GmPDH1 gene and application of protein coded by GmPDH1 gene in resisting soybean cyst nematode No.4 race

Abstract

The invention belongs to the technical field of molecular biology and crop germplasm resource identification, and provides an application of a GmPDH1 gene and a protein coded by the GmPDH1 gene in resisting soybean cyst nematode No.4 race. The nucleotide sequence of the GmPDH1 gene CDS is as shown in SEQ ID NO. 1, and the amino acid sequence coded by the gene is as shown in SEQ ID NO. 2. And the GmPDH1 positively regulates the resistance of the soybeans to the No.4 microspecies of the soybean cyst nematode. The method comprises the following steps: constructing an overexpression vector by using a pCAMBIA1302 vector, constructing a knockout vector by using a CRISPR-Cas9 editing technology, respectively transforming the overexpression vector and the knockout vector into soybean root systems, and inoculating the soybean root systems with soybean cyst nematode for identification. The result shows that the resistance of the soybean to the SCN4 is improved by overexpressing the soybean GmPDH1 gene, and the resistance of the soybean to the SCN4 is reduced by knocking out the GmPDH1 gene. And a gene resource is provided for soybean cyst nematode disease resistant genetic breeding of soybeans.

Description

Technical Field

[0001] The present invention belongs to the field of plant genetic engineering technology and specifically relates to GmPDH1 Application of genes and their encoded proteins in resistance to soybean cyst nematode race 4. Background Art

[0002] Soybean is an important economic crop and one of the world's most important grain and oil crops. Soybean cyst nematode (SCN) is a global disease that severely impacts soybean yield and quality. In the field, the disease exhibits a distinct spreading pattern, initially appearing as spots or patches, then gradually spreading, ultimately causing widespread plant dieback. This typically results in a 10% to 30% reduction in soybean yield, and in severe cases, can reach 70% to 80%.

[0003] GmPDH1 The gene encodes prephenate dehydrogenase (PDH), a key enzyme in the soybean plastidial tyrosine biosynthesis pathway. PDH participates in tyrosine synthesis and indirectly influences lignin synthesis by regulating phenylalanine synthesis, thereby enhancing root disease resistance. Unlike most plants, legumes possess both a prephenate dehydrogenase pathway and a pretyrosine pathway. Tyrosine (Tyr) is synthesized from chorismate, the final product of the shikimate pathway. Chorismate is first converted to prephenate by chorismate mutase (CM), a key step in the biosynthesis of tyrosine and phenylalanine (Phe). Prephenate is converted to tyrosine through oxidative decarboxylation and transamination. The HPP pathway and the arogenate pathway are common pathways for Tyr biosynthesis. Prephenate can be converted into 4-hydroxyphenylpyruvate (HPP) by prephenate dehydrogenase (PDH), and then produce Tyr through transamination, which is usually called the PDH pathway or HPP pathway. In another arogenate pathway, prephenate is transaminated to L-arogenate by prephenate aminotransferases (PPA-ATs), and then oxidatively decarboxylated to tyrosine by arogenate-specific tyrosine dehydrogenase (ADH), so it is also called the ADH pathway.

[0004] Previous studies have shown that Arabidopsis AT5G34930 The gene encoding arogenate dehydrogenase regulates tyrosine synthesis. AT5G34930 Two soybean homologs, Glyma.14g055300 and Glyma.18g023100, regulate two common pathways for tyrosine biosynthesis in soybean. Glyma.14g055300 encodes arogenate dehydrogenase (ADH2), which synthesizes tyrosine within the plastid, while Glyma.18g023100 encodes prephenate dehydrogenase (PDH1), which synthesizes tyrosine outside the plastid. Tyrosine synthesis by ADH is inhibited by tyrosine production. The soybean PDH pathway is unaffected by tyrosine and other intermediates and products of the pathway. This tyrosine biosynthesis pathway escapes competition with phenylalanine in the plastid, favoring the biosynthesis of phenylalanine, a precursor of lignin, a major cell wall component, and thus impacting soybean root disease resistance.

[0005] and GmPDH1 Whether this gene plays a role in soybean resistance to cyst nematodes has not been reported. Summary of the Invention

[0006] The present invention provides GmPDH1 The invention clones the soybean cyst nematode. GmPDH1 Gene, plant overexpression vector and knockout vector were constructed, transgenic soybean hairy roots were obtained, and the internal structure of soybean hairy roots was studied. GmPDH1 The results provide new insights into the molecular regulation of soybean resistance to cyst nematode disease.

[0007] The present invention is achieved by the following technical solutions: GmPDH1 Application of the gene and the protein encoded by the gene in resistance to soybean cyst nematode race 4, the GmPDH1 The nucleotide sequence of the gene CDS is shown in SEQ ID NO.1, and the amino acid sequence encoded by the gene is shown in SEQ ID NO.2.

[0008] Furthermore, the GmPDH1 The gene is overexpressed in soybean roots, promoting the synthesis of lignin in the cell walls of soybean roots and improving the resistance of soybean roots to soybean cyst nematode race 4.

[0009] described GmPDH1 The gene was overexpressed in soybean roots using the vector pCAMBIA1302- GmPDH1, It was constructed using the eukaryotic expression vector pCAMBIA1302 via CRISPR-Cas9 editing technology.

[0010] described GmPDH1 Application of gene overexpression vector in improving soybean resistance to soybean cyst nematode race 4.

[0011] described GmPDH1 Application of gene overexpression vector in genetic breeding of soybean cyst nematode resistance race 4.

[0012] The specific application method is: overexpression vector pCAMBIA1302- GmPDH1 Introduced into susceptible soybean varieties.

[0013] The soybean varieties described in the present invention are Jindou No. 23, Jack, and gray-skinned black beans.

[0014] Compared with the prior art, the present invention has the following beneficial effects: The present invention found soybean GmPDH1 The gene for resistance to soybean cyst nematode is positively regulated. GmPDH1 Overexpression of the gene in soybean roots can significantly improve the disease resistance of the plant; knockout GmPDH1 The gene significantly reduces soybean root resistance to soybean cyst nematode. GmPDH1 The study of gene function provides genetic resources for breeding soybean varieties resistant to soybean cyst nematodes. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a gel electrophoresis diagram of the RNA quality test of C. griseus in Example 1 of the present invention, wherein: M: Marker DL45000, I: C. griseus black bean RNA; Figure 2 For the gray skin black bean in Example 1 of the present invention GmPDH1 Gel electrophoresis of gene cloning; in the figure: M: Marker, DL2000; 1-2: GmPDH1 Gene; Figure 3 The electrophoresis diagram of the enzyme digestion of pCAMBIA1302 plasmid in Example 1 of the present invention; M in the figure: DNA Marker DL15000; 1, 2: plasmid pCAMBIA1302 Nco I and Spe I Double enzyme digestion; 3, 4: pCAMBIA1302 original plasmid; Figure 4 For pCAMBIA1302- in Example 1 of the present invention GmPDH1 Escherichia coli DHα Bacterial liquid PCR verification diagram; Figure 1, 2: pCAMBIA1302- GmPDH1 Positive monoclonal; M: DNA Marker III; Figure 5 This is a schematic diagram of the carrier in Example 1 of the present invention; Figure 6 pCAMBIA1302- GmPDH1PCR sequencing comparison of positive bacterial solution; Figure 7 pCAMBIA1302- GmPDH1 Transfection with K599 PCR validation diagram; in the figure, 0: negative control; 1-4: positive single clones; M: DNA Marker III; Figure 8 Schematic diagram of Agrobacterium infection; Figure 9 GFP fluorescence imaging of transgenic soybean hairy roots in Example 1 of the present invention; in the figure: AC: bright field corresponding to the fluorescence images of WT and OE; DF: WT and OE fluorescent hairy roots; Figure 10 For the transformation of pCAMBIA1302- GmPDH1 PCR detection diagram of gene-resistant hairy roots; M: DNA Marker III; 2: negative control; 1: positive control; 3-12: pCAMBIA1302- GmPDH1 Transformation of resistant hairy roots; Figure 11 for GmPDH1 Quality detection diagram of overexpressed hairy root RNA; Figure 12 The positive hairy roots in Example 1 of the present invention and the wild type GmPDH1 relative expression of genes; Figure 13 The number of cyst nematodes in the positive hairy roots and the wild type after infection by SCN4 in Example 1 of the present invention; Figure 14 For CRISPR / Cas9- GmPDH1 Schematic diagram of the target site of the gene knockout vector; Figure 15 In Example 2 of the present invention GmPDH1 Electrophoresis of target fragment; In the figure: M: Marker DL2000; 1-2: GmPDH1 Purpose fragment; Figure 16 The single enzyme digestion map of pHSE401 plasmid in Example 2 of the present invention; 1-2: Plasmid pHSE401 Bsa I Single enzyme digestion; 3-4: pHSE401 original plasmid; Figure 17 For CRISPR / Cas9- GmPDH1 Vector PCR verification image; M: DNA Marker III; 1-7: positive single clones; Figure 18 In Example 2 of the present invention GmPDH1Gene CRISPR / Cas9 vector sequencing results; PDH1-3, PDH1-8: positive single clones; Figure 19 This is an electrophoresis detection diagram of the CRISPR / Cas9 vector transformed into Agrobacterium rhizogenes in Example 2 of the present invention; M: DNA Marker III; 1-10: positive colonies; Figure 20 Transfer CRISPR / Cas9- GmPDH1 Gene positive identification PCR results; M: DNAMarker III; 2: wild type control; 1, 3-6: CRISPR / Cas9- GmPDH1 ; Figure 21 For CRISPR / Cas9- GmPDH1 Sequencing results of transgenic soybean hairy roots; in the figure: WT: wild soybean hairy roots; TArget1-50 / 82, TArget2-50 / 82: transformed CRISPR / Cas9- GmPDH1 soybean hairy roots of the carrier; Figure 22 The number of soybean cyst nematodes in the knockout-positive hairy roots and the control roots after being infected by SCN4 in Example 2 of the present invention. DETAILED DESCRIPTION

[0016] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, not all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0017] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and the disclosure and materials cited therein are hereby incorporated by reference.

[0018] Technical equivalents to the specific embodiments described that are apparent to those skilled in the art using no more than routine experimentation are intended to be encompassed by this application.

[0019] The experimental methods in the following examples, unless otherwise specified, are all conventional methods. The instruments and equipment used in the following examples, unless otherwise specified, are all conventional laboratory instruments and equipment; the experimental materials used in the following examples, unless otherwise specified, can all be purchased from conventional biochemical reagent stores.

[0020] The present invention provides soybean GmPDH1 Application of the gene and the protein encoded by the gene in regulating soybean resistance to SCN4. GmPDH1 The nucleotide sequence of the gene is shown in SEQ ID NO.1; the amino acid sequence of the protein encoded by it is shown in SEQ ID NO.2. GmPDH1 Gene or its encoded protein, improves soybean resistance to SCN4; by knocking out soybean GmPDH1 Gene or its encoded protein, so that soybean GmPDH1 The gene or the protein it encodes loses its biological function, reducing soybean resistance to SCN4.

[0021] Example 1: Overexpression GmPDH1 Genes improve soybean disease resistance one, GmPDH1 Gene cloning (1) Pick up black beans with full grains and cultivate them into seedlings on vermiculite. After the first three-leaf compound leaf unfolds, take 1 g of soybean root sample, place it in a 1.5 mL sampling tube, and place it in a -80℃ refrigerator for later use. The black bean root sample was ground into powder using a high-throughput grinder and cooled with liquid nitrogen. RNA was then extracted using the TaKaRa MiniBEST PlantRNA Extraction Kit. For specific methods, refer to the instructions. The integrity of the extracted RNA was tested by 1% agarose gel electrophoresis ( Figure 1 ).

[0022] (2) The extracted RNA solution was reverse transcribed using the PrimeScript™ RT reagent Kit (RR037A). The specific steps were referred to the instructions.

[0023] (3) Using the cDNA of black bean as a template, according to GmPDH1 The CDS sequence of the gene is designed with full-length primers containing homologous sequences (15-20 bp) at both ends of the vector and restriction enzyme cutting sites. GmPDH1 The primers for the CDS sequence of the gene are shown in Table 1. PCR amplification was performed using high-fidelity Taq enzyme. The PCR products were detected by 1% agarose gel electrophoresis. Figure 2 DNA was recovered from the gel using the Tiangen Agarose Gel DNA Recovery Kit (DP209), following the kit instructions. The PCR reaction system is shown in Table 2, and the reaction procedure is shown in Table 3.

[0024] Table 1: GmPDH1 Gene CDS sequence primers Table 2: PCR reaction system Table 3: PCR reaction program two, GmPDH1 Gene overexpression vector construction 1. Obtaining and purifying the target fragment: Using the cDNA of soybean variety gray-skinned black bean as template, according to GmPDH1 The CDS sequence of the gene is designed with full-length primers containing homologous sequences (15-20 bp) at both ends of the vector and restriction enzyme cutting sites. GmPDH1 Primers for the gene CDS sequence are shown in Table 4. PCR amplification was performed using high-fidelity Taq enzyme. PCR products were examined by 1% agarose gel electrophoresis. The products were then recovered and purified using a rapid gel purification kit, following the kit instructions. The PCR reaction system is shown in Table 5, and the reaction protocol is shown in Table 6. Steps 2 to 4 were set for 34 cycles.

[0025] Table 4: GmPDH1 Gene CDS sequence primers Table 5: PCR reaction system Table 6: Reaction Procedure B. Preparation and purification of linearized vector: Extract the pCAMBIA1302 vector plasmid using a plasmid extraction kit. Refer to the kit instructions for detailed steps. Load 1 μL of the extracted plasmid into an ultra-micro nucleic acid protein analyzer to measure the concentration of the extracted plasmid. Nco Ⅰ and Spe Ⅰ The plant expression vector pCAMBIA1302 was double-digested with restriction endonucleases. The digestion system is shown in Table 7. The reaction conditions were: 37°C for 3 h; then heated to 80°C for 20 min for inactivation. Figure 3 As shown, the circular plasmid digests faster than the enzyme-digested plasmid, indicating successful enzyme digestion. After detection, the digestion product is purified and recovered and can be used directly in the next step or stored at -20°C.

[0026] Table 7: Enzyme digestion system C. Recombination and transformation of the target fragment and linearized vector: The target fragment and linearized vector were recombined using seamless cloning. The reaction system is shown in Table 8. Reaction conditions are: 50°C for 5 min; then cool to 4°C or place on ice.

[0027] Table 8: Reaction system 2. Transformation of linearized vector and target fragment: A. Preparation of competent E. coli: (1) Thaw the E. coli (DH5α) culture from a -80°C ultra-low temperature freezer. Inoculate the culture into an antibiotic-free LB solid medium in a clean bench, seal the container, and place in a 37°C constant temperature incubator for overnight activation. (2) Pick a single colony of E. coli and inoculate it into 50 mL of LB liquid medium. Incubate the culture at 37°C, 200 rpm, and shake overnight until the OD 600 reaches 0.4 ≤ 0.6. (3) Incubate the E. coli on ice for 20 min. After cooling the culture to 0°C, transfer the culture into a pre-cooled 50 mL sterile centrifuge tube and centrifuge at 5000 rpm at 4°C for 8 min. Discard the supernatant. (4) Add 30 mL of pre-cooled sterile 0.1 M calcium chloride (CaCl2) solution to the precipitate. Use a pipette to suspend the cells. Centrifuge at 5000 rpm at 4°C for 8 min to collect the cells. Retain the precipitate. (5) Add 4 mL of pre-cooled sterile 0.1 M CaCl2 solution to resuspend the cells, add 1 mL of pre-cooled 80% sterile glycerol, mix with a pipette, and dispense 200 µL / tube into 1.5 mL sterile centrifuge tubes and store in a -80 °C ultra-low temperature refrigerator; B. Transform the recombinant product into competent E. coli. The recombinant plasmid was transformed into E. coli using the freeze-thaw method: (1) Add 5 µL of the recombinant plasmid to 200 µL of competent E. coli, gently blow with the pipette tip to mix, and place on ice for 30 min; (2) Heat shock in a 42°C metal bath for 90 s without shaking; (3) Quickly cool in an ice bath for 1-2 min; Add 800 µL of liquid LB culture medium, shake on a shaker at 37°C, 200 rpm for 45 min; (4) Take about 20 µL of the bacterial liquid and evenly spread it on LB solid culture medium containing 50 µg / mL kanamycin resistance, and culture in the dark at 37°C for 16 h.

[0028] C. Plant expression vector pCAMBIA- 1302-GmPDH1 Detection and extraction: Pick the single colony on the LB solid plate and add it to 1 mL of LB liquid medium (containing kanamycin 50ug / ml) and shake it at 200 rpm at 37℃ for 4-5 hours. GmPDH1-1302-F / R Primer 、 The bacterial solution was used as a template for PCR. The PCR reaction system is shown in Table 9, and the reaction program is shown in Table 10. Steps 2 to 4 were set for 30 cycles. The results are shown in Figure 4 Schematic diagram of the vector is shown in Figure 5 The fragment size is consistent with the expected size. This indicates that the plant overexpression vector was successfully constructed. The bacterial solution sequencing results are as follows Figure 6 shown.

[0029] Table 9: Reaction system Table 10: Reaction Procedure 3. pCAMBIA-1302- GmPDH1 Obtaining transgenic plants A. Preparation of competent Agrobacterium GV3101: (1) Pick the K599 strain stored at -80℃, streak it on a TY solid plate containing 50 μg / mL rifampicin, and incubate it in the dark at 28℃ for 48 h; (2) Pick a single colony and inoculate it into 150 mL of TY liquid medium containing 50 μg / mL rifampicin, shake it at 28℃ at 220 rpm for about 12 h, and shake it until the OD value is 0.4≤ 600 ≤ 0.6; centrifuge at 5000 rpm, 4℃ for 5 min, and discard the supernatant; (3) resuspend the precipitated bacteria in 10 mL of pre-cooled sterile NaCl solution with a concentration of 0.15 mol / L; centrifuge at 5000 rpm, 4℃ for 5 min, and discard the supernatant; (4) add 1 / 50 of the original volume of 20 mmol CaCl2 (containing 15% glycerol) to suspend the cells after precipitation, and divide them into sterile 1.5 mL centrifuge tubes, 200 μL / tube, and store in a -80℃ ultra-low temperature refrigerator.

[0030] B. K599 transformation and identification of recombinant plasmids: (1) Take 1-5 µL of recombinant plasmid and add it to 200 µL of competent cells. Mix thoroughly by gently pipetting and place on ice for 5 minutes. (2) Freeze in liquid nitrogen for 5 minutes; heat shock in a metal bath at 37°C for 5 minutes, then cool. (3) Add 800 µL of TY liquid medium containing 50 µg / mL rifampicin and slowly shake at 175 rpm for 3 hours at 28°C. Centrifuge at 3000 rpm for 4 minutes, discard part of the supernatant, and retain approximately 200 µL of the resuspended cells. (4) Spread 20 µL of the supernatant onto a TY solid plate containing 50 µg / mL kanamycin and 50 µg / mL rifampicin and incubate in the dark at 28°C for 48 hours. (5) Identification of positive clones: A single colony was picked and inoculated into TY liquid medium (containing 50 μg / mL kanamycin and 50 μg / mL rifampicin), and cultured at 28°C and 175 rpm for 48 h for colony PCR. The reaction system and reaction procedure were similar to those of E. coli recombinant plasmid colony PCR. The results of 1% agarose gel electrophoresis showed that the band size was consistent with the expected value, indicating that the transgenic strain was transformed and could be used for the next soybean infection experiment. The results are shown in Figure 2. Figure 7 shown.

[0031] C. Transformation of soybean hairy roots: The overexpression vector pCAMBIA1302 was introduced into the susceptible material Jindou No. 23 through Agrobacterium K599. The specific steps are as follows: (1) Select soybean seeds with full grains and uniform size, sterilize them with chlorine for 24 hours, and then germinate and grow seedlings in sterile vermiculite until the first true leaf of the seeds unfolds. (2) Spread the Agrobacterium K599 carrying the target vector on TY culture medium (with corresponding antibiotics, acetosyringa AS), and culture it in a 28℃ incubator without light for 48 hours. (3) Use a sterile scalpel to cut the hypocotyl of the plant obliquely and apply Agrobacterium to the wound. (4) Cut the plant seedlings coated with Agrobacterium into sterile vermiculite, and water the vermiculite with sterile water containing hygromycin in advance. (5) Place the seedling tray in the tissue culture room and spray the nutrient solution continuously until the soybean hairy roots grow. The process is as follows Figure 8 shown.

[0032] D. Overexpression GmPDH1 Identification of Gene-positive Soybean Hairy Roots GFP fluorescence verification of transgenic hairy roots: To detect pCAMBIA- 1302-GmPDH1 To determine whether the gene has been transferred into soybean hairy roots, soybean hairy roots were collected for GFP verification. Figure 9 The results showed that green fluorescence was detected in transgenic hairy roots under the excitation light, while no fluorescence was detected in the entire root of the control group under the excitation light. 4 positive plants were detected.

[0033] E. pCAMBIA- 1302-GmPDH1 transfection Sequencing detection of gene-positive soybean hairy roots (1) Extraction and PCR identification of hairy root DNA: 1) Take plant tissue (100 mg) and place it in a 2 mL centrifuge tube, freeze it in liquid nitrogen with sterile steel beads, and grind it using a high-throughput tissue grinder. 2) Use a plant DNA extraction kit to extract DNA to obtain a DNA solution. 3) PCR amplify the DNA of the hairy roots of the positive strains. The PCR primers are shown in Table 11, the PCR reaction system is shown in Table 12, and the reaction program is shown in Table 13. Set 34 cycles. The product size is 646 bp, and the results are as follows: Figure 10 .

[0034] Table 11: Primer sequences Table 12: PCR reaction system Table 13: Reaction Procedure (2)Extraction of hairy root RNA and reverse transcription into cDNA: The total RNA of positive hairy roots was extracted using a plant total RNA extraction kit. The specific operation steps are as follows: 1) Preparation: Sterilize the pipette tips; Wash, dry the mortar, pestle, and spatula, wrap them with tin foil and sterilize at 121 °C for 20 min; Treat the pipette tips with 0.1% DEPC, take them out after 12 h, and sterilize at 121 °C for 45 min to prepare DEPC-treated water and RNase-free pipette tips; 2) Grinding: Disinfect the mortar by igniting alcohol, and pre-cool it with liquid nitrogen for grinding the roots. Grind the tissue with a pestle, continuously add liquid nitrogen during the process until the sample is ground into powder, and quickly transfer it into a 1.5 mL RNase-free centrifuge tube; 3) Extract the total RNA according to the steps in the plant total RNA extraction kit instruction manual to obtain the RNA solution; 4) RNA detection: Take 1 μL of the RNA solution and measure the RNA concentration under an enzyme-labeling instrument. Determine the RNA quality based on 1.8 < OD260 / OD280 < 2.0; Use 1% agarose gel electrophoresis to detect the integrity of the RNA, and the result is as Figure 11 shown. 5) Reverse transcription into cDNA. Reverse transcribe the RNA according to the steps provided by the reverse transcription kit. The reverse transcription is carried out in a PCR instrument, and the reverse transcription program is: 45 °C for 60 min, 60 °C for 15 min, 4 °C forever.

[0035] (3)qRT-PCR amplification of pCAMBIA-1302- GmPDH1 transgenic positive soybean hairy root cDNA: Perform qRT-PCR reaction using a real-time fluorescence quantitative kit. Select GmActin as the internal reference gene, and the primers are qGmECR14-F and qGmECR14-R. The specific primers are shown in Table 14, the reaction system is shown in Table 15, and the reaction program is shown in Table 16. Set 30 cycles for steps 2 to 4.

[0036] Table 14: RT-PCR primers Table 15: Reaction system Table 16: Reaction program Through qRT-PCR detection of 4 GFP-positive plants, the results are as Figure 12 shown. The gene expression levels in the control group of soybean hairy roots and overexpressed transgenic soybean hairy roots, and the GmPDH1 expression levels of 4 soybean hairy roots GmPDH1 were significantly increased compared with those of the control group of soybean hairy roots.

[0037] 4. Disease resistance identification of positive transgenic plants Nematode egg suspensions were inoculated into the roots of overexpressing plants and wild-type plants. Roots transformed with an empty vector served as controls. Twenty-five days after nematode inoculation, soybean cysts were counted in hairy roots. WT hairy roots of JD23 control plants contained 39 cysts, while OE hairy roots contained 22 and 5 cysts, respectively. Jack control plants contained 119 cysts in their WT hairy roots, while OE hairy roots contained 65 and 71 cysts, respectively. Figure 13 ), indicating that when GmPDH1 When overexpressed in soybean roots, the number of soybean cysts was significantly reduced, indicating that this gene is involved in regulating soybean cyst nematode resistance.

[0038] Example 2: Knockout of soybean roots GmPDH1 Gene-reduced resistance of transformed plants to soybean cyst nematode race 4 1. Knockout soybeans GmPDH1 Construction of gene vector: through restriction enzyme cutting sites Bsa Ⅰ SgRNAs containing two target site sequences (19 bp) were constructed into CRISPR vectors to construct soybean GmPDH1 The gene knockout vector specifically includes the following steps: 1. CRISPR / Cas9 target selection and primer design: Targets were designed using the CRISPR / Cas9 target online website (http: / / crispr.tefor.net / ), with soybean as the target. GmPDH1 The CDS of the gene (Glyma.18g023100) was used as the target sequence. gRNA targets DT1 and DT2 were designed on the first and second exons of the target gene, respectively (Table 17). The first target site was the 19-nt before NGG (DT1-BsF / F0-gRNA1) with yellow bases; the second target site was the 19-nt before NGG (DT1-BsF / F0-gRNA2) with green bases. The underline represents the endonuclease. Bsa I The restriction enzyme cutting sites (Table 17) of the knockout vector target are as follows Figure 14 shown.

[0039] 2. Target gene amplification and gel recovery: Utilize the DT1 and DT2 primers designed in Table 17 and the previously constructed vector pCBC-DT1T2 as a template to clone the target gene by PCR. The reaction system is shown in Table 18. The reaction procedure is as follows: 98°C, 1 min; 98°C, 10 s; 62°C, 5 s; 72°C, 8 s; 34 cycles; 72°C, 1 min. Prepare a 1% agarose gel and perform gel electrophoresis on the PCR product at a voltage of 60-100 V. After the electrophoresis, observe under ultraviolet light. Figure 15 If the DNA is present, quickly cut the gel containing the target band under UV light and transfer it to a 2.0 mL centrifuge tube. Recover the target fragment according to the instructions of the Magen Gel Recovery Kit (HiPure Gel Pure DNA Mini Kit) and store it at -20°C until use.

[0040] Table 17: Primer sequences Table 18: Reaction system 3. Ligation of target fragment with pCBC-DT1T2 backbone vector: (1) Bbs I After enzyme digestion of the pHSE401 original plasmid and the target fragment, 0.8% agarose gel electrophoresis was performed, and the original plasmid pHSE401 without enzyme digestion was used as a negative control to compare the DNA band sizes to verify whether the pHSE401 vector plasmid was successfully digested ( Figure 16 The corresponding bands of the large fragments were recovered using the Magen gel extraction kit (HiPure Gel Pure DNA Mini Kit); the enzyme digestion reaction system is shown in Table 19. Reaction procedure: 37°C, enzyme digestion for 5 h; inactivation at 85°C for 20 min. (2) T4 DNA ligase was used to ligate the digested PCR products to the pHSE401 plasmid. The reaction system after ligation is shown in Table 19. Reaction procedure: 16°C, enzyme ligation for 6 h.

[0041] Table 19: Enzyme digestion reaction system 4. Transform the constructed plasmid vector into E. coli: The enzyme-linked product was transformed into E. coli DH5α competent cells, and after Kan and Spe screening and PCR identification ( Figure 17 ), pick the positive single clones and extract the plasmid for sequencing. The preparation method, transformation and identification of E. coli competent cells were the same as in Example 1. The primers used for bacterial testing were U626-IDF and U629-IDR, the vector sequencing primers were U626-IDF and U629-IDF, and the target site sequencing primers were gRNA-PDH1-F and gRNA-PDH1-R. The target site sequence was observed for base mutations. Table 20 shows the detection primer sequences and reaction conditions. The sequencing results are shown in Figure 2. Figure 18 .

[0042] Table 20: Detection primers and reaction conditions 2. Preparation and transformation of competent cells of Agrobacterium rhizogenes K599: The preparation method, transformation and identification of competent cells of Agrobacterium K599 were the same as those in Example 1. The detection primers were U626-IDF and U629-IDR (Table 20). Figure 19 .

[0043] 3. Soybeans GmPDH1 Obtaining gene knockout plants: The disease-resistant variety of soybean with gray skin and black beans was selected for gene knockout transgenic soybean. The specific operation is shown in Example 1.

[0044] Four, GmPDH1 Identification of positive soybean hairy roots with gene knockout To verify GmPDH1 To determine whether the gene has been transferred into soybean hairy roots, the genomic DNA of transgenic hairy roots was extracted. The transgenic hairy root DNA was used as a template and genomic DNA PCR amplification was performed using gRNA-specific primers. Detection by 1% agarose gel electrophoresis, as shown in Figure 20, showed that the gRNA gene was transferred into the soybean hairy roots. Next, we further identified whether the target site was effectively edited in the transgenic hairy roots. An amplification primer of about 800 bp containing the target sites Target1 and Target2 was designed. Then, using this primer and the positive transgenic hairy root genomic DNA as a template, PCR amplification was performed. The amplified target band was recovered and purified by gel tapping, sent for testing, and the base editing of the target site was counted. Figure 21 Compared with the wild type, transgenic plant No. 50 had a base A insertion in Target1, and No. 82 had a base A deletion in Target1, indicating that the editing was successful.

[0045] 5. Disease resistance identification of CRISPR / Cas9-GmPHD1 transgenic positive plants The hairy roots of transgenic soybean plants 50 and 82 and the hairy roots infected with empty bacteria of K599 were inoculated with soybean cyst nematode egg suspension, and the number of root cysts was observed after 25 days. Figure 22 As shown, 25 days after nematode inoculation, wild-type and knockout GmPDH1 The roots of all genotype materials have cysts. The average number of cysts in the roots of wild type is 8, while the number of cysts in the roots of positive plants is 16 and 14 respectively. Compared with the wild type, the number of cysts on the hairy roots of transgenic plants is significantly increased. GmPDH1 After the gene was generated, the resistance of gray-skinned black beans was affected, indicating that GmPDH1 The gene is involved in the regulatory process of SCN4 resistance in gray-skinned black beans.

[0046] The present invention overexpresses GmPDH1Genes positively regulate tyrosine synthesis. On the one hand, it can increase the production of tyrosine in the PDH pathway, provide more precursors for the synthesis of defense compounds, and enhance the stress resistance of soybean roots. On the other hand, because the soybean PDH1 enzyme is not inhibited by tyrosine and its intermediates, while ADH is sensitive to tyrosine, a large amount of tyrosine inhibits the function of the ADH enzyme, and more intermediate arogenate is used to synthesize phenylalanine, which is beneficial to the synthesis of lignin, a component of plant cell walls, and is used to resist nematode stress.

[0047] The disease-resistant strain was cloned from the disease-resistant variety of black bean. GmPDH1 By constructing the plant overexpression vector pCAMBIA1302- GmPDH1 accomplish GmPDH1 Overexpression in susceptible varieties and analysis of changes in plant disease resistance before and after overexpression. GmPDH1 It has been successfully integrated into soybean hairy roots and expressed smoothly. Phenotypic statistics were performed by nematode stress. GmPDH1 The number of cysts in overexpression-positive plants was reduced.

[0048] By designing target gene-specific target sites, constructing CRISPR / Cas9- GmPDH1 The knockout vector was transformed into soybean hairy roots using Agrobacterium rhizogenes K599 in a one-step method. The resistance of the knockout-positive hairy roots was reduced by nematode stress. GmPDH1 The gene responds to the stress of soybean cyst nematode No. 4 and is involved in the process of resisting SCN4, laying the foundation for the later breeding of disease-resistant soybean varieties.

[0049] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. GmPDH1 The application of the gene and the protein it encodes in resistance to soybean cyst nematode race 4 is characterized by: described GmPDH1 The nucleotide sequence of the gene CDS is shown in SEQ ID NO.1, and the amino acid sequence encoded by the gene is shown in SEQ ID NO.

2.

2. The use according to claim 1, characterized in that: described GmPDH1 The gene is overexpressed in soybean roots, promoting the synthesis of lignin in the cell walls of soybean roots and improving the resistance of soybean roots to soybean cyst nematode race 4.

3. The use according to claim 2, characterized in that: described GmPDH1 The gene was overexpressed in soybean roots using the vector pCAMBIA1302- GmPDH1, It was constructed using the eukaryotic expression vector pCAMBIA1302 via CRISPR-Cas9 editing technology.

4. The use according to claim 3, characterized in that: described GmPDH1 Application of gene overexpression vector in improving soybean resistance to soybean cyst nematode race 4.

5. The use according to claim 3, characterized in that: described GmPDH1 Application of gene overexpression vector in genetic breeding of soybean cyst nematode resistance race 4.

6. The use according to claim 5, characterized in that: The specific application method is: overexpression vector pCAMBIA1302- GmPDH1 Introduced into susceptible soybean varieties.

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