JPH07123982A - Peroxidase transcription regulatory gene and method of using the same - Google Patents

Abstract

(57) [Summary] [Purpose] Peroxidase gene pr derived from horseradish
To provide a transcriptional regulatory gene effective for xC2 transcriptional regulation and a method for using the same. [Structure] A peroxidase transcription regulatory gene having the nucleotide sequence shown in SEQ ID NO: 1.

Description

Detailed Description of the Invention

[0001]

[Industrial applications]

TECHNICAL FIELD The present invention relates to a transcriptional regulatory gene having a function of suppressing or promoting transcription of plant genes, and more particularly to a transcriptional regulatory gene effective for transcriptional regulation of horseradish-derived peroxidase gene prxC2 and a method of using the same.

[0002]

[Prior art]

With the development of plant molecular biology in recent years, in breeding of varieties and the like having useful traits such as disease resistance, a gene capable of expressing a gene having the trait of interest in a sense direction or an antisense direction can be expressed in a plant. , And the technology for promoting or suppressing its expression has become possible. For example, an example of introducing an insecticidal BT toxin gene derived from Bacillus thuringensis (DA Fischhoff et al .: BioTechnology,
vol.5, p.807-813,1987) and an example of introducing a polygalacturonase cDNA gene for overripening of tomato fruit in the antisense direction (CJSmith et al .: Nature, vol.334,
p.724-727, 1988) etc. have been reported.

These are all cauliflower mosaic virus (CaM
V) 35S promoter and the BT gene in the sense direction and the polygalacturonase cDNA gene in the antisense direction downstream of the 35S promoter, respectively, and the Agrobacterium tumefacien (Agrobacterium tumefacien
It has been confirmed that the expression of the transgene and the acquisition of a new trait by the introduction have been confirmed.

However, when the gene expression is suppressed by using the antisense method, the following problems occur. That is,
Many genes in plants have a small multi-gene fam
ily), and the homology of the nucleotide sequence of its structural gene region is high, expression of genes other than the target is also suppressed even when antisense is used, and sometimes adverse effects such as growth inhibition of the plant itself are suppressed. There was a possibility of affecting. Therefore, it has been desired to develop a method for specifically suppressing only the target gene.

[Problems to be Solved by the Invention]

Therefore, as a result of intensive studies on the above problems, the present inventors have found that good results can be obtained by using a transcriptional regulatory gene for the horseradish peroxidase gene, and completed the present invention. . Therefore, the first object of the present invention is to specifically and artificially
It is intended to provide a novel peroxidase transcriptional regulatory gene capable of promoting or repressing gene transcription. The second object of the present invention is to provide a recombinant plasmid having a novel structural gene. Furthermore, a third object of the present invention is to provide a method for producing a recombinant vector having excellent specificity.

[Means for Solving the Problems]

The above objects of the present invention have been achieved by a peroxidase transcription gene having the nucleotide sequence shown in SEQ ID NO: 1, a recombinant plasmid, and a method for producing a recombinant vector using the same. First, peroxidase as the background of the present invention will be described below.

Peroxidase is an enzyme that exists in plants, animals, and microorganisms, and functions as a catalyst for the oxidation reaction by hydrogen peroxide. In general, plant peroxidase is a glycoprotein that has many isozymes (having the same function,
It consists of enzymes with different genes) and isoforms (although the genes are the same, but with different structures due to post-transcriptional / translational processing, glycosylation, etc.), forming a small multigene family. Furthermore, it is known that peroxidase is widely involved in removal of hydrogen peroxide, lignin biosynthesis, auxin metabolism which is a plant hormone, and the like.

In recent years, the base sequence of the peroxidase structural gene in horseradish, Arabidopsis, rice, tobacco, tomato, wheat, potato, cucumber, etc. has been clarified. The homology of the peroxidase gene at the nucleotide level in these plants is relatively high, in particular the region that coordinates the heme is characterized by being very well conserved. Among them, 4 cloned in horseradish
The homology of each kind of peroxidase genomic gene at the nucleotide level exceeds 60%, and it has been found that the structures of peroxidase genes are very similar.

On the other hand, the antisense method is known as a method for suppressing the action of a specific gene in plants. In this method, an antisense RNA is produced by linking a gene to be suppressed to a plant-expressible promoter, for example, the cauliflower mosaic virus 35S promoter in the reverse direction (from 3 ′ side to 5 ′ side). Since this antisense RNA is complementary to the mRNA of the gene to be suppressed, it can form double-stranded RNA in the nucleus and consequently suppress the function of the gene. However, it is difficult to specifically suppress one gene by the antisense method in the case of a small multigene group having a similar structure like the above-mentioned peroxidase gene.

Therefore, the present inventors solved this problem by suppressing the action of a transcriptional regulatory gene involved in gene expression. Peroxidase is involved in lignin biosynthesis. Therefore, if the peroxidase that catalyzes this lignin biosynthesis can be specifically suppressed, the development of trees as raw materials for paper and pulp will be developed. It is highly expected to be used when doing.

The transcriptional regulatory gene of the present invention is specifically prepared by the following method. (1) Isolation and purification of tobacco mRNA (2) Preparation of tobacco cDNA library (3) Selection of peroxidase transcription regulatory gene (4) Determination of the nucleotide sequence of the selected transcription regulatory gene (5) Confirmation of expression transcription regulatory activity Construction of recombinant plasmid for (6) Confirmation of expression transcription regulation activity

The method for preparing the transcriptional regulatory gene of the present invention will be described in detail below. (1) Isolation and Purification of Tobacco mRNA Tobacco used as a material for tobacco mRNA in the present invention is
There is no particular limitation, and for example, tobacco leaves, stems, roots, etc. can be widely used. Tobacco mRNA can be obtained, for example, by freezing and crushing leaves with liquid nitrogen or the like, followed by the method of Chomcznski et al. (Chomcznski et al .: Annal. Bioche
m., vol.162, p.156-159, 1987), and after extracting total RNA, it is purified by an oligo dT cellulose column according to a conventional method.

(2) Preparation of Tobacco cDNA Library As a method for synthesizing cDNA from the mRNA obtained by the above method, the Hoffman method can be adopted. That is, oligo dT is added to the obtained mRNA, and a reverse transcriptase is used as a primer to obtain a DNA-RNA hybrid. The obtained DNA
-RNA hybrid is decomposed by RN-aseH method in RNA hybrid, and then DNA polymerase I is allowed to act to synthesize cDNA. Then the obtained cDNA
A tobacco cDNA library can be prepared by connecting restriction enzymes EcoRI, NotI adapters and the like to both ends of the plasmid.

Further, the obtained cDNA is ligated to a cloning vector in the presence of DNA ligase. As the cloning vector used in this case, known ones such as plasmids and phages can be widely used, but lambda gt11 and lambda ZAPII having promoters suitable for expression cloning are particularly preferable.

Next, the above recombinant DNA is introduced into a host. The host to be used may be any type of prokaryote, yeast, eukaryote, etc., and is determined by the type of cloning vector already used. In addition, as typical prokaryotes, Escherichia coli, Bacillus subtilis, etc.
Saccharomyces cerevisiae, Schizosaccharomyces pombe, and the like can be used, and as eukaryotes, plant cells such as tobacco that can be mass-cultured can be used.

The above cloning vector may be introduced into the host in a state where the foreign gene can be easily introduced into each host. For example,
When Escherichia coli is used as the host, it is preferable to use cells that have been collected in a logarithmic growth phase and treated with magnesium ions. Furthermore, when introducing the cloning vector, for example, the electric pulse method can be used.

(3) Selection of Peroxidase Transcription Control Gene It is necessary to select a strain having a peroxidase transcription control gene from the tobacco cDNA library obtained as described above. Can be used. For example, when using lambda gt11 as a vector, the Southwestern method (H. Singh et al .: Cell., Vol. 52,
p.415-423,1988) is adopted.

That is, recombinant lambda phage plaques were formed on a petri dish, and nitrocellulose or nylon membrane containing isopropylthiogalactoside (hereinafter abbreviated as “IPTG”) was placed on the surface of the plaque, and the protein was recovered from the recombinant. To produce. That is, lac of lambda gt11 by IPTG
The promoter is activated and a protein encoding the integrated cDNA is produced.

Next, the produced protein is adsorbed on a nitrocellulose or nylon membrane. The membrane was subjected to a ligation reaction with an oligonucleotide probe corresponding to a region important for expression of the horseradish peroxidase prxC2 gene, and recombinant lambda containing a cDNA encoding a protein capable of specifically binding to this region. Phage can be obtained as positive plaques.

Then, DNA is extracted from the obtained recombinant plaque derived from the positive plaque by a conventional method, and an appropriate restriction enzyme is used to insert the inserted D into the E. coli transformant.
NA fragments can be excised. In addition, PCR was performed using both ends of the cloning site of the phage vector as primers (RKSaiki et al .: Science, vo-l.239, p487-49).
1, 1988) to amplify the DNA fragment.

(4) Determination of Nucleotide Sequence of Selected Transcription Regulator Genes Regarding the structure of the DNA fragment containing the peroxidase transcription regulator gene confirmed as described above, for example, the dideoxy chain termination method (F. Sanger et al .:
Proc., Natl.Acad.Sci., USA, vol.74, p.5463-5469,1977)
It is possible to determine the nucleotide sequence and predict the amino acid sequence from the open reading frame.

(5) Construction of Recombinant Plasmid for Confirming Expression and Transcriptional Regulatory Activity In order to confirm that the region deduced by the above method is a transcriptional regulatory gene, first, the target in a specific plant is targeted. The effect must be expressed. For that purpose, the obtained transcriptional regulatory gene is ligated in a sense direction or an antisense direction to a plant-expressible promoter such as the cauliflower mosaic virus 35S promoter. At this time, this recombinant gene is used as an effector.

Furthermore, a reporter gene (eg, chloramphenicol acetyltransferase (CAT) or β-glucuronidase (GU
The method using S) or luciferase (LUC) gene is also advantageous. That is, the promoter region of the target gene can be linked to these reporter genes, and this recombinant gene can be used as a reporter.

These effectors and reporters are based on pBI101 (Clonta
ch)) and the like, which can be introduced into plant cells. In addition, the construction of such a recombinant plasmid is a known method, for example, the method shown in Moleculae Cloning (Maniat is et al .: Molecular Cloning, A Laboratory Manual, Cold.
Spring Harbor Laboratory, NY, 1982).

(6) Confirmation of expression transcription regulation activity The recombinant plasmid obtained as described above was introduced into plant cells by the Agrobacterium infection method, electroporation method, or particle gun method described above. By detecting the expression of the reporter, the expression and transcription regulation activity of the transcription regulation gene of the present invention can be confirmed. For example, when it is desired to increase the expression activity, a sense transcription regulatory gene is introduced as an effector, and conversely, when it is desired to decrease it, an antisense transcription regulatory gene is introduced.
By this method, artificial transcriptional regulation can be confirmed.

The gene whose expression and transcription regulation can be confirmed as described above is horseradish peroxidase in tobacco.
It is of course possible to control the prxC2 gene, but it is also possible to broaden the expression of other plant genes such as rice and Arabidopsis. The transcriptional regulatory gene of the present invention can, of course, be prepared from tobacco as described above.
For example, the phosphite triester method (M. Hunkapiller et al .:
It can also be chemically synthesized according to Nature, vol.310, p.105-111, 1984).

The gene of the present invention and horseradish peroxidase gene p
When the expression activity of a plant structural gene is increased using the rxC2 promoter, as described above, a chimeric gene in which the transcriptional regulatory gene of the present invention is linked downstream of a plant-expressible promoter, and a structural gene to be expressed To construct a chimeric gene ligated under the control of horseradish peroxidase prxC2 promoter. Then, a vector in which these two chimeric genes are separately or simultaneously incorporated can be constructed by a known method, and this can be introduced into a host depending on the type of vector and expressed. Detailed conditions for expression are individually determined depending on the type of plant structural gene, vector, or host. In addition, the expressed plant material can be isolated and purified by a usual method.

[0028]

【The invention's effect】

As described above in detail, since the peroxidase transcriptional regulatory gene of the present invention can artificially and specifically promote or suppress transcription of a plant structural gene, use a recombinant plasmid having the structural gene. Due to
It will be easier than ever to produce recombinant vectors.

[0029]

【Example】

Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited thereto.

Example 1. (1) Isolation and purification of tobacco mRNA About 10 g of tobacco leaves grown in a greenhouse for about 2 months were ground in the presence of liquid nitrogen, and total RNA was purified as completely as possible by the method of Chomcznski described above. Extracted. 1 mg of this total RNA was dissolved in sterilized water treated with diethyl dicarbonate and treated at 65 ° C for 5 minutes, and then equivalent to twice the equivalent loading buffer (20 mM Tris-HCl, pH7.6, 0.5M NaC
1, 1 mM EDTA, 0.1% SDS) was added and applied to an oligo dT cellulose column that had been activated in advance.

Next, the column was washed with 5 to 10 column volumes of the loading buffer, and then 5 volumes of the washing buffer (20 mM Tris-HCl, pH
It was washed with 7.6, 0.1 M NaCl, 1 mM EDTA, 0.1% SDS). Furthermore, a 2-3 times column amount of elution buffer (10 mM Tris-HCl,
The mRNA was eluted using pH 7.6, 1 mM EDTA, 0.05% SDS) to obtain 10 μg of mRNA.

(2) Preparation of Tobacco cDNA Library To the mRNA obtained as described above, an oligo dT primer was added using a cDNA synthesis kit (Amersham) to synthesize a double-stranded cDNA. This cDNA was inserted into the EcoRI site of lambda gt11. Then, the obtained recombinant DNA was used to coat the lambda phage coat protein (in v
(itro packaging kit Gigapack II Gold, Stratagene)
Recombinant lambda phage was obtained by mixing with and leaving it at 20 ° C. for 2 hours.

On the other hand, Escherichia coli Y-1990 strain was inoculated into 10 ml of LBMM medium (1% tryptone, 1% sodium chloride, 0.5% yeast extract, 10 mM magnesium sulfate and 0.4% maltose) and shake-cultured at 37 ° C. for 12 hours. . After the culturing, the bacterial cells were collected by centrifugation and suspended in 10 ml of 10 mM magnesium sulfate which had been cooled to 4 ° C. in advance to make them susceptible to lambda phage infection. The Escherichia coli thus obtained was mixed with the above recombinant lambda phage, allowed to stand at 37 ° C. for 15 minutes for infection, and suspended in LBMM medium containing 0.7% agar. Then spread on LBMM medium containing 1.5% agar, and at 37 ℃
It was cultured for 12 hours.

(3) Selection of Peroxidase Transcription Regulator Gene The recombinant lambda phage obtained as described above was infected with Escherichia coli Y-1090 and seeded at 2 × 10 4 cells / 130 cm ² , and when plaque appeared (about 4 hours), soak in a membrane filter that had been soaked in 10 mM IPTG and then air-dried,
After incubating at 37 ℃ for 2.5 hours, remove the membrane filter from the plate and use 5% skim milk in binding buffer (10 mM Hepes (pH7.5), 50 mM NaCl, 1 mM).
EDTA, 5% glycerol) was used for blocking at 4 ° C. for 1 hour.

Then, the membrane filter was lightly washed with a binding buffer, and a binding reaction was carried out at 4 ° C. for 1 hour in the binding buffer containing the probe. As a probe for selection, a synthetic double-stranded oligonucleotide-TAAACACGTGATAT corresponding to a region important for expression of the horseradish peroxidase prxC2 gene was used.
-Labeled with nick translation was used.

After that, the membrane filter was washed with binding buffer three times at room temperature for about 30 minutes, and autoradiography was performed. As a result of primary screening and secondary screening, 5 × 10 ⁵ phage plaques
Two positive clones were obtained. This was designated as a transcription regulatory gene TFHP-1.

(4) Determination of Nucleotide Sequence of Selected Transcriptional Regulatory Gene From the positive clone phage DNA thus obtained, the insert DNA was amplified by PCR using the primers of the lambda gt11 cloning site. As a result, a band of about 1 kbp was confirmed by agarose gel electrophoresis. After treating this insert DNA with T4 DNA polymerase and T4 DNA kinase, the plasmid pBluescrip
It was incorporated into the EcoRV site of t SK ⁺ .

SEQUE based on the dideoxy method using this plasmid
The nucleotide sequence of the obtained plasmid was determined using NASE ^™ version 2.0 (manufactured by USB), and the sequence is shown in SEQ ID NO: 1 in Table 1 below.

[Table 1] When the determined nucleotide sequence of SEQ ID NO: 1 was searched, it was estimated to encode a protein consisting of 234 amino acids. In addition, the secondary structure of the protein encoded by TFHP-1 was analyzed by Robson's method, and helix-lo
It was presumed to have an op-helix (HLH) structure. This HLH
The structure is composed of two amphipathic α sandwiching 9 to 20 amino acid residues.
-Helix structure, which binds to DNA
It is known for mammalian transcription genes. This second h
The elix domain is a myoD involved in mammalian muscle cell differentiation
Was found to be homologous.

(5) Construction of Recombinant Plasmid for Confirming Expression and Transcriptional Regulatory Activity Next, in order to express the obtained transcriptional regulatory gene TFHP-1 in plants, pBI221 (Clontach, Inc.) having a cauliflower mosaic virus 35S promoter was expressed. A DNA fragment containing TFHP-1 was incorporated at the position of the β-glucuronidase gene (in the product) in the sense direction or the antisense direction, and this was used as an effector.

As a reporter, horseradish peroxidase
Approximately 500 bp of promoter region of prxC2 gene was combined with β-glucuronidase (GUS) structural gene to obtain pUC19
I used it. The construction diagram is as shown in FIG. The obtained recombinant plasmid was designated as competent high HB1.
01 (Toyobo Co., Ltd.) was used to introduce into Escherichia coli HB101 to obtain a transformant. This transformant was cultured at 37 ° C. in 1 liter of LB medium (1% tryptone, 0.5% yeast extract, 0.5% sodium chloride) and then isolated and purified according to a conventional method to give about 1 mg of plasmid D.
I got NA.

(6) Confirmation of expression transcription regulation activity The expression transcription regulation ability of the transcription gene TFHP-1 cloned as described above was examined by an electroporation method using tobacco protoplasts. As described in detail below, the above method is a method in which protoplasts of cultured tobacco cells BY-2 are charged to incorporate DNA and the transient expression thereof is examined.

First, from the cultured tobacco cells BY-2, the method of Okada et al. (K. Okada
Et al .: Plant Cell. Physiol., Vol. 27, 619, 1986). This protoplast is about 3x
10 ⁶ cells were electroporated in buffer (5 mM MES, 70 mM
M KCl, 0.3M mannitol, pH 5.8) Adjust to 1 ml and
Combination of types (reporter 20μg, reporter 20μ
An electric pulse (300 V, 0.2 msec.) was applied twice to g + sense effector 20 μg, reporter 20 μg + antisense effector 20 μg) using the gene transfer device GTE-10 (manufactured by Shimadzu Corporation).

Then, the protoplasts to which the electric pulse was applied were washed with 0.4 M mannitol, and then a medium for protoplasts (mixed salts for Murashige-Skoog medium (manufactured by Nippon Shinyaku Co., Ltd., to which 0.4 M mannitol was added) was added, and a diameter of 6 cm The cells were cultured for 24 hours at 25 ° C in a plastic Petri dish. After that, the protoplasts are crushed and the method of Jefferson et al. (RAJeff
β-glucuronidase activity was measured by erson et al., EMBO. J., vol. 6, p. 3901-3907, 1987).

As a result, compared to the 20 μg reporter only, 20
It was confirmed that the expression of the GUS gene was induced in the case of simultaneous introduction of μg of the sense effector, and the activity was remarkably decreased in the case of protoplasts containing 20 μg of the antisense effector. This proved that the transcriptional regulatory gene of the present invention can transcriptionally regulate the induction and repression of the expression of horseradish peroxidase prxC2 gene.

[Brief description of drawings]

FIG. 1 is a construction diagram of plasmid pUC19.

Continuation of front page (51) Int.Cl. ⁶ Identification code Office reference number FI technical display area C12R 1:19) (C12N 9/08 C12R 1:91)

Claims (3)

[Claims] 1. A peroxidase transcriptional regulatory gene having the nucleotide sequence shown in SEQ ID NO: 1. 2. A recombinant plasmid containing a peroxidase transcriptional regulatory gene having the nucleotide sequence shown in SEQ ID NO: 1. 3. A method for producing a recombinant vector which utilizes a peroxidase transcriptional regulatory gene having the nucleotide sequence shown in SEQ ID NO: 1 and a plant-expressible promoter.