JP2005080570A - Method for introducing biological material, and transformation method using the method for introducing the biological material - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for efficiently introducing a biological material into a cell; and to provide a transformation method by using the method for introducing the biological material. <P>SOLUTION: The method for introducing the biological material comprises directly introducing the biological material into the cell by using a virulent protein as a carrier. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a method for introducing a biological substance, and a transformation method using the method for introducing a biological substance, and more particularly, a method for introducing a biological substance using a virulent protein, and the biology The present invention relates to a transformation method using a method for introducing a chemical substance.

  As a method for introducing a biological substance such as a gene, an indirect introduction method such as an Agrobacterium method and a direct introduction method represented by a particle gun method are known. The former Agrobacterium method uses the infectivity of living Agrobacterium to plants. For example, as an Agrobacterium method, a method for incorporating foreign DNA into the chromosome of a dicotyledonous plant is known (Japanese Patent Laid-Open No. 60-70080). Such an Agrobacterium method has the advantage of high nuclear transfer efficiency of the introduced DNA.

  Also, the latter particle gun method is a method in which a useful gene is applied to metal particles such as gold and the like is struck using air compressed in cells. For example, the particle gun method is an improved method for introducing biological substances into living cells (Japanese Patent Publication No. 5-508316). Such a particle gun method has the advantage of being applied to a wide range of plant species without being limited to specific plant species and animal species.

Japanese Patent Laid-Open No. 60-70080

JP-T-5-508316

  However, the above-mentioned Agrobacterium method (indirect introduction method) has the disadvantages that it is poor in versatility and low in commercial value because the host species to be infected are limited.

  Furthermore, the particle gun method described above has a problem that the efficiency of nuclear transfer of the introduced DNA is low. As described above, there are problems that the conventional transformation methods do not complement each other, and these problems are factors that reduce the transformation efficiency in cells. Therefore, a method for efficiently introducing biological materials such as RNA and DNA into cells has been desired. At the same time, it has been desired to develop a biological material introduction method that can be introduced regardless of species and a highly efficient transformation method by using such a biological material introduction method.

  Therefore, an object of the present invention is to provide a biological substance introduction method capable of efficiently introducing a biological substance into cells, and a transformation method using the biological substance introduction method. is there.

  In order to achieve the above object, the inventors focused on molecular biological mechanisms related to nuclear translocation of foreign DNA originally possessed by plant pathogenic bacteria (Agrobacterium), and introduced a novel and highly efficient method for introducing a biological substance and It came to find the transformation method.

  The biological substance introduction method of the present invention is characterized in that a virulent protein is used as a carrier for introducing a biological substance into a cell, and the biological substance is directly introduced into the cell. To do.

  In a preferred embodiment of the biological substance introduction method of the present invention, the virulent protein is a protein derived from soil bacteria.

  In a preferred embodiment of the biological substance introduction method of the present invention, the virulent protein is VirD2 or VirE2.

  In a preferred embodiment of the biological substance introduction method of the present invention, the soil bacterium is a bacterium belonging to Agrobacterium tumefaciens.

  In a preferred embodiment of the method for introducing a biological substance of the present invention, the biological substance is a protein or peptide in which a border sequence recognized by DNA, RNA, and a virulent protein is chemically bound. To do.

  In a preferred embodiment of the biological substance introduction method of the present invention, the biological substance is DNA, and a border sequence is added to the DNA.

  In a preferred embodiment of the biological substance introduction method of the present invention, the border sequence consists of the base sequence shown in SEQ ID NO: 1 in the sequence listing.

  In a preferred embodiment of the biological substance introduction method of the present invention, the biological substance is directly added using at least one selected from the group consisting of a microinjection method, a PEG method, a laser method, and a particle gun method. It is introduced in.

  The transformation method of the present invention is characterized by being obtained by introducing a foreign gene using the above-described biological substance introduction method.

  According to the present invention, there is an advantageous effect that a biological substance can be efficiently introduced into a cell. Moreover, according to the present invention, there is an advantageous effect that a highly efficient transformation method applicable to all plant species and animal species can be provided.

  Moreover, according to the present invention, there is an advantageous effect that the efficiency of nuclear transfer of introduced DNA can be dramatically increased.

  In addition, according to the present invention, since the transgene to be used may be not only single-stranded but also double-stranded, there is an advantageous effect that the preparation process of the gene for introduction can be remarkably simplified.

  In addition, because of its high nuclear transfer efficiency, it has been difficult to produce by the conventional Agrobacterium method and particle gun method, the field of molecular breeding of useful transformed plants, and industries related to the application development (kiting) of the present invention There is an advantageous effect that it can contribute to the field.

  In the method for introducing a biological substance of the present invention, a biological substance is directly introduced into a cell using a virulent protein as a carrier. As described above, when the virulent protein is used as a carrier for carrying a biological substance, the biological substance can be efficiently transferred into a desired cell as demonstrated by an indirect introduction method such as the Agrobacterium method. Can be introduced. The term “directly” is intended to distinguish it from the pure Agrobacterium method, etc., and does not use the ability to infect soil bacteria heterogeneous cells, and means directly.

  Here, examples of the biological substance include DNA, RNA, and proteins or peptides obtained by chemically binding border sequences recognized by virulent proteins.

In a preferred embodiment of the biological material introduction method of the present invention, the biological material is DNA, and a border sequence is added to the DNA.
Here, the border sequence is a pair of incomplete direct repeats (almost the same in the same direction) present on Agrobacterium Ti plasmid (tumor-inducing plasmid tumorigenic plasmid) or Ri plasmid (root-inducing plasmid root hair-forming plasmid). The sequence is repeated about 25 residues), the sequence on the 5 'side is called RB (right border), and the sequence on the 3' side is called LB (left border). In Agrobacterium, the sequence between RB and LB (T-reagion: T region) is usually transferred to infected plant cells. In other words, the border sequence determines the region to be transferred to the host (in this case, the plant cell) during infection, and binds VirD2 to the T sequence to serve as a tag for the transferred gene. Therefore, the border sequence means a specific sequence that is recognized and bound by VirD2 having nuclear translocation ability. Any border arrangement having such a function can be applied to the introduction method of the present invention without particular limitation.
By adding such a border sequence to a biological substance to be transferred to the nucleus, it is possible to introduce a biological substance such as a foreign gene into a cell with high efficiency.
If an example of a border sequence is mentioned, it will consist of the base sequence shown in SEQ ID NO: 1 in the sequence listing and / or a base sequence complementary to the base sequence. The base sequence shown in SEQ ID NO: 1 in the sequence listing is the sequence of the strand (DNA strand) to which VirD2 is directly covalently bonded. Even a base sequence complementary to the base sequence is possible even if it is a base sequence complementary to the base sequence shown in SEQ ID NO: 1 in the sequence listing, that is, a so-called complementary strand (antisense strand), PCR, etc. This is because VirD2 can be bound if it becomes a double-stranded state as a result of amplification with.

The binding between the virulent protein and the desired biological substance to be introduced into the target cell is performed by utilizing an appropriate affinity, for example, when the desired biological substance to which a border sequence is added is used. Can do.
The affinity may include a covalent bond, an ionic bond, a hydrogen bond, a thing using hydrophobicity, a thing by van der Waals force, and the like. For example, when the biological substance is a gene, the border sequence of the transgene and the virulent protein bind to each other, but the reaction of both can be easily performed by mixing in an appropriate buffer. it can.

  In a preferred embodiment, the virulent protein is a protein derived from soil bacteria. As a protein derived from soil bacteria, VirD2 or VirE2 can be mentioned from the viewpoint that it binds to the biological substance to be introduced and has the ability to transfer to the host nucleus and has the function of incorporation into the host cell. .

  As the soil bacteria, bacteria belonging to Agrobacterium tumefaciens are preferable. The focus is on the high introduction rate of the Agrobacterium method, and its principle, ie, the information on the Ti plasmid is used to create virulent proteins and other proteins necessary for infection, and the T region is cut out by these actions. This is because the T region is transferred to an infected plant and incorporated into the plant chromosome.

  That is, in the present invention, virulent proteins such as VirD2 and VirE2 are separately artificially prepared, and further, the reaction with the biological substance to be introduced is also performed in a test tube, so that the biological substance can be efficiently introduced into the cell. It is to introduce to.

  In particular, the use of the Vir protein has the advantage that a biological substance can be introduced regardless of the species.

  In a preferred embodiment, the biological substance is directly introduced using at least one selected from the group consisting of a microinjection method, a PEG method, a laser method, and a particle gun method. A biological substance can be introduced into a desired target cell using a so-called direct introduction method such as the microinjection method, the PEG method, the laser method, or the particle gun method.

  In the microinjection method, there are a case where a small amount of DNA solution is directly injected into the nucleus and a case where DNA is injected into the cytoplasm and a part of the nucleus is transferred to the nucleus, both of which are included.

  The PEG method (polyethylene glycol method) utilizes a phenomenon in which so-called protoplasts with cell walls removed by enzymatic treatment are treated with high calcium concentration and high pH in the presence of PEG, and part of the extracellular fluid is taken up by cells. Thus, a biological substance is introduced into cells. In the PEG method, when an introduction substance is encapsulated in a liposome and the PEG method is applied, membrane fusion occurs, and the substance in the liposome can be introduced into the cell. Methods are also widely included.

  The laser method is a method of introducing an exogenous biological substance from a place where the cell wall or cytoplasm is removed into a cell from which part or all of the cell wall or cell membrane has been removed by a laser.

  The particle gun method is a method in which fine particles having adsorbed DNA on cells are driven at a high speed.

  According to these direct introduction methods, it has been pointed out that the efficiency of nuclear transfer of the introduced gene is usually low because a naked gene is directly introduced. Since the rent protein is used as a carrier for the biological substance and the biological substance to be introduced together with the pilent protein is introduced, high-efficiency introduction is possible.

  The above-described microinjection method, PEG method, laser method, and particle gun method can be performed by a conventional method except that a biological substance bound to a carrier is used instead of using a naked gene.

  Next, the transformation method of the present invention will be described. The transformation method of the present invention can be obtained by introducing a foreign gene using the biological substance introduction method of the present invention described above. The biological introduction method of the present invention has been described above, and it is possible to obtain a transformant by introducing a biological substance into a cell in the manner described in the above description.

  The method for introducing a biological substance of the present invention makes it possible to increase the efficiency with which the biological substance to be introduced in obtaining a transformant reaches the nucleus and is incorporated after entering the cell.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not intended to be interpreted as being limited to the following examples.

  First, using VirD2 and VirE2 as virulent proteins, the nuclear translocation efficiency of single-stranded DNA in the presence of these was investigated.

As a result of investigating the nuclear translocation ability of VirD2 by injecting a complex of purified and refolded VirD2 and rhodamine-labeled oligo DNA into large amounts of E. coli into onion epidermal cells, VirD2 caused the oligo DNA to enter the nucleus over time. Confirmed that it was migrated. Moreover, from the result of the dual luciferase assay using Arabidopsis protoplasts, it was confirmed that the relative activity value of the reporter gene was significantly increased in the presence of VirD2. When used alone, an increase in the relative activity value of the reporter gene was confirmed in the presence of VirD2.
FIG. 1 shows the results of the dialucylase activity test. This test is performed using two different luciferase genes, one is a firefly-derived luciferase gene and the other is a Renilla luciferase gene. These two genes are used in order to offset factors other than nuclear translocation (for example, efficiency of gene introduction into cells).
When luciferase is expressed, it reacts with each specific luciferin as a substrate and emits light at different wavelengths.
In this example, a border sequence was added to the firefly-derived gene, and no border sequence was added to the Renilla-derived gene. If both firefly and Renilla genes are transferred to the nucleus and expressed with the same efficiency, both (light of either wavelength) will emit the same amount of light. (In this case, the relative activity is 1). On the other hand, if a firefly gene with a border is transferred and expressed in the nucleus with higher efficiency than the other, the firefly emits a stronger light.
In FIG. 1, the relative activity on the vertical axis is firefly luciferase activity / Renilla luciferase activity.
In the control, only the gene was introduced into the cell without forming a complex with the VirD protein. BSA is obtained by adding a completely irrelevant protein (bovine serum-derived albumin) in place of the VirD2 protein. The heat-denatured VirD2 protein was obtained by heat-treating and adding VirD2, and as a result, the heat-treated VirD2 protein did not significantly affect the activity of VirD2.
From the left of FIG. 1, it can be seen that the firefly luciferase gene to which the border sequence is added is expressed 3 to 4 times stronger.
From the right in FIG. 1, it can be seen that when the VirD2 protein is added, the nuclear translocation efficiency is higher than the control.

Next, the nuclear translocation efficiency of double-stranded DNA in the presence of VirD2 was examined. In the nuclear translocation model of VirD2, VirD2 is assumed to be attached to the 5 ′ end border sequence of single-stranded DNA. Therefore, it was considered that the efficiency of nuclear translocation is further increased with double-stranded DNA having border sequences at both ends. As a result of the experiment, VirD2 recognized double-stranded DNA as expected, and the nuclear translocation efficiency was higher than that of single-stranded DNA. It has also been found that the presence of a border sequence is effective for nuclear translocation of VirD2, and that the presence of VirE2 is not always necessary when double-stranded DNA is used.
FIG. 2 is a diagram showing the synergistic effect of VirD2 and VirE2 on the DNA nuclear translocation rate. In other words, it can be seen from this figure that nuclear transfer is possible with higher efficiency when used in combination with VirE2 than when VirD2 is used alone.

Confirmation of improved nuclear transfer efficiency of double-stranded DNA in the presence of VirD2 using particle gun method Double-stranded DNA was introduced into onion epidermis cells using the particle gun method, which is frequently used as an actual plant transformation method As a result of investigating the effect of VirD2 in the case, it was found that the nuclear translocation efficiency of double-stranded DNA increases in proportion to the amount of VirD2 coexisting.
FIG. 3 shows the dose-dependent effect of Vir2D on DNA nuclear translocation. As can be seen from FIG. 3, the nuclear translocation efficiency increases as the concentration of the VirD2 protein increases.

  Applied to the field of molecular breeding of useful transformed plants that were difficult to produce by conventional methods such as the Agrobacterium method and particle gun method, and the industries such as pharmaceuticals, agribusiness, foods, and organisms, and the reagent industry can do.

FIG. 1 shows the results of the dialucylase activity test. FIG. 2 shows the synergistic effect of VirD2 and VirE2 on the rate of DNA nuclear translocation. FIG. 3 shows the dose-dependent effect of Vir2D on DNA nuclear translocation.

Claims (9)

  A method for introducing a biological substance, wherein a virulent protein is used as a carrier for introducing the biological substance into a cell, and the biological substance is directly introduced into the cell.   2. The method according to claim 1, wherein the virulent protein is a protein derived from soil bacteria.   3. The method according to claim 2, wherein the virulent protein is VirD2 or VirE2.   3. The method according to claim 2, wherein the soil bacteria are bacteria belonging to Agrobacterium tumefaciens.   The biological substance is a protein or peptide in which a border sequence recognized by DNA, RNA, and a virulent protein is chemically bound, or any one of claims 1 to 4, Method.   The method according to claim 1, wherein the biological substance is DNA, and a border sequence is added to the DNA.   7. The method according to claim 6, wherein the border sequence comprises the base sequence shown in SEQ ID NO: 1 in the sequence listing and / or a base sequence complementary to the base sequence.   The biological substance is directly introduced using at least one selected from the group consisting of a microinjection method, a PEG method, a laser method, and a particle gun method. the method of.   The transformation method which introduce | transduced the foreign gene using the biological substance introduction | transduction method of any one of Claims 1-8.

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