JP5067688B2 - Method for inhibiting protein disulfide bond formation in plants - Google Patents

Description

  The present invention relates to a method for comprehensively inhibiting disulfide bond formation of an intracellular protein, characterized by suppressing the expression of an endogenous Ero1 gene in a plant cell.

  The nascent polypeptide chain is converted into a functional molecule with the correct folding structure in the lumen of the endoplasmic reticulum (hereinafter sometimes abbreviated as ER), but disulfide bonds are soluble protein protein disulfide isomerase (protein disulfide isomerase) formed by isomerase (PDI). PDI is the first thiol-disulfide oxidoreductase identified and has two thioredoxin-like domains containing a CxxC (C: cysteine residue) (SEQ ID NO: 10) active site, each of which forms disulfide bonds in the substrate protein and Catalyze isomerization (Non-Patent Documents 1 to 4). Regarding the molecular mechanism of disulfide bond formation, Ero1 (ER oxidoreductase) was recently identified as an enzyme protein responsible for supplying oxidative power to PDI by genetic and biochemical analysis of yeast (Saccharomyces cerevisiae) (non-patent literature) 5-7). Ero1 is a membrane protein localized in the ER lumen (Non-patent Documents 5 and 6) and retains flavin adenine dinucleotide (FAD) as a coenzyme (Non-patent Documents 8 and 9). Ero1 has two cysteine pairs (CxxxxC (SEQ ID NO: 11) and CxxCxxC (SEQ ID NO: 12) motifs) as active sites, and PDI by an thiol-disulfide exchange reaction between Ero1 (SS) and PDI (-SH) proteins. Introduce a disulfide bond.

  The Ero1 gene has only one gene in yeast (Non-patent Document 5) and two genes in human (Non-patent Documents 10 and 11). In yeast and humans, Ero1 supplies oxidative power to PDI to control disulfide bond formation in the nascent polypeptide chain, and PDI has been identified as a wide variety of homologues, each serving as a substrate It has been suggested that it exhibits specificity for nascent polypeptide chains (Non-Patent Documents 12 to 16).

On the other hand, in higher plants, two Ero1 genes (Non-patent Document 17) have been identified in Arabidopsis thaliana, but it is unclear whether the protein disulfide bond formation process is dependent on Ero1 protein in seed endosperm.
However, it has been reported that one seed storage protein, glutelin, accumulates as a precursor in rice (Oryza sativa) PDI1-1 gene deletion mutant esp2 (Non-patent Document 18). Glutelin is first synthesized as a precursor molecule (proglutelin: molecular weight of about 57 kD) in the ER, and after disulfide bonds are formed, it is finally transported to the storage vacuole and has an acidic subunit (molecular weight 37-39 kD) and Processing to the basic subunit (molecular weight 22-23 kD) suggests that PDI is essential for protein disulfide bond formation in rice seed endosperm cells. The PDI gene forms a multigene family, and so far, 19 orthologs have been identified in rice, 22 in maize (Zea mays), and 22 in Arabidopsis thaliana (Non-patent Document 19).

Food allergens are causative substances such as anaphylactic shock and atopic dermatitis. The main food allergen is a protein having a plurality of cysteine residues, forms a disulfide bond, takes a stable α-helix structure, and becomes a conformational epitope for an IgE antibody. Storage protein is one of the major plant food allergens. Development of plants with low allergenicity is required in the edible part.
Add water to the flour and knead to make dough. The characteristics of flour dough depend on the formation of gluten in protein complexes by forming disulfide bonds when the reduced thiol groups of cysteine residues are kneaded (Non-patent Documents 20 and 21). Wheat flour is classified into high-powered flour, medium-powered flour, and weak-powered flour from those with high protein content. Since strong flour contains a large amount of reduced thiol groups contained in protein, it is thought that when it is made into dough, it forms many disulfide bonds and increases gluten-forming ability. On the other hand, the reason why gluten is not formed even when dough is made by adding water to rice flour is thought to be due to the fact that the protein contained in rice flour has few reduced thiol groups. Therefore, when using rice flour as a substitute for wheat flour, it is considered desirable to use rice flour containing a large amount of reduced thiol groups, but no rice flour having such properties has been reported so far. Generally, when using rice flour as a substitute for wheat flour, it is necessary to add flour-derived powdered gluten to the rice flour (Patent Document 1).

Prior art document information related to the invention of the present application is shown below.
Patent 3075552 Goldberger, RF, et al., (1963) J. Biol. Chem. 238, 628-635 Givol, D., et al., (1965) Proc. Natl. Acad. Sci. USA 53, 676-684 Edman, JC, et al., (1985) Nature 317, 267-270 Kulp, MS, et al., (2006) J. Biol. Chem. 281, 876-884 Frand, AR, and Kaiser, CA (1998) Mol. Cell 1, 161-170 Pollard, MG, et al., (1998) Mol. Cell 1, 171-182 Frand, AR, and Kaiser, CA (1999) Mol. Cell 4, 469-477 Tu, BP, et al., (2000) Science 290, 1571-1574 Gross, E., et al., (2004) Cell 117, 601-610 Cabibbo, A., et al., (2000) J. Biol. Chem. 275, 4827-4833 Pagani, M., et al., (2000) J. Biol. Chem. 275, 23685-23692 Gunther, R., et al., (1993) J. Biol. Chem. 268, 7728-7732 Koivunen, P., et al., (1996) Biochem. J. 316, 599-605 Oliver, JD, et al., (1997) Science 275, 86-88 Molinari, M., and Helenius, A. (1999) Nature 402, 90-93 Norgaard, P., et al., (2001) J. Cell Biol. 152, 553-562 Dixon, DP, et al., (2003) Antioxid. Redox Signal. 5, 389-396 Takemoto, Y., et al., (2002). Plant Physiol. 128, 1212-1222 Houston, NL, et al., (2005) Plant Physiol. 137, 762-778 Shewry, PR, and Tatham, AS (1997) J. Cereal Sci. 25, 207-227 Shewry, PR, and Halford, NG (2002) J. Exp. Bot. 53, 947-958

  The present invention has been made in view of such circumstances, and its object is to suppress the expression of an endogenous Ero1 gene in a plant cell, and to form a disulfide bond in the intracellular protein. It is providing the method of inhibiting this comprehensively. Another object of the present invention is to provide a method for inhibiting the oxidation activity of protein disulfide isomerase, which comprises suppressing endogenous Ero1 gene expression in plant cells. Another object of the present invention is to provide a DNA having a function of suppressing the expression of the Ero1 gene.

  In order to solve the above-mentioned problems, the present inventors suppressed the enzyme activity of Ero1 protein in the substrate protein-PDI-Ero1 electron transport cascade as a method of comprehensively inhibiting protein disulfide bond formation in plant seed endosperm. We examined whether it was effective to do.

First, a transformant in which rice Ero1 gene expression was specifically suppressed by endosperm by RNA interference was prepared. As a result, in this Ero1 RNA interference transformant seed, glutelin was excessively accumulated as a precursor molecular species.
Furthermore, we investigated the effect of Ero1 gene expression suppression on α-globulin (Glb), which requires intramolecular disulfide bond formation for localization to vacuolar protein granules PBII. As a result, in the Ero1 RNA interference transformant, Glb was observed as a granule having a shape different from that of the wild type.

  That is, the present inventors have found that protein disulfide bond formation is inhibited by suppressing Ero1 gene expression specifically in seed endosperm in higher plants, and completed the present invention.

More specifically, the present invention provides the following [1] to [16].
[1] Comprehensive disulfide bond formation of intracellular proteins characterized by suppressing endogenous expression of DNA according to any one of (a) to (d) below in plant cells How to inhibit.
(A) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 2.
(B) DNA containing the coding region of the base sequence set forth in SEQ ID NO: 1.
(C) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence set forth in SEQ ID NO: 2.
(D) A DNA that hybridizes under stringent conditions with a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1.
[2] A method for inhibiting the oxidative activity of protein disulfide isomerase, which suppresses endogenous expression of DNA according to any one of (a) to (d) below in a plant cell.
(A) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 2.
(B) DNA containing the coding region of the base sequence set forth in SEQ ID NO: 1.
(C) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence set forth in SEQ ID NO: 2.
(D) A DNA that hybridizes under stringent conditions with a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1.
[3] The method according to [1] or [2], wherein the expression of the DNA is specifically suppressed in endosperm cells.
[4] The method according to any one of [1] to [3], wherein the plant is a monocotyledonous plant.
[5] The method according to any one of [1] to [4], including a step of introducing the DNA according to any one of (i) to (iv) below into a plant cell.
(I) [1] DNA encoding an antisense RNA complementary to the transcription product of the DNA according to any one of (a) to (d).
(Ii) [1] DNA encoding an RNA having a ribozyme activity that specifically cleaves the transcription product of the DNA according to any one of (a) to (d).
(Iii) [1] DNA encoding a dsRNA complementary to the transcription product of the DNA according to any one of (a) to (d).
(Iv) [1] DNA encoding an aptamer that specifically recognizes the protein encoded by the DNA according to any one of (a) to (d).
[6] The method according to [5], wherein DNA is introduced into plant cells by Agrobacterium.
[7] A plant in which the expression of the DNA according to any one of (1) (a) to (d) is suppressed by the method according to [1] to [6].
[8] The DNA according to any one of (i) to (iv) below.
(I) [1] DNA encoding an antisense RNA complementary to the transcription product of the DNA according to any one of (a) to (d).
(Ii) [1] DNA encoding an RNA having a ribozyme activity that specifically cleaves the transcription product of the DNA according to any one of (a) to (d).
(Iii) [1] DNA encoding a dsRNA complementary to the transcription product of the DNA according to any one of (a) to (d).
(Iv) [1] DNA encoding an aptamer that specifically recognizes the protein encoded by the DNA according to any one of (a) to (d).
[9] The DNA according to [8], which is used for comprehensively inhibiting protein disulfide bond formation in plant cells.
[10] The DNA according to [8], which is used to inhibit the oxidation activity of protein disulfide isomerase in plant cells.
[11] A vector comprising the DNA of [9] or [10].
[12] A transformed plant cell carrying the DNA of [8] or the vector of [11].
[13] A transformed plant comprising the transformed plant cell according to [12].
[14] A transformed plant that is a descendant or clone of the transformed plant according to [13].
[15] A propagation material for the transformed plant according to [13] or [14].
[16] A method for producing a transformed plant according to [13] or [14], wherein the DNA according to [8] or the vector according to [11] is introduced into a plant cell, A method comprising a step of regenerating a plant body.

The method of the present invention has made it possible to comprehensively inhibit the formation of disulfide bonds in the intracellular protein of plants.
The major food allergens include storage proteins, which are proteins having a plurality of cysteine residues and form a disulfide bond to form a stable α-helix structure and become a conformational epitope for an IgE antibody. By the method of the present invention, it is possible to comprehensively inhibit protein disulfide bond formation in the edible part and provide a plant with low allergenicity.
In addition, the endosperm of transformed rice produced by the method of the present invention inhibits Ero1-dependent protein disulfide bond formation, and as a result, it contains many reduced thiol groups. Therefore, in a dough produced by adding water to kneaded seeds and kneading, a disulfide bond is formed between thiol groups, and thus the viscosity, plasticity and elasticity of the dough increase. Rice flour having dough properties with increased viscosity, plasticity and elasticity can be utilized, for example, as a substitute for flour or mixed with flour.

[Embodiment of the Invention]
The present invention relates to a method for comprehensively inhibiting disulfide bond formation of an intracellular protein, characterized by suppressing the expression of an endogenous Ero1 gene in a plant cell. Furthermore, the present invention relates to a method for inhibiting the oxidative activity of protein disulfide isomerase (PDI) group, characterized by suppressing the expression of endogenous Ero1 gene in plant cells.

  The present inventors have clarified the relationship with protein disulfide bond formation and the nucleotide sequence of the Ero1 gene of rice, which has been shown to have a function of supplying oxidizing power to PDI in plants, SEQ ID NO: The amino acid sequence of the protein encoded by this gene is shown in SEQ ID NO: 2.

The Ero1 gene of the present invention specifically includes the DNA described in any one of the following (a) to (d), which encodes a protein that promotes the formation of a disulfide bond of the protein.
(A) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 2.
(B) DNA containing the coding region of the base sequence set forth in SEQ ID NO: 1.
(C) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence set forth in SEQ ID NO: 2.
(D) A DNA that hybridizes under stringent conditions with a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1.

  The Ero1 gene used in the present invention is not particularly limited as long as it can encode the Ero1 protein, and each Ero1 gene includes genomic DNA, chemically synthesized DNA, etc. in addition to cDNA. Moreover, as long as the Ero1 gene encodes the Ero1 protein, DNA having an arbitrary base sequence based on the degeneracy of the genetic code is included.

  Preparation of genomic DNA and cDNA can be performed by those skilled in the art using conventional means. For genomic DNA, for example, genomic DNA is extracted from a plant, a genomic library (vectors such as plasmids, phages, cosmids, BACs, PACs, etc. can be used), expanded, and Ero1 gene (for example, It is possible to prepare by performing colony hybridization or plaque hybridization using a probe prepared based on the DNA described in SEQ ID NO: 1. It is also possible to prepare by preparing a primer specific for the Ero1 gene and performing PCR using this primer. In addition, for example, cDNA is synthesized based on mRNA extracted from a plant, inserted into a vector such as λZAP to create a cDNA library, developed, and colony hybridization in the same manner as described above. Alternatively, it can be prepared by performing plaque hybridization or by performing PCR.

  Furthermore, since the Ero1 gene is considered to exist widely in the plant kingdom, the Ero1 gene includes homologous genes existing in various plants. Here, the “homologous gene” refers to a gene encoding a protein functionally equivalent to the Ero1 gene product in rice in various plants. Examples of such proteins include, but are not limited to, Ero1 protein mutants, alleles, variants, homologs, partial peptides of Ero1 protein, or fusion proteins with other proteins.

  The variant of Ero1 protein in the present invention is a naturally occurring protein consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 2. And a protein functionally equivalent to the protein consisting of the amino acid sequence set forth in SEQ ID NO: 2. A protein encoded by a naturally-occurring DNA that hybridizes under stringent conditions with a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1, wherein the protein comprises the amino acid sequence set forth in SEQ ID NO: 2 Functionally equivalent proteins can also be mentioned as mutants of Ero1 protein.

  In the present invention, the number of amino acids to be mutated is not particularly limited, but is usually within 30 amino acids, preferably within 15 amino acids, and more preferably within 5 amino acids (for example, within 3 amino acids). The amino acid residue to be mutated is preferably mutated to another amino acid in which the properties of the amino acid side chain are conserved. For example, amino acid side chain properties include hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), amino acids having aliphatic side chains (G, A, V, L, I, P), amino acids having hydroxyl group-containing side chains (S, T, Y), sulfur atom-containing side chains Amino acids having amino acids (C, M), amino acids having carboxylic acid and amide-containing side chains (D, N, E, Q), amino acids having base-containing side chains (R, K, H), aromatic-containing side chains The amino acids (H, F, Y, W) that can be used can be listed (the parentheses indicate the single letter of the amino acid). It is already known that a polypeptide having an amino acid sequence modified by deletion, addition and / or substitution by one or more amino acid residues to a certain amino acid sequence maintains its biological activity. Yes.

In the present invention, “functionally equivalent” means that the target protein has the same biological function or biochemical function as the Ero1 protein. In the present invention, examples of the biological function and biochemical function of the Ero1 protein include a function of introducing a disulfide bond into PDI and a function of promoting the formation of a disulfide bond of a protein. Biological properties include the specificity of the site to be expressed and the expression level.
Methods well known to those skilled in the art for isolating homologous genes include hybridization techniques (Southern, EM, Journal of Molecular Biology, Vol. 98, 503, 1975) and polymerase chain reaction (PCR) techniques (Saiki). , RK, et al. Science, vol. 230, 1350-1354, 1985, Saiki, RK et al. Science, vol. 239, 487-491, 1988). That is, for those skilled in the art, various nucleotide sequences of Ero1 gene (for example, DNA described in SEQ ID NO: 1) or a part thereof are used as probes, and oligonucleotides that specifically hybridize to Ero1 gene are used as primers. It is usually possible to isolate a homologous gene of Ero1 gene from a plant.

  In order to isolate DNA encoding such a homologous gene, a hybridization reaction is usually carried out under stringent conditions. Those skilled in the art can appropriately select stringent hybridization conditions. For example, in hybridization solution containing 25% formamide, 50% formamide under more severe conditions, 4 × SSC, 50 mM Hepes pH 7.0, 10 × Denhardt's solution, 20 μg / ml denatured salmon sperm DNA at 42 ° C. After overnight prehybridization, a labeled probe is added and hybridization is performed by incubating overnight at 42 ° C. The cleaning solution and temperature conditions for the subsequent cleaning are about 1xSSC, 0.1% SDS, 37 ° C, more severe conditions are about 0.5xSSC, 0.1% SDS, 42 ° C, and more severe conditions are about 0.2xSSC. , 0.1% SDS, about 65 ° C. ”. Thus, isolation of DNA having high homology with the probe sequence can be expected as the conditions for washing hybridization are more severe. However, combinations of the above SSC, SDS, and temperature conditions are exemplary, and those skilled in the art will understand the above or other factors that determine the stringency of hybridization (eg, probe concentration, probe length, hybridization reaction). It is possible to achieve the same stringency as above by appropriately combining the time and the like.

  The homology of the isolated DNA is at least 50% or more, more preferably 70% or more, more preferably 90% or more (for example, 95%, 96%, 97%, 98%, 99%) in the entire amino acid sequence. And the like). Sequence homology can be determined using BLASTN (nucleic acid level) and BLASTX (amino acid level) programs (Altschul et al. J. Mol. Biol., 215: 403-410, 1990). The program is based on the algorithm BLAST by Karlin and Altschul (Proc. Natl. Acad. Sci. USA, 87: 2264-2268, 1990, Proc. Natl. Acad. Sci. USA, 90: 5873-5877, 1993). Yes. When analyzing a base sequence by BLASTN, parameters are set to score = 100 and wordlength = 12, for example. Further, when the amino acid sequence is analyzed by BLASTX, the parameters are, for example, score = 50 and wordlength = 3. When the amino acid sequence is analyzed using the Gapped BLAST program, it can be performed as described in Altschul et al. (Nucleic Acids Res. 25: 3389-3402, 1997). When using BLAST and Gapped BLAST programs, the default parameters of each program are used. Specific methods of these analysis methods are known.

The present invention provides a method for comprehensively inhibiting disulfide bond formation of an intracellular protein, characterized by suppressing the expression of an endogenous Ero1 gene in a plant cell.
Furthermore, the present invention provides a method for inhibiting the oxidation activity of PDI, characterized by suppressing the expression of the endogenous Ero1 gene in plant cells.

The nascent polypeptide chain is converted into a functional molecule with the correct folding structure in the endoplasmic reticulum (ER) lumen (folding). Specifically, a linear peptide is folded by the interaction of amino acid residues (hydrogen bonding) to form a secondary structure such as an α helix structure or β sheet structure, which forms the three-dimensional structure of the protein. To do. Proteins exhibit their unique functions by being forged. The higher order structures are all affected by the primary structure. For example, hydrophobic amino acid residues form a hydrophobic bond and Cys form a disulfide bond to stabilize the higher order structure.
In a series of folding processes, disulfide bonds are formed by PDI, a soluble protein. PDI is a thiol-disulfide oxidoreductase that has two thioredoxin-like domains containing a CxxC (SEQ ID NO: 10) active site and catalyzes disulfide bond formation and isomerization, respectively.

In the present invention, “exclusive inhibition of disulfide bond formation in intracellular proteins” means that the substrate protein-PDI-Ero1 electron transfer cascade is suppressed by suppressing the expression of Ero1, an enzyme protein responsible for supplying oxidative power to PDI. And comprehensive inhibition of substrate protein disulfide bond formation. Accordingly, the present invention also provides a method of inhibiting the oxidative activity of PDI by inhibiting endogenous Ero1 expression.
Inhibition of disulfide bond formation according to the present invention is preferably carried out in the endoplasmic reticulum.
In the present invention, the disulfide bond of the protein to be inhibited may be a disulfide bond in a protein molecule or a disulfide bond between protein molecules.

In the present invention, the plant that suppresses the expression of the Ero1 gene is not particularly limited, and a desired plant that suppresses the formation of protein disulfide bonds in the plant can be used. However, crops are preferred from an industrial viewpoint. There are no particular restrictions on useful crops. And dicotyledonous plants such as soybean, cotton, tomato and potato.
The plant of the present invention is more preferably a monocotyledonous plant, most preferably rice.

  In the present specification, “suppression of Ero1 gene expression” includes suppression of gene transcription and suppression of protein translation. Moreover, not only complete cessation of expression of Ero1 gene but also decrease of expression is included.

  In the present invention, the part that suppresses the expression of the Ero1 gene may be the whole plant or a part of the plant body. When the expression of the Ero1 gene is completely stopped, it is preferably a part of the plant body. Examples of the plant body include, but are not limited to, seed endosperm, leaves, roots, and the like.

A plant in which formation of a disulfide bond of a substrate protein is inhibited by the method of the present invention is useful for prevention of diseases caused by ingestion of a substrate protein having a higher-order structure by the disulfide bond. Examples of such diseases include allergic diseases, more preferably anaphylactic shock, atopic dermatitis, allergic rhinitis, bronchial asthma and the like.
In addition, rice endosperm produced by the method of the present invention contains many reduced thiol groups. Therefore, in a dough produced by adding water to kneaded seeds and kneading, a disulfide bond is formed between thiol groups, and thus the viscosity, plasticity and elasticity of the dough increase. Examples of methods for using rice flour having dough characteristics with increased viscosity, plasticity and elasticity include, but are not limited to, use as a substitute for wheat flour, or use in combination with wheat flour.

  In the present invention, as a method for suppressing the expression of the Ero1 gene, introduction of RNA complementary to the transcription product of the Ero1 gene or DNA encoding a ribozyme that specifically cleaves the transcription product into a plant cell is possible. Can be mentioned.

One embodiment of the “RNA complementary to the transcript of the Ero1 gene” of the present invention is an antisense RNA complementary to the mRNA of the Ero1 gene.
There are several factors as described below for the action of an antisense nucleic acid to suppress the expression of a target gene. That is, transcription initiation inhibition by triplex formation, transcription inhibition by hybridization with a site where an open loop structure is locally created by RNA polymerase, transcription inhibition by hybridization with RNA that is undergoing synthesis, intron and exon Of splicing by hybrid formation at the junction with the protein, suppression of splicing by hybridization with the spliceosome formation site, suppression of transition from the nucleus to the cytoplasm by hybridization with mRNA, hybridization with capping sites and poly (A) addition sites Splicing suppression by formation, translation initiation suppression by hybridization with the translation initiation factor binding site, translation suppression by hybridization with the ribosome binding site near the initiation codon, peptization by hybridization with the mRNA translation region and polysome binding site For example, inhibition of gene chain elongation is prevented, and hybridization between nucleic acid and protein interaction sites is performed. They inhibit the expression of target genes by inhibiting transcription, splicing, or translation processes.

  The antisense sequence used in the present invention may suppress the expression of the target gene by any of the actions described above. In one embodiment, if an antisense sequence complementary to the untranslated region near the 5 ′ end of the mRNA of the Ero1 gene is designed, it is considered effective for inhibiting translation of the gene. However, sequences complementary to the coding region or the 3 ′ untranslated region can also be used. As described above, a DNA containing an antisense sequence of a non-translated region as well as a translated region of a gene is also included in the antisense DNA used in the present invention. The antisense DNA to be used is linked downstream of a suitable promoter, and preferably a sequence containing a transcription termination signal is linked on the 3 ′ side. The DNA thus prepared can be transformed into a desired plant by a known method. The sequence of the antisense DNA is preferably a sequence complementary to the endogenous gene or a part thereof possessed by the plant to be transformed, but may be not completely complementary as long as the gene expression can be effectively inhibited. Good. The transcribed RNA preferably has a complementarity of 90% or more, most preferably 95% or more, to the transcription product of the target gene. In order to effectively inhibit the expression of a target gene using an antisense sequence, the length of the antisense DNA is at least 15 bases or more, preferably 100 bases or more, more preferably 500 bases or more. is there. Usually, the length of the antisense DNA used is shorter than 5 kb, preferably shorter than 2.5 kb.

  Another embodiment of the “RNA complementary to the transcript of the Ero1 gene” is a dsRNA complementary to the transcript of the Ero1 gene. In RNA interference (RNAi), when a double-stranded RNA (hereinafter referred to as dsRNA) having the same or similar sequence as the target gene sequence is introduced into the cell, the expression of the introduced foreign gene and target endogenous gene are both suppressed. It is a phenomenon. When dsRNA of about 40 to several hundred base pairs is introduced into a cell, a RNaseIII-like nuclease called Dicer with a helicase domain is present in the presence of ATP, and dsRNA is about 21 to 23 base pairs from the 3 ′ end. Cut out and produce siRNA (short interference RNA). A protein specific to this siRNA binds to form a nuclease complex (RISC: RNA-induced silencing complex). This complex recognizes and binds to the same sequence as siRNA, and cleaves the mRNA of the target gene at the center of siRNA by RNaseIII-like enzyme activity. In addition to this pathway, the antisense strand of siRNA binds to mRNA and acts as a primer for RNA-dependent RNA polymerase (RsRP) to synthesize dsRNA. There is also a possibility that this dsRNA becomes Dicer's substrate again to generate new siRNA and amplify the action.

  The RNA of the present invention can be expressed from an antisense coding DNA that encodes an antisense RNA to any region of the target gene mRNA and a sense code DNA that encodes a sense RNA of any region of the target gene mRNA. . Moreover, dsRNA can also be produced from these antisense RNA and sense RNA.

  When the dsRNA expression system of the present invention is retained in a vector or the like, the antisense RNA and sense RNA are expressed from the same vector, and the antisense RNA and sense RNA are expressed from different vectors, respectively. There is. For example, antisense RNA and sense RNA can be expressed from the same vector as an antisense RNA in which a promoter capable of expressing a short RNA such as polIII is linked upstream of the antisense code DNA and the sense code DNA. It can be constructed by constructing an expression cassette and a sense RNA expression cassette, respectively, and inserting these cassettes into the vector in the same direction or in the opposite direction. It is also possible to construct an expression system in which the antisense code DNA and the sense code DNA are arranged in opposite directions so as to face each other on different strands. In this configuration, one double-stranded DNA (siRNA-encoding DNA) in which an antisense RNA coding strand and a sense RNA coding strand are paired is provided, and antisense RNA and sense RNA from each strand are provided on both sides thereof. A promoter is provided oppositely so that it can be expressed. In this case, in order to avoid adding extra sequences downstream of the sense RNA and antisense RNA, a terminator is added to the 3 ′ end of each strand (antisense RNA coding strand, sense RNA coding strand). It is preferable to provide. As this terminator, a sequence in which four or more A (adenine) bases are continued can be used. Moreover, in this palindromic style expression system, it is preferable that the types of the two promoters are different.

  In addition, as a configuration for expressing antisense RNA and sense RNA from different vectors, for example, antisense coding DNA and an antisense in which a promoter capable of expressing a short RNA such as polIII is linked upstream of sense coding DNA. An RNA expression cassette and a sense RNA expression cassette can be constructed, and these cassettes can be held in different vectors.

  In the RNAi of the present invention, siRNA may be used as dsRNA. “SiRNA” means a double-stranded RNA consisting of short strands that are not toxic in cells, for example, 15 to 49 base pairs, preferably 15 to 35 base pairs, and more preferably 21 Can be ~ 30 base pairs. Alternatively, the siRNA to be expressed is transcribed and the length of the final double-stranded RNA portion is, for example, 15 to 49 base pairs, preferably 15 to 35 base pairs, more preferably 21 to 30 base pairs. be able to.

  The DNA used for RNAi need not be completely identical to the target gene, but has at least 70% or more, preferably 80% or more, more preferably 90% or more, most preferably 95% or more sequence homology. .

  The part of the double-stranded RNA in which the RNAs in dsRNA are paired is not limited to a perfect pair, but mismatch (corresponding base is not complementary), bulge (no base corresponding to one strand) ) Or the like may include an unpaired portion. In the present invention, both bulges and mismatches may be included in the double-stranded RNA region where RNAs in dsRNA pair with each other.

  The “suppression of Ero1 gene expression” of the present invention can also be carried out using a DNA encoding a ribozyme. A ribozyme refers to an RNA molecule having catalytic activity. Some ribozymes have a variety of activities. Among them, research on ribozymes as enzymes that cleave RNA has made it possible to design ribozymes for site-specific cleavage of RNA. Some ribozymes have a group I intron type and a size of 400 nucleotides or more like M1RNA contained in RNaseP, but some have an active domain of about 40 nucleotides called hammerhead type or hairpin type.

  For example, the self-cleaving domain of hammerhead ribozyme cleaves on the 3 ′ side of C15 of G13U14C15, but it is important for U14 to form a base pair with A at position 9, and the base at position 15 is In addition to C, A or U is also shown to be cleaved. If the ribozyme substrate binding site is designed to be complementary to the RNA sequence near the target site, it is possible to create a restriction enzyme-like RNA-cleaving ribozyme that recognizes the UC, UU, or UA sequence in the target RNA. It is. For example, there are a plurality of sites that can be targets in the coding region of the enzyme of the present invention that is an inhibition target.

  Hairpin ribozymes are also useful for the purposes of the present invention. Hairpin ribozymes are found, for example, in the minus strand of tobacco ring spot virus satellite RNA (J. M. Buzayan Nature 323: 349, 1986). This ribozyme has also been shown to be designed to cause target-specific RNA cleavage.

In the present invention, examples of a method for suppressing the expression of the Ero1 gene include administration of a DNA encoding an aptamer that specifically recognizes the Ero1 protein to a subject.
The “aptamer” of the present invention can be used alone for treating diseases, but can also be used in a form in which other molecular species such as a fluorescently labeled dye is bound.

  The aptamer of the present invention can be produced using methods well known to those skilled in the art. Although not limited, for example, “in vitro selection method (SELEX method)” (Tuerk, C. and Gold, L., Science, 249, 505-510 (1990), Green, L. et al, Meths. Enzymol., 2, 75-86 (1991), Gold, L. et al, Annu. Rev. Biochem., 64,763-797 (1995), Uphoff, KW et al., Curr. Opin. Struct. Biol., 6, 281- 288 (1996)).

  The “in vitro selection method” is a method of selecting a molecule having affinity for the Ero1 protein from a pool of nucleic acid molecules containing a random sequence and excluding those molecules having no affinity. By repeating the cycle of amplifying only selected molecules by the PCR method and selecting by affinity, molecules having strong binding ability can be concentrated.

  Specifically, first, a single-stranded nucleic acid molecule containing a random base sequence of about 20 to 300 bases, preferably 30 to 150, more preferably about 30 to 100 bases, such as DNA or RNA, is prepared. These nucleic acid molecules may be synthesized directly, or in the case of RNA molecules, DNA molecules may be synthesized first and then prepared by a transcription reaction. When these nucleic acid molecules are DNA, in order to enable PCR amplification, they have base sequences to serve as primers at both ends. The primer binding sequence portion is not particularly limited, but may be prepared to have an appropriate restriction enzyme site so that the primer portion can be excised with a restriction enzyme after PCR amplification. The length of the primer binding sequence portion is not particularly limited, but is about 20-50, preferably about 20-30 bases. Further, in order to make it possible to separate the single-stranded DNA after PCR amplification by electrophoresis or the like, the 5 ′ end may be labeled with a radiolabel, a fluorescent label, or the like. Furthermore, when preparing an RNA molecule, an appropriate promoter such as a T7 promoter sequence may be placed on the 5 'end primer so that transcription from the DNA molecule to the RNA molecule is possible. Good.

  Next, random nucleic acid molecules obtained by PCR amplification and peptide fragments including Ero1 protein or a specific region of Ero1 protein are mixed at an appropriate concentration ratio, and incubated under appropriate conditions. After incubation, the mixture is subjected to electrophoresis to separate the nucleic acid molecule-Ero1 protein complex from the free nucleic acid molecule. Nucleic acid molecules forming a complex with Ero1 protein are extracted according to a standard method. When the recovered nucleic acid molecule is DNA, PCR amplification is further performed, and the amplified DNA is recovered as single-stranded DNA by heat denaturation or the like. When the recovered nucleic acid molecule is RNA, the RNA is reverse transcribed into cDNA, and then PCR amplified, and the amplified DNA is transcribed to prepare RNA.

  Mixing of the above-mentioned nucleic acid molecules with Ero1 protein, separation of nucleic acid molecules bound to Ero1 protein, PCR amplification (in the case of RNA, reverse transcription after amplification), and use of amplified nucleic acid molecule for binding to Ero1 protein again The series of operations up to is done several rounds. By repeating rounds, nucleic acid molecules that bind to Ero1 protein more specifically can be selected. The obtained nucleic acid molecule can be sequenced according to a conventional method.

  Based on the sequence of the nucleic acid molecule obtained by the above steps, the phosphate skeleton is modified, for example, phosphorothioate bond, phosphorodithioate bond, phosphoramidothioate bond, phosphoramidate bond, phosphoramidate An aptamer composed of a phosphate skeleton containing a bond and a methylphosphonate bond can also be obtained.

RNA complementary to the transcript of the Ero1 gene of the present invention, DNA encoding a ribozyme that specifically cleaves the transcript, and DNA encoding an aptamer that specifically recognizes Ero1 protein include the following (i): To (iv).
(I) DNA encoding an antisense RNA complementary to the transcription product of the DNA according to any one of (a) to (d) below.
(Ii) DNA encoding an RNA having a ribozyme activity that specifically cleaves the transcription product of the DNA according to any one of (a) to (d) below.
(Iii) DNA encoding a dsRNA complementary to the transcription product of DNA described in any of (a) to (d) below.
(Iv) DNA encoding an aptamer that specifically recognizes a protein encoded by the DNA described in any of (a) to (d) below.
(A) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 2.
(B) DNA containing the coding region of the base sequence set forth in SEQ ID NO: 1.
(C) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence set forth in SEQ ID NO: 2.
(D) A DNA that hybridizes under stringent conditions with a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1.

As an example of the DNA encoding the dsRNA of the present invention, more preferably, siRNA having a region amplified by the primers described in SEQ ID NOs: 3 and 5 in the DNA described in SEQ ID NO: 1.
These DNAs can be used to comprehensively inhibit the formation of protein disulfide bonds or to inhibit the oxidation activity of protein disulfide isomerase in plant cells.

The present invention also provides a vector containing a DNA that suppresses the expression of the Ero1 gene and a transformed plant cell.
The vector of the present invention includes a vector constructed for inducing the above-mentioned RNAi. Since the vector of the present invention contains the DNA of the present invention, when introduced into a cell, single-stranded RNA formed by transcription in the cell paired within the molecule to form dsRNA.

The vector used for the transformation of plant cells is not particularly limited as long as the inserted gene can be expressed in the cells. For example, a plasmid, a phage, or a cosmid can be exemplified.
The “plant cell” includes various forms of plant cells such as suspension culture cells, protoplasts, leaf sections, callus and the like.

  The vector of the present invention may contain a promoter for constitutively or inducibly expressing the DNA that suppresses the expression of the Ero1 gene of the present invention. The vector of the present invention may contain a promoter for expressing the DNA of the present invention in a site-specific manner. For example, in order to express DNA that suppresses the expression of the Ero1 gene of the present invention specifically in the endosperm, a promoter such as rice seed storage protein, starch synthase, etc. is expressed, and the DNA that suppresses the expression of the Ero1 gene of the present invention is expressed. Can be inserted into a vector. Specific examples include the promoters described in the Examples.

  Those skilled in the art can appropriately prepare a vector having a desired DNA by a general genetic engineering technique. Usually, various commercially available vectors can be used.

The vector of the present invention is also useful for retaining or expressing a DNA that suppresses the expression of the Ero1 gene of the present invention in a host cell.
The DNA that suppresses the expression of the Ero1 gene in the present invention is usually carried (inserted) into an appropriate vector and introduced into a host cell. That is, the present invention provides a host cell carrying a DNA or vector that suppresses the expression of the Ero1 gene. The vector is not particularly limited as long as it can stably hold the inserted DNA. An expression vector is particularly useful when a vector is used for the purpose of introducing and expressing the DNA of the present invention into a cell having an endogenous gene. The expression vector is not particularly limited as long as it is a vector that expresses a DNA that suppresses the expression of the Ero1 gene of the present invention in a test tube, a plant individual or the like. For example, a pBEST vector (manufactured by Promega) is used for in vitro expression. ), PBINPLUS vector (van Engelen, FA et al., (1995). PBINPLUS: an improved plant transformation vector based on pBIN19. Transgenic Res. 4, 288-290.) . The DNA of the present invention can be inserted into a vector by a conventional method (Molecular Cloning, 5.61-5.63), for example, by a ligase reaction using a restriction enzyme site.

As a method of expressing the “DNA that suppresses the expression of Ero1 gene” of the present invention in vivo, the DNA of the present invention is incorporated into an appropriate vector, for example, polyethylene glycol method, electroporation method, Agrobacterium method, Liposome method, cationic liposome method, calcium phosphate precipitation method, electric pulse perforation (electroporation) (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Section 9.1-9.9), lipofection Examples thereof include a method known to those skilled in the art, such as a method (GIBCO-BRL), a microinjection method, and a particle gun method.
Administration into a plant may be an ex vivo method or an in vivo method.

In addition, when the DNA that suppresses the expression of the Ero1 gene of the present invention is introduced into the plant body, the DNA can also be directly introduced into plant cells using a microinjection method, an electroporation method, a polyethylene glycol method, or the like. However, it can also be introduced into a plant cell indirectly via a virus or bacterium capable of infecting plants as a vector by incorporating it into a plasmid for gene transfer into a plant. Examples of such viruses include cauliflower mosaic virus, tobacco mosaic virus, gemini virus and the like as typical viruses, and Agrobacterium and the like as bacteria. When introducing a gene into a plant by the Agrobacterium method, a commercially available plasmid can be used. The method for introducing the DNA of the present invention into a plant using such a vector is preferably the method described in Toki (1997) Plant Mol. Biol. Rep. 15, 16-21. .
These transformation methods described above are preferably selected as appropriate depending on the type of plant or the like that serves as the host (for example, monocotyledonous plants or dicotyledonous plants).

The present invention also provides a plant cell carrying a DNA that suppresses the expression of the Ero1 gene or the vector of the present invention. Furthermore, this invention provides the transformed plant body containing the plant cell of this invention. The cells into which the DNA that suppresses the expression of the Ero1 gene or the vector of the present invention is introduced include plant cells for producing transformed plants. There is no restriction | limiting in particular as a plant cell.
The plant cells of the present invention include cultured cells as well as cells in the plant body. Also included are protoplasts, shoot primordia, multi-buds, and hairy roots.

  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 cells. For example, with regard to the technique for producing a transformed plant body, a method for regenerating a plant body by introducing a gene into protoplasts using polyethylene glycol, a method for regenerating a plant body by introducing a gene into protoplasts using an electric pulse, or a gene for cells by a particle gun method. Can be mentioned, and a method for regenerating a plant body and a method for regenerating a plant body by introducing a gene via Agrobacterium are not particularly limited. Some techniques have already been established and are widely used in the technical field of the present invention. In the present invention, these methods can be suitably used.

In order to efficiently select plant cells transformed by introduction of a vector containing DNA that suppresses the expression of Ero1 gene of the present invention, the recombinant vector contains an appropriate selection marker gene or contains a selection marker gene You may introduce | transduce into a plant cell with a plasmid vector. Selectable marker genes used for this purpose include, for example, the neomycin phosphotransferase gene that is resistant to the antibiotics kanamycin or gentamicin, the hygromycin phosphotransferase gene that is resistant to hygromycin, and the acetyl that is resistant to the herbicide phosphinothricin. Examples include transferase genes.
Plant cells into which the recombinant vector has been introduced are placed on a known selection medium containing a suitable selection agent according to the type of the introduced selection marker gene and cultured. As a result, transformed plant cultured cells can be obtained.

  Transformed plant cells can regenerate plant bodies by redifferentiation. The method of redifferentiation varies depending on the type of plant cell. For example, Fujimura et al. (Plant Tissue Culture Lett. 2:74 (1995)) can be used for rice, and Shillito et al. (Bio / Technology 7) for maize. : 581 (1989)) and Gorden-Kamm et al. (Plant Cell 2: 603 (1990)), and for potatoes, Visser et al. (Theor. Appl. Genet 78: 594 (1989)). In the case of tobacco, the method of Nagata and Takebe (Planta 99:12 (1971)) can be cited, and in the case of Arabidopsis, the method of Akama et al. (Plant Cell Reports 12: 7-11 (1992)) can be cited as eucalyptus. Then, the method of Toi et al. (Japanese Patent Laid-Open No. 8-89113) can be mentioned.

In addition, the presence of introduced foreign DNA in a transformed plant that has been regenerated and cultivated in this way can be analyzed by a known PCR method or Southern hybridization method, or by analyzing the base sequence of the DNA in the plant body. Can be confirmed.
In this case, extraction of DNA from the transformed plant body can be performed according to a known method of J. Sambrook et al. (Molecular Cloning, 2nd edition, Cold Spring Harbor Laboratory Press, 1989).

  When the foreign gene comprising the DNA of the present invention present in the regenerated plant body is analyzed using the PCR method, the amplification reaction is performed using the DNA extracted from the regenerated plant body as a template as described above. In addition, an amplification reaction is carried out in a reaction mixture in which the DNA of the present invention or a synthesized oligonucleotide having a base sequence appropriately selected according to the base sequence of the DNA modified according to the present invention is used as a primer. You can also. In the amplification reaction, DNA denaturation, annealing, and extension reactions are repeated several tens of times to obtain an amplification product of a DNA fragment containing the DNA sequence of the present invention. When the reaction solution containing the amplification product is subjected to, for example, agarose electrophoresis, it is possible to confirm that the amplified DNA fragments are fractionated and the DNA fragments correspond to the DNA of the present invention.

Further, whether or not the expression of the Ero1 gene is suppressed by introduction of the DNA that suppresses the expression of the Ero1 gene of the present invention into a plant can be confirmed by the method of the example.
Specifically, a method of analyzing the expression level of Ero1 protein by Western blotting and a method of comparative analysis of the accumulated amount of seed storage protein can be mentioned. In the present invention, when a decrease in Ero1 protein expression level and excessive accumulation of proglutelin are observed, it can be determined that the expression of Ero1 gene is suppressed. Furthermore, as a method for confirming whether or not the expression of the Ero1 gene is suppressed, an analysis of whether or not the generation amount of hydrogen peroxide, which is considered to result from the substrate protein-PDI-Ero1 electron transfer cascade, decreases, or It can also be analyzed whether α-globulin accumulates as vacuole-derived protein granules PBII.

  Once a transformed plant into which the DNA of the present invention is introduced into the genome is obtained, offspring can be obtained from the plant by sexual reproduction or asexual reproduction. It is also possible to obtain a propagation material (for example, seeds, fruits, cuttings, tubers, tuberous roots, strains, callus, protoplasts, etc.) from the plant body, its descendants or clones, and mass-produce the plant body based on them. Is possible. The present invention includes plant cells into which the DNA or vector of the present invention has been introduced, plant bodies containing the cells, progeny and clones of the plant bodies, and propagation materials for the plant bodies, their progeny, and clones. These plant cells, plants containing the cells, progeny and clones of the plants, and propagation materials of the plants, progeny and clones thereof can be used in a method for controlling seed dormancy of plants. .

  In the present invention, as described above, it is possible to comprehensively inhibit the formation of disulfide bonds in plant intracellular proteins by suppressing the expression of the Ero1 gene. Plants produced by the method of the present invention can inhibit protein disulfide bond formation in useful crops, for example.

  Furthermore, DNA encoding antisense RNA, DNA encoding dsRNA, and DNA encoding ribozyme activity are used to suppress the expression of endogenous Ero1 gene in plants based on the sequence information of Ero1 gene. Furthermore, it is also possible to prepare DNA or the like encoding an aptamer. The produced DNA can be used to inhibit disulfide bonds in plant proteins.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.

[Materials and methods]
(1) Construction of Ero1 RNA interference induction vector and rice transformation Total RNA was extracted from rice (Oryza sativa cv Nipponbare) seeds, and cDNA was prepared by reverse transcription reaction. PCR was performed using cDNA as a template, and a DNA fragment encoding Lys-410 was amplified from Val-119 of rice Ero1 (Os03g0733800). An inverted repeat fragment ligated with the sense strand and antisense strand was prepared and α-globulin of a binary vector containing hygromycin phosphotransferase (Kawagoe, Y., et al., (2005) Plant Cell 17, 1141-1153) Glb) gene was introduced downstream of the gene promoter using a restriction enzyme recognition site to prepare an Ero1 RNA interference induction vector (FIG. 1A).
A binary vector (BV) that expresses a GFP-Glb or GFP-GlbΔC fusion protein under induction of Ero1 RNA interference was calculated by recombination of the attachment site (att) between the following DV (Destination Vector) and EV (Entry Vector). Seed preparation (DV1xEV1; DV1xEV2; DV2xEV1; DV2xEV2) (Figure 3).
Specifically, DV is transferred to a binary vector (Kawagoe, Y., et al., (2005) Plant Cell 17, 1141-1153) containing sp-GFP-Glb or sp-GFP-GlbΔC downstream of the Glb promoter. -Cm ^R -ccdB-attR2 fragment (Invitrogen) was introduced and prepared (DV1 and DV2).
EV was prepared by introducing the inverted repeat fragment of the above Ero1 gene using a restriction enzyme site between attL1-attL2 of the pENTR vector (Invitrogen). Upstream of the inverted repeat of Ero1 gene are two promoters expressed in endosperm, Gt1 (Zheng, Z., et al., (1993) Plant J. 4, 357-366) (EV1) and APS2b promoter ( EV2) (Onda and Kawagoe, 2006 Agricultural Chemistry Society presentation; Ohdan, T., et al., (2005) J. Exp. Bot. 56, 3229-3244). The Gt1 promoter was amplified by PCR using 2530 bases upstream from the start codon of the Gt1 gene (AK107314), the APS2b promoter was 2048 bases containing the 5'-UTR of the APS2b gene (AK103906), and genomic DNA as a template. A Gt1 terminator was ligated downstream of the inverted repeat of the Ero1 gene, and 944 bases downstream from the stop codon of the Gt1 gene (AK107314) were similarly isolated.
Rice transformation was performed by the Agrobacterium method using the above vector (Goto, F., et al., (1999) Nat. Biotechnol. 17, 282-286).

(2) Confocal laser microscope observation
For the transformant co-expressed with the GFP-Glb fusion protein under the induction of Ero1 RNA interference (FIG. 3), a section (100 μm) was prepared from the T1 ripened seed. The GFP fluorescence image in the adjacent cell of the endosperm aleurone layer of this section was continuously acquired with a confocal laser microscope (TCS SP2 AOBS, Leica Microsystems). As a control, a transformant expressing only the GFP-Glb fusion protein was similarly analyzed.
Hydrogen peroxide generation in the endosperm cells was visualized using a hydrogen peroxide specific fluorescent probe. Wild-type matured seed slices (100 μm) were immersed in 1xPBS buffer containing 5 μM of hydrogen peroxide-specific fluorescent probe BES-H ₂ O ₂ (Wako), reacted at room temperature for 30 minutes, Observation was performed with a confocal laser microscope (excitation wavelength: 488 nm; fluorescence wavelength: 505-555 nm). In addition, in order to confirm the hydrogen peroxide specificity of BES-H ₂ O ₂ , a reaction was performed using a superoxide-specific fluorescent probe BES-So (Wako). As a result, unlike BES-H ₂ O ₂ , no fluorescence was detected (data not shown), and it was confirmed that BES-H ₂ O ₂ reacted specifically with hydrogen peroxide. PBI was visualized by rhodamine staining (Rhodamine B, Sigma).

(3) Seed storage protein total extraction and electrophoresis Wild-type or Ero1 RNA interference transformant T1 ripened seeds were disrupted in liquid nitrogen and SDS-Urea buffer (50 mM Tris-Cl, pH 6.8, 8 M Urea, 4% (w / v) SDS, 20% (v / v) glycerol, 5% (v / v) 2-mercaptoethanol) (seed 20 mg / 700 mL). After shaking at 25 ° C. for 4 hours, the total extracted protein was separated by SDS-polyacrylamide gel electrophoresis (PAGE) (acrylamide 14%).

(4) Western analysis The seed total protein extracted in (3) was separated by SDS-PAGE, electrically transferred to a polyvinyliden difluoride membrane (ATTO), and then reacted with an anti-rice Ero1 rabbit antibody. Antigen-antibody complexes were detected by ECL (Amersham Biosciences) using a Horseradish peroxidase labeled anti-rabbit antibody (donkey) (Amersham Biosciences). Antigen rice Ero1 protein used for antibody production was prepared by the following method.
PCR was performed using rice EST clone (OSJNEc05N03) as a template to amplify a DNA fragment encoding from Arg-57 to C-terminus of Ero1. The obtained PCR fragment was introduced into pQE30 (QIAGEN) using a restriction enzyme site to construct an N-terminal 6xHis-tagged recombinant protein expression vector (Ero1 / pQE30). Host E. coli BL21 was transformed with Ero1 / pQE30 and pre-cultured at 37 ° C. in an LB liquid medium containing Ampicillin (50 μg / mL). Isopropyl-β-D (-)-thiogaractopyranoside (1 mM) was added, followed by culturing at 37 ° C. for 2 hours, and collection by centrifugation (5000 rpm, 5 minutes).
E. coli cells were obtained by sonication in buffer A (20 mM Tris-Cl, pH 7.5, 2 mM EDTA, 0.2 M NaCl, 1 mg / mL lysozyme) followed by centrifugation (13000 rpm, 10 minutes). Precipitate fraction was added to buffer B (100 mM Tris-Cl, pH 7.5, 2 mM EDTA, 0.2 M NaCl, 1% (w / v) deoxycholic acid, 1% (v / v) Nonidet P40, 10 mM 2- After suspension washing in mercaptoethanol), the target protein was collected as inclusion bodies by centrifugation. Suspend the inclusion body in buffer C (50 mM phosphate buffer, pH 8.0, 250 mM KCl, 8 M Urea, 10% (v / v) glycerol), and centrifuge (10000 rpm, 20 minutes). The Ero1 protein was further purified using Ni-NTA Agarose (QIAGEN) resin.

[Example 1] Endosperm-specific suppression of rice Ero1 gene expression Rice Ero1 homologue is encoded by chromosome 3. In the present invention, rice Ero1 gene expression was suppressed by RNA interference method having extremely high sequence specificity. First, for the rice Ero1 gene, a DNA fragment encoding a region having a high primary homology with those of yeast and Arabidopsis thaliana was isolated. An RNA interference induction vector into which the inverted repeat fragment was introduced was constructed to produce transformed rice (FIG. 1A).
When oxidative folding via Ero1 protein is performed in higher plants, the Ero1 gene may be essential for plant growth. Therefore, in order to maintain the fertility of plant individuals, Ero1 gene expression was specifically suppressed in the seed endosperm. Specifically, we compared three different endosperm specific promoters as regulators of RNA interference induction. Isolate the promoters of rice seed storage protein α-globulin or glutelin (Glb and Gt1 respectively) and APS2b gene encoding the small subunit of cytoplasmic ADP-glucose pyrophosphorylase, upstream of the inverted repeat of Ero1 gene In this way, transformants were prepared. Seed fertility was highest when the APS2b promoter was used, and improved in the order of APS2b>Gt1> = Glb.
When total protein was extracted from the seeds of the produced transformed rice and the Ero1 protein expression level was compared and analyzed by Western method, the expression level of Ero1 protein was significantly reduced in the RNA interference transformant compared to the wild type (Fig. 1B). It was confirmed that the expression of Ero1 gene in seeds was suppressed by this method.

[Example 2] Ero1 protein-dependent electron transfer in rice endosperm cells When the accumulation of seed storage protein was compared and analyzed, glutelin was a precursor molecular species (proglutelin) in Ero1 RNA interference transformants compared to wild type. Overaccumulated (Figure 1C).
In yeast, electrons are transferred from the polypeptide chain to the Ero1 protein during the oxidative folding process of proteins in the ER, but oxygen molecules have been reported as the final electron acceptor of the substrate protein-PDI-Ero1 electron transfer cascade. (Tu, BP, and Weissman, JS (2002) Mol. Cell 10, 983-994). And it is suggested that reactive oxygen species such as hydrogen peroxide are generated as a result of electron donation to oxygen molecules (Tu, BP, and Weissman, JS (2002) Mol. Cell 10, 983-994; Gross, E., et al., (2006) Proc. Natl. Acad. Sci. USA 103, 299-304). In the present invention, attention has been focused on the generation of hydrogen peroxide as one index for examining the function of the electron transfer cascade via Ero1 in seed endosperm cells. When endosperm cells of wild-type matured seeds were reacted with a hydrogen peroxide-specific fluorescent probe and analyzed by confocal laser microscopy, strong fluorescence (BES fluorescence) was detected (Fig. 2A). PBI, an ER-derived protein granule, was visualized by rhodamine staining, but BES fluorescence co-localized with PBI, and BES fluorescence intensity showed a difference between PBI (Fig. 2B).
These results suggest that the substrate protein-PDI-Ero1 electron transport cascade via Ero1 protein functions in rice seed endosperm cells, and that the electron transfer is inhibited by the suppression of Ero1 gene expression. .

[Example 3] Effect of Ero1 RNA interference on intracellular accumulation of seed storage protein α-globulin is composed of three regions, A, B and C, each having a consensus sequence containing cysteine residues (regions A and L _45). xxC ₄₈ (SEQ ID NO: 7); region B, C ₇₈ C ₇₉ xQ ₈₁ L ₈₂ (SEQ ID NO: 8); region C, P ₁₆₈ xxC ₁₇₁ (SEQ ID NO: 9)). Previous studies have reported that α-globulin eventually accumulates as vacuolar-derived protein granules PBII, and that intradomain BC interstitial disulfide bond formation is essential for α-globulin PBII localization (Kawagoe , Y., et al., (2005) Plant Cell 17, 1141-1153).
Therefore, in order to investigate the effect of Ero1 gene expression suppression on α-globulin (Glb) intracellular accumulation, Ero1 RNA interference was induced under the control of Gt1 or APS2b promoter (PGt1 or PAPS2b, respectively) and GFP-Glb fusion protein was co-expressed (FIG. 3A, 3B and 3C upper stage).
When confocal laser analysis was performed on ripened seed slices, GFP fluorescence showed a different shape from the wild type in Ero1 RNA interference transformants. Specifically, in the wild type, GFP fluorescence is observed as a sphere with a diameter of about 4 μm, whereas in the PGt1 :: Ero1 RNA interference transformant, many small granules that are distinct from PBII are observed, and PAPS2b :: It was also observed as small granules in the Ero1 RNA interference transformant (FIG. 4).
On the other hand, α-globulin lacking region C (GlbΔC) is localized in PBI, which is dependent on intermolecular disulfide bond formation (Kawagoe, Y., et al., (2005) Plant Cell 17, 1141- 1153). Therefore, under the induction of Ero1 RNA interference, the GFP-GlbΔC fusion protein was coexpressed (FIG. 3A, 3B and 3C bottom) and analyzed in the same manner. As a result, GFP fluorescence was observed as large and small granules and as a network in PGt1 :: Ero1 RNA interference transformant and PAPS2b :: Ero1 RNA interference transformant (FIG. 5), which is different from typical PBI in wild type The shape was shown (Figure 2B, right panel rhodamine).
These results suggest that Ero1 is important for protein granule formation in endosperm cells, and that suppression of Ero1 gene expression inhibits the formation of disulfide bonds in seed proteins.

(A) shows the structure of an Ero1 RNA interference induction vector. An inverted repeat was prepared for a DNA fragment encoding Lys-410 from Val-119 of rice Ero1, and ligated downstream of the Glb promoter. (B) A photograph showing the expression level of Ero1 protein in wild type and Ero1 RNA interference transformant rice seeds. All proteins extracted from mature seeds of wild type (lane WT) and transformant (lane ero1) introduced with Ero1 RNA interference induction vector were subjected to SDS-PAGE separation and Western analysis was performed using anti-rice Ero1 antibody . Arrow indicates rice Ero1 protein. (C) A photograph showing storage protein accumulation in wild-type and Ero1 RNA interference-transformed rice seeds. Arrows indicate the glutelin precursor (proglutelin), the acidic subunit (α-glutelin) of the mature glutelin, and the basic subunit (β-glutelin) from the top. It is the photograph which visualized hydrogen peroxide generation | occurrence | production in a rice seed endosperm cell. (A) A wild-type matured seed slice was reacted with a hydrogen peroxide-specific fluorescent probe, and then observed with a confocal laser microscope. Bar = 10 μm. (B) A section of a wild type matured seed was double-stained with a hydrogen peroxide-specific fluorescent probe (panel BES) and rhodamine (panel rhodamine), and each fluorescence was analyzed by a confocal laser microscope. Bar = 5 μm. It is a figure which shows the structure of the co-expression vector of Ero1 RNA interference and a GFP-Glb fusion protein. A Gt1 (A) or APS2b (B) promoter was linked upstream of the inverted repeat of the Ero1 gene in FIG. 1A (PGt1 :: Ero1 RNAi and PAPS2b :: Ero1 RNAi, respectively). A Gt1 terminator was connected downstream of each. GFP-Glb full length (PBII localization) (C upper stage) or GFP-GlbΔC (PBI localization) (C lower stage) was introduced into the arrow site of each vector (A and B). It is a photograph which shows the intracellular localization of the GFP-Glb fusion protein in an Ero1 RNA interference transformant. For ripening seeds of wild type and rice (PGt1 :: Ero1 RNAi and PAPS2b :: Ero1 RNAi, respectively) transformed with a vector in which the full length GFP-Glb (upper part of FIG. 3C) was introduced into the vectors of FIGS. 3A and 3B. GFP fluorescence was analyzed with a confocal laser microscope. Bar = 2μm. It is a photograph showing the intracellular localization of the GFP-GlbΔC fusion protein in the Ero1 RNA interference induction transformant. Confocal GFP fluorescence for ripened seeds of rice (PGt1 :: Ero1 RNAi and PAPS2b :: Ero1 RNAi, respectively) transformed with a vector in which GFP-GlbΔC (bottom of Fig. 3C) was introduced into the vectors of Fig. 3A and Fig. 3B Analysis was performed with a laser microscope. Bar = 2μm.

Claims (13)

Comprehensively inhibits the formation of disulfide bonds of the intracellular protein characterized by suppressing the expression of the endogenous DNA described in any of (a) to (d) below in the plant cell Method.
(A) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 2.
(B) DNA containing the coding region of the base sequence set forth in SEQ ID NO: 1.
(C) SEQ ID NO: 2 amino acids within 30 amino acids in the amino acid sequence set forth in substitution, see containing deletion, insertion, and / or an amino acid sequence added, it has the function of promoting disulfide bond formation of intracellular proteins DNA encoding a protein.
(D) DNA encoding a protein having a sequence identity of 90% or more with the DNA comprising the base sequence set forth in SEQ ID NO: 1 and a function of promoting the formation of disulfide bonds in intracellular proteins . A method for inhibiting the oxidative activity of protein disulfide isomerase, comprising suppressing endogenous expression of the DNA according to any one of (a) to (d) below in a plant cell.
(A) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 2.
(B) DNA containing the coding region of the base sequence set forth in SEQ ID NO: 1.
(C) SEQ ID NO: 2 amino acid substitutions within 30 amino acids in the amino acid sequence set forth in, deletions, seen including insertion, and / or an amino acid sequence obtained by adding, encoding a protein having an oxidation activity of protein disulfide isomerase DNA .
(D) DNA encoding a protein having a sequence identity of 90% or more with the DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 1 and having protein disulfide isomerase oxidizing activity . The method according to claim 1 or 2, wherein the expression of the DNA is specifically suppressed in endosperm cells. The method according to any one of claims 1 to 3, wherein the plant is a monocotyledonous plant. The method according to any one of claims 1 to 4, further comprising the step of introducing the DNA according to any one of (i) to (iv) below into a plant cell.
(I) DNA encoding an antisense RNA complementary to the transcription product of the DNA according to any one of claims 1 (a) to (d).
(Ii) DNA encoding an RNA having a ribozyme activity that specifically cleaves the transcription product of the DNA according to any one of claims 1 (a) to (d).
(Iii) DNA encoding a dsRNA complementary to the transcription product of the DNA according to any one of claims 1 (a) to (d).
(Iv) DNA encoding an aptamer that specifically recognizes the protein encoded by the DNA according to any one of claims 1 (a) to (d).   6. The method according to claim 5, wherein the DNA is introduced into plant cells by Agrobacterium. A plant body in which expression of the DNA according to any one of claims 1 (a) to (d) is suppressed by the method according to any one of claims 1 to 6. In order to comprehensively inhibit protein disulfide bond formation in plant cells, including the DNA according to any one of (i) to (iv) below or a vector containing the DNA, or a protein disulfide isomerase An agent for inhibiting oxidative activity .
(I) DNA encoding an antisense RNA complementary to the transcription product of the DNA according to any one of claims 1 (a) to (d).
(Ii) DNA encoding an RNA having a ribozyme activity that specifically cleaves the transcription product of the DNA according to any one of claims 1 (a) to (d).
(Iii) DNA encoding a dsRNA complementary to the transcription product of the DNA according to any one of claims 1 (a) to (d).
(Iv) DNA encoding an aptamer that specifically recognizes the protein encoded by the DNA according to any one of claims 1 (a) to (d). Or the DNA holds the base compactors claim 8, in cells, disulfide bond formation of the protein has been comprehensively inhibited, or, oxidation activity of protein disulfide isomerase has been inhibited, the transformed plant cells. A transformed plant comprising the transformed plant cell according to claim 9 . A transformed plant that is a descendant or clone of the transformed plant according to claim 10 . The propagation material of the transformed plant body of Claim 10 or 11 . A method of manufacturing a transgenic plant according to claim 10 or 11, the DNA or base restrictor according to claim 8 introduced into a plant cell, comprising the step of regenerating a plant from the plant cell .