WO2009084439A1 - リグナン水酸化酵素 - Google Patents
リグナン水酸化酵素 Download PDFInfo
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- WO2009084439A1 WO2009084439A1 PCT/JP2008/072943 JP2008072943W WO2009084439A1 WO 2009084439 A1 WO2009084439 A1 WO 2009084439A1 JP 2008072943 W JP2008072943 W JP 2008072943W WO 2009084439 A1 WO2009084439 A1 WO 2009084439A1
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
- C12P17/181—Heterocyclic compounds containing oxygen atoms as the only ring heteroatoms in the condensed system, e.g. Salinomycin, Septamycin
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
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- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
- C07D493/02—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
- C07D493/04—Ortho-condensed systems
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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)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
- C12N15/8255—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving lignin biosynthesis
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
Definitions
- the present invention relates to an enzyme having an activity of transferring a hydroxyl group to lignan and a method of using the enzyme.
- Lignans are secondary metabolites of vascular plants (such as sesamin and sesamorin) and are widely distributed in plants. So far, lignans have been reported to be contained in plant seeds, fruits, cuttings, tubers, and / or tuberous roots, and are thought to contribute mainly to biological defense mechanisms in plants. . This lignan is attracting attention as having a wide range of physiological and pharmacological functions outside of plants due to its strong antioxidant properties and the like. Lignans have a structure in which two molecules of a phenylpropanoid compound having a C 6 -C 3 skeleton are polymerized, and the 8,8 ′ bond type is most common (Lignans, DC Ayres and JD. Loike (1990)).
- Representative lignans include (+)-pinoresinol, (+)-sesamin, (+)-sesaminol, (+)-sesamorin and (+)-sesamolin contained in sesame indicum; Forsythia intermedia (+)-Pinoresinol, (-)-Arctigenin and (-)-Mata Resinol; (-)-Pinoresinol and (-)-Larisiresinol; Linum usitatissimum contained in Daphne tangutica (+)-Secoisolariciresinol contained therein (Phytochemistry Rev. (2003) 2: 257-288). The molecular structures of these lignans are diverse and are classified into 8 subclasses based on the skeletal structure (see Phytochemistry Rev. (2003) 2: 371-390).
- pinoresinol is the first biosynthetic lignan synthesized by the polymerization of coniferyl alcohol, and it is diverse from (+)-pinoresinol through various biosynthetic pathways unique to individual plant species.
- Lignans have been reported to be synthesized (see J. Wood. Sci. 53, 273-284 (2007), Lignans: biosynthesis and function, Comprehensive natural products chemistry, (1999) 1: 640-713).
- Piperitol is synthesized by the action of piperitol synthase on (+)-pinoresinol.
- Pinoresinol is a major lignan that is distributed in many plants because it is the first lignan synthesized in the lignan biosynthetic pathway, for example, Asteraceae, Asteraceae, Asteraceae, Apiaceae, Dendrobaceae It is contained in magnoliaceae, liliaceae and pineaceae plants.
- Non-patent Document 6 Plant : Physiol., (2000) 123: 453 and patents.
- Reference 1 Refer to Japanese translations of PCT publication No. 2001-507931).
- forsythia pinoresinol-larisiresinol reductase gene see Non-Patent Document 7: J. Biol.
- Non-Patent Document 9 J. Biol. Chem., (2001) 276 : No. 12614 and Japanese Patent Application Laid-Open No. 2002-512790). Further, sesame has reported a cytochrome P450 enzyme gene having piperitol-sesamin synthesis activity and a method of use (Non-patent Document 10: Proc. Nat. Acad. Sci. USA, (2006) 103: 10116 and Patent Document 3). : Refer to Japanese Translation of PCT International Publication No. 2007-507201).
- Lignans are known to undergo a variety of modifications such as glycosylation, hydroxylation, methylation, and prenylation after skeleton formation, and have glycosylation activity against furofuran-type lignans such as sesamignan sesaminol.
- a glucosidase gene has been isolated and a method for using it has been reported (see Patent Document 1: Japanese Patent Application Laid-Open No. 2006-129728).
- Non-Patent Document 1 Ligularia kanaitizensis of the genus Metacaraceae native to China and Allamanda neriifolia of the genus Allamanda genus Oleander belong to the 9th hydroxylated derivative of furofuran-type lignan represented by pinoresinol (Non-Patent Document 1). : Lignans, D.C.Ayres and JD.Loike (1990); Non-patent document 2: Phytochemistry, (1988) 27,575, Non-patent document 3: Indian J. Chem., (1995) 34B, 975, etc. reference).
- 9-Hydroxypinoresinol a 9-position hydroxylated derivative
- 2-methyl-2-butenoic-pinoresinol which has been further modified by hydroxyl groups to become ester groups, has been found to have anti-HIV-1 reverse transcriptase (RT) activity. Elucidation of the route is awaited (Non-patent document 4: J.
- Non-patent Document 9 De Monteno
- P.R.O. Cytochrome P450-structure, mechanism, and biochemistry.3rd edition.Kluwer Academic / Plenum Publishers, NY. (2005)
- Non-Patent Document 10 J. Biol. reference).
- (+)-raleatricine hydroxylase is a lignan hydroxylase that does not have oxygen at the 9 (9 ') position of the furan ring, and still catalyzes the 9-position hydroxylation of furofuran-type lignans typified by pinoresinol.
- the enzyme is unknown. Therefore, further acquisition of lignan oxidase gene and functional analysis are desired.
- the present invention has been made in view of the above circumstances, and an object of the present invention is to provide an enzyme having lignan hydroxylation activity, particularly an enzyme that catalyzes a reaction for transferring a hydroxyl group to lignans, preferably from piperitol to 9-hydroxy. It is to provide an enzyme that catalyzes the hydroxylation reaction of piperitol, pinoresinol to 9-hydroxypinoresinol.
- the object of the present invention is to use a hydroxylated lignan (preferably, the 9-position is hydroxylated by metabolic engineering using an enzyme having an activity of transferring a hydroxyl group to the lignan (preferably at the 9-position). Lignan; hereinafter 9-hydroxylated lignan).
- a further object of the present invention is to provide a method for producing metabolically engineered plants in which the content ratio of lignan and hydroxylated lignan is increased or decreased in order to efficiently produce lignan or hydroxylated lignan. .
- the present invention includes the following polypeptides having lignan hydroxylation activity, polynucleotides encoding the same, vectors or transformants containing the polynucleotides, and polypeptides having lignan hydroxylation activity using the transformants.
- the present invention relates to a method for producing a peptide.
- a polypeptide having lignan hydroxylation activity (A) the amino acid sequence of SEQ ID NO: 26; (B) an amino acid sequence in which 1 to 15 amino acids are deleted, inserted, substituted and / or added in the amino acid sequence of SEQ ID NO: 26; or (c) at least 80 relative to the amino acid sequence of SEQ ID NO: 26 Amino acid sequence having% identity, A polypeptide containing (2) A polypeptide having lignan hydroxylation activity as described in (1) above, (A) the amino acid sequence of SEQ ID NO: 26; or (b ′) an amino acid sequence in which one to several amino acids are deleted, inserted, substituted and / or added in the amino acid sequence of SEQ ID NO: 26; A polypeptide containing (3) A polynucleotide which is any of the following (a) to (e): (A) a polynucleotide comprising the base sequence of SEQ ID NO: 27; (B) a polynucleotide encoding a protein
- the polynucleotide according to (3) which encodes a polypeptide having lignan hydroxylation activity and is any one of the following (f) to (i): (F) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 27; (G) a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 27; (H) a polynucleotide comprising a base sequence that is at least 90% identical to the base sequence of SEQ ID NO: 27; or (i) one to several bases deleted, inserted, substituted and replaced in the base sequence of SEQ ID NO: 27 Polynucleotide added.
- the polynucleotide according to (3) above comprising the base sequence of SEQ ID NO: 27.
- An oligonucleotide comprising the fragment of the polynucleotide according to any one of (3) to (6) above or a complementary sequence thereof.
- the oligonucleotide according to (7) which suppresses the expression of the polypeptide according to (1) or (2).
- a vector comprising the polynucleotide according to any one of (3) to (6) above.
- (10) A method for producing a polypeptide, wherein the vector according to (9) is used.
- (11) A transformant in which the polynucleotide according to any one of (3) to (6) is introduced.
- (12) The content ratio of hydroxylated lignan is modified by introducing the lignan and the polynucleotide according to any one of (3) to (6) above.
- (13) The transformant according to (11) or (12) above, which is an organism or a progeny thereof, or a tissue derived therefrom.
- (14) The transformant according to (13) above, wherein the organism is a plant.
- (15) The transformant according to (14) above, wherein the plant is sesame, forsythia or flax.
- a method for producing a polypeptide comprising using the transformant according to any one of (11) to (15) above.
- (17) A method for producing a hydroxylated lignan comprising using the transformant according to any one of (11) to (15) above.
- (18) The method for producing a hydroxylated lignan according to the above (17), wherein the hydroxylated lignan substrate is piperitol or pinoresinol.
- (19) A cell comprising the vector according to (9) above.
- (21) A method for producing a polypeptide, comprising using the cell according to (19) or (20).
- (22) A method for producing a hydroxylated lignan, wherein the cell according to (19) or (20) is used.
- (23) The method for producing a hydroxylated lignan according to the above (22), wherein the hydroxylated lignan substrate is piperitol or pinoresinol.
- (24) A method for producing a hydroxylated lignan, comprising using the polypeptide according to (1) or (2) above.
- (25) The method for producing a hydroxylated lignan according to the above (24), wherein the hydroxylated lignan substrate is piperitol or pinoresinol.
- a food or industrial product comprising a hydroxylated lignan obtained by the production method according to any one of (17), (22) or (24).
- the use of the polypeptide (lignan hydroxylase) according to the present invention produces an effect that the amount of lignan and hydroxylated lignan can be artificially adjusted in a living organism (particularly, a plant). Further, by introducing a new hydroxyl group into the lignan using the lignan hydroxylase according to the present invention, it becomes possible to further modify the hydroxyl group, for example, glycosylation or esterification becomes possible. . Based on these effects, the present invention can develop new physiologically functional substances.
- 9-hydroxypiperitol from piperitol and / or 9-hydroxypinoresinol from pinoresinol are artificially produced. There is an effect that it can be produced.
- a plant and / or microorganism in which the amount of lignan and hydroxylated lignan is artificially controlled can be produced by expressing the lignan hydroxylase according to the present invention in a desired organism using gene recombination technology. There is an effect that can be.
- SiD gene by RT-PCR is shown.
- 2 shows the HPLC analysis of the product of SiD6.
- Signal assignment by NMR analysis of P3 (9-hydroxypiperitol) is shown.
- the structure of P3 (9-hydroxypiperitol) is shown.
- a schematic diagram of lignan hydroxylation catalyzed by SiD6 is shown.
- the present inventors have probed a 2-oxoglutarate (hereinafter referred to as 2-OG) -dependent dioxygenase gene belonging to an oxidase family different from polyphenol oxidase which is a conventionally known lignan hydroxylase.
- 2-OG 2-oxoglutarate
- SiD gene sesame 2-OG-dependent dioxygenase-like gene group
- polypeptide having lignan hydroxylation activity according to the present invention, the polynucleotide encoding the polypeptide, and use thereof will be described in detail.
- polypeptide The present inventors have found a novel hydroxylase having lignan, particularly piperitol and / or pinoresinol as a main substrate, and have completed the present invention. Furthermore, the present inventors have found that the novel hydroxylase hydroxylates piperitol. So far, hydroxypiperitol has not been found.
- the present invention relates to a polypeptide having lignan hydroxylation activity, wherein (a) the amino acid sequence of SEQ ID NO: 26; (b) 1-15 amino acids are deleted in the amino acid sequence of SEQ ID NO: 26 A polypeptide comprising: an amino acid sequence inserted, substituted and / or added; or (c) an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 26.
- polypeptide is used interchangeably with “peptide” or “protein”.
- fragment of a polypeptide is intended to be a partial fragment of the polypeptide.
- the polypeptide according to the present invention may also be a polypeptide isolated from a natural source or chemically synthesized.
- isolated polypeptide or protein is intended to be a polypeptide or protein that has been removed from its natural environment.
- recombinantly produced polypeptides and proteins expressed in a host cell can be isolated in the same manner as natural or recombinant polypeptides and proteins that have been substantially purified by any suitable technique. It is thought that there is.
- Polypeptides according to the present invention include natural purified products, products of chemical synthesis procedures, and prokaryotic or eukaryotic hosts (eg, bacterial cells, yeast cells, higher plant cells, insect cells, and mammalian cells). ) From recombinantly produced products. Depending on the host used in the recombinant production procedure, the polypeptides according to the invention may be hydroxylated or non-hydroxylated. Furthermore, the polypeptides according to the invention may also contain an initiating modified methionine residue in some cases as a result of host-mediated processes.
- prokaryotic or eukaryotic hosts eg, bacterial cells, yeast cells, higher plant cells, insect cells, and mammalian cells.
- lignan hydroxylation activity is intended to mean the activity of hydroxylating lignans (preferably the activity of producing 9-hydroxylated lignans), ie the activity of transferring hydroxyl groups to lignans. Is done. That is, as used herein, hydroxylase and hydroxyltransferase are used interchangeably.
- the polypeptide according to the present invention is preferably a polypeptide consisting of the amino acid sequence of SEQ ID NO: 26.
- polypeptide according to the present invention is preferably a polypeptide having the amino acid sequence of SEQ ID NO: 26 as described above and having lignan hydroxylation activity.
- Such variants include deletions, insertions, inversions, repeats, and type substitutions (eg, replacement of one hydrophilic residue with another, but usually strongly hydrophilic residues strongly hydrophobic In which the residues are not substituted).
- “neutral” amino acid substitutions in a polypeptide generally have little effect on the activity of the polypeptide.
- Those skilled in the art can easily mutate one to several amino acids in the amino acid sequence of a polypeptide using well-known techniques. For example, according to a known point mutation introduction method, an arbitrary base of a polynucleotide encoding a polypeptide can be mutated. In addition, a deletion mutant or an addition mutant can be prepared by designing a primer corresponding to an arbitrary site of a polynucleotide encoding a polypeptide. Furthermore, if the method described in this specification is used, it can be easily determined whether the produced mutant has a desired activity.
- Preferred variants have conservative or non-conservative amino acid substitutions, deletions, or additions. Silent substitution, addition, and deletion are preferred, and conservative substitution is particularly preferred. These do not alter the polypeptide activity according to the invention.
- Conservative substitutions are substitutions of one amino acid for another in the aliphatic amino acids Ala, Val, Leu, and Ile; exchange of hydroxyl residues Ser and Thr, acidic residues Asp and Glu exchange, substitution between amide residues Asn and Gln, exchange of basic residues Lys and Arg, and substitution between aromatic residues Phe, Tyr.
- a preferred polypeptide in the present invention is a polypeptide having lignan hydroxylation activity, which is (a) the amino acid sequence of SEQ ID NO: 26; or (b ′) 1 to several amino acids in the amino acid sequence of SEQ ID NO: 26. Contains amino acid sequences with amino acids deleted, inserted, substituted and / or added. As described above, such a mutant polypeptide is not limited to a polypeptide having a mutation artificially introduced by a known mutant polypeptide production method, and a naturally occurring polypeptide is isolated and purified. It may be what you did.
- 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 be a complex polypeptide including a structure other than the polypeptide.
- examples of the “structure other than the polypeptide” include sugar chains and isoprenoid groups, but are not particularly limited.
- polypeptide according to the present invention may contain an additional polypeptide.
- additional polypeptide include epitope-tagged polypeptides such as His tag, c-Myc tag, and Flag.
- polypeptide according to the present invention may be in a state where a polynucleotide encoding the polypeptide according to the present invention is introduced into a host cell and the polypeptide is expressed in the cell, or a cell, tissue, etc. It may be isolated and purified from.
- the polypeptide according to the present invention may be chemically synthesized.
- the polypeptides of the invention can be expressed recombinantly in a modified form such as a fusion protein.
- additional amino acids (tags) of the polypeptides according to the invention particularly regions of charged amino acids, improve the stability and persistence in the host cell during purification or subsequent manipulation and storage.
- tags can be added to the N-terminus and / or C-terminus of the polypeptide.
- the polypeptide may have a plurality of tags, and the position of each tag may be separated or continuous.
- the polypeptide according to the present embodiment can be added to the N-terminus or C-terminus, for example, to a tag label (tag sequence or marker sequence) that is a sequence encoding a peptide that facilitates purification of the fused polypeptide. Such sequences can be removed prior to final preparation of the polypeptide.
- the tag amino acid sequence is a hexa-histidine peptide (eg, a tag provided in a pQE vector (Qiagen, Inc.), among many of them Are publicly and / or commercially available. For example, Gentz et al., Proc. Natl. Acad. Sci.
- hexahistidine provides convenient purification of the fusion protein.
- the “HA” tag is another peptide useful for purification corresponding to an epitope derived from influenza hemagglutinin (HA) protein, which is described in Wilson et al., Cell 37: 767 (1984) (herein). Which is incorporated by reference).
- Other such fusion proteins include a polypeptide according to this embodiment or a fragment thereof fused to Fc at the N or C terminus.
- polypeptide according to the present invention may be produced recombinantly or chemically synthesized as described in detail below.
- Recombinant production can be performed using methods well known in the art, for example, using vectors and cells as detailed below.
- Synthetic peptides can be synthesized using known methods of chemical synthesis. For example, Houghten is prepared for the synthesis of multiple peptides, such as 10-20 mg of 248 different 13-residue peptides that represent a single amino acid variant of the HA1 polypeptide segment prepared and characterized in less than 4 weeks. A simple method is described. Houghten, R.A. A. , Proc. Natl. Acad. Sci. USA 82: 5131-5135 (1985). This “Simultaneous Multiple Peptide Synthesis (SMPS)” process is further described in US Pat. No. 4,631,211 to Houghten et al. (1986).
- SMPS Simultaneous Multiple Peptide Synthesis
- polypeptides according to the present invention are useful in methods and kits for hydroxylating lignans to obtain hydroxylated lignans.
- the polypeptide according to the present invention can catalyze the hydroxylation reaction of lignans (particularly piperitol or pinoresinol).
- the polypeptide according to the present invention only needs to contain at least the amino acid sequence of SEQ ID NO: 26. That is, it should be noted that the polypeptide comprising the amino acid sequence of SEQ ID NO: 26 and any amino acid sequence having a specific function (eg, tag) is also included in the present invention. Moreover, the amino acid sequence of SEQ ID NO: 26 and the arbitrary amino acid sequence may be linked with an appropriate linker peptide so as not to inhibit the respective functions.
- polypeptide according to the present invention has an activity of hydroxylating piperitol in addition to the activity of hydroxylating pinoresinol
- the polypeptide can be used by hydroxylating pinoresinol for use in these polypeptides. It should not be limited only to producing hydroxide.
- an object of the present invention is to provide a polypeptide having an activity of hydroxylating lignan, and does not exist in the polypeptide production method specifically described in the present specification. Therefore, it should be noted that polypeptides having an activity of hydroxylating lignans obtained by methods other than the above methods also belong to the technical scope of the present invention.
- the present invention also provides a polynucleotide encoding the polypeptide according to the present invention having lignan hydroxylation activity.
- the present invention provides a polynucleotide characterized by any of the following (a) to (e): (A) a polynucleotide comprising the base sequence of SEQ ID NO: 27; (B) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 26; (C) a polypeptide in which 1 to 15 amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 26 and which encodes a polypeptide having lignan hydroxylation activity nucleotide; (D) a polynucleotide having at least 80% identity with the amino acid sequence of SEQ ID NO: 26 and encoding a polypeptide having lignan hydroxylation activity; or (e) a base of SEQ
- nucleic acid sequence As used herein, the term “polynucleotide” is used interchangeably with “gene”, “nucleic acid” or “nucleic acid molecule” and is intended to be a polymer of nucleotides.
- base sequence is used interchangeably with “nucleic acid sequence” or “nucleotide sequence” and refers to the sequence of deoxyribonucleotides (abbreviated A, G, C, and T). As shown.
- polynucleotide containing the base sequence of SEQ ID NO: 1 or a fragment thereof intends the polynucleotide containing the sequence represented by each deoxynucleotide A, G, C and / or T of SEQ ID NO: 1 or a fragment thereof. Is done.
- the polynucleotide according to the present invention may exist in the form of RNA (for example, mRNA) or in the form of DNA (for example, cDNA or genomic DNA).
- DNA can be double-stranded or single-stranded.
- Single-stranded DNA or RNA can be the coding strand (also known as the sense strand) or it can be the non-coding strand (also known as the antisense strand).
- oligonucleotide is intended to be a combination of several to several tens of nucleotides, and is used interchangeably with “polynucleotide”. Oligonucleotides are called dinucleotides (dimers) and trinucleotides (trimers) as short ones, and are represented by the number of nucleotides polymerized such as 30 mers or 100 mers as long ones. Oligonucleotides can be produced as fragments of longer polynucleotides or synthesized.
- a fragment of a polynucleotide according to the invention is a fragment of a length of at least 12 nt (nucleotide), preferably about 15 nt, and more preferably at least about 20 nt, even more preferably at least about 30 nt, and even more preferably at least about 40 nt. Is intended.
- a fragment having a length of at least 20 nt for example, a fragment comprising 20 or more consecutive bases from the base sequence of SEQ ID NO: 1 is contemplated. Since the base sequence of SEQ ID NO: 1 is provided with reference to this specification, those skilled in the art can easily prepare a DNA fragment based on SEQ ID NO: 1.
- restriction endonuclease cleavage or ultrasonic shearing can be readily used to create fragments of various sizes.
- fragments can be made synthetically.
- Appropriate fragments are synthesized by Applied Biosystems Incorporated (ABI, 850 Lincoln Center Centr Dr., Foster City, CA 94404) 392 synthesizer and the like.
- polynucleotide according to the present invention can be fused to the polynucleotide encoding the tag tag (tag sequence or marker sequence) on the 5 'side or 3' side.
- the polynucleotide according to the present invention is preferably a polynucleotide encoding a polypeptide having lignan hydroxylation activity or a variant thereof.
- the present invention provides a variant of a polynucleotide encoding a polypeptide having lignan hydroxylation activity.
- a “variant” can occur naturally, such as a natural allelic variant.
- allelic variant is intended one of several interchangeable forms of a gene occupying a given locus on the chromosome of an organism. Non-naturally occurring variants can be generated, for example, using mutagenesis techniques well known in the art.
- the polynucleotide according to the present invention is a mutant in which 1 to several bases are deleted, inserted, substituted or added in the base sequence of the polynucleotide encoding the polypeptide having lignan hydroxylation activity. Is preferred. Variants can be mutated in coding or non-coding regions, or both. Mutations in the coding region can generate conservative or non-conservative amino acid deletions, insertions, substitutions and / or additions.
- a preferred polynucleotide in the present invention is a polynucleotide that encodes a polypeptide having lignan hydroxylation activity and is any of the following (f) to (i).
- F a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 27;
- G a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 27;
- H a polynucleotide comprising a base sequence that is at least 90% identical to the base sequence of SEQ ID NO: 27; or (i) one to several bases deleted, inserted, or substituted in the base sequence of SEQ ID NO: 27 And / or added polynucleotides.
- the polynucleotide according to the present invention comprises a polynucleotide encoding a polypeptide having lignan hydroxylation activity or a polynucleotide that hybridizes to the polynucleotide under stringent hybridization conditions. Isolated polynucleotides are preferred.
- the most preferred polynucleotide in the present invention is a polynucleotide consisting of the base sequence of SEQ ID NO: 27.
- Hybridization is described in Sambrook et al., Molecular® Cloning, A • Laboratory • Manual, 2d • Ed. , Cold
- the appropriate hybridization temperature varies depending on the base sequence and the length of the base sequence. For example, when a DNA fragment consisting of 18 bases encoding 6 amino acids is used as a probe, a temperature of 50 ° C. or lower is preferable.
- the “polynucleotide hybridizing under stringent conditions” refers to a polynucleotide consisting of a base sequence complementary to the base sequence of SEQ ID NO: 27 or a polynucleotide encoding the amino acid sequence of SEQ ID NO: 26.
- a polynucleotide (for example, DNA) obtained by using a colony hybridization method, a plaque hybridization method, a Southern hybridization method, or the like using all or part of the probe as a probe.
- a hybridization method for example, methods described in Molecular® Cloning® 3rd Ed., Current Protocols® in Molecular Molecular Biology, John Wiley®, and Sons® 1987-1997 can be used.
- stringent conditions may be any of low stringent conditions, medium stringent conditions, and high stringent conditions.
- Low stringent conditions are, for example, conditions of 5 ⁇ SSC, 5 ⁇ Denhardt's solution, 0.5% SDS, 50% formamide, 32 ° C.
- the “medium stringent conditions” are, for example, conditions of 5 ⁇ SSC, 5 ⁇ Denhardt's solution, 0.5% SDS, 50% formamide, and 42 ° C.
- “High stringent conditions” are, for example, conditions of 5 ⁇ SSC, 5 ⁇ Denhardt's solution, 0.5% SDS, 50% formamide, 50 ° C.
- a polynucleotide having high homology eg, DNA
- a polynucleotide having high homology eg, DNA
- multiple factors such as temperature, probe concentration, probe length, ionic strength, time, and salt concentration can be considered as factors that affect hybridization stringency. Those skilled in the art will select these factors as appropriate. It is possible to achieve similar stringency.
- Alkphos Direct Labeling Reagents manufactured by Amersham Pharmacia
- Hybridized polynucleotides eg, DNA
- the polynucleotide encoding SEQ ID NO: 26 is 80% or more and 81%. Over 82%, Over 83%, Over 84%, Over 85%, Over 86%, Over 87%, Over 88%, Over 89%, Over 90%, Over 91%, Over 92%, Over 93%, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% Polynucleotides having an identity of 99.8% or more and 99.9% or more can be mentioned.
- the present invention provides an oligonucleotide comprising a fragment of the above polynucleotide or a complementary sequence thereof.
- the oligonucleotide according to the present invention does not encode a lignan hydroxylated polypeptide, the person skilled in the art will make it possible for the polynucleotide according to the present invention to produce the polypeptide according to the present invention as a primer for polymerase chain reaction (PCR). Easy to understand that can be used.
- PCR polymerase chain reaction
- FISH in situ hybridization
- the polynucleotide or oligonucleotide according to the present invention includes not only double-stranded DNA but also single-stranded DNA and RNA such as sense strand and antisense strand constituting the same.
- the polynucleotide or oligonucleotide according to the present invention can be used as a tool for gene expression manipulation by an antisense RNA mechanism. With antisense RNA technology, a decrease in the gene product derived from the endogenous gene is observed.
- the content of the polypeptide having lignan hydroxylation activity can be decreased, and as a result, the hydroxylated lignan content or content ratio in the plant can be controlled (increased or decreased). it can.
- the polynucleotide or oligonucleotide according to the present invention may contain a sequence such as an untranslated region (UTR) sequence or a vector sequence (including an expression vector sequence).
- Examples of the method for obtaining the polynucleotide or oligonucleotide according to the present invention include various known methods for isolating the DNA fragment containing the polynucleotide or oligonucleotide according to the present invention. For example, by preparing a probe that specifically hybridizes with a part of the base sequence of the polynucleotide according to the present invention and screening a genomic DNA library or cDNA library, the polynucleotide or oligonucleotide according to the present invention is obtained. Can be acquired.
- Such a probe may be a polynucleotide (oligonucleotide) that specifically hybridizes to at least part of the base sequence of the polynucleotide according to the present invention or its complementary sequence.
- Polynucleotides selected by such hybridization include natural polynucleotides (for example, polynucleotides derived from plants such as sesame plants and bryophytes), but are polynucleotides derived from other than plants. May be.
- Another method for obtaining the polynucleotide according to the present invention is a method using PCR.
- PCR amplification method for example, a step of preparing a primer using the 5 ′ side and / or 3 ′ side sequence (or its complementary sequence) of the cDNA of the polynucleotide of the present invention, using these primers It includes a step of PCR amplification using genomic DNA (or cDNA) or the like as a template. If this method is used, a large amount of DNA fragments containing the polynucleotide of the present invention can be obtained.
- the source for obtaining the polynucleotide according to the present invention is not particularly limited, but is preferably a biological material containing piperitol or pinoresinol.
- biological material intends a biological sample (a tissue sample or cell sample obtained from an organism). In the embodiment described below, sesame is used, but the present invention is not limited to this.
- polypeptide having lignan hydroxylation activity in a transformant or a cell can be synthesized.
- polynucleotide according to the present invention it is possible to easily detect an organism expressing a polypeptide having lignan hydroxylation activity by detecting a hybridizing polynucleotide.
- the oligonucleotide according to the present invention can be used as a hybridization probe for detecting a polynucleotide encoding a polypeptide having lignan hydroxylation activity or as a primer for amplifying the polynucleotide, thereby producing a polynucleotide having lignan hydroxylation activity.
- An organism or tissue expressing the peptide can be easily detected.
- the oligonucleotide can be used as an antisense oligonucleotide to suppress the expression of a polypeptide having lignan hydroxylation activity in the organism or its tissue or cell.
- the polynucleotide according to the present invention also has the activity of hydroxylating piperitol in addition to the activity of the polypeptide encoded by the polynucleotide to hydroxylate pinoresinol. Therefore, the use of the polynucleotide according to the present invention should not be limited to hydroxylation of only pinoresinol as a substrate to produce these hydroxides (for example, a 9th-position hydroxide).
- an object of the present invention is to provide a polynucleotide encoding a polypeptide having an activity of hydroxylating piperitol or pinoresinol, and an oligonucleotide that hybridizes with the polynucleotide. It does not exist in the method for producing polynucleotides and oligonucleotides specifically described therein. Therefore, it should be noted that a piperitol obtained by a method other than the above methods or a polynucleotide encoding a polypeptide having an activity of hydroxylating pinoresinol also belongs to the technical scope of the present invention.
- the present invention further controls the amount of lignan and hydroxylated lignan in an organism (preferably a plant) by using the polypeptide or polynucleotide according to the present invention. Methods for (increasing or decreasing) and utilization of the controlled organism (preferably plants) are provided.
- the present invention provides a vector used for producing a polypeptide having lignan hydroxylation activity.
- the vector according to the present invention may be a vector used for in vitro translation or a vector used for recombinant expression.
- the vector according to the present invention is not particularly limited as long as it contains the above-described polynucleotide according to the present invention.
- examples thereof include a recombinant expression vector into which a cDNA of a polynucleotide encoding a polypeptide having lignan hydroxylation activity has been inserted.
- a method for producing a recombinant expression vector includes, but is not limited to, a method using a plasmid, phage, cosmid or the like.
- the specific type of the vector is not particularly limited, and a vector that can be expressed in the host cell can be appropriately selected. That is, according to the type of the host cell, a promoter sequence is appropriately selected in order to reliably express the polynucleotide according to the present invention, and a vector in which this and the polynucleotide according to the present invention are incorporated into various plasmids is used as an expression vector. Use it.
- the expression vector according to the present invention contains an expression control region (for example, promoter, terminator, and / or replication origin, etc.) depending on the type of host to be introduced.
- Conventional promoters eg, trc promoter, tac promoter, lac promoter, etc.
- yeast promoters include glyceraldehyde 3-phosphate dehydrogenase promoter, PH05 promoter, etc.
- Examples of the promoter for filamentous fungi include amylase and trpC.
- animal cell host promoters include viral promoters (eg, SV40 early promoter, SV40 late promoter, etc.).
- the expression vector can be prepared according to a conventional technique using a restriction enzyme and / or ligase. Transformation of the host with the expression vector can also be performed according to conventional techniques.
- a host transformed with the above expression vector is cultured, cultivated or bred, and then subjected to conventional techniques (eg, filtration, centrifugation, cell disruption, gel filtration chromatography, ion exchange chromatography) from the culture. Etc.), the target protein can be recovered and purified.
- conventional techniques eg, filtration, centrifugation, cell disruption, gel filtration chromatography, ion exchange chromatography
- the expression vector preferably contains at least one selectable marker.
- markers include dihydrofolate reductase or neomycin resistance genes for eukaryotic cell culture, and E. coli. Examples of culture in E. coli and other bacteria include a tetracycline resistance gene or an ampicillin resistance gene.
- the polypeptide of the present invention may be expressed as a fusion polypeptide.
- GFP Green Fluorescent Protein
- the polypeptide of the present invention is used as a GFP fusion polypeptide. It may be expressed as
- the above host cells are not particularly limited, and various conventionally known cells can be suitably used. Specifically, for example, bacteria such as Escherichia coli, yeasts (budding yeast Saccharomyces cerevisiae, fission yeast Schizosaccharomyces pombe), nematodes (Caenorhabditis elegans), Xenopus laevis (Xenopus moth, Xenopus moth, etc.) However, it is not particularly limited. Appropriate culture media and conditions for the above-described host cells are well known in the art.
- a method for introducing the expression vector into a host cell that is, a transformation method is not particularly limited, and a conventionally known method such as an electroporation method, a calcium phosphate method, a liposome method, or a DEAE dextran method can be suitably used.
- a conventionally known method such as an electroporation method, a calcium phosphate method, a liposome method, or a DEAE dextran method can be suitably used.
- an expression system using baculovirus may be used.
- a polypeptide having lignan hydroxylation activity can be expressed in the organism or cell by introducing the polynucleotide into the organism or cell. Furthermore, if the vector according to the present invention is used in a cell-free protein synthesis system, a polypeptide having lignan hydroxylation activity can be synthesized.
- the vector according to the present invention should include at least a polynucleotide encoding the polypeptide according to the present invention. That is, it should be noted that vectors other than expression vectors are also included in the technical scope of the present invention.
- an object of the present invention is to provide a vector containing a polynucleotide encoding the polypeptide according to the present invention, and the individual vector species and cell species specifically described in the present specification.
- vector production methods and cell introduction methods As well as vector production methods and cell introduction methods. Therefore, it should be noted that vectors obtained using vector species and vector production methods other than those described above also belong to the technical scope of the present invention.
- Transformant or cell provides a transformant or cell into which a polynucleotide encoding the above-mentioned polypeptide having lignan hydroxylation activity is introduced.
- the term “transformant” is intended to include living organisms as well as tissues or organs.
- the method for producing a transformant or cell is not particularly limited, and examples thereof include a method for transformation by introducing the above-described recombinant vector into a host.
- the organism to be transformed is not particularly limited, and examples thereof include various microorganisms, plants and animals exemplified in the host cell.
- the transformant or cell according to the present invention is characterized in that the composition of lignan and / or hydroxylated lignan naturally contained in these transformant or cell is modified.
- the transformant or cell according to the present invention is preferably a plant or its progeny, or a tissue derived therefrom, and particularly preferably sesame, forsythia or flax.
- Such a transformant or cell can increase or decrease the content of hydroxylated lignan in an organism producing lignan by using the method for controlling the hydroxylated lignan content according to the present invention.
- the transformant according to the present invention may be a plant transformant.
- the plant transformant according to this embodiment is obtained by introducing a recombinant vector containing the polynucleotide according to the present invention into a plant so that the polypeptide encoded by the polynucleotide can be expressed.
- the recombinant expression vector used for plant transformation is not particularly limited as long as it is a vector capable of expressing the polynucleotide according to the present invention in the plant.
- a vector having a promoter that constitutively expresses a polynucleotide in a plant cell eg, cauliflower mosaic virus 35S promoter
- a promoter that is inducibly activated by an external stimulus e.g., cauliflower mosaic virus 35S promoter
- examples thereof include a vector having (for example, a metallothionein promoter).
- Plants to be transformed in the present invention include whole plants, plant organs (eg leaves, petals, stems, roots, seeds, etc.), plant tissues (eg epidermis, phloem, soft tissue, xylem, vascular bundle, It means any of a palisade tissue, a spongy tissue, etc.) or a plant culture cell, or various forms of plant cells (eg, suspension culture cells), protoplasts, leaf sections, callus, and the like.
- the plant used for transformation is not particularly limited, and may be any plant belonging to the monocotyledonous plant class or the dicotyledonous plant class.
- transformation methods known to those skilled in the art for example, Agrobacterium method, gene gun, PEG method, electroporation method, etc.
- Agrobacterium method for example, Agrobacterium method, gene gun, PEG method, electroporation method, etc.
- a method using Agrobacterium and a method for directly introducing it into plant cells are well known.
- the constructed plant expression vector is introduced into an appropriate Agrobacterium (for example, Agrobacterium tumefaciens), and this strain is introduced into the leaf disk method (Hirofumi Uchimiya).
- the plant genetic manipulation manual (1990), pages 27-31, Kodansha Scientific, Tokyo) can be used to infect sterile cultured leaf pieces to obtain transformed plants.
- an expression vector is first introduced into Agrobacterium, and then transformed Agrobacterium is transformed into a plant cell or a plant cell by the method described in Plant MolecularMoBiology Manual (SB Gelvin et al., Academic Press Publishers). It is a method of introducing into plant tissue.
- plant tissue includes callus obtained by culturing plant cells.
- a binary vector such as pBI121 or pPZP202 can be used.
- an electroporation method and a gene gun method are known.
- a plant body, a plant organ, and a plant tissue itself may be used as they are, or may be used after preparing a section, or a protoplast may be prepared and used.
- the sample thus prepared can be processed using a gene transfer apparatus (for example, PDS-1000 (BIO-RAD)).
- the treatment conditions vary depending on the plant or sample, but are usually performed at a pressure of about 450 to 2000 psi and a distance of about 4 to 12 cm.
- the cell or plant tissue into which the gene has been introduced is first selected for drug resistance such as hygromycin resistance, and then regenerated into a plant body by a conventional method. Regeneration of a plant body from a transformed cell can be performed by a method known to those skilled in the art depending on the type of plant cell.
- transformation is performed by introducing a recombinant vector into the cultured cells using a gene gun, electroporation method, or the like.
- Callus, shoots, hairy roots, etc. obtained as a result of transformation can be used as they are for cell culture, tissue culture or organ culture, and can be used at a suitable concentration using conventionally known plant tissue culture methods. It can be regenerated into plants by administration of plant hormones (auxin, cytokinin, gibberellin, abscisic acid, ethylene, brassinolide, etc.).
- a gene has been introduced into a plant can be confirmed by a PCR method, Southern hybridization method, Northern hybridization method or the like.
- DNA is prepared from a transformed plant, PCR is performed by designing DNA-specific primers. PCR can be performed under the same conditions as those used for preparing the plasmid. Thereafter, the amplification product is subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis, etc., stained with ethidium bromide, SYBR Green solution, etc., and the amplification product is detected as a single band, It can be confirmed that it has been transformed.
- PCR can be performed using a primer previously labeled with a fluorescent dye or the like to detect an amplification product. Furthermore, it is possible to employ a method in which the amplification product is bound to a solid phase such as a microplate and the amplification product is confirmed by fluorescence or enzyme reaction.
- the present invention also includes a plant body into which the polynucleotide according to the present invention is introduced so that it can be expressed, or a progeny of the plant body having the same properties as the plant body, or a tissue derived therefrom.
- transformant plant examples include sesame, rice, tobacco, barley, wheat, rapeseed, potato, tomato, poplar, banana, eucalyptus, sweet potato, tides, alfalfa, lupine, corn, cauliflower, rose, chrysanthemum.
- the transformant according to the present invention can be produced using sesame.
- a method for producing a sesame transformant for example, T. et al. Asamizu: Transformation of same plants using MAT vector system: introduction of fity acid desaturase genes. Examples include known methods as described in Sesame Newsletter 16: 22-25 (2002).
- For the transformation of Forsythia, Carlo Rosati, et al. Known methods such as those described in “Regeneration and Agrobacterium-mediated transformation of Forsyria ⁇ intermedia“ Spring Glory ”.
- hydroxylated lignan (hydroxypiperitol and / or) is produced in a low-cost and environmentally-friendly production process in order to produce hydroxylated lignan in the sesame. Hydroxypinoresinol) can be produced.
- tobacco can be suitably used as the transformant according to the present invention.
- Tobacco is a typical plant that can be easily transformed together with petunia and the like, and can be regenerated from one cell (protoplast) from which the cell wall has been removed to one individual plant. Since one regenerated plant does not fall into a chimera unlike one plant derived from multiple cells, a transformant can be produced efficiently.
- a preferable method for transformation of tobacco is the leaf disk method. This method is a method that is easy to operate and can obtain a number of independent transformants from one leaf section.
- the transformation method is described in, for example, “Guideline for Neoplastic Chemistry Experiment 3—Separation and Analysis of Nucleic Acids and Genetic Experiment Method—Chemical Dojin” 1996
- a leaf disc is cut out on a sterile petri dish from tobacco leaves grown aseptically, and the cut leaf disc is pre-cultured in NB medium.
- the pre-cultured leaf disc is soaked in an Agrobacterium infection solution and co-cultured.
- the leaf disc is embedded in NB medium supplemented with carbenicillin and kanamycin until callus is formed from the leaf disc and a shoot is made After passage, get a shoot.
- the shoot grows and the distinction between the foliage becomes clear, the shoot is cut from the stem and transferred to MS medium without antibiotics and hormones. After roots are produced from the cut shoots, they are grown in a greenhouse.
- the shoot is transferred to a hormone-containing medium to promote rooting, and at the same time, a part of the leaf is cut out from the shoot and transplanted to an assay medium supplemented with carbenicillin and kanamycin. About 10 days after transplanting, those with callus leaves are considered to be kanamycin resistant individuals and those that have turned brown are considered to be kanamycin sensitive individuals and discarded.
- the transformed tobacco thus obtained is used to produce hydroxylated lignans in the tobacco, the hydroxylated lignans (hydroxypiperitol and / or hydroxypino) are produced by a low-cost and environmentally friendly production process. Resinol) can be produced.
- rice can be suitably used as the transformant according to the present invention.
- One embodiment for producing rice transformants is described below.
- the polynucleotide according to the present invention is introduced into a binary vector pPZP202 containing a hygromycin resistance gene to construct a transformation vector.
- the polynucleotide according to the present invention is operably linked to the promoter CaMV35S in frame.
- the obtained transformation vector is used to transform Agrobacterium tumefaciens EHA101 strain by electroporation under the selection of 50 mg / l kanamycin and hygromycin.
- the resulting Agrobacterium strain is stored frozen until use.
- the wild-type seeds are crushed with brown rice, sterilized with 70% ethanol for 3 minutes, washed 3 times with sterilized distilled water, then sterilized with 50% sodium hypochlorite solution for 30 minutes, and sterilized distilled. Wash 5 times with water.
- 30 g / l sucrose, 0.3 g / l casamino acid, 2.8 g / l proline, 2.0 mg / l 2,4-D were added and solidified with 4.0 g / l gellite.
- callus induction medium containing N6 medium Chou et al., 1975, Sci. Sinica, 18, 659-668.
- the medium pH is adjusted to pH 5.8 before autoclaving.
- Brown rice is grown in a light place at 28 ° C. for 4 weeks to obtain a callus having a size of about 5 mm. This callus is used for Agrobacterium infection.
- the above-mentioned Agrobacterium cryopreserved in glycerol was adjusted to pH 7.2 containing 20 mg / l kanamashiin, 50 mg / l hygromycin, 100 mg / l spectinomycin, and solidified with 15 g / l agar. Cultivation is performed at 28 ° C. for 3 days in the dark on AB medium (Chilton et al., 1974, Proc. Natl. Acad. Sci. USA, 71, 3672-3676).
- Agrobacterium cells are collected and suspended in liquid AAM medium (Hiei et al., 1994) containing 10 mg / l acetone syringone (Hiei et al., 1994, Plant J., 6, 271-282). After immersing the above callus in the resulting suspension for 2 minutes, the excess water was removed with a sterilized paper towel, and this was placed in the above callus induction medium containing 10 mg / l acetone syringone, and in the dark. Incubate at 28 ° C for 3 days to infect Agrobacterium.
- the obtained infected callus is washed 10 times with sterilized distilled water, and finally washed once with sterilized distilled water containing 500 mg / l carbenicillin, and then excess water is removed with a sterilized paper towel.
- This callus was cultured at 28 ° C. for 2 weeks in the above callus induction medium containing 10 mg / l acetosyringone, 50 mg / l hygromycin and 300 mg / l carbenicillin, and 50 mg / l hygromycin and 100 mg / l carbenicillin were further added. Cultivate for 4 weeks in the callus induction medium.
- Hygromycin resistant callus selected, 30 g / l sucrose, 30 g / l sorbitol, 2 g / l casamino acid, 2.2 mg / l kinetin, 1.0 mg / l NAA, 100 mg / l carbenicillin, 50 mg Transfer to regeneration medium containing pH 5.8 MS basal medium (Murashige and Skoog, 1962, Physiol. Plant., 15, 473-497) containing / l hygromycin and 4 g / l gellite.
- the transformant thus obtained can be easily regenerated in a regeneration medium containing hygromycin and transferred to soil for cultivation.
- the transformed rice thus obtained is used to produce hydroxylated lignans in the rice, the hydroxylated lignans (hydroxypiperitol and / or hydroxypino) are produced in a low-cost and environmentally-friendly production process. Resinol) can be produced.
- the transformant according to the present invention is an organism containing lignan (particularly pinoresinol or piperitol)
- the lignan hydroxide can be produced by introducing the polynucleotide, regardless of the species. it can.
- a transformant introduced with a recombinant expression vector containing a polynucleotide encoding the polypeptide according to the present invention it is possible to catalyze the reaction of hydroxylating lignans present in living organisms such as plants. Large-scale preparation of hydroxylated lignans is possible with a production process that is costly and environmentally friendly. Furthermore, the present invention can provide an inexpensive food or industrial product by preparing a large amount of hydroxylated lignan.
- a polypeptide that catalyzes a lignan hydroxylation reaction can be provided at low cost and under a low environmental load.
- the cells according to the present invention may be various bacterial hosts.
- the cell according to the present embodiment is obtained by introducing a recombinant vector containing the polynucleotide according to the present invention into the cell so that the polypeptide encoded by the polynucleotide can be expressed.
- Prokaryotes or eukaryotes can be used as the host.
- a bacterium belonging to the genus Escherichia for example, Escherichia coli
- a bacterium belonging to the genus Bacillus for example, Bacillus subtilis, etc.
- Lower eukaryotes for example, eukaryotic microorganisms such as yeast or filamentous fungi
- yeast include microorganisms of the genus Saccharomyces (for example, Saccharomyces cerevisiae), and the filamentous fungi include the genus Aspergillus.
- microorganisms for example, Aspergillus oryzae, Aspergillus niger, etc.
- microorganisms belonging to the genus Penicillium and animal cells or plant cells.
- Animal cells include cells from mice, hamsters, monkeys, humans, etc.
- insect cells eg, silkworm cells, or adult silkworms
- hosts can also be used as hosts.
- a person skilled in the art can recognize lignans in a wide range of organisms from bacteria to higher plants if a recombinant expression vector containing a polynucleotide encoding a polypeptide having lignan hydroxylation activity is introduced. Easily understand that hydroxylation ability can be imparted.
- the cell according to the present invention can produce the hydroxy lignan by introducing the polynucleotide, regardless of the species, as long as it is an organism containing lignan (particularly pinoresinol or piperitol).
- the lignan hydroxylation reaction can be catalyzed in the cell, so that the cost is low and the environment is low.
- Large-scale preparation of hydroxylated lignans is possible in an intensive production process.
- the present invention can provide an inexpensive food or industrial product by preparing a large amount of hydroxylated lignan.
- a polypeptide that catalyzes a lignan hydroxylation reaction can be provided at low cost and under a low environmental load.
- the transformant or cell according to the present invention only needs to be introduced with at least a polynucleotide encoding the polypeptide according to the present invention. That is, it should be noted that transformants or cells produced by means other than recombinant expression vectors are also included in the technical scope of the present invention.
- the polypeptide according to the present invention has the activity of hydroxylating piperitol in addition to the activity of hydroxylating pinoresinol, so the use of the transformant or cell according to the present invention As such, it should not be limited to only hydroxylating pinoresinol to produce these hydrates.
- an object of the present invention is to provide a transformant or a cell in which a polynucleotide encoding the polypeptide according to the present invention is introduced, and is specifically described in the present specification. It does not reside in the particular vector species and method of introduction described. Therefore, it should be noted that vector species and cell types other than those described above, and transformants or cells obtained using vector production methods and cell introduction methods also belong to the technical scope of the present invention.
- the present invention provides a method for producing a polypeptide according to the present invention.
- a method for producing a polypeptide according to the present invention it is possible to provide a polypeptide that catalyzes a lignan hydroxylation reaction under low-cost and environmentally-friendly conditions.
- a polypeptide that catalyzes a lignan hydroxylation reaction can be easily produced.
- a vector containing a polynucleotide encoding the polypeptide of the present invention is used.
- the method for producing a polypeptide according to one embodiment of the present invention it is preferable to use the above vector for a cell-free protein synthesis system.
- a cell-free protein synthesis system various commercially available kits may be used.
- the method for producing a polypeptide according to this embodiment includes a step of incubating the vector and a cell-free protein synthesis solution.
- the method for producing a polypeptide according to another embodiment of the present invention it is preferable to use a recombinant expression system.
- a recombinant expression system When a recombinant expression system is used, the polynucleotide according to the present invention is incorporated into a recombinant expression vector, and then introduced into a host so that it can be expressed by a known method, and the polypeptide obtained by translation in the host is purified.
- the recombinant expression vector may or may not be a plasmid, as long as the target polynucleotide can be introduced into the host.
- the method for producing a polypeptide according to this embodiment includes a step of introducing the vector into a host.
- the expression vector when the foreign polynucleotide is introduced into the host, the expression vector preferably incorporates a promoter that functions in the host so as to express the foreign polynucleotide.
- the method for purifying a recombinantly produced polypeptide differs depending on the host used and the nature of the polypeptide, but the target polypeptide can be purified relatively easily by using a tag or the like.
- the method for producing a polypeptide according to the present embodiment preferably further includes a step of purifying the polypeptide from an extract of cells or tissues containing the polypeptide according to the present invention.
- the step of purifying a polypeptide is to prepare a cell extract from cells or tissues by a well-known method (for example, a method in which a cell or tissue is disrupted and then centrifuged to collect a soluble fraction).
- HPLC high performance liquid chromatography
- the polypeptide is purified from cells or tissues that naturally express the polypeptide according to the present invention.
- the method for producing a polypeptide according to this embodiment preferably includes a step of identifying a cell or tissue that naturally expresses the polypeptide according to the present invention using the oligonucleotide described above.
- the method for producing a polypeptide according to this embodiment further includes a step of purifying the above-described polypeptide.
- the method for producing a polypeptide according to the present invention is characterized in that the polypeptide according to the present invention is chemically synthesized.
- the polypeptide according to the present invention can be chemically synthesized by applying a well-known chemical synthesis technique based on the amino acid sequence of the polypeptide according to the present invention described herein. .
- the polypeptide obtained by the method for producing a polypeptide according to the present invention may be a naturally occurring mutant polypeptide or an artificially prepared mutant polypeptide.
- the method for producing the mutant polypeptide is not particularly limited.
- a site-directed mutagenesis method see, for example, Hashimoto-Gotoh, Gene 152, 271-275 (1995)
- a method of introducing a point mutation into a base sequence using a PCR method or a mutant polypeptide
- a mutant polypeptide production method such as a mutant strain production method by transposon insertion
- a commercially available kit may be used for the production of the mutant polypeptide.
- the method for producing a polypeptide according to the present invention is known based on at least the amino acid sequence of a polypeptide having lignan hydroxylation activity or the base sequence of a polynucleotide encoding a polypeptide having lignan hydroxylation activity. It can be said that conventional techniques may be used.
- an object of the present invention is to provide a method for producing a polypeptide having lignan hydroxylation activity, and a production method including steps other than the various steps described above is also within the technical scope of the present invention. It must be noted that it belongs.
- the present invention provides a method for producing hydroxylated lignans using organisms or cells that express a polypeptide according to the present invention.
- the organism may be a natural unmodified organism or a transformant using a recombinant expression system.
- the method for producing hydroxylated lignan according to the present invention can efficiently produce lignan (particularly pinoresinol or piperitol).
- a method for producing hydroxylated lignan according to an embodiment of the present invention is characterized in that hydroxylated lignan is produced using an organism transformed with a polynucleotide encoding the polypeptide according to the present invention or a tissue thereof.
- the organism is the above-mentioned transformant plant or bacterium, and particularly preferably Escherichia coli, sesame, forsythia or flax.
- the method for producing hydroxylated lignan according to a preferred embodiment of the present invention includes a step of introducing a polynucleotide encoding the polypeptide according to the present invention into the organism.
- the various gene introduction methods described above may be used.
- the organism has a different composition between a hydroxylated lignan produced before transformation and a hydroxylated lignan produced after transformation. Specifically, the content rate of the lignan obtained from the said organism and its hydroxide increases. It is preferable that the method for producing hydroxylated lignan according to this aspect of the present embodiment further includes a step of extracting the hydroxylated lignan from the organism.
- the method for producing hydroxylated lignan according to the present invention includes the step of introducing the oligonucleotide according to the present invention as an antisense oligonucleotide into an organism that naturally expresses the polypeptide according to the present invention. .
- the step of introducing the oligonucleotide into the organism the above-described antisense RNA technology may be used.
- the method for producing hydroxylated lignan according to the present embodiment further includes a step of identifying an organism that naturally expresses the polypeptide according to the present invention using the above-described oligonucleotide. It is preferable that the method for producing hydroxylated lignan according to this aspect of the present embodiment further includes a step of extracting the hydroxylated lignan from the organism.
- the organism has a different composition between the hydroxylated lignan produced before the introduction of the oligonucleotide and the hydroxylated lignan produced after the introduction. Specifically, the content of lignans and their hydroxides obtained from the organisms is reduced.
- the production method of the hydroxylated lignan according to the present invention may use at least an organism that expresses the polypeptide according to the present invention.
- an object of the present invention is to provide a method for producing hydroxylated lignan based on an organism in which the composition of the hydroxylated lignan is modified by the polypeptide according to the present invention. It should be noted that production methods using various cells also belong to the technical scope of the present invention.
- the present invention provides foodstuffs and industrial products produced using the hydroxylated lignans obtained by the above-described method for producing hydroxylated lignans.
- the food described in this section is produced by using the hydroxylated lignan extracted from the above-mentioned transformed plant even if it is the seed, fruit, cuttings, tubers, and / or tuberous root of the above-mentioned transformed plant. Or a processed food (eg, sesame, forsythia or flax, or a processed food thereof).
- the food or industrial product according to the present invention may contain a desired amount of lignan (particularly pinoresinol or piperitol).
- a hydroxylated lignan extract extracted from the transformed plant according to the present invention having an increased hydroxylated lignan content as described above is provided as a food having a high hydroxylated lignan content.
- the target for modifying the hydroxylated lignan composition is not particularly limited, and can be any organism other than plants, such as animals, bacteria, or yeasts.
- polypeptides or polynucleotides according to the present invention can be produced from industrial products (eg, laboratory reagents, films, biodegradable plastics, functional fibers, lubricants, or It can be used as a raw material for industrial products such as detergents.
- industrial products eg, laboratory reagents, films, biodegradable plastics, functional fibers, lubricants, or It can be used as a raw material for industrial products such as detergents.
- the present invention relates to all polypeptides having lignan hydroxylation activity and uses thereof.
- the lignan hydroxylase may be derived from any plant, animal or microorganism, and the amount of lignan can be controlled as long as it has lignan hydroxylase activity.
- the present invention relates to a plant, its progeny, or a tissue thereof in which the amount of lignan produced by introducing a polynucleotide encoding lignan hydroxylase, and the form thereof may be a cut flower. If the lignan hydroxylated polypeptide according to the present invention is used, the production of hydroxylated lignan can be promoted or suppressed.
- the polynucleotide is introduced into a plant to express the polynucleotide constitutively or tissue-specifically to increase the expression of the target polypeptide, and an antisense method Those skilled in the art will readily understand that the expression of the target polypeptide can be suppressed by using the simultaneous suppression method and the RNAi method.
- RNA was extracted from sesame seeds using the RNeasy Plant Mini Kit (Qiagen) according to the manufacturer's recommended method. Subsequently, Oligotex-MAG mRNA purification kit (TaKaRa) was used to obtain 5 ⁇ g of poly A (+) RNA.
- a cDNA library was made from this poly A (+) RNA using the ZAP Express cDNA Synthesis Kit and Zap Express cDNA Gigapack 3 Gold Cloning Kit (Stratagene) according to the manufacturer's recommended method. The prepared library was 1 ⁇ 10 7 pfu / ml (reference: Ono et al., Proc. Natl. Acad. Sci. USA 103, 10116-10121. 2006).
- Example 2 Preparation of hybridization probe
- RNA was synthesized from 1 ⁇ g of RNA.
- At3g13610 an Arabidopsis 2-oxoglutarate-dependent dioxygenase-like gene (hereinafter 2OG-dioxygenase) -like gene, was screened using At3g13610-Fw (SEQ ID NO: 1) and At3g13610-Rv primer (SEQ ID NO: 2) A probe was used.
- Sequence number 1 At3g13610-Fw: 5'-ATG GCT CCA ACA CTC TTG ACA ACC CAA-3 '
- Sequence number 2 At3g13610-Rv: 5'-TCA GAT CTT GGC GTA ATC GAC GGT TTT-3 '
- a non-radioisotope DIG-nucleic acid detection system (Roche) was used according to the manufacturer's recommended PCR conditions to introduce DIG label into the fragments obtained by RT-PCR.
- the PCR reaction solution (50 ⁇ l) consists of 1 ⁇ l of each cDNA, 1 ⁇ Taq buffer (TaKaRa), 0.2 nM dNTPs, primers (SEQ ID NOs: 1 and 2) each 0.4 pmol / ⁇ l, and rTaq polymerase 2.5U.
- the PCR reaction was carried out at 94 ° C. for 5 minutes, followed by amplification for 30 cycles of 94 ° C. for 1 minute, 53 ° C. for 1 minute, and 72 ° C. for 2 minutes. This DIG-labeled fragment was used in the following experiments as a hybridization probe.
- Example 3 Screening of sesame 2OG-dioxygenase gene
- Screening the cDNA library obtained in Example 1 with the probe obtained in Example 2 using a non-radioisotope DIG-nucleic acid detection system (Roche Diagnostics) according to the manufacturer's recommended method did.
- Hybridization buffer (5 ⁇ SSC, 30% formamide, 50 mM sodium phosphate buffer (pH 7.0), 1% SDS, 2% blocking reagent (Roche), 0.1% lauroyl sarcosine, 80 g / ml salmon sperm DNA)
- the probe obtained in Example 2 was added and further incubated overnight.
- the membrane was washed in 5 ⁇ SSC washing solution containing 1% SDS at 55 ° C. for 30 minutes. About 1 ⁇ 10 6 pfu plaques were screened to obtain about 200 positive clones.
- the above 500 clones were inserted into the pBK-CMV plasmid (Stratagene) using a cDNA library synthesis kit according to the method recommended by the manufacturer.
- the partial DNA sequence of the insert was determined using a primer pair of M13RV and M13M4 ( ⁇ 20).
- a database search by Blast ⁇ ⁇ x was performed using the estimated amino acid sequence obtained based on the obtained DNA sequence, and a partial sequence of sesame-derived 2OG-dioxygenase (hereinafter referred to as SiD) was obtained.
- the base sequence was determined by the primer walking method using a synthetic oligonucleotide primer using DNA®Sequencer®model®3100 (Appllied®Biosystems).
- SiD ⁇ 1--7 Seven kinds of SiD-like genes (SiD ⁇ 1-7) were finally obtained by Clustal-W analysis. Since SiD genes other than SiD4 and SiD 7 did not contain the 5 'or 3' region of the putative ORF in the clones obtained by library screening, Gene Racer kit (Invitorogen) was used according to the manufacturer's recommended method. Amplification of cDNA End (hereinafter, RACE) was performed to amplify the 5 ′ and 3 ′ regions of each Sid gene. For RACE, the following primer sets specific to each SiD gene were used (SEQ ID NOs: 3 to 15 and 46). The base sequence of each amplification product was determined by a primer walking method using a synthetic oligonucleotide primer, and a SiD sequence containing a full-length ORF was obtained.
- RACE Amplification of cDNA End
- Sequence number 3 GR-SiD1-Fw3: 5'-GGG GAA CGG GCG CAG CTT GCG GGA AGA TT Sequence number 4: GR-SiD1-Rv3: 5'-GGC ATC ACC ATC GGT TCC CCC ACC GTG AAA Sequence number 5: SiD1-nest-Fw2: 5'-TGA TCT CGT GCT GGG GTT GAA Sequence number 6: SiD1-nest-Rv: 5'-TGC TCA TAA TCT CCA TTT GGT Sequence number 7: GR-SiD2-Fw: 5'-GGT CGA CAC AAG GAC GGC GGG GCG TTA Sequence number 8: GR-SiD2-Rv: 5'-ACG CCC CGC CGT CCT TGT GTC GAC CTA Sequence number 9: SiD2-nest-Fw: 5'-ACT GAT GGC GAA TGG ATT CTT Sequence number 10: SiD2-n
- SEQ ID NO: 16 Sid1 protein MAGVASPPAEVLLSKRVQELVITGEDPSGPYVCRNDDDNGELDATTENSPIPVVNIGHFLSGKWSDDESVQELKKLHSALSTWGCFQGIGHGIPSCFLDEVRRVGREFFEQPMEEKNKYGKTVTEFQGYGADPVPEEGQSLDWSDRLFLELVPEDQRNYRFWPQNPSSFKGTLEEYSEKMKTVTEIISKSMARSLHLEETCFLKQFGERAQLAGRFNYYSPCRRPDLVLGLKPHADGSGYTVILQDEPGLQVLNHGKWYTVPKNPDALLVLMGDQMEIMSNGVFRSPVHRVLSNGERDRISVAVFYTPEVGKEIGPEEGLISAEAPRVFKMVKDYADIHVGYYQRGMRSLHTVRV
- SEQ ID NO: 18 Sid2 protein MGEVDPAFIQALEHRPKPHSVEAQGIPLIDLSPANSPDPDPGSLSALAAEIGDACEKWGFFQVINHGVPLHVREKIDLVSRKFFALPKEEKKKVSRDEVNPSGYYDTEHTKNVRDWKEVFDFTVGEPMVMPASHEPDDRELKEVINQWPENPSEMREVCEEYGAEMQKLGHKLLELIALSLGLARDRFNGFFKDQTTFIRLNYYAPCPIPDLALGVGRHKDGGALTILAQDDVGGLEVKRKTDGEWILVKPTPDAYIINVGDIIQVWSNDKYESVEHRVKVNSERERFSIPFFLNPAHYTMVEPLEELVNKQNPANYNPYNWGKFFSTRKRSNYKKLDVENIQIHHFKNY *
- SEQ ID NO: 20 Sid3 protein MSELLSEPDNLIDFMLNKGNGVKGLSQINLKQIPDRFIQPPEERLDHIQIATQESVPVIDVSRWDDPGIAESICEAAAKWGFFQIINHGIPDEVLENVKRAAHDFFELPVEERRRYLKENSPTHTVMLKTSFSPLAEKILEWKDYLMHYCDGQENEHSKFWPPLSRDQVLDYVNWIKPIIRKLLTVLLNGIKVEQIDKVKESALMGSPVVTLLYYPKCPNPNVAAGAGRHSDVSSITILLQDDVGGLYVRATEGDQWIHIAPTKGALVVNIGDVLQIMSNDRYKSIEHRVFVNGSKNRVSVPVFVNPSSDAIIGPLPEVLKAGEKPIYKHVVFSDYFNYFFSKGHDGKRSLDYAKI *
- SEQ ID NO: 22 Sid4 protein MEPKLTKLGSSLPVPIVQELAKEKLATVPPRYVRPDQHQHTILSALNSSFPQIPVIDMQKFSDIYIMDSELQALHNACQEWGFFQLINHGVDSAVMEKMKIEIQEFFNLPIEEKKKFKHEEGDIQGYGQAFVVSEDQKLDWGDVFAIVTSPIYLRKPHLIAKLPATFRDATEVYASELKVLAMKILKLMAKALDMKAEEMETLFAEGMHSMRMNYYPPCPQPELVTGLCPHSDADGLTILLQVNEMDGLQIKKDGVWIPVSPLPNAFTINIGDNLEILTNGAYRSIEHRATVNKEKERISIATFLGANLDGDMGPSPSLVTPQTPAKFKRIGVTQYLKELFSRELMGKSYLDLMRI *
- SEQ ID NO: 24 Sid5 protein MMSCLQSWPEPVVRVQHLSDSGIRVIPERYVKKLSDRPSFCDSLSGEVNIPVIDMKGLYSDDASVRKKTAGMISGACREWGFFQVVNHGVRQEVMGRAREAWREFFKLPLEEKQKYANSPSTYEGYGSRLGVEKGISLDWSDYFFLNYLPLALRDQNKWPALPLSCREMVGEYCREVVELGGRLMKILSSNLGLEEEYLQEAFGGEEFGACMRVNYYPKCPQPDLTLGLSPHSDPGGMTLLFPDENVSGLQVRRGEKWITVDPVPNAFIVNIGDQLEVLSNGNYKSVEHRVIVNSEKERVSIALFYNPRGDMLIKPADELVTEDRPPLYPPTVYDEYRLYMRTRGPRGKSQVHSLKSLQ *
- SEQ ID NO: 26 Sid6 protein MEVQTMKVHAYDRLSELKAFDDSKSGVKGLVDAGVTKIPRFFINDNDMPGSEPCNFNSEAIFPVIDLSGMHHAANRAGIVSRVKEACEKWGFFQIINHEMPLRVMDEMIAGVRRFHEQDAEVKKKYYGRDVTKKFQYNSNFDLYKTRAAMWRDTITCVMAPHPPDPQELPDVCRDIMFEYSKHVMRVGHTVYELLSEALGLNPSYLRDIGCIESNFIVGHYSPACPEPELTFGIRSHVDFGLLTILLQDQIGGLQVLHQNQWVDVSPLPGSLIINVGDFIQLISNDKFKSVKHRALSKRVGPRISVGVFIKPYYADGDNLRVYGPIKELLTEEEPAIYRETTYKDYERFYFANCDDGTTKLPYFRLGT *
- SEQ ID NO: 28 Sid7 protein MAWRSQTEANYDRASELKAFDDTKTGVKGLVDSGITQVPRIFITPRNDSDKNLKPSDSQLKFPIIDLENIDEDPIRFKKVVDEVRDASGTWGFFQVINHGIPGSVLEEMLDGVRKFYEQDPEERKKWYTRDRKRSVVYNSNFDLYSAPAANWRDTFFCKMAPHPPSPEELPAVCRDIMFEYTKQVLKLGTSLFKLLSEALGLDANHLGDMKCADGLALLCHYYPFCPQPELTMGASQHADSDFLTVLLNDNVTGLQVLYQNQWFDVPSVPGSLVVNVGDLLQLISNDRLISSEHRVLANNVRSRVSVACFFRSDIDKSDELYGPIQELLSEDNPPKYRATTMKEYVNYYNAKGLDGTSALLHFRV *
- Example 4 Construction of Escherichia coli expression vector of sesame 2OG-dioxygenase (SiD)
- a primer containing a BamHI or BglII site upstream of the start methionine codon (ATG) of each SiD cDNA and an XhoI site downstream of the stop codon (SEQ ID NO: 30 to SEQ ID NO: 43), and a fragment containing each SiD gene ORF was amplified by PCR.
- Sequence number 30 Bgl2NcoI-SiD1-Fw: 5'-TTT AGA TCT TCC ATG GCT GGA GTT GCA TCC CCA Sequence number 31: Sid1-endXhoI-Rv: 5'-TTG ACA TAT AAT TGA TTT AGA TCT Sequence number 32: BamNco-SiD2-Fw: 5'-AAA GGA TCC ATG GGA GAA GTC GAC CCT GCA TT Sequence number 33: SiD2-KpnXho-Rv: 5'-AAA CTC GAG GTA CCC AAC CTT CAG TAG TTC TTG AAG T Sequence number 34: Bgl2-SiD3-Fw: 5'-TTT AGA TCT ATG TCT GAA CTA CTC TCG GAA Sequence number 35: SiD3-KpnXho-Rv: 5'-AAA CTC GAG GTA CCA ACA CGT CAT ATT TTC GCA
- the PCR reaction solution (25 ⁇ l) consists of sesame seed cDNA as a template, each primer 0.2 pmol / ⁇ l, 1 ⁇ KOD plus buffer (TOYOBO), 0.2 mM dNTPs, 1 mM MgSO 4 , 1U KODplus polymerase. After reacting at 94 ° C. for 5 minutes, PCR was performed by performing 30 cycles of 94 ° C. for 1 minute, 55 ° C. for 1 minute, and 72 ° C. for 2 minutes, and then held at 72 ° C. for 3 minutes. Each PCR product obtained was inserted into the multiple cloning site of pCR4 Blunt-TOPO vector (Invitrogen) according to the method recommended by the manufacturer. It was confirmed by PCR that there was no error in the inserted PCR product.
- Example 5 Expression analysis of SiD gene in sesame
- the gene expression patterns of 7 kinds of SiD genes in sesame plants were analyzed by RT-PCR method. According to prior literature (Ono et al., Proc. Natl. Acad. Sci. USA. 103, 10116-10121 (2006)), sesame seeds are matured leaves, petals, stems, berries, seeds (stages 1 to 6), seedlings ( 1 day and 7 days after germination induction).
- RNA was extracted from 1 g of the separated organ using RNeasy Plant Mini Kit (QIAGEN). A reverse transcription reaction was performed using 1 ⁇ g of the obtained RNA as a template to obtain cDNA.
- SuperScript First-Strand Synthesis System for RT-PCR (GIBCO BRL) was used for cDNA synthesis, and the synthesis conditions followed the conditions recommended by the manufacturer of this system.
- PCR reaction was performed using the SiD gene-specific primers (SEQ ID NOs: 30 to 43) described in Example 4.
- sesame 18S ribosomal RNA was used as an internal standard gene to compare the SiD gene with the endogenous gene expression level, and Si18S-Fw (SEQ ID NO: 44) and Si18S-Rv primer (sequence) were used for amplification of this gene. No. 45) was synthesized.
- Example 6 Expression of SiD recombinant protein by E. coli
- IPTG isopropyl- ⁇ -D-thiogalactopyranoside
- a final concentration of 0.5 mM was added to the culture solution, and further cultured with shaking overnight at 30 ° C., followed by centrifugation at 3000 rpm for 10 minutes at 4 ° C. Collected.
- the cells are suspended in 10 ml of buffer solution (30 mM Tris-HCl (pH 7.5), 30 mM NaCl), sonicated to disrupt E. coli, and then centrifuged at 15,000 rpm for 10 minutes at 4 ° C. The obtained supernatant was used as a crude enzyme solution for the following activity measurement.
- Example 7 Enzyme analysis of SiD recombinant protein
- a lignan such as pinoresinol can be obtained by extraction and purification from sesame, for example, according to a known method (Japan Agricultural Chemical Society 67: 1693 (1993)).
- the substrate was dissolved in a small amount of DMSO and then dissolved in 70% ethanol to obtain a substrate solution (1 mg / ml). 5 ⁇ l of this substrate solution, 145 ⁇ l of the crude enzyme solution of each SiD expressed in E.
- the enzyme reaction was stopped by adding 100% acetonitrile (150 ⁇ l) to the reaction tube. After vigorously stirring the reaction tube with a vortex mixer, centrifuge at 15,000 rpm for 5 minutes at 4 ° C, and clean the resulting supernatant using a filter (pore size 0.45mm, 4mm Millex-LH, Millipore) The supernatant was analyzed using liquid high performance chromatography (hereinafter HPLC). Analytical conditions for lignan and its hydroxide are as follows.
- Liquid chromatography (Lc-2010C (Shimadzu Corporation) was performed using a C-30 column (Nomura Chemical C30-UG-5, 4.6 mm ⁇ 150 mm). As the mobile phase, 90% acetonitrile containing 0.1% TFA was used as solution A, and 0.1% TFA was used as solution B. After equilibrating the column with a mixed solution of A solution 65%: B solution 35% (20 minutes), linear concentration gradient (A solution 65%: B solution 35% ⁇ A solution 0%: B solution 100%) was eluted for 20 minutes (flow rate 0.6 ml / min) and 7 minutes was eluted with 100% solution B. Absorption at 287 nm was measured to detect compounds contained in the sample.
- each peak of the compound was measured for a spectrum of 190 nm to 400 nm using SPD-10AV (Shimadzu Corporation), and a substance having two absorption maxima (230 nm and 280 nm) characteristic of lignan was searched.
- the pinoresinol standard is detected at about 8.7 minutes
- the piperitol standard at about 12.8 minutes
- the syringarezinol standard at about 7.9 minutes
- the secoisolaricillinol standard at about 6.2 minutes.
- SiD6 showed the highest 47% sequence identity with Desacetoxyvindoline 4-hydroxylase, a hydroxylase of periwinkle indole alkaloid, but this alkaloid has never been reported in sesame seeds.
- the activity of hydroxylating lignans with the 2OG dioxygenase family of enzymes is novel.
- Example 8 LC-MS analysis of the product by SiD6
- the molecular weights of the products P1 and P3 by SiD6 in Example 7 were analyzed by LC-MS analysis.
- Diaion SEPABEADS HP20 resin Mitsubishi Chemical
- a column packed with 1 ml of Diaion HP-20 resin (Mitsubishi Chemical) was washed with 5 ml of 50% acetone and then equilibrated with 10 ml of water.
- the enzyme reaction solution containing the pinoresinol product P1 of Example 7 was loaded onto the column, the impurities were washed with 5 ml of water, and then the reaction product was eluted with 2 ml of 80% acetone.
- the eluate was evaporated to dryness with an evaporator and then dissolved in 50% acetonitrile (100 ⁇ l) containing 1% formic acid to obtain an LC-MS analysis sample.
- the LC conditions are shown below.
- a Develosil C30-UG-3 column (Nomura Chemical Co., Ltd., 3.0 mm ⁇ 150 mm) was used, and the mobile phase used was water containing 10 mM ammonium acetate as solution A and 100% acetonitrile as solution B. Elution was performed using a linear concentration gradient for 10 minutes (A solution 70%: B solution 30% ⁇ A solution 30%: B solution 70%), and then A solution 30%: B solution 70% for 5 minutes. Tick elution was performed. (Flow rate: 0.2 ml / min).
- Detection was performed by collecting 230-500 nm data with a photodiode array detector (SPD-M10A, Shimadzu Corporation) and measuring the A280 nm chromatogram.
- a TOF-MS detector (LCMS-IT-TOF, Shimadzu Corporation) was connected after the PDA detector, and the molecular weight was measured.
- the measurement conditions of MS were both negative and positive modes, the measurement molecular weight range was 100-1000 Da, and the fence voltage was 4.5 KV and ⁇ 3.5 KV, respectively.
- pinoresinol and pinoresinol reaction product P1 were eluted at 10.1 minutes and 7.9 minutes, respectively.
- Piperitol and piperitol product P3 were eluted at 13.3 minutes and 10.9 minutes, respectively.
- LC-MS analysis results P1 is in a positive mode m / z 375.13 [M + H ] +, m / z 373.13 [M + H] in negative mode - giving molecular ions, and the hydroxyl group are added to the pinoresinol Inferred.
- the P3 is positive mode at m / z 373.12 [M + H ] +, m / z 371.11 [M + H] in negative mode - giving molecular ion was assumed that the hydroxyl group is added to the piperitol.
- the sesame-derived SiD6 gene encodes a lignan hydroxylase having an activity of hydroxylating pinoresinol and piperitol.
- Example 9 Purification and NMR analysis of product by SiD6
- P1 and P2 which are hydroxylated products of pinoresinol and P3 which is a hydroxylated product of piperitol
- purification and NMR analysis were performed.
- a column packed with 100 ml of Diaion SEPABEADS HP20 resin (Mitsubishi Chemical) was washed with 200 ml of 50% acetone and then equilibrated with 500 ml of distilled water.
- this enzyme is an enzyme having 9-position hydroxylation activity of furofuran-type lignans (FIG. 6). From the hydroxylation positions of pinoresinol and piperitol, it is presumed that the reaction products P4 and P5 with syringaredinol are similarly hydroxylated at the 9-position or 9,9'-position.
- the polypeptides and polynucleotides according to the present invention are useful for hydroxylating lignans.
- the transformant or cell into which the polynucleotide according to the present invention has been introduced so as to be expressed is extremely useful for producing lignan hydroxylation or a product using the same in the food field and various industrial fields.
- the transformant is a plant body
- the plant body itself can be used as a food, which is very useful in the agricultural field.
- the present invention can be widely used in agriculture, food industry, pharmaceutical industry and related industries.
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Abstract
Description
(a)配列番号:26のアミノ酸配列;
(b)配列番号:26のアミノ酸配列において、1~15個のアミノ酸が欠失、挿入、置換および/または付加されたアミノ酸配列;または
(c)配列番号:26のアミノ酸配列に対して少なくとも80%の同一性を有するアミノ酸配列、
を含有するポリペプチド。
(2) 上記(1)に記載のリグナン水酸化活性を有するポリペプチドであって、
(a)配列番号:26のアミノ酸配列;または
(b’)配列番号:26のアミノ酸配列において、1~数個のアミノ酸が欠失、挿入、置換および/または付加されたアミノ酸配列;
を含有するポリペプチド。
(3) 下記の(a)~(e)のいずれかであることを特徴とするポリヌクレオチド:
(a)配列番号27の塩基配列を含有するポリヌクレオチド;
(b)配列番号:26のアミノ酸配列を含有するタンパク質をコードするポリヌクレオチド;
(c)配列番号:26のアミノ酸配列において、1~15個のアミノ酸が欠失、置換、挿入および/または付加されたポリペプチドであって、かつリグナン水酸化活性を有するポリペプチドをコードするポリヌクレオチド;
(d)配列番号:26のアミノ酸配列と少なくとも80%の同一性を有するポリペプチドであって、かつリグナン水酸化活性を有するポリペプチドをコードするポリヌクレオチド;または
(e)配列番号:27の塩基配列と相補的な塩基配列からなるポリヌクレオチドとストリンジェントな条件下でハイブリダイズし、かつリグナン水酸化活性を有するポリペプチドをコードするポリヌクレオチド。
(4) リグナン水酸化活性を有するポリペプチドをコードし、かつ下記の(f)~(i)のいずれかである上記(3)に記載のポリヌクレオチド:
(f)配列番号:27の塩基配列からなるポリヌクレオチド;
(g)配列番号:27の塩基配列と相補的な塩基配列からなるポリヌクレオチドとストリンジェントな条件下でハイブリダイズするポリヌクレオチド;
(h)配列番号27の塩基配列と少なくとも90%同一である塩基配列からなるポリヌクレオチド;または
(i)配列番号:27の塩基配列において、1~数個の塩基が欠失、挿入、置換および/または付加されたポリヌクレオチド。
(5) 上記(3)に記載のポリヌクレオチドであって、配列番号27の塩基配列からなるポリヌクレオチド。
(6) 前記リグナン水酸化活性がリグナンの9位に対する水酸化である、上記(3)~(5)のいずれかに記載のポリヌクレオチド。
(7) 上記(3)~(6)のいずれか1項に記載のポリヌクレオチドのフラグメントまたはその相補配列からなることを特徴とするオリゴヌクレオチド。
(8) 上記(1)または(2)に記載のポリペプチドの発現を抑制することを特徴とする上記(7)に記載のオリゴヌクレオチド。
(9) 上記(3)~(6)のいずれか1項に記載のポリヌクレオチドを含むことを特徴とするベクター。
(10) 上記(9)に記載のベクターを用いることを特徴とするポリペプチドの製造方法。
(11) 上記(3)~(6)のいずれか1項に記載のポリヌクレオチドが導入されていることを特徴とする形質転換体。
(12) リグナンおよび上記(3)~(6)のいずれか1項に記載のポリヌクレオチドを導入することによって水酸化リグナンの含有比が改変されることを特徴とする上記(11)に記載の形質転換体。
(13) 生物もしくはその子孫、またはこれら由来の組織であることを特徴とする上記(11)または(12)に記載の形質転換体。
(14) 上記生物が植物であることを特徴とする上記(13)に記載の形質転換体。
(15) 上記植物がゴマ、レンギョウまたはアマであることを特徴とする上記(14)に記載の形質転換体。
(16) 上記(11)~(15)のいずれか1項に記載の形質転換体を用いることを特徴とするポリペプチドを製造するための方法。
(17) 上記(11)~(15)のいずれか1項に記載の形質転換体を用いることを特徴とする水酸化リグナンの製造方法。
(18) 上記水酸化リグナンの基質が、ピペリトール、またはピノレジノールであることを特徴とする上記(17)に記載の水酸化リグナンの製造方法。
(19) 上記(9)に記載のベクターを含有することを特徴とする細胞。
(20) ゴマ、レンギョウまたはアマ由来の細胞であることを特徴とする上記(19)に記載の細胞。
(21) 上記(19)または(20)に記載の細胞を用いることを特徴とするポリペプチドを製造するための方法。
(22) 上記(19)または(20)に記載の細胞を用いることを特徴とする水酸化リグナンの製造方法。
(23) 上記水酸化リグナンの基質が、ピペリトール、ピノレジノールであることを特徴とする上記(22)に記載の水酸化リグナンの製造方法。
(24) 上記(1)または(2)に記載のポリペプチドを用いることを特徴とする水酸化リグナンの製造方法。
(25) 上記水酸化リグナンの基質が、ピペリトール、またはピノレジノールであることを特徴とする上記(24)に記載の水酸化リグナンの製造方法。
(26) 上記(17)、(22)または(24)のいずれか1項に記載の製造方法により得られた水酸化リグナンを含有することを特徴とする食品または工業製品。
(27) 上記水酸化リグナンの基質が、ピペリトール、またはピノレジノールであることを特徴とする上記(26)に記載の食品または工業製品。
(28) 上記(3)~(6)のいずれか1項に記載のポリヌクレオチドを、リグナンを産生する生物に導入する工程を包含することを特徴とする生物中の水酸化リグナンの含有量を増加させる方法。
(29) 上記リグナンを産生する生物がゴマ、レンギョウまたはアマであることを特徴とする上記(28に記載の方法。
(30) 上記リグナンがピペリトール、またはピノレジノールであることを特徴とする上記(28または29に記載の方法。
(31) 上記(8)に記載のオリゴヌクレオチドを、リグナンを産生する生物に導入する工程を包含することを特徴とする生物中の水酸化リグナンの含有量を減少させる方法。
(32) 上記リグナンを産生する生物がゴマ、レンギョウまたはアマであることを特徴とする上記(31)に記載の方法。
(33) 上記リグナンがピペリトール、またはピノレジノールであることを特徴とする上記(31)または(32)に記載の方法。
(34) 下記式:
を有する化合物(9-ヒドロキシピペリトール)。
本発明者らは、リグナン、特に、ピペリトールおよび/またはピノレジノールを主な基質にする新規水酸化酵素を見出し、本発明を完成するに至った。さらに本発明者らは、上記新規水酸化酵素がピペリトールを水酸化することを見出した。これまでに、ヒドロキシピペリトールは見出されていない。
また、本発明は、リグナン水酸化活性を有する本発明に係るポリペプチドをコードするポリヌクレオチドを提供する。具体的には、本発明は、下記の(a)~(e)のいずれかであることを特徴とするポリヌクレオチドを提供する。
(a)配列番号27の塩基配列を含有するポリヌクレオチド;
(b)配列番号:26のアミノ酸配列を含有するタンパク質をコードするポリヌクレオチド;
(c)配列番号:26のアミノ酸配列において、1~15個のアミノ酸が欠失、置換、挿入および/または付加されたポリペプチドであって、かつリグナン水酸化活性を有するポリペプチドをコードするポリヌクレオチド;
(d)配列番号:26のアミノ酸配列と少なくとも80%の同一性を有するポリペプチドであって、かつリグナン水酸化活性を有するポリペプチドをコードするポリヌクレオチド;または
(e)配列番号:27の塩基配列と相補的な塩基配列からなるポリヌクレオチドとストリンジェントな条件下でハイブリダイズし、かつリグナン水酸化活性を有するポリペプチドをコードするポリヌクレオチド。
(f)配列番号:27の塩基配列からなるポリヌクレオチド;
(g)配列番号:27の塩基配列と相補的な塩基配列からなるポリヌクレオチドとストリンジェントな条件下でハイブリダイズするポリヌクレオチド;
(h)配列番号:27の塩基配列と少なくとも90%同一である塩基配列からなるポリヌクレオチド;または
(i)配列番号:27の塩基配列において、1~数個の塩基が欠失、挿入、置換および/または付加されたポリヌクレオチド。
本発明はさらに、本発明に係るポリペプチドまたはポリヌクレオチドを用いることによって生物(好ましくは、植物)中のリグナンおよび水酸化リグナンの量を制御(増加または減少)するための方法および当該制御された生物(好ましくは、植物)の利用を提供する。
本発明は、リグナン水酸化活性を有するポリペプチドを生成するために使用されるベクターを提供する。本発明に係るベクターは、インビトロ翻訳に用いるベクターであっても組換え発現に用いるベクターであってもよい。
本発明は、上述したリグナン水酸化活性を有するポリペプチドをコードするポリヌクレオチドが導入された形質転換体または細胞を提供する。本明細書中で使用される場合、用語「形質転換体」は、組織または器官だけでなく、生物個体を含むことが意図される。
本発明は、本発明に係るポリペプチドを生産する方法を提供する。本発明に係るポリペプチドの生産方法を用いれば、リグナン水酸化反応を触媒するポリペプチドを低コストでありかつ環境に低負荷な条件下で提供することが可能となる。また、本発明に係るポリペプチドの生産方法を用いれば、リグナン水酸化反応を触媒するポリペプチドを容易に生産することが可能となる。
これまで、リグナンおよび水酸化リグナンの生産は植物からの抽出により行われているので、大量生産ができない等の問題点を有していたが、本発明に従えば、リグナンおよび水酸化リグナンを低コストで大量生産できる。
本発明は、上述の水酸化リグナン生産方法により得られた水酸化リグナンを用いて製造される食品および工業製品を提供する。本項で記載する食品は、上述した形質転換植物体の種子、果実、切穂、塊茎、および/または塊根であっても、上述した形質転換植物体から抽出された水酸化リグナンを用いて製造された食品(例えば、ゴマ、レンギョウまたはアマ、あるいはこれらの加工食品)であってもよい。本発明に係る食品または工業製品は、所望する量のリグナン(特に、ピノレジノール、またはピペリトール)を含有することができる。
RNeasy Plant Mini Kit (Qiagen) を製造業者が推奨する方法に従って使用して、ゴマの種子から総RNAを抽出した。続いて、オリゴテックス-MAG mRNA精製キット(TaKaRa)を使用して、ポリA(+)RNA 5μgを得た。ZAP Express cDNA Synthesis Kit and Zap Express cDNA Gigapack 3 Gold Cloning Kit(Stratagene)を製造業者の推奨する方法に従って使用して、このポリA(+)RNAからcDNAライブラリーを作製した。作製したライブラリーは1×107pfu/mlであった(参考文献:Ono et al., Proc. Natl. Acad. Sci. USA 103, 10116-10121. 2006)。
RNeasy Plant Mini Kit(QIAGEN)を使用して、アブラナ科シロイヌナズナから総RNAを抽出した後、SuperScriptTM Firtst-Strand Synthesis System for RT-PCR(Invitrogen)を製造業者が推奨する条件に従って使用して、総RNA 1μgからcDNAを合成した。シロイヌナズナの2-オキソグルタレート依存型ジオキシゲナーゼ(以下2OG-ジオキシゲナーゼ)様遺伝子であるAt3g13610をAt3g13610-Fw (配列番号1)およびAt3g13610-Rvプライマー(配列番号2)を用いて増幅した断片をスクリーニングプローブとした。
配列番号1:At3g13610-Fw: 5’-ATG GCT CCA ACA CTC TTG ACA ACC CAA-3’
配列番号2:At3g13610-Rv: 5’-TCA GAT CTT GGC GTA ATC GAC GGT TTT-3’
非ラジオアイソトープDIG-核酸検出システム(ロシュ社)を、製造者が推奨するPCR条件に従って使用して、RT-PCRによって得られたフラグメントにDIG標識を導入した。具体的にはPCR反応液(50μl)は、各cDNA 1μl、1×Taqバッファー(TaKaRa)、0.2nM dNTPs、プライマー(配列番号1および2)各0.4pmol/μl、rTaq ポリメラーゼ2.5Uからなる。PCR反応は94℃で5分間反応させた後、94℃で1分間、53℃で1分間、72℃で2分間の反応を30サイクルの増幅を行った。このDIG標識化フラグメントを、ハイブリダイゼーション用プローブとして以下の実験に用いた。
非ラジオアイソトープDIG-核酸検出システム(ロシュ・ダイアグノスティックス)を製造者の推奨する方法に従って使用して、実施例1で得たcDNAライブラリーを、実施例2で得たプローブを用いてスクリーニングした。
配列番号4:GR-SiD1-Rv3: 5’-GGC ATC ACC ATC GGT TCC CCC ACC GTG AAA
配列番号5:SiD1-nest-Fw2: 5’-TGA TCT CGT GCT GGG GTT GAA
配列番号6:SiD1-nest-Rv: 5’-TGC TCA TAA TCT CCA TTT GGT
配列番号7:GR-SiD2-Fw: 5’-GGT CGA CAC AAG GAC GGC GGG GCG TTA
配列番号8:GR-SiD2-Rv: 5’-ACG CCC CGC CGT CCT TGT GTC GAC CTA
配列番号9:SiD2-nest-Fw: 5’-ACT GAT GGC GAA TGG ATT CTT
配列番号10:SiD2-nest -Rv2: 5’-TTC CTT CCA GTC CCT GAC ATT
配列番号11:GR-SiD3-Rv: 5’-GGG GTT CGG ACA CTT GGG GTA GTA GA
配列番号12:SiD3-nest-Rv: 5’-CAA GGC GGA TTC TTT GAC CTT
配列番号13:GR-SiD5-Rv: 5’-TGA TCC ACT TCT CGC CCC GCC GGA CTT
配列番号14:SiD5-nest-Rv: 5’-TGA GGG CAT TTT GGG TAA TAG
配列番号15:GR-SiD6-Rv: 5’-TGC GGG TCA GGT GGA TGA GGG GCC ATT
配列番号46:SiD6-nest-Rv: 5’-CAT TAC ACA AGT GAT AGT AT
MAGVASPPAEVLLSKRVQELVITGEDPSGPYVCRNDDDNGELDATTENSPIPVVNIGHFLSGKWSDDESVQELKKLHSALSTWGCFQGIGHGIPSCFLDEVRRVGREFFEQPMEEKNKYGKTVTEFQGYGADPVPEEGQSLDWSDRLFLELVPEDQRNYRFWPQNPSSFKGTLEEYSEKMKTVTEIISKSMARSLHLEETCFLKQFGERAQLAGRFNYYSPCRRPDLVLGLKPHADGSGYTVILQDEPGLQVLNHGKWYTVPKNPDALLVLMGDQMEIMSNGVFRSPVHRVLSNGERDRISVAVFYTPEVGKEIGPEEGLISAEAPRVFKMVKDYADIHVGYYQRGMRSLHTVRV
ATGGCTGGAGTTGCATCCCCACCCGCTGAAGTATTGCTGTCCAAAAGAGTCCAAGAATTGGTCATCACCGGTGAGGACCCGTCGGGGCCATACGTGTGTAGAAACGACGACGACAACGGGGAATTAGACGCGACAACTGAGAATTCTCCGATTCCAGTTGTGAACATTGGACACTTCTTGTCGGGAAAATGGTCCGATGATGAAAGTGTACAAGAGCTGAAGAAACTCCACTCGGCTCTCTCCACATGGGGATGCTTTCAGGGCATAGGTCATGGGATCCCGAGTTGTTTCCTGGACGAGGTACGAAGAGTTGGGAGGGAGTTCTTCGAGCAGCCAATGGAGGAGAAGAACAAGTATGGGAAAACGGTGACGGAGTTTCAAGGGTATGGAGCTGATCCCGTCCCGGAAGAAGGGCAGTCGCTCGACTGGTCGGATCGTCTTTTCCTAGAGTTAGTCCCTGAAGATCAAAGAAATTACAGATTCTGGCCTCAGAACCCATCCTCTTTCAAAGGAACACTGGAAGAGTACTCGGAGAAGATGAAAACAGTGACTGAGATAATATCCAAATCCATGGCAAGATCACTTCATCTTGAGGAGACCTGCTTCTTGAAACAGTTCGGGGAACGGGCGCAGCTTGCGGGAAGATTCAACTACTATTCGCCTTGCAGGAGGCCTGATCTCGTGCTGGGGTTGAAGCCTCACGCCGACGGATCAGGCTACACCGTTATACTGCAGGATGAACCCGGCCTTCAAGTACTCAACCATGGCAAATGGTATACTGTCCCCAAGAACCCTGATGCCCTTCTAGTCCTCATGGGGGACCAAATGGAGATTATGAGCAACGGGGTGTTCAGAAGTCCGGTGCACAGGGTGCTGAGCAATGGGGAGAGGGACAGGATCTCTGTGGCTGTATTTTACACGCCGGAGGTGGGGAAGGAGATCGGGCCGGAAGAGGGGTTGATCAGTGCGGAGGCACCGAGAGTGTTCAAGATGGTGAAAGATTATGCTGACATTCACGTGGGGTACTATCAGAGGGGAATGAGATCGCTTCATACTGTCAGAGTTTGATGCTCTATATATATAGGGAAAGTTTAGTCCATCTCGAGTTTGGTCAGATCTAAATCAATTATATGTCAAGTCAATACATTTGTCGTGATTAGTGTATAATTTAAAAAATGACTAATCATGTGACAAATGTATCACACTTGCTCTATAATTGATTTAGTTCAATGAAAGCTGATATAGATAAAAATTTTGCATGTANATATGGNGTGTGTTGGATGCCTTTCCAATGTTTAAATAACCATATTGCTGCTTGGGATTTCTTTTG
MGEVDPAFIQALEHRPKPHSVEAQGIPLIDLSPANSPDPDPGSLSALAAEIGDACEKWGFFQVINHGVPLHVREKIDLVSRKFFALPKEEKKKVSRDEVNPSGYYDTEHTKNVRDWKEVFDFTVGEPMVMPASHEPDDRELKEVINQWPENPSEMREVCEEYGAEMQKLGHKLLELIALSLGLARDRFNGFFKDQTTFIRLNYYAPCPIPDLALGVGRHKDGGALTILAQDDVGGLEVKRKTDGEWILVKPTPDAYIINVGDIIQVWSNDKYESVEHRVKVNSERERFSIPFFLNPAHYTMVEPLEELVNKQNPANYNPYNWGKFFSTRKRSNYKKLDVENIQIHHFKNY*
ATGGGAGAAGTCGACCCTGCATTCATCCAAGCGCTCGAACACAGGCCTAAACCCCACAGCGTCGAGGCCCAAGGCATCCCGTTAATCGATCTCTCCCCCGCCAACTCCCCGGACCCCGATCCGGGTTCCCTGTCAGCTCTCGCCGCCGAAATTGGTGATGCGTGCGAGAAATGGGGATTTTTCCAGGTGATCAACCACGGGGTGCCGTTGCATGTTCGGGAGAAAATTGACCTGGTTTCCAGGAAATTTTTTGCTCTGCCGAAAGAGGAGAAGAAGAAGGTTTCCAGGGATGAGGTGAACCCGTCGGGGTATTACGACACTGAGCACACTAAGAATGTCAGGGACTGGAAGGAAGTGTTTGATTTCACGGTGGGGGAACCGATGGTGATGCCGGCTTCGCATGAGCCTGATGACAGGGAGCTGAAAGAAGTGATCAATCAGTGGCCTGAGAATCCTTCAGAAATGAGGGAAGTGTGTGAAGAATACGGTGCAGAAATGCAAAAATTGGGACACAAGTTGCTGGAACTCATAGCCCTGAGCCTAGGCTTGGCGAGAGATCGATTCAATGGGTTTTTCAAGGATCAAACCACCTTCATTCGGCTGAATTACTATGCGCCATGCCCGATCCCTGATCTAGCTCTTGGCGTAGGTCGACACAAGGACGGCGGGGCGTTAACAATTCTTGCTCAAGACGATGTAGGGGGGCTGGAGGTGAAGAGGAAAACTGATGGCGAATGGATTCTTGTGAAACCTACTCCTGATGCCTATATAATCAATGTTGGTGACATTATACAGGTTTGGAGCAACGATAAGTACGAGAGTGTGGAACACAGAGTGAAAGTGAATTCAGAGAGAGAGAGATTTTCGATTCCCTTCTTCCTCAACCCTGCACATTATACTATGGTAGAACCGCTGGAGGAGCTGGTGAACAAGCAGAATCCTGCCAACTACAATCCTTACAACTGGGGAAAGTTCTTCTCCACCCGAAAACGCAGTAACTACAAGAAGCTTGATGTGGAGAACATTCAAATACATCACTTCAAGAACTACTGAAGGTTGCCCTTTTGGGCCTAAGTGTTCACATTCTCAATGATTATGCTTACAGACTGATGGATTTGGCTCTCTTGACTGTGCATGTATTATGAATAAATAATTACTTTAGATATATTATAAAAAAAAAAAAAAAAAAAA
MSELLSEPDNLIDFMLNKGNGVKGLSQINLKQIPDRFIQPPEERLDHIQIATQESVPVIDVSRWDDPGIAESICEAAAKWGFFQIINHGIPDEVLENVKRAAHDFFELPVEERRRYLKENSPTHTVMLKTSFSPLAEKILEWKDYLMHYCDGQENEHSKFWPPLSRDQVLDYVNWIKPIIRKLLTVLLNGIKVEQIDKVKESALMGSPVVTLLYYPKCPNPNVAAGAGRHSDVSSITILLQDDVGGLYVRATEGDQWIHIAPTKGALVVNIGDVLQIMSNDRYKSIEHRVFVNGSKNRVSVPVFVNPSSDAIIGPLPEVLKAGEKPIYKHVVFSDYFNYFFSKGHDGKRSLDYAKI*
ATGTCTGAACTACTCTCGGAACCCGACAACCTCATAGATTTTATGCTGAACAAAGGAAATGGAGTGAAGGGTCTCTCTCAGATAAACCTTAAACAAATCCCAGATCGATTCATCCAGCCCCCTGAAGAAAGATTGGACCATATCCAAATTGCGACCCAAGAATCCGTACCCGTTATCGATGTGTCCAGATGGGATGACCCGGGAATTGCAGAATCAATCTGCGAGGCAGCAGCCAAGTGGGGTTTCTTTCAGATCATCAATCATGGAATCCCAGATGAGGTTCTTGAAAATGTGAAGAGGGCTGCTCATGATTTCTTTGAGTTGCCTGTTGAGGAGAGGAGGAGGTATTTGAAGGAGAATTCTCCCACTCACACTGTGATGTTGAAGACTAGCTTTAGTCCTCTTGCTGAGAAGATTTTGGAGTGGAAAGACTATCTTATGCACTACTGTGATGGCCAAGAAAATGAGCATTCCAAGTTCTGGCCACCTTTGTCTAGAGATCAAGTTTTGGACTACGTAAACTGGATAAAGCCCATTATCAGAAAGCTACTGACAGTGTTGCTCAATGGTATTAAGGTGGAACAAATTGACAAGGTCAAAGAATCCGCCTTGATGGGCTCACCAGTTGTCACCCTTCTCTACTACCCCAAGTGTCCGAACCCCAATGTTGCAGCTGGAGCTGGCCGTCACTCTGATGTGTCATCAATCACCATCCTCCTACAAGACGACGTAGGTGGACTCTACGTACGAGCAACTGAAGGCGACCAGTGGATCCATATAGCACCAACCAAAGGAGCTCTTGTTGTAAACATCGGAGATGTGCTGCAGATCATGAGCAACGACAGGTACAAAAGCATCGAGCATCGTGTATTTGTGAATGGGAGCAAGAACAGGGTTTCCGTGCCCGTCTTTGTCAACCCTTCAAGTGACGCCATCATTGGCCCTCTGCCGGAAGTGCTGAAGGCCGGAGAGAAACCAATCTATAAACATGTTGTCTTCTCGGATTACTTCAATTACTTCTTTAGTAAAGGTCATGATGGCAAACGATCGCTGGATTATGCGAAAATATGACGTGTTTGTGTTTTGTAGGATAGCTTATCTTCACAAGTCTTTGCTGTCTTCTTGCATAGGCTGTGTCATATACTCACAGATTTATCTCCG
MEPKLTKLGSSLPVPIVQELAKEKLATVPPRYVRPDQHQHTILSALNSSFPQIPVIDMQKFSDIYIMDSELQALHNACQEWGFFQLINHGVDSAVMEKMKIEIQEFFNLPIEEKKKFKHEEGDIQGYGQAFVVSEDQKLDWGDVFAIVTSPIYLRKPHLIAKLPATFRDATEVYASELKVLAMKILKLMAKALDMKAEEMETLFAEGMHSMRMNYYPPCPQPELVTGLCPHSDADGLTILLQVNEMDGLQIKKDGVWIPVSPLPNAFTINIGDNLEILTNGAYRSIEHRATVNKEKERISIATFLGANLDGDMGPSPSLVTPQTPAKFKRIGVTQYLKELFSRELMGKSYLDLMRI*
ATGGAACCAAAATTAACAAAGCTAGGCAGCTCTCTTCCGGTGCCTATCGTACAAGAATTGGCCAAGGAGAAATTAGCAACGGTTCCTCCAAGATACGTGCGCCCAGATCAACATCAACACACGATTCTCTCTGCTCTTAATTCTTCCTTCCCTCAAATTCCTGTCATCGATATGCAGAAGTTTTCAGACATCTATATAATGGATTCTGAGCTTCAGGCCCTACATAATGCATGCCAAGAATGGGGTTTCTTTCAGTTGATCAACCATGGGGTGGACTCTGCTGTAATGGAGAAAATGAAGATAGAAATTCAAGAATTCTTTAATCTCCCAATAGAGGAGAAGAAGAAATTTAAGCATGAGGAAGGGGACATACAGGGTTATGGGCAAGCCTTTGTTGTATCAGAAGATCAAAAGCTCGACTGGGGAGACGTGTTTGCCATTGTTACCTCACCAATTTACCTCAGAAAGCCTCACTTAATCGCCAAGCTTCCTGCTACCTTCAGGGACGCCACAGAAGTGTATGCATCGGAACTCAAAGTTCTCGCCATGAAGATTCTAAAGCTAATGGCAAAAGCCTTAGACATGAAAGCTGAAGAAATGGAAACGCTATTCGCAGAAGGGATGCATTCCATGAGGATGAACTACTATCCTCCGTGTCCCCAGCCCGAGCTCGTCACGGGCCTCTGCCCTCACTCCGATGCAGATGGGCTCACCATTCTCCTCCAAGTGAATGAAATGGATGGCCTCCAGATCAAGAAAGATGGAGTCTGGATTCCCGTTTCTCCACTCCCTAATGCCTTCACCATCAATATTGGAGATAACTTGGAGATTCTGACAAACGGTGCTTATAGGAGCATTGAGCATAGAGCAACTGTCAACAAGGAGAAAGAAAGAATCTCCATTGCCACATTTCTGGGCGCGAATCTAGATGGTGATATGGGTCCGTCGCCAAGCCTCGTCACTCCTCAGACTCCGGCAAAATTCAAGAGGATCGGGGTGACTCAATATTTGAAGGAACTATTCTCGCGGGAACTCATGGGGAAATCATATCTAGACCTTATGAGGATTTAGGGTGTAGTACTGGGGTATGGTAATAACACCAACATGAGTTTGTACCTAATAAGTTATCAACCATTAGATTACAAATAATACTATGATCATGTGTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
MMSCLQSWPEPVVRVQHLSDSGIRVIPERYVKKLSDRPSFCDSLSGEVNIPVIDMKGLYSDDASVRKKTAGMISGACREWGFFQVVNHGVRQEVMGRAREAWREFFKLPLEEKQKYANSPSTYEGYGSRLGVEKGISLDWSDYFFLNYLPLALRDQNKWPALPLSCREMVGEYCREVVELGGRLMKILSSNLGLEEEYLQEAFGGEEFGACMRVNYYPKCPQPDLTLGLSPHSDPGGMTLLFPDENVSGLQVRRGEKWITVDPVPNAFIVNIGDQLEVLSNGNYKSVEHRVIVNSEKERVSIALFYNPRGDMLIKPADELVTEDRPPLYPPTVYDEYRLYMRTRGPRGKSQVHSLKSLQ*
ATGATGAGCTGCTTGCAGAGCTGGCCCGAACCCGTTGTCAGGGTCCAACACCTCTCCGACAGCGGGATTCGGGTAATCCCCGAACGCTACGTGAAGAAACTCTCAGACAGGCCGAGCTTTTGCGACTCCCTCTCCGGCGAAGTTAACATTCCAGTCATCGACATGAAGGGGTTGTACTCGGACGACGCCTCAGTCCGGAAGAAGACGGCCGGGATGATCAGCGGGGCATGCCGCGAGTGGGGGTTCTTCCAGGTGGTGAACCACGGGGTGAGACAGGAGGTGATGGGGCGGGCCAGGGAGGCGTGGCGCGAGTTTTTTAAGCTGCCGCTGGAGGAGAAGCAGAAGTACGCGAATTCGCCGAGCACGTATGAGGGGTACGGCAGCCGCCTGGGTGTGGAGAAGGGAATATCACTGGATTGGAGTGACTACTTTTTCCTGAATTACCTTCCTCTCGCACTCAGAGACCAGAATAAGTGGCCTGCACTTCCTCTTTCATGCAGGGAAATGGTGGGAGAGTACTGTAGAGAAGTGGTTGAACTTGGTGGAAGATTGATGAAGATTCTGTCGAGCAATCTTGGGCTGGAAGAAGAGTATCTTCAAGAAGCATTTGGAGGAGAGGAGTTTGGGGCATGCATGAGGGTTAACTATTACCCAAAATGCCCTCAACCGGACCTCACACTCGGCCTTTCTCCTCATTCCGACCCGGGTGGGATGACCCTTCTCTTCCCCGACGAGAACGTATCGGGTCTCCAAGTCCGGCGGGGCGAGAAGTGGATCACCGTCGACCCAGTCCCCAATGCGTTTATCGTCAATATAGGAGATCAACTTGAGGTGTTAAGCAATGGGAATTACAAGAGTGTGGAGCATAGGGTGATTGTGAATTCAGAGAAAGAGAGAGTGTCAATCGCATTGTTCTACAATCCAAGGGGTGATATGCTGATAAAGCCGGCGGATGAGCTGGTGACGGAGGACCGCCCACCGCTCTACCCGCCCACCGTTTACGACGAGTATAGGCTGTACATGAGGACAAGGGGCCCTCGTGGCAAGTCCCAAGTCCATTCACTTAAATCACTTCAATAACTTAATTAATATTATATTTAAATAATAATTTTAGGTAGTATTGTTCCATGAATTGTAGTGTTGTTTGTTAATTAATTTCCGCATTTTATGTAATTTGGATTGTACTACACATATATATCATGACTACTACTCATCATGGGTTAATTAAAAAAAAAAAAAAAA
MEVQTMKVHAYDRLSELKAFDDSKSGVKGLVDAGVTKIPRFFINDNDMPGSEPCNFNSEAIFPVIDLSGMHHAANRAGIVSRVKEACEKWGFFQIINHEMPLRVMDEMIAGVRRFHEQDAEVKKKYYGRDVTKKFQYNSNFDLYKTRAAMWRDTITCVMAPHPPDPQELPDVCRDIMFEYSKHVMRVGHTVYELLSEALGLNPSYLRDIGCIESNFIVGHYSPACPEPELTFGIRSHVDFGLLTILLQDQIGGLQVLHQNQWVDVSPLPGSLIINVGDFIQLISNDKFKSVKHRALSKRVGPRISVGVFIKPYYADGDNLRVYGPIKELLTEEEPAIYRETTYKDYERFYFANCDDGTTKLPYFRLGT*
ATGGAAGTTCAGACAATGAAAGTTCATGCATACGATCGACTAAGTGAACTAAAAGCATTCGATGATTCAAAATCAGGCGTGAAGGGACTTGTTGATGCTGGTGTTACGAAGATCCCACGCTTCTTCATTAATGATAATGATATGCCTGGATCCGAACCGTGCAACTTCAACTCAGAAGCCATCTTTCCAGTCATAGATTTATCAGGCATGCACCATGCTGCAAACCGTGCTGGAATTGTCAGCAGAGTGAAAGAGGCATGTGAGAAGTGGGGATTCTTTCAGATAATCAATCATGAGATGCCGCTGCGAGTGATGGATGAAATGATTGCAGGGGTTCGAAGATTTCACGAGCAAGATGCTGAGGTTAAGAAGAAATACTACGGTCGTGATGTCACGAAAAAGTTTCAGTACAATAGCAATTTCGATCTTTACAAAACACGGGCGGCCATGTGGAGGGATACTATCACTTGTGTAATGGCCCCTCATCCACCTGACCCGCAGGAATTGCCAGATGTATGCAGAGACATCATGTTTGAATACTCTAAGCATGTCATGAGAGTGGGGCATACCGTGTATGAATTGCTGTCGGAGGCTTTGGGCCTCAATCCCAGCTACCTGAGAGACATTGGCTGTATTGAGTCGAATTTCATCGTGGGCCATTATTCTCCGGCTTGCCCAGAACCAGAACTGACCTTTGGCATCAGAAGCCACGTCGACTTCGGCTTGCTCACAATACTCTTGCAGGACCAGATTGGCGGTCTCCAGGTGCTTCACCAGAATCAGTGGGTCGACGTTTCTCCCTTGCCTGGAAGTCTAATAATAAATGTTGGGGACTTTATACAGCTGATCAGTAACGACAAATTCAAAAGCGTGAAACACAGAGCACTATCAAAAAGGGTAGGGCCAAGAATTTCAGTTGGTGTTTTCATTAAACCCTACTACGCTGATGGAGATAATTTGCGGGTGTACGGACCTATCAAGGAGCTGTTAACTGAAGAAGAGCCGGCTATCTACAGGGAAACAACTTATAAAGACTATGAAAGATTCTACTTCGCCAATTGTGATGACGGAACCACCAAGCTGCCGTATTTCAGGCTGGGCACCTGATCAATGGTCCTGCAGTGGCAGCTTGTCAAGTACTGGATAGTTGTGAACTGACCTTCTTCACCA
MAWRSQTEANYDRASELKAFDDTKTGVKGLVDSGITQVPRIFITPRNDSDKNLKPSDSQLKFPIIDLENIDEDPIRFKKVVDEVRDASGTWGFFQVINHGIPGSVLEEMLDGVRKFYEQDPEERKKWYTRDRKRSVVYNSNFDLYSAPAANWRDTFFCKMAPHPPSPEELPAVCRDIMFEYTKQVLKLGTSLFKLLSEALGLDANHLGDMKCADGLALLCHYYPFCPQPELTMGASQHADSDFLTVLLNDNVTGLQVLYQNQWFDVPSVPGSLVVNVGDLLQLISNDRLISSEHRVLANNVRSRVSVACFFRSDIDKSDELYGPIQELLSEDNPPKYRATTMKEYVNYYNAKGLDGTSALLHFRV*
ATGGCCTGGAGATCTCAGACAGAAGCAAACTACGACAGAGCAAGCGAACTAAAAGCTTTTGATGACACCAAAACTGGTGTCAAAGGCTTAGTTGACAGTGGTATAACCCAAGTCCCGAGAATCTTCATCACCCCACGAAATGATTCAGACAAGAACCTTAAACCTTCCGATTCACAACTCAAATTCCCAATAATTGACCTCGAAAACATCGATGAAGATCCAATCAGGTTTAAGAAGGTCGTGGACGAGGTTCGAGATGCTTCAGGGACATGGGGTTTCTTCCAGGTGATCAATCATGGGATTCCGGGTTCTGTTTTGGAGGAGATGCTAGATGGGGTCCGGAAATTCTATGAACAAGATCCTGAGGAGAGGAAAAAGTGGTACACAAGGGATAGAAAAAGAAGTGTTGTTTACAATAGCAACTTTGATTTGTATAGTGCACCAGCAGCTAATTGGAGGGACACTTTCTTCTGTAAAATGGCTCCTCATCCTCCAAGCCCTGAGGAGTTGCCCGCTGTGTGCAGAGATATAATGTTTGAGTACACAAAGCAAGTTTTGAAACTGGGAACAAGTTTGTTTAAATTGTTGTCCGAGGCCCTTGGTCTGGATGCCAACCACCTTGGGGACATGAAATGTGCTGACGGGCTTGCTCTCCTGTGCCATTACTACCCCTTCTGCCCTCAGCCGGAGTTAACTATGGGCGCCAGCCAGCACGCGGACAGTGACTTCCTGACGGTGCTCCTAAATGACAATGTAACCGGCCTGCAAGTTCTTTACCAAAACCAGTGGTTTGATGTTCCCTCAGTGCCCGGATCTCTGGTGGTAAATGTTGGAGATCTTCTACAGCTTATATCAAATGATAGGTTGATTAGTTCGGAGCATAGAGTACTAGCAAACAACGTTCGTTCAAGGGTATCAGTCGCATGTTTCTTTAGAAGCGACATAGATAAGTCGGACGAGCTCTACGGACCAATCCAGGAACTCTTGTCTGAAGATAATCCACCAAAATACAGGGCAACCACCATGAAAGAGTATGTGAACTACTACAACGCCAAGGGGTTGGACGGAACTTCTGCTTTGTTACATTTCCGCGTTTGAATTGAAATGATATGATGGGAAGATGTTACTTTCCATATTAATATAATCCGGGAAAACGGAACATTCGAAATGTAGTATGAAAGAAAAATGTGCGGTCTATTTCTATTTTATTAGTAAAACCATAACGAATGTTGATTAACTATGATTAAAATTAAGCTTTCACTTTAAAAAAAAAAAAAAAAA
各SiDのcDNAの開始メチオニンコドン(ATG)の上流にBamHIまたはBglII部位を、終始コドンの下流にXhoI部位を有するプライマーを設計し(配列番号30~配列番号43)、各SiD遺伝子ORFを含むフラグメントをPCRにより増幅した。
配列番号31:Sid1-endXhoI-Rv: 5’-TTG ACA TAT AAT TGA TTT AGA TCT
配列番号32:BamNco-SiD2-Fw: 5’-AAA GGA TCC ATG GGA GAA GTC GAC CCT GCA TT
配列番号33:SiD2-KpnXho- Rv: 5’-AAA CTC GAG GTA CCC AAC CTT CAG TAG TTC TTG AAG T
配列番号34:Bgl2-SiD3-Fw: 5’-TTT AGA TCT ATG TCT GAA CTA CTC TCG GAA
配列番号35:SiD3-KpnXho-Rv: 5’-AAA CTC GAG GTA CCA ACA CGT CAT ATT TTC GCA TA
配列番号36:BamNco-SiD4-Fw: 5’-AAA GGA TCC ATG GAA CCA AAA TTA ACA AAG CTA
配列番号37:SiD4-KpnXho-Rv: 5’-AAA CTC GAG GTA CCT ACT ACA CCC TAA ATC CTC ATA A
配列番号38:BamHI-SiD5-Fw: 5’-AAA GGA TCC ATG AGC TGC TTG CAG AGC T
配列番号39:SiD5-KpnXho-Rv: 5’-AAA CTC GAG GTA CCT TAA TTA AGT TAT TGA AGT GAT TT
配列番号40:Bgl2Nco-SiD6-Fw: 5’- AAA GAT CTT CCA TGG AAG TTC AGA CAA TGA AA
配列番号41:SiD6-KpnXho-Rv: 5’-AAA CTC GAG GTA CCT GAT CAG GTG CCC AGC CTG AAA TA
配列番号42:BamNco-SiD7-Fw: 5’-AAA GGA TCC ATG GCC TGG AGA TCT CAG ACA GAA
配列番号43:SiD7-KpnXho-Rv: 5’-AAA CTC GAG GTA CCT TCA ATT CAA ACG CGG AAA TGT AA
7種のSiD遺伝子のゴマ植物体における遺伝子発現パターンをRT-PCR法によって解析した。先行文献(Ono et al.,Proc. Natl. Acad. Sci. USA. 103, 10116-10121 (2006))に従ってゴマを成熟葉、花弁、茎、さく果、種子(ステージ1~6)、芽生え(発芽誘導後1日と7日)に分けた。分離した器官から1gからRNeasy Plant Mini Kit(QIAGEN)を用いてRNAを抽出した。得られたRNA 1μgを鋳型として逆転写反応を行い、cDNAを得た。cDNA合成にはSuperScript First-Strand Synthesis System for RT-PCR(GIBCO BRL)を利用し、合成条件は本システム製造業者が推奨する条件に従った。得られたステージ別のcDNAを鋳型に、実施例4に記載のSiD遺伝子特異的なプライマー(配列番号30~43)を用いてPCR反応を行った。また、SiD遺伝子と内在遺伝子発現量とを比較するために、内部標準遺伝子としてゴマの18SリボソームRNAを用い、本遺伝子増幅のためにSi18S-Fw(配列番号44)およびSi18S-Rvプライマ-(配列番号45)を合成した。
配列番号45:Si18D-Rv: 5’- AAC ATC TAA GGG CAT CAC AGA
実施例4で構築したSiD遺伝子の大腸菌発現ベクターを大腸菌BL21(DE3)株に定法により形質転換した。これらの組換え大腸菌を最終濃度20μg/mlのアンピシリンを含むLB培地中にて37℃で一晩前培養した。前培養液の一部をアンピシリン50μg/ml、カザミノ酸0.5%を含むM9培地(10ml)に添加して、A600=0.6~1.0に達するまで振盪培養した。次いで、培養液に最終濃度0.5mMのIPTG(イソプロピル-β-D-チオガラクトピラノシド)を加え、さらに30℃で一晩振盪培養した後、3000rpmにて4℃で10分間遠心分離を行って集菌した。菌体を10mlの緩衝液(30mM Tris-HCl(pH7.5)、30mM NaCl)に懸濁した後に超音波処理を行って大腸菌を破砕し、次いで、15,000rpmにて4℃で10分間遠心分離を行い、得られた上清を粗酵素液として以下の活性測定に用いた。
ピノレジノールなどのリグナンは、例えば、公知の方法(日本農芸化学会誌 67:1693(1993))に従ってゴマから抽出および精製して得ることができる。基質は少量のDMSOに溶解した後に70%エタノールに溶解して基質溶液(1mg/ml)とした。この基質溶液5μl、大腸菌で発現させた各SiDの上記粗酵素液145μl、0.1M アスコルビン酸ナトリウムを10μl、0.1M 2-オキソグルタル酸(2OG)を10μl、および10mM FeSO4を10μlを反応チューブ中で混合した後、30℃で1時間反応させた。
LC-MS分析によって、実施例7におけるSiD6による生成物P1およびP3の分子量の解析を行った。
ピノレジノールの水酸化生成物であるP1およびP2、ピペリトールの水酸化生成物であるP3の水酸化部位を同定する為に各々の精製およびNMR分析を行った。ピノレジノール反応液については、ダイアイオンSEPABEADS HP20樹脂(三菱化学)を100ml充填したカラムを200mlの50%アセトンで洗浄した後、500mlの蒸留水で平衡化した。引き続き、実施例7のSiD6による生成物P1およびP2を含む酵素反応液100mlをカラムに負荷し、さらに200mlの蒸留水で洗浄し、タンパク質を除去した。最後に200mlの50%アセトンでP1、P2を溶出したものをエバポレーターで減圧濃縮した後、凍結乾燥させた。ピペリトール水酸化生成物であるP3についても同様に精製した。ついで、これをカラム:Develosil C30-UG-5(20×250 mm, 野村化学)、A液: 蒸留水, B液: 90% アセトニトリル, 溶出条件:ピノレジノール水酸化物(P1・P2):20%-70%B液(60min)、ピペリトール水酸化物(P3):60-100%B液(60min)、流速:6 ml/min、検出波長:280 nmで分画精製を行った。生成した主ピーク画分を回収して凍結乾燥標品を調製した。これをNMR(BRUKER社、750MHz)にて解析した結果、以下の通り同定できた。
P2:9, 9’-ジヒドロキシピノレジノール(溶出時間22.9min画分):NMR:δppm (DMSO-d6);2.77 (2H, d), 3.76 (6H, s), 4.75 (2H, d), 5.39 (2H, d), 6.60 (2H, d), 6.73 (2H, d), 6.85 (2H, dd), 7.13 (2H, d)。
Claims (34)
- リグナン水酸化活性を有するポリペプチドであって、
(a)配列番号:26のアミノ酸配列;
(b)配列番号:26のアミノ酸配列において、1~15個のアミノ酸が欠失、挿入、置換および/または付加されたアミノ酸配列;または
(c)配列番号:26のアミノ酸配列に対して少なくとも80%の同一性を有するアミノ酸配列、
を含有するポリペプチド。 - 請求項1に記載のリグナン水酸化活性を有するポリペプチドであって、
(a)配列番号:26のアミノ酸配列;または
(b’)配列番号:26のアミノ酸配列において、1~数個のアミノ酸が欠失、挿入、置換および/または付加されたアミノ酸配列;
を含有するポリペプチド。 - 下記の(a)~(e)のいずれかであることを特徴とするポリヌクレオチド:
(a)配列番号27の塩基配列を含有するポリヌクレオチド;
(b)配列番号:26のアミノ酸配列を含有するタンパク質をコードするポリヌクレオチド;
(c)配列番号:26のアミノ酸配列において、1~15個のアミノ酸が欠失、置換、挿入および/または付加されたポリペプチドであって、かつリグナン水酸化活性を有するポリペプチドをコードするポリヌクレオチド;
(d)配列番号:26のアミノ酸配列と少なくとも80%の同一性を有するポリペプチドであって、かつリグナン水酸化活性を有するポリペプチドをコードするポリヌクレオチド;または
(e)配列番号:27の塩基配列と相補的な塩基配列からなるポリヌクレオチドとストリンジェントな条件下でハイブリダイズし、かつリグナン水酸化活性を有するポリペプチドをコードするポリヌクレオチド。 - リグナン水酸化活性を有するポリペプチドをコードし、かつ下記の(f)~(i)のいずれかである請求項3に記載のポリヌクレオチド:
(f)配列番号:27の塩基配列からなるポリヌクレオチド;
(g)配列番号:27の塩基配列と相補的な塩基配列からなるポリヌクレオチドとストリンジェントな条件下でハイブリダイズするポリヌクレオチド;
(h)配列番号27の塩基配列と少なくとも90%同一である塩基配列からなるポリヌクレオチド;または
(i)配列番号:27の塩基配列において、1~数個の塩基が欠失、挿入、置換および/または付加されたポリヌクレオチド。 - 請求項3に記載のポリヌクレオチドであって、配列番号27の塩基配列からなるポリヌクレオチド。
- 前記リグナン水酸化活性がリグナンの9位に対する水酸化である、請求項3~5のいずれかに記載のポリヌクレオチド。
- 請求項3~6のいずれか1項に記載のポリヌクレオチドのフラグメントまたはその相補配列からなることを特徴とするオリゴヌクレオチド。
- 請求項1または2に記載のポリペプチドの発現を抑制することを特徴とする請求項7に記載のオリゴヌクレオチド。
- 請求項3~6のいずれか1項に記載のポリヌクレオチドを含むことを特徴とするベクター。
- 請求項9に記載のベクターを用いることを特徴とするポリペプチドの製造方法。
- 請求項3~6のいずれか1項に記載のポリヌクレオチドが導入されていることを特徴とする形質転換体。
- リグナンおよび請求項3~6のいずれか1項に記載のポリヌクレオチドを導入することによって水酸化リグナンの含有比が改変されることを特徴とする請求項11に記載の形質転換体。
- 生物もしくはその子孫、またはこれら由来の組織であることを特徴とする請求項11または12に記載の形質転換体。
- 上記生物が植物であることを特徴とする請求項13に記載の形質転換体。
- 上記植物がゴマ、レンギョウまたはアマであることを特徴とする請求項14に記載の形質転換体。
- 請求項11~15のいずれか1項に記載の形質転換体を用いることを特徴とするポリペプチドを製造するための方法。
- 請求項11~15のいずれか1項に記載の形質転換体を用いることを特徴とする水酸化リグナンの製造方法。
- 上記水酸化リグナンの基質が、ピペリトール、またはピノレジノールであることを特徴とする請求項17に記載の水酸化リグナンの製造方法。
- 請求項9に記載のベクターを含有することを特徴とする細胞。
- ゴマ、レンギョウまたはアマ由来の細胞であることを特徴とする請求項19に記載の細胞。
- 請求項19または20に記載の細胞を用いることを特徴とするポリペプチドを製造するための方法。
- 請求項19または20に記載の細胞を用いることを特徴とする水酸化リグナンの製造方法。
- 上記水酸化リグナンの基質が、ピペリトール、ピノレジノールであることを特徴とする請求項22に記載の水酸化リグナンの製造方法。
- 請求項1または2に記載のポリペプチドを用いることを特徴とする水酸化リグナンの製造方法。
- 上記水酸化リグナンの基質が、ピペリトール、またはピノレジノールであることを特徴とする請求項24に記載の水酸化リグナンの製造方法。
- 請求項17、22または24のいずれか1項に記載の製造方法により得られた水酸化リグナンを含有することを特徴とする食品または工業製品。
- 上記水酸化リグナンの基質が、ピペリトール、またはピノレジノールであることを特徴とする請求項26に記載の食品または工業製品。
- 請求項3~6のいずれか1項に記載のポリヌクレオチドを、リグナンを産生する生物に導入する工程を包含することを特徴とする生物中の水酸化リグナンの含有量を増加させる方法。
- 上記リグナンを産生する生物がゴマ、レンギョウまたはアマであることを特徴とする請求項28に記載の方法。
- 上記リグナンがピペリトール、またはピノレジノールであることを特徴とする請求項28または29に記載の方法。
- 請求項8に記載のオリゴヌクレオチドを、リグナンを産生する生物に導入する工程を包含することを特徴とする生物中の水酸化リグナンの含有量を減少させる方法。
- 上記リグナンを産生する生物がゴマ、レンギョウまたはアマであることを特徴とする請求項31に記載の方法。
- 上記リグナンがピペリトール、またはピノレジノールであることを特徴とする請求項31または32に記載の方法。
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BRPI0821448-4A2A BRPI0821448A2 (pt) | 2007-12-28 | 2008-12-17 | Polipeptídeo, polinucleotídeo, oligonucleotídeo, vetor, método para produzir um polipeptídeo, transformante, método para produzir uma lignana hidroxilada, célula, gênero alimentício ou produto industrial, métodos para aumentar e diminuir o teor de uma lignana hidroxilada em um organismo, e, composto |
EP08866873.6A EP2246418A4 (en) | 2007-12-28 | 2008-12-17 | HYDROXYLASE OF LIGNANS |
US12/745,292 US8288613B2 (en) | 2007-12-28 | 2008-12-17 | Lignan hydroxylase |
CA2710549A CA2710549A1 (en) | 2007-12-28 | 2008-12-17 | Lignan hydroxylase |
JP2009547999A JP5638807B2 (ja) | 2007-12-28 | 2008-12-17 | リグナン水酸化酵素 |
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US (1) | US8288613B2 (ja) |
EP (1) | EP2246418A4 (ja) |
JP (1) | JP5638807B2 (ja) |
KR (1) | KR20100105864A (ja) |
CN (1) | CN101469324B (ja) |
AU (1) | AU2008344534A1 (ja) |
BR (1) | BRPI0821448A2 (ja) |
CA (1) | CA2710549A1 (ja) |
RU (2) | RU2010131468A (ja) |
WO (1) | WO2009084439A1 (ja) |
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CN105796546B (zh) * | 2011-10-18 | 2020-03-03 | 株式会社爱茉莉太平洋 | 包含丁香脂素的sirt1活化剂 |
CN117535316B (zh) * | 2024-01-04 | 2024-03-29 | 湖南工程学院 | 一种人参PgJOX4基因及其在调节人参皂苷生物合成中的应用 |
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WO2005030944A1 (en) * | 2003-09-30 | 2005-04-07 | Suntory Limited | A gene encoding an enzyme for catalyzing biosynthesis of lignan, and use thereof |
WO2006049315A1 (ja) * | 2004-11-02 | 2006-05-11 | Suntory Limited | リグナン配糖化酵素およびその利用 |
WO2007119639A1 (ja) * | 2006-03-29 | 2007-10-25 | Suntory Limited | リグナンメチル化酵素をコードする遺伝子 |
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US6121512A (en) * | 1997-10-10 | 2000-09-19 | North Carolina State University | Cytochrome P-450 constructs and method of producing herbicide-resistant transgenic plants |
US6781031B2 (en) * | 2002-07-03 | 2004-08-24 | Sesaco Corporation | Non-dehiscent sesame variety S26 |
US7569389B2 (en) * | 2004-09-30 | 2009-08-04 | Ceres, Inc. | Nucleotide sequences and polypeptides encoded thereby useful for modifying plant characteristics |
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2008
- 2008-12-17 US US12/745,292 patent/US8288613B2/en not_active Expired - Fee Related
- 2008-12-17 AU AU2008344534A patent/AU2008344534A1/en not_active Abandoned
- 2008-12-17 WO PCT/JP2008/072943 patent/WO2009084439A1/ja active Application Filing
- 2008-12-17 RU RU2010131468/10A patent/RU2010131468A/ru not_active Application Discontinuation
- 2008-12-17 JP JP2009547999A patent/JP5638807B2/ja active Active
- 2008-12-17 CA CA2710549A patent/CA2710549A1/en not_active Abandoned
- 2008-12-17 BR BRPI0821448-4A2A patent/BRPI0821448A2/pt not_active IP Right Cessation
- 2008-12-17 EP EP08866873.6A patent/EP2246418A4/en not_active Withdrawn
- 2008-12-17 KR KR1020107016746A patent/KR20100105864A/ko not_active Application Discontinuation
- 2008-12-24 CN CN2008101852696A patent/CN101469324B/zh not_active Expired - Fee Related
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2012
- 2012-08-31 RU RU2012137206/10A patent/RU2012137206A/ru not_active Application Discontinuation
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JP2001507931A (ja) | 1996-11-08 | 2001-06-19 | ワシントン ステート ユニバーシティ リサーチ ファウンデーション | 組換えピノレシノール/ラリシレシノールレダクターゼ、組換えディリジェントタンパク質、および使用方法 |
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Also Published As
Publication number | Publication date |
---|---|
BRPI0821448A2 (pt) | 2014-10-14 |
CN101469324A (zh) | 2009-07-01 |
US20110104360A1 (en) | 2011-05-05 |
RU2010131468A (ru) | 2012-02-10 |
JP5638807B2 (ja) | 2014-12-10 |
EP2246418A1 (en) | 2010-11-03 |
AU2008344534A1 (en) | 2009-07-09 |
EP2246418A4 (en) | 2013-07-31 |
US8288613B2 (en) | 2012-10-16 |
KR20100105864A (ko) | 2010-09-30 |
JPWO2009084439A1 (ja) | 2011-05-19 |
CN101469324B (zh) | 2013-04-03 |
RU2012137206A (ru) | 2014-03-10 |
CA2710549A1 (en) | 2009-07-09 |
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