JPH10248570A - Promoter for metallothionein gene - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject promoter useful for expressing a heterogene in transgenic plants, as a promoter of a metallothionein of paddy rice plant. SOLUTION: This promoter for metallothionein gene containing a base sequence of the formula is obtained by a method for cloning metallothionein genes and then carrying out an inverse PCR. The promoter can control not only expression of a homogeneous gene but also that of a heterogene. The promoter is expressed in immature embryo, foliage dividing tissue cell or aleurone layer cell.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a promoter of a metallothionein gene which is a metal binding protein of rice, a recombinant expression vector containing the promoter and a target gene, and a transformant transformed with the expression vector.

[0002]

2. Description of the Related Art Metallothionein is a protein whose synthesis is induced by binding to heavy metal ions such as copper, zinc and cadmium. Metallothionein is widely present in the living world, is involved in the transport and storage of zinc and copper, which are essential for living organisms, and plays a role in maintaining homeostasis in living organisms. It is also known that it has a detoxifying effect by binding to harmful metals such as cadmium and mercury.

The metallothionein gene is known to be expressed in a tissue-specific manner and has a high expression level, and is expected to be effective as a promoter material. About the base sequence of the metallothionein gene, some plants,
For example, wheat (Eur. J. Biochem. 209: 971-976, 199
2), corn (FEBS letters 290: 103-106, 199
It is already known in 1). However, detailed analysis of the promoter sequence has not yet been performed. In addition, it has been suggested that metallothionein protein is involved in detoxification of heavy metals and redistribution of essential metals in vivo, but the function of plants has not been fully elucidated. Further, it is expected that a promoter of the metallothionein gene exists in addition to the above-mentioned plants.

[0004]

An object of the present invention is to provide a metallothionein gene promoter, a recombinant expression vector containing the promoter and the target gene, and a transformant transformed with the recombinant vector. I do.

[0005]

Means for Solving the Problems As a result of intensive studies based on the above problems, the present inventors succeeded in isolating the promoter of the rice metallothionein gene, and have completed the present invention. That is, the present invention relates to a metallothionein gene promoter comprising the base sequence represented by SEQ ID NO: 1 or a base sequence having one or more bases deleted, substituted or inserted.

Further, the present invention is a recombinant expression vector containing the promoter and a target gene for expression. Further, the present invention is a transformant transformed by the above-mentioned recombinant expression vector. Hereinafter, the present invention will be described in detail.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION One of the techniques for isolating the promoter of the present invention is a method of performing so-called inverse PCR after cloning a rice metallothionein gene.

(1) Cloning of metallothionein gene In the present invention, the metallothionein gene is cloned according to a known method. That is, mRNA is extracted from suspension culture cells of rice subjected to salt stress treatment, and cloned as cDNA into a λ phage vector to prepare a cDNA library. Next, a metallothionein gene can be cloned by selecting and amplifying a desired clone from the library.

The basic method for cloning is described in "Molecular Cloning" by T. Maniatis et al.
Spring Harbor Laboratory) (1982).

First, the cDNA of the metallothionein gene
Perform library preparation. After 4 days from the subculture, the rice suspension cultured cells were subjected to salt stress by culturing them in 2% NaCl for 24 hours. Then, total RNA was extracted and oligo (dT) -Late
x Purify Poly (A) ⁺ RNA with particles. Further, cDNA is synthesized using reverse transcriptase to prepare a cDNA phage library. The synthesis of cDNA is carried out, for example, by ZAP-
It can be performed using a cDNA synthesis kit (Stratagene) or the like. The cDNA phage library obtained as described above is converted to a plasmid library.

As a method for converting a phage library into a plasmid library, a known method such as I
n vitro excision method (Stratagene) and the like. That is, the phage library is transformed into Escherichia coli which is a host holding the plasmid,
A cDNA plasmid library is prepared by inoculating an agar plate containing ampicillin and forming a colony. Plasmid DNA is prepared from an arbitrary colony, treated with alkali denaturation, ethanol precipitation, and the like, dried in vacuo, and used as a template for sequence analysis.

Next, the base sequence of the metallothionein gene is determined. Analysis of the nucleotide sequence is performed by a known method.
Alternatively, it is performed using a commercially available automatic base sequencer. The deciphered nucleotide sequence is sent to NCBI (National Center for Biotechnol
ogy Information) and connected to the Blastn Server to perform a primary search with a known sequence. Further, the base sequence with homology recognized at the DNA level is converted into an amino acid sequence, and commercially available base sequence or amino acid sequence search software (for example, “ DNASI
S "," GENETYX "etc.).

(2) Isolation of promoter The promoter of the present invention is obtained by grinding rice tissue in a urea extraction buffer, extracting with phenol / chloroform, and then preparing genomic DNA by ethanol precipitation.
It is isolated by performing so-called inverse PCR based on the sequence of a metallothionein cDNA clone obtained from a rice (Oryza sativa) salt stress cDNA library search.

[0014] Inverse PCR is a technique for amplifying an unknown sequence adjacent to a known region. In the present invention, a primer for PCR is designed based on a metallothionein gene (cDNA sequence) having a known sequence, and a promoter having an unknown sequence is amplified.

First, genomic DNA is cleaved with a restriction enzyme so as to include a desired promoter region.
In this case, the cleavage site by the restriction enzyme is located at one site within the 5 'region of the exon including the promoter region, and at the other site within the exon (FIG. 1 (1)). As the restriction enzyme, those having 6 or more bases such as PstI, BamHI, HindIII and the like are preferable.

Next, the restriction enzyme digested fragments are self-ligated (FIGS. 1 (2) and (3)), and PCR is performed. Primers (primers 1 and 2) for PCR are designed and synthesized based on the sequence of exons, and are arranged so as to face each other (FIG. 1 (4)). After performing the PCR, it is linked to an appropriate vector and cloned (FIGS. 1 (5) and (6)).
Cloning is so-called TA cloning (for example, TA cloning kit (Invitroge
n)) is preferred. In addition, T7 and M13RV primers and the like can be used as primers for determining the nucleotide sequence.

SEQ ID NO: 1 contains the promoter of the present invention
The base sequence of DNA is exemplified, but is not limited to this sequence as long as the base sequence has essentially promoter activity (transcription activity). Mutation of multiple base sequences by deletion, insertion, substitution, addition, etc. Is possible. The mutation of the nucleotide sequence can be induced by a known point mutagenesis method or the like. Further, once the nucleotide sequence represented by SEQ ID NO: 1 is determined, the promoter of the present invention can be obtained by chemical synthesis or by hybridization using a DNA fragment having the nucleotide sequence as a probe.

Furthermore, in the present invention, in addition to the above-mentioned inverse PCR, the metallothionein gene is cut in the direction of the promoter from the restriction enzyme site of the exon (coding region) by an exo-type DNA degrading enzyme, and the clones cut to an appropriate position Alternatively, a semi-synthetic promoter can be prepared by chemically synthesizing a part of the promoter region and utilizing the cloned genomic DNA and restriction enzyme sites.

(3) Production of Recombinant Expression Vector In the present invention, the promoter of the present invention is ligated to the upstream of the coding sequence of the gene to be expressed and ligated to an appropriate vector, whereby the expression vector is prepared. Can be made.

Since the promoter of the present invention can control the expression of not only the same species but also a heterologous gene, the expression vector of the present invention is essentially controlled by the promoter of the present invention as the gene to be expressed. Or a so-called chimeric gene construct incorporating a heterologous gene (foreign gene). Further, other promoters such as a cold-inducible gene can be further incorporated. These regulators include intron sequences and the like that can significantly enhance the expression of heterologous genes in monocotyledonous plants. As the heterologous gene among the target genes, for example, a bialaphos resistance gene (MurakamiT, et al., M. et al.
GG 205: 42-50 (1986)), a hygromycin resistance gene conferring antibiotic resistance (Gritz L. and Davies J.
Gene 25: 179-188 (1983)) and the like, but are not limited thereto.

To confirm the promoter activity of the present invention in a chimeric gene, β-glucuronidase (GUS)
A chimeric gene (gene expression cassette) linked to a reporter gene such as a gene is constructed (FIG. 2) and introduced into rice cells using a particle gun method. The expression of the gene is confirmed by GUS histochemical staining, GUS quantification using a fluorescent substrate (4-MUG), or the like.

(4) Preparation of Transformant The transformant of the present invention comprises the recombinant vector of the present invention,
It can be obtained by introducing into a host (for example, Escherichia coli) compatible with the expression vector used in producing the recombinant vector, or by introducing the recombinant vector into a plant cell or a seed.

When a bacterium such as Escherichia coli is used as a host, the recombinant vector of the present invention may be capable of autonomous replication in the host and may contain the gene expression cassette of the present invention and a transcription termination sequence. preferable. Examples of the vector include pBR322 and pUC-based plasmids, and examples of the method of introducing the recombinant vector into bacteria include a method using calcium ions, an electroporation method, and a lipofection method.

Transformation methods using plant cells or seeds as a host include, for example, electroporation into protoplasts, Agrobacterium infection using a binary vector, and particle gun introduction. In addition, the promoter of the present invention is expressed mainly in immature embryos, meristem meristem cells, or aleurone layer cells among rice plants.

[0025]

[Example 1]

(1) Isolation of Gene from cDNA Library Salt stress was applied by culturing suspension culture cells 4 days after subculture of rice (variety: Yamaboushi) in 2% NaCl for 24 hours. Next, total RNA was extracted and oligo (dT) -Latex
Poly (A) ⁺ RNA was purified by the particles. Furthermore, cDNA was synthesized using reverse transcriptase to prepare a ZAP-cDNA phage library (ZAP-cDNA synthesis kit; using Stratagene).

A cDNA phage library prepared from suspension-cultured rice cells to which salt stress has been added was used as a Stratagene
Using helper phage from λZAP II vector
The plasmid was introduced into Escherichia coli by the in vitro excision method. That is, Escherichia coli was simultaneously infected with the recombinant λZAP and a helper phage, cultured at 37 ° C, and the supernatant was heated at 70 ° C to recover the plasmid and introduced into Escherichia coli.

The transformed Escherichia coli was inoculated on a MacConkey agar plate containing ampicillin and cultured for 1 day to form a colony, which was used as a cDNA plasmid library. Then, alkaline SDS from any E. coli colony
Plasmid DNA was prepared by the method. That is, 0.2 NN
aOH: Alkaline denaturation was performed with a 1% SDS solution for 5 minutes, 5M ammonium acetate was added and mixed, and the supernatant was collected by centrifugation. Next, after the obtained supernatant was subjected to phenol / chloroform extraction, 5 M ammonium acetate was added to perform ethanol precipitation, followed by vacuum drying and use as a template for sequence analysis.

The nucleotide sequence was analyzed using an automatic fluorescence sequencer according to the Dye Terminater Cycle Sequence method of Applied Biosystems. The decoded partial nucleotide sequence can be found at NCBI (National Center for Biotechnolog
y Information) and connected to Blastn Server for primary search. Furthermore, the partial base sequence in which homology was recognized at the DNA level was converted to an amino acid sequence, and homology was confirmed using "DNASIS" or "GENETYX".

As a result, ATMT1 which is a metallothionein of Arabidopsis was selected from the salt stress cDNA library.
An SSK35 clone showing homology with the barley bZE40 gene (Plant Mol. Biol. 20: 255-266, 1992) showing expression specific to the gene and the zygotic embryo was found. Based on homology analysis, SSK35 was a novel rice metallothionein gene, and as a result of analyzing its nucleotide sequence, it contained a full-length coding sequence (SEQ ID NO: 2).

(2) Confirmation of transcripts Tissues (leaves, roots) and 1 g of callus of rice (Oryza sativa L. var. Total RNA
Was extracted. 20 μg of each sample was subjected to agarose electrophoresis, transferred to a Hybond + membrane filter, and subjected to Northern blot analysis. A DNA fragment was amplified by a PCR method using T3 (SEQ ID NO: 3) and T7 primer (SEQ ID NO: 4) with the SSK35 clone containing the full length coding sequence of the metallothionein gene as a template. PCR was performed using a PCR reaction solution (10 mM Tris-HCl (pH 8.3), 50 mM
KCl, 1.5 mM MgCl ₂ , 200 μM dNTP, 0.2 μM primer pair, 2.5 U / 100 μl Taq polymerase, 1 ng template
The reaction was carried out for 25 seconds at 94 ° C. for 20 seconds, 55 ° C. for 20 seconds and 72 ° C. for 1 minute.

The DNA concentration of the obtained amplified fragment was estimated by agarose electrophoresis, and [α- ³² P] -dCTP (A) was detected using a multiprime DNA labeling system (Amersham).
The DNA fragment was labeled with mersham) to prepare a probe. The probe was purified using a Nick column (Pharmacia). Northern blot analysis was performed according to a conventional method, and a signal was detected using BAS2000.

As a result of Northern blot analysis, it was confirmed that the SSK35 gene was also expressed in the leaf sheath, but was remarkably expressed in zygotic embryos 7 days after pollination (FIG. 6-).
A). In the callus before the induction of regeneration, the SSK35 gene was hardly expressed, but was found to be strongly expressed in the callus three weeks after induction of the regeneration (FIG. 6-B). The expression of the SSK35 gene was also observed in seeds from which the embryo was removed one day after immersion (FIG. 6-C). This suggests that gibberellin induces the expression of the SSK35 gene.

Further, the cultured cells were cultured on a solid medium at 40 ° C. for 1 hour.
As a result of a heat stress test performed by treating for a time, it was confirmed that the expression of the SSK35 gene was also induced by heat stress (FIG. 6-D).

(3) Isolation of Promoter Sequence In order to isolate the promoter sequence of the SSK35 gene, inverse PCR was applied as follows (FIG. 1). Rice (Or
Genomic DNA was extracted from 1 g of leaves of yza sativa L. var Sasanishiki) seedlings using the urea-phenol method (Cell 35: 225, 1983). Based on the sequence information of SSK35 cDNA, 1 μg of genomic DNA was digested for 12 hours with a restriction enzyme (PstI) that recognizes 6 bases and cuts at one place in the translation region.

After phenol / chloroform extraction, T4D
Self-ligation was performed with NA ligase (1 unit / μg DNA, 16 ° C., 16 hours). After phenol / chloroform extraction, it was subjected to a PCR reaction. That is, a PCR reaction was performed by synthesizing a pair of 35-mer primers facing the outside with respect to the base sequence upstream of the restriction enzyme cleavage site of SSK35 cDNA.

The base sequences of the primers used in the inverse PCR method are shown below. Primer 1: 5'-AACAAGGCGAGCTCTGGAGGAATGGAGATGGCG
GT-3 '(SEQ ID NO: 5) Primer 2: 5'-GAAGGAGATAGATGTTGTTGCTGATTGAGCTCA
AG-3 '(SEQ ID NO: 6) Using Ex Taq polymerase (TAKARA) at 94 ° C.
The reaction was carried out at (Tm value -5) ° C for 90 seconds and at 72 ° C for 2 minutes. inve
rse PCR products are subjected to agarose gel electrophoresis, agarose fragments containing amplified DNA fragments are cut out, and GENECLEAN
Purified using II (BIO101). These DNA fragments are
Cloning was performed using an RII vector (Invitrogen). Since a restriction enzyme XbaI recognition site was found inside the cloned 1.7 kb DNA fragment, each of the cut fragments was cloned again into Bluescript SK (-), and the DNA sequence was analyzed. Analysis of the DNA sequence was performed using a T7 primer (SEQ ID NO: 4) and an M13RV primer (SEQ ID NO: 7) and an automatic fluorescence sequencer (ABI).

As a result, the nucleotide sequence up to 1330 bp upstream from the ATG codon for translation initiation of the SSK35 gene could be determined. The determined sequence is shown in SEQ ID NO: 1.

The obtained nucleotide sequence was designated as "GENETYX" (ver. 6.
2.0) and “DNASIS” (ver.
An attempt was made to identify a promoter sequence controlling the expression of 35 genes.

As a result, 137 bp from the translation initiation ATG codon
A "TATA" box was found upstream, and a heavy metal response element (MRE) and a "CCAAT" box thought to be involved in transcriptional activation were found 108 bp upstream of the "TATA" box. Furthermore, the gibberellin response element (GARE), MYB binding site, MYC binding site and cA are located upstream of the “TATA” box.
Each of the MP response elements was found. Therefore, it was found that the nucleotide sequences from No. 1 to 1255 shown in SEQ ID NO: 1 contained a promoter sequence controlling gene expression in early embryonic development of rice.

Incidentally, metallothionein is a metal (Cd, Z
n, Cu, etc.), and are presumed to have functions such as detoxification of heavy metals or maintenance of homeostasis related to the redistribution of essential metals. It is also interesting in that it shows a stable expression.

Example 2 (1) Construction of Expression Vector in Plant Cell The promoter sequence obtained as in Example 1 was replaced with Clon.
A chimeric gene linked to the GUS gene of the tech pBI221 plasmid was constructed (FIG. 2). That is, a DNA fragment containing the promoter sequence of the SSK35 gene was inserted into a pCRII vector, and digested with a restriction enzyme XhoI. Klenow enzyme (TAKARA
After digestion with the restriction enzyme HindIII, a 1.7 kbp fragment was separated by agarose electrophoresis, and a DNA fragment was extracted from the gel piece (GENECLEAN II Kit: manufactured by BIO101). Restriction enzyme
The plasmid pBI221 digested with HindIII / SmaI and the extracted and purified fragment were ligated using Ligation Kit ver. II (TAKARA), and a chimeric gene of the promoter (SEQ ID NO: 1) of the present invention and the GUS gene was ligated. Was built.

(2) Introduction of expression vector into plant and GU
Detection of S activity The constructed plasmid containing the chimeric gene was introduced into rice embryogenic calli using the particle gun method. That is, the mature rice seeds (Oryza sativa L. var Sasanishiki) are disinfected with 1% hypochlorous acid, and then NC2
Medium (N6 medium (Sci. Sinica 18: 659, 1975) + 2 mg / l
The seeds were inoculated on 2,4-dichloroacetic acid (2,4-D), 0.9% agar solid medium), and the callus generated after 3 weeks was cultured in AA medium (Mol. Gene. Gen.
et 161: 67, 1978, 0.9% agar solid medium). Thereafter, the cells were cultured for 1 to 2 weeks, and the calli were transferred to an NP medium (N6 medium + 1 m
g / l 2,4-D, 3.6% Sorbitol, 3.6% Mannitol) and transfer it to a filter paper after 4 hours.
hbock CM Model 260). Installation conditions are 15
Pumping (150 kg / cm ² ), 2 μg DNA / 1 mgAu / shot.

Sixteen hours after the introduction, the calli were transferred to an NH-30 medium (N6 medium + 1 mg / l, 2,4-D 30 mg / l hygromycin, 0.9% agar solid medium), and two weeks later, hygromycin-resistant growth Cell clumps (1 mm) were selected. In addition, NH-50 medium (N6
Medium + 1 mg / l 2,4-D 50 mg / l hygromachine, 0.9% agar solid medium) for the second selection. The selected calli were N1 medium (N6 medium + 3% sorbitol, 1 mg / l
Regeneration was induced in abscisic acid (ABA), 1% agar solid medium). The redifferentiated individuals were transplanted to a medium for acclimation (1/2 N6 medium + 0.05 mg / l naphthaleneacetic acid (NAA), 0.25% gellan gum solid medium).

For histochemical detection of GUS activity, see Jeffe
The method was performed based on the method of rson et al. (EMBO. J. 6: 3901, 1987). That is, a tissue section from which chlorophyll has been removed by methanol, 50 mM NaPO ₄ (pH 7.0), 0.2 mM
5-Bromo-4-chloro-3-indolyl-glucuronide (XG
luc), 0.1 mM potassium ferrocyanide, and 0.1 mM
It was observed by treatment in a mixture containing potassium ferricyanide. Further, the fluorometric method for GUS activity was performed as follows. That is, a plant sample is extracted with an extraction buffer (50 mM-PO4, 10 mM-EDTA, 0.1% sarkosyl, 0.1% Triton X-100, and 10 mM β-mercaptoethanol).
And the triturated liquid was centrifuged. Add 2 mM 4-
An equal amount of methylumbelliferyl-γ-glucuronide (4-MUG) was added, and the mixture was treated at 37 ° C. for 1 hour. 0.2 M Na
After stopping with ₂ CO ₃ , 4-methylumbelliferone (4-M
The fluorescence of U) was measured using a fluorescence spectrophotometer (U-2000; manufactured by Hitachi).

As a result of histochemical observation of the expression of the GUS gene by the SSK35 promoter in the transgenic individual, the expression of the GUS gene was 12 times higher in the callus 3 weeks after the induction of regeneration than in the callus before the induction. It was recognized that. In addition, the SSK35 promoter is a tissue-specific GU in foliar meristem / seed aleurone layer tissue and immature embryos.
It was found to induce S gene expression (Fig. 3
~ 5). In FIG. 3, a to f represent shoot meristems derived from redifferentiation of the transformant, FIG. 4 represents callus 3 weeks after induction of regeneration of the transformant, and a and b in FIG. C shows the section of the seed tissue of the non-transformed body.

These results were consistent with the results of Northern blot analysis. From the above results, it was found that the promoter obtained in the present invention can control tissue-specific gene expression in at least one of the foliage meristem, the seed aleurone layer tissue and the immature embryo.

[0047]

According to the present invention, there are provided a metallothionein gene promoter, a recombinant expression vector containing the metallothionein gene and the promoter, and a transformant transformed with the recombinant expression vector. The promoter of the present invention is useful for expressing a heterologous gene in a transgenic plant in a tissue-specific manner.

[0048]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 1255 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: genomic DNA Origin Organism: Oryza sativa L. var Sasa-nishiki Sequence: TAAAAGCATG CTAGTCGGTT TGGTTGAGGC TTTTTCTTTC TTAATGAAAA GCCGTGGGGC 60 GGTAATTTCC CTAGTGCGTC GAGTTCTTGT TTTTATTTCG GCCAACACTT TTATGCCGTG 120 CGGAGGATTA TCCCCTCGTT TATTCAAAAA TCATCTGTAT AGTTATGAAA AAAAAATCGA 180 AAACAAAATG GACAACATTC ATACAACATA CATATACAAG TCCACAAAAT TTTACTAAAC 240 TCAACTCACA CATCAAGAAA TAAAAAGAAA AAAAAATCCA ACGTATGAAC AATGCTGGTG 300 ATATTCACAC TTAAAATTTG TCTTTCTTTC CCCTCTCAAT TTGTAGGTCA GGTTTGAATT 360 TATTTTTTTA GTAGATGCTT ACCATACTCT AGACTATTAA AAAGATTTAG AATTCTTTTA 420 AAACCATTTG TACTGAATTT AAGAGAAAAA TGTATCACGA GGGAATATCC TGGAGGGATT 480 ATAATTCATC TCCTTATTAT TTAGCACTTT TTTTTATCTA CAGAGTTGAA AGGACATGGC 540 ATCACAATTA CTTTCATCTT TCATGCTCAG CCTCAGACGT CTAATTGGCT AGTATACTAG 600 CTTGTTAGGT GCAAAGTCAA GCAAGATCAT GG GTCA CATCAAATGT TTAAATGTGG ACAAAAAAAA AATAATTGCA 720 CAGTTTGTAT GTAAATTGCG AAACGAATCT TGTGAGTCTA ATTACGCCAT GATTTGACAA 780 TGTTGTGCTA CAGTAAACAT GTGCTAATGA TGTATTAATT AGATTTAATA GATTCATCTC 840 GCAGTTTATA TGTGGAATCT ATATATTTTG TTATTAATCT ATATTTAATA CTTAATACTT 900 TTGTCCGTGT ATGTAAAAAA ATTTTGACCA AACAACTGAA CACGGCCTGT ATATGTAACC 960 AAAGAAAGAT CAAAGGAGAG GAGGTAGCTA CTCCTACAAA GAAAGAAGAA ACGATGAGAT 1020 TTGATTGAAT ACTACTCCAG CTAGCTAGTA TACAGTACTC ATACGTGTCT ATCCATATTC 1080 CTGCTCCCAA TGCAAAAATG CAATGGCGAG ATTGCAAGGT TGTGTGGGTG GTGGACCCTG 1140 GATCGATCAC CTCCATTCTT TCTCCGCATC TCGCCACAGT ACGCTTCGCT GCTCTCCGCC 1200 TATATATACC ACCTCCTCCT CGATCATCAG TTCATCAGCA ACCAAAGCAA GAAGC 1255

SEQ ID NO: 2 Sequence length: 650 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA Origin Organism: Oryza sativa L. var Sasa-nishiki Sequence: AATTC 5 TTGAGCTCAA TCAGCAACAA CATCTATCTC CTTCTTGATT AGTTAAGTCC TTCGCCCTCC 65 CACGACGAAG AAGATGTCTT GCTGCGGCGG CAACTGCGGC TGCGGCAGCG GCTGCCAGTG 125 MSCCGGNCGGCSGCQC CGGCGGCGGC TGCGGCGGAT GCAAGATGTT CCCTGATGTG GAGGCCACAG GCACCACCAA 185 GGGCGGCKMFPDVEATGTTK GACCTTCGTC CTCGCTGCTC CATCCAACAA GGCGAGCTCT GGAGGAATGG AGATGGCGGT 245 TFVLAAPSNKASSGGMEMAV GGAGAGCGGC GAGAACGGCG GCTGCGGCTG CAACACCTGC AAGTGTGGCA CCAGCTGCAG 305 ESGENGGCGCNTCKCGTSCS CGGCTGCTCC TGCTGCTCCT GCAACTGAAT CTATCGTCGT CGTCGCCCGG CTGCATGAGG 365 GCSCCSCN ATTTATCGTA TGGATGCTGC TACTGTCGAT CAGAGCTTTG GTCGAGGCCT TAATTTGCTT 425 GCATTAGTAC CCAGCTTATA TGTAGGCAGG CCTTGCCTTT TGCTCTGACG CCTAAATAAA 485 ACCGTCGTCG TCGTTGTCAG TGTGTGCGTGTGTCTGTCGATCAA TGTTGGATGG ATCCCTAGCT 545 AGCTTGGATG GATCATCTAT CATCATGGTG ATCTCATCAT GTACTCCTGC TCCATCTCCT 605 CATGCGTGCC AGGCTTCTTA ATATAATCTA CCTCTGCTTT CATCC 650

SEQ ID NO: 3 Sequence length: 20 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence: AATTAACCCT CACTAAAGGG 20

SEQ ID NO: 4 Sequence length: 22 Sequence type: number of nucleic acid chains: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: CGGGATATCA CTCAGCATAA TG 22

SEQ ID NO: 5 Sequence length: 35 Sequence type: number of nucleic acid chains: single strand Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: AACAAGGCGA GCTCTGGAGG AATGGAGATG GCGGT 35

SEQ ID NO: 6 Sequence length: 35 Sequence type: number of nucleic acid chains: single strand Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: GAAGGAGATA GATGTTGTTG CTGATTGAGC TCAAG 35

SEQ ID NO: 7 Sequence length: 19 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: GGAAACAGCT ATGACCATG 19

[Brief description of the drawings]

FIG. 1. Invers for isolating the promoter of the invention.
FIG. 3 is a diagram showing an outline of an ePCR method.

FIG. 2 is a diagram showing the construction of a chimeric gene.

FIG. 3 is a photograph showing the results of histochemical analysis of GUS in a rice transformant (morphology of an organism).

FIG. 4 is a photograph showing the result of histochemical analysis of GUS in a rice transformant (organism form).

FIG. 5 is a photograph showing the results of histochemical analysis of GUS in a rice transformant (morphology of an organism).

FIG. 6 is an electrophoretic photograph showing the results of Northern blot analysis of SSK35.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI // (C12N 15/09 ZNA C12R 1:91) (C12N 5/10 C12R 1:91)

Claims (3)

[Claims] 1. A metallothionein gene promoter comprising a base sequence represented by SEQ ID NO: 1 or a base sequence in which one or more bases have been deleted, substituted or inserted. 2. A recombinant expression vector comprising the promoter according to claim 1 and a target gene for expression. 3. A transformant transformed by the recombinant expression vector according to claim 2.