# Patent

## Title
Ehd1 gene promoting plant flowering, and utilization thereof

## Abstract

A detailed linkage analysis of the Ehd1 region was performed with a large segregating population, essential for map-based cloning. As a result, the Ehd1 gene promoting rice heading (flowering) was successfully isolated. Rice heading time was also found to be altered upon introduction of this gene. Based on these facts, the newly isolated and identified Ehd1 gene is expected to be useful in promoting plant flowering.

## Background
TECHNICAL FIELD
The present invention relates to Ehd1 genes that promote plant flowering, and utilization thereof.
BACKGROUND ART
Rice heading time (flowering time) is mainly determined by photoperiod sensitivity that depends on day length and other factors (basal vegetative growth or temperature sensitivity). Genetic analysis of heading time has been performed for some time, and to date, heading time-associated genes such as Se1 locus (chromosome 6), E1 locus (chromosome 7), E2 locus (unknown), E3 locus (chromosome 3), or Ef1 locus (chromosome 10) (Kinoshita, Rice Genetics Newsletter 15: 13-74, 1998; Nishida et al., Ann. Bot. 88: 527-536, 2001) have been discovered using mutations and variations inherent to rice cultivars. Recently, the use of DNA markers in rice genetic analyses has advanced the genetic analysis of characteristics such as heading time that exhibits complex inheritance (quantitative traits) (Yano et al., Plant Physiol. 127: 1425-1429, 2001). Genes associated with rice photoperiod sensitivity have been isolated based on this work (Yano et al., Plant Cell 12: 2473-2484, 2000; Takahashi et al., PNAS 98: 7922-7927, 2001; Kojima et al., Plant Cell Physiol. 43: 1096-1105, 2002; Yano, Curr. Opin. Plant Biol. 4: 130-135, 2001). Attempts to use these isolated rice-heading-time-associated genes to elucidate genetic control mechanisms are also progressing (Izawa et al., Gene Dev. 16: 2006-2020, 2002; Hayama et al., Nature 422: 719-722, 2003). On the other hand, many cases of isolation of genes associated with plant flowering have been reported in Arabidopsis thaliana (Simpson, G. G. and Dean, C., Science 296: 285-289 (2002); Mouradov, A. et al., Plant Cell (Suppl.) 14: S111-130 (2002)). Furthermore, methods for controlling flowering time in Arabidopsis thaliana (a plant) using these genes have been proposed (Published Japanese Translation of International Publication Nos.: 2002-511270; 2002-532069; 2002-537768; 2000-512845; Hei 11-512289; Hei 11-506001; and Hei 10-508481). At the same time, a method for using rice genes to alter the flowering time of Arabidopsis thaliana (a plant) has also been suggested (Published Japanese Translation of International Publication No. 2002-335970). However, a great number of the genes associated with rice heading time remain to be isolated.
DISCLOSURE OF THE INVENTION
The present invention was made under these circumstances. An objective of the present invention is to provide novel genes that regulate plant flowering. Another objective of this invention is to modify plant flowering time using these genes.
The Ehd1 locus is a quantitative trait locus (QTL) associated with heading time. It was detected using the progeny of a cross between japonica rice cultivar “Taichung 65” and West African region rice cultivar “O. glaberrima Steud.” (IRGC 104038). The Ehd1 locus has been proven to be located on the long arm of chromosome 10. Furthermore, analysis using a nearly isogenic line of the Ehd1 region (the IRGC 104038 allele), which comprises a “Taichung 65” genetic background, proved that the Ehd1 locus is associated with heading promotion. Genetic studies hitherto have also proved that the Ehd1 locus was mapped as a single Mendelian locus (formally called Ef(t)) in the interval between RFLP markers C234 and G37, and co-segregated with C1369. However, isolating genes using map-based cloning was difficult at the level of resolution of this linkage analysis.
The present inventors carried out a detailed linkage analysis of the Ehd1 region with a large segregating population essential for map-based cloning. A generation of progenies derived from backcrossing Taichung 65 and IRGC 104038 was used as the segregating population for linkage analysis. From these backcrossed progenies, those whose Ehd1 region was heterologous, and whose other genomic regions were mostly substituted with the Taichung 65 type genome were selected. From the 2500 progeny plants (F2 generation) produced by these selected plants by self-fertilization, plant having a chromosome with a recombination near the Ehd1 locus were selected using CAPS markers C1286 and G37, which flank the Ehd1. Genotype of Ehd1 locus was determined by a progeny test of the F3 generation. As a result of linkage analysis, it was demonstrated that the Ehd1 was mapped between RFLP markers C814A and C234, and identified eight recombinant plants for C814A and two recombinant plants for C234.
Since the nucleotide sequences of the RFLP markers flanking the Ehd1 gene were found to be included in the published genomic nucleotide sequence information, the nucleotide sequence of the Ehd1 candidate genomic region was obtained from the published nucleotide sequence data. Using information on the nucleotide sequence of the candidate genomic region, novel CAPS markers were created to narrow down the candidate gene region. As a result, the Ehd1 candidate region was proved to be about 16 kb, flanked by CAPS markers 26-28 and 12-14. Gene predictions and similarity searches were carried out against the nucleotide sequence of this candidate region proved the presence of three types of predicted genes. One type showed similarity to the two-component response regulator (ARR) gene of Arabidopsis. The other two types of predicted genes were highly similar to rice EST, but shared no similarity to known genes with established functions. However, since none of these predicted genes could be excluded as Ehd1 candidates, these three types of predicted genes were transformed to verify function as Ehd1 candidates.
For transformation, a BAC library was constructed from genomic DNAs derived from the indica rice cultivar Kasalath assumed to comprise a functional Ehd1 allele. A BAC clone KBM128G10 comprising the Ehd1 candidate gene was selected for use from the library. From the BAC clone KBM128G10, an 11.5 kb BamHI fragment comprising the ARR-like candidate gene and one of the predicted genes having a high similarity to the rice EST, and a 7.6 kb KpnI fragment comprising two predicted genes other than the ARR-like candidate gene, were excised. Each of these fragments was incorporated into a Ti-plasmid vector pPZP2H-lac, and introduced into Taichung 65 via Agrobacterium. Regenerated plants were immediately transferred into growth chambers and cultivated under short-day conditions to measure the number of days until their heading. Almost all of the transgenic plants (T0) that were introduced with the 11.5 kb BamHI fragment headed earlier than those with the vector alone. On the other hand, the number of days to heading in plants introduced with the 7.6 kb KpnI fragment was about the same as for plants with the vector alone. Furthermore, transcription of the ARR-like Kasalath-derived candidate gene was observed in almost all of the plants introduced with the 11.5 kb BamHI fragment. From these results the Ehd1 candidates could thus be narrowed down to the ARR-like candidate gene comprised in the 11.5 kb BamHI fragment.
Furthermore, to confirm that heading promotion was due to the transgene, plants with lower copy numbers of the gene were selected from those transgenic plants (T0) with earlier heading. The inbred progenies thereof were cultivated under short-day conditions to investigate the number of days until heading. Each of the three types of progeny populations were respectively divided into early-maturing plants and late-maturing plants. All the early-maturing plants retained the transformed Kasalath-derived ARR-like candidate gene, and the number of days to their heading was essentially the same as for the nearly isogenic line, T65(Ehd1), in which the Ehd1 region of Taichung 65 had been substituted with the Ehd1 gene of O. glaberrima. On the other hand, the number of days to Taichung 65 heading was the same as that of late-maturing plants. These results proved that the ARR-like candidate gene was the Ehd1 gene.
In rice cultivars O. glaberrima (IRGC 104038), Kasalath, Nipponbare, and Taichung 65, genomic nucleotide sequences of about 7.6 kb in their Ehd1 regions were analyzed to compare the amino acid sequences of their respective predicted translational products. It was revealed that seven amino acids were substituted between IRGC 104038 and Taichung 65, two between Kasalath and Taichung 65, and one between Nipponbare and Taichung 65. Of these, the substitution of the 219th amino acid glycine to arginine was a unique mutation occurring only in Taichung 65. This glycine was highly conserved among the known ARR gene family. This amino acid mutation was thus assumed to be associated with the reduced Ehd1 function of Taichung 65-derived plants.
Therefore, it is expected that the newly isolated Ehd1 gene can be utilized to promote plant flowering (heading), and that DNAs which elicit a reduced function of the Ehd1 gene can be used to delay plant flowering.
Namely, the present invention relates to the Ehd1 gene, which promotes plant flowering (heading), and to utilization thereof. More specifically, the present invention provides the following:
[1] A DNA according to any one of the following (a) through (d), wherein said DNA encodes a plant-derived protein comprising a function of promoting plant flowering:
(a) a DNA encoding a protein comprising the amino acid sequence of SEQ ID NO: 3, 6, or 9;
(b) a DNA comprising a coding region of a nucleotide sequence of SEQ ID NO: 1, 2, 4, 5, 7, or 8;
(c) a DNA encoding a protein comprising an amino acid sequence of SEQ ID NO: 3, 6, or 9, wherein one or more amino acids are substituted, deleted, inserted, and/or added; and
(d) a DNA hybridizing under stringent conditions with a DNA comprising the nucleotide sequence of SEQ ID NO: 1, 2, 4, 5, 7, or 8;
[2] The DNA of [1], derived from rice;
[3] A DNA according to any one of the following (a) through (d):
(a) a DNA encoding an antisense RNA complementary to the transcriptional product of the DNA of [1] or [2];
(b) a DNA encoding an RNA comprising ribosomal activity that specifically cleaves the transcriptional product of the DNA of [1] or [2];
(c) a DNA encoding an RNA that, due to an RNAi effect, suppresses the expression of the DNA of [1] or [2] upon expression in a plant cell; and
(d) a DNA encoding an RNA that, due to a co-suppression effect, suppresses the expression of the DNA of [1] or [2] upon expression in a plant cell;
[4] The DNA of [1] or [2], used to promote plant flowering;
[5] The DNA of [3], used to delay plant flowering;
[6] A vector comprising the DNA of any one of [1] through [5];
[7] A transformed plant cell retaining the DNA of any one of [1] through [5] or the vector of [6];
[8] A transgenic plant comprising the transformed plant cell of [7];
[9] A transgenic plant that is a progeny or a clone of the transgenic plant of [8];
[10] A breeding material of the transgenic plant of [8] or [9];
[11] A method for producing the transgenic plant of [8], comprising the steps of introducing the DNA of any one of [1] through [5] or the vector of [6] into a plant cell, and regenerating a plant from said plant cell;
[12] A method for promoting plant flowering, comprising expressing the DNA of [1] or [2] in plant cells;
[13] A method for delaying plant flowering, comprising suppressing the expression of the endogenous DNA of [1] or [2] in plant cells;
[14] The method of [13], comprising introducing the DNA of [3] into a plant; and
[15] The method of any one of [12] through [14], wherein the plant is rice.
The present invention provides DNAs encoding plant-derived Ehd1 proteins comprising the function of promoting plant flowering.
In this invention, the plants from which DNAs encoding the Ehd1 protein are derived include, but are not limited to, rice, Arabidopsis thaliana, soybean, maize, barley, wheat and morning glory, for example.
Furthermore, there is no particular limitation as to the types of plants whose flowering is promoted on transforming the above-described DNA. For example, such plants include useful crops and ornamental plants. Specifically, useful crops include monocotyledons such as rice, and dicotyledons such as soybean. Ornamental plants include flowering plants such as chrysanthemum, morning glory, poinsettia, and cosmos.
In the present invention, “flowering” usually means the blooming of flowers, but refers to heading in gramineous plants such as rice. In this invention, promotion of flowering refers to accelerating flowering time, while delay of flowering refers to delaying flowering time.
Furthermore, the day-length (photoperiod) conditions under which the DNAs of this invention promoted the flowering of the above-described plants are, for example, natural day-length conditions, long-day conditions, short-day conditions, and so on. Short-day conditions are preferred. In this invention, long-day conditions are conditions in which there are 14 or more daylight hours per day. In this example, the light period was set at 15 hours, while the dark period was set at nine hours; however, long-day conditions are not limited to this example. Short-day conditions are those conditions in which there are 11 or fewer daylight hours per day. In this example, the light period was set at ten hours and the dark period at 14 hours; however, short-day conditions are not limited to this example.
Furthermore, in this invention, examples of DNAs encoding Ehd1 proteins are those DNAs comprising the coding region of the nucleotide sequences set forth in SEQ ID NO: 1, 2, 4, 5, 7, or 8, and DNAs encoding proteins comprising the amino acid sequences set forth in SEQ ID NO: 3, 6, or 9.
The present invention also comprises DNAs encoding proteins which are structurally analogous to an Ehd1 protein comprising an amino acid sequence set forth in SEQ ID NO: 3, 6, or 9, and comprising the function of promoting plant flowering.
Whether or not a DNA encodes a protein that comprises the function of promoting plant flowering can be determined by, for example, observing whether or not the flowering of plants introduced with the DNA is promoted; or whether or not the flowering of plants is delayed when they are introduced with a DNA that suppresses the expression of the DNA. Examples of such DNAs include mutants, derivatives, alleles, variants, and homologues that encode proteins comprising an amino acid sequence of SEQ ID NO: 3, 6, or 9, in which one or more of the amino acids are substituted, deleted, added, and/or inserted.
Examples of methods for preparing DNAs that encode proteins comprising altered amino acid sequences are well known to those skilled in the art, and include site-directed mutagenesis (Kramer, W. and Fritz, H.-J., (1987) “Oligonucleotide-directed construction of mutagenesis via gapped duplex DNA.” Methods in Enzymology, 154: 350-367). A protein's amino acid sequence may also mutate naturally due to a nucleotide sequence mutation. DNAs encoding proteins comprising an amino acid sequence of an Ehd1 protein wherein one or more amino acids are substituted, deleted, added, and/or inserted are also included in the DNAs encoding Ehd1 proteins of the present invention, so long as they encode a protein functionally equivalent to a naturally occurring type of Ehd1 protein (SEQ ID NO.: 3, 6, or 9). In addition, nucleotide sequence mutations that do not give rise to changes in the amino acid sequence of the protein (degenerate mutations) are also included in the DNAs encoding Ehd1 proteins of the present invention.
DNAs encoding proteins functionally equivalent to Ehd1 proteins, which comprise an amino acid sequence described in SEQ ID NO: 3, 6, or 9, can be produced, for example, by methods well known to those skilled in the art, including hybridization techniques (Southern, E. M. (1975) Journal of Molecular Biology 98: 503.); and polymerase chain reaction (PCR) techniques (Saiki, R. K. et al. (1985) Science 230: 1350-1354; Saiki, R. K. et al. (1988) Science 239: 487-491). That is, it is routine for a person skilled in the art to isolate DNAs with high homology to a DNA encoding Ehd1 protein from rice and other plants, using the genomic sequence of an Ehd1 region (SEQ ID NO: 1, 4, or 7), an Ehd1 cDNA sequence (SEQ ID NO: 2, 5, or 8) or parts thereof as a probe, and oligonucleotides hybridizing specifically to the genomic sequence of the Ehd1 region and the Ehd1 cDNA sequence as a primer. Such DNAs encoding proteins functionally equivalent to an Ehd1 protein, obtainable by hybridization techniques or PCR techniques, are also included in the DNAs encoding Ehd1 proteins of this invention.
Hybridization reactions to isolate such DNAs are preferably conducted under stringent conditions. Stringent hybridization conditions of the present invention include conditions such as 6 M urea, 0.4% SDS, and 0.5×SSC, and those conditions yielding similar stringencies to these. DNAs with higher homology are expected to be isolated when hybridization is performed under more stringent conditions, for example, 6 M urea, 0.4% SDS, and 0.1×SSC. Herein, high homology means identity over the entire amino acid sequence of at least 50% or above, more preferably 70% or above, much more preferably 90% or above, and most preferably 95% or above.
The degree of homology of one amino acid sequence or nucleotide sequence to another can be determined using the BLAST algorithm by Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990; Proc. Natl. Acad. Sci. USA 90: 6873-5877, 1993). Programs such as BLASTN and BLASTX, developed based on the BLAST algorithm (Altschul et al. (1990) J. Mol. Biol. 215: 403-410), are also being used. To analyze a nucleotide sequence according to BLASTN, parameters are set, for example, at score=100 and word length=12. On the other hand, parameters used for the analysis of amino acid sequences by BLASTX are, for example, score=50 and word length=3. The default parameters for each program are used when using the BLAST and Gapped BLAST programs. Specific techniques for such analysis are known in the (see www.ncbi.nlm.nih.gov.)
DNAs of the present invention include genomic DNAs, cDNAs, and chemically synthesized DNAs. Genomic DNAs and cDNAs can be prepared according to conventional methods known to those skilled in the art. More specifically, genomic DNAs can be prepared, for example, by (1) extracting genomic DNAs from rice cultivars that comprise a DNA encoding an Ehd1 protein; (2) constructing a genomic library (utilizing a vector, such as a plasmid, phage, cosmid, BAC, PAC, and so on); (3) expanding the library; and (4) conducting colony hybridization or plaque hybridization using a probe prepared based on a DNA that encodes an Ehd1 protein of the present invention (e.g. SEQ ID NO.: 1, 2, 4, 5, 7, or 8). Alternatively, genomic DNAs can be prepared by PCR, using primers specific to a DNA encoding an Ehd1 protein of the present invention (e.g. SEQ ID NO.: 1, 2, 4, 5, 7, or 8). cDNAs can be prepared, for example, by (1) synthesizing cDNAs based on mRNAs extracted from rice cultivars that comprise a DNA encoding an Ehd1 protein; (2) preparing a cDNA library by inserting the synthesized cDNA into vectors, such as λZAP; (3) expanding the cDNA library; and (4) conducting colony hybridization or plaque hybridization as described above. Alternatively, cDNAs can be also prepared by PCR.
DNAs encoding an Ehd1 protein of the present invention can be used to promote plant flowering, for example. To prepare transgenic plants with promoted flowering, the above-described DNAs are inserted into appropriate vectors and introduced into plant cells using a method described below. Transgenic plants are regenerated from the transformed plant cells thus obtained. The present invention provides such methods for promoting plant flowering.
The present invention also provides methods for delaying plant flowering. Transgenic plants with delayed flowering can be obtained, for example, by inserting a DNA which suppresses the expression of an Ehd1 protein-encoding DNA into an appropriate vector; transforming the DNA construct into a plant cell using a method described below; and regenerating a plant from the resulting transformed plant cell. The step of suppressing the expression of Ehd1 protein-encoding DNAs includes suppressing the transcription of these DNAs, as well as their translation into proteins. In addition, it includes not only loss of DNA expression but also its reduction. It also includes the loss of an in vivo function of the translated proteins in plant cells.
Antisense techniques are the most commonly used methods in the art to suppress the expression of a specific endogenous gene in plants. Ecker et al. were the first to demonstrate the antisense effect of an antisense RNA introduced into plant cells by electroporation (J. R. Ecker and R. W. Davis (1986) Proc. Natl. Acad. Sci. USA 83: 5372). After that, expression of antisense RNAs reportedly reduced target gene expression in tobacco and petunias (A. R. van der Krol et al. (1988) Nature 333: 866). Antisense techniques have now been established as a means for suppressing target gene expression in plants.
Multiple factors act in the suppression of target gene expression by antisense nucleic acids. These include: inhibition of transcription initiation by triple strand formation; inhibition of transcription by hybrid formation at a site where the RNA polymerase has formed a local open loop structure; transcription inhibition by hybrid formation with the RNA being synthesized; inhibition of splicing by hybrid formation at an intron-exon junction; inhibition of splicing by hybrid formation at a site of spliceosome formation; inhibition of mRNA translocation from the nucleus to the cytoplasm by hybrid formation with mRNA; inhibition of splicing by hybrid formation at a capping site or poly A addition site; inhibition of translation initiation by hybrid formation at a translation initiation factor binding site; inhibition of translation by hybrid formation at a ribosome binding site near the initiation codon; inhibition of peptide chain elongation by hybrid formation in a translated region or at an mRNA polysome binding site; and inhibition of gene expression by hybrid formation at a site of interaction between nucleic acids and proteins. These antisense nucleic acids suppress target gene expression by inhibiting various processes such as transcription, splicing, or translation (Hirashima and Inoue, “Shin Seikagaku Jikken Koza (New Biochemistry Experimentation Lectures) 2, Kakusan (Nucleic Acids) IV, Idenshi No Fukusei To Hatsugen (Replication and Expression of Genes),” Nihon Seikagakukai Hen (The Japanese Biochemical Society), Tokyo Kagaku Dozin, pp. 319-347, (1993)).
The antisense sequences of the present invention can suppress target gene expression by any of the above mechanisms. In one embodiment, an antisense sequence designed to be complementary to an untranslated region near the 5′ end of the mRNA of a gene is thought to effectively inhibit translation of that gene. Sequences complementary to coding regions or to an untranslated region on the 3′ side can also be used. Thus, the antisense DNAs used in the present invention include both DNAs comprising antisense sequences against untranslated regions and against translated regions of the gene. The antisense DNAs to be used are conjugated downstream of an appropriate promoter, and are preferably conjugated to sequences containing the transcription termination signal on the 3′ side. DNAs thus prepared can be transformed into a desired plant by known methods. The sequences of the antisense DNAs are preferably sequences complementary to an endogenous gene of the plant to be transformed, or a part thereof, but need not be perfectly complementary so long as they can effectively suppress the gene's expression. The transcribed RNAs are preferably at least 90%, and more preferably at least 95% complementary to the transcribed product of the target gene. In order to effectively suppress the expression of a target gene by means of an antisense sequence, antisense DNAs should be at least 15 nucleotides long, more preferably at least 100 nucleotides long, and still more preferably at least 500 nucleotides long. However, the antisense DNAs to be used are generally shorter than 5 kb, and preferably shorter than 2.5 kb.
DNAs encoding ribozymes can also be used to suppress the expression of endogenous genes. A ribozyme is an RNA molecule comprising catalytic activity. There are many ribozymes comprising various activities, and among them, research focusing on ribozymes as RNA-cleaving enzymes has enabled the design of ribozymes that cleave RNAs site-specifically. While some ribozymes of the group I intron type or the M1 RNA contained in RNaseP consist of 400 nucleotides or more, others belonging to the hammerhead-type or the hairpin-type comprise an activity domain of about 40 nucleotides (Makoto Koizumi and Eiko Ohtsuka (1990) Tanpakushitsu Kakusan Kohso (Nucleic acid, Protein, and Enzyme) 35: 2191).
The self-cleavage domain of a hammerhead-type ribozyme cleaves at the 3′ side of C15 of the G13U14C15 sequence, and formation of a nucleotide pair between U14 and A9 at the ninth position is considered to be important for this ribozyme activity. It has been shown that cleavage may also occur when the 15th nucleotide is A15 or U15 instead of C15 (M. Koizumi et al. (1988) FEBS Lett. 228: 225). If a ribozyme is designed to comprise a substrate-binding site complementary to the RNA sequences adjacent to the target site, one can create a restriction-enzyme-like RNA-cleaving ribozyme which recognizes the UC, UU, or UA sequence within a target RNA (M. Koizumi et al. (1988) FEBS Lett. 239: 285; M. Koizumi et al. (1989) Nucleic Acids Res. 17: 7059). For example, in DNAs that encode Edh1 proteins (SEQ ID NO.: 2, 5, or 8), there are a number of sites that can be used as ribozyme targets.
Hairpin-type ribozymes are also useful in the present invention. These ribozymes can be found, for example, in the minus strand of satellite RNA in tobacco ringspot virus (J. M. Buzayan (1986) Nature 323: 349). Ribozymes that cleave RNAs target-specifically have also been shown to be produced from hairpin-type ribozymes (Y. Kikuchi and N. Sasaki (1992) Nucleic Acids Res. 19: 6751; Yo Kikuchi (1992) Kagaku To Seibutsu (Chemistry and Biology) 30: 112).
Transcription is enabled in plant cells by fusing a ribozyme, designed to cleave a target, with a promoter such as the cauliflower mosaic virus 35S promoter, and with a transcription termination sequence. If extra sequences have been added to the 5′ end or the 3′ end of the transcribed RNA, ribozyme activity can be lost. In such cases, one can place an additional trimming ribozyme, which functions in cis, on the 5′ or the 3′ side of the ribozyme portion, in order to precisely cut the ribozyme portion from the transcribed RNA containing the ribozyme (K. Taira et al. (1990) Protein Eng. 3: 733; A. M. Dzaianott and J. J. Bujarski (1989) Proc. Natl. Acad. Sci. USA 86: 4823; C. A. Grosshands and R. T. Cech (1991) Nucleic Acids Res. 19: 3875; K. Taira et al. (1991) Nucleic Acid Res. 19: 5125). Even greater effects can be achieved by arranging these structural units in tandem, enabling multiple sites within a target gene to be cleaved (N. Yuyama et al., Biochem. Biophys. Res. Commun. 186: 1271 (1992)). Thus, using these ribozymes, the transcription products of a target gene of the present invention can be specifically cleaved, thereby suppressing expression of the gene.
Endogenous gene expression can also be suppressed by RNA interference (RNAi), using double-stranded RNAs that comprise a sequence identical or similar to a target gene. RNAi refers to the phenomenon in which a double-stranded RNA comprising a sequence identical or similar to a target gene sequence is introduced into cells, thereby suppressing expression of both the exogenous gene introduced and the target endogenous gene. The details of the RNAi mechanism are unclear, but it is thought that an introduced double-stranded RNA is first degraded into small pieces, which somehow serve as a target gene indicator, resulting in degradation of the target gene. RNAi is known to be effective in plants as well (Chuang C F, Meyerowitz E M, Proc Natl Acad Sci USA 97: 4985, 2000). For example, in order to use RNAi to suppress the expression of DNAs that encode the Ehd1 protein in plants, Ehd1 protein-encoding DNAs (SEQ ID: 2, 5, or 8), or double-stranded RNAs comprising a sequence similar to these DNAs, can be introduced into the plants in question. Plants whose flowering is delayed compared to a wild-type plant can then be selected from the resulting plants. Genes used for RNAi need not be completely identical to a target gene; however, they should comprise sequence identity of at least 70% or above, preferably 80% or above, more preferably 90% or above, and most preferably 95% or above. Sequence identity can be determined by an above-described method.
Suppression of endogenous gene expression can be achieved by co-suppression, through transformation with a DNA comprising a sequence identical or similar to a target gene sequence. “Co-suppression” refers to the phenomenon wherein transformation is used to introduce plants with a gene comprising a sequence identical or similar to a target endogenous gene sequence, thereby suppressing expression of both the exogenous gene introduced and the target endogenous gene. Although the details of the co-suppression mechanism are unclear, at least a part is thought to overlap with the RNAi mechanism. Co-suppression is also observed in plants (Smyth DR, Curr. Biol. 7: R793, 1997; Martienssen R Curr. Biol. 6: 810, 1996). For example, if one wishes to obtain a plant in which a DNA encoding an Ehd1 protein is co-suppressed, the plant in question can be transformed with a vector DNA designed to express the DNA encoding the Ehd1 protein, or a DNA comprising a similar sequence. Plants whose flowering is delayed compared to a wild-type plant are then selected from the resultant plants. Genes for use in co-suppression do not need to be completely identical to a target gene, but should comprise sequence identity of at least 70% or above, preferably 80% or above, more preferably 90% or above, and most preferably 95% or above. Sequence identity may be determined by an above-described method.
The present invention provides methods for producing transgenic plants, wherein said methods comprise the steps of introducing a DNA of the present invention into plant cells, and regenerating plants from these cells.
In the present invention, cells can be derived from any plant, without limitation. Vectors used for the transformation of plant cells are not limited as long as they can express the inserted gene in the plant cells. Vectors that can be used include, for example, vectors comprising promoters (e.g., cauliflower mosaic virus 35S promoter) for constitutive gene expression in plant cells, and vectors comprising promoters that are inducibly activated by external stimuli. The term “plant cell” as used herein includes various forms of plant cells, such as culture cell suspensions, protoplasts, leaf sections, and calluses.
A vector can be introduced into plant cells by known methods, such as by using polyethylene glycol, electroporation, Agrobacterium-mediated transfer, and particle bombardment. Agrobacterium (for example, EHA101) mediated transfer can be carried out by, for example, the ultraspeed transformation of monocotyledon (Japanese Patent No. 3141084). Particle bombardment can be carried out by, for example, using equipment available from Bio-Rad. Plants can be regenerated from transformed plant cells by known methods and according to the type of the plant cell (see Toki et al., (1995) Plant Physiol. 100:1503-1507).
For example, methods for producing a transformed rice plant include: (1) introducing genes into protoplasts using polyethylene glycol, then regenerating the plant (suitable for indica rice cultivars) (Datta, S. K. (1995) in “Gene Transfer To Plants”, Potrykus I and Spangenberg Eds., pp 66-74); (2) introducing genes into protoplasts using electric pulses, then regenerating the plant (suitable for japonica rice cultivars) (Toki et al. (1992) Plant Physiol. 100: 1503-1507); (3) introducing genes directly into cells by particle bombardment, then regenerating the plant (Christou et al. (1991) Bio/Technology, 9: 957-962); (4) introducing genes using Agrobacterium, then regenerating the plant (Hiei et al. (1994) Plant J. 6: 271-282); and so on. These methods are already established in the art and are widely used in the technical field of the present invention. Such methods can be suitably used in the present invention.
Once a transgenic plant is obtained, in which a DNA of the present invention has been introduced into its genome, progenies can be derived from that plant by sexual or vegetative propagation. Alternatively, plants can be mass-produced from breeding materials obtained from the plant (for example, from seeds, fruits, ears, tubers, tubercles, tubs, calluses, protoplasts, etc.), as well as from progenies or clones thereof.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a diagram showing the Ehd1 locus on chromosomes.
FIG. 2 is a bar graph showing the number of days until heading of nearly isogenic rice line T65(Ehd1) of the O. glaberrima Ehd1 gene, and its recurrent parent line (Taichung 65) under short-day, long-day, and natural day-length conditions. Under short-day and long-day conditions, plants were cultivated without transplantation in a day-light Kyushu University glasshouse. The results for natural day-length conditions are those of early season cultivation at Kyushu University (Fukuoka City) (sowed on May 2 and transplanted on June 14).
FIG. 3 represents a diagram showing a high-resolution linkage map and physical map of the Ehd1 region:
A: a linkage map prepared using a segregating population of 2500 individual plants and RFLP markers. The labels above the line represent RFLP markers, and the numerals under the line indicate the number of recombinations detected in the intervals between respective markers;
B: a detailed linkage map using CAPS markers and prepared based on nucleotide sequence information; and
C: a putative gene in the candidate genomic region, and genomic DNA fragments used in the transformation.
FIG. 4 depicts bar graphs showing the frequency distribution of the number of days to heading of a transformed plant (T0) produced by introducing either the 11.5 kb BamHI fragment or 7.6 kb KpnI fragment of Kasalath into Taichung 65, and cultivating under short-day conditions (ten hours of daylight).
FIG. 5 is a photograph showing the presence or absence of Kasalath ARR-like gene expression in the individual transformed plants. Total RNA was extracted from each plant, reverse transcribed, PCR-amplified, and digested with DdeI. The sizes of products corresponding to the Kasalath and Taichung 65-derived mRNAs are shown with “K” and “T” respectively.
FIG. 6 is a diagram showing the Ehd1 gene structure and nucleotide sequence polymorphism.
FIG. 7 shows a comparison of Ehd1 protein amino acid sequences. The arrow indicates the position of the mutated amino acid found only in Taichung 65. The amino acid sequences of Taichung 65, Nipponbare, Kasalath, and IRGC 104038 are set forth in SEQ ID NOs. 12, 9, 6, and 3, respectively.
FIG. 8 represents a photograph and diagram showing changes in the amount of Ehd1 mRNA accumulated over a day. Four weeks after sowing, total RNA was extracted from the leaves of Taichung 65 (T) and nearly isogenic line T65(Ehd1) (N) of the Ehd1 gene of O. glaberrima Steud. (IRGC 104038), and RT-PCR analysis was performed over 30 PCR cycles.



BEST MODE FOR CARRYING OUT THE INVENTION
Herein below, the present invention is more specifically described with reference to Examples; however, it is not to be construed as being limited thereto.
EXAMPLE 1
The Heading-promoting Action of the Ehd1 Gene Under Short-day Conditions
The Ehd1 locus is a QTL associated with heading time and detected using the progeny of a cross between japonica rice cultivar “Taichung 65” and West African region rice cultivar O. glaberrima Steud. (IRGC 104038). The Ehd1 locus has been proved to be located on the long arm of chromosome 10 (Doi et al., Breeding Science 49: 395-399, 1999) (FIG. 1). The Ehd1 gene of O. glaberrima (IRGC 104038) has been shown to comprise a heading-promoting action, and acts dominantly over the allele of Taichung 65. In this Example, both a nearly isogenic rice line T65(Ehd1), in which the Ehd1 region of Taichung 65 had been substituted with the Ehd1 gene of O. glaberrima, and Taichung 65 were cultivated under different day-length conditions to investigate the number of days until their heading. T65(Ehd1) headed seven days earlier than Taichung 65 under natural day-length conditions, 14 days earlier under long-day conditions, and 33 days earlier under short-day conditions (FIG. 2). These results demonstrated that the Ehd1 gene comprised the function of promoting heading, and that its heading-promoting action becomes more prominent under short-day conditions.
EXAMPLE 2
High-resolution Linkage Analysis
Genetic analysis performed hitherto has proven that the Ehd1 locus is positioned as a single gene locus (old name: Ef(t)) between RFLP markers C234 and G37, and co-segregated with C1369 (Doi, Taguchi, and Yoshimura: Japanese Society of Breeding, 94th lecture, Japanese Journal of Breeding (Suppl.), p 104, 1998). However, at the resolution level of this linkage analysis, it was difficult to isolate and identify genes using map-based cloning. In this example, detailed linkage analysis of the Ehd1 region was performed with a large segregating population essential for map-based cloning. A generation of progenies derived from backcrossing Taichung 65 and IRGC 104038 was used as the segregating population for linkage analysis. From these backcrossed progeny, plants were selected whose Ehd1 region was heterologous, and whose other genomic regions were mostly substituted with Taichung 65 type genome. These selected plants underwent self-fertilization, producing 2500 progeny plants (the F2 population). Those plants that comprise a chromosome with a recombination near the Ehd1 locus were selected using Ehd1-flanking CAPS (Cleaved Amplified Polymorphic Sequence) markers C1286 (primers [SEQ ID NO: 13/5′-CCAATGAAGGGTAAGTATCG-3′] and [SEQ ID NO: 14/5′-TGTGCTTAAGATACACGGTAGTTCA-3′], restriction enzyme NruI); and G37 (primers [SEQ ID NO: 15/5′-CTGCAGCTTCCACCATGGCA-3′] and [SEQ ID NO: 16/5′-CAAGGGTGCATTCATTGCACCTCCTCTAGCCATGGCCTAATGATGCA-3′], restriction enzyme EcoT22I) The genotype of the Ehd1 locus was determined by a progeny test of the F3 generation. That is, 48 inbred progeny plants from the selected plants (F2) were cultivated in an experimental farm at Kyushu University to determine their Ehd1 genotype based on variations in the number of days until the heading of each line. The linkage analysis results located the Ehd1 locus between RFLP markers C814A and C234, identifying eight and two recombinant individuals between Ehd1 and these markers respectively (FIG. 3).
EXAMPLE 3
Identification of a Candidate Gene
The nucleotide sequences of the RFLP markers flanking the Ehd1 gene were found to be comprised in published genomic nucleotide sequences, such that the nucleotide sequence of Ehd1 candidate genomic region was obtained from published nucleotide sequence data (GenBank Accession No. AC027038). Using information on the nucleotide sequences of this candidate genomic region, novel CAPS markers were prepared for narrowing down the candidate genomic region. Ehd1 was found to have two recombinations with CAPS marker 26-28 (primers [SEQ ID NO: 17/5′-ACGCTGCAACAAAGAGCAGA-3′] and [SEQ ID NO: 18/5′-TTGTTGACGAAAGCCCATTG-3′], restriction enzyme MspI); and one recombination with 12-14 (primers [SEQ ID NO: 19/5′-GGAGATCATGCTCACGGATG-3′] and [SEQ ID NO: 20/5′-CAAGCAAACACGGAGCGACT-3′], restriction enzyme BamHI). Furthermore, the Ehd1 gene was co-segregated with CAPS markers 13-15 (primers [SEQ ID NO: 21/5′-CCTTGCATCCGTCTTGATTG-3′] and [SEQ ID NO: 22/5′-GGGCAAATTCCCTCCAGAGT-3′], restriction enzyme MspI); 19-21 (primers [SEQ ID NO: 23/5′-TTTGGATACGTACCCCTGCAT-3′] and [SEQ ID NO: 24/5′-GCGCAATCGCATACACAATAA-3′ ], restriction enzyme MspI); and 23-25 (primers [SEQ ID NO: 25/5′-GAGCCCGAGCCCATGTATAG-3′] and [SEQ ID NO: 26/5′-TGGCTAAGATGGAGGGACGA-3′], restriction enzyme MboI). Therefore, the Ehd1 candidate region was narrowed down to a region of about 16 kb, flanked by CAPS markers 26-28 and 12-14. Gene predictions and similarity searches carried out against the nucleotide sequence of this candidate region using GENSCAN (http://genes.mit.edu/GENSCAN.html) proved the presence of three types of predicted genes. One type showed similarity to the two-component response regulator (ARR) gene of Arabidopsis. The other two types showed high similarity to rice EST, but no resemblance to known genes whose functions had been established. However, since none of these predicted genes could be excluded as Ehd1 candidates, transformation of these three types of predicted genes was used to verify function as Ehd1 candidates.
EXAMPLE 4
Functional Verification of Ehd1 Candidate Gene
A genomic DNA fragment derived from the indica rice cultivar Kasalath, which is assumed to comprise a functional Ehd1 allele, was used for the transformation. That is, a BAC library constructed from the genomic DNA of Kasalath (Baba et al., Bulletin of the NIAR 14: 41-49, 2000) was screened using a pair of primers 10-12 (SEQ ID NO: 27/5′-ATTGGGCCAAACTGCAAGAT-3′ and SEQ ID NO: 28/5′-ACGAGCCTAATGGGGGAGAT-3′) capable of amplifying the nucleotide sequence near Ehd1 to select BAC clone KBM128G10, which comprises the Ehd1 candidate gene. An 11.5 kb BamHI fragment comprising the ARR-like candidate gene and one of the predicted genes showing high similarity to the rice EST, and a 7.6 kb KpnI fragment comprising the two predicted genes other than the ARR-like candidate genes (FIG. 3), were excised from the BAC clone KBM128G10. These fragments were each incorporated into the Ti-plasmid vector pPZP2H-lac (Fuse et al., Plant Biotechnology 18: 219-222, 2001), and these transformed vectors were then introduced into Taichung 65, mediated by Agrobacterium. Regenerated plants were immediately transferred to growth chambers under short-day conditions (ten hours of daylight) and cultivated to investigate the number of days until heading. Almost all of the transgenic plants (TO) (18 plants) introduced with the 11.5 kb BamHI fragment headed earlier than the plants introduced with the vector alone (six plants) (FIG. 4). On the other hand, the number of days to heading in the six plants introduced with the 7.6 kb KpnI fragment was about the same as for the five plants introduced with the vector alone (FIG. 4). The expression of the Kasalath-derived ARR-like candidate gene in plants introduced with the 11.5 kb BamHI fragment was also confirmed by RT-PCR using a gene-specific marker (primers [SEQ ID NO: 29/5′-GAGATCAACGGCCACCGAAG-3′] and [SEQ ID NO: 30/5′-GTCGAGAGCGGTGGATGACA-3′], restriction enzyme DdeI). Transcription of the Kasalath-derived ARR-like candidate gene was observed in almost all of the plants (FIG. 5). Thus, the Ehd1 candidate could be narrowed down to the ARR-like candidate gene contained in the 11.5 kb BamHI fragment. Furthermore, to confirm that heading promotion was due to the transgene, individual plants comprising low copy numbers of the gene were selected from those transformed plants (T0) with earlier heading. Their self-fertilized progenies were cultivated under short-day conditions to investigate the number of days until heading. Each of the three types of progeny populations were divided into early-maturing plants (41-70 days to heading) and late-maturing plants (81 days or more to heading). All of the early-maturing plants retained the introduced Kasalath-derived ARR-like candidate gene, and headed at about the same time as the nearly isogenic rice line T65(Ehd1). On the other hand, the number of days to heading of Taichung 65 was 81 days or more, about the same as for late-maturing plants (Table 1). From the above-described results, the ARR-like candidate gene was shown to comprise heading-promotion function under short-day conditions, and to be the Ehd1 gene.









TABLE 1







Progeny
Number of days to heading (days)


















population
41-45
46-50
51-55
56-60
61-65
66-70
71-75
76-80
>81
Total




















Population
4
21
 2
—
—
—
—
—
5*
32


1


Population
—
 1
 7
—
7
4
—
—
8*
27


2


Population
—
 2
17
—
1
—
—
—
5*
25


3


Taichung 65
—
—
—
—
—
—
—
—
5
 5


T65(Ehd1)
—
 1
 2
—
—
—
—
—
—
3





*The introduced gene was not retained.






EXAMPLE 5
Nucleotide Sequence Analysis of the Ehd1 Candidate Gene
The genomic nucleotide sequences of approximately 7.6 kb Ehd1 regions were analyzed in rice cultivars O. glaberrima (IRGC 104038), Kasalath, Nipponbare, and Taichung 65 (TA repeats of 200 bp or more exist in Taichung 65; the exact number of these has not been determined). Compared to Kasalath, 60 or more nucleotide substitutions, insertions, and deletions were detected in this region in Nipponbare and Taichung 65, and at 140 positions or more in IRGC 104038 (FIG. 6). The full-length cDNA of IRGC 104038 was also determined, and as a result of comparing the genomic nucleotide sequences, Ehd1 was predicted to comprise six exons, with a full-length transcriptional region of 1316 bp and encoding a protein comprising 341 amino acids (FIG. 7). Comparison of differences in nucleotide sequences within the predicted transcriptional region with that of Kasalath detected four single-nucleotide substitutions and two-nucleotide insertions in Nipponbare; five single-nucleotide substitutions and two-nucleotide insertions in Taichung 65; and fourteen single-nucleotide substitutions and four- and three-nucleotide deletions in IRGC 104038. On comparing the amino acid sequences of the predicted translational products of IRGC 104038, Kasalath, and Nipponbare, and Taichung 65, it was revealed that seven amino acids are substituted between IRGC 104038 and Taichung 65, two amino acids between Kasalath and Taichung 65, and one amino acid between Nipponbare and Taichung 65 (FIG. 7). Of these, the substitution of glycine at the 219th amino acid (G in IRGC 104038, Kasalath, and Nipponbare) to arginine (R in Taichung 65) was the only mutation occurring in Taichung 65 alone (FIG. 7). This glycine had been highly conserved among the known ARR gene families. These facts suggested that this amino acid mutation is associated with the reduced function of Ehd1 derived from Taichung 65.
The genomic nucleotide sequences of the Ehd1 regions of O. glaberrima (IRGC 104038), Kasalath, Nipponbare, and Taichung 65 are set forth in SEQ ID NOs: 1, 4, 7, and 10 respectively; the nucleotide sequences of cDNAs thereof are set out in SEQ ID NOs: 2, 5, 8, and 11 respectively; and the amino acid sequences of the proteins encoded by these DNAs are set out in SEQ ID NOs: 3, 6, 9, and 12, respectively.
EXAMPLE 6
Expression Analysis of the Ehd1 Candidate Gene
Variations of the Ehd1 gene expression level over a one-day period were examined for T65(Ehd1) in which the Ehd1 region of Taichung 65 had been substituted with a chromosomal fragment derived from IRGC 104038. T65(Ehd1) and Taichung 65 were sowed in experimental greenhouses (at Tsukuba City, Ibaraki Prefecture) during the last ten days of December (sunrise: 6:50 AM, sunset: 4:30 PM; short-day conditions), cultivated for four weeks, and then the leaves were collected from both rice lines every three hours over 24 hours for analysis. Total RNA was extracted from these materials, and RT-PCR was performed for the Ehd1 gene using a pair of primers (sense strand [SEQ ID NO: 31/5′-TGGATCACCGAGAGCTGTGG-3′] and an antisense strand [SEQ ID NO: 32/5′-ATTTCCTTGCATCCGTCTTG-3′]). As a result, the transcriptional product of this gene was found to accumulate in large amounts around dawn (3 and 6 o'clock), with a tendency to decrease to undetectable levels after sunset till midnight (18 and 21 o'clock) (FIG. 8). Such circadian fluctuation in gene expression level is a phenomenon often observed in genes associated with photoperiod sensitivity, indicating some sort of association of this Ehd1 gene with photosignal transduction. From the above-described results, the candidate gene identified by map-based cloning was judged to be the Ehd1 gene promoting rice heading.
INDUSTRIAL APPLICABILITY
In conventional rice breeding, flowering (heading) time has been altered by (1) selection of early-maturing and late-maturing varieties by crossing, (2) mutagenesis by radiation and chemicals, and so on. However, these procedures posed problems such as the requirement of long periods of time for successful breeding, and the inability to control the degree or direction of variation. The present invention has enabled the establishment of a novel method for modifying plant flowering time by utilizing the isolated Ehd1 gene. Therefore, by transforming a sense strand of Ehd1 gene into a plant cultivar in which the function of this gene has been lost, for example, into Taichung 65 rice, rice heading (flowering) can be promoted under short-day conditions. At the same time, by introducing the Ehd1 gene in the antisense direction into a plant cultivar in which the function of the Ehd1 gene is retained, for example, into Nipponbare or Kasalath rice, expression of the endogenous Ehd1 gene can be suppressed to delay heading (flowering). Since this alteration can be expected to occur not only under short-day conditions but also under long-day or natural day-length conditions, the present invention is effective in controlling heading time (flowering time) under various cultivation conditions. Since the period of time required for transformation is extremely short compared to that required for gene transfer by crossing, flowering time can be modified without altering other characteristics. The use of the isolated Ehd1 gene to promote flowering is expected to be useful in simple alteration of the flowering time of plants such as rice, and to contribute to the breeding of plants adapted to different locations.

## Claims

1. An isolated A DNA according to (a) or (b) below, wherein said DNA encodes a plant-derived protein that functions to promote plant flowering:
(a) a DNA encoding a protein comprising the amino acid sequence of SEQ ID NO:6;
(b) a DNA consisting of the nucleotide sequence of SEQ ID NO:4 or 5.

2. A vector comprising the DNA of claim 1.

3. A transformed plant cell comprising the DNA of claim 1.

4. A transgenic plant comprising the transformed plant cell of claim 3.

5. A transgenic plant that is a progeny or a clone of the transgenic plant of claim 4 and wherein the progeny or clone comprise an isolated DNA encoding a protein comprising the amino acid sequence of SEQ ID NO:6 or an isolated DNA consisting of the nucleotide sequence of SEQ ID NO:4 or 5.

6. A breeding material of the transgenic plant of claim 4 and wherein the breeding material comprises an isolated DNA encoding a protein comprising the amino acid sequence of SEQ ID NO:6 or an isolated DNA consisting of the nucleotide sequence of SEQ ID NO:4 or 5.

7. A method for producing a transgenic plant, comprising the steps of introducing the DNA of claim 1 into a plant cell, and regenerating a plant from said plant cell.

8. A method for promoting plant flowering, comprising expressing the DNA of claim 1 in plant cells.

9. The method of claim 8, wherein the plant is rice.

10. A transformed plant cell comprising the vector of claim 2.

11. A transgenic plant comprising the transformed plant cell of claim 10.

12. A transgenic plant that is a progeny or a clone of the transgenic plant of claim 11 and wherein the progeny or clone comprise an isolated DNA encoding a protein comprising the amino acid sequence of SEQ ID NO:6 or an isolated DNA consisting of the nucleotide sequence of SEQ ID NO:4 or 5.

13. A breeding material of the transgenic plant of claim 11 and wherein the breeding material comprises an isolated DNA encoding a protein comprising the amino acid sequence of SEQ ID NO:6 or an isolated DNA consisting of the nucleotide sequence of SEQ ID NO:4 or 5.

14. A breeding material of the transgenic plant of claim 12 and wherein the breeding material comprises an isolated DNA encoding a protein comprising the amino acid sequence of SEQ ID NO:6 or an isolated DNA consisting of the nucleotide sequence of SEQ ID NO:4 or 5.

15. A method for producing a transgenic plant, comprising the steps of introducing the vector of claim 2 into a plant cell, and regenerating a plant from said plant cell.

16. A breeding material of the transgenic plant of claim 5 and wherein the breeding material comprises an isolated DNA encoding a protein comprising the amino acid sequence of SEQ ID NO:6 or an isolated DNA consisting of the nucleotide sequence of SEQ ID NO:4 or 5.