Barley and method for producing barley

Abstract

To provide a barley plant that yields grains in which the combined content of β-glucan and fructan is increased relative to that of grains obtained from a wild-type barley plant, and also to provide a barley plant that yields grains in which a content of γ-aminobutyric acid (GABA) is increased relative to that of grains obtained from a wild-type barley plant.SOLUTION: A barley plant having loss of function in both the SSIIa gene and the GBSSI gene.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to barley and methods for producing barley. [Background technology]

[0002] Currently, barley grains containing large amounts of functional components such as β-glucan or fructan are produced around the world, and such barley grains are being sold in Asia, including Japan, and Europe.

[0003] For example, Patent Document 1 discloses such a barley plant, which contains a reduced level or activity of SSIIa protein and is capable of producing barley kernels having a starch content of at least 41% (w / w).It also discloses that the barley kernels have a β-glucan content of 5 to 9% (w / w) or more than 9% (w / w) and a fructan content of 3 to 11% (w / w) or 4 to 11% (w / w). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Special Publication No. 2013-500022 Summary of the Invention [Problem to be solved by the invention]

[0005] On the other hand, Patent Document 1 does not disclose the total content of β-glucan and fructan in barley grains. As such, at present, little progress has been made in research into the creation of barley plant lines with an increased total content of β-glucan and fructan in barley grains. Furthermore, there is still room for research into the creation of barley plant lines with an increased content of γ-aminobutyric acid in barley grains.

[0006] An object of the present invention is to provide a barley plant which can produce grain having an increased total content of β-glucan and fructan compared to grain derived from a wild-type barley plant. Another object of the present invention is to provide a barley plant which can produce grain having increased γ-aminobutyric acid (GABA) compared to grain derived from a wild-type barley plant. [Means for solving the problem]

[0007] As a result of extensive research, the present inventors have found that grains derived from barley plants into which nonsense mutations have been introduced into the SSIIa gene and the GBSSI gene have an increased total content of β-glucan and fructan compared to grains derived from wild-type barley plants, and that grains derived from barley plants into which nonsense mutations have been introduced into the SSIIa gene and the GBSSI gene have an increased content of γ-aminobutyric acid compared to grains derived from wild-type barley plants, leading to the completion of the present invention.

[0008] The present invention provides, for example, the following inventions. [1] A barley plant lacking the functions of the SSIIa gene and the GBSSI gene. [2] The barley plant according to [1], wherein the β-glucan content in grains derived from the barley plant is 10 w / w% or more. [2a] The barley plant according to [1] or [2], wherein the content of γ-aminobutyric acid in grains derived from the barley plant is 50 mg / 100 g or more. [3] A method for producing a barley plant, comprising the step of deleting the functions of the SSIIa gene and the GBSSI gene in a barley plant. [4] The method according to [3], wherein the β-glucan content in the grains derived from the barley plant is 10 w / w% or more. [4a] The method according to [3] or [4], wherein the content of γ-aminobutyric acid in grains derived from the barley plant is 50 mg / 100 g or more. [5] A method for increasing the total content of β-glucan and fructan in grain derived from a barley plant, the method comprising the step of deleting the functions of the SSIIa gene and the GBSSI gene in the barley plant. [6] A method for increasing the total content of β-glucan and fructan, as well as the content of γ-aminobutyric acid, in grain derived from a barley plant, the method comprising the step of deleting the functions of the SSIIa gene and the GBSSI gene in the barley plant. [7] A method for increasing the content of γ-aminobutyric acid in grain derived from a barley plant, comprising the step of deleting the functions of the SSIIa gene and the GBSSI gene in the barley plant. [Effects of the Invention]

[0009] According to the present invention, it is possible to provide a barley plant that can produce grains having an increased total content of β-glucan and fructan compared to grains derived from wild-type barley plants. [Brief explanation of the drawings]

[0010] [Figure 1] 1 is a graph showing the results of measuring the β-glucan content in barley grains. [Figure 2] 1 is a graph showing the results of measuring the fructan content in barley kernels. [Figure 3] 1 is a graph showing the results of measuring the total content of β-glucan and fructan in barley kernels. DETAILED DESCRIPTION OF THE INVENTION

[0011] Hereinafter, embodiments of the present invention will be described in detail, but the present invention is not limited to the following embodiments.

[0012] [Barley plant] The barley plant according to the present embodiment is deficient in the functions of the SSIIa gene and the GBSSI gene. Therefore, the barley plant according to the present embodiment has an increased total content of β-glucan and fructan in its grains compared to wild-type barley plants. Furthermore, the barley plant according to the present embodiment also has an increased content of γ-aminobutyric acid (GABA) in its grains compared to wild-type barley plants. Examples of wild-type barley plants include Yumesakiboshi.

[0013] As used herein, barley plants refer to the genus Hordeum vulgare of the family Poaceae. Unless otherwise specified, the term "plant" as used herein includes whole plants, plant cells, plant protoplasts, plant calluses, or plant parts such as embryos, pollen, ovules, gametes, grains, seeds, leaves, flowers, branches, fruits, stems, roots, anthers, etc.

[0014] The SSIIa and GBSSI genes each encode enzymes involved in starch synthesis. Starch is composed of amylose, a linear chain of glucose units, and amylopectin, a branched chain of amylose with side chains.

[0015] The SSIIa gene is a gene encoding the SSIIa protein (Starch Synthase IIa). The SSIIa protein is thought to have the function of extending the side chains of amylopectin. Examples of the SSIIa gene include a gene encoding the amino acid sequence shown in SEQ ID NO: 1 and a gene containing the nucleotide sequence shown in SEQ ID NO: 2.

[0016] The GBSSI gene encodes the GBSSI (Granule-bound starch synthase I) protein. The GBSSI protein is thought to have the function of extending linear amylose chains. Examples of the GBSSI gene include a gene encoding the amino acid sequence shown in SEQ ID NO: 3 and a gene containing the nucleotide sequence shown in SEQ ID NO: 4.

[0017] As used herein, "gene" refers to a DNA sequence capable of expressing a protein. A "gene" may include a region (coding region) that is transcribed into mRNA in a cell and then translated into a protein, an expression regulatory region located upstream and / or downstream of the coding region, and an untranslated region (5'UTR, 3'UTR). The coding region may include not only exons but also introns.

[0018] The expression regulatory region may be, for example, a transcription regulatory region or a translation regulatory region. Examples of the transcription regulatory region include a promoter, an enhancer, and a silencer. Examples of the translation regulatory region include a ribosome binding region.

[0019] As used herein, "gene function is deficient" means that the SSIIa gene and GBSSI gene have mutations, and the expression products (e.g., proteins) of the genes do not function in the cells of the barley plant.

[0020] Whether a barley plant is deficient in the function of the SSIIa gene can be analyzed, for example, by observing grain shrinkage during maturation, or whether a barley plant is deficient in the function of the GBSSI gene can be analyzed, for example, by starch iodine reaction.

[0021] When the functions of the SSIIa gene and the GBSSI gene are analyzed using the above-mentioned method, if the quantitative value of their function is less than 5%, less than 3%, less than 1%, or 0% of the quantitative value of the function of each wild-type gene, it can be determined that the genes are not functioning (the gene function is missing).

[0022] Specific examples of "gene function deficiency" include a case where the gene has a mutation and cannot be translated into a normal protein, or a case where the entire base sequence of the gene is deleted from the genome.

[0023] As used herein, the term "mutation" means that the SSIIa gene or GBSSI gene in a barley plant, or the protein encoded thereby, differs in at least one base or one amino acid residue from the SSIIa gene or GBSSI gene in a wild-type barley plant, or the protein encoded thereby. The mutation may also be a deletion, substitution, and / or addition (insertion) of a base in the base sequence of the SSIIa gene or GBSSI gene. Mutations that cause the loss of function of the SSIIa gene and GBSSI gene can be selected as desired by those skilled in the art. The barley plant according to this embodiment is a homozygote in which the gene having the above mutation (mutant gene) is present in both alleles.

[0024] The deletion of bases may be, for example, 1 to 1000 bases, 1 to 800 bases, 1 to 500 bases, 1 to 400 bases, 1 to 300 bases, 1 to 200 bases, 1 to 100 bases, 1 to 50 bases, 1 to 20 bases, 1 to 10 bases, 1 to 5 bases, 1 to 3 bases, 1 or 2 bases, or 1 base.

[0025] The insertion of bases may be, for example, an insertion of 1 to 1000 bases, 1 to 800 bases, 1 to 500 bases, 1 to 400 bases, 1 to 300 bases, 1 to 200 bases, 1 to 100 bases, 1 to 50 bases, 1 to 20 bases, 1 to 10 bases, 1 to 5 bases, 1 to 3 bases, 1 or 2 bases, or 1 base.

[0026] The substitution of bases may be, for example, a substitution of 1 to 1000 bases, 1 to 800 bases, 1 to 500 bases, 1 to 400 bases, 1 to 300 bases, 1 to 200 bases, 1 to 100 bases, 1 to 50 bases, 1 to 20 bases, 1 to 10 bases, 1 to 5 bases, 1 to 3 bases, 1 or 2 bases, or 1 base.

[0027] The deletion, insertion, and substitution may occur in consecutive bases or discontinuous bases. For example, in the case of a 200-base deletion, the deletion may be a continuous 200-base deletion, or two 100-base deletions may occur.

[0028] When a specific embodiment of the SSIIa gene function is one in which translation into a normal SSIIa protein is not possible, more specifically, the SSIIa gene may have a mutation in the nucleotide sequence of the coding region and / or expression regulatory region of the gene. When the nucleotide sequence of the coding region of the gene has a mutation, the mutation may be, for example, at least one mutation selected from the group consisting of a missense mutation, a nonsense mutation, a frameshift mutation, and a splicing site mutation, and preferably a nonsense mutation. The SSIIa gene function may be a mutation in the amino acid sequence of the protein encoded by the gene (SEQ ID NO: 1) in which the 320th amino acid residue (codon) and subsequent amino acid residues (codons) (the 320th codon in the nucleotide sequence shown in SEQ ID NO: 2) are deleted, or a mutation in which the 43rd amino acid residue (codon) and subsequent amino acid residues (codons) (the 43rd codon in the nucleotide sequence shown in SEQ ID NO: 2) are deleted.

[0029] When a specific embodiment of the GBSSI gene function is one in which translation into a normal GBSSI protein is not possible, the term "SSIIa gene" may be read as "GBSSI gene." However, the GBSSI gene function may be a mutation in the amino acid sequence (SEQ ID NO: 3) of the protein encoded by the gene, resulting in the deletion of amino acid residues (codons) subsequent to the 194th amino acid residue (codon 194 in the base sequence shown in SEQ ID NO: 4). A specific embodiment of the GBSSI gene function may be a mutation in the amino acid sequence shown in SEQ ID NO: 3, in which the glutamine residue at position 312 is replaced with a histidine residue, or a mutation in which the amino acid sequence shown in SEQ ID NO: 3 becomes the amino acid sequence shown in SEQ ID NO: 5, and in which the aspartic acid residue at position 287 in the amino acid sequence shown in SEQ ID NO: 5 is replaced with a valine residue, or a mutation in which the glycine residue at position 513 is replaced with a tryptophan residue.

[0030] The β-glucan content in grain derived from a barley plant according to one embodiment may be, for example, 5 w / w% or more, 5.5 w / w% or more, 6 w / w% or more, 6.5 w / w% or more, 7 w / w% or more, 7.5 w / w% or more, 8 w / w% or more, 8.5 w / w% or more, 9 w / w% or more, 9.5 w / w% or more, 10 w / w% or more, 10.5 w / w% or more, 11 w / w% or more, 11.5 w / w% or more, or 12 w / w% or more. The β-glucan content in grain derived from a barley plant according to one embodiment may be, for example, 20 w / w% or less, 18 w / w% or less, or 15 w / w% or less.

[0031] In this specification, the β-glucan content in grains derived from barley plants is measured by a method in which β-glucan is hydrolyzed to D-glucose using an enzyme such as lichenase, β-glucanase, or β-glucosidase, and the amount of D-glucose produced is measured (AOAC Method 995.16, AACC Method 32-23, and ICC Standard Method No. 166). This method can also be performed using a commercially available kit (e.g., Megazyme Mixed Linkage Beta-Glucan Assay Kit (Megazyme)).

[0032] The fructan content in grain derived from a barley plant according to one embodiment may be, for example, 2.5 w / w% or more, 3 w / w% or more, 3.5 w / w% or more, 4 w / w% or more, 4.5 w / w% or more, 5 w / w% or more, 5.5 w / w% or more, 6 w / w% or more, or 6.5 w / w% or more. The fructan content in grain derived from a barley plant according to one embodiment may be, for example, 15 w / w% or less, 12 w / w% or less, or 10 w / w% or less.

[0033] In this specification, the fructan content in grains derived from barley plants is measured by a method in which fructans are hydrolyzed with enzymes such as inulinase and levanase to produce D-glucose and D-fructose, and the amounts of D-glucose and D-fructose produced are measured. This method can also be performed using a commercially available kit (e.g., Fructan Assay Kit (Megazyme)).

[0034] The total content of β-glucan and fructan in grain derived from a barley plant according to one embodiment may be, for example, 7 w / w% or more, 7.5 w / w% or more, 8 w / w% or more, 8.5 w / w% or more, 9 w / w% or more, 9.5 w / w% or more, 10 w / w% or more, 10.5 w / w% or more, 11 w / w% or more, 11.5 w / w% or more, 12 w / w% or more, 12.5 w / w% or more, 13 w / w% or more, 13.5 w / w% or more, 14 w / w% or more, 14.5 w / w% or more, 15 w / w% or more, 15.5 w / w% or more, 16 w / w% or more, 16.5 w / w% or more, 17 w / w% or more, 17.5 w / w% or more, or 18.5 w / w% or more. The total content of β-glucan and fructan in grain derived from a barley plant according to one embodiment may be, for example, 40 w / w% or less, 35 w / w% or less, 30 w / w% or less, 25 w / w% or less, or 20 w / w% or less.

[0035] A barley plant according to one embodiment may have or not have a mutation in the amo1 gene. The amo1 gene is known to be involved in amylose synthesis. The nucleotide sequence of the amo1 gene itself has not been isolated, but linkage markers have been developed. For example, it has been reported that the amo1 line "high amylose Glacier (HAG)" has a single-base substitution (T to G) at the 8601st base of the ssIIIa gene (GenBank no. JN256947). Whether or not the amo1 gene has a mutation can be determined by analyzing such a marker as a linkage marker.

[0036] The amylose content in grain derived from a barley plant according to one embodiment may be, for example, 5 wt% or less, 4 wt% or less, or 3 wt% or less.The amylose content in grain derived from a barley plant according to one embodiment may be, for example, 0 wt% or more.

[0037] In this specification, the amylose content in grains derived from barley plants is measured by a method of measuring the color change when an iodine solution or the like is added to grains derived from barley plants, based on the fact that when an iodine solution or the like is added to amylose, iodine penetrates into the helical structure of the amylose, causing the amylose to turn blue-purple. This method can also be performed using commercially available iodine-containing mouthwash or the like.

[0038] The content of gamma-aminobutyric acid (GABA) in grain derived from a barley plant according to one embodiment may be, for example, 50 mg / 100 g or more, 100 mg / 100 g or more, 150 mg / 100 g or more, 200 mg / g or more, or 250 mg / 100 g or more, and may be 300 mg / 100 g or less, 350 mg / 100 g or less, or 400 mg / 100 g or less.

[0039] In this specification, the gamma-aminobutyric acid (GABA) content in grains derived from barley plants is measured as follows: The reaction in which L-glutamic acid and succinic semialdehyde are produced in the presence of GABA, α-ketoglutaric acid, and GABA transaminase in a sample is coupled with the reaction in which hydrogen peroxide is produced in the presence of L-glutamic acid and L-glutamate oxidase; a peroxidase reaction (color reaction) using hydrogen peroxide and a coloring agent as substrates is then simultaneously carried out, and the color of the sample is measured. This method can also be performed using a commercially available GABA measurement kit (e.g., GABA Miel (Enzyme Sensor)).

[0040] Grains derived from the barley plants according to the present embodiments can be used to produce food or beverage products such as breakfast cereals, biscuits, muffins, muesli bars, barley rice, barley miso, beer, shochu, barley tea, noodles, sweeteners, low-calorie additives, bulking agents, dietary fiber, anti-sticking agents, preservatives, probiotic agents, etc. Furthermore, leaves of the barley plants according to the present embodiments can be used to produce, for example, green juice and the like.

[0041] [Method for producing barley plants] The method for producing a barley plant according to this embodiment (hereinafter also referred to as the "production method according to this embodiment") comprises a step of deleting the functions of the SSIIa gene and the GBSSI gene in a barley plant (deletion step).

[0042] The deleting step may involve, for example, introducing mutations into the SSIIa gene and GBSSI gene in the barley plant, or deleting the entire nucleotide sequences of the SSIIa gene and GBSSI gene from the genome of the barley plant. The mutations to be introduced are as described above.

[0043] Methods for introducing mutations into the SSIIa gene and the GBSSI gene include, for example, methods involving the insertion of exogenous factors, methods involving mutagen treatment, and methods involving genome editing technology. Examples of methods involving the insertion of exogenous factors include the insertion of transposons or T-DNA. Examples of methods involving mutagen treatment include methods in which random mutations are introduced by mutagen treatment such as EMS (methyl methanesulfonate), DEB (diepoxybutane), X-ray treatment, ion beam treatment, and gamma-ray treatment, and barley plants lacking the function of each gene are selected from the resulting plants. Examples of methods involving genome editing technology include methods in which mutations are introduced by targeting each gene using zinc finger nucleases (ZFN), TALEN, or CRISPR / Cas9.

[0044] In addition, in the case of the method involving the insertion of an exogenous factor or the method involving mutagen treatment, barley plants lacking the function of each gene may be selected from, for example, a mutant library of soybean plants into which transposons or T-DNAs have been randomly inserted, or a mutant library of barley plants into which mutations have been randomly introduced by mutagen treatment.

[0045] Methods for deleting the entire nucleotide sequences of the SSIIa gene and the GBSSI gene from the genome of a barley plant include, for example, methods using the genome editing techniques described above.

[0046] The deleting step may be performed by crossing barley plants, or may be performed without crossing. When the deleting step is performed by crossing barley plants, for example, a method of crossing a barley plant lacking the function of the SSIIa gene with a barley plant lacking the function of the GBSSI gene may be used. That is, the deleting step may include preparing a barley plant lacking the function of the SSIIa gene (first barley plant), preparing a barley plant lacking the function of the GBSSI gene (second barley plant), and crossing the first barley plant with the second barley plant. The first barley plant and the second barley plant can be prepared, for example, by introducing a mutation into the above-mentioned gene or by deleting the entire nucleotide sequence of the gene from the genome of the barley plant.

[0047] The production method according to this embodiment may further include a step of growing a barley plant (growing step). The growth conditions for the barley plant in the growing step may be the usual conditions used by those skilled in the art to grow (cultivate) barley, and may be set appropriately depending on the state of the barley plant, the nature of the defecting step, etc. The temperature may be, for example, 20°C to 30°C. The relative humidity may be, for example, 60% to 90%. The photoperiod may be a 14-hour light period and a 10-hour dark period.

[0048] In one embodiment, the barley plant produced by the production method may have a β-glucan content in grain derived from the barley plant of, for example, 5 w / w% or more, 5.5 w / w% or more, 6 w / w% or more, 6.5 w / w% or more, 7 w / w% or more, 7.5 w / w% or more, 8 w / w% or more, 8.5 w / w% or more, 9 w / w% or more, 9.5 w / w% or more, 10 w / w% or more, 10.5 w / w% or more, 11 w / w% or more, 11.5 w / w% or more, or 12 w / w% or more. In one embodiment, the barley plant produced by the production method may have a β-glucan content in grain derived from the barley plant of, for example, 20 w / w% or less, 18 w / w% or less, or 15 w / w% or less.

[0049] In one embodiment of the production method, the barley plant produced may have a fructan content in grains derived from the barley plant of, for example, 2.5 w / w% or more, 3 w / w% or more, 3.5 w / w% or more, 4 w / w% or more, 4.5 w / w% or more, 5 w / w% or more, 5.5 w / w% or more, 6 w / w% or more, or 6.5 w / w% or more. In one embodiment of the production method, the barley plant produced may have a fructan content in grains derived from the barley plant of, for example, 15 w / w% or less, 12 w / w% or less, or 10 w / w% or less.

[0050] In one embodiment of the production method, the barley plant produced has a total content of β-glucan and fructan in grains derived from the barley plant of, for example, 7 w / w% or more, 7.5 w / w% or more, 8 w / w% or more, 8.5 w / w% or more, 9 w / w% or more, 9.5 w / w% or more, 10 w / w% or more, 10.5 w / w% or more, 11 w / w% or more, 11 In one embodiment, the barley plant produced by the method may have a total β-glucan and fructan content in grain derived from the barley plant of, for example, 40 w / w% or less, 35 w / w% or less, 30 w / w% or less, 25 w / w% or less, or 20 w / w% or less.

[0051] In one embodiment of the production method, the barley plant produced may have a gamma-aminobutyric acid (GABA) content in grain derived from the barley plant of, for example, 50 mg / 100 g or more, 100 mg / 100 g or more, 150 mg / 100 g or more, 200 mg / 100 g or more, or 250 mg / 100 g or more, or 300 mg / 100 g or less, 350 mg / 100 g or less, or 400 mg / 100 g or less.

[0052] [Method for increasing the total content of β-glucan and fructan in barley] The method for increasing the total content of β-glucan and fructan in grain derived from a barley plant according to this embodiment (hereinafter also referred to as the "method according to this embodiment") comprises a step of deleting the functions of the SSIIa gene and the GBSSI gene in a barley plant.

[0053] "Increasing the total content of β-glucan and fructan" means increasing the total content of β-glucan and fructan in grain derived from a barley plant compared to that in grain derived from a wild-type barley plant, the wild-type barley plant being as described above.

[0054] The step of deleting the functions of the SSIIa gene and the GBSSI gene is as described in the deleting step in the production method according to this embodiment.

[0055] As described above, the barley plants according to this embodiment also have an increased γ-aminobutyric acid (GABA) content in their grains compared to wild-type barley plants. Accordingly, the present invention also provides a method for increasing the total content of β-glucan and fructan, as well as the γ-aminobutyric acid (GABA) content, in grain derived from a barley plant, the method comprising the step of deleting the functions of the SSIIa gene and the GBSSI gene in the barley plant. The present invention further provides a method for increasing the γ-aminobutyric acid (GABA) content in grain derived from a barley plant, the method comprising the step of deleting the functions of the SSIIa gene and the GBSSI gene in the barley plant.

[0056] "Increasing the total content of β-glucan and fructan, and the content of γ-aminobutyric acid (GABA)" and "increasing the content of γ-aminobutyric acid (GABA)" are obtained by replacing "the total content of β-glucan and fructan" in the explanation of "increasing the total content of β-glucan and fructan" above with "the total content of β-glucan and fructan, and the content of γ-aminobutyric acid (GABA)" or "the content of γ-aminobutyric acid (GABA)." [Example]

[0057] The present invention will be described in more detail below with reference to examples, although the present invention is not limited to the following examples.

[0058] (Test Example 1) The barley plant with a mutation in the SSIIa gene was a cross between GSHO 3425, obtained from an overseas genebank (the National Small Grains Collection (NSGC) of the United States Department of Agriculture's Agricultural Research Service (USDA-ARS) in the United States), and the wild-type barley plant Yumesakiboshi (hereinafter also referred to as the "SSIIa mutant"). GSHO 3425 is a mutant in which the 43rd codon in the nucleotide sequence (SEQ ID NO: 2) of the coding region of the SSIIa gene has been replaced with a stop codon, resulting in a loss of function of the gene alone. The barley plant with a mutation in the GBSSI gene was a mutant (domestic line) in which the 194th codon in the nucleotide sequence (SEQ ID NO: 4) of the coding region of the GBSSI gene has been replaced with a stop codon, resulting in a loss of function of the gene alone (hereinafter also referred to as the "GBSSI mutant"). A cross between GSHO 3425 and the GBSSI mutant was used as a barley plant having mutations in the SSIIa gene and the GBSSI gene (hereinafter also referred to as the "SSIIa+GBSSI mutant"). Yumesakiboshi was used as a wild-type barley plant.

[0059] The above barley plants were grown under field conditions in Chikugo City with autumn 2022 sowing, and the grains were harvested. The barley grains were then crushed using a Cyclone Sample Mill CSM-S1 (Fujiwara Seisakusho), placed in a polyethylene bag with a zipper, and stored in a desiccator. The β-glucan content, fructan content, and total content of these in each barley grain were measured using the following methods. The results are shown in Figures 1 to 3 and Table 1.

[0060] Eighty milligrams of the ground barley grains was weighed and used to measure the β-glucan content in the barley grains. Measurement of the β-glucan content in the barley grains was performed using the Megazyme Mixed Linkage Beta-Glucan Assay Kit (Megazyme) according to the manufacturer's recommended protocol ((A) Assay Procedure for Oat and Barley Flour and Fiber Samples—Streamlined Method (AOAC Method 995.16, AACC Method 32-23, and ICC Standard Method No. 166)).

[0061] 25 mg of the ground barley grains was weighed and used to measure the fructan content in the barley grains, which was measured using a Fructan Assay Kit (Megazyme) according to the manufacturer's recommended protocol.

[0062] [Table 1]

[0063] The β-glucan content in barley grains was increased by approximately 1.4-fold in the GBSSI mutant, approximately 1.5-fold in the SSIIa mutant, and approximately 2.7-fold in the SSIIa+GBSSI mutant compared to the wild-type (Figure 1 and Table 1). The results of the SSIIa+GBSSI mutant demonstrated that the loss-of-function mutations in the SSIIa gene and the GBSSI gene synergistically increased the β-glucan content in barley grains. Test results for the SSIIa+amo1 mutant described in Patent Literature 1 showed that the β-glucan content in barley grains was 7.1±0.4% or 6.5±0.8%. The β-glucan content in the SSIIa+GBSSI mutant was 12.2%, which was higher than that of the SSIIa+amo1 mutant described in Patent Literature 1.

[0064] Compared with the wild type, the fructan content in barley grains was reduced by approximately 0.7-fold in the GBSSI mutant and increased by approximately 2.0-fold in the SSIIa mutant (Fig. 2 and Table 1). Contrary to expectations, the fructan content in the SSIIa+GBSSI mutant increased by 2.8-fold, the highest content.

[0065] The total content of β-glucan and fructan in barley grains was increased by approximately 1.2-fold in the GBSSI mutant, approximately 1.7-fold in the SSIIa mutant, and approximately 2.7-fold in the SSIIa+GBSSI mutant compared to the wild type (Fig. 3 and Table 1). The results for the SSIIa+GBSSI mutant demonstrated that the loss-of-function mutations in the SSIIa gene and the GBSSI gene synergistically increased the total content of β-glucan and fructan in barley grains.

[0066] (Test Example 2) The wild-type barley plants, SSIIa mutant, GBSSI mutant, and SSIIa+GBSSI mutant used in Experiment 1 were grown under field conditions in Chikugo City in the fall of 2022. The grains were harvested and crushed in a cyclomill. 1 g of crushed sample was added to 10 ml of sterile water and shaken for 1 hour. After stirring, 1 ml of the supernatant was transferred to a tube and centrifuged for approximately 5 minutes to obtain a sample from each barley plant. The GBSSI+SSIIa mutant sample was further diluted 2-fold with distilled water. The GABA content (mg / 100 g) of each barley plant was compared using a GABA measurement kit (GABA Miel; Enzyme Sensor) according to the manufacturer's recommended protocol. The results are shown in Table 2.

[0067] [Table 2]

[0068] As shown in Table 2, the GBSSI+SSIIa mutant had a higher GABA content compared to wild-type barley plants, the SSIIa mutant, and the GBSSI mutant.

Claims

1. A barley plant in which the functions of the SSIIa gene and the GBSSI gene are defective.

2. 2. The barley plant according to claim 1, wherein the β-glucan content in grain derived from said barley plant is 10 wt% or more.

3. 3. The barley plant according to claim 1, wherein the content of γ-aminobutyric acid in grains derived from said barley plant is 50 mg / 100 g or more.

4. A method for producing a barley plant, comprising the step of deleting the functions of the SSIIa gene and the GBSSI gene in a barley plant.

5. The method according to claim 4, wherein the β-glucan content in the grain derived from the barley plant is 10 wt% or more.

6. 6. The method according to claim 4 or 5, wherein the content of γ-aminobutyric acid in grains derived from the barley plant is 50 mg / 100 g or more.

7. A method for increasing the total content of β-glucan and fructan in grain derived from a barley plant, comprising the step of deleting the functions of the SSIIa gene and the GBSSI gene in the barley plant.

8. A method for increasing the total content of β-glucan and fructan, as well as the content of γ-aminobutyric acid, in grain derived from a barley plant, comprising the step of deleting the functions of the SSIIa gene and the GBSSI gene in the barley plant.

9. A method for increasing the content of γ-aminobutyric acid in grain derived from a barley plant, comprising the step of deleting the functions of the SSIIa gene and the GBSSI gene in the barley plant.

Images