Application of GmHAP5A protein in improving soybean resistance to soybean cyst nematode

Abstract

The present invention discloses the use of a GmHAP5A protein for improving soybean resistance to soybean cyst nematodes. This invention belongs to the technical field of plant disease resistance. The present invention provides a method for improving soybean resistance to soybean cyst nematodes. The present invention provides the use of a GmHAP5A protein for improving soybean resistance to soybean cyst nematodes. The sequence of the GmHAP5A protein is shown in SEQ ID NO. 4. This provides a new approach and method for breeding soybeans resistant to soybean cyst nematodes.

Description

Technical Field

[0001] The invention belongs to the technical field of plant disease resistance, and particularly relates to application of GmHAP5A protein in improving soybean resistance to soybean cyst nematode. Background Art

[0002] The soybean cyst nematode (Heterodera glycines Ichinohe) is a significant pest that damages soybean yields, causing severe economic losses worldwide. It parasitizes soybean roots, extracting nutrients, causing plant stunting, leaf yellowing, yield reduction, and even plant death. Therefore, research on the soybean cyst nematode has significant theoretical and applied value. The soybean cyst nematode's life cycle consists of five stages: egg, larva, nymph, adult, and cyst. Its parasitic life cycle in soybean roots also involves three key developmental stages: infection, vegetative cell formation, and cyst formation. During this process, the nematode infects soybean root cells and releases secretions that alter the host's biochemical, physiological, and genetic mechanisms, thereby providing the necessary nutrients and environment. Therefore, studying the soybean cyst nematode's life cycle and parasitic processes can provide insights into its infection mechanisms and host resistance mechanisms, providing an important theoretical foundation for the development of natural resistance and transgenic technologies.

[0003] Furthermore, soybean cyst nematodes have developed resistance to chemical pesticides and are insensitive to biological control measures, making traditional control methods ineffective. Therefore, studying the resistance mechanisms and biological control strategies of soybean cyst nematodes is crucial for developing new control technologies and methods to safeguard soybean yields. In the context of current transgenic and gene editing technologies, studying the functional genes and regulatory networks of soybean cyst nematodes can provide important genetic resources and molecular markers for breeding soybeans for disease resistance and nematode resistance. Furthermore, combined with gene editing technology, soybean resistance to soybean cyst nematodes can be enhanced by modifying key genes, further providing new approaches and methods for resistance breeding in soybeans. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for resisting soybean cyst nematodes.

[0005] The present invention provides an application of a GmHAP5A protein in improving soybean resistance to soybean cyst nematodes. The sequence of the GmHAP5A protein is shown in SEQ ID NO.4.

[0006] The present invention provides an application of a GmHAP5A gene in improving soybean resistance to soybean cyst nematodes. The sequence of the GmHAP5A gene is shown in SEQ ID NO.3.

[0007] The present invention provides an application of a recombinant vector containing the gene shown in SEQ ID NO. 3 in improving soybean resistance to soybean cyst nematodes.

[0008] The present invention provides an application of a recombinant microbial cell containing the gene shown in SEQ ID NO. 3 in improving soybean resistance to soybean cyst nematodes.

[0009] It is further defined that the starting cell of the recombinant microbial cell is a prokaryotic microbial cell or a eukaryotic microbial cell.

[0010] The present invention provides an application of a recombinant vector containing the gene shown in SEQ ID NO. 3 in improving soybean resistance to soybean cyst nematodes.

[0011] It is further defined that the starting vector of the recombinant vector is pCAMBIA3300.

[0012] The present invention provides a method for improving soybean resistance to bean cyst nematodes, wherein soybean plants overexpressing the gene shown in SEQ ID NO. 3 are infected by soybean cyst nematodes.

[0013] The present invention provides a breeding method for preparing soybeans with high resistance to bean cyst nematodes, and the specific steps of the method are as follows:

[0014] Step 1: Ligate the gene shown in SEQ ID NO. 3 to the vector pCAMBIA3300 to obtain a recombinant vector;

[0015] Step 2: Transform the recombinant vector described in step 1 into Agrobacterium to obtain recombinant Agrobacterium;

[0016] Step 3: The recombinant Agrobacterium described in step 2 is transferred into soybean to obtain transgenic soybean plants, and positive transgenic soybean plants are obtained after identification.

[0017] The present invention provides a soybean plant containing an overexpressed gene shown in SEQ ID NO. 3 for use in breeding for resistance to soybean cyst nematode disease, or a mutant soybean plant containing a mutant gene shown in SEQ ID NO. 12 for use in controlling soybean resistance to soybean cyst nematode disease.

[0018] Beneficial effects: The average number of female insects in the pCAMBIA3300-GmHAP5A transgenic-positive roots was 1.92 / cm, which was lower than that in the control group. The average number of female insects in the GmHAP5A mutant-positive roots was 3.83 / cm. Based on the female insect index, there was a significant difference between the transgenic-positive roots and the negative control roots, confirming that the GmHAP5A gene has obvious resistance to soybean cyst nematode disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1For the cloning of GmHAP5A gene; M: DL2000; 1-4 PCR product;

[0020] Figure 2 The expression pattern of GmHAP5A gene in roots of Dongnong 50 and Suinong 14 after nematode stress was analyzed;

[0021] Figure 3 PCR test results of Agrobacterium rhizogenes culture medium of pCAMBIA3300-GmHAP5A recombinant plasmid; M: DL 2000(+) DNA molecular weight standard; 1-6: recombinant positive transformants;

[0022] Figure 4 PCR detection results of Agrobacterium rhizogenes culture medium of CPF1 / CAS12-GmHAP5A recombinant plasmid; M:DL Figure 5 Bar test strips for genetically modified soybean plants;

[0023] Figure 6 is the mutation result diagram;

[0024] Figure 7 Statistical results of soybean cyst nematodes in transgenic positive roots and roots with empty vector pCAMBIA3300 and CPF1 / Cas12;

[0025] Figure 8 Phenotypic comparison of transgenic roots and roots carrying empty vector pCAMBIA3300 and CPF1 / Cas12; Note: a. Overexpression pCAMBIA3300 empty vector control; b. pCAMBIA3300-GmHAP5A transgenic roots; c. CPF1 / Cas12-GmHAP5A gene non-mutation positive roots; d. CPF1 / Cas12-GmHAP5A gene mutation positive roots. DETAILED DESCRIPTION

[0026] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The pharmaceutical reagents used in the following examples were purchased from conventional biochemical reagent stores unless otherwise specified.

[0027] Plant varieties: Dongnong L10 (resistant), Suinong 14 (susceptible), Dongnong 50 (susceptible).

[0028] The cyst nematode-resistant variety Dongnong L-10 and the cyst nematode-susceptible variety Suinong 14 are recorded in the article Wu Depeng, Zhao Yue, Sheng Bihan, et al. Analysis of the genetic model of Dongnong L-10 against soybean cyst nematode race 3 [J]. Soybean Science, 2016, 35(3): 6. DOI: 10.11861 / j.issn.1000-9841.2016.03.0367.

[0029] Diseased soil type: Soybean cyst nematode HG type 1.2.3.5.7 (physiological race 3) is recorded in the article "Analysis of the genetic model of soybean cyst nematode race 3 by Dongnong L-10" by Wu Depeng, Zhao Yue, Sheng Bihan, et al. [J]. Soybean Science, 2016, 35(3): 6. DOI: 10.11861 / j.issn.1000-9841.2016.03.0367, collected from the experimental field of the Soybean Research Institute of Northeast Agricultural University.

[0030] Example 1. Bioinformatics analysis of the GmHAP5A gene and its cloning

[0031] 1) ProtParam software predicted the hydrophilicity / hydrophobicity of the candidate gene GmHAP5A, with an overall average hydrophilicity of -0.393 and an instability index of 74.16, indicating an unstable protein. Analysis of conserved domains of the protein encoded by the GmHAP5A gene using Interproscan and NCBI revealed that amino acids 61 to 144 of the GmHAP5A protein contain the HFDNF-YC-like histone domain. GmHAP5A is a histone fold domain found in nuclear transcription factor Y subunit gamma (NF-YC) and similar proteins.

[0032] Phyre software was used to predict the tertiary structure of the protein encoded by the GmHAP5A gene, and Rasmol software was used to analyze the protein's tertiary structure. The results showed that the protein encoded by the GmHAP5A gene mainly has three structures: α-helix (56.65%), β-turn (2.65%), and random coil (40.70%). A 2000-bp sequence upstream of the ATG of the GmHAP5A gene was intercepted and analyzed for cis-acting elements in the promoter using Plant CARE online analysis software. Sixteen cis-acting elements were found upstream of the gene. Some of these elements are related to stress and coercion, while others play an important role in responding to abiotic stress. It is speculated that this gene may sense adverse stress and participate in stress resistance.

[0033] 2. Methods for obtaining the GmHAP5A gene

[0034] Cloning of the GmHAP5A gene

[0035] (1) The soybean cyst nematode-resistant variety 'Dongnong L-10' was used as the material. When the first set of three leaves grew, the material was taken, total RNA was extracted and reverse transcribed to synthesize the first chain cDNA.

[0036] (2) Based on the GmHAP5A gene sequence on Phytozome, gene cloning primers were designed using Primer5 software

[0037] F: 5'-AGAACACGGGGGACTATGGAGAACAACCAGCAA-3' (SEQ ID NO. 1);

[0038] R: 5'-ATCCTCTGTTTCTAGTCAGTGATGGTGATGGTGATGCTAGAATTCCATACTCTG C-3' (SEQ ID NO. 2);

[0039] PCR was performed using cDNA as a template. The reaction system (Table 1) was as follows: 98°C for 3 min; 37 cycles of 98°C for 10 s, 60°C for 5 s, and 68°C for 10 min; 12°C plus. After the reaction, the PCR product was subjected to 1% agarose gel electrophoresis and the target fragment was purified by gel recovery ( Figure 1 ).

[0040] Table 1 Reaction system of cloning gene system

[0041]

[0042] GmHAP5A gene coding sequence (SEQ ID NO.3):

[0043] ATGGAGAACAACCAGCAACAAGGCGCTCAAGCCCAATCGGGACCGTACCCCGGCGGCGCCGGTGGAAGTGCAGGTGCAGGTGCAGGTGCAGGCGCGGCCCCGTTCCAGCACCTGCTCCAGCAGCAGCAGCAGCAGCTGCAGATGTTCTGGTCGTACCAGCGGCAAGAGATCGAGCACGTGAACGACTTCAAGAACCACCAGCTCCCCTTGGCCCGCATCAAGAAGATCATGAAGGCCGACGAGGACGTCCGCATGATCTCCGCCGAGGCCCCCATCCTCTTCGCCAAGGCCTGCGAGCTCTTCATCCTCGAGCTCACCATCCGCTCCTGGCTCCACGCCGACGAGAACAAGCGCCGCACCCTCCAGAAGAACGACATCGCCGCCGCCATCACTCGCACCGACATTTTCGACTTCCTCGTCGACATCGTCCCCCGCGACGAGATCAAGGACGACGCCGCGCTCGTCGGGGCAACGGCCAGTGGGGTGCCTTACTACTACCCGCCCATTGGCCAGCCTGCCGGGATGATGATTGGCCGCCCCGCCGTCGATCCCGCCACCGGAGTTTATGTCCAGCCGCCCTCCCAGGCCTGGCAGTCCGTCTGGCAGTCCGCCGCCGAGGACACGCCCTACGGCACCGGTGCCCAGGGGAACCTTGATGGCCAGAGTATGGAATTCTAG;

[0044] Amino acid sequence of GmHAP5A (SEQ ID NO.4):

[0045] MENNQQQGAQAQSGPYPGGAGGSAGAGAGAGAAPFQHLLQQQQQQQLQMFWSY QRQEIEHVNDFKNHQLPLARIKKIMKADEDVRMISAEAPILFAKACELFILELTIRSWLHADENKRRTLQKNDIAAAITRTDIFDFLVDIVPRDEIKDDAALVGATASGVPYYYPPIGQPAGMMIGRPAVDPATGVYVQPPSQAWQSVWQSAAEDTPYGTGAQGNLDGQSMEF。

[0046] Example 2. Analysis of the expression pattern of the GmHAP5A gene

[0047] 1. Get materials

[0048] (1) Using the washing-sieving method, the obtained cyst soil sample is placed on a 40-mesh sieve, and a 60-mesh sieve is connected below. The soil sample on the filter is washed with turbulent water, and the diseased soil in the 40-mesh filter is washed until the water flow is clear, and the soybean cyst nematodes are washed onto the 60-mesh sieve. Using the 63% sucrose-resuspension method, the sample on the 60-mesh sieve is transferred to a 50 mL centrifuge tube with a small spoon, and the sucrose solution is poured in, shaken, and centrifuged at 3000 rpm for 5 minutes. The sucrose supernatant is filtered with filter paper, and the cysts are left on the filter paper. The separated cysts are broken by mechanical means, and the released eggs are incubated at 26°C for 7 days. The hatched second-instar larvae (J2) are mixed into an egg suspension of 2000 eggs / ml for the next step of inoculation and identification. Fine sand and soil were mixed in a ratio of 1:1, and the egg suspension was sprinkled into the sand-soil mixture. Resistant varieties were sown in vermiculite and transplanted into diseased soil after roots grew. Root samples were taken at 0d, 5d, 10d, and 15d, with three replicates at each time point.

[0049] 2. cDNA Synthesis

[0050] Total RNA was extracted from sample 1 (Trizol total RNA extraction reagent, TIANGEN BIOTECH, DP405-02), and reverse transcribed to synthesize the first-strand cDNA (Rever Tra Ace qPCR RT Master Mi with DNA remover, TOYOBO, FSQ-301).

[0051] 3. Fluorescence Quantitative PCR Analysis

[0052] (1) The reference gene was the soybean housekeeping gene GmActin 4 (Genbank No: AF049106), and the qRT-PCR

[0053] Methods Quantitative primers (SEQ ID NO.5 and SEQ ID NO.6) of the candidate gene GmHAP5A were designed and the fluorescence quantitative kit TB Premix Ex Taq TMThe experiment was carried out according to the instructions of the manufacturer. The reaction system is shown in Table 2. The test instrument used was Applied Biosystems 7500 Fast Real-Time PCR System (Lifetechnologies, USA). The reaction program was 95°C for 30 s, 95°C for 3 s, 60°C for 30 s, and 72°C for 30 s for 40 cycles. After the reaction program was completed, the amplification curve and melting curve were analyzed. 2- △△CT The relative expression of genes was calculated by the CT method, and the CT value was the average value of three repeated experiments.

[0054] F: 5'-AGTTTATGTCCAGCCGCCCTCCCA-3' (SEQ ID NO. 5);

[0055] R: 5'-TTCCATACTCTGGCCATCAAGGTT-3' (SEQ ID NO. 6);

[0056] Table 2 Reaction system

[0057]

[0058]

[0059] (2) Analysis of GmHAP5A gene-specific expression

[0060] Soybean cyst nematode (SCN) stress was applied to resistant and susceptible soybean varieties. The specific operation was as follows: 7-day-old soybean roots were inoculated with an SCN egg suspension at a concentration of 2000 20 larvae per ml (the treatment included two materials, the disease-resistant variety 'Dongnong L10' and the disease-susceptible variety 'Suinong 14', T was the treatment, and CK was the control). The corresponding uninoculated soybean roots served as the control. Fifteen days after inoculation, the soybean roots of the treatment and control groups were observed using acid fuchsin staining, and the expression level of the GmHAP5A gene in the resistant and susceptible soybean varieties was determined. The results are shown in Figure 2. Figure 2 The results show that the expression level of GmHAP5A gene increased significantly in the disease-resistant materials after SCN 3 physiological race stress, while the increase was smaller in the susceptible varieties. This indicates that GmHAP5A gene is involved in the soybean cyst nematode resistance response.

[0061] Example 3. Construction of plants overexpressing the GmHAP5A gene

[0062] (1) Referring to the instructions of the TaKaRa Mini BEST Agarose Gel DNA Extraction Kit Ver. 4.0 from Baori Biotechnology Co., Ltd., the gel-recovered product obtained above was ligated with the cloning vector pCAMBIA3300 and transformed into E. coli competent cells DH5α. Single clones were picked for bacterial culture. Cloning primers were used as primers for identifying positive clones in E. coli. 1 μL of the cultured bacterial solution was selected as the template for bacterial PCR amplification. The PCR reaction procedure was as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 sec; annealing at 60°C for 30 sec; extension temperature at 72°C for 1 min / kb for a total of 35 cycles; final extension at 72°C for 5 min, and storage at 4°C. After the reaction procedure was completed, the correct bands were detected and separated by 1% agarose gel electrophoresis. Approximately 500 μL of bacterial solution was aspirated and submitted to Beijing BGI Genomics Co., Ltd. for base sequencing. Finally, the target fragment of 678 bp of the GmHAP5A gene was obtained, indicating that the GmHAP5A gene was ligated to the expression vector and successfully transformed into E. coli. The E. coli solution that passed the sequencing was used to extract the E. coli plasmid according to the instructions of the TaKaRa Mini BEST Plasmid Purification Kit Ver.4.0.

[0063] (2) Preparation and transformation of competent Agrobacterium rhizogenes.

[0064] Preparation of competent Agrobacterium rhizogenes (K599) by electroporation. The electroporation transformation method for Agrobacterium rhizogenes (K599) specifically includes placing a 1mm ice bath in an electroporation cuvette for 30 minutes; adding 1μL of the GmHAP5A recombinant plasmid to a competent K599 cell (100μL) and gently pipetting it to the bottom of the 1mm cuvette before placing it in an electroporation tank; performing an electroporation reaction using the preset parameter Agrobacterium; then immediately transferring 500uL of SOC culture medium to the electroporation cuvette, gently mixing it, and aspirating the liquid to 1.5mL. EP tube; placed at 28 ° C, 150 rpm, renatured for 1-3 hours; aspirated 100 μL was evenly spread on a plate with kanamycin (kana) and streptomycin (str) resistance genes as screening markers, and incubated in a 28 ° C incubator for 24 hours in the dark, and then a single clone was picked for bacterial liquid PCR verification, and a target band of 678 bp was obtained, indicating that pCAMBIA3300-GmHAP5A had been successfully transformed into Agrobacterium rhizogenes K599. Agrobacterium rhizogenes K599 containing the pCAMBIA3300-GmHAP5A vector was obtained. Figure 3 ).

[0065] (3) Healthy Dongnong 50 soybean seeds were sown in silt and grown in a greenhouse at 24°C with a 12 / 12h photoperiod. A single Agrobacterium spot was picked and inoculated into 2 mL LB (containing 50 mg / Str) liquid medium and cultured overnight at 28°C and 200 rpm. The bacterial solution was transferred to 50 mL LB (containing 50 mg / Str) culture medium at a ratio of 1:100 and cultured at 28°C and 200 rpm until the OD reached about 0.8-1.0. The resuspended bacterial solution of 3-4 day old Dongnong 50 seedlings was collected using a 1 mL syringe. The bacterial solutions were Agrobacterium K599 containing the pCAMBIA3300-GmHAP5A recombinant vector and Agrobacterium K599 containing the CPF1 / Cas12-GmHAP5A recombinant vector, respectively. A cross wound was made at the cotyledon node or the proximal hypocotyl of the seedling and the bacterial solution was inoculated. After bacterial injection, incubate in the dark at 28°C, 150 rpm, for 40 minutes. Place the infected soybean seedlings in vermiculite, covering the wound with the vermiculite. After 2-3 weeks of growth, wait until the roots have reached a certain length and test positive for infection before inoculating with a suspension of cyst nematode eggs. This method can rapidly produce hairy roots overexpressing the GmHAP5A gene.

[0066] (4) Soybean genomic DNA extraction. In this study, the soybean genomic DNA was extracted using the SDS-minimum extraction method to extract the genomic DNA of the transgenic hairy roots. For GmHAP5A overexpressing hairy roots, Bar protein test strips were used for detection ( Figure 5 ).

[0067] Example 4. Construction of plants with mutant GmHAP5A genes

[0068] (1) Based on the full-length sequence of the GmHAP5A gene, the 20th base upstream of TTTV was selected as the target sequence. To improve the mutation efficiency, three targets were screened based on the on-target rate and specificity using the CCTop-CRISPR / Cas9 target online predictor website.

[0069] (2) Three targets (SEQ ID NO. 5) were synthesized by GenScript Biotech Co., Ltd. and linked to the CPF1 / Cas12 vector by seamless cloning;

[0070] Target 1: CGACTTCCTCGTCGACATCGTC; (SEQ ID NO. 7)

[0071] Target 2: TGTCCAGCCGCCCTCCCAGGCC; (SEQ ID NO.8)

[0072] Target 3: GACTTCCTCGTCGACATCGTCC; (SEQ ID NO. 9)

[0073] The synthesized CPF1 / Cas12-GmHAP5A recombinant plasmid was transformed into TOP10 competent cells, and the recombinant plasmid was extracted, identified, and transformed into K599.

[0074] (3) Preparation and transformation of competent Agrobacterium rhizogenes.

[0075] Preparation of electroporation competent cells of Agrobacterium rhizogenes (K599) was performed according to Chen Anle (2014). The electroporation transformation method of Agrobacterium rhizogenes (K599) specifically includes the following steps: placing a 1mm ice bath in an electroporation cup for 30 minutes; adding 1μL-GmHAP5A recombinant plasmid to a competent K599 (100μL), gently pipetting it with a pipette, moving it to the bottom of the 1mm point rotating cup, and then placing it in an electroporation tank; using the preset parameter Agrobacterium, the electroporation reaction was performed; then, 500uL of SOC culture medium was immediately transferred to the electroporation cup, gently mixed, and then the liquid was aspirated into a 1.5mL EP tube; placed at 28℃, 150rpm, and renatured for 1-3 hours; 100μL was evenly spread on a plate with kanamycin (kana) and streptomycin (str) resistance genes as screening markers, and after inverted in a dark incubator at 28℃ for 24 hours, a single clone was picked for bacterial liquid PCR verification, and SEQ ID NO.10 and SEQ ID The primer pair shown in NO.11 was used for PCR verification to obtain a target band of 503 bp in length. The CPF1 / Cas12-GmHAP5A recombinant plasmid was transformed into Agrobacterium rhizogenes K599 according to the above method ( Figure 4 ).

[0076] F: 5'-GTAAAACGACGGCCAGTG-3' (SEQ ID NO. 10);

[0077] R: 5'-TTAAGGACCCGGGAAAAAA-3' (SEQ ID NO. 11).

[0078] (4) Healthy Dongnong 50 soybean seeds were sown in silt and grown in a greenhouse at 24°C with a 12 / 12h photoperiod. A single Agrobacterium spot was picked and inoculated into 2 mL LB (containing 50 mg / Str) liquid medium and cultured overnight at 28°C and 200 rpm. The bacterial solution was transferred to 50 mL LB (containing 50 mg / Str) culture medium at a ratio of 1:100 and cultured at 28°C and 200 rpm until the OD reached about 0.8-1.0. The resuspended bacterial solution of 3-4 day old Dongnong 50 seedlings was collected using a 1 mL syringe. The bacterial solutions were Agrobacterium rhizogenes K599 containing the pCAMBIA3300-GmHAP5A recombinant vector and Agrobacterium rhizogenes K599 containing the CPF1 / Cas12-GmHAP5A recombinant vector, respectively. A cross wound was made at the cotyledon node or the proximal hypocotyl of the seedling and the bacterial solution was inoculated. After bacterial injection, incubate in the dark at 28°C, 150 rpm, for 40 minutes. Place the infected soybean seedlings in vermiculite, covering the wound with the vermiculite. After 2-3 weeks of growth, wait until the roots have reached a certain length and test positive for the root cyst nematode egg suspension before inoculating them. This method can quickly generate hairy roots with GmHAP5A gene mutations.

[0079] (5) Molecular detection of transgenic hairy roots and functional verification of candidate genes

[0080] 1) Soybean genomic DNA extraction. In this study, the SDS-miniprep method was used to extract the genomic DNA of transgenic hairy roots. For the GmHAP5A mutant hairy roots, PCR verification was performed using the primer pair shown in SEQ ID NO.13 and SEQ ID NO.14. The PCR amplification product with the correct target band was sequenced and verified. The sequencing results showed a double peak at the target site 3 position, indicating that target 3 was successfully edited and mutated ( Figure 6 ).

[0081] Mutated sequence (SEQ ID NO.12): ATGGAGAACAACCAGCAACAAGGCGCTCAAGCCC AATCGGGACCGTACCCCGGCGGCGCCGGTGGAAGTGCAGGTGCAGGTGCAGGTGCAGGCGCGGCCCCGTTCCAGCACCTGCTCCAGCAGCAGCAGCAGCAGCTGCAGATGTTCTGGTCGTACCAGCGGCAAGAGATCGAGCACGTGAACGACTTCAAGAACCACCAGCTCCCCTTGGCCCGCATCAAGAAGATCATGAAGGCCGACGAGGACGTCCGCATGATCTCCGCCGAGGCCCCCATCCTCTTCGCCAAGGCCTGCGAGCTCTTCATCCTCGAGCTCACCATCCGCTCCTGGCTCCACGCCGACGAGAACAAGCGCCGCACCCTCCAGAAGAACGACATCGCCGCCGCCATCACTCGCACCGACAT TTTCGACTTCCTCGT CGACAGCCAGTCCCGCGACGAGATCAAGGACGACGCCGCGCTCGTCGGGGCAACGGCCAGTGGGGTGCCTTACTACTACCCGCCCATTGGCCAGCCTGCCGGGATGATGATTGGCCGCCCCGCCGTCGATCCCGCCACCGGAGTTTATGTCCAGCCGCCCTCCCAGGCCTGGCAGTCCGTCTGGCAGTCCGCCGCCGAGGACACGCCCTACGGCACCGGTGCCCAGGGGAACCTTGATGGCCAGAGTATGGAATTCTAG;

[0082] Primers for detecting GmHAP5A gene mutation

[0083] F: 5’-GAACGACTTCAAGAACCACC-3’ (SEQ ID NO.13);

[0084] R: 5’-AAGAAAACAAGACAACGCAA-3’ (SEQ ID NO.14).

[0085] Example 5. Application of transgenic plants in resisting soybean cyst nematode disease

[0086] 1. Identification of transgenic root resistance to SCN by acid fuchsin staining. Histological examination of SCN infection showed that nematodes had begun feeding on the 15th day after inoculation. The international acid fuchsin staining method was used to observe nematode invasion and development in the roots. The method included 1) rinsing: the residual soil on the roots was rinsed with clean water;

[0087] 2. Soaking: Soak the roots in 3% NaClO solution for 1 hour (the decolorization time depends on the decolorization situation, the higher the NaClO concentration, the faster the decolorization), to ensure that the roots are completely decolorized;

[0088] 3. Root staining: Rinse the roots with running water to remove NaClO, then soak them in distilled water for 30 minutes. Boil the 30-fold diluted acid fuchsin staining solution (mother liquor: 3.5 g fuchsin, 250 mL glacial acetic acid, 750 mL distilled water), place the decolorized roots in the fuchsin and continue boiling for 45 seconds to 2 minutes (shorten the boiling time as much as possible if the coloring is good, the root system will be more compact). When taking out, wipe them with absorbent paper and observe.

[0089] 4. Fifteen plants with transgenic GmHAP5A gene-positive roots (pCAMBIA3300-GmHAP5A plants), GmHAP5A gene mutation-positive roots (CPF1-GmHAP5A plants), overexpression empty vector (pCAMBIA3300 plants) and edited empty vector-positive roots (CPF1 plants) were inoculated with No. 3 physiological egg suspension for identification. The eggs were counted under a 20× and 100× optical microscope using the acid fuchsin staining method. 5-6 lateral roots were taken from each plant for repeated analysis. The average number of female insects per plant was calculated and a paired t-test was performed. Figure 7 As shown, the average number of female insects in the roots of the wild-type control was 2.74 / cm, while the average number of female insects in the roots positive for the pCAMBIA3300-GmHAP5A transgene was 1.92 / cm, which were lower than those in the control group. The results of the paired sample t-test were 2.14 and 3.00 at the P=0.05 and 0.01 levels, respectively, and the two-tailed P value (Sig) was 0.007.

[0090] The average number of female insects in GmHAP5A mutation-positive roots was 3.83 / cm. The results of the paired sample t-test showed that the two-tailed t values ​​were 2.14 and 2.98 at the P=0.05 and 0.01 levels, respectively, and the two-tailed P value (Sig) was 0.004. Based on the female insect index, there was a significant difference between the transgenic positive roots and the negative control roots, preliminarily confirming that the GmHAP5A gene has significant resistance to soybean cyst nematode disease ( Figure 8 ).

Claims

1. Application of the GmHAP5A gene in improving soybean resistance to soybean cyst nematodes, characterized in that: The sequence of the GmHAP5A gene is shown in SEQ ID NO.

3.

2. Use of recombinant microbial cells containing the gene shown in SEQ ID NO. 3 in improving soybean resistance to soybean cyst nematodes.

3. The use according to claim 2, characterized in that The starting cell of the recombinant microbial cell is a prokaryotic microbial cell or a eukaryotic microbial cell.

4. Use of a recombinant vector containing the gene shown in SEQ ID NO. 3 in improving soybean resistance to soybean cyst nematodes.

5. The use according to claim 4, characterized in that The starting vector of the recombinant vector is pCAMBIA3300.

6. A breeding method for preparing soybeans with high resistance to soybean cyst nematodes, characterized in that: The specific steps of the method are as follows: Step 1: Ligate the gene shown in SEQ ID NO. 3 to the vector pCAMBIA3300 to obtain a recombinant vector; Step 2: Transform the recombinant vector described in step 1 into Agrobacterium to obtain recombinant Agrobacterium; Step 3: The recombinant Agrobacterium described in step 2 is transferred into soybean to obtain transgenic soybean plants, and positive transgenic soybean plants are obtained after identification.

7. Use of soybean plants containing overexpressed gene represented by SEQ ID NO. 3 in breeding for resistance to soybean cyst nematode disease.

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