CN102344487A - Plant resistance-related protein ATWRKY46 as well as encoding gene and application thereof - Google Patents
Plant resistance-related protein ATWRKY46 as well as encoding gene and application thereof Download PDFInfo
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Abstract
The invention discloses a plant resistance-related protein ATWRKY46 as well as an encoding gene and application thereof. The protein provided by the invention is named ATWRKY46 protein, which is from Arabidopsis thaliana and is any one of the following: (a) the protein formed by amino acid sequences as shown in a sequence 1; and (b) the protein which is derived from the sequence 1 in a way that the amino acid sequences as shown in the sequence 1 are subjected to replacement and/or deletion and/or addition of one or a plurality of amino acid residues, and has any function of the following: inducing a starting function of an ICS1 gene promoter, and inducing a starting function of a PR1 (pathogenesis-related protein) gene promoter, and the protein which is related to synthesis of salicylic acid in a plant and plant resistance and is derived from the sequence 1. The ATWRKY46 protein provided by the invention can increase disease resistance of the plant through regulating and controlling the synthesis of the salicylic acid in the plant. The invention is of great value to cultivate disease-resistant plants.
Description
Technical field
The present invention relates to a kind of plant resistance to environment stress associated protein ATWRKY46 and encoding sox and application.
Background technology
Plant is the various diseases of normal generation in the cultivation and production process, and some diseases constitute greatly threat, its output of grievous injury farm crop and quality to plant-growth.For Plant diseases, main at present employing medicine measure control.People begin to use abiotic inductor inducing plant disease resistance recently, and have been widely used in multiple important farm crop such as tobacco, potato, tomato, cucumber, Kidney bean, paddy rice.Though the effect of this measure is desirable, cost is higher, and contaminate environment.Therefore, press for the new biological means of exploitation 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 meeting was induced, and other position that can be diffused into plant rapidly is with protective plant.
Pathogenesis-related proteins PR is one type of stress protein; Be considered in plant disease-resistant, play an important role; The proteic gene of PR1 of wherein encoding often is used as the plant disease-resistant reaction and system obtains the indicator in the resistance, and the PR1 expression of gene plays an important role in the plant disease-resistant reaction.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 the startup function basically 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 extensively to be present in the intravital aldehydes matter of plant, is considered to bring out the signal of systemic acquired resistance, can induce the resistance to several diseases substances such as virus, bacterium, fungies.Research shows, execute Whitfield's ointment outward after, the PR1 gene transcription is significantly activated.Research finds that also after plant a part suffered the pathogenic agent attack, salicylic acid content can increase in the plant materials, and salicylic increase appears at before the PR1 genetic expression.Salicylic synthesizing is to act on the different chorismic acid of substrate (isochorismate) thereby salicylate through different chorismic acid synthetase (ICS, isochorismate synthase) ICS1 to a great extent in the plant.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 the startup function basically 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 that ETHYLENEINSENSITIVE3 (EIN3) and EIN3-LIKE1 can directly combine with SA biosynthesizing key gene ICS1 promotor; And negative synthetic level of regulating SA; Do not find as yet and can induce the ICS1 promotor to start ICS1 genetic expression, thereby increase the albumen of the synthetic level of Whitfield's ointment through forward.Discover that in addition SAR Deficient 1 (SARD1) and CBP60g albumen are to induce ICS1 genetic expression and SA synthetic important factor; But fail to find to induce the ICS1 promotor to start ICS1 genetic expression through 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 ATWRKY46 and encoding sox and application.Albumen provided by the invention, called after ATWRKY46 albumen, from Arabidopis thaliana (Arabidopsis thaliana), be following (a) and (b), (c), (d) and (e) in any one:
(a) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 1;
(b) with the aminoacid sequence of sequence 1 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have induce the ICS1 gene promoter start function by sequence 1 deutero-protein;
(c) with the aminoacid sequence of sequence 1 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have induce the PR1 gene promoter start function by sequence 1 deutero-protein;
(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 with Whitfield's ointment in the plant by sequence 1 deutero-protein;
(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 with plant resistance to environment stress by sequence 1 deutero-protein.
In order to make the protein in (a) be convenient to purifying, proteinic N-terminal or C-terminal that can the aminoacid sequence shown in the sequence 1 is formed 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 synthesize its encoding sox earlier, carry out biology again and express and obtain.Above-mentioned (b), (c), (d) or (e) in proteinic encoding sox can through will be in the dna sequence dna shown in the sequence in the 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 the table 1.
The gene of encoding said proteins (ATWRKY46 gene) also belongs to protection scope of the present invention.
Said gene can be following 1) to 10) in arbitrary described dna molecular:
1) sequence 2 of sequence table is from the dna molecular shown in 5 ' terminal the 73rd to 957 Nucleotide;
2) dna molecular shown in the sequence 2 of sequence table;
3) under stringent condition with 1) or 2) the dna sequence dna hybridization that limits and coding have and induce the ICS1 gene promoter to start the proteic dna molecular of function;
4) with 1) or 2) dna sequence dna that limits has 90% above homology and coding has the proteic dna molecular of inducing ICS1 gene promoter startup function;
5) under stringent condition with 1) or 2) the dna sequence dna hybridization that limits and coding have and induce the PR1 gene promoter to start the proteic dna molecular of function;
6) with 1) or 2) dna sequence dna that limits has 90% above homology and coding has the proteic dna molecular of inducing PR1 gene promoter startup function;
7) under stringent condition with 1) or 2) the dna sequence dna hybridization that limits and the dna molecular of coded plant Whitfield's ointment synthesis associated protein;
8) with 1) or 2) dna sequence dna that limits has the dna molecular of 90% above homology and coded plant Whitfield's ointment synthesis associated protein;
9) under stringent condition with 1) or 2) the dna sequence dna hybridization that limits and the dna molecular of coded plant resistance-associated protein;
10) with 1) or 2) 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 and is lower than about 1.5M, is generally about 0.01-1.0M, and pH is between about 7.0-8.3, and temperature is between about 30-60 degree centigrade.Also can be through adding destabilizing agent, for example methane amide is realized stringent hybridization condition.Above-mentioned stringent condition can be 0.1 * SSPE (or 0.1 * SSC), in the solution of 0.1%SDS, hybridization and wash film under 65 ℃ of conditions.
The nucleotide sequence of said ICS1 gene promoter is shown in the sequence 3 of sequence table.
The nucleotide sequence of said PR1 gene promoter is shown in the sequence 4 of sequence table.
The recombinant expression vector, expression cassette, transgenic cell line or the reorganization bacterium that contain said gene all belong to protection scope of the present invention.
Available existing plant expression vector construction contains the recombinant expression vector of said gene.Said plant expression vector comprises double base agrobacterium vector and the carrier etc. that can be used for the plant micropellet bombardment.Said plant expression vector also can comprise 3 ' end untranslated zone of foreign gene, promptly comprises the dna fragmentation of polyadenylic acid signal and any other participation mRNA processing or genetic expression.The bootable polyadenylic acid of said polyadenylic acid signal joins 3 ' end of mRNA precursor.When using said gene constructed recombinant plant expression vector, before its transcription initiation Nucleotide, can add any enhancement type promotor or constitutive promoter, they can use separately or be used in combination 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 enhanser zones 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 said translation control signal and initiator codon is widely, can be natural, also can be synthetic.Translation initiation region can be from transcription initiation zone or structure gene.For the ease of transgenic plant cells or plant being identified and screening; Can process used plant expression vector, can produce enzyme or the gene of luminophor, antibiotic marker thing or the anti-chemical reagent marker gene etc. of colour-change with resistance as adding the coding that in plant, to express.
Said 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; Said 326-FLAG expression vector is the recombinant plasmid that obtains at DNA shown in the sequence 6 of multiple clone site (as between PstI and the EcoRI restriction enzyme site) the insertion sequence table of pUC19 carrier.Said recombinant expression vector specifically can be: the recombinant plasmid that the ATWRKY46 gene shown in the sequence 2 of insertion sequence table obtains between the XbaI of said 326-FLAG expression vector and XhoI restriction enzyme site.
Increase total length or its arbitrary segmental primer of said gene to also belonging to protection scope of the present invention.
The present invention also protects said albumen or the application of said gene in inducing the startup function of ICS1 gene promoter; The nucleotide sequence of said ICS1 gene promoter is shown in the sequence 3 of sequence table.Said application specifically can be and in Arabidopis thaliana, induces recombinant plasmid 326-Pro
AtICS1:: the said ICS1 gene promoter among the GFP starts the GFP expression of gene; Said 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 said 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.Said GFP gene like the sequence 7 of sequence table from shown in 5 ' the terminal 862-1578 position Nucleotide.
The present invention also protects said albumen or the application of said gene in inducing the startup function of PR1 gene promoter; The nucleotide sequence of said PR1 gene promoter is shown in the sequence 4 of sequence table.Said application specifically can be and in Arabidopis thaliana, induces recombinant plasmid 326-Pro
AtPR1:: the said PR1 gene promoter among the GFP starts the GFP expression of gene; Said 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 said 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.Said GFP gene like the sequence 7 of sequence table from shown in 5 ' the terminal 862-1578 position Nucleotide.
The present invention also protects said albumen or the application of said gene in cultivating resistance plant.Said resistance is presented as that the Whitfield's ointment synthesis capability increases and/or the disease resistance ability increases.Said plant is dicotyledons or monocotyledons, like Arabidopis thaliana.The pathogenic bacterium of said disease can be 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 said disease also can be the rhizosphere germ that withers.
Said Arabidopis thaliana specifically can be the environmental Arabidopis thaliana of Colombia.
In one embodiment of the invention; With the ATWRKY46 gene fusion in the conversion expression vector that contains the FLAG reporter gene; Through 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.The result shows that the ATWRKY46 gene can be induced the startup function of ICS1 gene promoter, thereby impels the GFP reporter gene expression.
In another embodiment of the present invention; With the ATWRKY46 gene fusion in the conversion expression vector that contains the FLAG reporter gene; Through 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.The result shows that the ATWRKY46 gene can be induced the startup function of PR1 gene promoter, thereby impels the GFP reporter gene expression.
ATWRKY46 albumen provided by the invention can be through salicylic synthetic in the regulation and control plant, the disease resistance of enhancement of plant.The present invention all has great value for cultivating disease-resistant plants.
Description of drawings
Fig. 1 obtains the electrophorogram of ATWRKY46 gene for pcr amplification.
Fig. 2 is the structural representation of recombinant plasmid 326-ATWRKY46-FLAG.
Fig. 3 obtains the electrophorogram of ICS1 gene promoter for pcr amplification.
Fig. 4 is recombinant plasmid 326-Pro
AtICS1:: the structural representation of GFP.
Fig. 5 obtains the electrophorogram of PR1 gene promoter for pcr amplification.
Fig. 6 is recombinant plasmid 326-Pro
AtPR1:: the structural representation of GFP.
Fig. 7 is recombinant plasmid 326-ATWRKY46-FLAG and recombinant plasmid 326-Pro
AtICS1:: after GFP transforms protoplastis jointly, the microscopic result of GFP fluorescin.
Fig. 8 is recombinant plasmid 326-ATWRKY46-FLAG and recombinant plasmid 326-Pro
AtICS1:: after GFP transforms protoplastis jointly, the Western Blotting result of GFP fluorescin (confidential reference items albumen is the big subunit of Rubisco).
Fig. 9 is recombinant plasmid 326-ATWRKY46-FLAG and recombinant plasmid 326-Pro
AtICS1:: after GFP transforms protoplastis jointly, the proteic Western Blotting result of FLAG (confidential reference items albumen is the big subunit of Rubisco).
Figure 10 is recombinant plasmid 326-ATWRKY46-FLAG and recombinant plasmid 326-Pro
AtPR1:: after GFP transforms protoplastis jointly, the microscopic result of GFP fluorescin.
Figure 11 is recombinant plasmid 326-ATWRKY46-FLAG and recombinant plasmid 326-Pro
AtPR1:: after GFP transforms protoplastis jointly, the Western Blotting result of GFP fluorescin (confidential reference items albumen is the big subunit of Rubisco).
Figure 12 is recombinant plasmid 326-ATWRKY46-FLAG and recombinant plasmid 326-Pro
AtPR1:: after GFP transforms protoplastis jointly, the proteic Western Blotting result of FLAG (confidential reference items albumen is the big subunit of Rubisco).
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment; As there is not a specified otherwise; 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).Used test materials among the following embodiment like no specified otherwise, is to buy from routine biochemistry reagent shop and obtains.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 is provided with repeated experiments three times, results averaged.
The environmental Arabidopis thaliana (Arabidopsis thaliana) of Colombia; Claim Arabidopis thaliana or Arabidopis thaliana Col-0 again: 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. Arabidopis thaliana Biological resources center (ABRC) and obtain.
The pUC19 carrier: available from NEB company, cat. no is N3041S.
326-FLAG expression vector: the recombinant plasmid that DNA obtains shown in the sequence 6 of insertion sequence table between the PstI of pUC19 carrier and EcoRI restriction enzyme site; Among the DNA, be the 35S constitutive promoter from 5 ' terminal the 1st to 835 Nucleotide 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 DNA obtains shown in the sequence 7 of insertion sequence table between the PstI of pUC19 carrier and EcoRI restriction enzyme site; Among the DNA, be the 35S constitutive promoter from 5 ' terminal the 1st to 835 Nucleotide 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.
Used Anti-GFP antibody is the GFP Living Colors A.V.Monoclonal Antibody (article No. 632381) of Clontech company among the embodiment.
So that Anti-FLAG antibody is the Anti-FLAG M2 Monoclonal Antibody (article No. F3165) of Sigma company among the embodiment.
The acquisition of embodiment 1, ATWRKY46 albumen and encoding sox thereof
1, according to existing ncbi database and document, it is following that a pair of primer is set:
F1 (forward primer): 5 '-
TCTAGACACATCTCCCACCAATCTCA-3 ' (underscore mark XbaI enzyme cutting recognition sequence);
R1 (reverse primer): 5 '-
CTCGAGTCGACCACAACCAATCCTGTC-3 ' (underscore mark XhoI restriction endonuclease recognition sequence).
2, extract the genomic dna of the environmental Arabidopsis leaf of Colombia.
3, with the genomic dna be template, adopt step 1 designed primer right, carry out pcr amplification with the PrimeSTAR HS of TaKaRa company high-fidelity enzyme.
4, pcr amplification product is checked order, shown in the sequence 2 of sequence table.
With the albumen called after ATWRKY46 albumen shown in the sequence 1 of sequence table.With the proteic encoding sox called after of ATWRKY46 ATWRKY46 gene, its genomic dna is (is initiator codon from 5 ' terminal the 73rd to 75 Nucleotide) shown in the sequence 2 of sequence table.
The clone of embodiment 2, each gene and the structure of recombinant expression vector thereof
One, the structure of the acquisition of ATWRKY46 gene and recombinant plasmid 326-ATWRKY46-FLAG
1, extracts the genomic dna of the environmental Arabidopsis leaf of Colombia.
2, the genomic dna with step 1 is a template, and is right with the primer that F1 and R1 form, and under the effect of the PrimeSTAR HS of TaKaRa company high-fidelity enzyme, carries out pcr amplification, obtains 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,, reclaim enzyme and cut product with the pcr amplification product of restriction enzyme XbaI and XhoI double digestion step 2.
4,, reclaim the carrier framework of about 3827bp with restriction enzyme XbaI and XhoI double digestion 326-FLAG expression vector.
5, the carrier framework of the enzyme of step 3 being cut product and step 4 is connected, and obtains recombinant plasmid 326-ATWRKY46-FLAG (structural representation is seen Fig. 2).According to sequencing result, recombinant plasmid 326-ATWRKY46-FLAG is carried out the result be described below: between the XbaI of 326-FLAG expression vector and XhoI restriction enzyme site, inserted the ATWRKY46 gene shown in the sequence 2 of sequence table.
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), following 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, the genomic dna with step 2 is a template, and is right with the primer that F2 and R2 form, and under the effect of the PrimeSTAR HS of TaKaRa company high-fidelity enzyme, carries out pcr amplification, obtains 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,, reclaim enzyme and cut product with the pcr amplification product of restriction enzyme PstI and BamHI double digestion step 3.
5,, reclaim the carrier framework (having removed the 35S constitutive promoter in the 326-GFP expression vector) of about 3635bp with restriction enzyme PstI and BamHI double digestion 326-GFP expression vector.
6, the carrier framework of the enzyme of step 4 being cut product and step 5 is connected, and obtains recombinant plasmid 326-Pro
AtICS1:: GFP (structural representation is seen Fig. 4).According to sequencing result, to recombinant plasmid 326-Pro
AtICS1:: it is following 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), following 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, the genomic dna with step 2 is a template, and is right with the primer that F3 and R3 form, and under the effect of the PrimeSTAR HS of TaKaRa company high-fidelity enzyme, carries out pcr amplification, obtains 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,, reclaim enzyme and cut product with the pcr amplification product of restriction enzyme PstI and XhoI double digestion step 3.
5,, reclaim the carrier framework of about 3641bp with restriction enzyme PstI and XhoI double digestion 326-GFP expression vector.
6, the carrier framework of the enzyme of step 4 being cut product and step 5 is connected, and obtains recombinant plasmid 326-Pro
AtPR1:: GFP (structural representation is seen Fig. 6).According to sequencing result, to recombinant plasmid 326-Pro
AtPR1:: it is following 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 (is that T7 label, the 34th to 660 Nucleotide are the FLC gene from 5 ' terminal the 1st to 33 Nucleotide); 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 ATWRKY46 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-ATWRKY46-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-ATWRKY46-FLAG; With another the negative contrast of 326-FLAG expression vector as recombinant plasmid 326-ATWRKY46-FLAG), concrete steps are following:
1, on the MS substratum, sprouts the environmental Arabidopis thaliana seed of Colombia, treat to be transplanted in the soil when root grows to 1-3 centimetre 23 ℃ of hot-house cultures (illumination every day 12h, intensity of illumination is 150 μ E).
2, in the 90mm culture dish, add the 20ml distilled water, add 1.82g D-N.F,USP MANNITOL again and make its dissolving.
3, get in the step 1 and to cultivate the 4 weeks blade of bolting (about 90) not, be cut into wide rectangular of 1mm, place 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, using 1M KOH to transfer pH is 5.6.
5, with step 3 slice is pulled out, place the enzymatic hydrolysis system of step 4, enzymolysis 3 hours (23 ℃, 40-50rpm) under dark condition.
6, the enzymolysis solution that will finish step 5 is crossed 100-200 purpose sieve, and the green mixture after filtering is placed 15ml centrifuge tube (the about 1cm of diameter), 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 and softly washs, and centrifugal then (4 ℃, 100g) 1 minute collecting precipitation, every pipe add the ice-cold W5 solution of 4ml and softly wash; Placed on ice 30 minutes, centrifugal then (23 ℃, 100g) 1 minute collecting precipitation, every pipe deposition 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 using 1M KOH to transfer pH 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 using 1M KOH to transfer pH is 5.6.
8, with recombinant plasmid 326-ATWRKY46-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); Add 310ul PEG4000/Ca (NO then
3)
2Solution, soft mixing; Place 30 minutes (20-30 minute all can), add 1ml W5 solution then and put upside down mixing back and forth, centrifugal then (23 ℃, 100g) 1 minute collecting precipitation; Add 100ul W5 solution mixing, and then add 900ul W5 solution mixing.Be provided with a transfection 326-GFP expression vector over against photograph.
PEG4000/Ca (NO
3)
2Solution: PEG40004g, Mannitol (available from Sigma, article No. M1902 is 1M Mannitol 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 places in six orifice plates, hatches 12 hours for 23 ℃.
Two, fluorescence microscope
Draw the 30ul step 19 in hatch the mixed solution of completion, place on the slide glass, gently after the covered, be put in and observe under the ZEISS AXIOSKOP fluorescent microscope and take pictures.
The result sees Fig. 7.Under fluorescence observation: transform recombinant plasmid 326-Pro jointly
AtICS1:: in the protoplastis of GFP and recombinant plasmid 326-ATWRKY46-FLAG, occurred with transform the 326-GFP carrier over against take a picture with green fluorescence; 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 tangible green fluorescence.The result shows, ATWRKY46 albumen can with the effect of ICS1 promoter region, impel its follow-up GFP genetic expression, the ATWRKY46 gene has played promoted effect on the promotor of just regulating and control the synthetic key enzyme ICS1 of Whitfield's ointment.
Three, Western Blotting detects
Draw the 30ul step 19 in hatch the mixed solution of completion, (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), adopt the horseradish peroxidase HRP antibody of Anti-GFP antibody (or Anti-FLAG antibody) and sheep anti mouse to carry out Western Blotting then.
Adopt the result of Anti-GFP antibody hybridization to see Fig. 8.Transforming recombinant plasmid 326-Pro jointly
AtICS1:: in the protoplastis of GFP and recombinant plasmid 326-ATWRKY46-FLAG, occurred with transform the 326-GFP carrier over against take a picture with band, can confirm as the GFP band.The common recombinant plasmid 326-Pro that transforms
AtICS1:: GFP and recombinant plasmid 326-T
7-FLC (or 326-FLAG expression vector) does not show the GFP protein signal.
Adopt the result of Anti-FLAG antibody hybridization to see Fig. 9.Transforming recombinant plasmid 326-Pro jointly
AtICS1:: in the protoplastis of GFP and recombinant plasmid 326-ATWRKY46-FLAG; The ATWRKY46 gene has obtained expression really; This just further from protein level illustrated the expressed albumen of ATWRKY46 gene can with the promoter region effect of ICS1, and impel its follow-up GFP expression of gene.Therefore can judge that the ATWRKY46 gene has played promoted effect on the promotor of just regulating and control the synthetic key gene ICS1 of Whitfield's ointment.
Embodiment 4, ATWRKY46 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, other is with the step 1 of embodiment 3.
Two, fluorescence microscope
Step 2 with embodiment 3.
The result sees Figure 10.Under fluorescence observation, transforming recombinant plasmid 326-Pro jointly
AtPR1:: in the protoplastis of GFP and recombinant plasmid 326-ATWRKY46-FLAG, occurred with transform the 326-GFP carrier over against take a picture with green fluorescence; 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 tangible green fluorescence.The result shows that ATWRKY46 albumen has promoted initial the transcribing of disease-resistant gene PR1 promotor, and impels its follow-up GFP expression of gene.The ATWRKY46 gene possibly be through salicylic synthetic initial the transcribing that promotes disease-resistant gene PR1 promotor of regulation and control plant.
Three, Western Blotting detects
Step 3 with embodiment 3.
Adopt the result of Anti-GFP antibody hybridization to see Figure 11.Transforming recombinant plasmid 326-Pro jointly
AtPR1:: in the protoplastis of GFP and recombinant plasmid 326-ATWRKY46-FLAG, occurred with transform 326-GFP over against the same band of taking a picture, can confirm 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 result of Anti-FLAG antibody hybridization to see Figure 12.At the common recombinant plasmid 326-Pro that transforms
AtPR1:: in the protoplastis of GFP and recombinant plasmid 326-ATWRKY46-FLAG, the ATWRKY46 gene has obtained expression really, this also further illustrate ATWRKY46 protein induced initial the transcribing of disease-resistant gene PR1 promotor, impelled follow-up GFP expression of gene.The ATWRKY46 gene maybe be through the salicylic synthetic expression that promotes disease-resistant gene PR1 promotor of regulation and control plant.
Claims (10)
1. protein, be following (a) and (b), (c), (d) and (e) in any one:
(a) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 1;
(b) with the aminoacid sequence of sequence 1 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have induce the ICS1 gene promoter start function by sequence 1 deutero-protein; The nucleotide sequence of said ICS1 gene promoter is shown in the sequence 3 of sequence table;
(c) with the aminoacid sequence of sequence 1 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have induce the PR1 gene promoter start function by sequence 1 deutero-protein; The nucleotide sequence of said PR1 gene promoter is shown in the sequence 4 of sequence table;
(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 with Whitfield's ointment in the plant by sequence 1 deutero-protein;
(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 with plant resistance to environment stress by sequence 1 deutero-protein.
2. coding claim 1 said proteic gene.
3. gene as claimed in claim 2 is characterized in that: said gene is following 1) to 10) in arbitrary described dna molecular:
1) sequence 2 of sequence table is from the dna molecular shown in 5 ' terminal the 73rd to 957 Nucleotide;
2) dna molecular shown in the sequence 2 of sequence table;
3) under stringent condition with 1) or 2) the dna sequence dna hybridization that limits and coding have and induce the ICS1 gene promoter to start the proteic dna molecular of function;
4) with 1) or 2) dna sequence dna that limits has 90% above homology and coding has the proteic dna molecular of inducing ICS1 gene promoter startup function;
5) under stringent condition with 1) or 2) the dna sequence dna hybridization that limits and coding have and induce the PR1 gene promoter to start the proteic dna molecular of function;
6) with 1) or 2) dna sequence dna that limits has 90% above homology and coding has the proteic dna molecular of inducing PR1 gene promoter startup function;
7) under stringent condition with 1) or 2) the dna sequence dna hybridization that limits and the dna molecular of coded plant Whitfield's ointment synthesis associated protein;
8) with 1) or 2) dna sequence dna that limits has the dna molecular of 90% above homology and coded plant Whitfield's ointment synthesis associated protein;
9) under stringent condition with 1) or 2) the dna sequence dna hybridization that limits and the dna molecular of coded plant resistance-associated protein;
10) with 1) or 2) dna sequence dna that limits has the dna molecular of 90% above homology and coded plant resistance-associated protein.
4. the recombinant expression vector, expression cassette, transgenic cell line or the reorganization bacterium that contain claim 2 or 3 said genes.
5. recombinant expression vector as claimed in claim 4 is characterized in that: said recombinant expression vector is 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; Said 326-FLAG expression vector is the recombinant plasmid that obtains at DNA shown in the sequence 6 of the multiple clone site insertion sequence table of pUC19 carrier.
6. the application of the said albumen of claim 1 in inducing the startup function of ICS1 gene promoter; The nucleotide sequence of said ICS1 gene promoter is shown in the sequence 3 of sequence table.
7. the application of the said albumen of claim 1 in inducing the startup function of PR1 gene promoter; The nucleotide sequence of said PR1 gene promoter is shown in the sequence 4 of sequence table.
8. claim 2 or the application of 3 said genes in inducing the startup function of ICS1 gene promoter; The nucleotide sequence of said ICS1 gene promoter is shown in the sequence 3 of sequence table.
9. claim 2 or the application of 3 said genes in inducing the startup function of PR1 gene promoter; The nucleotide sequence of said PR1 gene promoter is shown in the sequence 4 of sequence table.
10. the said albumen of claim 1, and/or, claim 2 or 3 said genes, the application in cultivating resistance plant.
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| CN106543272A (en) * | 2015-09-18 | 2017-03-29 | 江苏众红生物工程创药研究院有限公司 | Multifunctional label fusion protein and its expression and application |
| CN107699572A (en) * | 2016-08-03 | 2018-02-16 | 南京农业大学 | A kind of identification and application of tomato SolyWRKY54 transcription factors regulation and control tomato yellow leaf curl virus |
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