JP2006204209A - Gene (ycf1) encoding yeast cadmium factor 1 or plant transformed by its modified gene, and method for sanitizing cadmium-contaminated soil by using the plant - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a plant with high tolerance to cadmium than a wild type, and to provide a method for sanitizing cadmium-contaminated soil by using the plant. <P>SOLUTION: The invention relates to the plant with a high tolerance to cadmium transformed with a polynucleotide selected from (I) a polynucleotide having a base sequence described in Sequence No 1 and (II) a polynucleotide which hybridizes in a stringent condition with a polynucleotide having a specific base sequence originated from yeasts, and encoding a protein having an activity of yeast cadmium factor 1 (YCF1). The invention relates to the method for sanitizing soil by removing cadmium from the cadmium contaminated soil comprising a process (1) culturing the plant in the cadmium contaminated soil, and a process (2) recovering whole or a part of the plant cultured in the process (1). <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention mainly relates to a plant transformed with a polynucleotide (Ycf1) encoding yeast cadmium factor 1 or a modified gene thereof, and a method for purifying cadmium-contaminated soil using the plant.

  Contamination of the biosphere with hazardous substances such as heavy metal compounds is a serious problem. Therefore, a method for efficiently and economically removing heavy metal compounds accumulated in the soil and water environment is required.

  As a solution to this problem, there is known a method (bioremediation) for purification using microorganisms. However, microorganisms are small and difficult to recover.

  In 1995, Salt et al. Proposed a method of purification using phytoremediation using plants that accumulate metal. In the United States where bioremediation is in practical use, the market for phytomediation already exists, and the estimated market size in 1999 is $ 30 to $ 49 million (Non-Patent Document 1).

On the other hand, the ABC superfamily is known as an ATP-driven transporter protein, and typical molecular species of the ABC superfamily include YCF1, MDR1, MRP1, CFTR, cMOAT, SUR, ABCR, and ABC1. In recent years, it has been reported that human-derived MRP1 functions in plant cells to impart cadmium resistance activity to the plant (Patent Document 1). However, it is considered more practical to use a gene derived from yeast, plant, algae or bacteria as a foreign gene used for transformation than a gene derived from human. Thus, there is a need for heavy metal resistant plants transformed with genes derived from yeast, plants, algae or bacteria.
Japanese Patent Application 2002-15510 Nikkei Biobusiness September 2001 issue

  This invention makes it a main subject to provide the plant which has higher cadmium tolerance than a wild type. Another object of the present invention is to provide a method for purifying contaminated soil using the plant.

  As a result of intensive studies, the present inventors transformed tobacco (Nicotiana tabacum cv. Samsun NN) using the full-length cDNA of yeast-derived cadmium factor 1 (YCF1), and the transformed tobacco was resistant to cadmium. As a result, the present invention has been completed.

The present invention mainly relates to the following matters.
[Item 1]
(I) a polynucleotide having the base sequence set forth in SEQ ID NO: 1, and
(II) a polynucleotide that hybridizes with the polynucleotide having the base sequence of SEQ ID NO: 1 under stringent conditions, and that encodes a protein having yeast cadmium factor 1 (YCF1) activity;
A cadmium-tolerant plant transformed with at least one polynucleotide selected from
[Item 2]
(i) a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2, and
(ii) A polypeptide having an amino acid sequence in which one or more amino acids are deleted, added or substituted in the amino acid sequence shown in SEQ ID NO: 2, and having a yeast cadmium factor 1 (YCF1) activity ,
A cadmium-tolerant plant capable of expressing at least one polypeptide selected from
[Item 3]
Or 2. The plant according to 2.
[Item 4]
(1) a step of cultivating the plant according to any one of claims 1 to 3 in cadmium-contaminated soil; and
(2) A step of collecting all or part of the plant cultivated in step (1),
A soil remediation method for removing cadmium from cadmium-contaminated soil.

  Hereinafter, the present invention will be described in more detail.

  The cadmium-tolerant plant of the present invention is basically obtained by transforming a plant with a polynucleotide encoding cadmium factor 1 (YCF1) derived from yeast or a modified protein thereof.

The polynucleotide encoding YCF1 or a modified protein thereof used in the present invention is:
(I) a polynucleotide having the base sequence set forth in SEQ ID NO: 1, and
(II) a polynucleotide that hybridizes with the polynucleotide having the base sequence of SEQ ID NO: 1 under stringent conditions, and that encodes a protein having yeast cadmium factor 1 (YCF1) activity;
At least one polynucleotide selected from.

As used herein, “polynucleotide hybridizing under stringent conditions” refers to a polynucleotide obtained by using a colony hybridization method, a plaque hybridization method, a Southern blot hybridization method, or the like. For example, a probe (for example, a polynucleotide having the 3171st to 4017th base sequences of SEQ ID NO: 1) is allowed to act on a support on which a polynucleotide to be detected is immobilized, and 0.7 to 1.0 M Pre-hybridization at 42 ° C. for 2 hours in the presence of NaCl, followed by hybridization at 42 ° C. for 12 to 16 hours in the presence of 0.7 to 1.0 M NaCl, followed by 0.1 to 2 About twice as much SSC solution (The composition of SSC solution of 1 times concentration is 150 mM salt. Sodium, it can be mentioned DNA or the like can be identified by washing the filter at 42 ° C. using a 15mM sodium citrate). Hybridization, Molecular Cloning:. A laboratory Mannual , 2 nd Ed, can be carried out according to the method described in such as (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989.).

  Examples of DNA that hybridizes under stringent conditions include DNA having a certain degree of homology with the base sequence of DNA used as a probe (for example, the 3171 to 4017th base sequence of SEQ ID NO: 1). The homology is, for example, 70% or more, preferably 80% or more, more preferably 90% or more, further preferably 93% or more, particularly preferably 95% or more, and most preferably 98% or more.

  The above-mentioned polynucleotides (I) to (II) encoding YCF1 or a modified protein thereof can be obtained by a conventional method. For example, using the genome extracted from yeast as a template, the polynucleotide can be amplified. It can be obtained by performing PCR (Polymerase Chain Reaction) using the designed oligonucleotide as a primer. At this time, if necessary, mutation may be introduced by a conventional method.

  The obtained polynucleotides (I) to (II) are introduced into plant cells by an appropriate gene introduction method. At this time, the polynucleotides (I) to (II) are usually linked to a suitable vector and introduced into plant cells.

  The vector used in the present invention is not particularly limited as long as it can express the target protein in plant cells, but is capable of autonomous replication of an expression vector containing the target oligonucleotide in plant cells or An expression cassette containing the target oligonucleotide can be incorporated into the chromosome, and a promoter containing a promoter at a position where the target polynucleotide can be transcribed is preferably used. The vector used in the present invention contains a selectable marker (for example, drug resistance gene, antibiotic resistance gene, reporter gene) as necessary.

  Examples of the vector used in the present invention include, but are not limited to, pBiEl2-GUS, pIG121-Hm, pBI121, pBiHyg-HSE, pB119, pBI101, pGV3850, and pABH-Hm1.

  The promoter used in the present invention is not particularly limited as long as it promotes transcription of the target polynucleotide in plant cells, and examples thereof include E12 promoter, CaMV35S promoter, Cab promoter, RuBisCo promoter, PR1 promoter and the like. Can do.

  As a method for linking the expression cassette containing the polynucleotides (I) to (II) to a vector, a conventional method such as ligation is used. At this time, the expression cassette containing the polynucleotides (I) to (II) may be indirectly linked to the vector via an appropriate sequence (for example, a restriction enzyme recognition site) or directly. It may be connected. In addition, a transcription termination sequence is not necessarily required, but it is preferably arranged immediately below the expression cassette containing the polynucleotide of the present invention.

  Any method capable of introducing a polynucleotide into a plant cell can be used as a method for introducing the expression vector containing the polynucleotide (I) to (II) thus obtained into a plant cell. For example, Agrobacterium method, protoplast method, particle gun method, electroporation method, lithium acetate method, method using calcium ion, etc. are preferable, preferably Agrobacterium method, protoplast method, particle gun method .

  Moreover, when obtaining the plant body in a desired growth (or growth) stage from the plant cell transformed as described above, a method usually used according to the plant species to be used may be performed.

  For example, when tobacco having a large plant mass is used, in a preferred embodiment of the present invention, tobacco cells can be transformed by the following method, and plants can be obtained from the transformed tobacco cells: 1 / 2. Prepare seedlings of tobacco (Nicotiana tabacum cv. Samsun NN) grown in Linsmaire-Skoog (1 / 2LS) medium. On the other hand, Agrobacterium (for example, Agrobacterium tumefaciens) introduced with a vector containing the polynucleotide used in the present invention is cultured in a liquid medium, and the tobacco leaves are immersed in this culture solution. Thereafter, the tobacco leaves are cut into pieces with a scalpel. Place the leaf back on a Murashige-Skoog (MS) medium with filter paper on the surface and co-cultivate in the light for 2 days at 25 ° C. Thereafter, the sections are transplanted into MS medium containing 250 μg / ml hygromycin and 300 μg / ml claforan. When callus or shoot formation begins from the cut end of the section, transplant to NAA-free medium with the same composition and promote rooting. Once rooted, transfer to hormone-free MS medium and grow plants in agripots. A transformed plant is selected using a medium supplemented with hygromycin.

  The plant transformed with the polynucleotides (I) to (II) is not particularly limited, and examples thereof include eggplants, cruciferous, mallow, redwood, leguminous, amaranthaceae, asteraceae, and gramineous. , Plants belonging to the willow family, magnoli family, boxwood family, citrus family, or cypress family. Specific examples include tobacco (Nicotiana tabacum), Arabidopsis thaliana, kenaf (Hibiscus cannabinus), maple oyster (Hibiscus coccineus), American squirrel (Hibiscus moscheutos), kingfish (Hibiscus hamabo), bissogus (sin) Mustard (Brassica juncea), thumpi rotundifolium, okra (Abelmoschus esculentus), tuna (Abelmoschus manihot), spinach (Spinacia oleracea), chard (Beta vulgaris var. Vulgaris) Raccoon bean (Crotalaria sessiliflora), Gakutanuki bean (Crotalaria calycina), Crotalaria juncea, sunflower (Helianthus annuus), rice (Oryza sativa), barley (Hordeum vulgare), oat ua (navenus) (Populus nigra), Liriodend ron tulipifera), Hondua (Buxus microphylla), Citrus unshiu, lemon (Citrus lemon), Hydrangea macrophylla, and willow (Salix myrsinites). Among them, tobacco, kenaf, mustard, okra , Raccoon bean, poplar and willow, particularly preferably tobacco.

  Thus, a plant transformed with the polynucleotides (I) to (II) is obtained.

The invention also relates to the following plants:
(i) a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2, and
(ii) A polypeptide having an amino acid sequence in which one or more amino acids are deleted, added or substituted in the amino acid sequence shown in SEQ ID NO: 2, and having a yeast cadmium factor 1 (YCF1) activity A cadmium-tolerant plant capable of expressing at least one polypeptide selected from.

  In the present specification, the “amino acid sequence in which one or more amino acids are deleted, added or substituted” is particularly limited as long as it is an amino acid sequence modified to have yeast cadmium factor 1 (YCF1) activity. Although not, for example, an amino acid sequence in which any number of amino acids, such as 1 to 100, preferably 1 to 60, more preferably 1 to 15, and more preferably 1 to 5, are deleted, added or substituted Say that.

  In addition, the plant of the present invention includes plants in every growth process, and includes, for example, seeds, callus, seedlings, young seedlings, middle seedlings, mature seedlings, propagation leaves, flowering, fruiting and the like. The plant of the present invention also includes a part of the plant of the present invention, and includes, for example, cells, tissue sections, roots, shoots and the like.

  The present invention further relates to phytomediation using the plant of the present invention. That is, the present invention includes (1) a step of cultivating the cadmium-resistant plant of the present invention in cadmium-contaminated soil, and (2) a step of recovering all or part of the plant cultivated in step (1). It also relates to the purification method.

  As a method for cultivating the cadmium resistant plant of the present invention, a method suitable for the plant species is used. For example, the plant of the present invention is transplanted to contaminated soil and cultivated while supplying a predetermined amount of water, fertilizer (eg, compost), uncontaminated soil, etc. according to the plant species, plant growth stage, climate, etc. be able to. When the plant of the present invention is in an initial growth stage, for example, when the plant of the present invention is a seed or a seedling, it grows to some extent (for example, until it becomes a seedling or a medium seedling) under appropriate conditions. Then, it is preferably transplanted to contaminated soil.

  As a method for recovering the cadmium-tolerant plant of the present invention, a method suitable for the plant species (for example, a method using a harvester) is used. At this time, only a part of the plant may be recovered, but it is preferable to recover the entire plant including the root part.

  ADVANTAGE OF THE INVENTION By this invention, the cadmium tolerance plant and the purification | cleaning method of contaminated soil using this cadmium tolerance plant are provided. The cadmium-tolerant plant of the present invention can grow well in the presence of cadmium at a high concentration (for example, 250 μM).

  The cadmium-tolerant plant of the present invention can be suitably used for phytomediation of soil containing cadmium.

  Moreover, since the cadmium resistant plant of the present invention is transformed with a yeast-derived cadmium factor 1 gene or a modified gene thereof, it is considered to be more practical than a cadmium resistant plant transformed with a human-derived gene. .

  The present invention will be described more specifically with reference to the following examples, but the present invention is not limited to the examples.

1. Construction of Binary Vector There are one restriction enzyme site that can be used for subcloning in the binary vector pBiEl2-GUS (pHM6), XbaI at the 5′-end and SacI at the 3′-end. Therefore, it was decided to introduce an XbaI site at the 5′-end and a SacI site at the 3′-end of the transgene. First plasmid with the 5'-end of YCF1 cDNA (pBS-T- (SK +) - pKmYCF1-5 ') a YCF1 cDNA 5'-end containing an XbaI site, was amplified by PCR using oligo YCF1 primers, AlwNI And BglII restriction enzyme treatment was performed to obtain an AlwNI / BglII fragment. Then 5 of YCF1 '-, was obtained from the plasmid (pIBI-YCF1) with cDNA lacking the 3'-end of the BglII / SalI fragment of about 4300b with BglII and SalI restriction enzyme treatment. Furthermore, a SalI / NotI fragment containing the 3'-end of YCF1 was obtained from a plasmid (pFastBacI-YCF1-6His) having 6'His derived from pQE70 downstream of the 3'-end of YCF1 cDNA by treating with SalI and NotI restriction enzymes. It was. This was ligated to the pBS-KS + plasmid using SalI / NotI restriction enzymes, and a base sequence with 6 × His added to the 3′-end of YCF1 was introduced to obtain a pBS-KS + -YCF1-6His plasmid. Further, this plasmid was treated with AlwNI and SalI restriction enzymes to obtain AlwnI / SalI fragments. The AlwNI / BglII fragment derived from pBS-T- (SK +)-pKmYCF1-5 ′ and the BglII / SalI fragment derived from pIBI-YCF1 were introduced by ligation to obtain pBS-pKm-YCF1-6His. By treating this plasmid with XbaI and SacI restriction enzymes, a full length YCF1 cDNA and a fragment containing 6 × His at its 3′-end were obtained. This was introduced into pHM6 by ligation using the XbaI and SacI restriction enzyme treatment sites, and the CaMV35S promoter, the HPG gene that is a hygromycin resistance gene, the YCF1 cDNA full length and 6 × His were linked to the selection marker (pHM6- pKmYCF1-6His) was prepared.

2. Transformation of Agrobacterium tumefaciens In order to introduce the binary vector (pHM6-pKmYCF1-6His) obtained above into plants, Agrobacterium tumefaciens (strain LBA4404) for plant infection was transformed. Transformation was performed by electroporation, and after plating, a plasmid was extracted from a kanamycin-resistant colony and treated with a restriction enzyme, and it was confirmed by agarose gel electrophoresis that a binary vector was introduced.

3. Tobacco transformation The seedlings of tobacco ( Nicotiana tabacum cv. Samsun NN) grown in 1/2 Linsmaire-Skoog (1 / 2LS) medium in an agripot were prepared. Agrobacterium tumefaciens (LBA4404 strain) transformed with a binary vector was pre-cultured in a liquid medium, and the true leaf of the agripot tobacco was immersed in the petri dish in this A. tumefaciens culture solution. This tobacco leaf is cut into sections of about 5-10 mm square using a scalpel, lightly dehydrated with a sterilized Kim towel, and placed on a Murashige-Skoog (MS) medium with filter paper on the surface with the back of the leaf up, The cells were cocultured at 25 ° C. for 2 days in a bright place. Sections were transplanted to MS medium containing 250 μg / ml hygromycin and 300 μg / ml claforan after co-culture. The medium was replaced with a new one every two weeks. Those that started to callus or shoot from the cut end of the section were transplanted to NAA-free medium with the same composition to promote rooting. Once rooted, they were transferred to hormone-free MS medium and the plants were grown in agripots. Plants were always subjected to selection by applying selective pressure using a medium supplemented with hygromycin until use in experiments.

[Results and discussion]
As a result of the transformation, 36 clones of tobacco YCF1 transformants were obtained. The transformant was not different from the wild type in its form.

4. Northern analysis In order to examine the expression level at the mRNA level of the clones obtained by transformation, Northern analysis was performed according to the following procedure.

4-1. Extraction of total RNA Tobacco YCF1 transformants grown in agripots and wild-type leaves (rapidly frozen in liquid nitrogen and stored at -80 ° C) from 200 mg to QIAGEN's RNeasy Plant Using the Mini Kit, 10 μg to 30 μg of total RNA was extracted.

4-2. Electrophoresis and transfer of RNA samples
Reagents used for electrophoresis and transfer of RNA samples are as follows.

20 x MOPS
(0.4M MOPS, 0.1M NaOAc, 0.02M EDTA)
The mixture was adjusted to pH 7.0 with KOH and autoclaved for 1 hour.
RNA sample buffer formaldehyde 1.6ml
Formamide 5.0ml
20 x MOPS 0.5ml
Glycerine pigment solution 1.6ml
total 8.7ml
Formaldehyde

Glycerine pigment solution Glycerin 5ml
1mg / ml Bromophenol blue 1ml
1mg / ml xylene cyanol 1ml
0.5M EDTA (pH8.0) 0.02ml
H ₂ O 2.98ml
total 10ml
Ethidium bromide solution (10 mg / ml in H ₂ O)
20 x SSC
(3M NaCl, 0.3M trisodium citrate dihydrate)
Mupid-2 (Cosmo Bio) was used for autoclaving water electrophoresis. First, a gel was prepared. Agarose (0.47 g) was placed in a 100 ml Erlenmeyer flask, and further 36 ml of autoclave water and 2 ml of 20 × MOPS were added and heated to completely dissolve the agarose. Then, when it was cooled down to the extent that it could be touched by hand, 2 ml of formaldehyde was added and stirred, poured into a gel cassette and combed to harden the gel. As the electrophoresis buffer, 20 × MOPS was diluted with autoclaved water and used as 1 × MOPS.

  Add 16 μl of RNA sample buffer to RNA solution containing 10 μg of total RNA, let stand at 65 ° C. for 10 minutes, then quench in ice, apply 20 μl each, run at 50 V for 5 minutes, then run 100 V Electrophoresis was performed. After electrophoresis, the electrophoresis image was confirmed by FAS (TOYOBO).

  A capillary transfer method was used for the transfer. 20 × SSC was used as the transfer buffer. Place the gel on the Whatman 3MM filter paper, which has been thoroughly moistened with 20 x SSC, and placed on the bottom of the gel so that air does not enter, and then a nylon membrane (Amersham Hybond N +) on the gel I put it on. On top of that, Whatman 3MM filter paper soaked in 2 × SSC, which is one size larger than the membrane, was placed, placed on Kim Towel and left at room temperature for 15 hours or more.

  After the transfer, the position of the well was filled in with a water-resistant colored pencil before peeling off the membrane from the gel. Thereafter, the membrane was transferred into 2 × SSC, shaken lightly for about 5 minutes, wrapped in a wrap film, and irradiated with 1200 count ultraviolet rays with a UV crosslinker (Funakoshi CL-1000) to immobilize RNA on the membrane.

4-3. Preparation of probe
YCF1 -fw primer (5'-TGAAGAGCTACAACCAGATTCG-3 '(SEQ ID NO: 3)) and YCF1-rv primer (5'-TTCCAGCTCTCCTTCTACAGG-3' (SEQ ID NO: 4) )) Was used to amplify a fragment of about 850b from the YCF1 cDNA internal sequence 3171b to 4017b by PCR. This PCR product was purified and used as a probe. The composition of the PCR reaction solution is as follows.

Composition of PCR reaction solution
Templatae DNA 100ng
10 × Taq buffer 5μl
2 mM dNTPs 5 μl
50μM YCF1-fw primer 1μl
50μM YCF1-rv primer 1μl
Taq polymerase (SIGMA) 1μl
+ H ₂ O 50μl
The PCR reaction cycle is as follows:
[1] 95 ° C for 1 minute
[2] 45 ° C for 30 seconds
[3] 72 ° C. for 1 minute The above [1] → [2] → [3] were performed 30 cycles.

. 4-4 probe about 50ng prepared by labeling the above probe [alpha] - ³² were labeled with P-CTP and Random Primer DNA Labeling Kit (Roche) . First, a total of 9 μl of about 50 ng of DNA serving as a probe and H ₂ O was used, treated at 100 ° C. for 5 minutes, and rapidly cooled in ice. Next, the remaining reagents in the following reaction mixture were added and incubated at 37 ° C. for 30 minutes or longer.

reagent
Reaction mixture
denatured DNA (50ng) + H ₂ O 9μl
dNTP mix (A, T, G) 3μl
reaction buffer 2μl
³² P-dCTP (3700kBq / μl) 5μl
Klenow enzyme (exo-) 1μl
+ H ₂ O 20μl
The reaction mixture was purified using a quick spin column (Roche), and the uptake rate of ³² P-dCTP was measured with a Geiger counter. The probe was heat denatured at 95 ° C. for 5 minutes, cooled on ice and used for hybridization.

4-5. Hybridization Place the membrane on the inside of the hybrid bottle with the RNA attached inside, and pre-warm the hybridization solution (1M NaCl, 10% Dextran Sulfate, 20mM Tris-HCl (pH7.5), 1% SDS, 5 × Denhardt's solution, 50% Formamide) and salmon sperm DNA heat-denatured at 95 ° C. for 5 minutes were added to 100 μg / ml. Prehybridization was performed at 42 ° C. for 2 hours. Then, the whole amount of the previously labeled probe was added, and hybridization was performed at 42 ° C. for 12 to 16 hours.

4-6. Wash
After completion of hybridization, the membrane was shaken in 2 × SSC / 0.2% SDS preheated to 60 ° C. for 5 minutes at 60 ° C. After discarding the washing solution and repeating this, the washing solution was discarded, and 2 × SSC / 0.2% SDS was further added, and shaking at 60 ° C. for 20 minutes was repeated twice. After this washing, the membrane count was measured and exposed to X-ray film (Kodak) to detect the signal. The exposure time was 16 hours at −80 ° C., and the detection was carried out by changing the exposure time again after seeing the film condition.

[Discussion]
As a result of Northern analysis, a band of about 4.5 kb corresponding to the full length of YCF1 mRNA was confirmed in 24 clones of tobacco YCF1 transformant, and since this band was not confirmed in the wild type, YCF1 introduced into tobacco was It was confirmed that it was expressed at the mRNA level.

5. Cadmium resistance test

5-1. Transplantation 36 specimens of Ycf1-introduced tobacco were grown in agripots and transplanted approximately every month.

5-2. Preparation of 1 / 2LS medium Sucrose 15g was dissolved in 1 L of pure water, and stock of Table 1 was added with a pipette to a predetermined concentration, and stirred well. The pH was adjusted to 5.8 using 1N-NaOH and 0.1N-NaOH, and the whole was made up to 1 L. Divided equally into two 500 ml sterilized bottles, 4 g of agar powder was added to each bottle and sterilized by autoclave. The medium was aseptically and evenly flowed and solidified in 20 clean-dried agripots in a clean bench.

5-3. Passage culture One planted tobacco line was aseptically planted in two agripots. Leave a few leaves from above, cut the leaves with scalpels and tweezers, cut the stems with scalpels and plant them in a new agripot with tweezers.

5-4. Storage The agripot was left under a fluorescent lamp in a 23 ° C. culture room and observation was continued.

5-5. Preparation of Cd-containing callus derivatization medium
Cytokinin 10 ⁻⁶ M and auxin 10 ⁻⁵ M were added to LS medium prepared in the same manner as described above (Stock6 was added twice) and sterilized. The medium was divided into two equal parts, 1 mM Cd aqueous solution Stock was added to 0 μM and 250 μM, and the mixture was poured into a petri dish and hardened.

5-6. Applying a leaf sample Three tobacco leaves from the top of the same culture stage are sampled, and a 9 mm square section is cut with a scalpel so as to include the veins. Sections were placed in a petri dish containing control (0 μM) and cadmium-containing (250 μM) medium.

5-7. Storage The petri dish was allowed to stand under a fluorescent lamp in a 23 ° C. culture room and observation was continued. As a control, the same operation was performed for the wild type strain. About 1 month later, the judgment was made.

5-8. Determination The growth state of tobacco callus cells in a control (0 μM) or cadmium-containing (250 μM) medium was compared with that of the wild strain to determine the presence or absence of resistance. Fresh weight of petri dish callus cells was measured.

[result]
Among transformants, Y39 and Y43 showed significantly higher resistance to cadmium (Cd) -containing compounds than the wild type. Table 2 shows the growth determination results of the transformants Y39 and Y43 in the presence of cadmium. In Table 2, ◎ indicates good, ○ indicates normal, and X indicates poor. Table 3 shows the fresh weight of the transformed strains Y39 and Y43.

SEQ ID NO: 1 is the nucleic acid sequence of Ycf1.
SEQ ID NO: 2 is the amino acid sequence of YCF1.
SEQ ID NO: 3 is a primer.
SEQ ID NO: 4 is a primer.

Claims (4)

(I) a polynucleotide having the base sequence set forth in SEQ ID NO: 1, and
(II) a polynucleotide that hybridizes with the polynucleotide having the base sequence of SEQ ID NO: 1 under stringent conditions, and that encodes a protein having yeast cadmium factor 1 (YCF1) activity;
A cadmium-tolerant plant transformed with at least one polynucleotide selected from (i) a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2, and
(ii) A polypeptide having an amino acid sequence in which one or more amino acids are deleted, added or substituted in the amino acid sequence shown in SEQ ID NO: 2, and having a yeast cadmium factor 1 (YCF1) activity ,
A cadmium-tolerant plant capable of expressing at least one polypeptide selected from The plant is a plant belonging to the family Solanumaceae, Brassicaceae, Aoiaceae, Rubiaceae, Leguminosae, Amaranthaceae, Asteraceae, Gramineae, Willowaceae, Magnoliaceae, Boxwood, Citrus or Yukinosita 2. The plant according to 2. (1) a step of cultivating the plant according to any one of claims 1 to 3 in cadmium-contaminated soil; and
(2) A step of collecting all or part of the plant cultivated in step (1),
A soil remediation method for removing cadmium from cadmium-contaminated soil.