JP2008043203A - Deoxymugineic acid synthase and utilization thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a DMAS ädeoxymugineic acid synthase [an enzyme reducing the keto form (3"-keto intermediate) of nicotianamine into 2'-deoxymugineic acid]} and to provide a gene encoding the enzyme. <P>SOLUTION: The keto form of the nicotianamine is synthesized from the nicotianamine with an NAAT (nicotianamine amino group transferase). A sample is incubated with the keto form of the nicotianamine to measure the amount of the synthesized 2'-deoxymugineic acid. Thereby, measurement is made to judge whether or not the sample has an enzyme activity for reducing the keto form of the nicotianamine into the 2'-deoxymugineic acid. The measurement is carried out on a candidate protein for the DMAS obtained by a microarray analysis to thereby identify the DMAS. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to an enzyme involved in the mechanism by which plants acquire iron from soil. More specifically, the present invention relates to 2'-deoxymugineic acid as a keto body of nicotianamine in the pathway for biosynthesis of mugineic acids of grasses. It relates to an enzyme that reduces it.

  Plants take up iron, which is an important element required for various events in cells (for example, respiration, chlorophyll biosynthesis, electron transfer in photosynthesis, etc.) from soil.

  Iron is abundantly contained in the soil, but since it exists mainly as a trivalent iron compound, it is hardly dissolved in the soil solution in neutral or alkaline soil. Therefore, plants have developed a mechanism for obtaining iron from the soil.

  In non-grown plants, first, the pH of the soil is lowered by enhancing the emission of protons into the rhizosphere. It also induces the expression of trivalent iron reductase and reduces iron to the iron (II) form on the root surface to further improve the solubility. The produced iron (II) ions are transported into the body through a divalent iron transporter present in the root plasma membrane.

  On the other hand, in gramineous plants, iron chelate substances called mugineic acid family phytosiderophores (MAs: mugineic acids) are secreted from the roots to solubilize iron in the soil. The iron (III) -MAs complex produced is reabsorbed to the root via a specific transporter. Here, the synthesis and secretion of mugineic acids increases markedly in response to iron deficiency. And iron deficiency tolerance in Gramineae plants strongly correlates with the quantity and quality of secreted mugineic acids. For example, rice, wheat and corn are sensitive to reduced iron supply because they secrete relatively small amounts of 2'-deoxymugineic acid (DMA) only. Conversely, barley secretes a large amount of various wheat acids, including wheat acid (MA), 3-hydroxy wheat acid (HMA) and 3-epihydroxy wheat acid (epi-HMA), so that available iron is available. It can grow even in small situations.

  The biosynthetic pathway for mugineic acids is shown in FIG. Muginic acids are synthesized from L-methionine. Nicotianamine synthase (NAS) catalyzes the trimerization of S-adenosyl-L-methionine (SAM) to form nicotianamine (NA). NA is transferred by nicotianamine aminotransferase (NAAT) and converted to a 3 "-keto intermediate. Subsequently, the 3" -carbon of the keto intermediate is reduced, so that Is produced. DMAs are MAs synthesized for the first time in the above path. The biosynthetic pathway of all mugineic acids is common from L-methionine to DMA, but the steps after DMA vary depending on the plant variety. Almost all genes involved in the biosynthetic pathway of mugineic acids have been identified in our laboratory, and to date, seven types of mugineic acids have been identified.

  The expression of all the genes isolated to date involved in the biosynthesis of barley barley acids induced by iron deficiency is almost restricted to the roots. This suggests that root-specific expression of these genes is important for barley's strong tolerance to iron deficiency. On the other hand, many rice genes involved in the biosynthesis of mugineic acids induced by iron deficiency are expressed in both roots and aboveground parts.

In the biosynthetic pathway of mugineic acids, the existence of an enzyme that converts a 3 ″ -keto intermediate into DMA has been proposed, and once this enzyme is identified, the biosynthetic pathway of mugineic acids is completely elucidated. The enzyme that converts the 3 ″ -keto intermediate to DMA has been reported to depend on NAD (P) H for its activity. In particular, Patent Document 1 describes an enzyme that converts the 3 ″ -keto intermediate into DMA, and the sequence information of this enzyme is registered in a database (Non-Patent Document 1-2).
International Publication WO2002 / 077240 Pamphlet (October 3, 2002) http: // srs. ddbj. nig. ac. jp / cgi-bin / wgetz? -Id + 4X1771StX6D + -e + [DADRLELEASE: 'BAC10594.1'] http: // srs. ddbj. nig. ac. jp / cgi-bin / wgetz? -Id + 4Xl771StX6l + -e + [DADRELEASE: 'BAC10595.1']

  However, the present inventors have confirmed that the enzyme described in Patent Document 1 does not have the activity of converting the 3 ″ -keto intermediate into DMA. That is, the enzyme having the activity has not yet been identified. Therefore, it has been impossible to use the enzyme and the gene encoding the enzyme.

  The present invention has been made in view of the above problems, and an object of the present invention is to provide an enzyme that reduces the keto form of nicotianamine to 2'-deoxymugineic acid.

  Until now, only an inaccurate measuring method has been known for the reduction activity of keto bodies of nicotianamine to 2'-deoxymugineic acid. This is because the 3 "-keto intermediate of nicotianamine is an unstable compound and is not easily chemically synthesized, and in the state where the enzyme is not obtained, the optimal reaction conditions for the enzyme Is unknown.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have established a method for measuring the enzyme activity for reducing the keto form of nicotianamine to 2′-deoxymugineic acid. The present invention was completed by identifying deoxymugineate synthase from the grass family.

  As used herein, “the keto form of nicotianamine” refers to an “intermediate in which 3” -carbon of nicotianamine is a ketone ”. In this specification,“ 3 ”-keto intermediate Used interchangeably with "body". Also, as used herein, “reducing the keto form of nicotianamine to 2′-deoxymugineic acid” means reducing the 3 ″ -carbon.

  That is, the polypeptide according to the present invention is characterized by comprising the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, 5 or 7.

  In addition, as long as the above polypeptide reduces the keto form of nicotianamine to 2′-deoxymugineic acid, one or several amino acids in the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, 5 and 7 May be a polypeptide comprising an amino acid sequence substituted, deleted or added.

  The polynucleotide according to the present invention is characterized by encoding the above polypeptide.

  Moreover, it is preferable that the said polynucleotide consists of a base sequence shown in either sequence number 2, 4, 6 or 8.

  Furthermore, if the polynucleotide encodes an enzyme that reduces the keto body of nicotianamine to 2′-deoxymugineic acid, the nucleotide sequence shown in any one of SEQ ID NOs: 2, 4, 6 or 8 is 1 or It may be a polynucleotide comprising a base sequence in which several bases are substituted, deleted or added.

  In addition, if the polynucleotide encodes an enzyme that reduces the keto body of nicotianamine to 2′-deoxymugineic acid, the polynucleotide has a complementary sequence of the base sequence represented by any of SEQ ID NOs: 2, 4, 6 or 8. The polynucleotide may be capable of hybridizing under stringent conditions.

  The vector according to the present invention is characterized by comprising the above-mentioned polynucleotide.

  The transformant according to the present invention is characterized in that the polynucleotide is introduced.

  The method for producing a plant having resistance to iron deficiency according to the present invention includes a step of introducing the polynucleotide into a plant.

  The method for producing a plant having iron deficiency tolerance includes introducing at least one polynucleotide selected from the group consisting of a nicotianamine aminotransferase gene, a nicotianamine synthase gene, and an S-adenosyl-L-methionine synthase gene into the plant. Preferably, the method further includes the step of:

  The antibody according to the present invention is characterized by specifically binding to the polypeptide.

  The method for synthesizing 2'-deoxymugineic acid according to the present invention is characterized by including a step of incubating the above polypeptide with a keto body of nicotianamine.

  The method for synthesizing 2'-deoxymugineic acid according to the present invention preferably includes a step of incubating the polypeptide with nicotianamine and nicotianamine aminotransferase.

  The enzyme composition for synthesizing 2'-deoxymugineic acid according to the present invention is characterized by containing the above-mentioned polypeptide.

  An enzyme kit for synthesizing 2'-deoxymugineic acid according to the present invention is characterized by comprising the above polypeptide.

  The enzyme kit preferably further comprises nicotianamine aminotransferase.

  The method for measuring enzyme activity according to the present invention is a method for measuring enzyme activity for reducing a keto body of nicotianamine to 2′-deoxymugineic acid, incubating the sample to be measured with the keto body of nicotianamine, And measuring the amount of 2′-deoxymugineic acid produced by the incubating step.

  The method for measuring enzyme activity according to the present invention is a method for measuring enzyme activity for reducing the ketoamine of cotianamine to 2'-deoxymugineic acid, and incubating the sample to be measured with nicotianamine and nicotianamine aminotransferase. Preferably, the method includes a step and a step of measuring the amount of 2′-deoxymugineic acid produced by the incubating step.

  The screening method for plants having resistance to iron deficiency according to the present invention includes a step of incubating a plant extract with the antibody.

  Preferably, the screening method further includes a step of incubating the extract with at least one antibody against nicotianamine aminotransferase, nicotianamine synthase or S-adenosyl-L-methionine synthase.

  The screening method for plants having resistance to iron deficiency according to the present invention may also include a step of incubating an extract of a plant with the polynucleotide.

  The screening method preferably further includes a step of incubating the extract with at least one of a nicotianamine aminotransferase gene, a nicotianamine synthase gene, and an S-adenosyl-L-methionine synthase gene.

  If this invention is used, the plant which has iron deficiency tolerance can be screened or produced easily.

[1: Deoxymugine acid synthase]
In one aspect, the present invention provides deoxymuginate synthase. As used herein, “deoxymugineate synthase” intends a polypeptide having the activity of reducing the keto form of nicotianamine to 2′-deoxymugineate.

  As used herein, the term “polypeptide” is used interchangeably with “peptide” or “protein”. In addition, “fragment” of a polypeptide is intended to be a partial fragment of the polypeptide. The polypeptides according to the invention may also be isolated from natural sources or chemically synthesized.

  The term “isolated” polypeptide or protein is intended to be a polypeptide or protein that has been removed from its natural environment. For example, 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.

  In order to facilitate understanding of the present invention, a synthetic route of 2'-deoxymugineic acid is shown in FIG. Referring to FIG. 1, a reader of this specification refers to deoxymumine acid synthase (DMAS), a keto body of nicotianamine (3 ″ -Keto intermediate), and 2′-deoxymugineic acid (2′-deoxymugineic acid). It can be easily understood that it is an enzyme that reduces to the above.

  The polypeptide according to the present invention is preferably a deoxymugineic acid synthase of a grass family, and more preferably a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, 3, 5 or 7 or a variant thereof. .

  As used herein with respect to a protein or polypeptide, the term “variant” is intended to mean a polypeptide that retains the specific activity of the polypeptide of interest, and is expressed in “SEQ ID NO: 1, 3, 5, or 7”. The term “polypeptide variant comprising the amino acid sequence shown” is intended to mean a polypeptide having deoxymumine acid synthase activity (ie, activity of reducing the keto nicotianamine to 2′-deoxymugineic acid).

  It is well known in the art that some amino acids in the amino acid sequence that make up a polypeptide can be easily modified without significantly affecting the structure or function of the polypeptide. Furthermore, it is also well known that there are variants in natural proteins that do not significantly alter the structure or function of the protein, not only artificially modified.

  One skilled in the art can readily mutate one or 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 deoxymugineic acid synthase activity.

  Preferred variants have conservative or non-conservative amino acid substitutions, deletions or additions. These do not change the deoxymugineic acid synthase activity of the polypeptides according to the invention.

  Thus, the polypeptide according to this embodiment is a polypeptide having deoxymugineic acid synthase activity, and (1) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1, 3, 5 or 7, or (2) A polypeptide comprising an amino acid sequence in which one or several amino acids are substituted, deleted or added in the amino acid sequence shown in SEQ ID NO: 1, 3, 5 or 7 is preferred.

  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 glycosylated or non-glycosylated. 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.

  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. As used herein, examples of the “structure other than the polypeptide” include sugar chains and isoprenoid groups, but are not particularly limited.

  The polypeptide according to the present invention may include an additional polypeptide. Examples of the additional polypeptide include epitope-tagged polypeptides such as His, Myc, and Flag.

  In another aspect, the present invention provides a method for producing deoxymugineic acid synthase (ie, a polypeptide having an activity of reducing a keto form of nicotianamine to 2'-deoxymugineic acid).

  In one embodiment, the method for producing a polypeptide according to the present invention is characterized by using a vector containing a polynucleotide encoding the polypeptide.

  In one aspect of this embodiment, it is preferable that the vector is used in a recombinant expression system in the method for producing a polypeptide according to this embodiment. When a recombinant expression system is used, a polynucleotide obtained by incorporating a polynucleotide encoding the polypeptide of the present invention into a recombinant expression vector, introducing it into a host capable of expression by a known method, and translating it in the host. For example, a method of purifying a peptide can be employed. The recombinant expression vector may or may not be a plasmid, as long as the target polynucleotide can be introduced into the host. Preferably, the method for producing a polypeptide according to this embodiment includes a step of introducing the vector into a host.

  Thus, when introducing a foreign polynucleotide into a 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 this embodiment preferably further includes a step of purifying the polypeptide from an extract of cells or tissues containing the polypeptide. 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). Known methods (eg ammonium sulfate precipitation or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography) The step of purifying by a) is preferred, but not limited thereto. Most preferably, high performance liquid chromatography (“HPLC”) is used for purification.

  In another aspect of the present embodiment, the method for producing a polypeptide according to the present embodiment is preferably such that the vector is used in a cell-free protein synthesis system. When using a cell-free protein synthesis system, various commercially available kits can be used. Preferably, 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 cell-free protein synthesis system is a technique widely used for identification of various proteins encoded by intracellular mRNA and cloned cDNA. The cell-free protein synthesis system (cell-free protein synthesis method, cell-free protein translation system) The cell-free protein synthesis solution is also used.

  Cell-free protein synthesis systems include systems using wheat germ extract, systems using rabbit reticulocyte extract, systems using Escherichia coli S30 extract, and cell component extracts obtained from plant devolatilized protoplasts. . Generally, eukaryotic genes are selected for translation of eukaryotic genes, ie, systems using wheat germ extract or systems using rabbit reticulocyte extract. In view of the origin of the gene (prokaryotes / eukaryotes) and the intended use of the protein after synthesis, it may be selected from the above synthesis system.

  Many virus-derived gene products express their activity after translation through complex biochemical reactions involving intracellular membranes such as the endoplasmic reticulum and Golgi apparatus. In order to reproduce the above, an intracellular membrane component (for example, a microsomal membrane) needs to be added. A cell component extract obtained from plant evacuated protoplasts is preferred because it can be used as a cell-free protein synthesis solution retaining an intracellular membrane component, and therefore does not require the addition of a microsomal membrane.

  As used herein, “intracellular membrane component” refers to an organelle composed of a lipid membrane present in the cytoplasm (ie, cells such as the endoplasmic reticulum, Golgi apparatus, mitochondria, chloroplast, vacuole and the like). Inner granules in general) are intended. In particular, the endoplasmic reticulum and the Golgi apparatus play an important role in post-translational modification of proteins, and are essential cellular components for the maturation of membrane proteins and secreted proteins.

  In another embodiment, the method for producing a polypeptide according to the present invention preferably purifies the polypeptide from cells or tissues that naturally express the polypeptide. 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 an antibody or oligonucleotide described below. In addition, the polypeptide production method according to the present embodiment preferably further includes a step of purifying the polypeptide.

  In still another embodiment, the method for producing a polypeptide according to the present invention is characterized by chemically synthesizing the polypeptide according to the present invention. Those skilled in the art will easily understand that 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. .

  As described above, 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.

  Thus, it can be said that the method for producing a polypeptide according to the present invention may use a known and conventional technique based on at least the amino acid sequence of the polypeptide or the base sequence of the polynucleotide encoding the polypeptide. That is, it should be noted that a production method including steps other than the various steps described above also belongs to the technical scope of the present invention.

  In addition, as a method for determining whether or not the variant of the polypeptide according to the present invention has a desired deoxymugineic acid synthase activity, the method for measuring deoxymugineate synthase activity described later in [7] is used. Can do.

[2: Deoxymuginate synthase gene]
In one aspect, the present invention provides a gene encoding deoxymugineate synthase. As used herein, “a gene encoding deoxymugineic acid synthase” intends a polynucleotide encoding a polypeptide having an activity of reducing the keto form of nicotianamine to 2′-deoxymugineic acid.

  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. As used herein, the term “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.

  The polynucleotide according to the present invention preferably encodes the polypeptide according to the present invention. When the amino acid sequence of a specific polypeptide is obtained, the base sequence of the polynucleotide encoding the polypeptide can be easily designed.

  The polynucleotide according to the present invention is preferably a gene encoding deoxymugineic acid synthase of a grass family, and is a polynucleotide comprising the base sequence shown in SEQ ID NO: 2, 4, 6 or 8, or a variant thereof. It is more preferable.

As used herein with respect to polynucleotides, the term “variant” is intended to refer to a polynucleotide that encodes a polypeptide that retains the same activity as that of a particular polypeptide, “SEQ ID NOs: 2, 4 , A polynucleotide variant consisting of the nucleotide sequence shown in either 6 or 8 "is a polypeptide having deoxymugineic acid synthase activity (that is, an activity of reducing the keto form of nicotianamine to 2'-deoxymugineic acid). Encoding polynucleotides are contemplated. That is, as used herein, a variant in terms of a polynucleotide is a polynucleotide that encodes a polypeptide that reduces the keto form of nicotianamine to 2′-deoxymugineic acid,
A polynucleotide comprising a base sequence in which one or several bases are substituted, deleted or added in the base sequence shown in any of SEQ ID NOs: 2, 4, 6 or 8; or , 6 or 8 may be a polynucleotide capable of hybridizing under stringent conditions with a complementary sequence of the base sequence shown in either of 6 or 8.

  The polynucleotide according to the present invention may exist in the form of RNA (eg, mRNA) or in the form of DNA (eg, 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).

  As used herein, the term “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 chemically synthesized.

  The polynucleotide according to the present invention can also be fused to the polynucleotide encoding the tag tag (tag sequence or marker sequence) described above on the 5 'or 3' side.

  Hybridization is described in Sambrook et al., Molecular Cloning, A Laboratory Manual, 2d Ed. , Cold Spring Harbor Laboratory (1989). Usually, the higher the temperature and the lower the salt concentration, the higher the stringency (harder to hybridize), and a more homologous polynucleotide can be obtained. 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.

  As used herein, the term “stringent hybridization conditions” refers to hybridization solution (50% formamide, 5 × SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate. (Containing pH 7.6), 5 × Denhart solution, 10% dextran sulfate, and 20 μg / ml denatured sheared salmon sperm DNA) overnight at 42 ° C., then 0.1 × at about 65 ° C. It is intended to wash the filter in SSC. Depending on the polynucleotide that hybridizes to a “portion” of the polynucleotide, at least about 15 nucleotides (nt) of the reference polynucleotide, and more preferably at least about 20 nt, even more preferably at least about 30 nt, and even more preferably about Polynucleotides (either DNA or RNA) that hybridize to polynucleotides longer than 30 nt are contemplated. Polynucleotides (oligonucleotides) that hybridize to “parts” of such polynucleotides are also useful as detection probes as discussed in more detail herein.

  As described above, the polynucleotide according to the present invention is a polynucleotide encoding a polypeptide having deoxymugineic acid synthase activity, and is preferably any of the following: (1) SEQ ID NOs: 2, 4, A polynucleotide comprising the nucleotide sequence represented by 6 or 8; (2) a nucleotide sequence in which one or several bases are substituted, deleted or added in the nucleotide sequence represented by SEQ ID NO: 2, 4, 6 or 8; Or (3) a polynucleotide comprising a complementary sequence of a base sequence in which one or several bases are substituted, deleted or added in the base sequence shown in SEQ ID NO: 2, 4, 6 or 8 A polynucleotide that hybridizes with a nucleotide under stringent conditions.

  The polynucleotide according to the present invention may contain a sequence such as a sequence of an untranslated region (UTR) or a vector sequence (including an expression vector sequence).

  The vector according to the present invention can be prepared by inserting the polynucleotide according to the present invention into a predetermined vector by a known gene recombination technique. The vector is not limited to this, but a cloning vector can be used in addition to the recombinant expression vector described below.

  Although it does not specifically limit as a supply source for acquiring the polynucleotide based on this invention, It is preferable that it is a biological material. As used herein, the term “biological material” is intended to be a biological sample (tissue sample or cell sample obtained from an organism), and in the examples described below, In particular, rice, barley, wheat and corn) are used, but not limited thereto.

[3: Antibody]
The present invention provides an antibody that specifically binds to deoxymumine acid synthase (ie, a polypeptide having an activity of reducing the keto form of nicotianamine to 2′-deoxymugineic acid). The antibody according to the present invention is not limited as long as it can specifically bind to the polypeptide, and may be a polyclonal antibody against the polypeptide, but is preferably a monoclonal antibody against the polypeptide. Monoclonal antibodies have advantages such as uniform properties, easy supply, and semipermanent storage as hybridomas.

As used herein, the term “antibody” refers to immunoglobulins (IgA, IgD, IgE, IgG, IgM and their Fab fragments, F (ab ′) ₂ fragments, Fc fragments), eg Examples include, but are not limited to, polyclonal antibodies, monoclonal antibodies, single chain antibodies, and anti-idiotype antibodies.

  “Antibodies” can be prepared according to various known methods. For example, monoclonal antibodies can be prepared by techniques known in the art (eg, hybridoma method (Kohler, G. and Milstein, C., Nature 256, 495-497 (1975)), trioma method, human B-cell hybridoma method (Kozbor). , Immunology Today 4, 72 (1983)) and the EBV-hybridoma method (see Monoclonal Antibodies and Cancer Therapy, Alan R Lis, Inc., 77-96 (1985)). .

  Peptide antibodies can also be obtained by methods known in the art (eg, Chow, M. et al., Proc. Natl. Acad. Sci. USA 82: 910-914; and Bittle, FJ et al., J. Gen. Virol. 66: 2347-2354 (1985), which is incorporated by reference herein, can be readily prepared.

It will be apparent to those skilled in the art that Fab and F (ab ′) ₂ and other fragments of the antibodies of the invention can be used in accordance with the methods disclosed herein. Such fragments can typically be produced by proteolytic cleavage using enzymes such as papain (resulting in Fab fragments) or pepsin (resulting in F (ab ′) ₂ fragments). Alternatively, fragments that bind to a polypeptide according to the invention can be produced by the application of recombinant DNA technology or by chemical synthesis.

As described above, the antibody according to the present embodiment only needs to have a fragment (for example, Fab fragment and F (ab ′) ₂ fragment) that specifically binds to the polypeptide according to the present invention. It should be noted that immunoglobulins consisting of Fc fragments are also included in the present invention.

  That is, an object of the present invention is to provide an antibody that specifically binds to the polypeptide according to the present invention and use thereof, and the types of individual immunoglobulins specifically described in the present specification. (IgA, IgD, IgE, IgG, or IgM) does not exist in an antibody production method or the like. Therefore, it should be noted that antibodies obtained by methods other than the above methods also belong to the scope of the present invention.

[4: Production of plants imparted with iron deficiency tolerance]
The present invention provides a method for producing a plant imparted with iron deficiency tolerance. The method for producing a plant according to the present invention only needs to include a step of introducing the polynucleotide according to the present invention into the plant. In one embodiment, the method for producing a plant according to the present invention includes the step of mating a plant that does not express the polynucleotide according to the present invention with a plant that expresses the polynucleotide according to the present invention. This is a preferable method for producing a plant that is not a transformant.

  In another embodiment, the method for producing a plant according to the present invention introduces 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 plant body to which the iron deficiency tolerance is imparted according to the method according to the present embodiment, which includes a process, can be a plant transformant.

  When a recombinant expression vector is used, the vector to be 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. Examples of such a vector include a vector having a promoter that constitutively expresses a polynucleotide in a plant cell (eg, cauliflower mosaic virus 35S promoter), or a promoter that is inducibly activated by an external stimulus. The vector which has is mentioned.

  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 cultured cell, or various forms of plant cells (eg, suspension cultured cells), protoplasts, leaf sections, callus and the like. Although it does not specifically limit as a plant used for transformation, It is preferable a Gramineae plant, and it is more preferable that they are a rice, a barley, a wheat, or corn.

  For the introduction of genes into plants, transformation methods known to those skilled in the art (for example, the Agrobacterium method, gene gun, PEG method, electroporation method, etc.) are used. It is divided roughly into the method of introducing into cells. When the Agrobacterium method is used, 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. Also, the method of Nagel et al. (Micibiol. Lett., 67, 325 (1990)) can be used. In this method, for example, an expression vector is first introduced into Agrobacterium, and then transformed Agrobacterium is transformed into a plant cell by the method described in Plant Molecular Biology Manual (SB Gelvin et al., Academic Press Publishers). It is a method of introducing into plant tissue. Here, “plant tissue” includes callus obtained by culturing plant cells. When performing transformation using the Agrobacterium method, pBI binary vectors (for example, pBIG, pBIN19, pBI101, pBI121, pBI221, and pPZP202) can be used.

  Electroporation and gene gun methods are known as methods for directly introducing genes into plant cells or plant tissues. When using a gene gun, the plant body, plant organ, plant tissue itself may be used as it is as a target for gene introduction, or it may be used after preparing a section, or a protoplast is prepared and used. Also good. The sample prepared in this manner 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.

  A cell or plant tissue into which a gene has been introduced is first selected for drug resistance such as hygromycin resistance, and then regenerated into a plant 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. The selection marker is not limited to hygromycin resistance, and examples thereof include drug resistance to bleomycin, kanamycin, gentamicin, chloramphenicol and the like.

  When plant cultured cells are used as a host, the recombinant vector can be obtained by, for example, microinjection, electroporation (electroporation), polyethylene glycol, gene gun (particle gun), protoplast fusion, calcium phosphate, etc. It is introduced into a cultured cell and transformed. Callus, shoots, hairy roots, etc. obtained as a result of transformation can be used for cell culture, tissue culture or organ culture as they are, and can be used at a suitable concentration using a conventionally known plant tissue culture method. Of plant hormones (auxin, cytokinin, gibberellin, abscisic acid, ethylene, brassinolide, etc.).

  Whether or not a gene has been introduced into a plant can be confirmed by PCR, Southern hybridization, Northern hybridization, or the like. For example, 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 amplified product is subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis or capillary electrophoresis, stained with ethidium bromide, SYBR Green solution, etc., and the amplified product is detected as a single band, It can be confirmed that it has been transformed. Further, 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.

  Once a transformed plant in which the polynucleotide of the present invention is integrated into the genome is obtained, offspring can be obtained by sexual reproduction or asexual reproduction of the plant. Further, for example, seeds, fruits, cuttings, tubers, tuberous roots, strains, callus, protoplasts, and the like can be obtained from the plant or its progeny, or clones thereof, and the plant can be mass-produced based on them. it can. Therefore, the present invention also includes a plant into which the polynucleotide according to the present invention is introduced so that it can be expressed, or a progeny of the plant having the same properties as the plant, or a tissue derived therefrom.

  In addition, as described above, several other enzymes are involved in the pathway for biosynthesis of 2'-deoxymugineic acid from methionine. By introducing together the polynucleotides encoding these enzymes into the plant, a plant with further improved resistance to iron deficiency can be produced. Specific examples of the enzyme include nicotianamine aminotransferase, nicotianamine synthase, and S-adenosyl-L-methionine synthase.

  These enzymes are well known in the art. For example, for rice, nicotianamine aminotransferase OsNAAT-1 is shown in SEQ ID NO: 19 (amino acid sequence) and SEQ ID NO: 20 (base sequence), and nicotianamine synthesis is performed. The enzymes OsNAS1, OsNAS2 and OsNAS3 are shown in SEQ ID NOs: 21, 23 and 25 (amino acid sequences) and SEQ ID NOs: 22, 24 and 26 (base sequences), respectively, and OsSAMS1 which is an S-adenosyl-L-methionine synthase. And OsSAMS2 are shown in SEQ ID NO: 27 and 29 (amino acid sequence) and SEQ ID NO: 28 and 30 (base sequence), respectively.

  In addition, HvSAMS which is a barley S-adenosyl-L-methionine synthetase is shown by sequence number 31 (amino acid sequence) and sequence number 32 (base sequence) as said enzyme in plants other than rice. In addition, referring to the accession number (DDBJ / EMBL / GenBank) shown below, the amino acid sequence and base sequence of a desired enzyme can be easily obtained: nicotianamine aminotransferase (for example, D88273 and AB005788 ( Nicotineamine synthase (for example, AB061270, AB062711, AB042551 (above corn), AB010086, AB011265, AB011264, AB011266, AB011268, AB011269, AB019525, AF136941, AF136694 (above A19, 342) Arabidopsis), AJ242045 (tomato)) and the like.

  The nicotianamine aminotransferase, nicotianamine synthase and S-adenosyl-L-methionine synthase are not limited to those having the sequence provided by the above-mentioned sequence number or accession number. It may be a mutant. For mutants, see the term definitions in this specification. Moreover, those skilled in the art who read this specification will easily understand that the method for introducing each polynucleotide into the plant may be the same as the method for introducing the polynucleotide according to the present invention into the plant.

[5: Utilization of deoxymugineate synthase and gene / screening of plants with resistance to iron deficiency]
The present invention provides a method for screening for plants having resistance to iron deficiency. The plant screening method according to the present invention only needs to include a step of detecting deoxymugineic acid synthase expressed in the plant body, and based on the presence or absence of expression of the polypeptide according to the present invention, iron deficiency tolerance A plant having the above is selected.

  As described above, the polypeptide according to the present invention synthesizes a compound having an important function when a plant acquires iron from soil. Therefore, plants that express the polypeptide have a high ability to acquire iron and are resistant to iron deficiency. The plant screened according to the method of the present invention may be a natural plant body or a transformant.

  In one embodiment, the method for screening a plant having resistance to iron deficiency according to the present invention detects a polypeptide according to the present invention by reacting an extract from a plant with an antibody according to the present invention. As described above, the antibody specifically binds to the polypeptide according to the present invention to form an immune complex. Therefore, by detecting the formation of the complex, synthesis of deoxymugineic acid expressed in the plant body is performed. Enzymes can be easily detected. Formation of the complex can be detected using, for example, a method in which the antibody is previously labeled with an isotope or a method using a secondary antibody against the antibody. Specifically, the screening method may be a well-known Western blot method, but is not limited thereto. Moreover, although the extract from the said plant can be produced using the freezing crushing method using liquid nitrogen, and a commercially available extraction kit, it is not limited to these. In addition, as used herein, the “extract” may be a crude product or a purified product that has undergone several purification steps.

  As described above, several other enzymes are involved in the pathway for biosynthesis of 2'-deoxymugineic acid from methionine. Therefore, it is preferable that the screening method according to the present invention further detects an enzyme other than deoxymugineic acid synthase. Specifically, the screening method according to the present invention includes a step of detecting expression of each enzyme in a plant body using an antibody against nicotianamine aminotransferase, nicotianamine synthase, S-adenosyl-L-methionine synthase and the like. Is further included. The method for detecting the expression of each enzyme follows the method for detecting the expression of the polypeptide according to the present invention described above.

  In another embodiment, the screening method according to the present invention includes a step of incubating an oligonucleotide comprising a fragment of the polynucleotide according to the present invention or a complementary sequence thereof with an extract from a plant. Preferably, the screening method according to the present invention is characterized by including a step of hybridizing an extract from a plant with genomic DNA, mRNA or cDNA for mRNA derived from the target plant.

  By using the screening method according to the present invention, a plant body that expresses a polypeptide having deoxymugineic acid synthase activity can be easily detected (screened) by detecting a hybridizing target polynucleotide.

  As used herein, the term “oligonucleotide” is intended to be a combination of several to several tens or hundreds of nucleotides, and is used interchangeably with “polynucleotide”. Oligonucleotides are called dinucleotides (dimers) and trinucleotides (trimers) for short ones, and 30 mers (also called 30 bases and 30 nucleotides) or 100 mer (also called 100 bases and 100 nucleotides) for long ones. And the number of nucleotides polymerized. Oligonucleotides can be produced as fragments of longer polynucleotides or chemically synthesized.

  The oligonucleotide used in the screening method according to the present invention can be used as a PCR primer or a hybridization probe for obtaining the polynucleotide according to the present invention or a fragment thereof.

  In addition, those skilled in the art are attributed to the hybridization that occurs between the oligonucleotide used in the screening method according to the present invention and the target gene (polynucleotide) in any of the above-described applications. It is easily understood that it is used to hybridize with a gene of interest (polynucleotide).

  A fragment of a polynucleotide according to the invention is at least 7 nt (nucleotide), 10 nt, 12 nt, 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. Although fragments of length are intended, those skilled in the art can appropriately set a preferred length according to the use described above. By “a fragment having a length of at least 20 nt”, for example, a fragment comprising 20 or more consecutive base sequences from the base sequence shown in SEQ ID NO: 2 or a complementary sequence thereof is intended. By referring to the present specification, the base sequence shown in SEQ ID NO: 2 is provided, so that those skilled in the art can easily prepare a DNA fragment based on SEQ ID NO: 2. For example, restriction endonuclease cleavage or ultrasonic shearing can be readily used to create fragments of various sizes. Alternatively, such fragments can be made synthetically. Appropriate fragments (oligonucleotides) are synthesized by Applied Biosystems Incorporated (ABI, 850 Lincoln Center Dr., Foster City, CA 94404) type 392 synthesizer and the like.

  As described above, several other enzymes are involved in the pathway for biosynthesis of 2'-deoxymugineic acid from methionine. Therefore, it is preferable that the screening method according to the present invention further detects a polynucleotide encoding each of the above enzymes other than deoxymugineic acid synthase. Specifically, the screening method according to the present invention comprises a target plant using an oligonucleotide comprising a fragment such as a nicotianamine aminotransferase gene, a nicotianamine synthase gene, an S-adenosyl-L-methionine synthase gene, or a complementary sequence thereof. The method further includes the step of further hybridizing with the genomic DNA, mRNA or cDNA derived from the derived mRNA.

  Oligonucleotides used in the screening method according to the present invention include not only double-stranded DNA but also single-stranded DNA and RNA such as sense strand and antisense strand constituting the same. The oligonucleotide can be used as a tool for gene expression manipulation by an antisense RNA mechanism. Antisense RNA technology reduces gene products derived from endogenous genes. By introducing the above-mentioned oligonucleotide, the content of the polypeptide having deoxymugineic acid synthase activity can be reduced, and as a result, iron deficiency tolerance in plants can be controlled.

  Thus, the oligonucleotide used in the screening method according to the present invention is used as a hybridization probe for detecting a polynucleotide encoding a polypeptide having deoxymugineic acid synthase activity or as a primer for amplifying the polynucleotide. Thus, a plant or tissue expressing a polypeptide having the activity can be easily detected (screened). Furthermore, the above-mentioned oligonucleotide can be used as an antisense oligonucleotide to control the expression of a polypeptide having deoxymugineic acid synthase activity in a plant or its tissue or cell.

[6: Method, enzyme composition and enzyme kit for synthesizing deoxymugineic acid]
The present invention further provides a method of synthesizing deoxymugineic acid.

  As described above, the polypeptide according to the present invention has the activity of reducing the keto form of nicotianamine to produce 2'-deoxymugineic acid in the pathway for biosynthesis of mugineic acids of gramineous plants. Therefore, 2'-deoxymugineic acid can be synthesized by incubating the polypeptide with a keto body of nicotianamine and a coenzyme. In addition, it is preferable to use NADH or NADPH as the coenzyme, and the incubation is preferably in the range of pH 8.0 to 9.5, and in the range of pH 8.5 to 9.0. More preferably. Moreover, it is preferable to perform the said incubation within the temperature range of 20 to 30 degreeC, and it is more preferable to carry out within the temperature range of 25 to 26 degreeC.

  The keto body of nicotianamine is an unstable compound. Therefore, in the method for synthesizing deoxymugineic acid according to the present invention, the keto body is preferably produced from nicotianamine rather than being provided as such. That is, in the method of synthesizing deoxymugineic acid according to the present invention, the step of incubating the polypeptide according to the present invention with the keto body of nicotianamine comprises the step of incubating the polypeptide according to the present invention with nicotianamine, nicotianamine aminotransferase, It may be a step of incubating with the substrate. As the coenzyme, pyridoxal 5'-phosphate (PLP) is preferably used, and 2-oxoglutarate is preferably used as the substrate. In the incubation, the pH is preferably in the range of 8.0 to 9.5, and more preferably in the range of 8.5 to 9.0. Moreover, it is preferable to perform the said incubation within the temperature range of 15 degreeC-35 degreeC, and it is more preferable to carry out within the temperature range of 24 degreeC-26 degreeC.

  The present invention further provides an enzyme composition and an enzyme kit for synthesizing deoxymugineic acid. The enzyme composition according to the present invention is characterized by containing the polypeptide according to the present invention, and the enzyme kit according to the present invention is characterized by comprising the polypeptide according to the present invention. Since the polypeptide according to the present invention has an enzyme activity for reducing the keto form of nicotianamine to 2′-deoxymugineic acid, a composition and kit for using this enzyme activity are described in the present specification. Are referred to as “enzyme composition” and “enzyme kit”.

  As used herein, “composition” is a form in which various components are contained in one substance, and “kit” contains at least one of various components in another substance. It is intended to be in form.

  As described above, in order to carry out the enzymatic reaction for synthesizing deoxymugineic acid, it is sufficient that the polypeptide (or polynucleotide) according to the present invention is present, and other enzymes described in the present specification. Coenzymes and substrates may be present as necessary. Therefore, the enzyme kit according to the present invention may be provided with the polypeptide (or polynucleotide) according to the present invention and other enzymes, coenzymes, etc. in the same container or separately.

[7: Method for measuring deoxymugineic acid synthase activity]
The present invention provides a method for measuring the enzyme activity of deoxymugineate synthase. The method for measuring enzyme activity according to the present invention comprises a step of incubating a sample to be measured with the keto body, and incubating the sample in order to measure the enzyme activity for reducing the keto body of nicotianamine to 2′-deoxymugineic acid. The process of measuring the quantity of 2'-deoxymugineic acid synthesize | combined by the process to perform should just be included. In addition, it is preferable to measure also about various controls with a sample. Examples of various controls include a negative control that clearly shows that the enzyme to be measured is not contained, a positive control that clearly shows that the polypeptide of the present invention is contained, and a chemical control that uses NaBH _4. However, it is not limited to these.

  Further, as described above, the keto body of nicotianamine is an unstable compound and is chemically very difficult to synthesize. Therefore, it is preferable to further include a step of generating a keto body of nicotianamine by incubating nicotianamine with nicotianamine aminotransferase and a coenzyme and a substrate. The conditions in the step of incubating the sample with the keto body and the step of producing a keto body of nicotianamine preferably follow the description described above in [6].

  The amount of 2'-deoxymugineic acid synthesized by the step of incubating the sample with the keto body can be measured using, for example, high performance liquid chromatography (HPLC), but is not limited thereto. Using the above measurement results, the enzyme activity for reducing the keto form of nicotianamine in each sample to 2'-deoxymugineic acid can be measured.

  The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope shown in the claims, and embodiments obtained by appropriately combining technical means disclosed in different embodiments. Is also included in the technical scope of the present invention.

  Moreover, all the academic literatures and patent literatures described in this specification are incorporated herein by reference.

[Example 1: Measurement of enzyme activity]
Nicotianamine aminotransferase (NAAT) and candidate proteins were purified and subjected to measurement of enzyme activity. See Example 2 for candidate proteins.

  A partial sequence of HvNAAT-A was cloned into pET15b (Novagen) as a His tag fusion protein. And it introduce | transduced into colon_bacillus | E._coli BL21 (Lys) stock | strain. Induction and purification of protein expression was in accordance with the manufacturer's instructions. The protein was quantified using the Bradford method. By removing the signal peptide, a large amount of nicotianamine aminotransferase (NAAT) protein could be obtained.

  Next, an ORF encoding the candidate protein was amplified using a primer having an XbaI site at the 5 'end and a HindIII site at the 3' end. Then, the amplified ORF was subcloned into pCR-BluntII-TOPO (Invitrogen, Carlsbad, CA), and the sequence was confirmed. The prepared plasmid was cleaved with XbaI and HindIII, and the fragment containing the ORF was subcloned into pMAL-c2 (New England Biolabs). These pMAL-c2 plasmids were introduced into E. coli XLI-Blue strain. The E. coli cells were induced to produce recombinant fusion proteins. Then, the obtained protein was purified. As described above, a large amount of purified protein could be obtained for the candidate protein.

Next, enzyme activity was measured for each protein. Here, NaBH ₄ was used as a chemical control because it can chemically reduce the keto form of nicotianamine to DMA.

5 μg of HvNAAT-A fusion protein per reaction was placed in a filter unit (Amicon Ultrafree-MC 30-kDa-cutoff Filter Unit, Millipore) and centrifuged at 6200 × g at 4 ° C. for 15 minutes. The effluent is discarded and 50 μl of N- [Tris (hydroxymethyl) methyl] -3-aminopropanesulfonic acid buffer (50 mM TAPS, 5 mM KCl, 5 mM MgCl ₂ , 10 mM 2-oxoglutarate, 10 μM pyridoxal 5′-phosphorus Acid (PLP) and 150 μM nicotianamine) were added to the filter unit. The solution was mixed several times by pipetting and then incubated at 26 ° C. for 30 minutes. Thus, a keto body of nicotianamine was generated. Subsequently, the filter unit was placed in a new Eppendorf tube and centrifuged at 6200 × g and 4 ° C. for 1 minute. The effluent was collected and NADPH was added to a final concentration of 25 μM.

  Here, 1 μg of the protein sample for measuring the activity was placed on a new filter unit, and centrifuged at 6200 × g and 4 ° C. for 1 minute. Each sample was adjusted separately.

Then, 46 μl of the effluent containing 3 ″ -keto intermediate and NADPH prepared as described above was added to the filter unit containing each sample. Subsequently, the mixture was mixed by pipetting twice or three times. Incubated for 30 minutes at 0 ° C. As a chemical control, ₄ μl of 0.25 M NaBH ₄ was added and the mixture was centrifuged for 1 minute and then kept at 26 ° C. for 5 minutes.

  Thereafter, the filter unit was placed on a new Eppendorf tube and centrifuged at 6200 × g and 4 ° C. for 5 minutes to collect the effluent. Each 50 μl sample was then analyzed by HPLC using DMA as a standard. By carrying out the above method on DMAS candidate proteins, it was possible to identify rice, barley, wheat and corn DMAS, as described later.

  It was found that the optimum pH of deoxymugineic acid synthase was in an unexpected range at the stage where the enzyme activity was measured as described above. Finding this optimum pH is a major factor that has established a measurement method.

  As shown in FIG. 5, the enzyme activity of deoxymugineic acid synthase at pH 8 was 6.5 to 14.9 times the enzyme activity at physiological pH (pH 7). The enzyme activities at pH 8 and pH 9 were almost the same. That is, the pH when DMAS is reacted is preferably greater than 7, and most preferably 8-9. These results indicate that the activity of DMAS is greatly reduced at neutral pH in the cytoplasm, reinforcing the hypothesis that DMA is synthesized in intracellular vesicles derived from rough endoplasmic reticulum. . The optimum pH for NAS is 9. NAAT is 8.5 to 9.

  Mugineic acids are thought to be synthesized in the vesicles derived from the rough endoplasmic reticulum in the root tissue. These vesicles are in an expanded state until the start of MAs secretion and contract by the end of secretion. The vesicular polarity migration is involved in the secretion of phytosiderophores in iron-deficient barley roots. NAS and NAAT are thought to localize in the vesicles. The optimum pH for DMAS activity suggests that the DMAS protein is localized in these intracellular vesicles that are stored until DMA is synthesized and secreted. As described later, from the homology of amino acid sequences, DMAS is considered to be a member of the AKR4 family belonging to the aldo-keto reductase superfamily (AKRs). Here, AKRs have been reported to have activity between pH 6.8 and 11, but have already been determined. vulgare aldehyde reductase, p. Like Somniferum codeinone reductase and Digitalis purpurea aldose reductase, AKR4 protein has an optimum pH around 7. That is, it has been difficult to predict the optimum pH of DMAS based on the homology of amino acid sequences with known proteins.

Example 2: Isolation of DMAS
Analysis of proteins induced under iron deficiency conditions was performed using rice 22k custom oligo DNA microarrays containing sequence data obtained from the rice full-length cDNA project. Among the obtained proteins, proteins presumed to belong to the aldo-keto reductase superfamily were used as candidate proteins for deoxymugineate synthase (DMAS).

  As described above, deoxymugineic acid synthase activity depends on NAD (P) H. This suggests that the enzyme may be a member of the aldo-keto reductase superfamily (AKRs). AKRs are an enzyme superfamily that includes NAD (P) (H) -dependent oxidoreductases, and are known to catalyze the reduction of aldehydes, ketones, monosaccharides, ketosteroids, and prostaglandins.

  This superfamily contains over 120 enzymes, which are divided into 15 families, 9 of which have multiple subfamilies. However, although classified as such, the homology within this family is very low and it is difficult to find an enzyme with the desired activity from sequence similarity. In addition, since AKRs of Gramineae have been hardly characterized, it is known whether a protein presumed to belong to the aldo-keto reductase superfamily is actually a member of AKRs or even DMAS. The only way to do this is to actually measure enzyme activity. However, until now there has been no effective method for measuring enzyme activity.

  For each candidate protein, an ORF was prepared using a cDNA library prepared from iron-deficient rice roots. Using each ORF, the deoxymugineic acid synthase activity of each candidate protein was measured by the method described in Example 1. As a result of the measurement, it was found that AK073738 (presumed NADPH-dependent oxidoreductase) has deoxymugineate synthase activity. This was named OsDMAS1. FIG. 4 b is a diagram showing a measurement result of HPLC in Example 1 of OsDMAS1. As shown in the figure, DMA is certainly synthesized by OsDMAS1.

  Next, barley candidate proteins were selected from various databases based on homology with Oryza sativa NADPH-dependent oxidoreductase (OsDMAS1, AK0773738). For each candidate protein, ORFs were prepared using a cDNA library prepared from iron-deficient barley roots. Using each ORF, the deoxymugineic acid synthase activity of each candidate protein was measured by the method described in Example 1. As a result of measurement, it was found that Unigene No. 6858 in the HarvEST database (Version 1.35; http://harvest.ucr.edu) has deoxymugineate synthase activity. This was named HvDMAS1. FIG. 4 c is a diagram showing a measurement result of HPLC in Example 1 of HvDMAS1. As shown in the figure, it can be seen that deoxymugineic acid (DMA) is synthesized by HvDMAS1.

  Furthermore, wheat and corn candidate proteins were selected from various databases based on homology with OsDMAS1. For each candidate protein, ORFs were prepared using cDNA libraries prepared from iron-deficient wheat or corn roots. Using each ORF, the deoxymugineic acid synthase activity of each candidate protein was measured by the method described in Example 1. As a result of the measurement, it was found that the candidate proteins of wheat and corn obtained by BLAST (www.ncbi.nlm.nih.gov/BLAST/) each have deoxymugineic acid synthase activity. These were named TaDMAS1 and ZmDMAS1.

  FIG. 4 is a diagram showing the results of the HPLC analysis described in Example 1 for the above four types of DMAS proteins. As shown in the figure, in the test tube, all four proteins were able to convert the 3 ″ -keto intermediate to DMA. It was TaDMAS1 that showed the highest enzyme activity. Followed by OsDMAS1, ZmDMAS1, and HVDMAS1.

  Thus, all DMAS proteins were able to synthesize DMA from 3 "-keto intermediates in vitro. TaDMAS1 showed the highest enzymatic activity, and barley had the most wheat. Secretes acids: In rice roots, the amount of NA is higher than that of barley root and the amount of DMA is lower than that of barley root, which is more active in HvDMAS1 and more NA for the production of DMA Interestingly, the activity of endogenous nicotianamine synthase (NAS) is also higher in wheat than in barley, and deoxymuginate synthase (DMAS) is nicotianamine. Apparently more active than aminotransferase (NAAT), ie, NAAT is 5 μ for each reaction. While used were, each DMAS was used 1μg Nevertheless, the TaDMAS1 and chemical control, 3 "-. Seems keto intermediate is a limiting factor (Figure 5).

Note that reduction of the 3 ″ -carbon of the 3 ″ -keto intermediate by NaBH ₄ is not considered to be stereospecific as in the case of enzymatic reduction. And not all chemically produced DMAs have the same conformation at the 3 ″ -carbon as natural DMA.

  As described above, by isolating the DMAS gene of Gramineae, it is involved in the biosynthesis pathway of mugineic acids including S-adenosyl-L-methionine synthase (SAMS), NAS, NAAT, IDS2 and IDS3. All of the genes encoding the enzyme have been isolated.

  In addition, the rice genome database (KOME) was searched using the cDNA sequence of OsDMAS1. This provided the structure and genomic position of the OsDMAS1 gene. Furthermore, the nucleotide sequence of the promoter region of the OsDMAS1 gene was analyzed.

  FIG. 2 a is a schematic diagram showing the structure of the OsDMAS1 gene. As shown in the figure, OsDMAS1 is located on the rice chromosome 3 and is composed of four exons and three introns.

  The length of the genome fragment is 2706 bp and the ORF is 957 bp. The promoter region of the above gene contains a sequence similar to iron deficiency response factor 2 (IDE2: Fe-defective response element 2).

[Example 3: Analysis of amino acid sequence]
From a cDNA library prepared from iron-deficient rice roots, the ORF of OsDMAS1 was amplified using the forward primer shown in SEQ ID NO: 9 and the reverse primer shown in SEQ ID NO: 10.

  The ORF of HvDMAS1 was amplified from a cDNA library prepared from iron-deficient barley roots using the forward primer shown in SEQ ID NO: 11 and the reverse primer shown in SEQ ID NO: 12.

  From the cDNA pool prepared from iron-deficient wheat roots, the TaDMAS1 ORF was amplified using the forward primer shown in SEQ ID NO: 13 and the reverse primer shown in SEQ ID NO: 14.

  From a cDNA library prepared from iron-deficient corn roots, the ORF of ZmDMAS1 was amplified using the forward primer shown in SEQ ID NO: 15 and the reverse primer shown in SEQ ID NO: 16.

  The DMAS cDNA amplified as described above is subcloned into pENTR / D-TOPO (Invitrogen, Carlsbad, Calif.), Thermo Sequence Cycle Sequencing Kit (Shimadzu Corporation, Kyoto, Japan) and DNA sequencer (DSQ-2000L, Shimadzu). Sequence).

  Here, amino acid sequences representing each subfamily of the AKR family were aligned. It also recognized the NADPH binding domain and putative DMAS active site. In addition, systematic affinity was determined. FIG. 2b shows a phylogenetic tree of the aldo-keto reductase superfamily (AKR4).

  OsDMAS1 shows 54.7% homology with Papaver soniferum codeinone reductase (AKR4B2-3), Medicago sativa chalcone polyketide reductase (AKR4A2) and Glycine max chalcone polyketide reductase (AKR481 and AKR4%), respectively. % Homology. Based on sequence homology, DMAS is a member of the AKR4 group. However, DMAS does not fall into any previously defined subfamily. That is, the sequence homology is 60% or less, and it was shown that DMAS belongs to a novel subfamily based on the amino acid sequence and the specificity of the substrate. This result supports the fact that it is difficult to find an enzyme with the desired activity from sequence similarity.

  FIG. 3 is a comparative view of the arrangement of OsDMAS1, HvDMAS1, TaDMAS1, and ZmDMAS1. As shown in the figure, HvDMAS1 is a homologue of rice NADPH-dependent oxidoreductase. OsDMAS1 and HvDMAS1 show 86% homology. According to the nucleotide sequence corresponding to each DMAS, OsDMAS1 encodes a polypeptide of estimated 318 amino acids, and the other DMAS clones encode a polypeptide of estimated 314 amino acids. The sequence of the DMAS gene in these grasses is highly conserved (82-97%). HvDMAS1 and TaDMAS1 show the highest homology (97%). All DMAS proteins have a NADPH binding domain like other AKRs. In ZmDMAS, lysine is substituted for threonine at position 123, but the sequence of the active site of the protein is highly conserved.

  Although there are some variations in grass DMAS, the substrate binding domain is highly conserved (FIG. 3). The role of amino acid at amino acid position 123 in ZmDMAS is not well understood, but isotope substitution of lysine to threonine (FIG. 3) has no effect on enzyme activity (FIG. 4). . The NADPH binding domain is also conserved. This is the largest feature point for AKRs.

  Thus, alignment of amino acid sequences between deoxymugineate synthase (DMAS) and known AKR members reveals that DMASs belong to the AKR4 group. The homology with somniferum codeinone reductase (AKR4B2-3) and Medicago sativa chalcone polyketide reductase (AKR4A2) and Glycine max chalcone polyketide reductase (AKR4A1) was revealed. However, grass DMAS is not classified into existing subfamilies. This indicates that a new subfamily can be defined within the AKR4 family.

  There are a plurality of genes for HvNAS, OsNAS, HvNAAT and OsNAAT. This suggests the possibility of multiple DMAS genes. However, the rice gene AK102609 (presumed NADPH-dependent oxidoreductase 1), which has 63% sequence homology with OsDMAS1, did not show the enzyme activity of DMAS. This also indicates the difficulty of finding DMAS from sequence homology.

[Example 4: Analysis by Northern blot method]
Barley (Hordeum Vulgare L. cv. Ehimehadaka no. 1), wheat (Triticum aestivum L. cv. Chinese spring), maize (Zea mays cv. Alice) and rice (Oryza p. Wet). Germination and culture on filter paper. In order to perform the iron deficiency treatment, the plant was transplanted to a culture medium lacking iron. Two weeks later, roots and leaves were taken, frozen in liquid nitrogen and stored at -80 ° C until use.

  Total RNA was extracted from roots and aerial parts. Then, using a 1.2% (w / v) agarose gel containing 0.66 M formaldehyde, 10 μg of electrophoresis was performed for each lane. Then, it was transferred to a Hybond-N + membrane (Amersham, USA). HvDMAS1 ORF labeled with digoxigenin (DIG) was incubated with the membrane at 68 ° C. and analyzed by Northern blotting. FIG. 6 is a diagram showing the results of the Northern blot method.

  As described above, OsDMAS1 was identified as a gene whose expression is promoted under iron-deficient conditions. The expression pattern of the gene was further characterized by Northern blot analysis. As shown in FIG. 6, under the condition of iron deficiency, the expression of all DMAS genes was promoted in the root, and only the expression of OsDMAS1 and TaDMAS1 was promoted in the above-ground part.

[Example 5: Transformation and growth conditions of rice]
A 1.3 kb region located 5 ′ upstream of the OsDMAS1 gene was amplified by PCR using genomic DNA as a template. As a primer, the forward primer shown to sequence number 17 and the reverse primer shown to sequence number 18 were used. Each primer contains restriction sites for XbaI and HindIII. The amplified fragment was incorporated into pBluescript II SK + vector to confirm the sequence. Subsequently, the 1.3 kb XbaI-HindIII fragment was subcloned upstream of the uidA ORF of the pIG121Hm vector. The uidA ORF encodes β-glucuronidase (GUS). Rice (Oryza sativa L. cv. Tsukinokari) was transformed with the Agrobacterium tumefaciens strain (C58) carrying the vector prepared as described above. As described above, 6 lines of transgenic rice introduced with the OsDMAS promoter-GUS fragment were obtained. Then, T1 seeds were germinated on MS medium containing 50 mg L ⁻¹ hygromycin. After 4 weeks, the plants were transferred to a medium with sufficient iron or deficiency and left for 2 weeks. Then, expression was analyzed by observing staining based on GUS activity. FIG. 7 is a diagram showing the staining result.

  OsDMAS gene expression was detected in iron-sufficient roots and was strongly induced in response to iron deficiency. In iron-sufficient plant roots, staining based on GUS activity derived from OsDMAS1 promoter activity was observed in the central column. However, no staining was observed in cells of the epidermis, integument and cortex (FIG. 7a). When expanded, strong staining was detected in inner sheath cells adjacent to the native and epigenetic xylem (FIG. 7b). In other words, the activity of the OsDMAS1 promoter in the roots of plants with sufficient iron is localized in cells involved in long-distance transport (FIG. 7c).

  In the roots of iron-deficient plants, the OsDMAS1 promoter is active in all tissues including the epidermis, rind, epidermis and all central columns (FIG. 7e). A particularly intense staining was detected in the inner sheath cells adjacent to the native and metamorphic xylem, as was the case with iron-sufficient roots (FIG. 7f). In addition, intense staining was observed in half cells. Interestingly, in both iron-deficient and iron-deficient roots, strong GUS activity was detected in the cells surrounding the epigenetic xylem I, ie in the area where the lateral roots grow.

  In the leaves of plants with sufficient iron, no GUS expression was observed (FIG. 7g). On the other hand, under iron-deficient conditions, GUS activity was detected in cells between epigenetic wood part I and sieve element (FIG. 7h). This suggests that DMA plays some role in iron homeostasis.

  It is known that mugineic acids are secreted in the morning by a clear daily rhythm. Similarly, genes involved in the biosynthesis of mugineic acids are also expressed diurnally. The promoter region of OsDMAS1 contains cis elements containing IDE2-like sequences (-1408 to -1434) and evening elements (-262 to -201, -667 to -734, -702 to -752; EE; AATACT). . This suggests that OsDMAS expression is diurnally controlled by iron deficiency.

  Like all other genes involved in the MAs biosynthetic pathway, expression is promoted in root tissues under iron deficiency. In the above-ground part under iron deficiency, the expression of DMAS is promoted in rice and wheat, and no expression is observed in barley and corn (FIG. 6). Interestingly, in rice, most genes involved in the biosynthetic pathway of MAs are expressed in both roots and aerial parts, whereas in barley most resistant to iron deficiency among gramineous plants. The expression of those genes is limited to the roots. The OsDMAS1 promoter has an IDE2-like sequence, suggesting that expression of the gene is regulated by iron deficiency. OsDMAS1 expression was detected in iron-sufficient roots (FIG. 7). This is consistent with reports that iron detected DMA in sufficient rice and barley roots. OsNAS1-2 and OsNAAT1 are similarly expressed in cells involved in long-distance transport under iron-sufficient conditions. In contrast, HvNAAT-A, which encodes a protein that converts NA to a 3 ″ -keto intermediate, is not expressed in the presence of iron, whereas HvNAAT-B exhibits basal levels of expression. Gene expression increases under iron-deficient conditions DMAS expression is promoted in root tissues under iron-deficient conditions (Figure 6), which is important for DMA production and secretion. In the aerial part, DMA is detected under iron-sufficient conditions and increases under iron-deficient conditions, while barley secretes a larger amount of barley acids, and in leaves, iron-sufficient conditions and In both iron-deficient conditions, more DMA was detected in rice than in barley, and OsDMAS1 promoter-GUS activity was not detected in the above-ground part of iron-enriched rice. DMA detected in iron-enriched rice leaves may have been transported from the root in the form of a complex with iron, and this hypothesis is that under iron-sufficient conditions, the expression of OsDMAS1 is This is enhanced by the fact that it is observed only in the part involved in long-distance transport.Under iron deficiency, the amount of DMAS increases in the above-ground part of rice. Suggests that this DMA is involved in iron homeostasis and not in the acquisition of iron from the soil.

  If this invention is used, since the plant which can grow also in alkaline soil with little solubilized iron content can be produced, it is useful. Moreover, if this invention is used, since the plant which has iron deficiency tolerance can be screened, it is useful.

It is a figure which shows a part of biosynthesis pathway of mugineic acid of Gramineae. a is a schematic diagram showing the structure of the gene of OsDMAS1, and b is a diagram showing a phylogenetic tree of the aldo-keto reductase superfamily (AKR4). It is a comparison figure of the amino acid sequence of deoxymugineic acid synthase of Gramineae. a is a graph showing measurement results of HPLC according to the NaBH _4, b is a graph showing measurement results of HPLC according to OsDMAS1, c is a graph showing measurement results of HPLC according to HvDMAS1, d is the TaDMAS1 It is a figure which shows the measurement result of such HPLC, e is a figure which shows the measurement result of HPLC which concerns on ZmDMAS1, f is a figure which shows the measurement result of HPLC which concerns on negative control, g is related with refined DMA It is a figure which shows the measurement result of HPLC. It is a graph which shows the influence of pH with respect to the activity of each deoxymugineic acid synthase which concerns on the Example of this invention. It is a figure which shows the result of the Northern blotting which concerns on the Example of this invention. a is a cross-sectional view of the root under the iron-sufficient condition, b is an enlarged cross-sectional view of the root under the iron-sufficient condition, c is a longitudinal cross-sectional view of the root under the iron-sufficient condition, and d is iron-deficient Fig. 2 is a longitudinal cross-sectional view of a root under conditions, e is a cross-sectional view of the root under iron-deficient conditions, f is an enlarged cross-sectional view of the root under iron-deficient conditions, and g is a leaf under iron-sufficient conditions. And h is a longitudinal sectional view of a leaf under iron deficiency conditions.

Claims (22)

  A polypeptide comprising the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, 5 and 7.   A polypeptide comprising an amino acid sequence in which one or several amino acids are substituted, deleted or added in the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, 5 or 7, comprising a keto body of nicotianamine A polypeptide characterized by being reduced to 2′-deoxymugineic acid.   A polynucleotide encoding the polypeptide of claim 1 or 2.   A polynucleotide comprising the base sequence represented by any of SEQ ID NOs: 2, 4, 6 or 8.   A polynucleotide consisting of a base sequence represented by any one of SEQ ID NOs: 2, 4, 6 or 8, wherein one or several bases are substituted, deleted or added, comprising a keto body of nicotianamine A polynucleotide encoding an enzyme that reduces to 2'-deoxymugineic acid.   A polynucleotide capable of hybridizing under stringent conditions with a complementary sequence of the nucleotide sequence shown in any one of SEQ ID NOs: 2, 4, 6 or 8, and reducing a keto form of nicotianamine to 2′-deoxymugineic acid A polynucleotide which encodes an enzyme which   A vector comprising the polynucleotide according to any one of claims 3 to 6.   A transformant into which the polynucleotide according to any one of claims 3 to 6 has been introduced.   A method for producing a plant having resistance to iron deficiency, comprising a step of introducing the polynucleotide according to any one of claims 3 to 6 into the plant.   The method according to claim 9, further comprising the step of introducing at least one of a nicotianamine aminotransferase gene, a nicotianamine synthase gene, or an S-adenosyl-L-methionine synthase gene into a plant.   An antibody that specifically binds to the polypeptide according to claim 1 or 2.   A method for synthesizing 2'-deoxymugineic acid, comprising the step of incubating the polypeptide of claim 1 or 2 with a keto body of nicotianamine.   A method for synthesizing 2'-deoxymugineic acid, comprising the step of incubating the polypeptide according to claim 1 or 2 with nicotianamine and nicotianamine aminotransferase.   An enzyme composition for synthesizing 2'-deoxymugineic acid, comprising the polypeptide according to claim 1 or 2.   An enzyme kit for synthesizing 2'-deoxymugineic acid, comprising the polypeptide according to claim 1 or 2.   The enzyme kit according to claim 15, further comprising nicotianamine aminotransferase. A method for measuring an enzyme activity for reducing a keto body of nicotianamine to 2′-deoxymugineic acid,
Incubating a sample to be measured with a keto body of nicotianamine;
Measuring the amount of 2′-deoxymugineic acid produced by the incubating step;
A method comprising the steps of: A method for measuring an enzyme activity for reducing a keto body of nicotianamine to 2′-deoxymugineic acid,
Incubating the sample to be measured with nicotianamine and nicotianamine aminotransferase;
Measuring the amount of 2′-deoxymugineic acid produced by the incubating step;
A method comprising the steps of:   A method for screening a plant having resistance to iron deficiency, comprising a step of incubating an extract from a plant with the antibody according to claim 11.   20. The method of claim 19, further comprising incubating the extract with at least one antibody against nicotianamine aminotransferase, nicotianamine synthase or S-adenosyl-L-methionine synthase.   A method for screening a plant having resistance to iron deficiency, comprising a step of incubating an extract from a plant with the polynucleotide according to any one of claims 3 to 6.   The method according to claim 21, further comprising the step of incubating the extract with at least one of a nicotianamine aminotransferase gene, a nicotianamine synthase gene, and an S-adenosyl-L-methionine synthase gene. .