JP2002272469A - A cis-regulatory sequence showing the ability to regulate DNA damage-responsive gene expression in higher plants - Google Patents

Abstract

(57) [Summary] (with correction) [PROBLEMS] To provide a cis-regulator derived from a higher plant, which activates transcription in response to DNA damage. SOLUTION: A DNA damage responsive promoter. This promoter is (a) Arabidopsis thaliana (Arabido
(b) DNA comprising a base sequence in which one or several bases have been deleted, substituted or added in the above base sequence and exhibiting DNA damage responsiveness; or And (c) a DNA that hybridizes with a DNA consisting of the above-mentioned nucleotide sequence under stringent conditions and shows DNA damage responsiveness.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a nucleotide sequence that functions as a cis-regulator that expresses the ability to activate transcription in response to DNA damage, and its use.

[0002]

2. Description of the Related Art Organisms are provided with a group of genes encoding DNA repair factors that are expressed in response to DNA damage.
DNA repair related factors were first found in microorganisms and animals. In higher plants, various DNA repair-related factors have been identified based on homology with known DNA repair-related factors in microorganisms and animals. Some of the higher plant DNA repair related factors identified from homology have been shown to respond to the stimulus of DNA damage. However, the expression of DNA repair-related factors
The mechanism induced by the injury is unknown. Furthermore, no information has been obtained on the regulatory factors of DNA repair-related genes involved in the induction of DNA damage responsiveness.

[0003] Studies on the mechanism of induction of DNA damage responsiveness for DNA repair-related factors have preceded higher microorganisms and animals than higher plants. But,
Mechanism of DNA damage responsiveness induction of microorganisms and animal DNA
Comparing with the mechanism of injury responsive induction, no commonalities between them are found. Therefore, it is extremely difficult to infer the mechanism of induction of DNA damage responsiveness in higher plants from knowledge of the mechanism of induction of DNA damage responsiveness in microorganisms and animals.

In higher plants, Arabidopsis RAD5
One gene has been shown to be transcriptionally activated in response to DNA damage (Doutriaux et al., Mo.
lGen Genet 257: 283-291 (19
98)). However, it is not known how the RAD51 gene responds to DNA damage.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to provide a
A To provide a cis-regulator derived from higher plants that results in transcriptional activation in response to A-damage. That is, to identify a DNA damage-responsive cis-regulatory sequence, and to construct a plant expression cassette and a recombinant plasmid for expressing a heterologous gene using the same, and to specifically express in response to DNA damage. The present invention provides a method for introducing a desired heterologous gene into a plant, and a plant cell and a plant transformed using this method.

[0006]

Means for Solving the Problems The present inventors have found that the RAD51 promoter of Arabidopsis thaliana expresses transcriptional activating ability in response to DNA damage stimulation,
From deletion / substitution analysis of 51 promoters, 44
The present inventors have found that a promoter fragment consisting of base pairs has DNA damage responsiveness, and based on this, completed the present invention.

[0007] The present inventors have found that an element responsive to DNA damage exists in the promoter region upstream of the RAD51 gene. For example, a DNA having the nucleotide sequence of SEQ ID NO: 1 is converted to a DNA that is unrelated to the DNA damage response.
T of cauliflower mosaic virus 35S promoter
DNA containing an ATA box, a DNA containing a base sequence of 46 base pairs upstream from the transcription start point with the transcription start point being 1, and DN linked to a DNA encoding a reporter gene
The A fragment is transformed into a plant cell, and the plant cell
The damage was shown to increase the reporter gene expression as compared to the case without DNA damage. Therefore, DNA having the sequence of SEQ ID NO: 1
It was revealed that it has a sufficient function as an A damage responsive cis regulator.

[0008] The DNA damage-responsive promoter of the present invention comprises: (a) a DNase having the nucleotide sequence of SEQ ID NO: 1;
A; (b) a DNA consisting of a base sequence in which one or several bases are deleted, substituted or added in the base sequence represented by SEQ ID NO: 1 and exhibiting DNA damage responsiveness; or (c) SEQ ID NO: 1 DN consisting of the base sequence represented by
A that contains DNA that hybridizes with A under stringent conditions and exhibits DNA damage responsiveness.

[0009] In one embodiment, the promoter of the present invention comprises a deletion or substitution of one or several bases in the base sequence of positions 1 to 11 and 18 to 44 of the base sequence shown in SEQ ID NO: 1. Alternatively, it may be a DNA consisting of an added base sequence and showing DNA damage responsiveness.

[0010] In one embodiment, the promoter of the present invention is obtained by deleting or substituting one or several bases in the base sequence at positions 19 to 24 and 38 to 44 of the base sequence shown in SEQ ID NO: 1. Alternatively, it may be a DNA consisting of an added base sequence and showing DNA damage responsiveness.

The plant expression cassette of the present invention is a plant expression cassette for expressing a heterologous gene in response to DNA damage. The plant expression cassette of the present invention comprises the above promoter; and a site for inserting the heterologous gene so that the heterologous gene is operably linked to the promoter.

[0012] The recombinant plasmid of the present invention is a recombinant plasmid for expressing a heterologous gene in response to DNA damage. The recombinant plasmid of the present invention comprises the above-mentioned promoter; and a heterologous gene operably linked to the promoter.

[0013] In one embodiment, the heterologous gene is
It may have a DNA damage resistance function. In another embodiment,
The heterologous gene may be a gene encoding a recombinase or a reporter gene.

The method of the present invention is a method for transforming a plant cell with a heterologous gene whose specific expression is desired in response to DNA damage. The method of the present invention includes a step of transforming a plant cell with the above-mentioned recombinant plasmid to obtain a transformed plant cell.

[0015] In one embodiment, the heterologous gene is
It may have a DNA damage resistance function. In another embodiment,
The heterologous gene may be a gene encoding a recombinase or a reporter gene.

In one embodiment, the plant cell may be a monocotyledonous plant cell.

[0017] In one embodiment, the plant cell may be a dicotyledonous plant cell.

The transformed plant cell of the present invention can be obtained by the above method.

The plant of the present invention can be obtained by redifferentiating the above plant cells.

[0020]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.

[0021] DNA damage responsive promoter of <DNA damage responsive promoter> The present invention includes a DNA having the nucleotide sequence represented by SEQ ID NO: 1. The sequence of SEQ ID NO: 1 is 5'-ACGTAATCGAGACTTGTTGA
AGAAGCCTTTGCCCTCATCGTCGT-
3 '.

The nucleotide sequence represented by SEQ ID NO: 1 was isolated from the upstream region of the RAD51 gene of Arabidopsis thaliana. The base sequence represented by SEQ ID NO: 1 is also referred to herein as a DNA damage responsive cis control sequence. The DNA damage responsive cis control sequence found in the present invention is D
In response to the stimulus of NA damage, it activates the expression of a gene operably linked to the sequence.

As used herein, the term "represents DNA damage responsiveness" refers to a promoter that is introduced into a plant in a natural plant or as an expression cassette linked to any structural gene. Damage to plant DNA means an increase in the amount of transcription of its structural gene.

[0024] "DNA damage" refers to a physical or chemical change in DNA caused by any factor. Such damage can occur either artificially or naturally. Examples of DNA damage include:
Base modification with an alkylating agent such as methyl methanesulfonic acid; abnormal bases such as uracil and hypoxanthine generated by deamination of cytosine and adenine; abasic sites generated as a result of a depurination reaction. These are examples of single base damage in DNA. DNA
Examples of the damages that cause damage to two bases include a dimer formed between adjacent pyrimidines on the same chain by ultraviolet light; a cross-linked structure between the two strands by mitomycin C, psoralen, or the like. Other examples of DNA damage include chain breakage due to phosphodiester bonds or sugar changes due to ionizing radiation such as gamma radiation. DNA
Examples of the agent that damages the above include bleomycin, aminobenzopyrene, acetylaminofluorene and the like in addition to the agents mentioned above. DNA damage also
It can also be caused by physical damage to cells, such as gene transfer by particle gun.

In the present specification, the promoter is "DN
The expression “A damage responsiveness” typically means that when DNA damage is given, the amount of a gene product linked to the promoter increases. In the promoter of the present invention, typically, the amount of a gene product is preferably about 10% or more, more preferably about 20% or more, and still more preferably about 30% by DNA damage.
Or more, more preferably about 40% or more, more preferably about 50% or more, more preferably about 60% or more, more preferably about 70% or more, and still more preferably about 80% or more.
As mentioned above, it is more preferably about 90% or more, more preferably about 100% or more. Here, “the amount of the gene product increases by 10%” means that the amount of the gene product becomes 110%, assuming that the amount of the gene product under the condition that DNA damage is not given is 100%. Here, the amount of the gene product refers to the amount of the gene product measured by any known method for measuring the amount of protein, for example, when the gene product is luciferase, the amount of luminescence measured by a luminometer.

The promoter of the present invention is not necessarily limited by the sequence of SEQ ID NO: 1, but consists of a base sequence in which one or several amino acids have been deleted, substituted or added to this sequence, and A promoter containing DNA exhibiting DNA damage responsiveness is also included in the present invention.

For example, in the nucleotide sequence represented by SEQ ID NO: 1 having a nucleotide sequence obtained by deleting or modifying a sequence that is not essential for exhibiting DNA damage responsiveness,
Promoters containing DNA that exhibits damage responsiveness are within the scope of the invention. Alternatively, for example, the sequence of SEQ ID NO: 1 can be modified to enhance the expression activity of the promoter. The variants thus obtained are also within the scope of the present invention, as long as they exhibit DNA damage responsiveness.

The promoter of the present invention preferably comprises one or several nucleotides in the nucleotide sequence of SEQ ID NO: 1 and the nucleotides at positions 1 to 11 and 18 to 44 in the nucleotide sequence of SEQ ID NO: 1. Has been deleted, substituted or added, or one or several bases have been deleted, substituted or added in the nucleotide sequence of positions 19 to 24 and 38 to 44 of the nucleotide sequence shown in SEQ ID NO: 1. It consists of the base sequence. It is preferable that the promoter of the present invention includes positions 12 to 17 of the nucleotide sequence represented by SEQ ID NO: 1. However, as long as the DNA damage responsiveness is exhibited, 1 at positions 12 to 17 of the nucleotide sequence represented by SEQ ID NO: 1
Alternatively, a promoter containing a DNA consisting of a base sequence in which several bases are deleted, substituted or added is also included in the scope of the present invention.

As used herein, the term "one or several" is not intended to mean only one or two, three, or five or six, but may include even more. One or several are preferably 1 to 40, more preferably 1 to 35, more preferably 1 to 30,
More preferably 1 to 25, more preferably 1 to 20
, More preferably 1 to 15, more preferably 1 to 1
0, more preferably 1 to 9, more preferably 1 to 8
, More preferably 1 to 7, more preferably 1 to 6
, More preferably 1 to 5, more preferably 1 to 4
, More preferably 1 to 3, more preferably 1 or 2.

Such a sequence can be obtained by performing a deletion / substitution experiment substantially according to Example 3 of the present invention.

The promoter of the present invention further includes, in addition to the above, a DNA which hybridizes with a DNA consisting of the nucleotide sequence of SEQ ID NO: 1 under stringent conditions and has a DNA damage responsiveness. A promoter is included.

As used herein, the term “stringent conditions” for hybridization refers to the nucleotide sequence of SEQ ID NO: 1 and other nucleotide sequences highly homologous thereto (for example, those present in plants other than Arabidopsis thaliana). RAD5
Conditions sufficient to form an oligonucleotide duplex with DNA encoding a homolog of one promoter). Representative examples of the stringent conditions applied to the present invention include the following conditions: 1M N
aCl, 1% SDS, 10% dextran sulfate, ³² P-labeled probe DNA (1 × 10 ⁷ cpm) and 50 μg
After hybridization at 60 ° C. for 16 hours in a solution containing / ml salmon sperm DNA, washing with 2 × SSC / 1% SDS at 60 ° C. for 30 minutes is repeated twice.

In order to isolate a DNA having the nucleotide sequence of SEQ ID NO: 1 and a promoter that hybridizes with the DNA under stringent conditions and exhibits DNA damage responsiveness, for example, in the present invention, for example, A primer pair corresponding to the nucleotide sequence shown in SEQ ID NO: 1 can be used. Using this primer pair, PCR is performed using plant cDNA or genomic DNA as a template, and then, using the amplified DNA fragment obtained as a probe, a cDNA library or genomic library of the same plant can be screened. . An example of such a primer pair is a 29-mer forward primer (AtR
AD51: 5'-GGAAGCTTCACCGGGTTT
GAACCCGGTTAG-3 ′ (SEQ ID NO: 2));
A 30-mer reverse primer (AtRAD51-D:
5'-GGCCATGGTCGTCATTTTCTCTC
Combination with AATCAGAG-3 ′ (SEQ ID NO: 3).

[0034] Thus, stringent hybridization conditions are applied to the present invention also can also be defined as a condition for PCR, in a typical example, the primers (SEQ ID NO: 2 and 3) may be used. At this time, the PCR reaction conditions were as follows: denaturation at 94 ° C. for 5 minutes;
4 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 1 second
The conditions may be such that 30 minutes are repeated for 30 minutes and finally incubation is performed at 72 ° C. for 7 minutes.

The PCR can be performed according to the manufacturer's guidelines for commercially available kits and devices, or by a method well known to those skilled in the art. Methods for preparing a gene library and cloning a gene are well known to those skilled in the art. For example, Sa
mbrook et al., Molecular Clonin
g: A Laboratory Manual, 2nd edition (Cold Spring Harbor Labor)
ateory, 1989). The nucleotide sequence of the obtained gene can be determined using a nucleotide sequence analysis method known in the art or a commercially available automatic sequencer.

The promoter isolated and identified by screening as described above (ie, SEQ ID NO: 1)
, And homologs) were confirmed to be responsive to DNA damage by generating plant cells transformed according to the disclosure herein and observing expression according to the assay described in Example 3. I can do it.

The promoter of the present invention also has at least 80% sequence identity, preferably at least 85% sequence identity, more preferably at least 85% sequence identity with the nucleotide sequence of SEQ ID NO: 1 as long as it can exhibit DNA damage responsiveness in plants. At least 90% sequence identity, even more preferably at least 95% sequence identity, most preferably at least 99%
And DNA containing a base sequence having the same sequence identity as described above. The term “sequence identity” means that the two base sequences compared are the same, and the percentage (%) of sequence identity between the two base sequences compared is the ratio of the two bases compared. After optimally aligning the sequences, the same number of identical nucleobases (eg, A, T, C, G, U, or I) occur in both sequences to obtain the number of matched positions, giving the number of matched positions, The number of positions determined is divided by the total number of comparison bases, and the result is multiplied by 100 to calculate. Sequence identity can be calculated, for example, using the following tools for sequence analysis: Unix
Base GCG Wisconsin Package
(Program Manual for the W
isconsin Package, Version
8, Genetics Comput, September 1994
er Group, 575Science Drive
Madison, Wisconsin, USA537
11; Rice, P. (1996) Program M
annual for EGCG Package, Pe
ter Rice, The SangerCentr
e, Hinxton Hall, Cambridge,
CB101Rq, Englamd) and the Ex
PASy World WideWeb Molecular Biology Server (Geneva University Hos
Pital and University of G
eneva, Geneva, Switzerland)
checking ...

[0038] The promoter in the present invention is not limited to those isolated from nature, and may include synthetic polynucleotides. A synthetic polynucleotide can be obtained, for example, by synthesizing or modifying a promoter sequenced as described above by a method well known to those skilled in the art. Synthetic polynucleotides include, for example, Applied
It can be synthesized using the BioSystems oligonucleotide synthesizer according to the specifications provided by the manufacturer.

<Measurement of Promoter Activity by Measurement of LUC Activity or GUS Activity>
It can be ligated to a recombinant plasmid. In order to evaluate the activity of the ligated promoter, a plasmid can be prepared in which a reporter gene, for example, a gene encoding an appropriate enzyme, is ligated downstream of the promoter. This plasmid is introduced into a plant cell, and the expression of the reporter gene is observed, for example, by measuring the enzyme activity.
When a plant is used as a host, for example, luciferase (LU
It is common to measure the expression of C) or β-glucuronidase (GUS) as an index. Measurements of luciferase can be performed without killing the cells, it is preferable to use a luciferase (LUC) as the reporter gene. Also in the present specification, a method of measuring the expression of LUC can be applied.

LUC activity is determined by the method of Matsuo, N .; Et al., P
lant Biotechnology 18, 71-
75 (2001) and Mill.
ar, A. Et al., Plant Mol. Biol. Rep
tr. , 10: 324-337 (1992). For example, 200 μl of cells were
Transfer to a 5 ml microcentrifuge tube and centrifuge for 1 minute at about 10,000 g and remove the liquid medium. Cells were washed with 250 μl of ice-cold passive lysis buffer (Prom
ega), homogenized with a pestle and clarified by centrifugation at 4 ° C. at 10,000 g for 1 minute to obtain a cell lysate. Other plant tissues,
Homogenize in 2 ml of passive lysis buffer using an ice-cold pestle and mortar. For measurement of luciferase activity, 10 μl of cell lysate was added to 50 μl LARII
Transfer to a luminometer containing the solution (Promega), mix by tapping, and place in luminometer. Then, 50 μl of Stop & Glo solution (Promega) is added to the reaction mixture for detection of Rluc activity. Each luciferase activity is measured for 10 seconds in a luminometer. In this way, luciferase activity can be measured.

GUS activity was determined by the method of Jefferson et al. (EMBO J 6: 3901-3907 (198).
7)) can be measured basically according to. For example, a protoplast extract equivalent to 50 μg in protein amount and 2
5 μl of 20 mM 4-methylumbelliferyl glucuronide (4MUG), plus extraction buffer, and 100 μl methanol to suppress GUS-like activity endogenous to plant tissue (Kosuki et al., Plant Science)
e 70: 133-140 (1990)) and make up to 500 μl and incubate at 37 ° C.
After 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. Enzyme activity was 4-MU pmol / min /
Express in mg protein.

Various variants of the promoter of the present invention can also be evaluated for their activity as promoters, as described above. Based on the findings described herein, for example, RAD51 deleted to various lengths from the 5 ′ upstream side
The promoter activity can be measured using a plasmid obtained by fusing the promoter region with the LUC gene or GUS gene, and a part essential for the activity can be identified. Techniques for identifying such active moieties per se are known to those skilled in the art.

<Construction of Expression Cassette and Recombinant Plasmid> The DNA damage-responsive promoter in the present invention is prepared by a method well known to those skilled in the art so that the heterologous gene is operably linked to the promoter. It can be used as an expression cassette containing a site for inserting a gene. This expression cassette is used to convert a heterologous gene into DNA.
It can be used to express in response to injury. This expression cassette can be used to transform a plant cell using a known gene recombination technique.

Similarly, the DNA damage responsive promoter of the present invention can be used as a recombinant plasmid containing the promoter and a desired heterologous gene operably linked to the promoter. This plasmid can be used to express a heterologous gene in response to DNA damage. This recombinant plasmid can be used to transform plant cells.

This expression cassette can also be linked to a second promoter to enhance the expression of the promoter according to the invention. "Second promoter" refers to any promoter expressed in plants, and may include any of a constitutive promoter, a tissue-specific promoter, and an inducible promoter.

"Constitutive promoter" refers to a promoter that allows a structural gene to be expressed at a certain level regardless of stimuli inside and outside a plant cell. The use of a constitutive promoter is convenient and preferred if the heterologous gene does not confer undesired traits on the plant when expressed in other tissues or organs of the plant. Examples of constitutive promoters include, but are not limited to, the cauliflower mosaic virus (CaMV) 35S promoter (P35S) and the nopaline synthase promoter (Tnos).

In the present invention, the term "tissue-specific promoter" refers to a promoter that causes a structural gene to be specifically expressed in at least a specific tissue. Examples of such tissue-specific promoters include the Arabidopsis thaliana CAB1 gene promoter, which exhibits both tissue specificity and photoresponsiveness;
B. , An, G .; 1988. Identificat
ion of upstream regulator
y elements involved inthe
developmental expression
of the Arabidopsis thalia
na cab1 gene. Proc. Natl. Ac
ad. Sci. USA. , 80: 8017-8021).
Therefore, the expression cassette itself containing the DNA damage responsive promoter and the site for inserting the heterologous gene so that the heterologous gene is operably linked to this promoter can be combined with other regulatory elements as necessary. Use within the scope of the present invention is also included in the scope of the present invention.

The “inducible promoter” refers to a promoter that causes a structural gene to be expressed when given a specific stimulus such as a chemical agent or physical stress, and does not show an expression activity in the absence of the stimulus. Examples of inducible promoters include the glutathione S-transferase (GST) promoter (van de
r Kop, D.E. A. Et al., Plant Mol. Bi
ol. , 39: 979, 1999), but is not limited thereto.

[0049] As used herein, the term "expression cassette"
Or "Plant expression cassette" includes a DNA damage responsive promoter of the present invention, the expressible heterologous gene and the promoter (i.e., as can control the expression of the DNA, i.e., "in-frame") And a site for inserting the heterologous gene so as to be linked.

As used herein, the term "site for inserting a heterologous gene" means any site that enables the inserted heterologous gene to be operably linked to the promoter of the present invention. Can be The site for inserting a heterologous gene can be, for example, any restriction enzyme site, for example, a multiple cloning site. The site for inserting a heterologous gene can be, for example, included in the linker or in a sequence that acts similarly to the linker. The linker can be preferably linked to the 3 ′ end of the DNA damage responsive promoter of the present invention.

The “heterologous gene” that can be inserted into the cassette of the present invention includes an endogenous gene in Arabidopsis thaliana other than the Arabidopsis thaliana RAD51 gene, an endogenous gene in a plant other than Arabidopsis thaliana, or a gene foreign to the plant (for example, (Genes derived from animals, insects, bacteria and fungi), the gene products of which it is desired to be expressed in response to DNA damage.

Preferred examples of the heterologous gene in the present invention are as follows: the gene product to be encoded exhibits a DNA repair function, an active oxygen scavenging function, and the like;
It is a gene showing NA damage resistance function. Specific examples of such a gene include, but are not limited to, a gene encoding a repair enzyme, a gene encoding an active oxygen scavenging enzyme, and a gene encoding an enzyme acting on recovery from cell damage.

Examples of the repair enzyme include photo-recovery enzyme, ultraviolet endonuclease, DNA glycosylase, AP-
Endonuclease, RecA protein, eukaryotic RecA-like protein group, RAD51-like protein, D
MC1-like protein, exonuclease V (recB
C enzyme), Ada protein, polymerase I, polymerase II, polymerase III, DNA polymerase β, DNA ligase, and 3 ′ → 5 ′ exonuclease, and homologs of these enzymes. The sequences of the genes encoding these repair enzymes are known to those skilled in the art and can be easily obtained.

An example of the active oxygen scavenging enzyme is superoxide dismutase (SOD). The sequences of the genes encoding these active oxygen elimination enzymes are as follows:
It is known to those skilled in the art and can be easily obtained.

Another example of a preferred heterologous gene in the present invention is a gene encoding a recombinant enzyme. Examples of such genes include recA, recB, rec
C, recE, recF, recJ, and recN. The sequences of these genes are known to those skilled in the art and can be easily obtained.

Another example of a preferred heterologous gene in the present invention is a reporter gene. Examples of reporter genes include luciferase (LUC) gene or β-
Glucuronidase (GUS) gene and green fluorescent protein (GFP) gene. The sequences of these genes are known to those skilled in the art and can be easily obtained. Luciferase and green fluorescent protein genes are preferable as reporter genes because their activities can be measured without killing cells. The luciferase gene is particularly preferred as a reporter gene.

Another example of a preferred heterologous gene in the present invention is a gene encoding a nuclease.

[0058] "Recombinant plasmid" refers to DNA in which, in addition to an expression cassette, various regulatory elements have been operably linked in cells of a host plant.
Suitably, it may include at least one of a terminator, a drug resistance gene, and an enhancer. It is well known to those skilled in the art that the type of recombinant plasmid and the type of regulatory element used can vary depending on the host cell. The recombinant plasmid used in the present invention may further have a T-DNA region. The T-DNA region increases the efficiency of gene transfer, particularly when transforming plants with Agrobacterium.

The “terminator” is located downstream of the region encoding the protein of the gene,
Is a DNA containing a sequence involved in termination of transcription when transcribed into E. coli and addition of a polyA sequence. Terminators are known to contribute to mRNA stability and affect gene expression. Therefore, in order to improve the expression efficiency of the heterologous gene preferably comprises a terminator. Examples of terminators include:
Nopaline synthase gene terminator (Tno
s), and cauliflower mosaic virus (CaM
V) 35S terminator, but is not limited thereto. This terminator sequence, via a linker sequence having the multiple cloning site,
Can be combined with a promoter sequence.

It is desirable that the “drug resistance gene” facilitates selection of a transformed plant. Neomycin phosphotransferase II (NPTII) gene for conferring kanamycin resistance, hygromycin phosphotransferase gene for conferring hygromycin resistance, and the like can be preferably used.
It is not limited to these.

An "enhancer" is a DNA containing a sequence having an action of promoting transcription from a promoter.
In general, an enhancer can have a transcription promoting effect even at a large distance from a promoter that exerts its action. Therefore, enhancers are thought to act on trans.

[0062] The recombinant plasmid of the present invention can be prepared using a gene recombination technique well known to those skilled in the art. The construction of recombinant plasmids, for example, although a vector of vectors or pUC-type pBI-type is preferably used, but not limited to.

<Transformation of Cells and Plants> Using the expression cassette constructed as described above or a recombinant plasmid containing the same, by a known gene recombination technique,
The desired plant cells can be transformed. The expression cassette used for the transformation is integrated into the DNA in the plant cell. The DNA in plant cells includes not only chromosomes but also DNAs contained in various organelles (eg, mitochondria, chloroplasts) contained in plant cells.

As used herein, the term “plant” includes both monocotyledonous plants and dicotyledonous plants. Preferred plants are dicotyledonous plants. The dicotyledonous plant includes both a subphylum and a joint subphylum. Monocotyledons include communis and lilies.

Examples of the eyes contained in the joint flower subclass are the orders of Gentianes, Eggplants, Lamies, Agonaceae, Psyllium,
Examples include the order Lepidoptera, Scrophulariformes, Rubiaceae, Pseumatoids, and Asteraceae. As an example of the order contained in the subphyllophyceae, there may be mentioned Euphorbiaceae. Preferred eyes are solanacea and euphoria. As an example of the order included in the communis, the order Cyperacea is included. Examples of the orders of the order Lilies include the orders Lilies and the Orchids.

Examples of the families contained in the order of the Solanaceae include the Solanaceae, the Parasitoids, the Hanashinobu, the Pondaceae, and the Convolvulaceae. An example of a family included in the order Euphorida includes Brassicaceae. Preferred families are Solanaceae and Brassicaceae. Examples of the family included in the order Cyperaceae include the Poaceae family. An example of a family included in the order Lilies includes the lily family.
An example of a family included in the order Orchid includes orchids.

Examples of the genera included in the Solanaceae include tobacco, tomato, eggplant, petunia, datura, capsicum, physalis, and wolfberry. Preferred genera are the genera Tobacco, Tomato, Eggplant, Petunia, and Datura, more preferably Tobacco, Tomato, and Eggplant.

Examples of the genera included in the Brassicaceae include Brassica, Arabidopsis and Wasabi, with the preferred genus being Brassica and Arabidopsis.

Examples of the genera included in the lily family include the genus Allium and the lily genus.

Examples of the genera included in the orchid family include the genus Cecoc, the genus Phalaenopsis, and the genus Cattleya.

Examples of preferred species in the genus Tobacco include:
Nicotiana tabacum species.

Examples of preferred species in the genus Tomato include:
Lycopersicon esculentum species.

Examples of preferred species in the genus Solanum include S
olanum melongena species.

Examples of preferred species of Brassica include Brassica napus, Brassic
a juncea species, Brassica campes
tris species and Brassica oleracea
Seeds.

Preferred examples of the Arabidopsis genus include Arabidopsis thaliana.

Examples of preferred species of the genus Allium include A
allium fistulosum species, Allium
fistulosum species, Allium chinen
sis species, Allium schoenoprasum
Species, Allium tuberosum species and All
ium cepa species.

Examples of preferred species in the genus Lily include L
ilium lancifolium species, Lilium
longiflorum species, Liium conc
color species, Lilium speciosum species and Lilium regulare species.

Examples of preferred species of the genus Cattleya include Cattleya bicolor.

Unless otherwise indicated, a “plant” refers to a plant in a seedling state, a plant in an immature state of development (ie, a plant before flower bud formation), a plant with flower buds,
Includes plants with seeds and seeds obtained from the plants.

Examples of “plant cells” include cells of various tissues in plant organs such as flowers, leaves, and roots, calli, and suspension cultured cells.

Methods for transforming plant cells using the recombinant plasmid include methods well known to those skilled in the art, for example,
Examples include an Agrobacterium-mediated method and a method of direct introduction into cells.

As a method via Agrobacterium, for example, the method of Nagel et al. (FEMS Micr)
obiol. Lett. , 67: 325 (1990)).
Can be used. In this method, Agrobacterium is first transformed with a recombinant plasmid (for example, by electroporation), and then the transformed Agrobacterium is introduced into a plant cell by a well-known method such as a leaf disk method. Is the way.

Examples of a method for directly transforming cells with the recombinant plasmid include an electroporation method, a particle gun method, a calcium phosphate method, and a polyethylene glycol method. These methods are well known in the art, and methods suitable for plants to be transformed are:
It can be appropriately selected by those skilled in the art.

The plant cells transformed with the recombinant plasmid are selected on the basis of, for example, drug resistance such as kanamycin resistance. The selected cells can be regenerated into a plant by a conventional method.

[0085] The function of the promoter of the present invention in the regenerated plant can be confirmed by a method well known to those skilled in the art. For example, confirmation of the expression of a heterologous gene
Usually, it can be performed using Northern blot analysis using RNA extracted from the target tissue as a sample. The procedure for this analysis is well known to those skilled in the art.

In addition, the fact that the heterologous gene introduced by the method of the present invention was expressed in response to DNA damage means that, for example, a plant expressed by a recombinant plasmid in which a promoter and an LUC gene are operably linked. In the body,
The distribution of LUC activity in any tissue can be confirmed using a luminometer according to a conventional method. Alternatively, the distribution of GUS activity in an arbitrary tissue in a plant obtained by transformation with a recombinant plasmid in which a promoter and a GUS gene are operably linked is subjected to histochemical staining by a conventional method. You can check it.

The transformed plant cell of the present invention can be used as a sensor for a chemical or physical factor that causes DNA damage. For example, the transformed plant cells of the present invention are cultured in a medium containing a substance that may cause DNA damage and in a medium not containing the substance, and the expression of the heterologous gene in each medium is measured. Can be determined to determine whether the substance causes DNA damage. If the expression of a heterologous gene is increased by the presence of the substance, the substance is
Damage. Alternatively, in a specific situation, the transformed plant cell of the present invention is put therein, cultured, and the expression of the heterologous gene in each medium is measured. By comparing the results, it is determined whether the situation causes DNA damage. May be determined. If the expression of a heterologous gene under that particular situation is increased compared to normal conditions, that situation causes DNA damage to the plant cells.

A DNA damage resistance gene was introduced and DN
Plant cells or plants that express the A damage resistance gene in response to DNA damage have increased resistance to DNA damage by expressing the DNA damage resistance gene. Such plant cells and plants have various uses. For example, it can be suitably used for the purpose of reducing gene instability due to UV-B.

A plant cell or a plant into which a gene encoding a recombinase has been introduced and which expresses the recombinase in response to DNA damage has improved recombination efficiency by expressing the recombinase. Such plant cells and plants have various uses. For example, in transformation,
And it can be suitably used as a breeding material.

The production of the above-mentioned transformed plant is also carried out by DNA
It can also be used to impart traits other than damage resistance to plants. For example, the use of a heterologous gene encoding a nuclease can be expected to increase the frequency of gene recombination.

The production of any useful plants and useful plant cells utilizing DNA damage responsive gene expression is within the scope of the present invention.

[0092]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below based on embodiments. The scope of the present invention is not limited only to the examples. Restriction enzymes used in the Examples, plasmid etc. are available from commercial sources.

<Example 1: Isolation of promoter of RAD51 gene> Sequence of known RAD51 gene of Arabidopsis thaliana (Doutriaux et al., Mol Gen Ge
net 257: 283-291 (1998)) and a pair of primers (AtRAD51-U; 5'-G
GAAGCTTCACCGGTTTTGAACCCGGT
TAG-3 '(SEQ ID NO: 1) and AtRAD51-
D; 5'-GGCCATGGTCGTCATTTTCTC
TCAATCAGAG-3 ′) was synthesized. AtRAD
51-U is a forward primer, which has an H
It has an indIII site (AAGCTT). AtRA
D51-D is a reverse primer and has an NcoI site (CCATGG) at the 5 ′ end. When this primer pair is used, positions −648 to +58 of RAD51 are amplified. Position +58 encodes the G of the start codon ATG.

According to a conventional method, Arabidopsis thaliana Col1-
O strain (Arabidopsis thaliana C
ol1-O) was prepared. Using the above primer pair and this DNA as a template, PC
R was performed to obtain an amplified DNA fragment. The PCR reaction conditions were as follows: denaturation at 94 ° C for 5 minutes, 30 cycles of 94 ° C for 30 seconds, 55 ° C for 30 seconds and 72 ° C for 1 minute, and finally incubation at 72 ° C for 7 minutes. Was performed.

The amplified DNA was sequenced according to a conventional method to confirm that the target site was amplified.

GUS of pBI221 (Clontech) often used as a gene expression vector for higher plants
By replacing the coding region with the LUC + fragment from pSPluc + (Promega), pBI2
21-LUC + was made. D obtained by PCR
The NA fragment is cut with HindIII and NcoI,
According to a conventional method, the expression vector plasmid p
BI221-LUC + (Luc at the cellular level
Method for Observing Activity Cell Engineering Separate Volume Plant Cell Engineering Series 680-84 (1997)) cauliflower mosaic virus 35S promoter site (ie, Hind)
III-NcoI site) to produce a recombinant plasmid (FIG. 1). This plasmid contains the Arabidopsis RAD51 promoter (AtRAD51 pr
o. ) Is a plasmid in which a firefly luciferase gene is linked to the 3 ′ side, and a nopaline synthase terminator is linked to the 3 ′ side of the firefly luciferase gene.

In the same manner, a plasmid for an internal standard was prepared (FIG. 1). First, pRL-null (Prome
ga) renilla luciferase coding region
Digest with Xbal Blunt and BglII, then p35SGFP (Clontech) SacI
It was excised by cloning into the blunt-BglII site. The intron sequence from pRL-null was then used to convert this plasmid into NheI and Sma.
After digestion with I, it was removed by self-ligation.
In this way, a plasmid was obtained in which the Renilla luciferase gene was ligated to the 3 'side of the 35S promoter and the nopaline synthase terminator was ligated to the 3' side of the Renilla luciferase gene.

As a control, ArLIM1 was used.
The firefly luciferase gene is ligated to the 3 'side of 5 (promoter of a meiosis-inducible gene encoding an E. coli RecA-like gene similar to RAD51) or Cab1 (light-inducible promoter from Arabidopsis thaliana), and A plasmid was constructed in which a nopaline synthase terminator was linked to the 3 'end of the firefly luciferase gene.

The thus prepared plasmid containing any of the luciferase genes was simultaneously introduced into cultured tobacco cells (BY-2) using a gene gun. The experiments were performed in six replicates. Specifically, the plasmid was introduced as follows. Liquid medium (Nagata, N., Oka
da, K .; Takebe, I .; , Matsui,
C. 1981. Delivery of tobac
co mosaic virus RNA into
plant protoplasts mediate
d by reverse-phase evapor
ation vehicles (liposome
s). Mol. Gen. Genet. , 184: 161
-165), 5 day tobacco BY-2 cells (2 ml) were spread on 3% agar plates and lightly dried to remove excess liquid to prepare plates of cultured tobacco cells. Gold particles (1.6 μm in diameter)
mg of plasmid DNA. The model PDS-1000 / He particle delivery system (Bio
Rad) was used to introduce DNA-coated gold particles into cultured tobacco cells. For gene transfer, a plate of cultured tobacco cells was placed 6-9 cm away from the stopping screen and 1100 ps
Bombardment was performed under reduced pressure of i. After bombardment, cells were collected using 2 ml of liquid medium and transferred to flasks.

The resulting culture containing the transformed tobacco cultured cells was divided into two, and 50 μg / ml of bleomycin was added to one of the flasks as a DNA damage treatment section, followed by culturing for 24 hours. The other flask was cultured for 24 hours without the addition of bleomycin to give an untreated section. After the culture, each transformed tobacco cultured cell
atsuo, N .; Et al., Plant Biotechno
Luciferase activity was measured according to the method described in J. Logy 18, 71-75 (2001).

Specifically, luciferase activity was measured as follows. First, a culture solution containing 200 μl of the transformed tobacco culture cells was transferred to a 1.5 ml microcentrifuge tube,
Then, centrifugation was performed at 10,000 g for 1 minute, and the liquid medium was removed. The cells are lysed in 250 μl of ice-cold passive lysis buffer (Promega), homogenized with a disposable plastic pestle (Kontes) and clarified by centrifugation at 10,000 g for 1 min at 4 ° C. To obtain a cell lysate. 2 ml of other plant tissue using an ice-cooled pestle and mortar
Homogenize in a passive lysis buffer. For measurement of luciferase activity, 10 μl of cell lysate was
l Luminometer containing LARII solution (Promega) (Model LB9501; Berthold)
And mix by tapping, then place in a luminometer. Then, 50 μl of Stop & Gl
o solution (Promega) is added to the reaction mixture for detection of Rluc activity. Each luciferase activity is measured for 10 seconds in a luminometer.

The results are shown in FIG. This figure shows the relative values when the average value of the untreated expression level in each promoter is set to 1. As a result, luciferase activity
Clear induction was observed in the bleomycin-treated group. No expression induction was observed for the promoters used as controls (35S promoter, ArLIM15 and Cab1) (FIG. 2).

Accordingly, this DNA fragment was found to be a promoter responsible for transcriptional activation in response to DNA damage.

Example 2 Responsiveness of RAD51 Gene Promoter to DNA Damage in Tobacco and Arabidopsis thaliana A plasmid having the same structure as that of Example 1 was used. For tobacco cells, see Matsuoka, K. et al. And Nakam
ura, K .; (1991) Proc. Natl. Aca
d. Sci. USA 88, 834-838, for Arabidopsis thaliana cells and Cloug.
h and Bent (1998) Plant J. 16:
According to the method described in 735-43, the cells were introduced into tobacco cells and Arabidopsis cells using the Agrobacterium method to obtain transformed tobacco cells and transformed Arabidopsis cells.

The luciferase activity of these cells was measured in the same manner as in Example 1. As a result, clear transcriptional activation was observed by bleomycin treatment.

Example 3 RAD Gene Promoter Deletion / Substitution Analysis A primer having any one of the following sequences (i) to (vii) was synthesized. (I) AtRAD51-248 (5'-GGAAGCT
TCTTAATTAATAATAAAAAATGAGGA
Ii) AtRAD51-198 (5'-GGAAGC
TTATAGAATAAGAATTTGGGCTTT-
3 ′; SEQ ID NO: 4; 30 mer); (iii) AtRAD51-183 (5′-GGAAG
CTTGGGCTTTAATAGCCCTTTAAAAG-
3 ′; SEQ ID NO: 5; 29 mer); (iv) AtRAD51-169 (5′-GGAAGC
TTCCTTTAAAGCCCATATGATCA-
3 ′; SEQ ID NO: 6; 30 mer); (v) AtRAD51-124 (5′-GGAAGCT
TACGTAATCGAGACTTGTTGAAG-
3 '; SEQ ID NO: 7; 30 mer); and (vi) AtRAD51-80 (5'-GGAAGCT
TCTTGTATAATAATTTTGGGTTGTG-
3 '; SEQ ID NO: 8 (31 mer).

One of these primers was used as a forward promoter, the sequence of SEQ ID NO: 3 was used as a reverse primer, and the RAD51 promoter sequence at positions −648 to +58 prepared in Example 1 was used. Example 1 using the recombinant plasmid containing
PCR was performed under the same conditions as described above to obtain various promoter fragments. When the primer (i) was used, -248
When the promoter fragment at positions +58 to +58 was used with the primer (ii), the promoter fragment at positions −198 to +58 was used, and when the primer at (iii) was used, the promoter fragment was −183 to +58. When the promoter fragment of (iv) used the primer of (iv),
When the promoter fragment at positions +58 to +58 is used with the primer (v), the promoter fragment at positions −124 to +58 is used, and when the primer at position (vi) is used, the promoter fragment at positions −80 to +58 is used. When the primer fragment of (vii) was used, the promoter fragment at positions −56 to +58 was amplified.

In order to obtain a promoter fragment at positions −56 to +58, the −64 prepared in Example 1 above was used.
The recombinant plasmid containing the RAD51 promoter sequence at positions 8 to +58 was digested with NcoI and HaeII to cut out the promoter fragment at positions −56 to +58.

In the same manner as in Example 1, a plasmid was prepared by linking each of these promoter fragments to the firefly luciferase gene and the nopaline synthase terminator, and the promoter activity was examined. The experiment was performed in six replicates.

FIG. 3 shows the case where promoter fragments from -124 to +58, -80 to +58 and -56 to +58 were used (respectively -124 and -58, respectively). 80 and -56). In FIG. 3, the position from -648 to +
The relative activity is shown assuming that the average activity in the untreated section using the promoter up to position 58 is 100. As a result, in the range from position -648 to position -124 upstream of the expected transcription start point, the promoter activity gradually decreased, but the ability to respond to DNA damage by bleomycin treatment was maintained. However, the deletion up to position -80 significantly reduced the promoter activity and at the same time reduced the ability to respond to DNA damage. This suggests that a sequence responsible for DNA damage responsiveness exists between the -124 and -80 positions.

To further confirm this, the promoter fragment at positions -124 to +58 and the promoter fragment at positions -80 to +58
Upstream of each of the 58th promoter fragment, 35S
A plasmid was prepared by ligating the B domain of the promoter. The activity of this plasmid was measured in the same manner as described above.

The results are shown in FIG. In FIG. 4, the average activity of the untreated section of the promoters at positions −648 to +58 is 1
The relative activity is shown as 00. As a result, in the promoter fragment at positions −124 to +58 (denoted as −124), a DNA damage response was observed at the same time as an increase in activity,
Only an increase in activity was observed for the promoter fragment at positions −80 to +58 (denoted as −80).

From the above findings, it was revealed that the cis factor necessary for DNA damage exists in the region from -124 to -80 (FIG. 5).

<Example 4: DN of promoter of the present invention>
A Damage responsiveness> The following pair of primers were synthesized.

AtRAD51-124 (5'-GGAA)
GCTTACGTAATCGAGACTTGTTGAA
G-3 ′; SEQ ID NO: 7; 30 mer); r51gWT R
v (5'-GGAGATCTACGACGATGAGG
GCAA-3 '; SEQ ID NO: 9; 24-mer).

AtRAD51-124 was used as the forward promoter, r51gWT Rv was used as the reverse primer, and -64 prepared in Example 1 above.
By performing PCR under the same conditions as in Example 1 above using a recombinant plasmid containing the RAD51 promoter sequence at positions 8 to +58 as a template, a 44 base pair promoter fragment from positions -124 to -80 was amplified.

This promoter fragment was ligated to a minimal promoter region containing only the TATA box region of the 35S promoter, which was unrelated to the DNA damage response.
T-N46 was obtained.

As a control, a plasmid N46 was prepared in which only the minimal promoter region was ligated to the firefly luciferase gene and the nopaline synthase terminator without ligating the promoter fragment.

The responsiveness to DNA damage of these plasmids was examined in the same manner as in Example 1. FIG. 6 shows the results. As a result, when the promoter fragment at positions -124 to +81 was used, DNA damage was caused, and clear transcriptional activation was shown. (FIG. 6). Therefore, it was shown that this 44 base pair DNA fragment was a cis control sequence having the ability to activate DNA damage-responsive transcription.

<Example 5: By site-directed mutagenesis
To examine the essential region in the promoter of the promoter of the analysis> invention of the present invention was performed site-directed mutagenesis. The details are as follows.

First, a primer for introducing a mutation was synthesized.

Six sequences C from positions -123 to -118
The following primer pairs were synthesized to mutate GTAAT to GGATCC. −124 Rv (5′-TG
GATCTCGAGCTTAAGCTTGGCGTAA
-3 '; SEQ ID NO: 10; 27 mer); and -123 /
-118 Fw (5'-GGATCCCGAGACTT
GTTTGAAGAAGCCTTTG-3 ′; SEQ ID NO: 1
1; 30 mer).

Six sequences G from -106 to -101
The following primer pairs were synthesized to mutate AAGAA to GGATCC. -105 / -101 Rv
(5'-GGATCCAACAAGTCTCCGATTA
CGTGGATCT-3 '; SEQ ID NO: 12; 30 mer); and -100 / N46 Fw (5'-GCCT
TTGCCCTCATCGGTCGTAGATCTCGC
-3 '; SEQ ID NO: 13; 29 mer).

Six sequences GC from positions -100 to -95
The following primer pairs were synthesized to mutate CTTT to AGATCT. -100 / -95 Rv (5 '
-AGATCTTTCTTCAACAAGTTCTCGA
SEQ ID NO: 14; 29 mer); and -94 / N46 Fw (5'-GCCCCATCGT
CGTAGATCTCGCAAGA-3 ′; SEQ ID NO: 1
5; 27 mer).

Six sequences GCC from positions -94 to -89
The following primer pairs were synthesized to mutate CTC to AGATCT. −94 / −89 Rv (5′-A
GATCTAAAGGGCTCTTCACACAAGTC
TCG-3 '; SEQ ID NO: 16; 29 mer); and -8
8 / N46 Fw (5'-ATCGGTCGTAGATC
TCGCAAGACCCTTC-3 ′; SEQ ID NO: 17;
27 mer).

Six sequences TCG from positions -87 to -82
The following primer pairs were synthesized to mutate TCG to GAATTC. r51gdWT Fw; 5'-G
GGGATCCACGTAATCGAGACTTG-
3 '; SEQ ID NO: 18; 24-mer); and r51 gdM
CB Rv; 5'-GGAGATCTAGAATTCT
GAGGGCAAAAGG-3 '; SEQ ID NO: 19; 27-mer).

Next, using any one of these primer pairs and the gWT-N46 plasmid obtained in Example 4 as a template, UPLOAD No.
Reverse PCR according to the protocol described at 59: 17-18.
(Inverse PCR) was performed. Inverse PCR is a method in which the entire circumference of a plasmid is PCR-amplified with two types of primers having no homologous portion in the reverse direction.
And deletion can be easily performed. The mutant promoter amplified in this way is designated BglII and Ba.
After cleavage with mHI, N4 as described in Example 4 respectively
Plasmids having mutant promoters were obtained by inserting and ligating into the BglII site of 6 plasmids.

In order to mutate the six sequences AGACTT from positions −115 to −110 to CTCGAG,
First, the following two primer pairs were synthesized: Part A
Primer pair for amplifying (3 'side): AtRAD
51-D; GGCCATGGTCGTCATTTTCTC
TCAATCAGAG (SEQ ID NO: 3; 30 mer); and AtRAD51-115 / -109XhoIFw; G
GCTCGAGGTTGAAGAAGCCTTTGCC
CTCA (SEQ ID NO: 20; 30 mer); and a primer pair for amplifying part B (5 ′ side): AtRA
D51-U; GGAAGCTTCACCGGTTTTGA
ACCCGGTTTAG (SEQ ID NO: 1; 29-mer); and AtRAD51-115 / -109XhoIRv; G
GCTCGAGCGATTACCTTTGGGTCAG
CTTT (SEQ ID NO: 21; 30 mer)).

Using a primer pair for amplifying part A, PCR was performed using the recombinant plasmid described in Example 1 as a template to obtain a 3'-side amplified DNA fragment. This fragment was cut with XhoI to obtain a part A containing a mutation at positions −115 to −110.

Next, PCR was carried out using a primer pair for amplifying part B and the recombinant plasmid described in Example 1 as a template to obtain a 5'-side amplified DNA fragment. This fragment was digested with XhoI, and
Part B containing the mutation at position 01 was obtained. Parts A and B were ligated using DNA ligase. The ligated fragment was cut with HindIII and NcoI and-
A DNA fragment containing a mutation from position 115 to position -101 was obtained. This DNA fragment was ligated with pBI221-
A recombinant plasmid containing a promoter at positions -648 to +58 containing a mutation from position -115 to -110 was produced by replacing the cauliflower mosaic virus 35S promoter site of LUC + (that is, a HindIII-NcoI site).

The following primer pair was then synthesized to generate a 44 base pair mutant: r51gdD1
Fw; 5'-GGGGATCCACGTAATCGC
TCGAG-3 '; SEQ ID NO: 22; 24-mer); and r51gWT Rv; 5'-GGAGATCTACCG
ACGATGAGGGCAA-3 ′; SEQ ID NO: 23; 2
4mer) was synthesized.

Next, using this primer pair, -64 containing a mutation from position -115 to -110 obtained above.
PCR was performed using a recombinant plasmid containing a promoter at positions 8 to +58 as a template. The mutant promoter amplified in this manner was replaced with BglII and BamHI.
And then ligated by inserting into the BglII site of the N46 plasmid described in Example 4 to obtain a plasmid having a mutant promoter of 44 bases containing a mutation from position -115 to position -110. .

In order to mutate the four sequences TTGA from position -108 to position -105 to CTCG, first, the following two pairs of primers were synthesized: part A (3 '
Side): A primer pair for amplifying: AtRAD51-
D; GGCCATGGTCGTCATTTTCTCCA
ATCAGAG (SEQ ID NO: 3; 30 mer); and At
RAD51-108 / -102XhoIFw; GGCT
CGAGAAGCCTTTGCCCTCATCGGTCG
T (SEQ ID NO: 24; 30 mer); and part B
Primer pair for amplifying (5 'side): AtRAD
51-U; GGAAGCTTCACCGGTTTGAA
CCCGGTTAG (SEQ ID NO: 1; 29-mer); and AtRAD51-108 / -102XhoIRv; GG
CTCGAGCAAGTCTCGAGTACGTTTTG
GGT (SEQ ID NO: 25; 30 mer)).

Using a primer pair for amplifying part A, PCR was performed using the recombinant plasmid described in Example 1 as a template to obtain a 3'-side amplified DNA fragment. This fragment was digested with XhoI to obtain Part A containing a mutation at positions −107 to −105.

Next, PCR was performed using a pair of primers for amplifying part B and the recombinant plasmid described in Example 1 as a template to obtain a 5 ′ amplified DNA fragment. This fragment was digested with XhoI, and
Part B containing the mutation at position 05 was obtained. Parts A and B were ligated using DNA ligase. The ligated fragment was cut with HindIII and NcoI, and
A DNA fragment containing a mutation from position 108 to position -105 was obtained. This DNA fragment was ligated with pBI221-
A recombinant plasmid containing a promoter from positions -648 to +58 containing a mutation from position -108 to -105 was prepared by replacing the cauliflower mosaic virus 35S promoter site of LUC + (that is, a HindIII-NcoI site).

The following primer pair was then synthesized to generate a 44 base pair mutant: r51gdWT
Fw; 5'-GGGGATCCACGTAATCGA
GACTTG-3 ′; SEQ ID NO: 18; 24-mer); and r51gWT Rv; 5′-GGAGATCTACCG
ACGATGAGGGCAA-3 ′; SEQ ID NO: 23; 2
4 mer).

Next, using this primer pair, -64 containing a mutation from position -108 to position -105 obtained above.
PCR was performed using a recombinant plasmid containing a promoter at positions 8 to +58 as a template. The mutant promoter amplified in this manner was replaced with BglII and BamHI.
And then ligated by inserting into the BglII site of the N46 plasmid described in Example 4 to obtain a plasmid having a mutant promoter of 44 bases containing a mutation from position -115 to position -110. .

The activity of each of the plasmids thus obtained was measured in the same manner as in Example 1.
FIG. 6 shows the results when a mutation was introduced from the −115 position to the −110 position. When the mutation was introduced from the −115 position to the −110 position, the relative activity in the untreated group and the relative activity in the bleomycin-treated group (that is, the activity in response to DNA damage) were drastically reduced. As a result, from −124 to −8
Of the sequence at position 1, the region at positions -115 to -110 was found to be an essential region for exhibiting DNA damage responsiveness.

When a mutation was introduced from the -108 position to the -105 position and when a mutation was introduced from the -87 position to the -82 position, the relative activity of the untreated group and the relative activity of the bleomycin-treated group were lower than the response. No clear loss of sex and expression level was seen, and DNA damage responsiveness was seen. Therefore, it was found that out of the sequence from positions -124 to -81, positions -108 to -105 and -87 to -82 are not so important for DNA damage responsiveness. Also,
Since positions -87 to -82 are not so important,-
The 81st place was also considered less important.

[0140]

According to the present invention, a DNA damage responsive promoter is provided. The promoter of the present invention is useful for expressing a heterologous gene in response to DNA damage. By linking a gene having a DNA damage resistance function to the promoter of the present invention and introducing the gene into a plant cell, the DNA damage resistance of the plant cell can be improved. Further, by linking a gene encoding a recombinase to the promoter of the present invention and introducing the gene into a plant cell, the efficiency of gene recombination of the plant cell can be controlled.

Transformed cells obtained by introducing a promoter of the present invention with an appropriate reporter gene ligated into plant cells can also be used as a sensor for detecting environmental factors that cause DNA damage.

Using the promoter of the present invention as a probe, Southwestern method or One-hybrid method
By d method or the like, it is possible to identify a DNA damage-responsive transcription control factor and obtain its gene. By overexpression, suppression of expression, modification, etc. of the obtained transcription control factor,
Artificial control of DNA damage response genes can be performed.

[0143]

[Sequence List] SEQUENCE LISTING <110> Yasuyuki Yamada, President, Nara Institute of Science and Technology <120> Higher plant cis control sequence which exhibits a capacity for controlling a gene expression in response to DNA damage <130> J1-00006735 <160> 25 <170> PatentIn version 3.0 <210> 1 <211> 44 <212> DNA <213> Arabidopsis thaliana <400> 1 acgtaatcga gacttgttga agaagccttt gccctcatcg tcgt 44 <210> 2 <211> 29 <212> DNA < 213> Artificial sequence: primer sequence <400> 2 ggaagcttca ccggtttgaa cccggttag 29 <210> 3 <211> 30 <212> DNA <213> Artificial sequence: primer sequence <400> 3 ggccatggtc gtcatttctc tcaatcagag 30 <210> 4 <211> 30 <212> DNA <213> Artificial sequence: primer sequence <400> 4 ggaagcttat agaataagaa tttgggcttt 30 <210> 5 <211> 29 <212> DNA <213> Artificial sequence: primer sequence <400> 5 ggaagcttgg gctttaatag cctttaaag 29 < 210> 6 <211> 30 <212> DNA <213> Artificial sequence: primer sequence <400> 6 ggaagcttcc tttaaagccc aatatgatca 30 <210> 7 <211> 30 <212> DNA < 213> Artificial sequence: primer sequence <400> 7 ggaagcttac gtaatcgaga cttgttgaag 30 <210> 8 <211> 31 <212> DNA <213> Artificial sequence: primer sequence <400> 8 ggaagcttct tgtataataa ttttggttgt g 31 <210> 9 <211 > 24 <212> DNA <213> Artificial sequence: primer sequence <400> 9 ggagatctac gacgatgagg gcaa 24 <210> 10 <211> 27 <212> DNA <213> Artificial sequence: primer sequence <400> 10 tggatctcga gcttaagctt ggcgtaa 27 <210> 11 <211> 30 <212> DNA <213> Artificial sequence: primer sequence <400> 11 ggatcccgag acttgttgaa gaagcctttg 30 <210> 12 <211> 30 <212> DNA <213> Artificial sequence: primer sequence <400 > 12 ggatccaaca agtctcgatt acgtggatct 30 <210> 13 <211> 29 <212> DNA <213> Artificial sequence: primer sequence <400> 13 gcctttgccc tcatcgtcgt agatctcgc 29 <210> 14 <211> 29 <212> DNA <213> Artificial sequence: primer sequence <400> 14 agatctttct tcaacaagtc tcgattacg 29 <210> 15 <211> 27 <212> DNA <213> Artificial sequence: primer sequence <400> 15 gccctcatcg tcgtagatct cgcaaga 27 <2 10> 16 <211> 29 <212> DNA <213> Artificial sequence: primer sequence <400> 16 agatctaaag gcttcttcaa caagtctcg 29 <210> 17 <211> 27 <212> DNA <213> Artificial sequence: primer sequence <400> 17 atcgtcgtag atctcgcaag acccttc 27 <210> 18 <211> 24 <212> DNA <213> Artificial sequence: primer sequence <400> 18 ggggatccac gtaatcgaga cttg 24 <210> 19 <211> 27 <212> DNA <213> Artificial sequence : primer sequence <400> 19 ggagatctag aattctgagg gcaaagg 27 <210> 20 <211> 30 <212> DNA <213> Artificial sequence: primer sequence <400> 20 ggctcgaggt tgaagaagcc tttgccctca 30 <210> 21 <211> 30 <212> DNA <213> Artificial sequence: primer sequence <400> 21 ggctcgagcg attacgtttg ggtcagcttt 30 <210> 22 <211> 24 <212> DNA <213> Artificial sequence: primer sequence <400> 22 ggggatccac gtaatcgctc gagg 24 <210> 23 < 211> 24 <212> DNA <213> Artificial sequence: primer sequence <400> 23 ggagatctac gacgatgagg gcaa 24 <210> 24 <211> 30 <212> DNA <213> Artificial sequence: primer sequence <400> 24 ggctcgagaa gcctttgc cc tcatcgtcgt 30 <210> 25 <211> 30 <212> DNA <213> Artificial sequence: primer sequence <400> 25 ggctcgagca agtctcgatt acgtttgggt 30

[Brief description of the drawings]

FIG. 1 is a schematic diagram of the structure of various promoter fusion gene constructs used for promoter expression analysis.
Rluc indicates a Renilla luciferase gene,
LUC + indicates the firefly luciferase gene.

FIG. 2 is a schematic diagram showing changes in promoter activity by bleomycin treatment. ArLIM15 is A
It is a promoter of a meiotically induced gene encoding an Escherichia coli RecA-like gene similar to tRAD51. CaMV35S is a constitutively expressed promoter, and Cab1 is a light-inducible promoter. The relative value when the average value of the untreated expression level is 1 is shown. Except for CaMV35S, all are derived from Arabidopsis thaliana. Error bars indicate standard deviation of 6 samples.

FIG. 3 is a graph showing the DNA damage inducing activity of a 5 ′ deletion mutant promoter of AtRAD51 gene. The relative activity is shown by setting the average activity of the untreated section of the promoter at positions −648 to +58 as 100. Error bars indicate standard deviation of 6 samples.

FIG. 4 is a graph showing the activity of a 5 ′ deletion mutant promoter of the AtRAD51 gene, to which a CaMV35S promoter B region was bound upstream. The average activity of the untreated section of the promoter at positions −648 to +58 was 10
The relative activity is shown as 0. Error bars represent the standard deviation of six samples.

Figure 5 shows a typical sequence of the promoter of the present invention.

FIG. 6 is a graph showing an experiment for acquiring the function of a DNA damage response region. gWT-N46 is a plasmid in which the promoter of the present invention is linked to the CaMV35S-46 promoter, gmutD1-N46 is a plasmid in which a 6-base mutation is introduced from position -115 to position -109, and N46 is CaMV35S-46. A plasmid using only 46 promoters and not ligating another promoter upstream is shown. The values indicate relative activities with the average activity of the untreated section of the promoter at positions −648 to +58 as 100. Error bars indicate standard deviation of 6 samples.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Tomohide Maeda 8916-5 Takayamacho, Ikoma, Nara Prefecture Graduate School Dormitory 6-309 F-term (Reference) 2B030 AA02 AB03 AD08 CA17 CA19 CB03 CD03 CD07 CD09 4B024 AA08 BA08 DA01 EA04 FA02 GA11 HA12 4B065 AA88X AA88Y AB01 AC14 BA02 BA25 CA53

Claims (10)

[Claims] 1. A DNA damage responsive promoter, which comprises: (a) a DNA having the nucleotide sequence of SEQ ID NO: 1;
(B) a DNA consisting of a base sequence in which one or several bases are deleted, substituted or added in the base sequence represented by SEQ ID NO: 1 and exhibiting DNA damage responsiveness; or (c) a DNA represented by SEQ ID NO: 1 Under stringent conditions with DNA consisting of the base sequence
A promoter comprising DNA exhibiting NA damage responsiveness. 2. The position from position 1 of the base sequence represented by SEQ ID NO: 1.
The promoter according to claim 1, wherein the promoter comprises a nucleotide sequence in which one or several bases have been deleted, substituted or added in the nucleotide sequences at positions 11 and 18 to 44, and is a DNA exhibiting DNA damage responsiveness. . 3. The base sequence of SEQ ID NO: 1 comprises a base sequence in which one or several bases have been deleted, substituted or added in the base sequences at positions 19 to 24 and positions 38 to 44, and The promoter according to claim 1, which is a DNA exhibiting DNA damage responsiveness. 4. A plant expression cassette for expressing a heterologous gene in response to DNA damage, comprising: a promoter according to claim 1; and a heterologous gene operably linked to said promoter. A plant expression cassette comprising a site for inserting the heterologous gene. 5. A recombinant plasmid for expressing a heterologous gene in response to DNA damage, comprising: a promoter according to claim 1; and a heterologous gene operably linked to the promoter. Replacement plasmid. 6. A method for obtaining a plant cell transformed with a heterologous gene whose specific expression is desired in response to DNA damage, comprising transforming a plant cell with the recombinant plasmid according to claim 5. Obtaining a transformed plant cell. 7. The plant cell is a monocot plant cell.
The method of claim 6. 8. The plant cell is a dicotyledonous plant cell,
The method of claim 6. 9. obtained by the method according to any one of claims 6-8, transformed plant cells. 10. A plant obtained by redifferentiating the plant cell according to claim 9.