JPH09252778A - Expression cassette capable of imparting medicine metabolizing function and use of the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an expression cassette containing a promoter and terminator functioning in plant cells and a fused gene of a chlorophyll transferring signal peptide gene with a cytohrome p450 derived from a non plant body and capable of imparting a medicine metabolizing function to a plant. SOLUTION: This new expression cassette has (A) a promotor capable of functioning in plant cells, (B) a fused gene of a gene encoding a signal peptide for chlorophyll transfer expressed by the formula with a gene of a cytochrome P450 derived from a non plant body and (C) a terminator capable of functioning in plant cells, and can efficiently impart a medicine metabolizing function to a plant by introducing the same into the plant cells. The expression cassette is obtained by functionally joining 35S promoter derived from cauliflower mosaic virus as the promotor capable of functioning in the plant cells, and a NOS terminator derived from T-DNA, etc., as the terminator capable of functioning in the above fused gene and in the plant cells.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an expression cassette imparting drug metabolizing ability.

[0002]

2. Description of the Related Art Cytochrome P450 is an enzyme widely distributed in the living world, from microorganisms to plants and mammals, and constitutes a group of heme enzyme families containing heme in the molecule. Cytochrome P450 catalyzes the reaction of adding monoatomic oxygen to a wide range of lipid-soluble compounds including drugs, poisons and pesticides taken up in the body, and detoxifies them by metabolizing these compounds. Have the ability to turn into.

[0003]

Recently, the presence of various cytochrome P450s in plants has been revealed. However, the content of cytochrome P450 in plants is lower than that in mammals. For this reason, plants have far less ability to metabolize drugs than mammals. Therefore, a means for efficiently imparting drug-metabolizing ability to plants has been desired.

[0004]

[Means for Solving the Problems] In view of the above situation, the present inventors have conducted extensive studies, and as a result, expressed a non-plant-derived cytochrome P450 in chloroplasts, thereby imparting a drug-metabolizing ability to plants. Succeeded in giving efficiently, it came to the present invention. That is, the present invention provides: (1) a promoter capable of functioning in plant cells, (2) a fusion gene of a gene encoding a chloroplast translocation signal peptide sequence and a non-plant-derived cytochrome P450 gene, and (3) capable of functioning in plant cells An expression cassette having a terminator in a functional form (hereinafter referred to as the expression cassette of the present invention), II. The chloroplast translocation signal peptide sequence is SEQ ID NO: 10.
The above item I, which is an amino acid sequence represented by
The described expression cassette, III. The non-plant-derived cytochrome P450 gene is derived from a mammal, IV. A microorganism containing the expression cassette according to the above item I, V. A plant transformed by introducing the expression cassette described in the above item I, VI. (1) a promoter capable of functioning in plant cells, (2)
A fusion gene of a gene encoding a chloroplast translocation signal peptide sequence and a non-plant-derived cytochrome P450 gene, and (3) an expression cassette having a functional terminator capable of functioning in a plant cell in a plant cell And a method for imparting drug-metabolizing ability in a plant characterized by expressing a non-plant-derived cytochrome P450 in a chloroplast,
Is provided.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail. The expression cassette of the present invention comprises (1) a promoter capable of functioning in plant cells, (2) a fusion gene of a gene encoding a chloroplast translocation signal peptide sequence and a non-plant-derived cytochrome P450 gene, and (3) a plant It contains terminators that can function in cells, and each has a form that allows them to function.

"A fusion gene of a gene encoding a chloroplast translocation signal peptide sequence and a non-plant-derived cytochrome P450 gene" means, for example, a gene encoding a chloroplast translocation signal peptide sequence and a non-plant-derived cytochrome P450.
It can be constructed by connecting genes.
To connect both genes, coordinate the gene encoding the chloroplast translocation signal peptide sequence on the 5'side and the non-plant-derived cytochrome P450 gene on the 3'side, so that a stop codon does not appear in the middle. It can be obtained by using a different linker so that the frames are connected without shifting.

The non-plant-derived cytochrome P450 is not particularly limited as long as it is non-plant-derived and has monoatomic oxygen addition activity, and various cytochrome P450 molecular species can be used. For example, I 1, I
Cytochrome P belonging to I, III, IV gene families
There are 450 molecular species. Specifically, mammal-derived cytochromes P450c, P450d, P450 1A1, P450
1A2, P450 2A6, P450 2B6, P450 2C8, P450 2C9, P450 2C1
8, P4502D6, P4502E1, P450 3A4, P450 3A5 and the like. Since these molecular species metabolize compounds having different chemical structures, the optimal molecular species can be selected according to the purpose. For example, rat liver-derived cytochrome P4
50c has activities such as ring hydroxylation of aromatic ring hydrocarbon compounds such as benzpyrene and O-deethylation of 7-ethoxycoumarin. The above-mentioned non-plant-derived cytochrome P450 gene whose structure has been clarified can be obtained by a known method described in the literature, or can be cloned by the PCR method based on a reported nucleotide sequence. is there. Alternatively, the target gene can be prepared by chemical synthesis.

Non-plant-derived cytochrome P450 is
It may be supplied in the form of a fusion enzyme gene with the gene for ADPH-P450 reductase. As the reductase used in this case, the reducing power supply activity (NA
It is not particularly limited as long as it has a function of transferring electrons from DPH to P450). For example, NADPH-P450 reductase derived from a microorganism such as yeast,
N derived from mammals such as human, rat, rabbit and pig
Examples thereof include ADPH-P450 reductase or plant-derived NADPH-P450 reductase. The genes of these NADPH-P450 reductases can be obtained by known methods described in the literature etc., if the structures are clear. In addition, it is also possible to clone the gene by the PCR method or the like based on the reported nucleotide sequence. Alternatively, the target gene can also be produced by chemical synthesis. The fusion enzyme gene with the gene for NADPH-P450 reductase is, for example, microsomal cytochrome P4.
50 genes containing at least a microsomal membrane binding region, a substrate binding region and a heme binding region, and NADPH-P
It can be constructed by connecting the genes containing the respective binding regions of 450 reductase flavin mononucleotide, flavin adenine dinucleotide and NADPH. In addition, when connecting both genes, the non-plant-derived cytochrome P450 gene was placed on the 5 ′ side and NADPH
-P450 reductase gene is coordinated to the 3'side respectively,
In order to prevent the termination codon from appearing on the way, both genes may be connected using an appropriate linker sequence so that the frames are not displaced.

"A gene encoding a chloroplast translocation signal peptide sequence" refers to various genes encoded by the nucleus, of which proteins whose transcription and translation products translocate to chloroplast and are localized in chloroplast. It means a gene encoding a signal peptide sequence. For example, a gene encoding a signal peptide sequence of chloroplast a / b binding protein, a signal peptide sequence of chloroplast type ω-3 desaturase, and a signal peptide sequence of chloroplast ferroketalase can be mentioned. . Specifically, for example, there is a gene encoding a chloroplast translocation signal peptide sequence which is the amino acid sequence represented by SEQ ID NO: 10. The genes encoding these chloroplast translocation signal peptide sequences can be obtained by known methods described in the literature etc., if the structures are clarified. In addition, based on the reported base sequence,
It is also possible to clone the gene by the PCR method or the like. Alternatively, the target gene can also be produced by chemical synthesis.

[Promoter capable of functioning in plant cells]
The term "gene" refers to a gene having a region involved in gene expression functions such as transcription and translation in plant cells, and includes, for example, the cauliflower mosaic virus-derived 35S promoter, the nopaline synthase gene promoter, and the octopine synthase gene. And a constitutive promoter derived from T-DNA of Agrobacterium tumefaciens, a tobacco-derived Par A gene promoter, a potato patatin gene promoter, and the like.

The term "terminator capable of functioning in plant cells" refers to a gene having a region involved in gene expression functions such as termination of transcription in plant cells.
Examples thereof include T-DNA-derived NOS terminator and potato patatin gene terminator.

"Having a functional form" means a gene encoding a chloroplast translocation signal peptide and cytochrome P.
In order for the fusion gene of 450 genes to be substantially transcribed and translated in plant cells, for example, from the 5 ′ end, (1) a promoter functional in plant cells, (2) a chloroplast translocation signal peptide sequence It means that the gene exists in the order of a fusion gene of the encoding gene and a non-plant-derived cytochrome P450 gene, and (3) a terminator capable of functioning in plant cells. The expression cassette thus constructed can be introduced into a plant cell by a usual method as described below. The plant (cell) used in the present invention
It is not particularly limited, and examples thereof include plants such as tobacco, tomato, potato, soybean, rice and wheat.

The constructed expression cassette can be used, for example, as a monocotyledonous plant such as rice, corn, sorghum, rye, barley, wheat and onion, and as dicotyledonous plants, legumes such as soybean, pea, green beans and alfalfa; tobacco. , Solanaceae plants such as tomato, eggplant and potato, Brassicaceae plants such as radish, rapeseed, cabbage and Arabidopsis; Cucurbitaceae plants such as melon, cucumber and pumpkin; Ceriaceae plants such as carrot and celery; Asteraceae such as lettuce The method of introduction into a plant varies depending on the plant species, but 1) a system utilizing infection with Agrobacterium, a soil bacterium, 2) protoplasting plant cells, and in the presence of polyethylene glycol (hereinafter referred to as PEG). PEG method of incorporating a plasmid into protoplasts by
3) An electroporation method in which a plasmid is incorporated into protoplasts by applying an electric pulse, 4), a microinjection method in which a plasmid is injected into protoplasts or plant cells by a microneedle, 5) a system for introducing a gene by the calcium phosphate method, 4) A usual method such as a system in which a gene is introduced into a plant tissue or a protoplast by a particle gun can be used. The amount of the plasmid of the present invention introduced into plant cells is, for example,
About 10 to about 100 pg per cell by the PEG method, about 0.5 to about 1 KV / cm by the electroporation method
About 10 to about 100p per cell by applying an electric pulse of
g, about 1 per cell by microinjection method
About pg is preferable. As the plant cells used for gene transfer, leaf disks, protoplast cells, cultured cells themselves, plant stems, seeds, etc. are appropriately selected and used according to various gene transfer methods. More specifically, for example, gene transfer into rice can be performed by the particle gun method. After threshing and hulling rice seeds, Linsmaier and auxin containing
Skoog (LS) medium (Physiol. Plant. 18, 100-127 (196
5)) Cultivate the cells and try to dedifferentiate the embryos. The dedifferentiated embryo is cut off and placed on LS agar medium containing auxin. The expression cassette constructed as described above and a plasmid for a drug resistance marker such as an antibiotic are mixed, and the gene is introduced into an embryo placed on an agar medium by a particle gun. The embryos are aseptically selected and cultured in an LS medium containing a drug such as an antibiotic and a plant hormone. Further, the expanded embryo-derived callus is selected on an LS medium containing an antibiotic to obtain a regenerated individual into which a drug resistance gene such as an antibiotic has been introduced. Genomic DNA is prepared from these selected plants, and plants in which the non-plant-derived cytochrome P450 monooxygenase gene is inserted are selected by PCR or Southern blot analysis.

A plant cell into which a gene has been introduced is usually
There is a selection marker that simultaneously introduced drug resistance genes such as kanamycin and hygromycin. Selection is performed based on this marker first, and then further selection is performed by analyzing the expression level of the gene of interest. be able to.

Then, the plant cell into which the constructed expression cassette has been introduced can be redifferentiated according to a method usually used in plant tissue culture technology to prepare a plant having the gene. Such a method
For example, SB Gelvin, RA Schilperoort and D.
PS Verma by Plant Molecular Biology
/ Manual (Plant Molecular Biology / Manual), Luer Academic Publishers (Kluwer Academic
Publishers), published in 1988, etc. For example, MS, White, B5, N6 containing plant hormones such as 1-naphthalene acetic acid and 6-benzyladenine,
Ordinary plant culture such as MS, White, B5, N6, NN agar medium containing hormone-free stems and leaves appeared from plant cells placed on a known agar medium used for ordinary plant culture such as NN agar medium It is transplanted to the well-known agar medium used for and rooted. After that, the plant can be obtained by repeating the shoot apical culture in the culture pot every month. The plants such as plants and plant cells thus obtained express non-plant-derived cytochrome P450 in the chloroplast and have excellent metabolic ability for drugs (particularly drugs that act on chloroplasts). As a result, the plant can be made resistant to the above compounds.

[0016]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the following examples, and ordinary modifications in the technical field of the present invention can be made. You can In the following, "Cytochrome P45
"0" is described as "CYP". The materials used in this example are as follows. That is, the oligonucleotide shown in the sequence listing is Model 39 of Applied Biosystems.
4 Synthesized by DNA Synthesizer. Restriction enzyme, T4 polynucleotide kinase, Klenow fragment, and
Takara Taq polymerase was purchased from Takara Shuzo. pfu polymerase was purchased from Stratagene. pBI 221
The plasmid vector was purchased from Clontech.
Anti-rat CYP1A1 antibody
Ig (G + L) -alkaline phosphatase antibody was purchased from Tagoimological.

Example 1 (Construction of plasmid vector pB2BS) As shown in FIG. 1, cauliflower mosaic virus 35
By changing the restriction enzyme recognition sites upstream and downstream of the S promoter (35S-P), a pB2BS plasmid vector useful for subsequent binary plasmid construction was constructed. First, pBI 221 plasmid vector was digested with Hind III and Pst I, and the synthetic linker A shown in Fig. 11-A was ligated to bind Hind III / Sph I / Pst I to Ssp.
I / Bam HI recognition site was changed. Then, the resulting pB2B was digested with Xba I and Sac I to delete the coding region of β-glucuronidase, and the synthetic linker B shown in FIG. 11-B was ligated to Xba I /
Replaced Sph I / Sac I with Ssp I / Bam HI recognition site, pB2BS
A plasmid vector was obtained. The linker is 500 pmol of synthetic DNA shown in SEQ ID NOs: 1, 2, 3 and 4.
The 5'end of each of these was phosphorylated by T4 polynucleotide kinase, and the paired SEQ ID NOS: 1 and 2 and SEQ ID NOS: 3 and 4 were heated at 65 ° C, and then at 25 ° C
The one annealed at was used. Using the Model 373 DNA Sequencer from Applied Biosystems, confirm that there is no mistake in the sequence of the inserted linker using Dye Deoxy * Te.
It was confirmed by the rminator method.

Example 2 (Construction of Binary Plasmid) Structures of the constructed eight kinds of binary plasmids are shown in FIG. Moreover, each construction method is described below. 1) Construction of pIQVA As shown in Fig. 3, tobacco Nicotiana plumbaginifolia
Ribulose 1, 5-bisphosphate carboxylase / oxygenase small subunit (RbcS) of chlorophyll transfer signal peptide (TP) Xba I immediately upstream of the initiation codon of cDNA
To create the recognition site, Poulsen et al.,
Mol. Gen. Genet., 205: 193-200 (1986), plasmid p8Bp18 containing RbcS promoter and TP.
As a template, using the primers shown in SEQ ID NO: 5 and SEQ ID NO: 6 with pfu polymerase for 94 ° C for 5 minutes, 94 ° C / 42 ° C / 75 ° C for 1 minute / 30 seconds / 2.5 minutes.
Perform PCR amplification under the condition of 30 cycles (PCR 1),
A bp fragment was obtained. I confirmed that this sequence is correct. This was inserted into plasmid vector pB2BS digested with Xba I and Pst I to obtain plasmid pB2TP. In addition, in order to construct a sequence encoding Sph I recognition site and glutamine, valine, and tryptophan upstream of the cDNA encoding rat CYP1A1 microsomal membrane anchor sequence, Oeda et al. (DNA, 4 ( 3): 20
3-210 (1985)) described rat CYP1A1
Using the plasmid pAMC1 containing the cDNA as a template, SEQ ID NO: 7,
And 94 ° C for 5 minutes at 94 ° C / 42 ° C / 75 ° C with pfu polymerase using the primer shown in SEQ ID NO: 8.
PCR for 30 minutes / 30 seconds / 2.5 minutes for 30 cycles
Amplification was performed (PCR 2) to obtain a fragment of about 240 bp. After all, I confirmed that this sequence was correct. This is S
It was inserted into pB2TP plasmid digested with ph I and Pst I to obtain plasmid pB2TPQV. On the other hand, about 1,400 pAMC1 digested with upstream Pst 1 (1) and Hind III
The bp fragment was similarly digested with Pst I and Hind III to give pB
It was inserted into 2TPQV to obtain plasmid pBQVA. The TP of pBQVA thus obtained and the region encoding rat CYP1A1 were excised with Xba I and Sac I, and Xba I
And Sac I digested binary vector pIG121-HM
(Onouchi et al., Nucleic. Acid Res., 19: 6373 -6378
(1991))
pIQVA was obtained.

2) Construction of pIQVR As shown in FIG. 4, Sakaki et al. (Biochemistry, 3
3: 4933 -4939 (1994)) described rat CYP.
Plasmid pAFCR containing 1A1 and the yeast NADPH-P450 reductase fusion gene was digested with Pst 1 (1) and Hind III, and the resulting approximately 3400 bp fragment was similarly digested with Pst I and Hid.
Inserted into pB2TPQV digested with nd III, plasmid pBQV
Got R. Next, pBQVR was cut out with Xba I and Sac I and inserted into the binary vector pIG121-HM which was similarly digested with Xba I and Sac I to obtain the binary plasmid pIQVR.

3) Construction of pIΔQA As shown in FIG. 5, the microsomal membrane anchor sequence of 31 amino acid residues of rat CYP1A1 was deleted and Sph I was added to it.
In order to prepare a sequence encoding the recognition site and glutamine, plasmid pAMC1 containing rat CYP1A1 cDNA was used as a template, and the primers shown in SEQ ID NO: 7 and SEQ ID NO: 9 were used to pfu polymerase at 94 ° C. for 5 minutes. 94
PCR amplification was performed under the conditions of 30 ° C for 1 minute / 30 seconds / 2.5 minutes at ℃ / 42 ° C / 75 ° C (PCR 3) to obtain a fragment of about 140 bp. I confirmed that this sequence is correct.
Insert this into pB2TP digested with Sph I and Pst I,
The plasmid pB2TPΔQ was obtained. On the other hand, pAMC1
About 1,400 b obtained by digestion with Pst 1 (1) and Hind III
The p fragment was similarly digested with Pst I and Hind III to give pB2T.
It was inserted into PΔQ to obtain plasmid pBΔQA. The TP of pBΔQA thus obtained and the region encoding rat CYP1A1 were excised with Xba I and Sac I, and Xba I
And Sac I digested binary vector pIG121-HM
A binary plasmid pIΔQA was obtained by inserting

4) Construction of pIΔQR As shown in FIG. 6, rat CYP1A1 and yeast NAD
Plasmid pAFCR containing PH-P450 reductase fusion gene
About 3,400 bp obtained by digestion with Pst 1 (1) and Hind III
Fragment of pB2T digested with Pst I and Hind III in the same manner.
It was inserted into PΔQ to obtain plasmid pBΔQR. Then pBΔD
The binary plasmid pIΔQR was obtained by excising QR with Xba I and Sac I and inserting it into the binary vector pIG121-HG which was also digested with Xba I and Sac I.

5) Construction of pIRQVA As shown in FIG. 7, the plasmid p8Bp18 containing the RbcS promoter and TP was digested with Bam HI and Sph I, and the resulting fragment of about 1,300 bp was similarly digested with Bam HI and Sph I. The plasmid pBRQVA was obtained by inserting it into the plasmid pBQVA digested with. Next, pBRQVA was digested with Bam HI and the binary vector pIG121-HM was digested with Hind III, and each end was blunted with Klenow fragment. They were digested with Sac I and ligated to obtain the binary plasmid pIRQVA.

6) Construction of pIRQVR As shown in FIG. 8, plasmid p8Bp18 was digested with Bam HI and Sph I, and the resulting fragment of about 1,300 bp was transformed into plasmid pBQVR similarly digested with Bam HI and Sph I. The plasmid pBRQVA was obtained by insertion. Next, pBRQVA is Bam HI, the binary vector pIG121-
HM was digested with Hind III and each end was blunted with Klenow fragment. Digest them with Sac I,
By ligation, the binary plasmid pIRQVR was obtained.

7) Construction of pIRΔQA As shown in FIG. 9, plasmid p8Bp18 was digested with Bam HI and Sph I, and the resulting fragment of about 1,300 bp was similarly digested with Bam HI and Sph I to obtain plasmid pBΔQA. The plasmid pBRΔQA was obtained by inserting the plasmid into pBRΔQA. Next, pBRΔQA was added to Bam HI, the binary vector pIG121-
HM was digested with Hind III and each end was blunted with Klenow fragment. Digest them with Sac I,
By ligation, the binary plasmid pIRΔQA was obtained.

8) Construction of pIR ΔQR As shown in FIG. 10, the plasmid p8Bp18 was digested with Bam HI and Sph I, and the resulting fragment of about 1,300 bp was similarly digested with Bam HI and Sph I to obtain plasmid pBΔQR. The plasmid pBRΔQR was obtained by inserting the plasmid into pBRΔQR. Next, pBRΔQR is added to Bam HI, the binary vector pIG121-
HM was digested with Hind III and each end was blunted with Klenow fragment. Digest them with Sac I,
By ligation, the binary plasmid pIRΔQR was obtained.

Example 3 (Introduction of binary plasmid into Agrobacterium) Binary plasmids pIΔQA and pIΔ prepared in Example 2
QR, pIQVA, pIQVR, pIR ΔQA, pIR ΔQR, pIRQVA, pI
Each of RQVR and binary vector pIG121-HM was prepared using a Bio-Rad 2 mm wide cuvette.
Electroporation (Gene P at 0 W, 2.5 kvolts, 25 mF)
ulser ^TM , Bio-Rad, Inc.
It was introduced into the cterium tumefaciens EHA101 strain. Agrobacterium carrying each binary plasmid was selected on YEB agar medium containing 50 mg / ml kanamycin and 50 mg / ml hygromycin. PCR reaction (94 ° C, 5 minutes, 94 ° C, 5 minutes, using CYP1A1 or a primer that anneals specifically to the yeast NADPH-P450 reductase gene using the plasmid prepared from it as a template
Their presence was confirmed by performing 94 ℃ / 60 ℃ / 74 ℃ for 1 minute / 1 minute / 2 minutes 30 cycles).

Example 4 (Infection of plants) Infection of plants with Agrobacterium was carried out by a leaf disk method using leaves of Nicotiana tabacum, which was germinated aseptically. About 5 mm square leaflets were soaked in Agrobacterium solution diluted 100-fold with LS liquid medium for about 3 hours in the dark, and then 0.5 mM 1-naphthalene acetic acid and 4 mM 6-benzyl were added. LS agar medium containing adenine (L
S-NB medium) and co-cultured in the dark at 25 ° C for 2 days. Infected tissue was 50 mg / ml kanamycin,
Hygromycin 50 mg / ml and 300 mg / ml
Was transferred to the LS-NB medium containing cefotaxime, and the medium was changed every week. Kanamycin and hygromycin-resistant foliage that appeared after about 1 month were transferred to hormone-free LS agar medium containing 50 mg / ml of kanamycin. After that, subculture was carried out by shoot apical culture every 30 days.

Example 5 (Preparation and PCR of plant chromosomal DNA
Confirmation of target gene by method) Tobacco leaf chromosomal DNA is ISOPL of Nippon Gene Co., Ltd.
Prepared using ANT. Place 0.1 g of leaves that had been cut into 3 mm squares beforehand into a 1.5 ml microcentrifuge tube, freeze in liquid nitrogen, and then use 300 μl of ISOPLAN.
T Solution I was added and the tissue was disrupted with a glass rod. Subsequent extraction of DNA is performed according to the ISOPLANT protocol, and TE is adjusted to a final concentration of 0.5 mg / ml.
Suspended in water. pIG121-HM, pIΔQA, pIQVA, pIRΔQA,
Also, CYP1A1 cD was found in the recombinant plant into which pIRQVA was introduced.
For the recombinant plants in which pIΔQR, pIQVR, pIRΔQR and pIRQVR were introduced into NA, primers that specifically anneal to the yeast NADPH-P450 reductase gene were used.
PCR reaction with Taq polymerase (94 ℃, 5 minutes, 94 ℃)
/ 60 ℃ / 74 ℃ for 1 minute / 1 minute / 2 minutes for 30 cycles) to obtain the target chromosomal DNA of all recombinant plants.
It was confirmed that the DNA was incorporated. The results are shown in Table 1.

Example 6 (Isolation of intact chloroplasts) The isolation of intact chloroplasts from the leaves of tobacco plants was carried out by the method of Bartlette et al. (Method in Chloroplast Mo
lecular Biology, pp.1081-1091 (1982) Elsevir, Amst
erdam). The leaves of the plant were soaked in an LS liquid medium at a wet weight of 3 g and protected from light for 1 hour.
Leaf pieces excluding these main veins were cut into 5 mm squares beforehand, and 12 ml of 50 mM HEPES-KOH (pH 7.5), 1 mM magnesium chloride, 1 mM manganese chloride, 2 mM EDTA, 0.33
The cells were disrupted in a disruption buffer containing M sorbitol and 5 mM ascorbic acid. A Polytron homogenizer was used for crushing, and crushing was performed for 5 seconds at level 3.5. 4 pieces of crushed liquid
After filtering through Miracloth overlaid on one another, it was centrifuged at 2,200 g for 30 seconds. The obtained precipitate was suspended in 1 ml of crushing buffer, overlaid with 36 ml of 5 to 45% Percoll linear density gradient solution, and centrifuged at 10,000 rpm for 10 minutes using Hitachi RPRS-10 Swing Rotor. . Of the two separated bands, the lower band was recovered. Add 3 volumes of 50 mM HEPES-KOH (pH 8.0) and 0.
Add wash buffer containing 33 M sorbitol at 3,300 g
It was centrifuged for 15 seconds to remove the Percoll remaining in the solution. This procedure was repeated several times, and the final precipitate was added to a suspension buffer containing 50 mM HEPES-KOH (pH 7.5), 2 mM EDTA, and 0.33 M sorbitol so that the amount of protein was 20 μg per 1 ml solution. Resuspended. The content of intact chloroplast was calculated by observation with an optical microscope, and the fractions with the value of 80% or more were used below.

Example 7 (Analysis of Expressed / Transported Proteins by Western Blot) The intact chloroplasts isolated from the leaves of the recombinant tobacco plant according to Example 6 were solubilized with a buffer containing 4% SDS. After that, it was subjected to SDS-polyacrylamide gel electrophoresis.
The proteins in the gel were then electrophoretically transferred to nitrocellulose membranes. Incubate the nitrocellulose membrane in PBS buffer containing 1% milk protein for 1 hour, wash it with PBS buffer containing 0.05% Tween 20 and 0.1% milk protein, and then add PBS buffer containing 0.1% milk protein. Anti-rat CYP1A1 diluted 1,000 times with the solution
Incubated with antibody for 1 hour. After washing the nitrocellulose membrane with PBS buffer containing 0.05% Tween 20 and 0.1% milk protein for 3 times for 15 minutes each,
With anti-goat Ig (G + L) -alkaline phosphatase antibody diluted 1,000-fold with PBS buffer containing 0.1% milk protein.
Incubated for 1 hour. Perform the same washing operation as above
Repeat 3 times, then add 10 mM Tris-HCl (pH: 100 mM sodium chloride and 5 mM magnesium chloride).
9.6) The plate was washed twice for 15 minutes each with a buffer solution. Bound antibody was detected by adding nitroblue tetrazolium and 5-bromo 4-chloro 3-indolyl phosphate. As a result, it was found that all recombinant tobacco plants reacted with the anti-rat CYP1A1 antibody at about 60 kilodaltons.
55 kilodalton (rat CYP1A1 expressing strain) or 13
A band was detected at the position of 0 kilodalton (rat CYP1A1 / yeast NADPH-P450 reductase-expressing strain), confirming that the target enzyme was produced in the recombinant tobacco plant and further transported to the chloroplast. Was done. Based on their size, of the two bands observed in the rat CYP1A1 expressing strain, about 60 kilodaltons are fusion proteins of TP and CYP1A1, and 55 kilodaltons are processed by the signal peptidase present in the chloroplast envelope. It is speculated that this is a mature protein that has undergone.

Example 8 (Metabolism of Drug by Recombinant Tobacco Plant-Chloroplast Fraction-) 2,200 prepared from leaves of the recombinant tobacco plant of Example 6
The g-precipitate was used as a chloroplast fraction, and the 7-ethoxycoumarin O-deethylation activity was measured in the dark. Add NADPH to the chloroplast fraction to a final concentration of 0.4 mM, and keep it at 37 ° C in the dark.
Pre-incubated for 10 minutes. Add 7-ethoxycoumarin to a final concentration of 0.4 mM, and add 0, 2
After 5 and 10 minutes, 500 ml of the reaction solution was collected, and 15 ml of 30% trichloroacetic acid was added to stop the reaction, followed by centrifugation. Extract the supernatant with 1 ml of chloroform and add another 2 ml of 0.01 N NaOH / 0.1 M NaCl to the chloroform layer.
Extracted. Excitation of the water layer at 366 nm, resulting
The amount of 7-hydroxycoumarin, a hydroxide of 7-ethoxycoumarin, produced was calculated by measuring the fluorescence intensity at 461 nm. Under all conditions, the protein weight was 0.9 to 1.5 mg containing 3 m of chloroplast fraction.
l of reaction solution was used. Recombinant tobacco plant SR-1 / pIG12
1-HM, SR-1 / pIQVA, SR-1 / pIΔQA, SR-1 / pIΔQR, SR
The results of the activity measurement of -1 / pIRΔQA and SR-1 / pIRQVR are shown in Tables 1 to 3. 7-Ethoxycoumarin is a control strain SR-1 / pIG in which only a binary vector was introduced.
Although it was hardly metabolized by 121-HM, the transformed tobaccos into which the gene encoding the fusion enzyme of CYP1A1 and CYP1A1 / NADPH-P450 reductase was introduced were all about 3 to 15 times more active than the control strain. showed that.

[0032]

[Table 1] ─────────────────────────────────── 7-Hydroxycoumarin production amount Plasmid strain mg protein ¹ mg Chlorophyll ² PCR amplification ³ ─────────────────────────────────── pIG121-HM A1 0.87 0.14-A3 1.43 0.10-B1 0.92 0.12-B1-3 1.68 0.18-pIQVA A2 2.33 0.13 + B2 2.42 0.14 + G1 2.15 0.11 + H1 2.58 0.07 + H3 3.33 0.13 + I 3.06 0.23 + R1 9.83 0.72 + S1 1.58 0.06 + T1 14.63 1.98 + pIΔQA A 5.77 0.29 + B1 4.76 0.63 + B2 4.86 0.27 + D1 6.72 0.35 + E1 7.47 0.45 + J2 15.61 0.39 + pIΔQR A1 5.24 0.65 + A2 5.95 0.36 + A3 10.60 3.81 + B2 5.63 0.24 + ────────── ──────────────────────────

[0033]

[Table 2] ─────────────────────────────────── 7-hydroxycoumarin production amount Plasmid strain mg protein ¹ mg Chlorophyll ² PCR amplification ³ ─────────────────────────────────── pIΔQR B3-2 10.00 0.64 + C1 5.23 0.18 + C2 3.42 0.26 + C2-1 2.80 0.33 + C2-2 4.88 0.35 + D1 5.12 0.25 + E1 15.90 0.62 + E2 6.41 0.19 + E3-1 10.00 1.18 + E3-2 3.33 0.29 + H1 2.03 0.23 + H2 6.30 0.40 + H3 3.05 0.09 + P1 4.22 0.16 + Q1 5.79 0.46 + V1 2.16 0.07 + W1 1.81 0.05 + Z 7.27 0.29 + pIR ΔQA A1 2.19 0.10 + B4 3.03 0.12 + C2-3 2.56 0.13 + D2 4.81 0.38 + E1 3.13 0.17 + ── ────────────────────────────────

[0034]

[Table 3] ─────────────────────────────────── 7-hydroxycoumarin production amount Plasmid strain mg protein ¹ mg Chlorophyll ² PCR amplification ³ ─────────────────────────────────── pIRQVR A4 9.04 0.32 + B2 4.31 0.16 + C1 7.50 0.36 + H1 1.32 0.08 + J1 1.76 0.11 + R1 0.72 0.10 + T1 12.19 0.52 + a1 14.71 1.03 + b1 14.29 0.60 + c1 7.11 1.44 + f1 2.42 0.18 + ────────────── ───────────────────── 1; pmol 7-hydroxycoumarin / min / mg protein 2; pmol 7-hydroxycoumarin / min / mg chlorophyll 3; + is amplified Was done, -indicates that it was not amplified

Example 9 (Metabolism of Drugs by Recombinant Tobacco Plants-Intact Chloroplasts) Using the intact chloroplasts isolated from the leaves of the recombinant tobacco plants according to Example 6, light irradiation and The 7-ethoxycoumarin O-deethylation activity was measured under light-shielded conditions. Under light conditions, the chloroplast fraction had a light intensity of 150 μmol photons / m ² / s and light of 400 nm to 700 nm (65
(Having a peak from 0 to 680 nm) at 25 ° C
After preincubating for 10 minutes at 7, the reaction was initiated by adding 7-ethoxycoumarin to a final concentration of 0.4 mM. Under light-shielded conditions, the chloroplast fraction was preincubated in the dark at 25 ° C for 10 minutes, and then the reaction was initiated by adding 7-ethoxycoumarin to a final concentration of 0.4 mM. 0, 2, and 5 minutes after starting the reaction, aliquot 500 μl of the reaction mixture,
By performing the same operation as in Example 8, the production amount of 7-hydroxycoumarin was calculated. In order to investigate the effect of the photosynthetic electron transfer inhibitor 3,4-dichlorophenyl 1,1-dimethylurea (DCMU), DCMU was added so that the final concentration was 50 μM during preincubation, and the same light was added. The activity was measured under irradiation conditions. In addition,
Protein weights of 25 to 7 under all conditions
3 ml of reaction solution containing 5 μg of intact chloroplast was used. Recombinant tobacco plant SR-1 / pIG121-HM (control strain)
, SR-1 / pIΔQA and SR-1 / pIΔQR results are shown in Table 4.
~ 5. As a result of the measurement, 7-ethoxycoumarin was hardly metabolized by SR-1 / pIG121-HM into which only the binary vector was introduced under any conditions. SR-1 / pI which is a CYP1A1 expressing strain under light irradiation
ΔQVA showed about 2-3 times higher activity than the control strain, while SR-1 / pIΔQVR, which is a strain expressing the gene encoding the fusion enzyme of CYP1A1 / NADPH-P450 reductase, showed about 10 to 16 times higher activity than the control strain. It showed high activity. On the other hand, under shading conditions, SR-1 / pIΔQVA hardly metabolized 7-ethoxycoumarin, showing no difference from the control strain. SR-1 / pIΔQVR showed a value slightly higher than that of the control strain, but was 1/4 or less of that under the light irradiation condition. In addition, in the reaction system to which DCMU was added, the values were as low as those under the light-shielding condition.

[0036]

[Table 4] ─────────────────────────────────── 7-hydroxycoumarin production amount Plasmid strain Reaction conditions nmol / Min / mg protein ─────────────────────────────────── pIG121-HM A1 light irradiation 0.45 light shielding 0.40 light Irradiation + DCMU 0.31 pIΔQA A1 Light irradiation 1.68 Light shielding 0.35 Light irradiation + DCMU 0.46 B2 Light irradiation 1.18 Light shielding 0.43 Light irradiation + DCMU 0.31 E1 Light irradiation 1.03 Light shielding 0.31 Light irradiation + DCMU 0.67 J1 Light irradiation 1.36 Light shielding 0.31 Light irradiation + DCMU 0.32 pIΔQR A3 light irradiation 6.62 light shielding 0.10 light irradiation + DCMU 3.14 B3-2 light irradiation 5.19 light shielding 0.92 light irradiation + DCMU 0.48 ─────────────────────────── ─────────

[0037]

[Table 5] ─────────────────────────────────── 7-Hydroxycoumarin production amount Plasmid strain Reaction conditions nmol / Min / mg Protein ─────────────────────────────────── pIΔQR E1 Light irradiation 6.20 Shading 0.76 Light irradiation + DCMU 1.24 H2 Light irradiation 7.70 Shading 2.11 Light irradiation + DCMU 4.06 ────────────────────────────────────

[0038]

[Sequence list]

 SEQ ID NO: 1 Sequence Length: 50 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence: TCTAGAGTAC TGCATGCGGT ACCTGCAGTC GACAAGCTTC CCGGGAGCTC 50

SEQ ID NO: 2 Sequence Length: 43 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence: AGGGCCCTTC GAACAGCTGA CGCGTCCATG GCGTACGTCA TGA 43

SEQ ID NO: 3 Sequence Length: 24 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence: AGCTAGAATA TTGGATCCAT TGCA 24

SEQ ID NO: 4 Sequence Length: 16 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence: TACCTAGGTT ATAAGA 16

SEQ ID NO: 5 Sequence length: 41 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence: AAAAAATCTA GAATGGCTTC CTCAGTTCTT TCCTCTGCAG C 41

SEQ ID NO: 6 Sequence length: 32 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence: AAAAAACTGC ATGCATTGCA CTCTTCCGCC GT 32

SEQ ID NO: 7 Sequence Length: 47 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence: AAATGCATGC AGGTGTGGCC TTCTGTGTAT GGATTCCCAG CCTTCAC 47

SEQ ID NO: 8 Sequence Length: 26 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence: ATACGGATCT GCAGCACGTC CCCATA 26

SEQ ID NO: 9 Sequence Length: 39 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid, Synthetic DNA Sequence: AAATGCATGC AGACAAGAAC CTGGGTTCCC AAAGGTCTG 39

SEQ ID NO: 10 Sequence length: 57 Sequence type: Amino acid Sequence: 1 5 10 15 Met Ala Ser Ser Val Leu Ser Ser Ala Ala Val Ala Thr Arg Ser Asn 20 25 30 Val Ala Gln Ala Asn Met Val Ala Pro Phe Thr Gly Leu Lys Ser Ala 35 40 45 Ala Ser Phe Pro Val Ser Arg Lys Gln Asn Leu Asp Ile Thr Ser Ile 50 55 Ala Ser Asn Gly Gg Arg Val Gln Cys

[Brief description of drawings]

FIG. 1 is a diagram showing a method for constructing a plasmid vector pB2BS.

FIG. 2 is a diagram showing structures of various binary plasmids.

FIG. 3 is a diagram showing a method for constructing a binary plasmid pIQVA.

FIG. 4 is a diagram showing a method for constructing a binary plasmid pIQVAR.

FIG. 5 is a diagram showing a method for constructing a binary plasmid pIC.

FIG. 6 is a diagram showing a method for constructing a binary plasmid pIΔQR.

FIG. 7 is a diagram showing a method for constructing a binary plasmid pIRQVA.

FIG. 8 is a diagram showing a method for constructing a binary plasmid pIRQVR.

FIG. 9 is a diagram showing a method for constructing a binary plasmid pIRΔQA.

FIG. 10 is a diagram showing a method for constructing a binary plasmid pIRΔQR.

FIG. 11 shows the nucleotide sequences of synthetic linkers and PCR primers. A to G are listed in the sequence listing as SEQ ID NOS: 1 to 7, respectively.

[Explanation of symbols]

Sh: Cleavage site by restriction enzyme Sph I Xa: Cleavage site by restriction enzyme Xba I Bm: Cleavage site by restriction enzyme Bam HI Pt: Cleavage site by restriction enzyme Pst I Hd: Cleavage site by restriction enzyme Hind III Sc: Restriction enzyme Sac Cleavage site by I NOS-P: Nopaline synthase promoter NOS-T: Nopaline synthase terminator 35S-P: Cauliflower mosaic virus 35S promoter RbsS-P: Ribulose bisphosphate carboxylase / oxygenase small subunit promoter NPT II: Neomycin phosphotransferase II Coding region HPT: Hygromycin phosphotransferase coding region GUS: b-glucuronidase coding region CYP1A1: Rat cytochrome P450 (CYP1A1) coding region cDNA Reductase: yeast NADPH-P450 reductase coding region Offspring TP: cDNA for chloroplast translocation signal of tobacco RbcS RA: Arginine and alanine RB connecting CYP1A1 and reductase fusion enzyme, LB: Border region required for transfer of T-DNA to plant genome Gray box: Tobacco RbcS Chloroplast transfer signal Shading: Rat cytochrome P450 (CYP1A1) microsomal membrane anchor Number below box: Rat cytochrome P450 (CYP1)
A1) and the amino acid number from the N-terminus of each reductase

Continuation of front page (51) Int.Cl. ⁶ Identification code Office reference number FI technical display area C12R 1:01)

Claims (6)

[Claims] (1) A promoter capable of functioning in a plant cell, (2) a fusion gene of a gene encoding a chloroplast translocation signal peptide sequence and a non-plant-derived cytochrome P450 gene, and (3) a plant cell An expression cassette, which comprises a terminator capable of functioning in a functional form. 2. The expression cassette according to claim 1, wherein the chloroplast translocation signal peptide sequence is the amino acid sequence represented by SEQ ID NO: 10. 3. The expression cassette according to claim 1, wherein the non-plant-derived cytochrome P450 gene is derived from a mammal. 4. A microorganism containing the expression cassette according to claim 1. 5. A plant transformed by introducing the expression cassette according to claim 1. 6. A promoter capable of functioning in a plant cell, (2) a fusion gene of a gene encoding a chloroplast translocation signal peptide sequence and a non-plant-derived cytochrome P450 gene, and (3) a plant cell. A method for imparting a drug-metabolizing ability in a plant, which comprises introducing into a plant cell an expression cassette having a functional terminator capable of functioning in the plant, and expressing a non-plant-derived cytochrome P450 in the chloroplast.