JP2001269176A - Peptides having the function of suppressing gene transcription - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a peptide derived from a higher plant having a function to bond to a DNA and suppress the transcription of gene, a gene coding for the peptide, a recombinant vector containing the gene and a transformant containing the recombinant vector. SOLUTION: The peptide derived from a higher plant has a motif composed of aspartic acid-leucine-asparagine and a function to suppress the transcription of gene.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a peptide having a function of suppressing gene transcription, a gene encoding the peptide, a recombinant vector containing the gene, and a transformant containing the recombinant vector.

[0002]

BACKGROUND OF THE INVENTION A protein factor ERF (Ethy
lene Responsive Element binding Factor) is the ERF
It is a transcription factor unique to plants having a DNA binding domain named domain. Recent studies have revealed that genes encoding ERF proteins constitute a multigene family. To date, cDNA encoding a protein factor having an ERF domain has been elucidated from tobacco and Arabidopsis plants. Reveals domains that will act as repressors,
The present invention has been completed.

[0003]

That is, the present invention relates to D
A peptide derived from a higher plant having a function of binding to NA and suppressing gene transcription, a gene encoding the peptide,
An object of the present invention is to provide a recombinant vector containing the gene and a transformant containing the recombinant vector.

[0004]

Means for Solving the Problems The present inventors have proposed tobacco,
As a result of functional analysis of cDNAs of rice and Arabidopsis thaliana, it was found that a region having a motif composed of aspartic acid-leucine-asparagine (DLN) in the carboxy terminal region of the ERF factor has a function of suppressing gene transcription. Thus, the present invention has been completed. Such a D
Examples of the peptide having an LN motif and having the function of suppressing gene transcription include a peptide contained in ERF3 derived from tobacco (SEQ ID NO: 1); AtERF3, AtERF4, AtERF7, and AtERF derived from Arabidopsis thaliana
A peptide included in RF8; a peptide included in osERF3 derived from rice.

[0005]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS A method for analyzing functions
A protein coding region is excised from the cDNA encoding the RF factor, ligated to a region encoding the DNA binding domain of the yeast GAL4 transcription factor, and further downstream of the cauliflower mosaic virus 35S promoter that functions in plant cells. To construct an effector plasmid. This was introduced by electroporation into tobacco cultured cells simultaneously with a reporter gene comprising a luciferase gene having a GAL4 protein binding site bound to a promoter region, and examined by measuring the activity of the luciferase gene as a reporter gene.

Next, in order to identify the repressor domain, which is a region having a repressor function, which is present in the repressor element, a deletion analysis method for deleting the coding region of each gene was used. Which region the repressor domain is in was examined.

[0007]

[Example] Isolation of ERF3 gene A nucleotide sequence AGCCGCC that has been shown to function as a plant hormone ethylene-responsive cis element from a tobacco cDNA library (Reference Plant Cell, 1995 vo
l. 7, p173-182) and a cDNA encoding a protein capable of binding were isolated using a DNA fragment containing AGCCGCC as a probe by a DNA-protein affinity method.

(Method for preparing cDNA library) Leaves are collected from a tobacco plant and ground in a liquid nitrogen using a mixer. For 10 g of the crushed leaf powder, 20 mL of
RNA extraction buffer (8 M guanidine hydrochloride, 20 mM
Disodium ethylenediaminetetraacetate (EDTA), 20 mM
2- (N-morpholino) ethanesulfonic acid (MES), 50
mM mercaptoethanol and 8 mL of TE solution (10 mM
Phenol saturated with Tris-Cl pH8.0, 1mM EDTA) and 2m
L of chloroform was added and stirred well, and centrifuged (10000).
g) and collect the supernatant. 0.2 times the volume of the supernatant
After adding M acetic acid and 0.7 volumes of ethanol, the mixture is allowed to stand at −20 ° C., and then centrifuged (10000 g). The precipitate was dissolved in 2 mL of a TE solution, and 500 mL of a 10 M lithium chloride solution was added.
And centrifuge. The precipitate was washed with 70% alcohol and air-dried to obtain a total RNA preparation. The total RAN was converted to total RNA using Polymega Tract System 1000 manufactured by Promega.
Only mRNA was purified from. Using this mRNA as a template, a double-stranded cDNA was synthesized using a cDNA direction cloning kit manufactured by Pharmacia. According to the protocol of Pharmacia, these were used for the Lambda gt11 expression vector EcoRI-NotI.
After integration into a restriction enzyme site, a library was completed using a lambda packaging kit (Pharmacia).

(Screening method) Tobacco cDNA prepared
An agent for infecting lambda phage into which the library has been incorporated by selecting about 40,000 plaques and infecting Escherichia coli 1090, which had been suspended in 0.5 cc mL of 10 mM magnesium sulfate solution, and infecting the cells. 50 mg / L sodium ampicillate LB agar medium (10 g tryptone, 10 g sodium chloride, 5 g yeast extra,
And 15g agar) and culture at 37 ° C. When the plaque becomes about 2 mm, 1 mM isopropyl-β-D-
A nitrocellulose membrane immersed in a thiogalactopyranoside (IPTG) solution is placed on an agar medium where plaques are growing, and further cultured at 37 ° C. for 4 hours. The filter was removed from the plate, the phage was transferred, and a binding buffer containing 5% (V / V) skim milk (25 mM HEPES [2- {4- (2-hydroxylethyl) -1-piperazinyl} ethanesulfonic acid] p
H7.5, 40 mM KCl, 5% v / v glycerol, 20ug / ml poly (dA-d
After immersion in T) (dA-dT), 0.5 mM EDTA) at 4 ° C. for 2 hours, it is further immersed in a binding buffer containing 0.5% (V / V) skim milk at 4 ° C. for 1 hour. After removing all buffer, ³² P
Double-stranded DNA (SEQ ID NO: 3) consisting of the following sequence end-labeled with a radioisotope: AGATCTCATAAGAGCCGCCACTAAA
Put the filter in a buffer (3 mL per filter) containing ATAAGACCGATCAAATAGAGCCGCCATG at 15 ° C.
After immersion in the same buffer without DNA three times, the plate was exposed to X-ray film, and the phage to which the probe DNA was bound was isolated from the original agar plate. The isolated phage is infected with Escherichia coli, and the above screening is repeated twice more. After isolation into a single plaque, DNA is extracted from the phage, and the cDNA region is replaced with the restriction enzyme Eco.
It was excised using RI and NotI. This DNA fragment was incorporated into a restriction enzyme EcoRI-NotI site of a plasmid pT7D3 (Pharmacia), and the nucleotide sequence of the plasmid was analyzed to obtain the entire nucleotide sequence of the cDNA of tobacco ERF3 (SEQ ID NO: 1). ER
A plasmid having the full-length sequence of F3 cDNA was designated as pERF3.

(Effector plasmid containing full-length ERF3
Construction of pGAL4DB-ERF3: See Fig. 1) Clontech (C
lontech, USA) with plasmids pBI221 and XhoI and S
After cutting with stI and blunt-end treatment with T4 polymerase,
Except for GUS gene by agarose gel electrophoresis, cauliflower mosaic virus 35S promoter (hereinafter referred to as CaMV35S)
35S-Nos plasmid fragment D containing the transcription termination region (Nos terminator, hereinafter Nos-ter) of the nopaline synthase gene
I got NA. Clonetech's pAS2-1 vector is digested with the restriction enzyme HindIII, and a 748 bp DNA coding region (1-147 amino acid residues) encoding the yeast GAL4 protein is digested.
After the DNA fragment (hereinafter referred to as GAL4DBD) was isolated by agarose gel electrophoresis, it was blunt-ended with T4 DNA polymerase. This DNA fragment containing GAL4DBD was converted to the 35S-No.
s DNA into the blunt-ended site between the 35S promoter and Nos terminator, and
A 35S-GAL4DBD vector (described as p35S-GALDB in FIG. 1) was selected by selecting those in which the ORFs of the DNA binding region of the GAL4 protein were arranged in the forward direction.

(Creation of full length and deleted coding region of ERF3 and cloning into p35S-GALDB) cD of ERF3
5-terminal upper primer primer1 designed so that the reading frame matches the NA plasmid pERF3 and GAL4DBD
(SEQ ID NO: 4: binds to ERF3 nucleotide sequence 1-19) GATGGCTGTCA
3rd terminal lower primer primer2 having AAAATAAGG and restriction enzyme SalI site (SEQ ID NO: 5: binds to ERF3 base sequence 661-678) CCAAATAACATTATCGGTCGACTCAAAATTCCATAGGTG using ERF3 whole protein coding region (SEQ ID NO: 1: ERF3 base sequence 1-678; amino acid sequence) 1-225) was amplified by the PCR method to obtain a DNA fragment. The conditions for the PCR reaction were as follows:
The annealing was carried out for 25 minutes at 47 ° C. for 1 minute and the elongation reaction at 74 ° C. for 1 minute. Hereinafter, all PCR reactions were performed under the same conditions. The obtained DNA fragment is subjected to restriction enzyme Sal.
After digestion with I, the target DNA fragment was isolated by agarose electrophoresis. The DNA fragment encoding this ERF3 was previously digested with restriction enzymes SmaI and SalI.
Incorporated into DBD plasmid, effector plasmid pGA
L-ERF3 was constructed. The above plasmid pBI221 to pGAL
The procedure for constructing 4DB-ERF3 is shown in FIG.

(Construction of ERF3 deletion effector plasmid pGAL4-1 / 25ERF3 containing ERF3 amino acids 1/25) pE
5-terminal upper primer prim designed to match the reading frame with the frame encoding RF3 plasmid and GAL4DBD
er3 (SEQ ID NO: 6: binding site ERF3 nucleotide sequence 1-19) GATGGCT
3-terminal lower primer primer4 having GTCAAAAATAAGG and restriction enzyme SalI site (SEQ ID NO: 7: binding site ERF3 nucleotide sequence 69-75) CTTCCTTACACCGTCGACTTAAACCTCC
Base sequence 1-75 corresponding to the amino acid sequence 1/25 coding region of
Was obtained by PCR. This DNA fragment was digested with restriction enzyme SalI, and the target DNA fragment was isolated by agarose electrophoresis. The DNA fragment of the DNA region 1-75 encoding the amino acid sequence 1/25 of this ERF3 was incorporated into the 35S-GAL4DBD plasmid which had been digested with restriction enzymes SmaI and SalI in advance, and the effector plasmid pGAL-1 / 25E
RF3 was built.

(Construction of ERF3 Deletion Effector Plasmid pGAL4-26 / 82ERF3 Containing ERF3 Amino Acids 26/82) A 5-terminal upper primer primer5 (SEQ ID NO: 5) designed so that the reading frame coincides with the frame encoding pERF3 plasmid and GAL4DBD 8: Binding site ERF3 nucleotide sequence 75-99)
TCACTACAGAGGTGTAAGGAAGAGG and 3-terminal lower primer primer6 having a restriction enzyme SalI site (SEQ ID NO: 9: binding site ERF3 base sequence 239-246) CTCTGATTCTCGTCGACTTAAGGGAAG
Using TTAG, a DNA fragment containing the region of the nucleotide sequence 75-246 corresponding to the amino acid sequence 26/82 coding region of ERF3 was obtained by PCR. This DNA fragment was digested with restriction enzyme SalI, and the target DNA fragment was isolated by agarose electrophoresis. This DNA fragment was incorporated into a 35S-GAL4DBD plasmid which had been digested with restriction enzymes SmaI and SalI in advance to construct an effector plasmid pGAL-26 / 82ERF3.

(Construction of ERF3 Deletion Effector Plasmid pGAL4-83 / 225ERF3 Containing ERF3 Amino Acids 83/225) A 5-terminal upper primer primer7 (SEQ ID NO: designed so that the reading frame matches the frame encoding pERF3 plasmid and GAL4DBD) 10: Binding site ERF3 base sequence 249-2
69) 3-terminal lower primer primer2 having TTCACCGACGGAGAATCAGAG and restriction enzyme SalI site (SEQ ID NO: 5: binding site ERF3 nucleotide sequence 661-678) CCAAATAACATTATCGGTCGACTCAAA
Using ATTCCATAGGTG, a DNA fragment containing the region of the nucleotide sequence 249-678 corresponding to the amino acid sequence 83/225 coding region of ERF3
Obtained by PCR method. This DNA fragment was digested with restriction enzyme SalI, and the target DNA fragment was isolated by agarose electrophoresis. This DNA fragment was incorporated into a 35S-GAL4DBD plasmid which had been digested with restriction enzymes SmaI and SalI in advance to construct an effector plasmid pGAL-83 / 225ERF3.

(Construction of ERF3 Deletion Effector Plasmid pGAL4-83 / 123ERF3 Containing ERF3 Amino Acids 83/123) A 5-terminal upper primer primer7 (SEQ ID NO: designed so that the reading frame coincides with the frame encoding pERF3 plasmid and GAL4DBD) 11: Binding site ERF3 base sequence 246-2
69) 3-terminal lower primer primer8 with TTCACCGACGGAGAATCAGAG and restriction enzyme SalI site (SEQ ID NO: 12: binding site ERF3 nucleotide sequence 356-369) GCCATCTGCAGCGTCGACTCAAAGA
PCR of a DNA fragment containing the region of the nucleotide sequence 246-369 corresponding to the amino acid sequence 83/123 coding region of ERF3 using CGGCGCG
Obtained by the method. This DNA fragment was digested with restriction enzyme SalI, and the target DNA fragment was isolated by agarose electrophoresis. This DNA fragment was incorporated into a 35S-GAL4DBD plasmid which had been digested with restriction enzymes SmaI and SalI in advance to construct an effector plasmid pGAL-83 / 123ERF3.

(Construction of ERF3 Release Effector Plasmid pGAL4-124 / 189ERF3 Containing ERF3 Amino Acids 124/189) Five-terminal upper primer primer9 (SEQ ID NO: 9) designed so that the reading frame coincides with the frame encoding pERF3 plasmid and GAL4DBD. 13: Binding site ERF3 base sequence 369
-391) CTCCGTTGCTGCAGATGGCCGGTG and 3-terminal lower primer primer10 having a restriction enzyme SalI site (SEQ ID NO: 14
Binding site ERF3 nucleotide sequence 559-567) TCCGACACAGTAGGGTCGACT
Using CAGGCGTTAAC a DNA fragment containing the region of the base sequence 369-567 corresponding to the amino acid sequence 83/123 coding region of ERF3
Obtained by PCR method. This DNA fragment was digested with restriction enzyme SalI, and the target DNA fragment was isolated by agarose electrophoresis. This DNA fragment was incorporated into a 35S-GAL4DBD plasmid which had been digested with restriction enzymes SmaI and SalI in advance to construct an effector plasmid pGAL-124-189ERF3.

(Construction of ERF3 Deletion Effector Plasmid pGAL4-191 / 225ERF3 Containing ERF3 Amino Acids 191/225) A 5-terminal upper primer primer11 (SEQ ID NO: designed so that the reading frame coincides with the frame encoding pERF3 plasmid and GAL4DBD) 15: Binding site ERF3 base sequence
569-593) AGTGGGTCCTACTGTGTCGGACTC and 3-terminal lower primer primer2 having a restriction enzyme SalI site (SEQ ID NO: 5: binding site ERF3 base sequence 661-678) CCAAATAACATTATCGG
Amino acid sequence 19 of ERF3 using TCGACTCAAAATTCCATAGGTG
A DNA fragment containing the nucleotide sequence 569-678 corresponding to the 1/225 coding region was obtained by PCR. This DNA fragment was digested with restriction enzyme SalI, and the target DNA fragment was isolated by agarose electrophoresis. This DNA fragment was combined with the restriction enzyme SmaI.
The effector plasmid pGAL-191 / 225ERF3 was constructed by incorporation into 35S-GAL4DBD plasmid which had been digested with SalI in advance.

(Construction of ERF3 Release Effector Plasmid pGAL4-204 / 225ERF3 Containing ERF3 Amino Acids 204/225) A five-terminal upper primer primer12 (SEQ ID NO: designed so that the reading frame coincides with the frame encoding pERF3 plasmid and GAL4DBD) 16: Binding site ERF3 base sequence 60
9-627) 3-terminal lower primer primer2 having AGAGAACCAATATGATGGG and a restriction enzyme SalI site (SEQ ID NO: 5: binding site ERF3 nucleotide sequence 661-678) CCAAATAACATTATCGGTCGACTCAAA
Using ATTCCATAGGTG, a DNA fragment containing the nucleotide sequence 609-678 corresponding to the amino acid sequence 204/225 coding region of ERF3 was obtained by PCR. This DNA fragment was digested with restriction enzyme SalI, and the target DNA fragment was isolated by agarose electrophoresis. This DNA fragment was incorporated into 35S-GAL4DBD plasmid which had been digested with restriction enzymes SmaI and SalI in advance,
The effector plasmid pGAL-204 / 225ERF3 was constructed.

Construction of Reporter Gene (See FIGS. 2 and 3) The plasmid pUC18 is digested with restriction enzymes EcoRI and SstI.
pBI221 (Clontech) with restriction enzymes EcoRI and Sst
Digest with I, Nos-ter (nopaline synthase terminator)
It is isolated by agarose gel electrophoresis in which a 270 bp DNA fragment containing the region is inserted. The obtained fragment is digested with the restriction enzyme EcoR.
EcoRI-Ss of plasmid pUC18 digested with I and SstI
Insert at the tI site. Cauliflower mosaic virus 35S
DNA 1 of complementary strand containing promoter TATA box (SEQ ID NO: 17) AGCTTAGATCTGCAAGACCCTTCCTCTATATAAGGAAGTTCAT
TTCATTTGGAGAGGACACGCTG and DNA2 (SEQ ID NO: 18)
GATCCAGCGTGTCCTCTCCAAATGAAATGAACTTCCTTATATAGAGGAAG
GGTCTTGCAGATCTA is synthesized. The synthesized DNA was heated at 90 ° C. for 2 minutes, then at 60 ° C. for 1 hour, and then at room temperature (25 ° C.).
C) for 2 hours to allow annealing to form a double strand. Restriction enzyme pUC18 plasmid with Nos-ter HindI
Digest with II and BamHI. The synthesized double-stranded DNA was
Insert into HindIII-BamHI site to construct a plasmid containing TATA-box and Nos-ter. The procedure described above is shown in FIG.

This plasmid is digested with the restriction enzyme SstI,
Perform blunt-end treatment with T4 DNA polymerase. Plasmid vector PGV-CS2 (manufactured by Toyo Ink) containing firefly luciferase gene (LUC) and restriction enzyme XbaI
After digestion with NcoI and blunt-end treatment with T4 DNA polymerase, a 1.65 kb DNA fragment containing the luciferase gene was isolated and purified by agarose gel electrophoresis. This DNA fragment was inserted into the above plasmid containing the TATA box and Nos terminator to construct a TATA-LUC reporter gene. DN of yeast GAL4 protein
Plasmid pG5CAT (Clontech
Was digested with restriction enzymes SmaI and XbaI and blunt-ended with T4 DNA polymerase.
A DNA fragment containing the DNA binding sequence of the GAL4 protein was purified by agarose gel electrophoresis. Digest the TATA-LUC vector with the restriction enzyme BglII and blunt-end it with T4 DNA polymerase. At this site, 5 copies of the DNA fragment containing the DNA binding sequence of the GAL4 protein, which has been blunt-ended, are inserted, and those having the DNA binding sequence of the GAL4 protein oriented in the forward direction are selected from the obtained plasmids. -Build LUC. (See Fig. 3)

(Construction of Reference Gene) A cassette vector pRL-null manufactured by Promega having a luciferase gene derived from Renilla was cut with restriction enzymes NheI and XbaI, and blunt-ended with T4 DNA polymerase, followed by agarose. Renilla mushrooms by gel electrophoresis
Purify a 948 bp DNA fragment containing the luciferase gene. This DNA fragment was used to construct the GUS of the pBI221 vector excluding the GUS gene used in the construction of the effector plasmid.
Insert into the region where the gene was. Among the obtained plasmids, those having the Renilla luciferase gene oriented in the forward direction are selected (construction of pPTRL).

(Measurement of Reporter Gene Activity) The reporter gene and the effector plasmid were introduced into tobacco cultured cell protoplasts by electroporation, and the effect of the effector was determined by measuring the activity of the reporter gene.

(Preparation method of protoplast) 100 mL of MS
Medium (Murasige-Skoog medium mixed salts, manufactured by Nippon Pharmaceutical Co., 3% sucrose, 0.2 g / L KH ₂ PO ₄ , 0.2 g / L m-inosito
l, 2 mg / L glycine, 1 mg / L Thiamine hydrochloride, 0.2 mg / L
2, 4-D, pH adjusted to 5.8) 7 ml of tobacco cultured cells cultured for 7 days Pre-cultured solution of BY-2 was added, and the cells were cultured in the dark at 26 ° C for 3 days. Opening is 125 mm, manufactured by Tokyo Screen Co., Ltd.)
Wash cells with MS medium containing mannitol. Wash cells with 25 mL of 0.4 M mannitol in MS medium
And left at room temperature for 10 minutes. The cells are collected by centrifugation at 3,000 rpm for 1 minute. The cells are mixed with 20 mL of 1% cellulase (Onodka RS,) and 0.1% pectylase Y-23.
Resuspend cells in MS medium containing
Digest the cell wall while rotating and shaking at 60 rpm in the dark at 26 ° C for 90 minutes. Then, centrifuge at 1,000 rpm for 5 minutes to collect protoplasts.

(Gene transfer by electroporation) The protoplasts obtained above were electroporated in an electroporation buffer so as to have a concentration of 2.5 × 10 6 cells / mL.
(5 mM MES pH 5.8, 70 mM KCl, 0.3 M mannitol). PGAL4-LUC reporter gene constructed in an electroporation cuvette (Gene Pulser cuvette 0.4 cm elecrode, manufactured by Bio-Rad) and pGALDB-ERF3 as effector plasmid or its delation series (pGALDB-1 / 25ERF3 to pGALDB-204)
-225ERF3), add 10 ug of each DNA and 1 ug of the reference gene plasmid to 100 uL of 2X electroporation buffer (10 mM MES pH5.8, 140 mM KCl, 0.6 M mannitol), and make up to 200 uL with sterile water. . Add 600 uL of the protoplast suspension to a cuvette, and use an electroporator (Genepulser II Electroporation System).
DNA is introduced under the conditions of 600 V and 25 mF using Stem Bio-Rad. After introduction, the protoplasts were collected from the cuvette by centrifugation at 1,000 rpm for 5 minutes, and 5 mL
The protoplasts were resuspended in MS medium containing 0.4 M mannitol, allowed to stand in the dark at 26 ° C. for 6 hours, and then the activity of the reporter gene was measured.

(Measurement of luciferase activity) 1 mL of protoplast suspension was allowed to stand for 6 hours, and 2,000 rpm
Centrifuge for 3 minutes to collect protoplasts, then
LuciferaseTM Reporter Assay System (Promega)
Suspend in 50 uL of Passive Lysis Buffer attached to. After crushing the protoplasts, centrifuge and collect the supernatant. Using 5 uL of this cell extract, Dual-Lucifer
Luciferase activity was measured using the aseTM Reporter Assay System (Promega) and a luminescence reader (BLR-201, Aloka). For measurement of firefly luciferase and Renilla luciferase activities, luminescence for 10 seconds was counted in the integration mode according to the measurement kit instructions. The activity value of the reference gene is divided by the activity value of the reporter gene, and the relative value, Rela
The tive luchifarase activity was determined as a measured value. Assuming that the relative value without the effector was 100, the effect of the effector was investigated by the change in the activity value of the reporter gene when the effector plasmid was simultaneously introduced into the cells. That is, since the activity value of the reporter when the pGAL4-LUC reporter gene and the pGALDB-ERF3 effector plasmid are introduced is 50, pHGALDB-ER
F3 has the effect of suppressing the activity of the reporter gene (repressor function). Hereinafter, the activity value of the reporter was measured, and the relative activity value of the reporter was 10
When the value is 0 or less, since the introduced effector has a repressor function, it was examined which effector functions as a repressor by measuring the activity of the reporter.

(Identification of Repressor Domain) Tobacco ER
To analyze the function of F3 gene involved in transcriptional regulation, reporter genes pGAL4-LUC and pGALDB-ERF3 were introduced into protoplasts prepared from cultured tobacco cells by electroporation, and the activity of the reporter genes was examined. As a result, the pGALDB-ERF3 effector reduced the activity of the reporter gene by 50% as compared to the case where only the reporter gene without the effector was introduced (control). This indicates that ERF3 functions as a repressor that suppresses transcription. In order to identify the repressor domain of ERF3, the protein coding region of ERF3 gene
Effector plasmid with N fragment, pGALDB-1 / 25ERF3,
Using pGALDB-26 / 82ERF3 and pGALDB-83 / 225ERF3, it was examined which region is a repressor domain having a function of suppressing reporter gene activity. The results are shown in FIG. 4B.

The individual effector plasmids described above were introduced into cultured tobacco cells together with a reporter gene, and the activity of the reporter gene was measured. As a result, pGALDB-83 / 225ERF3
The effector was shown to have a repressor function to suppress the activity of the reporter gene by about 50% as compared to the control, as in the case where pGALDB-ERF3 was introduced.
However, pGALDB-1 / 25ERF3 and pGALDB-26 / 82ERF3 had no effect of suppressing reporter activity. This indicates that the region corresponding to the amino acid sequence 83/225 of ERF3 has a repressor function. The other regions, 1/25 or 26/82, were found to have no repressor function (FIG. 4B).

Next, in order to identify in which region of the 83/225 amino acid sequence the repressor function is present, an effector plasmid, pGALDB-83 / 123ERF3, having the above-disclosed 83/225 region DNA, was specified. , pGALDB-
124 / 189ERF3 or pGALDB-191 / 225ERF3 was introduced into protoplasts together with the reporter gene pGAL4-LUC to measure the activity of the reporter gene. As a result, pGALDB-1
When 91 / 225ERF3 was introduced, the activity of the reporter gene was reduced by about 50%. However, when other effectors, pGALDB-83 / 123ERF3 or pGALDB-124 / 189ERF3, were introduced, the activity of the reporter did not decrease. From this result, the region having the repressor function of ERF3 (repression domain) is the amino acid sequence of ERF3, 191/225
To be present. Furthermore, since the effector plasmid pGALDB-204 / 225ERF3 having a region where this 191/225 region is deleted has the effect of similarly suppressing the activity of the reporter gene, the 22 amino acids of the amino acid sequence region 204/225 of ERF3 Region was the repressor domain of ERF3 (FIG. 4B).
The amino acid sequence of this 22 amino acid region is shown in SEQ ID NO: 2.

In order to determine which amino acids in the amino acid sequences of these repressor domains are involved in the repressor function, pGALDB-191 having a repressor function was examined.
/ 225ERF3 effector plasmid and primer 5'M1 (SEQ ID NO: 19: binds to ERF3 base sequence 571-589) CCGACACAGTA
GGACCCAC and 3′M1 (SEQ ID NO: 20: ERF3 nucleotide sequence 591-610)
A PCR was performed using GCTCGTCCTCTGCAGTGGAAG, and DN was obtained by substituting the codon of alanine for the codon of aspartic acid at amino acid number 197 of ERF3 shown in SEQ ID NO: 1.
The effector plasmid pM1-191 / 225ERF3 with A was constructed.

Similarly, the primer 5'M2 (SEQ ID NO: 21: ER
Binds to F3 nucleotide sequence 639-620) CAATTCCTCTTTTCCCATCA and 3'M
2 (SEQ ID NO: 22: binds to ERF3 nucleotide sequence 641-660) CTCTT
A PCR reaction was carried out using GCTCTTAACCTTGCT to construct an effector plasmid pM2-191 / 225ERF3 having a DNA in which the aspartic acid codons at amino acids 214 and 216 of ERF3 shown in SEQ ID NO: 1 were replaced with codons of alanine.
In addition, primer 5′M3 (SEQ ID NO: 23: ERF3 base sequence 65
1-670) CCATAGGTGGAGCAAGGTTA and 3'M3 (SEQ ID NO: 24: ERF3 nucleotide sequence 672-680) CATTTTGATGATGACGA
A PCR reaction was performed using TAA, and the ER
An effector plasmid pM3-191 / 225ERF3 having a DNA in which the glutamic acid codon at amino acid number 224 of F3 was replaced with an alanine codon was constructed.

Each of these plasmids was introduced into a protoplast together with a reporter gene pGAL4-LUC, and the activity of the reporter was measured to determine whether the substituted amino acids affected the repressor function. As a result, pM1-1 obtained by substituting the aspartic acid at position 197 with alanine
PM3-191 / 225ERF3 effector plasmid in which 91 / 225ERF3 and 224th glutamic acid were substituted with alanine,
Since the effect of suppressing the activity of the reporter gene is similar to that of pGALDB-191 / 225ERF3 in which no substitution is made, it is considered that these amino acid sites are not involved in the repression function. On the other hand, the effector plasmid pM2- in which the aspartic acids at positions 214 and 216 were replaced with alanine
191 / 225ERF3 lost the effect of suppressing reporter gene activity (FIG. 5). This revealed that the region containing aspartic acid at positions 214 and 216 is an important region for the repressor function. It was demonstrated that a peptide having this sequence is a novel sequence related to the function as a repressor.

The peptide of the present invention having the function of suppressing the transcription of the gene can be fused with a DNA-binding protein that specifically binds to the transcriptional regulatory region of the oncogene, and expressed in cells to express the oncogene. Expression can be efficiently suppressed. When the repressor function was examined, it was non-specific for the gene. However, since the repressor function requires binding to DNA, fusion with a DNA-binding domain that binds to a specific DNA makes the gene specific or non-specific. It is possible to suppress the transfer in an efficient manner. This makes it possible to control, for example, the expression of a gene encoding a pigment metabolic enzyme, and to create a flower having a petal of a different color that has not been obtained until now. In addition, by suppressing the expression of proteins that are allergens, it is possible to produce foods with less allergens.

[0033]

[Sequence List] SEQUENCE LISTING <110> Secretary of Agency of Industrial Science and Technology <120> Novel repression domain of plant specific transcription factor <130> <160> 24 <210> 1 <211> 678 <212> DNA <213> Nicotiana tobacum <400> 1 atg gct gtc aaa aat aag gtt agt aat ggc aat ctg aaa gga gga aat 48 Met Ala Val Lys Asn Lys Val Ser Asn Gly Asn Leu Lys Gly Gly Asn 5 10 15 gtg aaa aca gat gga gtt aag gag gtt cac tac aga ggt gta agg aag 96 Val Lys Thr Asp Gly Val Lys Glu Val His Tyr Ser Gly Val Ser Lys 20 25 30 agg cca tgg ggt cgg tat gca gct gaa atc cgt gac ccg ggt aag aag 144 Ser Pro Trp Gly Arg Tyr Ala Ala Glu Ile Arg Asp Pro Gly Lys Lys 35 40 45 agt cgg gtc tgg tta ggt act ttc gac acg gcg gaa gag gcg gct aag 192 Ser Arg Val Trp Leu Gly Thr Phe Asp Thr Ala Glu Glu Ala Ala Lys 50 55 60 gcg tac gac acc gcc gct cga gag ttt cgt gga ccc aaa gca aaa act 240 Ala Tyr Asp Thr Ala Ala Arg Glu Phe Arg Gly Pro Lys Ala Lys Thr 65 70 75 80 aac ttc cct tca ccg acg gag aat cag agc cca agt cac agc ag c acc 288 Asn Phe Pro Ser Pro Thr Glu Asn Gln Ser Pro Ser His Ser Ser Thr 85 90 95 gtg gag tcc tct agt gga gag aat ggt gtt cac gcg ccg cct cat gcg 336 Val Glu Ser Ser Ser Gly Glu Asn Gly Val His Ala Pro Pro His Ala 100 105 110 ccg ctc gag ctg gat ctc acg cgc cgt ctt ggc tcc gtt gct gca gat 384 Pro Leu Glu Leu Asp Leu Thr Arg Arg Leu Gly Ser Val Ala Ala Asp 115 120 125 ggc ggt gac aac tgt cgc cgt tct ggg gaa gtt ggg tac ccg att ttc 432 Gly Gly Asp Asn Cys Arg Arg Ser Gly Glu Val Gly Tyr Pro Ile Phe 130 135 140 cac cag cag ccg act gtg gcg gtt ctg cca aat ggc cag ccg gtt Gct Gln 480 Pro Thr Val Ala Val Leu Pro Asn Gly Gln Pro Val Leu 145 150 155 160 ctc ttt gat tct ttg tgg cgg gcg gga gtt gtt aac agg cct cag cct 528 Leu Phe Asp Ser Leu Trp Arg Ala Gly Val Val Asn Ser Pro Gln Pro 165 170 175 tac cat gta acg ccg atg ggg ttt aac ggc gtt aac gcc gga gtg ggt 576 Tyr His Val Thr Pro Met Gly Phe Asn Gly Val Asn Ala Gly Val Gly 180 185 190 cct act gtg tcg gac tcg tcc tct gca gtg gaa gag aac caa tat gat 624 Pro Thr Val Ser Asp Ser Ser Ser Ala Val Glu Glu Asn Gln Tyr Asp 195 200 205 ggg aaa aga gga att gat ctt gat ctt aac ctt gct cca cct atg gaa 672 Gly Lys Ser Gly Ile Asp Leu Asp Leu Asn Leu Ala Pro Pro Met Glu 210 215 220 ttt tga 678 Phe 225 <210> 2 <211> 22 <212> RPT <213> Nicotiana tobacum <400> 2 Glu Asn Gln Tyr Asp Gly Lys Ser Gly Ile Asp Leu Asp Leu Asn Leu 5 10 15 Ala Pro Pro Met Glu Phe 20 <210> 3 <211> 53 <212> DNA <213> Artificial Sequence <223> Description of Artificial Sequence: Synthetic DNA <400> 3 agatctcata agagccgcca ctaaaataag accgatcaaa tagagccgcc atg < 210> 4 <211> 19 <212> DNA <213> Artificial Sequence <223> Description of Artificial Sequence: Synthetic primer DNA <400> 4 gatggctgtc aaaaataagg <210> 5 <211> 37 <212> DNA <213> Artificial Sequence <223> Description of Artificial Sequence: Synthetic primer DNA <400> 5 ccaaataaca ttatcggtcg actcaaattcc ataggtg <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <223> Description of Artificial Sequence: S synthetic primer DNA <400> 6 gatggctgtc aaaaataagg <210> 7 <211> 28 <212> DNA <213> Artificial Sequence <223> Description of Artificial Sequence: Synthetic primer DNA <400> 7 cttccttaca ccgtcgactt aaacctcc <210> 8 <211 > 25 <212> DNA <213> Artificial Sequence <223> Description of Artificial Sequence: Synthetic primer DNA <400> 8 tcactacaga ggtgtaagga agagg <210> 9 <211> 31 <212> DNA <213> Artificial Sequence <223> Description of Artificial Sequence: Synthetic primer DNA <400> 9 ctctgattct cgtcgactta agggaagtta g <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <223> Description of Artificial Sequence: Synthetic primer DNA <400> 10 ttcaccgacg gagaatcaga g <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <223> Description of Artificial Sequence: Synthetic primer DNA <400> 11 ttcaccgacg gagaatcaga g <210> 12 <211> 32 <212> DNA <213> Artificial Sequence <223> Description of Artificial Sequence: Synthetic primer DNA <400> 12 gccatctgca gcgtcgactc aaagacggcg cg <210> 13 <211> 24 <2 12> DNA <213> Artificial Sequence <223> Description of Artificial Sequence: Synthetic primer DNA <400> 13 ctccgttgct gcagatggcc ggtg <210> 14 <211> 32 <212> DNA <213> Artificial Sequence <223> Description of Artificial Sequence : Synthetic primer DNA <400> 14 tccgacacag tagggtcgac tcaggcgtta ac <210> 15 <211> 24 <212> DNA <213> Artificial Sequence <223> Description of Artificial Sequence: Synthetic primer DNA <400> 15 agtgggtcct actgtgtcgg actc <210> 16 <211> 19 <212> DNA <213> Artificial Sequence <223> Description of Artificial Sequence: Synthetic primer DNA <400> 16 agagaaccaa tatgatggg <210> 17 <211> 65 <212> DNA <213> Artificial Sequence <223 > Description of Artificial Sequence: Synthetic DNA <400> 17 agcttagatc tgcaagaccc ttcctctata taaggaagtt catttcattt ggagaggaca 10 20 30 40 50 60 cgctg 65 <210> 18 <211> 65 <212> DNA <213> Artificial Sequence <223> Description of Artificial Sequence : Synthetic DNA <400> 18 gatccagcgt gtcctctcca aatgaaatga acttccttat atagaggaag ggtcttgcag 10 20 30 40 50 60 atcta 65 <210> 19 <211> 19 <212> DNA <213> Artificial Sequence <223> Description of Artificial Sequence: Synthetic primer DNA <400> 19 ccgacacagt aggacccac <210> 20 <211> 21 <212> DNA <213> Artificial Sequence <223> Description of Artificial Sequence: Synthetic primer DNA <400> 20 gctcgtcctc tgcagtggaa g <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <223> Description of Artificial Sequence: Synthetic primer DNA <400> 21 caattcctct tttcccatca <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <223> Description of Artificial Sequence: Synthetic primer DNA <400> 22 ctcttgctct taaccttgct <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <223> Description of Artificial Sequence: Synthetic primer DNA <400> 23 ccataggtgg agcaaggtta <210> 24 <211> 19 <212> DNA <213> Artificial Sequence <223> Description of Artificial Sequence : Synthetic primer DNA <400> 24 cattttgatg atgacgtaa

[Brief description of the drawings]

FIG. 1 is a diagram showing the procedure for constructing pGAL4DB-ERF3 from plasmid pBI221.

FIG. 2 shows the first half of the procedure for constructing the reporter gene GAL4-LUC.

FIG. 3 is a view showing the latter half of the procedure for constructing the reporter gene GAL4-LUC, following FIG. 2;

FIG. 4A is a view showing a reporter gene and an effector plasmid. In the figure, 5XGAL4: GAL4 transcription factor
DNA binding sequence, TATA: region containing CaMV35S promoter TATA box, LUC: luciferase gene, CaMV 35S:
Cauliflower mosaic virus 35S protein gene promoter, GAL4 DB: yeast GAL4 transcription factor DAN binding domain coding region, Nos: nopaline synthase gene transcription termination region. B shows the effect of ERF3 and ERF3 deletion on reporter gene activity (Relative Activity)
FIG. In the figure, each number on the left (such as 1/225)
Indicates the amino acid region of ERF3. The middle box indicates the amino acid sequence region corresponding to the number on the left. The graph on the right shows the activity of the reporter gene when the effector plasmid having the region on the left was introduced. The activity of the reporter gene without the effector was set to 100. E
Among the effectors of the RF3 and ERF3 deletions, the effector having the amino acid sequence 1/225, 83/225. 191/225, 201/225 reduces the activity of the reporter gene to 50% and has the effect of suppressing transcription. It is shown. The minimum area of suppression effect (repressor function) is 201/225
Thus, it was revealed that this region is a repressor domain.

FIG. 5 is a view showing the results of an amino acid substitution experiment of a repressor domain. This region is important for repressor function because only alanine (A) replaces 214 and 216 aspartic acids with alanine (A) within the 191/225 region that functions as the repressor domain of tobacco ERF3 This indicates that the amino acid sequence is present.

[Procedure amendment]

[Submission date] April 11, 2000 (2004.1.
1)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0019

[Correction method] Change

[Correction contents]

Construction of Reporter Gene (See FIGS. 2 and 3) The plasmid pUC18 is digested with restriction enzymes EcoRI and SstI.
pBI221 (Clontech) with restriction enzymes EcoRI and Sst
Digest with I, Nos-ter (nopaline synthase terminator)
It is isolated by agarose gel electrophoresis in which a 270 bp DNA fragment containing the region is inserted. The obtained fragment is digested with the restriction enzyme EcoR.
EcoRI-Ss of plasmid pUC18 digested with I and SstI
Insert at the tI site. Cauliflower mosaic virus 35S
DNA 1 of complementary strand containing promoter TATA box (SEQ ID NO: 17) AGCTTAGATCTGCAAGACCCTTCCTCTATATAAGGAAGTTCAT
TTCATTTGGAGAGGACACGCTG and DNA2 (SEQ ID NO: 18)
GATCCAGCGTGTCCTCTCCAAATGAAATGAACTTCCTTATATAGAGGAAG
GGTCTTGCAGATCTA is synthesized. The synthesized DNA was heated at 90 ° C. for 2 minutes, then at 60 ° C. for 1 hour, and then at room temperature (25 ° C.).
C) for 2 hours to allow annealing to form a double strand. Restriction enzyme pUC18 plasmid with Nos-ter HindI
Digest with II and BamHI. The synthesized double-stranded DNA was converted to H of pUC18 .
Insert into the indIII-BamHI site to construct a plasmid containing TATA-box and Nos-ter. The procedure described above is shown in FIG.

[Procedure amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0024

[Correction method] Change

[Correction contents]

(Gene transfer by electroporation) The protoplast obtained above was electroporated to a concentration of 2.5 × 10 ⁶ cells / mL in an electroporation buffer.
(5 mM MES pH 5.8, 70 mM KCl, 0.3 M mannitol). PGAL4-LUC reporter gene constructed in an electroporation cuvette (Gene Pulser cuvette 0.4 cm elecrode, manufactured by Bio-Rad) and pGALDB-ERF3 as effector plasmid or its delation series (pGALDB-1 / 25ERF3 to pGALDB-204)
-225ERF3), add 10 ug of each DNA and 1 ug of the reference gene plasmid to 100 uL of 2X electroporation buffer (10 mM MES pH5.8, 140 mM KCl, 0.6 M mannitol), and make up to 200 uL with sterile water. . Add 600 uL of the protoplast suspension to a cuvette, and use an electroporator (Genepulser II Electroporation System).
DNA is introduced under the conditions of 600 V and 25 mF using Stem Bio-Rad. After introduction, the protoplasts were collected from the cuvette by centrifugation at 1,000 rpm for 5 minutes, and 5 mL
The protoplasts were resuspended in MS medium containing 0.4 M mannitol, allowed to stand in the dark at 26 ° C. for 6 hours, and then the activity of the reporter gene was measured.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 5/10 C12R 1:91) // C12P 21/02) (C12N 15/09 ZNA C12N 15/00 ZNAA C12R 1:91) 5/00 C (C12N 5/10 C12R 1:91) C12R 1:91) F term (reference) 4B024 AA08 AA11 AA20 BA80 CA04 DA01 EA04 GA14 GA17 GA21 HA13 4B064 AG01 CA11 CA19 CC01 CC24 DA11 4B065 AA89X AA89Y AB01 AC14 AC16 AC20 BA03 BA10 BA16 BB01 BC01 BC03 BD50 CA24 CA46 CA53 4H045 AA10 AA20 BA10 CA30 EA05 EA50 FA74

Claims (6)

[Claims] 1. A peptide derived from a higher plant having a motif of aspartic acid-leucine-asparagine and having a function of suppressing gene transcription. 2. (a) a peptide having the amino acid sequence represented by SEQ ID NO: 1, or (b) an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence (a). The peptide according to claim 1, which is a peptide. 3. (a) a peptide having the amino acid sequence represented by SEQ ID NO: 2, or (b) an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence (a). The peptide according to claim 1, which is a peptide. A gene encoding the peptide according to claim 2 or 3. 5. A recombinant vector containing the gene according to claim 4. 6. A transformant containing the recombinant vector according to claim 5.