Application of GmNIC1 gene and GmNIC2 gene in regulating plant resistance to soybean cyst nematode

Abstract

This invention belongs to the field of biotechnology, specifically involving GmNIC1 Genes and GmNIC2 The application of genes in regulating the ability of plants to resist soybean cyst nematode, the aforementioned GmNIC1 The nucleotide sequence of the gene is shown in SEQ ID NO.1; GmNIC2 The nucleotide sequence of the gene is shown in SEQ ID NO.2. This invention discloses for the first time... GmNIC1 as well as GmNIC2 The negative regulatory role of genes in soybean resistance to cyst nematode (SCN), and through construction GmNIC1 and GmNIC2 The study identified gene silencing mutants and verified that the loss of function in this gene family significantly enhances soybean resistance to SCN. This application provides a new target for a deeper understanding of the soybean-nematode interaction mechanism and offers novel gene resources and technical pathways for molecular design breeding of resistant soybeans.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically involving GmNIC1 Genes and GmNIC2 Application of genes in regulating the ability of plants to resist soybean cyst nematode. Background Technology

[0002] Soybean Cyst Nematode (SCN) is one of the most devastating soil-borne pathogens in global soybean production. This nematode infects soybean roots as its second-stage larvae (J2), establishing specialized syncytia that disrupt root function, thereby inhibiting plant growth and development and significantly reducing yield and quality. Reports indicate that SCN causes an average annual soybean yield loss of 10%-30%, and in severe cases, even exceeding 50%. It is widely distributed in major soybean-growing regions in the Americas and Asia, posing a persistent threat to the global soybean supply chain.

[0003] Traditional SCN control strategies mainly include crop rotation and the selection of resistant varieties. However, these methods are limited by agricultural planting systems, the genetic diversity of SCN populations, and the homogeneity of resistance genes, making long-term effective control difficult. Of particular concern is that multiple regions have reported new physiological races of SCN that can overcome the barriers of mainstream resistance genes (such as Rhg1 and Rhg4), causing some resistant varieties to become ineffective and leading to a phenomenon known as "resistance fatigue." Furthermore, SCN sporangia can remain dormant in the soil for up to 10 years, making it difficult to completely eradicate the population through conventional crop rotation.

[0004] The rise of genetic engineering technology has provided a breakthrough for developing novel nematode-resistant soybean varieties. Strategies including gene editing, RNA interference, and transgenic expression of nematode-resistant proteins have all made phased progress. For example, using CRISPR-Cas9 to knock out certain negative regulatory genes significantly improved soybean resistance to SCN. Genome-wide association studies and QTL mapping have also revealed several new resistance loci, laying the foundation for molecular design breeding. However, identifying key genes that can both regulate endogenous metabolism and target SCN adaptation mechanisms remains a current research challenge and hot topic. Summary of the Invention

[0005] To address the need for novel SCN resistance genes in existing technologies, this invention provides... GmNIC1 Genes and GmNIC2 The specific technical solution for the application of genes in regulating the ability of plants to resist soybean cyst nematodes is as follows:

[0006] In a first aspect, the present invention provides GmNIC1 Genes and / or GmNIC2 The application of genes in regulating the ability of plants to resist soybean cyst nematode, the aforementioned GmNIC1The nucleotide sequence of the gene is shown in SEQ ID NO.1; GmNIC2 The nucleotide sequence of the gene is shown in SEQ ID NO.2.

[0007] Furthermore, the application is carried out through methods such as knocking out, knocking down, silencing, or interfering. GmNIC1 and / or GmNIC2 Genes that enhance a plant's resistance to soybean cyst nematode.

[0008] Secondly, this invention provides the application of GmNIC1 protein and / or GmNIC2 protein in regulating the ability of plants to resist soybean cyst nematode, wherein the amino acid sequence of the GmNIC1 protein is shown in SEQ ID NO.3; and the amino acid sequence of the GmNIC2 protein is shown in SEQ ID NO.4.

[0009] Thirdly, this invention provides the application of a recombinant vector in regulating the ability of plants to resist soybean cyst nematode, wherein the recombinant vector comprises GmNIC1 Genes and / or GmNIC2 Genes, the ones mentioned GmNIC1 The nucleotide sequence of the gene is shown in SEQ ID NO. 1; GmNIC2 The nucleotide sequence of the gene is shown in SEQ ID NO.2.

[0010] Fourthly, this invention provides the application of genetically engineered bacteria in regulating the ability of plants to resist soybean cyst nematodes, wherein the genetically engineered bacteria contain... GmNIC1 Genes and / or GmNIC2 Genes, the ones mentioned GmNIC1 The nucleotide sequence of the gene is shown in SEQ ID NO.1; GmNIC2 The nucleotide sequence of the gene is shown in SEQ ID NO.2.

[0011] Fifthly, the present invention provides a method for enhancing the resistance of soybeans to soybean cyst nematode, comprising: knocking out, knocking down, silencing, or interfering with the soybean's... GmNIC1 and / or GmNIC2 Genes were introduced to enhance soybean's resistance to soybean cyst nematode; GmNIC1 The nucleotide sequence of the gene is shown in SEQ ID NO.1; GmNIC2 The nucleotide sequence of the gene is shown in SEQ ID NO.2.

[0012] Furthermore, the silence GmNIC1 and / or GmNIC2 Genes, including:

[0013] (1) According to GmNIC1 and / or GmNIC2 The genome sequence, constructGmNIC1 and / or GmNIC2 RNAi vectors for genes;

[0014] (2) The CRISPR / Cas9 vector was transferred into Agrobacterium competent cells to construct cells containing the knockout vector. GmNIC1 and / or GmNIC2 Agrobacterium strains carrying RNAi gene vectors;

[0015] (3) The Agrobacterium strain was used to transform soybeans and a silent strain was cultivated. GmNIC1 and / or GmNIC2 A homozygous soybean line with the gene.

[0016] Furthermore, the knockout GmNIC1 and / or GmNIC2 The gene was generated from soybeans using CRISPR / Cas9 technology. GmNIC1 and / or GmNIC2 Genes are knocked out.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] This invention discloses for the first time GmNIC1 as well as GmNIC2 The negative regulatory role of genes in soybean resistance to cyst nematode (SCN), and through construction GmNIC1 and / or GmNIC2 The study identified gene silencing mutants and verified that the loss of function in this gene family significantly enhances soybean resistance to SCN. This application provides a new target for a deeper understanding of the soybean-nematode interaction mechanism and offers novel gene resources and technical pathways for molecular design breeding of resistant soybeans. Attached Figure Description

[0019] Figure 1 for GmNIC A schematic diagram of the phylogenetic tree of gene families in soybeans.

[0020] Figure 2 Day 8 (8 dpi) after soybean inoculation with soybean cyst nematode (SCN) GmNIC1 and GmNIC2 A schematic diagram showing the relative expression levels of genes in susceptible and resistant varieties; where Wm82 Mock represents soybeans not inoculated with SCN, and Wm82 SCN 8 dpi represents soybeans on day 8 after SCN inoculation. NIC1 express GmNIC1 Gene, NIC2 express GmNIC2 Gene.

[0021] Figure 3This is a schematic diagram showing the amino acid sequence alignment information of GmNIC1 and GmNIC2 proteins; where, NIC1 express GmNIC1 Gene, NIC2 express GmNIC2 Gene.

[0022] Figure 4 For silence GmNIC1 or GmNIC2 A schematic diagram illustrating the changes in transcriptional levels and overexpression of the corresponding gene in soybean hairy roots. GmNIC1 or GmNIC2 A schematic diagram illustrating the changes in transcriptional levels of the corresponding gene in the hairy roots of soybean; where A: Wm82 Control represents wild-type susceptible soybean plants, and Wm82 NIC1 RNAi represents silencing GmNIC1 The soybean plant with the gene Wm82 NIC2 RNAi represents silencing GmNIC2 Soybean plants with the gene; B: Forrest EV indicates a resistant soybean plant, Forrest OE- NIC1 Overexpression GmNIC1 Forrest OE- gene-based soybean plants NIC2 Overexpression GmNIC2 Soybean plants with genetically modified genes.

[0023] Figure 5 For silence GmNIC1 and GmNIC2 Genes and overexpression GmNIC1 or GmNIC2 A schematic diagram of the SCN resistance phenotype in soybean hairy roots, with representative root images stained with acid fuchsin (scale bar = 500 mm); where Wm82 represents wild-type susceptible soybean plants, and EV represents control soybean plants. NIC1 RNAi represents silencing GmNIC1 Genetically modified soybean plants, NIC2 RNAi represents silencing GmNIC2 Soybean plants with the gene; Forrest represents resistant soybean plants, EV represents control soybean plants, Forrest OE- NIC1 Overexpression GmNIC1 Forrest OE- gene-based soybean plants NIC2 Overexpression GmNIC2 Soybean plants with genetically modified genes.

[0024] For silence Figure 6Statistical analysis of the proportion of nematodes at different developmental stages; among them, the lower the proportion of nematodes in the J2, J3, and sporangium stages, the stronger the resistance. Wm82 EV represents soybean plants of the susceptible control variety. RNAi represents silencing NIC1 / 2 The soybean plant with the gene Wm82 RNAi represents silencing NIC1 Soybean plants with genetically modified genes.

[0025] For overexpression GmNIC1 Statistical analysis of the proportion of nematodes at different developmental stages; where a lower proportion of nematodes in the J2, J3, and sporangium stages indicates stronger resistance. Forrest EV represents soybean plants of the control group, resistant varieties. Overexpression NIC2 Forrest soybean plants with genetically modified genes Overexpression GmNIC2 Soybean plants with genetically modified genes.

[0026] To knock out Figure 7 and The graph shows the nicotinamide content in the roots of soybean plants treated with SCN and the control group. In this graph, Wm82 Mock represents wild-type soybean plants not treated with SCN, Wm82 SCN 8 dpi represents wild-type soybean plants on day 8 after SCN inoculation, and Wm82 NIC1 Crispr Mock represents plants knocked out without SCN inoculation. NIC1 / 2 In soybean plants, Wm82 NIC1 Crispr SCN 8 dpi indicates the knockout of the SCN gene on day 8 after inoculation. Soybean plants with the gene Wm82NIC2 Crispr Mock represent SCN knockout without inoculation. NIC1 OE In soybean plants, Wm82 NIC2 Crispr SCN8 dpi indicates a knockout on day 8 after inoculation with SCN. Soybean plants with genetically modified genes. Detailed Implementation

[0027] 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 in conjunction with 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. 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. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this application pertains. The experimental materials used in the embodiments of the present invention are all conventional experimental materials in the art and can be purchased through commercial channels. Experimental methods without detailed conditions are performed according to conventional experimental methods or according to the operating instructions recommended by the supplier. In the following embodiments, the soybean variety used is Wm82; the soybean cyst nematode used is from soybean cyst nematodes preserved in the Plant Nematode Laboratory of Zhejiang University ( GmNIC1 ,SCN,HgType 0) population.

[0028] In the following embodiments, The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the GmNIC1 protein is shown in SEQ ID NO.2. NIC2OE The nucleotide sequence of the gene is shown in SEQ ID NO.3, and the amino acid sequence of the GmNIC2 protein is shown in SEQ ID NO.4, used for silencing. The positive strand sequence of the gene, as shown in SEQ ID NO.5, is used for silencing. GmNIC2 The positive strand sequence of the gene, as shown in SEQ ID NO.6, is used for silencing. The antisense strand sequence of the gene, as shown in SEQ ID NO.7, is used for silencing. Figure 8 The antisense strand sequence of the gene is shown in SEQ ID NO.8.

[0029] SEQ ID NO.1:

[0030] ATGGTCTCACAGACAGTTGAACTCTTAAAGAATGAGATTCCTCTGGAGCAGGAATCAGTGGTTTTAGCTGAAGATGCTGTAAATGGTCTCGTTCTTGTGGACATCATAAATGGCTTTTGCACAGTTGGTGCTGGAAATCTGGCTCCAAGAGAATCCAATACGCAGATTTCGGGAATGATCAGTG AATCAGCAAGGCTGGCTAGAGTGTTCTGTGAGAAGAATTTGCCGGTTATGGCTTTCCTGGATTCTCACCATCCTAACAAGCCAGAGGACCCTTATCCCCCTCACTGTATTGTTGGCTCTGATGAATCAAATCTGGGTTCCAGCATTAAGATGGCTAGAGAATGAACCTAATGTAACAATCAGGCGA AAGGATTGTTTTGATGGATATTTGGGCTCAATACAAGAAGATGGTTCAAATGTTTTTGTAGATTGGGTGAAGAAGAATAAGATAACAACTCTGCTGGTAGTAGGTGTGTGCACAGATATCTGCGTTCTAGATTTTGTATGCTCCACAATGTCAGCTAAAAACCGTGGTTTTCTGGAGCCTCTAG AAATGTGGTGGTGTATTCCCGTGCCTGTGCTACCTTTAATGTCCCTCTGGAGGTAGCCAGAAATACCAAAGGAGCTTTGGCACATCCTCAGGAGTTTATGCATCATGTAGGCCTATATATGGCCAAAGAACGTGGAGCCAAGATAGCAAATGAAGTATTGTTTGGTGCAGCAGGGAAGGTTTAA

[0031] SEQ ID NO.2:

[0032] ATGGTTTCTTCCAACACTGCCGAGCTGCTGAGAGAGGAGATTCCTGTGAAGCAACAACCTCTGACTTTGTCTGCAGACATCATAACCGGCCTCGTACTCGTCGATGTTGTCAATGGCTTCTGCACCGTTGGAGCTGGCAATTTGGCGCCGAAAGAACCAGATGAACGAATTTCTCAGATGGTAAAAGAATCTCTGAGGCTGTCCAAAGCATTCTCTGAGAGAAAGTGGCCAATTTTTGCTTTCCTTGATTGCCATCACCCCGACAAACCCGAACCCCCTTATCCACCACACTGTATTATAGGATCAGGCGAAGAAAAATTGGTTCCTGATCTGCTGTGGTTGGAAAATGACCCAAATGCAACACTCAGGCAAAAAGAATGCATTGATGGATTCCTTGGGTCAACTGAGAAAGATGGCTCTAATGTCTTCATTGATTGGGTGAAAAATAATCAAATAAAACAAATTTTGGTTGCTGGGATATGCACTGATATATGTGTGCTGGATTTTGTCTCTTCTGTCTTGTCTGTGAGAAACCGTGGTTTCCTTACTCCTCTGGAAAATGTGATCGTGTCTTCCCAAGCTTGTGCTACTTATGATTTGCCATTGCATGTGGCCAAAACCAACAAGGATTTTGTATCTCATCCACAGGAGTTGATGCATCACGTTGGCCTTTACATAGCCAGTGGAAGGGGAGCCCATATAGCTTCAGAAGTGTTATTTGAATAA

[0033] SEQ ID NO.3:

[0034] MVSQTVELLKNEIPLEQESVVLAEDAVNGLVLVDIINGFCTVGAGNLAPRESNTQISGMISESARLARVFCEKNLPVMAFLDSHHPNKPEDPYPPHCIVGSDESNLVPALRWLENEPNVTIRRKDCFDGYLGSIQEDGSNVFVDWVKKNKITTLLVVGVCTDICVLDFVCSTMSAKNRGFLEPLENVVVYSRACATFNVPLEVARNTKGALAHPQEFMHHVGLYMAKERGAKIANEVLFGAAGKV

[0035] SEQ ID NO.4:

[0036] MVSSNTAELLREEIPVKQQPLTLSADIITGLVLVDVVNGFCTVGAGNLAPKEPDERISQMVKESLRLSKAFSERKWPIFAFLDCHHPDKPEPPYPPHCIIGSGEEKLVPDLLWLENDPNATLRQKECIDGFLGSTEKDGSNVFIDWVKNNQIKQILVAGICTDICVLDFVSSVLSVRNRGFLTPLENVIVSSQACATYDLPLHVAKTNKDFVSHPQELMHHVGLYIASGRGAHIASEVLFE。

[0037] SEQ ID NO.5:

[0038] CAGACAGTTGAACTCTTAAAGAATGAGATTCCTCTGGAGCAGGAATCAGTGGTTTTAGCTGAAGATGCTGTAAATGGTCTCGTTCTTGTGGACATCATAAATGGCTTTTGCACAGTTGGTGCTGGAAATCTGGCTCCAAGAGAATCCAATACGCAGATTTCGGGAATGATCAGTGAATC AGCAAGGCTGGCTAGAGTGTTCTGTGAGAAGAATTTGCCGGTTATGGCTTTCCTGGATTCTCACCATCCTAACAAGCCAGAGGACCCTTATCCCCCTCACTGTATTGTTGGCTCTGATGAATCAAATCTGGTTTCCAGCATTAAGATGGCTAGAGAATGAACCTAATGTAACAATCAGGCG AAAGGATTGTTTTGATGGATATTTGGGCTCAATACAAGAAGATGGTTCAAATGTTTTTGTAGATTGGGTGAAGAAGAATAAGATAACAACTCTGCTGGTAGTAGGTGTGTGCACAGATATCTGCGTTCTAGATTTTGTATGCTCCACAATGTCAGCTAAAAACCGTGGTTTTCTGGAGC CTCTAGAAAATGTGGTGGTGTATTCCCGTGCCTGTGCTACCTTTAATGTCCCTCTGGAGGTAGCCAGAAATACCAAAGGAGCTTTGGCACATCCTCAGGAGTTTATGCATCATGTAGGCCTATATATGGCCAAAGAACGTGGAGCCAAGATAGCAAATGAAGTATTGTTTGGTGCAGCAG

[0039] SEQ ID NO.6:

[0040] GAGAGAGGAGATTCCTGTGAAGCAACAACCTCTGACTTTGTCTGCAGACATCATAACCGGCCTCGTACTCGTCGATGTTGTCAATGGCTTCTGCACCGTTGGAGCTGGCAATTTGGCGCCGAAAGAACCAGATGAACGAATTTCTCAGATGGTAAAAGAATCTCTGAGGCTGTCCAAAGCATTCTCTGAGAGAAAGTGGCCAATTTTTGCTTTCCTTGATTGCCATCACCCCGACAAACCCGAACCCCCTTATCCACCACACTGTATTATAGGATCAGGCGAAGAAAAATTGGTTCCTGATCTGCTGTGGTTGGAAAATGACCCAAATGCAACACTCAGGCAAAAAGAATGCATTGATGGATTCCTTGGGTCAACTGAGAAAGATGGCTCTAATGTCTTCATTGATTGGGTGAAAAATAATCAAATAAAACAAATTTTGGTTGCTGGGATATGCACTGATATATGTGTGCTGGATTTTGTCTCTTCTGTCTTGTCTGTGAGAAACCGTGGTTTCCTTACTCCTCTGGAAAATGTGATCGTGTCTTCCCAAGCTTGTGCTACTTATGATTTGCCATTGCATGTGGCCAAAACCAACAAGGATTTTGTATCTCATCCACAGGAGTTGATGCATCACGTTGGCCTTTACATAGCCAGTGGAAGGGGAGCCCATA

[0041] SEQ ID NO.7:

[0042] CTGCTGCACCAAACAATACTTCATTTGCTATCTTGGCTCCACGTTCTTTGGCCATATATAGGCCTACATGATGCATAAACTCCTGAGGATGTGCCAAAGCTCCTTTGGTATTTCTGGCTACCTCCAGAGGGACATTAAAGGTAGCACAGGCACGGGAATACACCACCACATTTTCTAGAGGCTCCAGAAAACCACGGTTTTTAGCTGACATTGTGGAGCATACAAAATCTAGAACGCAGATATCTGTGCACACACCTACTACCAGCAGAGTTGTTATCTTATTCTTCTTCACCCAATCTACAAAAACATTTGAACCATCTTCTTGTATTGAGCCCAAATATCCATCAAAACAATCCTTTCGCCTGATTGTTACATTAGGTTCATTCTCTAGCCATCTTAATGCTGGAACCAGATTTGATTCATCAGAGCCAACAATACAGTGAGGGGGATAAGGGTCCTCTGGCTTGTTAGGATGGTGAGAATCCAGGAAAGCCATAACCGGCAAATTCTTCTCACAGAACACTCTAGCCAGCCTTGCTGATTCACTGATCATTCCCGAAATCTGCGTATTGGATTCTCTTGGAGCCAGATTTCCAGCACCAACTGTGCAAAAGCCATTTATGATGTCCACAAGAACGAGACCATTTACAGCATCTTCAGCTAAAACCACTGATTCCTGCTCCAGAGGAATCTCATTCTTTAAGAGTTCAACTGTCTG

[0043] SEQ ID NO.8:

[0044] TATGGGCTCCCCTTCCACTGGCTATGTAAAGGCCAACGTGATGCATCAACTCCTGTGGATGAGATACAAAATCCTTGTTGGTTTTGGCCACATGCAATGGCAAATCATAAGTAGCACAAGCTTGGGAAGACACGATCACATTTTCCAGAGGAGTAAGGAAACCACGG TTTCTCACAGACAAGACAGAAGAGACAAAATCCAGCACACATATATCAGTGCATATCCCAGCAACCAAAATTTGTTTTATTTGATTATTTTTCACCCAATCAATGAAGACATTAGAGCCATCTTTCTCAGTTGACCCAAGGAATCCATCAATGCATTCTTTTTGCCTG AGTGTTGCATTTGGGTCATTTTCCAACCACAGCAGATCAGGAACCAATTTTTCTCGCCTGATCCTATAATACAGTGTTGGTGGATAAGGGGGTTCGGGTTTGTCGGGGTGATGGCAATCAAGGAAAGCAAAAATTGGCCACTTTCTCTCAGAGAATGCTTTGGACAGC CTCAGAGATTCTTTTACCATCTGAGAAATTCGTTCATCTGGTTCTTTCGGCGCCAAATTGCCAGCTCCAACGGTGCAGAAGCCATTGACATCGACGAGTACGAGGCCGGTTATGATGTCTGCAGACAAAGTCAGAGGTTGTTGCTTCACAGGAATCTCCTCTCTC

[0045] Example 1: Screening of GmNIC family genes related to nematode resistance

[0046] NIC in soybeans Analyze the phylogenetic relationships of family genes and construct an evolutionary tree ( GmNIC1 ), revealing different Evolutionary relationships and conserved characteristics of family members in soybean.

[0047] Wild-type soybean variety Wm82 was selected as the experimental material, and two treatment groups were set up: one group was infected with SCN, and the other group was a mock (uninoculated with SCN) control group. The roots of soybeans in both groups were sampled 8 days (8 dpi) after infection with soybean cyst nematodes for subsequent gene expression analysis.

[0048] Total RNA was extracted from root samples from each treatment group, and cDNA was synthesized using a reverse transcription system. Subsequently, the cDNA was analyzed by real-time quantitative PCR (RT-qPCR). GmNIC2 Changes in gene expression levels after SCN infection; The expression levels of the gene were standardized relative to the control gene to ensure the accuracy and comparability of the data.

[0049] like GmNIC1 As shown, on day 8 (8 dpi) after SCN infection, the GmNICs gene showed significant upregulation in the infected soybean roots. Compared with the uninfected Mock group, the expression level of GmNICs in the SCN-infected group increased significantly, by about 20-fold, indicating that the expression of this gene was significantly enhanced in the early stage of SCN infection (p<0.01, indicating significant difference).

[0050] Further analysis of the GmNIC1 (Glyma.08G067600) and GmNIC2 (Glyma.08G279200) sequences was conducted using the Phytozome soybean genome database (https: / / phytozome-next.jgi.doe.gov / ). Amino acid sequence alignment of the proteins encoded by these two genes showed high homology and strong structural conservation (e.g., ...). As shown in the figure, it suggests that it may have a synergistic or redundant function in the soybean response to SCN.

[0051] Example 2 GmNIC1 , Gene cloning and vector construction

[0052] I. Overexpression GmNIC2 Construction of gene vectors

[0053] 1. Cloning of genes

[0054] Using cDNA from wild-type soybean Williams 82 (Wm82) to analyze soybean GmNIC2 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.

[0055] The PCR reaction system is as follows:

[0056] Table 1

[0057]

[0058] Primer I: c gac gac aag acc gt g acc ATGGTCTCACAGACAGTTGAACTC (where lowercase letters represent the homologous recombination vector sequence, used for homologous recombination ligation);

[0059] Primer II: ga gga gaa gag ccg TTAAACCTTCCCTGCTGCACC (where the lowercase letters represent the homologous recombination vector sequence, used for homologous recombination ligation).

[0060] Using cDNA from wild-type soybean Williams 82 (Wm82) to analyze soybean 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 as shown in Table 1. The amplified fragment yielded... Soybean Cyst Nematode and The full-length gene fragment.

[0061] Primer III: c gac gac aag acc gt g acc ATGGTTTCTTCCAACACTGCCGA (where the lowercase letters represent the homologous recombination vector sequence, used for homologous recombination ligation);

[0062] Primer IV: ga gga gaa gag ccg TTATTCAAATAACACTTCTGAAGCTATATGGGCTC (where lowercase letters represent the homologous recombination vector sequence, used for homologous recombination ligation).

[0063] 2. Construction of overexpression vectors

[0064] The amplified GmNIC1 and The gene fragment was subjected to homologous recombination and fused with the 35S promoter of cauliflower mosaic virus (CaMV), obtained in the previous step. GmNIC2 or 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.

[0065] The reaction system is as follows:

[0066] Table 2

[0067]

[0068] Reaction conditions: 50℃, 15min.

[0069] 2. Plasmid preparation

[0070] 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-. GmNIC1 and OE- .

[0071] (1) Escherichia coli transformation:

[0072] Table 3

[0073]

[0074] 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.

[0075] (2) Colony PCR:

[0076] Table 4

[0077]

[0078] The forward and reverse primers are the same as those used in Example 2.

[0079] Use a pipette tip to pick up the colonies obtained in step (1) and add them to the prepared system.

[0080] The PCR reaction conditions are as follows:

[0081] (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℃.

[0082] two, GmNIC2 and Carrier construction

[0083] 1. Select the positive sense fragment of the silent gene NIC1 / 2 in soybean (fragment sequence see SEQ ID NO.5 and SEQ ID NO.6), and use PCR to amplify the target gene NIC1 / 2 fragment NIC1+ or NIC2+ from the full-length product of NIC1 / 2 obtained above as a template.

[0084] The PCR reaction system is as follows:

[0085] Table 5

[0086]

[0087] Note: KOD one PCR Master Mix was purchased from Toyobo, product number KMM-201.

[0088] The primers for amplifying NIC1+ are:

[0089] Forward primer: tcactcggatccatttaaatCAGACAGTTGAACTCTTAAAGAATGAGATTC;

[0090] Reverse primer: GCcctaggaggcgcgcccCTGCTGCACCAAACAATACTTCATTTG;

[0091] The primers for amplifying NIC2+ are:

[0092] Forward primer: gaaagggatcttcactcgGAGAGAGGAGATTCCTGTGAAGCAACAACCTC;

[0093] Reverse primer: acgatctgcttgacTATGGGCTCCCCTTCCACTGGCTATGTAAAGG.

[0094] The PCR reaction conditions were: (1) pre-denaturation at 95℃ for 2 min; (2) denaturation at 95℃ for 30 sec, 55℃ for 30 sec, and 72℃ for 10 sec for a total of 35 cycles; (3) storage at 4℃.

[0095] 2. The vector pGRNAi2 was digested with Swa I overnight at 37°C to obtain the Swa I digestion product. The 5' end of the DNA was then dephosphorylated using Quick CIP at 37°C for 15 min, followed by 65°C for 20 min. The digested product was then purified using a DNA purification kit (purchased from Novizan, catalog number DC301).

[0096] The Swa I enzyme digestion system is as follows:

[0097] Table 6

[0098]

[0099] The Quick CIP dephosphorylation system is as follows:

[0100] Table 7

[0101]

[0102] 3. Using the Gibson homologous recombination method, the gene fragment NIC1+ or NIC2+ obtained in step 1 is ligated with the single enzyme digestion and purification product obtained in step 2. The ligation product is named GRNAi2-NIC1+ / GRNAi2-NIC2+.

[0103] The reaction system is as follows:

[0104] Table 8

[0105]

[0106] Reaction conditions: 50℃, 15 min.

[0107] 4. Further screening was conducted using E. coli transformation and colony PCR. Samples with the target band were further verified by PCR, and finally the successfully ligated vector GRNAi2-NIC1+ / GRNAi2-NIC2+ was obtained.

[0108] (1) Escherichia coli transformation:

[0109] Table 9

[0110]

[0111] 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 LB solid medium containing 50 mg / L Kan antibiotic, and incubate overnight at 37°C.

[0112] (2) Colony PCR:

[0113] Table 10

[0114]

[0115] Use a pipette tip to pick up the colonies obtained in step (1) and add them to the prepared system.

[0116] The PCR reaction conditions were: (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℃.

[0117] The vector GRNAi2-NIC1+ / GRNAi2-NIC2+ was obtained.

[0118] 5. Digest the vector GRNAi2-NIC1+ / GRNAi2-NIC2+ with BamHI overnight at 37°C, then dephosphorylate the 5' end of the DNA with QuickCIP at 37°C for 15 min, followed by 65°C for 20 min.

[0119] The BamHI enzyme digestion system is as follows:

[0120] Table 11

[0121]

[0122] The Quick CIP dephosphorylation system is as follows:

[0123] Table 12

[0124]

[0125] 6. Select the antisense fragment of the silent gene NIC1 / 2 of soybean (fragment sequence see SEQ ID NO.7 and SEQ ID NO.8), and use PCR to amplify the target gene NIC1 / 2 fragment NIC1- or NIC2- using the full-length product of NIC1 / 2 obtained above as a template.

[0126] The PCR reaction system is as follows:

[0127] Table 13

[0128]

[0129] The primers for amplifying NIC1- are:

[0130] Forward primer: ggtagatatcatttaaatCAGACAGTTGAACTCTTAAAGAATGAGATTC;

[0131] Reverse primer cgatacAatggcgcgcccCTGCTGCACCAAACAATACTTCATTTG;

[0132] The primers for amplifying NIC2- are:

[0133] Forward primer agcgggtagatatcatttGAGAGAGGAGATTCCTGTGAAGCAACAACCTC;

[0134] Reverse primer caattatcgatacAatggGCTCCCCTTCCACTGGCTATGTAAAGG.

[0135] The PCR reaction conditions are as follows:

[0136] (1) Pre-denaturation at 95℃ for 2 min; (2) Denaturation at 95℃ for 30 sec, 55℃ for 30 sec, and 72℃ for 10 sec, for a total of 35 cycles; (3) Storage at 4℃.

[0137] 7. Using Gibson homologous recombination again, ligate the gene fragment NIC1- or NIC2- obtained in step 6 and the GRNAi2+ Bam HI single enzyme digestion purification product obtained in step 5, respectively. Name the ligation product GRNAi2 NIC1+ / - or GRNAi2 NIC2+ / -.

[0138] The reaction system is as follows:

[0139] Table 14

[0140]

[0141] Reaction conditions: 50℃, 15min.

[0142] 8. Using E. coli transformation and antibiotic screening, and further molecular verification and sequencing were performed by PCR. Successfully ligated vectors GRNAi2 NIC1+ / - or GRNAi2 NIC2+ / - were obtained.

[0143] (1) Escherichia coli transformation:

[0144] Table 15

[0145]

[0146] 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; after removal, centrifuge at 6000 rpm for 2 min, remove the supernatant, and spread the remainder onto LB solid medium containing 50 mg / L Kan antibiotic, and incubate overnight at 37°C.

[0147] (2) Colony PCR:

[0148] Table 16

[0149]

[0150] Use a pipette tip to pick up the colonies obtained after the above-mentioned E. coli transformation and spread them onto Kan medium, and add them to the prepared system.

[0151] The PCR reaction conditions are as follows:

[0152] (1) Pre-denature at 95℃ for 5 min; (2) Denature at 95℃ for 30 sec, 55℃ for 30 sec, and 72℃ for 45 sec for a total of 35 cycles; (3) Store at 4℃. The vectors GRNAi2 NIC1+ / - or GRNAi2 NIC2+ / - were obtained.

[0153] Example 3: Silence GmNIC1 Gene, Genes and overexpression GmNIC2 Gene, Obtaining genetically modified soybean roots

[0154] 1. Preparation of Agrobacterium

[0155] The prepared GRNAi2 NIC1+ / - or GRNAi2 NIC2+ / - and OE- Nicotinamidase or OE- plasmid vector transformed into Agrobacterium rhizogenes ( Figure 1 In the ARqua1 strain. Two days later, positive clones were picked and inoculated into selective liquid culture 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.

[0156] 2. Transformation of soybean hairy root system

[0157] 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.

[0158] 3. Screening and validation of transgenic root systems

[0159] After 3 days of culture on the co-culture medium, the cotyledons were transferred to the rooting medium. After 14 days of culture at 28°C, the transformed roots were observed using a fluorescence microscope, and soybean roots that appeared red under the microscope were selected.

[0160] 4. RNAi and GmNIC RNAi gene silencing and OE- or OE- GmNIC Overexpression verification

[0161] 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:

[0162] The steps are as follows: Extraction of plant RNA: In this experiment, plant RNA was extracted using the Trizol method. The steps are 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 incubated at room temperature for 5 min. 200 μL of chloroform was added, and the mixture was vigorously vortexed for 15 sec and then incubated 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 incubated 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, and the mixture was centrifuged again. The ethanol was dried in a clean bench. Finally, 20-30 μL of RNase Freewater was added to dissolve the RNA. The concentration was measured, and the RNA was stored at -80 °C for later use.

[0163] 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.

[0164] Quantitative PCR: AceQ qPCR SYBR Green Master Mix was used. The reaction system is as follows:

[0165] Table 17

[0166]

[0167] After preparing the system, place the sample on the Bio-Rad CFX96™ Real-Time PCR Detection System for amplification.

[0168] like As shown in Figure A, compared with the control group, the root density in the gene silencing treatment group was significantly higher. GmNICs and Gene expression levels are significantly reduced; such as Figure 2 As shown in B, compared with the control group, overexpression of OE- and OE- Figure 3 In the root of quality or GmNIC1 Gene expression levels were significantly increased.

[0169] Example 4: Silence Gene or GmNIC2 Genes and overexpression Gene or GmNIC1 and GmNIC2 Gene-induced soybean resistance to nematodes

[0170] 1. Nematode culture: Soybean cyst nematodes preserved in the Plant Nematode Laboratory of Zhejiang University ( GmNIC1 and GmNIC2 A population of nematode SCN (Hg Type 0) was used to hatch nematode eggs using 3 mM ZnCl2 buffer and incubated at room temperature for 5 days until they reached 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.

[0171] 2. Nematode inoculation: Under greenhouse conditions, J2-stage nematodes were inoculated into the root tips of transgenic soybean plants and non-transgenic control plants. At least 500 nematodes were inoculated into each plant's root system. The control plants were transformed using Agrobacterium rhizogenes with an empty vector. The inoculated soybeans were then cultured in an incubator for 14 days. The developmental status of the nematodes was then observed.

[0172] 3. Observation and statistics of nematode development

[0173] The development of nematodes in soybean roots was observed and recorded using acid fuchsin staining. The specific steps were as follows: First, soybean roots were removed 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. After washing three times with clean water to remove excess sodium hypochlorite, acid fuchsin staining solution was added, and the roots were boiled in a water bath for 5 minutes. Finally, the staining solution was washed away with tap water. Nematodes in the transgenic and control groups were observed under a microscope, and their developmental stages and numbers were recorded.

[0174] Staining results as follows As shown, the statistical results are as follows: GmNIC1 As shown, compared with the control group, Gene or GmNIC1 Gene silencing significantly inhibited the development of root nematodes in the susceptible soybean variety Williams 82; for example As shown, compared with the control group, overexpression GmNIC1 Gene or It significantly promoted the development of root nematodes in the resistant soybean variety Forrest.

[0175] Example 5 GmNIC2 Genes and Research on the mechanisms that enhance plant resistance to nematodes

[0176] This embodiment uses the CRISPR / Cas9 system to knock out the proteins in soybeans. GmNIC1 and Genes were used to quantitatively detect the nicotinamide (NAM) content in soybean roots under different treatment conditions using high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS), thereby clarifying the gene content. GmNIC2 The regulatory role of genes in NAM metabolism and their physiological significance in the response to SCN infection.

[0177] (1) and GmNIC1 Functional annotation of the gene indicates that it encodes nicotinamide, a hydrolytic enzyme, and its catalytic reaction is: Nicotinamide + H2O → Nicotinate + NH3.

[0178] Its international enzyme designation is EC 3.5.1.19. In soybeans, this enzyme is responsible for metabolizing NAM into niacin.

[0179] (2) Wild-type soybean Williams 82 (Wm82) and its varieties were selected respectively. Crispr GmNIC2 Crispr knockout mutants were used as experimental materials. Soybean seedlings were cultured under sterile conditions, and the roots were inoculated with J2-stage soybean cyst nematode larvae, with no fewer than 500 nematodes inoculated per plant root system. A control group (uninoculated with SCN) and a treatment group (inoculated with SCN) were set up. Root samples were collected on day 8 (8 dpi) after treatment. All samples were immediately frozen in liquid nitrogen and ground, and stored at -80℃ for analysis.

[0180] (3) The NAM content in soybean roots was determined by HPLC-MS / MS.

[0181] The steps are as follows: (1) Take 0.5 g of fresh root tissue sample, add 0.01 mol / L hydrochloric acid extract and extract three times by ultrasonication, combine the supernatants and make up to volume. (2) After filtration through a 0.22 μm aqueous filter membrane, send it to a liquid chromatography-mass spectrometer for quantitative analysis. (3) The analytical conditions include: Waters ACQUITY UPLC BEH HSS T3 column, mobile phase is 0.1% formic acid water (A) and methanol (B), mass spectrometry monitoring mode is positive ion mode, [M+H]^+ = 123, daughter ions are 78 and 80, and collision energy is 18 V and 15 V.

[0182] like GmNIC1 GmNIC2 GmNIC1 RNAi GmNIC2 RNAi GmNIC1 GmNIC2 GmNIC1 GmNIC2 GmNIC1 GmNIC2 Agrobacterium rhizogenes GmNIC1 GmNIC2 GmNIC1 GmNIC2 Figure 4 GmNIC1 GmNIC2 Figure 4 GmNIC1 GmNIC2 GmNIC1 GmNIC2 GmNIC1 GmNIC2 GmNIC1 GmNIC2 Soybean Cyst Nematode Figure 5 Figure 6 GmNIC1 GmNIC2 Figure 6 GmNIC1 GmNIC2 GmNIC1 GmNIC2 GmNIC1 GmNIC2 GmNIC GmNIC1 GmNIC2 GmNIC1 GmNIC2 Figure 7 As shown, the NAM quantitative results indicate that under SCN infection conditions, the nicotinamide content in wild-type Wm82 soybeans did not change significantly; while... GmNIC1 and GmNIC2 In the gene knockout mutant, the NAM content was significantly increased, with an increase of more than 2 times.

Claims

1. GmNIC1 Genes and / or GmNIC2 The application of genes in regulating the ability of soybeans to resist soybean cyst nematode is characterized by, The GmNIC1 The nucleotide sequence of the gene is shown in SEQ ID NO.1; GmNIC2 The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

2. The application according to claim 1, characterized in that, The application methods are: knockout, knockdown, silencing, and interference. GmNIC1 and / or GmNIC2 One way in the genes enhances soybean's resistance to soybean cyst nematode.

3. The application of GmNIC1 and / or GmNIC2 proteins in regulating the ability of soybean to resist soybean cyst nematode, characterized in that, The amino acid sequence of the GmNIC1 protein is shown in SEQ ID NO.3; the amino acid sequence of the GmNIC2 protein is shown in SEQ ID NO.

4.

4. Knock out, knock down, silence, or interfere GmNIC1 Genes and / or GmNIC2 The application of gene recombinant vectors in enhancing soybean resistance to soybean cyst nematode is characterized by, The GmNIC1 The nucleotide sequence of the gene is shown in SEQ ID NO. 1; GmNIC2 The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

5. Knock out, knock down, silence, or interfere GmNIC1 Genes and / or GmNIC2 The application of genetically engineered bacteria in enhancing soybean resistance to soybean cyst nematode is characterized by, The GmNIC1 The nucleotide sequence of the gene is shown in SEQ ID NO. 1; GmNIC2 The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

6. A method for enhancing the resistance of soybeans to soybean cyst nematode, characterized in that, include: Knockout, knockdown, silence, and interference with soybeans GmNIC1 and / or GmNIC2 One approach in the genes to enhance soybean's resistance to soybean cyst nematode; GmNIC1 The nucleotide sequence of the gene is shown in SEQ ID NO.1; GmNIC2 The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

7. The method according to claim 6, characterized in that, The silence GmNIC1 and / or GmNIC2 Genes, including: (1) According to GmNIC1 and / or GmNIC2 The genome sequence, construct GmNIC1 and / or GmNIC2 RNAi vectors for genes; (2) The RNAi vector was transferred into Agrobacterium competent cells to construct cells containing the knockout vector. GmNIC1 and / or GmNIC2 Agrobacterium strains carrying RNAi gene vectors; (3) The Agrobacterium strain was used to transform soybeans and a silent strain was cultivated. GmNIC1 and / or GmNIC2 A homozygous soybean line with the gene.

8. The method according to claim 6, characterized in that, The knockout GmNIC1 and / or GmNIC2 The gene is: soybean genotype obtained through CRISPR / Cas9 technology. GmNIC1 and / or GmNIC2 Genes are knocked out.

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