Application of Panax ginseng transcription factor PgJAZ13 in enhancing tobacco brown spot disease resistance

Abstract

The present invention belongs to the field of biological genetic engineering and relates to the application of the ginseng transcription factor PgJAZ13 in enhancing tobacco brown spot disease resistance. The sequence of the PgJAZ13 gene is shown in SEQ ID NO.2. The present invention clones the PgJAZ13 gene and constructs a corresponding plant expression vector. Through an Agrobacterium-mediated transformation method, the gene is successfully transferred into wild-type tobacco leaves, and positive tobacco plants that stably overexpress the PgJAZ13 gene are screened. Experiments have shown that heterologous overexpression of the PgJAZ13 gene in tobacco can significantly increase the expression levels of genes related to disease resistance and key enzyme genes for terpenoid synthesis, thereby promoting the biosynthesis of disease-resistant proteins and terpenes, and ultimately enhancing the plant's disease resistance.

Description

Technical Field

[0001] The invention belongs to the field of biological genetic engineering and relates to the application of ginseng transcription factor PgJAZ13 in enhancing tobacco brown spot disease resistance. Background Art

[0002] JAZ proteins play an important role in responses to biotic and abiotic stresses. In soybean, overexpression of GmTIFY10e and GmTIFF10g (homologs of Arabidopsis AtJAZ1 and AtJAZ2) can increase proline content and POD and CAT enzyme activities, reduce malondialdehyde content, and lead to changes in the expression of genes related to the ABA signaling pathway, thereby enhancing salt tolerance. [1] In cotton, overexpression of the GhJAZ2 gene reduces the plant's sensitivity to jasmonic acid (JA) and decreases the expression of the JA-responsive genes GhPDF1.2 and GhVSP, thereby increasing the plant's susceptibility to Verticillium dahliae and insect herbivory. [2] However, the functions of JAZ proteins in ginseng have not been fully elucidated.

[0003] Tobacco brown spot disease is a fungal plant disease that primarily affects tobacco leaves. It is caused by Alternaria alternate and causes severe damage to tobacco leaf quality. [3] The leaves are the main site of occurrence of brown star disease. The disease initially manifests as yellow-brown spots, which often form concentric ring-like lesions on the leaves. When the brown star disease is severe, these concentric ring-like lesions will connect and merge with each other to form a continuous lesion area. In dry conditions, the lesion area may crack, causing the leaves to dry out and break, significantly reducing the commercial value of tobacco leaves. [4] Given that the widespread use of chemical pesticides has gradually led to food safety and environmental pollution problems, the use of genetic engineering technology to cultivate transgenic plants with antibacterial and antifungal properties has become an economical and environmentally friendly strategy.

[0004] References:

[0005] [1] LIU YL, ZHENG L, JIN LG, et al. Genome-wide analysis of the soybeanTIFY family and identification of GmTIFY10e and GmTIFY10g response to saltstress[J]. Front Plant Sci, 2022,13:17.

[0006] [2]HE

[0007] [3]LAMONDIA J A.Outbreak of Brown Spot of Tobacco Caused byAlternaria alternata in Connecticut and Massachusetts[J].Plant Disease,2001,85(2):230.

[0008] [4] YAKIMOVA ET, YORDANOVA ZP, SLAVOV S, et al. Alternaria alternata ATtoxin induces programmed cell death in tobacco [J]. J Phytopathol, 2009, 157(10): 592-601. Summary of the Invention

[0009] The purpose of the present invention is to provide an application of ginseng transcription factor PgJAZ13 in enhancing tobacco brown spot disease resistance.

[0010] In order to achieve the above object, the technical solution adopted by the present invention is:

[0011] The invention discloses an application of ginseng transcription factor PgJAZ13 in enhancing tobacco brown spot disease resistance. The sequence of the PgJAZ13 gene is shown in SEQ ID NO.2.

[0012] The sequence of the PgJAZ13 protein is shown in SEQ ID NO.1.

[0013] SEQ ID NO.1 is:

[0014] MSTSSVIVDSGKFSGLRTARSPADKSNFSQTCNLLSQYLKEKGTLGDLSLGMSSGFECNGMPEPFRRPATPQTAAATTMNLFPVAEKPVQGFGSIVSNQEVPAKSEPEAAQMTIFYGGQVIVFNDFPAEKAKEIMLLANKGNSPILNNLTQKPIDQPSNLIPSSPNVVPNFSNSIIQERAQRPPQPIVSDLPIARKASLTRFLEKRKDRITARAPYASPAAAPSKPAESKSWLGLAAQSPVKFESEL。

[0015] SEQ ID NO. 2 is as follows:

[0016] .

[0017] The invention relates to the use of the ginseng transcription factor PgJAZ13 in improving the expression level of at least one gene related to tobacco brown spot disease resistance, wherein the sequence of the PgJAZ13 gene is shown in SEQ ID NO.2.

[0018] In one preferred embodiment, the genes related to tobacco brown spot disease resistance include one or more of PR (1a, 1b, 1c, 4a, ob12), ERF5, LRR-RLK (1, 2), CIPK (1, 2), GsSRK (1, 2) and fungal cell wall decomposition-related genes Echit (3, B) and β-GLU (1, 2).

[0019] The invention relates to the use of the ginseng transcription factor PgJAZ13 in improving the expression level of at least one key enzyme gene for terpenoid synthesis related to tobacco brown spot disease resistance. The sequence of the PgJAZ13 gene is shown in SEQ ID NO.2.

[0020] In one preferred embodiment, the key enzyme genes for terpenoid synthesis related to brown star disease resistance include one or more of 3-hydroxy-3-methylglutaryl-CoA synthetase, 3-hydroxy-3-methylglutaryl-CoA reductase, 1-deoxy-D-xylulose-5-phosphate synthase, 1-deoxy-D-xylulose-5-phosphate isomerase, farnesyl pyrophosphate synthase and squalene synthase 3.

[0021] The invention discloses an application of the ginseng transcription factor PgJAZ13 in improving the expression level of at least one protein related to tobacco brown spot disease resistance. The sequence of the PgJAZ13 gene is shown in SEQ ID NO.2.

[0022] The invention discloses an application of the ginseng transcription factor PgJAZ13 in improving the expression level of at least one terpenoid substance related to tobacco brown spot disease resistance. The sequence of the PgJAZ13 gene is shown in SEQ ID NO.2.

[0023] A plasmid overexpressing the PgJAZ13 gene.

[0024] In a preferred embodiment, the vector is digested by enzymes to obtain a digested vector, and the PgJAZ13 gene is connected to the digested vector.

[0025] In a preferred embodiment, the vector is pCAMBIA1301s plasmid.

[0026] An Agrobacterium competent cell overexpressing the PgJAZ13 gene.

[0027] In a preferred embodiment, the Agrobacterium competent cells are obtained by transforming the plasmid into DH5α competent cells.

[0028] The overexpression Agrobacterium competent cell is used for enhancing tobacco brown spot disease resistance, and the overexpression Agrobacterium competent cell overexpresses the PgJAZ13 gene.

[0029] A tobacco plant, wherein the overexpression plant overexpresses the PgJAZ13 gene.

[0030] The overexpressed tobacco has significantly enhanced resistance to brown spot disease.

[0031] The present invention cloned the PgJAZ13 gene and constructed a corresponding plant expression vector. The gene was successfully transferred into wild-type tobacco leaves via Agrobacterium-mediated transformation. Beta-glucuronidase (GUS) staining and quantitative polymerase chain reaction (RT-qPCR) were used to identify positive tobacco plants stably overexpressing the PgJAZ13 gene at the genomic DNA and transcribed mRNA levels.

[0032] The present invention involves transcriptomic analysis of transgenic strains. The results showed that in tobacco strains overexpressing PgJAZ13, Gene Ontology (GO) enrichment analysis identified the "defense response" category as the most prominent, followed by the "stress response" and "biostimulus response" categories. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed the "plant hormone signaling" pathway as the most prominent, with enrichment observed for the "diterpenoid biosynthesis and metabolism" pathway.

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

[0034] Experiments have shown that heterologous overexpression of the PgJAZ13 gene in tobacco can significantly increase the expression levels of genes related to disease resistance and key enzyme genes for terpenoid synthesis, thereby promoting the biosynthesis of disease-resistant proteins and terpenes, and ultimately enhancing the plant's disease resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 For subcellular localization analysis of PgJAZ13;

[0036] Figure 2 Identification of PgJAZ13 transgenic tobacco plants; Figure A: PCR identification of the hygromycin gene in 35S::PgJAZ13 transgenic tobacco leaves; Lane 1: Marker 2000; Lanes 2-6: genomic DNA of transgenic lines (OE1, OE4, OE5, OE6 and OE9); Lane 7: positive control, empty vector plasmid; Lane 8: negative control, genomic DNA of the wild-type line; Figure B: GUS staining signals of transgenic tobacco and wild-type leaves.

[0037] Scale bar = 1 mm;

[0038] Figure 3 The expression levels of PgJAZ13 in WT and OE strains were detected by RT-qPCR. NtEF1α was used as the internal reference gene, and the significant differences were analyzed by t-test, *P<0.05, **P<0.01;

[0039] Figure 4Transcriptome analysis of transgenic tobacco overexpressing PgJAZ13; Figure A: Volcano plot of differential gene expression distribution; Figure B: Heat map of correlation coefficients between samples; Figure C: KEGG enrichment analysis of differential genes showing upregulated pathways; Figure D: KEGG enrichment analysis of differential genes showing downregulated pathways; Figure E: GO enrichment analysis of differential genes showing upregulated terms; Figure F: GO enrichment analysis of differential genes showing downregulated terms;

[0040] Figure 5 Expression analysis of key enzyme genes for terpenoid biosynthesis in transgenic tobacco overexpressing PgJAZ13; HMGS, 3-hydroxy-3-methylglutaryl-CoA synthetase; HMGR, 3-hydroxy-3-methylglutaryl-CoA reductase; DXS, 1-deoxy-D-xylulose-5-phosphate synthase; DXR, 1-deoxy-D-xylulose-5-phosphate isomerase; FPS, farnesyl pyrophosphate synthase; SS3, squalene synthase 3; NtEF1α was used as the internal reference gene, and significant differences were analyzed using the t-test, *P < 0.05, **P < 0.01;

[0041] Figure 6 Expression analysis of disease resistance genes in transgenic tobacco overexpressing PgJAZ13. RT-qPCR was used to detect the expression levels of disease resistance-related genes in leaves of WT and OE4 / OE5 / OE6 lines. NtEF1α was used as the internal reference gene, and significant differences were analyzed using the t-test. *P<0.05, **P<0.01.

[0042] Figure 7 PgJAZ13 can improve the resistance of transgenic tobacco to Alternaria alternata (A. alternata); disease symptoms of 30-day-old wild type (WT) and OE6 strains 10 days after inoculation with Alternaria alternata; scale bar = 1 cm. DETAILED DESCRIPTION

[0043] Example 1

[0044] Construction of gene PgJAZ13 overexpression vector

[0045] 1.1 PCR amplification of target fragments

[0046] 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′)

[0047] PgJAZ13+GFP-F: cgggggactgagctcggtaccATGTCCACCTCGTCGGTAATTG (SEQ IDNO.3)

[0048] PgJAZ13+GFP-R: gctcaccatgtcgactctagaCAATTCGCTTTCAAACTTGACTGG (SEQ ID NO. 4).

[0049] 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.

[0050] The reaction system is as follows:

[0051] Table 1 PCR amplification system

[0052]

[0053] The PCR reaction program was set up as follows:

[0054] Table 2 PCR reaction procedure

[0055]

[0056] 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.

[0057] 1.4 PCR product purification and expression vector linearization

[0058] The PCR products were subjected to gel electrophoresis to remove nonspecific amplification bands, and the target DNA fragments were purified using a gel recovery kit from Vazyme. The PCR products were mixed with 6x loading buffer and electrophoresed on an agarose gel for 30 minutes. Under ultraviolet light, the agarose gel area containing the target-sized DNA fragment was accurately cut and placed in a clean 1.5ml centrifuge tube. The gel was then weighed, and subsequent steps were performed according to the instructions for use of the kit. The final DNA solution was tested for concentration and quality using an ultramicro spectrophotometer and then stored in a -20°C refrigerator.

[0059] The expression vector was digested with BamHI and KpnⅠ restriction enzymes, and the same gel recovery experiment was performed. The construction method of the pCAMBIA1301s-GFP plasmid was referred to the existing technology (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.).

[0060] The enzyme digestion system is as follows:

[0061] Table 3 Endonuclease digestion reaction system

[0062] 1.5 Vector ligation and E. coli transformation

[0063] The homologous recombination reaction is as follows:

[0064] Table 4 Homologous recombination reaction system

[0065]

[0066] 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:

[0067] (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.

[0068] (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.

[0069] (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.

[0070] (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.

[0071] The LB medium formula is as follows (volume 1 L):

[0072] Table 5 LB medium formula

[0073]

[0074] To prepare LB solid medium, add 15 g / L of agar powder to the liquid medium and then autoclave at 121°C for 15 minutes. After sterilization, add the appropriate amount of antibiotics after the autoclaved medium cools to approximately 60°C. This medium can be stored at room temperature for one week.

[0075] 1.6 Identification of positive clones

[0076] 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:

[0077] Table 6 Bacterial liquid PCR reaction system

[0078]

[0079] The PCR reaction procedure is as follows:

[0080] Table 7 Bacterial liquid PCR reaction program

[0081]

[0082] 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.

[0083] 1.7 Plasmid extraction

[0084] Adopt Beijing Qingke Biotechnology Co., Ltd. Plasmid Mini Kit is used for small-scale plasmid DNA extraction. All steps are performed at room temperature:

[0085] (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;

[0086] (2) Add 250 μL of Buffer PA containing RNase A to the bacteria and mix thoroughly by pipetting until no obvious bacterial clumps remain;

[0087] (3) Add 250 μL of Buffer PB and gently invert the tube 6-8 times to fully lyse the cells.

[0088] (4) Add 350 μL of Buffer PC and gently invert 6-8 times to mix thoroughly. Centrifuge at 12,000 rpm for 10 min.

[0089] (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.

[0090] (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;

[0091] (7) Place the adsorption column back into the collection tube and centrifuge the empty tube at 12,000 rpm for 2 min;

[0092] (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.

[0093] (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.

[0094] Example 2

[0095] Subcellular localization analysis of PgJAZ13

[0096] 2.1 Preparation of chemically competent Agrobacterium cells

[0097] (1) Take out the GV3101 / LB4404 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:

[0098] Table 8 YEB medium formula

[0099]

[0100] 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.

[0101] (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.

[0102] (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.

[0103] (4) Place the centrifuge tube in a 4°C pre-cooled centrifuge and centrifuge at 4,000 rpm for 10 minutes.

[0104] (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.

[0105] (6) After removing the supernatant, add 1 mL of pre-chilled 20 mM CaCl₂ solution to the pellet and gently pipette over ice to resuspend the bacterial suspension. The suspension was then aliquoted into pre-chilled 1.5 mL sterile centrifuge tubes, rapidly frozen with liquid nitrogen, and stored at -80°C until needed.

[0106] 2.2 Transformation of Agrobacterium competent cells

[0107] (1) Remove a tube of GV3101 / LB4404 competent cells from a -80°C freezer and thaw on ice for 10 minutes. In a clean bench, add 2 μL of the plasmid extracted in Example 1 to the bacterial suspension and gently pipette to mix. Then, let it stand on ice for 30 minutes.

[0108] (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.

[0109] (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.

[0110] (4) Single colonies were picked for bacterial liquid PCR identification, and the method was the same as that in Example 1. Positive colonies were selected for expansion culture and the next step of genetic transformation or bacterial strain preservation was carried out.

[0111] 2.3 Tobacco cultivation

[0112] (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:

[0113] Table 9 Formula of MS medium

[0114]

[0115] Adjust the pH to 5.8, add 8 g / L agar powder, and sterilize by autoclaving at 121°C for 15 min.

[0116] (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.

[0117] 2.4 Activation and expansion of Agrobacterium GV3101

[0118] (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.

[0119] (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.

[0120] (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.

[0121] (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.

[0122] 2.5 Tobacco transient transformation

[0123] The pCAMBIA1301s-JAZ-GFP vector was transformed into Agrobacterium tumefaciens GV3101. The transformation method is detailed in 2.2. The epidermal cells of tobacco leaves were infected by injection infiltration, with three leaves infected per sample group. Two days after injection, 0.5 cm × 0.5 cm tobacco leaf specimens were cut and immersed in DAPI staining solution. Incubated at room temperature for 10 minutes. A 10 ml syringe was used to apply vacuum, and the specimens were then placed under a laser confocal scanning microscope (LSM880; ZEISS) for fluorescence observation.

[0124] like Figure 1 As shown in the figure, in the control experiment, that is, in samples containing only the empty 35S-GFP vector, GFP signals were observed to be distributed in both the cytoplasm and the nucleus. However, in the experimental group containing the PgJAZ13-GFP fusion protein, the fluorescent signal was specifically localized to the nucleus, indicating that PgJAZ13 is a nuclear transcription factor.

[0125] Example 3 Construction of transgenic tobacco overexpressing PgJAZ13

[0126] To construct tobacco plants stably overexpressing PgJAZ13, we used Agrobacterium tumefaciens LB4404 carrying the pCAMBIA1301s-35s-PgJAZ13 plasmid to infect common tobacco. The specific experimental method is as follows:

[0127] 3.1 Preparation of tobacco culture medium

[0128] Prepare the culture medium required for the experiment in advance. After preparing according to the formula, sterilize it at 121℃ for 25 minutes, cool it to 60℃, add antibiotics, and then divide it for use. The culture medium and formula involved are as follows:

[0129] Table 10: Formula of culture medium used in transgenic tobacco construction

[0130]

[0131] 3.2 Tobacco pre-cultivation

[0132] Nicotiana tabacum Xanthi was used, and the pre-culture method was the same as in Example 2 until six true leaves grew. 3.3 LB4404 Agrobacterium activation

[0133] (1) Take out the recombinant Agrobacterium tumefaciens LB4404 carrying the pCAMBIA1301s-PgJAZ13-GFP plasmid from the -80°C freezer. Use an inoculation loop to gently scrape a portion of the bacteria in a clean bench and inoculate it into 3 mL of YEB liquid medium containing Sm (125 μg / mL) and Kan (50 μg / mL) resistance. Incubate at 28°C with shaking at 200 rpm overnight.

[0134] (2) Secondary activation: inoculate the overnight bacterial solution into a new YEB liquid medium with the same resistance, and culture at 28°C and 200 rpm until the OD = 0.8-1.2. Do not over-culture (about 16 h to 24 h);

[0135] (3) Collecting bacteria: Centrifuge at 8000 rpm for 1 min at room temperature to collect bacteria;

[0136] (4) Resuspend the bacteria in a liquid co-culture medium containing AS (100 μM) at a 1:1 ratio of equal volume. Place the resuspended bacteria in a shaker (100-110 rpm) and continue dark culture for about 30 min.

[0137] 3.4 Tobacco genetic transformation

[0138] (1) Cut tobacco leaves into 5 × 5 mm leaf discs, taking care to avoid the veins and leaf edges as much as possible;

[0139] (2) Transfer the leaf disc to the resuspended bacterial solution and infect on a shaker (100-110 rpm) for 45-60 min;

[0140] (3) Discard the bacterial solution, spread the leaf disc on the co-culture plate, and incubate in the dark for 48 h;

[0141] (4) Transfer to screening medium (28°C, 16 h light culture / 8 h dark culture) and culture for about two weeks;

[0142] (5) After callus tissue grows on the leaf disc, transfer it to budding medium and culture it for about 3 weeks. After leaf buds appear on the leaf disc, cut the leaf buds and transfer them to rooting medium for culture until the seedlings grow;

[0143] (6) Transplant the seedlings into nutrient pots to obtain five transgenic lines: OE1, OE4, OE5, OE6, and OE9.

[0144] 3.5 Identification of transgenic tobacco by GUS staining

[0145] pCAMBIA1301s-GFP is a plasmid containing the β-D-glucuronidase (GUS) gene, which is not endogenous in tobacco. Transgenic tobacco leaves were stained with GUS using a Coolaber GUS staining kit to determine whether the pCAMBIA1301s-PgJAZ13-GFP plasmid is stably expressed in tobacco cells.

[0146] 3.6 PCR identification of transgenic tobacco

[0147] (1) Extraction of transgenic tobacco genomic DNA

[0148] 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.

[0149] (2) PCR verification, the primer sequences are as follows:

[0150] Primer Sequence (5′→3′)

[0151] Hyg-F: ACACTACATGGCGTGATTTCAT(SEQ ID NO.5)

[0152] Hyg-R:TCCACTATCGGCGAGTACTTCT(SEQ ID NO.6)

[0153] Table 11 PCR identification reaction system

[0154]

[0155] The reaction conditions are the same as in Example 1.

[0156] The PCR products were collected and subjected to agarose gel electrophoresis, and images were taken using a ChemiDoc XRS gel imager from Bio Rad.

[0157] like Figure 2 As shown in A, all five transgenic lines (OE1, OE4, OE5, OE6, and OE9) showed a blue staining reaction characteristic of GUS enzyme activity, among which the OE6 line showed significant GUS enzyme activity. Figure 2 As shown in Figure B, genomic DNA was amplified and verified by PCR using specific primers containing the hygromycin resistance gene selection marker (SEQ ID NO.5 and SEQ ID NO.6). The results showed that all transgenic positive plants produced an amplified band of the expected size of 550 bp, while the corresponding amplified fragment was not detected in the wild-type plants.

[0158] Example 4

[0159] Detection of PgJAZ13 gene expression in transgenic lines

[0160] 4.1 Total RNA extraction from tobacco leaf tissue

[0161] (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.

[0162] (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.

[0163] (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.

[0164] (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 minutes;

[0165] (5) The mixture was transferred to an adsorption column RA, centrifuged at 13,000 rpm for 2 min, and the filtrate was discarded;

[0166] (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;

[0167] (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;

[0168] (8) Place the adsorption column back into the empty collection tube and centrifuge at 13,000 rpm for 2 minutes;

[0169] (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.

[0170] 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.

[0171] 4.2 Reverse transcription synthesis of first-strand cDNA

[0172] (1) Removal of genomic DNA

[0173] In an RNase-free 0.2 mL PCR tube, add the following reagents in order:

[0174] Table 12 Reverse transcription reaction system

[0175]

[0176] 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.

[0177] (2) Synthesis of the first strand of cDNA

[0178] 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.

[0179] 4.3 RT-qPCR analysis

[0180] RT-qPCR analysis was performed using the Novozymes ChamQ Universal SYBR qPCR Master Mix (Q711-02) kit. The system is as follows:

[0181] Table 13 RT-qPCR reaction system

[0182]

[0183] The amplification procedure is as follows:

[0184] Table 14 RT-qPCR reaction procedure

[0185]

[0186] 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.

[0187] The primer sequences used are as follows:

[0188] Primer Sequence (5′→3′)

[0189] qRT-PgJAZ13-F:TTACGGCGGTCAAGTCATC (SEQ ID NO.7);

[0190] qRT-PgJAZ13-R:GAGGGAATCAAATTGGAAGG (SEQ ID NO. 8).

[0191] like Figure 3 As shown, analysis of five transgenic lines showed that the expression level of the PgJAZ13 gene in the OE6 line was significantly higher than that in the other lines.

[0192] Example 5

[0193] Transcriptomic Analysis of Transgenic Tobacco Overexpressing PgJAZ13

[0194] 5.1 Library construction and sequencing

[0195] In this experiment, leaf tissue samples from transgenic line OE-6 and wild-type control (WT) tobacco were collected. These samples were from three independent biological replicates and used for subsequent RNA extraction. 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, 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.

[0196] 5.2 RNA-seq data analysis

[0197] 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.

[0198] like Figure 4 (A, B) A total of 6485 differentially expressed genes (DEGs) were identified between OE6 and WT lines, of which 2783 were upregulated and 3702 were downregulated in OE6. The correlation between OE6 and WT samples was very low (r 2 =0.25). KEGG enrichment analysis and GO enrichment analysis were then performed on the DEGs between OE6 and WT lines.

[0199] like Figure 4 (C, D) In ​​KEGG pathway enrichment analysis of 6485 DEGs, "Plant hormone signaling" emerged as the most dominant pathway category, consistent with the key involvement of JAZ proteins in the jasmonic acid signaling pathway. Diterpenoid biosynthesis pathways were also enriched. In contrast, fatty acid elongation and steroid hormone biosynthesis pathways were downregulated. These results suggest that PgJAZ13 may regulate terpenoid biosynthesis metabolism.

[0200] like Figure 4 (E, F) GO enrichment analysis of 6485 DEGs shows the top 20 most significant GO functional terms. Among the upregulated functional terms, "defense response" was the most significant, followed by "stress response" and "biostimulus response." In contrast, genes related to amino acid metabolism were downregulated in the OE6 line. These results suggest that overexpression of PgJAZ13 in tobacco may affect the expression of genes related to pathogen resistance.

[0201] Example 6

[0202] Detection of the expression levels of disease resistance genes and key enzyme genes for terpenoid synthesis in transgenic lines

[0203] The experimental steps of tobacco leaf RNA extraction, reverse transcription, and RT-qPCR analysis were referred to Example 4.

[0204] Entertainment:

[0205] Primer Sequence(5′→3′)

[0206] NtEF1α-F:TGAGATGCACCACGAAGCTC(SEQ ID NO.9)

[0207] NtEF1α-R:CCAACATTGTCACCAGGAAGTG(SEQ ID NO.10)

[0208] NtHMGS-F:AACCGGCCACATTAATACCA(SEQ ID NO.11)

[0209] NtHMGS-R:TCACAAAGTCCTTACCACCA(SEQ ID NO.12)

[0210] NtHMGR-F:TTGAGGATGAAAACGATGAG(SEQ ID NO.13)

[0211] NtHMGR-R:ATTCCAAAGAGTATGACGGC(SEQ ID NO.14)

[0212] NtDXS-F:CTGCCATTGATGACAGACCA(SEQ ID NO.15)

[0213] NtDXS-R:AGCAACATGAGACCCGAAAC(SEQ ID NO.16)

[0214] NtDXR-F:CAGCTACAACCTTAACCACC(SEQ ID NO.17)

[0215] NtDXR-R:TTCTCAGCGACTATATCCAA(SEQ ID NO.18)

[0216] NtFPS-F:AGGTGCTGGGTAAGATTGGC(SEQ ID NO.19)

[0217] NtFPS-R:ACACTGCTTGCACTGCTTTG(SEQ ID NO.20)

[0218] NtSS3-F:TGGGTGCAGGAATGGCAA(SEQ ID NO.21)

[0219] <h2 style=";text-align:left;direction:ltr">NtSS3-R:TTCTTTCCCAGAGGCGCG(SEQ ID NO.22)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0220] <h2 style=";text-align:left;direction:ltr"> NtPR1a-F:ACTCTTGCCGTGCCCAAA(SEQ ID NO.23)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0221] <h2 style=";text-align:left;direction:ltr"> NtPR1a-R:TGCTACCTGGTCGTCCCA(SEQ ID NO.24)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0222] <h2 style=";text-align:left;direction:ltr"> NtPR1b-F:GGGACAACGGGGTAGCAG(SEQ ID NO.25)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0223] <h2 style=";text-align:left;direction:ltr"> NtPR1b-R:GGCCTTAGCAGCCGTCAT(SEQ ID NO.26)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0224] <h2 style=";text-align:left;direction:ltr"> NtPR1c-F:TGGGACGACCAGGTAGCA(SEQ ID NO.27)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0225] <h2 style=";text-align:left;direction:ltr"> NtPR1c-R:ATCGCCACTTCCCCAAGC(SEQ ID NO.28)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0226] <h2 style=";text-align:left;direction:ltr"> NtPR4a-F:GCACAGAGCGCCACAAAC(SEQ ID NO.29)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0227] <h2 style=";text-align:left;direction:ltr"> NtPR4a-R:GGCATCCCAAGTAGCGCA(SEQ ID NO.30)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0228] <h2 style=";text-align:left;direction:ltr"> NtPRob12-F:CTACACAGTCTGGGCCGC(SEQ ID NO.31)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0229] <h2 style=";text-align:left;direction:ltr"> NtPRob12-R:GACCCCAAATGCGAGCCT(SEQ ID NO.32)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0230] <h2 style=";text-align:left;direction:ltr"> NtEChiB-F:CGAGGATGGGCAACAGCA(SEQ ID NO.33)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0231] <h2 style=";text-align:left;direction:ltr"> NtEChiB-R:CCGACCAGGAGCACAAGG(SEQ ID NO.34)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0232] <h2 style=";text-align:left;direction:ltr"> NtEChit3-F:TGACAGAGCAGCGAACCG(SEQ ID NO.35)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0233] <h2 style=";text-align:left;direction:ltr"> NtEChit3-R:ACCCTAGCGTCAGAGCCA(SEQ ID NO.36)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0234] <h2 style=";text-align:left;direction:ltr"> NtERF5-F:GCGGCGGAAATTCGTGAC(SEQ ID NO.37)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0235] <h2 style=";text-align:left;direction:ltr"> NtERF5-R:ACGCCGCCCTGTCATATG(SEQ ID NO.38)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0236] <h2 style=";text-align:left;direction:ltr"> NtβGLU1-F:TGTTGCTCCTGCCATGCA(SEQ ID NO.39)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0237] <h2 style=";text-align:left;direction:ltr"> NtβGLU1-R:GGGCGGGTAGGTATTCGC(SEQ ID NO.40)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0238] <h2 style=";text-align:left;direction:ltr"> NtβGLU2-F:AGCAGCATCAGGGTTGCA(SEQ ID NO.41)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0239] <h2 style=";text-align:left;direction:ltr"> NtβGLU2-R:TGGGTGGGTAGGTGTTTGC(SEQ ID NO.42)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0240] <h2 style=";text-align:left;direction:ltr"> NtLRR-RLK1-F:GGGCGGTCCGTTCAAGAA(SEQ ID NO.43)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0241] <h2 style=";text-align:left;direction:ltr"> NtLRR-RLK1-R:AGTGGGGCATGGCAGAAC(SEQ ID NO.44)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0242] <h2 style=";text-align:left;direction:ltr"> NtLRR-RLK2-F:GTGCAACCTCGTCGGACC(SEQ ID NO.45)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0243] <h2 style=";text-align:left;direction:ltr"> NtLRR-RLK2-R:GCGTTGCCATCCAGTGCT(SEQ ID NO.46)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0244] <h2 style=";text-align:left;direction:ltr"> NtCIPK1-F:CGGCAGAGTCCCCTGAGA(SEQ ID NO.47)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0245] <h2 style=";text-align:left;direction:ltr"> NtCIPK1-R:ACCTCCGCCAGAAGCCTA(SEQ ID NO.48)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0246] <h2 style=";text-align:left;direction:ltr"> NtCIPK2-F:AGGAAGTGGCGAAGACGAC(SEQ ID NO.49)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0247] <h2 style=";text-align:left;direction:ltr"> NtCIPK2-R:TCTCCCACTCTCTTGCCCT(SEQ ID NO.50)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0248] NtGsSRK1-F: TGTCGGCGGAAGTGTGTC (SEQ ID NO.51)

[0249] NtGsSRK1-R:AACCTCCTCGTCCCCGAC (SEQ ID NO.52)

[0250] NtGsSRK2-F: TGGTGTTGGGCATCTCCG (SEQ ID NO.53)

[0251] NtGsSRK2-R: ACCACTGGTTGCCTGCTC (SEQ ID NO.54)

[0252] like Figure 6 As shown, the expression levels of biotic stress-related genes in tobacco, including PR (1a, 1b, 1c, 4a, ob12), ERF5, LRR-RLK (1, 2), CIPK (1, 2), and GsSRK (1, 2), as well as fungal cell wall decomposition-related genes Echit (3, B) and β-GLU (1, 2), were significantly upregulated after PgJAZ13 overexpression. These results indicate that PgJAZ13 overexpression in tobacco induces the expression of genes associated with pathogen resistance.

[0253] The experimental results of Example 5 showed that the diterpenoid biosynthesis metabolic pathway was significantly enriched. Based on this finding, the present invention further detected the expression levels of key enzyme genes in the terpenoid biosynthesis pathway in transgenic strains OE4, OE5, OE6 and wild-type control (WT). The operation steps refer to Example 4. Figure 5 As shown in the results, overexpression of the PgJAZ13 gene significantly upregulated the expression of key enzyme genes for terpenoid synthesis, indicating that PgJAZ13 may play an important role in promoting the biosynthesis of terpenoids.

[0254] Example 7

[0255] Alternaria infection experiment

[0256] 7.1 Collection of Alternaria spores

[0257] (1) In a clean bench, inoculate Alternaria spores onto PDA medium and culture in a 28°C incubator. Incubate in the dark for about one week until the mycelium covers the entire medium.

[0258] (2) Prepare a spore suspension of Alternaria alternata using a 0.05% Tween-80 solution. Add 3 mL of Tween-80 to the culture medium of Alternaria alternata and gently scrape the spores using a triangular applicator.

[0259] (3) Filter through two layers of filter paper to remove the mycelium and collect the Alternaria spores. Dilute to a concentration of about 5×10 5 Note that the clean bench cannot be ventilated during the entire experimental process.

[0260] 7.2 Alternaria spores infecting tobacco leaves

[0261] (1) Select mature tobacco leaves and cover the petioles with soaked cotton wool to moisten the leaves. Use a syringe needle to make four symmetrical wounds and inoculate each wound with 50 μL of spore suspension. Place in an incubator at 28°C with 16 h light / 8 h dark incubation.

[0262] (2) Observe the pathological status of tobacco leaves every day and keep the cotton wool moist;

[0263] (3) After 10 days, take photos of the lesions.

[0264] like Figure 7 As shown in the figure, compared with the wild type, the disease symptoms of the overexpression line were milder and the lesion area was significantly smaller than that of the wild type.

[0265] These results indicate that heterologous overexpression of the PgJAZ13 gene in tobacco can significantly increase the expression levels of genes related to disease resistance and key enzyme genes for terpenoid synthesis, thereby promoting the biosynthesis of disease-resistant proteins and terpenes, and ultimately enhancing the plant's disease resistance.

[0266] 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. Application of ginseng transcription factor PgJAZ13 in enhancing tobacco brown spot disease resistance, characterized in that: described PgJAZ13 The sequence of the gene is shown in SEQ ID NO.

2.

2. A plasmid, characterized in that The plasmid overexpressed PgJAZ13 Gene; the sequence of the PgJAZ13 gene is shown in SEQ ID NO.

2.

3. An Agrobacterium competent cell, characterized in that The Agrobacterium competent cells overexpress PgJAZ13 Gene; the sequence of the PgJAZ13 gene is shown in SEQ ID NO.

2.

4. Use of the plasmid according to claim 2 or the Agrobacterium competent cell according to claim 3 in enhancing resistance to tobacco brown spot disease.

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