Application of GmYHT gene in improving resistance of soybean to soybean cyst nematode disease

Abstract

The invention discloses application of a GmYHT 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 GmYHT gene in improving soybean cyst nematode disease resistance of soybeans. A nucleic acid marker of the GmYHT gene is as shown in SEQ ID NO.3. The invention also provides an application of the GmYHT gene in improving soybean cyst nematode disease resistance of the soybeans. The overexpression of the GmYHT gene can significantly improve the resistance of soybeans to the soybean cyst nematode disease, and the breeding application of the GmYHT gene is beneficial to breeding of a new soybean variety with excellent characters, and becomes an important research direction for comprehensive prevention and control of the soybean cyst nematode disease at present.

Description

Technical Field

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

[0002] Soybean (Glycine max [Linn.] Merr.) is native to China and has been cultivated for over 5,000 years. It is the fourth largest crop after rice, corn, and wheat, providing important oils and plant protein for humans and animals. Soybean cyst nematodes (SCN) are widespread in my country's major soybean-producing regions, causing annual economic losses of up to $120 million. SCN are obligate, sessile, and endoparasitic plant pathogens with a wide host range, diverse transmission pathways, robust reproductive capacity, and a long survival period. The soybean cyst nematode life cycle consists of the egg stage (J1), four larval instars (J2), and the adult stage (J3). The larval instar is the stage when soybean infestation is most severe. The soybean cyst nematode invades the roots of soybean seedlings through its own peristalsis, as well as through transmission vectors such as farming, water flow, and seeds. It parasitizes the root cortex, piercing the cell walls with its mouthparts and secreting digestive enzymes to destroy adjacent parenchyma cells in the root, forming a syncytium and thereby obtaining a stable nutrient source. Based on the differences in pathogenicity of different populations to soybean, the soybean cyst nematode disease is divided into 16 physiological races. Currently, the pathogenic races that cause the most economic losses in soybeans in my country are primarily races 1, 3, and 4. Race 3 is the dominant species and is primarily distributed in Northeast China. Race 4, with its extremely strong infectivity, is mostly found in the Huanghuaihai and Northeast regions of China. With the expansion of soybean planting areas and continuous cropping, the variation of physiological races has accelerated. Breeding disease-resistant varieties and discovering new loci and genes are the most cost-effective methods for preventing and controlling the disease.

[0003] Current research focuses on genes near the resistance loci, Rgh1 and Rgh4. However, these genes only explain approximately 60% of the genetic variation. Long-term use of these antigenic genes can lead to a gradual loss of resistance. Therefore, the discovery of new genes for resistance to soybean cyst nematodes is crucial. Studying their role in activating the plant's immune response to pathogens can provide candidate genes for soybean genetic improvement, facilitate the development of new soybean varieties with superior traits, and become a key research direction for the integrated prevention and control of soybean cyst nematode disease. Summary of the Invention

[0004] The purpose of the present invention is to improve the resistance of soybean to soybean cyst nematode disease.

[0005] The present invention provides an application of a protein encoded by a GmYHT gene in improving the resistance of soybean to soybean cyst nematode disease. The sequence of the protein encoded by the GmYHT gene is shown in SEQ ID NO.4.

[0006] The present invention provides an application of a GmYHT gene in improving the resistance of soybean to soybean cyst nematode disease. The nucleic acid molecule of the GmYHT gene is shown as SEQ ID NO.3.

[0007] Further defined is the use of overexpressing the GmYHT gene in soybean to improve the resistance of soybean to soybean cyst nematode disease.

[0008] The present invention provides an application of a recombinant vector containing a nucleic acid molecule shown in SEQ ID NO. 3 in improving the resistance of soybean to soybean cyst nematode disease.

[0009] It is further defined that the recombinant vector is an overexpression vector.

[0010] The present invention provides an application of a recombinant microbial cell containing a nucleic acid molecule shown in SEQ ID NO. 3 in improving the resistance of soybean to soybean cyst nematode disease.

[0011] It is further defined that the nucleic acid molecule shown in SEQ ID NO. 3 is overexpressed in a microbial cell.

[0012] The present invention provides a breeding method for preparing soybeans resistant to soybean cyst nematode disease, wherein the specific steps of the method are as follows:

[0013] Step 1: Ligate the nucleic acid molecule represented by SEQ ID NO. 3 with the vector pCAMBIA3300 to obtain a recombinant vector;

[0014] Step 2: Transform the recombinant vector described in step 1 into Agrobacterium to obtain recombinant Agrobacterium;

[0015] 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.

[0016] It is further defined that the primer combination for amplifying the nucleic acid molecule shown in SEQ ID NO. 3 in step 1 is SEQ ID NO. 1 and SEQ ID NO. 2.

[0017] The present invention provides a method for improving the resistance of soybean to soybean cyst nematode disease, which uses soybean cyst nematodes to infect soybeans over-expressing the nucleic acid molecule shown in SEQ ID NO.3.

[0018] Beneficial effects: The GmYHT gene was overexpressed in soybean to obtain pCAMBIA3300-GmYHT transgenic positive roots. Soybean seedlings were inoculated from soil infected with soybean cyst nematodes. The average number of female insects in the transgenic soybean roots was 2.767 / cm, which was lower than the number of female insects in the wild-type soybean roots (control group). The average number of female insects in the control group's roots was 5.193 / cm. There was a very significant difference between the transgenic positive roots and the control roots, confirming that increasing the expression of the GmYHT gene has obvious resistance to soybean cyst nematode disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the result of RNA extraction from the roots of Dongnong L10;

[0020] Figure 2 This is the result of GmYHT gene cloning; Note: M: DL2000; 1-4: PCR cloning products;

[0021] Figure 3 Plant expression vector was transformed into DH5α Escherichia coli; Note: M: DL2000(+) DNA molecular weight standard; 1-6: PCR products;

[0022] Figure 4 This is the result of PCR amplification of the pCAMBIA3300-GmYHT recombinant plasmid in Agrobacterium rhizogenes K599 culture; Note: M: DL2000(+) DNA molecular weight standard; 1-6: PCR amplification products;

[0023] Figure 5 Figure 1 shows the results of genetic transformation mediated by Agrobacterium rhizogenes. Notes: (A) Soybean seeds (Dongnong 50) germinated in vermiculite; (B) Agrobacterium K599 cells containing the overexpressed GmYHT gene were collected after culture at 28°C; (C) Soybean hypocotyls were cut obliquely; (D) Soybean hypocotyl explants (whole) and explants on hypocotyls (whole); (E) Cultured with a culture cover at 28°C for heat preservation and moisture retention; (F) GFP fluorescence field of soybean hairy roots.

[0024] Figure 6 Figure 1 shows the results of transgenic identification of soybean plants; Note: (A) PCR detection of the Bar gene; (B) Transgenic test strip identification, WT Dongnong 50, 1-4pCAMBIA3300-GmYHT transgenic positive root detection results;

[0025] Figure 7Figure 2 shows the results of soybean cyst nematode identification in overexpressing plants; Note: (A) Average number of nematodes per unit area of ​​SCN, GmYHT-OE: overexpressing plants, WT: wild type (Dongnong 50); (B) Distribution of nematodes in the root of pCAMBIA3300-GmYHT transgenic-positive plants and pCAMBIA3300 empty-load control group; white arrows indicate SCN. 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 L-10 (disease-resistant), Dongnong 50 (disease-susceptible).

[0028] The cyst nematode-resistant variety Dongnong L-10 and the susceptible variety Heinong 37 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. GmYHT gene cloning

[0031] 1) Using the soybean cyst nematode-resistant variety Dongnong-L10 as the material, when the first three-leaf compound leaf grew, a root tissue sample (about 1 g) was taken, RNA was extracted, and detected by agarose gel electrophoresis ( Figure 1 ), the RNA band pattern was clear, cDNA was extracted using a reverse transcription kit, and OD was measured using a UV spectrophotometer. 260 / OD 280 When the ratio is between 1.8, the cDNA has good integrity and high purity and can be stored at -20℃.

[0032] 3) Target gene CDS cloning. V13The database was used to search the sequence of soybean Williams 82 (Glycine maxWm82.a2.v1) gene, and blast alignment was performed to obtain the CDS sequence information of GmYHT. Specific primers were designed using Primer 5.0 software to amplify the gene sequence (primer 1). Using cDNA as the cloning template, the system was set up in 20 μL: GmYHT-F 1 μL, GmYHT-R 1 μL, template DNA 1 μL, KOD One TM PCR Master Mix (TOYOBO) 10 μL, ddH2O 7 μL. PCR program: 98°C pre-denaturation for 3 min; 98°C denaturation for 10 s, 55°C annealing for 5 s, 68°C extension for 5 s, 36 cycles; 68°C extension for 10 min, and storage at 4°C. Target bands were obtained using a gel imaging system ( Figure 2 ). Primer 1 Overexpression primer:

[0033] OE-GmYHT-F: TCGAGCTCCGTCGACAAGCTTATGGCTTATCAAGTGTTGCT (SEQ ID NO. 1);

[0034] OE-GmYHT-R: GCCCTTGCTCACCATAAGCTTAATAGTAGGGAATGGGTTAA (SEQ ID NO. 2);

[0035] GmYHT gene sequence (SEQ ID NO.3):

[0036]

[0037] GmYHT amino acid sequence (SEQ ID NO.4):

[0038] MAYQVLLICLVSTIVFAYILWRKQSKKNLPPSPKALPIIGHLHLVSPIPHQDFYKLSTRHGPIMQLFLGSVPCVVASTAEAAKEFLKTHEINFSNRPGQNVAVKGLAYDSQDFLFAFAPFGPYWKFM KKLCMSELLSGRMMDQFLPVRQQETKRFISRVFRKGVAGEAVDFGDELMTLSNNIVSRMTLSQKTSENDNQAEEMKKLVSNIAELMGKFNVSDFIWYLKPFDLQGFNRKIKETRDRFDVVVDGIIKQR QEERRKNKETGTAKQFKDMLDVLLDMHEDENAEIKLDKKNIKAFIMDIFVAGTDTSAVSIEWAMAELINNPDVLEKARQEIDAVVGKSRMVEESDIANLPYLQAIVRETLRLHPGGPLVVRESSKSA VVCGYDIPAKTRLFVNVWAIGRDPNHWEKPFEFRPERFIRDGQNQLDVRGQHYHFIPFGSGRRTCPGASLAWQVVPVNLAIIIQCFQWKLVGGNGKVDMEEKSGITLPRANPIICVPVPRINPFPTI.

[0039] Example 2. Vector construction and colon transformation

[0040] The HindⅢ-digested linearized vector pCAMBIA3300 was ligated with the target gene fragment using a homologous recombination kit (Vazyme, China). The homologous recombination system consisted of 20 μL of the following: 1.5 μL of the vector fragment, 0.5 μL of the target fragment, 2 μL of the homologous recombination enzyme, 4 μL of 5×CE Buffer, and 12 μL of ddH2O. The PCR instrument was set at 37°C for 30 min. The successfully recombined pCAMBIA3300-GmYHT plasmid was then transformed into DH5α competent cells, and a single clone was picked for bacterial culture. Cloning primers were used to identify positive clones in E. coli. 1 μL of the cultured bacterial solution was selected as a template for PCR amplification of the bacterial solution (the PCR procedure was the same as the CDS cloning procedure). After the reaction was completed, the target fragment was separated by agarose gel electrophoresis, and the target fragment band size was 1530 bp. Figure 3 ) The PCR products were sent to Ruibo Biotechnology Company for base sequencing using DANMANV8.0 The software was used to align the sequence with the CDS sequence, and the E. coli liquid with the correct sequence alignment was extracted with reference to the instructions of the Plasmid Mini Kit I kit (Omega, USA) to extract the E. coli plasmid DNA.

[0041] Example 3. Transformation of Agrobacterium rhizogenes with recombinant vector

[0042] The transformation method of Agrobacterium rhizogenes K599 includes: taking competent K599 (100 μL) stored at -80°C and bringing it to room temperature, placing it on ice when it is partially thawed and in an ice-water mixture state, adding 1 μg of pCAMBIA3300-GmYHT recombinant plasmid, and mixing it quickly and vigorously by pipetting. The mixture is then placed on ice for 5 minutes, in liquid nitrogen for 5 minutes, in a 37°C water bath for 5 minutes, and in an ice bath for 5 minutes. Then, 700 μL of LB solid medium without antibiotics was added, and the culture was shaken at 200 rpm at 28°C for 2 hours. The cells were then collected by centrifugation at 1,2000 rpm for 1 minute. 50 μL was evenly spread on a plate containing the kanamycin (Kan) and streptomycin (Str) resistance gene Bar as a selection marker. After inverted culture at 28°C in the dark for 24 hours, a single clone was picked for bacterial liquid PCR verification, and a target band of 1530 bp was obtained. The results showed that pCAMBIA3300-GmYHT had been successfully transformed into Agrobacterium rhizogenes K599 ( Figure 4 ).

[0043] Example 4. Agrobacterium rhizogenes-mediated genetic transformation

[0044] Select Dongnong 50 (a sensitive SCN variety) with uniform grain size, complete and no disease spots ( Figure 5 (A) in the figure was sown in vermiculite and cultured at 25°C with 16 hours of light and 8 hours of darkness. On the 6th day, the bacteria were enriched using a sterile surgical blade and then used ( Figure 5 (B)); will germinate to V E The seedlings were taken out and the vermiculite was cleaned with clean water. Then the soybean hypocotyl was cut at 45° with a sterile blade to create a certain wound surface ( Figure 5 200 μL of Agrobacterium K599 bacterial suspension was spread on LB solid medium containing Kan (50 mg / mL) and Str (50 mg / mL), and cultured in a 28°C incubator overnight. The colonies were gently scraped off and resuspended in liquid culture medium to an OD of 0. 600 =0.8, which is the bacterial infection solution. Select 3-4 day old seedlings and use a 1mL syringe to collect the resuspended bacteria. Inject the bacterial solution 2-3 times at the cotyledonary node or proximal hypocotyl of the seedling. Cut the wound approximately 1 cm below the wound.

[0045] Place the infected soybean seedlings back into the moistened vermiculite ( Figure 5 (E)), cover the culture pot with a fresh-keeping cover for 2-3 weeks, and wait for the hairy roots to grow to a certain length, and use PCR to identify ( Figure 6 (A)) and Bar test strips ( Figure 6 The plants were positively identified using the method in (B)), proving that the marker gene introduced together with the GmYHT gene was introduced and functioned, and that the GmYHT gene was overexpressed in soybean to obtain GmYHT-OX.

[0046] Soybean hairy roots were illuminated by LUYOR-3415RG dual-wavelength fluorescent protein excitation light source, and GFP green fluorescent hairy roots ( Figure 5 (F)). For transgenic plants, only the positive root tissue was retained, the excess part was cut off, and the plants were transplanted into diseased soil inoculated with soybean cyst nematode egg suspension and cultured for 14 days ( Figure 5 (A)-(F)).

[0047] Example 5. Functional verification of candidate genes

[0048] The root system of the transgenic plants was treated by acid fuchsin staining. First, Dongnong 50 soybean seedlings inoculated for 15 days were taken from the soil infected with soybean cyst nematodes, and the roots were rinsed with clean water. The rinsed soybean roots were then immersed in a 3%-5% sodium hypochlorite aqueous solution for 1-2 hours until the roots were completely decolorized. The residual NaClO solution on the soybean roots was washed with water, and then soaked in distilled water for 15 minutes. The decolorized roots were placed in a 30-fold diluted acid fuchsin solution (mother liquor: 0.175g fuchsin, 12.5mL glacial acetic acid, distilled water to 1.5L) and boiled for 120s. Finally, the roots were taken out and wiped with absorbent paper, and the roots were pressed and examined under a microscope. The number of soybean cyst nematodes was observed and recorded under a 20× stereo microscope ( Figure 7 (B) ) , soybean root length was measured, and five lateral roots per plant were taken as biological replicates. The number of nematodes per unit length of soybean root was calculated, and the statistical data were analyzed using SPSS software. The results showed that the average number of lateral nematodes in soybeans overexpressing hairy roots was 2.76 / cm, while the average number of lateral nematodes in wild-type soybeans transfected with the pCAMBIA3300 empty vector was 5.19 / cm ( Figure 7 (A) in the figure). The results of the t-test (Table 1) showed that the means of the two groups were 2.767 and 5.193 per cm, respectively, with standard deviations of 0.4398 and 0.7554, respectively. The t-value of the test result was 10.846, and the mean P value (two-tailed) was 0.003, which were extremely significantly lower than those of the control group (P < 0.01). This preliminarily confirmed that GmYHT had significant resistance to soybean cyst nematode race 3.

[0049] Table 1 t-test difference analysis

[0050]

Claims

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

4.

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

3.

3. The use according to claim 2, characterized in that Application of overexpression of GmYHT gene in soybean to improve the resistance of soybean to cyst nematode disease.

4. Use of a recombinant vector containing the nucleic acid molecule shown in SEQ ID NO. 3 in improving soybean resistance 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. 3 in improving soybean resistance 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. 3 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. 3 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.3 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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