KR101315345B1 - Flowering gene XsFTs from Cocklebur and the uses thereof - Google Patents

Abstract

The present invention is to control the flowering time XsFTs ( Xanthium strumarim flowering locus T) protein, a gene encoding the protein, a recombinant vector comprising the gene, a host cell transformed with the recombinant vector, a recombinant comprising the XsFTs gene A method of controlling the flowering time of a plant under a single condition including transforming the vector into a plant cell, and a flowering time of the plant under a single condition comprising transforming the plant cell with a recombinant vector comprising the XsFTs gene. The present invention provides a method for producing a controlled transgenic plant, and a composition for controlling the flowering time of a plant under a single condition comprising a transgenic plant whose flowering time is controlled under a single condition prepared by the above method, and seeds and XsFTs genes thereof.

Description

Flowering gene XsFTs from Cocklebur and the uses approximately

The present invention is a dog flowering control gene XsFT1 , XsFT2 And XsFT3 and uses thereof, and more particularly XsFTs ( Xanthium for controlling flowering time). strumarium flowering locus T) protein, a gene encoding said protein, a recombinant vector comprising said gene, a host cell transformed with said recombinant vector, and a recombinant vector comprising said XsFTs gene, comprising transforming plant cells into Method of controlling the flowering time of the plant under a single condition, Method of producing a transgenic plant in which the flowering time of the plant is controlled under a single condition comprising the step of transforming the plant cell with a recombinant vector comprising the XsFTs gene, the method The present invention relates to a transgenic plant having a flowering time adjusted under a single condition prepared by the present invention, and a seed and its seed and the XsFTs gene.

In many plant species, flowering is determined by a combination of environmental and developmental signals. Photoperiod is a common environmental signal that causes flowering (Garner and Allard, Monthly Weather Review 48, 415-415. 1920). For example, single plants (SDPs) induce flowers when the sun is shorter than the threshold, while flowering of long-day plants (LDP) is promoted when the sun is longer than a certain number. Long-life plants and single plants can be further classified into obligate plants and facultative plants. In order to blossom, absolute plants absolutely need a certain photoperiod. However, conditional plants can eventually bloom even under non-induced photoperiods. Graft experiments and local light exposure studies indicate that photoperiod perception predominates in the leaves and causes translocatable signals known as florigens, which cause flowering of apical meristem. Zeevaart, Curr Opin Plant Biol 11, 541-547. 2008).

Flowering locus T ( FLOWERING LOCUS T, FT ) is a floral integrator that promotes flowering in Arabidopsis. Inducible photoperiods result in the accumulation of CO (CONSTANS) and eventually activate FT transcription in the leaves. Putative transcription factor FD expressed in apical meristem interacts with FT (Wigge meat al . , Science 309, 1056-1059. 2005). As a result, it has been found that FT protein migrates to apical meristem in several plant species, including Arabidopsis, rice and gourds. Since FT migration to apical meristem is exactly the same as the concept of flowering hormone, FT protein is accepted as a major component of flowering hormone signal.

In the Arabidopsis, there are other FT -like genes such as TSF and TFL1 . TSF is thought to promote excessive flowering with FT , but it can be activated alone by cytokinin (D'Aloia meat al . , Plant Journal , 972-979. 2011). TFL1, on the other hand, inhibits flowering despite sharing high sequence similarity with FT and TSF . Thus, there is a functional diversity between FT -like genes in Arabidopsis. Various FT -like genes have been reported in many flowering plants such as soybeans, poplars, grapes, citrus fruits, sugar beets, apples, alfalfa, corn and rice, and diversity may be responsible for differences in flowering patterns.

In Arabidopsis, during photoperiodic conversion experiments or when using a thermal shock inducible promoter, transient expression of FT resulted in stable flowering, indicating that flowering is 'locked in' without sustained expression of FT (Notaguchi) et al ., Plant & Cell Physiology 49 , 1645-1658. 2008). This FT -independent, permanent flowering state is LEAFY and APETALA The apical meristem-identity genes of flowers, such as 1 , may be the result of a positive feedback loop that activates each other (Liu et al. al ., Development 134, 1901-1910. 2007).

Balsam ( Impatiens) In some plant species, such as balsamina ), floral reversion occurs during the non-induced photoperiod. A simple model for the inversion of flowers in this species is that flowering depends on the continuous production of flowering hormone signals, which are produced only during growth in inducible photoperiods (Pouteau meat al . , Development 124, 3343-3351. 1997). In other species, such as Perilla crispa , grafting studies have demonstrated that after exposure to inducible photoperiods, leaf hormone signals continue to be produced in non-induced photoperiods (Zeevaart, Mededeelingen). van de Landbouwhoogeschool Wageningen 58, 1-88. 1958). However, inversion of flowers can occur in the perennial phase under non-induced photoperiods, when the induced leaves no longer exist, indicating that the periphery require flowering hormone signals for sustained flowering, such as balsam.

Macaw ( Xanthium) g. Studies in strumarium, cocklebur) for example, another interesting aspect is a flowering indirect induction (Zeevaart, Annual Review of Plant Physiology 27, 321-348. 1976). Hawktail is an obligate short-day plant (Hamner and Bonner, Botanical Gazette 100, 388-431. 1938), a single juvenile guinea pig may induce flowering of an uninduced plant by grafting. In addition, acceptor plants that are not exposed to SD can also induce another flowering plant by grafting. This indicates that the transmitted flowering signal can be amplified in the tissues of the dogs without photoperiod induction. One possible model of indirect induction is that FT initially induced by inducible photoperiods can induce their own expression in non-induced tissues (Zeevaart, Plant Cell 18, 1783-1789. 2006). To investigate the pattern of flowering in macaw , we isolated and analyzed three genes encoding FT homologues ( XsFT1 , XsFT2 , and XsFT3 ). Expression of all XsFTs in the macaw was induced by a single treatment, all of which restored the delayed flowering phenotype of Arabidopsis ft mutants. However, the expression pattern of XsFTs was not consistent with the expression pattern of XsFTs which the present inventors to predict in indirect induction.

On the other hand, Korean Patent Publication No. 2001-0042756 discloses the 'genetically modified plant in which the flowering locus T (FT) and flowering is controlled', and Korean Patent Publication No. 2004-0097423 discloses a method for controlling flowering time derived from Arabidopsis. A novel protein, a DNA encoding the same, a recombinant vector, a transformant, and a method of controlling the flowering time of a plant using the same are disclosed. However, as in the present invention, there is no disclosure about the flowering time regulation gene XsFTs derived from docomari .

The present invention is derived from the above requirements, and the present inventors induce early flowering in Arabidopsis ft mutants transformed with a recombinant vector comprising a flowering control gene XsFT1 , XsFT2 or XsFT3 derived from a single plant, an absolute single plant. By confirming that the present invention has been completed, the present invention has been completed.

In order to solve the above problems, the present invention is to control the flowering time XsFTs ( Xanthium strumarim providing flowering locus T) protein.

The present invention also provides a gene encoding the protein.

The present invention also provides a recombinant vector comprising the gene.

The present invention also provides a host cell transformed with the recombinant vector.

In addition, the present invention provides a method for controlling the flowering time of a plant under a single condition comprising the step of transforming the plant cell with a recombinant vector comprising the XsFTs gene.

The present invention also provides a method for producing a transgenic plant in which the flowering time of the plant is controlled under a single condition including transforming the plant cell with the recombinant vector including the XsFTs gene.

The present invention also provides a transformed plant and its seed whose flowering time is controlled under a single condition produced by the above method.

In addition, the present invention provides a composition for regulating the flowering period of a plant under a single condition, comprising an XsFTs gene for regulating flowering period.

Snake ( Xanthium) according to the present invention FT ( flowering ) from strumarim locus T ) gene controls the flowering time under a single condition. Therefore, by developing the transgenic plants in which early flowering is induced under a single condition by controlling the expression of the gene in plants, the new plant has been shortened. Can provide. In addition, this will be useful for the development of various crop development fields such as food, feed, flowers, horticulture and energy crops, plant seed industry and agriculture, which can induce early flowering under a single condition, and thus can use transformed plants with improved productivity. It is expected.

Figure 1 shows the FT -like genes of macaw. A, amino acid sequence alignment; Genome structure of B, XsFT1 , HaFT2 , and FT . Black rectangles represent exons and lines represent introns. The number on the black box shows the length of the exon. The intron length is shown below the line.
FIG. 2 illustrates a variety of pizza plant branches that are divided according to codon locations using a General Time Reversible model, an invariant site, and a gamma distributed rate (GTR + I + G). Bayesian consensus phylogram of 47 FT-like genes from the line is shown. Schematic diagram of Baby Pole At1g18100 / MOTHER OF FT and tomato ( Solanum lycopersicon ) branched using AAO31791. Red branches = well-supported (posterior probability of at least 0.95); Purple = TFL1 / CEN / BFT Clade; Blue = monocotyledonous FT-like sequence; Green = true dicotyledonous FT -like sequence of Rosid and early branch; Orange = Asteraceae FT-like clade. Arrows indicate FT -like genes that have been newly isolated from dogtails . Abbreviation: Snapdragon ( Antirrhinum majus , Am ), Barberry ( Aquilegia) coerulea , Ac ), Arabidopsis thaliana , At ), Beat ( Beta vulgaris , Bv ), wild grass ( Brachypodium) distachyon , Bd ), papaya ( Carica papaya , Cp ), yellow thistle ( Centaurea solstitialis , Cs , Chenopodium rubrum , Cr ), chrysanthemum ( Chrysanthemum lavandulifolium , Cl ), tangerine ( Citrus unshiu ( Ci ), sweet pumpkin ( Cucurbita maxima , Cm ), Chunbi ( Cymbidium faberi , Cf ), fig ( Ficus carica , Fc ), sunflower ( Helianthus annuus , Ha ), morning glory ( Ipomoea nil , In ), wild lettuce ( Lactuca virosa , Lv ), apple ( Malus x domestica , Md ), alfalfa ( Medicago truncatula, Mt ), yellow monkey ( Mimulus guttatus , Mg ), paddy ( Oryza sativa , Os , Populus trichocarpa , Pt ), Peach ( Prunus) persica , Pp ), tomato ( Solanum lycopersicon , Sl ), wheat ( Triticum aestivum , Ta ), grapes ( Vitis vinifera , Vv ), macaw ( Xanthium strumarium , Xs ).
3 shows that overexpression of XsFTs restores the flowering delay phenotype of Arabidopsis ft mutants. A, ft -101 and 35S :: XsFT1 . The plant was grown in the day; B, flowering time of T2 plants with 35S :: XsFT1 , 35S :: XsFT2 , 35S :: XsFT3 in ft-101 plants ( ft- 101 background). Two or three independent T2 lines were examined. Flowering time was measured by counting the initial rosette leaf number. Values are mean ± standard deviation (N = 6 to 10).
4 shows the analysis of diurnal expression patterns of XsFTs by qRT-PCR. RNA was extracted every 4 hours from the leaves under LD (17 hour light / 7 hour cancer), first and fourth SD (8 hour light / 16 hour cancer). Samples were obtained from ZT8. Three technical iterations are shown here. Biological repetition showed similar expression patterns.
5 shows that XsFTs expression was detected in the induced leaves. Single leaves were treated with 10 inductive SDs (8 hours light / 16 hours cancer) and samples were taken from the leaves of ZT0. L6 is the induced leaf, and the smaller the number after L, the younger the leaf. LD and SD are negative control and positive control, respectively. Expression was analyzed by qRT-PCR. Three technical iterations are shown here. Biological repetition showed similar expression patterns.
6 shows a decrease in XsFTs expression induced in non- induced photoperiod . Samples were obtained at ZT0 for one week in the induced leaves. LD and SD are negative control and positive control, respectively. Expression was analyzed by qRT-PCR. Three technical iterations are shown here. Biological repetition showed similar expression patterns.
7 shows that no indirect XsFTs expression was detected. Total plants were treated with two weeks of inducible SD (8 hours / 16 hours cancer) and non-induced LD (17 hours / 7 hours cancer). Samples were obtained from leaves at ZT0 immediately after SD treatment or LD treatment and after 2 weeks (L1, L2 and L8). L8 is the same leaf obtained from SD and LD. Three technical iterations are shown here. Biological repetition showed similar expression patterns.
8 shows that XsFTs expression does not diffuse in the same leaf. Some of the leaves were covered while giving two inductive SDs (8 hours light / 16 hours cancer) and samples were taken from each part at ZT0. Covered areas are shown in black. LD and SD are negative control and positive control, respectively. Expression was analyzed by RT-PCR.

In order to achieve the object of the present invention, the present invention provides XsFTs proteins that control the flowering time.

XsFTs protein of the present invention preferably means XsFT1 (SEQ ID NO: 4), XsFT2 (SEQ ID NO: 5) and XsFT3 (SEQ ID NO: 6). The range of XsFTs proteins according to the present invention includes proteins having the amino acid sequence of SEQ ID NO: 4 to SEQ ID NO: 6 derived from docomari and functional equivalents of the proteins. By "functional equivalent" is at least 70%, preferably at least 80%, more preferably at least 90% of each amino acid sequence represented by SEQ ID NO: 4 to SEQ ID NO: 6 as a result of the addition, substitution or deletion of amino acids As mentioned above, most preferably, it is a protein which has 95% or more of sequence homology, and shows the substantially homogeneous conserved function of each protein represented by SEQ ID NO: 4-SEQ ID NO: 6. "Substantially preserved function" means control of the flowering time of a plant.

The invention also includes fragments, derivatives and analogues of the XsFTs protein. As used herein, the terms “fragment”, “derivative” and “analogue” refer to a polypeptide having biological function or activity substantially the same as the XsFTs polypeptide of the invention. Fragments, derivatives, and analogs of the present invention comprise (i) polypeptides substituted with one or more conservative or nonconservative amino acid residues (preferably conservative amino acid residues), wherein the substituted amino acid residues are encoded by a genetic code. Or (ii) a polypeptide having substituent (s) at one or more amino acid residues, or (iii) another compound (a compound capable of extending the half-life of a polypeptide, such as polyethylene glycol) A polypeptide derived from a bound mature polypeptide, or (iv) an additional amino acid sequence (e.g., a leader sequence, a secretory sequence, a sequence used to purify the polypeptide, a proteinogen sequence or a fusion protein) and It may be a polypeptide derived from said polypeptide bound. Such fragments, derivatives and analogs as defined herein are well known to those skilled in the art.

Polynucleotides encoding a mature polypeptide represented by SEQ ID NO: 4 to SEQ ID NO: 6 includes a coding sequence encoding only the mature polypeptide; Sequences encoding mature polypeptides and various additional coding sequences; Mature polypeptides (and any additional coding sequences) and sequences encoding noncoding sequences.

The term "polynucleotide encoding a polypeptide" refers to a polynucleotide encoding a polypeptide, or a polynucleotide further comprising additional coding and / or noncoding sequences.

The invention also relates to variants of said polynucleotides encoding polypeptides comprising the same amino acid sequences as described herein, or fragments, analogs, and derivatives thereof. Polynucleotide variants can be naturally occurring allelic variants or non-naturally occurring variants. The nucleotide mutant includes a substitution mutant, a deletion mutant, and an insertion mutant. As is known in the art, an allelic variant is an alternative to a polynucleotide, which may comprise one or more substituted, deleted or inserted nucleotides and which does not result in a substantial functional change in the polypeptide encoded by the variant Do not.

The present invention also provides a gene encoding the XsFTs protein. Genes of the present invention are each XsFT1 (SEQ ID NO: 1), XsFT2 (SEQ ID NO: 2) encoding the XsFTs proteins XsFT1, XsFT2 and XsFT3 proteins And XsFT3 (SEQ ID NO: 3) gene. The gene of the present invention may be DNA or RNA encoding XsFTs protein. DNA includes cDNA, genomic DNA or artificial synthetic DNA. DNA can be single stranded or double stranded. The DNA may be a coding strand or a non-coding strand.

Preferably, the XsFTs gene of the present invention may include a nucleotide sequence represented by SEQ ID NO: 1 to SEQ ID NO: 3. In addition, homologues of the nucleotide sequences are included within the scope of the present invention. Specifically, the gene has at least 70%, more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95% sequence homology with each of the nucleotide sequences of SEQ ID NO: 1 to SEQ ID NO: 3 Branches can include base sequences. The "% sequence homology" for a polynucleotide is determined by comparing the comparison regions with two optimally arranged sequences, wherein part of the polynucleotide sequence in the comparison region is the reference sequence (addition or deletion) for the optimal alignment of the two sequences. It may include the addition or deletion (ie, gap) compared to).

The invention also relates to a polynucleotide which hybridizes with a sequence having at least 50%, preferably at least 70%, more preferably at least 80% identity to each of the nucleotide sequences of SEQ ID NOs: 1 to 3 described above will be. The present invention particularly relates to polynucleotides that hybridize to the polynucleotides described herein under stringent conditions. In the present invention, "stringent conditions" are (1) hybridization and washing under lower ionic strength and higher temperature such as 0.2 x SSC, 0.1% SDS, 60 ° C; Or (2) hybridization in the presence of denaturing agents such as 50% (v / v) formamide, 0.1% bovine serum / 0.1% Ficoll and 42 ° C .; Or (3) at least 80%, preferably at least 90%, more preferably at least 95%. In addition, the biological function and activity of the polypeptide encoded by the hybridizable polynucleotide is the same as the biological function and activity of each mature polypeptide represented by SEQ ID NO: 4 to SEQ ID NO: 6.

In accordance with an embodiment of the present invention, it should be understood that the XsFTs gene is preferably derived from docomari . However, embodiments of the present invention have other dicotyledons that have high homology (eg, 60% or more, ie 70%, 80%, 85%, 90%, 95%, even 98% sequence identity) with the Docomari gene. It also includes other genes derived from plants. Sequencing methods and means for determining sequence identity or homology (eg, BLAST) are well known in the art.

The present invention also provides a recombinant vector comprising the XsFTs gene.

The recombinant plant expression vector of the present invention can be used as a transient expression vector that can be temporarily expressed in a plant into which a foreign gene has been introduced, and a plant expression vector that can be permanently expressed in a plant into which a foreign gene is introduced.

Binary vectors that can be used in the present invention may be any binary vector containing RB (right border) and LB (left border) of T-DNA capable of transforming plants when present with Ti plasmid of A. tumefaciens . However, it is preferable to use pBI101 (Cat #: 6018-1, Clontech, USA), pBIN19 (Genbank Accession No. U09365), pBI121, pCAMBIA vector, and the like, which are frequently used in the art.

The term "vector" is used to refer to a DNA fragment (s), nucleic acid molecule, which is transferred into a cell. The vector replicates the DNA and can be independently regenerated in the host cell. The term "carrier" is often used interchangeably with "vector ". The term "expression vector" means a recombinant DNA molecule comprising a desired coding sequence and a suitable nucleic acid sequence necessary for expressing a coding sequence operably linked in a particular host organism. Promoters, enhancers, termination signals and polyadenylation signals available in eukaryotic cells are known.

A preferred example of a plant expression vector is a Ti-plasmid vector that is capable of transferring a so-called T-region into a plant cell when it is present in a suitable host such as Agrobacterium tumefaciens. Other types of Ti-plasmid vectors (see EP 0 116 718 B1) are currently used to transfer hybrid DNA sequences to plant cells or protoplasts in which new plants capable of properly inserting hybrid DNA into the plant's genome can be produced have. A particularly preferred form of the Ti-plasmid vector is the so-called binary (binary) vector as claimed in EP 0 120 516 B1 and US Pat. No. 4,940,838. Other suitable vectors that can be used to introduce the genes according to the invention into a plant host are viral vectors, such as those which can be derived from double stranded plant viruses (eg CaMV) and single stranded viruses, gemini viruses, etc. For example, it may be selected from an incomplete plant viral vector. The use of such vectors may be particularly advantageous when it is difficult to transform the plant host properly.

The expression vector preferably comprises one or more selectable markers. The marker is typically a nucleic acid sequence having a property that can be selected by a chemical method, and includes all genes capable of distinguishing a transformed cell from a non-transformed cell. Examples include, but are not limited to, herbicide resistance genes such as glyphosate or phosphinothricin, antibiotic resistance genes such as kanamycin, G418, bleomycin, hygromycin, and chloramphenicol It doesn't happen.

In a plant expression vector according to an embodiment of the present invention, the promoter may be CaMV 35S, actin, ubiquitin, pEMU, MAS, or histone promoter, but is not limited thereto. The term "promoter " refers to the region of DNA upstream from the structural gene and refers to a DNA molecule to which an RNA polymerase binds to initiate transcription. A "plant promoter" is a promoter capable of initiating transcription in plant cells. A "constitutive promoter" is a promoter that is active under most environmental conditions and developmental conditions or cell differentiation. Constructive promoters may be preferred in the present invention because the choice of transformants can be made by various tissues at various stages. Therefore, the constant promoter does not limit the selectivity.

In the plant expression vector according to an embodiment of the present invention, the terminator may use a conventional terminator, such as nopalin synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, agro Bacterium tumerfaciens ( Agrobacterium) tumefaciens ), but not limited to the terminator of the octopine gene (Octopine) gene.

The present invention also provides a host cell transformed with the recombinant vector.

The host cell capable of continuously cloning and expressing the vector of the present invention in a stable manner can be used in any host cell known in the art, for example, E. coli JM109, E. coli BL21, E. coli RR1, E. strains of the genus Bacillus, such as coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus thuringiensis, and Salmonella typhimurium, Serratia marcensons, and various Pseudomonas species. Enterobacteria and strains.

In addition, when transforming a vector of the present invention to eukaryotic cells, yeast (Saccharomyce cerevisiae), insect cells, human cells (e.g., CHO cell line (Chinese hamster ovary), W138, BHK, COS-7, 293) as host cells , HepG2, 3T3, RIN and MDCK cell lines) and plant cells and the like can be used.

The method of carrying the vector of the present invention into a host cell is performed by the CaCl ₂ method (Cohen, SN et. al ., Proc . Natl . Acac . Sci . USA , 9: 2110-2114. 1973), Hanahan method (Hanahan, D., J. Mol . Biol . , 166: 557-580. 1983) and electroporation method (Dower, WJ et al . , Nucleic . Acids Res . , 16: 6127-6145. 1988) and the like. In addition, when the host cell is a eukaryotic cell, microinjection method (Capecchi, MR, Cell , 22: 479.1980), calcium phosphate precipitation method (Graham, FL et al. , Virology , 52: 456. 1973), electroporation method ( Neumann, E. et al . , EMBO J. , 1: 841. 1982), liposome-mediated transfection (Wong, TK et al . , Gene , 10: 87. 1980), DEAE-dextran treatment (Gopal, Mol . Cell Biol . , 5: 1188-1190. 1985), and gene balm (Yang et al . , Proc . Natl . Acad . Sci . 87: 9568-9572. 1990) and the like can be injected into the host cell.

In addition, the present invention provides a method for controlling the flowering time of a plant under a single condition comprising the step of transforming the plant cell with a recombinant vector comprising the XsFTs gene. Arabidopsis-derived FT genes induce early flowering in long-term conditions, while the XsFTs gene of the present invention shows a marked difference in inducing early flowering in a single condition.

In the method according to an embodiment of the present invention, the XsFTs gene may be composed of the base sequence of SEQ ID NO: 1 to SEQ ID NO: 3, respectively, but is not limited thereto.

Method according to an embodiment of the present invention the XsFTs By overexpressing the gene to induce early flowering is characterized by controlling the flowering time of the plant. The regulation of flowering time may be regulated by the gain or loss of function by overexpression of a gene or virus-induced gene silencing (VIGS), but is not limited thereto.

In addition, the present invention comprises the steps of transforming a plant cell with a recombinant vector comprising the XsFTs gene; And it provides a method for producing a transformed plant in which the flowering time of the plant is controlled under a single condition comprising the step of regenerating the plant from the transformed plant cells.

In a method according to an embodiment of the present invention, the plant produced by the method is characterized in that the transformed plant is controlled flowering time under a single condition.

The method of the present invention comprises the step of transforming plant cells with the recombinant vector according to the present invention, which transformation can be mediated by, for example, Agrobacterium tumerfaciens. In addition, the method of the present invention comprises regenerating a transgenic plant from the transformed plant cell. Any of the methods known in the art can be used for regeneration of transgenic plants from transgenic plant cells.

Transformed plant cells must be regenerated into whole plants. Techniques for the regeneration of mature plants from callus or protoplast cultures are well known in the art for a number of different species (Handbook of Plant Cell Culture, Vol. 1-5, 1983-1989, Momillan, N. Y.).

 The present invention also provides a transgenic plant and its seeds in which early flowering is induced under a single condition.

Preferably, the plant may be a dicotyledonous plant, but is not limited thereto. Preferably, the dicotyledonous plant is Arabidopsis edorea.

The dicotyledonous plants include Asteraceae (Diaphyllaceae, Diapensiaceae), Asteraceae (Clethraceae), Pyrolaceae, Ericaceae, Myrsinaceae, Primaceae and Primaceae (Plumbaginaceae), Persimmonaceae (Ebenaceae) , Styracaceae, Symplocaceae, Blackaceae, Oleaceae, Loganiaceae, Gentianaceae, Menyanthaceae, Oleaceae , Apocynaceae, Asclepiadaceae, Rubinaceae, Polemoniaceae, Convolvulaceae, Boraginaceae, Verbenaceae, Lamiaceae, Labiatae, Solanaceae (Scrophulariaceae), Bignoniaceae, Acanthaceae, Sesame (Pedaliaceae), Fructose (Orobanchaceae). Gesneriaceae, Lentibulariaceae, Phrymaceae, Plantaginaceae, Caprifoliaceae, Adoxaceae, Valerianaceae, Dipsacaceae, Campanaceae Campanulaceae, Compositae, Myricaceae, Sapaceae, Juglandaceae, Salicaceae, Birchaceae, Beechaceae, Fagaceae, Elmaceae, Mulaceae , Nettleaceae (Santalaceae), Mistletoe (Loranthaceae), Camphoraceae (Trifolium, Polygonaceae), Lichenaceae (Northaceae, Phytolaccaceae), Liliumaceae (Aycotinaceae), Pomegranate (Aizoaceae), Purslane (Portulacaceae), Caryophyllaceae, Cenopodiaceae, Amaranthaceae, Cactaceae, Magnoliaceae, Illiciaceae, Lauraceae, Cassia family, Cecidiphyllaceae, beauty Ranunculaceae, Berberidaceae, Lardizabalaceae, Agaraceae, Menispermaceae, Nymphaeaceae, Cryophyllaceae, Fishbane, Cabombaceae (Saururaceae), Piperaceae, Chloranthaceae, Aristolochiaceae, Actinidiaceae, Tea Tree (The Camellia family), Theaceae, Guttaiferae, Droseraceae, Papaveraceae, Capparidaceae, Cruciferaceae (Cruciferaceae, Cruciferae), Sycamoreaceae (Prunusaceae, Platanaceae), Evergreenaceae (Prunusaceae, Hamamelidaceae), Pheasant's Uralaceae, Crassulaceae, Panaxaceae, Saxifragaceae Eucommiaceae, Pittosporaceae, Rosaceae, Leguminosae, Oxalidaceae, Geraniaceae, Tropaeolaceae, Zygophyllaceae, Linaceae, Euphorbiaceae, Callitrichaceae, Rutaceae, Simaroubaceae, Meliaceae, Polygalaceae, Anacardiaceae, Mapleaceae , Aceraceae, Sapindaceae, Hippocastanaceae, Beechaceae (Sabiaceae), Balsam (Ballaceae, Balsaminaceae), Aquifoliaceae, Celastraceae, Chilliaceae (Staphyleaceae) , Boxuxaceae, Empetraceae, Rhamnaceae, Vitaceae, Ellaeocarpaceae, Tiliaceae, Malvaceae, Sterculiaceae, Cypressaceae , Thymelaeaceae, Elaeagnaceae, Frucourtiaceae, Violaceae, Passifloraceae, Tamaricanaceae, Elatinaceae, Begoniaceae, Pak (Cucurbitaceae), Panaxaceae (Lythraceae), Pomegranate (Punicaceae), Pinus (Onagraceae), Antaloea (Aaloiaceae), Bataceae (Alangiaceae), Dogwood (Hornaceae, Cornaceae), Arboraceae (Ogalpi) Tree family, Araliaceae) or umbel family (Umbelliferae (Apiaceae)), but is not limited thereto.

In another aspect, the present invention provides a composition for controlling the flowering time of a plant under a single condition, comprising the XsFTs gene consisting of the nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO: 3. The composition comprises each of the XsFTs gene consisting of the nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO: 3 as an active ingredient, and to promote or delay the flowering of the plant by transforming each of the genes in the plant overexpress or silencing through VIGS It can be done.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Experimental Method

Plant material

Hamner and Bonner ( Botanical Gazette 100, 388-431. Dokomari seeds of varieties used by 1938 (in Chicago) Was collected and called Xanthium pennsylvanicum from Carlos O. Miller (Indiana University) (Salisbury, Cambridge: Cambridge University Press. 1990). Seeds of the dogs were incubated overnight at 37 ° C. for dormant breakthrough and were treated with LD (17 hours / 7 hours cancer) or SD (8 hours under 100 μmol m ⁻² s ⁻¹ cool-white fluorescent light). Light / 16 hours dark) in a 27 ° C. incubator.

XsFT  And XsACT cDNA of Cloning

For XsFT cloning, total RNA was extracted from induced and non-induced leaves using the Nucleospin RNA Plant Kit (Macherey-Nagel), according to the manufacturer's protocol. Reverse transcription was performed for 90 minutes at 42 ° C. with Smart MMLV (Clontech) and oligo (dT). XsFT was amplified under the following conditions using degenerate primers (PefFT-F and PefFT-R2); 8 cycles of touchdown of 98 ° C. 30 sec, 64 ° C., 59 ° C. and 54 ° C. 30 sec (−0.5 ° C./cycle), 72 ° C. 1 minute; 34 cycles of 98 ° C. 30 sec, 60 ° C., 55 ° C. and 50 ° C. 30 sec, 72 ° C. per minute. Nested PCR was performed to clone the 3 'region of the XsFTs . After the first PCR was performed using XsFT-F1 and RACE-F, the resulting product was amplified using XsFT-F2 and RACE-F1. Amplified fragments were cloned into pGEM T-easy (Promega) and each clone was sequenced. 5 'region of the XsFTs was amplified using deXsFT_ATG-F and XsFT-R1, the amplified bands were cloned and sequence analysis. The upstream region of XsFTs was obtained using TAIL-PCR (Liu) using the primers listed in Table 1. meat al . , Plant Journal 8, 457-463. 1995). XsACT For cloning, degenerate primers ACT46S, ACT165S, ACT245A, and ACT284A were used (Mckinney et al. , Plant Journal 8, 613-622. 1995). PCR conditions were described in XsFT Same as the degenerate PCR method. Sequence information was registered in GenBank with the following registration numbers: XsFT1 Genome: JF434696, XsACT1 : JF434697, XsACT2 : JF434698, XsACT3 : JF434699, XsACT4 : JF434700, XsFT1 : JF434701, XsFT2 : JF434702, XsFT3 : JF434703.

Name Sequence (5 'to 3') Experiment PefFT-F TGG TTG GTN ACN GAY ATN CCN G (SEQ ID NO: 7) XsFT cloning PerFT-R2 AGR TTR TAN ANY TCN GCR AA (SEQ ID NO: 8) XsFT cloning XsFT-F1 CCA CAG GAG CAC GTT TTG GTC AAG (SEQ ID NO: 9) XsFT 3 'RACE XsFT-F2 GTA TGT TAT GAG AGT CCA AGA CCA TC (SEQ ID NO: 10) XsFT 3 'RACE RACE-F1 TTT TTT TTT TTT TTT TTT TTT TTT TTV NN (SEQ ID NO: 11) XsFT 3 'RACE XsFT-R1 CAT TGA TGG TCT TGG ACT CTC ATA AC (SEQ ID NO: 12) XsFT cloning deFT_ATG-F ATG WSN ATH AAY ATH MgN gAY CCN YTN (SEQ ID NO: 13) XsFT cloning XsFT1tail-R2 CCA TTG CTA ACT TCC CTA TCG TTG (SEQ ID NO: 14) XsFT1 TAIL-PCR XsFT2tail-R2 CCG TTG CTA ACT TCC ATA TCG TTG (SEQ ID NO: 15) XsFT2 TAIL-PCR XsFT1tail-R1 ATA TCA ACA CGA GGC TGG TTT ACA AC (SEQ ID NO: 16) XsFT1 TAIL-PCR XsFT2tail-R1 ATA TCG ACG TGA GGC TGG TTT ACA AC (SEQ ID NO: 17) XsFT2 TAIL-PCR XsFT2tail-R3 GAT CTG GTG AAA CTA TCA ACA ACA TC (SEQ ID NO: 18) XsFT2 TAIL-PCR XsFT3tail-R1 TCG TCA CCT CCG ATT TCA ACC CTG (SEQ ID NO: 19) XsFT3 TAIL-PCR XsFT3tail-R2 TTC ACA ACT TGA GAG GGT TTT AGC TC (SEQ ID NO: 20) XsFT3 TAIL-PCR XsFT3tail-R3 TGT AAC TTC CCT ATC GTT ATA AGA AAC (SEQ ID NO: 21) XsFT3 TAIL-PCR XsFT1 cORF-F CAC CAT GgC GAG GAG GGA GAG GGA CCC GTT G (SEQ ID NO: 22) 35S :: XsFT1 construction XsFT2 cORF-F CAC CAT GAC GAG GAG GGA GAG GGA CCC GTT G (SEQ ID NO: 23) 35S :: XsFT2 , XsFT3 construction XsFT1-3UTR-R AAT GTA AGC CCT TTG ACA CGC CTT TG (SEQ ID NO: 24) 35S :: XsFT1 construction, XsFT1 qPCR XsFT2-3UTR-R AGG TAT TCT TCC TTG TTA TGC CAT TC (SEQ ID NO: 25) 35S :: XsFT2 construction, XsFT1 qPCR XsFT3-3UTR-R AAA TGC CGT ACA CGC CTT ATA AAC (SEQ ID NO: 26) 35S :: XsFT3 construction, XsFT1 qPCR XsFT1realT-F GCT CTA CAA TCT TGG ATC TCC AGT G (SEQ ID NO: 27) XsFT1 qPCR XsFT2realT-F GCT CTA CAA CCT TGG ATC TCC TGT G (SEQ ID NO: 28) XsFT2 qPCR XsFT3realT-F1 GCT CTA TAA CCT TGG ATC GCC TG (SEQ ID NO: 29) XsFT3 qPCR ACT46S ATG GTN GGN ATG GGN CAR AA (SEQ ID NO: 30) XsACT cloning ACT165S GTN CCN ATN TAY GAR GG (SEQ ID NO: 31) XsACT cloning ACT245A GTN ATN ACY TGN CCR TCN GG (SEQ ID NO: 32) XsACT cloning ACT284A ATR TCN ACR TCR CAY TTC ATN AT (SEQ ID NO: 33) XsACT cloning XsACT-F2 AGG GAG AAG ATG ACA CAG ATC ATG (SEQ ID NO: 34) XsACT 1 qPCR XsACT-R2 GAT GGC ATG AGG AAG CGC ATA AC (SEQ ID NO: 35) XsACT 1qPCR

Tree tree analysis

FT -like and TFL1 / CEN -like genes include NCBI (http://www.blast.ncbi.nlm.nih.gov), Phytozome (http://www.phytozome.net), and CoGe (http: // synteny identified as a BLAST search in .cnr.berkeley.edu / CoGe /). Sequence alignments were translated and combined into amino acid sequences using Mesquite version 2.74 (http://mesquiteproject.org/mesquite/mesquite.html), and MUSCLE (Edgar, Nucleic) before manual correction using Mesquite 2.74. Acids Research 32, 1792-1797. 2004). Misaligned areas were excluded from the analysis. Once aligned, the sequence was identified by Mr. Grethor's parallel processing cluster at the University of St. Louis, Missouri. Bayes 3.2.1 (Ronquist and Huelsenbeck, Bioinformatics 19, 1572-1574. 2003) were analyzed using the base phylogenetic method. The analysis uses 20 million generations using a continuous GTR model (GTR + I + G) with a flat prior and a constant position and gamma distribution divided according to the codon position with a tree tree sampled every 1000 generations. It consists of two separate probes. Convergence between two runs was determined by measuring the mean standard deviation of the split frequency. After confirming convergence, 25% of the first sample was removed by "burn-in" and Mr. Bayes was used to summarize the phylogenetic tree with estimated credibility values and to generate a majority rule consensus tree. The phylogenetic tree obtained was visualized using FigTree version 1.3.1 ( http://tree.bio.ed.ac.uk / software / figtree / ) and Adobe Illustrator CS.

35S: XsFT  Preparation of Transgenic Plants

The total length of the cDNA is XsFTs for XsFT1 XsFT1-cORF-F and XsFT1-3UTR-R, for XsFT2 XsFT2-cORF-F and XsFT2-3UTR-R, for XsFT3 XsFT2-cORF-F and R-XsFT3-3UTR Amplification using primers. Amplification products were cloned into pENTR / D-TOPO (Invitrogen) and sequenced. Using LR clonase mix II (Invitrogen), inserts were introduced into binary vector pEG100 with a 35S CaMV promoter (Earley et al . , Plant Journal 45, 616-629. 2006). The binary vector was transformed into Agrobacterium tumerfaciens strain GV3101 ^{Rif +} . Transformation constructs are floral dip method (Clough and Bent, Plant Journal 16, 735-743. Arabidopsis Mutant ft- 101 (Takada and Goto, Plant) Cell 15, 2856-2865. 2003).

real time RT - PCR

Total RNA was extracted and reverse transcription was performed by the method described above. SYBR premix EX Taq II (Takara) was used for the PCR reaction in ABI PRISM 7500 (Applied Biosystems). For amplification, XsFT1 : XsFT1realT-F and XsFT1-3UTR-R, XsFT2 : XsFT2realT-F and XsFT2-3UTR-R, XsFT3 : XsFT3realT-F1 and XsFT3-3UTR-R, XsACT1 : XsACT-F2 and XsACT-R2 primers Was used. XsACT1 was used for standardization.

Example  1. from the Docomari FT Homologous  Separation and Analysis

Given the essential role of FT in promoting flowering, the present inventors investigated whether indirect induction in lizards could be related to indirect induction of FT homologues. To investigate this possibility, we first isolated the 'functional' FT homologue. Since the macaw is a single plant (SDP), FT homologues will be expressed in SD. Thus, the Docomari FT homologues (hereinafter referred to as XsFTs ; Xanthium strumarium Flowering Locus T ) was isolated for identification. RNA was extracted from the leaves of single cultivated plants and XsFTs cDNA was isolated by reverse transcription PCR (RT-PCR) using degenerate primers (Table 1). Three different FT -like partial cDNAs were identified and named XsFT1 , XsFT2 and XsFT3 . Total length sequence was determined by rapid amplification of cDNA ends (RACE) and thermal asymmetric interlaced-PCR (TAIL-PCR). Expected amino acid sequences of XsFTs were compared with amino acid sequences of Arabidopsis FT and other FT-like proteins (FIG. 1A). XsFTs had the following amino acid sequence similarities to Arabidopsis FT: 73.3%, 70.5% and 74.4% for XsFT1, XsFT2 and XsFT3, respectively. Very important for FT function (Hanzawa et al. , Proceedings of the National Academy of Sciences , USA 102, 7748-7753. 2005), two conserved amino acids Tyr85 and Glu140 of FT were conserved in all three XsFTs. Compared to XsFT1 and XsFT2, XsFT3 lacks one amino acid at the N-terminus and two additional amino acids at the C-terminus.

High sequence similarity and conserved gene structure suggest that XsFTs are homologues of Arabidopsis FT . A phylogenetic tree analysis was performed to determine the evolutionary flexibility of XsFT1, XsFT2 and XsFT3 along with other FT -like genes of various flowering plants. The soft relationship between the 47 FT -like genes identified by the BLAST search was estimated using the Bayesian phylogenetic method. This analysis estimates a valid FT -like bifurcation consisting of monocotyledonous and true dicotyledonous sequences, with three XsFTs containing portions of the Asteraceae subbranch (FIG. 2).

Example  2. XsFTs Expression pattern of

In Arabidopsis, the expression of FT and upstream regulator CO of FT is influenced by photoperiod and circadian clock (Valverde et al. , Science 303, 1003-1006. 2004). Therefore, it was important to determine the expression level of XsFT mRNA throughout the cycle, for which RNA samples were prepared from leaf tissues of plants grown in LD and plants converted to SD during the first and fourth cycles (SD1 and SD4). . Expression of each XsFT was examined by RT-qPCR using gene specific primers.

In LD, any XsFTs expression was minimally detected or absent during the 24 hour period. In the first inducible SD cycle, XsFT2 and XsFT3 expression was weakly induced during bright culture (FIG. 4). However, expression of XsFT1 in the first SD was similar to that in LD. Expression of XsFT1 was clearly detected in SD4 samples, demonstrating that at least one SD is required for expression of XsFT1 . The expression of XsFT2 and XsFT3 is increased in SD4 samples compared to the expression of SD1, so that the expression of all XsFTs is driven by multiple inducible cycles, consistent with the fact that a large number of inducible SDs in dogs lead to active flowering. Increased. Despite maximal expression in SD4 during culturing, mRNA levels of all XsFTs decreased to mRNA levels of non-induced LD samples in the early and middle stages of cancer culture, and after the required night length (8.5 hours), increased XsFTs expression. it started.

Example  3. Arabic Pole ft  Restoring the flowering delay phenotype of the mutant XsFTs

Sequence analysis and expression patterns indicated that the XsFTs isolated by the inventors may be functional FT homologues. To further investigate the functionality, XsFTs were linked to a potent CaMV 35S promoter ( 35S :: XsFT s) and transformed to Arabidopsis ft mutant plants ( ft- 101 ). T1 plants blooming earlier than ft mutants were selected, and T2 plants were analyzed for flowering time at long days (FIG. 3). All XsFTs were able to restore the delayed flowering phenotype of Arabidopsis ft mutant plants. Transformation constructs were identified by PCR and XsFTs Phenotypes co-separated with the presence of the constructs. These data indicate that all XsFTs that we isolated are functional FTs. It proposes to code homologues.

Example  4. Indirect XsFTs mRNA  Induction test

Indirect induction in lizards indicates that flowering signals are grafted and self-proliferating in plants that were not exposed to inducible photoperiods (Zeevaart, Plant Cell 18, 1783-1789. 2006). One possible model for the indirect induction is that the expression of FT mRNA can be induced by the FT FT mRNA or protein. We tested this hypothesis using another approach. The first is a single-lead induction experiment in which only a single-leaf of lizard is exposed to SD, which can induce flowering. If indirect induction is involved in inducing XsFT mRNA expression in tissues that are not exposed to inducible photoperiods, XsFT mRNA will also be detected in other leaves. Single leaves were exposed to SD until flowering and RNA was extracted from the leaves. As shown in FIG. 5, strong expression of XsFTs was detected in the induced leaves (L6), but not in the non-induced leaves (L1-L5, L7, L8) above and below the induced leaves. To investigate whether the induction of any XsFT s mRNA is maintained in non-inductive conditions, RNA was extracted from ZT0-derived leaves daily for one week after conversion to non-inducible sun (FIG. 6). MRNA levels of all XsFTs was reduced to the control level, the reduction of XsFT1 mRNA levels were reduced faster than the level of XsFT2 and XsFT3. Therefore, continuous exposure to inducible photoperiods is essential to maintain the expression of all XsFTs tested.

Although indirect induction of XsFT s mRNA was not observed in single-leaf induction experiments, it is possible that indirect induction was not detected because of the weak indirect effect of single leaves. Thus, we further experimented with the indirect induction potential associated with induction of XsFTs using total plants. Flowering induced young plants by placing them in SD for 3 days, which was then sufficient to induce permanently active flowering in non-induced LD. After treatment, the plants were transferred to LD, and after two weeks RNA was extracted from newly developing leaves that were not exposed to SD. However, there was no difference in XsFTs levels compared to LD control (FIG. 7). Thus, despite vigorous flowering, indirect induction of XsFTs mRNA was not observed in single leaf or total plant induction experiments.

To investigate if indirect induction could occur within a single leaf, part of one leaf was covered and only the rest of the leaf was exposed to SD (FIG. 8). After two inductions, the expression of XsFTs was examined. We could not detect XsFT s mRNA in the non- derived portion of the leaf. The last possibility is that indirect induction may occur by sustained FT expression in the shoot apex, but in this case it may not be expected that the FT produced in the apex will be delivered by grafting. We evaluated XsFTs mRNA expression at the apical of induced plants, but there was no detectable expression (data not shown).

Attach an electronic file to a sequence list

Claims (12)

An Xanthium strumarium flowering locus T (XsFT) protein regulating flowering time, comprising the amino acid sequence of any one of SEQ ID NOs: 4 to 6. A gene encoding the protein of claim 1. According to claim 2, wherein the gene is a gene, characterized in that consisting of the base sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 3. A recombinant vector comprising the gene of claim 2. Arabidopsis thaliana plant cells transformed with the recombinant vector of claim 4. A method of controlling the flowering time of Arabidopsis plants under a single condition, comprising transforming a recombinant vector comprising the gene of claim 2 into Arabidopsis thaliana plant cells. The method of controlling the flowering time of Arabidopsis plants according to claim 6, wherein early flowering is induced by overexpressing the gene. Transforming Arabidopsis thaliana plant cells with a recombinant vector comprising the gene of claim 2; And
A method for producing a transgenic Arabidopsis plant with a controlled flowering period under a single condition comprising the step of regenerating Arabidopsis plants from the transformed Arabidopsis plant cells. A Arabidopsis thaliana plant in which the flowering period is controlled under a single condition prepared by the method of claim 8. The transgenic Arabidopsis plant according to claim 9, wherein early flowering is induced under a single condition. delete Composition for controlling the flowering time of Arabidopsis thaliana plants under a single condition, comprising an XsFT gene consisting of any one of SEQ ID NO: 1 to SEQ ID NO: 3.

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