WO2005040374A1 - ゲノムdna断片の選抜方法 - Google Patents
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- WO2005040374A1 WO2005040374A1 PCT/JP2004/015743 JP2004015743W WO2005040374A1 WO 2005040374 A1 WO2005040374 A1 WO 2005040374A1 JP 2004015743 W JP2004015743 W JP 2004015743W WO 2005040374 A1 WO2005040374 A1 WO 2005040374A1
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Classifications
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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
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1079—Screening libraries by altering the phenotype or phenotypic trait of the host
-
- 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)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
-
- 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
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants
Definitions
- the present invention relates to a method for efficiently selecting a genomic DNA fragment that can produce a mutation that can be agriculturally beneficial to a plant.
- a plant genomic library is transformed by applying a shotgun 'claw jungle to a genomic library as a plant instead of a microorganism (Klee et al. Mol Gen Genet 210: 282-287, 1987).
- a genomic library was constructed from arabidopsis transformants produced by introducing a kanamycin-resistant gene derived from a microorganism, and a mixed strain containing a large number of agrobacterium strains containing genomic clones was discarded from a petunia leaf disc.
- petunia cells resistant to kanamycin that is, petunia cells containing a kanamycin resistance gene derived from an Arabidopsis transformant.
- the results showed that the microorganism-derived kanamycin resistance gene in the Arabidopsis genome can be captured even after transformation into petunia.
- Heterosis is the phenomenon in which the first generation of hybrids (F1) shows stronger viability than both parents when two parent varieties are crossed.
- F1 hybrid stress
- the whole plant has high vigor, large plants and organs, high yield, high growth rate, disease,
- Various traits such as resistance to insect damage, resistance to various environmental stresses such as drought, high temperature and low temperature, increase / decrease of specific components, increase / decrease of specific enzyme activity, etc. are observed. Many are very useful for agriculture.
- the first-generation hybrid breeding method which produces new varieties by crossing different parents, has been used for the improvement of cultivated plants for a long time, and greatly contributes to breeding excellent varieties in many crops including corn. Dedication.
- the first generation hybrid breeding method requires a number of steps, including breeding and improving breeding groups, breeding inbred lines, general combination ability test, specific combination ability test, and selection of F1 varieties. Each step requires a great deal of time and effort. In addition, heterosis often has a great effect when genetically distantly related parents are crossed, but on the other hand, when the relative relationship is distant, the mating often does not have fertility even when crossed. The range of species that can be used was limited.
- Heterosis traits are dominated by many loci in different linkage groups, in which alleles that favor survival and productivity are dominant and those that are disadvantageous are recessive. It is considered that there are many cases. Because of the large number of loci linked together, it is almost impossible to obtain a line in which the favorable allele is homozygous at all of the many loci. However, in F1, heterosis is caused by the combination of all the favorable alleles of the parents.
- each two alleles are heterozygous.Either of the alleles may be more favorable to survival and productivity than homozygous. Heterosis power is developed by summation.
- heterosis is governed by a large number of genetic factors, and it has been difficult to cultivate new varieties with improved traits whose expression is observed to be improved in heterosis by conventional techniques. .
- the traits whose expression is enhanced by heterosis are often so-called quantitative traits, but genetic analysis of the quantitative trait loci (QTL, Quantitative Trait Loci) that govern them is not easy.
- QTL quantitative trait loci
- molecular biological techniques it has become possible to perform genetic analysis of QTL using DNA markers.
- chromosomal sites containing QTLs that govern certain quantitative traits have been identified.
- research to clone agriculturally useful genes using molecular maps and molecular biology techniques is also being actively pursued.
- the site containing the QTL can only be roughly identified, and the DNA fragment containing a large number of genes theoretically becomes apparent as a DNA fragment containing the QTL. It ’s not good. Then, it is not easy to identify a fragment having a size that can be crawled or a fragment that can be introduced into a plant by transformation. In addition, it takes a long time and a lot of labor to create a detailed gene map, identify the desired gene based on the map information, and close the gene. In fact, there have been few cases in which DNA fragments that increase quantitative traits have been cloned based on QTL analysis.
- a vector that can clone a large DNA fragment of 40 to 80 kb and that can introduce a gene into a plant is known (Liu et al. Proc. Natl. Acad. Sci. USA 96: 6535-6540, 1999). ). Experiments have also been attempted to introduce individual, specific cloned genomic fragments from plants into higher plants. However, no attempt has been made to individually introduce a large number of genomic fragments constituting a genomic DNA library into plants with unknown functions.
- Patent Document 1 PCT International Publication WO 03/018808 A
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- the present invention provides a method for efficiently selecting a large number of genomic DNA fragments that can produce agriculturally beneficial mutations, and selecting and preparing them as cloned DNA fragments.
- the present invention provides a method for efficiently selecting and preparing a genomic DNA fragment that improves the expression of a trait whose expression is improved by a plurality of genetic factors. [0028] The present invention provides a method for efficiently selecting a large number of genomic DNA fragments capable of improving a trait or a quantitative trait expressed in heterosis, and selecting and preparing as a cloned DNA fragment. I will provide a.
- the present invention relates to a method for breeding group breeding, improvement, self-breeding, general combination ability test, specific combination ability test, selection of F1 varieties and the like, which cannot be avoided by techniques such as the first generation hybrid breeding method.
- Another object of the present invention is to provide a method for efficiently selecting and preparing genomic DNA fragments that can bring about a potentially agriculturally useful mutation to a plant without requiring many steps, each of which requires a long time.
- the present invention is based on only the phenotype of the plant into which the genomic DNA fragment has been introduced, even when the mechanism of the expression of the trait and the information on each gene that causes the trait to be expressed are almost unknown.
- a method for efficiently selecting and preparing a genomic DNA fragment that can bring about a mutation that can be agriculturally beneficial to a plant by selecting a superior individual.
- the present invention provides a genomic DNA that enables expression similar to the trait improvement observed in heterosis (hereinafter, referred to as "heterosis-like expression”) not only between the same plant varieties but also between different plant varieties.
- heterosis-like expression a genomic DNA that enables expression similar to the trait improvement observed in heterosis
- the present invention provides a method for efficiently selecting and preparing a large number of genomic DNA fragments that enable heterosis-like expression without requiring much time and labor.
- the present invention provides a method for transforming a plant with a genomic DNA fragment prepared by the method of the present invention, which is capable of causing a mutation that can be agriculturally beneficial to a plant, or a genomic DNA fragment capable of causing heterosis-like expression.
- the present invention provides a method for producing a plant having a mutation which may be agriculturally beneficial, and a plant produced by the method.
- the present invention relates to all or a part of a genomic DNA fragment capable of causing a mutation that can be agriculturally beneficial to a plant or a genomic DNA fragment capable of causing heterosis-like expression, prepared by the method of the present invention.
- the term "agriculturally beneficial mutation” refers to "a plant, particularly a cultivated plant or a plant under normal or favorable cultivation conditions for the plant or under conditions where some stress is applied to the plant. And Z or an ornamental plant, a mutation that causes a quantitative increase or decrease or an increase or decrease in the growth rate of at least a part of the plant. Stressful conditions include salt concentration, high and low temperatures, dryness, disease and the like in the cultivation area.
- Such a mutation causes high yield if the number of seeds and leaves increases under normal cultivation conditions, and does not die under the conditions of disease stress and the like. This is because an increase in the number of leaves and stems means resistance to disease stress and the like. Naturally, content components and enzymes contained in plants are also included in "parts of plants”. Decreasing the size of the whole or part of the plant is also often agriculturally beneficial because dwarf plants are actively bred and widely cultivated!
- the concept of "a mutation that causes a quantitative increase or decrease or an increase in the growth rate of at least a part of a plant under some cultivation conditions” is a concept of "vig.” high (vigor), large plants and organs, high yield, high growth rate, disease and insect resistance, strong against various environmental stresses such as drought, high temperature and low temperature, increase and decrease of specific components Includes many agriculturally beneficial mutations, such as increased or decreased specific enzyme activity, dwarfism, etc.
- selection includes the case where when a certain selected group is subjected to a certain selection step, the element of the selected group becomes zero, that is, the selected group includes: Includes cases where it is found that elements that meet the selection criteria are not included
- the present invention is a method for selecting a genomic DNA fragment that can bring about a mutation that can be agriculturally beneficial to a plant by the following steps (1) to (4) and, if desired, (5).
- genomic DNA fragment is isolated from a plant by a commonly used method. After strict restriction digestion and size fractionation, construct a genomic DNA library by a conventional method.
- the plant that supplies the genomic DNA fragment there is no particular limitation on the plant that supplies the genomic DNA fragment, but a preferred example is a plant that can generate heterosis by crossing with a plant into which the genomic DNA fragment is introduced.
- a preferred example is a plant that can generate heterosis by crossing with a plant into which the genomic DNA fragment is introduced.
- the plant to be introduced is japonica rice, Oryza 'Lfipogon, which is a kind of wild rice, and indica rice are preferable.
- the plant to be introduced is a specific corn variety, other corn varieties or wild-type teosinte are preferred examples of the source plant.
- larger heterosis is observed in plants with a closer relationship. Conventionally, when the relative relationship is distant, crossing becomes impossible, so that heterosis in combination with plants with distant relatives could not be used. Since the genomic DNA fragment of the original plant can be easily used, the plant is also preferred because of its close relationship, and can be used as the donor plant.
- Various vectors can be used as the closing vector used for the construction of the genome library.
- a vector that can be used directly for transformation of the plant into which it is introduced is used.
- PSB200 or pCLD04541 can be used to transform rice, tobacco, arabidopsis, etc.
- pSB25UNpHm can be used to transform corn. .
- the DNA fragment to be cloned may contain at least one gene, but each gene in the genome and a region required for controlling its expression are included. As much as possible, a size of 1 kb or more is preferred, a size of 10 kb or more is more preferred, more preferably 20 kb or more, more preferably 30-40 kb or more. In any case, there is no particular upper limit on the size of the DNA fragment as long as it can be introduced into the clawing vector. Methods for partial restriction digestion to obtain such DNA fragment sizes are known.
- the total number of clones constituting the genomic DNA library that is, the size of the library, is preferably large enough to include many genes of the plant genome.
- a restriction enzyme that recognizes four bases, such as MboI or Taql.
- Methods for determining appropriate degradation conditions are known, for example, Molecular Cloning, A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, Cold
- the total number of clones for an arbitrary genomic fragment to be included in the genomic DNA library with a certain probability is calculated by the following equation.
- ⁇ is the probability that an arbitrary genomic fragment is contained in the genomic DNA library
- f is the average length of the genomic fragment contained in the clone / genome size of the original plant
- N is the total number of genomic clones. is there).
- genomic DNA library After the construction of the genomic DNA library, some of the clones constituting the library are incorporated into Escherichia coli and cultured. Count the number of colonies that appear (for plasmid or cosmid vectors) and plaque (for phage vectors), and estimate the total number of clones contained in the library. Furthermore, DNA is prepared from a portion of the colonies and phages that have appeared, the size of the cloned DNA fragment is measured, and the average fragment length is estimated.
- a vector that can be used directly for plant transformation for example, a vector such as pSB200, pCLD04541, or pSB25UNpHm
- an individual clone can be used as it is in a transformation experiment. Otherwise, the transformation experiment can be performed after transferring all or a part of the DNA fragment contained in each clone to a transformation vector.
- the destination plant used for the transformation may be a plant different from the plant from which the genomic DNA is derived, may be a different variety of the same species, or may be a same species of the same species.
- preferable plants include plants for producing cereals such as rice, barley, wheat, and corn; plants for producing luxury products such as coffee, cocoa, tea, and tobacco; and ornamental plants such as vegetables, fruits, and flowers. There is a wide range of plants with virtually no restrictions.
- the transformation method may be any existing method! For example, as a biological introduction method, a force injection method such as an aglobatalyte method is used.As a physical introduction method, a microinjection method, an electoral port method, a particle gun method, a silicon carbide method, an air injection method, or the like is used. The polyethylene glycol method is known.
- a biological introduction method a force injection method such as an aglobatalyte method is used.
- a physical introduction method, a microinjection method, an electoral port method, a particle gun method, a silicon carbide method, an air injection method, or the like is used.
- the polyethylene glycol method is known.
- the genomic DNA is integrated into the genome of the target plant. According to the present invention, it was an advantage that the number of genomic DNA fragments capable of causing heterosis-like expression, which can provide one plant genome library, is not limited to one. Therefore, in order to select more genomic fragments, it is desirable to introduce more genomic fragments individually into
- the selection method of the present invention does not prevent the inclusion of any preliminary selection step, in order to eliminate the bias of the candidate fragments to be selected, the genomic DNA fragment to be introduced into the plant must be prepared before the introduction. In addition, it is desirable not to include a preliminary selection step as much as possible.
- genomic DNA fragment to be introduced what kind of phenotype the DNA fragment is involved in, especially in the original plant, is! do not need. Only by selecting the phenotype of the transformant is the power to identify useful genomic DNA fragments.
- each genomic clone to be introduced into a plant is amplified and Z or stored. Storage is carried out by a conventional method, such as purified DNA, a bacterium such as Escherichia coli containing a genomic clone, or a yeast.
- the regenerated transgenic plant and its progeny plant are plant
- vigor size of plant and individual organsWeight, yield, growth rate, disease 'resistance to insect damage, dryness'High temperature' resistance to various environmental stresses such as low temperature, increase of specific components', Evaluate various agriculturally useful traits, such as increasing or decreasing specific enzyme activities.
- vigor refers to the vitality and vigorous growth of the whole plant.
- the trait to be evaluated is not particularly limited as long as it is an agriculturally useful trait, irrespective of the characteristics of the genomic DNA fragment source plant or the introduced genomic DNA fragment. Is preferably a quantitative trait or a trait that is improved by heterosis, and is preferably a trait that is agriculturally useful when the target plant is regarded as a breeding target.
- a phenotypically mutated plant is selected for a trait evaluated in comparison with a plant into which the genomic fragment has not been introduced. For example, compared to a plant into which the genomic fragment has not been introduced, the vigor of the whole plant is higher and the vigor of the plant and individual organs is higher. High weight, high yield, high growth rate, high resistance to diseases and insects, dryness, high temperature High resistance to various environmental stresses such as low temperature, high and low specific components, specific enzyme activity Plants that show an increase, decrease, etc. can be selected. For each trait, the direction of mutation to be selected is not limited to one.
- the dwarf trait is an important agronomic trait that is a breeding target in various crops, so the size of plants and individual organs is higher than that of plants that have not introduced the genomic fragment. Plants that have become smaller can also be selected. The same applies to other traits.
- Such traits are mostly so-called quantitative traits, and greatly affect not only genetic factors but also environmental factors. Even in the case of a small plant into which a genomic fragment has not been introduced, the measured value shows a distribution having a certain variation due to environmental factors and the like. According to the present invention, in a population of plants into which a genomic DNA fragment has been randomly introduced, if the genomic DNA fragment causing the phenotypic variation is present, it is expected that the distribution of measured values will be broadened. Then, by selecting a plant showing a measurement value located at the end of this distribution, a small population including a plant containing a genomic DNA fragment that causes a phenotypic variation can be obtained.
- the progeny plants are evaluated, and repetitive surveys for various traits are performed. And other traits, etc., and can be evaluated in detail from the viewpoint of molecular biology, genetics, biochemistry and plant physiology. After various evaluations, it can be used agriculturally as a new variety. In addition, if a plant exhibiting more excellent traits can be obtained from these plants, the genomic DNA fragment introduced into such a plant and selected as a genomic DNA fragment of even higher value can be selected. be able to.
- genomic DNA fragments introduced into the selected plants are separately stored as genomic clones as described above, and may be propagated in Escherichia coli using a clawing vector, PCR, or LAMP.
- the required amount can be produced using a biochemical propagation method such as the method.
- the base sequence is determined, the contained genes, introns, etc.
- the analysis of the genetic element of the above can be investigated in detail.
- This genomic fragment can be introduced into any plant using a known transformation technique, so that it is possible to improve the breed of a plant different from the plant from which the genomic DNA fragment is derived, and to cultivate a different variety of the same plant. It can be used for improving and breeding the same varieties of plants of the same species.
- step (4) If necessary, all or part of the genomic DNA fragment thus selected is again introduced into the same or different plant, and the same evaluation is carried out. Can be subjected to a typical selection process. In this case, the transformation may be performed using the same clawing vector used in step (2), or another clawing vector may be used.
- the genomic DNA fragment selected in step (4) is subcloned into the vector. Since the restriction sites used for cloning in the cloning vector vary depending on the cloning vector, depending on the restriction enzyme used, only a part of the genomic DNA fragment selected in step (4) is subcloned. May be appropriate.
- the size of the DNA fragment that can be closed varies depending on the closing vector or the method of closing, and for this reason, it may be appropriate to subclone only a part of the DNA fragment.
- One of the effects of performing a secondary selection step using only a part of the DNA fragment is that transformation is performed using only a part of the DNA fragment, and the entire DNA fragment is used. If it is found that the same effect is obtained as in the case where the genomic DNA fragment is selected in step (4), an unnecessary portion is clarified.
- the transformed plant obtained by the secondary transformation and its progeny plants are vigor of the whole plant, the size of the plant and individual organs, weight, yield, growth rate, disease '' Evaluation of various agriculturally useful traits such as resistance to insect damage, resistance to various environmental stresses such as drought, high temperature and low temperature, increase / decrease of specific components, increase / decrease of specific enzyme activity, etc.
- genomic DNA fragments that resulted in a plant whose phenotype was mutated in comparison with a plant into which the genomic fragment had not been introduced were evaluated in the primary selection step. Irrespective of cultivation conditions, plant species, and other conditions in the plant, it is possible to cause a mutation of a preferred phenotype in a plant, and it is possible to select a particularly preferred genomic DNA fragment.
- progeny plants are evaluated, and demodulation of various traits is performed, and characteristics of the mutated trait, inheritance pattern, and association with other traits are evaluated. be able to. After various evaluations, they tend to use agriculturally as new varieties.
- Genomic DNA fragments that have been confirmed to be able to be selected will be selected. Therefore, a genomic DNA fragment of higher value is selected than in the case of only the primary selection.
- This selection step can be repeated any number of times, and as a result, a genomic DNA fragment of higher value can be selected.
- the present invention has been described centering on a method for selecting a genomic DNA fragment.
- the present invention relates to a genomic DNA fragment that can bring about a mutation that can be agriculturally beneficial to the plant thus selected.
- a plant having a potentially agriculturally useful mutation resulting from transformation with a genomic DNA fragment are also provided.
- Various methods have been known for introducing a specific DNA fragment into a plant cell or plant tissue, forming a callus on the cell or tissue, culturing the callus, and redistributing the callus into a complete plant. I have. See, for example, Hiei et al. Plant J. 6: 271-282, 1994.
- the plant may be regenerated from the transformed cells without significant callus formation depending on the plant.
- the present invention is also effective in this case. To fix the regenerated plant as a variety, the method of Maruta et al. Molecular Breeding 8: 273-284, 2001 is known. [0073] The present invention further provides the genomic DNA fragment of the present invention as a marker in plant breeding.
- a plant having the genomic DNA fragment of the present invention can be used to enhance the efficiency of plant breeding.
- Use for breeding refers to use as a genomic DNA fragment donating plant for introducing the genomic DNA fragment of the present invention into another plant, or breeding by a cross breeding method. It may be used as a parent plant for carrying out the process.
- genomic DNA is also prepared for a progeny plant, which is obtained by crossing a plant known to have the genomic DNA fragment of the present invention with a plant to be breeded, and It is known that a gene containing the genomic DNA fragment of the present invention is selected, and the selected progeny plant individual is used as a marker by using the sequence information of the specific genomic DNA fragment as a marker. al.
- the entire genomic DNA fragment may be used as a marker, and if a part of the genomic DNA fragment contains a specific sequence, the partial sequence may be used as the marker.
- the genomic DNA fragment of the present invention may be hybridized to the base sequence of the genomic fragment obtained by the method disclosed herein under mild or severe stringency conditions, and Also included are biologically active isolated DNA and RNA.
- Hybridization conditions under severe stringency include, for example, those described in Molecular Cloning, etc., at 65 ° C. in 0.5 M sodium phosphate pH 7.2, 1 mM EDTA, 7% SDS, 1% BSA.
- the genomic DNA fragment selection method of the present invention elucidates the functions of plant genes, such as a method of comparing and examining a base sequence with a known base sequence in conventional genomic analysis and a method of estimating the function of cDNA. Search for DNA fragments involved in heterosis with a new approach that does not need to be performed.
- the method for selecting a genomic DNA fragment of the present invention comprises the steps of:
- the method for selecting a genomic DNA fragment of the present invention can select a wide range of traits useful for agriculture where there are no restrictions on the traits to be selected.
- genomic DNA fragments are selected based on the phenotype of the target plant, and thus the selected genomic DNA fragments are used as they are for breeding the target plant. be able to.
- a genomic DNA fragment causing the same effect as heterosis is obtained as a cloned DNA fragment and as a DNA fragment that can be easily transformed into a plant. Unlike the use of heterosis by classical breeding methods, it does not require long time and much labor for breeding.
- the method of selecting a genomic DNA fragment of the present invention differs from the use of heterosis by the classical breeding method in that a genomic fragment of one cultivar is introduced into the other cultivar, so that the breeding between parent varieties can be performed.
- the combination of plants, which is not possible with the conventional hybrid breeding method, is possible without the limitation of segregation. Therefore, the DNA fragment of the present invention can be easily introduced into various plants by transformation methods, regardless of whether it is the same species or different from the plant derived from the DNA fragment, and used for breeding. Take advantage of the benefits of heterosis in the meantime It is possible.
- the method for selecting genomic DNA fragments of the present invention does not require searching for loci involved in agronomic traits, so that it can be performed efficiently without requiring a long time and a large amount of labor. It is possible to select genomic DNA fragments that increase or decrease the quantitative trait.
- genomic DNA fragment of the present invention since the genomic DNA fragment of the present invention has an effect of inducing expression similar to heterosis by transformation, it can be used as a marker in a normal cross breeding method to select progeny of the cross. Efficiency can be greatly increased.
- FIG. 1 is a genetic map of the closing vector pSB200.
- FIG. 2 is a genetic map of a closing vector pSB25UNpHm.
- Fig. 3 is a photograph showing an example of a transgenic plant selected based on external observation of ear size, number of grains per ear, and viga of the whole plant, and a control plant ( Generation: TO, selected lines: 5310).
- Fig. 4 is a view showing the results of a rice blast resistance test of transformed rice plants selected by introducing a genomic DNA fragment of Oryza 'rufipogon.
- FIG. 5 is a diagram showing the amount of leaf elongation under stress conditions of a transformed rice plant selected by introducing a genomic DNA fragment of Oryza 'rufipogon.
- FIG. 6 is a graph showing the effect of introducing a genomic DNA fragment in the growth of transformed tobacco callus selected by introducing a genomic DNA fragment of Oryza 'rufipogon.
- Fig. 7 is a photograph showing an example of a transgenic plant selected by introducing a genomic DNA fragment of theosinte and a control plant.
- FIG. 8 is a diagram showing amplification sites of a genomic DNA fragment of Oryza rufipogon by PCR.
- FIG. 9 is a photograph showing an example of the result of amplification of a genomic DNA fragment of Oryza rufipogon by PCR.
- FIG. 10 is a photograph showing an example of electrophoresis of a restriction enzyme fragment generated from a genomic DNA fragment of Oryza 'rufipogon.
- FIG. 11 is a photograph showing an example of electrophoresis of a restriction enzyme fragment of a transformation vector into which a genomic DNA fragment of Oryza 'rufipogon has been incorporated.
- Example 1 Extraction of genomic DNA from Oryza and Rufipogon Construction of Genomic DNA Library
- Oryza cultivated in a greenhouse using seeds of Oryza rufipogon, a closely related species of rice, obtained from the National Institute for Bioresources, Ministry of Agriculture, Forestry and Fisheries 'Genomic DNA was extracted from the leaves of Luffypogon by a conventional method.
- This genomic DNA was subjected to partial restriction digestion with the restriction enzyme Taql, and a 30 kb to 50 kb fraction was prepared by sucrose density gradient centrifugation. Using this fraction, cloning was performed at the Nsp (7524) V (sometimes simply referred to as NspV) cleavage site of the cosmid vector PSB200 to construct a genomic DNA library.
- Nsp 7524
- V sometimes simply referred to as NspV
- PSB200 is a cloning vector constructed from pSBll described in Komari et al. (Plant J. 10: 165-174, 1996). That is, the ubiquitin promoter derived from corn, the hygromycin resistance gene, the 3 'end signal of the NOS gene,
- the Nsp (7524) V cleavage site is added to pSBll.
- pSB200 a genomic DNA library having an average fragment length of about 40 kb can be constructed.
- PSB200 is also a vector for transformation of higher plants, and can introduce genes into various plants using the hygromycin resistance gene as a selection marker.
- Oryza 'Luffigong's genomic DNA library is a clone of japonica rice with clones
- the constituent clones of the genomic DNA library were individually introduced into Agrobacterium strain LBA4404 (pSBl) ( Komari et al. 1996).
- the method used for the introduction is Triparental mating (Ditta et al. Proc Natl Acad Sci. USA 77: 7347-7351, 1980).
- These agrobacterium were individually introduced into rice (cultivar Yukihikari).
- the transformation was carried out according to Hiei et al. 1994 by inoculating an immature embryo with agrobacterium.
- the immature embryos of the varieties Yukihikari are sold for food and obtained from plants sown with green rice and cultivated in a greenhouse or their progeny plants cultivated in a greenhouse.
- a transformed plant was obtained in which a total of 5310 genomic DNA fragments contained in the genomic DNA library were individually introduced. In addition, for each genomic DNA fragment, 115 independent transformants were obtained.
- a transformed current plant is referred to as a TO generation plant, and its progeny plants are referred to as a T1 generation plant, a T2 generation plant, and the like in order of generation.
- Example 3 Oriza 'Luffigon's genomic DNA fragments contained in a genomic DNA fragment contained in the library
- the transformed plants were cultivated in a greenhouse, and the individual plants were examined for the overall plant size, plant height, relative growth rate, number of ears, above-ground weight, ear weight, ear length, number of fertile grains, and yield.
- the relative growth rate refers to the amount of daily growth per unit plant height, and is calculated by ((Survey end day plant height Survey start day plant height) Z days of survey period) Z Survey start plant height.
- Tables 1 to 6 and FIG. 3 show examples of selected plants and names given to the introduced genomic DNA fragments. In these examples, multiple transformants in which the same genomic fragment was introduced showed similar mutations and were selected.
- the distribution of the measured values of the control plant was applied to a normal distribution.
- Rice (Yukihikari) transformed with GUS gene was used as a control plant.
- the probability of appearance of a line showing the measured value of the selected transformed plant line was calculated.
- the value of the probability of appearance for the number of selected lines is extremely small, and the expected value of the appearance of such selected lines is far below 1.0. Therefore, the assumption that the introduced fragment had no effect was rejected, and it was statistically proved that the selected line showed significant mutation.
- transgenic ⁇ S plants and control plants generation: T0, number of selected lines' ⁇
- Control plant 558 0.55 Standard deviation: 0.30 [Table 6] Examples of transformants selected based on spike length and whole plant viga and control plants
- plants of the progeny of the transgenic plants were cultivated, and the plants were evaluated in the same manner as described above.
- individuals containing the introduced genomic DNA fragment are expected to separate from individuals not containing the introduced genomic DNA fragment according to Mendel's law.
- the presence or absence of the introduced fragment is determined using the Polymerase Chain Reaction (PCR) method. Investigated.
- Tables 7 to 9 below show examples of the selected plants and names given to the introduced genomic DNA fragments.
- all plants in which the presence of the introduced fragment was confirmed by PCR showed the same mutation, and the introduced fragment was detected by PCR. None of the viable plants showed such mutations.
- selection was performed based on the viga of the whole plant and some measured values.
- transgenic plant selected based on plant height at 14th H after transplantation and viga of whole plant and control plant (generation: Tl, number of selected lines 114)
- transgenic plant selected based on plant height and whole plant viga 21 days after transplantation and paired plants (generation: ⁇ , number of selected lines 114)
- Example 4 Selection of target objects based on evaluation of disease resistance
- the T1 generation plant of the transformed line created in Example 2 was transformed and compared with V ⁇ , Yukihikari and Koshihikari as control plants, and evaluated for V and blast resistance. Plants having mutations in resistance-related traits were selected. In addition, genomic DNA fragments introduced into these plants were selected as genomic DNA fragments that could cause agriculturally beneficial mutations in the crop.
- the inoculation source was adjusted as follows.
- the flora of the rice blast strain TSU-01 was inoculated on an oatmeal agar medium (Difco) containing 10 g / 1 of sucrose, and cultured at 26 ° C in the dark for 3 weeks.
- 10 ml of sterile distilled water was added to the plate, the hypha was cut with a sterilized paintbrush, and the plate was cultured under illumination at 25 ° C for 3 days.
- 8 ml of LB liquid medium (Difco) diluted to 1/2 concentration with sterile distilled water was placed in the plate, and the conidia were suspended with a sterile paintbrush. After filtering the suspension through double gauze, the conidia concentration was adjusted to about 2 ⁇ 10 6 conidia / ml.
- Silwet L-77 was added to the inoculum so that the final concentration was 0.01%.
- Inoculation was performed by applying the inoculum with a paintbrush to the developed top leaf of the plant 19 days after transplantation. Immediately after the application, the inoculated leaves were passed through a plastic tube, the upper and lower openings were filled with absorbent cotton, and sufficiently moistened with distilled water. The plants were cultivated for one week at a photoperiod of 14 hours, 25 ° C during the day and 20 ° C at night. During this time, the cotton wool packed in the tube opening was moistened with distilled water once a day. Cut the inoculated leaves and reduce the number and area of lesions to 0 (no symptom)? Evaluation was made with a morbidity of 3 (lesions spread on most of the leaves).
- the presence or absence of the introduced fragment was also determined based on the individual and individual abilities of the collected leaf pieces based on their sensitivity to idalomycin. Individuals without hygromycin resistance, ie, introduced fragments ⁇ individuals were excluded as separate individuals, and the average morbidity of the transformed individuals was compared. Many of the transgenic lines showed the same degree of disease as the untransformed control “Yukihikari”. The following shows examples of 13 transformed lines. A014D1201, A020E0401, A023F0303 and A078C0102 showed significantly lower morbidity than the control “Yukihikari” (FIG. 4). In particular, the average morbidity of A078C0102 was 0, indicating that the introduced Oryza refipogon genomic fragment may have conferred a high degree of blast resistance.
- Example 5 Selection of an object to be shaped based on the evaluation of drying resistance
- the degree of drought tolerance of the transformed plants was evaluated.
- the evaluation was performed on a five-stage scale for each individual with the naked eye (0: one withered, 5: completely recovered). In order to correct the variation in data among the pots, the score of each test individual was subtracted by the score of Yukihikari in the same pot, and the value was used as the score of each individual. Based on the evaluation results, the top 10% were selected as plants containing genomic fragments that are more likely to confer drought tolerance to the crop.
- the leaf length of the top leaf of each individual was measured immediately before the drying treatment and one week after the treatment.
- Leaf length one week after treatment Force The value obtained by subtracting the leaf length immediately before the treatment was defined as the amount of leaf elongation under drought stress treatment conditions for each individual.
- re-watering treatment was performed. After 4 days, the degree of recovery was examined, and the amount of leaf elongation under stress treatment conditions was evaluated. Evaluation was performed as described above, except that Yukihikari (T2), in which only the GUS gene was introduced, was also added.
- Example 6 Evaluation of maize transformed by genomic DNA fragments contained in a genomic DNA library derived from Oryza rufipogon, selection of mutated cattle plant
- Maize was transformed using the Agrobacterium containing a genomic fragment derived from Oryza rufipogon prepared in Example 1. Transformation was performed according to Ishida et al 2003 (Plat Biotechnol. 20: 57-66). The introduced variety is inbred variety A188 (obtained from the Research Institute for Biological Resources, Ministry of Agriculture, Forestry and Fisheries). Similarly to Example 1, a genomic DNA library and an Agrobacterium containing a genomic DNA fragment derived from Oryza rufipogon were prepared using pSB25UNpHm as a vector. Using this, corn was transformed in the same manner as described above. As a result, transformants into which a total of 108 genomic DNA fragments contained in the genomic DNA library were individually introduced were obtained.
- pSB25UNpHm replaces the promoter of the bar gene of pSB25 described in Ishida et al. Nature Biotech 14: 745-750, 1996 with a maize-derived ubiquitin promoter, and further has an Nsp (7524) V cleavage site, -Better with Seel cleavage site and Ceul cleavage site.
- pSB25UNpHm has the same cloning ability as pSB200, and can introduce genes into various plants such as maize using the bar gene as a selection marker.
- Transgenic plants were cultivated in a greenhouse as in the case of rice, and plant height on the 28th day after transplantation, plant height on the 35th day, relative growth rate (((plant height on the 35th day after transplantation, 28 / 7) 28 days after Z transplantation), leaf height, maximum ear weight, maximum number of ear grains, maximum ear total grain weight, 1 grain weight (maximum total ear weight Z maximum)
- the effect of the introduced genomic DNA fragment was investigated by investigating the number of ear grains. In order to compensate for seasonal growth irregularities, the average value of all individuals on the same pot picking day was calculated, and the data was normalized using the formula of (value of each individual-average value) Z-average value and analyzed.
- Maize (A188) transformed with the GUS gene was used as a control plant.
- the variance was significantly greater than that of the control plants. This showed that the distribution of measured values in the plant population into which the genomic DNA fragment was introduced was broadened.
- the probability of appearance of a line showing the measured value of the selected transgenic plant line was calculated.
- the value of the probability of occurrence with respect to the number of selected strains is extremely small in each case, so the expected value of the appearance of such selected strains is significantly lower than 1.0.
- the results were below the expected value in 5 lines. Therefore, the assumption that the introduced fragment had no effect was rejected, and it was statistically proved that the selected lines showed significant mutation.
- the obtained transformed maize was cultivated in a greenhouse, and pollen was fed with pollen of a maize variety (A188) that was separately grown in a greenhouse to obtain seeds.
- the generation derived from this seed is referred to herein as the T1 generation.
- T1 generation 5 to 8 individuals per line were cultivated in a greenhouse and examined for traits.
- individuals containing the introduced genomic DNA fragment will be separated from those containing the introduced genomic DNA fragment according to Mendel's law.Therefore, the presence or absence of the introduced fragment is investigated using the Polymerase Chain Reaction (PCR) method. did. The presence or absence of the fragment can be checked using the PCR method. (Details are described in Example 16).
- transgenic plants in which one or more traits were mutated and their progeny were selected.
- genomic DNA fragments introduced into these plants were selected as genomic DNA fragments capable of causing maize to have agriculturally beneficial mutations.
- Example of transgenic plant selected based on relative growth rate
- transgenic ffi corn sorghum
- Transformation of tobacco was carried out using the agrobacterium containing the genomic DNA fragment derived from Oryza rufipogon prepared in Example 1. Transformation method is Komari Theor Appl The introduced variety is SRI (Kodama et al. Plant Physiol 105: 601-605, 1994).
- the transformed plant and its progeny are cultivated in a greenhouse in the same manner as in the case of rice corn, and the whole plant has a bigger, plant height, relative growth rate, leaf number, leaf length, leaf width, leaf weight.
- the effect of the introduced genomic DNA fragment was determined by examining the above-ground weight, yield, drought tolerance, salt tolerance, disease resistance, and the like. Then, for one or more of these traits, a mutated transgenic product and its progeny were selected. Furthermore, genomic DNA fragments introduced into these plants were selected as genomic DNA fragments capable of causing mutations that could be agriculturally beneficial in tobacco.
- the probability of appearance of an individual showing the measured value of the selected transgenic plant individual was calculated.
- the value of the probability of appearance of the plant exemplified here with respect to the number of selected individuals is extremely small, and thus the expected value of the appearance of such selected individuals is significantly lower than 1.0. Therefore, the assumption that the introduced fragment had no effect was rejected, and it was proved statistically that the selected individuals exhibited significant mutation.
- genomic DNA was isolated from Arabidopsis thaliana, a genomic DNA library was constructed, and genomic clones constituting the genomic DNA library were individually transformed into rice by a transformation method. (Yukihikari). As a result, a transgenic plant was obtained in which a total of 1477 genomic DNA fragments contained in the genomic DNA library were individually introduced.
- the Arabidopsis ecotype used was Columbia, and the seed was obtained from the international Arabidopsis Genetic Resource Bank (eg, RIKEN
- Bioresource Center Power is also available.
- Transgenic plants and their progeny are planted in a greenhouse as in the case of Oryza rufipogon-derived genomic DNA library, and the overall plant size, plant height, relative growth rate, number of panicles, Genomic DNA introduced by examining the above-ground weight, ear weight, ear length, fertile grain number, yield, leaf number, leaf length, leaf width, leaf weight, drought tolerance, salt tolerance, disease resistance, etc. The effect of the fragments was determined.
- the rice (Yukihikari) transformed with the GUS gene was used as a control plant. Then, among these traits, one or more traits were selected, and a transgenic plant having a mutation and its progeny were selected.
- the genomic DNA fragments introduced into the selected plants were selected as genomic DNA fragments derived from Arabidopsis, which can cause agriculturally beneficial mutations in the crop.
- Example 9 Evaluation of rice plants transformed by genomic DNA fragments contained in a genomic DNA library derived from rosegrass, selection of mutated bovine plants
- genomic DNA was isolated from rosegrass (Chloris gayana), a genomic DNA library was constructed, and the genomic clones constituting the genomic DNA library were individually transformed into rice by the transformation method. (Yukihikari). As a result, transgenic plants were obtained in which a total of 1450 genomic DNA fragments contained in the genomic DNA library were individually introduced.
- the variety of rosegrass used was commercially available as Riki Riede. Transgenic plants and their progeny are cultivated in a greenhouse, as in the case of the genomic DNA library derived from Oryza rufipogon.
- the rice plant (Yukihikari) transformed with the GUS gene was used as a control plant. Then, for one or more of these traits, a transgenic plant having a mutation and its progeny were selected.
- the genomic DNA fragment introduced into the selected plants was selected as a genomic DNA fragment derived from rosegrass that can cause agriculturally beneficial mutations in the crop.
- Example 10 Evaluation of rice and corn transformed by genomic DNA fragments contained in a genomic DNA library derived from sorghum, selection of mutated cattle-like plants
- genomic DNA was isolated from sorghum (Sorghum bicolor), a genomic DNA library was constructed, and genomic clones constituting this genomic DNA library were individually transformed into rice (Yuki Strain), and corn (A188). As a result, transformants into which 2560 and 200 genomic DNA fragments contained in the genomic DNA library were individually introduced were obtained.
- the sorghum variety used is commercially available as Gold Sorgo I. Transgenic plants and their progeny plants are cultivated in a greenhouse, as in the case of the genomic DNA library derived from Oryza rufipogon, and the whole plant is made up of viga, plant height, relative growth rate, number of ears, and aboveground part.
- Examples of the total panicle weight of rice are shown below (Table 21).
- the control plant The distribution of constant values was fitted to a normal distribution.
- the rice (Yukihikari) transformed with the GUS gene was used as a control plant. According to this normal distribution, assuming that the introduced fragment had no effect, the probability of appearance of a line showing the measured value of the selected transformed plant line was calculated. In each case, the value of the probability of occurrence with respect to the number of selected strains is extremely small in each case, and the expected value of the appearance of such selected strains is much lower than 1.0.
- Out of the 2504 lines investigated for the total panicle weight 43 lines showed results that were lower than expected. Therefore, the assumption that the introduced fragment had no effect was rejected, and it was statistically proved that the selected line showed significant mutation.
- the plant height on the 28th day after the transplantation of the transgenic plant the plant height on the 35th day, and the relative growth rate (((35 days after the transplantation, plant height on the 28th day after the transplantation) (Plant height) / 7) 28 days after Z transplantation), leaf height, maximum panicle weight, maximum panicle number, maximum panicle total grain weight, 1 panicle weight (maximum panicle total grain weight Z maximum panicle grain)
- the variance of the relative growth rate, the maximum number of panicles, and the weight of one grain was significantly larger than that of the control plant.
- the average value of all individuals on the same potting day was calculated, and the data was standardized and analyzed using the formula of (value of each individual-average value) Z average value.
- corn (A188) transformed with the GUS gene was used as a control plant. According to this normal distribution, assuming that the introduced fragment had no effect, the probability of appearance of a line showing the measured value of the selected transformant or line was calculated. In each of the strains exemplified here, the numerical value of the probability of occurrence with respect to the number of selected strains is extremely small. The expected value for the emergence of selected strains like this is well below 1.0. Of the 150 lines studied, the relative growth rate was 7 lines, and the maximum number of spikelets was 8 lines, and the expected value was less than 1.0. Therefore, the assumption that the introduced fragment had no effect was rejected, and it was statistically proved that the selection line showed significant mutation.
- genomic DNA fragment derived from sorghum was selected as a genomic DNA fragment capable of causing a corn to have an agriculturally beneficial mutation.
- Example 11 Evaluation of rice transformed by genomic DNA fragments contained in a genomic DNA library derived from theosinth
- genomic DNA was isolated from theosin (KZea diploperenis), a genomic DNA library was constructed, and the genomic clones constituting the genomic DNA library were individually transformed into rice (Yukihikari ).
- the teosinte varieties used are those that are sold as teosinte for pasture.
- the transgenic product and its progeny plants are cultivated in a greenhouse, as in the case of the genomic DNA library derived from Oryza rufipogon, and the whole plant has a viga, plant height, relative growth rate, number of ears, and above-ground weight.
- the effect of the introduced genomic DNA fragment was determined by investigating the cultivar, ear weight, ear length, number of fertile grains, yield, number of leaves, leaf length, leaf width, leaf weight, drought resistance, and disease resistance. . Then, for one or more of these traits, a transgenic plant having a mutation and its progeny were selected.
- the genomic DNA fragment introduced into the selected plant was selected as a teosinte-derived genomic DNA fragment capable of causing agriculturally beneficial mutations in the crop.
- the rice (Yukihikari) transformed with the GUS gene was used as a control plant.
- Figure 7 shows the rice cultivation status after the introduction of the teosinte genomic fragment.
- genomic DNA was isolated from Sorghum Sudanese, a genomic DNA library was constructed, and genomic clones constituting the genomic DNA library were individually transformed into rice by the transformation method. (Yukihikari). As a result, a transgenic plant was obtained in which a total of 2644 genomic DNA fragments contained in the genomic DNA library were individually introduced.
- the Sudangrass varieties used are those that are commercially available for pasture use. Transgenic plants and their progeny plants are cultivated in a greenhouse, as in the case of the genomic DNA library derived from Oryza rufipogon, and the whole plant is made up of viga, plant height, relative growth rate, number of ears, and aboveground part.
- genomic DNA was isolated from millet (Seteria italica), a genomic DNA library was constructed, and genomic clones constituting the genomic DNA library were individually transformed into rice ( (Yukihikari) was introduced. As a result, transformants were obtained in which a total of 2952 genomic DNA fragments contained in the genomic DNA library were individually introduced.
- the varieties of millet used are the very early Italian millet R, which is commercially available for pasture.
- Transgenic plants and their progeny are cultivated in a greenhouse as in the case of the genomic DNA library derived from Oryza's rufibongon, and the whole plant is bigger, plant height, relative growth rate, number of ears, and aboveground weight.
- the effect of the introduced genomic DNA fragment was determined by investigating, ear weight, ear length, number of fertile grains, yield, number of leaves, leaf length, leaf width, leaf weight, drought tolerance, disease resistance, etc. . Then, for one or more of these traits, a transformed plant in which a mutation occurred and a progeny plant thereof were selected.
- the genomic DNA fragment introduced into the selected plant was selected as a millet-derived genomic DNA fragment capable of causing agriculturally beneficial mutations in the crop.
- Example 4 Evaluation of ti products transformed by genomic DNA fragments contained in genomic DNA library from Giagrass ⁇ , selection of ti products from ⁇
- genomic DNA is isolated from girgrass (Panicum maximum), a genomic DNA library is constructed, and the genomic clones constituting this genomic DNA library are individually transformed by the transformation method. Introduced to rice, corn and tobacco. The girgrass varieties used were colored gingergrass commercially available for pasture use.
- Transgenic animals and their progeny plants are cultivated in a greenhouse, as in the case of the genomic DNA library derived from Oryza rufipogon, and the whole plant is made up of Viga, plant height, relative growth rate, number of ears, Introduced genomic DNA by investigating aboveground weight, ear weight, ear length, number of fertile grains, yield, number of leaves, leaf length, leaf width, leaf weight, drought tolerance, salt tolerance, disease resistance, etc. The effect of the fragment was determined. Then, for one or more of these traits, a transgenic plant having a mutation and its progeny were selected. The genomic DNA fragment introduced into the selected plant was selected as a genomic DNA fragment derived from girgrass, which can cause an agriculturally beneficial mutation in rice, corn, or tobacco.
- Selected Genomic DNA Fragments as Type I Selected genomic DNA fragments (AS4 (A011D07), AS8 (A014E08), AS19 (A010B03), AS20 (A011C02), AS22 ( A014D12), AS27 (A012D12), AS28 (A015C06),
- AS4 A011D07
- AS8 A014E08
- AS19 A010B03
- AS20 A011C02
- AS22 A014D12
- AS27 A012D12
- AS28 A015C06
- PCR PCR2 in Fig. 8
- the primer sequences used are as follows. AS4: 5 '-TGGGCTCCAGCAGAAACGAACCCT-3' and 5 '-CTTATATTTAGGAACGGAGTGAGT-3'
- PCRs were performed using Takara ExTaq (TAKARA) or Takara LA Taq (TAKARA), heat denaturation (94 ° C, 30 seconds), annealing (58 ° C, 30 seconds) and extension reaction (72 ° C). , 30 seconds) was repeated 30 times or 35 times.
- the PCR products were analyzed by agarose gel electrophoresis.
- the genomic DNA evaluated in Example 3 and mutated in rice was transformed into corn in Example 6.
- the obtained transformed corn was cultivated in a greenhouse, and pollen of the maize variety A188, which had been grown in a separate greenhouse, was pollinated. It is expected that the obtained progeny seeds will be separated from individuals containing the genomic DNA fragment of Oryza 'rufipogon and from individuals not containing the fragment. Therefore, using the border sequence of the T-DNA of the introduced genomic DNA fragment as a marker, amplification was performed by PCR and the presence or absence of a genomic DNA fragment derived from Oryza rufipogon was examined. As a result, plant individuals having the marker were determined to be suitable for breeding and usable for further breeding steps, and plant individuals not having the marker were determined to be unsuitable for breeding.
- Table 30 shows the relationship between the presence or absence of the introduced genomic DNA fragment and the measured value of the trait. [Table 30] Relationship between presence / absence of the fragment and traits based on markers created from the extracted genome fragments
- the plasmid was isolated from the bacterial pellet according to a conventional method (alkali method), and the plasmid DNA was dissolved in 40 ⁇ l to obtain a selected genomic DNA fragment and a genomic DNA fragment containing a cloning vector.
- Example 17 Using the plasmid DNA prepared in Example 17 (clone name: AS88, AS90, AS95-AS102, AS104-AS106), a reaction solution comprising the following components was prepared.
- Each plasmid DNA contains the following genomic DNA fragment. (A018D06,
- the reaction solution was mixed with 41 ⁇ 6 dye and mixed, and subjected to electrophoresis (100 V, 1 hour) using 0.71 on a 0.7% agarose gel. After the electrophoresis, the gel was stained with EtBr to obtain restriction enzyme fragments that also generated genomic cloning power. An example of this is shown in Fig. 10.19.Integration of a genomic DNA fragment reported in E. coli into a vector for transformation (DSB200).
- plasmid DNA genomic DNA fragment G001A03 isolated and purified from Escherichia coli
- a BP reaction 25. C,- ⁇
- the plasmid DNA was precipitated. After centrifugation (15000 rpm), the obtained precipitate was washed with 70% ethanol and dissolved with 10 ⁇ l of TE.
- the plasmid was introduced into Escherichia coli DB3.1 by electoporation, plated on LA (Sp50Cm30), and cultured at 28 ° C for 3 days.
- the single colony that grew was cultured in 2 ml of LB (Sp50Cm30), plasmid DNA was isolated according to a standard method (alkaline method), fragment analysis was performed using Hindlll and Sacl, and the target plasmid was selected.
- the recombinant plasmid (G001A03DEST) was selected using this as an index (Fig. 11, lane 2). .
- an LR reaction was performed under the same conditions as the BP reaction, and a clone G001A03bar in which the drug selection marker gene was replaced was selected.
- the aatRl-ccdB-Cm-aatR2 fragment was replaced by the aatBlbar-aatB2 fragment by the LR reaction, and the vector size changed from 10.4 kb to 9.3 kb.
- G001A03bar was selected using this as an index (Fig. 11, lane 3). ).
- the target selected fragment was successfully incorporated into a vector that can be transformed into a plant.
- Example 20 Analysis of Selected Genomic DNA Clones The nucleotide sequence of 280-500 bases at both ends of the selected genomic DNA fragment was investigated. The results are also shown by the SEQ ID numbers corresponding to the genomic DNA fragments in Tables 1 to 9 and FIG. In addition, Table 31 below shows the sequences of PCR primer pairs that can detect these fragments by the PCR method.
- the genomic DNA fragments selected in Examples 3 to 14 were introduced into rice, corn, and tobacco by the method described in Example 2, Example 6, or Example 7.
- the obtained transgenic plants and their progeny plants were evaluated in the same manner as in Example 3-14. And comment For one or more of the traits evaluated, a transgenic plant having a mutation and its progeny were selected.
- the genomic DNA fragments introduced into the selected plants were selected as genomic DNA fragments capable of causing agriculturally beneficial mutations in crops.
- Genomic DNA fragments confirmed to be feasible were selected. Therefore, a genomic DNA fragment of higher value was selected than in the case of only the primary selection.
- Table 32 shows examples of genomic DNA fragments thus selected.
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JP2008173011A (ja) * | 2007-01-16 | 2008-07-31 | Univ Nagoya | 形質転換植物体の作出方法及びその利用 |
JP5175188B2 (ja) * | 2006-06-23 | 2013-04-03 | 日本たばこ産業株式会社 | 植物形質転換用コスミドベクター及びその利用法 |
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US11965169B2 (en) * | 2020-08-20 | 2024-04-23 | AG Biomolecules LLC | Transgenic safflower event stack IND-1ØØØ3-4 x IND-1ØØ15-7 and methods to use it |
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US5733744A (en) * | 1995-01-13 | 1998-03-31 | Cornell Research Foundation, Inc. | Binary BAC vector |
AU694393B2 (en) | 1996-09-26 | 1998-07-16 | National Institute Of Agrobiological Resources, Ministry Of Agriculture, Forestry And Fisheries | New high capacity binary shuttle vector |
US6521408B1 (en) | 1997-09-25 | 2003-02-18 | National Institute Of Agrobiological Sciences | Method for assessing a function of a gene |
US7045679B1 (en) * | 1998-08-26 | 2006-05-16 | Stine Biotechnology, Inc. | Transgenic plants |
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US8298819B2 (en) | 2006-06-23 | 2012-10-30 | Japan Tobacco Inc. | Cosmid vector for plant transformation and use thereof |
JP5175188B2 (ja) * | 2006-06-23 | 2013-04-03 | 日本たばこ産業株式会社 | 植物形質転換用コスミドベクター及びその利用法 |
JP2008173011A (ja) * | 2007-01-16 | 2008-07-31 | Univ Nagoya | 形質転換植物体の作出方法及びその利用 |
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