CN102367274B - Plant resistance associated protein named as CRK 25 and its coding gene and use - Google Patents
Plant resistance associated protein named as CRK 25 and its coding gene and use Download PDFInfo
- Publication number
- CN102367274B CN102367274B CN 201110317238 CN201110317238A CN102367274B CN 102367274 B CN102367274 B CN 102367274B CN 201110317238 CN201110317238 CN 201110317238 CN 201110317238 A CN201110317238 A CN 201110317238A CN 102367274 B CN102367274 B CN 102367274B
- Authority
- CN
- China
- Prior art keywords
- sequence
- gene
- plant
- gfp
- recombinant plasmid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Landscapes
- Peptides Or Proteins (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a plant resistance associated protein named as CRK 25 and its coding gene and use. The plant resistance associated protein named as CRK 25 is extracted from arabidopsis thaliana. The plant resistance associated protein named as CRK 25 is one of 1), a protein composed of an amino acid sequence I, and 2), a sequence I derived protein composed of an amino acid sequence formed from the amino acid sequence I through one or more amino acid residue replacement and/or deletion and/or addition, wherein the sequence I derived protein has any one of characteristics of an ICS1 gene promoter starting induction function, a PR1 gene promoter starting induction function, plant salicylic acid synthesis relativity and plant resistance relativity. The plant resistance associated protein named as CRK 25 can improve disease resistance of a plant through controlling synthesis of salicylic acid in the plant. The plant resistance associated protein named as CRK 25 is very valuable to disease-resistant plant cultivation.
Description
Technical field
The present invention relates to a kind of plant resistance to environment stress associated protein CRK25 and encoding gene and application.
Background technology
Plant is the various diseases of normal generation in the cultivation and production process, and some diseases consist of great threat to plant-growth, its output of grievous injury farm crop and quality.For Plant diseases, main employing medicine measure control at present.People begin to use abiotic inductor inducing plant disease resistance recently, and have been widely used in the multiple important farm crop such as tobacco, potato, tomato, cucumber, Kidney bean, paddy rice.Although the effect of this measure is desirable, cost is higher, and contaminate environment.Therefore, in the urgent need to developing new biological means to improve the disease resistance of plant self.
Systemic acquired resistance is a kind of defensive raction of plant, and when plant was attacked by pathogenic agent and insect, this reaction can be induced, and other position that can be diffused into rapidly plant is with protective plant.
Pathogenesis-related proteins PR is a class stress protein, be considered to play an important role in plant disease-resistant, the gene of PR1 albumen of wherein encoding is often used as Plant defense responses and system and obtains indicator in resistance, and the expression of PR1 gene plays an important role in Plant defense responses.The PR1 gene is under the normal growth condition, and the background expression amount is very low, that is to say that the PR1 gene promoter do not bring into play start-up performance substantially under the normal growth condition, is subjected to adverse circumstance to induce the (expression of promotor gene when suffering the disease invasion and attack.
Whitfield's ointment is the aldehydes matter that extensively is present in plant materials, is considered to bring out the signal of systemic acquired resistance, can induce the resistance to multiple pathogens such as virus, bacterium, fungies.Studies show that, after executing Whitfield's ointment outward, transcribing of PR1 gene significantly activated.Research also finds, when plant a part suffers after pathogenic agent attacks, in plant materials, salicylic acid content can increase, and before salicylic increase appears at PR1 genetic expression.In plant, salicylic synthesizing is to act on the different chorismic acid of substrate (isochorismate) thereby salicylate by different chorismic acid synthetase (ICS, isochorismate synthase) ICS1 to a great extent.The ICS1 gene is under the normal growth condition, and the background expression amount is very low, that is to say that the ICS1 gene promoter do not bring into play start-up performance substantially under the normal growth condition, is subjected to adverse circumstance to induce the expression of (pathogen infection and uviolizing) promotor gene.Up to the present, only find ETHYLENE INSENSITIVE3 (EIN3) and EIN3-LIKE1 can with the direct combination of SA biosynthesizing key gene ICS1 promotor, and the synthetic level of negative regulator SA.Research finds that SAR Deficient 1 (SARD1) and CBP60g albumen are to induce ICS1 genetic expression and the synthetic important factor of SA in addition, but fail to find to induce the ICS1 promotor to start ICS1 genetic expression by forward more, thereby increase the albumen of the synthetic level of Whitfield's ointment.
Summary of the invention
The purpose of this invention is to provide a kind of plant resistance to environment stress associated protein CRK25 and encoding gene and application.
Albumen provided by the invention, called after CRK25 albumen, from Arabidopis thaliana (Arabidopsis thaliana), be following (a), (b), (c), (d) and (e) in any one:
(a) protein that is formed by the aminoacid sequence shown in sequence in sequence table 1;
(b) with the aminoacid sequence of sequence 1 through replacement and/or disappearance and/or the interpolation of one or several amino-acid residue and have induce ICS1 gene promoter start-up performance by the derivative protein of sequence 1;
(c) with the aminoacid sequence of sequence 1 through replacement and/or disappearance and/or the interpolation of one or several amino-acid residue and have induce PR1 gene promoter start-up performance by the derivative protein of sequence 1;
(d) with the aminoacid sequence of sequence 1 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and synthetic relevant to Whitfield's ointment in the plant protein that is derived by sequence 1;
(e) with the aminoacid sequence of sequence 1 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and relevant to the plant resistance to environment stress protein that is derived by sequence 1.
In order to make the protein in (a) be convenient to purifying, N-terminal or C-terminal that can the protein that the aminoacid sequence shown in sequence 1 forms in by sequence table connect label as shown in table 1.
The sequence of table 1 label
| Label | Residue | Sequence |
| Poly-Arg | 5-6 (being generally 5) | RRRRR |
| Poly-His | 2-10 (being generally 6) | HHHHHH |
| FLAG | 8 | DYKDDDDK |
| Strep-tag II | 8 | WSHPQFEK |
| c-myc | 10 | EQKLISEEDL |
Above-mentioned (b), (c), (d) but or the protein synthetic (e), also can first synthesize its encoding gene, then carry out biological expression and obtain.Above-mentioned (b), (c), (d) or (e) in protein encoding gene can by will be in the DNA sequence dna shown in sequence in sequence table 2 codon of one or several amino-acid residue of disappearance, and/or carry out the missense mutation of one or several base pair, and/or obtain at the encoding sequence that its 5 ' end and/or 3 ' end connects the label shown in table 1.
The gene of encoding said proteins (CRK25 gene) also belongs to protection scope of the present invention.
Described gene can be following 1) to 9) in arbitrary described DNA molecular:
1) DNA molecular shown in the sequence 2 of sequence table;
2) under stringent condition with 1) DNA molecular with the albumen of inducing ICS1 gene promoter start-up performance of the DNA sequence dna hybridization that limits and coding;
3) with 1) DNA sequence dna that limits has the DNA molecular that 90% above homology and coding have the albumen of inducing ICS1 gene promoter start-up performance;
4) under stringent condition with 1) DNA molecular with the albumen of inducing OR1 gene promoter start-up performance of the DNA sequence dna hybridization that limits and coding;
5) with 1) DNA sequence dna that limits has the DNA molecular that 90% above homology and coding have the albumen of inducing PR1 gene promoter start-up performance;
6) under stringent condition with 1) the DNA sequence dna hybridization that limits and the DNA molecular of coded plant Whitfield's ointment synthesis associated protein;
7) with 1) DNA sequence dna that limits has the DNA molecular of 90% above homology and coded plant Whitfield's ointment synthesis associated protein;
8) under stringent condition with 1) the DNA sequence dna hybridization that limits and the DNA molecular of coded plant resistance-associated protein;
9) with 1) DNA sequence dna that limits has the DNA molecular of 90% above homology and coded plant resistance-associated protein.
" stringent hybridization condition " is often referred to salt concn lower than about 1.5M, is generally approximately 0.01-1.0M, and pH is approximately between 7.0-8.3, and temperature is approximately between 30-60 degree centigrade.Also can be by adding destabilizing agent, for example methane amide, realize stringent hybridization condition.Above-mentioned stringent condition can be 0.1 * SSPE (or 0.1 * SSC), in the solution of 0.1%SDS, hybridize under 65 ℃ of conditions and wash film.
The nucleotide sequence of described ICS1 gene promoter is as shown in the sequence 3 of sequence table.
The nucleotide sequence of described PR1 gene promoter is as shown in the sequence 4 of sequence table.
The recombinant expression vector, expression cassette, transgenic cell line or the recombinant bacterium that contain described gene all belong to protection scope of the present invention.
Available existing plant expression vector construction contains the recombinant expression vector of described gene.Described plant expression vector comprises the double base agrobacterium vector and can be used for the carrier etc. of plant micropellet bombardment.Described plant expression vector also can comprise 3 ' of foreign gene and hold untranslated regional, namely comprises the DNA fragmentation of polyadenylic acid signal and any other participation mRNA processing or genetic expression.The bootable polyadenylic acid of described polyadenylic acid signal joins 3 ' end of mRNA precursor.When using described gene constructed recombinant plant expression vector, can add any enhancement type promotor or constitutive promoter before its transcription initiation Nucleotide, they can use separately or be combined with other plant promoter; In addition, when using gene constructed plant expression vector of the present invention, also can use enhanser, comprise translational enhancer or transcriptional enhancer, these enhansers zone can be ATG initiator codon or neighboring region initiator codon etc., but must be identical with the reading frame of encoding sequence, to guarantee the correct translation of whole sequence.The source of described translation control signal and initiator codon is widely, can be natural, also can synthesize.Translation initiation region can be from transcription initiation zone or structure gene.For the ease of transgenic plant cells or plant are identified and are screened, can process plant expression vector used, as add the coding that to express in plant can produce enzyme or the gene of luminophor, the antibiotic marker thing with resistance or the anti-chemical reagent marker gene etc. of colour-change.
Described recombinant expression vector specifically can be the recombinant plasmid that obtains at DNA fragmentation shown in the sequence 2 of the multiple clone site insertion sequence table of 326-FLAG expression vector; Described 326-FLAG expression vector is the recombinant plasmid that obtains at DNA as shown in the sequence 6 of multiple clone site (as between PstI and EcoRI restriction enzyme site) the insertion sequence table of pUC19 carrier.Described recombinant expression vector specifically can be: the recombinant plasmid that the CRK25 gene shown in the sequence 2 of insertion sequence table obtains between the XbaI of described 326-FLAG expression vector and BamH I restriction enzyme site.
The increase total length of described gene or the primer pair of its arbitrary fragment also belongs to protection scope of the present invention.
The present invention also protects described albumen or the application of described gene in the start-up performance of inducing the ICS1 gene promoter; The nucleotide sequence of described ICS1 gene promoter is as shown in the sequence 3 of sequence table.Described application specifically can be and induce recombinant plasmid 326-Pro in Arabidopis thaliana
AtICS1:: the described ICS1 gene promoter in GFP starts the expression of GFP gene; Described recombinant plasmid 326-Pro
AtICS1:: GFP is for replacing the small segment between 326-GFP expression vector PstI and BamHI restriction enzyme site (35S constitutive promoter) recombinant plasmid that obtains for the ICS1 gene promoter (ProAtICS1) shown in the sequence 3 of sequence table; The recombinant plasmid that described 326-GFP expression vector obtains for DNA shown in the sequence 7 of insertion sequence table between the PstI of pUC19 carrier and EcoRI restriction enzyme site.Described GFP gene as the sequence 7 of sequence table from as shown in 5 ' end 862-1578 position Nucleotide.
The present invention also protects described albumen or the application of described gene in the start-up performance of inducing the PR1 gene promoter; The nucleotide sequence of described PR1 gene promoter is as shown in the sequence 4 of sequence table.Described application specifically can be and induce recombinant plasmid 326-Pro in Arabidopis thaliana
AtPR1:: the described PR1 gene promoter in GFP starts the expression of GFP gene; Described recombinant plasmid 326-Pro
AtPR1:: GFP replaces the small segment between 326-GFP expression vector PstI and XhoI restriction enzyme site (35S constitutive promoter) for the PR1 gene promoter (Pro shown in the sequence 4 of sequence table
AtPR1) recombinant plasmid that obtains; The recombinant plasmid that described 326-GFP expression vector obtains for DNA shown in the sequence 7 of insertion sequence table between the PstI of pUC19 carrier and EcoRI restriction enzyme site.Described GFP gene as the sequence 7 of sequence table from as shown in 5 ' end 862-1578 position Nucleotide.
The present invention also protects described albumen or the application of described gene in cultivating resistance plant.Described resistance is presented as that the Whitfield's ointment synthesis capability increases and/or the disease resistance ability increases.Described plant is dicotyledons or monocotyledons, as Arabidopis thaliana.The pathogenic bacterium of described disease can be the pathogenic mutation (bacterium) of pseudomonas syringae tomato and/or rape downy mildew (fungi), and these two kinds of microorganisms all endanger blade.The pathogenic bacterium of described disease also can be the rhizosphere germ that withers.
Described Arabidopis thaliana specifically can be the environmental Arabidopis thaliana of Colombia.
In one embodiment of the invention, with the CRK25 gene fusion in the conversion expression vector that contains the FLAG reporter gene, by with the common instantaneous conversion plant protoplast of the conversion expression vector of the GFP reporter gene that is connected with the ICS1 gene promoter, whether produce green fluorescence according to the fluorescence microscope plant protoplast, and extracting the protoplastis total protein, the antibody with GFP and FLAG carries out Western Blotting test respectively.Result shows, the CRK25 gene can be induced the start-up performance of ICS1 gene promoter, thereby impels the GFP reporter gene expression.
In another embodiment of the present invention, with the CPK25 gene fusion in the conversion expression vector that contains the FLAG reporter gene, by with the common instantaneous conversion plant protoplast of the conversion expression vector of the GFP reporter gene that is connected with the PR1 gene promoter, whether produce green fluorescence according to the fluorescence microscope plant protoplast, and extracting the protoplastis total protein, the antibody with GFP and FLAG carries out Western Blotting test respectively.Result shows, the CPK25 gene can be induced the start-up performance of PR1 gene promoter, thereby impels the GFP reporter gene expression.
CRK25 albumen provided by the invention can by salicylic synthetic in regulating plant, strengthen the disease resistance of plant.The present invention all has great value for cultivating disease-resistant plants.
Description of drawings
Fig. 1 is the electrophorogram that pcr amplification obtains the CRK25 gene.
Fig. 2 is the structural representation of recombinant plasmid 326-CRK25-FLAG.
Fig. 3 is the electrophorogram that pcr amplification obtains the ICS1 gene promoter.
Fig. 4 is recombinant plasmid 326-Pro
AtICS1:: the structural representation of GFP.
Fig. 5 is the electrophorogram that pcr amplification obtains the PR1 gene promoter.
Fig. 6 is recombinant plasmid 326-Pro
AtPR1:: the structural representation of GFP.
Fig. 7 is recombinant plasmid 326-CRK25-FLAG and recombinant plasmid 326-Pro
AtICS1:: after GFP transforms protoplastis jointly, the microscopic result of GFP fluorescin.
Fig. 8 is recombinant plasmid 326-CRK25-FLAG and recombinant plasmid 326-Pro
AtICS1:: after GFP transforms protoplastis jointly, the Western Blotting result of GFP fluorescin (internal reference albumen is the large subunit of Rubisco).
Fig. 9 is recombinant plasmid 326-CRK25-FLAG and recombinant plasmid 326-Pro
AtICS1:: after GFP transforms protoplastis jointly, the Western Blotting result of FLAG albumen (internal reference albumen is the large subunit of Rubisco).
Figure 10 is recombinant plasmid 326-CRK25-FLAG and recombinant plasmid 326-Pro
AtPR1:: after GFP transforms protoplastis jointly, the microscopic result of GFP fluorescin.
Figure 11 is recombinant plasmid 326-CRK25-FLAG and recombinant plasmid 326-Pro
AtPR1:: after GFP transforms protoplastis jointly, the Western Blotting result of GFP fluorescin (internal reference albumen is the large subunit of Rubisco).
Figure 12 is recombinant plasmid 326-CRK25-FLAG and recombinant plasmid 326-Pro
AtPR1:: after GFP transforms protoplastis jointly, the Western Blotting result of FLAG albumen (internal reference albumen is the large subunit of Rubisco).
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, be ordinary method, concrete steps can be referring to " Molecular Cloning:A Laboratory Manual " (Sambrook J., Russell, David W., Molecular Cloning:A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring Harbor).Test materials used in following embodiment if no special instructions, is and purchases available from routine biochemistry reagent shop.The mensuration of the synthetic and DNA sequence dna of the primer in following examples, biological company limited carries out by Beijing English fine horse.Quantitative test in following examples all arranges repeated experiments three times, results averaged.
The environmental Arabidopis thaliana (Arabidopsis thaliana) of Colombia, claim again Arabidopis thaliana or Arabidopis thaliana Col-0: reference: Jin JB, Kim YA, Kim SJ, Lee SH, Kim DH, Cheong GW, Hwang is new dynamin-like protein I.2001.A, ADL6, is involved in traffickingfrom the trans-Golgi network to the central vacuole in Arabidopsis.Plant Cell 13:1511-1526.; The public can buy from U.S.'s Arabidopis thaliana Biological resources centers (ABRC) and obtain.
The pUC19 carrier: available from NEB company, cat. no is N3041S.
326-FLAG expression vector: the recombinant plasmid that shown in the sequence 6 of insertion sequence table, DNA obtains between the PstI of pUC19 carrier and EcoRI restriction enzyme site; In DNA, be the 35S constitutive promoter from the 1st to 835 Nucleotide of 5 ' end shown in the sequence 6 of sequence table, 862-933 position Nucleotide is 3 * FLAG gene, and 934-1192 position Nucleotide is the Nos terminator.
326-GFP expression vector: the recombinant plasmid that shown in the sequence 7 of insertion sequence table, DNA obtains between the PstI of pUC19 carrier and EcoRI restriction enzyme site; In DNA, be the 35S constitutive promoter from the 1st to 835 Nucleotide of 5 ' end shown in the sequence 7 of sequence table, 862-1578 position Nucleotide is the GFP gene, and 1579-1837 position Nucleotide is the Nos terminator.
In embodiment, Anti-GFP antibody used is the GFP Living Colors A.V.Monoclonal Antibody (article No. 632381) of Clontech company.So in embodiment, that Anti-FLAG antibody is the Anti-FLAG M2Monoclonal Antibody (article No. F3165) of Sigma company.
The acquisition of embodiment 1, CRK25 albumen and encoding gene thereof
1, according to existing ncbi database and document, pair of primers is set as follows:
F1 (forward primer): 5 '-
ACTAGTATGTCTTCTTGTTTCAAATCCT-3 ' (underscore mark Spe I restriction endonuclease recognition sequence);
R1 (reverse primer): 5 '-
GGATCCTACGAGGATAAACTATAGTGATG-3 ' (underscore mark BamH I restriction endonuclease recognition sequence).
2, extract the genomic dna of the environmental Arabidopsis leaf of Colombia.
3, take genomic dna as template, adopt the primer pair of step 1 design, carry out pcr amplification with the TaKaRa PrimeSTAR HS of company high-fidelity enzyme.
4, pcr amplification product is checked order, as shown in the sequence 2 of sequence table.
With the albumen called after CRK25 albumen shown in the sequence 1 of sequence table.With the encoding gene called after CRK25 gene of CRK25 albumen, its genomic dna is as shown in the sequence 2 of sequence table.
The structure of embodiment 2, each gene cloning and recombinant expression vector thereof
One, the structure of the acquisition of CRK25 gene and recombinant plasmid 326-CRK25-FLAG
1, extract the genomic dna of the environmental Arabidopsis leaf of Colombia.
2, take the genomic dna of step 1 as template, the primer pair with F1 and R1 form carries out pcr amplification under the effect of the TaKaRa PrimeSTAR HS of company high-fidelity enzyme, obtain pcr amplification product.The agarose gel electrophoresis figure of pcr amplification product sees Fig. 1 (M represents the marker of Nucleotide, is the DL2000 of TaKaRa company).
3, with the pcr amplification product of restriction enzyme Spe I and BamH I double digestion step 2, reclaim enzyme and cut product.
4, with restriction enzyme XbaI and BamH I double digestion 326-FLAG expression vector, reclaim the approximately carrier framework of 3853bp.
5, the enzyme of step 3 is cut the carrier framework connection (restriction enzyme Spe I and XbaI are isocaudarner) that product is connected with step, obtained recombinant plasmid 326-CRK25-FLAG (structural representation is seen Fig. 2).According to sequencing result, recombinant plasmid 326-CRK25-FLAG is carried out result be described below: inserted the CRK25 gene shown in the sequence 2 of sequence table between the XbaI of 326-FLAG expression vector and BamHI restriction enzyme site.
Two, the acquisition of ICS1 gene promoter (ProAtICS1) and recombinant plasmid 326-Pro
AtICS1:: the structure of GFP
1, retrieval ncbi database and document, obtain different chorismic acid synthetase gene sequence (accession number: AT1G74710), as follows according to the primer of a pair of targeted promotor of sequences Design:
F2 (forward primer): 5 '-AA
CTGCAGTTATCCACGCTTTGTCACACA-3 ' (underscore mark PstI restriction endonuclease recognition sequence);
R2 (reverse primer): 5 '-CG
GGATCCCCATTGCAGAAATTCGTAAAGT-3 ' (underscore mark BamHI restriction endonuclease recognition sequence).
2, extract the genomic dna of the environmental Arabidopsis leaf of Colombia.
3, take the genomic dna of step 2 as template, the primer pair with F2 and R2 form carries out pcr amplification under the effect of the TaKaRa PrimeSTAR HS of company high-fidelity enzyme, obtain pcr amplification product.The agarose gel electrophoresis figure of pcr amplification product sees Fig. 3 (M represents the marker of Nucleotide, is the DL2000 of TaKaRa company).
4, with the pcr amplification product of restriction enzyme PstI and BamHI double digestion step 3, reclaim enzyme and cut product.
5, with restriction enzyme PstI and BamHI double digestion 326-GFP expression vector, reclaim the approximately carrier framework of 3635bp (having removed the 35S constitutive promoter in the 326-GFP expression vector).
6, the enzyme of step 4 is cut the carrier framework connection that product is connected with step, obtained recombinant plasmid 326-Pro
AtICS1:: GFP (structural representation is seen Fig. 4).According to sequencing result, to recombinant plasmid 326-Pro
AtICS1:: it is as follows that GFP carries out structrual description: the small segment between 326-GFP expression vector PstI and BamHI restriction enzyme site (35S constitutive promoter) is replaced for the ICS1 gene promoter (ProAtICS1) shown in the sequence 3 of sequence table.
Three, PR1 gene promoter (Pro
AtPR1) acquisition and recombinant plasmid 326-Pro
AtPR1:: the structure of GFP
1, retrieval ncbi database and document, the sequence of acquisition AtPR1 (accession number: AT2G14610), as follows according to the primer of a pair of targeted promotor of sequences Design:
F3 (forward primer): 5 '-AA
CTGCAGTGGCAAATAAACAACGGACA-3 ' (underscore mark PstI restriction endonuclease recognition sequence);
R3 (reverse primer): 5 '-CCG
CTCGAGTAAAATTCATTTTTCTAAGTTGA-3 ' (underscore mark XhoI restriction endonuclease recognition sequence).
2, extract the genomic dna of the environmental Arabidopsis leaf of Colombia.
3, take the genomic dna of step 2 as template, the primer pair with F3 and R3 form carries out pcr amplification under the effect of the TaKaRa PrimeSTAR HS of company high-fidelity enzyme, obtain pcr amplification product.The agarose gel electrophoresis figure of pcr amplification product sees Fig. 5 (M represents the marker of Nucleotide, is the DL2000 of TaKaRa company).
4, with the pcr amplification product of restriction enzyme PstI and XhoI double digestion step 3, reclaim enzyme and cut product.
5, with restriction enzyme PstI and XhoI double digestion 326-GFP expression vector, reclaim the approximately carrier framework of 3641bp.
6, the enzyme of step 4 is cut the carrier framework connection that product is connected with step, obtained recombinant plasmid 326-Pro
AtPR1:: GFP (structural representation is seen Fig. 6).According to sequencing result, to recombinant plasmid 326-Pro
AtPR1:: it is as follows that GFP carries out structrual description: the small segment between 326-GFP expression vector PstI and XhoI restriction enzyme site (35S constitutive promoter) is replaced for the PR1 gene promoter (Pro shown in the sequence 4 of sequence table
AtPR1).
Four, recombinant plasmid 326-T
7The structure of-FLC
T7-FLC gene shown in the sequence 5 of the sequence of composition sequence table (being that T7 label, the 34th to 660 Nucleotide are the FLC gene from the 1st to 33 Nucleotide of 5 ' end), insert between the Spe I and BamH I restriction enzyme site of 326-FLAG expression vector, obtain recombinant plasmid 326-T
7-FLC.The FLC gene be one known not with Pro
AtICS1And Pro
AtPR1The gene of effect.
Embodiment 3, the application of CRK25 gene in inducing the expression of ICS1 gene promoter (ProAtICS1) promotor gene
One, the transient expression of recombinant plasmid in the Arabidopsis leaf protoplastis
Recombinant plasmid 326-CRK25-FLAG and recombinant plasmid 326-Pro with embodiment 2 structures
AtICS1:: the common arabidopsis thaliana transformation protoplastis of GFP is (with recombinant plasmid 326-T
7-FLC is as the negative contrast of recombinant plasmid 326-CRK25-FLAG; With another the negative contrast as recombinant plasmid 326-CRK25-FLAG of 326-FLAG expression vector), concrete steps are as follows:
1, sprout the environmental Arabidopis thaliana seed of Colombia on the MS substratum, be transplanted in soil 23 ℃ of hot-house cultures (illumination every day 12h, intensity of illumination is 150 μ E) when root grows to 1-3 centimetre.
2, add the 20ml distilled water in the 90mm culture dish, then add the 1.82g PEARLITOL 25C and make its dissolving.
3, get in step 1 and to cultivate the 4 weeks blade of bolting (approximately 90) not, be cut into wide rectangular of 1mm, be placed in the mannitol solution 0.5 hour of step 2.
4, at 100ml triangular flask configuration 15ml enzymatic hydrolysis system: Cellulase R-10 (available from YAKULT HONSHA) 150mg, Marcerozyme R-10 (available from YAKULT HONSHA) 37.5mg, MES (available from USB) 30mg, BSA (available from AMRESCO) 15mg, Mannitol are (available from Sigma, article No. M1902 is 1M Mannitol mother liquor) 7.5ml, CaCl
2(available from Sigma, article No. C-2536 is the 200mM mother liquor) 0.075ml, all the other are water, transferring pH with 1M KOH is 5.6.
5, with step 3 slice is pulled out, be placed in the enzymatic hydrolysis system of step 4, enzymolysis 3 hours (23 ℃, 40-50rpm) under dark condition.
The enzymolysis solution that 6, will carry out step 5 is crossed 100-200 purpose sieve, and the green mixture after filtering is placed in 15ml centrifuge tube (diameter 1cm), is divided into two pipes.
7, with 15 minutes collecting precipitations of the centrifuge tube of step 6 centrifugal (4 ℃, 60g), every pipe adds the ice-cold W5 solution of 4ml softly to wash, centrifugal (4 ℃, 100g) 1 minute collecting precipitation then, and every pipe adds the ice-cold W5 solution of 4ml softly to wash; Placed on ice 30 minutes, centrifugal (23 ℃, 100g) 1 minute collecting precipitation then, every pipe precipitation is resuspended with 0.5ml MaMg solution, is protoplastis solution.
W5 solution: NaCl (available from Junsei) 9g, CaCl
2(available from Sigma, article No. C-2661) 13.88g, MES (available from USB) 0.292mg, KCl (available from AMRESCO) 0.372mg, Glucose (available from AMRESCO) 0.902g, water is settled to 1L, and transferring pH with 1M KOH is 5.6.
MaMg solution: Mannitol (available from Sigma, article No. M1902) 7.288g, MgCl
2(available from AMRESCO, article No. 0288-500G) 0.305g, MES (available from USB) 100mg, water is settled to 100ml, and transferring pH with 1M KOH is 5.6.
8, with recombinant plasmid 326-CRK25-FLAG (or recombinant plasmid 326-T
7-FLC or 326-FLAG expression vector) and recombinant plasmid 326-Pro
AtICS1:: GFP respectively gets 15ug in the 10ml pipe, adds 300ul protoplastis solution, with the soft mixing of 200ul rifle head (cutting off front end); Then add 310ul PEG4000/Ca (NO
3)
2Solution, soft mixing; Place 30 minutes (20-30 minute all can), then add 1ml W5 solution to put upside down back and forth mixing, then centrifugal (23 ℃, 100g) 1 minute collecting precipitation; Add 100ul W5 solution mixing, and then add 900ul W5 solution mixing.The positive control of a transfection 326-GFP expression vector is set.
PEG4000/Ca (NO
3)
2Solution: PEG40004g, Mannitol (available from Sigma, article No. M1902 is the 1MMannitol mother liquor) 4ml, Ca (NO
3)
21mg, dH
2O 1.8ml, water is settled to 10ml.
9, the mixed solution that step 8 is obtained is placed in six orifice plates, hatches 12 hours for 23 ℃.
Two, fluorescence microscope
Draw the 30ul step 19 in hatch the mixed solution of completing, be placed on slide glass, after covered, be put under ZEISS AXIOSKOP fluorescent microscope and observe and take pictures gently.
The results are shown in Figure 7.Under fluorescence observation: jointly transform recombinant plasmid 326-Pro
AtICS1:: in the protoplastis of GFP and recombinant plasmid 326-CRK25-FLAG, the green fluorescence identical with the positive control that transforms the 326-GFP carrier appearred; The common recombinant plasmid 326-Pro that transforms
AtICS1:: GFP and recombinant plasmid 326-T
7The protoplastis of-FLC (or 326-FLAG expression vector) can not be observed obvious green fluorescence.Result shows, CRK25 albumen can with the effect of ICS1 promoter region, impel its follow-up GFP genetic expression, the CRK25 gene has played the effect that promotes on the promotor that just regulates and controls the synthetic key enzyme ICS1 of Whitfield's ointment.
Three, Western Blotting detects
Draw the 30ul step 19 in hatch the mixed solution of completing, (resolving gel concentration is 12%, and concentrated gum concentration is 4%, length is 1cm to carry out the SDS-PAGE electrophoresis; Electric current adopts 20mA), then adopt the horseradish peroxidase HRP antibody of Anti-GFP antibody (or Anti-FLAG antibody) and sheep anti mouse to carry out Western Blotting.
That adopts the hybridization of Anti-GFP antibody the results are shown in Figure 8.Jointly transforming recombinant plasmid 326-Pro
AtICS1:: in the protoplastis of GFP and recombinant plasmid 326-CRK25-FLAG, the band identical with the positive control that transforms the 326-GFP carrier occur, can be defined as the GFP band.The common recombinant plasmid 326-Pro that transforms
AtICS1:: GFP and recombinant plasmid 326-T
7In the protoplastis of-FLC (or 326-FLAG expression vector), do not have the GFP protein signal.
That adopts the hybridization of Anti-FLAG antibody the results are shown in Figure 9.Jointly transforming recombinant plasmid 326-Pro
AtICS1:: in the protoplastis of GFP and recombinant plasmid 326-CRK25-FLAG, the CRK25 gene has obtained expression really, this just further from protein level illustrated the expressed albumen of CRK25 gene can with the promoter region effect of ICS1, and impel the expression of its follow-up GFP gene.Therefore can judge, the CRK25 gene has played the effect that promotes on the promotor that just regulates and controls the synthetic key gene ICS1 of Whitfield's ointment.
Embodiment 4, CRK25 gene are being induced PR1 gene promoter (Pro
AtPR1) application of promotor gene in expressing
One, the transient expression of recombinant plasmid in the Arabidopsis leaf protoplastis
Use recombinant plasmid 326-Pro
AtPR1:: GFP replaces recombinant plasmid 326-Pro
AtICS1:: GFP, the step 1 of the other the same as in Example 3.
Two, fluorescence microscope
Step 2 with embodiment 3.
The results are shown in Figure 10.Under fluorescence observation, jointly transforming recombinant plasmid 326-Pro
AtPR1:: in the protoplastis of GFP and recombinant plasmid 326-CRK25-FLAG, the green fluorescence identical with the positive control that transforms the 326-GFP carrier appearred; The common recombinant plasmid 326-Pro that transforms
AtPR1:: GFP and recombinant plasmid 326-T
7The protoplastis of-FLC (or 326-FLAG expression vector) can not be observed obvious green fluorescence.Result shows, CRK25 albumen has promoted initial the transcribing of disease-resistant gene PR1 promotor, and impels the expression of its follow-up GFP gene.The CRK25 gene may be by salicylic synthetic initial the transcribing that promotes disease-resistant gene PR1 promotor of regulating plant.
Three, Western Blotting detects
Step 3 with embodiment 3.
Adopt the Figure 11 that the results are shown in of Anti-GFP antibody hybridization.Jointly transforming recombinant plasmid 326-Pro
AtPR1:: in the protoplastis of GFP and recombinant plasmid 326-CRK25-FLAG, occurred and the positive control same strap that transforms 326-GFP, can be defined as the GFP band.The common recombinant plasmid 326-Pro that transforms
AtPR1:: GFP and recombinant plasmid 326-T
7In the protoplastis of-FLC (or 326-FLAG expression vector), do not have the GFP protein signal.
Adopt the Figure 12 that the results are shown in of Anti-FLAG antibody hybridization.At the common recombinant plasmid 326-Pro that transforms
AtPR1:: in the protoplastis of GFP and recombinant plasmid 326-CRK25-FLAG, the CRK25 gene has obtained expression really, this also further illustrate CRK25 protein induced initial the transcribing of disease-resistant gene PR1 promotor, impelled the expression of follow-up GFP gene.The CRK25 gene may be by the salicylic synthetic expression that promotes disease-resistant gene PE1 promotor of regulating plant.
Claims (6)
1. the application of protein in the start-up performance of inducing the ICS1 gene promoter that is formed by the aminoacid sequence shown in sequence in sequence table 1; The nucleotide sequence of described ICS1 gene promoter is as shown in the sequence 3 of sequence table.
2. the application of protein in the start-up performance of inducing the PR1 gene promoter that is formed by the aminoacid sequence shown in sequence in sequence table 1; The nucleotide sequence of described PR1 gene promoter is as shown in the sequence 4 of sequence table.
3. the application of DNA molecular in the start-up performance of inducing the ICS1 gene promoter shown in the sequence 2 of sequence table; The nucleotide sequence of described ICS1 gene promoter is as shown in the sequence 3 of sequence table.
4. the application of DNA molecular in the start-up performance of inducing the PR1 gene promoter shown in the sequence 2 of sequence table; The nucleotide sequence of described PR1 gene promoter is as shown in the sequence 4 of sequence table.
5. the application of protein in cultivating resistance plant that is formed by the aminoacid sequence shown in sequence in sequence table 1.
6. the application of the DNA molecular shown in the sequence 2 of sequence table in cultivating resistance plant.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110317238 CN102367274B (en) | 2011-10-18 | 2011-10-18 | Plant resistance associated protein named as CRK 25 and its coding gene and use |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110317238 CN102367274B (en) | 2011-10-18 | 2011-10-18 | Plant resistance associated protein named as CRK 25 and its coding gene and use |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102367274A CN102367274A (en) | 2012-03-07 |
| CN102367274B true CN102367274B (en) | 2013-06-19 |
Family
ID=45759870
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201110317238 Active CN102367274B (en) | 2011-10-18 | 2011-10-18 | Plant resistance associated protein named as CRK 25 and its coding gene and use |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102367274B (en) |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100999549B (en) * | 2006-12-30 | 2010-12-08 | 中国科学技术大学 | Plant conversion-resisting resisting related protein and its coding gene and application |
| CN101775381B (en) * | 2010-01-12 | 2012-03-14 | 北京农业生物技术研究中心 | Plant stress tolerance correlative protein kinase, encoding gene and application thereof |
-
2011
- 2011-10-18 CN CN 201110317238 patent/CN102367274B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN102367274A (en) | 2012-03-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Fan et al. | Infection of Ustilaginoidea virens intercepts rice seed formation but activates grain‐filling‐related genes | |
| Jabnoune et al. | A rice cis-natural antisense RNA acts as a translational enhancer for its cognate mRNA and contributes to phosphate homeostasis and plant fitness | |
| Zhu et al. | The DnaJ OsDjA7/8 is essential for chloroplast development in rice (Oryza sativa) | |
| Ali et al. | An Arabidopsis ATPase gene involved in nematode‐induced syncytium development and abiotic stress responses | |
| CN105753953B (en) | Disease-resistant wheat albumen and encoding gene and its application in regulation disease resistance of plant | |
| Xiao et al. | An Agrobacterium-mediated transient expression method contributes to functional analysis of a transcription factor and potential application of gene editing in Chenopodium quinoa | |
| CN103588868A (en) | Wheat protein TaMYB1, and coding gene and application thereof | |
| CN102344487B (en) | Plant resistance-related protein ATWRKY46 as well as encoding gene and application thereof | |
| Moradyar et al. | Pathogen-induced expression of chimeric chitinase gene containing synthetic promoter confers antifungal resistance in transgenic canola | |
| CN102367273B (en) | Plant resistance related protein ATSAR 25, and encoding genes and application thereof | |
| CN102367272B (en) | Plant resistance related protein ANAC001, its encoding gene and application thereof | |
| CN102367274B (en) | Plant resistance associated protein named as CRK 25 and its coding gene and use | |
| CN102367275B (en) | Plant resistance related protein MAG, and coding gene and application thereof | |
| CN102367276B (en) | Plant resistance related protein ZIP11, its encoding gene and application thereof | |
| Park et al. | Characterization of the rice RbcS3 promoter and its transit peptide for use in chloroplast-targeted expression | |
| KR20200070357A (en) | Plant resistance | |
| CN102382184B (en) | Plant resistance-related protein ATSAR19 and coding gene and application thereof | |
| CN102367271B (en) | Chinese medicine composition anti-wrinkle multi-function liquid, its preparation and application | |
| Sierocka et al. | Novel genes specifically expressed during the development of the male thalli and antheridia in the dioecious liverwort Pellia endiviifolia | |
| Zhou et al. | The transmembrane protein LbRSG from the recretohalophyte Limonium bicolor enhances salt gland development and salt tolerance | |
| CN102465132B (en) | Application of WRKY polypeptide Glyma02g39870 in promotion of salicylic acid biosynthesis and enhancement of disease resistance of plants | |
| Thakare et al. | GFP tagging based method to analyze the genome editing efficiency of CRISPR/Cas9-gRNAs through transient expression in N. benthamiana | |
| CN103819547A (en) | Late blight resistance related protein, and related biomaterial and application thereof | |
| US11319553B2 (en) | Compositions and methods conferring resistance to fungal diseases | |
| US20220162622A1 (en) | A novel vector for gene transfer and gene copy proliferation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C41 | Transfer of patent application or patent right or utility model | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20160612 Address after: Siming District of Xiamen City, Fujian province 361009 Luling road 1721-1725 Patentee after: Fujian Zhongke biological Limited by Share Ltd Address before: 100093 Beijing City, Haidian District Xiangshan Nanxin Village No. 20 Patentee before: Plant Inst., Chinese Academy of Sciences |
|
| CP02 | Change in the address of a patent holder | ||
| CP02 | Change in the address of a patent holder |
Address after: 362000 Hengshan village photoelectric industry park, Hu tou Town, Anxi County, Quanzhou, Fujian Patentee after: FU'JIAN PROVINCE CHINESE ACADEMY OF SCIENCES BIOTECHNOLOGY, Inc. Address before: 1721-1725 Lvling Road, Siming District, Xiamen City, Fujian Province Patentee before: FU'JIAN PROVINCE CHINESE ACADEMY OF SCIENCES BIOTECHNOLOGY, Inc. |