JP2003159071A - Embryo-specific gene of plant and promoter of the gene, and their utilization - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a gene which expressed itself from an embryo-specific and embryo-expressing pole initial stage, and to provide a promoter which can induce an arbitrary gene from the embryo-specific and embryo-expressing pole initial stage. <P>SOLUTION: Gene expressions between a cell inducing the expression of microspore embryo and an ordinary cell continuing the formation of pollens are comprehensively analyzed by a rapeseed immature culture method. An upstream region estimated to be an expression control site in a cloned gene is cloned by an inverse PCR method using rapeseed genome DNA as a template. It is clarified that the cloned DNA fragment has a nature for strongly inducing the embryo tissue-specific expression of a foreign gene from an embryo- expressing pole early time in both a rapeseed pollen embryo-expressing process and an Arabidopsis thaliana zygotic embryo-expressing process. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a plant embryo-specific gene, a promoter of the gene, and uses thereof.

[0002]

2. Description of the Related Art Needless to say, plant seeds are used as grains and food materials, but they are produced and sold as the main products in the seed and seedling industry, and their quality and characteristics are controlled and improved. The importance of is undisputed. Based on the results of molecular biology research represented by genome research, it is considered that the molecular breeding of seed traits will become more important in the future. In order to enhance the agricultural production technology by artificially modifying seed traits by gene recombination, a technology for efficiently and transspecifically expressing a transgene is important.

Up to now, many genes have been isolated which are specifically expressed in association with the formation of seed tissues such as embryos and endosperms, and there are many reports on promoters that control their expression. The mechanism of action is analyzed in detail. There are many examples. However, they relate to genes whose expression is induced relatively late in seed development, represented by genes encoding seed storage proteins. A promoter capable of inducing seed-gene-specific gene expression from the very early stage of embryogenesis is a highly useful material from the viewpoint of molecular breeding, but the embryonic tissue at the very early stage of development is extremely small and is buried inside the floral organs. Since it is difficult to separate it from the vegetative tissue on the maternal side, analysis has not progressed so far.

Further, in recent years, during the embryonic development process, particularly in the early stage of embryogenesis from the fertilized egg through the spherical embryo to the heart-type embryo until the formation of apical meristems, genes in vegetative organs such as leaves and roots are expressed. It is becoming clear that a mechanism different from the expression control mechanism exists. For example, it has been clarified that the expression of nitrate reductase, which was thought to be uniquely induced by nitrate ions, is activated during embryogenesis in a developmental stage-specific manner regardless of the presence or absence of nitrate ions. (Fukuoka H, Ogawa T, Minami H,
Yano H, Ohkawa Y (1996) L. Plant Physiol. 111: 39-4
7). In addition, CaMV 3 derived from cauliflower mosaic virus has been widely used in many scenes of genetic engineering of plants and has been considered to induce constitutive expression in all tissues of plants.
The 5S promoter has been reported to have no activity in the very early stages of embryonic development (Custers JBM Snepvangers SC
HJ. Jansen H J. Zhang L. van Lookeren Campagne M
M. (1999) Protoplasma. 208: 257-264.). As described above, it is difficult to control the expression of an arbitrary transgene in an embryonic tissue-specific manner in the early stage of embryonic development by the conventional gene expression control technology. Technology that makes this possible is the development of gene manipulation technology that uses a single cell adventitious embryo induction system and the artificial induction technology of apomixis, and the use of the reproductive mechanism of higher plants to improve the efficiency and advanced efficiency of breed breeding and seedling production technology. It is considered to be extremely useful in achieving this. However, there have been no reports on promoters available for such purposes.

[0005]

SUMMARY OF THE INVENTION The present invention has been made in view of the above circumstances, and an object thereof is a gene that is embryo-specific and is expressed from the very early stage of embryogenesis in a plant, and It is intended to provide a promoter for the gene, which is embryo-specific and can induce any gene from the very early stage of embryogenesis.

[0006]

[Means for Solving the Problems] The inventors of the present invention have conducted extensive studies to solve the above problems. First of all, immature pollen culture method of rapeseed (Keller WA, Fan Z, Pechan P, Long
N, Grainger J (1987) In Proc. 7th Intl. Rapeseed Co
ngress. Poznan, Poland, pp 152-157 .; Fukuoka H, Og
awa T, Minami H, Yano H, Ohkawa Y (1996) L. Plant
Physiol. 111: 39-47) was used to comprehensively analyze gene expression between cells that induced microspore embryogenesis and cells that continued normal pollen formation. Specifically, the differential display method (Ito, T, Sakaki, Y. (1996) Method
S. Mol. Genet. 8, 229-245, 1996.), a gene whose expression is strongly induced by the induction of embryo development by high temperature treatment was detected and cloned. Analysis of the expression timing and organ specificity of the cloned gene by Northern blotting method and RT-PCR method,
It was revealed that the expression of the gene was strongly induced specifically in the embryonic tissue from the very early stage of embryo development, and was below the detection limit in pollen, leaves, callus and the like immediately after isolation. Next, the upstream region, which is considered to be the expression control site of this gene, was cloned by the inverse PCR method using rapeseed genomic DNA as a template. Whether or not the cloned DNA fragment has a promoter activity was examined by a gene recombinant plant production experiment using a reporter gene. As a result, the DNA fragment has a property of strongly inducing the expression of a foreign gene in an embryonic tissue-specific manner from the earliest stage of embryonic development in both processes of rapeseed pollen embryo development and Arabidopsis (Arabidopsis thaliana) zygote embryogenesis. It became clear.

Further, using the nucleotide sequence information of the DNA fragment,
When a homology search using the BLAST algorithm was performed on the publicly available gene database, no known nucleotide sequence with high homology was found. Therefore,
An amino acid sequence homologous to the amino acid sequence deduced from the cDNA sequence of the above gene was searched from the entire genome sequence information of Arabidopsis thaliana, and the upstream region of the region presumed to encode the amino acid sequence was cloned using the PCR method. . Whether or not the cloned DNA fragment has a promoter activity was examined by a gene recombinant plant production experiment using a reporter gene. As a result, it was revealed that, similar to the DNA fragment cloned from rapeseed, it exhibits an early embryo-specific gene expression induction pattern. This indicates that it is possible to obtain a DNA having a promoter activity having similar characteristics from plants of different species by using the above gene. From the above results, it was shown that the DNA having the promoter activity has an effect of causing gene expression specific to the embryonic tissue from the very early stage along with the induction of plant embryogenesis.

[0008] When the gene recombination technology is used for the breeding of new varieties and the production of substances using higher plants, it is not possible to control the expression of the introduced gene in a tissue-specific manner or in a development-time-specific manner. This is an important technology. The DNA having the promoter activity in the present invention strongly induces the expression of any gene specifically in the embryo and at the very early stage of embryogenesis when the seed of the plant is formed. for that reason,
Technology for producing and accumulating useful substances in embryo tissues by inducing expression of various metabolic enzyme genes, seed inactivation by forced expression of genes that cause embryonic lethality, embryo tissues of morphogenesis-related genes such as hormone synthesis system-related genes It is expected to be applicable to morphological modification of seed embryos by induction of expression in E. coli.

Furthermore, by using the DNA having the promoter activity of the present invention to control the expression of a selectable marker gene during gene recombination, an antibiotic resistance in a transformation technique utilizing an adventitious embryo induction system from a single cell can be obtained. It is expected that selectable marker genes such as E. coli can be expressed from the early stage of somatic embryogenesis.

Furthermore, since the expression of an arbitrary gene is specifically induced in the embryonic tissue, it is expected that the effect of the introduced gene on tissues other than the embryo can be reduced. For example, it is possible to prevent the production of a selectable marker gene product, which is desirable not to be accumulated in the harvested product, in agricultural products other than the seed embryo, which have edible parts such as leaves, roots and pulp. In addition, in the industrial field of genetic engineering in higher plants, it can be widely used when it is necessary to express a transgene specifically in embryonic tissues.

That is, the present invention relates to a gene which is expressed in a plant in an embryo-specific manner from the very early stage of embryo development, and
A promoter of the gene, which is embryo-specific and is capable of inducing an arbitrary gene from an extremely early stage of embryogenesis, more specifically, [1] which is expressed specifically in embryogenesis of a plant, (A) to (c) the naturally-occurring DNA, (a) a DNA encoding a protein consisting of the amino acid sequence of SEQ ID NO: 6, (b) the amino acid sequence of SEQ ID NO: 6 Which encodes a protein consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in
A, (c) DN comprising the nucleotide sequence set forth in SEQ ID NO: 5
D that hybridizes with A under stringent conditions
NA, [2] an artificial modified form of the DNA described in [1], [3] a protein encoded by the DNA described in [1] or [2], [4] the following (a) to
DN having the promoter activity according to any one of (c)
A, (a) DNA involved in the expression control of the gene encoding the protein described in [3], (b) SEQ ID NO: 7,
A DNA containing the nucleotide sequence according to any one of 8 and 9;
(C) a DNA containing a base sequence in which one or more bases have been substituted, deleted, added, and / or inserted in the base sequence of any of SEQ ID NOs: 7, 8 or 9;
[5] A vector containing the DNA according to any one of [1], [2] or [4], [6] the DNA according to any one of [1], [2] or [4], or [5] [7] A transformed plant cell carrying the vector according to [7].
A transformed plant comprising the transformed plant cell according to [6], [8] A transformed plant which is a progeny or a clone of the transformed plant according to [7],

[9] Reproduction material for the transformed plant according to [7] or [8], [1
0] The method for producing the transformed plant according to [7] or [8], which comprises the DNA according to any one of [1], [2] or [4], or the vector according to [5]. Is introduced into a plant cell, and a method comprising the step of regenerating a plant from the plant cell, [11] a test compound,
A method for evaluating whether or not to regulate the promoter activity of the DNA according to [4], which comprises (a) DN according to [4]
A test compound, which comprises a step of contacting a test compound with a cell or cell extract containing a DNA having a structure in which A and a reporter gene are functionally linked, and (b) measuring the expression level of the reporter gene. [12] a method of determining that the test compound regulates the promoter activity of DNA according to [4] when the expression level of the reporter gene is changed, [12] comprising the following steps (a) and (b) , [4] a method for screening a compound that regulates the promoter activity of DNA, (a) [1.
According to the evaluation method described in 1],
Evaluating whether or not to regulate the promoter activity of the DNA according to [4], (b) selecting from the test compound a compound evaluated to regulate the promoter activity of the DNA according to [4], Is provided.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION The present inventors have cloned a gene encoding a protein which is active from the very early stage of embryonic development of a plant and which is specifically expressed in embryonic tissues. The nucleotide sequence of the gene DNA is shown in SEQ ID NO: 5, and the amino acid sequence of the protein encoded by the DNA is shown in SEQ ID NO: 6. Furthermore, the present inventors utilize the gene,
We have succeeded in isolating a DNA having a promoter activity that is involved in the regulation of expression of the gene.

The present invention provides a DNA (BES1 (Brassica Embryogen) encoding a protein that is specifically expressed in plant embryogenesis.
esis Specific gene 1) gene) is provided. The above-mentioned DNA of the present invention can be used for obtaining (isolating) DNA involved in the expression control of an embryo-specific gene having a promoter activity. In a preferred embodiment of the present invention, first, the DNA of the present invention or a part thereof as a probe, or
The above-mentioned DNA from a desired plant is prepared using an oligonucleotide that hybridizes specifically with the DNA of the present invention as a primer
Isolate DNA with high homology to. Those skilled in the art can perform this isolation step according to well-known methods. Thus, the DNA obtained by using the above-mentioned DNA of the present invention is considered to express specifically in embryonic tissue, and thus the obtained DNA is involved in the regulation of the expression of embryo-specific genes. Regions (promoters) are expected to be included. Even when the expression control region is not included in the DNA, it is considered that the expression control DNA region exists near the genomic DNA in which the DNA is located. The expression control DNA region can be obtained by those skilled in the art by generally known methods using the above-mentioned DNA of the present invention. Furthermore, since the DNA of the present invention is expressed specifically in the embryonic development of plants, it can be used as a marker in the early stage of embryonic development of plants.

The above-mentioned DNA of the present invention is preferably a DNA encoding a protein consisting of the amino acid sequence of SEQ ID NO: 6. In addition, general hybridization techniques (Southern EM: J Mol Bio
l 98: 503, 1975) or PCR technology (Saiki RK, et al: S
cience 230: 1350, 1985, Saiki RK, et al: Science23
9: 487, 1988), which can be isolated, and which hybridize with the DNA consisting of the nucleotide sequence of SEQ ID NO: 5 is also included in the DNA of the present invention. That is, SEQ ID NO: 5
SEQ ID NO: 5 from another plant, using as a probe the DNA having the nucleotide sequence described in SEQ ID NO: 5 or a part thereof, and using an oligonucleotide that specifically hybridizes to the DNA having the nucleotide sequence described in SEQ ID NO: 5 as a primer. Isolation of a DNA having a high homology with the DNA having the nucleotide sequence described in 1 above can be usually performed by those skilled in the art.

In order to isolate such DNA, the hybridization reaction is preferably carried out under stringent conditions. In the present invention, stringent hybridization conditions include 6M urea, 0.4% SDS,
It refers to the condition of 0.5 × SSC or the hybridization condition of stringency equivalent thereto. More stringent conditions, such as 6M urea, 0.4% SDS, 0.
Under the condition of 1 × SSC, it is expected that more homologous DNA can be isolated. The DNA thus isolated is considered to have a high homology with the amino acid sequence of SEQ ID NO: 6 at the amino acid level. High homology means at least 50% or more, preferably 70% or more, more preferably 90% or more, and most preferably 95% or more sequence identity over the entire amino acid sequence.

The identity of amino acid sequences and nucleotide sequences is determined by the algorithm BLAST (Prol
c. Natl. Acad. Sei. USA 87: 2264-2268, 1990, Karlin.
S & Altschul SF: Proc Natl Acad Sci USA 90: 5873,
1993). Programs called BLASTN and BLASTX based on the BLAST algorithm have been developed (Altschul SF, et al: J Mol Biol 215: 403, 1
990). When BLASTN is used to analyze the nucleotide sequence, the parameters are, for example, score = 100 and wordlength = 12. When BLASTX is used to analyze the amino acid sequence, the parameters are, for example, score = 50, wordlength = 3.
And When using BLAST and Gapped BLAST programs, the default parameters for each program are used.
Specific methods of these analysis methods are known (http: /
/www.ncbi.nlm.nih.gov/).

The present invention also provides a DNA encoding a protein structurally similar to the above-mentioned protein expressed specifically in embryogenesis of plants (for example, SEQ ID NO: 6). As such DNA, 1 in the protein
Alternatively, a DNA encoding a protein having an amino acid sequence in which a plurality of amino acids are substituted, deleted, added, and / or inserted is included. Such DNA is also used for isolation of DNA having the promoter activity of the present invention.

Encodes a protein structurally similar to the above-mentioned protein expressed specifically in embryogenesis of plants
Methods well known to those skilled in the art for preparing DNA include the hybridization techniques or polymerase chain reaction (PCR) techniques described above. Also in the natural world, it is possible that the amino acid sequence of the encoded protein is mutated due to the mutation of the nucleotide sequence. Although the mutation may not be accompanied by the mutation of amino acid in the protein (degenerate mutation), such degenerate mutant DNA is also included in the present invention.

The present invention also provides an artificially modified DNA of the above-mentioned plant-encoding DNA encoding a protein specifically expressed in embryogenesis. The modified form means a DNA having a base sequence in which one or more bases are artificially substituted, deleted, added, and / or inserted in the above DNA. Examples of such a variant include a DN in which a base has been substituted in order to prepare a protein with modified activity.
His-tag and GST to facilitate purification of A and proteins
An example is a tag-added DNA or the like. The method for preparing the variant is not particularly limited, and for example, a method for producing a recombinant using a normal gene recombination technique, and further, a DNA encoding a protein specifically expressed in the embryogenesis of the plant of the present invention In contrast, the site-directed mutagenesis method (Kramer W & Fritz HJ: Methods Enzymol 154: 350, 1
987) and a method for introducing a mutation. Such a modified product is used in addition to the production of a transformed plant described below,
It can be used for the isolation of the DNA encoding the protein of the present invention that is specifically expressed in the embryonic development of the plant, or the DNA having the promoter activity of the present invention.

The DNA of the present invention includes genomic DNA, cDNA, and chemically synthesized DNA. The genomic DNA and cDNA can be prepared by a person skilled in the art using conventional means. The genomic DNA is, for example, genomic DNA extracted from a plant having a gene that is specifically expressed in the embryogenesis of the above-mentioned plant, and a genomic library (as a vector, plasmid, phage, cosmid, BAC, PAC, etc. can be used). It is possible to prepare by preparing colonies, developing this, and performing colony hybridization or plaque hybridization using a probe prepared based on the DNA encoding the protein.
It is also possible to prepare by preparing a primer specific to the DNA encoding the protein which is specifically expressed in the embryonic development of the above plant, and performing PCR using this primer. In addition, cDNA is, for example, cDNA is synthesized based on mRNA extracted from a plant having a gene encoding the protein, and this is inserted into a vector such as λZAP to prepare a cDNA library. It can be prepared by performing hybridization, plaque hybridization, or PCR.

Further, the present invention provides a protein encoded by the above DNA.

The present invention also provides a DNA having a promoter activity, which is involved in the expression control of the gene encoding the above protein. In the present invention, by introducing a DNA having the promoter activity into a plant, transcriptional activation can be carried out in an embryonic tissue-specific manner for any gene from the very early stage to the late stage of embryogenesis.

The above-mentioned DNA having a promoter activity can be isolated using, for example, a DNA encoding a protein expressed specifically in the embryonic development of the plant of the present invention. As this isolation method, for example, by the above-mentioned hybridization technique or PCR technique, after cloning the DNA encoding the protein that is specifically expressed in the embryonic development of the plant of the present invention, the upstream region of the DNA is determined by the PCR method or the like. The method of isolation etc. can be mentioned. Whether or not the isolated upstream region has a promoter activity can be examined by those skilled in the art by a well-known reporter assay or the like using a reporter gene. The reporter gene is not particularly limited as long as its expression can be detected, and examples thereof include CAT gene, lacZ gene, luciferase gene, β-glucuronidase gene (GUS) and GFP commonly used by those skilled in the art. A gene etc. can be mentioned. It is also possible to examine whether or not the upstream region of the gene has a promoter activity by examining the expression of the gene of the present invention as a reporter.

The expression level of the reporter gene can be measured by a method known to those skilled in the art depending on the kind of the reporter gene. For example, if the reporter gene
In the case of the CAT gene, by detecting the acetylation of chloramphenicol by the gene product,
The expression level of the reporter gene can be measured. When the reporter gene is the lacZ gene, by detecting the color development of the dye compound due to the catalytic action of the gene expression product, and when it is the luciferase gene, the fluorescent compound due to the catalytic action of the gene expression product By detecting fluorescence, and in the case of β-glucuronidase gene (GUS), Glucuron (ICN) emits light or 5-bromo-4-chloro-3-indolyl-catalyst of the gene expression product. β-glucuronide (X
The expression level of the reporter gene can be measured by detecting the color development of -Gluc) and, in the case of the GFP gene, by detecting the fluorescence from the GFP protein.

When the gene of the present invention is used as a reporter, the expression level of the gene can be measured by a method known to those skilled in the art. For example,
The transcription level of the gene can be measured by extracting the mRNA of the gene expressed from the DNA fragment according to a standard method and carrying out the Northern hybridization method or RT-PCR method using this mRNA as a template. In addition, DN
It is also possible to measure the transcription level of the gene using A array technology. It is also possible to measure the translation level of a gene by collecting a fraction containing the protein encoded by the gene according to a standard method and detecting the expression of the protein of the present invention by an electrophoresis method such as SDS-PAGE. it can. Furthermore, it is also possible to measure the translation level of a gene by performing Western blotting using an antibody against the protein encoded by the gene and detecting the expression of the protein. The antibody used for detecting the protein encoded by the gene is not particularly limited as long as it is a detectable antibody, and for example, both a monoclonal antibody and a polyclonal antibody can be used. The antibody can be prepared by a method known to those skilled in the art. The polyclonal antibody can be obtained, for example, as follows. A small animal such as a rabbit is immunized with the protein of the present invention or a recombinant protein expressed as a fusion protein with GST in a microorganism such as Escherichia coli or a partial peptide thereof to obtain serum. For example, ammonium sulfate precipitation, protein A, protein G column, DEAE
It is prepared by purification by ion exchange chromatography, an affinity column coupled with the protein of the present invention or a synthetic peptide, and the like. If it is a monoclonal antibody, for example, a small animal such as a mouse is immunized with the protein of the present invention or a partial peptide thereof,
The spleen was excised from the same mouse, the cells were crushed to separate the cells, and the cells were fused with mouse myeloma cells using a reagent such as polyethylene glycol. From the resulting fused cells (hybridomas), A clone is selected that produces an antibody that binds to the protein of the invention. Then, the obtained hybridoma was intraperitoneally transplanted to a mouse, ascites was collected from the mouse, and the obtained monoclonal antibody was, for example, ammonium sulfate precipitation, protein A, protein G column, DEAE ion exchange chromatography,
It can be prepared by purification with an affinity column or the like to which the protein or synthetic peptide of the present invention is coupled.

Specific examples of the DNA having a promoter activity of the present invention include DNAs containing the nucleotide sequence of any of SEQ ID NO: 7, 8 or 9. Further, the DNA is structurally similar to the DNA containing the nucleotide sequence set forth in SEQ ID NO: 7, 8 or 9 and includes the nucleotide sequence set forth in SEQ ID NO: 7, 8 or 9.
D with promoter activity equivalent to or improved with DNA
NA is also included in the “DNA having promoter activity” of the present invention. As such DNA, for example, SEQ ID NO ::
1 in the nucleotide sequence of any of 7, 8 or 9
Alternatively, a DNA containing a nucleotide sequence in which a plurality of bases are substituted, deleted, added, and / or inserted can be exemplified. The DNA is
It can be prepared by the above-mentioned hybridization technique, polymerase chain reaction (PCR) technique, site-directed mutagenesis method and the like.

The present invention also provides a vector containing the DNA of the present invention, a DNA having a promoter activity involved in the expression control of a gene encoding the protein of the present invention, and a trait carrying the DNA of the present invention or the above vector. Provided are a transformed plant cell, a transformed plant having the transformed plant cell, a transformed plant which is a progeny or a clone of the transformed plant, and a propagation material for the transformed plant.

Further, the present invention is a method for producing the above transformed plant, which encodes the protein of the present invention.
A step of introducing a DNA or a DNA having a promoter activity involved in the expression control of a gene encoding the protein of the present invention, or a vector containing the DNA into a plant cell to regenerate a plant from the plant cell Provide a way.

The introduction of the DNA of the present invention or the vector containing the DNA into plant cells is known to those skilled in the art,
For example, it can be carried out by the Agrobacterium method, electroporation method (electroporation method), particle gun method or the like.

When the above Agrobacterium method is used,
For example, the method of Nagel et al. (Microbiol. Lett. 67: 325, 199).
0) is used. According to this method, the recombinant vector is transformed into an Agrobacterium bacterium, and then the transformed Agrobacterium is introduced into plant cells by a known method such as the leaf disk method. In addition, the vector includes an expression promoter so that the DNA is expressed in the plant after being introduced into the plant, for example. Generally, the DNA of the present invention is located downstream of the promoter, and the terminator is located downstream of the DNA. The recombinant vector used for this purpose is appropriately selected by those skilled in the art depending on the method of introduction into the plant or the type of plant. Examples of the promoter include CaMV35S derived from cauliflower mosaic virus, corn ubiquitin promoter (Japanese Patent Laid-Open No. 2-79983), and the like.

The terminator may be, for example, a terminator derived from cauliflower mosaic virus, a terminator derived from the nopaline synthase gene, or the like, but is not limited thereto as long as it is a promoter or terminator that functions in a plant. .

The plant into which the DNA of the present invention or a vector containing the DNA is introduced may be an explant, and cultured cells may be prepared from these plants and introduced into the obtained cultured cells. Good. The “plant cell” of the present invention includes, for example, leaves, roots,
Examples include plant cells such as scutellum in stems, flowers and seeds, callus, suspension cultured cells and the like.

In order to efficiently select plant cells transformed by introducing the DNA of the present invention or a vector containing the DNA, the recombinant vector contains an appropriate selectable marker gene or contains a selectable marker gene. It is preferable to introduce it into a plant cell together with the plasmid vector containing it. Selectable marker genes used for this purpose include, for example, the hygromycin phosphotransferase gene resistant to the antibiotic hygromycin, the neomycin phosphotransferase resistant to kanamycin or gentamicin, and the acetyltransferase gene resistant to the herbicide phosphinothricin. Etc.

The plant cells into which the recombinant vector has been introduced are
According to the kind of the introduced selectable marker gene, it is placed in a known selective medium containing an appropriate selective drug and cultured. As a result, transformed plant cultured cells can be obtained.

Then, a plant is regenerated from a transformed cell into which the DNA of the present invention or a vector containing the DNA has been introduced.
Regeneration of a plant can be performed by a method known to those skilled in the art depending on the type of plant cell. The vector that can be used in the present invention is not particularly limited, and for example, pBI121 and the like used in Examples can be appropriately modified and used. Further, the vector of the present invention, for example, by introducing into Agrobacterium, by infecting callus or young plants, it is possible to produce a transformed plant, further,
It is possible to obtain seeds derived from such transformed plants. The transformation method of introducing the DNA of the present invention or a vector containing the DNA into a plant is not limited to the binary vector method using Agrobacterium, and a particle gun method, an electroporation method or the like can also be used. is there.

The plant body regenerated from the transformed cells is then cultured in an acclimation medium. Then, the acclimatized regenerated plant is cultivated under normal cultivation conditions.

The presence of the introduced exogenous DNA in the transformed and cultivated transformed plants was determined by the known PCR method or Southern hybridization method.
Alternatively, it can be confirmed by analyzing the base sequence of the nucleic acid in the plant. In this case, the nucleic acid extraction from the transformed plant can be performed by the method known by J. Sambrook et al. (Molecu
lar Cloning, 2nd Edition, Cold Spring Harbor laboratory P
ress, 1989).

When the foreign gene existing in the regenerated plant is analyzed by the PCR method, the amplification reaction is carried out using the nucleic acid extracted from the regenerated plant as a template as described above. Further, when the nucleic acid is DNA, a synthesized oligonucleotide having a base sequence appropriately selected according to the base sequence of the DNA is used as a primer, and an amplification reaction is performed in a reaction solution in which these are mixed. You can also
In the amplification reaction, the denaturation of DNA, the annealing, and the extension reaction are repeated tens of times to obtain an amplification product of a DNA fragment containing the DNA of the present invention. When the reaction solution containing the amplification product is subjected to, for example, agarose gel electrophoresis, various amplified DNA fragments are fractionated, and the DNA fragments are isolated by the DN of the present invention.
It is possible to confirm that it corresponds to A.

Once a transformed plant in which the DNA of the present invention has been introduced into the chromosome is obtained, it is possible to obtain progeny from the plant by sexual reproduction or asexual reproduction. In addition, propagation material (eg seeds, fruits, cuttings, tubers, tuberous roots, strains, callus, from the plant or its progeny or clones,
It is also possible to obtain protoplasts and the like) and mass-produce the plant based on them.

The present invention further provides a method for evaluating whether or not a test compound regulates the promoter activity of a DNA having the promoter activity of the present invention. The test compound used in the evaluation method of the present invention is not particularly limited, and examples thereof include single compounds such as natural compounds, organic compounds, inorganic compounds, proteins, peptides, and expression of compound libraries and gene libraries. Examples include products, cell extracts, cell culture supernatants, fermented microorganism products, marine organism extracts, plant extracts, prokaryotic cell extracts, eukaryotic single cell extracts or animal cell extracts.

In this method, first, a test compound is brought into contact with a cell or cell extract containing a DNA having a structure in which a DNA having a promoter activity of the present invention and a reporter gene are functionally linked.

In the present invention, "operably linked" means that the expression of the reporter gene is induced by binding of a transcription factor to the DNA having the promoter activity of the present invention. Means that the reporter gene is bound to a DNA having Therefore,
Even when the reporter gene is bound to another gene to form a fusion protein with another gene product, the transcription factor binds to the DNA having the promoter activity of the present invention to bind the fusion protein of the fusion protein. If expression is induced, it is included in the meaning of "functionally linked". The reporter gene used in this method is
It is possible to use the genes mentioned above.

As the "cells containing a DNA having a structure in which the DNA having the promoter activity of the present invention and a reporter gene are functionally linked" in the present method, for example, the above-mentioned DNA
A cell into which a vector containing is introduced.
The vector can be produced by a method well known to those skilled in the art. Introduction of a vector into a cell is a general method,
For example, calcium phosphate precipitation method, electric pulse perforation method,
It can be carried out by a lipofetamine method, a microinjection method or the like.

In addition, "having the promoter activity of the present invention
The "cell containing DNA having a structure in which DNA and a reporter gene are operably linked" also includes cells in which the structure is inserted into the chromosome. The insertion of the DNA structure into the chromosome can be performed by a method generally used by those skilled in the art, for example, a gene introduction method utilizing homologous recombination. Examples of the “cell” used include cells derived from rapeseed, Arabidopsis thaliana, etc., but the origin is not particularly limited.

The "cell extract containing a DNA having a structure in which the DNA having the promoter activity of the present invention and a reporter gene are functionally linked" in the present method is contained in, for example, a commercially available in vitro transcription / translation kit. In the cell extract,
Examples thereof include those to which a DNA having a structure in which a DNA having a promoter activity of the present invention and a reporter gene are functionally linked is added.

The "contact" in the present method is to add a test compound to the culture solution of cells containing DNA having a structure in which a DNA having a promoter activity of the present invention and a reporter gene are functionally linked, or It can be carried out by adding a test compound to the above-mentioned commercially available cell extract. When the test compound is a protein, for example, a vector containing DNA encoding the protein,
It can also be carried out by introducing into the cells or adding the vector to the cell extract.

In this method, the expression level of the reporter gene is then measured. The expression level of the reporter gene can be measured by the method described above depending on the type of the reporter gene.

In the present method, the test compound regulates the promoter activity of the DNA of the present invention when the test compound changes the expression level of the reporter gene, as compared with the case of the measurement in the absence of the test compound. It is judged that it did.

Furthermore, in the present invention, the above-mentioned evaluation method is utilized to analyze the DNA of the present invention for a plurality of test compounds.
It is possible to efficiently screen a compound that regulates the promoter activity by evaluating whether or not the promoter activity is regulated and selecting a compound that regulates the promoter activity. The present invention also provides a method for screening a compound that regulates such promoter activity.

[0050]

EXAMPLES The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[Example 1] Cloning of embryogenesis-specific gene Embryonic development of a plant proceeds deep in the flower tissue separated from the outside world, and it is not possible to artificially isolate a fertilized egg and a very early embryo. Generally difficult. Therefore, the immature pollen culture method of rape (Brassica napus) is used as an experimental system capable of synchronously and frequently inducing embryonic development from single cells. In this culture method, immature pollen in the late mononuclear stage is extracted from the bud before flowering and artificially cultured (Keller WA, Fan Z, Pechan
P, Long N, Grainger J (1987) In Proc. 7th Intl. Rap
eseed Congress. Poznan, Poland, pp 152-157 .; Fukuok
a H, Ogawa T, Minami H, Yano H, Ohkawa Y (1996) L.
Plant Physiol. 111: 39-47). By this operation, the mononuclear immature pollen deviates from the pathway of mature pollen formation and initiates embryonic development. Using this method, gene expression is comprehensively analyzed between cells that have induced microspore embryogenesis and cells that continue normal pollen formation, and genes that are strongly induced by embryogenesis induction are detected. Then, its cloning and structure elucidation were performed.

In the rapeseed immature pollen culture method used here, it is essential for the induction of embryogenesis that a high temperature treatment of 32 ° C. is performed for 24 to 96 hours after the start of culture. On the other hand, under the culture temperature condition of 20 ° C, immature pollen forms mature pollen as in natural conditions, and embryonic development is not observed. In other words, this temperature condition of 32 ° C plays a role of triggering the change in the developmental fate of immature pollen from mature pollen formation to embryogenesis, and the overall pattern of gene expression is also to initiate and progress embryogenesis. It is thought that it will change greatly. Therefore, a gene whose expression is induced in response to this high temperature treatment was isolated as follows.

The isolated immature pollen was cultured in the dark at 32 ° C. for 2 days (hereinafter referred to as sample 232), or at 20 ° C. for 2 days (sample 220), and total RNA was extracted from each in accordance with a standard method. Three single-stranded DNA primers (5'- GTTTTTTTTTTTTTTTA -3 'for each of the two RNA samples)
(SEQ ID NO: 1), 5'- GTTTTTTTTTTTTTTTC-3 '(SEQ ID NO: 2), 5'- GTTTTTTTTTTTTTTTG -3' (SEQ ID NO:
3), hereinafter GT15A, GT15C, GT15G, respectively, and these three are collectively referred to as GT primers)
NA was synthesized. By this operation, sample 220 + GT15A,
Sample 220 + GT15C, Sample 220 + GT15G, Sample 23
2 + GT15A, sample 232 + GT15C, sample 232 + GT15G,
A total of 6 types of cDNA mixture were obtained. Using these cDNA mixtures as templates, 12-mer arbitrary primers (manufactured by Wako Pure Chemical Industries) and individual GT primers (having the same sequences as those used for cDNA synthesis) were used, and cDNs were separately prepared by PCR reaction.
A was amplified. At that time, GT15A, GT15C, and GT15G are fluorescent dyes.
FITC was used with its 5'end labeled. This operation allows sample 220 and sample 232 for one arbitrary primer and one GT primer combination.
Two kinds of amplification products were obtained.

These two kinds of amplification products were separated by electrophoresis based on the difference in molecular weight, and the separated images were detected by a fluorescence image analyzer, and sample 220 and sample 232.
Compared between. The cDNA corresponding to the gene commonly expressed in both samples is commonly observed between the two samples, but is derived from the gene specifically expressed in embryogenesis.
cDNA was found only in sample 232. A method of detecting a gene specifically expressed in this way is generally called a differential display method, and in this example, (Ito, T, Sakaki, Y. (1996) Method
s Mol. Genet. 8, 229-245, 1996.).

Using this method, plural kinds of bands due to mRNA molecules existing only in cells subjected to high temperature treatment were detected on an electrophoretic gel. One of them, BES1, is GT15A and an arbitrary primer (5'-GCCTGCCTCACG -3 '(SEQ ID NO:
4)) was used in the reaction product. Its amplified DN
A product is extracted and purified from electrophoresis gel, inserted into cloning vector (pGEM-T, Promega), cloned,
The base sequence was determined. Further, based on the nucleotide sequence, a full-length cDNA was isolated using Lifetech Oriental's 5'RACE system, inserted into the above-mentioned pGEM-T vector and cloned, and the entire nucleotide sequence was determined. Its cD
The nucleotide sequence of NA is shown in SEQ ID NO: 5, and the amino acid sequence deduced from the nucleotide sequence is shown in SEQ ID NO: 6, respectively.
When the timing and organ specificity of the expression of the gene were analyzed by the Northern blotting method and the RT-PCR method, FIG.
As shown in Fig. 2 and Fig. 2, expression of BES1 gene transcript was strongly induced in embryonic tissue from the very early stage of embryogenesis, and was below the detection limit in pollen, leaves, callus, etc. immediately after isolation. Became clear.

Example 2 Cloning of Expression Control Site of Gene Specific to Embryonic Development The upstream region of the BES1 translation region, which is considered to be the BES1 expression control site, was cloned by the inverse PCR method using rapeseed genomic DNA as a template. Rapeseed genomic DNA restriction enzyme BamHI
After double digestion with EcoRI and blunting both ends with T4 DNA polymerase, self-circularization with T4 DNA ligase was performed. Using this as a template, PCR was carried out using two types of reverse PCR primers designed from the sequence of BES1 cDNA, and 2179 bp upstream of the translation initiation site of the BES1 gene was cloned by the PCR method from the nucleotide sequence data of the amplified product.
The entire base sequence was determined, and a homology search was performed using the BLAST algorithm on a known DNA sequence database. As a result, the portion of the obtained 2179 bp at the 5'side of about 1.1 kbp was Arabidopsis thaliana. It was judged that this region is not the transcriptional control region of BES1 because it showed extremely high homology with the protein coding region of the known gene of BES1. Cloned as. Its nucleotide sequence is shown in SEQ ID NO: 7, and this DNA fragment is hereinafter referred to as PBES1.
The DNA and amino acid sequence data of the present invention and the homology search using the data are used for the NCBI (National Center for Biological Information) published on the Internet.
(Othchnology Information) operated by a program on a computer system and a gene database published on the system.

[Example 3] Examination of specificity of gene expression control pattern The activity of PBES1 as a promoter was examined by a gene recombinant plant production experiment using a reporter gene. The vector used is pBI121 which is commonly used as a plant transformation gene vector. This vector has a β-glucuronidase gene (GUS) controlled by the CaMV-35S promoter derived from cauliflower mosaic virus as a reporter gene. In this experiment, this CaMV-35
A modified vector prepared by removing the S promoter and inserting PBES1 therein was used. This vector was transformed into rapeseed and Arabidopsis thaliana by the binary vector method via Agrobacterium. Using the expression induction pattern of GUS in the transformed plant as an index, the gene expression inducing ability as a promoter of PBES1 and its timing / tissue specificity were determined by 5-bromo-4-chloro-3-indolyl-β-glucuronide (X-Gluc ) Was used as a substrate for histochemical activity staining for detection. As a result, the DNA fragment PBES1
Revealed that in both the development of rapeseed pollen embryo and the development of Arabidopsis zygote embryo, it has a property of strongly inducing the expression of a foreign gene specifically in the embryonic tissue from the earliest stage of embryogenesis (Fig. 3-1, (See 2, 3 and 5).
Moreover, as shown in FIG. 3-4, no activity was detected in vegetative tissues such as leaves and roots. From the above results, it was shown that the promoter sequence has an action of inducing gene expression specific to embryonic tissue from the very early stages along with the induction of plant embryogenesis. It is clear that its temporal and spatial control is extremely strict and its specificity is extremely high.

For the purpose of identifying the region controlling such embryo-specific expression control, a similar analysis was carried out by a transformed plant using a deletion fragment obtained by deleting PBES1 from the 5 ′ side. Crude protein was extracted from immature seeds (4 days after flowering) of the transformant, and GUS activity was quantitatively measured by photon counting using luminescence intensity with Glucuron (ICN) as a substrate. As a result, as shown in Fig. 4, PBES1
It was revealed that there is a sequence that plays an important role in embryo-specific expression control of the promoter in the region of 104 bp in length between 297 and 400 bases from the 3'side. The nucleotide sequence of this region is shown in SEQ ID NO: 8.

Example 4 Cloning of Similar Promoters from Other Plant Species Using the nucleotide sequence information of PBES1, a homology search was performed using the BLAST algorithm against a public gene database, and high homology was found. No known base sequence with sex was searched. However, the cD of the previously isolated BES1 gene
Amino acid sequence deduced from NA sequence (SEQ ID NO: 6)
Was highly homologous to the putative protein F20O9.30 (GenBank accession number: T04605) deduced from the whole genome sequence information of Arabidopsis thaliana, which was revealed by database search using the BLAST algorithm. Therefore, the genome sequence of Arabidopsis thaliana with the F20O9.30 gene was acquired from the database (GenB
ank accession number: AL021749), upstream region 421 bp of the region presumed to encode F20O9.30 was cloned using the PCR method. Its base sequence is shown in SEQ ID NO: 9 and is hereinafter referred to as PAtBES1. PAtBES1 was ligated upstream of GUS and introduced into Arabidopsis using the binary vector method, and the same transformation experiment was performed. As a result, Fig. 3-
As shown in 6, it was revealed that PAtBES1 also exhibits an early embryo-specific gene expression induction pattern like PBES1. This indicates that it is possible to obtain a promoter sequence having similar characteristics from plants of different species by using the amino acid sequence information of BES1 protein.

[0060]

[Effects of the Invention] When the gene recombination technology is used for the breeding of new varieties and the production of substances using higher plants, the expression of the introduced gene is controlled in a tissue-specific or development-specific manner of the plant. That is an important technology. The DNA having the promoter activity in the present invention strongly induces the expression of any gene specifically in the embryo and at the very early stage of embryogenesis when the seed of the plant is formed. Therefore, technologies for producing and accumulating useful substances in embryonic tissues by inducing expression of various metabolic enzyme genes, seed inactivation by forced expression of genes that cause embryonic lethality, morphogenesis-related genes such as hormone synthesis-related genes, etc. It can be used to modify the morphology of seed embryos by inducing expression in embryo tissues.

Furthermore, by using the DNA having the promoter activity of the present invention to control the expression of a selectable marker gene during gene recombination, an antibiotic resistance can be obtained in a transformation technique utilizing a somatic embryo induction system from a single cell. It becomes possible to express a selectable marker gene such as from the early stage of somatic embryogenesis.

Further, since the expression of an arbitrary gene is specifically induced in the embryonic tissue, it is possible to reduce the influence of the introduced gene on tissues other than the embryo. For example, it is possible to prevent the production of a selectable marker gene product, which is desirable not to be accumulated in the harvested product, in agricultural products other than the seed embryo, which have edible parts such as leaves, roots and pulp. In addition, in the industrial field of genetic engineering in higher plants, it can be widely used when it is necessary to express a transgene specifically in embryonic tissues.

[0063]

[Sequence list]                                SEQUENCE LISTING        <110> National Agricultural Research Organization <120> Embryo specific genes of plants, promoters of the genes, and use t hereof <130> ARO-A0102 <140> <141> <160> 9 <170> PatentIn Ver. 2.1 <210> 1 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: An       Artificially Synthesized Primer Sequence <400> 1 gttttttttt tttttta 17 <210> 2 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: An       Artificially Synthesized Primer Sequence <400> 2 gttttttttt ttttttc 17 <210> 3 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: An       Artificially Synthesized Primer Sequence <400> 3 gttttttttt ttttttg 17 <210> 4 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: An       Artificially Synthesized Primer Sequence <400> 4 gcctgcctca cg 12 <210> 5 <211> 873 <212> DNA <213> Brassica napus <220> <221> CDS <222> (51) .. (680) <400> 5 acacatttca agagactgat ttatttgagg tctagttaaa gtttggaggg atg ggt 56                                                        Met Gly                                                          1 ttg atc aaa agg ttt gat gct tat ttg atg act gtg atg tta gtg agt 104 Leu Ile Lys Arg Phe Asp Ala Tyr Leu Met Thr Val Met Leu Val Ser           5 10 15 ttg ggg ttt gca att ggg ttt tca aat ggg tac aag ttc tat gtt ggt 152 Leu Gly Phe Ala Ile Gly Phe Ser Asn Gly Tyr Lys Phe Tyr Val Gly      20 25 30 ggg aga gat ggt tgg gtc ctt act cca tcc gag gac tat tct cat tgg 200 Gly Arg Asp Gly Trp Val Leu Thr Pro Ser Glu Asp Tyr Ser His Trp  35 40 45 50 tct tat cga agc cgg ttt cag gtc aat gac act ctt tat ttt aag tac 248 Ser Tyr Arg Ser Arg Phe Gln Val Asn Asp Thr Leu Tyr Phe Lys Tyr                  55 60 65 cct aag ggg aaa gat tca gtg ttg gag gtg agt gaa gaa gaa ttc aac 296 Pro Lys Gly Lys Asp Ser Val Leu Glu Val Ser Glu Glu Glu Phe Asn              70 75 80 aca tgc aac act act cac cca ata acc tcc ctc acg ggc gga gac tct 344 Thr Cys Asn Thr Thr His Pro Ile Thr Ser Leu Thr Gly Gly Asp Ser          85 90 95 ctc tat gtt ctt agc cgc tca ggt ccg ttc ttt ttt gtc agc ggc aac 392 Leu Tyr Val Leu Ser Arg Ser Gly Pro Phe Phe Phe Val Ser Gly Asn     100 105 110 tca gaa aat tgt ctc aaa ggt cag aag cta ccc gtg aag gtc atg tcc 440 Ser Glu Asn Cys Leu Lys Gly Gln Lys Leu Pro Val Lys Val Met Ser 115 120 125 130 gcc gcc cac cac agc cac agt cct cgt cag cca tct cca tcg ccc tca 488 Ala Ala His His Ser His Ser Pro Arg Gln Pro Ser Pro Ser Pro Ser                 135 140 145 ccg tca ccg act ctg tct cca agc cat cag gct ttg tca tct cca gcg 536 Pro Ser Pro Thr Leu Ser Pro Ser His Gln Ala Leu Ser Ser Pro Ala             150 155 160 cct tct ccg aga gtg gtt ctt tct gaa tct gaa gct ctt gct ccg gct 584 Pro Ser Pro Arg Val Val Leu Ser Glu Ser Glu Ala Leu Ala Pro Ala         165 170 175 cca gga cca gcg aaa gct cac aat tcg gcc ggt ttg gtg agt cct agg 632 Pro Gly Pro Ala Lys Ala His Asn Ser Ala Gly Leu Val Ser Pro Arg     180 185 190 gcg gtc tct cta gga ttg gtt ctc gtg gtt att ata agt ttc gtg ctt 680 Ala Val Ser Leu Gly Leu Val Leu Val Val Ile Ile Ser Phe Val Leu 195 200 205 210 tagagttata atggcgaggg taataaatcc tccataattg agtttaatat taatgttcct 740 tgaaccatct tattaattgt gtttgaactt tgttatgttt gtcctttttg ttatgttttt 800 catgtctcgt gtttttcttt actaaacaaa gtttgtattt atactaaaaa gagcatgtat 860 ttgttttgtg tct 873 <210> 6 <211> 210 <212> PRT <213> Brassica napus <400> 6 Met Gly Leu Ile Lys Arg Phe Asp Ala Tyr Leu Met Thr Val Met Leu   1 5 10 15 Val Ser Leu Gly Phe Ala Ile Gly Phe Ser Asn Gly Tyr Lys Phe Tyr              20 25 30 Val Gly Gly Arg Asp Gly Trp Val Leu Thr Pro Ser Glu Asp Tyr Ser          35 40 45 His Trp Ser Tyr Arg Ser Arg Phe Gln Val Asn Asp Thr Leu Tyr Phe      50 55 60 Lys Tyr Pro Lys Gly Lys Asp Ser Val Leu Glu Val Ser Glu Glu Glu  65 70 75 80 Phe Asn Thr Cys Asn Thr Thr His Pro Ile Thr Ser Leu Thr Gly Gly                  85 90 95 Asp Ser Leu Tyr Val Leu Ser Arg Ser Gly Pro Phe Phe Phe Val Ser             100 105 110 Gly Asn Ser Glu Asn Cys Leu Lys Gly Gln Lys Leu Pro Val Lys Val         115 120 125 Met Ser Ala Ala His His Ser His Ser Pro Arg Gln Pro Ser Pro Ser     130 135 140 Pro Ser Pro Ser Pro Thr Leu Ser Pro Ser His Gln Ala Leu Ser Ser 145 150 155 160 Pro Ala Pro Ser Pro Arg Val Val Leu Ser Glu Ser Glu Ala Leu Ala                 165 170 175 Pro Ala Pro Gly Pro Ala Lys Ala His Asn Ser Ala Gly Leu Val Ser             180 185 190 Pro Arg Ala Val Ser Leu Gly Leu Val Leu Val Val Ile Ile Ser Phe         195 200 205 Val Leu     210                                                         <210> 7 <211> 1035 <212> DNA <213> Brassica napus <400> 7 tattgtttga catacagcta aaacatacca caattatttg acaaaagatt caattagaaa 60 gcatggttcc acaaaggcca agcaattatc acagagcttc aatatagtca atggtgggca 120 tttcactaaa gtgtgatcag ctcaatagtg ataagaagat aaatctaaaa cacaaacctg 180 gtcgctccat tatcgagttt cgaactctga tcccaaaacc cctagaggtt ccagcttttc 240 catctcacaa cttctagtga aactagggtt tgggttttct gcagactgta gaggaagcag 300 tagaagattg ctggctgtat cagaagctaa atagacgtgg ctgaagaata ttttgaagct 360 tcacaattag ctcctctcta agctaggcat actgaagaaa gcatccatcc acttatagaa 420 atagatgggc tttaaacgta gaggatgaaa tatgttctgt tacaagaaaa tagtaaatag 480 tttattctaa tattgtccaa ctagttgggc tgcaaatata gaggataaaa tccttaaagc 540 ctttctttta acatacttga tcatttaaaa gcacattagt tttttaatta ttttgaactc 600 cttctagttg gtttttttag gcttttagct catttgtata cttttttttt cttatataaa 660 atgaagacaa tacgttacgc aagttatttt tttaagaaaa ttgaatttca gctttatatt 720 aaatgactga caaattagaa gatctagttt taaatggttg tttgtgattt gaggagggag 780 taaatgaaat gtgtgaaaag aagagagatg gccgttaagt ccattgcatg ttgaaacgtg 840 tgaagaaaac aaaagaggag caaaacttga aacgcttctt attaattttg catattctct 900 agagagtcga agttgatgca gccaaaagct aacggctatg acttctctac tttcactata 960 aatacgcata caagcactga agagcaacac acatttcaag agactgattt atttgaggtc 1020 tagttaaagt ttgga 1035 <210> 8 <211> 104 <212> DNA <213> Brassica napus <400> 8 gtatactttt tttttcttat ataaaatgaa gacaatacgt tacgcaagtt atttttttaa 60 gaaaattgaa tttcagcttt atattaaatg actgacaaat taga 104 <210> 9 <211> 421 <212> DNA <213> Arabidopsis thaliana <400> 9 gatatttatc gatttgtaaa ttgaaatata aaataaaaca atatgtaacg taagttttaa 60 ataataataa atactaaagg attacaattt tatatttgat ggctaaataa ttacaagatc 120 tgagtgagaa atgtttgttt gtgagaaaga aagtgaaaaa ttaaaggtag atagccgtta 180 agtccactgc atgtcgagac gtgttcaaaa gaagacagga gcaaaaccaa aacgcttcta 240 cttaattcgc atttctttag gtaatcgatg cacagcccaa aaagcttgac ctaacggcta 300 tgttttcttt actttcacca taaataagca cctcttgagg ttgcaaacac acacacacac 360 acacactcac ttcaaaagag ttagtaagaa gttggggttt gattaacgtt ttgcatcgga 420 g 421

[Brief description of drawings]

FIG. 1 is a photograph showing a rapeseed BES1 transcript detected by RT-PCR. 1 to 12 are immature pollen immediately after isolation, immature pollen after culturing at 20 ° C. for 2 days, immature pollen after culturing at 32 ° C. for 4 days, immature pollen after culturing at 32 ° C. for 4 days, embryo at several cell stages, spherical Embryo, heart / torpedo, cotyledon, ripe seed,
Leaf, callus (culture temperature 20 ℃), callus (culture temperature 32 ℃)
The sample prepared from is shown.

FIG. 2 is a photograph showing a rapeseed BES1 transcript detected by the Northern hybridization method. 1 ~ 7
Are immature pollen immediately after isolation, immature pollen after culturing at 20 ° C for 2 days, immature pollen after culturing at 32 ° C for 2 days, immature pollen after culturing at 32 ° C for 4 days, spherical embryos, heart-shaped / torpedo embryos, respectively. , Shows samples prepared from leaves.

FIG. 3 is a photograph showing a gene expression control pattern of a promoter detected by a gene recombination experiment. All samples were histochemically active stained with X-Gluc as a substrate. The black part shows the activity of GUS. 1 to 6 are PBES1 :: GUS-introduced pollen embryos isolated from the rapeseed, respectively, 4th day, 8th day, 14th day, and PBES1 :: G
Seedlings of Arabidopsis transformants introduced with US (after sowing)
7th day), immature embryo of the same transformant (4 days after flowering), PAtBES
1 is a photograph of immature embryos (4 days after flowering) of Arabidopsis transformants introduced with 1 :: GUS.

FIG. 4 is a diagram showing 7 types of fragments in which PBES1 is deleted from the 5 ′ side and promoter activity (activity of GUS) of the fragments.

Continued front page    F term (reference) 2B030 AA02 AB03 AD20 CA08 CD14                       HA01                 4B024 AA08 BA80 CA04 DA01 EA04                       FA02 FA10 GA11 GA17                 4B063 QA08 QA18 QQ32 QR58 QR59                       QR80 QS24 QS28 QX01                 4B065 AA88X AA88Y AB01 AC14                       BA02 CA24 CA53                 4H045 AA10 AA30 BA10 CA30 EA05                       FA74

Claims (12)

[Claims] 1. The naturally-derived DNA according to any one of the following (a) to (c), which is specifically expressed in plant embryogenesis. (A) A DNA encoding a protein consisting of the amino acid sequence of SEQ ID NO: 6. (B) A DNA encoding a protein consisting of the amino acid sequence of SEQ ID NO: 6 in which one or more amino acids are substituted, deleted, added and / or inserted. (C) DN which hybridizes with the DNA consisting of the nucleotide sequence of SEQ ID NO: 5 under stringent conditions
A. 2. An artificial modified form of the DNA according to claim 1. 3. A protein encoded by the DNA according to claim 1 or 2. 4. A DNA having a promoter activity according to any of the following (a) to (c). (A) A DNA involved in the expression control of the gene encoding the protein according to claim 3. (B) A DNA containing the nucleotide sequence of any of SEQ ID NOs: 7, 8 or 9. (C) A DNA containing a base sequence in which one or more bases have been substituted, deleted, added, and / or inserted in the base sequence of SEQ ID NO: 7, 8 or 9. 5. A vector containing the DNA according to claim 1, 2 or 4. 6. A transformed plant cell carrying the DNA according to claim 1, 2 or 4, or the vector according to claim 5. 7. A transformed plant comprising the transformed plant cell according to claim 6. 8. A transformed plant which is a progeny or a clone of the transformed plant according to claim 7. 9. A propagation material for the transformed plant according to claim 7 or 8. 10. A method for producing the transformed plant according to claim 7 or 8, wherein the DNA according to any one of claims 1, 2 or 4 or the vector according to claim 5 is added to a plant cell. And a step of regenerating a plant from the plant cell. 11. A method for evaluating whether or not a test compound regulates the promoter activity of the DNA according to claim 4, wherein (a) the DNA according to claim 4 and the reporter gene are functional. Comprising a step of contacting a test compound with a cell or cell extract containing DNA having a structure bound to, and (b) measuring the expression level of the reporter gene, the test compound expressing the expression level of the reporter gene. The method of determining that a test compound regulates the promoter activity of DNA according to claim 4, when it is changed. 12. A method for screening a compound that regulates the promoter activity of DNA according to claim 4, which comprises the following steps (a) and (b). (A) A step of evaluating whether or not the test compound regulates the promoter activity of the DNA according to claim 4 with respect to the test compound by the evaluation method according to claim 11. (B) A step of selecting, from the test compounds, a compound evaluated to regulate the promoter activity of the DNA according to claim 4.