noted that the following detailed descriptions are exemplary and are only some embodiments of the present invention, not all embodiments.

[0032] Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0033] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. The experimental materials used in the embodiments of this invention are all conventional experimental materials in the art and are commercially available. Experimental methods not specifying detailed conditions are performed according to conventional experimental methods or the operating instructions recommended by the supplier.

[0034] The culture medium used in the following examples: LB medium formulation: Table 1

[0035] Co-culture medium: Table 2

[0036] Rooting medium formula: Table 3

[0037] In this invention, GmEXO70 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the GmEXO70 protein is shown in SEQ ID NO.2.

[0038] SEQ ID NO.1: SEQ ID NO.2: MPEGLTTPHAQKSLFNLSPQLKTHISLDLYIPLFLSPSSTSQHPPIPRFKKRAVIVVTIQKMAGLVVSSQCFLKLLLVVSVFHVSFAARRLNELVQDQSQLLHYHNGPLLYGKIAVNLIWYGNFKPSQKAIITDFVTSLSSPASQSSQPSVATWWKTTEKYYHLSPRKASLSLSLGDQILDETYSLGKS LTGKNLVQLASKGGQRNSINVVLTSADVAVEGFCMSRCGTHGSSASHLKKNSKSYKFAYIWVGNSETQCPGQCAWPFHQPIYGPQSPPLVAPNNDVGLDGMVINLASLLAGTATNPFGNGYFQGPAEAPLEAASACPGVYGKGAYPGYAGDLLVDSTTGASYNVKGANGRKYLVPALYDPSTSSCSTPV.

[0039] Example 1: Nematode Resistance Gene GmEXO70 Screening and identification 1. Construction of a single-cell nuclear transcriptome map of soybean roots. Using soybean resistant varieties PI 88788 and Forrest, and susceptible variety Williams 82 as materials, samples were collected at 1, 3, and 7 days (dpi) after inoculation with soybean cyst nematode (SCN, physiological race 3). Root cell nuclei were extracted, and single-cell nuclear transcriptome sequencing (snRNA-seq) was performed using the 10x Genomics platform. Quality control, dimensionality reduction, and cluster analysis were performed on the sequencing data, utilizing known marker genes (such as...) GmSNAP18 Annotating cell populations, etc., successfully identified 10 cell types, including epidermis, cortex, endodermis, pericycle, phloem, xylem, meristem, and syncytium. Figure 1 (As shown).

[0040] 2. Discovery of Syncytium-Specific Resistance Genes: Targeting the syncytium, a key feeding site in nematodes, the expression characteristics of its internal genes were analyzed. First, by comparing the gene expression profiles of the syncytium with other root cell types, a set of genes specifically highly expressed in the syncytium was screened. Subsequently, the differences in gene expression within the syncytium of resistant varieties (PI 88788, Forrest) and susceptible varieties (Williams 82) were further compared. Single-cell transcriptome analysis revealed that the genes… Glyma.04G100400 (named) GmEXO70The basal expression level of this gene (encoding an EXORDIUM protein) in the syncytia of resistant varieties was significantly higher than that in susceptible varieties. More importantly, after nematode infection, the expression of this gene was specifically induced and remained at a high level in the later stages of infection (3 dpi and 7 dpi). Figure 2 ). GmEXO70 Gene( Glyma.04G100400 In the resistant variety Forrest, it is strongly induced by nematode infection, but in the susceptible variety Williams 82, it is almost not expressed. Figure 2 This infection-induced expression pattern strongly suggests... GmEXO70 Genes are directly involved in the defense response of resistant soybean varieties against nematodes.

[0041] Example 2 GmEXO70 Gene cloning and vector construction 1. GmEXO70 Cloning of genes Using cDNA from wild-type soybean Williams 82 (Wm82) to analyze soybean GmEXO70 PCR amplification was performed using the open reading frame (ORF) of the target fragment, employing primers I (SEQ ID NO. 3) and II (SEQ ID NO. 4) and Toyobo's KOD onePCR Master Mix. The PCR products were detected by agarose gel electrophoresis, and the target fragment was recovered by gel excision.

[0042] The PCR reaction system is as follows: Table 4

[0043] Primer Ⅰ (SEQ ID NO.3): cgacgacaagaccgtgaccATGCCAGAGGGGCTAACCAC (where the lowercase letters represent the homologous recombination vector sequence, used for homologous recombination ligation); Primer II (SEQ ID NO.4): gaggagaagagccgTCACACGGGCGTTGAGCAA (where the lowercase letters represent the homologous recombination vector sequence used for homologous recombination ligation); Amplification GmEXO70 Gene fragments.

[0044] 2. Carrier Construction The amplified GmEXO70 Gene fragments were used in homologous recombination and combined with the 35S promoter of cauliflower mosaic virus (CaMV) and the gene obtained in Example 2. GmEXO70The purified PCR product and the nifedipine synthase (NOS) terminator were integrated into the binary vector pAGM4673. This backbone vector also carries an RFP fluorescent marker gene for subsequent screening of transgenic plants.

[0045] The reaction system is as follows: Table 5

[0046] Reaction conditions: 50℃, 15min.

[0047] 3. Plasmid preparation Further screening was conducted using E. coli transformation and colony PCR. Samples yielding the target band were then validated using PCR, ultimately resulting in the successfully ligated vector OE-. GmEXO70 .

[0048] (1) Escherichia coli transformation: Table 6

[0049] Incubate on ice for 30 min; incubate in a 42°C water bath for 45 s; incubate on ice for 2 min; add 1 mL of antibiotic-free LB liquid medium, place in a 37°C, 200 rpm shaker for 1 h; remove and centrifuge at 6000 rpm for 2 min, remove the supernatant, spread the remainder onto Kan LB solid medium, and incubate overnight at 37°C.

[0050] (2) Colony PCR: Table 7

[0051] The forward primer and the reverse primer are primer I (SEQ ID NO.3) and primer II (SEQ ID NO.4) as described in step 1 of this example, respectively.

[0052] Use a pipette tip to pick up the colonies obtained in step (1) and add them to the prepared system.

[0053] The PCR reaction conditions are as follows: (1) Pre-denaturation at 95℃ for 5 min; (2) Denaturation at 95℃ for 30 sec, 55℃ for 30 sec, 72℃ for 45 sec, for a total of 35 cycles; (3) Storage at 4℃.

[0054] Example 3, Overexpression GmEXO70 Obtaining and validating genetically modified soybean root systems 1. Preparation of Agrobacterium The obtained OE- GmEXO70 plasmid vector transformed into Agrobacterium rhizogenes ( Agrobacterium rhizogenesIn the ARqua1 strain. Two days later, positive clones were picked and inoculated into selective liquid medium, cultured with shaking at 28°C, and the bacterial cells were collected and resuspended to OD500 using infiltration buffer (B5 medium 3.0~3.5 g / L, sucrose 28~32 g / L, MES 3.8~4.0 g / L, acetylsuccinone 38~42 mg / L, 6-BA 1.5~1.8 mg / L, gibberellin 0.02~0.03 mg / L, surfactant Silwet L-77 100 mL / L, pH=5.4). 600 =Approximately 0.7, incubate at room temperature for 2 hours.

[0055] 2. Transformation of soybean hairy root system Select robust, plump soybean cotyledons, disinfect them with 75% ethanol for 30 seconds, rinse once with water, then soak them in disinfectant solution for 4 minutes, and finally soak them in water three times for five minutes each time. In a clean bench, place the cotyledons in an Agrobacterium suspension, and cut off the bottom one-fifth of the soybean cotyledons with a blade for Agrobacterium-mediated genetic transformation. Transfer the treated cotyledons to a co-culture medium covered with sterile filter paper and incubate for 3 days.

[0056] 3. Screening and validation of transgenic root systems After 3 days of culture on co-culture medium, cotyledons were transferred to rooting medium. After 14 days of culture at 28℃, the transformed roots were observed using a fluorescence microscope, and soybean roots that appeared red under the microscope (e.g., ...) were selected. Figure 3 (As shown). Transformation was performed using Agrobacterium rhizogenes-mediated transformation. GmEXO70 Soybean hairy roots observed under a stereofluorescence microscope after overexpression of the vector (carrying the tdTomato red fluorescent reporter gene). The image below (excited at 546 nm) shows strong red fluorescence in the transgenic hairy roots under red fluorescence excitation wavelength (546 nm), indicating that the recombinant vector has been successfully introduced into soybean root cells and is stably expressed. The image above, as a control, shows no specific fluorescence signal in the same root system under ultraviolet excitation wavelength (358 nm), excluding autofluorescence interference.

[0057] 4. GmEXO70 Gene overexpression verification Total RNA was extracted from positive soybean hairy root samples 3 days after SCN infection. cDNA was synthesized using the HiScript Reverse Transcriptase Kit, and RT-qPCR was performed using AceQ qPCR SYBR Green Master Mix. Each treatment employed at least three biological replicates and three technical replicates. Relative gene expression levels were calculated. The soybean Actin gene was used as an internal control, and its amplification primer sequences were as follows: Actin-F: 5'-GCAAGTGGTCGTACAACTGG-3' (SEQ ID NO.5); Actin-R: 5'-ACCAGCCAGATCAAGACGAA-3' (SEQ ID NO. 6).

[0058] The specific method is as follows: Plant RNA extraction: Plant RNA was extracted using the Trizol method. Soybean tissue was ground in liquid nitrogen and collected into a 1.5 mL centrifuge tube. 1 mL of Trizol was added, vortexed, and incubated at room temperature for 5 min. 200 μL of chloroform was added, and the mixture was vigorously vortexed for 15 sec, then incubated at room temperature for 5 min. The tube was centrifuged at 12000 rpm for 15 min at 4 °C. Approximately 500 μL of the supernatant was transferred to a new centrifuge tube. An equal volume of isopropanol was added, and the tube was inverted to mix. The tube was incubated at room temperature for 10 min. The tube was centrifuged at 12000 rpm for 10 min at 4 °C, and the supernatant was discarded. The precipitate was washed with 1 mL of 75% ethanol, centrifuged at 7500 rpm for 5 min at 4 °C, and the supernatant was discarded. This washing process was repeated once, followed by empty centrifugation. The ethanol was then dried in a clean bench. Finally, 20-30 μL of RNase-free water was added to dissolve the RNA. The concentration was measured, and the tube was stored at -80 °C for later use.

[0059] Reverse transcription: Take 500 ng of extracted RNA, add 2 μL DNA Digester Mix, add RNA-free H2O to 14 μL, incubate in a 37℃ metal bath for 2 min; add 2 μL reverse transcriptase, set the PCR program to 55℃ for 5 min, 85℃ for 5 s, and store in a -20℃ refrigerator for later use.

[0060] Quantitative PCR: AceQ qPCR SYBR Green Master Mix was used. The reaction system is as follows: Table 8

[0061] After preparing the reaction system, the samples were amplified using a Bio-Rad CFX96™ Real-Time PCR Detection System. The reaction program was set as follows: pre-denaturation at 95℃ for 5 min; followed by cycling: denaturation at 95℃ for 10 sec, annealing and extension at 60℃ for 30 sec (fluorescence signals were acquired during this stage), for a total of 40 cycles; after cycling, melt curve analysis was performed: signals were acquired every 0.5℃ from 65℃ to 95℃.

[0062] like Figure 4As shown, RT-qPCR was used to analyze the empty vector control (EV) and overexpression. GmEXO70 (OE) The relative expression level of the target gene in hairy roots, compared with the control group, was higher in the roots of the overexpression group. GmEXO70 Gene expression levels were significantly increased (**) P <0.01).

[0063] Example 4, Overexpression GmEXO70 Evaluation of soybean resistance to nematodes based on genetic factors Nematode inoculation: Select positive transgenic hairy roots (OE-) with consistent growth status. GmEXO70 ) and control hairy roots (OE-EV). Approximately 400 J2 stage soybean cyst nematodes (SCN race 3) were inoculated around each root system.

[0064] Observation of root nematode development: Transgenic hairy roots inoculated with cyst nematodes for 14 days (14 dpi) were selected and stained with acid fuchsin (0.1% acid fuchsin dissolved in lactic acid:glycerol:water solution). After decolorization, the morphology of root nematodes was observed under a stereomicroscope. The number of nematodes in the J2 stage (slender, early development), J3 stage (sausage-shaped, mid-development), and J4 stage / adult (pod-shaped, mature) in each group was counted, and the proportion of mature nematodes was calculated.

[0065] Individual sporangium count: Another group of transgenic hairy roots cultured for 21 days after inoculation (at which point the sporangia had matured and broken through the epidermis) were taken, and the root substrate was gently washed away. The total number of visible white females (Cysts) formed on each root system was directly counted under a stereomicroscope, and the average number of sporangia per root system was calculated.

[0066] like Figure 5 As shown in Figure A, after acid fuchsin staining, a large number of swollen and deeply stained nematodes were observed in the roots of the empty vector control group (OE-EV), indicating that they had developed normally to the J3 or J4 stage (indicated by the red arrows in the figure); while in the overexpression... GmEXO70 Root system ( OE-GmEXO70 In the J2 stage, the nematodes are mostly thin and thread-like, without obvious swelling, indicating that development has stopped. Figure 5 Statistical data from B further confirmed that the proportion of nematodes that had developed to the mature stage (J3+J4) in the overexpression group was significantly lower than that in the control group. P <0.01). This indicates GmEXO70 The introduction of this substance effectively suppressed the early transformation of nematodes from J2 to J3 / J4 morphology.

[0067] Consistent with the developmental arrest results observed under microscopic examination, the final sporangium count results ( Figure 6The results showed that dense white mature cysts were observed on the root surface of the control group (OE-EV); while those overexpressing [the virus]... GmEXO70 Group( OE-GmEXO70 The number of cysts on the root surface was significantly reduced. Statistical analysis showed that overexpression... GmEXO70 The gene reduced the number of sporangia per plant on soybean hairy roots by about 60% compared with the control group, and the difference was extremely significant (P<0.05).

[0068] comprehensive Figure 5 and Figure 6 The results show that GmEXO70 The gene endows soybeans with significant resistance to nematodes. Its mechanism of action is as follows: by inhibiting the early development process of nematodes in the roots (causing nematodes to diapause at the J2 stage), the number of mature cysts that are eventually formed is directly reduced, effectively blocking the reproductive cycle of cyst nematodes.

[0069] Example 5: Detection using immunofluorescence in situ hybridization (FISH) technology GmEXO70 Spatiotemporal expression characteristics of genes To verify at higher resolution and sensitivity GmEXO70 In this embodiment, the spatiotemporal expression patterns of genes in soybean roots and syncytium were investigated. In situ hybridization was performed using a digoxigenin (DIG)-labeled RNA probe, and the hybridization signal was detected using an immunofluorescence antibody amplification system (digoxigenin antibody + fluorescent secondary antibody).

[0070] 1. Probe preparation: Using soybean (Forrest variety) cDNA as a template, PCR amplification was performed. GmEXO70 A specific fragment of the gene (approximately 300-500 bp) was used. T7 and SP6 RNA polymerase promoter sequences were introduced at the 5' end of the primers, respectively. Using a digoxigenin RNA labeling kit (Roche), digoxigenin (DIG)-labeled antisense RNA probes (Antisenseprobe) and sense RNA probes (Sense probe, as a negative control) were transcribed in vitro.

[0071] 2. Preparation of tissue sections: Tissues from the Forrest soybean root infection zone were selected 3 days (3 dpi) and 7 days (7 dpi) after inoculation with soybean cyst nematode (SCN). Root segments were immersed in 4% paraformaldehyde (PFA) fixative and fixed overnight at 4°C. After dehydration and sedimentation with a gradient of sucrose, the sections were cryo-embedded (using OCT embedding medium) and cut into 10 μm thick transverse sections using a cryostat. These sections were then mounted on positively charged, anti-detachment slides.

[0072] 3. Hybridization and Immunofluorescence Detection (1) Hybridization: After permeabilization, DIG-labeled immunosorbent assay (IRISA) was performed on the slides. GmEXO70Hybridization buffer for antisense or sense probes, hybridized overnight (12-16 hours) in a humidified chamber at 55°C. (2) Washing and blocking: Wash away unbound probes with SSC buffer, then block the slides with blocking buffer containing BSA for 1 hour to reduce nonspecific background. (3) Anti-digoxin antibody incubation (primary antibody): Discard the blocking buffer, add mouse anti-digoxin monoclonal antibody (Mouse anti-DIG antibody), and incubate overnight at 4°C. This antibody can specifically recognize and bind to digoxin molecules on the probe. (4) Fluorescent secondary antibody incubation (secondary antibody): After washing the slides 3 times with PBST, add goat anti-mouse IgG secondary antibody (Goat anti-Mouse IgG, Alexa Fluor 488 conjugate) labeled with green fluorescence, and incubate at room temperature in the dark for 1-2 hours. (5) Nuclear counterstaining: Mount the slides with mounting medium containing DAPI (4',6-diamidinyl-2-phenylindole). DAPI can stain the cell nuclei blue to indicate cell location and syncytial morphology (syncytial bodies have typical multinucleated features).

[0073] 4. Observation by laser confocal microscopy: Excite and acquire fluorescence signals using a laser confocal microscope (Confocal Laser Scanning Microscope).

[0074] like Figure 7 As shown, specialized cells with large volumes and multiple nuclei, namely syncytium, can be seen in the vascular bundle region. (Green channel) GmEXO70 The results showed that in the experimental group hybridized with the antisense probe, a strong green fluorescent signal was specifically enriched in the cytoplasm of the syncytium. The fluorescence signal was extremely weak in adjacent uninfected cortical or pericycle cells; no significant green fluorescent signal was observed in the control group hybridized with the sense probe. Merge (overlay plot) showed that the green fluorescence perfectly overlapped with the morphological outline of the syncytium.

[0075] Highly sensitive immunofluorescence in situ hybridization experiments confirmed that... GmEXO70 The gene was strongly induced at the transcriptional level by soybean cyst nematode, and its expression exhibited strict syncytial specificity. This is consistent with the results of single-cell sequencing analysis in Example 1 ( GmEXO70 The finding that the gene was highly expressed in the syncytial cell population was a perfect match, further confirming that the gene is a key factor involved in the development and maintenance of syncytial function.

[0076] Example 6 GmEXO70 ( Glyma.04G100400 )-mediated functional network model of nematode resistance First, specific data screening and preprocessing were performed on the syncytial cell subsets of the disease-resistant variety Forrest at 3 days post-inoculation (3 dpi) to ensure the accuracy of co-expression analysis. Given the target gene... GmEXO70 ( Glyma.04G100400 The expression level of this substance was significantly increased and specific in the syncytiomes of disease-resistant varieties during this period. This study specifically selected data from this sample and used... R The hdWGCNA software package (version 0.4.06) was used in the language for high-dimensional weighted gene co-expression network analysis. To eliminate statistical noise from low-abundance genes, the SetupForWGCNA function was used for strict data filtering before network construction, retaining only genes detected and expressed in at least 5% of cells for subsequent calculations.

[0077] Subsequently, a weighted gene co-expression network was constructed using optimized computational parameters. A metacell aggregation strategy significantly reduced the sparsity interference of single-cell data. Specifically, the `MetacellsByGroups` function was used to aggregate neighboring cells with the most similar transcriptional features, with parameter k set to 25, thus constructing a more stable metacell dataset. Based on this, by analyzing the scale-free topological fit index under different thresholds, the optimal soft-power threshold was determined to be 4, ensuring that the network connectivity distribution conforms to the power-law distribution characteristics of biological networks. Based on this threshold, the `ConstructNetwork` function was called to calculate the adjacency matrix between genes and further transformed into a topological overlap matrix (TOM), thereby completing the construction of the whole-genome co-expression network.

[0078] Through module division and functional enrichment analysis, it was clarified that GmEXO70 The co-expression modules of the genes and the core biological pathways they participate in. Hierarchical clustering based on the topological overlap matrix identified 19 gene co-expression modules, among which the core gene of this invention is... GmEXO70 It is located in the Turquoise module (turquoise color module). Figure 8 A). Gene Ontology (GO) enrichment analysis for this module showed ( Figure 8 (B) Genes within the module are significantly enriched in pathways such as ATP metabolism and biosynthesis, glucose metabolism, and cell wall biogenesis. This result confirms the findings at the transcriptome level. GmEXO70 It does not play an independent role in the resistance response, but rather exists within a co-regulatory functional module responsible for energy supply (ATP metabolism) and physical barrier construction (cell wall reinforcement).

[0079] Based on the analysis results of the above modules, the resistance functional model constructed in this invention is as follows: GmEXO70It is possible that by recruiting and regulating high-energy-consuming metabolic activities and cell wall remodeling-related genes, the malignant expansion of the syncytium can be limited and the nutrient uptake of nematodes can be blocked, ultimately causing the nematode development to arrest at the J2 stage, thereby conferring significant resistance to soybeans.

[0080] This invention is not limited to the specific textual description above. Various changes can be made to this invention within the scope outlined in the claims, and all such changes are within the scope of this invention.

Claims

1. GmEXO70 The use of genes or related biological materials in at least one of the following: A1) Regulating the ability of soybeans to resist nematodes or preparing products that regulate the ability of soybeans to resist nematodes. A2) Cultivate soybeans with improved nematode resistance or prepare products that enhance soybean nematode resistance; A3) Preparation of genetically modified soybeans; The GmEXO70 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The application as described in claim 1, characterized in that, The biomaterials include one or more of the following: The GmEXO70 The gene-encoded protein and / or containing the above GmEXO70 Gene recombinant vectors, recombinant microorganisms, or transgenic soybean cell lines.

3. The application as described in claim 1 or 2, characterized in that, The regulation of soybean resistance to nematodes includes: regulating the development of soybean cyst nematodes within soybean roots.

4. The application as described in claim 3, characterized in that, The regulation of soybean's resistance to nematodes includes: by upregulating... GmEXO70 Gene expression is used to enhance soybean's resistance to nematodes.

5. Able to increase GmEXO70 Application of gene-expressing biological materials in the cultivation of soybeans resistant to soybean cyst nematode; GmEXO70 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

6. The application as described in claim 5, characterized in that, The biomaterial includes: GmEXO70 Gene-encoded proteins and / or those containing upregulated GmEXO70 Recombinant vectors for gene expression, recombinant microorganisms, or transgenic soybean cell lines.

7. The application as described in claim 5, characterized in that, The enhanced resistance to nematodes includes inhibiting the development of soybean cyst nematodes within soybean roots.

8. A method for improving soybean's resistance to nematodes, characterized in that, include: Will be able to increase GmEXO70 Gene-expressing biological materials are transferred into soybeans.

9. A method for cultivating soybeans with enhanced nematode resistance, characterized in that, include: Will be able to increase GmEXO70 Gene-expressing biological materials are transferred into soybeans.

10. A soybean with enhanced resistance to nematodes, characterized in that, The soybean contains an upward adjustment GmEXO70 Biological materials for gene expression; The GmEXO70 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

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