WO2022234758A1 - Plant variant having improved glucosylceramide percentage content - Google Patents

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

Plant mutants with enhanced glucosylceramide content

The present invention relates to plant mutants with improved glucosylceramide content, plant-derived glucosylceramide-containing extracts extracted from the plant mutants, food compositions or pharmaceutical compositions containing them, and the like.

Glucosylceramide is a compound that exists widely in organisms such as animals, plants, and filamentous fungi, and is a type of glycolipid consisting of sphingosine bases, fatty acids, and glucose. Glucosylceramide is a compound that plays an important role in the body. In particular, when plant-derived glucosylceramide is ingested, it has the effect of improving skin moisturization, preventing skin damage caused by ultraviolet rays, and preventing colorectal cancer. (Non-Patent Documents 1-4), and is attracting attention as a functional material.

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

This plant-derived glucosylceramide is usually extracted from the plant body with an extraction solvent such as ethanol, and is produced by further purification if necessary. There is a problem that the cost becomes very high. In addition, even when the plant body is directly ingested without extracting glucosylceramide, such as cooked rice, the expected health effects cannot be sufficiently obtained because the glucosylceramide content in the plant body is extremely low. There is a problem.

Therefore, if a plant body (plant mutant) with a higher glucosylceramide content than before can be obtained, the production cost of plant-derived glucosylceramide can be reduced, and mass production becomes possible, and the industrial value is immeasurable. do not have. Furthermore, direct ingestion of plants such as cooked rice products has the great advantage that expected sufficient health effects are likely to be obtained, but there have been no reports of such plants yet.

Therefore, the object of the present invention is to provide a plant mutant with a higher glucosylceramide content than before.

In order to solve the above problems, the present inventor focused on glucocerebrosidase, an enzyme that hydrolyzes glucosylceramide and converts it into ceramide. Although this glucocerebrosidase is known to exist mainly in animals, it is not known to exist in plants, except for a glucocerebrosidase belonging to glycoside hydrolase family 116 (GH116), which has been found in Arabidopsis thaliana. I didn't.

The present inventors have made extensive studies and found a gene (GH1 glucocerebrosidase gene) that belongs to glycoside hydrolase family 1 (GH1) and encodes a protein having glucocerebrosidase activity in plants, and further lacks its function. The inventors have also found that the content of glucosylceramide in plants can be significantly increased by suppressing it, and thus completed the present invention. To date, there have been no reports of plant mutants in which the function of the GH1 glucocerebrosidase gene has been deleted or suppressed.

That is, the present invention is the following <1> to <12>.
<1> A plant mutant in which one or more genes encoding a protein belonging to glycoside hydrolase family 1 and having glucocerebrosidase activity are defective or suppressed in function.
<2> The plant mutant according to <1>, wherein the plant mutant is a seed plant mutant.
<3> The plant mutant according to <2>, wherein the plant mutant is a gramineous plant mutant.
<4> Any one of <1> to <3>, in which the function of one or more of the genes containing the DNA shown in (a), (b), or (c) below is deleted or suppressed Plant mutants as described.
(a) DNA consisting of the base sequence shown in SEQ ID NO: 1, base numbers 112 to 1566 of SEQ ID NO: 1, SEQ ID NO: 2, or base numbers 76 to 1533 of SEQ ID NO: 2 in the sequence listing.
(b) SEQ ID NO: 1 in the sequence listing, base numbers 112 to 1566 in SEQ ID NO: 1, SEQ ID NO: 2, or base numbers 76 to 1533 in SEQ ID NO: 2, one or several bases DNA consisting of base sequences with substitutions, deletions, insertions, or additions.
(c) DNA consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 1 in the sequence listing, base numbers 112 to 1566 of SEQ ID NO: 1, SEQ ID NO: 2, or base numbers 76 to 1533 of SEQ ID NO: 2 DNA that can hybridize with under stringent conditions.
<5> The plant mutant according to any one of <1> to <4>, wherein the function of the gene is deleted or suppressed by inserting a transposon endogenous in the genome into the gene.
<6> The plant mutant according to <5>, wherein the transposon is a retrotransposon.
<7> A method for producing a plant-derived glucosylceramide-containing extract, comprising a step of extracting a glucosylceramide-containing material from the plant mutant according to any one of <1> to <6> using an extraction solvent.
<8> A plant-derived glucosylceramide-containing extract extracted from the plant mutant according to any one of <1> to <6>.
<9> A food composition comprising the plant mutant according to any one of <1> to <6> and/or the plant-derived glucosylceramide-containing extract according to <8>.
<10> A pharmaceutical composition comprising the plant mutant according to any one of <1> to <6> and/or the plant-derived glucosylceramide-containing extract according to <8> as an active ingredient.

<11> The plant mutant according to any one of <1> to <6>, the plant-derived glucosylceramide-containing extract according to <8>, the food composition according to <9>, or <10> 3. A method for improving moisture retention, wrinkles, and firmness of the skin, or preventing and treating damage caused by ultraviolet rays, comprising feeding or administering at least one selected from the group consisting of the pharmaceutical composition described in 1. above.
<12> The plant mutant according to any one of <1> to <6>, the plant-derived glucosylceramide-containing extract according to <8>, the food composition according to <9>, or <10> A method for preventing or treating colorectal cancer, comprising feeding or administering at least one selected from the group consisting of the pharmaceutical compositions described in 1.

According to the present invention, it is possible to provide a plant mutant with a higher glucosylceramide content than before. A plant-derived glucosylceramide-containing extract extracted from this plant mutant can also be provided. Furthermore, food compositions and pharmaceutical compositions containing at least one of these can also be provided.

It is the base sequence of the Os3BGlu6 gene of rice (Nipponbare). It is the base sequence of the Os10BGlu34 gene of rice (Nipponbare). 1 shows the nucleotide sequences of PR1, PR2, PR3, and PR4 primers used for PCR in Example 2. FIG. 1 shows the nucleotide sequences of PR5, PR6, PR7, and PR8 primers used for PCR in Example 2. FIG. 1 shows the nucleotide sequences of primers PR9, PR10, PR11, and PR12 used for PCR in Example 2. FIG.

The present invention will be described.
The present invention provides a plant mutant in which one or more genes encoding proteins belonging to glycoside hydrolase family 1 and having glucocerebrosidase activity are functionally deficient or suppressed (hereinafter referred to as "the plant of the present invention (Also referred to as "mutants"), plant-derived glucosylceramide-containing extracts extracted from the plant mutants of the present invention (hereinafter also referred to as "plant-derived glucosylceramide-containing extracts of the present invention"), A food composition containing the plant mutant and/or the plant-derived glucosylceramide-containing extract of the present invention (hereinafter also referred to as the "food composition of the present invention"), the plant mutant of the present invention and/or the present A pharmaceutical composition containing the plant-derived glucosylceramide-containing extract of the invention as an active ingredient (hereinafter also referred to as "the pharmaceutical composition of the present invention"), and the like.

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 one or more genes encoding proteins belonging to glycoside hydrolase family 1 (GH1) and having glucocerebrosidase activity are functionally deficient or suppressed. In the following, this gene is also referred to as "GH1 glucocerebrosidase gene".

Here, the "glucocerebrosidase activity" is an enzymatic activity possessed by an enzyme (glucocerebrosidase) having an EC number of (EC 3.2.1.45), that is, the glucose and ceramide of the glycolipid glucosylceramide β-1,4-glycosyl bond is hydrolyzed to catalyze the reaction to produce ceramide. In addition, "Glycoside Hydrolase family 1" is a family of carbohydrate hydrolases classified into about 130 groups 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). ing.

The plant mutant of the present invention is a mutant in which the function of the GH1 glucocerebrosidase gene present in the plant genome is deleted or suppressed. In the case of plants having multiple GH1 glucocerebrosidase genes in the genome, mutants in which at least one of the functions is deleted or suppressed may be used. In particular, it is the main activity of glucocerebrosidase belonging to GH1 in the plant (involved in more than 50%, more than 60%, more than 70% of the total activity of glucocerebrosidase belonging to GH1 in the plant) ) Mutants in which the function of the GH1 glucocerebrosidase gene is deleted or suppressed are preferred. However, in the case of such plants, mutants in which two or more GH1 glucocerebrosidase genes are functionally deficient or suppressed, or all GH1 glucocerebrosidase genes present in the genome are functionally deficient or suppressed. A modified mutant is more preferable because it tends to have a higher glucosylceramide content.

This plant mutant includes not only mature plants, but also leaves, stems, roots, petals, calyx, pistils, anthers, pollen, seeds, germ of seeds, endosperm of seeds, bran on the surface of seeds, and temporary seeds. It may be at least one selected from the group consisting of roots and sporangia, and refers to all plant-derived substances.

In addition, "the function of the GH1 glucocerebrosidase gene is deficient" means a state in which the protein encoded by the GH1 glucocerebrosidase gene cannot be expressed (a state in which the gene cannot be transcribed or translated into a protein), or It means that the protein expressed from this gene has no glucocerebrosidase activity. Furthermore, "the function of the GH1 glucocerebrosidase gene is suppressed" means that the expression level of the protein encoded by the GH1 glucocerebrosidase gene (transcription level of the gene or the amount of translation into protein) or glucocerebrosidase of the expressed protein In a state where the amount of sidase activity is significantly decreased (for example, decreased to 40% or less, further decreased to 30% or less, further decreased to 20% or less, further decreased to 10% or less compared to before suppression) It means that there is

Since this mutant is a plant (eukaryote), "the function of one or more GH1 glucocerebrosidase genes is lost or suppressed" means that at least one GH1 glucocerebrosidase gene has an allele It means that both of the two genes (gene set) that make up the gene are functionally deficient or suppressed (homologous mutant). Therefore, heterozygous mutants in which the function of any one of the gene sets constituting one allele is deleted or suppressed are not included in the plant mutants of the present invention, and two or more GH1 glucocerebrosidase genes A function-deficient or suppressed mutant means a mutant in which two or more sets of alleles described above are functionally deficient or suppressed (eg, double homozygous mutant).

Here, with respect to the method of deleting or suppressing the function of the GH1 glucocerebrosidase gene (the method of producing the plant mutant of the present invention), known gene recombination techniques, genome editing techniques, crossing techniques used in plant breeding, etc. Although not particularly limited, the plant mutant of the present invention is produced by inserting a transposon into the GH1 glucocerebrosidase gene (its protein coding region or promoter region) to delete or suppress its function. preferably. Since the plant mutant of the present invention does not become a genetically modified plant, the plant mutant of the present invention has the function of the GH1 glucocerebrosidase gene inserted into the gene by transfer or the like of a transposon endogenous to the genome. In other words, a plant mutant in which a transposon endogenous in the genome of the individual (wild type, etc.) before becoming this mutant is inserted by transposition etc. and this gene is deleted or suppressed More preferably, this transposon is a retrotransposon. In the case of plant mutants in which two or more GH1 glucocerebrosidase genes are functionally deficient or suppressed, it is preferred that the above-mentioned transposons (especially retrotransposons) are inserted into any of the genes.

Transposons are mutagenizing 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), retrotransposon Tos17 (hereinafter, sometimes simply referred to as "Tos17"), which is one of the retrotransposons endogenous in the rice genome, is activated by tissue culture to initiate transposition. A large number of gene-disruption strains have been created by taking advantage of this property. Therefore, it is also possible to obtain a rice mutant (the plant mutant of the present invention) in which Tos17 is inserted into one or more GH1 glucocerebrosidase genes in rice to have its function deleted or suppressed.

It should be noted that transposons are classified into two classes according to their transposition mechanism. Transposons belonging to class II transfer in the form of DNA without replication (transposons themselves are excised from the genome and transferred to other sites), and are also called DNA-type. Examples of transposons belonging to class II include Ac/Ds, Spm/dSpm, and Mu of maize (Zea mays) (Cell 56, 181-191 (1989); Cell 35, 235-242 (1983); Proc. Natl. Acad. ), and nDart (Mol. Genet. Genomics 273, 150-157 (2005)).

Transposons belonging to class I are also called retrotransposons or retrotypes, and replicate and transpose via RNA intermediates (transcription, reverse transcription). Transposons belonging to this class I are ubiquitous and represent a significant portion of the plant genome, and some of these are activated under stress conditions such as wounding, pathogen attack and cell culture. Examples of transposons belonging to class I include Tnt1A and Tto1 of Nicotiana tabacum (Plant J., 5, 535-542 (1994); Plant Mol. Biol., 36, 365-376 (1988)), supra. and Tos 17 of fermented rice (Proc. Natl. Acad. Sci. USA, 93, 7783-7788 (1996)). Since the retrotransposon once inserted into the genome is never cut out, this retrotransposon is preferable because it can induce more stable mutations.

For example, the rice retrotransposon Tos17, described above, has a length of 4.3 kb and is complemented by two identical LTRs (long terminal repeats) that are 138 bp in length and the PBS (primer binding site). Transcription of Tos17 is strongly activated by tissue culture, and the copy number of Tos17 increases with culture time (in Nipponbare, which will be described later, the initial copy number of Tos17 is 2, but in plants regenerated after tissue culture This copy number is increased from 5 to 30). Tos17 then translocates in the rice genome and induces stable mutations. In addition, this Tos17 has the property of being easily inserted into the gene region. Furthermore, the mutants obtained are also very stable, since there is no Tos17 activation in conditions other than tissue culture and little Tos17 transfer occurs in other conditions or after redifferentiation.
Therefore, for example, in rice (such as Nipponbare), first, tissue culture is performed to induce the transfer of Tos17, and the obtained mutants are subjected to PCR screening, measurement of glucocerebrosidase activity, etc., and one or more GH1 glucocerebrosidase genes. The rice mutant according to the present invention can be easily produced by selecting a mutant in which the function of is deleted or suppressed by Tos17, regenerating it into a plant, and further self-pollinated.

In this way, it is possible to obtain the plant mutant of the present invention in which the function of one or more GH1 glucocerebrosidase genes is deleted or suppressed. The plant mutant of the present invention is preferably a seed plant (angiosperm or gymnosperm) mutant from the viewpoint of industrial ease of use.

Seed plants include, but are not limited to, mallow plants, Chenopodiaceae plants, Rubiaceae plants, Cannabis plants, Hydrangea plants, Brassicaceae plants, Iridaceae plants, Gramineae plants, Araliaceae plants, Cucurbitaceae plants of the family Anacardiaceae, plants of the family Anacardiaceae, plants of the family Cyperaceae, plants of the family Bellflower, Asteraceae, plants of the camphor family, plants of the family Moraceae, plants of the poppy family, plants of the family Araceae, plants of the family Cactus, plants of the Labiatae family, plants of the family Nymphae, plants of the family Apiaceae , knotweed plant, ericaceous plant, camellia plant, solanaceous plant, caryophyllaceous plant, elm family plant, lotus plant, rose plant, lotus plant, amaryllidaceous plant, convolvulaceous plant, grape family plant, beech plants of the family Peonies, plants of the family Peonies, plants of the family Leguminosae, plants of the family Rutaceae, plants of the family Apocynaceae, plants of the family Oleaceae, plants of the family Palm, plants of the family Salicaceae, plants of the family Liliaceae, plants of the family Orchidaceae, plants of the Yew family, plants of the Ginkgo family, plants of the Cedar family , Cycadaceous plants, Cupressaceae plants, Pinaceae plants, and the like are shown as specific examples.

More specific examples of seed plants include cotton, hibiscus, spinach, pigweed, beet, madder, gardenia, coffee tree, hemp, hop, hydrangea, Arabidopsis thaliana, rapeseed, Japanese radish, Chinese cabbage, cabbage, cauliflower, broccoli, komatsuna, bok choy, Wasabi, iris, Japanese iris, rice, timothy, wheat, corn, sorghum, barley, rye, sugar cane, oat, millet, foxtail millet, turf, reed, bamboo, bamboo grass, taranogi, udo, fatsia, cucumber, bitter gourd, melon, Watermelon, bitter gourd, pumpkin, loofah, sweet gourd, gourd, yugao, sumac, Japanese wax tree, star grass, bellflower, osmanthus, lettuce, gerbera, gazania, thistle, burdock, sunflower, cosmos, dandelion, calendula, butterbur, ragweed, camphor, camphor tree , mulberry, fig, poppy, botanical, taro, cactus, perilla, salvia, lavender, water lily, carrot, parsley, celery, buckwheat, knotweed, azalea, blueberry, rhododendron, camellia, tomato, eggplant, tobacco, petunia, hot pepper, Potato, dianthus, carnation, gypsophila, chickweed, zelkova, muku tree, lotus, rose, cherry, almond, apricot, strawberry, plum, apple, pear, loquat, peach, lotus, garlic, green onion, onion, bindweed, morning glory, sweet potato, Grapes, beech, konara oak, sawtooth oak, chestnut, button, soybean, broad bean, black bean, wisteria, Japanese wisteria, lupine, kidney bean, pea, alfalfa, peanut, sweet pea, Lotus japonicus, mandarin orange, Japanese pepper, natsumikan, orange, lime, lemon, Grapefruit, trifoliate, water hyacinth, olive, jasmine, coconut palm, oil palm, date palm, palm, poplar, willow, lily, sun lily, tulip, daffodil, moth orchid, cattleya, vanilla, yew, hanger, ginkgo, cedar, cycad, cypress, pine, etc. is shown.

The present inventor discovered the presence of the GH1 glucocerebrosidase gene in mallow, cruciferous, gramineous, cucurbitaceous, asteraceous, solanaceous, rosaceous, leguminous, and umbelliferous plants. (Japanese Patent Application No. 2020-88950). Plant mutants in which one or more GH1 glucocerebrosidase genes are deleted or suppressed in any of these plants are very useful because they tend to have a higher glucosylceramide content than conventional ones, and are particularly useful in rice. Plant variants of family plants are more preferred.

For example, the present inventors have discovered that one rice cultivar, Oryza sativa L. cv. Nipponbare, has Os3BGlu6 and Os10BGlu34 genes as GH1 glucocerebrosidase genes (patent application 2020-88950, etc.). The nucleotide sequence of this Os3BGlu6 gene is shown in SEQ ID NO: 1 of the sequence listing and FIG. 1, and the nucleotide sequence of the Os10BGlu34 gene is shown in SEQ ID NO: 2 of the sequence listing and FIG.

The plant mutant of the present invention comprises the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing, nucleotide numbers 112 to 1566 in SEQ ID NO: 1, SEQ ID NO: 2, or nucleotide numbers 76 to 1533 in SEQ ID NO: 2. Preferred are functionally deficient or suppressed mutants of one or more GH1 glucocerebrosidase genes comprising DNA. That is, at least one, more preferably all, of the GH1 glucocerebrosidase genes whose function is deleted or suppressed is preferably a gene containing any of the DNAs described above. For example, Nipponbare rice mutants are preferably mutants in which the functions of the Os3BGlu6 gene and/or the Os10BGlu34 gene are deleted or suppressed. In particular, the Nipponbare rice mutant is more preferably a mutant in which at least the function of the Os3BGlu6 gene is deleted or suppressed, and further a mutant in which the functions of both the Os3BGlu6 gene and the Os10BGlu34 gene are deleted or suppressed ( A double homozygous mutant) is very suitable because it tends to have a higher glucosylceramide content.

For example, a rice Nipponbare mutant in which a retrotransposon (Tos17) endogenous to the rice Nipponbare genome is inserted into at least one allele of the Os3BGlu6 gene or Os10BGlu34 gene, and the function of which is deleted or suppressed. , is suitable because it does not become a genetically modified crop. For example, when Tos17 is inserted into the protein coding region of the gene, the function of the gene is lost, and when Tos17 is inserted into the promoter region of the gene, the function of the gene is lost or suppressed. It will happen.

Furthermore, in the plant mutant of the present invention, in the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing, nucleotide numbers 112 to 1566 in SEQ ID NO: 1, SEQ ID NO: 2, or nucleotide numbers 76 to 1533 in SEQ ID NO: 2, One or more GH1 glucocerebrosidase genes containing a DNA consisting of a base sequence in which one or several bases are substituted, deleted, inserted or added may be functionally deficient or suppressed. That is, at least one of the GH1 glucocerebrosidase genes whose function is deleted or suppressed may be a gene containing any of the above DNAs. Here, "several" means 20 or less, preferably 10 or less, more preferably 6 or less.

Furthermore, the plant mutant of the present invention has the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing, nucleotide numbers 112 to 1566 in SEQ ID NO: 1, SEQ ID NO: 2, or nucleotide numbers 76 to 1533 in SEQ ID NO: 2. The function of one or more GH1 glucocerebrosidase genes containing a DNA capable of hybridizing with a DNA consisting of a complementary nucleotide sequence under stringent conditions may be deleted or suppressed. That is, at least one of the GH1 glucocerebrosidase genes whose function is deleted or suppressed may be a gene containing any of the above DNAs.
Here, "stringent conditions" are conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. For example, after hybridization at 65° C. in the presence of 0.7-1.0 M sodium chloride, 0.1-5×SSC, 0.1% SDS solution (composition of 1×SSC: 150 mM chloride sodium, 15 mM sodium citrate) and washing at 60° C., preferably 65° C., more preferably 68° C., one to three times.

As shown in the aforementioned Japanese Patent Application No. 2020-88950, the present inventors have found that at least one GH1 glucocerebrosidase gene is present in various seed plants. The gene is a DNA consisting of a base sequence in which one or several bases are substituted, deleted, inserted or added in the base sequence shown in SEQ ID NO: 1 or 2, or SEQ ID NO: 1 or It is very likely that it contains a DNA that can hybridize under stringent conditions with a DNA consisting of a nucleotide sequence complementary to the nucleotide sequence shown in 2. Furthermore, the present inventors have found that among the glucocerebrosidases in plants, those belonging to GH1 are dominant. Therefore, the GH1 glucocerebrosidase gene is identified using a homology search using the nucleotide sequence or amino acid sequence of the Os3BGlu6 gene or the Os10BGlu34 gene, screening by hybridization using at least a part of these DNAs, etc., and its function The plant mutant of the present invention can be easily obtained from various plants by deleting or suppressing .

In such plant mutants of the present invention, the function of one or more GH1 glucocerebrosidase genes is deficient or suppressed, so that the glucosylceramide content is higher than in the past. For example, at least one site selected from the group consisting of leaf, stem, root, petal, calyx, pistil, anther, pollen, seed, seed germ, seed endosperm, seed surface bran, rhizome, and sporangia The content of glucosylceramide is higher than before. In addition, the plant mutant of the present invention has a higher glucosylceramide content in at least a part of the above-described sites than in the past, and includes those isolated from such sites. A portion thereof may contain a portion having a glucosylceramide content of about the same level as the conventional one. In addition, although not limited, in the case of extracting a glucosylceramide-containing extract from the plant mutant of the present invention or a part thereof and using it as a functional material, The glucosylceramide content may be 100 μg/g or more (0.01% by mass or more), further 200 μg/g or more (0.02% by mass or more), and further 300 μg/g or more (0.02% by mass or more). 03% by mass or more), 400 μg/g or more (0.04% by mass or more), and further 500 μg/g or more (0.05% by mass or more). For example, if it is a seed germ of a gramineous plant mutant, the glucosylceramide content may be 450 μg/g or more (0.045% by mass or more), or 480 μg/g or more (0.048% by mass or more). and may be 550 μg/g or more (0.055% by mass or more). In addition, if it is the bran of the seed of the gramineous plant mutant, the glucosylceramide content rate may be 110 μg / g or more (0.011% by mass or more), and 150 μg / g or more (0.015% by mass or more). and may be 160 μg/g or more (0.016% by mass or more). In the case of a gramineous plant mutant, the mixture containing the germ and bran is preferably used as the raw material for extraction of the plant-derived glucosylceramide-containing extract, and in this case, the mixing ratio of the germ and the bran is also limited. However, it is preferable to use 2 to 4 parts by mass of rice bran per 1 part by mass of germ. When the plant mutant of the present invention or a part thereof is used as a functional food (food composition) in the form of ingestion, the glucosylceramide content in the part that can be used in this food composition is 20 μg / g or more (0.002% by mass or more), further may be 30 μg/g or more (0.003% by mass or more), and further 40 μg/g or more (0.004% by mass or more). may be 50 μg/g or more (0.005% by mass or more), further may be 52 μg/g or more (0.0052% by mass or more), and further 55 μg/g or more (0.0052% by mass or more). 0055% by mass or more), may be 60 μg/g or more (0.006% by mass or more), and further may be 65 μg/g or more (0.0065% by mass or more).

Next, the plant-derived glucosylceramide-containing extract of the present invention and the method for producing the same will be described in detail.

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 is performed by using an extraction solvent such as an extraction process using an aqueous solution containing an organic solvent such as alcohol or a surfactant from the plant mutant of the present invention (whole or part of the plant mutant of the present invention). It can be produced by a production method including a step of extracting a glucosylceramide-containing substance. The extraction solvent is not particularly limited, but is preferably ethanol from the viewpoint of use of the extract in food compositions and the like. This ethanol may be hydrous ethanol (for example, hydrous ethanol having an ethanol concentration of 80% by weight or more, further 90% by weight or more). In addition, in this extraction step, heat treatment (for example, heat treatment at 40 to 80° C.) may be performed as necessary. Further, if necessary, a crude purification step (eg, concentration, etc.) may be performed to remove at least part of the extraction solvent. The plant-derived glucosylceramide-containing extract of the present invention in the form of liquid, powder, granule, or the like obtained after such steps is obtained.

Here, the content of plant-derived glucosylceramide contained 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. 02% by mass or more, 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, further preferably 30% by mass or less, and 20% by mass. or less, more preferably 10% by mass or less.

In addition, the plant-derived glucosylceramide-containing extract of the present invention is considered to correspond to the case where the structure or characteristics of the substance are specified simply by indicating the state, but even if this is not the case, this Specific components other than glucosylceramide in the extract (other components derived from the plant mutant of the present invention) and composition ratios have not been clearly specified, and specifying all of these components is extremely costly. Circumstances exist where it is impossible or almost impractical to directly identify the structure or characteristics of the structure or characteristics of the structure or characteristics of the structure or characteristics.

By ingesting such a plant-derived glucosylceramide-containing extract of the present invention (for example, a plant-derived glucosylceramide-containing extract produced by the production method described above) or the above-described plant mutant of the present invention, Due to the action of plant-derived glucosylceramide, it can be expected to improve skin moisturization, prevent or treat skin damage caused by ultraviolet rays, prevent or treat colorectal cancer, etc. In addition, since the components other than the plant-derived glucosylceramide contained in these are also plant-derived, they can be safely used in food compositions and the like at low cost.

Next, the food composition of the present invention and the pharmaceutical composition of the present invention containing the plant mutant of the present invention and/or the plant-derived glucosylceramide-containing extract of the present invention will be described in detail.

A food composition containing the plant mutant of the present invention (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 the plant mutant of the present invention and the present By preparing a food composition containing both the plant-derived glucosylceramide-containing extract of the present invention, the food composition of the present invention rich in plant-derived glucosylceramide can be provided at a low cost. For example, from the rice mutant of the present invention, not only rice and brown rice, which are the seeds of the rice mutant, but also food compositions obtained by processing the brown rice into a food (food containing the rice mutant It is possible to provide polished rice, rice flour, cooked rice foods (such as brown rice and processed cooked rice obtained by cooking polished rice), rice flour processed foods, etc., which are compositions) at a low price. In addition, supplements, which are food compositions containing a rice-derived glucosylceramide-containing extract extracted from the bran on the surface of brown rice (the bran on the surface of seeds), can be provided at a low cost. Here, "polished rice" means brown rice from which the germ and surface bran have been removed (scraped off by processing), and also includes rice from which part of the endosperm has been removed. "Rice flour" is a product obtained by processing brown rice or polished rice into powder. In addition, the food composition of the present invention can optionally contain other known ingredients 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 contains the plant mutant of the present invention and/or the plant-derived glucosylceramide-containing extract of the present invention as an active ingredient for improving skin moisturization, improving skin wrinkles, and improving skin firmness. It is also possible to provide a food composition for one or more uses selected from skin improvement, prevention of skin damage caused by ultraviolet rays, and prevention of colon cancer.

In addition, the pharmaceutical composition of the present invention can be obtained by incorporating the plant mutant of the present invention (whole or 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. can provide. In addition, the pharmaceutical composition of the present invention may, if necessary, further contain known components in the pharmaceutical field (excipients, binders, disintegrants, etc.) as long as they do not significantly affect the pharmacological action of the plant-derived glucosylceramide. , medically acceptable ingredients such as lubricants) can optionally be included. Moreover, the form may be a form suitable for oral administration such as tablets, capsules, granules, fine granules, powders, and syrups. Alternatively, it may be a parenteral drug (drop infusion, etc.). The pharmaceutical composition of the present invention is selected from improving skin moisture retention, improving skin wrinkles, improving skin firmness, preventing or treating skin damage caused by ultraviolet rays, and preventing or treating colorectal cancer. It can be suitably used for one or more pharmaceutical applications.
Furthermore, in the same way, a product for improving skin moisturization, improving skin wrinkles, improving skin firmness, or preventing or treating skin damage caused by ultraviolet rays, containing the plant-derived glucosylceramide-containing extract of the present invention as an active ingredient. Cosmetic compositions can also be provided.

In other words, the present invention provides human with at least one selected from the group consisting of the plant mutant 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. It can be said that the present invention provides a therapeutic method for improving moisturization, wrinkle improvement, skin firmness improvement, or preventing damage caused by ultraviolet rays, which is characterized by feeding or administering to the skin. The same applies to the method of applying the above cosmetic compositions to human skin. Furthermore, the present invention provides human with at least one selected from the group consisting of the plant mutant 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. It can also be said to provide a colon cancer prevention or treatment method characterized by feeding or administration.

It should be noted that the embodiment described above is merely an example for facilitating understanding of the present invention, and does not limit the present invention.

Examples of the present invention will be described below, but the present invention is not limited to the following examples, and various modifications are possible within the technical concept of the present invention.

(Example 1: Production of rice mutant)
Using ripe seeds of Japonica rice (Oryza sativa L. cv. Nipponbare: Nipponbare) as a starting material, according to the method of Hirochika et al. (Proc. Natl. Acad. Sci. USA, 93, 7783-7788 (1996)) Callus initiation culture and cell suspension culture were performed. The culture conditions for activating the retrotransposon Tos17 for gene disruption followed the method of Ohtsuki (rice protoplast culture system, Agriculture, Forestry and Fisheries Technical Information Association (1990)).

Specifically, the fully-ripened 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. The resulting callus was cultured in N6 liquid medium supplemented with 2,4-D for 5 months, and then transferred to a regeneration medium to obtain regenerated rice plants (first generation (M1) plants).

Then, from the leaves of about 20 regenerated rice plants (second generation (M2) plants) of the next generation obtained from this first generation, genomic DNA is obtained for each line, and thermal asymmetric interlaced (TAIL) PCR method. (Genomics, 25, 674-681 (1995)) and the suppression PCR method (Nucleic Acids Research, 23, 1087-1088 (1995)) were used to obtain the insertion site flanking sequences of Tos17 inserted into each strain. (Plant Biotechnology, 15(4), 253-256 (1998)) and databased (Plant Cell, 15, 1771-1780 (2003)). Then, from this database, the Os3BGlu6 gene (gene containing DNA consisting of the nucleotide sequence shown in SEQ ID NO: 1 and FIG. 1 in the sequence listing), which is a gene encoding glucocerebrosidase belonging to GH1, and the Os10BGlu34 gene (SEQ ID NO: in the sequence listing) 2 and FIG. 2), strains (NE1537, ND8040) in which Tos17 was transferred to the Os3BGlu6 gene and Tos17 in the Os10BGlu34 gene were identified. A transferred line (NE4173) was selected.

In addition, since the rice lines obtained here (NE1537, ND8040, NE4173) are all heterozygous mutants, by self-pollination of each, Os3BGlu6 gene homozygous mutants (homozy NE1537, homozygous ND8040) and Os10BGlu34 A gene homozygous deletion mutant (homozygous NE4173) was obtained.

Furthermore, this Os3BGlu6 gene homozygous mutant (homo NE1537, homozygous ND8040) and Os10BGlu34 gene homozygous mutant (homo NE4173) were emasculated in warm water (http://www.naro.affrc.go.jp/laboratory/tarc). /contents/school/kouhai/index.html) to obtain Os3BGlu6 gene and Os10BGlu34 gene double heterozygous deletion mutants (heterogeneous NE1537 x heterozygous NE4173, heterozygous ND8040 x heterozygous NE4173). Furthermore, by self-pollination of these double heterozygous mutants (hetero NE1537 × heterozygous NE4173, heterozygous ND8040 × heterozygous NE4173), Os3BGlu6 gene and Os10BGlu34 gene double homozygous mutants (homo NE1537 × homozygous NE4173, homozygous ND8040 × homozygous NE4173) was obtained.

Here, the heterozygous deletion mutant refers to a mutant in which Tos17 is inserted only in one gene of a specific allele and the function of one gene is deleted, and the homozygous deletion mutant refers to a specific It refers to a mutant in which Tos17 is inserted in both alleles of , resulting in loss of function in both genes. Furthermore, a double heterozygous mutant is a mutant in which Tos17 is inserted only in one gene of each of the two specific alleles and the function of one of the genes is deleted. Point. In addition, the double homozygous mutant refers to a mutant in which Tos17 is inserted into both genes of each of the two specific alleles and the functions of both genes are deleted.

(Example 2: Confirmation of homozygous deletion mutants)
[Confirmation of Homo NE1537]
By performing PCR using four primers prepared from the nucleotide sequence in the Os3BGlu6 gene or the sequence in Tos17, it was confirmed that Tos17 was inserted in both Os3BGlu6 alleles of homo NE1537 prepared in Example 1. confirmed.

Specifically, about 5 mm of homozygous NE1537 rice leaves were cut off, 200 μL of 2×PCR buffer for KOD-FX (manufactured by Toyobo) and zirconia beads were placed in an Eppendorf tube with a screw cap in advance, and then subjected to FastPrep 24 Instrument. (manufactured by MP Biomedicals), followed by centrifugation at 10,000 rpm, 25° C., and 10 minutes, and the supernatant was recovered to obtain an extracted DNA solution. PR1 (nucleotide sequence shown in SEQ ID NO: 3 and shown in FIG. 3) and PR2 (nucleotide sequence shown in SEQ ID NO: 4 and shown in FIG. 3), or PR3 (SEQ ID NO: 5) was added to this extracted DNA solution as F-primer and R-primer. and the nucleotide sequence shown in FIG. 3) and PR4 (SEQ ID NO: 6 and the nucleotide sequence shown in FIG. 3) and KOD-FX (DNA polymerase, manufactured by Toyobo Co., Ltd.) were added and heated at 94° C. for 2 minutes. , 98° C. for 10 seconds, 55° C. for 30 seconds, and 68° C. for 3 minutes as one cycle were repeated 35 times for PCR.

The PR1 and PR2 are PCR primer sets for amplifying the DNA fragment in the region spanning the Os3BGlu6 gene and Tos17, and the PR3 and PR4 are the region in the Os3BGlu6 gene (the region spanning the insertion site of Tos17). A PCR primer set for amplifying DNA fragments. When Tos17 is inserted into the Os3BGlu6 gene, the set of PR1 and PR2 can amplify the DNA fragment, but the set of PR3 and PR4 makes it difficult to amplify the DNA fragment because Tos17 is a very large DNA.

As a result, amplification of the DNA fragment was clearly confirmed when PCR was performed with the set of PR1 and PR2, and amplification of the DNA fragment was not clearly confirmed when PCR was performed with the set of PR3 and PR4. Furthermore, the base sequence of the DNA fragment amplified by the set of PR1 and PR2 was analyzed by DNA sequencing, and homozygous NE1537 was found to be an Os3BGlu6 gene homozygous deletion mutant in which Tos17 was inserted into both alleles of the Os3BGlu6 gene. confirmed.

[Confirmation of Homo ND8040]
Similarly, homozygous ND8040 produced in Example 1 was also confirmed to have Tos17 inserted in both Os3BGlu6 alleles.
Specifically, Homo ND8040 rice leaves were used, and PR5 (nucleotide sequence shown in SEQ ID NO: 7 and FIG. 4) and PR6 (SEQ ID NO: 8 and (nucleotide sequence shown in FIG. 4) or PR7 (nucleotide sequence shown in SEQ ID NO: 9 and FIG. 4) and PR8 (nucleotide sequence shown in SEQ ID NO: 10 and FIG. PCR was performed by the same method.

The PR5 and PR6 are PCR primer sets for amplifying the DNA fragment in the region spanning the Os3BGlu6 gene and Tos17, and the PR7 and PR8 are the region in the Os3BGlu6 gene (the region spanning the insertion site of Tos17). A PCR primer set for amplifying DNA fragments.

As a result, amplification of the DNA fragment was clearly confirmed when PCR was performed with the PR5 and PR6 set, and no DNA fragment amplification was clearly confirmed when the PCR was performed with the PR7 and PR8 set. Furthermore, the base sequence of the DNA fragment amplified by the set of PR5 and PR6 was analyzed by DNA sequencing, and it was found that homozygous ND8040 is an Os3BGlu6 gene homozygous deletion mutant in which Tos17 is inserted in both alleles of the Os3BGlu6 gene. confirmed.

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

Specifically, homozygous NE4173 rice leaves were used, and as the F-primer and R-primer for PCR, PR9 (nucleotide sequence shown in SEQ ID NO: 11 and FIG. 5) and PR10 (SEQ ID NO: 12 and (nucleotide sequence shown in FIG. 5) or PR11 (nucleotide sequence shown in SEQ ID NO: 13 and FIG. 5) and PR12 (nucleotide sequence shown in SEQ ID NO: 14 and FIG. 5). PCR was performed by the same method.

The PR9 and PR10 are a PCR primer set for amplifying a DNA fragment in the region spanning the Os10BGlu34 gene and Tos17, and PR11 and PR12 are the region in the Os10BGlu34 gene (the region spanning the insertion site of Tos17). A PCR primer set for amplifying DNA fragments.

As a result, amplification of the DNA fragment was clearly confirmed when PCR was performed with the set of PR9 and PR10, and amplification of the DNA fragment was not clearly confirmed when PCR was performed with the set of PR11 and PR12. Furthermore, the base sequence of the DNA fragment amplified by the set of PR9 and PR10 was analyzed by DNA sequencing, and homozygous NE4173 was found to be an Os10BGlu34 gene homozygous deletion mutant in which Tos17 was inserted into both alleles of the Os10BGlu34 gene. confirmed.

(Example 3: Confirmation of double homozygous deletion mutant)
[Homo NE1537 x Homo NE4173]
As in Example 2, it was confirmed that Tos17 was inserted in both the Os3BGlu6 allele and the Os10BGlu34 allele in the homozygous NE1537×homozygous NE4173 produced in Example 1.
Specifically, using homo NE1537 × homo NE4173 rice leaves, and as F-primers and R-primers for PCR, PR1 and PR2, PR3 and PR4, PR9 and PR10, or PR11 and PR12 of Example 2 PCR was performed in the same manner as in Example 2, except for using a set of 4 primers.

As a result, the amplification of the DNA fragment was clearly confirmed when PCR was performed with the set of PR1 and PR2 and the set of PR9 and PR10, and the amplification of the DNA fragment was clearly confirmed when the set of PR3 and PR4 and the set of PR11 and PR12 was performed. Amplification was not clearly confirmed. Furthermore, the base sequences of the DNA fragments amplified by the set of PR1 and PR2 and the set of PR9 and PR10 were analyzed by DNA sequencing. It was confirmed to be an Os3BGlu6 gene and Os10BGlu34 gene double homozygous deletion mutant in which Tos17 was inserted in both gene alleles.

[Homo ND8040 x Homo NE4173]
Similarly, it was confirmed that homozygous ND8040x homozygous NE4173 produced in Example 1 also had Tos17 inserted in both Os3BGlu6 alleles and both Os10BGlu34 alleles.
Specifically, using homo ND8040 × homo NE4173 rice leaves, and as F-primers and R-primers for PCR, PR5 and PR6, PR7 and PR8, PR9 and PR10, or PR11 and PR12 of Example 2 PCR was performed in the same manner as in Example 2, except for using a set of 4 primers.

As a result, the amplification of the DNA fragment was clearly confirmed when PCR was performed with the set of PR5 and PR6 and the set of PR9 and PR10, and the amplification of the DNA fragment was clearly confirmed when the set of PR7 and PR8 and the set of PR11 and PR12 was performed. Amplification was not clearly confirmed. Furthermore, the base sequences of the DNA fragments amplified by the set of PR5 and PR6 and the set of PR9 and PR10 were analyzed by DNA sequencing, and homozygous ND8040 × homozygous NE4173 had Tos17 inserted into both alleles of the Os3BGlu6 gene and Os10BGlu34. It was confirmed to be an Os3BGlu6 gene and Os10BGlu34 gene double homozygous deletion mutant in which Tos17 was inserted in both gene alleles.

(Example 4: Comparison of amount of glucosylceramide contained in seed germ of rice mutant)
Five rice mutants prepared in Example 1 (homo NE1537, homo ND8040, homo NE4173, homo NE1537 x homo NE4173, homo ND8040 x homo NE4173) and a wild strain (Nipponbare) were cultivated in a greenhouse, and Seeds were harvested. Then, after peeling off the husk of each seed obtained, 20 embryos collected from each seed were finely ground in a mortar, and 8 mL of ethanol was added and shaken to obtain the content of the embryo. Glucosylceramide was extracted. Then, this extracted sample was centrifuged at 15,000 rpm for 20 minutes, and 0.25-fold volume of 0.2 M HCl was added to the resulting supernatant and mixed. This was centrifuged at 15000 rpm for 20 minutes, the supernatant was subjected to LC-ESI-MS/MS (Liquid chromatography-electrospray ionization-tandem mass spectrometry), and the method of Yumoto et al. , 205-210). That is, ESI-MS/MS uses Agilent 6460 with Agilent 1200 separation module, and the high-performance liquid chromatography column uses TSKgel ODS-120A column (2.1 mm id × 30 cm, manufactured by Tosoh Corporation, registered trademark), Various glucosylceramides were eluted with appropriate methanol concentrations containing 0.1% formic acid and detected in the ESI-positive ion mode. Precursor ion m/z [M+H] ⁺ , product ion m/z, collision energy (eV), fragmentor voltage (V ) are shown in Table 1 below. The amounts of various glucosylceramides detected were obtained by using rice-derived glucosylceramide purchased from Nagara Science as a standard. Finally, the amounts of 8 kinds of glucosylceramide were totaled, and the amount of glucosylceramide contained in 1 g of embryo was calculated as the average value±standard error of 5 individuals. The results are shown in Table 2 below. In Table 2 below, when the amount of glucosylceramide was significantly higher than that of the wild strain (significant by 5%), an asterisk (*) indicates that the amount of glucosylceramide was significantly higher than that of homo NE1537. If the glucosylceramide amount was significantly higher than that of homo ND8040 (5% significant), *** was indicated, and if it was compared with homo NE4173, *** was indicated. When the amount of glucosylceramide was significantly high (significant at 5%), *** was marked.

From the results in Table 2 above, the amount of glucosylceramide contained in the embryos of homozygous NE1537, homozygous ND8040, and homozygous NE4173 was higher than that of the wild strain (strain in which neither the functions of the Os3BGlu6 gene nor the Os10BGlu34 gene were deleted or suppressed). was also found to be significantly higher. It was also revealed that the amount of glucosylceramide contained in the seed germ of homo NE1537×homo NE4173 and homo ND8040×homo NE4173 was even higher than that of homo NE1537, homo ND8040 and homo NE4173.

(Example 5: Comparison I of the amount of glucosylceramide contained in the bran on the seed surface of rice mutants)
8 mL of ethanol was added to 20 grains of each of the 6 kinds of rice seeds from which the germ was removed in Example 4, and the seeds were shaken at 50°C to extract glucosylceramide contained in the bran on the surface of the seeds. . Then, this extracted sample was centrifuged at 15,000 rpm for 20 minutes, and the supernatant was concentrated to an appropriate concentration. analyzed. Then, the amount of glucosylceramide contained in the bran in one seed was calculated as the average value±standard error of 5 individuals. The results are shown in Table 3 below. Also in Table 3 below, when the amount of glucosylceramide was significantly higher than that of the wild strain (5% significance), the asterisk (*) indicates that the amount of glucosylceramide was significantly higher than that of homo NE1537. If the amount of glucosylceramide was significantly higher than that of homo ND8040 (significant by 5%), a *** mark was indicated, and if it was compared with homo NE4173 When the amount of glucosylceramide was significantly higher (significant by 5%), *** was marked.

From the results in Table 3 above, it was found that the amount of glucosylceramide contained in the bran of seeds of homo NE1537, homo ND8040, and homo NE4173 is clearly higher than that of the wild type. It was also revealed that the amount of glucosylceramide contained in the bran of seeds of homo NE1537×homo NE4173 and homo ND8040×homo NE4173 was even higher than homo NE1537, homo ND8040 and homo NE4173.

(Example 6: Comparison II of the amount of glucosylceramide contained in the bran on the seed surface of rice mutants)
The rice husks of each seed obtained from the five rice mutants produced in Example 1 (homo NE1537, homo ND8040, homo NE4173, homo NE1537 x homo NE4173, homo ND8040 x homo NE4173) and a wild strain (Nipponbare) were used. After peeling, the seeds (brown rice) are polished with a rice polishing machine (MK Seiko Co., Ltd., small rice polishing machine RICELON SM-201K), and the resulting mixture containing bran is sieved with a tea strainer to remove polished rice pieces and germ. The bran on the seed surface was collected. Next, 8 mL of ethanol was added to the bran and the mixture was 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. analyzed. Then, the amount of glucosylceramide contained in 1 g of rice bran was determined as the average value±standard error of 3 individuals. The results are shown in Table 4 below. In Table 4 below, when the amount of glucosylceramide was significantly higher than that of the wild strain (5% significant), the * mark is indicated, and the amount of glucosylceramide was significantly higher than that of homo NE1537. If the amount of glucosylceramide was significantly higher than that of homo ND8040 (significant by 5%), a *** mark was indicated, and if it was compared with homo NE4173 When the amount of glucosylceramide was significantly higher (significant by 5%), *** was marked.

From the results in Table 4 above, it was found that the amount of glucosylceramide contained in the bran of seeds of homo NE1537, homo ND8040, and homo NE4173 is clearly higher than that of the wild type. It was also revealed that the amount of glucosylceramide contained in the bran of seeds of homo NE1537×homo NE4173 and homo ND8040×homo NE4173 was even higher than homo NE1537, homo ND8040 and homo NE4173.

Because rice bran is inexpensive and contains a large amount of glucosylceramide, many of the plant-derived glucosylceramides used in health foods are extracted from rice bran. From the results of Examples 5 and 6, it is presumed that by using rice bran obtained from at least one of the rice mutants described above, it is possible to produce glucosylceramide at a lower cost than before. It can be said that it is a useful rice mutant.

(Example 7: Comparison of amount of glucosylceramide contained in parts other than bran and germ in seeds of rice mutants)
After glucosylceramide contained in the bran on the surface of the seeds was extracted in Example 5, ethanol was added to the 6 kinds of rice seeds, vigorously mixed, and the ethanol was discarded. This process was repeated three times to remove as much of the glucosylceramide remaining in the surface bran as possible. Then, 10 grains of each grain were finely ground in a mortar, 8 mL of ethanol was added, and the grains were shaken to extract glucosylceramide contained in portions other than the bran and germ in the rice seeds. Thereafter, the amount of glucosylceramide in the extract was analyzed by LC-ESI-MS/MS in the same manner as in Example 4. Then, the amount of glucosylceramide contained in parts other than bran and germ in 1 g of seed was calculated as the average value±standard error of 5 individuals. The results are shown in Table 5 below. Also in Table 5 below, when the amount of glucosylceramide was significantly higher than that of the wild strain (5% significant), the asterisk * indicates that the amount of glucosylceramide was significantly higher than that of homo NE1537. If the amount of glucosylceramide was significantly higher than that of homo ND8040 (significant by 5%), a *** mark was indicated, and if it was compared with homo NE4173 When the amount of glucosylceramide was significantly higher (significant by 5%), *** was marked.

From the results in Table 5 above, it was found that the amount of glucosylceramide contained in the parts other than the bran and germ of the seeds of homozygous NE1537 and homozygous ND8040 is clearly higher than that of the wild type. In addition, the amount of glucosylceramide contained in the parts other than the bran and germ of the seeds of homo NE1537×homo NE4173 and homo ND8040×homo NE4173 is higher than not only the wild type and homo NE4173 but also homo NE1537 and homo ND8040. also became clear.

This result indicates that the germ and bran-free endosperm of the seeds of these rice mutants also contain a large amount of glucosylceramide. Therefore, rice-derived glucosylceramide is abundantly contained in cooked rice foods (processed cooked rice, etc.) and processed rice products (rice flour, etc.) produced from seeds (mainly the endosperm portion) obtained from the above-described rice mutants. It is also useful as a health food (functional food).

(Example 8: Comparison of amount of glucosylceramide contained in cooked rice obtained by cooking polished rice obtained from seeds of rice mutant)
After dehulling each seed obtained from the two rice mutants prepared in Example 1 (homo NE1537×homo NE4173, homo ND8040×homo NE4173) and a wild strain (Nipponbare), the seeds (brown rice) was polished with a rice polishing machine (MK Seiko Co., Ltd., small rice polishing machine RICELON SM-201K), and the polished rice was cooked in a rice cooker (Sanko Co., Ltd., two-stage high-speed rice cooker) in a conventional manner. Ten grains of the obtained cooked rice were ground in a mortar, 8 mL of ethanol was added, and the mixture was shaken to extract glucosylceramide contained in the cooked rice. Then, this extracted sample was centrifuged at 15,000 rpm for 20 minutes, and the supernatant was concentrated to an appropriate concentration. analyzed. Then, the amount of glucosylceramide contained in 1 g of cooked rice was determined as the average value±standard error of 5 individuals. The results are shown in Table 6 below. In addition, in Table 6 below, when the amount of glucosylceramide was significantly higher than that of the wild strain (5% significant), the * mark was indicated.

From the results in Table 6 above, it was found that the amount of glucosylceramide contained in the cooked rice of homo ND8040 x homo NE4173 and homo NE1537 x homo NE4173 was significantly higher than that of the wild strain. From this result, it was found that the cooked rice obtained by cooking the polished rice obtained from at least one of the above rice mutants also has a higher glucosylceramide content than the conventional one, and is a cooked rice food (brown rice or polished rice that has been cooked, etc.). It can be said that it will be useful even if it is applied to boiled rice.

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

Claims (10)

1. A plant mutant in which the function of one or more genes encoding a protein belonging to glycoside hydrolase family 1 and having glucocerebrosidase activity is deleted or suppressed.
2. The plant mutant according to claim 1, wherein the plant mutant is a seed plant mutant.
3. The plant mutant according to claim 2, wherein the plant mutant is a gramineous plant mutant.
4. The plant mutant according to any one of claims 1 to 3, wherein the function of one or more of the genes comprising the DNA shown in (a), (b), or (c) below is deleted or suppressed. .
   (a) DNA consisting of the base sequence shown in SEQ ID NO: 1, base numbers 112 to 1566 of SEQ ID NO: 1, SEQ ID NO: 2, or base numbers 76 to 1533 of SEQ ID NO: 2 in the sequence listing.
   (b) SEQ ID NO: 1 in the sequence listing, base numbers 112 to 1566 in SEQ ID NO: 1, SEQ ID NO: 2, or base numbers 76 to 1533 in SEQ ID NO: 2, one or several bases DNA consisting of base sequences with substitutions, deletions, insertions, or additions.
   (c) DNA consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 1 in the sequence listing, base numbers 112 to 1566 of SEQ ID NO: 1, SEQ ID NO: 2, or base numbers 76 to 1533 of SEQ ID NO: 2 DNA that can hybridize with under stringent conditions.
5. The plant mutant according to any one of claims 1 to 4, wherein the function of the gene is deleted or suppressed by inserting a transposon endogenous to the genome into the gene.
6. The plant mutant according to claim 5, wherein said transposon is a retrotransposon.
7. A method for producing a plant-derived glucosylceramide-containing extract, comprising a step of extracting a glucosylceramide-containing material from the plant mutant according to any one of claims 1 to 6 using an extraction solvent.
8. A plant-derived glucosylceramide-containing extract extracted from the plant mutant according to any one of claims 1 to 6.
9. A food composition comprising the plant mutant according to any one of claims 1 to 6 and/or the plant-derived glucosylceramide-containing extract according to claim 8.
10. A pharmaceutical composition comprising the plant mutant according to any one of claims 1 to 6 and/or the plant-derived glucosylceramide-containing extract according to claim 8 as an active ingredient.

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