JP4788002B2 - Method for introducing nucleic acid into cells using Agrobacterium, including the use of reduced pressure treatment / pressurization treatment - Google Patents

Description

  The present invention is a method for producing 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 pressure treatment and Agrobacterium. About. Furthermore, the present invention introduces a nucleic acid transductant (transformant) by introducing a bacterium such as Agrobacterium containing a desired nucleic acid into a cell or tissue by combining reduced pressure treatment or pressurized treatment with electroporation. ).

  Major crops such as wheat, barley, rice, corn, and soybean are essential for the survival of mankind, but in order to secure food commensurate with the global population growth, crops with higher yields than current crops must be used. It is necessary to develop it. As one of the varietal improvements that increase the yield, development of transformed crops using recombinant DNA technology has been carried out.

  For example, vegetables such as Chinese cabbage, tomatoes, and cucumbers are essential crops in terms of nutrition and enrichment. However, these vegetables are vulnerable to various pests and diseases, and if the pest resistance can be imparted by using gene recombination techniques, the yield can be stabilized. Therefore, transformation methods using these genes have been developed along with the isolation of useful genes.

  In order to perform transformation, in the case of a plant, generally, a method of directly introducing a gene into a plant (direct gene transfer method) or a method of indirectly transferring a gene into a plant (indirect Gene transfer method).

  To date, as an indirect gene transfer method, a method using Agrobacterium has been widely used. For example, a method of infecting callus obtained after 3 weeks of culturing mature rice seeds with Agrobacterium (see Hiei et al., Plant Journal, 6: 271-282, 1994) or pre-culture for several days A method (see Tanaka et al., Japanese Patent No. 3141084) that can rapidly obtain a transformant by infecting the seeds with Agrobacterium can be mentioned. However, when the indirect gene transfer method is used, there is a disadvantage that if the culture time is shortened, the gene transfer cannot be performed or the gene transfer efficiency is lowered.

  As for wheat, conventional: (1) Regarding the transformation of wheat, there was a problem that the difference between cultivars was large and the period required for cultivation was long (Non-patent Document 2); (2) Characteristics of wheat The method using Agrobacterium in conversion was not successful (Non-patent Document 2); and (3) In the transformation of wheat, immature embryos were used for 10 to 2 weeks after flowering for the subject. In addition to the difficulty in preparing the material that must be present, transformation of wheat such as bread wheat is still apt to be avoided due to the low transformation frequency.

Therefore, it is necessary to improve these disadvantages of the indirect gene transfer method. In addition, by developing a simple and rapid indirect gene transfer method, it is possible to rapidly and in large quantities obtain a nucleic acid transfer body (particularly, a transformed plant body). Therefore, the present invention facilitates mass processing and 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 research advances, It leads to the development of innovative recombinant crops.
Tanaka et al., Japanese Patent No. 3141084 Hiei et al., Plant Journal, 6: 271-282, 1994. Biotechnology in Agricultural and Forestry 46, Transgenic Crops I, Y. et al. P. S. Edited by Bajaj, published by Springer, 2000, in particular T. 33 on page 33. A. Description by 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. is there.

  By using the simple and rapid method of the present invention, transformed plants can be obtained quickly and in large quantities using Agrobacterium. Therefore, the present invention facilitates mass processing and 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 research advances, It leads to the development of innovative recombinant crops.

The above issues
a) maintaining the cell under a pressure different from atmospheric pressure; and b) contacting the cell with Agrobacterium comprising the nucleic acid, optionally c) as in step (b) above. Later, this was achieved by a method for producing a transformed plant comprising the step of placing the cells and the Agrobacterium under conditions where electroporation occurs.

  In one embodiment, the methods of the invention are performed using plant cells (eg, mature seeds).

  Specifically, in the present specification, the inventors have made various attempts as a method for treating cells so that nucleic acids are easily taken up when a gene is introduced into a plant using Agrobacterium. As a result, (1) nucleic acid introduction method using Agrobacterium (Agrobacterium) is not effective for plants other than Arabidopsis, and (2) nucleic acid introduction using Agrobacterium Contrary to the conventional knowledge that plants do not need to be kept in a vacuum state (Bent, Plant Physiology, 124: 1540-1547, December 2000), In the introduction of plant nucleic acid to be used, it has been found that rapid gene introduction is possible by using a step 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 nucleic acid introduction into cells can be significantly improved by combining electroporation with the gene introduction method of the present invention.

  This method is very simple. Furthermore, since the method of the present invention does not require a culture process that is usually required after a nucleic acid transfer operation, the resulting transductant has the advantage that it does not contain a culture mutation. It is known that culture mutations inevitably occur in the culture process required after a conventional nucleic acid introduction operation. The 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 originally introduced by the cell into which the nucleic acid is introduced and / or introduced. Any sequence modification (eg, substitution, deletion, insertion, translocation, inversion, duplication, etc.) that occurs in a nucleic acid sequence. Unintended culture mutations often impart undesirable traits to the nucleic acid transductant into which the nucleic acid has been introduced. Therefore, the method of the present invention that can obtain a desired nucleic acid transductant without causing any culture mutation is very useful.

  The present invention also does not require 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, gene transfer can be performed more rapidly than the conventional method. Provides the advantage of being possible.

Accordingly, the present invention provides the following.
1. A method for introducing a nucleic acid into a cell, comprising the following steps:
a) maintaining the cell under a pressure different from atmospheric pressure; and b) contacting the cell with Agrobacterium comprising the nucleic acid.
2. Item 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. Item 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 pressurizing the cells.
4). Item 2. 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. Item 3. The method according to Item 2, wherein the depressurizing step is performed under a reduced pressure of 0.096 MPa lower than the atmospheric pressure.
6). The method according to item 1, further comprising: c) after the step (b), placing the cells and the Agrobacterium under conditions that cause electroporation;
Including the method.
7). 7. The method according to item 6, wherein the step c) includes applying voltage pulses to the cells and the Agrobacterium in at least two directions.
8). Item 2. The method according to Item 1, wherein the cell is a plant cell.
9. Item 9. The method according to Item 8, wherein the plant cell is a cell of a dormant tissue of a plant.
10. Item 10. The method according to Item 9, wherein the dormant tissue of the plant is a seed.
11. Item 11. The method according to Item 10, wherein the seed of the plant is a mature seed.
12 Item 9. The method according to Item 8, wherein the plant cell is derived from a monocotyledonous plant.
13. Item 13. The method according to Item 12, wherein the monocotyledonous plant is a gramineous plant.
14 14. The method according to item 13, wherein the gramineous plant is wheat.
15. 14. The method according to item 13, wherein the gramineous plant is rice.
16. 14. The method according to item 13, wherein the gramineous plant is corn.
17. Item 9. The method according to Item 8, wherein the plant cell is derived from a dicotyledonous plant.
18. Item 18. The method according to Item 17, wherein the dicotyledonous plant is a legume.
19. Item 19. The method according to Item 18, wherein the legume is soybean.
20. 18. A method according to item 17, wherein the dicotyledonous plant is a cruciferous plant.
21. 21. The method of item 20, wherein the cruciferous plant is Chinese cabbage.
22. 21. The method of item 20, wherein the cruciferous plant is Arabidopsis thaliana.
23. Item 18. The method according to Item 17, wherein the dicotyledonous plant is a convolvulaceae plant.
24. 24. A method according to item 23, wherein the convolvulaceae plant is morning glory.
25. Item 18. The method according to Item 17, wherein the dicotyledonous plant is a solanaceous plant.
26. 26. A method according to item 25, wherein the solanaceous plant is a tomato.
27. Item 18. The method according to Item 17, wherein the dicotyledonous plant is a Cucurbitaceae plant.
28. 28. A method according to item 27, wherein the cucurbitaceae plant is cucumber.
29. Item 2. The method according to Item 1, wherein the Agrobacterium is Agrobacterium tumefaciens.
30. A method for producing a plant into which a nucleic acid has been introduced into a cell, comprising the following steps:
a) maintaining the plant cell under a pressure different from atmospheric pressure; and b) contacting the plant cell with Agrobacterium comprising the nucleic acid.
31. The method according to item 30, further comprising: c) after the step (b), placing the plant cell and the Agrobacterium under conditions that cause electroporation;
Including the method.
32. 31. The method according to item 30, further comprising the step of differentiating, growing and / or expanding the plant cell.
33. 30. The method according to Item 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 cells. Contacting the seed with the Agrobacterium.
34. 34. A method according to item 33, wherein the seed is a monocotyledonous seed.
35. 35. A method according to item 34, wherein the monocotyledonous plant seed is a mature seed.
36. 35. The method of item 34, wherein the monocotyledonous plant seed is a grass seed.
37. 38. A method according to item 36, wherein the grass seed is wheat seed.
38. The method according to item 36, wherein the grass seed is a rice seed.
39. 38. A method according to item 36, wherein the grass seed is a corn seed.
40. 34. A method according to item 33, wherein the seed is a dicotyledonous seed.
41. 41. The method of item 40, wherein the dicotyledonous seed is a legume seed.
42. 42. A method according to item 41, wherein the legume is soybean.
43. 41. A method according to item 40, wherein the dicotyledonous seed is a cruciferous seed.
44. 44. The method of item 43, wherein the cruciferous plant is Chinese cabbage.
45. 44. The method of item 43, wherein the cruciferous plant is Arabidopsis thaliana.
46. 41. A method according to item 40, wherein the dicotyledonous seed is a convolvulaceae seed.
47. 47. A method according to item 46, wherein the convolvulaceae plant is a morning glory.
48. 41. The method of item 40, wherein the dicotyledonous seed is a solanaceous plant seed.
49. 49. A method according to item 48, wherein the solanaceous plant is a tomato.
50. 41. The method of item 40, wherein the dicotyledonous seeds are cucurbitaceae seeds.
51. 51. A method according to item 50, wherein the cucurbitaceae plant is cucumber.
52. Item 52. A plant produced by the method according to any one of items 30 to 51.
53. 53. The plant according to item 52, which does not contain a culture mutation.
54. An apparatus for automated introduction of nucleic acids into cells, the apparatus comprising:
a) a container for containing a mixture containing Agrobacterium containing nucleic acids and cells;
b) means for placing Agrobacterium containing nucleic acid in the container of a);
c) means for placing the cells in the container of a);
d) a container for maintaining the cells under a pressure different from atmospheric pressure, the container having the ability to withstand a pressure different from atmospheric pressure;
e) means for placing the cells in the container of d);
f) means for maintaining the container in d) at a pressure different from atmospheric pressure; and g) means for automating and executing the means in 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 or different .
55. An electroporation electrode having at least two pairs of electrodes.
56. 56. Electroporation electrode according to item 55, comprising at least three pairs of electrodes.

  The gene transfer method of the present invention enables rapid and highly efficient indirect gene transfer to a target cell (tissue).

  The simple method of the present invention facilitates large-scale processing and large-scale analysis important in development research in this field, and as a result, induces dramatic progress in research and leads to the development of innovative recombinant crops.

FIG. 1 is a diagram schematically showing a binary vector pCAMBIA1390-sGFP possessed on T-DNA. FIG. 2A shows the results of observing the seeds of rice with white light on the 6th day after infection treatment with Agrobacterium. The left is a seed that has been subjected to reduced pressure treatment, Agrobacterium infection, and electroporation, and the right is a seed that has been subjected to reduced pressure treatment only. FIG. 2B shows the result of observing the seed observed in FIG. 2A by irradiating it with excitation light. Cells expressing GFP are brightly shining green. FIG. 3A shows the result of observing the seeds of rice with white light on the 8th day after the infection treatment with Agrobacterium. The left is a seed that has been subjected to reduced pressure treatment, Agrobacterium infection, and electroporation, and the right is a seed that has been subjected to reduced pressure treatment only. FIG. 3B shows the result of observing the seed observed in FIG. 3A by irradiating it with excitation light. Cells expressing GFP are brightly shining green. FIG. 4A shows the results of observing the seeds of rice on the 14th day after infection treatment with Agrobacterium with white light. The left is a seed that has been subjected to vacuum treatment, Agrobacterium infection, and electroporation, and the right is a seed that has been soaked in a bacterial solution after vacuum treatment without performing electroporation. FIG. 4B shows the result of observing the seed observed in FIG. 4A by irradiating 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 subjected to decompression treatment, Agrobacterium infection, and electroporation, the center is a seed subjected to decompression treatment only, and the right is subjected to decompression treatment without performing electroporation, The seeds are only immersed in the fungus. FIG. 5B shows the result of observing the seed observed in FIG. 5A by irradiating excitation light. Cells expressing GFP are brightly shining green. FIG. 6A shows the results of observing the seeds of Indica rice with white light on day 7 after the infection treatment with Agrobacterium. The left is a seed that has been subjected to vacuum treatment, Agrobacterium infection, and electroporation, and the right is a seed that has been soaked in a bacterial solution after vacuum treatment without performing electroporation. FIG. 6B shows the result of observing the seed observed in FIG. 6A by irradiating it with excitation light. Cells expressing GFP are brightly shining green. FIG. 7A shows the results of observing seeds of Chinese cabbage with white light on the 4th day after infection treatment with Agrobacterium. The left is a seed that has been subjected to vacuum treatment, Agrobacterium infection, and electroporation, and the right is a seed that has been soaked in a bacterial solution after vacuum treatment without performing electroporation. FIG. 7B shows the result of observing the seed observed in FIG. 7A by irradiating it with excitation light. Cells expressing GFP are brightly shining green. FIG. 8A shows the result of observing the seeds of soybean on the fourth day after infection treatment with Agrobacterium with white light. The left is a seed that has been subjected to vacuum treatment, Agrobacterium infection, and electroporation, and the right is a seed that has been soaked in a bacterial solution after vacuum treatment without performing electroporation. 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 4th day after infection treatment with Agrobacterium. The left is a seed that has been subjected to vacuum treatment, Agrobacterium infection, and electroporation, and the right is a seed that has been soaked in a bacterial solution after vacuum treatment without performing electroporation. FIG. 9B shows the result of observing the seed observed in FIG. 9A by irradiating it with excitation light. Cells expressing GFP are brightly shining green. FIG. 10A shows the result of observation of seeds of morning glory with white light on the 4th day after infection treatment with Agrobacterium. The left is a seed that has been subjected to vacuum treatment, Agrobacterium infection, and electroporation, and the right is a seed that has been soaked in a bacterial solution after vacuum treatment without performing electroporation. FIG. 10B shows the result of observing the seed observed in FIG. 10A by irradiating it with excitation light. Cells expressing GFP are brightly shining green. FIG. 11 is a perspective view of an electroporation chamber having a hexagonal cross section. FIG. 12 is a cross-sectional view along the X-ray of the electroporation chamber shown in FIG. FIG. 13 is a perspective view of an electroporation electrode of the type inserted into a test tube. FIG. 14 is a diagram illustrating a state where the electroporation electrode illustrated in FIG. 13 is inserted into a test tube.

SEQ ID NO: 1: Forward primer for detecting NPT II gene.
SEQ ID NO: 2: Reverse primer for detecting the NPT II gene.
SEQ ID NO: 3: Forward primer for detecting the NPT II gene.
SEQ ID NO: 4: Reverse primer for detecting the NPT II gene.

  The present invention will be described below. Throughout this specification, it should be understood that the singular forms also include the plural concept unless specifically stated otherwise. In addition, it is to be understood that the terms used in the present specification are used in the meaning normally used in the art unless otherwise specified.

  Listed below are definitions of terms particularly used in the present specification.

  In this specification, the term “nucleic acid introduction” means that a nucleic acid is artificially introduced into a cell or tissue. The phenotype of the cell or tissue into which the 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. As used herein, 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 the present specification, the terms “nucleic acid introduction”, “gene introduction”, and “transformation” may be used interchangeably. In this specification, the meaning of these terms is clear from the context in which the terms are included.

  The terms “nucleic acid transductant”, “gene transfectant” and “transformant” refer to all or part of a living organism generated from a nucleic acid transduced, gene transduced and transformed cell or tissue, respectively. However, in the present specification, the terms “nucleic acid introduced body”, “gene introduced body”, and “transformant” may be used interchangeably. In this specification, the meaning of these terms is clear from the context in which the terms are included. The nucleic acid transductant, the gene transductant, and the transformant can be any living organism, and examples thereof include prokaryotic cells and eukaryotic cells (including plant cells and the like) or living organisms generated from tissues. A transformant is also referred to as a transformed cell, transformed tissue, transformed host, etc., depending on the subject, and encompasses all of these forms herein, but may refer to a particular form in a particular context. . 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, etc.), Fungi and the like) and 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.

  As used herein, “tissue” refers to a population of cells having substantially the same function and / or morphology in a multicellular organism. A “tissue” usually has the same origin, but even cell populations with different origins can be referred to as a tissue if they have the same function and / 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 are classified into single tissues and composite tissues according to the types of constituent cells. Animal tissues are classified into epithelial tissues, connective tissues, muscle tissues, nerve tissues, etc. based on morphological, functional or developmental basis.

  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, monocotyledonous plant)). Or a tissue derived from a dicotyledonous plant) or a fungus. Preferably, the tissue used in the present invention is a tissue containing a cell wall, particularly preferably a plant tissue. “Plant tissues” include, but are not limited to, dormant tissues, germplasm, growth points, and flower buds. Preferred dormant tissues include ripe seeds, immature seeds, winter buds, and tubers, particularly preferably ripe seeds, but are not limited thereto.

  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 tissues with a specific spatial arrangement, and a tissue consists of many cells. Such organs include roots, leaves, stems and flowers in the case of plants, and skin, heart, blood vessels, cornea, retina, kidney, liver, pancreas, intestine, in the case of animals. Examples include, but are not limited to, placenta, umbilical cord, lungs, brain, nerves, peripheral limbs, and the like.

  As used herein, the term “selection” refers to a nucleic acid transductant introduced with a nucleic acid by an antibiotic resistance test and / or genetic engineering techniques (eg, PCR, Southern blotting, Northern blotting, etc.). Is distinguished from that into which no nucleic acid has been introduced. In certain cases, the term “selection” distinguishes transformants transformed with a drug resistance gene from untransformed plants by culturing and / or growing the transformed plant in the presence of a drug. It means the process to do.

  The Agrobacterium used for transformation of the monocotyledonous plant can be any Agrobacterium, preferably Agrobacterium tumefaciens. Agrobacterium is transformed with a plant expression vector containing the desired recombinant gene (eg, by electroporation). A desired recombinant gene can be introduced into a plant by infecting seed with transformed Agrobacterium. The introduced recombinant gene is integrated in the genome of the plant. In addition, the genome in a plant includes not only a nuclear chromosome but the genome contained in various organelles (for example, a mitochondria, a chloroplast, etc.) in a plant cell.

  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 a plant expression vector can be prepared using gene recombination techniques well known to those skilled in the art. For the construction of a plant expression vector for use in the Agrobacterium transformation method, for example, a pBI-type or pPZP-type vector is preferably used, but is not limited thereto.

  As used herein, a “growth regulator” is a factor that affects multicellular growth, is synthesized in an organ or cell of a multicellular organism (for example, a plant), and other organs by body fluids. A chemical substance or a derivative thereof that is delivered to the body or part thereof and has a function of changing the function, activity and / 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, 2,4-D and IAA (indole acetic acid), NAA (naphthalene acetic acid), and IBA (indole butyric acid).

  As used herein, the term “electroporation” is used to physically puncture a cell (eg, a plant cell) using a direct current high voltage pulse from which a nucleic acid (eg, a gene is included). (Nucleic acid) is a method of introducing into a cell. Electroporation conditions can be appropriately selected by those skilled in the art depending on the species, tissue, cells, and the like used. Typical electroporation voltage conditions are 10 V / cm to 200 V / cm, preferably 20 V / cm to 150 V / cm, more preferably 30 V / cm to 120 V / cm, and even more preferably 40 V / cm to 100 V. / Cm, most preferably 50 V / cm to 100 V / cm, but is not limited thereto. Typical electroporation pulse width conditions are 1 microsecond to 90 milliseconds, preferably 10 milliseconds to 90 milliseconds, more preferably 10 milliseconds to 90 milliseconds, and even more preferably 20 milliseconds to 80 milliseconds. Milliseconds, even more preferred 30 milliseconds to 80 milliseconds, even more preferred 40 milliseconds to 70 milliseconds, and most preferred 50 milliseconds to 60 milliseconds, but not limited thereto. The pulse width of electroporation 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 to 40 microseconds. Although it is 70 microseconds and 50 microseconds-60 microseconds, it is not limited to these. The number of typical electroporation pulses is 1 to 200 times, preferably 10 to 150 times, more preferably 20 to 120 times, still more preferably 30 to 110 times, most preferably 40 times. Although it is -100 times, it is not limited to these.

  As used herein, the phrase “place cell (or tissue) and nucleic acid under conditions in which electroporation occurs” means that the cell (or tissue) and nucleic acid are electroporated between them. All conditions (voltage conditions, pulse width conditions, pulse frequency conditions, positional relationship between cells (or tissues) and nucleic acids that are essential for the formation of nucleic acids (ie, introduction of nucleic acids into cells (or tissues)) , Including the execution time of electroporation). Conditions essential for electroporation to occur will be readily apparent to those skilled in the art, and those skilled in the art can appropriately determine the conditions.

  Furthermore, when performing electroporation according to the present invention, it is preferable to apply voltage pulses to cells (or tissues) and nucleic acids in at least two directions. The simplest is to apply a voltage pulse to a cell (or tissue) and nucleic acid for a certain period of time, and then reverse the voltage pulse by reversing the anode and cathode of the electrode used for the voltage pulse treatment. Can be achieved. This can also be accomplished using electrode pairs located at different locations within the electroporation chamber. By applying voltage pulses in at least two directions in this way, the nucleic acid introduction efficiency is significantly increased.

  The electroporation chamber used for electroporation in the present invention may have any size as long as it can accommodate cells and / or tissues to which nucleic acid is introduced. Particularly preferably, the electroporation chamber is sized to accommodate plant tissue (eg, plant seeds). The electroporation chamber of the present invention can be of any shape. Examples of the shape include a cube, a rectangular parallelepiped, a cylinder, and a tube (for example, a shape having a uniform or non-uniform cross section of the body and a bottom that may or may not be tapered). However, it is not limited to these. In order to be able to accommodate plant seeds, the diameter of the largest inscribed circle that touches at least three points on the inner surface of the electroporation chamber of the present invention can be, for example, the following length: at least about 5 mm Or longer than about 5 mm, 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 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 at least about 3 cm or longer than about 3 cm. Or at least about 4 cm or longer than about 4 cm, preferably at least about 5 cm or longer than about 5 cm, preferably 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 about 9 cm or longer than about 9 cm, preferably at least It is about 10 cm or longer than about 10 cm, preferably at least about 15 cm or longer than about 15 cm, and preferably at least about 20 cm or longer than about 20 cm. The upper limit of the diameter of the largest inscribed circle that contacts at least three points on the inner surface of the electroporation chamber of the present invention 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 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 container. 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. However, typically, the thickness of the electrode used is negligibly thin (for example, 0.1 mm).

  In one aspect, the electroporation chamber of the present invention has a rectangular cross section and has an internal dimension (eg, length × width × height) of 1 cm × 2 cm × 2 cm. In another aspect, the electroporation chamber of the present invention has a circular cross section and has an internal dimension (eg, diameter x height) of 1 cm x 4 cm.

  In a further aspect, the electroporation chamber of the present invention has a hexagonal cross section. For example, as shown in FIGS. 11 and 12, the electroporation chamber 1 having a hexagonal cross section includes a bottomed cylinder 18 having an open top surface and electrodes provided on the inner surfaces of the cylinder 18. 11-16. A cord (not shown) for applying a high voltage pulse by electroporation is electrically connected to the electrodes 11-16. The cylindrical body 18 has a bottom portion 17 and introduces a sample therein to perform electroporation. In a preferred embodiment, the electrodes of the electroporation chamber each have a size of 10 mm x 15 mm, but are not limited thereto. In yet another aspect, the electroporation chamber of the present invention has an n-gonal (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 very thin enough to be ignored. In this specification, the transverse section refers to a section perpendicular to the major axis direction of the chamber. In the present specification, the internal dimension refers to the length connecting any two points on the inner surface of the chamber container. In particular, in the case of a chamber having a rectangular cross section, the length and width of the cross section, and the height And in the case of a chamber having a circular cross section, it refers to the diameter and height of the cross section.

  In one embodiment, the electroporation chamber of the present invention can vary in size to 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 electroporation chamber of the present invention can be made from any material. As the material of the electroporation chamber, any material capable of forming a solid can be used. Examples include, but are not limited to, glass, silica, silicon, ceramic, silicon dioxide, plastics, metals (including alloys), natural and synthetic polymers (eg, polystyrene, cellulose, chitosan, dextran, and nylon). Not. The chamber may be formed from a plurality of layers 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) polyphenylene oxide, polybutylene terephthalate, reinforced polyethylene terephthalate, polyethersulfone, polyphenylene sulfide, polyarylate, polyetherimide, polyetheretherketone, polyimide, polyester, polyethylene, polypropylene, polyisobutylene, unsaturated Polyester, fluorinated resin, polyvinyl chloride, polyvinylidene chloride, polyvinyl acetate, polyvinyl alcohol, polyvinyl acetal, acrylic resin, polyacrylonitrile, polystyrene, acetal resin, phenol resin, urea resin, epoxy resin, melamine resin, styrene / acrylonitrile Copolymer, acrylonitrile butadiene styrene copolymer, silicone resin, polysulfur And organic materials such emissions may also be used. Preferably, the electroporation chamber of the present invention has the ability to withstand pressures different from atmospheric pressure (eg, the ability to not break, crack and deform when exposed to pressures different from atmospheric pressure), particularly preferably Ability to withstand decompression. Particularly preferably, the electroporation chamber of the present invention is made of polypropylene, silicone resin, and glass and comprises an electrode made of platinum or stainless steel.

  Preferably, the electroporation chamber of the present invention comprises 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 may be any means, for example, means utilizing ice, cooling gel or the like.

  The electroporation chamber of the present invention includes at least a pair (two) of electrodes. Thus, in certain embodiments, the chamber of the present invention comprises more than one (two) electrodes (eg, two (four) electrodes, three (six) electrodes, four (eight) electrodes, Electrode, five pairs (ten), 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 the 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 a pair (two) of electrodes, a voltage may be applied simultaneously to each pair of electrodes, or a voltage may be applied sequentially to each pair of electrodes. In the case where a voltage is applied to the electrodes of each pair, the device for sequentially switching the applied voltage is commercially available. For example, it is possible to use CU901 of Neppagene Corporation (Ichikawa City, Chiba Prefecture, Japan). For example, when a voltage is sequentially applied to more than a pair (two) of electrodes, the voltage switching interval is 120 seconds to 1 millisecond, preferably 60 seconds to 10 milliseconds, more preferably 30 seconds to 100 milliseconds. More preferably 10 to 500 milliseconds, even more preferably 5 to 750 milliseconds, still more preferably 3 to 800 milliseconds, most preferably 1.1 to 900 milliseconds, It is not limited to.

  The distance between the electrodes disposed in the electroporation chamber of the present invention can be any distance, and can vary depending on the size of the cells and / or tissues to which the nucleic acid is to be introduced. Particularly preferably, the distance between the electrodes is a distance capable of accommodating a plant tissue (for example, a plant seed). In order to be able to accommodate plant seeds, the distance between the electrodes can be, for example, the following length: at least about 5 mm or longer than about 5 mm, 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 at least about 9 mm or about More than 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 at least about 3 cm or more than about 3 cm Longer, preferably at least about 4 cm or longer than about 4 cm, preferably less Is also about 5 cm or longer than about 5 cm, preferably 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 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 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 3 cm, about 2 cm, about 1 cm, about 9 mm, about 8 mm, about 7 mm, or about 6 mm. Of course, the distance between the electrodes can be a length between the values specified above (eg, 1.5 cm, etc.).

  In one embodiment, the distance between the electrodes can be varied such that the distance between the pair of electrodes is a distance that can accommodate a plant seed. 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 as long as it has the property of allowing current to flow. Examples of the material for 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 electroporation chamber is a 1 cm long, 2 cm wide, 2 cm high rectangular chamber with platinum electrodes, and the distance between the platinum electrodes is about 1 cm. This electroporation 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 electroporation chamber is a 1 cm internal diameter x 4 cm high microtube chamber with stainless steel electrodes, and the distance between the stainless steel electrodes is about 1 cm. This microtube type chamber can be easily manufactured by attaching a stainless steel foil (for example, about 5 × 40 mm (thickness: about 0.1 mm)) to the inner surface of a commercially available microtube with an adhesive or the like. This microtube chamber simplifies solution exchange because it can be centrifuged to precipitate cells, tissues and / or seeds at the bottom. For this reason, this microtube-type chamber is particularly useful when processing minute (about 0.1 to 5 mm) plant seeds (for example, Arabidopsis thaliana). By using this chamber, it is possible to process a large amount of minute-sized plant seeds.

  In another aspect of the present invention, the electroporation electrode may be, for example, an electrode that is inserted into a test tube as shown in FIG. The electroporation electrode 2 shown in FIGS. 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 plate-like electrodes 21 and 22 of the electroporation electrode 2 are inserted into a test tube 25 and used. If necessary, a vent may be provided in the cap 23 or the test tube 25 to increase or decrease the pressure in the test tube. A sample is put in the test tube 25 and electroporation is performed by the electroporation electrode 2.

  As used herein, “maintaining a cell / tissue (including plant tissue) under a pressure different from atmospheric pressure” means that the cell / tissue (including plant tissue) is at atmospheric pressure (usually, 1 atm = 101.325 kPa = about 0.1 MPa) A step of maintaining a pressure higher than (pressure treatment) or a step of maintaining a lower pressure (decompression treatment).

  Although not intending to be bound by theory, by maintaining cells / tissues (including plant tissues) under a pressure different from atmospheric pressure, the pressure in the environment received by the cells / tissues changes, and nucleic acids such as DNA Nucleic acid introduction by electroporation targeting cells and tissues (especially plant cells and plant tissues) having cell walls that have been impossible in the past. / It is considered that transformation has become possible.

  As used herein, the term “reduced pressure treatment” refers to a treatment that maintains cells / tissues (including plant tissues (eg, seeds)) into which nucleic acid is introduced / transformed, at a pressure lower than atmospheric pressure. . In the present invention, the pressure reduction treatment is 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. Although it is performed at a pressure as low as 0.096 MPa, it is not limited thereto. The time of the decompression treatment 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, and most preferably about 60 minutes. It is not limited to.

  As used herein, the term “pressure treatment” refers to a treatment that maintains cells / tissues (including plant tissues (eg, seeds)) to be introduced / transformed at a pressure higher than atmospheric pressure. Say.

  In one aspect, the present invention relates to an electroporation apparatus. The electroporation apparatus of the present invention can introduce nucleic acid into any cell or tissue with remarkably high efficiency, and in particular, a cell having a cell wall or Useful for introducing nucleic acids into tissues (eg, plant cells or plant tissues). In one embodiment, the electroporation device of the present invention comprises both a) means for maintaining cells under a pressure different from atmospheric pressure, and b) electroporation means.

  As a means for maintaining the cells under a pressure different from the atmospheric pressure, any means having the ability to reduce pressure and / or pressurize can be used. Commercially available decompression devices (eg, vacuum desiccators, etc.) and / or pressurization devices can also be utilized. Any electroporation means can be used as the electroporation means. Commercially available electroporation means (eg, CUY21 EDIT gene transfer device, Neppagene, Ichikawa City, Chiba, Japan) can also be used. Preferably, the distance between the two electrodes (first electrode and second electrode) arranged in the electroporation means is a distance capable of accommodating plant seeds as defined above.

  In another embodiment, the electroporation device of the present invention comprises two electrodes at a distance that can accommodate plant seeds, and the electroporation device maintains cells / tissue under a pressure different from atmospheric pressure. Used in combination with. In this embodiment, the electroporation device and the means for maintaining the cells / tissue under a pressure different from atmospheric pressure need not be in the same instrument.

  Since the conventional electroporation apparatus 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 of the conventional electroporation apparatus and the distance between the electrodes are typically about 1 mm or 2 mm, and at most 4 mm at most. Therefore, an electroporation apparatus including a chamber that is large enough to accommodate plant seeds and an electrode disposed at a long distance enough to accommodate plant seeds as in the present invention has not been known.

  Furthermore, the electroporation apparatus of the present invention can be automated. In the automated electroporation apparatus of the present invention, injection and / or exchange of a solution such as a buffer can be performed by an automatic dispenser. As the automatic dispensing machine, for example, a commercially available epMotion 5070 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 placing nucleic acid and / or cells in a container containing a mixture 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 acid and cells may be any Can be a container. These containers may be the same or different. As the material of the container, any material capable of forming a solid can be used. Examples include, but are not limited to, glass, silica, silicon, ceramic, silicon dioxide, plastics, metals (including alloys), natural and synthetic polymers (eg, polystyrene, cellulose, chitosan, dextran, and nylon). Not. The container may be formed from a plurality of layers 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) polyphenylene oxide, polybutylene terephthalate, reinforced polyethylene terephthalate, polyethersulfone, polyphenylene sulfide, polyarylate, polyetherimide, polyetheretherketone, polyimide, polyester, polyethylene, polypropylene, polyisobutylene, unsaturated Polyester, fluorinated resin, polyvinyl chloride, polyvinylidene chloride, polyvinyl acetate, polyvinyl alcohol, polyvinyl acetal, acrylic resin, polyacrylonitrile, polystyrene, acetal resin, phenol resin, urea resin, epoxy resin, melamine resin, styrene / acrylonitrile Copolymer, acrylonitrile butadiene styrene copolymer, silicone resin, polysulfur And organic materials such emissions may also be used.

  Preferably, the first container of the present invention is made of a material that has high transparency and facilitates material observation (for example, polystyrene). Preferably, the second container of the present invention is made of a material (for example, polyamide, polycarbonate, (modified) polyphenylene oxide, polybutylene terephthalate, reinforced polyethylene terephthalate, polyether) capable of withstanding a pressure different from atmospheric pressure (particularly reduced pressure). Material made of sulfone, polyphenylene sulfide, polyarylate, polyetherimide, polyetheretherketone, polyimide and epoxy resin, and more preferably a material having high transparency and easy material observation (for example, acrylic resin) Made from. Preferably, the third container of the present invention is made of a material (for example, polypropylene, silicone resin, and glass) showing affinity with cells, and is made of platinum, gold, stainless steel, carbon, or conductive polymer. The electrode made of is provided. These materials may be coated with any suitable material to impart desired properties (eg, to provide insulation to the container body or to increase the conductivity of the electrode).

  For the first and second containers described above, preferably these containers have the ability to withstand pressures different from atmospheric pressure, and particularly preferably have the ability to withstand vacuum processing. For example, the first container may be accommodated in the second container and / 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 / 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. However, 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 / discharge using an automatic pump or the like can also be used.

  The automated electroporation apparatus of the present invention includes a control device for automating and implementing each operation means. Furthermore, the electroporation device of the present invention includes a power supply device. Such a control apparatus and a power supply apparatus may be arrange | positioned in the same apparatus as an electroporation apparatus, or may be connected with a cord as separate apparatuses.

  When electroporation is performed on seeds, it is preferable to leave seeds subjected to reduced pressure treatment or pressurized treatment 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, and even more preferably 20 hours to 26 hours. Although it is preferably about 24 hours, it is not limited thereto.

  Exemplary conditions for introducing nucleic acid in the present invention are as follows: seeds are left in tap water at 25 ° C. overnight, placed in a vacuum device the next day, and subjected to a vacuum treatment at a pressure lower than atmospheric pressure by 0.096 MPa for 1 hour. Then, a high-voltage pulse (100 V, 50 milliseconds, where the distance between the electrodes is 1 cm) is applied about 50 times to the seeds subjected to reduced pressure treatment under the condition that nucleic acid introduction / gene introduction is performed. is there. The voltage and the number of pulses can vary depending on the crop, and those skilled in the art can appropriately select the electroporation conditions as necessary. Thereafter, a normal plant individual can be obtained by selecting in a medium containing antibiotics and then raising the pot (growing in a pot).

  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 tissues, 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 cell wall formation and anabolism by chloroplasts. “Plant” includes both monocotyledonous and dicotyledonous plants. Monocotyledonous plants include gramineous plants. Preferred monocotyledonous plants include corn, wheat, rice, oat, barley, sorghum, rye and millet, and more preferably, corn, wheat and rice are not limited thereto. Wheat includes wheat cultivar Norin 61, for which it was difficult to obtain transformants by conventional methods. Examples of dicotyledonous plants include cruciferous plants, legumes, eggplants, cucurbits and convolvulaceae, but are not limited thereto. Brassicaceae plants include but are not limited to Chinese cabbage, rapeseed, cabbage and cauliflower. Preferred cruciferous plants are Chinese cabbage and rapeseed. A particularly preferred cruciferous plant is rapeseed. Leguminous plants include, but are not limited to, soybean, azuki bean, kidney bean, and cowpea. 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. Cucurbitaceae plants include, but are not limited to, cucumber, cucumber, melon and watermelon. A preferred cucurbitaceae plant is cucumber. 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 can mean a plant.

  In another embodiment, examples of plant species that can be used in the present invention include solanaceae, gramineous, cruciferous, rose, leguminous, cucurbitaceae, perillaceae, liliaceae, red crustaceae, ciliaceae, bindweed Plants such as family and asteraceae are listed. Furthermore, examples of plant species that can be used in the present invention include any tree species, any fruit tree species, mulberry plants (eg, rubber), and mallow (eg, cotton).

  Conveniently, the method of the invention performs electroporation on plant tissue (including dormant tissue (including ripe seeds, immature seeds, winter buds, and tubers), germplasm, growth points, and flower buds), Most simply, electroporation is performed on the seed. The seed into which nucleic acid has been introduced by the electroporation method of the present invention can be easily converted into a nucleic acid introducer / transformant, for example, by planting and cultivating it in soil. Seeds usually consist of three parts: embryo, endosperm, and seed coat (supervised by Yoshikichi Noguchi and Shinichiro Kawada, see Hideo Chisaka, Encyclopedia of Agriculture, 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 nucleic acid was introduced by the electroporation method of the present invention, expression of the introduced nucleic acid was observed in the embryo part. Therefore, a nucleic acid-introduced plant / 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, horseradish, and tuna.

  Examples of gramineous 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.

  As used herein, “animal” is a collective term for organisms belonging to the animal kingdom, and is characterized by organisms that require oxygen and organic food and can move arbitrarily unlike plants and minerals. Animals are broadly classified into vertebrates and invertebrates. As the vertebrate, for example, larvae, lampreys, cartilaginous fish, teleosts, amphibians, reptiles, birds, mammals and the like are used, and more preferably mammals (for example, single pores, marsupials, poor Teeth, wings, wings, carnivores, carnivores, long noses, odd hoofs, even hoofs, rodents, scales, sea cattle, cetaceans, primates, rodents, Rabbit eyes, etc.) are used. More preferably, a primate (for example, chimpanzee, Japanese monkey, human) is used. Most preferably, human-derived cells or organs are used. As the invertebrates, for example, crustaceans, millipede, Edahi beetle, centipede, komcade, insects and the like are used. More preferably, insects (for example, Lepidoptera (including silkworms)) are used.

  As used herein, “transgenic 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, and similarly “transgenic animal” refers to an animal into which a specific gene has been incorporated.

  Cells and tissues that have been transfected / transformed by the methods of the present invention can be differentiated, grown and / or expanded by any method known in the art. In the case of plant species, the process of differentiating, growing and / or proliferating cells or tissues can be achieved, for example, by cultivating the plant cells or plant tissues or plants containing them. In the present specification, plant cultivation can be performed by any method known in the art. See, for example, supervised by Isao Shimamoto and Kiyoshi Okada, “Experimental Protocol for Model Plants—Edited by Rice and Arabidopsis thaliana”: Cell Engineering, Separate Volume, Plant Cell Engineering Series 4; 28-32, and Niwa Yasuo, Arabidopsis cultivation method, pp. 33-40 and can be easily implemented by those skilled in the art and need not be described in detail herein. For example, Arabidopsis can be cultivated by soil cultivation, rock wool cultivation, or hydroponics. When cultivated under a white fluorescent lamp (about 6000 lux) under constant light conditions, the first flower will bloom about 4 weeks after sowing, and the seed will ripen about 16 days after flowering. About 40 to 50 seeds can be obtained with one pod, and about 10,000 seeds can be obtained in 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. Therefore, for example, when cultivating wheat in an artificial environment (for example, a greenhouse or a gross chamber), the wheat seedlings are treated at a low temperature and short day (for example, 20 ° C., light period 8 hours (about 2000 lux) in the early stage of growth. ) And 8 ° C. treatment in the dark period of 16 hours). This process is called vernalization. The cultivation conditions required for each plant species are generally well known in the art, and therefore need not be described in detail herein.

  In the case of species other than plants (eg, animal species), the nucleic acid introduced / transformed cells and tissues can be differentiated, grown and / or propagated (eg, Izumi) by any method known in the art. Oji, et al., Biochemistry Experiments 4. See Animal and Tissue Experiments, Chemistry Dojin, 1987). For example, cells and / or tissues into which nucleic acid has been introduced by electroporation can be grown at room temperature (about 25 ° C.) while supplying a commercial feed.

  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 cells / tissues. Examples of the surfactant used in the present invention include, for example, a surfactant that is commercially available as a pesticide spreading agent from Loveland Industries under the trade name of Silwet L-77, and is a low-toxic surfactant for plants. Examples include, but are not limited to, alkylene oxide-modified heptamethyltrisiloxane (Polyalkylene Modified Modified Hexamethyltrisiloxane) and Tween 20® (polyoxyethylene (20) sorbitan monolaurate), a surfactant that is less toxic to animals and plants. .

  As used herein, the “vir region transcription inducer” of Ti plasmid refers to a substance that induces transcription of genes present in the vir region of Ti plasmid. The Vir region transcription inducer is originally derived from a plant endogenous phenol compound (including acetosyringone) that is induced when a plant is subjected to stress such as a wound. A typical example of a vir region transcription inducer is acetosyringone, but in addition to acetosyringone and its derivatives, syringaldehyde and ferulic acid, which are precursors of lignin synthesis isolated from exudates of tobacco leaf sections. Alternatively, a derivative that induces transcription of a gene group present in the vir region is also included. In addition, since plants have endogenous acetosyringone, it is not always necessary to add acetosyringone in the practice of the present invention. Even if acetosyringone is not used, nucleic acids and / or Or introduction of bacteria is possible.

  As used herein, a “Ti plasmid” is an approximately 200 kbp plasmid of Agrobacterium tumefaciens that is a causative factor for crown gall tumors. The Ti plasmid has a vir region, a T-DNA region, and the like.

  As used herein, a “vir region” is a region of a gene responsible for pathogenicity in a Ti plasmid, which is a six transcription unit of virA, B, G, C, D, and E. Have

  As used herein, the introduced gene consists of a polynucleotide.

  As used herein, the terms “polynucleotide”, “oligonucleotide”, and “nucleic acid” are used interchangeably herein and refer to a polymer of nucleotides of any length. The term also includes “derivative oligonucleotide” or “derivative polynucleotide”. A “derivative oligonucleotide” or “derivative polynucleotide” refers to an oligonucleotide or polynucleotide that includes a derivative of a nucleotide or that has an unusual linkage between nucleotides and is used interchangeably. Specific examples of such an oligonucleotide include, for example, 2′-O-methyl-ribonucleotide, a derivative oligonucleotide in which a phosphodiester bond in an oligonucleotide is converted to a phosphorothioate bond, and a phosphodiester bond in an oligonucleotide. Is an N3′-P5 ′ phosphoramidate bond derivative oligonucleotide, a ribose and phosphodiester bond in the oligonucleotide converted to a peptide nucleic acid bond, uracil in the oligonucleotide is C− A derivative oligonucleotide substituted with 5-propynyluracil, a derivative oligonucleotide where uracil in the oligonucleotide is substituted with C-5 thiazole uracil, and cytosine in the oligonucleotide is C-5 propynylcytosine Substituted derivative oligonucleotides, derivative oligonucleotides in which the cytosine in the oligonucleotide is replaced with phenoxazine-modified cytosine, derivative oligos in which the ribose in the oligonucleotide is replaced with 2′-O-propyl ribose Examples thereof include derivative oligonucleotides in which ribose in nucleotides and oligonucleotides is substituted with 2′-methoxyethoxyribose. Unless otherwise indicated, a particular nucleic acid sequence may also be conservatively modified (eg, degenerate codon substitutes) and complementary sequences, as well as those explicitly indicated. 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 / or deoxyinosine residue. (Batzer et al., Nucleic Acid Res., 19: 5081, 1991; Ohtsuka et al., J. Biol. Chem., 260: 2605-2608, 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 (another) 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.

  As used herein, “gene” refers to a factor that defines a genetic trait. Usually arranged in a certain order on the chromosome. 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 genes refers to the degree of identity of two or more gene sequences with respect to each other. Therefore, the higher the homology between two genes, the higher the sequence identity or similarity. Whether two genes have homology can be examined 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 99% are identical, the genes are homologous.

  In this specification, comparison of base sequence identity and calculation of homology are calculated using BLAST, which is a sequence analysis tool, using default parameters.

  In the present specification, “expression” of a gene, polynucleotide, polypeptide or the like means that the gene or the like undergoes a certain action in vivo to take another form. Preferably, a gene, polynucleotide or the like is transcribed and translated into a polypeptide form. However, transcription and production of mRNA can also be an aspect of expression. More preferably, such polypeptide forms may be post-translationally processed.

  In the present specification, the “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, phosphorothioates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNA). .

  In the present specification, the “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 is n). The length of the fragment can be appropriately changed according to the purpose. For example, the lower limit of the length is 3, 4, 5, 6, 7, 8, 9, 10, Examples include 15, 20, 25, 30, 40, 50 and more amino acids, and lengths expressed in integers not specifically listed here (eg, 11 etc.) are also suitable as lower limits. obtain. In the case of polynucleotides, examples include 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100 and more nucleotides. Non-integer lengths (eg, 11 etc.) may also be appropriate as a lower limit.

  General molecular biology techniques that can be utilized in the present invention include Ausubel F. et al. A. Et al., Current Protocols in Molecular Biology, Wiley, New York, NY, 1988; Sambrook J. et al. Et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1987, etc., can be easily implemented by those skilled in the art.

  In the present specification, when referring to a gene, a “vector” refers to one that can transfer a target polynucleotide sequence into a target cell. Such vectors can be autonomously replicated in prokaryotic cells, yeast, animal cells, plant cells, insect cells, host cells such as animal individuals and plant individuals, preferably plant cells, or into the chromosome. Examples are those that can be integrated and 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. Regulatory elements can preferably include terminators, selectable markers such as drug resistance genes, and enhancers. It is well known to those skilled in the art that the type of organism (eg, plant) expression vector and the type of regulatory elements 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 phosphotransferase that confers resistance to the antibiotic hygromycin The hyg gene (Gritz and Davies, Gene, 25: 179, 1983); and the bar gene (EP 242236) encoding phosphinothricin acetyltransferase conferring resistance to the herbicide phosphinothricin (EP 242236); Spt gene encoding transferase; streptomycin resistance gene; spectinomycin resistance residue Drug resistance genes such as offspring (eg HS Chawla, Introduction to Plant Biotechnology 2nd: 363, Science Publishers, Inc., Monograph, 2002); and the gus gene encoding β-glucuronidase (Jefferson et al., Procson et al., Procson et al. Acad.Sci.USA, 6: 3901, 1986) and the luciferase gene (Ow et al., Science, 234: 856, 1986) and the GFP (green fluorescent protein) coding gene (eg, Funakoshi Co., Ltd., Hongo, Bunkyo-ku, Tokyo) Screenable marker genes such as, but not limited to:

  Examples of the drug used for selection in the present invention include, but are not limited to, kanamycin, hygromycin, geneticin, gentamicin, streptomycin, and spectinomycin.

  “Recombinant vector” refers to a vector capable of transferring a target polynucleotide sequence to a target cell. Such vectors can be autonomously replicated in plant cells and host cells such as plant individuals, or can be integrated into the chromosome, and contain a promoter at a position suitable for transcription of the polynucleotide of the present invention. What is present is exemplified.

  Examples of “recombinant vectors” for plant cells include Ti plasmids, tobacco mosaic virus vectors, geminivirus vectors, and the like.

  A “terminator” 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 is involved in the stability of mRNA and affects the expression level of the gene. Examples of the terminator include, but are not limited to, CaMV35S terminator, terminator of nopaline synthase gene (Tnos), and terminator of tobacco PR1a gene. As used herein, the term “promoter” refers to a region on DNA that determines the transcription start site of a gene and directly regulates its frequency, and is a base sequence that initiates transcription when RNA polymerase binds. is there. 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 base sequence is predicted using DNA analysis software, The promoter region can be estimated. The putative promoter region is usually upstream of the structural gene, but preferably the putative promoter region is present within about 2 kbp upstream from the first exon translation start point.

  In the present specification, when used for expression of a gene, “site specificity” generally means a part of an organism (eg, a plant) (eg, in the case of a plant, a root, a stem, a stem, a leaf, a flower, a seed, Germ, embryo, fruit, etc.) the specificity of the gene expression. “Time specificity” refers to the specificity of expression of a gene according to the stage of development of an organism (eg, a plant) (eg, if it is a plant, the growth stage (eg, the number of days after germination)). Such specificity can be introduced into the desired organism by selecting an appropriate promoter.

  In the present specification, the expression of the promoter of the present invention is “constitutive” in an almost constant amount in all tissues of an organism, whether it is in the juvenile stage or the mature stage of the organism. It refers to the property that is expressed. Specifically, when Northern blot analysis is performed under the same conditions as in the examples of the present specification, for example, the same or corresponding at any time point (for example, 2 points or more (for example, 5th day and 15th day)) Expression is said to be constitutive by definition of the present invention when almost the same level of expression is observed at any of the sites. Constitutive promoters are thought to play a role in maintaining 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 “constitutive” expression because it is premised on that expression is observed in a normal state. These properties can be determined by extracting RNA from any part of the organism and analyzing the expression level by Northern blot analysis or quantifying the expressed protein by Western blot. 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 use a stimulating factor having inducible activity of the promoter. Thus, a specific gene can be expressed under certain conditions.

  An “enhancer” can be used to increase the expression efficiency of a target gene. When used in plants, an enhancer region containing an upstream sequence in the CaMV35S promoter is preferred as an enhancer. A plurality of enhancers may be used, but one may be used or may not be used.

  As used herein, “operably linked” refers to a transcriptional translational regulatory sequence (eg, promoter, enhancer, etc.) or a translational regulatory sequence that has the expression (operation) of a desired sequence. That means. In order for a promoter to be operably linked to a gene, the promoter is usually placed immediately upstream of the gene, but there may be a sequence intervening between the promoter and the structural gene. It does not necessarily have to be placed adjacent to the structural gene.

  The presence of the introduced gene can be confirmed by Southern blotting or PCR. Transcription of the introduced gene can be detected by Northern blotting or PCR. If necessary, the expression of a protein as a gene product can be confirmed by, for example, Western blotting.

  The present invention will be described below based on examples, but the following examples are provided for illustrative purposes only. Accordingly, the scope of the present invention is not limited to the above detailed description of the invention nor the following examples, but is limited only by the scope of the claims.

(Electroporation equipment)
As the electroporation apparatus used in the present invention, commercially available electroporation means (for example, CUY21 EDIT gene transfer apparatus, Neppagene, Ichikawa City, Chiba Prefecture) can be used. The electroporation chamber used for electroporation in this example may be any size as long as it can accommodate the plant tissue to be transformed, but can be cooled. A preferred chamber is preferred. In this example, a custom-made electroporation chamber with a platinum electrode of 1 cm long × 2 cm wide × 2 cm high was used.

(Electroporation)
After decompression, the seeds and buffer in the petri dish are transferred to a chamber and left on ice for 1 minute. Then, as shown in the examples, a rectangular wave with a voltage of 100 V or 50 V (however, the distance between the electrodes is 1 cm) and a pulse width of 50 milliseconds (a voltage is applied for 50 milliseconds, 50 milliseconds to 75 seconds). Repeat cycle of resting for milliseconds), 50 times or 99 times. Further, after allowing the chamber to stand on ice for 2 minutes, the buffer solution is discarded, and the treated seed is returned to the original petri dish. 2 ml of 0.5% polyvinylpyrrolidone (PVP) aqueous solution is put into the petri dish where the seeds are returned. 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 propagation of various bacteria.
The petri dish is stored at 4 ° C. for about 1 hour and then allowed to stand at 25 ° C. overnight.

(GUS analysis)
X-Gluc solution (100 mM pH 7.0 phosphate buffer, 0.05% X-Gluc (5-bromo-4-chloro-3-indolyl-β-D-glucuronide cyclohexylammonium salt), 0.5 mM potassium ferricyanide 2 ml of 0.5 mM potassium ferrocyanide, 0.3% Triton X-100, 20% methanol) was added and allowed to stand overnight at 25 ° C. The expression of the GUS gene was confirmed from the degree of staining.

(Selection with antibiotic medium)
Without GUS analysis, grow as follows.
a) Transfer the seeds to a 9 cm × 15 mm petri dish with 7 cm filter paper.
b) Add 10 ml of an aqueous solution of antibiotics dissolved in distilled water. When wheat seeds are selected with geneticin, a concentration of 2000 ppm or 1200 ppm is used. In addition, the density | concentration when selecting a rice seed with geneticin is 200 ppm.
c) Growing in a culture room under conditions of 25 ° C. light period 16 hours (approximately 2000 lux) dark period 8 hours or 20 ° C. light period 8 hours (approximately 2000 lux) and 8 ° C. dark period 16 hours. To do.

(Raising transformants)
After selection with antibiotic medium, the plant was used in a pot (flower pot) with a diameter of 8.5 cm and a height of 5.5 cm containing horticultural soil (New Magic Soil; Sakata Seed, Tsuzuki-ku, Yokohama). And grow in an isolated gross chamber (15 ° C light period 8 hours (sodium lamp, about 50000 lux), dark condition 16 hours or 20 ° C light period 8 hours (approximately 2000 lux) and 8 ° C dark period 16 Time condition).

(DNA extraction)
In order to perform PCR for confirming gene transfer, 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 Uchimiya “Plant Gene Manipulation Manual How to Make Transgenic Plants” Kodansha Scientific, pages 71-74, book, 1989).

(PCR analysis)
Using a pair of primers (5′-ctgcgtgcaatcccatcttg-3 ′: SEQ ID NO: 1, 5′-actcgtcaagaggcgatagaag-3 ′: SEQ ID NO: 2) for extracting DNA from the leaves of the individual after electroporation and detecting the NPT II gene PCR was performed. In addition, an additional pair of primers (5′-catgattgaacaagagatgattgcacgcaggttctc-3 ′: SEQ ID NO: 3, 5′-cagaagaactcgtcaagaagcccagagaggcgat-3 ′: SEQ ID NO: 4) can also be used. Since it can detect, it is more preferable. As the polymerase, AmpliTaq Gold manufactured by Perkin Elmer Japan Co., Ltd. (Nishi-ku, Yokohama) is used according to the manufacturer's instructions. The thermal cycler conditions used for amplification are:
(A) 1 cycle of 95 ° C. for 10 minutes;
(B) 50 cycles of 95 ° C. for 1 minute, 64 ° C. for 2 minutes, 72 ° C. for 2 minutes;
(C) 72 ° C. for 7 minutes for 1 cycle; and (d) then stored at 4 ° C.

(Southern blot analysis)
When the presence of the transgene is confirmed by the PCR analysis after electroporation, Southern blot analysis is performed using DNA derived from the individual. Southern blotting is well known in the art, and those skilled in the art can appropriately select the conditions as necessary. In the Southern blot 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.

(Genetic analysis)
If the transgene is confirmed by Southern blot analysis, the plant is further grown and a large number of fully mature seeds are obtained by self-pollination. About 10 seeds are randomly picked from them and cultivated in pots filled with soil. DNA is extracted from the leaves of the young plant, and PCR is performed under the conditions described in the PCR analysis using this DNA as a template.
(Example 1: Transformation of rice plant by the method of the present invention)
(material)
Brown rice of Koshihikari was used as a ripe seed of japonica rice. Agricultural forest No. 61 was used as wheat seeds. As a mature seed of Indica rice, brown rice of IR24 was used. As a Chinese cabbage seed, “Musou” was used. “Fayatté” was used as a soybean seed. As a tomato seed, “Mini Carol” was used. As the morning glory seeds, “Sun Smile White White Wheel” was used. The seeds grown at our laboratory or purchased from a seed company were used. About 10 to 30 appropriate mature seeds were visually selected (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 producing a transgenic plant of wheat, this water absorption was carried out at 10 ° C. for 2 nights. EHA150 was used as an Agrobacterium.

  In addition, it is also possible to replace with a water absorption process by lengthening the time which immerses a mature seed in an Agrobacterium liquid.

(Seed sterilization)
The mixture was sterilized with 70% ethanol for 30 seconds and then with a 2.5% sodium hypochlorite solution for 20 minutes, and then washed with sterile water.

(vector)
Agrobacterium tumefaciens EHA105 has a binary vector pCAMBIA1390-sGFP (FIG. 1) having a hygromycin resistance gene which is a marker gene for selecting transformed cells and a GFP (Green Fluorescent Protein) gene serving as a reporter for transformed cells on T-DNA. The fungus was transformed and used for the experiment.

(Transformation)
Agrobacterium suspended in distilled water and adjusted to OD ₆₀₀ = about 0.5 was allowed to stand for 1 hour. Next, 1 μL of Silwet L-77 and 4 μL of 10 mg / ml acetosyringone were added to 3995 μL of Agrobacterium solution. Seeds were put into this bacterial solution and placed under reduced pressure (0.096 MPa) for 1 hour. The following electroporation was performed as necessary.

(Electroporation)
After the decompression treatment, the seeds and the buffer in the petri dish were transferred to a chamber and left on ice for 1 minute. Then, as shown in the example, at a voltage of 50 V / cm (however, the distance between the electrodes is 1 cm), a rectangular wave having a pulse width of 50 milliseconds (applying a 50 microsecond voltage and repeating a period of resting for 50 milliseconds) The number of pulses was 99. Further, the chamber was allowed to stand on ice for 2 minutes, and then the buffer solution was discarded, and the treated seeds were returned to the original petri dish. 2 ml of a 0.5% polyvinylpyrrolidone (PVP) aqueous solution was placed in the petri dish where the seeds were returned. This PVP aqueous solution contained a sodium hypochlorite aqueous solution in order to suppress the propagation of various bacteria (adjusted so that the effective chlorine concentration was about 0.01%). The petri dish was stored at 4 ° C. for about 1 hour and then allowed to stand at 25 ° C. overnight.

(Treatment of seeds after gene transfer)
Four days after the infection treatment with Agrobacterium, the seeds were transferred to a 0.5% sodium hypochlorite solution and sterilized. The results of rice on the 6th, 8th, and 14th days after the infection treatment with Agrobacterium are shown in FIGS. FIG. 5 shows the results of wheat on the 8th day after the infection treatment with Agrobacterium.

(Results of Japonica rice)
The fluorescence by GFP was observed 6 days after the treatment with Agrobacterium (FIG. 2), 8 days (FIG. 3), and 14 days (FIG. 4). As a result of gene transfer combining Agrobacterium and electroporation, 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 individuals into which the gene was introduced. Conventionally, there is no example of combining a direct gene transfer method such as electroporation and an indirect gene transfer method such as the Agrobacterium method, but by combining these methods, as shown in this Example It was impossible to predict that the gene could be introduced with high efficiency. Also, a strong expression level as shown in this example was not expected at all. On the other hand, when only the decompression treatment was performed, the expression of GFP was not confirmed (individuals on the right side of each of FIGS. 2B and 3B).

  Without electroporation, the amount of GFP expressed when Agrobacterium infection and gene transfer were performed after the reduced pressure treatment was lower than when electroporation was performed (on the right side of FIG. 4B). individual). In addition, when transfection was performed by Agrobacterium after gene reduction and gene transfer was performed without using electroporation, the expression of GFP was visually confirmed 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 done. However, the result of this Example has a remarkable effect that could not be predicted in terms of rapidity and simplicity in that it does not require such pre-culture.

(Wheat result)
After electroporation, 100 wheat seeds were transferred to a 6 cm petri dish together with an electroporation buffer and placed in a 10 ° C. dark incubator. On the next day, the electroporation buffer was discarded, and 5 ml of 500 ppm carbenicillin solution was added to sterilize Agrobacterium. Subsequently, the growth was continued in a dark incubator at 10 ° C. On the next day (second day after electroporation), selection with geneticin was started with the aim of producing transformants. The carbenicillin solution was discarded and the seeds were transferred to a 9 cm petri dish. 8 ml of 750 ppm geneticin solution and 2 ml of x100 benrate solution were added. Benrate was added with a fungicide (agrochemical) to prevent the generation of mold.

  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.

  About 2 to 10% of the individuals who were able to confirm GFP fluorescence in the shoot portion were selected after about 2 weeks. The individual was transplanted and grown in a pot filled with soil to obtain 7 individuals. These individuals were grown in an isolated gloss chamber.

  On the other hand, when only the decompression treatment was performed, the expression of GFP was not confirmed (center of FIG. 5).

  Conventionally, there is no example of combining a direct gene transfer method such as electroporation and an indirect gene transfer method such as the Agrobacterium method, but by combining these methods, as shown in this Example It was impossible to predict that the gene could be introduced with high efficiency. Also, a strong expression level as shown in this example was not expected at all. In view of the technical level of wheat transformation, the effect of the present invention is remarkable.

  Without electroporation, the amount of GFP expressed when Agrobacterium infection and gene transfer were performed after the reduced pressure treatment was lower than when electroporation was performed (right side of FIG. 5). In addition, when transfection was performed by Agrobacterium after gene reduction and gene transfer was performed without using electroporation, the expression of GFP was visually confirmed 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 done. However, the result of this Example has a remarkable effect that could not be predicted in terms of rapidity and simplicity in that it does not require such pre-culture.

(Indicaine results)
The fluorescence by GFP was observed 7 days after the treatment with Agrobacterium (FIG. 6). As a result of gene introduction combining Agrobacterium and electroporation, strong fluorescence was confirmed (individual on the left side of FIG. 6B). In addition, the expression of GFP was confirmed with the naked eye at a high efficiency of about 60% of the individuals into which the gene was introduced. Conventionally, there is no example of combining a direct gene transfer method such as electroporation and an indirect gene transfer method such as the Agrobacterium method, but by combining these methods, as shown in this Example It was impossible to predict that the gene could be introduced with high efficiency. Also, a strong expression level as shown in this example was not expected at all. Considering the fact that transformation into indicaine has been difficult in the past, the effect of the present invention is remarkable. In addition, the results of this example have a remarkable effect that could not be predicted in terms of rapidity and simplicity in that pre-culture using a medium supplemented with 2,4-D or the like is not required.

(Hakusai results)
Four days after the treatment with Agrobacterium (FIG. 7), the fluorescence by GFP was observed. As a result of gene introduction combining Agrobacterium and electroporation, strong fluorescence was confirmed (individual on the left side of FIG. 7B). In addition, the expression of GFP was confirmed with the naked eye at a high efficiency of about 60% of the individuals into which the gene was introduced. Conventionally, there is no example of combining a direct gene transfer method such as electroporation and an indirect gene transfer method such as the Agrobacterium method, but by combining these methods, as shown in this Example It was impossible to predict that the gene could be introduced with high efficiency. Also, a strong expression level as shown in this example was not expected at all.

(Result of soybean)
Four days after the treatment with Agrobacterium (FIG. 8), the fluorescence by GFP was observed. As a result of gene introduction combining Agrobacterium and electroporation, strong fluorescence was confirmed (individual on the left side of FIG. 8B). In addition, the expression of GFP was confirmed with the naked eye at a high efficiency of about 40% of the individuals into which the gene was introduced. Conventionally, there is no example of combining a direct gene transfer method such as electroporation and an indirect gene transfer method such as the Agrobacterium method, but by combining these methods, as shown in this Example It was impossible to predict that the gene could be introduced with high efficiency. Also, a strong expression level as shown in this example was not expected at all.

(Result of tomato)
Four days after the treatment with Agrobacterium (FIG. 9), the fluorescence by GFP was observed. As a result of gene introduction combining Agrobacterium and electroporation, strong fluorescence was confirmed (individual on the left side of FIG. 9B). In addition, the expression of GFP was confirmed with the naked eye at a high efficiency of about 50% of the individuals into which the gene was introduced. Conventionally, there is no example of combining a direct gene transfer method such as electroporation and an indirect gene transfer method such as the Agrobacterium method, but by combining these methods, as shown in this Example It was impossible to predict that the gene could be introduced with high efficiency. Also, a strong expression level as shown in this example was not expected at all.

(Results of morning glory)
The fluorescence by GFP was observed 4 days after the treatment with Agrobacterium (FIG. 10). As a result of gene introduction combining Agrobacterium and electroporation, strong fluorescence was confirmed (individual on the left side of FIG. 10B). In addition, the expression of GFP was confirmed with the naked eye at a high efficiency of about 50% of the individuals into which the gene was introduced. Conventionally, there is no example of combining a direct gene transfer method such as electroporation and an indirect gene transfer method such as the Agrobacterium method, but by combining these methods, as shown in this Example It was impossible to predict that the gene could be introduced with high efficiency. Also, a strong expression level as shown in this example was not expected at all.

  As mentioned above, although this invention has been illustrated using preferable embodiment of this invention, this invention should not be limited and 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 documents cited herein should be incorporated by reference in their entirety, as if the contents themselves were specifically described herein. Understood.

  A simple and rapid plant transformation method is provided.

  The simple method of the present invention facilitates large-scale processing and large-scale analysis important in development research in this field, and as a result, induces dramatic progress in research and leads to the development of innovative recombinant crops.

Claims (49)

A method for introducing a nucleic acid into a cell, comprising the following steps:
step to maintain the suspension containing the a) cells with Agrobacterium Riku arm under reduced pressure; Contact and
b ) a step of performing electroporation after the step ( a );
Including the method. Step of maintaining under the reduced pressure is a step of maintaining the cell under a reduced pressure of 0.096MPa, The method of claim 1. The method of claim 1, wherein the step b ) comprises applying voltage pulses to the cells and the Agrobacterium in at least two directions. The method of claim 1, wherein the cell is a plant cell. 5. The method of claim 4 , wherein the plant cell is a plant dormant tissue cell. 6. The method of claim 5 , wherein the plant dormant tissue is a seed. The method of claim 6 , wherein the seed of the plant is a mature seed. The method according to claim 4 , wherein the plant cell is derived from a monocotyledonous plant. The method according to claim 8 , wherein the monocotyledonous plant is a gramineous plant. The method according to claim 9 , wherein the gramineous plant is wheat. The method according to claim 9 , wherein the gramineous plant is rice. The method according to claim 9 , wherein the gramineous plant is corn. The method according to claim 4 , wherein the plant cell is derived from a dicotyledonous plant. It said dicotyledonous plant is a leguminous plant, a method according to claim 1 3. The legume is soybean, the method according to claim 1 4. It said dicotyledonous plant is a crucifer The method of claim 1 3. The method of claim 16 , wherein the cruciferous plant is Chinese cabbage. The method of claim 16 , wherein the cruciferous plant is Arabidopsis thaliana. It said dicotyledonous plant is a family Convolvulaceae plants The method of claim 1 3. 20. The method of claim 19 , wherein the Convolvulaceae plant is a morning glory. It said dicotyledonous plant is a solanaceous plant, a method according to claim 1 3. The Solanaceae is tomato A process according to claim 2 1. It said dicotyledonous plant is a cucurbits The method of claim 1 3. The Cucurbitaceae is Uri The method according to claim 2 3. The method according to claim 1, wherein the Agrobacterium is Agrobacterium tumefaciens. A method for producing a plant into which a nucleic acid has been introduced into a cell, comprising the following steps:
a) a step of maintaining the suspension containing the plant cells and Agrobacterium Riku arm under reduced pressure; Contact and
b ) a step of performing electroporation after the step ( a );
Including the method. 27. The method of claim 26 , further comprising the step of differentiating, growing and / or proliferating the plant cell. The method according to claim 2 6, comprising the step of said a) step, to maintain the suspension containing the seed and Agrobacterium Riku beam comprising said plant cells under reduced pressure, a method. The seed method of claim 2 8 is a monocot seeds. 30. The method of claim 29 , wherein the monocotyledonous seed is a mature seed. 30. The method of claim 29 , wherein the monocotyledonous seed is a grass seed. The family Gramineae seeds are wheat seeds The method of claim 3 1. The family Gramineae seeds are rice seeds The method of claim 3 1. The family Gramineae seeds are corn seeds The method of claim 3 1. The seed method of claim 2 8 is a seed of dicotyledonous plants. Seeds of the dicotyledonous plant is a seed of leguminous plants, The method of claim 3 6. The legume is soybean, The method of claim 3 6. Seeds of the dicotyledonous plant is a seed of crucifer The method of claim 35. The cruciferous plant is Chinese cabbage The method of claim 3 8. The cruciferous plant is Arabidopsis method of claim 3 8. Seeds of the dicotyledonous plant is a seed of family Convolvulaceae plants The method of claim 35. The family Convolvulaceae plant is morning glory A method according to claim 4 1. Seeds of the dicotyledonous plant is a seed of Solanaceae, The method of claim 35. The Solanaceae is tomato A process according to claim 4 3. Seeds of the dicotyledonous plant is a seed of cucurbits The method of claim 35. The Cucurbitaceae is Uri The method of claim 4 5. An apparatus for automated introduction of nucleic acids into cells, the apparatus comprising:
a) a container for containing a mixture containing Agrobacterium containing nucleic acids and cells;
b) means for placing Agrobacterium containing nucleic acid in the container of a);
c) means for placing the cells in the container of a);
d) a container that maintains the cells under reduced pressure and has the ability to withstand the reduced pressure;
e) means for placing the cells in the container of d);
f) means for maintaining in the container of d) under reduced pressure;
g) means for performing electroporation; and h) means for automating the means of b), c), e), f), and g), wherein the means of b), c And the means of e) are the same or different, and the container of a) and the container of d) are the same or different. 48. The apparatus of claim 47, wherein the means for performing electroporation comprises at least two or more pairs of electrodes. 48. The apparatus of claim 47, wherein the means for performing electroporation comprises at least three pairs of electrodes.