WO2022220152A1 - 栄養成分リッチなステビア植物 - Google Patents
栄養成分リッチなステビア植物 Download PDFInfo
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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
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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
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
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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
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23F—COFFEE; TEA; THEIR SUBSTITUTES; MANUFACTURE, PREPARATION, OR INFUSION THEREOF
- A23F3/00—Tea; Tea substitutes; Preparations thereof
- A23F3/16—Tea extraction; Tea extracts; Treating tea extract; Making instant tea
- A23F3/163—Liquid or semi-liquid tea extract preparations, e.g. gels, liquid extracts in solid capsules
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- 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
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- 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
- A23L27/10—Natural spices, flavouring agents or condiments; Extracts thereof
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/04—Plant cells or tissues
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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
- 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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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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
- 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
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- 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
- 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
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
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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
- C12Q2600/13—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
- C12Q2600/156—Polymorphic or mutational markers
Definitions
- the present invention relates to a stevia plant whose genotype is C/C at the position corresponding to position 37 of SEQ ID NO: 1, its production method, screening method, and the like.
- Stevia is a perennial plant of the Asteraceae family native to Paraguay, South America. Stevia contains various sweetening components that are tens to hundreds of times sweeter than sugar, and these sweetening components are extracted and used as natural sweeteners (Patent Document 1). However, much is still unknown about the genetic information of Stevia and what genes are involved in the regulation of in vivo events.
- the present invention provides a stevia plant body rich in nutritional components, a method for producing the plant body, a screening method, and the like.
- the present invention provides: [1] A stevia plant whose genotype at the position corresponding to position 37 in SEQ ID NO: 1 is C/C. [2] containing at least one nutritional ingredient selected from iron, zinc, phosphorus, copper, molybdenum, amino acids, rebaudioside D and rebaudioside M with a genotype of C/T at the position corresponding to position 37 of SEQ ID NO: 1;
- the plant according to [1] which contains more FRO2 than a certain control Stevia plant and/or has a lower expression level of FRO2 than the control Stevia plant.
- the plant according to [1] or [2] which is a non-genetically modified plant.
- tissue, tissue culture or cell according to any one of [1] to [3].
- tissue, tissue culture or cells of [4] selected from seeds, embryos, meristematic cells, pollen, leaves, roots, root tips, petals, protoplasts, leaf segments and callus.
- Iron, zinc including the step of obtaining an extract from the plant body according to any one of [1] to [3], the tissue, tissue culture or cells according to [4] or [5] , phosphorus, copper, molybdenum, amino acids, rebaudioside D and rebaudioside M.
- the plant extract of any one of [1] to [3], the tissue, tissue culture or cell extract of [4] or [5], or [8] A method for producing a food, a sweetener composition, a flavor, or a pharmaceutical, comprising the step of providing the extract according to 1, and adding the extract to a raw material of the food, the sweetener composition, the flavor, or the pharmaceutical.
- any one of [1] to [3], including the step of detecting whether or not the genotype at the position corresponding to position 37 of SEQ ID NO: 1 is C/C from the test stevia plant A method for screening the stevia plant according to .
- the stevia according to any one of [1] to [3], which contains a reagent for detecting whether the genotype at the position corresponding to position 37 of SEQ ID NO: 1 is C/C.
- Plant body screening kit [16] The kit of [15], wherein the reagents include primers and/or probes used in the dCAPS method or the TaqMan PCR method.
- obtaining a stevia plant body rich in nutritional components means for producing such a plant body, leaves obtained from such a plant body, foods and beverages containing an extract obtained from this leaf, etc. can be provided.
- FIG. 1 shows the mutation site (37th position) in the base sequence of SEQ ID NO:1.
- the boxed base is the base (C or T) at the mutation site.
- FIG. 2 shows the contents of RebD and RebM in individuals of genotype C/C or C/T.
- FIG. 3 shows the expression levels of the FRO2 gene in individuals of genotype C/C or C/T.
- FIG. 4 is a graph showing the ratio of the average content of each mineral component in individuals of genotype C/C when the average content of each mineral component in individuals of genotype C/T is set to 1.
- FIG. FIG. 5 is a graph showing the magnification of the average content of each amino acid in individuals of genotype C/C when the average content of each amino acid in individuals of genotype C/T is set to 1.
- FIG. 6 is a graph showing the relationship between the total content of RebD and RebM and the FRO2 expression level.
- FIG. 7 is a graph comparing the total RebD and RebM contents in the lowest 15% FRO2 expression level and the highest 15% FRO2 expression levels.
- FIG. 8 is a graph showing the relationship between the ratio of the total content of RebD and RebM to the TSG content (RebDM/TSG) and the FRO2 expression level.
- the genotype at the position corresponding to position 37 of SEQ ID NO: 1 is C/C (that is, SEQ ID NO: 1).
- a stevia plant that is homozygous for an allele in which the base at the position corresponding to position 37 of 1 is C) (hereinafter sometimes referred to as "the plant of the present invention” or "the stevia plant of the present invention") I will provide a.
- the fact that the genotype at the position corresponding to position 37 of SEQ ID NO: 1 is C/C may be referred to as "the genetic characteristic of the present invention”.
- Stevia is a plant with the scientific name of Stevia Rebaudiana Bertoni.
- “Position corresponding to” means, if a sequence identical to the reference sequence (i.e., SEQ ID NO: 1) exists in the genome, the position in that sequence that exists in the genome (i.e., position 37); When the same sequence as the reference sequence does not exist in the genome, it means the position corresponding to the position in the reference sequence in the sequence corresponding to the reference sequence in the genome. Whether or not a sequence identical to or corresponding to the reference sequence exists in the genome is determined, for example, by amplifying the genomic DNA of the target stevia plant with primers capable of amplifying the reference sequence by PCR, and sequencing the amplified product. It can be determined by performing alignment analysis between the obtained sequence and a reference sequence.
- Non-limiting examples of sequences corresponding to the reference sequence include, for example, 60% or more, 70% or more, 75% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more of the reference sequence. , 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% or more of the sequence identity.
- the position corresponding to the position in the reference sequence in the sequence corresponding to the reference sequence in the genome can be determined in consideration of the base sequences before and after the position in the reference sequence. For example, alignment analysis of a reference sequence and a sequence corresponding to the reference sequence in the genome can determine positions in the sequence corresponding to the reference sequence in the genome that correspond to positions in the reference sequence.
- the "position corresponding to position 37 in SEQ ID NO: 1" has the same nucleotide sequence as SEQ ID NO: 1 in the genome. It is 37th from the 5' side of the part consisting of.
- the genome of a stevia plant has a portion consisting of a nucleotide sequence corresponding to SEQ ID NO: 1, although the genome is not identical to SEQ ID NO: 1, the genome does not have a portion consisting of a nucleotide sequence identical to SEQ ID NO: 1.
- the "position corresponding to position 37 of SEQ ID NO: 1" does not necessarily correspond to position 37 from the 5' side of the portion corresponding to SEQ ID NO: 1, but the base sequence before and after position 37 of SEQ ID NO: 1, etc. Taking into account, it is possible to identify "the position corresponding to position 37 of SEQ ID NO: 1" in the genome of such stevia plants. For example, the position corresponding to position 37 of SEQ ID NO: 1 in the stevia plant genome is identified by alignment analysis of the nucleotide sequence of the portion corresponding to SEQ ID NO: 1 in the genome of the stevia plant and the nucleotide sequence of SEQ ID NO: 1. can do.
- a portion consisting of a base sequence corresponding to SEQ ID NO: 1 is, for example, 60% or more, 70% or more, 75% or more, 80% or more, 81% or more, 82% or more of the base sequence of SEQ ID NO: 1, 83% or higher, 84% or higher, 85% or higher, 86% or higher, 87% or higher, 88% or higher, 89% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% 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% or more of the sequence identity means a part of
- the "portion consisting of a base sequence corresponding to SEQ ID NO: 1" includes a forward primer that hybridizes to a complementary sequence of a portion 15 to 25 bases long from the 5' end of SEQ ID NO: 1, and SEQ ID NO: A portion of the Stevia plant genome that can be amplified by PCR using a reverse primer that hybridizes to a portion 15 to 25 bases long from the 3' end of 1 is included.
- the "portion consisting of the nucleotide sequence corresponding to SEQ ID NO: 1" includes, for example, PCR using a forward primer containing the nucleotide sequence of SEQ ID NO: 2 and a reverse primer containing the nucleotide sequence of SEQ ID NO: 3 Included are portions of the stevia plant genome that can be amplified by.
- the “allele in which the base at the position corresponding to position 37 of SEQ ID NO: 1 is C” includes the base sequence of SEQ ID NO: 4, 5, 6 or 20.
- the position corresponding to position 37 of SEQ ID NO: 1 is sometimes referred to as "the polymorphic site of the present invention” or “the mutation site of the present invention”.
- the mutation from T to C at the position corresponding to position 37 of SEQ ID NO: 1 is sometimes referred to as "the polymorphism of the present invention” or "the mutation of the present invention”.
- FIG. 1 shows position 37 of SEQ ID NO:1.
- the above genetic characteristics are based on the PCR method, TaqMan PCR method, sequencing method, microarray method, Invader method, TILLING method, RAD (random amplified polymorphic DNA) method, restriction fragment length polymorphism (RFLP) method, and PCR-SSCP method.
- AFLP amplified fragment length polymorphism
- SSLP simple sequence length polymorphism
- CAPS cleaved amplified polymorphic sequence
- dCAPS derived cleaved amplified polymorphic sequence
- ASO allele-specific oligonucleotide
- ARMS denaturant concentration gradient gel electrophoresis
- CCM chemical cleavage of mismatch
- DOL DOL method
- MALDI-TOF/MS method MALDI-TOF/MS method
- TDI method padlock probe method
- molecular beacon method DASH (dynamic allele specific hybridization) method
- UCAN method ECA method
- PINPOINT method PROBE (primer oligo base extension) method
- VSET very short extension
- the genetic features of the present invention can be detected with the following primer sets and restriction enzyme combinations.
- a forward primer having the nucleotide sequence shown in SEQ ID NO: 8 and a reverse primer having the nucleotide sequence shown in SEQ ID NO: 9 are used for the genomic DNA of the candidate plant. Even if the PCR product (approximately 220 bp long, eg, SEQ ID NO: 9) is treated with the restriction enzyme AflIII, only a band of approximately 220 bp long (eg, SEQ ID NO: 9) is obtained.
- PCR amplification for example, a PCR product of SEQ ID NO: 10 (about 220 bp long) is obtained, and the restriction enzyme AflIII is used to obtain a restriction enzyme digestion product of about 34 bp (eg, SEQ ID NO: 11) and a restriction enzyme digestion product of about 186 bp. (eg, SEQ ID NO: 12), the candidate plant does not have the genetic characteristics of the present invention.
- "about” means ⁇ 5 bp. Restriction enzyme treatment can be performed according to the conditions recommended by the vendor of each restriction enzyme used.
- the plant body of the present invention contains minerals (e.g., iron, zinc, phosphorus, copper, molybdenum, etc.), amino acids (e.g., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, etc.), rebaudioside D and rebaudioside M corresponding to position 37 of SEQ ID NO: 1 It contains more than the control stevia plant whose genotype at the locus is C/T (hereinafter sometimes referred to as "the chemical feature A of the present invention").
- the chemical feature A of the present invention contains more than the control stevia plant whose genotype at the locus is C/T (hereinafter sometimes referred to as "the chemical feature A of the present
- Consing more than a control stevia plant means, for example, that when cultivated under the same cultivation conditions, the content of the above component (eg, the content in dry leaves) is higher than that of a control stevia plant. More specifically, for example, when cultivated under the same cultivation conditions, the content of the above components in dry leaves is more than 1, 1.01 times or more, 1.02 times or more, 1.03 times or more, 1.04 times the content in the control stevia plant.
- the content to be compared may be the average value of multiple individuals.
- Dried leaves refer to fresh leaves of the stevia plant of the present invention that have been dried to reduce the water content to 3 to 4% by weight.
- rebaudioside may be abbreviated as “Reb.” or “Reb.”
- Rebaudioside D is sometimes abbreviated as “Reb.D” or “RebD.”
- the content of minerals in dry leaves is higher than that in the control stevia plant (hereinafter referred to as “the chemical feature A1-a of the present invention ”).
- the mineral content in the dried leaves is 1.5 times or more, 1.6 times or more, 1.7 times or more, 1.8 times or more, or 1.9 times the content in the control stevia plant.
- the content of amino acids (free amino acids) in dry leaves is higher than the content in the control stevia plant (hereinafter referred to as “the chemical composition of the present invention. (sometimes referred to as “feature A1-b”).
- the content of amino acids in dry leaves is more than 1.0 times, 1.01 times or more, 1.02 times or more, 1.03 times or more, 1.04 times the content in the control stevia plant.
- the content of iron in the dried leaves is higher than that in the control stevia plant (hereinafter referred to as "chemical feature A1-1 of the present invention”). sometimes called).
- the iron content in the dried leaves is 1.5 times or more, 1.6 times or more, 1.7 times or more, 1.8 times or more, or 1.9 times that of the control stevia plant. 2.0 times or more, 2.1 times or more, 2.2 times or more, 2.3 times or more, 2.4 times or more, or 2.5 times or more.
- the content of zinc in dry leaves is higher than that in the control stevia plant (hereinafter referred to as "chemical feature A1-2 of the present invention”). sometimes called).
- the zinc content in the dry leaves is more than 1.0 times, 1.01 times or more, 1.02 times or more, 1.03 times or more, 1.04 times the content in the control stevia plant.
- the content of phosphorus (inorganic phosphorus) in the dry leaves is higher than that in the control stevia plant (hereinafter referred to as "the chemical characteristics of the present invention A1-3").
- the phosphorus content in the dry leaves is 1.5 times or more, 1.6 times or more, 1.7 times or more, 1.8 times or more, or 1.9 times that of the control stevia plant. 2.0 times or more, 2.1 times or more, 2.2 times or more, 2.3 times or more, 2.4 times or more, 2.5 times or more, or 2.6 times or more.
- the content of copper in dry leaves is higher than that in the control stevia plant (hereinafter referred to as "Chemical Characteristics A1-4 of the Present Invention"). sometimes called).
- the copper content in the dry leaves is 3.0 times or more, 3.1 times or more, 3.2 times or more, 3.3 times or more, or 3.4 times that of the control stevia plant.
- the content of molybdenum in dry leaves is higher than that in the control stevia plant (hereinafter referred to as “Chemical Characteristics A1-5 of the Present Invention”). sometimes called).
- the molybdenum content in the dry leaves is 3.0 times or more, 3.1 times or more, 3.2 times or more, 3.3 times or more, or 3.4 times that of the control stevia plant.
- the content of alanine in dry leaves is higher than that in the control stevia plant (hereinafter referred to as “chemical feature A1-6 of the present invention”). sometimes called).
- the alanine content in the dry leaves is more than 1.0 times, 1.01 times or more, 1.02 times or more, 1.03 times or more, 1.04 times the content in the control stevia plant.
- the content of arginine in dry leaves is higher than that in the control stevia plant (hereinafter referred to as “Chemical Characteristics A1-7 of the Present Invention”). sometimes called).
- the content of arginine in dry leaves is 2.5 times or more, 2.6 times or more, 2.7 times or more, 2.8 times or more, or 2.9 times that of the control stevia plant.
- the content of asparagine in the dried leaves is higher than that in the control stevia plant (hereinafter referred to as "chemical feature A1-8 of the present invention”). sometimes called).
- the content of asparagine in the dry leaves is 2.0 times or more, 2.1 times or more, 2.2 times or more, 2.3 times or more, or 2.4 times that of the control stevia plant.
- the content of aspartic acid in dry leaves is higher than that in the control stevia plant (hereinafter referred to as "Chemical Characteristics A1-9 of the Present Invention").
- the content of aspartic acid in the dried leaves is 1.5 times or more, 1.6 times or more, 1.7 times or more, 1.8 times or more, or 1.9 times the content in the control stevia plant.
- the content of glutamic acid in dry leaves is higher than that in the control stevia plant (hereinafter referred to as "Chemical Characteristics A1-10 of the Present Invention"). sometimes called).
- the content of glutamic acid in dry leaves is more than 1.0 times, 1.01 times or more, 1.02 times or more, 1.03 times or more, 1.04 times the content in the control stevia plant.
- the content of glycine in dry leaves is higher than that in the control stevia plant (hereinafter referred to as "Chemical Characteristics A1-11 of the Present Invention"). sometimes called).
- the content of glycine in dry leaves is 1.0 times or more, 1.01 times or more, 1.02 times or more, 1.03 times or more, or 1.04 times that of the control stevia plant.
- the content of histidine in dry leaves is higher than that in the control stevia plant (hereinafter referred to as “Chemical Characteristics A1-12 of the Present Invention”). sometimes called).
- the content of histidine in dry leaves is more than 1.0 times, 1.01 times or more, 1.02 times or more, 1.03 times or more, 1.04 times the content in the control stevia plant.
- the content of isoleucine in dry leaves is higher than the content in the control stevia plant (hereinafter referred to as “chemical characteristics A1-13 of the present invention”). sometimes called).
- the content of isoleucine in dry leaves is more than 1.0 times, 1.01 times or more, 1.02 times or more, 1.03 times or more, or 1.04 times the content in the control stevia plant.
- the content of leucine in dry leaves is higher than that in the control stevia plant (hereinafter referred to as "Chemical Characteristics A1-14 of the Present Invention"). sometimes called).
- the leucine content in the dry leaves is more than 1.0 times, 1.01 times or more, 1.02 times or more, 1.03 times or more, 1.04 times or more than the content in the control stevia plant.
- the content of lysine in dry leaves is higher than that in the control stevia plant (hereinafter referred to as "Chemical Characteristics A1-15 of the Present Invention"). sometimes called).
- the content of lysine in dry leaves is more than 1.0 times, 1.01 times or more, 1.02 times or more, 1.03 times or more, 1.04 times the content in the control stevia plant.
- the content of phenylalanine in dry leaves is higher than that in the control stevia plant (hereinafter referred to as “Chemical Characteristics A1-16 of the Present Invention”). sometimes called).
- the content of phenylalanine in dry leaves is more than 1.0 times, 1.01 times or more, 1.02 times or more, 1.03 times or more, 1.04 times the content in the control stevia plant.
- the content of proline in the dry leaves is higher than that in the control stevia plant (hereinafter referred to as “chemical feature A1-17 of the present invention”). sometimes called).
- the content of proline in dry leaves is more than 1.0 times, 1.01 times or more, 1.02 times or more, 1.03 times or more, or 1.04 times that of the control stevia plant.
- the content of serine in dry leaves is higher than that in the control stevia plant (hereinafter referred to as “Chemical Characteristics A1-18 of the Present Invention”). sometimes called).
- the content of serine in dry leaves is 2.0 times or more, 2.1 times or more, 2.2 times or more, 2.3 times or more, or 2.4 times that of the control stevia plant.
- the content of threonine in dry leaves is higher than that in the control stevia plant (hereinafter referred to as "Chemical Characteristics A1-19 of the Present Invention"). sometimes called).
- the threonine content in the dry leaves is more than 1.0 times, 1.01 times or more, 1.02 times or more, 1.03 times or more, 1.04 times the content in the control stevia plant.
- the content of tyrosine in dry leaves is higher than that in the control stevia plant (hereinafter referred to as "Chemical Characteristics A1-20 of the Present Invention"). sometimes called).
- the content of tyrosine in dry leaves is more than 1.0 times, 1.01 times or more, 1.02 times or more, 1.03 times or more, 1.04 times the content in the control stevia plant.
- the content of valine in dry leaves is higher than that in the control stevia plant (hereinafter referred to as "chemical feature A1-21 of the present invention”). sometimes called).
- the content of valine in the dry leaves is more than 1.0 times, 1.01 times or more, 1.02 times or more, 1.03 times or more, 1.04 times the content in the control stevia plant.
- the content of RebD in dry leaves is higher than that in the control stevia plant (hereinafter referred to as “chemical feature A1-22 of the present invention”). sometimes called).
- the content of RebD in dry leaves is 2.0 times or more, 2.1 times or more, 2.2 times or more, 2.3 times or more, or 2.4 times that of the control stevia plant.
- the content of RebM in dry leaves is higher than that in the control stevia plant (hereinafter referred to as “chemical feature A1-23 of the present invention”). sometimes called).
- the content of RebM in dry leaves is 1.2 times or more, 1.3 times or more, 1.4 times or more, 1.5 times or more, or 1.6 times that of the control stevia plant.
- 1.8 times or more 1.9 times or more, 2.0 times or more, 2.1 times or more, 2.2 times or more, 2.3 times or more, 2.4 times or more, 2.5 times or more, 2.6 times or more, 2.7 times or more, 2.8 times or more, 2.9 times or more, 3.0 times or more, 5.0 times or more, 7.0 times or more, 9.0 times or more, 11 times or more, 13 times or more, 15 times or more, 17 times or more, 19 times or more, 21 times or more, 23 times or more, 25 times 27 times or more, 29 times or more, 31 times or more, 33 times or more, 35 times or more, 37 times or more, 39 times or more, 41 times or more, 43 times or more, 45 times or more, 47 times or more, 49 times or more, It may be 51 times or more, or 53 times or more.
- the total content of RebD and RebM in dry leaves is higher than the content in the control stevia plant (hereinafter referred to as "chemical feature A1 of the present invention -24").
- the total content of RebD and RebM in the dry leaves is 1.8 times or more, 1.9 times or more, 2.0 times or more, 2.1 times or more than the content in the control stevia plant.
- the plant of the present invention has a weight ratio (RebD/TSG) of RebD to total steviol glycoside (TSG) in dry leaves of the control stevia plant when cultivated under the same cultivation conditions. larger than the weight ratio in the body (hereinafter sometimes referred to as "the chemical feature A1-25 of the present invention").
- the RebD/TSG in the dry leaves is 2.5 times or more, 2.6 times or more, 2.7 times or more, 2.8 times or more that of the control stevia plant.
- TSG is a generic term for measurable steviol glycosides, and does not include unknown steviol glycosides or steviol glycosides present in amounts below the detection limit.
- TSG is RebA, RebB, RebC, RebD, RebE, RebF, RebG, RebI, RebJ, RebK, RebM, RebN, RebO, RebQ, RebR, Dulcoside A, Rubusoside, Steviolmonoside, Steviolbioside and Stevioside Any combination of two or more selected from the group consisting of In certain embodiments, the TSG consists of RebA, RebB, RebC, RebD, RebE, RebF, RebG, RebM, RebN and stevioside.
- the weight ratio of RebM to TSG (RebM/TSG) in dry leaves is greater than that of a control stevia plant (hereinafter referred to as "this Chemical features of the invention A1-26”).
- this Chemical features of the invention A1-26 when the plant of the present invention is cultivated under the same cultivation conditions, the RebM/TSG in the dry leaves is 1.5 times or more, 1.6 times or more, 1.7 times or more, 1.8 times or more that of the control stevia plant.
- the plant of the present invention has a weight ratio of total RebD and RebM to TSG in dry leaves (RebDM/TSG) greater than the weight ratio in a control stevia plant when cultivated under the same cultivation conditions (
- the chemical feature A1-27 of the present invention” when the plant of the present invention is cultivated under the same cultivation conditions, the RebDM/TSG in dry leaves is 2.0 times or more, 2.1 times or more, 2.2 times or more, 2.3 times or more that of RebDM/TSG in the control stevia plant.
- the plant of the present invention may have two or more of the above chemical characteristics in combination.
- the plant of the present invention has the chemical features A1-a and A1-b of the present invention, at least two of the chemical features A1-1 to A1-27 (for example, chemical 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 from A1-1 to A1-27 , 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27), for example
- the mineral content in dry leaves is 2.2-4.4 times or more
- the free amino acid content is 1.1-3.8 times or more
- the total content of RebD and RebM is 2.4 compared to the control stevia plant. It may be double or more.
- the plant of the present invention has at least two or more of the following characteristics (A1-1′) to (A1-27′), for example, characteristics (A1-1′) to (A1-27 '), 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, Have 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27: (A1-1')
- the iron content in dry leaves is 2.2 times or more compared to the control stevia plant body
- the content of zinc in dry leaves is 1.7 times or more compared to the control stevia plant
- A1-3' When cultivated under the same cultivation conditions, the phosphorus content in dry leaves is 2.2 times or more compared to the control stevia plant
- the copper content in dry leaves is 4.2 times or more compared to the control stevia plant body
- A1-5' When cultivated under the same cultivation conditions, the content of molybdenum in
- the plant body of the present invention has an iron content in dry leaves of 3.0 ppb or more, 3.1 ppb or more, 3.2 ppb or more, 3.3 ppb or more, 3.4 ppb or more, 3.5 ppb or more, 3.6 ppb or more, or 3.7 ppb.
- the plant body of the present invention has a dry leaf zinc content of 55 ppb or more, 57 ppb or more, 60 ppb or more, 62 ppb or more, 65 ppb or more, 67 ppb or more, 70 ppb or more, 72 ppb or more, 75 ppb or more, 77 ppb or more, 80 ppb or more, 82 ppb or more, 85 ppb or more, 87 ppb or more, 90 ppb or more, 92 ppb or more, 95 ppb or more, 97 ppb or more, or 100 ppb or more (hereinafter sometimes referred to as "chemical feature A2-2 of the present invention”) .
- the plant body of the present invention has a phosphorus (inorganic phosphorus) content in dry leaves of 13000 ppb or more, 13500 ppb or more, 14000 ppb or more, 14500 ppb or more, 15000 ppb or more, 15500 ppb or more, 16000 ppb or more, 16500 ppb or more, 17000 ppb or more.
- the plant body of the present invention has a dry leaf copper content of 35 ppb or more, 40 ppb or more, 45 ppb or more, 50 ppb or more, 55 ppb or more, 60 ppb or more, 65 ppb or more, 70 ppb or more, 75 ppb or more, 80 ppb or more, 85 ppb or higher, 90 ppb or higher, 95 ppb or higher, 100 ppb or higher, 105 ppb or higher, 110 ppb or higher, 115 ppb or higher, 120 ppb or higher, 125 ppb or higher, 130 ppb or higher, 135 ppb or higher, 140 ppb or higher, 145 ppb or higher, or 150 ppb or higher (hereinafter referred to as "this Chemical features of the invention A2-4”).
- the plant body of the present invention has a molybdenum content in dry leaves of 0.30 ppb or more, 0.35 ppb or more, 0.40 ppb or more, 0.45 ppb or more, 0.50 ppb or more, 0.55 ppb or more, 0.60 ppb or more, 0.65 ppb 0.70ppb or more, 0.75ppb or more, 0.80ppb or more, 0.85ppb or more, 0.90ppb or more, 0.95ppb or more, 1.00ppb or more, 1.05ppb or more, 1.10ppb or more, 1.15ppb or more, 1.20ppb or more, 1.25ppb or more, 1.30ppb or more, 1.35ppb or more, 1.40ppb or more, 1.45ppb or more, 1.50ppb or more, 1.55ppb or more, 1.60ppb or more, 1.65ppb or more, 1.70ppb or more, 1.75ppb or more, 1.80ppb or more, 1.85ppb or more,
- the plant body of the present invention has an alanine content in dry leaves of 21.0 ppm or more, 21.3 ppm or more, 21.5 ppm or more, 21.8 ppm or more, 22.0 ppm or more, 22.3 ppm or more, 22.5 ppm or more, 22.8 ppm 23.0 ppm or more, 23.3 ppm or more, 23.5 ppm or more, 23.8 ppm or more, 24.0 ppm or more, 24.3 ppm or more, 24.5 ppm or more, 24.8 ppm or more, 25.0 ppm or more, 25.3 ppm or more, 25.5 ppm or more, 25.8 ppm or more, 26.0ppm or more, 26.3ppm or more, 26.5ppm or more, 26.8ppm or more, 27.0ppm or more, 27.3ppm or more, 27.5ppm or more, 27.8ppm or more, 28.0ppm or more, 28.3ppm or more
- the plant body of the present invention has an arginine content in dry leaves of 1.0 ppm or more, 1.1 ppm or more, 1.2 ppm or more, 1.3 ppm or more, 1.4 ppm or more, 1.5 ppm or more, 1.6 ppm or more, 1.7 ppm or more.
- 1.8ppm or more 1.9ppm or more, 2.0ppm or more, 2.1ppm or more, 2.2ppm or more, 2.3ppm or more, 2.4ppm or more, 2.5ppm or more, 2.6ppm or more, 2.7ppm or more, 2.8ppm or more, 2.9ppm or more, 3.0 ppm or more, 3.1 ppm or more, 3.2 ppm or more, 3.3 ppm or more, 3.4 ppm or more, 3.5 ppm or more, 3.6 ppm or more, 3.7 ppm or more, 3.8 ppm or more, 3.9 ppm or more, 4.0 ppm or more, 4.1 ppm or more, 4.2 ppm Above, it may be 4.3 ppm or more, 4.4 ppm or more, or 4.5 ppm or more (hereinafter sometimes referred to as "chemical feature A2-7 of the present invention").
- the plant body of the present invention has an asparagine content in dry leaves of 2.5 ppm or more, 2.8 ppm or more, 3.0 ppm or more, 3.3 ppm or more, 3.5 ppm or more, 3.8 ppm or more, 4.0 ppm or more, 4.3 ppm.
- the plant body of the present invention has an aspartic acid content in dry leaves of 0.5 ppm or more, 0.6 ppm or more, 0.7 ppm or more, 0.8 ppm or more, 0.9 ppm or more, 1.0 ppm or more, 1.1 ppm or more, 1.2 ppm or more. It may be ppm or more, 1.3 ppm or more, 1.4 ppm or more, or 1.5 ppm or more (hereinafter sometimes referred to as "chemical feature A2-9 of the present invention").
- the plant body of the present invention has a dry leaf glutamic acid content of 1.5 ppm or more, 1.6 ppm or more, 1.7 ppm or more, 1.8 ppm or more, 1.9 ppm or more, 2.0 ppm or more, 2.1 ppm or more, 2.2 ppm. 2.3 ppm or more, 2.4 ppm or more, 2.5 ppm or more, 2.6 ppm or more, 2.7 ppm or more, 2.8 ppm or more, 2.9 ppm or more, or 3.0 ppm or more (hereinafter, "Chemical Features A2-10 of the Present Invention ”).
- the dry leaves of the plant of the present invention have a glycine content of 0.4 ppm or more, 0.5 ppm or more, 0.6 ppm or more, 0.7 ppm or more, 0.8 ppm or more, 0.9 ppm or more, or 1.0 ppm or more. good (hereinafter sometimes referred to as "chemical features A2-11 of the present invention").
- the dry leaves of the plant of the present invention have a histidine content of 0.9 ppm or more, 1.0 ppm or more, 1.1 ppm or more, 1.2 ppm or more, 1.3 ppm or more, 1.4 ppm or more, or 1.5 ppm or more. good (hereinafter sometimes referred to as "chemical features A2-12 of the present invention").
- the plant body of the present invention has an isoleucine content in dry leaves of 3.1 ppm or more, 3.2 ppm or more, 3.3 ppm or more, 3.4 ppm or more, 3.5 ppm or more, 3.6 ppm or more, 3.7 ppm or more, or 3.8 ppm.
- the plant body of the present invention has a dry leaf leucine content of 7.6 ppm or more, 7.7 ppm or more, 7.8 ppm or more, 7.9 ppm or more, 8.0 ppm or more, 8.1 ppm or more, 8.2 ppm or more, 8.3 ppm 8.4ppm or more, 8.5ppm or more, 8.6ppm or more, 8.7ppm or more, 8.8ppm or more, 8.9ppm or more, 9.0ppm or more, 9.1ppm or more, 9.2ppm or more, 9.3ppm or more, 9.4ppm or more, 9.5ppm or more, 9.6ppm or more, 9.7ppm or more, 9.8ppm or more, 9.9ppm or more, 10.0ppm or more, 10.1ppm or more, 10.2ppm or more, 10.3ppm or more, 10.4ppm or more, 10.5ppm or more, 10.6ppm or more, 10.7ppm or more, 10.8ppm or more, 10.
- the plant of the present invention has a dry leaf lysine content of 2.9 ppm or more, 3.0 ppm or more, 3.1 ppm or more, 3.2 ppm or more, 3.3 ppm or more, 3.4 ppm or more, 3.5 ppm or more, 3.6 ppm or more.
- the plant body of the present invention has a dry leaf phenylalanine content of 11.4 ppm or more, 11.6 ppm or more, 11.8 ppm or more, 12.0 ppm or more, 12.2 ppm or more, 12.4 ppm or more, 12.6 ppm or more, 12.8 ppm or more.
- the plant of the present invention has a dry leaf proline content of 2.8 ppm or more, 2.9 ppm or more, 3.0 ppm or more, 3.1 ppm or more, 3.2 ppm or more, 3.3 ppm or more, 3.4 ppm or more, or 3.5 ppm.
- the plant body of the present invention has a serine content in dry leaves of 27 ppm or more, 30 ppm or more, 32 ppm or more, 35 ppm or more, 37 ppm or more, 40 ppm or more, 42 ppm or more, 45 ppm or more, 47 ppm or more, 50 ppm or more, 52 ppm or more, 55 ppm or more, 57 ppm or more, 60 ppm or more, 62 ppm or more, 65 ppm or more, 67 ppm or more, 70 ppm or more, 72 ppm or more, 75 ppm or more, 77 ppm or more, 80 ppm or more, 82 ppm or more, 85 ppm or more, 87 ppm or more, 90 ppm or more, 92 ppm or more , 95ppm or more, 97ppm or more, 100ppm or more, 102ppm or more, 105ppm or more, 107
- the plant body of the present invention has a dry leaf threonine content of 3.8 ppm or more, 3.9 ppm or more, 4.0 ppm or more, 4.1 ppm or more, 4.2 ppm or more, 4.3 ppm or more, 4.4 ppm or more, 4.5 ppm or more.
- the plant of the present invention has a dry leaf tyrosine content of 2.2 ppm or more, 2.3 ppm or more, 2.4 ppm or more, 2.5 ppm or more, 2.6 ppm or more, 2.7 ppm or more, 2.8 ppm or more, 2.9 ppm or more.
- it may be 3.0 ppm or more, 3.1 ppm or more, 3.2 ppm or more, 3.3 ppm or more, 3.4 ppm or more, or 3.5 ppm or more (hereinafter sometimes referred to as "chemical characteristics A2-20 of the present invention").
- the plant body of the present invention has a valine content in dry leaves of 3.7 ppm or more, 3.8 ppm or more, 3.9 ppm or more, 4.0 ppm or more, 4.1 ppm or more, 4.2 ppm or more, 4.3 ppm or more, 4.4 ppm.
- the plant body of the present invention has a RebD content in dry leaves of 0.5% by weight or more, 0.6% by weight or more, 0.7% by weight or more, 0.8% by weight or more, 0.9% by weight or more, 1.0% by weight or more, 1.1% by weight or more, 1.2% by weight or more, 1.3% by weight or more, 1.4% by weight or more, 1.5% by weight or more, 1.6% by weight or more, 1.7% by weight or more, 1.8% by weight or more, 1.9% by weight or more, 2.0% by weight or more, 2.1% by weight or more, 2.2% by weight or more, 2.3% by weight or more, 2.4% by weight or more, 2.5% by weight or more, 2.6% by weight or more, 2.7% by weight or more, 2.8% by weight or more, 2.9% by weight or more, 3.0% by weight or more, It may be 3.1% by weight or more, 3.2% by weight or more, 3.3% by weight or more, 3.4% by weight or more, or 3.5% by weight or more (hereinafter sometimes referred to as "chemical characteristics
- the plant body of the present invention has a RebM content in dry leaves of 0.27% by weight or more, 0.30% by weight or more, 0.35% by weight or more, 0.40% by weight or more, 0.45% by weight or more, 0.50% by weight or more, 0.55% by weight or more, 0.60% by weight or more, 0.65% by weight or more, 0.70% by weight or more, 0.75% by weight or more, 0.80% by weight or more, 0.85% by weight or more, 0.90% by weight or more, 0.95% by weight or more, 1.00% by weight or more, 1.05% by weight or more, 1.10% by weight or more, 1.15% by weight or more, 1.20% by weight or more, 1.25% by weight or more, 1.30% by weight or more, 1.35% by weight or more, 1.40% by weight or more, 1.45% by weight or more, or 1.50% by weight or more (hereinafter sometimes referred to as "Chemical Features A2-23 of the present invention").
- the plant body of the present invention has a total content of RebD and RebM in dry leaves of 0.8% by weight or more, 0.9% by weight or more, 1.0% by weight or more, 1.1% by weight or more, 1.2% by weight or more, 1.3% by weight or more.
- % or more 1.4 wt% or more, 1.5 wt% or more, 1.6 wt% or more, 1.7 wt% or more, 1.8 wt% or more, 1.9 wt% or more, 2.0 wt% or more, 2.1 wt% or more, 2.2 wt% or more, 2.3 wt% or more % or more, 2.4 wt% or more, 2.5 wt% or more, 2.6 wt% or more, 2.7 wt% or more, 2.8 wt% or more, 2.9 wt% or more, 3.0 wt% or more, 3.1 wt% or more, 3.2 wt% or more, 3.3 wt% or more % or more, 3.4 wt% or more, 3.5 wt% or more, 3.6 wt% or more, 3.7 wt% or more, 3.8 wt% or more, 3.9 wt% or more, or 4.0 wt% or more (her
- the plant body of the present invention has a weight ratio of RebD to TSG in dry leaves (RebD/TSG) of 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% 11% or more, 12% or more, 13% or more, 14% or more, 15% or more, 16% or more, 17% or more, 18% or more, 19% or more, 20% or more, 21% or more, 22% or more, It may be 23% or more, 24% or more, 25% or more, 26% or more, 27% or more, 28% or more, 29% or more, or 30% or more (hereinafter referred to as "chemical feature A2-25 of the present invention” sometimes referred to as).
- the plant body of the present invention has a weight ratio of RebM to TSG in dry leaves (RebM/TSG) of 1.0% or more, 1.5% or more, 2.0% or more, 2.5% or more, 3.0% or more, 3.5%.
- the plant body of the present invention has a weight ratio of RebD and RebM to TSG in dry leaves (RebDM/TSG) of 5.5% or more, 7.0% or more, 8.5% or more, 10.0% or more, and 11.5%. 13.0% or higher, 14.5% or higher, 16.0% or higher, 17.5% or higher, 19.0% or higher, 20.5% or higher, 22.0% or higher, 23.5% or higher, 25.0% or higher, 26.5% or higher, 28.0% or higher, 29.5% or higher, 31.0% or more, 32.5% or more, 34.0% or more, 35.5% or more, 37.0% or more, 38.5% or more, or 40.0% or more (hereinafter sometimes referred to as "chemical characteristics A2-27 of the present invention" ).
- the plant of the present invention may have two or more of the above features in combination.
- the plant body of the present invention has at least two of the chemical features A2-1 to A2-27 of the present invention (e.g., two, three, chemical features A2-1 to A2-27, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 , 21, 22, 23, 24, 25, 26 or 27).
- the plant of the present invention has at least two or more of the following characteristics (A2-1′) to (A2-27′), for example characteristics (A2-1′) to (A2-27 '), 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, Have 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27:
- (A2-1') the iron content in the dried leaves is 3.0 ppb or more;
- (A2-2') the content of zinc in dry leaves is 55 ppb or more;
- A2-3') the phosphorus content in the dried leaves is 13000 ppb or more,
- A2-4' the content of copper in dry leaves is 35 ppb or more,
- A2-5') the content of molybdenum in dried leaves is 0.30 ppb or more;
- (A2-6') the content of alanine in dry leaves is 21.0 ppm or more,
- (A2-7') the content of arginine in the dry leaves is 1.0 ppm or more,
- the content of RebM in dry leaves is 0.43% by weight or more.
- A2-24' the total content of RebD and RebM in the dry leaves is 0.8% by weight or more;
- A2-25' Weight ratio of RebD to TSG in dried leaves is 18.8% or more
- A2-26' weight ratio of RebM to TSG in dry leaves is 6.6% or more
- A2-27' The total weight ratio of RebD and RebM in dry leaves to TSG is 25.4% or more.
- the mineral content can be measured by any known method such as ICP mass spectrometry, atomic absorption spectrometry, and ICP emission spectrometry, which are described in Examples below.
- the amino acid content can be measured by any known method such as HPLC described in Examples below, a pre-column derivatization method, a post-column derivatization method, and the like.
- the content of steviol glycosides such as RebD and RebM can be determined by the method described in Ohta et al., J. Appl. Glycosci., Vol. 57, No. 3, 199-209 (2010) or WO2010/038911, or can be measured by the method described in Examples. More specifically, the content of steviol glycosides can be measured, for example, by sampling fresh leaves from a stevia plant and performing LC/MS-MS.
- the plant of the present invention has a lower expression level of FRO2 than a control stevia plant whose genotype is C/T at the position corresponding to position 37 of SEQ ID NO: 1 (hereinafter referred to as "the chemical composition of the present invention. Characteristic B”).
- the FRO2 expression level is lower than that of the control stevia plant means, for example, that the expression level of FRO2 is lower than that of the control stevia plant when cultivated under the same cultivation conditions.
- the FRO2 gene expression level (e.g., FPKM value) in fresh leaves is 5.0% or more than the FRO2 gene expression level in fresh leaves of the control stevia plant, 5.2% or higher, 5.4% or higher, 5.6% or higher, 5.8% or higher, 6.0% or higher, 6.2% or higher, 6.4% or higher, 6.6% or higher, 6.8% or higher, 7.0% or higher, 7.2% or higher, 7.4% or higher, 7.6% ⁇ 7.8%, ⁇ 8.0%, ⁇ 8.2%, ⁇ 8.4%, ⁇ 8.6%, ⁇ 8.8%, ⁇ 9.0%, ⁇ 9.2%, ⁇ 9.4%, ⁇ 9.6%, ⁇ 9.8% or ⁇ 10.0% lower means that The expression level to be compared may be the median value of multiple individuals.
- the FRO2 gene expression level in fresh leaves is 6.8% as an FPKM value compared to the FRO2 gene expression level in fresh leaves of a control stevia plant. or lower.
- the expression level of FRO2 can be determined by sequencing by NGS or the like described in Examples below, or by any known method for measuring the expression level of a gene, such as a nucleic acid molecule encoding FRO2 or a unique fragment thereof.
- Various hybridization methods using hybridizing nucleic acid molecules e.g., in situ hybridization
- Northern blotting e.g., Southern blotting
- various PCR methods e.g., various PCR methods, and substances capable of specifically recognizing FRO2, such as antibodies immunoprecipitation, EIA (enzyme immunoassay) (e.g., ELISA (emzyme-linked immunosorbent assay), etc.), RIA (radio immunoassay) (e.g., IRMA (immunoradiometric assay), RAST (radioallergosorbent test), RIST (radioimmunosorbent test) etc.), western blotting, immunohistochemistry, immunocytochemistry, flow cytometry, and MRI
- the plant of the present invention contains less RebA than a control stevia plant whose genotype is C/T at the position corresponding to position 37 of SEQ ID NO: 1 (hereinafter referred to as "the chemistry of the present invention. Characteristic feature C1”).
- “Containing less than the control stevia plant” means, for example, that the content of RebA (eg, content in dry leaves) is less than that of the control stevia plant when cultivated under the same cultivation conditions. More specifically, for example, when the plant of the present invention is cultivated under the same cultivation conditions, the content of RebA in the dry leaves is 0.70 times or less, 0.68 times or less, or 0.66 times or less than the content in the control stevia plant.
- the content of RebA in dry leaves is 10.0% by weight or less, 9.6% by weight or less, 9.2% by weight or less, 8.8% by weight or less, or 8.4% by weight.
- % or less 8.0 wt% or less, 7.6 wt% or less, 7.2 wt% or less, 6.8 wt% or less, 6.4 wt% or less, 6.0 wt% or less, 5.6 wt% or less, 5.2 wt% or less, 4.8 wt% or less, 4.4 wt% or less %, 4.0% by weight or less, 3.6% by weight or less, 3.2% by weight or less, 2.8% by weight or less, 2.4% by weight or less, or 2.0% by weight or less (hereinafter referred to as "chemical feature C2 of the present invention" Sometimes).
- the plant body of the present invention may have two or more of the above chemical characteristics in combination.
- the plant of the invention may have two or three of the chemical features A to C of the invention.
- the plant of the present invention has at least two of the chemical features A1-1 to A1-27, A2-1 to A2-27, B, C1 and C2 of the present invention, for example 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 of these chemical features, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 , 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 50, 51, 52, 53, 54, 55, 56, 57, 58 or 59.
- the plant body of the present invention includes not only the whole plant body, but also plant organs (eg, leaves, petals, stems, roots, seeds, etc.), plant tissues (eg, epidermis, phloem, parenchyma, xylem, vascular bundles, and palisade). tissue, spongy tissue, etc.) or various forms of plant cells (eg, suspension cultured cells), protoplasts, leaf segments, callus, and the like.
- the leaves may also be dried leaves as previously described.
- the plant body of the present invention may also include tissue cultures or plant cultured cells. This is because plants can be regenerated by culturing such tissue cultures or plant cultured cells.
- tissue cultures or plant cultured cells of the present invention include embryos, meristematic cells, pollen, leaves, roots, root tips, petals, protoplasts, leaf explants and callus. It is not limited.
- a method for producing the plant body of the present invention comprises a step of crossbreeding the Stevia plant body of the present invention with a second Stevia plant body, which comprises iron, zinc, phosphorus, copper, molybdenum, amino acids, Containing at least one component selected from RebD and RebM more than a control stevia plant whose genotype at the position corresponding to position 37 in SEQ ID NO: 1 is C/T, and/or the expression level of FRO2 is lower than that of the control stevia plant (hereinafter sometimes referred to as "the production method of the present invention").
- a stevia plant produced by the method may have the same genetic characteristics as the plant of the present invention.
- the range of magnification for the expression level in the control stevia plant and the like are as described above for the plant of the present invention.
- to cross means that the plant body of the present invention (first generation (S1)) and the second plant body (S1) are crossed to obtain a child plant (the plant body of the present invention). It means to obtain a plant body (second generation (S2)) produced by the production method of. Backcrossing is preferable as a crossing method.
- the plant body (S2) born between the plant body and the plant body of the present invention (that is, the plant body having the genetic characteristics of the present invention) (S1) is further crossed to obtain the genetic characteristics of the present invention.
- the second plant (S1) used in the production method of the present invention has the same genetic characteristics as the plant of the present invention, it is substantially backcrossed becomes.
- the plant body of the present invention can be produced by self-fertilization. Selfing can be carried out by allowing the pollen of the stamens of the plant of the present invention to self-pollinate the pistil of the plant of the present invention.
- the plant produced by the production method of the present invention has the same genetic characteristics as the plant of the present invention, the plant produced by the production method of the present invention is further crossed with a third stevia plant. It is also possible to produce a stevia plant having a phenotype equivalent to that of the plant of the present invention.
- the plant body of the present invention can be produced by regenerating the plant body by culturing the tissue culture or plant culture cells described above.
- the culture conditions are the same as those for culturing wild-type stevia plant tissue cultures or plant cultured cells, and are known (Protocols for In Vitro cultures and secondary metabolite analysis of aromatic and medicinal plants, Methods in molecular biology, vo. 1391, pp113-123).
- the plant of the present invention can be produced by introducing the mutation of the present invention into the genome of a stevia plant.
- Mutation may be introduced by a genetic recombination technique or a non-genetic recombination technique.
- non-genetic recombination techniques include methods of inducing mutations in genes of host cells (or host plants) without introducing foreign genes.
- Such a method includes a method involving the action of a plant cell mutagen.
- mutagens include ethyl methanesulfonate (EMS) and sodium azide.
- EMS can treat plant cells at concentrations such as 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%.
- Treatment time is 1-48 hours, 2-36 hours, 3-30 hours, 4-28 hours, 5-26 hours, 6-24 hours.
- the treatment procedure itself is known, but it can be carried out by immersing the water-absorbing seeds that have undergone the water-absorbing process in a treatment solution containing the mutagen at the above-mentioned concentration for the above-mentioned treatment time.
- a method of irradiating plant cells with radiation or light such as X-rays, ⁇ -rays, and ultraviolet rays can also be used.
- cells, calluses, and plants having desired traits can be selected after culturing cells irradiated with an appropriate amount of UV light (ultraviolet lamp intensity, distance, and time) in a selective medium or the like. .
- the irradiation intensity at that time is 0.01 to 100Gr, 0.03 to 75Gr, 0.05 to 50Gr, 0.07 to 25Gr, 0.09 to 20Gr, 0.1 to 15Gr, 0.1 to 10Gr, 0.5 to 10Gr, 1 to 10Gr, and the irradiation distance is 1cm to 200m. , 5 cm to 100 m, 7 cm to 75 m, 9 cm to 50 m, 10 cm to 30 m, 10 cm to 20 m, 10 cm to 10 m. ⁇ 1 month, 5 minutes to 2 weeks, 10 minutes to 1 week.
- the intensity, distance, and time of irradiation vary depending on the type of radiation and the state of the object to be irradiated (cell, callus, plant body), but can be adjusted as appropriate by those skilled in the art.
- plant cells may undergo mutation during culturing, so it is preferable to return them to individual plants for more stable trait maintenance.
- a plant body obtained by subsequent genetic recombination (for example, by genome editing) using the plant body of the present invention as a host for example, a plant body obtained by genetic recombination using the plant body of the present invention as a host to further develop another trait
- Additional plants are not excluded from the scope of the present invention.
- the plant body of the present invention is obtained by a non-genetically-recombinant method, even if it is obtained by a genetically-recombinant method or its progeny (hereinafter sometimes referred to as “genetically-modified plant body”). plants or their progeny (hereinafter sometimes referred to as “non-genetically modified plants”).
- the screening method of the plant of the present invention The plant of the present invention and the plant having the same genetic characteristics as the plant of the present invention can be screened by detecting the genetic characteristics of the present invention from the tissue of the test plant. can be done.
- screening means distinguishing the plant of the present invention from other plants and selecting the plant of the present invention. Therefore, in another aspect, the present invention relates to whether or not the genotype at the position corresponding to position 37 of SEQ ID NO: 1 is C/C from a test stevia plant (for example, the existence of the genetic characteristic of the present invention). and/or absence) of the present invention (hereinafter sometimes referred to as "screening method A of the present invention").
- the presence of the genetic characteristic of the present invention is an allele having a nucleotide other than C (eg, T) at the position corresponding to position 37 of SEQ ID NO: 1 (eg, an allele containing the nucleotide sequence of SEQ ID NO: 16, 17 or 18). Determined by detecting the absence and, optionally, detecting the presence of an allele in which the base at position corresponding to position 37 of SEQ ID NO: 1 is C (e.g., an allele comprising the base sequence of SEQ ID NO: 4, 5 or 6) can do.
- C nucleotide other than C
- the absence of the genetic characteristic of the present invention is detection of the absence of an allele in which the nucleotide at position 37 of SEQ ID NO: 1 is C (e.g., an allele containing the nucleotide sequence of SEQ ID NO: 5, 6 or 7), and / Alternatively, determining by detecting the presence of an allele (e.g., an allele containing the base sequence of SEQ ID NO: 16, 17 or 18) in which the base at the position corresponding to position 37 of SEQ ID NO: 1 is other than C (e.g., T) can be done.
- an allele e.g., an allele containing the base sequence of SEQ ID NO: 16, 17 or 18
- Specific examples of the method for detecting genetic characteristics of the present invention include PCR method, TaqMan PCR method, sequencing method, microarray method, Invader method, TILLING method, RAD method, RFLP method, PCR-SSCP method, AFLP method, and SSLP method.
- the genetic characteristics of the present invention can be detected by the dCAPS method using the following primer sets and restriction enzymes.
- ⁇ Primer set The sequence of SEQ ID NO: 19 located at the 3' end and any contiguous sequence (e.g., any length of and a sequence complementary to any continuous sequence of 20 bases or more located on the 3' side of position 39 of SEQ ID NO: 9 or 10 (e.g., SEQ ID NO: 8 or 20)
- a primer set comprising a reverse primer comprising The primer sequences can be optimized as long as the above conditions are satisfied. For optimization of primer design see, eg, Sambrook and Russell et al., supra.
- each of the above primers may have a length of 15 to 50 bases, a length of 18 to 48 bases, a length of 20 to 45 bases, a length of 30 to 65 bases, and the like.
- ⁇ Restriction enzyme: AflIII the genetic characteristics of the present invention can be detected by the dCAPS method using a forward primer consisting of the sequence of SEQ ID NO:7, a reverse primer consisting of the sequence of SEQ ID NO:8, and the restriction enzyme AflIII.
- the screening method A of the present invention may further comprise a step of evaluating the content of nutritional components and/or the expression level of FRO2 in the test stevia plant from which the genetic characteristics of the present invention have been detected.
- the content of nutritional components and the expression level of FRO2 were evaluated as described in the section on plants of the present invention.
- individuals with high nutrient content and/or low FRO2 expression levels are selected and used as other Stevia plants.
- the screening method A of the present invention may be applied to the seed plant obtained by crossing with the plant. Therefore, the screening method A of the present invention can include one or more of the following steps.
- Individuals with a high content of nutritional components to be selected are, for example, the top 50%, the top 40%, and the top 30 in terms of the high content of nutritional components among the test stevia plants in which the genetic characteristics of the present invention have been detected. %, top 25%, top 20%, top 15%, top 10%, top 5%, top 4%, top 3%, top 2%, or top 1% individuals, etc. can be Individuals with low FRO2 expression levels to be selected are, for example, the top 50%, the top 40%, the top 30 in terms of low FRO2 expression levels among the test stevia plants in which the genetic characteristics of the present invention have been detected.
- steps (iv)-(vii) can be repeated multiple times. In this way, Stevia plants with higher nutrient content and/or lower FRO2 expression levels can be screened.
- the stevia plant to be tested may be a natural plant, a non-genetically modified plant, or a genetically modified plant.
- the non-genetically modified plant is as described in the section on the plant production method of the present invention.
- the test stevia plant may include a mutagenized stevia plant and its progeny.
- the mutagenesis treatment is as described in the section on plants of the present invention, and includes treatment with a mutagenizing agent, treatment with radiation or light irradiation, and the like.
- the present invention also provides the primer set described above, for example, a primer set comprising a forward primer containing the nucleotide sequence of SEQ ID NO: 7 or 19 and a reverse primer containing the nucleotide sequence of SEQ ID NO: 8 or 20. .
- the present invention further provides a primer set capable of amplifying a region having the base sequence of SEQ ID NO: 1 by PCR, for example, a forward primer containing the base sequence of SEQ ID NO: 2 and a reverse primer containing the base sequence of SEQ ID NO: 3.
- a primer set capable of amplifying a region having the base sequence of SEQ ID NO: 1 by PCR, for example, a forward primer containing the base sequence of SEQ ID NO: 2 and a reverse primer containing the base sequence of SEQ ID NO: 3.
- probes of the present invention provides probes capable of detecting the presence and/or absence of the genetic characteristics of the present invention (hereinafter sometimes referred to as "probes of the present invention").
- Probes of the invention can have structures suitable for a variety of detection methods for the presence and/or absence of genetic features of the invention.
- probes of the present invention can comprise nucleotide sequences complementary to portions of the genome containing mutation sites of the present invention.
- Non-limiting examples of such probes include those comprising nucleotide sequences selected from SEQ ID NOS: 4-6, 16-18.
- SEQ ID NOS: 4-6 are specific for alleles containing mutations of the invention
- SEQ ID NOS: 16-18 are specific for alleles that do not contain mutations of the invention.
- the presence of the genetic characteristic of the invention can be detected by detection of alleles containing the mutation of the invention and/or non-detection of alleles not containing the mutation of the invention, and the absence of the genetic characteristic of the invention is It can be detected by non-detection of alleles containing the mutation of the present invention and/or detection of alleles not containing the mutation of the present invention.
- Probes of the invention preferably have a label.
- the probe of the present invention has a base sequence complementary to a base sequence selected from SEQ ID NOS: 4-6, 16-18, and a label.
- the present invention further provides the sequence of SEQ ID NO: 19 located at the 3′ end and any contiguous sequence continuing 5′ from position 304 of SEQ ID NO: 13 optionally added to the 5′ end of said sequence (e.g. , a continuous sequence of any length) and a sequence complementary to any continuous sequence of 20 bases or more located 3′ to position 39 of SEQ ID NO: 9 or 10 (e.g., sequence No. 8 or 20), for example, a primer set containing a forward primer consisting of the sequence of SEQ ID NO: 7 and a reverse primer consisting of the sequence of SEQ ID NO: 8, and the restriction enzyme AflIII Kits containing, eg, screening kits are provided.
- sequence of SEQ ID NO: 19 located at the 3′ end and any contiguous sequence continuing 5′ from position 304 of SEQ ID NO: 13 optionally added to the 5′ end of said sequence (e.g. , a continuous sequence of any length) and a sequence complementary to any continuous sequence of 20 bases or more located 3′ to position 39
- the present invention also provides a screening kit comprising a primer set capable of amplifying a region containing a nucleotide sequence selected from the group consisting of SEQ ID NOS: 4-6, 16-18 by PCR, and the probe of the present invention. .
- primer sets, probes and kits can be used to detect the genetic characteristics of the present invention and can be used in the screening method A of the present invention.
- these primer sets and kits contain instructions including descriptions of the detection of the genetic characteristics of the present invention and the screening method A of the present invention, e.g. For example, URL, two-dimensional code), media recording information on usage, such as flexible discs, CDs, DVDs, Blu-ray discs, memory cards, USB memories, etc., may be included.
- the present invention detects whether the genotype at position corresponding to position 37 of SEQ ID NO: 1 is C/C (e.g., the presence and/or absence of the genetic characteristic of the present invention).
- a screening kit for stevia plants of the present invention is provided, which contains reagents for screening. Reagents may include primers and/or probes for use in CAPS, dCAPS or TaqMan PCR methods.
- reagents for detecting the presence and/or absence of the genetic features of the present invention are combinations of primer sets and restriction enzymes for detecting the genetic features of the present invention by dCAPS methods, e.g. A combination of a primer set containing a forward primer consisting of the sequence of No.
- a primer set for amplifying a characteristic site eg, a site containing a sequence selected from SEQ ID NOs: 1, 4 to 6, 16 to 18
- a genetic characteristic site of the present invention eg, SEQ ID NO: 4 to 6, a site containing a sequence selected from 16 to 18
- a method for producing a plant-derived extract and a product using the extract minerals (e.g., iron, zinc, phosphorus, copper, molybdenum, etc.), amino acids (e.g., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine,
- a method for producing an extract containing at least one nutritional component selected from methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, etc.), RebD and RebM (hereinafter referred to as “method for producing the extract of the present invention ”) is provided.
- a method for producing a purified nutritional component comprising a step of purifying at least one of the above nutritional components from the extract obtained by the method for producing an extract of the present invention. (sometimes referred to as “manufacturing methods") are provided. Specifically, a step of obtaining an extract containing the nutritional components from the Stevia plant of the present invention, the Stevia plant selected by the screening method A of the present invention, or the Stevia plant produced by the method of the present invention, and A method for producing a purified nutritional component is provided, comprising the step of purifying the nutritional component from the obtained extract.
- the extract containing the above nutrients can be obtained by reacting fresh or dried leaves of the plant of the present invention with an appropriate solvent (for example, an aqueous solvent such as water or an organic solvent such as alcohol, ether, and acetone).
- an appropriate solvent for example, an aqueous solvent such as water or an organic solvent such as alcohol, ether, and acetone.
- the method described in Ohta et al. or WO2010/038911 can be referred to.
- the extract containing the above-mentioned nutritional components was subjected to a gradient of ethyl acetate or other organic solvent:water, HPLC, gas chromatography, TOF-MS, UPLC, and other known methods to obtain individual nutritional components, such as RebD and RebM can be purified.
- the genotype at the position corresponding to position 37 in SEQ ID NO: 1 is C/T. It contains a higher content of at least one of the above nutrients than the control stevia plant.
- the higher content compared to the control stevia plant is as described in the plant of the present invention.
- the extract of the present invention thus obtained and/or the refined nutritional product obtained by the method for producing the refined nutritional product of the present invention (e.g., minerals (e.g., iron, zinc, phosphorus, copper, molybdenum, etc.) , amino acids (e.g., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, etc.), RebD and /or RebM) and other ingredients can be mixed to produce pharmaceuticals, flavors, or food and drink containing the above nutritional ingredients.
- minerals e.g., iron, zinc, phosphorus, copper, molybdenum, etc.
- amino acids e.g., alanine, arginine, asparagine, as
- the present invention provides a pharmaceutical product comprising a step of mixing the extract of the present invention and/or the purified nutritional product obtained by the method for producing the purified nutritional product of the present invention with other ingredients. , to provide a method for producing flavorings or food and drink.
- the present invention provides pharmaceuticals, fragrances, or food and drink products containing the nutritional component obtained by the production method.
- food and drink means beverages and foods. Accordingly, in certain embodiments, the present invention provides pharmaceuticals, flavors, beverages or foods, and methods of making such pharmaceuticals, flavors, beverages or foods.
- the present invention also provides herbal tea products containing the dried plant body of the present invention.
- the part of the plant contained in the herbal tea product of the present invention is not particularly limited, and may be any part such as leaves, stems or roots, but dried leaves containing more nutrients are preferred.
- the herbal tea product may be in the form of a crushed product, powder or granules of a dried plant body (for example, dried leaves), or a tea pack in which the crushed product, powder or granules are filled in a water-permeable pack.
- the present invention provides a nucleotide sequence of the Stevia plant of the present invention.
- the nucleotide sequence of the Stevia plant of the present invention comprises or consists of a nucleotide sequence selected from SEQ ID NOS: 4-6, 21 and 22.
- the present invention provides a screening method for plants with high nutrient content, which comprises the step of measuring the expression level of FRO2 (hereinafter referred to as the "screening method of the present invention”). B”).
- the screening method of the present invention it has become clear that there is a negative correlation between the expression level of FRO2 and the content of nutrient components, so that plants with high nutrient content can be screened using the expression level of FRO2 as an index. became.
- a low FRO2 expression level may mean, for example, that the FRO2 expression level is equal to or lower than the median value of the test plant population.
- the expression level of FRO2 is low means that the expression level of FRO2 is in the top 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4% of the test plant population. %, 3%, 2% or 1%.
- the expression level of FRO2 is low, for example, the FPKM value of FRO2 analyzed by NGS sequencing is 3.2 or less, 3.1 or less, 3.0 or less, 2.9 or less, 2.8 or less, 2.7 or less, 2.6 or less, 2.5 ⁇ 2.4 ⁇ 2.3 ⁇ 2.2 ⁇ 2.1 ⁇ 2.0 ⁇ 1.9 ⁇ 1.8 ⁇ 1.7 ⁇ 1.6 ⁇ 1.5 ⁇ 1.4 ⁇ 1.3 ⁇ 1.2 ⁇ 1.1 ⁇ 1.0 ⁇ 0.9 It may mean less than or equal to 0.8, less than or equal to 0.7, less than or equal to 0.6 or less than or equal to 0.5.
- the expression level of FRO2 can be measured by any known method for measuring the expression level of a gene, such as sequencing by NGS, or a nucleic acid molecule that specifically hybridizes to a nucleic acid molecule encoding FRO2 or a unique fragment thereof.
- Various hybridization methods e.g., in situ hybridization
- Northern blotting e.g., Southern blotting
- various PCR methods e.g., immunoprecipitation using a substance capable of specifically recognizing FRO2 such as antibody, EIA (eg, ELISA), RIA (eg, IRMA, RAST, RIST, etc.), western blotting, immunohistochemistry, immunocytochemistry, flow cytometry, MRI, and the like.
- the genomic sequence, CDS sequence and amino acid sequence of Stevia FRO2 are shown in SEQ ID NOS: 13, 14 and 15, respectively.
- the sequences of FRO2 genes of other plants are also known (for example, Arabidopsis thaliana: NM_001331284 (nucleotide sequence: SEQ ID NO: 23, amino acid sequence: SEQ ID NO: 24), Alfalfa: XM_013589350 (nucleotide sequence: SEQ ID NO: 25, amino acid sequence: SEQ ID NO: 24) Sequence: SEQ ID NO: 26), sunflower: XM_022180093 (nucleotide sequence: SEQ ID NO: 27, amino acid sequence: SEQ ID NO: 28), cassava: XM_021755804 (nucleotide sequence: SEQ ID NO: 29, amino acid sequence: SEQ ID NO: 30)), plants other than these It is also possible to obtain the sequence of the FRO2 gene of , by homology analysis of the sequence obtained by sequencing. Any plant tissue
- Nutrients include, but are not limited to, minerals (e.g., iron, zinc, phosphorus, copper, molybdenum, etc.), amino acids (e.g., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, etc.). , isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, etc.), sweeteners such as RebD and RebM.
- minerals e.g., iron, zinc, phosphorus, copper, molybdenum, etc.
- amino acids e.g., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, etc.
- Screening method B of the present invention may further comprise a step of evaluating the content of nutritional components in test plants with low FRO2 expression levels.
- the evaluation of the content of nutritional components is as described in the section on plants of the present invention.
- an individual having a high content of nutritional components is selected and crossed with another plant to obtain a cocoon plant.
- screening method B of the present invention may be applied. Accordingly, the screening method B of the present invention may comprise one or more of the following steps.
- other plants to be crossed may or may not have a low FRO2 expression level.
- steps (iv)-(vii) can be repeated multiple times. In this way, it is possible to screen for plants with higher nutrient content.
- Example 1 Relation between RebD/RebM content and genotype C/C Based on commercially available stevia seeds, individuals were selected based on growth conditions, leaf morphology, RebA, RebD, RebM content, etc., and populations were selected. got an I. In population I, genomic DNA was extracted from fresh leaves of each individual and sequenced by NGS (HiSeq 2500, Illumina). In addition, the steviol glycoside content in fresh leaves of each individual was quantified by LC/MS-MS (Shimadzu LCMS8050). Specifically, 0.25 g of fresh leaves were dried by freeze-drying, and 0.05 g of crushed dry matter was put into pure water.
- Table 2 shows the multiplication factor of the corresponding value in genotype C/C individuals when the ratio of RebD and RebM content to the average content of steviol glycosides or TSG content is 1, and the dry leaves of each genotype individual. Distributions of RebD and RebM contents (%DW) in As shown by these results, many C/C genotypes were found in individuals with high RebD and RebM contents. For example, all individuals with a total content of RebD and RebM in dry leaves of 1.3% by weight or more have genotype C/C, and 75% of individuals with genotype C/C have RebD and RebM in dry leaves. The total content was 1.3% by weight or more.
- RebA content and TSG content tended to be lower in individuals with genotype C/C than in individuals with genotype C/T.
- the TSG content means the total content of RebA, RebB, RebC, RebD, RebE, RebF, RebG, RebM, RebN and stevioside, and RebDM means the total of RebD and RebM.
- Example 3 Relationship between mineral content and genotype C/C
- the mineral components contained in the dried leaf extract were quantified. Fresh leaves were collected from each individual and heat-dried at 65°C for about 17 hours. 1 mL of hot water (95° C. or higher) was added to 100 mg of the obtained dried leaves, and after mixing with a stirrer for 5 minutes, 10 ⁇ L of nitric acid was added per 1 mL and allowed to stand overnight. The resulting sample was subjected to ICP-MS (Agilent 7500cx, Agilent Technologies) to measure the concentration of each element (using the attached MassHunter Workstation software). Table 3 shows the average content of each mineral component in dried leaves of individuals of each genotype. The scale factors for the average content of the ingredients are shown in Table 4 and Figure 4, respectively. All mineral components were present in genotype C/C individuals higher than genotype C/T individuals.
- Example 4 Relationship between amino acid content and genotype C/C
- free amino acids contained in dried leaf extracts were quantified. Fresh leaves were collected from each individual and heat-dried at 65°C for about 17 hours. 5 mL of hot water (95° C. or higher) was added to 100 mg of the obtained dried leaves, mixed with a stirrer for 5 minutes, subjected to ultrasonic treatment for 5 minutes, centrifuged at 12000 rpm for 10 minutes, and the supernatant was collected. The resulting supernatant sample was subjected to HPLC (Chromaster 5210/5260, Hitachi High-Tech) to measure the concentration of each amino acid (Empower 3, Waters was used). Tables 5 and 6 show the average content of each amino acid in dried leaves of individuals of each genotype. The magnification of the average content of is shown in Table 7 and Figure 5, respectively. All amino acids were present in genotype C/C individuals higher than genotype C/T individuals.
- Example 5 Relation between RebD/RebM content and FRO2 expression level
- Table 8 shows the average RebD and RebM contents in the populations of the top/bottom 50%, 25%, 15% and 10% FRO2 expression levels. These results show that the population of individuals with low FRO2 expression levels had significantly lower average RebD and RebM contents than the population of individuals with high FRO2 expression levels.
- FIG. 7 shows the results of comparing the total content of RebD and RebM in the population with the lowest FRO2 expression level of 15% and the population with the highest FRO2 expression level. Furthermore, it was also revealed that there is a stronger correlation between the ratio of the total content of RebD and RebM to the TSG content (RebDM/TSG) and the FRO2 expression level (Fig. 8).
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Abstract
Description
[1] 配列番号1の37位に相当する位置の遺伝子型がC/Cである、ステビア植物体。
[2] 鉄、亜鉛、リン、銅、モリブデン、アミノ酸、レバウジオシドD及びレバウジオシドMから選択される栄養成分の少なくとも1つを、配列番号1の37位に相当する位置の遺伝子型がC/Tである対照ステビア植物体より多く含有する、及び/又は、FRO2の発現量が、前記対照ステビア植物体より低い、[1]に記載の植物体。
[3] 非遺伝子組換植物体である、[1]又は[2]に記載の植物体。
[4] [1]~[3]のいずれか一項に記載の植物体の組織、組織培養物又は細胞。
[5] 種子、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、[4]に記載の組織、組織培養物又は細胞。
[7] 第2の植物体が[1]~[3]のいずれか一項に記載の植物体である、[6]に記載の方法。
[8] 鉄、亜鉛、リン、銅、モリブデン、アミノ酸、レバウジオシドD及びレバウジオシドMから選択される栄養成分の少なくとも1つを含む、[1]~[3]のいずれか一項に記載の植物体、[4]又は[5]に記載の組織、組織培養物又は細胞の抽出物。
[9] [1]~[3]のいずれか一項に記載の植物体、[4]又は[5]に記載の組織、組織培養物又は細胞から抽出物を得る工程を含む、鉄、亜鉛、リン、銅、モリブデン、アミノ酸、レバウジオシドD及びレバウジオシドMから選択される栄養成分の少なくとも1つを含む抽出物の製造方法。
[10] [1]~[3]のいずれか一項に記載の植物体の抽出物、[4]又は[5]に記載の組織、組織培養物又は細胞の抽出物、或いは、[8]に記載の抽出物を提供する工程、及び
前記抽出物を、食品、甘味料組成物、香料又は医薬品の原料に添加する工程
を含む、食品、甘味料組成物、香料又は医薬品の製造方法。
[11] [1]~[3]のいずれか一項に記載の植物体の乾燥物を含むハーブ茶製品。
[12] 被験ステビア植物体から、配列番号1の37位に相当する位置の遺伝子型がC/Cであるか否かを検出する工程を含む、[1]~[3]のいずれか一項に記載のステビア植物体をスクリーニングする方法。
[13] 遺伝的特徴を検出する工程が、シーケンシング、dCAPS法又はTaqMan PCR法を用いて行われる、[12]に記載の方法。
[14] 被験ステビア植物組織における、鉄、亜鉛、リン、銅、モリブデン、アミノ酸、レバウジオシドD及びレバウジオシドMから選択される栄養成分の少なくとも1つの含有量を測定する工程をさらに含む、[12]又は[13]に記載の方法。
[15] 配列番号1の37位に相当する位置の遺伝子型がC/Cであるか否かを検出するための試薬を含む、[1]~[3]のいずれか一項に記載のステビア植物体のスクリーニングキット。
[16] 試薬が、dCAPS法又はTaqMan PCR法に使用するプライマー及び/又はプローブを含む、[15]に記載のキット。
[17] FRO2の発現量を測定する工程を含む、鉄、亜鉛、リン、銅、モリブデン、アミノ酸、レバウジオシドD及びレバウジオシドMから選択される栄養成分の少なくとも1つの含有量が高い植物体のスクリーニング方法。
なお、本明細書において引用した全ての文献、及び公開公報、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、2021年4月15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2021-069151号)の明細書及び図面に記載の内容を包含する。
本発明は、配列番号1の37位に相当する位置の遺伝子型がC/Cである(すなわち、配列番号1の37位に相当する位置の塩基がCであるアレルについてホモ接合性である)ステビア植物体(以下、「本発明の植物体」又は「本発明のステビア植物体」と称する場合がある)を提供する。以下、配列番号1の37位に相当する位置の遺伝子型がC/Cであることを、「本発明の遺伝的特徴」と称することがある。
ステビアは、ステビア・レバウディアナ・ベルトニー(Stevia Rebaudiana Bertoni)の学名を有する植物である。
特定の態様において、「配列番号1に相当する塩基配列からなる部分」には、例えば、配列番号2の塩基配列を含むフォワードプライマーと、配列番号3の塩基配列を含むリバースプライマーとを用いたPCRにより増幅され得るステビア植物のゲノムの部分が含まれる。
ここで、配列番号1の37位に相当する位置を、「本発明の多型部位」又は「本発明の変異部位」と称することがある。また、配列番号1の37位に相当する位置におけるTからCへの変異を、「本発明の多型」又は「本発明の変異」と称することがある。図1に、配列番号1の37位を示す。
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。
(A1-1')同じ栽培条件で栽培した場合、対照ステビア植物体に比べ、乾燥葉中の鉄の含量が2.2倍以上、
(A1-2')同じ栽培条件で栽培した場合、対照ステビア植物体に比べ、乾燥葉中の亜鉛の含量が1.7倍以上、
(A1-3')同じ栽培条件で栽培した場合、対照ステビア植物体に比べ、乾燥葉中のリンの含量が2.2倍以上、
(A1-4')同じ栽培条件で栽培した場合、対照ステビア植物体に比べ、乾燥葉中の銅の含量が4.2倍以上、
(A1-5')同じ栽培条件で栽培した場合、対照ステビア植物体に比べ、乾燥葉中のモリブデンの含量が4.4倍以上、
(A1-6')同じ栽培条件で栽培した場合、対照ステビア植物体に比べ、乾燥葉中のアラニンの含量が1.2倍以上、
(A1-7')同じ栽培条件で栽培した場合、対照ステビア植物体に比べ、乾燥葉中のアルギニンの含量が3.8倍以上、
(A1-8')同じ栽培条件で栽培した場合、対照ステビア植物体に比べ、乾燥葉中のアスパラギンの含量が3.1倍以上、
(A1-9')同じ栽培条件で栽培した場合、対照ステビア植物体に比べ、乾燥葉中のアスパラギン酸の含量が2.2倍以上、
(A1-10')同じ栽培条件で栽培した場合、対照ステビア植物体に比べ、乾燥葉中のグルタミン酸の含量が1.5倍以上、
(A1-11')同じ栽培条件で栽培した場合、対照ステビア植物体に比べ、乾燥葉中のグリシンの含量が1.5倍以上、
(A1-12')同じ栽培条件で栽培した場合、対照ステビア植物体に比べ、乾燥葉中のヒスチジンの含量が1.6倍以上、
(A1-13')同じ栽培条件で栽培した場合、対照ステビア植物体に比べ、乾燥葉中のイソロイシンの含量が1.5倍以上、
(A1-14')同じ栽培条件で栽培した場合、対照ステビア植物体に比べ、乾燥葉中のロイシンの含量が1.2倍以上、
(A1-15')同じ栽培条件で栽培した場合、対照ステビア植物体に比べ、乾燥葉中のリシンの含量が1.4倍以上、
(A1-16')同じ栽培条件で栽培した場合、対照ステビア植物体に比べ、乾燥葉中のフェニルアラニンの含量が1.0倍超、
(A1-17')同じ栽培条件で栽培した場合、対照ステビア植物体に比べ、乾燥葉中のプロリンの含量が1.8倍以上、
(A1-18')同じ栽培条件で栽培した場合、対照ステビア植物体に比べ、乾燥葉中のセリンの含量が3.7倍以上、
(A1-19')同じ栽培条件で栽培した場合、対照ステビア植物体に比べ、乾燥葉中のトレオニンの含量が1.5倍以上、
(A1-20')同じ栽培条件で栽培した場合、対照ステビア植物体に比べ、乾燥葉中のチロシンの含量が1.7倍以上、
(A1-21')同じ栽培条件で栽培した場合、対照ステビア植物体に比べ、乾燥葉中のバリンの含量が1.5倍以上、
(A1-22')同じ栽培条件で栽培した場合、対照ステビア植物体に比べ、乾燥葉中のRebDの含量が2.9倍以上、
(A1-23')同じ栽培条件で栽培した場合、対照ステビア植物体に比べ、乾燥葉中のRebMの含量が1.7倍以上、
(A1-24')同じ栽培条件で栽培した場合、対照ステビア植物体に比べ、乾燥葉中のRebDとRebMの合計含量が2.4倍以上、
(A1-25')同じ栽培条件で栽培した場合、対照ステビア植物体に比べ、乾燥葉中のTSGに対するRebDの重量比が3.8倍以上、
(A1-26')同じ栽培条件で栽培した場合、対照ステビア植物体に比べ、乾燥葉中のTSGに対するRebMの重量比が2.1倍以上、
(A1-27')同じ栽培条件で栽培した場合、対照ステビア植物体に比べ、乾燥葉中のTSGに対するRebDとRebMの合計の重量比が3.1倍以上。
(A2-1')乾燥葉中の鉄の含量が3.0ppb以上、
(A2-2')乾燥葉中の亜鉛の含量が55ppb以上、
(A2-3')乾燥葉中のリンの含量が13000ppb以上、
(A2-4')乾燥葉中の銅の含量が35ppb以上、
(A2-5')乾燥葉中のモリブデンの含量が0.30ppb以上、
(A2-6')乾燥葉中のアラニンの含量が21.0ppm以上、
(A2-7')乾燥葉中のアルギニンの含量が1.0ppm以上、
(A2-8')乾燥葉中のアスパラギンの含量が2.5ppm以上、
(A2-9')乾燥葉中のアスパラギン酸の含量が0.5ppm以上、
(A2-10')乾燥葉中のグルタミン酸の含量が1.5ppm以上、
(A2-11')乾燥葉中のグリシンの含量が0.4ppm以上、
(A2-12')乾燥葉中のヒスチジンの含量が0.9ppm以上、
(A2-13')乾燥葉中のイソロイシンの含量が3.1ppm以上、
(A2-14')乾燥葉中のロイシンの含量が7.6ppm以上、
(A2-15')乾燥葉中のリシンの含量が2.9ppm以上、
(A2-16')乾燥葉中のフェニルアラニンの含量が11.4ppm以上、
(A2-17')乾燥葉中のプロリンの含量が2.8ppm以上、
(A2-18')乾燥葉中のセリンの含量が27ppm以上、
(A2-19')乾燥葉中のトレオニンの含量が3.8ppm以上、
(A2-20')乾燥葉中のチロシンの含量が2.2ppm以上、
(A2-21')乾燥葉中のバリンの含量が3.7ppm以上、
(A2-22')乾燥葉中のRebDの含量が1.3重量%以上。
(A2-23')乾燥葉中のRebMの含量が0.43重量%以上。
(A2-24')乾燥葉中のRebDとRebMの合計含量が0.8重量%以上、
(A2-25')乾燥葉中のRebDのTSGに対する重量比が18.8%以上
(A2-26')乾燥葉中のRebMのTSGに対する重量比が6.6%以上、
(A2-27')乾燥葉中のRebDとRebMの合計のTSGに対する重量比が25.4%以上。
本発明は、別の側面において、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、鉄、亜鉛、リン、銅、モリブデン、アミノ酸、RebD及びRebMから選択される成分の少なくとも1つを、配列番号1の37位に相当する位置の遺伝子型がC/Tである対照ステビア植物体より多く含有する、及び/又は、FRO2の発現量が、前記対照ステビア植物体より低いステビア植物体を作出する方法(以下、「本発明の作出方法」と称する場合がある)を提供する。
当該方法により作出されるステビア植物体は、本発明の植物体と同じ遺伝的特徴を有し得る。
本発明の作出方法によって得られる植物体におけるミネラル、アミノ酸、RebD及びRebMの含量又はRebD及びRebMのTSGに対する重量比の範囲、対照ステビア植物における含量又は重量比に対する倍率の範囲、FRO2の発現量の対照ステビア植物における発現量に対する倍率の範囲等は、本発明の植物体について上記したとおりである。
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。
本発明の植物体を宿主として事後的に遺伝子組み換え(例えばゲノム編集等により)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。
本発明の植物体は、遺伝子組換的手法により得られたもの又はその子孫(以下、「遺伝子組換植物体」と称することがある)であっても、非遺伝子組換的手法により得られたもの又はその子孫(以下、「非遺伝子組換植物体」と称することがある)であってもよい。
本発明の植物体及び本発明の植物体と同じ遺伝的特徴を有する植物体は、被験植物体の組織から本発明の遺伝的特徴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から、配列番号1の37位に相当する位置の遺伝子型がC/Cであるか否か(例えば、本発明の遺伝的特徴の存在及び/又は不在)を検出する工程を含む、本発明の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A」と称する場合がある)を提供する。
本発明の遺伝的特徴の不在は、配列番号1の37位に相当する位置の塩基がCであるアレル(例えば、配列番号5、6又は7の塩基配列を含むアレル)の不在の検出、及び/又は、配列番号1の37位に相当する位置の塩基がC以外(例えばT)であるアレル(例えば、配列番号16、17又は18の塩基配列を含むアレル)の存在の検出により決定することができる。
・プライマーセット:
3'末端に位置する配列番号19の配列と、任意選択で前記配列の5'末端に付加される配列番号13の304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9又は10の39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8又は20)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、上記Sambrook and Russell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。
・制限酵素:AflIII
特定の態様において、本発明の遺伝的特徴は、配列番号7の配列からなるフォワードプライマー、配列番号8の配列からなるリバースプライマー、及び制限酵素AflIIIを用いたdCAPS法により検出することができる。
(i)被験ステビア植物のゲノムから、本発明の遺伝的特徴を検出する工程、
(ii)本発明の遺伝的特徴が検出された被験ステビア植物組織の栄養成分の含量及び/又はFRO2の発現量を評価する工程、
(iii)本発明の遺伝的特徴が検出された被験ステビア植物体のうち、栄養成分の含量が高い個体及び/又はFRO2の発現量が低い個体を選択する工程、
(iv)選択した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、本発明の遺伝的特徴を検出する工程、
(vi)本発明の遺伝的特徴が検出された子植物組織の栄養成分の含量及び/又はFRO2の発現量を評価する工程、
(vii)本発明の遺伝的特徴が検出された子植物体のうち、栄養成分の含量が高い個体及び/又はFRO2の発現量が低い個体を選択する工程。
本発明のスクリーニング方法A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。
本発明のさらなる態様において、本発明の植物体、又は当該植物体の種子若しくは葉(例えば、乾燥葉又は新鮮葉)から抽出物を得る工程を含む、ミネラル(例えば、鉄、亜鉛、リン、銅、モリブデンなど)、アミノ酸(例えば、アラニン、アルギニン、アスパラギン、アスパラギン酸、システイン、グルタミン、グルタミン酸、グリシン、ヒスチジン、イソロイシン、ロイシン、リシン、メチオニン、フェニルアラニン、プロリン、セリン、トレオニン、トリプトファン、チロシン、バリンなど)、RebD及びRebMから選択される栄養成分の少なくとも1つを含む抽出物の製造方法(以下、「本発明の抽出物の製造方法」と称する場合がある)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上記栄養成分の少なくとも1つを精製する工程を含む、栄養成分精製品の製造方法(以下、「本発明の栄養成分精製品の製造方法」と称する場合がある)が提供される。
具体的には、本発明のステビア植物体、本発明のスクリーニング方法Aにより選別されたステビア植物体又は本発明の方法により製造されたステビア植物体から上記栄養成分を含む抽出物を得る工程、及び、得られた抽出物から上記栄養成分を精製する工程を含む、栄養成分精製品の製造方法が提供される。
また、上記栄養成分を含む抽出物を酢酸エチルその他の有機溶媒:水の勾配、HPLC、ガスクロマトグラフィー、TOF-MS、UPLC等の公知の方法を用いることにより個々の栄養成分、例えば、RebDやRebMを精製することができる。
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に係る塩基配列を提供する。本発明のステビア植物体に係る塩基配列は、配列番号4~6、21及び22から選択される塩基配列を含む、又はそれからなる。
別の側面において、本発明は、FRO2の発現量を測定する工程を含む、栄養成分の含有量が高い植物体のスクリーニング方法(以下、「本発明のスクリーニング方法B」と称する場合がある)を提供する。本発明により、FRO2の発現量と栄養成分の含量との間に負の相関関係があることが明らかとなり、FRO2の発現量を指標に栄養成分含量の高い植物体をスクリーニングすることができるようになった。
(i)被験植物においてFRO2の発現量を測定する工程、
(ii)FRO2の発現量が低い被験植物組織の栄養成分の含量を評価する工程、
(iii)FRO2の発現量が低い被験植物体のうち、栄養成分の含量が高い個体を選択する工程、
(iv)選択した個体を他の植物体と交配する工程、
(v)交配により得られた子植物体において、FRO2の発現量を測定する工程、
(vi)FRO2の発現量が低い子植物組織の栄養成分の含量を評価する工程、
(vii)FRO2の発現量が低い子植物体のうち、栄養成分の含量が高い個体を選択する工程。
市販ステビア種子をベースに、生育状況、葉形態、RebA、RebD、RebMの含量等を基準に個体選抜を行い、個体群Iを得た。個体群Iにおいて、各個体の新鮮葉からゲノムDNAを抽出し、NGS(HiSeq 2500、Illumina)によるシーケンシングを行った。また、各個体の新鮮葉に含まれるステビオール配糖体含量をLC/MS-MS(島津LCMS8050)にて定量した。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、ステビオール配糖体(RebA、RebB、RebC、RebD、RebE、RebF、RebG、RebM、RebN及びステビオシド)の濃度を定量した。各遺伝子型の個体の乾燥葉におけるRebD、RebM、RebA及びTSGの平均含量(%DW)並びにTSG含量に占めるRebD、RebMの割合(%)を表1に、遺伝子型C/Tの個体における各ステビオール配糖体の平均含量又はTSG含量に占めるRebD、RebM含量の割合を1としたときの遺伝子型C/Cの個体における対応する数値の倍率を表2に、各遺伝子型の個体の乾燥葉におけるRebD、RebM含量(%DW)の分布を図2にそれぞれ示す。これらの結果が示す通り、RebD及びRebMの含量が高い個体に遺伝子型C/Cが多く見出された。例えば、乾燥葉中のRebD及びRebMの合計含量が1.3重量%以上の個体はすべて遺伝子型C/Cを有し、遺伝子型C/Cを有する個体の75%において乾燥葉中のRebD及びRebMの合計含量が1.3重量%以上であった。これに対し、遺伝子型C/Tを有する個体において、乾燥葉中のRebD及びRebMの合計含量が1.3重量%以上である個体は認められなかった。また、RebA含量及びTSG含量については、遺伝子型C/Cを有する個体の方が、遺伝子型C/Tを有する個体よりも低い傾向にあった。なお、TSG含量はRebA、RebB、RebC、RebD、RebE、RebF、RebG、RebM、RebN及びステビオシドの合計含量を、RebDMはRebDとRebMの合計をそれぞれ意味する。
遺伝子型C/Cに係るSNPはFRO2遺伝子内に位置する。個体群Iの各個体の新鮮葉から全RNAを抽出し、NGS(HiSeq 2500、Illumina)によるシーケンシングを行った。得られたデータから各個体におけるFRO2遺伝子のFPKM値を算出した。その結果、FRO2遺伝子発現量の低い個体に遺伝子型C/Cが多く見出された(図3)。例えば、乾燥葉中のFRO2遺伝子発現量(FPKM値)が2.5以下の個体の約69%が遺伝子型C/Cを有し、また、遺伝子型C/Cを有する個体の31%において乾燥葉中のFRO2遺伝子発現量(FPKM値)が2.5以下であった。これに対し、遺伝子型C/Tを有する個体において、乾燥葉中のFRO2遺伝子発現量(FPKM値)が2.5以下の個体は約21%であった。以上の結果から、遺伝子型C/Cを有することにより、FRO2遺伝子の発現量が遺伝子型C/Tを有する個体より低くなる傾向があることが示唆された。
個体群Iにおける各遺伝子型の代表的な2個体について、乾燥葉抽出物に含まれるミネラル成分を定量した。各個体から新鮮葉を採取し、65℃で約17時間熱乾燥させた。得られた乾燥葉100mgあたり1mLの熱湯(95℃以上)を加え、5分間スターラーで混和後、1mLあたり硝酸10μLを添加し、一晩放置した。得られたサンプルをICP-MS(Agilent 7500cx、Agilent Technologies)に供し、各元素の濃度を測定した(付属のMassHunter Workstation softwareを使用)。各遺伝子型の個体の乾燥葉における各ミネラル成分の平均含量を表3に、遺伝子型C/Tの個体における各ミネラル成分の平均含量を1としたときの遺伝子型C/Cの個体における各ミネラル成分の平均含量の倍率を表4及び図4にそれぞれ示す。いずれのミネラル成分も遺伝子型C/Cの個体において、遺伝子型C/Tの個体以上の含量を示した。
個体群Iにおける各遺伝子型の代表的な2個体について、乾燥葉抽出物に含まれる遊離アミノ酸を定量した。各個体から新鮮葉を採取し、65℃で約17時間熱乾燥させた。得られた乾燥葉100mgあたり5mLの熱湯(95℃以上)を加え、5分間スターラーで混和し、5分間超音波処理に供した後、10分間12000rpmで遠心処理し、上清を採取した。得られた上清サンプルをHPLC(Chromaster 5210/5260、Hitachi High-Tech)に供し、各アミノ酸の濃度を測定した(Empower 3、Watersを使用)。各遺伝子型の個体の乾燥葉における各アミノ酸の平均含量を表5~6に、遺伝子型C/Tの個体における各アミノ酸の平均含量を1としたときの遺伝子型C/Cの個体における各アミノ酸の平均含量の倍率を表7及び図5にそれぞれ示す。いずれのアミノ酸も遺伝子型C/Cの個体において、遺伝子型C/Tの個体以上の含量を示した。
実施例1~2で得た個体群Iの各個体のRebD及びRebMの合計含量とFRO2発現量を散布図にプロットしたところ、両者に負の相関が認められた(図6)。また、FRO2発現量の上位/下位50%、25%、15%及び10%の個体群におけるRebD及びRebMの含量の平均値を表8に示す。
Claims (17)
- 配列番号1の37位に相当する位置の遺伝子型がC/Cである、ステビア植物体。
- 鉄、亜鉛、リン、銅、モリブデン、アミノ酸、レバウジオシドD及びレバウジオシドMから選択される栄養成分の少なくとも1つを、配列番号1の37位に相当する位置の遺伝子型がC/Tである対照ステビア植物体より多く含有する、及び/又は、FRO2の発現量が、前記対照ステビア植物体より低い、請求項1に記載の植物体。
- 非遺伝子組換植物体である、請求項1又は2に記載の植物体。
- 請求項1~3のいずれか一項に記載の植物体の組織、組織培養物又は細胞。
- 種子、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4に記載の組織、組織培養物又は細胞。
- 請求項1~3のいずれか一項に記載の植物体と第2のステビア植物体とを交雑させる工程を含む、鉄、亜鉛、リン、銅、モリブデン、アミノ酸、レバウジオシドD及びレバウジオシドMから選択される栄養成分の少なくとも1つを、配列番号1の37位に相当する位置の遺伝子型がC/Tである対照ステビア植物体より多く含有する、及び/又は、FRO2の発現量が、前記対照ステビア植物体より低い、ステビア植物体を作出する方法。
- 第2の植物体が請求項1~3のいずれか一項に記載の植物体である、請求項6に記載の方法。
- 鉄、亜鉛、リン、銅、モリブデン、アミノ酸、レバウジオシドD及びレバウジオシドMから選択される栄養成分の少なくとも1つを含む、請求項1~3のいずれか一項に記載の植物体、請求項4又は5に記載の組織、組織培養物又は細胞の抽出物。
- 請求項1~3のいずれか一項に記載の植物体、請求項4又は5に記載の組織、組織培養物又は細胞から抽出物を得る工程を含む、鉄、亜鉛、リン、銅、モリブデン、アミノ酸、レバウジオシドD及びレバウジオシドMから選択される栄養成分の少なくとも1つを含む抽出物の製造方法。
- 請求項1~3のいずれか一項に記載の植物体の抽出物、請求項4又は5に記載の組織、組織培養物又は細胞の抽出物、或いは、請求項8に記載の抽出物を提供する工程、及び
前記抽出物を、食品、甘味料組成物、香料又は医薬品の原料に添加する工程
を含む、食品、甘味料組成物、香料又は医薬品の製造方法。 - 請求項1~3のいずれか一項に記載の植物体の乾燥物を含むハーブ茶製品。
- 被験ステビア植物体から、配列番号1の37位に相当する位置の遺伝子型がC/Cであるか否かを検出する工程を含む、請求項1~3のいずれか一項に記載のステビア植物体をスクリーニングする方法。
- 遺伝的特徴を検出する工程が、シーケンシング、dCAPS法又はTaqMan PCR法を用いて行われる、請求項12に記載の方法。
- 被験ステビア植物組織における、鉄、亜鉛、リン、銅、モリブデン、アミノ酸、レバウジオシドD及びレバウジオシドMから選択される栄養成分の少なくとも1つの含有量を測定する工程をさらに含む、請求項12又は13に記載の方法。
- 配列番号1の37位に相当する位置の遺伝子型がC/Cであるか否かを検出するための試薬を含む、請求項1~3のいずれか一項に記載のステビア植物体のスクリーニングキット。
- 試薬が、dCAPS法又はTaqMan PCR法に使用するプライマー及び/又はプローブを含む、請求項15に記載のキット。
- FRO2の発現量を測定する工程を含む、鉄、亜鉛、リン、銅、モリブデン、アミノ酸、レバウジオシドD及びレバウジオシドMから選択される栄養成分の少なくとも1つの含有量が高い植物体のスクリーニング方法。
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