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

Abstract

The invention belongs to the field of biological gene engineering, and relates to application of a ginseng transcription factor PgERF2 in improvement of plant resistance to alternaria alternata, and the sequence of the ginseng transcription factor PgERF2 protein is as shown in SEQ ID NO.1; the sequence of the ginseng transcription factor PgERF2 gene is as shown in SEQ ID NO. 2. According to the invention, the PgERF2 gene is cloned, a corresponding plant expression vector is constructed, and the gene is successfully transferred into wild type arabidopsis thaliana. A transcriptomics analysis result shows that the response to biological stress in a transgenic plant is activated, the expression level of the plant defense protein related gene AtPDF1.2 A is remarkably improved, and it is proved that PgERF2 can be directly combined with a GCC-box element to activate the expression of the plant defense protein related gene AtPDF1.2 A, so that the resistance to alternaria alternata is remarkably enhanced.

Description

Technical Field

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

[0002] Ginseng saponins accumulate in large amounts in the roots of ginseng, 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 an important role in resisting biotic stresses such as pathogenic fungi. In Arabidopsis, the stability of ERF6 protein in vivo is enhanced by phosphorylation by MPK3 / MPK6. Phosphorylated ERF6 can continuously activate defense-related genes, especially those involved in fungal resistance, such as PDF1.1 and PDF1.2, thereby improving plant resistance to Botrytis cinerea. In soybean, overexpression of GmERF113 can increase the expression levels of disease resistance genes GmPR1 and GmPR10-1, thereby 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, resulting in significant losses to the national economy every year. Summary of the invention

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

[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 discloses an application of ginseng transcription factor PgERF2 in preparing a reagent for positively regulating the promoter of 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 digesting the vector with enzymes to obtain a digested vector, and then connecting the PgERF2 gene to the 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] A competent Agrobacterium cell overexpressing a 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 cell is used for enhancing the resistance of plants to Alternaria alternata, and the Agrobacterium competent cell overexpresses 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 a 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 invention clones the PgERF2 gene and constructs a corresponding plant expression vector. The gene is successfully transferred into wild-type Arabidopsis thaliana by Agrobacterium-mediated floral transformation. PCR technology is used to identify the genomic DNA and transcribed mRNA levels to obtain positive Arabidopsis thaliana plants that stably overexpress the PgERF2 gene. The transcriptomic analysis results show that the response to biological stress in the transgenic plants is activated, and the expression level of the plant defense protein-related gene AtPDF1.2A is significantly increased.

[0032] The present invention verifies through dual-luciferase reporter gene assay (Dual-LUC) and electrophoresis 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 show that the PgERF2 transcription factor in ginseng can specifically bind to the promoter region of the Arabidopsis AtPDF1.2A gene, thereby enhancing the transcriptional activity of the AtPDF1.2A gene. As a key gene for plant defense, the increase in the expression level of AtPDF1.2A significantly enhances the resistance of plants to pathogenic fungi. In addition, the ginseng PgERF2 transcription factor, its encoding gene, and the overexpression recombinant vector containing the gene involved in the present invention can all be applied to the genetic transformation of economic crops, such as rice, cotton, etc., providing a reliable gene resource and theoretical basis for molecular breeding of plant resistance to adversity. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Identification of PgERF2 transgenic Arabidopsis plants; A is PCR identification of hygromycin resistance gene (550bp) in transgenic Arabidopsis leaf tissue; Lane 1: DNA Marker 2000; Lane 2: Negative control: WT genomic DNA; Lanes 3, 4, 5: genomic DNA of transgenic lines (OE-1, OE-4, OE-5); Lane 5: Positive control: empty vector plasmid; B is RT-qPCR verification of the expression level of PgERF2 in transgenic Arabidopsis; AtEF1α is an internal reference gene, and the 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 gene AtPDF1.2A for Dual-LUC experiment;

[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 fragment

[0043] PrimeSTAR Max DNA Polymerase produced by Takara was used for PCR amplification. In addition, the primers were designed by 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. The kits for RNA extraction and reverse transcription were: High-purity total RNA rapid extraction kit: Beijing Biotech Biotechnology Co., Ltd.; RP1202; Trizol: Vazyme catalog number: R401-01; reverse transcription kit: 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 program

[0053]

[0054] After the reaction was terminated, the liquid on the wall of the test tube was 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] The PCR product was subjected to gel electrophoresis to remove non-specific amplification bands, and the target DNA fragment was purified using a gel recovery kit from Vazyme. The PCR product was mixed with 6 times the loading buffer and electrophoresed on an agarose gel for 30 minutes. Under ultraviolet light, the agarose gel area containing the target size DNA fragment was accurately cut and placed in a clean 1.5 ml centrifuge tube. The gel was then weighed, and the subsequent steps were performed according to the instructions for use of the kit. The final DNA solution, i.e., the PgERF2 fragment, was obtained. The concentration and quality were tested by an ultra-micro spectrophotometer and then stored in a -20°C refrigerator.

[0057] After the expression vector was cut with BamHI and KpnⅠ restriction endonucleases, the same gel recovery experiment was performed to obtain a linearized vector. The gel recovery experiment operation steps were the same as above. The construction method of pCAMBIA1301s-GFP plasmid refers to 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 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 mixing with a micropipette, place the sample in a PCR instrument and set it to 37°C for incubation for 30 minutes. After the reaction is completed, take it out immediately and place it on ice, and then perform the E. coli transformation experiment. The specific steps are as follows:

[0066] (1) Take out a DH5α competent cell stored in a -80°C refrigerator, quickly transfer it to ice, and thaw it in ice for 10 minutes. Then, add 10 μL of the ligation product to the competent cell, mix well, and then 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 on ice 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 the supernatant, and resuspend the cells in the remaining approximately 100 μL of culture medium. After thorough pipetting and mixing, inoculate the culture medium onto LB solid culture medium containing 100 mg / L kanamycin (Kan), spread evenly using a spreader, and incubate in a 37°C constant temperature incubator for 12 hours.

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

[0071] Table 5 LB medium formula

[0072]

[0073] To prepare LB solid medium, add 15g / L agar powder to the liquid medium, and then sterilize it at 121℃ for 15 minutes. After sterilization, add an appropriate amount of antibiotics (kanamycin) when the medium after autoclaving cools to about 60℃. The medium can be stored for one week at room temperature.

[0074] 1.5 Identification of positive clones

[0075] Select 4 single colonies of appropriate size and inoculate them into 200 μL LB liquid culture medium containing 100 mg / L Kan. Perform shaking culture at 37°C and 200 rpm for 4 hours, and then take an appropriate amount of bacterial liquid for PCR identification. The configured 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] The PCR products were analyzed by agarose gel electrophoresis to verify whether the obtained bands were single and whether their sizes were consistent with expectations. If the identification results of the PCR products were correct, the corresponding bacterial samples were sent to Beijing Qingke Biotechnology Co., Ltd. for DNA sequencing. After the sequencing results were correct, the remaining bacterial solution was expanded and cultured. Subsequently, 300 μL of fresh bacterial solution was taken and mixed with 700 μL of sterile glycerol, quickly frozen in liquid nitrogen, and stored in a -80°C refrigerator.

[0082] 1.6 Plasmid extraction

[0083] Adopt Beijing Qingke Biotechnology Co., Ltd. Plasmid Mini Kit is used to extract plasmid DNA. 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 by pipetting until no obvious bacterial clumps remain;

[0086] (3) Add 250 μL of Buffer PB and gently invert 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, being careful not to aspirate 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 the residual ethanol;

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

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

[0094] 2.1 Preparation of Agrobacterium chemically competent cells

[0095] (1) Take out the GV3101 strain (purchased from Shanghai Weidi Biotechnology Co., Ltd.) from the -80℃ 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 the YEB solid medium supplemented with Rif (50μg / mL) to recover the strain. After that, invert and culture it in a constant temperature incubator at 28℃ for 36 hours. The following is the formula of YEB medium:

[0096] Table 8 YEB medium formula

[0097]

[0098] When preparing YEB solid medium, add 15g / L agar powder according to the recipe and sterilize by autoclave at 121℃ for 15 minutes. Add antibiotics after the medium is cooled to about 50℃ and can be stored at room temperature for one week.

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

[0100] (3) Inoculate 50 μL of the overnight culture into 50 mL of fresh YEB liquid medium and culture at 28°C and 200 rpm until the OD of the culture 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 precooled 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 solution, and then centrifuge at 4°C, 4,000 rpm for 10 min.

[0103] (6) After removing the supernatant, add 1 mL of pre-cooled 20 mM CaCl2 solution to the precipitate and gently pipette on ice to resuspend the bacterial solution. Then, divide the bacterial solution into pre-cooled 1.5 mL sterile centrifuge tubes, quickly freeze them with liquid nitrogen, and store them at -80°C for future use to obtain GV3101 competent cells.

[0104] 2.2 Transformation of Agrobacterium competent cells

[0105] (1) Take out a GV3101 competent cell from the -80℃ freezer and thaw it on ice for 10 minutes. In a clean bench, add 2 μL of plasmid to the bacterial solution and pipette gently to mix it, 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 an ultra-clean workbench, add 900 μL YEB liquid culture medium, then slowly shake at 28°C, 100 rpm to revive the cells for 4-6 hours.

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

[0108] (4) Pick a single colony for bacterial liquid PCR identification, the method is the same as Example 1, select the positive colony for expansion culture, and then carry out the next step of genetic transformation or strain preservation.

[0109] 2.3 Obtaining transgenic Arabidopsis

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

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

[0112] 2.4 PCR identification of transgenic tobacco

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

[0114] The 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 manual.

[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 for agarose gel electrophoresis, and photos were taken using the ChemiDoc XRS gel imager from Bio Rad.

[0123] The results are as follows Figure 1 As shown in A, the genomic DNA was amplified by PCR using specific primers containing the selection marker hygromycin resistance gene. The results showed that the expected amplification 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 it on ice. Grind the sample into powder using a mortar and pestle. During this process, liquid nitrogen needs to be continuously added to the mortar. Add 50 mg of powder to a centrifuge tube and mix well 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, place 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 leave it at room temperature for 2 min to evaporate the residual ethanol; add 30 μL of RNase-free water preheated to 70-90°C to the adsorption membrane, leave it at room temperature for 2 min, and centrifuge at 12,000 rpm for 1 min.

[0135] Take 2 μL of the RNA and use it for agarose gel electrophoresis to evaluate the integrity of the RNA. The unused RNA sample should be stored in a -80℃ refrigerator for subsequent reverse transcription or other purposes.

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

[0137] (1) Removal of genomic DNA

[0138] In a 0.2 mL PCR tube free of RNase contamination, 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 take it out and immediately place it on ice for subsequent use.

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

[0143] Add 4 μL of 5×HiScriptⅡEnzyme Mix to the PCR tube containing the above reaction solution, and perform the following reaction program in the PCR instrument: 50℃, 15min; 85℃, 5s. After the reaction is completed, the synthesized cDNA sample is diluted 10 times and stored in a -20℃ refrigerator.

[0144] 3.3 RT-qPCR analysis

[0145] The kit Novozyme ChamQ Universal SYBR qPCR Master Mix (Q711-02) was used for RT-qPCR analysis. The system is as follows:

[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 The method is used to measure the relative differences in gene transcription levels and analyze and compare the expression levels of individual genes based on this.

[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 B, RT-qPCR analysis showed that the expression level of PgERF2 in transgenic plants was significantly higher than that in wild type, among which the expression levels of PgERF2 in OE-1, OE-4, and OE-5 transgenic plants were 164 times, 1100 times, and 587 times higher than that in wild type, respectively. These data verified 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 steps were the same as before). The total RNA of the extracted samples was quality tested and analyzed using a NanoDrop micro-volume UV spectrophotometer and an Agilent 2100 bioanalyzer. Subsequently, 3 μg of total RNA from each sample was used to construct a cDNA library. These libraries were sequenced using the MGISEQ-T7 sequencing platform using a PE150 read length. In order to obtain clean reads suitable for subsequent analysis, the adapter sequences and low-quality reads were removed. Finally, the clean reads were aligned with the tobacco reference genome sequence using the HISAT2 software.

[0161] 4.2 RNA-seq data analysis

[0162] The featureCounts software was used to calculate the number of aligned fragments per million (FPKM) of each gene in each sample. The DESeq2 software was used to analyze the differences in gene expression between samples, and the screening criteria for differentially expressed genes were set as log2FC absolute value>1 and P-adjusted value<0.05. The detected differentially expressed genes (DEGs) were compared with each classification entry in the GO database, and the number of genes in each classification was counted. Then, the significance of protein enrichment in specific GO functional entries was evaluated by Fisher's exact test, and the threshold q value was set ≤0.05 to identify significantly enriched functional annotation GO entries. At the same time, the KEGG public database was used to perform functional annotation and significant enrichment analysis of DEGs, and the threshold q value was set ≤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 in biological processes mainly focused on response to stimulus, response to biotic stimulus, response to external bioticstimulus, response to other organisms, and response to stress ( Figure 2 A). These results suggest that PgERF2 may play an important role in regulating the response of Arabidopsis to biotic stresses. 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 the clean bench, Alternaria alternata was inoculated onto potato glucose medium and sealed with a sealing film. Subsequently, it was placed in a constant temperature incubator at 28°C for inverted culture. The culture was continued in the dark for about one week until the medium was completely covered with mycelium. After that, 0.05% Tween 80 solution was added to the medium, and the mycelium was gently scraped with a spore stick to prepare a spore suspension with a concentration of about 5×10 5 / mL. Select Arabidopsis leaves with a growth cycle of 4 weeks, place the petiole on a moist cotton ball to maintain the leaf humidity. Use a syringe needle to make four symmetrical wounds on the leaf, and inoculate 10μL of spore suspension in each wound. After 5 days, collect the lesion picture.

[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 to a centrifuge tube containing 1 mL of Alternaria alternata spore suspension, and the spore concentration was set at 5 × 10 5 The cells were cultured under gentle shaking for 48 h, 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 biological stress

[0169] In order to further identify the downstream target genes of PgERF2, the present invention analyzed the significantly up-regulated genes enriched in the GO pathway "response tobiotic stimulus". In this process, the plant defense protein PDF1.2A (Protein Defense Protein 1.2A) was screened as a candidate gene. In the present invention, one-week-old PgERF2 overexpression strain OE-4 and wild-type WT Arabidopsis seedlings were exposed to Alternaria spore suspensions, and the expression level of AtPDF1.2 gene was quantitatively analyzed after 0 hours, 6 hours, 12 hours, 24 hours and 48 hours.

[0170] The experimental steps of Arabidopsis seedling RNA extraction, reverse transcription, RT-qPCR analysis, etc. refer 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 was significantly higher than that in WT before treatment (0h). After treatment, OE-4 was able to respond quickly to Alternaria treatment, with the expression level of AtPDF1.2 increasing by more than 100 times, while the increase in the wild type was relatively slow. These experimental results further confirmed 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 of the pGreenⅡ0800-LUC reporter vector and the promoter fragment, and specific primers were designed to amplify the full-length 2000bp sequence of AtPDF1.2A-pro. The primers are named AtPDF1.2A-pro-F / R, and 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 for oscillation sterilization for 10 minutes, then rinsed with sterile water 6 times to remove residual sodium hypochlorite, and the sterile seeds were transferred to solid MS medium and cultured at 28° C., 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 high pressure at 121°C for 15 min.

[0189] (2) The tobacco seedlings were transferred to soil and continued to grow at 28°C, 16 h light / 8 h 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 under shaking conditions of 28° C. and 200 rpm for about 36 hours.

[0192] (2) Pick a single colony and inoculate it into 3 mL of YRK liquid culture 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 incubate 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 transformed into Agrobacterium GV3101, respectively. The transformation method is detailed in Example 2. The two successfully verified Agrobacterium were mixed in equal volumes and co-infected with the epidermal cells under tobacco leaves by injection penetration. Three tobacco leaves were infected in each sample group.

[0197] 7.5 Quantitative detection of dual luciferase

[0198] The dual luciferase assay kit from TransGen was used to quantitatively detect firefly luciferase and Renilla luciferase. The specific steps are as follows:

[0199] (1) Sample collection began 2 days after injection. A 2-cm inner diameter cork punch was used to sample, avoiding the large vein area. Each leaf 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 grind 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 steps in the instruction manual of Double-Luciferase Reporter Assay Kit (Beijing Quanshijin Biotechnology Co., Ltd.); then, the ratio of firefly luciferase to Renilla luciferase activity was calculated.

[0202] The results are as follows Figure 5 As shown in the results, PgERF2 overexpression can significantly enhance the promoter activity of AtPgDF1.2A by about 3.4 times. 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] Through 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 refer to 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 proteins

[0210] (1) The correctly identified BL21 / pCold / TF-PgERF2 expression strain was inoculated into 4 mL of LB liquid culture 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 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) 200 μL of 1 M IPTG was added to induce the expression of PgERF2 protein, and then the mixture was shaken and induced 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. Centrifuge the tube several times in batches and discard the supernatant to collect the bacterial cells.

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

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

[0216] (7) Use an ultrasonic cell disruptor on ice to lyse the bacteria. 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 min and transfer the supernatant to a new centrifuge tube to obtain the crude protein extract.

[0218] 8.3 Purification and concentration of recombinant proteins

[0219] (1) Place 500 μL of Ni-NTA suspension (pre-mixed thoroughly, column bed volume is 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 equilibration, 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 incubate at 4°C and 100 rpm for 60 min.

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

[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 at 4°C for 10 min.

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

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

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

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

[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 marked in the underlined part. The probe was labeled with biotin at the 5′ end and purified by HPLC. The synthesis, labeling and purification of the probe were completed by Beijing Qingke Biotechnology Co., Ltd.

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

[0243] 9.2 Gel migration retardation experiment

[0244] (1) Prepare 6% TBE gel according to the recipe. Pay special attention to prevent bubble formation during the process. After the gel solidifies, rinse the sample wells with 0.5× TBE solution and perform pre-electrophoresis at 100 V for 30 minutes. The electrophoresis buffer used is 0.5× TBE.

[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 TF-tagged protein, and Y represents PgERF2-TF fusion protein. Add various reagents one by one in the given order, mix thoroughly before adding the labeled probe, and then leave at room temperature for 10 minutes to eliminate possible nonspecific binding between the probe and the protein, or to preferentially react with the cold probe. Then add the labeled probe, mix thoroughly, and leave at room temperature for 20 minutes.

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

[0251] (4) Transfer: Soak the nylon membrane in 0.5×TBE for at least 10 minutes. When transferring, arrange the membrane in the order of cathode plate-sponge-filter paper-gel-membrane-filter paper-sponge-anode plate, and use a glass rod to remove bubbles. The transfer process is carried out in an ice bath, the current is set to 380 mA, and it lasts for 1 hour.

[0252] (5) UV crosslinking: Spread the membrane flat on clean filter paper (make sure the side containing bromophenol blue faces upward), 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 solution and mix thoroughly. Remove the blocking solution on the nylon membrane and replace it with new blocking solution containing Streptavidin-HRP Conjugate. Incubate slowly on a horizontal shaker for 15 minutes.

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

[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 inspection: Mix 5 ml of BeyoECL Moon A solution and 5 ml of BeyoECL Moon B solution to make a working solution. Take out the nylon membrane, remove the excess liquid, evenly apply the substrate working solution, and let it stand at room temperature for 3 minutes. Remove the excess substrate on the side of the membrane to avoid drying, and 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 solution pH 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 chemiluminescent EMSA kit (GS009) of Bio-Tech Company.

[0268] The results are as follows Figure 6 As shown, EMSA further studied the in vitro binding relationship between PgERF2 and the cis-acting element GCC-box. A GCC-box element labeled with biotin was designed as a probe, and EMSA experiments were performed in triple tandem to verify the binding ability of PgERF2 to this site. Compared with the empty vector TF protein, the TF-PgERF2 fusion protein can bind to the biotin-labeled probe, and the formed DNA-protein complex leads to a slowdown in electrophoretic migration, showing a hysteresis band. By using 50-fold and 200-fold unlabeled competitive probes and 200-fold mutant competitive probes for competitive experiments, with the increase of the concentration of unlabeled competitive probes, the hysteresis band was observed to gradually weaken until it disappeared. In contrast, when the mutant probe was introduced, the binding of the protein to the probe was less affected, reflecting the maintenance of specific binding. Therefore, these results fully confirmed that the PgERF2 protein can specifically bind to the GCC-box site in the AtPDF1.2A promoter.

[0269] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the scope of protection of the invention.

Claims

1. Application of ginseng transcription factor PgERF2 in improving plant resistance to Alternaria alternata, It is characterized in that The sequence of the ginseng transcription factor PgERF2 gene is shown in SEQ ID NO.

2.

2. Application of ginseng transcription factor PgERF2 in improving the expression level of genes related to plant resistance to Alternaria alternata, It is characterized in that The sequence of the PgERF2 gene is shown in SEQ ID NO.

2.

3. The use according to claim 2, It is characterized in that The gene related to the plant's resistance to Alternaria alternata is AtPDF1.2A.

4. Application of ginseng transcription factor PgERF2 in the preparation of reagents for positively regulating the promoter of AtPgDF1.2A gene, It is characterized in that The sequence of the PgERF2 gene is shown in SEQ ID NO.

2.

5. Application of ginseng transcription factor PgERF2 protein in the preparation of a reagent specifically binding to the GCC-box site in the AtPDF1.2A promoter, It is characterized in that The sequence of the PgERF2 protein is shown in SEQ ID NO.

1.

6. A plasmid, It is characterized in that The plasmid overexpresses the PgERF2 gene; preferably, the plasmid is obtained by enzymatically cutting a vector to obtain an enzymatically cut vector, and then connecting the PgERF2 gene to the enzymatically cut vector.

7. An Agrobacterium competent cell, It is 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.

8. Use of the plasmid according to claim 6 or the Agrobacterium competent cell according to claim 7 in enhancing the resistance of plants to Alternaria alternata.

9. A plant, It is characterized in that The plant is obtained by infecting plant cells with the Agrobacterium competent cells and then performing tissue culture.

10. The overexpression plant according to claim 9, It is characterized in that The plant is Arabidopsis thaliana, rice or cotton.

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