WO2017002977A1 - 遺伝的組換えを誘発する方法及びその利用 - Google Patents
遺伝的組換えを誘発する方法及びその利用 Download PDFInfo
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- WO2017002977A1 WO2017002977A1 PCT/JP2016/069721 JP2016069721W WO2017002977A1 WO 2017002977 A1 WO2017002977 A1 WO 2017002977A1 JP 2016069721 W JP2016069721 W JP 2016069721W WO 2017002977 A1 WO2017002977 A1 WO 2017002977A1
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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
- 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
- C12N15/8283—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 for virus 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
- 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
Definitions
- biomass Increasing the mass (biomass) of biological resources, especially the increase in plant biomass, can be said not only to increase food production, but also to protect the global environment, prevent global warming, and reduce greenhouse gas emissions. Therefore, the technology for increasing plant biomass production and the creation of useful plants are extremely important.
- microorganisms are effectively used in various industries. For example, in bioethanol production using polysaccharides such as cellulose, yeast with characteristics such as high temperature resistance, high alcohol concentration resistance, and high alcohol synthesis ability is expected to perform ethanol fermentation at a lower cost. .
- the characteristics of useful plants and microorganisms are generally quantitative traits that are influenced by the expression of a large number of genes, not a single gene.
- normal mutation treatment requires treatment for a large number of generations with small changes in traits due to a single operation.
- Patent Documents 1, 2, and 3 a so-called restriction enzyme is transiently expressed in a cell to induce DNA breakage of the entire genome, realizing a large number of genome rearrangements at the same time. It has been reported that the mutant population can be efficiently obtained.
- chromosome doubling has an important significance in improving plant varieties.
- wheat is cultivated from diploid single-grain wheat to hexaploid normal wheat.
- Such genome doubling is thought to have led to the acquisition of agriculturally suitable properties such as improved productivity and threshability.
- Creation of resistant plants by artificially inducing genome doubling (patent document 4) and creation of yeast with improved stress tolerance by artificially inducing chromosomal aneuploidy (non-patent document 1) have been reported. ing.
- Non-patent Document 2 chromosomal aneuploidy, which improves ploidy only in certain chromosomes, has been reported to cause various genetic diseases in animals, and to induce fertility reduction and growth abnormalities in plants.
- Non-patent Document 4 a tetraploid-specific lethal gene has been reported in the filing yeast (Non-patent Document 4).
- non-patent Document 5 in order to select yeast having excellent traits in yeast, continuous culture for 1000 generations or more is required (Non-patent Document 5).
- the present specification provides a method for inducing genetic recombination and use thereof.
- the present inventors focused on the combination of chromosome doubling and DNA double-strand break treatment in order to create a eukaryotic population having a higher degree of diversity. And it discovered that various genetic recombination was realizable by making the protein which has DNA double strand cutting
- a method for inducing genetic recombination in a eukaryotic organism A genome set modification step in which a protein having a DNA double-strand break activity is allowed to act in cells of a eukaryotic organism that is a polyploid exceeding the ploidy inherently possessed by the eukaryotic organism;
- a method comprising: (2) Genetic recombination involves gene mutation by substitution, insertion and deletion of one or more bases, as well as chromosome inversion, inequality crossover, crossover, translocation, duplication, deletion, size copy number
- the method according to (1) which is one or two or more selected from the group consisting of a decrease in the number of copies, an increase in copy number, chromosomal doubling and chromosomal aneuploidy.
- the modifying step is a step of artificially activating the DNA double-strand break activity of the protein.
- the modifying step is a step of using the protein obtained by expression of a foreign gene encoding the protein.
- the protein is a frequent restriction enzyme.
- the frequent restriction enzyme is a restriction enzyme derived from a thermophilic bacterium.
- the frequent restriction enzyme is TaqI.
- the induction method according to (15), wherein the doubling step uses a doubling inducer.
- a method for producing a modified eukaryotic organism A modification step in which a protein having DNA double-strand breakage activity is allowed to act in the cells of a parent eukaryote that is a polyploid that exceeds the ploidy inherently possessed by the eukaryote.
- a method comprising: (19) Furthermore, a step of selecting an intended eukaryotic organism based on an arbitrary index,
- the production method according to (18), comprising: (20) A method for producing a eukaryotic population, A modification step of modifying a genome set of a parent eukaryote by allowing a protein having a DNA double-strand break activity to act in the cells of the parent eukaryote that is a polyploid that exceeds the inherent ploidy; A production method for obtaining a population of eukaryotic organisms having a modified genome set.
- a production method comprising: (21) A breeding material that is a eukaryotic organism that retains a gene encoding a protein having a ploidy exceeding the inherent ploidy and having a DNA double-strand break activity. (22) A method for producing breeding materials, A step of obtaining an eukaryotic organism having a desired polyploidy by repeating an artificial genome expansion operation one or more times for a eukaryotic organism that is a polyploid exceeding the inherent ploidy; Transforming the desired polyploid eukaryote to express a gene encoding a protein having DNA double-strand break activity; A method comprising: (23) A method for producing breeding materials, Transforming a eukaryotic organism that is a polyploid that inherently has a polyploidy so as to express a gene encoding a protein having DNA double-strand cleavage activity; Repeating the artificial genome expansion operation for the transformed eukaryotic organism one or more times to obtain a desired euploid transformed euk
- FIG. 2 is a diagram showing an outline of the structure of a TaqI gene yeast expression vector. It is a figure which shows the construction
- the disclosure of this specification relates to a method for inducing genetic recombination and use thereof.
- An outline of the method for inducing genetic recombination disclosed in this specification is illustrated in FIG.
- this induction method is performed in cells of a eukaryotic organism having a ploidy that exceeds the ploidy inherent in a eukaryotic organism (also referred to herein as intrinsic ploidy). Then, the genome set of a eukaryote is altered by the action of a protein having DNA double-strand break activity.
- a ploidy set of eukaryotic organisms can include various genetic recombination as a result of genetic recombination resulting from cleavage at various sites by DNA double-strand break enzymes.
- each eukaryotic organism retains a different genome set by genetic recombination, it is possible to construct a eukaryotic population that is rich in genome set composition.
- the eukaryotic organism population rich in the genome set composition thus obtained becomes a eukaryotic organism population rich in the diversity of traits at the same time.
- a eukaryotic organism that is a polyploid exceeding the intrinsic ploidy since a eukaryotic organism that is a polyploid exceeding the intrinsic ploidy is used, lethal individuals are less likely to occur due to large-scale deletion of chromosomes.
- the increase in the number of chromosomes facilitates the occurrence of chromosomal aneuploidy and can increase the frequency with which large-scale genome rearrangements are induced.
- the genome set obtained by such genetic recombination can include various mutations in addition to large changes such as chromosomal aneuploidy and deletion regions. For this reason, according to this production method, a population composed of eukaryotic individuals having various traits can be obtained.
- polyploids exceeding the intrinsic ploidy such as tetraploids and the like have been said to be difficult to breed mutations such as mutagenic drugs and gamma rays because genes are duplicated and recessive mutations are easily concealed.
- a protein having DNA double-strand break activity to act in these polyploid cells, it is possible to suppress the conventional disadvantages of polyploids and to make use of their advantages to enable more diverse genetic recombination.
- eukaryotic populations having various genome set compositions and traits can be obtained.
- Such a method for producing a diverse eukaryotic population is a useful tool for promoting the breeding of eukaryotic organisms.
- This eukaryotic population consists of eukaryotic organisms that have acquired new traits that can occur in the course of evolution, loss of traits, eukaryotic organisms that are equipped with deterioration and improvement of traits, etc. It is considered to be a group of eukaryotic organisms that can be provided with various forms of loss, reduction, modification and the like.
- the intended eukaryotic organism can be efficiently obtained by performing selection based on a desired index for the eukaryotic organism population obtained by the method of the present disclosure.
- genomic set refers to DNA that exists as chromosomal DNA in eukaryotic cells, is capable of self-replication in eukaryotic cells, and is transmitted to daughter cells.
- “genetic recombination” means, in a broad sense, a DNA cleavage / recombination phenomenon that occurs between DNAs. Therefore, “genetic recombination” in the present specification includes homologous recombination and non-homologous recombination. Furthermore, “genetic recombination” in the present specification includes gene mutation and chromosomal inversion, inequality crossover, crossover, translocation, duplication, deletion by substitution of one or more bases, insertion and deletion, etc. Chromosomal mutations such as loss, copy number reduction, copy number increase, chromosome doubling and chromosomal aneuploidy.
- “Ploidy” generally means the number of genome sets that an organism has.
- the “polyploid” is generally a method for expressing an organism by the number of genome sets that the organism has, and includes haploids, diploids, triploids, and the like.
- the “polyploidy inherent in a eukaryotic organism” means the number of chromosomes corresponding to one set of genomes in the eukaryotic organism inherently in the eukaryotic organism (basic Number) of genome sets. In this specification, it is also referred to as “intrinsic ploidy”.
- intrinsic ploidy includes what is considered to be the number of genome sets inherently possessed in the same or related species of the eukaryotic organism. For example, in general, since animals are diploid, the intrinsic ploidy is diploid. The inherent ploidy of plants varies. In addition, microorganisms are, for example, haploid and diploid such as yeast, but the intrinsic ploidy is diploid considering the life cycle.
- a polyploid exceeding the intrinsic ploidy means a eukaryotic organism having a genome set having a multiple exceeding the intrinsic ploidy.
- Polyploid exceeding intrinsic ploidy generally includes polyploids exceeding diploid in animals. In plants, various polyploids exist.
- the “polyploid exceeding the intrinsic ploidy” may be a homogeneous polyploid or a heteroploid derived from a hybrid or the like.
- the “polyploid exceeding the intrinsic ploidy” may be an aneuploid having an aneuploidy in which the number of some chromosomes is mutated, in addition to an integer polyploid.
- the ploidy of a eukaryotic chromosome can be determined by a conventionally known method, or can be determined by a flow cytometer or a tiling array as shown in Examples described later.
- the method for inducing genetic recombination of the present disclosure (hereinafter simply referred to as an inducing method) is a DNA double-strand break activity in a cell of a eukaryotic organism that is a polyploid exceeding the intrinsic ploidy of the eukaryote.
- the present induction method allows various genetic recombination in individual eukaryotic organisms, and as a result, obtains eukaryotic populations that retain the modified genome set. can do.
- this protein when allowed to act in cells of a parent eukaryotic organism originally having a single genome set, a population consisting of a plurality of eukaryotic objects that are diverse in terms of genome set composition and traits can be obtained. Can do.
- This induction method is applicable to any eukaryotic organism.
- eukaryotic organisms to be applied to this induction method include animals, plants, and eukaryotic microorganisms.
- the animal is not particularly limited, and examples thereof include mammals and non-mammals such as various fishes.
- derivation method what is derived from an animal should just be used, and any forms, such as a cell, a structure
- the plant which belongs to a dicotyledonous plant a monocotyledonous plant, for example, a Brassicaceae, Gramineae, Solanum, Legume, Willowaceae etc. ).
- Brassicaceae Arabidopsis thaliana, Brassica rapa, Brassica napus, Cabbage (Brassica oleracea var. Capitata), Chinese cabbage (Brassica rapa var. Pekinensis), Chingen rhinoceros (Brassica rapa var. Chinensis), turnip rapa), Nozawana (Brassica rapa var. hakabura), Mizuna (Brassica rapa var. lancinifolia), Komatsuna (Brassica rapa var. peruviridis), Pakchoi (Brassica rapa var.
- Eggplant family tobacco (Nicotiana tabacum), eggplant (Solanum melongena), potato (Solaneum tuberosum), tomato (Lycopersicon lycopersicum), red pepper (Capsicum annuum), petunia (Petunia) and the like.
- Legumes Soybean (Glycine max), pea (Pisum sativum), broad bean (Vicia faba), wisteria (Wisteria floribunda), groundnut (Arachis. Hypogaea), Lotus corniculatus var.
- Cucurbitaceae Pumpkin (Cucurbita maxima, Cucurbita moschata, Cucurbita pepo), cucumber (Cucumis sativus), crow cucumber (Trichosanthes cucumeroides), gourd (Lagenaria siceraria var. Gourda), etc. Rosaceae: Almond (Amygdalus communis), Rose (Rosa), Strawberry (Fragaria), Sakura (Prunus), Apple (Malus pumila var. Domestica), etc. Dianthus: Carnation (Dianthus caryophyllus). Willow: Poplar (Populus trichocarpa, Populus nigra, Populus tremula).
- Gramineae corn (Zea mays), rice (Oryza sativa), barley (Hordeum vulgare), wheat (Triticum aestivum), bamboo (Phyllostachys), sugar cane (Saccharum officinarum), napiergrass (Pennisetum pupureum), elian raven (Erian) ), Miscanthus virgatum, Sorghum switch glass (Panicum), etc.
- Myrtaceae Eucalyptus (Eucalyptus camaldulensis, Eucalyptus grandis), etc.
- the plant applied to this induction method may be a plant derived from a plant, but any plant that retains the ability to regenerate into a complete plant is convenient for obtaining a modified plant. Accordingly, the plant may be in any form such as a cell, tissue, organ, seed, or callus.
- microorganisms are not particularly limited, but considering material production, microorganisms such as molds such as Neisseria gonorrhoeae and yeasts can be mentioned.
- molds such as Neisseria gonorrhoeae and yeasts
- Aspergillus include Aspergillus genus such as Aspergillus aculeatus and Aspergillus oryzae.
- yeast various known yeasts can be used, yeast of the genus Saccharomyces such as Saccharomyces cerevisiae, yeast of the genus Schizosaccharomyces pombe such as Saccharomyces cerevisiae, Yeast of Candida such as Candida shehatae, Yeast of Pichia such as Pichia stipitis, Yeast of Hansenula, Klocckera, Schwanniomyces And yeast of the genus Yarrowia, yeast of the genus Trichosporon, yeast of the genus Brettanomyces, yeast of the genus Pachysolen, yeast of the genus Yamadazyma, Kluyveromyces ), Kluyveloma Examples include yeasts belonging to the genus Kluyveromyces such as Kluveromyces lactis, and yeasts belonging to the genus Isatokenchia such as Issatchenkia orientalis. Of these, Saccharomyces
- a eukaryotic organism that is a polyploid exceeding the intrinsic ploidy is used.
- a eukaryotic organism that is a polyploid exceeding the intrinsic ploidy that induces genetic recombination can also be referred to as a parent eukaryotic organism.
- a polyploid exceeding the intrinsic ploidy can be used as the parent eukaryote.
- the polyploid exceeding the intrinsic ploidy for example, since the intrinsic ploidy of the animal is diploid, a polyploid exceeding the diploid can be used as the parent eukaryote.
- polyploids that exceed the ploidy of the plants can be used as parent eukaryotes. In microorganisms, for example, in yeast and the like, polyploids exceeding diploidy can be used as parent eukaryotes.
- the polyploid exceeding the intrinsic ploidy may be a wild type.
- a diploid is present as a wild type such as wheat
- a wild type wheat exceeding diploid that is, a tetraploid wheat and a hexaploid wheat are also present as a wild type, such tetraploid or higher Wheat
- a parent eukaryotic organism a eukaryotic organism obtained by artificially performing an operation for increasing the genome size can also be used.
- the parent eukaryote is preferably a tetraploid or higher polyploid.
- the tetraploid or higher polyploid include tetraploid, pentaploid, hexaploid, heploid, and octaploid. According to the present disclosure, even if the ploidy of the chromosome is high, genetic size recombination is avoided by avoiding inconveniences related to the size of the genome by genetic recombination through DNA double strand breaks. Diversified eukaryotic populations can be obtained.
- the intrinsic ploidy of the parent eukaryote is tetraploid or higher, the efficiency of genetic recombination increases, and it becomes possible to construct a eukaryote population that is remarkably excellent in diversity. More preferably, it is pentaploid, more preferably hexaploid, still more preferably heploid, and even more preferably octaploid or more.
- the parent eukaryotic organism may be a parent eukaryotic organism that has been artificially increased in genome size so as to exceed the intrinsic ploidy.
- the parent eukaryote obtained in advance or obtained by performing an artificial genome size increasing operation that already exists may be subjected to a modification step.
- a process for obtaining a polyploid parent eukaryote having a ploidy exceeding the intrinsic ploidy by performing a genome size increasing operation on the eukaryote to be a parent eukaryote is obtained.
- the modification step may be performed on the parent eukaryotic organism.
- Artificial genome expansion procedures include cell fusion using various cells, suppression of meiosis by temperature and pressure in fertilized and unfertilized eggs in animals, mating in plants, and supply of chromosome doubling inducers such as colchicine Etc.
- examples include selection and separation of low-frequency zygotes among general heterothalism strains, methods using a mating type conversion system, cell fusion methods, and the like (Biochemical Experimental Method Vol. 39, Yeast Molecular Genetics Experimental Method). (Taiji Oshima, Academic Publishing Center)
- a person skilled in the art can appropriately apply a known technique to a eukaryotic organism in order to obtain a parent eukaryotic organism having a ploidy exceeding the intrinsic ploidy.
- the present protein that is, a protein having DNA double-strand breaking activity is allowed to act in the cells of the parent eukaryote.
- the protein is not particularly limited, but a known DNA double-strand cleaving enzyme can be typically used. Since this induction method allows large-scale genome reorganization using ploidy parent eukaryotes exceeding the intrinsic ploidy, the characteristics of DNA double-strand break activity of this protein (recognition site (ie, recognition site (ie, , Frequency) and the like are avoided or suppressed, and can be appropriately selected from known DNA double-strand cleaving enzymes.
- the cleavage site (recognition site) possessed by the DNA double-strand cleavage enzyme is not particularly limited. From the viewpoint of the efficiency of genetic recombination, a DNA double-strand cleaving enzyme called a so-called frequent restriction enzyme having a recognition site of about 4 to 6 bases on DNA is preferable. Since the number of cleavage sites in the genome contributes to genome reorganization efficiency, chromosomal DNA can be cleaved at a preferred frequency by using such a recognition site for the number of bases. For example, a DNA double-strand cleavage enzyme having a 4-base or 5-base recognition site is more preferable, and a 4-base recognition site is more preferable.
- Such DNA double-strand cleavage enzyme is not particularly limited, and examples include ApeKI, BsrI, BssKI, BstNI, BstUI, BtsCI, FatI, FauI, PhoI, PspGI, SmlI, TaqI, TfuI, TseI, Tsp45I, and TspRI. It is done.
- various known frequent enzymes such as Sse9I, MseI, DpnI and CviAII can be mentioned.
- a restriction enzyme a restriction enzyme derived from a thermophilic bacterium, which has an optimal temperature for DNA double-strand breakage activity in a region higher than the culture temperature of the parent eukaryotic cell. Is more preferable. This is because it is possible to activate the protein at any timing and reduce the activity by temperature treatment, which is advantageous for temporary action of the protein. In addition, such a protein can regulate its activity by temperature. Furthermore, such a protein can act on a relatively gentle DNA double-strand break activity. As such a protein, for example, a restriction enzyme having an optimal temperature for DNA double-strand cleavage activity of 50 ° C. or more and 80 ° C. or less can be used.
- the optimum temperatures include 50 ° C., 55 ° C., 60 ° C., 65 ° C., and 75 ° C. (all catalog values).
- the optimum temperature of the restriction enzyme can be selected based on (catalog value) based on catalogs of various sales companies. If the optimum temperature is less than 50 ° C., the DNA double-strand break activity may become too strong. If the optimum temperature exceeds 80 ° C., the DNA double-strand break activity may become too weak.
- the optimum temperature is preferably 55 ° C. or higher, more preferably 60 ° C. or higher, 62 ° C. or higher, or about 65 ° C.
- the optimum temperature is preferably 75 ° C. or lower, more preferably 70 ° C. or lower, and may be 68 ° C. or lower.
- the present protein can be appropriately selected from the following known restriction enzymes.
- ApeKI, BsrI, BssKI, BstNI, BstUI, BtsCI, FatI, FauI, PhoI, PspGI, SmlI, Sse9I, TaqI, TfuI, TseI, Tsp45I, TspRI are suitable as this protein.
- a restriction enzyme As a restriction enzyme.
- this protein In order for this protein to act in the cells of the parent eukaryote, at least this protein is present in the cells.
- the protein is inherently present in the cell, but is preferably supplied from the outside.
- This protein may be supplied directly to the cells of the parent eukaryotic organism, or an expression vector capable of expressing the gene encoding the protein is supplied to the cells of the parent eukaryotic organism for transformation. Also good.
- the present protein may be allowed to act via expression induction in the cells of the parent eukaryote. By carrying out like this, the effect
- a vector for expressing the present protein in the cells of the parent eukaryote can construct a vector for expressing the present protein in the cells of the parent eukaryote according to the type of cell and the transformation method as appropriate by a conventionally known method.
- the base sequence encoding this protein can be obtained from various databases.
- it does not specifically limit It is preferable to accompany the nuclear translocation signal useful in the eukaryotic organism to be used.
- various conventionally known vectors can be used as a base vector for an expression vector for expressing a protein in a plant cell.
- a plasmid, phage, cosmid or the like can be used, and can be appropriately selected according to the plant cell to be introduced and the introduction method.
- Specific examples include pBR322, pBR325, pUC19, pUC119, pBluescript, pBluescriptSK, and pBI vectors.
- the method for introducing a vector into a plant is a method using Agrobacterium, it is preferable to use a pBI binary vector.
- the pBI binary vector include pBIG, pBIN19, pBI101, pBI121, pBI221, and the like.
- the promoter is not particularly limited as long as it is a promoter capable of expressing a restriction enzyme gene in a plant body, and a known promoter can be suitably used.
- promoters include cauliflower mosaic virus 35S promoter (CaMV35S), various actin gene promoters, various ubiquitin gene promoters, nopaline synthase gene promoter, tobacco PR1a gene promoter, tomato ribulose 1,5-diphosphate carboxylase Oxidase small subunit gene promoter, napin gene promoter, etc.
- the promoter has a lower expression intensity than the 35S promoter, such as an SIG2 (AtSIG2) promoter derived from an Arabidopsis sigma factor.
- the expression vector may further contain other DNA segments as appropriate in addition to the promoter and the restriction enzyme gene.
- the other DNA segment is not particularly limited, and examples thereof include a terminator, a selection marker, an enhancer, and a base sequence for improving translation efficiency.
- the recombinant expression vector may further have a T-DNA region.
- the T-DNA region can increase the efficiency of gene transfer particularly when Agrobacterium is used to introduce the recombinant expression vector into a plant body.
- the transcription terminator is not particularly limited as long as it has a function as a transcription termination site, and may be a known one.
- the transcription termination region (Nos terminator) of the nopaline synthase gene the transcription termination region of the cauliflower mosaic virus 35S (CaMV35S terminator) and the like can be preferably used.
- the Nos terminator can be more preferably used.
- a selection marker and a base sequence for improving translation efficiency known elements can be appropriately selected and used.
- the method for constructing the expression vector is not particularly limited, and necessary elements may be appropriately introduced into the appropriately selected parent vector.
- Such an expression vector is introduced into a plant cell so that the protein is expressed transiently or constantly.
- an expression vector is physically introduced into a plant cell as a plasmid or the like using a PEG method, an electroporation method and a particle gun method. For constant expression, it is integrated into the plant genome using the Agrobacterium method or the like.
- the Agrobacterium method is preferably applied to dicotyledonous plants, especially Arabidopsis thaliana.
- an expression vector suitable for yeast may be similarly constructed and introduced into yeast.
- An expression vector can be constructed by a person skilled in the art using a known method, using a promoter, a terminator, and other enhancers as appropriate.
- the expression cassette can be configured in a chromosomal transfer form, or can be configured in a form that is retained outside the chromosome.
- an inducible promoter that can intentionally determine the timing of protein expression.
- other control regions such as a promoter and a terminator.
- inducible promoters include galactose-inducible promoters such as GAL1 and GAL10, promoters used in induction systems by induction / removal by addition of doxycycline such as Tet-on system / Tet-off system, heat such as HSP10, HSP60, HSP90, etc.
- a promoter of a gene encoding a shock protein (HSP) or the like can be used.
- HSP shock protein
- a CUP1 promoter activated by addition of copper ions is used.
- the CUP1 promoter By using the CUP1 promoter, cells are cultured in a medium that contains a carbon source such as glucose and does not contain copper ions, and then a copper ion compound is added to the medium and cultured to induce expression of a DNA double-strand break enzyme. be able to.
- concentration of copper ion can be set suitably, it can be set as about 50 micromol or more and 300 micromol or less, for example.
- the culture time can be about 1 to 6 hours.
- the CUP1 promoter has the advantage that it can easily and rapidly execute the expression induction and activation of the intentional DNA double-strand break enzyme.
- parent eukaryotic organism a parent eukaryotic organism capable of expressing and retaining the gene encoding this protein can be used.
- a parent eukaryotic organism having the desired ploidy can be obtained by introducing such a gene into a parent eukaryotic candidate having the desired ploidy and transforming it.
- Such a parent eukaryotic candidate having the desired ploidy is obtained by repeating an artificial genome expansion operation once or twice or more for a eukaryotic organism having a ploidy exceeding the intrinsic ploidy. May be.
- the artificial genome expansion operation is repeated one or more times on the transformant. It is possible to obtain a prokaryotic organism having a ploidy of.
- the parent eukaryotic organism is also a breeding material for producing a eukaryotic organism or eukaryotic population for breeding. Therefore, according to the disclosure of the present specification, a prokaryotic organism that is also useful as a breeding material that can be suitably used in the induction method and an induction method thereof are provided. According to the present disclosure, there is provided a breeding material that is a eukaryote and has a ploidy exceeding the intrinsic ploidy and holds a gene encoding a protein having a DNA double-strand break activity in an expressible manner.
- an induction method suitable for producing the breeding material of the present disclosure includes repeating an artificial genome expansion operation once or twice or more for a eukaryotic organism that is a polyploid having intrinsic ploidy, Obtaining a desired ploidy eukaryote, and transforming the desired ploidy eukaryote to express a gene encoding a protein having DNA double-strand break activity. be able to.
- this induction method includes a step of transforming a eukaryotic organism that is a polyploid having intrinsic ploidy so that a gene encoding a protein having a DNA double-strand break activity can be expressed, and the transformed true
- eukaryote protein having DNA double strand break activity, ploidy or polyploid, protein having DNA double strand break activity, and gene encoding the protein
- the various embodiments described in the eukaryotic population induction method of the present disclosure can be applied to the expression of humans and artificial genome expansion operations.
- this protein is allowed to act in the cells of the parent eukaryote.
- the aspect of the parent eukaryotic organism is not particularly limited.
- the parent eukaryote is a plant body
- the form of the plant body is not limited.
- cells, tissues, organs as a part of the plant individual, seeds, seedlings, or subsequent grown plant individuals it means the cell in callus.
- the parent eukaryote is a microorganism such as yeast, it means that the present protein is allowed to act in the cell.
- a condition capable of exerting a DNA double-strand break activity may be imparted to the present protein present in the cell.
- the present protein may be caused to act constantly (constitutively) with low activity, and may be caused to act intentionally and / or temporarily.
- the protein is expressed under the control of a suitable promoter, typically a constitutive promoter, so that the DNA double-strand break activity of the protein becomes too high. It is preferable to adjust the activity of the protein by the action temperature and the action time to such an extent that they are not. This is because the cells always die due to excessive DNA double strand breaks due to the constant action of this protein.
- a restriction enzyme derived from thermophile is used as the present protein, and the working temperature is set to a temperature sufficiently lower than the optimum temperature of such restriction enzyme.
- a restriction enzyme derived from a thermophilic bacterium is used.
- the working temperature may be 18 ° C. or higher and 30 ° C. or lower, although it depends on the type of the protein, the type of eukaryotic organism, and the like.
- the lower limit is more preferably 20 ° C. or higher, and further preferably 22 ° C. or higher.
- the upper limit is preferably 28 ° C. or lower, and more preferably 25 ° C. or lower.
- this protein is allowed to act intentionally and temporarily. This is because the cells are damaged by excessive DNA double-strand breaks due to the constant action of this protein.
- it is typically preferable to express the protein with an inducible promoter and use the activation operation of the protein.
- the present protein can be expressed and acted in cells at an intended timing, and its action can be reduced or stopped roughly by stopping the induction.
- the present protein is expressed by an inducible promoter, it is preferable to grow or culture the eukaryotic organism at a temperature sufficiently lower than the optimum temperature for the DNA double-strand break activity of the present protein. For example, it can be about 15 ° C. or more and 25 ° C. or less.
- the present protein is expressed using an inducible promoter in the modification step in which the present protein is allowed to act, after the modification step, the eukaryotic organism is grown or cultured under the condition that the operation of the inducible promoter can be stopped. It is preferable to do.
- the DNA double-strand break activity of this protein is artificially activated and then preferably inactivated.
- this protein can be made to act more restrictively and effectively.
- the present protein is used in which conditions different from normal growth conditions (cultivation conditions) of the parent eukaryote such as a restriction enzyme derived from thermophile are optimal conditions for DNA double-strand cleavage activity.
- the present protein can be activated and stopped at an intentional timing.
- the parent eukaryotic organism is grown (cultured) at a temperature lower than the temperature at which the protein is sufficiently activated, and the parent eukaryotic organism is grown at a temperature higher than the activation temperature of the protein. By (culturing), the present protein can be easily and temporarily allowed to act.
- the protein In order to activate and act on the DNA double-strand break activity of the present protein, it is preferable to carry out it under conditions that are milder than the so-called optimum conditions that maximize the activity of the present protein. By doing so, it is possible to construct a cell population having a more diverse genome set composition.
- the protein should be activated within a temperature range that is lower than the optimum temperature for a DNA double-strand break active enzyme and that does not impair cell activity. Is preferred. More preferably, the protein is preferably activated at a temperature close to the lower limit at which the DNA double-strand cleavage activity can be expressed.
- the temperature near the lower limit at which the activity of the DNA double-strand cleavage enzyme can be expressed can be said to be a temperature at which the activity is about 5% to 30%, assuming that the activity at the optimum temperature is 100%.
- the temperature can be about 5% to 20%.
- such an action temperature may be 18 ° C. or higher and 45 ° C. or lower, although it depends on the type of the present protein or the type of eukaryotic organism.
- the lower limit is 20 ° C. or higher, more preferably 22 ° C. or higher, still more preferably 25 ° C. or higher, more preferably 30 ° C. or higher, and even more preferably 35 ° C. or higher. is there.
- the upper limit is preferably 45 ° C. or lower, more preferably 42 ° C. or lower, further preferably 40 ° C. or lower, still more preferably 37 ° C. or lower, and still more preferably 35 ° C. or lower.
- the above temperature conditions are particularly suitable for microorganisms such as yeast and plants.
- this protein When the working temperature of this protein matches or is close to a suitable growth temperature or culture temperature generally applied to eukaryotic organisms, it can be combined with expression control of this protein by the operation of an inducible promoter. This protein can be allowed to act transiently on eukaryotic organisms.
- the protein can be operated under mild conditions (for example, the DNA double strand of the protein).
- a relatively long time for example, 2 hours or more, 3 hours or more, more preferably 4 hours or more, more preferably 6 Hours or more, preferably 12 hours or more, more preferably 24 hours or more, even more preferably 36 hours or more, even more preferably 48 hours or more, still more preferably 60 hours or more, more preferably 72 hours or more.
- cells expressing TaqI are preferably 20 ° C. or higher and 45 ° C. or lower, more preferably 25 ° C. or higher and 42 ° C. or lower, still more preferably.
- the action time is preferably 2 hours or more, more preferably 4 hours or more, still more preferably 5 hours or more, still more preferably 6 hours or more, still more preferably 12 hours or more, still more preferably 24 hours or more, and even more preferably.
- Such conditions are particularly suitable for microorganisms such as yeast and plants.
- the working conditions such as the working temperature and working time of this protein are those skilled in the art by evaluating the expression status of this protein, the growth (proliferation) status of eukaryotic organisms, and the evaluation of genome reorganization efficiency (using reporter genes). Or an evaluation based on a ploidy histogram) can be appropriately determined in consideration of the present protein to be used, the eukaryotic organism, and the intended genome modification efficiency.
- the modification step for causing the protein to act in the cells of the parent eukaryotic organism is, for example, in a plant, a seed before sowing harvested from a parent eukaryotic organism which is a parent plant transformed to express the protein, Between sowing and germination, it is carried out for a certain period of time on the seedlings after germination and more grown plants.
- a parent yeast transformed so that the present protein can be expressed is allowed to express the present protein for a certain period of time or activate the expressed present protein.
- These modification steps are preferably seeds or seedlings when the parent eukaryote is a plant. This is because multi-analyte processing is easy and it is convenient to obtain a population of variants.
- a cycle in which budding is actively performed such as a somatic cell division period (that is, a cycle in which the ploidy of the parent eukaryote is maintained) is preferable.
- the parent eukaryote is a plant
- seeds, seedlings, grown plants and the like are grown under a temperature condition of 37 ° C. for 24 hours, and thereafter, at a lower temperature growth condition (for example, In the case of Arabidopsis thaliana, the temperature can be returned to about 20 ° C. to 25 ° C.).
- the yeast culture conditions are maintained at 37 ° C. for 24 hours, and then returned to the normal culture temperature (approximately 25 ° C. to 30 ° C.).
- the genomic set of eukaryotic organisms that make up the newly constructed eukaryotic population obtained by this induction method has or has increased chromosomal aneuploidy as a result of genetic recombination.
- eukaryotic genome sets tend to have deletions and / or duplications in part of the chromosome as a result of genetic recombination.
- the genome set of such eukaryotes tends to have a reduced genome size as a result of genetic recombination, a part of the genome set is dropped, or a part of the genome set overlaps. Tend to have an increased size.
- such eukaryotic genome sets tend to have one or more mutations.
- this protein is allowed to act on a diploid parent eukaryote by performing this modification step on a parent eukaryotic organism that is a polyploid of tetraploid or higher.
- a population with genome set diversity exceeding expectations can be obtained. That is, genetic recombination for chromosomal DNA is significantly increased by targeting tetraploids or more.
- the eukaryotic organism obtained has a remarkable tendency to change in genome size (decrease or increase in size) and an aneuploidy.
- the introduction of a gene encoding a protein having DNA double-strand cleavage activity such as TaqI tends to cause growth suppression of the parent eukaryotic organism.
- the organism is tetraploid or more
- the TaqI gene or the like is introduced into the diploid and allowed to act, the degree of inhibition is reduced compared to growth inhibition. That is, when the prokaryotic organism is tetraploid or more, resistance to introduction of TaqI or the like can be exhibited.
- eucaryotic organisms such as plants tend to decrease in mass due to ploidy, but surprisingly, such a tendency is suppressed in eukaryotic organisms obtained for tetraploids or more. This is probably because the resulting eukaryotic organism is accompanied by a decrease or increase in chromosome size aneuploidy and / or genome size. Such a tendency becomes more prominent as the ploidy of the parent eukaryote becomes higher.
- the growth is remarkably suppressed when the protein is not allowed to act, but the mass may be equal to or greater than that of the wild type due to the action of the protein. is there. This is also believed to be due to a decrease in the size of the genome set, an increase and / or an increase in chromosomal aneuploidy.
- a chromosome having a DNA recombination activity is allowed to act in a cell of a eukaryotic organism having a doubled genome, thereby causing a chromosome or It is possible to obtain a eukaryotic organism having a new genome set composition generated by genetic recombination while suppressing functional damage of the gene. As a result, a eukaryotic population having a variety of genome set compositions can be constructed. Therefore, according to this induction method, it is possible to simultaneously and efficiently realize avoidance of inconvenience associated with chromosome doubling (increase in genome size) and realization of the benefits of chromosome doubling in parent eukaryotes. . As a result, it is possible to construct a population of eukaryotic organisms rich in diversity in traits.
- this induction method repeats the modification process and the selection process, such as further performing a modification process, using one or more eukaryotes selected from the eukaryote population as a parent eukaryote. It can also be implemented.
- this induction method can also be implemented as a method for producing a population of eukaryotic organisms comprising the modified genome set, which comprises the above modification step.
- the method for producing a modified eukaryotic organism disclosed in the present specification allows a protein having DNA double-strand cleavage activity to act in cells of a eukaryotic organism that is a polyploid exceeding the intrinsic ploidy, A modification step of modifying the genome set of the eukaryote, and a step of selecting an intended eukaryote from a population of eukaryotes holding the genome set based on an arbitrary index. Can do. According to this method, since one or more intended eukaryotic organisms are selected from a population of eukaryotic organisms having excellent diversity, the intended eukaryotic organism can be efficiently obtained.
- this production method can also be implemented as a method for breeding eukaryotic organisms such as plants and yeast.
- conventionally well-known breeding techniques can be applied to further progeny breeding after useful plants and yeasts are obtained.
- the various steps of the modification step in the production method described above can be applied as they are to the modification step in the method.
- the modification step and the selection step may be repeated. That is, one or more selected eukaryotic organisms may be used as parent eukaryotic organisms to perform the modification step and the selection step.
- cultivation was further continued for 9 weeks at 22 ° C., 16 hours light period, 8 hours dark period, light intensity of about 30 to 45 ⁇ E / m 2 / s, and further cultivation was continued for 2 weeks after irrigation was stopped.
- the plant body after cultivation was weighed after air drying, and was taken as the plant body dry weight.
- TaqI gene plant expression vector As a TaqI gene plant expression vector, pBI 35S: TaqI-NLS in which the TaqI gene is arranged downstream of the cauliflower mosaic virus 35S promoter and Arabidopsis thaliana by the method disclosed in JP 2011-160798 A (paragraphs 0121 to 0142).
- PBI AtSIG2 TaqI-NLS was constructed in which the TaqI gene was placed downstream of the sigma factor AtSIG2 promoter. This plasmid also has a nuclear translocation signal (NLS) on the C-terminal side of the TaqI coding region.
- Agrobacterium (C58C1 strain) carrying pBI 35S: TaqI-NLS was prepared according to Japanese Patent Application Laid-Open No. 2011-160798.
- 35S: TaqI gene was introduced into Arabidopsis thaliana 1406 strain (EMBO Journal (2006) 25, 5579-5590).
- a GUS reporter gene having an inverted repeat structure is introduced into a wild-type Arabidopsis ecotype Col-0 strain, and is constructed so that the GUS gene is expressed when homologous recombination occurs in the GUS gene. This mechanism is used for quantitative analysis of homologous recombination.
- the transformation method was performed using the implanter method. That is, the Agrobacterium tumefaciens plant expression vector prepared in Example 1 was electroporated (Plant (Molecular Biology Mannal, Second Edition, B. G. Stanton and A. S. Robbert, Kluwer Acdemic Publishers 1994). It was introduced into (Agrobacterium ientumefaciens) C58C1 strain. Subsequently, Agrobacterium tumefaciens into which a plant expression vector has been introduced was transformed into the wild type by the infiltration method described by Clough et al. (Steven J. Clough and Andrew F. Bent, 1998, The Plan Journal 16, 735-743). Introduced to Arabidopsis Ecotype Col-0.
- the transformed Arabidopsis thaliana was grown at 22 ° C., 16 hours light period, 8 hours dark period, light intensity of about 30-50 ⁇ mol / m 2 / sec, and T1 seeds were collected.
- the collected T1 seeds were modified MS agar medium containing kanamycin (50 mg / L) [sucrose 10 g / l, MES (2-Morpholinoethanesul phonic acid) 0.5 g / L, agar (for bacterial medium; Wako Pure Chemicals) 8 g / L), and aseptically seeded at 22 ° C., 16 hours light period, 8 hours dark period, light intensity of about 30 to 45 ⁇ mol / m 2 / sec for 2 weeks to select kanamycin resistant individuals, Transformant TCL878 was obtained.
- Arabidopsis thaliana ecotype Col-0 wild strain, 1406 strain, pBI 35S: TaqI-NLS introduced line obtained in Example 2 was grown on a sterile medium containing 1% sucrose for 3 weeks, and then 0.05 It was immersed in a 0.01% colchicine solution containing% Triton-X for 1 to 10 minutes. After that, transplanted to a 50 mm diameter pot containing supermix (Takii seedling), 22 weeks, 16 hours light period 8 hours dark period, light intensity about 30-50 ⁇ mol / m 2 / sec in an artificial weather room for 8 weeks Seeds were obtained after growth.
- next-generation plants the nuclei were stained using DAPI, and the ploidy of the genome was examined using a method using a flow cytometer, and tetraploid plants were selected to obtain Col-0_P4, 1406_P4, and TCL878_P4C2, respectively. . Further, the obtained tetraploid plant was treated with a colchicine solution in the same manner to select the flower stems that had been made 8-fold, and Col-0_P8, 1406_P8, and TCL878_P8 were obtained.
- the measurement of the plant ploidy using the flow cytometer was performed as follows.
- the rosette leaves of the plant body 3 weeks after germination or the fresh leaves of flower stems extracted from the plant were excised and rosette in 400 ⁇ l of the mixture of the nuclear extract and the nuclear staining solution of CyStain UV plant DNA reagent kit (Partec).
- the leaves were dipped and cut finely with a razor (about 100 cuts / cm 2 ). Thereafter, plant cell residues in the extract were removed using CellTrics (registered trademark) 50 ⁇ m (Partec).
- the obtained nuclear solution was measured using Cell Lab Quanta SC MPL (Beckman).
- SIG2 Introduction of TaqI gene into Arabidopsis thaliana
- the SIG2: TaqI gene was introduced into the Col-0_P4 strain prepared in Example 3 using Agrobacterium having pBI AtSIG2: TaqI-NLS.
- the implanter method was used in the same manner as in Example 2. Seeds obtained after infection with Agrobacterium were sown on MS agar medium containing kanamycin (Murashige Scoog inorganic salt, 1% sucrose, 0.05% MES, 0.8% Agar, 50 mg / l kanamycin sulfate). After growing for 2 weeks in an artificial weather room at 22 ° C., 16 hours light period, 8 hours dark period, and light intensity of about 30-50 ⁇ mol / m 2 / sec, Kanaimycin resistant individuals were selected to obtain transformant TS3055.
- kanamycin Merashige Scoog inorganic salt, 1% sucrose, 0.05% MES, 0.8% Agar, 50 mg / l
- genomic DNA was extracted from the plant body. Using the extracted genomic DNA, genome copy number polymorphism by tiling array was analyzed. The copy number polymorphism analysis was performed as follows. The results are shown in FIG.
- the tiling array of Arabidopsis thaliana was designed using the Agilent eArray system. At_tilling_400K_v3.2 was designed in which 381815 strand super 60 mer probes were arranged on the Arabidopsis genome at an average spatial resolution of about 314 nt. Similarly, At_tilling_180K_v4 was designed in which 177170 super 60 mer probes were arranged on the Arabidopsis genome at an average spatial resolution of about 677 nt. Tiling array was performed according to Agilent protocol. Tiling array scanning was performed using an Agilent G2565CA microarray scanner (Agilent).
- Relative level Log 10 (Cy5 (sample) / Cy3 (control)), and the average value of the relative level of 20 consecutive probes on the chromosome was calculated and shown in the figure.
- the dry weight of the TaqI gene introduction line was greatly reduced to 0.65 with respect to the control strain.
- the dry weight of the TaqI gene-introduced line was 0.86 with respect to the control strain, and the decrease in the dry weight was suppressed compared to the diploid plant.
- the dry weight of the octaploid plants Col-0_P8 and 1406_P8 produced from the Col-0 wild strain and the 1406 control strain was significantly lower than that of the wild strain. These are considered polyploidy syndromes.
- the plant of the octaploid TCL878_P8 line prepared from the 35S: TaqI gene-transferred line showed a tendency to recover its dry weight compared to Col-0_P8 and 1406_P8, while the line of TCL878_P8-10 # 3 was diploid Col- 0 Dry weight increased from wild type.
- chromosomes 2, 2 and 3 have 6 copies, and chromosome 4 has 5 copies.
- Chromosome 5 had 7 copies and showed chromosomal aneuploidy with different copy numbers for each chromosome.
- the 522 kbp region was increased by 2 copies from the surrounding region to about 8 copies.
- TaqI gene yeast expression vector As a TaqI gene yeast expression vector, as shown in FIG. 10, the TaqI gene (TtTaqI opt) optimized for the yeast codon downstream of the galactose-inducible promoter (pGAL1), the 3′UTR (tDIT1) of Saccharomyces cerevisiae DIT1 protein PORF-pGAL1-TtTaqI-tDIT1 (AUR) was used.
- the GF-FP reporter gene for evaluating genome reorganization efficiency sandwiches the antibiotic Nourseothricin resistance marker (nat1) between about 600 bp on the N-terminal side and 600 bp on the C-terminal side of green fluorescent protein (GFP). Designed to be When this cassette is used, homologous recombination occurs between GF-FP due to double-stranded DNA cleavage by TaqI, and when the full-length GFP gene is reconstructed, the yeast emits green fluorescence. Can be used to detect genome reorganization (see Fig. 12).
- S. cerevisiae BY4741 and BY4742 strains into which the GF-FP reporter gene was introduced into the upstream region of the ADH3 gene (3000 bp to 2000 bp upstream from the transcription start point) were designated as BY4741 + GFP strain and BY4742 + GFP strain, respectively.
- the leu2 gene, his3 gene of each strain was derived from S. cerevisiae S288C-derived LEU2 gene, BY4741 + GFP (LEU) strain, BY4741 + GFP (HIS) strain, and BY4742 + GFP (LEU) strain complemented with HIS3 gene, BY4742 + GFP (HIS) strain was prepared.
- TaqI gene yeast expression vector pORF-pGAL1-TtTaqIopt-tDIT1 (AUR) prepared in Example 10 was transformed into the BY4741 + GFP (HIS) strain prepared in Example 11 (BY4741 + GFP (HIS) + TaqI).
- YPD medium (10 g / L Yeast extract, 20 g / L Peptone, 20 g / L Glucose) + 0.5 mg / L aureobasidin A (AbA) with glucose as a sugar source is cultured at 30 ° C. overnight.
- the medium was changed to YPG (10 g / L Yeast extract, 20 g / L Peptone, 20 g / L Galactose) +0.5 mg / L AbA medium containing galactose as a sugar source, 20, 22.5, 25, TaqI expression was induced by culturing at 27.5 and 30 ° C. overnight.
- YPG 10 g / L Yeast extract, 20 g / L Peptone, 20 g / L Galactose
- TaqI expression was induced by culturing at 27.5 and 30 ° C. overnight.
- SDS-PAGE was performed with the same amount of bacterial cells, and Western blotting was performed using an anti-TaqI antibody. The results are shown in FIG.
- TaqI expression was induced at 20 ° C. in the same manner as in Example 12 using BY4741 + GFP (HIS) + TaqI strain. After temporarily activating TaqI by heat treatment at 42 ° C for 30 minutes or 60 minutes, the ratio of cells with GFP fluorescence in 1 ⁇ 10 5 cells is measured with a flow cytometer. Genomic reorganization efficiency was calculated. The results are shown in FIG. 14A. As a result, genome reorganization efficiency was 0.03-0.04%. On the other hand, as shown in FIG. 14B, when the medium was changed to YPD medium after heat treatment and recovery culture was performed for 18 hours, the genome reorganization efficiency increased to 0.06 to 0.07%.
- the genome reorganization efficiency increased with time at any temperature.
- genome reorganization efficiency increased significantly, reaching about 10 to 20 times that of transient heat treatment.
- BY4741 + GFP (LEU) strain, BY4742 + GFP (LEU) strain, BY4741 + GFP (HIS) strain and BY4742 + GFP (HIS) strain were joined according to a standard method, and cultured on an SD-Met-Lys agar plate at 30 ° C. for 5 days. After growing colonies were singled, the amount of genomic DNA was measured with a flow cytometer, and the strains confirmed to be diploid were designated as BY4743 + GFP (LEU) strain and BY4743 + GFP (HIS) strain.
- BY4743 + GFP (LEU) and BY4743 + GFP (HIS) strains were cell-fused to produce tetraploid yeast.
- Cell fusion was performed using the protoplast-PEG method. Colonies grown on the SD-Leu-His agar plate were used as cell fusion candidate strains. After the grown colonies were singled, the amount of genomic DNA was measured with a flow cytometer, and the strain confirmed to be tetraploid was designated as BY4744 + GFP (LH) strain.
- the TaqI gene yeast expression vector pORF-pGAL1-TtTaqIopt-tDIT1 (AUR) prepared in Example 10 was transformed into each doubling yeast prepared in Examples 11 and 14. Using the transformant, the medium was changed to a YPG + 0.5 mg / L AbA medium in the same manner as in Example 12, and then TaqI was gently activated while inducing TaqI expression at 35 ° C. Yeast was recovered after 22 hours, and the genome reorganization efficiency was measured with a flow cytometer in the same manner as in Example 13. The results are shown in FIG.
- the genome reorganization efficiency of haploid yeast was 0.5 to 0.75%, whereas 1.0% for diploid yeast and 2.5% for tetraploid yeast. It has been shown that genome doubling improves genome reorganization efficiency.
- TaqI was gently activated while inducing expression of TaqI at 35 ° C. using each doubling yeast. After 0, 16, and 46 hours, the yeast was collected, fixed with 70% ethanol, and then genomic DNA was fluorescently stained by DAPI staining. Nuclear phase analysis was performed with a flow cytometer using each doubled yeast that was fluorescently stained. The results are shown in FIG. As shown in FIG. 17, in the doubled yeast, an increase in the appearance frequency of individuals with increased or decreased ploidy was observed.
- OC2-A strain in which a xylose utilization gene was introduced into wine yeast OC-2 strain was prepared. That is, based on the OC700 strain (JP-A-2014-193152) in which a xylose utilization gene was introduced into the wine yeast OC-2 strain, the ADH2 gene was destroyed and the ADH1 gene was enhanced, and the mhpF gene (derived from E. coli) The introduced OC2-A strain was acquired.
- OC2-A (KlURA3) strain in which the URA3 gene derived from Kluyveromyces lactis was introduced into the OC2-A strain and an OC2-A (KmTRP1) strain in which the TRP1 gene derived from Kluyveromyces marxianus was introduced were prepared. Using both strains, cell fusion by protoplast-PEG method was performed to prepare a cell fusion strain OC2-A (CF).
- the OC2-A (CF) strain was transformed with a TaqI gene yeast expression vector, pORF-pGAL1-TtTaqIopt-tDIT1 (AUR), to obtain an OC2-A (CF) + TaqI strain.
- AUR pORF-pGAL1-TtTaqIopt-tDIT1
- OC2-A (CF) + TaqI strain the medium was changed to YPG + 0.5 mg / L AbA medium in the same manner as in Example 12, and then the expression was induced by culturing overnight at 20 ° C. Thereafter, TaqI activation was carried out at 35 ° C. for 8 to 24 hours, and a sample subjected to recovery culture in a YPD medium + 0.5 mg / L AbA medium was used as a genome reorganized yeast library.
- Accumulation culture (35 ° C.) was performed by repeating culture and planting in a medium containing xylose as a main sugar source. After the start of enrichment culture, the culture was continued for about 300 hours in YPX medium (10 g / L Yeast extract, 20 g / L Peptone, 1 g / L Glucose, 20 g / L Xylose) with slight addition of glucose. Thereafter, the medium was changed to YPX medium (10 g / L Yeast extract, 20 g / L Peptone, 20 g / L Xylose) using only xylose as a sugar source, and further enrichment culture was performed for about 200 hours. The transition of the amount of cells in these culture steps is shown in FIG. The culture solution after enrichment culture was streaked on a YPD agar plate, and the single strain was designated as OC2A-C5 strain.
- the OC2A-C5 strain has a significantly improved ability to assimilate xylose compared to the parent strain OC2-A strain and its cell fusion strain OC2-A (CF) strain, In 48 hours, 50 g / L xylose was completely consumed, and 21.3 g / L ethanol was produced.
- the OC2A-C5 strain has improved xylose utilization capacity compared to the parent strain, OC2-A strain, and its cell fusion strain, OC2-A (CF) strain, for 72 hours. 80 g / L glucose and 100 g / L xylose were completely consumed to produce 76.2 g / L ethanol.
- the OC2A-C5 strain significantly increased the ability to assimilate xylose, and as a result, the ability to produce and ferment ethanol and the like was also increased.
- the fermentation capacity is about 1.3 times that of the OC2-A (CF) strain and about 1.4 times that of the OC2-A strain. It was found that the xylose utilization performance was excellent.
- the OC2-A (CF) + TaqI strain into which the TaqI gene was introduced was subjected to an accumulation change under the evolutionary pressure of a xylose-containing medium after the expression and activation of the TaqI gene. It was found that the strain OC2A-C5, which is a useful strain, can be obtained by drought. In other words, it was found that cells suitable for conditions can be efficiently obtained by growing cells in a state in which the DNA double-strand-cleaving enzyme is expressed and activated under arbitrary conditions.
- YPX medium (10 g / L Yeast extract, 20 g / L Peptone, 20 g / L Xylose) was cultured at 39 to 41 ° C., and repeated culture was repeated for about 500 hours. The culture solution after enrichment culture was streaked on a YPD agar plate, and the single strain was designated as OC2A-TT strain.
- Glucose + xylose mixed fermentation medium (10 g / L Yeast extract, 30 g / L Glucose, 30 g / L Xylose) contains OC2-A, OC2-A (CF), OC2A-C5 and OC2A-TT
- OD600 1.0
- a fermentation test was conducted at 40 ° C. Sampling was performed at 0, 16, 24, 48, and 65 hours, and glucose, xylose, and ethanol were measured by HPLC. The results are shown in FIG.
- the OC2A-TT strain had increased proliferation ability (heat resistance) at 40 ° C. compared to other strains.
- the heat resistance of the OC2A-TT strain is remarkably increased, showing good xylose assimilation ability even in good high-temperature culture, and the xylose assimilation ability is remarkably increased.
- the ability to produce and ferment ethanol was also increased.
- the OC2-A (CF) + TaqI strain into which the TaqI gene has been introduced is accumulated and cultured under the evolutionary pressure of high temperature after expression and activation of the TaqI gene. It was found that the OC2A-TT strain, which is a useful strain, can be obtained. That is, it has been found that by growing a cell in a state in which a DNA double-strand break enzyme is activated and activated under arbitrary conditions, a cell suitable for the condition can be efficiently obtained.
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Abstract
Description
本出願は、2015年7月2日に出願した日本国特許出願である特願2015-133902及び2015年12月2日に出願した日本国特許出願である特願2015-236072の関連出願であって、これらの出願に基づく優先権を主張するものであり、これらの出願に記載された全ての内容は引用により本明細書に組み込まれるものとする。
本明細書は、遺伝的組換えを誘発する方法及びその利用等に関する。
真核生物体が本来的に有する倍数性を超える倍数体である真核生物体の細胞内においてDNA二本鎖切断活性を有するタンパク質を作用させるゲノムセットの改変工程、
を備える、方法。
(2)前記遺伝的組換えは、1又は2以上の塩基の置換、挿入及び欠失による遺伝子突然変異並びに染色体の逆位、不等交叉、交叉、転座、重複、欠失、サイズコピー数の低下、コピー数の増大、染色体倍数化及び染色体異数化からなる群から選択される1種又は2種以上である、(1)に記載の方法。
(3)前記真核生物体は4倍数性以上の倍数体である、(1)又は(2)に記載の誘発方法。
(4)前記遺伝的組換えは、染色体異数化を含む、(1)~(3)のいずれかに記載の誘発方法。
(5)前記遺伝的組換えは、ゲノムサイズの低下又は増加を含む、(1)~(4)のいずれかに記載の誘発方法。
(6)前記遺伝的組換えは、染色体の一部に欠失又は重複を含む、(1)~(5)のいずれかに記載の誘発方法。
(7)前記改変工程は、前記タンパク質の前記DNA二本鎖切断活性を人為的に活性化する工程である、(1)~(6)のいずれかに記載の誘発方法。
(8)前記改変工程は、前記タンパク質の前記DNA二本鎖切断活性についての至適温度より低い温度で作用させる工程である、(1)~(7)のいずれかに記載の誘発方法。
(9)前記改変工程は、前記タンパク質をコードする外来遺伝子の発現によって得られる前記タンパク質を用いる工程である、(1)~(8)のいずれかに記載の誘発方法。
(10)前記タンパク質は多頻度制限酵素である、(1)~(9)のいずれかに記載の誘発方法。
(11)前記多頻度制限酵素は、好熱菌由来の制限酵素である、(10)に記載の誘発方法。
(12)前記多頻度制限酵素は、TaqIである、(11)に記載の誘発方法。
(13)前記真核生物体は、植物体である、(1)~(12)のいずれかに記載の誘発方法。
(14)前記真核生物体は、微生物である、(1)~(13)のいずれかに記載の誘発方法。
(15)前記改変工程に先立って、人為的にゲノムサイズ増大操作によって親真核生物体を取得する倍数化工程を備える、(1)~(14)のいずれかに記載の誘発方法。
(16)前記倍数化工程は、倍化誘発剤を用いる、(15)に記載の誘発方法。
(17)前記倍数化工程は、細胞融合を用いる、(14)に記載の誘発方法。
(18)改変された真核生物体の生産方法であって、
真核生物体が本来的に有する倍数性を超える倍数体である親真核生物体の細胞内においてDNA二本鎖切断活性を有するタンパク質を作用させる改変工程、
を備える、方法。
(19)さらに、任意の指標に基づいて意図する真核生物体を選抜する工程、
を備える、(18)に記載の生産方法。
(20)真核生物体集団の生産方法であって、
本来的に有する倍数性を超える倍数体である親真核生物体の細胞内においてDNA二本鎖切断活性を有するタンパク質を作用させて親真核生物体のゲノムセットを改変する改変工程を備え、
改変されたゲノムセットを保持する真核生物体の集団を取得する、生産方法。
を備える、生産方法。
(21)本来的に有する倍数性を超える倍数性を有し、DNA二本鎖切断活性を有するタンパク質をコードする遺伝子を発現可能に保持する、真核生物体である育種材料。
(22)育種材料の生産方法であって、
本来的に有する倍数性を超える倍数体である真核生物体に対して人為的なゲノム増大操作を1回又は2回以上繰り返して、所望の倍数性の真核生物体を得る工程と、
前記所望の倍数性の真核生物体をDNA二本鎖切断活性を有するタンパク質をコードする遺伝子を発現可能に形質転換する工程と、
を備える、方法。
(23)育種材料の生産方法であって、
本来的に有する倍数性を超える倍数体である真核生物体をDNA二本鎖切断活性を有するタンパク質をコードする遺伝子を発現可能に形質転換する工程と、
形質転換された前記真核生物体に対して人為的なゲノム増大操作を1回又は2回以上繰り返して、所望の倍数性の形質転換された真核生物体を得る工程と、
を備える、方法。
本開示の遺伝的組換えの誘発方法(以下、単に、誘発方法という。)は、真核生物体の固有倍数性を超える倍数体である真核生物体の細胞内においてDNA二本鎖切断活性を有するタンパク質(以下、本タンパク質という。)を作用させて真核生物体のゲノムセットを改変する改変工程、を備えることができる。本誘発方法は、このような改変工程を備えることにより、個々の真核生物体において種々の遺伝的組換えが許容され、その結果、改変されたゲノムセットを保持する真核生物体集団を取得することができる。すなわち、元々は単一組成のゲノムセットを備える親真核生物体の細胞内において本タンパク質を作用させると、ゲノムセット組成及び形質に関して多様性に富む複数の真核性物体からなる集団を得ることができる。
(真核生物体)
本誘発方法は、任意の真核細胞生物体に適用可能である。本誘発方法に適用される真核生物体としては、動物、植物、真核性微生物が挙げられる。動物としては特に限定しないで、哺乳動物及び各種の魚類などの非哺乳動物が挙げられる。また、本誘発方法に適用される動物としては、動物に由来するものであればよく、細胞、組織、器官、受精卵などいずれの形態であってもよい。受精卵など、完全な動物に再生する能力を保持しているものであれば、改変した動物を得るのに都合がよい。
ナス科:タバコ(Nicotiana tabacum)、ナス(Solanum melongena)、ジャガイモ(Solaneum tuberosum)、トマト(Lycopersicon lycopersicum)、トウガラシ(Capsicum annuum)、ペチュニア(Petunia)など。
マメ科:ダイズ(Glycine max)、エンドウ(Pisum sativum)、ソラマメ(Vicia faba)、フジ(Wisteria floribunda)、ラッカセイ(Arachis. hypogaea)、ミヤコグサ(Lotus corniculatus var. japonicus)、インゲンマメ(Phaseolus vulgaris)、アズキ(Vigna angularis)、アカシア(Acacia)など。
キク科:キク(Chrysanthemum morifolium)、ヒマワリ(Helianthus annuus)など。
ヤシ科:アブラヤシ(Elaeis guineensis、Elaeis oleifera)、ココヤシ(Cocos nucifera)、ナツメヤシ(Phoenix dactylifera)、ロウヤシ(Copernicia)など。
ウルシ科:ハゼノキ(Rhus succedanea)、カシューナットノキ(Anacardium occidentale)、ウルシ(Toxicodendron vernicifluum)、マンゴー(Mangifera indica)、ピスタチオ(Pistacia vera)など。
ウリ科:カボチャ(Cucurbita maxima、Cucurbita moschata、Cucurbita pepo)、キュウリ(Cucumis sativus)、カラスウリ(Trichosanthes cucumeroides)、ヒョウタン(Lagenaria siceraria var. gourda)など。
バラ科:アーモンド(Amygdalus communis)、バラ(Rosa)、イチゴ(Fragaria)、サクラ(Prunus)、リンゴ(Malus pumila var. domestica)など。
ナデシコ科:カーネーション(Dianthus caryophyllus)など。
ヤナギ科:ポプラ(Populus trichocarpa、Populus nigra、Populus tremula)など。
イネ科:トウモロコシ(Zea mays)、イネ(Oryza sativa)、オオムギ(Hordeum vulgare)、コムギ(Triticum aestivum)、タケ(Phyllostachys)、サトウキビ(Saccharum officinarum)、ネピアグラス(Pennisetum pupureum)、エリアンサス(Erianthus ravenae)、ミスキャンタス(ススキ)(Miscanthus virgatum)、ソルガム(Sorghum)スイッチグラス(Panicum)など。
ユリ科:チューリップ(Tulipa)、ユリ(Lilium)など。
フトモモ科:ユーカリ(Eucalyptus camaldulensis、Eucalyptus grandis)など。
本誘発方法においては、固有倍数性を超える倍数体である真核生物体を用いる。なお、遺伝的組換えを誘発させる、固有倍数性を超える倍数体である真核生物体を、親真核生物体ということもできる。こうした倍数体を親真核生物体として用いることで、DNA二本鎖切断による種々の遺伝的組換えを許容し、飛躍的かつ効率的にゲノムセット組成及び形質の多様性に富む集団を構築できる。
本工程では、親真核生物体の細胞内において、本タンパク質、すなわち、DNA二本鎖切断活性を有するタンパク質を作用させる。本タンパク質としては、特に限定しないが、典型的には、公知のDNA二本鎖切断酵素を用いることができる。本誘発方法では、固有倍数性を超える倍数性の親真核生物体を用いて、大規模なゲノム再編が許容されることから、本タンパク質のDNA二本鎖切断活性の特性(認識部位(すなわち、頻度)等)の影響が回避又は抑制されており、公知のDNA二本鎖切断酵素から適宜選択して用いることができる。
制限酵素としては、好熱菌由来の制限酵素であって、親真核生物体の細胞の培養温度よりも高温領域にDNA二本鎖切断活性についての至適温度を有する制限酵素を使用することがより好ましい。温度処理により任意のタイミングで本タンパク質を活性化するとともにその活性を低下させるなどが可能であり、一時的な本タンパク質の作用に好都合であるからである。また、こうしたタンパク質であると、温度により、その活性の調節も可能であるからである。さらに、こうしたタンパク質であると、比較的緩やかなDNA二本鎖切断活性を作用させることができるからである。こうした本タンパク質としては、例えば、DNA二本鎖切断活性の至適温度が50℃以上80℃以下の制限酵素を用いることができる。例えば、至適温度が50℃、55℃、60℃、65℃、及び75℃(いずれも、カタログ値)が挙げられる。制限酵素の至適温度は、各種の販売会社のカタログ等に基づいて(カタログ値)等に基づいて選択することができる。至適温度は50℃未満であると、DNA二本鎖切断活性が強くなりすぎる場合がある。至適温度が80℃を超えると、DNA二本鎖切断活性が弱くなりすぎる場合がある。概して、至適温度は、好ましくは、55℃以上であり、より好ましくは60℃以上であり、62℃以上であってもよく、65℃程度であってもよい。また、概して、至適温度は、75℃以下であることが好ましく、より好ましくは70℃以下であり、また、68℃以下であってもよい。
例えば、植物の細胞内において、タンパク質を発現させるための発現ベクターの母体となるベクターとしては、従来公知の種々のベクターを用いることができる。例えば、プラスミド、ファージ、またはコスミド等を用いることができ、導入される植物細胞や導入方法に応じて適宜選択することができる。具体的には、例えば、pBR322、pBR325、pUC19、pUC119、pBluescript、pBluescriptSK、pBI系のベクター等を挙げることができる。特に、植物体へのベクターの導入法がアグロバクテリウムを用いる方法である場合には、pBI系のバイナリーベクターを用いることが好ましい。pBI系のバイナリーベクターとしては、具体的には、例えば、pBIG、pBIN19、pBI101、pBI121、pBI221等を挙げることができる。
親真核生物体としては、本タンパク質をコードする遺伝子を保持して発現可能である親真核生物体を用いることができる。この場合には、所望の倍数性を備える親真核生物体候補に、こうした遺伝子を導入し形質転換して所望の倍数性を備える親真核生物体を得ることができる。かかる所望の倍数性を備える親真核生物体候補は、固有倍数性を超える倍数性を有する真核生物体に対して人為的なゲノム増大操作を1回又は2回以上繰り返すことで得るようにしてもよい。
次いで、親真核生物体の細胞内において本タンパク質を作用させる態様について説明する。以下の説明において、親真核生物体の細胞内というときには、親真核生物体の態様は特に限定するものではない。例えば、親真核生物体が植物体の場合には、植物体の態様は問わないで、植物個体の一部としての細胞、組織、器官のほか、種子、幼苗、あるいはその後の成長した植物個体のほか、カルスにおける細胞内を意味している。また、親真核生物体が、酵母など微生物の場合には、その細胞内で本タンパク質を作用させることを意味する。
本明細書に開示される改変された真核生物体の生産方法は、固有倍数性を超える倍数体である真核生物体の細胞内においてDNA二本鎖切断活性を有するタンパク質を作用させて、前記真核生物体のゲノムセットを改変する改変工程と、前記ゲノムセットを保持する真核生物体の集団から、任意の指標に基づいて意図する真核生物体を選抜する工程と、を備えることができる。本方法によれば、多様性に優れる真核生物体の集団から意図した1又は2以上の真核生物体を選抜するため、効率的に意図した真核生物体を得ることができる。また、本方法によれば、選抜した真核生物体を利用して効率的な育種が可能である。したがって、本生産方法は、植物体や酵母などの真核生物体の育種方法としても実施することができる。なお、有用な植物や酵母などが得られた後のさらに後代の育種については、従来公知の育種技術を適用することができる。
TaqI遺伝子植物発現用ベクターとして、特開2011-160798号公報(段落0121~0142)に開示された方法により、カリフラワーモザイクウィルス35Sプロモーターの下流にTaqI遺伝子を配置したpBI 35S:TaqI-NLSおよび、シロイヌナズナシグマ因子AtSIG2プロモーターの下流にTaqI遺伝子を配置したpBI AtSIG2:TaqI -NLSを構築した。また、このプラスミドには、TaqIのコード領域のC末端側には核移行シグナル(NLS)を備えている。
pBI 35S:TaqI-NLSを保持するアグロバクテリウム(C58C1株)を特開2011-160798号公報に準じて作製した。このアグロバクテリウムを用いてシロイヌナズナ1406株(EMBO Journal (2006) 25, 5579-5590)に35S:TaqI遺伝子を導入した。1406株はInverted repeat 構造をもつGUSレポーター遺伝子が野生型シロイヌナズナ エコタイプCol-0株に導入されており、GUS遺伝子内で相同組み換えが生じるとGUS遺伝子が発現するように構築されている。この仕組みにより相同組換えの定量解析に利用されている。
本実施例では、コルヒチンを用いてゲノム増大操作を行い、4倍数体及び8倍数体植物を得た。
発芽後3週間の植物体のロゼット葉または、抽台した花茎の茎生葉を切除し、CyStain UV 植物用DNA試薬キット(Partec社)の核抽出液と核染色溶液の混合液400 μl中にロゼット葉を浸し、剃刀により細かく切断した(約100回切断/cm2)。その後抽出液中の植物細胞残渣をCellTrics(登録商標)50 μm(Partec社)用いて除去した。得られた核溶液をCell Lab Quanta SC MPL(ベックマン社)を用いて測定した。
pBI AtSIG2:TaqI-NLSを保持するアグロバクテリウムを用いて実施例3で作製したCol-0_P4株にSIG2:TaqI遺伝子を導入した。形質転換方法としては、実施例2と同様にインプランタ法を用いて行った。アグロバクテリウムの感染後取得した種子を、カナマイシンを含むMS寒天培地(ムラシゲスクーグ無機塩, 1% ショ糖, 0.05% MES, 0.8% Agar, 50mg/l硫酸カナマイシン)に播種した。22℃、16時間明期8時間暗期、光強度約30~50μmol/m2/secの人工気象室で2週間生育後、カナイマイシン耐性個体を選抜し形質転換体TS3055を得た。
35S:TaqI遺伝子を有する4倍体系統であるTCL878_P4C2系統について、発芽後1週間の幼植物体を37℃1日間熱処理し、その後生育し種子を採種した。得られたTCL878_P4C2系統後代種子およびコントロール株として1406_P4株を播種・生育し形態を観察した。結果を図2に示す。
シロイヌナズナのタイリングアレイはアジレント社のeArrayシステムを用いて設計した。381815個の鎖超60 merプローブをシロイヌナズナゲノム上に平均空間解像度は約314 ntに配置したAt_tilling_400K_v3.2を設計した。また同様に177170個の鎖超60 merプローブをシロイヌナズナゲノム上に平均空間解像度は約677ntに配置したAt_tilling_180K_v4を設計した。タイリングアレイはアジレント社プロトコールに従い行った。タイリングアレイのスキャニングはAgilent G2565CA マイクロアレイスキャナ(アジレント社)を用いて行った。蛍光シグナルの抽出と数値化はFeature Extractionソフトウェアを用いて行った。Relative levelは以下の計算式で求め(Relative level = Log10(Cy5(サンプル)/Cy3(コントロール))、さらに染色体上の連続する20プローブのRelative levelの平均値を求め、図示した。
プロモーターの異なる4倍体植物での効果を確認するために、SIG2:TaqI遺伝子を有する4倍体系統であるTS3055系統を用いて実施例5と同様に形態変化を観察した。実施例4で取得したSIG2:TaqI遺伝子を有する4倍体系統であるTS3055系統の形質転換体のT2世代の植物を生育し、コントロール株としてCol-0_P4株と形態を比較した。結果を図4に示す。
実施例2で選抜したTaqI遺伝子導入シロイヌナズナ2倍体系統(TCL878)、実施例3で選抜した4倍体系統(PCL878_P4)およびコントロール2倍体系統(1406)、4倍体系統(1406_P4)を播種後1週間目に1日間37℃の熱処理を行い、その後14週栽培を継続し乾燥重量を測定した。結果を図5に示す。
TaqI遺伝子の発現によるDNA二本鎖鎖切断の誘発が、植物体の倍数性に与える影響を調べるために、Col-0、Col-0_P4、1406、TCL878、TS3055、TCL878_P4(ぞれぞれについての発芽後1週間37℃1日間熱処理したものの後代)について植物体の倍数性を実施例3と同様にフローサイトメーターによる方法で測定し、ヒストグラムを作成した。結果を、図6に示す。
実施例3で選抜したTaqI遺伝子導入シロイヌナズナ8倍体系統の花茎に結実した種子を播種、生育させた。これらの植物につき、生育状態を観察するとともに、各植物の倍数性および14週栽培後の乾燥重量を測定した。また、倍数性を、実施例2と同様にフローサイトメーターにより測定した。8倍体系統後代の1つであるTCL878_P8-5#3株について、実施例5と同様にしてタイリングアレイを用いて遺伝子のコピー数を調べた。これらの結果を図7~図9に示す。
TaqI遺伝子酵母発現用ベクターとして、図10に示すように、ガラクトース誘導型プロモーター(pGAL1)の下流に酵母コドンに最適化したTaqI遺伝子(TtTaqI opt)、Saccharomyces cerevisiae由来DIT1タンパク質の3’UTR(tDIT1)を配置したpORF-pGAL1-TtTaqI-tDIT1(AUR)を用いた。
図11に示すように、ゲノム再編効率を評価するGF-FPレポーター遺伝子は、緑色蛍光蛋白質(GFP)のN末端側約600 bpとC末端側600 bpで抗生物質Nourseothricin耐性マーカー(nat1)を挟むようにデザインされている。このカセットを用いることで、TaqIによる二本鎖DNA切断により、GF-FP間で相同組換えが起こり、完全長のGFP遺伝子が再構築された場合、酵母が緑色蛍光を発するため、フローサイトメーター等を用いる事でゲノム再編を検出することが出来る(図12参照)。
実施例10で作製したTaqI遺伝子酵母発現用ベクター、pORF-pGAL1-TtTaqIopt-tDIT1(AUR)を実施例11で作製したBY4741+GFP(HIS)株へ形質転換した(BY4741+GFP(HIS)+TaqI)。グルコースを糖源とするYPD培地(10 g/L Yeast extract、20 g/L Peptone、20 g/L Glucose)+0.5 mg/L オーレオバシジンA(AbA)を用いて、30℃で終夜培養後、ガラクトースを糖源とするYPG(10 g/L Yeast extract、20 g/L Peptone、20 g/L Galactose)+0.5 mg/L AbA培地に培地交換し、20、22.5、25、27.5、30℃で終夜培養することで、TaqIの発現誘導を行った。各温度でのTaqI発現量を測定するために、菌体量を揃えてSDS-PAGEを行い、抗TaqI抗体を用いて、ウエスタンブロッティングを行った。結果を図13に示す。
BY4741+GFP(HIS)+TaqI株を用いて、実施例12と同様に20℃でTaqIの発現誘導を行った。42℃で30分間、または60分間熱処理する事で、一過的にTaqIの活性化を促した後、フローサイトメーターで1×105細胞中のGFP蛍光を持つ細胞の割合を測定することでゲノム再編効率を算出した。結果を図14Aに示す。その結果、ゲノム再編効率は0.03~0.04%だった。一方、図14Bに示すように、熱処理後にYPD培地に培地交換し、回復培養を18時間行った場合、ゲノム再編効率は0.06~0.07%に上昇した。
BY4741+GFP(HIS)+TaqI株を用いて、実施例12と同様にYPG+0.5 mg/L AbA培地に培地交換した後、20、25、30、35℃でTaqIを発現誘導しながら、穏やかにTaqIの活性化を行った。5、23、46時間後に酵母を回収し、実施例13と同様にフローサイトメーターでゲノム再編効率を測定した。結果を図15に示す。
BY4741+GFP(LEU)株とBY4742+GFP(LEU)株、BY4741+GFP(HIS)株とBY4742+GFP(HIS)株を定法に従いそれぞれ接合し、SD-Met-Lys寒天プレートで30℃、5日間培養した。生育したコロニーをシングル化後、フローサイトメーターでゲノムDNA量を測定し、2倍体であることを確認した株をBY4743+GFP(LEU)株、BY4743+GFP(HIS)株とした。
実施例10で作製したTaqI遺伝子酵母発現用ベクター、pORF-pGAL1-TtTaqIopt-tDIT1(AUR)を実施例11、14で作製した各倍加酵母へ形質転換した。形質転換体を用いて、実施例12と同様にYPG+0.5 mg/L AbA培地に培地交換した後、35℃でTaqIを発現誘導しながら、穏やかにTaqIの活性化を行った。22時間後に酵母を回収し、実施例13と同様にフローサイトメーターでゲノム再編効率を測定した。結果を図16に示す。
実施例16と同様に、各倍加酵母を用いて35℃でTaqIを発現誘導しながら、穏やかにTaqIの活性化を行った。0、16、46時間後に酵母を回収し、70%エタノールで固定後、DAPI染色によりゲノムDNAを蛍光染色した。蛍光染色した各倍加酵母を用いてフローサイトメーターで核相解析を行った。結果を図17に示す。図17に示すように、倍加酵母では、倍数性が増加または減少した個体の出現頻度の上昇が認められた。
(OC2-A(CF)株の取得)
ワイン酵母OC-2株にキシロース資化遺伝子を導入したOC2-A株を作製した。すなわち、ワイン酵母OC-2株にキシロース資化遺伝子を導入したOC700株(特開2014-193152)をベースに、ADH2遺伝子を破壊するとともにADH1遺伝子を増強し、mhpF遺伝子(E.coli由来)を導入したOC2-A株を取得した。
OC2-A株を用いて、以下のとおり、ゲノム再編によるキシロース資化能力の進化育種を行った。OC2-A株にKluyveromyces lactis由来のURA3遺伝子を導入したOC2-A(KlURA3)株と、Kluyveromyces marxianus由来のTRP1遺伝子を導入したOC2-A(KmTRP1)株を作製した。両株を用いてプロトプラスト-PEG法による細胞融合を行い、細胞融合株OC2-A(CF)株を作製した。
キシロースを主な糖源とする培地で培養、植え継ぎを繰り返すことで集積培養(35℃)を行った。集積培養を開始後、約300時間までは僅かにグルコースを加えたYPX培地(10g/L Yeast extract、20 g/L Peptone、1 g/L Glucose、20 g/L Xylose)で培養した。その後、キシロースのみを糖源とするYPX培地(10 g/L Yeast extract、20 g/L Peptone、20 g/L Xylose)に変更し、さらに集積培養を約200時間行った。これらの培養工程における菌体量の推移を図18に示す。集積培養後の培養液をYPD寒天プレートにストリークし、シングル化した株をOC2A-C5株とした。
OC2A-C5株のキシロース資化能力を発酵試験により評価した。5%キシロース発酵培地(10 g/L Yeast extract、50 g/L Xylose)にOC2-A株、OC2-A(CF)株、OC2A-C5株をそれぞれOD600 = 1.0で植菌し、32℃で発酵試験を行った。0、16、24、48、72時間でサンプリングを行い、HPLCでキシロース及びエタノールを測定した。結果を図19に示す。
実施例18で作製したOC2A-C5株を用いて、実施例18と同様にしてTaqI処理を行い、ゲノム再編酵母ライブラリーを作製した。次に、YPX培地(10 g/L Yeast extract、20 g/L Peptone、20 g/L Xylose)、39~41℃で培養、植え継ぎを繰り返すことで集積培養を約500時間行った。集積培養後の培養液をYPD寒天プレートにストリークし、シングル化した株をOC2A-TT株とした。
Claims (23)
- 真核生物体における遺伝的組換えの誘発方法であって、
真核生物体が本来的に有する倍数性を超える倍数体である真核生物体の細胞内においてDNA二本鎖切断活性を有するタンパク質を作用させるゲノムセットの改変工程、
を備える、方法。 - 前記遺伝的組換えは、1又は2以上の塩基の置換、挿入及び欠失による遺伝子突然変異並びに染色体の逆位、不等交叉、交叉、転座、重複、欠失、サイズコピー数の低下、コピー数の増大、染色体倍数化及び染色体異数化からなる群から選択される1種又は2種以上である、請求項1に記載の方法。
- 前記真核生物体は4倍数性以上の倍数体である、請求項1又は2に記載の誘発方法。
- 前記遺伝的組換えは、染色体異数化を含む、請求項1~3のいずれかに記載の誘発方法。
- 前記遺伝的組換えは、ゲノムサイズの低下又は増加を含む、請求項1~4いずれかに記載の誘発方法。
- 前記遺伝的組換えは、染色体の一部に欠失又は重複を含む、請求項1~5のいずれかに記載の誘発方法。
- 前記改変工程は、前記タンパク質の前記DNA二本鎖切断活性を人為的に活性化する工程である、請求項1~6のいずれかに記載の誘発方法。
- 前記改変工程は、前記タンパク質の前記DNA二本鎖切断活性についての至適温度より低い温度で作用させる工程である、請求項1~7のいずれかに記載の誘発方法。
- 前記改変工程は、前記タンパク質をコードする外来遺伝子の発現によって得られる前記タンパク質を用いる工程である、請求項1~8のいずれかに記載の誘発方法。
- 前記タンパク質は多頻度制限酵素である、請求項1~9のいずれかに記載の誘発方法。
- 前記多頻度制限酵素は、好熱菌由来の制限酵素である、請求項10に記載の誘発方法。
- 前記多頻度制限酵素は、TaqIである、請求項11に記載の誘発方法。
- 前記真核生物体は、植物体である、請求項1~12のいずれかに記載の誘発方法。
- 前記真核生物体は、微生物である、請求項1~13のいずれかに記載の誘発方法。
- 前改変工程に先立って、人為的にゲノムサイズ増大操作によって親真核生物体を取得する倍数化工程を備える、請求項1~14のいずれかに記載の誘発方法。
- 前記倍数化工程は、倍化誘発剤を用いる、請求項15に記載の誘発方法。
- 前記倍数化工程は、細胞融合を用いる、請求項14に記載の誘発方法。
- 改変された真核生物体の生産方法であって、
真核生物体が本来的に有する倍数性を超える倍数体である親真核生物体の細胞内においてDNA二本鎖切断活性を有するタンパク質を作用させて、前記親真核生物体のゲノムセットを改変する改変工程、
を備える、方法。 - さらに、改変されたゲノムセットを保持する真核生物体の集団から、任意の指標に基づいて意図する真核生物体を選抜する工程、
を備える、請求項18に記載の生産方法。 - 真核生物体集団の生産方法であって、
本来的に有する倍数性を超える倍数体である親真核生物体の細胞内においてDNA二本鎖切断活性を有するタンパク質を作用させて親真核生物体のゲノムセットを改変する改変工程を備え、
改変されたゲノムセットを保持する真核生物体の集団を取得する、生産方法。 - 本来的に有する倍数性を超える倍数性を有し、DNA二本鎖切断活性を有するタンパク質をコードする遺伝子を発現可能に保持する、真核生物体である育種材料。
- 育種材料の生産方法であって、
本来的に有する倍数性を超える倍数体である真核生物体に対して人為的なゲノム増大操作を1回又は2回以上繰り返して、所望の倍数性の真核生物体を得る工程と、
前記所望の倍数性の真核生物体をDNA二本鎖切断活性を有するタンパク質をコードする遺伝子を発現可能に形質転換する工程と、
を備える、方法。 - 育種材料の生産方法であって、
本来的に有する倍数性を超える倍数体である真核生物体をDNA二本鎖切断活性を有するタンパク質をコードする遺伝子を発現可能に形質転換する工程と、
形質転換された前記真核生物体に対して人為的なゲノム増大操作を1回又は2回以上繰り返して、所望の倍数性の形質転換された真核生物体を得る工程と、
を備える、方法。
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