WO2022181478A1 - タバコ属植物体およびその製造方法 - Google Patents
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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
- A01H6/82—Solanaceae, e.g. pepper, tobacco, potato, tomato or eggplant
- A01H6/823—Nicotiana, e.g. tobacco
-
- A—HUMAN NECESSITIES
- A24—TOBACCO; CIGARS; CIGARETTES; SIMULATED SMOKING DEVICES; SMOKERS' REQUISITES
- A24B—MANUFACTURE OR PREPARATION OF TOBACCO FOR SMOKING OR CHEWING; TOBACCO; SNUFF
- A24B15/00—Chemical features or treatment of tobacco; Tobacco substitutes, e.g. in liquid form
- A24B15/10—Chemical features of tobacco products or tobacco substitutes
-
- 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
- A01H1/06—Processes for producing mutations, e.g. treatment with chemicals or with radiation
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/10—Processes for modifying non-agronomic quality output traits, e.g. for industrial processing; Value added, non-agronomic traits
- A01H1/101—Processes for modifying non-agronomic quality output traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine or caffeine
-
- 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
- A01H1/12—Processes for modifying agronomic input traits, e.g. crop yield
- A01H1/122—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- A01H1/1245—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, e.g. pathogen, pest or disease resistance
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
- A01H5/12—Leaves
-
- A—HUMAN NECESSITIES
- A24—TOBACCO; CIGARS; CIGARETTES; SIMULATED SMOKING DEVICES; SMOKERS' REQUISITES
- A24B—MANUFACTURE OR PREPARATION OF TOBACCO FOR SMOKING OR CHEWING; TOBACCO; SNUFF
- A24B13/00—Tobacco for pipes, for cigars, e.g. cigar inserts, or for cigarettes; Chewing tobacco; Snuff
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
-
- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
-
- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
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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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y306/00—Hydrolases acting on acid anhydrides (3.6)
- C12Y306/01—Hydrolases acting on acid anhydrides (3.6) in phosphorus-containing anhydrides (3.6.1)
- C12Y306/01003—Adenosine triphosphatase (3.6.1.3)
Definitions
- the present invention relates to a Nicotiana plant and a method for producing the same.
- Solavetivone is one of the aroma components contained in Nicotiana plants, and is known to have a citrus-like aroma (Non-Patent Document 1). It has also been reported that soravetibon has effects of sensitizing the flavor and taste of tobacco and suppressing irritation (Patent Document 1).
- Nicotiana tabacum which has a resistance factor (N factor)
- viruses such as tobacco mosaic virus (TMV)
- soravetivone contained in leaves of Nicotiana plants increases.
- N factor tobacco mosaic virus
- Leaves inoculated with the virus develop local lesions at the inoculated portion and produce soravetivone as a phytoalexin (antibacterial substance). Since the production of soravetivone is restricted to the lesion periphery, rapid programmed cell death (PCD) such as hypersensitivity reaction is thought to be closely related to the production of soravetivone.
- PCD programmed cell death
- An object of one aspect of the present invention is to provide a Nicotiana plant that can achieve a high soravetivone content without virus inoculation, and a method for producing the same.
- a Nicotiana plant encodes a polypeptide having 95% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2.
- An endogenous gene containing a polynucleotide as a coding region, a polynucleotide encoding a polypeptide having 95% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 4 as a coding region and at least an endogenous gene comprising, as a coding region, a polynucleotide encoding a polypeptide having a sequence identity of 95% or more to the amino acid sequence shown in SEQ ID NO: 6
- a mutation that causes any function suppression is introduced into the genome (however, the function suppression is promotion of degradation of mRNA transcribed from the endogenous gene by RNAi or VIGS when compared to wild-type plants. some, except Nicotiana benthamiana).
- a method for producing a Nicotiana plant encodes a polypeptide having a sequence identity of 95% or more to the amino acid sequence shown in SEQ ID NO: 2.
- an endogenous gene containing, as a coding region, a polynucleotide encoding a polypeptide having a sequence identity of 95% or more to the amino acid sequence shown in SEQ ID NO: 4
- An endogenous gene containing as a region and a polynucleotide encoding a polypeptide having 95% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 6 as a coding region.
- the leaf tobacco according to one aspect of the present invention contains a polynucleotide encoding a polypeptide having 95% or more sequence identity with the amino acid sequence shown in SEQ ID NO:2. , an endogenous gene containing as a coding region, an endogenous gene containing as a coding region a polynucleotide encoding a polypeptide having 95% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 4 and at least one of an endogenous gene containing, as a coding region, a polynucleotide encoding a polypeptide having 95% or more sequence identity to the amino acid sequence shown in SEQ ID NO:6.
- a Nicotiana plant in which a mutation that causes functional suppression has been introduced into the genome (however, the functional suppression promotes degradation of mRNA transcribed from the endogenous gene by RNAi or VIGS when compared to wild-type plants. If , the leaf tobacco (excluding Nicotiana benthamiana) has a higher content of soravetivone than the leaf tobacco of the wild-type Nicotiana plant.
- a dried leaf according to an aspect of the present invention is a polynucleotide encoding a polypeptide having a sequence identity of 95% or more to the amino acid sequence shown in SEQ ID NO: 2 as the coding region, a polynucleotide encoding a polypeptide having 95% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 4 as the coding region
- At least one of an endogenous gene and an endogenous gene containing, as a coding region, a polynucleotide encoding a polypeptide having a sequence identity of 95% or more to the amino acid sequence shown in SEQ ID NO: 6
- a Nicotiana plant in which a mutation that causes suppression of the function of Nicotiana is introduced into the genome (however, the suppression of the function is compared to a wild-type plant, the degradation of mRNA transcribed from the endogenous gene by RNAi or VIGS) (excluding Nicotiana benthamiana, if promoted)
- a Nicotiana plant with a high soravetivone content can be provided without performing a treatment such as virus inoculation.
- FIG. 1 is a graph showing the results of GC-MS analysis of each line in a plant produced in Examples. The vertical axis indicates intensity, and the horizontal axis indicates time (min.). 4 is a graph showing the results of MutMap analysis;
- FIG. 1 is a diagram showing a phylogenetic tree based on the amino acid sequences of each gene of Arabidopsis thaliana, Nicotiana tabacum "Tsukuba 1", Nicotiana sylvestris, and Nicotiana benthamiana.
- FIG. 1 is a diagram showing a phylogenetic tree based on the amino acid sequences of each gene of Arabidopsis thaliana, Nicotiana tabacum "Tsukuba 1", Nicotiana sylvestris, and Nicotiana benthamiana.
- FIG. 10 is a graph showing the amount of NtabCA1 transcript in the NtabCA1-RNAi line (upper left), the amount of NtabCA2 in the NtabCA1-RNAi line (upper right), and the amount of NtabChlH transcript in the NtabChlH-RNAi line (lower left). All values are shown as relative values when the transcript amount of the control strain E5 is set to 1. It is an image of leaves in NtabCA1-RNAi, NtabChlH-RNAi and control. Graph showing the results of GC-MS analysis of NtabCA1-RNAi lines. The vertical axis indicates intensity, and the horizontal axis indicates time (min.). Fig.
- Figure 3 is a graph showing the amount of transcripts of NsCA1 in NsCA1-RNAi lines (left) and the amount of transcripts of NsCA2 in NsCA1-RNAi lines (right).
- Figure 10 is an image of the appearance of individuals in wild-type Nicotiana sylvestris, LSC13, NsCA1-RNAi lines and controls. It is the image which image
- Nicotiana plants One embodiment of the present invention is an endogenous gene comprising, as a coding region, a polynucleotide encoding a polypeptide having 95% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 2; An endogenous gene comprising, as a coding region, a polynucleotide encoding a polypeptide having 95% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 4, and the amino acid shown in SEQ ID NO: 6
- a mutation that causes functional suppression of at least one of an endogenous gene that contains, as a coding region, a polynucleotide encoding a polypeptide having a sequence identity of 95% or more to the sequence is introduced into the genome.
- a Nicotiana benthamiana plant (however, when the suppression of function is promotion of degradation of mRNA transcribed from the endogenous gene by RNAi or VIGS compared to a wild-type plant, Nicotiana benthamiana ) are
- an endogenous gene containing, as a coding region, a polynucleotide encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2, 4 or 6 (herein also referred to as "CA1 gene") ) are highly conserved with high sequence identity.
- SEQ ID NO: 2 shows the amino acid sequence of the polypeptide encoded by the Nicotiana sylvestris CA1 gene (also referred to herein as "NsCA1").
- SEQ ID NO: 4 shows the amino acid sequence of the polypeptide encoded by the CA1 gene present in the S genome of Nicotiana tabacum (also referred to herein as “NtabCA1-S”).
- SEQ ID NO: 6 shows the amino acid sequence of the polypeptide encoded by the CA1 gene present in the T genome of Nicotiana tabacum (also referred to herein as "NtabCA1-T”).
- the polypeptide having the amino acid sequence shown in SEQ ID NO: 2 is encoded by, for example, a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 1 (cDNA of the CA1 gene of Nicotiana sylvestris).
- a polypeptide having the amino acid sequence shown in SEQ ID NO: 4 is, for example, a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 3 (cDNA of CA1 gene encoded in the S genome of Nicotiana tabacum).
- the polypeptide having the amino acid sequence shown in SEQ ID NO: 6 is, for example, a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 5 (cDNA of CA1 gene encoded in the T genome of Nicotiana tabacum). coded by In the present specification, although not limited, NsCA1 polynucleotides of wild-type Nicotiana sylvestris, NtabCA1-S polynucleotides of wild-type Nicotiana tabacum, and wild-type Nicotiana tabacum may be polynucleotides encoding the polypeptides of SEQ ID NOs: 2, 4 and 6, respectively.
- a “wild-type plant” refers to a plant into which a factor that suppresses the expression of the CA1 gene has not been introduced and the CA1 gene has no mutation.
- the CA1 gene shows high homology with the type IIB Ca 2+ -ATPase gene localized in the endoplasmic reticulum in Nicotiana benthamiana. Therefore, the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2, 4 or 6 is considered to have Ca 2+ -ATPase activity.
- transient (VIGS: Virus induced gene silencing) and constitutively (RNAi) expression suppression lines of type IIB Ca 2+ -ATPase gene localized in the endoplasmic reticulum It has been reported that inoculation with various viruses or elicitors accelerates the development of programmed cell death compared to inoculation of control plants.
- Naturala plant includes whole organisms (e.g. adults, seedlings and seeds), tissues (e.g. leaves, stems, flowers, roots, reproductive organs, embryos and parts thereof, etc.). ), and their dried products.
- the Nicotiana plant is not particularly limited as long as it belongs to the genus Nicotiana. Examples include Nicotiana acaulis, Nicotiana acuminata, and Nicotiana acuminata var. multzjlora), Nicotiana africana, Nicotiana alata, Nicotiana amplexicaulis, Nicotiana arentsii, Nicotiana attenuata, Nicotiana Nicotiana benavidesii, Nicotiana benthamiana, Nicotiana bigelovii, Nicotiana bonariensis, Nicotiana cavicola, Nicotiana clevelandii, Nicotiana Nicotiana cordifolia, Nicotiana corymbosa, Nicotiana debneyi, Nicotiana excelsior, Nicotiana forgetiana, Nicotiana fragrans, Nicotiana glauca, Nicotiana glutinosa, Nicotiana goodspeedii, Nicotiana gossei, Nicotiana ingulba, Nicotiana kawakamii,
- Nicotiana otophora Hesperis, Nicotiana otophora), Nicotiana paniculata, Nicotiana pauczjlora, Nicotiana petunioides, Nicotiana plumbaginifolia, Nicotiana quadrivalvis, Nicotiana raimondii, Nicotiana repanda, Nicotiana rosulata, Nicotiana rosulata subsp.
- Nicotiana rotundifolia Nicotiana rustica (Malva tobacco), Nicotiana setchellii, Nicotiana simulans, Nicotiana solanifolia, Nicotiana s pegauinii, Nicotiana stocktonii, Nicotiana suaveolens, Nicotiana sylvestris, Nicotiana tabacum, Nicotiana thyrsiflora, Nicotiana tomentosa tomentosa), Nicotiana tomentosiformis, Nicotiana trigonophylla, Nicotiana umbratica, Nicotiana undulata, Nicotiana velutina, Nicotiana Wigan Dioides (Nicotiana wigandioides), hybrids of Nicotiana plants, and the like.
- Nicotiana tabacum and Nicotiana rustica which are used as raw materials for leaf tobacco production, are particularly preferred. Nicotiana sylvestris can also be preferably used.
- the soravetivone content in the Nicotiana plant according to one embodiment of the present invention is increased compared to the wild-type Nicotiana plant.
- soravetivone content refers to the content of soravetivone in Nicotiana plants.
- the soravetivone content can be expressed in % by weight relative to the dried Nicotiana plant.
- the content of soravetivone is usually expressed in % by weight relative to dried leaves (dried leaves).
- high soravetivone content means increased soravetivone content compared to the soravetivone content in wild-type Nicotiana plants.
- the high or increased soravetivone content is, for example, 2-fold or more, 3-fold or more, 5-fold or more, preferably 10-fold or more, 20-fold or more, 30-fold or more that of the wild-type Nicotiana plant, 50 times or more, more preferably 100 times or more.
- Soravetivone can be quantified by, for example, gas chromatography/mass spectrometry (GC-MS) analysis.
- GC-MS gas chromatography/mass spectrometry
- endogenous gene used herein means a gene that is originally present on the genome of a Nicotiana plant. In other words, an endogenous gene is not a foreign gene present in plants other than Nicotiana plants.
- sequence identity of an amino acid sequence
- sequence identity means the proportion of the (amino acid) sequence referred to that matches the reference (amino acid) sequence.
- non-matching portion of the sequences is the portion in which (amino acid residue) substitutions, additions, deletions or insertions are present.
- polypeptide having 95% or more sequence identity with the amino acid sequence shown in ⁇ specifying a polypeptide using the amino acid sequence described in the sequence listing is commonly used in Nicotiana plants. It may be an existing polypeptide.
- polypeptide and protein have substantially the same meaning and may be used interchangeably.
- the specific polypeptide whose abundance is reduced in Nicotiana plants according to one aspect of the present invention has 95% or more sequence identity with each amino acid sequence shown in the sequence listing. Any polypeptide may be used, and the sequence identity is preferably at a higher percentage (eg, 96%, 97%, 98%, or 99% or more).
- endogenous gene function suppression means a state in which a gene on the genome does not exhibit its original function. Therefore, “suppression of endogenous gene function” includes “mutation of endogenous gene”, “disruption of endogenous gene”, and “suppression of expression of endogenous gene” by other than the endogenous gene (including exogenous gene). It is a term that includes “.
- the Nicotiana plant according to the present embodiment is realized based on the finding by the present inventors that the soravetivone content is increased by suppressing the function of the CA1 gene. The effect on the production of soravetivone by suppressing the function of the CA1 gene has not been known so far.
- Nicotiana tabacum is amphidiploid and has a genome (also referred to as the "S genome") from the parent plant Nicotiana sylvestris and Nicotiana tomento. It has a genome (also referred to as “T genome”) derived from Nicotiana tomentosiformis.
- S genome a genome from the parent plant Nicotiana sylvestris and Nicotiana tomento. It has a genome (also referred to as “T genome”) derived from Nicotiana tomentosiformis.
- T genome also referred to as “T genome”
- the soravetivone content is increased by specifically suppressing the function of the CA1 gene on at least one of the S genome and the T genome. Therefore, the function of the CA1 gene on either the S genome or the T genome may be specifically suppressed, but the function of the CA1 gene on both the S genome and the T genome is specifically suppressed. preferably.
- Endogenous gene mutations include mutations in genes that do not produce original functional polypeptides (including mutations that produce polypeptides with reduced or absent function), and those that produce functional polypeptides. , a gene mutation that reduces the amount produced, or a gene mutation that produces a functional polypeptide but reduces the stability of the polypeptide.
- Endogenous gene disruption means that the native gene on the genome is not present or no transcript is produced from the gene that is on the genome.
- Endogenous gene expression suppression means that (i) no change occurs in the base of the endogenous gene, but the transcription or translation function of the gene (from transcription to mRNA to subsequent translation to polypeptide) is A state in which the amount of polypeptide produced is reduced or no polypeptide is produced by being modified via other factors, and (ii) a change occurs in the base of the endogenous gene, nonsense described later. -means a state in which mRNA is degraded by mediated mRNA decay.
- Endogenous gene expression suppression can occur, for example, by degradation of mRNA transcribed from the endogenous gene.
- mutant has the meaning commonly understood in the art to which this application belongs, e.g. Any change (eg, substitution, deletion, insertion, addition, duplication, inversion or translocation) of an amino acid residue is meant. Therefore, “mutation of an endogenous gene” includes mutation of a gene that does not produce an original functional polypeptide, mutation of a gene that produces a polypeptide but reduces the amount produced, and mutation of a gene that produces a polypeptide However, mutations in the gene that reduce the stability of the polypeptide, loss of the gene (coding region or full length including untranslated regions), or mutations that suppress transcription from the gene (transcription control region or transcription initiation region) , etc.).
- transcription control sequences such as promoters and terminators (including sequences upstream (5' side) and sequences downstream (3' side) relative to the coding region), 5'
- Such substitutions can be in at least one of the untranslated and 3' untranslated regions, the conserved sequences flanking the intron (5'GT-AG3'), and the coding region.
- substitutions in nucleotide sequences important for regulation of gene expression in the promoter sequence, 5′ untranslated region and 3′ untranslated region of a gene may reduce the transcriptional activity of the gene or stabilize transcripts from the gene. result in decreased sex. Any of these reductions can result in a reduction in translation product, concomitant with a reduction in transcripts from the gene.
- Substitutions in conserved intronic sequences e.g., 5' GT, 3' AG of the intron
- cause abnormal splicing of the mRNA resulting in abnormal mRNAs with unwanted introns added or inserted.
- Abnormal mRNAs produce abnormal translation products, eg, by frameshifting, or do not terminate translation.
- Substitutions in the coding region can result in incomplete translation products or translation products that do not maintain their original function.
- An incomplete translation product results from the conversion of a codon encoding an amino acid into a stop codon by the substitution (nonsense mutation).
- An incomplete translation product lacks one or more contiguous amino acid residues, including the C-terminal amino acid residue, as compared to the native translation product.
- the nonsense mutation occurs in any codon upstream of the original stop codon, preferably upstream of the original stop codon across one or more codons.
- nonsense-mediated mRNA decay (Brogna and Wen (2009) Nat. Structural Mol. Biol. 16: 107-113) occurs when a gene has a nonsense mutation in its protein-coding region, resulting in transcript degradation.
- the translation product produced in this case will be of incomplete length.
- the target gene consists of multiple exons, it is preferable that at least one exon having a nonsense mutation exists in order to generate nonsense-mediated mRNA decay, and the exon having the nonsense mutation is More preferably, it is not the most downstream (3' side) exon that constitutes the gene. Amino acid substitutions result in translation products that impair their native function. In this case, the amount of translation product (polypeptide) and transcript may be comparable compared to wild-type plants.
- the translation product undergoes a conformational change, a decrease in function as a functional domain, or the like.
- One of the preferred embodiments of the mutations of the present invention are amino acid substitutions that result in such translation products impairing their native function.
- the above amino acid substitutions are non-conservative substitutions that have a high probability of altering the function of the translation product.
- Non-conservative substitutions are substitutions of amino acids that differ in nature, such as amino acids that differ in charge or hydrophobicity (e.g., acidic to uncharged neutral amino acids, basic to acidic, polar to non-conservative). substitution with polar amino acids, etc.) and substitution with amino acids having side chains with different bulks (three-dimensional sizes).
- Mutations other than substitutions occur within the promoter sequence, the 5′ untranslated region and the 3′ untranslated region, which, like substitutions, reduce transcript abundance due to decreased transcriptional activity or stability. A decrease, and a reduction in the amount of polypeptide can result. Mutations other than substitutions of introns for conserved sequences, like substitutions, can also result in translation of polypeptides having amino acid sequences that differ from the original. Mutations other than substitutions into the coding region may also have amino acid sequences that differ from the original by deletion or insertion of amino acid residues (caused by deletion or insertion of multiples of three consecutive bases) or by frameshifting. translation of the polypeptide can occur. Also, large deletions involving the entire gene or insertion of large fragments into the gene can result in loss of expression of the gene itself.
- RNA sequence In the Nicotiana plant, mutation or disruption of the gene occurs as a result of, for example, mutagen treatment, gene recombination, genome editing or gene knockout.
- the mutagen treatment of the gene can be carried out by artificially acting a mutagen on Nicotiana plants (and optionally in combination with suppression of gene repair function).
- Chemical agents such as ethyl methanesulfonic acid (EMS), sodium azide, ethidium bromide, and nitrous acid can be used as types of mutagens. However, it is not limited to these.
- mutagens include ⁇ -rays, heavy ion beams, X-rays, neutron beams, UV rays, and the like, but are not limited to these as long as they are radiations that cause mutations in the genomic DNA of Nicotiana plants. .
- EMS is preferred.
- the gene recombination can be carried out by homologously recombining part or all of the target gene with a recombination sequence according to known gene targeting techniques. Genome editing of the gene can be performed by known techniques (eg, zinc-finger nucleases: ZFN, transcription activator-like effector nucleases: TALEN, CRISPR/Cas9 system, etc.).
- the gene knockout can be performed by a known transposon (mobile genetic element), T-DNA insertion, or the like.
- said gene mutation or disruption is not the result of spontaneous mutation.
- the guide RNA and Cas9 protein in the CRISPR/Cas9 system, genome editing is possible if the guide RNA and Cas9 protein, and in TALENs and ZFNs, the fusion protein (DNA binding domain and nuclease are fused) are present in the target cell.
- both the guide RNA and Cas9 protein as well as the fusion protein can be directly introduced into target cells. Methods for directly introducing these into target cells include the PEG method, electroporation method, particle bombardment method, and the like.
- a construct in which a gene encoding a guide RNA and Cas9 protein is connected to a desired promoter and terminator is created on a vector, and this is introduced into target cells and tissues using Agrobacterium or the like.
- the complementary sequence of the nucleotide sequence immediately upstream of XGG on the genome forms a base pair with part of the guide RNA, and within the nucleotide sequence double-stranded genomic DNA is generated by Cas9 get cut.
- each of the pair of DNA-binding domains of the dimer-forming artificial nuclease binds to the nucleotide sequence present on both ends of the FokI cleavage domain via a 5-20 base spacer.
- the nucleotide sequences are present on one and the other strand of double-stranded genomic DNA, thus one of the pair of DNA-binding domains binds to the one strand and the other to the other strand.
- the DNA-binding domain is composed of a repeating unit (module) of 33 to 34 amino acid residues and a number of modules corresponding to the number of bases to be bound.
- each of the pair of DNA-binding domains of the dimer-forming artificial nuclease binds to the nucleotide sequence present at both ends of the FokI cleavage domain via 5-20 base spacers.
- the DNA-binding domain is composed of multiple zinc finger modules.
- Gene mutation or disruption can be determined by detecting the presence or absence of mutations in the gene.
- Methods for detecting mutations in genes include (1) a method in which a DNA sequence containing the mutation is amplified by PCR or the like, and then the DNA base sequence is directly decoded using a commercially available sequencer or the like; (3) a method of detecting SNP (Single Nucleotide Polymorphism) using the Cycleave PCR method; (4) T7 Endonuclease I, etc.
- CAPS Cosmetic Amplified Polymorphic Sequence
- dCAPS derived CAPS
- a method for determining the presence or absence of a mutation by detecting whether or not it was detected PCR method using a TaqMan probe
- (8) performing single base extension using a primer adjacent to the mutation, and depending on the mass difference of the incorporated base There are methods for detecting the presence or absence of mutations (MassARRAY analysis method), (9) in the case of deletions or insertions, methods for detecting mutations from differences in electrophoretic mobility, etc., which can determine the presence or absence of mutations.
- mutation or disruption of a gene can be determined by comparing the size or expression levels of the protein resulting from the gene modification to those of the wild-type protein. Specifically, such a comparison can be made, for example, by performing Western blotting.
- MutMap method is a technique that combines bulk segregant analysis (BSA) and whole genome sequencing (WGS) to identify the causative gene regions of mutations (Abe, A. et al. ., Nat. Biotechnol., 30(2): 174-178 (2012)). Compared to conventional map-based cloning, this method does not require the creation of markers and does not require the use of a large number of individuals, greatly reducing labor and time. This allows for more rapid gene identification.
- BSA bulk segregant analysis
- WGS whole genome sequencing
- MutMap method first, a mutant line having a desired trait is crossed with the parent variety (original line) used for mutagen treatment to obtain the F1 generation, and the F1 individual is selfed to obtain the F2 generation. obtain. Traits acquired by mutation are often thought to be caused by recessive mutations. Therefore, the phenotype of the F1 generation should be the wild type, and the phenotype of the F2 generation should be separated at a ratio of 3:1 (wild type:mutant type).
- the MutMap method has so far been used mainly for genetic analysis of rice, which has a relatively small genome size.
- the present invention demonstrated that the MutMap method can also be applied to genetic analysis of organisms with relatively large genome sizes, such as Nicotiana plants.
- Nicotiana tabacum is amphidiploid, and has a genome (also referred to as "S genome") derived from the parent plant Nicotiana sylvestris and Nicotiana tomentosi. It has both genomes from Nicotiana tomentosiformis (also called "T genome”).
- S genome a genome derived from the parent plant Nicotiana sylvestris and Nicotiana tomentosi. It has both genomes from Nicotiana tomentosiformis (also called "T genome”).
- Nicotiana tabacum genes denoted by the same name are mostly present in each of the S and T genomes.
- said mutants may have said mutations in either the S genome or the T genome.
- Both the S genome and the T genome may have the aforementioned mutations.
- one gene may have one type of mutation, or a plurality of mutations, and the type of mutation does not matter. Nicotiana tabacum may have mutations in any or all of four alleles, two in each of the S and T genomes, and if mutations are present in multiple alleles, these Mutations may be the same or different.
- Suppression of the expression of the gene includes suppression of transcription from the gene to mRNA, suppression of translation of the gene into a polypeptide via mRNA (e.g., degradation of the mRNA), and suppression of the function of the translated polypeptide.
- Suppression of transcription can be achieved by inhibiting transcription factors that promote transcription from the gene, inhibiting access of transcription initiation factors to the gene, and the like.
- Suppression of translation can be achieved using antisense RNA molecules, RNAi molecules, or co-suppression molecules.
- Inhibition of polypeptide function can be achieved by molecules (eg, decoy nucleic acids, ribozymes, antibodies and inhibitory peptides) that inhibit the function of the polypeptide upon binding to the functional polypeptide.
- Vectors used for transformation of Nicotiana plants for the purpose of suppressing gene expression or introducing mutations into genes are vectors capable of expressing polynucleotides inserted into the vector in plant cells.
- the vector pBI-based, pPZP-based, and pSMA-based vectors are preferably used, for example, which are capable of introducing a polynucleotide of interest into plant cells via Agrobacterium.
- plasmids of the binary vector system (such as pBIG, pBIN19, pBI101, pBI121 and pPZP202) are preferred.
- a trigger sequence used to suppress target gene expression by RNAi is inserted into the vector.
- the trigger sequence is, for example, part of a polynucleotide (which may have 0.1-1% substitution) encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 2, 4 or 6, or Contiguous at least 21-30 bases (eg, 21 bases or more, 22 bases) that are part of a polynucleotide having SEQ ID NO: 1, 3 or 5 (which may have 0.1-1% substitution) above, 23 or more bases, 24 or more bases, 25 or more bases, 26 or more bases, 27 or more bases, 28 or more bases, 29 or more bases, and 30 or more bases) polynucleotides (sense RNA portions) shown in the base sequences, and A polynucleotide (antisense RNA portion) having a nucleotide sequence complementary to the polynucleotide. More specifically, the above-mentioned base sequence of "at least
- the suppression is, for example, by directly introducing a molecule for achieving the suppression into a plant, or by introducing a nucleic acid molecule encoding the molecule into the plant. (transformation of plant body).
- the nucleic acid molecule is integrated into one or more arbitrary regions in the genome of the plant.
- the suppression of function may be a decrease in the amount of translation of the originally functional polypeptide, which is the expression product of the gene, compared to a wild-type plant.
- the translation of the polypeptide is mediated by a decrease in mRNA (due to the presence of mRNA such as instability of the mRNA itself, promotion of degradation of the mRNA, or suppression of transcription of the mRNA) or a decrease in the amount of translation from the mRNA (translation components ( tRNA and ribosomes) due to deficiency, inhibition of recruitment, or functional defects).
- the suppression of function may be a decrease in the amount of mRNA transcribed from the endogenous gene compared to a wild-type plant.
- a decrease in mRNA abundance is caused, for example, by suppression of transcription of the mRNA from the endogenous gene.
- Suppression of transcription can be achieved by, for example, inhibiting access of transcription factors to the endogenous gene as a result of the introduction of mutations into the endogenous gene.
- a decrease in the abundance of mRNA transcribed from an endogenous gene refers to 70% or less, 60 % or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, or 1% or less of the transcript.
- the suppression of function may be promotion of degradation of mRNA transcribed from the endogenous gene.
- mRNA degradation can be caused by nonsense-mediated mRNA decay when the gene has a nonsense mutation in the protein coding region, or the presence of exogenous factors that degrade mRNA, endogenous components that degrade mRNA or the presence of degradation promoting sequences in the mRNA.
- the mRNA in the Nicotiana plant decreases. That is, in the Nicotiana plant, the suppression of function may be a decrease in the amount of mRNA transcribed from the endogenous gene as compared to a wild-type plant.
- the mutation may be insertion of a polynucleotide expressing a factor that promotes degradation of mRNA transcribed from the endogenous gene, outside the region where the endogenous gene exists.
- the agent may be an antisense RNA molecule, an RNAi molecule or a co-suppression molecule.
- a Nicotiana plant according to one aspect of the present invention has an endogenous gene comprising, as a coding region, a polynucleotide encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2, 4 or 6. suppressed.
- a Nicotiana plant according to one aspect of the present invention has 95% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2, 4 or 6 and encodes a polypeptide having Ca 2+ -ATPase activity.
- the endogenous gene containing the polynucleotide as a coding region is suppressed in function.
- a polynucleotide encoding a polypeptide having 95% or more sequence identity to the amino acid sequence shown in SEQ ID NO:2. an endogenous gene containing as a coding region, an endogenous gene containing as a coding region a polynucleotide encoding a polypeptide having 95% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 4 and at least one of an endogenous gene containing, as a coding region, a polynucleotide encoding a polypeptide having a sequence identity of 95% or more to the amino acid sequence shown in SEQ ID NO: 6.” means any of (a combination of) endogenous genes (a) to (f) below.
- a “reduced abundance” of a native functional polypeptide is 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% relative to the abundance of the wild-type polypeptide. Below is meant the presence of 10% or less, 5% or less, or 1% or less of the polypeptide.
- the “decrease in abundance” of the original functional polypeptide refers to the case where the original functional polypeptide has lost its function due to mutation of an endogenous gene, etc., and the functional polypeptide does not exist at all. include.
- the abundance of the polypeptide, based on the abundance of the wild-type polypeptide can be appropriately selected from the above values that result in a high soravetivone content in Nicotiana plants.
- the reduction in at least one of the abundance of the above-mentioned mRNA and the abundance of the original functional polypeptide in the Nicotiana plant according to the present invention is obtained from the Nicotiana plant, cultured cells, callus, and protoplasts. , seeds, and progeny are genetically stably inherited. Therefore, the Nicotiana plant according to the present invention can be an individual generated from cultured cells, callus, protoplasts, seeds, and progeny produced through artificial manipulation, and these materials for obtaining the individual are included in the scope of the present invention.
- the Nicotiana plant according to the present invention can further include breeding progeny obtained by crossing.
- Many plant species, including rice, wheat, barley, and soybean are being bred using mutants.
- mutants isolated from a mutant population treated with a mutagen have many mutations in addition to the gene of interest. Therefore, backcrossing is generally performed to eliminate extraneous mutations.
- backcrossing is generally performed to eliminate extraneous mutations.
- a cultivar with higher added value can be obtained by introducing the traits of the mutant into the cultivar. Since the traits of mutants are derived from mutations, it is necessary to select individuals with mutations in order to proceed with backcrossing.
- the fewer the number of mutations that bring about the target trait (high soravetivone in the present invention), the fewer the number of mutations to be focused on, and the less the effort involved in backcrossing.
- a simple method for detecting the presence or absence of mutations and whether the mutations are homozygous or heterozygous is required. This method can be performed using a method for detecting mutation, which will be described later.
- MAS Marker Assisted Selection
- SNPs and SSRs Simple Sequence Repeat
- SSRs Simple Sequence Repeat
- new polymorphic markers can be obtained and used by deciphering the genome sequence of the tobacco to be used and specifying the difference in base sequence and the number of repeat sequences.
- a Nicotiana plant consists of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2, 4 or 6, and Ca 2+ -
- the function of an endogenous gene whose coding region is a polynucleotide encoding a polypeptide having ATPase activity is suppressed.
- the number of amino acids to be deleted, substituted or added in each amino acid sequence is, for example, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, or 1.
- a Nicotiana plant according to one aspect of the present invention has suppressed function of an endogenous gene comprising a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1, 3 or 5 as a coding region.
- a Nicotiana plant according to one aspect of the present invention hybridizes under stringent conditions with a polynucleotide consisting of a nucleotide sequence complementary to a polynucleotide consisting of a nucleotide sequence shown in SEQ ID NO: 1, 3 or 5,
- a polynucleotide encoding a polypeptide having Ca 2+ -ATPase activity is suppressed.
- Stringent conditions refer to conditions under which a so-called nucleotide sequence-specific double-stranded polynucleotide is formed, but the formation of a non-specific double-stranded polynucleotide is remarkably suppressed.
- Tm value melting temperature
- the range from the melting temperature (Tm value) of a hybrid between highly homologous nucleic acids, for example, a double-stranded polynucleotide that perfectly matches the probe, to a temperature 15°C, preferably 10°C, more preferably 5°C lower can be said to be the condition for hybridizing at a temperature of
- hybridization can be performed at 68° C. for 20 hours in a general hybridization buffer.
- a buffer solution consisting of 0.25M Na2HPO4, pH 7.2, 7% SDS, 1 mM EDTA, and 1x Denhardt's solution at a temperature of 60 to 68°C, preferably 65°C, more preferably 68°C. and hybridized for 16 to 24 hours under conditions of 60 to 68°C, preferably 65°C, more preferably 68°C in a buffer solution consisting of 20 mM Na2HPO4, pH 7.2, 1% SDS, and 1 mM EDTA. and washing for 15 minutes twice.
- Other examples include 25% formamide, more stringent 50% formamide, 4 ⁇ SSC (sodium chloride/sodium citrate), 50 mM Hepes pH 7.0, 10 ⁇ Denhardt's solution, 20 ⁇ g/ml denatured salmon sperm DNA. After prehybridization is performed overnight at 42°C in a hybridization solution, a labeled probe is added and hybridization is performed by incubating at 42°C overnight.
- the washing solution and temperature conditions for subsequent washing are about "1 x SSC, 0.1% SDS, 37°C", and more severe conditions are about "0.5 x SSC, 0.1% SDS, 42°C". As a more severe condition, it can be carried out at about "0.2 ⁇ SSC, 0.1% SDS, 65° C.”.
- the more stringent the washing conditions for hybridization the more likely it is to isolate DNA having higher homology with the probe sequence.
- the combinations of conditions of SSC, SDS and temperature described above are examples, and those skilled in the art will recognize the above or other factors that determine the stringency of hybridization (e.g., probe concentration, probe length, hybridization reaction, etc.). time, etc.), it is possible to achieve the same stringency as above.
- those skilled in the art should refer to Molecular Cloning (Sambrook, J. et al., Molecular Cloning: a Laboratory Manual 2nd ed., Cold Spring Harbor Laboratory Press, 10 Skyline Drive Plainview, NY (1989)). can easily obtain such genes.
- One embodiment of the present invention is an endogenous gene comprising, as a coding region, a polynucleotide encoding a polypeptide having 95% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 2; and an endogenous gene comprising, as a coding region, a polynucleotide encoding a polypeptide having 95% or more sequence identity to the amino acid sequence shown in at least one of SEQ ID NO: 4 and SEQ ID NO: 6
- a Nicotiana plant comprising a step of introducing a mutation that causes either functional suppression into the genome of a Nicotiana plant (however, the functional suppression is RNAi or VIGS when compared with a wild-type plant) Nicotiana benthamiana except Nicotiana benthamiana in the case of promotion of degradation of mRNA transcribed from the endogenous gene.
- the step of introducing the mutation into the genome of the Nicotiana plant may be a step of introducing the endogenous gene in the genome. Details of mutations to be introduced into Nicotiana plants are described in item [1. plants of the genus Nicotiana].
- individuals exhibiting the desired phenotype may be further selected from the mutant population of plants having mutations.
- selecting individuals a procedure for selecting desired individuals from a mutant population (panel) obtained when treated with a mutagen will be described.
- Mutants of functionally defective Nicotiana plants having mutations in two alleles are obtained by, for example, the following method. can be obtained.
- Nicotiana plants are treated with a mutagen to prepare a mutant population (panel) in which the entire genome is mutated, and genomic DNA is extracted.
- Target genes polynucleotides
- Nicotiana tabacum first, lines (M2) having homozygous mutations in the S genome and T genome are obtained, and crossed to produce F1. Further, the self-fertilized progeny (F2) is bred, and a line having homozygous mutations in both the S and T genomes is obtained.
- Selection of individuals exhibiting the desired phenotype may be performed by measuring soravetibon content.
- One embodiment of the present invention provides a method for determining Nicotiana plants with high soravetivone content.
- the determination method includes the following steps: obtaining a sample by harvesting a portion of the Nicotiana plant; A step of detecting a mutation that causes functional suppression of the endogenous gene on the genome contained in the sample; and determining the Nicotiana plant in which the mutation is detected as a Nicotiana plant having a high soravetivone content. process.
- One embodiment of the present invention provides a method for breeding Nicotiana plants.
- the method includes the step of crossing Nicotiana plants having a high soravetivone content determined by the determination method described above.
- One embodiment of the present invention is the Nicotiana plant, the Nicotiana plant obtained by the production method, the Nicotiana plant determined by the determination method, or the Nicotiana plant obtained by the breeding method. or the breeding progeny obtained by crossing the Nicotiana plants.
- functional suppression of only one CA1 gene results in an extremely high soravetivone content. Therefore, breeding by a single-factor recessive inheritance pattern using a mutation on the CA1 gene as a mark becomes possible.
- functional suppression of the two CA1 genes results in high soravetivone content. Therefore, it is possible to perform breeding by a one-factor recessive or two-factor recessive inheritance pattern using mutations on the CA1-S gene and/or mutations on the CA1-T gene as markers.
- mutants isolated from a mutant population treated with a mutagen have multiple mutations outside the gene of interest. Therefore, backcrossing is generally performed to eliminate extraneous mutations. In this crossing, by crossing a cultivar possessing an excellent trait with the above mutant, the desired trait possessed by the mutant can be introduced into an existing cultivar.
- the breeding progeny obtained in this way can be a cultivar that imparts high added value to existing cultivars.
- One embodiment of the present invention includes the Nicotiana plant, the Nicotiana plant obtained by the production method, the Nicotiana plant determined by the determination method, the Nicotiana plant obtained by the breeding method, or provide leaf tobacco harvested from said offspring or breeding progeny.
- Leaf tobacco refers to leaves harvested from Nicotiana plants and used in the manufacture of tobacco products.
- a leaf tobacco according to one embodiment of the present invention contains, as a coding region, a polynucleotide encoding a polypeptide having 95% or more sequence identity to the amino acid sequence shown in SEQ ID NO:2.
- a sex gene an endogenous gene comprising, as a coding region, a polynucleotide encoding a polypeptide having 95% or more sequence identity to the amino acid sequence shown in SEQ ID NO:4, and SEQ ID NO:6.
- One embodiment of the present invention provides dried leaves produced from the leaf tobacco.
- Dried leaves are obtained by drying leaf tobacco. Any drying method can be used, and examples thereof include, but are not limited to, natural drying, air drying, warm air drying, hot air drying, and the like.
- a dried leaf according to an embodiment of the present invention contains, as a coding region, a polynucleotide encoding a polypeptide having a sequence identity of 95% or more to the amino acid sequence shown in SEQ ID NO: 2.
- an endogenous gene an endogenous gene comprising, as a coding region, a polynucleotide encoding a polypeptide having 95% or more sequence identity to the amino acid sequence shown in SEQ ID NO:4, and SEQ ID NO:6
- a mutation that causes suppression of the function of at least one of the endogenous genes that contains as a coding region a polynucleotide encoding a polypeptide having a sequence identity of 95% or more to the amino acid sequence shown in Genome (However, when the suppression of function is promotion of degradation of mRNA transcribed from the endogenous gene by RNAi or VIGS when compared to wild-type plants, Nicotiana - excluding Nicotiana benthamiana), which has a higher content of so
- One embodiment of the present invention provides a tobacco product containing the dried leaves.
- Tobacco products may be in any form and include, but are not limited to, cut tobacco, cigars, cigarettes, electronic cigarettes, heat-not-burn tobacco.
- the tobacco product contains dried leaves with a high soravetivone content, it is possible to improve the yellowness (yellow leaf-like flavor) in the flavor and taste of the tobacco product.
- Nicotiana plants and their production methods please refer to the items mentioned above regarding Nicotiana plants and their production methods.
- an endogenous gene shown in SEQ ID NO: 4, containing, as a coding region, a polynucleotide encoding a polypeptide having 95% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2; an endogenous gene comprising, as a coding region, a polynucleotide encoding a polypeptide having a sequence identity of 95% or more to the amino acid sequence shown in SEQ ID NO: 6, and 95% or more to the amino acid sequence shown in SEQ ID NO: 6
- Nicotiana plant wherein a mutation that causes functional suppression of at least one of an endogenous gene containing, as a coding region, a polynucleotide encoding a polypeptide having the sequence identity of is introduced into the genome plants (except Nicotiana benthamiana, if said functional suppression is enhanced degradation of mRNA transcribed from said endogenous gene by RNAi or VIGS when compared to wild-type plants).
- a method for producing a Nicotiana plant comprising, as a coding region, a polynucleotide encoding a polypeptide having 95% or more sequence identity with the amino acid sequence shown in SEQ ID NO:2.
- an endogenous gene comprising, as a coding region, a polynucleotide encoding a polypeptide having 95% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 4, and the sequence A mutation that causes suppression of at least one function of an endogenous gene containing, as a coding region, a polynucleotide encoding a polypeptide having 95% or more sequence identity to the amino acid sequence shown in number 6 into the genome of a Nicotiana plant, provided that the suppression of function is transcribed from the endogenous gene by RNAi or VIGS when compared to a wild-type plant Nicotiana benthamiana except Nicotiana benthamiana when promoting the degradation of mRNA).
- the step of introducing includes inserting a polynucleotide that expresses a factor that promotes degradation of mRNA transcribed from the endogenous gene outside the region where the endogenous gene is present, ( 12) The production method according to any one of (15).
- an endogenous gene comprising, as a coding region, a polynucleotide encoding a polypeptide having 95% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 2, shown in SEQ ID NO: 4; an endogenous gene comprising, as a coding region, a polynucleotide encoding a polypeptide having a sequence identity of 95% or more to the amino acid sequence shown in SEQ ID NO: 6, and 95% or more to the amino acid sequence shown in SEQ ID NO: 6 Nicotiana plant whose genome contains a mutation that suppresses the function of at least one of the endogenous genes containing, as the coding region, a polynucleotide encoding a polypeptide having the sequence identity of (However, Nicotiana benthamiana is excluded when the suppression of function is promotion of degradation of mRNA transcribed from the endogenous gene by RNAi or VIGS compared to wild-type plants.)
- an endogenous gene comprising, as a coding region, a polynucleotide encoding a polypeptide having 95% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 2, shown in SEQ ID NO: 4; an endogenous gene comprising, as a coding region, a polynucleotide encoding a polypeptide having a sequence identity of 95% or more to the amino acid sequence shown in SEQ ID NO: 6, and 95% or more to the amino acid sequence shown in SEQ ID NO: 6 Nicotiana plant whose genome contains a mutation that suppresses the function of at least one of the endogenous genes containing, as the coding region, a polynucleotide encoding a polypeptide having the sequence identity of (except for Nicotiana benthamiana, if the suppression of function is promotion of degradation of mRNA transcribed from the endogenous gene by RNAi or VIGS compared to wild-type plants) and having a higher soravetivone content than dried
- Example 1 Isolation of Nicotiana sylvestris high soravetibon-producing mutant strain (LSC13 strain)]
- Nicotiana sylvestris is diploid, and a phenotype caused by mutation of one gene can be observed in the M2 generation following the initial generation (M1 generation) that caused the mutation. Therefore, we constructed a library of Nicotiana sylvestris mutants and tried to obtain mutants with altered leaf components.
- Nicotiana sylvestris seeds were immersed in 0.6% EMS for 16 hours and then washed with distilled water for 30 minutes eight times. The treated seeds were sown and plants of the treated generation (M1 generation) were cultivated. Selfed seeds (M2 generation) were obtained from each line. About 200 or more seeds (M2 lines) were obtained from each of 4,945 lines of EMS-mutated Nicotiana sylvestris lines (M1 lines).
- LSC13 a line in which soravetivone was remarkably increased compared to WT and which caused pseudo-lesions on leaves was included.
- Table 3 shows the analysis results of soravetibon for each individual of Nicotiana sylvestris (WT), LSC13, F1, and F2.
- the soravetibon content was shown as an internal standard ratio.
- An individual with pseudo-lesions was designated as F2_LSC13, and an individual without such lesions was designated as F2_WT.
- N.D. indicates below the detection limit. Soravetivone was detected in all 28 LSC13 and F2 individuals with pseudo-lesions, while soravetivone was not detected in 28 WT, F1, and F2 individuals in which pseudo-lesions were not observed. (Fig. 2, Table 3).
- F2_LSC13 individuals 20 individuals excluding F2_LSC13_1, F2_LSC13_2, F2_LSC13_3, F2_LSC13_4, F2_LSC13_5, F2_LSC13_7, F2_LSC13_8, and F2_LSC13_11 (8 individuals in total) were subjected to MutMap analysis.
- RNA was extracted from a total of 16 types of axillary bud organs (n 3) using RNeasy Plant Mini Kit (Qiagen) and subjected to RNA-seq using NextSeq500 (Illumina). TruSeq (registered trademark) Stranded mRNA Library Prep (Illumina) was used to construct the RNA-seq library.
- Gene region prediction was performed by mapping RNA-seq reads to the aforementioned reference genome and combining machine prediction with CDS prediction based on the protein sequences of closely related plants.
- the Nicotiana sylvestris genome sequence constructed above was used as the reference genome.
- CLC Genomic Workbench (Qiagen) was used for quality control of sequence reads, mapping of sequence reads to the reference genome, and extraction of mutations.
- Fig. 3 shows the results of MutMap analysis.
- the region where the average value of SNP-index exceeded the upper limit of the 99% confidence interval was only in the range of approximately 2.55 Mb (24.47 to 27.02 Mb) on scaffold23.
- a total of 4.37 Mb (23.43 to 27.80 Mb) including this region and regions exceeding the 95% confidence interval existing before and after was considered as a candidate region (Fig. 3), and causative mutations were searched for.
- nsv1s000023m05199 and nsv1s000023m05424 were extracted as candidate genes.
- Table 4 shows the mutations present in the candidate genes and their expression levels. Mutation was present in nsv1s000023m05199 within 5 kb upstream of the gene, and the expression level was lower in LSC13 than in WT (Table 4).
- nsv1s000023m05424 had a mutation on the CDS, and the 692nd aspartic acid in the deduced amino acid sequence was replaced with asparagine (Table 4).
- nsv1s000023m05199 showed 100% sequence homology with XM_009795182.1 (Nicotiana sylvestris). Since XM_009795182.1 was annotated Magnesium (Mg)-chelatase subunit H (ChlH) in Nicotiana sylvestris, nsv1s000023g05199 was called NsChlH. In addition, when the genome sequence of tobacco cultivar (Nicotiana tabacum) "Tsukuba No.
- NsChlH-S two homologous genes corresponding to NsChlH (nttv1s031m00542 (referred to as NtabChlH-S, derived from the S subgenome) and nttv1s749m00829 (NtabChlH - termed T. derived from the T subgenome)) was identified.
- the CDS sequence of NtabChlH-S had 100% sequence identity with XM_016613992.1 (Nicotiana tabacum) and the CDS sequence of NtabChlH-T had 99.73% sequence identity with NM_001325713.1 (Nicotiana tabacum).
- Mg-chelatase is an enzyme responsible for the initial reaction of chlorophyll synthesis, and is known to coordinate magnesium ions to protoporphyrin IX to synthesize magnesium-protoporphyrin IX (Walker, J. C. et al. (1997) Biochem. J., 327(2), 321-333). Mg-chelatase is a complex protein consisting of three subunits (I, D, H) (Masuda, T. (2008) Photosyn. Res., 96(2), 121-143).
- Knockout or knockdown mutants of the unit-encoding gene are known to show a marked decrease in chlorophyll content, and the whole plant exhibits a white or pale green color (Zhang, D., et al. 2018) Front. Plant Sci. 9, 720).
- nsv1s000023m0542 has a CDS sequence (SEQ ID NO: 1) with 100% sequence identity with XM_009802651.1 (Nicotiana sylvestris) and 98.3% with GU361620.1 (Nicotiana benthamiana NbCA1). showed homology. Therefore, nsv1s000023m05424 was called NsCA1.
- a BLAST search was performed using the NsCA1 CDS sequence and amino acid sequence (SEQ ID NO: 2), and Tables 5 and 6 show the gene IDs of Nicotiana plants that resulted in hits.
- Amino acid sequence ID “XP — 016480446.1” in Table 6 corresponds to CDS sequence ID “XM — 016624960.1” in Table 5. Further, when the amino acid sequence of NsCA1 (SEQ ID NO: 2) was BLAST-searched on TAIR (The Arabidopsis Information Resource, https://www.arabidopsis.org/index.jsp), autoinhibited Ca 2+ -ATPase11 (ACA11 : At3g57330) had the highest sequence identity (70% amino acid sequence identity).
- ACA is a transmembrane protein that is localized in various intracellular membranes such as cell membrane, endoplasmic reticulum membrane, and tonoplast membrane, and transports calcium using energy from ATP hydrolysis (Baxter, I. (2003) Plant Physiol. 132(2) 618-628). It is known that there are 10 ACAs in Arabidopsis thaliana and 11 in rice (Baxter, I., et al. (2003) Plant Physiol. 132(2) 618-628).
- nsv1s000146m00783 was hit with 81% sequence identity and was considered to be a paralog of NsCA1. Therefore, nsv1s000146m00783 was named NsCA2.
- the CDS sequence and amino acid sequence of NsCA2 were BLAST-searched on the NCBI database, and the gene IDs of Nicotiana plants hit were shown in Tables 7 and 8. Amino acid sequence ID “XP — 016457617.1” in Table 8 corresponds to CDS sequence ID “XM — 016602132.1” in Table 7.
- NtabCA1-S nttv1s031m00301
- NtabCA1-T nttv1s749m00398
- the CDS sequence (SEQ ID NO: 3) and amino acid sequence (SEQ ID NO: 4) of NtabCA1-S were BLAST-searched on the NCBI database, and the gene IDs of Nicotiana plants that were hits are shown in Tables 9 and 10. Furthermore, the CDS sequence (SEQ ID NO: 5) and amino acid sequence (SEQ ID NO: 6) of NtabCA1-T were BLAST-searched on the NCBI database, and the gene IDs of Nicotiana plants that were hits are shown in Tables 11 and 12.
- NtabCA1-T (cultivar: Tsukuba No. 1) did not completely match the tobacco sequence XM_016624960.1 (cultivar: TN90) on the DB, but this was considered to be a difference between cultivars.
- NsCA2-S nttv1s264m00662
- NtabCA2-T nttv1s227m01082
- FIG. ACA2 (At4g37640) was used as an outgroup. ACA2 is known to be localized in the endoplasmic reticulum in Arabidopsis thaliana, and ACA11 and ACA4 are known to belong to phylogenetically different clades (Baxter, I., et al. (2003) Plant Physiol. 132 (2) 618-628).
- NsCA1 was considered to be a powerful causative gene that causes pseudo-lesion formation and hypersoravetivone in LSC13.
- RNAi strain Production of CA1 expression-suppressing strain (RNAi strain)
- NtabCA1 (NtabCA1-S and NtabCA1-T) and NtabChlH (NtabChlH-S and NtabChlH-T) expression-suppressing lines were produced by RNAi using Nicotiana tabacum "K326”.
- Total RNA was extracted from mature leaves of Nicotiana sylvestris using RNeasy Plant Mini Kit (Qiagen), and cDNA was synthesized using PrimeScriptTM RT reagent kit with gDNA Eraser (Takara Bio).
- the trigger sequence was amplified using PrimeSTAR (registered trademark) Max DNA Polymerase (Takara Bio Inc.). Table 17 shows the primer sequences used for the amplification of the RNAi trigger.
- a sequence (5'-CACC-3') necessary for cloning the amplified fragment is added to the 5' end of the forward primer (indicated with F at the end of the primer name).
- After confirming the sequence of the amplified fragment it was cloned into pENTRTM/D-TOPOTM Vector (Thermo Fisher).
- GatewayTM LR ClonaseTM Enzyme mix Thermo Fisher
- the trigger sequence was introduced into the binary vector pSP231 for RNAi.
- pSP231 is a pHellsgates12 (see Wesley et al., Plant J., 27, 581-590 (2001)) based vector and contains an expression unit for GFP.
- pSP231 is a binary vector that transcribes an RNAi sequence in which pdk/cat intron is flanked by inverted repeats of the trigger sequence using the cauliflower mosaic virus 35S RNA gene promoter.
- the pSP231 vector after sequence confirmation of the trigger portion was introduced into Agrobacterium LBA4404 by electroporation. After confirming the presence of the RNAi trigger sequence in the resulting transformed Agrobacterium by PCR, the Agrobacterium was used to transform plants. As a control, Agrobacterium into which pSP231 vector containing no trigger sequence was introduced was also used for transformation.
- Leaf segments were infected with Agrobacterium and cultured on Linsmaier and Skoog (LS) medium containing kanamycin (50 ⁇ g/mL). From the resulting callus, kanamycin-resistant redifferentiated individuals were obtained. Regarding the redifferentiated individuals, individuals showing GFP fluorescence in the entire leaf were grown in a plant box, and when they had grown sufficiently in the plant box, they were transplanted to a No. 4 pot and grown in a greenhouse (24° C.).
- TaqMan registered trademark
- Fast Advanced Master Mix Thermo Fisher
- the target genes were NtabCA1 and NtabCA2 in the NtabCA1-RNAi line, and NtabChlH in the NtabChlH-RNAi line.
- Table 18 shows the sequences of primers and probes used for quantitative PCR of each gene of NtabCA1, NtabCA2, NtabChlH, and elf ⁇ (internal standard gene). The primers and probes used were designed to detect both the S and T genes of each gene together.
- the transcript amount of NtabCA1 was reduced to about half compared to the control (line into which the pSP231 vector containing no trigger sequence was introduced), but the transcript amount of NtabCA2 was lower than that of the control.
- Four identical individuals were obtained (upper part of FIG. 5). All of the expression-suppressed individuals developed pseudo-lesions about one month after transplantation, and continued to develop pseudo-lesions thereafter (left side of FIG. 6). In addition, 3 individuals were obtained in which the expression of NtabChlH-RNAi was lower than that of the control (Fig. 5, bottom). In these three individuals, the entire plant body including leaves turned white (center of FIG. 6).
- NtabCA1-RNAi individuals 1 NtabChlH-RNAi individual and 3 control individuals, 1 to 5 true leaves or interleaved leaves were collected from each individual before flowering, and after removing the backbone, freeze-drying.
- Add 4 mL of methanol to 100 mg of freeze-dried leaves add 80 ⁇ L (0.4 mg / mL) of eicosane as an internal standard, extract the components by shaking at 130 rpm for 1 hour in a 15 mL centrifuge tube, and filter. .
- the three control plants were similarly extracted at a triple concentration (300 mg of freeze-dried leaves were extracted with 4 mL of methanol).
- GC-MS analysis was performed under the conditions shown in Table 2 to measure the soravetibon content.
- soravetivone was detected only in one individual extracted at a 3-fold concentration (internal standard ratio 0.058), and soravetivone was not detected in the other individuals.
- NtabCA1-RNAi detected soravetivone at an internal standard ratio of 0.109 to 0.377 (Fig. 7, Table 19).
- soravetivone was not detected in NtabChlH-RNAi individuals.
- Example 5 Production of CA1 expression-suppressing lines (RNAi lines) in Nicotiana sylvestris]
- NsCA1-RNAi line an NsCA1 expression suppression line (NsCA1-RNAi line) was produced by RNAi.
- the expression levels of NsCA1 and NsCA2 in redifferentiated individuals selected by the presence or absence of GFP fluorescence were confirmed according to the procedure of Example 4.
- the NsCA1 transcript level was reduced to less than half, but the NsCA2 transcript level was equivalent to that of the control. Two individuals were obtained (Fig. 8).
- NsCA1-RNAi lines were grown in a greenhouse (24° C.) in No. 4 pots. All of the NsCA1-RNAi lines developed pseudo-lesions similar to LSC13 about one month after transplantation to pots (upper right and lower right of FIG. 9). None of the three control (vector control) individuals formed pseudo-lesions like wild-type Nicotiana sylvestris (upper left and lower left in FIG. 9).
- the soravetivone content of the NsCA1-RNAi lines was increased 190- to 429-fold over the control (Table 20).
- the soravetivone content of the NsCA1-RNAi line was approximately the same as that of LSC13.
- the soravetivone content of the control was approximately the same as that of the wild-type Nicotiana sylvestris (Table 20).
- This library is a mixture of mutant inbred progeny seeds (M2 seeds) obtained for each 2000 EMS-treated current generation (M1 generation) and DNA extracted from 8 seedlings for each line grown from M2 seeds. Consists of bulk DNA.
- NtabCA1-s-1 and NtabCA1-t-1 were grown in a greenhouse, and NtabCA1-s-1 and NtabCA1-t-1 were crossed to obtain the F1 generation (NtabCA1-F1-1).
- NtabCA1-s-2 and NtabCA1-t-2 were grown in a greenhouse, and NtabCA1-s-2 and NtabCA1-t-2 were crossed to obtain the F1 generation (NtabCA1-F1-2).
- Two lines of F2 generation derived from different mutant lines were obtained by growing each F1 generation in the greenhouse and selfing (NtabCA1-F2-1 and NtabCA1-F2-2).
- the seeds of the F2 generation of the two lines were sown, DNA was extracted from the seedlings, and the sequences around the mutation were amplified by PCR using the primers in Table 23.
- This amplification product is further subjected to PCR to add the sequences (P7 sequence, P5 sequence and barcode sequence for each individual) used in sequencing using iSeq 100 (Illumina) to the amplification product, and the product is iSeq 100 (Illumina) to confirm the genotype of each individual.
- the NtabCA1-sstt individual had a maximum 44-fold increase in the soravetivone content compared to the control NtabCA1-SSTT individual. That is, in Nicotiana tabacum, suppression of the functions of both the NtabCA1-S gene and the NtabCA1-T gene was found to increase the soravetivone content.
- a Nicotiana plant with a high soravetivone content is provided.
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Abstract
Description
本発明の一実施形態は、配列番号2に示されるアミノ酸配列に対する95%以上の配列同一性を有しているポリペプチドをコードしているポリヌクレオチドを、コード領域として含んでいる内在性遺伝子、配列番号4に示されるアミノ酸配列に対する95%以上の配列同一性を有しているポリペプチドをコードしているポリヌクレオチドを、コード領域として含んでいる内在性遺伝子、および配列番号6に示されるアミノ酸配列に対する95%以上の配列同一性を有しているポリペプチドをコードしているポリヌクレオチドを、コード領域として含んでいる内在性遺伝子の少なくともいずれかの機能抑制を引き起こす変異がゲノムに導入されている、タバコ属植物体(ただし、上記機能抑制が、野生型植物と比べたときの、RNAiまたはVIGSによる上記内在性遺伝子から転写されたmRNAの分解の促進である場合には、ニコチアナ・ベンサミアナを除く)を提供する。
(a):配列番号2に示されるアミノ酸配列に対する95%以上の配列同一性を有しているポリペプチドをコードしているポリヌクレオチドを、コード領域として含んでいる内在性遺伝子;
(b):配列番号4に示されるアミノ酸配列に対する95%以上の配列同一性を有しているポリペプチドをコードしているポリヌクレオチドを、コード領域として含んでいる内在性遺伝子;
(c):配列番号6に示されるアミノ酸配列に対する95%以上の配列同一性を有しているポリペプチドをコードしているポリヌクレオチドを、コード領域として含んでいる内在性遺伝子;
(d):(a)および(b)の組合せ
(e):(a)および(c)の組合せ
(f):(b)および(c)の組合せ
本発明の一実施形態は、配列番号2に示されるアミノ酸配列に対する95%以上の配列同一性を有しているポリペプチドをコードしているポリヌクレオチドを、コード領域として含んでいる内在性遺伝子、ならびに配列番号4および配列番号6の少なくとも一方に示されるアミノ酸配列に対する95%以上の配列同一性を有しているポリペプチドをコードしているポリヌクレオチドを、コード領域として含んでいる内在性遺伝子のいずれか一方の機能抑制を引き起こす変異を、タバコ属植物体のゲノムに導入する工程を含んでいる、タバコ属植物体(ただし、上記機能抑制が、野生型植物と比べたときの、RNAiまたはVIGSによる上記内在性遺伝子から転写されたmRNAの分解の促進である場合には、ニコチアナ・ベンサミアナを除く)の製造方法を提供する。限定されるものではないが、上記変異をタバコ属植物体のゲノムに導入する工程は、ゲノム中の当該内在性遺伝子に導入する工程であってよい。タバコ属植物体に導入される変異の詳細は、項目〔1.タバコ属植物体〕に記載されている。
本発明の一実施形態は、高ソラベチボン含量のタバコ属植物体の決定方法を提供する。当該決定方法は以下の工程を包含する:
タバコ属植物体の一部を採取することによって、サンプルを入手する工程;
当該サンプルに含まれているゲノム上にある上記内在性遺伝子の機能抑制を引き起こす変異を検出する工程;および
上記変異が検出されたタバコ属植物体を、高ソラベチボン含量のタバコ属植物体として決定する工程。
以上の各実施形態をまとめると、本発明は、以下のように要約され得る。
栽培タバコ、ニコチアナ・タバカム(染色体数:2n=4x=48)は二倍体のタバコ野生種ニコチアナ・シルベストリス(染色体数:2n=2x=24)およびこれもまた二倍体のタバコ野生種ニコチアナ・トメントシフォルミス(染色体数:2n=2x=24)の交雑に由来する複二倍体であり、それぞれの親に由来する2つのサブゲノム(ニコチアナ・シルベストリス由来のサブゲノムおよびニコチアナ・トメントシフォルミス由来のサブゲノムをそれぞれ「S」または「Sゲノム」および「T」または「Tゲノム」と称する)を有する。そのため、ニコチアナ・タバカムにおいて、多くの遺伝子について同じ機能を有する遺伝子が2コピー存在しており、標的とする遺伝子の機能喪失変異を表現型として観察するためには、一般に機能が重複する2つの遺伝子の両方を変異させる必要がある。
LSC13の遺伝様式を調べるため、WT(ニコチアナ・シルベストリス)と交配したF1世代を作出し、さらに自殖してF2世代を得た。120個体のF2個体を温室にて育成し、表現型の分離比を確認した。対照としてWT、LSC13、およびF1をそれぞれ5個体、同条件で育成した。播種から3週間後にビニポットへ仮植し、さらに1か月後に3号鉢へ移植した。移植後およそ6週間経過した時点で、WTおよびF1の各5個体には擬似病斑が確認されなかったが、LSC13の5個体にはすべて擬似病斑が確認された(図1の上段)。120個体のF2個体では、28個体で擬似病斑が確認され、残りの92個体では擬似病斑は確認されなかった(図1の下段)。ソラベチボン含量を分析するため、WT、LSC13、およびF1の各5個体、およびF2個体のうち擬似病斑の確認された28個体および擬似病斑が確認されなかった92個体からランダムに選んだ28個体をGC-MS分析に供試した。各個体の葉を直径4cmの金属製円筒を用いて採取し、20mLのメタノールを加え130rpmで1時間振とうした。内部標準として400μLのエイコサン(eicosane)(0.4mg/mL)を加えた。抽出液をフィルターろ過後、表2の条件でGC-MS分析を行った。
3-1.リファレンスゲノムの構築
MutMap解析に先立ち、ニコチアナ・シルベストリスのリファレンスゲノムを構築した。ニコチアナ・シルベストリスの葉からCTAB(cetyl trimethyl ammonium bromide)法に従ってゲノムDNAを抽出した。このゲノムDNAを、次世代シーケンサーを用いたヌクレオチド配列解析に供して、ゲノム配列を構築し、これをリファレンスゲノムとして使用した。構築したリファレンスゲノムは、3,518本のスカフォールド(scaffold)から構成され、N50は48.7Mbであった。
上記の通り作製したLSC13とニコチアナ・シルベストリスとを交雑したF2集団120個体は、28個体がLSC13型の表現型を示した。この28個体のうち、上位葉においてより擬似病斑の発生が顕著な20個体のゲノムDNAをそれぞれ等量ずつ混合し、バルクDNAを作製した。バルクDNAを、HiSeq X(イルミナ社)を用いたペアエンドシーケンスに供し、およそ50.48Gbのシーケンスデータを得た。得られたシーケンスデータを用いてMutMap解析を実施した。
ニコチアナ・タバカム「K326」を用いてRNAiによるNtabCA1(NtabCA1-SおよびNtabCA1-T)およびNtabChlH(NtabChlH-SおよびNtabChlH-T)の発現抑制系統を作出した。ニコチアナ・シルベストリスの成熟期の葉からRNeasy Plant Mini Kit(キアゲン社)を用いてtotal RNAを抽出し、PrimeScript(商標)RT reagent kit with gDNA Eraser(タカラバイオ社)を用いてcDNAを合成した。合成したcDNAを鋳型に、PrimeSTAR(登録商標)Max DNA Polymerase(タカラバイオ社)を用いてトリガー配列を増幅した。RNAiのトリガーの増幅に用いたプライマー配列を表17に示した。なお、フォワードプライマー(プライマー名の最後にFと表記)の5’末端には増幅した断片のクローニングに必要な配列(5’-CACC-3’)が付与されている。増幅断片の配列確認後、pENTR(商標)/D-TOPO(登録商標)Vector(サーモフィッシャー社)にクローニングした。さらに、Gateway(商標)LR Clonase(商標)Enzyme mix(サーモフィッシャー社)を用いて、トリガー配列をRNAi用のバイナリーベクターpSP231に導入した。pSP231は、pHellsgates12(Wesley et al., Plant J., 27, 581-590 (2001)を参照のこと)を基礎としたベクターであり、GFPの発現ユニットを含む。pSP231は、トリガー配列の逆位反復配列がpdk/catイントロンを挟む型のRNAi配列を、カリフラワーモザイクウイルス35SRNA遺伝子プロモーターで転写するバイナリーベクターである。トリガー部分の配列確認後のpSP231ベクターをエレクトロポレーションによりアグロバクテリウムLBA4404に導入した。得られた形質転換アグロバクテリウムにおいて、RNAiトリガー配列の存在をPCRによって確認した後に、当該アグロバクテリウムを用いて植物の形質転換を実施した。対照としてトリガー配列を含まないpSP231ベクターを導入したアグロバクテリウムも形質転換に用いた。
実施例4の手順に従い、ニコチアナ・シルベストリスを用いて、RNAiによるNsCA1の発現抑制系統(NsCA1-RNAi系統)を作出した。GFP蛍光の有無により選抜した再分化個体におけるNsCA1およびNsCA2の発現量の確認を、実施例4の手順に従い実施した。その結果、対照(Control;トリガー配列を含まないpSP231ベクターが導入された系統)と比較してNsCA1の転写物量は半分以下に低下しているが、NsCA2の転写物量は対照と同等である個体を2個体取得した(図8)。これらNsCA1-RNAi系統2個体、対照系統3個体、野生型ニコチアナ・シルベストリス5個体、および4個体のLSC13を、4号鉢を用いて温室(24℃)にて育成した。NsCA1-RNAi系統はいずれも、鉢への移植後1か月程度経過した頃からLSC13と同様の擬似病斑が発生した(図9右上・右下)。対照(ベクターコントロール)の3個体はいずれも野生型のニコチアナ・シルベストリスと同様に擬似病斑を形成しなかった(図9左上・左下)。
タバコ栽培種(ニコチアナ・タバカム)「つくば1号」の種子にEMS処理を行って作製した2000系統のタバコ変異体ライブラリー(田島ら、平成23年度日本植物病理学会大会、P234、タバコ変異体パネルの作出)についてNtabCA1-S遺伝子およびNtabCA1-T遺伝子の遺伝子領域のヌクレオチド配列を解析し、いずれかの遺伝子に変異のある変異体を同定した。本ライブラリーは2000個体のEMS処理当代(M1世代)ごとに得られた変異体自殖後代種子(M2種子)と、M2種子から育成した各系統当たり8個体の幼苗から抽出したDNAを混和したバルクDNAからなる。
Claims (25)
- 配列番号2に示されるアミノ酸配列に対する95%以上の配列同一性を有しているポリペプチドをコードしているポリヌクレオチドを、コード領域として含んでいる内在性遺伝子、
配列番号4に示されるアミノ酸配列に対する95%以上の配列同一性を有しているポリペプチドをコードしているポリヌクレオチドを、コード領域として含んでいる内在性遺伝子、および
配列番号6に示されるアミノ酸配列に対する95%以上の配列同一性を有しているポリペプチドをコードしているポリヌクレオチドを、コード領域として含んでいる内在性遺伝子の少なくともいずれかの機能抑制を引き起こす変異がゲノムに導入されている、タバコ属植物体(ただし、上記機能抑制が、野生型植物と比べたときの、RNAiまたはVIGSによる上記内在性遺伝子から転写されたmRNAの分解の促進である場合には、ニコチアナ・ベンサミアナを除く)。 - 上記機能抑制は、野生型植物と比べたときの、上記内在性遺伝子から転写されたmRNAの存在量の減少である、請求項1に記載のタバコ属植物体。
- 上記機能抑制は、上記内在性遺伝子から転写されたmRNAの分解の促進である、請求項1または2に記載のタバコ属植物体。
- 上記変異は、上記内在性遺伝子に導入されている、請求項1~3のいずれか1項に記載のタバコ属植物体。
- 上記変異は、変異原処理、ゲノム編集または遺伝子ノックアウトによって導入されている、請求項4に記載のタバコ属植物体。
- 上記変異は、上記変異原処理によって導入されている、請求項5に記載のタバコ属植物体。
- 上記変異は、上記mRNAの分解を促進する因子を発現するポリヌクレオチドの、上記内在性遺伝子の存在する領域外への挿入である、請求項3に記載のタバコ属植物体。
- 上記因子は、アンチセンスRNA分子、RNAi分子または共抑制分子である、請求項7に記載のタバコ属植物体。
- 上記機能抑制は、野生型植物と比べたときの、本来の機能的な上記ポリペプチドの存在量の減少である、請求項1~8のいずれか1項に記載のタバコ属植物体。
- 上記機能抑制は、野生型植物と比べたときの、本来の機能的な上記ポリペプチドの翻訳量の減少である、請求項9に記載のタバコ属植物体。
- ニコチアナ・タバカム、ニコチアナ・シルベストリスまたはニコチアナ・ルスチカに属する、請求項1~10のいずれか1項に記載のタバコ属植物体。
- タバコ属植物体の製造方法であって、
配列番号2に示されるアミノ酸配列に対する95%以上の配列同一性を有しているポリペプチドをコードしているポリヌクレオチドを、コード領域として含んでいる内在性遺伝子、
配列番号4に示されるアミノ酸配列に対する95%以上の配列同一性を有しているポリペプチドをコードしているポリヌクレオチドを、コード領域として含んでいる内在性遺伝子、および
配列番号6に示されるアミノ酸配列に対する95%以上の配列同一性を有しているポリペプチドをコードしているポリヌクレオチドを、コード領域として含んでいる内在性遺伝子の少なくともいずれかの機能抑制を引き起こす変異を、タバコ属植物体のゲノムに導入する工程を含んでいる、タバコ属植物体(ただし、上記機能抑制が、野生型植物と比べたときの、RNAiまたはVIGSによる上記内在性遺伝子から転写されたmRNAの分解の促進である場合には、ニコチアナ・ベンサミアナを除く)の製造方法。 - 上記機能抑制は、野生型植物と比べたときの、上記内在性遺伝子から転写されたmRNAの存在量の減少である、請求項12に記載の製造方法。
- 上記機能抑制は、上記内在性遺伝子から転写されたmRNAの分解の促進である、請求項12または13に記載の製造方法。
- 上記導入する工程が、上記内在性遺伝子に上記変異を導入することを含んでいる、請求項12~14のいずれか1項に記載の製造方法。
- 上記導入する工程が、変異原処理、遺伝子の組換え、ゲノム編集または遺伝子ノックアウトによって実施される、請求項15に記載の製造方法。
- 上記変異は、上記変異原処理によって導入されている、請求項16に記載のタバコ属植物体。
- 上記導入する工程が、上記内在性遺伝子から転写されたmRNAの分解を促進する因子を発現するポリヌクレオチドを、当該内在性遺伝子の存在する領域外に挿入することを含んでいる、請求項12~15のいずれか1項に記載の製造方法。
- 上記因子が、アンチセンスRNA分子、RNAi分子または共抑制分子である、請求項18に記載の製造方法。
- 上記機能抑制は、野生型植物と比べたときの、本来の機能的な上記ポリペプチドの存在量の減少である、請求項12~19のいずれか1項に記載の製造方法。
- 上記機能抑制は、野生型植物と比べたときの、本来の機能的な上記ポリペプチドの翻訳量の減少である、請求項20に記載の製造方法。
- 請求項1~11のいずれか1項に記載のタバコ属植物体、または請求項11~19のいずれか1項に記載の製造方法によって得られたタバコ属植物体の、子孫または当該タバコ属植物体の交配によって得られた育種後代。
- 配列番号2に示されるアミノ酸配列に対する95%以上の配列同一性を有しているポリペプチドをコードしているポリヌクレオチドを、コード領域として含んでいる内在性遺伝子、
配列番号4に示されるアミノ酸配列に対する95%以上の配列同一性を有しているポリペプチドをコードしているポリヌクレオチドを、コード領域として含んでいる内在性遺伝子、および
配列番号6に示されるアミノ酸配列に対する95%以上の配列同一性を有しているポリペプチドをコードしているポリヌクレオチドを、コード領域として含んでいる内在性遺伝子の少なくともいずれかの機能抑制を引き起こす変異がゲノムに導入されているタバコ属植物体(ただし、上記機能抑制が、野生型植物と比べたときの、RNAiまたはVIGSによる上記内在性遺伝子から転写されたmRNAの分解の促進である場合には、ニコチアナ・ベンサミアナを除く)の葉たばこであって、
野生型のタバコ属植物体の葉たばこよりもソラベチボン含量が高い、葉たばこ。 - 配列番号2に示されるアミノ酸配列に対する95%以上の配列同一性を有しているポリペプチドをコードしているポリヌクレオチドを、コード領域として含んでいる内在性遺伝子、
配列番号4に示されるアミノ酸配列に対する95%以上の配列同一性を有しているポリペプチドをコードしているポリヌクレオチドを、コード領域として含んでいる内在性遺伝子、および
配列番号6に示されるアミノ酸配列に対する95%以上の配列同一性を有しているポリペプチドをコードしているポリヌクレオチドを、コード領域として含んでいる内在性遺伝子の少なくともいずれかの機能抑制を引き起こす変異がゲノムに導入されているタバコ属植物体(ただし、上記機能抑制が、野生型植物と比べたときの、RNAiまたはVIGSによる上記内在性遺伝子から転写されたmRNAの分解の促進である場合には、ニコチアナ・ベンサミアナを除く)の乾燥葉であって、
野生型のタバコ属植物体の乾燥葉よりもソラベチボン含量が高い、乾燥葉。 - 請求項24に記載の乾燥葉を含んでいる、たばこ製品。
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