CN101250221A - Plant ABA signal transduction regulatory protein as well as coding gene and use thereof - Google Patents
Plant ABA signal transduction regulatory protein as well as coding gene and use thereof Download PDFInfo
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Abstract
The invention discloses a plant ABA signal conduction modulation protein, a relative encoding gene and an application, wherein the protein is provided with one following amino acid residue sequence as (1) the amino acid residue sequence of SEQ ID No: 3 in sequence list, (2) the protein whose amino acid residue sequence of SEQ ID No: 3 in the sequence list whose one or several amino acid residues are substituted and/or deleted and/or added and provided with plant ABA signal conduction modulation protein activity. The protein has ubiquitin ligase activity, which is one positive modulation factor of ABA signal conduction route, wherein the modulation protein can adjust the ABA sensitivity of plant, thereby providing an important method for researching the separation and function of plant gene.
Description
Technical field
The present invention relates to a kind of plant ABA signal transduction regulatory protein and encoding gene and application, particularly relate to ABA signal transduction regulatory protein and an encoding gene and an application that derives from Arabidopis thaliana.
Background technology
(abscisic acid is a kind of very important natural phant tethelin ABA) to dormin, is one of five big class plant hormones that obtain the earliest confirming.Dormin has participated in many plant-growths and growth course, as seed germination, growth of seedling, blade stomatal movement etc., be a kind of important degeneration-resistant inducible factor in addition, mediated plant to the resistant function of adverse circumstance, so the research of abscisic acid signal transduction has crucial meaning to the understanding of regulation of plant growth and development, the raising of crop quality as arid, saline and alkaline, low temperature etc.
At present, in the ABA signal transduction path, the functional study report of two different RING FINGER ubiquitin-like ligase enzymes has been arranged, they are AIP2 and KEG, be two negative regulatory factors, the ABA signal pathway two basic points of degrading respectively important component ABI3 and ABI5.
At present, existing a plurality of Arabidopis thaliana ABA mutant obtain identifying that the while is existing separation the in many species about the synthetic of ABA and signal conduction gene, and their molecule mechanism and mechanism of action have obtained research extensively and profoundly.
Summary of the invention
The purpose of this invention is to provide a kind of plant ABA signal transduction regulatory protein and an encoding gene thereof.
The purpose of this invention is to provide a kind of plant ABA signal transduction regulatory protein and an encoding gene thereof.
Plant ABA signal transduction regulatory protein provided by the present invention, name is called RHA2a, derives from Arabidopis thaliana (Arabidopsis thaliana), is following (a) or protein (b):
(a) protein of forming by the amino acid residue sequence of sequence in the sequence table 3;
(b) with the amino acid residue sequence of sequence in the sequence table 3 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have plant ABA signal transduction regulatory function by (a) deutero-protein.
Wherein, the sequence in the sequence table 3 is made up of 155 amino-acid residues.
The encoding gene (RHA2a) of above-mentioned plant ABA signal transduction regulatory protein also belongs to protection scope of the present invention.
The cDNA gene of above-mentioned plant ABA signal transduction regulatory protein can have one of following nucleotide sequence:
1) dna sequence dna of sequence 2 in the sequence table;
2) polynucleotide of protein sequence shown in the sequence 3 in the code sequence tabulation;
3) nucleotide sequence of the dna sequence dna hybridization that under the rigorous condition of height, can limit with sequence in the sequence table 2.
Wherein, sequence 2 is made up of 468 deoxynucleotides in the sequence table, and 5 of sequence 2 ' end 1-468 position nucleotides sequence is classified encoding sequence (ORF) as in sequence table, and coding has the protein of the amino acid residue sequence of sequence 3 in the sequence table.
The genomic gene of above-mentioned plant ABA signal transduction regulatory protein can have one of following nucleotide sequence:
1) dna sequence dna of sequence 1 in the sequence table;
2) polynucleotide of protein sequence shown in the sequence 3 in the code sequence tabulation;
3) nucleotide sequence of the dna sequence dna hybridization that under the rigorous condition of height, can limit with sequence in the sequence table 2.
Wherein, sequence 1 is made up of 3110 deoxynucleotides in the sequence table, and 5 of sequence 1 ' end 1766-2233 position nucleotides sequence is classified encoding sequence (ORF) as in sequence table, and coding has the protein of the amino acid residue sequence of sequence 3 in the sequence table.
The rigorous condition of above-mentioned height can be 0.1 * SSPE (or 0.1 * SSC), in the solution of 0.1%SDS, under 65 ℃, hybridize and wash film.
The expression vector, transgenic cell line and the host bacterium that contain the encoding gene of above-mentioned plant ABA signal transduction regulatory protein all belong to protection scope of the present invention.
Utilize plant expression vector, the gene (RHA2a) of regulation and control of the present invention ABA signal conduction is imported vegetable cell or tissue, can obtain the plant that seed germination and early growth stage are raise to ABA susceptibility.
When using RHA2a to make up plant expression vector, before its transcription initiation Nucleotide, can add any enhancement type promotor or inducible promoter.For the ease of transgenic plant cells or plant being identified and screening, can process used plant expression vector, as adding selected marker's (gus gene, luciferase genes etc.) that can in plant, express or antibiotic marker thing (gentamicin marker, kantlex marker etc.) with resistance.From the security consideration of transgenic plant, can not add any selected marker, directly with adverse circumstance screening transformed plant.
Carry RHA2a of the present invention plant expression vector can Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, microinjection, electricity be led, conventional biological method transformed plant cells or tissue such as agriculture bacillus mediated by using, and the plant transformed tissue cultivating is become plant.By the plant transformed host both can be monocotyledonss such as paddy rice, corn, wheat, also can be dicotyledonss such as tomato, Arabidopis thaliana, tobacco, cotton.
Plant ABA signal transduction regulatory protein of the present invention is to have the active albumen of ubiquitin ligase, and its encoding gene is the simple relatively new gene of structure in the RING FINGER family.Functional study shows that plant ABA signal transduction regulatory protein of the present invention is a positive regulatory factor of ABA signal transduction path.Plant ABA signal transduction regulatory protein of the present invention can be regulated plant to ABA susceptibility, provides important means to the separation and the functional study of gene in the plant.
Description of drawings
Fig. 1 is the active detection of the ubiquitin ligase of RHA2a.The 1st to the 5th swimming lane is respectively the and 1. organizes to the 5. after the group reaction among the figure, electrophoresis, change film, with the westernblot result of HIS antibody.
Fig. 2 is for changeing the ABA susceptibility experiment of RHA2a gene Arabidopis thaliana.
Embodiment
Method therefor is ordinary method if no special instructions among the following embodiment.
The acquisition of embodiment 1, plant ABA signal transduction regulatory protein and encoding gene (RHA2a) thereof
1, acquisition plant ABA signal transduction regulatory protein and encoding gene (RHA2a)
Primer 1:5 '-ACT
AAGCTTTCTCTCCCGGTAATGGATGC-3 ' (upstream primer, line part base is the HindIII recognition site)
Primer 2: 5 '-AGA
GAATTCCTCGTCATTCGGAGCCAA-3 ' (downstream primer, line part base is the EcoRI recognition site)
Extract total DNA of Arabidopis thaliana (the Col-0 ecotype), with it is template, under the guiding of primer 1 and primer 2, carry out pcr amplification, reaction finishes the back PCR product is carried out purifying, shows, amplification obtains the fragment of 3110bp, show that through order-checking this fragment has the nucleotide sequence of sequence 1 in the sequence table, i.e. the encoding gene of plant ABA signal transduction regulatory protein is with its called after RHA2a; The 1766-2333 position nucleotide sequence (being the nucleotide sequence shown in the sequence 2 in the sequence table) of 5 of sequence 1 ' end is an encoding sequence (cDNA sequence) in sequence table, coding has the protein of sequence 3 described amino acid residue sequences in the sequence table, be plant ABA signal transduction regulatory protein of the present invention, with its called after RHA2a.
2, obtain the cDNA sequence of Arabidopis thaliana RHA2a with the method for PCR
According to Arabidopis thaliana database sequence result, utilize the method for PCR to obtain the nucleotide sequence of RHA2a.
Genomic dna with Arabidopis thaliana (the Col-0 ecotype) is a template, carries out pcr amplification with following primer:
Primer 3:5 '-AAGATGGGGCTACAAGGTCAG-3 ' (upstream primer),
Primer 4:5 '-GTGGAGAGAGAAACACGAGA-3 ' (downstream primer).
Amplification obtains the fragment of about 465bp, order-checking shows, this fragment has the 1766-2333 position nucleotide sequence (being the nucleotide sequence shown in the sequence 2 in the sequence table) of 5 ' end of sequence 1 in sequence table, encoding sequence (cDNA sequence) for RHA2a, coding has the protein of sequence 3 described amino acid residue sequences in the sequence table, plant ABA signal transduction regulatory protein RHA2a promptly of the present invention.
The functional verification of embodiment 2, plant ABA signal transduction regulatory protein and encoding gene (RHA2a) thereof
1, can express the structure of the recombinant vectors of plant ABA signal transduction regulatory protein RHA2a
Dna sequence dna and the suitable restriction enzyme site of carrier pCAMBIA1300 of the RHA2a that obtains according to the step 1 of embodiment 1, structure can be expressed the recombinant vectors of plant ABA signal transduction regulatory protein RHA2a, and concrete grammar is as described below:
The RHA2a fragment that the described amplification of the step 1 of embodiment 1 obtains is carried out double digestion with restriction enzyme HindIII and EcoRI, the carrier pCAMBIA1300 that purified enzyme is cut product and cut through the same enzyme enzyme (HindIII and EcoRI double digestion) then is connected, to connect product and check order, will show the correct recombinant vectors called after pCAMBIA1300-RHA2a that contains RHA2a through order-checking.
PCAMBIA1300-RHA2a is transformed RHA2a mutant N378380 (available from NASC under the mediation of agrobacterium tumefaciens, The Nottingham Arabidopsis Stock Centre), use selected marker's Totomycin (containing 50mg/L Totomycin MS substratum) to carry out resistance screening then, screening obtains the commentaries on classics pCAMBIA1300-RHA2a plant that can grow on the substratum that contains the 50mg/L Totomycin.The commentaries on classics pCAMBIA1300-RHA2a plant that resistance screening obtains carries out pcr amplification with primer 1 and primer 2 to it and identifies, obtains PCR and identifies correct commentaries on classics pCAMBIA1300-RHA2a plant.
PCR is identified that correct commentaries on classics pCAMBIA1300-RHA2a plant carries out the ABA sensitivity test, and concrete grammar is as described below:
To plant simultaneously, Shou Huo RHA2a mutant N378380 (orders from NASC simultaneously, TheNottingham Arabidopsis Stock Centre) and the above-mentioned PCR of obtaining identify that the planting seed of correct commentaries on classics pCAMBIA1300-RHA2a plant is on the MS substratum that contains 0.5 μ M ABA, 4 ℃ of dark cultivations 3 days, be transferred to 22 ℃ of illumination conditions then, adding up germination rate, cotyledon every day and turn the indexs such as elongation of green and root, is contrast with Arabidopis thaliana (the Col-0 ecotype).
The result shows that on the substratum that does not contain ABA, RHA2a mutant N378380, PCR identify that correct commentaries on classics pCAMBIA1300-RHA2a does not have difference at elongation and the Arabidopis thaliana (the Col-0 ecotype) that germination rate, cotyledon turn green and root; On the MS substratum of 0.5 μ M ABA, Arabidopis thaliana (the Col-0 ecotype), PCR identify that correct commentaries on classics pCAMBIA1300-RHA2a plant turns green at germination rate, cotyledon, the profound ABA of being subjected to of root system obviously suppresses, and RHA2a mutant N378380 can sprout normal growth at the MS substratum of 0.5 μ M ABA.Illustrate that this commentaries on classics pCAMBIA1300-RHA2a plant has recovered the insensitivity of RHA2a mutant N378380 to ABA, thereby conclude that the RHA2a gene is exactly the gene that causes RHA2a mutant N378380 that ABA susceptibility is reduced.
1, MBP-RHA2a fusion rotein, the proteic purifying of MBP
According to RHA2a cDNA sequence and the suitable restriction enzyme site design primer amplification RHA2a of carrier pMal-c2 that embodiment 1 obtains, primer sequence is as follows:
Primer 5:5 '-ACT
GAATTCAAGATGGGGCTACAAGGTCAG-3 ' (upstream primer, line part base is the EcoRI recognition site)
Primer 6:5 '-ACT
GGATCCGTGGAGAGAGAAACACGAGA-3 ' (downstream primer, line part base is the BamHI recognition site)
Extracting total DNA of Arabidopis thaliana Col-0, is template with it, under the guiding of primer 5 and primer 6, carry out pcr amplification, after reaction finishes the PCR product is carried out purifying and show, amplification obtains the fragment about 465bp, shows that through order-checking this fragment has the nucleotide sequence of sequence 2 in the sequence table.
The fragment that above-mentioned amplification obtains is carried out double digestion with restriction enzyme EcoRI and BamHI to purified PCR product, then purified enzyme is cut product and be connected with carrier pMal-c2 (available from NEB company) behind the BamHI double digestion through EcoRI, to connect product and carry out the enzyme evaluation of cutting and check order, evaluation be shown the correct recombinant vectors called after pMal-c2-RHA2a that contains RHA2a.
With pMal-c2-RHA2a transformed into escherichia coli BL21, screening has changed the e. coli bl21 of pMal-c2-RHA2a over to, will contain the e. coli bl21 called after BL-pMal-c2-RHA2a of pMal-c2-RHA2a.
BL-pMal-c2-RHA2a is inoculated in the LB substratum, selects mono-clonal and shake training at 37 ℃ and spend the night, be transferred in the LB substratum according to 1/100 in second day, shake bacterium when being cultured to OD600 and being 0.5, add final concentration and be 1 mmole/rise IPTG to induce, continue to cultivate 3 hours, collect thalline.According to pMal-c2 carrier specification sheets purifying RHA2a albumen, express the pMal-c2 empty carrier simultaneously according to the method described above and be contrast, go up sample SDS-PAGE gel electrophoresis, coomassie brilliant blue staining post analysis behind the purifying.The result shows according to electrophoretic migration speed, and what the pMal-c2-RHA2a thalline purifying behind the inducing culture obtained is the MBP-RHA2a fusion rotein, expresses pMal-c2 empty carrier purifying and obtains MBP albumen.
2, the acquisition of the MBP-RHA2a Δ fusion rotein of RING FINGER district (the aminoterminal 86-127 amino acids residue of sequence 3 in a sequence table) amino acid mutation (aminoterminal the 89th amino acids residue of sequence 3 in sequence table).
Synthetic in sequence table the 265th Nucleotide of Nucleotide of sequence 2 sport the RHA2acDNA fragment (called after RHA2a Δ) of A by T, add the EcoRI enzyme recognition site when synthetic and at its 5 ' end, and at its 3 ' end adding BamHI recognition site, this synthetic sequence encoding RING FINGER district (the aminoterminal 86-127 amino acids residue of sequence 3 in sequence table) amino acid (aminoterminal the 89th amino acids residue of sequence 3 in sequence table) sports the MBP-RHA2a fusion rotein of Ser, called after MBP-RHA2a Δ fusion rotein by Cys.
Utilize the RHA2a cDNA of the sudden change of above-mentioned acquisition purified PCR product to be carried out double digestion then with restriction enzyme EcoRI and BamHI, then purified enzyme is cut product and be connected with carrier pMal-c2 (available from NEB company) behind the BamHI double digestion through EcoRI, to connect product and carry out the enzyme evaluation of cutting and check order, evaluation be shown the correct recombinant vectors called after pMal-c2-RHA2a Δ that contains the RHA2a Δ.
According to the method for step 1, the pMal-c2-RHA2a Δ is transferred to e. coli bl21 is expressed and purifying obtains MBP-RHA2a Δ fusion rotein.
3, RHA2a is the functional verification experiment with active ubiquitin ligase
Carry out following 5 group reactions experiment, the ubiquitin ligase activity of checking RHA2a:
1. the ubiquitin activating enzyme E1 (GI:136632) of the MBP albumen that 1ug step 1 is obtained, 40ng wheat, ubiquitin binding enzyme E2 (UbcH5b), 2ug that 40ng derives from the people ubiquitin protein Ubiquitin that merges the HIS label adds 40ul ubiquitin reaction solution (50mM Tris (pH 7.4) together, 2mMATP, 5mMMgCl2,2mMDTT.) in, 30 ℃ were reacted 2 hours;
2. the MBP-RHA2a albumen that 1ug step 1 is obtained, ubiquitin binding enzyme E2 (UbcH5b), 2ug that 40ng derives from the people ubiquitin protein Ubiquitin that merges the HIS label adds 40ul ubiquitin reaction solution (50mMTris (pH 7.4) together, 2mMATP, 5mMMgCl2,2mMDTT.) in, 30 ℃ were reacted 2 hours;
3. the 1ugMBP-RHA2a albumen that step 1 is obtained, ubiquitin activating enzyme E1 (GI:136632), the 40ng of 40ng wheat), the 2ug ubiquitin protein Ubiquitin that merges the HIS label adds 40ul ubiquitin reaction solution (50mM Tris (pH 7.4) together, 2mMATP, 5mMMgCl2,2mMDTT.) in, 30 ℃ were reacted 2 hours;
4. the ubiquitin activating enzyme E1 (GI:136632) of the MBP-RHA2a albumen that 1ug step 1 is obtained, 40ng wheat, ubiquitin binding enzyme E2 (UbcH5b), 2ug that 40ng derives from the people ubiquitin protein Ubiquitin that merges the HIS label adds 40ul ubiquitin reaction solution (50mM Tris (pH 7.4) together, 2mMATP, 5mMMgCl2,2mMDTT.) in, 30 ℃ were reacted 2 hours;
5. the ubiquitin activating enzyme E1 (GI:136632) of the MBP-RHA2a Δ fusion rotein that 1ug step 2 is obtained, 40ng wheat, ubiquitin binding enzyme E2 (UbcH5b), 2ug that 40ng derives from the people ubiquitin protein Ubiquitin that merges the HIS label adds 40ul ubiquitin reaction solution (50mM Tris (pH 7.4) together, 2mMATP, 5mMMgCl2,2mMDTT.) in, 30 ℃ were reacted 2 hours;
Get above-mentioned 5 groups reaction solution and carry out the SDS-PAGE gel electrophoresis, change film behind the electrophoresis, use HIS antibody westernblot respectively.
The result as shown in Figure 1, the result show the 1. group (MBP contrast), the 2. group (not adding E1), the 3. organize in the reaction solution of (not adding E2) and all can not detect the MBP-RHA2a that is modified by ubiquitinization, have only 4. to organize under the situation that E1, E2 and RHA2a exist, can detect the protein band ((Ub) n-MBP-RHA2a) that RHA2a can be obtained after the ubiquitinization significantly.The 5. group can not detect the MBP-RHA2a Δ fusion rotein of being modified by ubiquitinization, the RHA2a albumen of the amino acid mutation in usefulness RING FINGER district be described, can not ubiquitinization.Wherein among Fig. 1, the 1st to the 5th swimming lane is respectively and 1. organizes to the 5. HIS antibody westernblot result of group reaction liquid.
The above results shows that RHA2a has the ubiquitin ligase activity, and complete RING FINGER district is necessary to the proteic function of RHA2a.
Embodiment 4, RHA2a overexpression can obviously improve the susceptibility of plant to ABA.
According to RHA2a dna sequence dna and the suitable restriction enzyme site design primer amplification RHA2a of carrier pBI121 that embodiment 1 obtains, primer sequence is as follows:
Primer 7:5 '-ACT
GGATCCAAGATGGGGCTACAAGGTCAG-3 ' (upstream primer, line part base is the BamHI recognition site)
Primer 8:5 '-ACT
GAGCTCTACTCAGTGGAGAGAGAAAC-3 ' (downstream primer, line part base is the SacI recognition site)
Extract total DNA of Arabidopis thaliana (the Col-0 ecotype), with it is template, under the guiding of primer 7 and primer 8, carry out pcr amplification, reaction finishes the back PCR product is carried out purifying, show that amplification obtains the fragment about 465bp, shows that through order-checking this fragment has the nucleotide sequence of sequence 2 in the sequence table.
The fragment that above-mentioned amplification obtains is carried out double digestion with restriction enzyme BamHI and SacI to purified PCR product, then purified enzyme being cut product is connected with the carrier pBI121 that cuts through the same enzyme enzyme, to connect product and carry out the enzyme evaluation of cutting and check order, evaluation be shown the correct recombinant vectors called after pBI121-RHA2a that contains RHA2a.
With pBI121-RHA2a arabidopsis thaliana transformation wild-type (the Col-0 ecotype) under the mediation of agrobacterium tumefaciens, use selected marker's kantlex (containing the MS substratum of 50mg/L kantlex) to carry out resistance screening then, screening obtains the commentaries on classics pBI121-RHA2a plant that can grow on the MS substratum of 50mg/L kantlex containing.The commentaries on classics pBI121-RHA2a plant that resistance screening obtains carries out pcr amplification with primer 7 and primer 8 to it and identifies, obtains PCR and identifies correct commentaries on classics pBI121-RHA2a plant.
PCR is identified that correct commentaries on classics pBI121-RHA2a plant carries out the ABA sensitivity test, and concrete steps are as described below:
To plant simultaneously, Shou Huo RHA2a mutant N378380 (orders from NASC simultaneously, TheNottingham Arabidopsis Stock Centre) (RHA2a among Fig. 2), the seed of Arabidopis thaliana (the Col-0 ecotype) (Col-0 among Fig. 2) or commentaries on classics pBI121-RHA2a plant (35S:RHA2a among Fig. 2) is sowed respectively and is being contained different concns (0 μ M, 0.2 μ M, 0.4 μ M, 0.5 μ M, 0.8 μ M and 1.0 μ M (as shown in Figure 2)) the MS substratum of ABA on, 4 ℃ of dark cultivations 3 days, be transferred to 22 ℃ of illumination conditions then, add up germination rate every day, cotyledon turns the index such as profound of green and root.
The result as shown in Figure 2, with respect to Arabidopis thaliana Col-0 wild-type (Col-0 among Fig. 2), commentaries on classics pBI121-RHA2a plant (35S:RHA2a among Fig. 2) is improved greatly to the susceptibility of ABA, on the ABA substratum of unusual lower concentration, the commentaries on classics pBI121-RHA2a plant of RHA2a overexpression just can not normal growth, it is green that cotyledon can not turn, root system is profound also to be subjected to obvious inhibition, and RHA2a mutant N378380 (rha2a among Fig. 2) can grow under the ABA of higher concentration condition to the susceptibility reduction of ABA.These explanations, the RHA2a overexpression can obviously improve the susceptibility of plant to ABA, that is to say that RHA2a is a new positive regulatory factor in the ABA signal pathway.
Sequence table
Claims (9)
1, a kind of plant ABA signal transduction regulatory protein is the protein with one of following amino acid residue sequences:
1) amino acid residue sequence of the SEQ ID № .3 in the sequence table;
2) with the SEQ ID № .3 amino acid residue sequence in the sequence table through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have the active protein of plant ABA signal transduction regulatory protein.
2, the encoding gene of the described plant ABA of claim 1 signal transduction regulatory protein.
3, encoding gene according to claim 2 is characterized in that: the cDNA gene of described plant ABA signal transduction regulatory protein has one of following nucleotide sequence:
1) SEQ ID № in the sequence table: 2 nucleotide sequence;
2) SEQ ID № in the code sequence tabulation: the polynucleotide of 3 protein sequences;
3) under the rigorous condition of height can with SEQ ID № in the sequence table: the nucleotide sequence of the 2 dna sequence dnas hybridization that limit.
4, encoding gene according to claim 3 is characterized in that: the genomic gene of described plant ABA signal transduction regulatory protein has one of following nucleotide sequence:
1) SEQ ID № in the sequence table: 1 nucleotide sequence;
2) SEQ ID № in the code sequence tabulation: the polynucleotide of 3 protein sequences;
3) under the rigorous condition of height can with SEQ ID № in the sequence table: the nucleotide sequence of the 1 dna sequence dna hybridization that limits.
5, the recombinant expression vector that contains any described plant ABA signal transduction regulatory protein encoding gene among the claim 2-4.
6, the transgenic cell line that contains any described plant ABA signal transduction regulatory protein encoding gene among the claim 2-4.
7, the host bacterium that contains any described plant ABA signal transduction regulatory protein encoding gene among the claim 2-4.
8, any described plant ABA signal transduction regulatory protein encoding gene is improving plant to the application in the susceptibility of dormin among the claim 2-4.
9, application according to claim 8 is characterized in that: described plant is an Arabidopis thaliana.
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