# Patent

## Title
Control of plant flowering time by regulation of phytochrome c expression

## Abstract

Rice phyC mutants were isolated using a mutant panel isolation method. When the mutants were grown under long-day photoperiodic conditions, it was found that they flowered (exposed their panicles (heads)) about one week earlier than the control rice. The results indicate that suppression of PHYC gene expression can promote plant flowering under long-day conditions. Utilization of the PHYC gene for promoting plant flowering will contribute substantially to breed improvement, for example, by facilitating the creation of useful agricultural crops and decorative plants that have a new characteristic adaptable for other cultivation areas and times. The rice phyC mutants described herein, which promote flowering under long-day conditions, will be highly prized as a new early-harvest rice cultivar.

## Background
RELATED APPLICATIONS
This application is a 35 U.S.C. §371 national stage filing of International Application No. PCT/JP02/08824, filed 30 Aug. 2002, which claims priority to Japanese Patent Application No. 2001-266330 filed on 03 Sep. 2001 in Japan. The contents of the aforementioned applications hereby incorporated by reference.
TECHNICAL FIELD
The present invention relates to utilization of the PHYC gene, which is involved in the regulation of plant flowering (heading) time.
BACKGROUND ART
Rice is a short-day plant, meaning it flowers (exposes its panicle (head)) when day-length becomes short. Photoreceptors that sense the day-length are called phytochrome (phy), pigment binding molecules. In rice there are three phytochrome encoding genes, PHYA, PHYB, and PHYC (Kay, S. A. et al., Nucleic Acids Res. 17: 2865-2866, 1989, Dehesh, K. et al., Mol. Gen. Genet. 225: 305-313, 1991, Tahir, M. et al., Plant Physiol. 118: 1535, 1998).
As a phytochrome mutant in monocots such as rice, a phyB mutant (ma3R) has been isolated from sorghum (Childs, K. L. et al., Plant Physiol. 113: 611-619, 1997). The ma3R mutant shows the early-flowering phenotype as well as characteristic phenotypes such as reduced chlorophyll content, stem elongation, and acceleration of apical dominance, which are obviously different from the normal plant type. Recently, the present inventors isolated phyA mutants from rice and analyzed their detailed phenotypes. As a result, no significant difference was observed in the flowering time of the mutant rice as compared to the control rice, Nipponbare, under either long-day or short-day photoperiodic conditions (Takano, M. et al., Plant Cell 13: 521-534, 2001). The se5 mutant of rice, in which levels of all phytochromes are reduced to below detectable levels, showed an early-flowering phenotype regardless of day-length photoperiod conditions (Izawa, T. et al., Plant J. 22: 391-339, 2000). However, in this mutant, all phytochrome genes, PHYA, PHYB, and PHYC, were unaltered though a mutation was found in the plastid heme oxidase encoding gene (Izawa, T. et al., Plant J. 22: 391-339, 2000).
Thus far, there has been no report of the isolation of a phyC mutant, even from the well-known experimental model plant Arabidopsis thaliana, and no report of a functional analysis of PHYC gene in relation to plant flowering time.
DISCLOSURE OF THE INVENTION
The present invention has been developed through contemplation of the background as mentioned above. An objective of the present invention is to isolate phyC mutants and analyze their phenotypes to provide a method for PHYC gene utilization. Specifically, the present invention aims at providing phyC mutants that hasten the onset of flowering (heading) and a method to accelerate flowering by suppressing the PHYC gene expression.
The present inventors have conducted exhaustive research to achieve the above objectives. To elucidate the function of the PHYC gene, phyC mutants were obtained using a mutant isolation method with mutant panels (Hirochika, H. In: Molecular Biology of Rice, Springer-Verelag (Tokyo), pp. 43-58, 1999).
In this system, insertional mutant lines were obtained by activating retrotransposon Tos17 in tissue cultures of rice, a short-day plant. It is known that when rice seeds are tissue cultured, a retrotransposon in the rice genome called Tos17 becomes activated and disrupts genes by transposition into other chromosomal regions. By forming calluses from the seeds to regenerate mature plants, a large number of independent mutant lines can easily be generated.
The mutants of interest were isolated among such generated mutant lines. Specifically, each collection of 980 mutant lines was placed in a three dimensional matrix called a mutant panel that resembles ten microtiter plates piled to form a Z-axis of eight rows, a Y-axis of-twelve rows, and an X-axis of ten rows. DNA was extracted from a pool of mutant lines on each axis, and used in screening for mutants of interest. These pooled DNAs were used as templates for PCR that contained a set of primers specific to the PHYC gene and to the LTR regions of Tos17. An amplified band was obtained only when the Tos17 retrotransposon was present near the primer specific to the PHYC gene (i.e., Tos17 was inserted within the PHYC gene). In this way, only eight PCR amplifications were needed to determine whether a mutant of interest was contained in a panel, i.e., the 980 mutant lines. When a specific band was amplified, the same combination of primers was utilized for PCR on X and Y-axes in the same panel. A row where amplification was found was confirmed on each axis. The location of the mutant of interest was determined from the crossing point of such rows within the three dimensional matrix.
From the experiments, a phyC mutant was successfully isolated in which Tos17 was inserted in the coding region at the first exon of the PHYC gene. Furthermore, it was found that the phyC mutant initiated flowering (heading) approximately one week earlier as compared to the control rice when grown under long-day conditions. Therefore, it was elucidated for the first time that the PHYC gene product is involved in sensing long-day photoperiods to delay flowering. To date, there has been no report of involvement of the PHYC gene in the determination of plant flowering time. The results herein suggest that suppressing the PHYC gene expression will enable the promotion of plant flowering under long-day conditions. Notably, there are no significant differences in phenotype, with the exception of flowering time, in the phyC mutant as compared to the wild-type, so it has an advantage of specifically driving early flowering through the suppression of the gene expression. Utilization of the PHYC gene to promote flowering will contribute substantially to breed improvement, for example, by facilitating the creation of useful agricultural crops and decorative plants that have a new characteristic adaptable for other cultivation areas and times. In addition, the rice phyC mutant, which flowers earlier under long-day conditions, will be highly prized as a new early-harvesting rice cultivar.
Namely, the present invention relates to the utilization of the PHYC gene, which controls plant flowering (heading) time under long-day conditions, to promote flowering, specifically,
(1) a nucleic acid that promotes flowering of a plant, wherein said nucleic acid is selected from any one of (a) to (c):

    (a) an antisense nucleic acid complementary to a plant PHYC transcript;
    (b) a nucleic acid having ribozyme activity that specifically cleaves the plant PHYC transcript; and
    (c) a nucleic acid that inhibits a plant phyC gene expression through co-suppression;


(2) the nucleic acid of (1), wherein the plant is a short-day plant;
(3) the nucleic acid of (2), wherein the short-day plant is rice;
(4) a vector comprising the nucleic acid of any one of (1) to (3);
(5) a transformed plant cell carrying the nucleic acid of any one of (1) to (3) or the vector of (4);
(6) a transgenic plant comprising the transformed plant cell of (5);
(7) a transgenic plant that is a progeny or a clone of the transgenic plant of (6);
(8) a reproducing material of the transgenic plant of (6) or 7;
(9) a method for producing the transgenic plant of (6) or (7) wherein the method comprises the step of introducing the nucleic acid of any one of (1) to (3) or the vector of (4) into a plant cell, and regenerating a plant from the plant cell;
(10) a method for promoting flowering-of a plant, wherein the method comprises suppressing endogenous PHYC gene expression in cells of the plant;
(11) the method of (10), wherein the method comprises introducing the nucleic acid of any one of (1) to (3) or the vector of (4) into the plant;
(12) the method of any one of (9) to (11), wherein the plant is a short-day plant;
(13) the method of (12), wherein the short-day plant is rice;
(14) a rice phyC mutant;
(15) a rice phyC mutant that is a progeny or clone of the mutant of (14); and
(16) a reproducing material of the rice phyC mutant of (14) or (15).
The present inventors have elucidated that the rice phyC mutation can promote rice flowering (heading) under long-day conditions. This indicates that it is possible to promote plant flowering under long-day conditions by suppressing plant PHYC gene expression.
The present invention provides nucleic acids that promote plant flowering. In a preferred embodiment of the present invention, plant flowering is promoted under long-day conditions by suppressing PHYC gene expression.
Generally, the term “flowering” means that flowers open; however, in the context of the present invention, as the term “flowering” applies to plants of the rice family including rice, for example, the term flowering means that panicles (heads) emerge. In the present invention the phrase “promote flowering” means to advance the onset of flowering. In the present invention, the phrase “long-day conditions” refers to photoperiodic conditions where a dark period in a day is shorter than a threshold dark period required for photoperiodic responses (critical dark period) Specifically, a 14-hour light/10-hour dark photoperiod is generally used as an example.
In the present invention, the above-mentioned PHYC gene is a gene encoding a phyC protein, which is one of plant pigment binding proteins, phytochtome. The PHYC gene is found in various plants. Therefore, in the present invention, it is possible to promote flowering in a desired plant by suppressing PHYC gene expression of the plant. In the present invention, the plant chosen to have flowering promoted under long-day conditions by regulating PHYC gene expression is preferably a short-day plant and more preferably a plant belonging to the rice family. A specifically preferred example is rice. A short-day plant is a plant that forms flower buds or whose flower bud formation is promoted under photoperiodic conditions where an uninterrupted dark period is longer than the critical dark period. Preferably, plants whose flowering may be promoted by this invention are, for example, useful agricultural crops and decorative plants. In concrete terms, useful agricultural crops may be monocot plants, such as rice, or dicot plants, such as soybean. Decorative plants may be flowering plants, such as chrysanthemum, morning glory, poinsettia, and cosmos.
The PHYC genes in the present invention include rice PHYC gene (Genbank accession number: AB018442) and Arabidopsis thaliana PHYC gene (Genbank accession number: Z32538) as working examples.
Furthermore, using methods known to one skilled in the art, such as hybridization technique (Southern, E. M. et al., Journal of Molecular Biology 98: 503, 1975) or polymerase chain reaction (PCR) techniques (Saiki, R. K. et al., Science 230: 1350-1354, 1985, Saiki, R. K. et al., Science 239: 487-491, 1988), homologues of the above-mentioned PHYC gene can be isolated, and the nucleotide sequence information of such genes can be obtained. For example, hybridization technique using the rice PHYC gene nucleotide sequence (Genbank accession number: AB018442) or its partial sequence as a probe, or PCR technique using specific oligonucleotide as a primer to hybridize to the PHYC gene enables one to isolate DNA highly homologous to the PHYC gene from a desired plant.
To isolate such DNA, the hybridization reaction is generally carried out under stringent conditions. Hybridization conditions of 6 M urea, 0.4% SDS, and 0.5×SSC, or equivalent conditions can be used as stringent conditions. Employing even higher stringency conditions, such as 6 M urea, 0.4% SDS, and 0.1×SSC, may enable one to obtain highly homologous DNA. The sequence of the isolated DNA can be determined by a known method.
Generally, the determination of whether an isolated DNA encodes a phyC protein is made based on-sequence homology. Sequence homology can be searched using programs called BLASTN (nucleic acid level) or BLASTX (amino acid level) (Altschul, S. F. et al., J. Mol. Biol. 215: 403-410, 1990). These programs are based on Karlin and Altschul's BLAST algorithm (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990, Proc. Natl. Acad. Sci. USA 90: 5873-5877, 1993). If a nucleotide sequence is analyzed by BLASTN, parameters may be set, for example, to score=100 and wordlength=12. If an amino acid sequence is analyzed by BLASTX, parameters may be set, for example, to score=50 and wordlength=3. Amino acid sequences may be analyzed using Gapped BLAST program as described in Altschul et al. (Nucleic Acids Res. 25: 3389-3402, 1997). When BLAST and Gapped BLAST programs are used for sequence analysis, the default parameters of the respective programs are used. These specific analytical methods are widely known.
In a method of the present invention, to generate a plant with promoted flowering, DNA that suppresses PHYC gene expression is inserted into an appropriate vector and introduced into plant cells from which whole plants are regenerated. The phrase “suppression of PHYC gene expression” encompasses both transcriptional and translational suppression, and includes not only complete cessation of the DNA expression but also a reduction in the expression level.
In the present invention, to suppress expression of an endogenous plant gene, one skilled in art may utilize, for example, an antisense technique. The efficacy of the antisense technique in plant cells was proven by Ecker et al. for the first time when antisense RNA was introduced by electroporation using transient gene expression (Ecker, J. R. and Davis, R. W. Proc. Natl. Acad. Sci USA 83: 5372, 1986). Later, the antisense effect was also observed in tobacco and petunia for reduction of expression of a target gene (Krol, A. R. et al., Nature 333: 866, 1988). Today, it is an established technique for suppressing plant gene expression. There are multiple mechanisms for suppressing target gene expression by antisense nucleic acids, namely: inhibition of transcription initiation by triple strand formation; transcription suppression caused by hybrid formation at a site where an RNA polymerase has formed a local open loop structure; transcription inhibition caused by hybridization to RNA being synthesized; splicing suppression caused by hybrid formation at a junction between an intron and exon; splicing suppression caused by hybrid formation at a spliceosome site; suppression of mRNA translocation from the nucleus to cytoplasm by hybrid formation with mRNA; splicing suppression by hybridization at a capping site or poly A addition site; suppression of translation initiation by hybrid formation at a translation initiation factor binding site; translation suppression caused by hybridization to a ribosome binding site near an initiation codon; peptide extension inhibition by hybridizing within a translated region or polysome binding site of mRNA; suppression of gene expression by hybrid formation at a site where nucleic acid and protein interact; etc. These various mechanisms inhibit the processes of transcription, splicing, and translation of the target gene to suppress gene expression (Hirashima and Inoue, New Biochemistry Experiment Vol. 2 Nucleic acid IV, Replication and Expression of Gene, Japanese Biochemical Society ed., Tokyo Kagaku Dojin, pp. 319-347, 1993). Therefore, the present invention provides antisense nucleic acids that are complementary to plant PHYC gene transcription products. The above-mentioned antisense nucleic acids include antisense DNA, antisense RNA, and DNA encoding the antisense RNA.
In the present invention, antisense nucleic acids can be used for any of the above-mentioned mechanisms to suppress target gene expression. In one embodiment, an antisense nucleic acid designed to be complementary to the 5′ untranslated region of mRNA allows for effective for translation inhibition. However, nucleic acids complementary to the coding region or 3′ untranslated region may be also utilized. Antisense DNA designed from not only translated regions but also untranslated regions may be included in the present invention. Employed antisense DNA is ligated downstream of an appropriate promoter and, preferably, a sequence comprising transcription termination signal is conjugated at its 3′ end.
Antisense nucleic acids of the present invention may be prepared, for example, by the phosphorothioate method using sequence information from the DNA of SEQ ID NO: 3 (Stein, C. A. et al., Nucleic Acids Res. 16: 3209-3221, 1988). Prepared nucleic acids may be used for transfecting a desired plant by any known method. Preferably, the antisense nucleic acid sequence is complementary to the endogenous gene or a part of the gene of the transfected plant. However, so long as the gene expression can effectively be inhibited, it does not have to be completely complementary. Antisense nucleic acids of the present invention are preferably 90% or more, and most preferably 95% or more, complementary to the target gene transcript. To inhibit the target gene expression effectively using an antisense nucleic acid, the antisense nucleic acid should be at least 15 or more nucleotides in length, preferably 100 or more nucleotides, and most preferably 500 or more nucleotides in length. Antisense nucleic acids to be used are usually shorter than 5 kb and preferably shorter than 2.5 kb.
Another method for suppressing endogenous gene expression is the ribozyme technique. A ribozyme is an RNA molecule with catalytic activity. Ribozymes with various kinds of activities are known in the art. Research on ribozymes as RNA-cleaving enzymes has enabled the design of a ribozyme that cleaves RNA at a specific site. Therefore, the present invention provides nucleic acids with ribozyme activity that specifically cleave plant PHYC gene transcripts. The above-mentioned nucleic acids of the present invention include RNA with ribozyme activity and DNA that encodes such RNA.
While ribozymes, such as those of the group I intron type and m1RNA contained in RnaseP, can be large, with 400 nucleotides or more, there are smaller ones as well, including the hammerhead type and hairpin type having an activity domain of approximately 40 nucleotides (Koizumi, M. and Otsuka, E., Tanpakushitsu Kakusan Koso 35: 2191, 1990). For example, it is known that the self cleavage domain of a hammerhead type ribozyme cleaves at the 3′ side of C15 of sequence G13U14C15. It is considered important for cleaving activity that A at 9th position forms a base pair with U14. Furthermore, it has been shown that the cleavage also occurs when the 15th base is A or U instead of C (Koizumi, M. et al., FEBS Lett. 228: 225, 1988). Therefore, if one designs a ribozyme to have a substrate binding site complementary to an RNA sequence close to the target site, the ribozyme can be utilized as a restriction enzyme-like RNA cleaving ribozyme to recognize the sequence UC, UU, or UA in the target RNA (Koizumi, M. et al., FEBS Lett. 239: 285, 1988, Koizumi, M. and Otsuka, E., Tanpakushitu Kakusan Koso 35: 2191, 1990, Koizumi, M. et al., Nucleic Acids Res. 17: 7059, 1989).
In addition, hairpin type ribozymes are useful in the context of the present invention. A hairpin type ribozyme can be found, for example, in the minus strand of satellite RNA in tobacco ringspot virus (Buzayan, J. M. Nature 323: 349, 1986). This ribozyme can also be designed to target-specifically cleave RNA (Kikuchi, Y and Sasaki, N. Nucleic Acids Res. 19: 6751, 1992, Kikuchi, H. Chemistry and Biology, 30: 112, 1992).
A ribozyme designed to cleave a target may be, for example, ligated to a promoter, such as cauliflower mosaic virus 35S promoter, and a transcription terminator sequence to be transcribed in plant cells. However, if unnecessary sequences are added to the 5′ or 3′ end of the transcribed RNA, ribozyme activity may be lost. In such a case, to accurately cut out only the ribozyme portion from a transcribed RNA comprising the ribozyme sequence, one can place another cis-acting trimming ribozyme at the 5′ or 3′ side of the ribozyme (Taira, K. et al., Protein Eng. 3: 733, 1990, Dzianott, A. M. and Bujarski, J. J. Proc. Natl. Acad. Sci. USA 86: 4823, 1989, Grosshans, C. A. and Cech, R. T. Nucleic Acids Res. 19: 3875, 1991, Taira, K. et al., Nucleic Acids Res. 19: 5125, 1991). In addition, multiple sites within a target gene can be cleaved by arranging such structural units in tandem to achieve greater effects (Yuyama, N. et al., Biochem. Biophys. Res. Commun. 186: 1271, 1992). Thus, one can use such ribozymes to specifically cleave a target transcript of the present invention to thereby suppress the gene expression.
Endogenous gene expression may also be suppressed by co-suppression, through transformation with a nucleic acid comprising a sequence identical or similar to the target gene sequence. “Co-suppression” refers to a phenomenon in which, when a gene having a sequence identical or similar to the target endogenous gene is introduced into plants by transformation, expression of both the target endogenous gene and introduced exogenous gene becomes suppressed. The mechanism of co-suppression is not well understood, but it is often seen in plants (Curr. Biol. 7: R793, 1997, Curr. Biol. 6: 810, 1996). Therefore, the present invention provides nucleic acids that have an inhibitory effect on plant PHYC gene expression through co-suppression. The nucleic acids of the present invention include DNA and RNA that have inhibitory effect by co-suppression.
To obtain a plant whose PHYC gene is co-suppressed using the above-mentioned nucleic acids of the present invention, for example, a vector DNA that expresses DNA comprising the PHYC gene sequence or a sequence similar to the gene is introduced into target plants followed by selection of a plant having the phyC mutant phenotype, that is, the ability to promote flowering under long-day conditions. Genes used for co-suppression do not have to be completely identical but should be at least 70% or more, preferably 80% or more, and most preferably 90% or more (for example 95% or more) identical to the target gene sequence.
Moreover, the endogenous gene suppression in the present invention may be achieved by introducing into a plant a gene that causes a dominant negative phenotype to a target gene. The “gene causing a dominant negative phenotype” refers to a gene whose expression can eliminate or reduce an activity of an endogenous wild-type gene originally present in a plant.
In addition, the present invention provides the above mentioned nucleic acids, vectors comprising the nucleic acids, transformed plant cells having the nucleic acids or vectors comprising the nucleic acids, transgenic plants containing the transformed plant cells, transgenic plants that are progeny or clones of the above transgenic plants, and breeding materials from the transgenic plants.
Moreover, the present invention provides a method for producing the above-mentioned transgenic plants that includes the process of introducing a nucleic acid of the present invention into plant cells, and regenerating plant bodies from the plant cells.
A nucleic acid of the present invention can be introduced into plant cells by one skilled in the art using known methods, for example, the agrobacterium method, electroporation method, and particle gun method.
The method of Nagel et al., for example, is used for the agrobacterium method (Microbiol. Lett. 67: 325, 1990). According to this method, agrobacterium is transformed by a recombinant vector and introduced to plant cells by a known method such as the leaf disc method. When a nucleic acid of the present invention is a DNA, the above vector comprises, for example, a promoter to express the DNA in a plant subsequent to introduction into the plant. Generally, a DNA of the present invention is placed downstream of such a promoter and, moreover, a terminator sequence is placed downstream of the DNA. A recombinant vector used for this purpose is suitably determined by one skilled in the art depending on the transfection method or the type of a plant. The above-mentioned promoter may be, for example, a cauliflower mosaic virus derived CaMV35S promoter or the ubiquitin promoter from maize (Unexamined Published Japanese Patent Application No. (JP-A) Hei 2-79983).
The above-mentioned terminator may be, for example, a cauliflower mosaic virus derived terminator or nopalin synthase terminator. However, so long as they function as a promoter or terminator in a plant, there is no limitation on them.
Plants transfected by nucleic acids of the present invention may be explants. Alternatively, cultured cells may be prepared from these plants, and such nucleic acids may be introduced into the cultured cells. “Plant cells” in the present invention may be, for example, cells from leaves, roots, stems, flowers, seed scutella, calluses, and cultured cell suspensions.
In addition, to efficiently select transformed plant cells into which a nucleic acid of the present invention has been introduced, the above recombinant vector preferably harbors an appropriate selective marker gene or is introduced into plant cells together with a plasmid vector harboring a selective marker gene. Selective marker genes used for this purpose include, for example, the hygromycin phosphotransferase gene, which confers resistance to the antibiotic hygromycin; the neomycin phosphotransferase gene, which confers resistance to kanamycin or gentamycin; and the acetyltransferase gene, which confers resistance to an herbicide, phosphinothricin.
Plant cells transfected with a recombinant vector are plated and cultured on a known selective medium containing an appropriate selective drug, depending on the type of the introduced selective marker gene. In this way, one can obtain transformed plant cultured cells.
Next, a plant body is regenerated from the transformed plant cells into which a nucleic acid of the present invention has been introduced. Regeneration of a plant can be carried out by methods known to one skilled in the art depending on the plant cell type (Toki et al., Plant Physiol. 100: 1503-1507, 1995). Several techniques have already been established to generate transformed rice plants, and those techniques are widely used in the field of the present invention. For example, rice plants can be regenerated after (1) genes are introduced into protoplasts using polyethylene glycol (suitable for Indica rice varieties) (Datta, S. K. et al., In Gene Transfer To Plants (Potrykus I and Spangenberg Eds.) pp 66-74, 1995); (2) genes are introduced into protoplasts using electric pulse (suitable for Japonica rice varieties) (Toki et al., Plant Physiol. 100: 1503-1507, 1992); (3) genes are introduced directly into cells using the particle gun method (Christou et al., Bio/technology, 9: 957-962, 1991); or (4) genes are introduced using agrobacteria (Hiei et al., Plant J. 6: 271-282, 1994). In the present invention, these methods can preferably be used.
The plants regenerated from transformed plant cells are subsequently cultured in acclimatization medium. Then, after the acclimatized regenerated plants are grown under the normal cultivation conditions, flowering-promoted plants can be obtained. Seeds can also be obtained when these plants mature and fruit.
The exogenously introduced nucleic acid in a thus regenerated and grown transgenic plant can be confirmed by known methods, such as PCR or Southern hybridization, or by analyzing the nucleotide sequence of the nucleic acid from the plant. To extract nucleic acid from a transgenic plant, the known method of J. Sambrook et al. may be used (Molecular Cloning, 2nd edition, Cold Spring Harbor laboratory Press, 1989).
To conduct PCR analysis of the exogenous gene comprising a nucleic acid of the present invention, which exists in the regenerated plant body, an amplification reaction is carried out using the template nucleic acid that was extracted from the regenerated plant by the above-mentioned method. When the nucleic acid of the present invention is DNA, the amplification reaction may be carried out in a reaction mixture containing as primers synthesized oligonucleotides having nucleotide sequences that are appropriately selected according to a nucleotide sequence of the DNA. An amplified DNA fragment comprising a DNA sequence of the present invention may be obtained by repeating the denaturation, annealing, and extension steps for DNA several ten cycles in the amplification reaction. The respective amplified DNA fragments can be separated by, for example, electrophoresing the reaction solution containing amplified products on agarose gel. It is then possible to confirm the DNA fragment corresponding to DNA of the present invention.
Once a transgenic plant in which a nucleic acid of the present invention has been inserted into the chromosomes is obtained, one can obtain the plant progeny by sexual or non-sexual reproduction. Also, it is possible to mass-produce such plants by obtaining reproductive materials (such as seeds, fruits, cuttings, stem tubers, root tubers, shoots, calluses, and protoplasts) from the above plant, or its progeny or clone.
In the present invention, as mentioned above, by suppressing PHYC gene expression, plant flowering can be promoted.
Moreover, the present invention provides rice phyC mutants, their progeny or clones, and reproductive materials of the phyC mutants.
The rice phyC mutants in the present invention include not only the rice phyC mutants that can promote flowering under long-day conditions (homozygous mutants), but also heterozygous mutants. The heterozygous mutants are useful for generating homozygous mutants. In addition, the rice phyC mutants in the present invention include, besides the phyC mutants (homozygous and heterozygous mutants) isolated in the present invention, phyC mutants (homozygous and heterozygous mutants) that will newly be isolated by using the mutant panel method described in the Examples.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the insertion site of the Tos17 retrotransposon in the phyC mutant. The black frame in panel A indicates the border where the Tos17 is inserted; Tos17 is inserted between histidine (H) residue 224 and glutamic acid (E) residue 245. Panel B illustrates the insertion site of Tos17 in the chromosome.
FIG. 2 shows a photograph demonstrating the isolation of phyC mutants. A heterozygous mutant of the phyC mutation was self fertilized, and resulting offspring (#1-9) were analyzed by Southern hybridization using a probe detecting a 3.8 kb fragment containing PHYC gene, which would be yielded by XhoI-treatment. (+/+): wild-type, (+/−): heterozygous phyC mutant, and (−/−): homozygous phyC mutant.
FIG. 3 shows the expression level of the PHYC gene transcript using the competitive RT-PCR method. log(Comp) indicates logarithms of competitor concentrations. PhyC indicates PHYC transcripts, and Comp indicates amplified products derived from competitor sequence. (+/+) wild-type, (+/−): heterozygous phyC mutant, and (−/−): homozygous phyC mutant.
FIG. 4 shows the flowering (heading) time of the phyC mutants grown in a field. osphyA-2: phyA mutant, (+/±): wild-type or heterozygous mutant segregated after self fertilization, osphyC-1: phyC mutant.
FIG. 5 shows the relationship between flowering time and genotypes of the F2 group segregants of phyC mutant backcrossed with Nipponbare. 
FIG. 6 shows the flowering (heading) time of phyC mutant under long-day and short-day photoperiodic conditions. 14L/1OD and 10.5L/13.5D in the horizontal axis indicate long-day and short-day conditions, respectively.



BEST MODE FOR CARRYING OUT THE INVENTION
The present invention will be explained in detail below with reference to the examples below but is not intended to be limited to these examples.
EXAMPLE 1
Isolation of phyC Mutant
The present inventors isolated phyC mutants using the mutant panel method. PHYC gene primers were designed based on the rice PHYC cDNA sequence (Genbank Accession number: AB018442). Six primers were designed to cover the whole sequence of the phyC gene since it is a large sized gene (BR, DR, EF, FR, GF, and HR) (Table 1). Also, two primers (LTR1 and LTR4) were designed to face outward within LTR sequences at both ends of Tos17 (Table 2).










TABLE 1





phyC-
phy-C
Primer



specific primer*
specific primer sequence
site**







BR
GTGATGGCAGACCATCAACC
1412-1393



(SEQ ID NO: 1)


 


BR1
CCAGTGTCTCCATCATCATCC
1367-1347


(SEQ ID NO: 2)


 


DR
CATACCTAAGCGGGAAAGGGAC
1449-1428


(SEQ ID NO: 3)


 


DR1
AAAGGGACAAACCTCGGGCTTG
1435-1414


(SEQ ID NO: 4)


 


EF
CGTACAAGTTCCATGAGGATGAGC
1018-1041


(SEQ ID NO: 5)


 


EF1
GAGGTGATTGCTGAGTGCAAGAG
1047-1069


(SEQ ID NO: 6)


 


FR
ATCGACCAGAGGCTTCCCTATG
2306-2285


(SEQ ID NO: 7)


 


FR1
TGGCTTCCATGACAGGTAATCC
2286-2265


(SEQ ID NO: 8)


 


GF
GCCTAATTGAGACAGCAACTGCG
2176-2198


(SEQ ID NO: 9)


 


GF1
TTGGCTGTTGACATCACTGG
2205-2224


(SEQ ID NO: 10)


 


HR
ACGAGCTTCTGGCTTATGTAAAGG
3586-3563


(SEQ ID NO: 11)


 


HR1
TGGCGGAACATCTCTTGTATCAG
3532-3510


(SEQ ID NO: 12)





*BR1, DR1, EF1, FR1, GF1, and HR1 indicate nested PCR primers for BR, DR, EF, FR, GF, and HR, respectively.


**Primer site in the rice PHYC gene cDNA sequence (Genbank Accession number: AB018442)















TABLE 2






Tos17



Tos17 specific primer*
specific primer sequence







LTR1 (SEQ ID NO: 13)
TTGGATCTTGTATCTTGTATATAC



 


LTR2 (SEQ ID NO: 14)
GCTAATACTATTGTTAGGTTGCAA


 


LTR4 (SEQ ID NO: 15)
CTGGACATGGGCCAACTATACAGT


 


LTR5 (SEQ ID NO: 16)
ATTAGCTTGTATATATATTTAACA





*LTR2 and LTR5 indicate nested PCR primers for LTR1 and LTR4, respectively.






9600 individuals on mutant panels were PCR screened by using 12 primer sets ((6 PHYC specific primers)×(2 Tos17 specific primers)). A specific amplification was observed when the BR and LTR4 primers were used. In a DNA pool for the mutant panel, DNAs derived from 80 (X-axis) to 120 (Z-axis) individuals are included, which means that the concentration of each template is very low since DNA of each individual is diluted 1/120 to 1/80. In addition, it is difficult to detect a specific amplification by one time PCR because the LTR sequence of Tos17 has a high AT content and Tm of the designed primers is low. Therefore, to increase sensitivity and specificity, additional primers (BR1, DR1, EF1, FR1, GF1, HR1, LTR2, and LTR5) were designed to correspond downstream of the above respective primers, and PCR was done twice (nested PCR) to detect amplification. As a result, a number of non-specific bands were seen, and therefore, Southern hybridization was carried out to identify the specific band. The identified band was excised and the DNA fragment was extracted. The DNA fragment was sequenced to determine the Tos17 insertion site. The Tos17 was inserted upstream of the chromophore (opened tetrapyrrole) binding site (244th amino acid) (FIG. 1), suggesting that it is a null mutant.
Also, Tos17 insertion was confirmed by Southern hybridization. When rice genomic DNA is digested with XHoI, it is expected to yield a 3.8 kb band from the wild-type rice genome based on the reported rice PHYC genomic DNA sequence (D. Basu et al. Plant Mol. Biol. 44: 27-42, 2000). In contrast, Tos17 insertional mutants are expected to yield two bands at 7.5 kb and 0.4 kb, respectively. DNA was extracted from progeny (#1-9) of self fertilized phyC heterozygous mutants and subjected to Southern hybridization using a probe that recognizes the 3.8 kb fragment containing the PHYC gene, which would be yielded by XhoI treatment (for mutants, using a probe comprising the full-length phyC cDNA, which recognizes the 7.5 kb fragment). The expected results were obtained. In addition, it was found that the Tos17 insertional mutation was segregated into homozygous mutants (#2, 5, 6, and 7) and heterozygous mutants (#1, 4, 8, and 9), and that the Tos17 was missing in #3 (FIG. 2). As known from these results, one phyC mutant line (osphyC-1) was obtained.
EXAMPLE 2
Confirmation of PHYC Gene Transcript by RT-PCR
To confirm a lack of PHYC gene expression in the isolated phyC mutants, a heterozygous phyC mutant was self-pollinated, and DNAs were extracted from its progeny (#1-9) to determine their genotypes. In addition, RNAs were extracted to conduct competitive RT-PCR using BR and EF primers described in Table 1 (FIG. 3). As a competitor DNA, a DNA fragment which harbors BR and EF sequences at both ends and, between them, a 340 bp sequence unrelated to phyC cDNA, was used. As a result, the PHYC gene was amplified from wild-type (#3) or heterozygous phyC mutants (#1, 4, 8, and 9) but homozygous phyC mutants (#2, 5, 6, and 7) did not show a band at the expected size, indicating no PHYC gene expression.
EXAMPLE 3
Effect of phyC Mutation on Flowering (Heading) Time
To investigate flowering time, Nipponbare and phyC mutant seeds were sowed on Jun. 28, 2000 and transplanted into a field on Jul. 14, 2000. These rice plants were grown under natural day lengths and the time of flowering was observed. As shown in FIG. 4, a phyA mutant (osphyA-2) control, and wild-type or heterozygous mutant segregants from the self fertilized phyC mutant flowered on the 60th to 61st day after sowing, while, the phyC mutants flowered on the 54th day, about a week earlier.
In addition, to confirm the linkage between the phyC mutation and earlier flowering time, the phyC mutant was backcrossed with Nipponbare, and the genotype and flowering time of its F2 group segregants were investigated. The F2 group (n=54) and control Nipponbare group (n=63) were sown on May 15, 2002 and transplanted into a field on Jun. 5, 2002. As shown in FIG. 5, the segregated heterozygous and wild-type individuals (n=45) flowered after an average of 93.2 days and no earlier than the 91st day after sowing. On the other hand, the phyC mutants (n=9) flowered after an average of 83.1 days and no later than the 84th day. The genotype completely correlated with the flowering time in this group. The control Nipponbare flowered after 94.1 days on average, which was almost identical to the flowering time of heterozygous and wild-type individuals.
Furthermore, to test flowering time under short-day and long-day conditions, the plants were grown in a climate-control incubator (short-day: 10.5-hour light/13.5-hour dark, long-day: 14-hour light/10-hour dark) (FIG. 6). Under short-day conditions, there was no difference among Nipponbare, phyA mutants, and phyC mutants. All of them flowered after approximately 50 days. Under long-day conditions, Nipponbare and the phyA mutants took about 90 days to flower, while, the phyC mutants flowered after about 83 days, about a week earlier. Therefore, the PHYC gene is considered to sense a long-day photoperiod and function to slow flowering.
In addition, the phyC mutants were investigated for phenotypic differences, apart from flowering time, such as plant height, plant feature, chlorophyll content, and chlorophyll a/b ratio; however, no significant difference was observed.
INDUSTRIAL APPLICABILITY
The present invention provides a method for promoting flowering (heading) using a plant PHYC gene. A phyC mutant that does not show phenotypic changes (for example changes of plant height, plant feature, chlorophyll content, and chlorophyll a/b ratio) other than flowering time is described herein. As such, it appears that suppression of PHYC expression can specifically promote flowering. Utilization of the PHYC gene to promote flowering will contribute substantially to breed improvement, for example, by facilitating the creation of useful agricultural crops and decorative plants that have a new characteristic adaptable for other cultivation areas and times. The present invention also provides a plant phyC mutant whose flowering is promoted under long-day photoperiodic conditions. The rice phyC mutant will be highly prized as a new early-harvest rice cultivar.

## Claims

1. A method for promoting flowering of a rice plant, wherein the method comprises introducing an isolated nucleic acid into the plant, wherein the nucleic acid inhibits the expression of the rice PHYC gene of SEQ ID NO:17, thereby promoting flowering of the plant, and is:
(a) an antisense nucleic acid selected from the group consisting of:
(i) the antisense nucleic acid that is complementary to the rice PHYC gene of SEQ ID NO:17 transcript; and
(ii) an antisense nucleic acid that is the complement of a nucleic acid that is 95% identical to the rice PHYC gene of SEQ ID NO:17 transcript; or

(b) a nucleic acid that inhibits a rice plant phyC gene expression through co-suppression selected from the group consisting of:
(i) an isolated rice PHYC gene of SEQ ID NO:17; and
(ii) a nucleic acid that is at least 95% identical to the rice PHYC gene of SEQ ID NO:17.

2. The method of claim 1, wherein the plant is a short-day plant.

3. A transgenic plant produced by the method of claim 1.

4. A transgenic plant that is a progeny or a clone of the parent transgenic plant of claim 3, wherein the progeny or clone comprises the nucleic acid that was introduced into the parent plant.

5. A reproducing material of the transgenic plant of claim 3 or 4, wherein the reproducing material comprises the nucleic acid that was introduced into the transgenic plant.