JP3172766B2 - Method for producing protein in recombinant plant - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for efficiently producing a foreign useful protein in a plant. The present invention also relates to a method for expressing a foreign protein to impart a new trait to a plant, and to breeding of a plant.

[0002]

2. Description of the Related Art It has been reported that when a foreign gene is introduced into the nucleus of a plant cell, the amount of peptide expressed as the foreign gene product is 0.1 to 0.3% of the total protein when the amount of expression is large. In particular, the product of the foreign gene has a molecular weight of 7,000
In the case of the following small peptides (hereinafter referred to as “short peptides”), there is a high possibility that they will be degraded in plants, and it is very difficult to accumulate them in large quantities. Therefore, in the plant,
A method for efficiently expressing a short peptide is desired.

[0003] When a foreign gene is introduced into a plant and expressed, in most cases, such as the CaMV35S promoter,
We use a constantly expressed (constitutive) promoter. However, this expression method may at least burden the protein production capacity of the plant, and the expressed protein may adversely affect the plant. Attempts to produce proteins that actually affect plant growth, such as genes involved in plant hormone synthesis, often fail. Such an effect is considered to be particularly serious as the production amount of the protein introduced into the plant is increased.

[0004]

DISCLOSURE OF THE INVENTION The present invention has been made to solve the above-mentioned problems, and has the following objects: (1) A foreign protein (peptide) is used as a fusion protein with another protein in plants. By expressing
(2) an expression cassette that constitutively expresses the recombinant gene encoding the fusion protein at a constant level and that inducibly increases the expression level. Or providing an expression vector containing the expression cassette to increase the amount of the foreign protein produced in the plant; (3) cutting the protein once expressed as a fusion protein under the inducing condition with the fusion portion, And (4) providing a plant transformed with the above expression cassette or expression vector; and (5) using a pathogen-resistant protein as the foreign protein. Providing plants that are resistant to pathogens.

[0005]

Means for Solving the Problems The present invention relates to a recombinant gene in which a foreign gene and a plant gene are linked via the hinge region of tobacco chitinase.

In a preferred embodiment, the plant gene is a gene encoding a tobacco PR1a protein.

[0007] In a preferred embodiment, the foreign gene is linked to a signal sequence of a plant protein.

[0008] In a preferred embodiment, the foreign gene is a gene encoding an antibacterial peptide derived from a dipteran insect, and more preferably a gene encoding Sarcotoxin1a.

[0009] The present invention further relates to an expression cassette containing a plant gene promoter and the recombinant gene.

[0010] In a preferred embodiment, the plant gene promoter is an inducible tobacco PR1a gene promoter.

In a preferred embodiment, the expression cassette has a terminator from the tobacco PR1a gene.

In a preferred embodiment, the expression gene is an expression cassette in which the foreign gene is linked to a signal sequence of a plant protein.

[0013] The present invention also relates to an expression vector for introducing the above expression cassette into a plant.

[0014] In a preferred embodiment, the expression vector further has a T-DNA region and a drug resistance gene.

In a preferred embodiment, cauliflower mosaic virus 35S expressing the drug resistance gene
The promoter constitutively expresses an inducible promoter for expressing a foreign gene, and increases the activity of the inducible promoter by necrotic lesion formation associated with salicylic acid or pathogen infection (eg, tobacco mosaic virus infection). .

The present invention also relates to a plant transformed with the recombinant gene, the expression cassette or the expression vector.

Further, the present invention provides the following steps: (1) a group consisting of the recombinant gene, an expression cassette, or a gene having the expression cassette and a drug resistance gene linked to a constitutively expressed plant promoter; Obtaining a plant cell transformed with a gene having a foreign gene selected from the group consisting of: (2) culturing the transformed plant cell under inducing conditions; and (3) encoding the foreign gene Recovering the peptide to be obtained, and a method for producing a peptide encoded by the foreign gene.

In a preferred embodiment, the plant promoter for constitutively expressing the drug resistance gene is a cauliflower mosaic virus 35S promoter, and the cauliflower mosaic virus 35S promoter constitutes an inducible promoter for expressing a foreign gene. It increases the expression level of a plant exogenous gene that is specifically expressed and has resistance to pathogenic bacteria due to necrotic lesion formation associated with salicylic acid or pathogen infection.

The present invention also relates to a plant having resistance to a pathogenic bacterium.
A recombinant gene in which a gene encoding an antimicrobial peptide and a plant gene are linked via the hinge region of tobacco chitinase, an expression cassette in which the recombinant gene is linked to a plant gene promoter, or the expression cassette And a gene having a drug resistance gene linked to a constitutively expressed plant promoter.

In a preferred embodiment, the transformed plant is resistant to wildfire or soft rot fungi.

In a preferred embodiment, the antimicrobial peptide is Sarcotoxin1a derived from a dipteran insect.

[0022]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail.

The present invention relates to a recombinant gene in which a foreign gene and a plant gene are linked via the hinge region of tobacco chitinase, an expression cassette in which a plant gene promoter and a recombinant gene are linked, and the expression cassette in plants. It relates to an expression vector to be introduced.

The term "plant" as used herein includes both monocotyledonous plants and dicotyledonous plants. Particularly preferred plants include tobacco, citrus, Chinese cabbage, lettuce, peach,
Rice, potatoes, tomatoes, barley, wheat and apples.

As used herein, the term “plant gene promoter” refers to a known promoter expressed in plants. For example, the tobacco infection-specific protein PR1a
Promoters (hereinafter referred to as tobacco PR1a promoter), whose expression is induced by a certain stress, cauliflower mosaic virus 35S promoter (hereinafter referred to as CaMV35S promoter), nopaline synthase promoter, and the like. Not done. An enhancer can be used for high expression. As the enhancer, an enhancer region containing the sequence upstream of the 35S promoter is preferable. A plurality of enhancers may be used.

The term "plant gene" as used herein
Means a gene possessed by a plant. As the plant gene, any gene can be used. Suitable examples include, but are not limited to, the tobacco infection-specific protein PR1a gene.

As used herein, the term “hinge region of tobacco chitinase” refers to a region that separates different functional regions (chitin-binding region and catalytic region) in tobacco chitinase protein. (H. Shinshi et al., Plant Mole. Bi
ol. 14, 357-368 (1990). The presence of the hinge region of tobacco chitinase at the junction between the plant gene and the foreign gene is expected to avoid steric hindrance of each protein.

As used herein, the term "foreign gene"
The term refers to a gene encoding a protein that is heterologous to the host plant. Plant genes, genes of microorganisms such as bacteria and yeasts, genes of insects, genes of animals and the like can be suitably used. When breeding pathogenic resistant plants,
Antimicrobial peptides can be used as foreign genes.

For example, it refers to a gene encoding an antimicrobial peptide derived from a dipteran insect, but is not limited thereto. The antibacterial peptide derived from dipteran insects is preferably, but not limited to, sarcotoxin 1A, zapesin, or an antifungal protein derived from the common fly.
A signal sequence can be added to a foreign gene to be introduced into the expression cassette, and the resulting fusion protein can be secreted.

For the preparation of a recombinant gene of the present invention in which a foreign gene and a plant gene are linked via the hinge region of tobacco chitinase, known gene recombination techniques can be used.

As used herein, the term "expression cassette" refers to the expression of the above-mentioned recombinant gene and various regulatory elements such as promoters and enhancers that regulate its expression so that the above-mentioned recombinant gene can be expressed. A nucleic acid sequence that is operably linked in a cell. The expression cassette can be constructed according to a conventional method. An appropriate promoter, such as the tobacco PR1a promoter, is ligated to the recombinant gene. The tobacco PR1a promoter is known as an inducible and high-expression promoter.
More preferably, a terminator may be linked to the recombinant gene.

As used herein, the term "terminator" is a sequence located downstream of a region encoding a protein of a gene and involved in terminating transcription when DNA is transcribed into mRNA and adding a polyA sequence. It is. It is known that the terminator is involved in mRNA stability and affects the gene expression level. Examples of the terminator include a cauliflower mosaic virus 35S terminator, a nopaline synthase gene terminator, and a tobacco PR1a gene terminator. It has been confirmed that the tobacco PR1a gene terminator enhances gene expression efficiency.

[0033] As used herein, the term "expression vector" refers to a medium that transfers the above expression cassette into a host cell. It is well known to those skilled in the art that the type of expression vector and the type of regulatory element used may vary depending on the host cell. The expression vector of the present invention may further have a T-DNA region and a drug resistance gene.

As the drug resistance gene, it is desirable that selection of a transformed plant is easy. Neomycin phosphotransferase (NP
The T) gene, the hygromycin phosphotransferase gene and the like can be suitably used.

As the promoter for expressing the drug resistance gene, the above-mentioned plant gene promoter, for example, tobacco
Examples include, but are not limited to, PR1a promoter, CaMV35S promoter, nopaline synthesis promoter, and the like. Preferably, a CaMV35S promoter that constitutively expresses high may be used. When an inducible tobacco PR1a promoter is used as a promoter for expressing a foreign gene in the expression cassette, the CaMV35S promoter constitutively expresses the inducible PR1a promoter, and necrotic lesions associated with salicylic acid or pathogen infection. Formation increases the expression of the PR1a promoter. This is probably because the CaMV35S promoter and the PR1a promoter are adjacent to each other, so that the properties of the PR1a promoter are affected by the cis element on the 35S promoter.

The vector used for the construction of the expression vector may be a pBI vector, a pUC vector or pTRA.
A system vector can be suitably used. A pBI-based vector can introduce an expression cassette into a plant via Agrobacterium such as a binary vector system or an intermediate vector system. For example, pBI121, pBI101, pBI101.2, pBI10
1.3 and the like. A pUC-based vector can directly introduce an expression cassette into a plant. For example, pUC18, pUC19, pUC9
And the like. In this application, the pTRA system (Ohshima et al,
Plant Cell, Vol. 2, pp. 95-106 (1990)) can be suitably used. This expression vector contains an expression cassette containing a fusion gene in a region (T-region) that can be introduced into a plant, and an NPT gene expressed under the control of the CaMV35S promoter as a marker gene.

When the expression vector of the present invention has a T-region, the structure of the T-region is as follows: (1) When transferring from Agrobacterium to a plant, the foreign gene to be introduced moves first, An expression cassette containing a foreign gene can be arranged on the right side of the T-region so that the marker gene is transferred. Therefore, transgenic plants selected by drug resistance can contain a foreign gene with extremely high probability. (2) The CaMV35S promoter that controls the expression of the marker gene and the PR1a gene promoter that controls the expression of the foreign gene are placed side by side. It is arranged. Thus, when a foreign gene was originally expressed in a plant using the PR1a promoter, almost no expression was observed under non-inducible conditions, but it probably showed constitutive expression even in non-inducible states due to the influence of the CaMV35S promoter, and It is characterized in that it shows a high level of expression upon induction.

When the PR1a promoter is used, the expression level of the above expression vector is inducibly increased by salicylic acid or necrotic lesion formation associated with pathogen infection.

The expression vector of the present invention can be prepared by using a gene recombination technique well known to those skilled in the art.

A method well known to those skilled in the art can be used by introducing a recombinant gene, an expression cassette or an expression vector into a plant cell, and electroporation.

The above-obtained expression cassette or expression vector can be introduced into cells by the above-described method via Agrobacterium or directly into cells.

Agrobacterium-mediated methods are described, for example, in the method of Nagel et al. (Micribiol. Lett., 67, 325 (1990)).
Can be used. In this method, first, for example, an expression vector is transformed into Agrobacterium by electroporation, and then the transformed Agrobacterium is transformed into a plant molecular biology manual (SB Gelv.
in et al., Academic Press Publishers). Methods for directly introducing an expression cassette and an expression vector into cells include an electroporation method and a gene gun method.

Cells into which the expression cassette and the expression vector have been introduced can be first selected for drug resistance such as kanamycin resistance, and then regenerated into plants by a conventional method.

For confirming the expression of a foreign gene in a transformed plant, a method well known to those skilled in the art can be used.
For example, a soluble protein is extracted from a transformed plant, and the protein extracted by SDS-polyacrylamide gel electrophoresis is separated and transferred to a PVDF membrane as described in Analytical Biochemistry 166, 368-379. Expression can be confirmed by reacting with an antibody against the target protein and immunochemically detecting the resulting band.

The pathogen resistance is measured by inoculating the leaves of the transformed plant and control plant, which have been regenerated into a plant, with the pathogen using a fine needle bundle, and after an appropriate time, for example, 6 days, the size of the necrotic lesion. The degree of the symptom is determined from the change in leaf color. As the pathogen, wildfire fungus (P. sylingae pv. T
abaci), soft rot fungus (E. cartovara subsp. carotovora)
And the like.

In one embodiment of the present invention, a tobacco PR1a protein gene is used as a plant gene, and a Sarcotoxin1a gene, which is an antibacterial peptide derived from a dipteran insect, is used as a foreign gene, and is linked via the hinge region of tobacco chitinase. The following is a description of what has been made.

Sarcotoxin 1a is a peptide that has widespread antibacterial activity against bacteria, and it has already been confirmed that introduction of this peptide into tobacco can produce plants that exhibit a certain level of resistance to pathogenic bacteria (particularly). Kaihei 7-250685). However, since short peptides such as Sarcotoxin1a are expected to be unstable in plants, they are expected to be stabilized by producing them as fusion proteins with PR1a, an infection-specific protein possessed by tobacco. You. The hinge region of tobacco chitinase is a region that separates different functional regions (chitin-binding region and catalytic region) in tobacco chitinase protein. Tobacco PR1a
By inserting the hinge region of tobacco chitinase into the junction between the protein and Sarcotoxin, steric hindrance of each protein can be avoided. Further, a signal sequence of tobacco PR1a protein is added to the N-terminus of the fusion protein, whereby the fusion protein can be secreted. It is expected that the antibacterial property against pathogens will be improved, because the fusion protein is secreted into the intercellular space, thereby preventing damage to plant cells and further preventing pathogens from entering the intercellular space.

[0048]

The present invention will be specifically described below with reference to examples. Restriction enzymes, plasmids and the like used in this example can be obtained from Takara Shuzo Co., Ltd., Toyobo Co., Ltd. or the like.

Example 1 Preparation of Recombinant Gene and Expression Cassette A recombinant gene in which a foreign gene and a plant gene are linked via the hinge region of tobacco chitinase was prepared as follows. A description will be given based on FIG.

(1) Preparation of P + S PR1a Fragment (DNA Fragment Having Part of PR1a Promoter and Sequence Encoding Signal Peptide of PR1a Protein) Plasmid pPR-γ having a genomic clone of the tobacco PR1a gene shown in FIG. (M. Oshima et al., FEBS Letters 2
55, 243-246 (1987)), a DNA fragment (P + S PR1a fragment) was amplified from the XhoI site in the PR1a promoter region to the region encoding the signal peptide using the template.
I got The P + SPR1a fragment was prepared by using a primer PPR1a51 having the sequence of SEQ ID NO: 1 and a primer SPR1a31 having the sequence of SEQ ID NO: 2 as primers, using Pfu polymerase (st
ratagene). Among the primers used in this PCR, SPR1a31 has a sequence complementary to the sequence encoding the signal peptide of the PR1a gene, but has a partial base substitution as shown in FIG. Although the restriction enzyme site Pst1 was newly introduced by this base substitution, the encoded amino acid sequence was not changed.

(2) Preparation of TPR1a Fragment (DNA Fragment Having Terminator Region of PR1a Gene) Using the plasmid pPR-γ having the genomic clone of the tobacco PR1a gene of FIG. 1 as a template, a DNA fragment having a terminator region of the PR1a gene ( TPR1a fragment). TPR
The 1a fragment is a DNA fragment obtained by amplifying the terminator region of PR1a by PCR using the primer TPR1a51 of SEQ ID NO: 3 and the primer BKSBE of SEQ ID NO: 4. this
Among the primers used for PCR, TPR1a51 had Sarcot at the 5 'end.
Has a sequence homologous to the 3 'end of the cDNA encoding oxin 1a,
In addition, immediately after the stop codon, it has a recognition sequence for SacI. Immediately before the stop codon, a base is partially substituted as shown in FIG. Although the restriction enzyme recognition site PmaCI was newly introduced by this base substitution, the encoded amino acid sequence was not changed. On the other hand, the primer BKSBE
-Has a sequence homologous from the EcolI site to the BamHI site in the vector portion of -γ. This site is adjacent to the 3 'end of the portion corresponding to the PR1a gene terminator of pPR-γ.

(3) HMPR1a fragment (tobacco chitinase
Preparation of DNA fragment having hinge region and region encoding PR1a mature protein) DNA having region encoding mature PR1a protein using plasmid pPR-γ having a genomic clone of tobacco PR1a gene in FIG. 1 as a template A fragment (H MPR1a fragment) was obtained. The HMPR1a fragment was prepared using the primer HMPPR1a5 of SEQ ID NO: 5.
PCR using primers MPR1a31 of SEQ ID NO: 1 and SEQ ID NO: 6
It is a fragment that has been blessed by. Among the primers used in this PCR, HMPR1a51 contains the same sequence as the 12-base sequence of the PmaCI site of TPR1a51 at the 5 ′ end and a region encoding the hinge region of tobacco chitinase on the 3 ′ side. Has the same sequence as

[0053] MPR1a31 has a recognition site for the restriction enzyme SacI added immediately after the 3'-side stop codon.

(4) Preparation of MSARCO Fragment (DNA Fragment Having Region Encoding Sarcotoxin Mature Peptide) Known plasmid pTO19 (N.M.) containing the cDNA of Sarcotoxin1A
Using atsumoto et al., BioChem. J., 239 , 717 (1986)) as a template, a DNA fragment (MSARCO fragment) having a region encoding a mature Sarcotoxin peptide was obtained. This MSARCO
The fragment is a DNA fragment obtained by amplifying a region encoding the mature peptide of Sarcotoxin by PCR using primer MSARCO51 of SEQ ID NO: 7 and primer MSARCO31 of SEQ ID NO: 8. Primers MSARCO51 and MSARCO31
Are shown in FIGS. 4 and 5, respectively. Primer MS
ARCO51 has a sequence complementary to the PstI site of SPR1a31 and its surrounding 14 bases near the 5 ′ end. As shown in FIG. 5, MSARCO31 has recognition sites for restriction enzymes PmaCI and SacI introduced around a sequence complementary to a region encoding a stop codon. This restriction enzyme site was introduced in the same manner as in TPR1a51.

(5) MSARCO-TPR fragment (MSARCO fragment and TPR1
Preparation of DNA Fragment Linked with Fragment a) Using the MSARCO DNA fragment obtained in (4) above and the TPR1a fragment obtained in (2) as templates, primers MSARCO31 and BSAR
Amplification was performed by a recombinant PCR method using KSBE to obtain a MSARCO-TPR fragment.

(6) Preparation of Recombinant Gene (Connection of P + SPR1a Fragment, H MPR1a Fragment and MSARCO-TPR1a Fragment) P + SPR1a Fragment Obtained in (1) above, Obtained in (3)
The HMPR1a fragment and the MSARCO-TPR1a fragment, which is the binding fragment of (2) and (4) obtained in (5), were cloned into plasmid pUC18, respectively.
1a, pUC H MPR1a and pUC MSARCO-TPR1a (see FIG. 6). Correct insertion of the target sequence into these plasmids was confirmed using a base sequence determination kit and a base sequence analyzer manufactured by Applied Biosystems. These DNAs, which were cloned into pUC18 and whose nucleotide sequence was confirmed, were used as a material for subsequent gene recombination. SEQ ID NO: 7 shows the nucleotide sequence of the sequenced PR1a terminator.

Hereinafter, the production of the recombinant gene of the present invention will be described with reference to FIG.

First, an upstream (5 ′ side) portion of the XhoI site of the promoter region of the PR1a gene of the plasmid pPR-γ was digested with restriction enzymes EcoRI and XhoI, and the obtained fragment of about 1.6 kb was digested.
Purification was performed using Bio101 Gene Clean kit. On the other hand, P + S PR
The plasmid pUC P + SPR1a into which the 1a fragment was cloned
digested with oRI-XhoI.
A 1.6 kb fragment was inserted. The resulting plasmid (pUC P + S
PR (2.4)) is a DN encoding the full-length PR1a gene promoter and signal sequence of the PR1a gene as inserts.
A region is included. This plasmid is cut out with EcoRI,
It has a cassette for the tobacco PR1a promoter.

PUC MSARCO-TPR1a was digested with restriction enzyme PstI, and a DNA fragment corresponding to MSARCO-TPR1a was purified. This fragment was inserted into the PstI site of pUC P + SPR (2.4). The inserted DNA sequence of the obtained plasmid is correct P + SP
R1a- Plasmid inserted in the direction of MSARCO-TPR1a
(pUC P + SPR (2.4) MSARCO-TPR1a) was selected. This plasmid contains an expressible non-fused Saracotoxi, excised with EcoRI and driven by the tobacco PR1a promoter.
It has a cassette for the gene encoding n.

Next, plasmids pUC H MPR1a to H MPR
1a was cut out with restriction enzymes PmaCI and SacI. On the other hand, pUC P + SPR (2.4) MSARCO-TPR1a was digested with PmaCI-SacI, and the excised HMPR1a portion was inserted into the site (pUC
P + S PR1a (2.4) MSARCO H MPR1aTPR). This plasmid contains the gene for Sarcotoxin1a, an antimicrobial peptide derived from dipteran insects as a foreign gene, the tobacco PR1a protein gene as a plant gene, and the tobacco chitinase hing.
It contains the recombinant gene of the present invention linked via the e region. Furthermore, this plasmid has a recombinant gene encoding the Sarcotoxin1a-PR1a fusion protein controlled by the tobacco PR1a promoter, and when this plasmid is cut with EcoRI, the expressible Sarcotoxin1a is controlled by the tobacco PR1a promoter. -h
a recombinant gene encoding the inge-PR1a fusion protein,
P + SPR1a (2.4) MSARCO H MPR1a TPR, that is, an expression cassette that expresses the recombinant gene of the present invention.

Example 2 Construction of Expression Vector Binary vector pTRA415 (FIG. 7) (Ohshima et al, Plant
Cell 2, 95-106 (1990)) has a sequence in which the NPT gene is bound downstream of the CaMV35S promoter in a BglII-BglII fragment. This pTRA415 was cut with BglII, religated with ligase, and then introduced into E. coli JM109. From the transformants, a plasmid (pTRA415 (R)) in which the BglII-BglII fragment of pTRA415 was inserted in the reverse direction was selected. pTRA415 (R) has an NPT gene governed by the CaMV35S promoter as a marker gene inside the T-region,
On the right side, it has an EcoRI site as a (unique) restriction enzyme cleavage site that cuts only one place in pTRA415 (R). this
When a foreign gene is introduced into the EcoRI site, the marker gene is transferred after the transfer of the foreign gene into the plant.

The above plasmid pUC P + S PR1a (2.4) MSARCO
TPR (plasmid expressing unfused Sarcotoxin) with EcoR
After digestion with I, the P + SPR1a (2.4) MSARCO TPR fragment was cut out and inserted into the EcoRI site of pTRA415 (R). From the obtained plasmid, those in which the PR1a promoter of the inserted expression cassette was inserted so as to be adjacent to the CaMV35S promoter of the marker gene were selected (plasmid PS
S).

Similarly, the plasmid pUC P + S PR1a (2.4) MS
ARCO H MPR1a TPR (plasmid expressing fusion Sarcotoxin-PR1a protein) is cut with EcoRI and P + SPR1a (2.4)
The MSARCO H MPR1a TPR fragment was cut out and pTRA415 (R) Eco
Inserted at RI site. From the obtained plasmid, the PR1a promoter of the inserted expression cassette was
Those inserted so as to be adjacent to the CaMV35S promoter were selected (plasmid PSP).

Example 3 Introduction of Expression Cassette into Tobacco (Transformation of Agrobacterium tumefaciens)
um tumefaciens with 250 μg / ml streptomycin and 50
Culture in a medium containing μg / ml rifampicin at 28 ° C and Na
According to the method of gel et al. (Micribiol. Lett., 67, 325 (1990)), a cell culture solution was prepared and an expression vector (plasmid PS
P) was introduced into the bacteria by electroporation. Similarly, plasmid PSS (this is the unfused Sarcoto
xin) and JP-A-7-25068
Plasmid PST10 according to 5, which is constitutively unfused Sa
rcotoxin) was introduced into the bacteria. FIG. 8 shows a schematic diagram of each plasmid of PSP, PSS and PST10 used in the present invention.

(Transformation of tobacco) Agrobacterium transformed with the plasmid PSP by the above method was obtained, and a YEB medium (DN
After shaking culture in Acloning, Vol. 2, p.
The mixture was diluted twice and co-cultured with leaf pieces of tobacco (Nicotiana tabacum Samsun NN). After 2-3 days, the bacteria are removed with a medium containing antibiotics, and subcultured with a selection medium every two weeks.
Transformed tobacco cells were selected and re-differentiated by a standard method. As a result, 29 independent transformed individuals were obtained.

Similarly, a transformed individual transformed with the plasmid PSS or the plasmid PST10 was obtained.

Example 4 Detection of Fusion Protein in Transformed Plant by Western Blotting Transformed plant transformed with PSP obtained, plasmid PST
Soluble protein extracts were prepared from the plants transformed in step 10 and wild-type Samsun NN plants, respectively.
Four leaf pieces of about 7 mm in diameter were cut from each tobacco leaf, and cultured for 2 days in a suspended state on a 0.5 mM aqueous solution of salicylic acid and, as a target, on pure water.
0 mM Na-PO ₄ (pH 7.0), 1 mM Na-EDTA, 10 μM A-PMSF, 0.5
Grind in μg / ml Leupeptin, 2mM DTT, 10,000rpm
And centrifuged at for 10 minutes to recover the supernatant to obtain a protein extract. 2.5 μl of this extract is used for Analytical Biochemistr
y After separation by SDS-polyacrylamide gel electrophoresis indicated by 166, 368-379, transfer to PVDF membrane, anti-Sarcot
The band reacting with the oxin antibody was detected immunochemically. FIG. 9 shows the results of Western blotting.

In the plant into which the fusion gene was introduced (PSP plant), a band considered to be a fusion protein was detected regardless of the presence or absence of salicylic acid treatment. However, the amount of fusion protein detected was significantly increased by induction with salicylic acid. Salicylic acid treatment resulted in the appearance of free (unfused) Sarcotoxin bands. No band reacting with anti-Sarcotoxin antibody was detected in Samsun NN plants, but Sarcotoxi in PST10 plants.
A band presumed to be derived from n was detected, which was consistent with the band appearing on the low molecular side of the PSP plant. Similar experiments were performed with anti-PR1a antibody instead of anti-Sarcotoxin antibody. As a result, it was confirmed that the high-molecular-side band observed in the PSP plant was also recognized by the anti-PR1a antibody, and that the high-molecular-side band was a fusion protein.

Example 5 Assay of Wild Plant Blight Resistance of Transformed Plant Introduced with Fusion Gene Four leaves were wounded on the leaves of transformed tobacco and control plants using fine needles.
2 × 10 ⁷ ml of a suspension of wildfire fungus (P. sylingae pv. Tabaci) was inoculated. The same treatment was performed four times for one plant. Six days later, the degree of the symptom was determined based on the size of the necrotic lesion and changes in leaf color.

The evaluation of necrotic lesions was represented by the following numbers.

0 Necrotic site is not recognized 1 Necrotic site diameter is 3 mm or less 2 Necrotic site diameter is 5 mm or less 3 Necrotic site diameter is 10 mm or less 4 Necrotic site diameter is 10 mm or more Expressed by numbers.

0 No yellowing site is observed 1 There is a pale yellowing site with a diameter of about 5 mm 2 There is a yellowing site with a diameter of more than 5 mm 3 There is a yellowing site with a diameter of 10 mm or more Necrosis due to yellowing The result is shown in FIG. Some of the tobacco plants into which the fusion gene had been introduced showed reduced symptom caused by wildfire compared with control plants.

[0073] It can be seen that the use of the expression cassette of the present invention enhances the resistance compared to expressing sarcotoxin alone.

Example 6 Test for Resistance to Soft Rot of Transgenic Plant Introduced with Fusion Gene A leaf piece of 7 mm in diameter was cut out from the leaves of the transformed tobacco and control plants.
Suspension of soft rot fungus (E. carotovara subsp. Carotovora) 4
× 10 ⁷ ml, and suspended on pure water as a control. Four leaf pieces for one plant line were used in the experiment. Three days later, the degree of symptom was determined by the change in leaf color, and the bacterial growth was determined by the change in turbidity of the suspension. The results are shown in FIG.

As a result, the symptom caused by the soft rot fungus was reduced in some of the tobacco plants into which the fusion gene was introduced, as compared with the control plant, and at the same time, the growth of the soft rot fungus was suppressed.

From this, it was found that the introduction of the present fusion protein resulted in plants resistant to wildfire and soft rot fungi.

[0077]

As a result of introducing the fusion protein of the present invention into a plant using the above expression vector and expressing it, the fusion protein was constitutively produced and its production was increased by inducing salicylic acid and the like. In addition, the unidentified proteolytic activity induced by salicylic acid / TMV infection allowed the inducible production of each free protein as a result of the cleavage of the fusion protein at its junction.

By using the recombinant gene, expression cassette or expression vector of the present invention, a foreign protein can be constitutively produced as a fusion protein and the respective free proteins can be produced inductively. By this method, an antimicrobial peptide can be expressed as a fusion protein with a plant gene, and a plant resistant to pathogenic bacteria can be produced.

[0079]

[Sequence list]

[0080]

[SEQ ID NO: 1] Sequence length: 32 Sequence type: nucleic acid Topology: linear Sequence type: primer Sequence AGTAGAATTC TTAAAACACC CTCGAGGATT TC 32

[0081]

[SEQ ID NO: 2] Sequence length: 23 Sequence type: Nucleic acid Topology: Linear Sequence type: Primer Sequence: GGGCTCTGCA GGAGTGGGAT ATT 23

[0082]

[SEQ ID NO: 3] Sequence length: 43 Sequence type: Nucleic acid Topology: Linear Sequence type: Primer Sequence: ACAGCACGTG GTTAATTGAG CTCGAAACGA CCTACGTCCA TTC 43

[0083]

[SEQ ID NO: 4] Sequence length: 21 Sequence type: nucleic acid Topology: Linear Sequence type: Primer Sequence: TCCCCCGGGC TGCAGGGAATT C
21

[0084]

[SEQ ID NO: 5] Sequence length: 72 Sequence type: nucleic acid Topology: linear Sequence type: primer Sequence: TACACACGTG GTCCCACACC TACACCCCCC ACCCCACCCG GTGGTGGGCA AAATTCTCAA CAAGACTATT TG 72

[0085]

[SEQ ID NO: 6] Sequence length: 30 Sequence type: nucleic acid Topology: linear Sequence type: primer Sequence: TATCGAGCTC AATTAGTATG GACTTTCGCC 30

[0086]

[SEQ ID NO: 7] Sequence length: 36 Sequence type: nucleic acid Topology: linear Sequence type: primer Sequence: CACTCCTGCA GAGCCGGTTG GTTGAAAAAG ATTGGC 36

[0087]

[SEQ ID NO: 8] Sequence length: 33 Sequence type: nucleic acid Topology: linear Sequence type: primer Sequence: TTTCGAGCTC AATTAACCAC GTGCTGTAGC AGC 33

[0088]

[SEQ ID NO: 9] Sequence length: 508 Sequence type: Nucleic acid Topology: Linear Sequence type: Tobacco PR1a Terminator Sequence: TTGAAACGAC CTACGTCCAT TTCACGTTAA TATGTATGGA TTGTTCTGCT TGATATCAAG 60 AACTTAAATA ATTGATATA TGACTATAG 120A TGA TGA TGA TGA TGA TGA TGA TGA GGGGAAAAAT 180 ATGAGTTGAT GATCAGCTTG ATGTATGATC TGATATTATT ATGAACAGCG TTTTGTACTC 240 ATACGAATCA TGTGTTGATG GTCTAGCTAC TTGCGATATT ACGAGCAAAA TTCTTAACTA 300 CATGCCTTAG GAACAAGCTT ACACAGTTCA TATAATCTAC TAGAGGGCCA AAAACATGAA 360 AATTACCAAT TTAGATGGTA GGAGGATATT GAAAGTGGAG CAGCTAGTTT TAATAACTGA 420 CCGTTAGTCT TAAAATTGAC GGTATAAAAA TATTTACATA ATCAGGTCAT TTATAAGGTA 480 ATTATAGGTA AATATTTATG ACGAATTC 508

[Brief description of the drawings]

FIG. 1 is a diagram showing a plasmid pPR-γ having a genomic clone of the tobacco PR1a gene.

FIG. 2 is a view showing a substituted sequence of a primer SPR1a31.

FIG. 3 is a view showing a substituted sequence of a primer TPR1a51.

FIG. 4 is a view showing the sequence of a primer MSARCO51.

FIG. 5 is a view showing a substituted sequence of a primer MSARCO31.

FIG. 6 is a diagram illustrating the creation of a recombinant gene of the present invention.

FIG. 7: Binary vectors pTRA415 and pTRA415
It is a figure showing (R).

FIG. 8 is a diagram showing plasmids PSP, PSS and PST10 used in the present invention.

FIG. 9 shows the expression of Sarcotoxin in plants transformed with PSP and PST10.

FIG. 10 is a view showing resistance of tobacco wildfire to transformed plants.

FIG. 11 is a graph showing resistance to soft rot fungi of transformed plants.

──────────────────────────────────────────────────続 き Continuing on the front page (72) Yoshimasa Matsukawa 221-10 Yamaguchi Nogi, Aomori Prefecture Green Bio Center (72) Inventor Shunji Natori 2208-19 Nagawa, Tone-cho, Kitasoma-gun, Ibaraki Inventor Yuko Ohashi 2-1-2 Kannondai, Tsukuba, Ibaraki Pref. Ministry of Agriculture, Forestry and Fisheries, National Institute for Agricultural and Biological Resources (58) Field surveyed (Int. Cl. ⁷ , DB name) C12N 15/00-15/84 A01H 5/00 C12N 5/10 C12P 21/02 BIOSIS (DIALOG) WPI (DIALOG)

Claims (22)

(57) [Claims] 1. A recombinant gene in which a foreign gene and a plant gene are linked via a hinge region of tobacco chitinase. 2. The recombinant gene according to claim 1, wherein the plant gene is a gene encoding a tobacco PR1a protein. 3. The recombinant gene according to claim 2, wherein the foreign gene is linked to a signal sequence of a plant protein. 4. The recombinant gene according to claim 3, wherein the foreign gene is a gene encoding an antibacterial peptide derived from a dipteran insect. 5. The recombinant gene according to claim 4 , wherein the antimicrobial peptide derived from the dipteran insect is Sarcotoxin 1a. 6. An expression cassette wherein the recombinant gene according to any one of claims 1 to 5 is linked to a plant gene promoter. 7. The plant gene promoter according to claim 6, wherein the plant gene promoter is an inducible tobacco PR1a gene promoter.
An expression cassette according to item 1. 8. The expression cassette according to claim 7, further comprising a terminator derived from the tobacco PR1a gene. 9. An expression vector for introducing the expression cassette according to any one of claims 6 to 8 into a plant. 10. The expression vector according to claim 9, further comprising a T-DNA region and a drug resistance gene. 11. The expression vector according to claim 9, wherein the drug resistance gene is expressed by a cauliflower mosaic virus 35S promoter. 12. A cauliflower mosaic virus 35S promoter that expresses the drug resistance gene,
The expression vector according to claim 11, wherein an inducible promoter for expressing a foreign gene is constitutively expressed, and the activity of the inducible promoter is increased by necrotic lesion formation associated with salicylic acid or pathogen infection. A tobacco transformed with the recombinant gene according to any one of claims 1 to 5. 14. A tobacco transformed with the expression cassette according to any one of claims 6 to 8. A tobacco transformed with the expression vector according to any one of claims 9 to 12. 16. The following steps: (1) the recombinant gene according to any one of claims 1 to 5, the expression cassette according to any one of claims 6 to 8, or a configuration comprising the expression cassette Obtaining a tobacco cell transformed with a gene having an exogenous gene selected from the group consisting of a gene having a drug resistance gene linked to a plant promoter which is expressed in a plant; (2) the transformed tobacco A method for producing a peptide encoded by the foreign gene, comprising: culturing the cell under induction conditions; and (3) recovering the peptide encoded by the foreign gene. 17. The tobacco PR1a wherein the foreign gene is inducible.
17. The method of claim 16, wherein the method is linked to a promoter. 18. The method of claim 16, wherein said constitutively expressed plant promoter is the cauliflower mosaic virus 35S promoter. 19. A cauliflower mosaic virus 35S promoter that expresses the drug resistance gene,
19. The method according to claim 18, wherein the inducible promoter for expressing the foreign gene is constitutively expressed, and the activity of the inducible promoter is increased by necrotic lesion formation associated with salicylic acid or pathogen infection. 20. A recombinant gene in which a gene encoding an antimicrobial peptide and a plant gene are linked via a hinge region of tobacco chitinase, and the recombinant gene is linked to a plant gene promoter. A tobacco resistant to pathogenic bacteria, comprising a gene selected from the group consisting of an expression cassette, or a gene having a drug resistance gene linked to a plant promoter constitutively expressing the expression cassette. 21. The tobacco according to claim 20, wherein the pathogenic bacterium is a wildfire bacterium or a soft rot fungus. 22. The antibacterial peptide is Sarcotoxin 1a derived from a dipteran insect.
The cigarette described in.