Application of GmXTH2 gene and GmXTH19 gene in enhancing nematode resistance of plants

Abstract

The invention belongs to the technical field of biology, and particularly relates to application of a GmXTH2 gene and a GmXTH19 gene in enhancing the nematode resistance of plants, the nucleotide sequence of the GmXTH2 gene is shown as SEQ ID NO.1, and the nucleotide sequence of the GmXTH19 gene is shown as SEQ ID NO.3. The invention further discloses a preparation method of the GmXTH2 gene and the GmXTH19 gene. Up-regulated expression of XTH family genes in soybean roots after nematode infection is found for the first time, the effects of the GmXTH2 genes and the GmXTH19 genes in soybean SCN resistance are verified, it is found that overexpression of the GmXTH2 genes and the GmXTH19 genes can thicken cell walls of soybean cells, the effect of enhancing soybean SCN resistance is achieved, and a new gene resource and a new implementation approach are provided for breeding of resistant soybeans.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to the application of GmXTH2 gene and GmXTH19 gene in enhancing the nematode resistance of plants. Background Art

[0002] Soybean Cyst Nematode (SCN) is one of the most destructive pathogens in global soybean production. Its threat to the soybean industry is not only reflected in large-scale yield losses, but also increases production costs by reducing the market value of soybeans, thereby affecting the stability of the global soybean supply chain. SCN mainly parasitizes on the soybean roots, and by destroying the water and nutrient absorption systems of the roots, it causes the plants to grow slowly, the leaves to turn yellow, dwarf, and even lead to the death of the whole plant in severe cases. Research shows that in areas severely affected by soybean cyst nematodes, the yield reduction can be as high as 30%, which poses a huge threat to the economic benefits of soybean cultivation and the income of farmers.

[0003] As the origin of soybeans, China's traditional demand for soybeans is not only limited to being a food source of high-quality plant protein, but also includes its wide application in feed and vegetable oils. However, currently 85% of China's soybean demand relies on imports, and the main source countries include the United States, Brazil, and Argentina. The high incidence of soybean cyst nematode disease and complex factors in the global soybean supply chain have led to a year-on-year increase in the cost of soybean imports. Public data shows that China imported 100 million tons of soybeans in 2020, which makes the global dependence of the soybean industry more prominent. The United States is the largest soybean producer in the world and also one of the countries most severely affected by soybean cyst nematode disease. Each year, soybean cyst nematode disease alone causes economic losses of more than 1 billion US dollars to the US agriculture. With the gradual expansion of the soybean planting scale in China, the risk of soybean cyst nematode disaster in China has also increased significantly. According to statistics, the affected area of soybean cyst nematodes in the main soybean producing areas of China has exceeded 2.67 million hectares, and the soybean yield reduction ranges from 10% to 30%, seriously affecting soybean yield and industrial development. Due to the small size of soybean cyst nematodes and their parasitism on soybean roots, it is often difficult to detect them in time, and the symptoms they cause are similar to those of nutrient deficiency, which is extremely easy to be misdiagnosed, further increasing the difficulty of prevention and control.

[0004] At present, the prevention and control of soybean cyst nematodes mainly rely on the breeding of disease-resistant varieties and crop rotation in farmland. However, with the increase in the diversity and adaptability of nematode populations, the resistance of disease-resistant varieties gradually loses, and the implementation of rotation is also restricted by the crop planting cycle, so the control effect is unstable and difficult to maintain for a long time. With the continuous pressure on global food and oil supply, it is particularly urgent to develop more efficient and long-lasting nematode-resistant breeding strategies.

[0005] As an important source of global food and vegetable protein, soybeans are irreplaceable in the livestock, catering, and oilseed industries. Therefore, developing innovative anti-nematode technologies and cultivating new varieties with strong disease resistance have become an urgent need in the current agricultural science and technology field. Effective anti-nematode breeding strategies will not only help improve the yield and quality of soybeans but also reduce the cost of international soybean trade, enhance China's competitiveness in the global soybean market, and thus promote the sustainable development of the soybean industry. Summary of the Invention

[0006] In response to the need for soybean anti-nematode breeding in the prior art, the present invention provides the application of GmXTH2 gene and GmXTH19 gene in enhancing the nematode resistance of plants. The specific technical solutions are as follows:

[0007] First, the present invention provides the application of GmXTH2 gene and / or GmXTH19 gene in enhancing the nematode resistance of plants. 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.

[0008] Furthermore, the way of the application is:

[0009] By overexpressing the GmXTH2 gene and / or GmXTH19 gene, the cell wall of the plant is thickened to enhance the nematode resistance of the plant.

[0010] Furthermore, the plant is soybean.

[0011] Furthermore, the soybean is Williams 82 or Forrest.

[0012] Second, the present invention provides the application of GmXTH2 protein and / or GmXTH19 protein in enhancing the nematode resistance of plants. The amino acid sequence of the GmXTH2 protein is shown in SEQ ID NO.2, and the amino acid sequence of the GmXTH19 protein is shown in SEQ ID NO.4.

[0013] Third, the present invention provides the application of a recombinant vector in enhancing the nematode resistance of plants. The recombinant vector contains the GmXTH2 gene and / or GmXTH19 gene; 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.

[0014] Furthermore, the plant is soybean.

[0015] Furthermore, the soybean is Williams 82 or Forrest.

[0016] Fourthly, the present invention provides an application of a genetically engineered bacterium in enhancing the nematode resistance of plants, wherein the genetically engineered bacterium contains the GmXTH2 gene and / or the GmXTH19 gene; the nucleotide sequence of the GmXTH2 gene is as shown in SEQ ID NO.1, and the nucleotide sequence of the GmXTH19 gene is as shown in SEQ ID NO.3.

[0017] Furthermore, the plant is soybean.

[0018] Furthermore, the soybean is Williams 82 or Forrest.

[0019] Fifthly, the present invention provides a method for enhancing the nematode resistance of soybean, by overexpressing the GmXTH2 gene and / or the GmXTH19 gene to thicken the cell wall of soybean, so as to enhance the nematode resistance of soybean.

[0020] Furthermore, the soybean is Williams 82 or Forrest.

[0021] Furthermore, the overexpression of the GmXTH2 gene and / or the GmXTH19 gene includes the following steps:

[0022] (1) Design primer sequences using the cDNA of the GmXTH2 gene and / or the GmXTH19 gene as a template, and construct an overexpression vector;

[0023] (2) Transfer the vector into Agrobacterium competent cells to obtain Agrobacterium overexpressing the GmXTH2 gene and / or the GmXTH19 gene;

[0024] (3) Transfer the Agrobacterium containing the overexpression vector of the GmXTH2 gene and / or the GmXTH19 gene into the explants of soybean, obtain seedlings through tissue culture, and screen to obtain soybean plants overexpressing the GmXTH2 gene and / or the GmXTH19 gene.

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

[0026] The present invention firstly discovers the up-regulated expression of XTH family genes in soybean roots after nematode infection, verifies the role of the GmXTH2 gene and the GmXTH19 gene in soybean SCN resistance, and finds that the overexpression of the GmXTH2 and GmXTH19 genes can thicken the cell wall of soybean, playing a role in enhancing soybean SCN resistance, providing new gene resources and implementation ways for the breeding of resistant soybeans. Description of the Drawings

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

[0028] Figure 2 Schematic diagram of the changes in the relative expression levels of GmXTH2 gene and GmXTH19 gene after inoculating soybean with soybean cyst nematode.

[0029] Figure 3 Schematic diagram of the sequence information of GmXTH2 and GmXTH19 proteins.

[0030] Figure 4 Typical Tdtomato / RFP fluorescence image of OE - GmXTH2 transgenic hairy roots; among them, the fluorescently labeled hairy roots indicate the successful overexpression of GmXTH2.

[0031] Figure 5 Schematic diagram of the transcriptional levels of GmXTH2 and GmXTH19 in OE - GmXTH2 and OE - GmXTH19 transgenic roots.

[0032] Figure 6 Schematic diagram of the nematode - resistant phenotypes of OE - GmXTH2 and OE - GmXTH19 overexpressing soybean hairy roots; among them, A is a representative picture stained with acid fuchsin, bar = 250μm, and B is the statistical analysis result of the nematode - resistant level of transgenic roots. The vertical axis represents the proportion of nematodes in the J2 and J3 stages and cyst stages among the total number of nematodes. The lower the value, the stronger the nematode - resistant ability.

[0033] Figure 7 Transmission electron microscopy section results of the cell walls of OE - GmXTH2 and OE - GmXTH19 overexpressing soybean hairy roots; among them, A is a representative transmission electron microscopy section picture of the cell wall, and B is a schematic diagram of the statistical results of the cell wall thickness in the overexpressing lines. Detailed implementation manners

[0034] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments. It should be noted that the following detailed description is exemplary and only a part of the embodiments of the present invention, rather than all of the embodiments.

[0035] Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts shall 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 technical field to which this application belongs. The experimental materials used in the embodiments of the present invention are all conventional experimental materials in the art and can be obtained through commercial channels. The experimental methods without specified detailed conditions are carried out according to conventional experimental methods or according to the operation manuals recommended by the suppliers.

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

[0038] LB culture medium formula:

[0039] Table 1

[0040]

[0041] Co-culture medium:

[0042] Table 2

[0043]

[0044] Rooting culture medium formula:

[0045] Table 3

[0046]

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

[0054] SEQ ID NO.4:

[0055] MAQIEKLLVALFLFAVAQSIILVDATFSKSMYITWGSQHASMQGEDLQLVLDQTSGSAAQTKKAFLFGSIESRIKLVPGNSAGTVTAYYLSSTGSQHDEIDFEFLGNISGQPYIVHTNIYTQGNGSREQQFYLWFDPTSDFHNYTIHWNPIEVVWYIDSIPIRVYRNYENEGIAYPNKQGMRVYTSLWNADDWATRGGLVKTNWSGAPFIARFNHFRARACKWNGAVSINQCALNFPANWWTSPTYKQLSYAKLGQMNWVRNNYMIYDYCRDTKRFNGMMPPECFKSQF。

[0056] Example 1: Screening of nematode resistance genes

[0057] (1) Analyzed the evolutionary relationship of the XTH family in soybean, constructed an evolutionary tree, and revealed the evolutionary relationship between different XTH family genes and their conservation in soybean (as Figure 1 shown).

[0058] (2) Conducted an in-depth study on the gene expression changes in soybean hairy roots after infection with soybean cyst nematode (SCN) using transcriptome analysis method.

[0059] First, quantitatively compared the gene expression of the SCN infection treatment group and the control group inoculated only with SCN through transcript analysis software. The results showed that the expression of multiple XTH family genes was significantly up-regulated in the treatment group (as Figure 2 shown).

[0060] Subsequently, using the gene sequences of XTH, two genes that responded most significantly to SCN were identified by retrieving through the Phytozome soybean genome database (https: / / phytozome-next.jgi.doe.gov / ), namely XTH2 (Glyma.01G146000) and XTH19 (Glyma.09G193500). Further DNA sequence alignment results showed that the amino acid sequence similarity between XTH2 and XTH19 was as high as 96.4%, indicating that these two genes were homologous genes (as Figure 3 shown).

[0061] Example 2 Gene Cloning and Vector Construction of GmXTH2 and GmXTH19

[0062] 1. Cloning of GmXTH2 / GmXTH19 Genes

[0063] The open reading frame (ORF) of soybean GmXTH2 was PCR amplified using the cDNA of wild-type soybean Williams 82 (Wm82), with primers I and II, and the reagent was KOD one PCR Master Mix from Toyobo Company. The PCR product was detected by agarose gel electrophoresis, and the target fragment was recovered by cutting the gel.

[0064] The PCR reaction system was as follows:

[0065] Table 4

[0066]

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

[0068] Primer II: ga gga gaa gag ccg TCAAGAGATGTTGCATTCTGGAGGAAG (where the lowercase letters are part of the sequence of the homologous recombination vector, used for homologous recombination ligation)

[0069] The open reading frame (ORF) of soybean GmXTH19 was PCR amplified using the cDNA of wild-type soybean Williams 82 (Wm82), with primers III and IV, and the reagent was KOD one PCR Master Mix from Toyobo Company. The PCR product was detected by agarose gel electrophoresis, and the target fragment was recovered by cutting the gel. The PCR reaction system was the same as above.

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

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

[0072] Gene fragments of GmXTH2 and GmXTH19 were amplified.

[0073] Example 3 Obtaining of Soybean Roots Overexpressing GmXTH2 Gene and GmXTH19 Gene

[0074] 1. Vector construction

[0075] The amplified gene fragments of GmXTH2 and GmXTH19 were integrated into the binary vector pAGM4673 by homologous recombination method, together with the cauliflower mosaic virus (CaMV) 35S promoter, the purified PCR products of GmXTH2 or GmXTH19 obtained in Example 2, and the nopaline synthase (NOS) terminator. This backbone vector also carries the 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 °C, 15 min.

[0080] 2. Plasmid preparation

[0081] By means of Escherichia coli transformation and colony PCR screening, the samples with the target bands were further verified by PCR, and finally the successfully ligated vectors OE-GmXTH2 and OE-GmXTH19 were obtained.

[0082] (1) Escherichia coli transformation:

[0083] Table 6

[0084]

[0085] Let it stand on ice for 30 min; incubate in a 42 °C water bath for 45 s; let it stand on ice for 2 min; add 1 mL of antibiotic-free LB liquid medium, place it at 37 °C, and shake it in a shaker at 200 rpm for 1 h; after taking it out, centrifuge it at 6000 rpm for 2 min, remove the supernatant, and spread the remainder on Kan LB solid medium and culture it overnight at 37 °C.

[0086] (2) Colony PCR:

[0087] Table 7

[0088]

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

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

[0091] The PCR reaction conditions are:

[0092] (1) Pre-denaturation at 95 °C for 5 min; (2) Denaturation at 95 °C for 30 sec, annealing at 55 °C for 30 sec, extension at 72 °C for 45 sec, for a total of 35 cycles; (3) Store at 4 °C.

[0093] 3. Agrobacterium preparation

[0094] Transform the prepared OE-GmXTH2 or OE-GmXTH19 plasmid vector into the Agrobacterium rhizogenes ARqua1 strain. After 2 days, pick positive clones and inoculate them into selective liquid medium, shake and culture them at 28 °C, collect the bacteria, and resuspend them with infiltration buffer (3.0 - 3.5 g / L of B5 medium, 28 - 32 g / L of sucrose, 3.8 - 4.0 g / L of MES, 38 - 42 mg / L of acetosyringone, 1.5 - 1.8 mg / L of 6-BA, 0.02 - 0.03 mg / L of gibberellin, 100 mL / L of surfactant Silwet L-77, pH = 5.4) to an OD 600 = about 0.7 and incubate at room temperature for 2 h.

[0095] 4. Transformation of soybean hairy roots

[0096] Select healthy and plump soybean cotyledons, disinfect them with 75% ethanol for 30 s, rinse them once with water, then soak them in disinfectant for 4 min for disinfection, and finally soak them in water three times, 5 minutes each time. Place the cotyledons in the Agrobacterium suspension in a laminar flow hood, cut off one-fifth of the bottom of the soybean cotyledons with a blade, and perform Agrobacterium-mediated genetic transformation. Transfer the treated cotyledons to the co-culture medium covered with sterile filter paper and culture them. Culture in an incubator for 3 d.

[0097] 5. Screening and Verification of Transgenic Roots

[0098] Transfer the cotyledons cultured on the co-culture medium for 3 days to the rooting medium. After culturing at 28 °C for 14 days, observe the transformed roots using a fluorescence microscope, and screen out the soybean roots that appear red under the microscope (as shown in Figure 4 ).

[0099] 6. Verification of Overexpression of GmXTH2 Gene

[0100] Extract total RNA from positive soybean hairy root samples 3 days after infection with SCN, synthesize cDNA using HiScript Reverse Transcriptase Kit, and perform RT-qPCR using AceQ qPCR SYBR Green Master Mix. Each treatment has at least 3 biological replicates and 3 technical replicates. Calculate the relative expression level of the gene. Soybean Actin gene is used as an internal reference. The specific method is as follows:

[0101] The steps are as follows:

[0102] Extraction of plant RNA: In this experiment, Trizol method is used to extract plant RNA. The steps are as follows: Add liquid nitrogen to the soybean tissue and grind it, collect it into a 1.5 mL centrifuge tube, add 1 mL of Trizol and mix well by shaking, and let it stand at room temperature for 5 min; add 200 μL of chloroform, shake vigorously for 15 sec and then let it stand at room temperature for 5 min; centrifuge at 4 °C, 12,000 rpm for 15 min; pipette about 500 μL of the supernatant into a new centrifuge tube; add an equal volume of isopropanol, invert and mix well, and let it stand at room temperature for 10 min; centrifuge at 4 °C, 12,000 rpm for 10 min, discard the supernatant; wash the precipitate with 1 mL of 75% ethanol, centrifuge at 4 °C, 7,500 rpm for 5 min, discard the supernatant, repeat the washing once and then centrifuge without supernatant, and dry the alcohol in a laminar flow hood; finally, add 20 - 30 μL of RNase Free water to dissolve the RNA, measure the concentration and store it in a -80 °C refrigerator for later use.

[0103] Reverse transcription: Take 500 ng of the extracted RNA, add 2 μL of DNA Digester Mix, and make up to 14 μL with RNA free H 2 2O, incubate in a metal bath at 37 °C for 2 min; add 2 μL of reverse transcriptase, set the PCR program as 55 °C for 5 min, 85 °C for 5 s, and store it in a -20 °C refrigerator for later use.

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

[0105] Table 8

[0106]

[0107] After the system was prepared, the samples were placed on a Bio-Rad CFX96 Real-Time PCR Detection System for amplification. TM Real-Time PCR Detection System) for amplification.

[0108] As Figure 5 shown, compared with the control group, the expression level of the GmXTH2 gene in the roots of the overexpression group was significantly increased.

[0109] Example 4 Resistance of Soybeans Overexpressing GmXTH2 Gene or GmXTH19 Gene to Nematodes

[0110] 1. Nematode Cultivation

[0111] From the soybean cyst nematode (Soybean Cyst Nematode, SCN, Hg Type 0) population stored in the Plant Nematology Laboratory of Zhejiang University, nematode eggs were hatched using 3 mM ZnCl2 buffer solution and incubated at room temperature for 5 days to develop to the J2 stage. The J2-stage nematodes were treated with a solution containing 0.1 g / L HgCl2 and 0.01% sodium azide for 3 minutes, then rinsed twice with sterile water. The J2-stage SCNs were suspended in a 0.05% aqueous solution of sterile agarose and prepared for root inoculation.

[0112] 2. Nematode Inoculation

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

[0114] 3. Observation and Statistics of Nematode Development

[0115] The acid fuchsin staining method was used to observe and record the development of nematodes in soybean roots. The specific steps are as follows:

[0116] First, the soybean roots were taken out from the culture medium and washed with clean water. Then, sodium hypochlorite was diluted with water at a ratio of 1:4, and the soybean roots were soaked in the sodium hypochlorite solution for 5 minutes, washed three times with clean water to remove the excess sodium hypochlorite, acid fuchsin staining solution was added, and boiled in a water bath for 5 minutes. Finally, the staining solution was washed off with tap water. The nematodes in the roots of the transgenic group and the control group were observed under a microscope, and the developmental stages and developmental numbers of the nematodes were recorded.

[0117] The results are as Figure 6 A and Figure 6As shown in B, compared with the control group, the overexpression of GmXTH2 gene and GmXTH19 gene significantly inhibited the development of root-knot nematodes in Wm82 and For soybeans. This indicates that GmXTH2 and GmXTH19 play a crucial role in enhancing resistance to soybean cyst nematode infection.

[0118] Example 5 Research on the mechanism of GmXTH2 gene and GmXTH19 in enhancing plant resistance to nematodes

[0119] By the same method as in Example 3, the constructed overexpression vectors of GmXTH2 and GmXTH19 were transferred into soybeans, and positive soybean hairy roots were screened by fluorescence. The transformed soybean roots were embedded in resin for ultra-thin 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 in the dark at room temperature for 4 hours, and then fixed with 1% osmium tetroxide in 0.1M phosphate buffer at pH 7.0 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 samples were dehydrated at room temperature with a series of different concentrations of ethanol (50%, 70%, 80%, 90%, 95% and 100%) and pure acetone, each concentration for 20 minutes.

[0122] 3. Embedding: The dehydrated samples were embedded in LR White resin and heated and polymerized at 60 °C to prepare a solid block retaining fine structure.

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

[0124] 5. Staining: The ultrathin sections were double-stained, first stained with uranyl acetate for 15 minutes, and then stained 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, focusing on the regions with changes in cell wall structure.

[0126] As Figure 7As shown, through ultra-thin sectioning and transmission electron microscopy, a significant thickening of the cell walls in the roots of GmXTH2- and GmXTH19-overexpressing soybeans can be clearly observed.

Claims

1. Use of GmXTH2 gene and / or GmXTH19 gene in enhancing plant resistance to nematodes, characterized in that: 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.

2. The use according to claim 1, characterized in that: The application pathways are: By overexpressing the GmXTH2 gene and / or the GmXTH19 gene, the cell wall of the plant is thickened, thereby enhancing the plant's ability to resist nematodes.

3. The use according to any one of claims 1 or 2, characterized in that: The plant is soybean.

4. Use 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.2, and the amino acid sequence of the GmXTH19 protein is shown in SEQ ID NO.

4.

5. The use of a recombinant vector in enhancing the nematode resistance of a plant, characterized in that: The recombinant vector comprises the GmXTH2 gene and / or the GmXTH19 gene; 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.

6. The use of genetically engineered bacteria in enhancing the nematode resistance of plants, characterized in that: The genetically engineered bacteria comprises the GmXTH2 gene and / or the GmXTH19 gene; 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.

7. A method for enhancing soybean nematode resistance, characterized in that: By overexpressing the GmXTH2 gene and / or the GmXTH19 gene, the cell wall of soybean is thickened to enhance the nematode resistance of soybean.

8. The method according to claim 7, characterized in that The overexpression of GmXTH2 gene and / or GmXTH19 gene comprises the following steps: (1) Designing primer sequences using the cDNA of the GmXTH2 gene and / or the GmXTH19 gene as a template to construct an overexpression vector; (2) transferring the vector into Agrobacterium competent cells to obtain Agrobacterium overexpressing the GmXTH2 gene and / or the GmXTH19 gene; (3) The Agrobacterium containing the GmXTH2 gene and / or GmXTH19 gene overexpression vector is transferred into soybean explants, tissue culture is performed to obtain seedlings, and soybean plants overexpressing the GmXTH2 gene and / or GmXTH19 gene are screened and obtained.

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