JP2008306983A - Gene associated with plant cadmium resistance and method for preparing cadmium-sensitive plant - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a gene associated with plant cadmium resistance in order to contribute to practical application of purification of soil pollution caused by a heavy metal such as cadmium etc., by using a plant (phytoremediation). <P>SOLUTION: The gene comprises a DNA that is any DNA of (a) a DNA encoding a protein composed of an amino acid sequence represented by sequence number: 1, (b) a DNA encoding a protein composed of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted and/or added in the amino acid sequence represented by the sequence number: 1, (c) a DNA containing a coding region of a base sequence represented by sequence number: 2 or sequence number: 3, and (d) a DNA to be hybridized with a DNA containing the coding region of a base sequence represented by the sequence number: 2 or the sequence number: 3 under a stringent condition, and encodes a protein associated with plant cadmium resistance. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a gene involved in cadmium resistance of a plant and a method for producing a cadmium-sensitive plant by destroying or suppressing expression of the gene.

  In recent years, research has been actively conducted on methods for purifying soil contamination (phytomediation) with heavy metals such as cadmium, copper, and lead using plants. However, the amount that plants can absorb and detoxify heavy metals per unit area and per hour is small, and with a few exceptions, full-scale practical use has not been achieved. Thus, if the ability to absorb and accumulate heavy metals can be enhanced by genetic recombination, it can be expected to supplement conventional methods. For this purpose, firstly, the isolation of genes involved in the ability to absorb and accumulate heavy metals is required. To date, phytocheratin, metallothoinin, transporter, etc. are known as factors involved in resistance and accumulation of heavy metals, but in plants into which these genes have been introduced, resistance and accumulation capacity against heavy metals may have increased, It may not be the case and the evaluation is not fixed. The reason for this is that there are still many unclear points regarding the mechanism of absorption and accumulation of heavy metals rooted in complex physiology, and the possibility of the involvement of complex factors at the gene expression level and protein level. In order to overcome such a current situation, it is considered that a comprehensive analysis of metal absorption / accumulation mechanism is effective (see Non-Patent Document 1), and such research is desired to proceed. is the current situation.

Lahner et al. (2003) Nature Biotechnology 21: 1215-1221.

  An object of the present invention is to solve various problems in the prior art and achieve the following objects. That is, an object of the present invention is to provide a gene involved in cadmium resistance of plants in order to contribute to the practical use of phytomediation of soil contamination by heavy metals such as cadmium using plants. Furthermore, this invention also aims at providing the method of producing a cadmium sensitive plant using the said gene.

First, the present inventors used a T-DNA tagging method (see Koncz C et al., (1989) Proc Natl Acad Sci USA. 86 (21): 8467-71) to identify mutant populations of Arabidopsis (Arabidopsis thaliana). A mutant having strong sensitivity to cadmium (Cd) was selected. Next, in the mutant, by analyzing the base sequence of the gene destroyed by T-DNA, a gene involved in cadmium tolerance in plants was identified, and the present invention was completed.
The T-DNA tagging method is a method for estimating the function of a gene by inserting T-DNA from the outside into a genomic gene and suppressing the expression of the gene. The above method is a powerful method because the correlation between a gene and its function, that is, a phenotype is clear, and once a phenotype is found, it has an advantage that a related gene can be immediately identified. .
Moreover, since the genome size of Arabidopsis (Arabidopsis thaliana) is very small, the scale of the mutant population by T-DNA tagging itself can be reduced. Therefore, it is advantageous in that the target mutant can be selected efficiently. In Arabidopsis, the entire base sequence has already been determined. By decoding the sequence of the genome into which T-DNA has been inserted, it is possible to instantly identify which gene has been destroyed by T-DNA. This is also advantageous in that quick data acquisition is possible.

  The gene involved in plant cadmium tolerance identified by the present inventors is a gene conventionally known as a gene encoding triose phosphate isomerase (TPI). TPI is known to be an enzyme that isomerizes from dihydroxyacetone phosphate to glyceraldehyde 3-phosphate in glycolysis, but has not been known to be involved in resistance to cadmium. This is a new finding by the inventors. In addition, it is a new finding by the present inventors that a plant exhibiting a strong sensitivity to cadmium can be obtained by suppressing the expression of the TPI gene. These findings are based on phytomediation (using plants). It is considered to be very useful for research and development in the field of remediation of contaminated soil by heavy metals such as cadmium.

This invention is based on the said knowledge by this inventor, and as a means for solving the said subject, it is as follows. That is,
<1> A DNA according to any one of (a) to (d) below, characterized in that it comprises a DNA encoding a protein involved in plant cadmium resistance.
(A) DNA encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 1
(B) DNA encoding a protein comprising an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence represented by SEQ ID NO: 1.
(C) DNA containing the coding region of the base sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3
(D) DNA that hybridizes under stringent conditions with DNA containing the coding region of the nucleotide sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3.
<2> A method for producing a cadmium-sensitive plant, wherein the gene according to <1> is destroyed or expression-suppressed in a plant.
<3> The method for producing a cadmium-sensitive plant according to <2>, wherein the gene according to <1> is disrupted or expression is suppressed by base substitution, deletion, and / or insertion.
<4> The method for producing a cadmium-sensitive plant according to any one of <2> to <3>, wherein the gene according to <1> is disrupted or expression is suppressed by inserting T-DNA.
<5> A cadmium-sensitive plant obtained by the method for producing a cadmium-sensitive plant according to any one of <2> to <4>.
<6> The cadmium-sensitive plant according to <5>, wherein the plant is Arabidopsis thaliana.
<7> A gene obtained by inserting T-DNA into the gene according to <1>.
<8> The gene according to <7>, including the base sequence represented by SEQ ID NO: 4.

  According to the present invention, conventional problems can be solved, a gene involved in cadmium resistance in a plant can be provided, and a method for producing a cadmium-sensitive plant using the gene is provided. can do. INDUSTRIAL APPLICABILITY The present invention greatly contributes to the practical use of soil contamination purification (phytomediation) with heavy metals such as cadmium using plants.

(Gene involved in cadmium tolerance in plants)
The gene of the present invention is the DNA described in any of (a) to (d) below, and is characterized by comprising a DNA encoding a protein involved in plant cadmium resistance.
(A) DNA encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 1
(B) DNA encoding a protein comprising an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence represented by SEQ ID NO: 1.
(C) DNA containing the coding region of the base sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3
(D) DNA that hybridizes under stringent conditions with DNA containing the coding region of the nucleotide sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3.

The present inventors have identified a novel gene (hereinafter, sometimes simply referred to as “gene”) that is involved in cadmium tolerance in plants. In addition, the amino acid sequence of the protein of the gene in Arabidopsis thaliana identified by the present inventors is SEQ ID NO: 1, and the nucleotide sequence of the DNA encoding the protein is SEQ ID NO: 2 (genomic sequence). And SEQ ID NO: 3 (cDNA sequence).
The protein consisting of the amino acid sequence represented by SEQ ID NO: 1 is a protein conventionally known as triose phosphate isomerase (TPI). The TPI is known to be an enzyme having a function of isomerizing dihydroxyacetone phosphate to glyceraldehyde 3-phosphate in a glycolysis system.

  In the present invention, the gene encoded by the gene is involved in plant resistance to cadmium. Here, the term “involved in plant tolerance to cadmium” means that, for example, the protein is related in some way to the absorption and accumulation mechanisms of cadmium in plants, the detoxification mechanism of cadmium, and the like. It means that the plant is given resistance to cadmium. In addition, there is no restriction | limiting in particular as a kind of said plant, According to the objective, it can select suitably, For example, Brassicaceae plants, such as Arabidopsis thaliana, Asteraceae plant, Labiatae plant, Gramineae plant etc. are mentioned.

  Whether or not the protein encoded by the gene is involved in plant tolerance to cadmium is determined by, for example, examining whether or not the plant sensitivity to cadmium is increased by destroying or suppressing expression of the gene in the plant. Can be confirmed. The method for disrupting or suppressing expression of the gene is not particularly limited, and is, for example, as described in the item of the method for producing a cadmium-sensitive plant of the present invention described later. Moreover, there is no restriction | limiting in particular also as the confirmation method of the sensitivity with respect to the cadmium of the said plant, For example, it can carry out using the cadmium sensitivity test etc. which were described in the Example mentioned later.

  In addition, the gene is a protein that is structurally similar to the protein consisting of the amino acid sequence represented by SEQ ID NO: 1, and may be a gene consisting of DNA encoding a protein involved in plant cadmium resistance. Good. Examples of such a gene include a gene comprising a DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted and / or added in the protein. Here, the “plural amino acids” means that even if the number of amino acids is substituted, deleted, inserted, and / or added, the property that the protein is involved in plant resistance to cadmium is maintained. Although there is no particular limitation, it is usually within 1 to 10 amino acids, preferably within 1 to 5 amino acids, and more preferably within 1 to 3 amino acids. In the protein, a region where amino acids can be substituted, deleted, inserted, and / or added is not particularly limited as long as the above characteristics are maintained, and can be appropriately selected according to the purpose.

Further, the form of the gene is not particularly limited, and examples thereof include genomic DNA, cDNA, chemically synthesized DNA, etc. The preparation method of these DNAs is not particularly limited, and examples thereof include hybridization techniques, polymerases, and the like. It can be prepared using techniques known to those skilled in the art, such as chain reaction (PCR) technique and reverse transcription reaction (RT) technique.
For genomic DNA, for example, genomic DNA is extracted from a plant having the gene, a genomic library is prepared, and the genomic DNA is developed to obtain the nucleotide sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3 or on the genome. It can be prepared by performing colony hybridization or plaque hybridization using a probe prepared based on the base sequence in the vicinity thereof. It is also possible to prepare a primer specific for the nucleotide sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3 or a neighboring nucleotide sequence on the genome and perform PCR using this primer. .
In addition, for example, cDNA is synthesized based on mRNA extracted from a plant having the gene, and PCR is performed using primers prepared based on the nucleotide sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3. Etc. can be prepared. In addition, cDNA was inserted into a vector such as λZAP to prepare a cDNA library, which was developed, and using a probe prepared based on the base sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3, It can also be prepared by performing colony hybridization or plaque hybridization.

Further, not only a DNA containing the coding region of the nucleotide sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3 but also a gene comprising a DNA that hybridizes with the DNA under stringent conditions, which can be prepared in this manner. Moreover, as long as the protein encoded by the DNA maintains the characteristic that it is involved in plant tolerance to cadmium, it is included in the gene of the present invention.
In the present invention, “under stringent conditions” means, for example, that the sodium concentration is usually 25 to 500 mM, and preferably 25 to 300 mM. Moreover, it means that temperature is 42-68 degreeC normally, and it is preferable that it is 42-65 degreeC.
DNA that hybridizes under such stringent conditions is considered to have high homology with the amino acid sequence described in SEQ ID NO: 1 in the amino acid sequence of the protein encoded by the DNA. Here, high homology means that the homology is at least 70% or more in the entire amino acid sequence, preferably 80% or more, more preferably 90% or more, and particularly preferably 95% or more. In addition, the homology of the amino acid sequence or the base sequence as described above uses, for example, means known to those skilled in the art, such as BLAST (http://www.ddbj.nig.ac.jp/Welcome-j.html). Can be obtained.

(Method for producing cadmium-sensitive plants)
The method for producing a cadmium-sensitive plant of the present invention is characterized by at least including destroying or suppressing expression of the gene of the present invention (a gene involved in cadmium resistance of a plant) in the plant.

<Gene disruption or expression suppression>
Since the gene of the present invention encodes a protein involved in cadmium resistance, by destroying or suppressing expression of the gene in the plant, the protein involved in cadmium resistance is not produced or produced in the plant. The amount is reduced, which can increase the plant's sensitivity to cadmium. In addition, there is no restriction | limiting in particular as a kind of plant used as the object which performs destruction or expression suppression of the said gene, According to the kind of cadmium sensitive plant to produce, it can determine suitably.
The method for disrupting or suppressing expression of the gene is not particularly limited and may be appropriately selected depending on the intended purpose. For example, base substitution or deletion in the structural gene region or regulatory gene region of the gene And / or a method of suppressing the expression by destroying the function of the gene by causing an insertion; by expressing an antisense RNA strand complementary to at least a part of the mRNA of the gene in a plant; And a method for suppressing translation into protein.
Here, the “gene disruption or expression suppression” may be one that inhibits the transcription process of the gene from DNA to mRNA, or that inhibits the translation process of the gene from mRNA to protein. It may be. Moreover, there is no restriction | limiting in particular also as the grade of the inhibition, As a result, the plant which received the destruction or expression suppression of the said gene should just show the sensitivity with respect to cadmium.

-Gene disruption by base substitution, deletion, and / or insertion-
--T-DNA insertion--
There is no particular limitation on the method for causing base insertion in the structural gene region of the gene or the regulatory gene region (for example, promoter, etc.), and methods known to those skilled in the art can be used as appropriate. The T-DNA tagging method (see Koncz C et al. (1989) Proc Natl Acad Sci USA. 86 (21): 8467-71) can be used. The T-DNA tagging method is a method of suppressing the expression of a gene by inserting T-DNA into the genome gene from the outside.
Here, the T-DNA only needs to contain a certain boundary region (repeated sequence of about 25 bp called RB and LB in the same direction) at both ends, and the internal gene is It may be replaced with another gene. The vector used for the T-DNA tagging method is not particularly limited and can be appropriately selected according to the purpose. For example, pGPTV-bar (Becker D et al., (1992) as shown in Examples described later. Plant Mol Biol. 20: 1195-1197) and the like can be used. The vector can be introduced into a plant cell by a technique known to those skilled in the art. For example, the vector is introduced into a plant cell by incorporating the vector into Agrobacterium and infecting the plant cell with the vector. Can do.
By randomly inserting T-DNA into the genome of the target plant by the T-DNA tagging method, and selecting a mutant exhibiting susceptibility to cadmium from the obtained multiple types of mutants, Cadmium sensitive plants can be obtained. In addition, there is no restriction | limiting in particular as a method of selecting the variant which shows the sensitivity with respect to cadmium, For example, selection can be performed using a cadmium sensitivity test etc. which were described in the Example.

-Substitution, deletion-
There is no particular limitation on the method for causing base substitution or deletion in the structural gene region or regulatory gene region (for example, promoter) of the gene, and methods known to those skilled in the art can be appropriately used. For example, techniques such as radiation irradiation and neutron beam irradiation can be used. A cadmium-sensitive plant can be obtained by selecting a mutant showing sensitivity to cadmium from a plurality of types of mutants obtained by these techniques.

-Translation suppression by antisense RNA strand-
A method for expressing an antisense RNA strand complementary to at least a part of the mRNA of the gene in a plant is not particularly limited, and methods known to those skilled in the art can be appropriately used. For example, the gene Constructing an expression cassette in which at least a part of the coding region of the base sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3 or a partial region thereof is linked downstream of the promoter in the antisense direction, Examples thereof include a method for introducing an expression cassette into a plant cell.
The method for constructing the expression cassette is not particularly limited and can be appropriately selected depending on the purpose. For example, a promoter sequence that can be transcribed in plants, a DNA fragment encoding the antisense RNA strand, and necessary Depending on the case, it can be constructed by appropriately linking terminator sequences and the like.
Further, the method for introducing the expression cassette into plant cells is not particularly limited and can be appropriately selected according to the purpose. For example, those skilled in the art such as microinjection method, electroporation method, particle gun method, etc. A known method can be used. The expression cassette may be introduced into plant cells via a vector. For example, the expression cassette can be introduced into plant cells by cloning the expression cassette into an appropriate vector, incorporating the vector into Agrobacterium, and infecting it with plant cells.

Plant cells in which the gene is destroyed or expression-suppressed as described above can be regenerated into plant individuals by techniques known to those skilled in the art. For example, there is a method in which callus-like transformed cells are transferred to a medium with different types and concentrations of hormones and cultured to form somatic embryos to obtain complete plants. Examples of the medium to be used include LS medium and MS medium.
Once a transformed plant body (cadmium-sensitive plant) in which the gene in the chromosome is destroyed or suppressed is obtained, progeny can be obtained from the plant body by sexual reproduction or asexual reproduction. It is also possible to obtain seeds from the plant, its progeny, or clones, and mass-produce the plant based on them.

(Cadmium sensitive plant)
The cadmium-sensitive plant of the present invention is a plant obtained by the method for producing the cadmium-sensitive plant of the present invention, wherein the gene of the present invention (a gene involved in cadmium tolerance of the plant) is destroyed or expression-suppressed. Features.

There is no restriction | limiting in particular as a kind of said plant, According to the objective, it can select suitably, For example, Brassicaceae plants, such as Arabidopsis thaliana, Asteraceae plants, Lamiaceae plants, Gramineae plants, etc. are mentioned.
The cadmium-sensitive plant includes not only the entire plant body but also a part of the plant body or a propagation material (for example, a plant organ (for example, seed, leaf, petal, stem, root, etc.), plant tissue (for example, , Epidermis, phloem, soft tissue, xylem, vascular bundle, etc.), plant cultured cells, etc.).

It can be confirmed that the plant exhibits cadmium sensitivity, for example, by using a cadmium sensitivity test as shown in Examples described later. For example, the seed of the plant is grown in the presence of cadmium at a concentration of about 25 μM to 100 μM, and the amount of root elongation is compared with that of the wild type. In the presence of cadmium at the above-mentioned concentration, when the amount of root growth of the plant is, for example, about 2/3 or less of the amount of wild-type root elongation, the plant is confirmed to exhibit cadmium sensitivity. be able to.
In addition, there is no restriction | limiting in particular as a grade of the cadmium sensitivity of the said cadmium sensitive plant, It can select suitably according to the objective, What is necessary is just to show cadmium sensitivity stronger than a wild type plant at least.

(Gene with T-DNA inserted)
The present invention also relates to a gene obtained by inserting T-DNA into the gene of the present invention (a gene involved in plant cadmium resistance).
The gene into which the T-DNA has been inserted is obtained by disrupting the function of the gene of the present invention (the function of being involved in plant cadmium resistance). The method for inserting T-DNA into the gene of the present invention is as described in the item of the method for producing a cadmium-sensitive plant of the present invention. The insertion site is not particularly limited as long as it is an insertion site that destroys the function of the gene, and can be appropriately selected.
Specific examples of the gene into which the T-DNA has been inserted include, for example, a gene containing the base sequence shown in SEQ ID NO: 4. The gene containing the base sequence represented by SEQ ID NO: 4 is one obtained by inserting T-DNA into the seventh intron of the Arabidopsis thaliana TPI gene, which is the gene of the present invention, as shown in the Examples described later. The function of the gene of the present invention (the function of being involved in cadmium tolerance in plants) has been destroyed.
Here, in the base sequence shown in SEQ ID NO: 4, the 1st to 236th base is a part of the genome sequence of the Arabidopsis TPI gene (corresponding to the 1875th to 2110th bases of SEQ ID NO: 2). 237 to 352 bases indicate a part of the inserted T-DNA sequence. Among them, the 260th to 352rd bases indicate the LB region of T-DNA, and the 331st to 352rd bases indicate the LB3 primer site (see Example 2 below).

  EXAMPLES Examples of the present invention will be described below, but the present invention is not limited to these examples, and includes ordinary alterations and modifications to these performed in the technical field.

(Example 1: Selection of plant mutants exhibiting cadmium sensitivity)
In this Example 1, for the purpose of searching for genes involved in cadmium tolerance of plants, Arabidopsis (Arabidopsis thaliana) mutant populations were prepared using the T-DNA tagging method, from which sensitivity to cadmium (Cd) was observed. Increased mutants were selected as follows.

<Plant transformation>
-Construction of T-DNA vector (binary vector)-
The vector 35sp-sGFP-PUC18 (Shizuoka Prefectural University) was added to the T-DNA vector (binary vector) pGPTV-bar (see Becker D et al., (1992) Plant Mol Biol. 20: 1195-1197) digested with the restriction enzymes HindIII and EcoRI. A fragment containing sGFP cut out by HindIII and EcoRI from the Graduate School of Life and Health Sciences Department of Food and Nutrition Chemistry (Food Cell Engineering Laboratory) was used. . The obtained new vector was cloned into Escherichia coli and then introduced into Agrobacterium (Agrobacterium thmefaciens GV3101) by an electroporation apparatus (manufactured by Biorad). The conditions for electroporation are as follows. Cuvette interval 0.1 cm, voltage 1.7 kv, resistance 200Ω, capacitance 25 μF.
Agrobacterium was cultured at 28 ° C. for 2 days in a YEP medium containing 100 mg / l kanamycin and 80 mg / l rifampicin. A single colony was picked up and the presence of the constructed vector was confirmed by PCR, and then cultured in a YEP liquid medium. After collection, the cells were suspended in a suspension (5% sucrose, 0.025% Silwet L77 (OSI specialties, UK)) so that OD600 = 1.5 to 2.0.

-Infection with Arabidopsis-
On the other hand, Arabidopsis thaliana Col 0 gl-1 was cultivated in a closed greenhouse (22 ° C., 16 hours light period) and used as a material for transformation. Immature seeds and open flowers were removed from the Arabidopsis strain and the Agrobacterium suspension was sprayed onto the Arabidopsis buds. Plants infected with Agrobacterium were placed in semi-sealed containers for 24 hours to maintain humidity. Thereafter, the plant was washed away in order to remove excess suspension and sugar contained therein. Five to six strains of Arabidopsis were used in one operation. The transformed plants were grown until the seeds were fully matured, after which the seeds (T1 generation) were harvested.

<Selection>
-Selection with herbicides-
Seeds (T1) in the pod that matures after the genetic recombination operation are mixed with those that have not been transformed. Therefore, it is necessary to select only transformed seeds. Since the transformed plant has a herbicide (bialaphos) resistance gene bar, it was decided to use a herbie solution containing bialaphos for selection of transformants. However, since the appropriate concentration to use for selection is unknown, the optimal concentration of the Herbie solution used to select the transformed individuals was first determined. Herbie solution was diluted to various bialaphos concentrations (0-1,000 mg / l) and sprayed every 2 days on known transformed Arabidopsis with wild type and bar 2 weeks after germination. The wild type was sensitive to 30 mg / l of the herbicide and completely died at 60 ml / l 2 weeks after spraying. However, in the transformant having bar, growth inhibition was not observed up to 240 ml / l, and it almost died at 1,000 mg / l (FIG. 2). From these results, it was decided to use a Herbie solution with a concentration of 100 mg / l for selection of transformants in T1.

  T1 seeds, 5,000 to 7,500 grains, were mixed with fine-grained sand and sown in a rice seedling tray (300 × 600 × 30 mm). Watering, wrapping in wraps, sprouting treatment at 4 ° C for 7 days, and cultivating in a closed greenhouse. In addition, the soil which mixed vermiculite, perlite, planter soil, and peat moss in the ratio of 1: 1: 1: 3 was used for cultivation. In order to select transgenic individuals having the herbicide (bialafos) resistance gene bar, seedlings 2 weeks after germination were treated with a herbie solution (Sankei Chemical; containing 18% bialaphos as an active ingredient) with a bialaphos concentration of 50 mg / After adjusting to 1 l, 200 ml was sprayed per tray. Thereafter, every 4 days, a Herbie solution with a concentration of 100 mg / l bialaphos was sprayed until the non-transformed plants were completely dead. Surviving individuals, 5 strains were collected and transplanted to a new tray. After ripening, T2 generation seeds were collected.

  FIG. 3 shows a state where seeds (T1) obtained by actual transformation were sown on a tray and the transformants were selected. One week after the application of the herbicide, a difference began to appear in the growth of the plant (T1) considered to be a transformant and the plant considered to be a wild type. The difference appeared clearly at the second week, and the wild type almost stopped growing and the leaves became yellow to brown. The surviving individuals had thick leaves and no delay in growth. By this method, about 13,000 transformants could be finally obtained. In addition, although the difference (0.3-0.8%) of transformation efficiency was seen between the lots of the transformation experiment, the average transformation efficiency was 0.51% (Table 1). Transformants were collected from 5 strains and seeds (T2) were collected.

-Primary selection by Cd-
As a method for obtaining a Cd-sensitive mutant, it is considered to be effective to place the seedling of the transformant in a medium containing Cd and select an individual whose root elongation is suppressed as a mutant. However, the optimal Cd concentration to enter the medium is unknown. Therefore, in order to first obtain the optimum Cd concentration for selection of sensitive individuals, a Cd sensitivity test as shown in Example 3 described later was performed using the wild type. As a result, as the Cd concentration increased, the amount of root elongation gradually decreased and the occurrence of lateral roots increased. In particular, the suppression of root elongation in the case of 75 μM or more was increased (FIG. 4). This experiment was repeated 5 times, all with similar results.
Based on these facts, the amount of root elongation is suppressed by Cd, survival after the experiment is possible, and 50 μM is a reasonable concentration that is expected to increase the difference in the amount of root elongation between the wild type and the mutant. It was thought that.

Based on the above results, T2 seeds were sown in a medium containing 50 μM Cd, and Cd-sensitive mutants were selected as follows.
Since T2 generation seeds are collected by mixing five seeds, the handling unit was set as a subpool. Furthermore, 5 sub-pools were designated as 1 super pool. That is, there are 25 seeds in one super pool. About 500 grains per super pool were placed in a 1.5 ml tube, water was added, and then germination treatment was performed at 4 ° C. for 4 days. Sterilization was performed by immersing the seed in a sodium hypochlorite solution having an effective concentration of 1% for 15 minutes. The sterilized seeds were washed several times with distilled water and sown on 5 ½ concentration MS media (diameter 90 mm) containing 50 μM CdCl ₂ . After culturing at 24 ° C for 16 hours under illumination for 7 days, the petri dish is observed and individuals whose root elongation is suppressed to about 2/3 or less compared to wild-type roots, or individuals whose leaf color has become lighter Were visually selected and subcultured to a ½ concentration MS medium without CdCl ₂ . After 2-3 weeks, potting was performed and T3 generation seeds were collected.

  As a result of selecting Cd-sensitive mutants by the primary selection, 4,985 individuals could be obtained. The next generation (T3) seeds were obtained by potting them up, but it was 2,851 individuals that died during the cultivation due to poor growth or were actually seeded due to sterility. When the seeds derived from the same line were duplicated, the number of lines obtained independently as a result of the primary selection was estimated to be about 1,600 lines.

-Secondary selection by Cd-
In the line obtained by the primary selection, in addition to the Cd-sensitive mutant, it is considered that there are a considerable number of individuals with little root elongation due to poor germination. Therefore, the second selection was performed as follows.
First, about 20 T3 generation seeds and non-transformed seeds obtained by primary selection were placed in 1.5 ml tubes, respectively, and sprouting treatment and sterilization treatment were performed in the same manner as in primary selection. The seeds were sown in a half-concentration MS medium (containing 3% sucrose and 1.2% agar) without CdCl ₂ .
After 4 days of cultivation, individuals with good growth and similar root lengths were selected, and each strain was added to a half-concentration MS medium (containing 3% sucrose and 1.2% agar) containing 50 μM CdCl _2. Five individuals were placed. And I marked with a marker from the back of the petri dish at the root. After 6 days, the extended root tip was marked with a marker. Mutant and wild type root elongation were compared visually, and as a result, mutants in which root elongation was suppressed to about 2/3 or less compared to wild type roots were selected.

  The state of the secondary selection is shown in FIG. Of the lines obtained by the primary selection, secondary selection was performed on 740 lines, and 30 mutants could be obtained. Among these, a line (41-3 line; FIG. 6) showing particularly strong sensitivity to cadmium was selected, and genes were identified in Example 2 below.

(Example 2: Identification of genes involved in cadmium resistance)
In Example 2, a line (41-3 line; FIG. 6) showing strong sensitivity to cadmium was selected from the mutants obtained by the secondary selection in Example 1, and T-DNA insertion was performed. The disrupted gene (expression was suppressed) was identified. In a mutant showing strong sensitivity to cadmium, it can be presumed that a gene that has been disrupted by T-DNA insertion (expression is suppressed) is a gene involved in cadmium resistance in plants.

<Amplification by TAIL-PCR and determination of nucleotide sequence>
In order to detect a gene disrupted by T-DNA tagging by extracting DNA from the 41-3 line (FIG. 6) showing strong sensitivity to cadmium among mutants obtained by secondary selection, TAIL-PCR (thermal asymmetry interpolated PCR) was performed. TAIL-PCR is a method in which a primer specific to a border sequence of T-DNA and a non-specific primer are combined to amplify the boundary region between T-DNA integrated into the genome and the genome by PCR (Liu et al. (1995) Plant J 8: 457-463).
Regenerate primer DEG1 and specific primers LB1 to LB3 were designed as shown in Table 2. PCR (polymerase chain reaction) was performed under the conditions shown in FIG. The reaction volume was 20 μl, and final concentrations were dNTP 0.2 mM, regenerated primer 1 μM, specific primer 0.2 μM, and polymerase (Ex Taq (Takara)) 0.05 unit / μl. As the template for the second and third reactions, 1 μl of the first and second reaction solutions diluted 50-fold was used. The amplified DNA was confirmed by agarose gel electrophoresis. The results are shown in FIG.

Subsequently, DNA amplified by TAIL-PCR was excised from the gel after agarose gel electrophoresis, and direct sequencing was performed. The fluorescent reagent used was Bigdye terminator 3.1 (Applied Biosystems), and the DNA was labeled according to the manufacturer's protocol. The sequencer used was made by ABI. If the sequencing result was poor, the DNA was excised from the gel, and then TA cloning (Invitrogen, USA) was performed for sequencing. The result of the sequence is shown in SEQ ID NO: 4. The obtained base sequence information was searched by BLAST of DNA data bank of Japan (http://www.ddbj.nig.ac.jp/Welcome-j.html). Furthermore, the T-DNA insertion site was determined based on the nucleotide sequence information.
As a result, the base sequence (SEQ ID NO: 4) of the DNA amplified by TAIL-PCR is a part of the genome sequence of the triose phosphate isomerase (TPI) gene (positions 1,875 to 2,110 of SEQ ID NO: 2). Corresponding to the base) and a part of the T-DNA sequence (up to the LB3 primer site) (see FIG. 9A). That is, as shown in FIG. 9B, it was presumed that T-DNA was inserted into the seventh intron of the TPI gene located on the third chromosome. Triose phosphate isomerase (TPI) is conventionally known to be an enzyme that isomerizes dihydroxyacetone phosphate to glyceraldehyde 3-phosphate in a glycolytic system, and its amino acid sequence is SEQ ID NO: 1. The DNA sequences are as shown in SEQ ID NO: 2 (genomic sequence) and SEQ ID NO: 3 (cDNA sequence).

<Confirmation of T-DNA insertion site>
Next, in order to check whether the T-DNA insertion site determined above was correct, PCR was performed using 41-3 strains of genomic DNA as a template. The primers used (41-3F / R / LB) are shown in Table 3. Moreover, the schematic diagram of the site | part which the used primer anneals to genomic DNA is shown to FIG. 10A. The PCR conditions are as follows. After 94 ° C / 5 minutes, 94 ° C / 30 seconds, 56 ° C / 1 minute, and 72 ° C / 1 minute were repeated 25 times. Further, after 72 ° C / 5 minutes, the temperature was kept at 4 ° C.
The result of PCR is shown in FIG. 10B. In all individuals tested, a band was obtained when the combination of F and LB primers was used, and the mutant type showed that this insertion site was correct and the insertion site was homozygous. It was strongly suggested that there was.

<Confirmation of TPI gene expression suppression>
Next, semi-quantitative PCR was performed on the 41-3 strain for the purpose of confirming whether the expression of the TPI gene was suppressed by the insertion of T-DNA. The primers used (ACT F / R, TPI F / R) are shown in Table 3. RNA was extracted from wild type and mutant (41-3 strain) using Trisol, and reverse transcription reaction was performed using it as a template. Further, after the reaction, PCR was performed using the obtained cDNA as a template.
As a result, expression of the TPI gene was observed in the wild type but not in the 41-3 line (FIG. 11). This indicates that the TPI gene is not transcribed by the introduction of T-DNA, or that the transcript is rapidly degraded. In any case, transcripts of 41-3 strains of TPI gene did not accumulate, strongly suggesting that no TPI was produced.

(Example 3: Cadmium sensitivity of selected plant mutants)
In Example 3, the sensitivity of the plant mutant (41-3 strain) selected in Example 1 to cadmium (Cd) was confirmed.

<Cd sensitivity test>
In order to grasp the characteristics of the 41-3 line, a root elongation test was conducted in the presence of Cd. Germination treatment and sterilization treatment were performed on the 41-3 seeds in the same manner as in the primary selection, and the seeds were sown in a ½ concentration MS medium. Four days later, seedlings germinated in MS medium containing CdCl ₂ at various concentrations of 0 to 150 μM were subcultured. Then, as in the case of the secondary selection, the root end is marked with a marker, and after 6 days, the image is captured with a scanner, and the amount of root elongation is analyzed with the analysis software Scion image (http://www.scioncorp.com/). It was measured.

  As a result, when Cd was 0 μM, there was no difference in the elongation between the mutant and the wild-type root, but the root elongation of the mutant was suppressed as compared with the wild-type, particularly at a Cd concentration of 25 μM to 100 μM. (FIG. 12). In addition, in the case of 125 μM or more, root elongation almost stopped in both cases.

  As described above, Examples 1 to 3 suggested that the triose phosphate isomerase (TPI) gene is a gene involved in resistance to cadmium in plants. TPI is known to be an enzyme that isomerizes from dihydroxyacetone phosphate to glyceraldehyde 3-phosphate in glycolysis, but has not been known to be involved in resistance to cadmium. This is a new finding by the inventors. Moreover, it is a new finding by the present inventors that plants exhibiting a strong sensitivity to cadmium can be obtained by suppressing the expression of this TPI gene. These findings include, for example, phytomediation (plant It is considered to be very useful for research and development in the field of (contamination of contaminated soil with heavy metals such as cadmium).

  The gene involved in resistance to cadmium identified in the present invention and the method for producing a cadmium-sensitive plant using the gene are, for example, in the field of phytomediation (purification of contaminated soil with heavy metals such as cadmium using plants) It can be suitably used for research and development in Japan.

1 is a schematic diagram of a T-DNA vector (binary vector) constructed in Example 1. FIG. GUS was cut out from pGPTV-bar-GUS, and sGFP was introduced therein. Nos3 ': Nos terminator, CaMV35S: Cauliflower mosaic virus 35S promoter, LB: Left border, RB: Right border. FIG. 2 examined the difference in the growth of recombinants due to the difference in the concentration of bialaphos in order to determine the optimal concentration of the bialaphos-containing herbicide (Herbie solution) used to select transformed individuals in Example 1. It is a figure which shows a result. Recombinant seeds having the wild type and the bar gene were sown in soil, and bialaphos (0-1,000 mg / l) was sprayed every two days. FIG. 3 is a diagram showing a state of selection of seeds (T1) obtained by transformation in Example 1. Individuals at 2 weeks after germination were selected with a herbicide containing 50 mg / l bialaphos and then selected with a herbicide containing 100 mg / l bialaphos. A tray having a height of 30 cm, a width of 60 cm, and a height of 3 cm was used, and 200 ml of herbicide was sprayed every two days on one tray, and selection was performed over 2 weeks. Individuals marked with arrows are transformants. FIG. 4 is a diagram showing the results of conducting a Cd sensitivity test using a wild type in order to obtain an optimal cadmium (Cd) concentration for selection of sensitive individuals in Example 1. Wild-type seeds were sown in 1/2 MS medium and exposed 3 days later in 1/2 MS medium containing CdCl ₂ for 6 days. n = 15, vertical lines indicate standard deviation. FIG. 5 is a diagram showing a state of secondary selection of transformants with cadmium (Cd) in Example 1. The seedlings on the 4th day after germination were placed in a medium containing 50 μM Cd and photographed after 6 days. The upper right circle is a wild type, the upper left, the middle, and the lower middle circle are sensitive mutants. 6 is a graph showing inhibition of root elongation by cadmium (Cd) in the cadmium-sensitive mutant (41-3 strain) selected in Example 1. FIG. A comparison was made of the extent of root elongation between the wild type and the 41-3 line when exposed to 0 μM and 50 μM Cd for 6 days. FIG. 7 is a diagram showing the PCR conditions for TAIL-PCR performed in Example 2. FIG. 8 is an electrophoretic image showing the results of TAIL-PCR performed in Example 2. 1: Molecular weight marker (100 bp ladder), 2: 1st TAIL PCR product, 3: 2nd TAIL PCR product, 4: 3rd TAIL PCR product (second reaction diluted 50-fold was used as template) ) 5: Third TAIL PCR product (second reaction diluted 500-fold was used as template). FIG. 9A is a diagram showing amplification sites by TAIL-PCR performed in Example 2. Sequencing was performed for the third amplification product (product amplified using LB3 primer) (SEQ ID NO: 4). FIG. 9B is a diagram showing a T-DNA insertion site in the cadmium-sensitive mutant (41-3 strain) determined in Example 2. T-DNA was inserted into the seventh intron of the triose phosphate isomerase (TPI) gene. Lines and boxes indicate introns and exons, respectively. FIG. 10A is a schematic diagram of a site where the primer used in the PCR performed for confirmation of the T-DNA insertion site in Example 2 anneals. PCR was performed using 41-3 strains of genomic DNA as a template. LB, F, and R represent primers. FIG. 10B is an electrophoretic image showing the results of PCR performed for confirming the T-DNA insertion site in Example 2. 1: F, R primer combination, 2: F, LB primer combination. FIG. 11 is an electrophoretic image showing the results of semi-quantitative PCR performed on 41-3 lines in Example 2 for the purpose of confirming whether TPI gene expression is suppressed by T-DNA insertion. . RNA was extracted from plants grown in the greenhouse, and the expression of the TPI gene was confirmed by RT-PCR. WT: Wild type, 41-3: 41-3 strain (cadmium sensitive mutant). FIG. 12 is a diagram showing the results of confirming the sensitivity of the plant mutant (41-3 strain) selected in Example 1 to cadmium (Cd) in Example 3. 41-3 line (●) and wild type (■) seedlings were placed on a medium containing 0 to 150 μM Cd, and the amount of root elongation was measured on the 6th day. n = 15, vertical lines indicate standard errors.

Claims (8)

A gene according to any one of (a) to (d) below, comprising a DNA encoding a protein involved in plant cadmium resistance:
(A) DNA encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 1
(B) DNA encoding a protein comprising an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence represented by SEQ ID NO: 1.
(C) DNA containing the coding region of the base sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3
(D) DNA that hybridizes under stringent conditions with DNA containing the coding region of the nucleotide sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3.   A method for producing a cadmium-sensitive plant, comprising destroying or suppressing expression of the gene of claim 1 in a plant.   The method for producing a cadmium-sensitive plant according to claim 2, wherein the gene according to claim 1 is disrupted or expression is suppressed by base substitution, deletion, and / or insertion.   The method for producing a cadmium-sensitive plant according to any one of claims 2 to 3, wherein the gene according to claim 1 is disrupted or expression is suppressed by inserting T-DNA.   A cadmium-sensitive plant obtained by the method for producing a cadmium-sensitive plant according to any one of claims 2 to 4.   The cadmium-sensitive plant according to claim 5, wherein the plant is Arabidopsis thaliana.   A gene comprising T-DNA inserted into the gene according to claim 1.   The gene according to claim 7, comprising the base sequence represented by SEQ ID NO: 4.