JP2002176984A - Method for locally controlling expression of gene - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method, for example, for drawing a pattern that can be visually recognized on a plant. SOLUTION: This method is a method for locally controlling the expression of a gene in a host by locally stimulating the host into which a stimulation- inducible promoter was transduced.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for controlling gene expression locally. In particular, the present invention relates to a method for drawing a visually identifiable pattern on a plant, and a transformed plant in which a visually identifiable pattern is locally expressed by stimulation.

[0002]

2. Description of the Related Art It is known that a specific gene can be highly expressed by genetic recombination to change the color of petals and leaves (Lloyd AM et al., (1992) Science 25).
8, 1773-1775; Holton TA et al., (1993) Nature 366, 276-
279). It is also possible to generate a reverting patch-like sector in the petal by inserting a transposon into an endogenous gene that determines flower color, and to produce a certain pattern (Sommer H et al., (1988) Mol. Gen. Genet. 211,
49-55). Furthermore, there has been a report that a flower color is produced only on the outside or inside of a petunia petal by utilizing the fact that cosuppression by a foreign gene occurs unevenly depending on the state of a cell (van der Krol).
(1990) Plant Cell 2, 291-299).

On the other hand, when greening peas sprouts,
ELIP2 (Early Light Ind
ucible Protein) has been identified (Scharnhorst C et al.,
(1984) Plant Mol Biol 4, 241-245). Later, it was revealed that the ELIP gene also exists in barley, Arabidopsis, and Lansou, and that expression was induced by stress such as strong light, UV, and drought (Adamska I.
(1997) Physiol. Plant. 100, 794-805).

[0004]

However, it is very difficult to artificially design a pattern generated by such a method, and there is no such report at present. If a pattern such as a character or a symbol can be artificially drawn on a plant body, for example, a leaf or a petal, a flower or an ornamental plant,
In addition, it is possible to add value to commodities such as fruits, and large demand can be expected in the market. Also, in animals and the like, gene expression has not been locally controlled so far.

[0005]

Means for Solving the Problems In view of the above-mentioned circumstances, the present inventors have conducted various studies to apply a pattern locally to a plant by genetic engineering techniques, and as a result, the presence or absence of expression in the plant was determined. It has been found that suitable results can be obtained by combining a reporter gene that can be visualized with a promoter that responds to a specific stimulus. The present inventors have found that it is possible to locally control gene expression in a host other than a plant such as an animal by using a promoter that responds to the specific stimulus, and have completed the present invention.

That is, the present invention provides the following (1) to (1)
0). (1) A method for locally controlling gene expression in a host by locally applying a stimulus to a host into which a stimulus-inducible promoter has been introduced. (2) The method according to the above (1), wherein the host is a plant. (3) A method of drawing a visually distinguishable pattern on a plant body, comprising the following (a) to (c): (a) upstream of a gene capable of visually distinguishable expression in a plant A stimulus-inducible promoter is operably linked and incorporated into an expression vector, (b) the expression vector is introduced into a plant cell to obtain a transformed plant, and (c) a local stimulus is applied to the transformed plant. The above method, comprising giving. (4) The method according to any one of (1) to (3), wherein the stimulus-inducible promoter induces gene expression by intense light, ultraviolet light, and / or contact stimulus. (5) An expression vector comprising a stimulus-inducible promoter and a gene capable of visually discriminating expression in plants.

(6) A transgenic plant comprising the expression vector according to (5). (7) A transgenic plant that locally expresses a visually identifiable pattern upon stimulation. (8) The transformed plant according to (7), which is obtained by the method according to (3) or (4). (9) A transgenic plant capable of locally expressing a visually discernable pattern upon stimulation. (10) The transformed plant according to any of (6) to (9), wherein the stimulus is strong light, ultraviolet light, and / or contact stimulus.

[0008]

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method for locally controlling gene expression in a host, and in particular, a method for drawing a visually discernible pattern on a plant. The plant to which the method of the present invention can be applied is not particularly limited as long as the local expression can be visually identified, and may be any of monocotyledonous plants and dicotyledonous plants. .
Plant parts to be patterned include leaves, petals, fruits and the like, but are not limited to these.
For example, patterns on petals of flowers such as cyclamen, petunias, tulips, roses, carnations, leaves of houseplants such as poinsettia and ivy, vegetables such as turnips and lettuce, and fruits such as fruits such as apples, bananas and oranges. The plant can be used to attach

The term "visually identifiable" means that it can be identified by human vision. Specifically, the color of a portion where the introduced gene is expressed and the color of the portion where the introduced gene is not expressed,
That is, it means that the difference in hue, saturation, and / or lightness can be visually identified.

The term "pattern" includes letters and symbols such as alphabets and the like which can be identified on a plant, symbols, figures, and stripes, polka dots and the like which are generally recognized as patterns, and is not particularly limited. . For example, a pattern having a clear outline and distinguishable as a character or the like, and a pattern having an indistinct outline but recognizable as a pattern as a whole can be included as a pattern in this specification. Therefore,
One embodiment of the present invention is a method of drawing a pattern that can be clearly recognized as a character as described in an example to be described later, and in some cases, a method of transmitting a message. In another embodiment, an individual having a plurality of colors with one branch can be obtained by locally stimulating a small flower such as gypsophila.

[0011] The gene capable of visually distinguishing expression in a plant is not particularly limited.
For example, chalcone synthase that induces anthocyanin synthesis
CHS and CSL1 genes, chlorophyllase gene that degrades chlorophyll, and other genes whose expression can be visually identified directly, luciferase gene, β-glucuronidase gene, anthocyanin synthesis gene, etc. And a gene that can be visually identified by suppressing the expression of an endogenous gene of a host plant, such as a gene that can be used and an antisense of a phytoene desaturase gene.
The gene may be a tag to which FLAG, His, myc, or the like is added, or may encode a fusion protein.

The stimulus-inducible promoter that can be used in the present invention is a promoter that induces the expression of a controlled gene by a locally applied stimulus (stress) such as strong light, ultraviolet light, or contact stimulus. here,
"High light stimulation" is light that exceeds the photosynthetic ability,
For example, this means irradiation with light of 200 to 2000 μE / m ² / s or more for about 30 minutes or more. The term “ultraviolet stimulation” refers to irradiation of ultraviolet light having a wavelength of 100 to 400 nm for several seconds or more. Examples of the promoter that can be suitably used in the present invention include ELIP2 promoter (SEQ ID NO: 3) discovered by the present inventors, a heat shock promoter that induces gene expression by thermal stimulation, and a TCH promoter (Touch that induces gene expression by contact stimulation). -induced genes, TCH1-3)
And the like. Further, it may be a fragment of the nucleotide sequence represented by SEQ ID NO: 3 containing the functional region of the ELIP2 promoter. For example, in the nucleotide sequence represented by SEQ ID NO: 3, using a sequence containing 300 continuous nucleotide sequences on the downstream side Is also good. In addition, a sequence in which one or several nucleotides are deleted, substituted and / or added to the nucleotide sequence, and which can induce gene expression by strong light stress and / or chloroplast disruption. May be used. Here, "several" means the number of nucleotides that can be deleted, substituted and / or added by a well-known method such as site-directed mutagenesis.

[0013] ELIP2 is expressed under the above-mentioned stress conditions, and is thought to play a role in protecting the reaction center responsible for the photosynthetic light reaction in some form. The present inventors have focused on this ELIP2 expression mode, revealing that there is a control region that responds to strong light and chloroplast destruction in the 5 ′ promoter region supposed to control ELIP2 expression, By using this region, it was shown that the expression of genes located downstream can be made to have strong light inducibility and inducibility by chloroplast destruction.

The features of the ELIP2 promoter are as follows: 1) the stimulus is given arbitrarily high; and 2) the signal is cell-autonomous, and only the stimulated cells respond to the stimulus, and the stimulated cells Does not signal unstimulated cells. Promoters having these two characteristics are particularly preferably used in the method of the present invention.

That is, the above-mentioned promoter induces expression of downstream genes by intense light stimulus, but does not receive stimulus under room light, and responds only by positively applying intense light. This has the advantage that there are few technical restrictions on the location and shape of the stimulus. Compared with hormone treatment or the like, intense light treatment has a higher degree of freedom in giving a stimulus, and is very advantageous when the purpose is to give a stimulus locally. Even when there is no intercellular signal, when a pattern is induced locally on a plant to draw a visually identifiable pattern, a pattern with a sharp outline can be drawn.

The stimulus-inducible promoter and the above gene need to be operably linked and incorporated into an expression vector. One skilled in the art would normally perform functional ligation between the promoter and the gene, specifically, so that the translation initiation codon of the gene located downstream would be the first ATG that comes downstream of the promoter. In order to insert a promoter and a gene into an expression vector, first, an appropriate restriction enzyme site is added to the promoter and the gene as necessary, followed by cutting with a restriction enzyme, and insertion into a restriction enzyme site or a multiple cloning site of the expression vector and ligation. And the like.

The expression vector used in the present invention is not particularly limited as long as it can be replicated in a host, and examples thereof include a plasmid vector, a virus vector, and a phage vector. Examples of the plasmid vector include E. coli-derived plasmids (eg, pBR322, pBR325, pUC118, pU118).
C119, pUC18, pUC19, pBluescript, etc.), Bacillus subtilis-derived plasmids (eg, pUB110, pTP5, etc.), yeast-derived plasmids (eg, YEp13, YCp50, etc.), and the like.
A, EMBL3, EMBL4, λgt10, λgt11, λZAP, etc.). Furthermore, animal viruses such as retrovirus, adenovirus, adeno-associated virus or vaccinia virus, and insect virus vectors such as baculovirus can also be used. In the present invention, in particular, pUC119, pBIN
19 and the like are preferably used. In addition to the stimulus-inducible promoter and the above genes, regulatory sequences such as translation enhancers and the like, commonly used in the art, and selection markers can be appropriately incorporated into the expression vector.

The above-mentioned expression vector is then introduced into a host cell so that the promoter of the present invention can function to obtain a transformant. The host may be any of plants and animals, and is not particularly limited, but is preferably a plant. When the host is a plant, a transformed plant can be obtained as follows.

The plant cells to be transduced can be any cells, such as whole plants, plant organs (eg leaves, petals, stems, roots,
Seeds, etc.), plant tissues (eg, epidermis, phloem, parenchyma, xylem, vascular bundle, etc.) or cultured plant cells may be used, and are not particularly limited.

Transformation of a plant may be performed by any method commonly used in the art, and is not particularly limited. For example, Agrobacterium method, electroporation method, particle gun method, PEG method and the like may be used. Can be.

When the Agrobacterium method is used, for example, the constructed plant expression vector is introduced into an appropriate Agrobacterium, for example, Agrobacterium tumefaciens, and this strain is subjected to a vacuum infiltration method (Bechtold et al., (1993) CR Acad. Sci. Ser.
III Sci. Vie, 316, 1194-1199), etc., and infecting the aseptically cultured leaf of the host to obtain a transformed plant. It is possible to transform calli, roots, leaf pieces and the like by Agrobacterium infection.

The vacuum infiltration method is a method in which, for example, a plant inflorescence is immersed in an Agrobacterium suspension, subjected to a reduced pressure treatment for several minutes, and then grown normally to thereby include a transformed seed. is there. When the electroporation method is used, for example, by an electroporation apparatus equipped with a pulse controller, a voltage of 500 to 600 V, 1000
The gene can be introduced into a host by treating under the conditions of μF and 20 msec.

When the particle gun method is used, the plant body, plant organ, or plant tissue itself may be used as it is, may be used after preparing a section, or may be used after preparing a protoplast. Is also good. The sample prepared in this manner is transferred to a gene transfer device (eg, BIOLISTIC PO
S1000 / He; BioRad, etc.). Treatment conditions vary depending on the plant or sample, but usually 10
The pressure is about 00 to 1100 psi and the distance is about 5 to 10 cm.

The tumor tissue, shoot, hairy root and the like obtained as a result of the transformation can be used for cell culture, tissue culture or organ culture as it is, or by using a conventionally known plant tissue culture method. Plants can be regenerated by administering plant hormones (auxin, cytokinin, gibberellin, abscisic acid, ethylene, brassinolide, etc.) at an appropriate concentration.

Cultivation is carried out using a usual plant culture medium, for example, MS
Basic medium (Murashige, T. & Skoog, F. (1962) Physiol.
Plant. 15: 473), LS basic medium (Linsmaier, EM & S
koog, F. (1965) Physiol. Plant. 18: 100), protoplast culture medium (modified LS medium) and the like. As a culture method, any of a general solid culture method and a liquid culture method can be adopted.

[0026] The method of the present invention also relates to a bacterium belonging to the genus Escherichia such as Escherichia coli, Bacillus subtilis.
s), Pseudomonas putida (Pseudom)
bacterium belonging to the genus Pseudomonas such as P. onas putida, the genus Rhizobium such as Rhizobium meliloti, and Saccharomyce.
s cerevisiae), Schizosaccharomyces pombe (Schizo
Saccharomyces pombe), animal cells such as COS cells and CHO cells, or insect cells such as Sf9 can also be used as hosts. When using Escherichia coli, yeast or the like as a host, the expression vector of the present invention is capable of autonomous replication in the bacterium, and at the same time, the promoter of the present invention, a ribosome binding sequence, a gene for controlling expression, a transcription termination sequence, Preferably, it is configured.

As Escherichia coli, for example, Escherichia
Coli (Escherichia coli) DH5α, HB101 and the like,
Examples of Bacillus subtilis include, but are not limited to, Bacillus subtilis and the like. The method for introducing the expression vector of the present invention into bacteria is not particularly limited. For example, a method using calcium ions (Co
hen et al., Proc. Natl. Acad. Sci. USA, 69: 2110 (197
2)), electroporation and the like.

When yeast is used as a host, for example, Saccharomyces cerevisiae, Pichia pastoris (Pichea pas
tris) is used. The method for introducing the expression vector of the present invention into yeast is not particularly limited, and examples thereof include an electroporation method, a spheroplast method, and a lithium acetate method.

When an animal cell is used as a host, monkey cell CO
S-7, Vero, Chinese hamster ovary cells (CHO cells), mouse L cells and the like are used. The method for introducing the expression vector of the present invention into animal cells is not particularly limited, and examples thereof include an electroporation method, a calcium phosphate method, and a lipofection method. Examples of a medium for culturing a transformant obtained by using an animal cell as a host include a commonly used RPMI1640 medium, a DMEM medium, or a medium obtained by adding fetal bovine serum or the like to such a medium. Culture is usually performed at 37 ° C in the presence of 5% CO ₂ for 1 hour.
Do for ~ 30 days. During the culture, antibiotics such as kanamycin and penicillin may be added to the medium as needed.

When insect cells are used as hosts, Sf9 cells and the like are used. The method for introducing the expression vector of the present invention into insect cells is not particularly limited, and examples thereof include a calcium phosphate method, a lipofection method, and an electroporation method.

Confirmation that the vector has been introduced into the host can be confirmed by PCR, Southern hybridization, Northern hybridization, or the like. For example, DNA can be prepared from a host into which a vector has been introduced, a DNA-specific primer can be designed, and PCR can be performed by a conventional method. After PCR, the amplification product is subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis or capillary electrophoresis, etc., and ethidium bromide, SYBR
Transformation can be confirmed by staining with Green solution or the like and detecting the amplification product as a single band. Alternatively, amplification products can be detected by performing PCR using primers that have been labeled with a fluorescent dye or the like in advance. Furthermore, a method of binding the amplification product to a solid phase such as a microplate and confirming the amplification product by fluorescence or enzyme reaction may be used. The method of the present invention comprises locally stimulating the transformed plant obtained as described above.

Examples of a method of locally applying a stimulus include a method of applying a stimulus only to a portion where the introduced gene is desired to be expressed, and a method of providing a stimulus entirely after shielding a portion where the introduced gene is not to be expressed. In the case of stimulation by strong light or ultraviolet light, for example, in the case of a plant, a stainless sheet or aluminum seal is cut out to cover the plant body, and a stimulus such as strong light is applied from above, or the output from the strong light irradiation device is A method of irradiating a plant body with a desired pattern shape, or limiting an irradiation area using an optical fiber, microscopic irradiation light, or the like can be given. In the case of a contact stimulus, a method of giving a subject a specific shape with a needle or a seal may be used.

[0033] The present invention also relates to a transgenic plant which locally expresses a visually identifiable pattern upon stimulation. As the stimulus, the above strong light, ultraviolet light, and / or contact stimulus can be used alone or in combination.

In the first embodiment, the transformed plant of the present invention is characterized by comprising the above-mentioned expression vector, that is, a stimulus-inducible promoter and a gene capable of visually discriminating expression in plants. It is characterized by containing a vector.

In another embodiment, the transgenic plant of the present invention is a transgenic plant that locally expresses a visually discernable pattern upon stimulation. The transformed plant of the present invention can be obtained, for example, by the above-described method of the present invention.

Alternatively, the present invention also provides a transformed plant capable of locally expressing a visually identifiable pattern upon stimulation. In a transformed plant of this type, for example, a stimulus-inducible promoter is operably linked upstream of a gene capable of visually discriminating expression in a plant, incorporated into an expression vector, and the expression vector is introduced into a plant cell. Can be obtained by: At this stage, the transformed plant is in a state before stimulation, and cannot be visually distinguished from a wild-type plant. In the transformed plant in this state, a pattern can be arbitrarily drawn by giving a local stimulus to a store or a consumer according to the wishes of the seller and the consumer. Stimuli that can be used in this embodiment include, for example, strong light stimulation, ultraviolet light stimulation, contact stimulation, and the like.

One example of the method of the present invention, which is described in detail in the Examples below, uses ELIP2 as a stimulus-inducible promoter.
A promoter is used, and a gene placed downstream is a luciferase gene. In this case, after the expression of luciferase is locally induced by intense light irradiation, luminescence can be emitted from the intensely light-treated portion by spraying luciferin, which is a substrate, or the like.

The ELIP2 promoter was used as a stimulus-inducible promoter, and the gene located downstream was replaced with a chalcone synthase, CH, which induces anthocyanin synthesis to turn leaves red.
When using the S gene, intense light irradiation induces the expression of chalcone synthase, and activation of chalcone synthase increases anthocyanin synthesis activity and accumulates anthocyanins. Can be dyed.

When the ELIP2 promoter is used as a stimulus-inducible promoter and the downstream gene is an antisense RNA of a gene encoding a kind of carotenoid synthase, phytoene desaturase, the antisense RNA is irradiated by intense light. The expression of sense RNA is induced, the photosensitivity of chloroplasts contained in the cells is increased, chloroplast destruction occurs under normal growth conditions, and the cells turn white. Therefore, the part irradiated with strong light can be changed to white.

As a stimulus-inducible promoter, a TCH promoter (Touch-i
In the case of using nduced genes, TCH1-3), it is possible to construct a system by contact stimulation by identifying a TCH expression control region and preparing a chimeric construct containing the region.

Furthermore, using a microinjection technique for animal cells and plant cells, for example, a gene introduced into only specific cells is expressed to analyze the effect on surrounding cells, or the specific gene is fused with GFP. It is possible to introduce the fusion protein expressed in a specific cell into a visualizable form of a protein or the like, and to analyze whether or not the fusion protein expressed in the cell moves between cells. Therefore, the present invention is very useful also in the field of basic science.

[0042]

EXAMPLES The present invention will be further described below with reference to examples, but the present invention is not limited to the following examples. [Example 1] Creation of PELIP2 :: LUC construct A GenBank was searched to find an Arabidopsis ELIP2 gene (Hedd
ad M. and Adamska I. (2000) Proc. Natl. Acad. Sci. U
SA 97, 3741-3746) was found in the ATFCA1 contig. Primer ELIP-FSAL (5'-CGC GTC
GAC ATA ATA TTT ATT TAT TTA GTG ATT C-3 ′: SEQ ID NO: 1) and primer ELIP-RSAL (5′-CGC GTC GAC TGA TTA
Using GGT TTT CTA AAA GCC GA-3 ': SEQ ID NO: 2), a PCR reaction was carried out by a standard method using Arabidopsis genomic DNA as a template. PCR reaction conditions Genomic DNA 1μg Primer 0.2μM each dNTP 0.35mM each 1x buffer (Boehringer ExpandHiFiderity kit) Enzyme 0.5μl / 50μl Reaction solution

The obtained product was digested with SalI, and p6GLUC (Aoyama T. and Chua NH) containing the luciferase gene was digested.
(1997) Plant J. 11, 605-612). The resulting plasmid was digested with ScaI / PvuII, and
2 :: A fragment containing LUC was recovered, blunt-ended using Klenow enzyme, and then pUC119 (J. Sambrook et al., "Molecular Clon
ing ", 1989, Cold Spring Harbor Laboratory Press, C
old Spring Harbor). The obtained plasmid (yy211) was digested with HindIII, and a region from base -2 to -1907 bases from the translation initiation point of ELIP2 (SEQ ID NO: 3)
The HindIII fragment containing the fusion gene between the ELIP2 promoter and the LUC reporter containing
LJ755I5) was introduced into HindIII. The obtained plasmid (yy210) was obtained by combining the BASTA resistance gene and the ELIP2 promoter (PELIP2) / LUC reporter fusion gene (FIG. 1) with T-DNA.
Have in.

Example 2 Transformation of Arabidopsis thaliana The plasmid yy210 obtained in Example 1 was replaced with Agrobacterium GV3101pMP90 (C. Koncz et al., Plant Molecular Biolo
gy Manual, SB Gelvin & RA Schilperoort, B
2: 1-22, Kluwer Academic Publishers, Dordrecht, 199
This Agrobacterium was infected into Arabidopsis thaliana by a vacuum wetting method. Arabidopsis independent transformants # 52, # 58, # 62, # 69, # 72, and # 75 were grown to obtain T2 generation seeds. In the obtained plants of the ELIP2 :: LUC line, luminescence derived from luciferase is induced by intense light treatment.

Example 3 Characters were drawn on the leaves of the plant obtained in Example 2. Cut two leaves of Arabidopsis thaliana (one month after germination) of ELIP2 :: LUC line,
It was placed on a GM plate containing 1% sucrose and 0.8% agar, sprayed with 5 mM luciferin (containing 0.1% Trinton X100), and the next day, one of the two plates was subjected to intense light treatment in the form of the letter "Y". . Specifically, the stainless sheet was hollowed out into a “Y” shape, the leaves were covered with the sheet from above, and intense light irradiation was performed from above. For the intense light treatment, a xenon light source was subjected to a filter treatment, and irradiated with 150 W / m ² intense light including a wavelength of 400 to 700 nm excluding ultraviolet rays and a heat ray part for 3 hours.

5 mM luciferin (containing 0.1% Trinton X100) was sprayed on the seedlings that had been subjected to intense light treatment and the seedlings that had not been treated, and luciferase luminescence was measured in vivo using an Argus camera system (Hamamatsu Photonics, Hamamatsu) (Millar). AJ et al., (1992) Plant Mol. Biol. Rep.
10, 324-337).

As a result, almost no luciferase luminescence was observed from the untreated leaves (left in FIG. 2), but the leaves (right in FIG. 2) subjected to intense light treatment in the shape of the letter “Y” Y "
Light emission was observed in the shape of the letter The upper part of FIG. 2 was used for processing.
A photograph of the ELIP2 :: LUC leaf, and the lower part shows a luminescence image due to its luciferase activity.

[0048]

As described in detail above, according to the present invention, patterns such as characters and symbols can be artificially drawn on plants, for example, leaves and petals, and can be used for products such as flowers, houseplants, and fruits. It became possible to add value to

[0049]

[Sequence List] SEQUENCE LISTING <110> RIKEN <120> A method for locally controlling gene expressions <130> RJH12-080T <160> 3 <170> PatentIn Ver. 2.0 <210> 1 <211> 34 <212> DNA < 213> Arabidopsis thaliana <400> 1 cgcgtcgaca taatatttat ttatttagtg attc 34 <210> 2 <211> 32 <212> DNA <213> Arabidopsis thaliana <400> 2 cgcgtcgact gattaggttt tctaaaagcc ga 32 <210> 3 <211> 1909 <212> DNA <213> Arabidopsis thaliana <400> 3 aagcttatgt tttgcttgct tggccttgtg gccggctaaa taacctaagt atataatcaa 60 taaattacat aaacgaccgg ataaatcaga acttaattat aatgttaaaa gtaattaaat 120 gaatgatgac cctcgtcgtc tctcctctta aaacacaata atctcataaa tacaattttt 180 atatgaagtc ttcacaaact actataaatt tcccaaagtt gttctctctc tttgtccact 240 cgaagactca tggggataag aaggcactcg tgttatgggc ttcggggctt tttattgttc 300 gccctttcga ggaccttctc acatgtttgg acttttgagt ctaaatgcct tatttcctca 360 cgaatgacgt gaacattttt tcgtcctgaa ctcaaaagta tcaattacac acttcacact 420 actaatggtc aagttttttc gtatttgaaa ctacaaatgt aagtaatata tgcgataaat 480 ttattatcct ctgctagtaa a atccattaa aaaaaactac caatgtgggt gtatgttttc 540 tttcagtata tataaagagt tttcgcagag agcacatatc gatggtggta catcaaatct 600 gatcattcat atctaaataa ttgttgttga gttttctata tgtgtggaac gttagattgc 660 ttaagctaga aattgacaca agagtttgat caccgagttc ctgcctcctg gcctctaacc 720 ggtacgtctc gggtgggaac tgtctcccac aacttttcac tatcaagcaa aaagtattac 780 aagcactaat agaggtttct cttctccttg ttacaaattt agaaattaac tcacacacaa 840 aagacaatag aaaaggagac tataccgaca cgaagactat gcccatatct tcacaattgt 900 gctattattt accgttctaa tctggaaaaa aaaacagcgc acgtaggaga attgggcacg 960 aagacatgaa tgggacacat aaaggatcaa aagagtcatc atgggtctgc catgatgaaa 1020 gttaagaaag ttccaatacg cgcgaaacaa gattgaatgc gacttctctc tataccaaaa 1080 acgaatctcc caagtcccaa gtgaatattg tgagccacta acttgttatt aacattttga 1140 gacaaaaaaa aaatttgtac gtgtttttgc caaacccact tggccatgac caatcagaaa 1200 aagacaagga agatgtgtat ctatgtgtaa tgaggaggcc acgccatcag tttacttgca 1260 cttttccaac tagacgtggc ctctcaccga tctcaacctc actttcctct catttctcaa 1320 attttattgg ctacgtgttt ttgtgtttag cgt tcaaccc aaatatcgat atattcttct 1380 ttttttttca catttttaac gatttcgagc aaaataattc gatttatttg tataatttta 1440 atatggtagt tttacaaata atgaaacgaa tgaccaactg atttgttagg tgttacaata 1500 aatatggaaa aaatctcata agtttgaaaa catttattct gaggaaactt tttcttcccc 1560 aaaagaaaaa aaaagtttaa agtggaaaaa agaaaaaaaa ggaataaaaa gtttgaattc 1620 agtttttttc ttttctttga tagatttctt tctacttatt tattactcta ctacacacca 1680 cacaaaaaca aaaataaaat aagtaatcat agtatcccat aaatcagtaa agataaataa 1740 aatccagaaa atactgggcc tatcattttc cttcaccaac tctataaatg aagagataat 1800 cctacagtta cacctcaaac caactccatc tcacttctca agtcttataa tttattcatt 1860 tctctcttct tcatcgatct tcggctttta gaaaacctaa tcagaaatg 1909

[Brief description of the drawings]

FIG. 1 shows a schematic diagram of an ELIP2 :: LUC construct.

FIG. 2 shows photographs before and after intense light irradiation (150 W / m ² , 180 minutes) of leaves of the ELIP2 :: LUC line grown in low light in the shape of the letter “Y” and a luminescence image due to luciferase activity. .

 ────────────────────────────────────────────────── ─── Continued on the front page F term (reference) 2B030 AA02 AB03 AD20 CA06 CA17 CA19 CB02 CD03 CD07 CD10 4B024 AA08 BA80 CA04 DA01 EA04 FA02 GA11 4B065 AA88Y AA89X AA90Y AB01 AC14 BA02 CA53

Claims (10)

[Claims] 1. A method for locally controlling gene expression in a host by locally applying a stimulus to a host into which a stimulus-inducible promoter has been introduced. 2. The method according to claim 1, wherein the host is a plant. 3. A method for drawing a visually identifiable pattern on a plant body, comprising the following steps (a) to (c): (a) a gene capable of visually identifiable expression in a plant; A stimulus-inducible promoter is operably linked upstream and incorporated into an expression vector, and (b) the expression vector is introduced into a plant cell to obtain a transformed plant. Providing a stimulus. 4. The method according to any one of claims 1 to 3, wherein the stimulus-inducible promoter induces gene expression by intense light, ultraviolet light, and / or contact stimulus. 5. An expression vector comprising a stimulus-inducible promoter and a gene capable of visually discriminating expression in plants. 6. A transgenic plant comprising the expression vector according to claim 5. 7. A transformed plant wherein a pattern visually recognizable by stimulation is locally expressed. 8. The transformed plant according to claim 7, obtained by the method according to claim 3 or 4. 9. A transformed plant capable of locally expressing a visually discernable pattern upon stimulation. 10. The transformed plant according to claim 6, wherein the stimulus is strong light, ultraviolet light, and / or contact stimulus.