# Patent

## Title
Bzip type transcription factors regulating the expression of rice storage protein

## Abstract

cDNAs (RISBZ1, RISBZ4, and RISBZ5) encoding bZIP transcription factors were isolated from a cDNA library originating in rice plant seed. The cDNAs encode novel proteins and have binding activity to the GCN4 motif. Among them, RISBZ1 activated transcription mediated by the GCN4 motif by 100-fold or more. Since the expression of RISBZ1 precedes the expression of a seed storage protein gene and is expressed only in maturing seeds, it is suggested that RISBZ1 controls the expression of rice seed storage proteins. In addition, by linking the recognition sequence of the transcription factor, the GCN4 motif, in tandem and introducing it into the promoter for a gene encoding seed storage protein to facilitate its binding to the transcription factor RISBZ1, expression of a foreign gene under the control of the modified promoters is greatly enhanced.

## Background
TECHNICAL FIELD
The present invention relates to a novel transcription factor and its use pertaining to the endosperm-specific expression of the storage protein in the rice plant seed.
BACKGROUND ART
Seed storage protein is expressed in seeds only during the maturing stage, and the expression of genes encoding this protein is analyzed as a suitable model for investigating the transcription regulatory mechanism of plant genes (Goldberg, R. B. et al., Science 266: 605–614, 1994). The expression of a gene that codes for a seed storage protein is known to be regulated by the cooperation of a plurality of cis factors in a promoter. The binding of a transcription factor to a specific cis regulatory factor is important in the initiation of transcription and the tissue- and time-specific expression. It can be explained that the expression of a seed storage protein is induced by several types of cis regulatory factors relating to the regulation of seed-specific expression when transcription factors that recognize specific cis regulatory factor bind and aggregate. Functional analyses of cis regulatory factors and transcription factors of crop storage protein genes have been conducted in order to elucidate the molecular mechanism of the expression of seed storage proteins (Thomas, T. L., Plant Cell 5: 1401–1410, 1993; Morton, R. L. et al., in Seed Development and Germination, pp. 103–138, Marcel Dekker, Inc., 1995).
However, despite considerable research, analyses using transformed plants failed to identify the cis regulatory factors essential for gene expression regulation in nearly all crops studied, and the gene expression regulatory mechanism has still not been clearly understood. In the case of monocotyledons in particular, the promoter analyses using stable transformed plants has been performed in only the seed storage protein, glutelin, of the rice plants. On the other hand, in the case of maize, wheat and barley, analyses have been conducted using particle guns or tobacco transformants (Muller, M. and Knudsen, S., Plant J. 6:343–355, 1993; Albani, D. et al., Plant Cell 9: 171–184, 1997; Marzabal, P. M. et al., Plant J. 16: 41–52, 1998).
It has been shown that the endosperm-specific expression of the seed storage protein gene of grains is controlled by the collaborative action of several types of cis regulatory factors. The Prolamin box (TGTAAAG), GCN4 motif (TGA(G/C)TCA), AACA motif (AACAAAA), and ACGT motif, which are conserved in the seed storage protein gene promoters of numerous grains, have been characterized as cis regulatory factors involved in endosperm-specific expression by loss-of-function and gain-of-function analyses (Morton, R. L. et al., In: Seed Development and Germination, pp. 103–138, Marcel Dekker Inc., 1995).
The GCN4 motif has been frequently found not only from seed storage protein gene, but also from promoters of genes involved in the metabolism (Muller, M. and Knudsen, S., Plant J. 6: 343–355, 1993). Recently, a polymer of the GCN4 motif of rice plant glutelin gene has been found to reproduce endosperm-specific expression in transformed rice plants, and remarkable decrease in promoter activity and changes in its expression pattern have been found due to the substitution or deletion of nucleotides in the GCN4 motif. These facts prove that the GCN4 motif plays an important role in endosperm-specific expression (Wu, C. Y. et al., Plant J. 14: 673–683, 1998). The GCN4 motif is coupled to a Prolamin box (TGTAAAG) via a plurality of bases in many cases, and is one of the constituents of the two-factor endosperm box found in the prolamin gene promoters of nearly all grains, including wheat glutenin, barley hordein, rye secalin, sorghum cafulin and adlay coixin. The AACA motif is involved in the expression of nearly all rice glutelin genes. Although the combination of two motifs (GCN4 motif and Prolamin box or GCN4 motif and AACA motif) is required for gene expression, in order to adequately function as an endosperm-specific promoter, an additional motif is essential (Takaiwa, F. et al., Plant Mol. Biol. 30: 1207–1221, 1996; Yoshihara, T. et al., FEBS Letts. 383: 213–218, 1996; Wu, C. Y. et al., Plant J. (in press)). Recently, it has been demonstrated that, in order to function as aminimum promoter capable of reproducing endosperm-specific expression in glutelin genes (GluB1) of rice plant, at least three constituents, the GCN4 motif, the AACA motif, and the ACGT motif, present in the −197 bp promoter region, are essential (Wu, C. Y. et al., Plant J. 14: 673–683, 1998; Wu, C. Y. et al., Plant J. 23: 415–421, 2000).
Opaque2 (O2) of maize is an endosperm-specific transcription factor of the bZIP type, and this O2 binds to the ACGT motif in the 22 kDa α-zein gene promoter of maize to activate transcription (Schmidt, R. J. et al., Plant Cell 4: 689–700, 1992). O2 has been reported to be involved in endosperm-specific transcription of b-32 ribosome deactivating protein gene by binding to the (Ga/tTGAPyPuTGPu) sequence (Lohmer, S. et al., EMBO J. 10: 617–624, 1991). O2 is thus considered to have a wide-ranging binding capability. Reportedly, the GCN4 motif is recognized by O2, and transcription is activated through the binding of O2 to the GCN4 motif (Wu, C. Y. et al., Plant J. 14: 673–683, 1998; Holdsworth, M. J. et al., Plant Mol. Biol. 29: 711–720, 1995). In seeds, during the maturing stage, in vivo footprint analysis showed that the nuclear protein binds to the GCN4 motif and Prolamin box present in wheat low molecular weight glutenin gene promoter (Vicente-Carbajos, J. et al., Plant J. 13: 629–640, 1998) and maize γ-zein gene promoter (Marzabal, P. M. et al., Plant J. 16: 41–52, 1998). In addition, the results of an in vitro DNaseI footprint analysis showed that the nuclear protein of maturing rice plant seeds as well as GST-O2 fused protein specifically recognize the GCN4 motif of the rice glutelin gene promoter (Wu, C. Y., et al., Plant J. 14: 673–683, 1998; Kim, S. Y. and Wu, R., Nucl. Acids Res. 18: 6845–6852, 1990). These findings indicate that an O2-like transcription factor is present in grain seeds, and that it controls the endosperm-specific expression of numerous seed storage protein genes mediated by the GCN4 motif.
Recently, cDNA clones of transcription factors that recognize the GCN4 motif have been isolated in wheat (Albani, D. et al., Plant Cell 9: 171–184, 1997) and barley (Vicente-Carbajos, J. et al., Plant J. 13: 629–640, 1998; Onate, L. et al., J. Biol. Chem. 274: 9175–9182, 1999), and have been named SPA, BLZ1 and BLZ2. These transcription factors have been determined to activate the transcription of seed storage protein genes mediated by the GCN4 motif in wheat low molecular weight glutenin and barley B1 hordein gene promoter. Interestingly, these transcription factors were expressed seed-specifically. Although cDNA that codes for a transcription factor having a high homology with the bZIP domain of O2 has previously been isolated from rice plants, it remains to be confirmed whether or not it activates transcription of seed storage protein gene mediated by the GCN4 motif (Izawa, T. et al., Plant Cell 6: 1277–1287, 1994; Nakase, M. et al., Plant Mol. Biol. 33: 513–522, 1997).
DISCLOSURE OF THE INVENTION
An object of the present invention is to provide a novel transcription factor that regulates the expression of rice seed storage protein by binding to the GCN4 motif, a gene that codes for the factor, plant cells and plant bodies in which the gene has been introduced, and a method for production and use thereof.
The present inventors conducted research to resolve the above problems. As mentioned above, the GCN4 motif is a sequence that is highly conserved in the promoters of grain seed storage protein genes, and plays a central role in the endosperm-specific expression of the genes. This GCN4 motif is recognized by the bZIP transcription factor family that is closely related to the Opaque2 (O2) protein of maize. Therefore, the present inventors thought that, by isolating bZIP transcription factor from the rice seeds, it would be possible to identify the transcription factor that binds to the GCN4 motif to control the expression of rice seed storage protein.
First, the present inventors screened a cDNA library originating in rice seed and isolated cDNA that codes for five types of bZIP transcription factors (RISBZ1, RISBZ2, RISBZ3, RISBZ4, and RISBZ5). Based on the homology of the presumed amino acid sequences, RISBZ2 and RISBZ3 were identical to RITA1 (Izawa, T. et al., Plant Cell 6: 1277–1287, 1994) and REB (Nakase, M. et al., Plant Mol. Biol. 33: 513–522, 1997), respectively, and the remaining RISBZ1, RISBZ4; and RISBZ5 were revealed to code for novel proteins. When the binding ability of RISBZ1, RISBZ2, RISBZ3, RISBZ4, and RISBZ5 to GCN4 motif was investigated, they all exhibited binding activity to the GCN4 motif. Furthermore, the transcription activation ability of the five proteins by binding to the GCN4 motif was investigated. As a result, only RISBZ1 activated transcription 100-fold or more by binding to the GCN4 motif. In addition, an analysis using the GAL4 DNA binding domain of yeast revealed that proline-rich, 27 amino acid residues of the N-terminal side of RISBZ1 functioned as a the transcription-activating domain. The difference in transcription activation ability between RISBZ1 and the other RISBZ proteins was primarily due to the mutation of 7 amino acid residues (for RISBZ2) or deletion of the transcription-activating domain (for RISBZ3, RISBZ4, and RISBZ5). This finding suggests that the difference in transcription activation ability between RISBZ1 and other RISBZ proteins occur due to a structural mutation of the transcription activating domain. In addition, RISBZ1 was found to form not only a homodimer, but also heterodimers with other RISBZ proteins. Since the expression of RISBZ1 precedes the expression of seed storage protein gene and is expressed only in maturing seeds, RISBZ1 may control the expression of seed storage protein. In order to investigate the expression of RISBZ1 gene, the promoter of the RISBZ1 gene was coupled to a GUS reporter gene, and this construct was introduced into a rice plant. In this rice plant the GUS gene was strongly expressed in the aleurone layer.
As described above, the present inventors demonstrated that the novel proteins RISBZ1, RUSBZ4, and RISBZ5 actually bind to the GCN4 motif, and clarified that RISBZ1 is a transcription activation factor involved in endosperm-specific expression of the rice seed storage protein gene.
The present inventors also produced a transformed plant that contained a DNA construct in which the RISBZ1 of the present invention was connected downstream of a promoter and a DNA construct in which a reporter gene was connected downstream of a promoter containing the target sequence of RISBZ1. The inventors then succeeded in measuring the transcription activity of RISBZ1 in the transformed plant by using the expression of the reporter gene as an indicator. These findings enable high level expression of a useful, highly value-added foreign gene within the transformed plant cells in which the foreign gene is connected downstream of a promoter containing the target sequence of RISBZ1 instead of the above reporter gene.
The present invention relates to a novel transcription factor that regulates the expression of rice seed storage protein by binding to the GCN4 motif, a gene encoding the factor, plant cells and plants in which the gene has been introduced, and methods for production and use thereof. More specifically, the present invention provides the following:
[1] a DNA selected from the following (a) through (d):

    
    
        (a) a DNA encoding a protein comprising the amino acid sequence set forth in any one of SEQ ID NOs: 2, 5, and 7;
        (b) a DNA comprising a coding region of the nucleotide sequence set forth in any one of SEQ ID NOs: 1, 3, 4, and 6;
        (c) a DNA comprising the amino acid sequence set forth in any one of SEQ ID NOs: 2, 5, and 7, in which one or more amino acids are substituted, deleted, added, and/or inserted, and encoding a protein that is functionally equivalent to a protein comprising the amino acid sequence set forth in any one of SEQ ID NOs: 2, 5, and 7; and
        (d) a DNA hybridizing under stringent conditions with a DNA comprising the nucleotide sequence set forth in any one of SEQ ID NOs: 1, 3, 4, and 6, and encoding a protein functionally equivalent to a protein comprising the amino acid sequence set forth in any one of SEQ ID NOs: 2, 5, and 7;
    
    


[2] the DNA according to [1], which encodes a protein that binds to the GCN4 motif or activates expression of rice seed storage protein;
[3] the DNA according to [1] or [2], which is derived from rice plant;
[4] a DNA encoding antisense RNA complementary to a transcription product of the DNA according to any one of [1] through [3];
[5] a DNA encoding an RNA having ribozyme activity that specifically cleaves a transcription product of the DNA according to any one of [1] through [3];
[6] a DNA encoding an RNA that suppresses the expression of the DNA according to any one of [1] through [3] in plant cells by co-inhibition effects, and having 90% or more homology with the DNA according to any one of [1] through [3];
[7] a DNA encoding a protein having a dominant negative phenotype of a protein encoded by the DNA according to any one of [1] through [3] which is endogenous in plant cells;
[8] a vector containing the DNA according to any one of [1] through [3];
[9] a transformed cell retaining the DNA according to any one of [1] through [3] or the vector according to [8];
[10] a protein that is encoded by the DNA according to any one of [1] through [3];
[11] a method of producing the protein according to [10], the method comprising steps of culturing the transformed cell according to [9] and collecting the expressed protein from said transformed cell or their culture supernatant;
[12] a vector containing the DNA according to any one of [4] through [7];
[13] a transformed plant cell retaining the DNA according to any one of [1] through [7] or the vector according to [8] or [12];
[14] a transformed plant containing the transformed plant cell according to [13];
[15] a transformed plant that is a progeny or clone of the transformed plant according to [14];
[16] a reproductive material of the transformed plant according to [14] or [15];
[17] an antibody that binds to the protein according to [10];
[18] a plant having on its genome a DNA construct in which the DNA according to [1] is operably connected downstream of an expression control region and a DNA construct in which a foreign gene is operably connected downstream of an expression control region having the target sequence of the protein according to [10];
[19] the plant according to [18], wherein the target sequence is a sequence containing the GCN4 motif;
[20] the plant according to [19], wherein the GCN4 motif has the sequence set forth in any one of SEQ ID NOs: 8, 13, and 14;
[21] the plant according to [18], wherein the target sequence is a sequence containing a G/C box; and,
[22] a method of producing the plant according to any one of [18] through [21], the method comprising a step of crossing a plant having on its genome a DNA construct in which the DNA according to [1] is operably connected downstream of an expression control region, with a plant having on its genome a DNA construct in which a foreign gene is operably connected downstream of an expression control region containing the target sequence of the protein according to [10].
The present invention provides DNAs encoding RISBZ1, RISBZ4, and RISBZ5 protein originating in the rice plant. The nucleotide sequence of the cDNA of RISBZ1 is shown in SEQ ID NO: 1, the amino acid sequence of the protein encoded by the cDNA is shown in SEQ ID NO: 2, and the nucleotide sequence of the genome DNA is shown in SEQ ID NO: 3 (the genome DNA sequence set forth in SEQ ID NO: 3 contains introns and is composed of six exons). The nucleotide sequences of the cDNAs of RISBZ4 and RISBZ5 proteins are shown in SEQ ID NO: 4 and 6, respectively, while the amino acid sequences of the proteins encoded by the cDNAs of RISBZ4 and RISBZ5 proteins are shown in SEQ ID NO: 5 and 7, respectively. In the present specification, the RISBZ1, RISBZ4, and RISBZ5 of the present invention are collectively referred to as RISBZ.
The RISBZ proteins of the present invention are thought to be bZIP transcription factors having the ability to bind the GCN4 motif. Among these, RISBZ1 remarkably activates transcription by binding to the GCN4 motif. Since the promoter of the RISBZ1 gene is activated in the aleurone layer of rice seeds, RISBZ1 is thought to be a transcription-activating factor that controls the expression of rice seed storage protein.
In addition, it has been reported that bZIP transcription factors form various homo/heterodimers through the combination of various factors belonging to the bZIP transcription factor family. As a result, control factors with various functions are formed, which control gene transcription. In the Examples described below, RISBZ2 and RISBZ3 were shown to form a heterodimer with RISBZ1. In addition, RISBZ4 and RISBZ5 have extremely high homology (96% and 82.7%, respectively) with the bZIP domain of RISBZ3, and these factors would also form heterodimers with RISBZ1. These facts suggest that RISBZ4 and RISBZ5 of the present invention would form, with the RISBZ1 and other RISBZ members of the present invention, heterodimers having various transcription activating abilities and DNA binding properties depending on the maturation stage and tissue to control the expression of seed storage protein.
Thus, the DNA encoding the RISBZ protein of the present invention, or a molecule that controls the expression of the DNA, would be useful in, for example, regulating the expression of seed storage protein. Regulation of the expression of seed storage protein has various industrial advantages. For example, it would be possible to accumulate abundant foreign gene products in the endosperm by deleting seed storage protein in the endosperm. On the other hand, by highly accumulating seed storage protein in the endosperm, it would be possible to produce seeds (e.g., rice) having greater nutritional value.
The DNA encoding the RISBZ protein of the present invention includes genomic DNA, cDNA, and chemically synthesized DNA. A genomic DNA and cDNA can be prepared according to conventional methods known to those skilled in the art. More specifically, a genomic DNA can be prepared, for example, as follows: (1) extracting genomic DNA from plant cells or tissues; (2) constructing a genomic library (utilizing a vector, such as plasmid, phage, cosmid, BAC, PAC, and so on); (3) spreading the library; and (4) conducting colony hybridization or plaque hybridization using a probe prepared based on the DNA encoding the protein of the present invention (e.g. SEQ ID NO: 1, 3, 4, or 6). Alternatively, a genomic DNA can be prepared by PCR, using primers specific to the DNA encoding the protein of the present invention (e.g. SEQ ID NO: 1, 3, 4, or 6). On the other hand, cDNA can be prepared, for example, as follows: (1) synthesizing cDNAs based on mRNAs extracted from plant cells or tissues; (2) preparing a cDNA library by inserting the synthesized cDNA into vectors, such as λZAP; (3) spreading the cDNA library; and (4) conducting colony hybridization or plaque hybridization as described above. Alternatively, cDNA can also be prepared by PCR.
The present invention includes DNAs encoding proteins functionally equivalent to the RISBZ protein of SEQ ID NO: 2, 5, or 7. Herein, the term “functionally equivalent to the RISBZ protein” means that the object protein has the biological function equivalent to those of RISBZ protein of SEQ ID NO: 2, 5, or 7, such as the function of binding to GCN4 motif and/or regulating the expression of rice seed storage proteins. The rice seed storage proteins include, for example, rice glutelins.
Examples of such DNAs include those encoding mutants, derivatives, alleles, variants, and homologues comprising the amino acid sequence of. SEQ ID NO: 2, 5, or 7 wherein one or more amino acids are substituted, deleted, added, and/or inserted.
Examples of methods for preparing a DNA encoding a protein comprising altered amino acids well known to those skilled in the art include the site-directed mutagenesis (Kramer, W. and Fritz, H. -J., Oligonucleotide-directed construction of mutagenesis via gapped duplex DNA. Methods in Enzymology, 154: 350–367, 1987). The amino acid sequence of a protein may also be mutated spontaneously due to the mutation of a nucleotide sequence. A DNA encoding proteins having the amino acid sequence of a natural RISBZ protein (SEQ ID NOs: 2, 5, or 7) wherein one or more amino acids are substituted, deleted, and/or added are also included in the DNA of the present invention, so long as they encode a protein functionally equivalent to the natural RISBZ protein. Additionally, nucleotide sequence mutants that do not give rise to amino acid sequence changes in the protein (degeneracy mutants) are also included in the DNA of the present invention. The numbers of nucleotide mutations in the object DNA at amino acid level is typically 100 amino acids or less, preferably 50 amino acids or less, more preferably 20 amino acids or less, and most preferably 10 amino acids or less (for example, amino acids or less or 3 amino acids or less).
Whether or not a certain DNA codes for a protein having the function of binding to the GCN4 motif can be determined by, for example, gel shift assay usually used by those skilled in the art. More specifically, this assay can be carried out as follows: First, the detected DNA is incorporated into a vector so that its gene product forms a fused protein with GST and the vector is allowed to express the fused protein. The expression product is purified using GST as an indicator followed by mixing with a labeled DNA probe containing the GCN4 motif. This mixed solution is analyzed by electrophoresis using nondenaturing acrylamide gel. Binding activity can then be evaluated based on the locations of the detected bands on the gel.
In addition, whether or not a certain DNA codes for a protein having the function of activating expression of rice seed storage protein can be determined by, for example, a reporter assay. More specifically, this assay can be carried out as follows. First, a vector is constructed so that a reporter gene is connected to and downstream of the promoter of rice seed storage protein. This vector and a vector that expresses the gene product of a test DNA are introduced into the cells for the reporter assay, and the transcription activity of the test DNA gene product is evaluated by measuring the activity of the reporter gene product. An example of the promoter of rice seed storage protein that can be used for the reporter assay is the rice glutelin gene promoter. There are no particular restrictions to the reporter gene provided its expression can be detected, and any reporter gene that are usually used in various assay systems by those skilled in the art, can be used. A preferable example of the reporter gene is the β-glucuronidase (GUS) gene.
A DNA encoding a protein functionally equivalent to the RISBZ protein set forth in SEQ ID NO: 2, 5, or 7 can be produced by, for example, methods well known to those skilled in the art including: methods using hybridization techniques (Southern, E. M., Journal of Molecular Biology, Vol. 98, 503, 1975); and 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). It is routine for a person skilled in the art to isolate a DNA with high homology to the RISBZ gene from rice and so forth using the RISBZ gene (SEQ ID NO: 1, 3, 4, or 6) or parts thereof as a probe, and oligonucleotides hybridizing specifically to the gene as a primer. Such a DNA encoding a protein functionally equivalent to the RISBZ protein, isolable by hybridization techniques or PCR techniques, is included in the DNA 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 which yield a similar stringency to the conditions. DNAs with higher homology are expected to be isolated efficiently when hybridization is performed under conditions with higher stringency, for example, 6 M urea, 0.4% SDS, and 0.1×SSC. These DNAs isolated under such conditions are expected to encode a protein having a high amino acid level homology with RISBZ protein (SEQ ID NO: 2, 5, or 7). Herein, high homology means an identity of at least 50% or more, more preferably means an identity of at least 70% or more, and most preferably means an identity of at least 90% or more (e.g., 95% or more) throughout the entire amino acid sequence. The degree of sequence identity can be determined by FASTA search (Pearson W. R. and D. J. Lipman Proc. Natl. Acad. Sci. USA. 85:2444–2448, 1988) or BLAST search.
The DNA of the present invention can be used, for example, to prepare recombinant proteins and to produce transgenic plants as described above.
A recombinant protein is usually prepared by inserting a DNA encoding a protein of the present invention into an appropriate expression vector, introducing the vector into an appropriate cell, culturing the transformed cells, and purifying expressed proteins. A recombinant protein can be expressed as a fusion protein with other proteins so as to be easily purified, for example, as a fusion protein with maltose binding protein in Escherichia coli (New England Biolabs, USA, vector pMAL series), as a fusion protein with glutathione-S-transferase (GST) (Amersham Pharmacia Biotech, vector pGEX series), or tagged with histidine (Novagen, pET series). The host cell is not limited so long as the cell is suitable for expressing the recombinant protein. It is possible to utilize, for example, yeast, plant, insect cells or various other animal cells besides the above-described E. coli. A vector can be introduced into a host cell by a variety of methods known to one skilled in the art. For example, a transformation method using calcium ions (Mandel, M. and Higa, A. Journal of Molecular Biology, 53, 158–162,1970; Hanahan, D. Journal of Molecular Biology, 166, 557–580, 1983) can be used to introduce a vector into E. coli. A recombinant protein expressed in the host cells can be purified and recovered from the host cells or the culture supernatant thereof by known methods in the art. When a recombinant protein is expressed as a fusion protein with maltose binding protein or other partners, the recombinant protein can be easily purified via affinity chromatography.
The resulting protein can be used to prepare an antibody that binds to the protein. For example, a polyclonal antibody can be prepared by immunizing immune animals, such as rabbits, with a purified protein of the present invention or its portion, collecting blood after a certain period, and removing clots. A monoclonal antibody can be prepared by fusing myeloma cells with the antibody-forming cells of animals immunized with the above protein or its portion, isolating a monoclonal cell expressing a desired antibody (hybridoma), and recovering the antibody from the cell. The antibody thus obtained can be utilized to purify or detect a protein of the present invention. Accordingly, the present invention includes antibodies that bind to proteins of the invention.
A plant transformant expressing DNAs of the present invention can be created by inserting a DNA encoding a protein of the present invention into an appropriate vector, introducing this vector into a plant cell, and then, regenerating the resulting transformed plant cell.
On the other hand, a plant transformant in which the expression of the DNA of the present invention is suppressed can be created using a DNA that suppresses the expression of a DNA encoding a protein of the present invention: wherein the DNA is inserted into an appropriate vector, the vector is introduced into a plant cell, and then, the resulting transformed plant cell is regenerated. The phrase “suppression of expression of a DNA encoding a protein of the present invention” includes suppression of gene transcription as well as suppression of translation to protein. Furthermore, it also includes the complete inability of expression of DNA as well as reduction of expression.
The expression of a specific endogenous gene in plants can be suppressed by methods utilizing antisense technology conventional to the art. Ecker et al. were the first to demonstrate the antisense effect of an antisense RNA introduced by electroporation into plant cells by using the transient gene expression method (J. R. Ecker and R. W. Davis Proc. Natl. Acad. Sci. USA 83: 5372, 1986). Thereafter, the target gene expression was reportedly reduced in tobacco and petunias by expressing antisense RNAs (A. R. van der Krol et al. Nature 333: 866, 1988). The antisense technique has now been established as a means of suppressing target-gene expression in plants.
Multiple factors cause antisense nucleic acid to suppress the target-gene expression. These include the following: inhibition of transcription initiation by triple strand formation; suppression of transcription by hybrid formation at the site where the RNA polymerase has formed a local open loop structure; transcription inhibition by hybrid formation with the RNA being synthesized; suppression of splicing by hybrid formation at the junction between an intron and an exon; suppression of splicing by hybrid formation at the site of spliceosome formation; suppression of mRNA translocation from the nucleus to the cytoplasm by hybrid formation with mRNA; suppression of splicing by hybrid formation at the capping site or at the poly(A) addition site; suppression of translation initiation by hybrid formation at the binding site for the translation initiation factors; suppression of translation by hybrid formation at the site for ribosome binding near the initiation codon; inhibition of peptide chain elongation by hybrid formation in the translated region or at the polysome binding sites of mRNA; and suppression of gene expression by hybrid formation at the sites of interaction between nucleic acids and proteins. These factors suppress the target gene expression by inhibiting the process of 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)).
An antisense sequence of the present invention can suppress the target gene expression by any of the above mechanisms. In one embodiment, if an antisense sequence is designed to be complementary to the untranslated region near the 5′ end of the gene's mRNA, it will effectively inhibit translation of a gene. It is also possible to use sequences complementary to the coding regions or to the untranslated region on the 3′ side. Thus, the antisense DNA used in the present invention includes a DNA having antisense sequences against both the untranslated regions and the translated regions of the gene. The antisense DNA to be used is connected downstream of an appropriate promoter, and, preferably, a sequence containing the transcription termination signal is connected on the 3′ side. The DNA thus prepared can be transfected into the desired plant by known methods. The sequence of the antisense DNA is preferably a sequence complementary to the endogenous gene of the plant to be transformed or a part thereof, but it need not be perfectly complementary so long as it can effectively inhibit the gene expression. The transcribed RNA is preferably 90% or more, and most preferably 95% or more complementary to the transcribed products of the target gene. The complementary of sequences can be determined by the above-described search methods. In order to effectively inhibit the expression of the target gene by means of an antisense sequence, the antisense DNA should be at least 15 nucleotides long or more, preferably 100 nucleotides long or more, and still more preferably 500 nucleotides long or more. The antisense DNA to be used is generally shorter than 5 kb, and preferably shorter than 2.5 kb.
DNA encoding ribozymes can also be used to suppress the expression of endogenous genes. A ribozyme means an RNA molecule that has catalytic activities. There are many ribozymes having various activities. Research on the ribozymes as RNA cleaving enzyme has enabled the design of a ribozyme that site-specifically cleaves RNA. While some ribozymes of the group I intron type or the M1RNA contained in RNaseP consist of 400 nucleotides or more, others belonging to the hammerhead type or the hairpin type have an activity domain of about 40 nucleotides (Makoto Koizumi and Eiko Ohtsuka Tanpakushitsu Kakusan Kohso (Nucleic acid, Protein, and Enzyme) 35: 2191, 1990).
The self-cleavage domain of a hammerhead type ribozyme cleaves at the 3′ side of C15 of the sequence G13U14C15. Formation of a nucleotide pair between U14 and A at the ninth position is considered important for the ribozyme activity. It has been shown that the cleavage also occurs when the nucleotide at the 15th position is A or U instead of C (M. Koizumi et al. FEBS Lett. 228: 225, 1988). If the substrate binding site of the ribozyme is designed to be complementary to the RNA sequences adjacent to the target site, one can create a restriction-enzyme-like RNA cleaving ribozyme which recognizes the sequence UC, UU, or UA within the target RNA (M. Koizumi et al. FEBS Lett. 239: 285, 1988; Makoto Koizumi and Eiko Ohtsuka Tanpakushitsu Kakusan Kohso (Protein, Nucleic acid, and Enzyme), 35: 2191, 1990; M. Koizumi et al. Nucleic Acids Res. 17: 7059, 1989). For example, in the coding region of the RISBZ gene (SEQ ID NO: 1, 3, 4, or 6), there are pluralities of sites that can be used as the ribozyme target.
The hairpin-type ribozyme is also useful in the present invention. A hairpin-type ribozyme can be found, for example, in the minus strand of the satellite RNA of tobacco ringspot virus (J. M. Buzayan, Nature 323: 349,1986). This ribozyme has also been shown to target-specifically cleave RNA (Y. Kikuchi and N. Sasaki (1992) Nucleic Acids Res. 19: 6751; Yo Kikuchi (1992) Kagaku To Seibutsu (Chemistry and Biology) 30: 112).
The ribozyme designed to cleave the target is fused with a promoter, such as the cauliflower mosaic virus 35S promoter, and with a transcription termination sequence, so that it will be transcribed in plant cells. If extra sequences have been added to the 5′ end or the 3′ end of the transcribed RNA, the ribozyme activity can be lost. In this case, one can place an additional trimming ribozyme, which functions in cis to perform the trimming 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). Multiple sites within the target gene can be cleaved by arranging these structural units in tandem to achieve greater effects (N. Yuyama et al., Biochem. Biophys. Res. Commun. 186: 1271 (1992)). By using such ribozymes, it is possible to specifically cleave the transcription products of the target gene in the present invention, thereby suppressing the expression of the gene.
Endogenous gene expression can also be suppressed by co-suppression through the transformation by DNA having a sequence identical or similar to the target gene sequence. “Co-suppression” refers to the phenomenon in which, when a gene having a sequence identical or similar to the target endogenous gene sequence is introduced into plants by transformation, expression of both the introduced exogenous gene and the target endogenous gene becomes suppressed. Although the detailed mechanism of co-suppression is unknown, it is frequently observed in plants (Curr. Biol. 7: R793, 1997, Curr. Biol. 6: 810, 1996). For example, if one wishes to obtain a plant body in which the RISBZ gene is co-suppressed, the plant in question can be transformed via a vector DNA designed so as to express the RISBZ gene or DNA having a similar sequence to select a plant having the RISBZ mutant character, for example, a plant with modified expression level of storage proteins in seeds, among the resultant plants. The gene to be used for co-suppression need not be identical to the target gene, but it should have at least 70% or more sequence identity, preferably 80% or more sequence identity, and more preferably 90% or more (e.g., 95% or more) sequence identity. Sequence identity can be determined by using the above-described search.
In addition, endogenous gene expression in the present invention can also be suppressed by transforming the plant with a gene encoding a protein having the dominant negative phenotype of the expression product of the target gene. “A DNA encoding a protein having the dominant negative phenotype” as used herein means a DNA encoding a protein, which upon expression, can eliminate or reduce the activity of the protein encoded by endogenous gene inherent to the plant. An example thereof is a DNA that codes for a peptide having GCN4 binding ability and having no transcription activating domain of the protein of the present invention (for example, the peptide missing the 1st to 40th amino acids of the amino acid sequence of SEQ ID NO: 2 or a peptide of other proteins corresponding thereto).
The vector used to transform plant cells is not particularly restricted as long as it is capable of expressing an inserted gene in the cells. For example, a vector having a promoter for performing constitutive gene expression in plant cells (e.g., the 35S promoter of cauliflower mosaic virus), or a vector having a promoter that is inductively activated by an external stimulus can be used. In addition, a promoter that guarantees tissue-specific expression can also be suitably used. Examples of tissue-specific promoters include a promoter of glutelin gene (Takaiwa, F. et al., Plant Mol. Biol. 17: 875–885, 1991) or a promoter of the RISBZ1 of the present invention for the expression in the seeds of rice plants, and a promoter of glycinin gene for the expression in the seeds of leguminous crops such as kidney beans, broad beans and green peas or oil seed crops such as peanuts, sesame seeds, rape seeds, cottonseeds, sunflower seeds and safflower seeds, or a promoter of the major storage protein of each of the above crops such as a promoter of phaseolin gene in the case of kidney beans (Murai, N. et al., Science 222: 476–482, 1993) or a promoter of the gluciferrin gene in the case of rape seed (Rodin, J. et al., Plant Mol. Biol. 20: 559–563, 1992), a promoter of the patatin gene (Rocha-Sosa, M. et al., EMBO J. 8: 23–29, 1989) for the expression in the root tuber of potatoes, a promoter of the sporamin gene for the expression in the root tuber of sweet potatoes (Hattori, T. and Nakamura, K., Plant Mol. Biol. 11: 417–426, 1988), and a promoter of the ribulose-1,5-bisphosphate decarboxylase gene for the expression in the leaves of spinach and other vegetables (Orozco, B. M. and Ogren, W. L., Plant Mol. Biol. 23: 1129–1138, 1993).
The plant cell to which a vector is introduced used herein includes various forms of plant cells, such as cultured cell suspensions, protoplasts, leaf sections, and callus.
A vector can be introduced into plant cells by known methods, such as the polyethylene glycol method, electroporation, Agrobacterium-mediated transfer, and particle bombardment. Plants can be regenerated from transformed plant cells by known methods depending on the type of the plant cell (Toki et al., (1995) Plant Physiol. 100:1503–1507). For example, transformation and regeneration methods for rice plants include: (1) introducing genes into protoplasts using polyethylene glycol and regenerating the plant body (suitable for indica rice cultivars) (Datta, S. K. (1995) in “Gene Transfer To Plants”, Potrykus I and Spangenberg Eds., pp66–74); (2) introducing genes into protoplasts using electric pulse, and regenerating the plant body (suitable for japonica rice cultivars)(Toki et al (1992) Plant Physiol. 100, 1503–1507); (3) introducing genes directly into cells by the particle bombardment, and regenerating the plant body (Christou et al. (1991) Bio/Technology, 9: 957–962); (4) introducing genes using Agrobacterium, and regenerating the plant body (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 for the present invention.
Once a transformed plant with the DNA of the present invention integrated into the genome is obtained, it is possible to gain progenies from that plant body by sexual or vegetative propagation. Alternatively, plants can be mass-produced from breeding materials (for example, seeds, fruits, ears, tubers, tubercles, tubs, callus, protoplast, etc.) obtained from the plant, as well as progenies or clones thereof. Plant cells transformed with the DNA of the present invention, plant bodies including these cells, progenies and clones of the plant, as well as breeding materials obtained from the plant, its progenies and clones, are all included in the present invention. The plant body of the present invention is preferably a monocotyledon, more preferably a plant of the Poaceae, and most preferably a rice plant.
In addition, the present invention provides a plant body in which a foreign gene product has been highly expressed using the RISBZ gene of the present invention. The plant body of the present invention has in its genome a DNA construct in which the DNA of the present invention is operably connected downstream of an expression control region, and a DNA construct in which a foreign gene is operably connected downstream of an expression control region having a target sequence.
The DNA of the present invention or a foreign gene being “operably connected” downstream of an expression control region means that the DNA of the present invention or a foreign gene binds to an expression control region so as to induce the expression of the DNA of the present invention or a foreign gene by the binding of a transcription factor to the expression control region.
The target sequence refers to a DNA sequence to which the RISBZ protein of the present invention, which is a transcription factor, binds, and is preferably a DNA sequence that contains the GCN4 motif or G/C box. Examples of the GCN4 motif include the sequences shown below which have been found in various genes:
* GCN4 Motif (name of gene containing GCN4 motif)











GCTGAGTCATGA/(GluB-1)
SEQ ID NO: 8




 



CATGAGTCACTT/(GluA-1)
SEQ ID NO: 9



 



AGTGAGTCACTT/(GluA-3)
SEQ ID NO: 10



 



GGTGAGTCATAT/(LMWG)
SEQ ID NO: 11



 



GGTGAGTCATGT/(Hordein)
SEQ ID NO: 12



 



GATGAGTCATGC/(Gliadin)
SEQ ID NO: 13



 



AATGAGTCATCA/(Secalin).
SEQ ID NO: 14






Preferable GCN4 motif sequences for use as target sequences include “GCTGAGTCATGA/SEQ ID NO: 8”, GATGAGTCATGC/SEQ ID NO: 13” and “AATGAGTCATCA/SEQ ID NO: 14”. Specific examples of a G/C box include the sequence, “AGCCACGTCACA/SEQ ID NO: 15”. Sequences in which the above GCN4 motif or G/C box is repeated in tandem are also included in the target sequence of the present invention, and a preferable example is a sequence in which the GCN4 motif or G/C box are repeated in tandem four times.
Examples of foreign genes include genes coding for antibodies, enzymes, and physiologically active peptides.
Moreover, the present invention provides a method of producing a plant body in which a foreign gene product is highly expressed using the RISBZ gene of the present invention. Examples of the methods for producing the plant body include a method of crossing “a plant body having a DNA construct in its genome, in which the DNA of the present invention is operably connected downstream of an expression control region,” and “a plant body having a DNA construct in its genome, in which a foreign gene is operably connected downstream of an expression control region having the target sequence of the protein of the present invention.”
The above-described “DNA construct in which the DNA of the present invention is operably connected downstream of an expression control region,” and “the DNA construct in which a foreign gene is operably connected downstream of an expression control region having a target sequence” can be introduced into the plant genome by a conventional method by those skilled in the art, such as a method that uses the above-mentioned agrobacterium. 
In addition, crossing of plant bodies can be carried out by a conventional method for those skilled in the art. For example, in order to prevent self-propagation, only the pollen is sterilized by demasculating using the tip shearing method on the day of crossing or by demasculating using hot water on the day of crossing to shake pollinate the ear of the pollen mother.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a drawing representing a genealogical tree based on the homology of the amino acid sequence of RISBZ protein and O2-like bZIP protein. The entire amino acid sequences of these proteins are compared to understand the similarity and the evolutionary relationship of these proteins.
FIG. 2 compares the amino acid sequences of RISBZ protein and O2-like bZIP protein. Outline letters on a black background shows the amino acids that retained 50% or more. The presumed nuclear migration signal (NLSA: SV40-like motif) (Varagona, M. J. et al., Plant Cell 4: 1213–1227, 1992) and the serine-rich phosphorylation sites are indicated with double lines and broken lines, respectively. The bold lines indicate the basic domain, which has a two-factor nuclear migration signal (NLSB) structure. Downward arrows indicate the leucine repeats. The primer used for the production of the rice bZIP probe was designed based on the amino acid sequences indicated by rightward and leftward arrows. BLZ1 (Vicente-Carbojos, J. et al., Plant J. 13: 629–640, 1998) and BLZ2 (Onate, L. et al., J. Biol. Chem. 274: 9175–9182, 1999) represent O2-like bZIP proteins isolated from barley, O2 (Hartings, H. et al., EMBO J. 8: 2795–2801, 1989) and OHP1 (Pysh, L. D. et al., Plant Cell 5: 227–236, 1993) from maize, SPA from wheat (Albani D. et al., Plant Cell9: 171–184, 1997), O2-sorg from sorghum (Pirovano, L. et al., Plant Mol. Biol. 24: 515–523, 1994), and O2-coix from adlay (Vettore, A. L. et al., Plant Mol. Biol. 36: 249–263, 1998).
FIG. 3 is a continuation of FIG. 2.
FIG. 4 shows the structure of a gene that codes for O2-like bZIP protein. The structures of the intron/exon region of the BLZ1 gene of barley and the Opaque2 gene of maize (O2) (Hartings, H. et al., EMBO J. 8: 2795–2801, 1989), sorghum (O2-sorg) and adlay (O2-coix) are shown. The thick bars and thin lines represent exons and introns, respectively. The numbers indicate the number of nucleotides of the exons and introns.
FIG. 5 is a photograph representing the result of a Northern blot showing the transcription patterns of the RISBZ genes. Northern blotting analysis was performed on the whole RNA extracted from the root, seedling, and maturing seeds (5, 10, 15, 20, and 30 DAF) using a unique nucleotide sequence of a region downstream of the bZIP domain for the probe. In order to compare transcription patterns, the analysis was also conducted using the. GluB-1 gene-coding region as the probe. The stained images of 25S rRNA obtained using ethidium bromide are shown as a control.
FIG. 6 represents the results of histological analysis of the RISBZ1 promoter/GUS reporter gene in a transformed rice plant.
(A) is a schematic drawing of the RISBZ1 promoter/GUS reporter gene. (a) and (b) show the sequence from the −1674th to +4th nucleotides counting from the transcription initiation point of the RISBZ1 gene and the sequence from the −1674th to +213th gene that contains uORF, respectively, both connected to the GUS reporter gene on a binary vector. (c) shows the GluB1 promoter (−245 to +18) sequence binding to the GUS reporter gene on a plasmid vector.
(B) are photographs showing the expression of GUS reporter gene in a seed during the maturation process. After cutting the seed (10 DAF) of a rice plant, into which the reporter gene was introduced, in the longitudinal direction, the cut seed was immersed in X-gluc solution and incubated at 37° C. EN indicates the endosperm, while EM indicates the embryo.
(C) is a graph showing the GUS activity of a seed extract of a transformed rice plant. 15 DAF seeds were used for analysis. The promoter structures of the introduced genes are as shown in (a) and (b) of (A), respectively. Vertical lines indicate the mean value. MU represents 4-methylumbelliferone.
FIG. 7 shows photographs of gel electrophoretic patterns as determined from a methylation interference experiment for identifying the RISBZ1 protein-binding site on the GluB1 promoter. Each of the strands (top and bottom) of the promoter fragment of the GluB1 gene (−245 to +18) was labeled. After partially methylating each strand, they were incubated with GST-RISBZ1 protein, the fragments that did not bind to the protein and the fragment that bound to the protein were each collected and subjected to electrophoresis after chemically cleaved by piperidine. The sites (indicated by asterisks) that were not cleaved by piperidine were only found in the GCN4 motif.
FIG. 8 shows the result of electrophoresis in gel shift analysis to investigate the binding capability of RISBZ1 protein to the GCN4 motif.
(A) shows 21-bp DNA fragments that contain the GCN4 motif of a WILD:GluB-1 promoter sequence (−175 to −155) of an oligonucleotide used as the probe and competitor. M1 to M7 are a series of 21-bp DNA fragments that were mutated every 3 bp. The GCN4 motif is underlined.
(B) through (F) show the results of gel shift analysis of the GST-RISBZ fused protein. A 21-bp DNA fragment (WILD) was added as the probe. (B) is for GST-RISBZ1, (C) for GST-RISBZ2, (D) for GST-RISBZ3, (E) for GST-RISBZ4, and (F) for GST-RISBZ5. The competitor was added to a stoichiometric ratio of 100 times or more against the probe. Lane 1: No protein; Lane 2: No competitor; and Lanes 3 to 10: With Competitor (wild type (W) and M1 to M7).
FIG. 9 represents heterodimer forming ability of RISBZ1 with other RISBZ proteins.
(A) shows the vector structure used as the in vitro transcription/translation reaction template. The vectors contain DNA coding for full-length RISBZ1 protein, short-form RISBZ2 protein (sRISBZ2: 218 to 329), or short-form RISBZ3 protein (sRISBZ3: 126 to 237).
(B) shows photographs of gel electrophoretic patterns representing the results of a DNA binding assay. In lanes 2, 4, 6, and 8, DNA complexes that bound to the full length or short-form protein were detected. In lanes 3 and 7, DNA complexes that bound to the heterodimer of full length RISBZ1 protein and short-form protein were detected.
FIG. 10 shows the results of identification of the transcription-activating domain determined by transient analysis.
(A) shows the structure of the reporter and effector plasmid. A GUS gene in which 9 copies of GAL4-DNA binding sites and CaMV35S core promoter sequence are linked was used for the reporter. The effector plasmid contained DNA coding for a protein in which the GAL4 DNA binding domain was linked to the N-terminal side of truncated RISBZ1 protein.
(B) is a graph showing GUS activity when the reporter and effector plasmid were used.
FIG. 11 shows the hydropathy patterns of the N-terminal region of RISBZ1 (WT) and mutant RISBZ1 (M1 to 8) proteins determined by the formula of Kyte and Doolittle (Kyte, J. and Doolittle, R. F. J., Mol. Biol. 157: 105–132, 1982). Positive values indicate hydropathy.
FIG. 12 schematically shows the transcription activity measurement system of RIZBZ1 using GUS activity as the indicator, photographs of Northern blot analysis, and a graph showing GUS activity measurement results. The ordinate of the graph represents GUS activity that is the indicator of the strength of the transcription activity of each transcription factor.
FIG. 13 is a graph showing the recognition sequences of transcription factors RISBZ1, Opaque2, SPA, and RISBZ3 (RITA1). The ordinate of the graph represents GUS activity that is the indicator of the strength of the transcription activity of each transcription factor. The sequences used in the experiment are shown below the graph.
FIG. 14 is a graph showing the transcription activating ability of the RISBZ1 of the present invention relative to GCN4 motifs originating in various genes. The ordinate of the graph represents GUS activity, which is the indicator of the strength of the transcription activity of each transcription factor. The nucleotide sequences of the GCN4 motifs used in the experiment are shown below the graph.



MODE FOR CARRYING OUT THE INVENTION
The present invention will be described in more detail below with reference to Examples, but is not to be construed as being limited thereto.
EXAMPLE 1
Isolation of cDNA Clones Encoding the bZIP Transcription Factor From Seed cDNA Libraries
Fourteen-day leaves and roots of rice plant (Oryza sativa L. c. v. Mangetumochi) cultivated by hydroponics were frozen in liquid nitrogen and kept at  ̃80° C. until use. Maturing rice seeds were collected from rice plants cultivated in the fields.
Using oligonucleotide primers designed from highly conserved amino acid sequences (SNRESA and KVKMAED) within the bZIP domain of the Opaque 2 (O2)-like protein, RT-PCR was performed by using poly(A)+ mRNA as a template, which was prepared from the rice seeds. From poly (A) RNA extracted from seeds at 6 to 16 days after flowering (DAF) (Takaiwa F. et al. Mol. Gen. Genet. 208: 15–22, 1987), single-stranded cDNA was synthesized by reverse transcription using oligo(dT)20 as a primer and Superscript reverse transcriptase (Gibco BRL, Paisly, UK). Next, cDNA was amplified using a pair of primers (5′-TCC AAC/T A/CGI GAA/G A/TCI GC-3′; SEQ ID NO: 16, and 5′-GTC CTC C/TGC CAT CTT CAC CTT-3′; SEQ ID NO: 17). These primers were designed based on highly conserved amino acid sequences within the bZIP-type transcription factors that were expressed in cereal seeds. After dissolving the single-stranded cDNA in a PCR reaction mixture containing 10 mM Tris-HCl pH 8.3, 1.5 mM MgCl2, 50 mM KCl, 0.01% (w/v) gelatin, 200 μM dNTPs, 1 μM oligonucleotide primers, TaqI polymerase was added to the mixture and the resulting mixture was incubated in a thermal cycler at 94° C. for 5 min. cDNA was then synthesized and amplified by three-cycle PCR (for 1 min at 94° C., for 1 min at 40° C., and then for 2 mins at 72° C.) followed by 30-cycle PCR (for 1 min at 94° C., for 1 min at 55° C., and then for 2 mins at 72° C.). The amplified DNA fragment was cloned into a TA cloning vector (pCR2.1; Invitrogen), and subjected to sequencing by using the ABI PRISM dye terminator sequence system. The reaction products were analyzed by ABI PRISM 310 Genetic Analyzer (Perkin Elmer-Applied Biosystems) to determine the nucleotide sequences of at least 50 clones. The obtained nucleotide sequence data was analysed and searched on databases by using the GENETYX and BLAST algorisms. As a result, five distinct DNA fragments with 213-bp were found. Two of these were identical to the bZIP domain sequences of REB (Izawa T. et al. Plant Cell 6: 1277–1287, 1994) and the RITA1 (Nakase M. et al. Plant Mol. Biol. 33: 513–522, 1997). Using the five DNA fragments with 213-bp as primers, a cDNA library was prepared from RNA of maturing (6-16DAF) seeds (ZAPII; STRATAGENE) This was then screened to obtain their full-length cDNAs corresponding to each of the fragments under high stringent conditions. [α-32P]-dCTP was incorporated into the DNA fragments by random priming (Amersham Pharmacia Biotech) and the resulting fragments were used as probes. As a pre-hybridisation solution, a mixture containing 5×SSC, 5× Denhard's solution, 0.1% SDS, 50% formamide, 100 μg/ml salmon sperm DNA was used. After hybridization, filters were washed once at 55° C. with a mixture consisting of 2×SSC and 0.1% SDS, and then twice at 55° C. with a mixture consisting of 0.1×SSC and 0.1% SDS.
Based on the homologies to each nucleotide sequence, the cDNA clones obtained were termed as RISBZ1 (rice seed b-Zipper 1) (SEQ ID NO: 1), RISBZ2, RISBZ3, RISBZ4 (SEQ ID NO: 4), and RISBZ5 (SEQ ID NO: 6). Among them, RISBZ2 and RISBZ3 were identical to REB (Izawa T. et al. Plant Cell 6: 1277–1287, 1994) and RITAL (Nakase M. et al. Plant Mol. Biol. 33: 513–522, 1997), respectively, which have previously been isolated from cDNA libraries of seeds and leaves.
EXAMPLE 2
Identification of RISBZ cDNA
The newly identified RISBZ cDNAs (RISBZ1, RISBZ4, and RISBZ5) were characterized in detail as described below. RISBZ1 cDNA was the longest, which had 1742 bp in length excluding poly(A), and contained a reading frame encoding 436 amino acids that had 46,491 Dal of an estimated molecular weight. RISBZ4 and RISBZ5 have reading frames encoding 278 and 295 amino acids; their estimated molecular weights are 29,383 Dal and 31,925 Dal respectively.
RISBZ1 mRNA has a longer leader sequence (245 bases long) than average leader sequences. Interestingly, a small open reading frame, encoding 31 amino acid residues, was found within the leader sequence in the upstream of the actual initiation codon of the RISBZ1 protein. Similar small upstream open reading frames (UORF) have previously been found in maize Opaque 2 (O2) (Hartings H. et al. EMBO J. 8: 2795–2801, 1989), wheat SPA (Albani D. et al. Plant Cell 9: 171–184, 1997), and barley BLZ1 and BLZ2 (Vincente-Carbojos J. et al. Plant J. 13: 629–640, 1998; Onate L. et al. J. Biol. Chem. 274: 9175–9182, 1999), but these uORFs have little homology with each other. It has previously been reported that uORF of the maize O2 mRNA is involved in translational control. uORF was found only in RISBZ1 mRNA but not in other RISBZ mRNA.
The flanking sequence of the initiation codon is GCAATGG. This sequence coincided with eukaryotic translational initiation sequence, c(a/c) (A/G) (A/C)cAUGGCG, derived from monocotyledonous plants. There were 100 bps between the initiation codon and uORF. The open reading frame encoding RISBZ1 had two identical termination codons (TAG). There were 229 bps between the termination codon and poly (A) sequence. The polyadenylation signal sequence (AATATA) was found in the region at −19 to −24 from the site to which poly(A) was added.
RISBZ1 is closely related to rice REB (Nakase M. et al. Plant Mol. Biol. 33: 513–522, 1997), maize OHP-1 and OHP-2 (Pysh L. D. et al. Plant Cell 5: 227–236, 1993), and barley BLZ1 (Vincente-Carbojos J. et al. Plant J. 13: 629–640, 1998) (FIG. 1), and showed the homologies of 48.2% (rice REB), 45.7% (barley BLZ1), and 46.6% (maize OHPL), respectively, at the amino acid level. Furthermore, these bZIP domains were highly conserved (73.7% to 76.3%). At the amino acid level, the homologies of RITA1 (RISBZ3) with RISBZ4 and RISBZ5 were 88.8% and 47.6% respectively. By contrast, the homology of RISBZ4 with RISBZ5 was 48.2%. RISBZ3, RISBZ4, and RISBZ5 comprise a unique group among the O2-like transcription factors that were previously reported. Furthermore, the five RISBZ cDNAs isolated from the seed cDNA library could be classified into two groups based upon the amino acid homology (FIG. 1). The RISBZ3, RISBZ4, and RISBZ5 lacked the N- and C-terminal regions present in RISBZ1 and RISBZ2, and their sizes reduced about 100 to 150 amino acid residues compared with those of RISBZ1 and RISBZ2 (FIGS. 2 and 3).
RISBZ1 and RISBZ2 were rich in proline residues at their N-terminal region, which lacked in other RISBZ proteins (FIGS. 2 and 3). RISBZ1 and RISBZ2 were also rich in acidic amino acids at the peripheral region of the 60th amino acid residue from their N-termini and at the intermediate region located in the upstream of their bZIP domains. These proline-rich or acidic amino acid-rich regions were found in other O2-like transcription factors.
Since serine-rich sequence (SGSS) was found in the region ranging from 207th to 210th residues of RISBZ1, the protein was considered to be a target sequence of casein kinase II (Hunter T. and Karin M. Cell 70: 375–387, 1992) (FIGS. 2 and 3) Similar sequence (SSSS) was also found in RISBZ2. However, it was missing in the other RISBZ proteins (FIGS. 2 and 3).
So far, two nuclear transition signals (NLSA: an SV-40-like motif and NLSB: a 2-factor motif) have been identified, which are involved in transport of maize Opaque2 (O2) proteins from cytoplasm into nucleus (Varagona M. J. et al. Plant Cell 4: 1213–1227, 1992). These motifs were searched on RISBZ1 and sequences homologous to NLSA and NLSB were found at the same sites as O2 (101 to 135 and 232 to 264).
EXAMPLE 3
Genomic Structure of the RISBZ1 Gene
Using primers designed from the nucleotide sequence of the RISBZL cDNA, the genomic region encoding promoter and RISBZL protein was isolated. The PCR reaction was performed using rice genomic DNA as a template and two pairs of oligonucleotide primers (RIS1f: 5′-ATGGGTTGCGTAGCCGTAGCT-3′/SEQ ID NO: 18 and RELr5: 5′-TTGCTTGGCATGAGCATCTGT-3′/SEQ ID NO: 19) and (RELf2: 5′-GAGGATCAGGCCCATAT-3′/SEQ ID NO: 20 and RIS1r: 5′-TCGCTATATTAAGGGAGACCA-3′/SEQ ID NO: 21). DNA fragments were amplified using TAKARALA Taqpolymerase (TAKARA) in a thermal cycler through 30-cycle reactions for 10 sec at 98° C., for 30 sec at 56° C. and for 5 min at 68° C. The promoter region of the RISBZ1 gene was also amplified by thermal asymmetric interlaced (TAIL) PCR, based on the method by Liu et al, in which three oligonucleotides were used as specific primers, tail1: 5′-TGCTCCATTGCGCTCTCGGACGAG-3′/SEQ ID NO: 22, tail2: 5′-ATGAATTCGCGAGGGGTTTTCGA-3′/SEQ ID NO: 23, and tail3: 5′-GTTTGGGAGAAATTCGATCAAATGC-3′/SEQ ID NO: 24.
The results revealed that the RISBZ1 gene comprises of six exons and five introns (FIG. 4). The constitution of exon/intron in this RISBZ1 gene was identical to that of the maize O2 (Hartings H. et al. EMBO J. 8: 2795–2801, 1989), Sorghum O2 (Pirovano L. et al. Plant Mol. Biol. 24: 515–523, 1994), adlay O2 (Vettore A. L. et al. Plant Mol. Biol. 36: 249–263, 1998), and barley BLZ1 (Vicente-Carbojos J. et al. Plant J. 13: 629–640, 1998) genes (FIG. 4).
The transcription initiation site of the RISBZ1 gene was determined by the primer extension analysis according to the method of Sambrook et al. (Sambrook J. et al. Molecular Cloning: A Laboratory Manual, 2nd Ed., pp. 7.79–7.83, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). Specifically, a primer, 5′-ATGGTATGGTGTTCCTAGCACAGGTGTAGC-3′ (SEQ ID NO: 25), was produced by labelling with T4 kinase, the 5′ end of the oligonucleotide comprising 30 nucleotides, which was complementary to a sequence immediately downstream of a desired region. Reverse transcription reaction was conducted using this primer and 5 μg of mRNA as a template, and a Superscript reverse transcriptase kit (Gibco BRL, Paisly, UK). This reaction was carried out in a mixture comprising 20 mM Tris-HCl, 50 MM MgCl2, 10 mM DTT, 500 μM dNTP, 100,000 cpm primer, 5 μg mRNA, and 200-unit Superscript reverse transcriptase (Gibco BRL, Paisly, UK), for 50 min at 42° C.
As a result, the transcription initiation site was mapped to the 245-nt upstream region from the translation initiation codon of the RISBZ1 gene. A ‘TATA’ box was localized at −30 to −35-nt from the transcription initiation site. Three ‘ACGT’ motifs were found in the 63-, 123-, and 198-bp upstream regions from the transcription initiation site but none of motifs responsible for expression of seed-specific genes, such as, GCN4 and ‘AACA’ were found. In contrast, a number of the recognition sequences for Dof domain protein, ‘AAAG’, were found. These motifs may be involved in stage- and/or tissue-specific expression of the RISBZ1 gene. For example, if the ‘ACGT’ motif is a target sequence of the RISBZ1 protein, the RISBZ1 gene may be autoregulated by itself. However, when the RISBZ1 promoter/GUS reporter gene and the 35S CaMV promoter/RISBZ1 gene were introduced into protoplast cells, no transcriptional activation of the reporter gene was observed. These data suggest that the RISBZ1 promoter has no target sequence for the RISBZ1 protein; namely, the ‘ACGT’ motif found in the RISBZ1 promoter is not a target sequence of the protein. Therefore, the RISBZ1 gene is probably not autoregulated. In contrast, upon overexpression of the rice prolamin box binding factor (RPBF) gene (which recognizes the Dof domain) transcription of the RISBZ1 promoter/GUS reporter gene is activated. This suggests that the recognition sequences of the Dof domain proteins are involved in specific expression of the RISBZ1 gene.
EXAMPLE 4
Tissue-Specificity of the RISBZ mRNA
Northern blotting was carried out to analyze the expression of the RIABZ gene. According to the method by Takaiwa et al. (Varagona M. J. et al. Plant Cell 4: 1213–1227, 1992), total RNA was extracted from 5 to 30 DAF seeds, roots, and seedling (5-, 10-, 15-, 20- and 30-DAF), and was transferred to membrane filters after fractionation by agarose gel electrophoresis. As probes, the following DNA fragments ranging from the downstream sequence of the bZIP domain-encoding region to the 3′ non-coding region in the RISBZ cDNA were used: RISBZ1, 354-bp ranging from 1388th to 1742nd nucleotides; RISBZ2, 346-bp ranging from 1351st to 1696th nucleotides; RISBZ3, 486-bp ranging from 741st to 1226th nucleotides; and RISBZ5, 621-bp ranging from 742nd to 1362nd nucleotides.
Hybridization was carried out in a solution containing 5×SSC, 5× Denhard's solution, 0.1% SDS, and 50% formamide, at 45° C. After the hybridization, the membrane filters were washed twice for 30 min with a mixed solution comprising 2×SSC and 0.1% SDS, and then twice for 30 min with a mixture comprising 0.1×SSC and 0.1% SDS.
As shown in FIG. 5, the RISBZ1 gene was expressed only in seeds, not in other tissues analyzed. The largest amount of the RISBZ1 mRNA was accumulated in seeds harvested from 5 DAF to 10 DAF. Such a high accumulation of mRNA was maintained until 15 DAF, and gradually decreased towards maturing. The peak of the RISBZ1 gene expression appeared at an earlier stage than that of the glutelin gene. The glutelin mRNA expression was detected from 5 DAF, had a peak at 15 DAF, and was then gradually decreased (FIG. 5). This result suggests that the RISBZ1 acts as an activator of the glutelin gene. Similar expression patterns have also been reported in the maize O2 (Hartings H. et al. EMBO J. 8: 2795–2801, 1989), wheat SPA (Albani D. et al. Plant Cell 9: 171–184, 1997), and barley BLZ2 genes (Onate L. et al. J. Biol. Chem. 274: 9175–9182, 1999).
The RISBZ2 was expressed in all the tissues analyzed. The RISBZ3 and RISBZ 4 were expressed specifically in seeds at later stages of maturing (FIG. 5). The RISBZ3 and RISBZ 4 mRNA levels gradually increased until 20DAF and then decreased. The expression level of RISBZ5 was extremely low, compared with other RISBZ genes, and its mRNA peak was at 10 DAF.
EXAMPLE 5
Expression of the RISBZ1 Promoter/GUS Reporter Gene Construct in Transformants
To examine an expression pattern of the RISBZ1 gene, the sequence fragment ranging from −1674 to +213 nt numbering from the transcription initiation site, was ligated upstream of GUS gene. This reporter gene was introduced into rice plant by using Agrobacterium (FIG. 6A). Transformed rice plant (Oryza sativa L. c. v. kitaake) was constructed as follows. Two oligonucleotide primers with the PstI or BamHI restriction site at its 5′ end, 5′-AAAACTGCAGTTTTCTGA-3′ (SEQ ID NO: 26) and 5′-AATGGATCCGCGAGGGGTTTTCGAA-3′ (SEQ ID NO: 27), were used to amplify the 5′-end regions (from −1674th to +4th and from −1674th to +213rd) of the RISBZ1 gene by PCR. The PCR reaction was carried out in a reaction mixture (10 mM Tris-HCl pH 8.3, 1.5 mM MgCl2, 50 mM KCl, 0.01% (w/v) gelatine, 200 μM dNTPs, 1 μM primers, 0.5 μg template DNA, and 2.5-unit TaqI polymerase) by 30 cycles of incubation for 1 min at 94° C., for 1 min at 50° C. and for 2 min at 72° C. After digestion with restriction enzymes, PstI and BamHI, the PCR product was cloned into the plasmid vector pBI201, and was cleaved with restriction enzymes, PstI and SacI. The resulting DNA fragment containing the RISBZ1 promoter/GUS gene was inserted between the Sse8387I and SacI sites of the binary vector p8cHm, which contains the CaMV35S promoter/hygromycin phosphotransferase (HPT) gene. Transformation was performed according to the method described in Goto F. et al. Nature Biotech. 17: 282–286.
The reporter plasmid was constructed as follows. 1×21 bp, 3×21 bp, and 5×21 bp of GCN4 motifs/GUS genes, as constructed by Wu et al. (Wu C. Y. et al. Plant J. 14: 673–683, 1998), were used as the reporter. A pair of 48-bp oligonucleotides with overhanged (ACGT) 5′ ends, which were complementary to each other, was associated to construct tetramers comprising 12-bp wild-type GCN4 motif (GCTGAGTCATGA/SEQ ID NO: 8) and mutant GCN4 motif (GCTTCCTCATGA/SEQ ID NO: 28). These double-stranded oligonucleotide were inserted into the SalI and StuI sites of the −46CaMV/GUS reporter gene.
Transient assay for rice callus protoplast was carried out according to the method described by Wu et al. The GUS activity was measured according to the method of Jefferson (Jefferson R. A. Plant Mol. Biol. Rep. 5: 387–405, 1987), by measuring fluorescence intensity of 4-methyl-umbelliferone derived from the glucuronide precursor. Using Bio Rad Kit, the concentration of proteins was measured. Bovine serum albumin was used as a standard protein.
As shown in FIG. 6B, high GUS activities was observed in the aleulon and sub aleulon layers of maturing seeds, but not in germs. The GUS activity was not detected in roots, leaves, and stems even by highly sensitive fluorescence measurement. These results indicate that the RISBZ1 gene is expressed exclusively in the aleulon and sub aleulon layers. To examine the role of the 5′-end untranslated region and uORF, the GUS activity was compared with that of a plant, which lacked uORF ranging from −1674th to +4th numbering from the transcription initiation site (FIG. 6A). As a result, no change in the expression site was observed due to the lack of uORF (FIG. 6B), but 5- to 10-fold weaker promoter activities were observed (FIG. 6C). These data suggest that the 5′ untranslated region may play a role in upregulation of the translation, in contrast to the results in the maize O2 in which uORF functions as a suppressor of the translation (Lohmer S. et al. Plant Cell 5: 65–73).
EXAMPLE 6
Transcription Activating Ability of Five RISBZ Proteins Through Their Binding to the GCN4 Motif
Transcription activating ability of the five RISBZ proteins through their binding to the GCN4 motif was measured by transient assay. The plasmids, into which each RISBZ1 protein-encoding sequences were ligated downstream of CaMV35S promoter as an effector, were prepared. Effector plasmids were prepared as follows. The plasmid that encodes RISBZ1 lacking its N-terminal region was prepared by PCR. In order to amplify cDNA encoding the regions ranging from 41st, 81st, 121st, and 161st amino acids numbering from the N-terminus of RISBZ1 to its C-terminus the following primers were designed:









Forward primers




RIS1-1:


5′-AACCATGGTGCTGGAGCGGTGCCCGT-3′
(SEQ ID NO: 29)


 


RIS1-2:


5′-AACCATGGCGGCGGAGGCGGCGGCG-3′
(SEQ ID NO: 30)


 


RIS1-3:


5′-CCCCATGGAGTACAACGCGATGC-3′
(SEQ ID NO: 31)


 


RIS1-4:


5′-AACCATGGTTGGTTCCATCCTGAGT-3′
(SEQ ID NO: 32)


 


RIS1-5:


5′-AACCATGGCTCATGCCAAGCAAGCT-3′
(SEQ ID NO: 33)


 


RIS1-6:


5′-AACCATGGATGAAGAAGATAAAGTGAAG-3′
(SEQ ID NO: 34)


 


Reverse primer


BRIS1R:


5′-TAGGATCCGCTCCTACTACTGAAGCT-3′.
(SEQ ID NO: 35)






These primers were designed to have an NcoI or BamHI restriction site at their 5′ end. Since a translational initiation codon was lost by deletion of its N-terminal region, ATG of the NcoI restriction site was utilized. cDNAs were amplified by PCR comprising incubation for 2 min at 94° C., 30-cycle reaction for 1 min at 94° C., for 1 min at 50° C., and for 2 min at 72° C., followed by incubation for 5 min at 72° C. The PCR products were digested with restriction enzymes, NcoI and BamHI, and then purified through agarose gel electrophoresis. The purified cDNA fragments were finally inserted into the pRT100 vector (Topfer R. et al. Nucl. Acids Res. 15: 5890, 1987).
Plasmids encoding the fusion proteins comprising GAL4 DNA-binding domain (amino acid residues from 1st to 147th) and the RISBZ1 or RISBZ2 gene were also constructed. In order to amplify the cDNA region encoding various N-terminal region of RISBZ1 and RISBZ2 by PCR using Pfu Taq polymerase (STRATAGENE), the following reverse primers, to which a BamHI site, a terminal codon, and an SstI site were added at its 5′-end, were prepared as well as the following forward primers:








Forward primers











RISBZ1-F1:
5′-AAGGATCCAATGGAGCACGTGTTCGCC-3′
(SEQ ID NO: 36)



 


RISBZ1-F2:
5′-AAGGATCCGGCGGCGGAGGCGGCGCG-3′
(SEQ ID NO: 37)


 


RISBZ1-F3:
5′-GCCGGATCCAGTTGGTTCCATCCTGAG-3′
(SEQ ID NO: 38)


 


RISBZ1-F4:
5′-AAGGATCCTGATGAAGAAGATAAAGT-3′
(SEQ ID NO: 39)


 


RISBZ1-F1-2:
5′-AAGGATCCAGGAGTAGATGACGTCGGC-3′
(SEQ ID NO: 40)


 


RISBZ1-F1-3:
5′-AAGGATCCAGACGAGATCCCCGACCCGCT-3′
(SEQ ID NO: 41)


 








Reverse primers











RISBZ1-R1:
5′-TAGAGCTCTACGCCGCCGGCATCGGGCT-3′
(SEQ ID NO: 42)



 


RISBZ1-R2:
5′-TAGAGCTCTAAAGGATCATATTTCCCAT-3′
(SEQ ID NO: 43)


 


RISBZ1-R1-1:
5′-TAGAGCTCTAGGCGGCCGCCGCCGGCTG-3′
(SEQ ID NO: 44)


 


RISBZ1-R1-2:
5′-TAGAGCTCTACGGCGGCGGCGGAGCCCA-3′.
(SEQ ID NO: 45)






cDNAs encoding various N-terminal regions of RISBZL and RISBZ2 were amplified by PCR comprising incubation for 2 min at 94° C., 30 cycles of reaction for 1 min at 94° C., for 1 min at 50° C., and for 1 min at 72° C., and then incubation for 5 min at 72° C., using the above-described primers. The amplified cDNAs were digested with BamHI and SacI restriction enzymes, and were purified by 2% agarose gel electrophoresis. The purified cDNA fragments were ligated downstream of the GAL4 DNA domain-encoding region in the 35S-564 vector digested with the same restriction enzymes so that their reading frames were matched. Mutations were also introduced into the N-terminal regions of RISBZ1 by PCR mutagenesis. The cDNA sequences were confirmed, and their partial sequence from 1st to 57th amino acid residues was amplified by PCR. The products were ligated downstream of the GAL4 DNA domain-encoding region in their reading frames.
In addition, reporter plasmids, into which the GUS gene, and one or three repeat(s) of the 12-bp GCN4 motif or one or five repeat(s) of the 21-bp GCN4 motif were inserted, were constructed. For negative control experiments, a reporter plasmid comprising four repeats of a mutant 12-bp GCN4 motif and the GUS reporter gene was used. The mutant 12-bp GCN4 motif has a mutation in the target sequence that is recognized by the RISBZ1 and O2. These plasmid constructs were introduced alone or in combination with other reporter or effector plasmid into rice protoplast cells prepared from its callus culture, and the GUS activity was assayed. When the reporter plasmid or effector plasmid was introduced alone into the protoplast, the GUS activity was detected at a low level. As shown in Table 1, however, in the presence of 35S/RISBZ1 or 35S/O2, which were introduced as effector plasmids, the transcription of the reporter gene was activated. Even in the presence of these effector plasmids, the transcriptional activity of the GUS gene downstream of the mutant 12-bp GCN4 motif was the same level as that of background. These results indicate that the RISBZ1 gene product activates the reporter gene mediated by the GCN4 motif. The transcriptional activity of the reporter gene induced by the RISBZ1 gene product was slightly higher than that induced by the O2 gene product. As shown in Table 2, the activity induced by RISBZ1 was enhanced depending on the copy number of the GCN4 motif. 1 to 12 copies of 21-bp GCN motif were assayed, and the transcriptional activity was enhanced proportionately up to 9 copies. However, even though the other RISBZ genes were expressed under the control of the 35S CaMV promoter, the transcriptional activity of the reporter gene was less than or equal to 1.4% of that induced by the RISBZ1 or O2 gene product. Thus, it was revealed that only the RISBZ1 protein can activate the transcription through its binding to the GCN4 motif.










TABLE 1







Effector
GUS activity (pM 4-MU/min/mg protein)









35S/Opaque2
2658 ± 318



35S/RISBZ1
2994 ± 157



35S/RISBZ2
44 ± 7



35S/RISBZ3
 1.3 ± 1.2



35S/RISBZ4
17.3 ± 0.9



35S/RISBZ5
  31 ± 8.8










The 4×12-bp GCN4 motifs/GUS reporter gene was introduced into protoplast cells together with the effector plasmid, and the GUS activity was measured. Data were obtained from three independent measurements.









TABLE 2









Effector GUS Activity



(pM 4-MU/min/mg protein)










Reporter
(−)
(+) RISBZ1
(+) Opaque2





1 × 12-bp GCN4
32 ± 1.5
295 ± 4.5 
182 ± 6 




(9.2*)
(5.6*)


4 × 12-bp GCN4
21
 604 ± 24.5
452 ± 7.5 




(28.7*)
(21.5*)


1 × 21-bp GCN4
30 ± 3  
1318 ± 55.5 
1139 ± 22.5 




(43.9*)
(37.9*)


5 × 21-bp GCN4
104
13222 ± 1094 
11932 ± 22.5  




(127.1*)
(114.7*)









As a reporter, the 1×12-bp, 4×12-bp, 1×21-bp or 5×21-bp GCN4 motif/GUS gene was used. This table shows the GUS activity induced by the expression of RISBZ1 (+RISBZ1) gene or by Opaque2 (+Opaque2) gene.

EXAMPLE 7
Binding Site of the RISBZ1 Protein
The present inventors have previously discovered that the O2 protein recognizes the GCN4 motif (TGAGTCA) that is present in the promoter region ranging from −165th to −160th of GluB-1, a glutelin gene (Wu C. Y. et al. Plant J. 14: 673–683, 1998). By a methylation interference experiment, the present inventors have also determined the binding site of the RISBZ1 protein in the promoter region of the GluB-1 gene.
Production and purification of the GST-RISBZ1 fusion protein were performed as follows. Five coding regions from RISBZ1 cDNA were amplified by PCR using oligonucleotide primers to which the following appropriate restriction enzyme sites were added at their 5′ end; BamHI-blunt ends for RISBZ1, BamHI-XhoI for RISBZ2, BamHI-SalI for RISBZ3, BamHI-SalI for RISBZ4, and BamHI-XhoI for RISBZ5. After digestion with the restriction enzymes, the PCR products were ligated into the cloning sites of the pGEX-4T-3 vector (Amersham Pharmacia Biotech). The GST-RISBZ fusion protein was expressed according to the method of Suzuki et al. (Suzuki A. et al. Plant Cell Physiol. 39: 555–559, 1998). After affinity purification, the GST fusion protein was dialyzed against a binding buffer comprising 20 mM HEPES-KOH pH 7.9, 50 mM KCl, 1 mM EDTA, and 10% glycerol, for four hours, and immediately stored at −80° C.
Methylation interference experiment was performed as described by Weinberger et al. (Weinberger J. et al. Nature 322: 846–849, 1986). The 5′-flanking region (from −245th to +18th nucleotides) of the GluB1 gene was digested with restriction enzymes, SalI and BamHI, and the ends of the fragment was labeled with [α-32P] dCTP by a ‘fill-in’ reaction. The labelled fragment was methylated by treating it with dimethyl sulphate, mixed with GST-RISBZ1, and then incubated. Using non-denaturing acrylamide gel (5%, 0.25×TBE) electrophoresis, the DNA fragment complexed with GST-RISBZ1 and free DNA fragments were separated from each other. These DNA fragments were further purified by DEAE Sepharose column chromatography, were treated with piperidine, and were fractionated by 6% denaturing acrylamide gel electrophoresis.
As shown in FIG. 7, the GST-RISBZ1 fusion protein protected guanine residues that locate in the −165th to −160th region of the GluB-1 promoter. The guanine residues protected were the same residues protected in the O2 promoter (Albani D. et al. Plant Cell 9: 171–184, 1997). A guanine residue present in the ‘ACGT’ motif (also termed as an A/G hybrid box) at the −79th to −76th residues in the promoter region ranging from −197th to +18th, was not protected.
Furthermore, gel shift assay was conducted as described below to examine whether the RISBZ1 protein can recognize the GCN4 motif.
A pair of oligonucleotides complementary to each other, which was prepared by adding TCGA sequence was added to 21-nt fragment of GluB1 promoter region (from −175th to −155th), was labeled at its ends with [α-32P] dCTP by ‘fill-in’ reaction for use as a probe. Seven pairs of complementary oligonucleotides with mutations every three contiguous nucleotides (FIG. 8A) were also synthesized for use as mutant competitor fragments and were annealed. Gel shift analysis using the GST fusion protein was carried out by a method described by Wu et al. (Wu C. Y. et al. Plant J. 14: 673–683, 1998) and by Suzuki et al. (Suzuki A. et al. Plant Cell Physiol. 39: 555–559, 1998). The labeled oligonucleotide probe was mixed with 0.5 μg of the GST-RISBZ fusion protein, and incubated for 20 min at room temperature. In competition experiments, the competitor fragment was added to the mixture at the 100-fold or higher molecular weight ratio. The reacted mixture was analyzed by non-denaturing acrylamide gel (5%, 0.25×TBE) electrophoresis.
The detection of shift bands showed that the GST-RISBZ1 protein was able to bind to the 21-bp DNA fragment containing the GCN4 motif (FIG. 8B). Furthermore, as shown FIG. 8A, the 21-bp DNA fragments with mutation in every three contiguous nucleotides were used as competitors and examined. When the DNA fragments with the mutations in the GCF motif were added as the competitor, the binding of the DNA fragments that were added as probes was hardly or not inhibited at all (FIGS. 8B to F). By contrast, when the DNA fragments with mutations in the franking sequence of the GCN4 motif were added as the competitor, the shift bands disappeared (FIGS. 8B to F). Since the mutation of the GCN4 motif markedly affects the binding of the RISBZ1 protein to the motif, it was revealed that the RISBZ1 protein recognizes the GCN4 motif sequence specifically. The similar experiments carried out using the other RISBZ proteins revealed that all the RISBZ proteins could specifically recognize the GCN4 motif. As shown in FIGS. 8B to F, the affinity of each RISBZ proteins for the GCN4 motif slightly varies. In the cases of RISBZ2 and RISBZ5, when the DNA fragments with mutations in the franking sequence of the GCN motif were used as the competitor, the shift bands were not disappeared completely (FIGS. 8C and F).
From these results, it was revealed that the RISBZ proteins specifically recognize the GCN4 motif with slightly variable affinities.
EXAMPLE 8
Ability of RISBZ1 Protein to Form a Heterodimer
It was considered that the RISBZ1 protein, a bZIP-type transcription factor, binds to the GCN4-like motif upon forming a heterodimer with other RISBZ proteins. Therefore, the ability of RISBZ1 to heterodimerize with RISBZ2 or RISBZ3 was examined. The full-length RISBZ1 protein, and short-form-RISBZ2 protein (sRISBZ2) and short-form RISBZ3 protein (sRISBZ3) were prepared using wheat germ extracts (FIG. 9A), and were used for DNA binding assay. The in vitro translation was carried out as follows. The coding region of RISBZ1 cDNA and the bZIP domain-encoding regions of RISBZ2 cDNA and RISBZ3 cDNA were amplified using the following forward primers with the NcoI site at their 5′ ends and reverse primers encoding a terminator codon and the BamHI site;








For RISBZ1











R1F:
5′-AAACCATGGAGCACGTGTTCGCCGT-3′ and
(SEQ ID NO: 46)



BRIS1r:
5′-TAGGATCCGCTCCTACTACTGAAGCT-3′;
(SEQ ID NO: 47)


 








For sRISBZ2











dR2-1:
5′-AAACCATGGAGGGAGAAGCTGAGACC-3′ and
(SEQ ID NO: 48)



R2ra1:
5′-AAAGGATCCTACATATCAGAAGCGGCGGGA-3′; and
(SEQ ID NO: 49)


 








For sRISBZ3,











dR3-1:
5′-AAACCATGGATATAGAGGGCGGTCCA-3′ and
(SEQ ID NO: 50)



R3ral:
5′-AAAGGATCCTACAGCCCGCCCAGGTGGCCG-3′.
(SEQ ID NO: 51)






PCR amplification was carried out in a reaction mixture comprising 10 mM Tris-HCl pH 8.3, 1.5 mM MgCl2, 50 mM KCl, 0.01% (w/v) gelatine, 200 μM dNTPs, 1 μM primers, 0.5 mg template DNA, and 2.5-unit TaqI polymerase by 30 cycles of incubation for 1 min at 94° C., for 1 min at 50° C. and for 2 min at 72° C.
The PCR products were digested with restriction enzymes, NcoI and BamHI, and were ligated into the pET8c cloning vector (Novagen) to construct plasmids. Using these plasmids as templates, in vitro transcription/translation (TNT coupled wheat germ extract systems; Promega) was performed for the production of the full-length RISBZ1 protein, and short-form-RISBZ2 (RISBZ2s) and -RISBZ3 (RISBZ3s). For gel shift assay, 4 μl of the wheat germ extract that was used in the above reaction was used.
Gel shift assay was employed to separate homodimers and heterodimers bound to the 21-bp GCN4 motifs. After pre-incubating RISBZ1 with sRISBZ2 and sRISBZ3, the DNA probes comprising the GCN4 motif were added to the incubation mixture. The results indicate that RISBZ homodimers as well as heterodimers can bind to the GCN4 motif. Therefore, it was demonstrated that the RISBZ proteins form heterodimers with the other members of the RISBZ family.
EXAMPLE 9
Involvement of the N-Terminal Region of the RISBZ1 Proteins in the Transcriptional Activation
Transient assay was performed to identify the domain of the RISBZ1 protein involved in transcription activation. The GUS gene, to which three copies of the 21-bp GCN4 motif and the core promoter sequence of CaMV35S were connected, was prepared as a reporter. Various domains of the RISBZ1 proteins were expressed using the CaMV35S promoter in order to examine if these domains can activate the reporter gene.
A series of effector plasmids encoding RISBZ1 proteins in which every 40 amino acids from N-terminus to the basic domain were deleted (encoding the amino acids region ranging from 41st to 436th, 81st to 436, 121st to 436th, 161st to 436th, 201st to 436th, or 235th to 436th in the amino acid sequence set forth in SEQ ID NO: 2), were constructed. When the effector plasmid encoding the full-length RISBZ1 protein and the reporter plasmid (the GUS gene to which four copies of the 12-bp GCN motif and the core promoter sequence of CaMV35S were linked) were introduced into protoplasts, approximately 30-fold higher activity of GUS was detected compared to that of protoplast into which the reporter plasmid alone was introduced. When the transcriptional activity of this reporter gene was set as 100%, the activity of the gene with deletion of the first 40-amino acid was decreased to 20%. Furthermore, the activity of the reporter gene was decreased gradually to 10% by deleting each 40 amino acids. Hence, it was suggested that the N-terminal 40 amino acid residues of RISBZ1 are mainly involved in the transcription activation.
To further analyze the association of the N-terminal 40 amino acids of RISBZ1 with its transcription activating ability, various fusion proteins between the DNA binding domain of the yeast transcriptional activating factor GAL4 and various portions of the RISBZ1 protein were constructed and expressed for the gain-of-function assay. As shown in FIG. 10, a plasmid, in which the coding sequences of fused proteins comprising the GAL4-DNA binding domain and various regions of RISBZ1 were connected downstream of the CaMV35S promoter, was constructed and used as an effector. These effector plasmids were introduced into protoplast together with a reporter construct (the GUS gene, to which nine copies of the GAL4-DNA binding site and CaMV35S core promoter were connected).
The significant difference was not found in transcription activating ability of the fusion protein comprising the GAL4-DNA binding domain and the partial amino acid sequence from 1st to 235th amino acids of RISBZ1, compared with that of a series of the fused proteins in which amino acids were deleted towards the 27th residue from the C-terminal residue of RISBZ1 (FIG. 10). The transcription activating ability of the fusion protein with the first 20 amino acid residues were dramatically decreased (FIG. 10). A fusion protein with deletion of the N-terminal eight residues of RISBZ1 lost the transcriptional activity. In contrast, fusion proteins comprising the GAL4-DNA binding domain and other region of RISBZ1 (from 27th through 57th, 81st through 234th, 161st through 234th, or 235th through 436th in SEQ ID NO: 2) had no effect on the transcriptional activity of the reporter gen. These results suggest that the proline-rich domain within the N-terminal 27 amino acid residues of the RISBZ1 protein, rather than the acidic domain, involves in the transcription activation.
EXAMPLE 10
Difference Between RISBZ1 and Other RISBZ Proteins in Transcription Activating Ability Analyzed by Domain Swapping
Although all the members of the RISBZ protein family have similar affinity for the GCN4 motif sequence, only the RISBZ1 has the transcription activating ability. To find out the reason of this difference, domain swapping between RISBZ1-, and RISBZ2- or RISBZ3-protein was carried out. The N-terminal region at 1st through 299th of RISBZ1, which resides upstream of the bZIP domain, was replaced with the N-terminal region, 1st through 229th of RISBZ2 or 1st through 137th of RISBZ3.
Fusion proteins that have the N-terminal region of RISBZ1 together with the DNA binding domain of RISBZ2 or RISBZ3 showed only approximately 15% or 38% of the transcription activating ability, respectively, compared with that of the full-length RISBZ1. In contrast, fusion proteins that have the N-terminal region of RISBZ2 or RISBZ3 together with the RISBZ1 DNA binding domain showed a slightly higher transcription activity than that induced by the RISBZ1 DNA binding domain alone.
These results indicate that the N-terminal region is mainly involved in the transcription activation. The lower level of the RISBZ2 or RISBZ3 transcription activating ability may be due to deletion or mutation of the region corresponding to RISBZ1 transcription activating domain during evolution. Alternatively, the formation process of transcription activating domain may be responsible for that. It is highly possible that the lower activity of RISBZ3 is due to the lack of the proline-rich domain present in RISBZ1. This applies to RISBZ4 and RISBZ5. The results of the gel shift assay probably exclude the possibility that the differences of affinity with GCN4 motif raise the differences of transcription activating ability.
The proline-rich domain of RISBZ1 was also highly conserved in RISBZ2, but the transcription activating ability of RISBZ2 was extremely low compared to that of RISBZ1. When an effector plasmid that encodes a fused protein comprising the N-terminal 27 amino acid residues of RISBZ2 including proline-rich domain and the GAL4-DNA binding domain was introduced together with a reporter plasmid encoding the GCN4 motif connected to the GUS gene into protoplast, no increased activity of GUS was observed.
Since only eight-residue differences among the N-terminal 27 residues were observed between RISBZ1 and RISBZ2, the present inventors have examined which of the residues among the eight are responsible for the difference in transcription activating ability. The eight amino acid residues of RISBZ1, which were different from RISBZ2, were replaced one by one with the residues of RISBZ2, and the resulting chimeric N-terminal sequences comprising 40 amino acids were fused with the GAL4-DNA binding domain to construct effector plasmids encoding the fused proteins. These effector plasmids were introduced into protoplast together with the reporter plasmid in which the GCN4 motif was fused with the GUS gene. Among eight effector plasmids, all the effector constructs, except for those encoding a protein with replacement of the seventh residue counting from the N-terminus of RISBZ1, did not activate the transcription of the reporter gene. It was presumed using the Kyte and Doolittle formula that all these seven substitutions of amino acids, which were lost transcription activating ability, would induce the change of a hydropacy pattern (FIG. 11).
EXAMPLE 11
Use of the Transcription Factor RISBZ1 for Plant Breeding
The present inventors have examined the possibility to use the transcription factor, RISBZ1, which has a transcription activating ability for plant breeding. In order to specifically overexpress the transcription factor in seeds, rice-plants were transformed with a plasmid construct that encodes the RISBZ1 gene under the control of the promoter of the rice prolamin gene, which encodes a seed storage protein, with 13-kDa molecular masses. The DNA fragment ranging from the EcoRI site, located at the −29th position, to the poly (A) addition site of the RISBZ1 gene was linked to the prolamin promoter encompassing from the −652nd through −13th from the translation initiation site ATG of the gene. The construct was inserted into the binary pGTV-Bar vector, and the resulting vector was introduced into rice plants using Agrobacterium. By this approach, 28 independent transformed lines were established. Screening of rice plants that overexpress the RISBZ1 mRNA was carried out by Northern hybridization of RNA extracted from maturing seeds using cDNA of RISBZ1 as a probe (FIG. 12). These lines overexpressing RISBZ1 were crossed with the transformed rice plants, in which a plasmid construct encoding five tandem repeats of the 21-bp GCN4 motif (5′-GTTTGTCATGGCTGAGTCATG-3′/SEQ ID NO: 52), a target of the RISBZ1 protein, linked to the minimum promoter/GUS reporter had been introduced.
As a result, it was revealed that the expression level of GUS reporter genes were, due to overexpression of RISBZ1 enhanced by 400-times or more (450 to 750 nmol/min/mg protein) than that of controls, 5×GCN4 lines (11 to 14 nmol/min/mg protein) (FIG. 12). These results suggest that the transcription of foreign genes can be highly activated by connecting the foreign genes downstream of the target sequence of the transcription factor RISBZ1 with transcription activating ability and overexpressing RISBZ1.
The RISBZ1 proteins can activate not only the glutelin gene but also other storage protein genes. The 35S CaMV promoter/RISBZ1 fusion construct together with the glutelin promoter/GUS, glutelin promoter (−980th to ATG)/GUS, or 13-Kd prolamin promoter (from −652nd to −29th)/GUS, was introduced into rice protoplast using electroporation, and the transient expressions of them were examined.
The results indicated that the RISBZ1 protein bound to the target sequences containing GCN4 motifs in these promoters and activated the transcription of the foreign genes. It was revealed that the transcriptions were activated 5 to 10-fold in the case of the 13-Kd prolamin promoter and 20 to 30-fold in the case of the globulin promoter, higher than that of the background. Therefore, methylation interference reaction was used to determine how RISBZ1 recognizes the nucleotide sequences of these genes.
The results showed that three GCN4 motifs (TGACACA/SEQ ID NO:86, GATGACTCA/SEQ ID NO:87, and TGACTCAC/SEQ ID NO:88) of the prolamin gene and three motifs different from the GCN4 motif (GGTGACAC SEQ ID NO:89, GTATGTGGC /SEQ ID NO:90, and GATCCATGTCAC/SEQ ID NO:91) of the globulin gene were recognized by the RISBZ1 protein. To determine specific sequences in the promoters that are recognized by the RISBZ1, transient expression of the GUS gene was examined by using a chimeric promoter sequence in which the G, A, C, G/C, MG, or C/A box, GCN4, 22-Kd zein binding site and four repeats of 12-bp sequence including the b-32 binding site were inserted in tandem into the 46 CaMV 35S core promoter/GUS reporter gene. The results indicate that the RISBZ1 protein preferentially recognizes the G/C box and GCN4 motif (FIG. 13).
Furthermore, it was studied to see if the RISBZ1 protein recognized various distinct GCN4 motifs present in the promoter for the storage protein genes. The results indicate that the flanking sequences of the core sequence ‘TGAGTCA’ of GCN4 motif influence transcription activating ability, and that the GCN4 motifs of the wheat gliadin gene and rye secalin gene have high transcription activating ability (FIG. 14).
INDUSTRIAL APPLICABILITY
The present invention provides novel transcription factors that regulate the expression of rice seed storage proteins, and genes that encode the transcription factors. It is expected that the expression of many seed storage proteins regulated by the RISBZ1 protein of the present invention having transcription activating ability can be enhanced by introducing the gene encoding the RISBZ1 protein into cells to overexpress it. The present invention also provides novel gene expression systems in which a useful foreign gene, encoding such as an antibody and an enzyme, can be highly expressed using the transcription factor of the present invention, by linking the recognition sequence of the transcription factor, the GCN4 motif, in tandem and introducing it into the promoter for a gene encoding a seed storage protein to facilitate its binding to the transcription factor. Thus, expression of the gene encoding storage protein and the useful foreign gene can be greatly enhanced under the control of the modified promoter. This enables abundant accumulation of a seed storage protein in endosperm, and more nutritious seeds (e.g. rice) and production of seeds in which useful proteins are highly accumulated.

## Claims

1. An isolated nucleic acid molecule comprising:
a) the coding region of SEQ ID NO:1, or SEQ ID NO:3; or
b) a DNA encoding SEQ ID NO:2.

2. The nucleic acid molecule of claim 1, which encodes a protein that binds to the GCN4 motif or activates expression of a rice seed storage protein.

3. The nucleic acid molecule of claim 1, which is derived from a rice plant.

4. A vector comprising the nucleic acid molecule of claim 1.

5. An isolated nucleic acid molecule encoding an antisense RNA complementary to the full-length transcription product of a nucleic acid sequence, and wherein said nucleic acid sequence comprises:
a) the coding region of SEQ ID NO:1, or SEQ ID NO:3; or
b) a DNA encoding SEQ ID NO:2.

6. A method of producing a protein, comprising the steps of culturing a transformed cell, and collecting the protein expressed from said transformed cell or from the supernatant of said transformed cell, and wherein said protein comprises:
a) the amino acid sequence encoded by SEQ ID NO:1, or SEQ ID NO:3; or
b) the amino acid sequence of SEQ ID NO:2.

7. A transformed cell comprising a nucleic acid sequence comprising:
a) the coding region of SEQ ID NO:1, or SEQ ID NO:3; or
b) a DNA encoding SEQ ID NO:2.

8. The transformed cell of claim 7, which is a plant cell.

9. A plant which is derived from the transformed cell of claim 8, wherein said plant comprises said nucleic acid sequence.

10. A plant derived from a progeny of, or a clone of the plant of claim 9, wherein said derived plant comprises said nucleic acid sequence.

11. A reproductive material of the plant of claim 9, wherein said reproductive material comprises said nucleic acid sequence.

12. A reproductive material of the plant of claim 10, wherein said reproductive material comprises said nucleic acid sequence.

13. A plant which comprises
a) a first nucleic acid construct comprising;
i) a first expression control region, and
ii) a first nucleic acid sequence downstream of said first expression control region, wherein said first nucleic acid sequence comprises:
A) the coding region of SEQ ID NO:1, or SEQ ID NO:3; or
B) a DNA encoding SEQ ID NO:2; and


b) a second nucleic acid construct comprising;
i) a second expression control region having the target sequence of a protein encoded by said first nucleic acid sequence, and
ii) a foreign nucleic acid sequence downstream of said second expression control region.

14. The plant of claim 13, wherein said target sequence comprises the GCN4 motif.

15. The plant of claim 14, wherein said GCN4 motif comprises the sequence as set forth in SEQ ID NO:8, SEQ ID NO:13, or SEQ ID NO:14.

16. The plant of claim 13, wherein said target sequence comprises a G/C box.

17. A method of producing the plant of claim 13, wherein said method comprises a step of crossing a first plant with a second plant, wherein said first plant comprises:
a) a first nucleic acid construct comprising;
i) a first expression control region, and
ii) a first nucleic acid sequence downstream of said first expression control region, wherein said first nucleic acid sequence comprises:
A) the coding region of a DNA as set forth in SEQ ID NO:1, or SEQ ID NO:3; or
B) a DNA encoding SEQ ID NO:2; and



wherein said second plant comprises:
b) a second nucleic acid construct comprising;
i) a second expression control region having the target sequence of a protein encoded by said first nucleic acid sequence, and
ii) a foreign nucleic acid sequence downstream of said second expression control region.

18. An isolated nucleic acid encoding a protein having a sequence at least 95% identical to SEQ ID NO:2, wherein said protein binds to the GCN4 motif and activates expression of a rice seed storage protein.