CN116536347A - Application of calcium-dependent protein kinase gene GhCAMK in verticillium wilt resistance of plants - Google Patents
Application of calcium-dependent protein kinase gene GhCAMK in verticillium wilt resistance of plants Download PDFInfo
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- CN116536347A CN116536347A CN202310334048.5A CN202310334048A CN116536347A CN 116536347 A CN116536347 A CN 116536347A CN 202310334048 A CN202310334048 A CN 202310334048A CN 116536347 A CN116536347 A CN 116536347A
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- C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance
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Abstract
The invention discloses application of a calcium-dependent protein kinase gene GhCCAK in verticillium wilt resistance of plants. Belongs to the technical field of genetic engineering. The invention constructs a VIGS plant expression vector by using the GhCAMK gene to transform upland cotton TM-1, and the obtained transgenic cotton shows resistance to verticillium after being inoculated with cotton verticillium wilt bacteria V991, which shows that the GhCAMK gene is highly related to the resistance to cotton verticillium wilt. The invention lays an application foundation for molecular mechanism research of verticillium wilt resistance and cultivation of new varieties of new verticillium wilt-resistant plants, and provides new reference and reference for screening new disease-resistant genes and cultivating resistant plants.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to application of a calcium-dependent protein kinase gene GhCAMK in plant verticillium wilt resistance.
Background
Cotton verticillium wilt is a soil-borne disease caused by the soil filamentous fungus verticillium dahliae (Verticillium dahliae), and is also called as "cancer" of cotton because of rapid onset and difficult healing. In the 90 s, verticillium wilt of China spreads rapidly, and occurs in all cotton areas of China, so that 10% -20% of cotton yield reduction of China is caused throughout the year, and 70% of cotton yield reduction in partial cotton areas is achieved, so that the loss is huge, and the method is one of main obstacles restricting high and stable cotton yield of China. Practice proves that the cultivation of cotton disease-resistant varieties is an effective economic strategy for restricting verticillium wilt spreading. Due to the common non-disease resistance of the existing variety resources, lack of germplasm resources, poor broad spectrum and the like, breeders do not cultivate cultivars with excellent resistance to cotton verticillium wilt at present. In recent years, the development of candidate genes for resisting verticillium wilt of cotton based on genetic engineering has become another strategy for alleviating verticillium wilt invasion, and a series of genes such as GhSINAs, ghVLN2, ghSWEET42 and GhTIFY9 have been reported.
Calcium ions are important second messengers in plant cell signaling. When plants are subjected to external stress, calcium signals in cells are transmitted to downstream components through calmodulin, such as calmodulin, calmodulin-like and caldependent protein kinase (CDPK), etc., thereby regulating the expression of the related genes. In plants, the modulation of calcium ion concentration in the cytoplasm can be in response to a variety of endogenous and exogenous signals, including changes in hormone levels, abiotic stresses (e.g., drought, high, low temperature, or light, etc.), biotic stresses (e.g., pathogens and pathogenic microorganisms, etc.). Five calcium ion sensors in plants are available, including calcium pattern (CAM), calmodulin (CML), calmodulin B protein (CBL), calcium/calmodulin-dependent protein kinase (CCAMK) and calcium-dependent protein kinase (CDPK). Ca (Ca) 2+ CaM-dependent protein kinase (CCAMK) is a plant-specific protein kinase, first discovered from lily, in the smokeExpression is also found in grass, maize, rice and wheat. Studies have shown that CCAMK is expressed in roots and developing nodules, and that activation of CCAMK is the primary regulatory mode of plant root symbiosis, which can enhance tolerance of plants to environmental stresses.
CCAMK is involved in responding to plant abiotic stress processes. In arabidopsis, overexpression of TaCCAMK reduces sensitivity to ABA during seed germination and increases tolerance to salt stress during seed germination. In maize, zmCCAMK is in turn involved in ABA-induced antioxidant protection processes by phosphorylating ZmNAC 84. Recent studies have found that over-expression of ZmCCAMK and rice CCAMK enhances the antioxidant protective capacity of plants, positively regulating the tolerance of maize and rice plants to drought and oxidative stress.
In summary, how to provide a calpain gene that regulates cotton verticillium resistance is a problem that needs to be solved by those skilled in the art.
Disclosure of Invention
In view of this, the present invention provides the use of the calcium-dependent protein kinase gene GhCAMK in plants against verticillium wilt.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
application of calcium-dependent protein kinase gene GhCAMK in regulating and controlling verticillium wilt resistance of plants, wherein the nucleotide sequence of the gene GhCAMK is shown in SEQ ID NO: 1.
ATGGGACAAGATAAAGCGAAACTAGTAGAAGAATACGAAATCCTAGATATACTAGGACGAGGTGGATTCTCAGTCGTAAGAAAAGGTATAAAAAGAAAGAACGGATCAGATCATGAGAAAACACAAGTCGCCATTAAAACACTGAAACGATTCGGAACGACGCCGTCACCAGCTCGAGTCGAGAAAACAATTGCTTCGATGGCGGCGTTATTGCCGACGCGTAACCAGGTTTCCATCTCTGACGCGCTGTTGACGAACGAGATCCTCGTCATGAGGAAGATCGTCGAGAACGTTTCGCCGCATCCGAACGTGATCGATCTCTACGACGTTTACGAAGATCAAGCCGGGGTTCATTTGGTGCTGGAATTGTGCTCCGGCGGGGAGTTGTTCGATCGGATAGTGGCGGAAACACGGTACTCGGAAGCCGGTGCGGCAGCGGTGGTACGGCAGATCGCGGGAGGATTAGCGGCGATCCATAAGGCGAACATTGTTCATAGAGATCTGAAACCGGAGAACTGCTTGTTCTTGAATAAAAATAAAGATTCAACGTTGAAGATCATGGACTTTGGATTGAGTTCGGTGGAAGAATTTACGGATCCGGTTATCGGGTTGTTTGGATCCATAGATTATGTTTCACCTGAGGCGCTTTCTCAGGGGCAAATCACAGCAAAGAGCGATATGTGGTCTTTGGGTGTCATCTTATTCATCTTGCTTTCGGGGTATCCACCATTTATTGCTCAATCTAATCGTCAGAAACAACAGATGATAATGGCTGGAGAATACAATTTCGATGAGAGGACATGGAAAAACATTTCTTCATCAGCAAAGCATTTGATTTCTAATCTGTTGCAAGTTGATCCTGATAGAAGACCTAGTGCTGAACAACTTCTAGCTCATCCATGGGTCATCGGGGATTCAGCAAAGCAAGAACAAATAGATGCGGAGGTTGTTTCCAGATTGCAGAGTTTTAACGCGCGTCGTAAGCTACGTGCTGCCGCCATAGCCAGTGTGTTGAGCAGCAAGGTTTTACTAAGGACGAAGAGGTTAAGAAGTTTGCTTGGCTCCCATGACCTTTCAAAGGACGAAATTGATAACCTCAAGTCGAACTTTAAGAAAATATGTGCTAACGGTGACAATGCTACTTTACCCGAATTTGAGGAGGTGCTAAAAGCAATGAACATGTCTTCACTACTTCCTTTGGCTACTCGTATCTTTGACCTATTCGATAGCAATCGAGATGGTACTGTTGATATGAGAGAAATTGTGTGTGGATTTTCCAGTCTTAAGAATTCTAAAGGAGATGACGCTCTTCGCCTGTGCTTCGAGATGTATGATACAGATCGATCTGGATGCATTACTAAAGAAGAACTAGCATCAATGTTAAGAGCATTGCCCGATGACTGTCTTCCACCAGATATTACAGAACCTGGAAAGTTAGATGAAATATTCGATCGAATGGATGCAAACAGTGATGGGAAGGTTACGTTCGAAGAATTTAAGGATGCCATGCAAAGAGACAGCTCTCTCCAAGACGTAGTCCTCTCTTCTCTTCGACAACAGTAA,SEQ ID NO:1。
Application of protein or a substance for regulating and controlling expression of a protein coding gene in verticillium resistance of plants, wherein the amino acid sequence of the protein is shown in SEQ ID NO: 2.
MGQDKAKLVEEYEILDILGRGGFSVVRKGIKRKNGSDHEKTQVAIKTLKRFGTTPSPARVEKTIASMAALLPTRNQVSISDALLTNEILVMRKIVENVSPHPNVIDLYDVYEDQAGVHLVLELCSGGELFDRIVAETRYSEAGAAAVVRQIAGGLAAIHKANIVHRDLKPENCLFLNKNKDSTLKIMDFGLSSVEEFTDPVIGLFGSIDYVSPEALSQGQITAKSDMWSLGVILFILLSGYPPFIAQSNRQKQQMIMAGEYNFDERTWKNISSSAKHLISNLLQVDPDRRPSAEQLLAHPWVIGDSAKQEQIDAEVVSRLQSFNARRKLRAAAIASVLSSKVLLRTKRLRSLLGSHDLSKDEIDNLKSNFKKICANGDNATLPEFEEVLKAMNMSSLLPLATRIFDLFDSNRDGTVDMREIVCGFSSLKNSKGDDALRLCFEMYDTDRSGCITKEELASMLRALPDDCLPPDITEPGKLDEIFDRMDANSDGKVTFEEFKDAMQRDSSLQDVVLSSLRQQ*,SEQ ID NO:2。
The application of the gene GhCAMK or the protein related biological material in plant verticillium wilt resistance is that the biological material is any one of the following:
a: a nucleic acid molecule having a nucleotide sequence as described above or a nucleic acid molecule encoding the above protein;
b: an expression cassette comprising silencing of the nucleic acid molecule of a;
c: an expression vector comprising the nucleic acid molecule of A, or a recombinant vector comprising the expression cassette of B;
d: a recombinant microorganism comprising the nucleic acid molecule of A, a recombinant microorganism comprising the expression cassette of B, or a recombinant microorganism comprising the recombinant vector of C.
The term "expression cassette" refers to a DNA capable of expressing a protein as described in the above applications in a host cell, which DNA may include not only a promoter for initiating transcription of a gene encoding the protein, but also a terminator for terminating transcription of the gene encoding the protein. Further, the expression cassette may also include an enhancer sequence.
Furthermore, the gene GhCAMK or the protein related biological material is applied to breeding plant varieties with enhanced or reduced verticillium wilt resistance.
Furthermore, the gene GhCAMK or the protein related biological material is applied to plant breeding.
Further, the plant is a cotton plant.
Further, the plant is cotton.
Further, the plant is upland cotton TM-1.
A method for improving verticillium wilt resistance of plants by silencing or inhibiting expression of the above gene GhCAMK or activity of the above protein in plants.
The silencing or inhibiting of the expression level of the protein-encoding gene in the plant may be achieved by any means known in the art to effect deletion, insertion or base-change mutation of the gene, thereby achieving reduced or lost gene function.
Compared with the prior art, the invention has the beneficial effects that: the invention utilizes the GhCAMK gene sequence information to amplify the gene and constructs a VIGS plant expression vector to transform upland cotton TM-1, and the obtained transgenic cotton shows resistance to verticillium after being inoculated with cotton verticillium wilt bacteria V991, which shows that the GhCAMK gene is highly relevant to the resistance of cotton verticillium wilt. The invention lays an application foundation for molecular mechanism research of verticillium wilt resistance and cultivation of new varieties of new verticillium wilt-resistant plants, and provides new reference and reference for screening new disease-resistant genes and cultivating resistant plants.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.
FIG. 1 is a diagram showing PCR products of the GhCAMK gene in example 1 of the present invention, wherein the Marker is a 2000bp Laddermarker;
FIG. 2 is a graph showing qRT-PCR results of expression amounts of GhCAMK gene in different cotton tissues (root, stem, leaf, petal, bract and sepal) in example 2 of the present invention;
FIG. 3 is a drawing showing the signal molecules (jasmonic acid JA, salicylic acid SA, H) in example 2 of the present invention 2 O 2 Dip-dyeing) to induce qRT-PCR results of the expression amount of the GhCAMK gene;
FIG. 4 is a graph showing the results of qRT-PCR analysis of the expression pattern of the yellow wilt bacteria V991 infection-induced GhCAMK gene in example 2 of the present invention;
FIG. 5 is a graph showing phenotype of upland cotton TM-1 plants after injection of the VIGS vector in example 3 according to the present invention, wherein: a is the phenotype of the plant after 2 weeks of the VIGS vector injected with the ghca 1 gene; TRV 00 (control) and TRV GhCAMK (10) of upland cotton TM-1 were inoculated with verticillium wilt V991 spore solution by root dipping 7 condiia/mL) 21 days of morbidity;
FIG. 6 is a graph showing qRT-PCR results of expression amounts of the GhCAMK gene in three-leaf stage leaves of cotton plants of the invention in TRV 00 and TRV of upland cotton TM-1 in example 3;
FIG. 7 is a graph showing the statistical result of the disease index of transgenic upland cotton TM-1 inoculated with verticillium in example 3 of the present invention;
FIG. 8 is a graph showing cotton stalk slitters of a silent plant and a control plant of the result of analysis of resistance of a VIGS-interfered upland cotton TM-1 plant to verticillium wilt in example 3 of the present invention;
FIG. 9 is a graph showing comparison of the results of fungus recovery experiments performed on silenced plants of the VIGS-interfered upland cotton TM-1 plants of example 3 according to the results of analysis of resistance to verticillium wilt with surface sterilized stems of control plants;
FIG. 10 is a graph showing statistics of cotton verticillium recovery from a silent plant and a control plant of the analysis of resistance of a VIGS-interfered upland cotton TM-1 plant to verticillium in example 3 of the present invention;
FIG. 11 is a graph showing the relative abundance of verticillium DNA in cotton stems of a silent plant and a control plant of VIGS-interfered upland cotton TM-1 plants of example 3 according to the invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The cotton material comprises:
upland cotton TM-1, supplied by cotton institute of China academy of agricultural sciences;
plant VIGS silencing expression vector:
verticillium V991: the laboratory is stored, and the public can be obtained from the institute of biotechnology of nuclear sciences of agricultural sciences of Xinjiang (the institute of biotechnology of Uygur autonomous region of Xinjiang);
primer sequence: are provided by Shanghai biochemical engineering;
other biological materials include: the escherichia coli DH5 alpha, the agrobacterium tumefaciens GV3101, the BP reaction entry vector, the VIGS interference technology vectors pTRV1 and pTRV2 and the like belong to commercial strains or vectors, and are not repeated;
experimental reagent: the plasmid extraction kit, oligo (dT) 18, RNAase inhibitor, dNTP, pMD18-T Vector, T4-DNA ligase, endonuclease EcoRI and KpnI, exTaq enzyme, PCR product recovery kit and the like are all products of TaKaRa company, and the fluorescent quantitative PCR kit is a product of TOYOBO company;
the special plate for fluorescent quantitative PCR is manufactured by Labwares company;
RNA extraction kit was purchased from TIANGEN (Beijin, china);
the culture medium and solution used: LB liquid (solid) medium, YEP liquid (solid) medium, czapek medium, PDA medium, 50 xTAE Buffer (Na) 2 EDTA·2H 2 37.2g of O, 57.1mL of glacial acetic acid, pH adjustment by NaOH=8.3, water addition and volume fixation to 1L) and the like, and are prepared according to the conventional preparation method in the field;
other undescribed antibiotics, hormones and other reagents are common in the art and will not be described again.
Example 1
Acquisition of upland cotton GhCAMK Gene
Planting upland cotton TM-1, extracting RNA of cotton seedling leaves, and performing reverse transcription to obtain cDNA;
RNA extraction procedure referring to TIANGEN plant RNA rapid extraction kit, reverse transcription system is shown in Table 1:
TABLE 1 reverse transcription system
Reagent(s) | Dosage of |
TotalRNA | 1μg |
Oligo(dT)18(0.5μg/μL) | 1μL |
2×TSReactionMix | 10μL |
TransScriptRT/EnzymeMix | 1μL |
gDNARemover | 1μL |
RNase-freeWater | Supplement to 20 mu L |
Executing the program: 42 ℃ for 30min and 85 ℃ for 5s.
cDNA with the ID number of Gh_D06G1869 gene (the nucleotide sequence is shown as SEQ ID NO:1, the amino acid sequence is shown as SEQ ID NO: 2) in a cottonFGD website (https:// CottonFGD. Org) is used as a template, primers are designed and amplified, and the primer sequences are as follows:
GhCCAMK-F:5’-ATGGGACAAGATAAAGCGAAAC-3’,SEQ ID NO:3;
GhCCAMK-R:5’-TTACTGTTGTCGAAGAGAAGAGAG-3’,SEQ ID NO:4。
the PCR amplification system is shown in Table 2:
TABLE 2PCR amplification System
Reagent(s) | Dosage of |
GhCCAMK-F(10μM) | 1μL |
GhCCAMK-R(10μM) | 1μL |
5×TransStartFastPfuBuffer | 10μL |
2.5mMdNTPs | 4μL |
TransStartFastPfuDNAPolymerase(2.5U/μL) | 1μL |
Nuclease-freeWater | Supplement to 50 mu L |
PCR amplification procedure: 95 ℃ for 2min;95℃20s,56℃30s,72℃120s,35 cycles; 7min at 72 ℃.
The PCR electrophoresis products were subjected to 1% agarose gel electrophoresis, and the results are shown in FIG. 1. As can be seen from FIG. 1, the gene GhCAMK has a size of about 1563bp.
Example 2
1. Tissue expression pattern of GhCAMK gene under natural condition
Respectively taking leaves, roots, stems, flowers, sepals and bracts of cotton in the full-bloom stage of the cotton, extracting RNA of the cotton, obtaining cDNA by using a reverse transcription kit, and carrying out qPCR amplification by adopting the following quantitative PCR primers:
QRT-PCR-GhCCAMKF:5’-GGCGATCCATAAGGCGAACA-3’,SEQ ID NO:5;
QRT-PCR-GhCCAMKR:5’-CTCTTTGCTGTGATTTGCCCC-3’,SEQ ID NO:6。
the reference gene is Ubiquitin7 (Ubiquitin 7, UB 7), and the primer sequence is designed as follows:
UBQ7F:5’-GAAGGCATTCCACCTGACCAAC-3’,SEQ ID NO:7;
UBQ7R:5’-CTTGACCTTCTTCTTCTTGTGCTTG-3’,SEQ ID NO:8。
qRT-PCR was performed on an ABI 7500Real-time PCR sequence detection system and software (AppliedBiosystems, USA).
The 20. Mu.L reaction system was designed as follows:
the reaction system was 10. Mu. LSYBR Green Realtime PCR Master Mix, 1. Mu.L of cDNA product, 0.4. Mu.L (10. Mu.M) of each of the upstream and downstream primers, and the nucleic-free Water was supplemented to a total reaction volume of 20. Mu.L. 4 technical replicates per gene.
The corresponding procedure is:
the quantitative procedure was 94℃for 30s;95℃5s,57℃15s,72℃31s,40 cycles.
The specificity of the amplified product after 40 cycles was detected by dissolution profile analysis. Each reaction included at least three replicates. Primer amplification efficiency was measured by plotting the log of the template dilution against the Ct value of each diluted sample, diluted with a single template to different concentrations.
The detection results are shown in fig. 2, and analysis shows that the GhCCAK gene is expressed in all tissues of cotton, but the transcription level is obviously different, wherein the expression of the gene in leaves and bracts is higher than that of other tissues, and the gene is the stem; the expression level in roots, flowers and sepals is significantly lower than in other tissues, and the transcription level in flowers is slightly higher than in roots and sepals.
2. Is stressed by signal molecules (jasmonic acid JA, salicylic acid SA and H) 2 O 2 Dip dyeing) expression pattern of the gene GhCAMK
The TM-1 cotton seeds are soaked in water for 24 hours and then sowed in nutrient soil, and are subjected to illumination for 12 hours or dark for 12 hours, and are subjected to illumination culture at 26-28 ℃. The humidity is kept at 60% or above, water is poured once for 4-5 d, cotton seedlings with consistent growth vigor and size are selected when two leaves of the seedlings are centered, and 200 mu M jasmonic acid, 2mM salicylic acid and 1mM H are respectively used 2 O 2 Soaking cotton seedling root system for 10min, and planting cotton seedling in the nutrition pot. And respectively taking cotton root systems at the time points of 0.5h, 1h and 3h after the treatment, extracting RNA, and reversely transcribing cDNA for expression pattern analysis.
The qRT-PCR analysis related primers and the PCR reaction procedure were the same as in example 1, section "tissue expression pattern of the gene GhCAMK under natural conditions".
The qRT-PCR detection results are shown in FIG. 3, and the expression level of GhCAMK is shown in JA and H 2 O 2 The treatment time is highest at 0h, and then drops significantly; the expression level of ghvcmk was down-regulated after SA treatment. It was demonstrated that the expression level of GhCAMK was affected by the stress of the signal molecule.
3. Expression pattern of GhCCAK under verticillium infection condition
Root injury is carried out by selecting two-leaf and one-heart upland cotton TM-1, and the concentration of verticillium wilt V991 conidium liquid is 1 multiplied by 10 7 Spores/mL with water treatment as negative control. After the cotton seedling root system is soaked in the bacterial liquid for 10min, the cotton seedling root system is planted in a nutrition pot, and the cotton seedling root system is taken at the time points of 0h, 0.5h, 2h, 6h, 12h, 24h and 48h after inoculation, RNA is extracted, and cDNA is reversely transcribed for expression pattern analysis.
The qRT-PCR analysis related primers and the PCR reaction procedure were the same as in example 1, section "tissue expression pattern of the gene GhCAMK under natural conditions".
The detection result of qRT-PCR is shown in figure 4, after verticillium wilt V991 conidium liquid treatment, the expression level of GhCAMK is obviously reduced after inoculating verticillium wilt pathogen for 2 hours, and the expression level tends to be lowest after 48 hours. After 0.5 hour of water treatment, the significance was down-regulated. The result shows that after the root system of upland cotton TM-1 is infected by verticillium, the expression level of the gene GhCAMK is obviously changed.
Example 3
And a VIGS interference vector is constructed by using a VIGS technology, so that the gene GhCCAK is silenced. The phenotypic change of cotton after gene silencing during verticillium infection is observed, and further proves that the gene GhCAMK is highly related to verticillium resistance.
1. Construction of recombinant expression vectors
(1) Primer design and PCR amplification
Primers are designed in a non-conserved region of the gene GhCAMK, and the design principle of the primers is as follows: the restriction enzyme EcoRI cleavage site and the protecting base are added to the upstream primer, and the restriction enzyme KpnI cleavage site and the protecting base are added to the downstream primer, and the primers are designed as follows:
GhCCAMK-1F:5’-CGGAATTCTTGGTGCTGGAATTGTGCTC-3’,SEQ ID NO:9;
GhCCAMK-1R:5’-GGGGTACCCTTTGCTGTGATTTGCCCCT-3’,SEQ ID NO:10。
extracting total RNA of upland cotton TM-1, reversely transcribing the total RNA into cDNA, and carrying out PCR amplification by using the cDNA as a template and adopting a primer pair consisting of a primer GhCCAK-1F and a primer GhCCAK-1R to obtain a PCR amplification product.
The PCR amplification system is shown in Table 3:
TABLE 3PCR amplification System
Reagent(s) | Dosage of |
GhCCAMK-1F(10μM) | 1μL |
GhCCAMK-1R(10μM) | 1μL |
5×TransStartFastPfuBuffer | 10μL |
2.5mMdNTPs | 4μL |
TransStartFastPfuDNAPolymerase | 1μL |
Nuclease-freeWater | Supplement to 50 mu L |
PCR amplification procedure: 95 ℃ for 2min;95℃for 20s,52℃for 30s,72℃for 30s,35 cycles; and at 72℃for 5min.
Amplifying a target sequence (about 318 bp) by PCR; the amplified product was subjected to 1% agarose gel electrophoresis.
(2) Sequencing to obtain recombinant expression vector
The PCR amplified product was recovered by gel ligation with pEASY-Blunt Zero Cloning Kit (cloning vector), and the positive transformants identified by PCR using the GhCCAK-1F/R primer were sequenced.
The positive transformants which were sequenced successfully were selected to extract plasmids, and the plasmids and empty vector pTRV2 were digested with the enzymes EcoRI and KpnI, respectively, in a water bath at 37℃and the digestion system was as shown in Table 4:
table 4 enzyme digestion System
Reagent(s) | Dosage of |
GhCCAK plasmid | 3μL |
EcoRI | 1μL |
KpnI | 1μL |
10×FlyCutBuffer | 2μL |
Nuclease-freeWater | Supplement to 20 mu L |
And (3) enzyme cutting: 37 ℃ for 1h; 10min at 80 ℃.
The enzyme cutting product is subjected to 1% agarose gel electrophoresis, and is connected by using T4 DNA ligase, the plant expression vector recombinant plasmid TRV 2-GhCAMK is used for transforming agrobacterium GV3101, and the GhCAMK-1F/R primer is used for obtaining the VIGS interference vector TRV2: ghCAMK after PCR screening and identification.
2. Acquisition of transgenic cotton
(1) Bacterial liquid culture
Agrobacterium was transformed with pTRV1 and TRV 2-GhCAMK, respectively, and the resulting mixture was transformed into a vector containing kanamycin (50. Mu.g. Multidot.mL) -1 ) Gentamicin (50. Mu.g.mL) -1 ) And rifampicin (25. Mu.g.mL) -1 ) Resistant LB flask was incubated at 28℃to OD 600 Reaching 0.6 to 0.8.
(2) Resuspension obtaining
The cells were collected by centrifugation at 4000rpm for 5min and resuspended in the appropriate volume (formulation: 10 mmol.L) -1 MgCl 2 ,10mmol·L -1 MES and 200. Mu. Mol.L -1 Acetosyringone) was resuspended to a final concentration of OD 600 1.5. And standing the heavy suspension at room temperature in dark for more than 3 hours. Obtaining heavy suspension containing recombinant plasmid TRV 2-GhCAMK. The preparation method is similar to that of a heavy suspension containing pTRV1 vector, a heavy suspension containing TRV2-GhCLA1 vector of GhCLA 1gene fragment and a heavy suspension containing pTRV2 empty vector.
Mixing the heavy suspension containing pTRV1 vector and the heavy suspension containing target gene fragment TRV 2-GhCAMK in the volume ratio of 1:1 to obtain TRV, injecting GhCAMK solution into cotton cotyledon for obtaining gene silencing transformant; mixing the heavy suspension containing pTRV1 vector and the heavy suspension containing GhCLA 1gene fragment of TRV2-GhCLA1 vector uniformly according to the volume ratio of 1:1 to obtain TRV, wherein the TRV is GhCLA1 solution for injecting cotton cotyledons for detecting whether a gene silencing system is correct or not; the suspension containing pTRV1 carrier and suspension containing pTRV2 empty carrier are mixed uniformly according to the volume ratio of 1:1 to obtain TRV:00 solution, and the solution is injected into cotton cotyledon to be used as genetic transformation control strain.
(3) Recipient culture
The TM-1 cotton seeds are sowed in nutrient soil after being soaked in water for 24 hours, and are subjected to illumination for 12 hours or dark for 12 hours, and are subjected to illumination culture at the temperature of 26-28 ℃. The humidity is kept at 60% and above, water is poured once for 4-5 d, and the two cotyledons can be used for VIGS operation when the two cotyledons are flat and unfolded and the true cotyledons are not yet developed.
(4) Acquisition of GhCAMK Gene silencing transformants
Firstly, lightly puncturing the back of cotyledons by using a syringe needle to cause micro-wounds, and injecting the prepared heavy suspension mixed according to the volume ratio of 1:1 from the wounds by using a syringe without the needle to obtain the cotton GhCCAK gene silencing transformant. Light is prevented from 24 hours, 12 hours of illumination/12 hours of darkness, and the temperature is 26-28 ℃ of illumination culture.
A GhCLA 1gene silencing test strain and pTRV2 empty vector genetic transformation control strain were obtained in a similar manner.
Agrobacteria VIGS specific methods reference: gao, X., shan, L.functional genomic anal ysis ofcotton genes with agrobacterium-treated virus-reduced gene sizing.methods Mol Biol,2013,975:157-165.
3. Detection of VIGS genetic transformation System
(1) After 2 weeks, the phenotype of the different treated cotton was observed, and the VIGS system was examined using the upland cotton GhCLA 1gene (cloroplastos alterados gene) as a marker gene. The gene participates in chloroplast development process, codes 1-deoxyxylulose 5-phosphate synthase protein, is highly conserved in evolution, and cotton plants have obvious albino phenotype after GhCLA 1gene is silenced, thus being a marked character which is easy to identify. As shown in FIG. 5, 2 weeks after infection with VIGS, the true leaves of the plants injected with TRV1 and TRV2-GhCLA1 were almost completely whitened (FIG. 5A), while leaves injected with the empty vectors pTRV1 and pTRV2 as controls (TRV: 00) were unchanged (FIG. 5B TRV: 00). Demonstrating the successful establishment of a TRV-mediated VIGS system in upland cotton TM-1.
(2) Fluorescent quantitative Real time-PCR detection
RNA (preferably new long true leaves) is extracted from the treated cotton leaves, then qRT-PCR is performed to detect whether the target gene is expressed down or not, and the expression of the target gene is detected. For each material 30 individual plants were treated.
And extracting total RNA of the leaves of the VIGS-impregnated cotton plants by using the RNA extraction kit. The expression of the post-silencing GhCCAK gene which is disturbed and silenced is detected by fluorescent quantitative Real time-PCR by using cotton UBQ7 as an internal reference gene.
qRT-PCR analysis of the relevant primers and PCR procedure refer to example 2 part 1, "tissue expression pattern of the Gene GhCAMK in nature".
As shown in FIG. 6, compared with the empty vector (TRV: 00) control, the expression level of the GhCAMK gene in the randomly selected GhCAMK gene VIGS-infected plants is remarkably reduced, and the silencing effect is remarkable.
4. Inoculation and resistance identification for verticillium wilt resistance of cotton
(1) Inoculating verticillium wilt of cotton
The stored verticillium dahliae strain V991, a verticillium dahliae pathogen, is activated on a PDA culture medium. The selected thalli are cultured for 3-5 d in Czapek culture solution at 25 ℃ and 200 rpm. Filtering the pathogenic bacteria culture solution with 4 layers of gauze, counting pathogenic bacteria concentration with blood cell counting plate, and regulating final concentration to 1.0X10 with sterilized double distilled water 7 spores/mL, and Tween-20 was added to a final concentration of 0.001% (volume percent). The obtained transformant after 2 weeks of VIGS gene silencing was inoculated with verticillium V991 spore liquid by root injury.
(2) Verticillium wilt disease incidence statistics
And (5) investigation and statistics of verticillium wilt morbidity when the true leaves begin to turn yellow and wilt, and statistics of disease grade by adopting a 0-4 grade method. For each material 30 individual plants were treated. Let 3 biological replicates.
Disease index statistical reference: xu Li, zhu Longfu, zhang Xianlong. Research progress on the mechanism of cotton verticillium wilt resistance, crop theory, 2012,38:1553-1560; xu L, zhu L F, zhang X L.research on resistance mechanism ofcotton to Verticillium wilt. ActaAgron Sin,2012,38:1553-1560.
Disease index = [ (number of disease plants at each stage x corresponding grade)/survey total number of plants x highest grade of disease (4) ]100
And (3) test result statistics: the disease of verticillium dahliae after 2 weeks of infection with VIGS, with respect to the empty vector control (TRV: 00) plants, was inoculated with the silenced plant of TRV: ghvcak, after 21d with verticillium dahliae strain V991. As can be seen in FIG. 5B, the control (TRV: 00) plants showed more pale yellow plaques and a larger area in the leaves and more pronounced curling of the edges down than the GhCAMK gene-silenced plants. The results of the statistical analysis of 3 biological repeated observation show that the average disease index of the control plants injected with the empty vector is 72%, and the disease resistance of the plants after GhCAMK gene silencing is obviously enhanced, and the average disease index is 56%. The GhCAMK gene is shown to be involved in verticillium-induced allergic reaction. After the GhCAMK gene is silenced in upland cotton TM-1, the incidence rate and disease indication of cotton are obviously reduced when the cotton is infected by verticillium wilt bacteria, which indicates that the silencing of the GhCAMK gene improves the resistance of the cotton to verticillium wilt.
(3) Verification of the involvement of the GhCAMK Gene in the verticillium-induced allergic reaction
In order to verify the accuracy of the phenotype result, the invention continues to perform verticillium restoration culture experiments, disease plant splitting rod treatment and plant verticillium DNA relative abundance detection.
The verticillium recovery culture experimental method comprises the following steps: cutting the stem of cotton seedling with 21d after verticillium wilt treatment into segments with the length of 1cm from 6cm of cotyledons by scissors, soaking for 1min by 75% alcohol, soaking for 30min by 30% hydrogen peroxide, washing for 4-5 times by sterile water, and finally placing the segments of cotton stem after treatment on a PDA culture medium for culturing for 3d at 25 ℃, thus observing the growth condition of verticillium wilt bacteria recovery culture.
The treatment method of the disease planting dissecting rod comprises the following steps: taking a plurality of cotton stalks of 21d inoculated with verticillium wilt, longitudinally cutting the cotton stalks by using a sharp blade to expose the longitudinal section of the cotton stalks, observing the phenotype of the vascular bundle tissue of the cotton stalks, wherein if the vascular bundle tissue of the cotton stalks is brown, the vascular bundle tissue of the cotton stalks is infected with verticillium wilt and is ill, and if the vascular bundle tissue of the cotton stalks is not brown and the phenotype of the vascular bundle tissue of the cotton stalks is normal, the vascular bundle tissue of the cotton stalks is not ill.
The method for the relative abundance of the verticillium dahliae DNA of the plants comprises the following steps: RNA of the stem of cotton seedlings after verticillium treatment is extracted, inverted into cDNA and subjected to qPCR amplification.
The primers used were:
ITS1-F:5’-AAAGTTTTAATGGTTCGCTAAGA-3’,SEQ ID NO:11;
ST-VE1-R:5’-CTTGGTCATTTAGAGGAAGTAA-3’,SEQ ID NO:12。
reverse transcription and qPCR reaction procedure conditions and procedures refer to example 2, part 1, "tissue expression pattern of the naturally occurring gene gccamk".
As shown in fig. 8-11, after the stalks are longitudinally cut, the stalk vascular bundles of the control plants injected with the empty vector become light brown, while the stalk vascular bundles of the plants with the silence GhCCAK genes are normal, and the browning degree of the stalk cutting material of the GhCCAK plant materials is lower than that of the control plant materials injected with the empty vector (fig. 8); the bacterial colony of verticillium bacteria cultured by the GhCCAK plant material in the PDA culture medium is less than that of the TRV 00 plant material (figure 9); the recovery rate of verticillium of the TRV 00 control plants reaches 83%, and the recovery rate of verticillium of the plants after the TRV GhCAMK gene silencing reaches 27% (figure 10); the relative abundance of the verticillium DNA of the TRV 00 plant is about 3 times that of the plant with the TRV GhCAMK gene silenced (figure 11).
These results also indicate that cotton has increased resistance to verticillium after silencing the gccamk gene, indicating that the gccamk gene is involved in regulating verticillium resistance.
In the present specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, and identical and similar parts between the embodiments are all enough to refer to each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (5)
1. The application of the calcium-dependent protein kinase gene GhCAMK in regulating and controlling verticillium wilt resistance of plants is characterized in that the nucleotide sequence of the gene GhCAMK is shown in SEQ ID NO: 1.
2. The application of protein or a substance for regulating the expression of a protein coding gene in plant verticillium wilt resistance is characterized in that the amino acid sequence of the protein is shown as SEQ ID NO: 2.
3. Use of the gene ghvcak according to claim 1 or the protein-related biomaterial according to claim 2 for plant verticillium wilt resistance, characterized in that the biomaterial is any of the following:
a: a nucleic acid molecule according to claim 1 or a nucleic acid molecule encoding a protein according to claim 2;
b: an expression cassette comprising silencing of the nucleic acid molecule of a;
c: an expression vector comprising the nucleic acid molecule of A, or a recombinant vector comprising the expression cassette of B;
d: a recombinant microorganism comprising the nucleic acid molecule of A, a recombinant microorganism comprising the expression cassette of B, or a recombinant microorganism comprising the recombinant vector of C.
4. Use according to any one of claims 1 to 3, wherein the plant is a cotton plant.
5. A method for increasing verticillium wilt resistance in a plant, comprising increasing verticillium wilt resistance in a plant by silencing or inhibiting expression of the gene ghvcak according to claim 1 or activity of the protein according to claim 2.
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Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20080034453A1 (en) * | 1999-05-06 | 2008-02-07 | Nordine Cheikh | Annotated Plant Genes |
CN110343157A (en) * | 2019-07-31 | 2019-10-18 | 新疆农业科学院核技术生物技术研究所(新疆维吾尔自治区生物技术研究中心) | Cotton verticillium wilt related gene GhBONI and its coding albumen and application |
CN114790449A (en) * | 2022-05-11 | 2022-07-26 | 新疆农业科学院核技术生物技术研究所(新疆维吾尔自治区生物技术研究中心) | Application of calcium-dependent protein kinase gene GhCPK4 in resisting verticillium wilt of plants |
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Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20080034453A1 (en) * | 1999-05-06 | 2008-02-07 | Nordine Cheikh | Annotated Plant Genes |
CN110343157A (en) * | 2019-07-31 | 2019-10-18 | 新疆农业科学院核技术生物技术研究所(新疆维吾尔自治区生物技术研究中心) | Cotton verticillium wilt related gene GhBONI and its coding albumen and application |
CN114790449A (en) * | 2022-05-11 | 2022-07-26 | 新疆农业科学院核技术生物技术研究所(新疆维吾尔自治区生物技术研究中心) | Application of calcium-dependent protein kinase gene GhCPK4 in resisting verticillium wilt of plants |
Non-Patent Citations (3)
Title |
---|
HU Q 等: ""GhCPK33 Negatively Regulates Defense against Verticillium dahliae by Phosphorylating GhOPR3"", PLANT PHYSIOLOGY, vol. 178, no. 2, 27 August 2018 (2018-08-27), pages 876 * |
ZENG H 等: ""Calmodulin and calmodulin‐like protein‐mediated plant responses to biotic stresses"", PLANT, CELL & ENVIRONMENT, vol. 46, no. 12, 14 August 2023 (2023-08-14), pages 3680 - 3703 * |
邓亚辉: ""陆地棉黄萎病抗病基因的挖掘及功能鉴定"", 中国博士学位论文全文数据库 农业科技辑, no. 3, 15 March 2023 (2023-03-15), pages 046 - 8 * |
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