An AhRabF1 gene and its application in resistance to peanut leaf spot disease

Abstract

The present invention discloses an AhRabF1 gene and its application in peanut leaf spot resistance. The present invention uses peanut leaf spot resistance germplasm PI 196622 as experimental material, clones the AhRabF1 gene, and verifies the expression pattern of the AhRabF1 gene in different tissues by fluorescent quantitative PCR, and the gene has a higher expression abundance in leaves. The present invention constructs an AhRabF1 gene overexpression vector and obtains AhRabF1 overexpression transgenic plants by using Agrobacterium-mediated genetic transformation technology. Experimental verification shows that the AhRabF1 overexpression peanut plants have significantly higher resistance to leaf spot than wild-type plants. The identification and functional analysis of the peanut AhRabF1 gene enriches the gene resources for peanut disease resistance breeding, and has important theoretical significance and application value for the breeding and screening of disease-resistant plants.

Description

Technical Field

[0001] The invention belongs to the technical field of plant disease prevention and control gene engineering, and specifically relates to a peanut AhRabF1 gene and an application thereof in resistance to peanut leaf spot disease. Background Art

[0002] Leaf spot is the main leaf disease of peanuts, which is divided into early spot (also known as brown spot) and late spot (also known as black spot). Different types of leaf spot can occur simultaneously on a plant or a leaf. The disease is common in all peanut production areas and has become one of the main reasons for the reduction in peanut production worldwide. The disease is caused by pathogenic bacteria, mainly forming lesions on leaves, petioles, stipules and stems can be affected. In severe cases, a large number of leaves can fall, affecting the photosynthesis of plants, which can lead to a 5%-15% reduction in peanut production, and even a reduction of more than 30% in some years.

[0003] The application of chemical agents such as fungicides is an important measure for the prevention and control of leaf spot disease in the field. The prevention and control effect of peanut leaf diseases is generally around 60%, but it increases production costs, increases the economic burden on farmers, and also has a harmful impact on human health and the environment. Therefore, breeding and promoting disease-resistant peanut varieties is a fundamental and effective measure against peanut leaf spot disease. However, traditional hybrid breeding not only has a long cycle, but also the varieties selected are difficult to have resistance, yield and quality. At present, it is possible to combine with genomics to find resistance genes and markers to accelerate the breeding of leaf spot disease-resistant peanut varieties.

[0004] In the previous study of genome-wide association analysis of leaf spot resistance genes, a gene related to disease resistance was located, which was annotated as a GTP-binding protein gene. Many studies have shown that GTP-binding proteins have many functions, such as regulating cell polarity, cell growth, morphological development, cytoplasmic division, hormone signaling, and plant-pathogen interaction. The present invention cloned this gene in peanuts, named it AhRabF1 gene, and conducted in-depth research on its role in resistance to peanut leaf spot disease. Summary of the invention

[0005] In view of the above problems existing in the prior art, the purpose of the present invention is to provide an AhRabF1 gene and its application in resistance to peanut leaf spot disease. The present invention proves through experiments that the AhRabF1 gene has the function of resisting peanut leaf spot disease, providing candidate genes and theoretical support for disease-resistant breeding of peanut.

[0006] In order to solve the above technical problems, the technical solution to be adopted in the present invention is:

[0007] The present invention provides a peanut AhRabF1 gene, the nucleotide sequence of the AhRabF1 gene is shown as SEQ ID NO.1.

[0008] Furthermore, the amplification primers of the AhRabF1 gene are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0009] Furthermore, the AhRabF1 gene is located in the cytoplasm.

[0010] The present invention also provides the AhRabF1 gene-encoded protein, whose amino acid sequence is shown in SEQ ID NO.2.

[0011] The present invention also provides application of the peanut AhRabF1 gene in resistance to peanut leaf spot disease.

[0012] Furthermore, the application comprises the following steps:

[0013] (1) PCR amplification and cloning of AhRabF1 gene;

[0014] (2) Connecting the cloned full-length CDS of AhRabF1 gene to the expression vector to obtain an overexpression recombinant vector;

[0015] (3) Transforming the overexpression recombinant vector into Agrobacterium to obtain an overexpression recombinant strain;

[0016] (4) Transform the overexpression recombinant strain into plants, screen and obtain the AhRabF1 gene overexpression strain.

[0017] Furthermore, the PCR amplification system in step (1) is: 2×Taq Master Mix (Dye Plus) 10 μL; RabF1-1F 1 μL; RabF1-1R 1 μL; cDNA template 1 μL; ddH2O 7 μL.

[0018] Furthermore, the PCR amplification program in step (1) is: 94°C for 5 min; 94°C for 30 s, 61°C for 30 s, 72°C for 60 s, repeated for 35 cycles; 72°C for 5 min.

[0019] Furthermore, the AhRabF1 gene overexpression strain can enhance the ability of peanut to resist leaf spot disease.

[0020] The present invention also provides the application of the peanut AhRabF1 gene in breeding peanut varieties with leaf spot resistance.

[0021] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows: the present invention screened and cloned an AhRabF1 gene from peanut germplasm PI 196622, and verified the expression pattern of the AhRabF1 gene in different tissues by fluorescence quantitative PCR, and the gene was expressed more abundantly in leaves. The present invention constructed an AhRabF1 gene overexpression vector and obtained overexpression transgenic plants by using Agrobacterium-mediated genetic transformation technology. Experimental verification showed that the resistance of overexpression peanut plants to leaf spot disease was significantly higher than that of wild-type control plants. The identification and functional analysis of the peanut AhRabF1 gene enriched the genetic resources for disease-resistant breeding of peanuts, and had important theoretical significance and application value for the breeding and screening of disease-resistant plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0023] Figure 1 This is the PCR amplification diagram of peanut AhRabF1 gene, lane M is D2000 Marker; lanes 1-4 are amplified products;

[0024] Figure 2 This is the phylogenetic tree of peanut AhRabF1 protein and RabF1 protein sequences of other species. Note: Arachis hypogaea: peanut; Cicer arietinum: chickpea; Glycine max: soybean; Lotus japonicus: Japanese lotus; Lupinus angustifolius: lupine; Prosopis alba: white horse alfalfa; Trifolium pratense: clover; Vitis vinifera: grape;

[0025] Figure 3 The subcellular localization results of AhRabF1 gene;

[0026] Figure 4 The relative expression of AhRabF1 gene in leaves of VIGS (virus-induced gene silencing) plants and control plants is shown on the ordinate, and the abscissa is the test plant number, where 1 is the ck control plant, 2-9 are gene silenced plants, and the experimental material is PI 196622;

[0027] Figure 5 This is a comparison of the leaves of the gene-silenced plants and the control plants 4 days after inoculation with pathogens in vitro. The left side of the figure -: leaves of the control plants, the right side of the figure +: leaves of the VIGS-silenced plants;

[0028] Figure 6is the relative expression of AhRabF1 gene in the leaves of overexpression plants and control plants, as shown on the ordinate, and the abscissa is the test plant number, where 1 is the ck control plant, 2-6 are the overexpression plants, and the experimental material is Huayu 967;

[0029] Figure 7 This is a comparison of the overexpression plant and the control plant 15 days after inoculation with pathogens. The left side of the figure is -: control plant, and the right side of the figure is +: overexpression plant;

[0030] Figure 8 This is a comparison of the leaves of the overexpression plant and the control plant 21 days after inoculation with the pathogen. The left side of the figure shows the leaves of the control ck plant, and the right side shows the leaves of the overexpression plant. DETAILED DESCRIPTION

[0031] The present invention is further described in detail below in conjunction with embodiments. The following embodiments are for explanation of the present invention but the present invention is not limited to the following embodiments.

[0032] Example 1 Cloning and expression pattern analysis of peanut AhRabF1 gene

[0033] 1. Experimental Materials

[0034] 1.1 Plant materials

[0035] The material used in the present invention is peanut germplasm PI 196622, which originates from Cote d'Ivoire and can be obtained by applying from the USDA-ARSGermplasm Resources Information Network (GRIN) (ars-grin.gov) website.

[0036] 1.2 Experimental Reagents

[0037] RNA was extracted from peanut leaves using the plant RNA extraction kit from Nanjing Novezan Biotechnology Co., Ltd., and RNA was extracted from peanut seeds using the plant total RNA extraction kit from Beijing Tiangen Biotechnology Co., Ltd. cDNA was synthesized using the Evo M-MLV Plus cDNA synthesis kit from Acre Bio and the ReScript Ⅱ RT Super Mix for qPCR kit from Nobel Bio. The fluorescent quantitative PCR kit was purchased from Acre Biotechnology Co., Ltd., and the target gene was recovered using the agarose gel DNA recovery kit from Beijing Tiangen Biotechnology Co., Ltd.

[0038] D2000 Marker, TaqMix, etc. were purchased from Beijing Tiangen Biotechnology Co., Ltd.

[0039] 50×TAE buffer, PDA solid culture medium, DNA loading buffer, DNA Marker and Gelred were purchased from Qingdao Shenggong Biotechnology Co., Ltd.

[0040] 2. Experimental methods and results

[0041] 2.1 RNA extraction and cDNA synthesis

[0042] Different grinding methods were selected according to the different characteristics of peanut seeds and leaves. When grinding seeds, the seeds were cut into thin slices in advance, placed in a mortar that had been soaked in DEPC water and baked in an oven at 180°C, and liquid nitrogen was poured in for rapid grinding until it became a fine powder. A pre-cooled medicine spoon was used to take soybean-sized powder and put it into a 1.5 mL centrifuge tube with lysis solution added in advance. Cotton gloves were worn throughout the process to prevent frostbite. When grinding leaves, 1 to 2 healthy leaves of similar leaf age were selected and placed in a 1.5 mL centrifuge tube. Two 3 mm steel balls dried after treatment with DEPC water were added together. The centrifuge tube and adapter were pre-cooled in liquid nitrogen for 2-3 minutes, and the adapter was immediately installed. The centrifuge tube was placed on a frozen sample grinder for full grinding, and the lysis solution was added immediately after grinding. After the seeds and leaves were added with lysis solution, the total RNA of peanut seeds and leaves was extracted according to the instructions of the plant RNA extraction kit.

[0043] After RNA was obtained, the integrity of the RNA was checked by electrophoresis using 1% agarose gel.

[0044] RNA of the peanut germplasm PI 196622 was extracted, and the RNA was reverse transcribed to synthesize cDNA. The complete coding region of the AhRabF1 gene was amplified by PCR using the cDNA as a template. The sequence of the AhRabF1 gene is as follows (SEQ ID NO. 1), with a total of 603 bases.

[0045] ATGGGCTGTGGCTCCTCCACTCTAGGTAGGGATTCGAGACCGCTTGGTCGAGACAATTCTGAGAATGGTGGAGGGCAGGACGCCAAGAACCTTCGTGTTAAGCTTGTTCTCTTAGGTGATTCTGGCGTCGGTAAAAGCTGTATTGTTCTACGGTTTGTCCGTGGTCAATTCGATCCAACATCCAAGGTAACTGTTGGAGCATCTTTTTTGTCGCAAACGATAGCACTGCAAGACTCTACAACAGTTAAGTTTGAAATATGGGATACTGCTGGTCAAGAGAGGTATGCTGCATTGGCACCCCTATATTATCGTGGTGCAGCGGTTGCAGTTATTGTCTATGATATTACAAGCCCGGAATCTTTCAGCAAAGCACAATACTGGGTTAAGGAGCTACAAAAGCACGGAAGCCCTGAAATAGTTCTGGCATTGGTTGGTAATAAAGCTGATCTTCATGAGAAGCGAGAGGTGGCTGTTCAGGATGGTATTGACTATGCAGAGAAGAACGGAATGTTCTTTATAGAGACATCTGCAAAGACAGCAGACAACATAAATGAACTCTTTGAGGAAATTGCGAAAAGACTGCCTCGCCCTTCAGCTTCTTGA

[0046] The amino acid sequence of the protein encoded by the AhRabF1 gene is as follows (SEQ ID NO.2):

[0047] MGCGSSTLGRDSRPLGRDNSENGGGQDAKNLRVKLVLLGDSGVGKSCIVLRFVRGQFDPTSKVTVGASFLSQTIALQDSTTVKFEIWDTAGQERYAALAPLYYRGAAVAVIVYDITSPESFSKAQYWVKELQKHGSPEIVLALVGNKADLHEKREVAVQDGIDYAEKNGMFFIETSAKTADNINELFEEIAKRLPRPSAS*

[0048] The primers for amplifying the AhRabF1 gene are RabF1-1F and RabF1-1R:

[0049] RabF1-1F: ATGGGCTGTGGCTCCTCCA (SEQ ID NO.3);

[0050] RabF1-1R: TCAAGAAGCTGAAGGGCGAGG (SEQ ID NO. 4).

[0051] The AhRabF1 gene amplification system was as follows: 2×Taq Master Mix (Dye Plus) 10μL; RabF1-1F 1μL; RabF1-1R 1μL; cDNA template 1μL; ddH2O 7μL. The AhRabF1 gene PCR amplification program was as follows: 94℃ 5min; 94℃ 30s; 61℃ 30s; 72℃ 60s; repeat 35 cycles, 72℃ 5min. The PCR amplification results are shown in Figure 1. Figure 1 The phylogenetic tree of peanut AhRabF1 protein and RabF1 protein sequences in other species is shown in Figure 2 shown.

[0052] 2.2 Analysis of expression patterns of peanut AhRabF1 gene in different tissues

[0053] RT-qPCR primers were designed based on the cloned AhRabF1 gene sequence, and actin was selected as the internal reference gene. The cDNA of seeds and leaves was used as the template for RT-qPCR to analyze the expression pattern of the AhRabF1 gene in different tissues. Each sample was repeated 3 times. The samples were placed in the AB7500 fluorescent quantitative PCR instrument, and the program was 95℃ 30s; 95℃ 5s, 60℃ 30s, 72℃ 20s, and repeated 40 cycles. After the program is completed, the software will generate a melting curve and peak number to calculate the relative expression of the gene and clarify its expression pattern in different tissues.

[0054] RT-qPCR results showed that the expression level of AhRabF1 gene in leaves was 3.72 times that in seeds. Because peanut leaf spot is a leaf disease, the expression level of this gene in leaves is higher than that in seeds.

[0055] 2.3 Isolation and inoculation of peanut leaf spot pathogen

[0056] 2.3.1 Isolation of pathogens

[0057] Take leaves with peanut leaf spot lesions, cut off tissue with a diameter of 5 mm at the junction of the diseased and healthy parts, soak them in 75% ethanol solution for 2 minutes, rinse them with sterilized ddH2O 3 times, soak them in 1% sodium hypochlorite solution for 1 minute, and then rinse them with sterilized ddH2O 3 times. Use sterile filter paper to absorb the moisture on the surface of the leaves, place them face up on PDA culture medium, and culture them in a constant temperature incubator at 28°C to obtain the pathogen of peanut leaf spot.

[0058] 2.3.2 Plant inoculation

[0059] Take the peanut leaf spot pathogen, prepare a leaf spot pathogen suspension, smear it on the front and back of peanut leaves of the same leaf age, cultivate it in a constant temperature artificial climate box at 28°C, cover it with plastic film, maintain relative humidity >90%, and observe the growth of the plants from the 3rd day of inoculation.

[0060] 2.3.3 Detached leaf inoculation

[0061] Take the 3rd to 4th true leaves of VIGS plants and control plants respectively, soak them in 75% ethanol solution for 1 min on a clean bench, then rinse with sterilized ddH2O, use sterile filter paper to dry the surface moisture of the leaves, and place them face up in PDA medium. Pick the mycelium of the pathogen and place it on the front of the peanut leaf at a position two-thirds of the distance from the leaf base to the midrib, then place the culture dish in a light incubator with a relative humidity of 90% and a temperature of 28°C, with 16 h light / 8 h dark. Inoculate 6 peanut leaves for each material, and observe the disease condition of the leaves after 3 days.

[0062] 2.4 Analysis of peanut AhRabF1 gene expression patterns before and after inoculation

[0063] 7 days after inoculation, RNA was extracted from plant leaves, reverse transcribed to synthesize cDNA, and then the cDNA concentration was measured. Each sample was repeated 3 times, RabF1-q-F1 and RabF1-q-R1 were used as primers for amplifying the AhRabF1 gene, and actin-F and action-R were used as internal reference gene primers. The primer sequences are as follows:

[0064] RabF1-q-F1:AGAGGTATGCTGCATTGGCAC (SEQ ID NO.5);

[0065] RabF1-q-R1: CAGGGCTTTCCGTGCTTTTGTA (SEQ ID NO. 6);

[0066] actin-F:TTGGAATGGGTCAGAAGGATGC (SEQ ID NO.7);

[0067] actin-R: AGTGGTGCCTCAGTAAGAAGC (SEQ ID NO. 8).

[0068] After the reaction system was configured, it was placed in an AB7500 fluorescent quantitative PCR instrument and RT-qPCR was performed according to the program of 95℃ 30s; 95℃ 5s, 60℃ 30s, 72℃ 20s, and repeated 40 cycles. After the program was completed, the relative expression level was calculated to compare the expression difference of the gene in peanut leaves before and after pathogen infection.

[0069] The experimental results showed that the expression of AhRabF1 gene was upregulated after peanut leaves were infected with leaf spot pathogen, indicating that AhRabF1 gene was involved in the molecular regulation of peanut leaf spot resistance pathway.

[0070] Example 2: Subcellular localization

[0071] 1. Design and synthesize primers for amplifying target genes

[0072] RabF1-F3: CTCTCGAGCTTTCGCGAGCTCATGGGCTGTGGCTCCTCC;

[0073] RabF1-R3: GCCCTTGCTCACCATGGATCCAGAAGCTGAAGGGCGAGGC.

[0074] 2. Amplification of target fragment

[0075] Using pRI101-AhRabF1 plasmid as template, Prime STAR HS DNA polymerase was used to amplify the full length of the target gene. The PCR reaction system was as follows:

[0076] 5×buffer 10μL

[0077] dNTP (2.5 mM each) 4 μL

[0078] Primer RabF1-F3 (10 μM / L) 1.5 μL

[0079] Primer RabF1-R3 (10 μM / L) 1.5 μL

[0080] Prime STAR HS DNA polymerase (2.5U / μL) 0.5μL

[0081] cDNA 1 μL

[0082] ddH2O 31.5 μL

[0083] The PCR reaction conditions were: 98°C for 10s, 55°C for 5s, 72°C for 40s (30 cycles); 72°C for 5min. After the PCR was completed, electrophoresis was performed, and the gel was cut and the target fragment was recovered.

[0084] 3. Double enzyme digestion expression vector

[0085] The 1300sGFP vector plasmid was double-digested with SacI and BamHI, incubated in a 37°C water bath for 15 min, and then detected by agarose gel electrophoresis and the target fragment was recovered by gel excision.

[0086] 4. Ligation, transformation, screening and sequencing

[0087] The recombinant ligation kit was used for ligation. After reacting at 50°C for 30 minutes, the competent DH5α was transformed. The LB plate (kanamycin sulfate, 50 μg / mL) was coated and cultured overnight. After the colonies grew, the positive clones were screened by PCR and the positive bacterial solution was sent for sequencing. The sequencing results confirmed that the vector was successfully constructed.

[0088] Screening primers:

[0089] RabF1-258F:ATGGGATACTGCTGGTCAAG;

[0090] eGFP-R:ACTTGTGGCCGTTTACGTCG;

[0091] The following is the sequencing result (the underlined sequence is the full-length sequence), SEQ ID NO.9:

[0092] TCACCAAAGGGTAATTCGGGAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTCATCAAAAGGACAGTAGAAAAGGAAGGTGGCACCTCCAAATGCCATCATTGCGATAAAGGAAAGGCTATCGTTCAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAA AGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTCGCAAGACCCTTCCTCTATAAGGAAGTTTCATTTCATTTGGAGAGGACACGCTGAAATCACCAGTCTCTCTCTACAAATCTATCTCTCTCGAGCTTTCGCGAGCTC ATGGGCTGTGGCTCCTCCACTCTAGGTAGGGATTCGAGACCGCTTGGTCGAGACAATTCTGAGAATGGTGGAGGGCAGGACGCCAAGAACCTTCGTGTTAAGCTTGTTCTCTTAGGTGATTCTGGCGTCGGTAAAAGCT GTATTGTTCTACGGTTTGTCCGTGGTCAATTCGATCCAACATCCAAGGTAACTGTTGGAGCATCTTTTTTGTCGCAA ACGATAGCACTGCAAGACTCTACAACAGTTAAGTTTGAAATATGGGATACTGCTGGTCAAGAGAGGTATGCTGCATT GGCACCCCTATATTATCGTGGTGCAGCGGTTGCAGTTATTGTCTATGATATTACAAGCCCGGAATCTTTCAGCAAAG CACAATACTGGGTTAAGGAGCTACAAAAGCACGGAAGCCCTGAAATAGTTCTGGCATTGGTTGGTAATAAAGCTGAT CTTCATGAGAAGCGAGAGGTGGCTGTTCAGGATGGTATTGACTATGCAGAGAAGAACGGAATGTTCTTTATAGAGAC ATCTGCAAAGACAGCAGACAACATAAATGAACTCTTTGAGGAAATTGCGAAAAGACTGCCTCGCCCTTCAGCTTCTT GA GGATCCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGGTGGTGCCCATCCCTGTCGAA

[0093] 5. Subcellular localization results

[0094] AhRabF1 and empty vector 1300S-GFP were propagated and plasmids were extracted. The plasmids of AhRabF1 and empty vector 1300S-GFP were transformed into Arabidopsis protoplasts with nuclear markers, cultured under weak light for 8-10 h, and the subcellular localization of AhRabF1 was observed under a laser confocal microscope. Figure 3 As shown, the results showed localization in the cytoplasm.

[0095]

[0096] The excitation and emission wavelengths of fluorescent proteins are as follows:

[0097] Red fluorescent protein RFP: excitation wavelength is 532nm, emission wavelength is 588nm;

[0098] Green fluorescent protein GFP: excitation wavelength is 488nm and emission wavelength is 507nm.

[0099] Example 3: Peanut VIGS (Virus-Induced Gene Silencing) Experiment

[0100] 1. Reagent preparation

[0101] 500mM MgCl2 (magnesium chloride): 10.1g MgCl2·6H2O dissolved in 100mL sterile distilled water, sterilized by 0.22μm filtration, stored at 4℃, working concentration 10 mM;

[0102] 100mM MES (2-N-morpholinoethanesulfonic acid): 2.12g MES (anhydrous) solid powder was dissolved in 100mL sterile distilled water, filtered through 0.22μm for sterilization, and stored at 4℃. The working concentration was 10mM.

[0103] 74mM AS (acetosyringone): 145mg AS powder dissolved in 10mL anhydrous ethanol, stored at 4℃, working concentration 200μM;

[0104] MM buffer: 2 mL MgCl2 and 10 mL MES are required for every 100 mL buffer, and the remaining volume is made up with sterile distilled water;

[0105] MMA buffer: Add AS to MM buffer, add 270 μL per 100 mL, and adjust the pH to 5.2-5.7 with HCl or Mg(OH)2;

[0106] 2. Bacteria activation

[0107] Take out GV3101 bacteria (containing pTRV2:: AhRabF1 plasmid) from -80℃, put it on ice to thaw, take 100μL of bacterial solution and add it to 5mL LB liquid medium (containing kanamycin Kan and rifampicin Rif with a final concentration of 50μg / mL), and culture at 28℃ and 200rpm until the bacterial solution OD reaches 600 When the value is about 0.3-1.8, it is stored at 4°C for future use; the same method is used to activate the GV3101 strain containing the empty vector pTRV2 plasmid.

[0108] 3. Prepare bacterial suspension (the amount is determined according to the experimental requirements and should not be stored for a long time)

[0109] Add 100 μL of the activated bacterial solution to 5 mL of the above-mentioned LB liquid medium containing antibiotics, and culture at 28°C, 200 rpm for 12 h; then take out 5 mL of the bacterial solution and add it to 100 mL of LB liquid medium (containing 50 μL Kan, 100 μL Rif, 10 mL MES and 27 μL AS) and culture at 28°C, 200 rpm for 12 h. 600 When the value reaches 1.5-2.0, collect the bacteria by centrifugation at 6000 rpm for 10 min at room temperature, discard the supernatant, and resuspend the bacteria with a small amount of MMA buffer until it is completely mixed and there is no bacterial clumps. This way, a bacterial suspension is obtained.

[0110] 4. Infection method

[0111] The pTRV1 and pTRV2:: AhRabF1 bacterial suspensions of equal concentration and volume were mixed, and the mixture was placed in a dark place at room temperature for 3-4 hours, and then poured into a beaker. Thirty peanut seedlings that had germinated for 14 days were taken and completely immersed in the bacterial suspension. The beaker was placed in a vacuum tank, and the vacuum tank was connected to the vacuum pump with a rubber hose. When evacuating the vacuum, the vent valve of the vacuum tank was first opened, and then the vacuum pump was turned on. When the pressure gauge of the vacuum pump reached 0.6-0.8, the timing was started. After 7 minutes, the vent valve of the vacuum tank and the vacuum pump were closed in turn, and the vent valve of the vacuum tank was slowly opened. After the air pressure inside and outside the vacuum tank was balanced, the beaker was taken out. The bacterial suspension was discarded, and the bacteria on the surface of the seedlings were rinsed with sterile deionized water. The seedlings were placed in a hydroponic box containing 1 / 5 Hoagland's nutrient solution in turn, and cultured in the dark for 16 hours. After that, the artificial climate box was adjusted to 16 hours (light) / 8 hours (darkness). After 7 days, all the seedlings were transferred to nutrient soil for further culture.

[0112] The disease-resistant peanut PI 196622 was used as the experimental material. The control group was injected with bacterial solution containing the pTRV2 empty vector plasmid, and the experimental group was injected with bacterial solution containing the pTRV2::AhRabF1 plasmid.

[0113] 5. Identification method of transformed plants

[0114] Seven days after infection, DNA from leaves of gene-silenced plants and control plants were extracted, and primers pTRV2-F: TTGTTACTCAAGGAAGCACGAT; AhRabF1-278R: TCTTGACCAGCAGTATCCC AT were used to detect positive gene-silenced plants, and the amplified product was approximately 357 bp.

[0115] 6. Identification of the expression level of AhRabF1 gene in silenced plants

[0116] Seven days after infection, RNA was extracted from the leaves of gene-silenced plants and control plants, and the expression of AhRabF1 gene was detected by RT-qPCR after reverse transcription into cDNA.

[0117] The detection primer sequences are as follows:

[0118] RabF1-q-F1: AGAGGTATGCTGCATTGGCAC;

[0119] RabF1-q-R1: CAGGGCTTTCCGTGCTTTTGTA;

[0120] actin-F:TTGGAATGGGTCAGAAGGATGC;

[0121] actin-R: AGTGGTGCCTCAGTAAGAAGC.

[0122] 7. Experimental results

[0123] Through VIGS experiments, the expression levels of the AhRabF1 gene in the leaves of the gene-silenced plants of the present invention were lower than those of the control plants. The expression level of the AhRabF1 gene in the control plant ck was 1, and the expression levels of the AhRabF1 gene in the silenced plants ranged from 0.26 to 0.82 ( Figure 4 ).

[0124] from Figure 5 It can be seen intuitively that 4 days after inoculation, the leaves with silenced AhRabF1 gene showed lesions, while the control ck leaves showed no lesions. The above results prove that the inhibition of AhRabF1 gene expression significantly reduces the resistance of peanut to leaf spot disease.

[0125] Example 4: Peanut overexpression experiment

[0126] 1. Reagent preparation

[0127] 500mM MgCl2 (magnesium chloride): 10.1g MgCl2·6H2O dissolved in 100mL sterile water, sterilized by 0.22μm filtration, stored at 4℃, working concentration 10 mM;

[0128] 500mM MES (2-N-morpholinoethanesulfonic acid): 10.6g MES (anhydrous) solid powder was dissolved in 100mL distilled water, sterilized by 0.22μm filtration, and stored at 4℃. The working concentration is 10mM.

[0129] 100mM AS (acetosyringone): 196mg AS powder dissolved in 10mL anhydrous ethanol, stored at 4℃, working concentration 100μM;

[0130] 1L infection buffer MMA: 959ml sterile water + 1mL 100mM acetosyringone + 20mL 500mM MES + 20mL 500mM MgCl2

[0131] 2. Bacteria activation

[0132] Take out the GV3101 strain (containing the pRI101:: AhRabF1 plasmid) from -80℃, put it on ice to thaw, take 100μL of the bacterial solution and add it to 5mL LB liquid medium (containing kanamycin Kan and rifampicin Rif at a final concentration of 50μg / mL), and culture at 28℃ and 200rpm until the bacterial solution OD reaches 600 The concentration is about 0.3-1.8, and it is stored at 4°C for future use. The same method is used to activate the GV3101 strain containing the pRI101 empty vector plasmid.

[0133] 3. Preparation of bacterial suspension

[0134] Add 50 μL of the activated bacterial solution to 50 mL of the above LB liquid medium containing antibiotics, and culture at 28°C, 200 rpm until the bacterial solution OD reaches 600 When the value is 0.6-0.8, centrifuge at 6000 rpm for 10 min at room temperature to collect the precipitate, then add 50 mL of infection buffer to resuspend the bacteria until completely mixed to obtain a bacterial suspension.

[0135] 4. Infection method

[0136] The calyx tube injection method is used to infect peanut plants. The injection time is generally carried out before 8:00 in the morning. A 1mL medical syringe is used to draw up the bacterial suspension and inject it into the calyx tube of the flower. The injected flower and the new fruit needles thereafter are marked to ensure that the transgenic pods are harvested.

[0137] The susceptible peanut variety Huayu 967 was used as the experimental material. The control group was injected with bacterial solution containing pRI101 empty vector plasmid, and the experimental group was injected with bacterial solution containing pRI101::AhRabF1 plasmid.

[0138] 5. Identification of overexpressing transgenic plants

[0139] After harvesting the pods, a small cotyledon (about 5ug) was cut from the far embryo end of the peanut seeds, and genomic DNA was extracted by SDS method. Transgenic seeds were screened by identification primers, and the identification primers were 35S: GACGCACAATCCCACTATCC; AhRabF1-278R: TCTTGACCAGCAGTATCCCAT. The amplified product was about 443bp.

[0140] 6. Identification of the expression level of AhRabF1 gene in overexpression plants

[0141] The transgenic and control seeds were planted in an artificial climate chamber. After 21 days, RNA was extracted from the leaves of the transgenic and control plants. After reverse transcription into cDNA, the expression of the AhRabF1 gene was detected by RT-qPCR. The detection primer sequences are as follows:

[0142] RabF1-q-F1: AGAGGTATGCTGCATTGGCAC;

[0143] RabF1-q-R1: CAGGGCTTTCCGTGCTTTTGTA;

[0144] actin-F:TTGGAATGGGTCAGAAGGATGC;

[0145] actin-R: AGTGGTGCCTCAGTAAGAAGC.

[0146] 7. Experimental results

[0147] Through overexpression experiments, the expression level of the AhRabF1 gene in the leaves of the overexpressed plants of the present invention was increased compared with that of the control. The expression level of the AhRabF1 gene in the control plant ck was 1, and the expression level of the AhRabF1 gene in the overexpressed plants ranged from 1.95 to 3.43.

[0148] from Figure 7 and Figure 8 It can be seen that after a period of time of inoculation with pathogens, AhRabF1 overexpressing plants grew robustly and had no lesions on leaves, while the control plants had diseased leaves and lesions on the control leaves. The experimental results of this example further prove that overexpressing the AhRabF1 gene can enhance the resistance of peanut to leaf spot disease.

[0149] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. AhRabF1 The application of the gene in resistance to peanut leaf spot disease is characterized in that: Said AhRabF1 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. according to claim 1 AhRabF1 The application of the gene in resistance to peanut leaf spot disease is characterized in that: The application comprises the following steps: (1) PCR amplification cloning AhRabF1 Gene; (2) The cloned AhRabF1 The full-length CDS of the gene is connected to the expression vector to obtain an overexpression recombinant vector; (3) Transforming the overexpression recombinant vector into Agrobacterium to obtain an overexpression recombinant strain; (4) Transform the overexpression recombinant strain into plants, screen and obtain AhRabF1 Gene overexpression lines.

3. The use according to claim 2, characterized in that: The PCR amplification system in step (1) is: 2×TaqMaster Mix Dye Plus 10 μL; RabF1-1F 1 μL; RabF1-1R 1 μL; cDNA template 1 μL; ddH2O 7 μL.

4. The use according to claim 2, characterized in that: The PCR amplification program in step (1) is: 94°C for 5 min; 94°C for 30 s, 61°C for 30 s, 72°C for 60 s, repeated for 35 cycles; 72°C for 5 min.

5. The use according to claim 1, characterized in that: AhRabF1 Gene overexpression strains can enhance the ability of peanut to resist leaf spot disease.

6. Peanuts AhRabF1 The application of the gene in breeding peanut varieties with leaf spot resistance is characterized in that: Said AhRabF1 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

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