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WO2006038571A1 - Procédé de transfert d’acide nucléique en cellule à l’aide d’une agrobactérie impliquant un traitement à pression réduite/à pression élevée - Google Patents

Procédé de transfert d’acide nucléique en cellule à l’aide d’une agrobactérie impliquant un traitement à pression réduite/à pression élevée Download PDF

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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
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WO
WIPO (PCT)
Prior art keywords
plant
seed
cell
cells
agrobacterium
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Application number
PCT/JP2005/018198
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English (en)
Japanese (ja)
Inventor
Takashi Hagio
Original Assignee
National Institute Of Agrobiological Sciences
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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/ja
Publication of WO2006038571A1 publication Critical patent/WO2006038571A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—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.

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Abstract

L’invention concerne un procédé permettant de transformer une plante rapidement et de façon pratique par rapport aux procédés existants. Le procédé comprend les phases suivantes : a) la phase de maintien d’une cellule à une pression différente de la pression atmosphérique ; et b) la phase de mise en contact de la cellule avec une agrobactérie contenant un acide nucléique ; suivi, le cas échéant, c) du placement de la cellule et de l’agrobactérie dans des conditions permettant une électrophorèse. Du fait de son côté pratique, ce procédé facilite des traitements à grande échelle et des analyses à grande échelle, qui sont importants pour la recherche et le développement dans ce domaine, et permet des avancées rapides dans les études, permettant le développement de récoltes modifiées génétiquement marquant leur époque.
PCT/JP2005/018198 2004-10-01 2005-09-30 Procédé de transfert d’acide nucléique en cellule à l’aide d’une agrobactérie impliquant un traitement à pression réduite/à pression élevée WO2006038571A1 (fr)

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JP2014502840A (ja) * 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 植物のバキュームインフィルトレーション方法およびシステム
JP2016063828A (ja) * 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 電気穿孔用の反応器装置

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EP2418283A1 (fr) * 2010-08-07 2012-02-15 Nomad Bioscience GmbH Procédé de transfection de plantes

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WO1999067357A2 (fr) * 1998-06-23 1999-12-29 Aventis Cropscience S.A. Transformation de monocotyledones fondee sur agrobacterium
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2016063828A (ja) * 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 電気穿孔用の反応器装置
JP2014502840A (ja) * 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 植物のバキュームインフィルトレーション方法およびシステム

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