KR20110085732A - Ostmt2 gene involved in vacuolar sugar transport from rice - Google Patents

Abstract

PURPOSE: An Oryza sativa L.-derived OsTMT2 gene which transports sugar into a vacuole is provide dot detect a novel gene relating to plant growth and environmental stress. CONSTITUTION: An Oryza sativa L.-derived OsTMT2(Oryza sativa tonoplast monosaccharide transporter 2) protein relating to vacuolar sugar transport contains an amino acid of sequence number 2. A gene encoding OsTMT2 protein contains a base sequence of sequence number 1. A recombinant vector contains OsTMT2 gene. The transgenic plant is prepared by the recombinant vector containing OsTMT2 gene. The plant is Oryza sativa L. or Arabidopsis thaliana. A composition for enhancing vacuolar sugar transport contains OsTMT2 gene.

Description

OsTMT2 gene involved in vacuolar sugar transport from rice}

The present invention relates to the rice gene OsTMT2 , which serves to transport sugar into vacuoles during plant growth and development, and more particularly, to the OsTMT2 protein derived from rice, which serves to transport sugar into vacuoles during plant growth and development, Genes encoding the protein, recombinant vectors comprising the genes, plants transformed with the recombinant vectors and seeds thereof, methods of transforming plants with the recombinant vectors to increase sugar transport into vacuoles, prepared by the method A composition for increasing sugar transport into a vacuole of a plant having increased sugar in the vacuole and a vegetation comprising the gene.

In higher plants, sugar is produced in the mesophyll cells of mature leaves known as feeders. Heterotrophic cells such as roots and seeds are sink organs and their nutrition depends on the supply of sugar. Therefore, proper production, storage and transport of sugars are essential for maintaining plant growth and development.

Plants have a variety of sugar transport systems. Sugars are transported to surrounding cells either through extracellular transport pathways by sugar transporters across membranes or through intracellular transport pathways through plasmodesmata. In phloem sieve cells, sugars are loaded into the extracellular and / or intracellular pathways by sugar transporters, depending on the plant species, and also transported over long distances from the source to the recipient organs (Williams et. al, 2000, Trends in Plant Science 5, 283-290; Lim et al, 2006, Physiologia Plantarum 126, 572-584. In addition to these short and long distance transports, sugar molecules are distributed to other subcellular organelles within the cell, depending on the needs of the nutrients. For example, transient starch is broken down into maltose in chloroplasts, and then maltose is transported into the cytoplasm at night through a maltose transporter (Weise et. al, 2006, Plant Physiology 141, 879-886).

In many plants, disaccharide sucrose is the main form of sugar for long-distance transport in phloem somatic cells, while the major monosaccharide sugar forms are glucose and fructose. Sugar transporters make up a large family of genes. For example, 53 putative monosaccharide transporter genes and more than 10 disaccharide transporters belonging to 7 different subfamilies have been identified in Arabidopsis (Buttner, 2007, FEBS Letters 581, 2318-2324; Lalonde et al, 2004, Annual Review of Plant Biology 55, 341-372). In addition, rice has more than 20 monosaccharide transporter genes and five disaccharide transporters (Lim et al, 2006, Physiologia). Plantarum 126, 572-584).

In mature plant cells, vacuoles occupy the largest part of the cell volume and are separated from the cytoplasm by tonoplast, a kind of semi-permeable vacuolar membrane. Vacuoles play a major role as long-term and temporary storage spaces in cells. That is, over-produced extra metabolites are transported into vacuoles and also released on metabolic demand (Wormit et al, 2006, Plant Cell 18, 3476-3490). In this process, specific tonoplasmic sugar transporters are required for vacuole transport of these nutrients. On the other hand, sugar transporters located in many plasma membranes are well characterized in various plants (Lalonde et al, 2004, Annual Review). of Plant Biology 55, 341-372; Lim et al, 2006, Physiologia Plantarum 126, 572-584), the presence of transporters per tonoplasm has only recently been identified in Arabidopsis and barley tonoplasm proteome (Carter et al, 2004, Plant Cell 16, 3285-3303; Endler et al, 2006, Plant Physiology 141, 196-207). Moreover, so far there are only two types of vacuole sugar transporters, Arabidopsis thaliana ) Tonoplasmic monosaccharide transporter (AtTMT) and vacuole glucose transporter (AtVGT) are functionally known in detail (Aluri et al, 2007, PNAS of USA 104, 2537-2542).

Tonoplasm localization of AtTMT and AtVGT was previously investigated by transient expression analysis of GFP fusion constructs using protoplasts, and the active glucose transport capacity of AtVGT1 was isolated. Proven in yeast vacuoles. In addition, gene expression and mutation analysis of AtVGT1 suggested a role in seed germination and flowering (Aluri et al, 2007, PNAS of USA 104, 2537-2542). In contrast, AtTMT1 and AtTMT2, which have very long hydrophilic central loops, are known to be overexpressed in response to stress treatments such as drought, salinity and low temperature. Sugar transport activity of AtTMT1 was demonstrated using vacuoles isolated from cold-treated tmt1 mutants. These data collectively suggest a major role for AtTMT that differs from AtVGT1 during stress response in Arabidopsis (Wormit et al, 2006, Plant). Cell 18, 3476-3490). Also, unlike AtTMT, bacteria that act as glucose transporters Synechocystis sp. SsGTR, a TMT homolog of PCC6803, is present in the plasma membrane of cyanobacteria. SsGTR catalyzes proton-coupled sugar transport into bacterial cells (Schmetterer, 1990, Plant) Molecular Biology 14, 697-706). In other plants, the role of TMT is unknown and the identification of these genes remains an important issue.

To explain the possible role of TMT protein during rice growth and development, the present invention has isolated and identified two TMTs , OsTMT1 and OsTMT2 , from rice. The subcellular location of the OsTMT-GFP fusion protein was examined in Arabidopsis. Detailed expression pattern is OsTMT RT-PCR, histochemical analysis, and additional in situ hybridization in transformed rice expressing promoter - GUS fusion analysis by situ hybridization). Buttocks transportability experiment was to separate vacuoles in transformed Arabidopsis thaliana lines TMT- deficient mutant (tmt1 -2-3) expressing OsTMT1, replacement experiment the glycemic level also the transformed Arabidopsis thaliana lines each passing through the plast Was carried out. Finally, based on the data obtained in the present invention, the relationship of OsTMT in the recovery of sugars during copper transport was investigated.

The present invention is derived by the request as described above, the present inventors have found that rice (Oryza The present invention was completed by discovering that OsTMT2 , a TMT gene derived from sativa ), functions to transport sugar into vacuoles during plant growth and development.

In order to solve the above problems, the present invention provides a rice-derived OsTMT2 protein that serves to transport sugar into vacuoles during plant growth and development.

The present invention also provides a gene encoding the protein.

The present invention also provides a recombinant vector comprising the gene.

The present invention also provides a plant transformed with the recombinant vector and seeds thereof.

The present invention also provides a method of increasing sugar transport into vacuoles by transforming a plant with the recombinant vector.

The present invention also provides a plant with increased sugar in the vacuoles produced by the method.

The present invention also provides a composition for increasing sugar transport into the vacuoles of a plant comprising the gene.

The rice-derived OsTMT2 of the present invention is a gene involved in the transport of sugars in vacuoles during rice growth and development, and has been successfully isolated and identified from rice for the first time. Therefore, it may be useful to search for new genes involved in plant growth, development and environmental stress and to study the lineage.

1 shows the phylogenetic tree of the TMT gene constructed using the neighbor-joining method. The scale bar corresponds to a distance of 10 changes per 100 amino acid positions. The number of branches represents the bootstrap value as a percentage. Access numbers are described in Materials and Methods.
2 shows OsTMT1 and OsTMT2 The genome structure of the gene is shown. Exons are shown as black rectangles and introns are shown as lines. The number of rectangles represents the number of exons.
3 shows a comparison of deduced amino acid sequences for the rice tonoplasmic monosaccharide transporters (OsTMTs) OsTMT1, OsTMT2, OsTMT3 and OsTMT4. These proteins have 12 putative transmembrane regions as shown by the underlined sequences (1-12). Identical and conserved residues are indicated by asterisks, dots and colons.
4 is a transgenic rice callus and OsTMT1 - indicates the cell and is located in the transformed Arabidopsis OsTMT1-GFP fusion in the mesophyll protoplasts isolated from thaliana protein expressing GFP. (a, b) Location of OsTMT1-GFP fusion protein in transgenic rice callus. Plasma membranes stained with FM4-64 do not overlap OsTMT1-GFP. (c) Location of OsTMT1-GFP fusion protein in Arabidopsis protoplasts. Chlorophyll autofluorescence and FM4-64 were used as markers of chloroplast and plasma membrane, respectively. The GFP signal is shown in green and the plasma membrane stained with FM4-64 is shown in red.
5 shows the subcellular location of OsTMT2-GFP fusion protein in transgenic Arabidopsis expressing transformed rice callus and OsTMT2 - GFP . (a) Location of OsTMT2-GFP fusion protein in transgenic rice callus. (b) OsTMT2-GFP fusion proteins in the leaves of the transgenic Arabidopsis expressing OsTMT2-GFP. Chlorophyll autofluorescence was used as chloroplast marker.
6 shows expression analysis of OsTMT1 and OsTMT2 genes. (a) Expression patterns of OsTMT1 and OsTMT2 in rice tissue comprising leaves, roots, flowers and immature seed mixtures of 1-12 DAF caryopses . (b) Expression patterns of OsTMT1 and OsTMT2 in endosperm and seed shells. (c) Changes in expression levels of OsTMT1 and OsTMT2 genes during rice seed development. Rice seeds were collected hourly up to 15 DAF. Act1 is a PCR control. (d) Effect of sugars on the expression of OsTMT1 and OsTMT2 genes in rice leaves. Truncated leaves were treated with MS medium containing sucrose, glucose, fructose or mannitol and collected after 12 and 24 hours. OsUBQ5 is a PCR control.
MS: MS medium, MS + S: MS medium + 175mM sucrose, MS + G: MS medium + 175mM glucose, MS + F: MS medium + 175mM fructose, MS + M: MS medium + 175mM mannitol
FIG. 7 shows the expression of OsTMT1 and OsTMT2 genes for low temperature (a), drought (b) and saline (c) treatments in two week old rice seedlings under continuous light conditions of 150 μmol / m ² s. (a) For cold stress, seedlings were grown at 4 ° C. for up to 6 hours. (b, c) For drought and high salinity stress stimulation, seedlings were each dried in air and grown at 28 ° C. for up to 6 hours in 250 mM NaCl. This experiment was carried out by Jang et al. (2003, Plant Physiology 131, 516-524).
8 shows pOsTMT1 - GUS or pOsTMT2 - GUS Histochemical location of GUS expression in the leaves, roots and flowers of transgenic rice with constructs. (a, b) pOsTMT1 -GUS ( a) or pOsTMT2 - GUS (b) Cross section of leaf of line. (c, d) Cross section of the root of pOsTMT1 - GUS (c) or pOsTMT2-GUS (d) lines. (e, f) pOsTMTl - spikelet of GUS . (g, h) Hydrophobicization of pOsTMT2 - GUS .
BS: bundle sheath cells, CC: companion cells, MC: lamellar cells, X: metataxylem, SE: sieve element, VP: vascular parenchyma
9 shows pOsTMT1 - GUS or pOsTMT2 - GUS Histochemical position and in situ hybridization of the GUS expression in immature seeds of the transgenic rice plant having a construct (in situ hybridization) analysis. (a, b) GUS expression in 7-8 DAF immature seeds of pOsTMT1 - GUS (a) or pOsTMT2 - GUS (b). (c, d) longitudinal section of 5 DAF immature seeds of pOsTMT1 - GUS (c) or pOsTMT2 - GUS (d). (e, f) Cross sections (images of dark areas) of 5 DAF immature seeds of pOsTMT1 - GUS (e) or pOsTMT2 - GUS (f). (g, h) Longitudinal section of 12-15 DAF immature seeds of pOsTMT1 - GUS (g) or pOsTMT2-GUS (h). Arrows indicate GUS expression on the dorsal side. (i) Detection of OsTMT2 transcripts by in situ hybridization of cross sections of 3 DAF seeds. (j) Similar sections hybridized with sense probes did not show positive labeling. (k) Detection of OsTMT2 transcript by in situ hybridization of the longitudinal section of 5 DAF seeds. (l) Similar sections hybridized with sense probes did not show positive labeling.
C: cross cell layers, E: epidermis, Em: embryo, En: embryo, I: integument, M: mesorp, N: nucellar epidermis, NP: nucellar projecion, T: tube cell layers, V: main vascular bundle
Figure 10 illustrates the sugar content of Arabidopsis tmt1 -2-3 triple glucose transport recovery (complementation) and these plants into vacuoles isolated from the (triple) mutant plants overexpressing OsTMT1. (a) OsTMT1, OsTMT1 - OX1 and OsTMT1 - transgenic expression of tmt1 OsTMT1 in the -2 line expressing OX2. UBQ is a PCR control. (b) Arabidopsis thaliana wild-type, mutant tmt1 -2-3, OsTMT1-OX1 and time slot analysis of ¹⁴ C- glucose uptake into isolated from vacuoles OsTMT1-OX2 line. The gray dashed line with open squares, a black straight line in the open rhombus with black dotted line, the closed triangle gray line and closed circle, respectively tmt1 -2-3 triple mutant, wild type, OsTMT1-OX1 and OX2 represents an OsTMT1- . (c) Recovery of sugar content in transgenic Arabidopsis tmt1-2-3 lines expressing OsTMT1 . The dark gray bars represent wild type, the white bars represent the tmt1-2-3 triple mutation, the black bars represent OsTMT1-OX1, and the light gray bars represent OsTMT1-OX2. The tip of each bar represents the mean ± standard deviation from three experiments.

In order to achieve the object of the present invention, the present invention, consisting of the amino acid sequence represented by SEQ ID NO: 2, OsTMT2 ( Oryza derived from rice, which serves to transport sugar into vacuoles during plant growth and development) sativa tonoplast monosaccharide transporter 2) protein.

The range of OsTMT2 protein according to the present invention includes a protein consisting of the amino acid sequence represented by SEQ ID NO: 2 isolated from rice and functional equivalents of the protein. "Functional equivalent" means at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 70% of the amino acid sequence represented by SEQ ID NO: 2 as a result of the addition, substitution, or deletion of the amino acid Is 95% or more of sequence homology, and refers to a protein that exhibits substantially homogeneous physiological activity with the protein represented by SEQ ID NO: 2. "Substantially homogeneous physiological activity" means the activity of transporting sugar into the vacuoles of a plant.

The present invention also provides a gene encoding the OsTMT2 protein. Genes of the invention include both genomic DNA and cDNA encoding OsTMT2 protein. Preferably, the gene of the present invention may include the nucleotide sequence shown in SEQ ID NO: 1.

Variants of the above base sequences are also included within the scope of the present invention. Specifically, the gene has a base sequence having a sequence homology of at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% with the nucleotide sequence of SEQ ID NO: 1, respectively. It may include. The "% sequence homology" for a polynucleotide is identified by comparing two optimally arranged sequences with a comparison region, wherein part of the polynucleotide sequence in the comparison region is the reference sequence (addition or deletion) for the optimal alignment of the two sequences. It may include the addition or deletion (ie, gap) compared to).

The present invention also provides a recombinant vector comprising the OsTMT2 gene according to the present invention.

The term "recombinant" refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a protein encoded by a peptide, heterologous peptide or heterologous nucleic acid. The recombinant cell can express a gene or a gene fragment that is not found in the natural form of the cell in one of the sense or antisense form. In addition, the recombinant cell can express a gene found in a cell in its natural state, but the gene has been modified and reintroduced intracellularly by an artificial means.

The term "vector" is used to refer to a DNA fragment (s), nucleic acid molecule, which is transferred into a cell. The vector replicates the DNA and can be independently regenerated in the host cell. The term "carrier" is often used interchangeably with "vector". The term “expression vector” refers to a recombinant DNA molecule comprising a coding sequence of interest and a suitable nucleic acid sequence necessary to express a coding sequence operably linked in a particular host organism. Promoters, enhancers, termination signals and polyadenylation signals available in eukaryotic cells are known.

Vectors of the invention can typically be constructed as vectors for cloning or expression. In addition, the vector of the present invention can be constructed using prokaryotic or eukaryotic cells as hosts. For example, when the vector of the present invention is an expression vector and the prokaryotic cell is a host, a strong promoter (for example, a pLλ promoter, a trp promoter, a lac promoter, a T7 promoter, a tac promoter, etc.) capable of promoting transcription, It is common to include ribosomal binding sites and transcription / detox termination sequences for initiation of translation. When E. coli is used as a host cell, a promoter and an operator site of the E. coli tryptophan biosynthetic pathway, and a phage λ left promoter (pLλ promoter) can be used as regulatory sites.

On the other hand, vectors that can be used in the present invention are plasmids (eg, pSC101, ColE1, pBR322, pUC8 / 9, pHC79, pGEX series, pET series and pUC19, etc.) which are often used in the art, phage (e.g. λgt4.λB , λ-Charon, λΔz1 and M13, etc.) or viruses (eg SV40, etc.).

On the other hand, when the vector of the present invention is an expression vector and the eukaryotic cell is a host, a promoter derived from the mammalian cell genome (for example, metallothionine promoter) or a promoter derived from a mammalian virus (for example, adeno) Late viral promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter and tk promoter of HSV) can be used and generally have a polyadenylation sequence as a transcription termination sequence.

The recombinant vector may preferably be a recombinant plant expression vector, with a preferred example of the recombinant vector being capable of transferring a portion of itself, a so-called T-region, to plant cells when present in a suitable host such as Agrobacterium tumerfaciens. Ti-plasmid vector. Other types of Ti-plasmid vectors (see EP 0 116 718 B1) are currently used to transfer hybrid DNA sequences to plant cells or protoplasts in which new plants capable of properly inserting hybrid DNA into the plant's genome can be produced have. A particularly preferred form of the Ti-plasmid vector is a so-called binary vector as claimed in EP 0 120 516 B1 and U.S. Patent No. 4,940,838. Other suitable vectors that can be used to introduce the DNA according to the invention into the plant host include viral vectors such as those that can be derived from double-stranded plant viruses (e. G., CaMV) and single- For example, from non -complete plant virus vectors. The use of such vectors may be particularly advantageous when it is difficult to transform the plant host properly.

The expression vector will preferably comprise one or more selectable markers. The marker is typically a nucleic acid sequence having properties that can be selected by a chemical method, which corresponds to all genes capable of distinguishing transformed cells from non-transformed cells. Examples include herbicide resistance genes such as glyphosate or phosphinothricin, kanamycin, G418, bleomycin, hygromycin, and chloramphenicol. Resistance genes include, but are not limited to.

In the recombinant vector of the present invention, the promoter may be, but is not limited to, CaMV 35S, actin, ubiquitin, pEMU, MAS or histone promoter. The term "promoter " refers to the region of DNA upstream from the structural gene and refers to a DNA molecule to which an RNA polymerase binds to initiate transcription. A "plant promoter" is a promoter capable of initiating transcription in plant cells. A "constitutive promoter" is a promoter that is active under most environmental conditions and developmental conditions or cell differentiation. Constructive promoters may be preferred in the present invention because the choice of transformants can be made by various tissues at various stages. Thus, the constitutive promoter does not limit the selection possibilities.

In the recombinant vectors of the present invention, conventional terminators can be used, for example nopalin synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, Agrobacterium tumefaciens (Agrobacterium tumefaciens) Terminator of the octopine gene, but is not limited thereto. Regarding the need for terminators, it is generally known that such regions increase the certainty and efficiency of transcription in plant cells. Therefore, the use of a terminator is highly desirable in the context of the present invention.

The present invention also provides a host cell transformed with the recombinant vector of the present invention. The host cell capable of continuously cloning and expressing the vector of the present invention in a prokaryotic cell while being stable can be used in any host cell known in the art, for example, E. coli JM109, E. coli BL21, E. coli RR1. , Bacillus genus strains, such as E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus thuringiensis, and Salmonella typhimurium, Serratia marcensons, and various Pseudomonas Enterobacteria such as species and strains.

In addition, when transforming a vector of the present invention to eukaryotic cells, yeast ( Saccharomyce) as a host cell cerevisiae ), insect cells, human cells (eg, CHO cell lines (Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK cell lines), and plant cells. The host cell is preferably a plant cell, more preferably rice or Arabidopsis.

The method of carrying the vector of the present invention into a host cell is performed by using the CaCl ₂ method or one method (Hanahan, D., J. Mol. Biol., 166: 557-580 (1983)) when the host cell is a prokaryotic cell. And the electroporation method. When the host cell is a eukaryotic cell, the vector is injected into the host cell by microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, DEAE-dextran treatment, and gene bombardment .

The present invention also provides plants and seeds thereof transformed with the recombinant vector of the present invention. The plant is Arabidopsis, potato, eggplant, tobacco, pepper, tomato, burdock, garland chrysanthemum, lettuce, bellflower, spinach, chard, sweet potato, celery, carrot, buttercup, parsley, cabbage, cabbage, gatchi, watermelon, melon, cucumber, It may be a dicotyledonous plant such as pumpkin, gourd, strawberry, soybean, green beans, kidney beans, peas or monocotyledonous plants such as rice, barley, wheat, rye, corn, sugarcane, oats, onions, and preferably Arabidopsis or rice to be.

The present invention also provides a method of increasing sugar transport into vacuoles comprising transforming a plant with a recombinant vector of the invention to overexpress the OsTMT2 gene. Since the OsTMT2 gene is present in tonolast, it carries out sugar transport into the vacuole, so overexpressing the gene can increase the transport of sugar into the vacuole.

Transformation of a plant means any method of transferring DNA to a plant. Such transformation methods do not necessarily have a regeneration and / or tissue culture period. Transformation of plant species is now common for plant species, including both terminal plants as well as dicotyledonous plants. In principle, any transformation method can be used to introduce hybrid DNA according to the invention into suitable progenitor cells. Method is calcium / polyethylene glycol method for protoplasts (Krens, FA et al., 1982, Nature 296, 72-74; Negrutiu I. et al., June 1987, Plant Mol. Biol. 8, 363-373), protoplasts Electroporation (Shillito RD et al., 1985 Bio / Technol. 3, 1099-1102), microscopic injection into plant elements (Crossway A. et al., 1986, Mol. Gen. Genet. 202, 179-185 ), (DNA or RNA-coated) particle bombardment of various plant elements (Klein TM et al., 1987, Nature 327, 70), Agrobacterium tumulopasis by plant infiltration or transformation of mature pollen or vesicles And infection with (incomplete) virus (EP 0 301 316) in en mediated gene transfer. A preferred method according to the present invention comprises Agrobacterium mediated DNA delivery. Especially preferred is the use of the so-called binary vector technology as described in EP A 120 516 and US Pat. No. 4,940,838.

The present invention also provides a plant with increased sugar in vacuoles produced by the method.

The invention also provides a composition for increasing sugar transport into the vacuoles of a plant comprising the OsTMT2 gene of the invention. The composition of the present invention comprises the OsTMT2 gene as an active ingredient, the gene is present in tonoplast, while performing sugar transport into the vacuoles, overexpressing the gene can increase sugar transport into the vacuoles. will be.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Materials and methods

Plant material

Wild type rice (Oryza sativa L. cv. Dongjin) grown in a greenhouse at 30 ° C. during the day of the 14 hour-light / 10 hour-dark cycle and 20 ° C. at night was used in the present invention. Immature seeds were collected at different stages of development and flowers were collected just before heading. Flag leaves of mature plants were collected, while roots were collected at the four-leaf seedling stage. Wild-type Arabidopsis thaliana (ecotype Columbia) and tmt1 -2-3 mutant plants (Wormit et al, 2006, Plant Cell 18, 3476-3490) was used for transformation. All Arabidopsis plants were grown on 22 ° C. soils of 14 h-light / 10 h-dark, unless otherwise specified.

OsTMT  Battlefield cDNA  Clone Clone

OsTMT1 and OsTMT2 Full-length cDNAs of genes include gene specific primers including translation initiation codons and 3 ′ untranslated region (UTR); OsTMT1 : 5'-AAATCTCCCCTAAAAGCTTCC-3 '(SEQ ID NO: 3) and 5'-GAACTAGTACTCGGCTATGCTAA-3' (SEQ ID NO: 4), OsTMT2 : 5'-AAGAGGTGGAAGAAGAGGGAT-3 '(SEQ ID NO: 5) and 5'-TATTATGAACCCCAACATAGTAGC-3' (SEQ ID NO: 6) was used to isolate by PCR. All synthesized cDNAs were subcloned into pGEM-T easy vectors (Promega, Madison, Wis.) And then sequenced. The cDNA sequences of OsTMT1 and OsTMT2 were registered in the NCBI database, respectively (registration numbers: GU066765 and GU066766).

Sequence alignment ( sequence alignment ) And tree tree ( phylogenetic tree ) Production

The deduced amino acid sequence of the OsTMT gene is shown in the CLUSTAL W program (Thompson et al, 1994, Nucleic). Acids Research 22, 4673-4680) was used to sequence alignment with previously reported genes in other species. The phylogenetic tree is derived from MEGA software version 4.0 (Tamura et al, 2007, Molecular Biology and Evolution 24, 1596-1599). Bootstrap analysis was performed with 1,000 replicates and the bootstrap value was expressed as a percentage. The registration numbers of the sequences used for the generation of the phylogenetic tree were AtTMT1 (At1g20840; Z50752), AtTMT2 (At4g35300; AJ532570), AtTMT3 (At3g51490; AJ532571) from Arabidopsis; HvSTP1 (AJ534445), HvSTP2 (AJ534446) from barley; PST type 2a from sugar cane (AY165599); It is VvHT6 (AY861386) derived from grapes.

OsTMT - GFP  Of fusion proteins Subcellular  location

To investigate the subcellular locations of OsTMT1 and OsTMT2 in rice and Arabidopsis, each full-length cDNA fragment except for stop codon and 3'UTR was amplified and then JJ461 binary vector (Cho et al, 2009, Plant Physiology 149, 745-759) was cloned into the region between the CaMV35S promoter and sGFP. Primers used in the preparation of OsTMT-GFP fusion vectors were OsTMT1 : 5'-GCTCTAGACTTGGTGGTAAGATTCGCCG-3 '(SEQ ID NO: 7) and 5'-CCCTCGAGAGTCCTCCTTGGCCTGCTTTGC-3' (SEQ ID NO: 8), OsTMT2 : 5'-GCTCTAGAGGGGGGACTAT ' SEQ ID NO: 9) and 5'-CCCTCGAGAGGCCTTTGTAGCCTGCATTTG-3 '(SEQ ID NO: 10). Transformed callus and plants expressing OsTMT - GFP fusion constructs and protoplasts isolated from the transformed Arabidopsis were examined using confocal microscopy (LSM 510 META, Carl Zeiss, Jena, Germany). . Chlorophyll autofluorescence was used as the chloroplast marker. In transformed rice callus, the plasma membrane was stained using lipophilic styryl dye FM4-64 (Molecular Probes, Carlsbad, Calif.).

Promoter Cloning and GUS  Preparation of Fusion Vectors

Using rice genomic DNA as a template, 5 'upstream regions of the OsTMT1 and OsTMT2 genes, 2445 bp and 2481 bp in length, respectively, were amplified by PCR. Proof-reading DNA polymerase pfu (SolGent, Daejeon, Korea) was used to reduce PCR errors, and primers used for amplification were OsTMT1 : 5'-CCCTCGAGAAACCTAAACTTGAAATGTGCTA-3 '(SEQ ID NO: 11) and 5'-CGCCTAGGCGAATCTTACCACTGCACAAGAA- 3 '(SEQ ID NO: 12), OsTMT2 : 5'-GCGTCGACATCGTGATTCTTTCTTTCAAAC-3' (SEQ ID NO: 13) and 5'-GCTCTAGACTTCACCCCTCTCCGATCCC-3 '(SEQ ID NO: 14). The amplified products were cloned into pGEM T-easy vector and then sequenced. Cloned inserts were cleaved with Xho I / Avr II or Sal I / Xba I and then promoter-free β- glucuronidase from the binary vector pC1300 intC (Ouwerkerk et al, 2001, Planta 213, 370-378). ( GUS ) was subcloned to Sal I and Xba I sites of JJ376 vector containing reporter gene. As a result, the promoter region of OsTMT1 OsTMT2 GUS gene is linked to the terminator of the nopaline synthase (nos) gene Fused to the gene. These constructs, including the OsTMT1 promoter-GUS and OsTMT2 promoter-GUS fusions, were named pOsTMT1 - GUS and pOsTMT2 - GUS , respectively.

Transformation of Rice

CaMV35S - OsTMT - GFP or pOsTMT - for the production of transgenic rice plants expressing the GUS fusion constructs, each of the Agrobacterium having a vector Solarium tyumeo Pacific Enschede (Agrobacterium tumefaciens ) LBA4404 strain was incubated at 28 ° C. for 3 days in AB medium supplemented with 25 mg / L kanamycin, and transformed callus was obtained through the previously described Agrobacterium-mediated coculture method (Jeon et. al, 2000, The Plant Journal 22, 561-570). Transformed rice was re-differentiated from the callus transformed in a selection medium containing 50 mg / L hygromycin and 250 mg / L cefotaxime.

Transformation of Arabidopsis

CaMV35S - OsTMT - GFP, CaMV35S - 28 Agrobacterium tyumeo Pacific Enschede (A. tumefaciens) GV3101 strains having one of the structures is OsTMT2 in an LB liquid medium containing 25mg / L of kanamycin to the standing position (stationary phase) - OsTMT1 or CaMV35S It was incubated with shaking at 250 rpm at ℃. For CaMV35S - OsTMT - GFP , wild-type Arabidopsis (ecotype Columbia) was transformed by the previously described floral dip method (Clough & Bent, 1998, The Plant Journal 16, 735-743). CaMV35S - OsTMT1 And CaMV35S-OsTMT2 for the construct, were used for switching the Arabidopsis thaliana mutant lines transfected tmt1 -2-3. All transgenic plants were selected in Gamborg B5 medium containing 25 mg / L hygromycin and grown on 22 ° C. soil at 14 hours-light / 10 hours-light. To investigate the subcellular location of OsTMTs in Arabidopsis, as in Sheen's method previously described, protoplasts were isolated from leaves of well-expanded 3-4 week transgenic plants (Sheen, 2001, Plant Physiology 127, 1466-1475).

Histochemical GUS  analysis

Various organs were collected from the transgenic plants, 0.2 M sodium phosphate (pH 7.0), 10 mM EDTA, 20% methanol, 2% DMSO, 0.1% 5-bromo-4-chloro-3-indol β-glucopyranoside (X- In a GUS reaction solution containing Gluc) as previously described (Jeon et al, 1999, Plant Molecular Biology 39, 35-44). Samples were incubated for 1 hour to overnight at 37 ° C. until staining was sufficient, then transferred to 70% ethanol to remove chlorophyll. After staining, the samples were fixed in FAA solution containing 50% ethanol, 5% acetic acid and 3.7% formaldehyde. Flowers were observed with a dissecting microscope (SZX12; Olympus, Tokyo, Japan) and photographed with ImagePro Express (Olympus, Tokyo, Japan). Stained samples were embedded in paraplast plus (Sigma, St Louis, MO) for observation of longitudinal and cross sections, and 10 μm sections were prepared using microtome (Microtome, Microm HM360, Woldorf, Germany). The sections were then mounted with slides and observed under a compound microscope.

Party treatment

For the sugar treatment, the four leaf stage seedlings were incubated for 48 hours under dark conditions to remove their endogenous sugars. The leaves were then cut into 1 cm pieces, as described previously (Cho et al, 2006, Planta 224, 598-611; Dian et al, 2003, Planta 218, 261-268) and 175 mM sucrose, glucose, fruc Treatment was under 28 ° C. dark conditions in liquid MS medium containing toss or mannitol. The samples were collected 12 or 24 hours after treatment.

RT - PCR  analysis

For RT-PCR analysis, total RNA was obtained from samples collected using Trizol reagent and reverse transcription was carried out using an oligo-dT primer with First-Strand cDNA Synthesis Kit (Roche, Mannheim, Germany). did. First-Strand cDNA is the housekeeping gene Act1 (McElroy et al, 1990, Plant Cell 2, 163-171), OsUBQ5 (Jain et al, 2006, BBRC 345, 645-651), and the sugar-inducible gene OsGBSSII (Dian et al, 2003, Planta 218, 261-268). Used in PCR reactions with gene specific primers and control primers. Gene specific primers were OsTMT1 : 5'-GAAGTCATCACCGAGTTCTTC-3 '(SEQ ID NO: 15) and 5'-TACAGGGAGAAGAATTCCAAA-3' (SEQ ID NO: 16), OsTMT2 : 5'-GTCTTTGGTATATACGCAGTTGTT-3 '(SEQ ID NO: 17) and 5' -AGTGGACATGAATAACATGAAAAT-3 '(SEQ ID NO: 18), OsGBSSII : 5'-TAGGTGTCACTGAATGGCTAGAAC-3' (SEQ ID NO: 19) and 5'-TGGCCCACATCTCTAAGTAACATC-3 '(SEQ ID NO: 20), Act1 : 5'-GGAACTGGTATGGTCAAGGC-3' (SEQ ID NO: 19) Numbers 21) and 5'-AGTCTCATGGATACCCGCAG-3 '(SEQ ID NO: 22), and OsUBQ5 : 5'-GACTACAACATCCAGAAGGAGTC-3' (SEQ ID NO: 23) and 5'-TCATCTAATAACCAGTTCGATTTC-3 '(SEQ ID NO: 24). For transgenic Arabidopsis plants, UBQ (At5g20620) Primers: 5'-GTGGTGCTAAGAAGAGGAAGA-3 '(SEQ ID NO: 25) and 5'-TCAAGCTTCAACTTCTTCTTT-3' (SEQ ID NO: 26) were used as PCR controls (Cho et al, 2009, Plant Physiology 149, 745-759). ). RT-PCR analysis was performed by the previously described method (Cho et al, 2005, Plant Cell Reports 24, 225-236) and repeated at least three times to obtain similar results.

In Situ Hybridization ( in situ hybridization )

Collected 3 and 5 days after fertilization (DAF) fruit were fixed in 50 mM sodium phosphate buffer containing 4% paraformaldehyde, 0.25% glutaraldehyde for 1 hour, then dehydrated with a graded series of ethanol, and finally embedded in paraplast plus (Sigma). Embedded samples were then carefully cut into longitudinal or transverse sections using a Microtome (Microm HM360). The cut 7 μm sections were mounted on slides and printed using modified methods of Kouchi & Hata (1993, Molecular & General Genetics 238, 106-119) and Hirose et al. (2002, Plant & Cell Physiology 38, 1389-1396). Used for situ hybridization. To make hybridization probes, a 523 bp fragment of the central loop region of OsTMT2 cDNA was subcloned into the Bam HI and Eco RI sites of pBluescript II SK (+) (Stratagene, La Jolla, Calif.). This vector was linearized by cleavage with Bam HI or Eco RI and labeled by in vitro transcription using digoxigenin (DIG) -labeled mix (Roche) and T3 or T7 polymerase. Sense and antisense probes labeled with DIG were synthesized from the T3 or T7 promoter of the construct, respectively, and then hydrolyzed to about 100 to 200 bases. The sections were hybridized for 16 hours at 42 ° C. with sense and antisense probes labeled with DIG. Hybridized probes were detected immunologically using anti-DIG alkaline phosphatase conjugate (Roche). Nitro blue tetrazolium chloride (NBT) / BCIP (5-Bromo-4-chloro-3-indolyl phosphate, toluidine salt) ready-to-use tablets (Roche) were used as substrates.

Separated leaves Leaf flesh  Into vacuole Glucose  Quantification of Absorption and Soluble Sugars

To examine the glucose transport capacity of OsTMT1, the mesophyll of the intact wild-type leaves the vacuole, tmt1 -2-3 and OsTMT1 It was isolated from the transgenic plant. Plants were grown for 4-5 weeks in 22 ° C. soil under photoperiod (90 μmol / m ² s) with 8 hours-light / 16 hours of cancer, and for 5 days under 50 μmol / m ² s bright conditions before vesicle separation. It was raised. Separation of vacuoles and their use in ¹⁴ C-glucose transport experiments were reported by Wormit et al. (2006, Plant Cell 18, 3476-3490). Transport 125 μM ¹⁴ C- glucose: using a (specific activity 300 mCi mmol ^-1) and 1 mM Mg ^{2 +} -ATP was done for 15 minutes.

For the measurement of soluble sugar content, wild, tmt1 -2-3 and OsTMT1 Arabidopsis leaves collected from transgenic plants were frozen into fine powder using TissueLyser (Qiagen-Retsch GmbH, Haan, Germany) with pre-cooled adapters. A total of 100 mg of leaf powder was then extracted using 80% ethanol at 80 ° C. for 1 hour, and the extract was dissolved in sterile water after evaporating the water. Spectroscopic quantification of sugar content was performed by the method of Lee et al. (2008, Plant , Cell & Environment 31, 1851-1863).

Example  One: OsTMT1 and OsTMT2 of Cloning

Reference sequence (Wormit et al, 2006, Plant Cell 18, 3476-3490) as a systematic blast search of sequence databases AtTMT (annotated with the annotation rice Using isoforms (isoform)) (BLAST search) is four rice (Oryza sativa ) TMT gene; OsTMT1 (LOC_Os10g39440), OsTMT2 (LOC_Os02g13560), OsTMT3 (LOC_Os03g03680), and OsTMT4 (LOC_Os11g40540) were identified. In order to compare the similarity between OsTMTs and AtTMTs, and to identify TMT sequences from other plant species, the present invention has made phylogenetic tree using the conjugation method based on amino acid sequence comparison. This analysis showed that among the four OsTMTs identified, OsTMT1 and OsTMT2 showed a closer evolutionary relationship with AtTMTs than OsTMT3 or OsTMT4 (FIG. 1). In fact, OsTMT1 and OsTMT2 form a monocot subgroup with two putative sugar transporters from barley, HvSTP1 and HvSTP2, in the phylogenetic tree of the present invention, which are found between the maternal and magnetic tissues of fruiting during early seed development. It has been suggested to be involved in regulating sugar ratios (Weschke et al, 2003, The Plant Journal 33, 395-411). This monocotyledonous subgroup also includes PST type 2a, an estimated sugar transporter derived from sugarcane, which is homologous to AtTMTs (Casu et al, 2003, Plant Molecular Biology 52, 371-386). The tonoplasmic positions of HvSTP1, HvSTP2, and PST type 2a and the sugar transporter function have not yet been determined. AtTMT and grape derived VvHT6, a putative sugar transporter, form a dicot subgroup (FIG. 1). This finding further elucidated the location and function of OsTMT1 and OsTMT2. In addition, OsTMT1 and OsTMT2 did not show any significant homology with the Arabidopsis VGT group, known as the vacuole glucose transporter subfamily, or the Arabidopsis STP group, a sugar transporter subfamily of plasma membranes (data not shown).

The full-length cDNAs of OsTMT1 and OsTMT2 were isolated by RT-PCR using gene specific primers comprising the upstream region of the translation initiation codon and the downstream region of the stop codon. Sequence analysis and comparison of their genomic clones showed that the two genes had a genomic structure consisting of six exons and included an intron at the 5'UTR (FIG. 2). The deduced amino acid sequences have a very large hydrophilic central loop of about 340 amino acid residues between the 12 expected transmembrane domains and the transmembrane domains 6 and 7 which are characteristic of the TMT family, 740 and 746, respectively. OsTMT1 (GenBank Accession No. GU066765) and OsTMT2 (Accession No. GU066766) of the amino acids were shown (FIG. 3). Their actual structural similarity to AtTMTs suggests that OsTMT1 and OsTMT2 are members of monosaccharide transporters located in tonoplasm.

Example  2: OsTMT - GFP  Of fusion proteins Subcellular  location

To determine the subcellular location of OsTMT1 and OsTMT2, the present invention is directed to OsTMT1 - GFP or OsTMT2 - GFP under the control of the CaMV35S promoter. Transgenic rice callus expressing the constructs was made. Analysis of this transgenic callus shows that OsTMT1-GFP is located in the tonoplasm of large and small vacuoles in the callus (FIG. 4A). To distinguish tonoplasm-localized GFP signals from the plasma membrane, the transformed rice callus mainly stains the plasma membrane and FM4, a lipophilic styryl dye known to follow the endocytic pathway in various eukaryotic cells, including plants. Stained at -64 (Ueda et al, 2001, The EMBO journal 20, 4730-4741). However, the GFP signal did not merge well with the signal of FM4-64 in many regions, indicating that OsTMT1 was not located in the plasma membrane (FIG. 4B). In addition, the tonoplasmic position of OsTMT1 has been demonstrated in protoplasts isolated from the mesenchymal cells of transgenic Arabidopsis plants that overexpress OsTMT1 - GFP . This was clearly seen by the presence of vacutononoplasm indented by chloroplasts with chlorophyll autofluorescence (FIG. 4C). Similar results were found in rice and Arabidopsis cells expressing OsTMT1 - GFP (FIG. 5). These results clearly show that OsTMT1 and OsTMT2 are present in tonoplasm and are not targeted to the plasma membrane.

Example  3: OsTMT1 and OsTMT2 Expression Analysis

Semi-quantitative RT-PCR was performed to investigate the spatiotemporal expression profiles of OsTMT1 and OsTMT2 genes in rice. The results showed significant levels of OsTMT1 and OsTMT2 transcripts in all rice organs used in the experiment, including leaves, roots, flowers and immature seeds (FIG. 6A). Analysis of immature seeds showed that OsTMT1 and OsTMT2 are preferentially expressed in the seed shells, but not in embryos (FIG. 6B). RT-PCR analysis of OsTMT1 and OsTMT2 was performed on a series of rice seeds collected at different developmental stages up to 15 days after fertilization (DAF). Rice seeds are stretched longitudinally mainly using the cellularization and proliferation of the mammary gland cells during the pre-storage phase (1-6 DAF), followed by the starch-filling milky phase (7-15). Thickening during DAF) (Hirose et al, 2002, Plant & Cell) Physiology 43, 452-459). The expression of both OsTMTs was found to be higher during the pre-storage phase compared to the starch-filling phase, suggesting the role of OsTMT1 and OsTMT2 in all organs, especially seed shells, during early seed development (FIG. 6C).

Next, to investigate the effect of soluble sugars on OsTMT gene expression, the cut leaves depleted of endogenous sugars were treated with MS medium containing 175 mM sucrose, glucose, fructose or mannitol. RT-PCR analysis reveals the well-known sugar-inducible gene OsGBSSII Similar to the expression of (Dian et al, 2003, Planta 218, 261-268), levels of OsTMT1 and OsTMT2 were increased by 12 and 24 hours treatment of sucrose, glucose or fructose, while mannitol had no effect There was no (Figure 6d).

It is already known that the AtTMT gene is significantly overexpressed by environmental stress such as salinity, drought and low temperature. The present invention examined whether the expression of OsTMT1 and OsTMT2 is changed by such stress stimulation. The present invention did not observe any significant or reproducible changes in the expression of OsTMT1 and OsTMT2 in cold (4 ° C.), salinity (250 mM NaCl) or drought-exposed rice samples, which is probably clearly distinguished from AtTMT. It has a function (FIG. 7). The public microarray or massively parallel signature sequencing (MPSS) data available also consistently did not show any significant changes in these genes in response to these environmental stresses (data not shown).

Example  4: OsTMT  Promoter GUS Transgenic rice Histochemical analysis

The expression of OsTMT1 and OsTMT2 is expressed by OsTMT1 promoter- GUS ( pOsTMT1 - GUS ) and OsTMT2 promoter- GUS ( pOsTMT2 - GUS ) were examined by histochemical GUS analysis of transformed rice expressing each of the fusion constructs. Approximately 2.5 kb 5 'upstream fragments of OsTMT1 and OsTMT2 were fused transcriptionally to the GUS promoter gene. Among transgenic rice plants expressing pOsTMT1 - GUS or pOsTMT2 - GUS No significant change in GUS expression was found (data not shown). Each representative line with the highest GUS intensity was used primarily for the next analysis. By histochemical analysis, the present invention showed that GUS activity is detectable in all organs of rice used in experiments involving leaves, roots, flowers and immature seeds (FIGS. 8 and 9).

In immature leaves of the pOsTMT1 - GUS and pOsTMT2 - GUS transgenic rice lines, GUS activity was observed in lobules , ductal sheath cells and vascular bundles (eg duct cells and phloem companion cells but mature metaxylem cells). cells) but not in most cells (FIG. 8). The highest OsTMT1 expression was observed in the coronary soybean cells. In epidermal cells, GUS expression was very marginal in the pOsTMT1 - GUS line (FIG. 8A). However, in the pOsTMT2 - GUS line, the level of reporter expression was higher in the parenchymal cells and phloem semi-cells when compared to other regions, including lobules and epidermal cells (Figure 8b).

pOsTMT1 - GUS and pOsTMT2 - GUS In the roots of the lines, strong GUS activities were observed in the half cells of the vascular tissue (FIGS. 8C and 8D), whereas in flowers, the expression of the reporter was weakly detected only in the palea and lemma, and vein ) Showed relatively high expression. GUS activity was determined by pOsTMT1 - GUS including stamens And not in other internal organs of the pOsTMT2-GUS line (FIGS. 8E-8H).

pOsTMT1 - GUS Wow pOsTMT2 - GUS In immature seeds developing in two transgenic rice lines, expression of GUS was found abundantly in the tubular tissue of 7-8 DAF seed shells (FIGS. 9A and 9B). To further investigate the expression of OsTMT1 and OsTMT2 , pOsTMT1 - GUS And immature seeds of the pOsTMT2-GUS transgenic rice line were cut into longitudinal or transverse sections. In 5 DAF immature seeds, OsTMT1 and OsTMT2 are the dorsal side of the main vascular cells (FIGS. 9C and 9D) and nucellar projections of the maternal portion, cross cell layers and tube cell layers. It was highly expressed in (tube cell layers), but was not expressed in aleurone or subaleurone cells of the fibrous part (FIGS. 9E and 9F). GUS activity was highest in the dorsal (black arrow) dorsal tissue of 12-15 DAF immature seeds in both transformation lines (FIGS. 9G and 9H). Notably, the expression of GUS in the embryos of these immature seeds was only pOsTMT2 - GUS Only observed in line (FIG. 9H).

Endogenous expression of OsTMT2 was further investigated by in situ hybridization studies of immature rice seeds. These experiments showed that OsTMT2 is expressed mainly in the dorsal bundle of dorsal and nucellar projections of the 3 DAF immature seeds and in the lateral flanking tissue surrounding the envelope (FIGS. 9I and 9J). Also in 5 DAF immature seeds, OsTMT2 expression was observed in the lateral cross cell layer and tube cell layer of the maternal part (FIGS. 9K and 9L). This is in good agreement with the results of the RT-PCR (FIG. 6B) and histochemical analysis of GUS expression (FIGS. 9A-9H) which were performed first.

Example  5: OsTMT1 Transgenic Arabidopsis mutants expressing tmt1 -2- 3  From  Using separated vacuoles Monosaccharides  Analysis of transportation

Several failed attempts have been made to demonstrate the activity of sugar transporters belonging to the TMT family through functional complementation of yeast hexose transporter-deficient mutants (Wormit et al, 2006, Plant Cell). 18, 3476-3490). Similarly, the present invention has shown that OsTMT1 and OsTMT2 do not complement glucose transport activity in yeast mutant RE700A lacking an endogenous glucose transporter. Cold treated Arabidopsis tmt1 -2-3 triple knockout mutant in a separate body from the vacuole absorption of glucose is not significantly lower than wild type controls appeared in the previous report (Wormit et al, 2006, Plant Cell 18, 3476-3490). Accordingly, the present invention CaMV35S promoter-using transgenic Arabidopsis lines expressing tmt1 -2-3 OsTMT1 was designed replenishment (complementation) experiments on OsTMT1, Two independent homozygous transformation lines with moderate ( OsTMT1 - OX1 ) or strong ( OsTMT1 - OX2 ) transgene expression were selected in RT-PCR analysis (FIG. 10A).

tmt1 -2-3 to determine whether the OsTMT1 expressed in mutant background to recover the glucose transport activity decreased within tmt1 -2-3 mutant vacuole, the present invention is a wild type, transgenic tmt1 -2-3 and OsTMT1 The foliar vacuoles were separated from the plants. Glucose absorption is very low (0.5 pmolμL ^-1 vacuoles in tmt1 -2-3 min ^-1 , R ² = 0.364), which was significantly higher in Arabidopsis wild-type plants (2.4 pmol μL ^-1 vacuoles) min ⁻¹ , R ² = 0.75) (FIG. 10B). This is consistent with previous data showing that the tmt1 -2-3 mutant line is the ability to transport glucose into the vacuoles were markedly lower (Wormit et al, 2006, Plant Cell 18, 3476-3490). Importantly, OsTMT1 - OX1 and OsTMT1 - OX2 Vegetation vacuoles were observed in wild-type Arabidopsis ( OsTMT1 - OX1) For and OsTMT1-OX2 , respectively 3.0 pmol μL ^-1 vacuole min ^-1 , R ² = 0.766 and 4.0 pmol μL ^-1 vacuoles min ⁻¹ , R ² = 0.979). Also look for the highest expression of OsTMT1 OsTMT1 - OX2 Absorption of glucose into vacuoles of the line shows OsTMT1 - OX1 showing moderate expression of OsTMT1 It is noteworthy that it is significantly higher than plants (FIG. 10B). These results strongly suggest that OsTMTs transport monosaccharide sugars into vacuoles in rice in a manner similar to AtTMTs.

It has previously been reported that vacuole glucose transport is strongly overexpressed during cold incubation in Arabidopsis (Wormit et al, 2006, Plant Cell 18, 3476-3490). For transformants expressing OsTMT1 tmt1 to investigate whether the content is restored to wild type levels under cold stress conditions at -2 per mutant lines, the wild-type, tmt1 -2, and OsTMT1 transgenic Arabidopsis plants low-temperature chamber ( 9 ° C.) and incubated for 24 hours with continuous light. In cold-treated wild type plants, glucose, fructose and sucrose were found to accumulate at high concentrations (7.18, 4.41 and 9.50 μmol g fresh weight (fw) ⁻¹ , FIG. 10C, respectively). However, tmt1 -2-3 mutant Arabidopsis plants it is significantly further contains a low-hexyl source level compared to the wild type. In contrast, the transgenic lines expressing tmt1 -2-3 OsTMT1 showed a hexyl source level increases over a low temperature process as compared to 24 hours tmt1 -2-3 mutant plants. The OsTMT1 - OX 2 line also contained glucose and fructose at levels similar to the wild type. Transgenic tmt1 -2-3 of hexyl source level in mutant plants significantly increased expressing OsTMT1 under cold stress supports the evidence that the in OsTMT1 tmt1 -2-3 mutants can recover the role of the gene AtTMT more.

<110> University-Industry Cooperation Group of Kyung Hee University <120> OsTMT2 gene involved in vacuolar sugar transport from rice <130> PN10017 <160> 26 <170> KopatentIn 1.71 <210> 1 <211> 2597 <212> DNA <213> Oryza sativa <400> 1 aagaggtgga agaagaggga tcggagaggg gtgaagatgt cgggtgctgc tctcgtcgca 60 atcgcggcct ccatcggcaa cctgctgcag ggatgggaca atgccaccat tgcaggtgct 120 gtactataca taaagaagga attcaagcta gaaagtgagc ccactgtgga ggggctaatc 180 gtggccatgt cactgattgg tgcaaccatc atcacaacat tctcagggcc ggtatcagac 240 tggatcggcc ggcgccctat gctcattctc tcttcaattc tctacttcct cagcagcctc 300 atcatgctat ggtccccgaa tgtctatgtg ttactgctcg cacgccttat agatggattc 360 ggcatcggct tggctgtcac acttgtacct ttgtacatct cagagacagc tccttcagag 420 atcaggggtt tgctgaatac actgccacag ttcagtgggt caggagggat gttcttgtcc 480 tactgcatgg tgtttgggat gtcactgttg ccgtcacctg actggagaat tatgcttggt 540 gttctcgcaa tcccttcgtt gtttttcttc ggattgacaa tattctatct gccagaatca 600 ccaagatggc ttgtcagcaa agggcggatg gctgaggcaa agaaggtatt acaaaaatta 660 cgtgggagag aggatgtctc aggagaaatg gctcttcttg ttgaaggttt ggaggttgga 720 gctgacactt ccattgaaga gtacatcatc ggaccggcta tagagccagc tgatgagcat 780 gttgttgatg gcgataagga ccaaataaca ctgtacgggc ctgaagaggg ccaatcatgg 840 attgctcgac cttccaaggg acccagcatt cttggaagtg tgctttctct tacatctcgt 900 catggcagca tggtgaacca gagcgtgcca cttatggatc ctatagtcac gcttttcggc 960 agtgtccatg agaacatgcc tcatgctgga ggaagcatgc ggagtacatt gtttcccaac 1020 tttggtagta tgtttagtgt gacagatcag caccccaagg ttgatcaatg ggatgaggag 1080 aaccttcata gggatgatga ggaatacgcg tctgatggtg ctggaggtga ctatgaagac 1140 aatgtccaca gcccgttgct gtcccgacag accacgagcg cagaagggaa ggacattgca 1200 caccatgctc atcgtggaag tgctctgagt atgagaagaa gaagcctctt ggaagagggt 1260 ggcgaggcgg tgagcagcac tggcattggt gggggatggc agcttgcatg gaaatggtca 1320 gagcgagaag gcgaggatgg taagaaggaa ggaggattta aaaggatcta cttgcaccaa 1380 gaggaagtgc caggatcaag aaggggctca gttatttcac ttcctggtgg aggggacgct 1440 cctgaaggca gcgaattcat acatgctgcc gctttggtaa gccaaccagc actttactcc 1500 aaggatatca ttgaacagcg tatgtccggt ccagccatga ttcatccatc agaggcagct 1560 gccaaaggtt caagctggaa agatttgttt gaacctggag tgaggcgtgc gttgttagtc 1620 ggtgttggaa ttcaaatcct tcaacagttt gctggaataa atggggttct ctattacact 1680 ccacaaatac ttgagcaagc gggggtggca gttctccttt ccaatcttgg cctcagttca 1740 gcatcagctt ccatcttgat cagttctctg accaccctac tgatgcttcc tagcattggt 1800 ttagccatga gacttatgga catctctgga agaaggtttc tgcttctggg cacaattcca 1860 gtcttgatag catctctagt tgtcttggtt gtgtccaatg ttatcgacct gggtacagtg 1920 gcccacgccg cactctccac aatcagcgtc atcatctact tctgctgctt cgtcatggga 1980 ttcggtccga tccccaacat tctgtgtgcg gagatcttcc ccactagggt ccgcggcatc 2040 tgcattgcca tctgcgccct gacattctgg attggtgata tcatagtcac ctacagcctc 2100 cctgtgatgc tgaatgccat cggcctagca ggtgtctttg gtatatacgc agttgtttgc 2160 tcgattgcct tcgtgttcgt cttcctcaag gtccccgaga cgaagggcat gccgctcgaa 2220 gtcatcaccg agttcttcgc tgttggtgca aagcaaatgc aggctacaaa ggcctaattc 2280 ttcagcaact ctgctttcaa tctttatata acactgtaaa ttggaatctg caagatcaca 2340 ccatgaggct ggaggagctt tttgagttgt aaatgagaga agaggattgc tattcatgct 2400 ccttatgcaa gggaaaagag ttcattactg gcacagggca gctgtaaagt aggaaatttt 2460 catgttattc atgtccactt cttgtttatt tagtactgtg ttaggtaatg ttgttattca 2520 tcagtagttg ctgctaggga tgttcaaaaa taagcttgct aatctgattt tgagctacta 2580 tgttggggtt cataata 2597 <210> 2 <211> 746 <212> PRT <213> Oryza sativa <400> 2 Met Ser Gly Ala Ala Leu Val Ala Ile Ala Ala Ser Ile Gly Asn Leu   1 5 10 15 Leu Gln Gly Trp Asp Asn Ala Thr Ile Ala Gly Ala Val Leu Tyr Ile              20 25 30 Lys Lys Glu Phe Lys Leu Glu Ser Glu Pro Thr Val Glu Gly Leu Ile          35 40 45 Val Ala Met Ser Leu Ile Gly Ala Thr Ile Ile Thr Thr Phe Ser Gly      50 55 60 Pro Val Ser Asp Trp Ile Gly Arg Arg Pro Met Leu Ile Leu Ser Ser  65 70 75 80 Ile Leu Tyr Phe Leu Ser Ser Leu Ile Met Leu Trp Ser Pro Asn Val                  85 90 95 Tyr Val Leu Leu Leu Ala Arg Leu Ile Asp Gly Phe Gly Ile Gly Leu             100 105 110 Ala Val Thr Leu Val Pro Leu Tyr Ile Ser Glu Thr Ala Pro Ser Glu         115 120 125 Ile Arg Gly Leu Leu Asn Thr Leu Pro Gln Phe Ser Gly Ser Gly Gly     130 135 140 Met Phe Leu Ser Tyr Cys Met Val Phe Gly Met Ser Leu Leu Pro Ser 145 150 155 160 Pro Asp Trp Arg Ile Met Leu Gly Val Leu Ala Ile Pro Ser Leu Phe                 165 170 175 Phe Phe Gly Leu Thr Ile Phe Tyr Leu Pro Glu Ser Pro Arg Trp Leu             180 185 190 Val Ser Lys Gly Arg Met Ala Glu Ala Lys Lys Val Leu Gln Lys Leu         195 200 205 Arg Gly Arg Glu Asp Val Ser Gly Glu Met Ala Leu Leu Val Glu Gly     210 215 220 Leu Glu Val Gly Ala Asp Thr Ser Ile Glu Glu Tyr Ile Ile Gly Pro 225 230 235 240 Ala Ile Glu Pro Ala Asp Glu His Val Val Asp Gly Asp Lys Asp Gln                 245 250 255 Ile Thr Leu Tyr Gly Pro Glu Glu Gly Gln Ser Trp Ile Ala Arg Pro             260 265 270 Ser Lys Gly Pro Ser Ile Leu Gly Ser Val Leu Ser Leu Thr Ser Arg         275 280 285 His Gly Ser Met Val Asn Gln Ser Val Pro Leu Met Asp Pro Ile Val     290 295 300 Thr Leu Phe Gly Ser Val His Glu Asn Met Pro His Ala Gly Gly Ser 305 310 315 320 Met Arg Ser Thr Leu Phe Pro Asn Phe Gly Ser Met Phe Ser Val Thr                 325 330 335 Asp Gln His Pro Lys Val Asp Gln Trp Asp Glu Glu Asn Leu His Arg             340 345 350 Asp Asp Glu Glu Tyr Ala Ser Asp Gly Ala Gly Gly Asp Tyr Glu Asp         355 360 365 Asn Val His Ser Pro Leu Leu Ser Arg Gln Thr Thr Ser Ala Glu Gly     370 375 380 Lys Asp Ile Ala His His Ala His Arg Gly Ser Ala Leu Ser Met Arg 385 390 395 400 Arg Arg Ser Leu Leu Glu Glu Gly Gly Glu Ala Val Ser Ser Thr Gly                 405 410 415 Ile Gly Gly Gly Trp Gln Leu Ala Trp Lys Trp Ser Glu Arg Glu Gly             420 425 430 Glu Asp Gly Lys Lys Glu Gly Gly Phe Lys Arg Ile Tyr Leu His Gln         435 440 445 Glu Glu Val Pro Gly Ser Arg Arg Gly Ser Val Ile Ser Leu Pro Gly     450 455 460 Gly Gly Asp Ala Pro Glu Gly Ser Glu Phe Ile His Ala Ala Ala Leu 465 470 475 480 Val Ser Gln Pro Ala Leu Tyr Ser Lys Asp Ile Ile Glu Gln Arg Met                 485 490 495 Ser Gly Pro Ala Met Ile His Pro Ser Glu Ala Ala Ala Lys Gly Ser             500 505 510 Ser Trp Lys Asp Leu Phe Glu Pro Gly Val Arg Arg Ala Leu Leu Val         515 520 525 Gly Val Gly Ile Gln Ile Leu Gln Gln Phe Ala Gly Ile Asn Gly Val     530 535 540 Leu Tyr Tyr Thr Pro Gln Ile Leu Glu Gln Ala Gly Val Ala Val Leu 545 550 555 560 Leu Ser Asn Leu Gly Leu Ser Ser Ala Ser Ala Ser Ile Leu Ile Ser                 565 570 575 Ser Leu Thr Thr Leu Leu Met Leu Pro Ser Ile Gly Leu Ala Met Arg             580 585 590 Leu Met Asp Ile Ser Gly Arg Arg Phe Leu Leu Leu Gly Thr Ile Pro         595 600 605 Val Leu Ile Ala Ser Leu Val Val Leu Val Val Ser Asn Val Ile Asp     610 615 620 Leu Gly Thr Val Ala His Ala Ala Leu Ser Thr Ile Ser Val Ile Ile 625 630 635 640 Tyr Phe Cys Cys Phe Val Met Gly Phe Gly Pro Ile Pro Asn Ile Leu                 645 650 655 Cys Ala Glu Ile Phe Pro Thr Arg Val Arg Gly Ile Cys Ile Ala Ile             660 665 670 Cys Ala Leu Thr Phe Trp Ile Gly Asp Ile Ile Val Thr Tyr Ser Leu         675 680 685 Pro Val Met Leu Asn Ala Ile Gly Leu Ala Gly Val Phe Gly Ile Tyr     690 695 700 Ala Val Val Cys Ser Ile Ala Phe Val Phe Val Phe Leu Lys Val Pro 705 710 715 720 Glu Thr Lys Gly Met Pro Leu Glu Val Ile Thr Glu Phe Phe Ala Val                 725 730 735 Gly Ala Lys Gln Met Gln Ala Thr Lys Ala             740 745 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> OsTMT1 forward primer <400> 3 aaatctcccc taaaagcttc c 21 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> OsTMT1 reverse primer <400> 4 gaactagtac tcggctatgc taa 23 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> OsTMT2 forward primer <400> 5 aagaggtgga agaagaggga t 21 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> OsTMT2 reverse primer <400> 6 tattatgaac cccaacatag tagc 24 <210> 7 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> OsTMT1 forward primer <400> 7 gctctagact tggtggtaag attcgccg 28 <210> 8 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> OsTMT1 reverse primer <400> 8 ccctcgagag tcctccttgg cctgctttgc 30 <210> 9 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> OsTMT2 forward primer <400> 9 gctctagagg ggtgaagatg tcgggtgct 29 <210> 10 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> OsTMT2 reverse primer <400> 10 ccctcgagag gcctttgtag cctgcatttg 30 <210> 11 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> OsTMT1 forward primer <400> 11 ccctcgagaa acctaaactt gaaatgtgct a 31 <210> 12 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> OsTMT1 reverse primer <400> 12 cgcctaggcg aatcttacca ctgcacaaga a 31 <210> 13 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> OsTMT2 forward primer <400> 13 gcgtcgacat cgtgattctt tctttcaaac 30 <210> 14 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> OsTMT2 reverse primer <400> 14 gctctagact tcacccctct ccgatccc 28 <210> 15 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> OsTMT1 forward primer <400> 15 gaagtcatca ccgagttctt c 21 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> OsTMT1 reverse primer <400> 16 tacagggaga agaattccaa a 21 <210> 17 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> OsTMT2 forward primer <400> 17 gtctttggta tatacgcagt tgtt 24 <210> 18 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> OsTMT2 reverse primer <400> 18 agtggacatg aataacatga aaat 24 <210> 19 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> OsGBSSII forward primer <400> 19 taggtgtcac tgaatggcta gaac 24 <210> 20 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> OsGBSSII reverse primer <400> 20 tggcccacat ctctaagtaa catc 24 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Act1 forward primer <400> 21 ggaactggta tggtcaaggc 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Act1 reverse primer <400> 22 agtctcatgg atacccgcag 20 <210> 23 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> OsUBQ5 forward primer <400> 23 gactacaaca tccagaagga gtc 23 <210> 24 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> OsUBQ5 reverse primer <400> 24 tcatctaata accagttcga tttc 24 <210> 25 <211> 21 <212> DNA <213> Artificial Sequence <220> 223 UBQ forward primer <400> 25 gtggtgctaa gaagaggaag a 21 <210> 26 <211> 21 <212> DNA <213> Artificial Sequence <220> 223 UBQ reverse primer <400> 26 tcaagcttca acttcttctt t 21

Claims (10)

OsTMT2 ( Oryza derived from rice) involved in the transport of sugars in the vacuoles of sugars, consisting of the amino acid sequence represented by SEQ ID NO: 2 sativa tonoplast monosaccharide transporter 2) protein. Gene encoding the protein of claim 1. The gene according to claim 2, wherein the gene consists of a nucleotide sequence represented by SEQ ID NO: 1. A recombinant vector comprising the gene of claim 2. Plant transformed with the recombinant vector of claim 4. 6. The transformed plant of claim 5, wherein the plant is rice or Arabidopsis. Seed of the transformed plant of claim 5. A method of increasing sugar transport into a vacuole, comprising overexpressing an OsTMT2 gene by transforming a plant with the recombinant vector of claim 4. Plants with increased sugar in vacuoles produced by the method of claim 8. A composition for increasing sugar transport into a vacuole of a plant, comprising the gene of claim 2.

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