Application of GmPP2C-37like gene in improving soybean cyst nematode disease resistance of soybeans

Abstract

The invention discloses application of a GmPP2C-37like gene in improving soybean cyst nematode disease resistance of soybeans, and belongs to the technical field of plant disease resistance breeding. The invention aims to improve the soybean cyst nematode disease resistance of soybeans. The invention provides an application of a GmPP2C-37like gene in improving the capability of soybeans for resisting soybean cyst nematode disease. A nucleic acid small marker of the GmPP2C-37like gene is shown as SEQ ID NO.7. The invention further provides a preparation method of the GmPP2C-37like gene. The invention focuses on a new approach of resisting soybean cyst nematode disease, and excavates a new gene resource for improving soybean variety resistance.

Description

Technical Field

[0001] This invention belongs to the field of plant disease resistance breeding technology, specifically involving the application of the GmPP2C-37like gene in improving the ability of soybean to resist soybean cyst nematode disease. Background Technology

[0002] Soybean (Glycine max (L.) Merr.) originated in China and is one of the world's major food and economic crops, as well as a major source of plant protein and edible oil in my country. Soybean cyst nematode (SCN) is a global soil-borne disease and a major biological stress disease affecting soybeans. SCNs have unique life habits and parasitic methods, with a life cycle consisting of three stages: J1 (egg), J2 (larva), and J3 (adult). In the J2 stage, the nematode invades the soybean root protoplast through its mouthparts, forming a syncytium and feeding on the soybean's nutrients. Within the root, the SCN differentiates into females and males. After mating, the female forms a brown cyst that falls into the soil, while the male dies within the root. Cultivation practices and planting structures significantly impact the population size of soybean cyst nematodes. Current control methods primarily rely on crop rotation, planting resistant varieties, and chemical nematicides, but these methods can easily lead to loss of varietal resistance and environmental problems. To better control soybean cyst nematode (SCN) damage, genetic modification technology is a novel approach to cultivating resistant varieties, overcoming the drawbacks of long breeding cycles in traditional methods and avoiding the environmental and food safety issues associated with biological and chemical control. This study focuses on new pathways to SCN resistance and explores new genetic resources for improving soybean variety resistance. Summary of the Invention

[0003] The purpose of this invention is to improve the resistance of soybeans to soybean cyst nematode disease.

[0004] This invention provides the application of a protein encoded by the GmPP2C-37like gene in improving the resistance of soybean to soybean cyst nematode disease, the sequence of which is shown in SEQ ID NO.8.

[0005] This invention provides an application of the GmPP2C-37like gene in improving the resistance of soybean to soybean cyst nematode disease, wherein the nucleic acid molecules of the GmPP2C-37like gene are shown in SEQ ID NO.7.

[0006] To further limit the application of overexpressing the GmPP2C-37like gene in soybean to enhance the soybean's resistance to soybean cyst nematode disease.

[0007] This invention provides the application of a recombinant vector containing the nucleic acid molecule shown in SEQ ID NO.8 in improving the ability of soybean to resist soybean cyst nematode disease.

[0008] To further specify, the recombinant vector is an overexpression vector.

[0009] This invention provides the application of recombinant microbial cells containing the nucleic acid molecule shown in SEQ ID NO.7 in enhancing the ability of soybeans to resist soybean cyst nematode disease.

[0010] Further specified, the nucleic acid molecule shown in SEQ ID NO.7 is overexpressed in microbial cells.

[0011] This invention provides a breeding method for preparing soybeans resistant to soybean cyst nematode disease, the specific steps of which are as follows:

[0012] Step 1: The nucleic acid molecule shown in SEQ ID NO.7 is ligated to the pCAMBIA3300 vector to obtain the recombinant vector;

[0013] Step 2: Transform the recombinant vector described in Step 1 into Agrobacterium to obtain recombinant Agrobacterium;

[0014] Step 3: The recombinant Agrobacterium described in Step 2 is transferred into soybeans to obtain transgenic soybean plants, and positive transgenic soybean plants are obtained after identification.

[0015] To further specify, the primer combination for amplifying the nucleic acid molecule shown in SEQ ID NO.7 in step 1 is SEQ ID NO.1 and SEQ ID NO.2.

[0016] This invention provides a method for improving the resistance of soybean to soybean cyst nematode disease by infecting soybeans with soybean cyst nematode that overexpress the nucleic acid molecule shown in SEQ ID NO.3.

[0017] Beneficial Effects: Overexpression of the GmPP2C-37like gene in soybean yielded the pCAMBIA3300-GmPP2C-37like transgenic soybean. Soybean seedlings inoculated with soybean cyst nematode disease were collected. The average number of female nematodes on the positive roots of the transgenic soybean (10× microscope) was 2.3 / cm, while the average number of female nematodes on the roots of the control group (wild-type soybean) was 4 / cm. These results indicate that the female nematode index on the positive roots was significantly lower than that in the control group, confirming the significant effect of the GmPP2C-37like gene on soybean resistance to soybean cyst nematode disease. Attached Figure Description

[0018] Figure 1The image shows the cloning results of the GmPP2C-37like gene, where M is the DL2000 Marker and 1-6 are PCR products.

[0019] Figure 2 The image shows the PCR results of bacterial culture transformed with plant expression vectors: A is PCR of DH5α bacterial culture; B is PCR of EH105 bacterial culture; M is DL2000 Marker; 1-5 are PCR products.

[0020] Figure 3 Figure showing the subcellular localization analysis results of GmPP2C-37like protein;

[0021] Figure 4 The image shows the results of the root expression pattern analysis of the GmPP2C-37like gene after SCN stress, where T represents the experimental group and CK represents the control group.

[0022] Figure 5 Figure 1 shows the identification results of the root system of Dongnong 50 overexpression transgenic plants: A is the detection by Bar test strip; B is 2% agarose gel electrophoresis; C is the SCN phenotype of GmMYB29-OX and WT roots; D is the average number of nematodes per unit area in the roots of pCAMBIA3300-GmMYB29 transgenic plants and WT roots. Detailed Implementation

[0023] The following examples are provided to better understand the present invention, but do not limit the invention. Unless otherwise specified, the experimental methods in the following examples are conventional methods. Unless otherwise specified, the reagents used in the following examples were purchased from conventional biochemical reagent stores.

[0024] Plant varieties: Dongnong L-10 (disease resistant), Dongnong 50 (disease susceptible).

[0025] The cyst nematode resistant variety Dongnong L-10 is described in the article by Wu Depeng, Zhao Yue, Sheng Bihan, et al. Genetic model analysis of Dongnong L-10 against physiological race 3 of soybean cyst nematode [J]. Soybean Science, 2016, 35(3):6. DOI:10.11861 / j.issn.1000-9841.2016.03.0367.

[0026] Soil type of diseased soil: The soybean cyst nematode disease used was soybean cyst nematode HGtype 1.2.3.5.7 (physiological race 3), which is recorded in the article by Wu Depeng, Zhao Yue, Sheng Bihan, et al. Genetic model analysis of soybean cyst nematode physiological race 3 by Dongnong L-10 [J]. Soybean Science, 2016, 35(3):6.DOI:10.11861 / j.issn.1000-9841.2016.03.0367, and was collected from the experimental field of the Soybean Research Institute of Northeast Agricultural University.

[0027] Example 1. Cloning of the GmPP2C-37like gene

[0028] 1) Target gene CDS cloning. Phytozome was used. v13 The gene sequence of soybean Williams 82 (Glycine maxWm82.a2.v1) was retrieved from the database and subjected to BLAST alignment to obtain GmPP2C-37like CDS sequence information. Primers for gene cloning (primer 1) were designed using Primer 5.0 software. Using cDNA as a template, PCR was performed with the following program: pre-denaturation 94℃, 1 min; denaturation 98℃, 10 sec; annealing 58℃, 5 sec; extension temperature: 68℃, 30 sec / kb, for a total of 30 cycles, stored at 12℃. The PCR product was obtained and detected by agarose gel electrophoresis; the fragment size was 1203 bp, and the result was correct. Figure 1 The target fragment was obtained by gel recovery and purification. Fragment 1 was obtained using primer 1, and fragment 2 was obtained using primer 2.

[0029] The working system was set as follows (20 μL): cDNA: 1 μL, KODOne (high-fidelity amplification enzyme containing dNTPs, sucrose, etc.) 10 μL, OE-PP2C-F 1 μL, OE-PP2C-R 1 μL, ddH2O 7 μL;

[0030] Primer 1 overexpression primer:

[0031] GmPP2C-37like-37-F:TCGAGCTCCGTCGACAAGCTTATGGCTGGAATTTGCTGTGG (SEQ IDNO.1);

[0032] GmPP2C-37like-37-R:GCCCTTGCTCACCATAAGCTTACTACAACGACGTTAATTAA(SEQ IDNO.2);

[0033] Primer 2 Subcellular Primer:

[0034] subPP2C-37-F: TCGAGCTCCGTCGACAAGCTTATGGCTGGAATTTGCTGTGG (SEQ ID NO.3);

[0035] subPP2C-37-R: GCCCTTGCTCACCATAAGCTTACTACAACGACGTTAATTAA (SEQ ID NO.4);

[0036] Primer 3: Primers for real-time PCR.

[0037] RTPP2C-37-F: TGTTGCGACTATGTGTAAGGAG (SEQ ID NO.5);

[0038] RTPP2C-37-R: CCGTTCTCCATCGTCAGTTT (SEQ ID NO.6);

[0039] Gene sequence of GmPP2C-37like (SEQ ID NO.7):

[0040] ATGGCTGGAATTTGCTGTGGTGTTGTTGGAGAAGGTGACACTCCGGCTCCACTCGAGC

[0041] CCACTCCTCGCCCCTCCAGGCGCCGGAGTTTGGACATCTTACCTTTAAAATATATCGCCG

[0042] ACATGCCCATGCCGCCGCCGGAGGCTTTACGGAAGCGTCCCAAGCTCAACCTCAGGGA

[0043] CTGCGATAACGCAATTGAAAATTGCGACGAATCCACTGGACACAAGGTGACGAAGAAG

[0044] GAATCCAAAGTAGACTACGACGACGACGTCGTTTCGGAAACTAAGAACGTAACTGTTT

[0045] CGGAAGTTGAAGAAGAATCTCCCAAGTTCGGCGTGACGTCCGTTTGCGGCAGGAGAA

[0046] GAGACATGGAAGATTCCGTCTCGGTGCGGCCTTGCTTCACCCAAGGCTTCCACTACTTC

[0047] GGCGTCTTCGACGGTCACGGTTGCTCTCATGTTGCGACTATGTGTAAGGAGCGGCTACA

[0048] CGAGATCGTGAATGAAGAAATTGAAAGCGCGCGCGAGAATTTGGAGTGGAAACTGAC

[0049] GATGGAGAACGGATTCGCTCGCATGGACGACGAGGTTCATCGCCGGAGCCAGAGCAAC

[0050] CAGACCTTCACCTGCAGGTGTGAGCTCCAGACTCCTCACTGCGACGCCGTCGGATCCA

[0051] CCGCCGTCGTCGCCGTCGTCACGCCGGACAAAATCGTCGTCTCTAACTGCGGCGACTC

[0052] CCGCGCCGTCCTCTGCCGCAACGGCGTCGCCATCCCTCTCTCCTCCGATCACAAGCCGG

[0053] ATCGACCCGACGAATTACTCCGAGTCCAATCCAAGGGAGGGCGCGTGATTTACTGGGA

[0054] CGGTCCGAGAGTGCTTGGTGTGTTAGCAATGTCTCGAGCCATAGGTGACAATTATCTGA

[0055] AGCCGTACGTGATTTCAGAACCGGAGGTGATGGTGACGGAGCGGACGGAGGAGGACG

[0056] AGTGTTTGATACTGGCGAGTGATGGGTTGTGGGATGTGGTATCGAATGAGACCGCATGT

[0057] GGGGTGGTGAGGATGTGCCTCAAGGCGCAGAAGCCGCCGGGGTCTCCGGGGAGTGAC

[0058] GTGGCGGCTGACGGTTCCGACCGTGCTTGCTCCGATGCGTCGATTCTGTTGACCAAGTT

[0059] GGCGCTGGCAAGGCATAGTTCGGATAATGTGAGCGTGGTGGTGGTTGATTTGAGGAGGGATCAACGACAATCATCAAACTACAACGACGTTAATTAA;

[0060] GmPP2C-37like-37like amino acid sequence (SEQ ID NO.8):

[0061] MAGICCGVVGEGDTPAPLEPTPRPSRRRSLDILPLKYIADMPMPPPEALRKRPKLNLRDCD

[0062] NAIENCDESTGHKVTKKESKVDYDDDVVSETKNVTVSEVEEESPKFGVTSVCGRRRDME

[0063] DSVSVRPCFTQGFHYFGVFDGHGCSHVATMCKERLHEIVNEEIESARENLEWKLTMENGF

[0064] ARMDDEVHRRSQSNQTFTCRCELQTPHCDAVGSTAVVAVVTPDKIVVSNCGDSRAVLCRN

[0065] GVAIPLSSDHKPDRPDELLRVQSKGGRVIYWDGPRVLGVLAMSRAIGDNYLKPYVISEPEV

[0066] MVTERTEEDECLILASDGLWDVVSNETACGVVRMCLKAQKPPGSPGSDVAADGSDRACSDASILLTKLALARHSSDNVSVVVVDLRRDQRQSSNYNDVN.

[0067] Example 2. Obtaining transgenic recombinant vectors and recombinant microbial cells

[0068] 1) Following the instructions of the Seamless Cloning Kit from Beyotime Biotechnology Co., Ltd., the target fragment obtained in Example 1 was ligated into the pCAMBIA3300 and pCAMBIA1302 vectors using homologous recombinase to construct the pCAMBIA3300-GmPP2C-37like and pCAMBIA1302-GmPP2C-37like recombinant vectors.

[0069] 2) The recombinant vector obtained in step 1 was transformed into DH5α competent E. coli cells. Single clones were picked and cultured. The cloning primers (GmPP2C-37like-37-F:TCGAGCTCCGTCGACAAGCTTATGGCTGGAATTTGCTGTGG; GmPP2C-37like-37-R:GCCCTTGCTCACCATAAGCTTACTACAACGACGTTAATTA A) were used as positive cloning primers for the bacterial culture. 1 μL of the cultured bacterial culture was selected as a template for PCR amplification. The PCR reaction program was as follows: pre-denaturation 94℃, 5 min; denaturation 94℃, 30 sec; annealing 58℃, 30 sec; extension temperature: 72℃, 1 min / kb, for a total of 36 cycles; final extension 72℃, 5 min, and storage at 4℃. The results are shown in Figure (A in 2). After separation by 2% agarose gel electrophoresis, approximately 500 μL of bacterial culture with the correct bands was collected and sent to Shanghai Sangon Biotech Co., Ltd. for base sequencing. The sequencing results were processed using DNAman software, ultimately yielding the 1203 bp GmPP2C-37like gene, indicating that the GmPP2C-37like gene was successfully ligated into the expression vector and transformed into *E. coli*. Following the instructions of the TaKaRa MiniBEST Plasmid Purification Kit Ver. 4.0, plasmid DNA was extracted from the sequenced *E. coli* culture.

[0070] 3) Add 1000 ng of pCAMBIA3300-GmPP2C-37like recombinant plasmid to EHA105 competent cells, mix vigorously by pipetting, place on ice for 5 min, place in liquid nitrogen for 5 min, water bath at 37℃ for 5 min, and ice bath for 5 min; add 700 μL of antibiotic-free LB medium, place at 28℃, 200 rpm, and anneal for 2-3 h; take 100 μL and spread it on a selection plate containing kanamycin (Kan) and rifampicin (Rif), incubate in the dark at 28℃ for 24 h, pick positive single clones for bacterial PCR verification, the result is shown in Figure (B in 2), proving that pCAMBIA3300-GmPP2C-37like has been successfully transformed into Agrobacterium.

[0071] Example 3. Subcellular localization and RT-qPCR

[0072] 1) Subcellular localization

[0073] The recombinant pCAMBIA1302-GmPP2C-37like vector plasmid was transformed into GV3101 (p-soup19) Agrobacterium competent cells. Single colonies were picked and added to 1 mL of LB broth containing rifampicin (Kan) and kanamycin (Rif), and cultured at 28°C and 200 rpm for 1-2 days. The cells were then propagated in LB broth with shaking until the OD600 reached 0.8-1.0. The cells were resuspended in a suspension (100 mmol / L MES, 100 mmol / L MgCl2, 150 μmol / L AS). The bacterial suspension was injected into leaves of *Nicotiana benthamiana* using a 1 mL syringe. The distribution of GFP (green fluorescent tag) protein in the plants was observed under an inverted microscope. The results are shown below. Figure 3 As shown in the figure, the control group containing pCAMBIA1302-GFP was expressed in the cell membrane, cytoplasm and nucleus, while GmPP2C-37like protein was expressed in the nucleus, which was basically consistent with the subcellular localization prediction results.

[0074] 2) RT-qPCR

[0075] Quantitative primers were designed using the NCBI online website, with the Actin (AF049106) gene used as an internal reference gene for quantitative PCR. RT-qPCR results showed that ( Figure 4 In the susceptible soybean variety Dongnong 50, under SCN3 stress, the expression level of the GmPP2C-37like gene in the roots generally showed a trend of first increasing, then decreasing, and then increasing again, with the highest expression level observed in Dongnong 50 at 18 days. In the resistant variety Dongnong-L10, under SCN3 stress, the expression level of the GmPP2C-37like gene reached its maximum at 12 days. Nematode infection of soybean roots generally peaks around 14 days, thus preliminarily confirming that the GmPP2C-37Like gene is involved in the nematode stress response.

[0076] Example 4. Obtaining plants overexpressing the GmPP2C-37Like gene

[0077] Agrobacterium-mediated soybean transformation was used to construct overexpression lines by infecting recipient soybeans with Agrobacterium tumefaciens containing the pCAMBIA3300-GmPP2C-37like recombinant plasmid via Agrobacterium tumefaciens EHA105. Initially, 1000 explants were constructed, and genomic DNA was extracted from the plants. The GmPP2C-37like gene-specific primer PCR is shown in the figure. Figure 5 (A) and the expression of bar protein was detected, indicating that the protein was successfully expressed in the root system. Figure 5(B) The above results indicate that the pCAMBIA3300-GmPP2C-37Like overexpression line was successfully constructed. Three transgenic lines were screened and their seeds were harvested for further propagation to a genetically stable generation, yielding GmPP2C-37like-OX plants.

[0078] Example 5. Identification of disease resistance function of GmPP2C-37Like gene

[0079] The number of root nematodes in transgenic plants was identified using acid fuchsin staining. Based on the tissue examination patterns of SCN-infected plants, the number of nematodes was identified at the peak of nematode invasion, 15 days after inoculation. The international acid fuchsin staining method was used to observe the invasion and development of nematodes in the roots. Specific steps:

[0080] 1) Rinsing: Slowly remove the plant without damaging the roots and rinse it with clean water;

[0081] 2) Decolorization: Soak the roots in a 3% NaClO aqueous solution for 1-1.5 hours (the NaClO concentration can be increased appropriately to accelerate the decolorization efficiency of the roots) until the roots are completely white and transparent.

[0082] 3) Root staining: Rinse the roots with tap water to remove any NaClO residue, then soak them in distilled water for 15 minutes. Prepare an acidic fuchsin solution (stock solution: 3.5g fuchsin, 250mL glacial acetic acid, 750mL distilled water), bring it to a boil in a water bath, and then put the decolorized roots into the fuchsin solution and continue boiling for 2 minutes (this time can be extended as needed). Remove the roots, wipe them dry with absorbent paper, and observe the number of nematodes under a microscope.

[0083] Fifteen transgenic positive plants (GmPP2C-37like-OX plants) and fifteen wild-type plants (WT) of pCAMBIA3300-GmPP2C-37like were inoculated with soybean cyst nematode race 3, with 1000 nematodes per pot. The plants were then planted in soybean cyst nematode-infected soil. After 15 days, the nematode counts were performed under a 10× optical microscope using acid fuchsin staining. Five to six lateral roots from each plant were used for replication, and the average number of female nematodes per plant was calculated. Analysis of the nematode counts in the transgenic positive plants and wild-type plants revealed that the average number of female nematodes in the roots of the pCAMBIA3300-GmPP2C-37like transgenic positive plants under a 10× optical microscope was 2.3 nematodes / cm, while the average number of female nematodes in the roots of the control group was 4 nematodes / cm, a highly significant decrease (P<0.01), indicating that GmPP2C-37like has an inhibitory effect on soybean cyst nematodes. Figure 5 (C and D in the text).

Claims

1. Application of the protein encoded by the GmPP2C-37like gene in improving the resistance of soybean to soybean cyst nematode disease, characterized in that: The sequence of the GmPP2C-37like protein is shown in SEQ ID NO.

8.

2. Application of the GmPP2C-37like gene in improving soybean resistance to soybean cyst nematode disease, characterized in that: The nucleic acid molecule of the GmPP2C-37like gene is shown in SEQ ID NO.

7.

3. The use according to claim 2, characterized in that Application of overexpression of GmPP2C-37like gene in soybean to improve soybean resistance to soybean cyst nematode disease.

4. Use of a recombinant vector containing the nucleic acid molecule shown in SEQ ID NO. 8 in improving the resistance of soybean to soybean cyst nematode disease.

5. The use according to claim 4, characterized in that The recombinant vector is an overexpression vector.

6. Use of a recombinant microbial cell containing the nucleic acid molecule represented by SEQ ID NO. 7 in improving the resistance of soybean to soybean cyst nematode disease.

7. The use according to claim 6, characterized in that Overexpression of the nucleic acid molecule shown in SEQ ID NO.7 in microbial cells.

8. A breeding method for preparing soybeans resistant to soybean cyst nematode disease, characterized in that: The specific steps of the method are as follows: Step 1: Ligate the nucleic acid molecule represented by SEQ ID NO. 7 with 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.

9. The use according to claim 8, characterized in that The primer combination for amplifying the nucleic acid molecule shown in SEQ ID NO.7 in step 1 is SEQ ID NO.1 and SEQ ID NO.

2.

10. A method for improving soybean resistance to soybean cyst nematode disease, characterized in that: Soybean cyst nematodes are used to infect soybeans over-expressing the nucleic acid molecule shown in SEQ ID NO. 3.

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