JP4783123B2 - Genes that enhance plant iron transport - Google Patents

Description

  The present invention relates to a gene that enhances the iron transport ability of a plant. More specifically, the present invention relates to a plant whose iron transport ability is enhanced by transformation with a gene encoding an iron transport carrier protein derived from Hebino negosa, and an environmental remediation method using the same.

  In recent years, phytomediation (environmental restoration by plants) has attracted attention as a new technology that can gently treat a wide variety of pollutants having a relatively low concentration over a long period of time at low cost. Theoretically, it is thought that heavy metals can be absorbed and removed using several types of existing plants called hyperaccumulators (special element high concentration accumulation species). Various studies using gene recombination techniques that can impart completely different traits, which are difficult with conventional breeding methods, have been conducted with the aim of expanding applicable plant species and improving treatment capacity.

  For example, in Patent Document 1, a heavy metal compound or a cytotoxic organic compound is introduced into a plant by introducing a gene encoding MRP1 (multidrug resistance-associated protein 1), which is one of transporter proteins belonging to the ABC transporter, into the plant. A technique is disclosed in which heavy metal compounds and cytotoxic organic compounds are recovered from a contaminated environment by imparting resistance and growing the resistant plant in the contaminated environment. Patent Document 2 discloses a technique for purifying soil contaminated with heavy metals using a plant body in which the ability to accumulate heavy metals is enhanced by introducing a yeast-derived zrcl gene.

On the other hand, due to recent progress in molecular biology, various reports on the absorption mechanism of heavy metals, especially cadmium, which has a large impact on the amount of diffusion in rivers and soils and on human health, have been made. Yes. For example, in general plants, it has been reported that absorption of iron is inhibited by cadmium and falls into an iron deficiency state (cadmium-induced iron deficiency) (Non-patent Document 1). It has also been reported that iron transport carrier proteins encoded by IRT and Nramp, which are one of iron transport carrier genes, absorb cadmium together with iron, which is an element essential for plant growth (Non-patent Document 2, Non-patent document 3).
JP2003-210057 JP 2004-275051 A Marschner H (1995) Mineral Nutrition of Higher Plants, 2nd edn, Academic press, London, UK Korshnova YO et al. (1999) Plant Mol Biol 40 p37-44 Thomine S et al. (2000) Proc Natl Acad Sci USA 97 p4991-4996

  By the techniques disclosed in Patent Document 1 and Patent Document 2, it is possible to impart resistance to heavy metals and accumulation ability such as cadmium to plants. However, if sufficient iron essential for plant growth cannot be taken in, the plant will fall into an iron-deficient state, and the growth state will be adversely affected.

  In addition, the iron transporter protein encoded by IRT and Nramp absorbs cadmium together with iron, which is an essential element for plant growth, so that a sufficient amount of iron cannot be taken in, and the growth state is adversely affected. There is a fear.

  Therefore, the present invention provides a gene encoding a carrier protein capable of specifically transporting iron without being inhibited by a cation metal such as cadmium, and a cadmium or the like transformed with the gene. An object of the present invention is to provide a plant body that absorbs enough iron for growth even in the presence of metal and grows healthy and has a reduced amount of cadmium accumulation.

  Furthermore, an object of the present invention is to provide a method for carrying out environmental repair using a transformed plant body.

  In order to achieve such an object, the inventor of the present application has conducted extensive research to establish a tissue culture method and elucidate the function of Athyrium yokoscense having high cadmium resistance. As a result, it was found that the high cadmium tolerance of Hebnonegoza is due to the mechanism that the iron transport carrier protein has a very low probability of absorbing cadmium due to its specific affinity for iron. .

The present invention is based on such knowledge, and the invention according to claim 1
(A) a protein comprising the amino acid sequence of SEQ ID NO: 2 or 4 , or (b) the amino acid sequence of SEQ ID NO: 2 , comprising an amino acid sequence in which 1 to 51 amino acids have been deleted, substituted or added, and iron A protein that functions as a transport carrier having specific affinity , or a transport that consists of an amino acid sequence in which 1 to 39 amino acids are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 4 and that has specific affinity for iron A gene encoding a protein that functions as a carrier .

  Here, “specifically affinity with iron” means specifically with iron with almost no affinity with these metals, even under conditions exposed to metals such as cadmium, nickel, manganese, zinc and copper. It means to function as an affinity transport carrier.

The invention described in claim 2
(A) a protein comprising the amino acid sequence of SEQ ID NO: 2 or 4 , or (b) the amino acid sequence of SEQ ID NO: 2 , comprising an amino acid sequence in which 1 to 51 amino acids have been deleted, substituted or added, and iron A protein that functions as a transport carrier having specific affinity , or a transport that consists of an amino acid sequence in which 1 to 39 amino acids are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 4 and that has specific affinity for iron A protein that functions as a carrier .

  The above protein functions as an iron transport carrier, and even when metals such as cadmium, nickel, manganese, zinc, and copper are present, it specifically has an affinity for iron with little affinity for them.

The invention according to claim 3
(C) DNA comprising the nucleotide sequence of SEQ ID NO: 1 or 3 or (d) DNA comprising the nucleotide sequence of SEQ ID NO: 1 or 3 or DNA complementary thereto under stringent conditions and iron DNA encoding a protein that functions as a transport carrier that specifically has an affinity for
It is a gene consisting of

  The gene encodes a protein that functions as an iron transport carrier. Therefore, plants with this gene have transport carriers that have specific affinity for iron with little affinity for metals such as cadmium, nickel, manganese, zinc, and copper, and are essential. It becomes easy to take in the element iron.

  The invention according to claim 4 is a transformed plant cell containing the gene according to claim 1 or 3. From the plant cell, the plant according to claim 5 and a part thereof, and a propagation body and offspring thereof can be obtained. Therefore, even in the presence of metals such as cadmium, nickel, manganese, zinc, copper, etc., the amount of cadmium, an element that is very harmful to the human body, is maintained in a good growth state by taking in enough iron for growth. It becomes possible to provide a plant with less.

  The invention described in claim 6 is a plant cell further having an ability to accumulate environmental pollutants, and from the plant cell, the plant according to claim 7 and its part, and its propagation and progeny are obtained. can get.

  Even if it has only the ability to accumulate environmental pollutants, it may not be able to sufficiently take in iron, which is an essential element, in the presence of metals such as cadmium, nickel, manganese, zinc, copper, etc. By containing the gene of the present invention, a transport carrier that specifically has an affinity for iron functions even in the presence of metals such as cadmium, nickel, manganese, zinc, copper, etc., and a sufficient amount of iron for plant growth can be obtained. It is possible to capture. Therefore, it is possible to provide a plant that maintains a good growth state even in the presence of a metal such as cadmium, nickel, manganese, zinc, copper, and also has an ability to accumulate environmental pollutants.

  Here, environmental pollutants are cadmium, cobalt, nickel, copper, lead, zinc, mercury, chromium, arsenic, manganese, selenium, tin, and antimony.

  The invention described in claim 8 is to cultivate a host cell obtained by transformation with a vector incorporating the gene described in claim 1 or 3, and collect a protein accumulated in the culture. Thereby, the protein of Claim 2 can be obtained.

  The invention according to claim 9 is an environmental remediation method characterized by removing environmental pollutants from soil using the plant according to claim 7. As described above, the plant according to claim 7 has an ability to accumulate environmental pollutants and a function of taking in enough iron for plant growth even in the presence of metals such as cadmium, nickel, manganese, zinc and copper. It is possible to efficiently perform phytomediation by using this plant as a hyperaccumulator.

  According to the present invention, it is possible to provide a gene encoding a transport carrier protein that has specific affinity for iron and hardly has affinity for iron even in the presence of metals such as cadmium, nickel, manganese, zinc, and copper. Is possible. In addition, the transformed plant containing the gene does not inhibit the absorption of iron, which is an essential element for plants, even if metals such as cadmium, nickel, manganese, zinc and copper are present in the soil. Therefore, it is possible to provide a plant that maintains a good growth state even in the presence of metals such as cadmium, nickel, manganese, zinc, and copper, and that has a low accumulation amount of cadmium, which is a very harmful element for the human body. It becomes. Furthermore, the transformed plant body having the ability to accumulate environmental pollutants and containing the gene of the present invention is superior in that it absorbs environmental pollutants while absorbing a sufficient amount of iron for its growth. It becomes a hyper accumulator, and it becomes possible to efficiently remove environmental pollutants from the soil by the hyper accumulator.

  In addition, the iron transporter protein of the present invention has a specific affinity for iron even in the presence of metals such as cadmium, nickel, manganese, zinc, copper, etc., so a sensor for measuring iron concentration or a molecular switch based on iron concentration ( It can be used as an ion condenser.

  Hereinafter, embodiments of the present invention will be described in detail.

The present invention relates to (a) a protein consisting of the amino acid sequence of SEQ ID NO: 2 or 4 or (b) an amino acid sequence of SEQ ID NO: 2 obtained from Hevinonegoza , wherein 1 to 51 amino acids are deleted, substituted or added. A protein that functions as a transport carrier that specifically has an affinity for iron , or an amino acid sequence of SEQ ID NO: 4, wherein 1 to 39 amino acids are deleted, substituted, or added; It is a gene that encodes a protein that functions as a transport carrier specifically having an affinity for iron .

Here, the term "several" in SEQ ID NO: 2, preferably from 5 1 or less, particularly preferably 10 or less. In SEQ ID NO: 4, preferably 3 9 or less, particularly preferably 10 or less.

  Here, the protein (a) is a protein that functions as a transport carrier that specifically has an affinity for iron, and the protein (b) has one or several amino acids of the protein (a) deleted, substituted, or substituted. Of those having an added amino acid sequence, it means a protein that functions as a transport carrier that specifically has an affinity for iron.

  The above proteins can be expressed in appropriate hosts such as Escherichia coli, yeast, insect cells, etc. by inserting the gene encoding them or the gene of SEQ ID NO: 1, 3, 5 or 7 into an appropriate expression vector. Can be produced. Here, examples of the vector system to be used include a pET system (Novagen), a pYES system (Invitrogen), a pIZT system (Novagen), a BacVector system (Novagen), a ViraPower adenovirus expression system (Invitrogen), and the like. Examples of the host include Rosetta-gami (DE3) pLys (Novagen), INVSc (Invitrogen), Sf9 (Novagen) and the like. Moreover, a cell-free protein expression system is mentioned as another protein production method. Examples of cell-free protein expression systems include rapid translation systems (Roche).

  The protein thus obtained can be used as a sensor for measuring the iron concentration or a molecular switch (ion capacitor) based on the iron concentration by fixing it on an artificial substrate or a membrane.

  Next, the gene of the present invention can be obtained, for example, as follows. First, total RNA extraction is performed from Hevinonegoza or Hevinonegoza callus, and after using this to purify mRNA, a cDNA library is constructed using a commercially available kit. Examples of the total RNA extraction method include a high-quality total RNA extraction method (Jaakola L. et al. (2001) Mol Biotechnol. 19 p201-3) from a polysaccharide material of Jaakola et al. using CTAB and PVP. It is not limited to this. On the other hand, it can be obtained by preparing a DIG probe or a radioactive probe with a commercially available kit based on the nucleotide sequence of SEQ ID NO: 1, 3, 5 or 7 and performing colony hybridization under stringent conditions.

Here, the term "hybridizes under stringent conditions", only specific hybridization occurs, refers to conditions such as not to cause non-specific hybridization, DNA's having high homology, immediate Chi Examples include conditions in which DNAs having 90 % or more homology hybridize and DNAs having low homology do not hybridize. Specifically, general presence smell of Hybridization Buffer Te of 6 0 ° C. to 70 ° C. conditions like including salts and the like.

  Next, a method for obtaining a transformed plant containing the gene of the present invention will be described.

  As a promoter or enhancer which is an expression regulatory element for expressing the gene of the present invention, any promoter or enhancer capable of expressing the gene of the present invention in a plant cell, for example, a gene expressed in a plant cell such as Agrobacterium or Rhizobium Promoters derived from plants, plant viruses, and bacteria containing Examples of such promoters include Agrobacterium tumefaciens T-DNA-derived promoter, Smas promoter, cinnamon alcohol dehydrogenase promoter, Nos promoter, ribulose diphosphate carboxylase oxygenase (Rubisco) promoter, GRP1-8 promoter, cauliflower mosaic virus (CaMV) 35S promoter, promoters or enhancers such as plant-derived actin and histone, and other transcription initiation regions from various known plant genes. For example, by appropriately selecting a plant-derived promoter or enhancer, the gene of the present invention can be expressed in the whole plant or in specific organs such as leaves and roots.

In order to efficiently express the gene of the present invention, it is preferable to include a poly (A) ⁺ sequence at the 3 ′ end of the polynucleotide coding region of the coding region of the gene. Poly (A) ⁺ sequences may be derived from various plant genes or T-DNA, but are not limited thereto. In addition, other sequences useful for expressing a desired gene at a high level, for example, an intron sequence of a specific gene, a 5 'untranslated region sequence, and the like can be included in the recombinant expression vector.

  The recombinant expression vector preferably includes sequences encoding various antibiotic resistance genes and other marker genes as selection marker genes. Examples of marker genes include anti-spectinomycin gene, streptomycin resistance gene (streptomycin phosphotransferase (SPT) gene), kanamycin or geneticin resistant neomycin phosphotransferase (NPTII) gene, hygromycin resistant hygromycin phosphotransferase (HPT) Examples include genes, genes resistant to herbicides that inhibit acetolactate synthase (ALS), genes resistant to herbicides that inhibit glutamine synthase (eg, the bar gene), β-glucuronidase genes, luciferase genes, and the like.

  In addition, representative vectors useful for gene expression in higher plants are well known in the art, and are described in Agrobacterium tumefaciens described in Rogers et al., Methods Enzymol., 153: pp.253-277 (1987). A vector that can be integrated into the genome of a host plant when a part of a vector DNA such as a vector derived from Ti plasmid is introduced into a plant cell, such as pKYLX6, pKYLX7, pBI101, pBH2113, pBI121 (Clontech Laboratories, Inc. , Palo Alto, CA) and recombinant plasmids that can be derived therefrom using standard techniques in the art.

  Recombinant expression vectors are known methods for introducing foreign genes into plant cells, such as particle gun method, electroporation method, polyethylene glycol (PEG) method, calcium phosphate method, DEAE dextran method, microinjection, lipofection method. And can be introduced into desired plant cells using microorganism-mediated transfection methods such as the Agrobacterium method. In plant cells, the particle gun method, electroporation method, polyethylene glycol (PEG) method and Agrobacterium method are preferred, and the Agrobacterium method is particularly preferred (Bechtold N. & Pelletier G., Methods Mol. Biol. 82 , pp.259-266, 1998).

  As described above, the gene of the present invention introduced into a plant is integrated into the genome of the host plant cell and is stably maintained until a later generation. Therefore, the present invention includes a plant regenerated from the plant cell and a part thereof, and a propagation body (eg, seed or asexual reproductive body) and offspring.

  Moreover, cell fusion is mentioned as another transformation method. In this case, the cell wall of the plant cell to be fused with the Hevinonegoza cell is decomposed by an enzyme and converted into a protoplast. Next, polyethylene glycol is added as a cell fusion promoter, and the two protoplasts are efficiently fused to fuse the two cell nuclei into one. Thereby, the nucleus which has the gene of Hebinonegoza and the plant cell gene to fuse | melt is produced, and a desired plant body is obtained by culture | cultivating and growing this fused cell.

  In addition, without using genetic recombination or cell fusion, hebinonegoza can be crossed with other close relatives, and finally crossed with the desired plant. You may make it obtain the plant body containing the gene which codes the protein which functions as a support | carrier.

  Plants transformed by the above-described method can absorb a sufficient amount of iron for growth without being inhibited by metals such as cadmium, nickel, manganese, zinc and copper. Therefore, even in the presence of metals such as cadmium, nickel, manganese, zinc, and copper, it is possible to obtain a plant body that absorbs a sufficient amount of iron and maintains a good growth state without being affected by these effects. It becomes. Moreover, since the amount of cadmium accumulated, which is a very harmful element for the human body, decreases, the cadmium-contaminated land is not purified, and even if it is used as farmland, crops with low cadmium accumulation are produced and used for food. It becomes possible to provide. Furthermore, it is possible to reduce the cadmium accumulation amount of only a specific part of the plant body. For example, it is possible to reduce the cadmium accumulation amount of the edible part.

  In addition, by growing the plant body in environmentally contaminated soil, it is possible to prevent scattering of the contaminated soil and to prevent the diffusion of environmental pollutants. Furthermore, by covering the environmentally polluted soil with green, the landscape can be kept good and people can feel safe.

  Next, the plant body which has an environmental pollutant accumulation ability and contains the gene of this invention is demonstrated.

  Plants having the ability to accumulate environmental pollutants include, for example, the method for selecting plant species for phytomediation of Japanese Patent Application No. 2004-260091 previously filed by the present inventor, Yoshihara, T., Shoji K., Goto F., Screening for the Cd hyperaccumulator / Ultra-low-accumulator from major herbal angiosperms in Japan.In: Plant nutrition for food security, human health and environmental protection (Eds. Li, CJ et al., Proc. 2005 IPNC, Beijing, China) pp.750-751. Selected by the method described in Dentsu Institute report U03521 (Toshikazu Yoshihara, Fumiyuki Goto, Kazuhiro Shoko, search for herbaceous angiosperms resistant to cadmium and boron) can do.

  Specifically, the seeds of various plants are mixed with nutrients necessary for plant growth, trace metal elements that are thought to affect the absorption and accumulation of environmental pollutants, and concentrations that inhibit growth in most plants. After seeding in the medium or soil containing the environmental pollutants, and the nutrient salts necessary for plant growth and the medium or soil containing trace metal elements that are thought to affect the absorption and accumulation of environmental pollutants. Measure the accumulated amount of environmental pollutants in the above-ground part and root part of the plant, and accumulate the environmental pollutant from the ratio of the accumulated amount of environmental pollutant in the above-ground part and root part of the plant with and without the environmental pollutant Evaluate ability and select.

  As an example of the case where the environmental pollutant is cadmium, according to the above selection method, the species having a high cadmium accumulation ability in the above-ground part is an Asteraceae plant such as Achinonokeshi, and the species having a high cadmium accumulation ability in the root part is rush. Although it has been confirmed, these plants have also been confirmed to have low cadmium tolerance. Therefore, by transforming these plants to contain the gene of the present invention by the above-mentioned method, while absorbing a large amount of cadmium, it also absorbs iron essential for growth and improves the growth state. It is possible to obtain a very good hyperaccumulator to keep.

  Moreover, you may use the technique disclosed by Unexamined-Japanese-Patent No. 2004-016130 which the inventor applied earlier. Specifically, by constructing a nucleic acid molecule encoding a polypeptide containing an oligo or polypeptide capable of binding to environmental pollutants and an apoplasma transport signal, and using this to create a transgenic plant, A plant having a high ability to accumulate environmental pollutants can be obtained. Examples of oligos or polypeptides having an ability to bind to environmental pollutants include metallothioinene, ferritin, transferrin, polyhistidine, nickelin, phytochelin, Zα4 artificial peptide, mammalian plasma metal transport protein, and metal-binding fragments thereof. It is done.

  Therefore, by further transforming the above plant body to contain the gene of the present invention, or the plant body transformed to contain the gene of the present invention has an ability to bind to environmental pollutants. Or by further transformation with a nucleic acid molecule that encodes a polypeptide and a polypeptide containing an apoplasma transport signal, it absorbs a large amount of environmental pollutants and also sufficiently absorbs iron essential for growth. It is possible to obtain a very excellent hyperaccumulator that keeps the state good.

  The above-described embodiment is an example of a preferred embodiment of the present invention, but is not limited thereto, and various modifications can be made without departing from the scope of the present invention. For example, a plant having an ability to accumulate environmental pollutants may be obtained by a method other than the above.

  The following examples further illustrate the present invention, but the present invention is not limited to these examples.

<1: Callus and redifferentiated plant culture>
Athyrium yokoscense was collected at the foot of the Ashio copper mine in Ashio, Tochigi Prefecture. As a control plant, tobacco (Nicotiana tabacum L. BY-2), which is considered to have general properties with respect to cadmium tolerance and accumulation ability, was used.

  The test plant, Hevinonegoza, used one that had been subcultured by liquid shaking culture since September 2001 after inducing callus, or one that had been re-differentiated into a plant body from the induced callus ( Yoshihara, T. et al. (2005a) Plant Cell Rep 23 p579-585). Tobacco, which is a control plant, sterilizes the plant body, induces callus by placing leaf pieces of about 5 mm square on MS solid medium and culturing for about 1 month, and then removes gellan gum from MS liquid medium (for growth What was grown by shaking culture (100 rpm) using MS medium) was used. The culture environment conditions were all periodic light irradiation (about 4000 lux, 12 hours light period / 12 hours dark period), and the temperature was 25 ± 1 ° C. The medium was changed about once every 10 days to 2 weeks. The MS solid medium was prepared by adding 0.1 ppm kinetin, 2,4-D 1 ppm, 3% sucrose and 0.2% gellan gum to pH 6.0 (Murashige T, Skoog F ( 1962) Physiol Plant 15 p473-497).

<2: Callus growth under cadmium exposure>
FIG. 1 shows the results of culturing Hevinonegoza callus and tobacco callus obtained by the above-described culture method for 6 weeks in two types of MS medium for growth with CdSO ₄ added at 0 μM and 100 μM, respectively, and investigating the amount of proliferation. The amount of callus at the start of the culture was uniform for 3 g of fresh weight for Hebnone negosa callus, and uniform for 20 ml for tobacco callus because of extremely soft and high water content. The growth amount was calculated by weighing Hevinone Negoza callus as a fresh weight, cigarette callus as a volume by centrifugation at 15000 g, or as a fresh weight after suction filtration, and drying the obtained callus at 65 ° C. for 3 days. Weighed again as dry weight. The culture environment conditions were the same as those described above.

  In FIG. 1, ◇ indicates the relative growth of Hevinone negza callus, and ■ indicates the relative growth of tobacco callus. Here, the relative growth amount means the growth amount when the amount grown on the proliferation MS medium not added with cadmium is converted to 1. In addition, the data in this Example calculated | required the average value and the standard deviation, respectively, and the significance was determined about the difference of each experimental section by either the Student's t test method or Tukey's multiple comparison test method. Statistical calculation software (KyplotR ver.3.0, Cayence Co., Ltd.) was used for these calculations. Tobacco callus used as a control was remarkably inhibited by cadmium exposure, and after 6 weeks, the relative growth was 0.2, that is, only about 20% of the growth compared to the case without cadmium exposure. On the other hand, even after 6 weeks, Hebinonegoza callus showed the same amount of growth as when no cadmium exposure was performed. Therefore, it was confirmed that the induced and proliferated Hebinonegoza callus was not affected by cadmium at all in its growth.

<3: Effect of cadmium on expression of phytocheletin synthase (PCS) gene>
Hevinone goza callus and tobacco callus grown in 0, 1, 3, 5, 7 and 12 days in medium supplemented with 100 μM CdSO ₄ or 0 μM, 100 μM, 300 μM, 500 μM, 1000 μM and 3000 μM CdSO ₄ Total RNA was extracted from Hevinone gossa callus and tobacco callus grown for 1 week in each added medium, and the influence of cadmium on the expression of phytocheletin synthase (PCS) gene was examined by RT-PCR method. Phytocheletin is a low-molecular non-protein polypeptide capable of chelating cadmium and is a polypeptide involved in cadmium resistance. The total RNA extraction of Hevinone nega callus was performed by the high quality total RNA extraction method from the polysaccharide material of Jaakola et al. Using CTAB and PVP (Jaakola L. et al. (2001) Mol Biotechnol. 19 p201-3). Tobacco callus total RNA extraction was performed with a commercially available Trizol reagent (Invitrogen, USA). Using these total RNA and Oligo-dT primer, reverse transcription reaction was performed with a commercially available kit (Rever-tra Ace kit, TOYOBO) to obtain each cDNA, and these cDNAs and known sequences registered in GenBank PCR was performed using a primer set specific to each PCS gene designed from AY235426 and AB057412, which were used for analysis of the expression level. For the analysis of the expression level, an image analyzer (Image Reader LAS-2000, FUJIFILM) was used. In addition, the expression level of each actin (ACT) gene was analyzed separately in the same manner, and the expression level of the PCS gene was standardized. The specific primer set sequences for analyzing the expression levels of each PCS gene and ACT gene are listed below.
AyPCs1F 5'-GCAAGGTTGCTGCCAAGGTG-3 '(SEQ ID NO: 9)
AyPCs1R 5'-CTAGGTTTCCTCACTGCATGACC-3 '(SEQ ID NO: 10)
NtPCs1F 5'-GCTTTTCGCCCTAATCATAGTA-3 '(SEQ ID NO: 11)
NtPCs1R 5'-TGACCCAACTCTCATGTTTA-3 '(SEQ ID NO: 12)
AyAc1F 5'-CGACATACAGGTGTGATGGGT-3 '(SEQ ID NO: 13)
AyAc1R 5'-GCTTGAATAGCGACATACAT-3 '(SEQ ID NO: 14)
NtAc1F 5'-TCAGAAAGATGCCTATGTGGGA-3 '(SEQ ID NO: 15)
NtAc1R 5'-TGGCAACGTACATAGCTGGG-3 '(SEQ ID NO: 16)

  FIG. 2 shows the effect of cadmium (100 μM) on the expression of the PCS gene in Hebnonegoza callus and tobacco callus. In FIG. 2, (B) is the expression level data obtained by the image analyzer, and (A) is the data digitized based on the image of (B). □ indicates tobacco callus, and ♦ indicates PCS expression level (relative PCS expression level) when the ACT gene expression level of Hebinonegoza callus is 1. Under the conditions of exposure to 100 μM cadmium, almost no change was observed in the expression level of PCS gene in Hebnonegoza callus, whereas in tobacco callus, the expression level of PCS gene increased more than twice on the first day after the start of exposure. On the twelfth day after gradual decrease, it showed a change that almost returned to the original expression level.

  FIG. 3 shows the expression level of the PCS gene of Hebnonegoza callus cultured for 1 week under the cadmium exposure conditions of 0 μM, 100 μM, 300 μM, 500 μM, 1000 μM and 3000 μM. Even when cultured for 1 week under cadmium exposure conditions of 0 μM, 100 μM, 300 μM, 500 μM, 1000 μM, and 3000 μM, almost no change was observed in the expression level of PCS gene in Hebnonegoza. Therefore, in Hebnonegoza, it is unlikely that PCS is involved in cadmium resistance, and it was considered that cadmium resistance was obtained by another mechanism other than PCS.

<4: Content of essential metal elements in callus under cadmium exposure>
Hevinonegoza callus and tobacco callus cultured by the above-described method are each stored in 0 μM (MS), 10 μM (Cd10), 100 μM (Cd100) MS medium for growth with CdSO ₄ and MS medium for growth without addition of Fe (ΔFe) for 1 week FIG. 4 shows the results of analyzing the contents of essential metal elements (calcium, copper, iron, magnesium, manganese, zinc) in the grown Hebino negosa callus and tobacco callus using ICP (P4000, Hitachi, Japan). The culture environment conditions were the same as those described above. In addition, as for the sample solution for ICP measurement, calli grown under the above-mentioned conditions are each dried at 65 ° C./3 days or more and wet ashing is performed after measuring the dry weight (Yoshihara et al., U97042). The residue obtained after evaporation to dryness was dissolved in 15 mL of 1N hydrochloric acid.

In FIG. 4, the relative concentration on the vertical axis is a value calculated by assuming that the value in callus grown in a medium (MS) not containing cadmium is 1. Further, (a) shows the results for calcium, (b) for copper, (c) for iron, (d) for magnesium, (e) for manganese, and (f) for zinc. In Hebinonegoza callus, (a) calcium, (d) magnesium, and (e) manganese content decreased significantly by cadmium exposure, whereas in tobacco callus, only (c) iron content showed a significant decrease, and cadmium It became clear that the elements affected by exposure differed. That is, (a) calcium is reduced by about 20-25%, (d) magnesium is reduced by about 15-25%, and (e) manganese is reduced by about 30-35%, whereas (c) iron is used in tobacco callus. Only decreased by about 50-60%. In a typical plant it has been reported that iron absorption is inhibited falling into iron deficiency by cadmium (cadmium-induced iron deficiency) (Marschner H (1995) Mineral Nutrition of Higher Plants, 2 nd edn, Academic press, (London, UK), the decline in the iron content of tobacco callus demonstrates this markedly. However, cadmium exposure reduced the content of calcium, magnesium and manganese, not iron, in Hebnonegoza callus. This suggests that cadmium inhibits the absorption of magnesium, calcium and manganese in Hebnone negosa callus. However, because Hebino Negoza callus grew vigorously regardless of the presence or absence of cadmium, the absorption was not inhibited to the extent that these elements were deficient. Therefore, the iron transport carrier in Hebnone negosa callus has a very high specificity for iron compared to the iron transport carrier of other plants, and even in the presence of cadmium, it is not inhibited by cadmium and is a sufficient amount of iron for growth. It was found that can be absorbed.

<5: Effect of cadmium on the isolation and expression of iron absorption-related genes in Hebinonegoza>
Total RNA was extracted from Hebinonegoza callus and converted to cDNA to prepare a cDNA library. Hebinonegoza total RNA was extracted from Hebinonegoza callus grown for 3 weeks in a medium excluding iron according to the method of total RNA extraction of Jaakola et al. Described above, followed by reverse transcription reaction to isolate cDNA for partial sequence isolation of each gene homolog. Got. In addition, in order to isolate the full-length sequence of each gene homologue, a cDNA library was prepared from Hebinone nega total RNA obtained in the same manner. In preparation of the cDNA library, mRNA was purified from the obtained total RNA using MACS Column Type μ (Daiichi Kagaku), and then a Creator SMART cDNA library preparation kit (BD Bioscience Clontech) was used.

Next, known sequences of element transport carrier genes registered in GenBank (IRT; NM_118089, AF065444, which have been reported to be related to the transport of both iron and cadmium whose sequences are already known in Arabidopsis, etc.) AF246266, AB126086, AB126087, AB070226, AJ320253: Nramp; L41217, NM_106731, NM_103618, NM_101464, AY196091: YSL; AY5155566, AY515565, AY515560, AY512583, AP003936, CA758396) A rate primer was designed and produced. The degenerate primers are listed below.
degAyIRTF 5'-CCKTGGYMTAARTTYCCTTTYBCBGG-3 '(SEQ ID NO: 17)
degAyIRTR 5'-GARTGWRMYAHRATBCCAASYTCCARNACC-3 '(SEQ ID NO: 18)
degAyNrampF 5'-ATGCCBCAYAAYVTSTWYYTVCAYTCRGC-3 '(SEQ ID NO: 19)
degAyNrampR 5'-ATVGTVSWRCTCTGHCCMGMWGC-3 '(SEQ ID NO: 20)
degAyYSLF 5'-MMRCCNTTYACWMRNCARGAGAAYAC-3 '(SEQ ID NO: 21)
degAyYSLR 5'-CWGTDGCNRWWCCACTNGGRTA-3 '(SEQ ID NO: 22)

  PCR was performed using these primers and the cDNA for partial sequence isolation shown above, and the resulting fragment was cloned into the pCR-TOPO vector using a TA cloning kit (Invitrogen). The obtained fragments were analyzed by using a DNA sequencer (ABI model 3100) as a partial sequence, and then a sequence whose similarity was found compared to a known gene was used as a partial sequence for each gene homolog in Hebnonegoza. Used to create probes for full-length sequence isolation from libraries. Probes for full-length sequence isolation were prepared by PCR-DIG labeling using primers redesigned from the partial sequences shown above (DIG DNA Labeling and Detection Kit, Roche). The positions (sequences) of the probes, RT-PCR and real-time PCR expression analysis primers are shown in each full-length sequence. 5 to 8, 1F and 1R are primers for real-time PCR, and 2F and 2R are primers for probe preparation and RT-PCR. In FIGS. 5 to 6, the portions enclosed by squares are the start codon and the termination codon, and the portions enclosed by shaded squares indicate a common sequence thought to be related to iron transport. Isolation of the full-length cDNA from the cDNA library was performed using this probe in accordance with a standard method (Kazuhiro Makabe (1997) Bio-Experiment Illustrated Volume 4 Cloning without Effort, Shujunsha). The obtained clone was subjected to sequence analysis by a DNA sequencer (ABI model 3100) and compared with the known homologue shown above.

Expression analysis under cadmium exposure conditions was carried out using RT-PCR method or real-time PCR method for three types of element transport carrier gene homologs isolated from Hebonia negosa. Specifically, total RNA of Hevinonegoza callus grown for 3 weeks in a medium supplemented with 100 μM or 400 μM of two types of CdSO ₄ , and total RNA of the above-mentioned Jaakola et al. Extraction was performed according to the extraction method, and cDNA obtained by reverse transcription reaction was used as a template. In addition, the same operation was carried out using Hebinonegoza callus separately grown in an iron-deficient medium for 3 weeks and total RNA obtained from adult plants, and compared with the expression of these genes under cadmium exposure conditions.

  Among the isolated homologues, AyNramp1 (SEQ ID NO: 1 full length 1903 bp, ORF1530 bp, 510AA) and AyIRT1 (SEQ ID NO: 3 full length 1878 bp, ORF1152 bp, 384AA) were full-length genes. Both AyYSL1 (SEQ ID NO: 5: full length 301 bp) and AyYSL2 (SEQ ID NO: 7: full length 295 bp) were considered to be partial genes of the ORF. Among these, AyIRT1 and AyNramp1, in which the full length could be isolated, were compared in terms of amino acid sequence homology with homologs AtIRT1 and AtNramp3 whose sequences were revealed in Arabidopsis, respectively. The results are shown in FIGS. In FIG. 9, (a) shows the protein membrane structure of Hevinonegoza AyIRT1, and (b) shows the Arabidopsis AtIRT1 protein membrane structure. In FIG. 10, (a) shows the protein membrane structure of Hevinonegoza AyNramp1, and (b) shows the Arabidopsis AtNramp1 protein membrane structure. In FIG. 11, (A) shows a comparison with plant-derived Nramp, and (B) shows a comparison with animal-derived Nramp. In FIG. 11, GmDMT is soybean, LeNramp is tomato, OsNramp is rice, HsDMT is human, MmNramp is mouse, OmNramp is rainbow trout, and TrDMT is trough. The homology between the amino acid sequence of AtIRT1 and the amino acid sequence of AyIRT1 was 44.0%, the homology of the amino acid sequence of AtNramp1 and the amino acid sequence of AyNramp1 was 67.3%, and the homology was not so high, but in Arabidopsis Except for D100, E103 and C109, all are shown in Arabidopsis (Rogers EE et al. (2000) PNAS 97 p12356-12360) and mice (Lam-Yuk-Tseung et al. (2003) BLOOD 101 p3699-3707) All amino acid sequences strongly related to iron absorption were preserved. As shown in FIG. 11, the amino acid sequence of AyNramp1 is more similar to that in animals than in plants, and the sequence has been clarified so far as indicated by the arrows in FIG. The amino acid, which is all isoleucine in the plant Nramp, was changed to the same methionine as the animal in Hevinonegoza.

  Next, with respect to the protein membrane structure, the Arabidopsis AtIRT1 protein membrane structure shown in FIG. 9 has 8 transmembrane regions, whereas the Hemone Negoza AyIRT1 protein membrane structure has 6 transmembrane regions, which is two fewer. It was. In the Arabidopsis AtNramp1 protein membrane structure shown in FIG. 10, there are 11 transmembrane regions, whereas there are 9 transmembrane regions in the Hebinonegoza AyNramp1 protein membrane structure, two fewer. In FIGS. 9 and 10, reference numeral 1 denotes an extramembrane region, 2 denotes a transmembrane region, and 3 denotes an intramembrane region. Further, when focusing on the above-mentioned “amino acid sequence strongly related to conserved iron absorption”, for example, as shown in H236, S237, H263, E267 of AyIRT and H197, S198, H224, E228 of AtIRT, Many also opposed the existence inside and outside.

  Next, Hebnonegoza callus and adult plants were treated with iron deficiency and exposed to cadmium, and expression changes of these four isolated genes were examined. The results are shown in FIGS. As shown in FIG. 12, a significant increase in expression was observed in all genes in the snake nonegoza callus due to a strong iron deficiency treatment (two weeks of iron deficiency treatment). That is, it was confirmed that there was iron deficiency response. In FIG. 12, (a) is when grown on a normal growth MS medium, (b) is when grown for one week after removing iron from the growth MS medium, and (c) is a growth MS medium. In this case, the iron was removed from the plant and allowed to grow for 2 weeks. Next, as shown in FIG. 13, even when cadmium was exposed, increased expression was observed for AyYSL1 and AyYSL2, but no significant increase in expression was observed for AyIRT1 and AyNramp1. In FIG. 13, (a) shows a case where the medium is grown on a normal MS medium for growth, (b) shows a case where the medium is grown for one week in a medium containing 100 μM cadmium, and (c) shows a medium containing 400 μM cadmium. In the case of growing for 1 week. Moreover, as shown in FIG. 14, in the experiment using adult plants, significant expression was observed in all genes by iron deficiency treatment (ΔFe) or cadmium exposure (+ Cd) for one week for both roots and plant ground parts. No change was observed. In FIG. 14, (a) AyIRT1, (b) is AyYSL1, (c) is AyYSL2, and (d) is AyNramp1.

<6: Improvement of cadmium resistance in transformed yeast overexpressing Hebinonegoza iron absorption-related gene>
The ORF part of two genes (AyIRT1, AyNramp1), which were able to isolate the full length from Hebonia negosa, was amplified by PCR, confirmed the sequence, and then yeast (BY4741 strain) via yeast expression vector (pYES2.1vector / Invtrogen) ). Transformation of yeast was performed according to the method shown by Dohlman (Dohlman HG (2002) "Lazy Bones" Plasmid Transformation, A quick method for plasmid transformation into yeast. Http://www.med.unc.edu/~hdohlman/ lazybones.html). Specifically, yeast is cultured on an SD agar medium (BD bioscience / Clonetech) to isolate a single strain, a plasmid DNA containing carrier DNA and the isolated gene, and a transformation solution (40% polyethylene glycol, 0 .1M Lithium acetate, 10 mM Tris-HCl (pH 7.5), 1 mM EDTA) were mixed and allowed to stand overnight, and then transformed by heat shock at 42 ° C. Furthermore, this mixed solution was cultured in an SD agar medium excluding uracil, and the transgene was confirmed by colony PCR for the obtained strain. Primers having the same sequence as those used in the RT-PCR method were used.

The cadmium resistance of the strains obtained as described above (AyIRT1, AyNramp1) and the non-transformed strain (WT) was investigated. For the cadmium resistance test, an SD agar medium excluding uracil was used as a basic medium, and a medium supplemented with 0 μM and 30 μM CdSO ₄ was used, and 1/1, 1/10, 1/100 and 1/1000 were used. Diluted strains (dilution rate confirmed by absorbance at 600 nm) were added dropwise, and after culturing at 30 ° C. for 2 to 5 days, the colony formation state was compared with non-transformed strains. As for the accumulation ability, these transformed yeast strains were grown in a liquid medium having the same composition to which 30 μM CdSO ₄ was added (100 ml medium was dispensed into 500 ml Kolben, shaking strength was 140 rpm, and cultured at 30 ° C. for 5 days). After collection, the cells were suspended and washed in 500 μM CaCl ₂ buffer, and then collected again by centrifugation to obtain a sample for analysis of metal concentration by ICP (P4000, Hitachi, Japan).

  FIG. 15 shows the results obtained by inoculating several transformants (AyIRT1, AyNramp1) and non-transformed strains (WT) obtained in the above into a medium containing cadmium and examining their growth. (A) is the result of growing for 2 days in a cadmium-free medium, and (b) is the result of growing for 5 days in a medium to which 30 μM of cadmium is added. In any of the transformed strains, the growth was clearly better than that of the yeast strain not transformed. Moreover, the result of having investigated the cadmium and iron content in these stock | strains is shown in FIG. 16, (a) is iron concentration (content), (b) is cadmium concentration (content), (c) is cadmium with respect to iron. It is the figure which represented the quantity by molar ratio. In FIGS. 15 and 16, WT5 represents a control line in which only the pYES yeast transformation vector was introduced, AyIRT1-5 represents a line transformed with AyIRT1, and AyNramp1-5 represents a line transformed with AyNramp1. ing. The cadmium content is about 90 μg / g-FW in the AyIRT1-5 transformant and about 20 μg / g-FW in the AyNramp1-5 transformant, whereas it is about 200 μg / g− in the non-transformant WT5. It was FW. The iron content is 8 μg / g-FW for WT5, 7 μg / g-FW for AyIRT1-5 transformant, 10 μg / g-FW for AyNramp1-5 transformant, and in the transformed and non-transformed strains. There was no significant difference in the amount of iron absorbed. This is considered to be due to the sufficient supply of iron because iron was sufficiently present in the medium used in this example. Moreover, regarding the Cd / Fe value, the transformed strain was smaller. That is, it was clarified that the number of cadmium molecules to be mixed in order to obtain one molecule of iron in any transformed strain was smaller than that in the non-transformed strain. This tendency was particularly remarkable in the AyNramp1-5 transformant. From the above, by transforming with AyNramp1 and AyIRT1, even in the presence of cadmium, it has specific affinity for iron essential for growth and grows well. That is, it is possible to enhance the cadmium resistance of the plant body. Furthermore, the amount of cadmium accumulated is also reduced.

It is a figure which shows the growth of Hebinonegoza callus and tobacco callus under cadmium (100 micromol) exposure conditions. ◇ indicates the relative growth of Hevinone Negoza callus, and ■ indicates the relative growth of tobacco. It is a figure which shows the influence of cadmium (100 micromol) on the expression of the PCS gene in Hebinonegoza callus and tobacco callus. Figure 2 shows the expression of the PCS gene in Hevinonegoza callus grown under various cadmium exposure conditions. It is a figure which shows the content change of the metal element by cadmium exposure in Hebinonegoza callus and tobacco callus. (A) is calcium, (b) is copper, (c) is iron, (d) is magnesium, (e) is manganese, and (f) is the result for zinc. It is the sequence of the Hebinonegoza iron transport carrier gene (AyNramp1: SEQ ID NO: 1). This is the sequence of the Hebinonegoza iron transporter gene (AyIRT1: SEQ ID NO: 3). This is the sequence of the Hebinonegoza iron transporter gene (AyYSL1: SEQ ID NO: 5). This is the sequence of the Hebinonegoza iron transporter gene (AyYSL2: SEQ ID NO: 7). It is a figure which shows IRT protein membrane structure comparison. It is a figure which shows Nramp protein membrane structure comparison. It is a figure which shows the comparison of the amino acid sequence in Nramp of various organisms. It is a figure which shows the expression of an iron transport carrier gene in iron deficiency conditions in Hebino Negoza callus. It is a figure which shows the expression of the iron transport carrier gene under the cadmium exposure in Hebinonegoza callus. It is a figure which shows the expression of the iron transport carrier gene under the cadmium exposure in a Hebinonegoza adult plant. It is a figure which shows the cadmium tolerance in the yeast transformed by AyIRT1 and AyNramp1. It is a figure which shows (a) iron concentration, (b) cadmium concentration, and (c) Cd / Fe (mol ratio) in yeast transformed with AyIRT1 and AyNramp1.

Claims (9)

A gene encoding a protein shown in the following (a) or (b).
(A) a protein comprising the amino acid sequence of SEQ ID NO: 2 or 4 (b) in the amino acid sequence of SEQ ID NO: 2 , comprising an amino acid sequence in which 1 to 51 amino acids have been deleted, substituted or added, and specific to iron A protein that functions as a transport carrier that has an affinity for or a transport carrier that has an amino acid sequence in which 1 to 39 amino acids have been deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 4 and that specifically has an affinity for iron Functioning protein The protein shown in the following (a) or (b).
(A) a protein comprising the amino acid sequence of SEQ ID NO: 2 or 4 (b) in the amino acid sequence of SEQ ID NO: 2 , comprising an amino acid sequence in which 1 to 51 amino acids have been deleted, substituted or added, and specific to iron A protein that functions as a transport carrier that has an affinity for or a transport carrier that has an amino acid sequence in which 1 to 39 amino acids have been deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 4 and that specifically has an affinity for iron Functioning protein A gene comprising the following DNA (c) or (d):
(C) DNA comprising the nucleotide sequence of SEQ ID NO: 1 or 3
(D) DNA encoding a protein that hybridizes under stringent conditions with DNA consisting of the nucleotide sequence of SEQ ID NO: 1 or 3 or DNA complementary thereto, and that functions as a transport carrier specifically having an affinity for iron   A transformed plant cell containing the gene according to claim 1 or 3.   The plant body obtained from the plant cell of Claim 4, its part, its propagation body, and offspring.   The plant cell according to claim 4, further having an ability to accumulate environmental pollutants.   The plant body obtained from the plant cell of Claim 6, its part, its propagation body, and offspring.   The protein production according to claim 2, wherein a host cell obtained by transformation with a vector incorporating the gene according to claim 1 or 3 is cultured, and the protein accumulated in the culture is collected. Method.   An environmental remediation method comprising removing environmental pollutants from soil using the plant according to claim 7.