JP2018164436A - Complex for introducing gene into plant cell - Google Patents

Abstract

PROBLEM TO BE SOLVED: To develop means for efficiently introduce a desired gene to a wide range of plant species.SOLUTION: A complex contains the following proteins: (a) single VirD2 protein; (b) one or more VirE2 proteins; or (c) single VirD2 protein and one or more VirE2 proteins; and a gene introduced into a plant cell, in which cell membrane-permeable peptide (CPP) is fused into either or both of VirD2 protein and VirE2 protein.SELECTED DRAWING: None

Description

  The present invention relates to a complex of DNA and protein for introducing a gene into a plant cell.

  Gene transfer technology is being used in many fields regardless of basic and applied research, and is an essential basic technology in modern times. However, the conventional gene transfer technology for plants has two fatal problems: low efficiency because the transfer efficiency is not always high, and low versatility where applicable species are limited. Is present.

  The most important reason for this is that the conventional gene transfer technique uses gene transfer into plant cells via infection of plant cells by Agrobacterium as an essential process. In the conventional method, a complex of DNA and protein (VirD2, VirE2) (called T-DNA complex) is expressed in Agrobacterium cells, and Agrobacterium is infected with plant cells. A technique for introducing this T-DNA complex into plant cells has been taken (see Non-Patent Document 1).

Gelvin SB. Agrobacterium-mediated plant transformation: the biology behind the "gene-jockeying" tool. Microbiol. Mol. Biol. Rev., 67, 16-37. (2003)

  Plant species that are difficult to infect Agrobacterium due to the defense reaction on the plant cell side, or plant species that cannot be infected with Agrobacterium in the first place, have a problem that the gene introduction efficiency is poor or the gene introduction itself cannot be performed.

  The inventors of the present invention have made extensive studies to solve the above problems, and fused a cell membrane permeation peptide (CPP) to either or both of the VirD2 protein and the VirE2 protein forming the T-DNA complex. The present inventors have found that a desired gene can be directly introduced into a plant cell without the need for infection with um.

That is, the present invention provides the following:
(1) The following proteins:
(A) one VirD2 protein,
(B) one or more VirE2 proteins, or (c) a complex comprising one VirD2 protein and one or more VirE2 proteins and a gene to be introduced into a plant cell, either the VirD2 protein or the VirE2 protein Or a complex in which a cell membrane permeation peptide (CPP) is fused to both;
(2) The complex according to (1), wherein 80% or more of the amino acid residues of CPP are histidine residues, and the length of CPP is 8 or more amino acids;
(3) The complex according to (2), wherein the CPP has a length of 16 amino acids or more;
(4) The complex according to any one of (1) to (3), wherein all amino acid residues of CPP are histidine residues;
(5) The following two plasmids:
(A) one VirD2 protein,
(B) one or more VirE2 proteins, or (c) a plasmid containing a gene encoding one VirD2 protein and one or more VirE2 proteins; and a plasmid containing a gene to be introduced into a plant cell. A method for producing a complex according to claim 1, which comprises introducing the bacterium of the genus Agrobacterium into which the plasmid is introduced, and then obtaining the complex from the bacterium of the genus Agrobacterium. A method wherein a polynucleotide encoding CPP is fused to either or both of a gene encoding VirD2 protein, a gene encoding VirE2 protein;
(6) The following plasmid:
(A) a plasmid containing a gene encoding one VirD2 protein and a gene to be introduced into a plant cell;
(B) a plasmid containing a gene encoding one or more VirE2 proteins and a gene introduced into a plant cell, or (c) a gene encoding one VirD2 protein and one or more VirE2 proteins and introduced into a plant cell Introducing a plasmid containing the gene to be introduced into an Agrobacterium bacterium, culturing the Agrobacterium bacterium into which the plasmid has been introduced, and then obtaining the complex from the Agrobacterium bacterium, The method for producing a complex according to claim 1, wherein a polynucleotide encoding CPP is fused to either or both of a gene encoding VirD2 protein and a gene encoding VirE2 protein;
(7) The method according to (5) or (6), wherein 80% or more of the amino acid residues of CPP are histidine residues, and the length of CPP is 8 amino acids or more;
(8) The method according to (7), wherein the length of CPP is 16 amino acids or more;
(9) The method according to any one of (5) to (8), wherein all amino acid residues of CPP are histidine residues;
(10) A method for introducing a gene into a plant cell, comprising introducing the complex according to any one of (1) to (4) into the plant cell.

  According to the present invention, a desired gene can be directly introduced into a plant cell without requiring Agrobacterium infection. Therefore, a gene can be efficiently introduced into a plant species that is not easily infected by Agrobacterium due to a defense reaction on the plant cell side, or a plant species that cannot be infected by Agrobacterium in the first place. That is, by using the complex of the present invention, a desired gene can be efficiently and rapidly introduced into a wide variety of plant species.

FIG. 1 is a schematic diagram of a plasmid used for production of the complex of the present invention. A indicates plasmid # 1, B indicates plasmid # 2, and C indicates formation of a complex in Agrobacterium cells into which these plasmids are introduced. In the figure, H0 to H20 mean CPP (polyhistidine) having 0 to 20 histidine residues. FIG. 2 is a photograph showing the identification results of the complex obtained by the present invention by agarose gel electrophoresis. FIG. 3 is a graph showing the results of measuring the luciferase activity in plant cells after introducing the complex of the present invention (using luciferase as a reporter gene) into which the polyhistidines of various lengths were fused. It is. VirD2-Hn indicates that polyhistidine consisting of n histidine residues is fused to the VirD2 protein. VirE2-Hn indicates that polyhistidine consisting of n histidine residues is fused to the VirE2 protein. The vertical axis shows luciferase activity.

In one aspect, the present invention provides the following protein:
(A) one VirD2 protein,
(B) one or more VirE2 proteins, or (c) a complex comprising one VirD2 protein and one or more VirE2 proteins and a gene to be introduced into a plant cell, either the VirD2 protein or the VirE2 protein Alternatively, the present invention relates to a complex in which cell membrane permeation peptide (CPP) is fused to both (hereinafter referred to as “complex of the present invention”).

  VirD2 and VirE2 proteins are encoded by genes in the toxic region of the Ti plasmid. These proteins can form complexes with genes that are introduced into plants.

  In one embodiment, the complex of the present invention comprises one VirD2 protein and a gene to be introduced into the plant. In this specific example, Vir proteins other than VirD2 (VirA, VirB, VirC, VirD, VirE, and VirG proteins are known) may be included in the complex. In this specific example, the order of the Vir protein in the complex is arbitrary, but the gene introduced into the plant is bound to the VirD2 protein.

  In a further embodiment, the complex of the invention comprises one or more VirE2 proteins and a gene that is introduced into the plant. The number of VirE2 proteins may be 1 to several or several tens. In this specific example, a Vir protein other than VirE2 may be included in the complex. In this specific example, the order of the Vir protein in the complex is arbitrary, but the gene introduced into the plant is bound to the VirE2 protein.

  In a further embodiment, the complex of the invention comprises one VirD2 protein and one or more VirE2 proteins and a gene to be introduced into the plant. The number of VirE2 proteins may be 1 to several or several tens. In this specific example, a Vir protein other than VirD2 and VirE2 may be included in the complex. In this embodiment, the order of the Vir proteins in the complex is arbitrary. The gene introduced into the plant is linked to the VirD2 protein or the VirE2 protein.

  In the complex of the present invention, CPP is fused to either or both of VirD2 protein and VirE2 protein. Fusion refers to the binding of CPP to VirD2 protein or VirE2 protein by chemical bonding. The binding position of CPP in VirD2 protein or VirE2 protein may be any position, but preferably CPP is bound to the N-terminus or C-terminus of these proteins.

  The CPP in the complex of the present invention provides a peptide having a length of several amino acids or more, preferably 8 amino acids or more, and half or more of the constituent amino acids being histidine. The complex of the present invention in which such a CPP is fused has high gene transfer efficiency into plant cells.

  The constituent amino acids of CPP in the complex of the present invention are rich in histidine, preferably about 80% or more of the constituent amino acid residues are histidine residues, and more preferably about 90% or more of the constituent amino acid residues are histidine. A residue, more preferably all constituent amino acid residues are histidine residues.

  The length of CPP in the complex of the present invention is 8 amino acids or more, preferably 16 amino acids or more, more preferably 20 amino acids or more. Therefore, the length of the peptide of the present invention may be, for example, in the range of 8 amino acids to several tens of amino acids, preferably 16 amino acids to several tens of amino acids, more preferably 20 amino acids to several tens of amino acids. Here, several tens of amino acids mean any number of amino acids in the range of less than 10-100.

  A particularly preferred example of CPP in the complex of the present invention is CPP in which all constituent amino acid residues are histidine residues.

  The amino acid residue other than histidine constituting the peptide of the present invention may be any amino acid residue, natural amino acid residue, non-natural amino acid residue, modified amino acid residue, or synthetic amino acid residue. Good. A person skilled in the art can appropriately synthesize and modify amino acids. Preferably, the amino acid residues other than histidine constituting the peptide of the present invention are basic amino acid residues such as arginine and lysine, or amino acid residues having properties similar to histidine.

  One or more genes may be introduced into the plant. There may be one kind of gene introduced into a plant, or a plurality of kinds.

  By using the complex of the present invention, a desired gene can be directly introduced into a plant cell without going through a process of infection with Agrobacterium. Therefore, a desired gene can be efficiently and rapidly introduced into a wide range of plant species.

The complex of the present invention may be produced by genetic engineering techniques. Accordingly, the present invention, in a further aspect, comprises the following two plasmids:
(A) one VirD2 protein,
(B) one or more VirE2 proteins, or (c) a plasmid containing a gene encoding one VirD2 protein and one or more VirE2 proteins; and a plasmid containing a gene to be introduced into a plant cell. A method for producing the complex of the present invention, comprising culturing an Agrobacterium bacterium into which the plasmid has been introduced, and then obtaining the complex from the Agrobacterium bacterium. A polynucleotide encoding CPP is fused to either or both of a gene encoding VirD2 protein, a gene encoding VirE2 protein, or both.

  As a plasmid containing a gene encoding a protein used in the method of the present invention, a known plasmid containing an Agrobacterium origin of replication, such as pBIN19, pPZP111, pGreen0029, or a plasmid having these as a backbone, is used as the backbone. Can be built. Elements such as a promoter in the plasmid can be appropriately selected by those skilled in the art.

  A plasmid containing a gene encoding a gene to be introduced into a plant used in the method of the present invention is constructed using a known plasmid, for example, pBIN19, pPZP111, pGreen0029, or a plasmid having these as a backbone. Can do. Elements such as a promoter in the plasmid can be appropriately selected by those skilled in the art.

In the method for producing a complex of the present invention, the gene encoding the Vir protein and the gene introduced into the plant cell may be contained in one plasmid. Accordingly, the present invention in a further aspect provides the following plasmid:
(A) a plasmid containing a gene encoding one VirD2 protein and a gene to be introduced into a plant cell;
(B) a plasmid containing a gene encoding one or more VirE2 proteins and a gene introduced into a plant cell, or (c) a gene encoding one VirD2 protein and one or more VirE2 proteins and introduced into a plant cell Introducing a plasmid containing the gene to be introduced into an Agrobacterium bacterium, culturing the Agrobacterium bacterium into which the plasmid has been introduced, and then obtaining the complex from the Agrobacterium bacterium, The method for producing a complex according to claim 1, wherein a polynucleotide encoding CPP is fused to either or both of a gene encoding VirD2 protein and a gene encoding VirE2 protein.

  In the method for producing a complex of the present invention, a polynucleotide encoding CPP is fused to either or both of a gene encoding VirE2 protein and a gene encoding VirD2 protein. The fusion may be performed on the 3 'side or 5' side of the gene encoding the VirE2 protein or the gene encoding the VirD2 protein. The fusion may be performed via a spacer sequence or not.

  The plasmid is then introduced into Agrobacterium bacteria. Introduction of the plasmid can be performed by a known method. For example, a method using mating using a helper plasmid, a method using heat shock, or a method using electroporation may be used.

  Agrobacterium bacteria into which the plasmid has been introduced are cultured. The culture can be performed by a known method. For example, Agrobacterium bacteria may be cultured in an LB medium at 28 ° C. for an appropriate time. During the cultivation, the Vir protein expressed from the plasmid and the gene introduced into the plant form a complex within the bacterium of the genus Agrobacterium.

  Subsequently, the complex of the present invention can be obtained by separating bacteria belonging to the genus Agrobacterium from the culture using a known method such as centrifugation and crushing the cells. The disruption of the microbial cells can be performed using known means such as ultrasonic treatment, a homogenizer, a mill or the like.

  If necessary, the complex of the present invention may be purified using a known means such as column chromatography.

  The complex of the present invention can be produced by a genetic engineering technique as described above, but may be produced by a chemical synthesis method or by combining a genetic engineering technique and a chemical synthesis method.

Further, the complex of the present invention may be produced by mixing VirD2 protein and / or VirE2 protein and a gene introduced into a plant cell in vitro (either VirD2 protein, VirE2 protein or both). CPP is fused). For example, the following recombinant protein:
(A) a recombinant protein comprising one VirD2 protein,
(B) a recombinant protein containing one or more VirE2 proteins, or (c) a recombinant protein containing one VirD2 protein and one or more VirE2 proteins and a gene introduced into a plant cell in vitro. Thus, the complex of the present invention may be produced (CPP is fused to either or both of VirD2 protein and VirE2 protein).

  In a further aspect, the present invention relates to a method for introducing a gene into a plant cell comprising introducing the complex of the present invention into the plant cell. By incubating the complex of the present invention and a plant cell in a medium, the gene can be directly introduced into the plant cell. The method of introducing the complex into the plant cell, the type of medium and the incubation conditions can be appropriately selected and changed by those skilled in the art according to the type of plant cell.

  Since the gene introduction method of the present invention eliminates the bottleneck of the conventional method of Agrobacterium infection, it overcomes the poor introduction efficiency and low versatility of plant cells, which were the problems of the conventional method. Is.

  The present invention will be described in more detail and specifically below with reference to examples, but the examples are not intended to limit the present invention.

1) Test cells GV3101 strain was used as an Agrobacterium for expressing a polyhistidine-fused T-DNA complex. On the other hand, the BY-2 strain (rpc00001) which is a tobacco cultured cell was used as a plant cell.

2) Construction of Recombinant Agrobacterium A plasmid “plasmid # 1” expressing polyhistidine-fused VirD2 and VirE2 in which polyhistidine was fused to both VirD2 and VirE2 proteins constituting the T-DNA complex was constructed (FIG. 1). 1A). At this time, combinations of VirD2 and VirE2 were fused with polyhistidines of 5 chain lengths (histidines 0, 8, 12, 16, and 20 residues), so there were 25 types of plasmid # 1 (5 types x 5 types). Type). Moreover, a plasmid “plasmid # 2” into which a luciferase gene (Luc gene) was introduced was constructed as a reporter gene for evaluating gene transfer (FIG. 1B). By introducing these plasmids # 1 and # 2 together into Agrobacterium, recombinant Agrobacterium (25 types in total) was constructed.

  Plasmid # 1 was constructed as follows. The pGreen0229 plasmid was opened at position 536 by inverse PCR. Subsequently, a VirE2 expression cassette (VirE promoter-VirE2-CPP-NOS terminator) is placed on the 5 ′ side and a VirD2 expression cassette (VirD promoter-VirD2-CPP-NOS terminator) is placed on the 3 ′ side of the ring-opening site. Plasmid # 1 was constructed by introducing it by the Fusion Cloning method.

  Plasmid # 2 was constructed as follows. Rings between 1165 and 2906 of the pGWB402Ω plasmid having pPZP111 as the backbone were opened and removed by inverse PCR. Subsequently, a plasmid # 2 was constructed by introducing a gene encoding luciferase into this open circle site by the In-Fusion Cloning method.

  The plasmid was introduced into Agrobacterium as follows. Agrobacterium competent cells were dissolved at room temperature, and 50 ng of plasmid # 1 and plasmid # 2 were added to 20 μL of competent cells and tapped several times. The mixed solution of the competent cell and the plasmid was transferred to a cuvette that had been cooled on ice in advance, and electroporation was performed using a Gene Pulser electroporation system (manufactured by BioRad). At this time, the setting of Gene Pulser was performed under the conditions of 25 μF, 200Ω, and 2,500 V. After electroporation, 1 mL of SOC medium was quickly added to the cuvette. After pipetting about 3 times and transferring to a 1.5 mL tube, incubation was performed with shaking at 28 ° C. for 2 hours. Thereafter, the LB agar medium (containing 100 μg / mL spectinomycin and 50 μg / mL kanamycin) was inoculated and cultured at 28 ° C. overnight.

3) Preparation of polyhistidine fusion T-DNA complex (FIG. 1C)
All 25 types of recombinant Agrobacterium constructed as described above were inoculated into 5 mL of LB liquid medium and cultured overnight at 28 ° C. with shaking (preculture). 4 mL of the pre-culture solution cultured overnight was added to 50 mL of LB liquid induction medium [kanamycin 50 μg / mL, spectinomycin 100 μg / mL, acetosyringone 20 μM, MES-KOH (pH 5.7) 10 mM] at 28 ° C. Cultured overnight with shaking. Thereafter, the precipitate was recovered by centrifugation (5,000 g × 20 minutes, room temperature), and then OD600 = 0 in Agro-infiltration Buffer [MgCl ₂ 10 mM, MES-KOH (pH 5.7) 10 mM, acetosyringone 200 μM]. Suspended to be .5. Then, it was incubated overnight at room temperature in the dark. Thereafter, 10 mL was transferred to a 15 mL falcon tube, and the precipitate was collected by centrifugation (4,000 rpm × 15 minutes, 4 ° C.). 1 mL of LS medium was added to the precipitate and suspended. The suspension was subjected to ultrasonic disruption to obtain a cell disruption solution. The cell disruption solution was subjected to centrifugation (13,000 rpm × 10 minutes, 4 ° C.), and the supernatant was passed through a 0.22 μm syringe filter to obtain a polyhistidine fusion T-DNA complex extract.

  Confirmation of the produced complex (VirD2 protein + VirE2 protein + gene) was performed using affinity purification using Ni resin and PCR.

  First, a complex in which polyhistidine is not fused (VirD2-H0 + VirE2-H0 + Luc gene) and a complex in which polyhistidine is fused only to VirD2 (VirD2-H8 + VirE2-H0) in the same manner as described above. + Luc gene), a complex in which polyhistidine is fused only to VirE2 (VirD2-H0 + VirE2-H8 + Luc gene), a complex in which polyhistidine is fused to both VirD2 and VirE2 (VirD2-H8 + VirE2- H8 + Luc gene) was prepared.

Next, these complexes were subjected to affinity purification using Ni resin, and the complexes fused with polyhistidine were recovered. Affinity purified His Mag Sepharose Ni (GE Healthcare Japan Ltd.) was used.
Finally, it was confirmed by PCR whether the recovered complex contains the Luc gene (whether VirD2 or VirE2 fused with polyhistidine and the Luc gene form a complex). PCR was performed using the recovered complex as a template and a primer selective for the Luc gene.

  As a result, a complex in which polyhistidine is fused only to VirD2 (VirD2-H8 + VirE2-H0 + Luc gene), a complex in which polyhistidine is fused only to VirE2 (VirD2-H0 + VirE2-H8 + Luc gene) , Polyhistidine fused to both VirD2 and VirE2 (VirD2-H8 + VirE2-H8 + Luc gene) was subjected to affinity purification using Ni resin, and PCR was performed. As a result, the Luc gene was detected. confirmed. That is, it was confirmed that VirD2 and VirE2 fused with polyhistidine form a complex with the Luc gene (FIG. 2).

4) Evaluation of intracellular introduction of polyhistidine-fused T-DNA complex BY-2 cells (1 mL) on passage 3 were dispensed into 2.0 mL Eppendorf tubes. 1 mL of the polyhistidine fusion T-DNA complex extract obtained above was added to 1 mL of this BY-2 cell, and cultured with shaking at 28 ° C. overnight. Subsequently, BY-2 cells are collected by centrifugation (4,000 rpm × 10 minutes, 4 ° C.), suspended in 100 μL of × 1 Passive lysis buffer (Promega product), and then incubated at room temperature for 10 minutes. BY-2 cells were disrupted. The cell lysate was centrifuged (15,000 rpm × 10 minutes, room temperature), coelenterazine was added to the supernatant to a final concentration of 2 μM, and the mixture was incubated at room temperature for 10 minutes. Using a luminometer (GENE LIGHT GL-220A), the introduction of the polyhistidine fusion T-DNA complex into BY-2 cells was evaluated by measuring the luciferase activity in the cell lysate.

5) Results With respect to polyhistidine-fused T-DNA complexes prepared from all 25 types of recombinant Agrobacterium, direct gene introduction into BY-2 cells was evaluated. As a result, five types of polyhistidine fusion T-DNA complexes (VirD2-H8 + VirE2-H0, VirD2-H16 + VirE2-H0, VirD2-H8 + VirE2-H8, VirD2-H16 + VirE2-H12, VirD2-H20) Since luciferase activity was detected in BY-2 cells treated with + VirE2-H20), these five types of polyhistidine-fused T-DNA complexes were introduced into BY-2 cells and are functioning (Luc It was confirmed that the gene was expressed (FIG. 3). In particular, the highest luciferase activity was detected in the polyhistidine fusion T-DNA complex composed of VirD2-H20 + VirE2-H20. In the case of the above notation, for example, “VirD2-H20 + VirE2-H20”, polyhistidine comprising 20 histidine residues is fused to the VirD2 protein, and polyhistidine comprising 20 histidine residues to the VirE2 protein. Means a T-DNA complex in which is fused.

  From these results, the effect of the gene introduction method using the complex of the present invention was demonstrated.

  In the conventional gene transfer method using Agrobacterium infection, it is known that several days (3 days or more) are required from the Agrobacterium infection until the expression of the transgene is confirmed. However, in the gene introduction method using the complex of the present invention, the expression of the transgene was confirmed one day after the treatment with the polyhistidine-fused T-DNA complex. The reason why such immediate effect was confirmed in the direct introduction method is considered to be that the direct introduction method did not require the process of Agrobacterium infection and that efficient (rapid) gene introduction was realized.

  The present invention can be used for plant research, breeding, breed improvement, and the like.

Claims (10)

The following proteins:
(A) one VirD2 protein,
(B) one or more VirE2 proteins, or (c) a complex comprising one VirD2 protein and one or more VirE2 proteins and a gene to be introduced into a plant cell, either the VirD2 protein or the VirE2 protein Or a complex in which a cell membrane permeation peptide (CPP) is fused to both.   The complex according to claim 1, wherein 80% or more of the amino acid residues of CPP are histidine residues, and the length of CPP is 8 or more amino acids.   The complex according to claim 2, wherein the length of the CPP is 16 amino acids or more.   The complex according to any one of claims 1 to 3, wherein all amino acid residues of CPP are histidine residues. The following two plasmids:
(A) one VirD2 protein,
(B) one or more VirE2 proteins, or (c) a plasmid containing a gene encoding one VirD2 protein and one or more VirE2 proteins; and a plasmid containing a gene to be introduced into a plant cell. A method for producing a complex according to claim 1, which comprises introducing the bacterium of the genus Agrobacterium into which the plasmid is introduced, and then obtaining the complex from the bacterium of the genus Agrobacterium. A method wherein a polynucleotide encoding CPP is fused to either or both of a gene encoding VirD2 protein, a gene encoding VirE2 protein. The following plasmids:
(A) a plasmid containing a gene encoding one VirD2 protein and a gene to be introduced into a plant cell;
(B) a plasmid containing a gene encoding one or more VirE2 proteins and a gene introduced into a plant cell, or (c) a gene encoding one VirD2 protein and one or more VirE2 proteins and introduced into a plant cell Introducing a plasmid containing the gene to be introduced into an Agrobacterium bacterium, culturing the Agrobacterium bacterium into which the plasmid has been introduced, and then obtaining the complex from the Agrobacterium bacterium, The method for producing a complex according to claim 1, wherein a polynucleotide encoding CPP is fused to either or both of a gene encoding VirD2 protein and a gene encoding VirE2 protein.   The method according to claim 5 or 6, wherein 80% or more of the amino acid residues of CPP are histidine residues, and the length of CPP is 8 amino acids or more.   The method according to claim 7, wherein the length of the CPP is 16 amino acids or more.   The method according to any one of claims 5 to 8, wherein all amino acid residues of CPP are histidine residues.   A method for introducing a gene into a plant cell, comprising introducing the complex according to any one of claims 1 to 4 into the plant cell.