Plant mutants with improved glucosylceramide content

Abstract

Provided is a plant variant having a higher glucosylceramide percentage content than conventional strains.　In the plant variant, the function of at least one gene encoding a protein that belongs to the glycoside hydrolase family-1 and that has a glucocerebrosidase activity is deficient or suppressed.

Description

Technical Field

[0001] The present invention relates to a plant mutant with an improved glucosylceramide content, a plant-derived glucosylceramide-containing extract extracted from the plant mutant, a food composition or a pharmaceutical composition containing them, etc.

Background Art

[0002] Glucosylceramide is a compound widely present in organisms such as animals, plants, and filamentous fungi, and is a kind of glycolipid composed of sphingosine base, fatty acid, and glucose. This glucosylceramide is a compound that plays an important role in vivo. In particular, it is known that when plant-derived glucosylceramide is ingested, effects such as improving skin moisturization, preventing skin damage by ultraviolet rays, and preventing colorectal cancer are exerted (Non-Patent Documents 1-4), and it has attracted attention as a functional material.

[0003] For example, as plant-derived glucosylceramide, glucosylceramide derived from rice bran and glucosylceramide derived from konjac have already been commercialized as functional materials such as improving skin moisturization, and have formed a large market.

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Summary of the Invention

[0005] This plant-derived glucosylceramide is typically extracted from plants using extraction solvents such as ethanol, and then purified as needed. However, a challenge exists in that the production cost is very high due to the extremely low glucosylceramide content in plants. Furthermore, even when plants are consumed directly without extracting glucosylceramide, such as in rice-based foods, the extremely low glucosylceramide content in plants means that the expected health benefits cannot be fully obtained.

[0006] Therefore, if plant bodies (plant mutants) with a higher glucosylceramide content than conventional plants can be obtained, the manufacturing cost of plant-derived glucosylceramide can be reduced, and mass production will also become possible, resulting in immeasurable industrial value. Furthermore, when plant bodies are ingested directly, such as in rice-based foods, there is a significant advantage in that the expected health benefits can be more easily obtained, but there have been no reports of such plant bodies yet.

[0007] Therefore, the present invention aims to provide plant mutants in which the glucosylceramide content is higher than that of conventional plants. [Means for solving the problem]

[0008] To solve the above problems, the inventors focused on glucocerebrosidase, an enzyme that hydrolyzes glucosylceramide and converts it to ceramide. While glucocerebrosidase is mainly known to exist in animals, its existence in plants was unknown except for the discovery of a glucocerebrosidase belonging to glycoside hydrolase family 116 (GH116) in Arabidopsis thaliana.

[0009] The inventors diligently conducted research and discovered a gene (GH1 glucocerebrosidase gene) in plants that encodes a protein belonging to the glycoside hydrolase family 1 (GH1) and possessing glucocerebrosidase activity. Furthermore, they found that deleting or suppressing its function significantly increased the glucosylceramide content in plants compared to conventional methods, thus completing the present invention. To date, there have been no reports of plant mutants in which the function of the GH1 glucocerebrosidase gene is deficient or suppressed.

[0010] In other words, the present invention is as follows <1> ~ <12> That is the case. <1> A plant mutant in which the function of one or more genes encoding proteins belonging to the glycoside hydrolase family 1 and possessing glucocelerosidase activity is lost or suppressed. <2> The aforementioned plant mutant is a mutant of a seed plant. <1> Plant mutants as described above. <3> The aforementioned plant mutant is a mutant of a grass. <2> Plant mutants as described above. <4> The function of one or more of the aforementioned genes, including the DNA shown in (a), (b), or (c) below, is lost or suppressed. <1> ~ <3> A plant mutant described in any one of the following: (a) DNA consisting of the nucleotide sequence shown in Sequence ID No. 1, nucleotide numbers 112-1566 of Sequence ID No. 1, Sequence ID No. 2, or nucleotide numbers 76-1533 of Sequence ID No. 2 in the sequence listing. (b) DNA consisting of a sequence in which one or more bases have been substituted, deleted, inserted, or added in the sequence shown in Sequence ID No. 1, Sequence ID No. 1 with base numbers 112-1566, Sequence ID No. 2, or Sequence ID No. 2 with base numbers 76-1533 of the sequence listing. (c) DNA that can be hybridized under stringent conditions with DNA consisting of a nucleotide sequence complementary to the nucleotide sequence shown in Sequence ID No. 1, nucleotide numbers 112-1566 of Sequence ID No. 1, Sequence ID No. 2, or nucleotide numbers 76-1533 of Sequence ID No. 2 in the sequence listing. <5> The function of the aforementioned gene is deleted or suppressed when a transposon inherent in the genome is inserted into the gene. <1> ~ <4> A plant mutant described in any one of the following: <6> The aforementioned transposon is a retrotransposon. <5> Plant mutants as described above. <7> <1> ~ <6> A method for producing a plant-derived glucosylceramide-containing extract, comprising the step of extracting a glucosylceramide-containing substance from a plant mutant described in any one of the above using an extraction solvent. <8> <1> ~ <6> A plant-derived glucosylceramide-containing extract obtained from any one of the plant mutants described in [the relevant document]. <9> <1> ~ <6> Any one of the plant mutants and / or <8> A food composition containing a plant-derived glucosylceramide-containing extract as described above. <10> <1> ~ <6> Any one of the plant mutants and / or <8> A pharmaceutical composition containing a plant-derived glucosylceramide-containing extract as described above as an active ingredient.

[0011] <11> <1> ~ <6> A plant mutant described in any one of the following: <8> Plant-derived glucosylceramide-containing extract as described above, <9> The food composition described above, or <10> A treatment method for improving skin hydration, wrinkles, firmness, or preventing damage caused by ultraviolet rays, characterized by administering or giving at least one selected from the group consisting of the pharmaceutical compositions described above. <12> <1> ~ <6> A plant mutant described in any one of the following: <8> Plant-derived glucosylceramide-containing extract as described above, <9> The food composition described above, or <10> A method for preventing or treating colorectal cancer, characterized by administering or giving at least one selected from the group consisting of the pharmaceutical compositions described above. [Effects of the Invention]

[0012] According to the present invention, it is possible to provide a plant mutant with a higher content of glucosylceramide than before. And it is also possible to provide a plant-derived glucosylceramide-containing extract extracted from this plant mutant. Furthermore, it is possible to provide a food composition or a pharmaceutical composition containing at least one of these.

Brief Description of the Drawings

[0013] [Figure 1] It is the nucleotide sequence of the Os3BGlu6 gene of rice (Nipponbare). [Figure 2] It is the nucleotide sequence of the Os10BGlu34 gene of rice (Nipponbare). [Figure 3] It is the nucleotide sequence of primers PR1, PR2, PR3, and PR4 used in the PCR in Example 2. [Figure 4] It is the nucleotide sequence of primers PR5, PR6, PR7, and PR8 used in the PCR in Example 2. [Figure 5] It is the nucleotide sequence of primers PR9, PR10, PR11, and PR12 used in the PCR in Example 2.

Modes for Carrying Out the Invention

[0014] The present invention will be described. The present invention relates to a plant mutant in which the function of a gene encoding one or more proteins belonging to glycoside hydrolase family 1 and having glucocerebrosidase activity is defective or suppressed (hereinafter also referred to as "the plant mutant of the present invention"), a plant-derived glucosylceramide-containing extract extracted from the plant mutant of the present invention (hereinafter also referred to as "the plant-derived glucosylceramide-containing extract of the present invention"), a food composition containing the plant mutant of the present invention and / or the plant-derived glucosylceramide-containing extract of the present invention (hereinafter also referred to as "the food composition of the present invention"), a pharmaceutical composition containing the plant mutant of the present invention and / or the plant-derived glucosylceramide-containing extract of the present invention as an active ingredient (hereinafter also referred to as "the pharmaceutical composition of the present invention"), and the like.

[0015] First, the plant mutant of the present invention will be described in detail. The plant mutant of the present invention is a mutant in which the function of a gene encoding one or more proteins belonging to glycoside hydrolase family 1 (GH1) and having glucocerebrosidase activity is defective or suppressed. In the following, this gene is also referred to as "GH1 glucocerebrosidase gene".

[0016] Here, "glucocerebrosidase activity" refers to the enzymatic activity of the enzyme (glucocerebrosidase) with EC number (EC3.2.1.45), specifically the activity that catalyzes the hydrolysis of the β-1,4-glycosyl bond between glucose and ceramide in the glycolipid glucosylceramide to produce ceramide. Furthermore, "Glycoside Hydrolase family 1" is one of the families of carbohydrate hydrolases classified into approximately 130 categories by the Carbohydrate Active enzyme database (CAZy database, http: / / www.cazy.org / ), and glucocerebrosidases are known to belong to glycoside hydrolase family 1 (GH1), glycoside hydrolase family 30 (GH30), and glycoside hydrolase family 116 (GH116).

[0017] Furthermore, the plant mutant of the present invention is a mutant in which the function of the GH1 glucocerebrosidase gene present in the genome of the plant is lost or suppressed. In the case of a plant in which multiple GH1 glucocerebrosidase genes are present in the genome, it is sufficient if the mutant has a lost or suppressed function in at least one of them. In particular, it is preferable that the mutant has a lost or suppressed function in the GH1 glucocerebrosidase gene that is the active component of the glucocerebrosidase belonging to GH1 in that plant (involved in more than 50%, more than 60%, and more than 70% of the total activity of the glucocerebrosidase belonging to GH1 in that plant). However, in the case of such a plant, it is even more preferable that the mutant has a lost or suppressed function in two or more GH1 glucocerebrosidase genes, or a mutant in which the function of all GH1 glucocerebrosidase genes present in the genome is lost or suppressed, because this tends to result in a higher glucosylceramide content.

[0018] Furthermore, this plant mutant refers to all plant-derived components, including not only mature plants, but also at least one selected from the group consisting of leaves, stems, roots, petals, calyxes, pistils, anthers, pollen, seeds, seed embryos, seed endosperm, seed surface bran, rhizoids, and sporangia.

[0019] Furthermore, "deficiency of the GH1 glucocerebrosidase gene function" means that the protein encoded by the GH1 glucocerebrosidase gene cannot be expressed (i.e., the gene cannot be transcribed or translated into a protein), or that the protein expressed from this gene has no glucocerebrosidase activity at all. In addition, "suppression of the GH1 glucocerebrosidase gene function" means that the expression level of the protein encoded by the GH1 glucocerebrosidase gene (the amount of gene transcription or translation into a protein) or the amount of glucocerebrosidase activity of the expressed protein has been significantly reduced (for example, reduced to 40% or less, further reduced to 30% or less, further reduced to 20% or less, and further reduced to 10% or less compared to before suppression).

[0020] Furthermore, since this mutant is a plant (eukaryote), "the function of one or more GH1 glucocerebrosidase genes is lost or suppressed" means that in at least one GH1 glucocerebrosidase gene, both of the two genes (gene sets) constituting its allele are either lost or suppressed (i.e., it is a homozygous mutant). Therefore, heterozygous mutants in which the function of one of the gene sets constituting a single allele is lost or suppressed are not included in the plant mutants of this invention, and a mutant in which the function of two or more GH1 glucocerebrosidase genes is lost or suppressed means a mutant in which the function of two or more sets of the above alleles is lost or suppressed (for example, a double homozygous mutant).

[0021] Here, the method for deleting or suppressing the function of the GH1 glucocerebrosidase gene (the method for producing the plant mutant of the present invention) is not particularly limited and can be any known genetic engineering technology, genome editing technology, or crossbreeding technology used in plant breeding. However, it is preferable to produce the plant mutant of the present invention by deleting or suppressing its function by inserting a transposon into the GH1 glucocerebrosidase gene (its protein coding region or promoter region). Furthermore, since the plant mutant of the present invention does not become a genetically modified plant, it is more preferable that the plant mutant of the present invention is a plant mutant in which the function of the GH1 glucocerebrosidase gene is deleted or suppressed by the insertion of a transposon inherent in its genome through transposition or the like, that is, a plant mutant in which the function of this gene is deleted or suppressed by the insertion of a transposon inherent in the genome of the individual before it became a mutant (such as a wild type) through transposition or the like, and it is even more preferable that this transposon is a retrotransposon. In the case of plant mutants in which the function of two or more GH1 glucocerebrosidase genes is deficient or suppressed, it is preferable that the transposons (especially retrotransposons) described above are inserted into each of the genes.

[0022] Transposons are mutagenic genes that are ubiquitous not only in plant genomes but also in animal, yeast, and bacterial genomes, and are also called mobile genes or transposable elements. For example, in rice (Oryza sativa), numerous gene knockout lines have been created by utilizing the property of the retrotransposon Tos17 (hereinafter sometimes simply referred to as "Tos17"), one of the retrotransposons inherent in the rice genome, to be activated and transpose by tissue culture. Therefore, in rice, it is also possible to obtain rice mutants (plant mutants of the present invention) in which the function of Tos17 is lost or suppressed by inserting it into one or more GH1 glucocerebrosidase genes.

[0023] Transposons are classified into two classes based on their transposition mechanism. Class II transposons transpose in the form of DNA without replicating (the transposon itself is excised from the genome and transposed to another site), and are also called DNA transposons. Examples of transposons belonging to class II include Ac / Ds, Spm / dSpm, and Mu (Cell 56, 181-191 (1989); Cell 35, 235-242 (1983); Proc. Natl. Acad. Sci. USA 82, 4783-4787 (1985)) in maize (Zea mays), Tam (EMBO J, 3, 1015-1019 (1984)) in snapdragons (Antirrhinum majus), mPing (Nature 421, 163-167 (2003)) and nDart (Mol. Genet. Genomics 273, 150-157 (2005)) in rice.

[0024] Furthermore, transposons belonging to class I are also called retrotransposons or retrotransposons, and they replicate and transpose via RNA intermediates (transcription and reverse transcription). These class I transposons are ubiquitous in the plant genome, occupying a significant portion of it, and some of them are activated under stress conditions such as wounds, pathogen attacks, and cell culture. Examples of class I transposons include Tnt1A and Tto1 from tobacco (Nicotiana tabacum) (Plant J., 5, 535-542 (1994); Plant Mol. Biol., 36, 365-376 (1988)), and Tos17 from rice (Proc. Natl. Acad. Sci. USA, 93, 7783-7788 (1996)). Since retrotransposons, once inserted into the genome, cannot be excised, they are preferable because they can induce more stable mutations.

[0025] For example, the aforementioned rice retrotransposon Tos17 has a length of 4.3 kb and contains two identical LTRs (long terminal repeats) of 138 bp length, as well as a PBS (primer binding site) complementary to the 3' end of the initiating methionine tRNA. Tos17 transcription is strongly activated by tissue culture, and the copy number of Tos17 increases with culture time (in the Nipponbare variety, described later, the initial copy number of Tos17 is 2, but this copy number increases to 5-30 in plants regenerated after tissue culture). Tos17 then transposes within the rice genome, causing stable mutations. Furthermore, Tos17 has a property that makes it easily inserted into gene regions. In addition, there is no Tos17 activation under conditions other than tissue culture, and Tos17 transposition hardly occurs under other conditions or after redifferentiation, so the resulting mutants are very stable. Therefore, for example, in rice (such as Nipponbare), the rice mutant according to the present invention can be easily produced by first performing tissue culture to induce Tos17 translocation, then performing PCR screening or measuring glucocerebrosidase activity on the obtained mutants to select mutants in which the function of one or more GH1 glucocerebrosidase genes is deficient or suppressed by Tos17, regenerating these into plants, and further using methods such as self-pollination.

[0026] In this way, plant mutants of the present invention can be obtained in which the function of one or more GH1 glucocerebrosidase genes is deficient or suppressed. Furthermore, from the viewpoint of ease of industrial use, the plant mutants of the present invention are preferably mutants of seed plants (angiosperms or gymnosperms).

[0027] Seed plants are not limited to the following, but include Malvaceae, Chenopodiaceae, Rubiaceae, Cannabaceae, Hydrangeaceae, Brassicaceae, Iridaceae, Poaceae, Araliaceae, Cucurbitaceae, Anacardiaceae, Cyperaceae, Campanulaceae, Asteraceae, Lauraceae, Moraceae, Papaveraceae, Araceae, Cactaceae, Lamiaceae, Nymphaeaceae, Apiaceae, Polygonaceae, Ericaceae, Specific examples include plants from the Camellia family, Solanaceae family, Caryophyllaceae family, Ulmaceae family, Nelumbonaceae family, Rosaceae family, Nelumbonaceae family, Amaryllidaceae family, Convolvulaceae family, Vitaceae family, Fagaceae family, Paeoniaceae family, Fabaceae family, Rutaceae family, Pontederiaceae family, Oleaceae family, Palm family, Salicaceae family, Liliaceae family, Orchidaceae family, Taxaceae family, Ginkgoaceae family, Cupressaceae family, Cycadaceae family, Cupressaceae family, and Pinaceae family.

[0028] To give more specific examples of seed plants, there are cotton, hibiscus, spinach, goosefoot, beet, madder, gardenia, coffee plant, hemp, hops, hydrangea, Arabidopsis thaliana, rapeseed, radish, Chinese cabbage, cabbage, cauliflower, broccoli, komatsuna, bok choy, wasabi, iris, Japanese iris, Japanese iris, rice, timothy, wheat, corn, sorghum, barley, rye, sugarcane, oats, barnyard millet, foxtail millet, grass, reed, bamboo, dwarf bamboo, and tadpoles. Lani tree, Udo, Fatsia japonica, Cucumber, Bitter melon, Melon, Watermelon, Goya, Pumpkin, Loofah, Winter melon, Gourd, Moonflower, Poison ivy, Japanese wax tree, Star grass, Balloon flower, Coreopsis, Lettuce, Gerbera, Gazania, Thistle, Burdock, Sunflower, Cosmos, Dandelion, Calendula, Butterbur, Ragweed, Camphor tree, Bay laurel, Mulberry, Fig, Poppy, Water hyacinth, Taro, Cactus, Perilla, Salvia, Lavender, Water lily, Carrot, Water dropwort, Celery Buckwheat, knotweed, azalea, blueberry, rhododendron, camellia, tomato, eggplant, tobacco, petunia, chili pepper, potato, carnation, baby's breath, chickweed, zelkova, hackberry, lotus, rose, cherry blossom, almond, apricot, strawberry, plum, apple, pear, loquat, peach, lotus, garlic, leek, onion, bindweed, morning glory, sweet potato, grape, beech, sawtooth oak, chestnut, peony, soybean, broad bean, black bean, wisteria, Japanese wisteria, rupee The list includes eggplant, green beans, peas, alfalfa, peanuts, sweet peas, lotus, Satsuma mandarin, Japanese pepper, summer orange, orange, lime, lemon, grapefruit, trifoliate orange, water hyacinth, olive, jasmine, coconut palm, oil palm, date palm, palm tree, poplar, willow, lily, dwarf lily, tulip, narcissus, phalaenopsis orchid, cattleya, vanilla, yew, Japanese iris, ginkgo, cedar, cycad, cypress, pine, and others.

[0029] The inventors have discovered the presence of the GH1 glucocerebrosidase gene in plants of the Malvaceae family, Brassicaceae family, Poaceae family, Cucurbitaceae family, Asteraceae family, Solanaceae family, Rosaceae family, Fabaceae family, Apiaceae family, etc. (Japanese Patent Application No. 2020-88950). Plant mutants in which one or more of these GH1 glucocerebrosidase genes are deficient or suppressed tend to have a higher glucosylceramide content than conventional plants, making them very useful, and plant mutants of Poaceae plants are particularly preferable.

[0030] For example, the inventors have discovered that the rice variety Nipponbare (Oryza sativa L. cv. Nipponbare) contains the Os3BGlu6 gene and the Os10BGlu34 gene as GH1 glucocerebrosidase genes (e.g., Japanese Patent Application No. 2020-88950). The nucleotide sequence of the Os3BGlu6 gene is shown in Sequence ID No. 1 of the sequence listing and in Figure 1, and the nucleotide sequence of the Os10BGlu34 gene is shown in Sequence ID No. 2 of the sequence listing and in Figure 2.

[0031] Furthermore, the plant mutant of the present invention is preferably a mutant in which the function of one or more GH1 glucocerebrosidase genes containing DNA consisting of the base sequence shown in Sequence ID No. 1, base numbers 112 to 1566 of Sequence ID No. 1, Sequence ID No. 2, or base numbers 76 to 1533 of Sequence ID No. 2 is lost or suppressed. In other words, it is preferable that at least one, more preferably all, of the GH1 glucocerebrosidase genes whose function is lost or suppressed are genes containing any of the above-mentioned DNA. For example, in the case of a mutant of the rice variety Nipponbare, it is preferable that the function of the Os3BGlu6 gene and / or the Os10BGlu34 gene is lost or suppressed. In particular, it is more preferable that this Nipponbare rice mutant is a mutant in which the function of at least the Os3BGlu6 gene is lost or suppressed, and it is even more preferable that it is a mutant in which the function of both the Os3BGlu6 gene and the Os10BGlu34 gene is lost or suppressed (double homozygous mutant), as this tends to result in a higher glucosylceramide content.

[0032] For example, a variant of the rice variety Nipponbare in which a retrotransposon (Tos17), which is endogenous in the genome of Nipponbare rice, is inserted into at least one allele of the Os3BGlu6 gene or the Os10BGlu34 gene, resulting in a loss or suppression of its function, is preferable because it does not constitute a genetically modified crop. For example, if Tos17 is inserted into the protein-coding region of the above gene, the function of the gene will be lost, and if Tos17 is inserted into the promoter region of the above gene, the function of the gene will be lost or suppressed.

[0033] Furthermore, the plant mutants of the present invention may also have a defect or suppression of the function of one or more GH1 glucocerebrosidase genes containing DNA consisting of a nucleotide sequence in which one or more nucleotides have been substituted, deleted, inserted, or added in the nucleotide sequence shown in Sequence ID No. 1, nucleotide numbers 112 to 1566 of Sequence ID No. 1, Sequence ID No. 2, or nucleotide numbers 76 to 1533 of Sequence ID No. 2 in the sequence listing. In other words, at least one of the GH1 glucocerebrosidase genes whose function is defect or suppressed may be a gene containing any of the above-mentioned DNAs. Here, "a few" means 20 or fewer, preferably 10 or fewer, and more preferably 6 or fewer.

[0034] Furthermore, the plant mutants of the present invention may have a deficiency or suppression of the function of one or more GH1 glucocerebrosidase genes that contain DNA that can hybridize under stringent conditions with DNA consisting of a base sequence complementary to the base sequence shown in Sequence ID No. 1, base numbers 112-1566 of Sequence ID No. 1, Sequence ID No. 2, or base numbers 76-1533 of Sequence ID No. 2 in the sequence listing. In other words, at least one of the GH1 glucocerebrosidase genes whose function is deficiency or suppressed may be a gene containing any of the above-mentioned DNAs. Here, "stringent conditions" refer to conditions under which so-called specific hybrids are formed, and nonspecific hybrids are not formed. For example, one such condition is when hybridization is performed at 65°C in the presence of 0.7-1.0M sodium chloride, followed by washing 1-3 times at 60°C, preferably 65°C, more preferably 68°C using a 0.1-5×SSC, 0.1% SDS solution (composition of 1×SSC: 150mM sodium chloride, 15mM sodium citrate).

[0035] As described in the aforementioned Japanese Patent Application No. 2020-88950, the inventors have found that at least one GH1 glucocerebrosidase gene exists in various seed plants. Furthermore, it is highly probable that this gene contains DNA consisting of a nucleotide sequence in which one or more nucleotides have been substituted, deleted, inserted, or added to the nucleotide sequence shown in Sequence ID No. 1 or 2, or DNA that can hybridize under stringent conditions with DNA consisting of a nucleotide sequence complementary to the nucleotide sequence shown in Sequence ID No. 1 or 2. The inventors have also found that in plants, GH1-type glucocerebrosidases are dominant. Therefore, by identifying the GH1 glucocerebrosidase gene using homology searches using the nucleotide or amino acid sequences of the Os3BGlu6 gene or the Os10BGlu34 gene, or by screening by hybridization using at least some of these DNAs, and by deleting or suppressing its function, plant mutants of the present invention can be easily obtained from various plants.

[0036] Such plant mutants of the present invention have a higher glucosylceramide content than conventional plants because the function of one or more GH1 glucocerebrosidase genes is deficient or suppressed. For example, the glucosylceramide content of at least one part selected from the group consisting of leaves, stems, roots, petals, calyx, pistil, anther, pollen, seeds, seed embryo, seed endosperm, seed surface bran, rhizoids, and sporangia is higher than conventional plants. The plant mutants of the present invention have a higher glucosylceramide content in at least some of the above-mentioned parts, and this includes isolated parts, but it is also possible that some parts have a glucosylceramide content similar to that of conventional plants. Furthermore, although not limited thereto, when a glucosylceramide-containing extract is extracted from the plant mutant or a part thereof of the present invention and used as a functional material, the glucosylceramide content in the part that can be used as the raw material for extraction may be 100 μg / g or more (0.01% by mass or more), more may be 200 μg / g or more (0.02% by mass or more), more may be 300 μg / g or more (0.03% by mass or more), more may be 400 μg / g or more (0.04% by mass or more), and more may be 500 μg / g or more (0.05% by mass or more). For example, in the case of the embryo of a grass plant mutant, the glucosylceramide content may be 450 μg / g or more (0.045% by mass or more), more may be 480 μg / g or more (0.048% by mass or more), and more may be 550 μg / g or more (0.055% by mass or more). Furthermore, if the bran is from the seeds of a grass species mutant, its glucosylceramide content may be 110 μg / g or more (0.011% by mass or more), 150 μg / g or more (0.015% by mass or more), or 160 μg / g or more (0.016% by mass or more). In the case of a grass species mutant, it is preferable to use a mixture containing the germ and bran as the raw material for extracting plant-derived glucosylceramide-containing extracts. In this case, the mixing ratio of germ to bran is not limited, but it is preferable to use 2 to 4 parts by mass of bran per 1 part by mass of germ.Furthermore, when the plant mutant or a part thereof of the present invention is used as a functional food (food composition) in which it is ingested, the glucosylceramide content in the part that can be used in this food composition may be 20 μg / g or more (0.002% by mass or more), more may be 30 μg / g or more (0.003% by mass or more), more may be 40 μg / g or more (0.004% by mass or more), more may be 50 μg / g or more (0.005% by mass or more), more may be 52 μg / g or more (0.0052% by mass or more), more may be 55 μg / g or more (0.0055% by mass or more), more may be 60 μg / g or more (0.006% by mass or more), and more may be 65 μg / g or more (0.0065% by mass or more).

[0037] Next, the plant-derived glucosylceramide-containing extract of the present invention and its manufacturing method will be described in detail.

[0038] The plant-derived glucosylceramide-containing extract of the present invention is a plant extract containing plant-derived glucosylceramide extracted from the plant mutant of the present invention as described above. This can be produced by a manufacturing method that includes a step of extracting glucosylceramide-containing material from the plant mutant of the present invention (the whole or a part thereof) using an extraction solvent, such as an extraction step using an aqueous solution containing an organic solvent such as alcohol or a surfactant. The extraction solvent is not limited, but from the viewpoint of using the extract in food compositions, etc., ethanol is preferred. This ethanol may be aqueous ethanol (for example, aqueous ethanol with an ethanol concentration of 80% by weight or more, or even 90% by weight or more). Furthermore, in this extraction step, heat treatment (for example, heat treatment at 40 to 80°C) may be performed as needed. Furthermore, a crude purification step (for example, concentration) may be performed as needed to remove at least a part of the extraction solvent. The substance obtained after such steps, in liquid, powder, granular form, etc., is the plant-derived glucosylceramide-containing extract of the present invention.

[0039] Here, the plant-derived glucosylceramide content in the plant-derived glucosylceramide-containing extract of the present invention is not limited, but the lower limit is preferably 0.01% by mass or more, more preferably 0.02% by mass or more, even more preferably 0.05% by mass or more, even more preferably 0.1% by mass or more, and even more preferably 0.2% by mass or more. The upper limit is preferably 70% by mass or less, more preferably 50% by mass or less, even more preferably 40% by mass or less, even more preferably 30% by mass or less, even more preferably 20% by mass or less, and even more preferably 10% by mass or less.

[0040] Furthermore, while the plant-derived glucosylceramide-containing extract of the present invention is considered to be merely a case where the structure or properties of a substance are identified by describing its state, even if this were not the case, the specific components other than glucosylceramide in this extract (other components derived from the plant mutant of the present invention) and their composition ratios have not been clearly identified. Moreover, identifying all of these components would require an excessively large economic expenditure and amount of time, so it can be said that it is either impossible or impractical to directly identify them based on their structure or properties.

[0041] By ingesting such plant-derived glucosylceramide-containing extracts of the present invention (for example, plant-derived glucosylceramide-containing extracts produced by the manufacturing method described above) or the plant mutants of the present invention described above, it is expected that the plant-derived glucosylceramide contained therein will improve skin hydration, prevent or treat skin damage caused by ultraviolet rays, and prevent or treat colorectal cancer. Furthermore, since the components other than plant-derived glucosylceramide contained therein are also derived from plants, they can be used safely and at low cost in food compositions and the like.

[0042] Next, the food composition and pharmaceutical composition of the present invention, which include the plant variant and / or plant-derived glucosylceramide-containing extract of the present invention, will be described in detail.

[0043] By providing a food composition containing the plant mutant of the present invention (the whole or a part of the plant mutant of the present invention), a food composition containing the plant-derived glucosylceramide-containing extract of the present invention, or a food composition containing both the plant mutant of the present invention and the plant-derived glucosylceramide-containing extract of the present invention, a food composition of the present invention rich in plant-derived glucosylceramide can be provided at low cost. For example, from the rice mutant of the present invention, not only the seeds of this rice mutant, such as paddy rice and brown rice, but also food compositions (food compositions containing this rice mutant) that have been processed from this brown rice for food use can be provided at low cost, such as polished rice, rice flour, cooked rice products (processed cooked rice such as brown rice or polished rice), and processed rice flour products. Furthermore, supplements containing a rice-derived glucosylceramide-containing extract extracted from the bran on the surface of brown rice (bran on the surface of the seeds) can also be provided at low cost. Here, "polished rice" refers to brown rice from which the germ and surface bran have been removed (shaved off by processing), and also includes rice from which part of the endosperm has been removed. Furthermore, "rice flour" refers to brown rice or polished rice processed into a powder. The food composition of the present invention may optionally contain other known components in the food field, as long as they do not significantly affect the functionality of the plant-derived glucosylceramide. The food composition of the present invention may also provide one or more uses selected from those for improving skin hydration, improving skin wrinkles, improving skin firmness, preventing skin damage caused by ultraviolet rays, and preventing colorectal cancer, all containing the plant variant of the present invention and / or the plant-derived glucosylceramide-containing extract of the present invention as active ingredients.

[0044] Furthermore, the pharmaceutical composition of the present invention can be provided by including the plant mutant of the present invention (the whole or a part of the plant mutant of the present invention) and / or the plant-derived glucosylceramide-containing extract of the present invention as an active ingredient. The pharmaceutical composition of the present invention may also optionally contain other known components in the pharmaceutical field (medically acceptable components such as excipients, binders, disintegrants, and lubricants), as long as they do not significantly affect the pharmacological effects of the plant-derived glucosylceramide. The form may be a form suitable for oral administration, such as tablets, capsules, granules, fine granules, powders, or syrups. Alternatively, it may be a parenteral preparation (such as an intravenous drip). The pharmaceutical composition of the present invention can be suitably used for one or more pharmaceutical applications selected from the following: improving skin hydration, improving skin wrinkles, improving skin firmness, preventing or treating skin damage caused by ultraviolet rays, and preventing or treating colorectal cancer. Furthermore, in a similar manner, cosmetic compositions for improving skin hydration, improving skin wrinkles, improving skin firmness, or preventing or treating skin damage caused by ultraviolet rays can also be provided, which contain the plant-derived glucosylceramide-containing extract of the present invention as an active ingredient.

[0045] In other words, the present invention provides a method for improving skin hydration, reducing wrinkles, improving firmness, or preventing damage caused by ultraviolet rays, characterized by administering or providing to a human being at least one selected from the group consisting of the plant variant of the present invention, the plant-derived glucosylceramide-containing extract of the present invention, the food composition of the present invention, or the pharmaceutical composition of the present invention. The same applies to methods of applying the above cosmetic composition to human skin. Furthermore, the present invention also provides a method for preventing or treating colorectal cancer, characterized by administering or providing to a human being at least one selected from the group consisting of the plant variant of the present invention, the plant-derived glucosylceramide-containing extract of the present invention, the food composition of the present invention, or the pharmaceutical composition of the present invention.

[0046] The embodiments described above are merely examples to facilitate understanding of the present invention and do not limit it.

[0047] The following describes embodiments of the present invention, but the present invention is not limited to these embodiments, and various modifications are possible within the technical concept of the present invention. [Examples]

[0048] (Example 1: Preparation of rice mutants) Mature seeds of the Japonica rice variety (Oryza sativa L. cv. Nipponbare) were used as the starting material, and callus initiation culture and cell suspension culture were performed according to the method of Hirochika et al. (Proc. Natl. Acad. Sci. USA, 93, 7783-7788 (1996)). The culture conditions for activating the retrotransposon Tos17 to cause gene disruption were followed according to the method of Otsuki (rice protoplast culture system, Japan Agricultural, Forestry and Fisheries Technology Information Association (1990)).

[0049] Specifically, the mature seeds described above were cultured in MS medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) (25°C, for 1 month) to induce callus formation. The resulting callus was cultured in N6 liquid medium supplemented with 2,4-D for 5 months, and then transferred to a redifferentiation medium to obtain redifferentiated rice (first generation (M1) plants).

[0050] Then, from the leaves of approximately 20 redifferentiated rice plants (second-generation (M2) plants) obtained from this first generation, genomic DNA was obtained for each lineage. Using Thermal asymmetric interlaced (TAIL) PCR (Genomics, 25, 674-681 (1995)) and Suppression PCR (Nucleic Acids Research, 23, 1087-1088 (1995)), the sequences adjacent to the insertion site of Tos17 inserted into each lineage were obtained (Plant Biotechnology, 15(4), 253-256 (1998)), and compiled into a database (Plant Cell, 15, 1771-1780 (2003)). Then, from this database, we searched for adjacent nucleotide sequences that matched the nucleotide sequences of the Os3BGlu6 gene (a gene containing DNA consisting of the nucleotide sequence shown in Sequence ID No. 1 of the sequence listing and Figure 1) and the Os10BGlu34 gene (a gene containing DNA consisting of the nucleotide sequence shown in Sequence ID No. 2 of the sequence listing and Figure 2), which are genes encoding glucocerebrosidases belonging to GH1. As a result, we selected lines in which Tos17 was transferred to the Os3BGlu6 gene (NE1537, ND8040) and lines in which Tos17 was transferred to the Os10BGlu34 gene (NE4173).

[0051] Furthermore, since the rice lines obtained here (NE1537, ND8040, NE4173) are all heterozygous knockout mutants, we obtained homozygous knockout mutants of the Os3BGlu6 gene (homozygous NE1537, homozygous ND8040) and homozygous knockout mutants of the Os10BGlu34 gene (homozygous NE4173) by self-pollinating each of them.

[0052] Furthermore, these homozygous Os3BGlu6 gene knockout mutants (homo NE1537, homo ND8040) and Os10BGlu34 gene knockout mutant (homo NE4173) were cross-pollinated using the hot water emasculation method (http: / / www.naro.affrc.go.jp / laboratory / tarc / contents / school / kouhai / index.html) to obtain Os3BGlu6 gene and Os10BGlu34 gene double heterozygous knockout mutants (hetero NE1537 × hetero NE4173, hetero ND8040 × hetero NE4173), respectively. Furthermore, by self-pollinating these double heterozygous knockout mutants (hetero NE1537 × heterozygous NE4173, heterozygous ND8040 × heterozygous NE4173), we obtained double homozygous knockout mutants of the Os3BGlu6 gene and Os10BGlu34 gene (homozygous NE1537 × homozygous NE4173, homozygous ND8040 × homozygous NE4173).

[0053] Here, a heterozygous knockout mutant refers to a mutant in which Tos17 is inserted into only one of a specific allele, resulting in a loss of function in that one gene. A homozygous knockout mutant refers to a mutant in which Tos17 is inserted into both of a specific allele, resulting in a loss of function in both genes. Furthermore, a double heterozygous knockout mutant refers to a mutant in which, in two specific alleles, Tos17 is inserted into only one of the two alleles, resulting in a loss of function in one of those genes. Additionally, a double homozygous knockout mutant refers to a mutant in which, in two specific alleles, Tos17 is inserted into both of the two alleles, resulting in a loss of function in both genes.

[0054] (Example 2: Confirmation of homozygous knockout mutants) [Confirmation of Homo NE1537] By performing PCR using four primers created from the nucleotide sequence within the Os3BGlu6 gene or the sequence within Tos17, it was confirmed that the homozygous NE1537 produced in Example 1 had Tos17 inserted into both Os3BGlu6 alleles.

[0055] Specifically, approximately 5 mm of rice leaves from homo NE1537 were cut, placed in Eppendorf tubes with screw caps pre-filled with 200 μL of 2× PCR buffer for KOD-FX (Toyobo Co., Ltd.) and zirconia beads, and disrupted according to the DNA disruption and extraction protocol of FastPrep 24 Instrument (MP Biomedicals). After centrifugation at 10,000 rpm, 25°C, and 10 min, the supernatant was collected and used as the extracted DNA solution. To this extracted DNA solution, two primer sets, PR1 (nucleotide sequence shown in SEQ ID NO: 3 and Figure 3) and PR2 (nucleotide sequence shown in SEQ ID NO: 4 and Figure 3), or PR3 (nucleotide sequence shown in SEQ ID NO: 5 and Figure 3) and PR4 (nucleotide sequence shown in SEQ ID NO: 6 and Figure 3), along with KOD-FX (DNA polymerase, Toyobo Co., Ltd.), were added as F-primers and R-primers. PCR was then performed by repeating the reaction conditions, which consisted of 94°C for 2 minutes, 98°C for 10 seconds, 55°C for 30 seconds, and 68°C for 3 minutes, for 35 cycles.

[0056] PR1 and PR2 are PCR primer sets for amplifying DNA fragments in the region spanning the Os3BGlu6 gene and Tos17, while PR3 and PR4 are PCR primer sets for amplifying DNA fragments in the region within the Os3BGlu6 gene (the region spanning the Tos17 insertion site). When Tos17 is inserted into the Os3BGlu6 gene, the DNA fragment can be amplified using the PR1 and PR2 set, but amplification of the DNA fragment becomes difficult with the PR3 and PR4 set because Tos17 is a very large DNA molecule.

[0057] As a result, clear amplification of DNA fragments was observed when PCR was performed using the PR1 and PR2 sets, but not when PCR was performed using the PR3 and PR4 sets. Furthermore, DNA sequencing analysis of the base sequences of the DNA fragments amplified by the PR1 and PR2 sets confirmed that homozygous NE1537 is a homozygous Os3BGlu6 gene knockout mutant in which Tos17 is inserted into both alleles of the Os3BGlu6 gene.

[0058] [Confirmation of Homo ND8040] Similarly, we confirmed that the homozygous ND8040 synthesized in Example 1 also had Tos17 inserted into both Os3BGlu6 alleles. Specifically, PCR was performed using the same method as for confirming homozygous NE1537, except that the leaves of homozygous ND8040 rice were used, and two sets of primers were used as the F-primers and R-primers: PR5 (nucleotide sequence shown in SEQ ID NO: 7 and Figure 4) and PR6 (nucleotide sequence shown in SEQ ID NO: 8 and Figure 4), or PR7 (nucleotide sequence shown in SEQ ID NO: 9 and Figure 4) and PR8 (nucleotide sequence shown in SEQ ID NO: 10 and Figure 4).

[0059] PR5 and PR6 are PCR primer sets for amplifying DNA fragments in the region spanning the Os3BGlu6 gene and Tos17, while PR7 and PR8 are PCR primer sets for amplifying DNA fragments in the region within the Os3BGlu6 gene (the region spanning the insertion site of Tos17).

[0060] As a result, clear amplification of DNA fragments was observed when PCR was performed using the PR5 and PR6 sets, but not when PCR was performed using the PR7 and PR8 sets. Furthermore, DNA sequencing analysis of the base sequences of the DNA fragments amplified by the PR5 and PR6 sets confirmed that homozygous ND8040 is a homozygous Os3BGlu6 gene knockout mutant in which Tos17 is inserted into both alleles of the Os3BGlu6 gene.

[0061] [Confirmation of Homo NE4173] By performing PCR using four primers created from the nucleotide sequence within the Os10BGlu34 gene or the sequence within Tos17, it was confirmed that the homozygous NE4173 produced in Example 1 had Tos17 inserted into both Os10BGlu34 alleles.

[0062] Specifically, PCR was performed using rice leaves of homozygous NE4173, and the same method as for confirming homozygous NE1537 was used, except that two sets of primers were used as the F-primers and R-primers: PR9 (nucleotide sequence shown in SEQ ID NO: 11 and Figure 5) and PR10 (nucleotide sequence shown in SEQ ID NO: 12 and Figure 5), or PR11 (nucleotide sequence shown in SEQ ID NO: 13 and Figure 5) and PR12 (nucleotide sequence shown in SEQ ID NO: 14 and Figure 5).

[0063] PR9 and PR10 are PCR primer sets for amplifying DNA fragments in the region spanning the Os10BGlu34 gene and Tos17, while PR11 and PR12 are PCR primer sets for amplifying DNA fragments in the region within the Os10BGlu34 gene (the region spanning the insertion site of Tos17).

[0064] As a result, clear amplification of DNA fragments was observed when PCR was performed using the PR9 and PR10 sets, but not when PCR was performed using the PR11 and PR12 sets. Furthermore, DNA sequencing analysis of the base sequences of the DNA fragments amplified by the PR9 and PR10 sets confirmed that homozygous NE4173 is a homozygous Os10BGlu34 gene knockout mutant in which Tos17 is inserted into both alleles of the Os10BGlu34 gene.

[0065] (Example 3: Confirmation of double homozygous knockout mutant) [HomoNE1537×HomoNE4173] Similar to Example 2, it was confirmed that the homozygous NE1537 × homozygous NE4173 cells prepared in Example 1 had Tos17 inserted into both Os3BGlu6 alleles and both Os10BGlu34 alleles. Specifically, PCR was performed using leaves of homozygous NE1537 × homozygous NE4173 rice, and the same method as in Example 2 was used, except that the F-primers and R-primers were sets of four primers from Example 2: PR1 and PR2, PR3 and PR4, PR9 and PR10, or PR11 and PR12.

[0066] As a result, clear amplification of DNA fragments was observed when PCR was performed using the PR1 and PR2 sets and the PR9 and PR10 sets, but not when PCR was performed using the PR3 and PR4 sets and the PR11 and PR12 sets. Furthermore, the base sequences of the DNA fragments amplified by the PR1 and PR2 sets and the PR9 and PR10 sets were analyzed by DNA sequencing, and it was confirmed that homozygous NE1537 × homozygous NE4173 is a double homozygous knockout mutant of the Os3BGlu6 and Os10BGlu34 genes, in which Tos17 is inserted into both alleles of the Os3BGlu6 gene and Tos17 is inserted into both alleles of the Os10BGlu34 gene.

[0067] [Homo ND8040 × Homo NE4173] Similarly, we confirmed that the homozygous ND8040 × homozygous NE4173 strain produced in Example 1 also had Tos17 inserted into both the Os3BGlu6 allele and both the Os10BGlu34 allele. Specifically, PCR was performed using the same method as in Example 2, except that the leaves of homozygous ND8040 × homozygous NE4173 rice plants were used, and the four primer sets used for the F-primer and R-primer were PR5 and PR6, PR7 and PR8, PR9 and PR10, or PR11 and PR12 from Example 2.

[0068] As a result, clear amplification of DNA fragments was observed when PCR was performed using the PR5 and PR6 sets and the PR9 and PR10 sets, but not when PCR was performed using the PR7 and PR8 sets and the PR11 and PR12 sets. Furthermore, the base sequences of the DNA fragments amplified by the PR5 and PR6 sets and the PR9 and PR10 sets were analyzed by DNA sequencing, and it was confirmed that homozygous ND8040 × homozygous NE4173 is a double homozygous knockout mutant of the Os3BGlu6 and Os10BGlu34 genes, in which Tos17 is inserted into both alleles of the Os3BGlu6 gene and Tos17 is inserted into both alleles of the Os10BGlu34 gene.

[0069] (Example 4: Comparison of glucosylceramide content in the seed embryo of rice mutants) Five rice mutants (Homo NE1537, Homo ND8040, Homo NE4173, Homo NE1537 × Homo NE4173, Homo ND8040 × Homo NE4173) and a wild strain (Nipponbare) prepared in Example 1 were cultivated in a greenhouse, and seeds were collected from each. After removing the hulls from each seed, 20 embryos from each seed were finely ground in a mortar, and glucosylceramide contained in the embryo was extracted by adding 8 mL of ethanol and shaking. The extracted samples were then centrifuged at 15,000 rpm for 20 minutes, and 0.25 times the volume of 0.2 M HCl was added to the supernatant and mixed. This was centrifuged at 15,000 rpm for 20 minutes, and the supernatant was subjected to LC-ESI-MS / MS (Liquid chromatography-electrospray ionization-tandem mass spectrometry) and analyzed according to the method of Yumoto et al. (Bioscience, Biotechnology, and Biochemistry (2021) 85, 205-210). Specifically, ESI-MS / MS was performed using an Agilent 6460 with an Agilent 1200 separation module, and the high-performance liquid chromatography column was a TSKgel ODS-120A column (2.1 mm id × 30 cm, Tosoh Corporation, registered trademark). Various glucosylceramides were eluted with an appropriate methanol concentration containing 0.1% formic acid and detected in ESI-positive ion mode. Precursor ion m / z [M+H] for detecting eight glucosylceramides that are thought to be present in high amounts in rice. +The m / z of the product ion, collision energy (eV), and fragmentation voltage (V) are shown in Table 1 below. The amount of each detected glucosylceramide was determined using rice-derived glucosylceramide purchased from Nagara Science Co., Ltd. as a standard. Finally, the amounts of the eight types of glucosylceramide were totaled, and the amount of glucosylceramide contained in 1 g of germ was determined as the mean ± standard error of 5 individuals. These results are shown in Table 2 below. In Table 2 below, an asterisk (*) is used if the amount of glucosylceramide was significantly higher than that of the wild strain (5% significance), a ** is used if the amount of glucosylceramide was significantly higher than that of homo NE1537 (5% significance), a *** is used if the amount of glucosylceramide was significantly higher than that of homo ND8040 (5% significance), and a **** is used if the amount of glucosylceramide was significantly higher than that of homo NE4173 (5% significance).

[0070] [Table 1]

[0071] [Table 2]

[0072] The results in Table 2 above show that the amount of glucosylceramide in the embryos of homozygous NE1537, homozygous ND8040, and homozygous NE4173 is significantly higher than in the wild-type strain (a strain in which the functions of both the Os3BGlu6 and Os10BGlu34 genes are not deficient or suppressed). Furthermore, it was revealed that the amount of glucosylceramide in the embryos of seeds from homozygous NE1537 × homozygous NE4173 and homozygous ND8040 × homozygous NE4173 is even higher than that of homozygous NE1537, homozygous ND8040, and homozygous NE4173.

[0073] (Example 5: Comparison of glucosylceramide content in rice bran on the seed surface of rice mutants I) In Example 4, 8 mL of ethanol was added to 20 seeds each of the six types of rice seeds from which the germ had been removed, and the mixture was shaken at 50°C to extract glucosylceramide contained in the bran on the surface of the seeds. The extracted samples were then centrifuged at 15,000 rpm for 20 minutes, and the supernatant was concentrated to an appropriate concentration. The amount of glucosylceramide in the concentrated solution was then analyzed by LC-ESI-MS / MS using the same method as in Example 4. The amount of glucosylceramide contained in the bran of one seed was then determined as the mean value ± standard error of 5 samples. The results are shown in Table 3 below. In Table 3 below, an asterisk (*) is used to indicate a significantly higher amount of glucosylceramide compared to the wild-type strain (5% significance), a ** is used to indicate a significantly higher amount of glucosylceramide compared to homozygous NE1537 (5% significance), a *** is used to indicate a significantly higher amount of glucosylceramide compared to homozygous ND8040 (5% significance), and a **** is used to indicate a significantly higher amount of glucosylceramide compared to homozygous NE4173 (5% significance).

[0074] [Table 3]

[0075] The results in Table 3 above show that the amount of glucosylceramide in the seed bran of homo NE1537, homo ND8040, and homo NE4173 is significantly higher than that of the wild strain. Furthermore, it was revealed that the amount of glucosylceramide in the seed bran of homo NE1537 × homo NE4173 and homo ND8040 × homo NE4173 is even higher than that of homo NE1537, homo ND8040, and homo NE4173.

[0076] (Example 6: Comparison of glucosylceramide content in rice bran on the seed surface of rice mutants II) After removing the hulls from the seeds obtained from the five rice mutants (Homo NE1537, Homo ND8040, Homo NE4173, Homo NE1537 × Homo NE4173, Homo ND8040 × Homo NE4173) and the wild strain (Nipponbare) prepared in Example 1, these seeds (brown rice) were milled using a rice mill (MK Seiko Co., Ltd., small rice mill RICELON SM-201K). The resulting mixture containing the bran was sieved through a tea strainer to remove white rice fragments and germ, and the bran from the seed surface was collected. Next, 8 mL of ethanol was added to this bran and shaken to extract glucosylceramide contained in the bran. Then, this extracted sample was centrifuged at 15,000 rpm for 20 minutes, and the supernatant was concentrated to an appropriate concentration. The amount of glucosylceramide in the concentrated solution was then analyzed by LC-ESI-MS / MS using the same method as in Example 4. The amount of glucosylceramide contained in 1g of rice bran was then calculated as the mean ± standard error of the three individuals. The results are shown in Table 4 below. In Table 4 below, an asterisk (*) is used to indicate that the amount of glucosylceramide was significantly higher than that of the wild strain (5% significance), a ** is used to indicate that the amount of glucosylceramide was significantly higher than that of homozygous NE1537 (5% significance), a *** is used to indicate that the amount of glucosylceramide was significantly higher than that of homozygous ND8040 (5% significance), and a **** is used to indicate that the amount of glucosylceramide was significantly higher than that of homozygous NE4173 (5% significance).

[0077] [Table 4]

[0078] The results in Table 4 above show that the amount of glucosylceramide in the seed bran of homo NE1537, homo ND8040, and homo NE4173 is significantly higher than that of the wild strain. Furthermore, it was revealed that the amount of glucosylceramide in the seed bran of homo NE1537 × homo NE4173 and homo ND8040 × homo NE4173 is even higher than that of homo NE1537, homo ND8040, and homo NE4173.

[0079] Rice bran is inexpensive and contains a large amount of glucosylceramide, so many plant-derived glucosylceramides used in health foods are extracted from rice bran. Based on the results of Examples 5 and 6, it is estimated that glucosylceramide can be produced more cheaply than before by using rice bran obtained from at least one of the above-mentioned rice mutants, and these can be said to be useful rice mutants.

[0080] (Example 7: Comparison of glucosylceramide content in parts of rice mutant seeds other than bran and germ) In Example 5, ethanol was added to the six rice seeds from which glucosylceramide had been extracted from the bran on the surface of the seeds. After vigorous mixing, the ethanol was discarded. This process was repeated three times to remove as much glucosylceramide as possible from the bran on the surface. Then, 10 seeds of each variety were finely ground in a mortar, 8 mL of ethanol was added, and the mixture was shaken to extract glucosylceramide from the parts of the rice seeds other than the bran and germ. Subsequently, the amount of glucosylceramide in the extract was analyzed by LC-ESI-MS / MS using the same method as in Example 4. The amount of glucosylceramide in the parts of the seeds other than the bran and germ per 1 g was then determined as the mean value ± standard error of 5 samples. The results are shown in Table 5 below. In Table 5 below, an asterisk (*) is used to indicate a significantly higher amount of glucosylceramide compared to the wild-type strain (5% significance), a ** is used to indicate a significantly higher amount of glucosylceramide compared to homozygous NE1537 (5% significance), a *** is used to indicate a significantly higher amount of glucosylceramide compared to homozygous ND8040 (5% significance), and a **** is used to indicate a significantly higher amount of glucosylceramide compared to homozygous NE4173 (5% significance).

[0081] [Table 5]

[0082] The results in Table 5 above show that the amount of glucosylceramide in the parts of the seeds other than the bran and germ of Homo NE1537 and Homo ND8040 is significantly higher than that of the wild strain. Furthermore, it was revealed that the amount of glucosylceramide in the parts of the seeds other than the bran and germ of Homo NE1537 × Homo NE4173 and Homo ND8040 × Homo NE4173 is even higher than that of Homo NE1537 and Homo ND8040, as well as the wild strain and Homo NE4173.

[0083] These results indicate that glucosylceramide is also abundant in the endosperm portion of the seeds of these rice mutants, even after removing the germ and bran. Therefore, rice products (processed rice, etc.) and rice products (rice flour, etc.) produced from the seeds (mainly the endosperm portion) obtained from the above-mentioned rice mutants are rich in rice-derived glucosylceramide and are useful as health foods (functional foods).

[0084] (Example 8: Comparison of glucosylceramide content in cooked rice obtained from polished rice seeds of rice mutants) After removing the hulls from the seeds obtained from the two rice mutants (homo NE1537 × homo NE4173, homo ND8040 × homo NE4173) and the wild strain (Nipponbare) prepared in Example 1, these seeds (brown rice) were milled using a rice mill (MK Seiko Co., Ltd., small rice mill RICELON SM-201K). The milled white rice was then cooked using a rice cooker (Sanko Co., Ltd., two-stage high-speed rice cooker) according to the conventional method. Ten grains of the cooked rice were ground in a mortar, and glucosylceramide contained in the cooked rice was extracted by adding 8 mL of ethanol and shaking. The extracted sample was then centrifuged at 15,000 rpm for 20 minutes, and the supernatant was concentrated to an appropriate concentration. The amount of glucosylceramide in the concentrated solution was then analyzed by LC-ESI-MS / MS using the same method as in Example 4. The amount of glucosylceramide contained in 1g of cooked rice was then calculated as the mean ± standard error of 5 samples. The results are shown in Table 6 below. In Table 6 below, an asterisk (*) is used to indicate cases where the amount of glucosylceramide was significantly higher (5% significance) compared to the wild-type strain.

[0085] [Table 6]

[0086] From the results in Table 6 above, it was found that the amount of glucosylceramide in cooked rice from homo-ND8040 × homo-NE4173 and homo-NE1537 × homo-NE4173 was significantly higher than that of the wild strain. Based on these results, it can be said that cooked rice obtained from polished rice obtained from at least one of the above rice mutants also has a higher glucosylceramide content than conventional rice, and is useful when applied to rice-based foods (such as processed rice products made from cooked brown rice or polished rice).

[0087] This application claims priority based on Japanese Patent Application No. 2021-78534, filed on May 6, 2021, and incorporates all of its disclosures herein.

Claims

1. A grass mutant in which the function of two or more genes encoding a protein belonging to glycoside hydrolase family 1 and having glucocerebrosidase activity is lost or suppressed, including the DNA shown in (a) or (b) below. (a) DNA consisting of the base sequence shown in Sequence ID No. 1, base numbers 112 to 1566 of Sequence ID No. 1, Sequence ID No. 2, or base numbers 76 to 1533 of Sequence ID No. 2 in the sequence listing. (b) DNA consisting of a sequence in which one or more bases have been substituted, deleted, inserted, or added in the sequence shown in Sequence ID No. 1, Sequence ID No. 1 with base numbers 112 to 1566, Sequence ID No. 2, or Sequence ID No. 2 with base numbers 76 to 1533 of the sequence listing.

2. A grass plant mutant according to claim 1, wherein the glucosylceramide content of the seed embryo is 550 μg / g or more, the glucosylceramide content of the seed bran is 150 μg / g or more, or the glucosylceramide content of the part of the seed other than the bran and embryo is 55 μg / g or more.

3. The grass plant mutant according to claim 1 or 2, wherein the function of two or more of the aforementioned genes is deleted or suppressed by the insertion of a transposon inherent in the genome into the aforementioned genes.

4. The grass plant mutant according to claim 3, wherein the transposon is a retrotransposon.

5. A method for producing a plant-derived glucosylceramide-containing extract, comprising the step of extracting a glucosylceramide-containing substance from the seeds or a portion thereof of a grass plant mutant according to claim 1 or 2 using an extraction solvent.

6. A food composition comprising seeds or a portion thereof of a grass plant mutant according to claim 1 or 2.

7. The food composition according to claim 6, comprising the seeds or a part thereof of the aforementioned grass plant mutant as an active ingredient, for one or more uses selected from the group consisting of improving skin moisture, improving skin wrinkles, improving skin firmness, preventing skin damage caused by ultraviolet rays, and preventing colorectal cancer.

8. A pharmaceutical composition comprising the seeds or a portion thereof of the grass plant mutant described in claim 1 or 2 as an active ingredient.

9. The pharmaceutical composition according to claim 8, wherein the pharmaceutical use is one or more selected from the group consisting of improving skin hydration, improving skin wrinkles, improving skin firmness, preventing or treating skin damage caused by ultraviolet rays, and preventing or treating colorectal cancer.

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