Application of GmNIC1 gene and GmNIC2 gene in regulation of soybean cyst nematode resistance of plants

Abstract

The invention belongs to the technical field of biology, and particularly relates to application of a GmNIC1 gene and a GmNIC2 gene to regulation and control of soybean cyst nematode resistance of plants, and the nucleotide sequence of the GmNIC1 gene is shown as SEQ ID NO.1; the nucleotide sequence of the GmNIC2 gene is as shown in SEQ ID NO. 2. The invention discloses the negative regulation effect of GmNIC1 and GmNIC2 genes in the resistance of soybean to cyst nematode (SCN) for the first time, and by constructing gene silencing mutants of GmNIC1 and GmNIC2, it is verified that the resistance of soybean to SCN can be remarkably enhanced due to function deficiency of the gene family. The application provides a new target for deep understanding of a soybean-nematode interaction mechanism, and provides a brand new gene resource and technical path for molecular design breeding of resistant soybeans.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and specifically relates to GmNIC1 Genes and GmNIC2 The application of genes in regulating plant resistance to soybean cyst nematode. Background Art

[0002] Soybean cyst nematode (SCN) is one of the most devastating soil-borne pathogens in global soybean production. The second-stage larvae (J2) of this nematode infect soybean roots, establishing specialized vegetative syncytia. This nematode causes root dysfunction, inhibiting plant growth and development, and significantly reducing yield and quality. SCN reportedly causes annual soybean yield losses ranging from 10% to 30%, and in severe cases, exceeding 50%. SCN is widely distributed in major soybean-growing regions, including the Americas and Asia, posing a persistent threat to the global soybean supply chain.

[0003] Traditional SCN control strategies primarily involve crop rotation and the selection of resistant varieties. However, these methods are limited by factors such as agricultural cropping systems, the genetic diversity of SCN populations, and the homogeneity of resistance genes, making long-term effective control difficult. Of particular concern is the emergence of new SCN races that have been reported in multiple regions, capable of overcoming the barriers of mainstream resistance genes (such as Rhg1 and Rhg4), rendering some resistant varieties ineffective and leading to a phenomenon known as "resistance fatigue." Furthermore, SCN cysts can remain dormant in the soil for up to 10 years, making it difficult to completely eliminate them through conventional crop rotation.

[0004] The rise of genetic engineering technology has provided a breakthrough for the development of new nematode-resistant soybean varieties. Strategies including gene editing, RNA interference, and transgenic expression of nematode-resistant proteins have all achieved phased progress. For example, knocking out certain negative regulatory genes using CRISPR-Cas9 significantly improved soybean resistance to SCN. Genome-wide association analysis and QTL mapping have also revealed multiple 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 difficult and hot topic in current research. Summary of the Invention

[0005] In response to the demand for new SCN resistance genes in the prior art, the present invention provides GmNIC1 Genes and GmNIC2 The application of genes in regulating plant resistance to soybean cyst nematode, the specific technical solutions are as follows: In a first aspect, the present invention provides GmNIC1 Genes and / or GmNIC2 Application of genes in regulating plant resistance to soybean cyst nematode, the 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.

[0006] Furthermore, the application method is: by knocking out, knocking down, silencing, or interfering GmNIC1 and / or GmNIC2 Genes that enhance plant resistance to soybean cyst nematode.

[0007] In a second aspect, the present invention provides the use of GmNIC1 protein and / or GmNIC2 protein in regulating the resistance of plants to soybean cyst nematodes, wherein 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. In a third aspect, the present invention provides an application of a recombinant vector in regulating the ability of a plant to resist soybean cyst nematodes, wherein the recombinant vector comprises GmNIC1 Genes and / or GmNIC2 gene, 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.

[0008] In a fourth aspect, the present invention provides the use of genetically engineered bacteria in regulating the ability of plants to resist soybean cyst nematodes, wherein the genetically engineered bacteria comprises GmNIC1 Genes and / or GmNIC2 gene, 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.

[0009] In a fifth aspect, the present invention provides a method for enhancing the resistance of soybean to soybean cyst nematodes, comprising: knocking out, knocking down, silencing, or interfering with the GmNIC1 and / or GmNIC2 Gene to enhance soybean 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.

[0010] Furthermore, the silence GmNIC1 and / or GmNIC2 Genes, including: (1) According to GmNIC1 and / or GmNIC2 The genome sequence of GmNIC1 and / or GmNIC2RNAi vectors targeting genes; (2) The CRISPR / Cas9 vector is transferred into Agrobacterium competent cells to construct a knockout GmNIC1 and / or GmNIC2 Agrobacterium strains carrying gene RNAi vectors; (3) Transforming soybeans with the Agrobacterium strain to obtain silenced GmNIC1 and / or GmNIC2 Soybean homozygous lines of the gene.

[0011] Furthermore, the knockout GmNIC1 and / or GmNIC2 The gene was modified by CRISPR / Cas9 technology in soybean GmNIC1 and / or GmNIC2 Gene knockout.

[0012] Compared with the prior art, the present invention has the following beneficial effects: The present invention discloses for the first time GmNIC1 as well as GmNIC2 The negative regulatory role of genes in soybean resistance to cyst nematode (SCN) was investigated by constructing GmNIC1 and / or GmNIC2 The study of gene-silencing mutants of the nematode family confirmed that functional loss of this gene family can significantly enhance soybean resistance to SCN. This application provides a new target for a deeper understanding of the soybean-nematode interaction mechanism and provides a new genetic resource and technical path for the molecular design and breeding of resistant soybeans. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 for GmNIC Schematic diagram of the phylogenetic tree of gene families in soybean.

[0014] Figure 2 8 days after soybean cyst nematode (SCN) inoculation (8 dpi) GmNIC1 and GmNIC2 Schematic diagram of the relative expression level changes of genes in susceptible and resistant varieties; Wm82 Mock represents soybeans not inoculated with SCN, and Wm82 SCN 8 dpi represents soybeans inoculated with SCN on the 8th day. NIC1 express GmNIC1 Gene, NIC2 express [[ID=�48]]GmNIC2 Gene.

[0015] Figure 3 Schematic diagram of the amino acid sequence alignment information of GmNIC1 and GmNIC2 proteins; NIC1 express GmNIC1 Gene, NIC2 express GmNIC2 Gene.

[0016] Figure 4 For silence GmNIC1 or GmNIC2 Schematic diagram of the transcriptional level changes and overexpression of the corresponding genes in soybean hairy roots GmNIC1 or GmNIC2 Schematic diagram of the changes in transcriptional levels of the corresponding genes in soybean hairy roots; A: Wm82 Control represents the wild-type susceptible soybean plant, Wm82 NIC1 RNAi silencing GmNIC1 Genetic soybean plant, Wm82 NIC2 RNAi silencing GmNIC2 gene soybean plants; B: Forrest EV indicates resistant soybean plants, Forrest OE- NIC1 Overexpression GmNIC1 Genetic soybean plants, Forrest OE- NIC2 Overexpression GmNIC2 Genetically modified soybean plants.

[0017] Figure 5 For silence GmNIC1 and GmNIC2 Genes and overexpression GmNIC1 or GmNIC2 Schematic diagram of the SCN-resistant phenotype of soybean hairy roots expressing the gene, and representative root images after acid fuchsin staining (scale bar = 500 mm); Wm82 represents wild-type susceptible soybean plants, and EV represents control soybean plants. NIC1 RNAi silencing GmNIC1 genetically modified soybean plants, NIC2 RNAi silencing GmNIC2 gene soybean plants; Forrest represents resistant soybean plants, EV represents control soybean plants, Forrest OE- NIC1 Overexpression GmNIC1 Genetic soybean plants, Forrest OE- NIC2 Overexpression GmNIC2 Genetically modified soybean plants.

[0018] Figure 6 For silence NIC1 / 2 Statistical analysis of the proportion of nematodes at different developmental stages; the lower the proportion of nematodes in the J2, J3, and cyst stages, the stronger the resistance. Wm82 EV represents the soybean plants of the susceptible variety in the control group, and Wm82 NIC1 RNAi silencing GmNIC1 Genetic soybean plant, Wm82 NIC2 RNAi silencing GmNIC2Genetically modified soybean plants.

[0019] Figure 7 For overexpression NIC1 / 2 Statistical analysis of the proportion of nematodes at different developmental stages; the lower the proportion of nematodes in the J2, J3, and cyst stages, the stronger the resistance. Forrest EV represents the resistant soybean plants in the control group, and Forrest NIC1 OE Overexpression GmNIC1 Genetically modified soybean plants, Forrest NIC2OE Overexpression GmNIC2 Genetically modified soybean plants.

[0020] Figure 8 Knockout GmNIC1 and GmNIC2 Nicotinamide content in roots of soybeans inoculated with SCN and in the control treatment; Wm82 Mock represents wild-type soybean plants not inoculated with SCN, Wm82 SCN 8 dpi represents wild-type soybean plants on the 8th day after inoculation with SCN, and Wm82 NIC1 Crispr Mock represents knockout plants not inoculated with SCN. GmNIC1 Soybean plants with the gene, Wm82 NIC1 Crispr SCN 8 dpi indicates knockout on the 8th day after inoculation of SCN GmNIC1 Genetic soybean plants, Wm82NIC2 Crispr Mock represents the knockout of uninoculated SCN GmNIC2 Soybean plants with the gene, Wm82 NIC2 Crispr SCN8 dpi indicates knockout on the 8th day after inoculation of SCN GmNIC2 Genetically modified soybean plants. DETAILED DESCRIPTION

[0021] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme of the present invention is clearly and completely described below in conjunction with specific embodiments. It should be pointed out that the following detailed descriptions are exemplary and are only embodiments of a part of the present invention, not all embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work should fall within the scope of protection of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by ordinary technicians 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 field and can be purchased through commercial channels. Experimental methods without detailed conditions are carried out in accordance with conventional experimental methods or in accordance with the operating instructions recommended by the supplier. In the following examples, the soybean variety used is Wm82; the soybean cyst nematode used is from the soybean cyst nematode preserved in the Plant Nematology Laboratory of Zhejiang University ( Soybean Cyst Nematode, SCN, HgType 0) population.

[0022] In the following examples, GmNIC1 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. GmNIC2 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, which is used for silencing GmNIC1 The positive strand sequence of the gene is shown in SEQ ID NO.5, which is used for silencing GmNIC2 The positive strand sequence of the gene is shown in SEQ ID NO.6, which is used for silencing GmNIC1 The antisense sequence of the gene is shown in SEQ ID NO.7, which is used to silence GmNIC2 The antisense sequence of the gene is shown in SEQ ID NO.8.

[0023] SEQ ID NO.1: ATGGTCTCACAGACAGTTGAACTCTTAAAGAATGAGATTCCTCTGGAGCAGGAATCAGTGGTTTTAGCTGAAGATGCTGTAAATGGTCTCGTTCTTGTGGACATCATAAATGGCTTTTGCACAGTTGGTGCTGGAAATCTGGCTCCAAGAGAATCCAATACGCAGATTTCGGGAATGATCAGTG AATCAGCAAGGCTGGCTAGAGTGTTCTGTGAGAAGAATTTGCCGGTTATGGCTTTCCTGGATTCTCACCATCCTAACAAGCCAGAGGACCCTTATCCCCCTCACTGTATTGTTGGCTCTGATGAATCAAATCTGGGTTCCAGCATTAAGATGGCTAGAGAATGAACCTAATGTAACAATCAGGCGA AAGGATTGTTTTGATGGATATTTGGGCTCAATACAAGAAGATGGTTCAAATGTTTTTGTAGATTGGGTGAAGAAGAATAAGATAACAACTCTGCTGGTAGTAGGTGTGTGCACAGATATCTGCGTTCTAGATTTTGTATGCTCCACAATGTCAGCTAAAAACCGTGGTTTTCTGGAGCCTCTAG AAATGTGGTGGTGTATTCCCGTGCCTGTGCTACCTTTAATGTCCCTCTGGAGGTAGCCAGAAATACCAAAGGAGCTTTGGCACATCCTCAGGAGTTTATGCATCATGTAGGCCTATATATGGCCAAAGAACGTGGAGCCAAGATAGCAAATGAAGTATTGTTTGGTGCAGCAGGGAAGGTTTAA SEQ ID NO.2: ATGGTTTCTTCCAACACTGCCGAGCTGCTGAGAGAGGAGATTCCTGTGAAGCAACAACCTCTGACTTTGTCTGCAGACATCATAACCGGCCTCGTACTCGTCGATGTTGTCAATGGCTTCTGCACCGTTGGAGCTGGCAATTTGGCGCCGAAAGAACCAGATGAACGAATTTCTCAGATGGTAAAAGAATCTCTGAGGCTGTCCAAAGCATTCTCTGAGAGAAAGTGGCCAATTTTTGCTTTCCTTGATTGCCATCACCCCGACAAACCCGAACCCCCTTATCCACCACACTGTATTATAGGATCAGGCGAAGAAAAATTGGTTCCTGATCTGCTGTGGTTGGAAAATGACCCAAATGCAACACTCAGGCAAAAAGAATGCATTGATGGATTCCTTGGGTCAACTGAGAAAGATGGCTCTAATGTCTTCATTGATTGGGTGAAAAATAATCAAATAAAACAAATTTTGGTTGCTGGGATATGCACTGATATATGTGTGCTGGATTTTGTCTCTTCTGTCTTGTCTGTGAGAAACCGTGGTTTCCTTACTCCTCTGGAAAATGTGATCGTGTCTTCCCAAGCTTGTGCTACTTATGATTTGCCATTGCATGTGGCCAAAACCAACAAGGATTTTGTATCTCATCCACAGGAGTTGATGCATCACGTTGGCCTTTACATAGCCAGTGGAAGGGGAGCCCATATAGCTTCAGAAGTGTTATTTGAATAA SEQ ID NO.3: MVSQTVELLKNEIPLEQESVVLAEDAVNGLVLVDIINGFCTVGAGNLAPRESNTQISGMISESARLARVFCEKNLPVMAFLDSHHPNKPEDPYPPHCIVGSDESNLVPALRWLENEPNVTIRRKDCFDGYLGSIQEDGSNVFVDWVKKNKITTLLVVGVCTDICVLDFVCSTMSAKNRGFLEPLENVVVYSRACATFNVPLEVARNTKGALAHPQEFMHHVGLYMAKERGAKIANEVLFGAAGKV SEQ ID NO.4: MVSSNTAELLREEIPVKQQPLTLSADIITGLVLVDVVNGFCTVGAGNLAPKEPDERISQMVKESLRLSKAFSERKWPIFAFLDCHHPDKPEPPYPPHCIIGSGEEKLVPDLLWLENDPNATLRQKECIDGFLGSTEKDGSNVFIDWVKNNQIKQILVAGICTDICVLDFVSSVLSVRNRGFLTPLENVIVSSQACATYDLPLHVAKTNKDFVSHPQELMHHVGLYIASGRGAHIASEVLFE。

[0024] SEQ ID NO.5: CAGACAGTTGAACTCTTAAAGAATGAGATTCCTCTGGAGCAGGAATCAGTGGTTTTAGCTGAAGATGCTGTAAATGGTCTCGTTCTTGTGGACATCATAAATGGCTTTTGCACAGTTGGTGCTGGAAATCTGGCTCCAAGAGAATCCAATACGCAGATTTCGGGAATGATCAGTGAATC AGCAAGGCTGGCTAGAGTGTTCTGTGAGAAGAATTTGCCGGTTATGGCTTTCCTGGATTCTCACCATCCTAACAAGCCAGAGGACCCTTATCCCCCTCACTGTATTGTTGGCTCTGATGAATCAAATCTGGTTTCCAGCATTAAGATGGCTAGAGAATGAACCTAATGTAACAATCAGGCG AAAGGATTGTTTTGATGGATATTTGGGCTCAATACAAGAAGATGGTTCAAATGTTTTTGTAGATTGGGTGAAGAAGAATAAGATAACAACTCTGCTGGTAGTAGGTGTGTGCACAGATATCTGCGTTCTAGATTTTGTATGCTCCACAATGTCAGCTAAAAACCGTGGTTTTCTGGAGC CTCTAGAAAATGTGGTGGTGTATTCCCGTGCCTGTGCTACCTTTAATGTCCCTCTGGAGGTAGCCAGAAATACCAAAGGAGCTTTGGCACATCCTCAGGAGTTTATGCATCATGTAGGCCTATATATGGCCAAAGAACGTGGAGCCAAGATAGCAAATGAAGTATTGTTTGGTGCAGCAG SEQ ID NO.6: GAGAGAGGAGATTCCTGTGAAGCAACAACCTCTGACTTTGTCTGCAGACATCATAACCGGCCTCGTACTCGTCGATGTTGTCAATGGCTTCTGCACCGTTGGAGCTGGCAATTTGGCGCCGAAAGAACCAGATGAACGAATTTCTCAGATGGTAAAAGAATCTCTGAGGCTGTCCAAAGCATTCTCTGAGAGAAAGTGGCCAATTTTTGCTTTCCTTGATTGCCATCACCCCGACAAACCCGAACCCCCTTATCCACCACACTGTATTATAGGATCAGGCGAAGAAAAATTGGTTCCTGATCTGCTGTGGTTGGAAAATGACCCAAATGCAACACTCAGGCAAAAAGAATGCATTGATGGATTCCTTGGGTCAACTGAGAAAGATGGCTCTAATGTCTTCATTGATTGGGTGAAAAATAATCAAATAAAACAAATTTTGGTTGCTGGGATATGCACTGATATATGTGTGCTGGATTTTGTCTCTTCTGTCTTGTCTGTGAGAAACCGTGGTTTCCTTACTCCTCTGGAAAATGTGATCGTGTCTTCCCAAGCTTGTGCTACTTATGATTTGCCATTGCATGTGGCCAAAACCAACAAGGATTTTGTATCTCATCCACAGGAGTTGATGCATCACGTTGGCCTTTACATAGCCAGTGGAAGGGGAGCCCATA SEQ ID NO.7: CTGCTGCACCAAACAATACTTCATTTGCTATCTTGGCTCCACGTTCTTTGGCCATATATAGGCCTACATGATGCATAAACTCCTGAGGATGTGCCAAAGCTCCTTTGGTATTTCTGGCTACCTCCAGAGGGACATTAAAGGTAGCACAGGCACGGGAATACACCACCACATTTTCTAGAGGCTCCAGAAAACCACGGTTTTTAGCTGACATTGTGGAGCATACAAAATCTAGAACGCAGATATCTGTGCACACACCTACTACCAGCAGAGTTGTTATCTTATTCTTCTTCACCCAATCTACAAAAACATTTGAACCATCTTCTTGTATTGAGCCCAAATATCCATCAAAACAATCCTTTCGCCTGATTGTTACATTAGGTTCATTCTCTAGCCATCTTAATGCTGGAACCAGATTTGATTCATCAGAGCCAACAATACAGTGAGGGGGATAAGGGTCCTCTGGCTTGTTAGGATGGTGAGAATCCAGGAAAGCCATAACCGGCAAATTCTTCTCACAGAACACTCTAGCCAGCCTTGCTGATTCACTGATCATTCCCGAAATCTGCGTATTGGATTCTCTTGGAGCCAGATTTCCAGCACCAACTGTGCAAAAGCCATTTATGATGTCCACAAGAACGAGACCATTTACAGCATCTTCAGCTAAAACCACTGATTCCTGCTCCAGAGGAATCTCATTCTTTAAGAGTTCAACTGTCTG SEQ ID NO.8: TATGGGCTCCCCTTCCACTGGCTATGTAAAGGCCAACGTGATGCATCAACTCCTGTGGATGAGATACAAAATCCTTGTTGGTTTTGGCCACATGCAATGGCAAATCATAAGTAGCACAAGCTTGGGAAGACACGATCACATTTTCCAGAGGAGTAAGGAAACCACGG TTTCTCACAGACAAGACAGAAGAGACAAAATCCAGCACACATATATCAGTGCATATCCCAGCAACCAAAATTTGTTTTATTTGATTATTTTTCACCCAATCAATGAAGACATTAGAGCCATCTTTCTCAGTTGACCCAAGGAATCCATCAATGCATTCTTTTTGCCTG AGTGTTGCATTTGGGTCATTTTCCAACCACAGCAGATCAGGAACCAATTTTTCTCGCCTGATCCTATAATACAGTGTGGTGGATAAGGGGGTTCGGGTTTGTCGGGGTGATGGCAATCAAGGAAAGCAAAAATTGGCCACTTTCTCTCAGAGAATGCTTTGGACAGC CTCAGAGATTCTTTTACCATCTGAGAAATTCGTTCATCTGGTTCTTTCGGCGCCAAATTGCCAGCTCCAACGGTGCAGAAGCCATTGACATCGACGAGTACGAGGCCGGTTATGATGTCTGCAGACAAAGTCAGAGGTTGTTGCTTCACAGGAATCTCCTCTCTC Example 1 Screening of GmNIC family anti-nematode related genes NIC in soybean ( Nicotinamidase ) family genes to analyze the phylogenetic relationship and construct an evolutionary tree ( Figure 1 ), revealing different GmNIC The evolutionary relationships of family members in soybean and their conserved characteristics.

[0025] Wild-type soybean (Wm82) was used as the experimental material. Two treatment groups were set up: one group was infected with SCN, and the other was a mock (uninoculated SCN) control group. Roots of both groups were sampled 8 days after soybean cyst nematode infection (dpi) for subsequent gene expression analysis.

[0026] Total RNA was extracted from the root samples of each treatment group and cDNA was synthesized using a reverse transcription system. Subsequently, real-time quantitative PCR (RT-qPCR) analysis was performed. GmNIC Changes in gene expression levels after SCN infection; GmNICs The expression levels of the genes were normalized to those of the control genes to ensure the accuracy and comparability of the data.

[0027] like Figure 2 As shown in the data, at 8 days after SCN infection (8 dpi), the GmNICs gene showed significant up-regulated expression in the infected soybean root system. Compared with the uninfected Mock group, the GmNICs expression level in the SCN-infected group increased significantly, increasing by about 20 times, indicating that the expression of this gene was significantly enhanced in the early stage of SCN infection (p < 0.01, indicating a significant difference).

[0028] The sequences of GmNIC1 (Glyma.08G067600) and GmNIC2 (Glyma.08G279200) were further studied 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 that they had high homology and high structural conservation (e.g. Figure 3 The results indicate that they may have synergistic or redundant functions in soybean response to SCN.

[0029] Example 2 GmNIC1 、 GmNIC2 Gene cloning and vector construction 1. Overexpression GmNIC1 and GmNIC2 Construction of gene vector 1. GmNIC1 and GmNIC2 Gene cloning The wild-type soybean Williams 82 (Wm82) cDNA was used to clone soybean GmNIC1 PCR amplification of the open reading frame (ORF) of the gene was performed using Primers I and II and Toyobo's KOD one PCR Master Mix. PCR products were detected by agarose gel electrophoresis, and the target fragment was recovered by gel excision.

[0030] The PCR reaction system is: Table 1

[0031] Primer I: c gac gac aag acc gt g acc ATGGTCTCACAGACAGTTGAACTC (the lowercase letters represent the partial sequence of the homologous recombination vector, used for homologous recombination ligation); Primer II: ga gga gaa gag ccg TTAAACCTTCCCTGCTGCACC (the lowercase letters represent part of the homologous recombination vector sequence, used for homologous recombination ligation).

[0032] The wild-type soybean Williams 82 (Wm82) cDNA was used to clone soybean GmNIC1 The open reading frame (ORF) of the gene was amplified by PCR using primers III and IV and Toyobo's KOD one PCR Master Mix. The PCR product was detected by agarose gel electrophoresis and the target fragment was recovered by gel excision. The PCR reaction system was the same as that shown in Table 1. GmNIC1 and GmNIC2 full-length gene fragment.

[0033] Primer III: c gac gac aag acc gt g acc ATGGTTTCTTCCAACACTGCCGA (the lowercase letters represent the partial sequence of the homologous recombination vector, used for homologous recombination ligation); Primer IV: ga gga gaa gag ccg TTATTCAAATAACACTTCTGAAGCTATATGGGCTC (the lowercase letters represent part of the homologous recombination vector sequence, used for homologous recombination ligation).

[0034] 2. Overexpression vector construction The amplified GmNIC1 and GmNIC2 The gene fragment was synthesized by homologous recombination and fused with the 35S promoter of double cauliflower mosaic virus (CaMV). GmNIC1 or GmNIC2 The purified PCR product and the nopaline synthase (NOS) terminator were integrated into the binary vector pAGM4673. This backbone vector also carries the RFP fluorescent marker gene for subsequent transgenic plant screening.

[0035] The reaction system is: Table 2

[0036] Reaction conditions: 50°C, 15 min.

[0037] 2. Plasmid Preparation The target bands were further screened by E. coli transformation and colony PCR, and the samples were further verified by PCR, and finally the successfully connected vector OE- GmNIC1 and OE- GmNIC2 .

[0038] (1) E. coli transformation: Table 3

[0039] Place on ice for 30 minutes; in a 42°C water bath for 45 seconds; place on ice for 2 minutes; add 1 mL of antibiotic-free LB liquid medium and place at 37°C, shaker at 200 rpm for 1 hour; remove and centrifuge at 6000 rpm for 2 minutes, remove the supernatant, spread the remainder on Kan LB solid medium, and culture at 37°C overnight.

[0040] (2) Colony PCR: Table 4

[0041] The forward primer and reverse primer are the same as those used in Example 2.

[0042] Use the tip of the pipette to pick up the colonies obtained in step (1) and add them into the prepared system.

[0043] PCR reaction conditions are: (1) Pre-denaturation at 95°C for 5 min; (2) Denaturation at 95°C for 30 sec, 55°C for 30 sec, and 72°C for 45 sec, for a total of 35 cycles; (3) Storage at 4°C.

[0044] two, GmNIC1 RNAi and GmNIC2 RNAi Vector construction 1. Select the positive sense fragment of the soybean NIC1 / 2 silent gene (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+ using the full-length product of NIC1 / 2 obtained above as a template.

[0045] The PCR reaction system is: Table 5

[0046] Note: KOD one PCR Master Mix was purchased from Toyobo Company, catalog number KMM-201.

[0047] The primers for amplifying NIC1+ are: Forward primer: tcactcggatccatttaaatCAGACAGTTGAACTCTTAAAGAATGAGATTC; Reverse primer: GCcctaggaggcgcgcccCTGCTGCACCAAACAATACTTCATTTG; The primers for amplifying NIC2+ are: Forward primer: gaaagggatcttcactcgGAGAGAGGAGATTCCTGTGAAGCAACAACCTC; Reverse primer: acgatctgcttgacTATGGGCTCCCCTTCCACTGGCTATGTAAAGG.

[0048] The PCR reaction conditions were as follows: (1) pre-denaturation at 95°C for 2 min; (2) denaturation at 95°C for 30 sec, 55°C for 30 sec, and 72°C for 10 sec, for a total of 35 cycles; (3) storage at 4°C.

[0049] 2. Digest the pGRNAi2 vector with Swa I overnight at 37°C to obtain the Swa I digestion product. Dephosphorylate the 5' end of the DNA using Quick CIP at 37°C for 15 minutes and then at 65°C for 20 minutes. Purify the digestion product using a DNA purification kit (purchased from Novozymes, Cat. No. DC301).

[0050] The Swa I enzyme digestion system is: Table 6

[0051] The Quick CIP dephosphorylation system is: Table 7

[0052] 3. Using the Gibson homologous recombination method, the gene fragment NIC1+ or NIC2+ obtained in step 1 and the single enzyme digestion and purification product obtained in step 2 were connected. The connection product was named GRNAi2-NIC1+ / GRNAi2-NIC2+.

[0053] The reaction system is: Table 8

[0054] Reaction conditions: 50°C, 15 min.

[0055] 4. Further screening was performed using E. coli transformation and colony PCR. Samples with target bands were further verified by PCR, and finally the successfully connected vector GRNAi2-NIC1+ / GRNAi2-NIC2+ was obtained.

[0056] (1) E. coli transformation: Table 9

[0057] Place on ice for 30 min; in a 42°C water bath for 45 s; place on ice for 2 min; add 1 mL of antibiotic-free LB liquid medium and shake at 37°C and 200 rpm for 1 h; remove the culture and centrifuge at 6000 rpm for 2 min, remove the supernatant, and spread the remainder on LB solid medium containing 50 mg / L Kan antibiotics and culture at 37°C overnight.

[0058] (2) Colony PCR: Table 10

[0059] Use the tip of the pipette to pick up the colonies obtained in step (1) and add them into the prepared system.

[0060] The PCR reaction conditions were as follows: (1) pre-denaturation at 95°C for 5 min; (2) denaturation at 95°C for 30 sec, 55°C for 30 sec, and 72°C for 45 sec, for a total of 35 cycles; (3) storage at 4°C.

[0061] The vector GRNAi2-NIC1+ / GRNAi2-NIC2+ was obtained.

[0062] 5. Digest the vector GRNAi2-NIC1+ / GRNAi2-NIC2+ with BamHI and incubate at 37°C overnight. Dephosphorylate the 5' end of the DNA using QuickCIP at 37°C for 15 min and 65°C for 20 min.

[0063] The Bam HI enzyme digestion system is: Table 11

[0064] The Quick CIP dephosphorylation system is: Table 12

[0065] 6. Select the antisense fragment of the soybean NIC1 / 2 silent gene (fragment sequence see SEQ ID NO.7 and SEQ ID NO.8), use PCR to use the full-length product of NIC1 / 2 obtained above as a template, and amplify the target gene NIC1 / 2 fragment NIC1- or NIC2-.

[0066] The PCR reaction system is: Table 13

[0067] The primers for amplifying NIC1- are: Forward primer ggtagatatcatttaaatCAGACAGTTGAACTCTTAAAGAATGAGATTC; Reverse primer: cgatacAatggcgcgcccCTGCTGCACCAAACAATACTTCATTTG; The primers for amplifying NIC2- are: Forward primer agcgggtagatatcatttGAGAGAGGAGATTCCTGTGAAGCAACAACCTC; Reverse primer: caattatcgatacAatggGCTCCCCTTCCACTGGCTATGTAAAGG.

[0068] PCR reaction conditions are: (1) Pre-denaturation at 95°C for 2 min; (2) Denaturation at 95°C for 30 sec, 55°C for 30 sec, and 72°C for 10 sec, for a total of 35 cycles; (3) Storage at 4°C.

[0069] 7. Use Gibson homologous recombination again to connect the gene fragment NIC1- or NIC2- obtained in step 6 and the gRNAi2+ Bam HI single enzyme digestion and purification product obtained in step 5, respectively. The ligation product is named gRNAi2 NIC1+ / - or gRNAi2 NIC2+ / -.

[0070] The reaction system is: Table 14

[0071] Reaction conditions: 50°C, 15 min.

[0072] 8. Using E. coli transformation and antibiotic selection, followed by molecular verification using PCR and sequencing, the successfully linked vectors GRNAi2 NIC1+ / - or GRNAi2 NIC2+ / - were obtained.

[0073] (1) E. coli transformation: Table 15

[0074] Place on ice for 30 minutes; in a 42°C water bath for 45 seconds; place on ice for 2 minutes; add 1 ml of antibiotic-free LB liquid medium and place on a shaker at 37°C and 200 rpm for 1 hour; remove and centrifuge at 6000 rpm for 2 minutes, remove the supernatant, and spread the remainder on LB solid medium containing 50 mg / L Kan antibiotics, and culture at 37°C overnight.

[0075] (2) Colony PCR: Table 16

[0076] Use a pipette tip to pick up the colonies obtained after the above large intestine transformation and smearing on Kan medium and add them into the prepared system.

[0077] PCR reaction conditions are: (1) Pre-denaturation at 95°C for 5 min; (2) Denaturation at 95°C for 30 sec, 55°C for 30 sec, and 72°C for 45 sec, for a total of 35 cycles; (3) Storage at 4°C. The vectors GRNAi2 NIC1+ / - or GRNAi2 NIC2+ / - were obtained.

[0078] Example 3 Silence GmNIC1 Gene, GmNIC2 Genes and overexpression GmNIC1 Gene, GmNIC2 Obtaining genetically modified soybean roots 1. Agrobacterium Preparation The prepared GRNAi2 NIC1+ / - or GRNAi2 NIC2+ / - and OE- GmNIC1 or OE- [[ID=1۳4]]GmNIC2 The plasmid vector was transformed into Agrobacterium rhizogenes ( Agrobacterium rhizogenes ) strain ARqua1. After 2 days, positive clones were picked and inoculated into selective liquid culture medium. Cultured with shaking at 28°C, the cells were collected and resuspended in infiltration buffer (B5 medium 3.0-3.5 g / L, sucrose 28-32 g / L, MES 3.8-4.0 g / L, acetosyringone 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) to an OD of 0. 600 = about 0.7, incubate at room temperature for 2 h.

[0079] 2. Soybean hairy root transformation Select healthy, plump soybean cotyledons and disinfect them with 75% ethanol for 30 seconds. Rinse once with water, then soak in disinfectant for 4 minutes. Finally, soak three times in water for five minutes each. Place the cotyledons in an Agrobacterium suspension in a laminar flow hood. Use a razor blade to cut off the bottom fifth of the soybean cotyledons for Agrobacterium-mediated genetic transformation. Transfer the treated cotyledons to a co-culture medium covered with sterile filter paper and incubate in an incubator for 3 days.

[0080] 3. Screening and verification of transgenic root systems 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.

[0081] 4. GmNIC1 RNAi and GmNIC2 RNAi gene silencing and OE- GmNIC1 or OE- GmNIC2 Overexpression verification Total RNA was extracted from positive soybean hairy root samples 3 days after SCN infection. cDNA was synthesized using the HiScript Reverse Transcriptase Kit. RT-qPCR was performed using AceQ qPCR SYBR Green Master Mix. At least three biological replicates and three technical replicates were used for each treatment. Relative gene expression levels were calculated. The soybean Actin gene was used as an internal control. The specific method is as follows: The following steps are used for plant RNA extraction: The Trizol method was used to extract plant RNA. The following steps were performed: soybean tissue was ground in liquid nitrogen, collected in a 1.5 mL centrifuge tube, and 1 mL of Trizol was added and shaken thoroughly. The tube was then allowed to stand at room temperature for 5 minutes. 200 μL of chloroform was added, the tube was shaken vigorously for 15 seconds, and then allowed to stand at room temperature for 5 minutes. The tube was centrifuged at 12,000 rpm at 4°C for 15 minutes. Approximately 500 μL of the supernatant was transferred to a new centrifuge tube. An equal volume of isopropanol was added, the tube was mixed by inversion, and allowed to stand at room temperature for 10 minutes. The tube was centrifuged at 12,000 rpm at 4°C for 10 minutes, and the supernatant was discarded. The pellet was washed with 1 mL of 75% ethanol, centrifuged at 7,500 rpm at 4°C for 5 minutes, and the supernatant was discarded. The wash was repeated once, and the tube was emptied and the alcohol was dried in a laminar flow hood. Finally, 20-30 μL of RNase-free water was added to dissolve the RNA. The concentration was measured and the tube was stored at -80°C until further use.

[0082] Reverse transcription: Take 500 ng of extracted RNA, add 2 μL DNA Digester Mix, make up to 14 μL with RNA-free H2O, and incubate at 37°C in a metal bath for 2 min; add 2 μL reverse transcriptase, set the PCR program to 55°C for 5 min, 85°C for 5 s, and place in a -20°C refrigerator until use.

[0083] Quantitative PCR: Use AceQ qPCR SYBR Green Master Mix. The reaction system is as follows: Table 17

[0084] After the system was prepared, the sample was placed on a Bio-Rad CFX96™ Real-Time PCR Detection System for amplification.

[0085] like Figure 4 As shown in A, compared with the control group, the roots of the gene silencing treatment group GmNIC1 and GmNIC2 The expression of genes was significantly reduced; Figure 4 As shown in B, compared with the control group, overexpression of OE- GmNIC1 and OE- GmNIC2 The root of the quality GmNIC1 or GmNIC2 The expression of genes was significantly increased.

[0086] Example 4 Silence GmNIC1 Gene or GmNIC2 Genes and overexpression GmNIC1 Gene or GmNIC2 Genetic resistance to nematodes in soybeans 1. Nematode culture: Soybean cyst nematodes ( Soybean Cyst Nematode For the Hg Type 0 (SCN) population, nematode eggs were hatched in 3 mM ZnCl₂ buffer and incubated at room temperature for 5 days to allow them to develop to the J2 stage. J2 nematodes were treated with a solution containing 0.1 g / L HgCl₂ and 0.01% sodium azide for 3 minutes and then rinsed twice with sterile water. J2 SCNs were suspended in a 0.05% sterile agarose aqueous solution in preparation for root inoculation.

[0087] 2. Nematode Inoculation: Under greenhouse conditions, J2-stage nematodes were inoculated into the root tips of transgenic soybean plants and non-transgenic control plants, with a minimum of 500 nematodes per root. Control plants were transformed with Agrobacterium rhizogenes containing an empty vector. The inoculated soybeans were cultured in an incubator for 14 days. The nematode development was then observed.

[0088] 3. Observation and statistics of nematode development Acid fuchsin staining was used to observe and record nematode development in soybean roots. The specific steps were as follows: First, the soybean roots were removed from the culture medium and rinsed with clean water. Sodium hypochlorite was then diluted with water in a 1:4 ratio and soaked in the sodium hypochlorite solution for 5 minutes. The roots were then rinsed three times with clean water to remove excess sodium hypochlorite. After adding the acid fuchsin stain, the roots were boiled in a waterbath for 5 minutes and then rinsed with tap water to remove the stain. Nematodes in the roots of the transgenic and control groups were observed under a microscope, and the developmental stage and number of nematodes present were recorded.

[0089] The staining results are as follows Figure 5 As shown in the following statistics, Figure 6 As shown, compared with the control group, GmNIC1 Gene or GmNIC2 Gene silencing significantly inhibited the development of root nematodes in the susceptible soybean variety Williams 82; Figure 6 Compared with the control group, overexpression GmNIC1 Gene or GmNIC2 Significantly promoted the development of root nematodes in the resistant soybean variety Forrest.

[0090] Example 5 GmNIC1 Genes and GmNIC2 Research on the mechanism of enhancing plant resistance to nematodes In this example, the CRISPR / Cas9 system was used to knock out the GmNIC1 and GmNIC2 The nicotinamide (NAM) content in soybean roots under different treatment conditions was quantitatively detected by combining liquid chromatography-mass spectrometry (HPLC-MS / MS) to clarify GmNIC The regulatory effects of genes on NAM metabolism and its physiological significance in the response to SCN infection.

[0091] (1) GmNIC1 and GmNIC2 The functional annotation of the gene shows that its encoded product is Nicotinamidase, which belongs to the hydrolytic enzyme class and catalyzes the reaction: Nicotinamide + H2O → Nicotinate + NH3 Its international enzymatic number is EC 3.5.1.19. In soybeans, this enzyme is responsible for metabolizing NAM into nicotinic acid.

[0092] (2) Select wild type soybean Williams 82 (Wm82) and its GmNIC1 Crispr, GmNIC2 Crispr knockout mutants were used as experimental materials. Soybean seedlings were cultured under sterile conditions and their roots were inoculated with at least 500 J2 larvae of the soybean cyst nematode (SGC Nematode). A control group (no SCN inoculation) and a treatment group (SCN inoculation) were set up. Root samples were collected 8 days after treatment (dpi). All samples were immediately frozen in liquid nitrogen, ground, and stored at -80°C until analysis.

[0093] (3) The NAM content in soybean roots was determined by HPLC-MS / MS.

[0094] The steps are as follows: (1) Take 0.5 g of fresh root tissue sample, add 0.01 mol / L hydrochloric acid extract solution and ultrasonically extract three times, combine the supernatants and adjust the volume. (2) Filter through a 0.22 μm aqueous filter membrane and send it to liquid chromatography-mass spectrometry for quantitative analysis. (3) The analytical conditions include: Waters ACQUITY UPLC BEH HSS T3 column, 0.1% formic acid water (A) and methanol (B) as mobile phases, mass spectrometry monitoring mode is positive ion mode, [M+H]^+ = 123, product ions are 78 and 80, and collision energy is 18 V and 15 V.

[0095] like Figure 7 As shown in Figure 2, the NAM quantitative results showed that the nicotinamide content in wild-type Wm82 soybean did not change significantly under SCN infection conditions; GmNIC1 and GmNIC2 In the gene knockout mutant, the NAM content increased significantly, by more than 2 times.

Claims

1. GmNIC1 Genes and / or GmNIC2 The application of a gene in regulating the resistance of a plant to soybean cyst nematodes is characterized in that: described 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 use according to claim 1, characterized in that The application method is: by knocking out, knocking down, silencing, interfering GmNIC1 and / or GmNIC2 A way in which genes enhance plant resistance to soybean cyst nematode.

3. Use of GmNIC1 protein and / or GmNIC2 protein in regulating plant resistance to 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. Use of a recombinant vector in regulating plant resistance to soybean cyst nematode, characterized in that: The recombinant vector comprises GmNIC1 Genes and / or GmNIC2 gene, characterized in that 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. The use of genetically engineered bacteria in regulating plant resistance to soybean cyst nematode, characterized in that: The genetically engineered bacteria comprises GmNIC1 Genes and / or GmNIC2 gene, characterized in that 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 soybean resistance to soybean cyst nematode, characterized in that: include: Knockout, knockdown, silencing, interference with soybean GmNIC1 and / or GmNIC2 A method for enhancing soybean resistance to soybean cyst nematodes; 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 of GmNIC1 and / or GmNIC2 RNAi vectors targeting genes; (2) The CRISPR / Cas9 vector is transferred into Agrobacterium competent cells to construct a knockout GmNIC1 and / or GmNIC2 Agrobacterium strains carrying gene RNAi vectors; (3) Transforming soybeans with the Agrobacterium strain to obtain silenced GmNIC1 and / or GmNIC2 Soybean homozygous lines of the gene.

8. The method according to claim 6, characterized in that The knockout GmNIC1 and / or GmNIC2 Gene: soybean modified by CRISPR / Cas9 technology GmNIC1 and / or GmNIC2 Gene knockout.

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