JPH0630780A - Method for transducing adventitious gene to plant - Google Patents

Abstract

PURPOSE:To manifest the whole plant of an adventitious gene by inoculating a plant virus vector integrated with an adventitious gene and a specific plant virus into a plant. CONSTITUTION:A plant virus vector integrated with an adventitious gene and a plant virus capable of producing a wild type coat protein, having low particle formation ability, are inoculated into a plant. The plant virus vector is transferred to the whole plant and the adventitious gene can be manifested in the whole plant.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for introducing a foreign gene into a plant, improving the properties of the plant, and producing a useful protein in the whole plant.

[0002]

2. Description of the Related Art When a plant virus is infected with a plant, a certain plant virus, such as tobamovirus, proliferates in the plant and rapidly spreads in the plant while producing a large amount of a protein called coat protein. It is known.

Several gene manipulation systems using these plant viruses have already been constructed, and attempts have been made to introduce foreign genes into plants using this system. For example, a method of substituting a coat protein gene with a foreign gene (JP-A-63-14693) or a method of directly connecting the coat protein gene and a foreign gene to produce a fusion protein (JP-A-2-49591). Etc.

[0004] However, all of the viral vectors used in these known methods have a major drawback that they do not have the ability to spread throughout the plant (systemic infectivity), and they introduce useful properties into the entire plant. Alternatively, it was impossible to produce useful proteins in the whole plant.

In order for the plant virus to have systemic infectivity,
Particle formation by coat protein is required (T.Saito
et al .: Virology, 176, p. 329, 1990). The existing viral vector does not produce the coat protein (substitution type vector), or the coat protein is a fusion protein, and its properties change significantly (direct type vector). It is probable that he did not have systemic infectivity.

The present inventors inoculated plants with a wild-type plant virus or an attenuated plant virus together with the plant virus vector in order to supply the wild-type coat protein to the plant virus vector. However, in neither case did the plant virus vector spread throughout the plant. This is probably because the wild-type coat protein was used for particle formation of the wild-type or attenuated plant virus, not the plant virus vector.

[0007]

Therefore, an object of the present invention is to establish a method for expressing a foreign gene in a whole plant.

[0008]

[Means for Solving the Problems] The above-mentioned object is to provide a method of infecting a plant with a plant virus vector incorporating a foreign gene, wherein the plant virus vector has a wild-type coat protein producing ability and a particle forming ability. A plant gene is inoculated with a plant virus having a low virus content.

The present invention will be described in detail below, but prior to that, the terms used in the present specification will be explained.

Systemic infection: The transfer from a leaf inoculated with virus to a leaf not inoculated.

Particle formation: The viral genome is covered by the coat protein to become viral particles.

Examples of the plant virus used in the present invention include a group belonging to DNA viruses such as calimovirus and a group belonging to RNA viruses such as tobamovirus and bromovirus. Specific virus species include cauliflower mosaic virus belonging to calimovirus, tobacco mosaic virus belonging to tobamovirus, brom mosaic virus belonging to bromovirus, and the like.

The plant virus vector incorporating a foreign gene used in the present invention is a replicable recombinant virus, for example, one in which a foreign gene is linked downstream of a coat protein gene, or a coat protein gene. And some of which are replaced with foreign genes.

The plant virus capable of producing a wild type coat protein and having a low particle forming ability used in the present invention is a replicable plant virus, for example, a virus in which the particle formation initiation site is deleted or mutated. As a specific example, a mutant strain (QAD strain, Tetsuo Meshi et al .: The EMBO J in which a part of the 30K protein gene of tobacco mosaic virus belonging to the Tobamovirus group is deleted.
ournal vol.6, pages 2557-2563, 1987) and 30K
A mutant strain (FAD strain, Saito et al.) In which a hairpin structure (which is considered to be a particle formation initiation site) existing in the protein gene was introduced with a mutation so as not to change the amino acid sequence of the 30K protein to reduce the particle formation rate. al.:Virology, 1
76, p. 329, 1990). Hereinafter, a plant virus having a wild-type coat protein-producing ability and a low particle-forming ability is referred to as a helper virus.

The foreign gene used in the present invention includes
There are protein and peptide genes having pharmacology and physiological activity, protein genes that impart stress resistance to plants and pest resistance, protein genes that change plant morphology and flower color, and specifically, enkephalin, calcitonin, corticotropin, and human. Epidermal growth factor (EG
F) and genes encoding the angiotensin converting enzyme inhibitory (ACEI) peptides as shown in Table 1 and the like.

[0016]

[Table 1]

These genes can be obtained from genomic DNA or cDNA of organisms, plasmid DNA, or the like, but can also be synthesized using a DNA synthesizer or the like.

The method of the present invention can be carried out, for example, as follows.

When the plant virus is an RNA virus, R
The cDNA prepared from NA is incorporated into the downstream of the promoter of the plasmid having the promoter for in vitro transcription. Then, according to a commonly used gene manipulation method, a plasmid encoding a plant virus vector is constructed by incorporating a foreign gene into the downstream of the coat protein gene of this plasmid or by replacing a part or all of the coat protein gene with the foreign gene. To construct.

Next, according to a commonly used gene manipulation method, a plasmid encoding a helper virus is obtained by deleting the viral particle formation initiation site of the plasmid incorporating the viral cDNA or introducing a mutation into the particle formation initiation site. To construct.

Two types of RNA used in the present invention are prepared from the above constructed two types of plasmids by an in vitro transcription reaction. Both the plant virus vector and the helper virus can be used in the form of RNA, but the plant virus vector may be used in the form of particles.

The RNA thus prepared can be inoculated into a plant by inoculating it with a method such as rubbing the leaves of the plant together with carborundum.

When the plant virus is a DNA virus,
The DNA virus is directly integrated into the plasmid and genetic manipulation is performed. A plant virus vector and a helper virus can be prepared by a method of cutting out a DNA virus portion from a genetically engineered plasmid, and these can be inoculated and infected in a plant by the same method as in the case of RNA virus.

[0024]

The plant virus vector according to the method of the present invention is
Since it can be transferred to the whole plant, the foreign gene can be expressed in the whole plant. It is considered that this is because the plant virus vector forms particles by the wild-type coat protein supplied from the helper virus.

[0025]

The present invention will be described in more detail with reference to the following examples.

Example 1, Comparative Example 1 1. Construction of Plant Virus Vector Transcription Plasmid Incorporating ACEI Sequence

1) Preparation of synthetic DNA having ACEI sequence 0.2 μmol using 380A synthetic DNA machine manufactured by ABI
On the scale, two synthetic DNAs shown in FIG. 1 were prepared.

The vector constructed this time has a sequence coding for arginine on the 5'side of the ACEI sequence so that the ACEI peptide can be isolated from the expressed fusion protein by digestion with trypsin. Synthesized DNA
Was purified by ABI OPC cartridge.

2) Phosphorylation and Annealing of Synthetic DNA The two DNAs synthesized in 1) were prepared at a concentration of 0.1 μg / μl, phosphorylated with 0.2 mM ATP, and purified by phenol treatment and ether washing. Two purified DNAs were prepared at 0.02 μg / μl, mixed and treated at 65 ° C. for 5 minutes. After the treatment, annealing was performed by slowly cooling to room temperature.

3) Preparation of fragment for plasmid construction Known plasmid pTLW in which full-length cDNA of tobacco mosaic virus L strain was connected after T7 phage promoter.
3 (Watanabe et al., National Institute of Genetics Research Meeting, Japanese Patent Application No. 4-108628, March 11, 1992) was digested with the combination of restriction enzymes shown in Table 2 to prepare fragments of each size. .
The positional relationship of each fragment in pTLW3 is shown in Fig. 2-a.

[0031]

[Table 2]

The respective fragments are prepared by separation by agarose gel electrophoresis, followed by GENE CLEAN kit (manufactured by BIO 101).
Was used according to the attached instructions.

Of the above fragments, the 7.1 kbp fragment of KpnI and MluI was treated with alkaline phosphatase to remove the terminal phosphate, and used in the following reaction.

4) Construction and preparation of plasmid The annealed synthetic DNA solution prepared in 2) and the four DNA fragment solutions derived from pTLW3 prepared in 3) were prepared at the composition shown in Table 3 at 15 ° C. for 15 hours. By reacting each other, they were ligated to each other to prepare a plasmid. The resulting plasmid is shown in Figure 3-a.

[0035]

[Table 3]

Table 4 shows the composition of the buffer for the x10 binding reaction.

[0037]

[Table 4]

The reaction solution was used as it was for E. coli ・ H
B101 competent cells (Takara Shuzo Co., Ltd.) were transformed, and carbenicillin-resistant colonies were selected. The target plasmid was extracted and purified from the selected colonies of Escherichia coli.

2. Construction of plasmid for helper virus transcription

1) Preparation of Fragment for Plasmid Construction Known plasmid pTLW3 shown in FIG. 2 and virus c in which mutation was introduced at the particle formation initiation site of tobacco mosaic virus L strain without changing the amino acid sequence of 30K protein.
A known plasmid pLFAD (Sait
o et al .: Virology, 76, 329-336, 1990) was digested with a combination of restriction enzymes shown in Table 5 to prepare fragments of each size. The position of the fragment in pTLW3 is shown in Figure 2-a.
The position of the fragment in the FAD is shown in Figure 2-b.

[0041]

[Table 5]

Each fragment was prepared by separation by agarose electrophoresis and then GENECLEAN kit (manufactured by BIO 101).
Was used according to the attached instructions.

Of the above fragments, the 7.1 kbp fragment of KpnI and MluI was treated with alkaline phosphatase to remove the terminal phosphate and used in the following reaction.

2) Construction and preparation of plasmid The DNA fragment solution derived from pTLW3 and pLFAD prepared in 1) was treated with DNA Ligation Kit [Takara Shuzo Co., Ltd.].
Was manufactured according to the attached instruction manual.

The reaction solution was used as it was for E. coli ・ H
B101 competent cells (Takara Shuzo Co., Ltd.) were transformed, and carbenicillin-resistant colonies were selected. The target plasmid was extracted and purified from the selected colonies of Escherichia coli. The obtained plasmid is shown in Fig. 3-b.

3. Construction of plant virus vector and helper virus by in vitro transcription reaction

The two types of plasmids prepared in 1 and 2 above were digested with the restriction enzyme MluI and then purified by phenol treatment and ethanol precipitation to obtain template DNA. Using this template DNA in the reaction system shown in Table 6, at 37 ° C. for 2 hours,
In vitro transcription reaction was performed to prepare a plant virus vector and a helper virus (RNA). The prepared plant virus vector and helper virus (RNA) were purified by phenol treatment and ethanol precipitation.

[0048]

[Table 6]

4. Introduction of foreign gene into plants by inoculation of plant virus vector and helper virus

3. Using the plant virus vector and the helper virus prepared in the above, a virus mixture was prepared with the composition shown in Table 7. Samsung seed tobacco (Ni) 5 weeks after seeding with this virus mixture or plant virus vector
(Cottiana tabacum cv. Samusun) leaves were inoculated with 30 μl each.

[0051]

[Table 7]

5. Confirmation of replication and transfer to upper leaf of plant virus vector and helper virus

1) Preparation of sample 10 days after inoculation with the solution of Table 6, 2 mg sections were cut from each of the inoculated leaf and the non-inoculated leaf in the same individual. After adding 25 μl of SDS sampling buffer having the composition shown in Table 8 and homogenizing, the mixture was treated at 100 ° C. for 3 minutes. After cooling, centrifugation was carried out at 12,000 rpm for 5 minutes, and the supernatant was used as an SDS protein sample.

[0054]

[Table 8]

2) Detection of plant virus vector and helper virus in each sample
It was judged by the presence or absence of virus coat protein and fusion protein. 1 μl of the SDS-modified protein sample prepared in 1) was subjected to SDS-PAGE (12.5% polyacrylamide) to separate the protein. Separated protein is separated from gel by PV
Transferred to DF film. Next, anti-TMV rabbit serum was added to TB.
6 with S buffer (Tris-HCl (pH = 7.6) 20 mM and NaCl, 137 mM)
The 4,000-fold diluted solution was used as a primary antibody staining solution, and antibody staining was performed using a blotting detection kit for rabbit antibody (manufactured by Amersham) according to the attached instruction. The results are shown in Fig. 4.

As is clear from FIGS. 1 and 2, the plant virus vector was detected in the inoculated leaves of the plants inoculated with only the plant virus vector, but transfer to non-inoculated leaves was not observed ( Comparative example 1).

On the other hand, as can be seen from 3 and 4 in FIG. 4, in plants inoculated with both plant virus vector and helper virus, both plant virus vector and helper virus were detected in both inoculated leaves and non-inoculated leaves. It was confirmed that the leaves were transferred (systemic infection) (Example 1).

6. Confirmation of ACEI production in plants

5. Using the same sample as above, perform electrophoresis and transfer to the membrane in the same manner, and add anti-ACEI rabbit serum to TB.
4. Diluted 128,000 times with S buffer as primary antibody staining solution. Antibody staining was performed in the same manner as in. Results are shown in FIG.

Plants inoculated with only the plant virus vector had only inoculated leaves (1, 2 in FIG. 5), and plants inoculated with both the plant virus vector and helper virus had both inoculated leaves and non-inoculated leaves (see FIG. 5). 3, 4) It was confirmed that ACEI was produced in the form of fusion protein.

[0061]

INDUSTRIAL APPLICABILITY As described above, the method of the present invention enables systemic infection of a plant virus vector in a plant, expression of a foreign gene in the whole plant, preparation of a plant having improved properties, and usefulness in the plant. It became possible to produce proteins.

[Brief description of drawings]

FIG. 1 is a diagram showing the base sequence of synthetic DNA used in preparing plant virus vectors in Examples.

FIG. 2 DNA used for plasmid construction in Examples
It is a figure which shows the positional relationship in a plasmid pTLW3 (a) and pLFAD (b) of a fragment.

FIG. 3 (a) is a diagram showing a plasmid for transcription of a plant virus vector, and FIG. 3 (b) is a diagram showing a plasmid for transcription of a helper virus.

FIG. 4 is a diagram showing the presence or absence of a plant virus vector and a helper virus in inoculated leaves and non-inoculated leaves in the plant infection experiment conducted in the example.

FIG. 5 is a diagram showing the presence or absence of ACEI in inoculated leaves and non-inoculated leaves in the plant infection experiment conducted in the example.

 ─────────────────────────────────────────────────── ─── Continuation of front page (72) Inventor Yuji Matsunaga 171 Iwahara, Minamiashigara City, Kanagawa Prefecture (72) Inventor Yoshimi Okada 2-6-14 Tokiwadaira, Matsudo City, Chiba Prefecture

Claims (6)

[Claims] 1. A method for infecting a plant with a plant virus vector incorporating a foreign gene, wherein the plant virus vector is inoculated into a plant together with a plant virus having a wild-type coat protein-producing ability and a low particle-forming ability. A method for introducing a foreign gene into a plant, which is characterized in that 2. The method for introducing a foreign gene into a plant according to claim 1, wherein each of the plant viruses is an RNA virus. 3. The method for introducing a foreign gene into a plant according to claim 1, wherein the plant viruses are all tobamoviruses. 4. The method for introducing a foreign gene into a plant according to claim 1, wherein the plant viruses are the same virus species. 5. A plant virus vector in which a foreign gene is incorporated is constructed by substituting a foreign gene downstream of the coat protein gene of the plant virus genome or by replacing a part or all of the coat protein region with the foreign gene. 5. The method for introducing an exogenous gene into a plant according to claim 1, wherein the vector is a vector. 6. The method for introducing a foreign gene into a plant according to any one of claims 1 to 5, wherein the plant virus having a low particle forming ability is one in which the particle formation initiation site of genomic RNA is deleted or mutated.