WO2005012520A1 - 植物の再分化能を付与する遺伝子、並びにその利用 - Google Patents
植物の再分化能を付与する遺伝子、並びにその利用 Download PDFInfo
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- WO2005012520A1 WO2005012520A1 PCT/JP2004/011307 JP2004011307W WO2005012520A1 WO 2005012520 A1 WO2005012520 A1 WO 2005012520A1 JP 2004011307 W JP2004011307 W JP 2004011307W WO 2005012520 A1 WO2005012520 A1 WO 2005012520A1
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
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- 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)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8209—Selection, visualisation of transformants, reporter constructs, e.g. antibiotic resistance markers
- C12N15/821—Non-antibiotic resistance markers, e.g. morphogenetic, metabolic markers
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
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- 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)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- 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)
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
- C12N9/0044—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on other nitrogen compounds as donors (1.7)
Definitions
- the present invention relates to the isolation and identification of a gene that confers plant regeneration ability, the increase in regeneration ability using the gene, and a method for selecting transformed cells. According to the present invention, it is possible to improve the culture characteristics of a plant and to develop a transformation method in consideration of safety. Background art
- the differentiated tissue of a plant under appropriate conditions, dedifferentiates and undergoes cell division to form callus (dedifferentiated cell group). Callus can also regenerate under certain conditions to regenerate whole plants.
- the ability of such differentiated or dedifferentiated cells to regenerate individuals is called totipotency, and was first demonstrated in culture studies of tobacco and tomato in the 1930s-1950s.
- Tissue culture technology is a technology based on this totipotency, and is widely used especially in the field of plant breeding, such as creating new varieties by cell fusion and ovule culture, shortening the breeding years, and fixing genetic traits. Have been. In recent years, it has become an indispensable technology for molecular breeding and basic plant research as a fundamental technology in artificial gene transfer (transformation) for the purpose of gene function analysis.
- totipotency is considered to be the ability of all plants to retain, but in fact, it is known that some plants can easily exert that ability depending on plant species, varieties, and organs, and others can hardly.
- monocots such as rice, wheat, and corn, which are the main crops, are more difficult to tissue culture and regenerate, so much more trial and error is required for analysis using transformation and other cultures. is there.
- rice relatively easy culture systems have been established by using mature seeds of specific varieties, but varieties with sufficient regeneration ability are limited. "Especially tasting varieties Koshihikari, Sasanishiki, tropical Varieties of IR lines cultivated in rural areas have low regeneration ability and it is difficult to regenerate plant matter by tissue culture. If the ability to regenerate these varieties can be improved, it will not only be useful for studying the characteristics of breeding genes, but also for elucidating the mechanism of the regeneration process. Improvement is also expected.
- An object of the present invention is to provide isolation and identification of a gene involved in the regeneration capability of a plant, a method for improving a plant using the gene, and a transformation method using the gene as a selection marker. It is in. '
- a power salas BAC library (average length 120 kb) was prepared, and a BAC clone (BHAL15) containing a 57 ⁇ region was isolated by PCR screening.
- a Kasalath genomic fragment containing each target gene region was prepared using the appropriate restriction enzyme sites in the BHAL15 clone, and introduced into Koshihikari.
- the Kasalath genome containing the gene (NiR) predicted to encode fredoxin nitrite reductase was obtained. Only when the fragment (3F in Fig. 3) was introduced, the regeneration ability of Koshihikari was found to increase (Fig.
- Ferredoxin Nitrite reductase is a nitrite reductase that functions with ferredoxin as an electron donor and has the effect of converting nitrite ions to ammonia.
- FIG. 5 the expression level of this gene mRNA in calli was examined by semi-quantitative RT-PCR and real-time quantitative PCR. It was found that about 2.5 times the amount of mRNA was present (Fig. 6, upper left, middle ', and right graphs).
- Kasalath / gene can be used as a selection marker when transforming Koshihikari.
- a vector in which the Kasalath social gene and the target gene are inserted in parallel is introduced into Koshihikari, only the cells into which the 5-7 gene has been introduced will acquire the regeneration capability. Is also expected to be introduced.
- the 7? Gene could be used as a marker in the transformation to a highly regenerative variety.
- a rice high expression promoter One of them is to introduce a vector that overexpresses the 7? Gene under the control of the actin promoter into a highly redifferentiated cultivar Kasalath. Cultured. Only the transformed cells grew due to the effect of the overexpressed gene, and GUS staining was observed only in the grown cells (FIG. 9).
- the present invention relates to the isolation and identification of a gene that increases the regeneration potential of a plant, and to the improvement of plant culture characteristics using the gene, and further to a transformation method using the gene as a selection marker.
- the following [1;] to [22] are provided.
- a transformed plant which is a progeny or a clone of the transformed plant according to [8].
- a method for producing a transformed plant comprising a step of introducing the DNA according to [1] or [2] into a plant cell and regenerating the plant from the plant cell.
- a polynucleotide comprising at least 15 contiguous bases complementary to the base sequence of SEQ ID NO: 1 or 2 or a complementary sequence thereof.
- a method for increasing the regeneration capability of a plant comprising the step of expressing the DNA according to [1] or [2] in cells of a plant.
- An agent for modifying a plant regeneration ability comprising the DNA of [1] or [2] or the vector of [4] as an active ingredient.
- a method for determining a regeneration ability in a plant cell comprising the steps of:
- a method comprising detecting the expression of the DNA of [1] or the protein of [12].
- a method for determining the regeneration ability of a plant cell comprising detecting the activity of the protein according to [12] in the plant cell.
- a method for improving the regeneration ability of a plant comprising controlling the activity of the protein according to [12], which is endogenous in the plant.
- [2 1] A method for selecting a transformed plant cell, comprising:
- a method for modifying the regeneration ability of a plant comprising replacing the endogenous DNA according to [1] or [2] in the plant by crossing.
- the present invention provides a DNA encoding a rice-derived NiR protein.
- the nucleotide sequence of the genomic DNA of "Power Salas” is shown in SEQ ID NO: 1
- the nucleotide sequence of the cDNA of "Kasalath” is shown in SEQ ID NO: 2
- the amino acid sequence of the protein encoded by the DNA is shown in SEQ ID NO: 3- Show.
- the nucleotide sequence of the genomic DNA of "Koshihikari” is shown in SEQ ID NO: 4, the nucleotide sequence of the cDNA of "Koshihikari” is shown in SEQ ID NO: 5, and the amino acid sequence of the protein encoded by the DNA is shown in SEQ ID NO: 6. Shown in
- the regenerative ability of a plant can be improved by controlling the expression or activity of the ⁇ 3 ⁇ 4 gene of the plant. This has enabled the cultivation of difficult-to-culture varieties such as Koshihikari, and has also enabled the production of high-regeneration varieties with stable and high regeneration potential.
- “improving the regeneration ability” means only increasing the regeneration ability of a plant under culture conditions, and does not cause a change in the morphology of a regenerated individual. By improving the regeneration ability, a desired variety can be subjected to various culture experiments, and as a result, a new variety can be efficiently developed and gene function analysis can be performed.
- plant / gene means a gene encoding a plant redoxin nitrite reducing enzyme.
- Plant social genes include rice genes (Fig. 5) and genes derived from other plants.
- PSR1 DNA encoding a protein includes genomic DNA, cDNA, and chemically synthesized DNA. Preparation of genomic DNA and cDNA can be performed by a person skilled in the art using conventional means. Genomic DNA can be obtained, for example, by extracting genomic DNA from rice varieties having the ⁇ gene (eg,“ Koshihikari J ”) and genomic libraries (plasmid, phage, cosmid, BAC, 'PAC Can be used) and expand it to produce DNA encoding the protein of the present invention.
- ⁇ gene eg,“ Koshihikari J ”
- genomic libraries plasmid, phage, cosmid, BAC, 'PAC Can be used
- cDNA for example, cDNA is synthesized based on mRNA extracted from a rice cultivar (eg, “Koshihikari”) having the gene, and this is inserted into a vector such as LZAP to create a cDNA library. Then, it can be prepared by developing this and performing colony hybridization or plaque hybridization in the same manner as described above, or by performing PCR.
- the present invention encompasses a DNA encoding a protein functionally equivalent to the PSR1 protein (“Power Salas”) ′ set forth in SEQ ID NO: 3.
- “has a function equivalent to that of the PSR1 protein” means that it has a function of increasing the regeneration capability by modifying the expression level or activity of the target protein.
- Such DNAs include, for example, mutants, derivatives, and the like, which encode proteins consisting of an amino acid sequence in which one or more amino acids have been substituted, deleted, added and / or inserted in the amino acid sequence of SEQ ID NO: 3. Includes alleles, variants and homologs.
- Methods well known to those skilled in the art for preparing DNA encoding a protein having an altered amino acid sequence include, for example, the site-directed mutagenesis method. (Kramer, W. & Fritz, H.-J. (1987) Oligonucleotide-directed construction of mutagenesis via gapped duplex DNA. Methods in Enzymology,-154: 350-367).
- mutation of the amino acid sequence of the encoded protein due to mutation of the base sequence can occur in nature.
- the DNA encodes a protein having an amino acid sequence in which one or more amino acids are substituted, deleted or added in the amino acid sequence encoding the natural PSR1 protein
- the natural PSR1 protein (SEQ ID NO: As long as it encodes a protein having the same function as in 3), it is included in the DNA of the present invention.
- the base sequence is mutated, it may not be accompanied by mutation of an amino acid in a protein (degenerate mutation). Such a degenerate mutant is also included in the DNA of the present invention. .
- the base sequence of the PSR1 gene (SEQ ID NO: 2) or a part thereof is used as a probe, and an oligonucleotide that specifically hybridizes to the R1 gene (SEQ ID NO: 2) is used as a primer. Isolation of DNA having high homology with the PSR1 gene from rice and other plants is normally possible.
- the DNA of the present invention also includes a DNA encoding a protein having a function equivalent to that of the PSR1 protein, which can be isolated by the hybridizing technique or the PCR technique.
- a hybridization reaction is preferably performed under stringent conditions.
- the stringent hybridization conditions refer to the conditions of 6M urea, 0.4% SDS, 0.5 ⁇ SSC or the same stringency hybridization conditions. More stringing High ency conditions, eg, 6M urea, 0.4 ° /.
- SDS and 0.1xSSC isolation of DNA with higher homology can be expected.
- the DNA isolated thereby is considered to have high homology at the amino acid level with the amino acid sequence of the PSR1 protein (SEQ ID NO: 3 or 6).
- High homology means that at least 50% or more, more preferably 70% 'or more, and even more preferably 90% or more (for example, 95 ° 96 ° 97 ° 98 ° 99% Above).
- the identity of the amino acid sequence and nucleotide sequence was determined by using the analytic BLAST (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990, Proc Natl. Acad Sci USA 90: 5873, 1993) by Carlin and Artil. Can be determined.
- a program called BI STN or BLASTX based on the BLAST algorithm has been developed.
- Whether or not a certain DNA encodes a protein involved in redifferentiation ability can be evaluated as follows.
- the most common method is a method in which cultivation is performed after deleting the function of the DNA, and the regeneration ability is examined.
- this is a method in which the cells are cultured under conditions where the function of the DNA is maintained and under conditions where the function of the DNA is deleted, and the regeneration ability is compared.
- the regeneration ability does not change or is almost the same, it is determined that the DNA does not participate in the regeneration ability.
- the regeneration rate is further increased, and the difference can be regarded as the degree of the regeneration ability.
- the DNA of the present invention can be used, for example, for the preparation of recombinant proteins and for the production of transformed plants with altered regeneration ability.
- the DNA encoding the protein of the present invention should usually be expressed appropriately.
- the vector is introduced into an appropriate cell, and the expressed protein is purified by culturing the transformed cell.
- the recombinant protein can be expressed as a fusion protein with another protein, for example, to facilitate purification.
- a method for preparing a fusion protein with maltose binding protein using E is a method for preparing a fusion protein with maltose binding protein using E.
- the host cell is not particularly limited as long as it is a cell suitable for expressing the recombinant protein.
- Escherichia coli for example, yeast, various animal and plant cells, insect cells, and the like can be used.
- a vector into a host cell Various methods known to those skilled in the art can be used for introducing a vector into a host cell. For example, introduction into Escherichia coli4, introduction method using calcium ion (Mandel, M. & Higa, A. (1970) Journal of Molecular Biology, 53, 158-162, Hanahan, D. (1983) Journal of Molecular Biology, 166, 557-580) can be used.
- the recombinant protein expressed in the host cell can be purified and recovered from the host cell or a culture supernatant thereof by a method known to those skilled in the art. When the recombinant protein is expressed as a fusion protein with the above-mentioned maltose binding protein or the like, affinity purification can be easily performed.
- a transformed plant into which the DNA of the present invention has been introduced can be prepared by the method described below, and the protein of the present invention can be prepared from the plant. Therefore, the transformed plant of the present invention includes not only a plant into which the DNA of the present invention has been introduced to modify the regeneration capability, but also a DNA of the present invention for the preparation of the protein of the present invention. Also, a plant into which is introduced is included.
- an antibody that binds thereto can be prepared.
- a polyclonal antibody is used to immunize an immunized animal such as a heron with the purified protein of the present invention or a partial peptide thereof, and collect blood after a certain period of time.
- a monoclonal antibody is obtained by fusing antibody-producing cells of an animal immunized with the above-mentioned protein or peptide with bone tumor cells, and isolating a single clone of cells (hybridoma) producing the desired antibody. It can be prepared by obtaining an antibody from the cells.
- the antibody thus obtained can be used for purification and detection of the protein of the present invention.
- the present invention includes an antibody that binds to the protein of the present invention.
- a DNA encoding the protein of the present invention is inserted into an appropriate vector, and this is introduced into plant cells.
- the transformed plant cells thus obtained are regenerated.
- the plant cell into which the vector 1 is introduced is preferably a plant cell having low expression of the DNA of the present invention.
- plant cell includes various forms of plant cells, for example, suspension culture cells, protoplasts, leaf sections, and calli.
- the vector used for the transformation of a plant cell is not particularly limited as long as it can express the transgene in the cell.
- the plasmids “pBI121”, “pBI221”, and “pBI101” can be mentioned.
- the vector of the present invention may contain a promoter for constitutively or inducibly expressing the protein of the present invention. Examples of promoters for constitutive expression include cauliflower mosaic virus 35S promoter (Odell et al. 1985 Nature 313: 810), rice actin promoter (Zhang et al. 1991 Plant Cell 3: 1155), Corn ubiquitin promoter (Cornejo et al. 1993 Plant Mol. Biol. 23: 567).
- Promoters for inducible expression include, for example, infection and invasion of filamentous fungi, bacteria, and viruses, low temperature, high temperature, drying, ultraviolet irradiation, And promoters that are known to be expressed by external factors such as spraying.
- Such promoters include, for example, the promoter of the rice chitinase gene (Xu et al. 1996 Plant Mol. Biol. 30: 387) expressed by infection and invasion of filamentous fungi, bacteria, and viruses, and the PR protein gene of tobacco.
- Promoter I Ohshima et al. 1990 Plant Cell 2: 95
- Rice “lipl9” gene promoter induced by low temperature Arabit al. 1993 Mol. GenGenet.
- promoter of chalcone synthase gene of parsley induced by ultraviolet irradiation (Schulze-Lefert et al. 1989 EMB0 J. 8: 651), induced under anaerobic conditions Corn alcohol dehydrogenase gene promoter (Walker et al. 1987 Proc. Natl. Acad. Sci. USA 84: 6624).
- promoter of the rice chitinase gene and the promoter of the PR protein gene of tobacco are induced by specific compounds such as salicylic acid, and “rabl6” is induced by spraying the plant hormone abscisic acid.
- the vector may have a promoter of a DNA encoding the protein of the present invention.
- the promoter region of the DNA encoding the protein of the present invention can be obtained, for example, by screening a genomic DNA library using a DNA consisting of the nucleotide sequence of SEQ ID NO: 1 or 2 or a part thereof as a probe. It is possible.
- the present invention also provides a transformed cell into which the vector of the present invention has been introduced.
- Cells into which the vector of the present invention is introduced include, in addition to the above-described cells used for production of a recombinant protein, plant cells for producing a transformed plant.
- the plant cells are not particularly limited, and include, for example, cells of Arabidopsis, rice, corn, potato, tobacco and the like.
- the plant cells of the present invention include, in addition to cultured cells, Cells in plants are also included. It also includes protoplasts, shoot primordia, multiple shoots, and hairy roots.
- Transformation Plants can be regenerated from plant cells by a method known to those skilled in the art fc according to the type of plant cells (see Toki et al. (1995) Plant Physiol. 100: 1503-1507). ).
- a technique for producing a transformed plant is to introduce a gene into protoplasts with polyethylene glycol and regenerate the plant (Indian rice varieties are suitable) (Datta, SK (1995) In Gene
- progeny can be obtained from the plant by sexual or asexual reproduction.
- Propagation materials eg, seeds, fruits, cuttings, tubers, tubers, strains, calli, protoplasts, etc.
- the present invention includes plant cells into which the DNA of the present invention has been introduced, plants containing the cells, progeny and clones of the plants, and propagation materials of the plants, progeny thereof, and clones.
- the plant thus produced with altered regeneration ability is different from the wild-type plant. In comparison, its regeneration potential has changed.
- an increase in the regeneration ability is expected.
- the use of the method of the present invention can increase the regeneration ability of rice, which is a useful agricultural crop, and is extremely useful in breeding high-regeneration rice varieties.
- the present invention also provides a polynucleotide comprising at least 15 consecutive bases complementary to the base sequence of SEQ ID NO: 1 or 2 or a complementary sequence thereof.
- acquisition sequence refers to the sequence of one strand of a double-stranded DNA consisting of A: T, G: C base pairs with respect to the other strand.
- complementary is not limited to a completely complementary sequence in at least 15 contiguous nucleotide regions, but is at least 70%, preferably at least 80%, more preferably 90%, and still more preferably It suffices to have a nucleotide sequence identity of 95% or more (for example, 96% or more, 97% or more, 98% or more, 99% or more).
- DNA is useful as a prop for detecting or isolating the DNA of the present invention, and as a primer for performing amplification.
- the present invention provides a genetic diagnosis method for determining whether or not a plant has a regeneration ability.
- "determining the presence or absence of regeneration ability of a plant” is not only effective for determining the presence or absence of regeneration ability in cultivars that have been cultivated until now, but also in determining the presence or absence of regeneration ability in new varieties by crossing and gene recombination techniques The determination of the presence or absence of the regeneration ability is also included. This method is particularly effective for judging whether or not the Japanese rice cultivar has regeneration ability.
- the method of the present invention for evaluating the ability of a plant to regenerate is characterized by detecting the DNA encoding the PSR1 protein and the expression level of the PSR1 protein in the plant. For example, if the expression of DNA encoding PSR1 or the protein of PSR1 is higher than the gene and protein of Koshihikari, the test plant is diagnosed as a cultivar capable of redifferentiation.
- the present invention also provides a method of using the PSR1 gene as a selection marker in plant transformation.
- Selection of transformed plant cells used so far Marker genes include, for example, the hygromycin phosphotransferase gene, which is resistant to the antibiotic hygromycin, the neomycin phosphotransferase, which is resistant to kanamycin or gentamicin, the acetyltransferase gene, which is resistant to the herbicide phosphinothricin, and bialafos. And a bialaphos-resistant gene.
- transformed plant culture cells are obtained by culturing on a known selection medium containing an appropriate selection agent according to the type of the selection marker gene.
- the plant cells to be transformed do not have the ability to redifferentiate, such as Koshihikari, use special drugs for selection.
- Transformants can be selected without obtaining the regeneration ability as a marker trait. In other words, non-transformants cannot be re-differentiated. Individuals that have been re-differentiated by the effect of the 3 ⁇ 4-7 gene are considered to be transformants.
- the gene is used as a selection marker for plant cells having re-differentiating ability, the transformed cells can be selected by adding nitrite at a concentration that inhibits the growth of non-transformants to the selection medium. Can be.
- FIG. 1 is a graph and a photograph showing the phenotypes of Koshihikari and Kasalath.
- the photo shows Koshihikari on the left and Kasalath on the right.
- the graph shows the redifferentiation ability of Koshihikari and Salas in terms of the number of redifferentiated individuals per g of callus.
- FIG. 2 is a diagram showing the location of a regeneration-specific QTL on a chromosome.
- 'FIG. 3 is a diagram showing a high-precision linkage MAP of the regeneration capability QTL.
- - Figure 4 is a photograph showing the results of the complementation test. The left side shows the case where only the vector was introduced into Koshihikari, and the right side shows the state of regeneration when the 3F fragment of Kasalath was introduced into Koshihikari.
- FIG. 5 is a diagram showing mutation sites in the Kasalath_z genome relative to the Koshihikari J genome sequence.
- Arabic numerals in the schematic diagram indicate the number of inserted or deleted bases.
- Black squares indicate coding regions.
- Vertical lines indicate substitution sites.
- the sequence in the frame is a diagram comparing the gene sequences of Koshihikari (upper) and Kasalath (lower). Boxes indicate amino acids that differed between Koshihikari and Kasalath.
- the region shown in bold italics indicates the chloroplast transit peptide domain, the region underlined with a dotted line indicates the ferredoxin binding region, and the region underlined indicates the 4Fe-4S cluster.
- - Figure 6 is a photograph and a diagram comparing the expression levels of the ⁇ 3 ⁇ 4? Gene and NiR protein in Kashihikari and Kasalath karyu.
- the gene was detected by semi-quantitative RT-PCR
- the inubiquitin 1 gene (Rubql) was detected by semi-quantitative RT-PCR as an expression control
- the lower photo was NiR protein.
- the NiR protein was detected by Western blot haploidization using the antibody shown in Fig. 1.
- the graph on the right shows the results of measuring the expression level of the 7 ⁇ ? Gene by real-time quantitative RT-PCR using the gene expression level as an internal standard. Show.
- Figure 5 shows the primer sites for RT-PCR.
- FIG. 7 is a graph comparing the enzyme activities of the Koshihikari and Kasalath NiR recombinant proteins.
- FIG. 8 is a diagram and a photograph showing the results of an experiment for confirming the effectiveness of the? Gene as a selection marker.
- the schematic diagram is a diagram of the T-DNA region of the binary vector used for the transformation. The photograph shows the state of redifferentiation when each vector was introduced into Koshihikari. The table shows the percentage of GUS-stained individuals in the regenerated individuals.
- FIG. 9 is a photograph showing callus selection results when a vector overexpressing the? Gene by the actin promoter was introduced into Kasalath. The upper photograph shows the callus selection results.
- Nitrite was added to the medium, and the cells grew due to the effect of overexpression of the a ⁇ ⁇ NiR gene in the transformant, whereas growth of virulent was suppressed in the non-transformant b) .
- the lower photograph is the GUS of a and b callus.
- C The best mode for carrying out the invention showing the staining results. 'The present invention will be described more specifically with reference to the following examples, but the present invention is limited to these examples. It is not done.
- BC2F1 From the BC2F1 population, 30 individuals were selected from which the social region was replaced by Kasalath, and DNA was extracted from callus using 10 seeds each of these seeds (BC2F2 seeds), and the genotype was determined by molecular force. In addition, the regenerative ability was investigated, and linkage analysis was performed. Then, in order to identify a more detailed sitting area, genotype by molecular markers was investigated using about 3,800 BC3F2 seeds separated by Society, and high-precision linkage analysis was performed. As a result, it was found that is located within a region of about 50.8 kb between the molecular markers 3132 and P182 (Fig. 3). Predicting the genes present in this region suggested the presence of four genes, including Hypothetical Protein.
- a Kasalath BAC library (average length: 120 kb) was prepared, and a BAC clone (BHAL15) containing the region was isolated by PCR screening.
- a Kasalath genomic fragment containing each of the target gene regions was prepared using appropriate restriction enzyme sites in the BHAL15 clone, and introduced into Koshihikari.Then, a Kasalath genomic fragment containing the gene NiR predicted to encode ferredoxin nitrite reductase (NiR) was prepared. Only when 3F) in Fig. 3 was introduced, it was found that the ability to re-divide Koshihikari was possible (Fig. 4). When the base sequences of Kasalas and Koshihikari were determined and compared about 2 kb upstream of the gene region predicted to be ferredoxin nitrite reductase, many base sequence mutations were found (Fig. 5). ).
- the Kasalath PSR1 gene can be used as a selection marker when transforming Koshihikari.
- a vector in which the Kasalath / 3 ⁇ 43 ⁇ 4 gene and the target gene are incorporated in parallel is introduced into Koshihikari, only the cells into which the PSR1 gene has been introduced acquire the redifferentiation ability. Are also expected to be introduced.
- the J7? Gene which has the property of metabolizing toxic nitrite, the J7? Gene could be used as a marker in the transformation to highly reproductive varieties. Specifically, 7 ⁇ 3 ⁇ 4? Under the control of actin promoter, one of the high expression promoters in rice.
- the vector for overexpressing the gene was introduced into a highly redifferentiated cultivar, Salas, and cultured on a medium supplemented with nitrite at a concentration that would inhibit growth of the normal wild type. Due to the effect of the overexpressed gene, only the transformed cells proliferated, and GUS staining was observed only in the proliferating cells (FIG. 9).
- Transformation methods which mainly use crossing and selection, can use genes beyond species that cannot be bred in the past, and may be able to produce new plants.
- gene transfection and expression control analysis for the purpose of elucidating the function of each gene have been performed using the transformation method.
- a plasmid vector containing both the gene to be introduced and a drug resistance marker gene such as an antibiotic is introduced into plant cells by the agrobacterium method ⁇ electroporation method (electroporation method). Transformed cells are selected by drug treatment.
- the selected transformed cells are redifferentiated into plants through cell proliferation.
- establishment of tissue culture technology is indispensable to use such a transformation method.
- Tissue culture technology is not only a transformation method, It is also very effective for producing mutants by culture mutation, breeding varieties by cell fusion and ovule culture, fixing genetic traits and shortening the breeding years.
- -Rice is one of the major cereals, which is one of the most advanced cultivation technologies.
- the cultivation characteristics vary greatly between varieties.
- the main varieties in Japan such as Koshihi Riki Akitakomachi and many other indian varieties cultivated in the tropics, are difficult to culture, and these varieties cannot be used as materials for tissue culture.
- This kind of cultivar difference in cultivation characteristics is a phenomenon that is common not only to rice but also to many plants, but the clarification of the cause is still in progress.
- Cereals such as rice, corn, wheat, and wheat are the main sources of energy for humans and are the most important plants for humans. All of these cereals belong to the Poaceae family and are thought to have evolved from the same ancestor, and have high gene homology (genomic synteny) to each other. Among them, rice with the smallest genome size is rice, which is why rice is used as a model plant for cereals. Rice genes are also present in their related wheat and sorghum genomes, and genes isolated from rice can be easily isolated from wheat and corn directly into grain breeding such as wheat and sorghum. Since this gene can be applied, it is thought that this gene can be widely applied to plants as well as rice.
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KR1020067002052A KR101192258B1 (ko) | 2003-07-31 | 2004-07-30 | 식물에 재분화능을 부여하는 유전자 및 그 이용 |
DE602004019783T DE602004019783D1 (de) | 2003-07-31 | 2004-07-30 | Das rückdifferenzierungsvermögen einer pflanze verleihendes gen und nutzung davon |
JP2005512596A JP3979431B2 (ja) | 2003-07-31 | 2004-07-30 | 植物の再分化能を付与する遺伝子、並びにその利用 |
AU2004261892A AU2004261892B2 (en) | 2003-07-31 | 2004-07-30 | Genes that confer regeneration ability to plants and uses thereof |
US10/566,593 US7790957B2 (en) | 2003-07-31 | 2004-07-30 | Genes that confer regeneration ability to plants, and uses thereof |
EP04771310A EP1650301B8 (en) | 2003-07-31 | 2004-07-30 | Genes that confer regeneration ability to plants, and uses thereof |
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Cited By (9)
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US7751835B2 (en) | 2005-10-04 | 2010-07-06 | Airvana, Inc. | Non-circular paging areas |
US8085696B2 (en) | 2006-07-14 | 2011-12-27 | Airvana Networks Solutions, Inc. | Dynamic modification of route update protocols |
US8094630B2 (en) | 2005-12-16 | 2012-01-10 | Airvana Network Solutions, Inc. | Radio frequency dragging prevention |
US8099504B2 (en) | 2005-06-24 | 2012-01-17 | Airvana Network Solutions, Inc. | Preserving sessions in a wireless network |
US8145221B2 (en) | 2005-12-16 | 2012-03-27 | Airvana Network Solutions, Inc. | Radio network communication |
US8195187B2 (en) | 2001-06-25 | 2012-06-05 | Airvana Network Solutions, Inc. | Radio network control |
US8619702B2 (en) | 2005-12-16 | 2013-12-31 | Ericsson Evdo Inc. | Radio network control |
US8843638B2 (en) | 2007-12-13 | 2014-09-23 | Ericsson Evdo Inc. | Handing off active connections |
US9019935B2 (en) | 2001-06-25 | 2015-04-28 | Ericsson Evdo Inc. | Radio network control |
Families Citing this family (4)
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JP4368391B2 (ja) * | 2007-05-17 | 2009-11-18 | 本田技研工業株式会社 | イネおよびその作出方法 |
GB0715916D0 (en) * | 2007-08-15 | 2007-09-26 | Cambridge Advanced Tech | Production of modified plants |
CN105132441B (zh) * | 2015-09-29 | 2018-07-06 | 浙江农林大学 | 版纳甜龙竹NiR基因及其应用 |
EP4317430A1 (en) * | 2021-03-25 | 2024-02-07 | Suzhou Qi Biodesign biotechnology Company Limited | Method for improving plant genetic transformation and gene editing efficiency |
Family Cites Families (3)
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JPH07236486A (ja) * | 1994-03-02 | 1995-09-12 | Toyota Motor Corp | ポプラ属植物の亜硝酸還元酵素遺伝子 |
DE60022369T2 (de) * | 1999-10-04 | 2006-05-18 | Medicago Inc., Sainte Foy | Verfahren zur regulation der transkription von fremden genen in gegenwart von stickstoff |
WO2002036786A2 (en) * | 2000-10-31 | 2002-05-10 | Medicago Inc. | Method of selecting plant promoters to control transgene expression |
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Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
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US8195187B2 (en) | 2001-06-25 | 2012-06-05 | Airvana Network Solutions, Inc. | Radio network control |
US9019935B2 (en) | 2001-06-25 | 2015-04-28 | Ericsson Evdo Inc. | Radio network control |
US8099504B2 (en) | 2005-06-24 | 2012-01-17 | Airvana Network Solutions, Inc. | Preserving sessions in a wireless network |
US7751835B2 (en) | 2005-10-04 | 2010-07-06 | Airvana, Inc. | Non-circular paging areas |
US8094630B2 (en) | 2005-12-16 | 2012-01-10 | Airvana Network Solutions, Inc. | Radio frequency dragging prevention |
US8145221B2 (en) | 2005-12-16 | 2012-03-27 | Airvana Network Solutions, Inc. | Radio network communication |
US8619702B2 (en) | 2005-12-16 | 2013-12-31 | Ericsson Evdo Inc. | Radio network control |
US8085696B2 (en) | 2006-07-14 | 2011-12-27 | Airvana Networks Solutions, Inc. | Dynamic modification of route update protocols |
US8843638B2 (en) | 2007-12-13 | 2014-09-23 | Ericsson Evdo Inc. | Handing off active connections |
Also Published As
Publication number | Publication date |
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US20070186302A1 (en) | 2007-08-09 |
EP1650301B8 (en) | 2009-11-18 |
CN1842592A (zh) | 2006-10-04 |
EP1650301A1 (en) | 2006-04-26 |
CN100462435C (zh) | 2009-02-18 |
KR101192258B1 (ko) | 2012-10-17 |
US7790957B2 (en) | 2010-09-07 |
KR20060052938A (ko) | 2006-05-19 |
JP3979431B2 (ja) | 2007-09-19 |
EP1650301A4 (en) | 2006-12-06 |
JPWO2005012520A1 (ja) | 2006-09-21 |
AU2004261892A1 (en) | 2005-02-10 |
DE602004019783D1 (de) | 2009-04-16 |
EP1650301B1 (en) | 2009-03-04 |
AU2004261892B2 (en) | 2010-11-11 |
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