JP2955644B2 - Rice CatB gene promoter - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to production of useful substances or breeding of useful plants using a plant gene promoter. More specifically, the present invention relates to production of useful substances or breeding of useful plants using a rice catalase gene B (hereinafter referred to as CatB) promoter gene.

[0002]

2. Description of the Related Art Conventionally, various promoters used for the expression of plant genes are known. For example, an Agrobacterium genus T-DNA promoter, a nopaline synthetase (NOS) promoter, an octopine promoter and the like are known. The cauliflower mosaic virus (CaMV) promoter is also widely used as a plant gene promoter. However, its activity is
There is a problem that it is low for practical use.

[0003] A state in which the concentration of reactive oxygen species (superoxide, hydrogen peroxide, hydroxyl radical, singlet oxygen, etc.) in a cell is increased is called oxidative stress. This includes peroxidation, ultraviolet and radiation, abnormalities in the electron transport system of cytochromes, abnormal increases in peroxisomes, abiotic factors such as high and low temperatures, chemical substances, and air pollutants such as ozone and sulfur dioxide. The “oxidation state” that occurs and the “antioxidant defense mechanism (superoxide dismutase (SO
D), catalase (CAT), peroxidase, vitamins E, C and A, etc.).

[0004] It has long been known that eukaryotes increase SOD and CAT activity when exposed to oxidative stress. In addition, it has been observed that when SOD or CAT is induced in advance, cells show some resistance to oxidative stress.

[0005] It is known that oxidative stress also occurs in plants. For example, various conditions that inhibit photosynthesis (low temperature under light irradiation, herbicide treatment, etc.) cause abnormal generation of reactive oxygen species due to photosynthetic reaction pathways, etc., and plants are exposed to low temperatures even in dark places. When there is an increase in the concentration of reactive oxygen species such as hydrogen peroxide, active oxygen species are involved in the mechanism of action of herbicides (paraquat, etc.) and ultraviolet rays, and when drought stress and heavy metals cause oxidative stress, It is considered.

[0006] In order to combat oxidative stress, plants carry out various defense reactions such as an antioxidant defense enzyme system cooperating to eliminate these reactive oxygen species. Recently, experimental results have been reported that strongly suggest the involvement of catalase in cold tolerance.

[0007] On the other hand, it is known that plant cells rapidly generate reactive oxygen species called Oxidative burst on the surface thereof in response to biological stimuli such as infection with pathogenic bacteria. This phenomenon has traditionally been interpreted as attacking pathogens. However, it has recently been found that this phenomenon also functions as a signal for starting a cascade that induces the expression of various defense genes inside cells. Hydrogen peroxide has also recently been shown to be involved in defense protein induction during plant virus infection. These new findings are drawing attention as suggesting that catalase, which controls the concentration of reactive oxygen species such as hydrogen peroxide, is involved in the control of signal transduction.

As one of the mechanisms, induction of catalase (Cat3) gene expression by accumulation of a small amount of hydrogen peroxide during the training process has been reported as a mechanism of acquiring cold tolerance by chilling acclimation of corn seedlings. In addition, PR protein (Pa
As a mechanism of inducing thogenesis-related proteins, it has been reported that salicylic acid inhibits catalase and increases the concentration of hydrogen peroxide in cells to induce PR proteins. These suggest that the reactive oxygen species itself may be directly involved in the induction of reactive oxygen scavenging genes in plant cells, and that the scavenging enzymes that regulate the concentration of hydrogen peroxide play a role in signal control. May have.

[0009] On the other hand, in rice, a correlation between low-temperature germination of rice seeds and high catalase activity in the seeds has been suggested (Yata et al., 1990 Annual Meeting of the Japanese Society of Agricultural Chemistry). It has also recently been revealed that catalase gene is specifically expressed when the oxygen partial pressure is rapidly increased by exposing rice seedlings germinated in water under hypoxic conditions to the air. (Morita et al., Proceedings of the 1994 Plant Physiological Society, p. 115).

[0010] Therefore, the rice catalase gene promoter is highly likely to be responsive to various signals, and if a promoter having high activity can be found, it is considered to be very useful. For example, in combination with the catalase structural gene, it is introduced into plants with low cold resistance (such as indica rice or vegetables) to impart cold resistance. In combination with the catalase structural gene, the most cultivated japonica in Japan Introduce to rice seeds to enhance cold resistance, in combination with catalase structural gene, to accumulate catalase in high concentration in seeds, increase low-temperature germination or underwater germination, and make rice suitable for direct sowing It is possible to suppress photorespiration and enhance photosynthesis by introducing a gene encoding peroxisomal-localized catalase into C3 plants in combination with a catalase structural gene and accumulating more in peroxisomes. Genes known to be expressed during plant pathogen infection (P Protein, chitinase, etc.), a DNA fragment containing the exon portion of the rice catalase gene is inserted downstream in the antisense direction and introduced into a plant to suppress the expression of the endogenous catalase gene. There is a possibility that the expression of defense-related genes can be enhanced by increasing the concentration of moving hydrogen peroxide, and a general-purpose vector that can stably express the target gene with high efficiency in transformants It is expected to have usefulness such as expected development.

[0011] Therefore, from the rice gene, the activity is high,
If a promoter that can be used practically can be obtained,
In addition to breeding of crops such as rice, a general-purpose vector for substance production in other plant cells can be developed, and it is considered that this can greatly contribute to substance production using plant cells.

[0012] Catalase A (CatA), CatB, and catalase C (CatC) genes are known as structural genes of rice catalase, and each has a sequence with high homology. The sequence of CatB cDNA is known (Plant Physiol. (1994) 105: 1015-10).
16).

However, at present, the promoters of these genes have not been analyzed at all, and the positions of the promoters have not been specified.

[0014]

DISCLOSURE OF THE INVENTION The object of the present invention is to solve the above problems and to breed in rice and other crops, and has an activity much higher than that of the conventional CaMV promoter. An object of the present invention is to provide a plant gene promoter.

[0015]

Means for Solving the Problems The present invention has been completed by finding that the promoter activity of catalase B (CatB) gene of rice is very high.

The present invention relates to a sequence represented by SEQ ID NO: 1,
Or a sequence having a part thereof and having a promoter activity equivalent to that of the sequence of SEQ ID NO: 1.

The present invention relates to a sequence represented by SEQ ID NO: 2,
Alternatively, the present invention relates to a rice CatB gene promoter which is a sequence having a part thereof and which has a promoter activity equivalent to the sequence of SEQ ID NO: 2. This sequence is a sequence containing the first intron of the rice CatB structural gene.

The present invention relates to a plant expression cassette having the rice CatB gene promoter. In a preferred embodiment, the cassette comprises the sequence of SEQ ID NO: 2. In a further preferred embodiment, the plant expression cassette of the present invention comprises a nopaline synthetase gene terminator sequence.

Furthermore, the present invention relates to a recombinant plasmid comprising the rice CatB gene promoter and a foreign gene operably connected.

[0020] The present invention also relates to a plant cell and a plant transformed with the above-mentioned recombinant plasmid.

The present invention also relates to a method for producing a recombinant protein, comprising the following steps: a step of preparing a recombinant plasmid having a rice CatB gene promoter; a step of transforming a plant cell with the recombinant plasmid; Culturing the transformed plant cell.

Further, the present invention relates to a method for producing a recombinant plant, comprising the following steps: a step of preparing a recombinant plasmid having a rice CatB gene promoter; a step of transforming a plant cell with the recombinant plasmid; Culturing the transformed plant cell.

[0023]

BEST MODE FOR CARRYING OUT THE INVENTION The rice CatB gene promoter of the present invention has the sequence shown in SEQ ID NO: 1.

The rice CatB gene promoter can be screened from a rice genomic library. CLONTECH Laboratories In
c., Palo Alto, CA), a rice genomic DNA library (RICE Genomic Library) can be used.

As the probe, rice CatA cDNA isolated by the present inventors can be used. Rice Cat
Since the A structural gene and the rice CatB structural gene have portions similar in structure, a common portion thereof can be used.
The nucleotide sequence of the genomic CatA gene can be found in the Abstracts of the 1992 Annual Meeting of the Japanese Society of Agricultural Chemistry: Journal of the Japanese Society of Agricultural Chemistry 66
(3), 488 (1992)).

First, a rice genomic library is prepared using phage λ to form plaques. The plaque is transferred to a membrane such as nitrocellulose according to a conventional method, and hybridized with a labeled probe. After completion of hybridization, the plate is washed and subjected to autoradiography to prepare DNA from phage confirmed to be hybridized.

The phage DNA thus prepared is digested with a suitable restriction enzyme, and the digest is separated by agarose gel electrophoresis. The separated DNA fragment is transferred to a nylon membrane, the above probe is hybridized, and screening is performed based on the difference between the signal intensity and the band pattern.

The signal intensity and band pattern are Ca
A clone almost identical to that of the tA gene is considered to correspond to the CatA gene. Therefore, a clone whose band pattern only partially matches may be a CatA gene-deficient clone or a clone of another catalase gene other than CatA.
Using atA cDNA as a probe, hybridization is performed under the following loose and severe conditions, and the patterns are compared. By comparing this pattern,
It is presumed that it is another catalase gene other than CatA. Determine the method such as PCR or the base sequence,
It can be confirmed that they are different catalase genes.

Thus, genomic catalase genes other than CatA can be isolated. In the present application, two different genomic catalase genes, Ca
tB and CatC were isolated.

The promoter region can be specified based on the information of these sequences. When having an intron, the promoter sequence may include not only the promoter itself but also a region such as the first intron.

When the CatB gene promoter sequence is specified, the sequence can be cut out and inserted into a plant expression vector. The activity of the tethered promoter is determined by connecting a gene encoding an appropriate enzyme downstream of the promoter and measuring the expression of the gene, for example, by measuring the enzyme activity. When using a plant as a host,
For example, using a plasmid such as pBI221,
It is common to measure the expression of β-glucuronase (GUS) as an index.
A method of measuring the expression of S can be applied.

GUS activity was determined by the method of Jefferson et al. (EMBO
J 6: 3901-3907 (1987)). That is, a protoplast extract equivalent to 50 μg in protein amount and 25 μl of 20 mM 4-methyl um
belliferyl glucuronide (4MUG), an extraction buffer, and 100 μl methanol (Kosugi et al., Plant
Sci 70: 133-140 (1990)) to make a total volume of 500 μl and incubate at 37 ° C. After 0 hour and 2 hours, 200 μl was collected from the reaction solution and 0.2 M
The reaction is stopped by adding 0.8 ml of Na ₂ CO ₃ . Fluorescence at 455 nm is measured with 365 nm excitation light by a fluorescence spectrophotometer. The value of the 0-hour sample is subtracted from the value of the 2-hour sample, and the enzyme activity is expressed as 4-MU pmoles / min / mg protein.

Once the promoter region of the CatB gene is specified, it is within the scope of the present invention that the sequence is further modified to increase the promoter activity. For example, a sequence obtained by removing or partially modifying an unnecessary sequence in the CatB gene promoter region and having an activity equal to or higher than that before modification is also within the scope of the present invention.

The CatB gene promoter whose activity has been confirmed can be incorporated into an appropriate plant expression vector.
By inserting an appropriate linker sequence, for example, a linker having a multiple cloning site, at the 3 ′ end of the promoter sequence incorporated into the plant expression vector, an expression cassette suitable for a plant host can be prepared. The expression cassette may include a terminator sequence for the purpose of improving expression efficiency and the like. This terminator sequence can be linked to a promoter sequence via a linker sequence having the multiple cloning site.

An exogenous gene is connected to the multicloning site of the above-mentioned expression cassette for plants or downstream of the promoter so that the foreign gene can be expressed. The resulting recombinant plasmid can be transformed by protoplasting a plant cell according to a conventional method. Production of protoplasts of plant cells is described in Mol Gen. Genet 206: 408-413 (198
It can be performed according to the method described in 7). For example, the callus cell suspension 5 days after the passage is transferred to a plastic Petri dish having a diameter of 10 cm, the medium is removed with an aspirator, and the enzyme solution (eg, 4% Cellulase RS (Yakult), 1%
Macerozyme R10 (Yakult), 0.4M mannitol,
0.5% CaCl ₂ .2H ₂ O, and 0.5% Potassium Dextran Sulf
ate) Add 20 ml, parafilm and leave in the dark at 28 ° C. After about 5 hours, the mixture is filtered through a nylon mesh to remove undigested substances, and the washing operation is repeated, and then used for electroporation or the like.

The transformed plant cells can be used as they are for the production of useful substances encoded by foreign genes.
Recovery of useful substances from outside the cell or from inside the cell
It can be performed according to a conventional method. Furthermore, the transformed plant cells can be differentiated into transformed plant tissues or plants.

Furthermore, the rice CatB gene can be incorporated into a binary vector that can be expressed in both bacterial and plant hosts. Such binary vectors are well-known to those skilled in the art. For example, when an Agrobacterium expression system is used, a system for infection by microorganisms can be used. And, for example, it can be introduced into plants such as tobacco. Therefore, it can also be used for plant transformation.

The rice CatB gene has a high possibility of responding to various signals such as oxidative stress. Therefore, it can be expressed at various stages according to the signal. That is, it is considered that the expression can be freely regulated.

Therefore, from the rice gene, the activity is high,
If a promoter that can be used practically can be obtained,
In addition to rice breeding, it is possible to develop a general-purpose vector for substance production in other plant cells, which is expected to greatly contribute to substance production using plant cells. It can also be used for gene transfer into various plants.

Hereinafter, the present invention will be described based on examples, but the scope of the present invention is not limited to the examples.

[0041]

【Example】

(Isolation of Rice CatB Genomic Gene: Screening from Genomic Library) A part of CatA cDNA was used for cloning of CatB gene. Ca
Lambda containing incomplete length cDNA obtained during the cloning process of tA cDNA (3. 5 kbp of the 3 'end which lacks a total of about 450 bp of the 5' untranslated region and some coding regions of the total length of about 1.8 kbp) Phage (clone # 51)
Was amplified by PCR. DNA was prepared from the same phage and used as a template. As primers, λgt11-Forward Primer and λgt11 Reverse Primer
er (Toyobo) was used. The product is sentricon-100
(Amicon), and the concentration was adjusted to 25 ng / 10 μl to prepare a probe for the multiprime labeling method (Amersham).

[0042] CLONTECH Laborat
CatB gene was screened using a rice genomic DNA library (RICE Genomic Library) purchased from ories Inc., Palo Alto, CA. The phage λEMBL-3 constituting the genomic library was transformed into E. coli according to a conventional method. E. coli NM538 strain was infected to form a phage plaque. The phage plaque was transferred to a nylon membrane and hybridized with the above probe under the following standard conditions. The probe was labeled with ³² P.

Hybridization solution: 6 × SSC-0.1% SDS, 5
x Denhardt's, 100 μg / ml salmon sperm DNA; hybridization temperature: 65 ° C .; hybridization time: overnight.

Next, the membrane was washed under the following relatively mild conditions. Washing conditions: 2x SSC-0.1% SDS room temperature 5 minutes plus 30 minutes; 1x SSC-0.1% SDS 68 ° C, 1 hour.

After washing, the nylon membrane was subjected to autoradiography according to a conventional method to detect clones hybridized with the probe. DNA was prepared from each of the phages confirmed to hybridize.

The DNA of the above phage was prepared using SalI and Sca.
The restriction enzyme I was digested in combination, and the digest was separated by agarose gel electrophoresis. The separated DNA fragment was transferred to a nylon membrane, and the above-described CatA cD
Hybridization was performed using the NA fragment as a probe. Hybridization conditions were the same as above, and membrane washing was performed under the following conditions. Washing condition: 2x SSC room temperature 5-
10 minutes twice; 2x SSC-0.1% SDS 65 ° C, 30 minutes.

After washing, the nylon membrane was subjected to autoradiography according to a conventional method to detect a DNA fragment hybridized with the probe.

There are a group of clones having almost the same signal intensity and band pattern, and thus presumed to have the same structure, and a group of clones whose band patterns only partially match.

The clone in which the former signal intensity and band pattern almost coincided with each other was considered to correspond to the CatA gene. In fact, no. 74 clones are CatA
It was a gene corresponding to the cDNA.

On the other hand, a clone whose pattern of the latter band only partially matches may be a CatA gene-deficient clone or a clone of another catalase gene other than CatA. Therefore, CatA cDN
Using A as a probe, hybridization was performed under the following loose and severe conditions to compare patterns.

Loose hybridization conditions: 6x SSC-0.1% SDS, 5x Denhardt's, 100 with 50% formamide
μg / ml salmon sperm DNA; Hybridization temperature: 37
° C; Hybridization time: overnight.

The membrane washing conditions are the same as above.
2x SSC-0.1% SDS 5 minutes at room temperature plus 30 minutes; 1x SSC-0.1%
SDS 68 ° C., 1 hour.

Stringent hybridization conditions: 6x SSC-0.1% SDS, 5x Denhardt's, supplemented with 50% formamide.
100 μg / ml salmon sperm DNA; Hybridization temperature: 4
2 ° C; Hybridization time: overnight.

The membrane washing conditions were 2x SSC-0.1% SDS
Room temperature 5 minutes plus 30 minutes; 1x SSC-0.1% SDS 68 ° C, 1 hour.

As a result of hybridization under severe conditions, the same number of bands as in the standard hybridization pattern were detected. On the other hand, hybridization under loose conditions showed more bands than the pattern of hybridization under severe conditions. From these results, it is found that a clone having only a partial band pattern is not a CatA gene-deficient clone, but a DNA similar to CatA but having a different nucleotide sequence, that is, another catalase gene other than CatA. Was found to be likely.

Approximately 20 clones whose band patterns only partially matched were further analyzed as catalase isozyme gene candidates. No. of these Seven clones were subjected to detailed Southern analysis, partial nucleotide sequence analysis, CatA
It was concluded from Cat analysis based on the base sequence that it is a catalase gene different from CatA. Also, Cat
The sequence was found to be different from that of BcDNA.
C. The nucleotide sequence of CatC was announced at the 1994 meeting of the Japanese Society of Agricultural Chemistry (Abstract of lecture: Journal of the Japanese Society of Agricultural Chemistry 68, 404 (1994)).

Both CatA and No. 7 clones (Cat
C), there is a clone showing a pattern different from that of No. C). Six clones were analyzed in more detail. No.
The nucleotide sequences of the DNAs of the six clones were also partially analyzed. This sequence was consistent with the cDNA sequence of the CatB gene (Plant Physiol. (1994) 105: 1015-1016).

Based on these information, two DNA oligomers were synthesized to obtain the 5 ′ untranslated region of CatB gene.

GGTCGATTCTCATCTCTCCCACAACAAATC: SEQ ID NO: 4 (corresponding to CatB cDNA 102-131) CAATGTCTCAGGGCTTCCACGCTCATGCAC: SEQ ID NO: 5 (corresponding to CatB cDNA 484-455) On the other hand, a cDNA library was prepared from rice callus. PCR was performed under standard conditions using the DNA of this cDNA library as a template and the above two synthetic DNAs as primers. A DNA fragment of about 400 bp was amplified. Using the 400 bp DNA fragment as a probe, a clone group presumed to be the CatB gene was screened again to obtain several hybridizing clones.

The above No. 6 clones are PC
The upstream of the R-amplified 400 bp, 5 ′ of about 1 kbp or more
It was found that it had an upstream region and had a total length of about 5 kbp or more. This clone was named clone U6. A restriction map of clone U6 was made. FIG. 1 shows the restriction map.

From clone U6, the 5 'upstream region was about 1 kb.
A fragment containing p (Sal I-Eco RI digested fragment) and a fragment downstream thereof (Eco RI-Eco RI digested fragment) were cut out, inserted into a sequence analysis vector (pUC18), and from the 5 ′ side and from the 3 ′ side. A number of stepwise-deleted plasmids were prepared and their nucleotide sequences were determined. 4985b
The sequence of p was determined. SEQ ID NO: 3 shows the entire sequence.

(Preparation of a Recombinant Plasmid Having a Cat Gene Promoter)
It was 85 bp. And from the 1073rd position of SEQ ID NO: 3
Exon 1 at 1216, 2nd from 1333 to 1429
Exon, 2341 to 2618, 3rd exon, 2970
Exons 4 to 3746, Exon 5 from 3968 to 4057, Exon 6 from 4252 to 4319, Exon 7 from 4410 to 4503, and 4616
It was found that exons 8 to 4881 were present, and that introns 1 to 7 were present between each exon.

From the above sequence, the promoter region is 1073
It was presumed to be the sequence before the th.

The promoter region can be cut out based on this sequence information.

FIG. 2 shows the construction of a plasmid having a rice CatB gene promoter, CatB-GUS- △ 0.

Clone U6 was replaced with SalI and EcoRI.
And a 2.3 Kbp fragment was cut out. This fragment was ligated to pUC18 cut with SalI-EcoRI. The obtained recombinant plasmid was digested with Eco47I (manufactured by Toyobo Co., Ltd.) to recover a fragment of about 2 kbp.
This fragment was digested with the Klenow fragment of DNA polymerase and then with PstI. 7 of clone U6
A fragment A having a sequence from the 1st to the 1370th (SEQ ID NO: 2) was obtained. This fragment A contained a sequence upstream of the first exon, the first exon, the first intron, and a part of the second exon. 5 'of fragment A
The terminal side is a PstI site, and the 3 'end is a blunt end.

The plant cell expression vector pBI221 (Clontech) is a cauliflower mosaic virus (CaM
V) 35S promoter, β-glucuronase (GU
S) It has a coding region and a nopaline synthetase terminator (NOS · T). This pBI
221 was digested with PstI and SmaI and PstI
-Recover the SmaI large fragment B. This large fragment B is pB
This fragment is obtained by deleting the 35S promoter region of CaMV from I221. The large fragment B was ligated to the fragment A having the sequence from the 7th to the 1370th position of the clone U6 to prepare CatB-GUS- $ 0.

Similarly, a plasmid having the promoter regions of the CatA and CatC genes connected to the GUS gene,
tA-GUS- $ 0 and CatC-GUS- $ 0 were made. For CatA-GUS- $ 0, from the 1606th
At the 1893th, CatC-GUS- $ 0 has an intron at the 1229th to 1324th.

The resulting plasmid, CatA-GUS-
△ 0, CatB-GUS- △ 0, and CatC-GU
S-Δ0 was introduced into rice Oc cells. PBI221 was used as a control.

(Transformation of Rice Cells and Expression of GUS Gene) 70 ml of AA medium having the following composition was placed in a 300 ml flask, and rice Oc cells were gently stirred (90 rpm).
pm) and suspension culture was performed at 28 ° C.

Composition of AA medium (Mol. Gen. Genet. 161:
67-76 (1978)) 1) inorganic salt concentrations _{(mg / l) MnSO 4 .4-6H} 2 O 10 H 3 BO 3 3 ZnSO 4 .7H 2 O 2 Na 2 MoO 4 .2H 2 O 0.25 CuSO 4. _{2H 2 O 0.025 CoCl 2 .6H 2} O 0.025 KI 0.75 CaCl 2 .2H 2 O 150 MgSO 4 .7H 2 O 250 NaH 2 PO 4 .H 2 O 150 KCl 3,000 2) iron Fe-EDTA 40 3) vitamin organics Sucrose 20,000 nicotinic acid 1 thiamine hydrochloride 10 pyridoxine hydrochloride 1 myo-inositol 100 L-arginine 177 L-glycine 7.5 L-glutamine 900 L-aspartic acid 300 4) Hormonic acid 2,4-D 1 kinetin 0.2 GA3 0.1 every week 10 ml of the culture was transferred to 70 ml of fresh medium. Protoplasts were prepared using cells 5 days after transfer. Protoplasts were prepared using Mol Gen.
Genet 206: 408-413 (1987). Callus cell suspension 5 days after passage, 10 cm in diameter
Transfer to a plastic petri dish, remove the medium with an aspirator and remove the enzyme solution (4% Cellulase RS (Yakult), 1%
Macerozyme R10 (Yakult), 0.4 M _{mannitol, 0.5% CaCl 2 .2H 2 O} , 0.5% Potassium Dextran Su
lfate) was added. 28 ° C with parafilm
In a dark place. After about 5 hours, undigested matter was removed by filtration through a nylon mesh, and the washing operation was repeated.

The obtained protoplasts were added to an EP buffer (5 m
M MgCl ₂ , 70 mM KCl, 0.1% MES, 0.4M mannitol,
Add distilled water to pH 5.6 and sterilize with filter: Nat
ure338: 274-276 (1989)) and a part of it (0.5
ml) and the above plasmid DNA 20 μg / ml and salmon sperm DNA 30 μg /
ml. Next, Gene-Pulser (Bi
o-Rad, CA) at 300 V / cm with a capacitance of 500 μF and a resistance of 100Ω in a disposable cuvette with a 0.4 cm interelectrode distance. After incubation on ice for 10 minutes, 1.5 ml of R2P culture medium was added to the protoplasts, placed in Millicell (Millipore, CA), and the mixture was added to 5 ml of R2P medium (Plant Cell Physiol. 14: 1113-11).
21 (1973)). Add a nurse cell to the outside of the Millicell, 28 ° C,
Culture was performed under dark conditions. The presence or absence of a nurse cell is determined by CatA-G
It did not affect the expression of US-B0, CatB-GUS- △ 0, and CatC-GUS- △ 0.

[0073] Composition of R2P medium (1) Major elements (mg / l) KNO 3 4000 (NH 4) 2 SO 4 335 MgSO 4 .7H 2 O 250 CaCl 2 .2H 2 O 150 NaH 2 PO 4 .H 2 O 273 (2) Minor elements (mg / ml) MnSO 4 .4H 2 O 1.6 ZnSO 4 .7H 2 O 2.2 CuSO 4 .5H 2 O 0.125 H 3 BO 3 3.0 NaMoO 4 .2H 2 O 0.125 Na 2 EDTA 7.5 FeSO 4 .7H ₂ O 5.5 (3) Organic component (mg / ml) inositol 100 nicotinic acid 1.0 pyridoxine hydrochloride 1.0 thiamine hydrochloride 10 sucrose (g / l) 137 2,4-D (mg / l) 2.0 pH 5.6 For GUS activity The processing of protoplasts is basically
Lab Manual: Functional Analysis of Plant Genes (Masaki Iwabuchi and Norio Shimura, Ed., Maruzen, 1992) p. Performed according to 55 descriptions. After electroporation, 4 days, 28 ° C,
The protoplasts cultured in the dark were placed in a 1.5 ml microtube using a 1 ml Falcon pipette (# 7521), centrifuged at 9,000 rpm for 3 minutes at room temperature, and the pellet was stored at -80 degrees until analysis.

Add 200 μl of extraction buffer (50 mM sodium phosphate buffer (pH 7.0), 10 mM Na ₂ E
DTA, 0.1% TritonX-100, 0.1% Sodium Lauryl Sarco
sine, 10 mM β-mercaptoethanol), and the frozen pellet was thawed in ice water for 5 minutes. Using an ultrasonic processor (Branson Sonifier model 250), output c
Ontrol 2, Duty cycle 10% treatment for 1 minute. After the treatment, the mixture was centrifuged at 15,000 rpm for 20 minutes at 4 ° C. The supernatant (protoplast extract) was taken and used as a sample for GUS activity measurement. The protein concentration in the sample was determined by the Bradford method (AnalBiochem 72: 248-254 (1976)).

GUS activity followed the method of Jefferson et al., Supra. The endogenous GUS activity is 20% (V
/ V) of methanol. The experimental results were the results of at least two experiments, and each experiment included three samples (triplicates) of the same sample.

FIG. 5 shows the results. The result is CatB
The gene promoter is a control CaMV,
It showed very high activity compared to CatA and CatC promoters.

[0077]

Industrial Applicability The CatB gene promoter of the present invention has a much higher activity than CaMV which has been conventionally used as a general-purpose promoter for plants. Therefore, it is useful for producing useful substances using plant cells and improving varieties such as rice.

[0078]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 1072 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: Genomic DNA Sequence characteristics Characteristic symbol: Promoter Location: 1..1072 determined method: E SEQ GTCGACCTGC AGGTCAACGG ATCTTATTTT TCCATTATAA TATATATAAT AAATAAATAT 60 ATGTTTACTT TTATTATAGT ACTTTAAAAG ATAAATCTAT ATATGTTGTT CTAGTTCCTT 120 TAAACTAAAT ATTATTAAAG TTATTAATGG TTAAAGTTAT AAAAGTTTGA TATCAAACTC 180 GTCCAAAATG TCGATTAATA TCGAACCGGA GCGAGTACAG TATTAGTAGC AAGTCAGCCA 240 CATGGGACAT GGCCCACATG CATGCACGTC GTATGAACAC ACCGTGATTC TTTGCCACTT 300 GCATAATATT CTAGCACTGC TATACTACAC GACGACTGAC GGCGACGTCA GTTCAGTTTA 360 GTTTGCCGCA TCCATCGCGA AGGCTACTCT ACCCATCCCA TTTTTTTTTA AAAAAAAATA 420 CTATAAATCT AAATATCTTA CATTAGATTT GTATATTTTA AAGCAAAGAG AATAATATGT 480 AGATATAAGT ATGTACCTAC TCGCTCGAGC ACAAGATCAC TGCAACAAGC ATTGAAGATC 540 GCTTCTAG TGATCTATCAGTCGA TGATCGATCGATCGATCGATCGATCGATCTAGCATGATGATCATAGTGATCGATCATAGTGATCGATCATAGTGATCGATCATAGTGATCGATCATAGTGATCGATCATAGTGATCTAGTCATGTCATCAGCATGATGATCTAGTCATCGATCATC GAGAAA ACACATATAG AAATTATACA 660 GGATTTAGCA TTTGGGATGC CGGCCGGATT CCTGATTTCC CAGTCTCTGG CTTTCTTTTT 720 AAACAAAAAC GAAAAAAGCA GTGATCCGAT CGATCACGAT GAGCGAGCTA GTAAGCTCCA 780 AAACAAAATA GAGTACGTAC GTATAATCCT AGAGTCCGGA TAATAATAAT CCGTTTGGTT 840 CGCGTTAAAA AAGTCTTATC TCCTCGTGAT CCCTTTTTTT GGATCGATCC ATGTTCGTAG 900 TACGTGACAA GCACGCGCAC CAACCGAAGC AGGTACCTGT GTCGCTGCCT GTGGGCCCCA 960 CACACCCCAA GACGGCCATT AATAAACAAA CACGACGTGG ACGAAGAGAA GGGAGGCCGG 1020 CAAGAAGCAT ACTAGCACGC TACGAAACCC CCCTTCTCTT CGTCCCCAAA TT 1072 SEQ ID NO: 2 Sequence length: 1364 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Genomic DNA Sequence characteristics Characteristic symbol: Promoter Location: 1. .1364 Method for Characterization: E Sequence CTGCAGGTCA ACGGATCTTA TTTTTCCATT ATAATATATA TAATAAATAA ATATATGTTT 60 ACTTTTATTA TAGTACTTTA AAAGATAAAT CTATATATGT TGTTCTAGTT CCTTTAAACT 120 AAATATTATT AAAGTTATTA ATGGTTAAATGTATAG ATT AATATCGAAC CGGAGCGAGT ACAGTATTAG TAGCAAGTCA GCCACATGGG 240 ACATGGCCCA CATGCATGCA CGTCGTATGA ACACACCGTG ATTCTTTGCC ACTTGCATAA 300 TATTCTAGCA CTGCTATACT ACACGACGAC TGACGGCGAC GTCAGTTCAG TTTAGTTTGC 360 CGCATCCATC GCGAAGGCTA CTCTACCCAT CCCATTTTTT TTTAAAAAAA AATACTATAA 420 ATCTAAATAT CTTACATTAG ATTTGTATAT TTTAAAGCAA AGAGAATAAT ATGTAGATAT 480 AAGTATGTAC CTACTCGCTC GAGCACAAGA TCACTGCAAC AAGCATTGAA GATCGCTCCT 540 AGCAATGGTC TCAACTTACC ATGTAAACTA AGAGCAACTA TAATGTTTTT CTTTTATTAG 600 GAATGGTTGC ATCTTATATT TTGAGATTGA GAAAACACAT ATAGAAATTA TACAGGATTT 660 AGCATTTGGG ATGCCGGCCG GATTCCTGAT TTCCCAGTCT CTGGCTTTCT TTTTAAACAA 720 AAACGAAAAA AGCAGTGATC CGATCGATCA CGATGAGCGA GCTAGTAAGC TCCAAAACAA 780 AATAGAGTAC GTACGTATAA TCCTAGAGTC CGGATAATAA TAATCCGTTT GGTTCGCGTT 840 AAAAAAGTCT TATCTCCTCG TGATCCCTTT TTTTGGATCG ATCCATGTTC GTAGTACGTG 900 ACAAGCACGC GCACCAACCG AAGCAGGTAC CTGTGTCGCT GCCTGTGGGC CCCACACACC 960 CCAAGACGGC CATTAATAAA CAAACACGAC GTGGACGAAG AGAAGGGAGG CCGGCAAGAA 1020 GCATACTAGC ACGCTACGAA ACCC CCCTTC TCTTCGTCCC CAAATTGCAC TACAAAAAAG 1080 GCCGCCCCTT TCTTCTCTCC TCGTCCTTAT CACCACCAAT CCGATCCTCT TCTCTTCTCT 1140 TCTCTTCTTC CCCACATCCA GTTCGATTCT CATCTCTCCC ACAACAAATC ACGCCATGGA 1200 TCCCTACAAG GTGCCGCCTT TTCCTGATTT TCTTTTCTTC TAGATCGATC GTCGATTTGG 1260 TTTGGTTTGG TTTCTTGATG CGCTCATCCC AATCTGACTG ACTCACTGGA TTCCTCCTCC 1320 TTGCAGCATC GGCCGTCCAG CGGGAGCAAT TCCACCTTCT GGAC 1364 SEQ ID NO: 3 sequence Length: 4985 sequence Type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Genomic DNA Origin Organism name: Rice Sequence characteristics Characteristic symbol: Exon 1 Location: 1073..1216 Method for determining the characteristics: Characteristic of the S sequence Symbol representing the characteristic: Exon 2 Location: 1333..1429 Method for determining the characteristic: S Sequence characteristic Symbol representing the characteristic: Exon 3 Location: 2341.2618 Method determining the characteristic: S sequence Characteristic symbol: Exon 4 Location: 2970..3746 Determine characteristics Method: Characteristic of S sequence Characteristic symbol: Exon 5 Location: 3968..4057 Characteristic determination method: Symbol of S sequence characteristic Characteristic: Exon 6 Location: 4252..4319 Characteristic determination method : Characteristic of S sequence Characteristic symbol: Exon 7 Location: 4410..4503 Method for determining characteristics: S Sequence characteristic Characteristic character: Exon 8 Location: 4616.4881 Method for determining characteristics: S SEQ GTCGACCTGC AGGTCAACGG ATCTTATTTT TCCATTATAA TATATATAAT AAATAAATAT 60 ATGTTTACTT TTATTATAGT ACTTTAAAAG ATAAATCTAT ATATGTTGTT CTAGTTCCTT 120 TAAACTAAAT ATTATTAAAG TTATTAATGG TTAAAGTTAT AAAAGTTTGA TATCAAACTC 180 GTCCAAAATG TCGATTAATA TCGAACCGGA GCGAGTACAG TATTAGTAGC AAGTCAGCCA 240 CATGGGACAT GGCCCACATG CATGCACGTC GTATGAACAC ACCGTGATTC TTTGCCACTT 300 GCATAATATT CTAGCACTGC TATACTACAC GACGACTGAC GGCGACGTCA GTTCAGTTTA 360 GTTTGCCGCA TCCATCGCGA AGGCTACTCT ACCCATCCCA TTTTTTTTTA AAAAAAAATA 420 CTATAAATCT AAATATCTTA C ATTAGATTT GTATATTTTA AAGCAAAGAG AATAATATGT 480 AGATATAAGT ATGTACCTAC TCGCTCGAGC ACAAGATCAC TGCAACAAGC ATTGAAGATC 540 GCTCCTAGCA ATGGTCTCAA CTTACCATGT AAACTAAGAG CAACTATAAT GTTTTTCTTT 600 TATTAGGAAT GGTTGCATCT TATATTTTGA GATTGAGAAA ACACATATAG AAATTATACA 660 GGATTTAGCA TTTGGGATGC CGGCCGGATT CCTGATTTCC CAGTCTCTGG CTTTCTTTTT 720 AAACAAAAAC GAAAAAAGCA GTGATCCGAT CGATCACGAT GAGCGAGCTA GTAAGCTCCA 780 AAACAAAATA GAGTACGTAC GTATAATCCT AGAGTCCGGA TAATAATAAT CCGTTTGGTT 840 CGCGTTAAAA AAGTCTTATC TCCTCGTGAT CCCTTTTTTT GGATCGATCC ATGTTCGTAG 900 TACGTGACAA GCACGCGCAC CAACCGAAGC AGGTACCTGT GTCGCTGCCT GTGGGCCCCA 960 CACACCCCAA GACGGCCATT AATAAACAAA CACGACGTGG ACGAAGAGAA GGGAGGCCGG 1020 CAAGAAGCAT ACTAGCACGC TACGAAACCC CCCTTCTCTT CGTCCCCAAA TTGCACTACA 1080 AAAAAGGCCG CCCCTTTCTT CTCTCCTCGT CCTTATCACC ACCAATCCGA TCCTCTTCTC 1140 TTCTCTTCTC TTCTTCCCCA CATCCAGTTC GATTCTCATC TCTCCCACAA CAAATCACGC 1200 CATGGATCCC TACAAGGTGC CGCCTTTTCC TGATTTTCTT TTCTTCTAGA TCGATCGTCG 1260 ATTTGGTTTG GTTTGGTTTC TTGATGCGCT CATCC CAATC TGACTGACTC ACTGGATTCC 1320 TCCTCCTTGC AGCATCGGCC GTCCAGCGGG AGCAATTCCA CCTTCTGGAC CACCAACTCC 1380 GGCGCCCCCG TCTGGAACAA CAACTCCGCC CTCACCGTCG GAGAGCGAGG TACTGAGCTG 1440 CTCCATCCTC CATCTTCCTC ATCTCTTCCT CCACGAATCT ATACTTCCTA TCGATCCATG 1500 TGCTTTATGT CTGCTTAGAG GCGTATTTGG GCTTGCTTCC ATCATGATCC AATGTTAACT 1560 TTGAGCGATC AGATGCTTAA TAATTTGTCT GTGTCTGGTA GCAACAGAAA AGGTCCCTGG 1620 ATCTGTGTGA ATTGGACTGT AGCACGACGT GTGCGATCTT GCCTTACTAT TGTTCCTTTA 1680 GAGCTATTCA AAGTTTGCAT CTTTGGTGTG GGGGGGTTTT CAGCCTTTTG CTTTGAGTAA 1740 CATGTCCTTG TCACTACCTT GTGATTCATC TGTGCTGCTT ACACACAAAG AGGAAGATCT 1800 CTAGTTTGGT TGCACTATGT TCTTGTTATT GCTCGGTTAC ACTCCTTCAT CAACGCTAAA 1860 AGACGCAGTC TTGATATTTT TCTCTGTCGC TGTCAATCTG TCATACCATT AATAAAGAAC 1920 GTATAAAAAT TGACCTTTTC CCCATTCATA GTAGTGATTA TCTCACTTGT CCTCAGTTCT 1980 AAAACAGCAT TCTTGTTTTT TGGGGGTTTT TTTACCATTA TCGGTTCACT TTACAAAGTA 2040 ATCTGATGAC GATCAAATTT ACTGGAGTAC CAGAGAAGCA CAAGTCTATT TCATACATAT 2100 ATCCAACATG ACTCAAGTTT CGAAAGTGAC TGGAAGTCAT GTTCCAAATG TCATGGTCAC 2160 TAAAAGTACC AAACTCTGAC AAAGATGTAT TTGCATCTAG AACTAAGTGC TTCTATCATC 2220 GATCTGTGAA GTTTTCTTTC TAGTGTTTTC CCTTTATTTA TACTCAGCAA CTCCTGTATT 2280 TTTGCAATCT GTAATCATGG CTGTTTTGTG GTTTGACATG ATGAATTCTT CAATCGACAG 2340 GCCCTATCCT CCTTGAGGAC TATCATCTGA TTGAAAAGCT TGCACAGTTT GACAGGGAGC 2400 GTATCCCTGA ACGTGTCGTT CATGCAAGGG GAGCCAGTGC CAAGGGATTT TTTGAGGTTA 2460 CTCATGATAT TTCTCACCTC ACATGTGCTG ATTTTCTCCG TGCTCCTGGT GTTCAGACCC 2520 CAGTTATTGT TCGGTTCTCC ACAGTCGTGC ATGAGCGTGG AAGCCCTGAG ACATTGAGGG 2580 AACCACGTGG TTTTGCTGTC AAGTTTTACA CTAGAGAGGT ACTTGCTCTT TCGCTTCTTT 2640 CTTTCATAGG ATTGTAGGAG GGAGACATTC AGTATAATGT ATTCTTCAAG CATAAGGCTG 2700 TAGAATCAAA TGTCTAGTTG TTCTCAGTTG GTTAAGTAGA ACATGAAAGA TTGGTTGTCC 2760 TTTACCTCCA CACTCCATAG GTCCAGTGCA TTGCTTAATC TTATATCTAC TAAAGCAAAT 2820 GAGGGTAATT TGGTCTTATA TATCTCAAAA GTCTCACACA TTGGACATAT ATCTAACCAG 2880 TGTCACCTAC AACTTTGTCT TACTGTTTAT TTACTGATTT GCATTCAGTG CTGCCATATT 2940 TTGATATTTT CTGAGTCAAT GCTTTTCAGG GTAATTTTGA TCTTGT TGGG AACAATATGC 3000 CTGTCTTTTT TATCCGAGAT GGGATGAAAT TCCCTGACAT GGTCCATGCT TTCAAGCCAA 3060 GTCCAAAGAC CAATATGCAG GAGAACTGGA GAATAGTTGA TTTCTTTTCA CACCACCCAG 3120 AGAGCCTGCA CATGTTCTCC TTCCTCTTTG ACGATGTAGG CATCCCACTC AACCACAGGC 3180 ACATGGAGGG TTTTGGTGTC AACACCTACA CCCTAATCAA TAAGGATGGA AAGCCTCACC 3240 TTGTCAAATT CCACTGGAAG CCTACCTGTG GTGTCAAATG CCTGTTGGAT GATGAAGCTG 3300 TGAVTGTTGG CGGCACCTGC CACAGCCATG CCACGAAGGA CTTGACTGAT TCTATTGCAG 3360 CAGGGAATTA CCCAGAGTGG AAGCTTTACA TCCAGACTAT TGATCCTGAT CATGAGGACA 3420 GATTTGACTT CGATCCTCTT GATGTCACCA AGACATGGCC AGAGGATATC ATCCCCCTGC 3480 AGCCAGTTGG ACGGATGGTC CTGAACAAAA ACATTGATAA CTTCTTTGCA GAAAATGAAC 3540 AGCTTGCTTT CTGCCCAGCG ATAATTGTCC CTGGAATCCA TTACTCTGAT GATAAGCTGC 3600 TCCAGACAAG AATTTTCTCC TATGCTGATA CCCAAAGGCA CCGTCTTGGC CCAAACTATT 3660 TGATGCTTCC TGTGAATGCA CCAAAATGTG CATACCACAA CAACCACCAC GATGGCTCCA 3720 TGAATTTCAT GCACAGGGAT GAAGAGGTAC TGTGTGTATA TACTTTCAGA GATACATCTC 3780 CTGCATTCAG TTGTTGTGAT GCATCTTTCT GTTTTTGTCC ATTACATATT G TTTCTTCCA 3840 GTCAACACAA ACAGAATGGG ACTATCATTC AGTTTATTGC ATTTACATCT ATTTGCCTTG 3900 TTTTTAGGTT AACTACTTCC CTTCAATTCA GTTTATTGCA TTTACATCTA TTTGCCTTGT 3960 TTTTTAGGTT AACTACTTCC CTTCAAGGTT TGATGCTGCA CGTCATGCTG AGAAGGTCCC 4020 TATTCCTCCT CGTGTTCTAA CAGGCTGTCG GGAAAAGGTG TGTAACTTGG TCCACTTGAA 4080 CTCCTTGCGC TGTTACCTTG TGAGCATGGT TTTGTCCCGC TACATTAGGA GACTATTTGC 4140 TGATTTGGAA TGCGATGAAA TATGTATTAT AGATGTGGTA CCCTGGAAAG TACAATGACC 4200 ACATGCATTT GACACAATGT TTTCTGCCTC TCTTTTTTGT TGGAAATGCA GTGTGTCATT 4260 GACAAGGAGA ACAATTTCCA ACAGGCTGGT GAGAGATACC GGTCATTTGA CCCTGCCAGG 4320 TTTGTTCTTG TTCAATTTAA TTCGTGTGAA CACATCGAAG AGTTTGAGCA ACAACGCTAA 4380 TTAAACTTTT CTTTTTGTAT GTAACACAGG CAAGATCGTT TTCTCCAGCG GTGGGTTGAT 4440 GCTCTCTCAG ATCCTCGTAT TACACATGAA CTCCGTGGCA TCTGGATCTC CTACTGGTCG 4500 CAGGTAACAT AATTTCTTCG TGGGTGCAAA GTGCTTATCA GTTGTCAGTG AGAGATCATG 4560 TACAATTGTA CCTTGTATTG ACACACTGAA ACCATATATT TGTGTGTTGT TGCAGTGTGA 4620 TGCGTCCCTT GGGCAGAAGC TGGCTTCACG TCTCAACCTG AAACCAAACA TGTAGAT CGG 4680 CCAGGAGGAA TCCAGTGGTG GTGCTATGTT GGACAGTCAA ACATGAACTG TAATGTGTCG 4740 ACCAGCCGTA GTCGTGAATA AAATGTGATA CGGTGATATG TATACTGGTG ACGCAAGTTG 4800 TGAAACTGTA TCTGGAATCC TGAAAATATG CCTTGCTGTG TCTTGGGAAA TGAAACTGTA 4860 TCTGGAATCC TGAAAATATG CCTTGCTGTG TCTTGGGAAA ACATCTGCCA TCTCTTGTTT 4920 GCCTTTGTGT TAAGATGGCT TTAGAGACTG GAACGAACAA CCAGCTGTTT GCCTTTGTGC 4980 CTTTG 4985 SEQ ID NO: 4 sequence Length: 30 sequence Type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic DNA Antisense: No Fragment type: Intermediate fragment Origin: Rice sequence GGTCGATTCT CATCTCTCCC ACAACAAATC 30 SEQ ID NO: 5 Sequence length: 30 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic DNA Antisense: Yes Fragment type: Intermediate fragment Origin: Rice Sequence CAATGTCTCA GGGCTTCCAC GCTCATGCAC 30

[Brief description of the drawings]

FIG. 1 is a restriction map of clone U6.

FIG. 2 is a schematic diagram showing the construction of plasmid CatB-GUS- $ 0.

FIG. 3 is a schematic diagram showing the construction of plasmid CatA-GUS- $ 0.

FIG. 4 is a schematic diagram showing the production of plasmid CatC-GUS- $ 0.

FIG. 5 is a diagram comparing the activity of each promoter.

Claims (7)

(57) [Claims] 1. A rice CatB gene promoter comprising a sequence represented by SEQ ID NO: 2 or a sequence having a part thereof and having a promoter activity equivalent to that of SEQ ID NO: 2. 2. A plant expression cassette having the rice CatB gene promoter according to claim 1. 3. A recombinant plasmid comprising the rice CatB gene promoter according to claim 1 and a gene operably connected. 4. A plant cell transformed with the recombinant plasmid according to claim 3 , wherein the rice CatB gene is
A plant cell in which the offspring promoter can show activity . 5. A plant transformed with the recombinant plasmid according to claim 3 , wherein the rice CatB gene plant is transformed.
A plant in which a lomotor can exhibit activity . 6. A step of preparing the recombinant plasmid according to claim 3 , wherein the rice CatB is used with the recombinant plasmid.
A method for producing a recombinant protein, comprising a step of transforming a plant cell in which a gene promoter can exhibit activity ; and a step of culturing the transformed plant cell. 7. a step of preparing a recombinant plasmid having the rice CatB gene promoter according to claim 1;
The rice CatB gene promoter is
Transforming a plant cell in which the protein can exhibit activity ;
And a method for producing a recombinant plant, comprising a step of culturing the transformed plant cell.