JP2013158327A - Cesium transporter and cesium low-absorbent rice plant - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cesium low-absorbent rice plant and a cesium transporter.SOLUTION: There is disclosed a cesium transporter having a specific amino acid sequence. There is also disclosed a gene encoding the protein, or a cesium low-absorbent rice plant in which a specific sequence of DNA is characteristically deleted or knocked out.

Description

  The present invention relates to a cesium transporter and a low cesium absorption rice.

  Since the Fukushima accident, cesium (Cs) has been regarded as a problem as a radioactive element with a large amount of scattering and a long half-life. In particular, if it is present in food, it may cause internal exposure, so it cannot be shipped in the area adjacent to the nuclear power plant, and a sampling survey is conducted in a specific area adjacent to the restricted area to confirm that it is below the standard value. Is currently being shipped.

  Among food products, agricultural products absorb nutrients from the soil, so there is a particular concern about the possibility of accumulating indigenous Cs. In addition, since Cs is in the same family as potassium, which is an essential element of plants, the above possibility is further concerned.

  Among agricultural products, rice is cultivated nationwide, and the cultivation area is overwhelmingly large compared to other crops. And since paddy rice basically draws water from the upstream, it is necessary to monitor not only the cultivated land but also a wide area for a long time.

  On the other hand, studies on absorption of Cs and other radioactive elements or their stable isotopes into plants are not always required, so there is little knowledge and the situation is fumbling about rice. It is a fact.

  From the perspective of consumer sentiment, there is a potential demand for rice that does not or does not incorporate cesium with scientific evidence rather than phenomenological support.

ChoiY.H. Et al. Journal of Environmental Radioactivity, 80, 45-58 (2005) 'Transferof 137Cs to rice plants from various paddy soils contaminatedunder flooded conditions at different growth stages'

  The present invention has been studied in view of the above, and an object of the present invention is to provide rice with low cesium absorbability and to provide a cesium transporter and the like that contribute to future basic research.

The invention according to claim 1 is a protein shown in A1 or A2 below.
A1: A cesium transporter having the amino acid sequence set forth in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32.
A2: one or several amino acids in the amino acid sequence described in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32 A protein having an amino acid sequence containing a substitution, deletion, insertion, addition or inversion, and having a cesium transport activity.

The invention according to claim 2 is a DNA encoding a protein shown in B1 or B2 below, or a DNA shown in B3 or B4 below.
B1: A cesium transporter having the amino acid sequence set forth in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32.
B2: One or several amino acids in the amino acid sequence described in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32 A protein having an amino acid sequence containing a substitution, deletion, insertion, addition or inversion, and having a cesium transport activity.
B3: DNA described in SEQ ID NO: 33.
B4: a DNA comprising the DNA of SEQ ID NO: 33 containing a substitution, deletion, insertion, addition or inversion in the sequence and translated into a protein having cesium transport activity in rice.

The invention according to claim 3 is a vector containing a DNA encoding a protein shown in C1 or C2 below, or a vector containing a DNA shown in C3 or C4 below.
C1: A cesium transporter having the amino acid sequence set forth in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32.
C2: one or several amino acids in the amino acid sequence described in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32 A protein having an amino acid sequence containing a substitution, deletion, insertion, addition or inversion, and having a cesium transport activity.
C3: DNA described in SEQ ID NO: 33.
C4: DNA comprising a DNA having a substitution, deletion, insertion, addition, or inversion in the sequence described in SEQ ID NO: 33, and translated into a protein having cesium transport activity in rice.

The invention according to claim 4 is a cesium-low-absorbing rice, wherein the gene encoding the protein shown in D1 or D2 below or the DNA shown in D3 or D4 is deleted or deleted.
D1: A cesium transporter having the amino acid sequence set forth in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32.
D2: one or several amino acids in the amino acid sequence described in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32 A protein having an amino acid sequence containing a substitution, deletion, insertion, addition or inversion, and having a cesium transport activity.
D3: DNA set forth in SEQ ID NO: 33.
D4: DNA which is composed of a DNA having a substitution, deletion, insertion, addition or inversion in the sequence described in SEQ ID NO: 33 and which is translated into a protein having cesium transport activity in rice.

  ADVANTAGE OF THE INVENTION According to this invention, the cesium transporter, DNA, or vector which provides rice with low cesium absorption property and contributes to future basic research can be provided.

It is a list of accession numbers indicating transporter candidate genes. It is three types of obtained plasmid maps. It is a map of pYES2 plasmid. It is the figure which showed the growth curve of the yeast which has a cesium transporter in a liquid medium. It is the photograph which showed the culture result of the yeast which has a cesium transporter in the solid medium containing various concentrations of cesium. It is the experimental result which investigated the expression site | part of each cesium transporter gene by RT-PCR. It is the figure which showed the time-dependent change of the cesium absorption amount in yeast at the time of introduce | transducing the gene of accession number AK241580 into yeast.

  In the present embodiment, rice cultivation in which management technology including agricultural equipment is widespread, i.e., focusing on rice, focusing on identifying cesium transporters, and by losing cesium transporters, An embodiment for producing cesium low-absorption rice will be described.

Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.
<Selection of candidate genes>
First, we decided to construct a yeast library. Approximately 32,000 rice full-length cDNAs have been isolated, and of these, there are approximately 1300 proteins that transport nutrients and the like, that is, transporters or candidates thereof.

  Therefore, in addition to the previous research results by the present inventors, about 1350 rice candidate transporter genes (full-length cDNA) were obtained from the Rice Genome Resource Center (National Institute of Agrobiological Sciences). A list of accession numbers is shown in FIG.

  These genes are distributed in the form of plasmids that express recombinant proteins in the prokaryotic E. coli. There are three types of plasmids distributed, and plasmid maps are shown in FIG.

<Recombination of candidate genes into pYES2>
For the purpose of expressing the recombinant protein in yeast, it was decided to perform the work of incorporating the gene into the yeast protein expression vector pYES2 plasmid (Invitrogen). Since this plasmid also has a uracil synthetic gene, the expression of the introduced gene can be evaluated by using a yeast that does not synthesize uracil. Further, since the gene is inserted downstream of the galactose-inducible GAL1 promoter, it is possible to control the expression of the transgene in yeast. This plasmid map is shown in FIG. The sequences used for homologous recombination are shown in SEQ ID NOs: 34 and 35.

<Amplification of target gene>
The cDNA fragment to be inserted into pYES2 was prepared by amplifying only the target portion by PCR using a plasmid obtained from the Rice Genome Resource Center as a template. Although there are three types of plasmids as described above, since two sequences near the inserted gene are the same in pME18FL-3 and pCMVSPORT6, two sets of primer sets were designed to amplify the entire sequence near the gene by PCR. The forward primer and reverse primer for pFLC1 are shown in SEQ ID NOs: 36 and 37, respectively, and the forward primer and reverse primer for pME18FL-3 and pCMVSPORT6 are shown in SEQ ID NOs: 38 and 39, respectively.

  In addition, Prime Star Max (Takara) was used for PCR in consideration of the synthesis speed and high accuracy. To the 5 μl reaction system, 2.5 μl of 2 × Prime Star Max buffer, 0.125 μl of primer (prepared so that the forward primer and the reverse primer were each 10 pmol), and 4.875 μl of MQ were added. The template DNA was obtained by immersing the tip of the chip in a plasmid solution and attaching it slightly to the tip of the chip. PCR was performed at 98 ° C. for 2 minutes, followed by 30 cycles of 98 ° C. for 10 seconds, 55 ° C. for 5 seconds, 72 ° C. for 30 seconds, and finally reacted at 72 ° C. for 3 minutes.

<Preparation for pYES2>
pYES2 was previously cleaved with restriction enzymes XbaI, KpnI, and HindIII (all of Takara) and linearized. For the cleavage reaction, 9 μl of 10 × M Buffer and 3 μl of XbaI, KpnI, and HindIII were added to 72 μl (10 μg) of pYES2 solution and reacted at 37 ° C. overnight. Subsequently, after separation by 1% agarose gel electrophoresis, the target band was cut out from the gel. For extraction of DNA from the gel, Mag Extract (Toyobo) was used. The method followed the manual.

<Insertion of PCR product into pYES2>
For subcloning, the Gap-repair cloning method [Ma H, Kunes S, Schatz PJ, Botstein D (1987) “Plasmid construction
by homologous recombination in yeast ”Gene; 58 (2-3): 201-16.] About 100 ng of the DNA fragment obtained by the previous PCR and about 5 ng of linearized pYES2 were simultaneously added to the yeast (W303- 1A strain Genotype: MATa (leu2-3,112 trp1-1 can1-100
ura3-1 ade2-1 his3-11,15}) to obtain the desired plasmid.

<Confirmation of insertion of target gene>
In order to confirm that the target gene was correctly inserted into the plasmid, the sequence was confirmed using a DNA sequencer. First, a plasmid was extracted from yeast, the resulting plasmid was transformed into E. coli, and E. coli was further cultured to extract the plasmid.

Plasmid extraction from yeast was performed by the following method.
For extraction, a plasmid extraction kit HiYeld Plasmid Mini Kit (RBC) was used. Yeast cultured overnight in 3 ml of SC-ura liquid medium was transferred to a round bottom 2 ml tube (eppendorf) and collected by centrifugation at 3000 rpm for 3 minutes at room temperature. 200 μl of PDA1 was added, 10 μl of Zymolase was added thereto, and reacted at 37 ° C. for 1 hour. After adding 200 μl of PDA2 and stirring well, 300 μl of PDA3 was added and stirring well. After centrifugation at 15000 rpm for 10 minutes at room temperature, the supernatant was transferred to a filter column. After the solution was filtered by suction, 400 μl of W1 buffer was added and suction filtered. Subsequently, 600 μl of Wash buffer was added and suction filtered. The filter column was transferred to an empty column and centrifuged at 15000 rpm for 1 minute at room temperature. The filter column was transferred to a new 1.5 ml microtube, 30 μl of Elution buffer was added and allowed to stand for 1 minute. The solution was centrifuged at 15000 rpm for 1 minute at room temperature, and the filtrate was used as a plasmid solution.

The resulting plasmid was transformed into E. coli DH5α strain.
Competent cells were created by the method of Hanahan et al. [Hanahan, D (1983) “Studies on transformation of Escherichia coli
with plasmids ”Journal of Molecular Biology 166 (4): 557-580]. Transformation was performed using the Freeze-Though method. 10 μl of a plasmid solution extracted from yeast before 100 μl of competent cells were completely dissolved. The mixture was lightly stirred and allowed to stand on ice for 30 minutes, followed by reaction at 42 ° C. for 45 seconds and then left on ice for 3 minutes, and then 800 μl of SOC medium kept at 37 ° C. was added. After stirring, the mixture was allowed to stand for 30 minutes in an incubator at 37 ° C. After standing, the cells were collected by centrifugation at 5000 rpm for 2 minutes, and then the supernatant was discarded by decantation. And inoculated overnight.

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１２１℃２０分間のオートクレーブ後、６０℃程度まで培地温度が下がったところで
終わり濃度１００ｕｇ／ｍｌとなるようにアンピシリンナトリウム（Ｗａｋｏ）を加
えた。 The composition of the LA medium used is shown in Table 1.

The pH is adjusted to 7.2-7.5 with NaOH.
After autoclaving at 121 ° C. for 20 minutes, ampicillin sodium (Wako) was added to a final concentration of 100 ug / ml when the medium temperature dropped to about 60 ° C.

  For plasmid extraction from E. coli, a plasmid extraction kit HiYield Plasmid Mini Kit (RBC) was used. E. coli cultured overnight in 3 ml of LA liquid medium was transferred to a round bottom 2 ml tube (eppendorf) and collected by centrifugation at 3000 rpm for 3 minutes at room temperature. 200 μl of PDA1 was added and completely suspended. Subsequently, 200 μl of PDA2 was added and the mixture was tumbled 10 times, and then allowed to stand for 2 minutes. Then, 300 μl of PDA3 was added and stirred well. After centrifugation at 15000 rpm for 10 minutes at room temperature, the supernatant was transferred to a filter column. After the solution was filtered by suction, 400 μl of W1 buffer was added and suction filtered. Subsequently, 600 μl of Wash buffer was added and suction filtered. The filter column was transferred to an empty column and centrifuged at 15000 rpm for 1 minute at room temperature. The filter column was transferred to a new 1.5 ml microtube, 30 μl of Elution buffer was added and allowed to stand for 1 minute. The solution was centrifuged at 15000 rpm for 1 minute at room temperature, and the filtrate was used as a plasmid solution.

  The sequencer used was an ABI 3130 DNA sequencer. For the PCR reaction solution, 2 μl (200 to 400 ng) of plasmid DNA solution, 0.3 μl of BigDye (ABI), 4 μl of 5 × Sequence Buffer (ABI), 0.25 μl of Primer (12.5 pmol / μl), The amount of ultrapure water was 13.45 μl. The PCR reaction was performed using a thermal cycler 9700 (ABI). PCR was performed by denaturing at 96 ° C. for 2 minutes, followed by 35 cycles of 96 ° C. for 10 seconds, 50 ° C. for 5 seconds, and 60 ° C. for 4 minutes.

  Unreacted BigDye was removed using shephadex G-50 (GE Healthcare). 3 g of shephadex G-50 (Fine Grade) was placed in a 50 ml falcon tube, and 50 ml of ultrapure was added and allowed to stand overnight. Immediately before use, the Shepadex solution was mixed well and 300 μl was added to the multiscreen (GE Healthcare). The multiscreen was placed on a PCR plate and centrifuged at 1000 rpm at room temperature for 5 minutes. After centrifuging for 5 minutes, the center side and the outside of the plate were reversed and centrifuged at 1000 rpm at room temperature for 5 minutes. The multiscreen containing the gel was placed on a new PCR plate, and the sequence reaction product was applied to the center of the gel. Centrifugation was performed at 1000 rpm at room temperature for 5 minutes. The filtrate was subjected to a sequencer.

<Introduction of a plasmid in which the target gene has been inserted into yeast>
Plasmid transformation into yeast is described in H Ito, Y Fukuda, K Murata, and A Kimura (1983) “Transformation of
intact yeast cells treated with alkali cations. ”Journal of
bacteriology, 163-168 was partially modified.

First, yeast (W303-1A strain Genotype: MATa (leu2-3,112 trp1-1 can1-100
ura3-1 ade2-1 his3-11,15}) was cultured overnight at 30 ° C. in 3 ml of YPD medium to obtain a preculture solution. 3 ml of the preculture solution was added to 300 ml of Kolben (IWAKI) containing 100 ml of YPAD medium, and rotationally cultured at 30 ° C. and 90 rpm until OD ₆₀₀ = about 0.4 to 0.6. When OD ₆₀₀ was about 0.4 to 0.6, the cells were transferred to two 50 ml tubes (Labcon), and centrifuged at 3000 rpm for 2 minutes at room temperature to collect bacteria. The supernatant was discarded, rinsed once with 25 ml of sterilized water, and further collected to remove the supernatant completely. 2 ml of 0.1 M lithium acetate was added and suspended, and the cells were collected by centrifugation at 3000 rpm for 2 minutes at room temperature.

  After removing the supernatant, 6 ml of 50% PEG 4000, 900 μl of 1M lithium acetate, 625 μl of carrier DNA (2.0 mg / ml), 1 ml of dimethyl sulfoxide and 1.25 ml of sterilized water were added and vortexed. The two were combined and dispensed into a 96-well plate at 200 μl, and the DNA solution was added to each. Sealed to prevent leakage, and then allowed to stand at 30 ° C. for 30 minutes. Next, heat shock was performed at 42 ° C. for 20 minutes using a thermostatic device (TAITEC). During heat shock, mixing was performed by tumbling up and down every 3 to 5 minutes. The mixture was centrifuged at 3000 rpm for 10 minutes at room temperature, the supernatant was completely removed, 200 μl of YPAD liquid medium was added and suspended, and the mixture was kept at 30 ° C. for 1 hour. The mixture was centrifuged at 3000 rpm for 10 min at room temperature, the supernatant was completely removed, suspended in 20 μl of sterilized water, and all of the SC-ura solid medium was inoculated.

<Library screening>
Yeast strain and culture conditions:
Yeast (W303-1A strain Genotype: MATa (leu2-3,112 trp1-1 can1-100
ura3-1 ade2-1 his3-11,15}) were cultured in SC-ura medium. During liquid culture, rotary culture at 180 rpm was performed with a shaker (NR-3, TAITEC). SC-ura medium was used during preculture, and SG-ura + Gal medium was used during main culture. All were cultured at 30 ° C.

In addition, the composition and preparation in 100 ml of SC-ura or SG-ura medium are as follows.
Yeast Nitrogen Base (Japan BD): 0.67 g
Drop out mix: 0.182g
adenine (Wako): 0.04g
Succinic acid buffer: 5 ml added This was prepared with deionized water to 90 ml, 2 g of agarose (Wako) was added, and autoclaved at 121 ° C. for 20 minutes.
Add 2g of glucose to SC-ura medium Add 2g of galactose to SG-ura medium

・ Screening conditions:
In screening of the cesium transporter, evaluation was performed using cesium chloride (Wako).
Cesium chloride was dissolved in sterilized water to prepare a 4M cesium solution. After autoclaving, a cesium-containing medium was prepared by adding to SG-ura medium or SC-ura medium. To the selective medium, succinic acid / Tris buffer was added to a final concentration of 20 mM for the purpose of keeping the pH in the medium constant. At the time of screening, pre-culture was performed in a 96-well microtiter plate (BM instrument). Dispense 100 μl of SC-ura liquid medium into each well, inoculate the yeast using a pin replicator (Watson) or toothpick, and then culture at 30 ° C. overnight at 180 rpm using a constant temperature shaker (Tytec). did. Subsequently, 200 μl of SC-ura medium was put in each well of a 1.5 ml deep well plate, 20 μl of precultured yeast was inoculated there, and cultured at 30 ° C. for 4 hours 30 minutes. After culture, 100 μl was dispensed into a new 96-well microtiter plate, and the absorbance was measured (Model 680, BIO-RAD).

Moreover, _{OD 595} The addition of the culture solution 10μl was added with sterile water so that 0.01 to deep well plates 1.5 ml. After 10 μl of the main culture solution was added to the sterilized water to make the turbidity of all the bacterial solutions uniform, the solution was covered with surgical tape (3M) and stirred well for 3 minutes with a desktop mixer. 150 μl of the suspension was dispensed into a new 96-well microtiter plate. When this bacterial solution was inoculated into a solid medium, a bell blotter 96-well replicator (Samplatech) was used. Culturing was performed at 30 ° C. for 40 to 48 hours.

Cesium is not necessary for plant growth. The same applies to yeast. When the concentration of cesium in yeast increases, yeast growth and the like are suppressed. In both the absorbance measurement by liquid culture and the colony evaluation by solid medium, it was confirmed that 17 strains in the library had cesium transport activity. FIG. 4 is a diagram showing a growth curve of yeast having a cesium transporter in a liquid medium. FIG. 5 is a photograph showing the results of culturing yeast having a cesium transporter in a solid medium containing various concentrations of cesium.
In addition, galactose-inducibility and uracil requirement were examined together, and the appropriateness of screening of these 17 strains was guaranteed.

  Based on the above experiment, accession numbers AK107688, AK107088, AK111959, AK058856, AK066194, AK068574, AK102180, AK058673, AK059035, AK0658891, AK07118, AK070844, AK100504, AK10508, AK15687, It was confirmed that the gene In addition, each DNA was shown to sequence number 1,3,5,7,9,11,13,15,17,19,21,23,25,27,29,31,33. Further, except for AK12021 (SEQ ID NO: 33), the proteins to be translated are SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, respectively. It was shown to.

<Cesium transporter expression site>
Next, the expression site in rice was examined using a semi-quantitative RT-PCR method. Total RNA was extracted from rice at the 3 leaf stage cultivated in an environment of 28 ° C. for 16 hours and 24 ° C. for 8 hours using RNeasy Plant Mini Kit (Qiagen). The extraction method followed the manual attached to the kit. The only changes are as follows. After performing the Buffer RW1 treatment, 10 μl of DNase I (Nippon Gene) and 70 μl of Buffer RDD mixture were added to the column and left at room temperature for 15 minutes to decompose the DNA.

  Reverse transcription reaction was performed using 5 μg of extracted total RNA as a template. For the reaction, PrimeScript RT-PCR Kit (TaKaRa) was used. The method followed the attached manual. 240 μl of TE was added to 60 μl of the reaction solution to dilute 5 times, and stored at −30 degrees until PCR. Taq DNA Polymerase (BioBasic) was used for PCR. The composition of the reaction solution was in accordance with the manual.

  The synthetic oligos listed were used. The PCR reaction was carried out at 94 ° C. for 2 minutes, followed by 24-29 cycles of 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 30 seconds. PCR products were separated by 1.5% agarose electrophoresis, stained with ethidium bromide, and then a band was photographed with a digital camera. A photograph is shown in FIG. From this, it can be confirmed that a cesium transporter is formed at the root, except for strains with accession numbers AK1007078 and AK068574.

Table 2 shows the relationship between the primer set used for PCR and the sequence number.

<Transport activity in yeast>
Next, the transport activity of the cesium transporter in the 17 strains of yeast was measured. Yeast was inoculated into 3 ml of SD-gal-Ura liquid medium, and pre-cultured overnight with shaking culture at a growth chamber temperature of 30 ° C. and 150 rpm. The next morning, 3 ml of SD-gal-Ura liquid medium was added to the pre-cultured liquid to make a total volume of 6 ml, and the main culture was carried out for 3 hours by shaking culture at a growth chamber temperature of 30 ° C. and 150 rpm. A 135 ml SD-gal-Ura medium was charged with a bacterial solution to be added later, and 1050 μl of 4M CsCl was added and mixed well so that the CsCl concentration became 30 mM. 9 ml of this medium was dispensed into 50 ml Falcon tubes. 1 ml of the main culture broth was inoculated into a medium in a falcon tube, and cultured with shaking at a growth chamber temperature of 30 ° C and 150 rpm for 30 minutes to 5 hours.

  While shaking, 20 mM CaCl 2 was allowed to cool on ice. The mixture was centrifuged at 3500 rpm at 4 ° C. for 3 minutes with a swing rotor, and the supernatant was gently removed by decantation. Thereafter, the operation was performed on ice until the bacterial solution was transferred to a Teflon tube. The tube cultured for the above time was placed on ice to stop the culture. After centrifuging at 3500 rpm and 4 ° C. for 3 minutes with a swing rotor, 5 ml of ice-cooled 20 mM CaCl 2 was added and suspended by vortexing. The rinse was performed 3 times. After the final rinse, leave only a small amount of the supernatant, suspend the pellet by pipetting, transfer to a 1.5 ml Eppendorf tube, centrifuge at 10000 rpm, 4 ° C. for 1 minute, completely remove the supernatant, 1 ml Of MQ was added to each Eppendorf tube.

  10 μl was taken from each Eppendorf tube, and the number of cells was counted 3 times with a cell counter and the average was taken. The remaining tube of 990 μl was centrifuged with a centrifuge at 10,000 rpm at 4 ° C. for 1 minute, and the supernatant was removed by decantation. The pellet was suspended by pipetting, the bacterial solution was transferred to a Teflon tube, and completely dried at 80 ° C. for 30 minutes with a heat block. 125 μl of nitric acid was added to a Teflon tube and left at 120 ° C. for 2 hours to acid-decompose the yeast. After decomposition, 50 μl of hydrogen peroxide was added to each Teflon tube, centrifuged at 10000 rpm at 4 ° C. for 1 minute and allowed to stand at room temperature for 10 minutes. After standing, MQ was added to 2950 μl of each Teflon tube and made up to 3 ml. After setting the Teflon tube on the tube rack, it was suspended for 10 minutes by an ultrasonic cleaner. The suspension was transferred to a new 15 ml tube and stored at 4 ° C. until measurement. The concentration of cesium in the sample was measured with an atomic absorption meter (manufactured by Hitachi).

  The results are shown in FIG. Here, the time-dependent change in the amount of cesium absorbed in the yeast when the gene of accession number AK241580 was introduced into the yeast was shown, but the same tendency was observed in the other 16 strains. It can be confirmed that cesium transporter is actually formed in yeast.

<Cesium low absorption rice>
If the cesium transporter is lacking, cesium will not be absorbed into the plant body, so that rice that can be distributed even in soil with radioactive cesium attached can be grown. That is, accession numbers AK107688, AK107088, AK111959, AK058856, AK06664, AK068574, AK102180, AK058673, AK059035, AK065891, AK071181, AK070844, AK105084, AK105088, AK105167, AK2415 Alternatively, rice that is deficient in the gene can be produced and used to efficiently produce rice with low cesium absorption. Preferably, the rice is deficient in 2 or more genes, more preferably 5 or more deficient rice, more preferably 10 or more deficient rice, most preferably 17 or all of them. This is rice that has a defective gene.

  As an example of rice production, a cesium transporter is deleted using a mutation source such as γ-ray irradiation, and this is backcrossed and selected, so that it tastes good and does not take up cesium. Can produce difficult rice. General techniques can be used for backcrossing and selection methods.

By using the cesium transporter of the present invention, its transport activity can be enhanced, cesium can be stocked and concentrated in the whole rice plant or in a specific site, and gradual dyeing of fields and other grounds can be performed. Stocks and concentrates can be appropriately concentrated in a later step and separated from the environment.

Claims (4)

Protein shown in the following A1 or A2.
A1: A cesium transporter having the amino acid sequence set forth in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32.
A2: one or several amino acids in the amino acid sequence described in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32 A protein having an amino acid sequence containing a substitution, deletion, insertion, addition or inversion, and having a cesium transport activity.
DNA encoding the protein shown in B1 or B2 below or DNA shown in B3 or B4 below.
B1: A cesium transporter having the amino acid sequence set forth in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32.
B2: an amino acid sequence according to any one of SEQ ID NOs: 1 to 16, comprising an amino acid sequence including substitution, deletion, insertion, addition or inversion of one or several amino acids, and cesium transport activity A protein having
B3: DNA described in SEQ ID NO: 33.
B4: a DNA comprising the DNA of SEQ ID NO: 33 containing a substitution, deletion, insertion, addition or inversion in the sequence and translated into a protein having cesium transport activity in rice.
A vector containing a DNA encoding a protein shown in C1 or C2 below, or a vector containing a DNA shown in C3 or C4 below.
C1: A cesium transporter having the amino acid sequence set forth in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32.
C2: one or several amino acids in the amino acid sequence described in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32 A protein having an amino acid sequence containing a substitution, deletion, insertion, addition or inversion, and having a cesium transport activity.
C3: DNA described in SEQ ID NO: 33.
C4: DNA comprising a DNA having a substitution, deletion, insertion, addition, or inversion in the sequence described in SEQ ID NO: 33, and translated into a protein having cesium transport activity in rice.
A cesium-low-absorbing rice, wherein a gene encoding a protein shown in D1 or D2 below or a DNA shown in D3 or D4 is deleted or deleted.
D1: A cesium transporter having the amino acid sequence set forth in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32.
D2: one or several amino acids in the amino acid sequence described in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32 A protein having an amino acid sequence containing a substitution, deletion, insertion, addition or inversion, and having a cesium transport activity.
D3: DNA set forth in SEQ ID NO: 33.
D4: DNA which is composed of a DNA having a substitution, deletion, insertion, addition or inversion in the sequence described in SEQ ID NO: 33 and which is translated into a protein having cesium transport activity in rice.

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