WO2013065517A1 - カドミウム吸収制御遺伝子、タンパク質、及びカドミウム吸収抑制イネ - Google Patents
カドミウム吸収制御遺伝子、タンパク質、及びカドミウム吸収抑制イネ Download PDFInfo
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- the present invention relates to a transporter gene and a transporter protein involved in regulation of cadmium (hereinafter abbreviated as Cd) absorption by roots, a rice mutant in which Cd absorption is suppressed, and a low Cd absorption rice variety.
- Cd cadmium
- the present inventors irradiated rice seeds with a heavy ion beam and selected low Cd absorption rice mutants from the obtained plant bodies. Furthermore, the causative gene of the obtained mutant was identified, and the present invention was achieved. That is, the present invention is as follows. ⁇ 1> The present invention is a gene encoding a transporter protein involved in the control of cadmium absorption including any one of the following base sequences (A) to (C).
- A The DNA base sequence represented by SEQ ID NO: 2.
- B A base sequence in which one or more bases are deleted, substituted, or added in the base sequence described in SEQ ID NO: 2, and a DNA base sequence of a gene encoding a protein that controls cadmium absorption.
- (C) A DNA base sequence of a gene that hybridizes with the base sequence described in SEQ ID NO: 2 under a stringent condition and encodes a protein that controls cadmium absorption.
- the present invention is a gene encoding a transporter protein involved in the control of cadmium absorption including any one of the following base sequences (D) to (F).
- (D) DNA base sequence represented by SEQ ID NO: 3.
- (E) A DNA base sequence of a gene encoding a protein that controls cadmium absorption, wherein one or a plurality of bases are deleted, substituted, or added in the base sequence set forth in SEQ ID NO: 3.
- (F) A DNA base sequence of a gene that hybridizes with the base sequence described in SEQ ID NO: 3 under a stringent condition and encodes a protein that controls cadmium absorption.
- the present invention is a gene encoding a transporter protein involved in the control of cadmium absorption including any one of the following base sequences (G) to (I).
- (G) A DNA base sequence represented by SEQ ID NO: 4.
- (H) A base sequence in which one or more bases are deleted, substituted or added in the base sequence set forth in SEQ ID NO: 4, and a DNA base sequence of a gene encoding a protein that controls cadmium absorption.
- (I) A DNA base sequence of a gene that hybridizes with the base sequence described in SEQ ID NO: 4 under a stringent condition and encodes a protein that controls cadmium absorption.
- the present invention is a transporter protein involved in the control of cadmium absorption including any one of the following amino acid sequences (J) to (L).
- (J) The amino acid sequence set forth in SEQ ID NO: 1.
- (K) An amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence set forth in SEQ ID NO: 1, and the amino acid sequence of a protein that controls cadmium absorption.
- the present invention is a transporter protein involved in the regulation of cadmium absorption including any one of the following amino acid sequences (M) to (O).
- the present invention is a transporter protein involved in the control of cadmium absorption including any one of the following amino acid sequences (P) to (R).
- P The amino acid sequence set forth in SEQ ID NO: 6.
- Q An amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence set forth in SEQ ID NO: 6, and the amino acid sequence of a protein that controls cadmium absorption.
- R An amino acid sequence having at least 90% homology with the amino acid sequence set forth in SEQ ID NO: 6, and an amino acid sequence of a protein that controls cadmium absorption.
- the present invention is a recombinant vector comprising the DNA according to any one of ⁇ 1> to ⁇ 3>.
- the present invention is a transformant containing the DNA according to the above ⁇ 2> or ⁇ 3> or a transformant lacking the DNA according to claim 1.
- the present invention is a transformant using the recombinant vector according to ⁇ 7>.
- the present invention is a gene marker that can identify the DNA base sequence according to any one of ⁇ 1> to ⁇ 3>.
- this invention is a cadmium absorption suppression rice in which the function of the protein described in said ⁇ 4> is suppressed.
- the present invention is cadmium absorption-suppressed rice expressed by the protein encoded by the gene described in ⁇ 2> or ⁇ 3>.
- the present invention includes rice cultivar Koshihikari and heading, yield, The cadmium absorption-suppressed rice according to ⁇ 11> or ⁇ 12>, wherein the taste is comparable.
- the present invention is the cadmium absorption-suppressed rice described in the above ⁇ 11> or ⁇ 12>, wherein heading is about 2 weeks earlier than the rice variety Koshihikari.
- the present invention provides the cadmium absorption-suppressing rice mutation according to any one of ⁇ 11> to ⁇ 14>, which comprises the following steps (a) and (b), or steps (a) and (c): It is a body selection method.
- A) a step of cultivating a first generation seed irradiated with a heavy ion beam in a field and sowing a second generation seed;
- B) The second generation seeds obtained in the step (a) above are cultivated with a Cd-containing hydroponic solution, the stalks and leaves are excised, the conduit fluid secreted is collected, and the Cd concentration contained in the solution is collected.
- this invention is a cadmium absorption suppression rice obtained by crossing the cadmium absorption suppression rice mutant in any one of said ⁇ 11> thru
- the present invention provides a highly practical low Cd absorption rice variety. Furthermore, by using the low Cd-absorbing rice cultivar of the present invention as a parent, a new low Cd-absorbing rice cultivar can be cultivated without performing genetic recombination, and by using the DNA marker of the present invention, efficiency It is possible to select low Cd absorption rice varieties.
- FIG. 1 shows the cadmium concentration of brown rice when grown in a Cd-contaminated field.
- FIG. 2 is an explanatory diagram showing the growth state of Koshihikari and low Cd mutant lines.
- FIG. 3 is a graph showing the frequency distribution of the foliage Cd concentration of F2 individuals (92 individuals) mated with # 3-6-4 and Kasalath.
- FIG. 4 is a diagram showing the location of a low Cd gene estimated from gene mapping.
- FIG. 5 is a diagram showing base deletions and insertion positions on genomic DNAs of # 7-3-6 and # 3-6-4.
- FIG. 6 is a result of electrophoresis showing the difference in DNA amplified fragment length by PCR between Koshihikari and the mutant.
- FIG. 7 is a result chart showing the degree of yeast growth when each yeast mutant introduced with the TRECA gene and treca-1 gene is treated with an SD agar medium.
- FIG. 8 is a fluorescence observation diagram showing localization of TRECA protein and treca-1 protein.
- FIG. 9 is a result of electrophoresis showing the difference in DNA amplified fragment length between Koshihikari and mutant (# 3-6-4, # 3-5-20) using gene markers.
- FIG. 10 is an electrophoretogram showing the difference in DNA amplified fragment length between Koshihikari and mutant (# 7-3-6) using gene markers.
- the present invention relates to a transgene involved in the control of Cd absorption obtained by analyzing a gene of a low Cd absorption rice cultivar obtained by irradiating rice with a heavy ion beam often used in flower breeding and the like.
- Porter Transporter regulating cadmium absorption, hereinafter abbreviated as TRECA
- TRECA Transporter regulating cadmium absorption
- treca Transporter regulating cadmium absorption
- treca Transporter regulating cadmium absorption
- TRECA gene and mutant gene of the TRECA gene hereinafter abbreviated as treca
- a low Cd-absorbing rice variety containing the mutant gene and the low It is a new low Cd absorption rice variety obtained using Cd absorption rice varieties.
- “controlling cadmium absorption” means participating in the promotion or suppression of cadmium absorption. The present invention is described in detail below.
- the TRECA gene of the present invention is a gene shown in SEQ ID NO: 2 that encodes a transporter protein involved in the regulation of cadmium absorption shown in SEQ ID NO: 1.
- the base sequence shown in SEQ ID NO: 2 was identified by analyzing the Cd absorption-suppressed rice gene obtained by irradiation with the heavy ion beam.
- the present invention is a so-called open reading frame (hereinafter referred to as ORF) from the start codon to the stop codon of the heavy metal transporter gene derived from the TRECA gene.
- the TRECA gene is a database containing genetic information such as RAP-DB (The Rice Annotation Project Database) and NCBI (The National National Center for Biotechnology Information), because of its homology with known Arabidopsis base sequences. It is a kind of Nramp gene that is annotated as a gene that has the function of transporting heavy metals. Although it is described as “similar to OsNramp1” in RAP-DB and “OsNramp5” in NCBI, it has never been known about its function, in particular, its ability to control cadmium absorption in rice. .
- the TRECA gene according to the present invention is preferably an ORF consisting of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 2, but may be any gene as long as a portion corresponding to this portion is included. For example, a gene obtained by adding 5 ′ and 3 ′ UTR to the ORF is also included in the present invention.
- the TRECA gene of the present invention includes, for example, a mutant or derivative encoding a protein comprising an amino acid sequence in which one or several amino acids are substituted, deleted, added and / or inserted in the amino acid sequence shown in SEQ ID NO: 1. Is included.
- the base sequence is mutated, it may not be accompanied by amino acid mutation in the protein (degenerate mutation), and such a degenerate mutant is also included in the gene of the present invention.
- the method for obtaining the gene is not particularly limited, and a general method is adopted.
- the gene may be excised from a genomic DNA or a genomic DNA library of an organism having the gene with an appropriate restriction enzyme and purified. That is, the heavy metal transporter gene of the present invention includes genomic DNA and chemically synthesized DNA.
- Genomic DNA can be prepared by those skilled in the art using conventional means. For genomic DNA, for example, genomic DNA is extracted from the target organism, a genomic library (plasmids, phages, cosmids, BACs, PACs, etc. can be used as vectors) is developed and expanded, It can be prepared by performing colony hybridization or plaque hybridization using a probe prepared based on DNA encoding the protein (SEQ ID NO: 2).
- the gene isolated as described above is considered to have high homology with the amino acid sequence of the protein of the present invention (SEQ ID NO: 1) at the amino acid level encoded by the gene.
- High homology refers to a sequence identity of 80% or more, more preferably 90% or more, particularly preferably 95% or more in the entire amino acid sequence. Sequence identity can be determined by FASTA search (Pearson®W.R. And D.J. Lipman (1988) Proc. Natl. Acad. Sci. USA. 85: 2444-2448) and BLASTP search.
- the TRECA protein according to the present invention is a protein that is encoded by the TRECA gene, exists on the cell membrane, and controls the absorption of cadmium and manganese. Lines in which the gene encoding the TRECA protein of the present invention described later is completely deleted (# 7-2-13) and lines in which partial mutation has occurred (# 3-6-4, # 7-3-6, etc.) ), The absorption of cadmium and manganese is greatly suppressed, so it is clear that the protein controls the absorption of cadmium and manganese.
- TRECA protein shows iron transport activity in the system in which TRECA gene is introduced into yeast, and TRECA protein is also involved in iron absorption because yeast with mutant treca protein does not show iron transport activity. .
- deletion lines and mutant lines have no change in iron concentration (Table 2), IRT and ZIP families (Bughio N. et al. (2002 ) Journal of Experimental Botany. 53: 1677-1682), iron absorption capacity is expected to be low.
- the TRECA protein according to the present invention is a protein comprising the amino acid sequence represented by SEQ ID NO: 1, or an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 1. And a protein having the heavy metal transport function of TRECA protein, or a protein consisting of the amino acid sequence represented by SEQ ID NO: 1 and at an amino acid level of 80% or more, more preferably 90% or more, still more preferably 95% or more It is a protein that shows homology and has the heavy metal transport function of TRECA protein.
- the present invention is a mutant gene of the TRECA gene represented by SEQ ID NO: 3 (hereinafter referred to as treca-1 gene).
- the base sequence shown in SEQ ID NO: 3 is a gene derived from Cd absorption-suppressed rice # 3-6-4 obtained by irradiation with the heavy ion beam.
- the TRECA gene shown in SEQ ID NO: 2 It is a 50 bp by replacing the end site of exon, 32 bp from base number 1025 to 1056.
- the present invention is an ORF from the start codon to the stop codon of the treca-1 gene shown in SEQ ID NO: 3, but any gene may be used as long as a portion corresponding to this portion is included.
- a gene obtained by adding 5 ′ and 3 ′ UTR to the ORF is also included in the present invention.
- the treca-1 gene of the present invention includes, for example, a mutation encoding a protein consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, added and / or inserted in the amino acid sequence shown in SEQ ID NO: 5 Body and derivatives.
- the treca-1 protein encoded by the treca-1 gene according to the present invention has an amino acid sequence that is mutated from the TRECA protein as shown in SEQ ID NO: 5 and the Cd absorption function is suppressed.
- treca-1 protein consists of an amino acid sequence represented by SEQ ID NO: 5 or an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 5,
- a protein in which the heavy metal transport function of the TRECA protein is lost or a protein consisting of the amino acid sequence represented by SEQ ID NO: 5 and an amino acid level of 80% or more, more preferably 90% or more, and still more preferably 95% or more It is a protein that shows homology and has lost the heavy metal transport function of the TRECA protein.
- the present invention is a mutant gene of the TRECA gene shown in SEQ ID NO: 4 (hereinafter referred to as treca-2 gene).
- the base sequence shown in SEQ ID NO: 4 is a gene derived from Cd absorption-suppressed rice # 7-3-6 obtained by irradiation with the heavy ion beam, and the base sequence in the TRECA gene (cDNA) shown in SEQ ID NO: 2 A single base deficiency (cytosine deficiency) appears at number 915.
- the treca-2 gene according to the present invention is preferably an ORF consisting of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 4, but may be any gene as long as a portion corresponding to this portion is included. For example, a gene obtained by adding 5 ′ and 3 ′ UTR to the ORF is also included in the present invention.
- the treca-2 gene of the present invention includes, for example, a mutant encoding a protein comprising an amino acid sequence in which one or several amino acids are substituted, deleted, added and / or inserted in the amino acid sequence shown in SEQ ID NO: 6 Derivatives.
- the treca-2 protein encoded by the treca-2 gene according to the present invention is one in which the amino acid sequence is mutated from the TRECA protein as shown in SEQ ID NO: 6 and the Cd absorption function is suppressed.
- treca-2 protein consists of an amino acid sequence represented by SEQ ID NO: 6 or an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 6,
- a protein in which the heavy metal transport function of the TRECA protein is lost or a protein consisting of the amino acid sequence represented by SEQ ID NO: 6 and 80% or more, more preferably 90% or more, more preferably 95% or more at the amino acid level. It is a protein that shows homology and has lost the heavy metal transport function of the TRECA protein.
- the recombinant vector according to the present invention is one in which either the TRECA gene, the mutant treca-1 gene or the treca-2 gene is incorporated.
- the vector can be expressed into a target plant by a known transformation method so that the gene or gene fragment incorporated in the plant can be expressed to obtain the protein according to the present invention.
- the recombinant vector according to the present invention is preferably a binary vector, particularly preferably a “large-capacity binary shuttle vector” described in JP-A-10-155485.
- the vector into which the gene has been inserted is introduced into a target plant cell.
- a known method can be used. For example, an Agrobacterium method, an electroporation method, a particle gun method, a microinjection method, etc. are used. The method is most preferred.
- the transformant expressing the gene of the present invention is not limited to rice and is not limited as long as it is a plant having a protein or gene that controls Cd absorption by roots.
- the gene marker in the present invention is for identifying an individual from the difference in base sequence between the TRECA gene and the mutant treca gene.
- the gene marker serves as a marker for judging whether the gene of the present invention has been introduced into other rice varieties by mating or transformation based on the difference in the base sequence.
- Genomic DNA extracted from rice is used as a template, and a synthetic oligonucleotide with a base sequence appropriately selected according to the base sequence of the genomic DNA is used as a primer.
- a primer used as a primer.
- the reaction solution containing the product is subjected to, for example, agarose electrophoresis, various amplified DNA fragments are fractionated, and it can be confirmed that the DNA fragments correspond to the genomic DNA of the present invention.
- genomic DNA is extracted from rice cultivars such as Koshihikari and # 3-6-4 mutants, and a primer set that can amplify the target base part (the part with the inserted base) is used. Accordingly, when the extracted DNA amplified fragment by PCR is subjected to electrophoresis, it is fractionated into 200 to 500 bp and 600 to 800 bp, respectively, which can be used for identification of each individual.
- the # 7-2-13 mutant can be distinguished from other varieties by the absence of amplified DNA fragments.
- the primer is not limited as long as it has a base sequence capable of amplifying the mutated region.
- a primer set capable of amplifying a base portion including a single base deletion region [eg, [Os7g2572_F2976g (5'- TATATTCAGCCTGGGCAGATCGAG -3 ': SEQ ID NO: 7), Os7g2572_R3815g (5'- TGATGTACTGTCCAGCGTATGTGC- 3 ': SEQ ID NO: 8)], etc., amplifying the DNA fragment by PCR reaction, and cleaving the amplified DNA fragment having a restriction enzyme site newly generated by a single base deletion with a specific restriction enzyme (for example, FspI) By doing so, it is possible to distinguish from other varieties.
- the primer is not limited as long as it can amplify the mutated region.
- the restriction enzyme is not limited as long as it can cleave the mutated region.
- the DNA amplified fragment is purified by a spin column method, a glass bead adsorption method, or the like, and the DNA base sequence of the amplified portion is read by a sequencer, so that the DNA base sequence of the present invention is obtained. It is possible to identify individuals by detecting single nucleotide deficiency contained in and developing SNP (single nucleotide polymorphism) markers based on this information.
- SNP single nucleotide polymorphism
- the dose of the heavy ion beam for producing the cadmium absorption-suppressed rice mutant is not particularly limited as long as it does not damage the rice seeds and can induce the mutation, but if it is a carbon ion beam that can be induced at a high frequency, The range of 20 to 60 Gray is preferable.
- first generation seed hereinafter abbreviated as M1; the same applies to the second generation and beyond
- M1 first generation seed
- the obtained M2 seed is a low Cd mutant.
- M2 seeds There are the following two methods for selecting low Cd mutants using M2 seeds.
- next generation seeds can be secured by growing new shoots (hikobae) that emerge from the cut stumps. Furthermore, by allowing the seeds (M3 seeds) to self-grow, low Cd-absorbing rice having advanced generations such as M4 and M5 can be obtained.
- Low Cd-absorbing rice (M3 seed) can be obtained by analyzing the Cd concentration of brown rice and selecting individuals with low brown rice Cd concentration. Furthermore, the point which can obtain the low Cd absorption rice which generations, such as M4 and M5 advanced, can be obtained by self-propagating the seed is the same as that of the said hydroponics.
- Examples of a method for obtaining a gene related to the present invention include a hybridization technique and a polymerase chain reaction (PCR) technique.
- the former prepares, for example, a probe that specifically hybridizes with the nucleotide sequence of the gene of the present invention or a part thereof, and screens a genomic DNA library or a cDNA library.
- the latter uses, for example, the well-known Nipponbare sequence information to design a primer set (5 ′ side and 3 ′ side) for amplifying the gene in the present invention by PCR, perform PCR using cDNA as a template,
- a gene related to the present invention can be obtained by amplifying a DNA region sandwiched between both primers.
- the protein according to the present invention can determine the base sequence of the gene isolated above by a cycle sequencing method, and convert the base sequence into an amino acid sequence according to a codon.
- the amino acid sequence can be collated with the rice genome database (RAP-DB) to identify the protein.
- the production method includes the following procedure. 1) Crossing low Cd-absorbing rice (A plant) with existing rice varieties (B plant) to produce F1 2) Crossing F1 and B plant 3) Having low Cd gene from plant after crossing Select individuals with genetic markers
- the low Cd gene (for example, treca-1 gene or treca-2 gene) possessed by the A plant is intentionally introduced into the B plant by “backcrossing” in which the B plant is crossed. 5) At that time, genetic markers can be used to select only individuals having a low Cd gene from a large number of backcross individuals.
- the gene marker of the present invention can distinguish an individual having a low Cd gene from other individuals.
- genomic DNA extracted from the leaves and roots at the seedling stage may be used.
- genomic DNA extracted from the leaves and roots at the seedling stage may be used.
- 7) By repeating hybridization to the B plant and selecting only individuals with low Cd genes with markers, the genome structure is mostly B plants, but only Cd absorption is a new variety with the A plant genetic traits. (For example, low Cd Akitakomachi) can be created.
- B plant may be japonica or indica.
- Example 1-Selection of rice mutants with low Cd absorption> Rice (variety Koshihikari) seeds irradiated with heavy ion beam (carbon ion, 320MeV, 40Gy) using TIARA of Japan Atomic Energy Agency, Takasaki Quantum Applied Research Institute
- M1 the second and third generations are referred to as M2, M3, etc.
- M2 the second and third generations are referred to as M2, M3, etc.
- M2 seeds were obtained from each individual under the customary cultivation management at the National Institute for Agricultural Environment Technology.
- About 100,000 grains of the obtained M2 seeds were all mixed and subjected to the following low C
- Seedlings 10 days after sowing were treated with hydroponic solution containing 0.1 ppm of Cd for 4 days. After the treatment, the stem part of each individual was cut at 2 cm above the plate, the conduit fluid coming out from the cut surface was soaked in absorbent cotton, and only the conduit fluid was collected by centrifugation (2,000 rpm, 1 minute). .
- the plate for collection was made by making a 2 mm diameter hole in the bottom of the 96-well PCR plate, filling with absorbent cotton, and then overlapping the plate with no holes. The conduit liquid was soaked in cotton and centrifuged (2,000 rpm, 1 minute) to recover the lower plate.
- the conduit fluid collected as described above was diluted 50-200 times with 0.1 M nitric acid, and the Cd concentration was measured with an atomic absorption photometer (trade name: spectrAA-220Z, manufactured by Agilent Technologies). Only individuals with a Cd concentration of 1/5 or less than the Cd concentration of the conduit fluid collected from Koshihikari were collected, and Hikobae (new shoots emerged from the stubble) were grown. In order to secure generation seeds (M3), the seeds were transplanted and cultured in culture soil.
- the selection method using the Cd concentration of conduit fluid as an index is a space-saving and simple method, but it cannot be evaluated whether an individual with a low brown rice Cd concentration has been selected. Therefore, for the purpose of selecting mutants with low brown rice Cd concentration, direct analysis of brown rice Cd concentration is good, but in order to cultivate many mutants in Cd contaminated fields until the ripening stage, it is necessary to Can only be implemented.
- Cd-contaminated soil soil Cd concentration of 0.1M hydrochloric acid extracted: 1.8 mg kg -1
- One individual was transplanted to each other (cm diameter, with a hole on the bottom surface), and 288 Koshihikari non-treated with heavy ion beams were transplanted in the same manner as a control.
- 40 pots were placed in plastic containers (new TO trays) and flooded during the booting period. After that, Cd contained in the soil was eluted by dropping water, and tap water was continuously given until the ripening period to such an extent that the soil did not dry.
- Fertilization gave each pot 0.05gN / pot (300g soil) at the maximum tillering stage.
- Harvesting was carried out for each individual, and M3 generation brown rice was obtained through the processes of drying, threshing and rice bran.
- the obtained M3 brown rice is completely decomposed with nitric acid-perchloric acid, and the decomposed solution is appropriately diluted with mill-Q water, and the Cd concentration in the diluted solution is determined by an inductively coupled plasma mass spectrometer (manufactured by PerkinElmer, Inc. Name: ELAN DRC-e).
- Example 2-Confirmation of stability of low Cd mutation trait> (Evaluation by hydroponics) M3 lines of low Cd mutants (# 3-6-4, # 7-3-6, # 7-2-13) selected using brown rice Cd concentration as an index and Koshihikari without heavy ion beam treatment (hereinafter simply referred to as Koshihikari) The seedlings that had passed 10 days as in Example 1 were treated with Cd by hydroponics. After 4 days from the Cd treatment, the plants were harvested and dried by dividing them into stems and leaves and roots. The dried product was decomposed with a strong acid in the same manner as the brown rice of Example 3.
- the decomposition solution contains manganese (Mn), copper (Cu), iron (Fe), and zinc (Zn), which are essential elements for plants. All these elements are inductively coupled plasma. Simultaneous measurement was performed with an emission spectrometer (ICP-OES) (manufactured by Agilent Technologies, trade name: Vista-Pro). The results are shown in Table 2.
- ICP-OES emission spectrometer
- the Cd concentration in the mutant line was about 1/7 compared to Koshihikari.
- the root Cd concentration was about 1/4 of Koshihikari in all mutant strains. This indicates that the cause of low Cd in all mutant strains is due to low root Cd absorption.
- the mutant strains were characterized by extremely low manganese concentrations in both the foliage and roots, and the same phenomenon was observed in all mutant strains.
- a reverse transcription reaction was performed from the extracted total RNA (400 ng) using T7promoter®primer (Agilent) to synthesize cDNA.
- T7promoter®primer Agilent
- Cy3 Cyanine3
- Cyanine5 hereinafter referred to as Cy5
- labeled cRNA was synthesized by reacting in Transcription® Mix solution (Agilent) containing T7 RNA polymerase.
- the labeled cRNA extracted and synthesized from each individual was purified with Qiagen®RNeasy® Kit (manufactured by Qiagen), and then the RNA concentration and quality were checked using the Agilent Bioanalyzer 2100. Cy3 and Cy5 labeled cRNA were mixed in a cRNA target solution, and fragmentation buffer (Agilent) was added for target fragmentation and incubated at 60 ° C. for 30 minutes.
- fragmentation buffer Agilent
- the solution was dropped onto the probe surface on which 43,803 synthetic oligonucleosides of rice oligo DNA microarray 4 ⁇ 44 RAP-DB (Agilent) were placed, covered with a cover glass, and then in a thermostatic chamber (60 ° C., 17 hours) , 10 rpm).
- the low Cd mutant (# 3-6-4) selected according to Example 2 was considered to have a mutation in the transporter that carries cadmium and manganese into cells. Therefore, the results of the above microarray experiments have shown that Arabidopsis thaliana (Thomine et al., 2000, PNAS, 97, 4991-4996) and yeast (Cohen et al., 2000, J. Biol. Chem., 275, 33388- 33394), there was a report example, and we paid particular attention to the gene expression of the Nramp family, which has been identified as a transporter of divalent cations (Fe 2+ , Mn 2+, etc.).
- the rice Nramp family has been found from OsNramp1 to OsNramp7 (Takahishi et al., 2011, J. Exp. Bot., 62,4843-4850), in which the gene expression of OsNramp5 (Os07g0257200) is There was a 2.5-fold difference in expression between the mutant and Koshihikari, and there was no significant difference in expression in other Nramp families (1.0 to 1.7-fold difference).
- the genotypes of 92 individuals were examined using 97 microsatellite (SSR) markers.
- SSR microsatellite
- the linkage map was constructed with MAPMAKER / EXP / ver. 3.0 software.
- Gene mapping was performed with software (QTL Cartographer ver.2.5) based on Cd concentration and genotype data of 92 individuals.
- primer sets CNPorf5 (5′-CAC CAT GGA GAT TGA GAG AGA GAG CAG TG-3 ′: SEQ ID NO: 9) and CNPrt3 (5′-ACA CCC TTG TCG ATC GAT CGA TCT G-3 ′: SEQ ID NO: 10) (manufactured by Operon) was used for PCR, and the amplified fragment containing the full-length ORF was cloned into the pENTR / D-TOPO vector (manufactured by Invitrogen).
- genomic DNA was extracted according to the method of Xuet et al. (2005, “Plant Molecular Molecular” Reporter 23, 291-295).
- the primer which inserts the region where the mutation is recognized in ORF sequence is designed [CNP_GcheckFW (5′-GCA AGT CGA GTG CGA TCG TG-3 ′ : SEQ ID NO: 13) ⁇ CNP_GcheckRV (5'- CGC CGA TGA TGG AGA CGA TG-3 ′: SEQ ID NO: 14)], genomic DNA was amplified by PCR, and the base sequence was determined by direct sequence analysis.
- the length of the Koshihikari / Nipponbare ORF was 1617 bp.
- the ORF of # 7-3-6 a single base deletion (cytosine deletion) was observed at position 915 in the sequence of Koshihikari / Nipponbare.
- the ORF of # 3-6-4 changed the insertion of the ORF of Koshihikari / Nipponbare from 1025 to 1056, 32bp into 50bp, and the ORF length was 1635bp.
- SEQ ID NO: 1 shows the amino acid sequence of Koshihikari / Nipponbare. # 7-3-6 has a single base deletion, which causes a frame shift (shift in reading frame), greatly changes the 306th and subsequent amino acids, and a stop codon appears before Koshihikari. Translation stopped at 358th.
- the amino acid sequence of the mutant (# 7-3-6) is shown in SEQ ID NO: 6.
- # 3-6-4 had base insertion, so 11 amino acids (TGTYAGQYIMQ) were replaced with 17 amino acids (RPVTMGVSLVCHAHLIG) from the 341st to 352rd positions. Even if there was insertion, the reading frame did not shift, and the amino acids after that were exactly the same as Koshihikari.
- the amino acid sequence of the mutant (# 3-6-4) is shown in SEQ ID NO: 5.
- the base deletion and insertion position on the genomic DNA of # 7-3-6 and # 3-6-4 are schematically shown in FIG. # 7-3-6 was deficient in the 138th cytosine (C) in the 9th exon.
- a 433 bp base was inserted after the 73rd adenine (A) in the 10th exon.
- A adenine
- 50 bp were inserted into the exon and the remaining 383 bp were inserted into the intron.
- the inserted 433 bp was a transposon (translocation factor) called mPingA1.
- the protein encoded by the OsNramp5 gene regulates Cd absorption
- the Koshihikari-derived OsNramp5 gene was named TRECA (Transporter regulating cadmium absorption) gene
- its mutant type was treca -1 (derived from # 3-6-4) gene, treca-2 (derived from # 7-3-6) gene, and treca-3 (derived from # 7-2-13) gene.
- Example 4-Functional analysis using yeast> Primer sets CNPorf5 (5′-CAC CAT GGA GAT TGA GAG AGA GAG CAG TG-3 ′: SEQ ID NO: 9) and CNPrt3 (5′-) using Koshihikari and its mutant (# 3-6-4) cDNA as templates PCR was performed using ACA CCC TTG TCG ATC GAT CGA TCT G-3 ′ (SEQ ID NO: 10) (manufactured by Operon), and the amplified fragment containing the full-length ORF was cloned into pENTR / D-TOPO.
- each of the multiple cloning sites was inserted into a yeast expression vector pDR195 (Rentsch et al., 1995), which had been gate-activated by an LR reaction.
- pDR195 vector control
- lithium acetate (1) Cd sensitive mutant ⁇ ycf1 (MATalpha trp1-63 leu2-3, 112 gcn4-101 his3-609 ura3-52 ycf1 :: TRP1), (2) Mn Sex mutant ⁇ smf1 (MATa, his3 ⁇ 1; leu2 ⁇ 0; met15 ⁇ 0; ura3 ⁇ 0; YOL122c :: kanMX4) and (3) Fe Requirement mutant ⁇ fet3fet4 (MATa / MATalpha ade2 / + can1 / can1 his2 / t3rp2 / ura3 fet3-2 :: His3 / fet3-2 :: HIS3 fet4-1
- FIG. 7 shows the difference in heavy metal transport ability of the proteins encoded by the two genes based on the degree of growth of the mutant yeast strain.
- the ⁇ ycf1 strain into which the treca-1 gene was introduced was deficient in Cd absorption capacity, so that the influence of Cd toxicity was small and the growth was almost the same as VC.
- the treca-1 gene-introduced strain grew as much as VC. This seems to be because Mn absorption was restored by the introduction of TRECA gene.
- ⁇ fet3fet4 is a yeast strain deficient in iron transport ability.
- the TRECA transgenic yeast strain showed higher growth than VC.
- the treca-1 gene-introduced strain had the same degree of growth as VC. From this, the TRECA protein derived from Koshihikari has a function to transport Cd, Mn and Fe, and the treca-1 protein derived from # 3-6-4 has lost the transport activity to Cd, Mn and Fe. Became clear.
- the amplified fragment was cloned into pENTR / D-TOPO (manufactured by Invtrogen) to obtain each entry clone.
- LR clonase Each ORF was inserted into pH7FWG2,0 (Karimi et al., 2002) by LR reaction using II (manufactured by Invtrogen).
- the prepared construct was introduced into onion epidermal cells by the particle bombardment method and allowed to stand in the dark. Five to six hours later, fluorescence was observed using an LSM5 Pascal laser-scanning confocal microscope (Carl Zeiss). The results are shown in FIG.
- the fusion protein with ⁇ ⁇ GFP linked to the C terminal side of TRECA protein from Koshihikari and treca-1 protein from # 3-6-4 is expressed in onion epidermis cells, and the green fluorescence of GFP is observed. Then, the intracellular localization of TRECA protein and treca-1 protein was examined. All proteins were localized in the cell membrane. In addition, there was no significant difference in the fluorescence intensity between the two.
- Example 6 Method of discriminating low Cd rice using DNA marker> About the mutant (# 3-5-20), mutant (# 3-6-4), Koshihikari, and mutant (# 3-6-4) and Koshihikari F1 individuals, genome from their roots or leaves DNA was extracted and the concentration was measured with a spectrophotometer (trade name: NanoDrop 1000, manufactured by Thermo Fisher Scientific).
- a primer set [Os7g2572_F3711g (5'-TTC AGA ACG TGC TGG GCA AGT CG-3 ': SEQ ID NO: 11), Os7g2572_R3951g (5'-ACG GAT TAA CAA ATT AAT TATGTG GCA G-3 ′: SEQ ID NO: 12)] was designed. DNA fragments were amplified by PCR using genomic DNA as a template using KAPA2G Fast PCR Kit (manufactured by KAPA BIOSYSTEMS). The obtained PCR product was placed on a 3% agarose gel and subjected to electrophoresis. The results are shown in FIG.
- Koshihikari showed an amplified fragment of DNA at 240 bp, but # 3-5-20 and # 3-6-4 showed an amplified fragment of DNA around 700 bp.
- the body can now be distinguished.
- genomic DNA was extracted in the same manner as described above, Design a primer set [Os7g2572_F2976g (5'- TATATTCAGCCTGGGCAGATCGAG -3 ': SEQ ID NO: 7), Os7g2572_R3815g (5'- TGATGTACTGTCCAGCGTATGTGC -3': SEQ ID NO: 8) using KAPA2G Fast was amplified.
- the obtained PCR product was cleaved with the restriction enzyme FastDigest FspI (manufactured by Thermo Scientific).
- M is a size marker (Size marker)
- LK2 is # 7-3-6
- WT is Koshihikari
- F1 is # 7-3-6 ⁇ Koshihikari (# 7-3-6 x Koshihikari).
- an innovative low Cd absorption variety with high practicality is provided. Furthermore, by using the low Cd-absorbing variety of the present invention as a parent, a new low Cd-absorbing variety can be bred without performing genetic recombination, and by using the DNA marker of the present invention, it can be efficiently produced. Low Cd absorption individuals can be selected.
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Abstract
Description
<1> 本発明は、下記(A)乃至(C)のいずれかの塩基配列を含むカドミウム吸収の制御に関与するトランスポータータンパク質をコードする遺伝子である。
(A)配列番号2で表されるDNA塩基配列。
(B)配列番号2に記載の塩基配列において1若しくは複数の塩基が欠失、置換または付加されている塩基配列であって、カドミウム吸収を制御するタンパク質をコードする遺伝子のDNA塩基配列。
(C)配列番号2に記載の塩基配列とストリンジェントな条件下でハイブリダイズし、且つカドミウム吸収を制御するタンパク質をコードする遺伝子のDNA塩基配列。
<2> さらに本発明は、下記(D)乃至(F)のいずれかの塩基配列を含むカドミウム吸収の制御に関与するトランスポータータンパク質をコードする遺伝子である。
(D)配列番号3で表されるDNA塩基配列。
(E)配列番号3に記載の塩基配列において1若しくは複数の塩基が欠失、置換または付加されている塩基配列であって、カドミウム吸収を制御するタンパク質をコードする遺伝子のDNA塩基配列。
(F)配列番号3に記載の塩基配列とストリンジェントな条件下でハイブリダイズし、且つカドミウム吸収を制御するタンパク質をコードする遺伝子のDNA塩基配列。
<3> さらに本発明は、下記(G)乃至(I)のいずれかの塩基配列を含むカドミウム吸収の制御に関与するトランスポータータンパク質をコードする遺伝子である。
(G)配列番号4で表されるDNA塩基配列。
(H)配列番号4に記載の塩基配列において1若しくは複数の塩基が欠失、置換または付加されている塩基配列であって、カドミウム吸収を制御するタンパク質をコードする遺伝子のDNA塩基配列。
(I)配列番号4に記載の塩基配列とストリンジェントな条件下でハイブリダイズし、且つカドミウム吸収を制御するタンパク質をコードする遺伝子のDNA塩基配列。
<4> さらに本発明は、下記(J)乃至(L)のいずれかのアミノ酸配列を含むカドミウム吸収の制御に関与するトランスポータータンパク質である。
(J)配列番号1に記載のアミノ酸配列。
(K)配列番号1に記載のアミノ酸配列において1若しくは複数のアミノ酸が欠失、置換または付加されているアミノ酸配列であって、カドミウム吸収を制御するタンパク質のアミノ酸配列。
(L)配列番号1に記載のアミノ酸配列と少なくとも90%以上の相同性を有するアミノ酸配列であって、カドミウム吸収を制御するタンパク質のアミノ酸配列。
<5> さらに本発明は、下記(M)乃至(O)のいずれかのアミノ酸配列を含むカドミウム吸収の制御に関与するトランスポータータンパク質である。
(M)配列番号5に記載のアミノ酸配列。
(N)配列番号5に記載のアミノ酸配列において1若しくは複数のアミノ酸が欠失、置換または付加されているアミノ酸配列であって、カドミウム吸収を制御するタンパク質のアミノ酸配列。
(O)配列番号5に記載のアミノ酸配列と少なくとも90%以上の相同性を有するアミノ酸配列であって、カドミウム吸収を制御するタンパク質のアミノ酸配列。
<6> さらに本発明は、下記(P)乃至(R)のいずれかのアミノ酸配列を含むカドミウム吸収の制御に関与するトランスポータータンパク質である。
(P)配列番号6に記載のアミノ酸配列。
(Q)配列番号6に記載のアミノ酸配列において1若しくは複数のアミノ酸が欠失、置換または付加されているアミノ酸配列であって、カドミウム吸収を制御するタンパク質のアミノ酸配列。
(R)配列番号6に記載のアミノ酸配列と少なくとも90%以上の相同性を有するアミノ酸配列であって、カドミウム吸収を制御するタンパク質のアミノ酸配列。
<8> さらに本発明は、前記<2>又は<3>に記載のDNAを含む形質転換体又は請求項1に記載のDNAを欠失した形質転換体である。
<9> さらに本発明は、前記<7>に記載の組み換えベクターを用いた形質転換体である。
<10> さらに本発明は、前記<1>乃至<3>のいずれかに記載のDNA塩基配列を識別できる遺伝子マーカーである。
<11> さらに本発明は、前記<4>に記載されたタンパク質の機能が抑制されるカドミウム吸収抑制イネである。
<12>さらに本発明は、前記<2>又は<3>に記載された遺伝子がコードするタンパク質が発現するカドミウム吸収抑制イネである
<13> さらに本発明は、イネ品種コシヒカリと出穂、収量、食味が同程度である、前記<11>又は<12>に記載のカドミウム吸収抑制イネである。
<14> さらに本発明は、イネ品種コシヒカリと比べて、出穂が約2週間程度早い、前記<11>又は<12>に記載のカドミウム吸収抑制イネである。
<15> さらに本発明は、下記(イ)と(ロ)の工程、又は(イ)と(ハ)の工程からなる前記<11>乃至<14>のいずれかに記載のカドミウム吸収抑制イネ変異体の選抜方法である。
(イ)重イオンビームを照射した第1世代種子を圃場に栽培し、第2世代種子を採種する工程、
(ロ)前記(イ)の工程で得られた第2世代種子を、Cd含有水耕液で栽培し、茎葉を切除して分泌される導管液を採取し、その液に含まれるCd濃度によって、Cd吸収抑制イネ変異体を選抜するとともに第3世代種子を採種する工程、
(ハ)前記(イ)の工程で得られた第2世代種子の幼植物個体をCd汚染土壌で栽培し、第3世代種子のCd濃度によってカドミウム吸収抑制イネ変異体を選抜する工程、
<16> さらに本発明は、前記<11>乃至<14>のいずれかに記載のカドミウム吸収抑制イネ変異体と既存のイネ品種との交配により得られるカドミウム吸収抑制イネである。
本発明のTRECA遺伝子は、配列番号1に示されるカドミウム吸収の制御に関与するトランスポータータンパク質をコードする、配列番号2に示される遺伝子である。配列番号2に示す塩基配列は、前記重イオンビームを照射して得られたCd吸収抑制イネの遺伝子を解析することにより特定されたものである。本発明は、TRECA遺伝子に由来する重金属トランスポーター遺伝子の開始コドンから終始コドンまでの、いわゆるオープンリーディングフレーム(以下ORFといる。)である。
本発明に係るTRECAタンパク質は、TRECA遺伝子によってコードされ、細胞膜上に存在し、カドミウム及びマンガンの吸収を制御するタンパク質である。後記の本発明のTRECAタンパク質をコードする遺伝子を全部欠失している系統(#7-2-13)や一部変異が生じた系統(#3-6-4や#7-3-6等)において、カドミウム及びマンガンの吸収が大幅に抑制されることから、該タンパク質がカドミウム及びマンガンの吸収を制御することが明らかである。また、酵母にTRECA遺伝子を導入した系ではTRECAタンパク質は鉄の輸送活性を示し、変異型のtrecaタンパク質を持つ酵母では鉄の輸送活性を示さないことから、TRECAタンパク質は鉄の吸収にも関与する。しかし、上述の欠失系統や変異系統は鉄の濃度に変化がない(表2)ことから、これまで報告されている鉄吸収関連タンパク質であるIRTやZIPファミリー(Bughio N. et al. (2002) Journal of Experimental Botany. 53:1677-1682)に比べ、鉄吸収能力が低いと予想される。
本発明は、配列番号3に示されるTRECA遺伝子の変異遺伝子(以下treca-1遺伝子という。)である。配列番号3に示す塩基配列は、前記重イオンビームを照射して得られたCd吸収抑制イネ#3-6-4に由来する遺伝子であり、前記配列番号2に示すTRECA遺伝子においてcDNAの第10エキソンの末端部位、塩基番号1025から1056番目の32bpに置き換わって 50bpとなったものである。
本発明は、配列番号4に示されるTRECA遺伝子の変異遺伝子(以下treca-2遺伝子という。)である。配列番号4に示す塩基配列は、前記重イオンビームを照射して得られたCd吸収抑制イネ#7-3-6に由来する遺伝子であり、前記配列番号2に示すTRECA遺伝子(cDNA)における塩基番号915番目に一塩基欠損(シトシンの欠損)がみらる。
本発明による組換えベクターは、前記TRECA遺伝子、変異型のtreca-1遺伝子又はtreca-2遺伝子のいずれかが組み込まれたものである。前記ベクターは、公知の形質転換方法によって目的植物に発現可能に導入されることにより、当該植物において組み込まれた遺伝子あるいは遺伝子断片を発現させて本発明に係るタンパク質を得ることが出来るものである。
本発明の遺伝子を発現する形質転換体を作製する場合には、前記遺伝子が挿入された前記ベクターを目的とする植物細胞に導入する。該植物細胞へのベクターの導入は、公知の方法を用いることが可能であり、例えば、アグロバクテリウム法、エレクトロポレーション法、パーティクルガン法、マイクロインジェクション法等が用いられるが、中でもアグロバクテリウム法が最も好ましい。
本発明における遺伝子マーカーは、前記TRECA遺伝子と変異型treca遺伝子との塩基配列の違いから個体を識別するものである。
イネ種子に重イオンビームを照射する。カドミウム吸収抑制イネ変異体を作出する重イオンビームの線量としては、イネ種子に損傷を与えず、変異を誘発できる範囲内であれば特に限定されないが、高頻度で誘発できる炭素イオンビームであれば、20~60Grayの範囲が好ましい。
本発明に関わる遺伝子を獲得する方法として、ハイブリダイゼーション技術やポリメラーゼ連鎖反応(PCR)技術が挙げられる。前者は、例えば本発明における遺伝子の塩基配列もしくはその一部と特異的にハイブリダイズするプローブを調製し、ゲノムDNAライブラリーやcDNAライブラリーをスクリーニングする。後者は、例えば公知の日本晴の配列情報を用いて、本発明における遺伝子をPCR法によって増幅させるためのプライマーセット(5’側と3’側)を設計し、cDNAを鋳型にしてPCRを行い、両プライマーに挟まれるDNA領域を増幅させることで、本発明に関わる遺伝子を取得することができる。
本発明に関わるタンパク質は、上記で単離した遺伝子の塩基配列をサイクルシークエンス法によって決定し、塩基配列をコドンに従ってアミノ酸配列に変換することができる。そのアミノ酸配列をイネゲノムデータベース(RAP-DB)で照合し、タンパク質を同定することが可能である。
本発明によって得られたDNAを有する低Cd吸収イネ変異体と既存の品種を交配し、本発明による遺伝子マーカーを利用することで、効率良く新たな低Cd吸収イネ品種を作出することが可能である。
該作出方法として、以下のような手順がある。
1)低Cd吸収イネ(A植物)と既存のイネ品種(B植物)を交配し、F1を作出する
2)前記F1と前記B植物を交雑させる
3)交雑後の植物から低Cd遺伝子を有する個体を遺伝子マーカーで選抜する
5)その際、多数の戻し交雑個体の中から、低Cd遺伝子を有する個体のみを選抜するため、遺伝子マーカーを活用することができる。本発明の遺伝子マーカーは、低Cd遺伝子を有する個体とそれ以外の個体を識別できる。
7)B植物に交雑を繰り返し、低Cd遺伝子が入った個体のみをマーカーで選抜することで、ゲノム構造のほとんどがB植物であるが、Cd吸収のみA植物の遺伝形質の入った新たな品種(例えば、低Cdのあきたこまち)が作出できる。
8)B植物はジャポニカ種でもインディカ種でもかまわない。
<実施例1-Cd吸収の少ないイネ変異体の選抜>
(重イオンビーム照射)
独立行政法人日本原子力研究開発機構 高崎量子応用研究所のTIARAを用いて、重イオンビーム照射(炭素イオン、320MeV, 40Gy)したイネ(品種コシヒカリ(Koshihikari))種子(本種子が第1世代種子で、以下M1と略記し、第2、第3世代等をM2、M3等という。)3500粒を、培養土(住友化学社製、商品名:ボンソル1号)に播種し、得られた幼苗を(独)農業環境技術研究所が所有する水田圃場に一個体づつ移植し、(独)農業環境技術研究所における慣行の栽培管理で各個体からM2種子を得た。得られたM2種子約100,000粒をすべて混合し、以下の低Cd変異体の選抜工程に供試した。
M2の催芽種子を、底面に3mm口径の穴を開けた96穴のPCRプレートに播種し、スタイロホームで作成した浮遊台にプレートを乗せ、木村B水耕液(1/2濃度)の入った20L容コンテナ容器に置いた。
導管液のCd濃度を指標にした選抜法は省スペースで、簡便に実施できる方法である一方、玄米Cd濃度の低い個体が選ばれたかどうかについては評価できない。それゆえ、玄米Cd濃度の低い変異体を選抜する目的では、玄米Cd濃度を直接分析する方法がよいが、多数の変異体を登熟期までCd汚染圃場で栽培するには、特定の場所でしか実施できない。ここでは、少量のCd汚染土壌と温室等の比較的省スペースがあれば、多数の変異体を栽培でき、玄米Cd濃度によって低Cd変異体を選抜できる方法を考案した。
(水耕栽培での評価)
玄米Cd濃度を指標に選抜された低Cd変異体(#3-6-4, #7-3-6, #7-2-13)のM3系統と重イオンビーム無処理のコシヒカリ(以下単にコシヒカリという。)の種子を播種し、実施例1と同様10日経過した苗を水耕法でCd処理した。Cd処理してから4日間経過後、収穫して、茎葉部と根部に分けて乾燥した。該乾燥物を実施例3の玄米と同様の方法で強酸で分解した。分解液中にはCdの他、植物にとっては必須元素であるマンガン(Mn)、銅(Cu)、鉄(Fe)、亜鉛(Zn)含量も含まれており、これら全ての元素は誘導結合プラズマ発光分析装置(ICP-OES)(アジレント・テクノロジー社製、商品名;Vista-Pro)にて、同時に測定した。結果を表2に示す。
実施例1の玄米Cd濃度で選抜された低Cd変異体(#3-6-4, #7-3-6, #7-2-13)の世代の進んだM4系統とコシヒカリをCd汚染圃場(Cd濃度1.8mg kg-1)で栽培した。水管理は水稲栽培の慣行法に従い、中干しと間断灌漑で行った。施肥は元肥として5kg-N/10a, 8kg-P2O5/10a, 8kg-K2O/10aを与え、穂肥として2kg-N/10aと施用した。登熟期の玄米を収穫し、強酸による分解後、溶液中に含まれるCdをICP-MS(パーキンエルマー社製、商品名;ELAN DRC-e)で、他の重金属をICP-OES(アジレント・テクノロジー社製、商品名;Vista-Pro)で測定した。結果を図1に示し、栽培時の生育状況を図2に示した。
(マイクロアレイ実験)
前記変異体(#3-6-4)とコシヒカリの根から、RNA精製キット(株式会社リーゾ製、商品名:RNAすいすい-S)のプロトコールに従ってRNAを抽出した。RNA濃度を分光光度計(Thermo Fisher Scientific製、商品名:ナノドロップNanoDrop 1000)で、抽出されたRNAが分解されていないかどうか、その品質をAgilent社のバイオアナライザー2100でチェックした。
低Cd変異体(#3-6-4)が持つ低Cd吸収遺伝子の同定のため、遺伝子マッピングを行った。#3-6-4とインディカ品種であるカサラス(Kasalath)の交配から得られたF2種子を播種し、92個体の幼苗を0.02ppmのCd濃度が入った木村B水耕液(1/2濃度)で4日間栽培し、各個体の茎葉Cd濃度を測定した。結果を図3に、Cd濃度を4mgkg-1毎にクラス分けして示した。
前記(マイクロアレイ実験と遺伝子マッピング)の結果から、OsNramp5にターゲットを絞り、変異の挿入の有無を確認した。変異体(#3-6-4、# 7-3-6、#7-2-13)とコシヒカリの根からRNAを前記RNA精製キット(RNAすいすい-S )で抽出し、その後、逆転写酵素(TOYOBO 社製、商品名:ReverTra Ace)により、1本鎖cDNAを合成した。そのcDNA を鋳型として,プライマーセット CNPorf5 (5′-CAC CAT GGA GAT TGA GAG AGA GAG CAG TG-3′:配列番号9)および CNPrt3 (5′-ACA CCC TTG TCG ATC GAT CGA TCT G-3′:配列番号10) (オペロン社製)を用いて PCR を行い,全長 ORF を含む増幅断片をpENTR/D-TOPOベクター(Invitrogen社製)にクローニングした。本ベクターに含まれるUniversal M13 シークエンシングサイト[M13 Forward (-20)(5′-GTA AAA CGA CGG CCA G-3′:配列番号11)、M13 Reverse(5′-CAG GAA ACA GCT ATG AC-3′:配列番号12)]およびTRECA遺伝子特異的プライマー[CNP_GcheckFW(5′-GCA AGT CGA GTG CGA TCG TG-3′:配列番号13)、CNP_GcheckRV(5’- CGC CGA TGA TGG AGA CGA TG-3’ :配列番号14)]を用いて、pENTR/D-TOPOベクター(Invitrogen社製)にクローニングされたTRECA遺伝子の塩基配列をDNAシークエンサABI3130xl(Applied Biosystems社製)で決定した。
コシヒカリおよびその変異体 (#3-6-4) の cDNA を鋳型として,プライマーセット CNPorf5 (5′-CAC CAT GGA GAT TGA GAG AGA GAG CAG TG-3′:配列番号9)および CNPrt3 (5′-ACA CCC TTG TCG ATC GAT CGA TCT G-3′:配列番号10) (オペロン社製)を用いて PCR を行い,全長 ORF を含む増幅断片をpENTR/D-TOPO にクローニングした。
コシヒカリおよびその変異体 (#3-6-4) の cDNA を鋳型として,プライマーセット CNPorf5 (5′-CAC CAT GGA GAT TGA GAG AGA GAG CAG TG-3′:配列番号9)および CNPorf3 (5′- CCT TGG GAG CGG GAT GTC GGC CAG G-3′:配列番号10) (オペロン社製)を用いて、 PCR を行い ORF 領域を増幅した。
前記変異体(#3-5-20)、変異体( #3-6-4)、コシヒカリ、及び変異体(#3-6-4)とコシヒカリのF1個体について、それらの根または葉からゲノムDNAを抽出し、分光光度計(Thermo Fisher Scientific社製、商品名:ナノドロップNanoDrop 1000)で濃度を測定した。実施例3の塩基配列データを基に、変異が挿入された箇所を挟み込むプライマーセット[Os7g2572_F3711g(5’-TTC AGA ACG TGC TGG GCA AGT CG-3’ :配列番号11)、Os7g2572_R3951g(5’-ACG GAT TAA CAA ATT AAT TATGTG GCA G-3’ :配列番号12)] を設計した。KAPA2G Fast PCR Kit(KAPA BIOSYSTEMS社製)を使って、ゲノムDNAを鋳型としてPCRによるDNA断片の増幅を行った。得られたPCR産物を3%アガロースゲル上に乗せ、電気泳動を行った。結果を図9に示した。
Claims (16)
- 下記(A)乃至(C)のいずれかの塩基配列を含むカドミウム吸収の制御に関与するトランスポータータンパク質をコードする遺伝子。
(A)配列番号2で表されるDNA塩基配列。
(B)配列番号2に記載の塩基配列において1若しくは複数の塩基が欠失、置換または付加されている塩基配列であって、カドミウム吸収を制御するタンパク質をコードする遺伝子のDNA塩基配列。
(C)配列番号2に記載の塩基配列とストリンジェントな条件下でハイブリダイズし、且つカドミウム吸収を制御するタンパク質をコードする 遺伝子のDNA塩基配列。 - 下記(D)乃至(F)のいずれかの塩基配列を含むカドミウム吸収の制御に関与するトランスポータータンパク質をコードする遺伝子。
(D)配列番号3で表されるDNA塩基配列。
(E)配列番号3に記載の塩基配列において1若しくは複数の塩基が欠失、置換または付加されている塩基配列であって、カドミウム吸収を制御するタンパク質をコードする遺伝子のDNA塩基配列。
(F)配列番号3に記載の塩基配列とストリンジェントな条件下でハイブリダイズし、且つカドミウム吸収を制御するタンパク質をコードする遺伝子のDNA塩基配列。 - 下記(G)乃至(I)のいずれかの塩基配列を含むカドミウム吸収の制御に関与するトランスポータータンパク質をコードする遺伝子。
(G)配列番号4で表されるDNA塩基配列。
(H)配列番号4に記載の塩基配列において1若しくは複数の塩基が欠失、置換または付加されている塩基配列であって、カドミウム吸収を制御するタンパク質をコードする遺伝子のDNA塩基配列。
(I)配列番号4に記載の塩基配列とストリンジェントな条件下でハイブリダイズし、且つカドミウム吸収を制御するタンパク質をコードする遺伝子のDNA塩基配列。 - 下記(J)乃至(L)のいずれかのアミノ酸配列を含むカドミウム吸収の制御に関与するトランスポータータンパク質。
(J)配列番号1に記載のアミノ酸配列。
(K)配列番号1に記載のアミノ酸配列において1若しくは複数のアミノ酸が欠失、置換または付加されているアミノ酸配列であって、カドミウム吸収を制御するタンパク質のアミノ酸配列。
(L)配列番号1に記載のアミノ酸配列と少なくとも90%以上の相同性を有するアミノ酸配列であって、カドミウム吸収を制御するタンパク質のアミノ酸配列。 - 下記(M)乃至(O)のいずれかのアミノ酸配列を含むカドミウム吸収の制御に関与するトランスポータータンパク質。
(M)配列番号5に記載のアミノ酸配列。
(N)配列番号5に記載のアミノ酸配列において1若しくは複数のアミノ酸が欠失、置換または付加されているアミノ酸配列であって、カドミウム吸収を制御するタンパク質のアミノ酸配列。
(O)配列番号5に記載のアミノ酸配列と少なくとも90%以上の相同性を有するアミノ酸配列であって、カドミウム吸収を制御するタンパク質のアミノ酸配列。 - 下記(P)乃至(R)のいずれかのアミノ酸配列を含むカドミウム吸収を制御に関与するトランスポータータンパク質。
(P)配列番号6に記載のアミノ酸配列。
(Q)配列番号6に記載のアミノ酸配列において1若しくは複数のアミノ酸が欠失、置換または付加されているアミノ酸配列であって、カドミウム吸収を制御するタンパク質のアミノ酸配列。
(R)配列番号6に記載のアミノ酸配列と少なくとも90%以上の相同性を有するアミノ酸配列であって、カドミウム吸収を制御するタンパク質のアミノ酸配列。 - 前記請求項1乃至請求項3のいずれかに記載のDNAを含む組み換えベクター。
- 請求項2又は請求項3に記載のDNAを含む形質転換体、又は請求項1に記載のDNAを欠失した形質転換体。
- 前記請求項7に記載の組み換えベクターを用いた形質転換体。
- 前記請求項1乃至請求項3のいずれかに記載のDNAを含む遺伝子マーカー。
- 前記請求項4に記載されたタンパク質の発現が抑制されるカドミウム吸収抑制イネ。
- 前記請求項2又は請求項3に記載された遺伝子がコードするタンパク質が発現するカドミウム吸収抑制イネ。
- イネ品種コシヒカリと出穂、収量、食味が同程度である請求項11又は請求項12に記載のカドミウム吸収抑制イネ。
- イネ品種コシヒカリと比べて、出穂が約2週間程度早い請求項11又は請求項12に記載のカドミウム吸収抑制イネ。
- 下記(イ)と(ロ)の工程、又は(イ)と(ハ)の工程からなる請求項11乃至請求項14のいずれかに記載のカドミウム吸収抑制イネ変異体の選抜方法。
(イ)重イオンビームを照射した第1世代種子を圃場に栽培し、第2世代種子を採種する工程。
(ロ)前記(イ)の工程で得られた第2世代種子を、Cd含有水耕液で栽培し、茎葉を切除して分泌される導管液を採取し、その液に含まれるCd濃度によって、Cd吸収抑制イネ変異体を選抜するとともに第3世代種子を採種する工程。
(ハ)前記(イ)の工程で得られた第2世代種子の幼植物個体をCd汚染土壌で栽培し、第3世代種子のCd濃度によってカドミウム吸収抑制イネ変異体を選抜する工程。 - 前記請求項11乃至請求項14のいずれかに記載のカドミウム吸収抑制イネ変異体と既存のイネ品種との交配により得られるカドミウム吸収抑制イネ。
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CN109694876A (zh) * | 2017-10-24 | 2019-04-30 | 中国科学院植物研究所 | 培育低镉积累水稻的方法及其相关材料的用途 |
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Also Published As
Publication number | Publication date |
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US20140259233A1 (en) | 2014-09-11 |
JPWO2013065517A1 (ja) | 2015-04-02 |
CN103917646B (zh) | 2019-07-26 |
CN103917646A (zh) | 2014-07-09 |
JP5850475B2 (ja) | 2016-02-03 |
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