WO2006092882A1 - イネのアントラニル酸合成酵素遺伝子oasa2の新規改変遺伝子およびその利用 - Google Patents
イネのアントラニル酸合成酵素遺伝子oasa2の新規改変遺伝子およびその利用 Download PDFInfo
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- WO2006092882A1 WO2006092882A1 PCT/JP2005/018708 JP2005018708W WO2006092882A1 WO 2006092882 A1 WO2006092882 A1 WO 2006092882A1 JP 2005018708 W JP2005018708 W JP 2005018708W WO 2006092882 A1 WO2006092882 A1 WO 2006092882A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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)
- 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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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
Definitions
- the present invention relates to a novel modified gene of rice anthralate synthase gene OASA2 and its use, and in particular, has an enzyme activity equivalent to or higher than that of wild-type rice anthranilate synthase,
- the present invention relates to a novel modified gene encoding a modified rice anthranilate synthase having resistance to feedback inhibition by tribtophan and use thereof.
- Tryptophan is one of the amino acids that make up proteins and is essential for living organisms. Since animals cannot synthesize trybutophane in the body, they must be taken from food. Grains such as rice, corn, and wheat have a very low tryptophan content, so it is usually necessary to add industrially produced trybutophane to grain feed.
- O ASA1 and OASA2 exist as rice anthralate synthase alpha subunit genes, and have isolated them (see Patent Document 1).
- OASA2 is a gene that encodes a protein that mainly functions as an anthranilate synthase alpha subunit in rice with a high level of gene expression.
- the inventors have reported that the OASA2 protein is extremely sensitive to the concentration of tributophan and decreases its activity as the intracellular concentration of tributophan increases (see Non-Patent Document 3).
- the function of the OASA2 protein can be modified, it is considered that a new variety of plants in which a high concentration of tributophan is accumulated can be produced.
- the present invention has been made in view of the above-mentioned problems, and an object of the present invention is to make rice anthranil functionally modified so that the enzyme activity does not decrease even when the intracellular concentration of tributophan increases.
- the object is to provide an acid synthase and a novel modified gene encoding the enzyme, and to realize a new variety of plants containing a high concentration of triftophan.
- the present inventors have introduced a mutation into the rice anthralate synthase gene OASA2, thereby allowing a protein having resistance to feedback inhibition by tributophan ⁇ ilj put out. Furthermore, the inventors introduced a plurality of mutations in the wild type rice anthranilate synthase gene OAS A2 and combined them to have resistance to feedback inhibition by tributophan and to wild type. The present inventors have succeeded in producing a protein having an enzyme activity equal to or higher than that of rice anthroleate synthase, thereby completing the present invention.
- the polypeptide according to the present invention is a polypeptide having a mutation in at least one of positions 126, 367, or 369 of the amino acid sequence shown in SEQ ID NO: 1, It is characterized by resistance to feedback inhibition by tributophan in the synthetic pathway. Furthermore, in addition to the above mutation, a polypeptide having a mutation in at least one of positions 351, 522 or 530 of the amino acid sequence shown in SEQ ID NO: 1, wherein the wild-type rice anthroleic acid It is preferable that the enzyme activity is at least 0.7 times higher than that of the synthetic enzyme.
- Tributophane can be synthesized, and a plant expressing the polypeptide is useful as a highly nutritious plant containing a high concentration of tributophane.
- the mutation at position 126 in the amino acid sequence shown in SEQ ID NO: 1 is a mutation that replaces serine with furanine, and the mutation at position 367 in the amino acid sequence shown in SEQ ID NO: 1.
- the difference is a mutation that replaces tyrosine with alanine or isoleucine, and the mutation at position 369 in the amino acid sequence shown in SEQ ID NO: 1 is preferably a mutation that replaces alanine with leucine.
- the mutation at position 351 of the amino acid sequence shown in SEQ ID NO: 1 is a mutation that substitutes asparagine with aspartic acid, and the mutation at position 522 of the amino acid sequence shown in SEQ ID NO: 1 shows that glycine is a tyrosine.
- the mutation at position 530 of the amino acid sequence shown in SEQ ID NO: 1 is preferably a mutation that replaces leucine with alanine or aspartate. Due to the amino acid substitutions listed above, mutant rice anthroleate synthase having resistance to feedback inhibition by tributophan, or resistance to feedback inhibition by tributophan and at least 0 compared to wild rice anthranilate synthase. A mutant rice anthranilate synthase having an enzyme activity 7 times or more can be realized.
- mutant rice anthroleate synthase examples include SEQ ID NOs: 2 to 7 and SEQ ID NOs: 29 and 32; In the amino acid sequence shown in any one of 7 and SEQ ID NO: 29 or 32, a polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted, or added can be mentioned.
- the mutant rice anthroleate synthase having resistance to feedback inhibition by tributophan, or the resistance to feedback inhibition by tributophan and at least 0 compared to wild-type rice anthranilate synthase.
- a mutant rice anthranilate synthase with over 7 times the enzyme activity can be realized.
- the polypeptide according to the present invention is a polypeptide having a mutation in the tributophan binding region of anthralate synthase, and has a mutation at the fifth position of the amino acid sequence shown in SEQ ID NO: 26. It is characterized by having resistance to feedback inhibition by tributophan in the tributophan biosynthetic pathway.
- the mutation at position 5 of the amino acid sequence shown in SEQ ID NO: 26 is preferably a mutation that substitutes tyrosine for alanine or isoleucine.
- Polypeptides that are resistant to feedback inhibition by tributophan can synthesize tributophan even when the intracellular concentration of tributophan is increased, and plants expressing the polypeptide have a high concentration of tributophan. High nutritional value makes it useful as a plant.
- the polynucleotide according to the present invention is a polynucleotide encoding the polypeptide according to the present invention.
- the polynucleotide according to the present invention is a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 9 to 14, SEQ ID NO: 33, or 36, or SEQ ID NO: 9 !, 14 and SEQ ID NO: 33. It is preferable that it is a polynucleotide that is hybridized under stringent conditions with a polynucleotide that is complementary to a polynucleotide that also has a base sequence force that is shown in any case.
- the marker gene for selecting a transformant according to the present invention also has the polynucleotide power according to the present invention. Since the polypeptide encoded by the polynucleotide according to the present invention confers resistance to the trybutophane analog on the cell in which it is expressed, the transformation expressing the resistance to the trybutophane analog It can be used as a marker gene for selecting cells.
- the recombinant expression vector according to the present invention comprises the polynucleotide according to the present invention.
- the recombinant expression vector can be used as a recombinant expression vector for introducing the polynucleotide according to the present invention into cells, and when the polynucleotide according to the present invention is used as a selection marker, other genes can be used. It can also be used as a recombinant expression vector for introducing a cell into a cell.
- the transformant according to the present invention is introduced with the polynucleotide according to the present invention or the recombinant expression vector according to the present invention, and against feedback inhibition by tributophan in the tributophan biosynthesis pathway. It is a transformant in which a polypeptide having resistance is expressed.
- the transformant is preferably a plant cell or a plant body.
- a transgenic plant expressing a polypeptide having resistance to feedback inhibition by tribtophan is useful as a highly nutritious plant containing a high concentration of tributophan.
- the present invention also includes seeds obtained from the transformed product.
- the method for selecting transformed cells according to the present invention comprises a marker gene or By introducing the recombinant expression vector according to the present invention into a cell, the cell is imparted with resistance to a trybutophane analog that inhibits cell growth, and further exhibits resistance to the tributophane analog.
- a selection method comprising selecting cells that are present.
- the marker gene is a rice-derived gene, the protein encoded by this gene is expected to be low antigenic in rice transformants.
- the transformation kit according to the present invention is at least the polynucleotide according to the present invention! / Is characterized in that it contains any of the recombinant expression vectors according to the present invention.
- a transformant expressing the polypeptide of the present invention can be obtained simply and efficiently.
- the screening method according to the present invention is a method for screening a substance that binds to only one of the polypeptide according to the present invention and the wild-type inetranate synthase, and comprises the polypeptide according to the present invention. And a step of screening a substance that binds to wild type rice anthranilate synthase, and a step of comparing the results of the above two steps.
- substances involved in feedback inhibition by tributophan in the tributophan biosynthesis pathway can be screened, and the resistance to feedback inhibition may lead to the creation of a stronger mutant rice anthra-lulate synthase. is there.
- the screening kit according to the present invention is a kit for realizing the screening method according to the present invention, and includes at least one of the polypeptides according to the present invention and wild-type rice anthroleate synthase. It is characterized by that.
- the screening method according to the present invention can be carried out simply and efficiently by using the above-mentioned monitoring kit.
- the polypeptide according to the present invention has resistance to feedback inhibition by tributophan in the tributophan synthesis pathway, and has an enzyme activity equivalent to or higher than that of wild-type rice anthranilate synthase. Yes. Therefore, even if tributophan can be synthesized even in the presence of a high concentration of tryptophan, there is an effect.
- the polynucleotide according to the present invention encodes the polypeptide according to the present invention.
- the transformed plant according to the present invention has an effect that it can be used as food or feed having a high tributophane content and excellent nutritional value. Furthermore, the production of rice for feed with a high tributophane content has the effect of reducing the cost of livestock feed, and also the effective use of paddy fields to increase the self-sufficiency of feed in Japan. Play.
- FIG. 1 Anthrallic acid in a measurement system using lOOmM NHC1 as an amide group donor substrate
- FIG. 3 is a graph showing the degree of feedback inhibition of mutants and wild type having resistance to feedback inhibition by tributophan.
- FIG. 4 (A) is a schematic diagram of wild-type OASA2 protein.
- FIG. 4 (B) The amino acid sequence of the tributophan binding region of an organism having a tributophan synthesis system. It is the figure compared.
- FIG. 5 Transgenic OASA2 gene (Y367A, Y367A / L530D, S126F / L530D) or wild-type (wt) OASA2 gene-containing set for transformation of rice callus by agrobatatarum method It is a schematic diagram of a replacement vector.
- the polypeptide according to the present invention is a polypeptide having a mutation in the tributophane binding region of an anthralate synthase, and has a mutation at the 5th position of the amino acid sequence shown in SEQ ID NO: 26.
- the mutation at position 5 of the amino acid sequence shown in SEQ ID NO: 26 is preferably a mutation that replaces tyrosine with alanine or isoleucine.
- Fig. 4 (A) shows rice anthranilate synthase encoded by rice anthralate synthase gene OASA2 (hereinafter referred to as "OASA2 gene” as appropriate) (hereinafter referred to as “OASA2 protein” as appropriate) or A schematic diagram of “wild-type OASA2 protein” is shown.
- the numbers in Fig. 4 (A) indicate the positions of amino acids.
- cTP represents a chloroplast translocation signal, and I, II, and III represent domains in which amino acid residues located in the tryptophan binding region exist.
- Fig. 4 (B) shows a comparison of the amino acid sequences of tryptophan binding regions I, II and III of organisms having various tryptophan synthesis systems.
- Osl is rice OASA1 (Accession no. AB022602)
- Os2 is rice OASA2 (Accession no. AB022603)
- Atl is Arabidopsis ASA1 (Accession no. M92353)
- At2 is Arabidopsis ASA2 (Accession no. M92354)
- Ss is a thermophilic archaea (Sulfolobus solfataricus) TrpE (Accession no. 1QD L—A)
- Sti Salmonella tryphimurium TrpE Accession no. 1I1Q—A
- Sm is a ceftia bacterium (Serratia marcescens) Represents TrpE (Accession no. 1I7Q—A).
- N PSPYM amino acid sequence shown in SEQ ID NO: 26 is conserved in all the exemplified organisms in the tryptophan binding region II. The Therefore, it is considered to play a very important role in the binding region of tributophan.
- biological species having such a conserved amino acid sequence include those other than those exemplified in Fig. 4 (B)-Catharanthus roseus a subunit (Accession no.
- CAC29060 Henruda (Ruta graveolens) ) AS ⁇ 1 (Accession no.L34343), Heno Radar (Ruta graveolens) AS ⁇ 2 (Accession no.L34344), Tanoko (tobacco) ASA2 (Accession no.T01990), Yeast (Saccharomyces cerevisiae) TRP2 (Accession no. X683 27), the ability to include Escherichia coli TrpE (Accession no. V00368), Bacillus subtilis TrpE (Accession no. P03963), but is not limited thereto.
- the amino acid sequence shown in SEQ ID NO: 26 is, for example, in positions 363 to 367 in the amino acid sequence of the wild-type OASA2 protein encoded by rice anthralate synthase gene OASA2 shown in SEQ ID NO: 1. Applicable. Then, by replacing tyrosine in the conserved amino acid sequence shown in SEQ ID NO: 26 with another amino acid, preferably alanine or isoorcin, resistance to feedback inhibition by tributophan is obtained.
- the polypeptide according to the present invention is a polypeptide having a mutation in at least one of positions 126, 367 or 369 of the amino acid sequence shown in SEQ ID NO: 1, wherein tryptophan biosynthesis Any polypeptide that is resistant to feedback inhibition by tributophan in the pathway!
- the polypeptide having the amino acid sequence ability shown in SEQ ID NO: 1 is a wild-type intraanthrate synthase encoded by rice anthralate synthase gene OASA2.
- any polypeptide having a mutation and having resistance to feedback inhibition by tributophan may be used.
- mutations include, but are not limited to, amino acid substitutions, deletions, additions, and the like.
- an OASA2 protein having a mutation is referred to as a “mutant OASA2 protein” regardless of the position, type, number, etc. of the mutation.
- chorismate acidity is also anthralic acid.
- tributophane is produced from anthranilic acid via indole through a six-stage enzymatic reaction (see Non-Patent Document 1).
- Anthralic acid synthase catalyzes the production of chorismate acid anthranilic acid in the above pathway.
- anthralate synthase is feedback-inhibited by the final product tryptophan, and as the tryptophan concentration in the cell increases, the enzyme activity decreases. Therefore, if tributophane accumulates in a certain amount of cells, no more tributophane is synthesized. Since the polypeptide according to the present invention is resistant to feedback inhibition by tributophan as described above, it can synthesize tributophan even when the concentration of tributophan in the cell is increased. The body can be created.
- Resistance to feedback inhibition by tribtophan is, for example, measured in the in vitro enzyme activity measurement system when the enzyme activity of wild-type OASA2 protein is measured and the enzyme activity in the absence of tributophan is defined as 100%. Judgment can be made on the basis of the ratio (percentage) of the enzyme activity when tribtophan is added. More specifically, for example, as shown in FIG. 3, the enzyme activity of the wild-type OASA2 protein and the mutant OASA2 protein when no tributofane is added is defined as 100%, and the enzyme activity when tributofane-added caro is added.
- the ratio (percentage) When the ratio (percentage) is compared, if the ratio (percentage) of the enzyme activity of the wild-type OASA2 protein when sorbed with tributophan is exceeded, it can be said that it is resistant to feedback inhibition by tributophan.
- the percentage of the enzyme activity of the wild-type OASA 2 protein with tributofane it is not particularly limited, but it must be at least twice as high. Most preferably, it is at least 3 times or more, more preferably at least 4 times or more, and most preferably at least 5 times or more.
- the enzyme activity without addition of tributophan when enzyme activity is measured using the activity measurement system shown in Example 3 (2) ⁇ Enzyme activity measurement 1> described later, the enzyme activity without addition of tributophan ( When the amount of anthranilic acid synthesis is 100%, the enzyme activity (anthranilic acid synthesis amount) of 20% or more when 100 M tryptophan is added is preferred, and more than 30% is more preferred. Those with 40% or more remaining are particularly preferable, and those with 50% or more remaining are most preferable. [0036]
- the amino acid substituted with serine at position 126 of the OAS A2 protein is not particularly limited as long as it satisfies the above-mentioned resistance condition against feedback inhibition.
- Ferallanine is preferred. Specifically, a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 29 (serine at position 126 is substituted with phenylalanin), or SEQ ID NO: 2
- the polypeptide has an amino acid sequence ability in which one or several amino acids are deleted, substituted, or added, and has resistance to feedback inhibition by tryptophan. Is preferred.
- the amino acid substituted with tyrosine at position 367 of the OASA2 protein is not particularly limited as long as it satisfies the above-mentioned resistance condition against feedback inhibition, but it is not limited to alanine, isoleucine, and feasibility. -Lalanin and isoleucine are preferred, and luranin and isoleucine are more preferred.
- a polypeptide having the amino acid sequence ability shown in SEQ ID NO: 2 replaces tyrosine at position 367 with alanine
- a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 3 replaces tyrosine at position 367.
- a polypeptide having resistance to feedback inhibition is preferred.
- the amino acid substituted with alanine at position 369 of the OASA2 protein is not particularly limited as long as it satisfies the above-described resistance condition against feedback inhibition.
- the polypeptide having the amino acid sequence shown in SEQ ID NO: 30 (substitution of alanine at position 369 with leucine) or the amino acid sequence shown in SEQ ID NO: 30 lacks one or several amino acids.
- the polypeptide according to the present invention includes at least one of mutations at positions 351, 522, and 530 in addition to at least one mutation at positions 126, 367, and 369 of the OASA2 protein.
- a polypeptide having a mutation in one is preferable.
- Mutant OASA2 protein with a mutation combining any one of the above 351, 522, and 530, or a combination of two or more has an enzyme activity compared to wild-type OASA2 protein. Have been confirmed by the inventors (see Fig. 1). By combining these mutations that increase enzyme activity with the mutations that acquire resistance to feedback inhibition by tributophan, the enzyme has resistance to tributephan feedback inhibition and has the same enzyme activity as wild-type OASA2 protein.
- the enzyme activity may be, for example, the amount of anthracic acid synthesized measured using the activity measurement system shown in Example 3 (2) ⁇ Enzyme activity measurement 1> described later.
- the method for measuring enzyme activity is not limited to this, and any known enzyme activity measuring method or modification method thereof can be used that can measure anthranilic acid synthase activity.
- the enzyme activity of the same level or higher means that the enzyme activity of the mutant OASA2 protein is at least 0.7 times the enzyme activity of the wild-type OASA2 protein, for example, in an in vitro enzyme activity measurement system (without addition of tributophan). It is most preferable that the ratio is at least 0.8 times that it is preferable to be at least 0.8 times that it is more preferable that it is at least above. More specifically, for example, when the enzyme activity was measured using the activity measurement system shown in Example 3 (2) Enzyme activity measurement 1> described below (without addition of tributophan), an anthracite of the mutant OASA2 protein was used.
- the amount of synthesized luric acid is at least 0.7 times the amount of anthranilic acid synthesized in the wild-type OASA2 tank, and at least 0, more preferably at least 0.8 times. It is particularly preferred that it is 9 times or more, most preferably at least 1 time U.
- position 351 is asparagine
- position 522 is glycine
- position 530 is leucine.
- the amino acids that are substituted for these amino acids are tributophane when each mutation, or a combination thereof, and at least one mutation at positions 126, 367, or 369 are combined. It is not particularly limited as long as it has resistance to feedback inhibition by, and has an enzyme activity 0.7 times or more that of wild-type OASA2 protein.
- As the amino acid substituted with asparagine at position 351 aspartic acid is preferred.
- amino acid substituted with glycine at position 522 tyrosine is preferred.
- alanine and aspartic acid are preferred, and aspartic acid is more preferred. preferable.
- polypeptides having the following combinations of mutations are preferred.
- the polypeptide having the combination of mutations shown in vi) is most preferable. This is because, as shown in Examples described later, when a gene encoding the above-described polypeptide was introduced into rice callus, it was confirmed that the content of free tributophan increased to about 00 times.
- the polypeptide according to the present invention is a polypeptide having SEQ ID NO: 4 or 7 and SEQ ID NO: 30! 7 and SEQ ID NO: 30 !, 32 !, and the amino acid sequence shown in one of them is a polymorphic amino acid sequence with one or several amino acids deleted, substituted, or added. It is preferably a polypeptide that is resistant to feedback inhibition by tributophan and has an enzyme activity 0.7 times or more that of wild-type OASA2 protein.
- mutant polypeptide production method such as a mutagenesis method (e.g., 20 or less, preferably 10 or less, more preferably 7 or less, even more preferably). Means that 5 or less amino acids, particularly preferably 3 or less amino acids are substituted, deleted, or added.
- a mutant polypeptide is not limited to a polypeptide having a mutation artificially introduced by a known mutant polypeptide production method, but a similar naturally occurring mutant polypeptide is isolated and purified. It may be
- the polypeptide according to the present invention may be a polypeptide in which amino acids are peptide-bonded, but is not limited thereto, and may contain a structure other than a polypeptide.
- structures other than the polypeptide herein include sugar chains and isoprenoid groups, but are not particularly limited.
- polypeptide according to the present invention may include an additional polypeptide.
- additional polypeptide examples include a case where it is labeled with an epitope by His, Myc, Flag or the like.
- the polypeptide according to the present invention introduces a polynucleotide according to the present invention (polynucleotide encoding the polypeptide according to the present invention), which will be described later, into a host cell and encodes it.
- the peptide may be expressed in the cell, or may be isolated and purified from cells, tissues, and the like.
- the polypeptide according to the present invention may be a fusion protein linked to another polypeptide.
- the polypeptide according to the present invention may be chemically synthesized.
- the method (production method) for obtaining the polypeptide according to the present invention is not particularly limited, and the force
- the polypeptide according to the present invention has one or more mutations introduced into the wild-type OASA2 protein. It is optimal to obtain a mutant gene in which a mutation has been artificially introduced into the base sequence of the OASA2 gene and to express the polypeptide encoded by the mutant gene.
- Examples of known methods for introducing a mutation into a base sequence include the Kunkel method and the method for introducing a mutation into a base sequence using PCR, which are used by the inventors in the examples described later. Can do.
- kits eg, Mutan-K, Mutan—Super Express Km, LA—PCR in vitro mutagenesis Kit (all manufactured by Tacarano) ) Etc.
- OASA2 gene By inserting the OASA2 gene into which an artificial mutation has been introduced in this way into an appropriate expression vector, introducing it into an appropriate host cell, and producing a product (polypeptide) obtained by translation in the host, The polypeptide according to the present invention can be obtained.
- a product (polypeptide) of the O ASA2 gene into which the mutation is introduced is obtained using a known cell-free protein synthesis system such as wheat germ cell-free system. You can also One example of the method for obtaining a polypeptide according to the present invention will be described in detail in the following examples.
- the polynucleotide according to the present invention may be one that encodes the polypeptide according to the present invention! Since the polypeptide according to the present invention has been described in detail in the above (1), the power to omit the description here.
- the polypeptides described in the following [1] and [2] Any polynucleotide encoding a peptide may be used.
- a polypeptide having a mutation in at least one of position 126, position 367, or position 369 of the amino acid sequence shown in SEQ ID NO: 1, and against feedback inhibition by tributophan in the tributophan biosynthesis pathway A polypeptide having resistance.
- nucleotide sequence shown in SEQ ID NO: 8 is used as a polynucleotide encoding a polypeptide in which the amino acid sequence shown in SEQ ID NO: 2, ie, the 367th tyrosine of the amino acid sequence of OASA2 protein is substituted with alanine.
- the nucleotide sequence at positions 1099, 1100, and 1101 (ta tyrosine) of the polynucleotide consisting of (the OASA2 gene open reading frame region base sequence) is a codon corresponding to alanine, ie any of gct, gcc, gca or gcg.
- any polynucleotide that has been mutated may be used.
- the base sequence other than the bases at positions 1099 to 1101 may not be the same as the base sequence shown in SEQ ID NO: 8, for example, the encoded amino acid may not be mutated or the polynucleotide may have a base substitution.
- the polynucleotide according to the present invention is preferably a polynucleotide encoding the polypeptide described in [3] and [4] below.
- Polypeptide consisting of the amino acid sequence shown in any of SEQ ID NOs: 4 and 7, and SEQ ID NO: 30 and 32, or SEQ ID NO: 4 and 7 and SEQ ID NO: 30! 32 !, a polypeptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, or added in the amino acid sequence shown in any one of the amino acids, and is resistant to feedback inhibition by tributophan. And a polypeptide having an enzyme activity 0.7 times or more that of wild-type OASA2 protein.
- the specific base sequence is not particularly limited as long as it is a polynucleotide comprising a base sequence encoding any one of the amino acid sequences shown in SEQ ID NOs: 2 to 7 and SEQ ID NOs: 29 to 32. It is not a thing. However, it is preferable that the polynucleotide is highly homologous to the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 8, ie, the nucleotide sequence of the open reading frame region of the OASA2 gene, and of course. The homology may be, for example, at least 90% identity, preferably at least 95% identity, and most preferably at least 97% identity.
- the polynucleotide according to the present invention is a polynucleotide having the nucleotide sequence shown in any of SEQ ID NO: 9, 14 and SEQ ID NO: 33 or 36, or SEQ ID NO: 9! 14 and SEQ ID NO: 33 !, and 36 !, under a stringent condition with a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence shown in a misalignment and a nucleotide sequence complementary to the polynucleotide. Mention may be made of polynucleotides that hybridize. However, it is not limited to these! /.
- polynucleotides consisting of the nucleotide sequences shown in SEQ ID NO: 9, 14 and SEQ ID NO: 33, and 36 are the amino acid sequences shown in SEQ ID NO: 2, 7 and SEQ ID NO: 29, and 32, respectively. It is a polynucleotide having a nucleotide sequence that encodes a polypeptide.
- Polynucleotides comprising the nucleotide sequences shown in SEQ ID NOs: 9 to 14 and SEQ ID NOs: 33 to 36 are polynucleotides prepared by the inventors using the methods described in the examples described later.
- stringent conditions refer to hybrids only when at least 90% identity, preferably at least 95% identity, most preferably at least 97% identity exists between sequences. This means that a sesion occurs, for example, binding at 60 ° C under 2 X SSC wash conditions.
- the above hybridization can be performed by a conventionally known method such as the method described in “Molecular Cloning (Third Edition)” (J. bambrook & DW Russell, old Spring Harbor Laboratory Press, 2001). . Generally, the higher the temperature and the lower the salt concentration, the higher the stringency.
- Polynucleotides according to the present invention include DNA and RNA, which may be single-stranded or double-stranded. It may also contain sequences such as untranslated region (UTR) sequences and vector sequences (including expression vector sequences)! /.
- UTR untranslated region
- vector sequences including expression vector sequences
- the method (production method) for obtaining the polynucleotide according to the present invention is not particularly limited, and examples thereof include a method of artificially introducing a mutation into the base sequence of the OASA2 gene.
- a method for introducing a mutation into a base sequence a method for introducing a mutation into a base sequence using the Kunkel method or PCR can be exemplified.
- Commercially available kits such as Mutan-K, Mutan-uper Express Km, LA-P and R in vitro mutagenesis Kit (both manufactured by Takara Bio Inc.) can also be used.
- One example of the method for obtaining a polynucleotide according to the present invention will be described in detail in the Examples below.
- the polynucleotide according to the present invention can be used as a marker gene for transformation. That is, the marker gene for selecting a transformant according to the present invention should be composed of the polynucleotide according to the present invention described in (2) above! Poly according to the present invention Since cells into which nucleotides have been introduced become resistant to trybutophane, it is possible to select only those cells into which the polynucleotide has been introduced by adding a trybutophane analog compound to the medium.
- tryptophan analogs that inhibit cell growth include 5-methyltryptophan (5MT), 4-methyltryptophan (4MT), 6-methyltryptophan (6MT), 7-Methyltryptophan (7MT), 6-Methyl anthroleic acid (6MA), 5-Methyl anthroleic acid (5MA), 3-Methyl anthroleic acid (3MA), 5-Fluoroanthralic acid (5FA) 6-Fluoroanthral acid (6FA) and the like.
- an expression vector incorporating the polynucleotide is constructed, and the expression vector is introduced into a target cell. Since the cell into which the expression vector is introduced and the polypeptide encoded by the polynucleotide according to the present invention expresses tributophane resistance, it grows even in a medium supplemented with the above-described selective agent. On the other hand, the growth of cells into which the expression vector has not been introduced or cells in which the polypeptide encoded by the polynucleotide of the present invention is not expressed are inhibited. Therefore, it becomes possible to select only transformed cells into which the expression vector has been introduced and the polypeptide encoded by the polynucleotide according to the present invention is expressed.
- the polynucleotide according to the present invention is used for both the purpose of expressing the polynucleotide in the transformed cell and the marker gene, but the polynucleotide according to the present invention is used only as a single gene. It is also possible. Further, for example, by using a transcription promoter specific to plant callus cells, it is possible to control the expression time as a selection marker of the polynucleotide according to the present invention. In this case, an expression vector into which a gene encoding a protein to be expressed in the target cell is inserted may be constructed and transformed using the expression vector. It is also possible to introduce the polynucleotide according to the present invention alone into a target cell without constructing an expression vector incorporating the polynucleotide according to the present invention.
- the polynucleotide of the present invention as a selection marker, the problem that the types of markers that can be used in monocotyledonous plants such as rice are limited is solved. Also this The problem that the types of selectable markers that can be used when introducing a plurality of genes into cells is limited can be solved by using the genes. In other words, in addition to resistance to antibiotics such as hygromycin, which is usually used as a selectable marker, multiple target DNAs were introduced due to resistance to tributophan analogs such as 5-methyltributophan. Cell selection becomes possible. Furthermore, since the marker gene is a rice-derived gene, it is expected that the protein encoded by this gene has low antigenicity compared to rice transformants.
- resistance to tributophan-related compounds that inhibit cell proliferation is introduced by introducing the marker gene according to the present invention or the recombinant expression vector described below into the cells. Also included is a method for selecting transformed cells, which is applied to cells and further selected for cells expressing resistance to the tributophan analog.
- the recombinant expression vector according to the present invention is not particularly limited as long as it contains the polynucleotide according to the present invention described in (2) above.
- a recombinant expression vector into which cDNA is inserted can be mentioned.
- a plasmid, phage, cosmid or the like can be used, but it is not particularly limited.
- a manufacturing method may be performed using a known method.
- the specific type of vector is not particularly limited, and a vector that can be expressed in a host cell may be appropriately selected. That is, according to the type of host cell, a promoter sequence is appropriately selected in order to reliably express the gene, and this and the polynucleotide according to the present invention incorporated into various plasmids or the like may be used as an expression vector. ,.
- the recombinant expression vector can be used to express the polypeptide according to the present invention, but the polynucleotide according to the present invention is used as a marker gene, and others. And can be used as a recombinant expression vector for expressing a protein encoded by the other gene.
- the host cell is not particularly limited as long as it is a cell or organism having a tributophan synthesis system, and various conventionally known cells can be preferably used. Specifically, for example, the ability to include bacteria such as Escherichia coli, yeast (budding yeast Saccharomyces cerevisiae, fission yeast Schizosaccharomyces pombe) and the like is not particularly limited.
- the transformation kit according to the present invention only needs to contain at least one of the polynucleotide according to the present invention described in (2) above or the recombinant expression vector according to the present invention.
- Other specific configurations are not particularly limited, and the kit may be configured by appropriately selecting necessary reagents and instruments. By using the transformation phase kit, transformed cells can be obtained simply and efficiently.
- the transformant according to the present invention is introduced with the polynucleotide according to the present invention described in (2) above or the recombinant expression vector according to (4) above, and in the tributophane biosynthetic pathway.
- the “transformant” means not only a cell / tissue “organ” but also an individual organism.
- a method for producing a transformant is not particularly limited, and examples thereof include a method for introducing the above-described recombinant expression vector into a host cell for transformation.
- the organism to be transformed is not particularly limited, and examples include various microorganisms exemplified as the host cell in the above (4).
- the transformant according to the present invention is preferably a plant cell or a plant. This Such transformed plants have a high use value as food materials and feeds with high nutritional value due to their high tributophane content.
- the recombinant expression vector used for transformation of a plant is not particularly limited as long as it can express the inserted gene in the plant cell.
- a binary vector such as pBi.
- Specific examples of binary vectors include pBIG, ⁇ 19, ⁇ 01, ⁇ 21, and ⁇ 221. Also
- a vector having a promoter capable of expressing a gene in a plant is preferable.
- a known promoter can be preferably used as the promoter.
- cauliflower mosaic virus 35S promoter CaMV35S
- ubiquitin promoter ubiquitin promoter
- actin promoter can be mentioned.
- the plant cells include various types of plant cells such as suspension culture cells, protoplasts, leaf slices, canores and the like.
- a recombinant expression vector For introduction of a recombinant expression vector into plant cells, various known methods such as polyethylene glycol method, electroporation method (elect mouth-position method), agrobacterium method, and particle gun method are used. Can be used. In addition, regeneration of a plant body from a transformed cell can be performed by a known method according to the type of plant cell.
- the polynucleotide is also introduced into the seed obtained from the plant strength.
- cereal grains such as rice transformed with the polynucleotide of the present invention
- cereal grains having a high tributophane content can be obtained.
- the present invention also includes seeds obtained from the transformed product.
- the screening method according to the present invention binds to only one of the polypeptide according to the present invention described in (1) (variant OAS A2 protein) and wild-type rice anthroleate synthase (OASA2 protein).
- a method for screening a substance wherein a process for screening a substance that binds to a variant OASA2 protein is compared with a process for screening a substance that binds to a wild-type OASA2 protein, and the results of both the above steps are compared. The process may be included.
- the mutant OASA2 protein has resistance to feedback inhibition by tributophan in the tryptophan biosynthesis pathway
- the mutant OASA2 protein and the wild-type OASA2 protein A substance that binds to only one of them is likely to be a signal substance involved in feedback inhibition in the tributophan biosynthesis pathway. If such a substance force is found by this screening method, it is expected that elucidation of the mechanism of feedback inhibition will progress and it will contribute to the development of a mutant OASA2 protein with stronger resistance to feedback inhibition.
- the screening kit according to the present invention is a kit for carrying out the above-described screening method, and includes at least one of the polypeptides according to the present invention and wild-type rice anthroleate synthase. That's fine.
- Other specific configurations are not particularly limited, and necessary kits may be selected by appropriately selecting necessary reagents and instruments.
- the screening method according to the present invention can be carried out simply and efficiently.
- Rice anthranilate synthase gene OASA2 (ACCESSION NO. AB022603) was inserted into the EcoRI site of the cloning vector pBluescript SK + (manufactured by Stratagene) to construct pBS-OASA2.
- the inserted gene has the restriction enzyme site Kpnl force of the multicloning site inserted in the Sad direction!
- a sense (or antisense) oligonucleotide for introduction of mutation at the position where the mutation is to be introduced Set and use.
- two primers ie, a sense and an antisense oligonucleotide
- one of the mutation-introducing primers can be the same as the mutation-introducing oligonucleotide used in the Kunkel method.
- the mutations i) to vi) below were introduced using the Kunkel method, and the mutations vii) and viii) below were introduced using PCR.
- the 351st asparagine residue was substituted with an aspartic acid residue, and a primer for mutagenesis was designed to introduce a restriction enzyme site (BsiWI) at the position where the encoded amino acid did not change.
- the base sequence of the mutagenesis primer is as follows. The underline indicates the restriction enzyme site, and the lower case letter indicates the mutation introduction codon.
- N351D-F 5'—GTTTGAGAGGCGAACGTACGCCgatCCATTTGAAGTCT—3 '(SEQ ID NO: 15)
- Primers N351D-F, 23rd to 25th gat (aspartic acid, D) and 15th to 20th CGTACG are derived from the wild-type OASA2 AAT (asparagine, N) and CATACG sequences, respectively. Designed. By changing AAT to GAT, the 351st asparagine residue is replaced with the aspartic acid residue. In addition, by making CATACG a CGT ACG, a restriction enzyme site (BsiWI: CGTACG) is introduced without amino acid substitution (both ACA and ACG are threonine), making selection after mutation introduction easier.
- the mutation primer is used so that the 367th tyrosine residue is substituted with an alanine residue and a restriction enzyme site (SnaBI: TACGTA) is introduced at a position where the encoded amino acid does not change.
- a restriction enzyme site SnaBI: TACGTA
- the base sequence of the mutagenesis primer is as follows. The underline indicates the restriction enzyme site, and the lower case letter indicates the mutation introduction codon.
- Y367A-F 5'— GTGAACCCAAGTCCAgccATGGCATACGTACAGGCAAGAGGC— 3 '(SEQ ID NO: 16) iii) Substituting the 367th tyrosine residue with an isoleucine residue (Y367I) In the same manner as in i) above, the 367th tyrosine residue is replaced with an isoleucine residue, and the encoded amino acid does not change.
- a mutagenesis primer was designed to introduce a restriction enzyme site (SnaBI: TACGTA). The nucleotide sequence of the mutagenesis primer is as follows. The underline indicates the restriction enzyme site, and the lower case letter indicates the mutagenesis codon. (SEQ ID NO: 17)
- the leucine residue at position 530 is replaced with an alanine residue, and the amino acid to be encoded is mutated to introduce a restriction enzyme site (NheI: GCTAGC) at the same position.
- Introduced primers were designed.
- the base sequence of the mutagenesis primer is as follows.
- the underline indicates the restriction enzyme site, and the lower case letter indicates the mutation introduction codon.
- L530A- F 5,-ACGGAGACATGgctATCGCGCTAGCACTCCGCACCATT-3 '(SEQ ID NO: 18)
- the 530 th leucine residue is substituted with an aspartic acid residue, and a mutation primer is introduced so that a restriction enzyme site (NheI: GCTAGC) is introduced at a position where the encoded amino acid does not change.
- a restriction enzyme site NheI: GCTAGC
- the base sequence of the mutagenesis primer is as follows. The underline indicates the restriction enzyme site, and the lower case letter indicates the mutagenesis codon.
- L530D- F 5 ACGGAGACATGgacATCGCGCTAGCACTCCGCACCATT-3 '(SEQ ID NO: 19)
- the 522nd glycine residue is replaced with a tyrosine residue
- the 530th leucine residue is replaced with an aspartic acid residue
- the restriction enzyme site BciVI: GTATCC / GGATAC
- the base sequence of the mutation-introducing primer is as follows. The underline indicates the restriction enzyme site, and the lowercase letter indicates the mutagenesis codon.
- a mutagenesis primer was designed so that the 126th serine residue was substituted with a ferulalanine residue.
- Sense and antisense oligonucleotides were created because mutations were introduced using PCR in the mutagenesis method.
- the base sequence of the mutagenesis primer is as follows. Lower case letters indicate mutation-introducing codons.
- S126F- F 5,-GCTTCCTCTTCGAGttcGTCGAGCAGGGGCC-3 '(SEQ ID NO: 37)
- S126F- R 5,-GGCCCCTGCTCGACgaaCTCGAAGAGGAAGC-3' (SEQ ID NO: 38) 15th to 17th ttc of primer S126F-F (Ferulalanin, F ) was designed from wild-type OAS A2 TCC (serine, S). By changing TCC to TTC, the 126th serine residue is replaced with a ferrolanine residue.
- Primer S126F-R is the complementary strand of primer S126F-F.
- a mutation-introducing primer was designed so that the 369th alanine residue was amino acid substituted with a leucine residue.
- Sense and antisense oligonucleotides were created because mutations were introduced by PCR using the mutagenesis method.
- the base sequence of the mutation-introducing primer is as follows. Lower case letters indicate mutation-introducing codons.
- A369L-F 5'-CAAGTCCATACATGctaTATGTACAGGCAA-3 '(SEQ ID NO: 39)
- A369L-R 5'- TTGCCTGTACATAtagCATGTATGGACTTG-3' (SEQ ID NO: 40)
- Primer A369L-R is the complementary strand of primer A369L-F.
- the Kunkel method was performed according to the method described in Reference Document 13 below.
- Reference 1 Kunkel, TA (198b; Rapid and efticient site-specific mutagenesis with out phenotypic selection. Proceedings of the National Academy of Science of the US A, 82: 488-492.
- Reference 2 Kunkel, TA, Bebenek, K. and McClary, J. (1991) Efficient site-dire cted mutagenesis using uracU—containing DNA. Methods in Enzymology, 204: 125—1 39.
- the above-described PBS-OASA2 vector is used as a cage, and mutation is performed by performing the Kunkel method using the mutagenesis primer described in i) above. Introduced. When using the mutagenesis primers described in ii), iv), v), and vi) above, the Kunkel method was performed using the same saddle type as described above. N351D, Y367A, L530A, L530D, and G522Y + L530D mutant DNA were prepared by the above operation.
- Double mutant DNA of N351D + L530A was prepared by repeating the mutagenesis step twice. That is, first, the above-described pBS-OASA2 vector was used as a cage, and N351D mutant DNA was prepared using the mutation-introducing primer described in i) above. Next, the plasmid DNA (pBS-OASA2 (N351D) vector) into which the N351D mutation has been introduced is used as a saddle, and the mutant DNA into which two mutations have been introduced using the mutation-introducing primers described in iv) above. (N351D + L 530A) was obtained.
- a plasmid DNA (pBS-OASA2 (N35 ID) vector) into which the N351D mutation has been introduced is used as a saddle type mutant DNA into which two mutations have been introduced using the mutation introduction primer described in V) above ( N351D + L530D), using the plasmid for introduction of Y367A mutation (pBS-OASA2 (Y367A) vector) as a saddle type and introducing the two mutations using the mutagenesis primers described in v) above Obtained mutant DNA (Y367A + L530D)
- N351D + Y367A + L530A is a plasmid DNA (pBS-OASA2 (N351D + L530A) vector) into which a mutation of N351D + L530A has been introduced in advance, using the mutation-introducing primer described in ii) above.
- mutant DNA N351D + Y367A + L530A into which three mutations were introduced was obtained.
- the plasmid DNA (pBS-OASA2 (G522Y + L530D) vector) into which the mutation of G522Y + L530D has been introduced is used as a saddle type, and three mutations are introduced using the primers for mutation introduction described in ii) above.
- Mutant DNA Y367A + G522Y + L530A.
- the plasmid DNA (pBS-OASA2 (G522Y + L530D) vector) into which the mutation of G522Y + L530D has been introduced is used as a saddle type, and three mutations are introduced using the mutation introduction primers described in iii) above.
- a mutant DNA (Y367I + G522Y + L53 OA) was obtained.
- Mutagenesis by PCR was performed according to the method described in the following reference.
- PCR was performed separately for the side region and the 3 'side region so that the OASA2 gene overlapped with the primer region for mutagenesis.
- the 5 'region is a PBS-OASA2 vector
- the cloning sense primer (5 '-AAAA CTAGTATGGAGTCCATCGCCGCCGCCACG-3': SEQ ID NO: 41, underlined indicates the restriction enzyme Spel site) and primer S126F-R
- PCR was performed, and the 3 'region was pBS-OASA2 vector
- primer S126F- PCR was performed in combination with F and anti-sense primer for cloning (5 AAAGTCGACTGAGAGAGACTCTATTCCTTGTC-3 ': SEQ ID NO: 42, underlined indicates the restriction enzyme Sail site).
- a pBS-OASA2 vector is used as a saddle-type mutation by replacing the 530th leucine residue of OASA2 with an aspartic acid residue by amino acid substitution using the Kunkel method described above.
- the already introduced plasmid DNA was used. Therefore, by performing PCR using the mutation-introducing primers (S126F-F and S126F-R) described in vii) above, the 126th serine residue of OA SA2 was replaced with a phalalanin residue. A mutant gene into which two mutations for amino acid substitution of the second leucine residue to the aspartic acid residue are introduced is obtained.
- the combination of primers is the same as described above.
- the double mutant DNA of A369L + L5 30D was prepared in the same type as above and the mutation introduction program described in viii) above.
- the mutation introduction program described in viii) above By performing PCR using a lymer (A369L-F and A369L-R), the 369th alanine residue of OASA2 was replaced with a leucine residue, and the 530th leucine residue was replaced with an aspartic acid residue.
- a mutant gene into which two mutations are introduced is obtained.
- the composition of the PCR reaction was 1 X Pyrobest buffer II (TaKaRa), 0.2 mM dNTP, 0.5 M sense and antisense primer, 0.025 units / ⁇ 1 Pyrobest DNA polymerase (TaKaRa), lOng ⁇ DNA
- a PCR reaction device TeKaRa PCR Thermal Cycler MP TP3000 type, manufactured by Takara Shuzo Co., Ltd.
- the reaction for 3 minutes was repeated 25 cycles, held at 72 ° C for 10 minutes, and then cooled to 4 ° C.
- the amplified PCR product (5 'region fragment and 3' region fragment) was diluted 20-fold, and the PCR reaction solution [1
- X Pyrobest buffer II (TaKaRa), 0.2 mM dNTP, 0.025 units / ⁇ 1 Pyrobest DNA polymerase (TaKaRa)] is added at a rate of 1 / zl, and 2 times at 95 ° C using the above PCR reactor. After holding the reaction for 5 minutes, the reaction at 98 ° C for 20 seconds, 60 ° C for 35 seconds, and 72 ° C for 3 minutes was repeated 5 cycles to carry out extension reaction. By this reaction, a DNA fragment ligated from the cloning sense primer to the cloning antisense primer was synthesized.
- a cloning sense primer and a cloning antisense primer are added so that the primer concentration is 0.2 ⁇ each, and the PCR device is used at 95 ° C. Then, the reaction was repeated 20 cycles at 98 ° C for 20 seconds, 60 ° C for 35 seconds and 72 ° C for 3 minutes, held at 72 ° C for 10 minutes, and then cooled to 4 ° C.
- the DNA fragment linked to the above-mentioned cloning sense primer and cloning anti-sense primer was amplified, and this was amplified into the cloning vectors pBluescript KS + (Stratagene) Spel and Sail of the multicloning site.
- PBS-OASA2 S126F
- pBS-OASA2 A369L
- pBS-OASA2 S126F / L530D
- pBS-OASA2 A369L / L530D
- Example 2 Protein synthesis by wheat germ cell-free system
- the OASA2 gene is encoded on the rice nuclear genome and is a force-synthesized gene. It turns out that proteins migrate to the chloroplast and function in the chloroplast.
- the synthesized protein has a signal sequence for transfer to chloroplasts in the N-terminal region, and when this sequence is removed, it becomes a mature enzyme and exhibits enzyme activity. Therefore, in order to synthesize OA SA2 protein as a mature enzyme in a wheat germ cell-free synthesis system, the primer was designed so that it was synthesized in a form in which the 49-residue signal sequence present in the N-terminal region was removed.
- the ATG initiation codon was placed downstream of the linker sequence that enables split-PCR in the wheat germ cell-free synthesis system, and a 36-mer primer with the 148th to 164th base sequence strength of the OASA2 gene was designed.
- two types of antisense primers for Split-PCR were set on the complementary strand side of the above-mentioned sense Split-PCR primer on a vector (pBluescript SK +) in which the OASA2 gene was inserted.
- Antisense Split-PCR primer 1 and antisense Split-PCR primer 2 were used in order from the position of the inserted DNA.
- the base sequences of the primers for Split-PCR are as follows. Split-PCR will be described later.
- Antisense Split-PCR Primer 1 5'- GGAGAAAGGCGGACAGGTAT-3 '(SEQ ID NO: 22)
- Antisense Split-PCR Primer 2 5'- GGGGAAACGCCTGGTATCTT-3 '(SEQ ID NO: 23)
- the composition of the PCR reaction solution was 1 X Pyrobest buffer II (TaKaRa), 0.2 mM dNTP, 0.5 ⁇ M sense Split-PCR primer and antisense Split-PCR primer 1, 0.025 un its / ⁇ 1 Pyrobest DNA polymerase ( TaKaRa), lOng ⁇ type DNA (pBS-OASA2 vector into which each mutation has been introduced), and kept at 95 ° C for 2 minutes using a PCR reactor (TaKaRa PCR Thermal Cycler MP TP3000 type, manufactured by Takara Shuzo) And then at 98 ° C for 20 seconds and at 60 ° C for 35 seconds The reaction for 3 minutes at 72 ° C was repeated 25 cycles, held at 72 ° C for 10 minutes, and then cooled to 4 ° C.
- the DNA fragment ligated to the above-described primer for antisense Split-PCR and primer 1 for antisense Split-PCR was amplified, and this was used as the split-PCR cage type to be performed next
- split-PCR type DNA for transcription PCR into a transcription type DNA
- an SP6 RNA polymerase promoter and a tobacco mosaic virus (TMV) -derived translation enhancement sequence (omega sequence) are added upstream of the 5 'end of the fragment. I need to hesitate. Therefore, split-PCR was performed according to the method of Sawasaki et al. (Sawasaki et a 1. (2002) Proc Natl Acad Sci USA 99, 14652-14657).
- the above split-PCR vertical DNA was diluted 50 times, and PCR reaction solution [1 X EX Taq buffer (TaKaRa), 0.2 mM dNTP, 0.025 units / ⁇ 1 TaKaRa EX Taq (TaKaRa), 0.2 ⁇ M Add 1 to SP6 promoter primer, InM TMV-Omega primer, 0.2 ⁇ M antisense Split-PCR primer 2] and hold at 95 ° C for 2 minutes using the above PCR reactor. The reaction was repeated for 35 cycles at 20 ° C for 20 seconds, 60 ° C for 35 seconds, and 72 ° C for 3 minutes, held at 72 ° C for 10 minutes, and then cooled to 4 ° C.
- the nucleotide sequences of SP6 promoter primer and TMV-Omega primer are as follows. The underline indicates the SP6 promoter sequence, and the lower case letters indicate overlapping portions of both primers.
- SP6 promoter primer 5'- GCGTAGCATTTAggtgacact-3 '(SEQ ID NO: 24)
- the primer is divided into an upstream primer and a downstream primer, and the sequences of the 3 ′ end of the upstream primer and the 5 ′ end of the downstream primer are the same.
- a primer designed to overlap partially is called a split primer.
- a PCR reaction using such primers is called Split-PCR.
- MRNA was synthesized (transcribed) using the PCR product obtained in (1) above directly as a saddle type.
- the PCR product obtained in (1) above was transferred to the transcription reaction solution [80 mM HEPES-KOH (pH 7.8), 16 mM Mg (OAc) 2, lOmM spermidine ⁇ lOmM DTT ⁇ 3 mM NTP ⁇ 1 unit / ⁇ 1 RNasin RNase inhibitor (Promega), 1 unit / ⁇ 1 SP6 RNA polymerase (Promega)] was added in an amount of 1/10. After 2 hours of reaction at 37 ° C, ethanol precipitation and 70% ethanol washing were performed, and the product was dissolved in an appropriate amount of sterile water. The amount of RNA was calculated by measuring the absorbance at 260 °.
- the protein synthesized using the mRNA synthesized in (3) above as a saddle type was synthesized. That is, the above mRNA (about 30-35 ⁇ g) was added to a dialysis cup containing 50 1 wheat germ cell-free protein synthesis solution, and the dialysis force was added to a 24-well plate containing 1 ml of substrate solution per well. Soaked and incubated at 26 ° C for 24 hours.
- the OASA2 protein which is a subunit of rice anthralate synthase, also produces an anthrates with chorismate using ammonia derived from the amide group of glutamine supplied by the 13 subunit.
- AS activity an anthralic acid synthesis activity
- Addition also shows enzyme activity.
- the activity of OASA2 protein is feedback-suppressed by tryptophan. Therefore, it is essential to remove tributophane from the protein synthesis reaction solution, and it is necessary to change the environment of the expressed protein to a buffer component for measuring AS activity. Therefore, the component of the protein synthesis reaction solution was buffer A (50 mM Tris—HCl, pH7.6; 0.05 mM EDTA; 2 mM MgC 1; 0.05 mM DTT; 5% using Microspin G-25 columns (Amersham Biosciences) (Glycerol).
- buffer A 50 mM Tris—HCl, pH7.6; 0.05 mM EDTA; 2 mM MgC 1; 0.05 mM DTT; 5% using Microspin G-25 columns (Amersham Biosciences) (Glycerol).
- the reaction was stopped by adding 10 ⁇ l of IN HCl, and the synthesized anthralic acid was extracted by adding 300 ⁇ l of ethyl acetate.
- the synthesis amount of anthralic acid was measured using Wallac 1420 ARVOsx-FL (manufactured by PerkinElmer Life Science Japan) at an excitation wavelength of 355 nm / fluorescence (emmision) of 460 nm.
- trybutophane was added to the reaction system to a final concentration of 10 ⁇ or 100 ⁇ .
- OASA2 proteins A369L, S126F / L530D, Y367A / L530D, A36 9L / L530D, N351D / Y367A / L530D, Y367A / G522Y / L530D and Y367I / G522Y / L5
- the enzyme activity in the absence of tributophan was equal to or greater than that of the wild type, and resistance to tributophan feedback inhibition was obtained.
- the reaction was stopped by adding 10 ⁇ l IN HC1, and the synthesized anthraeric acid was extracted by adding 300 ⁇ l ethyl acetate.
- the synthesis amount of anthralic acid was measured using Wallac 1 420 ARVOsx-FL (manufactured by PerkinElmer Life Science Japan) at an excitation wavelength of 355 nm / fluorescence (emmision) of 460 nm.
- trybutophane was added to the reaction system to a final concentration of 10 M, M, or M.
- A369L is about 1 time
- S126F / L530D is about 2.5 times
- Y367A / L5 300 is about 2.2 times
- A369L / L530D is about 1.8 times
- the enzyme activity was approximately 1.3 times that of L530D, approximately 1.2 times that of N351D / Y367A / L530D, and approximately 0.8 times that of Y367I / G522Y / L530D.
- Figure 3 shows the enzyme activity of wild-type and mutated OASA2 protein enzyme activity at 100% when no tryptophan is added, and the enzyme activity when each concentration of tributophan is added is compared with the enzyme activity when no tribute is added. It is a thing.
- the wild type When tryptophan was added in an amount of 50 ⁇ ⁇ or more, the enzyme activity was less than 10% of that with no additive, but nine mutant OASA2 proteins (S126F, Y367A, A369L, S126F / L530D, Y367A / L530D, A369L / L530D) N351D / Y367A / L530D, Y367A / G522Y / L530D and Y36 7I / G522Y / L530D) have an enzyme activity of 30% or more when no tryptophan is added even when 50 ⁇ M is added. It was.
- Example 4 Analysis of free tryptophan content of yeast TRP2 gene-deficient mutant strain (MAT alpha his3 ⁇ 1 leu2 ⁇ 0 metl5 ⁇ 0 ura3 ⁇ 0 trp2 :: KANMX) expressing a mutant OASA2 gene]
- Yeast lacking the TRP2 gene corresponding to the OASA2 gene requires tryptophan for growth. Therefore, if the OASA2 gene is introduced into this defective strain and it can grow in the absence of tributophan, the introduced OASA2 gene can complement the function of TRP2.
- a mutant OASA2 gene resistant to tributophan feedback inhibition can be expressed in this yeast, and the content of free tributophan accumulated in the living body can be measured to test the effect of the gene.
- mutant OASA2 genes (S126F, Y367A, A369L, S126F / L530D, Y367A / L530D, A369L / L530D, N351D / Y367A / L530D) resistant to tributophan feedback inhibition were constructed.
- Y367A / G522Y / L530D, Y367I / G522Y / L530D pBluescript vector into which the wild type (Wt) OASA2 gene was inserted, and PCR was performed using the following primers.
- the chloroplast translocation signal is present in the N-terminal region of OASA2 protein, it is expressed in a form in which the signal sequence is removed in the same manner as the “Split-PCR primer” design described in (2) above.
- I designed the primer.
- restriction sites Kpnl / The sense primer as incorporated into EcoRI restriction enzyme site Kpnl (GGTACC) of the yeast expression vector pYES2 (I n vitr made 0 g en, Inc.), limit the antisense primer enzyme sites EcoRI (GAATTC ) was introduced. These restriction enzyme sites are underlined.
- the pYES2 vector has the URA3 gene, and the induction of protein expression can be controlled by galactose.
- Sense primer 5, -AAAGGTACCATGTGCTCCGCGGGGAAGCC-3, (SEQ ID NO: 27)
- Antisense primer 5'-AAAGAATTCTGAGAGAGACTCTATTCCTTGTC-3 '(SEQ ID NO: 28)
- the composition of the PCR reaction solution was 1 X Pyrobest buffer II (TaKaRa), 0.2 mM dNTP, 0.5 M sense and antisense primer, 0.025 units / ⁇ 1 Pyrobest DNA polymerase (TaKa Ra), lOng ⁇ type DNA (pBS -OASA2 vector) and hold it at 95 ° C for 2 minutes using a PCR reactor (TaKaRa PCR Thermal Cycler MP TP3000 type, manufactured by Takara Shuzo), then at 98 ° C for 20 seconds and at 60 ° C for 35 seconds The reaction for 3 minutes at 72 ° C was repeated 25 cycles, held at 72 ° C for 10 minutes, and then cooled to 4 ° C.
- the yeast transformants were prepared on a synthetic complete agar medium containing 2% glucose (0.67% amino acid-free! Batata yeast nitrogen base, excluding uracil, 0.2% dropout mixture, 2% butatoguar). A single colony is isolated, and then the cells are synthesized in a synthetic agar medium containing 2% galactose (0.67% free of amino acids! /, Batato yeast nitrogen base, 0.2% drop excluding uracil and tryptophan) Out mixture, 2% buttowager) and incubated at 30 ° C for 2 days. 20 mg of the bacterial cells picked up from the agar medium were suspended in 105 ⁇ l of distilled water and treated at 100 ° C. for 20 minutes.
- HPLC high performance liquid chromatography
- Example 5 Production of rice callus transformant expressing mutant OASA2 gene expression and analysis of free tributophan content of the transformed callus
- Construction of a recombinant vector for introducing a foreign gene was performed according to the method described in the following reference.
- the recombinant vector for exogenous gene transfer pUB-Hm (UrusWbara et al., Breeding Science 5 1: 33-38 (2001)) is a maize ubiquitin promoter, first intron, restriction enzyme Sse8387I site, NOS
- a recombinant vector for rice transformation can be constructed by inserting a terminator and a hygromycin resistance gene and inserting a foreign gene of interest in the Sse8387I site.
- the wild-type (Wt) OASA2 gene consisting of the full length was amplified by PCR using the pBS-OASA2 described in [Example 1] as a saddle and the sense primer and antisense primer shown below.
- the restriction enzyme site SpeI (ACTAGT) was introduced into the sense primer
- the restriction enzyme site Sall (GTCGAC) was introduced into the antisense primer.
- Sense primer 5 AAAACTAGTATGGAGTCCATCGCCGCCGCCACG— 3 '(SEQ ID NO: 43)
- Antisense primer 5 and AAAGTCGACTGAGAGAGACTCTATTCCTTGTC— 3 '(SEQ ID NO: 44)
- the full-length mutant OASA2 gene (Y367A, Y367A / L530D, S126F / L530D) has pBS-OASA2 as a saddle type and the above-mentioned sense primer (SEQ ID NO: 43) and antisense primer (SEQ ID NO: 44).
- SEQ ID NO: 43 sense primer
- SEQ ID NO: 44 antisense primer
- Expression vector for wheat germ cell-free synthesis system Improved expression vector pEU3b for wheat germ cell-free synthesis system and introduced Sse8387I site at both ends of the multi-cloning site Spel / Sall site. Inserted into / Sall site.
- the above-mentioned mutant OASA2 gene (Y367A, Y367A / L530D, S126F / L530D) or Digested the pEU3s plasmid vector containing the wild-type (Wt) OASA2 gene with the restriction enzyme Sse83871 to obtain a 1.8 kb DNA fragment (2).
- the pUB-Hm plasmid vector digested with the above restriction enzyme Sse8387I and the DNA fragment (2) were treated with DNA ligation 'kit ver.2 (manufactured by Takara Bio Inc.) to carry out a DNA ligation reaction. Thereby, a circular recombinant vector was constructed.
- This recombinant vector has a first intron region downstream of the ubiquitin promoter region, and a mutant OASA2 gene (Y367A, Y367A / L530D, S126F / L5 30D) between the first intron region and the NOS terminator region.
- a wild-type (Wt) OASA2 gene was inserted and linked, and had a structure containing a hygromycin resistance gene expressed in plants.
- the resulting recombinant vectors for introduction of foreign genes have pUB-OASA2 (wt) -Hm (having wild type OASA2 gene) and pUB-OASA2 (Y367A) -Hm (mutant OASA2 gene (Y367A), respectively) ), PUB-0 ASA2 (Y367A / L530D) -Hm (having mutant OASA2 gene (Y367A / L530D)) and pUB-OASA2 (S126F / L530D) -Hm (having mutant OASA2 gene (S126F / L530D)) ).
- Figure 5 shows a schematic diagram of pUB-OASA2 (wt) -Hm, pUB-OASA2 (Y367A) -Hm, pUB-OASA2 (Y367A / L53 0D) -Hm and pUB-OASA2 (S126F / L530D) -Hm Indicated.
- PUbi represents the ubiquitin promoter region
- P35S represents the cauliflower mosaic virus 35S promoter region
- hpt represents the hygromycin resistance gene region
- Sse8387I represents the restriction enzyme site.
- RB represents the right border array
- LB represents the left border array.
- YEB medium Bact Yeast Esteract lg / l, Sucrose 5g / l, 2mM MgCl
- Agrobacterium (Agrobacterium tumefaciens EHA101) was transplanted to 50 ml of a liquid medium of pH 7), and cultured with shaking at 30 ° C for 16 hours. Then, the culture solution was centrifuged at 4 ° C to obtain bacterial precipitates. The bacterial precipitate was suspended in ice-cooled 10 mM Tris-HCl pH 7.5, and the precipitate was obtained by centrifugation. The precipitate was suspended in 0.5 ml of YEB medium. To 0.2 ml of this bacterial suspension, the above three kinds of recombinant vector transfection solutions (5 g) were introduced and mixed well.
- Rice (variety: Nipponbare) was threshed from ripened seeds.
- the obtained seeds with a coat were immersed in a 70% ethanol solution for 60 seconds and then immersed in a sodium hypochlorite solution containing about 4% of effective chlorine for 6 minutes to sterilize the seeds. Furthermore, the seeds were washed with sterilized water.
- a 2N6 solid medium comprising 30g / l of sucrose, 2,4-D 2mg / l, casamino acid lg / 1 and gellite 2g / l, and N6 vitamin added to the inorganic salt composition of N6 medium ( UrusWbara et al., Breeding Science 51: 33-38 (2001)) was laid with rice seeds sterilized as described above. Callus was formed by incubating at 28 ° C for 3 weeks. The callus was also excised from the endosperm, and the callus was transferred to a medium having the same composition as described above and cultured for 7 days.
- the transformed agropacterium obtained as described above was introduced into the AA medium composition (Hiei et al., Plant Journal 6: 271-282 (1994)) and sucrose 20 g / l, 2 , 4-D 2 mg / l, force rice 0.2 mg / l and acetosyringone 10 mg / l were suspended in 30 ml of a liquid medium to obtain a bacterial solution. This suspension was placed in a 9 cm petri dish, and 100 rice calluses obtained as described above were added, and then immersed for 5 minutes.
- the inorganic salt composition of N6 medium is 30 g / l sucrose, 10 g / l glucose, 2,4-D 2 mg / l, lg / l casamino acid, 2 g gellite. Twenty soaked calli were placed on each petri dish on a 2N6CO solid medium supplemented with 10 mg / l of acetosyringone / l. Cultured in the dark at 24 ° C for 3 days, rice callus was infected with Agrobataterium.
- the transformed callus into which the recombinant vector was introduced was washed with sterilized water supplemented with 500 mg / l carbecillin to remove agrobacterium. After removing excess water from the callus, the callus was mixed with the mineral salt composition of N6 medium at 30 g / l sucrose, 2,4-D 2 mg / l, 20 cells were transferred per petri dish onto 2N6SE solid medium supplemented with 500 mg / l phosphorus, 30 mg / l hygromycin, lg / l casamino acid, and 2 mg / l gellite. By culturing in the dark at 28 ° C for 3 weeks, transformed calli resistant to idaromomycin was obtained. Furthermore, the growth callus was transplanted onto an individual medium having the same composition, and the hygromycin-resistant transformed callus was cultured at 28 ° C. for 3 weeks.
- Sense primer 5'-GGATGGCACCCGCAGCAGATCG-3 '(SEQ ID NO: 45)
- Antisense primer 5'- GTACTCATCACTTGTCATGGTTG-3' (SEQ ID NO: 46)
- the composition of the PCR reaction solution was 1 X Pyrobest buffer II (TaKaRa), 0.2 mM dNTP, 0.5 ⁇ M sense primer (SEQ ID NO: 45) and antisense primer (SEQ ID NO: 46), 0.025 units / ⁇ 1 Pyrobest Use DNA polymerase (TaKaRa), lOng ⁇ type DNA (callus DNA) and hold it at 95 ° C for 2 minutes using a PCR reaction device (TaKaRa PCR Thermal Cycler MP TP3000 type, manufactured by Takara Shuzo), then 98 ° C The reaction was repeated for 20 cycles at 60 ° C for 35 seconds and at 72 ° C for 3 minutes for 25 cycles, held at 72 ° C for 10 minutes, and then cooled to 4 ° C.
- Mutant OASA2 gene (Y367A, Y367A / L530D) or wild type (Wt) OASA2 gene
- lOOmg of each rice callus was ground in liquid nitrogen.
- Transfer the pulverized product to a 1.5 ml tube, and then add 500 1 chloroform: methanol: water mixture (5: 12: 3 volume ratio), mix well, then centrifuge at 5,000 Xrpm Centrifuged for 10 minutes at acceleration.
- the obtained supernatant was transferred to a new tube, 375 1 distilled water and 250 1 black mouth form were added, mixed vigorously, and then centrifuged for 10 minutes at a centrifugal acceleration of 5,000 X rpm.
- the aqueous layer thus extracted was transferred to a new tube, dried under reduced pressure, and then dissolved in 2 ml of 10 mM NaOH to obtain a tryptophan extract.
- HPLC high performance liquid chromatography
- the power ram used for this HPLC was Xterra RP18 column (4.6 X 150 mm) (Waters), and the developing solvent was acetonitrile-0.1M H3P04 aqueous solution (pH 4.0) with a concentration of 5% to 45% of acetonitrile.
- the gradient was used, the flow rate was 1 ml / min, the fluorescence wavelength was 348 nm at the excitation wavelength of 278 nm, and the tryptophan content was evaluated.
- Table 3 shows the measurement results obtained. As is clear from Table 3, the content of free tributophan produced in rice calli that expressed mutant OASA2 genes (Y367A, Y367A / L530D) resistant to inhibition of tributophan feedback was higher than that in the control rice callus. It was confirmed that it accumulated (5.5 to 38.8 times that of the wild type).
- Table 4 shows the measurement results obtained. As is clear from Table 4, the content of free tributophan produced in rice plants expressing mutant OASA2 gene (S126F / L530D) resistant to inhibition of tributophan feedback was higher than that in the control rice callus. It was confirmed that Specifically, it was 123 to 467 times that of the wild type.
- the present invention it is possible to produce a plant having a high tributophan accumulation. Therefore, the present invention can be widely used in agriculture, and the high tributophane-accumulating plant is suitable for livestock feed and food raw material, so that the present invention can also be used in the livestock and food industries.
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Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
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EP05793125A EP1870469A4 (en) | 2005-02-28 | 2005-10-11 | NOVEL MODIFIED GENE OF OASA2 GENE OF ANTHRANILIC ACID SYNTHASE OF RICE AND USE THEREOF |
BRPI0520119-5A BRPI0520119A2 (pt) | 2005-02-28 | 2005-10-11 | gene alterado do gene sintase de Ácido antranÍlico de arroz oasa2 e uso do mesmo |
CA002595529A CA2595529A1 (en) | 2005-02-28 | 2005-10-11 | Novel altered gene from rice anthranilic acid synthase gene oasa2 and use thereof |
AU2005328634A AU2005328634B2 (en) | 2005-02-28 | 2005-10-11 | Novel altered gene from rice anthranilic acid synthase gene OASA2 and use thereof |
US11/795,389 US20080134370A1 (en) | 2005-02-28 | 2005-10-11 | Novel Altered Gene from Rice Anthranilic Acid Synthase Gene Oasa2 and Use Thereof |
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JP2005055165A JP4651410B2 (ja) | 2004-04-13 | 2005-02-28 | イネのアントラニル酸合成酵素遺伝子oasa2の新規改変遺伝子およびその利用 |
JP2005-055165 | 2005-02-28 |
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US (1) | US20080134370A1 (ja) |
EP (1) | EP1870469A4 (ja) |
CN (1) | CN101128584A (ja) |
AU (1) | AU2005328634B2 (ja) |
BR (1) | BRPI0520119A2 (ja) |
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US5965727A (en) * | 1996-07-26 | 1999-10-12 | Board Of Trustees Of The University Of Illinois | For selectable markers and promoters for plant tissue culture transformation |
WO2002090497A2 (en) * | 2001-05-04 | 2002-11-14 | Renessen Llc | Transgenic high tryptophan plants |
WO2003092363A2 (en) * | 2002-05-03 | 2003-11-13 | Monsanto Technology, Llc | Transgenic high tryptophan plants |
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JP2005245336A (ja) * | 2004-03-04 | 2005-09-15 | Japan Science & Technology Agency | 変異タンパク質の機能変化のスクリーニング方法およびその利用 |
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2005
- 2005-10-11 EP EP05793125A patent/EP1870469A4/en not_active Withdrawn
- 2005-10-11 WO PCT/JP2005/018708 patent/WO2006092882A1/ja active Application Filing
- 2005-10-11 AU AU2005328634A patent/AU2005328634B2/en not_active Ceased
- 2005-10-11 CA CA002595529A patent/CA2595529A1/en not_active Abandoned
- 2005-10-11 CN CNA2005800487026A patent/CN101128584A/zh active Pending
- 2005-10-11 BR BRPI0520119-5A patent/BRPI0520119A2/pt not_active IP Right Cessation
- 2005-10-11 US US11/795,389 patent/US20080134370A1/en not_active Abandoned
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5965727A (en) * | 1996-07-26 | 1999-10-12 | Board Of Trustees Of The University Of Illinois | For selectable markers and promoters for plant tissue culture transformation |
WO2002090497A2 (en) * | 2001-05-04 | 2002-11-14 | Renessen Llc | Transgenic high tryptophan plants |
WO2003092363A2 (en) * | 2002-05-03 | 2003-11-13 | Monsanto Technology, Llc | Transgenic high tryptophan plants |
Non-Patent Citations (4)
Title |
---|
AKIRA WAKASA ET AL: "Idenshi kumikae Gijutsu ni yoru Amino Acid Seibum no Zoka shita Ine to Shinki Marker Idenshi no Kaihatsu", NOGYO KENKYU CENTER NEWS, no. 72, 1998, pages 8 - 9, XP003002048 * |
NIYOGI K.K ET AL: "Two Anthranilate synthase genes in arabidosis: defense-related regulation of the tryptophan pathway", THE PLANT CELL, vol. 4, 1992, pages 721 - 733, XP002032324 * |
See also references of EP1870469A4 * |
SONG H.S. ET AL: "Tissue culture-specific expression of a naturally occurring tobacco feedback-insensitive anthranilate synthase", PLANT PHYSIOL., vol. 117, 1998, pages 533 - 543, XP003002047 * |
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BRPI0520119A2 (pt) | 2009-04-22 |
EP1870469A4 (en) | 2009-03-25 |
US20080134370A1 (en) | 2008-06-05 |
AU2005328634B2 (en) | 2010-01-28 |
CN101128584A (zh) | 2008-02-20 |
CA2595529A1 (en) | 2006-09-08 |
AU2005328634A1 (en) | 2006-09-08 |
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