CN115948460B - 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 epidemic disease resistance related gene CaWRKY66 and application thereof. The nucleotide sequence of the CaWRKY66 gene is shown as SEQ ID No.1, and the encoded protein sequence is shown as SEQ ID No. 2. The CaWRKY66 gene has a key effect on plant disease resistance, particularly phytophthora capsici resistance. Silencing the CaWRKY66 gene can reduce the disease resistance of the capsicum to phytophthora capsici, and over-expression of the CaWRKY66 gene can promote the disease resistance of the capsicum and arabidopsis to phytophthora capsici. Therefore, the CaWRKY66 gene is used as a forward 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 epidemic disease resistance related gene CaWRKY66 and application thereof.
Background
Capsicum (Capsicum annuum) is an important vegetable and industrial feedstock crop worldwide. In recent years, the sowing area of the chillies in China is stabilized to be more than 200 ten thousand hm 2 throughout the year, and the planting area and the yield benefit are all the first vegetables in the country. In agricultural production, the pepper epidemic disease caused by phytophthora capsici (Phytophthoracapsici) frequently occurs, the yield is reduced by 20% -30% in light weight, the yield is reduced by more than 80% in heavy weight, even the yield is stopped, and the development of the pepper industry is severely restricted. P, capsici has a very wide host range, and can infect not only pepper, tomato and other solanaceae plants, but also cucurbitaceae (such as watermelon, cucumber, pumpkin and the like) and beans (such as lima beans) and other hundreds of crops, thereby causing destructive disasters in agricultural production.
In the natural habitat of the plants, a complex and efficient regulation network is formed and developed through the co-evolution of the plants and pathogenic bacteria, and the effective regulation of the antipathogenic response is realized through the fine regulation of the expression of the antipathogenic defense related genes. In this process, the transcription factor acts as a regulatory factor integrating the upstream signal and regulating the expression of a series of defense-related genes downstream, playing an important role in plant disease-resistant response. WKRY is an important family of transcription factors in plants, the different members of which contain 1-2 conserved WRKY domains that recognize and bind to the W-box (TTGACT/C) element on the target gene promoter, thereby regulating the expression of the target gene. WKRY transcription factors are widely involved in the response or resistance process of plants to biotic stress such as bacteria, fungi, oomycetes, viruses and the like, abiotic stress such as drought, high temperature, freeze injury and the like. However, little research has been done on the disease resistance function of the capsicum WRKY transcription factor response p. capsici.
Disclosure of Invention
The invention aims to provide a capsicum epidemic disease resistance related gene CaWRKY66 and application thereof, and provides gene resources for capsicum disease resistance genetic improvement.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a pepper epidemic disease resistance related gene CaWRKY66 comprises the following cloning steps:
according to RNA-seq transcriptome analysis and fluorescent quantitative PCR verification of interaction of capsicum and phytophthora capsici (P. capsici), a capsicum WRKY transcription factor coding gene CaWRKY66 which is remarkably up-regulated and expressed in the infection host process is discovered; the cDNA of a P. capsici infected pepper root system sample is used as a template, KOD high-fidelity enzyme PCR is used for amplifying the full-length sequence of a target gene open reading frame, the full-length sequence is constructed into a plant expression vector pBinGFP2 by an In-fusion method, sequencing analysis is carried out, and finally a pepper epidemic disease resistance related gene CaWRKY66 is obtained, the nucleotide sequence of the CaWRKY66 gene is shown as SEQ ID No.1, and the coded protein sequence is shown as SEQ ID No. 2.
A recombinant expression vector, a transgenic cell line or a recombinant bacterium containing the pepper epidemic disease resistance related gene CaWRKY 66.
A primer pair for amplifying the pepper epidemic disease resistance related gene CaWRKY66, wherein the primer pair has the following sequence:
CaWRKY66-F：5’-ctgtacaagggtacccccATGGAGGCTTCTTTCAA-3’，
CaWRKY66-R：5’-agaggatccgtcgaccccTCAGAGTGTGTAATCTTTGT-3’。
The real-time fluorescent quantitative primer pair of the pepper epidemic disease resistance related gene CaWRKY66 has the following sequence:
CaWRKY66-qF：5’-CAACGATGAACGATGGATGC-3’，
CaWRKY66-qR：5’-TGATGTGGCTGAAAGAGGAA-3’。
The application of the gene CaWRKY66 related to pepper epidemic disease resistance in regulating and controlling the resistance of plants to phytophthora capsici (Phytophthoracapsici, P. capsici);
Specifically, by using a virus-mediated gene silencing technology, silencing the CaWRKY66 gene can reduce the disease resistance of the capsicum to P. capsici; the CaWRKY66 gene is transiently overexpressed in the capsicum leaves, so that the disease resistance of the capsicum to P. capsici can be improved; the stable overexpression of the CaWRKY66 gene in Arabidopsis thaliana can improve the disease resistance of Arabidopsis thaliana plants to P. capsici.
The invention has the remarkable advantages that:
The invention provides a pepper epidemic disease resistance related gene CaWRKY66 and application thereof. The invention discovers that the disease resistance of the capsicum to P. capsici can be obviously reduced by utilizing the virus-mediated gene silencing technology to silence CaWRKY66 gene, and the disease resistance of the capsicum to P. capsici can be improved by transient overexpression of the gene. The stable overexpression of the CaWRKY66 gene in Arabidopsis thaliana can also significantly improve the resistance of Arabidopsis thaliana plants to P. capsici. Therefore, the CaWRKY66 gene is used as a forward regulatory factor, and can endow different plants with disease resistance to P. capsici.
Drawings
Fig. 1: the CaWRKY66 gene significantly up-regulates expression when P. capsici infects host 3 h.
Fig. 2: silencing the CaWRKY66 gene reduces the disease resistance of capsicum to P. capsici.
Fig. 3: the transient overexpression of the CaWRKY66 gene improves the disease resistance of the capsicum to P. capsici.
Fig. 4: stable overexpression of CaWRKY66 gene improves the disease resistance of Arabidopsis thaliana to P. capsici.
Detailed Description
In order to make the contents of the present invention more easily understood, the technical scheme of the present invention will be further described with reference to the specific embodiments, but the present invention is not limited thereto.
Example 1: cloning of Capsici fructus CaWRKY66 gene.
The invention clones a capsicum WRKY transcription factor coding gene CaWRKY66 which is obviously up-regulated and expressed in the process of infecting a host by P. capsici. The method comprises the following specific steps:
1) Infecting a pepper variety CM334 by using a P. capsici strain JX1, respectively taking pepper root system samples at different infection time points (0 h,3 h,6 h,12 h,24 h,48 h,72 h), and extracting total RNA of each sample by using a Trizol extraction method;
2) The first strand cDNA was synthesized using HiScript [ III ] 1: 1st Strand cDNA Synthesis Kit (+ GDNA WIPER) kit according to the reaction procedure recommended in the specification;
3) Designing a specific primer PCR to amplify a development reading frame sequence of CaWRKY66, constructing the sequence into a plant expression vector pBinGFP by utilizing an In-fusion technology to obtain a pBinGFP2-CaWRKY66 recombinant expression vector, and carrying out sequencing analysis. Finally, a capsicum WRKY transcription factor encoding gene CaWRKY66 is obtained, the nucleotide sequence of the CaWRKY66 gene is shown as SEQ ID No.1, and the encoded protein sequence is shown as SEQ ID No. 2.
Wherein, the specific primer pair sequences are as follows:
CaWRKY66-F：5’-ctgtacaagggtacccccATGGAGGCTTCTTTCAA-3’，
CaWRKY66-R：5’-agaggatccgtcgaccccTCAGAGTGTGTAATCTTTGT-3’。
4) Fluorescent quantitative PCR identification
The first strand of the synthesized cDNA is diluted 10 times as a template, and a primer pair consisting of CaWRKY66-qF and CaWRKY66-qR is adopted to detect the CaWRKY66 gene; the CaActin gene is used as an internal reference gene, and a primer pair consisting of CaActin-qF and CaActin-qR is used for amplifying the CaActin gene. Fluorescent quantitative PCR was performed according to the reaction system and amplification procedure recommended in the SYBR Premix ExTaqTM kit instructions. The reaction system is as follows: 2 mu L of template; forward and reverse primers were each 0.2 μl;2 XSYBR 5. Mu.L; ddH 2 O2.6. Mu.L. The reaction procedure is: 94 ℃ 5 s;94℃for 30 s,60℃for 34 s,40 cycles; 3 samples were repeated, each sample was amplified in parallel for the target gene CaWRKY66 and the capsicum internal reference gene CaActin, and the transcript level was analyzed relatively quantitatively by the 2 -△△CT method, and the fluorescent quantitative primer sequences were as follows:
CaWRKY66-qF：5’-CAACGATGAACGATGGATGC-3’，
CaWRKY66-qR：5’-TGATGTGGCTGAAAGAGGAA-3’；
CaActin-qF：5’-AGGGATGGGTCAAAAGGATGC-3’，
CaActin-qR：5’-GAGACAACACCGCCTGAATAGC-3’。
The results showed that the CaWRKY66 gene significantly up-regulated expression when p. capsici infects host 3 h by fluorescent quantitative PCR analysis (fig. 1).
Example 2: silencing the CaWRKY66 gene can reduce the disease resistance of capsicum to P. capsici.
The invention utilizes virus-mediated gene silencing (VIGS) technology to silence CaWRKY66 gene in pepper plants, and can reduce the disease resistance of pepper to P. capsici. The method comprises the following specific steps:
1) Construction of VIGS vectors using gateway technology
(1) Two specific sequence fragments iW-1 and iW-2 (FIG. 2A) of the CaWRKY66 gene are respectively amplified by PCR with KOD high-fidelity enzyme by using cDNA of P. capsici infected with capsicum 3h as a template, and the primer sequences are as follows:
iW1-F： 5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCTTCCATGCTATTGTCCGGT-3’，
iW1-R：
5’-GGGGACCACTTTGTACAAGAAAGCTGGGTCCCAATGATAGATGTGAGAG-3’；
iW2-F：5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCGTCCAATTAAATGTTCTCC-3’，
iW2-F：5’-GGGGACCACTTTGTACAAGAAAGCTGGGTCATCAGCAAAGCGAAGTGTC-3’；
(2) The PCR products iW-1 and iW-2 are respectively recovered and purified by a DNA purification recovery kit, and the recovery fragment is connected to pDONR207 by BP reaction;
(3) After the escherichia coli is transformed, a clone is selected, positive clones are screened out through PCR verification, and plasmids are extracted for sequencing;
(4) Connecting a target fragment to a target vector pTRV2 through LR reaction by using plasmids with correct comparison results after sequencing, and extracting plasmids after correct verification by using escherichia coli transformation screening and PCR verification;
(5) The plasmids extracted in the previous step are subjected to agrobacterium transformation screening and PCR verification to obtain GV3101 agrobacterium strains respectively containing pTRV2:iW-1 and pTRV2:iW-2 plasmids.
2) Culturing and infecting agrobacterium
(1) Adding the GV3101 agrobacterium strain containing pTRV2:iW-1, pTRV2:iW-2, pTRV2 (empty), pTRV2:PDS (indicating that after the PDS gene is silenced, the new leaves of the plant are bleached) and pTRV1 into a test tube containing 0.2wt% of rifampicin, 0.2wt% of kanamycin and 0.1wt% of gentamicin according to the proportion of 1:50 (v/v), sealing the test tube with a sealing film, and placing the test tube on a constant temperature shaking table at 28 ℃ for 200 rpm shake overnight culture;
(2) The next day, the bacterial liquid was centrifuged at 4000 rpm for 10: 10 min, and the supernatant was removed. The cells were resuspended in infection buffer (10 mM MgCl 2, 10mM MES, 200. Mu.M acetosyringone; pH 5.6) and the concentration was adjusted to OD 600 =0.8 with a spectrophotometer;
(3) GV3101 agrobacterium strain containing pTRV2:iw-1, pTRV2:iw-2, pTRV2:pds was mixed with equal volumes (V: v=1:1) of GV3101 agrobacterium strain containing pTRV1, respectively, and two cotyledons of three-leaf stage pepper seedlings were injected.
(4) And (3) placing the injected pepper plants in a dark culture condition of 56 h at 16 ℃, transferring to normal growth conditions (25 ℃,75% humidity, 60-70mmol photoprotes, 16h light/8 h dark photoperiod), and carrying out corresponding molecular identification and disease resistance phenotype analysis on the VIGS pepper plants after 20-25 d.
3) P, capsici vaccination and disease resistance analysis
When PDS gene silencing indicates that 3-4 new leaves of the control plant are bleached, the CaWRKY66 gene silencing plants (TRV: iW-1 and TRV: iW-2) and the control plant (TRV: 0) are subjected to disease resistance identification by in-vitro leaf inoculation and root irrigation inoculation respectively.
The method for inoculating the in-vitro leaves comprises the following steps: the capsicum leaves are cut, placed in a tray paved with moist filter paper, 3 mu L of zoospore suspension (5X 10 5/mL) is sucked and inoculated on the leaves, sealed by a preservative film and placed in a dark incubator at 25 ℃. After 3 h inoculation, the materials are used for extracting RNA, a cDNA first strand is synthesized, caActin gene is used as an internal reference gene, and relative quantitative analysis is carried out on the expression quantity of a target gene CaWRKY66 by utilizing fluorescent quantitative PCR (FIG. 2B). The leaves of 24 h and 48h after inoculation are respectively taken, genomic DNA of the sample is extracted by using a CTAB method, and the diluted DNA is taken as a template after 10 times. The primer pair consisting of PcActin-qF and PcActin-qR was used to amplify PcActin gene representing biomass of P. capsici, the primer pair consisting of CaActin-qF and CaActin-qR was used to amplify CaActin gene representing biomass of capsicum, and fluorescent quantitative PCR was used to detect relative biomass of pathogenic bacteria (FIG. 2C). After inoculation, 3D pieces of pepper leaves were observed and photographed for lesions under an ultraviolet inspection lamp (fig. 2D).
Wherein, the fluorescent quantitative PCR primer is 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: 5mL zoospore suspensions (5X 10 5/mL) were pipetted into the rhizosphere soil of pepper plants with a pipette gun and about 20 plants per treatment. Periodically watering the inoculated pepper plants, and keeping the soil humidity nearly saturated. Investigation of disease index was performed at 3 d, 5 d and 7 d post inoculation respectively (fig. 2E and 2F), and the grading standard was found in the root-irrigation inoculation grading standard (NY/T2060.1-2011) issued by the agricultural department in 2011.
The results of the study showed that CaWRKY66 gene-silenced plants (TRV: iW-1 and TRV: iW-2) had significantly reduced disease resistance to P. capsici compared to the TRV:0 control plants.
Example 3: the transient overexpression of the CaWRKY66 gene can improve the disease resistance of the capsicum to P. capsici.
According to the invention, the disease resistance of the capsicum to P. capsici can be improved by transient overexpression of the CaWRKY66 gene in capsicum leaves. The method comprises the following specific steps:
1) Adding agrobacterium GV3101 containing pBinGFP-CaWRKY 66, pBinGFP2 (no-load control) vector into a test tube containing 0.2wt% of rifampicin, 0.2wt% of kanamycin and 0.1wt% of gentamicin in a ratio of 1:50 (v/v), sealing the test tube, and placing the test tube on a shaking table at a constant temperature of 28 ℃ for 200 rpm shake overnight culture;
2) The next day, the bacterial liquid was centrifuged at 4000 rpm for 10: 10min, and the supernatant was removed. The cells were resuspended in infection buffer (10 mMMgCl 2, 10mm mes,200 μm acetosyringone, pH 5.6) and the concentration was adjusted to OD 600 = 0.8 with a spectrophotometer;
3) When the pepper plants grow to 6-8 leaf periods, an aseptic disposable injector (with needles removed) is used for sucking agrobacterium liquid, and pepper leaves with similar growth vigor and the same size are selected for full-leaf injection. The injected pepper plants are placed in a growth chamber with 25 ℃ and 75% humidity, 60-70mmol photoprotons and a photoperiod of 16h light/8 h darkness for cultivation, 36 h post-cut leaves are placed in a tray paved with moist filter paper, 3 mu L of zoospore suspension (5X 10 5/mL) is sucked and inoculated on the leaves. After inoculation, the tray is sealed with a preservative film to preserve moisture, and the tray is placed in a constant temperature incubator at 25 ℃ for dark culture, and after inoculation, the spot size is measured and photographed under an ultraviolet flaw detection lamp at 3 d (fig. 3A and 3B). Meanwhile, the materials are taken for extracting genome DNA, the materials are diluted 10 times and then used as templates, a primer pair consisting of PcActin-qF and PcActin-qR is used for amplifying PcActin genes to represent biomass of P. capsici, a primer pair consisting of CaActin-qF and CaActin-qR is used for amplifying CaActin genes to represent biomass of capsicum, and fluorescent quantitative PCR is used for detecting relative biomass of phytophthora capsici (figure 3C).
The research result shows that compared with the no-load control, the size of the lesion and the relative biomass of P. capsici of the pepper leaf with the transient over-expression CaWRKY66 are respectively reduced by 41.0 percent and 77.5 percent after being inoculated with P. capsici. Therefore, transient overexpression of CaWRKY66 can effectively inhibit p. capsici infection.
Example 4: the stable overexpression of the CaWRKY66 gene can improve the disease resistance of arabidopsis thaliana to P. capsici.
The invention can stably and over-express the CaWRKY66 gene in the Arabidopsis thaliana, and can improve the disease resistance of the Arabidopsis thaliana plant to P. capsici. The method comprises the following specific steps:
1) Obtaining transgenic Arabidopsis plants
(1) And introducing the CaWRKY66 gene into the PK7WG2 vector by using gateway technology to obtain the PK7WG2-CaWRKY66 recombinant expression vector. Adding agrobacterium GV3101 containing PK7WG2-CaWRKY66 super-expression vector into a test tube containing 0.2wt% of rifampicin, 0.2wt% of spectinomycin and 0.1wt% of gentamicin according to the proportion of 1:50 (v/v), sealing the test tube, and placing the test tube on a constant temperature shaking table at 28 ℃ for 200 rpm shake overnight culture;
(2) The next day, the bacterial liquid was centrifuged at 4000 rpm for 10: 10 min, and the supernatant was removed. The OD of the agrobacteria was adjusted to OD 600 =0.8 with the infection solution (1/2MS+10 g/L sucrose), after which 0.02% Silweet-77 was added for infecting the Arabidopsis inflorescence. At the time of transformation, the arabidopsis inflorescence was back-soaked in agrobacterium suspension for 30 seconds. After the transformation is finished, wrapping the arabidopsis plants with a preservative film, putting the arabidopsis plants in a black garbage bag horizontally, taking the arabidopsis plants apart after the black garbage bag is placed in the dark, taking the arabidopsis plants apart after the dark treatment of 18 h, growing the arabidopsis plants until the keratan fruits turn yellow under the conditions of 22 ℃,75% humidity, 60-70mmol photoprotic, 16h illumination/8 h darkness, and then harvesting seeds.
2) Screening and subculturing transgenic Arabidopsis plants
The harvested mature arabidopsis seeds are poured into a sterilized centrifuge tube, and a proper amount of disinfectant (commercially available 84: sterilized water=1:1) is added, and the mixture is fully inverted and uniformly mixed for about 5 min. Then the mixture is fully washed by sterile water for 5 times, and each time is about 5 min. Finally, the sterile water is removed, and 0.1 to 0.15 percent agarose (agrose) after sterilization and cooling is added to enable the arabidopsis seeds to be just suspended. The sterilized 200 uL gun heads with the heads cut off were used for sowing, and the seeds were sown one by one on the medium (1/2 MS+100 mg/L kanamycin+300 mg/L timentin). After two weeks, the selected green seedlings were transferred to pots containing nutrient soil vermiculite=2:1 and grown in a greenhouse at 22 ℃,75% humidity, 60-70mmol photoprotons, 16h light/8 h darkness. And carrying out PCR identification on the T 1 -generation transgenic arabidopsis plant and the progeny plant thereof, thereby obtaining a homozygous T 4 -generation transgenic plant line.
3) P, capsici vaccination and disease resistance analysis
The obtained T 4 generation transgenic lines (OE 1 and OE 2), the isolated negative control line (Null) and the wild type plant (WT) are subjected to disease resistance identification by respectively adopting in-vitro leaf inoculation and root irrigation inoculation.
The method for inoculating the in-vitro leaves comprises the following steps: arabidopsis leaves with similar growth vigor and same size are selected, placed in a tray paved with wet filter paper, 3 mu L of zoospore suspension (5×10 5/mL) is sucked and inoculated on the leaves, sealed by a preservative film, placed in a dark incubator at 25 ℃, and observed and photographed under an ultraviolet flaw detection lamp after inoculation of 3d (figure 4A). Meanwhile, the materials are taken for extracting genome DNA, the diluted materials are taken as templates, a primer pair consisting of PcActin-qF and PcActin-qR is used for amplifying PcActin genes to represent biomass of P. capsici, a primer pair consisting of AtActin-qF and AtActin-qR is used for amplifying the biomass of Arabidopsis thaliana, fluorescent quantitative PCR is used for detecting the relative biomass of phytophthora capsici, and the fluorescent quantitative PCR primer sequences are 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 zoospore suspensions (5X 10 5/mL) were pipetted into the rhizosphere soil of Arabidopsis plants with a pipette gun and approximately 20 plants were inoculated per treatment. The inoculated arabidopsis plants were periodically watered, kept nearly saturated with soil humidity, and phenotypically observed at 7 d a after inoculation (fig. 4C).
The research results show that: under the condition of in vitro leaf inoculation, compared with wild type plants (WT) and negative control plants (Null), the leaf spots of the leaves of the CaWRKY66 transgenic Arabidopsis strains (OE 1 and OE 2) are obviously reduced, and the relative biomass of pathogenic bacteria is reduced by about 63.4 percent. Under root irrigation inoculation conditions, the resistance level of the CaWRKY66 transgenic Arabidopsis strain to epidemic diseases is also obviously improved.
The foregoing description is only of the preferred embodiments of the invention, and all changes and modifications that come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Claims (1)
1. The application of the overexpression of the pepper epidemic disease resistance related gene CaWRKY66 in enhancing the resistance of plants to phytophthora capsici is characterized in that: the nucleotide sequence of the CaWRKY66 gene is shown as SEQ ID No.1, and the encoded protein sequence is shown as SEQ ID No. 2; the plant is capsicum and/or arabidopsis thaliana.
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| NCBI Reference Sequence: XP_047265727.1;Genbank;Genbank;20220405;第1-2页 * |
| 响应辣椒疫霉菌诱导的CaWRKY转录因子筛选及其信号通路分析;徐晓美等;中国农学通报;20221115;第38卷(第32期);第22-31页 * |
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