JP6192031B2 - Genes involved in accumulation of plant storage proteins and use thereof - Google Patents

Description

  The present invention relates to genes involved in the accumulation of plant seed storage proteins and the modification of plant seed storage proteins using the genes. Modification of plant seed storage proteins is useful in fields such as plant breeding and in improving the processing characteristics of rice flour.

  Rice seed storage protein glutelin is a digestible protein that accounts for about 60% of the total storage protein. However, rice has a lower content of lysine and tryptophan, which are essential amino acids than other plants (Encyclopedia of Nutrition and Nutrients http://www.fjkw33.com/eiyou/e32-3.html). In order to increase the lysine content of rice, studies have been attempted to express phaseolin, a bean protein, in rice seeds (Non-patent Document 1), but its accumulation efficiency is low. In addition, rice with low glutelin content is suitable for diabetic patients, and therefore, growing rice with low glutelin content is required. Although attempts have been made to cultivate low-glutelin rice, details such as the causative gene have not been clarified.

Rice glutelin is synthesized as a precursor on the endoplasmic reticulum and then transported to the storage vacuole via the Golgi apparatus. In storage vacuoles, it is cleaved into two subunits and accumulates as mature glutelin. The glup6 ( glutelin precursor 6 ) mutant (Non-patent Documents 2 and 3) accumulates more glutelin precursors than the wild type, and the accumulation amount of mature glutelin decreases. The GLUP6 gene has already been found to sit on chromosome 3 (Non-Patent Document 4), but has not yet been identified.

  On the other hand, in Arabidopsis thaliana, GEF (GEF: Guanine nucleotide Exchange Factor) is known to control activation of two different types of Rab5 (Non-patent Document 5). Rha1, a Rab5 family, is localized in the Golgi apparatus in tobacco leaf epithelial cells and is involved in vacuolar transport (Non-Patent Document 6). However, it is unclear how GEF is involved in intracellular transport of glutelin precursors in rice endosperm.

Zheng, Z., Sumi, K., Tanaka, K., and Murai, N. Plant Physiol. 109, 777-786 (1995) Tian, H.D., Kumamaru, T., and Satoh, H. Rice Genet. Newslet. 18, 48-50 (2001) Ueda, Y., Satoh-Cruz, M., Matsusaka, H., Takemoto-Kuno, Y., Fukuda, M., Okita, TW, Ogawa, M., Satoh, H., and Kumamaru, T. Breed Sci . 60, 568-574 (2010) Sato et al. Rice Genetics Newsletter 20: 43-45 (2003) Goh, T., Uchida, W., Arakawa, S., Ito, E., Dainobu, T., Ebine, K., Takeuchi, M., Sato, K., Ueda, T., and Nakano, A. Plant Cell 19, 3504-3515 (2007) Kotzer, A.M., Brandizzi, F., Neumann, U., Paris, N., Moore, I., and Hawes, C. J. Cell Sci. 117, 6377-6389 (2004)

glup6 is expected to be a factor involved in the transport of glutelin precursor to storage vacuoles. An object of the present invention is to provide a novel plant seed storage protein accumulation-related gene. Another object of the present invention is to modify the seed storage protein composition of plants using the gene, thereby modifying the characteristics of plant seed components.

  The present inventors focused on rice for which development of a simple method for modifying seed components is desired among plants, and in particular, a gene involved in the accumulation of storage protein glutelin, which is nutritionally good, is identified. We conducted earnest research to separate them. The inventors have succeeded in isolating the gene and found that the composition of the plant storage protein can be altered using the gene, thereby completing the present invention.

The present invention provides the following.
[1] A plant having the following polynucleotide (a), (b), (c), (d), (e), or (f) functionally and accumulating storage proteins in storage-type vacuoles A method for producing a plant with altered storage protein accumulation by disabling the polynucleotide in
(A) a polynucleotide comprising the base sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 11, or SEQ ID NO: 12;
(B) a polynucleotide comprising a nucleotide sequence complementary to a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 11 or SEQ ID NO: 12 A polynucleotide encoding a protein that hybridizes with a nucleotide under stringent conditions and has a function of accumulating the storage protein in a storage vacuole;
(C) having at least 90% identity with and storing the nucleotide sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 11 or SEQ ID NO: 12 A polynucleotide encoding a protein having a function of accumulating the protein in storage vacuoles;
(D) a polynucleotide encoding the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 6;
(E) a storage type vacuole comprising a storage protein consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, added and / or inserted in the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 6, and A polynucleotide encoding a protein having a function of accumulation in
(F) Coding a protein comprising an amino acid sequence having at least 97% identity with the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 6, and having a function of accumulating the storage protein in a storage type vacuole Polynucleotide.
[2] To a plant in which the polynucleotide of (a), (b), (c), (d), (e), or (f) defined in [1] is nonfunctional, A method for producing a plant with altered storage protein accumulation by introducing a defined polynucleotide (a), (b), (c), (d), (e), or (f) .
[3] The production method according to [1] or [2], wherein the storage protein is glutelin.
[4] A transformed plant transformed with a vector comprising a polynucleotide as defined in [1], or a vector comprising a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 7 or SEQ ID NO: 9, or its progeny.
[5] The transformed plant or its progeny according to [4], wherein the plant is rice, soybean, oat, pumpkin, or Arabidopsis thaliana.
[6] A crop obtained from the plant obtained by the production method according to any one of [1] to [3], or obtained from the plant according to [4] or [5] or its progeny , Breeding materials or processed products.

According to the present invention, a GLUP6-GEF gene involved in storage protein accumulation was provided.
The gene of the present invention is involved in intracellular transport of plant seeds. By regulating the expression and function of this gene, the amount of mature glutelin accumulated in the seed can be regulated. Therefore, the gene of the present invention is useful for breeding rice varieties with a reduced amount of mature glutelin. Traditionally, rice varieties have been improved by (1) selecting promising lines by crossing, and (2) mutagenesis by radiation or chemicals. These operations have problems such as requiring a long time and inability to control the degree and direction of mutation. Breeding rice varieties using the gene of the present invention is advantageous over conventional methods in that the target plant can be obtained with high certainty in a short period of time.

According to the present invention, selection and inspection of varieties can be performed easily and quickly. When glup6 mutants are used as a mating mother and glup6 type is selected from the progeny of the cross, the glup6 type should be efficiently selected at the seedling stage using a DNA polymorphism marker prepared from the nucleotide sequence of the mutation site Became possible. Furthermore, the identification of the GLUP6-GEF gene made it possible to select other amino acid substitution mutants and amino acid deletion mutants for glup6 using the Tilling method.

FIG. 1 shows the results of SDS-PAGE of mature seeds with glup6 mutation and Western blot analysis using glutelin antibodies. The samples used were A: wild type (Taichung 65), B: EM939 ( glup6 mutant), C: EM1327 ( glup6 mutant). The left figure shows SDS-PAGE images of wild-type and glup6 mutant seed storage proteins. The right figure shows the results of Western blotting analysis using an anti-glutelin antibody. FIG. 2 shows a diagram showing a high-density linkage map of GLUP6 gene and a candidate genomic region. A GLUP6 gene linkage map was constructed using RFLP, STS and CAPS markers. The marker name at the top of the linkage map, the genetic distance from the chromosome short arm end in parentheses, and the number of recombination individuals at each marker at the bottom of the map. The OJ number indicates a BAC clone derived from Nipponbare. A and B are linked maps constructed using 41 and 234 glup6 homozygous individuals, respectively. C is a 1122 map based on physical maps of BAC clones aligned within the candidate region of the GLUP6 gene and markers generated in the BAC clones. In the linkage map constructed using individual populations, D indicates a predicted gene in the candidate region with an arrow. Double arrows from A to D indicate candidate regions for the GLUP6 gene. C169, R216: CAPS marker; E50818, R10784, C53358, R10784, C63279: STS marker; R4996: Unknown. FIG. 3 shows a comparison of the nucleotide sequences of the coding sequences of the GLUP6 gene. FIG. 3 shows a comparison of the nucleotide sequences of the coding sequences of the GLUP6 gene. FIG. 3 shows a comparison of the nucleotide sequences of the coding sequences of the GLUP6 gene. FIG. 3 shows a comparison of the nucleotide sequences of the coding sequences of the GLUP6 gene. FIG. 3 shows a comparison of the nucleotide sequences of the coding sequences of the GLUP6 gene. 3 to 5, the asterisk (*) below the sequence indicates a common sequence, and the period (.) Below the sequence indicates a mutation site. The results of analyzing the nucleotide sequences of the GLUP6 candidate genes of glup6 mutant lines “EM939”, “EM1327” and their original varieties “Taichung 65” and “Nippon Hare” are shown (SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9). As a result, one base substitution was observed in one of the two predicted strains of glup6 mutant. An asterisk (*) below the sequence indicates a common sequence. “EM939” and “EM1327” were shown to have C418 replaced with T and C784 replaced with T, respectively. The underline of the 814th and 927th bases indicates the polymorphism between “Nihonbare” and “Taichung 65”. Nipponbare: Coding sequence in the genome sequence of Os03g0262900, JO3309P13: Base sequence of cDNA clone JO3309P13 (excluding introns), cDNA: Single-stranded cDNA obtained by reverse transcription of mRNA extracted from Taichung 65 seed , Base sequence of cDNA amplified by GLUP6-GEF specific primer, Taichung No. 65: coding sequence in genomic DNA of the gene of Taichung 65, EM939: coding sequence in genomic DNA of the gene of EM939, EM1327 : Each coding sequence in the genomic DNA of the gene of EM1327 is shown. FIG. 4 shows a comparison of the base sequences of genomic DNA of the GLUP6 gene. FIG. 4 shows a comparison of the base sequences of genomic DNA of the GLUP6 gene. FIG. 4 shows a comparison of the base sequences of genomic DNA of the GLUP6 gene. FIG. 4 shows a comparison of the base sequences of genomic DNA of the GLUP6 gene. FIG. 4 shows a comparison of the base sequences of genomic DNA of the GLUP6 gene. FIG. 4 shows a comparison of the base sequences of genomic DNA of the GLUP6 gene. FIG. 4 shows a comparison of the base sequences of genomic DNA of the GLUP6 gene. FIG. 4 shows a comparison of the base sequences of genomic DNA of the GLUP6 gene. FIG. 4 shows a comparison of the base sequences of genomic DNA of the GLUP6 gene. FIG. 4 shows a comparison of the base sequences of genomic DNA of the GLUP6 gene. The nucleotide sequences of the genomic DNAs of the GLUP6 candidate genes of glup6 mutant lines “EM939” and “EM1327” and their original varieties “Tainaka 65” and “Nippon Hare” are shown (SEQ ID NO: 11 to SEQ ID NO: 14). An asterisk (*) below the sequence indicates a common sequence. FIG. 5 shows the nucleotide sequence (including intron) of cDNA clone JO3309P13. FIG. 6 is a diagram showing a comparison of amino acid sequences of GEF proteins. The amino acid sequences estimated from the GLUP6 candidate genes of glup6 mutant lines "EM939", "EM1327" and their original varieties "Taichung 65" and "Nippon Hare" (SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 10). An asterisk (*) below the sequence indicates a common sequence, a bar (-) indicates a deletion site, and a period (.) Below the sequence indicates a mutation site. “EM” of “EM939” and “EM1327”, respectively, “-” at 140th and 262th indicate the formation of stop codon due to mutation of each strain. The underline of the 272nd amino acid indicates a polymorphism between “Nipponbare” and “Taichung 65”. "Nippon Hare", "Taichung 65", "EM939", "EM1327" are the amino acid sequences deduced from the genomic base sequence, and "cDNA" is reverse transcribed from mRNA extracted from the ripening seeds of "Taichung 65" The amino acid sequence deduced from the base sequence of the cDNA of the gene is shown. FIG. 7 is a diagram showing the results of complementation assay in which glup6 mutant was transformed with GFP-GEF gene. Specifically, the results of SDS-PAGE (A) and Western blot analysis (B) of the seeds of T1 individuals transformed with GEF ORF designed to be expressed with the GEF promoter in EM939 were shown. Figure. For Western blot analysis, GEF antibody and GFP antibody were used. The arrow indicates a band of 57-kD glutelin precursor that decreases with glup6 mutation. The numbers indicate the individual number of T1, and 6 grains were tested from each individual. 1-6: Seeds obtained by self-breeding of transformed individuals, asterisks (2, 3, 4): Seed of glutelin precursor at the same level as Taichung No. 65, GFP protein expressed seeds, unmarked ( 1, 5, 6): Seeds that accumulate the same level of glutelin precursor as EM939 and do not express GFP protein. An asterisk (*) indicates a seed that has been restored by transformation. FIG. 8 shows the results of analysis of GEF expression in ripened seeds by Western blot using a GLUP6-GEF antibody. FIG. 9 is a diagram showing the results of observation of the endosperm cell tissue of the glup6 mutant using a fluorescence microscope. A and B: Wild type (Taichung 65), C and D: EM939 ( glup6 mutant), E and F: EM1327 ( glup6 mutant). The site labeled with green (dark ash) indicates that the glutelin antibody has reacted, and the site labeled with red (light ash) indicates that the glutelin antibody has reacted. The upper row (A, C and E) shows the endosperm tissue in the early stage of ripening (1 week after flowering), and the lower row (B, D and F) shows the endosperm tissue in the late stage of ripening (3 weeks after flowering). . FIG. 10 shows the localization of β-glucan and glutelin in the glup6 mutant using a fluorescence microscope. AC: Wild type (Taichung 65), DF: EM939 ( glup6 mutant), GI: EM1327 ( glup6 mutant). Sites labeled with green (dark ash) (A, D, G) indicate that β-glucan antibody has reacted, and sites labeled with red (light ash) (B, E, H) indicate that glutelin antibody has reacted. . C, F, and I are merged images of A and B, D and E, and G and H, respectively. FIG. 11 shows the localization of β-glucan and glutelin in the glup4 mutant using a fluorescence microscope. AC: Wild type (Taichung 65), DF: EM956 ( glup4 mutant). The sites labeled with green (dark ash) (A, D) indicate that the β-glucan antibody has reacted, and the sites labeled with red (light ash) indicate that the (B, E) glutelin antibody has reacted. C and F are merged images of A and B and D and E, respectively. FIG. 12 is a diagram showing a comparison of the baking characteristics of wild type (Taichung 65) and glup6 using GOPAN.

[Mode for Carrying Out the Invention]

  The present inventors focused on rice for which development of a simple method for modifying seed components is desired among plants, and in particular, a gene involved in the accumulation of storage protein glutelin, which is nutritionally good, is identified. We conducted earnest research to separate them.

  Rice glutelin is synthesized as a precursor on the endoplasmic reticulum and then transported to the storage vacuole via the Golgi apparatus. In storage vacuoles, it is cleaved into two subunits and accumulates as mature glutelin. The glup6 (glutelin precursor 6) mutant accumulates more glutelin precursors than the wild-type and reduces the accumulation of mature glutelin (Figure 1). Therefore, although it is expected to be a factor involved in the transport of the glutelin precursor to the storage-type vacuole, the GLUP6 gene causing the mutation has not yet been identified.

  It has already been shown that the GLUP6 gene sits on chromosome 3 (Sato et al. Rice Genet. Newslet., 20: 43-45, 2003). Therefore, we constructed a genetic linkage map near the GLUP6 gene by linkage analysis using a chromosome 3 marker. GLUP6 gene linkage map was prepared using STS and CAPS markers. From homozygous F2 seeds of the glup6 mutant line EM939 and the Indian cultivar Kasalath, a glup6 gene homozygous individual that accumulates a large amount of glutelin precursor was selected by SDS-PAGE analysis and used for linkage analysis.

  By linkage analysis using 41 glup6 homozygous individuals, the GLUP6 gene was located in the 10.4 cM region between the STS marker R10784 and the CAPS marker R216 (FIG. 2A: Sato et al. Rice Genet. Newslet., 20: 43-45, 2003). Furthermore, the GLUP6 gene was narrowed down to a region of 1.6 cM by linkage analysis using 234 glup6 gene homozygous individuals (FIG. 2B). This gene region was covered by 4 BAC clones (FIG. 2C).

  The GLUP6 gene was narrowed down to a region of about 16 kb in the BAC clone OJ1041F02 by linkage analysis using newly created DNA markers based on the sequences in these BAC clones and 1122 homozygous glup6 genes (Fig. 2C and 2D). When gene prediction and similarity search were performed using RiceGAAS (Rice Genome Annotation Database; http://RiceGAAS.dna.affrc.go.jp/rgadb/) for the genome sequence of this candidate region, three genes were found. Was predicted (Figure 2D).

  Based on the genomic sequences of these predicted genes, the genomic nucleotide sequences of the GLUP6 gene candidate regions of glup6 mutant lines “EM939” and “EM1327” and their original cultivar “Taichung 65” were analyzed. As a result, one of the predicted genes showed a single base substitution for the base sequence of “Taichung 65” in the two glup6 mutants. Specifically, when compared with “Taichung 65” and the glup6 mutant, C418 was replaced with T in EM939, and C784 was replaced with EM1327, respectively. No substitution was found for other bases in the candidate region. Furthermore, the base sequence of cDNA reverse transcribed from the mRNA of the predicted gene isolated from the ripened seed of “Taichung 65” was analyzed. Fig. 3 shows their base sequences.

  The genome base sequence of each strain showed only the coding region sequence corresponding to the cDNA (FIG. 4). In addition, a 6-base polymorphism was observed in the genomic base sequence of the gene between “Taichung 65” and “Nippon Hare” (FIG. 4). This predicted gene has high homology in amino acid sequence with guanine nucleotide exchange factor (GEF). This result suggests that the GLUP6 gene encodes GEF.

  In “Nipponbare”, the full-length cDNA sequence of the predicted gene was searched by KOME (Knowledge-based Oryza Molecular biological Encyclopedia; http://cdna01.dna.affrc.go.jp/cDNA/) As a result of comparison, and as a result of comparing the genomic base sequence of “Taichung 65” with cDNA, the GLUP6 candidate gene was composed of 5 exons. The total length of the coding region of this gene was 1443 bp, and it was predicted to encode a protein consisting of 480 deduced amino acids (FIG. 6: Comparison of amino acid sequences of GEF protein). In glup6 mutant lines “EM939” and “EM1327”, the 139th and 261st glycines were replaced with stop codons, respectively (FIG. 6). These results suggest that the GLUP6 gene encodes GEF.

  In order to confirm that this GEF gene has a function for transport and accumulation of glutelin, a transformation experiment was carried out in which a GLUP6 candidate gene was introduced into a plant of “glup6 mutant”. GLUP6 candidate gene GEF promoter is subcloned from the genomic DNA of Taichung 65, and the GEF sequence (JO33091P13: distributed from Tsukuba Genome Resource Center) is used as a template to amplify the GEF sequence by PCR and include the constructed GFP The vector was introduced into the DV9 binary vector and introduced into “EM939” via Agrobacterium (EHA105 strain). As a result, the produced transformant had a glutelin precursor content reduced to almost the same as that of the original variety (FIG. 7A). Furthermore, Western blotting using a GFP antibody in the individual revealed a GFP band (FIG. 7B), indicating that the glup6 mutant was restored to the wild type by the introduction of the GEF gene. . From the above results, it was revealed that the GLUP6 gene encodes GEF.

  The presence or absence of GEF protein expression in the glup6 locus was investigated (FIG. 8). In “Taichung 65”, two bands were detected. On the other hand, in “EM939” and “EM1327”, the 63 kDa band of the two bands was missing. The lack of GEF protein in the glup6 mutant suggests that the function of the GEF has been lost due to the nonsense mutation.

  The localization of glutelin in the endosperm cell tissue of GEF-deficient mutant glup6 was investigated by fluorescence microscopy. In the wild type, a granule accumulating glutelin (PB-II) and a prolamin accumulating granule (PB-I) were observed alone, and the size of both PBs increased with ripening (Figures 9A and 9B). ). On the other hand, in the glup6 mutation, a large structure (paramural body: PMB) labeled with a glutelin antibody was observed. PMB has existed since the beginning of ripening, and its size increased with ripening (FIGS. 9C, 9D, and 9F). The glup6 mutant PB-II was almost constant in size from early to late ripening. These results indicate that glup6 mutation inhibits the transport of glutelin precursor to storage vacuoles. Furthermore, a signal indicating the presence of glutelin was also observed in the cell wall. From this result, it is considered that the intracellular transport of β-glucan, which is a constituent of the cell wall, may also be inhibited. Therefore, the localization of β-glucan and glutelin in the glup6 mutant was investigated by fluorescence microscope analysis. In the wild type, β-glucan was detected only in the cell wall (Figs. 10A and 10C), but in the glup6 mutant, β-glucan was present not only in the cell wall but also around and within the PMB (Fig. 10D to FIG. 10I). In the glup6 mutant, cell wall-like substances containing β-glucan were found to accompany PMB. These results suggest that glup6 mutation also inhibits the transport of β-glucan to the cell wall, possibly changing the structure of the cell wall, and increasing the accumulation of β-glucan.

  The glup4 mutation that accumulates many glutelin precursors is a structural gene mutation of Small GTPase Rab5a. Similarly, in the glup4 mutant, high accumulation of β-glucan was observed around PMB (FIGS. 11D to 11F). Using a mixed-linkage β-glucan assay kit from Megazyme, β-glucan contained in brown rice of wild type varieties (Taichung 65) and glup6 (EM939) was quantitatively compared. -Glucan was 48 mg (± 14, n = 3) per 100 g (dry weight), whereas 97 mg (± 49, n = 3) accumulated about twice as much in brown rice of glup6 It was.

  In order to investigate how the high accumulation of β-glucan affects the bread-making characteristics of rice flour and the taste of rice flour bread, we used the new product GOPAN (Breadmaker, registered trademark) of Sanyo Electric, glup6 We made bread using mutant (EM939) and wild-type (Taichung 65) rice flour and compared them (Fig. 12). As a result of repeated bread-making tests, bread dough made from wild-type rice flour was soft immediately after baking, but the dough started to harden after a few hours and became vomited after 2 days, while glup6 ( EM939), it was found that under similar conditions, the dough is less likely to be harder than the wild type. In general, rice flour bread has a problem of becoming harder with time. This problem is not limited to rice flour bread but is an important evaluation item in the quality of normal wheat bread, and is called “bread aging”. The cause of “rice flour bread that is difficult to age” shown by glup6 mutant (EM939) is the moisturizing characteristics of β-glucan accumulated in PMB, the aging characteristics of starch itself, or water-soluble β-glucan is glue There is a possibility of inhibiting aging by binding to modified starch.

  The present inventors have succeeded in isolating a new gene involved in the accumulation of plant storage protein, and using the gene, rice flour in which the composition of the plant storage protein and β-glucan is changed is excellent. It has been found that it has the characteristics of making bread, and this led to the completion of this study.

The present invention provides a GLUP6-GEF gene, that is, the following polynucleotide (or a gene comprising a polynucleotide), and a method for using the same, related to accumulation of plant storage proteins in storage vacuoles.
(A) a polynucleotide comprising the base sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 11, or SEQ ID NO: 12;
(B) a polynucleotide comprising a nucleotide sequence complementary to a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 11 or SEQ ID NO: 12 A polynucleotide encoding a protein that hybridizes with a nucleotide under stringent conditions and has a function of accumulating the storage protein in a storage vacuole;
(C) having at least 90% identity with and storing the nucleotide sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 11 or SEQ ID NO: 12 A polynucleotide encoding a protein having a function of accumulating the protein in storage vacuoles;
(D) a polynucleotide encoding the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 6;
(E) a storage type vacuole comprising a storage protein consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, added and / or inserted in the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 6, and A polynucleotide encoding a protein having a function of accumulation in
(F) Coding a protein comprising an amino acid sequence having at least 97% identity with the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 6, and having a function of accumulating the storage protein in a storage type vacuole Polynucleotide.

The present invention relates to proteins relating to accumulation of plant storage proteins in storage-type vacuoles, namely the following proteins, and uses thereof.
(E ′) a protein comprising the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 6;
(F ′) an amino acid sequence in which one or more amino acids are deleted, substituted, added and / or inserted in the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 6, and glutelin is accumulated in plant seeds A protein having a function of
(G ′) A protein comprising an amino acid sequence having at least 97% identity with the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 6, and having a function of accumulating glutelin in plant seeds.

In the present invention, when referring to “stringent conditions” with respect to a polynucleotide, it refers to moderately or highly stringent conditions, unless otherwise specified.
Moderately stringent conditions can be easily designed by those skilled in the art based on, for example, the length of the polynucleotide of interest. Basic conditions are shown in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd edition, chapters 6-7, Cold Spring Harbor Laboratory Press, 2001. Typically, moderately stringent conditions are 5 × SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0) pre-wash conditions for nitrocellulose filters; about 50-50 ° C. at about 50-50 ° C. Hybridization conditions of% formamide, 2-6 × SSC (or other similar hybridization solutions such as Stark's solution in about 50% formamide at about 42 ° C.); and about 40 ° C.-60 ° C., 0.5- Includes 6 x SSC, 0.1% SDS wash conditions. The moderately stringent conditions preferably include hybridization conditions of about 50 ° C. and 6 × SSC, and may include the aforementioned washing conditions and / or washing conditions.

  Highly stringent conditions (high stringent conditions) can also be easily designed by those skilled in the art based on, for example, the length of the polynucleotide of interest. High stringency conditions include higher temperatures and / or lower salt concentrations than moderately stringent conditions. Typically, hybridization conditions of about 65 ° C., 0.2-6 × SSC, preferably 6 × SSC, more preferably 2 × SSC, and even more preferably 0.2 × SSC are included. In any case, it is preferable to include washing conditions of about 65 to 68 ° C., 0.2 × SSC, and 0.1% SDS.

In either case, SSPE (1 × SSPE is 0.15 in place of SSC (1 × SSC is 0.15 M NaCl and 15 mM sodium citrate) as a buffer for hybridization, prewash and wash. M NaCl, 10 mM NaH ₂ PO ₄ , and 1.25 mM EDTA, pH 7.4) can be substituted. In either case, washing can be performed for about 15 minutes after hybridization is complete.

  Further, when hybridization is performed under stringent conditions for the present invention, a commercially available hybridization kit that does not use a radioactive substance as a probe, for example, ECL direct labeling & detection system (manufactured by Amersham) can be used. . When using this kit, stringent hybridization typically involves adding 5% (w / v) blocking reagent and 0.5 M NaCl to the hybridization buffer in the kit, Hybridization for 4 hours, followed by two 20 minute washes in 55 ° C., 0.4% SDS, 0.5 × SSC, and a further 5 minutes wash in 2 × SSC at room temperature.

  In addition, regarding the protein in the present invention, the number of amino acids to be substituted when “one or more amino acids are deleted, substituted, added and / or inserted” indicates the desired function of the protein comprising the amino acid sequence. Although it is not specifically limited as long as it has, it is about 1-9 pieces or 1-4 pieces. Since substitution of amino acids with similar properties will not lose the desired function, substitution of more amino acids may be possible. Means for preparing polynucleotides or proteins in which nucleotides or amino acids are substituted include, for example, the site-directed mutagenesis method (Kramer W & Fritz HJ: Methods Enzymol 154: 350, 1987).

  In the present invention, when the identity (sometimes referred to as homology) is high with respect to the base sequence, it is 90% or more, preferably 92% or more, more preferably 94% or more, even more preferably, unless otherwise specified. Refers to sequence identity of 96% or more, most preferably 98% or more. In the present invention, when amino acid sequences have high identity (sometimes referred to as homology), except for special cases, 97% or more, preferably 98% or more, more preferably 99% or more. Refers to identity. The identity of the base sequence or amino acid sequence is determined using the algorithm BLAST (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990, Proc Natl Acad Sci USA 90: 5873, 1993) by Carlin and Arthur. it can. Programs called BLASTN and BLASTX based on the BLAST algorithm have been developed (Altschul SF, et al: J Mol Biol 215: 403, 1990). When analyzing a base sequence using BLASTN, parameters are set to, for example, score = 100 and wordlength = 12. In addition, when an amino acid sequence is analyzed using BLASTX, the parameters are, for example, score = 50 and wordlength = 3. When using BLAST and Gapped BLAST programs, the default parameters of each program are used. Specific techniques for these analysis methods are well known to those skilled in the art (for example, http://www.ncbi.nlm.nih.gov/).

  In the present invention, the identity of a base sequence can also be calculated using BLAST 2, and the identity can be determined by searching for the amino acid sequence with blastp. In the present invention, when referring to identity with respect to a base sequence or amino acid sequence, it is a value obtained by these means under normal settings, except in special cases.

The polynucleotide of the present invention can be prepared from natural plant tissue material. In order to prepare the polynucleotide of the present invention, a hybridization technique or a polymerase chain reaction (PCR) technique can be used. For example, DNA having high identity with the GLUP6-GEF gene can be isolated from rice or other plants using a part of the GLUP6-GEF gene (SEQ ID NO: 3) as a probe or primer. The polynucleotide of the present invention includes genomic DNA, cDNA and chemically synthesized DNA. The polynucleotide of the present invention can be single-stranded DNA and double-stranded DNA.

In the present invention, “having a function” or “capable of function” with respect to a polynucleotide means that the target function is fulfilled by the presence of the polynucleotide, except in special cases. This includes transcription of the polynucleotide, translation of the transcript, and expression of the protein with the desired function. For example, if a mutant of the GLUP6-GEF gene has a mutation in a relatively important part, and thus the inherent function is reduced or lost, the mutant polynucleotide is said to be “functional” Neither can it be said that it is “functional”.

In the present invention, “storage protein” includes glutelin and globulin unless otherwise specified.
In the present invention, the term “glutelin” is one of pure proteins, unless otherwise specified, is contained in cereals, is not soluble in pure water / neutral salt solution and alcohol, and is diluted with dilute acid / diluted alkali. Means soluble protein (generic name). In the present invention, the term “storage protein” or “glutelin” simply refers to a mature form. In rice, glutelin accounts for about 60% of the total protein. Glutelin contains glutenin in gluten of wheat, and includes α-glutelin (molecular weight 37-40 kD in rice) and β-glutelin (molecular weight 20-23 kD in rice).

  In the present invention, the term “glutelin precursor” refers to a glutelin precursor having a molecular weight of about 57 kD, unless otherwise specified. From the definition of glutelin, the 57 kDa glutelin precursor is not included in glutelin. This precursor belongs to the salt-soluble “globulin”. In the specification, as an example of storage protein, glutelin in rice may be described as an example, but unless otherwise specified, explanations thereof include beans such as soybean, globulin such as oat, pumpkin, or Arabidopsis thaliana. The same applies to globulins in plants that accumulate in storage vacuoles.

  Mature glutelin can be quantified by extraction methods. For example, the extraction and quantification method of Kumamaru et al. Can be referred to (Kumamaru, T., Satoh, H., Iwata, N., Omura, T., Ogawa, M., and Tanaka, K. (1988). Mutants for rice storage proteins. 1. Screening of mutants for rice storage proteins of protein bodies in the starchy endosperm. Theor. Appl. Genet. 76, 11-16.

  To quantify glutelin precursors, glutelin and standards can be processed by SDS-PAGE, stained, and based on the intensity of the appearing band. More specifically, after pulverizing each of the matured brown rice of the control rice and the matured brown rice of the improved rice improved from the evaluation target rice, the SDS solution (50 mM Tris-HCl, pH 6. 8, 0.5% (w / v) SDS), apply to SDS-PAGE gel, and stain with CBB after electrophoresis. For each content, a calibration curve is prepared in advance if necessary, and the concentrations of the bands are compared with an image analyzer.

In the present specification, regarding the accumulation of storage protein, “modify” or “control” refers to modifying or regulating the amount of accumulation of storage protein in seeds, unless otherwise specified. Includes adjusting to increase the accumulation amount and adjusting to reduce the accumulation amount. When adjusting the amount of stored protein, factors related to other seeds may be adjusted at the same time. According to the study by the present inventors, the glup6 mutation inhibited the transport of the glutelin precursor to the storage-type vacuole, and a signal indicating the presence of glutelin was also observed in the cell wall. From this result, it was considered that the intracellular transport of β-glucan, which is a constituent of the cell wall, may also be inhibited. When the localization of β-glucan and glutelin in the glup6 mutant was investigated by fluorescence microscopy, β-glucan was detected only in the cell wall in the wild type, but in the glup6 mutant, β-glucan was found only in the cell wall. It was also present around and within the PMB. That is, these results suggest that glup6 mutation also inhibits the transport of β-glucan to the cell wall, possibly changing the structure of the cell wall, and increasing the accumulation of β-glucan .

In order to adjust to reduce the accumulation amount of the storage protein, the storage protein is reduced by reducing or losing the function of the gene involved in the storage protein accumulation (for example, the GLUP6-GEF gene of the present invention). Is included. In order to regulate the increase in glutelin accumulation, a gene involved in glutelin accumulation (for example, the GLUP6-GEF gene of the present invention) is introduced into a plant to exert its function, thereby increasing the glutelin content. included.

In the present specification, when the accumulation of storage protein is modified with respect to plant traits, the amount of mature storage protein is decreasing and increasing, unless otherwise specified. Including both. For example, the present invention, mutated GLUP6-GEF gene with rice from varieties with enabling function GLUP6-GEF gene, rice obtained by reducing or losing its function, compared to rice normal varieties Thus, the content of mature glutelin in the seed is reduced. Such a state can be referred to as “low glutelin (sex)” or “glutelin low accumulation (sex)”.

  In some embodiments of the invention, mature glutelin is reduced by at least about 5%, preferably by about 10%, more preferably by about 20%, compared to the original variety. If it is reduced by about 25%, it can be referred to as “low glutelin (sex)” or “glutelin low accumulation (sex)”. In this case, typically the amount of globulin precursor is increased by at least 5%, preferably increased by about 10%, more preferably if reduced by about 20%, more preferably about 25%. Has been reduced.

  In the present invention, the term “plant” is used to mean a plant individual or a part thereof, except for special cases, and the term “part” refers to a seed (a germinated seed, Including immature seeds), organs or parts thereof (including leaves, roots, stems, flowers, stamens, pistils, fragments thereof), plant culture cells, callus, protoplasts. Plants include genetically engineered plants and transformed plants. Plants include “crop” and “breeding material”. The “reproduction material” as used in the present invention refers to a material (or a “seed seedling”) that is used for propagation in all or part of a plant body, except for special cases. There are seedlings, cells, callus, shoots. The term “harvested product” as used in the present invention is used in a normal sense except in special cases. All or part of the plant body is not used for breeding, for example, the plant belongs to the genus Rice. The harvest includes harvested rice and fir.

The plant of the present invention includes a transformed plant (T ₀ ) obtained by obtaining a recombinant plant cell by the method of the present invention and regenerating the plant cell into a plant body. In the present invention, with respect to a plant, the term “its progeny” refers to a plant having the plant as at least one genetic parent and / or ancestor unless otherwise specified. As long as the target trait is inherited using the polynucleotide of the present invention, the progeny includes either a progeny (T ₁ etc.) obtained from the parent transformed plant or its progeny, T ₀ or T ₁ The progeny of the cross (eg, F ₁ ) and its descendants that are parents of

  In the present invention, “processed product” refers to a processed product produced directly or indirectly from a harvested product, unless otherwise specified. The scope of the present invention includes at least processed products that reflect the characteristics of the harvest of the present invention. For example, when the plant is rice, the rice as a raw material is used as a raw material, and the processability, taste, texture, smell, etc. are different from conventional ones due to the control of glutelin accumulation, Polished rice, cooked rice, rice flour, breads using rice flour, confectionery, buns, pizza, and sake are included in the scope of the present invention. Bread is a particularly preferred example of the processed product of the present invention (JP 2009-213370).

The present invention also provides a recombinant vector comprising the polynucleotide of the present invention, and a transformed plant transformed with such a vector.
The vector into which the polynucleotide of the present invention is inserted is not particularly limited as long as it can exhibit the function of the insert in plant cells. For example, using a vector having a promoter (eg, cauliflower mosaic virus 35S promoter) for constitutive expression of the gene in plant cells, or a vector having a promoter inducibly activated by an external stimulus. You can also. As used herein, “plant cells” include various forms of plant cells such as seeds, suspension culture cells, protoplasts, leaf sections, callus and the like.

  For introduction of a vector into a plant cell, various methods known to those skilled in the art such as polyethylene glycol method, electroporation method (electroporation method), Agrobacterium-mediated method, particle gun method and the like can be used. In the method using Agrobacterium (for example, EHA101), for example, an ultra-rapid monocotyl transformation method (Japanese Patent No. 314084) can be used. Further, in the particle gun method, for example, a product from Bio-Rad can be used.

  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, as a method for producing a transformed plant body in rice, a method of regenerating a plant body (suitable for Indian rice varieties) by introducing a gene into protoplasts using polyethylene glycol (Datta SK: In Gene Transfer To Plants (Potrykus I and Spangenberg, Eds) pp.66-74, 1995), a method of regenerating plants (Japanese rice varieties are suitable) by introducing genes into protoplasts by electric pulses (Toki S, et al: Plant Physiol 100: 1503, 1992), a method of introducing a gene directly into a cell by the particle gun method and regenerating the plant body (Christou P, et al: Biotechnology 9: 957, 1991), and gene via Agrobacterium Several techniques have already been established and widely used in the technical field of the present invention, such as a method for introducing plant and regenerating plant bodies (Hiei Y, et al: Plant J 6: 271, 1994). In the present invention, these methods can be suitably used.

  Once a transformed plant having a desired gene introduced into its genome is obtained, offspring can be obtained from the plant by sexual reproduction or asexual reproduction. It is also possible to obtain propagation materials (for example, seeds, fruits, cuttings, tubers, tuberous roots, strains, callus, protoplasts, etc.) from the plant and its descendants or clones, and mass-produce plants based on them. It is.

  The plant to be transformed according to the present invention is not particularly limited as long as it can control the accumulation of glutelin by the action of the gene comprising the polynucleotide of the present invention, but is preferably an angiosperm, more Preferably, it is a plant belonging to the monocotyledonous network, more preferably a plant belonging to the Cyprus subnet, most preferably a plant belonging to the family Gramineae (Poaceae (Gramineae)), for example, rice (Oryza), barley, rye, bread wheat. It is a plant belonging to any of the genera, barley, pearl barley, sugar cane, corn, sorghum, millet, millet and millet. Among the plants belonging to the family Gramineae, the plant is preferably a plant belonging to the genus Graminea, and more preferably rice (Oryza sativa L.). From the viewpoint that the plant to be transformed of the present invention is a plant that accumulates storage proteins in storage vacuoles, in particular, soybean, corn, rye, oat, wheat, barley, sorghum, pumpkin, Arabidopsis thaliana, Suitable for use on rice.

  Among rice varieties, the present invention is considered to be particularly suitable for “Koshihikari” and “Hinohikari” and their related species. Koshihikari relatives are those that use “Koshihikari” as the breeding parent in their breeding lineage, such as “Hinohikari” and “Kinuhikari”. Furthermore, the present invention is also applicable to good-tasting varieties such as “Sasanishiki” other than Koshihikari-related species, Mochi varieties such as “Shigaha Du Mochi”, “Habataki”, “Ochikara”, etc. It is considered suitable. The present invention is also considered to be suitable for high-yielding varieties such as “Mizhotachikara” and Indian varieties such as IR64.

  The present invention also provides a method for controlling storage protein accumulation in plants, which comprises using the polynucleotide of the present invention. As used herein, “utilizing (polynucleotide)” means that a plant having a polynucleotide functionally is allowed to reduce or lose the function of the polynucleotide, and a plant in which the polynucleotide is not functioning. Functionally introducing the polynucleotide is included.

  To generate a phenotype that reduces or eliminates the function of the polynucleotide, for example, by suppressing the expression of a gene comprising the polynucleotide of the present invention. As used herein, “suppression of expression” includes suppression of gene transcription, suppression of transcript splicing, and suppression of protein translation. Also included is a decrease in expression as well as complete cessation of gene expression. Moreover, suppression of the translated protein exerting its original function in plant cells is also included.

  According to the present invention, there is provided a “glup6 deletion mutation (body)” (which has reduced or lost the function of a gene comprising the polynucleotide of the present invention, resulting in a phenotype that is less accumulative in glutelin). The method for producing a deletion mutant is not particularly limited as long as a mutant in which the expression of the target gene or the function of the target protein is reduced or lost can be prepared. For example, mutagenesis is applied to a plant by chemical mutagen treatment or radiation treatment. , Viral infection, transposon transfer, spontaneous mutation. In addition, there are gene disruption by introduction of DNA fragments, gene expression suppression by antisense nucleic acid and the like.

  Suppression of gene expression can also be performed using a polynucleotide encoding a ribozyme. It is possible to design ribozymes that cleave RNA in a site-specific manner. In addition, gene expression can also be suppressed by RNA interferance (RNAi) using double-stranded RNA having the same or similar sequence as the target gene sequence. RNAi is known to have an effect in plants (Chuang CF & Meyerowitz EM: Proc Natl Acad Sci USA 97: 4985, 2000). Furthermore, suppression of gene expression can also be achieved by co-suppression caused by transformation with DNA having the same or similar sequence as the target gene sequence. “Co-suppression” refers to a phenomenon in which the expression of a foreign gene and a target endogenous gene are both suppressed when a gene having the same or similar sequence as the target endogenous gene is introduced into a plant by transformation. Point to. The details of the mechanism of co-suppression are not clear, but at least part of the mechanism is thought to overlap with that of RNAi. Co-suppression is also observed in plants (Smyth DR: Curr Biol 7: R793, 1997, Martienssen R: Curr Biol 6: 810, 1996).

[Materials and methods]
Plant material:
The glup6 mutant lines "EM939" and "EM1327" induced by fertilized egg treatment using the rice cultivar "Taichung 65" and the same N- methyl- N- nitrosourea were used as materials (Satoh et al, 2010). , Ueda et al, 2010). Ripe seeds and mature seeds of these lines were used for analysis. For the construction of the gene linkage map, hybrid F2 seeds of glup6 mutant line “EM939” and Indian variety “Kasalath” were used as material.

SDS-polyacrylamide gel electrophoresis (PAGE) analysis:
Grind 1 or half seed and add 200 μl sample buffer (0.125 M Tris-HCl, 4% SDS, 4 M urea, and 5% β-mercaptoethanol, pH 6.8) to 10 mg of seed The protein was extracted by shaking overnight. Extracted proteins were separated by SDS-PAGE. After completion of electrophoresis, the gel was stained with 0.5% Coomassie blue containing 50% ethanol and 7% acetic acid, and the stained gel was decolorized with 25% ethanol and 7% acetic acid.

Western blot analysis:
The protein was transferred from the gel after completion of SDS-PAGE to a nitrocellulose membrane at 10 V for 1 hour using a semi-driving apparatus. The transferred nitrocellulose membrane was immersed in a blocking buffer (Tris buffer saline containing 5% skim milk (TBS: 10 mM Tris-HCl, 0.15 M NaCl, pH 7.5)) for blocking treatment for 1 hour. After blocking, the nitrocellulose membrane was reacted overnight with a primary antibody diluted with a blocking buffer, and then washed three times with TBST (TBS containing 0.05% Tween 20) for 10 minutes. After washing, the mixture was reacted for 1 hour with a secondary antibody diluted with a blocking buffer, and then washed 3 times with TBST for 10 minutes. The target band was detected on the nitrocellulose membrane using an ECL reagent kit (GE healthcare, Buckinghamshire, UK). As the primary antibody, anti-α-gluterin-rabbit antibody and anti-GEF (VPS9) -rabbit antibody were used. As the secondary antibody, anti-rabbit IgG-goat antibody and anti-mouse IgG-goat antibody were used.

Gene linkage map construction:
In order to determine the genotype of the hybrid F2 seed of “EM939” and “Kasalath”, each offspring was cut in half, and the protein extracted from the site not containing the embryo was analyzed by SDS-PAGE. Seeds with a large amount of glutelin precursor accumulated by SDS-PAGE analysis were homozygous for the glup6 gene. The part containing the seed embryo that was glup6 homozygous was sown and DNA was extracted from the leaves of the grown seedlings and used for linkage analysis.

DNA extraction:
About 0.2 g of fresh leaves were dried and pulverized using a multi-bead shocker (manufactured by Yasui Kikai Co., Ltd.). Total DNA was extracted from the crushed leaves using the CTAB (cetyltrimethylammonium bromide) method (Murray and Thompson, 1980).

Subcloning of promoter and GLUP6 candidate genes:
Using the genomic DNA of “Taichung 65” as a template, a region about 2 kb upstream from the start codon of the GLUP6 candidate gene was amplified by a PCR reaction using the primers shown below, and used as a promoter for the GLUP6 candidate gene.
5 'primer: 5'-ata gtcgac cagtgga cacgtagtgg cttattg-3' (SEQ ID NO: 15)
( Gtcgac is the cleavage sequence by restriction enzyme SalI)
3 'primer: 5'-tat gaattcagatct ccggattcggtcacgttttgtg-3' (SEQ ID NO: 16)
( Gaattc and agatct are sequences cut by restriction enzymes EcoRI and Bgl II)
To obtain the coding sequence of GLUP6 candidate gene, using cDNA clone (J033091P13) as a template, 94 ℃ -1min (1 cycle), 94 ℃ -30sec → 61 ℃ -30sec → 72 ℃ -2min (30cycles) ), PCR was performed at 72 ° C. for 2 minutes (1 cycle). The GLUP6 candidate gene promoter and the coding sequence of the gene were introduced into an entry vector (EV (zeo) 126) containing GFP by restriction enzyme treatment and ligation reaction with SalI / EcoRI and BglII / XhoI, respectively. Thereafter, the constructed entry vector was inserted into a binary vector (DV9) by LR reaction (LRclonase).

Transformation to rice by the Agrobacterium method:
Ripe seeds of “EM939” were sterilized and then sown in a callus induction medium. Callus 1 month after sowing was used for transformation. The constructed construct was introduced into callus "EM939" by Agrobacterium (EHA105 strain).

Genomic sequence analysis:
Template preparation PCR: Using a genomic DNA extracted from leaves as a template, a sequence template was prepared using LA Taq polymerase (Takara) according to the attached protocol. Prepare a PCR reaction solution using 1 ng genomic DNA (100 ng / μl), 94 ℃ -30 seconds (1 cycle), 94 ℃ -30 seconds → 61 ℃ -30 seconds → 72 ℃ -2 minutes (30 cycles) ), PCR was performed at 72 ° C. for 2 minutes (1 cycle). Thereafter, the PCR product was purified by ethanol precipitation and concentrated.

Sequence reaction: 1 μl DNA solution (200-500 ng), 1 μl Big dye, 1.5 μl sequencing reaction solution (5 ×), 1.5 μl dH ₂ O, 5 μl 1/100 μM primer solution are mixed, then 96 ° C. for 3 minutes ( PCR was carried out under the conditions of 1 cycle), 94 ° C.-15 seconds → 50 ° C.-20 seconds → 60 ° C.-1 minute (30 cycles).

Base sequence analysis: Genome base sequence and cDNA base sequence were analyzed using a gene analyzer, ABI PRISMR3100 Genetic Analyzer (Applied Biosystems). Each nucleotide sequence obtained was converted into the nucleotide sequence between the wild type and the mutant using the CLUSTALW analysis software of DNA Data Bank of Japan (DDBJ) (http://www.ddbj.nig.ac.jp/Welcome-j.html). The sequence and deduced amino acid sequence were compared.

Seed fixation and embedding:
Ripened seeds were cut to a thickness of about 1 mm, soaked in fixative (50 mM Pipes containing 25% glutaraldehyde, 2% paraformaldehyde), 0.25 M sucrose, pH 7.24), and cells at 4 ° C overnight. Fixed. The fixed sections were washed 3 times for 10 minutes with 50 mM Pipes, 0.25 M sucrose (pH 7.24) buffer. After washing, dehydration was performed for 10 minutes at each concentration using an ethanol dilution series of 30, 50, 60, 70, 80, 90, 95, 100% (V / V). After dehydration, use LR-White resin concentration series (LR-White resin: ethanol = 1: 3 → 1: 2 → 1: 1 → 3: 1 → LR-White resin 100% x 3 days) in the sample. The resin was infiltrated. After penetration of the resin, the sample was poured into a gelatin capsule, poured into the resin, and then filled with nitrogen. The mixture was placed in a thermostatic chamber and polymerized by heating at 60 ° C. for 24 hours or more.

Fluorescent staining method:
All work was performed in a wet room and at room temperature. PBS (10 mM Na ₂ HPO ₄ , 10 mM NaH ₂ PO ₄ , 150 mM NaCl, pH 7.4) was added dropwise to the sections, allowed to stand for 10 minutes, and blocking buffer (0.8% BSA, 0.1% gelatin, 2 mM NaN) _The reaction was carried out for 10 minutes with PBS containing ₃ at pH 7.4). After the blocking reaction, the primary antibody solution diluted with a blocking buffer was reacted for 2 hours. After the reaction, the section was washed 5 times with PBS for 10 minutes and then reacted with a blocking buffer for 10 minutes. The reaction was carried out for 1 hour with a secondary antibody solution diluted with blocking buffer for 1 hour. After the reaction, the sections were washed 5 times with PBS for 10 minutes and then washed 3 times with distilled water for 1 minute. After drying, an anti-fading agent was dropped onto the sections, covered with a cover glass and sealed with nail polish. As primary antibodies, anti-β-gluterin-mouse antibody, anti-α-gluterin-rabbit antibody, anti-14 prolamin-rabbit antibody and anti-β-glucan-mouse antibody were used. As secondary antibodies, FITC-labeled anti-rabbit IgG-goat antibody, rhodamine-labeled anti-mouse IgG-goat antibody, FITC-labeled anti-mouse IgG-goat antibody, and rhodamine-labeled anti-rabbit IgG-goat antibody were used.

Example 1: Identification of candidate genes
The glup6 ( glutelin precursor 6 ) mutant accumulates more glutelin precursors than the wild type and reduces the amount of mature glutelin accumulated. It is a factor involved in the transport of glutelin precursors to storage-type vacuoles. Expected to be. It has already been shown that the GLUP6 gene sits on chromosome 3 (Sato et al. Rice Genet. Newslet., 20: 43-45, 2003). Therefore, we constructed a genetic linkage map near the GLUP6 gene by linkage analysis using a chromosome 3 marker. GLUP6 gene linkage map was prepared using STS and CAPS markers. From homozygous F2 seeds of the glup6 mutant line EM939 and the Indian cultivar Kasalath, a glup6 gene homozygous individual that accumulates a large amount of glutelin precursor was selected by SDS-PAGE analysis and used for linkage analysis.

  The high-density linkage map and candidate genomic region of the GLUP6 gene obtained by this example are shown in FIG. In this figure, a GLUP6 gene linkage map was created using RFLP, STS, and CAPS markers. The marker name at the top of the linkage map, the genetic distance from the chromosome short arm end in parentheses, and the number of recombination individuals at each marker at the bottom of the map. The OJ number indicates a BAC clone (Nihonbare). A and B are linked maps constructed using 41 and 234 glup6 homozygous individuals, respectively. C is a 1122 map based on physical maps of BAC clones aligned within the candidate region of the GLUP6 gene and markers generated in the BAC clones. In the linkage map constructed using individual populations, D indicates a predicted gene in the candidate region with an arrow. Double arrows from A to D indicate candidate regions for the GLUP6 gene. C169, R216: CAPS marker; E50818, R10784, C53358, R10784, C63279: STS marker; R4996: Unknown.

  By linkage analysis using 41 glup6 homozygous individuals, the GLUP6 gene was located in the 10.4 cM region between the STS marker R10784 and the CAPS marker R216 (FIG. 2A: Sato et al. Rice Genet. Newslet., 20: 43-45, 2003). Furthermore, the GLUP6 gene was narrowed down to a region of 1.6 cM by linkage analysis using 234 glup6 gene homozygous individuals (FIG. 2B). This gene region was covered by 4 BAC clones (FIG. 2C).

  The GLUP6 gene was narrowed down to a region of about 16 kb in the BAC clone OJ1041F02 by linkage analysis using newly created DNA markers based on the sequences in these BAC clones and 1122 homozygous glup6 genes (Fig. 2C and 2D). When gene prediction and similarity search were performed using RiceGAAS (Rice Genome Annotation Database; http://RiceGAAS.dna.affrc.go.jp/rgadb/) for the genome sequence of this candidate region, three genes were found. Was predicted (Figure 2D).

Based on the genome base sequences of these predicted genes obtained from Rice annotation project database (RAP-DB: http://rapdb.dna.affrc.go.jp/), glup6 mutant lines “EM939” and “EM1327 ”, And the genome sequence of the predicted gene in the GLUP6 gene candidate region of the original cultivar“ Taichung 65 ”. The genome base sequence of each strain showed only the coding region sequence corresponding to the cDNA (FIG. 4). As a result, in the coding region of one of the predicted genes, Os03g0262900, “Taichung 65” and glup6 mutant, C418 in EM939 and C784 in EM1327 were both replaced with T (FIG. 3). Since no base substitution was observed in other predicted genes, Os03g0262900 was selected as a GLUP6 candidate gene (FIG. 5). The gene is guanine nucleotide exchange factor (guanine nucleotide exchange factor of RAB protein belonging to superfamily having Vacuolar Protein Sorting 9 domain by RiceGAAS and Rice genome annotation project database (RGAP: http://rice.plantbiology.msu.edu/index.shtml). exchange factor (GEF).

Next, a single band was obtained by amplifying with a primer specific to the gene, using cDNA obtained by reverse transcription of mRNA extracted from ripening seeds of Taichung 65 as a template. The results of direct base sequence analysis of this band are shown in FIG. 3 as cDNA. As a result of comparing the genome base sequence of “Taichung 65” with the full-length genome base sequence of “Nippon Hare” in “Nippon Hare” obtained from RAP-DB, Two nucleotide polymorphisms were found in the fifth exon in the coding region of the sequence (nucleotides 814 and 927 in FIG. 3). As a result of comparing the genomic base sequence and the cDNA base sequence of the gene in “Taichung 65”, the GLUP6-GEF gene was composed of 5 exons and the total length of the coding region was 1443 bp (FIG. 3 and FIG. 3). Four). It also encoded a protein consisting of 480 deduced amino acids (Figure 6). This sequence completely matched LOC_Os03g15650.1 in the RGAP database. In contrast, in the glup6 mutant lines “EM939” and “EM1327”, the 140th and 262th glycines were replaced with stop codons, respectively (FIG. 6). From these results, it is considered that the GLUP6-GEF protein is not expressed in the glup6 mutant.

  In FIG. 6, Taichung No. 65 (cDNA) shows the base sequence of cDNA reverse transcribed from mRNA isolated from ripened seeds. An asterisk (*) below the sequence indicates a common sequence, and a bar (-) indicates a mutation site. : Indicates a polymorphism between "Taichung 65" and "Nihonbare".

Example 2: Function check
To demonstrate that GLUP6 candidate gene is a causative gene of glup6 variants performs a transformation experiment introducing GLUP6 candidate genes into plants of "glup6 variant", was complementation assay results. We received a lot of cDNA clone JO33091P13 from the Rice Genome Resource Center. As a result of analyzing the base sequence of the clone, the coding region sequence completely matched the Nipponbare base sequence (Fig. 3), but the 3 'end of the 4th exon was 618G and the 5' end of the 5 'end was 619G. The intron contains a 924 bp intron, and this sequence also completely matched the genomic base sequence of “Nipponbare” (data not shown). Using the cDNA clone JO33091P13 (SEQ ID NO: 3, FIG. 5) as a template, the sequence of the candidate gene was amplified by PCR. Furthermore, the promoter of the GLUP6 candidate gene was subcloned from the genomic DNA of “Taichung 65”. The candidate gene sequence and promoter were introduced into an entry vector containing GFP. The constructed entry vector was introduced into a DV9 binary vector and introduced into “EM939” via Agrobacterium (EHA105 strain).

FIG. 7 shows the result of complementation assay in which the glup6 mutant is transformed with the GEF gene. 1 to 6 represent 6 ripened seeds obtained by self-breeding of the transformed individuals.
As a result of SDS-PAGE analysis of the protein extracted from 6 seeds grown on the seeds of the produced transformant, among 6 seeds, 3 seeds whose glutelin precursor content is almost the same as the original variety, Three seeds that accumulated a large amount of the precursor were separated (FIG. 7A). As a result of Western blot analysis using GFP antibody of protein of the same seed, GFP band was detected in 3 seeds whose glutelin precursor content was almost the same as that of the original varieties, but the remaining 3 that accumulated a large amount of precursor The same band was not detected in the seeds of the grains (FIG. 7B). These results that glup6 variants by introduction of the candidate gene was restored to wild type, that is, the, the glup6 mutant was complemented by GEF gene. The gene was found to have 53% homology in amino acid sequence with Arabidopsis GEF (VPS9: At3g19770), indicating that the GLUP6 gene encodes GEF. Hereinafter, the gene is referred to as GLUP6-GEF gene, and the protein is referred to as GLUP6-GEF.

A large amount of GLUP6-GEF protein was expressed in E. coli and purified by a conventional method. The purified GLUP6-GEF protein was used as an antigen to immunize mice and rabbits. The presence or absence of expression of GLUP6-GEF proteins in seeds of glup6 entanglement variants using the obtained specific antibodies was investigated by Western blot analysis using the GEF antibody seed protein glup6 mutants (Figure 8). In “Taichung 65”, two bands were detected. On the other hand, in “EM939” and “EM1327”, a band of 53.8 kDa was lost among the two bands. The lack of GLUP6-GEF protein in the glup6 mutant suggests that the function of GLUP6-GEF was lost due to nonsense mutation.

Example 3: Study on localization of glutelin
The localization of glutelin in the endosperm cell tissue of GLUP6-GEF deficient mutant glup6 was investigated by fluorescence microscope analysis. FIG. 9 shows the results of observation of the endosperm cell tissue of the glup6 mutant using a fluorescence microscope. In FIG. 9, A and B: wild type (Taichung No. 65), C and D: EM939 (glup6 mutant), E and F: EM1327 (glup6 mutant) are shown. The site labeled with green was labeled with a glutelin antibody, and the site labeled with red was labeled with a prolamin antibody. The upper row (A, C and E) shows the endosperm tissue in the early stage of ripening (1 week after flowering), and the lower row (B, D and F) shows the endosperm tissue in the late stage of ripening (3 weeks after flowering). .

In the wild type, a granule that accumulates glutelin (PB-II) and a granule that accumulates prolamin (PB-I) were observed alone, and the size of both PBs increased with ripening (Figures 9A and 9B). ). On the other hand, in the glup6 mutation, a large paramural body (PMB) labeled with a glutelin antibody was observed. PMB has existed since the beginning of ripening, and its size increased with ripening (FIGS. 9C, 9D, and 9F). The glup6 mutant PB-II was almost constant in size from early to late ripening. PB-I was not different from the wild type. These results indicate that glup6 mutation inhibits the transport of glutelin precursor labeled with glutelin antibody to storage vacuoles. It was also found that the size of PB-II did not change through ripening. Furthermore, a signal indicating the presence of glutelin was also observed in the cell wall. From this result, it is considered that the intracellular transport of β-glucan, which is a constituent of the cell wall, may also be inhibited.

Therefore, the localization of β-glucan and glutelin in the glup6 mutant was investigated by fluorescence microscopy. FIG. 10 shows the results showing the localization of β-glucan and glutelin in the glup6 mutant using a fluorescence microscope. FIG. 10 shows AC: wild type (Taichung 65), DF: EM939 (glup6 mutant), and GI: EM1327 (glup6 mutant). The site labeled with green was labeled with β-glucan antibody, and the site labeled with red was labeled with glutelin antibody. In the wild-type, β-glucan was detected only in the cell wall (Figs. 10A and 10C), but in the glup6 mutant, β-glucan was present not only in the cell wall but also around and within the PMB (Fig. 10D to FIG. 10I). These results suggest that glup6 mutation also inhibits the transport of β-glucan to the cell wall, possibly altering the structure of the cell wall and increasing β-glucan accumulation.

The glup4 mutation that accumulates many glutelin precursors is a structural gene mutation of Small GTPase Rab5a. FIG. 11 shows the results showing the localization of β-glucan and glutelin in the glup4 mutant using a fluorescence microscope. In FIG. 11, AC: wild type (Taichung No. 65), DF: EM956 (glup4 mutant) are shown. In the wild type, β-glucan was detected only in the cell wall (FIGS. 11A and 11C). Similarly to the glup6 mutation, the β-glucan accumulated not only in the cell wall but also around the PMB as in the glup4 mutant (FIGS. 11D and 11F).

Example 4: Effect of β-glucan on rice flour bread In order to investigate how the high accumulation of β-glucan affects the bread-making characteristics of rice flour and the taste of rice flour bread, Using the product GOPAN (Breadmaker, registered trademark), glup6 mutant (EM939) and wild type (Taichung 65) rice flour, using 220 g of polished rice and 50 g of wheat gluten as the main ingredients I made bread and compared the two (Figure 12).

  As a result of repeated bread-making tests, bread dough made from wild-type rice flour was soft immediately after baking, but the dough started to harden after a few hours and became vomited after 2 days, while glup6 ( EM939), it was found that under similar conditions, the dough is less likely to be harder than the wild type. In general, rice flour bread has a problem of becoming harder with time. This problem is not limited to rice flour bread but is an important evaluation item in the quality of normal wheat bread, and is called “bread aging”. The cause of “rice flour bread that is difficult to age” shown by glup6 mutant (EM939) is the moisturizing characteristics of β-glucan accumulated in PMB, the aging characteristics of starch itself, or water-soluble β-glucan is glue There is a possibility of inhibiting aging by binding to modified starch.

  Such differences are apparent in appearance, and at 2 to 3 days after baking, wild-type (Taichung No. 65) rice flour bread is more crushed compared to glup6 rice flour bread It became clear (Figure 12).

  According to the present invention, genes involved in the accumulation of novel storage proteins are provided. The gene of the present invention is involved in intracellular transport of plant seeds. By regulating the expression and function of this gene, the amount of mature glutelin accumulated in the seed can be regulated. Therefore, the gene of the present invention is useful for breeding rice varieties with a reduced amount of mature glutelin. In addition, rice cultivar breeding using the gene of the present invention is more advantageous than conventional methods in that a target plant can be obtained with high certainty in a short period of time.

SEQ ID NO: 1: cDNA sequence of GLUP6-GEF gene derived from Nipponbare
SEQ ID NO: 2: amino acid sequence corresponding to SEQ ID NO: 1
SEQ ID NO: 3: cDNA clone JO33091P13 base sequence (including intron portion) for GLUP6-GEF gene
SEQ ID NO: 4: Amino acid sequence corresponding to SEQ ID NO: 3
SEQ ID NO: 5: cDNA sequence amplified with GLUP6-GEF- specific primers using a single-stranded cDNA obtained by reverse transcription of mRNA extracted from Taichung No. 65 seeds as a template
SEQ ID NO: 6: Amino acid sequence corresponding to SEQ ID NO: 5
SEQ ID NO: 7: Coding sequence in the genomic DNA of the gene of EM939
SEQ ID NO: 8: Amino acid sequence corresponding to SEQ ID NO: 7
SEQ ID NO: 9: coding sequence in genomic DNA of the gene of EM1327
SEQ ID NO: 10: Amino acid sequence corresponding to SEQ ID NO: 9
SEQ ID NO: 11: Nipponbare genomic DNA sequence
SEQ ID NO: 12: Taichung 65 genomic DNA sequence
SEQ ID NO: 13: EM939 genomic DNA sequence
SEQ ID NO: 14: Sequence of genomic DNA of EM1327
SEQ ID NO: 15: PCR primer
SEQ ID NO: 16: PCR primer

Claims (6)

The following polynucleotide (a), (b), (c), (d), or (e ) is functionally used in a plant that accumulates a storage protein in a storage type vacuole. A method for producing a plant with altered storage protein accumulation by disabling:
(A) a polynucleotide comprising the base sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 11, or SEQ ID NO: 12;
( B ) has at least 90% identity and storage with the base sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 11, or SEQ ID NO: 12. A polynucleotide encoding a protein having a function of accumulating the protein in storage vacuoles;
( C ) a polynucleotide encoding the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 6;
( D ) an amino acid sequence in which 1 to 9 amino acids are deleted, substituted, added and / or inserted in the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 6, and A polynucleotide encoding a protein having a function of accumulating in the vesicle;
( E ) encoding a protein comprising an amino acid sequence having at least 97% identity with the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 6, and having a function of accumulating the storage protein in a storage type vacuole Polynucleotide. To a plant in which the polynucleotide of (a), (b), (c), (d) or (e ) as defined in claim 1 is nonfunctional, (a) as defined in claim 1; A method for producing a plant in which the accumulation property of a storage protein is modified by introducing the polynucleotide of (b), (c), (d), or (e ) .   The production method according to claim 1 or 2, wherein the storage protein is glutelin. A vector comprising the defined polynucleotides to claim 1 or SEQ ID NO,: 7 or SEQ ID NO: transformed with a vector comprising a polynucleotide consisting of 9 base sequence, a transformant plant, except rice varieties called No. 65 and Nipponbare Taichung, the transformed plant. Plants, rice, soybean, oat or pumpkin, transformation plant according to claim 4,. According obtained from plants obtained by the method of production according to any one of claims 1 to 3, or obtained Ri by plants according to claim 4 or 5, crop, propagation material or the workpiece.