JP2013513381A - Rice varieties with increased trace element content and their uses - Google Patents

Abstract

  This is a functional food containing a transformed plant in which the content of trace elements by activation of rice OsNAS2 gene or OsNAS3 gene is increased as compared to the wild type, and a product produced from the transformed plant.

Description

  The present invention relates to a transformed plant in which the content of trace elements is increased by the activation of OsNAS2 gene or OsNAS2 gene in the plant, and a functional food containing a product generated from the transformed plant.

Iron accounts for the fourth largest amount of elements present on Earth, but is not readily available to plants. This is because iron in soil is mostly present in oxyhydrate form and has very low solubility. Moreover, about 30% of the world soil shows basicity. In soil having such properties, iron exists in the form of Fe ₂ O ₃ with very low solubility, making plant growth difficult (Non-patent Document 1). Thus, the lack of iron has a fatal effect on plants. In particular, iron deficiency affects chlorophyll synthesis and chloroplast development, reducing plant production.

  Iron also has important implications for human health. Iron is one of the indispensable trace metal elements necessary for various functions in cells. According to the World Health Organization (WHO), about 30% of the world's population suffers from anemia due to iron deficiency, most of which is concentrated in developing countries. Zinc deficiency inhibits plant growth, resistance to stress and chlorophyll production. Zinc is also known to play an important role in human immune responses, increase resistance to infection, and participate in cell growth and wound healing.

Plants are known to overcome iron deficiency in two ways (Non-Patent Document 2). Most plants absorb Fe ³⁺ after reduction to Fe ²⁺ , while gramineous plants such as rice and wheat, when iron deficiency, puts an iron-binding substance called phytosiderophore in the soil It secretes and binds Fe ³⁺ to increase solubility and absorb the complex of iron and phytosideophore.

  Rice is an important staple food for humans, and the development of rice varieties containing large amounts of iron has important implications for promoting human health. Therefore, the development of many rice varieties of iron by genetic engineering has been presented as an alternative to overcome the limitations of traditional breeding methods and solve human health problems caused by iron deficiency (non-patented) Reference 3).

  As a part of such efforts, a method for increasing the iron content by increasing the amount of ferritin, which is an iron storage protein originally present in plants, has been proposed (Non-Patent Document 4). In this method, a bean ferritin gene was transformed using a rice glutelin promoter showing endosperm-specific expression, and the produced rice seeds were used as a control group. In comparison, the iron content increased three times (Non-Patent Document 5). Moreover, as a result of overexpressing the ferritin gene of common bean (Phaseolus vulgaris) in rice seeds, the iron content increased two-fold (Non-patent Document 6).

  According to Patent Document 1, a ferritin-expressing gene is isolated from beans and rice, and then combined with an endosperm-specific promoter isolated from rice and corn seed storage proteins globulin, glutelin, and zein. An rice expression vector that expresses an expression vector and is transformed with the expression vector and has a high concentration of iron producing ability is described. However, this method utilizes genes derived from crops other than rice such as beans and corn.

According to Patent Document 2, a gene encoding nicotianamine aminotransferase (NAAT) which is an enzyme in the biosynthesis pathway of mugineic acids is introduced into a gramineous plant to improve iron water absorption. Is described. However, this method utilizes a gene coding for nicotianamine aminotransferase.
According to Patent Document 3, a gene encoding a reductase that reduces a keto body of nicotianamine to 2′-deoxymugineic acid is introduced to produce a plant with enhanced iron deficiency tolerance. However, this method utilizes a gene encoding a reductase that reduces the keto form of nicotianamine to 2′-deoxymugineic acid.

  On the other hand, nicotianamine (NA) is a chelator that binds to a metal element present in the plant body, and plays an important role in the absorption and homeostasis of the metal element (Non-patent Document 7). NA is made from three S-adenosyl-L-methionine molecules by the nicotianamine synthase (NAS) gene present in plants.

  Hypertension is the most frequently occurring chronic circulatory disease, and angiotensin-converting enzyme (RAS) is an important enzyme in the renin-angiotensin system (RAS), which is an important mechanism for increasing blood pressure. ACE (angiotensin converting enzyme) is an enzyme that converts angiotensin I (AngI: angiotensin I) to angiotensin II (Ang II: angiotension II), and it is known that the converted AngII contracts blood vessels to increase blood pressure. Yes. Therefore, although an ACE inhibitor has been developed and commercialized for the purpose of improving hypertension, there have been various reports showing that nicotianamine acts as an ACE inhibitor (Non-patent Document 8).

  The present inventor has clarified that the content of trace elements increases when the OsNAS2 gene or OsNAS2 gene originally present in rice is activated, thereby completing the present invention.

Korean Registered Patent No. 10-471679 Korean Registered Patent No. 10-454083 Korean Published Patent No. 10-2003-84892

Mori, Curr Opin Plant Biol 2: 250-253, 1999 Marschner et al., J.A. Plant Nutr 9: 3-7, 1986 Qu et al., Planta 222: 225-233, 2005 Theil et al., Annu Rev Biochem 56: 289-315, 1997 Goto et al., Nat Biotech 17: 282-286, 1999 Lucca et al., Theor Appl Genet 102: 392-397, 2001 Douchkov et al., Plant Cell Environ 28: 365-374, 2005 Usuda et al., Plant Biotech J 7: 87-95, 2009

  The present invention provides a transformed plant in which the content of a trace element due to activation of a gene present in the plant itself is increased compared to a wild type, and a functionality containing a product generated from the transformed plant. It provides food.

Exemplary embodiments of the present invention provide plants with increased trace element content.
Another exemplary embodiment of the present invention includes a step of growing a plant according to the above-described exemplary embodiment of the present invention under a condition in which one or more selected from the group consisting of iron and zinc is deficient. A method for growing a plant body is provided.
Another exemplary embodiment of the present invention provides a method for growing a plant body comprising the step of growing the plant body according to the above-described exemplary embodiment of the present invention in a condition where an excessive amount of heavy metal is present.
Another exemplary embodiment of the present invention provides a functional food containing the plant or containing a product produced therefrom.
Another exemplary embodiment of the present invention provides a pharmaceutical composition comprising a product comprising or produced from said plant.
Another exemplary embodiment of the present invention provides a method for producing a plant with increased trace element content.
Another exemplary embodiment of the present invention provides a method for growing a plant comprising the step of growing the plant as described above under high pH conditions.

According to the plant according to an embodiment of the present invention, it can have an increased trace element content as compared to the wild type (WT).
In addition, according to the functional food according to an embodiment of the present invention, it has an increased trace element content compared to the wild type (WT), and has high agricultural and nutritional benefits. it can.
Moreover, according to the method for producing a plant body having an increased trace element content according to an embodiment of the present invention, a plant body having an increased trace element content can be produced effectively.
In addition, according to the method for growing a plant according to an embodiment of the present invention, the plant is grown under growth conditions containing an amount lacking heavy metals such as iron or zinc, or copper, zinc or nickel. Plants can be grown under growth conditions containing heavy metals in excess.

1 is a drawing showing an embodiment of a DNA structure into which T-DNA is inserted. The expression pattern of OsNAS2 gene in rice leaves or roots grown for 7 days with 0,1,10,500 μM Fe (III) -EDTA added to MS medium, respectively, using quantitative real-time RT-PCR It is the analyzed graph. Rice seeds were grown for 7 days in MS medium without addition of 100 μM Fe (III) -EDTA, then transferred to medium supplemented with 100 μM Fe (III) -EDTA, and grown at a certain time. It is the result of measuring the expression of OsNAS2 from RNA extracted from leaves and roots. It is the graph which compared the expression level of OsNAS2 about OsNAS2-D1 and its wild type (WT). MS solid medium (MS) containing both iron and zinc, MS solid medium containing no iron (Fe-), wild type (WT) grown on MS solid medium containing no zinc (Zn-) and OsNAS2-D1 It is a phenotype. MS medium adjusted to pH 8.5 (MS / pH 8.5), wild type (WT) and OsNAS2-D1 grown on MS medium (Fe− / pH 8.5) containing 8.5 and no iron FIG. Wild type (WT) and OsNAS2-D1 phenotypes grown in respective solid MS media supplemented with excess amounts of zinc (5 mM), copper (0.3 mM) or nickel (0.5 mM). 1 is a drawing showing an embodiment of a DNA structure into which T-DNA is inserted. It is the graph which analyzed the expression aspect of OsNAS1 gene using the quantitative real-time RT-PCR in the leaf or root of the rice grown on the culture medium which does not contain Fe, Zn, Cu, or Mn in MS culture medium and MS culture medium. It is the graph which analyzed the expression aspect of OsNAS2 gene using the quantitative real-time RT-PCR in the leaf or root of the rice grown on the culture medium which does not contain Fe, Zn, Cu, or Mn in MS culture medium and MS culture medium. It is the graph which analyzed the expression aspect of OsNAS3 gene using the quantitative real-time RT-PCR in the leaf or root of the rice grown on the culture medium which does not contain Fe, Zn, Cu, or Mn in MS culture medium. It is the graph which compared the expression level of OsNAS3-D1 and its wild type (WT), OsNAS3-D2 and its wild type (WT). MS solid medium (MS) containing both iron and zinc, MS solid medium (Fe-) not containing iron, wild type (WT) and OsNAS3-D1 grown in MS solid medium (Zn) not containing zinc It is an image showing leaves for knowing phenotype and whitening phenomenon. OsNAS3-D1 showed phenotype and elongation of wild-type (WT) grown on each solid MS medium supplemented with excessive amounts of zinc (5 mM), copper (0.3 mM) or nickel (0.5 mM) It is a drawing. MS medium adjusted to pH 8.5 (MS / pH 8.5), wild type (WT) grown on MS medium (Fe− / pH 8.5) containing 8.5 and iron and OsNAS3-D1 2 is a drawing showing a phenotype and elongation.

Exemplary embodiments of the present invention provide plants with increased trace element content.
The plant body may be one in which the content of one or more metals selected from iron, zinc, and copper is increased compared to a wild-type plant body. For example, the plant may be one in which iron and / or zinc is increased as compared to a wild-type plant. The degree of increase in the metal content may be 10% or more, 20% or more, 30% or more, 50% or more, or 70% or more.

  The plant body may be one in which expression of the OsNAS2 gene or the OsNAS2 gene is increased. The OsNAS2 gene may be a gene encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2 (GenBank permission number: AB023818). For example, the gene may have the nucleotide sequence of SEQ ID NO: 1. However, the OsNAS2 gene includes any gene that encodes a polypeptide having OsNAS2 protein activity. For example, a variant of the protein such as SEQ ID NO: 2 having OsNAS2 protein activity may be included, but is not limited thereto. The OsNAS3 gene may be a gene encoding a polypeptide having the amino acid sequence of SEQ ID NO: 11 (GenBank permission number: AB023819). For example, the gene may have the nucleotide sequence of SEQ ID NO: 10. However, the OsNAS3 gene includes any gene that encodes a polypeptide having OsNAS3 protein activity. For example, a mutant of the protein such as SEQ ID NO: 11 having OsNAS3 protein activity may be included, but is not limited to these examples.

  The plant body may be a monocotyledonous plant. For example, rice, wheat and corn. The plant body may be the whole plant body, roots, leaves, seeds, and cells isolated from the plant body. The plant body also includes seeds produced from the plant body.

  In the plant body, increasing the expression of the OsNAS2 gene or the OsNAS3 gene may be performed by any method known in the field related to increasing the expression of an endogenous gene present in a cell. . For example, a transposable element (TE) or an insertion mutation method that introduces an insertion element such as T-DNA into the genome of a plant may be used. The insertion mutation method has an advantage that the inserted element acts as a tag for gene confirmation. A modified insertion mutation method may also be used. An example of a modified insertion mutation method is a gene trap system that comprises fusing the tagged gene with a reporter gene such as β-glucuronidase (GUS) or green fluorescent protein (GFP). ). According to this system, the gene can be confirmed based on the expression aspect. Insertion of a reporter without a promoter not only disrupts normal gene function, but if the direction of the inserted reporter gene is matched, the reporter is expressed and reflects the expression of the inserted gene, so that useful promoter separation is also possible. Is possible. Another example of a modified insertion mutagenesis is the activation tagging system. This system uses T-DNA or TE (transposable element) containing a multimerized CaMV 35S enhancer. Enhancers, regardless of orientation, can function at significant distances from the coding region, causing transcriptional activation of adjacent genes and the formation of dominant gain-of-function mutations. Can do. The plant body can be obtained by selecting individuals with increased expression of the OsNAS2 gene among the mutants formed by the above method (Jung et al., Plant Physiology 130: 1636-1644). The CaMV 35S enhancer may have, for example, the nucleotide sequence of SEQ ID NO: 12. The plant may be, for example, an enhancer-introduced one or an amplified copy number of the gene in order to increase the expression of the OsNAS2 gene or the OsNAS3 gene. Examples of the enhancer include a CaMV 35S enhancer (Cauliflower mosaic virus 35S enhancer) or a ligated product thereof (for example, 4 ligated products), and the enhancer is a vector such as pGA2715 (Jung et al., Those introduced into the genome of the plant by activation tagging vectors such as Plant Physiology 130: 1636-1644) and pGA2772 (Jung et al., Plant J 45: 123-132, 2006) It may be. The enhancer may be placed at an appropriate position with the OsNAS2 gene or the OsNAS3 gene depending on the characteristics of the selected enhancer. For example, the OsNAS2 gene or the OsNAS3 gene may be located adjacent to the 5 ′, 3 ′ or 5 ′ and 3 ′ ends of the OsNAS2 gene or the OsNAS3 gene. For example, the enhancer is a CaMV 35S enhancer, and may be introduced within about 10 kb, for example, about 2.06 kb downstream of the OsNAS2 gene, for example, downstream from the gene stop codon. Good. The enhancer is a CaMV 35S enhancer, and may be introduced upstream of the OsNAS2 gene, for example, within about 10 kb, for example, about 2.06 kb upstream from the start codon of the gene. . For example, the enhancer is a CaMV 35S enhancer, which is introduced within 10 kb, for example, 1.5 kb and / or 1.9 kb downstream of the OsNAS3 gene, for example, downstream from the gene termination codon. It may be. The enhancer is a CaMV 35S enhancer, and is introduced upstream of the OsNAS3 gene, for example, within 10 kb, for example, 1.5 kb and / or 1.9 kb upstream from the start codon of the gene. There may be. The enhancer may be a single enhancer or a combination of multiple enhancers. For example, the enhancer may be a concatenation of four copies of CaMV 35S enhancer.

  Methods for introducing a foreign nucleic acid structure, such as an enhancer, into a plant body are known. For example, the structure may be introduced via an Agrobacterium tumefaciens Ti plasmid, electroporation and bombardment. In addition, the introduction of a foreign nucleic acid structure into a plant may be performed by introducing a site-specifically at a specific position or by randomly introducing a plant having a specific trait after introduction at random.

  An example of the plant body may be rice deposited under a seed accession number: KACC 98008P, a seed accession number: KACC 98004P or KACC 98005P. Another example of the plant may be OsNAS2-D1 rice (Oryza sativa) deposited under seed deposit number KCTC 11597BP, or OsNAS3-D1 rice (Oryza sativa) deposited under seed deposit number KCTC 11598BP. . The rice has an increased iron and zinc content compared to wild-type rice. In addition, the rice can grow with resistance under conditions where zinc, copper or nickel is increased.

  Other exemplary embodiments of the present invention provide plants that are resistant to heavy metals. The plant body is as described above. The heavy metal may be one or more selected from zinc, copper and nickel. The degree of tolerance may be one that can grow with 5 mM or more zinc, 0.3 mM or more copper, or 0.5 mM or more nickel.

  Another exemplary embodiment of the present invention includes a step of growing a plant according to the above-described exemplary embodiment of the present invention under a condition in which one or more selected from the group consisting of iron and zinc is deficient. A method for growing a plant body is provided. The concentration of iron and zinc may be, for example, a culture medium containing no iron and zinc, or cultured under similar conditions.

  Another exemplary embodiment of the present invention provides a method of growing a plant body comprising the step of growing the plant body according to the above-described exemplary embodiment of the present invention in a condition where an excessive amount of heavy metal is present. The heavy metal may be 5 mM or more zinc, 0.3 mM or more copper, or 0.5 mM or more nickel.

  Another exemplary embodiment of the present invention provides a functional food containing the plant or containing a product produced therefrom. In the functional food, one or more selected from iron, zinc and copper is increased. The functional food may be a product containing or processed from seeds of the plant body, for example, rice.

  Another exemplary embodiment of the present invention provides a pharmaceutical composition comprising a product comprising or produced from said plant. The plant may be selected from the group consisting of roots, leaves, stems, seeds, and combinations thereof. The product may be obtained by processing a plant selected from the group consisting of roots, leaves, stems, seeds and combinations thereof. For example, it may be obtained by physically or chemically treating the plant body. For example, the product may be obtained through one or more processes selected from maturation, pulverization, extraction, separation and purification, and combinations thereof.

  The pharmaceutical composition may include a pharmaceutically acceptable diluent or carrier. The carrier or diluent may be any carrier known in the art. As the carrier or diluent, one or more of general excipients, disintegrants, binders, and lubricants can be selectively used. For example, when the composition is produced into a solid dosage form such as a tablet or a hard capsule, amorphous cellulose, lactose, low-substituted hydroxycellulose, etc. are used as excipients, and starch glycolic acid is used as a disintegrant. Sodium, anhydrous calcium monohydrogen phosphate, etc. may be used. As the binder, polyvinyl pyrrolidone, low-substituted hydroxypropyl cellulose, hydroxypropyl cellulose or the like is used. As the lubricant, magnesium stearate, silicon dioxide, talc or the like can be selected and used.

  The composition can be formulated into oral preparations such as granules, powders, liquids, tablets, capsules or dry syrups, or parenteral dosage forms such as injections. It is not limited. Desirably, the composition is in the form of granules, tablets or capsules, or in the form of a solution or injection.

  The therapeutically effective amount of the plant or product thereof used in the composition is the content of trace elements in the plant and the age, sex, disease weight and weight of the individual in need thereof. The situation can be easily determined by those skilled in the art. For example, when the plant or product is rice, the amount is 100 g to 500 g per day, for example, 100 g to 400 g, 100 g to 300 g, 200 g to 300 g, or 150 to 300 g.

  The composition can be administered orally or parenterally. For example, the composition can be administered orally, eg, orally via a common meal. In addition, the administration can be performed once or divided into several times a day.

The composition may be for treating a disease caused by a deficiency of a substance selected from iron, zinc, copper and combinations thereof. The disease may be iron deficiency symptoms. The iron deficiency symptoms include, for example, anemia, fatigue, pallor, hair loss, irritability, weakness, phagocytosis (pica), limbs Includes brittle or grooved nails, Plummer-Vinson syndrome and impaired immune function. The disease includes symptoms caused by zinc deficiency. The disease is also hypozincemia. Hypozincemia refers to a condition where there is not enough zinc necessary for metabolic demand. Zinc deficiency symptoms are hair damage, skin lesions, diarrhea, wasting of body tissue. Includes acrodermatitis enteropathica, anorexia, cognitive and motor function impairment, and dysmenorrhea. The disease is a disease caused by copper deficiency. Copper deficiency can be an syndrome of anemia or pancytopenia, neurodegeration, Menkes disease, spasticity, ataxia and neuropathy It is.
Another exemplary embodiment of the present invention provides a method for producing a plant with increased trace element content.

  An example of the method includes a step of introducing a structure containing a foreign enhancer into a plant body to obtain a plant body in which expression of the OsNAS2 gene or the OsNAS3 gene is increased. The enhancer, the plant body, the method for introducing the structure into the plant body, and the OsNAS2 gene or the OsNAS3 gene are as described above. Whether the expression of the OsNAS2 gene or the OsNAS3 gene is increased may be determined by a method known in the art. For example, direct enzyme activity measurement, measurement of mRNA level of OsNAS2 gene or OsNAS3 gene by a nucleic acid amplification method, or measurement of the amount of expressed protein is performed. The nucleic acid amplification method includes PCR including RT-PCR. An immunological method such as ELISA or Western blotting may be used to measure the amount of the protein. The method includes determining whether the expression of the OsNAS2 gene or the OsNAS3 gene is increased and determining whether one or more selected from iron, zinc, and copper are increased compared to the wild type. Further, it may be included. Methods for measuring the iron and / or zinc content are known.

For example, the method includes the step of introducing a Ti plasmid into which a 35S enhancer derived from a promoter of flower cabbage mosaic virus is inserted, and upstream and downstream of a gene encoding the OsNAS2 activity or OsNAS3 activity. Selecting a plant inserted in one or more positions.
The Ti plasmid is a circular plasmid contained in Agrobacterium tumefaciens, and is used to introduce genetic material into a plant body. The introduction of the 35S enhancer into the Ti plasmid can be easily produced by those skilled in the art using appropriate restriction enzymes and ligases. The 35S enhancer can be located at the left and / or right border of the T-DNA of the Ti plasmid. One or more, for example, four of the 35S enhancers may be adjacent. For example, it may be inserted by a pGA2715 vector or a pGA2772 vector. The introduction of the Ti plasmid into a plant may be performed by introducing the Ti plasmid into Agrobacterium tumefaciens and infecting the plant with Agrobacterium tumefaciens into which the Ti plasmid has been introduced. Generally, the T-DNA region of the Ti plasmid is inserted into the plant chromosome.

  The method includes a step of selecting a plant in which the 35S enhancer is inserted at one or more positions among upstream and downstream of a gene encoding OsNAS2 activity or OsNAS3 activity. “OsNAS2 activity” means an activity that catalyzes a reaction of converting three molecules of S-adenosyl-L-methionine to 3 S-methyl-5′-thioadenosine + nicotianamine or vice versa. . Desirably, the OsNAS2 activity may be due to a protein having the amino acid sequence of SEQ ID NO: 2. The gene encoding the OsNAS2 activity may be a gene encoding a protein having the amino acid sequence of SEQ ID NO: 2, for example, the nucleotide sequence of SEQ ID NO: 1. Whether the 35S enhancer has been inserted at one or more positions upstream and downstream of the gene encoding OsNAS2 may be determined by sequence analysis or nucleic acid amplification. The plant body is preferably rice (oryza sativa). For example, the 35S enhancer may be inserted about 2.06 kb downstream of the base sequence of SEQ ID NO: 1 encoding OsNAS2 activity.

  “OsNAS3 activity” means an activity that catalyzes a reaction of converting three molecules of S-adenosyl-L-methionine to 3 S-methyl-5′-thioadenosine + nicotianamine or vice versa. . Desirably, the OsNAS3 activity may be due to a protein having the amino acid of SEQ ID NO: 11. The gene encoding the OsNAS3 activity may be a gene encoding a protein having the amino acid of SEQ ID NO: 11, for example, the nucleotide sequence of SEQ ID NO: 10. Whether the 35S enhancer is inserted at one or more positions upstream and downstream of the gene encoding OsNAS3 may be determined by sequence analysis or nucleic acid amplification. The plant body is preferably rice (oryza sativa). For example, the 35S enhancer may be inserted about 1.5 kb or about 1.9 kb downstream of the base sequence of SEQ ID NO: 10 encoding OsNAS3 activity.

  FIG. 1 shows an embodiment of a DNA structure into which T-DNA has been inserted. In FIG. 1, the part indicated by the box from ATG to TGA indicates an exon of the OsNAS2 gene. T-DNA can be inserted approximately 2.06 kb after the TGA termination codon of the OsNAS2 gene (OsNAS2-D1 transformed plant).

  FIG. 6 shows one form of a DNA structure into which T-DNA has been inserted. In FIG. 6, the part indicated by the box from ATG to TGA indicates an exon of the OsNAS3 gene. T-DNA can be inserted approximately 1.5 kb after the TGA termination codon of the OsNAS3 gene (OsNAS3-D1 transformed plant). Moreover, T-DNA can be inserted in about 1.9 kb after the TGA termination codon of the OsNAS3 gene (OsNAS3-D2 transformed plant).

Another exemplary embodiment of the present invention provides a method for growing a plant comprising the step of growing the plant as described above under high pH conditions.
The pH may be 8.5 to 14, for example 8.5 to 12.

Hereinafter, the present invention will be described in detail by way of examples. However, the present invention is not limited by the following examples.
Example 1: Isolation of transformed plant in which expression of OsNAS2 gene was activated (1) Expression analysis of nicotianamine synthase 2 (OsNAS2) gene present in rice Three nicotianamine synthases were used in rice (OsNAS1, OsNAS2, OsNAS3) are present and reported to have enzymatic activity (Inoue et al., Plant J 36: 366-381, 2003). The expression level of the OsNAS2 gene according to the concentration of iron supplied from the outside was examined. Rice seeds were prepared with MS medium (containing 0.1 μM CuSO ₄ , 100 μM Fe (III) -EDTA, 30 μM ZnSO ₄ , 10 μM MnSO ₄ ), and Fe (III) -EDTA at 0 μM, 1 μM, 10 μM and 500 μM, respectively. Rice seeds were germinated with the added MS medium and grown for 7 days. In addition, rice seeds were grown for 7 days in an MS medium without addition of 100 μM Fe (III) -EDTA, then transplanted and grown in an MS medium with addition of 100 μM Fe (III) -EDTA to express OsNAS2. The change was confirmed.
RNA was prepared from rice roots and leaves, and the expression level was analyzed by quantitative real-time RT-PCR. PCR conditions were maintained at 95 ° C for 1 minute, and then repeated 45 times at 94 ° C for 15 seconds, 56 ° C for 10 seconds and 72 ° C for 10 seconds. The used primers are as follows. As an expression control group, rice actin 1 gene was amplified.

Primer sequence OsNAS2-forward primer: 5′-CGTCTGAgtgcgtgcattagta-3 ′ (SEQ ID NO: 3)
OsNAS2-reverse primer: 5′-GAAGCACAAACACAAACCGAATA-3 ′ (SEQ ID NO: 4)
OsAct1-forward primer: 5′-TGGAAGCTGCGGGTATCCAT-3 ′ (SEQ ID NO: 5)
OsAct1-reverse primer: 5′-TACTCAGCCCTTGGCAATCCACA-3 ′ (SEQ ID NO: 6)

The results are shown in FIGS. 2A and 2B. 2A and 2B, the vertical axis indicates the expression level based on the expression level of the rice actin 1 gene.
As can be seen from FIG. 2A, the OsNAS2 gene was strongly induced in leaves when rice was grown in a medium containing no iron, but not expressed in a medium containing 1 μM or more of Fe (III) -EDTA. In the roots, the expression was highest when grown in a medium containing no iron, and the expression decreased as the iron concentration increased. FIG. 2B shows the expression of OsNAS2 over time when 100 μM Fe (III) -EDTA was supplied to the medium. The rice used in FIGS. 2A and 2B is Tonjin rice.

(2) Production and isolation of transformed plant in which expression of OsNAS2 gene is activated The pGA2715 vector, which is a T-DNA vector, was used as a structure for producing transformed rice (Jeong et al., Plant Physiol 130: 1636-1644, 2002). The vector is adjacent to the right border and contains a β-glucuronidase (GUS) reporter gene, and is adjacent to the left border and contains a tetramerized transcription enhancer derived from the CaMV 35S promoter. The pGA2715 vector was transformed into Agrobacterium, transformed into wild-type Tonjin rice using the transformed Agrobacterium, and a rice plant population transfected with pGA2715 was produced (Lee et al.). al., J Plant Biol 42: 310-316, 1999; Jeong et al., Plant Physiol 130: 1636-1644, 2002; Jeong et al., Plant Journal 45: 123-132, 2006).

Involved in genomic DNA isolated from the transduced rice plant body, the position where the T-DNA was inserted in the pGA2715 vector was confirmed via inverse PCR. As a result, one transformed plant body (OsNAS2-D1) in which T-DNA was inserted around the OsNAS2 gene was isolated.
In the OsNAS2-D1 transformed plant body, T-DNA containing a 35S enhancer at the left boundary site is inserted about 2.06 kb downstream of the OsNAS2 gene. FIG. 1 shows the insertion position of T-DNA in OsNAS2-D1.

Example 2: Increased expression of OsNAS2 in transformed plants The genotype of OsNAS2-D1 selected in Example 1 was determined via PCR. Genomic DNA was extracted from leaves of Ts generation individuals of OsNAS2-D1, and used as template DNA for PCR. In connection with OsNAS2-D1, two gene-specific primers F (5′-ACCCTACTCCCCCCGAACTAA-3 ′: SEQ ID NO: 7) and primer R (5′-CAAGGAGCTGTCCGTCTAAC-3 ′: SEQ ID NO: 8), and T-DNA The specific primer RB (5′-CAAGGTTAGCATGTAATTAGCCAC-3 ′: SEQ ID NO: 9) was used. In order to know the expression level, the OsNAS2-D1 homozygote and each wild-type rice, that is, Tonjin rice (WT), in a medium to which 100 μM Fe (III) -EDTA was added or not added, 7 RNA was isolated from seedling leaves and roots, flag leaves, 10 cm size flowers and immature seeds grown for days, and reverse transcriptase was used for each cDNA. Manufactured. Real-time RT-PCR was performed using the prepared cDNA as a template and the primers of SEQ ID NOs: 1 and 2, and the amount of OsNAS2 gene transcription was compared. The results are shown in FIG. In FIG. 3, the vertical axis shows the relative transcription amount related to the rice actin 1 gene transcription amount.
As in FIG. 3, OsNAS2 gene expression was increased in OsNAS2-D1 under all conditions tested compared to wild type (WT).

Example 3: Increase in the amount of nicotianamine in the seed of the transformed plant In order to know the influence of the increase in the expression of the OsNAS2 gene on the increase in the amount of nicotianamine, which is a product of the active enzyme, the amount of nicotiamine in the seed was changed. It was measured. After pulverizing the seeds using Multi Bead Shocker (registered trademark) (Yasui Kikai Co., Ltd., Osaka), nicotianamine was added 3 times for 15 minutes from 20 mg powder using 400 μl of 80% ethanol. Extracted. The extracted nicotianamine was quantitatively analyzed using LC-ESI-TOF-MS. The results are shown in Table 1 below.

  As can be seen from Table 1, the amount of nicotianamine in OsNAS2-D1 seeds increased by about 19.8 times compared to the wild type.

Example 4: Measurement of trace element content in transformed plant body To know the effect of OsNAS2 gene activation on trace element accumulation in rice, wild type (WT), transformed plant body, OsNAS2- Trace elements were measured at D1. Using mature seeds and flag leaves of wild type and transformed plants as samples, the trace element measurement method is the method described in Lee et al., Plant Physiol 150: 786-800, 2009. It was helpful. The sample was dried at 70 ° C. for 2 days, the dryness was recorded, and the resulting sample was decomposed with 1 ml of 11N HNO ₃ in a 180 ° C. oven for 3 days. After dilution, the contents of trace element iron and zinc in the sample were measured by atomic absorption spectroscopy (AAS) (SpectrAA-800, Varian, Palo Alto, CA, USA). Table 2 shows the content of trace elements in the transformed plant body.

  As can be seen from Table 2, in mature seeds, OsNAS2-D1 increased iron by 3.0 times, zinc by 2.7 times, and copper by 1.2 times compared to the wild type. In the branches and leaves, when OsNAS2-D1 was compared with the wild type, iron and zinc increased 1.4 times and copper increased 1.5 times.

Example 5: Seedling phenotype analysis of transformed plant under conditions lacking iron and zinc The effect of OsNAS2 gene activation under conditions lacking iron and zinc was examined. Germination of wild type (WT) and OsNAS2-D1 with MS solid medium containing both iron and zinc, MS solid medium without iron (Fe-), MS solid medium without zinc (Zn-), The seedling phenotype was observed after growing for 8 days. The results are shown in Table 3. The phenotypic elongation and chlorophyll content were measured and shown in Table 3. To measure chlorophyll, leaves were collected, weighed, and extracted using 80% acetone. After freezing using liquid nitrogen, thoroughly mix finely crushed leaves and 80% acetone (add 1ml 80% acetone per 100mg of leaves), leave on ice for 15 minutes, centrifuge at 15,000g, The supernatant was divided and the absorbance was measured at 666 nm and 646 nm, and then the amount of chlorophyll containing chlorophyll a and chlorophyll b was measured by the method described in Arnon, Plant Physiol 24: 1-15, 1949.

As can be seen from FIG. 4 and Table 3, the medium (MS) conditions containing both iron and zinc do not show a difference in phenotype as described above. However, in the medium containing no iron (Fe-), OsNAS2-D1 is higher than wild type (WT), and chlorophyll is increased by 50% or more compared to wild type (WT). The chlorosis, which is a typical symptom of iron deficiency, has progressed further. Therefore, it can be seen that OsNAS2-D1 is more resistant to growing conditions lacking iron than the wild type. Even under medium conditions (Zn-) containing no zinc, OsNAS2-D1 had a higher growth rate than wild-type (WT). Therefore, it can be seen that OsNAS2-D1 grows more desirably even under growth conditions lacking zinc as compared to wild type (WT).
In order to verify the experimental results, wild type (WT) and OsNAS2-D1 stems and roots were collected under the respective conditions, and the concentrations of iron and zinc were the same as in Example 4. Measured with The results are shown in Table 4.

  As can be seen from FIG. 3, there was no phenotypic difference in the medium (MS) conditions containing both iron and zinc, as examined above. However, as can be seen from Table 4, OsNAS2-D1 increases the concentration of iron by 1.6 and 1.4 times in the stem and root, respectively, compared to the wild type (WT), and the concentration of zinc is The stem and root increased 1.8 times and 1.4 times, respectively. Also, under medium conditions that do not contain iron, OsNAS2-D1 increases the iron concentration in the stem and root by 1.9 times and 1.8 times, respectively, compared to the wild type (WT). Increased 1.7 times and 1.5 times in the stem and root. In addition, even under medium conditions that do not contain zinc, OsNAS2-D1 has a 1.7-fold increase in zinc concentration in the stem and root compared to the wild type (WT), and the iron concentration is The roots increased 1.5 times and 1.7 times, respectively. Thus, it can be seen that OsNAS2-D1 is more resistant to growth conditions lacking iron or zinc.

Example 6: Phenotypic observation of transformed plants under high pH conditions The soil pH has a number of effects on the solubility of elements required for the plant. For example, if pH is 8.0 or more, iron will change to Fe ₂ O ₃ form with low solubility, and the plant will not be able to use and absorb normally. As a result, iron shortage will occur.
Wild type (WT) and OsNAS2-D1 were germinated in MS medium adjusted to pH 8.5 (MS / pH 8.5) and grown for 10 days to observe the phenotype. Moreover, the same method was implemented and the phenotype was observed also in MS culture medium (Fe- / pH8.5) which does not contain iron. The phenotype and plant elongation are shown in FIG. As can be seen from FIG. 5, in the MS medium adjusted to pH 8.5 (MS / pH 8.5), it can be seen that OsNAS2-D1 has a larger elongation and is more resistant to wild type (WT). Such a result was more clearly shown in MS medium without iron (Fe− / pH 8.5).

Example 7: Phenotypic analysis of transformed plants under conditions that contained excessive amounts of heavy metals. The effect of OsNAS2 activation on toxicity generated during treatment with excessive amounts of heavy metals was examined. Germination of wild type (WT) and OsNAS2-D1 in solid MS medium supplemented with excess amounts of zinc (5 mM), copper (0.3 mM) or nickel (0.5 mM), respectively, grown for 10 days, seedling phenotype Was observed. The phenotype, plant elongation and heavy metal concentrations are shown in FIG.

  As can be seen from FIG. 6 and Table 5, OsNAS2-D1 was higher in height than the wild type, and the whitening phenomenon proceeded more slowly. These results indicate that OsNAS2-D1 has improved resistance to toxicity due to excessive amounts of heavy metals compared to wild type (WT).

  In particular, when grown in a solid MS medium containing excessive amounts of zinc, copper and nickel, the concentrations of zinc, copper and nickel in the stem and root were measured in the same manner as in Example 4 and the results are shown in Table 6. Indicated.

  As can be seen from Table 6, OsNAS2-D1 is 1.4 times and 1.5 times as much as zinc and 1.4 times as much as nickel and 1.4 times as much as copper, compared to wild type (WT). They increased by 1.4 times and 1.2 times, respectively.

Example 7: Iron bioavailability study In order to know the increase in iron bioavailability of the seeds of OsNAS2-D1 absorbed and utilized during seed intake, mice A feeding analysis was performed.
Female Balb / c mice at 3 weeks of age were fed with iron-rich diet (AIN-93G Purified Diet-45 mg Fe / kg diet, Control Diet (CD)) and iron-deficient diet (modified AIN-93G diet containing Iron 3 mg / kg feed, Iron-depleted Diet (ID)) was supplied. Two weeks later, blood was collected from these two groups of mice. This is because blood hemoglobin (Hb) and hematocrit (Hct) are measured to induce anemia and to know the degree of anemia. As a result of the measurement, the hemoglobin (Hb) and hematocrit (Hct) values of the mice fed with the feed deficient in iron decreased to 75 and 65%, respectively, compared with the mice fed with the diet sufficient with iron. (Table 7). It was found that anemia was induced when feeding on iron-deficient feed. After this, the rats fed the feed lacking iron were divided into two groups, the first group (wild type, WT, n = 10) fed the rice made of wild type (WT) seeds, The group (OsNAS2-D1, n = 9) was fed rice made of OsNAS2-D1 seeds. Then, a mouse group fed with a normal diet from the beginning was measured as a control group (control group, CD). After eating rice, blood was collected after one week, two weeks, and four weeks, and Hb and Hct were measured. The results are shown in Table 8. As shown in Table 8, Hb and Hct of mice did not show a significant difference before feeding on rice consisting of wild type (WT) or OsNAS2-D1 seeds. As a result of measuring Hb and Hct of two groups after one week, the second group showed a slight increase in Hb and Hct compared to the first group. Furthermore, as a result of measuring Hb and Hct after one week, the second group showed a significant increase in Hb and Hct compared to the first group, and the group fed normal diet Similar values were shown, and the Hb and Hct of the second group after two weeks passed were significantly higher than those of the first group, and were similar to the group fed with sufficient dietary iron.

Example 8: Zinc Bioavailability Study To determine if the zinc increase in the seed of OsNAS2-D1 is absorbed and utilized during seed intake, the mouse A feeding analysis was performed.
A mouse with a body weight of about 13 g, a diet with sufficient zinc (30 mg Zn / kg diet, Control Diet (CD)) and a diet with insufficient zinc (modified AIN-93G diet containing zinc 3 mg / kg diet, Zinc-depleted) Diet (ZD)) was supplied. Two weeks later, blood was collected from these two groups of mice and the zinc content was measured. As a result of the measurement, the blood zinc value of the mice fed with the feed deficient in zinc was reduced to 66% compared to the rats fed the feed with sufficient zinc (Table 9).

  Thereafter, the rats fed the diet deficient in zinc were divided into 3 groups, the first group was fed a normal diet (Zn− / CD, n = 10) with sufficient zinc, and the second group was wild type. (WT) Eating rice made of seeds (Zn- / WT, n = 10), the third group fed rice made of OsNAS2-D1 seeds (Zn- / OsNAS2-D1). As a control group, the zinc value of a mouse group fed with a normal diet from the beginning was measured. After feeding the normal diet and rice, blood was collected on the 2nd, 6th, 10th, 15th, 20th and 30th days, and the degree of recovery of the numerical value of zinc was measured. The results are shown in Table 10. As shown in Table 10, it was found that the value of zinc increased from the second day after feeding normal feed and rice. However, looking at the recovery rate, the group fed the OsNAS2-D1 seeds was similar in zinc value to the group fed the normal diet in 10 days, but the group fed the wild type seeds was fed on the 30th. Even after lapse, it still shows a low number. In addition, the group that had been fed a diet lacking zinc and changed to a normal diet showed a zinc value similar to the group that had been fed a normal diet from the beginning only after about 20 days.

The inventors obtained the rice cultivar OsNAS2-D1 having an increased trace metal content obtained from the above-mentioned example as a patent seed to the National Agricultural Science Resource Center on April 30, 2009. Deposited and given seed deposit number KACC 98008P.
In addition, the present inventors obtained the rice cultivar OsNAS2-D1 obtained from the above-mentioned examples and having an increased trace metal content, as of November 18, 2009, from the International Depositary under the Budapest Treaty. The seeds were deposited at the Korea Research Institute of Bioscience and Biotechnology (KRIBB) of the Authority, and the seed deposit number KCTC 11597BP was assigned.

Example 9: Isolation of transformed plant in which expression of OsNAS3 gene was activated (1) Expression analysis of three nicotianamine synthase genes present in rice Three nicotianamine synthases (rice) OsNAS1, OsNAS2, OsNAS3) exist and are reported to be enzymatically active (Inoue et al., Plant J 36: 366-381, 2003). The expression levels of the three genes were examined depending on the presence or absence of trace elements. Rice seeds were germinated in MS (Murashige and Skoog) medium (containing 0.1 μM CuSO ₄ , 100 μM Fe (III) -EDTA, 30 μM ZnSO ₄ , 10 μM MnSO ₄ ) and grown for 7 days. In the same manner, rice seeds were germinated in MS medium without 0.1 μM CuSO ₄ , 100 μM Fe (III) -EDTA, 30 μM ZnSO ₄ , or 10 μM MnSO ₄ and grown for 7 days. RNA was prepared from rice roots and leaves, and the expression level was analyzed by quantitative real-time RT-PCR. PCR conditions were maintained at 95 ° C for 1 minute, and then repeated 45 times at 94 ° C for 15 seconds, 56 ° C for 10 seconds and 72 ° C for 10 seconds. The used primers are as follows. As an expression control group, rice actin 1 gene was amplified.

Primer sequence OsNAS1-forward primer: 5′-ACTCCATTTGGTTGTCATTTTT-3 ′ (SEQ ID NO: 13)
OsNAS1-reverse primer: 5′-GGTTGACGTAGTTGCCCTAGTAG-3 ′ (SEQ ID NO: 14)
OsNAS2-forward primer: 5′-GTGATCAACTCCGTCATCGT-3 ′ (SEQ ID NO: 15)
OsNAS2-reverse primer: 5′-TGACAAACACCCTCTGCTTTC-3 ′ (SEQ ID NO: 16)
OsNAS3-forward primer: 5′-GTGATCAACTCCGTCATCATC-3 ′ (SEQ ID NO: 17)
OsNAS3-reverse primer: 5′-TCAGTCCTCATCATGGGAAAAA-3 ′ (SEQ ID NO: 18)
OsAct1-forward primer: 5′-TGGAAGCTGCGGGTATCCAT-3 ′ (SEQ ID NO: 23)
OsAct1-reverse primer: 5′-TACTCAGCCCTTGGCAATCCACA-3 ′ (SEQ ID NO: 24)

The results are shown in FIG. 8, FIG. 9 and FIG. 8, 9, and 10, the vertical axis indicates the expression level based on the expression level of the rice actin 1 gene.
As can be seen from FIG. 8, FIG. 9 and FIG. 10, the OsNAS1 gene and the OsNAS2 gene are relatively more leaf-like when rice is grown on a medium that does not contain iron than when it is grown on a medium that contains enough iron. In the roots, the expression was strongly induced, and in the roots, the degree of induction was weakly increased. On the other hand, the expression of the OsNAS3 gene was induced twice more strongly in the roots when rice was grown in a medium that did not contain iron, compared to when it was grown in a medium that contained enough iron. On the other hand, the expression decreased instead. This is presumed that OsNAS3 plays a different role from OsNAS1 and OsNAS2, and OsNAS3 was selected as a target gene. The rice used in FIGS. 8, 9 and 10 is Tonjin rice.

(2) Production and isolation of transformed plant in which expression of OsNAS3 gene is activated As a structure for producing transformed rice, pGA2715 vector, which is a T-DNA vector, was used (Jeong et al., Plant Physiol 130: 1636-1644, 2002). The vector is adjacent to the right border and contains a β-glucuronidase (GUS) reporter gene, and is adjacent to the left border and contains a tetramerized transcription enhancer derived from the CaMV 35S promoter. The pGA2715 vector was transformed into Agrobacterium, transformed into wild-type Tonjin rice using the transformed Agrobacterium, and a rice plant population transfected with pGA2715 was produced (Lee et al.). al., J Plant Biol 42: 310-316, 1999; Jeong et al., Plant Physiol 130: 1636-1644, 2002).

  The position where the T-DNA in the pGA2715 vector was inserted was confirmed with respect to the genomic DNA isolated from the transduced rice plant body via inverse PCR. As a result, two transformed plants into which T-DNA was inserted around the OsNAS3 gene were isolated.

  Among them, the OsNAS3-D1 transformed plant has about 1.5 kb downstream of the OsNAS3 gene inserted with T-DNA containing a 35S enhancer at the left boundary site, and the OsNAS3-D2 transformed plant has about 1 downstream. The T-DNA containing the 35S enhancer at the left boundary site is inserted at .9 kb. FIG. 7 shows the insertion positions of T-DNA in OsNAS3-D1 and OsNAS3-D2.

Example 10: Increased expression of OsNAS3 in transformed plants The genotypes of OsNAS3-D1 and OsNAS3-D2 selected in Example 9 were determined via PCR. Genomic DNA was extracted from leaves of T2 generation individuals of OsNAS3-D1 and OsNAS3-D2, and used as template DNA for PCR. For OsNAS3-D1, two gene specific primers F2 (5′-TTTAGGGGAAATGGAGGTTACT-3 ′, SEQ ID NO: 19) and R2 (5′-CTGTAACACTTTACACGCACCAA-3 ′, SEQ ID NO: 20), and T-DNA specific primer RB (5'-CAAGGTTAGCATTAGTAATTAGCCAC-3 ', SEQ ID NO: 21) was used. For OsNAS3-D2, two gene-specific primers F2 (SEQ ID NO: 19) and R3 (5′-GGCTTGCTCTTGTCATAGGGC-3 ′, SEQ ID NO: 22) and T-DNA specific primer RB (SEQ ID NO: 21) are used. did.

  In order to know the expression level, RNA was isolated from the 10-day-old leaves of homozygous OsNAS3-D1 and OsNAS3-D2 and their respective wild-type rice, that is, Dongjin rice (WT), and reverse transcriptase was used. Thus, each cDNA was prepared. Real-time RT-PCR was performed using the prepared cDNA as a template and primers of SEQ ID NOs: 17 and 18, and the amount of OsNAS3 gene transcription was compared. The results are shown in FIG. In FIG. 11, the vertical axis indicates the relative transcription amount related to the rice actin 1 gene transcription amount.

  As shown in FIG. 11, OsNAS3-D1 expression was increased about 60-fold for OsNAS3-D1 compared to wild-type (WT), and about 30-fold for OsNAS3-D2 compared to wild-type (WT). .

Example 11: Measurement of trace element content in transformed plant body In order to know the effect of activation of OsNAS3 gene on trace element accumulation in rice, wild type (WT), transformed plant body, OsNAS3- Trace elements in D1 and OsNAS-D2 were measured. Using mature seeds and flag leaves of wild type and transformed plants as samples, the trace element measurement method is the method described in Kim et al., Physiol Plant 116: 368-372, 2002. It was helpful. The sample was dried at 70 ° C. for 2 days, the amount of dryness was recorded, and the resulting sample was decomposed with 1 ml of 11N HNO ₃ in a 180 ° C. oven for 3 days. After dilution, the contents of iron, zinc, copper, and manganese as trace elements in the sample were measured by atomic absorption spectroscopy (AAS) (Solaar 989, Unicam Atomic Abosorption, Cambridge, UK). The results are shown in Table 11 below.

  As in Table 11, the amounts of copper and manganese do not show significant changes. In the case of OsNAS3-D1, iron is increased 1.5 times and zinc is increased 1.4 times compared to wild type (WT). In the case of OsNAS3-D2, iron and zinc are compared with wild type (WT). 1.2 times. In mature seeds, the amounts of iron and zinc were clearly increased in the branches and leaves of OsNAS3-D1 and OsNAS3-D2. In seeds, in the case of OsNAS3-D1, iron increased 2.9 times, zinc increased 2.2 times, and copper increased 1.7 times compared to the wild type (WT). In seeds, in the case of OsNAS3-D2, iron increased 1.7 times, zinc 1.5 times, and copper 1.3 times that of the wild type.

Example 12: Seedling phenotype analysis of transformed plants under conditions lacking iron and zinc The effect of OsNAS3 gene activation under conditions lacking iron and zinc was examined. In MS solid medium containing both iron and zinc, MS solid medium not containing iron, MS solid medium not containing zinc, wild type (WT) and OsNAS3-D1 were germinated and grown for 8 days. Observed. The results are shown in FIG. The phenotypic elongation and chlorophyll content were measured and are shown in Table 12 below. For measurement of chlorophyll, leaves were collected and weighed, and extracted using 80% acetone. After freezing using liquid nitrogen, mix finely crushed leaves with 80% acetone (add 1ml 80% acetone per 100mg leaf), leave on ice for 15 minutes, centrifuge at 15,000g, supernatant After the liquid was dispensed and the absorbance was measured at 666 nm and 646 nm, the amount of chlorophyll containing chlorophyll a and chlorophyll b was measured by the method described in Arnon, Plant Physiol 24: 1-15, 1949.

  As shown in FIG. 12 and Table 13, in the medium (MS) condition containing both iron and zinc, the height of the plant body is similar, and the difference in phenotype is not shown. However, in the medium containing no iron (Fe-), OsNAS3-D1 is higher than wild type (WT) and chlorophyll is increased by more than 50% compared to wild type (WT), resulting in iron deficiency. The chlorosis, which is a typical symptom, is progressing even less. Therefore, it can be seen that OsNAS3-D1 is more resistant to growth conditions lacking iron than the wild type. Even under medium conditions (Zn-) containing no zinc, OsNAS3-D1 had a higher growth rate than wild-type (WT). Therefore, it can be seen that OsNAS3-D1 grows even under growth conditions in which zinc is deficient compared to wild type (WT).

  In order to verify the experimental results, wild type (WT) and OsNAS3-D1 stems and roots were collected under the respective conditions, and the concentrations of iron and zinc were determined in the same manner as in Example 11. It was measured. The results are shown in Table 13.

  As shown in Table 13, in the medium (MS) condition containing both iron and zinc, no difference in phenotype is observed as examined above. However, compared to the wild type (WT), OsNAS3-D1 increases the iron concentration in the stem and root by 1.7 times and 1.6 times, respectively, and the zinc concentration in the stem and root, respectively. It increased by 2 times and 1.6 times. In addition, in the medium condition not containing iron, OsNAS3-D1 increased the iron concentration by 2.2 times and 2 times in the stem and root, respectively, compared to the wild type (WT). In addition, even under medium conditions that did not contain zinc, OsNAS3-D1 had a 1.4-fold increase in both zinc and stem concentrations compared to wild-type (WT). Thus, it can be seen that OsNAS3-D1 is more resistant to growth conditions lacking iron or zinc.

Example 13: Phenotypic analysis of transformed plants under conditions that contained excessive amounts of heavy metals. The effect of OsNAS3 activation on toxicity generated during treatment with excessive amounts of heavy metals was examined. Germination of wild type (WT) and OsNAS3-D1 in solid MS medium supplemented with excess amounts of zinc (5 mM), copper (0.3 mM) or nickel (0.5 mM), respectively, and grown for 10 days. Seedling phenotype Was observed. The phenotype and plant elongation are shown in FIG.

  As shown in FIG. 13, OsNAS3-D1 is higher in height than the wild type, and the whitening phenomenon is progressing further. These results indicate that OsNAS3-D1 has improved resistance to toxicity due to excessive amounts of heavy metals compared to wild type (WT). In particular, when grown in a solid MS medium containing an excessive amount of zinc, the zinc concentrations of stems and roots were measured by the same method as in Example 11, and the results are shown in Table 14.

  As in Table 14, OsNAS3-D1 showed 2.3 times higher zinc concentration in the stem than wild type (WT).

Example 14: Phenotypic observation of transformed plants under high pH conditions The soil pH has a number of effects on the solubility of the elements required for the plant. For example, if the pH is 8 or more, the iron changes to a low-solubility Fe ₂ O ₃ form, which makes the plant unable to use and absorb normally. As a result, iron shortage will occur.

  Wild type (WT) and OsNAS3-D1 were germinated in MS medium (MS) adjusted to pH 8.5 and grown for 10 days to observe the phenotype. Moreover, the same method was implemented and the phenotype was observed also in MS culture medium (Fe-) (pH 8.5) which does not contain iron. The phenotype and plant elongation are shown in FIG.

  As shown in FIG. 14, it can be seen that in MS medium adjusted to pH 8.5 (MS / pH 8.5), OsNAS3-D1 has a larger elongation than wild type (WT) and can be further tolerated. Such a result was further clearly shown in MS medium without iron (Fe− / pH 8.5).

Example 15: Bioavailability study To confirm the effect of increased iron content in OsNAS3-D1 seeds on iron absorption in animals, the seeds were fed and fed to the mouse. The bioavailability of iron was confirmed through analysis (mouse feeding assay).

  Female Balb / c mice that are 3 weeks old have enough iron (AIN-93G Purified Diet-45 mgFe / kg feed) and iron-deficient feed (modified AIN-93G diet containing iron 3 mg / kg feed) Supplied. Two weeks later, blood was collected from these two groups of mice. This is because blood hemoglobin (Hb) and hematocrit (Hct) are measured to induce anemia and to know the degree of anemia. As a result of the measurement, the hemoglobin (Hb) and hematocrit (Hct) values of the mice fed with the feed deficient in iron decreased by 77% and 79%, respectively, compared with the mice fed with the diet sufficient in iron ( Table 15). This means that anemia is induced when feeding on iron-deficient feed.

  After this, the rats fed the feed lacking iron were divided into two groups, the first group (wild type, WT, n = 10) fed the rice made of wild type (WT) seeds, The group (OsNAS3-D1, n = 9) was fed rice made of OsNAS3-D1 seeds. After 2 weeks, blood was collected and Hb and Hct were measured. Rice was eaten in the same manner, and blood was collected after another 2 weeks, and Hb and Hct were measured. The results are shown in Table 16.

  As in Table 16, Hb and Hct in mice did not show a significant difference before feeding rice consisting of wild type (WT) or OsNAS3-D1 seeds. As a result of measuring Hb and Hct of two groups after two weeks, the second group showed a significant increase in Hb and Hct compared to the first group. As a result of measuring Hb and Hct after a further two weeks, the second group also showed a significant increase in Hb and Hct compared to the first group.

  The inventors of the present invention used rice varieties OsNAS3-D1 and OsNAS3-D2 with increased trace metal content obtained from the above-mentioned Examples to the National Institute of Agrobiological Sciences on July 14, 2008. Deposited and given seed deposit numbers KACC 98004P and KACC 98005P, respectively.

  The present inventors also obtained the rice cultivar OsNAS3-D1 with an increased trace metal content obtained from the above example from the International Depositary Authority of the Budapest Treaty dated November 18, 2009. The seeds were deposited at the Korea Research Institute of Bioscience and Biotechnology (KRIBB) and given the seed accession number KCTC 11598BP.

  The sequences of SEQ ID NOS: 1 to 24 cited in the present specification are submitted through a sequence catalog file, and the contents of the sequence catalog file are incorporated herein by reference in their entirety.

Claims (21)

  A plant body in which the content of trace elements is increased, wherein the trace element is selected from the group consisting of iron, zinc, and copper.   The plant according to claim 1, wherein the plant is rice, wheat or corn.   The plant body according to claim 1, wherein the plant body has an increased expression of an OsNAS2 gene or an OsNAS3 gene.   The expression increase of the OsNAS2 gene or the OsNAS3 gene is performed by inserting an enhancer at one or more positions selected from the group consisting of upstream and downstream of the OsNAS2 gene or the OsNAS3 gene. Plant body.   5. The plant according to claim 4, wherein the structure in which four CaMV 35S enhancers are linked is inserted at a position within about 10 kb downstream of the OsNAS2 gene or the OsNAS3 gene.   The plant body is OsNAS2-D1 rice (oryza sativa) deposited under seed deposit number KCTC 11597BP or OsNAS3-D1 rice (oryza sativa) deposited under seed deposit number KCTC 11598BP. The plant according to 1.   The functional food containing the product produced from the plant body of Claim 1.   A pharmaceutical composition comprising a product produced from the plant according to claim 1.   9. The composition of claim 8, wherein the composition is for treating a disease caused by a deficiency of a substance selected from the group consisting of iron, zinc, copper, and combinations thereof.   The diseases are caused by iron deficiency, anemia, fatigue, pallor, hair loss, irritability, weakness, phagocytosis (pica) Caused by zinc deficiency, brittle or grooved nails, plummer-vinson syndrome, impaired immune function and combinations thereof; Hypozincemia, hair damage, skinlesion, diarrhea, wasting of body tissue, acrodermatitis enteropathica, anorexia, cognitive function and Cognitive and motor function impairment, dysmenorrhea and combinations thereof; syndrome of anemia, pancytopenia caused by copper deficiency Claims selected from the group consisting of pancytopenia, neurodegeration, Menkes disease, spasticity, ataxia, neuropathy and combinations thereof Item 9. The composition according to Item 8.   Increased content of trace elements selected from the group consisting of iron, zinc and copper, including the step of introducing a structure containing an exogenous enhancer into a plant to obtain a plant with increased expression of the OsNAS2 or OsNAS3 gene A method for producing a plant.   The step of introducing the structure into a plant is a step of introducing a Ti plasmid into which a 35S enhancer derived from a promoter of flower cabbage mosaic virus is inserted into the plant, and the step of obtaining the plant includes the 35S enhancer. The trace element content according to claim 11, wherein is a step of selecting a plant inserted at one or more positions among upstream and downstream of a gene encoding OsNAS2 activity or OsNAS3 activity. A method for producing an increased plant.   The plant with increased trace element content according to claim 12, wherein the 35S enhancer is inserted at a position within about 10 kb downstream of the gene encoding the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 11. Method of manufacturing the body.   The method for producing a plant body having an increased trace element content according to claim 12, wherein the 35S enhancer is a copy of 4 copies.   The method according to claim 11, wherein the plant body is rice (oryza sativa).   The plant body is OsNAS2-D1 rice (oryza sativa) deposited under seed deposit number KCTC 11597BP or OsNAS3-D1 rice (oryza sativa) deposited under seed deposit number KCTC 11598BP. 11. A method for producing a plant having an increased trace element content according to 11.   A plant body comprising the step of growing the plant body according to any one of claims 1 to 6 under a condition in which one or more selected from the group consisting of iron and zinc is deficient. How to grow.   A method for growing a plant body comprising the step of growing the plant body according to any one of claims 1 to 6 under a condition in which an excessive amount of heavy metal is present.   The method for growing a plant according to claim 18, wherein the heavy metal is zinc of 5 mM or more, copper of 0.3 mM or more, or nickel of 0.5 mM or more.   A method for growing a plant comprising the step of growing the plant according to any one of claims 1 to 6 under conditions of high pH.   The method for growing a plant according to claim 20, wherein the pH is 8.5 or more.