Barley with gluten-forming ability, its production method and evaluation method

Abstract

To provide barley with gluten-forming capacity.SOLUTION: It has been discovered that cysteines at positions 459, 473, and 534 in the D-hordein protein of barley are responsible for inhibiting gluten formation. It has also been elucidated that gluten-forming capacity can be imparted to barley by inducing the deletion or other modification of any one of these cysteines.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to barley having gluten-forming ability, and methods for producing and assessing the same. More specifically, the present invention relates to a method for imparting gluten-forming ability to barley by modifying the amino acid sequence of a D-hordein, and to barley having gluten-forming ability obtained by the method. The present invention also relates to a method for assessing the gluten-forming ability of barley using the amino acid sequence of a D-hordein as an indicator, and oligonucleotides (primers, probes, etc.) that target nucleotides encoding D-hordeins and that are used in the assessment method. [Background technology]

[0002] According to the United Nations' "World Population Prospects 2019," the world population is expected to increase from 7.7 billion in 2019 to 8.5 billion in 2030, and is expected to continue to grow thereafter. Securing food, such as agricultural crops, to feed this growing population is an urgent issue. Meanwhile, the amount of arable farmland is steadily decreasing due to frequent extreme weather events, the progression of desertification, and water resource constraints. Given this situation, Japan has also formulated its "Basic Plan for Agriculture, Forestry and Fisheries Research," which sets out the goals of improving land use efficiency and ensuring a stable food supply in the agriculture, forestry, fisheries and food sectors.

[0003] In this regard, barley is suitable for double cropping because its harvest time is earlier than wheat, and it can be harvested before the rainy season, which increases land use efficiency, thereby improving self-sufficiency and increasing farm profitability.It also has the advantage of being able to suppress diseases and other damage caused by continuous wheat cultivation.

[0004] Furthermore, barley contains a large amount of beta-glucan, a functional component that has the effect of lowering total blood cholesterol and preventing post-meal blood sugar levels from rising. Therefore, barley is extremely useful in the research and development of high-quality foods to create new demand.

[0005] On the other hand, barley cannot be made into a viscoelastic dough due to gluten like wheat, and is therefore not suitable for processing. For this reason, barley is limited to lightly processed foods (miso, pressed barley, rice grain barley, barley tea, beer, shochu, etc.) and is rarely used as flour.

[0006] To take advantage of the advantages and usefulness mentioned above and further expand demand, it is desirable to improve barley's processing suitability so that it can be used for a wider variety of purposes like wheat, but such a variety does not yet exist. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Japanese Patent Application Laid-Open No. 2014-54231 Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention has been made in view of the problems associated with the prior art, and has as its object to provide barley having gluten-forming ability, as well as methods for producing and determining barley having gluten-forming ability, which will enable the provision of such barley. [Means for solving the problem]

[0009] As a result of extensive research to achieve the above-mentioned object, the present inventors have concluded that the reason why barley does not produce gluten like wheat does is due to structural differences between seed storage proteins (barley hordein and wheat glutenin).

[0010] More specifically, barley is genetically closely related to wheat, and its seed storage proteins are also classified as prolamins, which are homoeologous proteins with high similarity to wheat (Figure 1). In wheat gluten, cysteines contained in high-molecular-weight glutenin and low-molecular-weight glutenin form disulfide (SS) bonds to form a giant mesh structure, which is the source of gluten elasticity. Barley does not form such a structure, and as mentioned above, its processing suitability is poor and its uses are limited.

[0011] Barley hordeins, which correspond to wheat glutenins, include B-hordeins and D-hordeins. The present inventors have previously demonstrated that the introduction of a barley chromosome site (1H short arm) containing B-hordeins into wheat improves the physical properties of wheat dough (Patent Document 1). Based on this finding, further investigation led to the idea that barley B-hordeins may be involved in network structure formation, while D-hordeins may inhibit network structure formation.

[0012] As shown in Figure 2, wheat high-molecular-weight glutenin (y-type), the homologous protein of barley D-hordein, contains seven cysteines, three of which are thought to form the gluten network structure through intermolecular bonds. On the other hand, barley D-hordein (e.g., a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2) contains the same seven cysteines as wheat y-type glutenin, as well as a barley-specific region containing three cysteines (corresponding to the region consisting of amino acids 454 to 540 in SEQ ID NO: 2), for a total of 10 cysteines (Cys10).

[0013] We hypothesized that, as shown in Figure 3, three cysteines present in barley D-hordein but not in wheat form new intermolecular disulfide bonds, resulting in the formation of aggregates due to excessive intermolecular bonds, preventing the formation of a meshwork structure.

[0014] To test this hypothesis, the present inventors first searched for barley with a mutation in any of the three cysteines (corresponding to cysteines at positions 459, 473, and 534 in SEQ ID NO: 2). As a result, they found "Kantokawa No. 35," which has a D-hordein mutation (Cys9) in which one cysteine ​​(corresponding to cysteine ​​at position 459 in SEQ ID NO: 2) is mutated to tyrosine. Gluten polymers (hydrated barley flour) were then prepared from the barley and observed under a scanning electron microscope. Image data demonstrating the formation of a network structure were obtained. Furthermore, analysis of the gluten polymers by size-exclusion high-performance liquid chromatography (HPLC) yielded data demonstrating an increase in polymer size.

[0015] Furthermore, by using genome editing to delete the barley-specific region (resulting in Cys7) in the barley variety "Hokurikukawa No. 71," which has the D-hordein at Cys10, they succeeded in producing a strain with a high degree of protein polymerization in the seeds and gluten-forming ability, which led to the completion of the present invention.

[0016] That is, the present invention provides the following aspects.

[0017] [1] A method for producing gluten-forming barley, comprising the following steps (1) and (2): (1) introducing at least one of the following modifications (a) and (b) into a D-hordein protein in a barley cell: (a) a modification in which a cysteine ​​at at least one site selected from the group consisting of position 459 of SEQ ID NO: 2 or a site corresponding to said position, position 473 of SEQ ID NO: 2 or a site corresponding to said position, and position 534 of SEQ ID NO: 2 or a site corresponding to said position in the D-hordein protein is substituted with another amino acid or deleted; (b) a modification in a D-hordein protein in which the number of cysteines in a region consisting of amino acids 454 to 540 of SEQ ID NO: 2 or a region corresponding to said region is reduced to two or less; (2) A step of regenerating a barley plant from the cells into which the modification has been introduced in step (1).

[0018] [2] Barley having gluten-forming ability, in which at least one of the following modifications (a) and (b) has been introduced into a D-hordein protein: (a) a modification in which a cysteine ​​at at least one site selected from the group consisting of position 459 of SEQ ID NO: 2 or a site corresponding to said position, position 473 of SEQ ID NO: 2 or a site corresponding to said position, and position 534 of SEQ ID NO: 2 or a site corresponding to said position in the D-hordein protein is substituted with another amino acid or deleted; (b) A modification in the D-hordein protein in which the number of cysteines in the region consisting of amino acids 454 to 540 of SEQ ID NO: 2 or a region corresponding to said region is reduced to two or less.

[0019] [3] A method for determining gluten-forming ability in barley, comprising the following steps (1) and (2): (1) detecting cysteine ​​in a region consisting of amino acids 454 to 540 of SEQ ID NO: 2 or a region corresponding to said region in a test barley D-hordein protein; (2) (a) The amino acid at at least one site selected from the group consisting of position 459 of SEQ ID NO: 2 or a site corresponding to said site, position 473 of SEQ ID NO: 2 or a site corresponding to said site, and position 534 of SEQ ID NO: 2 or a site corresponding to said site is an amino acid other than cysteine ​​or is deleted, and / or (b) if the number of cysteines in the region is two or less, determining that the test barley is a barley having gluten-forming ability.

[0020] [4] A reagent for use in the method according to [3], comprising the oligonucleotide (i) or (ii) below: (i) a pair of oligonucleotides designed to flank nucleotides encoding an amino acid at at least one site selected from the group consisting of position 459 of SEQ ID NO: 2 or a site corresponding to said position, position 473 of SEQ ID NO: 2 or a site corresponding to said position, and position 534 of SEQ ID NO: 2 or a site corresponding to said position; (ii) An oligonucleotide that hybridizes to a nucleotide encoding an amino acid at at least one position selected from the group consisting of position 459 of SEQ ID NO: 2 or a site corresponding to said position, position 473 of SEQ ID NO: 2 or a site corresponding to said position, and position 534 of SEQ ID NO: 2 or a site corresponding to said position. [Effects of the Invention]

[0021] According to the present invention, it is possible to provide barley having gluten-forming ability. [Brief explanation of the drawings]

[0022] [Figure 1] FIG. 1 is a schematic diagram showing the homology of seed storage proteins in barley and wheat and their locations on each chromosome. [Figure 2] FIG. 1 is a schematic diagram showing a comparison of barley D-hordins with wheat high molecular weight glutenins. [Figure 3] FIG. 1 is a schematic diagram showing a concept of a method for forming gluten in barley. [Figure 4] This is a photograph of an electrophoresis gel showing the results of PCR detection of the D-foldin mutant (Cys9) gene, which has nine cysteines, in an F4 generation population obtained by crossing "New Line J039" with "Yumesakiboshi." In the figure, the leftmost lane shows a size marker, lanes 1 and 6 show lines that do not contain Cys9, and lanes 2 to 5 show lines that do contain Cys9. [Figure 5]Photographs showing the results of scanning electron microscopic observation of gluten polymers (hydrated barley flour) for the barley line Kanto-Ku 35, which has Cys9, and its wild-type variety (Sachiho Golden, a D-Hordin with 10 cysteines (Cys10)). [Figure 6] This is a chromatogram showing the results of analyzing the insoluble fraction of gluten polymers using size-exclusion high-performance liquid chromatography for the Cys9 line (Kanto Skin No. 35) and the Cys10 variety (Sachiho Golden). [Figure 7] FIG. 1 is a schematic diagram showing the steps of genome editing according to the present invention. [Figure 8] Photographs showing the results of scanning electron microscopy of gluten polymers in the barley cultivar Hokurikukawa No. 71, which has Cys10, and its genome-edited line (HK11.29, which has D-hordin (Cys7) with seven cysteines). [Figure 9] This is a chromatogram showing the results of size-exclusion high-performance liquid chromatography analysis of the insoluble fraction of gluten polymers for the Cys7 line (HK11.29, "HK11.29-07" in the figure), the Cys10 variety (Hokurikukawa No. 71, "WT-03" in the figure), and the Cys9 line (a hybrid line of Shinkei J039 and Yumesakiboshi, "Cys9 line" in the figure). [Figure 10] 1 is a chromatogram showing the results of analyzing the insoluble fraction of gluten polymers by size-exclusion high-performance liquid chromatography for a Cys7 line (HK11.29), a Cys10 variety (Hokurikukawa 71), and wheat. DETAILED DESCRIPTION OF THE INVENTION

[0023] (Method of producing gluten-forming barley) As shown in the Examples below, the present inventors have demonstrated that gluten-forming ability can be imparted to barley by substituting, with another amino acid, or deleting, any of three cysteines that are present in barley D-hordeins but not in the corresponding high-molecular-weight glutenins of wheat. Thus, the method of the present invention for producing barley with gluten-forming ability is characterized by targeting the cysteines in D-hordeins, and more specifically provides the following:

[0024] A method for producing gluten-forming barley, comprising the following steps (1) and (2): (1) introducing at least one of the following modifications (a) and (b) into a D-hordein protein in a barley cell: (a) a modification in which a cysteine ​​at at least one site selected from the group consisting of position 459 of SEQ ID NO: 2 or a site corresponding to said position, position 473 of SEQ ID NO: 2 or a site corresponding to said position, and position 534 of SEQ ID NO: 2 or a site corresponding to said position in the D-hordein protein is substituted with another amino acid or deleted; (b) a modification in a D-hordein protein in which the number of cysteines in a region consisting of amino acids 454 to 540 of SEQ ID NO: 2 or a region corresponding to said region is reduced to two or less; (2) A step of regenerating a barley plant from the cells into which the modification has been introduced in step (1).

[0025] In the present invention, "barley" refers to a plant of the grass family belonging to the genus Hordeum, particularly Hordeum vulgare. Examples include two-rowed barley (H. vulgare f. distichon), four-rowed barley (H. vulgare subsp. vulgare), six-rowed barley (H. vulgare f. hexastichon), naked barley (H. vulgare var. nudum Hook. f.), and wild barley (H. vulgare subsp. spontaneum). Barley may be a wild species or a cultivated species (e.g., practical varieties such as fiber snow). Furthermore, it may be a genetically modified or genome-edited version of these barley species (e.g., disease-resistant crops, herbicide-resistant crops, pest-resistant crops, crops with improved taste, improved shelf life, or crops with improved yield).

[0026] In the present invention, "gluten" refers to a protein formed when barley seed storage proteins (such as D-hordein) are hydrated and associated in a network-like manner by disulfide bonds. Whether or not barley has the ability to form gluten can be determined, for example, by preparing hydrated barley flour and analyzing it by size-exclusion high-performance liquid chromatography (HPLC), as shown in the Examples below. More specifically, if the ratio of the area of ​​the peak attributable to gluten to the total peak area in a chromatogram obtained by this analysis is 0.04 or more, the barley can be determined to have the ability to form gluten. Furthermore, this determination is not limited to HPLC analysis, but can also be made by observing hydrated barley flour with an electron microscope and detecting the network structure, as shown in the Examples below. Furthermore, gluten can also be evaluated by other known gluten detection methods. Such known methods include, for example, the SDS precipitation test (Takata, K. et al., Prediction of bread-making quality by prolonged swelling SDS-sedimentation test. Breed. Sci., 1999, 49:221-223) and the gluten index method.

[0027] Furthermore, as will be described later, the barley targeted by the method of the present invention includes not only barley that is not capable of forming gluten (barley having 10 cysteines in D-hordein, as described below), but also barley that has some ability to form gluten (for example, barley having 9 or fewer cysteines in D-hordein, as described below). Therefore, the "barley capable of forming gluten" of the present invention can also be rephrased as "barley in which gluten-forming ability has been improved" by the modification according to the present invention, as described below, compared to before the modification.

[0028] A typical example of a "D-hordein" that can be modified in the present invention is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2. Other examples of D-hordeins according to the present invention include proteins consisting of the amino acid sequence set forth in SEQ ID NOs: 4, 6, 8, 10, 12, and 14. Furthermore, D-hordeins according to the present invention are not limited to those having 10 cysteines. By introducing the modifications described below, the number of cysteines retained may be 9 or less (e.g., 8 or less, 7 or less, 6 or less, 5 or less, or 4), as long as gluten-forming ability is improved. Examples of D-hordeins having 9 or less cysteines include proteins consisting of the amino acid sequence set forth in SEQ ID NO: 16 or 18. It is desirable to have at least one cysteine ​​in the amino acid sequence of positions 454 to 540 of SEQ ID NO: 2 or in a region corresponding to said sequence. Furthermore, in the amino acid sequence of D-hordein, the cysteines at or corresponding to position 41 in SEQ ID NO: 2, 692 in SEQ ID NO: 2, and 745 in SEQ ID NO: 2 may be involved in gluten formation. Therefore, it is desirable that the D-hordein of the present invention have cysteines at these three positions.

[0029] The term "corresponding" means that when the amino acid sequence of a D-hordein of another variety is aligned with the amino acid sequence of SEQ ID NO: 2 using, for example, BLAST (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi) or DNA analysis software (GENETYX-MAC, Sequencher, etc.), the sequence is aligned with the site or region of SEQ ID NO: 2.

[0030] Furthermore, mutations in nucleotide sequences can occur in nature, and the encoded amino acids can also change accordingly. Therefore, the D-hordeins of the present invention are not limited to the amino acid sequences exemplified above, but also include proteins consisting of amino acid sequences in which one or more amino acids have been substituted, deleted, added, and / or inserted in the amino acid sequences set forth in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18, for example.

[0031] Here, "multiple" typically means within 150 amino acids, preferably within 80 amino acids, more preferably within 70 amino acids, even more preferably within 60 amino acids, more preferably within 50 amino acids, even more preferably within 40 amino acids, more preferably within 35 amino acids, even more preferably within 30 amino acids (e.g., within 25 amino acids, within 20 amino acids, within 15 amino acids), and particularly preferably within 10 amino acids (e.g., within 9 amino acids, within 8 amino acids, within 7 amino acids, within 6 amino acids, within 5 amino acids, within 4 amino acids, within 3 amino acids, within 2 amino acids).

[0032] Furthermore, given the current state of the art, once a specific gene is obtained, a person skilled in the art can use the nucleotide sequence information of that gene to identify its homologous genes in the same or other plants. Methods for identifying homologous genes include, for example, hybridization techniques (Southern, EM, J. Mol. Biol., 98:503, 1975) and polymerase chain reaction (PCR) techniques (Saiki, RK, et al., Science, 230:1350-1354, 1985; Saiki, RK, et al., Science, 239:487-491, 1988). To identify homologous genes, hybridization reactions are typically performed under stringent conditions. Examples of stringent hybridization conditions include 6M urea, 0.4% SDS, and 0.5x SSC, or hybridization conditions of equivalent stringency. The use of more stringent conditions, such as 6 M urea, 0.4% SDS, and 0.1x SSC, is expected to result in the isolation of genes with higher homology. Thus, the D-hordeins of the present invention also include proteins consisting of an amino acid sequence encoded by DNA that hybridizes under stringent conditions with DNA consisting of a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, or 18 (e.g., the nucleotide sequence set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, or 13).

[0033] The protein encoded by the identified homologous gene typically has high homology (high similarity), preferably high identity, to that encoded by the specific gene. Here, "high" means at least 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more (e.g., 96% or more, 97% or more, 98% or more, 99% or more). Thus, D-hordeins according to the present invention include genes encoding amino acid sequences having 80% or more homology (similarity) or identity to the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, or 18.

[0034] Sequence homology can be determined using the BLAST program (Altschul et al. J. Mol. Biol., 215:403-410, 1990). This program is based on the BLAST algorithm by Karlin and Altschul (Proc. Natl. Acad. Sci. USA, 87:2264-2268, 1990; Proc. Natl. Acad. Sci. USA, 90:5873-5877, 1993). For example, when analyzing an amino acid sequence using BLAST, parameters are set to, for example, score = 50 and word length = 3. Furthermore, when analyzing an amino acid sequence using the Gapped BLAST program, the procedure can be 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 techniques for these analysis methods are known.

[0035] In such D-hordeins, the modifications introduced in the present invention are at least one of the following modifications (a) and (b): (a) a modification in which a cysteine ​​at at least one site selected from the group consisting of position 459 of SEQ ID NO: 2 or a site corresponding to said position, position 473 of SEQ ID NO: 2 or a site corresponding to said position, and position 534 of SEQ ID NO: 2 or a site corresponding to said position in the D-hordein protein is substituted with another amino acid or deleted; (b) A modification in the D-hordein protein in which the number of cysteines in the region consisting of amino acids 454 to 540 of SEQ ID NO: 2 or a region corresponding to said region is reduced to two or less.

[0036] In the modification (a), the "other amino acid" replacing the cysteine ​​is not particularly limited as long as it is an amino acid other than cysteine. Such "substitution" and "deletion" may not only be a substitution or deletion of the cysteine ​​alone, but also a substitution or deletion of an amino acid sequence containing the cysteine. Furthermore, in the three sites, the modification may be one cysteine ​​modification (a modification in which the cysteine ​​at position 459 in SEQ ID NO: 2 or a site corresponding to this position is replaced with another amino acid or deleted, a modification in which the cysteine ​​at position 473 in SEQ ID NO: 2 or a site corresponding to this position is replaced with another amino acid or deleted, or a modification in which the cysteine ​​at position 534 in SEQ ID NO: 2 or a site corresponding to this position is replaced with another amino acid or deleted), or two cysteines modification (a modification in which the cysteine ​​at position 459 in SEQ ID NO: 2 or a site corresponding to this position is replaced with another amino acid or deleted, and a modification in which the cysteine ​​at position 473 in SEQ ID NO: 2 or a site corresponding to this position is replaced with another amino acid or deleted). a modification in which the cysteine ​​at position 459 in SEQ ID NO: 2 or a site corresponding to said position is substituted with or deleted by another amino acid; a modification in which the cysteine ​​at position 459 in SEQ ID NO: 2 or a site corresponding to said position is substituted with or deleted by another amino acid, and a modification in which the cysteine ​​at position 534 in SEQ ID NO: 2 or a site corresponding to said position is substituted with or deleted by another amino acid; or a modification in which the cysteine ​​at position 473 in SEQ ID NO: 2 or a site corresponding to said position is substituted with or deleted by another amino acid, and a modification in which the cysteine ​​at position 534 in SEQ ID NO: 2 or a site corresponding to said position is substituted with or deleted by another amino acid), or modifications at all three cysteines.

[0037] Regarding modification (b), D-hordeins can be divided into six regions. There are six regions: a signal peptide (corresponding to the amino acid sequence of positions 1 to 21 in SEQ ID NO: 2), an N-terminal region (corresponding to the amino acid sequence of positions 22 to 131 in SEQ ID NO: 2), an R1 region (a region containing a repeating sequence with PGQGQQGYYPSATSPQQ as a repeating unit, corresponding to the amino acid sequence of positions 132 to 453 in SEQ ID NO: 2), an R2 region (corresponding to the amino acid sequence of positions 454 to 540 in SEQ ID NO: 2), an R3 region (a region containing a repeating sequence with PEQGQQTTVS as a repeating unit, corresponding to the amino acid sequence of positions 541 to 715 in SEQ ID NO: 2), and a C-terminal region (corresponding to the amino acid sequence of positions 716 to 757 in SEQ ID NO: 2) (Yong Qiang Gu et al., Genome, December 2003, 46(6), pp. 1084-1097; Xinkun Hu et al., Genetica. June 2018, 146(3), pp. 255-264, etc. Please refer to it).

[0038] In the present specification, the region referred to as "the region consisting of amino acids 454 to 540 in SEQ ID NO: 2 or a region corresponding to said region," "barley-specific region," or "Cys region" is the R2 region, and as shown in the Examples below, if the number of cysteines in this region is two or less, barley can have gluten-forming ability. As shown in the amino acid sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12, or 14, there are usually three cysteines in this region in barley. Therefore, modification (a) usually results in two or less cysteines in the R2 region. However, barley can be produced that has cysteines in the R2 region other than the three cysteine ​​sites (cysteines at positions 459 to 540 in SEQ ID NO: 2 or a position corresponding to said position, 473 in SEQ ID NO: 2 or a position corresponding to said position, and 534 in SEQ ID NO: 2 or a position corresponding to said position). Therefore, in such barley, it is conceivable to carry out modifications not only at the above three sites but also in the relevant region so that the number of cysteines is reduced to 2 or less. In such a case, the number of cysteines in the R2 region after the modification is preferably 1, and more preferably 0.

[0039] Such an amino acid sequence modification can be achieved by introducing a mutation into the gene encoding D-hordein. Known methods for introducing a mutation into a gene include, but are not limited to, genome editing, physical mutagenesis, and methods using chemical mutagens.

[0040] Genome editing is a method of modifying target genes using site-specific nucleases (e.g., DNA double-strand break enzymes such as zinc finger nucleases (ZFNs), transcription activation-like effector nucleases (TALENs), and CRISPR-Cas enzymes). For example, fusion proteins such as ZFNs (U.S. Patent Nos. 6,265,196, 8,524,500, 7,888,121, European Patent No. 1,720,995), TALENs (U.S. Patent Nos. 8,470,973, 8,586,363), and nuclease domain-fused PPR (pentatricopeptide repeat) (Nakamura et al., Plant Cell Physiol 53:1171-1179 (2012)), CRISPR-Cas9 (U.S. Patent No. 8,697,359, International Publication No. 2013 / 176772), CRISPR-Cpf1 (Zetsche B. et al., Cell, 163(3):759-71, (2015)), and Target-AID (K. Nishida et al., Targeted nucleotide editing using hybrid prokaryotic and Examples include methods that use guide RNA and protein complexes, such as those described in "Vertebrate Adaptive Immune Systems, Science, DOI: 10.1126 / science.aaf8729, (2016)," or protein complexes.

[0041] The "Cas enzyme" is not particularly limited and can be selected appropriately depending on the purpose, and examples thereof include type I CRISPR enzymes, type II CRISPR enzymes, and type III CRISPR enzymes, with the type II CRISPR enzyme Cas9 being preferred. The "Cas9" is not particularly limited and can be selected appropriately depending on the purpose, and examples thereof include Streptococcus pneumoniae Cas9, Streptococcus pyogenes Cas9, Streptococcus thermophilus Cas9, and Staphylococcus aureus Cas9, with Streptococcus pyogenes Cas9 (SpCas9) being preferred. Alternatively, it may be a mutant of Cas9 derived from these organisms, or the D10A mutant of Cas9, which is known to function as a nickase (a DNA cleaving enzyme that nicks only one DNA strand), or a Cas9 homolog or ortholog.

[0042] Physical mutagenesis methods include, for example, heavy ion beam (HIB) irradiation, fast neutron irradiation, gamma ray irradiation, and ultraviolet irradiation (see Hayashi et al., Cyclotrons and Their Applications, 2007, 18th International Conference, pp. 237-239, and Kazama et al., Plant Biotechnology, 2008, Vol. 25, pp. 113-117).

[0043] Examples of methods using chemical mutagens include treating seeds with chemical mutagens (see, for example, Zwar and Chandler, Planta, 1995, vol. 197, pp. 39-48). Chemical mutagens are not particularly limited, but include N-methyl-N-nitrosourea (MNU), ethyl methanesulfonate (EMS), N-ethyl-N-nitrosourea (ENU), sodium azide, sodium bisulfite, hydroxylamine, N-methyl-N'-nitro-N-nitroguanidine (MNNG), N-methyl-N'-nitrosoguanidine (NTG), O-methylhydroxylamine, nitrous acid, formic acid, and nucleotide analogs.

[0044] For barley into which a mutation has been introduced by the above method, it can be confirmed by known methods that the modification (mutation) of the present invention has been introduced into the D-hordein gene. Such known methods include, for example, DNA sequencing (next-generation sequencing, etc.), PCR, microarray analysis, Southern blotting, and Northern blotting. Using these methods, it can be determined whether a mutation (deletion) has been introduced into the nucleotide sequence encoding the R2 region of D-hordein by comparing the sequence or its length before and after the mutation.

[0045] Another method for confirming that a mutation according to the present invention has been introduced into a D-hordein gene is TILLING (Targeting Induced Local Lesions IN Genomes) (see Slade et al., Transgenic Res., 2005, Vol. 14, pp. 109-115, and Comai et al., Plant J., 2004, Vol. 37, pp. 778-786). In particular, when a non-selective mutation has been introduced into the barley genome using the aforementioned heavy ion beam irradiation or chemical mutagen, the D-hordein gene or a portion thereof can be amplified by PCR, and then individuals having a mutation in the amplification product can be selected by the aforementioned TILLING method.

[0046] Furthermore, by crossing barley into which a mutation has been introduced by the above-mentioned method with barley before the mutation was introduced (e.g., wild-type barley) and then performing backcrossing, it is also possible to remove mutations introduced into genes other than the gene of interest.

[0047] Barley into which the mutation of the present invention has been introduced into a D-hordein gene may be heterozygous for the D-hordein gene. In such cases, for example, such heterozygotes are crossed with each other to obtain F1 plants, and then homozygotes having the D-hordein gene into which the mutation has been introduced are selected from the F1 plants. In this case, "wheat that is homozygous having the D-hordein gene into which the mutation has been introduced" includes not only barley having two alleles of the D-hordein gene having identical mutations, but also barley having a first D-hordein gene having a first mutation and a second D-hordein gene having a second mutation.

[0048] In the present invention, the introduction of the mutations of the present invention into the D-hordein gene can be carried out on barley plants, seeds, or plant cells using the methods described above. Plant cells include not only cultured cells derived from barley, but also cells within the plant. Various forms of barley-derived cells are also included, such as germline cells, suspension-cultured cells, protoplasts, leaf segments, callus, immature embryos, and pollen.

[0049] Furthermore, in the present invention, the above-mentioned DNA encoding the site-specific nuclease, fusion protein, or guide RNA-protein complex may be introduced into barley cells in the form of a vector inserted therein.

[0050] The vector into which the DNA for introducing a mutation into the D-hordein gene is inserted is not particularly limited as long as it is capable of expressing the inserted gene in barley cells, and may contain a promoter for constitutive or inducible expression of the DNA. Examples of promoters for constitutive expression include the rice ubiquitin promoter, the cauliflower mosaic virus 35S promoter, the rice actin promoter, and the maize ubiquitin promoter. Examples of promoters for inducible expression include promoters known to be expressed in response to external factors such as infection or invasion by filamentous fungi, bacteria, or viruses, low temperature, high temperature, drought, ultraviolet radiation, and spraying with specific compounds. Furthermore, pol III promoters such as the U6 promoter are preferably used as promoters for expressing DNA encoding short RNAs such as guide RNAs as DNA according to the present invention.

[0051] Methods for introducing the DNA or a vector into which the DNA has been inserted into barley cells include, for example, particle bombardment methods such as in planta particle bombardment (iPB), Agrobacterium-mediated methods (Agrobacterium methods), polyethylene glycol methods, electroporation, liposome methods, microinjection methods, whisker methods, plasma methods, and laser injection methods. The iPB method is disclosed, for example, in JP-A Nos. 2017-205104 and 2017-205103.

[0052] Mutations can also be introduced into barley cells without taking the form of DNA, by introducing the above-mentioned site-specific nucleases and fusion proteins as proteins, or the above-mentioned guide RNA as RNA.

[0053] Thus, in the present invention, gluten-forming ability can be imparted to barley by using a substance that targets the D-hordein gene, such as the DNA, a vector into which the DNA has been inserted, the protein, or the RNA. Therefore, the present invention can also provide an agent for imparting gluten-forming ability to barley, which comprises as an active ingredient at least one substance that targets the D-hordein gene of barley, selected from the group consisting of the DNA, a vector into which the DNA has been inserted, the protein, and the RNA.

[0054] Such a pharmaceutical preparation may be one in which the two active ingredients are contained in a single composition, or may be one in which the two active ingredients are contained in separate compositions (a so-called kit).In addition to the above substances, the pharmaceutical preparation of the present invention may also contain other ingredients such as a buffer solution, a stabilizer, a preservative, an antiseptic, etc.

[0055] Furthermore, barley having gluten-forming ability can be obtained by regenerating a barley plant from cells in which the above-described modification of the present invention has been introduced into D-hordein by the above-mentioned method or the like.

[0056] For example, methods for producing transformed wheat plants include those described by Tingay et al. (Tingay S. et al. Plant J. 11:1369-1376, 1997), Murray et al. (Murray F et al. Plant Cell Report 22:397-402, 2004), and Travalla et al. (Travalla S et al. Plant Cell Report 23:780-789, 2005). Transformation and plant regeneration can also be performed using the method described by Tabei et al. (Tabei Yutaka, ed., "Transformation Protocols [Plant Edition]," Kagaku Dojin Co., Ltd., published September 20, 2012).

[0057] (Barley with gluten-forming ability) By using the above-mentioned method, it is possible to obtain barley having gluten-forming ability, in which the above-mentioned modification according to the present invention has been introduced into D-hordein. Provided is barley having gluten-forming ability, in which at least one of the following modifications (a) and (b) has been introduced into a D-hordein protein: (a) a modification in which a cysteine ​​at at least one site selected from the group consisting of position 459 of SEQ ID NO: 2 or a site corresponding to said position, position 473 of SEQ ID NO: 2 or a site corresponding to said position, and position 534 of SEQ ID NO: 2 or a site corresponding to said position in the D-hordein protein is substituted with another amino acid or deleted; (b) A modification in the D-hordein protein in which the number of cysteines in the region consisting of amino acids 454 to 540 of SEQ ID NO: 2 or a region corresponding to said region is reduced to two or less.

[0058] D-hordein, modifications in the protein, barley to which gluten-forming ability is imparted by the modifications, etc. are as described above. Furthermore, the barley of the present invention excludes existing barley that inherently has gluten-forming ability (for example, barleys that were first shown by the present inventors to have gluten-forming ability, such as "Kantokawa 35," "Nigrinudum," "Hungarian," "Ethiopia 1," "Ethiopia 7," "Ethiopia 59," "Ethiopia 63," "COL / NEPAL / 1985 / IBPGR / 23," and "New Line J039," as shown in the Examples below).

[0059] Furthermore, once a barley (plant) to which gluten-forming ability has been imparted is obtained, it is possible to obtain progeny from the plant through sexual or asexual reproduction. Furthermore, it is also possible to obtain propagation materials (e.g., seeds, cuttings, stumps, callus, protoplasts, etc.) from the plant, its progeny, or clones, and use these to mass-produce the plant. Therefore, the present invention includes progeny and clones of barley with gluten-forming ability, as well as their propagation materials. Examples of propagation materials include seeds, stumps, callus, and protoplasts.

[0060] Furthermore, like wheat, gluten-forming barley exhibits high processing suitability. That is, the gluten-forming barley of the present invention has high dough properties (at least one property selected from the strength, hardness, mixing resistance, and stability of dough prepared from wheat flour) and therefore high secondary processing suitability (suitability for producing wheat processed foods such as bread).

[0061] Therefore, the present invention can also provide barley seeds having gluten-forming ability of the present invention, barley flour prepared from the seeds (barley flour obtained by milling the seeds), and processed barley foods containing the barley flour.

[0062] In the present invention, "barley flour" may be any flour obtained by milling barley seeds, and there are no particular limitations on the part of the barley used for milling, the grinding method, etc. Examples of barley flour include strong flour, medium-strength flour, weak flour, whole wheat flour, graham flour, and semolina flour.

[0063] Furthermore, the "barley processed foods" produced from the barley flour are not particularly limited, and examples include breads such as white bread, rolls, buns, croissants, Danish pastries, French bread, bagels, scones, muffins, sweet rolls, savory rolls, and hardtack; noodles such as Chinese noodles, udon, spaghetti, macaroni, Okinawa soba, somen, hiyamugi, and soba; gyoza wrappers; wheat gluten; and sweets such as pies, donuts, cookies, biscuits, crackers, karinto, castella cakes, cakes, and manju.

[0064] <Method for determining gluten-forming ability in barley> As shown in the Examples below, the present inventors have demonstrated that a cysteine ​​present in a barley-specific region of barley D-hordein, which is not present in the corresponding wheat high-molecular-weight glutenin, inhibits gluten formation. Thus, the method of the present invention for assessing gluten-forming ability in barley is characterized by using cysteine ​​present in the specific region as an indicator, and more specifically provides the following:

[0065] A method for determining gluten-forming ability in barley, comprising the following steps (1) and (2): (1) detecting cysteine ​​in a region consisting of amino acids 454 to 540 of SEQ ID NO: 2 or a region corresponding to said region in a test barley D-hordein protein; (2) (a) The amino acid at at least one site selected from the group consisting of position 459 of SEQ ID NO: 2 or a site corresponding to said site, position 473 of SEQ ID NO: 2 or a site corresponding to said site, and position 534 of SEQ ID NO: 2 or a site corresponding to said site is an amino acid other than cysteine ​​or is deleted, and / or (b) if the number of cysteines in the region is two or less, determining that the test barley is a barley having gluten-forming ability.

[0066] In the present invention, the "test barley" is not particularly limited, and as described above, it may be a grass plant belonging to the genus Hordeum, particularly Hordeum vulgare. Furthermore, the "D-hordein protein," "the region consisting of amino acids at positions 454 to 540 in SEQ ID NO: 2 or said region," "position 459 in SEQ ID NO: 2 or a site corresponding to said site, position 473 in SEQ ID NO: 2 or a site corresponding to said site, and position 534 in SEQ ID NO: 2 or a site corresponding to said site," and "gluten-forming ability" in the determination method of the present invention are as described above.

[0067] In the present invention, "detection of cysteine ​​in a region consisting of amino acids 454 to 540 of SEQ ID NO: 2 in a D-hordein protein or a region corresponding to said region" can be carried out using genomic DNA encoding the D-hordein protein, a transcription product from the genomic DNA, or a translation product from the transcription product (D-hordein protein).

[0068] Known techniques can be used for detection in the present invention. When "genomic DNA encoding D-hordein protein" is the target, a DNA sample is first prepared from the test barley. Barley from which DNA is extracted can be not only grown plants, but also seeds or young plants. Tissues from which DNA samples can be extracted include leaves, for example. There are no particular limitations on the method for extracting genomic DNA from barley, and any known method can be appropriately selected and used, including the SDS-phenol method, the CTAB method, and the alkali treatment method. Alternatively, commercially available kits such as the DNeasy Plant mini kit (QIAGEN, Germany) can be used.

[0069] Furthermore, detection in the present invention can be performed by those skilled in the art using known polymorphism detection methods. Examples of such polymorphism detection methods include the PCR-SSP (PCR-sequence-specific primer) method, as shown in the Examples below. When using the PCR-SSP method to detect whether position 459 of SEQ ID NO: 2 or a site corresponding to said position is a cysteine ​​(hereinafter simply referred to as a "polymorphism"), one of a pair of oligonucleotides constituting a primer is designed so that its 3' end contains a base type complementary to the specific base type at the polymorphic site. Amplification by PCR using such a pair of oligonucleotides is limited to when genomic DNA derived from test barley having the specific base type at said site is used as a template, but is not amplified when genomic DNA derived from test barley having a different base type at said site is used as a template. Therefore, polymorphisms can be detected by using such a pair of oligonucleotides.

[0070] Another method for detecting polymorphisms is the PCR-SSCP (PCR-single-strand conformation polymorphism) method. Specifically, double-stranded DNA amplified by PCR using a pair of oligonucleotides designed to flank the polymorphic site is denatured by treatment with heat, alkali, or the like to form single-stranded DNA. When this single-stranded DNA is then subjected to denaturant-free polyacrylamide gel electrophoresis, the single-stranded DNA folds in the gel due to intramolecular interactions to form a higher-order structure. Since the interactions of the folded structure vary depending on the base species, the separated single-stranded DNA can be detected by silver staining or radioisotopes, and the mobility of the single-stranded DNA on the gel can be compared with that of a control to detect polymorphisms.

[0071] Another method for detecting polymorphisms is to use an intercalator. In this method, genomic DNA is first prepared from test barley as described above. Next, a region containing the polymorphic site is amplified using the genomic DNA as a template in a reaction system containing an intercalator that emits fluorescence when inserted into a DNA double strand. The temperature of the reaction system is then changed, and changes in the intensity of the fluorescence emitted by the intercalator are detected. The detected changes in the intensity of the fluorescence due to the temperature change are compared with a control. An example of such a method is high-resolution melting (HRM) analysis.

[0072] Another method for detecting polymorphisms utilizes an oligonucleotide probe that hybridizes to a region containing the polymorphic site. In this method, genomic DNA is first prepared from test barley as described above. An oligonucleotide probe that specifically hybridizes to a region containing the polymorphic site and is labeled with a reporter fluorescent dye and a quencher fluorescent dye is then prepared. The oligonucleotide probe is then hybridized to the genomic DNA, and the DNA containing the polymorphism is amplified using the genomic DNA hybridized with the oligonucleotide probe as a template. The fluorescence emitted by the reporter fluorescent dye is then detected as a result of degradation of the oligonucleotide probe during the amplification. Examples of such methods include the double-dye probe method, also known as the TaqMan® probe method.

[0073] Another method for detecting a polymorphism involves isolating DNA containing the polymorphic site and determining the sequence of the isolated DNA. The DNA can be isolated, for example, by PCR using a pair of oligonucleotides designed to flank the polymorphic site and genomic DNA as a template. The isolated DNA can be sequenced by methods known to those skilled in the art, such as the Maxam-Gilbert method or the Sanger method. Alternatively, the sequence can be determined by next-generation sequencing (NGS). Based on the sequence information thus determined, the number of cysteines in the region can be detected.

[0074] The next-generation sequencing method is not particularly limited, but examples include synthetic sequencing (sequencing-by-synthesis, for example, sequencing using Illumina's Solexa genome analyzer, Hiseq, or Miseq), pyrosequencing (for example, sequencing using Roche Diagnostics (454)'s GSLX or FLX sequencer (so-called 454 sequencing)), and ligase reaction sequencing (for example, sequencing using Life Technologies' SoliD or 5500xl).

[0075] Furthermore, the present invention is not limited to the above-mentioned methods, and other known techniques for detecting polymorphisms, such as RFLP (Restriction Fragment Length Polymorphism), CAPS (PCR-RFLP), denaturing gradient gel electrophoresis (DGGE), Invader, single nucleotide primer extension (SNuPE), allele-specific oligonucleotide (ASO) hybridization, RNase A mismatch cleavage, and DNA arrays, may also be used in the present invention.

[0076] Furthermore, when the target is "transcription products from the genomic DNA," in addition to the polymorphism detection methods described above, for example, RT-PCR, direct sequencing, Northern blotting, dot blotting, and cDNA microarray analysis can be used.

[0077] Furthermore, in the present invention, methods for detecting the translation product (D-hordein protein) include, for example, immunostaining, Western blotting, ELISA, flow cytometry, immunoprecipitation, and antibody array analysis. These methods use an antibody against D-hordein whose epitope is a region containing an amino acid at at least one site selected from the group consisting of position 459 in SEQ ID NO: 2 or a site corresponding thereto, position 473 in SEQ ID NO: 2 or a site corresponding thereto, and position 534 in SEQ ID NO: 2 or a site corresponding thereto.

[0078] Those skilled in the art can prepare such antibodies against D-hordeins by appropriately selecting known methods, such as inoculating an animal to be immunized with a polypeptide or the like comprising a region containing at least one amino acid sequence, activating the animal's immune system, and then recovering the animal's serum (polyclonal antibodies), or methods for producing monoclonal antibodies, such as the hybridoma method, recombinant DNA method, and phage display method.

[0079] Furthermore, when detecting translation products, if an antibody conjugated with a labeling substance is used, D-hordein can be directly detected by detecting the label. The labeling substance is not particularly limited as long as it can be bound to an antibody and is detectable, and examples include peroxidase, β-D-galactosidase, microperoxidase, horseradish peroxidase (HRP), fluorescein isothiocyanate (FITC), rhodamine isothiocyanate (RITC), alkaline phosphatase, biotin, and radioactive substances. Furthermore, in addition to the method of directly detecting D-hordein using an antibody conjugated with a labeling substance, a method of indirectly detecting D-hordein using a secondary antibody, protein G, protein A, or the like conjugated with a labeling substance can also be used.

[0080] <Agents for determining gluten-forming ability in barley> The present invention also provides a reagent for use in the aforementioned determination method, which comprises the oligonucleotide described in (i) or (ii) below. (i) a pair of oligonucleotides designed to flank nucleotides encoding an amino acid at at least one site selected from the group consisting of position 459 of SEQ ID NO: 2 or a site corresponding to said position, position 473 of SEQ ID NO: 2 or a site corresponding to said position, and position 534 of SEQ ID NO: 2 or a site corresponding to said position; (ii) An oligonucleotide that hybridizes to a nucleotide encoding an amino acid at at least one position selected from the group consisting of position 459 of SEQ ID NO: 2 or a site corresponding to said position, position 473 of SEQ ID NO: 2 or a site corresponding to said position, and position 534 of SEQ ID NO: 2 or a site corresponding to said position.

[0081] These polynucleotides have a nucleotide sequence complementary to a specific nucleotide sequence of a D-hordein gene. Here, "complementary" does not necessarily mean perfect complementarity, as long as they hybridize. These polynucleotides typically have 80% or more, preferably 90% or more, more preferably 95% or more, and particularly preferably 100% homology to the specific nucleotide sequence.

[0082] (i) With regard to a pair of oligonucleotides (primer set) according to the present invention designed to sandwich nucleotides encoding the amino acid at least at one site, the length of the oligonucleotides is generally 15 to 100 nucleotides, preferably 17 to 30 nucleotides, and more preferably 17 to 22 nucleotides. Depending on the detection method described above, one of the pair of oligonucleotides may contain a nucleotide sequence complementary to the nucleotides encoding the amino acid at least at one site.

[0083] An example of a pair of oligonucleotides of the present invention is the following primer set shown in the Examples below. the oligonucleotide set forth in SEQ ID NO: 21 and the oligonucleotide set forth in SEQ ID NO: 22; The oligonucleotide set forth in SEQ ID NO: 25 and the oligonucleotide set forth in SEQ ID NO: 26.

[0084] (ii) An oligonucleotide (oligonucleotide probe) according to the present invention that hybridizes to a nucleotide encoding the amino acid at at least one site. The oligonucleotide probe is preferably one that specifically hybridizes to a region containing a polymorphism under normal hybridization conditions, preferably under stringent hybridization conditions.

[0085] The oligonucleotide of the present invention may be appropriately labeled with an isotope, a fluorescent dye, biotin, etc., and then used. As a labeling method, the 5' end of the oligonucleotide may be cleaved using T4 polynucleotide kinase. 32 Labeling by phosphorylation with P, or by using a DNA polymerase such as Klenow enzyme and a random hexamer oligonucleotide as a primer. 32 Examples of such methods include a method of incorporating a substrate base labeled with an isotope such as P, a fluorescent dye, or biotin (random prime method, etc.).

[0086] The oligonucleotides of the present invention can be prepared, for example, using a commercially available oligonucleotide synthesizer. Oligonucleotide probes can also be prepared as double-stranded DNA fragments obtained by restriction enzyme treatment or the like. Furthermore, the oligonucleotides of the present invention do not need to be composed solely of natural nucleotides (deoxyribonucleotides (DNA) or ribonucleotides (RNA)) but may be composed partially or entirely of non-natural nucleotides. The non-natural nucleotides used in the present invention are not particularly limited as long as they have the same functions as natural nucleotides. However, from the viewpoints of increasing the efficiency of hybridization to regions containing polymorphisms and shortening the chain length of oligonucleotide primers and oligonucleotide probes, PNA (polyamide nucleic acid), LNA (registered trademark, locked nucleic acid), ENA (registered trademark, 2'-O,4'-C-Ethylene-bridged nucleic acids), and complexes thereof are preferred. PNA is a DNA or RNA main chain consisting of phosphate and pentose, replaced with a polyamide chain. LNA, also known as BNA (Bridged Nucleic Acid), is RNA in which the 2' oxygen and 4' carbon of a nucleotide are bridged.

[0087] In addition to the oligonucleotide, which is the active ingredient, the reagent may contain, as necessary, for example, sterilized water, physiological saline, vegetable oil, surfactant, lipid, solubilizing agent, buffer, preservative, etc.

[0088] Furthermore, the present invention can provide a kit for use in the method for determining gluten-forming ability in barley, which kit contains the oligonucleotide. The kit of the present invention can also contain a standard other than the oligonucleotide. Examples of such a standard include reagents for extracting genomic DNA from the test barley and reagents necessary for PCR reactions (e.g., deoxyribonucleotides, thermostable DNA polymerase, etc.). The kit can also include instructions for use that describe the determination method, etc.

[0089] Furthermore, as described above, an antibody against D-hordein having an epitope comprising a region containing at least one amino acid at the aforementioned site can also be used in the determination method of the present invention. Thus, similar to the above-mentioned oligonucleotide, the present invention can also provide an agent or kit for determining gluten-forming ability in barley, which comprises the antibody.

[0090] <Method for breeding barley with gluten-forming ability> The present invention provides a method for breeding barley having gluten-forming ability, which comprises the steps of: (a) crossing a barley having gluten-forming ability with an arbitrary variety; (b) selecting barley plants determined to have gluten-forming ability by the above-mentioned method from among the individuals obtained by the crossbreeding in step (a).

[0091] The "barley capable of gluten formation" is not particularly limited as long as it has the ability to form gluten, and examples thereof include "Kantokawa 35," "Nigrinudum," "Hungarian," "Ethiopia 1," "Ethiopia 7," "Ethiopia 59," "Ethiopia 63," "COL / NEPAL / 1985 / IBPGR / 23," and "New Line J039," as shown in the Examples below. Furthermore, examples of "any variety" to be crossed with the barley include, but are not limited to, barley varieties that do not have the ability to form gluten. By using the breeding method of the present invention, it is possible to appropriately select barley capable of gluten formation at the seedling stage, etc., and to develop varieties with the trait in a short period of time. [Example]

[0092] The present invention will be described in more detail below based on examples, but the present invention is not limited to the following examples.

[0093] [Example 1] Analysis of barley D-hordein As described above in [Means for Solving the Problems], the present inventors hypothesized that the inability of barley to produce gluten like that of wheat may be due to structural differences in the seed storage proteins (barley hordein and wheat glutenin). More specifically, they hypothesized that three cysteines present in barley D-hordein but absent in wheat form additional intermolecular disulfide bonds, which result in the formation of aggregates due to excessive intermolecular bonds, preventing the formation of gluten (mesh structure) (Figure 3).

[0094] To verify this hypothesis, the present inventors first searched for barley strains in which any of the three cysteines had been mutated. As a result, they discovered the "Kantokawa 35" strain, which has a D-hordein mutation (Cys9) in which one cysteine ​​(corresponding to cysteine ​​at position 459 in SEQ ID NO: 2) had been mutated to tyrosine. In addition to this strain, the inventors also discovered the following barley strains with nine cysteines in the D-hordein: "Nigrinudum," "Hungarian," "Ethiopia 1," "Ethiopia 7," "Ethiopia 59," "Ethiopia 63," "COL / NEPAL / 1985 / IBPGR / 23," and "New Line J039."

[0095] First, we attempted to detect the Cys9 mutant using the F4 generation population obtained by crossing "Shinkei J039" with "Yumesakiboshi (Cys10 line)" using the method described below. We also prepared gluten polymers (hydrated barley flour) using "Kantokawa 35" and its wild-type (Sachiho Golden, Cys10) strains. Furthermore, we observed these gluten polymers using a scanning electron microscope and analyzed them using size-exclusion high-performance liquid chromatography (HPLC).

[0096] (method) DNA extraction The embryos of two barley seeds were scraped off with a grinder, and 500 μL of DNA extraction buffer (1.25% (w / v) SDS, 500 mM NaCl, 100 mM Tris-Cl, EDTA, pH 8.0; see Dellaporta, SLet et al. (1983) A plant DNA minipreparation: Version II. Plant Mol Biol Rep 1:19-21) was added and suspended by vortexing. After incubation at 65°C for 15 minutes, 160 μL of 5 M potassium acetate was added, mixed well, and the mixture was allowed to stand on ice for 10 minutes. The mixture was centrifuged at 12,500 rpm for 5 minutes, and 500 μL of the supernatant was added to 330 μL of isopropanol and mixed well. The mixture was allowed to stand on ice for 5 minutes, and then centrifuged at 12,500 rpm for 5 minutes. The supernatant was discarded, the DNA was washed with 500 μL of 70% ethanol, dried, and then dissolved in 50 μL of TE containing RNase (7 μg / mL), incubated at 65° C. for 10 minutes, and used as template DNA for the PCR described below.

[0097] PCR 25 μL of reaction solution (1.5 mM MgCl, 0.1 mM each dNTP, 5 pmol each primer (Dh-C9-3: 5′-GCCGGGCTGCTGACCCTGG-3′ (SEQ ID NO: 21), Dh-C9-4: 5′-GGTCGGTGCAAGGGGCGTA-3′) (SEQ ID NO: 22), 0.5 U Hotstar Plus DNA polymerase (Qiagen), 1x PCR buffer (Qiagen), and 50 ng template DNA were prepared. Using a GeneAmp PCR System 9700 (Applied Biosystems), the reaction mixture was heat denatured at 94°C for 5 minutes, followed by 35 cycles (94°C for 30 seconds, 55°C for 30 seconds, 72°C for 30 seconds) and 72°C for 7 minutes. The resulting PCR product was subjected to electrophoresis on a 1.0% (w / v) agarose gel using TAE buffer and fractionated. The results are shown in Figure 4.

[0098] 1.3 Preparation of gluten polymer Barley seeds were milled to produce barley flour using a flour mill (Takarata Kogyo M-300A, a home flour mill, and Satake SRG05, a small rice flour mill). 1 ml of extraction buffer (0.5% SDS, 50 mM NaH2PO4-Na2HPO4 pH 6.9) was added to 0.010 g of the resulting barley flour and vortexed for 5 minutes. After centrifugation at 12,500 rpm for 1 minute, the supernatant was drawn up into a syringe (3 ml) with a needle. The needle was removed and the supernatant was filtered through a filter (ADVANTEC DISMIC-13HP PTFF 0.45 μm). The mixture was placed in an Eppendorf tube, heated at 80°C for 5 minutes, and stored as a soluble fraction. The pellet was added with 1 ml of extraction buffer, sonicated (Tokyo Rika, VCX-130, 130W, 3 mm probe diameter, 30% output, 30 seconds), and centrifuged at 12,500 rpm for 1 minute. The supernatant was then drawn up using a syringe with a needle, the needle removed, and filtered through a 0.45 μm filter. The mixture was placed in an Eppendorf tube, heated at 80°C for 5 minutes, and stored as an insoluble fraction.

[0099] 1.4. Scanning electron microscope observation A barley flour sample was attached to carbon tape, and a drop of water was placed on it. The sample was then observed using a scanning electron microscope (Hitachi High-Technologies Corporation, S-3400N scanning electron microscope with Type II cool stage) at a vacuum of 80 Pa and -25°C. The results are shown in Figure 5.

[0100] 1.5. Gluten Polymer Analysis by Size-Exclusion HPLC The pre-column was a TSK-GUARD Column 7.5*75 (Tosoh) and the column was a TSK-GEL G4000SW 7.5*300 (Tosoh). The buffer was a 1:1 mixture of 0.1% TFA and 100% acetonitrile at 0.25 ml / min. The conditions were a column temperature of 35°C, a measurement wavelength of 214 nm, and a run time of 40 minutes.

[0101] (Results and Discussion) 1.6. Mutant detection by PCR We investigated whether strains with the Cys9 mutation could be identified in the F4 generation of a cross between "Shinkei J039" and "Yumesakiboshi." "Shinkei J039" is a Cys9 strain, and "Yumesakiboshi" is a Cys10 strain. The D-hordein gene from "Shinkei J039" and the amino acid sequence it encodes are shown in SEQ ID NOs: 13 and 14, respectively. The D-hordein gene from "Yumesakiboshi" and the amino acid sequence it encodes are shown in SEQ ID NOs: 17 and 18, respectively. For this PCR analysis, we used a forward primer (Dh-C9-4) that hybridizes to the nucleotide encoding the tyrosine (the amino acid corresponding to cysteine ​​at position 459 in SEQ ID NO: 2) and a reverse primer (Dh-C9-3) that hybridizes to the nucleotide encoding the amino acids corresponding to positions 682 to 688 in SEQ ID NO: 2.

[0102] As a result, as shown in Figure 4, the PCR resulted in the amplification of a band (approximately 0.6 kbp) specific to the Cys9 mutant (lanes 2 to 5 in Figure 4). On the other hand, no such band was observed for Cys10 (lanes 1 and 6 in Figure 4), demonstrating that the use of these primers allows for the specific detection and discrimination of Cys9 mutants.

[0103] 1.7. Gluten Polymer Analysis by Size-Exclusion HPLC As shown in Figure 5, the Cys9 strain showed a tendency for the ratio of the peak of insoluble polymer 2 (UPP2), which is thought to form a network structure, to the peak of insoluble polymer 1 (UPP1), which is thought to form aggregates, to be higher compared to the wild-type strain (Cys10). Specifically, it was revealed that cysteines present in barley D-hordein but absent in wheat glutenin form additional intermolecular disulfide bonds, resulting in excessive intermolecular bonds, leading to the formation of aggregates and preventing the formation of a network structure (Cys10 strain). On the other hand, it was also revealed that substitution of these cysteines with other amino acids suppressed the formation of aggregates and facilitated the formation of a network structure (gluten) (Cys9 strain).

[0104] [Example 2] Genome editing Using the method described below, we attempted to create a mutant of the barley line "Hokurikukawa 71" (Cys10 line, wild-type) that contained D-hordein lacking the three cysteines (cysteines at positions 459, 473, and 534 of SEQ ID NO: 2).

[0105] (method) 2.1. Determination of target sequence Based on the sequence of the D-hordein gene of "Hokuriku Skin No. 71" (sequence set forth in SEQ ID NO: 1), a 20-bp target sequence to be included in the guide RNA was searched for. CRISPRdirect (https: / / crispr.dbcls.jp / ) was used to search for the target sequence. As a result, 5'-TCTTCACAGGGGTCGGTGCA-3' (SEQ ID NO: 23), upstream of the nucleotide encoding cysteine ​​at position 459 in the amino acid sequence of D-hordein (sequence set forth in SEQ ID NO: 2), was selected as target sequence 1. Similarly, 5'-AGACAGTGGTTTGCTGGCAA-3', downstream of the nucleotide encoding cysteine ​​at position 534, was selected as target sequence 2. (SEQ ID NO: 24) was designated as target sequence 2.

[0106] 2.2. Preparation of barley shoot apex samples Dried seeds of "Hokurikukawa 71" were shaken in a 20% aqueous solution of sodium hypochlorite for 30 minutes to sterilize the seed surface. Sterilized seeds were sown on a paper towel soaked in sterile water in a plastic petri dish. The sterilized seeds were imbibed in the dark at 4°C for 3 days and then used to prepare shoot apex samples. The coleoptile, first, second, and third leaves were removed from the imbibed seed embryos using a Nanopass Needle II 34G (TERUMO) to expose the tip of the shoot apical meristem. The embryos were then separated from the endosperm and placed on MS medium [MS salt (Sigma) 4.3 g / L, Maltose 30 g / L, MES 9.8 g / L, Phytagel 7 g / L]. 30–40 shoot apex samples were placed in the center of the medium to form a 1 cm diameter donut-shaped circle.

[0107] 2.3. Preparation of RNP complexes The recombinant SpCas9 protein (2 μg / μL) was provided by the National Agriculture and Food Research Organization (NARO). The guide RNAs consisted of crRNA and tracrRNA based on the target sequences described above and were chemically synthesized by Fasmac.

[0108] To a 1.5 mL tube, 5 μL of 50 μM crRNA, 5 μL of 50 μM tracrRNA, 5 μL of SpCas9 solution, 2 μL of 10× CutSmart buffer, 0.5 μL of Recombinant RNase Inhibitor (TaKaRa), and 2.5 μL of nuclease-free water were added and allowed to stand for 10 minutes to form RNP complexes. After preparing RNP complex solutions for each target sequence, they were combined into a single 1.5 mL tube.

[0109] 2.4. Preparation of RNP-bound gold particles 0.6 μm Gold microcarriers (Bio-Rad) were suspended in nuclease-free water at 180 μg / μL to prepare a gold suspension. 5 μL of TransIT-LT1 reagent (Mirus Bio) was added to a 1.5 mL tube containing the two RNP complexes and allowed to stand for 5 minutes. 15 μL of gold suspension was then added, mixed gently, and allowed to stand for 10 minutes. The mixture was centrifuged at 2,500 G for 15 seconds to precipitate the RNP-bound gold particles. The supernatant was removed, and the RNP-bound gold particles were resuspended in 24 μL of nuclease-free water.

[0110] 2.5. Particle bombardment Six microliters of the prepared RNP-bound gold particle suspension was spread onto a hydrophilic film (3M) and allowed to dry at room temperature. The suspension was then fired four times onto shoot apex samples on MS medium using a particle gun. The particle gun used was a PDS-1000 / He Particle Delivery System (Bio-Rad), and the gold particle injection pressure was 1,350 psi.

[0111] 2.6. DNA extraction 100 mg of plant tissue was flash-frozen in liquid nitrogen and disrupted using a multi-beads shocker. 400 μL of extraction solution [100 mM Tris-HCl (pH 9.0), 40 mM EDTA, 1.67% SDS] was added to the disrupted tissue and allowed to stand for 3 minutes. The extract was centrifuged at 5,000 G for 3 minutes, and 200 μL of the supernatant was collected. 500 μL of 99.5% EtOH and 20 μL of 3 M sodium acetate were added, and the mixture was centrifuged at 12,000 G for 10 minutes to precipitate DNA. The precipitated DNA was washed with 70% EtOH and then dissolved in 100 μL of ultrapure water.

[0112] 2.7. Genotyping A partial sequence of the D-hordein gene was amplified by PCR. The primers used for amplification were 5'-TGCAGCAAGGAGGATGGTGG-3' (SEQ ID NO: 25) and 5'-GGCTGCTCCACGCTAACATG-3' (SEQ ID NO: 26), and the PCR enzyme was KOD-One (Toyobo). The two-step PCR conditions consisted of 30 cycles of heat denaturation at 98°C for 10 seconds and extension at 68°C for 20 seconds. The amplified products were subjected to in vitro cleavage reactions and then analyzed using a microchip electrophoresis system. The in vitro cleavage reactions were performed using the RNP complex used to create the genome-edited individuals or HindIII (TaKaRa). DNA fragments that were not cleaved by the reaction were subjected to sequence analysis.

[0113] DNA Sequencing The DNA fragments were introduced into the Zero Blunt TOPO PCR Cloning Kit, and sequence analysis was performed using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and a SeqStudio Genetic Analyzer (Applied Biosystems).

[0114] (Results and Discussion) As described above, we designed CRSPR / Cas9 target sequences upstream and downstream of the three cysteines in D-hordein, and attempted to create genome-edited individuals in which the region containing the three cysteines (Cys region) was deleted by simultaneous cleavage at these two sites.

[0115] Specifically, an RNP complex consisting of guide RNA and SpCas9 was first introduced into 1,616 shoot apex samples by particle bombardment. These individuals were grown to the fifth leaf stage and genotyped. Specifically, DNA was extracted from the fifth leaf blade and used as a template to amplify the D-Hor partial sequence. The amplified product was then subjected to in vitro cleavage reactions and analyzed using a microchip electrophoresis system.

[0116] As a result, a D-hordein gene lacking the Cys domain was detected in one individual, and this individual was named HK11.29. The resulting D-hordein gene mutant lacked 252 bases, including the Cys domain, and the upstream and downstream regions of the deletion site were recombined in frame (SEQ ID NO: 19). The Cys-deleted D-hordein gene encoded a protein shorter than that of the wild-type (SEQ ID NO: 20). Furthermore, the genotype of the T1 individual obtained from HK11.29 was analyzed. As a result, a T1 individual homozygous for the Cys-deleted D-hordein gene was obtained.

[0117] The obtained T1 individuals were analyzed in the same manner as described above in "1.4. Observation with a scanning electron microscope" and "1.5. Analysis of gluten polymers by size-exclusion HPLC." The results are shown in Figures 8 to 10 and Table 1.

[0118] [Table 1]

[0119] As shown in Figures 8 and 9 and Table 1, compared to Hokurikukawa No. 71 (Cys10 line), the genome-edited individuals (deleted Cys7 mutant line) exhibited a mesh structure when observed with a scanning electron microscope, and an increased proportion of UPP2-derived peaks was observed when analyzed by size-exclusion HPLC. In particular, compared to the Cys9 line, the Cys7 line tended to have less UPP1 and more UPP2, revealing a higher gluten-forming ability. Furthermore, as shown in Figure 10, the elution pattern of the Cys7 line was similar to that of gluten-forming wheat, suggesting that the line possesses functions more similar to those of wheat high-molecular-weight glutenin. [Industrial Applicability]

[0120] As explained above, according to the present invention, it is possible to impart gluten-forming ability to barley. It is also possible to determine the gluten-forming ability of barley. Furthermore, barley with gluten-forming ability provided in this manner exhibits high processing suitability, like wheat. In other words, it exhibits high dough properties and, in turn, high secondary processing suitability (suitability for the production of wheat processed foods such as bread), and is therefore useful not only in the agricultural field but also in the food field, etc.

Claims

1. A method for producing gluten-forming barley, comprising the following steps (1) and (2): (1) introducing the following modification (a) into a D-hordein protein in a barley cell: (a) a modification in a D-hordein protein in which the cysteine ​​at position 459 of SEQ ID NO: 2 or a site corresponding to said position is substituted with another amino acid or deleted; (2) Regenerating a barley plant from the cells into which the modification has been introduced in step (1).

2. A method for determining gluten-forming ability in barley, comprising the following steps (1) and (2): (1) detecting an amino acid at position 459 of SEQ ID NO: 2 or a site corresponding to said position in a test barley D-hordein protein; (2) A method comprising a step of determining that the test barley is capable of forming gluten when the amino acid at position 459 of SEQ ID NO: 2 or a position corresponding to said position is an amino acid other than cysteine ​​or is deleted.

3. A reagent for use in the method according to claim 2, comprising the following oligonucleotide (i) or (ii): (i) a pair of oligonucleotides designed to flank a nucleotide encoding an amino acid at position 459 of SEQ ID NO: 2 or a site corresponding to said position; (ii) An oligonucleotide that hybridizes to a nucleotide encoding the amino acid at position 459 of SEQ ID NO: 2 or a position corresponding to said position.

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