JP2003319787A - Method for absorbing heavy metal - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method with which usefulness for environmental clean-up such as phytoremediation is obtained and a heavy metal such as cadmium, cobalt, copper or zinc is efficiently absorbed. <P>SOLUTION: The method for absorbing the heavy metal comprises incorporating the cadmium, cobalt, copper, zinc and/or arsenic into a recombinant cell in which any gene selected from the group consisting of an merC gene, an merF gene and an merT gene is transduced. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for absorbing heavy metals using transformed cells, which can be used for phytoremediation, monitoring soil contamination, and the like.

[0002]

2. Description of the Related Art As one of the serious environmental pollution problems, when considering the purification of heavy metal pollution in soil, the most effective method is to incinerate the heavy metal after removing it from the soil to remove the heavy metal from the soil. It is considered to be a means that is rational and can be applied to a wide range in terms of cost (see Non-Patent Document 1).

On the other hand, it is known that the gene existing in the Mer operon is involved in the transport of mercury (merC, merF, merT, merP) and the reduction of mercury (merA) and the decomposition of organic mercury (merB). For example, iron-oxidizing bacteria (Thiobacillus ferro
It is known that the merC gene found from oxidans strain E-15) is involved in the uptake of mercury ions (Hg (II)) into cells (see Non-Patent Document 2).

[0004]

[Non-Patent Document 1] Salt, DE et al., BIO / TECHNOLO
GY (1995) Vol.13, p.468-474

[Non-Patent Document 2] Biosci. Biotech. Biochem. (1996)
Vol.60, p.1289-1292

[0005]

Conventionally, studies on the transport of heavy metals and isolation of genes associated therewith have been carried out, but these have been studied for transport mechanisms centered on the essential element such as zinc. Mainly elucidated, applicable to phytoremediation, cadmium, cobalt, copper,
At present, the isolation of genes and the elucidation of the mechanism for the transfer of lead, arsenic, etc. are delayed. For this reason, there has not yet been known a method for widely and actively incorporating heavy metals such as cadmium, cobalt, copper, lead, and arsenic into cells.

[0006]

Means for Solving the Problems As a result of intensive studies for solving the above problems, the present inventors have found that genes into which genes involved in mercury transport such as the merC gene have been introduced have cadmium, cobalt, copper and zinc. The inventors have found that the ability to take in heavy metals other than mercury such as lead, arsenic, etc. is improved, and have completed the present invention. That is, according to the present invention, a recombinant cell into which any gene selected from the group consisting of the merC gene, the merF gene and the merT gene has been introduced, and cadmium, cobalt, copper, zinc and / or arsenic is introduced into the cell. A method for absorbing heavy metals is provided.

According to another aspect of the present invention, a recombinant cell into which any gene selected from the group consisting of the following (a), (b), (c) and (d) is introduced is used, and cadmium is used. There is provided a method for absorbing heavy metals, characterized by incorporating cobalt, copper, copper, zinc and / or arsenic into the cells. (A) a gene encoding a protein having the amino acid sequence set forth in SEQ ID NO: 1 (b) mercury having one to a plurality of substitutions, deletions and / or insertions in the protein having the amino acid sequence set forth in SEQ ID NO: 1 Gene encoding a protein capable of transferring ions into cells (c) Gene having the nucleotide sequence set forth in SEQ ID NO: 2 (d) Hybridizing with any of the above genes under stringent conditions, and mercury ion cells Genes involved in internal transfer

According to another aspect of the present invention, a recombinant yeast into which any gene selected from the group consisting of merC gene, merF gene and merT gene is introduced,
And a recombinant plant is provided. According to another aspect of the present invention, in a method for absorbing heavy metal in soil by a plant and accumulating it in the plant to purify soil contaminated with heavy metal, the plant comprises at least a merC gene and a merF gene. And a merT gene, which is a recombinant plant into which any gene selected from the group consisting of genes is introduced.

According to another aspect of the present invention, in a method for purifying soil contaminated with heavy metals by absorbing heavy metals in the soil into the plants and accumulating in the plants, the plants are at least The following (a), (b),
There is provided a method for remediating soil, which is a recombinant plant into which any gene selected from the group consisting of (c) and (d) has been introduced. (A) a gene encoding a protein having the amino acid sequence set forth in SEQ ID NO: 1 (b) mercury having one to a plurality of substitutions, deletions and / or insertions in the protein having the amino acid sequence set forth in SEQ ID NO: 1 Gene encoding a protein capable of transferring ions into cells (c) Gene having the nucleotide sequence set forth in SEQ ID NO: 2 (d) Hybridizing with any of the above genes under stringent conditions, and mercury ion cells Genes involved in internal transfer

The present invention will be described in detail below.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION The method of the present invention uses mercury ions (Hg
M, which is a gene involved in the intracellular uptake of (II))
It is characterized by using a transformed cell into which any one selected from the group consisting of the erC gene, the merF gene and the merT gene is introduced into the cell.

MerC gene, merF gene and m
The erT gene is contained in a microorganism resistant to mercury.
It can be optionally isolated from the er operon. Also,
The above-mentioned microorganism having resistance to mercury can be easily obtained by screening with a medium containing mercury, or a known substance may be optionally used.

Specific examples of the bacterium having the merC gene and the merT gene include Acinetobacter (Acinet).
microorganism belonging to the genus bacterium (Plasmid 30, 303-308, 19
93), microorganisms belonging to the genus Alcaligenes (Mol. Microbiol. 24, 321-329, 1997), microorganisms belonging to the genus Citrobacter (J. Mol. Evo).
l. 51, 607-622, 2000), Enterobacter (Enteroba
Microorganisms belonging to the genus cter (Mol. Microbiol. 24, 321-32
9, 1997), a microorganism belonging to the genus Escherichia (Microbiol. Mol. Biol. Rev. 63, 507-522, 199).
9 and J. Mol. Evol. 51, 607-622, 2000) A microorganism belonging to the genus Klebsiella (J. Mol. Evol. 5)
1, 607-622, 2000), microorganisms belonging to the genus Leclercia (J. Mol. Evol. 51, 607-622, 2000), microorganisms belonging to the genus Pantoea (Mol. Microbio)
l. 24, 321-329, 1997), Pseudomonas (Pseudomona)
s) microorganisms belonging to the genus (J. Mol. Evol. 51, 607-622, 20)
00, J. Mol. Evol. 51, 607-622, 2000, Gene 166, 77-
82, 1995, Macrobiology 143, 2549-2556, 1997, J. Mo.
l. Evol. 51, 607-622, 2000 and FEBS letter 427 (200
0) 78-82) Microorganism belonging to the genus Shewanella (Mol. Gen. Genet. 228, 294-299, 1991) Microorganism belonging to the genus Sphingomonas (J. Mol. Ev
ol. 51, 607-622, 2000), Thiobacillus (Thiobacillu
s) microorganisms belonging to the genus (Gene 96, 115-120, 1990) and microorganisms belonging to the genus Xanthomonas (Re
s. Microbiol. 151, 1-12, 2000).

As the bacterium having the merF gene, a microorganism belonging to the genus Pseudomonas (Gene 146, 73-78, 1994), Xanthomonas (Xanthom) is used.
microorganism belonging to the genus onas) (J. Mol. Biol. 230, 1103-11
07, 1993), a microorganism belonging to the genus Escherichia (Appl. Environ. Microbiol. 63, 1066-1076, 1)
997) etc. are raised.

The nucleotide sequences of these genes existing in the above-mentioned microorganisms are known, and based on such information, it can be arbitrarily isolated from the microorganism by using a general genetic engineering technique. It can also be synthesized by using a DNA synthesizer or the like. Specific examples of preferred genes include:
A gene encoding the protein (MerC) shown in SEQ ID NO: 1, 1 in the protein shown in SEQ ID NO: 1
To a gene encoding a protein having a plurality of substitutions, deletions and / or insertions, capable of transporting mercury ions into cells, a gene having the nucleotide sequence of SEQ ID NO: 2 (merC), and Examples include genes that hybridize with any of the genes under stringent conditions and are involved in mercury ion intracellular transport.

Here, in the present specification, the "protein having substitution, deletion and / or insertion of 1 to a plurality of bases" is, for example, 1 to 40, preferably 1 to 20,
More preferably, it refers to a protein having an amino acid sequence in which any number of amino acids of 1 to 10 is substituted, deleted and / or inserted. In addition, "a gene that hybridizes under stringent conditions" means a base of DNA obtained by using a DNA as a probe and using a colony hybridization method, a plaque hybridization method, a Southern blot hybridization method, or the like. Sequence means, for example, 0.7 to 1.0 using a filter on which DNA derived from a colony or plaque or a fragment of the DNA is immobilized.
After hybridization at 65 ° C. in the presence of M NaCl, 0.1-2 × SSC solution (1 × SSC composition is 150 mM sodium chloride, 15 mM sodium citrate) was used to filter under 65 ° C. conditions. Examples thereof include DNA that can be identified by washing.

Hybridization is performed using Molecular Cl
oning: A laboratory Mannual, 2ndEd., Cold Spring H
arbor Laboratory, Cold Spring Harbor, NY., 1989. Hereinafter, it can be performed according to the method described in "Molecular cloning 2nd edition"). Examples of the DNA that hybridizes under stringent conditions include DNAs having a certain homology with the base sequence of the DNA used as a probe. The homology above a certain level here means, for example, 55% or more, preferably 6
It is 0% or more, more preferably 70% or more, and further preferably 90% or more. It should be noted that the DNA having a certain homology or more as referred to herein includes both the above-mentioned polynucleotide having homology and its complementary strand polynucleotide.

In addition, the above-mentioned genes whose codons are changed according to the host to be transformed are also included in the scope of the gene of the present application. As a method for transforming cells with the above gene, the gene may be ligated to an expression vector suitable for the host, and the host may be transformed by a general transformation method. Examples of the host include Corynebacte and Escherichia.
rium), Brevibacterium genus,
Genus Bacillus, Pseudomona
Saccharomyces c., a bacterium used as a host for general gene recombination of genus etc.
erevisae), Schizosaccharomyces pombe (Schizosaccha)
romyces pombe), Kluyveromyces lactis (Kluyv
eromyces lactis), Trichosporon pullulans (Trich
osporon pullulans), Schwanniomyces arubius (S
chwanniomyces alluvius) and kenaf (Hibi
scus cannabinus), mustard (Brassica juncea), sunflower (Helianthus annuus), rice (Oryza sativa), rapeseed (Brassica napus), poplar (Populus nigra),
Plant cells such as Liriodendron tulipifera can be mentioned.

Of these, preferably yeast such as Saccharomyces cerevisae and kenaf (Hibiscus cannabinus), mustard (Brassica junce).
a), sunflower (Helianthus annuus), poplar (Populus n
plant cells such as igra) and lily tree (Liriodendron tulipifera). The expression vector can be selected in consideration of the combination with the host, and is preferably capable of autonomous replication in the host cell or can be integrated into the chromosome and contains a promoter at a position where the gene of the present invention can be transcribed. Is used.

When bacteria are used as host cells, DN
The expression vector for expressing A is preferably a recombinant vector capable of autonomous replication in the bacterium and at the same time composed of a promoter, a ribosome binding sequence, the above DNA and a transcription termination sequence. A gene that controls the promoter may be included. Examples of expression vectors for bacteria include pUC118 (Takara Shuzo) and pUC19 [G
ene, 33, 103 (1985)] and pGEMEX-1 (Promeg
a company's) pGEM system, pKK223-2 (Pharmacia company), p
BluescriptII SK (+), pBluescriptII SK (-) (Stratagene
Known products or commercially available products such as those manufactured by K.K. can be exemplified.

[0021] Examples of the promoter for bacteria include
trp promoter (P trp), lac promoter (P lac), PL
Various promoters derived from Escherichia coli, phage, etc. such as promoter, PR promoter, PSE promoter, etc.
Examples thereof include SP01 promoter, SP02 promoter, penP promoter and the like. Examples of the method for introducing the recombinant vector into the bacterial host include a method using calcium ions and an electroporation method.

As an expression vector for yeast, for example, YE
p13 (ATCC37115), YEp24 (ATCC37051), Ycp5O (ATCC374
19), pHS19, pHS15, pYPR3831X (Gene 84 (1989) 47-5
4) etc. can be illustrated. Examples of the promoter for yeast include PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, gal1 promoter, gal10 promoter, heat shock protein promoter, MFα1 promoter, CUP1 promoter and the like.

As a method for introducing the recombinant vector into the yeast host, any method can be used as long as it is a method for introducing DNA into yeast, and examples thereof include electroporation method, spheroblast method and lithium acetate method. be able to.

As a method for transforming a yeast host, for example, plasmid pYPR3831X (Gene 84 (1989) 47-54) is used.
Recombinant plasmid containing erC gene (pYPR3831
X-merC) by the lithium acetate method (Ito, H et al. (1983)
J. Bacteriol. 153 (1), 163-168) etc. to transform yeast strains such as Saccharomyces cerevisiae etc., spread on YNBD agar plate and allowed to grow at optimal growth temperature, then confirm introduction of plasmid.

Furthermore, when an inducible promoter is used, it becomes possible to express the transgene after the recombinant yeast has grown appropriately or after the recombinant plant has grown to a target size. Is preferred.

Furthermore, regarding yeast cells into which the above-mentioned plasmid has been introduced, whether or not the merC gene is expressed in a galactose-dependent manner is analyzed by Northern hybridization, whereby the gene functions in the above-mentioned yeast cells. You can check whether it is. The heavy metal uptake ability of the transformant can be analyzed by the following method. For example, the yeast having the pYPR3831X-merC obtained above is cultured in a YNBD liquid medium with shaking, and then galactose is added to half of the yeast to induce the expression of the merC gene. Also, as a control, mer
Yeast transformed with the pYPR3831X plasmid into which the C gene has not been introduced is also prepared in the same manner as described above, and then plates of these four yeasts are agar-fixed. CdCl ₂ , CoCl ₂ , CuCl ₂ , ZnCl ₂ on these plates
The amount of heavy metal uptake can be determined by placing an antibiotic assay disc (diameter 6 mm) on which a certain amount of heavy metal-containing compound of interest is placed, and measuring and comparing the diameter of the inhibition circle after culturing. Monitor. That is, for example, when the heavy metal uptake activity was increased by the function of the introduced gene, it was confirmed that the diameter of the inhibition circle was increased by inhibiting the growth of yeast, and the heavy metal uptake ability of yeast was increased. can do.

In the method of the present invention, the recombinant thus obtained is brought into contact with a heavy metal-containing medium to incorporate the heavy metal into the cells. As the heavy metal-containing medium, various media such as heavy metal-containing medium, soil or sludge can be used, and as the contact method, the recombinant sufficiently grown or grown as described in the above-mentioned function confirmation section. The method of contacting with the medium can be mentioned.

Further, the recombinant obtained by the above method is
For example, in the case of recombinant yeast, it can be used as a recombinant yeast that is extremely sensitive to heavy metals to select genes involved in heavy metal resistance. That is, it was synthesized from bacteria, yeast, plants, and animals that are resistant to heavy metals such as cadmium.
Genes involved in heavy metal resistance can be screened by inserting the cDNA into a general yeast expression vector and confirming the heavy metal resistance of the recombinant yeast after retransformation into the recombinant yeast of the present invention. .

As an expression vector for plant cells, for example, pIG121-Hm [Plant Cell Report, 15, 809-814 (199
5)), pBI121 [EMBO J. 6, 3901-3907 (1987)], pLAN41
1 and pLAN421 (Plant Cell Reports 10 (1991) 286-290)
Can be illustrated.

Examples of promoters for plant cells include the cauliflower mosaic virus 35S promoter (Mol. Gen. Genet (1990) 220, 389-392) and the like. Further, a promoter of a maize-derived alcohol dehydrogenase (Maydica 35 (1990) 353-357) and a promoter of an IRE gene derived from Arabidopsis thaliana (Japanese Unexamined Patent Application Publication No. 2002-242242).
When a merC gene, a merF gene or merT is introduced using a known root-specific promoter (eg, 0-270873), it is considered that heavy metal uptake in soil can be efficiently carried out. Examples of methods for introducing a recombinant vector into plants include particle gun method, electroporation method, Agrobacterium method and the like.

Further, in the case of a recombinant plant using the above-mentioned plant as a host, for example, as a heavy metal highly accumulating plant which efficiently absorbs them from soil with a low concentration of heavy metals and accumulates them in plants to purify them. It can also be used as a monitor for soil contaminated with a trace amount of heavy metals by using it in paddy fields or by growing the plant in contaminated soil. When plants absorb too much heavy metals, such as cadmium, and accumulate above their detoxification mechanisms, damage occurs, and in Arabidopsis, whitening of the green leaves occurs. At that time, it was shown that the cadmium content increased (G. Vert et al., ThePlant Cell (2002).
14, 1223-1233), and it is possible to produce a transgenic plant as described above.

The recombinant plant of the present invention having an increased heavy metal absorption ability is further cultivated by introducing a heavy metal resistance gene, or by crossing with a plant having a high heavy metal resistance, to thereby accumulate heavy metal in the plant body. Can be increased. Examples of genes capable of imparting heavy metal tolerance to plants include the metallothionein gene, which is a heavy metal binding protein of animals and yeasts. By introducing the gene into plants, harmful heavy metals were detoxified, and heavy metal resistant plants against cadmium and the like were produced. Hasegawa introduced the yeast metallothionein gene cup1 into mustard (B. juncea) and produced a plant that could grow normally even in a cadmium aqueous solution at a concentration of 400 μM (Hase
gawa, I. 2002. Phytoremediation: a novel strategy
for removing toxic heavy metals from contaminated
soils using plants. Farming Japan. 36: 10-15).
In addition, by enhancing the activity of the metabolic system that synthesizes glutathione (GSH), a component of phytokeratin, which is one of the metallothioneins, heavy metal-resistant plants that are resistant to high-concentration heavy metal media have been created (Zhu et a
l. 1999 Overexpression of glutathione synthetase i
n Indian mustard enhances cadmium accumulation and
tolerance. Plant Physiol. 119: 73-79; Zhu et a
l. 1999 Cadmium tolerance and accumulation in Indi
an mustard is enhanced by overexpressing γ-glutam
ylcysteine synthetase. Plant Physiol. 121: 1169-11
77).

Furthermore, by using the recombinant plant obtained in the above, which has an increased ability to accumulate heavy metals in the plant,
Soil polluted with heavy metals can be purified by the phytoremediation method. The state of pollution of the soil to be purified is, for example, the value regulated by law, etc., for plant products that are tested in the form of being edible or ingestible by humans regardless of whether they are crops or natural or artificial means. , Or the state of soil, etc., which contains pollutants at a concentration that may cause more pollutants to be contained in them than the values that are medically problematic or that are socially problematic. Show. Specific soils for purification include agricultural land, that is, paddy soil, soil of all agricultural land where agricultural crops such as upland soil are cultivated, and where suitable treatment is performed, a residential area and a factory site can be grown. , Soils that are not normally used as agricultural land, such as non-residential areas, can also be targeted. In addition, sludge, sludge, etc. that can grow plants by appropriate treatment are also targeted.

As the above-mentioned heavy metals, cadmium, zinc,
Examples of the metal species and compounds containing these metals, which are problematic as heavy metal pollution such as lead, chromium, copper, mercury and / or arsenic, and preferred metal species among them are cadmium, chromium, zinc and lead. Alternatively, it is copper.

The content of the above-mentioned heavy metals in the soil is not particularly limited as long as it is within the range where plants grow, but specifically, it is usually 1% (10000 ppm) or less, preferably the content is 0.1 to 2000 ppm. , Especially for agricultural land, 0.5 to 100 ppm, preferably 0.5 to 5
It is desirable to apply to ppm soil. When it is contained at a concentration exceeding this, it is desirable to reduce the concentration in advance by an appropriate method such as a chemical treatment method and then grow the plant. Incidentally, even if the soil to be purified contains a metal other than the above-mentioned heavy metals, there is no particular problem as long as it does not inhibit the growth of plants.

The plants used in the method of the present invention include:
It is not particularly limited as long as it absorbs / accumulates the above-mentioned heavy metals, and specific examples thereof include plants such as Brassicaceae, Malvaceae, Leguminaceae, Acalyptaceae, Solanaceae, Asteraceae, Amaranthaceae, and Gramineae. . The above-mentioned plants are preferably Brassicaceae such as mustard, rapeseed and nozawana; Mallowaceae such as okra, troll mallow and kenaf; Leguminaceae such as crotalaria and sesbania; Pedicaceae such as beet for feed and sugar beet; Solanaceae such as tobacco; Asteraceae such as sunflower and safflower; Amaranthaceae such as amaranth; Plants belonging to grass family such as sorghum, sugar cane, rice, and wheat are mentioned, and more preferably mustard, Nozawana, kenaf, okra, beet for feed, Examples include tobacco, amaranth, sunflower, sorghum or crotalaria. As the plant, it is also possible to use a recombinant plant in which a gene that promotes absorption and accumulation of heavy metals has been introduced into a selected plant species containing these. Examples of the gene include metallothionein synthesis genes such as MT-1, CUP1, and PsMTA (Kr Mmer, U. and Chardonnens, AN, Appl.
Microbiol. Biotechnol. 2001, vol.55, p661-672, Kar
enlampi, S., Schat H., Vangronsveld J., Verkleij
JAC, van der Lelie D., Mergeay M., and Tervahau
ta AI, Environmental Pollution. 2000, vol.107, p
225-231, Hasegawa, I. Farming Japan 2002, vol.36,
p10-15), phytokeratin synthase genes such as carboxypeptidase, and enzyme genes involved in glutathione synthesis system. The plant species and genes used in the present invention are not limited to the species described here.

In order to grow a plant on a target soil, a method of directly purifying the soil while sowing and growing it at a suitable time, a seedling by a nursery, a seedling by a nursery box, a cell seedling, a pot seedling, a plug seedling, Examples include a method in which a separately grown plant such as a paper pot seedling or a vegetatively propagated plant is transplanted to a target soil and cultivated to purify it. These can take any method depending on the plant species and the state of the polluting medium. For example, when sowing plant seeds directly, it is suitable to sow 100 to 50,000 seeds per 10 ares to be purified. The pH of the soil in which the plant is cultivated is appropriately in the range of 3 to 10. However, depending on the soil condition, the pH value greatly affects the absorbability of heavy metals, so it is also necessary to adjust the pH according to the cultivation conditions. If necessary, a biodegradable chelating agent, a soil acidifying agent, a heavy metal absorption promoter such as salts, or a nutritional ingredient, a fertilizer or the like can be applied.

[0038] Plant harvesting can be directed to both above-ground plant parts, including stems and leaves, and below-ground plant parts, including roots. As a method of harvesting a plant body, a method of mowing only the above-ground part, a method of mowing the above-ground part that re-grows from the remaining plant body once or several times, a plant above-ground part and an underground part respectively Examples include a method of separately harvesting and a method of simultaneously harvesting the above-ground part and the underground part of the plant. Regarding the harvesting time of plants, efficient purification can be performed by carrying out at a time when the heavy metal absorption amount, production amount, morphology, etc. are optimal depending on the plant species to be cultivated.

The harvested plants can then be lightened and / or reduced in volume and treated by incineration or other suitable method. As weight reduction and / or volume reduction processing,
It is carried out by herbicide treatment, on-site drying treatment, composting treatment, squeezing treatment, microbial treatment, decomposition treatment, pulverization treatment, etc., and a combination of two or more thereof. Of these, the herbicide treatment can be carried out not only after harvesting the plants but also before harvesting,
In this case, the plants can be harvested in a dry state in a lightweight state.

The on-site drying treatment is a method in which dead plants are left standing in a napped state for an appropriate period to naturally dry and reduce the weight, and then harvested. The squeezing treatment is a method of squeezing the harvested plant with a squeezing machine to separate juice and residue. The composting treatment is a method in which the harvested plants are directly or after being crushed, and then deposited in the open air or in an appropriate facility and left to stand naturally to form compost. When left to stand, soil, a fermentation accelerator, etc. can be added and mixed as needed. Although the period of composting varies depending on the conditions, it is desirable that it is shorter than the purification target period. The microbial treatment is a method of depositing and decomposing by the force of microorganisms, like the above-mentioned composting treatment. The decomposition treatment focuses on the decomposition action of sedimentary plants, and is substantially the same as the composting treatment, microbial treatment, putrefaction and the like.

The present invention will be described below with reference to examples.
The present invention is not limited to this range.

[0042]

Examples Example 1: Mer derived from Thiobacillus ferrooxidans strain E-15
Plasmid pTM314 (J. Bacteriol. 171) containing the C gene
(1989) 3458-3464) as a template, the 5'-side primer having the sequence shown in SEQ ID NO: 3 (5'-GCGAATTCAT GTCAGCCATA ACCCGCATCA TC-3 '), and the sequence shown in SEQ ID NO: 4 (5'
-GCGTCGACTC AGCTACGCGG GGCGGGCGTT TC-3 ') was prepared and PCR was performed using ExTaq DNA polymerase (manufactured by Takarasha) (96 ° C 2min-(94 ° C 30sec-64 ° C).
30 sec-72 ° C. 30 sec) 30 cycles -72 ° C. 5 min) was performed to amplify a 451 bp fragment having the nucleotide sequence of SEQ ID NO: 2.

The merC fragment recovered by GENECLEAN II (manufactured by BIO101) was ligated to pGEM T-EasyVector (manufactured by Promega), and Escherichia coli DH5α (manufactured by Toyobo) was transformed. The obtained white colonies were analyzed by colony PCR, a plasmid was prepared from the colonies containing the target fragment length by the alkali-SDS method, and the nucleotide sequence was confirmed by a sequencer.
After confirming that the sequence is correct, pGEM T-Easy Vector (P
The merC gene portion was excised with Eco RI and Sal I from Romega) and recovered with GENECLEAN II (BIO101), and the DNA fragment containing this merC gene was named merC / E, S.

Yeast plasmid pYPR3831X (Gene 84 (1989) 47-54) having a galactose-inducible gal1 promoter and a tryptophan synthesis gene as a marker was prepared by
Cut it out with EcoRI and Sal I and add it to the above merC /
E and S were ligated to create pYPR3831X-merC (Fig. 1). In the plasmid, the merC gene was galactose-inducible gal1.
Placed under the control of a promoter.

The pYPR3831X-merC obtained above was transformed into a Saccharomyces cerevisiae mutant strain (EH13-15) lacking the ability to synthesize tryptophan in YPD liquid medium (2% bacto peptone, 1% y).
eastextract, and 2% glucose) and then YNBD agar plate (0.67% yeast nitrogen) according to the standard method.
It was applied to base without amino acid, and 2% glucose) and cultured at 30 ° C. for 88 hours.

Here, the introduction of the plasmid into the grown yeast can be confirmed by a conventional method by collecting the plasmid and then performing electrophoresis. According to the method described above, the yeast into which pYPR3831X-merC had been introduced was added to 5 mL of YNBD liquid medium, and shake-cultured at 30 ° C. for 16 hours. The culture was divided in half and each was warmed to 30 ° C YN
B liquid medium (0.67% yeast nitrogen base without amino
acid) and washed twice, one in 2.5 mL YNBD medium and the other in 2.5 mL YNBG medium (0.67% yeast nitrog
en base without amino acid, and 2% galactose), and then shake culture was performed at 30 ° C. for 4 hours.

3 mL YNBD Top Agarose (0.67% yeast
nitrogen base without amino acid, 2% glucose and
0.7% agarose) was melted at 45 ° C., 100 μL of the above cells cultured in YNBD medium were added and mixed well, and then spread on YNBD lower layer agar medium (1.5% Agar). In addition, regarding the above-mentioned bacterial cells cultured in YNBG medium, 3 mL of YNBG Top Aga
rose (0.67% yeast nitrogen base without amino aci
d, 2% galactose and 0.7% agarose) was melted at 45 ° C, and the above-mentioned cells cultured in YNBG medium were mixed well with 100 μL, and then YNBG lower agar medium (1.5% Agar)
Sprinkled on.

Further, as a control, plasmid
The pYPR3831X transformed into Saccharomyces cerevisiae EH13-15 in the same manner as above was also cultured in the same manner as above to prepare a total of four types of microorganism-containing medium. After these four types of upper agar solidified, heavy metal uptake activity was confirmed for each. That is, a compound for heavy metal-containing compounds is used for antibiotics testing (diameter 6 mm)
After placing on a garlic plate and culturing at 30 ° C. for 36 hours, the diameter of the inhibition circle was measured and compared.

The above measurement was carried out by using CdCl ₂ , CuCl ₂ , ZnCl ₂ and CoC.
FIG. 2 shows the results obtained by changing the concentration of the heavy metal-containing compound for each of the four compounds of l ₂ . From this figure, the yeast in which the merC gene has been introduced and whose function has been expressed by galactose has a larger diameter of the inhibition circle than the other three yeasts and shows high sensitivity to the above heavy metals. It can be seen that this increases the heavy metal uptake capacity.

Total RNA was extracted from the above four yeasts, size-fractionated with formaldehyde-denatured agar gel, and transferred to Hybond-N (manufactured by Amersham) using the capillary method. ^The 32P-labeled merC fragment was used as a probe for hybridization, washed by a conventional method, and an autoradiograph was obtained to analyze the expression of the merC gene. As a result, yeast cells containing the pYPR3831X-merC vector were found to be galactose. M only when guided by
The erC gene transcript could be detected. Almost equal amount of all
The fact that RNA was used for analysis was confirmed by staining the gel with ethidium bromide.

Example 2: pKK223-3 (Pharmacia)
Plasmid pTMC527 (Biosci. Biotech. Biochem. 60 (1996) in which merC was ligated to the downstream of the tac promoter of
1289-12921) was transformed into E. coli JM109 (manufactured by Toyobo Co., Ltd.). 0.3% of the cells containing 50 μg / ml of ampicillin
It was shake-cultured overnight at 37 ° C. in an LB medium containing% glucose. After washing the cells with LB liquid medium preheated to 37 ℃,
It was resuspended in 3 ml of LB liquid medium warmed to room temperature. 0.5% LB To
p Agar (3 mL) was pre-dissolved at 48 ℃ and resuspended in the above LB liquid medium warmed to 37 ℃ 10
0 μL and 1 mM IPTG (30 μL) were added and mixed, and spread on an LB plate.

As a control, a plate prepared by the same procedure as above was used by transforming E. coli JM109 (manufactured by Toyobo Co.) with pTG223-free plate and pKK223-3. The same operation as in Example 1 was performed on the above three types of plates to confirm the heavy metal uptake activity into E. coli. The results are shown in Fig. 3. From these results, it was found that in E. coli, the merC gene was expressed in Cd as in yeast.
It was highly sensitive to Cl ₂ , CuCl ₂ , ZnCl ₂ and CoCl ₂ .

Example 3: Arabidopsis
The merC gene was introduced into thaliana) by the following method. SEQ ID NO: 5 (GC designed to add restriction enzyme site BamHI to the start codon side of ORF (SEQ ID NO: 2) of the merC gene and SacI to the stop codon side (GC
GGATCCAT GTCAGCCATA ACCCGCATCA TC) and a DNA primer having the sequence shown in SEQ ID NO: 6 (GCGAGCTC
PCR was performed using a DNA primer having a sequence represented by TC AGCTACGCGG GGCGGGCGTT TC). After confirming the sequence of the obtained PCR product, the restriction enzyme BamH
I, SacI treated, BamHI, SacI treated expression vector
An integrated pBImerC was prepared downstream of the cauliflower mosaic virus-derived 35S promoter of pBI121 (Clontech). pBImerC to Agrobacterium EHA105 (Hood
EE et al. J Bacteriol. (1986) 158, 383-385) was transformed by the electroporation method. Using the obtained transformed Agrobacterium, the infiltration method (infiltrate
ion method) (Clough SJ, Bent AF. Plant J. (1998) 16: 735
-743) to the Arabidopsis thaliana (Colombia)
The C gene was introduced. The gene is introduced by seeding on an MS medium containing 50 mM kanamycin for selection, extracting DNA from the leaves, and using this as a template, a synthetic DNA having the sequence shown in SEQ ID NO: 5 and a sequence shown in SEQ ID NO: 6 Confirmed by PCR using synthetic DNA as a primer.

The susceptibility of the transformed Arabidopsis thus produced to cadmium was measured by the following method. 875
Post-germination 3 selected with kanamycin in 1/2 MS (Murashige-Skoog) liquid medium (10 ml) containing μM cadmium
On day 0, one rosette leaf was floated for 4 days, and the change in the leaf condition of the wild type and the transformant was visually examined. As a result, on the second day, whitening occurred in the transformants and 4 in the wild type.
Bleaching was confirmed on the day. From this, it was suggested that the transformed Arabidopsis having the merC gene inserted therein had increased susceptibility to cadmium as compared with the wild type.

[0055]

According to the method of the present invention, E. coli, yeast,
A wide range of heavy metals such as cadmium, cobalt, copper and zinc can be taken up into cells by a host such as a plant.

[0056]

[Sequence list] SEQUENCE LISTING <110> Mitsubishi Chemical Corporation <120> A method to increase heavy metal accumulation in plants and the ot her organisms <130> J10041 <160> 4 <210> 1 <211> 144 <212> PRT <213> Thiobacillus ferrooxidans E-15 <300> <303> Gene <304> 84 <306> 47-54 <307> 1989 <400> 1 Met Ser Ala Ile Thr Arg Ile Ile Asp Lys Ile Gly Ile Val Gly Thr   1 5 10 15 Ile Val Gly Ser Phe Ser Cys Ala Met Cys Phe Pro Ala Ala Ala Ser              20 25 30 Leu Gly Ala Ala Ile Gly Leu Gly Phe Leu Ser Gln Trp Glu Gly Leu          35 40 45 Phe Val Gln Trp Leu Ile Pro Ile Phe Ala Ser Val Ala Leu Leu Ala      50 55 60 Thr Leu Ala Gly Trp Phe Ser His Arg Gln Trp Gln Arg Thr Leu Leu  65 70 75 80 Gly Ser Ile Gly Pro Val Leu Ala Leu Val Gly Val Phe Gly Leu Thr                  85 90 95 His His Phe Leu Asp Lys Asp Leu Ala Arg Val Ile Phe Tyr Thr Gly             100 105 110 Leu Val Val Met Phe Leu Val Ser Ile Trp Asp Met Val Asn Pro Ala         115 120 125 Asn Arg Arg Cys Ala Thr Asp Gly Cys Glu Thr Pro Ala Pro Arg Ser     130 135 140

[0057] <210> 2 <211> 435 <212> DNA <213> Thiobacillus ferrooxidans E-15 <300> <303> Gene <304> 84 <306> 47-54 <307> 1989 <400> 2 atg tca gcc ata acc cgc atc atc gac aaa att ggc ata gtc ggt acc 48 Met Ser Ala Ile Thr Arg Ile Ile Asp Lys Ile Gly Ile Val Gly Thr                   5 10 15 atc gtc ggt agt ttc agt tgc gcc atg tgt ttc ccc gca gca gcg agc 96 Ile Val Gly Ser Phe Ser Cys Ala Met Cys Phe Pro Ala Ala Ala Ser              20 25 30 ctc ggc gct gca atc gga ttg ggc ttt ctc agc cag tgg gaa ggc ctg 144 Leu Gly Ala Ala Ile Gly Leu Gly Phe Leu Ser Gln Trp Glu Gly Leu          35 40 45 ttc gtg cag tgg ctg att ccg att ttc gcc agc gtg gca tta ttg gcg 192 Phe Val Gln Trp Leu Ile Pro Ile Phe Ala Ser Val Ala Leu Leu Ala      50 55 60 acc ttg gcg ggc tgg ttc tcg cac cgc caa tgg caa cgc acg ctg ctg 240 Thr Leu Ala Gly Trp Phe Ser His Arg Gln Trp Gln Arg Thr Leu Leu  65 70 75 80 ggc tcg atc ggt ccg gtg cta gcg ctt gtc ggg gtg ttt ggg tta acg 288 Gly Ser Ile Gly Pro Val Leu Ala Leu Val Gly Val Phe Gly Leu Thr                  85 90 95 cat cac ttt ctg gac aag gac ctg gcg cgc gta att ttt tat acc gga 336 His His Phe Leu Asp Lys Asp Leu Ala Arg Val Ile Phe Tyr Thr Gly             100 105 110 ttg gtg gtg atg ttc ctt gtc tcc atc tgg gac atg gtc aat ccg gcg 384 Leu Val Val Met Phe Leu Val Ser Ile Trp Asp Met Val Asn Pro Ala         115 120 125 aac cgc cgc tgc gcg acc gac ggc tgc gaa acg ccc gcc ccg cgt agc 432 Asn Arg Arg Cys Ala Thr Asp Gly Cys Glu Thr Pro Ala Pro Arg Ser     130 135 140 tga 435 stop 145

[0058] <210> 3 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 3 GCGAATTCAT GTCAGCCATA ACCCGCATCA TC 32

[0059] <210> 4 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 4 GCGTCGACTC AGCTACGCGG GGCGGGCGTT TC 32

[0060] <210> 5 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 5 GCGGATCCAT GTCAGCCATA ACCCGCATCA TC 32

[0061] <210> 6 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 6 GCGAGCTCTC AGCTACGCGG GGCGGGCGTT TC 32

[Brief description of drawings]

FIG. 1 is a schematic diagram showing the constitution of plasmid pYPR3831X-merC.

FIG. 2 is a diagram showing the correlation between the heavy metal concentration and the diameter of the inhibition circle in the yeast transformed with the plasmid pYPR3831X-merC and its control.

FIG. 3 is a diagram showing the correlation between heavy metal concentration and diameter of inhibition circle in Escherichia coli transformed with plasmid pTMC527 and its control.

Continued front page    F term (reference) 2B030 AA02 AB04 AD20 CA17 CB01                       CG05                 4B024 AA08 CA02 CA11 DA01 DA06                       DA12 EA04 GA14                 4B065 AA01Y AA26X AA80X AA89X                       AB01 AC20 BA02 BA03 BB15                       BB37 BC03 CA53 CA54                 4D004 AA41 AB03 CA17 CC20

Claims (6)

[Claims] 1. A recombinant cell into which any gene selected from the group consisting of the merC gene, the merF gene and the merT gene has been introduced, and cadmium, cobalt, copper, zinc and / or arsenic is introduced into the cell. A method for absorbing heavy metals, which is characterized in that 2. A recombinant cell into which any gene selected from the group consisting of the following (a), (b), (c) and (d) is introduced, and cadmium, cobalt, copper,
A method for absorbing heavy metals, which comprises incorporating zinc and / or arsenic into the cells. (A) a gene encoding a protein having the amino acid sequence set forth in SEQ ID NO: 1 (b) mercury having one to a plurality of substitutions, deletions and / or insertions in the protein having the amino acid sequence set forth in SEQ ID NO: 1 Gene encoding a protein capable of transferring ions into cells (c) Gene having the nucleotide sequence set forth in SEQ ID NO: 2 (d) Hybridizing with any of the above genes under stringent conditions, and mercury ion cells Genes involved in internal transfer 3. A recombinant yeast into which any gene selected from the group consisting of merC gene, merF gene and merT gene has been introduced. 4. A recombinant plant into which any gene selected from the group consisting of merC gene, merF gene and merT gene has been introduced. 5. A method for purifying soil contaminated with heavy metals by allowing plants to absorb heavy metals in the soil and accumulate the heavy metals in the plants, wherein the plants are at least m.
A method for remediating soil, which is a recombinant plant into which any gene selected from the group consisting of erC gene, merF gene and merT gene has been introduced. 6. A method for purifying soil contaminated with heavy metals by allowing plants to absorb heavy metals in the soil and accumulating in the plants, wherein the plants contain at least the following (a) and (b): A method for remediating soil, which is a recombinant plant into which any gene selected from the group consisting of (c) and (d) is introduced. (A) a gene encoding a protein having the amino acid sequence set forth in SEQ ID NO: 1 (b) mercury having one to a plurality of substitutions, deletions and / or insertions in the protein having the amino acid sequence set forth in SEQ ID NO: 1 Gene encoding a protein capable of transferring ions into cells (c) Gene having the nucleotide sequence set forth in SEQ ID NO: 2 (d) Hybridizing with any of the above genes under stringent conditions, and mercury ion cells Genes involved in internal transfer