Application of GmXTH2 and GmXTH19 genes in enhancing plant resistance to nematodes

Abstract

This invention belongs to the field of biotechnology, specifically relating to the application of the GmXTH2 and GmXTH19 genes in enhancing plant resistance to nematodes. The nucleotide sequence of the GmXTH2 gene is shown in SEQ ID NO.1, and the nucleotide sequence of the GmXTH19 gene is shown in SEQ ID NO.3. This invention is the first to discover the upregulation of XTH family genes in soybean roots after nematode infection and verifies the roles of the GmXTH2 and GmXTH19 genes in soybean SCN resistance. It was found that overexpression of the GmXTH2 and GmXTH19 genes can thicken the cell wall of soybean cells, thereby enhancing soybean SCN resistance, providing new gene resources and pathways for breeding resistant soybeans.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically involving GmXTH2 Genes and GmXTH19 Application of genes in enhancing plant resistance to nematodes. Background Technology

[0002] Soybean cyst nematode (SCN) is one of the most destructive pathogens in global soybean production. Its threat to the soybean industry extends beyond large-scale yield losses; it also increases production costs by reducing the market value of soybeans, thereby impacting the stability of the global soybean supply chain. SCN primarily parasitizes the soybean root system, disrupting the root's water and nutrient absorption system, leading to slow plant growth, yellowing leaves, stunted growth, and even, in severe cases, the death of the entire plant. Studies have shown that in areas severely infested with soybean cyst nematodes, yield losses can reach up to 30%, posing a significant threat to the economic benefits of soybean cultivation and farmers' income.

[0003] As the origin of soybeans, my country has traditionally demanded soybeans not only as a source of high-quality plant protein but also for their widespread use in animal feed and vegetable oils. The high incidence of soybean cyst nematode disease and the complexities of the global soybean supply chain have led to a year-on-year increase in soybean import costs. With the gradual expansion of soybean cultivation, the risk of soybean cyst nematode infestation has also increased significantly. Because soybean cyst nematodes are small and parasitize the roots of soybeans, they are often difficult to detect in a timely manner, and the symptoms they cause are similar to those of nutrient deficiencies, making misdiagnosis very easy, which further increases the difficulty of prevention and control.

[0004] Currently, the control of soybean cyst nematodes mainly relies on the breeding of resistant varieties and crop rotation. However, as nematode populations become more diverse and adaptable, the resistance of resistant varieties gradually diminishes, and crop rotation is limited by the crop planting cycle, resulting in unstable control effects that are difficult to maintain in the long term. With the continued pressure on global food and oilseed supplies, developing more efficient and sustainable nematode-resistant breeding strategies is particularly urgent.

[0005] Soybeans are a vital source of global food and plant protein, playing an irreplaceable role in livestock farming, the catering industry, and the oilseed industry. Therefore, developing innovative nematode-resistant technologies and breeding new, disease-resistant varieties have become urgent needs in the field of agricultural science and technology. Effective nematode-resistant breeding strategies will not only help improve soybean yield and quality but also reduce international soybean trade costs, enhance my country's competitiveness in the global soybean market, and thus promote the sustainable development of the soybean industry. Summary of the Invention

[0006] To address the existing need for soybean nematode-resistant breeding, this invention provides... GmXTH2 Genes and GmXTH19 The specific technical solutions for applying genes to enhance plant resistance to nematodes are as follows:

[0007] In a first aspect, the present invention provides GmXTH2 Genes and / or GmXTH19 The application of genes in enhancing plant resistance to nematodes, the aforementioned GmXTH2 The nucleotide sequence of the gene is shown in SEQ ID NO.1. GmXTH19 The nucleotide sequence of the gene is shown in SEQ ID NO.2.

[0008] Furthermore, the application method is as follows:

[0009] Through overexpression GmXTH2 Genes and / or GmXTH19 Genes that thicken the cell walls of plants to enhance their resistance to nematodes.

[0010] Furthermore, the plant in question is soybean.

[0011] Furthermore, the soybeans are Williams 82 or Forrest.

[0012] Secondly, the present invention provides the application of GmXTH2 protein and / or GmXTH19 protein in enhancing plant resistance to nematodes, wherein the amino acid sequence of the GmXTH2 protein is shown in SEQ ID NO.3 and the amino acid sequence of the GmXTH19 protein is shown in SEQ ID NO.4.

[0013] Thirdly, the present invention provides the application of a recombinant vector in enhancing plant resistance to nematodes, the recombinant vector comprising... GmXTH2 Genes and / or GmXTH19 Genes; the stated GmXTH2 The nucleotide sequence of the gene is shown in SEQ ID NO.1. GmXTH19 The nucleotide sequence of the gene is shown in SEQ ID NO.2.

[0014] Furthermore, the plant in question is soybean.

[0015] Furthermore, the soybeans are Williams 82 or Forrest.

[0016] Fourthly, the present invention provides the application of genetically engineered bacteria in enhancing plant resistance to nematodes, wherein the genetically engineered bacteria contain... GmXTH2 Genes and / or GmXTH19 Genes; the stated GmXTH2The nucleotide sequence of the gene is shown in SEQ ID NO.1. GmXTH19 The nucleotide sequence of the gene is shown in SEQ ID NO.2.

[0017] Furthermore, the plant in question is soybean.

[0018] Furthermore, the soybeans are Williams 82 or Forrest.

[0019] Fifthly, the present invention provides a method for enhancing the resistance of soybeans to nematodes by overexpressing... GmXTH2 Genes and / or GmXTH19 Genes that thicken the cell walls of soybeans to enhance their resistance to nematodes.

[0020] Furthermore, the soybeans are Williams 82 or Forrest.

[0021] Furthermore, the overexpression GmXTH2 Genes and / or GmXTH19 Genes, including the following steps:

[0022] (1) with GmXTH2 Genes and / or GmXTH19 Primer sequences were designed using the gene's cDNA as a template to construct an overexpression vector;

[0023] (2) The vector was transferred into Agrobacterium competent cells to obtain overexpression. GmXTH2 Genes and / or GmXTH19 Agrobacterium gene;

[0024] (3) containing GmXTH2 Genes and / or GmXTH19 Agrobacterium, a gene overexpression vector, was transferred into soybean explants, and seedlings were obtained through tissue culture. Seedlings were then selected for overexpression. GmXTH2 Genes and / or GmXTH19 Soybean plants with genetically modified genes.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] This invention is the first to discover the presence of nematodes in soybean roots after nematode infection. XTH The upregulation of family genes was verified. GmXTH2 Genes and GmXTH19 The role of genes in soybean SCN resistance was discovered. GmXTH2 and GmXTH19 Overexpression of the gene can thicken the cell wall of soybean cells, thereby enhancing soybean SCN resistance and providing new gene resources and pathways for breeding resistant soybeans. Attached Figure Description

[0027] Figure 1 This is a schematic diagram of the evolutionary tree of the XTH family in soybean.

[0028] Figure 2 After inoculating soybeans with soybean cyst nematode GmXTH2 Genes and GmXTH19 A schematic diagram illustrating the relative changes in gene expression levels.

[0029] Figure 3 This is a schematic diagram showing the sequence information of the GmXTH2 and GmXTH19 proteins.

[0030] Figure 4 Typical Tdtomato / RFP fluorescence images of hairy roots from the OE-GmXTH2 transgenic organism; in which fluorescently labeled hairy roots indicate GmXTH2 Successful expression.

[0031] Figure 5 For OE-GmXTH2 and OE-GmXTH19 transgenic roots GmXTH2 and GmXTH19 A schematic diagram of transcription levels.

[0032] Figure 6 This is a schematic diagram of the nematode resistance phenotype of soybean hairy roots overexpressing OE-GmXTH2 and OE-GmXTH19. In the diagram, A is a representative image stained with acid fuchsin, bar=250μm, B is the statistical analysis results of the nematode resistance level of transgenic roots, and the vertical axis represents the proportion of nematodes in the J2 and J3 stages and the cyst stage to the total number of nematodes. The lower the value, the stronger the nematode resistance.

[0033] Figure 7 The images show transmission electron microscopy (TEM) sections of soybean hairy root cells overexpressing OE-GmXTH2 and OE-GmXTH19. In the images, A is a representative TEM section of the cell wall, and B is a schematic diagram of the statistical results of cell wall thickness in the overexpression lines. Detailed Implementation

[0034] 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.

[0035] 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.

[0036] 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.

[0037] The culture medium used in the following examples:

[0038] LB medium formulation:

[0039] Table 1

[0040]

[0041] Co-culture medium:

[0042] Table 2

[0043]

[0044] Rooting medium formula:

[0045] Table 3

[0046]

[0047] In the following embodiments, GmXTH2 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the GmXTH2 protein is shown in SEQ ID NO.3. GmXTH19 The nucleotide sequence of the gene is shown in SEQ ID NO.2, and the amino acid sequence of the GmXTH19 protein is shown in SEQ ID NO.4.

[0048] SEQ ID NO.1:

[0049] ATGGCACAAATTGAGAAATTTTTGGTAGCTTTGTTCCTCTTTGCAATGGCACACAGTATAATCCTCGTAGATGCCACTTTTTCCAAAAGCATGTACATCACTTGGGGTTCCCGACATGCATCAATGCAGGGTGAAGACCTCCAACTTGTGTTGGATCAAACCTCAGGATCCGCAGCACAAACAAAGAAAGCATTCTTATTTGGAAGTATTGAATCGAGAATCAAGCTAGTGCCTGGTAATTCTGCAGGAACAGTTACTGCCTACTATCTATCTTCTACCGGAAGCCAGCATGATGAGATAGATTTTGAGTTCTTAGGCAACATTTCAGGACAACCATACATTGTCCATACAAACATATATACACAAGGAAATGGAAGCAGGGAGCAACAATTTTACCTCTGGTTTGACCCAACTGCTGATTTTCACAATTACACCATTCATTGGAACCCCATTGAAGTTGTGTGGTACATTGATAGTATACCAATTAGGGCGTATCGAAACTATGAAAATGAAGGCATTGCTTACCCAAACAAGCAAGGAATGAGGGTTTATACTAGCCTGTGGAATGCAGATGATTGGGCCACTAGAGGTGGGCTTGTTAAGACCAATTGGAGTGGTGCACCATTCATAGCCAGATTTAATCATTTTAGAGCAAGGGCTTGTAAGTGGAATGGAGCAGTTAGTATCAACCAATGTGCATTAAATTTCCCTGCCAATTGGTGGACCTCTCCCACATACAAGCAATTGAGTTATGCCAAACTGGGCCAGATGAATTGGGTCAGGAACAATTACATGATCTATGATTATTGCAGAGATACCAAAAGATTCAATGGACGGATGCCTCCTGAATGCTTCAAGTCACAATTCTAA

[0050] SEQ ID NO.2:

[0051] ATGGCACAAATTGAGAAATTGTTGGTAGCTTTGTTCCTTTTTGCAGTGGCACAAAGCATAATCCTCGTAGATGCCACCTTTTCCAAAAGCATGTACATCACTTGGGGTTCTCAACATGCATCAATGCAGGGTGAAGACCTCCAACTTGTGTTGGATCAAACCTCAGGATCTGCAGCTCAAACAAAGAAAGCATTCTTATTTGGAAGTATTGAATCGAGAATCAAGCTGGTCCCTGGTAATTCTGCAGGAACAGTTACTGCCTACTATCTATCCTCTACAGGAAGCCAACATGATGAGATAGATTTCGAGTTCTTAGGCAACATTTCAGGACAACCATACATTGTCCATACGAACATATACACACAAGGAAATGGAAGCAGGGAGCAACAATTTTACCTCTGGTTTGACCCAACTTCTGACTTTCACAATTACACCATTCATTGGAACCCCATTGAAGTTGTGTGGTATATTGATAGTATACCAATTAGGGTGTACCGAAACTATGAAAATGAGGGCATTGCTTACCCAAACAAGCAAGGAATGAGGGTTTATACTAGCCTGTGGAATGCAGATGATTGGGCCACTAGGGGTGGGCTTGTTAAGACCAATTGGAGTGGTGCACCATTCATAGCCAGATTTAATCATTTTAGAGCAAGGGCTTGTAAGTGGAATGGAGCAGTTAGTATCAACCAATGTGCCTTAAATTTCCCTGCCAATTGGTGGACCTCTCCCACATACAAGCAATTGAGTTATGCCAAATTGGGACAGATGAATTGGGTCAGGAACAATTACATGATCTATGATTATTGCAGAGATACTAAAAGATTCAATGGAATGATGCCTCCTGAATGCTTCAAGTCACAATTCTAA

[0052] SEQ ID NO.3:

[0053] MAQIEKFLVALFLFAMAHSIILVDATFSKSMYITWGSRHASMQGEDLQLVLDQTSGSAAQTKKAFLFGSIESRIKLVPGNSAGTVTAYYLSSTGSQHDEIDFEFLGNISGQPYIVHTNIYTQGNGSREQQFYLWFDPTADFHNY TIHWNPIEVVWYIDSIPIRAYRNYENEGIAYPNKQGMRVYTSLWNADDWATRGGLVKTNWSGAPFIARFNHFRARACKWNGAVSINQCALNFPANWWTSPTYKQLSYAKLGQMNWVRNNYMIYDYCRDTKRFNGRMPPECFKSQF

[0054] SEQ ID NO.4:

[0055] MAQIEKLLVALFLFAVAQSIILVDATFSKSMYITWGSQHASMQGEDLQLVLDQTSGSAAQTKKAFLFGSIESRIKLVPGNSAGTVTAYYLSSTGSQHDEIDFEFLGNISGQPYIVHTNIYTQGNGSREQQFYLWFDPTSDFHNYT IHWNPIEVVWYIDSIPIRVYRNYENEGIAYPNKQGMRVYTSLWNADDWATRGGLVKTNWSGAPFIARFNHFRARACKWNGAVSINQCALNFPANWWTSPTYKQLSYAKLGQMNWVRNNYMIYDYCRDTKRFNGMMPPECFKSQF.

[0056] Example 1: Screening for nematode resistance genes

[0057] (1) An evolutionary relationship of the XTH family in soybean was analyzed, and a phylogenetic tree was constructed, revealing the evolutionary relationships between different XTH family genes and their conservation in soybean (e.g., Figure 1 (As shown).

[0058] (2) Transcriptome analysis was used to study the changes in gene expression in soybean hairy roots after infection by soybean cyst nematode (SCN).

[0059] First, gene expression in the SCN-infected group and the SCN-inoculated control group was quantitatively compared using transcriptional analysis software. The results showed that the expression of several XTH family genes was significantly upregulated in the treatment group (e.g., Figure 2 (As shown).

[0060] Subsequently, using the XTH gene sequence, a search was conducted in the Phytozome soybean genome database (https: / / phytozome-next.jgi.doe.gov / ) to identify two genes that showed the most significant response to SCN: XTH2 (Glyma.01G146000) and XTH19 (Glyma.09G193500). Further DNA sequence alignment showed that XTH2 and XTH19 had an amino acid sequence similarity of 96.4%, indicating that these two genes are homologous (e.g., ...). Figure 3 (As shown).

[0061] Example 2 GmXTH2, GmXTH19 Gene cloning and vector construction

[0062] 1. GmXTH2 / GmXTH19 Cloning of genes

[0063] Using cDNA from wild-type soybean Williams 82 (Wm82) to analyze soybean GmXTH2 PCR amplification was performed using the open reading frame (ORF) of the target fragment, with primers I and II, and Toyobo's KOD one PCR Master Mix as the reagent. The PCR products were detected by agarose gel electrophoresis, and the target fragment was recovered by gel excision.

[0064] The PCR reaction system is as follows:

[0065] Table 4

[0066]

[0067] Primer I: c gac gac aag acc gt g acc ATGGCACAAATTGAGAAATTTTTGGTAGC (where lowercase letters represent the homologous recombination vector sequence used for homologous recombination ligation)

[0068] Primer II: ga gga gaa gag ccg TCA AGAGATGTTGCATTCTGGAGGAAG (where lowercase letters represent the homologous recombination vector sequence, used for homologous recombination ligation)

[0069] Using cDNA from wild-type soybean Williams 82 (Wm82) to analyze soybean GmXTH19 PCR amplification was performed using the open reading frame (ORF) of the target fragment, with primers III and IV, and Toyobo's KOD one PCR Master Mix as the reagent. The PCR products were detected by agarose gel electrophoresis, and the target fragment was recovered by gel excision. The PCR reaction system was the same as above.

[0070] Primer III: c gac gac aag acc gt g acc ATGGCACAAATTGAGAAATTGTTGGTAGCTTTGTTCCTT (where lowercase letters represent the homologous recombination vector sequence, used for homologous recombination ligation)

[0071] Primer IV: ga gga gaa gag ccg TTAGAATTGTGACTTGAAGCATTCAGGAGGCATCAT (where lowercase letters represent the homologous recombination vector sequence, used for homologous recombination ligation)

[0072] Amplification GmXTH2 and GmXTH19 Gene fragments.

[0073] Example 3 Overexpression GmXTH2 Gene GmXTH19 Obtaining genetically modified soybean roots

[0074] 1. Carrier Construction

[0075] The amplified GmXTH2 and GmXTH19 The gene fragment was synthesized using homologous recombination and combined with the 35S promoter of cauliflower mosaic virus (CaMV) and the gene obtained in Example 2. GmXTH2 or GmXTH19 The 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.

[0076] The reaction system is as follows:

[0077] Table 5

[0078]

[0079] Reaction conditions: 50℃, 15min.

[0080] 2. Plasmid preparation

[0081] 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-. GmXTH2 and OE- GmXTH19 .

[0082] (1) Escherichia coli transformation:

[0083] Table 6

[0084]

[0085] 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.

[0086] (2) Colony PCR:

[0087] Table 7

[0088]

[0089] The forward and reverse primers are the same as those used in Example 2.

[0090] Use a pipette tip to pick up the colonies obtained in step (1) and add them to the prepared system.

[0091] The PCR reaction conditions are as follows:

[0092] (1) Pre-denaturation at 95℃ for 5 min; (2) Denaturation at 95℃ for 30 sec, 55℃ for 30 sec, and 72℃ for 45 sec for a total of 35 cycles; (3) Storage at 4℃.

[0093] 3. Preparation of Agrobacterium

[0094] The obtained OE- GmXTH2 or OE- GmXTH19 plasmid vector transformed into Agrobacterium rhizogenes ( Agrobacterium rhizogenes In 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.

[0095] 4. Transformation of soybean hairy root system

[0096] 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.

[0097] 5. Screening and validation of transgenic root systems

[0098] After 3 days of culture on the 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 4 (As shown).

[0099] 6. GmXTH2 Gene overexpression verification

[0100] 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. The specific methods are as follows:

[0101] The steps are as follows:

[0102] Plant RNA extraction: Plant RNA was extracted using the Trizol method in this experiment. The steps were as follows: Soybean tissue was ground in liquid nitrogen and collected into a 1.5 mL centrifuge tube. 1 mL of Trizol was added and vortexed to mix well, and the mixture was allowed to stand at room temperature for 5 min. 200 μL of chloroform was added, and the mixture was vigorously vortexed for 15 sec and then allowed to stand at room temperature for 5 min. The mixture was centrifuged at 12,000 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 mixture was inverted to mix well and allowed to stand at room temperature for 10 min. The mixture was centrifuged at 12,000 rpm for 10 min at 4 °C, and the supernatant was discarded. The precipitate was washed with 1 mL of 75% ethanol, centrifuged at 7,500 rpm for 5 min at 4 °C, and the supernatant was discarded. The washing was repeated once, followed by empty centrifugation. The ethanol was 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 RNA was stored at -80 °C for later use.

[0103] 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.

[0104] Quantitative PCR: AceQ qPCR SYBR Green Master Mix was used. The reaction system is as follows:

[0105] Table 8

[0106]

[0107] After preparing the system, place the sample on the Bio-Rad CFX96™ Real-Time PCR Detection System for amplification.

[0108] like Figure 5 As shown, compared with the control group, the root of the overexpression group had higher concentrations of [unclear text - possibly related to root canal or stem cells]. GmXTH2 Gene expression levels were significantly increased.

[0109] Example 4 Overexpression GmXTH2 Gene or GmXTH19 Gene-induced soybean resistance to nematodes

[0110] 1. Nematode culture

[0111] Soybean cyst nematodes preserved from the Plant Nematode Laboratory of Zhejiang University Soybean Cyst Nematode A population of SCN, Hg Type 0 was used to hatch nematode eggs using 3 mM ZnCl2 buffer and incubated at room temperature for 5 days to allow them to develop to the J2 stage. The J2-stage nematodes were then treated for 3 min with a solution containing 0.1 g / L HgCl2 and 0.01% sodium azide, followed by two rinses with sterile water. The J2-stage SCNs were then suspended in 0.05% sterile agarose solution for root inoculation.

[0112] 2. Nematode inoculation

[0113] Under greenhouse conditions, J2-stage nematodes were inoculated onto the root tips of transgenic soybean plants and non-transgenic control plants, with at least 500 nematodes inoculated per plant. The inoculated soybeans were then cultured in an incubator for 14 days. The developmental status of the nematodes was subsequently observed.

[0114] 3. Observation and statistics of nematode development

[0115] The development of nematodes in soybean roots was observed and recorded using acid fuchsin staining. The specific steps are as follows:

[0116] First, remove the soybean roots from the culture medium and wash them with clean water. Then, dilute sodium hypochlorite with water at a ratio of 1:4 and soak the soybean roots in the sodium hypochlorite solution for 5 minutes. Wash the roots three times with clean water to remove excess sodium hypochlorite. Add acidic fuchsin staining solution and boil in a water bath for 5 minutes. Finally, wash away the staining solution with tap water. Observe the nematodes in the roots of the transgenic and control groups under a microscope and record the developmental stages and numbers of the nematodes.

[0117] The results are as follows Figure 6 A and Figure 6 As shown in Figure B, compared with the control group... GmXTH2 Genes and GmXTH19 Overexpression of the gene significantly suppressed the development of Wm82 and For soybean root nematodes. This indicates that... GmXTH2 and GmXTH19 It plays a crucial role in enhancing resistance to soybean cyst nematode infection.

[0118] Example 5 GmXTH2 Genes and GmXTH19 Research on the mechanisms that enhance plant resistance to nematodes

[0119] Using the same method as in Example 3, the constructed GmXTH2 and GmXTH19 The overexpression vector was transformed into soybean, and positive soybean hairy roots were selected by fluorescence screening. The transformed soybean roots were then embedded in resin for ultrathin section preparation and transmission electron microscopy (TEM) observation. The specific steps are as follows:

[0120] 1. Fixation: The treated soybean roots were fixed with 2.5% glutaraldehyde at room temperature in the dark for 4 hours, and then fixed with 1% osmium tetroxide in 0.1 M pH 7.0 phosphate buffer for 2 hours. After fixation, the samples were rinsed with 100 mM phosphate buffer (pH 7.0) for 15 minutes each time, for a total of 3 times, to remove excess fixative.

[0121] 2. Dehydration: The product was treated sequentially with different concentrations of ethanol (50%, 70%, 80%, 90%, 95% and 100%) and pure acetone, for 20 minutes at each concentration, and the dehydration process was carried out at room temperature.

[0122] 3. Embedding: The dehydrated sample was embedded in LR White resin and then heated and polymerized at 60°C to prepare a solid block that retains the fine structure.

[0123] 4. Sectioning: The resin block was sectioned into ultrathin sections (70 nm) using a Leica UC 6 microtome (Leica, Vienna, Austria) and a Diatome diamond blade (Diatome, Switzerland), and the sections were placed on a 200-mesh copper grid.

[0124] 5. Staining: Double staining is performed on the ultrathin sections. First, stain with uranium acetate for 15 minutes, and then stain with lead citrate for 15 minutes.

[0125] 6. TEM observation: The sections were observed using an H-7650 transmission electron microscope (Hitachi, Ibaraki, Japan) at an accelerating voltage of 80 kV, with a focus on areas of change in cell wall structure.

[0126] like Figure 7 As shown, through ultrathin sections and transmission electron microscopy, it can be clearly observed that... GmXTH2 and GmXTH19 Significant thickening of soybean root cell walls was observed with overexpression.

Claims

1. GmXTH2 Genes and / or GmXTH19 The application of genes in enhancing plant resistance to nematodes is characterized by, The GmXTH2 The nucleotide sequence of the gene is shown in SEQ ID NO.

1. GmXTH19 The nucleotide sequence of the gene is shown in SEQ ID NO.2, and the plant is soybean.

2. The application according to claim 1, characterized in that, The application method is as follows: Through overexpression GmXTH2 Genes and / or GmXTH19 Genes to enhance a plant's resistance to nematodes.

3. The application of GmXTH2 protein and / or GmXTH19 protein in enhancing plant resistance to nematodes, characterized in that, The amino acid sequence of the GmXTH2 protein is shown in SEQ ID NO.3, and the amino acid sequence of the GmXTH19 protein is shown in SEQ ID NO.

4. The plant is soybean.

4. The application of recombinant vectors in enhancing plant resistance to nematodes, characterized in that, The recombinant vector contains GmXTH2 Genes and / or GmXTH19 Genes; the stated GmXTH2 The nucleotide sequence of the gene is shown in SEQ ID NO.

1. GmXTH19 The nucleotide sequence of the gene is shown in SEQ ID NO.2, and the plant is soybean.

5. The application of genetically engineered bacteria in enhancing plant resistance to nematodes, characterized in that, The genetically engineered bacteria contain GmXTH2 Genes and / or GmXTH19 Genes; the stated GmXTH2 The nucleotide sequence of the gene is shown in SEQ ID NO.

1. GmXTH19 The nucleotide sequence of the gene is shown in SEQ ID NO.2, and the plant is soybean.

6. A method for enhancing soybean's resistance to nematodes, characterized in that, Through overexpression GmXTH2 Genes and / or GmXTH19 Genes to enhance soybean's resistance to nematodes.

7. The method according to claim 6, characterized in that, The overexpression GmXTH2 Genes and / or GmXTH19 Genes, including the following steps: (1) with GmXTH2 Genes and / or GmXTH19 Primer sequences were designed using the gene's cDNA as a template to construct an overexpression vector; (2) The vector was transferred into Agrobacterium competent cells to obtain overexpression. GmXTH2 Genes and / or GmXTH19 Agrobacterium gene; (3) containing GmXTH2 Genes and / or GmXTH19 Agrobacterium, a gene overexpression vector, was transferred into soybean explants, and seedlings were obtained through tissue culture. Seedlings were then selected for overexpression. GmXTH2 Genes and / or GmXTH19 Soybean plants with genetically modified genes.

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