JP2001352851A - Transgenic plant and method for producing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing transgenic plants of major crops or garden plants such as expensive flowering plants, capable of controlling not only sufficient growth of roots, leaves and stems but also bolting and flowering time, and to provide transgenic plants obtained by the method. SOLUTION: This transgenic plant overexpresses blue light receptor protein and is produced by transforming a plant with an expression vector connected with a blue light receptor protein LPK1 gene through a promoter of the LPK1 having LOV domain and kelch repetition downstream of cauliflower mosaic virus 35S promoter. In the above vector, the blue light receptor protein gene can be connected downstream of promoter of the above-mentioned blue light receptor protein through a reporter gene such as β-glucuronidase gene.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a transgenic plant capable of controlling flowering time irrespective of day length and temperature change, a method for producing the same, and a method for cultivating the transgenic plant.

[0002]

BACKGROUND OF THE INVENTION Since plants cannot move, they respond to environmental stimuli and utilize them as signals for development and differentiation, thereby causing physiological and biochemical changes at the solid and cellular levels. And gene expression. Light is not only an energy source for plants, but also a very important signal. Unlike animals, plants sense mainly red and blue. These wavelengths are
End of dormancy, germination, cell elongation, regression reaction, chloroplast movement,
It regulates many plant responses such as gap opening, flavonoid biosynthesis, and flowering. The red signal is detected by phytochrome, and in the case of Arabidopsis, the phytochrome photoreceptor is encoded by five genes: phyA, phyB, phyC, phyD, and phyE. These phytochromes are known to have many duplicate functions and some distinct functions (Genes De
v, 1989,3,1745-1757).

The blue response of plants has been studied for many years,
Until recently, the structures of the blue receptors and their signaling pathways were unknown. Ahmad and Cashmo
re is the blue receptor cryptochrome 1 (CRY
The sequence of 1) was first reported (Nature, 1993, 306, 162-16).
6). CRY1 is composed of 5,10-methenyltetrahydrofolate (MTHF) and flavin adenine dinucleotide (F
AD) is a 75 kD protein that binds two cofactors. This indicates sequence similarity to class I light-recovery enzymes, but lacks DNA photorepair activity (Scie
nce, 1995, 269,968-970; Biochemistry, 1995, 34, 68
92-6899). CRY1 elongates the hypocotyl, petiole and stem,
Regulates blue-dependent phenomena such as cotyledon and leaf expansion, anthocyanin accumulation and flowering (Plant J, 1995,
8, 653-658; Planta, 1995, 197, 233-239). Arabidopsis also has another cryptochrome, CRY2. The function of CRY2 somewhat overlaps that of CRY1 (Plant Cell, 1988, 10, 197-207). CRY
Both 1 and CRY2 are involved in preventing hypocotyl elongation, anthocyanin formation, flowering, and suppressing blue induction of stem growth (Plant J, 1996, 10,893-902; Proc. Natl. Acad. Sci. 19
98, 95, 2686-2690). In addition, it has been shown that CRY1 plays a role in sensing an optical signal acting on an oscillator causing a circadian rhythm (Plant Cell, 1997, 9, 947-955; Scie).
nce, 1998, 282, 1488-1490). Overexpression of CRY1 significantly shortens the free running cycle at high fluences of blue or white light. On the other hand, the elimination of CRY1 results in the extension of the period in both the high and low light intensity regions (Science, 1
998, 282, 1488-1490).

[0004] NPH1 or phototropin, which is involved in phototropism, is a recently sequenced blue receptor (Sceince, 1997, 278, 2120-2123). NPH1 is a plasma membrane-associated serine / threonine protein kinase whose activity is induced by light (Sciencem, 1998, 282,
1698-1702). NPH1 has two domains found in the eag family of photosensors, oxygen sensors, and voltage-gated potassium channel proteins and named LOV (Science, 1997, 278, 2120-2123). NPH1
Both LOV domains associate flavin mononucleotide (FMN) with a flavin / protein ratio of 1 (acting as a UV-A / blue absorbing chromophore) (Science, 199).
8, 282, 1698-1701). The crystal structures of the human HERG LOV domain and the Bradyrhizobium FixL LOV domain have been determined, and the LOV domain has an α that creates a hydrophobic pocket capable of binding ligand.
Forming a β-sheet core sandwiched between helices. The hydrophobic pocket is accessible for ligand binding by hydrophilic invasion (Cell, 1998, 95, 649-655; PNA
S, 1988, 95, 15177-15182).

[0005] An NPH1-related sequence encoding a protein having two LOV domains in Arabidopsis thaliana (N
(PL1 or NPH1L) has been reported, but its function is unknown (Plant Physiol, 1998, 117, 719).
Zeaxanthin is also considered to be a photoreceptor chromophore for opening the gap, but the apoprotein of the receptor is not yet known (Plant Cell, 1998, 10,1121-1134). Since so many phenomena are regulated by blue, these four receptors, CRY1, CRY2, NP
Blue light receptors for H1 and gap response may not be involved in all phenomena. Blue receptors associated with chloroplast movement have been reported to be different from these four receptors (Plant Cell Physiol, 2000, 41, 84-9).
3).

[0006] More recently, two reports have been made on genes that regulate flowering time in Arabidopsis. Key et al. (Cell, 101, 319-329, 2000) reported that a transgenic bioluminescence phenotype was used to screen for a late-flowering mutant of Arabidopsis thaliana. The discovery of protein ZTL and the amino acid sequence of such a quality ZTL and the blue light receptor protein LKP2 by the present inventors are described. Bartel et al. (Cell,
101, 331-340, 2000) contains blue light receptor protein F
It describes that ZTL and FKL1 were found as homologs of KF1, and that a ZTL cDNA bound downstream of the cauliflower mosaic virus 35S promoter was expressed.

[0007]

[0005] Flowering of plants is an important phenomenon in plant production, breeding and horticulture. Flower differentiation,
At present, flowering control is mainly performed by adjusting artificial daylength conditions and temperature conditions. However, there are problems in terms of costs such as equipment costs such as a light irradiation device and a temperature control device, lighting costs, and heating costs. Although the application of transgenic plants has been started, the types thereof are limited, and creation of useful transgenic plants in a wider range is desired. An object of the present invention is to provide a method for producing a transgenic plant of a horticultural plant such as a main crop or an expensive flower, which can control the stage of drawing and the time of flowering as well as sufficient growth of roots, leaves, and stems. To provide a transgenic plant obtained by the method.

[0008]

The growth of plants, such as germination and flowering, depends on various natural conditions such as changes in daylight hours and temperature changes. Plants have the function of sensing their natural conditions, and the substances responsible for that function are expressed and produced by genetically controlled mechanisms, and each substance regulates each stage of growth. The present inventors have determined that the long-day plant Arabidopsis thaliana has a blue light receptor protein LK.
The P1 or LKP2 gene was isolated and placed downstream of the cauliflower mosaic virus 35S promoter.
Transformation with an expression vector linked to the LKP1 gene via the P1 promoter
By overexpressing No. 1, it has been found that a transgenic plant in which flowering is induced at about the same time as under short-day conditions even under long-day conditions, which is a long-day condition under which flowering is induced, is obtained. Reached.

That is, the present invention provides a method for producing a transgenic plant overexpressing a blue light receptor protein having an LOV domain and Kelch repeats, wherein the method comprises the steps of:
A method for producing a transgenic plant, characterized by transforming with an expression vector to which the gene is linked via the blue light receptor protein promoter (Claim 1); The method for producing a transgenic plant according to claim 1, wherein a blue light receptor protein gene is linked downstream via a reporter gene (claim 2), and the reporter gene is β-glucuronidase. The method for producing a transgenic plant according to claim 2, which is a gene (claim 3), wherein the blue light receptor protein is LKP1 having an amino acid sequence represented by SEQ ID NO: 2. The method for producing a transgenic plant according to any one of claims 1 to 3 (claim 4); The method for producing a transgenic plant according to claim 4, wherein the 1.5 kb 5 'non-coding region of LKP1 is used as a promoter of the receptor protein (claim 5). It is LKP2 having the amino acid sequence represented by SEQ ID NO: 4, wherein the method for producing a transgenic plant according to any one of claims 1 to 3 (claim 6), and wherein the promoter derived from a plant virus is cauliflower mosaic The method for producing a transgenic plant according to any one of claims 1 to 6, which is a virus 35S promoter (claim 7), and using an expression vector pBE2113 as a cauliflower mosaic virus 35S promoter. A method for producing the transgenic plant according to claim 7. Law (Claim 8)
The transgenic plant is a transgenic Arabidopsis thaliana, the method for producing a transgenic plant according to any one of claims 1 to 8 (claim 9), and the transgenic plant according to any one of claims 1 to 9. A transgenic plant (Claim 10) obtainable by a method for producing a plant, or having a rosette-shaped upper stem leaf and capable of forming an extended primary inflorescence with many rosette leaves. The transgenic plant according to claim 10 or claim 11, wherein flowering is not induced even when the seed is subjected to vernalization treatment at a low temperature (claim 12). And the transgenic plant according to any one of claims 10 to 12 (claim 13), It relates claim 13 transgenic plants, wherein the propagation material is a seed (claim 14).

The present invention also provides a method for cultivating a plant, which comprises cultivating the transgenic plant according to any one of claims 10 to 14 (claim 15), and controlling the flowering time of the plant. The method for cultivating a plant according to claim 15 (claim 16), the method for cultivating a crop according to claim 16 (claim 17), wherein the control of the flowering time is delaying the flowering time. The present invention relates to a method for cultivating a plant according to any one of claims 15 to 17, wherein the drawer is delayed.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION As a method for producing a transgenic plant of the present invention, an LOV domain and Kerch (ke
1ch) A method for producing a transgenic plant that overexpresses a repeatable blue light receptor protein,
The method is not particularly limited as long as it is a method of transforming with a blue light receptor protein gene-linked expression vector downstream of a plant virus-derived promoter via the blue light receptor protein promoter. No, as the blue light receptor protein gene,
All or part of the cDNA encoding the blue light receptor protein or all or part of the structural gene of the blue light receptor protein can be exemplified. Further, a blue light receptor protein gene can be linked to the downstream of the promoter of the blue light receptor protein via a reporter gene. As the reporter gene, any known reporter gene can be used, but a gene not normally present in plants, for example, β-glucuronidase gene or GFP (green fluor
escent protein) gene is preferred.

The blue light receptor protein is not particularly limited as long as it is a known blue light receptor protein, but is not limited to SEQ ID NO: 1 found by the present inventors.
LKP1 having an amino acid sequence represented by SEQ ID NO: 2 or a translation product of LKP2 cDNA represented by SEQ ID NO: 3, which is a translation product of LKP1 cDNA represented by
LKP2 having the amino acid sequence represented by SEQ ID NO: 4 can be specifically exemplified. The LKP1 cDNA comprises 2297 bp and 610 amino acid residues.
KP2 cDNA is composed of 2265 bp and consists of 612 amino acid residues. Both LKP1 and LKP2 proteins have one LOV domain found in blue light receptor,
It has one ch domain. Hereinafter, the blue signal function and the circadian clock function of LKP1 will be described.

LKP1 is involved in the photoaffinity response of yellowed seedlings to blue light at low light intensity in Arabidopsis thaliana.
It has a LOV domain with very high homology (84% similarity) to the domain. The LOV domain in NPH1 is
FMN is non-covalently bound at a flavin / protein ratio of 1 (Science, 1998, 282, 1698-1701). N
The LOV domain of PH1 is thought to be the light-sensitive chromophore of this blue receptor kinase. 35S :: LK of the present invention
P1 transgenic plants show a long hypocotyl phenotype due to cell elongation in white light. In Arabidopsis, phytochromes A and B and cryptochrome C
Photoreceptors such as RY1 and CRY2 have been shown to contribute to the inhibition of hypocotyl cell elongation. LKP1 overexpression absorbs most of the blue signal in the cell and reduces its entry into CRYs, resulting in no inhibition of hypocotyl elongation by cryptochrome. In addition, 35 of the present invention
S :: LKP1 transgenic plants also show delayed flowering under long day conditions. PHYA, CRY1 and CRY
Two photoreceptor variants also affect the long-day pathway of flowering time in Arabidopsis. LKP1 is a nucleocytoplasmic protein whose nuclear cytoplasmic localization is also found in photoreceptors such as phytochrome and cytochrome.

Second, the LOV domain also functions in protein-protein interactions. The NPH1LOV1 domain is a signaling element that works downstream of NPH1, NPH3
(Science, 1999, 2
86, 961-964). The LOV domain is related to the PAS domain (Trends Biochem Sci., 1997, 22, 331-33
3). The PAS domain is not only involved in light sensing and signaling like phytochrome, but also WC-2, PE
It is also related to clock related proteins like R and CLOCLK. The PAS domains of these proteins also function in protein-protein interactions. LKP1 motif search finds LK with E-value 0.0045
The PAS domain motif in the P1LOV domain was picked up. A long hypocotyl phenotype is also found in LHY and CCA1 mutants, where the myb-related gene was activated at the 35S promoter (Cell, 1998, 93, 1207-121).
7). LHY and CCA1 are thought to be closely related to the central oscillator of the circadian clock in Arabidopsis. Also, 35S :: LKP1 transgenic seedlings show clear cotyledon movement, whereas wild-type seedlings do not (Cell, 1998, 93, 1219-1229; Science,
1999, 285, 1579-1582). Delayed flowering in the long day pathway is also seen in LHY and CCA1 dominant mutants, where circadian clock regulation of gene expression and leaf movement is disrupted (Cell, 1998, 93, 1219-1229; Cell, 199
8, 93, 1207-1217). When the GFP-LKP1-expressing plant tissue culture in the dark was transferred to white light, the GFP signal did not transfer to the nucleus, indicating that the nuclear cytoplasmic localization of GFP-LKP1 was not controlled only by light.

It is highly likely that LKP1 is the same substance as ZTL in the report of Key et al. And Bartel et al., And LKP2 is the same substance as FKL1 in the report of Bartel et al. Key's group is Zeitlu, a long-term mutant
pe1 has been characterized. This mutant has a very long time in hazy blue, suggesting that the blue signaling to the clock has been altered. Ze
The itlupe1 mutant has a mutation in the Kerch repeat coding region of the LKP1 gene, and their data support our view on LKP1 function. LKP1 /
ZTL1 appears to be a blue signal converting molecule and is thought to control flowering time and circadian rhythm. On the other hand, Bartel
Team also developed a delayed Arabidopsis flowering mutant (fkf
The chromosome 1 was found to reach the gene deletion in 1).
The part of the defect at the end of is separated. This mutant has a hypocotyl elongation defect and the FKF1 transcript is regulated in a circadian fashion. FKF1 is LKP1 / ZTL1
Is homologous (65.9% in amino acids), and LKP1 /
ZTL1 and FKF1 belong to the same small gene family. However, in our RT-PCR experiments,
The LKP1 transcript was not clearly controlled in circadian fashion. Each mutant is a delayed flowering phenotype, although there is no perfect complementation of flowering among the three genes. Although the targets for these proteins may be different, LKP1 / ZTL1 is an effective regulator of flowering time in higher plants because mutations and overexpression of LKP1 / ZTL1 affect flowering time.

The promoter for the blue light receptor protein may be any promoter as long as it is a promoter present in the non-coding region 5 'to the coding region of the blue light receptor protein or a sequence containing the same. , LKP1 can be specifically exemplified by a 1.5 kb 5 'non-coding region. The plant virus-derived promoter is not particularly limited, but includes a cauliflower mosaic virus 35S promoter, a corn ubiquitin promoter, a nopaline synthase (NOS) gene promoter, an octopine (synthetic enzyme) gene promoter, and a rice actin gene. (A
ct1) A gene promoter and the like can be specifically exemplified. Among them, the cauliflower mosaic virus 35S promoter is preferable in view of the strength of expression. As such a cauliflower mosaic virus 35S promoter,
For example, the expression vector pBE2113 (Mitsuhara et a
l., Plant Cell Physiol. 37, 49-59, 1996) can be used. Examples of transgenic plants include grains such as rice, wheat, corn and soybeans, vegetables such as lettuce and cabbage, horticultural plants such as flowers, and research plants such as Arabidopsis. These are not limiting.

Next, a method for producing a transgenic plant that overexpresses the blue light receptor protein of the present invention will be described. An expression vector in which the gene is linked via a blue light receptor protein promoter having a LOV domain and Kelch repeat downstream of a promoter derived from a plant virus, is used to prepare an Arabidopsis thaliana, tobacco, corn, wheat, rice, carrot,
By introducing it into plant cell lines established from soybeans, etc., and regenerating this transfected cell line into plants,
Transgenic plants can be obtained. As a method for introducing a vector into a host plant cell, the Agrobacterium method is suitable, but in addition, for example, a polyethylene glycol method, an electroporation method, a particle gun method, or the like can be used (experiment with a model plant). Protocol, Shujunsha (1996)).

The transgenic plant of the present invention is not particularly limited as long as it can be obtained by the above-described method for producing a transgenic plant. In addition to transgenic plants that can control flowering time, transgenic plants that have rosette-shaped upper leaves and can form many rosette leaves and extended primary flower stems, and seeds that have low temperature vernalization Transgenic plants that do not induce flowering even after the treatment can be suitably exemplified. In addition, as a form of the transgenic plant, besides plants, protoplasts,
Examples include plant material parts such as callus, leaf disk, and hypocotyl, and propagation materials such as seeds, tubers, cuttings, and meliclones.

The method for cultivating the plant of the present invention is not particularly limited as long as it is a method for cultivating the above-mentioned transgenic plant. It is possible to delay the flowering time and the delay of the drawing of plants. LKP2 can also be prepared in the same manner as LKP1.
It has been confirmed that P2 has the same function as LKP1.

[0020]

EXAMPLES The present invention will be described more specifically below with reference to examples, but the technical scope of the present invention is not limited to these examples. First, materials and experimental methods in Examples will be described below.

(Plant Material) Arabidopsis (Arabidopsis)
s thaliana (Columbia ecotype) seeds are aseptically 0.
Germination medium containing 8% agar and 0.9M sucrose (Plan
t J., 1997, 12, 851-861) and incubated for 3 days at 4 ° C in the dark to break dormancy. Then 1
22 ° C under long-day conditions of 6 hours light irradiation and 8 hours darkness
Grown in. Rosette seedlings were transferred to soil and grown under the same conditions. For the flowering test, seeds are sown on soil and incubated at 4 ° C. for 3 days in the dark, and then for long days (16 hours light / 8 hours dark) or short day conditions (8 hours light). / 16 hours dark) at 22 ° C. Seeds are sown on soil for springing, and short-day at 4 ° C
For 30 days. Vernalization-sensitive delayed flowering mutant fve-2 (Landsberg background) (Mol. Gen. Gene
t., 1991, 229, 57-66; Cold Spring Harbor Lab. Press, NY,
1994, pp403-433) was used as a control.

(Screening of cDNA library)
cDNA library [CD4-13 (0.5-1k
b), CD4-14 (1-2 kb), CD4-15 (2
-3 kb) and CD4-16 (3-6 kb)] (Cell, 1
993,72,427-441) was screened by the plaque hybrid method (Maniatis et al., "Molecular Cloning", 1982), using a 4.15k from Arabidopsis genomic DNA.
bPCR fragment (6771-1 of MSF19P1 clone)
0920 region) as a probe. In addition, primers consisting of the nucleotide sequences shown in SEQ ID NOs: 5 and 6 were used. Each cDNA library was plated as 6 × 5,000 plaques and screened.

(DNA Sequencing and Analysis) Plasmid DNA templates for sequencing were generated using an automated plasmid separation system (model PI-100, Kurabo, Oosaka). The DNA sequence is a DNA sequencer
l 317; Applied Biosystems, Foster City, CA) using the BigDye-Terminator sequencing method. GENETYX
(Software Development, Tokyo) and DNASIS (HI
TACHI Software Engineering, Tokyo) software system was used for DNA and amino acid sequence analysis.
BLAST search is available on the NCBI website (http: //www.n
cbi.nlm.nih.gov/). GENSCAN is on the Stanford website (http: //genomic.stanf)
ord.edu/GENSCANW.html). Motif search website (http://www.motif.genome.ad.jp/)
Went through.

(RNA method) Total RNA is phenol / SD
S method (Kiyosue et al., Plant. Mol. Biol. 19, 239-249,
1992). One-step RT-PCR is 0.0
Starting from 2-2 μg of RNA, Ready-To
-Go RT-PCR beads (Amersham Pharmacia Biot)
ech). The primers used for LKP1 RNA amplification were the nucleotide sequence shown in SEQ ID NO: 7 corresponding to the first exon and SEQ ID NO: 8 corresponding to the second exon.
Is the base sequence shown in FIG. 18S ribosomal RNA
Was amplified using a Quantum RNA 18S internal standard primer for plants (Ambion, Austin, TX). The reaction volume for each reaction was 50 μl, and 10 μl of each reaction was placed in each lane.

(Transgenic Plant) A 1.5-kb 5 'non-coding region having a 21b overlap with the LKP1 coding sequence from the start codon of ATG is composed of Pfu Turbo DNA polymerase (Stratagen) from genomic DNA of Arabidopsis thaliana, Colombia ecotype.
PCR amplified by e). The primer used was
This is the nucleotide sequence shown in SEQ ID NOS: 9 and 10 in which an XbaI site and a SmaI site have been introduced into the respective ends. The PCR fragment was pCR-Blunt II-TOPO
(Invitrogen), sequenced to identify sequence, promoterless β-glucuronidase (GUS) expression vector pBI101 (Clonte
ch, Pa Alto, CA). The resulting construct was sequenced and confirmed in-frame fusion of the LKP1 7 amino acid region to the GUS protein region.

Cauliflower mosaic virus 35S
The expression vector pBE2113 containing the promoter is
Used for overexpression of KP1. The LKP1 coding sequence is Pfu Turbo or LA-TagDN.
SEQ ID NOS: 11 and 1 amplified by A polymerase (Takara, Kyoto) and having BamHI sites introduced at each end
The nucleotide sequence shown in No. 2 was used as a primer.
The PCR fragment was pCR-BluntII-TOPO or pC
R2.1-TPOPO (Invitrogen), sequenced to identify sequence,
Connected to BE2113. The sense orientation construct was selected and used for transformation.

An S65T modification version of GFP (smRS-GFP) (Davis and Vierstr
a, Plant Mol. Biol. 36, 521-528, 1998)
Used to make KP1 constructs. The GFP coding region is Arabidopsis Biological
psmR from al Resource Center
Primers having the nucleotide sequences shown in SEQ ID NOs: 13 and 14 which were amplified by S-GFP and had XbaI and StuI sites introduced at their respective ends were used. The LKP1 coding region was PCR amplified, StuI site and Ba
SEQ ID NOS: 15 and 16 with mHI sites introduced at each end
Were used. Both PCs
The R fragment was subcloned, sequenced to identify the sequence, ligated at the StuI site, and then GFP
-The LKP1 fragment was introduced into pBE2113. Transformation of Arabidopsis thaliana via Agrobacterium
It was performed by a simplified implanta infiltration method (Plant J, 16, 735-743, 1998). Transformed lines were selected on the germination agar medium containing 50 μg / ml kanamycin.

(GUS Staining and GFP Observation) Histochemical localization of GUS activity in transgenic plants was performed by the method of Nakajima et al. (Plant J, 12, 851-861, 1997). Removing the roots and leaves of the 35S :: GFP-LKP1 plant,
The cells were incubated in the dark for 30 minutes and immersed in water, after which the cells were observed with a Zeiss Axioplan microscope.

Example 1 [Separation of LKP1 sequence] In order to identify a new blue light receptor, NPH1LOV
A search of the Arabidopsis thaliana database for domain sequences revealed several sequences. One sequence was located in the PI clone of MSF19 in chromosome 5. The DNA sequence was analyzed by the GENSCAN program to identify the sequence encoding the protein containing the LOV domain. The cDNA for the protein was screened from a cDNA library from 3 day old hypocotyls (Cell, 1993, 72, 427-
441). Eighteen clones were isolated from 4 × 300,000 phage and analyzed. Since terminal sequencing of these clones revealed that the nine cDNAs corresponded to the open reading frame (ORF), the nucleotide sequence of all nine of these clones was determined. The longest clone has a molecular weight of 65905.17 Da.
Encoding a protein consisting of 610 amino acids
229 with 830 bp ORF and nuclear localization signal
It had a 7 bp insert (see FIG. 1).

By BLAST search analysis, this protein has a LOV domain at the N-terminal part and a Kelch family (Cell, 1993, 72,
681-693; J. Med. Biol, 1994, 236, 1277-1282) had six internal repeats (see FIGS. 2 and 3). Therefore, this protein was converted to LKP1 (LOV
It was named Kerch protein 1). The LOV domain of LKP1 is the LOV domain of the blue signaling molecule of Arabidopsis NPH1 and Neurospora white collar-1 (EM
BO J, 1996, 15, 1650-1657) and Arabidopsis NPH
1L (NPL1) (Plant Physiol, 1998,117,719), A
Diantum PHY3 (PNAS, 1998, 95, 15826-15830) and the LOV domain of Halobacterium bacterio-opsin activator (J. Bacteriol, 1988, 170, 4903), which are considered photoreceptors, have significant sequence correlation. (48.8-58.1% identity, 74.
4-86.0% similarity). Kerch repetition of LKP1 is caused by yeast tip elongation abnormal protein 1 (yeast tip elonga
tion aberrant protein1: YEA1) and homology (25-29% identity, 37-45% similarity)
Human and mouse host-cell factor C1 (Cell, 1993,
74, 115-125) is defined as a homology (25-31% identity, 39-46% similarity) with Physarium actin-
Fragmin kinase (EMBO J, 1996, 15, 5547-5556)
Has homology (26-27% identity, 39-44% similarity), respectively. Kerch repeats were first identified in the Kerch gene product, a cyclic water tube component that regulates cytoplasmic flow between nurse and oocytes during Drosophila oocyte maturation (Cell, 1993, 72,681).
-693), which is present in proteins known to bind to F-actin (Cell, 1993, 72,681-693; EMBO J,
1996, 15, 5547-5556; Genes Dev., 1995, 9, 1074-108
6). Seven Kerch repeats of the fungus Dactylium dendroides
From the analogy with the established three-dimensional structure of galactose oxidase from
It is assumed to be a one-domain super-barrel structure consisting of β-strands.

Example 2 Organ-Specific Expression of LKP1 Gene and Promoter Activity Organ-specific LKP1 mRNA levels were tested by RT-PCR. 2 μg of total RNA was used in each reaction and 18S rRNA was amplified as a control. The results are shown in FIG. 4 (see Reference Photo 1). LKP1 mRNA
Was found in all organs and at different levels. Dried seed (DS) <stem (St) <silique (Si)
<Root (R) <Flower (F) and leaf (CL, RL). Since the mRNA levels of some blue receptors have been reported to change during the day, LKP1 mRN in rosette leaves
A levels were checked every 4 hours, but no clear rhythmicity in RNA expression could be detected. The promoter activity of the LKP1 gene was determined histochemically by the GUS activity of transgenic Arabidopsis thaliana having the LKP1P :: GUS construct. The GUS activity when a transgenic Arabidopsis thaliana (T3) plant having the LKP1 promoter / GUS fusion gene was sown on an agar germination medium containing 50 μg / ml kanamycin was measured for seedling (A), rosette leaf (B), and flower (C). Fig. 5 (see Reference Photo 2) shows the results of examination on the stalk upper leaves (D) and the silique fruit (E).
Shown in Strong signals were detected in cotyledons (FIG. 5A), rosette leaves (FIG. 5B), and upper stem leaves (FIG. 5D). GUS activity was also detected in roots, calyxes, siliques and siliques with floral axes, but activity was minimal in hypocotyls, petiole bases, petals, young floral axes and major axes. This promoter activity was consistent with the LKP1 mRNA level shown in FIG. 4, indicating that the 1.5 kb promoter region plays an important role in regulating LKP1 gene expression.

Example 3 [Properties of transgenic Arabidopsis thaliana having 35S-linked LKP1] (Elongation of hypocotyl and petiole) In order to examine the function of LKP1,
The LKP1 gene knockout line was PCR-screened from the 50,000 T-DNA pools made at the Kazusa DNA Research Center, but no knockout line was found. The transgenic Arabidopsis thaliana expressed in was prepared as described above (PlantCell Physiol, 1996, 3
7, 49-59, Method Enzymol, 1987, 153, 253-277). As shown in FIG. 6 (see Reference Photo 3), this transgenic plant overexpresses the transformed LKP1 and has a phenotype with hypocotyls and petiole extended under long and short days. (FIGS. 6A and C show 6 day old T2 seedlings and 2 week old 35S :: LKP1 rosette plants).
No elongated hypocotyl and petiole phenotype of 35S :: LKP1 seedlings was observed in plants grown in the dark. Three
Epidermal cells of the hypocotyl of 5S :: LKP1 plants were 3-5 times longer than those of control or wild type (FIGS. 6B and D).
Shows that 35S :: LKP1 and 35S grown under long-day conditions in agar germination medium containing 50 μg / ml kanamycin.
FIG. 6E shows a vector-only control transformant.
S :: LKP1 seedling epidermal cells, and FIG. 6F is a target epidermal cell. ), But the total number of epidermal cells in the hypocotyl was the same as in the axis. The petiole of 35S :: LKP1 seedlings also had elongated epidermal cells.

(Cotyledon Movement) Another characteristic of the 35S :: LKP1 seedling was clear cotyledon movement. Five-day-old seedlings were grown on agar germination medium under long-day conditions and photographed every 4 hours. ZT is the number of hours since Akatsuki. The cotyledon of 35S :: LKP1 seedling is midnight (ZT20-
0) vertically and horizontally in the afternoon (ZT8-16). On the other hand, the control and wild-type seedlings did not show such clear cotyledon movement under long-day conditions (FIG. 7; see Reference Photo 4). This is presumably because in 35S :: LKP1 seedlings, the cotyledon petioles grow at different cell expansion rates between the inner epidermal cells and the outer cells. In Arabidopsis grown at relatively high light intensity, the LKP1 gene promoter activity was not detected in the cotyledon petiole (FIG. 5A), so that clear cotyledon movement was due to ectopically expressed LKP1 in cotyledons. Maybe. It is known that cotyledon and leaf movements of Arabidopsis show a circadian rhythm (Science, 1996, 274, 790-792; Cell, 1998, 93, 1219).
-1229). GIGANTEA (GI) and delayed elongation hypocotyl (LHY) genes are known to be involved in the process (Science, 1999, 285, 1579-1582). Therefore, LKP1 may interact with these regulatory molecules in some way.

(Delayed Flowering) The 35S :: LKP1 transgenic Arabidopsis thaliana of the present invention is largely characterized by a delayed flowering phenotype. FIG. 8 (see Reference Photo 5) shows the experimental results of delayed flower formation when grown under long-day conditions. In FIG. 8, A is 4.5 weeks old of wild type (Columbia), and BF is 35S ::
LKP1 Arabidopsis thaliana, B is 4.5 weeks old, C is 11 weeks old, D is 12.5 weeks old, E is 8.5 weeks old, F is 1 week old
One week old, G shows 12 weeks old. Under long-day conditions, the Colombian wild-type plants flower about four weeks after sowing, whereas
5S :: LKP1 transgenic plants do not flower 4 weeks after sowing (FIGS. 8A and B). Also, the 35S :: LKP1 transgenic plant of the present invention exhibited two types of delayed flowering phenotype. Type 1 plants have a large number of rosette leaves (57.1 ± 6.
6) was formed. The lottery started 11 weeks after sowing (Fig. 8
C and D). Type 2 plants began to draw 6 weeks after sowing and had 17.4 ± 2.5 rosette leaves at that time. Type 2 plants have rosette-shaped stem upper leaves,
Many rosette leaves and extended primary floral inflorescences formed (FIGS. 8E and F). Type 1 and type 2 like phenotypes are found in wild plants grown under high light intensity and low light intensity, short day conditions, respectively.

The expression levels of the LKP1 gene of these type 1 and type 2 transgenic plants and wild type plants were measured by RT-PCR. 0.02-2 μg total R
LKP1 expression levels in 35S :: LKP1 transgenic and wild-type Arabidopsis
The results of the T-PCR are shown in FIG. 9 (see Reference Photo 6). Type 2 plants accumulate much higher levels of LKP1 mRNA than wild type, while type 1 plants accumulate higher levels of LKP1 mRNA than type 2 plants. Analysis of the phenotypes of these two different types of T3 plants showed that the kanamycin-resistant T3 plants had a 1: 2 ratio of both type 1 and type 2 phenotypes (18: 4
1, x ² = 0.22, P> 0.95). The different LKP-OX phenotypes may be due to differences in LKP1 expression levels and / or copy number of the transformed gene.

In Arabidopsis thaliana, more than 50 flowering-time regulator genes have been identified and characterized by a genetic method using mutants, and several genetic pathways may form part of the regulatory process. Is considered (Pl
ant Cell, 1998, 10, 1973-1989; Plant Physiol, 199
8, 117, 1-98). The autonomous pathway regulates flowering independently of the photoperiod. Mutants such as fca and ld flower longer and shorter than wild-type under long and short days and exhibit a reduced flowering period in response to vernalization, so that the corresponding genes are autonomous pathways It is thought to work with. The FCA gene encodes an RNA linking the protein to the protein-protein interaction domain (Cell, 1997, 89, 737-74).
5) The LUMINIDEPENDENS gene encodes a glutamine-rich homeobox transcription factor (Pl
ant Cell, 1994, 6, 75-83). The long day pathway regulates flowering specifically corresponding to long day conditions. Mutations or deletions in long-day pathway genes, such as co, fha and gi, cause delayed flowering under long-day conditions, but do not occur under short-day conditions, and suddenly Mutants have little or no response to vernalization. CO encodes a putative transcription factor with two zinc fingers (Cell, 1995, 80, 847-857), and FHA encodes cryptochrome 2 (CRY2) (Science, 1).
998, 279, 1360-1363), GI encodes a novel membrane protein (Science, 1999, 285, 1579-1582; EMBO J, 1).
999, 18, 4679-4688). The short day or GA pathway regulates flowering under short day conditions mediated by gibberellic acid. Vernalization promotes flowering, presumably because it reduces DNA methylation.

In order to classify the LKP1 functional pathway in 35S :: LKP1 plants, the effects of light conditions and vernalization on the type 1 delayed flowering phenotype of 35S :: LKP1 plants were determined by the number of rosette leaves before the drawer. The analysis was performed (Fig. 1
0). Flowering time was measured by the total number of leaves of the plant before the lottery.
Colombian wild species, 35S :: LKP1 transgenic plant, fve-2 mutant plant were incubated with moistened seeds at 4 ° C. for 30 days under short-day conditions for vernalization, and under long-day conditions. (18 hours light irradiation, 6 hours dark),
Alternatively, the cells were grown under short-day conditions (light irradiation for 9 hours, darkness for 15 hours). The delayed flowering of the 35S :: LKP1 plant was clearly observed under the long day condition, but did not occur under the short day condition. Because the fve mutants flower slower than wild-type plants under both long and short day conditions and show reduced flowering time in response to vernalization, fve is an autonomous pathway. (Cold Spring Ha
rborLab. Press, NY, 1994, pp403-433; Plant Physio
l, 1998, 117, 1-8). In fact, a mutant fv susceptible to vernalization
The delayed flowering phenotype of e-2 is affected by vernalization under long and short day conditions. On the other hand, against the delayed flowering phenotype of 35S :: LKP1, vernalization had no effect on either long day or short day conditions. Therefore, it is concluded that LKP1 functions in the long-day pathway of flowering in Arabidopsis thaliana (Plant Cell, 1998, 10, 1973-1989; Plant Physi
l, 1998, 117, 1-8).

(Intracellular localization of GFP-LKP1) LK
To visualize the subcellular localization of P1, GFP-LKP
After the 35S promoter derived from one fusion construct was introduced into Arabidopsis thaliana, it was observed (FIG. 11; see Reference Photo 7). FIG. 11A shows control T2 plants containing only the 35S vector, and FIG. 11B shows 35S :: LKP1 transgenic plants grown on agar GM containing 50 μg / ml kanamycin under long-day conditions. 35S :: GFP-LKP1
The plant roots were observed with a light microscope or a fluorescence microscope.
Transgenic plants exhibited a delayed flowering phenotype under long day conditions (FIG. 11B), indicating that GFP-LKP
1 indicates that it is functionally active. Afternoon, 35
When the root cells of the S :: GFP-LKP1 plant were viewed with a fluorescence microscope, it was found that the GFP-related fluorescence was distributed in the nucleus but not in the nucleolus (FIGS. 11C and D). This nuclear localization of GFP-LKP1 was also detected in leaf cell thread and epidermal cells. On the other hand, the signal was detected in the early morning cytosol under long day conditions (FIG. 11).
F). This cytosol localization of GFP-LKP1 did not change during a 1 hour incubation under white light or dark conditions.

[0039]

The transgenic plant makes it possible to carry out planned plant cultivation irrespective of fluctuations in harvest time and amount due to temperature changes. In other words, the flowering time can be kept constant regardless of the photoperiod or temperature change. In other words, the flowering can be delayed without using a light irradiation device or the like that controls the photoperiod, thereby reducing lighting costs and equipment costs. It is possible to do. Further, by delaying the lottery and flowering, it is possible to improve the leaf production per unit plant. In particular, the transgenic plants of the present invention growing under long-day conditions continue to grow vegetatively when the wild type is performing flower bud formation, resulting in many rosette leaves per individual, And horticultural varieties can make the flowering time constant regardless of the photoperiod,
Furthermore, it can be expected that leaf production per unit plant will be improved. Also, if applied to short-term plants growing in areas with long-term warm climates, leaves, flowers,
Fruit and seed production can be increased. Furthermore, since the present transgenic plant takes a form different from the original plant, it is possible to create a commercial plant useful for appreciation. In addition, flowering of Arabidopsis is usually
It is induced by the low-temperature vernalization treatment even under short-day conditions, but flowering induction by the low-temperature treatment is not observed in LKP1 and LKP2 overexpression Arabidopsis thaliana. If this is used for crops, it is possible to prevent undesired crops due to temperature changes and flowering that reduces the commercial value.

[0040]

[Sequence List] SEQUENCE LISTING <110> JAPAN SCIENCE AND TECHNOLOGY CORPORATION <120> Transgenic Plants <130> 12-023 <140> <141> <160> 16 <170> PatentIn Ver. 2.1 <210> 1 <211> 2297 <212> DNA <213> Arabidopsis thaliana <220> <221> CDS <222> (135) .. (1964) <400> gac agt ggt tcc gat cta agc gcc gat 170 Met Glu Trp Asp Ser Gly Ser Asp Leu Ser Ala Asp 1 5 10 gat gct tcg tca ctg gcg gat gat gaa gag gga ggt ctt ttt ccc gga 218 Asp Ala Ser Ser Leu Ala Asp Asp Glu Glu Gly Gly Leu Phe Pro Gly 15 20 25 ggt gga cca att cca tat ccc gtt ggg aat ttg ctt cac acg gcg cct 266 Gly Gly Pro Ile Pro Tyr Pro Val Gly Asn Leu Leu His Thr Ala Pro 30 35 40 tgt gga ttc gtt gtt act gat gcc gtt gag ccg gac caa cct att ata 314 Cys Gly Phe Val Val Thr Asp Ala Val Glu Pro Asp Gln Pro Ile Ile 45 50 55 60 tat gtc aac acc gtc ttc gaa atg gtt aca ggg tat cg t gct gag gaa 362 Tyr Val Asn Thr Val Phe Glu Met Val Thr Gly Tyr Arg Ala Glu Glu 65 70 75 gtt ctc gga gga aat tgc cgc ttc ttg caa tgt aga gga ccg ttt gct 410 Val Leu Gly Gly Asn Cys Arg Phe Leu Gln Cys Arg Gly Pro Phe Ala 80 85 90 aaa aga agg cat cca tta gtt gac tct atg gtt gtt tcc gag ata aga 458 Lys Arg Arg His Pro Leu Val Asp Ser Met Val Val Ser Glu Ile Arg 95 100 105 aaa tgt att gat gag ggc att gaa ttt caa ggc gag ttg ttg aac ttt 506 Lys Cys Ile Asp Glu Gly Ile Glu Phe Gln Gly Glu Leu Leu Asn Phe 110 115 120 agg aaa gat gga tct ccg ttg atg aat aga ttg cgg ttg act Lys Asp Gly Ser Pro Leu Met Asn Arg Leu Arg Leu Thr Pro Ile 125 130 135 140 tat gga gac gat gat act att acc cat ata att gga atc cag ttc ttt 602 Tyr Gly Asp Asp Asp Thr Ile Thr His Ile Ile Gly Ile Gln Phe Phe 145 150 155 atc gag aca gac att gac ctt gga cct gtt ctt gga tct tca aca aaa 650 Ile Glu Thr Asp Ile Asp Leu Gly Pro Val Leu Gly Ser Ser Thr Lys 160 165 170 gaa aag tca att gat ggg ata tac tca gct tta gct gct ggg gag cgg 698 Glu Lys Ser Ile Asp Gly Ile Tyr Ser Ala Leu Ala Ala Gly Glu Arg 175 180 185 aat gtt tcc cga gga atg tgt ggg tta ttc cag ctg agt gac gag gtt 746 Asn Val Ser Arg Gly Met Cys Gly Leu Phe Gln Leu Ser Asp Glu Val 190 195 200 gtg tct atg aag ata ttg tca cgc ctg aca cca aga gac gtt gca tct 794 Val Ser Met Lys Ile Leu Ser Arg Leu Thr Pro Arg Asp Val Ala Ser 205 210 215 220 gtt agc tct gtg tgc cgc agg cta tat gtg ctg aca aag aat gag gat 842 Val Ser Ser Val Cys Arg Arg Leu Tyr Val Leu Thr Lys Asn Glu Asp 225 230 235 ctt tgg cga agg gtt tgt cag aat gca tgg gga agt cgg aca aca 890 Leu Trp Arg Arg Val Cys Gln Asn Ala Trp Gly Ser Glu Thr Thr Arg 240 245 250 gta tta gag act gtt cct ggt gct aaa aga ctc ggt tgg ggc cgc ttg 938 Val Leu Glu Thr Val Pro Gly Ala Lys Arg Leu Gly Trp Gly Arg Leu 255 260 265 gct aga gaa ctg acc acc ctt gaa gct gca gct tgg aga aaa ctt tcg 986 Ala Arg Glu Leu Thr Thr Leu Glu Ala Ala Ala Trp Arg Lys Leu Ser 270 275 280 gtt gga ggt tct gtt gag cct tctcgc tgt aat ttt agt gct tgt gct 1034 Val Gly Gly Ser Val Glu Pro Ser Arg Cys Asn Phe Ser Ala Cys Ala 285 290 295 300 gtc ggg aac aga gtt gtt ctg ttt gga gga gaa ggt gtg aat atg cag 1082 Val Gly Asn Arg Val Val Leu Phe Gly Gly Glu Gly Val Asn Met Gln 305 310 315 cct atg aat gat acc ttt gtt tta gat ctg aat tct gat tac cct gaa 1130 Pro Met Asn Asp Thr Phe Val Leu Asp Leu Asn Ser Asp Tyr Pro Glu 320 325 330 tgg caa cat gtc aaa gtg agc tcc cct cct cca gga cgc tgg ggc cac 1178 Trp Gln His Val Lys Val Ser Ser Pro Pro Pro Gly Arg Trp Gly His 335 340 345 acg ctc act tgt gta aat ggc tcc aat ctg gtt gta ttt ggt ggc tgt 1226 Thr Leu Thr Cys Val Asn Gly Ser Asn Leu Val Val Phe Gly Gly Cys 350 355 360 ggt cag caa ggt ttg ctg aac gat gtg ttt gtc ttg aat ctc gat gct 1274 Gly Gln Gln Gly Gly Leu Leu Asn Asp Val Phe Val Leu Asn Leu Asp Ala 365 370 375 380 aag ccg cct act tgg agg gaa att tcg ggt ttg gct ccg cct cta cca 1322 Lys Pro Pro Thr Trp Arg Glu Ile Ser Gly Leu Ala Pro Pro Leu Pro 385 390 395 395 aga tca tgg cac agc tca tgc acc ctt gac gga acc aaa ctg ata gtc 1370 Arg Ser Trp His Ser Ser Cys Thr Leu Asp Gly Thr Lys Leu Ile Val 400 405 410 tct gga ggc tgt gca gat tcc ggc gtt ctt cta agt gac acg ttc ctc 1418 Ser Gly Gly Cys Ala Asp Ser Gly Val Leu Leu Ser Asp Thr Phe Leu 415 420 425 ctc gac ctt tca ata gag aaa cca gtg tgg aga gag att cca gct gcc 1466 Leu Asp Leu Ser Ile Glu Lys Pro Val Trp Arg Glu Ile Pro Ala Ala 430 435 440 tgg act ccg cct tcc cgg tta ggc cac aca cta tcg gtt tat gga gga 1514 Trp Thr Pro Pro Ser Arg Leu Gly His Thr Leu Ser Val Tyr Gly Gly 445 450 450 455 460 aga aag atc ttg atg ttt ggt ggt ctt gct aag agc ggg cct ttg aaa 1562 Arg Lys Ile Leu Met Phe Gly Gly Leu Ala Lys Ser Gly Pro Leu Lys 465 470 475 ttc cgt tcg agt gat gtc ttc aca atg gat cta agc gaa gag gag Serc 16 Serg Asp Val Phe Thr Met Asp Leu Ser Glu Glu Glu Pro 480 485 490 tgt tgg agg tgt gtg acc ggt agc gga atg cct gga gca gga aac cca 1658 Cys Trp Arg Cys Val Thr Gly Ser Gly Met Pro Gly Ala Gly Asn Pro 4 95 500 505 gga gga gta gca cca cca cca agg cta gat cac gtt gca gtt aac ctc 1706 Gly Gly Val Ala Pro Pro Pro Arg Leu Asp His Val Ala Val Asn Leu 510 515 520 cct gga ggc aga atc ttg ata ttt ggc ggc tca gtg gca ggg ctt cac 1754 Pro Gly Gly Arg Ile Leu Ile Phe Gly Gly Ser Val Ala Gly Leu His 525 530 535 540 tca gcg tct cag ctt tat cta ctt gac cca acg gag gac aaa ccg act 1802 Ser Ala Ser Gln Leu Tyr Leu Leu Asp Pro Thr Glu Asp Lys Pro Thr 545 550 555 tgg aga ata cta aac att cca gga aga ccg cct cgg ttt gct tgg gga 1850 Trp Arg Ile Leu Asn Ile Pro Gly Arg Pro Pro Arg Phe Ala Trp Gly 560 565 570 570 cat ggc act tgt gtt gtg gga gga aca cga gcg ata gtg cta ggt ggt 1898 His Gly Thr Cys Val Val Gly Gly Thr Arg Ala Ile Val Leu Gly Gly 575 580 585 cag acc gga gaa gaa tgg atg cta agt gag cta cac gaa tta tca c 1946 Gln Thr Gly Glu Glu Trp Met Leu Ser Glu Leu His Glu Leu Ser Leu 590 595 600 gcg agc tat ctc acg taa ccaaagagag cctatgtatg aaagagaacc 1994 Ala Ser Tyr Leu Thr 605 610 cagccataga agaagatgaa aaa gggttct cggttaggat actggagctt taggggagag 2054 cgactcagtg gtgggacttt ggtactgctc atatttttta agtccttctt cttctttctt 2114 cctcattagg aggactttta gtgcttatat aatatatata gtgaaaatgt catctctctc 2174 tttctctctg tagtgcttgt ttctgttgtt ctctttgaag tttgagaagt gcatttgtgt 2234 tcatattctg taaattttat ttatgattac aaactttaat aagatccttt ctcttcttat 2294 gct 2297 <210> 2 <211> 609 <212> PRT <213> Arabidopsis thaliana <400> 2 Met Glu Trp Asp Ser Gly Ser Asp Leu Ser Ala Asp Asp Ala Ser Ser 1 5 10 15 Leu Ala Asp Asp Glu Glu Gly Gly Leu Phe Pro Gly Gly Gly Pro Ile 20 25 30 Pro Tyr Pro Val Gly Asn Leu Leu His Thr Ala Pro Cys Gly Phe Val 35 40 45 Val Thr Asp Ala Val Glu Pro Asp Gln Pro Ile Ile Tyr Val Asn Thr 50 55 60 Val Phe Glu Met Val Thr Gly Tyr Arg Ala Glu Glu Val Leu Gly Gly 65 70 75 80 Asn Cys Arg Phe Leu Gln Cys Arg Gly Pro Phe Ala Lys Arg Arg His 85 90 95 Pro Leu Val Asp Ser Met Val Val Ser Glu Ile Arg Lys Cys Ile Asp 100 105 110 Glu Gly Ile Glu Phe Gln Gly Glu Leu Leu Asn Phe Arg Lys Asp Gly 115 120 125 Ser Pro Leu Met Asn Arg Leu Arg Leu Thr Pro Ile Tyr Gly Asp Asp 130 135 140 Asp Thr Ile Thr His Ile Ile Gly Ile Gln Phe Phe Ile Glu Thr Asp 145 150 155 160 Ile Asp Leu Gly Pro Val Leu Gly Ser Ser Thr Lys Glu Lys Ser Ile 165 170 175 Asp Gly Ile Tyr Ser Ala Leu Ala Ala Gly Glu Arg Asn Val Ser Arg 180 185 190 Gly Met Cys Gly Leu Phe Gln Leu Ser Asp Glu Val Val Ser Met Lys 195 200 205 Ile Leu Ser Arg Leu Thr Pro Arg Asp Val Ala Ser Val Ser Ser Val 210 215 220 Cys Arg Arg Leu Tyr Val Leu Thr Lys Asn Glu Asp Leu Trp Arg Arg 225 230 235 240 Val Cys Gln Asn Ala Trp Gly Ser Glu Thr Arg Val Leu Glu Thr 245 250 255 Val Pro Gly Ala Lys Arg Leu Gly Trp Gly Arg Leu Ala Arg Glu Leu 260 265 270 Thr Thr Leu Glu Ala Ala Ala Trp Arg Lys Leu Ser Val Gly Gly Ser 275 280 285 Val Glu Pro Ser Arg Cys Asn Phe Ser Ala Cys Ala Val Gly Asn Arg 290 295 300 Val Val Leu Phe Gly Gly Glu Gly Val Asn Met Gln Pro Met Asn Asp 305 310 315 320 Thr Phe Val Leu Asp Leu Asn Ser Asp Tyr Pro Glu Trp Gln His Val 325 330 335 Ly s Val Ser Ser Pro Pro Pro Gly Arg Trp Gly His Thr Leu Thr Cys 340 345 350 Val Asn Gly Ser Asn Leu Val Val Phe Gly Gly Cys Gly Gln Gln Gly 355 360 365 Leu Leu Asn Asp Val Phe Val Leu Asn Leu Asp Ala Lys Pro Pro Thr 370 375 380 Trp Arg Glu Ile Ser Gly Leu Ala Pro Pro Leu Pro Arg Ser Trp His 385 390 395 400 Ser Ser Cys Thr Leu Asp Gly Thr Lys Leu Ile Val Ser Gly Gly Cys 405 410 415 Ala Asp Ser Gly Val Leu Leu Ser Asp Thr Phe Leu Leu Asp Leu Ser 420 425 430 Ile Glu Lys Pro Val Trp Arg Glu Ile Pro Ala Ala Trp Thr Pro Pro 435 440 445 Ser Arg Leu Gly His Thr Leu Ser Val Tyr Gly Gly Arg Lys Ile Leu 450 455 460 Met Phe Gly Gly Leu Ala Lys Ser Gly Pro Leu Lys Phe Arg Ser Ser 465 470 475 480 Asp Val Phe Thr Met Asp Leu Ser Glu Glu Glu Pro Cys Trp Arg Cys 485 490 495 Val Thr Gly Ser Gly Met Pro Gly Ala Gly Asn Pro Gly Gly Val Ala 500 505 510 Pro Pro Pro Arg Leu Asp His Val Ala Val Asn Leu Pro Gly Gly Arg 515 520 525 Ile Leu Ile Phe Gly Gly Ser Val Ala Gly Leu His Ser Ala Ser Gln 530 535 540 Leu Ty r Leu Leu Asp Pro Thr Glu Asp Lys Pro Thr Trp Arg Ile Leu 545 550 555 560 Asn Ile Pro Gly Arg Pro Pro Arg Phe Ala Trp Gly His Gly Thr Cys 565 570 575 Val Val Gly Gly Thr Arg Ala Ile Val Leu Gly Gly Gln Thr Gly Glu 580 585 590 Glu Trp Met Leu Ser Glu Leu His Glu Leu Ser Leu Ala Ser Tyr Leu 595 600 605 Thr <210> 3 <211> 2265 <212> DNA <213> Arabidopsis thaliana <220> <221> CDS <222> (230) .. (2065) <400> 3 ctcttttctt cgctcgattc catttcaaaa tctgttcttc tatctcctgt tcctgaagaa 60 gacgatttct gggtttgtct aaagtttgga actttttttc ctgtggagac ttcgattacc 120 tacagatatc agattcctcg attcaaaatc aaactttgtt cttctgggtc tcttctcctg 180 ttgctgagga agaagattag tgggcttaag ctttattttg tgggtgtgt atg caa aat 238 Met Gln Asn 1 caa atg gag tgg gat agt gat tcc gat ctc agc ggt gga gat gaa gtg 286 Gln Met Glu Trp Asp Ser Asp Ser Asp Leu Ser Gly Gly Asp Glu Val 5 10 15 gcg gag gat gga tgg ttc ggt gga gat aac ggagg atg ttt ccg 334 Ala Glu Asp Gly Trp Phe Gly Gly Asp Asn Gly Ala Ile Pro Phe Pro 20 25 30 35 gta ggt agt ctt c ct ggc acg gcg ccg tgt ggg ttt gtg gtc agc gat 382 Val Gly Ser Leu Pro Gly Thr Ala Pro Cys Gly Phe Val Val Ser Asp 40 45 50 gct ctt gaa cct gac aac cct atc att tat gtt aac act gtt ttt gag 430 Ala Leu Glu Pro Asp Asn Pro Ile Ile Tyr Val Asn Thr Val Phe Glu 55 60 65 att gtt act ggt tat cga gct gaa gaa gtt att ggt cga aat tgc cgt 478 Ile Val Thr Gly Tyr Arg Ala Glu Glu Val Ile Gly Arg Asn Cys Arg 70 75 80 ttc ttg caa tgt aga ggg cca ttt act aaa aga agg cat cca atg gta 526 Phe Leu Gln Cys Arg Gly Pro Phe Thr Lys Arg Arg His Pro Met Val 85 90 95 gat tct aca att gta gcg aag atg cga caa tgt cta gaa aat ggc atc 574 Asp Ser Thr Ile Val Ala Lys Met Arg Gln Cys Leu Glu Asn Gly Ile 100 105 110 115 gag ttt caa ggc gag ttg ctc aac ttt cgg aaa gat gga tct ccc ttg 622 Glu Phe Gln Glu Glu Leu Asn Phe Arg Lys Asp Gly Ser Pro Leu 120 125 130 atg aac aag ctg cgt cta gtc cct atc cgt gaa gaa gac gaa atc act 670 Met Asn Lys Leu Arg Leu Val Pro Ile Arg Glu Glu Asp Glu Ile Thr 135 140 145 cat ttc ata ggc g tt ctc tta ttt aca gat gca aaa att gat ctt ggc 718 His Phe Ile Gly Val Leu Leu Phe Thr Asp Ala Lys Ile Asp Leu Gly 150 155 160 cca tct cct gat tta tct gca aaa gaa att cct aga ata tct cgc tca766 Ser Pro Asp Leu Ser Ala Lys Glu Ile Pro Arg Ile Ser Arg Ser 165 170 175 ttt act tct gct tta cca atc gga gag cgt aat gtt tct cgt gga ctc 814 Phe Thr Ser Ala Leu Pro Ile Gly Glu Arg Asn Val Ser Arg Gly Leu 180 185 190 195 tgt ggg ata ttc gaa ctg agt gat gaa gta ata gct atc aag ata ttg 862 Cys Gly Ile Phe Glu Leu Ser Asp Glu Val Ile Ala Ile Lys Ile Leu 200 205 210 tca cag ttg act cct ggt gat gca tcg gtt ggt tgt gta tgt agg 910 Ser Gln Leu Thr Pro Gly Asp Ile Ala Ser Val Gly Cys Val Cys Arg 215 220 225 cgg ctc aat gag ttg aca aag aat gat gat gtg tgg aga atg gtc tgt 958 Arg Leu Asn Glu Leu Thr Lys Asn Asp Asp Val Trp Arg Met Val Cys 230 235 240 caa aac aca tgg ggt acc gag gca aca cgt gtt ctt gag agt gtt ccc 1006 Gln Asn Thr Trp Gly Thr Glu Ala Thr Arg Val Leu Glu Ser Val Pro 245 250 255 ggt gca aaa agg att ggt tgg gtg cga ctg gcc cga gag ttc acc act 1054 Gly Ala Lys Arg Ile Gly Trp Val Arg Leu Ala Arg Glu Phe Thr Thr 260 265 270 275 275 cac gaa gca aca gca tgg agg aag ttt tca gtt gga gact gtt gag 1102 His Glu Ala Thr Ala Trp Arg Lys Phe Ser Val Gly Gly Thr Val Glu 280 285 290 cct tct cgg tgt aat ttc agc gca tgt gca gtt ggg aac agg att gtt 1150 Pro Ser Arg Cys Asn Phe Ser Ala Cys Ala Val Gly Asn Arg Ile Val 295 300 305 atc ttt ggc ggg gaa ggt gtg aat atg cag ccg atg aat gat aca ttt 1198 Ile Phe Gly Gly Glu Glu Gly Val Asn Met Gln Pro Met Asn Asp Thr Phe 310 315 320 gtg ttg gac ctt gg agt agt ccc gag tgg aaa tct gtt ctg gtc 1246 Val Leu Asp Leu Gly Ser Ser Ser Pro Glu Trp Lys Ser Val Leu Val 325 330 335 agc tct cct cct ccg ggt cgc tgg ggt cac aca ctt tct tgt gtc aac 1294 Ser Ser Pro Pro Pro Gly Arg Trp Gly His Thr Leu Ser Cys Val Asn 340 345 350 355 ggt tcc cgt tta gta gtg ttt gga ggt tac ggg agc cac gga tta ctc 1342 Gly Ser Arg Leu Val Val Phe Gly Gly Tyr Gly Ser His Gly Leu Leu 360 365 370 aat gat gtg ttc tta tta gac ctt gat gca gac cca cca agc tgg agg 1390 Asn Asp Val Phe Leu Leu Asu Leu Asp Ala Asp Pro Pro Ser Trp Arg 375 380 385 gaa gta tcc gga ttg gcc cctca cca aga tca tgg cac agt tcg 1438 Glu Val Ser Gly Leu Ala Pro Pro Ile Pro Arg Ser Trp His Ser Ser 390 395 400 tgc aca ctt gat ggc act aag ctg att gta tct ggt ggt tgt gct gat 1486 Cys Thr Leu Asp Gly Thr Lys Leu Ile Val Ser Gly Gly Cys Ala Asp 405 410 415 tca gga gct cta ctc agc gat aca ttc ctg ctt gac cta tcg atg gat 1534 Ser Gly Ala Leu Leu Ser Asp Thr Phe Leu Leu Asp Leu Ser Met Asp 420 425 430 435 ata cct gct tgg agg gag ata ccg gtt cca tgg act ccc cca tct cgc 1582 Ile Pro Ala Trp Arg Glu Ile Pro Val Pro Trp Thr Pro Pro Ser Arg 440 445 450 ctc ggg cat act cta act gtc tat ggt gac cgc aag att ctt atg ttt 1630 Leu Gly His Thr Leu Thr Val Tyr Gly Asp Arg Lys Ile Leu Met Phe 455 460 465 ggc ggt cta gct aaa aac ggt act ttg aga ttc cgc tct aac gat gta 1678 Gly Gly Leu Ala Lys Asn Gly Thr Leu A rg Phe Arg Ser Asn Asp Val 470 475 480 tac acg atg gat ctt agc gag gat gaa ccg agt tgg aga cct gtg att 1726 Tyr Thr Met Asp Leu Ser Glu Asp Glu Pro Ser Trp Arg Pro Val Ile 485 490 490 495 ggg tat gga tct agc ctc ccc gga ggc atg gca gct cca cca cca agg 1774 Gly Tyr Gly Ser Ser Leu Pro Gly Gly Met Ala Ala Pro Pro Pro Arg 500 505 510 510 515 ctg gac cat gtg gca atc agc ctt ccc ggt ggt aga atc ttg atc ttt Leu Asp His Val Ala Ile Ser Leu Pro Gly Gly Arg Ile Leu Ile Phe 520 525 530 ggt ggt tcg gtt gca ggg ctt gac tcg gct tct cag ctt tat ctt ctt 1870 Gly Gly Ser Val Ala Gly Leu Asp Ser Ala Ser Gln Leu Tyr Leu Leu 535 540 545 gat cca aat gag gag aaa ccg gca tgg aga ata ctg aat gtt cag gga 1918 Asp Pro Asn Glu Glu Lys Pro Ala Trp Arg Ile Leu Asn Val Gln Gly 550 555 560 ggt ccg ccg agg ttt gca tgg aca aca tgt gtg gtc gga gga 1966 Gly Pro Pro Arg Phe Ala Trp Gly His Thr Thr Cys Val Val Gly Gly 565 570 575 act cga ttg gtc gta tta ggt ggt cag acc ggt gaa gag tgg atg tta 2014 Thr Arg Leu Val V al Leu Gly Gly Gln Thr Gly Glu Glu Trp Met Leu 580 585 590 595 aat gaa gca cat gaa ctg tta cta gca act tct acc act gca agt act 2062 Asn Glu Ala His Glu Leu Leu Leu Ala Thr Ser Thr Thr Ala Ser Thr 600 605 610 tga tcaagtgatg aagttaaaaa ggcatttaga tgtgttggac aaaagatcta 2115 tacattgatc aaaaacatgt tcatattgtc tgtacatggt gagtttgata cgtccatgtt 2175 ttgttcatct gttgacttaa tgtttttttt ttgtatatgc aatattacga attggattgt 2235 acataacgtc ttttgtagtg caagtggcca 2265 <210> 4 <211> 611 <212> PRT <213> Arabidopsis thaliana <400> 4 Met Gln Asn Gln Met Glu Trp Asp Ser Asp Ser Asp Leu Ser Gly Gly 1 5 10 15 Asp Glu Val Ala Glu Asp Gly Trp Phe Gly Gly Asp Asn Gly Ala Ile 20 25 30 Pro Phe Pro Val Gly Ser Leu Pro Gly Thr Ala Pro Cys Gly Phe Val 35 40 45 Val Ser Asp Ala Leu Glu Pro Asp Asn Pro Ile Ile Tyr Val Asn Thr 50 55 60 Val Phe Glu Ile Val Thr Gly Tyr Arg Ala Glu Glu Val Ile Gly Arg 65 70 75 80 Asn Cys Arg Phe Leu Gln Cys Arg Gly Pro Phe Thr Lys Arg Arg His 85 90 95 Pro Met Val Asp Ser Thr Ile Val Ala Lys Met Arg Gln Cys Leu Glu 100 105 110 Asn Gly Ile Glu Phe Gln Gly Glu Leu Leu Asn Phe Arg Lys Asp Gly 115 120 125 Ser Pro Leu Met Asn Lys Leu Arg Leu Val Pro Ile Arg Glu Glu Asp 130 135 140 Glu Ile Thr His Phe Ile Gly Val Leu Leu Phe Thr Asp Ala Lys Ile 145 150 155 160 Asp Leu Gly Pro Ser Pro Asp Leu Ser Ala Lys Glu Ile Pro Arg Ile 165 170 175 Ser Arg Ser Phe Thr Ser Ala Leu Pro Ile Gly Glu Arg Asn Val Ser 180 185 190 Arg Gly Leu Cys Gly Ile Phe Glu Leu Ser Asp Glu Val Ile Ala Ile 195 200 205 Lys Ile Leu Ser Gln Leu Thr Pro Gly Asp Ile Ala Ser Val Gly Cys 210 215 220 Val Cys Arg Arg Leu Asn Glu Leu Thr Lys Asn Asp Asp Val Trp Arg 225 230 235 240 Met Val Cys Gln Asn Thr Trp Gly Thr Glu Ala Thr Arg Val Leu Glu 245 250 255 Ser Val Pro Gly Ala Lys Arg Ile Gly Trp Val Arg Leu Ala Arg Glu 260 265 270 Phe Thr Thr His Glu Ala Thr Ala Trp Arg Lys Phe Ser Val Gly Gly 275 280 285 Thr Val Glu Pro Ser Arg Cys Asn Phe Ser Ala Cys Ala Val Gly Asn 290 295 300 Arg Ile Val Ile Phe Gly Gly Glu Gly Val Asn Me t Gln Pro Met Asn 305 310 315 320 Asp Thr Phe Val Leu Asp Leu Gly Ser Ser Ser Pro Glu Trp Lys Ser 325 330 335 Val Leu Val Ser Ser Pro Pro Pro Gly Arg Trp Gly His Thr Leu Ser 340 345 350 Cys Val Asn Gly Ser Arg Leu Val Val Phe Gly Gly Tyr Gly Ser His 355 360 365 Gly Leu Leu Asn Asp Val Phe Leu Leu Asp Leu Asp Ala Asp Pro Pro 370 375 380 Ser Trp Arg Glu Val Ser Gly Leu Ala Pro Pro Ile Pro Arg Ser Trp 385 390 395 400 His Ser Ser Cys Thr Leu Asp Gly Thr Lys Leu Ile Val Ser Gly Gly 405 410 415 Cys Ala Asp Ser Gly Ala Leu Leu Ser Asp Thr Phe Leu Leu Asp Leu 420 425 430 Ser Met Asp Ile Pro Ala Trp Arg Glu Ile Pro Val Pro Trp Thr Pro 435 440 445 Pro Ser Arg Leu Gly His Thr Leu Thr Val Tyr Gly Asp Arg Lys Ile 450 455 460 Leu Met Phe Gly Gly Leu Ala Lys Asn Gly Thr Leu Arg Phe Arg Ser 465 470 475 475 480 Asn Asp Val Tyr Thr Met Asp Leu Ser Glu Asp Glu Pro Ser Trp Arg 485 490 495 Pro Val Ile Gly Tyr Gly Ser Ser Leu Pro Gly Gly Met Ala Ala Pro 500 505 510 Pro Pro Arg Leu Asp His Val Ala Ile Ser Leu Pr o Gly Gly Arg Ile 515 520 525 Leu Ile Phe Gly Gly Ser Val Ala Gly Leu Asp Ser Ala Ser Gln Leu 530 535 540 Tyr Leu Leu Asp Pro Asn Glu Glu Lys Pro Ala Trp Arg Ile Leu Asn 545 550 555 555 560 Val Gln Gly Gly Pro Pro Arg Phe Ala Trp Gly His Thr Thr Cys Val 565 570 575 Val Gly Gly Thr Arg Leu Val Val Leu Gly Gly Gln Thr Gly Glu Glu 580 585 590 Trp Met Leu Asn Glu Ala His Glu Leu Leu Leu Ala Thr Ser Thr Thr 595 600 605 Ala Ser Thr 610 <210> 5 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 5 gagttttatg gttttatcta cttgacccga 30 <210> 6 < 211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 6 actagcgatt acgctcggcc ggtagaggac 30 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence < 220> <223> Description of Artificial Sequence: Primer <400> 7 tcatggagtg ggacagtggt 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 8 caacaactcg c cttgaaatt 20 <210> 9 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 9 gagatctaga tagttgcccc atatcggtaa 30 <210> 10 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 10 tctccccggg gaaccactgt cccactccat 30 <210> 11 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 11 gagaggatcc tcatggagtg ggacagtggt 30 <210> 12 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 12 tctcggatcc ggttacgtga gatagctcgc 30 <210> 13 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 13 gagatctaga caatgagtaa aggagaagaa 30 <210> 14 <211> 30 <212> DNA < 213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 14 tctcaggcct ttgtatagtt catccatgcc 30 <210> 15 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Desc ription of Artificial Sequence: Primer <400> 15 gagaaggcct catggagtgg gacagtggt 29 <210> 16 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 16 tctcggatcc ggttacgtga gatagctcgc 30

[Brief description of the drawings]

FIG. 1 is a view showing the structure of LKP1 cDNA.

FIG. 2 is a diagram showing the similarity between the LOV domain of LKP1 and similar domains of other proteins.

FIG. 3 shows the alignment of LKP1 Kelch repeats.

FIG. 4 is a diagram showing an accumulation analysis of LKP1 RNA using RT-PCR.

FIG. 5 shows GUS histochemical localization in the transgenic Arabidopsis thaliana of the present invention.

FIG. 6 shows the long hypocotyl and petiole phenotype of 35S :: LKP1 transgenic Arabidopsis thaliana of the present invention.

FIG. 7 is a diagram showing cotyledon movement in the transgenic Arabidopsis thaliana of the present invention.

FIG. 8 shows a delayed flowering phenotype of 35S :: LKP1 transgenic Arabidopsis thaliana of the present invention.

FIG. 9 shows LKP1 expression levels in 35S :: LKP1 transgenic and wild-type Arabidopsis thaliana of the present invention.

FIG. 10 shows the flowering time of 35S :: LKP1 transgenic Arabidopsis thaliana of the present invention.

FIG. 11: Delayed flowering phenotype and GFP of 35S :: LKP1 transgenic Arabidopsis thaliana of the present invention
FIG. 2 shows the subcellular localization of the LKP1 fusion protein.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12R 1:91) C12N 5/00 C (C12P 21/02 15/00 ZNAA C12R 1:91) C12R 1: 91) F term (reference) 2B030 AA02 AB03 AD20 CA17 CA19 CB02 CD03 CD07 CD10 CD13 CD14 4B024 AA08 BA63 CA04 DA01 EA01 FA02 FA10 HA01 4B064 AG20 CA11 CA19 CC24 DA11 4B065 AA89X AA89Y AB01 AC14 AC16 BA02 CA24 CA31 CA53

Claims (18)

[Claims] 1. The LOV domain and kelch (kelc)
h) A method for producing a transgenic plant that overexpresses a repeatable blue light receptor protein, wherein the gene is ligated downstream of a plant virus-derived promoter via the blue light receptor protein promoter. A method for producing a transgenic plant, characterized by transforming with an expression vector. 2. The method for producing a transgenic plant according to claim 1, wherein a blue light receptor protein gene is linked via a reporter gene downstream of the blue light receptor protein promoter. 3. The method for producing a transgenic plant according to claim 2, wherein the reporter gene is a β-glucuronidase gene. 4. The blue light receptor protein has SEQ ID NO: 2.
The method for producing a transgenic plant according to any one of claims 1 to 3, which is LKP1 having an amino acid sequence represented by: 5. The method for producing a transgenic plant according to claim 4, wherein a 1.5 kb 5 ′ non-coding region of LKP1 is used as a promoter of the blue light receptor protein. 6. The blue light receptor protein is SEQ ID NO: 4.
The method for producing a transgenic plant according to any one of claims 1 to 3, which is LKP2 having an amino acid sequence represented by: 7. The method for producing a transgenic plant according to claim 1, wherein the plant virus-derived promoter is a cauliflower mosaic virus 35S promoter. 8. The method for producing a transgenic plant according to claim 7, wherein the expression vector pBE2113 is used as the cauliflower mosaic virus 35S promoter. 9. The method for producing a transgenic plant according to claim 1, wherein the transgenic plant is a transgenic Arabidopsis thaliana. 10. A transgenic plant obtainable by the method for producing a transgenic plant according to any one of 1 to 9. 11. The transgenic plant according to claim 10, which has a rosette-shaped upper leaf and is capable of forming an extended primary flower inflorescence with many rosette leaves. 12. The transgenic plant according to claim 10, wherein flowering is not induced even when the seed is subjected to vernalization treatment at a low temperature. 13. The transgenic plant according to claim 10, which is a propagation material. 14. The transgenic plant according to claim 13, wherein the propagation material is a seed. 15. A method for cultivating a plant, comprising culturing the transgenic plant according to any one of 10 to 14. 16. The method for cultivating a plant according to claim 15, wherein the flowering time of the plant is controlled. 17. The method for cultivating a crop according to claim 16, wherein the control of the flowering time is a delay of the flowering time. 18. The method for cultivating a plant according to claim 15, wherein the drawing of the plant is delayed.