JP3407033B2 - Peptides having the function of suppressing gene transcription - Google Patents

Description

Detailed Description of the Invention

[0001]

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

[0002]

BACKGROUND OF THE INVENTION The protein factor ERF (Ethy, which binds to cis-regulatory elements in response to the plant hormone ethylene)
lene Responsive Element binding Factor) is the ERF
It is a plant-specific transcription factor that has a DNA-binding domain named domain. Recent studies have revealed that the genes encoding the ERF proteins make up the multigene family. To date, cDNAs encoding protein factors having an ERF domain have been clarified from tobacco and Arabidopsis plants, but the present inventors have further performed functional analysis on cDNAs of tobacco, rice, and Arabidopsis thaliana to show intracellular Reveal the domain that acts as a repressor in
The present invention has been completed.

[0003]

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

[0004]

The present inventors have found that tobacco,
As a result of functional analysis of cDNA of rice and Arabidopsis thaliana, it was discovered that a region having a motif of aspartic acid-leucine-asparagine (DLN) in the carboxy-terminal region of ERF factor has a function of suppressing gene transcription. The present invention has been completed. D like this
Examples of the peptide having an LN motif and having a function of suppressing gene transcription include, for example, peptides contained in ERF3 derived from tobacco; AtERF3 derived from Arabidopsis thaliana.
(SEQ ID NO: 1), AtERF4, AtERF7 and At
Peptide contained in ERF8; osERF3 derived from rice
Examples include peptides included in.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION
A protein coding region was excised from the cDNA encoding the RF factor, ligated to the 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. Construct an effector plasmid. This was investigated by measuring the activity of the luciferase gene, which is a reporter gene, by introducing it into the tobacco cultured cells or Arabidopsis thaliana leaves by the electroporation method at the same time as the reporter gene consisting of the luciferase gene having the GAL4 protein-binding site bound to the promoter region. It was

[0006] Next, in order to identify a repressor domain, which is a region having a repressor function existing in the repressor factor, the ERF factor was determined by using the deletion analysis method, which is a method of deleting the coding region of each gene. We investigated in which region of the protein the repressor domain resides.

[0007]

[Example] Arabidopsis (Japanese name Arabidopsis) AtER
Isolation of F3 gene (preparation of probe) A plasmid cloned from the cDNA of tobacco ERF3 gene whose nucleotide sequence has been determined.
pERF3 was digested with restriction enzymes EcoRI and NotI, and a 950 bp DNA fragment containing ERF3 cDNA was isolated by agarose gel electrophoresis. After denaturing about 50 ng of this DNA fragment at 100 ° C for 10 minutes, DN
A labeling kit (Amersham Pharmacia Ready
-To-GoDNA Labeling Beads) and 5uL of alpha ³² P-dCT
A probe was prepared by labeling with P (Amersham Pharmacia AA0005).

(Preparation and screening of cDNA) Leaves are collected from Arabidopsis plants and ground into powder using a mixer in liquid nitrogen. 20 mL of RNA extraction buffer (8M guanidine hydrochloride,
20 mM disodium ethylenediaminetetraacetic acid (EDT
A), 20 mM 2- (N-morpholino) ethanesulfonic acid
(MES), 50 mM mercaptoethanol) and 8 mL TE
Phenol saturated with a solution (10 mM Tris-Cl pH8.0, 1 mM EDTA) and 2 mL of chloroform are added, stirred well, and centrifuged (10000 g) to collect the supernatant. 0.2 volumes of 1M acetic acid and 0.7 volumes of ethanol are added to the supernatant, and the mixture is left standing at -20 ° C and then centrifuged (10000g). Dissolve the precipitate in 2 mL of TE solution, add 500 mL of 10M lithium chloride solution, and
Let stand for 2 hours and centrifuge. The precipitate was washed with 70% alcohol and then air-dried to obtain a total RNA preparation. This total RAN was converted to total RNA using Poly A Tract System 1000 manufactured by Promega.
Only mRNA was purified from. Double-stranded cDNA is synthesized using this mRNA as a template and the Pharmacia cDNA Direction Cloning Kit. These are EcoRI-NotI of the lambda gt11 expression vector according to the Pharmacia protocol.
After incorporating into the restriction enzyme site, the library was completed using the lambda packaging kit (Pharmacia).

Lambda phage incorporating the prepared Arabidopsis cDNA library was plated (10 cm x 14 cm)
In advance, the capacity to obtain about 40,000 plaques
Suspend and infect Escherichia coli 1090 strain that had been suspended with 0.5 ccmL of 10 mM magnesium sulfate solution, 50 mg / L sodium ampicillate LB agar medium (10 g tryptone, 10 g per 1 L, which is a drug for selection) Develop in sodium chloride, 5g yeast extract, 15g agar) and incubate at 37 ℃. When the plaque is about 2 mm, it is transferred to a nitrocellulose membrane (S & S) and the denaturing solution (0.2 M NaOH,
After allowing to stand for 5 minutes on a filter paper (Whatman 3MM) soaked in 1.5M NaCl) and for 5 minutes on a filter paper soaked in a neutralizing solution (0.4M Tris-HCl, pH7.5, 2xSSC), add 5x to a 2xSSC solution. Soak for 1 minute and air dry on filter paper for 20 minutes.

The plaque DNA is fixed on a nitrocellulose membrane by drying at 80 ° C. for 120 minutes. Soak in 2mL of hybridization buffer (6xSSC, 0.05% skim milk) for each nitrocellulose membrane (10cmx14cm) 60
After incubating at 0 ° C. for 2 hours, it was heated at 100 ° C. for 10 minutes, replaced with a new buffer containing ERF3 probe denatured by rapid cooling, and incubated at 60 ° C. for 12 hours. Then, each filter has 5 mL of 2% SDS solution containing 0.1% SDS (sodium dodecyl sulfate) for 15 minutes, containing 0.1% SDS.
It was washed with 0.5xSSC for 15 minutes and 0.2xSSC containing 0.1% SDS for 15 minutes, and exposed to an X-ray film. ERF3 probe-bound plaques were isolated from agar plates and 1 mL phage buffer (10 mM Tris-HCl pH7.5, 10 mM MgSO4)
After the suspension, the phages were collected and the above screening was repeated 3 times to obtain a single plaque. This plaque was again infected with E. coli, amplified, DNA was extracted from the phage, and the cDNA region was excised using restriction enzymes EcoRI and NotI. This DNA fragment was inserted into the restriction enzyme EcoRI-NotI site of the plasmid pT7D3 (Pharmacia), and the plasmid nucleotide sequence was analyzed. AtERF3 shown in SEQ ID NO: 1
The plasmid having the full-length sequence of the cDNA of was named pAtERF3.

Identification of AtERF3 repression domain Construction of effector plasmid (effector plasmid containing the full length AtERF3 pGAL4DB-At
Construction of ERF3: Figure 1) Clontech (Clontech, U
(SA) plasmid pBI221 is cleaved with restriction enzymes XhoI and SstI, blunt-ended with T4 polymerase, the GUS gene is removed by agarose gel electrophoresis, and transcription of cauliflower mosaic virus 35S promoter (CaMV35S) and nopaline synthase genes is performed. End area (Nos terminator,
35S-Nos plasmid fragment DNA containing the following Nos-ter) was obtained.
Restriction enzyme HindII for pAS2-1 vector manufactured by Clontech
Digested with I, DNA binding region of yeast GAL4 protein (1-1
A 748 bp DNA fragment (hereinafter referred to as GAL4DBD) encoding 47 amino acid residues) was isolated by agarose gel electrophoresis and blunt-ended with T4 DNA polymerase.
The DNA fragment containing this GAL4DBD was used to convert the 35S-Nos DNA
The p35S-GAL4DBD vector was constructed by inserting into the blunt end site between the 35S promoter and the Nos terminator and selecting the one in which the ORF of the DNA binding region of the yeast GAL4 protein was aligned in the forward direction with respect to the 35S promoter.

AtERF3 cDNA plasmids pAtERF3 and GAL4DBD
5 terminal upper primer primer1 (SEQ ID NO: 3: bound to AtERF3 nucleotide sequence 1-20) GATGAGGAGAGGAAGAGGCTC and restriction enzyme SalI designed to match the reading frame
3rd lower primer having a site primer2 (SEQ ID NO: 4: bound to AtERF3 nucleotide sequence 651-678) GTCGACTTAGAGACGTA
Using GATCGGTACAGTGAAG, AtERF3 whole protein coding region (SEQ ID NO: 1: AtERF3 nucleotide sequence 1-678; amino acid sequence 1
-225) was amplified by the PCR method to obtain a DNA fragment. The conditions of PCR reaction are denaturation reaction 94 ° C for 1 minute, annealing reaction 47 ° C 2
The extension reaction was carried out for 25 cycles by setting the extension reaction at 74 ° C. for 1 minute as one cycle. Hereinafter, all PCR reactions were performed under the same conditions. The obtained DNA fragment was digested with the restriction enzyme SalI, and then the target DNA fragment was isolated by agarose gel electrophoresis.
The DNA fragment encoding this AtERF3 was digested with the restriction enzymes SmaI and Sal.
The effector plasmid pGALDB-AtERF3 was constructed by incorporating it into the 35S-GAL4DBD plasmid previously digested with I. The procedure for constructing this effector plasmid pGALDB-AtERF3 is shown in FIG.

(Construction of Effector Plasmid pGAL-166 / 225AtERF3 Containing AtERF3 Amino Acids 166/225) pAtERF3 plasmid and the 5th terminal upper primer designed to match the reading frame with GAL4DBD primer3
(SEQ ID NO: 5: binding site AtERF3 nucleotide sequence 495-518) TCCGG
Lower terminal primer primer2 with AGGATTGTCACAGCGATTG and restriction enzyme SalI site (SEQ ID NO: 4: binding site AtERF
3 nucleotide sequence 651-678) GTCGATTAGAGACGTAGATCGGTACAGTGAA
Using G, a DNA fragment containing the region of nucleotide sequence 496-678 corresponding to the amino acid sequence 166/225 coding region of AtERF3 was obtained by the PCR method. This DNA fragment was digested with the restriction enzyme SalI, and the target DNA fragment was isolated by agarose electrophoresis. D coding for this AtERF3 amino acid sequence 166/225
The NA fragment (DNA region 496-678) was incorporated into the 35S-GAL4DBD plasmid that had been previously digested with the restriction enzymes SmaI and SalI to construct the effector plasmid pGAL-166 / 225AtERF3.

(Construction of Reporter Gene: FIG. 2 and FIG. 3) The plasmid pUC18 is digested with restriction enzymes EcoRI and SstI.
pBI221 (Clontech) with restriction enzymes EcoRI and Sst
Digested with I, Nos-ter (nopaline synthase terminator)
It is isolated by agarose gel electrophoresis which inserts a 270 bp DNA fragment containing the region. The resulting fragment is the restriction enzyme EcoR
EcoRI-Ss of plasmid pUC18 digested with I and SstI
Insert at tI site. Cauliflower mosaic virus 35S
Complementary strand DNA1 containing the promoter TATA box (SEQ ID NO: 6) AGCTTAGATCTGCAAGACCCTTCCTCTATATAAGGAAGTTCA
TTTCATTTGGAGAGGACACGCTG and DNA2 (SEQ ID NO: 7) GATC
CAGCGTGTCCTCTCCAAATGAAATGAACTTCCTTATATAGAGGAAGGGTC
Synthesize TTGCAGATCTA. The synthesized DNA is heated at 90 ° C. for 2 minutes, then at 60 ° C. for 1 hour, and then left standing at room temperature (25 ° C.) for 2 hours to anneal to form a double strand.
The pUC18 plasmid containing Nos-ter was digested with restriction enzymes HindIII and B
Digest with amHI. Synthesized double-stranded DNA was used for HindIII of pUC18
-Insert at the BamHI site to construct a plasmid containing TATA-box and Nos-ter. The above procedure is shown in FIG.

This plasmid was digested with the restriction enzyme SstI,
Blunt end with T4 DNA polymerase. Plasmid vector PGV-CS2 (manufactured by Toyo Ink Co.) containing the firefly luciferase gene (LUC) was used as 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, which has 5 copies of A-binding sequence)
Digested with restriction enzymes SmaI and XbaI and blunt-ended with T4 DNA polymerase.
The DNA fragment containing the DNA binding sequence of 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. A blunt-ended 5 copies DNA fragment containing the DNA-binding sequence of GAL4 protein was inserted into this site, and those having the DNA-binding sequence of GAL4 protein in the forward direction were selected from the obtained plasmids. -Builded LUC. (See Figure 3)

(Construction of Reference Gene) A cassette vector pRL-null manufactured by Promega which has a luciferase gene derived from Renilla is cut with restriction enzymes NheI and XbaI and blunt-ended with T4 DNA polymerase, and then agarose. Purify a 948 bp DNA fragment containing the Renilla luciferase gene by gel electrophoresis. This DNA fragment was used in the construction of the effector plasmid.
Insert in the region where the US gene was. From the obtained plasmids, those in which the Renilla luciferase gene is oriented in the forward direction are selected (construction of pPTRL).

(Method for measuring the activity of reporter gene) The reporter gene and effector plasmid were introduced into protoplasts of cultured tobacco cells by electroporation, and the effect of the effector was examined by measuring the activity of the reporter gene. (Protoplast preparation method) 100 mL of MS medium (mixed salts for Murashige-Skoog medium, Nippon Pharmaceutical Co., Ltd., 3% sucrose, 0.2 g / L KH2PO4, 0.2 g / L m-inositol, 2 mg / L g
lycine, 1 mg / L thiamine hydrochloride, 0.2 mg / L 2, 4-D, pH
To 5.8) cultured tobacco cells for 7 days
By adding 3 ml of BY-2 preculture medium and culturing the cells in the dark at 26 ° C for 3 days, the cells were made of a metal mesh (openness of 125 mm,
(Tokyo Screen) Collect by filtration and wash the cells with MS medium containing 0.4M mannitol. Washed cells 25
Suspend in 1 mL of MS medium containing 0.4 M mannitol.
Leave at room temperature for 0 minutes Centrifuge at 3,000 rpm for 1 minute to collect cells. The cells were resuspended in 20 mL of MS medium containing 1% cellulase (Onozuka RS,) and 0.1% pectolyase Y-23 (manufactured by Seishin) and spun at 60 rpm in the dark for 90 minutes at 26 ° C. Digest the cell wall with shaking. Then, centrifuge at 1,000 rpm for 5 minutes to collect protoplasts.

(Gene Transfer by Electroporation) The protoplasts obtained above were used at a concentration of 2.5 × 10 ⁶ cells /
Electroporation buffer (5 m
Resuspend in M MES pH5.8, 70 mM KCl, 0.3 M mannitol). PGAL4-LUC reporter gene constructed in a cuvette for electroporation (Gene Pulcer cuvette 0.4 cm elecrode, manufactured by Bio-Rad) and pGALDB-AtERF3 as an effector plasmid or its deletion series (pGALDB-1 / 25AtERF3 to pGALDB-204-225)
AtERF3) DNA was added at 10 ug and reference gene plasmid 1 ug at 100 uL of 2X electroporation buffer (10 mM MES pH5.8, 140 mM KCl, 0.6 M mannitol), and the total volume was made up to 200 uL with sterilized water. To do. Add 600 uL of protoplast suspension to the cuvette and place it in an electroporator (Genepulser II Electroporation).
System Bio-Rad) is used to introduce DNA under the conditions of 600 V and 25 mF. After the introduction, collect the protoplasts from the cuvette by centrifugation at 1,000 rpm for 5 minutes, and collect 5 mL.
The protoplasts were resuspended in MS medium containing 0.4 M mannitol, and the reporter gene activity was measured after standing at 26 ° C. for 6 hours in the dark.

(Measurement of luciferase activity) 1 mL of the protoplast suspension, which had been allowed to stand for 6 hours, was sampled at 2,000 rpm.
After collecting the protoplasts by centrifugation for 3 minutes at
LuciferaseTM Reporter Assay System (Promega)
Suspend in 50 uL of Passive Lysis Buffer attached to. After crushing the protoplasts, centrifuge and collect the supernatant. Use 5 uL of this cell extract for Dual-Lucifer
Luciferase activity was measured using aseTMReporter Assay System (Promega) and luminescence reader (BLR-201, Aloka). For the measurement of firefly luciferase and Renilla luciferase activities, luminescence for 10 seconds was counted in integration mode according to the instruction of the measurement kit. 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 obtained as a measurement value. When the effector plasmid was not introduced, the relative value was set to 100, and the effect of the effector was investigated by the fluctuation of 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-AtERF3 effector plasmid was introduced was reduced, pGALDB
-AtERF3 has the effect of suppressing the activity of the reporter gene (repressor function). Less than,
When the relative activity value of the reporter is 100 or less by measuring the activity value of the reporter, the introduced effector has a repressor function, so the activity of the reporter is measured by determining which effector functions as the repressor. Investigated by doing.

(Identification of repressor domain) In order to analyze the function involved in the transcriptional regulation of Arabidopsis AtERF3 gene, reporter genes pGAL4-LUC and pGALDB-AtERF3 were introduced into protoplasts prepared from cultured tobacco cells by electroporation. , The activity of the reporter gene was investigated. As a result, the pGAL-AtERF3 effector reduced the activity of the reporter gene to 80% as compared with the case of using the reporter gene alone without introducing the effector (control). AtERF3 performed as a control experiment
P35S-GALDBD, which does not contain the coding region of Escherichia coli, did not affect the activity of the reporter gene. This is AtER
It shows that F3 functions as a repressor that suppresses transcription.

Next, it was examined whether pGAL-166 / 225AtERF3, an effector plasmid having a DAN fragment in which the protein coding region of the AtERF3 gene was deleted, has a function of suppressing the activity of the reporter gene. pGAL-166 / 225AtER
The F3 effector plasmid was introduced into tobacco cultured cells together with the reporter gene, and the activity of the reporter gene was measured.As a result, pGAL-166 / 225AtERF3 effector was identified as pGALDB.
Similar to the case of introducing -AtERF3, it was shown that there is a repressor function that suppresses the activity of the reporter gene to about 50% as compared with the control. (Fig. 4B). From this result, the region having the repressor function of AtERF3 (repression domain) is the amino acid sequence of AtERF3, 166/225.
It was revealed that it exists in (Fig. 4B). The amino acid sequence of this 60 amino acid region is shown in SEQ ID NO: 2.

The peptide having the function of suppressing the transcription of the gene of the present invention is fused with a DNA-binding protein that specifically binds to the transcriptional regulatory region of the oncogene, and expressed in the cell to express the oncogene. The expression can be efficiently suppressed. In addition, the repressor function was nonspecific to the gene when examined, but it is necessary to bind to DNA. Therefore, by fusion with a DNA binding domain that binds to specific DNA, the repressor function may be gene-specific or non-specific. It is possible to suppress the transfer. This makes it possible, for example, to control the expression of a gene encoding an enzyme of the pigment metabolism system, and to create a flower having petals of different colors, which has never been obtained before. In addition, by suppressing the expression of the protein that becomes an allergen, it becomes possible to produce foods that are low in allergen.

[0023]

[Sequence list] SEQUENCE LISTING    <110> Secretary of Agency of Industrial Science and Technology    <120> Novel repression domain of plant specific transcription factor    <130>    <160> 7    <210> 1 <211> 678 <212> DNA <213> Arabidopsis thaliana    <400> 1 atg agg aga gga aga ggc tct tcc gcc gtc gcc gga cct acc gtc gtt 48 Met Arg Arg Gly Arg Gly Ser Ser Ala Val Ala Gly Pro Thr Val Val                 5 10 15    gcc gcc atc aac gga tct gta aaa gaa atc aga ttc aga ggc gta agg 96 Ala Ala Ile Asn Gly Ser Val Lys Glu Ile Arg Phe Arg Gly Val Arg             20 25 30    aag aga cct tgg gga cga ttc gca gct gag atc cgt gat cca tgg aaa 144 Lys Arg Pro Trp Gly Arg Phe Ala Ala Glu Ile Arg Asp Pro Trp Lys         35 40 45    aaa gct cgt gtt tgg tta ggt act ttc gat tcc gcc gaa gaa gct gct 192 Lys Ala Arg Val Trp Leu Gly Thr Phe Asp Ser Ala Glu Glu Ala Ala      50 55 60    cgc gct tac gac tcc gcc gct cgt aac ctc cgt ggt cct aaa gcc aaa 240 Arg Ala Tyr Asp Ser Ala Ala Arg Asn Leu Arg Gly Pro Lys Ala Lys 65 70 75 80    act aat ttc ccc atc gat tct tct tct cct cct cct cct aat ctc cga 288 Thr Asn Phe Pro Ile Asp Ser Ser Ser Pro Pro Pro Pro Asn Leu Arg                 85 90 95    ttt aat cag att cgt aat caa aat caa aac caa gtc gat ccg ttt atg 336 Phe Asn Gln Ile Arg Asn Gln Asn Gln Asn Gln Val Asp Pro Phe Met             100 105 110    gac cac cgg tta ttc acc gac cat caa caa cag ttc ccg att gtt aac 384 Asp His Arg Leu Phe Thr Asp His Gln Gln Gln Phe Pro Ile Val Asn         115 120 125    cgg cct act agt agc agc atg agc agc acc gtt gaa tcg ttt agc gga 432 Arg Pro Thr Ser Ser Ser Met Ser Ser Thr Val Glu Ser Phe Ser Gly     130 135 140    ccc aga cct acg acg atg aaa ccg gcc acg acg aag aga tat cct aga 480 Pro Arg Pro Thr Thr Met Lys Pro Ala Thr Thr Lys Arg Tyr Pro Arg 145 150 155 160    act cca ccg gtt gtt ccg gag gat tgt cac agc gat tgc gat tcg tcg 528 Thr Pro Pro Val Val Pro Glu Asp Cys His Ser Asp Cys Asp Ser Ser                 165 170 175    tcg tct gta atc gac gac gac gac gat atc gca tcg tct tca cgg cga 576 Ser Ser Val Ile Asp Asp Asp Asp Asp Ile Ala Ser Ser Ser Arg Arg             180 185 190    cgg aat ccg ccg ttt caa ttc gat ctt aat ttt cca ccg ttg gat tgt 624 Arg Asn Pro Pro Phe Gln Phe Asp Leu Asn Phe Pro Pro Leu Asp Cys         195 200 205    gtt gac ttg ttc aat ggc gct gat gat ctt cac tgt acc gat cta cgt 672 Val Asp Leu Phe Asn Gly Ala Asp Asp Leu His Cys Thr Asp Leu Arg     210 215 220    ctc taa 678 Leu 225    <210> 2 <211> 60 <212> RPT <213> Arabidopsis thaliana    <400> 2 Pro Glu Asp Cys His Ser Asp Cys Asp Ser Ser Ser Ser Val Ile Asp                     5 10 15 Asp Asp Asp Asp Ile Ala Ser Ser Ser Arg Arg Arg Asn Pro Pro Phe                20 25 30 Gln Phe Asp Leu Asn Phe Pro Pro Leu Asp Cys Val Asp Leu Phe Asn         35 40 45 Gly Ala Asp Asp Leu His Cys Thr Asp Leu Arg Leu         50 55 60    <210> 3 <211> 21 <212> DNA <213> Artificial Sequence    <223> Description of Artificial Sequence: Synthetic primer DNA    <400> 3 gatgaggaga ggaagaggct c    <210> 4 <211> 33 <212> DNA <213> Artificial Sequence    <223> Description of Artificial Sequence: Synthetic primer DNA    <400> 4 gtcgacttag agacgtagat cggtacagtg aag    <210> 5 <211> 24 <212> DNA <213> Artificial Sequence    <223> Description of Artificial Sequence: Synthetic primer DNA    <400> 5 tccggaggat tgtcacagcg attg    <210> 6 <211> 65 <212> DNA <213> Artificial Sequence    <223> Description of Artificial Sequence: Synthetic primer DNA    <400> 6 agcttagatc tgcaagaccc ttcctctata taaggaagtt catttcattt ggagaggaca         10 20 30 40 50 60 cgctg     65    <210> 7 <211> <212> DNA <213> Artificial Sequence    <223> Description of Artificial Sequence: Synthetic primer DNA    <400> 7 gatccagcgt gtcctctcca aatgaaatga acttccttat atagaggaag ggtcttgcag         10 20 30 40 50 60 atcta     65

[Brief description of drawings]

FIG. 1 shows the procedure for constructing pGAL4DB-AtERF3 from plasmid pBI221.

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

FIG. 3 is a diagram showing the latter half of the procedure for constructing the reporter gene GAL4-LUC subsequent to FIG.

FIG. 4A is a diagram showing a reporter gene and an effector plasmid. In the figure, 5XGAL4: GAL4 transcription factor
DNA binding sequence, TATA: CaMV 35S promoter region containing 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 is a figure which shows the influence which the deletion of AtERF3 and AtERF3 has on the activity (Relative Activity) of a reporter gene. In the figure, the number on the left (166/22
5) shows the amino acid region of AtERF3. The box in the middle 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 an effector plasmid having the region on the left is introduced. The activity of the reporter gene when no effector was added was set to 100. It has been shown that the AtERF3 and AtERF3 deletion effectors having the amino acid sequence 166/225 reduce the activity of the reporter gene to 50% and have the effect of suppressing transcription.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁷ Identification code FI C12N 5/10 C12N 5/00 A (56) References The Plant Cell, 1995, Vol. 7, p. 173-182 Biochem. J. , 1999, Vol. 339, p. 111-117 The Journal of Biological Chemistry, 1999, Vol. 274, No. 41, p. 29500-259504 Mol Gen Genet, 1997, Vol. 253, No. 5, p. 553-561 (58) Fields surveyed (Int.Cl. ⁷ , DB name) C12N 15/09 C12N 15/29 C07K 14/415 BIOSIS / WPI (DIALOG) PubMed SwissProt / PIR / GeneS eq GenBank / EMBL / DDBJ / GeneSeq

Claims (5)

(57) [Claims] 1. A peptide comprising (a) the amino acid sequence represented by SEQ ID NO: 1, or (b) an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence (a). A peptide having the function of suppressing gene transcription. 2. A peptide comprising (a) the amino acid sequence represented by SEQ ID NO: 2, or (b) an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence (a). A peptide having the function of suppressing gene transcription. 3. A gene encoding the peptide according to claim 1 or 2. 4. A recombinant vector containing the gene according to claim 3. 5. A transformant containing the recombinant vector according to claim 4.