CN116445504A - Clone of marigold disease-resistant gene TeWRKY2 and application thereof - Google Patents

Abstract

The invention discloses a marigold disease-resistant gene TeWRKY2, the nucleotide sequence of which is shown as SEQ ID NO 1 in a sequence table. According to the invention, the TeWRKY2 gene is cloned from marigold and a vector is constructed to transform the arabidopsis, and the disease resistance function of the TeWRKY2 is verified through phenotypic character and disease analysis of the arabidopsis transgenic and wild plants after being infected by the alternaria marigold. The invention provides powerful theoretical guidance for fundamentally preventing and treating black spot and has direct application value.

Description

Clone of marigold disease-resistant gene TeWRKY2 and application thereof

Technical Field

The invention relates to the field of molecular biology, in particular to cloning and application of a marigold disease-resistant gene TeWRKY 2.

Background

Tagetes erecta L. is an important ornamental plant and is also a functional flower for lutein (lutein) extraction. The marigold black spot disease severely restricts the development of the marigold industry, and mainly damages flowers and stems and leaves, so that plants die early. The tagetes alternaria (Alternaria tagetica) is one of pathogenic fungi of the tagetes alternaria, and research on candidate genes for disease resistance of the tagetes alternaria infection is carried out, so that powerful theoretical guidance is provided for fundamentally preventing and treating the alternaria, and the tagetes alternaria is directly applied to the application value.

Disclosure of Invention

The invention aims at cloning a marigold disease-resistant gene TeWRKY2 and researching the disease-resistant function thereof, and discovers that the marigold disease-resistant gene TeWRKY2 can inhibit the growth of the marigold alternaria. The nucleotide sequence of the marigold disease-resistant gene TeWRKY2 is shown as SEQ ID NO. 1 in a sequence table.

The primer pair for cloning the disease resistance gene TeWRKY2 is TeWRKY2-F and TeWRKY2-R, and the nucleotide sequences of the primer pair are shown as SEQ ID NO. 2 and SEQ ID NO. 3 in a sequence table.

The primer pair for constructing the over-expression vector comprises C85-TeWRKY2-F and C85-TeWRKY2-R, and the nucleotide sequences are shown as SEQ ID NO. 4 and SEQ ID NO. 5 in a sequence table; the nucleotide sequence of the ORF fragment of TeWRKY2 with the enzyme cutting site is shown as SEQ ID NO. 6 in the sequence table. The amino acid coded by the marigold disease-resistant gene TeWRKY2 has an amino acid sequence shown in SEQ ID NO. 7 in a sequence table.

Application of marigold disease-resistant gene TeWRKY2 in improving plant disease resistance is provided.

The application of the invention, wherein the marigold disease-resistant gene TeWRKY2 can inhibit the growth of the marigold Alternaria alternata.

The application of the invention, wherein the plant is arabidopsis thaliana or marigold.

According to the invention, the TeWRKY2 gene is cloned from marigold and a vector is constructed to transform the arabidopsis, and the disease resistance function of the TeWRKY2 is verified through phenotypic character and disease analysis of the arabidopsis transgenic and wild plants after being infected by the alternaria marigold. The invention provides powerful theoretical guidance for fundamentally preventing and treating black spot and has direct application value.

The TeWRKY2 factor of the present invention and its function will be further described with reference to the accompanying drawings.

Drawings

FIG. 1 is a diagram showing the detection of the cloning electrophoresis of the TeWRKY2 gene of the present invention; wherein M: DL2000 DNA markers; 1. 2: a TeWRKY2 amplified fragment;

FIG. 2 is a diagram showing the double cleavage verification of the over-expression vector of the present invention; wherein, M is DL2000 DNA Marker;1:TeWRKY2 gene;

FIG. 3 is a PCR electrophoresis detection chart of the transformed Agrobacterium tumefaciens bacterial liquid in the present invention; wherein M: DL2000 DNA markers; 1: water control; 2. 3: converting the TeWRKY2 into agrobacterium tumefaciens bacterial liquid;

FIG. 4 is a diagram showing the collection and screening process of TeWRKY2 transgenic seeds of the present invention; wherein, the seeds selected in a; b seeds inoculated onto the culture medium; c germinated seeds and long-rooted seeds;

FIG. 5 shows the PCR detection of GFP in Arabidopsis thaliana; m: DL2000 DNA markers; 1. 2: a TeWRKY2 gene band;

FIG. 6 shows a pathogenic bacteria activation process according to the present invention, wherein a: a strain; b: spores; c: bacterial liquid.

Detailed Description

1. Cloning of Tagetes TeWRKY2 Gene

1.1 test materials

1.1.1 plant Material

The disease resistant material and the disease sensitive material Tagetes Ts used in this test were taken from the Japanese greenhouse of the national academy of sciences of agriculture and forestry in Beijing city. Planting in flowerpot (vermiculite: turf=1:2) and normal water and fertilizer management. In the seedling stage, 3-5 well-grown, pest-free and fully developed leaves are collected from 9 to 10 am, and three biological replicates are collected for each sample.

1.1.2 strains and vectors

Strains: coli competent cells DH 5. Alpha. Were purchased from Tiangen Biochemical technology (Beijing) Co.

And (3) a carrier: the cloning vector is Promega companyA carrier.

1.1.3 major reagents, kits and instrumentation

Reagent: RNA extraction kit (MiniBEST Plant RNA Extraction Ki, takara Co., ltd.) and reverse transcription kitIII All-in-one RT SuperMix Perfect for qPCR kit, norwegian Co.), high-fidelity enzyme PCR amplification reagent (2× ++>Max Master Mix, vazyme company), recovery and purification kit of target fragment (/ -for the purpose of purification>SV Grl and PCR Clean-Up System, promega, ligation kit of target fragment and vector (Promega T4 ligase kit, promega), plasmid miniprep kit (AxyPrepTM Plasmid Miniprep Kit, axygen), plant genomic DNA extraction kit (MiniBEST Universal Genomic DNA Extraction Kit, takara), fluorescent quantitative PCR kit (Takara)>Green PCR Kit, qiagen), fluorescent quantitative reverse transcription Kit (HiScript II Q RT SuperMix for qPCR, vazyme), takara LA Taq, DL2000 DNA Marker, etc., purchased from Takara; ampicillin (Amp) was purchased from Solarbio corporation.

Instrument apparatus: PCR instruments (Bio-rad, T100), microcentrifuges (Thermo Scientific Heraeus, pico 17), ultra-low temperature refrigerators at-80 ℃ (SANYO, MDF-U53V), refrigerators at-20 ℃ (Haier, DW-25L 262), refrigerators at 4 ℃ (Haier, BCD-225 TMPM), vertical pressure steam sterilizers (Jiangyin coast medical equipment Co., ltd., LS-B75-II), ultra-micro spectrophotometers (Food ALYT Photometer), ultraviolet gel imaging systems (Tanon, 1600), full temperature concussion boxes (Suzhou Pein laboratory equipment Co., THZ-C-1), fluorescent quantitative PCR instruments (Roche 9700), ion sputtering equipment (Tokyo Hitachi, japan, ID-5), scanning electron microscopes (S-400 n), gel electrophoresis instruments (Beijing Lily Hi instruments, JY-SPCT), water baths (Shanghai Heng, DK-8D), ultra-clean benches (Suzhou purification, SW-CJ-1), metal-C-1, japan (Japan-3, japan-35H, japan-35, etc.), biological systems (PRO-3, japan-35H, etc.

1.1.4 preparation of Medium

Configuration of LB medium (1L for example):

1.1.5 PCR primers required for the assay

Primers were designed using Primer premier 5.0 software, see Table 1, and were synthesized by Shanghai Biotechnology Co., ltd. After centrifugation at 4000rpm, a certain amount of sterile water was added according to the instructions to prepare a 10. Mu.M concentration, and the resulting primers were stored at-20℃for further use.

TABLE 1 primer sequences

1.2 test methods

1.2.1 Extraction of RNA and Synthesis of cDNA

(1) Extraction of RNA

With reference to the Takara MiniBEST Plant RNA Extraction Kit kit instruction, the specific method of operation is as follows:

(1) before the start of the experiment, the tools used for the bench, the pipette, etc. were wiped with 75% alcohol.

(2) Preparing liquid: solution I:1L Buffer RL, 20. Mu.L 50 XDTT Buffer; solution II: 5. Mu.L of 10 XDNase I Buffer, 4. Mu. L Recombinant DNase T, 41. Mu.L of RNase free dH2O.

(3) Fresh plant material or plant material stored at low temperature and taken out from a refrigerator at-80 ℃ below zero, 0.1g of the plant material is taken, and after vibration grinding at low temperature by a grinding instrument, 450 mu L of solution I is added, and the mixture is uniformly mixed up and down and centrifuged at 12000rpm for 5min.

(4) Taking out the supernatant, transferring to a new 1.5mL centrifuge tube, adding 1/2 volume of absolute ethyl alcohol into the supernatant, reversing the supernatant upside down uniformly, rapidly transferring into RNA spin column, standing for 1min, centrifuging at 12000rpm for 1min, and discarding the filtrate.

(5) To RNA spin column, 500. Mu.L Buffer RWA was added, centrifuged at 12000rpm for 30s, and the filtrate was discarded.

(6) 600. Mu.L Buffer RWB (the addition of a specified amount of alcohol was confirmed before use) was added to the RNA spin column, and the mixture was centrifuged at 12000rpm for 30s, and the filtrate was discarded.

(7) 50. Mu.L of solution II was added to the center of the membrane, allowed to stand at room temperature for 15min, DNA was completely digested, then 350. Mu.L of Buffer RWB was added, and centrifuged at 12000rpm for 30s, and the filtrate was discarded.

(8) In RNA spin column, 600. Mu.L Buffer RWB was added, centrifuged at 12000rpm for 30s, and the filtrate was discarded.

(9) After centrifugation for 2min, RNAspin column was placed on a new 1.5mL centrifuge tube and allowed to stand at room temperature for 5min to allow alcohol to evaporate sufficiently.

50. Mu.L of RNase Free H was added ₂ O was placed in the center of the membrane, and the membrane was left to stand at room temperature for 5min, and centrifuged at 12000rpm for 2min, thereby eluting RNA.

The solution in the centrifuge tube of 1.5mL was added to the center of the membrane and allowed to stand for 5min at 12000rpm for 2min to obtain a total RNA solution. 1 μl of each was measured for RNA concentration and quality by ultra-micro spectrophotometry and 1% agarose gel electrophoresis, and the RNA solution was stored in a-80℃refrigerator for use.

(2) cDNA Synthesis

Synthesis of first strand cDNA with reference to Reverse Transcription System kit instructions, the following reagents were added sequentially to a 0.5mL centrifuge tube:

then 80. Mu.L of RNA-free water is added, and after being mixed uniformly, the mixture is preserved at-20 ℃ for standby.

1.2.2 amplification of TeWRKY2 full Length

Amplification primers TeWRKY2-F and TeWRKY2-R of TeWRKY2 gene are designed by using Primer premier 5.0 software; cloning TeWRKY2 with cDNA of Tagetes leaf Ts as template, using 2×Max Master Mix amplified the ORF of TeWRKY 2. The reaction system is as follows:

after light mixing, the mixture was placed in a Bio-rad T100 PCR apparatus and reacted as follows:

the denaturation to extension step was set 34 cycles. After amplification, 2. Mu.L of PCR product was obtained 1% agarose gel electrophoresis was performed.

(1) Recovery and purification of the fragment of interest

The method comprises the following specific steps:

(1) the DNA bands were excised on agarose gels and the gel pieces placed in a 1.5mL sterile centrifuge tube.

(2) Adding binding solution (Membrane Binding Solution) at a ratio of 100mg to 100 μl, standing in a metal bath at 65deg.C for 10min, mixing, standing upside down for 3 times, dissolving gel block completely, taking out, and naturally cooling to room temperature.

(3) Taking a centrifugal adsorption column, sleeving the column into a collecting pipe, moving the solution into the adsorption column, standing for 1min at normal temperature, centrifuging at 12000rpm for 30s, and discarding the filtrate.

(4) mu.L of the rinse solution (Membrane Wash Solution) was added, centrifuged at 12000rpm for 30s, and the waste liquid was discarded.

(5) mu.L of the rinse solution was added, centrifuged at 12000rpm for 30s, and the filtrate was discarded.

(6) After the centrifugal separation at 12000rpm for 1min, the adsorption column is placed in a clean centrifuge tube, and the centrifuge tube is opened and placed in an ultra-clean bench to blow for 10min so as to fully volatilize residual alcohol.

(7) 30 mu L Nuclease Free Water was added dropwise to the center of the adsorption column membrane, and the mixture was allowed to stand at room temperature for 3min and centrifuged at 12000rpm for 1min to elute the DNA solution.

(2) In vitro ligation for recovery of fragments of interest and sequencing vectors

Due to 2×The PCR product of Max Master Mix high-fidelity enzyme is blunt end, unable to directly connect with T carrier, and needs to add A to its end. The end addition A of the recovered product was performed using TakaraTaq enzyme, and the following reagents were added in order:

after being evenly mixed, the mixture is kept at 72 ℃ for 30min and then is preserved at-20 ℃ for standby.

Cloning of the vectors using Promega T4 ligase kitAdding the product obtained by adding A at the tail end, and sequentially adding the following reagents:

after the samples are evenly mixed, the mixture is centrifuged for a short time and is connected with the mixture for 8 to 12 hours at the temperature of 4 ℃.

(3) Conversion and sequencing of ligation products

The transformation of the escherichia coli DH5 alpha is carried out by adopting a heat shock method:

(1) in a sterile operating platform, an LB solid medium with the final concentration of Amp of 50mg/L is prepared and poured into a culture dish.

(2) mu.L of DH 5. Alpha. E.coli competent was thawed on ice. mu.L of ligation product was added to competent cells, gently mixed and then ice-bathed for 30min.

(3) Placing the mixture on ice for 3-5 min immediately after heat shock for 90s in a water bath kettle at 42 ℃, and not shaking a centrifuge tube in the process.

(4) 500. Mu.L of LB liquid medium preheated at 37℃is added, and after light mixing, the mixture is put into a shaking table at 37℃for activation at 180rpm for 60min.

(5) 200. Mu.L of LB liquid medium, 40. Mu.L of 20mg/mL X-Gal and 4. Mu.L of 200g/L IPIG solution were mixed and spread evenly on LB (containing 50mg/L Amp) dishes.

(6) 200 mu L of activated bacterial liquid is evenly coated on a culture dish, and is inversely and light-shielded at 37 ℃ for culturing for 10-14 hours.

(7) In a sterile operation table, 10 single colonies were picked up by a gun head and placed in 2mL of a liquid medium containing LB (containing 50mg/L Amp), shake-cultured at 180rpm for 14-16 h, and then sent to a company for sequencing.

(8) Comparing the sequencing results by DNAMAN, and selecting a sample with correct sequence for plasmid extraction.

(4) Small extraction of plasmids

Referring to the instructions of the AxyPrepTM Plasmid Miniprep Kit kit, the following steps were performed:

(1) into a 1.5mL centrifuge tube, 1mL of the bacterial liquid was poured, and the bacterial cells were collected by centrifugation at 10000rpm for 2min, and the supernatant was discarded.

(2) After 250. Mu.L of Buffer S1 is added to resuspend the bacterial solution, 250. Mu.L of Buffer S2 is added, and the mixture is gently mixed by reversing the solution for 4 to 6 times until the solution is transparent.

(3) 350. Mu.L Buffer S3 was added, gently mixed by reversing the top and bottom for 6 times, and centrifuged at 12000rpm for 10min.

(4) The preparation tube was placed on a 2mL centrifuge tube, the supernatant from step (3) was aspirated into the preparation tube, centrifuged at 12000rpm for 1min, and the filtrate was decanted.

(5) 500buffer W1 was added to the preparation tube, centrifuged at 12000rpm for 1min, and the filtrate was decanted.

(6) 700buffer W2 was added and centrifuged at 12000rpm for 1min, and the filtrate was decanted. This operation was repeated once.

(7) Centrifuging at 12000rpm for 30min, placing the preparation tube into a new centrifuge tube, standing at a super clean bench for 10min to completely evaporate alcohol, adding 50 μl of Eluent into the center of the preparation tube membrane, standing at room temperature for 1min, centrifuging at 12000rpm for 1min, and storing the obtained plasmid solution in a refrigerator at-20deg.C.

1.3 results and analysis

PCR amplification of the TeWRKY2 gene ORF was performed using cDNA of the Tagetes leaves obtained by reverse transcription as a template and TeWRKY2-F and TeWRKY2-R as primers. The PCR product is subjected to electrophoresis detection to obtain 2 specific bands. The specific bands are recovered and purified and then connected with a cloning vector, and the sequencing is carried out, and the sequencing result shows that: the full-length 1600bp sequence of the TeWRKY2 gene (FIG. 1). The sequence was found to have an open reading frame (Open Reading Frame, ORF) of 1236bp, encoding 411 amino acids.

TeWRKY2 sequence:

CAACTTACCTATAAGGGGGCGAATTGGGCCCGACGTCGCATGCTCCCGGCCGCCATGGCGGCCGCGGGAATTCGATTGGTGCTGATACCAAGGAAAATTAAATCACAATGCAACCAAGGTGGTATTGAAATTACACTAATGGACAAGTCATCTGACAATGTGGAGTTGACCAATGACTCCAACAATAGAGACCTGTCTCATCAAGAAACAAATTCAGAGTCTATAAAAGTTAAGGAGCCTCATGATAATTCTAATCAGGAAGGAAGTTCCACAACCGTACTATCTCACAAAGAGTTGGATGATCAAAATGACAAACCTACTCTTCATACGGAAAGGGTTGCTGGATCAGAATCTTTTCAAGAAAAGGTCACTAACACTTCACAGCAAACACCTGGATCAGAACCTGATAATGAAAATAATAGTGTGCTATTAAGGACCGAGAAAGGGCTTGATAAATTACCACTAAGACGTAGTGCTGACAGTGTTACAGTTGCACAATCAGCACCTTCTGATCAAGGTGTCACTTTCTCAAAACTACCTGAAAAACCTACTGGTGATGGATATAACTGGAGAAAATATGGTCAAAAGCTTGTAAAAGGGAATACATTTGTACGAAGCTATTATAAATGTACATTTGCTAATTGCCCGGCAAGAAAACAGGTGGAACGTTCAAATGATGGGATTATTACAGAAATAAATTACTTATGGAAGCATGAACACTCTAAACCTTCGCATACACTTGTTAAAGGCTCGGCTTTTGCTCTTCCTGTTTCTGATAAGCCTTCAGAAGACTATTCATCTGTGCTTCCTGCAACAACTCATGATCATAAGGTGTCAGAAACCGACACACGTCTGCTTGTGGTGGTTCCTGTAAGTGACAAAAATGTGGAAACTTCTATTAAGAGCAATGAAATGAAAAGTGAAGTTGATAATGATATATCATCAGGCTCAAAGAGACAGAAGAGAGAGACTTGTAGTGTGAACGAAGGTATTTCAACCAAAACAAACTGTGAGCCGCGAGTAGTTGTTCAGACAACAAGTGTAGTTGATATTGTAAATGATGGCTATCGGTGGCGCAAATATGGGCAGAAATTAGTGAAAGGCAATCCTAACCCAAGGAGTTACTATCGGTGCACAAGCGCTGGTTGTGCTGCCAAAAAGCATGTAGAACGGGCATCTCATGATGAAAAGGTGGTAATTACAACTTATGAGGGGCGCCATGATCATGACATGCCTTCTGGTGGTCGAACTGTTACTCAAAACATGCCAGGGACTGGGACTGGGAGTGGCCCAGCATCTATTGAAAAGGATGGTTCAAGACCTCAGCCAGAATCTAGTGGTATGGAAATGGTTCTTCATGTTAGTGCTACTTGAGGCGCAAGTACTACAGTTGAGTAATTTATCATGCCGTCGACTTGTAGATCATAATGATAGTCACATAGTGTTGTTATATACCTATAAATCTTGATGATTTGCAAATTAAAGATTGGTGTATGTGGGTGAATCACTAGTGAATTCGCGGCCGCCTGCAGGTCGACCATATGGGAGAGCTCCCAACGCGTTGGATGCATAGCTTGAGTATTCTTATAGGGTTCACCCC

TeWRKY2-ORF

ATGGACAAGTCATCTGACAATGTGGAGTTGACCAATGACTCCAACAATAGAGACCTGTCTCATCAAGAAACAAATTCAGAGTCTATAAAAGTTAAGGAGCCTCATGATAATTCTAATCAGGAAGGAAGTTCCACAACCGTACTATCTCACAAAGAGTTGGATGATCAAAATGACAAACCTACTCTTCATACGGAAAGGGTTGCTGGATCAGAATCTTTTCAAGAAAAGGTCACTAACACTTCACAGCAAACACCTGGATCAGAACCTGATAATGAAAATAATAGTGTGCTATTAAGGACCGAGAAAGGGCTTGATAAATTACCACTAAGACGTAGTGCTGACAGTGTTACAGTTGCACAATCAGCACCTTCTGATCAAGGTGTCACTTTCTCAAAACTACCTGAAAAACCTACTGGTGATGGATATAACTGGAGAAAATATGGTCAAAAGCTTGTAAAAGGGAATACATTTGTACGAAGCTATTATAAATGTACATTTGCTAATTGCCCGGCAAGAAAACAGGTGGAACGTTCAAATGATGGGATTATTACAGAAATAAATTACTTATGGAAGCATGAACACTCTAAACCTTCGCATACACTTGTTAAAGGCTCGGCTTTTGCTCTTCCTGTTTCTGATAAGCCTTCAGAAGACTATTCATCTGTGCTTCCTGCAACAACTCATGATCATAAGGTGTCAGAAACCGACACACGTCTGCTTGTGGTGGTTCCTGTAAGTGACAAAAATGTGGAAACTTCTATTAAGAGCAATGAAATGAAAAGTGAAGTTGATAATGATATATCATCAGGCTCAAAGAGACAGAAGAGAGAGACTTGTAGTGTGAACGAAGGTATTTCAACCAAAACAAACTGTGAGCCGCGAGTAGTTGTTCAGACAACAAGTGTAGTTGATATTGTAAATGATGGCTATCGGTGGCGCAAATATGGGCAGAAATTAGTGAAAGGCAATCCTAACCCAAGGAGTTACTATCGGTGCACAAGCGCTGGTTGTGCTGCCAAAAAGCATGTAGAACGGGCATCTCATGATGAAAAGGTGGTAATTACAACTTATGAGGGGCGCCATGATCATGACATGCCTTCTGGTGGTCGAACTGTTACTCAAAACATGCCAGGGACTGGGACTGGGAGTGGCCCAGCATCTATTGAAAAGGATGGTTCAAGACCTCAGCCAGAATCTAGTGGTATGGAAATGGTTCTTCATGTTAGTGCTACTTGA

amino acid sequence:

MDKSSDNVELTNDSNNRDLSHQETNSESIKVKEPHDNSNQEGSSTTVLSH

KELDDQNDKPTLHTERVAGSESFQEKVTNTSQQTPGSEPDNENNSVLLRT

EKGLDKLPLRRSADSVTVAQSAPSDQGVTFSKLPEKPTGDGYNWRKYGQK

LVKGNTFVRSYYKCTFANCPARKQVERSNDGIITEINYLWKHEHSKPSHT

LVKGSAFALPVSDKPSEDYSSVLPATTHDHKVSETDTRLLVVVPVSDKNV

ETSIKSNEMKSEVDNDISSGSKRQKRETCSVNEGISTKTNCEPRVVVQTT

SVVDIVNDGYRWRKYGQKLVKGNPNPRSYYRCTSAGCAAKKHVERASHDE

KVVITTYEGRHDHDMPSGGRTVTQNMPGTGTGSGPASIEKDGSRPQPESS

GMEMVLHVSAT

2. construction of overexpression vector and functional verification of transformed Arabidopsis thaliana

2.1 test materials

2.1.1 plant Material

The plant material used for the transgene is marigold Ts which is preserved in a holly flower germplasm resource preservation center of the national academy of sciences of agriculture and forestry in Beijing city. Planting in a field, managing normal water and fertilizer, and collecting full seeds.

2.1.2 strains and vectors

Strains: agrobacterium tumefaciens EHA105 electrocuting competent cells were purchased from Beijing Bomaide Gene technologies Inc., and Alternaria alternata strain was saved for the present laboratory.

And (3) a carrier: the plant over-expression vector pMDC85 plasmid is stored in the laboratory.

2.1.3 apparatus and instruments

PCR instrument (Bio-rad, T100), microcentrifuge (Thermo Scientific)Heraeus, pico 17), -80℃ultra-low temperature refrigerator (SANYO, MDF-U53V), -20℃refrigerator (Haier, DW-25L 262), -4℃refrigerator (Haier, BCD-225 TMPM), vertical pressure steam sterilizer (LS-B75-II, jiangyin coastal medical devices Co., ltd.), ultra-micro spectrophotometer (Food ALYT Photometer), confocal laser scanning System (Nikon, ECLIPSE Ti), electric shock instrument (Bio-rad, gene Pulser Xcell) ^TM ) Ultraviolet gel imaging system (Tanon, 1600), biological microscope (BU 200 i) gel electrophoresis apparatus (Beijing Liuyi apparatus, JY-SPCT), ultra clean bench (Suzhou purification, SW-CJ-1 FD), water bath (Shanghai Yiheng, DK-8D), electronic balance (JA 2003), etc.

2.1.4 test reagents and Medium preparation

2.1.4.1 main reagent

Seamless cloning kitHD Cloning Kit User Manual, takara corporation), the target fragment is recovered and purified by using a recovery kit (/ -a ]>SV Grl and PCR Clean-Up System, promega Co.) and PCR Rapid amplification kit (2X Taq Plus Master Mix, norpraise Co.). DL2000 DNAMaroker was purchased from Takara, restriction enzymes Pac I and Asc I were purchased from England Biotechnology, N.Y., kanamycin (Kanamycin, kan), ampicillin (Ampicillin, amp), rifampin (Rrifampiciin, rif) were purchased from Solaro, hygromycin (Hygromycin, hyg) were purchased from Roche, 75% alcohol was purchased from Li Erkang, and corn meal medium was purchased from soraro.

2.1.4.2 main culture medium

(1) Configuration of YEP medium (1L for example):

the pH value is adjusted to 5.8-6.0, and the mixture is placed in a vertical pressure steam sterilizer for sterilization at 117 ℃ for 17min.

(2) MS solid medium (1L for example):

the pH value is adjusted to 5.8-6.0, and the mixture is placed in a vertical pressure steam sterilizer for sterilization at 117 ℃ for 17min.

2.1.5 PCR primers required for the assay

TABLE 2 primer sequences

2.2 test methods

2.2.1 construction of the overexpression vector

2.2.1.1 Double enzyme digestion of pMDC85 vector

According to the characteristics of multiple cloning sites of the pMDC85 expression vector and the characteristics of the TeWRKY2 gene sequence, respectively adding upstream and downstream primers of Pac I and Asc I recognition site sequences: C85-TeWRKY2-F and C85-TeWRKY2-R (Table 2); and the sequencing correct TeWRKY2 plasmid product is diluted 1000 times as a template, and the reaction system and the procedure amplify the ORF fragment of TeWRKY2 with the enzyme cutting site.

Double digestion treatment is carried out on the pMDC85 empty expression vector which is sequenced correctly by using restriction enzymes Pac I and Asc I, and the digestion system is as follows:

and (3) performing enzyme digestion at 37 ℃ for 6-8 hours, and performing 1% agarose gel electrophoresis to detect, wherein the expected band is cut and recovered.

2.2.1.2 Amplification of the TeWRKY2 fragment of interest

TeWRKY2 target fragment amplification System:

program system:

2.2.1.3 seamless cloning techniques construction of an overexpression vector

Seamless cloning technology reaction system:

the solution was mixed with a pipette, the metal bath was set at 50℃for 15min, and then placed on ice.

2.2.1.4 conversion

And (3) transforming the competent cells of the escherichia coli DH5 alpha by adopting a ligation product heat shock method, and shaking the transformed bacterial liquid at 180rpm at 37 ℃ for 1h. After the bacterial liquid is flicked and mixed evenly, 300 mu L of bacterial liquid is sucked and evenly coated on LB plate culture medium containing Kan resistance by a coater, and the bacterial liquid is cultured for 12 to 16 hours at 37 ℃ in an inverted way.

2.2.1.5 positive clone selection and bacterial liquid PCR verification:

(1) The next day the plate was removed, 5 monoclonal colonies were picked from the plate with a 2.5. Mu.L gun head on an ultra clean bench, and placed into a 1.5mL centrifuge tube containing 600. Mu.L LB+Kan, and shaken at 37℃for 3-5 h. Then Pac I and Asc I double enzyme digestion detection is carried out by taking 1 mu L of mixed bacterial liquid as a template.

The reaction system and the reaction procedure are as follows: 1 mu L of bacterial liquid; 10×Cutsmart Buffer 5. Mu.L; pac I1. Mu.L; asc I1 μl; ddH ₂ O was replenished to 50. Mu.L. And performing metal bath enzyme digestion for 3 hours at 37 ℃.

(2) Detecting whether a target strip exists or not by 1% agarose gel electrophoresis, sucking 200 μl of bacterial liquid with the strip in an ultra-clean workbench, putting the bacterial liquid into a 1.5mL centrifuge tube, and sending the bacterial liquid to Shanghai to carry out sequencing.

(3) After the sequencing result is obtained, software is used for comparison, and a sample with correct sequence is selected for agrobacterium transformation.

2.2.2 transformation and identification of Agrobacterium competent cells

2.2.2.1 transformation of Agrobacterium competent cells with the overexpression vector

Transformation of Agrobacterium EHA105 competent cells by electric shock transformation:

(1) After the agrobacterium competent cells are thawed on ice, 1 mu L of recombinant plasmid is added into 40 mu L of bacterial liquid, and the mixture is transferred into a precooled electric shock cup after being mixed by flicking with fingers, and the cup cover is covered.

(2) The electric shock cup is quickly put into the electric shock groove for electric shock after being wiped dry, and the electric shock instrument sets parameters: c=25μf, pc=200ohm, v=2400V.

(3) After the electric shock is finished, the bacteria liquid in the cup is completely transferred into a clean 1.5mL centrifuge tube by adding 700 mu L of YEP liquid culture medium, and the bacteria liquid is oscillated for 1-2 hours at 28 ℃ at 150 rpm.

(4) After 10. Mu.L of the culture medium was diluted with 200mL of LB liquid medium, 200. Mu.L of the diluted liquid was spread on a YEP solid medium (containing 50mg/L Kan and 50mg/L Rif) by a spreader, and the culture was inverted and light-shielded at 28℃for 2 to 3 days until colonies were grown.

2.2.2.2 identification of recombinant Agrobacterium

Single colonies were picked into 5mL YEP liquid medium containing 50mg/L Kan and 50mg/L Rif and shake cultured at 28℃for 36-48h at 150 rpm. Taking bacterial liquid as a template, taking water as a negative control, and taking primers C85-TeWRKY2-F/C85-TeWRKY2-R (table 2) as bacterial liquid for PCR detection of positive clones, wherein the reaction system is as follows:

after mixing, the mixture was placed in a PCR apparatus under the following reaction conditions:

denaturation to extension step was performed for 34 cycles. The PCR products are detected by 1% agarose gel electrophoresis, 25% sterile glycerol is added into the agrobacterium strain amplified to obtain the expected band, and the agrobacterium strain is preserved at-80 ℃ for standby.

2.2.3 Agrobacterium transformation of Arabidopsis thaliana

2.2.3.1 over-expression vector inflorescence dip-dyeing method for transforming Arabidopsis thaliana

(1) The Agrobacterium tumefaciens bacteria solution containing the recombinant plasmid stored at-80℃was streaked on YEP solid medium (containing Kan 50mg/L, rif 25 mg/L), and cultured at 28℃for 1-2d until colonies were grown.

(2) The monoclonal colonies were picked and placed in 5mL of YEP medium (Kan 50mg/L, rif 25 mg/L) and cultured at 28℃for 36-48 hours at 180 rpm.

(3) Inoculating new YEP liquid culture medium (Kan 50mg/L, rif 25 mg/L) into bacterial liquid 1:50, and continuously culturing until OD ₆₀₀ ＝1.0-1.2。

(4) The cells were collected by centrifugation at 5000rpm at 4 ℃. The transformed solution (2.2 g/L MS salt, 50g sucrose, 0.01 mg/L6-BA, 1MKOH adjusted pH=5.7-5.8, 50uL/L silwet) was added to resuspend the cells and diluted to OD ₆₀₀ ＝0.8-1.0。

(5) Cutting off the pod and the flowers of Arabidopsis thaliana, soaking the buds with the bacterial liquid for 10-30s (conditional preferably vacuumized, negative pressure is beneficial to the entry of the bacterial liquid of agrobacterium into pollen tubes).

The pressure is favorable for the agrobacterium tumefaciens bacteria liquid to enter the pollen tube.

(6) Placing the arabidopsis soaked in the bacterial liquid in a basin, shading and culturing for 24 hours, and then placing the arabidopsis in the right place for normal culture.

(7) After the seed pods immersed with partial rosettes of the bacterial liquid are mature, collecting seeds, drying in the shade and placing for 7d, and then screening.

2.2.3.2 screening of Arabidopsis seeds

(1) The culture medium and 0.1% agarose solution were prepared, and autoclaved in sterile water. On an ultra clean bench, 25. Mu.L of hygromycin (5 mg/mL) was added to 250mL of the sterilized medium (about 60 ℃) and then mixed and dispensed into sterile 2cm high dishes. Seeds were dispensed into 1.5mL sterile centrifuge tubes.

(2) The unsealed 75% ethanol was sucked up by a pipette and added into a centrifuge tube filled with seeds in 1mL, the mixture was shaken up and down, sterilized for 30s, and rinsed 1 time with sterile water.

(3) The steps are the same, disinfection is carried out for 10min, and 10% sodium hypochlorite solution is used for washing for 5 times.

(4) 1mL of 0.1% agarose solution is sucked by a pipette and added into a centrifuge tube filled with seeds, and the centrifuge tube is inverted after shaking up and down uniformly.

(5) The tube lid was opened and seeds and solution were spread on the medium. The dish was shaken by hand to allow even distribution of seeds.

(6) Sealing the culture dish with sealing film, and inverting in the illumination incubator. The illumination time of 16h/d is set, the temperature is 25 ℃, the dark time of 8h/d is set, and the temperature is 22 ℃.

2.2.4 activation and inoculation of Alternaria alternata pathogens

(1) Storing the single spore paper dish in an ultralow temperature refrigerator at-80 ℃, sterilizing an ultra-clean workbench, uniformly spraying 75% alcohol, sterilizing by an ultraviolet lamp for 30min, and exhausting for 10min.

(2) Preparing corn meal culture medium, corn meal 75g, purified water 1000mL, and pH in natural state.

(3) Heating for 3-4 min in a microwave oven for 3min 10 s-3 min 13s, and sterilizing in an autoclave if solid is needed to be reheated.

(4) Firstly, sterilizing the glove, then igniting the alcohol lamp, pouring the culture medium, slightly pushing about 2/3 of the culture dish to uniformly spread the culture dish, and drying for about 20 minutes.

(5) Forceps were sterilized with 75% alcohol and each single dish was sterilized. The colony is clamped out and placed in the middle of a culture dish, a sealing film is covered on a culture cover for sealing, the date and the bacterial number are recorded, the culture dish is placed in an incubator for culture, and the growth condition is observed after one week.

(6) Until the whole culture dish is full, 3mL of sterile water is dripped into the culture dish by a pipetting gun, hyphae are brushed by an oil painting brush sterilized by an autoclave in one direction, and the brushed bacterial liquid is split-packed by a 5mL centrifuge tube.

(7) 30 mu L of the brushed bacterial liquid is observed on a glass slide by a biological microscope to see whether spores are generated in the brushed bacterial liquid, and the bacterial liquid with the spores can be stored in a refrigerator at the temperature of minus 80 ℃ for a long time.

(8) After counting by a blood cell counting plate, centrifuging, re-suspending and diluting, the conidium suspension with the concentration of 1X 104CFUmL-1 is prepared for standby.

(9) Selecting strong Arabidopsis seedlings (CK) with consistent growth, C85-TeWRKY2 Arabidopsis seedlings transferred into an overexpression vector, and uniformly spraying spore suspension on the surfaces of seedling leaves, wherein clear water is smeared as a control.

(10) The inoculated seedlings are placed in an artificial illumination incubator for 40 hours in the dark at 25 ℃, then are cultivated under the condition of 18/6 hours (illumination/darkness), the shooting record is observed after 2 days, and the number of the leaf diseases is counted by SPSS 22.0 software.

2.3 results and analysis

2.3.1 construction of pMDC85-TeWRKY2 expression vector

According to the cloned cDNA sequence of the TeWRKY2 gene, primers for identifying base sequences and protecting bases by Pac I and Asc I restriction enzymes are added at two ends of the complete ORF, and the TeWRKY2 ORF is amplified to obtain an expected band.

Double digestion of the empty pMDC85 vector with Pac I and Asc I restriction enzymes, recovery of pMDC85 restriction fragments, ligation using a seamless cloning kit and transformation of the ligation product into E.coli DH 5. Alpha. Competent, plasmid extraction of the resistant colonies and double digestion with Pac I and Asc I, detection of the digested product by agarose gel electrophoresis, as shown in FIG. 2, gave bands of identical expected sizes for pMDC85 and TeWRKY2, respectively, indicating successful construction of the expression vector pMDC85-TeWRKY2, further sequencing of the digested correct clone, confirmation of no base errors for subsequent testing.

2.3.2 transfer of recombinant vector pMDC85-TeWRKY2 into Agrobacterium

The constructed pMDC85-TeWRKY2 is transferred into an agrobacterium competent cell EHA105 by an electric shock method, a resistant clone is obtained preliminarily by Kan resistance plate screening, single colonies are selected and subjected to bacterial liquid PCR verification by using C85-TeWRKY2-F and C85-TeWRKY2-R primers, water is used as a contrast, the result is shown in figure 3, the PCR product of the resistant colonies is electrophoresed to obtain an expected amplified band, and water is used as a template without the band, so that the expression vector pMDC85-TeWRKY2 is successfully introduced into agrobacterium.

2.3.3 over-expression vector mediated Agrobacterium transformation Arabidopsis thaliana and seed selection and Collection

After arabidopsis seeds were selected and soaked, the full mature seeds were selected and stored in a 1.5mL centrifuge tube (fig. 4 a). After seed sterilization, the seeds were inoculated on MS medium (hygromycin containing) and cultured (FIG. 4 b) 140-160 seeds per vector transformation, after 4 days of culture, some seeds were observed to germinate, about 50-80. After the germinated seeds were further cultured, part of the leaves of the seeds were brown, and no longer grown, but there were also seeds with growing roots (fig. 4 c), about 10 to 20 grains. Plants rooted in the flask were transplanted and 2 35S:: teWRKY 2::: GFP T were obtained by hygromycin screening and GFP green fluorescent protein PCR detection (FIG. 5) ₁ And (5) strain substitution. Randomly selecting 2 lines from the 2 transgenic lines, sowing, cultivating and harvesting seeds in the same way, and obtaining T ₂ Generation positive lines.

3.3.4 activation and inoculation of Alternaria alternata pathogens

Placing the strain on a corn culture medium, placing the corn culture medium on an illumination incubator for culturing as shown in fig. 6a, adding a sterile water brush pen to the super clean bench after the surface of the strain is full of mycelia, gently brushing the mycelia, and placing the strain on the sterile incubator for uncapping and culturing until spores grow out as shown in fig. 6b. Alternaria alternata spores are prepared into spore suspension with the concentration of 1000 spores/mL (figure 6 c), and the spore suspension is uniformly sprayed on leaves of arabidopsis seedling for inoculation, wherein each strain is 2mL, and inoculated sterile water is used as a control. After inoculation, the plants are moisturized to cause natural disease, and the humidity is 100%. Moisturizing for 40h, photographing 48h after inoculation, and recording and observing the disease condition of arabidopsis leaves.

3.3.5 analysis of leaf lesions and phenotypes of the TeWRKY2 transgenic Arabidopsis positive lines

Statistical and phenotypic analysis of the number of leaves of T2 generation Arabidopsis lines of wild type and transgenic varieties was performed. The results show that after the dark culture for 40h after inoculating the tagetes erecta alternan and the dark alternate culture for 18h light/6 h, 35S:: teWRKY2:: GFP transgenic arabidopsis thaliana and wild type arabidopsis thaliana have few leaves which start to turn yellow and dry up, and the change of the number of leaf lesions is not obvious. After 5d, the number of leaf dry yellowing of WT was significantly higher than 35S:: teWRKY2:: GFP transgenic Arabidopsis. After 7d, the WT leaves were significantly more dry and yellow than transgenic arabidopsis. Furthermore, there was no significant difference in flowering time and growth rate by recording the statistics of transgenic arabidopsis thaliana and wild type arabidopsis thaliana. The above results speculate that the TeWRKY2 gene has a function of combating infection of alternaria marigold in arabidopsis.

TABLE 3 statistics of lesion number after Vaccination of wild type and transgenic Arabidopsis leaves with Aztec marigold

The above examples are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solution of the present invention should fall within the scope of protection defined by the claims of the present invention without departing from the spirit of the present invention.

Claims (7)

1. A marigold disease-resistant gene TeWRKY2, which is characterized in that: the nucleotide sequence of TeWRKY2 is shown as SEQ ID NO. 1 in the sequence table.

2. A primer pair for cloning the disease resistance gene TeWRKY2 of claim 1, characterized in that: the primer pairs are TeWRKY2-F and TeWRKY2-R, and the nucleotide sequences of the primer pairs are shown as SEQ ID NO. 2 and SEQ ID NO. 3 in a sequence table.

3. An overexpression vector containing the disease-resistant gene TeWRKY2 according to claim 1, characterized in that: the primer pairs for constructing the over-expression vector are C85-TeWRKY2-F and C85-TeWRKY2-R, and the nucleotide sequences of the primer pairs are shown as SEQ ID NO. 4 and SEQ ID NO. 5 in a sequence table; the nucleotide sequence of the ORF fragment of TeWRKY2 with the enzyme cutting site is shown as SEQ ID NO. 6 in the sequence table.

4. An amino acid encoded by the marigold disease-resistant gene TeWRKY2 of claim 1, which is characterized in that: the sequence of the amino acid is shown as SEQ ID NO. 7 in the sequence table.

5. Application of marigold disease-resistant gene TeWRKY2 in improving plant disease resistance is provided.

6. The use according to claim 5, characterized in that: the marigold disease-resistant gene TeWRKY2 can inhibit the growth of Alternaria marigold.

7. The use according to claim 6, characterized in that: the plant is Arabidopsis thaliana or Tagetes erecta.