CN115948460A - Pepper epidemic disease resistance related gene CaWRKY66 and application thereof - Google Patents
Pepper epidemic disease resistance related gene CaWRKY66 and application thereof Download PDFInfo
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Abstract
The invention discloses a pepper phytophthora disease resistance related geneCaWRKY66And applications thereof. The above-mentionedCaWRKY66The nucleotide sequence of the gene is shown as SEQ IDNo.1, and the coded protein sequence is shown as SEQ ID No. 2. The above-mentionedCaWRKY66The gene has a key effect on plant disease resistance, particularly phytophthora capsici resistance. SilencingCaWRKY66The gene can reduce the disease resistance of pepper to phytophthora capsici and over-expressCaWRKY66The gene can promote the disease resistance of pepper and arabidopsis to phytophthora capsici. Therefore, the temperature of the molten metal is controlled,CaWRKY66the gene is used as a positive regulatory factor and can endow different plants with disease resistance to phytophthora capsici.
Description
Technical Field
The invention belongs to the technical field of agricultural biology, and particularly relates to a pepper phytophthora blight resistance related geneCaWRKY66And applications thereof.
Background
Hot pepper (A)Capsicum annuum) Is an important vegetable and industrial raw material crop worldwide. The seeding area of the pepper in China is stabilized at 200 ten thousand hm all year round in recent years 2 In the above way, the planting area and the yield benefit are the first vegetable in China. In agricultural production, the plant is Phytophthora capsici (A)Phytophthoracapsici) The pepper epidemic disease caused frequently occurs, the yield is reduced by 20-30% if the pepper epidemic disease is slight, the yield is reduced by more than 80% if the pepper epidemic disease is severe, and even the pepper epidemic disease is completely absorbed, so that the development of the pepper industry is severely restricted.P. capsiciThe host range is very wide, and besides solanaceous plants such as hot peppers, tomatoes and the like, the host can also infect hundreds of crops such as cucurbits (such as watermelons, cucumbers, pumpkins and the like) and beans (such as lima beans) and the like, thereby causing destructive disasters in agricultural production.
The plant forms and develops a set of complex and efficient regulation and control network through the coevolution with pathogenic bacteria in the natural habitat of the plant, and realizes the effective regulation and control of disease-resistant reaction by finely regulating the expression of disease-resistant defense related genes. In the process, the transcription factor is used as a regulation factor for integrating an upstream signal and regulating the expression of a series of downstream defense-related genes, and plays an important regulation role in the disease resistance response of plants. WKRY is an important transcription factor family in plants, and different members of the WKRY contain 1-2 conserved WRKY functional domains, and can recognize and combine with W-box (TTGACT/C) elements on a target gene promoter so as to regulate and control the expression of a target gene. WKRY transcriptionThe factors are widely involved in the response or resistance process of plants to biotic stress such as bacteria, fungi, oomycetes and viruses and abiotic stress such as drought, high temperature and freeze injury. However, with respect to pepper WRKY transcription factor responseP. capsiciThe research on disease resistance is still few.
Disclosure of Invention
The invention aims to provide a pepper phytophthora blight resistance related geneCaWRKY66And the application thereof provides gene resources for the disease-resistant genetic improvement of the pepper.
In order to achieve the purpose, the invention adopts the following technical scheme:
phytophthora resistance related gene of capsicumCaWRKY66The cloning method comprises the following steps:
according to the species Capsici fructus and Phytophthora capsici (P. capsici) The interactive RNA-seq transcriptome analysis and the fluorescent quantitative PCR verification discover a pepper WRKY transcription factor coding gene which is significantly up-regulated and expressed in the host infection processCaWRKY66(ii) a To be provided withP. capsiciUsing cDNA infecting pepper root system sample as template, amplifying target gene open reading frame full-length sequence by KOD high fidelity enzyme PCR, constructing the sequence into plant expression vector pBinGFP2 by using In-fusion method, sequencing and analyzing to obtain pepper disease-resistant related geneCaWRKY66SaidCaWRKY66The nucleotide sequence of the gene is shown as SEQ ID No.1, and the coded protein sequence is shown as SEQ ID No. 2.
The pepper epidemic disease resistance related gene is containedCaWRKY66The recombinant expression vector, the transgenic cell line or the recombinant strain of (1).
Used for amplifying the related gene of the pepper disease resistanceCaWRKY66The primer pair of (4), the sequences of the primer pair are as follows:
CaWRKY66-F：5’-ctgtacaagggtacccccATGGAGGCTTCTTTCAA-3’，
CaWRKY66-R：5’-agaggatccgtcgaccccTCAGAGTGTGTAATCTTTGT-3’。
the pepper epidemic disease resistance related geneCaWRKY66The real-time fluorescent quantitative primer pair has the following sequences:
CaWRKY66-qF：5’-CAACGATGAACGATGGATGC-3’，
CaWRKY66-qR：5’-TGATGTGGCTGAAAGAGGAA-3’。
the pepper epidemic disease resistance related geneCaWRKY66In regulating phytophthora capsici of plants (Phytophthoracapsici，P. capsici) Use in resistance;
in particular, silencing is achieved by using virus-mediated gene silencing techniquesCaWRKY66The gene can reduce the pepper pairP. capsiciThe disease resistance of the composition; transient overexpression in pepper leavesCaWRKY66Gene capable of improving pepper pairsP. capsiciDisease resistance of (1); stable overexpression in ArabidopsisCaWRKY66Gene capable of improving arabidopsis thaliana plant pairP. capsiciThe disease resistance of (1).
The invention has the following remarkable advantages:
the invention provides a pepper epidemic disease resistance related geneCaWRKY66And applications thereof. The present inventors have discovered that silencing is achieved by using a virus-mediated gene silencing techniqueCaWRKY66The gene can obviously reduce the pepper pairsP. capsiciThe disease resistance of the pepper can be improved by transiently over-expressing the geneP. capsiciThe disease resistance of (1). Stable overexpression in ArabidopsisCaWRKY66The gene can also obviously improve the arabidopsis plant pairP. capsiciResistance of (2). Therefore, the temperature of the molten metal is controlled,CaWRKY66the gene is used as a positive regulatory factor and can endow different plant pairs withP. capsiciThe disease resistance of (1).
Drawings
FIG. 1:CaWRKY66the gene is inP. capsiciWhen the host is infected for 3 h, the expression is obviously up-regulated.
FIG. 2: silencingCaWRKY66Genetically reduced pepper pairsP. capsiciThe disease resistance of (1).
FIG. 3: transient overexpressionCaWRKY66Gene-enhanced pepper pairP. capsiciThe disease resistance of (1).
FIG. 4: stable overexpressionCaWRKY66Gene-enhanced Arabidopsis thaliana pairP. capsiciThe disease resistance of (1).
Detailed Description
In order to make the content of the present invention more comprehensible, the technical solutions of the present invention are further described below with reference to specific embodiments, but the present invention is not limited thereto.
Example 1: chili pepperCaWRKY66Cloning of the gene.
The invention clonesP. capsiciHot pepper WRKY transcription factor coding gene capable of realizing significant up-regulation expression in host infection processCaWRKY66. The method comprises the following specific steps:
1) By usingP. capsiciThe strain JX1 infects a pepper variety CM334, pepper root system samples of different infection time points (0 h,3 h, 6h, 12 h,24 h,48 h and 72 h) are respectively taken, and the total RNA of each sample is extracted by using a Trizol extraction method;
2) Synthesizing a first cDNA chain by using HiScript III 1st Strand cDNA Synthesis Kit (+ gDNA wiper) kits according to a reaction program recommended by the instruction;
3) Design of specific primers for PCR amplificationCaWRKY66The reading frame sequence is developed, and the reading frame sequence is constructed into a plant expression vector pBinGFP2 by utilizing an In-fusion technology to obtain a pBinGFP2-CaWRKY66 recombinant expression vector, and sequencing analysis is performed. Finally obtaining a pepper WRKY transcription factor coding geneCaWRKY66SaidCaWRKY66The nucleotide sequence of the gene is shown as SEQ ID No.1, and the coded protein sequence is shown as SEQ ID No. 2.
Wherein, the specific primer pair has the following sequences:
CaWRKY66-F：5’-ctgtacaagggtacccccATGGAGGCTTCTTTCAA-3’，
CaWRKY66-R：5’-agaggatccgtcgaccccTCAGAGTGTGTAATCTTTGT-3’。
4) Fluorescent quantitative PCR identification
The first strand of the synthesized cDNA is diluted by 10 times and used as a template, and a primer pair consisting of CaWRKY66-qF and CaWRKY66-qR is adopted for detectionCaWRKY66A gene; to be provided withCaActinThe gene is used as reference geneCaActin-qF andCaActinamplification of primer pairs consisting of-qRCaActinA gene. The fluorescent quantitative PCR was performed according to the reaction system and amplification program recommended by the SYBR Premix ExTaqTM kit instructions. The reaction system is as follows: 2 mu L of template; forward and reverse primers are 0.2 mu L respectively;2×SYBR 5 μL；ddH 2 o2.6. Mu.L. The reaction procedure is as follows: 5 s at 94 ℃; 30 s at 94 ℃, 34 s at 60 ℃,40 cycles; 3 sample replicates, each sample amplifying the target gene in parallelCaWRKY66Internal reference gene of hot pepperCaActinBy means of 2 -△△CT The method carries out relative quantitative analysis of transcription level, and the fluorescent quantitative primer sequences are as follows:
CaWRKY66-qF：5’-CAACGATGAACGATGGATGC-3’，
CaWRKY66-qR：5’-TGATGTGGCTGAAAGAGGAA-3’；
CaActin-qF：5’-AGGGATGGGTCAAAAGGATGC-3’，
CaActin-qR：5’-GAGACAACACCGCCTGAATAGC-3’。
the result is shown by the fluorescent quantitative PCR analysisCaWRKY66The gene is inP. capsiciExpression was significantly upregulated 3 h upon infestation of the host (FIG. 1).
Example 2: silencingCaWRKY66The gene can reduce the hot pepper pairP. capsiciThe disease resistance of (1).
The invention uses virus-mediated gene silencing (VIGS) technology to silence in pepper plantsCaWRKY66Gene, can reduce pepper pairsP. capsiciThe disease resistance of (1). The method comprises the following specific steps:
1) Construction of VIGS vector by gateway technology
(1) To be provided withP. capsiciUsing cDNA infecting pepper for 3 h as template, using KOD high fidelity enzyme to respectively PCR amplifyCaWRKY66Two specific sequence fragments of the gene, iW-1 and iW-2 (FIG. 2A), the primer sequences used were as follows:
iW1-F：5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCTTCCATGCTATTGTCCGGT-3’，
iW1-R：
5’-GGGGACCACTTTGTACAAGAAAGCTGGGTCCCAATGATAGATGTGAGAG-3’；
iW2-F：5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCGTCCAATTAAATGTTCTCC-3’，
iW2-F：5’-GGGGACCACTTTGTACAAGAAAGCTGGGTCATCAGCAAAGCGAAGTGTC-3’；
(2) Respectively recovering and purifying PCR products iW-1 and iW-2 by a DNA purification recovery kit, and connecting the recovered fragments to pDONR207 by BP reaction;
(3) After transforming the escherichia coli, selecting clone, screening out positive clone through PCR verification, extracting plasmid and sequencing;
(4) Connecting the target fragment to a target vector pTRV2 through LR reaction on the plasmid with correct comparison result after sequencing, and extracting the plasmid after the verification is correct by using escherichia coli transformation screening and PCR verification;
(5) And carrying out agrobacterium transformation screening and PCR verification on the plasmids extracted in the last step to obtain the GV3101 agrobacterium strain containing pTRV2: iW-1 and pTRV2: iW-2 plasmids respectively.
2) Cultivation and infection of Agrobacterium
(1) The samples containing pTRV2: iW-1, pTRV2: iW-2, pTRV2 (no-load), pTRV2: PDS (indicated control,PDSafter genes are silenced, newly grown leaves of plants are bleached), adding GV3101 agrobacterium strain of pTRV1 into a test tube of liquid LB culture medium containing 0.2wt% rifampicin, 0.2wt% kanamycin and 0.1wt% gentamicin according to the proportion of 1 (v/v), sealing the test tube with a sealing membrane, and placing the test tube on a constant temperature shaking table at 28 ℃ for shaking overnight at 200 rpm;
(2) The next day, the supernatant was removed after centrifugation of the broth at 4000 rpm for 10 min. With infection buffer (10 mM MgCl) 2 10mM MES, 200. Mu.M acetosyringone; pH 5.6) resuspension of the cells, adjustment of the concentration to OD with a spectrophotometer 600 =0.8；
(3) The GV3101 agrobacterium strain containing pTRV2: iW-1, pTRV2: iW-2, pTRV2: PDS was mixed with the GV3101 agrobacterium strain containing pTRV1 in equal volume (V: V = 1.
(4) Placing the injected pepper plants at 16 ℃ for dark culture for 56 h, transferring to normal growth conditions (25 ℃,75% humidity, 60-70mmol of photoproton, 16h of illumination/8 h of dark photoperiod), and performing corresponding molecular identification and disease-resistant phenotype analysis on the VIGS pepper plants after 20-25 d.
3）P. capsiciInoculation and disease resistance analysis
When in usePDSGene silencing indicates emergence of new leaves in control plantsWhen 3 to 4 leaves are bleached, the pairCaWRKY66The disease resistance identification of the gene silencing plants (TRV: iW-1 and TRV: iW-2) and the control plants (TRV: 0) is carried out by adopting in vitro leaf inoculation and root irrigation inoculation respectively.
The method for inoculating the in vitro leaves comprises the following steps: cutting Capsici fructus leaves, placing in a tray covered with wet filter paper, and sucking 3 μ L zoospore suspension (5 × 10) 5 One per mL) on the leaf, sealing with a preservative film, and placing in a dark incubator at 25 ℃. Inoculating for 3 h, collecting materials, extracting RNA, and synthesizing cDNA first strand to obtain final productCaActinThe gene is used as an internal reference gene, and the fluorescent quantitative PCR is utilized to target the geneCaWRKY66The expression level of (2) was analyzed quantitatively (FIG. 2B). And taking leaves 24 h and 48 h after inoculation respectively, extracting the genome DNA of the sample by using a CTAB method, and diluting by 10 times to be used as a template. Amplification with primer pair consisting of PcActin-qF and PcActin-qRPcActinRepresentation of a GeneP. capsiciThe biomass of (1) is amplified by a primer pair consisting of CaActin-qF and CaActin-qRCaActinThe genes represent the biomass of pepper, and the relative biomass of pathogenic bacteria was detected by fluorescent quantitative PCR (FIG. 2C). And 3D after inoculation, observing and photographing the lesion of the pepper leaves under an ultraviolet flaw detection lamp (figure 2D).
Wherein, the fluorescent quantitative PCR primers are as follows:
CaWRKY66-qF：5’-CAACGATGAACGATGGATGC-3’，
CaWRKY66-qR：5’-TGATGTGGCTGAAAGAGGAA-3’；
PcActin-qF：5’- ACTGCACGTTCCAGACGAT-3’，
PcActin-qR：5’-CCACCACCTTGATCTTCATG -3’；
CaActin-qF：5’-AGGGATGGGTCAAAAGGATGC-3’，
CaActin-qR：5’-GAGACAACACCGCCTGAATAGC-3’。
the root irrigation inoculation method comprises the following steps: 5 mL of zoospore suspension (concentration 5X 10) were aspirated by pipette 5 one/mL) were inoculated into the rhizosphere soil of pepper plants, and about 20 plants were inoculated per treatment. The inoculated pepper plants are watered regularly, and the soil humidity is kept to be nearly saturated. Disease indices were investigated at 3 d, 5 d and 7 d post-inoculation, respectively (FIGS. 2E and 2F)The grading standard is the grading standard of root irrigation inoculation method issued by the department of agriculture in 2011 (NY/T2060.1-2011).
The research result shows that compared with the TRV:0 control plant,CaWRKY66gene silent plant (TRV: iW-1 and TRV: iW-2) pairP. capsiciThe disease resistance of (2) is significantly reduced.
Example 3: transient overexpressionCaWRKY66The gene can improve the hot pepper pairP. capsiciThe disease resistance of (1).
The invention is through transient overexpression in pepper leavesCaWRKY66Gene capable of improving pepper pairsP. capsiciThe disease resistance of (1). The method comprises the following specific steps:
1) Adding agrobacterium GV3101 containing pBinGFP2-CaWRKY66 and pBinGFP2 (no-load control) vectors into a test tube of liquid LB culture medium containing 0.2wt% rifampicin, 0.2wt% kanamycin and 0.1wt% gentamicin according to the proportion of 1;
2) The next day, the supernatant was removed after centrifugation of the broth at 4000 rpm for 10 min. With infection buffer (10 mM MgCl) 2 10mM MES, 200. Mu.M acetosyringone, pH 5.6) and the concentration was adjusted to OD using a spectrophotometer 600 =0.8；
3) When the pepper plants grow to 6-8 leaf stages, sucking agrobacterium liquid by using a sterile disposable syringe (needle head is removed), and selecting pepper leaves with similar growth vigor and the same size for whole-leaf injection. Culturing the injected Capsici fructus plant in growth room with 25 deg.C, 75% humidity, 60-70mmol of light proton, and photoperiod of 16h light/8 h dark, shearing the injected leaf blade after 36 h, placing in a tray paved with wet filter paper, and sucking 3 μ L zoospore suspension (5 × 10) 5 one/mL) were inoculated onto the leaves. After inoculation, the trays were sealed with a preservative film to preserve moisture, placed in a constant temperature incubator at 25 ℃ for dark culture, and 3 d after inoculation, the size of the lesion was measured under an ultraviolet inspection lamp and photographed (fig. 3A and 3B). Meanwhile, the materials are obtained for extracting genome DNA, diluted by 10 times and used as a template, and a primer pair consisting of PcActin-qF and PcActin-qR is used for amplificationPcActinRepresentation of a GeneP. capsiciBiomass of (2) as CaAcAmplification of primer pair consisting of tin-qF and CaActin-qRCaActinThe gene represents the biomass of pepper, and the relative biomass of phytophthora capsici was detected by fluorescent quantitative PCR (fig. 3C).
The results of the study show that transient overexpression is compared with the unloaded controlCaWRKY66Inoculating the pepper leavesP. capsiciThe size of the posterior lesionP. capsiciThe relative biomass was reduced by 41.0% and 77.5%, respectively. Thus, transient overexpressionCaWRKY66Can effectively inhibitP. capsiciInfection of (2).
Example 4: stable overexpressionCaWRKY66The gene can improve the arabidopsis thaliana pairP. capsiciThe disease resistance of (1).
The invention stably overexpresses in Arabidopsis thalianaCaWRKY66Gene capable of improving arabidopsis thaliana plant pairP. capsiciThe disease resistance of (1). The method comprises the following specific steps:
1) Obtaining transgenic Arabidopsis plants
(1) Will utilize gateway technologyCaWRKY66The gene is transferred into a PK7WG2 vector to obtain a PK7WG2-CaWRKY66 recombinant expression vector. Adding agrobacterium GV3101 containing PK7WG2-CaWRKY66 overexpression vector into a test tube of a liquid LB culture medium containing 0.2wt% of rifampicin, 0.2wt% of spectinomycin and 0.1wt% of gentamicin according to the proportion of 1 (v/v), sealing the test tube with a sealing membrane, and placing the test tube on a constant temperature shaking table at 28 ℃ for overnight culture by shaking at 200 rpm;
(2) The next day, the supernatant was removed after centrifugation of the broth at 4000 rpm for 10 min. The OD value of the agrobacterium is adjusted to OD by using the infection liquid (1/2MS +10g/L sucrose) 600 =0.8, after which 0.02% Silweet-77 was added for infecting arabidopsis inflorescences. For transformation, arabidopsis inflorescences were inverted and immersed in Agrobacterium suspension for 30 seconds. After the transformation is finished, wrapping the arabidopsis plant by using a preservative film, flatly placing the arabidopsis plant, putting the arabidopsis plant into a black garbage bag, performing dark treatment for 18 hours, then disassembling the arabidopsis plant, growing the arabidopsis plant under the conditions of 22 ℃,75% humidity, 60-70mmol of photoproton and 16 hours of illumination/8 hours of darkness until the siliques turn yellow, and then harvesting seeds.
2) Screening and subculture of transgenic Arabidopsis plants
Pouring the obtained mature seeds of Arabidopsis thaliana into a sterilized centrifuge tube, and adding a proper amount of disinfectantVenom (commercial 84: sterile water = 1), mix well by inversion for about 5 min. Then, the mixture is fully washed by sterile water for 5 times, and each time lasts for about 5 min. Finally, sterile water is removed, and sterilized and cooled 0.1% -0.15% agarose (agrose) is added to suspend the Arabidopsis seeds exactly. The seeds are sown with a sterilized 200 uL of the head-cut tips, one line by one line on the culture medium (1/2 MS +100 mg/L kanamycin +300 mg/L timentin). After two weeks, the grown green seedlings were selected and transferred to pots containing nutrient soil vermiculite =2 1 and grown in a greenhouse under conditions of 22 ℃,75% humidity, 60-70mmol of photoprotons, 16h of light/8 h of dark. For T 1 Carrying out PCR identification on transgenic arabidopsis plants and progeny plants thereof to obtain homozygous T 4 Generation transgenic lines.
3）P. capsiciInoculation and disease resistance analysis
For the obtained T 4 The generation transgenic lines (OE 1 and OE 2), the separated negative control line (Null) and the wild type plant (WT) are respectively subjected to isolated leaf inoculation and root irrigation inoculation for disease resistance identification.
The method for inoculating the in vitro leaves comprises the following steps: selecting leaves of Arabidopsis thaliana with similar growth and same size, placing in a tray paved with wet filter paper, and sucking 3 μ L zoospore suspension (5 × 10) 5 one/mL) was inoculated on leaves, sealed with a preservative film, placed in a dark incubator at 25 ℃ and observed and photographed under an ultraviolet inspection lamp 3 d after inoculation (FIG. 4A). Meanwhile, the materials are obtained for extracting genome DNA, diluted by 10 times and used as a template, and a primer pair consisting of PcActin-qF and PcActin-qR is used for amplificationPcActinRepresentation of a GeneP. capsiciThe biomass of (2), amplified with a primer pair consisting of AtActin-qF and AtActin-qRAtActinThe gene represents the biomass of arabidopsis thaliana, and the relative biomass of phytophthora capsici is detected by using fluorescent quantitative PCR (figure 4B), wherein the fluorescent quantitative PCR primer sequence is as follows:
PcActin-qF：5’-ACTGCACGTTCCAGACGAT-3’，
PcActin-qR：5’-CCACCACCTTGATCTTCATG-3’ ；
AtActin-qF：5’-TGTTCCCTGGAATTGCTGACCGTA-3’，
AtActin-qR：5’-TGCGACCACCTTGATCTTCATGCT-3’。
the root irrigation inoculation method comprises the following steps: 5 mL of zoospore suspension (concentration 5X 10) were aspirated by pipette 5 one/mL) were inoculated into the arabidopsis plant rhizosphere soil, approximately 20 plants per treatment were inoculated. The inoculated Arabidopsis plants were periodically watered to keep the soil moisture near saturation, and phenotype observations were made 7 d after inoculation (FIG. 4C).
The research result shows that: compared with wild type plants (WT) and negative control plants (Null) under the condition of in vitro leaf inoculation,CaWRKY66the lesion of the leaves of the transgenic Arabidopsis lines (OE 1 and OE 2) is obviously reduced, and the relative biomass of pathogenic bacteria is reduced by about 63.4 percent. Under the condition of root-irrigation and inoculation,CaWRKY66the resistance level of the transgenic arabidopsis strain to epidemic diseases is obviously improved.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
Claims (5)
1. Phytophthora-resistant gene of capsicumCaWRKY66The application of the plant resistance to phytophthora capsici is characterized in that: the above-mentionedCaWRKY66The nucleotide sequence of the gene is shown in SEQ ID No.1, and the protein sequence coded by the gene is shown in SEQ ID No. 2.
2. The pepper phytophthora blight-resistant related gene according to claim 1CaWRKY66The application of the plant resistance to phytophthora capsici is characterized in that: the plant is pepper and/or arabidopsis thaliana.
3. A pepper disease resistance-associated gene comprising the pepper disease resistance-associated gene as set forth in claim 1CaWRKY66The recombinant expression vector, the transgenic cell line or the recombinant strain of (1).
4. A method for amplifying the gene related to the phytophthora blight of hot pepper as claimed in claim 1CaWRKY66The primer set of (2), characterized in that: the sequences of the primer pairs are as follows:
CaWRKY66-F：5’-ctgtacaagggtacccccATGGAGGCTTCTTTCAA-3’，
CaWRKY66-R：5’-agaggatccgtcgaccccTCAGAGTGTGTAATCTTTGT-3’。
5. a pepper disease resistance-associated gene as set forth in claim 1CaWRKY66The real-time fluorescent quantitative primer pair is characterized in that: the real-time fluorescent quantitative primer pair has the following sequences:
CaWRKY66-qF：5’-CAACGATGAACGATGGATGC-3’，
CaWRKY66-qR：5’-TGATGTGGCTGAAAGAGGAA-3’。
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019096896A1 (en) * | 2017-11-15 | 2019-05-23 | Heinrich Heine Universität Düsseldorf | Method for inducing acquired resistance in a plant |
| CN110408626A (en) * | 2018-04-28 | 2019-11-05 | 南京农业大学 | It is a kind of improve soyabean phytophthora resistance gene GmWRKY148 and its application |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019096896A1 (en) * | 2017-11-15 | 2019-05-23 | Heinrich Heine Universität Düsseldorf | Method for inducing acquired resistance in a plant |
| CN110408626A (en) * | 2018-04-28 | 2019-11-05 | 南京农业大学 | It is a kind of improve soyabean phytophthora resistance gene GmWRKY148 and its application |
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