Application of Panax ginseng transcription factor PgERF2 in improving plant resistance to Alternaria alternata

Abstract

The present invention belongs to the field of biological genetic engineering and relates to the use of ginseng transcription factor PgERF2 in improving plant resistance to Alternaria alternata. The sequence of the ginseng transcription factor PgERF2 protein is shown in SEQ ID NO.1; the sequence of the ginseng transcription factor PgERF2 gene is shown in SEQ ID NO.2. The present invention clones the PgERF2 gene and constructs a corresponding plant expression vector, successfully transferring the gene into wild-type Arabidopsis thaliana. Transcriptomic analysis results show that the response to biotic stress is activated in the transgenic plants, and the expression level of the plant defense protein-related gene AtPDF1.2A is significantly increased. It is also demonstrated that PgERF2 can directly bind to the GCC-box element to activate the expression of the plant defense protein-related gene AtPDF1.2A, thereby significantly enhancing resistance to Alternaria alternata.

Description

Technical Field

[0001] The invention belongs to the field of biological genetic engineering and relates to the application of ginseng transcription factor PgERF2 in improving the resistance of plants to Alternaria alternata. Background Art

[0002] Ginseng roots accumulate high levels of ginsenosides, which may confer resistance to various potential biotic stresses, such as antimicrobial activity against pathogens and anti-herbivory activity against insects and other herbivores. ERF transcription factors play a crucial role in defense against biotic stresses, including fungal pathogens. In Arabidopsis, phosphorylation of the ERF6 protein by MPK3 / MPK6 enhances its stability. Phosphorylated ERF6 can consistently activate defense-related genes, particularly those involved in fungal resistance, such as PDF1.1 and PDF1.2, thereby enhancing plant resistance to Botrytis cinerea. In soybean, overexpression of GmERF113 elevates the expression of the disease-resistance genes GmPR1 and GmPR10-1, enhancing resistance to the soybean blight pathogen Phytophthora sojae.

[0003] However, the functions of ERF transcription factors in ginseng have not been fully elucidated.

[0004] Alternaria alternata is a saprophytic pathogenic fungus. Its multiple pathological species can infect a variety of economic crops including potatoes, pears, citrus and tobacco, causing a series of serious agricultural diseases and causing significant losses to the national economy every year. Summary of the Invention

[0005] The purpose of the present invention is to provide an application of ginseng transcription factor PgERF2 in improving the resistance of plants to Alternaria alternata.

[0006] In order to achieve the above object, the technical solution of the present invention is as follows:

[0007] The application of Panax ginseng transcription factor PgERF2 in improving plant resistance to Alternaria alternata. The sequence of Panax ginseng transcription factor PgERF2 gene is shown in SEQ ID NO.2.

[0008] The sequence of the transcription factor PgERF2 protein is shown in SEQ ID NO.1.

[0009] SEQ ID NO.1 is:

[0010] MIKENCSFDSDDFAFLESIRRHLLDESDDISVDLRWMTSVSDTIVVKCEPEIEAASSEFVNILAVQPKIEAEEVAPPKERHYRGVRRRPWGKFAAEIRDPAKNGARVWLGTFETAEDAALAYDRAAFRMRGSRAMLNFPLRVNSGEPEPVRITSKRSAGNMLNSSSSISSSS YSDSSTSVSKGKRRKTVAPVVVLERMGSVEVDSFKNWLMDDDFFNSLVKNVDTRGIPFPRSMN。

[0011] SEQ ID NO.2 is:

[0012] .

[0013] The invention discloses an application of ginseng transcription factor PgERF2 in improving the expression level of genes related to plant resistance to Alternaria alternata. The sequence of the PgERF2 gene is shown in SEQ ID NO.2.

[0014] In a preferred embodiment, the gene related to the plant's resistance to Alternaria alternata is AtPDF1.2A.

[0015] In a preferred embodiment, the plant is Arabidopsis thaliana, rice or cotton.

[0016] The invention relates to the use of ginseng transcription factor PgERF2 in preparing a reagent for positively regulating the promoter of the AtPgDF1.2A gene, wherein the sequence of the PgERF2 gene is shown in SEQ ID NO.2.

[0017] Application of ginseng transcription factor PgERF2 protein in preparing a reagent binding to the GCC-box site in the AtPDF1.2A promoter, wherein the sequence of the PgERF2 protein is shown in SEQ ID NO.1.

[0018] A plasmid, wherein the overexpression plasmid overexpresses the PgERF2 gene.

[0019] In a preferred embodiment, the plasmid is obtained by enzymatically digesting a vector to obtain an enzyme-digested vector, and then connecting the PgERF2 gene to the enzyme-digested vector.

[0020] In a preferred embodiment, the vector is pCAMBIA1301s plasmid.

[0021] The plasmid is used for enhancing the resistance of plants to Alternaria alternata, and the plasmid overexpresses the PgERF2 gene.

[0022] An Agrobacterium competent cell overexpressing the PgERF2 gene.

[0023] In a preferred embodiment, the Agrobacterium competent cells are obtained by transforming the plasmid into DH5α competent cells.

[0024] The Agrobacterium competent cells are used in enhancing the resistance of plants to Alternaria alternata, and the Agrobacterium competent cells overexpress the PgERF2 gene.

[0025] A plant is obtained by infecting plant cells with the Agrobacterium competent cells and then conducting tissue culture.

[0026] In a preferred embodiment, the plant cells are leaf cells.

[0027] In a preferred embodiment, the plant cells are leaf epidermal cells.

[0028] A plant overexpressing the PgERF2 gene.

[0029] In a preferred embodiment, the plant is Arabidopsis thaliana, rice or cotton.

[0030] The resistance of the plant to Alternaria alternata is significantly enhanced.

[0031] The present invention cloned the PgERF2 gene and constructed a corresponding plant expression vector. The gene was successfully transferred into wild-type Arabidopsis thaliana through Agrobacterium-mediated floral transformation. PCR was used to identify the transgenic plants at the genomic DNA and transcribed mRNA levels, yielding positive Arabidopsis plants that stably overexpressed the PgERF2 gene. Transcriptomic analysis revealed that the transgenic plants activated responses to biotic stresses, with significantly increased expression of the plant defense protein-related gene AtPDF1.2A.

[0032] The present invention verifies through dual-luciferase reporter gene assay (Dual-LUC) and electrophoretic mobility shift assay (EMSA) that PgERF2 can directly bind to the GCC-box element to activate the expression of the plant defense protein-related gene AtPDF1.2A, thereby enhancing resistance to pathogens.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] Experimental data demonstrate that the PgERF2 transcription factor in ginseng specifically binds to the promoter region of the Arabidopsis thaliana AtPDF1.2A gene, thereby enhancing its transcriptional activity. As a key gene for plant defense, increased expression of AtPDF1.2A significantly enhances plant resistance to pathogenic fungi. Furthermore, the ginseng PgERF2 transcription factor, its encoding gene, and recombinant overexpression vectors containing this gene, described in the present invention, can be applied to the genetic transformation of economic crops such as rice and cotton, providing a reliable genetic resource and theoretical foundation for molecular breeding of plant stress resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Identification of PgERF2 transgenic Arabidopsis plants; A: PCR identification of the hygromycin resistance gene (550 bp) in transgenic Arabidopsis leaf tissue; Lane 1: DNA Marker 2000; Lane 2: Negative control: WT genomic DNA; Lanes 3, 4, and 5: Genomic DNA of transgenic lines (OE-1, OE-4, and OE-5); Lane 5: Positive control: empty vector plasmid; B: RT-qPCR verification of the expression level of PgERF2 in transgenic Arabidopsis; AtEF1α is an internal reference gene, and error bars represent mean ± SD (n = 3, t-test, *P < 0.05 and **P < 0.01);

[0036] Figure 2 GO enrichment analysis of differentially expressed genes between OE-4 and WT samples; A indicates up-regulated differentially expressed genes; B indicates down-regulated differentially expressed genes;

[0037] Figure 3PgERF2 enhances resistance of Arabidopsis to Alternaria alternata; Disease symptoms of 30-day-old wild type (WT) and OE-4 strains at 5 days after inoculation with Alternaria alternata; Scale bar = 1 cm;

[0038] Figure 4 AtPDA1.2A gene rapidly responds to Alternaria treatment; AtEF1α is an internal reference gene; error bars represent mean ± SD (n = 3, t test, *P < 0.05 and **P < 0.01);

[0039] Figure 5 To analyze the effect of PgERF2 on the promoter activity of the gene AtPDF1.2A for Dual-LUC experiments;

[0040] Figure 6 EMSA experiments showed that PgERF2 binds to the GCC-box element. DETAILED DESCRIPTION

[0041] Example 1 Construction of gene PgERF2 overexpression vector

[0042] 1.1 PCR amplification of target fragments

[0043] PCR amplification was performed using PrimeSTAR Max DNA Polymerase from Takara. Primers were designed using Primer Premier 5.0 software and synthesized by Beijing Qingke Biotechnology Co., Ltd. The primer sequences are as follows: Primer Sequence (5′→3′)

[0044] PC1301s-PgERF2-F: agctttcgcgagctcggtaccATGATTAAAGAAAATTGCAGT (SEQ IDNO.3);

[0045] PC1301s-PgERF2-R: caggtcgactctagaggatccCTAGTTCATCGATCGAGGAA (SEQ IDNO.4);

[0046] The cDNA template was obtained by reverse transcription of total RNA from ginseng callus tissue. RNA extraction and reverse transcription kits were: High-Purity Total RNA Rapid Extraction Kit (Beijing Biotech Biotechnology Co., Ltd.); RP1202; Trizol (Vazyme, catalog number R401-01); and HiScript II 1st Strand cDNA Synthesis Kit (+gDNAwiper); R212-01.

[0047] The reaction system is as follows:

[0048] Table 1 PCR amplification system

[0049]

[0050]

[0051] The PCR reaction program was set up as follows:

[0052] Table 2 PCR reaction procedure

[0053]

[0054] After the reaction is terminated, the liquid on the wall of the test tube is collected by instant centrifugation and then placed on ice for the next agarose gel purification step or temporarily stored in a -20°C refrigerator.

[0055] 1.2 PCR product purification and expression vector linearization

[0056] PCR products were subjected to gel electrophoresis to remove nonspecific amplification bands, and the target DNA fragment was purified using a gel recovery kit from Vazyme. The PCR product was mixed with 6x loading buffer and electrophoresed on an agarose gel for 30 minutes. Under ultraviolet light, the region of the agarose gel containing the target DNA fragment was precisely cut and placed in a clean 1.5ml centrifuge tube. The gel was then weighed, and subsequent steps were performed according to the kit's instructions. The resulting DNA solution, the PgERF2 fragment, was then tested for concentration and quality using an ultramicrospectrophotometer and stored in a -20°C refrigerator.

[0057] After the expression vector was cut with BamHI and KpnⅠ restriction enzymes, the same gel recovery experiment was performed to obtain the linearized vector. The gel recovery experiment procedure was the same as above. The construction method of the pCAMBIA1301s-GFP plasmid was referenced from the prior art (JIANG T, ZHANG Y, ZUO GG, et al. Transcription factor PgNAC72 activates DAMMARENEDIOL SYNTHASE expression to promote ginseng saponin biosynthesis [J]. Plant Physiology, 2024, 195(4): 2952-2969.).

[0058] The enzyme digestion system is as follows:

[0059] Table 3 Endonuclease digestion reaction system

[0060]

[0061] 1.3 Vector ligation and E. coli transformation

[0062] The homologous recombination reaction is as follows:

[0063] Table 4 Homologous recombination reaction system

[0064]

[0065] After gently pipetting and mixing using a micropipette, place the sample in a PCR instrument and incubate at 37°C for 30 minutes. After the reaction is complete, remove the sample immediately and place it on ice before performing the E. coli transformation experiment. The specific steps are as follows:

[0066] (1) Take out a tube of DH5α competent cells stored at -80°C, quickly transfer it to ice, and thaw it in ice for 10 minutes. Then, add 10 μL of the ligation product to the competent cells, mix well, and let it stand on ice for 15 minutes.

[0067] (2) Place the centrifuge tube in step (1) in a 42°C water bath for 1 minute of heat shock treatment, then quickly move to ice and place in an ice bath for 2 minutes.

[0068] (3) Add 700 μL of LB liquid medium without resistance to the centrifuge tube, shake gently to mix, and then perform shaking culture at 37°C and 200 rpm for 60 minutes.

[0069] (4) Centrifuge at 1,0000 × g for 1 minute to collect the cells. Remove 700 μL of supernatant and resuspend the cells in approximately 100 μL of culture medium. After thorough pipetting and mixing, inoculate the culture onto LB solid medium containing 100 mg / L kanamycin (Kan). Spread the culture evenly using a spreader and incubate in a 37°C incubator for 12 hours.

[0070] The LB medium formula is as follows (volume 1 L):

[0071] Table 5 LB culture medium formula

[0072]

[0073] To prepare LB solid medium, add 15g / L agar powder to the liquid medium and then autoclave at 121°C for 15 minutes. After sterilization, add the appropriate amount of antibiotic (kanamycin) when the autoclaved medium cools to approximately 60°C. This medium can be stored at room temperature for one week.

[0074] 1.5 Identification of positive clones

[0075] Select four single colonies of appropriate size and inoculate them into 200 μL of LB liquid medium containing 100 mg / L Kan. Incubate at 37°C and 200 rpm for 4 hours, shaking. Then, take an appropriate amount of the bacterial solution for PCR identification. The reaction system is as follows:

[0076] Table 6 Bacterial liquid PCR reaction system

[0077]

[0078] The PCR reaction procedure is as follows:

[0079] Table 7 Bacterial liquid PCR reaction program

[0080]

[0081] PCR products were analyzed by agarose gel electrophoresis to verify that the resulting bands were single and of the expected size. If the PCR product was identified correctly, a sample of the corresponding bacterial culture was sent to Beijing Qingke Biotechnology Co., Ltd. for DNA sequencing. After sequencing results were confirmed, the remaining bacterial culture was expanded. Subsequently, 300 μL of the fresh bacterial culture was mixed with 700 μL of sterile glycerol, snap-frozen in liquid nitrogen, and stored at -80°C.

[0082] 1.6 Plasmid extraction

[0083] Adopt Beijing Qingke Biotechnology Co., Ltd. Plasmid Mini Kit is used for small-scale plasmid DNA extraction. All steps are performed at room temperature:

[0084] (1) Take 4 mL of overnight culture and centrifuge at 12,000 × g for 1 min to collect the cells and discard the supernatant as much as possible;

[0085] (2) Add 250 μL of Buffer PA containing RNase A to the bacteria and mix thoroughly by pipetting until no obvious bacterial clumps remain;

[0086] (3) Add 250 μL of Buffer PB and gently invert the tube 6-8 times to fully lyse the cells.

[0087] (4) Add 350 μL of Buffer PC and gently invert 6-8 times to mix thoroughly. Centrifuge at 12,000 rpm for 10 min.

[0088] (5) Transfer the supernatant to the adsorption column, taking care not to remove the precipitate. Centrifuge at 12,000 rpm for 1 min, discard the filtrate, and return the adsorption column to the collection tube.

[0089] (6) Add 600 μL of Buffer PW containing anhydrous ethanol along the wall of the adsorption column, centrifuge at 12,000 rpm for 1 min, discard the waste liquid, and repeat this step once;

[0090] (7) Place the adsorption column back into the collection tube and centrifuge the empty tube at 12,000 rpm for 2 min;

[0091] (8) Place the adsorption column in a new, clean 1.5 mL centrifuge tube, open the tube cap, and leave at room temperature for 2 min to evaporate any residual ethanol.

[0092] (9) Add 35-50 μL of Elution Buffer preheated to 60°C to the center of the adsorption membrane, let it stand at room temperature for 2 minutes, and centrifuge at 12,000 rpm for 2 minutes. Store the resulting plasmid solution in a -20°C refrigerator.

[0093] Example 2 Construction of transgenic Arabidopsis thaliana overexpressing PgERF2

[0094] 2.1 Preparation of chemically competent Agrobacterium cells

[0095] (1) Take out the GV3101 strain (purchased from Shanghai Weidi Biotechnology Co., Ltd.) from the -80°C freezer and place it in liquid nitrogen for rapid freezing protection. Then, use an inoculation loop to take an appropriate amount of bacterial liquid and spread it on YEB solid medium supplemented with Rif (50 μg / mL) to recover the strain. Then, invert and culture it in a constant temperature incubator at 28°C for 36 hours. The following is the formula of YEB medium:

[0096] Table 8 YEB culture medium formula

[0097]

[0098] To prepare YEB solid medium, add 15g / L agar powder according to the recipe and autoclave at 121°C for 15 minutes. Add antibiotics after cooling the medium to approximately 50°C. The medium can be stored at room temperature for one week.

[0099] (2) Select a single colony of appropriate size and inoculate it into 5 mL of YEB liquid medium containing Rif (50 μg / mL), and culture it at 28°C and 200 rpm with shaking overnight.

[0100] (3) Inoculate 50 μL of overnight cultured bacteria into 50 mL of fresh YEB liquid medium and culture at 28°C and 200 rpm until the OD value of the bacterial solution reaches 0. 600 The value reached about 0.6 to 0.8. Subsequently, the bacterial solution was transferred to a 50 mL sterile centrifuge tube and placed in an ice bath for 30 minutes.

[0101] (4) Place the centrifuge tube in a 4°C pre-cooled centrifuge and centrifuge at 4,000 rpm for 10 minutes.

[0102] (5) Remove the supernatant and add 10 mL of pre-cooled 0.15 M NaCl solution to the precipitate. Gently pipette on ice to resuspend the bacterial suspension, and then centrifuge at 4°C, 4,000 rpm for 10 minutes.

[0103] (6) After removing the supernatant, add 1 mL of pre-chilled 20 mM CaCl2 solution to the pellet and gently pipette on ice to resuspend the bacterial solution. The bacterial solution is then divided into pre-chilled 1.5 mL sterile centrifuge tubes, rapidly frozen with liquid nitrogen, and stored at -80°C until ready for use. This yields GV3101 competent cells.

[0104] 2.2 Transformation of Agrobacterium competent cells

[0105] (1) Take out a tube of GV3101 competent cells from a -80°C freezer and thaw on ice for 10 minutes. In a clean bench, add 2 μL of plasmid to the bacterial suspension and gently pipette to mix. Then, let it stand on ice for 30 minutes.

[0106] (2) Quickly freeze in liquid nitrogen for 2-5 minutes, then heat shock in a 37°C water bath for 5 minutes. In a clean bench, add 900 μL of YEB liquid culture medium and slowly shake at 28°C, 100 rpm to resuscitate the cells for 4-6 hours.

[0107] (3) Centrifuge at 8,000 rpm for 1 minute, remove 800 μL of supernatant, and resuspend the cells in the remaining culture medium. Spread the resuspended cells evenly on the surface of YRK (YEB containing 50 mg / L Rif and 50 mg / L Kan) solid culture medium and incubate inverted in a 28°C incubator for 2 days.

[0108] (4) Single colonies were picked for bacterial liquid PCR identification, using the same method as in Example 1. Positive colonies were selected for expansion culture and then used for the next step of genetic transformation or bacterial strain preservation.

[0109] 2.3 Obtaining transgenic Arabidopsis

[0110] Pick a single positive colony and inoculate it into 3 ml of YEB liquid medium containing the selected antibiotic. Incubate at 28°C and 200 rpm for 12 hours. Then, transfer 300 μL of the bacterial solution into 100 ml of YRK liquid medium and continue to expand the culture until the OD600 value reaches 0.8. At this point, the bacterial solution can be used for genetic transformation experiments in Arabidopsis thaliana.

[0111] Using Zhang et al.[6] Using the proposed floral transformation method, the PgERF2 overexpression vector was transformed into the genome of wild-type Arabidopsis Col-0 plants. The transformed plants were then incubated in an incubator (23°C, 16-hour light / 8-hour dark cycle, 10,000 lux) until T0-generation transgenic Arabidopsis seeds were harvested. After hygromycin selection and PCR identification, T1-generation transgenic Arabidopsis seedlings were successfully obtained. These transgenic plants will be used for subsequent transcriptomic analysis.

[0112] 2.4 PCR identification of transgenic tobacco

[0113] (1) Extraction of transgenic tobacco genomic DNA

[0114] Transgenic tobacco gDNA was extracted using the Steady Pure Plant Genomic DNA Extraction Kit from Acryl. For detailed experimental steps, please refer to the kit instructions.

[0115] (2) PCR verification, the primer sequences are as follows:

[0116] Primer Sequence (5′→3′)

[0117] Hyg-F:ACACTACATGGCGTGATTTCAT(SEQ ID NO.5)

[0118] Hyg-R:TCCACTATCGGCGAGTACTTCT(SEQ ID NO.6)

[0119] Table 9 PCR identification reaction system

[0120]

[0121] The reaction conditions are the same as in Example 1.

[0122] The PCR products were collected and subjected to agarose gel electrophoresis, and images were taken using a ChemiDoc XRS gel imager from Bio Rad.

[0123] The results are as follows Figure 1 As shown in A, genomic DNA was amplified by PCR using specific primers containing the selection marker hygromycin resistance gene. The results showed that the expected amplified product of 550 bp was obtained in the transgenic positive plants OE-1, OE-4, and OE-5, while the corresponding fragment was not detected in the wild-type plants.

[0124] Example 3 Detection of PgERF2 gene expression in transgenic lines

[0125] 3.1 Total RNA extraction from Arabidopsis leaf tissue

[0126] (1) Add 1 ml of Trizol to an RNase-free centrifuge tube and place on ice. Grind the sample into a powder using a mortar and pestle. Continuously add liquid nitrogen to the mortar. Add 50 mg of the powder to the centrifuge tube and mix thoroughly using a vortexer. Let stand at room temperature for 5 minutes.

[0127] (2) Centrifuge at 12,000 × g for 5 min at 4°C. Carefully pipette 950 μL of the supernatant into a new RNase-free centrifuge tube. Add chloroform (1 / 5 the volume of Trizol) and mix thoroughly. Let stand at room temperature for 5 min.

[0128] (3) Centrifuge at 12,000 × g for 10 min at 4°C. The homogenate will separate into three layers: the supernatant containing RNA, the middle protein layer, and the lower organic phase.

[0129] (4) Transfer 450 μl of supernatant to another new RNase-free centrifuge tube (do not aspirate the intermediate protein layer); add 450 μl of isopropanol equal to the volume of Trizol, mix thoroughly, and let stand at room temperature for 10 min;

[0130] (5) The mixture was transferred to an adsorption column RA, centrifuged at 13,000 rpm for 2 min, and the filtrate was discarded;

[0131] (6) Add 500 μL of deproteinized solution RW1, let stand at room temperature for 3 min, centrifuge at 13,000 rpm for 30 s, and discard the filtrate;

[0132] (7) Add 500 μL of rinsing solution RW pre-added with anhydrous ethanol, centrifuge at 13,000 rpm for 30 s, and discard the filtrate; repeat this operation once;

[0133] (8) Place the adsorption column back into the empty collection tube and centrifuge at 13,000 rpm for 2 minutes;

[0134] (9) Place the adsorption column in a new RNase-free centrifuge tube, open the lid and let it stand at room temperature for 2 minutes to evaporate the residual ethanol; add 30 μL of RNase-free water preheated to 70-90°C to the adsorption membrane, let it stand at room temperature for 2 minutes, and centrifuge at 12,000 rpm for 1 minute.

[0135] After the RNA is collected, 2 μL is used for agarose gel electrophoresis to assess the integrity of the RNA. The remaining RNA sample should be stored in a -80°C refrigerator for subsequent reverse transcription or other uses.

[0136] 3.2 Reverse transcription synthesis of first-strand cDNA

[0137] (1) Removal of genomic DNA

[0138] In an RNase-free 0.2 mL PCR tube, add the following reagents in order:

[0139] Table 10 Reverse transcription reaction system

[0140]

[0141] After mixing the above reagents, incubate the sample at 42°C for 2 minutes in a PCR instrument, then remove it and immediately place it on ice for subsequent use.

[0142] (2) Synthesis of the first strand of cDNA

[0143] Add 4 μL of 5× HiScript II Enzyme Mix to the PCR tube containing the above reaction solution and perform the following reaction in a PCR instrument: 50°C for 15 minutes; 85°C for 5 seconds. After the reaction is complete, dilute the synthesized cDNA sample 10-fold and store in a -20°C freezer.

[0144] 3.3 RT-qPCR analysis

[0145] RT-qPCR analysis was performed using the kit Novozymes ChamQ Universal SYBR qPCR Master Mix (Q711-02) using the following system:

[0146] Table 11 RT-qPCR reaction system

[0147]

[0148] The amplification procedure is as follows:

[0149] Table 12 RT-qPCR reaction procedure

[0150]

[0151]

[0152] PgACT was used as the internal reference gene, and each sample was tested in triplicate. -ΔΔCt This method was used to measure the relative differences in gene transcription levels and to analyze and compare the expression levels of individual genes accordingly.

[0153] The primer sequences used are as follows:

[0154] Primer Sequence (5′→3′)

[0155] qRT-PgERF2-F: CTTCGTCATCAATATCGTCGTC (SEQ ID NO.7)

[0156] qRT-PgERF2-R: CCACTTCTACACTCCCCATCC (SEQ ID NO.8)

[0157] The results are as follows Figure 1 As shown in Figure B, RT-qPCR analysis showed that the expression level of PgERF2 in transgenic plants was significantly higher than that in the wild type. In OE-1, OE-4, and OE-5 transgenic plants, the expression levels of PgERF2 were increased by 164-fold, 1100-fold, and 587-fold, respectively, compared to the wild type. These data confirmed that the PgERF2 gene had been successfully integrated and overexpressed in Arabidopsis.

[0158] Example 4 Transcriptomic Analysis of Transgenic Arabidopsis Overexpressing PgERF2

[0159] 4.1 Library construction and sequencing

[0160] Leaf tissue samples of transgenic line OE-4 and wild-type control (WT) Arabidopsis were collected. These samples were from three independent biological replicates and used for subsequent RNA extraction (RNA extraction procedures were the same as above). The total RNA of the extracted samples was quality tested and analyzed using a NanoDrop micro-UV spectrophotometer and an Agilent 2100 bioanalyzer. Subsequently, cDNA libraries were constructed using 3 μg of total RNA from each sample. These libraries were sequenced using the MGISEQ-T7 sequencing platform using a PE150 read length. To obtain clean reads suitable for subsequent analysis, adapter sequences and low-quality reads were removed. Finally, the clean reads were aligned with the tobacco reference genome sequence using HISAT2 software.

[0161] 4.2 RNA-seq data analysis

[0162] FeatureCounts software was used to calculate the number of fragments per kilobase of transcript per million (FPKM) of each gene in each sample. Differential gene expression between samples was analyzed using DESeq2 software, with the criteria for differentially expressed genes set as an absolute log2FC value >1 and an adjusted P value <0.05. The detected differentially expressed genes (DEGs) were compared with the GO database for each category, and the number of genes in each category was counted. Subsequently, Fisher's exact test was used to assess the significance of protein enrichment in specific GO functional terms, with a threshold q value of ≤0.05 to identify significantly enriched functional annotation GO terms. Furthermore, functional annotation and significant enrichment analysis of DEGs were performed using the KEGG public database, with a threshold q value of ≤0.05.

[0163] The results are as follows Figure 2 As shown in the figure, GO enrichment analysis of differentially expressed genes revealed that the upregulated genes mainly focused on response to stimulus, response to biotic stimulus, response to external bioticstimulus, response to other organisms, and response to stress in biological processes ( Figure 2 A). These results suggest that PgERF2 may play an important role in regulating the response of Arabidopsis to biotic stress. In contrast, the down-regulated genes were mainly concentrated in the cellular response to hypoxia, the cellular response to decreased oxygen levels, the cellular response to oxygen levels, the response to hypoxia, and the response to decreased oxygen levels ( Figure 2 B).

[0164] Example 5 Alternaria infection experiment

[0165] In a clean bench, Alternaria alternata was inoculated onto potato dextrose medium and sealed with parafilm. Subsequently, it was placed in an incubator at 28°C for inverted culture. Culture was continued in the dark for about a week until the medium was completely covered with mycelium. Afterwards, a 0.05% Tween 80 solution was added to the medium, and mycelium was gently scraped off using a spore stick to prepare a spore suspension with a concentration of approximately 5 × 10 5 spores / mL. Four-week-old Arabidopsis leaves were selected. The petioles were placed on moistened cotton balls to maintain leaf moisture. Four symmetrical wounds were made on the leaves using a syringe needle, and 10 μL of spore suspension was inoculated into each wound. Images of the lesions were collected after 5 days.

[0166] For the RT-qPCR analysis of differentially expressed genes in response to biotic stress (RT-qPCR analysis steps were the same as above), 10-day-old Arabidopsis seedlings grown on MS medium were transferred into a centrifuge tube containing 1 mL of Alternaria spore suspension, and the spore concentration was set at 5 × 10 5 The cells were cultured at 48 h under gentle shaking conditions, and samples were collected at 0, 6, 12, 24, and 48 h.

[0167] like Figure 3 As shown, the overexpression lines showed milder disease symptoms compared with the wild type.

[0168] Example 6 RT-qPCR analysis of differentially expressed genes in response to biotic stress

[0169] To further identify downstream target genes of PgERF2, the present invention analyzed significantly upregulated genes enriched in the GO pathway "response tobiotic stimulus." This process identified the plant defense protein PDF1.2A (Protein Defense Protein 1.2A) as a candidate gene. One-week-old PgERF2-overexpressing strain OE-4 and wild-type WT Arabidopsis seedlings were exposed to a suspension of Alternaria alternata spores. The expression levels of the AtPDF1.2 gene were quantitatively analyzed after 0, 6, 12, 24, and 48 hours.

[0170] The experimental steps of Arabidopsis seedling RNA extraction, reverse transcription, RT-qPCR analysis, etc. were referred to Example 3.

[0171] The primer sequences are as follows:

[0172] Primer Sequence (5′→3′)

[0173] AtEF1α-F: CACCCTTGGTGTCAAGCAGATGA (SEQ ID NO.9)

[0174] AtEF1α-R:TTGTCTCCCTCGAATCCAGAGATTG (SEQ ID NO.10)

[0175] AtPDF1.2A-F:TTGCTGCTTTCGACGCAC(SEQ ID NO.11)

[0176] AtPDF1.2A-R:CCGCAAACCCCTGACCAT(SEQ ID NO.12)

[0177] The results are as follows Figure 4 The expression level of AtPDF1.2A in OE-4 cells was significantly higher than that in the wild-type cells before treatment (0 h). After treatment, OE-4 cells rapidly responded to Alternaria alternata treatment, with AtPDF1.2 expression levels increasing by more than 100-fold, while the wild-type cells experienced a more gradual increase. These experimental results further confirm that PgERF2 can enhance defense against pathogens by regulating the expression of the AtPDF1.2A gene.

[0178] Example 7 Dual luciferase reporter gene experiment

[0179] 7.1 Construction of reporter gene vector

[0180] The promoter sequence of AtPDF1.2A was searched in the ginseng genome database. BamHⅠ and KpnⅠ were selected as the restriction sites for the pGreenⅡ0800-LUC reporter vector and promoter fragment. Specific primers were designed to amplify the full-length 2000bp sequence of AtPDF1.2A-pro. The primers are named AtPDF1.2A-pro-F / R. The detailed information is shown below. The vector construction process refers to Example 1.

[0181] Primer Sequence (5′→3′)

[0182] AtPDF1.2A-pro-F: ctatagggcgaattgggtaccTCTTCATCAGTTCACTATAAATAGAGGTTG (SEQ ID NO.13)

[0183] AtPDF1.2A-pro-R: atcgataccgtcgacctcgagGATGATTATTACTATTTTGTTTTCAATGTATAGA (SEQ ID NO. 14) 7.2 Cultivation of tobacco

[0184] (1) Wild-type Nicotiana benthamiana seeds were placed in 1% sodium hypochlorite and sterilized by shaking for 10 minutes. The seeds were then rinsed six times with sterile water to remove residual sodium hypochlorite. The sterile seeds were transferred to solid MS medium and cultured at 28°C under 16 hours of light / 8 hours of darkness for 5-7 days until the seedling stage. The formula of MS medium is as follows:

[0185] Table 13 Tobacco seedling culture medium formula

[0186]

[0187]

[0188] Adjust the pH to 5.8, add 8 g / L agar powder, and sterilize by autoclaving at 121°C for 15 min.

[0189] (2) The tobacco seedlings were transferred to soil and continued to grow at 28°C, 16 hours light / 8 hours dark conditions until 3-4 strong leaves were grown.

[0190] 7.3 Activation and expansion of Agrobacterium GV3101

[0191] (1) The constructed recombinant Agrobacterium strain was inoculated into YRK liquid culture medium and cultured with shaking at 28°C and 200 rpm for approximately 36 hours.

[0192] (2) Pick a single colony and inoculate it into 3 mL of YRK liquid medium. Incubate the culture overnight at 28°C and 200 rpm with shaking.

[0193] (3) Take 200 μL of bacterial solution and add it to 5 mL of YRK medium containing 10 mM MES (pH = 5.6) and 200 μM AS. Cultivate at 28°C and 200 rpm until OD600 = about 1.5.

[0194] (4) Centrifuge at 5000 × g for 10 min, discard the supernatant, resuspend the cells with an equal volume of 10 mM MgCl2 solution, then add 200 μM AS and let it stand in the dark for 3 h.

[0195] 7.4 Tobacco transient transformation

[0196] The pCAMBIA1301s-PgERF2 effector vector constructed in Example 1 and the reporter gene vector constructed in step 7.1 were separately transformed into Agrobacterium GV3101. The transformation method is detailed in Example 2. Equal volumes of the two successfully validated Agrobacterium strains were mixed and co-infected with the epidermal cells beneath tobacco leaves by injection infiltration. Three tobacco leaves were infected in each sample group.

[0197] 7.5 Quantitative detection of dual luciferase

[0198] Firefly luciferase and Renilla luciferase were quantitatively detected using the TransGen Dual Luciferase Assay Kit. The specific steps are as follows:

[0199] (1) Sample collection began 2 days after injection. Samples were taken using a 2 cm inner diameter punch, avoiding the large vein area. Each sample was sampled once and quickly placed in a 1.5 mL centrifuge tube and quickly frozen using liquid nitrogen.

[0200] (2) Use an electric grinder to quickly crush the sample, and add 100 μL of protein extract (PBS buffer containing 1 mM DTT and pH 7.8) twice, homogenize, and centrifuge at 4°C and 12,000 rpm for 10 minutes. The supernatant is the crude enzyme extract.

[0201] (3) Collect the supernatant and The enzymatic activities of firefly luciferase and Renilla luciferase were determined according to the instructions of the Double-Luciferase Reporter Assay Kit (Beijing Quanshijin Biotechnology Co., Ltd.); then, the ratio of firefly luciferase to Renilla luciferase activities was calculated.

[0202] The results are as follows Figure 5 As shown in the results, overexpression of PgERF2 significantly enhanced the promoter activity of AtPgDF1.2A by about 3.4-fold. This result indicates that the PgERF2 transcription factor has a positive regulatory effect on the promoter of the AtPgDF1.2A gene and can significantly increase its transcriptional activity.

[0203] Example 8 Prokaryotic expression and purification of PgERF2 protein

[0204] 8.1 Construction of prokaryotic expression vector

[0205] By homologous recombination technology, the present invention successfully constructed the pCold / TF-PgERF2 prokaryotic expression vector using the pCold / TF vector and the target fragment of Example 1 to express the PgERF2 protein, and selected BamHI and KpnⅠ as the restriction endonuclease sites of the pCold / TF vector (purchased from Miaoling Biotechnology). The specific vector construction steps are shown in Example 1. The successfully constructed vector was transformed into BL21 competent cells and subsequently processed according to the screening and identification method described in Example 1 to obtain the BL21 / pCold / TF-PgERF2 expression strain. The primer sequences are as follows:

[0206] Primer Sequence (5′→3′)

[0207] pCold-TF-PgERF2-F: aggcatatggagctcggtaccATGATTAAAGAAAATTGCAGTTTTGAT (SEQ ID NO. 15)

[0208] pCold-TF-PgERF2-R: agcagagattacctatctagaCTAGTTCATCGATCGAGGAAAAGG (SEQ ID NO. 16)

[0209] 8.2 Inducible Expression of Recombinant Protein

[0210] (1) The correctly identified BL21 / pCold / TF-PgERF2 expression strain was inoculated into 4 mL of LB liquid medium (containing 50 mg / L Amp), and then cultured with shaking at 37°C and 200 rpm overnight.

[0211] (2) Transfer all of the above bacterial suspension to 200 mL of LB (containing 50 mg / L Amp) liquid culture medium for expansion culture. Cultivate under the same conditions for 2 hours until the OD600 value reaches 0.6-0.8.

[0212] (3) Add 200 μL of 1 M IPTG to induce the expression of PgERF2 protein, and then shake and induce at 37°C and 200 rpm for 4 hours.

[0213] (4) Transfer the induced bacterial solution to a 50 mL centrifuge tube and centrifuge at 4°C and 4,900 × g for 10 min. Discard the supernatant after multiple batch centrifugations to collect the bacterial cells.

[0214] (5) Resuspend the cells in 15 mL of pre-chilled PBS buffer, centrifuge at 4°C, 4,900 × g for 10 min, and discard the supernatant. Repeat this step once to collect the cells.

[0215] (6) Resuspend the cells by adding 2-5 mL of Lysis buffer per gram of wet weight, then add lysozyme (final concentration 1 mg / mL), mix well, and place on ice for 30 minutes.

[0216] (7) Use an ultrasonic cell disruptor on ice to lyse the cells. Set the power to 250 W, sonicate for 5 seconds, and cool for 5 seconds for a total of 5 minutes until the cells are fully lysed.

[0217] (8) Centrifuge at 4°C and 10,000 × g for 20 minutes and transfer the supernatant to a new centrifuge tube to obtain the crude protein extract.

[0218] 8.3 Purification and Concentration of Recombinant Protein

[0219] (1) Transfer 500 μL of Ni-NTA suspension (pre-mixed thoroughly, column bed volume 250 μL) into a 2 mL centrifuge tube, centrifuge briefly, and discard the supernatant.

[0220] (2) Add 1 mL of Lysis buffer to the Ni-NTA matrix for equilibrium, shake gently, centrifuge briefly, and discard the supernatant.

[0221] (3) Add the crude protein extract to the equilibrated Ni-NTA matrix, shake gently to mix, and then incubate at 4°C and 100 rpm for 60 minutes.

[0222] (4) Slowly add the mixture to the purification column, and after filling, open the bottom cap to drain the waste liquid.

[0223] (5) After the liquid flows out, slowly add 1 mL of pre-cooled wash buffer to the column to wash away the impurities. Repeat 8 times.

[0224] (6) Slowly add 1 mL of elution buffer to the column, collecting the eluate. Repeat 4 times.

[0225] (7) Transfer the eluate into a 5 mL ultrafiltration tube and centrifuge at 4000 × g for 10 minutes at 4°C.

[0226] (8) Discard the filtrate and add 4 mL of 20 mM Tris·HCl (pH = 7.2) to the ultrafiltration tube. Centrifuge at 4°C, 4,000 × g for 10 minutes. Discard the filtrate and repeat this step once.

[0227] (9) Add 2 mL of 20 mM Tris·HCl to the ultrafiltration tube and gently pipette to dissolve the protein on the membrane. Take 10 μL to measure the protein concentration. Dispense the remaining protein solution into new 1.5 mL tubes and store at -80°C.

[0228] 8.4 Preparation of buffers related to protein expression and purification experiments

[0229] In the process of preparing Lysis buffer, Wash buffer and Elution buffer, the required basic components are the same, but the content of imidazole in each buffer will be different:

[0230] Table 14 Formulas of buffers related to protein expression and purification experiments

[0231]

[0232] Amount of imidazole required for each buffer:

[0233] Table 15 Formulas of buffers related to protein expression and purification experiments (continued)

[0234]

[0235] The pH of the buffer was adjusted to 8.0 using 1 M NaOH and stored at room temperature.

[0236] Example 9 Gel mobility experiment

[0237] 9.1 Probe Labeling and Annealing

[0238] (1) The following probes were designed based on the GCC-box site sequence in the AtPgDF1.2A promoter:

[0239] Table 16 Probe names and sequences

[0240]

[0241] The predicted GCC-box and its mutant sequence are indicated by underlined sections. The probe was biotin-labeled at the 5′ end and purified by high-performance liquid chromatography. Probe synthesis, labeling, and purification were performed by Beijing Qingke Biotechnology Co., Ltd.

[0242] (2) Mix the forward and reverse strands in an equimolar ratio and dilute the probe to a final concentration of 10 μM using Tris buffer containing 10 mM Tris, 1 mM EDTA, and 50 mM NaCl. Anneal in a PCR instrument using the following protocol: 95°C for 5 min, followed by 1°C decrease per cycle for 70 cycles, each lasting 1 min. After completion, store the probe at −20°C.

[0243] 9.2 Gel shift retardation assay

[0244] (1) Prepare a 6% TBE gel according to the recipe, taking special care to prevent bubble formation. After the gel solidifies, rinse the sample wells with 0.5× TBE solution and perform pre-electrophoresis at 100 V for 30 minutes using 0.5× TBE running buffer.

[0245] (2) Under low temperature conditions, prepare the protein-probe binding reaction system according to the following formula (volume unit is μL):

[0246] Table 17 EMSA experimental reaction system

[0247]

[0248]

[0249] X represents the TF-tagged protein, and Y represents the PgERF2-TF fusion protein. Add each reagent one by one in the specified order, mixing thoroughly before adding the labeled probe. Then, let the mixture stand at room temperature for 10 minutes to eliminate any nonspecific binding between the probe and the protein, or to prioritize the reaction of the cold probe. Then, add the labeled probe, mix thoroughly, and let it stand at room temperature for 20 minutes.

[0250] (3) Electrophoresis: After the binding reaction is complete, add 1 μl of EMSA / Gel-Shift loading buffer (colorless, 10×), mix well, and immediately load the sample. Use 0.5× TBE as the electrophoresis buffer and set the voltage to 100 V. Stop electrophoresis when the indicator reaches 3 / 4 of the gel.

[0251] (4) Transfer: Soak the nylon membrane in 0.5× TBE for at least 10 minutes. Arrange the membrane in the order of cathode plate, sponge, filter paper, gel, membrane, filter paper, sponge, and anode plate, using a glass rod to remove air bubbles. The transfer was performed in an ice bath with a current of 380 mA for 1 hour.

[0252] (5) UV crosslinking: Lay the membrane flat on clean filter paper (make sure the side containing bromophenol blue faces upwards), then quickly transfer it to a UV light source, keep a distance of 10 cm from the UV lamp, and irradiate for 15 minutes.

[0253] (6) Blocking: Select an appropriate container, add 15 mL of blocking solution, and place the UV-crosslinked nylon membrane. Then, incubate on a horizontal shaker at a slow speed for 15 minutes.

[0254] (7) Hybridization: Add 7.5 μl of Streptavidin-HRP Conjugate (1:2000 dilution) to 15 mL of blocking buffer and mix thoroughly. Remove the blocking buffer from the nylon membrane and replace it with new blocking buffer containing Streptavidin-HRP Conjugate. Incubate slowly on a horizontal shaker for 15 minutes.

[0255] (8) Rinse: Mix 25 ml of washing solution (5×) with 100 ml of pure water to make 125 ml of washing solution. Transfer the nylon membrane to a container containing 15 ml of washing solution and rinse for 1 minute. After changing the washing solution, repeat three times for 5 minutes each time.

[0256] (9) Equilibration: Transfer the nylon membrane to a container containing 20 ml of detection equilibration solution and incubate slowly on a horizontal shaker for 5 minutes.

[0257] (10) Luminescence examination: Mix 5 ml of BeyoECL Moon A solution and 5 ml of BeyoECL Moon B solution to prepare the working solution. Remove the nylon membrane, remove the excess liquid, and evenly coat it with the substrate working solution. Let it stand at room temperature for 3 minutes. Remove the excess substrate from the side of the membrane by aspirating it to prevent it from drying out. Then, place it in a chemiluminescence imager for development.

[0258] 9.3 Buffers and reagents required for EMSA experiments

[0259] (1) 5×TBE formula:

[0260] Table 18 TBE buffer formula

[0261]

[0262]

[0263] Adjust the pH of the solution to 8.3 and dilute to 0.5× with deionized water before use;

[0264] (2) 6% non-denaturing polyacrylamide gel formula:

[0265] Table 19 EMSA non-denaturing polyacrylamide gel formula

[0266]

[0267] (3) Other buffers were from the chemiluminescence EMSA kit (GS009) of Beyotime.

[0268] The results are as follows Figure 6 As shown, EMSA further investigated the in vitro binding relationship between PgERF2 and the cis-acting GCC-box element. A biotin-labeled GCC-box element was designed as a probe, and EMSA experiments were performed in a triple tandem format to verify PgERF2 binding to this site. Compared to the empty TF protein vector, the TF-PgERF2 fusion protein bound to the biotin-labeled probe. The resulting DNA-protein complex resulted in a slower electrophoretic migration velocity, manifesting as a hysteresis band. Competition experiments using 50- and 200-fold concentrations of the unlabeled competitive probe and a 200-fold concentration of the mutant competitive probe revealed a gradual weakening and eventual disappearance of the hysteresis band with increasing concentrations of the unlabeled competitive probe. In contrast, introduction of the mutant probe significantly reduced protein-probe binding, reflecting the maintenance of specific binding. These results confirm that PgERF2 specifically binds to the GCC-box site in the AtPDF1.2A promoter.

[0269] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. Use of ginseng transcription factor PgERF2 in improving plant resistance to Alternaria alternata, characterized in that: The sequence of the ginseng transcription factor PgERF2 gene is shown in SEQ ID NO. 2; the plant is Arabidopsis thaliana or ginseng.

2. The application of ginseng transcription factor PgERF2 in increasing the expression level of genes related to plant resistance to Alternaria alternata, characterized in that: The sequence of the PgERF2 gene is shown in SEQ ID NO.2; the gene related to the plant's resistance to Alternaria is AtPDF1.2A; and the plant is Arabidopsis thaliana or ginseng.

3. The use of ginseng transcription factor PgERF2 in the preparation of a reagent for positively regulating the AtPgDF1.2A gene promoter, characterized in that: The sequence of the PgERF2 gene is shown in SEQ ID NO.

2.

4. Preparation and characterization of ginseng transcription factor PgERF2 protein AtPDF1.2A The invention relates to a GCC-box site-specific binding reagent in a promoter, characterized in that: The sequence of the PgERF2 protein is shown in SEQ ID NO.

1.

5. A plasmid, characterized in that The plasmid overexpresses the PgERF2 gene; the sequence of the PgERF2 gene is shown as SEQ ID NO.

2.

6. An Agrobacterium competent cell, characterized in that The Agrobacterium competent cells overexpress the PgERF2 gene; the Agrobacterium competent cells are obtained by transforming the plasmid into DH5α competent cells; the sequence of the PgERF2 gene is shown in SEQ ID NO.

2.

7. Use of the plasmid according to claim 5 or the Agrobacterium competent cell according to claim 6 in enhancing resistance of a plant to Alternaria alternata; the plant is Arabidopsis thaliana or ginseng.

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