WO2020027155A1 - 甘味成分高含有型ステビア植物及びそのスクリーニング方法 - Google Patents
甘味成分高含有型ステビア植物及びそのスクリーニング方法 Download PDFInfo
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- 238000004226 microchip electrophoresis Methods 0.000 description 1
- 235000021096 natural sweeteners Nutrition 0.000 description 1
- 235000013615 non-nutritive sweetener Nutrition 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000002751 oligonucleotide probe Substances 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- KCXFHTAICRTXLI-UHFFFAOYSA-N propane-1-sulfonic acid Chemical compound CCCS(O)(=O)=O KCXFHTAICRTXLI-UHFFFAOYSA-N 0.000 description 1
- 238000012175 pyrosequencing Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 229930000044 secondary metabolite Natural products 0.000 description 1
- 239000006152 selective media Substances 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 238000004114 suspension culture Methods 0.000 description 1
- 235000019605 sweet taste sensations Nutrition 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
Images
Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
- A01H6/14—Asteraceae or Compositae, e.g. safflower, sunflower, artichoke or lettuce
- A01H6/1488—Stevia
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/10—Processes for modifying non-agronomic quality output traits, e.g. for industrial processing; Value added, non-agronomic traits
- A01H1/101—Processes for modifying non-agronomic quality output traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine or caffeine
- A01H1/102—Processes for modifying non-agronomic quality output traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine or caffeine involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis
- A01H1/103—Non-starch polysaccharides, e.g. cellulose, fructans or levans
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
- A01H5/12—Leaves
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
- A23L2/52—Adding ingredients
- A23L2/60—Sweeteners
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
- A23L27/30—Artificial sweetening agents
- A23L27/33—Artificial sweetening agents containing sugars or derivatives
- A23L27/36—Terpene glycosides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/28—Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/46—Ingredients of undetermined constitution or reaction products thereof, e.g. skin, bone, milk, cotton fibre, eggshell, oxgall or plant extracts
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/13—Plant traits
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
Definitions
- the present invention relates to a stevia plant having a high content of a sweet component and a screening method thereof.
- Patent Document 1 discloses a functional sweetener composition containing a vitamin, a high-intensity sweetener, and a sweet taste improving composition.
- Rebaudioside (hereinafter also referred to as “Reb”) is known as a sweet component contained in stevia extract.
- Stevia extract is extracted and purified from stevia dried leaves.
- Stevia is a perennial plant of the Chrysanthemum family originating in Paraguay, South America, and its scientific name is Stevia @ Rebaudiana @ Bertoni. Since stevia contains a component having a sweetness about 300 times or more that of sugar, stevia is cultivated to extract this sweet component and use it as a natural sweetener.
- As Reb the existence of various glycosides such as RebA, RebB, RebC, RebD, RebE, and RebM has been reported (Table 2012-504552).
- RebA for example, has been evaluated as a sweetener having a high degree of sweetness and good sweetness, and is widely used. Other Rebs are also being found to have their own sweetness and associated taste.
- Patent Documents 2 to 4 stevia plants that are considered to have a high content of a specific rebaudioside are known.
- a method for screening a sweet component-rich type stevia plant comprising a step of detecting a mutation in a portion corresponding to SEQ ID NO: 1 from the genome of a test stevia plant.
- a portion of the genome of a test Stevia plant amplified by PCR using a forward primer selected from SEQ ID NOs: 2 to 8 and a reverse primer selected from SEQ ID NOs: 9 to 15 in a portion corresponding to SEQ ID NO: 1
- the method according to [1] wherein [3]
- the method according to [1] or [2], wherein the mutation comprises a C to A mutation at a position corresponding to position 49 of SEQ ID NO: 1.
- [4] The method according to any one of [1] to [3], wherein the step of detecting the mutation is performed using a dCAPS method or a TaqMan PCR method.
- [5] The method according to any one of [1] to [4], further comprising a step of measuring a content of a sweet component of the test Stevia plant tissue in which the mutation is detected.
- the test stevia plant belongs to an isolated population obtained from a hybrid parent having at least one heterozygous mutation in a portion corresponding to SEQ ID NO: 1, and contains a sweet component contained in a sweet component-rich stevia plant.
- a primer set comprising: a forward primer comprising a sequence; and a reverse primer comprising a sequence complementary to any continuous sequence of 20 or more bases located 3 ′ to position 50 of SEQ ID NO: 1.
- a kit comprising the primer set according to [10] and a restriction enzyme, wherein the restriction enzyme is DdeI when the forward primer contains SEQ ID NO: 47, and the restriction enzyme is DdeI when the forward primer contains SEQ ID NO: 48.
- the enzyme is MaeI or SpeI, if the forward primer contains SEQ ID NO: 49, the restriction enzyme is AflII or MseI, if the forward primer contains SEQ ID NO: 50, the restriction enzyme is Bce83I, and the forward primer is SEQ ID NO: 51.
- restriction enzyme is BseMII
- the restriction enzyme is BsiI
- the forward primer contains SEQ ID NO: 53
- the restriction enzyme is BspHI or Hpy178III
- the forward primer is When SEQ ID NO: 54 is included, the restriction enzyme is SfeI and the forward primer is SEQ ID NO: 5, the restriction enzyme is SmlI, if the forward primer contains SEQ ID NO: 56, the restriction enzyme is EcoP15I, if the forward primer contains SEQ ID NO: 57, the restriction enzyme is AvaI, and the forward primer is No.
- the restriction enzyme is BclI; if the forward primer contains SEQ ID NO: 59, the restriction enzyme is BseRI; if the forward primer contains SEQ ID NO: 60, the restriction enzyme is CviRI or PstI; If the primer comprises SEQ ID NO: 61, the restriction enzyme is DrdII; if the forward primer comprises SEQ ID NO: 62, the restriction enzyme is Eco57I; if the forward primer comprises SEQ ID NO: 63, the restriction enzyme is GsuI; If the forward primer contains SEQ ID NO: 64, the restriction enzyme is HphI and the forward primer Contains SEQ ID NO: 65, the restriction enzyme is Hpy188I; if the forward primer contains SEQ ID NO: 66, the restriction enzyme is MboII; if the forward primer contains SEQ ID NO: 67, the restriction enzyme is Pfl1108I; If the primer comprises SEQ ID NO: 68, the restriction enzyme is PsiI; if the forward primer comprises SEQ ID NO: 69, the restriction enzyme
- [12] A Stevia plant with a high content of sweet components, which has a mutation in a portion of the genome corresponding to SEQ ID NO: 1.
- the tissue, tissue culture or cell according to [15] which is selected from an embryo, meristem cell, pollen, leaf, root, root tip, petal, protoplast, leaf section and callus.
- a method for producing a sweet component-rich Stevia plant comprising the step of crossing the Stevia plant according to any one of [12] to [14] with a second Stevia plant. [18] The method according to [17], wherein the second plant is the stevia plant according to any one of [12] to [14]. [19] A method for producing a sweet component-rich stevia plant, comprising a step of introducing a mutation from C to A at a position corresponding to position 49 of SEQ ID NO: 1 in the genome of the stevia plant. [20] The method according to [19], wherein the introduction of the mutation is performed by a mutagenesis treatment.
- the present invention has one or more of the following effects.
- Stevia plants having a high content of sweet components can be selected at low cost.
- the time required for developing a stevia plant having a high content of a sweet component can be shortened.
- the success rate of development of Stevia plants having a high content of sweet components can be increased.
- the production efficiency of a sweet component derived from a stevia plant can be increased.
- the production cost of a sweet component derived from a stevia plant can be reduced.
- FIG. 1 is a diagram showing the frequency distribution of the sweet component content in M1 generation individuals.
- the vertical axis indicates the number of individuals, and the horizontal axis indicates the concentration (%) of the sweetness component in the dried leaves.
- FIG. 2 is a diagram showing the distribution of the sweet component content in the mutant C49A positive individuals (C49A + ) and negative individuals (C49A ⁇ ) of the isolated population A.
- the vertical axis indicates the sweet component concentration (%) in the dried leaves, and the dotted line indicates the average sweet component concentration of all individuals belonging to the separated population A.
- FIG. 3 is a diagram showing the distribution of the sweet component content in mutant C49A positive individuals (C49A + ) and negative individuals (C49A ⁇ ) of the segregating population B.
- the vertical axis indicates the sweet component concentration (%) in the dried leaves, and the dotted line indicates the average of the sweet component concentrations of all the individuals belonging to the separated population B.
- Sweetening component-rich type Stevia plants present invention has a mutation in the portion of the genome which corresponds to SEQ ID NO: 1, sweetening component-rich type Stevia plants (hereinafter, the "plant of the present invention” May be referred to).
- the nucleotide sequence represented by SEQ ID NO: 1 is as follows.
- the plant of the present invention is a species derived from a wild-type Stevia plant, but has a gene mutation so that the sweetness component content is increased (hereinafter referred to as “mutation of the present invention” There is).
- “Having a mutation in a portion of the genome corresponding to SEQ ID NO: 1” means a portion consisting of the nucleotide sequence of SEQ ID NO: 1 in the genome of Stevia plant or a portion consisting of the nucleotide sequence substantially the same as SEQ ID NO: 1. Has a mutation.
- the nucleotide sequence substantially the same as SEQ ID NO: 1 means, for example, 60% or more, 70% or more, 75% or more, 80% or more, 81% or more, 82% or more, 83% 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 98.1% or more, 98.4% or more, 98.7% or more, 99% or more, 99.2% or more, 99.5% or more, or 99.8% It means a base sequence having the above sequence identity.
- a portion of the genome corresponding to SEQ ID NO: 1 includes a forward primer that hybridizes to a portion having a length of 15 to 25 bases from the 5 ′ end of SEQ ID NO: 1 and a 3 ′ end from SEQ ID NO: 1 It contains a portion of the genome of a Stevia plant that is amplified by a reverse primer that hybridizes to a complementary sequence of a portion having a length of 15 to 25 bases.
- the portion of the genome corresponding to SEQ ID NO: 1 includes a forward primer having a base sequence selected from SEQ ID NOs: 2 to 8, and a reverse primer having a base sequence selected from SEQ ID NOs: 9 to 15.
- “Having a mutation in a genome portion” means that one or more bases differ from a major base sequence (for example, SEQ ID NO: 1) of a predetermined genome portion of a Stevia plant. Mutations include deletions, substitutions and / or additions of nucleotides. In addition, the mutation may be located in the coding region or non-coding region of the gene, and when located in the coding region, the mutation may or may not be accompanied by an amino acid mutation.
- the mutation is a position corresponding to position 11 of SEQ ID NO: 16, a position corresponding to position 21 of SEQ ID NO: 17, a position corresponding to position 31 of SEQ ID NO: 18, the sequence of the Stevia plant genome. It includes a mutation at a position corresponding to position 41 of SEQ ID NO: 19, or a position corresponding to position 49 of SEQ ID NO: 1. Even though the genome of the Stevia plant has a portion consisting of the same nucleotide sequence as SEQ ID NO: 16, 17, 18, 19 or 1, it has a portion consisting of the nucleotide sequence corresponding to SEQ ID NO: 16, 17, 18, 19 or 1. May have.
- the mutation is 11th, 21st, 31st, 41st, 41th, or 49th from the 5 'side of the portion consisting of the same nucleotide sequence as SEQ ID NO: 16, 17, 18, 19 or 1 in the genome of the Stevia plant. Present at the position nucleotide.
- the mutation is not necessarily SEQ ID NO: 16, 17, 18, 19 or 1.
- SEQ ID NO: 16, 17, 18, 19, or 1 are not present at positions 11, 21, 31, 41, or 49 from the 5 ′ side of the portion corresponding to the sequence of SEQ ID NO: 16, 17, 18, 19, or 1, Considering the nucleotide sequence around position 31, 41, or 49, etc., SEQ ID NO: 16, 17, 18, 19, or 1, 11, 21, 31, 31, 41 or 41 in the Stevia plant genome, or The position corresponding to the 49th position can be specified.
- the alignment analysis of the nucleotide sequence of a portion corresponding to SEQ ID NO: 16, 17, 18, 19 or 1 in the genome of the Stevia plant with the nucleotide sequence of SEQ ID NO: 16, 17, 18, 19 or 1 indicates that the Stevia plant has Positions corresponding to positions 11, 21, 31, 31, 41, or 49 of SEQ ID NO: 16, 17, 18, 19 or 1 in the genome can be identified.
- the mutation is a position corresponding to position 11 of SEQ ID NO: 16, a position corresponding to position 21 of SEQ ID NO: 17, a position corresponding to position 31 of SEQ ID NO: 18, SEQ ID NO: 19 in the genome of the Stevia plant.
- the mutation is a position corresponding to position 11 of SEQ ID NO: 16, a position corresponding to position 21 of SEQ ID NO: 17, a position corresponding to position 31 of SEQ ID NO: 18, the sequence of the Stevia plant genome.
- SEQ ID NO: 20 shows a sequence in which position 49 of SEQ ID NO: 1 has been substituted with C to A.
- the plant of the present invention is of a high sweetness component-containing type.
- the high-sweet component-containing Stevia plant means that the content of the sweet component is higher than that of the Stevia plant having no mutation of the present invention.
- the high content of the sweet component refers to, for example, a plant of the present invention (that is, a plant of the present invention) in an isolated population obtained by crossing two Stevia plants having at least one heterozygous mutation of the present invention.
- the average or median content of the sweet component in the population of Stevia plants having the mutation of the present invention is the average or median content of the sweet component in the population of Stevia plants having no mutation of the present invention.
- the plant of the present invention has a sweet component content that is equal to or higher than the average value of all individuals in the segregating population.
- the average value of the content of the sweet component in the population of the plant of the present invention belonging to the separated population is the content of the sweet component in the population of the Stevia plant having no mutation of the present invention belonging to the same separated population.
- the median content of the sweet component in the population of the plant of the present invention belonging to the separated population is the sweet component in the group of the Stevia plant having no mutation of the present invention belonging to the same separated population.
- the sweet component is not limited as long as it is a sweet component contained in a Stevia plant, and includes, for example, one or more steviol glycosides or a combination thereof.
- Steviol glycosides include, for example, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside I, rebaudioside J, rebaudioside K, rebaudioside N, rebaudioside M, rebaudioside O, rebaudioside R, , Dulcoside A, rubusoside, steviol, steviol monoside, steviol bioside, stevioside and the like.
- the sweetening component is rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside I, rebaudioside J, rebaudioside K, rebaudioside N, rebaudioside M, rebaudioside O, rebaudioside O, rebaudioside Q R, dulcosid A, rubusoside, steviol, steviol monoside, steviol bioside, and one or more components selected from stevioside.
- the sweetener comprises rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside F, rebaudioside N, rebaudioside M and rebaudioside O, or consists of these steviol glycosides.
- the Stevia plant of the present invention is a species derived from a wild Stevia plant, and has the above-described mutation in the gene so that the content of the sweet component is increased.
- the mutation of the gene may be caused by a genetic recombination technique or a non-genetical recombination technique.
- the Stevia plant of the present invention may have the gene mutation in a heterozygous or homozygous form.
- the gene mutation is performed by PCR, TaqMan PCR, sequencing, microarray, invader, TILLING, RAD (random amplified polymorphic DNA), restriction enzyme fragment length polymorphism (RFLP), PCR-SSCP, AFLP (amplified fragment length polymorphism) method, SSLP (simple sequence length polymorphism) method, CAPS (cleaved amplified polymorphic sequence) method, dCAPS (derived fragmented amplified polymorphic sequence) method, allele-specific oligonucleotide (ASO) method, ARMS method Denaturing gradient gel electrophoresis (DGGE), CCM (chemical cleavage of mismatch), DOL, MALDI-TOF / MS, TDI, padlock probe, molecular beacon, DASH (dynamic allele specific hybridization) , UCAN method, ECA method, PINPOI NT method, PROBE (primer oligo base extension) method, VSET (very short extension) method, Survivor assay
- examples of the “non-genetical recombination technique” include a method of inducing a mutation in a gene of a host cell (or host plant) without introducing a foreign gene.
- examples of such a method include a method of causing a mutagen of a plant cell to act.
- mutagens include ethyl methanesulfonic acid (EMS) and sodium azide.
- EMS is 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%
- Plant cells can be treated at a concentration such as 1.0%.
- Processing time is about 1 hour to about 48 hours, about 2 hours to about 36 hours, about 3 hours to about 30 hours, about 4 hours to about 28 hours, about 5 hours to about 26 hours, about 6 hours to about 24 hours It is.
- the procedure of the treatment itself is publicly known, it can be carried out by immersing the water-absorbed seed that has undergone the water-absorbing process in the treatment solution containing the mutagen at the above-mentioned concentration for the above-mentioned treatment time.
- Non-genetical recombination method is a method of irradiating plant cells with radiation or light such as X-rays, ⁇ -rays, and ultraviolet rays.
- radiation or light such as X-rays, ⁇ -rays, and ultraviolet rays.
- cells irradiated with an appropriate amount of ultraviolet light are cultured in a selective medium or the like, and then cells, calli, and plants having a desired trait are extracted. You can choose.
- the irradiation intensity at that time is 0.01 to 100 Gr, 0.03 to 75 Gr, 0.05 to 50 Gr, 0.07 to 25 Gr, 0.09 to 20 Gr, 0.1 to 15 Gr, 0.1 to 10 Gr, 0.1 to 10 Gr, and 0.1 to 10 Gr.
- the intensity, distance, and time of irradiation vary depending on the type of radiation or light beam and the state to be irradiated (cells, calli, plants), but can be appropriately adjusted by those skilled in the art.
- plant cells may be mutated during culturing, so it is preferable to return them to plant individuals for more stable trait maintenance.
- Plants obtained by ex-post genetic modification using a non-genetically modified Stevia plant as a host for example, a plant obtained by genetically modifying a plant of the present invention as a host and further adding another trait may also be used. , Is not excluded from the scope of the present invention.
- the sweet component can be extracted in the form of an extract by reacting fresh or dried leaves of the plant of the present invention with an appropriate solvent (aqueous solvent such as water or organic solvent such as alcohol, ether and acetone). It can.
- aqueous solvent such as water or organic solvent such as alcohol, ether and acetone
- Dried leaf means that the water content is reduced to 10% by weight or less, 7% by weight or less, 5% by weight or less, 4% by weight or less, 3% by weight or less, 2% by weight or less and 1% by weight or less by drying fresh leaves It is the one that has been reduced to Preferably, the water content of the dried leaves of the plant of the present invention is 3-4% by weight.
- the extract thus obtained is subjected to a gradient of ethyl acetate and other organic solvents: water, high performance liquid chromatography (High Performance Liquid Chromatography: HPLC), gas chromatography, time-of-flight mass spectrometry (Time- The sweet component can be purified by using a known method such as of-Flight mass spectrometry (TOF-MS) and ultra high performance liquid chromatography (UPLC).
- HPLC High Performance Liquid Chromatography
- TOF-MS time-of-flight mass spectrometry
- UPLC ultra high performance liquid chromatography
- the content of the sweet component according to the present invention can be measured by the method described in JP-T-2012-504552 or the method described in Examples described later. Specifically, for example, it can be measured by sampling fresh leaves from the stevia plant of the present invention and performing LC / MS-MS.
- the plant of the present invention includes not only the whole plant but also plant organs (eg, leaves, petals, stems, roots, seeds, etc.), plant tissues (eg, epidermis, phloem, parenchyma, xylem, vascular bundle, fence) Tissue, spongy tissue, etc.) or various forms of plant cells (eg, suspension culture cells), protoplasts, leaf sections, callus, and the like.
- plant organs eg, leaves, petals, stems, roots, seeds, etc.
- plant tissues eg, epidermis, phloem, parenchyma, xylem, vascular bundle, fence
- Tissue e.g, spongy tissue, etc.
- plant cells eg, suspension culture cells
- protoplasts eg, protoplasts, leaf sections, callus, and the like.
- the plant of the present invention may also include a tissue culture or a plant culture cell. This is because a plant can be regenerated by culturing such a tissue culture or plant culture cell.
- tissue cultures or plant culture cells of the plant of the present invention include embryos, meristem cells, pollen, leaves, roots, root tips, petals, protoplasts, leaf sections and callus, and the like. It is not limited.
- the plant of the present invention also comprises, in addition to the mutation of the present invention, other mutations such as WRKY-02-XbaI, WRKY-08-KpnI, WRKY-09-AflII, WRKY-14 and WD40-01-PvuI. It may have a mutation that is positive for at least one dCAPS marker selected from the group. Plants positive for these markers have a higher RebM content than negative plants (hereinafter, this mutation may be referred to as “high RebM-containing mutation”).
- Positive for WRKY-02-XbaI means that PCR amplification was performed on the genomic DNA of the candidate plant using a forward primer having the base sequence shown in SEQ ID NO: 21 and a reverse primer shown in SEQ ID NO: 22, and the resulting PCR was performed.
- the XbaI restriction enzyme treatment of the product (about 383 bp in length: for example, SEQ ID NO: 23 or 24) means that only a band of about 383 bp (about 383 bp in length: for example, SEQ ID NO: 23 or 24) is obtained.
- the candidate plant becomes WRKY-02-XbaI-negative.
- Positive for WRKY-08-KpnI means that PCR amplification was performed on the genomic DNA of the candidate plant using a forward primer having the base sequence shown in SEQ ID NO: 26 and a reverse primer shown in SEQ ID NO: 27, and the obtained PCR was obtained.
- a product treated with a restriction enzyme of about 258 bp eg, SEQ ID NO: 30
- the candidate plant is negative for WRKY-08-KpnI.
- Positive for WRKY-09-AflII means that PCR amplification was performed on the genomic DNA of the candidate plant using a forward primer having the base sequence shown in SEQ ID NO: 31 and a reverse primer shown in SEQ ID NO: 32, and the obtained PCR was obtained.
- the AflII restriction enzyme treatment of the product (about 390 bp in length) (for example, SEQ ID NO: 33 or 34) means that only a band of about 390 bp (for example, SEQ ID NO: 33 or 34) is obtained.
- a restriction enzyme-treated product of about 347 bp eg, SEQ ID NO: 35
- the candidate plant will be WRKY-09-AflII negative.
- Positive for WRKY-14 is a PCR of about 140 bp when PCR was performed on genomic DNA of a candidate plant using a forward primer having the nucleotide sequence of SEQ ID NO: 36 and a reverse primer of SEQ ID NO: 37.
- Generating only a product means producing a PCR product of 140 bp (eg, SEQ ID NO: 38) and 158 bp (eg, SEQ ID NO: 39), meaning negative.
- Positive for WD40-01-PvuI means that PCR amplification was performed on the genomic DNA of the candidate plant using a forward primer having the base sequence shown in SEQ ID NO: 40 and a reverse primer shown in SEQ ID NO: 41, and the resulting PCR was performed.
- a PvuI restriction enzyme treatment of the product (about 288 bp in length) (for example, SEQ ID NO: 42 or 43) results in only a band of about 288 bp in length (for example, SEQ ID NO: 42 or 43).
- a restriction enzyme-treated product of about 240 bp eg, SEQ ID NO: 44
- the candidate plant will be WD40-01-PvuI negative.
- “about” means ⁇ 5 bp.
- the restriction enzyme treatment can be performed under the conditions recommended by the vendor of each restriction enzyme to be used.
- the plant of the present invention also includes the mutation shown in SEQ ID NO: 45, that is, the nucleotide sequence of the corresponding wild-type allele (SEQ ID NO: 46), in addition to the aforementioned mutation and / or the genetic characteristic of dCAPS marker positive. It may have a mutation from wild type A to T in the 60th base sequence (Patent Document 4).
- a plant having the mutation has a higher RebC content than a plant not having the mutation (hereinafter, also referred to as “high RebC-containing mutation”).
- the plant having the mutation of the present invention tends to increase the content of the whole sweet component, these plants are combined with the other mutations described above (for example, the high RebM-containing mutation and / or the high RebC-containing mutation) to obtain these components.
- the other mutations described above for example, the high RebM-containing mutation and / or the high RebC-containing mutation.
- the effects of other mutations can be enhanced.
- the present invention produces a sweet component-rich type stevia plant comprising a step of crossing a stevia plant of the present invention with a second stevia plant.
- a method hereinafter, referred to as “the production method of the present invention”.
- “Stevia plant with high sweetness component” produced by the method has the same phenotype and genetic properties as the plant of the present invention.
- the phenotype of the plant produced by the production method of the present invention is the phenotype of the high sweetness component-containing type described in the section of the plant of the present invention.
- the genetic property of the plant produced by the production method of the present invention is to have the mutation of the present invention.
- the plant produced by the production method of the present invention may have the mutation in a heterozygous or homozygous form. The method for detecting these mutations is as described above and below.
- crossing refers to crossing a plant of the present invention with a second plant to produce a progeny plant thereof (a plant produced by the production method of the present invention). Means to get.
- backcrossing refers to crossing a progeny produced between a plant of the present invention and a second plant with a plant of the present invention (that is, a plant having a genetic mutation of the present invention). This is a technique for producing a homozygous plant having the gene mutation of the present invention.
- the second plant used in the production method of the present invention has the same phenotype and genetic properties as the plant of the present invention, it is substantially backcrossed.
- the gene mutation of the present invention is inherited according to Mendel's law, and accordingly, a phenotype correlated with the gene mutation, that is, a phenotype of high sweetness content is also inherited according to Mendel's law.
- the plant of the present invention can be produced by selfing.
- Self-breeding can be performed by self-pollinating the stamen pollen of the plant of the present invention to the pistil of the plant of the present invention.
- the plant produced by the production method of the present invention has the same phenotype and genetic properties as the plant of the present invention, the plant produced by the production method of the present invention is further transformed into a third Stevia plant It is also possible to produce a sweet component-rich type stevia plant by crossing.
- the plant of the present invention can be produced by regenerating the plant by culturing the above-described tissue culture or plant culture cell.
- the culture conditions are the same as those for culturing a tissue culture or a plant culture cell of a wild-type Stevia plant, and are known (Protocols for In Vitro cultures and secondary metabolite analysis of aromatic and medicinal plants, Method in molecular biology, vo. 1391, pp113-123.).
- the plant of the present invention can be produced by introducing a mutation from C to A at a position corresponding to position 49 of SEQ ID NO: 1 in the genome of a Stevia plant.
- Mutation may be introduced by a genetic recombination technique or a non-genetical recombination technique.
- the non-genetical recombination technique includes a mutagenesis treatment such as a treatment with a mutagenic agent or a treatment with radiation or light irradiation described in the section of the plant of the present invention.
- the plant of the present invention can be screened by detecting the mutation of the present invention from the tissue of a test plant.
- “screening” means that the plant of the present invention is distinguished from other plants, and the plant of the present invention is selected. Therefore, in another aspect, the present invention provides a method for screening a sweet component-rich Stevia plant comprising a step of detecting a mutation in a portion corresponding to SEQ ID NO: 1 from the genome of a test Stevia plant (hereinafter, referred to as “The screening method of the present invention”).
- the “mutation in the portion corresponding to SEQ ID NO: 1” (mutation of the present invention) and the “sweet sweet component-rich Stevia plant” are as described above for the plant of the present invention.
- mutation detection method of the present invention include PCR, TaqManqPCR, sequencing, microarray, invader, TILLING, RAD, RFLP, PCR-SSCP, AFLP, SSLP, and CAPS.
- Method dCAPS method, ASO method, ARMS method, DGGE method, CCM method, DOL method, MALDI-TOF / MS method, TDI method, padlock probe method, molecular beacon method, DASH method, UCAN method, ECA method, PINPOINT method, PROBE method, VSET method, Survivor assay, Sniper assay, Luminex assay, GOOD method, LCx method, SNaPshot method, Mass ARRAY method, pyrosequencing method, SNP-IT method, melting curve analysis method, and the like, but are not limited thereto. It is not something to be done.
- a primer whose 3 ′ terminal has a sequence complementary to the mutation site of the present invention.
- the primer designed in this way, if the template sample has a mutation, the primer completely hybridizes to the template, so that the polymerase extension reaction proceeds, but the template does not have the mutation of the present invention. In such cases, the extension reaction does not occur because the nucleotide at the 3 'end of the primer causes a mismatch with the template. Therefore, PCR amplification is performed using such primers, and the amplified product is analyzed by agarose gel electrophoresis or the like. If an amplified product of a predetermined size can be confirmed, the sample template has a mutation.
- a primer sequence is designed so that the mutation of the present invention does not overlap with the primer sequence, and the gene mutation of the present invention can be amplified by PCR, and the nucleotide sequence of the amplified nucleotide fragment is sequenced.
- the gene mutation of the present invention can be detected.
- PCR and agarose gel electrophoresis see Sambrook, Fritsch and Maniatis, "Molecular Cloning: A Laboratory Manual” 2nd Edition (1989), Cold Spring Harbor Laboratory Press.
- the TaqMan PCR method is a method using a fluorescently labeled allele-specific oligo and a PCR reaction with Taq DNA polymerase (Livak, KJ Genet. Anal. 14, 143 (1999); Morris T. et al., J Clin. Microbiol. 34, 2933 (1996)).
- the sequencing method is a method for analyzing the presence or absence of a mutation by amplifying a region containing a mutation by PCR and sequencing the DNA sequence using Dye Terminator or the like (Sambrook, Fritsch and Maniatis, "Molecular Cloning: A Laboratory Manual ”2nd Edition (1989), Cold Spring Harbor Laboratory Press).
- a DNA microarray is one in which one end of a nucleotide probe is fixed in an array on a support, and includes a DNA chip, a Gene chip, a microchip, a bead array, and the like.
- a probe containing a sequence complementary to the gene mutation of the present invention By using a probe containing a sequence complementary to the gene mutation of the present invention, the presence or absence of the gene mutation of the present invention can be comprehensively detected.
- Examples of a DNA microarray assay such as a DNA chip include a GeneChip assay (Affymetrix; see US Pat. Nos. 6,045,996, 5,925,525, and 5,858,659).
- the GeneChip technology utilizes a miniaturized high-density microarray of oligonucleotide probes attached to a chip.
- the invader method is a special type of hybridization in which two types of reporter probes and one type of invader probe specific to each allele of a gene mutation such as SNP are hybridized to a template DNA, and the DNA structure is recognized and cut.
- This method combines DNA cleavage with a Cleavase enzyme having endonuclease activity (Livak, KJ Biomol. Eng. 14, 143-149 (1999); Morris T. et al., J. Clin. Microbiol. 34, 2933). (1996); Lyamichev, V. et al., Science, 260, 778-783 (1993), etc.).
- the TILLING (Targeting Induced Local Lesions IN Genomes) method is a method for screening mutation mismatches in the genome of a mutated mutant population by PCR amplification and CELI nuclease treatment.
- the mutation of the present invention can be detected by the dCAPS method using the following primer set and restriction enzyme.
- ⁇ Primer set A sequence selected from SEQ ID NOs: 47 to 70 located at the 3 'end, and any contiguous sequence optionally following from position 28 to 5' of SEQ ID NO: 1 added to the 5 'end of the sequence (eg, , A continuous primer of any length, and a sequence complementary to a sequence of at least 20 consecutive nucleotides located 3 ′ to position 50 of SEQ ID NO: 1 (eg, SEQ ID NO: 15). , 71), and a reverse primer comprising:
- the primer sequence can be optimized within a range that satisfies the above conditions.
- Each of the primers may be 15 to 50 bases, 18 to 48 bases, 20 to 45 bases, 30 to 65 bases, or the like.
- Restriction enzymes corresponding to each of SEQ ID NOs: 47 to 70 are shown below. In the following sequence, “R” represents A or G, and “Y” represents C or T.
- the mutation of the present invention can be detected by the dCAPS method using the following primer set and restriction enzyme.
- the screening method of the present invention may further include a step of measuring the content of the sweet component of the test Stevia plant tissue in which the mutation has been detected.
- the measurement of the sweet component content is as described in the section of the plant of the present invention. Further, in this embodiment, among the test Stevia plants in which the mutation was detected, an individual having a high sweetness component content was selected, and this was crossed with another Stevia plant, and the resulting progeny plants were obtained.
- the screening method of the present invention may be applied. Therefore, the screening method of the present invention may include one or more of the following steps.
- Individuals having a high content of the sweet component to be selected are, for example, among test Stevia plants in which mutations are detected, up to the top 50%, to the top 40%, to the top 30% with respect to the high content of the sweet component, The individuals may be the top 20%, the top 10%, the top 5%, the top 4%, the top 3%, the top 2% or the top 1%.
- Other Stevia plants to be crossed may or may not contain the mutation of the present invention.
- steps (iv) to (vii) can be repeated a plurality of times. In this way, Stevia plants having a higher sweet component content can be screened.
- the test Stevia plant may be a natural plant or a non-genetically modified plant.
- the non-genetically modified plant is as described in the section of the plant of the present invention.
- the test Stevia plant may include a Stevia plant subjected to a mutagenesis treatment and a progeny plant thereof.
- the mutagenesis treatment is as described in the section of the plant of the present invention, and includes treatment with a mutagen, treatment with radiation or light irradiation, and the like.
- At least one of the mating parents of the test Stevia plant in the screening method of the present invention has the mutation of the present invention heterozygously, and any of the mating parents of the test Stevia plant of the present invention The mutation is not homozygous.
- the test Stevia plant belongs to an isolated population generated by crossing two Stevia plants at least one of which has the mutation of the present invention heterozygously.
- a segregated population is obtained by crossing, and individuals with a high sweet component content are selected from the segregated population.However, quantifying the sweet component content of all individuals in the segregated population requires a great deal of time and cost. It takes effort. On the other hand, detection of a genetic variation can be performed with less time, cost, and effort than detection of the content of a sweet component.
- the mutation of the present invention is experimentally found to be included in individuals having a sweetness component content above the average value in an isolated population resulting from the crossing of two Stevia plants at least one of which has the mutation of the present invention heterozygously. (See Examples), and by detecting the mutation of the present invention, individuals having a sweet component content equal to or higher than the average value can be selected from the separated population. As a result, the number of individuals whose sweetness component content is to be measured can be significantly reduced, and breeding development of Stevia plants with a high sweetness component content can be reduced in cost, speed, efficiency, and success rate improved. And so on.
- the present invention provides a probe capable of detecting the presence or absence of the mutation of the present invention (hereinafter, may be referred to as “probe of the present invention”).
- the probe of the present invention may have a structure suitable for various detection methods of the mutation of the present invention.
- the probe of the present invention may include a nucleotide sequence that is complementary to a portion of the genome or transcript containing the mutation site of the present invention.
- Non-limiting examples of such probes include those that specifically hybridize to a base sequence selected from SEQ ID NOs: 1, 16 to 20, and 99 to 102.
- SEQ ID NOS: 20, 99 to 102 are nucleotide sequences containing the mutation of the present invention
- SEQ ID NOs: 1, 16 to 19 are nucleotide sequences containing no mutation of the present invention.
- the probe capable of detecting the presence of the mutation of the present invention hybridizes to a nucleotide sequence selected from SEQ ID NOs: 20, 99 to 102, but to a nucleotide sequence selected from SEQ ID NOs: 1, 16 to 19 Have hybridization conditions that do not hybridize.
- the probe capable of detecting the absence of the mutation of the present invention hybridizes to a nucleotide sequence selected from SEQ ID NOs: 1 and 16 to 19, but hybridizes to a nucleotide sequence selected from SEQ ID NOs: 20 and 99 to 102. Have hybridization conditions that do not hybridize.
- the presence of the mutation of the present invention can be detected by detecting the nucleotide sequence containing the mutation of the present invention and / or not detecting the nucleotide sequence containing no mutation of the present invention. And / or by detecting a nucleotide sequence containing no mutation of the present invention.
- the probe of the present invention preferably has a label. Non-limiting examples of such labels include fluorescent labels, luminescent labels, radioactive labels, dyes, enzymes, quencher, binding sites to detectable labels, and the like.
- the probe of the present invention has a base sequence that specifically hybridizes to a base sequence selected from SEQ ID NOs: 1, 16 to 20, and 99 to 102, and a label.
- the present invention also provides a primer set including the combination of the forward primer and the reverse primer, and a kit including the primer set and a corresponding restriction enzyme.
- the present invention further provides a kit comprising a primer set capable of amplifying, by PCR, a region having a base sequence selected from SEQ ID NOs: 1, 16 to 20, and 99 to 102, and a probe of the present invention.
- These primer sets and kits can be used for detecting the mutation of the present invention, and can be used for the screening method of the present invention.
- these primer sets and kits include the detection of the mutation of the present invention and instructions including a description of the screening method of the present invention, for example, instructions for use, and a medium on which information on the method of use is recorded, such as a flexible disk. , CD, DVD, Blu-ray disc, memory card, USB memory, and the like.
- an extract containing a stevia sweet component comprising a step of obtaining an extract from the plant of the present invention, or a seed or a dried leaf of the plant,
- a method for producing a product (hereinafter, referred to as “the method for producing the extract of the present invention”) is provided.
- a method for producing a stevia sweet component (hereinafter, referred to as “a method for producing a stevia sweet component of the present invention”) including a step of purifying a stevia sweet component from an extract obtained by the method for producing an extract of the present invention is described.
- the method for producing a stevia sweet component of the present invention includes a method for producing individual steviol glycosides.
- the stevia sweet component-containing extract can be obtained by reacting a fresh or dried leaf of the plant of the present invention with a suitable solvent (aqueous solvent such as water or organic solvent such as alcohol, ether and acetone).
- a suitable solvent aqueous solvent such as water or organic solvent such as alcohol, ether and acetone.
- the method described in JP-A-2012-504552 or the method described in Examples described later can be referred to.
- the extract containing stevia sweetener is extracted from ethyl acetate and other organic solvents: water gradient, high performance liquid chromatography (HPLC), gas chromatography, time-of-flight mass spectrometry. : TOF-MS), and a known method such as ultra high performance liquid chromatography (UPLC) can be used to purify the stevia sweet component.
- HPLC high performance liquid chromatography
- TOF-MS time-of-flight mass spectrometry.
- UPLC ultra high performance liquid chromatography
- extract of the present invention An extract obtained by the method for producing an extract of the present invention (hereinafter referred to as “extract of the present invention”) is compared with an extract obtained by a similar method from a Stevia plant not containing the mutation of the present invention. , Characterized by a high content of sweet components. Here, “the content of the sweet component is high” is as described in the section of the plant of the present invention.
- the stevia sweet component is obtained by mixing the extract of the present invention and / or the stevia sweet component obtained by the method for producing the stevia sweet component of the present invention (hereinafter referred to as “the stevia sweet component of the present invention”) with other components.
- Novel pharmaceuticals, flavors or foods and drinks with an increased content of can be produced. Therefore, in another aspect, the present invention provides a method for producing a medicine, a flavor, or a food or drink, comprising a step of mixing the extract of the present invention and / or the stevia sweet component of the present invention with another component.
- the present invention provides a novel drug, flavor, or food or beverage, which has an increased content of a sweet component of stevia, obtained by the production method.
- the food and drink means beverages and foods.
- the present invention provides novel pharmaceuticals, flavors, beverages, or foods, and provides methods for making such pharmaceuticals, flavors, beverages, or foods.
- nucleotide Sequence of Plant of the Present Invention provides a nucleotide sequence of the Stevia plant of the present invention.
- a specific embodiment of the nucleotide sequence of the Stevia plant having the mutation of the present invention comprises or consists of a nucleotide sequence selected from SEQ ID NOs: 20, 99 to 102.
- This extract was subjected to LC / MS-MS analysis in LCMS8050 ion mode (Shimadzu LCMS8050), and the concentrations of RebA, RebB, RebC, RebD, RebF, RebM, RebN and RebO were quantified. And An individual having a sweetness component concentration of about 20% was designated as parent individual 1 (P1). As parent individual 2 (P2), an individual having a sweet component concentration of 5% in the dried leaves, which was derived from another Stevia plant group, was selected.
- the amount of genomic information is sufficient (sequence coverage is ⁇ 5 or more), the mutations are not continuous, and for each of 306 mutations with no sequence insertion or deletion, It was examined whether it was present in the individual. As a result, it was revealed that the mutation from C to A at position 49 of SEQ ID NO: 1 (C49A) was present in individuals with high sweetness components but not in individuals with low sweetness components.
- FIGS. 2 and 3 show the distribution of the sweetness component content in the mutant C49A positive individuals and negative individuals in each segregating population. From these results, it can be seen that the sweet component content in the mutant C49A-positive individual is higher than the average value of the sweet component content of each whole separated population.
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Abstract
Description
[1] 被験ステビア植物体のゲノムから、配列番号1に相当する部分における変異を検出する工程を含む、甘味成分高含有型ステビア植物体をスクリーニングする方法。
[2] 配列番号1に相当する部分が、配列番号2~8から選択されるフォワードプライマー及び配列番号9~15から選択されるリバースプライマーを用いたPCRで増幅される被験ステビア植物のゲノムの部分である、[1]に記載の方法。
[3] 変異が、配列番号1の49位に相当する位置におけるCからAへの変異を含む、[1]又は[2]に記載の方法。
[4] 変異を検出する工程が、dCAPS法又はTaqMan PCR法を用いて行われる、[1]~[3]のいずれか一項に記載の方法。
[5] 変異が検出された被験ステビア植物組織の甘味成分の含有量を測定する工程をさらに含む、[1]~[4]のいずれか一項に記載の方法。
[6] 被験ステビア植物体が、少なくとも一方が配列番号1に相当する部分における変異をヘテロで有する交配親から得られた分離集団に属し、甘味成分高含有型ステビア植物体に含まれる甘味成分含有量が、前記分離集団に属する全個体の甘味成分含有量の平均値より高い、[1]~[5]のいずれか一項に記載の方法。
[8] 甘味成分高含有型ステビア植物体が、非遺伝子組換植物体である、[1]~[7]のいずれか一項に記載の方法。
[9] 被験ステビア植物体が、突然変異誘発処理を行ったステビア植物体及びその子孫植物体を含む、[1]~[8]のいずれか一項に記載の方法。
[10] 3’末端に位置する配列番号47~70から選択される配列と、任意選択で前記配列の5’末端に付加される配列番号1の28位から5’側に続く任意の連続する配列とを含むフォワードプライマーと、配列番号1の50位より3’側に位置する任意の連続する20塩基以上の配列に相補的な配列を含むリバースプライマーとを含む、プライマーセット。
[11] [10]に記載のプライマーセットと制限酵素とを含むキットであって、フォワードプライマーが配列番号47を含む場合、制限酵素はDdeIであり、フォワードプライマーが配列番号48を含む場合、制限酵素はMaeI又はSpeIであり、フォワードプライマーが配列番号49を含む場合、制限酵素はAflII又はMseIであり、フォワードプライマーが配列番号50を含む場合、制限酵素はBce83Iであり、フォワードプライマーが配列番号51を含む場合、制限酵素はBseMIIであり、フォワードプライマーが配列番号52を含む場合、制限酵素はBsiIであり、フォワードプライマーが配列番号53を含む場合、制限酵素はBspHI又はHpy178IIIであり、フォワードプライマーが配列番号54を含む場合、制限酵素はSfeIであり、フォワードプライマーが配列番号55を含む場合、制限酵素はSmlIであり、フォワードプライマーが配列番号56を含む場合、制限酵素はEcoP15Iであり、フォワードプライマーが配列番号57を含む場合、制限酵素はAvaIであり、フォワードプライマーが配列番号58を含む場合、制限酵素はBclIであり、フォワードプライマーが配列番号59を含む場合、制限酵素はBseRIであり、フォワードプライマーが配列番号60を含む場合、制限酵素はCviRI又はPstIであり、フォワードプライマーが配列番号61を含む場合、制限酵素はDrdIIであり、フォワードプライマーが配列番号62を含む場合、制限酵素はEco57Iであり、フォワードプライマーが配列番号63を含む場合、制限酵素はGsuIであり、フォワードプライマーが配列番号64を含む場合、制限酵素はHphIであり、フォワードプライマーが配列番号65を含む場合、制限酵素はHpy188Iであり、フォワードプライマーが配列番号66を含む場合、制限酵素はMboIIであり、フォワードプライマーが配列番号67を含む場合、制限酵素はPfl1108Iであり、フォワードプライマーが配列番号68を含む場合、制限酵素はPsiIであり、フォワードプライマーが配列番号69を含む場合、制限酵素はTaqI又はXhoIであり、フォワードプライマーが配列番号70を含む場合、制限酵素はStySKIであるキット。
[13] 変異が、配列番号1の49位に相当する位置におけるCからAへの変異を含む、[12]に記載の植物体。
[14] 非遺伝子組換植物体である、[12]又は[13]に記載の植物体。
[15] [12]~[14]のいずれか一項に記載の植物体の種子、組織、乾燥葉、組織培養物又は細胞。
[16] 胚、分裂組織細胞、花粉、葉、根、根端、花弁、プロトプラスト、葉の切片及びカルスから選択される、[15]に記載の組織、組織培養物又は細胞。
[17] [12]~[14]のいずれか一項に記載のステビア植物体と第2のステビア植物体とを交雑させる工程を含む、甘味成分高含有型ステビア植物体を作出する方法。
[18] 第2の植物体が[12]~[14]のいずれか一項に記載のステビア植物体である、[17]に記載の方法。
[19] ステビア植物体のゲノムの配列番号1の49位に相当する位置にCからAへの変異を導入する工程を含む、甘味成分高含有型ステビア植物体を作出する方法。
[20] 変異の導入が、突然変異誘発処理によって行われる、[19]に記載の方法。
[21] [12]~[14]のいずれか一項に記載の植物体、[15]に記載の種子、組織、乾燥葉、組織培養物又は細胞、あるいは、[16]に記載の組織、組織培養物又は細胞の抽出物を提供する工程、及び
前記抽出物を、飲食品、甘味組成物、香料又は医薬品の原料に添加する工程
を含む、飲食品、甘味組成物、香料又は医薬品の製造方法 。
(1)甘味成分の含有量が高いステビア植物の選抜を低コストで行うことができる。
(2)甘味成分の含有量が高いステビア植物の開発に要する時間を短縮化できる。
(3)甘味成分の含有量が高いステビア植物の開発の成功率を高めることができる。
(4)ステビア植物由来の甘味成分の生産効率を高めることができる。
(5)ステビア植物由来の甘味成分の生産コストを低減することができる。
なお、本明細書において引用した全ての文献、および公開公報、特許公報その他の特許文献は、参照として本明細書に組み込むものとする。また、本明細書は、2018年7月31日に出願された本願優先権主張の基礎となる日本国特許出願(特願2018-144512号)の明細書および図面に記載の内容を包含する。
本発明は、1つの側面において、配列番号1に相当するゲノムの部分に変異を有する、甘味成分高含有型ステビア植物体(以下、「本発明の植物体」と称することがある)を提供する。配列番号1が表す塩基配列は以下のとおりである。
本発明の植物体は、野生種のステビア植物体から派生した種であるが、甘味成分含量が高くなるように遺伝子に変異が生じたものである(以下、「本発明の変異」と称する場合がある)。
一般に、植物細胞は、培養の間に変異を伴うことがあるため、より安定した形質維持のために植物個体に戻すことが好ましい。
非遺伝子組み換えステビア植物体を宿主として事後的に遺伝子組み換えを行って得られた植物体(例えば、本発明の植物体を宿主として遺伝子組み換えを行って、さらに別の形質を付加した植物体)も、本発明の範囲から除外されるものではない。
さらに、このようにして得られた抽出液に対し、酢酸エチルその他の有機溶媒:水の勾配、高速液体クロマトグラフィー(High Performance Liquid Chromatography:HPLC)、ガスクロマトグラフィー、飛行時間型質量分析(Time-of-Flight mass spectrometry:TOF-MS)、超高速液体クロマトグラフィー (Ultra (High)Performance Liquid chromatography:UPLC) 等の公知の方法を用いることにより甘味成分を精製することができる。
上記bp長に関し、「約」とは、±5bpを意味する。制限酵素処理は、使用する各制限酵素の販売元が推奨する条件に従って行うことができる。
本発明の変異を有する植物体は、甘味成分全体の含量が高まる傾向にあるため、上述の他の変異(例えば、高RebM含有型変異及び/又は高RebC含有型変異)と組み合わせることで、これら他の変異による効果を増強することができる。
本発明は、別の実施態様において、本発明のステビア植物体と第2のステビア植物体とを交雑させる工程を含む、甘味成分高含有型ステビア植物体を作出する方法(以下、「本発明の作出方法」という)を提供する。
当該方法により作出される「甘味成分高含有型ステビア植物体」は、本発明の植物体と同じ表現型と遺伝的性質を有する。
さらに別の態様として、本発明の植物体は、ステビア植物体のゲノムの配列番号1の49位に相当する位置にCからAへの変異を導入することで作出することも可能である。変異の導入は、遺伝子組換え的手法により行っても、非遺伝子組換え的手法により行ってもよい。非遺伝子組換え的手法は、本発明の植物体の項に記載した、変異誘発剤による処理や、放射線又は光線の照射による処理等の突然変異誘発処理を含む。
本発明の植物体は、被験植物体の組織から本発明の変異を検出することによりスクリーニングすることができる。ここで、「スクリーニング」とは、本発明の植物体とそれ以外の植物体とを識別し、本発明の植物体を選択することを意味する。
したがって、本発明は、別の側面において、被験ステビア植物体のゲノムから、配列番号1に相当する部分における変異を検出する工程を含む、甘味成分高含有型ステビア植物体をスクリーニングする方法(以下、「本発明のスクリーニング方法」という)を提供する。「配列番号1に相当する部分における変異」(本発明の変異)、及び「甘味成分高含有型ステビア植物体」については、本発明の植物体について上記したとおりである。
あるいは、本発明の変異とプライマー配列とは重複させず、かつ本発明の遺伝子変異をPCR増幅させることが可能なようにプライマー配列を設計し、増幅されたヌクレオチド断片の塩基配列をシーケンスすることにより、本発明の遺伝子変異を検出することができる。
PCR及びアガロースゲル電気泳動については、以下を参照:Sambrook, Fritsch and Maniatis, ”Molecular Cloning: A Laboratory Manual” 2nd Edition (1989), Cold Spring Harbor Laboratory Press。
シークエンス法とは、変異を含む領域をPCRにて増幅させ、Dye Terminatorなどを用いてDNA配列をシークエンスすることで、変異の有無を解析する方法である(Sambrook, Fritsch and Maniatis, ”Molecular Cloning: A Laboratory Manual” 2nd Edition (1989), Cold Spring Harbor Laboratory Press)。
DNAマイクロアレイは、ヌクレオチドプローブの一端が支持体上にアレイ状に固定されたものであり、DNAチップ、Geneチップ、マイクロチップ、ビーズアレイ等を包含する。本発明の遺伝子変異と相補的な配列を含むプローブを用いることで、網羅的に本発明の遺伝子変異の有無を検出することができる。DNAチップなどのDNAマイクロアレイアッセイとしてはGeneChipアッセイが挙げられる(Affymetrix社;米国特許第6,045,996号、同第5,925,525号、及び同第5,858,659号参照)。GeneChip技術は、チップに貼り付けたオリゴヌクレオチドプローブの小型化高密度マイクロアレイを利用するものである。
TILLING(Targeting Induced Local Lesions IN Genomes)法とは、変異導入した突然変異体集団のゲノム中の変異ミスマッチをPCR増幅とCEL Iヌクレアーゼ処理によってスクリーニングする方法である。
・プライマーセット:
3’末端に位置する配列番号47~70から選択される配列と、任意選択で前記配列の5’末端に付加される配列番号1の28位から5’側に続く任意の連続する配列(例えば、任意の長さの連続する配列)とを含むフォワードプライマーと、配列番号1の50位より3’側に位置する任意の連続する20塩基以上の配列に相補的な配列(例えば、配列番号15、71)を含むリバースプライマーとを含む、プライマーセット。プライマーの配列は、上記の条件を満たす範囲で最適化することができる。プライマー設計の最適化については、例えば、Sambrook and Russell, ”Molecular Cloning: A Laboratory Manual” 3rd Edition (2001), Cold Spring Harbor Laboratory Press等を参照のこと。また、上記各プライマーは、15~50塩基長、18~48塩基長、20~45塩基長、30~65塩基長等であってよい。
(i)被験ステビア植物のゲノムから、配列番号1に相当する部分における変異を検出する工程、
(ii)変異が検出された被験ステビア植物組織の甘味成分の含有量を測定する工程、
(iii)変異が検出された被験ステビア植物体のうち、甘味成分の含有量が高い個体を選択する工程、
(iv)選択した甘味成分の含有量が高い個体を他のステビア植物体と交配する工程、
(v)交配により得られた子植物体のゲノムから、配列番号1に相当する部分における変異を検出する工程、
(vi)変異が検出された子植物組織の甘味成分の含有量を測定する工程、
(vii)変異が検出された子植物体のうち、甘味成分の含有量が高い個体を選択する工程。
本発明のスクリーニング方法において、被験ステビア植物体は、突然変異誘発処理を行ったステビア植物及びその子孫植物を含んでもよい。突然変異誘発処理については、本発明の植物体の項に記載したとおりであり、変異誘発剤による処理や、放射線又は光線の照射による処理等を含む。
本発明のプローブは、好ましくは標識を有する。かかる標識の非限定例としては、蛍光標識、発光標識、放射性標識、色素、酵素、クエンチャー、検出可能な標識との結合部分等が挙げられる。特定の態様において、本発明のプローブは、配列番号1、16~20、99~102から選択される塩基配列に特異的にハイブリダイズする塩基配列と、標識とを有する。
本発明のさらなる側面において、本発明の植物体、又は当該植物体の種子若しくは乾燥葉から抽出物を得る工程を含む、ステビア甘味成分含有抽出物の製造方法(以下、「本発明の抽出物の製造方法」という)が提供される。さらに、本発明の抽出物の製造方法により得られた抽出物からステビア甘味成分を精製する工程を含む、ステビア甘味成分の製造方法(以下、「本発明のステビア甘味成分の製造方法」という)が提供される。ステビア甘味成分には、上述のとおり、種々のステビオール配糖体が包含されるため、本発明のステビア甘味成分の製造方法は、個々のステビオール配糖体の製造方法を包含する。
また、ステビア甘味成分含有抽出物を酢酸エチルその他の有機溶媒:水の勾配、高速液体クロマトグラフィー(High Performance Liquid Chromatography:HPLC)、ガスクロマトグラフィー、飛行時間型質量分析(Time-of-Flight mass spectrometry:TOF-MS)、超高速液体クロマトグラフィー (Ultra (High)Performance Liquid chromatography:UPLC) 等の公知の方法を用いることによりステビア甘味成分を精製することができる。
本発明は、別の側面において、本発明のステビア植物体に係る塩基配列を提供する。本発明の変異を有するステビア植物体に係る塩基配列の特定の態様は、配列番号20、99~102から選択される塩基配列を含む、又はそれからなる。
野生型ステビア種子(市販品種、2014年8月に導入)約2000個(重量換算)を3つに分けて、各々に対して0.1%、0.2%又は0.3%のエチルメタンスルホン酸(EMS)処理を行って遺伝子改変を行った。
このEMS処理済み種子及び無処理の種子を、サントリー研究センター内温室にて播種し、EMS処理当代(M0世代)苗を得た。処理濃度間での発芽率差異は見られなかった。
EMS処理当代(M0世代)及び無処理個体より適量の新鮮葉をサンプリングし、LC/MS-MS(島津LCMS8050)にて甘味成分の濃度を定量した。具体的には、0.25gの新鮮葉をフリーズドライにより乾燥し、破砕物乾物0.05gを純水中に投入した。超音波処理20分にて抽出し、遠心・濾過した後に0.33mlの抽出液を得た。この抽出液をLCMS8050イオンモード(島津LCMS8050)にてLC/MS-MS分析を行い、RebA、RebB、RebC、RebD、RebF、RebM、RebN及びRebOの濃度を定量し、その合計を甘味成分の濃度とした。甘味成分濃度が約20%の個体を親個体1(P1)とした。また、親個体2(P2)として、別のステビア植物体の集団に由来する、乾燥葉中の甘味成分濃度が5%の個体を選定した。
親個体1(P1)と親個体2(P2)との交配により処理第一代(M1世代)種子を採種し、サントリー研究センター内の温室内で播種し、M1世代苗を得た(1603個体の分離集団)。上記M1世代個体より適量の新鮮葉をサンプリングし、上記(1)と同様にLC/MS-MS(島津LCMS8050)にて甘味成分を定量した。結果を図1に示す。
甘味成分の含有量が最も高い30個体(甘味成分高含有個体)と、最も低い30個体(甘味成分低含有個体)の新鮮葉からゲノムDNAを抽出し、両個体群のいずれか一方のみに存在する変異について調査した。ゲノム解析で検出した変異のうち、ゲノム情報量が十分あり(シーケンスカバレッジが×5以上)、変異が連続しておらず、配列の挿入や欠失のない306ヵ所の変異について、各変異がどの個体に存在するかを検討した。その結果、配列番号1の49位におけるCからAへの変異(C49A)が、甘味成分高含有個体には存在するが、低含有個体には存在しないことが明らかとなった。
変異C49Aをヘテロで有するステビア植物体と、変異C49Aを有しないステビア植物体とを交配し、2つの分離集団(分離集団A(443個体)及び分離集団B(446個体))を得た。両分離集団の各個体における変異C49Aの有無と、甘味成分含有量とを調査した。変異C49Aの有無の調査にはdCAPS法を用いた。各個体からゲノムDNAを抽出し、以下のプライマーを用いてPCRを行い、PCR産物に制限酵素(SpeI)を添加し、37℃で酵素反応を行った。制限酵素処理後、マイクロチップ型電気泳動装置LabChip GX Touch HT(PerkinElmer)により電気泳動を行い、電気泳動後のバンドパターンによってマーカーの識別を行った。
フォワードプライマー:5’-TTATTTAATGATCCAATGGAGGGGGTGATTCAGGTAATAAAAGGCACT-3’(配列番号71)
リバースプライマー:5’-TGAGGGTTCTCAATTGATTTCCGATTGG-3’(配列番号15)
得られた約367bpPCR産物(例えば、配列番号96又は97)に対しSpeI制限酵素処理を行い、約321bpの制限酵素処理産物(例えば、配列番号98)を生じたものを変異C49A陽性とした。
甘味成分含有量の定量は(1)と同様に行った。
各分離集団の変異C49A陽性個体及び陰性個体における甘味成分含有量の分布を図2~3に示す。これらの結果から、変異C49A陽性個体における甘味成分含有量は、各分離集団全体の甘味成分含有量の平均値より高いことが分かる。
Claims (21)
- 被験ステビア植物体のゲノムから、配列番号1に相当する部分における変異を検出する工程を含む、甘味成分高含有型ステビア植物体をスクリーニングする方法。
- 配列番号1に相当する部分が、配列番号2~8から選択されるフォワードプライマー及び配列番号9~15から選択されるリバースプライマーを用いたPCRで増幅される被験ステビア植物のゲノムの部分である、請求項1に記載の方法。
- 変異が、配列番号1の49位に相当する位置におけるCからAへの変異を含む、請求項1又は2に記載の方法。
- 変異を検出する工程が、dCAPS法又はTaqMan PCR法を用いて行われる、請求項1~3のいずれか一項に記載の方法。
- 変異が検出された被験ステビア植物組織の甘味成分の含有量を測定する工程をさらに含む、請求項1~4のいずれか一項に記載の方法。
- 被験ステビア植物体が、少なくとも一方が配列番号1に相当する部分における変異をヘテロで有する交配親から得られた分離集団に属し、甘味成分高含有型ステビア植物体に含まれる甘味成分含有量が、前記分離集団に属する全個体の甘味成分含有量の平均値より高い、請求項1~5のいずれか一項に記載の方法。
- 甘味成分がステビオール配糖体を含む、請求項1~6のいずれか一項に記載の方法。
- 甘味成分高含有型ステビア植物体が、非遺伝子組換植物体である、請求項1~7のいずれか一項に記載の方法。
- 被験ステビア植物体が、突然変異誘発処理を行ったステビア植物体及びその子孫植物体を含む、請求項1~8のいずれか一項に記載の方法。
- 3’末端に位置する配列番号47~70から選択される配列と、任意選択で前記配列の5’末端に付加される配列番号1の28位から5’側に続く任意の連続する配列とを含むフォワードプライマーと、配列番号1の50位より3’側に位置する任意の連続する20塩基以上の配列に相補的な配列を含むリバースプライマーとを含む、プライマーセット。
- 請求項10に記載のプライマーセットと制限酵素とを含むキットであって、フォワードプライマーが配列番号47を含む場合、制限酵素はDdeIであり、フォワードプライマーが配列番号48を含む場合、制限酵素はMaeI又はSpeIであり、フォワードプライマーが配列番号49を含む場合、制限酵素はAflII又はMseIであり、フォワードプライマーが配列番号50を含む場合、制限酵素はBce83Iであり、フォワードプライマーが配列番号51を含む場合、制限酵素はBseMIIであり、フォワードプライマーが配列番号52を含む場合、制限酵素はBsiIであり、フォワードプライマーが配列番号53を含む場合、制限酵素はBspHI又はHpy178IIIであり、フォワードプライマーが配列番号54を含む場合、制限酵素はSfeIであり、フォワードプライマーが配列番号55を含む場合、制限酵素はSmlIであり、フォワードプライマーが配列番号56を含む場合、制限酵素はEcoP15Iであり、フォワードプライマーが配列番号57を含む場合、制限酵素はAvaIであり、フォワードプライマーが配列番号58を含む場合、制限酵素はBclIであり、フォワードプライマーが配列番号59を含む場合、制限酵素はBseRIであり、フォワードプライマーが配列番号60を含む場合、制限酵素はCviRI又はPstIであり、フォワードプライマーが配列番号61を含む場合、制限酵素はDrdIIであり、フォワードプライマーが配列番号62を含む場合、制限酵素はEco57Iであり、フォワードプライマーが配列番号63を含む場合、制限酵素はGsuIであり、フォワードプライマーが配列番号64を含む場合、制限酵素はHphIであり、フォワードプライマーが配列番号65を含む場合、制限酵素はHpy188Iであり、フォワードプライマーが配列番号66を含む場合、制限酵素はMboIIであり、フォワードプライマーが配列番号67を含む場合、制限酵素はPfl1108Iであり、フォワードプライマーが配列番号68を含む場合、制限酵素はPsiIであり、フォワードプライマーが配列番号69を含む場合、制限酵素はTaqI又はXhoIであり、フォワードプライマーが配列番号70を含む場合、制限酵素はStySKIであるキット。
- 配列番号1に相当するゲノムの部分に変異を有する、甘味成分高含有型ステビア植物体。
- 変異が、配列番号1の49位に相当する位置におけるCからAへの変異を含む、請求項12に記載の植物体。
- 非遺伝子組換植物体である、請求項12又は13に記載の植物体。
- 請求項12~14のいずれか一項に記載の植物体の種子、組織、乾燥葉、組織培養物又は細胞。
- 胚、分裂組織細胞、花粉、葉、根、根端、花弁、プロトプラスト、葉の切片及びカルスから選択される、請求項15に記載の組織、組織培養物又は細胞。
- 請求項12~14のいずれか一項に記載のステビア植物体と第2のステビア植物体とを交雑させる工程を含む、甘味成分高含有型ステビア植物体を作出する方法。
- 第2の植物体が請求項12~14のいずれか一項に記載のステビア植物体である、請求項17に記載の方法。
- ステビア植物体のゲノムの配列番号1の49位に相当する位置にCからAへの変異を導入する工程を含む、甘味成分高含有型ステビア植物体を作出する方法。
- 変異の導入が、突然変異誘発処理によって行われる、請求項19に記載の方法。
- 請求項12~14のいずれか一項に記載の植物体、請求項15に記載の種子、組織、乾燥葉、組織培養物又は細胞、あるいは、請求項16に記載の組織、組織培養物又は細胞の抽出物を提供する工程、及び
前記抽出物を、飲食品、甘味組成物、香料又は医薬品の原料に添加する工程
を含む、飲食品、甘味組成物、香料又は医薬品の製造方法 。
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