JPH10215880A - Plant promoter gene, vector containing the same and transformant - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new plant promoter gene excellent in transcriptive activity, having strong activity for carrying out foreign gene expression even in a plant cell, comprising a promoter gene of a garlic alliin lyase having a specific base sequence. SOLUTION: This promoter gene of a garlic alliin lyase containing a base sequence of the formula or its equivalent sequence shows transcriptive activity equal to or more excellent than that of 35S promoter and rice plant actin gene and is useful as a plant promoter gene having strong activity in carrying out foreign gene expression in a plant. The promoter gene is obtained by immersing garlic scale in water, germinating it to give a seedling leaf, extracting a chromosome DNA from the leaf by a conventional method, treating the DNA with a restriction enzyme, adding an oligonucleotide complementary with the formed cohesive end to both ends of the formed fragment and performing a PCR reaction by using a primer and subjecting the obtained proliferated material to a second PCR reaction by using a primer different from the former primer.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION Field of the Invention The present invention relates to plant promoters. More specifically,
The present invention relates to a promoter gene derived from an alliin lyase (Alliin lyase: EC4.4.1.4) chromosomal gene cloned from garlic (Allium sativum L), a vector containing the same, and a plant cell transformed with the same.

[0002] The development of gene transfer methods to the background art plant cells, it has become possible production of a variety of transformed plant. Good-lasting tomatoes using antisense technology, herbicide-resistant soybeans and rapeseed, and insect-resistant potatoes have already been produced and sold as commodities. One of the important problems in producing such a transformed plant is to efficiently express the introduced gene in plant cells. A promoter sequence is a major factor that determines the transcription level of a gene in a plant cell. In general, the expression level of a target gene can be increased by using a promoter sequence having strong transcription activity.

[0003] However, it is known that the promoter sequence has specificity based on the difference in the RNA polymerase present in each cell. Therefore, when gene expression is to be carried out in plant cells, Must be used. Attempts have been made to select a gene having a strong promoter activity from such a viewpoint, but the promoters that can be used at a practical level are limited, and in most cases, the 35S promoter derived from the cauliflower mosaic virus is used. .

It is also known that there is a difference in transcription efficiency between dicotyledonous plants and monocotyledonous plants, and it is reported that the cauliflower mosaic virus 35S promoter also has low transcriptional activity in monocotyledonous rice. Have been. Under such circumstances, providing a promoter having a strong activity is extremely important in performing molecular breeding of a plant using a transformation method, and particularly simple plants to which major grains such as rice, wheat, and maize belong. Promoters that function efficiently in cotyledons are of particular importance.

[0005] Garlic is a plant belonging to the genus Allium belonging to the lily family, which is a monocotyledonous plant, and has been used as a nourishing tonic for a long time. In addition, antibacterial action and anticancer action,
It has also been shown to have various physiological actions such as a cholesterol lowering action and an anticoagulant action, and is widely used as a pharmaceutical or health food in various parts of the world.

[0006] Organic sulfur compounds are representative of the characteristic components of Allium plants such as garlic.
It is known that Alliin lyase (EC4.4.1.4), which is one of the organic sulfur compound metabolizing enzymes, is expressed in garlic. Alliin lyase is an enzyme involved in the production of irritating odor components of allium plants such as garlic and onion, and produces alkylthiosulfinate from S-alkyl-L-cysteine sulfoxide. Alliin lyase is also known to have a dimeric structure of a glycoprotein with a molecular weight of about 55,000 and to require pyridoxal pentaphosphate as a coenzyme.

Recently, a garlic lyne lyase cDNA was cloned by Van Damme et al. And Rabinkov et al., And its nucleotide sequence was revealed (Van Damme et al., Eur. JB).
iochem., 209, 751-757, 1992; Rabinkov et al., Appl. Bioc.
hem. Biotechnol. 48, 149-171, 1994).

[0008] However, as far as the present inventors know, the control sequence of the garlic allyin lyase gene, particularly the promoter sequence, has not been reported.

[0009]

SUMMARY OF THE INVENTION The present inventors cloned the 5'-terminal region containing the promoter sequence of the alliin lyase gene from a garlic chromosome gene and determined its nucleotide sequence. Subsequently, a chimeric plasmid in which a β-glocuronidase gene was linked as a reporter gene downstream of the cloned gene fragment was prepared, introduced into garlic tissue, and β-glocuronidase activity was measured.
It has been found that the isolated gene fragment exhibits a transcription activity comparable to or superior to that of the 35S promoter or rice actin gene promoter. The present invention is based on this finding.

That is, the present invention provides a promoter gene having a strong activity for expressing a foreign gene in monocotyledonous plants, an expression vector into which the promoter gene has been introduced,
It is another object of the present invention to provide plant cells transformed by these (including transgenic plants obtained by redifferentiation).

The promoter gene according to the present invention is a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 1 or an equivalent sequence thereof, and a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 2 or an equivalent sequence thereof.

[0012] The promoter gene according to the present invention is useful for expression of a protein or antisense RNA in a host (particularly a plant).

[0013]

DETAILED DESCRIPTION OF THE INVENTION Promoter gene According to the present invention, a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 1 or 2 or an equivalent sequence thereof is provided.

The nucleotide sequences of SEQ ID NOS: 1 and 2 are a part of the nucleotide sequences of SEQ ID NOs: 3 and 4 isolated as a promoter gene of garlic inline lyase. Specifically, the nucleotide sequence of SEQ ID NO: 1 corresponds to 69 of SEQ ID NO: 3.
The nucleotide sequence of SEQ ID NO: 2 corresponds to the nucleotide sequence of Nos. 691 to 1062 of SEQ ID NO: 4. As shown in the Examples below, the nucleotide sequences of SEQ ID NOS: 1 and 2 are considered to be basic promoter regions of the alliin lyase gene.

As used herein, the term "equivalent sequence" means a base sequence in which a modification such as addition, insertion, deletion or substitution of a base which is not essential for the promoter gene of the present invention has occurred. Specifically, it refers to a modification such as addition, insertion, deletion or substitution of a base which does not substantially impair the promoter activity.

For example, the equivalent sequence of the base sequence of SEQ ID NO: 3 includes the base sequence of SEQ ID NO: 4, and the equivalent sequence of the base sequence of SEQ ID NO: 4 includes the base sequence of SEQ ID NO: 3.

The promoter gene according to the present invention can be obtained by using the PCR method as described in Examples below (see FIG. 1).

That is, after cleaving the garlic chromosome gene with an arbitrary restriction enzyme, an arbitrary double-stranded oligonucleotide (hereinafter abbreviated as a cassette) having a cohesive end complementary to the generated cohesive end at both ends of the generated fragment. Is added. Using the thus obtained DNA fragment mixture as a template, a specific primer (ALPRO-3) (Example 1) and a primer sequence (C1) homologous to the cassette sequence were used to obtain 1
A second PCR reaction is performed.

Subsequently, using the obtained PCR amplification product as a template, a second time using a specific primer (ALPRO-2) having a different sequence from the primer used in the first PCR and a cassette sequence homologous sequence primer (C2) Is performed. The primers used at this time are C
1. It has a sequence present inside the ALPRO-3 sequence. Therefore, the second PCR is a so-called Neste
d PCR reaction. By performing such PCR, an alliin lyase-specific amplification product is obtained.

Expression Vector The expression vector according to the present invention contains the above promoter gene. Here, the expression vector refers to a carrier DNA that has a role of transferring a foreign gene (a gene encoding a desired protein) into a host cell and expressing the gene in the cell. The expression vector according to the present invention,
It may include restriction sites for joining foreign genes. At this restriction site, the foreign gene can be operably linked to a promoter gene according to the present invention.

In the present invention, the foreign gene includes β
-Glucuronidase gene, alliin lyase, γ-
Enzyme genes related to sulfur compound metabolism such as glutamyl transpeptidase, cysteine synthase, and glutathione synthase, antisense genes (antisense R
NA), and plant virus genes.

Examples of expression vectors include pUC vectors (eg, pUC118 and pUC119), pBR vectors (eg, pBR322), pBI vectors (eg, pBI112, pBI221), pGA vectors (pGA492, pGAH) and the like. Also,
In addition, viral vectors and the like can also be mentioned.

The vector according to the present invention comprises desired control sequences (eg, a terminator sequence, an enhancer sequence, an intron sequence) for expressing any protein in a host cell in addition to the promoter gene and the foreign gene according to the present invention. ) Or a marker gene for selecting a host cell into which the vector has been introduced (for example, a neomycin resistance gene, a kanamycin resistance gene, a hygromycin resistance gene), and the like. In addition, this vector may contain the nucleotide sequence of the gene according to the present invention in a repeated form. These may be present in a vector according to a conventional method. The procedure and method for constructing the vector according to the present invention may be those commonly used in the field of genetic engineering.

Transformant A transformant can be obtained by introducing the expression vector of the present invention into a host cell by a conventional method. In the obtained transformant, a gene encoding a desired protein or an antisense RNA gene can be expressed under the control of the gene according to the present invention.

[0025] Host cells include mainly plant cells, but Agrobacterium, Escherichia coli, yeast, viruses and the like can also be used as host cells.

As plant cells, monocotyledonous plants (for example,
Allium plants such as garlic, leek, onion, and grasses such as rice, wheat, corn).

As a method for introducing a vector into a host cell, a so-called biological introduction method using Agrobacterium or a virus (Methods in Enzymology, 118, 627,
1986. and Methods in Enzymology, 153, 253, 198
7, EMBO J., 6, 307, 1987, Nature, 310, 511, 1984,
Nature 334, 179, 1988, EMBO J., 7, 1588, 1987.
), A physical introduction method using electroporation or a particle gun, or a method using a chemical reagent such as a calcium phosphate method (Methods in Enzymology, 15
3, 313, 1987, Proc. Natl. Acad. Sci. USA, 82, 582
4, 1985, Nature, 296, 72, 1982, EMBO J., 3, 2712,
1984, Mol.Gen. Genet., 202, 179, 1986, Nature, 32
5, 192, 1987, Nature, 327, 70, 1987.).

Transgenic plants Transgenic plants that express the introduced gene in their cells by re-differentiating transformed cells obtained by introducing the vector provided by the present invention into host cells (characters) Transformed plant) can be obtained.

Regeneration of the transformed cells can be performed according to a conventional method.

When the host is garlic, for example, Plant Ce
ll Report 15, 17-21 (1995) and In Vitro Cell Dev. Biol.
30P, 150-155 (1994).

The transgenic plant can also be prepared by directly introducing the vector provided by the present invention into a plant,
Alternatively, it can be obtained by introducing into germ cells such as pollen cells.

[0032] In this specification, the term "transformed plant cell" includes the above-mentioned transgenic plant.

Evaluation of Promoter Activity The fact that the cloned DNA fragment contains a promoter sequence is based on the fact that a reporter gene, for example, β-glucuronidase gene is ligated downstream of the candidate DNA fragment, and garlic is produced using a method such as particle gun. After introduction into the tissue, it can be confirmed by measuring β-glucuronidase activity.

Similarly, a cauliflower mosaic virus 35S promoter and a gene having a reporter gene downstream of a rice actin gene promoter exhibiting a strong activity in monocotyledonous plant cells such as rice and maize are introduced into garlic tissue, and β-glucuronidase is obtained. By comparing the activities, the activity intensity of the cloned alliin lyase promoter can be estimated as a relative activity with the cauliflower mosaic virus 35S promoter or the rice actin gene promoter.

Further, a promoter consisting of a series of DNA fragments lacking a part of the cloned DNA fragment,
A promoter in which a plurality of promoter regions are linked can be prepared. Also, an inducible promoter (chimeric promoter) which can control the expression of promoter activity arbitrarily by linking the promoter gene according to the present invention to another promoter sequence or operator sequence.
Etc. can also be produced.

[0036]

The present invention will be described with reference to the following examples, but the present invention is not limited to these examples.

Example 1 Design of Specific Primers Garlic Aliin Lieace c reported by Van Damme et al.
With reference to the DNA base sequence, the following primer ALPR
O-2 and ALPRO-3 were designed and synthesized. In this design, GG was used to minimize the possibility that the primer sequence region would be cleaved by introns.
Conditions such as not selecting a region having an array were considered.

The primer designed in this manner is as follows: ALPRO-2: 5-CGGGTGTAGCAAGGTGT
TGCACTCGCATTTAGGAGAG-3 (SEQ ID NO: 5) ALPRO-3: 5-GCTGACTGGGTTGAA
AAAAGTAGCTCATTCTGG-3 (SEQ ID NO: 6).

Example 2 Preparation of garlic chromosome DNA 27 g of young plant leaves germinated by immersion of garlic (Fukuchi white) phosphorus pieces in water were ground in a mortar under freezing with liquid nitrogen, and extracted with an extraction buffer (100 mM Tris-HCl). , pH
8, 100 mM EDTA, 250 mM NaCl, 10
108 ml of 0 μg / ml Proteinase K) was added, and extraction was performed while gently stirring. 10% (wt / vol) Sark so that the final concentration is 1%
The osyl solution was added and incubated at 55 ° C. for 2 hours. The precipitate was removed by centrifugation.
A volume of isopropyl alcohol was added and mixed gently, and allowed to stand at -20 ° C for 30 minutes to precipitate nucleic acid components. The precipitated nucleic acid component was separated by centrifugation, and 9 ml of T
E buffer (10 mM Tris-HCl, pH 8, 1 mM
Suspended in EDTA) solution. After removing insolubles by centrifugation, 9.7 g of CsCl was added, and the mixture was dissolved with gentle stirring. Completely remove insoluble matter by centrifugation,
0.5 ml of a 10 mg / ml ethidium bromide solution
Was added and left on ice for 30 minutes, followed by centrifugation again.
The obtained supernatant was purified using a Hitachi RPS-40 rotor for 35 minutes.
The mixture was subjected to density gradient centrifugation at krpm for 16 hours to fractionate a DNA band. The resulting DNA fraction is dialyzed against TE buffer to remove ethidium bromide and CsCl. Thus, 412.5 μg of garlic chromosomal DNA was finally obtained.

Example 3 Cloning by LA-PCR
Grayed LA-PCR is Takara LA-PCR in vi
The test was performed using a tro Cloning Kit (Takara Shuzo Co., Ltd.) according to the attached operation method.

3 μg of garlic chromosomal DNA was digested with restriction enzyme S
Cleavage with peI, XbaI cassette 5 '-(OH) GTACATATTGTCGTTAGAACGCGTAATACGACTCACTATAGGG
AGAT-3 '3' -CATGTATAACAGCAATCTTGCGCATTATGCTGAGTGATATCCC
TCTAGATC (OH) -5 'was ligated to both ends of the DNA fragment using ligase. L
After the iigation reaction, DNA is precipitated by ethanol precipitation.
And the first PCR reaction is performed. The PCR reaction was performed using the cassette primer C1 (5′-GTACATATTGTTCGTTA) attached to the kit as a primer.
GAACCGCGTAATACGACTCA-3 ′) and the alliin lyase specific primer ALPRO-3 were performed under the conditions that two steps of 98 ° C., 20 seconds and 68 ° C., 15 minutes were repeated 30 cycles. The amplification mixture was diluted 1/10, and 1 μl thereof was used as a template, and the attached cassette primer C2 (5′-CGTTAG) was used.
AACCGCGTAATACGACTCACTATAGG
GAGA-3 ′) and Aliin Liece specific primer ALPRO-2 as a primer for the second PC
An R reaction was performed. The PCR conditions were the same as in the first round.

By such PCR, a specific amplified product of about 1.2 kb was obtained. The amplified product was purified by agarose gel electrophoresis and cloned using a pT7Blue-T plasmid vector (Novagen).

The same operation was performed using restriction enzymes such as HindIII and BglII, and the resulting specific PCR product was cloned. These cloned DNA fragments are subjected to PCR for the 5′-terminal region of the alliin liease structural gene (5 ′ end side of the ALPRO-2 complementary sequence) to obtain the desired alliin liease gene 5 'A clone containing the terminal control region was screened.

As a result, the longest 5 ′ terminal control region DNA fragment of about 1.2 kb was cloned when SpeI was used.

Example 4 Determination of Nucleotide Sequence The nucleotide sequence of the obtained DNA fragment of about 1.2 kb was
It was determined using the NA sequencer DSQ1. Analysis of the nucleotide sequence revealed that there were two types of LA-PCR amplification products having different lengths of 1328 bp and 1318 bp, and ALP-Spe15 and AL
It was named P-Spe18. The determined base sequence is as shown in SEQ ID NOs: 3 and 4.

ALP-Spe15 and ALP-Spe
Comparing the 18 base sequences, the 385 bp sequence upstream of the initiation codon ATG is very similar,
94.3% are homologous sequences, whereas the upstream 72
The 8 bp homology was low, showing only 47.0% homology. In the region of high homology, a CAT sequence characteristic of a eukaryotic promoter and a sequence similar to the TATA sequence are present, and a basic promoter region is present in a region of about 400 bp upstream of the initiation codon. It was considered likely. On the other hand, it was presumed that the region of low homology may contain a regulatory sequence that controls the expression of the alliin lyase gene.

As a result of the above-mentioned nucleotide sequence determination, it was revealed that garlic lichen lyase was encoded by at least two kinds of genes.

Example 5 Evaluation Vector for Promoter Activity
In order to evaluate the promoter activity of the obtained garlic inline ACE promoter sequence, pBI221 was first used.
(Clontech Laboratories) was modified to construct evaluation vectors pBI221-StuI and pBI221-Hpa.

These modified plasmids were prepared by performing a PCR reaction using primers into which mutations had been introduced, and pBI221-StuI was pBI221-StuI.
And the restriction enzyme StuI recognition sequence AGGCCT was introduced without replacement of amino acid (arginine) by deleting the SmaI recognition sequence of A. and replacing the third codon CGT of the β-glucuronidase gene as a reporter gene with AGG. It is. On the other hand, pBI2
21-Hpa is a sequence surrounding the BamHI and SmaI recognition sequences located upstream of the β-glucuronidase gene of pBI221, and 5′-GGATCCCCGGGTCA.
GTCCCTTATG-3 to 5′-GGATCCGT
By converting to TAACGAGGAGTCCCTTATAG-3, the SmaI recognition sequence was deleted and H
This is one into which a paI recognition sequence has been introduced.

On the other hand, for the cloned alliin lacease promoter region, a primer having a StuI recognition sequence introduced downstream of the initiation codon ATG of the alliin ligase gene, AL-Stu-M;
-GTAAGGCCTTACATAGCCAATTAA
TTAGCT-3 'and cassette primer C2 described above; 5'-TAATACGACTCACTATAGG
By performing PCR using GAGA-3 ′, Sp
The entire region of the promoter cloned by hI and StuI could be taken out as a cassette.

The PCR was performed using the nucleotide sequence of Example 4 (the nucleotide sequences of SEQ ID NOS: 3 and 4) as a template.

The promoter cassette thus prepared was inserted into pBI using the SphI / StuI recognition sequence.
221 was introduced into the modified vector to construct a vector for evaluating the promoter activity.

The base sequences around the translation start point of each vector thus prepared are as shown in Table 1.

[0054]

[Table 1]

Example 6 Evaluation of Promoter Activity Promoter activity was evaluated by introducing each evaluation vector into garlic slices using a particle gun and expressing β-protein.
Glucuronidase (GUS) activity was used as an index.
That is, 0.8 μg of each vector DNA was coated on 0.2 mg of gold particles, and then introduced into garlic tissue using a particle gun manufactured by Reebok Co., Ltd. under the conditions of 15 pumping times and a distance of 10 cm from the stopper to the sample. After repeating this operation three times, β-glucuronidase activity was measured by a conventional method. At this time, the activity was evaluated by two methods, a method of qualitatively examining the degree of coloring of X-glucuronide and a method of quantitatively counting the number of coloring spots.

Tables 2 and 3 show the qualitative evaluation (GUS activity) and the quantitative evaluation (number of coloring spots).

[0057]

[Table 2]

[0058]

[Table 3]

In the above, “mGUS” indicates a modified plasmid in which a StuI recognition sequence has been introduced into a β-glucuronidase structural gene. "GUS (HpaI)"
Modifies the β-glucuronidase 5 ′ untranslated region to
The modified plasmid into which the aI recognition sequence has been introduced is shown. The number of coloring spots is an average value of an experiment using 10 sections.

From the above results, the cloned DNA
That the fragment has activity as a promoter, its activity is stronger than the rice actin promoter,
Cauliflower mosaic virus 35, currently widely used as the strongest promoter as a plant promoter
It was shown to have activity comparable to the S promoter.

The 35S promoter has already been used in practical breeding of transgenic plants, and it is considered that the alliin lyase promoter gene cloned here can also be used as a practical promoter.

[0062]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 382 Sequence type: Nucleic acid Sequence type: Single-stranded Topology: Linear Sequence type: Genomic DNA sequence AACTCTGTAT AAAACAATTT ACCTGTTGGT TTGTTAACAA CATATTAGAA ACCTAGGTAA 60 CTATATAAGG GTATGCAATA ATTAAAATGC ATGTGTATAGTGATCTATGATTCATGATCTAGATCATCATGATCATAGATCATC ATGAATAAAG CAAAAAAAGC TTTATACATA 180 ATTACATATA TGATATGTCC ATACGGTAAA AAGTTAAAAC AAAATGTATG AATGATATAA 240 TGCCATGCAT CCATTGTATG ATCCTCCCCT GTCATCTGAG ATATTTTCTG GTTCCTAT 300 AAAAGGAGGC CGCATTACAG TAGGATCATTAG TAGGACAATTAG TAG

SEQ ID NO: 2 Sequence length: 372 Sequence type: nucleic acid Sequence type: single-stranded Topology: linear Sequence type: Genomic DNA sequence AACTCTGTAT AAAACAAATT TACCTGTTGG TTAACAACAT ATTAGAAACC TTGTTAACTG 60 AGGGTATGCA ATAATTAAAA TGCATGTGTA TAATTTTATA TATGATAG AAAGTTGCGT TTGAGATACA CTCATGAATA AAGCAAAAAA AGCTTTATAC ATAATTACAT 180 ATATGATATG TCCATACGGT AAAAGTTAAA ACAAAATGTA TGAATGATAT AATGCCATGC 240 ATCCATTGTA TGATCCTCCC CTGTCATCTG AGATATTTCTC TGGTTCAGCT TAGAAGATCGATC TG

SEQ ID NO: 3 Sequence length: 1076 Sequence type: nucleic acid Sequence type: single-stranded Topology: linear Sequence type: Genomic DNA sequence CTAGTCCCCA TCAGCATCAC ATGGAGGAGG ACTTTCCAGC CTAGTGTCAG TGAAAAGGGT 60 CGCCAGGTCC ACAACGAAGT TCGAAGATTT CGGGCCGT GTACAG GAATCACTCA AAGTTTTCAC ATTGTTAAGA CCATCCACAC ATTCTGTTGC CAAAATCATC 180 TCATTACCAA CAATATGATA CATTAGATGT CTAGATTCCT CGTCTTCCCC GACGTACATG 240 TAATACTTAT TAGTACCCGG TCGTGTCGCA TTTCTGTCTT ACCATGACCC ATGTTTCTCA 300 CCATATTCCA TAGAAGTATG TCATGTACAT CGCTCTCTCA CTAGGATATC CATAGCGCGG 360 GTAGGCCACT AGGACTATAG GCGGACTGGA TCAACAGCGG GCCCAGGGTA ATAGTAGCGG 420 GGAAGAACGG AAGCCTGCAG CTTAGGTACC CATTGCAAGT AGGCTTACGA AGAGGTACCT 480 TGACGACATT GGTAGTAGCA GAGCAGGTAT CAAAAGTATG CCCAAAGGAC TCTTCGTTGT 540 GCTTTTAAAT GAGAACTCTT CGTCTGTTTG CTGATAAAGT AATTTTAGAA CACAGTATAA 600 GATAACCCAT GTTGCTATGC AGAATTTTAA AGCACGGTAC TATAAATTAA CACAGTACAC 660 ATCAGCTAGC CCTGTATAAA TCAAACTTCT TTCAAACTCT GTATAAA ACA ATTTACCTGT 720 TGGTTTGTTA ACAACATATT AGAAACCTAG GTAACTATAT AAGGGTATGC AATAATTAAA 780 ATGCATGTGT ATAATTTTAT ATATGGAATA TCTCCAAATA CAAAGTTGCG TTTGAGATGC 840 ACTCATGAAT AAAGCAAAAA AAGCTTTATA CATAATTACA TATATGATAT GTCCATACGG 900 TAAAAAGTTA AAACAAAATG TATGAATGAT ATAATGCCAT GCATCCATTG TATGATCCTC 960 CCCTGTCATC TGAGATATTT TCTGGTTCCT CTATAAAAGG AGGCCGCATT ACTTTGGACA 1020 AAGCAGAAGC AGATCATATA TATCCATAGC TAGCAATTAA AGCTAATTAA TTGGCT 1076

SEQ ID NO: 4 Sequence length: 1062 Sequence type: nucleic acid Sequence type: single-stranded Topology: linear Sequence type: Genomic DNA sequence CTAGTAGAAT ATAAATACTG AATTACTCAG GAAAGGAATA AACACCGTTC AAAACTTTCC 60 ATCTTTATTT AAAGACAAAT AAAACCAAAA CAATATTAATTCACCTCGGTT 120 CTAAGCACAC TCTAATAATC TGTCTTTTAA AATTTGAGAC CATTCAAAAA CAACAGATAC 180 ATCTCTGTTT TGGTCTTTGT CCACAAATAA ATGCATTTAA TGACCCCTTG GCTCCTTTAA 240 TTGTGGCGTC ATTGCACAAC TGTACCACCC TCACTAACAT TAAACCGTAC TGTCTACGCT 300 AAGCAATAAA TTAATTGTCA GTTTTTTACC CATCCACCCT ATTAGGACTT CACTTCCTCT 360 TTTAAAGTCT GTGCCCTAGT TCATTCCCTT CCTATTTCAA AATCTAATGC CGCAAATTAT 420 CCGAAATAGC ATATTCCTTT TCGAAGCTCT CTTGCACCAT CAACCTATAC AGATATGACA 480 AAATTCAAGC AAAGAAATTA GGAAAAAGAA AAAAAAAGGG CAATATCTCT CTTTTTCACA 540 GCATTAGGAG TAATCTGAGA ATTTTAAGCA TATGTACACT TTCTTGTAAG CCAAGCAATA 600 TATTACTTTG GGAAATTTAA GGAACAGTGA AAAATAACAA CCAAAAATCA TGCACAATTT 660 TTTGATTCAT GCTAAGCAAT AAACTTAGGT AACTCTGTAT AAAACAA ATT TACCTGTTGG 720 TTAACAACAT ATTAGAAACC TTGTTAACTG AGGGTATGCA ATAATTAAAA TGCATGTGTA 780 TAATTTTATA TATGGAATAT CTCCAAATGC AAAGTTGCGT TTGAGATACA CTCATGAATA 840 AAGCAAAAAA AGCTTTATAC ATAATTACAT ATATGATATG TCCATACGGT AAAAGTTAAA 900 ACAAAATGTA TGAATGATAT AATGCCATGC ATCCATTGTA TGATCCTCCC CTGTCATCTG 960 AGATATTTTC TGGTTCCTCT ATAAAAGGAG GCCGCATTAC TTTGGACAAA GCAGAAGCAG 1020 ATCTCATATA TATCCATAGC TAGCAAAGCT AATTAATTAG CT 1062

SEQ ID NO: 5 Sequence length: 35 Sequence type: nucleic acid Sequence type: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence CGGTGTAGCA AGTGTTGCAC TCGCATTTAG GAGAG 35

SEQ ID NO: 6 Sequence length: 33 Sequence type: nucleic acid Sequence type: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence GCTGACTGGG TTGAAAAAGT AGCTCATTCT GTG 33

[Brief description of the drawings]

FIG. 1 is a diagram showing an outline of a promoter gene isolation step according to the present invention.

Claims (8)

[Claims] 1. A promoter gene for garlic inline ACE. 2. The gene according to claim 1, which comprises the nucleotide sequence of SEQ ID NO: 1 or an equivalent sequence thereof. 3. The gene according to claim 1, which comprises the nucleotide sequence of SEQ ID NO: 2 or an equivalent sequence thereof. 4. The gene according to claim 1, which comprises the nucleotide sequence of SEQ ID NO: 3 or an equivalent sequence thereof. 5. The gene according to claim 1, which comprises the nucleotide sequence of SEQ ID NO: 4 or an equivalent sequence thereof. 6. An expression vector comprising the gene according to any one of claims 1 to 5. 7. The expression vector according to claim 6, wherein a foreign gene can be operably linked to the gene, and the foreign gene can be expressed in a plant cell. [8] A plant cell transformed with the expression vector according to [6] or [7].