JP2008295454A - Seed-specific promoter and use thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a promoter having seed-specific activity and to provide a method for expressing a foreign protein in a seed. <P>SOLUTION: A plurality of promoters of rice seed expression genes are isolated; a binary vector is prepared by inserting each promoter to the upstream of GUS reporter gene and rice is transformed by an Agrobacterium method. An expression site by each promoter, an expression in a seed maturation process and an expression strength in a seed are examined by using an expression amount of GUS as an index and thereby a promoter that has specific expression activity at a specific site of seed and higher activity than those of a constant promoter and a known seed-specific promoter has been found. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a seed-specific promoter and use thereof.

  Genetic recombination technology has been applied as a method for improving plant varieties. Plants to which functions such as herbicide resistance and pest resistance have been added have already been created by genetic recombination technology and are being put to practical use. However, plant varieties improvement by genetic recombination technology is not limited to adding functions to plants, but R & D using plants as useful protein factories, in other words, introducing foreign genes into plants and expressing useful proteins in plants. Has been made.

  Plant production of recombinant proteins has many advantages, most notably the cost savings compared to animal transgenic lines and the ease of adjusting the production scale according to the market scale, May not be contaminated with animal-derived pathogens such as viruses and prions (Daniell et al .; Trends Plant Sci. 6, 219-226 (2001), Fischer and Emans; Transgenic Research 9,279-299 (2000), Giddings et al .; Nature Biotech. 18, 1151-1156 (2000)).

  It has recently been shown that seed systems have advantages over leaf or root systems for the production of recombinant proteins in plants (Delaney, 2002; Plants as Factories for Protein Production (Hood, EE and Howard, EN pp. 139-158 (2002). Netherlands: Kluwer Academic, Howard and Hood; Plants as Factories for Protein Production (Hood, EE and Howard, JA) pp. vii-x (2002). Netherlands: Kluwer Academic). Seeds are storage organs, in which a small number of storage proteins are stably accumulated in a special organelle called protein granules. Utilizing these properties, seed has been used as an ideal bioreactor for the production of recombinant proteins. Recombinant proteins accumulated in seeds are very stable and can be delivered orally as they are without the need for processing or purification. It has been reported that when an antibody or vaccine is expressed in seeds, it is highly stable and does not degrade for many years when stored at room temperature. Furthermore, vaccines delivered through seeds are believed to induce antibody production by the mucosal immune system without purification and processing (Walmsley and Arntzen; Curr. Opin. Biotech. 11, 126-129 ( 2000)).

  By the way, in the protein production by gene recombination, the production amount of the target protein is influenced by many factors such as transcription factors. The most important and easy to control factor is the choice of promoter. In order to use rice seed as a platform for the production of recombinant proteins, the promoter controls not only the time and place of expression but also its level, so it is tailored to the requirements of individual proteins and biotechnology applications. It is important to use a promoter.

  However, analysis of cis-regulators involved in endosperm-specific expression is limited to a few glutelin genes using heterologous transgenic tobacco and allogeneic transgenic rice (Croissant-Sych and Okita; Plant Sci. 116, 27 -35 (1996), Takaiwa et al .; Plant Mol. Biol. 16, 49-58 (1991a), Takaiwa et al .; Plant Mol. Biol. 30, 1207-1221 (1996), Wu et al .; Plant J. 14, 673- 983 (1998a), Wu et al .; Plant J. 23, 415-421 (2000), Yoshihara et al .; FEBS Lett. 383, 213-218 (1996), Zhao et al .; Plant Mol. Biol. 25, 429-436 (1994) ), Zheng et al .; Plant J. 4, 357-366 (1993)). For a few other rice storage protein promoters, only a spatial expression pattern has been observed (Wu et al .; Plant Cell Physiol., 39, 885-889 (1998b)).

JP2002-058492 Trends Plant Sci. 6, 219-226 (2001) Transgenic Research 9, 279-299 (2000) Nature Biotech. 18, 1151-1156 (2000) Plants as Factories for Protein Production (Hood, E.E. and Howard, J.A) pp. 139-158 (2002). Netherlands: Kluwer Academic Plants as Factories for Protein Production (Hood, E.E. and Howard, J.A) pp. Vii-x (2002). Netherlands: Kluwer Academic Curr. Opin. Biotech. 11, 126-129 (2000) Plant Sci. 116, 27-35 (1996) Plant Mol. Biol. 16, 49-58 (1991a) Plant Mol. Biol. 30, 1207-1221 (1996) Plant J. 14, 673-983 (1998a) Plant J. 23, 415-421 (2000) FEBS Lett. 383, 213-218 (1996) Plant Mol. Biol. 25, 429-436 (1994) Plant J. 4, 357-366 (1993) Plant Cell Physiol., 39, 885-889 (1998b)

  The present invention has been made in view of such circumstances, and an object of the present invention is to provide a promoter having specific activity in seeds and a method for expressing foreign proteins in seeds. It is another object of the present invention to provide a promoter having specific activity at a specific site such as seed endosperm, embryo, or aleurone tissue.

  In order to solve the above-mentioned problems, the present inventors have isolated a plurality of rice seed expression promoters, prepared binary vectors in which the respective promoters are inserted upstream of the GUS reporter gene, and obtained rice by the Agrobacterium method. Transformed. Using the expression level of GUS as an index, we examined the expression site by each promoter, the expression during seed maturation, and the expression intensity in seeds. We found a promoter with higher activity compared to the seed-specific promoter. The usefulness of ingesting seeds expressing a foreign gene product as food is as described above. For this purpose, it is necessary that the foreign gene is expressed in the edible part of the seed. For example, in rice, which is one of the main cereals, endosperm is a normal edible part. Therefore, for rice, the above purpose is realized by an endosperm-specific promoter. Furthermore, a promoter specific to a seed specific site enables expression of a foreign gene at an arbitrary place of the seed, and can be an important tool in performing metabolic engineering using the seed. For example, fatty acid metabolism can be controlled using a promoter expressed in aleurone tissue or embryo.

That is, the present inventors relate to a seed-specific site-specific promoter and use thereof, more specifically,
(1) DNA having promoter activity in seeds according to any one of (a) to (c) below
(A) DNA comprising the base sequence described in any one of SEQ ID NO: 1 to SEQ ID NO: 7
(B) DNA comprising a sequence in which one or more bases are added, deleted, substituted or inserted into the base sequence described in any one of SEQ ID NO: 1 to SEQ ID NO: 7.
(C) DNA that hybridizes under stringent conditions with DNA comprising the sequence described in any of SEQ ID NO: 1 to SEQ ID NO: 7,
(2) DNA in which any gene is functionally linked downstream of the DNA described in (1) above
(3) a vector comprising the DNA described in (1) or (2) above,
(4) A transformed plant cell retaining the DNA described in (2) above,
(5) A transformed plant cell introduced with the vector described in (3) above,
(6) Transformed plant holding the cells described in (4) or (5) (7) Propagating material of the plant described in (6) (8) The seed (7) (9) A method for expressing an arbitrary gene in seeds of plant cells, comprising the following steps (a) and (b): (a) the DNA described in (2) above or (3) (B) A step of regenerating a plant from the plant cell is provided.

  Seeds are essentially storage tissues and have a large space for accumulating foreign gene products. Enzymes and antibodies accumulated in seeds are stable for more than one year even when stored at room temperature. There is no need to refine if you eat. Useful substances can be produced at extremely low cost. Substances can be produced only in the field without the need for special facilities, and there is no contamination with animal viruses.

  Use of these promoters is extremely useful as a promoter for producing useful substances using seeds. For example, it enables mass production of pharmaceuticals (vaccines, antibodies, blood products, interferons, etc.) and industrial enzymes in seeds. In addition, by expressing the allergen epitope, it can be used for the development of allergy treatment crops that can treat hay fever and house dust allergy by eating. In addition, it is possible to improve seed nutrition by expressing a highly nutritious foreign gene in the seed. Moreover, functional seeds can be produced by highly expressing functional peptides and functional proteins having a lowering effect on hypertension, serum cholesterol, blood sugar level, etc. in the seeds using the above promoter.

  In addition, the promoter set that enables the expression of the seed according to the present invention at an arbitrary place and at the seed expression time can be used as an important tool in performing metabolic engineering using the seed. For example, in the metabolism of fatty acids, a target metabolic process can be controlled using a promoter expressed in aleurone or embryo.

  The present invention provides a novel DNA having promoter activity in seeds. In the present invention, as described above, the present inventors have found a promoter having a specific expression activity at a specific site of seeds and a higher activity compared to a constitutive promoter or a known seed-specific promoter. It is based on.

Specific examples of the DNA of the present invention include DNA having promoter activity comprising the sequence described in any one of SEQ ID NOs: 1 to 7. The DNA was newly found by the present inventors as rice-derived DNA having promoter activity, and can be classified into the following three groups.
(A) Endosperm-specific promoter DNA group (the base sequence of each DNA is described in SEQ ID NOs: 1 to 4)
(B) Embryo or aleurone tissue-specific promoter DNA group (the base sequence of each DNA is described in SEQ ID NOs: 5 and 6)
(C) Promoter DNA group expressed in the whole seed (the base sequence of each DNA is described in SEQ ID NO: 7)

  The endosperm-specific promoter DNA group of (A) includes the rice glutelin gene GluB-1 promoter (SEQ ID NO: 1), the rice glutelin gene GluB-4 promoter (SEQ ID NO: 2), and the 10 kDa prolamin promoter (SEQ ID NO: 3). ), 16 kDa prolamin promoter (SEQ ID NO: 4). The expression sites of this group are already found on the 7th day after flowering in the aleurone and subaleurone tissues, and spread to the endosperm site during the ripening process. This expression pattern does not change throughout the ripening process.

  The promoters listed as the embryo or aleurone tissue-specific promoter DNA group in (B) are the rice embryoglobulin gene promoter (SEQ ID NO: 5) and the rice oleosin promoter (SEQ ID NO: 6). In this group, expression is observed in the aleurone tissue at the early stage of ripening (7th day), and the expression spreads to the aleurone tissue and embryo during the ripening process, but no expression is observed in the endosperm site.

  The promoter mentioned as the promoter expressed in the whole seed of (C) is the promoter of the rice ADP glucose pyrophosphate enzyme gene (SEQ ID NO: 7). This promoter is first expressed in the embryo at the early stage of ripening and is expressed in the whole seed during the ripening process (highest expression in the embryo even in the late ripening stage)

  Preparation of these seed-specific promoter DNAs (hereinafter abbreviated as “DNA of the present invention”) of (A) to (C) can be carried out by those skilled in the art using conventional means. For example, a suitable primer pair (for example, SEQ ID NOs: 9 to 22) is designed from the nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 7, and PCR is performed using rice genomic DNA as a template. Can be prepared by screening a genomic library. Furthermore, if a commercially available DNA synthesizer is used, the target DNA can be prepared by synthesis.

  The DNA of the present invention can also be used for obtaining (isolating) DNA having promoter activity. In this DNA isolation, first, the DNA of the present invention or a part thereof is used as a probe, or an oligonucleotide that specifically hybridizes to the DNA of the present invention is used as a primer, and high homology with the above DNA from a desired organism. Can be isolated. General hybridization techniques (Southern, EM., J Mol Biol, 1975, 98, 503.) or PCR techniques (Saiki, RK. Et al., Science, 1985, 230, 1350., Saiki, RK. Et. al., Science, 1988, 239, 487.), which is hybridizable to DNA consisting of the nucleotide sequence of any one of SEQ ID NOs: 1 to 7 is also included in the DNA of the present invention. . Specifically, the DNA consisting of the base sequence described in any one of SEQ ID NOs: 1 to 7 or a part thereof as a probe, and specifically to the DNA consisting of the base sequence described in any one of SEQ ID NOs: 1 to 7 Isolation of DNA having high homology with the DNA consisting of the nucleotide sequence of any one of SEQ ID NOs: 1 to 7 from a desired organism using a hybridizing oligonucleotide as a primer can be usually performed by those skilled in the art. That is. In order to isolate such DNA, the hybridization reaction is preferably carried out under stringent conditions. As stringent hybridization conditions in the present invention, hybridization conditions of 6M urea, 0.4% SDS, 0.5 × SSC, or 0.1% SDS (60 ° C., 0.3 mol NaCl, 0.03M sodium citrate), or these Refers to hybridization conditions of the same stringency. It is expected that DNA with higher homology can be isolated under conditions of higher stringency, for example, conditions of 6M urea, 0.4% SDS, 0.1 × SSC. High homology refers to sequence identity that is preferably 50% or more, more preferably 70% or more, and most preferably 90% or more (for example, 95, 96, 97, 98, 99%) over the entire base sequence. .

  The identity of the nucleotide sequence is determined by the algorithm BLAST (Proc. Natl. Acad. Sci. USA, 1990, 87, 2264-2268., Karlin, S. & Altschul, SF., Proc NatlAcad Sci USA, 90 , 5873.). A program called BLASTN based on the BLAST algorithm has been developed (Altschul, SF. Et al., J Mol Biol, 1990, 215, 403.). When analyzing a base sequence using BLASTN, parameters are set to, for example, score = 100 and wordlength = 12. When using BLAST and Gapped BLAST programs, the default parameters of each program are used. Specific methods of these analysis methods are known (https://www.ncbi.nlm.nih.gov/).

  The DNA of the present invention is usually derived from a plant, preferably from a monocotyledonous plant, more preferably from a grass family, but the origin thereof is not particularly limited as long as it has seed-specific promoter activity.

  The present invention also provides DNA that is structurally similar to the above DNA and has promoter activity. Examples of such DNA include a base sequence in which one or more bases are substituted, deleted, added, and / or inserted in the base sequence described in any one of SEQ ID NOs: 1 to 7, and seeds Mention may be made of DNA having specific promoter activity. Such DNA can also be used for isolation of DNA having promoter activity of the present invention. Methods well known to those skilled in the art for preparing such DNA include, for example, the above-described hybridization technique, polymerase chain reaction (PCR) technique, and the like. Furthermore, the above DNA is obtained by, for example, site-directed mutagenesis method (Kramer, W. & Fritz, HJ., Methods Enzymol, 1987, 154) , 350.) can also be prepared by introducing mutations.

  Whether or not the DNA prepared as described above has promoter activity can be examined by those skilled in the art by a well-known reporter assay using a reporter gene. The reporter gene is not particularly limited as long as its expression can be detected. For example, a CAT gene, a lacZ gene, a luciferase gene, a β-glucuronidase (hereinafter, GUS) gene generally used by those skilled in the art. And the GFP gene.

  The expression level of the reporter gene can be measured by methods known to those skilled in the art depending on the type of the reporter gene. For example, when the reporter gene is a CAT gene, the expression level of the reporter gene can be measured by detecting acetylation of chloramphenicol by the gene product. When the reporter gene is a lacZ gene, by detecting the color development of the dye compound catalyzed by the gene expression product, and when the reporter gene is a luciferase gene, the fluorescent compound catalyzed by the gene expression product By detecting fluorescence, and in the case of the GUS gene, Glucuron (ICN) luminescence or 5-bromo-4-chloro-3-indolyl-β-glucuronide (X In the case of a GFP gene, the expression level of the reporter gene can be measured by detecting the fluorescence of GFP protein.

  When a gene other than the above is used as a reporter, the expression level of the gene can be measured by methods known to those skilled in the art. For example, the level of transcription of the gene can be measured by extracting the mRNA of the gene according to a standard method and performing Northern hybridization or RT-PCR using the mRNA as a template. Furthermore, it is also possible to measure the transcription level of the gene using DNA array technology. In addition, a fraction containing a protein encoded by the gene may be collected according to a standard method, and the expression level of the protein of the present invention may be detected by electrophoresis such as SDS-PAGE to measure the translation level of the gene. 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 detection of the protein encoded by the gene is not particularly limited as long as it is a detectable antibody. For example, both a monoclonal antibody and a polyclonal antibody can be used. The antibody can be prepared by methods known to those skilled in the art.

  The present invention also provides DNA in which any gene is functionally linked downstream of the promoter DNA. The DNA of the present invention can seed-specifically express a desired protein or peptide encoded by the arbitrary gene through activation of the promoter DNA.

  In the present invention, “functionally linked” means that the DNA of the present invention and the gene can be expressed so that expression of a downstream gene is induced by binding of a transcription factor to the DNA having the promoter activity of the present invention. And are connected. Therefore, even when the gene is bound to another gene and forms a fusion protein with another gene product, the expression of the fusion protein is caused by binding of the transcription factor to the DNA of the present invention. Anything that is induced is included in the meaning of “functionally linked”.

  The present invention provides a vector comprising the above promoter DNA or DNA to which an arbitrary gene is operably linked downstream (hereinafter, abbreviated as the above DNA). The vector of the present invention is useful for retaining the DNA in a host cell or transforming a plant to express a desired protein or the like.

  The vector for inserting the DNA is not particularly limited as long as the inserted gene can be expressed in plant cells. For example, using a vector having a promoter (eg, cauliflower mosaic virus 35S promoter) for constitutive gene expression in plant cells or a vector having a promoter that is inducibly activated by external stimuli Is also possible. The vector into which the DNA is inserted may be introduced into plant cells by methods known to those skilled in the art, such as polyethylene glycol method, electroporation (electroporation), Agrobacterium-mediated method, particle gun method, etc. Can do. In the method using Agrobacterium, for example, according to the method of Nagel et al. (Microbiol. Lett., 67: 325 (1990)), the expression vector into which the DNA is inserted is introduced into Agrobacterium, and this Agrobacterium is introduced. The above DNA can be introduced into plant cells by infecting plant cells with a direct infection method or leaf disk method.

  The present invention also provides a transformed plant cell into which the above DNA or vector has been introduced. The transformed plant cell in the present invention may be in any form as long as it is a plant cell or a collection of cells introduced with the DNA or vector and can regenerate the plant body. For example, suspension culture cells, protoplasts, leaf sections, callus and the like are included in the plant cells of the present invention.

  For introduction of a vector into a plant cell, various methods known to those skilled in the art such as polyethylene glycol method, electroporation (electroporation), Agrobacterium-mediated method, and particle gun method can be used.

  Furthermore, this invention provides the transformed plant body which hold | maintained the cell mentioned above. The plant of the present invention can be used as a production system for a desired gene product.

  Regeneration of plant bodies from transformed plant cells can be performed by methods known to those skilled in the art depending on the type of plant. For rice, the method of Fujimura et al. (Plant Tissue Culture Lett. 2:74 (1995)) can be mentioned, and for corn, the method of Shillito et al. (Bio / Technology 7: 581 (1989)) or Gorden-Kamm et al. ( Plant Cell 2: 603 (1990)), for potato, Visser et al. (Theor. Appl. Genet 78: 594 (1989)), for Arabidopsis, Akama et al. (Plant Cell Reports 12). : 7-11 (1992)), and for Eucalyptus, the method of Toi et al. (JP-A-8-89113) can be used.

  In addition, the present invention provides not only a plant body that holds cells into which the DNA has been introduced, but also a propagation material thereof. Once transformed plants with the above DNA or vector introduced into the genome are obtained, breeding materials (eg, seeds, fruits, cuttings, tubers, tuberous roots, strains, callus, protoplasts, etc.) are obtained, It is also possible to mass-produce the plant based on the above. In particular, the seed is a propagation material, and at the same time a site where foreign gene products are highly accumulated by the introduced promoter.

  Furthermore, the present invention provides a method for expressing any gene in the seed of a plant cell. The method of the present invention comprises a step of introducing a DNA in which an arbitrary gene is functionally linked downstream of the promoter DNA of the present invention or the vector of the present invention into a plant cell, and regenerating a plant from the plant cell. Process. The introduction step into the plant cell and the regeneration step of the plant body can be performed by the above-described methods. The method of the present invention can be used for the purpose of obtaining a desired gene product from a plant or for the purpose of obtaining seeds in which the desired gene product has been accumulated. Since the plant body regenerated using the desired gene by the method of the present invention bears seeds accumulating the desired gene product, the desired gene product can be obtained by purifying the seed. In addition, when the desired product is a pharmaceutical or the like, the seed can be ingested as it is, so that the purification step of the gene product can be omitted and the seed can be used as a final form.

  Among the promoters isolated by the present inventors, an interesting finding was obtained for the 10 kDa rice prolamin promoter. In the process of studying seed-specific promoter activity, when a Nos terminator is bound downstream of the 10 kDa prolamin promoter, expression is observed not only in the seed but also in the phloem of the root, stem, etc. When the 3 ′ untranslated region (0.3 kb; SEQ ID NO: 8) was bound, it was revealed that expression other than seeds was suppressed. That is, the present inventors suppress the expression of the 3′-untranslated region sequence of the rice 10 kDa prolamin promoter at sites other than endosperm, and the sequence of the 3′-side untranslated region is required for endosperm-specific expression. I found out. Previously, the present inventors have been able to highly express foreign gene products in seeds by inserting a 5 ′ untranslated region between a promoter that ensures expression in seeds and a foreign gene. Although it has been clarified (Japanese Patent Laid-Open No. 2002-58492), the seed storage protein gene is the first to reveal that not only the 5 ′ side but also the 3 ′ side sequence is necessary for seed-specific expression. .

  The 3 'untranslated region should be used to bind to the downstream of a promoter that is active in other tissues in addition to endosperm, thereby suppressing expression outside the endosperm and enabling endosperm-specific expression. Can be considered.

  Therefore, the present invention provides (1) DNA of 3 ′ untranslated region described in SEQ ID NO: 8, (2) vector containing the 3 ′ untranslated region, (3) arbitrary promoter and the 3 ′ untranslated region (4) a vector containing any promoter, any gene, and the 3 ′ untranslated region, (5) a cell and a transformed plant carrying the vector of (4), and (6) the transformation The present invention also provides a plant propagation material and (7) a method for inducing endosperm-specific expression using the 3 ′ untranslated region.

EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not restrict | limited to these Examples.
[Example 1] Characterizing the expression patterns and promoter activities of multiple genes expressed in seeds, rather than investigating the construction of promoter-GUS chimeric genes and presumed isolation regulators of transgenic plants did. Fifteen promoters ranging in size from 0.8 to 2.4 kb were isolated by PCR using genomic DNA or genomic clones as templates.

  The size of the gene and the corresponding promoter is as follows: rice 10 kDa prolamin, 0.8 kb; rice 13 kDa prolamin (PG5a), 0.9 kb; rice 16 kDa prolamin, 0.9 kb; rice glutelin GluB-4, 1.4 kb; rice embryo globulin (REG2), 1.3 kb; rice 18 kDa oleosin (Ole18), 1.3 kb; rice glutamate synthase gene (GOGAT), 0.8 kb; rice pyruvate, normal phosphate dikinase (PPDK), 0.8 kb; rice ADP-glucose pyrophosphorylase ( AGPase), 2.0 kb; rice starch branching enzyme (SBE1), 2.0 kb; and soybean β-conglycinin, 1.0 kb. Rice glutelin GluB-1: 1.3 kb, 2.3 kb, rice glutelin GluB-2; 2.4 kb and rice alanine aminotransferase (AlaAT); 1 kb, rice 26 kDa globulin (Glb-1); 1.0 kb, maize ubiquitin promoter: 2 kb.

  Among these, 2.3 kb GluB-1, GluB-4, 10 kDa prolamin, 16 kDa prolamin, rice embryo globulin, rice oleosin, rice ADP glucose pyrophosphorylase promoter sequences are represented by SEQ ID NOs: 1 to 7, respectively. The sequence of the primer pair used to release was shown in SEQ ID NOs: 9 to 22).

  Various promoter fragments were inserted into a modified binary vector pGPTV-35S-HPT containing the hygromycin phosphotransferase (HPT) gene as a selection marker (FIG. 1). The modified vector was constructed from the pGPTV-HPT binary vector (Becker et al. 1992) using the Nos promoter as the HPT gene promoter instead of the 0.8 kb CaMV 35S promoter. The seed promoter to be studied was introduced upstream of the UdiA gene encoding β-glucuronidase (GUS) in a modified binary vector.

  Transgenic rice plants (Oryza sativa cv Kitaake) were produced by Agrobacterium-mediated transformation. The plasmid constructed as described above was introduced into Agrobacterium tumefaciens EHA105 strain by electroporation. Five week callus callus derived from mature rice seeds was treated for 3 days with transformed A. tumefaciens. Each of the infected calli was continuously cultured for 4 weeks in N6 selection medium and MS regeneration medium containing hygromycin. The regenerated seedlings were transferred to the greenhouse (Goto et al., Nature Biotech. 17, 282-286 (1999)).

  For each construct, over 20 independent transgenic plants were created. The presence of the desired promoter fusion was confirmed by PCR using genomic DNA isolated from leaves of an independent transgenic rice line, and positive lines were used for analysis of promoter properties.

Example 2 To identify the site of GUS reporter gene expression guided by the active seed storage protein promoter in the seed of the seed storage protein gene promoter , transgenic rice seeds were examined by histochemical staining. For histochemical analysis, mature seeds at the 17th day after flowering (DAF) were sectioned longitudinally with a razor blade and 0.5 mM X-Gluc (5-bromo-4-chloro) -3-Indoylglucuronide) and 20% methanol in 50 mM sodium phosphate buffer (pH 7.0) at 37 ° C. The optimal incubation time for the staining reaction varied from 30 minutes to overnight depending on the extent of GUS activity.

  The detected expression pattern is shown in FIG. Rice glutelin promoters (1.3 kb and 2.3 kb GluB-1, GluB-2 and GluB-4, FIGS. 2-a-d) and prolamin promoters (10 kDa, 13 kDa and 16 kDa, FIGS. 2-e-g) in endosperm Guided the expression of GUS gene. GUS expression from the glutelin and prolamin promoters was observed in aleurone and suballeurone but not in embryos. Further examination of the maturing seeds of transgenic rice containing the glutelin and prolamin promoters revealed that GUS activity was highest in the outer part of the endosperm and weak inside. The GluB-1 promoter (both 1.3 kb and 2.3 kb) showed much higher activity in the endosperm region near the embryo. GUS expression guided by the 13kDa prolamin promoter (PG5a) was strictly restricted to the outer part of the endosperm. The 26 kDa globulin Glb-1 promoter led to GUS expression in internal starchy endosperm tissue (FIG. 2-h). GUS expression produced by the embryo storage protein promoter (REG2, Ole18 and β-conglycinin, Fig. 2-i-k) was confined to the embryo and aleurone, and no expression was seen in the endosperm. Little difference was observed between the expression patterns of GUS mediated by these embryo storage protein promoters. Interestingly, despite the many reports on the expression difference between dicotyledonous and monocotyledonous plants (Chowdhury et al., Plant Cell Rep. 16, 277-281 (1997), Rathaous et al., Plant Mol. Biol. 23, 613-618 (1993)), β-conglycinin promoter derived from soybean, a dicotyledonous plant, maintained embryo-specific expression even in rice, a monocotyledonous plant. It is also noted that the GUS expression induced by the β-conglycinin promoter is extremely weak in rice, in contrast to the high expression in embryos and cotyledons when the same promoter was introduced into tobacco, a dicotyledonous plant. there were.

  Overall, no GUS activity was detected in any of the leaves, leaf sheaths, stems and roots in transgenic rice with a fusion with the seed storage protein promoter (data not shown). The only exception is the 10 kDa prolamin promoter, which was expressed to some degree in vegetative organs (Figure 3). These results support the conclusion that endosperm storage protein genes (except for 10 kDa prolamin) are expressed in an endosperm-specific manner, and expression of embryo storage protein genes is restricted to the embryo and aleurone layers.

  Although the seed storage protein promoter showed specific expression in either endosperm or embryo, the non-storage protein promoter exhibited a different expression pattern (Figure 2). The GUS gene under the direction of the AlaAT promoter was expressed in the central part of starchy endosperm, and the activity was higher in the endosperm near the embryo (Fig. 2-l). The expression pattern of PPDK-GUS transgene was similar to that of endosperm storage protein (Fig. 2-o). The GUS gene directed by the AGPase promoter was expressed throughout the seed, including the pericarp, but was particularly high in internal starchy endosperm and embryos (Fig. 2-n). In contrast, the GOGAT promoter and the SBE promoter led to the expression of the GUS gene mainly in the scutellum (embryo-endosperm boundary) (FIGS. 2-m and 2-p).

[Example 3] GUS expression pattern in vegetative organs Most promoters examined showed endosperm-specific or embryo-specific expression of the GUS gene. However, GUS activity is observed in the vegetative tissues of transgenic rice with 10 kDa prolamin, PPDK and AGPase promoters (Fig. 3), as well as the AlaAT promoter (Kikuchi et al., Plant Mol. Biol. 39, 149-159 (1999)). It was detected among them. In these transgenic rice, GUS activity was detected not only in the endosperm or whole seed, but also in the phloem of the vascular bundle in leaves, leaf sheaths and stems (FIGS. 3-ac). In these transgenic rice plants, GUS activity was also detected in the root endothelium. However, the expression pattern produced by the AGPase promoter was somewhat different from that of the PPDK and 10 kDa prolamin promoters, especially the former promoter caused high expression of GUS in the apical meristem, whereas the latter two The promoter resulted in uniform staining of root tissue. Furthermore, GUS activity in the roots was also different, and the AGPase promoter was stronger than the PPDK promoter and the 10 kDa prolamin promoter.

The native 10 kDa prolamin gene is normally expressed in mature endosperm and is not detected in vegetative tissue. The observed ectopic expression of the GUS fusion could be restored to normal endosperm-specific expression by replacing the Nos terminator with a 0.3 kb region downstream of the stop codon of the native 10 kDa prolamin gene ( Unpresented data). Note that this substitution of the 3 'transcription termination region had little effect on promoter activity. These results indicate that the endosperm-specific expression of the 10 kDa prolamin gene requires both 5 'and 3' flanking regions.
The primer pairs used for isolation of the 3 ′ transcription termination region are shown in SEQ ID NOs: 23 and 24.

[Example 4] The distribution of transgene expression in seeds in the process of generating promoter activity during seed development was examined by tissue staining of longitudinal sections of seeds collected in 7, 12 and 17 DAF. Specifically, mature seeds at 7th, 12th, and 17th days after flowering (DAF) were sectioned with a razor blade in the longitudinal direction, and 0.5 mM X-Gluc (5-bromo-4- Chloro-3-indoyl glucuronide) and 20% methanol in 50 mM sodium phosphate buffer (pH 7.0) at 37 ° C. The optimal incubation time for the staining reaction varied from 30 minutes to overnight depending on the extent of GUS activity.

  The expression pattern in the seed maturation process was examined in all transgenic lines, and the results of one representative line with respect to various offspring promoters are shown in FIG. 4 and FIG. Interestingly, the site where GUS expression was first detected varied from construct to construct. In the case of the glutelin and prolamin promoters, blue GUS staining was first observed in the peripheral region of the endosperm, ie, aleurone and subaleurone. For the glutelin and 16kDa prolamin promoters, staining with seed maturation (17 DAF) subsequently spread to the internal starchy endosperm, but this was not seen with the 10kDa and 13kDa prolamin promoters (Figure 4-a). ~ G). This expression pattern was in stark contrast to the 26 kDa Glb-1 promoter, where blue GUS staining was first observed in internal starch endosperm cells near the embryo and the expression pattern did not change during the seed development process (Fig. 4-h).

  Expression of the GUS gene guided by the promoters of REG2, Ole18 and β-conglycinin genes was observed by 7 DAF. This activity first occurred in the aleurone layer and then tended to be expressed in the embryo. Expression of these promoters was restricted to aleurone and embryos (Figs. 4-i, j and Fig. 5-k).

  The temporal patterns during seed maturation of representative transgenic lines of non-storage protein promoters are shown in FIGS. GUS expression from the AlaAT promoter first appeared in internal starchy endosperm tissue and eventually spread throughout the endosperm, but the embryo remained unstained (FIG. 5-l). The GUS activity produced by the SBE1 promoter was also limited to internal starchy endosperm tissues, especially tissues near the embryo (Fig. 5-p). However, due to the very low GUS activity, blue staining could not be detected up to 12 DAF. In contrast, when the AGPase gene promoter fusion was introduced, GUS staining started from the embryo and then spread to the central part of the endosperm. Blue GUS staining was eventually observed throughout the seed tissue toward maturation, with strong staining in the embryo (FIG. 5-n). This expression profile during seed development was very similar to that observed with the ubiquitin promoter (Figure 5-q). In contrast, the PPDK promoter showed an expression pattern similar to that of the glutelin promoter and the prolamin gene promoter (FIG. 5-o). The GUS activity induced by the GOGAT promoter was confined to the scutellum and there was little change in the seed development process except that GUS activity was not detected at 7 DAF (FIG. 5-m).

Example 5: Quantitative analysis of promoter strength To assess the strength of various promoters, a fluorometric assay for GUS activity was performed according to Jefferson (1987). 17 DAF maturing seeds were homogenized in GUS extraction buffer (50 mM NaPO ₄ , pH 7.0, 10 mM 2-mercaptoethanol, 10 mM Na ₂ -EDTA, 0.1% SDS, 0.1% Triton X-100). After centrifugation, 10 μl of the supernatant was mixed with 90 μl of assay buffer containing 1 mM 4-methylumbelliferyl β-D-glucuronide (MUG). After 1 hour incubation at 37 ° C., the reaction was stopped by adding 900 μl of 0.2M Na ₂ CO ₃ . The fluorometer value was compared with the 4-methylumbelliferone (4MU) dilution series. The protein content was evaluated using the Bio-Rad Protein Assay kit and serum albumin as a standard substance. Three seeds from each transgenic plant were assayed.

  As shown in FIG. 6, there was a large difference in promoter activity. The seed promoters analyzed this time can be classified into four groups based on promoter strength. The following four groups belong to the high GUS activity group: GluB-4, 10 kDa prolamin, 16 kDa prolamin and Glb-1. The average GUS activities of these promoters were 44.8 ± 16.5, 38.8 ± 10.8, 27.1 ± 12.7 and 28.6 ± 11.8 pmol 4 MU / min / μg protein, respectively. The moderate GUS activity group includes the following two: 2.3 kb GluB-1 and AGPase gene promoters. Their GUS activity is lower than that observed in the high activity group, but much higher than others. The average GUS activity of the 2.3 kb GluB-1 promoter and AGPase promoter was 21.3 ± 7.0 and 10 ± 4.7 pmol 4MU / min / μg protein, respectively. Seven promoters, namely 1.3 kb GluB-1, GluB-2, 13 kD prolamin, REG-2, Ole18, AlaAT and PPDK, were provisionally classified into relatively low GUS activity groups. The average GUS activities of these promoters were 2.1 ± 1.2, 5.5 ± 2.2, 7.4 ± 5.5, 2.4 ± 1.2, 2 ± 4.6, 5.9 ± 4.0 and 4.0 ± 3.0 pmol 4 MU / min / μg protein, respectively. The remaining three promoters, GOGAT, SBE1, and β-conglycinin gene promoters were classified into low GUS activity groups. The GUS expression guided by these promoters was negligible and the activity was less than 1 pmol 4 MU / min / μg protein. The GUS activity from the ubiquitin promoter used as a control averaged 7.4 ± 8.5 pmol 4 MU / min / μg protein (in mature seeds). Although the ubiquitin promoter is used as a general-purpose promoter for many purposes, this level was comparable to that obtained from a promoter of a relatively low GUS activity group.

  For comparison, the activities of PPDK promoter and AGPase promoter in vegetative tissues were also examined. Mean GUS activity in leaves, stems and sheaths is 8.7 ± 6.8, 3.7 ± 3.6 and 16.3 ± 13.9 pmol 4 MU / min / μg protein for PPDK, respectively, and 12.5 ± 5.0, 40.2 ± 28.5 and 23.2 for AGPase, respectively. The protein was ± 16.6 pmol 4 MU / min / μg protein. These promoter activities were similar to or higher than those obtained from mature seeds. In contrast, for the 10 kDa prolamin promoter, although expression in vegetative tissue was observed, GUS activity (leaf 3.1 ± 1.1; stem 6.0 ± 2.9; sheath 2.3 ± 1.0 pmol 4 MU / min / μg protein) is still mature It was much lower than that observed in seeds. PPDK, AGPase and 10 kDa prolamin genes were constitutively expressed, but expression levels in different tissues varied from gene to gene.

It is the schematic regarding the construction of a chimeric gene for rice transformation. The 5 'flanking regions of various rice seed storage protein and non-storage protein genes were fused between two of the three restriction sites, Hind III, Sal I and Sma I, followed by the GUS reporter gene and Nos terminator. Fused. The promoters are 1.3 kb GluB-1, 2.3 kb GluB-1, GluB-2, GluB-4, 10 kDa prolamin, 13 kDa prolamin (PG5), 16 kDa prolamin, 26 kDa Glb-1, REG2, Ole18, soybean β-conglycinin, AlaAT , GOGAT, PPDK, AGPase and SBE1 genes. FIG. 3 is a photograph showing the results of histochemical analysis of GUS expression guided by gene promoters of various seed storage proteins and non-storage proteins. GUS protein was detected by incubating a manual longitudinal section of the transgenic seed in X-Gluc solution. a, 1.3 kb GluB-1 promoter; b, 2.3 kb GluB-1 promoter; c, GluB-2 promoter; d, GluB-4 promoter; e, 10 kDa prolamin promoter; f, 13 kDa prolamin (PG5a) promoter; g, 16 kDa H, 26 kDa Glb-1 promoter; i, REG2 promoter; j, Ole18 promoter; k, β-conglycinin promoter; l, AlaAT promoter; m, GOGAT promoter; n, AGPase promoter; o, PPDK promoter; p , SBE1 promoter. This is a photograph showing the results of histochemical GUS assay in vegetative tissues. lf, leaf; ls, leaf sheath; sk, stem; rt, root; ed, eudodersis, a, 10 kDa prolamin promoter; b, PPDK promoter; c, AGPP promoter. It is a photograph showing the time course of development of GUS activity induced by the seed promoter during the seed maturation process. X-Gluc histochemical staining of longitudinal sections of 7, 12 and 17 DAF transgenic rice seeds. a, 1.3 kb GluB-1 promoter; b, 2.3 kb GluB-1 promoter; c, GluB-2 promoter; d, GluB-4 promoter; e, 10 kDa prolamin promoter; f, 13 kD prolamin promoter (RG5a); g, 16 kDa Prolamin promoter; h, 26 kDa Glb-1 promoter; i, REG2 promoter; j, Ole18 promoter. It is a photograph which shows the continuation of FIG. k, β-conglycinin promoter; l, AlaAT promoter; m, GOGAT promoter; n, AGPase promoter; o, PPDK promoter; p, SBE1 promoter; q, ubiquitin promoter. 17 shows the results of GUS activity expressed from various promoters in mature seeds of DAF. FIG. GUS activity was expressed in pmol 4 MU / min / μg protein unit.

Claims (9)

The DNA having promoter activity in seeds according to any one of (a) to (c) below.
(A) DNA comprising the base sequence described in any one of SEQ ID NO: 1 to SEQ ID NO: 7
(B) DNA comprising a sequence in which one or more bases are added, deleted, substituted or inserted into the base sequence described in any one of SEQ ID NO: 1 to SEQ ID NO: 7.
(C) DNA that hybridizes under stringent conditions with DNA comprising the sequence described in any one of SEQ ID NO: 1 to SEQ ID NO: 7. A DNA in which an arbitrary gene is operably linked downstream of the DNA of claim 1. A vector comprising the DNA according to claim 1 or 2. A transformed plant cell retaining the DNA according to claim 2. A transformed plant cell into which the vector according to claim 3 has been introduced. A transformed plant body, which holds the cell according to claim 4 or 5. The propagation material of the plant body described in Claim 6. The propagation material according to claim 7, which is a seed. A method of expressing an arbitrary gene in plant cell seeds comprising the following steps (a) and (b): (a) introducing the DNA described in claim 2 or the vector described in claim 3 into a plant cell; (B) regenerating the plant body from the plant cell.