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

Abstract

The invention discloses application of GmHAP5A protein in improving soybean cyst nematode resistance of soybeans. Belongs to the technical field of plant disease resistance. The invention aims to provide a method for resisting soybean cyst nematode by soybeans. The invention provides application of a GmHAP5A protein in improving soybean cyst nematode resistance of soybeans. The sequence of the GmHAP5A protein is shown as SEQ ID NO.4. The invention also provides application of the GmHAP5A protein in improving soybean cyst nematode resistance of soybeans. And a new way and a new method are provided for breeding the resistant soybean cyst nematode of the soybeans.

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] Soybean cyst nematode (Heterodera glycines Ichinohe) is an important pest that harms soybean production and causes serious economic losses in soybean production worldwide. It parasitizes on the roots of soybeans to extract nutrients, causing plant stunting, leaf yellowing, reduced yields, and even death. Therefore, the study of soybean cyst nematodes has important theoretical and applied value. The life history of soybean cyst nematodes includes five stages: eggs, larvae, nymphs, adults, and cysts. Its parasitic mode on soybean roots also goes through three important developmental stages: infection, vegetative cell formation, and cyst formation. In this process, the nematode infects soybean root cells and releases secretions to change the biochemical, physiological, and genetic mechanisms in the host, thereby providing it with the required nutrition and environment. Therefore, studying the life history and parasitic process of soybean cyst nematodes can provide a deep understanding of its infection mechanism and host resistance mechanism, and provide an important theoretical basis for the research and development of natural resistance and transgenic technology.

[0003] In addition, soybean cyst nematodes have developed resistance to chemical pesticides and are insensitive to biological control measures, so traditional control methods have been unable to control their damage. Therefore, studying the resistance mechanism and biological control strategy of soybean cyst nematodes is of great significance for developing new control technologies and methods and ensuring soybean yield. Under the background of current transgenic technology and gene editing technology, studying the functional genes and regulatory networks of soybean cyst nematodes can provide important gene resources and molecular markers for disease-resistant breeding and nematode-resistant transgenic breeding of soybeans. At the same time, combined with gene editing technology, the resistance of soybeans to soybean cyst nematodes can be improved by changing key genes, further providing new ways and methods for soybean resistance breeding. 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 a gene shown in SEQ ID NO.3 in improving the resistance of soybean to soybean cyst nematodes.

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

[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: Connect the gene shown in SEQ ID NO.3 to the vector pCAMBIA3300 to obtain a recombinant vector;

[0015] Step 2: transforming 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 an application of a soybean plant containing a gene overexpressed as shown in SEQ ID NO.3 in breeding for resistance to soybean cyst nematode disease or an application of a mutant soybean plant containing a mutant gene of SEQ ID NO.12 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. According to 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 1It is the clone of GmHAP5A gene; M: DL2000; 1-4 PCR product;

[0020] Figure 2 The expression pattern of GmHAP5A gene in the 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 The results of PCR detection 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 The statistical results of soybean cyst nematodes in transgenic positive roots and roots with empty vector pCAMBIA3300 and CPF1 / Cas12;

[0025] Figure 8 The phenotypes of transgenic positive roots and empty vector pCAMBIA3300 and CPF1 / Cas12 roots are compared; Note: a. Overexpression pCAMBIA3300 empty vector control; b. pCAMBIA3300-GmHAP5A transgenic positive roots, c. CPF1 / Cas12-GmHAP5A gene non-mutated positive roots; d. CPF1 / Cas12-GmHAP5A gene mutated positive roots. DETAILED DESCRIPTION

[0026] The following examples are provided for 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 are 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 Wu Depeng, Zhao Yue, Sheng Bihan, et al. Analysis of genetic model of Dongnong L-10 against soybean cyst nematode physiological race 3 [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) The hydrophilicity / hydrophobicity of the candidate gene GmHAP5A was predicted by ProtParam software, with an overall average hydrophilicity of -0.393 and an instability index of 74.16, indicating that it is an unstable protein. The conservative domain analysis of the protein encoded by the GmGmHAP5A gene combined with Interproscan and NCBI website revealed that the amino acids from position 61 to position 144 of the GmHAP5A protein are the HFDNF-YC-like histone domain, and GmHAP5A is a histone folding domain found in nuclear transcription factor Y subunit γ (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 graphical representation of the tertiary structure of the protein. The results showed that the protein encoded by the GmHAP5A gene mainly had three structures, namely α helix (56.65%), β turn structure (2.65%) and random coil (40.70%). The 2000bp sequence upstream of the ATG of the GmHAP5A gene was intercepted, and the cis-acting elements of the promoter were analyzed in the Plant CARE online analysis software. It was found that there were 16 cis-acting elements upstream of the gene. Some of these elements were stress-related elements, and some played an important role in responding to abiotic stress. It is speculated that the gene may sense adverse stress and participate in the stress resistance process.

[0033] 2. Method for obtaining GmHAP5A gene

[0034] Cloning of GmHAP5A gene

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

[0036] (2) Based on the GmHAP5A gene sequence on Phytozome, the Primer5 software was used to design gene cloning primers

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

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

[0039] The PCR reaction was carried out with cDNA as template. The reaction system (Table 1) was as follows: 98°C for 3 min; 37 cycles: 98°C for 10 s, 60°C for 5 s, 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) The cyst soil sample obtained was placed on a 40-mesh sieve by the washing-sieving method, and a 60-mesh sieve was connected below. The soil sample on the filter was washed with turbulent water flow, and the diseased soil in the 40-mesh filter was washed until the water flow was clear, and the soybean cyst nematodes were washed to the 60-mesh sieve. The 63% sucrose-resuspended method was used to transfer the sample on the 60-mesh sieve into a 50 mL centrifuge tube with a small spoon, pour in the sucrose solution, shake well, centrifuge at 3000 rpm for 5 minutes, and filter the sucrose supernatant with filter paper, leaving the cyst on the filter paper. The separated cysts were broken by mechanical method, and the released eggs were incubated at 26°C for 7 days. The hatched second-instar larvae (J2) were 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] The total RNA of sample 1 was extracted (Trizol total RNA extraction reagent, TIANGEN BIOTECH, DP405-02), and reverse transcribed to synthesize the first chain of cDNA (Rever Tra Ace qPCR RT Master Mi with DNAremover, TOYOBO, FSQ-301).

[0051] 3. Fluorescence quantitative PCR analysis

[0052] (1) The reference gene was the soybean housekeeping gene GmActin 4 (Genbank No: AF049106).

[0053] Methods The 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 TMII manual, the reaction system is shown in (Table 2). The test instrument used was Applied Biosystems 7500 Fast Real-Time PCR System (Life technologies, USA), and the reaction program was 95°C for 30 s, 95°C for 3 s, 60°C for 30 s, 72°C for 30 s, and 40 cycles. After the reaction program was completed, the amplification curve and melting curve were analyzed, and 2- △△CT The relative expression of genes was calculated by the CT method, and the CT value was averaged from 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] The soybean resistant and susceptible varieties were subjected to SCN 3 (Soybean Cyst Nematode (SCN disease) stress treatment. The specific operation was as follows: the soybean roots grown for 7 days were inoculated with SCN egg suspension with 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 were used as the control. After 15 days of inoculation, the soybean roots of the treatment and control groups were observed using the acid fuchsin staining method, and the expression level of the GmHAP5A gene in the soybean resistant and susceptible varieties was determined. The results are shown in Figure 2. Figure 2 As shown, the expression level of GmHAP5A gene in disease-resistant materials increased significantly after SCN 3 physiological race stress, while the increase was smaller in susceptible varieties. This indicates that GmHAP5A gene is involved in the soybean resistance to cyst nematodes.

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

[0062] (1) Referring to the instructions of TaKaRa Mini BEST Agarose Gel DNA Extraction Kit Ver.4.0 of Baori Biotechnology Co., Ltd., the gel recovery product obtained above was connected with the cloning vector pCAMBIA3300, and transformed into the competent E. coli cell DH5α, and the single clone product was picked for bacterial culture. The cloning primers were used as primers for identifying positive clones of E. coli liquid, and 1 μL of the cultured bacterial liquid was selected as the template for bacterial liquid PCR amplification. The PCR reaction program was: pre-denaturation at 94°C, 5min; denaturation at 94°C, 30sec; annealing at 60°C, 30sec; extension temperature: 72°C, 1min / kb, a total of 35 cycles; final extension at 72°C, 5min, and storage at 4°C. After the reaction program was completed, after 1% agarose gel electrophoresis detection and separation, about 500 μL of bacterial liquid with the correct band was aspirated and handed over to Beijing BGI Genomics Co., Ltd. for base sequencing. Finally, the target fragment size of 678 bp of GmHAP5A gene was obtained, indicating that the GmHAP5A gene was connected to the expression vector and successfully transformed into E. coli. The qualified E. coli solution was extracted from E. coli plasmid according to the instructions of TaKaRa Mini BEST Plasmid Purification Kit Ver.4.0.

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

[0064] Preparation of electroporation competent cells of Agrobacterium rhizogenes (K599). The electroporation transformation method of Agrobacterium rhizogenes (K599) specifically includes placing the electroporation transformation cup in an ice bath at 1mm for 30 minutes; adding 1μL-GmHAP5A recombinant plasmid to a competent K599 (100μL), gently blowing with a pipette, moving to the bottom of the 1mm point transfer cup, and then placing it in the electroporation tank; using the preset parameter Agrobacterium, the electroporation reaction is performed; then immediately aspirating 500uL of SOC culture medium into the electroporation cup, gently mixing and aspirating the liquid to 1.5mL EP tube; placed at 28°C, 150rpm, renaturation for 1-3h; 100μL was evenly spread on a plate with kanamycin (kana) and streptomycin (str) resistance genes as screening markers, inverted and dark cultured in a 28°C incubator for 24h, and a single clone was picked for bacterial liquid PCR verification to obtain a target band of 678bp in length, proving that pCAMBIA3300-GmHAP5A has been successfully transferred into Agrobacterium rhizogenes K599 to obtain Agrobacterium rhizogenes K599 containing the pCAMBIA3300-GmHAP5A vector ( Figure 3 ).

[0065] (3) Healthy Dongnong 50 soybean seeds were selected and sown in siltstone and placed in a greenhouse for growth at 24°C and 12 / 12h photoperiod. A single Agrobacterium spot was selected and inoculated into 2mL LB (containing 50mg / Str) liquid medium and cultured overnight at 28°C and 200rpm. The bacterial solution was transferred to 50mL LB (containing 50mg / Str) culture medium at a ratio of 1:100 and cultured at 28°C and 200rpm until the OD reached about 0.8-1.0. The resuspended bacterial solution of 3-4d Dongnong 50 seedlings was collected using a 1mL syringe. The bacterial solutions were Agrobacterium K599 containing the pCAMBIA3300-GmHAP5A recombinant vector and Agrobacterium K599 containing the CPF1 / Cas12-GmHAP5A recombinant vector. A cross wound was made at the cotyledon node or the proximal hypocotyl of the seedling for bacterial inoculation. After the bacterial solution is injected, the incubation is carried out at 28°C, 150rpm, and dark culture for 40 minutes. The infected soybean seedlings are placed in vermiculite and the wounds are covered with vermiculite. After 2-3 weeks of growth, the roots are tested positive when they grow to a certain length and are ready to be inoculated with cyst nematode egg suspension. This method can quickly obtain hairy roots overexpressing the GmHAP5A gene.

[0066] (4) Extraction of soybean genomic DNA. In this study, the SDS-small-volume extraction method was used to extract the genomic DNA of 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 gene

[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 Agrobacterium rhizogenes competent cells.

[0075] Preparation of electroporation competent cells of Agrobacterium rhizogenes (K599) was performed by referring to Chen Anle (2014). The electroporation transformation method of Agrobacterium rhizogenes (K599) specifically includes placing an electroporation transformation cup at 1mm in ice bath for 30 minutes; adding 1μL-GmHAP5A recombinant plasmid to a competent K599 (100μL), gently blowing with a pipette, moving to the bottom of the 1mm point rotating cup, and then placing it in the electroporation tank; using the preset parameter Agrobacterium, the electroporation reaction was performed; then 500uL SOC culture medium was immediately transferred to the electroporation cup, and the liquid was aspirated into a 1.5mL EP tube after gently mixing; placed at 28℃, 150rpm, and renatured for 1-3h; 100μL was evenly smeared on a plate with kanamycin (kana) and streptomycin (str) resistance genes as screening markers, and after inverted dark culture in a 28℃ incubator for 24h, 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. According to the above method, the CPF1 / Cas12-GmHAP5A recombinant plasmid was transferred into Agrobacterium rhizogenes K599 ( 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 selected and sown in siltstone and placed in a greenhouse for growth at 24°C and 12 / 12h photoperiod. A single Agrobacterium spot was selected and inoculated into 2mL LB (containing 50mg / Str) liquid medium and cultured overnight at 28°C and 200rpm. The bacterial solution was transferred to 50mL LB (containing 50mg / Str) culture medium at a ratio of 1:100 and cultured at 28°C and 200rpm until the OD reached about 0.8-1.0. The resuspended bacterial solution of 3-4d Dongnong 50 seedlings was collected using a 1mL syringe. The bacterial solutions were Agrobacterium K599 containing the pCAMBIA3300-GmHAP5A recombinant vector and Agrobacterium K599 containing the CPF1 / Cas12-GmHAP5A recombinant vector. A cross wound was made at the cotyledon node or the proximal hypocotyl of the seedling for bacterial inoculation. After the bacterial solution is injected, the incubation is carried out at 28°C, 150rpm, and dark culture for 40 minutes. The infected soybean seedlings are placed in vermiculite and the wounds are covered with vermiculite. After 2-3 weeks of growth, the roots are tested positive when they grow to a certain length and are ready to be inoculated with cyst nematode egg suspension. This method can quickly obtain GmHAP5A gene mutant hairy roots.

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

[0080] 1) Extraction of soybean genomic DNA. In this study, the SDS-small-scale extraction 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 site 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 the invasion and development of nematodes in the roots. The method included 1) flushing: flush the residual soil on the roots with clean water;

[0087] 2. Soaking: Soak the roots in 3% NaClO aqueous 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 NaClO on the roots with running water, soak them in distilled water for 30 minutes, boil the 30-fold diluted acid fuchsin staining solution (mother liquor: 3.5g fuchsin, 250mL glacial acetic acid, 750mL distilled water), put the decolorized roots into the fuchsin and continue boiling for 45s-2min (try to shorten the time if the coloring is good, the root system will be tighter), wipe them with absorbent paper when taking them out, and observe.

[0089] 4. The 15 plants with positive roots of transgenic GmHAP5A gene (pCAMBIA3300-GmHAP5A plants), positive roots of GmHAP5A gene mutation (CPF1-GmHAP5A plants), positive roots of overexpression empty vector (pCAMBIA3300 plants) and edited empty vector (CPF1 plants) were inoculated with No. 3 physiological egg suspension for identification, and counted under 20× and 100× optical microscopes by acid fuchsin staining. 5-6 lateral roots were taken from each plant for replication, and the average value of female insects in each plant was calculated, and paired t test was used. Figure 7 As shown, the average number of female insects in the roots of the wild-type control was 2.74 / cm, and the average number of female insects in the roots of the pCAMBIA3300-GmHAP5A transgenic positive was 1.92 / cm, which was lower than that of 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 values ​​(Sig) were 0.007;

[0090] The average number of female insects in the 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. According to the female insect index, there was a significant difference between the transgenic positive roots and the negative control roots, which preliminarily confirmed that the GmHAP5A gene had significant resistance to soybean cyst nematode disease ( Figure 8 ).

Claims

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

4.

2. 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.

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

4. Use of recombinant microbial cells 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 cell of the recombinant microbial cell is a prokaryotic microbial cell or a eukaryotic microbial cell.

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

7. The use according to claim 6, characterized in that: The starting vector of the recombinant vector is pCAMBIA3300.

8. A method for improving soybean resistance to bean cyst nematodes, characterized in that: The soybean plant overexpressing the gene shown in SEQ ID NO.3 was infected with soybean cyst nematode.

9. A breeding method for preparing soybeans with high resistance to bean cyst nematodes, characterized in that: The specific steps of the method are as follows: Step 1: Connect the gene shown in SEQ ID NO.3 to the vector pCAMBIA3300 to obtain a recombinant vector; Step 2: transforming 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.

10. Use of soybean plants containing overexpressed gene shown in SEQ ID NO. 3 in breeding for resistance to soybean cyst nematode disease or use of mutant soybean plants containing mutant gene shown in SEQ ID NO. 12 in controlling soybean resistance to soybean cyst nematode disease.

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