WO2006038571A1 - Method of transferring nucleic acid into cell with the use of agrobacterium involving treatment under reduced pressure/elevated pressure - Google Patents

Method of transferring nucleic acid into cell with the use of agrobacterium involving treatment under reduced pressure/elevated pressure Download PDF

Info

Publication number
WO2006038571A1
WO2006038571A1 PCT/JP2005/018198 JP2005018198W WO2006038571A1 WO 2006038571 A1 WO2006038571 A1 WO 2006038571A1 JP 2005018198 W JP2005018198 W JP 2005018198W WO 2006038571 A1 WO2006038571 A1 WO 2006038571A1
Authority
WO
WIPO (PCT)
Prior art keywords
plant
seed
cell
cells
agrobacterium
Prior art date
Application number
PCT/JP2005/018198
Other languages
French (fr)
Japanese (ja)
Inventor
Takashi Hagio
Original Assignee
National Institute Of Agrobiological Sciences
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by National Institute Of Agrobiological Sciences filed Critical National Institute Of Agrobiological Sciences
Priority to JP2006539274A priority Critical patent/JP4788002B2/en
Publication of WO2006038571A1 publication Critical patent/WO2006038571A1/en

Links

Classifications

    • 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
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8205—Agrobacterium mediated transformation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/02—Electrical or electromagnetic means, e.g. for electroporation or for cell fusion
    • 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
    • C12N15/8206—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated

Definitions

  • Decompression treatment Z Method for introducing nucleic acid into cells using agrobacterium, including the use of pressure treatment
  • the present invention introduces a nucleic acid transductant (including a transformant) by introducing a desired nucleic acid into a cell or tissue (including a plant tissue) using reduced pressure treatment or pressurized treatment and agrobacterium. ) On how to create. Furthermore, the present invention introduces a bacterium such as agrobacterium containing a desired nucleic acid into a cell or tissue by combining a reduced pressure treatment or a pressurized treatment with electrovola- tion, and a nucleic acid transductant ( The present invention relates to a method for producing a transformant (including a transformant).
  • vegetables such as roe, crocodiles, tomatoes and cucumbers are rich in diet and are essential for nutrition.
  • these vegetables can be made resistant to disease and pests by using gene recombination techniques that are vulnerable to various diseases and pests, the yield can be stabilized. Therefore, transformation methods using these genes have been developed along with the isolation of useful genes.
  • the present invention facilitates large-scale processing and large-scale analysis not only in the industrial field where it is necessary to obtain plant transformants but also in development research using plants, and thus induces dramatic progress in research. This will lead to the development of technological yarn and changeable crops.
  • Patent Document 1 Tanaka et al., Japanese Patent No. 3141084
  • Non-patent literature l Hiei et al., Plant Journal, 6: 271-282, 1994
  • Non-patent document 2 Biotechnology in Agriculture and Forestry 46, Trangenic Crops I, edited by Y.P.S. Bajaj, published by Springer, 2000, especially T. A. Loeb
  • the problem to be solved by the present invention is to provide a rapid and highly efficient indirect gene transfer method in the field in view of the above situation that a rapid and highly efficient indirect gene transfer method has not been established. It is to be.
  • Transformation m3 ⁇ 4 objects can be obtained quickly and in large quantities. Therefore, the present invention makes not only the industrial field where it is necessary to obtain plant transformants, but also makes it easy to conduct large-scale processing and large-scale analysis even for development research using plants. Induces the research progress
  • step (b) After the step (b), the step of arranging the cells and the agrobacterium under the condition that the electopore por- tion occurs.
  • the method of the present invention is carried out using plant cells (eg, mature seeds).
  • the inventors easily incorporate a nucleic acid when a gene is introduced into a plant using agrobacterium.
  • (1) keeping the plant in a vacuum state and the nucleic acid introduction method using agrobacterium is not effective for plants other than Arabidopsis, and (2) Contrary to the conventional knowledge that the plant does not need to be kept in vacuum for the introduction of the nucleic acid to be used (Bent, Plant Physiology, 124: 1540-1547, December 2000), as shown in the examples.
  • This method is extremely simple. Furthermore, since the method of the present invention does not require a culture process that is usually required after the nucleic acid introduction operation, the resulting transductant does not contain a culture mutation. There are also advantages. It is known that culture mutations inevitably occur in the culture process required after conventional nucleic acid introduction procedures. Culture mutation means a genetic mutation that occurs during the culture process, as can be generally understood by those skilled in the art. During the culture process, the nucleic acid sequence and Z or transfer originally introduced by the cell into which the nucleic acid is introduced This refers to any sequence modification (for example, substitution, deletion, insertion, translocation, inversion, duplication, etc.) that occurs over the entire nucleic acid sequence.
  • the present invention also does not require the pre-culture of target cells (tissues) for several days to several weeks, which has been essential in the conventional indirect gene transfer method, and as a result, more rapidly than the conventional method.
  • the potential for gene transfer provides the advantage.
  • the present invention provides the following.
  • a method for introducing a nucleic acid into a cell comprising the following steps:
  • step of maintaining the cells under a pressure different from atmospheric pressure is a step of subjecting the cells to reduced pressure treatment.
  • step (b) after the step (b), the step of placing the cells and the agrobacterium under conditions that cause electopore poration;
  • step c) includes applying a voltage pulse to the cell and the agrobacterium in at least two directions.
  • leguminous plant m is soybean.
  • a method for producing a plant into which a nucleic acid has been introduced into a cell comprising the following steps:
  • step (b) after the step (b), placing the plant cell and the agrobacterium under conditions where electo-portion occurs,
  • step a) includes a step of maintaining the seed containing the plant cell under a pressure different from atmospheric pressure
  • step b) includes the plant cell.
  • leguminous plant is soybean.
  • An apparatus for automated introduction of nucleic acids into cells comprising: a) a container for containing a mixture of agrobacterium containing nucleic acids and cells;
  • a container for maintaining cells under a pressure different from atmospheric pressure the container having the ability to withstand a pressure different from atmospheric pressure
  • An electrode for electo-poration having at least two pairs of electrodes.
  • the gene introduction method of the present invention enables rapid and highly efficient indirect gene introduction into a target cell (tissue).
  • the simple method of the present invention facilitates large-scale processing and large-scale analysis, which are important in development research in this field, and thus induces dramatic progress in research and development of innovative recombinant crops. Leads to.
  • FIG. 1 is a diagram schematically showing a binary vector pCAMBIA1390-sGFP on T-DNA.
  • FIG. 2A shows the result of observation of seeds with white light on the 6th day after the treatment with agrobacterium. The left is a seed that has been subjected to decompression, agrobacterium infection, and electoporation, and the right is a seed that has been subjected to decompression only.
  • Figure 2B is the seed observed in Figure 2A Shows the result of observation by irradiating excitation light. Cells expressing GFP are brightly shining green.
  • FIG. 3A shows the result of white seed observation of rice seeds on the 8th day after the treatment with agrobacterium.
  • the left is a seed that has been subjected to decompression, agrobacterium infection, and electoporation, and the right is a seed that has undergone only a decompression treatment.
  • Figure 3B is the seed observed in Figure 3A. Shows the result of observation by irradiating excitation light. Cells expressing GFP are brightly shining green.
  • FIG. 4A shows the results of observation of white seeds on the 14th day of rice after infection with agrobacterium.
  • the left is a seed that has been subjected to decompression treatment, agrobacterium infection, and electo-poration, and the right is a seed that has been soaked in a bacterial solution after depressurization without erect-mouth poration.
  • FIG. 4B shows the results of observing the seeds observed in FIG. 4A by irradiating them with excitation light. Cells expressing GFP are brightly shining green.
  • FIG. 5A shows the results of observing white seeds of wheat on the 8th day after infection treatment with agrobacterium.
  • the left is a seed that has undergone decompression, agrobacterium infection, and electo-poration
  • the center is a seed that has undergone depressurization only
  • the right is a seed that has undergone de-electoration without decompression. Later, the seeds were just immersed in the fungus solution.
  • FIG. 5B shows the results of observing the seeds observed in FIG. 5A by irradiating them with excitation light. Cells expressing GFP are brightly shining green.
  • FIG. 6A shows the results of observing seeds of white rice in 7 days after infection treatment with agrobataterium for indy power rice. On the left are the seeds that have undergone decompression treatment, agrobacterium infection, and electo-poration, and on the right is the electo-poration. Without seeding, the seeds were only immersed in the bacterial solution after the reduced pressure treatment.
  • FIG. 6B shows the results of observing the seeds observed in FIG. 6A by irradiating them with excitation light. Cells expressing GFP are brightly shining green.
  • FIG. 7A shows the results of observation of white and white Species seeds on day 4 after infection with agrobacterium.
  • the left is a seed that has been subjected to decompression, agro-acterium infection, and electo-poration, and the right is a seed that has been soaked in a bacterial solution after depressurization without erect-poration. It is.
  • FIG. 7B shows the results of observing the seeds observed in FIG. 7A by irradiating them with excitation light. Cells expressing GFP are brightly shining green.
  • FIG. 8A shows the results of observation of seeds with white light on the 4th day after the infection treatment with agrobacterium.
  • the left is a seed that has been subjected to decompression treatment, agrobacterium infection, and electo-poration, and the right is a seed that has been soaked in a bacterial solution after depressurization without erect-mouth poration.
  • FIG. 8B shows the results of observing the seeds observed in FIG. 8A by irradiating them with excitation light. Cells expressing GFP are brightly shining green.
  • FIG. 9A shows the results of observing seeds of tomatoes with white light on the fourth day after infection treatment with agrobacterium.
  • the left is a seed that has been subjected to decompression, agrobacterium infection, and electoral poration
  • the right is a seed that has been soaked in a bacterial solution after depressurizing without performing electoral poration. .
  • FIG. 9B shows the results of observing the seeds observed in FIG. 9A by irradiating them with excitation light. Cells expressing GFP are brightly shining green.
  • FIG. 10A shows the results of observation of seeds of morning glory with white light on the fourth day after infection treatment with agrobata trumum.
  • the left is a seed that has been subjected to decompression, agro-acterium infection, and electo-poration, and the right is a seed that has been soaked in a bacterial solution after depressurization without e-poor poration. .
  • FIG. 10B shows the results of observing the seeds observed in FIG. 10A by irradiating them with excitation light. Cells expressing GFP glow brightly in green.
  • FIG. 11 is a perspective view of an erect-portion chamber having a hexagonal cross section.
  • FIG. 12 is a cross-sectional view of the electrification chamber shown in FIG. 11 along the X-ray.
  • FIG. 13 is a perspective view of an erect-portion polarization electrode of a type to be inserted into a test tube.
  • FIG. 14 is a view showing a state in which the electrification polarization electrode shown in FIG. 13 is inserted into a test tube.
  • SEQ ID NO: 1 A forward primer for detecting the NPT II gene.
  • SEQ ID NO: 2 Reverse primer for detecting the NPT II gene.
  • SEQ ID NO: 3 Forward primer for detecting NPT II gene.
  • SEQ ID NO: 4 Reverse primer for detecting the NPT II gene.
  • nucleic acid introduction means that a nucleic acid is artificially introduced into a cell or tissue.
  • the expression type of a cell or tissue into which nucleic acid has been introduced by “nucleic acid introduction” may or may not change.
  • gene transfer means that a nucleic acid containing a gene, which is a factor defining a genetic trait, is artificially introduced into a cell or tissue.
  • the phenotype of a cell or tissue into which a nucleic acid containing a gene has been introduced by “gene transfer” may or may not change.
  • transformation means that a phenotype of a cell or tissue is changed by introducing a nucleic acid containing a gene into the cell or tissue.
  • nucleic acid introduction means that a nucleic acid containing a gene into the cell or tissue.
  • gene introduction means that a nucleic acid containing a gene into the cell or tissue.
  • nucleic acid transductant refers to all or one part of a living organism generated from a nucleic acid transduced, gene transduced, and transformed cell or tissue, respectively. Part. However, in the present specification, the terms “nucleic acid introduced body”, “gene introduced body”, and “transformant” may be used interchangeably. In the present specification, the meaning of these terms is obvious from the contextual power in which the terms are included. Nucleic acid transductants, gene transfectants, and transformants can be any organism, for example, prokaryotic cells and eukaryotic cells (including plant cells, etc.) or living organisms originating from tissues. Is done.
  • Transformants are also referred to as transformed cells, transformed tissues, transformed hosts, etc., depending on the subject, and include all of these forms in this specification, but in certain contexts a particular form Can point to. The same applies to nucleic acid transducers and gene transfectants.
  • the term "cell” refers to a cell from any organism (eg, any type of multicellular organism (eg, animal (eg, vertebrate, invertebrate), plant (eg, monocotyledonous, dicotyledonous). Plant, etc.), fungi, etc.) or unicellular organisms (eg, cells derived from bacteria (eg, E. coli), etc.).
  • the cell used in the present invention is a cell having a cell wall, particularly preferably a plant cell.
  • tissue refers to a cell population having substantially the same function and Z or morphology in a multicellular organism.
  • tissue can be referred to as a tissue even if it is a population of cells with the same origin, even if they have the same function and Z or morphology.
  • tissue constitutes part of an organ. Plants are roughly classified into meristems and permanent tissues according to the stage of development of the constituent cells, and divided into single tissues and composite tissues according to the type of constituent cells. Animal tissues are classified into epithelial tissue, connective tissue, muscle tissue, nerve tissue, etc. based on morphological, functional or developmental basis.
  • tissue refers to any tissue from any organism (eg, any type of multicellular organism (eg, animal (eg, vertebrate, invertebrate), plant (eg, Or monocotyledonous plants, dicotyledonous plants, etc.), fungi, etc.)).
  • the tissue used in the present invention is a tissue containing a cell wall, and particularly preferably a plant tissue.
  • Plant tissue includes, but is not limited to, dormant tissue, germplasm, growth points, and flower buds.
  • Preferred dormant tissues include ripe seeds, immature seeds, winter buds, and tubers, particularly preferably the ability to be a ripe seed.
  • organ refers to a structure in which a function of an individual organism is localized and operates in a specific part of the individual, and that part is morphologically independent.
  • an organ in general, in multicellular organisms (eg animals, plants, fungi), an organ consists of several forces with a specific spatial arrangement, and a tissue also has many cellular forces.
  • Such organs include roots, leaves, stems, and flowers in the case of plants, and skin, heart, blood vessels, cornea, retina, kidneys, liver, spleen, intestines, in the case of animals. Examples include, but are not limited to, placenta, umbilical cord, lungs, brain, nerves, and extremities.
  • selection refers to introduction of nucleic acid by antibiotic resistance testing and Z or genetic engineering techniques (eg, PCR, Southern blotting, Northern blotting, etc.). This means that the introduced nucleic acid is distinguished from the non-nucleic acid-introduced one.
  • ⁇ selection '' means that transformants transformed with a drug resistance gene have been transformed by culturing and Z or growing the transformed sickle in the presence of the drug! / ⁇ It means the process of distinguishing ⁇ plants.
  • the agrobacterium used for transformation of monocotyledonous plants can be any bacterium belonging to the genus Agrobacterium, preferably Agrobacterium tumefaciens.
  • Agrobataterium is transformed with a plant expression vector containing the desired recombinant gene (eg, by electrovolution).
  • a desired recombinant gene can be introduced into a plant by infecting seeds with the transformed agrobacterium.
  • the introduced recombinant gene is integrated in the genome of the plant.
  • the genome in plants includes not only nuclear chromosomes but also genomes contained in various organelles (eg, mitochondria, chloroplasts, etc.) in plant cells.
  • an appropriate plant expression vector containing the desired recombinant gene is constructed.
  • Such plant expression vectors are known in the art. It can be produced using genetic recombination techniques well known to those skilled in the art.
  • the construction of a plant expression vector for use in the agrobacterium transformation method is not limited to, for example, the ability to suitably use a pBI or pPZP vector.
  • a "growth regulator” is a factor that affects multicellular growth, and is synthesized in organs and cells of a multicellular organism (for example, a plant) and into body fluids. Thus, a chemical substance or derivative thereof that is transported to other organs or parts and that has the effect of altering the function, activity and Z or structure of one or many organs. Growth regulators for plant cells are referred to as plant growth regulators. Plant growth regulators include, but are not limited to, plant hormones. Plant hormones include, but are not limited to, auxin, gibberellin, cytokinin, abscisic acid, and ethylene. Auxins include, but are not limited to, 2,400 juice (indole acetic acid), NAA (naphthalene acetic acid), and IBA (indole butyric acid).
  • the term "elect mouth polarization” is used to physically puncture a cell (eg, a plant cell) using a direct-current high-voltage pulse, and force the nucleic acid (eg, A nucleic acid containing a gene) is introduced into cells.
  • the conditions of the electopore polarization can be appropriately selected by those skilled in the art depending on the species, tissues, cells, etc. used.
  • the voltage conditions of a typical electrification pole position are 10VZcm to 200VZcm, preferably 20VZcm to 150VZcm, more preferably 30VZcm to 120VZcm, even more preferably 40V / cm to 1 OOVZcm, most preferably 50V / cm to 1 OOV / cm, but not limited to.
  • the pulse width condition of a typical electopore polarization is 1 microsecond to 90 milliseconds, preferably 10 milliseconds to 90 milliseconds, more preferably 10 milliseconds to 90 milliseconds, still more preferably 20 milliseconds to 80 milliseconds.
  • Milliseconds even more preferably 30 milliseconds to 80 milliseconds, still more preferably 40 milliseconds to 70 milliseconds, and most preferably 50 milliseconds to 60 milliseconds, but is not limited thereto.
  • the pulse width of the electo-portion may be less than 1 millisecond, for example, 10 microseconds to 90 microseconds, 20 microseconds to 80 microseconds, 30 microseconds, to 80 microseconds, 40 Microseconds, 70 microseconds, 50 microseconds, 60 microseconds, but not limited to these.
  • the pulse of a typical electo porch Number of times ⁇ 1 to 200 times, preferably ⁇ 10 to 150 times, more preferably ⁇ 20 to 120 times, more preferably 30 to 110 times, most preferably 40 to 100 times Power is not limited to these.
  • the phrase “place cells (or tissues) and nucleic acids under conditions that cause electoporation occurs” means that cells (or tissues) and nucleic acids are placed between them. All conditions (voltage condition, pulse width condition, pulse frequency condition, cell (or tissue) and nucleic acid between the cells (or tissue) that are essential for electoral polarization to occur between the cells (or tissue). (Including the positional relationship between them and the execution time of the electo-portion). The conditions essential for the occurrence of the electrification are readily apparent to those skilled in the art, and those skilled in the art can appropriately determine the conditions.
  • voltage pulses to cells (or tissues) and nucleic acids in at least two directions when performing electophoresis according to the present invention.
  • the simplest is to apply a voltage pulse to a cell (or tissue) and a nucleic acid for a certain period of time, and then reapply the voltage pulse by reversing the anode and force sword of the electrode used to process the voltage. This can thus be achieved.
  • This can also be accomplished using electrode pairs located at different locations within the electopore polarization chamber.
  • the size of the electopore chamber used when performing the electoporation in the present invention can be any size as long as it can accommodate cells and ⁇ or tissue to which nucleic acid is introduced. May be. Particularly preferably, the electoporation chamber has a size that can accommodate plant tissue (eg, plant seeds).
  • the elect port polarization chamber of the present invention can be of any shape. This shape can be a cube, cuboid, cylinder, tube (e.g., a force that is uniform on the body or a uniform
  • V having a cross-section and having a tapered bottom or not!
  • the maximum diameter of the inscribed circle that contacts at least three points on the inner surface of the electrification chamber of the present invention can be, for example, the following length: : A force that is at least about 5mm or more than about 5mm Long, preferably at least about 6 mm or longer than about 6 mm, preferably at least about 7 mm or longer than about 7 mm, preferably at least about 8 mm or longer than about 8 mm, Preferably, the force is at least about 9 mm or longer than about 9 mm, preferably at least about 1 cm or longer than about 1 cm, preferably at least about 2 cm or longer than about 2 cm.
  • the force is at least about 3 cm or longer than about 3 cm, preferably at least about 4 cm or longer than about 4 cm, preferably at least about 5 cm or longer than about 5 cm.
  • a force that is at least about 6 cm or longer than about 6 cm preferably a force that is at least about 7 cm or longer than about 7 cm, preferably at least about 8 a force that is cm or longer than about 8 cm, preferably at least about 9 cm or longer than about 9 cm, preferably at least about 10 cm, or longer than about 10 cm, preferably at least about 15 cm
  • the upper limit of the diameter of the maximum inscribed circle that contacts at least three points of the inner surface of the electrification chamber of the present invention may be, for example, 1S, but is not limited to: about 25 cm, about 20 cm, about 15 cm, About 10 cm, about 9 cm, about 8 cm, about 7 cm, about 6 cm, about 5 cm, about 4 cm, about 3 cm, about 2 cm, about 1 cm, about 9 mm, about 8 mm, about 7 mm, or about 6 mm. Of course, this length can be a length between the values specified above (eg, 1.5 cm, etc.).
  • the “inscribed circle” refers to an arbitrary circle drawn so as to touch at least three arbitrary points on the inner surface of the chamber.
  • the electrode disposed in the chamber is also regarded as a part of the container, and therefore, the inner surface of the chamber container also includes the electrode surface.
  • the thickness of the electrode used is negligibly thin (eg, 0.1 mm).
  • the electrification chamber of the present invention has a rectangular cross section and has an internal dimension (for example, vertical X horizontal X height) of 1 cm X 2 cm X 2 cm.
  • the electrification chamber of the present invention has a circular cross section and an internal dimension (eg, diameter X height) of 1 cm X 4 cm.
  • the electrification chamber of the present invention has a hexagonal cross section.
  • the electrification polarization chamber 11 has a bottomed cylindrical body 18 whose upper surface is open, and electrodes 11 to 16 provided on each inner surface of the cylindrical body 18.
  • the electrodes 11 to 16 are electrically connected to a cord (not shown) for applying a high-voltage noise by electo-portion polarization.
  • the cylindrical body 18 has a bottom portion 17, and a sample is introduced into the cylindrical body 18 so as to perform electo-mouth polarization.
  • the electrodes of the electo-portion chamber are not limited to forces each having a size of lOmm x 15 mm.
  • the area occupied by the electrode is excluded from the internal dimensions of the chamber.
  • the thickness of the electrode used is negligibly thin.
  • the transverse section refers to a section perpendicular to the major axis direction of the chamber.
  • the internal dimension refers to the length connecting any two points on the inner surface of the chamber.
  • the length and width of the cross section As well as, in the case of a chamber having a circular cross section, the diameter and height of the cross section.
  • the electrification chamber of the present invention can be varied to a size that can accommodate plant seeds.
  • the size variation can be achieved by any means. For example, it can be adjusted to an appropriate size using a screw or the like.
  • the electrification chamber of the present invention can be made of any material. Any material capable of forming a solid can be used as the material for the electoral polarization chamber. Examples include glass, silica, silicon, ceramic, silicon dioxide, plastics, metals (including alloys), natural and synthetic polymers (eg, polystyrene, cellulose, chitosan, dextran, and nylon). Not limited.
  • the chamber 1 may also be formed with laminar forces of a plurality of different materials. For example, an inorganic insulating material such as glass, quartz glass, alumina, sapphire, forsterite, silicon oxide, silicon carbide, or silicon nitride can be used.
  • the electrification chamber of the present invention has the ability to withstand pressures different from atmospheric pressure (for example, the ability to not break, crack and deform when exposed to pressures different from atmospheric pressure), in particular Preferably, it has the ability to withstand decompression.
  • the electrification chamber of the present invention is made of polypropylene, silicone resin, and glass cover, and comprises an electrode made of platinum or stainless steel.
  • the electrification chamber of the present invention includes a temperature control means.
  • the temperature control means senses a temperature change with a sensor or the like, and can control the temperature of the chamber manually or automatically.
  • the temperature control means is typically a cooling means that acts to cool the temperature of the chamber.
  • the cooling means can be any means, for example, means utilizing ice, cooling gel, or the like.
  • the electrification polarization chamber 1 of the present invention is provided with at least a pair (two) of electrodes.
  • the chamber of the present invention has more than one (two) electrodes (eg, two (four) electrodes, three (six) electrodes, four (eight) electrodes Electrode, five pairs (ten electrodes), or more pairs of electrodes).
  • two electrodes (four electrodes) can be attached by arranging electrodes along the inner surfaces facing each other.
  • a chamber having a hexagonal cross section it is possible to attach three pairs (six) of electrodes by placing electrodes along the opposing inner surfaces.
  • the number of electrode pairs arranged in the chamber of the present invention can be any number and can take any spatial positional relationship.
  • a device that switches the applied voltage sequentially when applying a voltage to each pair of electrodes is commercially available.
  • CU901 of Neppagene Co., Ltd. Koashikawa, Chiba, Japan.
  • a pair ( 2) When applying voltage sequentially to more electrodes, the voltage switching interval is 120 to 1 milliseconds, preferably 60 to 10 milliseconds, more preferably 30 to 100 milliseconds. Forces that are preferably 10 to 500 milliseconds, even more preferably 5 to 750 milliseconds, even more preferably 3 to 800 milliseconds, and most preferably 1.1 to 900 milliseconds. Not.
  • the distance between the electrodes disposed in the electopore chamber of the present invention can be any distance, and varies depending on the size of the cell and Z or tissue to which the nucleic acid is introduced. obtain. Particularly preferably, the distance between the electrodes is a distance that can accommodate plant tissue (eg, plant seeds). In order to be able to accommodate plant seeds, the distance between the electrodes can be, for example, the following lengths: at least about 5 mm or longer than about 5 mm, preferably at least about 6 mm. Or a force greater than about 6 mm, preferably at least about 7 mm or longer than about 7 mm, preferably at least about 8 mm or longer than about 8 mm, preferably at least about 9 mm.
  • the upper limit of the distance between the electrodes can be, for example, but is not limited to: about 25 cm, about 20 cm, about 15 cm, about 10 cm, about 9 cm, about 8 cm, about 7 cm, about 6 cm, about 5 cm, about 4 cm , About 3cm, about 2cm, about lcm, about 9mm, about 8mm, about 7mm, or about 6mm.
  • the distance between the electrodes can be a length between the values specified above (eg 1.5 cm).
  • the distance between the electrodes is such that the distance between the pair of electrodes is a plant seed.
  • the distance can be changed so that the distance can be accommodated.
  • Variation in the distance between the electrodes can be achieved by any means. For example, it can be adjusted to an appropriate distance using a screw or the like.
  • the electrode can be made of any material force as long as it has properties that allow current to flow.
  • the material of the electrode include, but are not limited to, platinum, gold, stainless steel, carbon, conductive polymer, and the like. Particularly preferably, the electrode is a platinum electrode.
  • An exemplary electo-portion chamber chamber is a rectangular chamber with vertical electrodes 1 cm x 2 cm x height 2 cm with platinum electrodes, and the distance between the platinum electrodes is about lcm.
  • the electopore chamber is particularly useful when treating medium-sized (about 5-15 mm) plant seeds (eg, wheat, rice, corn, etc.). By using this chamber, it is possible to process a large amount of medium-sized plant seeds (for example, about 10 to 30 grains).
  • Another exemplary electopore chamber is a microtube chamber with an inner diameter lcm x height 4cm with stainless steel electrodes, and the distance between the stainless steel electrodes is about lcm.
  • This micro tube type chamber is easily manufactured by attaching a stainless steel foil (for example, about 5 X 40 mm (thickness: about 0.1 mm)) to the inner surface of a commercially available micro tube with an adhesive. obtain.
  • This microtube chamber simplifies solution exchange because it can be centrifuged to precipitate cells, tissues and Z or seeds at the bottom. For this reason, this microtube-type chamber is particularly useful when processing minute plant seeds (eg, about 0.1 to 5 mm) (eg, Arabidopsis thaliana). By using this chamber, it is possible to process a large amount of small-sized plant seeds.
  • the electrification electrode may be an electrode of a type inserted into a test tube as shown in FIG. 13 to 14 includes a pair of flat electrodes 21 and 22, a cap 23 and an electric cord 24.
  • the flat plate-like electrodes 21 and 22 of the electrification electrode 2 are inserted into a test tube 25 and used. If necessary, vents may be provided in the cap 23 or the test tube 25 to increase or decrease the pressure in the test tube. Place the sample in the test tube 25, and then use the electo-pore polarization electrode 2 to -Perform a cission.
  • the term "maintaining cell Z tissue (including plant tissue) under a pressure different from atmospheric pressure” refers to cell Z tissue (including plant tissue), Atmospheric pressure (usually
  • maintaining the cellular Z tissue (including plant tissue) under a pressure different from atmospheric pressure changes the environmental pressure experienced by the cellular Z tissue.
  • Buffers containing nucleic acids such as DNA can easily penetrate between tissues and cells, and as a result, target cells and tissues with cell walls (especially plant cells and plant tissues) that were not possible before Nucleic acid introduction by electoporation Z transformation is considered possible.
  • the term "reduced pressure treatment” refers to nucleic acid-introduced Z transformed cells Z tissues (including plant tissues (eg, seeds)) below atmospheric pressure and under atmospheric pressure.
  • the decompression treatment is performed at a pressure 0.02 MPa lower than the atmospheric pressure, preferably 0.04 MPa lower pressure, more preferably 0.06 MPa lower pressure, even more preferably 0.08 MPa lower pressure, most preferably 0. 096 MPa Forces applied at low pressures are not limited to these.
  • the pressure treatment time is 1 minute to 120 minutes, preferably 10 minutes to 100 minutes, more preferably 15 minutes to 90 minutes, even more preferably 30 minutes to 70 minutes, most preferably about 60 minutes. It is not limited.
  • pressure treatment refers to nucleic acid-introduced Z-transformed cells Z tissues (including plant tissues (eg, seeds)) above atmospheric pressure and under atmospheric pressure. This is the process to maintain.
  • the present invention relates to an electo-portion polarization device.
  • the electoporation apparatus of the present invention can introduce nucleic acid into any cell or yarn and weave with a remarkably high and efficiency, and in particular, the cell wall, which until now has been impossible to introduce nucleic acid by electoporation. It is useful for introducing a nucleic acid into a cell or tissue (eg, a plant cell or plant tissue).
  • the electo-portionation device of the present invention comprises: a) means for maintaining the cell under a pressure different from atmospheric pressure; and b) the elect Both means of tropo-ration means are provided.
  • any means having the ability to reduce pressure and Z or pressurize can be used.
  • Commercially available decompression devices eg, vacuum desiccators
  • Z or pressurization devices can also be utilized.
  • Any electo-portioning means can be used as the electo-portioning means.
  • Commercially available electroporation means e.g., CUY21EDIT gene transfer device, Nepagene, Sasakawa, Chiba, Japan
  • the distance between the two electrodes (first electrode and second electrode) arranged in the above-mentioned electopore polarization means is a distance capable of accommodating plant seeds as defined above. .
  • the electoporation device of the present invention includes two electrodes at a distance that can accommodate plant seeds, and the electoporation device has a pressure different from atmospheric pressure for cell Z tissue. Used in combination with keeping down. In this embodiment, it is not necessary that the electopore polarization device and the means for maintaining the cell Z yarn and weave under a pressure different from the atmospheric pressure are present in the same device.
  • the conventional electopore polarization device was intended to apply a voltage pulse to extremely small cells, it was necessary to make the inner dimensions of the chamber and the distance between the electrodes as small as possible. For this reason, the inner dimension of the chamber and the distance between the electrodes of the conventional electrification device are typically about 1 mm or 2 mm, and at most 4 mm at most. Therefore, an electoral polarization device including a chamber that is large enough to accommodate plant seeds and an electrode that is disposed at a long distance enough to accommodate plant seeds, as in the present invention, has been known so far. Guess! /.
  • the electo-portion polarization device of the present invention can be executed in an automated manner.
  • injection and Z or replacement of a solution such as a buffer solution can be performed by an automatic dispenser.
  • an automatic dispenser for example, a commercially available ep Motion5070 workstation (Eppendorf Co., Ltd. 3 Higashi Kanda, Chiyoda-ku, Tokyo, Japan) can be used, but is not limited thereto.
  • ep Motion5070 workstation Eppendorf Co., Ltd. 3 Higashi Kanda, Chiyoda-ku, Tokyo, Japan
  • such an automatic dispenser can be advantageously used as a means for putting nucleic acid and Z or cells in a container containing a mixed solution containing nucleic acid and cells.
  • a first container for containing a mixture containing nucleic acid and cells, a second container for maintaining cells under a pressure different from atmospheric pressure, and a third container for applying a voltage pulse to the mixture containing nucleic acids and cells can be any container. These containers may be the same or different. As the material of this container, any material capable of forming a solid can be used. Examples include glass, silica, silicon, ceramics, silicon dioxide, plastics, metals (including alloys), natural and synthetic polymers (eg, polystyrene, cellulose, chitosan, dextran, and nylon). It is not limited. The container may be formed of a plurality of layers of different materials.
  • inorganic insulating materials such as glass, quartz glass, alumina, sapphire, forsterite, silicon oxide, silicon carbide, and silicon nitride can be used.
  • the first container of the present invention is also produced with a material force (for example, polystyrene) that has high transparency and facilitates material observation.
  • the second container of the present invention is made of a material capable of withstanding a pressure different from atmospheric pressure (especially reduced pressure) (for example, polyamide, polycarbonate, (modified) polyphenylene oxide, polybutylene terephthalate, reinforced polyethylene terephthalate).
  • Polyethersulfone, polyphenylene sulfide, polyarylate, polyetherimide, polyetheretherketone, polyimide and epoxy resin and more preferably a material with high transparency and easy material observation (for example, , Ryo Krill Resin).
  • the third container of the present invention is made of a material showing affinity with cells (for example, polypropylene, silicone resin, and glass), and is made of platinum, gold, stainless steel, carbon, or conductive.
  • An electrode made of a conductive polymer is provided. These The material may be coated with any suitable material to impart the desired properties (eg, to provide insulation to the container body or to increase the conductivity of the electrode).
  • these containers preferably have the ability to withstand a pressure different from the atmospheric pressure, and particularly preferably have the ability to withstand a decompression process.
  • the first container may be housed in the second container and Z or the third container.
  • the mixed solution containing the nucleic acid and the cells is transferred from the first container to the second container (or another container accommodated therein) and Z or the third container (or the container). It may be injected into a separate container).
  • a belt conveyor or the like can be advantageously used for the means for placing the cells in the second container and the means for placing the mixed solution containing the nucleic acid and the cells in the third container.
  • the present invention is not limited to this, and any means can be used.
  • a method of moving the liquid disposed in the first container, the second container, and the third container by suction Z discharge using an automatic pump or the like can also be used.
  • the automated rect port por- tion device of the present invention includes a control device for automating and implementing each operation means. Further, the elect port polarization device of the present invention includes a power supply device. Such a control device and a power supply device may be arranged in the same device as the elect port polarization device or may be connected as a separate device by a cord.
  • the seeds to be subjected to the decompression treatment or the calorie pressure treatment are left in water (for example, tap water) before the treatment.
  • the standing time is 6 hours to 48 hours, preferably 12 hours to 36 hours, more preferably 18 hours to 30 hours, even more preferably 20 hours to 26 hours,
  • the power that is most preferably about 24 hours is not limited to these!
  • Exemplary nucleic acid introduction conditions in the present invention are that the seeds are allowed to stand in tap water overnight at 25 ° C, and placed in a vacuum apparatus the next day for 1 hour at a pressure lower than atmospheric pressure by 0.096 MPa. After the vacuum treatment, the high-pressure pulse (100 V, 50 milliseconds, but the distance between the electrodes is about 1 cm) by electo-portion is applied about 50 times to the seed that has been subjected to the vacuum treatment. If introduced, it is a drought condition. Voltage and number of pulses depend on the crop Those skilled in the art can appropriately select the electopore polarization conditions as needed. Thereafter, selection is performed in a medium containing antibiotics, and then normal plant individuals can be obtained by potting (growing in pots).
  • plant is a general term for organisms belonging to the plant kingdom, characterized by chloroplasts, hard cell walls, the presence of abundant permanent embryonic tissue, and organisms that are not capable of motility. It is done. Plant types are broadly classified in, for example, “Primary Color Makino Botanical Encyclopedia” (Kitatakakan (1982)), and all types of plants described therein can be used in the present invention. Typically, a plant refers to a flowering plant having an assimilation effect by chloroplast formation. “Plant” includes both monocotyledonous and dicotyledonous plants. Monocotyledonous plants include gramineous plants.
  • Preferred monocotyledonous plants include corn, wheat, rice, embata, barley, sorghum, rye and wheat, and more preferably, power including corn, wheat and rice.
  • Wheat includes wheat cultivar Nori 61, which had been difficult to obtain transformants by conventional methods.
  • Dicotyledonous plants include, but are not limited to, cruciferous plants, legumes, solanaceous plants, cucurbitaceous plants, and convolvulaceae plants.
  • Brassicaceae plants include, but are not limited to, cabbage, rapeseed, cabbage and cauliflower.
  • Preferred cruciferous plants are roe, kusasai and rapeseed.
  • a particularly preferred cruciferous plant is rapeseed.
  • Leguminous plants include, but are not limited to, soybeans, oysters, kidney beans, and cowpeas.
  • a preferred legume is soybean.
  • solanaceous plants include, but are not limited to, tomatoes, eggplants, and potatoes.
  • a preferred solanaceous plant is tomato.
  • the cucurbitaceae plants include, but are not limited to, power of matawali, cucumber, melon, and watermelon.
  • a preferred cucurbitaceae plant is mcwauli.
  • Convolvulaceae plants include, but are not limited to, morning glory, sweet potato and convolvulus.
  • a preferred convolvulaceae plant is morning glory. Unless otherwise indicated, a plant means any plant, plant organ, plant tissue, plant cell, and seed.
  • Examples of plant organs include roots, leaves, stems and flowers.
  • Examples of plant cells include callus and suspension culture cells.
  • a plant may mean a plant body.
  • examples of plant species that can be used in the present invention include solanaceae, gramineous, cruciferous, rose, legume, cucurbitaceae, perilla, lily, akaza, Plants such as celery family, urchinaceae and asteraceae are included.
  • examples of plant species that can be used in the present invention include any vine species, any fruit cultivar, cucurbitaceae plants (eg, rubber), and mallow (eg cotton). .
  • the method of the present invention can be applied to plant tissues (including dormant tissues (including ripe seeds, immature seeds, winter buds, and tubers), germplasm, growth points, and flower buds). Conduct poration and, most conveniently, perform erect mouth poration on the seeds.
  • the seed into which nucleic acid has been introduced by the electoporation method of the present invention can be easily converted into a nucleic acid introducer Z transformant by, for example, planting it in soil and growing it.
  • Seeds are usually composed of three parts: embryo, endosperm, and seed coat (supervised by Yoshikichi Noguchi II, Shinichiro IIda, for the relevant part, written by Hideo Chisaka, Agricultural University Dictionary, Yokendo, p. 896, 1987) .
  • the embryo is the part that contains all the genetic information of the plant and grows into the plant body. All monocotyledonous and dicotyledonous plants have embryos.
  • the nucleic acid was introduced by the electoporation method of the present invention, expression of the introduced nucleic acid was observed in the embryo part. Accordingly, a nuclear acid-introduced plant Z-transformed plant can be easily obtained from any plant having seeds including embryos by this simplest method of the present invention.
  • Examples of cruciferous plants include plants belonging to Raphanus, Brassica, Arabidopsis, Wasabia, or Capsella, and include, for example, radish, rape, Arabidopsis thaliana, coral rust, thrips and the like.
  • grass family plants include plants belonging to Oryza, Triticum, Hordeum, Secale, Saccharum, Sorghum, or Zea, and include, for example, rice, barley, rye, sugarcane, sorghum, corn, and the like.
  • Animal as used in this specification is a general term for organisms belonging to the animal kingdom. They require oxygen and organic food, and can be moved freely unlike plants and minerals. More characterized. Animals are broadly classified into vertebrates and invertebrates. As the vertebrate, for example, metal eels, shark eels, cartilaginous fish, teleosts, amphibians, reptiles, birds, mammals, etc.
  • mammals for example, single pores, Marsupial, rodent, winged, winged, carnivorous, carnivorous, long-nosed, odd-hoofed, cloven-hoofed, rodent, scale, sea cattle, cetacean, primate , Rodents, maggots, etc.
  • primates for example, chimpanzees, dihonds, humans
  • human-derived cells or organs are used.
  • invertebrates for example, crustaceans, millipedes, edible beetles, centipedes, komcades, insects and the like are used.
  • insects for example, Lepidoptera (including silkworms) are used.
  • transgene organism refers to an organism into which a specific gene has been incorporated.
  • Transgenic plant refers to a plant into which a specific gene has been incorporated.
  • transgene animal refers to an animal into which a specific gene has been incorporated.
  • Nucleic acid introduction by the method of the present invention Z-transformed cells and tissues can be differentiated, grown and Z or proliferated by any method known in the art. In the case of plant species, the process of differentiating, growing and Z or proliferating cells or tissues is for example
  • plant cultivation can be performed by any method known in the art. Plant cultivation methods are, for example, supervised by Isao Shimamoto and Kiyoshi Okada, “Experimental Protoco of Model Plants,“ Rouine Arabidopsis ”edited by: Cell Engineering, Supplementary Plant Cell Engineering Series 4; Rice Cultivation Method (Toshitoshi Okuno) pp. 28 — 32, and Yasuo Niwa, Arabidopsis Cultivation Method, pp. 33-40, and those skilled in the art can easily carry out the method and need not be described in detail herein.
  • Arabidopsis can be cultivated by soil cultivation, rock wool cultivation, or hydroponics. If cultivated under constant fluorescent conditions under white fluorescent lamps (about 60000 lux), the first flowers will bloom about 4 weeks after sowing, and the seeds will mature about 16 days after flowering. About 40-50 seeds can be obtained with one pod, and about 10,000 seeds can be obtained within 2 to 3 months after sowing. In addition, for example, in the cultivation of wheat, it is well known that it does not head and bloom unless it is exposed to low-temperature short-day conditions for a certain period after sowing.
  • low temperature short-day treatment for example, 20 ° C light period 8 hours (about 20 ° C) 2000 lux
  • 8 ° C dark period 16 hours This process is called vernalization and be called.
  • the cultivation conditions required for each plant species are generally well known in the art and therefore need not be described in detail herein.
  • nucleic acid-introduced Z-transformed cells and tissues can be differentiated, grown and Z- or propagated by any method known in the art. Obtain (see, for example, Meiji Izumi et al., bibliography of Biochemical Experiments, 4. Animal-Organization Experiments, Chemical Dojin, 1987)
  • cells and Z or tissue into which nucleic acid has been introduced by electopore position can be grown at room temperature (about 25 ° C.) while supplying commercially available feed.
  • surfactant refers to a soluble compound that reduces the surface tension of a liquid and reduces the interfacial tension between liquids or between a liquid and a solid.
  • the surfactant preferably has an action of promoting the penetration of bacteria (for example, agrobacterium) into the cell 'tissue.
  • surfactant used in the present invention for example, a surfactant with low toxicity to plants which is commercially available as a pesticide spreading agent from Loveland Industries under the trade name Silw et L-77, polyalkylene O sulfoxides modified heptene Tame Chino Les trisiloxane (polyalkyleneoxide modified Heptamethyltrisiloxane), and T W een20 a low surfactant toxic for animals and plants (TM) (poly Okishiechiren (20) sorbitan monolaurate), but may be mentioned It is not limited to these.
  • Silw et L-77 polyalkylene O sulfoxides modified heptene Tame Chino Les trisiloxane
  • T W een20 a low surfactant toxic for animals and plants (TM) (poly Okishiechiren (20) sorbitan monolaurate), but may be mentioned It is not limited to these.
  • vir region transcription inducer of Ti plasmid refers to a substance that induces transcription of genes present in the vir region of Ti plasmid.
  • Vir domain transcription inducers are originally derived from plant endogenous phenolic compounds (including acetosyringone) that are induced when plants are subjected to stress such as wounds.
  • a typical example of a Vir region transcription inducer is acetosyringone, but in addition to acetosyringone and its derivatives, exudate of tapaco leaf sections, isolated lignin synthesis precursors such as syringaldehyde and ferulic acid, or these And derivatives that induce transcription of genes present in the vir region.
  • Ti plasmid is an approximately 200 kbp plasmid of Agrobacterium tumefaciens, which is a causative factor of crown gall tumor. Ti plasmid has vir region, T-DNA region and so on.
  • a "vir region” is a region of a gene responsible for pathogenicity in a Ti plasmid, and includes virA, B, G, C, D, And 6 transcription units E and E.
  • the introduced gene consists of a polynucleotide.
  • polynucleotide As used herein, the terms “polynucleotide”, “oligonucleotide” and “nucleic acid” are used interchangeably herein and refer to a nucleotide polymer of any length. The term also includes “derivative oligonucleotide” or “derivative polynucleotide”. “Derivative oligonucleotide” or “derivative polynucleotide” refers to an oligonucleotide or polynucleotide that includes a derivative of a nucleotide or that has unusual linkages between nucleotides, and is used interchangeably.
  • oligonucleotides include, for example, 2 ′ O-methyl-ribonucleotides, derivative oligonucleotides in which a phosphodiester bond in an oligonucleotide is converted to a phosphoroate bond, and phosphorous in an oligonucleotide.
  • a particular nucleic acid sequence also includes conservatively modified variants (e.g., degenerate codon substitutes) and complementary sequences, as well as those explicitly indicated. It is contemplated. Specifically, a degenerate codon substitute creates a sequence in which the third position of one or more selected (or all) codons is replaced with a mixed base and a Z or doxyinosine residue. (Batzer et al., Nucleic Acid Res., 19: 5081, 1991; Ohtsuka3 ⁇ 4, J. Biol. Chem., 260: 26 05-2608, 1985; Rossolini et al., Mol. Cell. Probes, 8: 91 — 98, 1994).
  • nucleic acid is also used herein interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.
  • Particular nucleic acid sequences also include “splice variants”.
  • a particular protein encoded by a nucleic acid implicitly encompasses any protein encoded by a splice variant of that nucleic acid.
  • a “splice variant” is the product of alternative splicing of a gene. After transcription, the initial nucleic acid transcript can be spliced such that different (alternate) nucleic acid splice products encode different polypeptides.
  • the production mechanism of splice variants varies, but includes exon alternative splicing.
  • Other polypeptides derived from the same nucleic acid by read-through transcription are also encompassed by this definition. Any product of a splicing reaction (including recombinant forms of splice products) is included in this definition.
  • gene refers to a factor that defines a genetic trait. Usually arranged on a chromosome in a certain order. A gene that defines the primary structure of a protein is called a structural gene, and a gene that affects its expression is called a regulatory gene. As used herein, “gene” may refer to “polynucleotide”, “oligonucleotide”, and “nucleic acid”. As used herein, “homology” of a gene refers to the degree of identity of two or more gene sequences to each other. Therefore, the higher the homology between two genes, the higher the sequence identity or similarity.
  • the ability of two genes to have homology can be determined by direct sequence comparison or, in the case of nucleic acids, hybridization methods under stringent conditions.
  • the DNA sequence between the gene sequences is typically at least 50% identical, preferably at least 70% identical, more preferably at least 80%, 90% 95%, 96%, 97%, 98% or If they are 99% identical, they have homology.
  • expression of a gene, polynucleotide, polypeptide or the like means that the gene or the like is subjected to a certain action in vivo to take another form.
  • it refers to force transcription and translation of genes, polynucleotides, and the like to form a polypeptide, but transcription and production of mRNA may also be an embodiment of expression. More preferably, such polypeptide forms may be post-translationally processed.
  • nucleotide may be natural or non-natural.
  • “Derivative nucleotide” or “nucleotide analog” refers to a nucleotide that is different from a naturally occurring nucleotide but has the same function as the original nucleotide. Such derivative nucleotides and nucleotide analogs are well known in the art. Examples of such derivative nucleotides and nucleotide analogs include, but are not limited to, phosphoroates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2-0-methyl ribonucleotides, peptide nucleic acids (PNA).
  • PNA peptide nucleic acids
  • fragment refers to a polypeptide or polynucleotide having a sequence length of 1 to n-1 with respect to a full-length polypeptide or polynucleotide (length n). Say Chido.
  • the length of the fragment can be changed as appropriate according to its purpose. For example, the lower limit of the length is 3, 4, 5, 6, 7, 8, 9, 10 in the case of a polypeptide. , 15, 2, 0, 25, 30, 40, 50 and more, and lengths expressed in integers not specifically listed here (for example, 11 etc.) are also suitable as lower limits. It can be.
  • examples include 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100 and more nucleotides. !, NA! /, An integer length (eg 11) may also be appropriate as a lower limit.
  • a "vector” refers to a vector that can transfer a target polynucleotide sequence to a target cell.
  • vectors include prokaryotic cells, yeast, animal cells, plant cells, insect cells, host cells such as animal individuals and plant individuals, preferably those capable of autonomous replication in plant cells, or chromosomes. Examples thereof include those that contain a promoter at a position suitable for transcription of the polynucleotide of the present invention.
  • “Expression vector” refers to a nucleic acid sequence in which various regulatory elements are operably linked in a host cell in addition to a structural gene and a promoter that regulates its expression.
  • the regulatory element may preferably include a terminator, a selectable marker such as a drug resistance gene, and an enhancer. It is well known to those skilled in the art that the type of organism (eg, plant) expression vector and the type of regulatory element used can vary depending on the host cell.
  • the neo gene encoding the enzyme neomycin phosphotransferase that confers resistance to the antibiotic kanamycin (Beck et al., Gene, 19: 327, 1982; the enzyme hygromycin that confers resistance to the antibiotic hygromycin.
  • the hyg gene encoding mycin phosphotransferase (Gritz and Davies, Gene, 25: 179, 1983); and the phosphinotricin acetyltransferase that gives metaphysics to the herbicide phosphi nothricin bar Genes ( ⁇ 242236); spt genes that code for streptomycin phosphotransferase; streptomycin resistance genes; pharmacological genes such as spectinomycin resistance genes (eg, HS Chawla, Introduction to Plant Biotec hnology 2nd: 363, Science Publishers, Inc., Book, 20 02); and the gus gene (Jefferson et al., Proc. Natl. Acad. Sci.
  • Examples of the drug used for selection in the present invention include, but are not limited to, kanamycin, no, idaromomycin, dienetin, gentamicin, streptomycin, and spectinomycin.
  • Recombinant vector refers to a vector capable of transferring a target polynucleotide sequence into a target cell.
  • a vector can be autonomously replicated in a plant cell and a host cell such as a plant individual, or can be integrated into a chromosome, and contains a promoter at a position suitable for transcription of the polynucleotide of the present invention. Examples are shown.
  • Recombinant vectors for plant cells include Ti plasmids, tobacco mosaic virus vectors, diemi-virus vectors, and the like.
  • Terminal 1 is a sequence that is located downstream of a region encoding a protein of a gene, and is involved in termination of transcription when DNA is transcribed into mRNA and addition of a poly A sequence. It is known that the terminator influences the expression level of a gene by being involved in mRNA stability. Examples of the terminator include, but are not limited to, a CaMV35S terminator, a nopaline synthase gene terminator (Tnos), and a tobacco PRla gene terminator.
  • promoter refers to a region on DNA that determines the start site of gene transcription and directly regulates its frequency, and is the base on which RNA polymerase binds and initiates transcription. Is an array.
  • the promoter region is usually a region within about 2 kbp upstream of the first exon of the putative protein coding region, if the protein coding region in the genomic nucleotide sequence is predicted using DNA analysis software, The promoter region can be estimated.
  • the putative promoter region is usually a force upstream of the structural gene.
  • the putative promoter region is present within about 2 kbp upstream from the first exon translation start point.
  • site specificity generally means a part of an organism (for example, a plant) (for example, in the case of a plant, a root, stem, stem, leaf, flower). , Seed, germ, embryo, fruit, etc.).
  • time specificity refers to the specificity of expression of a gene in accordance with the stage of development of an organism (eg, a plant) (eg, if a plant, the growth stage (eg, days after germination)). Idiosyncratic Sex can be introduced into a desired organism by selecting an appropriate promoter.
  • the expression of the promoter of the present invention is "constitutive" means that, in all yarns and weaves of an organism, the growth of the organism is in the juvenile or mature stage. Even so, it is a property that is expressed in an almost constant amount.
  • Northern Plot analysis is performed under the same conditions as in the examples of the present specification, for example, at the same time (for example, two or more points (for example, 5th day and 15th day))
  • Expression is said to be constitutive by the definition of the present invention when almost the same level of expression is observed at any of the corresponding sites.
  • Constitutive promoters are thought to play a role in maintaining the homeostasis of organisms in normal growth environments.
  • the expression of the promoter of the present invention being “stress responsive” refers to the property that the expression level changes when at least one stress is applied to an organism.
  • stress inducibility the property of increasing the expression level
  • stress suppression the property of decreasing the expression level.
  • the expression of “stress suppression” is a concept that overlaps with the “constitutive” expression because it is assumed that the expression is observed in the normal state.
  • Plants or plant parts (specific cells, tissues, etc.) transformed with a vector incorporating a stress-inducible promoter together with a nucleic acid encoding the polypeptide of the present invention contain a stimulating factor having inducibility of the promoter. By using it, a specific gene can be expressed under certain conditions.
  • the enhancer is preferably an enzyme region containing an upstream sequence in the CaMV35S promoter.
  • a plurality of sensors can be used, but one may or may not be used.
  • operably linked means a transcriptional translational regulatory sequence (eg, promoter, henno, sensor, etc.) that has the expression (operation) of a desired sequence or It means being placed under the control of the translation arrangement.
  • a promoter In order for a promoter to be operably linked to a gene, it is usually necessary to place the promoter immediately upstream of that gene. Since there may be a sequence intervening between the motor and the structural gene, the promoter and the structural gene are not necessarily arranged adjacent to each other.
  • the presence of the introduced gene can be confirmed by Southern plotting or PCR. Transcription of the introduced gene can be detected by Northern plotting or PCR. If necessary, the expression of a protein as a gene product can be confirmed by, for example, a Western plot method.
  • a commercially available electopore polarization means (for example, CUY21EDIT gene transfer device, Neppagene, Inc., Yodogawa, Chiba) can be used for the device for the electopore polarization used in the present invention.
  • the electopore polarization chamber used for performing the electopore polarization may have any size as long as it can accommodate the plant tissue to be transformed.
  • a coolable chamber is preferred.
  • a custom-made electroporation chamber having a platinum electrode and a length of 1 cm ⁇ width 2 cm ⁇ height 2 cm was used.
  • X Glue solution (100 mM pH 7.0, phosphate buffer, 0.05% X— Gluc (5-Bromo —4—Black mouth 3—Indolyl 1 ⁇ —D—Glucuroro cyclohexylammonum salt) 2 ml) of 0.5 mM ferricyanium potassium, 0.5 mM ferrocyan potassium, 0.3% triton X-100, 20% methanol), and allowed to stand at 25 ° C. The expression of the GUS gene was confirmed from the degree of staining.
  • GUS analysis is carried out as follows.
  • the plant After selection with antibiotic medium, the plant is horticultural soil (New Magic Soil; Sakata Seed Co., Ltd., Sakai Tsuzuki-ku, Yokohama).
  • a pot of 8.5 cm in diameter and 5.5 cm in height (flowerpot) Cultivated in an isolated dalos chamber (15 ° C light period 8 hour thorium lamp, approximately 50,000 lux), dark period 16 hours condition or 20 ° C light period 8 hours (approximately 2000 lux) And 8 ° C dark period 16 hours).
  • the leaves and stems of the transformed plant are collected and DNA is extracted. Any known method may be used for DNA extraction.
  • a typical DNA extraction method is the CTAB method (Hirofumi Uchibuchi “Plant Gene Manipulation Manual How to Make Transgenic Plants” Kodansha Scientific, pages 71-74, book, 1989).
  • a pair of primers (5 '-ctgcgtgcaatccatcttg-3': Cat No. 1 and 5'—actcgtc) that extract DNA from the leaves of individuals after electoral poration and detect the NPT II gene PCR was performed using aagaaggcgatagaag-3 ′: SEQ ID NO: 2).
  • An additional pair of Phuima ⁇ (5-catgattgaacaagatggattgcacgcaggttctc-3: eye ti column number d, 5-cagaagaactcgtcaagaaggcgatagaaggcgat-: ⁇ Self column 3 ⁇ 4> 4) can also be used, and this primer pair is more specifically NPT II Since a gene can be detected, it is more preferable.
  • AmpliTaq Gold from Perkin Elma Japan Co., Ltd. (Yokohama Shaanxi Ward) is used according to the manufacturer's instructions.
  • the conditions of the thermal cycler used for amplification are:
  • the plant is further grown and a large number of mature seeds are obtained by self-pollination.
  • the medium strength is also randomly picked from about 10 seeds and cultivated in pots filled with soil.
  • the leaf force DNA of the young plant is extracted, and PCR is performed under the conditions described in the above PCR analysis using this DNA as a template.
  • Brown rice of Koshihikari was used as a ripe seed of japonica rice.
  • Agricultural forest No. 61 was used as a wheat seed.
  • IR24 brown rice was used as a ripe seed of indy power rice.
  • As a Chinese cabbage seed “Musou” was used.
  • “Fayatt e ” was used as soybean seeds.
  • Toma As a seed “Mini Carol” was used.
  • As the morning glory seeds “Sun Smile White Circle” was used. The seeds grown at this laboratory or purchased from a seed company were used. About 10-30 appropriate mature seeds were selected by visual inspection (depending on the size of the seeds), and water was absorbed overnight at 25 ° C in tap water.
  • the tap water used for water absorption contains an aqueous sodium hypochlorite solution (adjusted so that the effective chlorine concentration is about 0.01%) in order to suppress the growth of various bacteria.
  • the water absorption was conducted at 10 ° C for two days. EHA150 was used as agrobacterium.
  • the seeds and buffer in the petri dish were transferred to the chamber 1 and left on ice for 1 minute. And, as shown in the example, with a voltage of 50VZcm (however, the distance between the electrodes is lcm), and a rectangular wave with a pulse width of 50 milliseconds (capacitance of 50 microsecond voltage, repeating a period of resting for 50 milliseconds) The number of pulses was 99.
  • the buffer solution was discarded, and the treated seeds were returned to the original petri dish. 2 ml of 0.5% polybulurpyrrolidone (PVP) aqueous solution was placed in the petri dish where the seeds were returned.
  • PVP polybulurpyrrolidone
  • This PVP aqueous solution contained an aqueous sodium hypochlorite solution to suppress the growth of bacteria (adjusted so that the effective chlorine concentration was about 0.01%).
  • Figures 2 to 4 show the results of rice on the 6th, 8th, and 14th days after infection with agrobacterium.
  • Figure 5 shows the results of wheat on the eighth day after infection with agrobacterium.
  • GFP fluorescence was observed 6 days (Fig. 2), 8 days (Fig. 3), and 14 days (Fig. 4) after treatment with agrobacterium.
  • strong fluorescence was confirmed (the individual on the left side of each of FIGS. 2B, 3B, and 4B).
  • the expression of GFP was confirmed with the naked eye at a high efficiency of about 50% of the transgenic individuals.
  • a strong expression level as shown in the present example was completely unexpected.
  • the expression of GFP was not confirmed (the individual on the right side of each of FIGS. 2B and 3B).
  • the expression level of GFP in the case of gene transfer after infection with agrobacterium after decompression without using erect mouth poration is lower than that in the case where elect mouth poration is performed. It was strong (individual on the right side of Fig. 4B) In addition, when transfection was carried out with agrobacterium after depressurization without using erect mouth poration, the expression of GFP was confirmed with the naked eye in about 5% of individuals. In the conventional agrobacterial method, the target cells (tissues) are removed before infection with agrobacterium. It was considered essential to treat (pre-culture) with a plant hormone such as D. However, the results of this example have a significant effect that cannot be predicted in rapidity and simplicity in that no such pre-culture is required.
  • the petri dish was transferred to a lighted incubator to grow plants.
  • the conditions are as follows: fluorescent lighting approximately 3000 lux, temperature 10 ° C, lighting on for 8 hours, lighting off for 16 hours.
  • GFP fluorescence was observed 7 days after treatment with agrobacterium (Fig. 6).
  • agrobacterium agrobacterium
  • strong fluorescence was confirmed (individual on the left in Fig. 6B).
  • the expression of GFP was confirmed with the naked eye at a high efficiency of about 60% of the transgenic individuals.
  • the direct gene transfer method such as the elect mouth poration
  • the indirect gene transfer method such as the agrobatterium method.
  • the gene transfer with high efficiency was quite unpredictable.
  • a strong expression level as shown in this example was not expected at all. Considering that it has been difficult to transform to indy rice in the past, the effect of the present invention is remarkable.
  • the results of this Example have a remarkable effect that could not be predicted in terms of rapidity and convenience in that pre-culture using a medium supplemented with 2, 4-D or the like is not required.
  • GFP fluorescence was observed 4 days after treatment with agrobacterium (Fig. 7).
  • agrobacterium agrobacterium
  • strong fluorescence was confirmed (individual on the left in FIG. 7B).
  • the expression of GFP was confirmed with the naked eye at a high efficiency of about 60% of the transgenic individuals.
  • GFP fluorescence was observed 4 days after treatment with agrobacterium (Fig. 8).
  • agrobacterium agrobacterium
  • strong fluorescence was confirmed (individual on the left in FIG. 8B).
  • the expression of GFP was confirmed with the naked eye at a high efficiency of about 40% of the transgenic individuals.
  • the direct gene transfer method such as the electopore poration
  • the indirect gene transfer method such as the agrobatterium method.
  • the gene transfer with high efficiency was quite unpredictable.
  • a strong expression level as shown in this example was not expected at all.
  • GFP fluorescence was observed 4 days after treatment with agrobacterium (Fig. 9).
  • agrobacterium agrobacterium
  • strong fluorescence was confirmed (individual on the left in Fig. 9B).
  • the expression of GFP was confirmed with the naked eye at a high efficiency of about 50% of the transgenic individuals.
  • the direct gene transfer method such as the elect mouth poration
  • the indirect gene transfer method such as the agrobatterium method.
  • the gene transfer with high efficiency was quite unpredictable.
  • a strong expression level as shown in this example was not expected at all.
  • GFP fluorescence was observed 4 days after treatment with agrobacterium (Fig. 10).
  • agrobacterium As a result of gene transfer combining agrobacterium and electoporation, strong fluorescence was confirmed (individual on the left in Fig. 10B).
  • the expression of GFP was confirmed with the naked eye at a high efficiency of about 50% of the transgenic individuals.
  • the elect mouth bole There is no example of combining a direct gene transfer method such as Cyon and an indirect gene transfer method such as an agrobacterium, but combining these methods is expensive as shown in this example. The fact that we were able to introduce genes efficiently was a completely unpredictable result.
  • the strong expression level shown in the present example is completely unexpected.
  • the simple method of the present invention facilitates large-scale processing and large-scale analysis that are important in development research in this field, and as a result, induces breakthrough research and leads to the development of innovative recombinant crops. Connected.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Electromagnetism (AREA)
  • Sustainable Development (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

To establish a method of conveniently and quickly transforming a plant compared with the existing methods, it is intended to provide a method of very conveniently and quickly transforming a plant. The above problem can be solved by a method comprising: a) the step of maintaining a cell under a pressure different from the atmospheric pressure; and b) the step of bringing the cell into contact with an agrobacterium containing a nucleic acid; followed by, if desired, c) setting the cell and the agrobacterium under such conditions as allowing the occurrence of electroporation. Because of being convenient, this method facilitates mass-scale treatments and mass-scale analyses, which are important in research and development in this field and, in its turn, induces rapid advances in studies, thereby enabling the development of epoch-making genetically modified crops.

Description

明 細 書  Specification
減圧処理 Z加圧処理の使用を含む、ァグロバタテリゥムを用いる細胞へ の核酸導入方法  Decompression treatment Z Method for introducing nucleic acid into cells using agrobacterium, including the use of pressure treatment
技術分野  Technical field
[0001] 本発明は、減圧処理または加圧処理と、ァグロバタテリゥムを用いて、所望の核酸 を細胞または組織 (植物組織を含む)へ導入し、核酸導入体 (形質転換体を含む)を 作出する方法に関する。さらに、本発明は、減圧処理または加圧処理と、エレクトロボ 一レーシヨンを組み合わせることによって、所望の核酸を含むァグロバタテリゥムのよ うな細菌を細胞または組織に導入して、核酸導入体 (形質転換体を含む)を作出する 方法に関する。  [0001] The present invention introduces a nucleic acid transductant (including a transformant) by introducing a desired nucleic acid into a cell or tissue (including a plant tissue) using reduced pressure treatment or pressurized treatment and agrobacterium. ) On how to create. Furthermore, the present invention introduces a bacterium such as agrobacterium containing a desired nucleic acid into a cell or tissue by combining a reduced pressure treatment or a pressurized treatment with electrovola- tion, and a nucleic acid transductant ( The present invention relates to a method for producing a transformant (including a transformant).
背景技術  Background art
[0002] コムギ、ォォムギ、イネ、トウモロコシ、ダイズなどの主要穀物は人類の生存にとって 必須のものであるが、世界的な人口増加に見合った食料を確保していくためには、 現在より収量の多い作物を開発していく必要がある。収率を上げる品種改良の一つ として、組換え DNA技術を用いた形質転換作物の開発が行われて 、る。  [0002] Major crops such as wheat, barley, rice, corn, and soybean are essential for the survival of mankind. Many crops need to be developed. One of the varietal improvements that increase yield is the development of transformed crops using recombinant DNA technology.
[0003] また、例えば、ノ、クサイ、トマト、キユウリなどの野菜類は食生活を豊かにし、栄養面 でも必須の作物である。し力しながら、これらの野菜は様々な病虫害に弱ぐ遺伝子 組換え技術の利用により耐病虫害性を付与することができるならば、収穫量を安定さ せることが可能となる。そのため、有用な遺伝子の単離と共にこれらの遺伝子を用い る形質転換方法が開発されてきた。 [0003] Further, for example, vegetables such as roe, crocodiles, tomatoes and cucumbers are rich in diet and are essential for nutrition. However, if these vegetables can be made resistant to disease and pests by using gene recombination techniques that are vulnerable to various diseases and pests, the yield can be stabilized. Therefore, transformation methods using these genes have been developed along with the isolation of useful genes.
[0004] 形質転換を行うためには、植物の場合、一般に、植物に対して直接的に遺伝子導 入を行う方法 (直接的遺伝子導入法)、または植物に対して間接的に遺伝子導入を 行う方法 (間接的遺伝子導入法)が行われる。 [0004] In order to carry out transformation, in the case of plants, generally, gene transfer directly to plants (direct gene transfer method) or gene transfer to plants indirectly. The method (indirect gene transfer method) is performed.
[0005] 現在までに、間接的な遺伝子導入法として、ァグロバタテリゥムを利用した方法が広 く利用されている。例えばイネの完熟種子を培養して 3週間後に得られたカルスに対 してァグロバタテリゥムを感染させる方法（Hieiら， Plant Journal, 6 : 271 - 282, 1 994を参照）あるいは、数日間、前培養した種子に対してァグロバタテリゥムを感染さ せて形質転換体を迅速に得ることができる方法（田中ら、特許第 3141084号を参照 )を挙げることができる。しかし、間接的遺伝子導入法を用いた場合、培養時間を短く すると、遺伝子導入ができなくなるか、または遺伝子導入効率が低下するという欠点 がある。 [0005] To date, a method using agrobacterium has been widely used as an indirect gene transfer method. For example, a method of infecting callus obtained after 3 weeks of cultivating a mature rice seed (see Hiei et al., Plant Journal, 6: 271-282, 1 994) or several Infects agrobataterum on seeds pre-cultured for days And a method (see Tanaka et al., Japanese Patent No. 3141084) that can rapidly obtain transformants. However, when the indirect gene transfer method is used, there is a drawback that if the culture time is shortened, the gene transfer cannot be performed or the gene transfer efficiency is lowered.
[0006] また、コムギについては、従来：（1)コムギの形質転換については、品種間差が大き ぐ培養に必要な期間も長いという問題があったこと (非特許文献 2)； (2)コムギの形 質転換においてァグロパクテリゥムを利用する方法は成功していな力つたこと (非特 許文献 2)；ならびに、（3)コムギの形質転換では、その対象に開花後 10日から 2週 間程度の未熟胚を用いなければならな 、と 、う材料調製の困難性に加え、形質転換 頻度が低いことなど力 未だにパンコムギ等のコムギの形質転換は敬遠されがちであ るという現状がある。  [0006] In addition, conventional wheat: (1) The transformation of wheat had a problem that the difference between cultivars was large and the period required for cultivation was long (Non-patent Document 2); (2) The method of using agrobacterium for the transformation of wheat has been unsuccessful (Non-Patent Document 2); and (3) In addition to the difficulty of preparing raw materials and the low frequency of transformation, the current situation is that transformation of wheat, such as bread wheat, still tends to be avoided. is there.
[0007] そこで、間接的遺伝子導入法のこれら欠点を改善する必要がある。また、簡便かつ 迅速な間接的遺伝子導入法を開発することによって、核酸導入体 (特に、形質転換 植物体)を、迅速かつ大量に得ることが可能になる。従って、本発明は、植物形質転 換体を得ることが必要な産業分野のみならず、植物を用いる開発研究においても大 量処理 ·大量解析を容易にせしめ、ひいては飛躍的な研究の進歩を誘発し、画期的 な糸且換え作物の開発につながる。  [0007] Therefore, it is necessary to improve these disadvantages of the indirect gene transfer method. In addition, by developing a simple and rapid method for indirect gene transfer, it is possible to obtain a nucleic acid transduct (particularly a transformed plant) rapidly and in large quantities. Therefore, the present invention facilitates large-scale processing and large-scale analysis not only in the industrial field where it is necessary to obtain plant transformants but also in development research using plants, and thus induces dramatic progress in research. This will lead to the development of groundbreaking yarn and changeable crops.
特許文献 1：田中ら、特許第 3141084号  Patent Document 1: Tanaka et al., Japanese Patent No. 3141084
非特許文献 l :Hieiら， Plant Journal, 6 : 271 - 282, 1994  Non-patent literature l: Hiei et al., Plant Journal, 6: 271-282, 1994
非特干文献 2： Biotechnology in Agriculture and Forestry 46、 Trangeni c Crops I、 Y. P. S. Bajaj編、 Springer社発行、 2000年、特に 33頁の T. A. L oeb〖こよる 己載  Non-patent document 2: Biotechnology in Agriculture and Forestry 46, Trangenic Crops I, edited by Y.P.S. Bajaj, published by Springer, 2000, especially T. A. Loeb
発明の開示  Disclosure of the invention
発明が解決しょうとする課題  Problems to be solved by the invention
[0008] 本発明の解決しょうとする課題は、迅速かつ高効率な間接的遺伝子導入法が確立 されていないという上記現状に鑑み、当該分野において、迅速かつ高効率な間接的 遺伝子導入法を提供することである。 [0008] The problem to be solved by the present invention is to provide a rapid and highly efficient indirect gene transfer method in the field in view of the above situation that a rapid and highly efficient indirect gene transfer method has not been established. It is to be.
[0009] 簡便かつ迅速な本発明の方法を用いることによって、ァグロバタテリゥムを用いて、 形質転 m¾物体を、迅速かつ大量に得ることが可能になる。従って、本発明は、植 物形質転換体を得ることが必要な産業分野のみならず、植物を用いる開発研究にお V、ても大量処理 ·大量解析を容易にせしめ、ひ 、ては飛躍的な研究の進歩を誘発し[0009] By using simple and rapid method of the present invention, Transformation m¾ objects can be obtained quickly and in large quantities. Therefore, the present invention makes not only the industrial field where it is necessary to obtain plant transformants, but also makes it easy to conduct large-scale processing and large-scale analysis even for development research using plants. Induces the research progress
、画期的な組換え作物の開発につながる。 , Leading to the development of groundbreaking recombinant crops.
課題を解決するための手段  Means for solving the problem
[0010] 上記課題は、  [0010] The above problem is
a)細胞を大気圧と異なる圧力下に維持する工程;および  a) maintaining the cells under a pressure different from atmospheric pressure; and
b)該細胞を、核酸を含むァグロパクテリゥムと接触させる工程  b) contacting the cell with an agrobacterium containing nucleic acid
を包含し、必要に応じて、さらに  Including, if necessary, further
c)前記工程 (b)の後に、前記細胞と前記ァグロパクテリゥムとを、エレクト口ポーレー シヨンが起きる条件下に配置する工程  c) After the step (b), the step of arranging the cells and the agrobacterium under the condition that the electopore por- tion occurs.
を包含する形質転換植物を作製する方法によって達成された。  This has been achieved by a method for producing a transformed plant comprising
[0011] 一つの実施形態では、本発明の方法は、植物細胞 (例えば、完熟種子）を用いて 実施される。 [0011] In one embodiment, the method of the present invention is carried out using plant cells (eg, mature seeds).
[0012] 具体的には、本願明細書において発明者らは、植物に対してァグロバタテリゥムを 用いる遺伝子導入を行う際に、核酸が取り込まれやす！ヽように細胞を処理する方法と して各種試みた。その結果、（1)植物を真空状態に保ち、ァグロパクテリゥム (Agrob acterium)を用いる核酸導入法は、 Arabidopsis以外の植物には効果的でないこと 、および（2)ァグロパクテリゥムを用いる核酸導入のためには、植物を真空状態に保 つ必要がないこと（Bent, Plant Physiology, 124 : 1540—1547, 2000年 12月 )、という従来の知見に反して、実施例に示すように、ァグロパクテリゥムを用いる植物 核酸導入において、植物組織を大気圧と異なる圧力下に維持する工程を用いること によって、迅速な遺伝子導入が可能であることを見出した。この本発明の方法は、植 物細胞のみならず任意の細胞に対して行われ得る。さらに、本発明の遺伝子導入方 法に、エレクト口ポーレーシヨンを組み合わせることによって、細胞への核酸導入効率 を顕著に改善し得る。  [0012] Specifically, in the present specification, the inventors easily incorporate a nucleic acid when a gene is introduced into a plant using agrobacterium. Various attempts were made as a method of treating cells. As a result, (1) keeping the plant in a vacuum state and the nucleic acid introduction method using agrobacterium is not effective for plants other than Arabidopsis, and (2) Contrary to the conventional knowledge that the plant does not need to be kept in vacuum for the introduction of the nucleic acid to be used (Bent, Plant Physiology, 124: 1540-1547, December 2000), as shown in the examples. In addition, in the introduction of plant nucleic acid using agrobacterium, it was found that rapid gene transfer is possible by using a process of maintaining plant tissue under a pressure different from atmospheric pressure. This method of the present invention can be performed not only on plant cells but also on arbitrary cells. Furthermore, the efficiency of introducing a nucleic acid into a cell can be remarkably improved by combining the method for introducing a gene of the present invention with the electopore population.
[0013] 本方法は極めて簡便である。さらに、本発明の方法は、核酸導入操作後に通常必 要な培養過程を必要としな ヽため、得られる形質導入体が培養変異を含まな ヽと ヽぅ 利点も有する。従来の核酸導入操作後に必要とされた培養過程では、必然的に、培 養変異が起こることが知られている。培養変異とは、当業者が通常理解し得る通り、 培養過程で生じる遺伝学的変異を意味し、培養過程の間に、核酸導入される細胞が もともと有して ヽた核酸配列および Zまたは導入する核酸配列にぉ ヽて生じる任意 の配列改変 (例えば、置換、欠失、挿入、転座、逆位、重複など)をいう。意図されな 、培養変異は、核酸導入した核酸導入体に望ましくな 、形質を付与することが多!ヽ 。従って、この培養変異を全く起こさずに所望の核酸導入体を得ることができる本発 明の方法は、非常に有益である。 [0013] This method is extremely simple. Furthermore, since the method of the present invention does not require a culture process that is usually required after the nucleic acid introduction operation, the resulting transductant does not contain a culture mutation. There are also advantages. It is known that culture mutations inevitably occur in the culture process required after conventional nucleic acid introduction procedures. Culture mutation means a genetic mutation that occurs during the culture process, as can be generally understood by those skilled in the art. During the culture process, the nucleic acid sequence and Z or transfer originally introduced by the cell into which the nucleic acid is introduced This refers to any sequence modification (for example, substitution, deletion, insertion, translocation, inversion, duplication, etc.) that occurs over the entire nucleic acid sequence. Unintentionally, culture mutations often impart a desirable trait to a nucleic acid transducer into which a nucleic acid has been introduced. Therefore, the method of the present invention which can obtain a desired nucleic acid transductant without causing any culture mutation is very useful.
[0014] 本発明はまた、従来の間接的遺伝子導入法で必須とされていた、数日〜数週間の 対象細胞 (組織)の前培養を必要とせず、その結果、従来法よりも迅速に遺伝子導入 が可能であると 、う利点を提供する。  [0014] The present invention also does not require the pre-culture of target cells (tissues) for several days to several weeks, which has been essential in the conventional indirect gene transfer method, and as a result, more rapidly than the conventional method. The potential for gene transfer provides the advantage.
[0015] 従って、本発明は以下を提供する。  Accordingly, the present invention provides the following.
1.核酸を細胞内に導入する方法であって、以下の工程：  1. A method for introducing a nucleic acid into a cell, comprising the following steps:
a)細胞を大気圧と異なる圧力下に維持する工程;および  a) maintaining the cells under a pressure different from atmospheric pressure; and
b)該細胞を、核酸を含むァグロパクテリゥムと接触させる工程  b) contacting the cell with an agrobacterium containing nucleic acid
を包含する方法。  Including the method.
2.前記細胞を大気圧と異なる圧力下に維持する工程が、前記細胞を減圧処理する 工程である、項目 1に記載の方法。  2. The method according to item 1, wherein the step of maintaining the cells under a pressure different from atmospheric pressure is a step of subjecting the cells to reduced pressure treatment.
3.前記細胞を大気圧と異なる圧力下に維持する工程が、前記細胞を加圧処理する 工程である、項目 1に記載の方法。  3. The method according to item 1, wherein the step of maintaining the cells under a pressure different from atmospheric pressure is a step of pressurizing the cells.
4.前記細胞を大気圧と異なる圧力下に維持する工程が、該細胞を、前記核酸を含 むァグロバタテリゥムと接触させる工程の前に行われる、項目 1に記載の方法。  4. The method according to item 1, wherein the step of maintaining the cell under a pressure different from atmospheric pressure is performed before the step of contacting the cell with agrobacterium containing the nucleic acid.
5.前記減圧処理する工程が、大気圧よりも 0. 096MPa低い減圧下で行われる、項 目 2に記載の方法。  5. The method according to Item 2, wherein the decompressing step is performed under a reduced pressure of 0.096 MPa lower than the atmospheric pressure.
6.項目 1に記載の方法であって、さらに  6. The method according to item 1, further comprising:
c)前記工程 (b)の後に、前記細胞と前記ァグロパクテリゥムとを、エレクト口ポーレー シヨンが起きる条件下に配置する工程、  c) after the step (b), the step of placing the cells and the agrobacterium under conditions that cause electopore poration;
を包含する方法。 7.項目 6に記載の方法であって、前記 c)工程が、少なくとも二種類の方向で、前記 細胞と前記ァグロバタテリゥムとに電圧パルスをかけることを含む、方法。 Including the method. 7. The method according to item 6, wherein the step c) includes applying a voltage pulse to the cell and the agrobacterium in at least two directions.
8.前記細胞が植物細胞である、項目 1に記載の方法。  8. The method according to item 1, wherein the cell is a plant cell.
9.前記植物細胞が、植物の休眠組織の細胞である、項目 8に記載の方法。  9. The method according to item 8, wherein the plant cell is a cell of a dormant tissue of a plant.
10. m記植物の休眠組織が種子である、項目 9に記載の方法。  10. The method according to item 9, wherein the dormant tissue of the plant is m seed.
、  ,
11. m記植物の種子が完熟種子である、項目 10に記載の方法。  11. The method according to item 10, wherein the seeds of the plant m are ripe seeds.
、  ,
12. m記植物細胞が、単子葉植物由来である項目 8に記載の方法。  12. The method according to item 8, wherein the plant cell is derived from a monocotyledonous plant.
、  ,
13. m記単子葉植物がイネ科植物である、項目 12に記載の方法。  13. The method according to item 12, wherein the monocotyledonous plant m is a gramineous plant.
、  ,
14. m記イネ科植物がコムギである、項目 13に記載の方法。  14. The method according to Item 13, wherein the Gramineae plant is wheat.
、  ,
15. m記イネ科植物力 Sイネである、項目 13に記載の方法。  15. The method according to item 13, wherein the plant power is S rice.
、  ,
16. m記イネ科植物がトウモロコシである、項目 13に記載の方法。  16. The method according to item 13, wherein the Gramineae plant is corn.
、  ,
17. m記植物細胞が、双子葉植物由来である項目 8に記載の方法。  17. The method according to item 8, wherein the plant cell is derived from a dicotyledonous plant.
、  ,
18. m記双子葉植物がマメ科植物である、項目 17に記載の方法。  18. The method according to item 17, wherein the dicotyledon m is a legume.
、  ,
19. m記マメ科植物がダイズである、項目 18に記載の方法。  19. The method according to item 18, wherein the leguminous plant m is soybean.
、  ,
20. m記双子葉植物がアブラナ科植物である、項目 17に記載の方法。  20. The method according to item 17, wherein the dicotyledonous m plant is a cruciferous plant.
、  ,
21. m記アブラナ科植物がハクサイである、項目 20の方法。  21. The method of item 20, wherein the cruciferous plant is Chinese cabbage.
、  ,
22. m記アブラナ科植物がシロイヌナズナである、項目 20の方法。  22. The method of item 20, wherein the cruciferous plant is Arabidopsis thaliana.
、  ,
23. m記双子葉植物がヒルガオ科植物である、項目 17に記載の方法。  23. The method according to item 17, wherein the dicotyledonous plant m is a convolvulaceae plant.
、  ,
24. m記ヒルガオ科植物がアサガオである、項目 23に記載の方法。  24. The method according to Item 23, wherein the Convolvulaceae plant is masao.
、  ,
25. m記双子葉植物がナス科植物である、項目 17に記載の方法。  25. The method according to item 17, wherein the dicotyledonous plant m is a solanaceous plant.
、  ,
26. m記ナス科植物がトマトである、項目 25に記載の方法。  26. The method according to item 25, wherein the solanaceous plant is tomato.
、  ,
27. m記双子葉植物がゥリ科植物である、項目 17に記載の方法。  27. The method according to item 17, wherein the dicotyledonous m plant is a cucurbitaceae plant.
、  ,
28. m記ゥリ科植物がゥリである、項目 27に記載の方法。  28. The method according to item 27, wherein the cucurbitaceae plant is cucumber.
、  ,
29. m記ァグロバタテリゥム力 Agrobacterium tumefaciensである、項目 1に記載の方 法。  29. The method according to item 1, which is Agrobacterium tumefaciens.
30.核酸を細胞内に導入した植物を作製する方法であって、以下の工程:  30. A method for producing a plant into which a nucleic acid has been introduced into a cell, comprising the following steps:
a)植物細胞を大気圧と異なる圧力下に維持する工程；および a) maintaining the plant cell under a pressure different from atmospheric pressure; and
b)該植物細胞を、核酸を含むァグロパクテリゥムと接触させる工程 を包含する方法。 b) contacting the plant cell with agrobacterium containing nucleic acid Including the method.
31.項目 30に記載の方法であって、さらに  31. The method according to item 30, further comprising:
c)前記工程 (b)の後に、前記植物細胞と前記ァグロパクテリゥムとを、エレクト口ポー レーシヨンが起きる条件下に配置する工程、 c) after the step (b), placing the plant cell and the agrobacterium under conditions where electo-portion occurs,
を包含する方法。 Including the method.
32.項目 30に記載の方法であって、前記植物細胞を、分化、成長および Zまたは増 殖させる工程をさらに包含する、方法。  32. The method according to item 30, further comprising the step of differentiating, growing and Z or growing the plant cell.
33.項目 30に記載の方法であって、前記 a)工程が、前記植物細胞を含む種子を大 気圧と異なる圧力下に維持する工程を含み、かつ、前記 b)工程が、該植物細胞を含 む該種子を、該ァグロパクテリゥムと接触させる工程を含む、方法。  33. The method according to item 30, wherein the step a) includes a step of maintaining the seed containing the plant cell under a pressure different from atmospheric pressure, and the step b) includes the plant cell. Contacting the seeds containing with the agrobacterium.
34.前記種子が、単子葉植物の種子である項目 33に記載の方法。  34. The method according to item 33, wherein the seed is a monocotyledonous seed.
35.前記単子葉植物の種子が完熟種子である、項目 34に記載の方法。  35. The method according to item 34, wherein the seeds of monocotyledonous plants are mature seeds.
36.前記単子葉植物の種子がイネ科の種子である、項目 34に記載の方法。  36. The method according to item 34, wherein the monocotyledonous plant seed is a grass seed.
37.前記イネ科種子がコムギ種子である、項目 36に記載の方法。  37. The method according to item 36, wherein the grass seed is a wheat seed.
38.前記イネ科種子がイネ種子である、項目 36に記載の方法。  38. A method according to item 36, wherein the grass seed is a rice seed.
39.前記イネ科種子がトウモロコシ種子である、項目 36に記載の方法。  39. The method according to item 36, wherein the grass seed is corn seed.
40.前記種子が、双子葉植物の種子である項目 33に記載の方法。  40. A method according to item 33, wherein the seed is a dicotyledonous seed.
41.前記双子葉植物の種子がマメ科植物の種子である、項目 40に記載の方法。 41. The method of item 40, wherein the dicotyledonous seed is a legume seed.
42.前記マメ科植物がダイズである、項目 41に記載の方法。 42. The method according to item 41, wherein the leguminous plant is soybean.
43.前記双子葉植物の種子がアブラナ科植物の種子である、項目 40に記載の方法  43. The method according to item 40, wherein the dicotyledonous plant seed is a cruciferous plant seed.
44.前記アブラナ科植物がハクサイである、項目 43の方法。 44. The method of item 43, wherein the cruciferous plant is Chinese cabbage.
45.前記アブラナ科植物がシロイヌナズナである、項目 43の方法。  45. The method of item 43, wherein the cruciferous plant is Arabidopsis thaliana.
46.前記双子葉植物の種子がヒルガオ科植物の種子である、項目 40に記載の方法  46. The method according to item 40, wherein the dicotyledonous seed is a seed of a convolvulaceae plant.
47.前記ヒルガオ科植物がアサガオである、項目 46に記載の方法。 47. The method according to item 46, wherein the convolvulaceae plant is morning glory.
48.前記双子葉植物の種子がナス科植物の種子である、項目 40に記載の方法。 48. A method according to item 40, wherein the dicotyledonous plant seed is a solanaceous plant seed.
49.前記ナス科植物がトマトである、項目 48に記載の方法。 50.前記双子葉植物の種子がゥリ科植物の種子である、項目 40に記載の方法。49. The method according to Item 48, wherein the solanaceous plant is tomato. 50. A method according to item 40, wherein the dicotyledonous seeds are cucurbitaceae seeds.
51.前記ゥリ科植物がゥリである、項目 50に記載の方法。 51. The method according to item 50, wherein the cucurbitaceae plant is cucumber.
52.項目 30〜51のいずれか 1項に記載の方法によって作製された、植物。  52. A plant produced by the method according to any one of items 30 to 51.
53.培養変異を含まない、項目 52に記載の植物。  53. The plant according to item 52, which does not contain a culture mutation.
54. 自動化して核酸を細胞内に導入する装置であって、該装置は、以下： a)核酸を含むァグロパクテリゥムと細胞とを含む混合液を入れる容器；  54. An apparatus for automated introduction of nucleic acids into cells, the apparatus comprising: a) a container for containing a mixture of agrobacterium containing nucleic acids and cells;
b)該 a)の容器中に核酸を含むァグロパクテリゥムを入れる手段；  b) Means for placing agrobacterium containing nucleic acid in the container of a);
c)該 a)の容器中に細胞を入れる手段；  c) means for placing the cells in the container of a);
d)細胞を大気圧と異なる圧力下に維持する容器であって、大気圧と異なる圧力に耐 える能力を有する、容器；  d) a container for maintaining cells under a pressure different from atmospheric pressure, the container having the ability to withstand a pressure different from atmospheric pressure;
e)該細胞を、該 d)の容器中に配置する手段；  e) means for placing the cells in the container of d);
f )該 d)の容器中を、大気圧と異なる圧力に維持する手段；および  f) means for maintaining the container in d) at a pressure different from atmospheric pressure; and
g)該 b)、 c；)、 e)、および f)の手段を自動化して実行する手段  g) means for automating and executing the means of b), c;), e), and f)
を備え、ここで該 b)の手段、該 c)の手段、および該 e)の手段は、同一であるかまたは 異なり、そして該 a)の容器、および該 d)の容器は、同一である力または異なる、装置  Wherein the means of b), the means of c) and the means of e) are the same or different, and the container of a) and the container of d) are the same Power or different, equipment
55.少なくとも 2つ以上の対の電極を有する、エレクト口ポーレーシヨン用電極。55. An electrode for electo-poration having at least two pairs of electrodes.
56.少なくとも 3対の電極を有する、項目 55に記載のエレクト口ポーレーシヨン用電極 発明の効果 56. The electrode for electo-poration according to item 55, which has at least three pairs of electrodes Effect of the invention
[0016] 本発明の遺伝子導入方法によって、対象となる細胞 (組織）に対して、迅速かつ高 効率な間接的遺伝子導入が可能となる。  [0016] The gene introduction method of the present invention enables rapid and highly efficient indirect gene introduction into a target cell (tissue).
[0017] 簡便な本発明の方法は、この分野の開発研究において重要な大量処理 ·大量解 析を容易にせしめ、ひいては飛躍的な研究の進歩を誘発し、画期的な組換え作物の 開発につながる。 [0017] The simple method of the present invention facilitates large-scale processing and large-scale analysis, which are important in development research in this field, and thus induces dramatic progress in research and development of innovative recombinant crops. Leads to.
図面の簡単な説明  Brief Description of Drawings
[0018] [図 1]図 1は、 T— DNA上に有するバイナリーベクター pCAMBIA1390— sGFPを 模式的に示した図である。 [図 2A]図 2Aは、イネについて、ァグロバタテリゥムによる感染処理後 6日目の種子を 白色光で観察した結果を示す。左は、減圧処理、ァグロパクテリゥム感染、およびェ レクト口ポーレーシヨンを行った種子であり、右は、減圧処理のみを行った種子である 圆 2B]図 2Bは、図 2Aで観察した種子を、励起光を照射して観察した結果を示す。 G FPを発現する細胞が、緑色に明るく光っている。 [0018] FIG. 1 is a diagram schematically showing a binary vector pCAMBIA1390-sGFP on T-DNA. [FIG. 2A] FIG. 2A shows the result of observation of seeds with white light on the 6th day after the treatment with agrobacterium. The left is a seed that has been subjected to decompression, agrobacterium infection, and electoporation, and the right is a seed that has been subjected to decompression only. 2B] Figure 2B is the seed observed in Figure 2A Shows the result of observation by irradiating excitation light. Cells expressing GFP are brightly shining green.
[図 3A]図 3Aは、イネについて、ァグロバタテリゥムによる感染処理後 8日目の種子を 白色光で観察した結果を示す。左は、減圧処理、ァグロパクテリゥム感染、およびェ レクト口ポーレーシヨンを行った種子であり、右は、減圧処理のみを行った種子である 圆 3B]図 3Bは、図 3Aで観察した種子を、励起光を照射して観察した結果を示す。 G FPを発現する細胞が、緑色に明るく光っている。  [FIG. 3A] FIG. 3A shows the result of white seed observation of rice seeds on the 8th day after the treatment with agrobacterium. The left is a seed that has been subjected to decompression, agrobacterium infection, and electoporation, and the right is a seed that has undergone only a decompression treatment. 3B] Figure 3B is the seed observed in Figure 3A. Shows the result of observation by irradiating excitation light. Cells expressing GFP are brightly shining green.
[図 4A]図 4Aは、イネについて、ァグロバタテリゥムによる感染処理後 14日目の種子 を白色光で観察した結果を示す。左は、減圧処理、ァグロパクテリゥム感染、および エレクト口ポーレーシヨンを行った種子であり、右は、エレクト口ポーレーシヨンを行わ ずに、減圧処理後、菌液に浸したのみの種子である。  [FIG. 4A] FIG. 4A shows the results of observation of white seeds on the 14th day of rice after infection with agrobacterium. The left is a seed that has been subjected to decompression treatment, agrobacterium infection, and electo-poration, and the right is a seed that has been soaked in a bacterial solution after depressurization without erect-mouth poration.
圆 4B]図 4Bは、図 4Aで観察した種子を、励起光を照射して観察した結果を示す。 G FPを発現する細胞が、緑色に明るく光っている。 [4B] FIG. 4B shows the results of observing the seeds observed in FIG. 4A by irradiating them with excitation light. Cells expressing GFP are brightly shining green.
[図 5A]図 5Aは、コムギについて、ァグロバタテリゥムによる感染処理後 8日目の種子 を白色光で観察した結果を示す。左は、減圧処理、ァグロパクテリゥム感染、および エレクト口ポーレーシヨンを行った種子であり、中央は、減圧処理のみを行った種子で あり、右は、エレクト口ポーレーシヨンを行わずに、減圧処理後、菌液に浸したのみの 種子である。  [FIG. 5A] FIG. 5A shows the results of observing white seeds of wheat on the 8th day after infection treatment with agrobacterium. The left is a seed that has undergone decompression, agrobacterium infection, and electo-poration, the center is a seed that has undergone depressurization only, and the right is a seed that has undergone de-electoration without decompression. Later, the seeds were just immersed in the fungus solution.
圆 5B]図 5Bは、図 5Aで観察した種子を、励起光を照射して観察した結果を示す。 G FPを発現する細胞が、緑色に明るく光っている。 [5B] FIG. 5B shows the results of observing the seeds observed in FIG. 5A by irradiating them with excitation light. Cells expressing GFP are brightly shining green.
[図 6A]図 6Aは、インディ力イネについて、ァグロバタテリゥムによる感染処理後 7日目 の種子を白色光で観察した結果を示す。左は、減圧処理、ァグロパクテリゥム感染、 およびエレクト口ポーレーシヨンを行った種子であり、右は、エレクト口ポーレーシヨン を行わずに、減圧処理後、菌液に浸したのみの種子である。 [FIG. 6A] FIG. 6A shows the results of observing seeds of white rice in 7 days after infection treatment with agrobataterium for indy power rice. On the left are the seeds that have undergone decompression treatment, agrobacterium infection, and electo-poration, and on the right is the electo-poration. Without seeding, the seeds were only immersed in the bacterial solution after the reduced pressure treatment.
圆 6B]図 6Bは、図 6Aで観察した種子を、励起光を照射して観察した結果を示す。 G FPを発現する細胞が、緑色に明るく光っている。 [6B] FIG. 6B shows the results of observing the seeds observed in FIG. 6A by irradiating them with excitation light. Cells expressing GFP are brightly shining green.
[図 7A]図 7Aは、ノ、クサイについて、ァグロバタテリゥムによる感染処理後 4日目の種 子を白色光で観察した結果を示す。左は、減圧処理、ァグロパクテリゥム感染、およ びエレクト口ポーレーシヨンを行った種子であり、右は、エレクト口ポーレーシヨンを行 わずに、減圧処理後、菌液に浸したのみの種子である。  [FIG. 7A] FIG. 7A shows the results of observation of white and white Species seeds on day 4 after infection with agrobacterium. The left is a seed that has been subjected to decompression, agro-acterium infection, and electo-poration, and the right is a seed that has been soaked in a bacterial solution after depressurization without erect-poration. It is.
圆 7B]図 7Bは、図 7Aで観察した種子を、励起光を照射して観察した結果を示す。 G FPを発現する細胞が、緑色に明るく光っている。 [7B] FIG. 7B shows the results of observing the seeds observed in FIG. 7A by irradiating them with excitation light. Cells expressing GFP are brightly shining green.
[図 8A]図 8Aは、ダイズについて、ァグロバタテリゥムによる感染処理後 4日目の種子 を白色光で観察した結果を示す。左は、減圧処理、ァグロパクテリゥム感染、および エレクト口ポーレーシヨンを行った種子であり、右は、エレクト口ポーレーシヨンを行わ ずに、減圧処理後、菌液に浸したのみの種子である。  [FIG. 8A] FIG. 8A shows the results of observation of seeds with white light on the 4th day after the infection treatment with agrobacterium. The left is a seed that has been subjected to decompression treatment, agrobacterium infection, and electo-poration, and the right is a seed that has been soaked in a bacterial solution after depressurization without erect-mouth poration.
圆 8B]図 8Bは、図 8Aで観察した種子を、励起光を照射して観察した結果を示す。 G FPを発現する細胞が、緑色に明るく光っている。 [8B] FIG. 8B shows the results of observing the seeds observed in FIG. 8A by irradiating them with excitation light. Cells expressing GFP are brightly shining green.
[図 9A]図 9Aは、トマトについて、ァグロバタテリゥムによる感染処理後 4日目の種子を 白色光で観察した結果を示す。左は、減圧処理、ァグロパクテリゥム感染、およびェ レクト口ポーレーシヨンを行った種子であり、右は、エレクト口ポーレーシヨンを行わず に、減圧処理後、菌液に浸したのみの種子である。  [FIG. 9A] FIG. 9A shows the results of observing seeds of tomatoes with white light on the fourth day after infection treatment with agrobacterium. The left is a seed that has been subjected to decompression, agrobacterium infection, and electoral poration, and the right is a seed that has been soaked in a bacterial solution after depressurizing without performing electoral poration. .
圆 9B]図 9Bは、図 9Aで観察した種子を、励起光を照射して観察した結果を示す。 G FPを発現する細胞が、緑色に明るく光っている。 [9B] FIG. 9B shows the results of observing the seeds observed in FIG. 9A by irradiating them with excitation light. Cells expressing GFP are brightly shining green.
[図 10A]図 10Aは、アサガオについて、ァグロバタテリゥムによる感染処理後 4日目の 種子を白色光で観察した結果を示す。左は、減圧処理、ァグロパクテリゥム感染、お よびエレクト口ポーレーシヨンを行った種子であり、右は、エレクト口ポーレーシヨンを 行わずに、減圧処理後、菌液に浸したのみの種子である。  [FIG. 10A] FIG. 10A shows the results of observation of seeds of morning glory with white light on the fourth day after infection treatment with agrobata trumum. The left is a seed that has been subjected to decompression, agro-acterium infection, and electo-poration, and the right is a seed that has been soaked in a bacterial solution after depressurization without e-poor poration. .
圆 10B]図 10Bは、図 10Aで観察した種子を、励起光を照射して観察した結果を示 す。 GFPを発現する細胞が、緑色に明るく光っている。 [10B] FIG. 10B shows the results of observing the seeds observed in FIG. 10A by irradiating them with excitation light. Cells expressing GFP glow brightly in green.
[図 11]図 11は、六角形の横断面を有するエレクト口ポーレーシヨンチャンバ一の斜視 図である。 [Fig. 11] Fig. 11 is a perspective view of an erect-portion chamber having a hexagonal cross section. FIG.
[図 12]図 12は、図 11に示したエレクト口ポーレーシヨンチャンバ一の X線に沿った断 面図である。  [FIG. 12] FIG. 12 is a cross-sectional view of the electrification chamber shown in FIG. 11 along the X-ray.
[図 13]図 13は、試験管に挿入するタイプのエレクト口ポーレーシヨン電極の斜視図で ある。  [FIG. 13] FIG. 13 is a perspective view of an erect-portion polarization electrode of a type to be inserted into a test tube.
[図 14]図 14は、図 13に示したエレクト口ポーレーシヨン電極を、試験管に挿入した様 子を示す図である。  FIG. 14 is a view showing a state in which the electrification polarization electrode shown in FIG. 13 is inserted into a test tube.
配列表フリーテキスト  Sequence listing free text
[0019] 配列番号 1 :NPT II遺伝子を検出するための正方向プライマー。 [0019] SEQ ID NO: 1: A forward primer for detecting the NPT II gene.
配列番号 2： NPT II遺伝子を検出するための逆方向プライマー。  SEQ ID NO: 2: Reverse primer for detecting the NPT II gene.
配列番号 3 :NPT II遺伝子を検出するための正方向プライマー。  SEQ ID NO: 3: Forward primer for detecting NPT II gene.
配列番号 4： NPT II遺伝子を検出するための逆方向プライマー。  SEQ ID NO: 4: Reverse primer for detecting the NPT II gene.
発明を実施するための最良の形態  BEST MODE FOR CARRYING OUT THE INVENTION
[0020] 以下、本発明を説明する。本明細書の全体にわたり、単数形の表現は、特に言及 しない限り、その複数形の概念をも含むことが理解されるべきである。また、本明細書 において使用される用語は、特に言及しない限り、当該分野で通常用いられる意味 で用いられることが理解されるべきである。 [0020] Hereinafter, the present invention will be described. Throughout this specification, it should be understood that expression in the singular also includes the concept of the plural unless specifically stated otherwise. In addition, it is to be understood that the terms used in this specification are used in the meaning normally used in the art unless otherwise specified.
[0021] 以下に本明細書において特に使用される用語の定義を列挙する。 [0021] The definitions of terms particularly used in the present specification are listed below.
[0022] 本明細書において、用語「核酸導入」とは、人為的に核酸を細胞内または組織内に 導入することを意味する。「核酸導入」によって核酸を導入した細胞または組織の表 現型は、変化しても変化しなくても良い。本明細書において、用語「遺伝子導入」とは 、遺伝形質を規定する因子である遺伝子を含む核酸を、人為的に細胞内または組織 内に導入することを意味する。「遺伝子導入」によって遺伝子を含む核酸を導入した 細胞または組織の表現型は、変化しても変化しなくても良い。本明細書において、用 語「形質転換」とは、細胞内または組織内に、遺伝子を含む核酸を導入することによ つて、その細胞またはその組織の表現型に変化を生じることを意味する。ただし本明 細書では、用語「核酸導入」と「遺伝子導入」と「形質転換」とが互換可能に使用され る場合がある。本明細書中において、これらの用語が指す意味は、その用語が含ま れる文脈から明らかである。 In the present specification, the term “nucleic acid introduction” means that a nucleic acid is artificially introduced into a cell or tissue. The expression type of a cell or tissue into which nucleic acid has been introduced by “nucleic acid introduction” may or may not change. In this specification, the term “gene transfer” means that a nucleic acid containing a gene, which is a factor defining a genetic trait, is artificially introduced into a cell or tissue. The phenotype of a cell or tissue into which a nucleic acid containing a gene has been introduced by “gene transfer” may or may not change. In this specification, the term “transformation” means that a phenotype of a cell or tissue is changed by introducing a nucleic acid containing a gene into the cell or tissue. However, in this specification, the terms “nucleic acid introduction”, “gene introduction”, and “transformation” may be used interchangeably. In this specification, the meaning of these terms includes the terms. It is clear from the context.
[0023] 用語「核酸導入体」、「遺伝子導入体」および「形質転換体」とは、それぞれ、核酸 導入、遺伝子導入、および形質転換された細胞または組織から発生する生命体の全 部または一部をいう。ただし本明細書では、用語「核酸導入体」と「遺伝子導入体」と「 形質転換体」とが互換可能に使用される場合がある。本明細書中において、これらの 用語が指す意味は、その用語が含まれる文脈力 明らかである。核酸導入体、遺伝 子導入体、および形質転換体は、任意の生命体であり得、例えば、原核細胞、およ び真核細胞 (植物細胞等を含む)または組織から発生する生命体が例示される。形 質転換体は、その対象に依存して、形質転換細胞、形質転換組織、形質転換宿主 などともいわれ、本明細書においてそれらの形態をすベて包含するが、特定の文脈 において特定の形態を指し得る。同様のことが、核酸導入体および遺伝子導入体に おいても当てはまる。  [0023] The terms "nucleic acid transductant", "gene transfectant" and "transformant" refer to all or one part of a living organism generated from a nucleic acid transduced, gene transduced, and transformed cell or tissue, respectively. Part. However, in the present specification, the terms “nucleic acid introduced body”, “gene introduced body”, and “transformant” may be used interchangeably. In the present specification, the meaning of these terms is obvious from the contextual power in which the terms are included. Nucleic acid transductants, gene transfectants, and transformants can be any organism, for example, prokaryotic cells and eukaryotic cells (including plant cells, etc.) or living organisms originating from tissues. Is done. Transformants are also referred to as transformed cells, transformed tissues, transformed hosts, etc., depending on the subject, and include all of these forms in this specification, but in certain contexts a particular form Can point to. The same applies to nucleic acid transducers and gene transfectants.
[0024] 用語「細胞」は、どの生物由来の細胞 (たとえば、任意の種類の多細胞生物（例え ば、動物 (たとえば、脊椎動物、無脊椎動物）、植物 (たとえば、単子葉植物、双子葉 植物など）、真菌類など）、単細胞生物 (例えば、細菌 (例えば、大腸菌)など）由来の 細胞)でもよい。好ましくは、本発明において使用される細胞は、細胞壁を有する細 胞であり、特に好ましくは、植物細胞である。  [0024] The term "cell" refers to a cell from any organism (eg, any type of multicellular organism (eg, animal (eg, vertebrate, invertebrate), plant (eg, monocotyledonous, dicotyledonous). Plant, etc.), fungi, etc.) or unicellular organisms (eg, cells derived from bacteria (eg, E. coli), etc.). Preferably, the cell used in the present invention is a cell having a cell wall, particularly preferably a plant cell.
[0025] 本明細書において「組織」（tissue)とは、多細胞生物において、実質的に同一の 機能および Zまたは形態をもつ細胞集団をいう。通常「組織」は、同じ起源を有する 力 異なる起源を持つ細胞集団であっても、同一の機能および Zまたは形態を有す るのであれば、組織と呼ばれ得る。通常、組織は、器官の一部を構成する。植物では 、構成細胞の発達段階によって***組織と永久組織とに大別され、また構成細胞の 種類によって単一組織と複合組織とに分けるなど、 、ろ 、ろな分類が行われて 、る。 動物の組織は，形態的、機能的または発生的根拠に基づき、上皮組織、結合組織、 筋肉組織、神経組織などに区別される。  [0025] As used herein, "tissue" refers to a cell population having substantially the same function and Z or morphology in a multicellular organism. Usually a “tissue” can be referred to as a tissue even if it is a population of cells with the same origin, even if they have the same function and Z or morphology. Usually, tissue constitutes part of an organ. Plants are roughly classified into meristems and permanent tissues according to the stage of development of the constituent cells, and divided into single tissues and composite tissues according to the type of constituent cells. Animal tissues are classified into epithelial tissue, connective tissue, muscle tissue, nerve tissue, etc. based on morphological, functional or developmental basis.
[0026] 本明細書中において、用語「組織」は、どの生物由来のどの組織 (たとえば、任意の 種類の多細胞生物 (例えば、動物 (たとえば、脊椎動物、無脊椎動物）、植物 (たとえ ば、単子葉植物、双子葉植物など)、真菌類など）由来の組織)でもよい。好ましくは、 本発明において使用される組織は、細胞壁を含む組織であり、特に好ましくは、植物 組織である。「植物組織」としては、休眠組織、生殖質、生長点、および花芽が挙げら れるが、これに限定されない。好ましい休眠組織としては、完熟種子、未熟種子、冬 芽、および塊茎が挙げられ、特に好ましくは完熟種子である力 これに限定されない [0026] As used herein, the term "tissue" refers to any tissue from any organism (eg, any type of multicellular organism (eg, animal (eg, vertebrate, invertebrate), plant (eg, Or monocotyledonous plants, dicotyledonous plants, etc.), fungi, etc.)). Preferably, The tissue used in the present invention is a tissue containing a cell wall, and particularly preferably a plant tissue. “Plant tissue” includes, but is not limited to, dormant tissue, germplasm, growth points, and flower buds. Preferred dormant tissues include ripe seeds, immature seeds, winter buds, and tubers, particularly preferably the ability to be a ripe seed.
[0027] 用語「器官」は、生物個体のある機能が個体内の特定の部分に局在して営まれ，か つその部分が形態的に独立性をもっている構造体をいう。一般に多細胞生物 (例え ば、動物、植物、真菌類)では器官は特定の空間的配置をもついくつ力の組織から なり、組織は多数の細胞力もなる。そのような器官としては、植物の場合には、根、葉 、茎、および花などが挙げられ、動物の場合には、皮膚、心臓、血管、角膜、網膜、 腎臓、肝臓、脾臓、腸、胎盤、臍帯、肺、脳、神経、四肢末梢などが挙げられるが、そ れらに限定されない。 [0027] The term "organ" refers to a structure in which a function of an individual organism is localized and operates in a specific part of the individual, and that part is morphologically independent. In general, in multicellular organisms (eg animals, plants, fungi), an organ consists of several forces with a specific spatial arrangement, and a tissue also has many cellular forces. Such organs include roots, leaves, stems, and flowers in the case of plants, and skin, heart, blood vessels, cornea, retina, kidneys, liver, spleen, intestines, in the case of animals. Examples include, but are not limited to, placenta, umbilical cord, lungs, brain, nerves, and extremities.
[0028] 本願明細書において使用する場合、用語「選抜」とは、抗生物質に対する耐性検 定および Zまたは遺伝子工学的手法 (例えば、 PCR、サザンブロット法、ノーザンブ ロット法など）によって、核酸導入された核酸導入体を、核酸導入されていないものと 区別することを意味する。特定の場合では、用語「選抜」は、形質転鎌物を薬物存 在下で培養および Zまたは育成することによって、薬物耐性遺伝子によって形質転 換された形質転換体を、形質転換されて!/ヽな!ヽ植物とを区別する工程を意味する。  [0028] As used herein, the term "selection" refers to introduction of nucleic acid by antibiotic resistance testing and Z or genetic engineering techniques (eg, PCR, Southern blotting, Northern blotting, etc.). This means that the introduced nucleic acid is distinguished from the non-nucleic acid-introduced one. In certain cases, the term `` selection '' means that transformants transformed with a drug resistance gene have been transformed by culturing and Z or growing the transformed sickle in the presence of the drug! / ヽIt means the process of distinguishing ヽ plants.
[0029] 単子葉植物の形質転換に用いられるァグロバタテリゥムは、任意のァグロバクテリウ ム属細菌であり得、好ましくは Agrobacterium tumefaciensである。ァグロバタテリ ゥムは、所望の組換え遺伝子を含む植物発現用ベクターで (例えば、エレクトロボレ ーシヨンによって)形質転換される。形質転換されたァグロパクテリゥムで種子を感染 することにより、所望の組換え遺伝子を植物に導入し得る。導入された組換え遺伝子 は、植物中のゲノムに組み込まれて存在する。なお、植物中のゲノムとは、核染色体 のみならず、植物細胞中の各種オルガネラ（例えば、ミトコンドリア、葉緑体など）に含 まれるゲノムを含んで 、う。  [0029] The agrobacterium used for transformation of monocotyledonous plants can be any bacterium belonging to the genus Agrobacterium, preferably Agrobacterium tumefaciens. Agrobataterium is transformed with a plant expression vector containing the desired recombinant gene (eg, by electrovolution). A desired recombinant gene can be introduced into a plant by infecting seeds with the transformed agrobacterium. The introduced recombinant gene is integrated in the genome of the plant. The genome in plants includes not only nuclear chromosomes but also genomes contained in various organelles (eg, mitochondria, chloroplasts, etc.) in plant cells.
[0030] 単子葉植物に所望の組換え遺伝子を導入するために、所望の組換え遺伝子を含 む適切な植物発現用ベクターが構築される。このような植物発現用ベクターは、当業 者に周知の遺伝子組換え技術を用いて作製され得る。ァグロパクテリゥム形質転換 法において使用するための植物発現用ベクターの構築には、例えば、 pBI系または p PZP系のベクターが好適に用いられる力 これらに限定されない。 [0030] In order to introduce a desired recombinant gene into a monocotyledonous plant, an appropriate plant expression vector containing the desired recombinant gene is constructed. Such plant expression vectors are known in the art. It can be produced using genetic recombination techniques well known to those skilled in the art. The construction of a plant expression vector for use in the agrobacterium transformation method is not limited to, for example, the ability to suitably use a pBI or pPZP vector.
[0031] 本明細書において使用する場合、「成長調節因子」とは、多細胞の成長に影響を 与える因子であって、多細胞生物（例えば、植物）の器官や細胞で合成され、体液に よって他の器官や部分に運ばれる化学物質またはその誘導体であって、 1つまたは 数多くの器官の機能、活性および Zまたは構造を変える作用を有する物質をいう。 植物細胞に対する成長調節因子を、植物成長調節因子という。植物成長調節因子と しては、植物ホルモンが挙げられる力 これに限定されない。植物ホルモンとしては、 オーキシン、ジベレリン、サイトカイニン、アブシジン酸、およびエチレンが挙げられる 力 これらに限定されない。オーキシンとしては、 2, 4 0ぉょび1八八(ィンドール酢 酸）、 NAA (ナフタリン酢酸）、および IBA (インドール酪酸）が挙げられるが、これに 限定されない。 [0031] As used herein, a "growth regulator" is a factor that affects multicellular growth, and is synthesized in organs and cells of a multicellular organism (for example, a plant) and into body fluids. Thus, a chemical substance or derivative thereof that is transported to other organs or parts and that has the effect of altering the function, activity and Z or structure of one or many organs. Growth regulators for plant cells are referred to as plant growth regulators. Plant growth regulators include, but are not limited to, plant hormones. Plant hormones include, but are not limited to, auxin, gibberellin, cytokinin, abscisic acid, and ethylene. Auxins include, but are not limited to, 2,400 juice (indole acetic acid), NAA (naphthalene acetic acid), and IBA (indole butyric acid).
[0032] 本明細書において使用する場合、用語「エレクト口ポーレーシヨン」とは、直流の高 電圧パルスを用いて物理的に細胞 (例えば、植物細胞）に小孔をあけ、そこ力 核酸 (例えば、遺伝子を含む核酸)を細胞内に導入する方法をいう。エレクト口ポーレーシ ヨンの条件は、使用する種、組織、細胞などに依存して、当業者が適宜選択し得る。 代表的なエレクト口ポーレーシヨンの電圧の条件は、 10VZcm〜200VZcm、好ま しくは 20VZcm〜150VZcm、より好ましくは 30VZcm〜120VZcm、なおより好 ましくは 40V/cm〜 1 OOVZcm、最も好ましくは 50V/cm〜 1 OOV/cmであるが 、これらに限定されない。代表的なエレクト口ポーレーシヨンのパルス幅の条件は、 1 マイクロ秒〜 90ミリ秒、好ましくは 10ミリ秒〜 90ミリ秒、より好ましくは 10ミリ秒〜 90ミリ 秒、なお好ましくは 20ミリ秒〜 80ミリ秒、なおより好ましくは 30ミリ秒〜 80ミリ秒、さらに なおより好ましくは 40ミリ秒〜 70ミリ秒、最も好ましくは 50ミリ秒〜 60ミリ秒であるが、 これらに限定されない。また、エレクト口ポーレーシヨンのパルス幅は、 1ミリ秒未満で あってもよぐ例えば、 10マイクロ秒〜 90マイクロ秒、 20マイクロ秒〜 80マイクロ秒、 3 0マイクロ秒、〜 80マイクロ秒、、 40マイクロ秒、〜 70マイクロ秒、、 50マイクロ秒、〜 60マイク 口秒であるが、これらに限定されない。代表的なエレクト口ポーレーシヨンのパルスの 回数 ίま、 1回〜 200回、好ましく ίま 10回〜 150回、より好ましく ίま 20回〜 120回、な おより好ましくは 30回〜 110回、最も好ましくは 40回〜 100回である力 これらに限 定されない。 [0032] As used herein, the term "elect mouth polarization" is used to physically puncture a cell (eg, a plant cell) using a direct-current high-voltage pulse, and force the nucleic acid (eg, A nucleic acid containing a gene) is introduced into cells. The conditions of the electopore polarization can be appropriately selected by those skilled in the art depending on the species, tissues, cells, etc. used. The voltage conditions of a typical electrification pole position are 10VZcm to 200VZcm, preferably 20VZcm to 150VZcm, more preferably 30VZcm to 120VZcm, even more preferably 40V / cm to 1 OOVZcm, most preferably 50V / cm to 1 OOV / cm, but not limited to. The pulse width condition of a typical electopore polarization is 1 microsecond to 90 milliseconds, preferably 10 milliseconds to 90 milliseconds, more preferably 10 milliseconds to 90 milliseconds, still more preferably 20 milliseconds to 80 milliseconds. Milliseconds, even more preferably 30 milliseconds to 80 milliseconds, still more preferably 40 milliseconds to 70 milliseconds, and most preferably 50 milliseconds to 60 milliseconds, but is not limited thereto. Further, the pulse width of the electo-portion may be less than 1 millisecond, for example, 10 microseconds to 90 microseconds, 20 microseconds to 80 microseconds, 30 microseconds, to 80 microseconds, 40 Microseconds, 70 microseconds, 50 microseconds, 60 microseconds, but not limited to these. Of the pulse of a typical electo porch Number of times ί, 1 to 200 times, preferably ί 10 to 150 times, more preferably ί 20 to 120 times, more preferably 30 to 110 times, most preferably 40 to 100 times Power is not limited to these.
[0033] 本明細書において使用する場合、句「細胞 (または組織）と核酸とを、エレクト口ポー レーシヨンが起きる条件下に配置する」とは、細胞 (または組織)と核酸とを、それらの 間でエレクト口ポーレーシヨンが起きる（すなわち、細胞 (または組織)への核酸導入 が起こる）ために必須のすべての条件 (電圧条件、パルス幅条件、パルス回数条件、 細胞 (または組織）と核酸との間の位置関係、エレクト口ポーレーシヨンの実行時間な どを含む）を備えた状態に配置することを意味する。エレクト口ポーレーシヨンが起きる ために必須の条件は、当業者に容易に明らかであり、当業者はその条件を適宜決定 し得る。  [0033] As used herein, the phrase "place cells (or tissues) and nucleic acids under conditions that cause electoporation occurs" means that cells (or tissues) and nucleic acids are placed between them. All conditions (voltage condition, pulse width condition, pulse frequency condition, cell (or tissue) and nucleic acid between the cells (or tissue) that are essential for electoral polarization to occur between the cells (or tissue). (Including the positional relationship between them and the execution time of the electo-portion). The conditions essential for the occurrence of the electrification are readily apparent to those skilled in the art, and those skilled in the art can appropriately determine the conditions.
[0034] さらに、本発明に従ってエレクト口ポーレーシヨンを行う際に、細胞 (または組織）と 核酸とに、少なくとも二種類の方向で電圧パルスをかけることが好ましい。最も簡単に は、細胞 (または組織）と核酸とに一定時間電圧パルスをかけた後で、その電圧ノ ル ス処理に使用した電極のアノードと力ソードとを逆にして電圧パルスをかけ直すことに よって、これは達成され得る。さらにこれは、エレクト口ポーレーシヨンチャンバ一内で 別の位置に配置された電極対を使用しても達成され得る。このようにして少なくとも二 種類の方向で電圧パルスをかけることによって、核酸導入効率が顕著に高まる。  [0034] Furthermore, it is preferable to apply voltage pulses to cells (or tissues) and nucleic acids in at least two directions when performing electophoresis according to the present invention. The simplest is to apply a voltage pulse to a cell (or tissue) and a nucleic acid for a certain period of time, and then reapply the voltage pulse by reversing the anode and force sword of the electrode used to process the voltage. This can thus be achieved. This can also be accomplished using electrode pairs located at different locations within the electopore polarization chamber. By applying voltage pulses in at least two directions in this way, the nucleic acid introduction efficiency is significantly increased.
[0035] 本発明においてエレクト口ポーレーシヨンを行う際に使用されるエレクト口ポーレー シヨンチャンバ一は、核酸導入の対象となる細胞および ζまたは組織を収容できるも のであれば、どのような大きさであってもよい。特に好ましくは、エレクト口ポーレーショ ンチャンバ一は、植物組織 (例えば、植物種子）を収容し得る大きさを有する。本発明 のエレクト口ポーレーシヨンチャンバ一は、任意の形状であり得る。この形状としては、 立方体、直方体、円筒形、チューブ形 (例えば、胴体が均一である力または均一でな [0035] The size of the electopore chamber used when performing the electoporation in the present invention can be any size as long as it can accommodate cells and ζ or tissue to which nucleic acid is introduced. May be. Particularly preferably, the electoporation chamber has a size that can accommodate plant tissue (eg, plant seeds). The elect port polarization chamber of the present invention can be of any shape. This shape can be a cube, cuboid, cylinder, tube (e.g., a force that is uniform on the body or a uniform
V、横断面を有し、かつ底が先細りしてもしなて 、なくてもよ!、形状)などが挙げられるV, having a cross-section and having a tapered bottom or not!
1S これらに限定されない。植物種子を収容し得る大きさであるために、本発明のェ レクト口ポーレーシヨンチャンバ一の内面の少なくとも 3点に接する最大の内接円の直 径は、例えば、以下の長さであり得る：少なくとも約 5mmである力または約 5mmよりも 長い、好ましくは、少なくとも約 6mmである力または約 6mmよりも長い、好ましくは、 少なくとも約 7mmであるかまたは約 7mmよりも長い、好ましくは、少なくとも約 8mm であるかまたは約 8mmよりも長い、好ましくは、少なくとも約 9mmである力または約 9 mmよりも長い、好ましくは、少なくとも約 lcmである力または約 lcmよりも長い、好ま しくは、少なくとも約 2cmである力または約 2cmよりも長い、好ましくは、少なくとも約 3 cmである力または約 3cmよりも長い、好ましくは、少なくとも約 4cmである力または約 4cmよりも長い、好ましくは、少なくとも約 5cmであるかまたは約 5cmよりも長い、好ま しくは、少なくとも約 6cmである力または約 6cmよりも長い、好ましくは、少なくとも約 7 cmである力または約 7cmよりも長い、好ましくは、少なくとも約 8cmである力または約 8cmよりも長い、好ましくは、少なくとも約 9cmであるかまたは約 9cmよりも長い、好ま しくは、少なくとも約 10cmである力または約 10cmよりも長い、好ましくは、少なくとも 約 15cmである力または約 15cmよりも長い、そして好ましくは、少なくとも約 20cmで あるかまたは約 20cmよりも長い。本発明のエレクト口ポーレーシヨンチャンバ一の内 面の少なくとも 3点に接する最大の内接円の直径の上限は、例えば、以下であり得る 1S これらに限定されない：約 25cm、約 20cm、約 15cm、約 10cm、約 9cm、約 8c m、約 7cm、約 6cm、約 5cm、約 4cm、約 3cm、約 2cm、約 lcm、約 9mm、約 8mm 、約 7mm、または約 6mm。当然ながら、この長さは、上記に明示した数値の間の長 さ（例えば、 1. 5cmなど)であり得る。本明細書中において「内接円」とは、チャンバ 一容器の内面上における少なくとも 3つの任意の点に接するように描かれる任意の円 を指す。ここで、チャンバ一内に配置された電極も容器の一部とみなされ、従って、チ ヤンバー容器の内面は、電極表面も含む。ただし、代表的に、使用される電極の厚 みは、無視できるほど非常に薄い（例えば、 0. 1mmなど)。 1S It is not limited to these. Since the size can accommodate plant seeds, the maximum diameter of the inscribed circle that contacts at least three points on the inner surface of the electrification chamber of the present invention can be, for example, the following length: : A force that is at least about 5mm or more than about 5mm Long, preferably at least about 6 mm or longer than about 6 mm, preferably at least about 7 mm or longer than about 7 mm, preferably at least about 8 mm or longer than about 8 mm, Preferably, the force is at least about 9 mm or longer than about 9 mm, preferably at least about 1 cm or longer than about 1 cm, preferably at least about 2 cm or longer than about 2 cm. Preferably, the force is at least about 3 cm or longer than about 3 cm, preferably at least about 4 cm or longer than about 4 cm, preferably at least about 5 cm or longer than about 5 cm. Or a force that is at least about 6 cm or longer than about 6 cm, preferably a force that is at least about 7 cm or longer than about 7 cm, preferably at least about 8 a force that is cm or longer than about 8 cm, preferably at least about 9 cm or longer than about 9 cm, preferably at least about 10 cm, or longer than about 10 cm, preferably at least about 15 cm Or a force greater than about 15 cm, and preferably at least about 20 cm or greater than about 20 cm. The upper limit of the diameter of the maximum inscribed circle that contacts at least three points of the inner surface of the electrification chamber of the present invention may be, for example, 1S, but is not limited to: about 25 cm, about 20 cm, about 15 cm, About 10 cm, about 9 cm, about 8 cm, about 7 cm, about 6 cm, about 5 cm, about 4 cm, about 3 cm, about 2 cm, about 1 cm, about 9 mm, about 8 mm, about 7 mm, or about 6 mm. Of course, this length can be a length between the values specified above (eg, 1.5 cm, etc.). In the present specification, the “inscribed circle” refers to an arbitrary circle drawn so as to touch at least three arbitrary points on the inner surface of the chamber. Here, the electrode disposed in the chamber is also regarded as a part of the container, and therefore, the inner surface of the chamber container also includes the electrode surface. Typically, however, the thickness of the electrode used is negligibly thin (eg, 0.1 mm).
[0036] 一つの局面において、本発明のエレクト口ポーレーシヨンチャンバ一は、四角形の 横断面を有し、かつ内寸 (例えば、縦 X横 X高さ）が、 1cm X 2cm X 2cmである。別 の局面では、本発明のエレクト口ポーレーシヨンチャンバ一は、円形の横断面を有し、 かつ内寸（例えば、直径 X高さ）が、 1cm X 4cmである。  [0036] In one aspect, the electrification chamber of the present invention has a rectangular cross section and has an internal dimension (for example, vertical X horizontal X height) of 1 cm X 2 cm X 2 cm. In another aspect, the electrification chamber of the present invention has a circular cross section and an internal dimension (eg, diameter X height) of 1 cm X 4 cm.
[0037] さらなる局面において、本発明のエレクト口ポーレーシヨンチャンバ一は、六角形の 横断面を有する。例えば、図 11および図 12に示すように、六角形の横断面を有する エレクト口ポーレーシヨンチャンバ一 1は、上面が開口する有底の筒体 18と、その筒 体 18の各内面に備えられた電極 11〜16とを有する。電極 11〜16には、エレクト口 ポーレーシヨンによる高電圧ノ ルスをかけるためのコード (示さず）が電気的に連結す る。筒体 18は、底部 17を有し、その内部に試料を導入して、エレクト口ポーレーシヨン を行う。好ましい実施形態において、このエレクト口ポーレーシヨンチャンバ一の電極 は、各々が lOmm X 15mmの大きさを有する力 これに限定されない。なお別の局 面において、本発明のエレクト口ポーレーシヨンチャンバ一は、 n角开 (n=整数）の横 断面を有する。ここで、電極の占める領域は、チャンバ一の内寸から除かれる。ただ し上記の通り、代表的に、使用される電極の厚みは、無視できるほど非常に薄い。本 明細書中において横断面とは、チャンバ一の長軸方向に直交する断面をいう。本明 細書中において内寸とは、チャンバ一容器内面上の任意の 2点を結ぶ長さを指し、 特に、四角形の横断面を有するチャンバ一の場合には、その横断面の縦および横、 ならびに高さを指し、そして円形の横断面を有するチャンバ一の場合には、その横断 面の直径、および高さを指す。 [0037] In a further aspect, the electrification chamber of the present invention has a hexagonal cross section. For example, as shown in FIGS. 11 and 12, it has a hexagonal cross section. The electrification polarization chamber 11 has a bottomed cylindrical body 18 whose upper surface is open, and electrodes 11 to 16 provided on each inner surface of the cylindrical body 18. The electrodes 11 to 16 are electrically connected to a cord (not shown) for applying a high-voltage noise by electo-portion polarization. The cylindrical body 18 has a bottom portion 17, and a sample is introduced into the cylindrical body 18 so as to perform electo-mouth polarization. In a preferred embodiment, the electrodes of the electo-portion chamber are not limited to forces each having a size of lOmm x 15 mm. In another aspect, the electrification chamber of the present invention has an n-square (n = integer) cross section. Here, the area occupied by the electrode is excluded from the internal dimensions of the chamber. However, as described above, typically, the thickness of the electrode used is negligibly thin. In this specification, the transverse section refers to a section perpendicular to the major axis direction of the chamber. In this description, the internal dimension refers to the length connecting any two points on the inner surface of the chamber. In particular, in the case of a chamber having a square cross section, the length and width of the cross section, As well as, in the case of a chamber having a circular cross section, the diameter and height of the cross section.
[0038] 一つの実施形態では、本発明のエレクト口ポーレーシヨンチャンバ一は、植物種子 を収容し得る大きさに変動可能である。大きさの変動は、任意の手段によって達成さ れ得る。例えば、ネジなどを利用して適切な大きさに調整され得る。  [0038] In one embodiment, the electrification chamber of the present invention can be varied to a size that can accommodate plant seeds. The size variation can be achieved by any means. For example, it can be adjusted to an appropriate size using a screw or the like.
[0039] 本発明のエレクト口ポーレーシヨンチャンバ一は、任意の材料から作製され得る。ェ レクト口ポーレーシヨンチャンバ一の材料としては、固体を形成し得る任意の材料が使 用され得る。例えば、ガラス、シリカ、シリコン、セラミック、二酸化珪素、プラスチック、 金属 (合金も含まれる）、天然および合成のポリマー（例えば、ポリスチレン、セルロー ス、キトサン、デキストラン、およびナイロン)などが挙げられるがそれらに限定されな い。チャンバ一は、複数の異なる材料の層力も形成されていてもよい。例えば、ガラス 、石英ガラス、アルミナ、サファイア、フォルステライト、酸化珪素、炭化珪素、窒化珪 素などの無機絶縁材料を使用することができる。ポリアミド、ポリカーボネート、（変性） ポリフエ-レンオキサイド、ポリブチレンテレフタレート、強化ポリエチレンテレフタレー ト、ポリエーテルスルホン、ポリフエ-レンスルフイド、ポリアリレート、ポリエーテルイミド 、ポリエーテルエーテルケトン、ポリイミド、ポリエステル、ポリエチレン、ポリプロピレン 、ポリイソブチレン、不飽和ポリエステル、含フッ素榭脂、ポリ塩化ビニル、ポリ塩化ビ -リデン、ポリ酢酸ビュル、ポリビュルアルコール、ポリビュルァセタール、アクリル榭 脂、ポリアクリロニトリル、ポリスチレン、ァセタール榭脂、フエノール榭脂、ユリア榭脂 、エポキシ榭脂、メラミン榭脂、スチレン'アクリロニトリル共重合体、アクリロニトリルブ タジエンスチレン共重合体、シリコーン榭脂、ポリスルホンなどの有機材料なども使用 され得る。好ましくは、本発明のエレクト口ポーレーシヨンチャンバ一は、大気圧と異な る圧力に耐える能力（例えば、大気圧と異なる圧力にさらされても、破壊、亀裂および 変形しない能力）を有し、特に好ましくは、減圧処理に耐える能力を有する。特に好 ましくは、本発明のエレクト口ポーレーシヨンチャンバ一は、ポリプロピレン、シリコーン 榭脂、およびガラスカゝら作製され、そして白金製またはステンレス製の電極を備える。 [0039] The electrification chamber of the present invention can be made of any material. Any material capable of forming a solid can be used as the material for the electoral polarization chamber. Examples include glass, silica, silicon, ceramic, silicon dioxide, plastics, metals (including alloys), natural and synthetic polymers (eg, polystyrene, cellulose, chitosan, dextran, and nylon). Not limited. The chamber 1 may also be formed with laminar forces of a plurality of different materials. For example, an inorganic insulating material such as glass, quartz glass, alumina, sapphire, forsterite, silicon oxide, silicon carbide, or silicon nitride can be used. Polyamide, Polycarbonate, (Modified) Polyethylene oxide, Polybutylene terephthalate, Reinforced polyethylene terephthalate, Polyethersulfone, Polyphenylene sulfide, Polyarylate, Polyetherimide, Polyetheretherketone, Polyimide, Polyester, Polyethylene, Polypropylene , Polyisobutylene, unsaturated polyester, fluorine-containing resin, polyvinyl chloride, polyvinylidene chloride, poly (vinyl acetate), poly (bull alcohol), poly (bullacetal), acrylic resin, polyacrylonitrile, polystyrene, acetal resin, phenol Organic materials such as resin, urea resin, epoxy resin, melamine resin, styrene 'acrylonitrile copolymer, acrylonitrile butadiene styrene copolymer, silicone resin, and polysulfone can also be used. Preferably, the electrification chamber of the present invention has the ability to withstand pressures different from atmospheric pressure (for example, the ability to not break, crack and deform when exposed to pressures different from atmospheric pressure), in particular Preferably, it has the ability to withstand decompression. Particularly preferably, the electrification chamber of the present invention is made of polypropylene, silicone resin, and glass cover, and comprises an electrode made of platinum or stainless steel.
[0040] 好ましくは、本発明のエレクト口ポーレーシヨンチャンバ一は、温度制御手段を備え る。温度制御手段は、例えば、センサーなどで温度変化を感知し、手動または自動 で、チャンバ一の温度を制御し得る。温度制御手段は、代表的に、チャンバ一の温 度を冷却するように作用する冷却手段である。冷却手段は任意の手段であり得、例 えば、氷、冷却ゲルなどを利用した手段であり得る。  [0040] Preferably, the electrification chamber of the present invention includes a temperature control means. The temperature control means senses a temperature change with a sensor or the like, and can control the temperature of the chamber manually or automatically. The temperature control means is typically a cooling means that acts to cool the temperature of the chamber. The cooling means can be any means, for example, means utilizing ice, cooling gel, or the like.
[0041] 本発明のエレクト口ポーレーシヨンチャンバ一には、少なくとも一対（二個）の電極が 備えられる。従って、特定の実施形態では、本発明のチャンバ一は、一対 (二個）より 多い電極 (例えば、二対（四個）の電極、三対 (六個）の電極、四対 (八個）の電極、五 対 (十個）の電極、またはより多い対の電極)を備え得る。例えば、四角形の横断面を 有するチャンバ一において、向かい合う内面に沿って電極を配置すると、二対（四個 )の電極を取り付けることが可能である。さらなる例として、六角形の横断面を有する チャンバ一において、向かい合う内面に沿って電極を配置すると、三対（六個）の電 極を取り付けることが可能である。このように、本発明のチャンバ一内に配置される電 極対は、任意の数であり得、そして任意の空間的位置関係をとり得る。一対 (二個)よ り多い電極を用いる場合、各対をなす電極に同時に電圧を印加しても、各対をなす 電極に順番に電圧を印加してもよ！ヽ。各対をなす電極に十番に電圧を印加する場合 、印加する電圧を順次切り替える装置は、市販されており。例えば、ネッパジーン株 式会社（日本国千葉県巿川巿)の CU901を用いることが可能である。例えば、一対（ 二個）より多い電極に順次電圧を印加する場合、電圧を切り替える間隔としては、 12 0秒から 1ミリ秒、好ましくは 60秒から 10ミリ秒、より好ましくは 30秒から 100ミリ秒、な お好ましくは 10秒から 500ミリ秒、なおより好ましくは 5秒から 750ミリ秒、さらになおよ り好ましくは 3秒から 800ミリ秒、最も好ましくは 1. 1秒から 900ミリ秒である力 これら に限定されない。 [0041] The electrification polarization chamber 1 of the present invention is provided with at least a pair (two) of electrodes. Thus, in certain embodiments, the chamber of the present invention has more than one (two) electrodes (eg, two (four) electrodes, three (six) electrodes, four (eight) electrodes Electrode, five pairs (ten electrodes), or more pairs of electrodes). For example, in a chamber having a rectangular cross section, two electrodes (four electrodes) can be attached by arranging electrodes along the inner surfaces facing each other. As a further example, in a chamber having a hexagonal cross section, it is possible to attach three pairs (six) of electrodes by placing electrodes along the opposing inner surfaces. Thus, the number of electrode pairs arranged in the chamber of the present invention can be any number and can take any spatial positional relationship. When using more than one (two) electrodes, you can apply a voltage to each pair of electrodes simultaneously, or you can apply a voltage to each pair of electrodes in turn!ヽ. A device that switches the applied voltage sequentially when applying a voltage to each pair of electrodes is commercially available. For example, it is possible to use CU901 of Neppagene Co., Ltd. (Kashikawa, Chiba, Japan). For example, a pair ( 2) When applying voltage sequentially to more electrodes, the voltage switching interval is 120 to 1 milliseconds, preferably 60 to 10 milliseconds, more preferably 30 to 100 milliseconds. Forces that are preferably 10 to 500 milliseconds, even more preferably 5 to 750 milliseconds, even more preferably 3 to 800 milliseconds, and most preferably 1.1 to 900 milliseconds. Not.
[0042] 本発明のエレクト口ポーレーシヨンチャンバ一内に配置される電極の間の距離は任 意の距離であり得、核酸導入の対象となる細胞および Zまたは組織の大きさに応じ て変動し得る。特に好ましくは、電極間の距離は、植物組織 (例えば、植物種子）を収 容し得る距離である。植物種子を収容し得る距離であるために、電極間の距離は、例 えば、以下の長さであり得る：少なくとも約 5mmであるかまたは約 5mmよりも長い、好 ましくは、少なくとも約 6mmである力または約 6mmよりも長い、好ましくは、少なくとも 約 7mmであるかまたは約 7mmよりも長い、好ましくは、少なくとも約 8mmであるかま たは約 8mmよりも長い、好ましくは、少なくとも約 9mmであるかまたは約 9mmよりも 長い、好ましくは、少なくとも約 lcmである力または約 lcmよりも長い、好ましくは、少 なくとも約 2cmである力または約 2cmよりも長い、好ましくは、少なくとも約 3cmである かまたは約 3cmよりも長い、好ましくは、少なくとも約 4cmである力または約 4cmよりも 長い、好ましくは、少なくとも約 5cmである力または約 5cmよりも長い、好ましくは、少 なくとも約 6cmである力または約 6cmよりも長い、好ましくは、少なくとも約 7cmである かまたは約 7cmよりも長い、好ましくは、少なくとも約 8cmである力または約 8cmよりも 長い、好ましくは、少なくとも約 9cmである力または約 9cmよりも長い、好ましくは、少 なくとも約 10cmであるかまたは約 10cmよりも長い、好ましくは、少なくとも約 15cmで ある力または約 15cmよりも長い、そして好ましくは、少なくとも約 20cmである力また は約 20cmよりも長い。電極間の距離の上限は、例えば、以下であり得るが、これらに 限定されない：約 25cm、約 20cm、約 15cm、約 10cm、約 9cm、約 8cm、約 7cm、 約 6cm、約 5cm、約 4cm、約 3cm、約 2cm、約 lcm、約 9mm、約 8mm、約 7mm、 または約 6mm。当然ながら、電極間の距離は、上記に明示した数値の間の長さ（例 えば、 1. 5cmなど）であり得る。  [0042] The distance between the electrodes disposed in the electopore chamber of the present invention can be any distance, and varies depending on the size of the cell and Z or tissue to which the nucleic acid is introduced. obtain. Particularly preferably, the distance between the electrodes is a distance that can accommodate plant tissue (eg, plant seeds). In order to be able to accommodate plant seeds, the distance between the electrodes can be, for example, the following lengths: at least about 5 mm or longer than about 5 mm, preferably at least about 6 mm. Or a force greater than about 6 mm, preferably at least about 7 mm or longer than about 7 mm, preferably at least about 8 mm or longer than about 8 mm, preferably at least about 9 mm. A force that is or is longer than about 9 mm, preferably at least about lcm or longer than about lcm, preferably at least about 2 cm, or longer than about 2 cm, preferably at least about 3 cm. Is or is longer than about 3 cm, preferably at least about 4 cm or longer than about 4 cm, preferably at least about 5 cm or longer than about 5 cm, preferably less A force that is at least about 6 cm or longer than about 6 cm, preferably at least about 7 cm or longer than about 7 cm, preferably at least about 8 cm or longer than about 8 cm, preferably at least A force that is about 9 cm or longer than about 9 cm, preferably at least about 10 cm or longer than about 10 cm, preferably at least about 15 cm or longer than about 15 cm, and preferably A force that is at least about 20 cm or longer than about 20 cm. The upper limit of the distance between the electrodes can be, for example, but is not limited to: about 25 cm, about 20 cm, about 15 cm, about 10 cm, about 9 cm, about 8 cm, about 7 cm, about 6 cm, about 5 cm, about 4 cm , About 3cm, about 2cm, about lcm, about 9mm, about 8mm, about 7mm, or about 6mm. Of course, the distance between the electrodes can be a length between the values specified above (eg 1.5 cm).
[0043] 一つの実施形態では、電極間の距離は、対になった電極間の距離が植物種子を 収容し得る距離となるように変動可能である。電極間の距離の変動は、任意の手段 によって達成され得る。例えば、ネジなどを利用して適切な距離に調整され得る。 [0043] In one embodiment, the distance between the electrodes is such that the distance between the pair of electrodes is a plant seed. The distance can be changed so that the distance can be accommodated. Variation in the distance between the electrodes can be achieved by any means. For example, it can be adjusted to an appropriate distance using a screw or the like.
[0044] 電極は、電流を流し得ると ヽぅ性質を有する限りにお 、て任意の材料力 作製され 得る。電極の材料としては、例えば、白金、金、ステンレス、炭素、導電性ポリマーな どが挙げられる力 これらに限定されない。特に好ましくは、電極は白金電極である。  [0044] The electrode can be made of any material force as long as it has properties that allow current to flow. Examples of the material of the electrode include, but are not limited to, platinum, gold, stainless steel, carbon, conductive polymer, and the like. Particularly preferably, the electrode is a platinum electrode.
[0045] 例示的なエレクト口ポーレーシヨンチャンバ一は、白金電極を備える縦 lcm X横 2c m X高さ 2cmの直方形チャンバ一であり、その白金電極間の距離は、約 lcmである 。このエレクト口ポーレーシヨンチャンバ一は、中程度の大きさ（約 5〜 15mm程度）の 植物種子 (例えば、コムギ、イネ、トウモロコシなど）を処理する際に特に有用である。 このチャンバ一を使用することにより、中程度の大きさの植物種子を大量にまとめて（ 例えば、約 10〜30粒）処理することが可能である。  [0045] An exemplary electo-portion chamber chamber is a rectangular chamber with vertical electrodes 1 cm x 2 cm x height 2 cm with platinum electrodes, and the distance between the platinum electrodes is about lcm. The electopore chamber is particularly useful when treating medium-sized (about 5-15 mm) plant seeds (eg, wheat, rice, corn, etc.). By using this chamber, it is possible to process a large amount of medium-sized plant seeds (for example, about 10 to 30 grains).
[0046] 他の例示的なエレクト口ポーレーシヨンチャンバ一は、ステンレス電極を備える内径 lcm X高さ 4cmのマイクロチューブ型チャンバ一であり、ステンレス電極間の距離は 、約 lcmである。このマイクロチューブ型チャンバ一は、市販のマイクロチューブの内 面に、ステンレス箔（例えば、約 5 X 40mm (厚さ約 0. lmm) )を接着剤などで貼り付 けることによって、容易に作製され得る。このマイクロチューブ型チャンバ一は、遠心 分離にかけて、細胞、組織および Zまたは種子を底に沈殿させることができるため、 溶液の交換を簡単にする。このため、このマイクロチューブ型チャンバ一は、微小な（ 約 0. l〜5mm程度の)植物種子 (例えば、シロイヌナズナなど）を処理する際に特に 有用である。このチャンバ一を使用することにより、微小な大きさの植物種子を大量に まとめて処理することが可能である。  [0046] Another exemplary electopore chamber is a microtube chamber with an inner diameter lcm x height 4cm with stainless steel electrodes, and the distance between the stainless steel electrodes is about lcm. This micro tube type chamber is easily manufactured by attaching a stainless steel foil (for example, about 5 X 40 mm (thickness: about 0.1 mm)) to the inner surface of a commercially available micro tube with an adhesive. obtain. This microtube chamber simplifies solution exchange because it can be centrifuged to precipitate cells, tissues and Z or seeds at the bottom. For this reason, this microtube-type chamber is particularly useful when processing minute plant seeds (eg, about 0.1 to 5 mm) (eg, Arabidopsis thaliana). By using this chamber, it is possible to process a large amount of small-sized plant seeds.
[0047] 本発明の別の局面において、エレクト口ポーレーシヨン電極は、例えば、図 13に示 すように、試験管に挿入するタイプの電極であってもよい。図 13〜 14に示すエレクト 口ポーレーシヨン電極 2は、一対の平板状電極 21および 22、ならびに、キャップ 23お よび電気コード 24を備える。図 14に示すように、エレクト口ポーレーシヨン電極 2の平 板状電極 21および 22は、試験管 25に挿入して用いる。必要に応じて、キャップ 23ま たは試験管 25に通気口を設けて、試験管内の圧力を、加圧、減圧してもよい。試験 管 25内に試料を入れて、エレクト口ポーレーシヨン電極 2によって、エレクト口ボーレ ーシヨンを行う。 [0047] In another aspect of the present invention, the electrification electrode may be an electrode of a type inserted into a test tube as shown in FIG. 13 to 14 includes a pair of flat electrodes 21 and 22, a cap 23 and an electric cord 24. As shown in FIG. 14, the flat plate-like electrodes 21 and 22 of the electrification electrode 2 are inserted into a test tube 25 and used. If necessary, vents may be provided in the cap 23 or the test tube 25 to increase or decrease the pressure in the test tube. Place the sample in the test tube 25, and then use the electo-pore polarization electrode 2 to -Perform a cission.
[0048] 本願明細書にぉ ヽて使用する場合、細胞 Z組織 (植物組織を含む)を、「大気圧と 異なる圧力下に維持する」とは、細胞 Z組織 (植物組織を含む)を、大気圧 (通常は、 [0048] As used herein, the term "maintaining cell Z tissue (including plant tissue) under a pressure different from atmospheric pressure" refers to cell Z tissue (including plant tissue), Atmospheric pressure (usually
1気圧 = 101. 325kPa =約 0. IMPa)よりも高い圧力下に維持する工程 (加圧処理 )、または低!ヽ圧力下に維持する工程 (減圧処理)を!ヽぅ。 1 atm = 101. 325kPa = approx. 0. IMPa) The process of maintaining under a pressure higher than (pressure treatment) or the process of maintaining under a low pressure (decompression process).
[0049] 理論に拘束されることを意図しな ヽが、細胞 Z組織 (植物組織を含む)を大気圧と 異なる圧力下に維持することによって、細胞 Z組織が受ける環境中の圧力が変化し[0049] Although not intended to be bound by theory, maintaining the cellular Z tissue (including plant tissue) under a pressure different from atmospheric pressure changes the environmental pressure experienced by the cellular Z tissue.
、 DNAなどの核酸を含む緩衝液が組織間 ·細胞間に浸透しやすくなり、その結果、 従来不可能であった細胞壁を有する細胞および組織 (特に、植物細胞および植物組 織)を標的とするエレクト口ポーレーシヨンによる核酸導入 Z形質転換が可能になった ものと考えられる。 Buffers containing nucleic acids such as DNA can easily penetrate between tissues and cells, and as a result, target cells and tissues with cell walls (especially plant cells and plant tissues) that were not possible before Nucleic acid introduction by electoporation Z transformation is considered possible.
[0050] 本明細書において使用する場合、用語「減圧処理」とは、核酸導入 Z形質転換さ れる細胞 Z組織 (植物組織 (例えば、種子)を含む)を大気圧より低!ヽ気圧下に維持 する処理をいう。本発明において、減圧処理は、大気圧よりも、 0. 02MPa低い圧力 、好ましくは 0. 04MPa低い圧力、より好ましくは 0. 06MPa低い圧力、さらにより好ま しくは 0. 08MPa低い圧力、最も好ましくは 0. 096MPa低い圧力で行われる力 こ れらに限定されない。減圧処理の時間は、 1分〜 120分、好ましくは 10分〜 100分、 より好ましくは 15分〜 90分、さらにより好ましくは 30分〜 70分、最も好ましくは約 60 分である力 これらに限定されない。  [0050] As used herein, the term "reduced pressure treatment" refers to nucleic acid-introduced Z transformed cells Z tissues (including plant tissues (eg, seeds)) below atmospheric pressure and under atmospheric pressure. The process to be maintained. In the present invention, the decompression treatment is performed at a pressure 0.02 MPa lower than the atmospheric pressure, preferably 0.04 MPa lower pressure, more preferably 0.06 MPa lower pressure, even more preferably 0.08 MPa lower pressure, most preferably 0. 096 MPa Forces applied at low pressures are not limited to these. The pressure treatment time is 1 minute to 120 minutes, preferably 10 minutes to 100 minutes, more preferably 15 minutes to 90 minutes, even more preferably 30 minutes to 70 minutes, most preferably about 60 minutes. It is not limited.
[0051] 本明細書において使用する場合、用語「加圧処理」とは、核酸導入 Z形質転換さ れる細胞 Z組織 (植物組織 (例えば、種子)を含む)を大気圧より高!ヽ気圧下に維持 する処理をいう。 [0051] As used herein, the term "pressure treatment" refers to nucleic acid-introduced Z-transformed cells Z tissues (including plant tissues (eg, seeds)) above atmospheric pressure and under atmospheric pressure. This is the process to maintain.
[0052] 一つの局面において、本発明は、エレクト口ポーレーシヨン装置に関する。本発明 のエレクト口ポーレーシヨン装置は、顕著に高 、効率で任意の細胞または糸且織に対し て核酸導入し得、そして特に、これまでエレクト口ポーレーシヨンによる核酸導入が不 可能であった、細胞壁を有する細胞または組織 (例えば、植物細胞または植物組織） に核酸導入するために有用である。一つの実施形態では、本発明のエレクト口ボーレ ーシヨン装置は、 a)細胞を大気圧と異なる圧力下に維持する手段、および、 b)エレク トロポーレーシヨン手段の両方の手段を備える。 [0052] In one aspect, the present invention relates to an electo-portion polarization device. The electoporation apparatus of the present invention can introduce nucleic acid into any cell or yarn and weave with a remarkably high and efficiency, and in particular, the cell wall, which until now has been impossible to introduce nucleic acid by electoporation. It is useful for introducing a nucleic acid into a cell or tissue (eg, a plant cell or plant tissue). In one embodiment, the electo-portionation device of the present invention comprises: a) means for maintaining the cell under a pressure different from atmospheric pressure; and b) the elect Both means of tropo-ration means are provided.
[0053] 細胞を大気圧と異なる圧力下に維持する手段としては、減圧および Zまたは加圧 する能力を有する任意の手段が利用され得る。市販の減圧装置 (例えば、真空デシ ケーターなど）および Zまたは加圧装置もまた利用され得る。エレクト口ポーレーショ ン手段としては、任意のエレクト口ポーレーシヨン手段が利用され得る。市販のエレク トロポーレーシヨン手段（例えば、 CUY21EDIT遺伝子導入装置、ネッパジーン社、 日本国千葉県巿川巿)もまた利用され得る。好ましくは、上記のエレクト口ポーレーシ ヨン手段に配置される 2つの電極 (第一電極および第二電極)の間の距離は、上記で 定義付けられたような、植物種子を収容し得る距離にある。  [0053] As a means for maintaining the cells under a pressure different from the atmospheric pressure, any means having the ability to reduce pressure and Z or pressurize can be used. Commercially available decompression devices (eg, vacuum desiccators) and Z or pressurization devices can also be utilized. Any electo-portioning means can be used as the electo-portioning means. Commercially available electroporation means (e.g., CUY21EDIT gene transfer device, Nepagene, Sasakawa, Chiba, Japan) can also be used. Preferably, the distance between the two electrodes (first electrode and second electrode) arranged in the above-mentioned electopore polarization means is a distance capable of accommodating plant seeds as defined above. .
[0054] 別の実施形態では、本発明のエレクト口ポーレーシヨン装置は、植物種子を収容し 得る距離にある二つの電極を含み、そしてこのエレクト口ポーレーシヨン装置は、細胞 Z組織を大気圧と異なる圧力下に維持することと組み合わせて使用される。この実施 形態では、このエレクト口ポーレーシヨン装置と、細胞 Z糸且織を大気圧と異なる圧力下 に維持する手段とが、同一の機器の中に存在する必要はな 、。  [0054] In another embodiment, the electoporation device of the present invention includes two electrodes at a distance that can accommodate plant seeds, and the electoporation device has a pressure different from atmospheric pressure for cell Z tissue. Used in combination with keeping down. In this embodiment, it is not necessary that the electopore polarization device and the means for maintaining the cell Z yarn and weave under a pressure different from the atmospheric pressure are present in the same device.
[0055] 従来のエレクト口ポーレーシヨン装置は、極めて小さい細胞に対して電圧パルスを かけることを目的としていたため、チャンバ一の内寸および電極間の距離をできるだ け小さくする必要があった。このため、従来のエレクト口ポーレーシヨン装置のチャン バーの内寸および電極間の距離は、代表的に、 1mmまたは 2mm程度であり、長く てもせいぜい 4mmであった。従って、本発明のような、植物種子をも収容し得るほど 大きなチャンバ一および植物種子をも収容し得るほど長い距離で配置された電極を 備えたエレクト口ポーレーシヨン装置は、これまで知られて 、な！/、。  [0055] Since the conventional electopore polarization device was intended to apply a voltage pulse to extremely small cells, it was necessary to make the inner dimensions of the chamber and the distance between the electrodes as small as possible. For this reason, the inner dimension of the chamber and the distance between the electrodes of the conventional electrification device are typically about 1 mm or 2 mm, and at most 4 mm at most. Therefore, an electoral polarization device including a chamber that is large enough to accommodate plant seeds and an electrode that is disposed at a long distance enough to accommodate plant seeds, as in the present invention, has been known so far. Yeah! /.
[0056] さらに、本発明のエレクト口ポーレーシヨン装置は、自動化して実行され得る。本発 明の自動化工レクト口ポーレーシヨン装置において、緩衝液などの溶液の注入および Zまたは交換は、自動分注機により行われ得る。 自動分注機は、例えば、市販の ep Motion5070 ワークステーション（エツペンドルフ株式会社 日本国東京都千代田 区東神田 3)などが利用され得るが、これに限定されない。特に、核酸と細胞とを含む 混合液を入れる容器中に、核酸および Zまたは細胞を入れる手段として、このような 自動分注機が有利に使用され得る。 [0057] 核酸と細胞とを含む混合液を入れる第一容器、細胞を大気圧と異なる圧力下に維 持する第二容器、および核酸と細胞とを含む混合液に電圧パルスをかける第三容器 は、任意の容器であり得る。これらの容器は、同一であっても異なってもよい。この容 器の材質としては、固体を形成し得る任意の材質が使用され得る。例えば、ガラス、 シリカ、シリコン、セラミック、二酸化珪素、プラスチック、金属 (合金も含まれる）、天然 および合成のポリマー（例えば、ポリスチレン、セルロース、キトサン、デキストラン、お よびナイロン)などが挙げられるがそれらに限定されない。容器は、複数の異なる材 料の層カゝら形成されていてもよい。例えば、ガラス、石英ガラス、アルミナ、サファイア 、フォルステライト、酸化珪素、炭化珪素、窒化珪素などの無機絶縁材料を使用する ことができる。ポリアミド、ポリカーボネート、（変性)ポリフエ-レンオキサイド、ポリブチ レンテレフタレート、強化ポリエチレンテレフタレート、ポリエーテルスルホン、ポリフエ 二レンスルフイド、ポリアリレート、ポリエーテルイミド、ポリエーテルエーテルケトン、ポ リイミド、ポリエステル、ポリエチレン、ポリプロピレン、ポリイソブチレン、不飽和ポリエ ステル、含フッ素榭脂、ポリ塩化ビュル、ポリ塩ィ匕ビユリデン、ポリ酢酸ビュル、ポリビ -ルアルコール、ポリビュルァセタール、アクリル榭脂、ポリアクリロニトリル、ポリスチ レン、ァセタール榭脂、フエノール榭脂、ユリア榭脂、エポキシ榭脂、メラミン榭脂、ス チレン.アクリロニトリル共重合体、アクリロニトリルブタジエンスチレン共重合体、シリコ ーン榭脂、ポリスルホンなどの有機材料なども使用され得る。 [0056] Further, the electo-portion polarization device of the present invention can be executed in an automated manner. In the automated rectifying port polarization apparatus of the present invention, injection and Z or replacement of a solution such as a buffer solution can be performed by an automatic dispenser. As the automatic dispensing machine, for example, a commercially available ep Motion5070 workstation (Eppendorf Co., Ltd. 3 Higashi Kanda, Chiyoda-ku, Tokyo, Japan) can be used, but is not limited thereto. In particular, such an automatic dispenser can be advantageously used as a means for putting nucleic acid and Z or cells in a container containing a mixed solution containing nucleic acid and cells. [0057] A first container for containing a mixture containing nucleic acid and cells, a second container for maintaining cells under a pressure different from atmospheric pressure, and a third container for applying a voltage pulse to the mixture containing nucleic acids and cells Can be any container. These containers may be the same or different. As the material of this container, any material capable of forming a solid can be used. Examples include glass, silica, silicon, ceramics, silicon dioxide, plastics, metals (including alloys), natural and synthetic polymers (eg, polystyrene, cellulose, chitosan, dextran, and nylon). It is not limited. The container may be formed of a plurality of layers of different materials. For example, inorganic insulating materials such as glass, quartz glass, alumina, sapphire, forsterite, silicon oxide, silicon carbide, and silicon nitride can be used. Polyamide, Polycarbonate, (Modified) Polyethylene oxide, Polybutylene terephthalate, Reinforced polyethylene terephthalate, Polyethersulfone, Polyphenylene sulfide, Polyarylate, Polyetherimide, Polyetheretherketone, Polyimide, Polyester, Polyethylene, Polypropylene, Poly Isobutylene, unsaturated polyester, fluorine-containing resin, polychlorinated butyl, polysalt-vinylidene, polyacetate butyl, polyvinyl alcohol, polybutylacetal, acrylic resin, polyacrylonitrile, polystyrene, acetal resin, Phenolic resin, urea resin, epoxy resin, melamine resin, styrene, acrylonitrile copolymer, acrylonitrile butadiene styrene copolymer, silicone resin, police And organic materials such as phone may also be used.
[0058] 好ましくは、本発明の第一容器は、透明度が高く材料観察を容易にする材料 (例え ば、ポリスチレン)力も作製される。好ましくは、本発明の第二容器は、大気圧と異なる 圧力（特に、減圧）に耐える能力を有する材料 (例えば、ポリアミド、ポリカーボネート、 (変性）ポリフエ-レンオキサイド、ポリブチレンテレフタレート、強化ポリエチレンテレ フタレート、ポリエーテルスルホン、ポリフエ二レンスルフイド、ポリアリレート、ポリエー テルイミド、ポリエーテルエーテルケトン、ポリイミドおよびエポキシ榭脂）力も作製され 、より好ましくは、透明度が高く材料観察を容易にする性質も備えた材料 (例えば、了 クリル樹脂)カゝら作製される。好ましくは、本発明の第三容器は、細胞との親和性を示 す材料 (例えば、ポリプロピレン、シリコーン榭脂、およびガラス)から作製され、白金 製、金製、ステンレス製、炭素製、または導電性ポリマー製の電極を備える。これらの 材料は、所望の性質を付与するために (例えば、容器本体に絶縁性を付与するため に、または電極の導電性を高めるために）、任意の適切な材料でコーティングされて ちょい。 [0058] Preferably, the first container of the present invention is also produced with a material force (for example, polystyrene) that has high transparency and facilitates material observation. Preferably, the second container of the present invention is made of a material capable of withstanding a pressure different from atmospheric pressure (especially reduced pressure) (for example, polyamide, polycarbonate, (modified) polyphenylene oxide, polybutylene terephthalate, reinforced polyethylene terephthalate). , Polyethersulfone, polyphenylene sulfide, polyarylate, polyetherimide, polyetheretherketone, polyimide and epoxy resin), and more preferably a material with high transparency and easy material observation (for example, , Ryo Krill Resin). Preferably, the third container of the present invention is made of a material showing affinity with cells (for example, polypropylene, silicone resin, and glass), and is made of platinum, gold, stainless steel, carbon, or conductive. An electrode made of a conductive polymer is provided. these The material may be coated with any suitable material to impart the desired properties (eg, to provide insulation to the container body or to increase the conductivity of the electrode).
[0059] 上記の第一容器および第二容器にっ 、て、好ましくは、これらの容器は、大気圧と 異なる圧力に耐える能力を有し、特に好ましくは、減圧処理に耐える能力を有する。 例えば、上記の第一容器は、上記の第二容器内および Zまたは上記の第三容器内 に収容されてもよい。または、核酸と細胞とを含む混合液は、上記の第一容器から、 上記の第二容器 (または、その内部に収容される別の容器）内および Zまたは上記 の第三容器 (または、その内部に収容される別の容器）内に注入されてもよい。  [0059] According to the first container and the second container described above, these containers preferably have the ability to withstand a pressure different from the atmospheric pressure, and particularly preferably have the ability to withstand a decompression process. For example, the first container may be housed in the second container and Z or the third container. Alternatively, the mixed solution containing the nucleic acid and the cells is transferred from the first container to the second container (or another container accommodated therein) and Z or the third container (or the container). It may be injected into a separate container).
[0060] 細胞を、上記の第二容器中に配置する手段、および核酸と細胞とを含む混合液を 、上記の第三容器中に配置する手段には、ベルトコンベアなどが有利に使用され得 る力 これに限定されず、任意の手段が利用され得る。自動ポンプなどを利用して、 第一容器、第二容器および第三容器中に配置された液体を、吸引 Z排出することに よって移動させる方法もまた利用され得る。  [0060] A belt conveyor or the like can be advantageously used for the means for placing the cells in the second container and the means for placing the mixed solution containing the nucleic acid and the cells in the third container. However, the present invention is not limited to this, and any means can be used. A method of moving the liquid disposed in the first container, the second container, and the third container by suction Z discharge using an automatic pump or the like can also be used.
[0061] 本発明の自動化工レクト口ポーレーシヨン装置は、各操作手段を自動化して実施す るための制御装置を含む。さらに本発明のエレクト口ポーレーシヨン装置は、電源装 置を含む。このような制御装置および電源装置は、エレクト口ポーレーシヨン装置と同 一の機器内に配置されても、別々の機器としてコードで接続されてもよい。  [0061] The automated rect port por- tion device of the present invention includes a control device for automating and implementing each operation means. Further, the elect port polarization device of the present invention includes a power supply device. Such a control device and a power supply device may be arranged in the same device as the elect port polarization device or may be connected as a separate device by a cord.
[0062] 種子に対してエレクト口ポーレーシヨンを行う場合、好ましくは、減圧処理またはカロ 圧処理をする種子を、処理前に、水（例えば、水道水）中に放置する。処理前の種子 を水中に放置する場合、その放置時間は、 6時間〜 48時間、好ましくは 12時間〜 3 6時間、より好ましくは 18時間〜 30時間、さらにより好ましくは 20時間〜 26時間、最 も好ましくは約 24時間である力 これらに限定されな!、。  [0062] In the case of performing the erect mouth poration on the seeds, preferably, the seeds to be subjected to the decompression treatment or the calorie pressure treatment are left in water (for example, tap water) before the treatment. When the seed before treatment is left in water, the standing time is 6 hours to 48 hours, preferably 12 hours to 36 hours, more preferably 18 hours to 30 hours, even more preferably 20 hours to 26 hours, The power that is most preferably about 24 hours is not limited to these!
[0063] 本発明における例示的な核酸導入の条件は、種子を 25°Cで一晩水道水中に放置 し、翌日、真空装置の中に置き、大気圧より 0. 096MPa低い圧力にて 1時間減圧処 理を行い、その後、減圧処理した種子に対して、エレクト口ポーレーシヨンによる高電 圧パルス（100V、 50ミリ秒、ただし、電極間の距離は lcm)を 50回程度かけ、核酸 導入 Z遺伝子導入を行うと ヽぅ条件である。電圧およびパルス回数は作物によって 変化し得、当業者は、必要に応じて、そのエレクト口ポーレーシヨン条件を適宜選択し 得る。その後、抗生物質を含む培地において選抜し、その後鉢上げ (ポットでの育成 )により通常の植物個体を得ることができる。 [0063] Exemplary nucleic acid introduction conditions in the present invention are that the seeds are allowed to stand in tap water overnight at 25 ° C, and placed in a vacuum apparatus the next day for 1 hour at a pressure lower than atmospheric pressure by 0.096 MPa. After the vacuum treatment, the high-pressure pulse (100 V, 50 milliseconds, but the distance between the electrodes is about 1 cm) by electo-portion is applied about 50 times to the seed that has been subjected to the vacuum treatment. If introduced, it is a drought condition. Voltage and number of pulses depend on the crop Those skilled in the art can appropriately select the electopore polarization conditions as needed. Thereafter, selection is performed in a medium containing antibiotics, and then normal plant individuals can be obtained by potting (growing in pots).
本明細書において用いられる「植物」とは、植物界に属する生物の総称であり、葉 緑体、硬い細胞壁、豊富な永続性の胚的組織の存在，および運動する能力がない 生物により特徴付けられる。植物の種類は、例えば、「原色牧野植物大図鑑」（北隆 館（1982) )などにおいて広範に分類されており、そこに記載されるすべての種類の 植物が、本発明において使用され得る。代表的には、植物は、細胞壁の形成'葉緑 体による同化作用をもつ顕花植物をいう。「植物」は、単子葉植物および双子葉植物 のいずれも含む。単子葉植物としては、イネ科植物が挙げられる。好ましい単子葉植 物としては、トウモロコシ、コムギ、イネ、ェンバタ、ォォムギ、ソルガム、ライムギ及びァ ヮが挙げられ、さらに好ましくは、トウモロコシ、コムギ、イネが挙げられる力 これらに 限定されない。コムギには、従来法では形質転換体を得ることが困難であったコムギ 品種農林 61号も含まれる。双子葉植物としては、アブラナ科植物、マメ科植物、ナス 科植物、ゥリ科植物、ヒルガオ科植物が挙げられる力 これらに限定されない。ァブラ ナ科植物としては、ハクサイ、ナタネ、キャベツ、カリフラワーが挙げられる力 これら に限定されない。好ましいアブラナ科植物は、ノ、クサイおよびナタネである。特に好ま しいアブラナ科植物は、ナタネである。マメ科植物としては、ダイズ、ァヅキ、インゲン マメ、ササゲが挙げられるが、これらに限定されない。好ましいマメ科植物は、ダイズ である。ナス科植物としては、トマト、ナス、バレイショが挙げられる力 これらに限定さ れない。好ましいナス科植物は、トマトである。ゥリ科植物としては、マタワゥリ、キユウリ 、メロン、スイカが挙げられる力 これらに限定されない。好ましいゥリ科植物は、マク ワウリである。ヒルガオ科植物としては、アサガオ、カンショ、ヒルガオが挙げられるが、 これらに限定されない。好ましいヒルガオ科植物は、アサガオである。特に他で示さな い限り、植物は、植物体、植物器官、植物組織、植物細胞、および種子のいずれをも 意味する。植物器官の例としては、根、葉、茎、および花などが挙げられる。植物細 胞の例としては、カルスおよび懸濁培養細胞が挙げられる。特定の実施形態では、 植物は、植物体を意味し得る。 [0065] 別の実施形態において、本発明において使用され得る植物種の例としては、ナス 科、イネ科、アブラナ科、バラ科、マメ科、ゥリ科、シソ科、ユリ科、ァカザ科、セリ科、ヒ ルガォ科、キク科などの植物が挙げられる。さらに、本発明において使用され得る植 物種の例としては、任意の榭木種、任意の果榭種、クヮ科植物（例えば、ゴム）、およ びァオイ科植物（例えば、綿花）が挙げられる。 As used herein, “plant” is a general term for organisms belonging to the plant kingdom, characterized by chloroplasts, hard cell walls, the presence of abundant permanent embryonic tissue, and organisms that are not capable of motility. It is done. Plant types are broadly classified in, for example, “Primary Color Makino Botanical Encyclopedia” (Kitatakakan (1982)), and all types of plants described therein can be used in the present invention. Typically, a plant refers to a flowering plant having an assimilation effect by chloroplast formation. “Plant” includes both monocotyledonous and dicotyledonous plants. Monocotyledonous plants include gramineous plants. Preferred monocotyledonous plants include corn, wheat, rice, embata, barley, sorghum, rye and wheat, and more preferably, power including corn, wheat and rice. Wheat includes wheat cultivar Nori 61, which had been difficult to obtain transformants by conventional methods. Dicotyledonous plants include, but are not limited to, cruciferous plants, legumes, solanaceous plants, cucurbitaceous plants, and convolvulaceae plants. Brassicaceae plants include, but are not limited to, cabbage, rapeseed, cabbage and cauliflower. Preferred cruciferous plants are roe, kusasai and rapeseed. A particularly preferred cruciferous plant is rapeseed. Leguminous plants include, but are not limited to, soybeans, oysters, kidney beans, and cowpeas. A preferred legume is soybean. Examples of solanaceous plants include, but are not limited to, tomatoes, eggplants, and potatoes. A preferred solanaceous plant is tomato. The cucurbitaceae plants include, but are not limited to, power of matawali, cucumber, melon, and watermelon. A preferred cucurbitaceae plant is mcwauli. Convolvulaceae plants include, but are not limited to, morning glory, sweet potato and convolvulus. A preferred convolvulaceae plant is morning glory. Unless otherwise indicated, a plant means any plant, plant organ, plant tissue, plant cell, and seed. Examples of plant organs include roots, leaves, stems and flowers. Examples of plant cells include callus and suspension culture cells. In certain embodiments, a plant may mean a plant body. [0065] In another embodiment, examples of plant species that can be used in the present invention include solanaceae, gramineous, cruciferous, rose, legume, cucurbitaceae, perilla, lily, akaza, Plants such as celery family, urchinaceae and asteraceae are included. In addition, examples of plant species that can be used in the present invention include any vine species, any fruit cultivar, cucurbitaceae plants (eg, rubber), and mallow (eg cotton). .
[0066] 簡便には、本発明の方法は、植物組織 (休眠組織 (完熟種子、未熟種子、冬芽、お よび塊茎を含む）、生殖質、生長点、および花芽を含む）に対してエレクト口ポーレー シヨンを行い、最も簡便には、種子に対してエレクト口ポーレーシヨンを行う。本発明の エレクト口ポーレーシヨン方法により核酸導入された種子はそのまま、例えば、土に植 えて栽培することにより、容易に核酸導入体 Z形質転換体となり得る。種子は通常、 胚、胚乳および種皮の三部分から構成される（野口弥吉り II田信一郎監修，該当部 分は千坂英夫著，農学大事典，養賢堂， 896頁， 1987を参照のこと)。胚が、植物の すべての遺伝情報を備え、そして植物体へと生育する部分である。すべての単子葉 植物および双子葉植物は胚を有する。本発明のエレクト口ポーレーシヨン方法により 核酸導入すると、胚の部分において、導入した核酸の発現が認められた。従って、胚 を含む種子を有するあらゆる植物で、本発明のこの最も簡便な方法により、容易に核 酸導入植物体 Z形質転換植物体を得ることができる。  [0066] Conveniently, the method of the present invention can be applied to plant tissues (including dormant tissues (including ripe seeds, immature seeds, winter buds, and tubers), germplasm, growth points, and flower buds). Conduct poration and, most conveniently, perform erect mouth poration on the seeds. The seed into which nucleic acid has been introduced by the electoporation method of the present invention can be easily converted into a nucleic acid introducer Z transformant by, for example, planting it in soil and growing it. Seeds are usually composed of three parts: embryo, endosperm, and seed coat (supervised by Yoshikichi Noguchi II, Shinichiro IIda, for the relevant part, written by Hideo Chisaka, Agricultural University Dictionary, Yokendo, p. 896, 1987) . The embryo is the part that contains all the genetic information of the plant and grows into the plant body. All monocotyledonous and dicotyledonous plants have embryos. When the nucleic acid was introduced by the electoporation method of the present invention, expression of the introduced nucleic acid was observed in the embryo part. Accordingly, a nuclear acid-introduced plant Z-transformed plant can be easily obtained from any plant having seeds including embryos by this simplest method of the present invention.
[0067] アブラナ科の植物の例としては、 Raphanus、 Brassica、 Arabidopsis、 Wasabia, または Capsellaに属する植物が挙げられ、例えば、大根、アブラナ、シロイヌナズナ 、ヮサビ、ナズナなどを含む。  [0067] Examples of cruciferous plants include plants belonging to Raphanus, Brassica, Arabidopsis, Wasabia, or Capsella, and include, for example, radish, rape, Arabidopsis thaliana, coral rust, thrips and the like.
[0068] イネ科の植物の例としては、 Oryza、 Triticum、 Hordeum、 Secale、 Saccharum 、 Sorghum,または Zeaに属する植物が挙げられ、例えば、イネ、ォォムギ、ライムギ 、サトウキビ、ソルガム、トウモロコシなどを含む。  [0068] Examples of the grass family plants include plants belonging to Oryza, Triticum, Hordeum, Secale, Saccharum, Sorghum, or Zea, and include, for example, rice, barley, rye, sugarcane, sorghum, corn, and the like.
[0069] 本明細書において用いられる「動物」とは、動物界に属する生物の総称であり、酸 素と有機性食物を必要とし，植物や鉱物と違って任意的に動くことができる生物によ り特徴付けられる。動物は、大きく脊椎動物と無脊椎動物とに分類される。脊椎動物 としては、例えば、メタラウナギ類、ャッメゥナギ類、軟骨魚類、硬骨魚類、両生類、爬 虫類、鳥類、哺乳動物などが用いられ、より好ましくは、哺乳動物 (例えば、単孔類、 有袋類、貧歯類、皮翼類、翼手類、食肉類、食虫類、長鼻類、奇蹄類、偶蹄類、管 歯類、有鱗類、海牛類、クジラ目、霊長類、齧歯類、ゥサギ目など)が用いられる。さら に好ましくは、霊長類 (たとえば、チンパンジー、二ホンザル、ヒト）が用いられる。最も 好ましくはヒト由来の細胞または器官などが用いられる。無脊椎動物としては、例えば 、甲殻綱、ヤスデ綱、エダヒゲムシ綱、ムカデ綱、コムカデ綱、昆虫綱などが用いられ る。より好ましくは、昆虫 (例えば、チョウ目（カイコなどを含む))が用いられる。 [0069] "Animal" as used in this specification is a general term for organisms belonging to the animal kingdom. They require oxygen and organic food, and can be moved freely unlike plants and minerals. More characterized. Animals are broadly classified into vertebrates and invertebrates. As the vertebrate, for example, metal eels, shark eels, cartilaginous fish, teleosts, amphibians, reptiles, birds, mammals, etc. are used, more preferably mammals (for example, single pores, Marsupial, rodent, winged, winged, carnivorous, carnivorous, long-nosed, odd-hoofed, cloven-hoofed, rodent, scale, sea cattle, cetacean, primate , Rodents, maggots, etc.). More preferably, primates (for example, chimpanzees, dihonds, humans) are used. Most preferably, human-derived cells or organs are used. As invertebrates, for example, crustaceans, millipedes, edible beetles, centipedes, komcades, insects and the like are used. More preferably, insects (for example, Lepidoptera (including silkworms)) are used.
[0070] 本明細書にぉ 、て、「トランスジヱニック生物」とは、特定の遺伝子が組み込まれた 生物をいう。「トランスジエニック植物」とは、特定の遺伝子が組み込まれた植物をいい 、同様に「トランスジエニック動物」とは、特定の遺伝子が組み込まれた動物をいう。  As used herein, “transgene organism” refers to an organism into which a specific gene has been incorporated. “Transgenic plant” refers to a plant into which a specific gene has been incorporated. Similarly, “transgene animal” refers to an animal into which a specific gene has been incorporated.
[0071] 本発明の方法により核酸導入 Z形質転換された細胞および組織は、当該分野に おいて公知の任意の方法によって、分化、成長および Zまたは増殖され得る。植物 種の場合、細胞または組織を分化、成長および Zまたは増殖させる工程は、例えば [0071] Nucleic acid introduction by the method of the present invention Z-transformed cells and tissues can be differentiated, grown and Z or proliferated by any method known in the art. In the case of plant species, the process of differentiating, growing and Z or proliferating cells or tissues is for example
、その植物細胞もしくは植物組織またはそれらを含む植物体を栽培することによって 達成され得る。本明細書では、植物の栽培は当該分野において公知の任意の方法 により行うことができる。植物の栽培方法は、例えば、監修 島本功および岡田清， 「 モデル植物の実験プロトコ一ルーイネ ·シロイヌナズナ編 」：細胞工学別冊植物細 胞工学シリーズ 4 ;イネの栽培法 (奥野員敏) pp. 28— 32、ならびに、丹羽康夫著， シロイヌナズナの栽培法， pp. 33— 40に例示されており、当業者であれば容易に実 施することができることから本明細書では詳述する必要はない。例えば、シロイヌナズ ナの栽培は土耕、ロックウール耕、水耕いずれでも行うことができる。白色蛍光灯（60 00ルクス程度)の下、恒明条件で栽培すれば播種後 4週間程度で最初の花が咲き、 開花後 16日程度で種子が完熟する。 1さやで約 40〜50粒の種子が得られ、播種後 2〜3ヶ月で枯死するまでの間に 10000粒程度の種子が得られる。また、例えば、コ ムギの栽培においては、播種後に一定期間の低温短日条件にさらされなければ、出 穂および開花しないことが周知である。従って、例えば、人工環境下 (例えば、温室 やダロスチャンバ一）においてコムギを栽培する場合には、生育初期段階で、コムギ 幼植物に低温短日処理 (例えば、 20°C 明期 8時間（約 2000ルクス）および 8°C 暗 期 16時間での処理など）を行う必要がある。この処理は春化処理 (vernalization)と 呼ばれる。このような各植物種ごとに必要とされる栽培条件は、当該分野において一 般に広く知られており、従って、本明細書中で詳述する必要はない。 It can be achieved by cultivating the plant cell or plant tissue or the plant body containing them. In the present specification, plant cultivation can be performed by any method known in the art. Plant cultivation methods are, for example, supervised by Isao Shimamoto and Kiyoshi Okada, “Experimental Protoco of Model Plants,“ Rouine Arabidopsis ”edited by: Cell Engineering, Supplementary Plant Cell Engineering Series 4; Rice Cultivation Method (Toshitoshi Okuno) pp. 28 — 32, and Yasuo Niwa, Arabidopsis Cultivation Method, pp. 33-40, and those skilled in the art can easily carry out the method and need not be described in detail herein. For example, Arabidopsis can be cultivated by soil cultivation, rock wool cultivation, or hydroponics. If cultivated under constant fluorescent conditions under white fluorescent lamps (about 60000 lux), the first flowers will bloom about 4 weeks after sowing, and the seeds will mature about 16 days after flowering. About 40-50 seeds can be obtained with one pod, and about 10,000 seeds can be obtained within 2 to 3 months after sowing. In addition, for example, in the cultivation of wheat, it is well known that it does not head and bloom unless it is exposed to low-temperature short-day conditions for a certain period after sowing. Thus, for example, when cultivating wheat in an artificial environment (for example, in a greenhouse or Daros chamber), low temperature short-day treatment (for example, 20 ° C light period 8 hours (about 20 ° C) 2000 lux) and 8 ° C dark period 16 hours). This process is called vernalization and be called. The cultivation conditions required for each plant species are generally well known in the art and therefore need not be described in detail herein.
[0072] 植物以外の種 (例えば、動物種)の場合においても、核酸導入 Z形質転換された 細胞および組織は、当該分野において公知の任意の方法によって、分化、成長およ び Zまたは増殖され得る（例えば、泉美治ら編，生物化学実験のてびき 4.動物-組 織実験法，化学同人， 1987年などを参照のこと)。例えば、エレクト口ポーレーシヨン により核酸導入した細胞および Zまたは組織は、市販の飼料を供給しながら常温下（ 約 25°C)で育成され得る。  [0072] In the case of species other than plants (eg, animal species), the nucleic acid-introduced Z-transformed cells and tissues can be differentiated, grown and Z- or propagated by any method known in the art. Obtain (see, for example, Meiji Izumi et al., Bibliography of Biochemical Experiments, 4. Animal-Organization Experiments, Chemical Dojin, 1987) For example, cells and Z or tissue into which nucleic acid has been introduced by electopore position can be grown at room temperature (about 25 ° C.) while supplying commercially available feed.
[0073] 本明細書において使用される場合、「界面活性剤」とは、液体の表面張力を減らし ,また液体間または液体と固体間の界面張力を減じる可溶性の化合物をいう。界面 活性剤は、好ましくは、細胞'組織への細菌（例えば、ァグロパクテリゥム）の浸透を促 進する作用を有する。本発明にもいて用いられる界面活性剤としては、例えば、 Silw et L— 77の商品名で Loveland Industries社より農薬の展着剤として市販されて いる、植物に対する毒性の低い界面活性剤である、ポリアルキレンォキシド修飾ヘプ タメチノレトリシロキサン (Polyalkyleneoxide Modified Heptamethyltrisiloxane )、および動植物に対する毒性の低い界面活性剤である TWeen20 (登録商標）（ポリ ォキシエチレン（20)ソルビタンモノラウレート）、が挙げられるがこれらに限定されな い。 [0073] As used herein, "surfactant" refers to a soluble compound that reduces the surface tension of a liquid and reduces the interfacial tension between liquids or between a liquid and a solid. The surfactant preferably has an action of promoting the penetration of bacteria (for example, agrobacterium) into the cell 'tissue. As the surfactant used in the present invention, for example, a surfactant with low toxicity to plants which is commercially available as a pesticide spreading agent from Loveland Industries under the trade name Silw et L-77, polyalkylene O sulfoxides modified heptene Tame Chino Les trisiloxane (polyalkyleneoxide modified Heptamethyltrisiloxane), and T W een20 a low surfactant toxic for animals and plants (TM) (poly Okishiechiren (20) sorbitan monolaurate), but may be mentioned It is not limited to these.
[0074] 本明細書にぉ 、て使用される場合、 Tiプラスミドの「vir領域転写誘導物質」とは、 T iプラスミドの vir領域に存在する遺伝子群の転写を誘導する物質を ヽぅ。 Vir領域転 写誘導物質は、本来は植物が傷などのストレスを受けたときに誘導される植物内在性 のフエノールイ匕合物 (ァセトシリンゴンを含む)に由来する。 Vir領域転写誘導物質の 代表例は、ァセトシリンゴンであるが、ァセトシリンゴンおよびその誘導体の他に、タパ コ葉切片の滲出液力 単離されたリグニン合成の前駆体であるシリングアルデヒドや フェルラ酸あるいは、これらの誘導体であって、 vir領域に存在する遺伝子群の転写 を誘導する物質も含まれる。また、植物には内在性のァセトシリンゴンがあるため、本 発明の実施においては、ァセトシリンゴンの添カ卩は必ずしも必要ではなぐァセトシリ ンゴンを用いなくても、細胞 '組織への核酸および Zまたは細菌の導入は可能である [0075] 本明細書にぉ 、て使用される場合、「Tiプラスミド」とは、 Agrobacterium tumefacien sの約 200kbpのプラスミドであって、クラウンゴール腫瘍の原因因子である。 Tiプラス ミドは、 vir領域、 T— DNA領域などを有する。 [0074] As used herein, "vir region transcription inducer" of Ti plasmid refers to a substance that induces transcription of genes present in the vir region of Ti plasmid. Vir domain transcription inducers are originally derived from plant endogenous phenolic compounds (including acetosyringone) that are induced when plants are subjected to stress such as wounds. A typical example of a Vir region transcription inducer is acetosyringone, but in addition to acetosyringone and its derivatives, exudate of tapaco leaf sections, isolated lignin synthesis precursors such as syringaldehyde and ferulic acid, or these And derivatives that induce transcription of genes present in the vir region. In addition, since plants have endogenous acetosyringone, in the practice of the present invention, it is not always necessary to use acetosyringone. Is possible [0075] As used herein, "Ti plasmid" is an approximately 200 kbp plasmid of Agrobacterium tumefaciens, which is a causative factor of crown gall tumor. Ti plasmid has vir region, T-DNA region and so on.
[0076] 本明細書にぉ 、て使用される場合、「vir領域」とは、 Tiプラスミド中の病原性を担つ ている遺伝子の領域であって、 virA、 B、 G、 C、 D、および Eの 6つの転写単位を有 する。 [0076] As used herein, a "vir region" is a region of a gene responsible for pathogenicity in a Ti plasmid, and includes virA, B, G, C, D, And 6 transcription units E and E.
[0077] 本明細書において使用される場合、導入される遺伝子は、ポリヌクレオチドからなる  [0077] As used herein, the introduced gene consists of a polynucleotide.
[0078] 本明細書において使用される用語「ポリヌクレオチド」、「オリゴヌクレオチド」および「 核酸」は、本明細書において同じ意味で使用され、任意の長さのヌクレオチドのポリ マーをいう。この用語はまた、「誘導体オリゴヌクレオチド」または「誘導体ポリヌクレオ チド」を含む。「誘導体オリゴヌクレオチド」または「誘導体ポリヌクレオチド」とは、ヌク レオチドの誘導体を含む力、またはヌクレオチド間の結合が通常とは異なるオリゴヌク レオチドまたはポリヌクレオチドをいい、互換的に使用される。そのようなオリゴヌタレ ォチドとして具体的には、例えば、 2' O—メチルーリボヌクレオチド、オリゴヌクレオ チド中のリン酸ジエステル結合がホスホロチォエート結合に変換された誘導体オリゴ ヌクレオチド、オリゴヌクレオチド中のリン酸ジエステル結合が N3， 一 P5，ホスホロアミ デート結合に変換された誘導体オリゴヌクレオチド、オリゴヌクレオチド中のリボースと リン酸ジエステル結合とがペプチド核酸結合に変換された誘導体オリゴヌクレオチド、 オリゴヌクレオチド中のゥラシルが C 5プロピ-ルゥラシルで置換された誘導体オリ ゴヌクレオチド、オリゴヌクレオチド中のゥラシルが C— 5チアゾールゥラシルで置換さ れた誘導体オリゴヌクレオチド、オリゴヌクレオチド中のシトシンが C— 5プロピニルシト シンで置換された誘導体オリゴヌクレオチド、オリゴヌクレオチド中のシトシンがフエノ キサジン修飾シトシン（phenoxazine— modified cytosine)で置換された誘導体 オリゴヌクレオチド、オリゴヌクレオチド中のリボースが 2， 一 O プロピルリボースで置 換された誘導体オリゴヌクレオチドおよびオリゴヌクレオチド中のリボースが 2，ーメトキ シエトキシリボースで置換された誘導体オリゴヌクレオチドなどが例示される。他にそう ではないと示されなければ、特定の核酸配列はまた、明示的に示された配列と同様 に、その保存的に改変された改変体 (例えば、縮重コドン置換体)および相補配列を 包含することが企図される。具体的には、縮重コドン置換体は、 1またはそれ以上の 選択された (または、すべての）コドンの 3番目の位置が混合塩基および Zまたはデ ォキシイノシン残基で置換された配列を作成することにより達成され得る（Batzerら， Nucleic Acid Res. , 19 : 5081, 1991 ; Ohtsuka¾, J. Biol. Chem. , 260 : 26 05 - 2608, 1985 ;Rossoliniら， Mol. Cell. Probes, 8 : 91— 98, 1994)。用語「 核酸」はまた、本明細書において、遺伝子、 cDNA、 mRNA、オリゴヌクレオチド、お よびポリヌクレオチドと互換可能に使用される。特定の核酸配列はまた、「スプライス 改変体」を包含する。同様に、核酸によりコードされた特定のタンパク質は、その核酸 のスプライス改変体によりコードされる任意のタンパク質を暗黙に包含する。その名が 示唆するように「スプライス改変体」は、遺伝子のオルタナティブスプライシングの産物 である。転写後、最初の核酸転写物は、異なる（別の)核酸スプライス産物が異なるポ リペプチドをコードするようにスプライスされ得る。スプライス改変体の産生機構は変 化するが、ェキソンのオルタナティブスプライシングを含む。読み過し転写により同じ 核酸に由来する別のポリペプチドもまた、この定義に包含される。スプライシング反応 の任意の産物 (組換え形態のスプライス産物を含む）がこの定義に含まれる。 [0078] As used herein, the terms "polynucleotide", "oligonucleotide" and "nucleic acid" are used interchangeably herein and refer to a nucleotide polymer of any length. The term also includes “derivative oligonucleotide” or “derivative polynucleotide”. “Derivative oligonucleotide” or “derivative polynucleotide” refers to an oligonucleotide or polynucleotide that includes a derivative of a nucleotide or that has unusual linkages between nucleotides, and is used interchangeably. Specific examples of such oligonucleotides include, for example, 2 ′ O-methyl-ribonucleotides, derivative oligonucleotides in which a phosphodiester bond in an oligonucleotide is converted to a phosphoroate bond, and phosphorous in an oligonucleotide. Derivative oligonucleotide in which acid diester bond is converted to N3, P5, phosphoramidate bond, derivative oligonucleotide in which ribose and phosphodiester bond in oligonucleotide are converted to peptide nucleic acid bond, uracil in oligonucleotide is C Derivative oligonucleotides substituted with 5-propyluracil, uracil in oligonucleotide replaced with C-5 thiazoleuracil, cytosine in oligonucleotide replaced with C-5 propynylcytosine Derivative oligonucleotides, derivative oligonucleotides in which the cytosine in the oligonucleotide is replaced with phenoxazine-modified cytosine oligonucleotides, derivative oligonucleotides in which the ribose in the oligonucleotide is replaced with 2, 1 O-propyl ribose, and Examples include derivative oligonucleotides in which the ribose in the oligonucleotide is substituted with 2, -methoxyethoxy ribose. Else Unless otherwise indicated, a particular nucleic acid sequence also includes conservatively modified variants (e.g., degenerate codon substitutes) and complementary sequences, as well as those explicitly indicated. It is contemplated. Specifically, a degenerate codon substitute creates a sequence in which the third position of one or more selected (or all) codons is replaced with a mixed base and a Z or doxyinosine residue. (Batzer et al., Nucleic Acid Res., 19: 5081, 1991; Ohtsuka¾, J. Biol. Chem., 260: 26 05-2608, 1985; Rossolini et al., Mol. Cell. Probes, 8: 91 — 98, 1994). The term “nucleic acid” is also used herein interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide. Particular nucleic acid sequences also include “splice variants”. Similarly, a particular protein encoded by a nucleic acid implicitly encompasses any protein encoded by a splice variant of that nucleic acid. As the name suggests, a “splice variant” is the product of alternative splicing of a gene. After transcription, the initial nucleic acid transcript can be spliced such that different (alternate) nucleic acid splice products encode different polypeptides. The production mechanism of splice variants varies, but includes exon alternative splicing. Other polypeptides derived from the same nucleic acid by read-through transcription are also encompassed by this definition. Any product of a splicing reaction (including recombinant forms of splice products) is included in this definition.
本明細書において「遺伝子」とは、遺伝形質を規定する因子をいう。通常染色体上 に一定の順序に配列して ヽる。タンパク質の一次構造を規定する遺伝子を構造遺伝 子といい、その発現を左右するものを調節遺伝子という。本明細書では、「遺伝子」は 、「ポリヌクレオチド」、「オリゴヌクレオチド」および「核酸」をさすことがある。本明細書 において遺伝子の「相同性」とは、 2以上の遺伝子配列の、互いに対する同一性の程 度をいう。従って、ある 2つの遺伝子の相同性が高いほど、それらの配列の同一性ま たは類似性は高い。 2種類の遺伝子が相同性を有する力否かは、配列の直接の比 較、または核酸の場合ストリンジェントな条件下でのハイブリダィゼーシヨン法によって 調べられ得る。 2つの遺伝子配列を直接比較する場合、その遺伝子配列間で DNA 配列が、代表的には少なくとも 50%同一である場合、好ましくは少なくとも 70%同一 である場合、より好ましくは少なくとも 80%、 90%、 95%、 96%、 97%、 98%または 99%同一である場合、それらの遺伝子は相同性を有する。 As used herein, “gene” refers to a factor that defines a genetic trait. Usually arranged on a chromosome in a certain order. A gene that defines the primary structure of a protein is called a structural gene, and a gene that affects its expression is called a regulatory gene. As used herein, “gene” may refer to “polynucleotide”, “oligonucleotide”, and “nucleic acid”. As used herein, “homology” of a gene refers to the degree of identity of two or more gene sequences to each other. Therefore, the higher the homology between two genes, the higher the sequence identity or similarity. The ability of two genes to have homology can be determined by direct sequence comparison or, in the case of nucleic acids, hybridization methods under stringent conditions. When directly comparing two gene sequences, the DNA sequence between the gene sequences is typically at least 50% identical, preferably at least 70% identical, more preferably at least 80%, 90% 95%, 96%, 97%, 98% or If they are 99% identical, they have homology.
[0080] 本明細書では塩基配列の同一性の比較および相同性の算出は、配列分析用ツー ルである BLASTを用いてデフォルトパラメータを用いて算出される。  [0080] In the present specification, comparison of base sequence identity and homology calculation are performed using BLAST, which is a tool for sequence analysis, using default parameters.
[0081] 本明細書にぉ 、て遺伝子、ポリヌクレオチド、ポリペプチドなどの「発現」とは、その 遺伝子など力 Sインビボで一定の作用を受けて、別の形態になることをいう。好ましくは 、遺伝子、ポリヌクレオチドなど力 転写および翻訳されて、ポリペプチドの形態にな ることをいうが、転写されて mRNAが作製されることもまた発現の一態様であり得る。 より好ましくは、そのようなポリペプチドの形態は、翻訳後プロセシングを受けたもので あり得る。  [0081] As used herein, "expression" of a gene, polynucleotide, polypeptide or the like means that the gene or the like is subjected to a certain action in vivo to take another form. Preferably, it refers to force transcription and translation of genes, polynucleotides, and the like to form a polypeptide, but transcription and production of mRNA may also be an embodiment of expression. More preferably, such polypeptide forms may be post-translationally processed.
[0082] 本明細書にぉ 、て「ヌクレオチド」は、天然のものでも非天然のものでもよ 、。「誘導 体ヌクレオチド」または「ヌクレオチドアナログ」とは、天然に存在するヌクレオチドとは 異なるがもとのヌクレオチドと同様の機能を有するものをいう。そのような誘導体ヌクレ ォチドおよびヌクレオチドアナログは、当該分野において周知である。そのような誘導 体ヌクレオチドおよびヌクレオチドアナログの例としては、ホスホロチォエート、ホスホ ルアミデート、メチルホスホネート、キラルメチルホスホネート、 2— 0—メチルリボヌク レオチド、ペプチド 核酸 (PNA)が含まれる力 これらに限定されない。  [0082] As used herein, "nucleotide" may be natural or non-natural. “Derivative nucleotide” or “nucleotide analog” refers to a nucleotide that is different from a naturally occurring nucleotide but has the same function as the original nucleotide. Such derivative nucleotides and nucleotide analogs are well known in the art. Examples of such derivative nucleotides and nucleotide analogs include, but are not limited to, phosphoroates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2-0-methyl ribonucleotides, peptide nucleic acids (PNA).
[0083] 本明細書にぉ 、て、「フラグメント」とは、全長のポリペプチドまたはポリヌクレオチド（ 長さが n)に対して、 l〜n— 1までの配列長さを有するポリペプチドまたはポリヌクレオ チドをいう。フラグメントの長さは、その目的に応じて、適宜変更することができ、例え ば、その長さの下限としては、ポリペプチドの場合、 3、 4、 5、 6、 7、 8、 9、 10、 15, 2 0、 25、 30、 40、 50およびそれ以上のアミノ酸が挙げられ、ここの具体的に列挙して いない整数で表される長さ (例えば、 11など)もまた、下限として適切であり得る。また 、ポリヌクレオチドの場合、 5、 6、 7、 8、 9、 10、 15, 20、 25、 30、 40、 50、 75、 100 およびそれ以上のヌクレオチドが挙げられ、ここの具体的に列挙して!/、な!/、整数で表 される長さ（例えば、 11など)もまた、下限として適切であり得る。  [0083] As used herein, "fragment" refers to a polypeptide or polynucleotide having a sequence length of 1 to n-1 with respect to a full-length polypeptide or polynucleotide (length n). Say Chido. The length of the fragment can be changed as appropriate according to its purpose. For example, the lower limit of the length is 3, 4, 5, 6, 7, 8, 9, 10 in the case of a polypeptide. , 15, 2, 0, 25, 30, 40, 50 and more, and lengths expressed in integers not specifically listed here (for example, 11 etc.) are also suitable as lower limits. It can be. In the case of polynucleotides, examples include 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100 and more nucleotides. !!, NA! /, An integer length (eg 11) may also be appropriate as a lower limit.
[0084] 本発明において利用され得る一般的な分子生物学的手法としては、 Ausubel F.  [0084] General molecular biological techniques that can be used in the present invention include Ausubel F. et al.
A.り編, し urrent Protocols in Molecular Biology, "Wiley, New YorK, N Y, 1988 ; Sambrook J.ら, Molecular Cloning： A Laboratory Manual, 2n d Ed. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1987などを参酌して当業者であれば容易に実施をすることができる。 A. Rihen, urrent Protocols in Molecular Biology, "Wiley, New YorK, NY, 1988; Sambrook J. et al., Molecular Cloning: A Laboratory Manual, 2n A person skilled in the art can easily carry out the operation with reference to Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1987 and the like.
[0085] 本明細書において遺伝子について言及する場合、「ベクター」とは、 目的のポリヌク レオチド配列を目的の細胞へと移入させることができるものをいう。そのようなベクター としては、原核生物細胞、酵母、動物細胞、植物細胞、昆虫細胞、動物個体および 植物個体等の宿主細胞、好ましくは植物細胞において自律複製が可能である力、ま たは染色体中への糸且込みが可能で、本発明のポリヌクレオチドの転写に適した位置 にプロモーターを含有して 、るものが例示される。 [0085] In the present specification, when referring to a gene, a "vector" refers to a vector that can transfer a target polynucleotide sequence to a target cell. Such vectors include prokaryotic cells, yeast, animal cells, plant cells, insect cells, host cells such as animal individuals and plant individuals, preferably those capable of autonomous replication in plant cells, or chromosomes. Examples thereof include those that contain a promoter at a position suitable for transcription of the polynucleotide of the present invention.
[0086] 「発現ベクター」は、構造遺伝子およびその発現を調節するプロモーターに加えて 種々の調節エレメントが宿主の細胞中で作動し得る状態で連結されて 、る核酸配列 をいう。調節エレメントは、好ましくは、ターミネータ一、薬剤耐性遺伝子のような選択 マーカーおよび、ェンハンサーを含み得る。生物（例えば、植物）の発現ベクターのタ イブおよび使用される調節エレメントの種類が、宿主細胞に応じて変わり得ることは、 当業者に周知の事項である。選抜のための選択マーカーとしては、抗生物質カナマ イシンに対する耐性を与える酵素ネオマイシンホスホトランスフェラーゼをコードする n eo遺伝子（Beckら， Gene, 19 : 327, 1982 ;抗生物質ハイグロマイシンに対する耐 性を与える酵素ハイグロマイシンホスホトランスフェラーゼをコードする hyg遺伝子 (G ritz及び Davies, Gene, 25 : 179, 1983)；及び除草剤ホスフィノトリシン（phosphi nothricin)に対する而性を与えるホスフィノトリシンァセチルトランスフェラーゼをコ一 ドする bar遺伝子（ΕΡ 242236)；ストレプトマイシンフォスフォトランスフェラーゼをコ ードする spt遺伝子；ストレプトマイシン耐性遺伝子；スぺクチノマイシン耐性遺伝子な どの薬剤而ォ性遺伝子（例えば、 H. S. Chawla, Introduction to Plant Biotec hnology 2nd: 363, Science Publishers, Inc. ,単行本， 2002)；ならびに j8 - グノレクロ-ダーゼをコードする gus遺伝子 (Jeffersonら， Proc. Natl. Acad. Sci. U SA, 6 : 3901, 1986)およびルシフェラーゼ遺伝子（Owら， Science, 234 : 856, 1 986)、および GFP (緑色蛍光タンパク質)コード遺伝子 (例えば、フナコシ株式会社 、東京都文京区本郷から入手可能)のようなスクリーン可能なマーカー遺伝子が挙げ られる力 これらに限定されない。 [0087] 本発明において選抜に使用する薬剤としては、カナマイシン、ノ、イダロマイシン、ジ エネティシン、ゲンタマイシン、ストレプトマイシン、スぺクチノマイシンが挙げられるが これらに限定されない。 [0086] "Expression vector" refers to a nucleic acid sequence in which various regulatory elements are operably linked in a host cell in addition to a structural gene and a promoter that regulates its expression. The regulatory element may preferably include a terminator, a selectable marker such as a drug resistance gene, and an enhancer. It is well known to those skilled in the art that the type of organism (eg, plant) expression vector and the type of regulatory element used can vary depending on the host cell. As a selection marker for selection, the neo gene encoding the enzyme neomycin phosphotransferase that confers resistance to the antibiotic kanamycin (Beck et al., Gene, 19: 327, 1982; the enzyme hygromycin that confers resistance to the antibiotic hygromycin. The hyg gene encoding mycin phosphotransferase (Gritz and Davies, Gene, 25: 179, 1983); and the phosphinotricin acetyltransferase that gives metaphysics to the herbicide phosphi nothricin bar Genes (ΕΡ 242236); spt genes that code for streptomycin phosphotransferase; streptomycin resistance genes; pharmacological genes such as spectinomycin resistance genes (eg, HS Chawla, Introduction to Plant Biotec hnology 2nd: 363, Science Publishers, Inc., Book, 20 02); and the gus gene (Jefferson et al., Proc. Natl. Acad. Sci. U SA, 6: 3901, 1986) and the luciferase gene (Ow et al., Science, 234: 856, 1 986) encoding j8-gnorecrodase. ), And GFP (green fluorescent protein) encoding gene (for example, available from Funakoshi Co., Ltd., Hongo, Bunkyo-ku, Tokyo), and the like. [0087] Examples of the drug used for selection in the present invention include, but are not limited to, kanamycin, no, idaromomycin, dienetin, gentamicin, streptomycin, and spectinomycin.
[0088] 「組換えベクター」とは、 目的のポリヌクレオチド配列を目的の細胞へと移入させるこ とができるベクターをいう。そのようなベクターとしては、植物細胞、および植物個体 等の宿主細胞において自立複製が可能、または染色体中への組込みが可能で、本 発明のポリヌクレオチドの転写に適した位置にプロモーターを含有しているものが例 示される。  [0088] "Recombinant vector" refers to a vector capable of transferring a target polynucleotide sequence into a target cell. Such a vector can be autonomously replicated in a plant cell and a host cell such as a plant individual, or can be integrated into a chromosome, and contains a promoter at a position suitable for transcription of the polynucleotide of the present invention. Examples are shown.
[0089] 植物細胞に対する「組換えベクター」としては、 Tiプラスミド、タバコモザイクウィルス ベクター、ジエミ-ウィルスベクターなどが例示される。  [0089] "Recombinant vectors" for plant cells include Ti plasmids, tobacco mosaic virus vectors, diemi-virus vectors, and the like.
[0090] 「ターミネータ一」は、遺伝子のタンパク質をコードする領域の下流に位置し、 DNA が mRNAに転写される際の転写の終結、ポリ A配列の付加に関与する配列である。 ターミネータ一は、 mRNAの安定性に関与して遺伝子の発現量に影響を及ぼすこと が知られている。ターミネータ一としては、 CaMV35Sターミネータ一、ノパリン合成 酵素遺伝子のターミネータ一（Tnos)、タバコ PRla遺伝子のターミネータ一が挙げら れるが、これに限定されない。本明細書において用いられる「プロモーター」とは、遺 伝子の転写の開始部位を決定し、またその頻度を直接的に調節する DNA上の領域 をいい、 RNAポリメラーゼが結合して転写を始める塩基配列である。プロモーターの 領域は、通常、推定タンパク質コード領域の第 1ェキソンの上流約 2kbp以内の領域 であることが多いので、 DNA解析用ソフトウェアを用いてゲノム塩基配列中のタンパ ク質コード領域を予測すれば、プロモータ領域を推定することはできる。推定プロモ 一ター領域は、通常構造遺伝子の上流にある力 好ましくは、推定プロモーター領域 は、第一ェキソン翻訳開始点から上流約 2kbp以内に存在する。  [0090] "Terminator 1" is a sequence that is located downstream of a region encoding a protein of a gene, and is involved in termination of transcription when DNA is transcribed into mRNA and addition of a poly A sequence. It is known that the terminator influences the expression level of a gene by being involved in mRNA stability. Examples of the terminator include, but are not limited to, a CaMV35S terminator, a nopaline synthase gene terminator (Tnos), and a tobacco PRla gene terminator. As used herein, the term “promoter” refers to a region on DNA that determines the start site of gene transcription and directly regulates its frequency, and is the base on which RNA polymerase binds and initiates transcription. Is an array. Since the promoter region is usually a region within about 2 kbp upstream of the first exon of the putative protein coding region, if the protein coding region in the genomic nucleotide sequence is predicted using DNA analysis software, The promoter region can be estimated. The putative promoter region is usually a force upstream of the structural gene. Preferably, the putative promoter region is present within about 2 kbp upstream from the first exon translation start point.
[0091] 本明細書において、遺伝子の発現について用いられる場合、一般に、「部位特異 性」とは、生物 (例えば、植物)の部位 (例えば、植物の場合、根、茎、幹、葉、花、種 子、胚芽、胚、果実など）におけるその遺伝子の発現の特異性をいう。「時期特異性」 とは、生物 (たとえば、植物）の発達段階 (例えば、植物であれば生長段階 (例えば、 発芽後の芽生えの日数）に応じたその遺伝子の発現の特異性をいう。そのような特異 性は、適切なプロモーターを選択することによって、所望の生物に導入することがで きる。 [0091] In the present specification, when used for gene expression, "site specificity" generally means a part of an organism (for example, a plant) (for example, in the case of a plant, a root, stem, stem, leaf, flower). , Seed, germ, embryo, fruit, etc.). “Time specificity” refers to the specificity of expression of a gene in accordance with the stage of development of an organism (eg, a plant) (eg, if a plant, the growth stage (eg, days after germination)). Idiosyncratic Sex can be introduced into a desired organism by selecting an appropriate promoter.
[0092] 本明細書において、本発明のプロモーターの発現が「構成的」であるとは、生物の すべての糸且織にぉ 、て、その生物の生長の幼若期または成熟期の 、ずれにあって もほぼ一定の量で発現される性質をいう。具体的には、本明細書の実施例と同様の 条件でノーザンプロット分析したとき、例えば、任意の時点で (例えば、 2点以上 (例え ば、 5日目および 15日目））の同一または対応する部位のいずれにおいても、ほぼ同 程度の発現量がみられるとき、本発明の定義上、発現が構成的であるという。構成的 プロモーターは、通常の生育環境にある生物の恒常性維持に役割を果たしていると 考えられる。本発明のプロモーターの発現が「ストレス応答性」であるとは、少なくとも 1つのストレスが生物体に与えられたとき、その発現量が変化する性質をいう。特に、 発現量が増加する性質を「ストレス誘導性」といい、発現量が減少する性質を「ストレ ス抑制性」という。「ストレス抑制性」の発現は、正常時において、発現が見られること を前提としているので、「構成的」な発現と重複する概念である。これらの性質は、生 物の任意の部分から RNAを抽出してノーザンプロット分析で発現量を分析することま たは発現されたタンパク質をウェスタンプロットにより定量することにより決定すること ができる。ストレス誘導性のプロモーターを本発明のポリペプチドをコードする核酸と ともに組み込んだベクターで形質転換された植物または植物の部分 (特定の細胞、 組織など）は、そのプロモーターの誘導活性をもつ刺激因子を用いることにより、ある 条件下での特定の遺伝子の発現を行うことができる。  [0092] In the present specification, the expression of the promoter of the present invention is "constitutive" means that, in all yarns and weaves of an organism, the growth of the organism is in the juvenile or mature stage. Even so, it is a property that is expressed in an almost constant amount. Specifically, when Northern Plot analysis is performed under the same conditions as in the examples of the present specification, for example, at the same time (for example, two or more points (for example, 5th day and 15th day)) Expression is said to be constitutive by the definition of the present invention when almost the same level of expression is observed at any of the corresponding sites. Constitutive promoters are thought to play a role in maintaining the homeostasis of organisms in normal growth environments. The expression of the promoter of the present invention being “stress responsive” refers to the property that the expression level changes when at least one stress is applied to an organism. In particular, the property of increasing the expression level is called “stress inducibility”, and the property of decreasing the expression level is called “stress suppression”. The expression of “stress suppression” is a concept that overlaps with the “constitutive” expression because it is assumed that the expression is observed in the normal state. These properties can be determined by extracting RNA from any part of the organism and analyzing the expression level by Northern plot analysis or quantifying the expressed protein by Western plot. Plants or plant parts (specific cells, tissues, etc.) transformed with a vector incorporating a stress-inducible promoter together with a nucleic acid encoding the polypeptide of the present invention contain a stimulating factor having inducibility of the promoter. By using it, a specific gene can be expressed under certain conditions.
[0093] 「ェンノヽンサ一」は、目的遺伝子の発現効率を高めるために用いられ得る。植物に おいて使用する場合、ェンハンサ一としては、 CaMV35Sプロモーター内の上流側 の配列を含むェンノヽンサ一領域が好ましい。ェンノヽンサ一は複数個用いられ得るが 1個用いられてもよいし、用いなくともよい。  “0093” can be used to increase the expression efficiency of a target gene. When used in plants, the enhancer is preferably an enzyme region containing an upstream sequence in the CaMV35S promoter. A plurality of sensors can be used, but one may or may not be used.
[0094] 本明細書にぉ 、て「作動可能に連結された (る）」とは、所望の配列の発現 (作動） がある転写翻訳調節配列（例えば、プロモーター、ェンノ、ンサ一など)または翻訳調 節配列の制御下に配置されることをいう。プロモーターが遺伝子に作動可能に連結 されるためには、通常、その遺伝子のすぐ上流にプロモーターが配置される力 プロ モーターと構造遺伝子との間に介在する配列が存在してもよいため、プロモーターと 構造遺伝子とは必ずしも隣接して配置される必要はない。 [0094] As used herein, "operably linked" means a transcriptional translational regulatory sequence (eg, promoter, henno, sensor, etc.) that has the expression (operation) of a desired sequence or It means being placed under the control of the translation arrangement. In order for a promoter to be operably linked to a gene, it is usually necessary to place the promoter immediately upstream of that gene. Since there may be a sequence intervening between the motor and the structural gene, the promoter and the structural gene are not necessarily arranged adjacent to each other.
[0095] 導入した遺伝子の存在は、サザンプロット法または PCR法によって確認し得る。導 入した遺伝子の転写は、ノーザンプロット法または PCR法により、検出し得る。必要に 応じて、遺伝子産物たるタンパク質の発現を、例えば、ウェスタンプロット法により確認 し得る。 [0095] The presence of the introduced gene can be confirmed by Southern plotting or PCR. Transcription of the introduced gene can be detected by Northern plotting or PCR. If necessary, the expression of a protein as a gene product can be confirmed by, for example, a Western plot method.
[0096] 以下に、実施例に基づいて本発明を説明するが、以下の実施例は、例示の目的の みに提供される。従って、本発明の範囲は、上記発明の詳細な説明にも下記実施例 にも限定されるものではなぐ請求の範囲によってのみ限定される。  [0096] Hereinafter, the present invention will be described based on examples, but the following examples are provided for illustrative purposes only. Accordingly, the scope of the present invention is limited only by the scope of the claims which are not limited to the above detailed description of the invention nor the following examples.
実施例  Example
[0097] (エレクト口ポーレーシヨンの装置）  [0097] (Elect Mouth Polarization Device)
本発明に用いたエレクト口ポーレーシヨンの装置には、市販のエレクト口ポーレーシ ヨン手段 (例えば、 CUY21EDIT遺伝子導入装置、ネッパジーン社、千葉県巿川巿 )を使用し得る。本実施例においてエレクト口ポーレーシヨンを行う際に使用されるェ レクト口ポーレーシヨンチャンバ一は、形質転換の対象となる植物組織を収容できるも のであれば、どのような大きさであってもよいが、冷却可能なチャンバ一が好ましい。 本実施例では、白金電極を備える縦 lcm X横 2cm X高さ 2cmの特注のエレクトロポ 一レーシヨンチャンバ一を使用した。  A commercially available electopore polarization means (for example, CUY21EDIT gene transfer device, Neppagene, Inc., Yodogawa, Chiba) can be used for the device for the electopore polarization used in the present invention. In the present embodiment, the electopore polarization chamber used for performing the electopore polarization may have any size as long as it can accommodate the plant tissue to be transformed. A coolable chamber is preferred. In this example, a custom-made electroporation chamber having a platinum electrode and a length of 1 cm × width 2 cm × height 2 cm was used.
[0098] (エレクト口ポーレーシヨン）  [0098] (Elect mouth polarization)
減圧処理の後、シャーレの中の種子と緩衝液をチャンバ一に移し、氷上に 1分間静 置する。そして、実施例に示すように、 100Vあるいは 50Vの電圧で (ただし、電極間 の距離は lcm)、パルス幅 50ミリ秒の矩形波（50ミリ秒の間、電圧を加え、 50ミリ秒〜 75ミリ秒休む周期を繰り返す）、パルス回数 50回あるいは 99回行う。さらに氷上にチ ヤンバーを 2分間静置した後、緩衝液を捨て、元のシャーレに処理した種子を戻す。 この種子を戻したシャーレに、 2mlの 0. 5%ポリビュルピロリドン（PVP)水溶液を入 れる。この PVP水溶液は、雑菌の繁殖を抑えるために次亜塩素酸ナトリウム水溶液を 含む (有効塩素濃度が約 0. 01%となるように調整した)。  After decompression, transfer seeds and buffer in the petri dish to chamber 1 and leave on ice for 1 minute. Then, as shown in the examples, at a voltage of 100 V or 50 V (however, the distance between the electrodes is 1 cm), a rectangular wave with a pulse width of 50 milliseconds (applied voltage for 50 milliseconds, 50 milliseconds to 75 seconds) Repeat the cycle of resting for milliseconds), and repeat the pulse 50 times or 99 times. After leaving the chamber on ice for 2 minutes, discard the buffer and return the treated seed to the original petri dish. Into the petri dish where these seeds have been returned, 2 ml of 0.5% polybulurpyrrolidone (PVP) aqueous solution is added. This aqueous PVP solution contains an aqueous sodium hypochlorite solution (supplied so that the effective chlorine concentration is about 0.01%) in order to suppress the growth of bacteria.
シャーレを 4°Cで約 1時間保存した後、 25°Cでー晚静置する。 [0099] (GUS分析） Store the petri dish at 4 ° C for about 1 hour, then leave it at 25 ° C. [0099] (GUS analysis)
X— Glue液（100 mM pH7. 0 リン酸緩衝液、 0. 05% X— Gluc (5—ブロモ —4—クロ口一 3—インドリル一 β—D—グルクロ-ド シクロへキシルアンモ -ゥム塩） 、 0. 5mM フェリシアンィ匕カリウム、 0. 5mM フエロシアンィ匕カリウム、 0. 3% トリト ン X—100、 20% メタノール）を 2mlカ卩え、 25°Cでー晚静置した。染色の程度から G US遺伝子の発現を確認した。  X—Glue solution (100 mM pH 7.0, phosphate buffer, 0.05% X— Gluc (5-Bromo —4—Black mouth 3—Indolyl 1 β—D—Glucuroro cyclohexylammonum salt) 2 ml) of 0.5 mM ferricyanium potassium, 0.5 mM ferrocyan potassium, 0.3% triton X-100, 20% methanol), and allowed to stand at 25 ° C. The expression of the GUS gene was confirmed from the degree of staining.
[0100] (抗生物質培地による選抜） [0100] (Selection with antibiotic medium)
GUS分析を行わず、以下のように生育させる。  GUS analysis is carried out as follows.
a) 7cmのろ紙を敷いた 9cm X 15mmのシャーレに、種子を移す。  a) Transfer the seeds to a 9cm x 15mm petri dish with 7cm filter paper.
b)蒸留水に溶力した抗生物質の水溶液を 10ml添加する。コムギの種子をジエネティ シンで選抜するときは濃度 2000ppmまたは 1200ppmを用いる。なお、イネの種子 をジエネティシンで選抜するときの濃度は、 200ppmである。  b) Add 10 ml of an aqueous solution of antibiotics dissolved in distilled water. When selecting wheat seeds with dienetin, use a concentration of 2000 ppm or 1200 ppm. The concentration of rice seeds selected by dieneticin is 200 ppm.
c)培養室で、 25°C 明期 16時間（約 2000ルクス） 暗期 8時間の条件下または 20 °C 明期 8時間（約 2000ルクス）および 8°C 暗期 16時間の条件下にて、育成する。  c) In a culture room, 25 ° C, light period 16 hours (approx. 2000 lux), dark period 8 hours, or 20 ° C light period 8 hours (approx. 2000 lux) and 8 ° C, dark period 16 hours. And nurture.
[0101] (形質転換体の育成）  [0101] (Raising transformants)
抗生物質培地による選抜の後、植物を、園芸用培土 (ニューマジックソィル;株式会 社サカタのタネ、横浜巿都筑区）を入れた直径 8. 5cm、高さ 5. 5cmのポット (植木鉢 )を用いて、隔離型のダロスチャンバ一で育成する（15°C 明期 8時間けトリウムラン プ、約 50000ルクス）、暗期 16時間の条件または 20°C 明期 8時間（約 2000ルクス） および 8°C 暗期 16時間の条件)。  After selection with antibiotic medium, the plant is horticultural soil (New Magic Soil; Sakata Seed Co., Ltd., Sakai Tsuzuki-ku, Yokohama). A pot of 8.5 cm in diameter and 5.5 cm in height (flowerpot) Cultivated in an isolated dalos chamber (15 ° C light period 8 hour thorium lamp, approximately 50,000 lux), dark period 16 hours condition or 20 ° C light period 8 hours (approximately 2000 lux) And 8 ° C dark period 16 hours).
[0102] (DNAの抽出）  [0102] (DNA extraction)
遺伝子導入を確認する PCRを行うために、形質転換植物の葉および茎を採取して 、 DNAを抽出する。 DNAの抽出には、任意の周知の方法を使用し得る。代表的な DNA抽出方法は、 CTAB法である（内宫博文「植物遺伝子操作マニュアル トランス ジエニック植物の作り方」講談社サイエンティフィック， 71— 74頁，単行本， 1989)。  In order to perform PCR to confirm gene transfer, the leaves and stems of the transformed plant are collected and DNA is extracted. Any known method may be used for DNA extraction. A typical DNA extraction method is the CTAB method (Hirofumi Uchibuchi “Plant Gene Manipulation Manual How to Make Transgenic Plants” Kodansha Scientific, pages 71-74, book, 1989).
[0103] (PCR分析）  [0103] (PCR analysis)
エレクト口ポーレーシヨン後の個体の葉から DNAを抽出し、 NPT II遺伝子を検出 する一対のプライマー（5 ' - ctgcgtgcaatccatcttg - 3 '：配歹幡号 1、 5'— actcgtc aagaaggcgatagaag- 3 '：配列番号 2)を用いて PCRを行った。また、さらなる一対 のプフイマ ~~ (5 ― catgattgaacaagatggattgcacgcaggttctc― 3 ：目 ti列番 d、 5 ― cagaagaactcgtcaagaaggcgatagaaggcgat— ：酉己列 ¾> 4)もまた使用され得 、このプライマー対は、より特異的に NPT II遺伝子を検出し得るので、より好ましい。 ポリメラーゼは、株式会社パーキンエルマ一ジャパン（横浜巿西区）の AmpliTaq G oldを、製造業者の指示に従って、使用する。増幅に使用するサーマルサイクラ一の 条件は： A pair of primers (5 '-ctgcgtgcaatccatcttg-3': Cat No. 1 and 5'—actcgtc) that extract DNA from the leaves of individuals after electoral poration and detect the NPT II gene PCR was performed using aagaaggcgatagaag-3 ′: SEQ ID NO: 2). An additional pair of Phuima ~~ (5-catgattgaacaagatggattgcacgcaggttctc-3: eye ti column number d, 5-cagaagaactcgtcaagaaggcgatagaaggcgat-: 酉 Self column ¾> 4) can also be used, and this primer pair is more specifically NPT II Since a gene can be detected, it is more preferable. As the polymerase, AmpliTaq Gold from Perkin Elma Japan Co., Ltd. (Yokohama Shaanxi Ward) is used according to the manufacturer's instructions. The conditions of the thermal cycler used for amplification are:
(a) 95°C 10分間を 1サイクル；  (a) 1 cycle at 95 ° C for 10 minutes;
(b) 95°C 1分間、 64°C 2分間、 72°C 2分間を 50サイクル；  (b) 50 cycles of 95 ° C for 1 minute, 64 ° C for 2 minutes, 72 ° C for 2 minutes;
(c) 72°C 7分間を 1サイクル；および  (c) one cycle of 72 ° C for 7 minutes; and
(d)その後 4°Cにて保存  (d) Then store at 4 ° C
である。  It is.
[0104] (サザンブロット分析）  [0104] (Southern blot analysis)
エレクト口ポーレーシヨン後、上記 PCR分析により導入遺伝子の存在が確認される 場合、その個体由来の DNAを用いて、サザンプロット分析を行う。サザンプロット法 は当該分野において周知であり、当業者は、その条件を必要に応じて適宜選択し得 る。本実施例のサザンプロット分析では、導入した NPT II遺伝子に特異的な配列（ 配列表において、配列番号 5として示される）を、プローブとして使用する。  If the presence of the transgene is confirmed by PCR analysis after electoporation, perform Southern plot analysis using DNA from the individual. Southern plotting is well known in the art, and those skilled in the art can appropriately select the conditions as necessary. In the Southern plot analysis of this example, a sequence specific to the introduced NPT II gene (shown as SEQ ID NO: 5 in the sequence listing) is used as a probe.
[0105] (遺伝解析） [0105] (Genetic analysis)
サザンプロット分析により導入遺伝子が確認される場合、その植物については、さら に生育させ、自家受粉により多数の完熟種子を得る。その中力もランダムに約 10個 の種子を取り、土を詰めたポットで栽培する。幼植物の葉力 DNAを抽出し、この D NAをテンプレートとして上記 PCR分析に記載の条件下で PCRを行う。  If the transgene is confirmed by Southern plot analysis, the plant is further grown and a large number of mature seeds are obtained by self-pollination. The medium strength is also randomly picked from about 10 seeds and cultivated in pots filled with soil. The leaf force DNA of the young plant is extracted, and PCR is performed under the conditions described in the above PCR analysis using this DNA as a template.
(実施例 1：本発明の方法によるイネ植物の形質転換）  (Example 1: Transformation of rice plant by the method of the present invention)
(材料）  (Material)
ジャポニカイネの完熟種子として、コシヒカリの玄米を使用した。コムギ種子として、 農林 61号を使用した。インディ力イネの完熟種子として、 IR24の玄米を使用した。ハ クサイ種子として、「無双」を使用した。ダイズ種子として、「Fayatte」を使用した。トマ ト種子として、「ミニキャロル」を使用した。アサガオ種子として、「サンスマイル曜白大 輪」を使用した。種子は当研究所で生育したもの、あるいは種苗会社から購入したも のを用いた。目視により適当な完熟種子を約 10〜30粒選択し (種子の大きさにより 異なる）、水道水中にて 25°Cで一晩吸水させた。好ましくは、吸水に使用される水道 水は、雑菌の繁殖を抑えるために次亜塩素酸ナトリウム水溶液を含む (有効塩素濃 度が約 0. 01%となるように調整する)。コムギの形質転 ^¾物体作出を目的とした実 験においては、この吸水は 10°Cでニ晚にわたつて行った。ァグロバタテリゥムとして、 EHA150菌を使用した。 Brown rice of Koshihikari was used as a ripe seed of japonica rice. Agricultural forest No. 61 was used as a wheat seed. IR24 brown rice was used as a ripe seed of indy power rice. As a Chinese cabbage seed, “Musou” was used. “Fayatt e ” was used as soybean seeds. Toma As a seed, “Mini Carol” was used. As the morning glory seeds, “Sun Smile White Circle” was used. The seeds grown at this laboratory or purchased from a seed company were used. About 10-30 appropriate mature seeds were selected by visual inspection (depending on the size of the seeds), and water was absorbed overnight at 25 ° C in tap water. Preferably, the tap water used for water absorption contains an aqueous sodium hypochlorite solution (adjusted so that the effective chlorine concentration is about 0.01%) in order to suppress the growth of various bacteria. In an experiment aimed at the transformation of wheat, the water absorption was conducted at 10 ° C for two days. EHA150 was used as agrobacterium.
[0106] なお、完熟種子をァグロバタテリゥム菌液に浸す時間を長くすることによって、吸水 工程に代えることも可能である。  [0106] It is possible to replace the water-absorbing step by increasing the time for immersing the mature seed in the agrobacterium bacterium solution.
[0107] (種子の滅菌）  [0107] (Seed sterilization)
70%エタノールで 30秒、続けて 2. 5%次亜塩素酸ナトリウム溶液で 20分間滅菌し た後、滅菌水で洗浄した。  Sterilized with 70% ethanol for 30 seconds, followed by 2.5% sodium hypochlorite solution for 20 minutes, and then washed with sterile water.
[0108] (ベクター）  [0108] (Vector)
形質転換細胞を選抜するマーカー遺伝子であるハイグロマイシン耐性遺伝子と、 形質転換細胞のレポーターとなる GFP (Green Fluorescent Protein)遺伝子を T— DNA上に有するバイナリーベクター pCAMBIA1390— sGFP (図 1)を Agroba cterium tumefaciens EHA105菌に形質転換し実験に用いた。  A binary vector pCAMBIA1390—sGFP (Fig. 1) containing a hygromycin resistance gene, a marker gene for selecting transformed cells, and a GFP (Green Fluorescent Protein) gene, which serves as a reporter for transformed cells, on T-DNA. Agroba cterium tumefaciens EHA105 bacteria were transformed and used for experiments.
[0109] (形質転換）  [0109] (Transformation)
蒸留水中に懸濁し、 OD =約 0. 5に調製したァグロバタテリゥムを、 1時間静置し  Suspend agrobataterum in OD = approx. 0.5 for 1 hour.
600  600
た。次に、 3995 μ Lのァグロノくクテリゥム菌液に、 1 μ Lの Silwet L— 77および 4 μ Lの lOmg/mlァセトシリンゴンを添カ卩した。この菌液に種子を入れ、 1時間、減圧下（ 0. 096MPa)に置いた。必要に応じて、以下のエレクト口ポーレーシヨンを行った。  It was. Next, 39 μL of agronocuterum solution was supplemented with 1 μL of Silwet L-77 and 4 μL of lOmg / ml acetosyringone. Seeds were put into this bacterial solution and placed under reduced pressure (0.096 MPa) for 1 hour. The following electo-portionation was performed as necessary.
[0110] (エレクト口ポーレーシヨン） [0110] (Elect Mouth Poration)
減圧処理の後、シャーレの中の種子と緩衝液をチャンバ一に移し、氷上に 1分間静 置した。そして、実施例に示すように、 50VZcmの電圧で (ただし、電極間の距離は lcm)、パルス幅 50ミリ秒の矩形波（50マイクロ秒電圧をカ卩え、 50ミリ秒休む周期を 繰り返す）、パルス回数 99回行った。さらに氷上にチャンバ一を 2分間静置した後、 緩衝液を捨て、元のシャーレに処理した種子を戻した。この種子を戻したシャーレに 、 2mlの 0. 5%ポリビュルピロリドン（PVP)水溶液を入れた。この PVP水溶液は、雑 菌の繁殖を抑えるために次亜塩素酸ナトリウム水溶液を含んだ (有効塩素濃度が約 0 . 01%となるように調整した)。シャーレを 4°Cで約 1時間保存した後、 25°Cでー晚静 し 7こ。 After decompression, the seeds and buffer in the petri dish were transferred to the chamber 1 and left on ice for 1 minute. And, as shown in the example, with a voltage of 50VZcm (however, the distance between the electrodes is lcm), and a rectangular wave with a pulse width of 50 milliseconds (capacitance of 50 microsecond voltage, repeating a period of resting for 50 milliseconds) The number of pulses was 99. After leaving the chamber on ice for 2 minutes, The buffer solution was discarded, and the treated seeds were returned to the original petri dish. 2 ml of 0.5% polybulurpyrrolidone (PVP) aqueous solution was placed in the petri dish where the seeds were returned. This PVP aqueous solution contained an aqueous sodium hypochlorite solution to suppress the growth of bacteria (adjusted so that the effective chlorine concentration was about 0.01%). Store the petri dish at 4 ° C for about 1 hour, then calm down at 25 ° C.
[0111] (遺伝子導入後の種子の処理）  [0111] (Seed treatment after gene transfer)
ァグロバタテリゥムによる感染処理の 4日後、 0. 5%次亜塩素酸ナトリウム溶液に種 子を移して、滅菌処理をした。イネについての、ァグロバタテリゥムによる感染処理後 6日目、 8日目、および 14日目の結果を図 2〜4に示す。コムギについての、ァグロバ クテリゥムによる感染処理後 8日目の結果を図 5に示す。  Four days after infection with agrobacterium, the seeds were transferred to a 0.5% sodium hypochlorite solution and sterilized. Figures 2 to 4 show the results of rice on the 6th, 8th, and 14th days after infection with agrobacterium. Figure 5 shows the results of wheat on the eighth day after infection with agrobacterium.
[0112] (ジャポニカイネの結果）  [0112] (Results of Japonica rice)
ァグロバタテリゥムによる処理の 6日後（図 2)、 8日後（図 3)、および 14日後（図 4) に GFPによる蛍光の観察を行った。ァグロバタテリゥムとエレクト口ポーレーシヨンを組 み合わせた遺伝子導入の結果、強い蛍光が確認された（図 2B、図 3B、および図 4B の各々の左側の個体）。また、遺伝子導入を行った個体中、約 50%の個体という高 い効率で GFPの発現を肉眼で確認した。従来、エレクト口ポーレーシヨンのような直 接的遺伝子導入法と、ァグロパクテリゥム法のような間接的遺伝子導入法を組み合わ せた例はないが、これらの方法を組み合わせることで、本実施例に示されるように高 い効率で、遺伝子導入ができたことは、全く予測され得な力つたことである。また、本 実施例に示されるような強い発現量も、全く予想されな力つたことである。その一方で 、減圧処理のみを行った場合は、 GFPの発現は確認されな力つた（図 2B、および図 3Bの各々の右側の個体)。  GFP fluorescence was observed 6 days (Fig. 2), 8 days (Fig. 3), and 14 days (Fig. 4) after treatment with agrobacterium. As a result of gene transfer combining agrobacterium and electoporation, strong fluorescence was confirmed (the individual on the left side of each of FIGS. 2B, 3B, and 4B). In addition, the expression of GFP was confirmed with the naked eye at a high efficiency of about 50% of the transgenic individuals. Conventionally, there has been no example of combining direct gene transfer methods such as electopore por- tion with indirect gene transfer methods such as agro-acterium method. As shown in Fig. 2, the gene transfer with high efficiency was quite unpredictable. In addition, a strong expression level as shown in the present example was completely unexpected. On the other hand, when only the decompression treatment was performed, the expression of GFP was not confirmed (the individual on the right side of each of FIGS. 2B and 3B).
[0113] エレクト口ポーレーシヨンを用いずに、減圧処理後にァグロバタテリゥムによる感染を 行 、遺伝子導入を行った場合の GFP発現量は、エレクト口ポーレーシヨンを行った場 合と比較して、低力つた（図 4Bの右側の個体)。また、エレクト口ポーレーシヨンを用い ずに、減圧処理後にァグロバタテリゥムによる感染を行い遺伝子導入を行った場合に は、約 5%の個体において、 GFPの発現を肉眼で確認した。従来のァグロバクテリウ ム法では、ァグロパクテリゥムによる感染を行う前に、対象となる細胞 (組織)を 2, 4— Dのような植物ホルモンによって処理 (前培養)することが必須であると考えられてい た。しかし、本実施例の結果は、そのような前培養を必要としない点において、迅速 性'簡便性において予測され得な力つた顕著な効果を有する。 [0113] The expression level of GFP in the case of gene transfer after infection with agrobacterium after decompression without using erect mouth poration is lower than that in the case where elect mouth poration is performed. It was strong (individual on the right side of Fig. 4B) In addition, when transfection was carried out with agrobacterium after depressurization without using erect mouth poration, the expression of GFP was confirmed with the naked eye in about 5% of individuals. In the conventional agrobacterial method, the target cells (tissues) are removed before infection with agrobacterium. It was considered essential to treat (pre-culture) with a plant hormone such as D. However, the results of this example have a significant effect that cannot be predicted in rapidity and simplicity in that no such pre-culture is required.
[0114] (コムギの結果）  [0114] (Wheat result)
エレクト口ポレーシヨン後、コムギ種子 100粒をエレクト口ポレーシヨン用バッファーと 共に 6cmシャーレに移し、 10°C暗黒の培養器に入れた。翌日、エレクト口ポレーショ ン用バッファーを捨てて、 500ppmカルべ-シリン液を 5mlカ卩えてァグロバタテリゥム の除菌を行った。引き続き 10°C暗黒の培養器で育成を続けた。さらに翌日（エレクト 口ポレーシヨン後 2日目）、形質転換体作出を目指してジエネティシンによる選抜を開 始した。カルべ-シリン液を捨てて、種子を 9cmシャーレに移した。 750ppmジエネテ イシン液 8mlと X 100ベンレート液を 2ml加えた。ベンレートは殺菌剤（農薬）でカビ の発生を予防する目的で添加した。  After electoral positioning, 100 wheat seeds were transferred to a 6 cm petri dish together with the buffer for electoric positioning and placed in a 10 ° C dark incubator. On the next day, the buffer for electoporation was discarded, and 5 ml of 500 ppm carbesilin solution was added to sterilize agrobataterum. The growth was continued in a dark incubator at 10 ° C. The next day (second day after electoral position), selection with geneticin was started with the aim of producing transformants. The carbe-silin solution was discarded and the seeds were transferred to a 9 cm petri dish. 8 ml of 750 ppm dienetine solution and 2 ml of X100 benrate solution were added. Benrate is a fungicide (agrochemical) added to prevent mold formation.
[0115] シャーレを照明付きの培養器に移して植物体を育成した。その条件は、以下のとお りである：蛍光灯照明 約 3000ルックス、温度 10°C、照明オンを 8時間、照明オフを 1 6時間。  [0115] The petri dish was transferred to a lighted incubator to grow plants. The conditions are as follows: fluorescent lighting approximately 3000 lux, temperature 10 ° C, lighting on for 8 hours, lighting off for 16 hours.
[0116] 約 2週間後に苗条部分に GFP蛍光が確認できる個体を選び出した、苗条部分に G FP蛍光が確認できる個体は約 5— 10%程度であった。その個体を、土を詰めたポッ トに移植して育成させ、 7個体を得た。これらの個体を、隔離ダロスチャンバ一内で育 成した。  [0116] About 2-10% of individuals were selected to identify GFP fluorescence in the shoot portion after about 2 weeks, and GFP fluorescence was confirmed in the shoot portion. The individuals were transplanted and grown in pots filled with soil to obtain 7 individuals. These individuals were raised in an isolated Daros chamber.
[0117] その一方で、減圧処理のみを行った場合は、 GFPの発現は確認されな力つた（図 5 中央)。  [0117] On the other hand, when only the decompression treatment was performed, the expression of GFP was not confirmed (center of FIG. 5).
[0118] 従来、エレクト口ポーレーシヨンのような直接的遺伝子導入法と、ァグロバタテリゥム 法のような間接的遺伝子導入法を組み合わせた例はな 、が、これらの方法を組み合 わせることで、本実施例に示されるように高い効率で、遺伝子導入ができたことは、全 く予測され得な力つたことである。また、本実施例に示されるような強い発現量も、全く 予想されなかったことである。コムギの形質転換につ 、ての技術レベルを考慮すれ ば、本発明の効果は顕著なものである。  [0118] Conventionally, there is no example of combining a direct gene transfer method such as elect mouth por- tion with an indirect gene transfer method such as agro-batterium method. Thus, as shown in the present example, the gene transfer with high efficiency was completely unpredictable. Also, a strong expression level as shown in this example was not expected at all. The effect of the present invention is remarkable in view of the technical level of wheat transformation.
[0119] エレクト口ポーレーシヨンを用いずに、減圧処理後にァグロバタテリゥムによる感染を 行 、遺伝子導入を行った場合の GFP発現量は、エレクト口ポーレーシヨンを行った場 合と比較して、低力つた（図 5右)。また、エレクト口ポーレーシヨンを用いずに、減圧処 理後にァグロバタテリゥムによる感染を行い遺伝子導入を行った場合には、約 5%の 個体において、 GFPの発現を肉眼で確認した。従来のァグロバタテリゥム法では、ァ グロパクテリゥムによる感染を行う前に、対象となる細胞 (組織)を 2, 4— Dのような植 物ホルモンによって処理 (前培養)することが必須であると考えられていた。しかし、本 実施例の結果は、そのような前培養を必要としない点において、迅速性'簡便性にお V、て予測され得な力つた顕著な効果を有する。 [0119] Infection caused by agrobacterium after decompression without using erect mouth poration On the other hand, the GFP expression level when the gene was introduced was lower than that when the electoral poration was performed (Fig. 5, right). In addition, when transfection was carried out by agrobaterium after decompression without using erect mouth poration, the expression of GFP was confirmed with the naked eye in about 5% of individuals. In the conventional agrobacterium method, it is essential to treat (pre-culture) the target cells (tissue) with a plant hormone such as 2, 4-D before infection with agrobacterium. It was thought. However, the results of this example have a significant effect that can be predicted from V speed and simplicity in that no such pre-culture is required.
[0120] (インディ力イネの結果） [0120] (Result of indy power rice)
ァグロバタテリゥムによる処理の 7日後（図 6)に GFPによる蛍光の観察を行った。ァ グロバタテリゥムとエレクト口ポーレーシヨンを組み合わせた遺伝子導入の結果、強 ヽ 蛍光が確認された（図 6Bの左側の個体)。また、遺伝子導入を行った個体中、約 60 %の個体という高い効率で GFPの発現を肉眼で確認した。従来、エレクト口ポーレー シヨンのような直接的遺伝子導入法と、ァグロバタテリゥム法のような間接的遺伝子導 入法を組み合わせた例はないが、これらの方法を組み合わせることで、本実施例に 示されるように高い効率で、遺伝子導入ができたことは、全く予測され得な力つたこと である。また、本実施例に示されるような強い発現量も、全く予想されなかったことで ある。従来、インディ力イネへの形質転換が困難であったことを考慮すれば、本発明 の効果は、顕著なものである。しかも、本実施例の結果は、 2, 4— Dなどを添加した 培地を用いる前培養を必要としない点において、迅速性'簡便性において予測され 得なかった顕著な効果を有する。  GFP fluorescence was observed 7 days after treatment with agrobacterium (Fig. 6). As a result of gene transfer combining globataterium and electoporation, strong fluorescence was confirmed (individual on the left in Fig. 6B). In addition, the expression of GFP was confirmed with the naked eye at a high efficiency of about 60% of the transgenic individuals. Conventionally, there is no example of combining the direct gene transfer method such as the elect mouth poration and the indirect gene transfer method such as the agrobatterium method. As shown in Fig. 2, the gene transfer with high efficiency was quite unpredictable. Also, a strong expression level as shown in this example was not expected at all. Considering that it has been difficult to transform to indy rice in the past, the effect of the present invention is remarkable. Moreover, the results of this Example have a remarkable effect that could not be predicted in terms of rapidity and convenience in that pre-culture using a medium supplemented with 2, 4-D or the like is not required.
[0121] (ハクサイの結果） [0121] (Results of Chinese cabbage)
ァグロバタテリゥムによる処理の 4日後（図 7)に GFPによる蛍光の観察を行った。ァ グロバタテリゥムとエレクト口ポーレーシヨンを組み合わせた遺伝子導入の結果、強 ヽ 蛍光が確認された（図 7Bの左側の個体)。また、遺伝子導入を行った個体中、約 60 %の個体という高い効率で GFPの発現を肉眼で確認した。従来、エレクト口ポーレー シヨンのような直接的遺伝子導入法と、ァグロバタテリゥム法のような間接的遺伝子導 入法を組み合わせた例はないが、これらの方法を組み合わせることで、本実施例に 示されるように高い効率で、遺伝子導入ができたことは、全く予測され得な力つたこと である。また、本実施例に示されるような強い発現量も、全く予想されなかったことで ある。 GFP fluorescence was observed 4 days after treatment with agrobacterium (Fig. 7). As a result of gene transfer combining globataterium and electoporation, strong fluorescence was confirmed (individual on the left in FIG. 7B). In addition, the expression of GFP was confirmed with the naked eye at a high efficiency of about 60% of the transgenic individuals. Conventionally, there is no example of combining the direct gene transfer method such as the electopore poration with the indirect gene transfer method such as the agrobatterium method. In As shown, the fact that gene transfer was possible with high efficiency was quite unpredictable. Also, a strong expression level as shown in this example was not expected at all.
[0122] (ダイズの結果）  [0122] (Results of soybean)
ァグロバタテリゥムによる処理の 4日後（図 8)に GFPによる蛍光の観察を行った。ァ グロバタテリゥムとエレクト口ポーレーシヨンを組み合わせた遺伝子導入の結果、強 ヽ 蛍光が確認された（図 8Bの左側の個体)。また、遺伝子導入を行った個体中、約 40 %の個体という高い効率で GFPの発現を肉眼で確認した。従来、エレクト口ポーレー シヨンのような直接的遺伝子導入法と、ァグロバタテリゥム法のような間接的遺伝子導 入法を組み合わせた例はないが、これらの方法を組み合わせることで、本実施例に 示されるように高い効率で、遺伝子導入ができたことは、全く予測され得な力つたこと である。また、本実施例に示されるような強い発現量も、全く予想されなかったことで ある。  GFP fluorescence was observed 4 days after treatment with agrobacterium (Fig. 8). As a result of gene transfer combining globataterium and electoporation, strong fluorescence was confirmed (individual on the left in FIG. 8B). In addition, the expression of GFP was confirmed with the naked eye at a high efficiency of about 40% of the transgenic individuals. Conventionally, there is no example of combining the direct gene transfer method such as the electopore poration with the indirect gene transfer method such as the agrobatterium method. As shown in Fig. 2, the gene transfer with high efficiency was quite unpredictable. Also, a strong expression level as shown in this example was not expected at all.
[0123] (トマトの結果）  [0123] (Tomato results)
ァグロバタテリゥムによる処理の 4日後（図 9)に GFPによる蛍光の観察を行った。ァ グロバタテリゥムとエレクト口ポーレーシヨンを組み合わせた遺伝子導入の結果、強 ヽ 蛍光が確認された（図 9Bの左側の個体)。また、遺伝子導入を行った個体中、約 50 %の個体という高い効率で GFPの発現を肉眼で確認した。従来、エレクト口ポーレー シヨンのような直接的遺伝子導入法と、ァグロバタテリゥム法のような間接的遺伝子導 入法を組み合わせた例はないが、これらの方法を組み合わせることで、本実施例に 示されるように高い効率で、遺伝子導入ができたことは、全く予測され得な力つたこと である。また、本実施例に示されるような強い発現量も、全く予想されなかったことで ある。  GFP fluorescence was observed 4 days after treatment with agrobacterium (Fig. 9). As a result of gene transfer combining globataterium and electoporation, strong fluorescence was confirmed (individual on the left in Fig. 9B). In addition, the expression of GFP was confirmed with the naked eye at a high efficiency of about 50% of the transgenic individuals. Conventionally, there is no example of combining the direct gene transfer method such as the elect mouth poration and the indirect gene transfer method such as the agrobatterium method. As shown in Fig. 2, the gene transfer with high efficiency was quite unpredictable. Also, a strong expression level as shown in this example was not expected at all.
[0124] (アサガオの結果）  [0124] (Results of morning glory)
ァグロバタテリゥムによる処理の 4日後（図 10)に GFPによる蛍光の観察を行った。 ァグロバタテリゥムとエレクト口ポーレーシヨンを組み合わせた遺伝子導入の結果、強 い蛍光が確認された（図 10Bの左側の個体)。また、遺伝子導入を行った個体中、約 50%の個体という高い効率で GFPの発現を肉眼で確認した。従来、エレクト口ボーレ ーシヨンのような直接的遺伝子導入法と、ァグロバタテリゥム法のような間接的遺伝子 導入法を組み合わせた例はないが、これらの方法を組み合わせることで、本実施例 に示されるように高い効率で、遺伝子導入ができたことは、全く予測され得なカゝつたこ とである。また、本実施例に示されるような強い発現量も、全く予想されなカゝつたことで ある。 GFP fluorescence was observed 4 days after treatment with agrobacterium (Fig. 10). As a result of gene transfer combining agrobacterium and electoporation, strong fluorescence was confirmed (individual on the left in Fig. 10B). In addition, the expression of GFP was confirmed with the naked eye at a high efficiency of about 50% of the transgenic individuals. Conventionally, the elect mouth bole There is no example of combining a direct gene transfer method such as Cyon and an indirect gene transfer method such as an agrobacterium, but combining these methods is expensive as shown in this example. The fact that we were able to introduce genes efficiently was a completely unpredictable result. In addition, the strong expression level shown in the present example is completely unexpected.
[0125] 以上のように、本発明の好ましい実施形態を用いて本発明を例示してきた力 本発 明は、この実施形態に限定して解釈されるべきものではない。本発明は、特許請求 の範囲によってのみその範囲が解釈されるべきであることが理解される。当業者は、 本発明の具体的な好ましい実施形態の記載から、本発明の記載および技術常識に 基づいて等価な範囲を実施することができることが理解される。本明細書において引 用した特許、特許出願および文献は、その内容自体が具体的に本明細書に記載さ れているのと同様にその内容が本明細書に対する参考として援用されるべきであるこ とが理解される。  [0125] As described above, the power of the present invention exemplified by the preferred embodiment of the present invention. The present invention should not be construed as being limited to this embodiment. It is understood that the scope of the present invention should be construed only by the claims. It is understood that those skilled in the art can implement an equivalent range based on the description of the present invention and the common general technical knowledge from the description of specific preferred embodiments of the present invention. Patents, patent applications, and literature references cited in this specification should be incorporated by reference as if the contents themselves were specifically described in the present specification. Is understood.
産業上の利用可能性  Industrial applicability
[0126] 簡便かつ迅速な植物形質転換方法が提供される。 [0126] A simple and rapid plant transformation method is provided.
[0127] 簡便な本発明の方法はこの分野の開発研究において重要な大量処理 ·大量解析 を容易にせしめ、ひいては飛躍的な研究の進歩を誘発し、画期的な組換え作物の開 発につながる。  [0127] The simple method of the present invention facilitates large-scale processing and large-scale analysis that are important in development research in this field, and as a result, induces breakthrough research and leads to the development of innovative recombinant crops. Connected.

Claims

請求の範囲 The scope of the claims
[I] 核酸を細胞内に導入する方法であって、以下の工程：  [I] A method for introducing a nucleic acid into a cell, comprising the following steps:
a)細胞を大気圧と異なる圧力下に維持する工程;および  a) maintaining the cells under a pressure different from atmospheric pressure; and
b)該細胞を、核酸を含むァグロパクテリゥムと接触させる工程  b) contacting the cell with an agrobacterium containing nucleic acid
を包含する方法。  Including the method.
[2] 前記細胞を大気圧と異なる圧力下に維持する工程が、前記細胞を減圧処理するェ 程である、請求項 1に記載の方法。  [2] The method according to claim 1, wherein the step of maintaining the cells under a pressure different from atmospheric pressure is a step of subjecting the cells to reduced pressure treatment.
[3] 前記細胞を大気圧と異なる圧力下に維持する工程が、前記細胞を加圧処理するェ 程である、請求項 1に記載の方法。 [3] The method according to claim 1, wherein the step of maintaining the cells under a pressure different from atmospheric pressure is a step of pressurizing the cells.
[4] 前記細胞を大気圧と異なる圧力下に維持する工程が、該細胞を、前記核酸を含むァ グロパクテリゥムと接触させる工程の前に行われる、請求項 1に記載の方法。 [4] The method according to claim 1, wherein the step of maintaining the cell under a pressure different from atmospheric pressure is performed before the step of contacting the cell with an agrobacterium containing the nucleic acid.
[5] 前記減圧処理する工程が、大気圧よりも 0. 096MPa低い減圧下で行われる、請求 項 2に記載の方法。  [5] The method according to claim 2, wherein the decompression step is performed under a reduced pressure of 0.096 MPa lower than the atmospheric pressure.
[6] 請求項 1に記載の方法であって、さらに [6] The method of claim 1, further comprising:
c)前記工程 (b)の後に、前記細胞と前記ァグロパクテリゥムとを、エレクト口ポーレー シヨンが起きる条件下に配置する工程、  c) after the step (b), the step of placing the cells and the agrobacterium under conditions that cause electopore poration;
を包含する方法。  Including the method.
[7] 請求項 6に記載の方法であって、前記 c)工程が、少なくとも二種類の方向で、前記 細胞と前記ァグロバタテリゥムとに電圧パルスをかけることを含む、方法。  7. The method according to claim 6, wherein the step c) includes applying a voltage pulse to the cell and the agrobacterium in at least two directions.
[8] 前記細胞が植物細胞である、請求項 1に記載の方法。 8. The method according to claim 1, wherein the cell is a plant cell.
[9] 前記植物細胞が、植物の休眠組織の細胞である、請求項 8に記載の方法。 9. The method according to claim 8, wherein the plant cell is a cell of a dormant tissue of a plant.
[10] 前記植物の休眠組織が種子である、請求項 9に記載の方法。 10. The method according to claim 9, wherein the dormant tissue of the plant is a seed.
[II] 前記植物の種子が完熟種子である、請求項 10に記載の方法。  [II] The method according to claim 10, wherein the seed of the plant is a mature seed.
[12] 前記植物細胞が、単子葉植物由来である請求項 8に記載の方法。 12. The method according to claim 8, wherein the plant cell is derived from a monocotyledonous plant.
[13] 前記単子葉植物がイネ科植物である、請求項 12に記載の方法。 13. The method according to claim 12, wherein the monocotyledonous plant is a gramineous plant.
[14] 前記イネ科植物がコムギである、請求項 13に記載の方法。 14. The method according to claim 13, wherein the gramineous plant is wheat.
[15] 前記イネ科植物がイネである、請求項 13に記載の方法。  15. The method according to claim 13, wherein the gramineous plant is rice.
[16] 前記イネ科植物がトウモロコシである、請求項 13に記載の方法。 16. The method according to claim 13, wherein the gramineous plant is corn.
[17] 前記植物細胞が、双子葉植物由来である請求項 8に記載の方法。 17. The method according to claim 8, wherein the plant cell is derived from a dicotyledonous plant.
[18] 前記双子葉植物がマメ科植物である、請求項 17に記載の方法。  18. The method according to claim 17, wherein the dicotyledonous plant is a legume.
[19] 前記マメ科植物がダイズである、請求項 18に記載の方法。  [19] The method according to claim 18, wherein the legume is soybean.
[20] 前記双子葉植物がアブラナ科植物である、請求項 17に記載の方法。  [20] The method of claim 17, wherein the dicotyledonous plant is a cruciferous plant.
[21] 前記アブラナ科植物がハクサイである、請求項 20の方法。  21. The method of claim 20, wherein the cruciferous plant is Chinese cabbage.
[22] 前記アブラナ科植物がシロイヌナズナである、請求項 20の方法。  [22] The method of claim 20, wherein the cruciferous plant is Arabidopsis.
[23] 前記双子葉植物がヒルガオ科植物である、請求項 17に記載の方法。  [23] The method according to claim 17, wherein the dicotyledonous plant is a convolvulaceae plant.
[24] 前記ヒルガオ科植物がアサガオである、請求項 23に記載の方法。  24. The method of claim 23, wherein the convolvulaceae plant is a morning glory.
[25] 前記双子葉植物がナス科植物である、請求項 17に記載の方法。  [25] The method according to claim 17, wherein the dicotyledonous plant is a solanaceous plant.
[26] 前記ナス科植物がトマトである、請求項 25に記載の方法。  26. The method according to claim 25, wherein the solanaceous plant is tomato.
[27] 前記双子葉植物がゥリ科植物である、請求項 17に記載の方法。  [27] The method according to claim 17, wherein the dicotyledonous plant is a cucurbitaceae plant.
[28] 前記ゥリ科植物がゥリである、請求項 27に記載の方法。  28. The method according to claim 27, wherein the cucurbitaceae plant is cucumber.
[29] 前記ァグロバタテリゥムが、 Agrobacterium tumefaciensである、請求項 1に記載の方 法。  [29] The method according to claim 1, wherein the agrobacterium is Agrobacterium tumefaciens.
[30] 核酸を細胞内に導入した植物を作製する方法であって、以下の工程：  [30] A method for producing a plant into which a nucleic acid is introduced into a cell, comprising the following steps:
a)植物細胞を大気圧と異なる圧力下に維持する工程；および  a) maintaining the plant cell under a pressure different from atmospheric pressure; and
b)該植物細胞を、核酸を含むァグロパクテリゥムと接触させる工程  b) contacting the plant cell with agrobacterium containing nucleic acid
を包含する方法。  Including the method.
[31] 請求項 30に記載の方法であって、さらに  [31] The method of claim 30, further comprising
c)前記工程 (b)の後に、前記植物細胞と前記ァグロパクテリゥムとを、エレクト口ポー レーシヨンが起きる条件下に配置する工程、  c) after the step (b), placing the plant cell and the agrobacterium under conditions where electo-portion occurs,
を包含する方法。  Including the method.
[32] 請求項 30に記載の方法であって、前記植物細胞を、分化、成長および Zまたは増 殖させる工程をさらに包含する、方法。  32. The method of claim 30, further comprising the step of differentiating, growing and Z or growing the plant cell.
[33] 請求項 30に記載の方法であって、前記 a)工程が、前記植物細胞を含む種子を大気 圧と異なる圧力下に維持する工程を含み、かつ、前記 b)工程が、該植物細胞を含む 該種子を、該ァグロパクテリゥムと接触させる工程を含む、方法。  [33] The method according to claim 30, wherein the step a) includes a step of maintaining seeds containing the plant cells under a pressure different from atmospheric pressure, and the step b) includes the plant. A method comprising contacting the seed comprising cells with the agrobacterium.
[34] 前記種子が、単子葉植物の種子である請求項 33に記載の方法。 34. The method according to claim 33, wherein the seed is a monocotyledonous seed.
[35] 前記単子葉植物の種子が完熟種子である、請求項 34に記載の方法。 35. The method according to claim 34, wherein the monocotyledonous seed is a mature seed.
[36] 前記単子葉植物の種子がイネ科の種子である、請求項 34に記載の方法。  36. The method according to claim 34, wherein the monocotyledonous seed is a grass seed.
[37] 前記イネ科種子がコムギ種子である、請求項 36に記載の方法。  [37] The method of claim 36, wherein the grass seed is a wheat seed.
[38] 前記イネ科種子がイネ種子である、請求項 36に記載の方法。  [38] The method of claim 36, wherein the grass seed is a rice seed.
[39] 前記イネ科種子がトウモロコシ種子である、請求項 36に記載の方法。  39. The method of claim 36, wherein the grass seed is corn seed.
[40] 前記種子が、双子葉植物の種子である請求項 33に記載の方法。  40. The method according to claim 33, wherein the seed is a dicotyledonous seed.
[41] 前記双子葉植物の種子がマメ科植物の種子である、請求項 40に記載の方法。  41. The method of claim 40, wherein the dicotyledonous seed is a legume seed.
[42] 前記マメ科植物がダイズである、請求項 41に記載の方法。  [42] The method of claim 41, wherein the legume is soybean.
[43] 前記双子葉植物の種子がアブラナ科植物の種子である、請求項 40に記載の方法。  43. The method of claim 40, wherein the dicotyledonous plant seed is a cruciferous plant seed.
[44] 前記アブラナ科植物がノ、クサイである、請求項 43の方法。  [44] The method according to claim 43, wherein the cruciferous plant is a rhinoceros.
[45] 前記アブラナ科植物がシロイヌナズナである、請求項 43の方法。  45. The method of claim 43, wherein the cruciferous plant is Arabidopsis.
[46] 前記双子葉植物の種子がヒルガオ科植物の種子である、請求項 40に記載の方法。  46. The method of claim 40, wherein the dicotyledonous plant seed is a convolvulaceae seed.
[47] 前記ヒルガオ科植物がアサガオである、請求項 46に記載の方法。  [47] The method of claim 46, wherein the convolvulaceae is a morning glory.
[48] 前記双子葉植物の種子がナス科植物の種子である、請求項 40に記載の方法。  48. The method of claim 40, wherein the dicotyledonous plant seed is a solanaceous plant seed.
[49] 前記ナス科植物がトマトである、請求項 48に記載の方法。  [49] The method according to claim 48, wherein the solanaceous plant is tomato.
[50] 前記双子葉植物の種子がゥリ科植物の種子である、請求項 40に記載の方法。  50. The method of claim 40, wherein the dicotyledonous plant seed is a cucurbitaceae plant seed.
[51] 前記ゥリ科植物がゥリである、請求項 50に記載の方法。  51. The method of claim 50, wherein the cucurbitaceae plant is cucumber.
[52] 請求項 30〜51のいずれか 1項に記載の方法によって作製された、植物。  [52] A plant produced by the method according to any one of claims 30 to 51.
[53] 培養変異を含まない、請求項 52に記載の植物。  [53] The plant according to claim 52, which does not contain a culture mutation.
[54] 自動化して核酸を細胞内に導入する装置であって、該装置は、以下：  [54] An apparatus for automated introduction of nucleic acid into cells, the apparatus comprising:
a)核酸を含むァグロパクテリゥムと細胞とを含む混合液を入れる容器；  a) a container for containing a mixture of agrobacterium containing nucleic acids and cells;
b)該 a)の容器中に核酸を含むァグロパクテリゥムを入れる手段；  b) Means for placing agrobacterium containing nucleic acid in the container of a);
c)該 a)の容器中に細胞を入れる手段；  c) means for placing the cells in the container of a);
d)細胞を大気圧と異なる圧力下に維持する容器であって、大気圧と異なる圧力に耐 える能力を有する、容器；  d) a container for maintaining cells under a pressure different from atmospheric pressure, the container having the ability to withstand a pressure different from atmospheric pressure;
e)該細胞を、該 d)の容器中に配置する手段；  e) means for placing the cells in the container of d);
f )該 d)の容器中を、大気圧と異なる圧力に維持する手段；および  f) means for maintaining the container in d) at a pressure different from atmospheric pressure; and
g)該 b)、 c；)、 e)、および f)の手段を自動化して実行する手段 を備え、ここで該 b)の手段、該 c)の手段、および該 e)の手段は、同一であるかまたは 異なり、そして該 a)の容器、および該 d)の容器は、同一である力 たは異なる、装置 g) means for automating and executing the means of b), c;), e), and f) Wherein the means of b), the means of c) and the means of e) are the same or different, and the container of a) and the container of d) are the same Force or different, equipment
[55] 少なくとも 2つ以上の対の電極を有する、エレクト口ポーレーシヨン用電極。 [55] An electrode for electo-portioning having at least two pairs of electrodes.
[56] 少なくとも 3対の電極を有する、請求項 55に記載のエレクト口ポーレーシヨン用電極。  [56] The electrode for electo-poration according to claim 55, having at least three pairs of electrodes.
PCT/JP2005/018198 2004-10-01 2005-09-30 Method of transferring nucleic acid into cell with the use of agrobacterium involving treatment under reduced pressure/elevated pressure WO2006038571A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2006539274A JP4788002B2 (en) 2004-10-01 2005-09-30 Method for introducing nucleic acid into cells using Agrobacterium, including the use of reduced pressure treatment / pressurization treatment

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2004-290839 2004-10-01
JP2004290839 2004-10-01

Publications (1)

Publication Number Publication Date
WO2006038571A1 true WO2006038571A1 (en) 2006-04-13

Family

ID=36142645

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2005/018198 WO2006038571A1 (en) 2004-10-01 2005-09-30 Method of transferring nucleic acid into cell with the use of agrobacterium involving treatment under reduced pressure/elevated pressure

Country Status (2)

Country Link
JP (1) JP4788002B2 (en)
WO (1) WO2006038571A1 (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2014502840A (en) * 2010-12-22 2014-02-06 フィリップ・モーリス・プロダクツ・ソシエテ・アノニム Plant vacuum infiltration method and system
JP2016063828A (en) * 2009-12-23 2016-04-28 ズートツッカー アクチェンゲゼルシャフト マンハイム／オクセンフルト Reactor device for electroporation

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2418283A1 (en) * 2010-08-07 2012-02-15 Nomad Bioscience GmbH Process of transfecting plants

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997048814A2 (en) * 1996-06-21 1997-12-24 Monsanto Company Methods for the production of stably-transformed, fertile wheat employing agrobacterium-mediated transformation and compositions derived therefrom
WO1999067357A2 (en) * 1998-06-23 1999-12-29 Aventis Cropscience S.A. Transformation of monocots by co-cultivating an inflorescence with agrobacterium
WO2004007736A1 (en) * 2002-07-16 2004-01-22 National Institute Of Agrobiological Sciences Electroporation method including the use of depressurization/pressurization

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997048814A2 (en) * 1996-06-21 1997-12-24 Monsanto Company Methods for the production of stably-transformed, fertile wheat employing agrobacterium-mediated transformation and compositions derived therefrom
WO1999067357A2 (en) * 1998-06-23 1999-12-29 Aventis Cropscience S.A. Transformation of monocots by co-cultivating an inflorescence with agrobacterium
WO2004007736A1 (en) * 2002-07-16 2004-01-22 National Institute Of Agrobiological Sciences Electroporation method including the use of depressurization/pressurization

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
AMOAH B.K. ET AL: "Factors influencing Agrobacterium-mediated transient expression of uidA in wheat inflorescence tissue", J. EXP. BOT., vol. 52, no. 358, May 2001 (2001-05-01), pages 1135 - 1142, XP002995060 *
BECHTOLD N. AND PELLETIER G.: "In planta Agrobacterium-mediated transformation of adult Arabidopsis thaliana plants by vacuum infiltration", METHODS IN MOLECULAR BIOLOGY, vol. 82, 1998, pages 259 - 266, XP000914126 *
FELDMANN K.A.: "T-DNA insertion mutagenesis in Arabidopsis: mutational spectrum", PLANT J., vol. 1, no. 1, 1991, pages 71 - 82, XP002995061 *
SONG G.-Q. AND YAMAGUCHI K.-I.: "Efficient Agroinfiltration-mediated transient GUS expression system for assying different promoters in rice", PLANT BIOTECHNOL., vol. 20, no. 3, 2003, pages 235 - 239, XP002995059 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2016063828A (en) * 2009-12-23 2016-04-28 ズートツッカー アクチェンゲゼルシャフト マンハイム／オクセンフルト Reactor device for electroporation
JP2014502840A (en) * 2010-12-22 2014-02-06 フィリップ・モーリス・プロダクツ・ソシエテ・アノニム Plant vacuum infiltration method and system

Also Published As

Publication number Publication date
JP4788002B2 (en) 2011-10-05
JPWO2006038571A1 (en) 2008-05-15

Similar Documents

Publication Publication Date Title
JP4273231B2 (en) Electroporation method including the use of reduced pressure / pressurization
CN107459565A (en) Application of the soybean drought resisting GAP-associated protein GAP in regulating and controlling soybean drought resistance
CN108192920B (en) Method for improving plant disease resistance by using NDR1 gene
KR100781059B1 (en) An inducible promoter activated by environmental stresses and a method for the generation of transgenic plants producing a target protein specifically in plant guard cells using the promoter
CN109266647B (en) Rice stem borer-killing inducible promoter and application thereof
CN108823239B (en) Carrier for improving disease resistance of plants and application thereof
WO2006038571A1 (en) Method of transferring nucleic acid into cell with the use of agrobacterium involving treatment under reduced pressure/elevated pressure
CN109734784B (en) Application of SlDALR1 gene in enhancing resistance to bacterial leaf spot of tomato
US6835868B1 (en) Transgenic plants expressing dermaseptin peptides providing broad spectrum resistance to pathogens
CN116083445A (en) CrBZR1 gene and application thereof
US20080189805A1 (en) Novel genes and rna molecules that confer stress tolerance
JP5201497B2 (en) Ultrasonic transformation
KR20020067303A (en) Efficient method for the development of transgenic plants by gene manipulation
AU772964B2 (en) Transgenic plants that are resistant to a broad spectrum of pathogens
CN113980986B (en) Application of CRK22 gene and encoding protein thereof in potato stress-resistant breeding
US20080189804A1 (en) Novel genes and rna molecules that confer stress tolerance
CN114230649B (en) Tn1 protein related to rice tillering force, related biological material and application thereof
US20040064847A1 (en) Transgenic plants exhibiting resistance to a spectrum of pathogens
KR100951062B1 (en) BrD1 Gene coding the insect-resistant Defensin from Brassica rapa, transgenic plants using that and method enhancing the insect-resistant by expression of said gene
CN110117314B (en) Artificially synthesized Bt insecticidal protein gene Cry1Ab-GM1, expression vector thereof and application thereof
CN117987428A (en) Bacterial wilt-resistant related gene CaARF and application thereof
KR101570761B1 (en) A method for producing marker-free transgenic rice and the insect resistance rice plant produced by the marker-free transformation
WO2014025137A1 (en) Uip1 gene for increasing resistance of plants to drought stress and use thereof
CN116023453A (en) Application of protein IbRING in regulation and control of salt tolerance and drought resistance of plants
AU2012201139A1 (en) Electroporation method including the use of depressurization/pressurization

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KM KP KR KZ LC LK LR LS LT LU LV LY MA MD MG MK MN MW MX MZ NA NG NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SM SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LT LU LV MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 2006539274

Country of ref document: JP

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 05788038

Country of ref document: EP

Kind code of ref document: A1