Cloning of a disease resistance gene tewrky33 from tagetes erecta and application thereof

Abstract

The application discloses a marigold disease-resistant gene TeWRKY33, and a nucleotide sequence is shown as SEQ ID NO:1 in a sequence table. The application clones the TeWRKY33 gene from marigold, constructs a vector, and transforms Arabidopsis thaliana, and through phenotype and disease analysis of the Arabidopsis thaliana transgenic and wild type plants after being infected by Alternaria alternata, the disease-resistant function of the TeWRKY33 is verified. The application provides strong theoretical guidance for fundamentally preventing and treating black spot disease, and has direct application value.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and in particular to the cloning and application of a marigold disease resistance gene TeWRKY33. Background Technology

[0002] Marigold (Tagetes erecta L.) is an important ornamental plant and also a functional flower used for extracting lutein. Black spot disease in marigolds severely restricts the development of the marigold industry, mainly damaging flowers and stems and leaves, causing premature withering and death of the plant. Alternaria tagetica is one of the pathogenic fungi causing marigold black spot disease. Research on candidate genes for resistance to Alternaria tagetica infection in marigolds will provide strong theoretical guidance for the fundamental prevention and control of black spot disease and has direct application value. Summary of the Invention

[0003] The purpose of this invention is to clone the marigold disease resistance gene TeWRKY33 and study its disease resistance function, discovering that it can inhibit the growth of Alternaria rubra. The nucleotide sequence of the marigold disease resistance gene TeWRKY33 of this invention is shown in SEQ ID NO:1 of the sequence listing.

[0004] The primer pair used in this invention for cloning the disease resistance gene TeWRKY33 is TeWRKY33-F and TeWRKY33-R, and its nucleotide sequence is shown in SEQ ID NO:2 and SEQ ID NO:3 in the sequence listing.

[0005] An overexpression vector containing the disease resistance gene described in this invention, wherein the primer pair used to construct the overexpression vector is C85-TeWRKY33-F and C85-TeWRKY33-R, and the nucleotide sequences are shown in SEQ ID NO:4 and SEQ ID NO:5 in the sequence listing; the nucleotide sequence of the ORF fragment of TeWRKY33 with restriction enzyme sites is shown in SEQ ID NO:6 in the sequence listing. An amino acid sequence encoding the marigold disease resistance gene TeWRKY33 described in this invention is shown in SEQ ID NO:7 in the sequence listing.

[0006] Application of the marigold disease resistance gene TeWRKY33 in improving plant disease resistance.

[0007] In the application described in this invention, the marigold disease resistance gene TeWRKY33 can inhibit the growth of Alternaria marigoldii.

[0008] In the application described in this invention, the plant is Arabidopsis thaliana or marigold.

[0009] This invention clones the TeWRKY33 gene from marigolds and constructs a vector to transform Arabidopsis thaliana. Phenotypic traits and disease incidence in transgenic and wild-type Arabidopsis plants infected with Alternaria rubra are analyzed to verify the disease resistance function of TeWRKY33. This invention provides strong theoretical guidance for the fundamental prevention and control of black spot disease and has direct application value.

[0010] The TeWRKY33 gene and its function of the present invention will be further explained below with reference to the accompanying drawings. Attached Figure Description

[0011] Figure 1 This is an electrophoresis detection image of the TeWRKY33 gene clone of the present invention; wherein, M: DL2000 DNA Marker; 1, 2, 3: TeWRKY33 amplified fragments;

[0012] Figure 2 This is a double enzyme digestion verification diagram of the overexpression vector of the present invention; wherein, M: DL2000 DNA Marker; 2: TeWRKY33 gene;

[0013] Figure 3 This is a PCR electrophoresis detection image of Agrobacterium-transformed bacterial solution in this invention; where M: DL2000 DNA Marker; 5: water control; 4: Agrobacterium-transformed bacterial solution using TeWRKY33.

[0014] Figure 4 This diagram illustrates the collection and screening process of TeWRKY33 transgenic seeds in this invention; a: Selected seeds; b: Seeds inoculated onto the culture medium; c: Germinated seeds and seeds with roots.

[0015] Figure 5 This is an image of Arabidopsis thaliana GFP PCR electrophoresis detection in this invention; M: DL2000 DNA Marker; 3, 4: TeWRKY33 gene bands;

[0016] Figure 6 This is the pathogen activation process in the present invention, wherein a: strain; b: spore; c: bacterial solution. Detailed Implementation

[0017] 1. Cloning of the marigold TeWRKY33 gene

[0018] 1.1 Test Materials

[0019] 1.1.1 Plant materials

[0020] The disease-resistant and susceptible marigold materials used in this experiment were obtained from the Japanese greenhouse of the Beijing Academy of Agricultural and Forestry Sciences. They were planted in pots (with a substrate of vermiculite: peat moss = 1:2) and managed with normal water and fertilizer. During the seedling stage, 3-5 healthy, disease-free, and fully expanded leaves were collected between 9:00 AM and 10:00 AM, with three biological replicates for each sample.

[0021] 1.1.2 Strains and Vectors

[0022] Strains: Escherichia coli competent cells DH5α were purchased from Tiangen Biotech (Beijing) Co., Ltd.

[0023] Vector: The cloning vector used was from Promega. Easy carrier.

[0024] 1.1.3 Main Reagents, Kits and Instruments

[0025] Reagents: RNA extraction kit (MiniBEST Plant RNA Extraction Ki, Takara), reverse transcription kit ( III. All-in-one RT SuperMix Perfect for qPCR Kit (Novizan), High-fidelity enzyme PCR amplification reagent ( Max Master Mix (Vazyme), a kit for the recovery and purification of the target fragment ( SV Grl and PCR Clean-Up System (Promega), ligation kit for target fragment and vector (Promega T4 ligase kit, Promega), plasmid mini-extraction kit (AxyPrep™ Plasmid Miniprep Kit, Axygen), plant genomic DNA extraction kit (MiniBEST Universal Genomic DNA Extraction Kit, Takara), real-time PCR kit (… Green PCR Kit (Qiagen), Real-time Reverse Transcription Kit (HiScript II Q RT SuperMix for qPCR, Vazyme), Takara LA Taq, DL2000 DNA Marker, etc. were purchased from Takara; Ampicillin (Amp) was purchased from Solarbio.

[0026] Instruments and Equipment: PCR instrument (Bio-rad, T100), microcentrifuge (Thermo Scientific Heraeus, Pico 17), -80℃ ultra-low temperature freezer (SANYO, MDF-U53V), -20℃ freezer (Haier, DW-25L262), 4℃ freezer (Haier, BCD-225TMPM), vertical pressure steam sterilizer (Jiangyin Binjiang Medical Equipment Co., Ltd., LS-B75-II), ultra-micro spectrophotometer (Food ALYT Photometer), ultraviolet gel imaging system (Tanon, 1600), full-temperature shaking incubator (Suzhou Peiying Experimental Equipment Co., Ltd., THZ-C-1), real-time PCR instrument (Roche). Equipment included: 9700, ion sputtering equipment (Tokyo Hitachi, ID-5), scanning electron microscope (Tokyo Hitachi, S-400n), gel electrophoresis apparatus (Beijing Liuyi Instruments, JY-SPCT), water bath (Shanghai Yiheng, DK-8D), clean bench (Suzhou Purification, SW-CJ-1FD), metal bath (Yixing Yukadi Biotechnology Co., Ltd., H2O3-PROIII), shaker (Thermolyne, M37610-33), electronic balance (JA2003), etc.

[0027] 1.1.4 Culture medium preparation

[0028] Preparation of LB medium (using 1L as an example):

[0029]

[0030] 1.1.5 PCR primers required for the experiment

[0031] Primers were designed using Primer Premier 5.0 software (see Table 1). The primers were synthesized at Shanghai Sangon Biotech Co., Ltd. (Shanghai Sangon). After centrifugation at 4000 rpm, the synthesized primers were diluted to a concentration of 10 μM with sterile water according to the manufacturer's instructions and stored at -20℃ for later use.

[0032] Table 1 Primer sequences

[0033]

[0034] 1.2 Test Methods

[0035] 1.2.1 RNA extraction and cDNA synthesis

[0036] (1) RNA extraction

[0037] The following is a slightly modified version of the Takara MiniBEST Plant RNA Extraction Kit instructions:

[0038] ① Before the experiment begins, wipe the surfaces of the workbench and pipettes with 75% alcohol.

[0039] ② Solution preparation: Solution I: 1L Buffer RL, 20μL 50×DTT Buffer; Solution II: 5μL 10×DNase I Buffer, 4μL Recombinant DNase T, 41μL RNase free dH2O.

[0040] ③ Take 0.1g of fresh plant material or plant material that has been stored at low temperature after being taken from a -80℃ freezer, grind it with a grinder at low temperature, add 450μL of solution I, mix well by shaking, and centrifuge at 12000rpm for 5min.

[0041] ④ Remove the supernatant and transfer it to a new 1.5 mL centrifuge tube. Add 1 / 2 volume of anhydrous ethanol to the supernatant, invert the tube to mix well, and then quickly transfer it into an RNA spin column. Let it stand for 1 min, then centrifuge at 12000 rpm for 1 min and discard the filtrate.

[0042] ⑤ Add 500 μL of Buffer RWA to the RNA spin column, centrifuge at 12000 rpm for 30 seconds, and discard the filtrate.

[0043] ⑥ Add 600 μL of Buffer RWB to the RNA spin column (confirm the specified amount of ethanol before use), centrifuge at 12000 rpm for 30 seconds, and discard the filtrate.

[0044] ⑦ Add 50 μL of Solution II to the center of the membrane, let it stand at room temperature for 15 min until the DNA is completely digested, then add 350 μL of Buffer RWB, centrifuge at 12000 rpm for 30 s, and discard the filtrate.

[0045] ⑧ Add 600 μL of Buffer RWB to the RNA spin column, centrifuge at 12000 rpm for 30 seconds, and discard the filtrate.

[0046] ⑨ After centrifuging for 2 minutes, place the RNA spin column onto a new 1.5 mL centrifuge tube and let it stand at room temperature for 5 minutes to allow the alcohol to evaporate completely.

[0047] ⑩ Add 50 μL of RNase-Free H2O to the center of the membrane, let it stand at room temperature for 5 min, centrifuge at 12000 rpm for 2 min, and elute the RNA.

[0048] Add another 1.5 mL of liquid from a centrifuge tube to the center of the membrane and let stand for 5 min. Centrifuge at 12000 rpm for 2 min to obtain the total RNA solution. Take 1 μL and use a micro spectrophotometer and 1% agarose gel electrophoresis to detect the concentration and mass of RNA. Store the RNA solution at -80℃ for later use.

[0049] (2) cDNA synthesis

[0050] The synthesis of the first strand of cDNA was performed according to the instructions of the Reverse Transcription System kit. The following reagents were added sequentially to a 0.5 mL centrifuge tube:

[0051]

[0052] Add 80 μL of RNA-free water, mix well, and store at -20°C for later use.

[0053] 1.2.2 Amplification of full-length TeWRKY33

[0054] Primers TeWRKY33-F and TeWRKY33-R for amplifying the TeWRKY33 gene were designed using Primer Premier 5.0 software. TeWRKY33 was cloned using cDNA from marigold leaves as a template. Max Master Mix was used to amplify TeWRKY33 ORF. The reaction system is as follows:

[0055]

[0056] After gently mixing, place the mixture in a Bio-rad T100 PCR instrument and react as follows:

[0057]

[0058] The denaturation to extension step was repeated 34 times. After amplification, 2 μL of the PCR product was collected for 1% agarose gel electrophoresis.

[0059] (1) Recovery and purification of the target fragment

[0060] The specific steps are as follows:

[0061] ① Cut DNA bands from an agarose gel and place the gel block in a 1.5 mL sterile centrifuge tube.

[0062] ② Add the binding solution (Membrane Binding Solution) at a ratio of 100mg:100μL, place in a 65℃ metal bath for 10 minutes, inverting the container three times during the process to mix thoroughly until the gel block is completely dissolved, then remove and allow to cool naturally to room temperature.

[0063] ③ Take a centrifugal adsorption column, insert it into the collection tube, transfer the above solution into the adsorption column and let it stand at room temperature for 1 min, centrifuge at 12000 rpm for 30 s, and discard the filtrate.

[0064] ④ Add 700 μL of Membrane Wash Solution, centrifuge at 12000 rpm for 30 seconds, and discard the waste liquid.

[0065] ⑤ Add 500 μL of rinsing solution, centrifuge at 12000 rpm for 30 seconds, and discard the filtrate.

[0066] ⑥ After centrifugation at 12000 rpm for 1 min, place the adsorption column in a clean centrifuge tube, open the cap and place it in a clean bench for 10 min to allow the residual alcohol to evaporate fully.

[0067] ⑦ Add 30 μL of Nuclease Free Water to the center of the adsorption column membrane, let stand at room temperature for 3 min, and then centrifuge at 12000 rpm for 1 min to elute the DNA solution.

[0068] (2) In vitro ligation of the target fragment and sequencing vector

[0069] because The PCR products of Max Master Mix high-fidelity enzymes have blunt ends and cannot be directly ligated into T-vectors; therefore, they require end-capping (A) treatment. A-capping is performed on the recovered products using TakaraTaq enzyme, with the following reagents added sequentially:

[0070]

[0071] After mixing, keep at 72℃ for 30 minutes, then store at -20℃ for later use.

[0072] Using the Promega T4 ligase kit, in the cloning vector Add the product with terminal A to Easy, and then add the following reagents in sequence:

[0073]

[0074] After mixing the samples, briefly centrifuge them and then connect them at 4℃ for 8–12 h.

[0075] (3) Transformation and sequencing of the ligation product

[0076] Transformation of Escherichia coli DH5α using the heat shock method:

[0077] ① In a sterile operating table, prepare LB solid culture medium with a final concentration of 50 mg / L for Amp and pour it into a petri dish.

[0078] ② Thaw 50 μL of DH5α E. coli competent cells on ice. Add 5 μL of ligation product to the competent cells, mix gently, and incubate on ice for 30 min.

[0079] ③ After heat shocking in a 42℃ water bath for 90 seconds, immediately place on ice for 3-5 minutes. Do not shake the centrifuge tubes during this process.

[0080] ④ Add 500 μL of LB liquid medium preheated at 37℃, mix gently, and then place in a shaker at 37℃ at 180 rpm for 60 min to activate.

[0081] ⑤ Take 200 μL of LB liquid culture medium, 40 μL of 20 mg / mL X-Gal and 4 μL of 200 g / L IPIG solution, mix well and spread evenly on LB (containing 50 mg / L Amp) culture dish.

[0082] ⑥ Take 200 μL of the activated bacterial solution and spread it evenly on a petri dish. Incubate at 37°C upside down in the dark for 10–14 h.

[0083] ⑦ In a sterile operating table, pick 10 single colonies with a pipette tip and place them into 2 mL of liquid culture medium containing LB (containing 50 mg / L Amp). After shaking at 180 rpm for 14–16 h, send them to the company for sequencing.

[0084] ⑧ Use DNAMAN to compare the sequencing results and select samples with correct sequences for plasmid extraction.

[0085] (4) Small-scale extraction of plasmids

[0086] Referring to the instructions for the AxyPrep™ Plasmid Miniprep Kit, the operating steps are as follows:

[0087] ① Pour 1 mL of bacterial culture into a 1.5 mL centrifuge tube, centrifuge at 10000 rpm for 2 min to collect the bacterial cells, and discard the supernatant.

[0088] ② Add 250 μL of Buffer S1 to resuspend the bacterial culture, then add 250 μL of Buffer S2. Gently invert the container 4 to 6 times to mix until the solution is clear.

[0089] ③ Add 350 μL Buffer S3, gently invert 6 times to mix, and centrifuge at 12000 rpm for 10 min.

[0090] ④ Place the preparation tube onto a 2mL centrifuge tube, take the supernatant from step ③ into the preparation tube, centrifuge at 12000rpm for 1min, and discard the filtrate.

[0091] ⑤ Add 500 mL of Buffer W1 to the preparation tube, centrifuge at 12,000 rpm for 1 min, and discard the filtrate.

[0092] ⑥ Add 700 mL of Buffer W2, centrifuge at 12,000 rpm for 1 minute, and discard the filtrate. Repeat this operation once.

[0093] ⑦ Centrifuge at 12000 rpm for 30 min, place the preparation tube in a new centrifuge tube, let it stand in a clean bench for 10 min to allow the alcohol to evaporate completely, then add 50 μL of Eluent to the center of the membrane in the preparation tube, let it stand at room temperature for 1 min, centrifuge at 12000 rpm for 1 min, and store the plasmid solution in a -20℃ refrigerator for later use.

[0094] 1.3 Results and Analysis

[0095] Using cDNA from marigold leaves obtained by reverse transcription as a template, PCR amplification of the TeWRKY33 gene ORF was performed using TeWRKY33-F and TeWRKY33-R primers. The PCR product was detected by electrophoresis, yielding two specific bands. The specific bands were recovered, purified, ligated into a cloning vector, and sequenced. Sequencing results showed that the full-length 987 bp sequence of the TeWRKY33 gene (…) Figure 1 The open reading frame (ORF) of this sequence is 873 bp and encodes 290 amino acids.

[0096] TeWRKY33 sequence:

[0097] CTACGCCTACGACATTAACCCTCACTAAAGGTACTAGTCCTGCAGGTTTAAACGAATTCGCCCTTTAATGGCATCTTCTGGTGGCAGCTTGAATACATCAACTACAACTTCACAATACCCATCTTTTTCTTTTTCAACATCTTTTAGTGAACTTATTGGCTCTAATAACAACAATCAAGATTCAAATTGGGGTTTGTTTAGTGGGATTGAAAGTCCAAAAATACCCTTTTCTCCATCTTCTTTTCTTTCTCTACACCAACTGGGTTGAACCAAAATCAGTTCTTGGACTCTCCTTTCTTTCCTTTCAATTCAAATATTGTTCCATCTCCAACAACTGGAAGTTTTTCTAGTTTGGATTTGAAAGATGATGAAAGAAAGTTTTCTAATTTCTCATTTCAACCACAACCCATTTCTTCTGTGTTTTTCTGCAGCCAAAATGGAAGAACAAGTGAAAAGTCAACCACAAGAATGGAGTTT GACTAAACAAAATAATTTCTTATCAGAAATGAACAAACCCAAATCAGAATCAACACAATCTATTCAAACCCAAACCCAATATGTTTCTGATCAATCTCAAGCCATTAGAGATAGAACAAGATTAGAGGATGGTTACAATTGGAGAAAATATGGTCAAAAAACAAGTCAAAGGAAGCGAAAACCCGCGAAGTTATTACAAATGTACATACCCAAACTGTCCAACGAAGAAAAAAGTCGAAAGAAACTTAGAGGGACATATCACCGAGATCGTTTACAAAGGAAATCATAGTCATTCTAAGCCTCAAAATGCCAAGAAATCATCTTCAAACAATTTCATTGAGGCACCAACTGAAAACAGTCAGTTCGAATCTTCGGCCTCGTTTGGGGATGATGACTTTGAGCAAGCCTCGTCGATTAGTAAATCCGGAGATGACCATGAAAACGAGCCTGAAGCCAAGAGATGGAAGTGA

[0098] WRKY33-ORF

[0099] ATGGCATCTTCTGGTGGCAGCTTGAATACATCAACTACAACTTCACAATACCCATCTTTTTCTTTTTCAACATCTTTTAGTGAACTTATTGGCTCTAATAACAACAATCAAGATTCAAATTGGGGTTTGTTTAGTGGGATTGAAAGTCCAAAAATACCCTTTTCTCCATCTTCTTTTCTTTCTACACCAACTGGGTTGAACCAAAATCAGTTCTTGGACTCTCCTTTCTTTCCTTTCAATTCAAATATTGTTCCATCTCCAACAACTGGAAGTTTTTCTAGTTTGGATTTGAAAGATGATGAAAGAAAGTTTTCTAATTTCTCATTTCAACCACAACCCATTTCTTCTGTTTCTGCAGCCAAAATGGAAGAACAAGTGAAAAGTCAACCACAAGAATGGAGTTTGACTAAACAAAATAATTTCTTATCAGAAATGAACAAACCCAAATCAGAATCAACACAATCTATTCAAACCCAAACCCAATATGTTTCTGATCAATCTCAAGCCATTAGAGATAGAACAAGATTAGAGGATGGTTACAATTGGAGAAAATATGGTCAAAAACAAGTCAAAGGAAGCGAAAACCCGCGAAGTTATTACAAATGTACATACCCAAACTGTCCAACGAAGAAAAAAGTCGAAAGAAACTTAGAGGGACATATCACCGAGATCGTTTACAAAGGAAATCATAGTCATTCTAAGCCTCAAAATGCCAAGAAATCATCTTCAAACAATTTCATTGAGGCACCAACTGAAAACAGTCAGTTCGAATCTTCGGCCTCGTTTGGGGATGATGACTTTGAGCAAGCCTCGTCGATTAGTAAATCCGGAGATGACCATGAAAACGAGCCTGAAGCCAAGAGATGGAAGTGA

[0100] Amino acid sequence:

[0101] MASSGGSLNTSTTTSQYPSFSFSTSFSELIGSNNNNQDSNWGLFSGIESP

[0102] KIPFSPSFLSTPTGLNQNQFLDSPFFPFNSNIVPSPTTGSFSSDLKDD

[0103] ERKFSNFSFQPQPISSVSAAKMEEQVKSQPQEWSLTKQNNFLSEMNKPKS

[0104] ESTQSIQTQTQYVSDQSQAIRDRTRLEDGYNWRKYGQKQVKGSENPRSYY

[0105] KCTYPNCPTKKKVERNLEGHITEIVYKGNHSHSKPQNAKKSSSNNFIEAP

[0106] TENSQFESSASFGDDDFEQASSISKSGDDHENEPEAKRWK

[0107] 2. Construction of overexpression vector and functional verification in Arabidopsis thaliana

[0108] 2.1 Test Materials

[0109] 2.1.1 Plant materials

[0110] The transgenic plant material used was marigold (Ma) preserved at the Beijing Academy of Agricultural and Forestry Sciences' Sijiqing Flower Germplasm Resource Conservation Center. It was planted in the field under normal water and fertilizer management, and plump seeds were collected.

[0111] 2.1.2 Strains and Vectors

[0112] Strains: Agrobacterium tumefaciens EHA105 electrocompetent cells were purchased from Beijing Bomed Gene Technology Co., Ltd., and Alternaria strains were preserved in our laboratory.

[0113] Vector: The plant overexpression vector pMDC85 plasmid was preserved in our laboratory.

[0114] 2.1.3 Equipment and Instruments

[0115] PCR instrument (Bio-rad, T100), microcentrifuge (Thermo Scientific Heraeus, Pico 17), -80℃ ultra-low temperature freezer (SANYO, MDF-U53V), -20℃ freezer (Haier, DW-25L262), 4℃ freezer (Haier, BCD-225TMPM), vertical pressure steam sterilizer (Jiangyin Binjiang Medical Equipment Co., Ltd., LS-B75-II), ultra-micro spectrophotometer (Food ALYT Photometer), laser confocal scanning system (Nikon, ECLIPSE Ti), electroshock device (Bio-rad, Gene Pulser Xcell). TM The equipment includes an ultraviolet gel imaging system (Tanon, 1600), a biological microscope (BU200i), a gel electrophoresis apparatus (Beijing Liuyi Instruments, JY-SPCT), a clean bench (Suzhou Purification, SW-CJ-1FD), a water bath (Shanghai Yiheng, DK-8D), and an electronic balance (JA2003).

[0116] 2.1.4 Preparation of test reagents and culture media

[0117] 2.1.4.1 Main Reagents

[0118] Seamless Cloning Kit HD Cloning Kit User Manual (Takara), recovery and purification of the target fragment using a recovery kit ( SV Grl and PCR Clean-Up System (Promega), PCR rapid amplification kit (2×Taq Plus Master Mix, Novizan). DL2000 DNA Marker was purchased from Takara, restriction endonucleases Pac I and Asci I were purchased from New England Biotech, Kanamycin (Kan), Ampicillin (Amp), and Rifampicin (Rif) were purchased from Solarbio, Hygromycin (Hyg) was purchased from Roche, 75% ethanol was purchased from Lylecon, and corn flour medium was purchased from Solarbio.

[0119] 2.1.4.2 Main Culture Medium

[0120] ① Preparation of YEP medium (taking 1L as an example):

[0121]

[0122] Adjust the pH to 5.8–6.0 and sterilize in a vertical pressure steam sterilizer at 117°C for 17 minutes.

[0123] ②MS solid culture medium (1L as an example):

[0124]

[0125] Adjust the pH to 5.8–6.0 and sterilize in a vertical pressure steam sterilizer at 117°C for 17 minutes.

[0126] 2.1.5 PCR primers required for the experiment

[0127] Table 2 Primer sequences

[0128]

[0129] 2.2 Test Methods

[0130] 2.2.1 Construction of overexpression vectors

[0131] 2.2.1.1 Double digestion of pMDC85 vector

[0132] Based on the multiple cloning site characteristics of the pMDC85 expression vector and the TeWRKY33 gene sequence characteristics, upstream and downstream primers for Pac I and Asc I recognition site sequences, namely C85-TeWRKY33-F and C85-TeWRKY33-R (Table 2), were added respectively. The correctly sequenced TeWRKY33 plasmid product was diluted 1000 times as a template, and the reaction system and program were used to amplify the ORF fragment of TeWRKY33 with restriction sites.

[0133] The correctly sequenced empty pMDC85 expression vector was double-digested using restriction endonucleases Pac I and Asc I. The digestion system is as follows:

[0134]

[0135] Enzyme digestion at 37℃ for 6-8 hours, followed by detection by 1% agarose gel electrophoresis, with expected band recovery via gel cutting.

[0136] 2.2.1.2 Amplification of the TeWRKY33 target fragment

[0137] TeWRKY33 target fragment amplification system:

[0138]

[0139] Program structure:

[0140]

[0141] 2.2.1.3 Construction of overexpression vectors using seamless cloning technology

[0142] Seamless cloning technology reaction system:

[0143]

[0144]

[0145] Mix the solution with a pipette, incubate in a metal bath at 50°C for 15 minutes, and then place on ice.

[0146] 2.2.1.4 Transformation

[0147] Escherichia coli DH5α competent cells were transformed using the ligation product heat shock method. The transformed bacterial solution was incubated at 37°C and 180 rpm for 1 hour. After gently mixing the bacterial solution, 300 μL of the solution was evenly spread onto LB agar plates containing Kan resistance using a spreader and incubated upside down at 37°C for 12–16 hours.

[0148] 2.2.1.5 Screening of positive clones and verification by bacterial culture PCR:

[0149] (1) The next day, take out the plate from the previous step and, on a clean bench, use a 2.5 μL pipette tip to pick 5 single colonies from the plate and place them into a 1.5 mL centrifuge tube containing 600 μL LB+Kan. Incubate at 37°C for 3–5 h. Then, use 1 μL of the mixed bacterial solution as a template for double digestion with Pac I and Asc I.

[0150] The reaction system and procedure were as follows: 1 μL bacterial culture; 5 μL 10×Cutsmart Buffer; 1 μL Pac I; 1 μL Asc I; ddH2O to bring the total to 50 μL. Enzyme digestion was performed in a metal bath at 37℃ for 3 h.

[0151] (2) 1% agarose gel electrophoresis was used to detect the presence of the target band. 200 μl of the bacterial solution with the band was taken from the ultra-clean workbench and placed into a 1.5 mL centrifuge tube and sent to Shanghai Sangon Biotech for sequencing.

[0152] (3) After obtaining the sequencing results, use software to compare them and select samples with correct sequences for Agrobacterium transformation.

[0153] 2.2.2 Transformation and Identification of Agrobacterium-Competent Cells

[0154] 2.2.2.1 Transformation of Agrobacterium competent cells with overexpression vector

[0155] Agrobacterium EHA105 competent cells were transformed using electroporation transformation:

[0156] (1) After thawing Agrobacterium competent cells on ice, take 1 μL of recombinant plasmid and add it to 40 μL of bacterial solution. Gently tap the mixture with your fingers and then transfer it to a pre-cooled electroporation cup and cover it with the lid.

[0157] (2) Dry the shock cup and quickly place it into the shock tank for shock. Set the parameters of the shock device as follows: C = 25μF, PC = 200ohm, V = 2400V.

[0158] (3) After the electrocution is completed, quickly put it back into ice, add 700 μL of YEP liquid culture medium, transfer all the bacterial solution in the cup to a clean 1.5 mL centrifuge tube, and shake at 28℃ and 150 rpm for 1 to 2 hours.

[0159] (4) Take 10 μL and dilute it with 200 mL of LB liquid medium. Take 200 μL of the diluted liquid and spread it on YEP solid medium (containing 50 mg / L Kan and 50 mg / L Rif) using a spreader. Incubate at 28°C inverted and in the dark for 2-3 days until colonies grow.

[0160] 2.2.2.2 Identification of recombinant Agrobacterium tumefaciens

[0161] Single colonies were picked and placed into 5 mL of YEP liquid medium containing 50 mg / L Kan and 50 mg / L Rif, and incubated at 28°C with shaking at 150 rpm for 36–48 h. Using the bacterial culture as a template and water as a negative control, positive clones were detected by PCR using primers C85-TeWRKY33-F / C85-TeWRKY33-R (Table 2). The reaction system is as follows:

[0162]

[0163] After mixing, place the mixture in a PCR instrument under the following reaction conditions:

[0164]

[0165] The denaturation to extension steps were performed for 34 cycles. PCR products were detected by 1% agarose gel electrophoresis. Agrobacterium strains that amplified the expected bands were added to 25% sterile glycerol and stored at -80°C for later use.

[0166] 2.2.3 Agrobacterium-mediated transformation of Arabidopsis thaliana

[0167] 2.2.3.1 Transformation of Arabidopsis thaliana using overexpression vector inflorescence inoculation method

[0168] (1) Agrobacterium tumefaciens containing recombinant plasmid stored at -80℃ was streaked onto YEP solid medium (containing 50 mg / L Kan and 25 mg / L Rif) for activation and cultured at 28℃ for 1-2 days until colonies grew.

[0169] (2) Pick a single colony and place it in 5 mL of YEP medium (Kan 50 mg / L, Rif 25 mg / L) and incubate at 28℃ and 180 rpm for 36-48 h.

[0170] (3) Inoculate the bacterial culture at a 1:50 ratio with fresh YEP liquid medium (Kan 50 mg / L, Rif 25 mg / L) and continue culturing until OD500. 600 =1.0-1.2.

[0171] (4) Collect bacterial cells by centrifugation at 5000 rpm at 4℃. Resuspend the bacterial cells in conversion buffer (2.2 g / L MS salt, 50 g sucrose, 0.01 mg / L 6-BA, 1 M KOH to adjust pH to 5.7-5.8, then add 50 μL / L silwet) and dilute to OD. 600 =0.8-1.0.

[0172] (5) Cut off the Arabidopsis thaliana pods and open flowers, and soak the flower buds in bacterial solution for 10-30 seconds (if possible, vacuuming is best, as negative pressure helps the Agrobacterium tumefaciens bacterial solution enter the pollen tube).

[0173] Pressure facilitates the entry of Agrobacterium tumefaciens bacterial solution into the pollen tube.

[0174] (6) Place the Arabidopsis thaliana soaked in bacterial solution in a basin, incubate in the dark for 24 hours, then place it upright and carry out normal culture.

[0175] (7) After the seed pods that have been soaked in the bacterial solution have matured, collect the seeds, dry them in the shade for 7 days, and then screen them.

[0176] 2.2.3.2 Screening of Arabidopsis thaliana seeds

[0177] (1) Prepare the culture medium, 0.1% agarose solution, and sterilize with sterile water by autoclaving. On a laminar flow hood, add 25 μL of hygromycin (5 mg / mL) to 250 mL of sterilized culture medium (approximately 60°C), mix well, and dispense into sterile 2 cm high petri dishes. Dispense the seed culture into 1.5 mL sterile centrifuge tubes.

[0178] (2) Use a pipette to add 1 mL of unopened 75% ethanol to a centrifuge tube containing seeds, shake well, sterilize for 30 seconds, and rinse once with sterile water.

[0179] (3) Follow the same steps as above, disinfect for 10 minutes, and rinse 5 times with 10% sodium hypochlorite solution.

[0180] (4) Use a pipette to add 1 mL of 0.1% agarose solution to the centrifuge tube containing the seeds, shake it up and down to mix, and then invert it.

[0181] (5) Open the tube cap and sprinkle the seeds and solution onto the culture medium. Shake the petri dish by hand to distribute the seeds evenly.

[0182] (6) Seal the culture dish with sealing film and invert it in the light incubator. Set the light time to 16 h / d and the temperature to 25℃, and the dark time to 8 h / d and the temperature to 22℃.

[0183] 2.2.4 Activation and inoculation of Alternaria solani pathogen

[0184] (1) The single-spore paper discs were stored in an ultra-low temperature freezer at -80℃, disinfected in a clean bench, sprayed evenly with 75% alcohol, sterilized with ultraviolet light for 30 minutes, and then vented for 10 minutes.

[0185] (2) Prepare corn flour culture medium: 75g corn flour, 1000mL purified water, pH under natural conditions.

[0186] (3) Microwave heating for 3 to 4 minutes, generally between 3 minutes and 10 seconds and 3 minutes and 13 seconds. If there is solid, it needs to be heated again. Then sterilize with an autoclave.

[0187] (4) First disinfect the gloves and then light the alcohol lamp. Pour the culture medium, about 2 / 3 of the culture dish, and gently push it to spread it evenly. Blow it dry in about 20 minutes.

[0188] (5) Disinfect the tweezers with 75% alcohol. Disinfect the paper plate after each single spore inoculation. Take out the colony and place it in the middle of the petri dish. Cover with the culture cap and seal with the sealing film. Record the date and bacterial number. Place it in an incubator for cultivation. Observe the growth after one week.

[0189] (6) Continue until the culture dish is completely covered. Add 3 mL of sterile water with a pipette. Use an autoclaved oil paintbrush to brush the mycelium in one direction. Dispense the brushed bacterial solution into 5 mL centrifuge tubes.

[0190] (7) Place 30 μL of the brushed bacterial solution on a glass slide and observe with a biological microscope whether spores have been produced in the brushed bacterial solution. The bacterial solution that has produced spores can be stored in a -80℃ refrigerator for a long time.

[0191] (8) After counting with a hemocytometer, centrifugation, resuspension and dilution, a conidial suspension with a concentration of 1×104 CFU mL-1 was prepared for use.

[0192] (9) Select healthy Arabidopsis thaliana seedlings with consistent growth (CK) and Arabidopsis thaliana seedlings transformed with the overexpression vector C85-TeWRKY33. Spray the spore suspension evenly onto the surface of the seedling leaves, and use water as a control.

[0193] (10) After inoculation, the seedlings were placed in an artificial light incubator and cultured in the dark at 25°C for 40 hours. Then, they were cultured under 18 / 6 hours (light / dark) conditions. After 2 days, the seedlings were observed, photographed and recorded. The number of diseased leaves was counted using SPSS 22.0 software.

[0194] 2.3 Results and Analysis

[0195] 2.3.1 Construction of the pMDC85-TeWRKY33 expression vector

[0196] Based on the cloned TeWRKY33 gene cDNA sequence, primers containing Pac I and Asc I restriction endonucleases for recognizing base sequences and protecting bases were added to both ends of its complete ORF. The TeWRKY33 ORF was then amplified, yielding the expected band.

[0197] The empty pMDC85 vector was double-digested with Pac I and Asc I restriction endonucleases. The pMDC85 digested fragments were recovered, and ligation was performed using a seamless cloning kit. The ligation products were transformed into E. coli DH5α competent cells. Plasmids were extracted from resistant colonies and double-digested with Pac I and Asc I. The digestion products were detected by agarose gel electrophoresis. The results are as follows: Figure 2 As shown, bands of the same size as expected were obtained for pMDC85 and TeWRKY33, indicating that the expression vector pMDC85-TeWRKY33 was successfully constructed. Clones with correct enzyme digestion were further sequenced, and after confirming the absence of base errors, they were used for subsequent trials.

[0198] 2.3.2 Recombinant vector pMDC85-TeWRKY33 was transformed into Agrobacterium.

[0199] The constructed pMDC85-TeWRKY33 was transformed into Agrobacterium tumefaciens competent cells EHA105 via electroporation. Anti-resistant clones were initially obtained through Kansas resistance plate selection. Single colonies were picked and subjected to culture PCR verification using C85-TeWRKY33-F and C85-TeWRKY33-R primers, with water as a control. Results are shown below. Figure 3 Electrophoresis of the PCR product of the resistant colony yielded the expected amplified band, while no such band was observed when water was used as a template, indicating that the expression vector pMDC85-TeWRKY33 was successfully introduced into Agrobacterium.

[0200] 2.3.3 Agrobacterium-mediated transformation of Arabidopsis thaliana using overexpression vectors and seed screening and collection

[0201] After selection and soaking, plump and mature Arabidopsis seeds were selected and stored in 1.5 mL centrifuge tubes. Figure 4 a). After sterilization, the seeds were inoculated onto MS medium (containing hygromycin) and cultured. Figure 4b) 140-160 seeds were inoculated for each vector transformation. After 4 days of culture, germination was observed in approximately 50-80 seeds. Of the germinated seeds, some developed brown leaves and ceased growth, while others developed roots and continued to grow. Figure 4 c), approximately 10-20 seeds. Plantlets that rooted in the culture flasks were transplanted and screened by hygromycin and GFP green fluorescent protein PCR detection (…). Figure 5 Two 35S::TeWRKY33::GFP T1 generation lines were obtained. Two lines were randomly selected from each of the two transgenic lines, and the seeds were sown, cultivated and harvested in the same manner to obtain T2 generation positive lines.

[0202] 3.3.4 Activation and inoculation of Alternaria solani pathogen

[0203] The strain was placed on corn culture medium and incubated in a light incubator. Figure 6 As shown in a, after the surface is covered with mycelium, sterile water is added to the laminar flow hood, and the mycelium is gently brushed off with a brush. The laminar flow hood is then placed in a sterile incubator with the lid off and cultured until spores appear. Figure 6 b. Prepare a spore suspension of Alternaria spores at a concentration of 1000 spores / mL. Figure 6 c) Inoculate Arabidopsis seedlings by evenly spraying spore suspension onto the leaves, 2 mL per plant, with sterile water used as a control. After inoculation, keep the plants moist to allow natural disease development at 100% humidity. Maintain humidity for 40 hours, and photograph and record the disease development on the Arabidopsis leaves 48 hours after inoculation.

[0204] 3.3.5 Analysis of the number and phenotypic number of leaf lesions in positive TeWRKY33 transgenic Arabidopsis thaliana lines

[0205] The number of diseased leaves in T2 generation Arabidopsis thaliana lines of wild type and transgenic varieties was statistically analyzed and phenotypic. The results showed that after inoculation with *Alternaria truncatula* pathogen and dark culture for 40 h, followed by alternating 18 h light / 6 h dark culture, a small number of leaves in both 35S::TeWRKY33::GFP transgenic and wild-type Arabidopsis thaliana began to turn yellow and wither after 3 days, but the number of leaf lesions did not change significantly. After 5 days, the number of yellowing and withering leaves in WT was significantly higher than in 35S::TeWRKY33::GFP transgenic Arabidopsis thaliana. After 7 days, the number of yellowing and withering leaves in WT was significantly higher than in transgenic Arabidopsis thaliana. Furthermore, there was no significant difference in flowering time and growth rate between transgenic and wild-type Arabidopsis thaliana. These results suggest that the TeWRKY33 gene has a function of resisting *Alternaria truncatula* infection in Arabidopsis thaliana.

[0206] Table 3. Statistics on the number of lesions after leaf inoculation of wild-type and transgenic Arabidopsis thaliana with Alternaria rubra var. marigold.

[0207]

[0208] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A disease-resistant gene in marigolds TeWRKY33 Its features are: Its nucleotide sequence is shown in SEQ ID NO:1 in the sequence listing.

2. For cloning the disease resistance gene of claim 1 TeWRKY33 The primer pair is characterized by: The primer pair is TeWRKY33-F and TeWRKY33-R, Its nucleotide sequence is shown in SEQ ID NO:2 and SEQ ID NO:3 in the sequence listing.

3. A device containing the disease-resistant gene as described in claim 1 TeWRKY33 The overexpression vector is characterized by: The primer pair used to construct the overexpression vector is: C85-TeWRKY33-F and C85-TeWRKY33-R The nucleotide sequences are shown in SEQ ID NO:4 and SEQ ID NO:5 in the sequence listing; [The text abruptly ends here, likely due to an incomplete sentence or missing information.] TeWRKY33 The nucleotide sequence of the ORF fragment is shown in SEQ ID NO:6 in the sequence listing.

4. A marigold disease-resistant gene as described in claim 1 TeWRKY33 The encoded amino acid is characterized by: Its amino acid sequence is shown in SEQ ID NO:7 in the sequence listing.

5. The marigold disease-resistant gene as described in claim 1 TeWRKY33 Its application in improving plant disease resistance is characterized by: The marigold disease resistance gene TeWRKY33 It can inhibit the growth of Alternaria rubra, the plant being Arabidopsis thaliana or marigold.

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