KR100990118B1 - Use of OsHXK5 gene as glucose sensor - Google Patents

Abstract

The present invention relates to a plant transformed with a recombinant vector comprising an OsHXK5 (Oryza sativa Hexokinase 5) gene and its seeds, a composition for detecting glucose in a plant comprising the gene, and an OsHXK5 gene according to a plant by transforming the gene into a plant. The present invention relates to a method for determining the glucose sensing ability of the method and to control the growth of plants using the gene.

OsHXK5, Glucose Detection, Plant Growth, Glucose Sensor

Description

Use of OsHXK5 gene as glucose sensor

The present invention relates to the use of the OsHXK5 gene as a glucose sensor, and more particularly, for the detection of glucose in a plant transformed with a recombinant vector comprising an OsHXK5 (Oryza sativa Hexokinase 5) gene, seeds thereof, and a plant comprising the gene. The present invention relates to a composition, a method of transforming a gene into a plant to determine glucose sensing ability of an OsHXK5 gene according to a plant, and a method of controlling plant growth using the gene.

In higher plants, sugar is not only used as a basic source of carbon supply and energy metabolism, but also regulates physiological processes during the growth cycle from germination to flowering and old age, and during the protective response to biological and abiotic stresses. Is known as a signaling material (Jang and Sheen) (1994) Plant Cell 6: 1665-1679. Therefore, precise sugar recognition and signaling systems are important for many essential metabolic regulation to maintain normal plant growth and development.

Glucose, one of the main products of photosynthesis, is the most widely known sugar component that regulates many signaling pathways in plants (Koch KE (1996) Annu Rev Plant Physiol Plant Mol Biol) 47: 509-540). In unicellular microbial yeast, several glucose sensors, such as hexokinase ScHXK2, glucose transporter-like proteins Snf3 and Rgt2, and G protein-coupled receptor Gpr1, detect cell internal and external glucose states to regulate cell growth and gene expression. (Rolland et al. (2002) Sugar sensing and signaling in plants. 14: S185-205). Similarly, recent plant studies have revealed a sugar detection and signaling system mediated by hexokinase as an internal glucose sensor or GPCR, a G protein-coupled receptor as an external glucose sensor (Rolland et al. (2001) Trends Biochem Sci 26: 310-317).

In addition to the catalytic function of hexokinase, which phosphorylates hexose to form hexose 6-phosphate, hexokinase function as an evolutionarily conserved glucose sensor in plants is characterized by Arabidopsis hexokinase1 ( AtHXK1 ) transgenic plants and glucose insensitive ( gin2 ) Has been importantly recognized from biochemical, genetic, and molecular studies of mutants (Jang et al. (1997) Plant Cell 9: 5-19). Transgenic plants expressing the AtHXK1 mutant allele with depleted enzyme activity in the gin2 mutant line provided strong evidence that AtHXK1's enzymatic activity and sugar cognitive function were not coupled in Arabidopsis plants. In addition, proteomics and yeast two hybrid interaction experiments showed that AtHXK1 regulates 19S of vacuole H ⁺ -ATPase B1 (VHA-B1) and proteasome subunit (RPT5B) to directly regulate expression of specific photosynthetic genes in the nucleus. It was shown that it interacts with two partners, molecules, and that the interaction of AtHXK1 with VHA-B1 or RPT5B does not require the enzymatic activity of AtHXK1. In tomatoes, AtHXK1 expression is known to cause induction of photosynthesis, growth inhibition and rapid aging that are characteristic of sugar recognition and signaling in photosynthetic tissue. With the exception of Arabidopsis HXK1, the function of hexokinase as a glucose sensor has not yet been demonstrated in other plant species.

Hexokinase has been shown to be associated with various organelles such as mitochondria, chloroplasts, Golgi apparatus, endoplasmic reticulum, actin filaments and plasma membranes (Schleucher et al. (1998) Plant Physiol 118: 1439-1445). In yeast, the glucose sensor ScHXK2 is found to be partially present in the nucleus as well as in the cytoplasm and has a nuclear localization signal in the N-terminal domain (Herrero et al. (1998) FEBS Lett 434: 71-76). Moreover, nuclear localization of ScHXK2 is essential for glucose inhibition of several genes such as SUC2 , HXK1 and GLK1 . Part of AtHXK1, which is highly related to mitochondria, has been found to be located in the nucleus. Thus, under glucose-excess conditions, nuclear AtHXK1 binds to its substrate and is known to cause inhibition of expression of the gene of interest (Cho et al. (2006) Cell 127: 579-589).

Since the inventors have previously OsHXK1 (O ryza s ativa H x o e k inase 1) to the OsHXK10 Ten rice hexokinases were isolated and all OsHXKs were identified to have hexokinase activity (Cho et al. (2006) Planta 224: 598-611). In addition, it was confirmed that OsHXK4 and OsHXK7 are present in the chloroplast strom and cytoplasm, respectively.

Korean Patent Registration No. 10-0660616 discloses a method for regulating the growth and development of plants using Bacillus glucokinase, but is different from the gene of the present invention.

The present invention has been made in accordance with the above requirements, and the present inventors have identified rice homologous hexokinases of OsHXK5 and OsHXK6 based on sequence similarity and intracellular location, and these genes have sugar recognition and signaling functions. In order to investigate whether the functions of OsHXK5 and OsHXK6 in corn and rice leaf protoplasts and transgenic rice plants were observed, Arabidopsis gin2-1 mutants were identified as wild OsHXK5, OsHXK6 and alleles from which the enzyme activities of these genes were removed. The invention was completed by transforming and analyzing sugar recognition and signaling properties.

In order to solve the above problems, the present invention provides a plant transformed with a recombinant vector comprising OsHXK5 (Oryza sativa Hexokinase 5) gene and its seed.

The present invention also provides a composition for detecting glucose in a plant comprising the gene.

In addition, the present invention provides a method for determining the glucose sensing ability of the OsHXK5 gene according to the plant by transforming the gene into a plant.

The present invention also provides a method of controlling the growth of plants using the gene.

According to the present invention, the OsHXK5 gene of the present invention has a dual targeting ability against mitochondria and the nucleus, and can also serve as a glucose sensor in plants.

In order to achieve the object of the present invention, the present invention provides a plant transformed with a recombinant vector comprising OsHXK5 (Oryza sativa Hexokinase 5) gene.

OsHXK5 gene included in the transformed plant can be used as a glucose sensor. That is, the growth of the plant can be controlled according to the glucose content in the plant.

The plant may be inhibited growth in a glucose-containing medium, the growth inhibition may be through inhibition of photosynthesis related gene expression. Photosynthesis related genes can be, for example, but not limited to, RbcS , chlorophyll a / b binding protein 2 ( CAB2 ), sedoheptulose-biphosphatase ( SBP ), carbonic anhydrase ( CAA ), and the like. It doesn't work.

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. Recombinant cells can also express genes found in natural cells, but the genes have been modified and reintroduced into cells by 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.

Preferred examples of plant expression vectors are Ti-plasmid vectors which, when present in a suitable host such as Agrobacterium tumerfaciens, can transfer part of themselves, the so-called T-region, into plant cells. Another type of Ti-plasmid vector (see EP 0 116 718 B1) is used to transfer hybrid DNA sequences to protoplasts from which current plant cells or new plants can be produced that properly insert hybrid DNA into the plant's genome. 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 a plant host are viral vectors, such as those which can be derived from double stranded plant viruses (eg CaMV) and single stranded viruses, gemini viruses, etc. For example, it may be selected from an incomplete plant viral vector. The use of such vectors can be advantageous, especially when it is difficult to properly transform a plant host.

The expression vector preferably comprises 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, but are not limited to, herbicide resistance genes such as glyphosate or phosphinothricin, antibiotic resistance genes such as kanamycin, G418, bleomycin, hygromycin, and chloramphenicol It doesn't happen.

In the plant expression vector according to an embodiment of the present invention, the promoter may be CaMV 35S, actin, ubiquitin, pEMU, MAS or histone promoter, but is not limited thereto. 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.

The terminator may be a conventional terminator, and examples thereof include nopaline synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, agrobacterium tumefaciens (ocrobacterium tumefaciens) Terminator of the Fine (Octopine) gene, etc., but is not limited thereto. With regard to 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 terminators is highly desirable in the context of the present invention.

The plant according to the present invention is a monocotyledonous plant such as rice, barley, corn, wheat, rye, oats, grass, forage, millet, sugar cane, rice, orchardgrass, or Arabidopsis, potato, eggplant, tobacco, pepper, tomato , Burdock, garland chrysanthemum, lettuce, bellflower, spinach, chard, sweet potato, celery, carrot, buttercup, parsley, cabbage, cabbage, mustard, watermelon, melon, cucumber, pumpkin, gourd, strawberry, soybean, green bean, kidney bean, or pea It may be a dicotyledonous plant, but is preferably Arabidopsis or rice.

The hexokinase gene according to the present invention may be preferably composed of a nucleotide sequence represented by SEQ ID NO: 1. In addition, variants of such sequences are included within the scope of the present invention. A variant is a nucleotide sequence that changes in base sequence but has similar functional properties to that of SEQ ID NO: 1. Specifically, the hexokinase gene may comprise a nucleotide sequence having at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% homology with the nucleotide sequence of SEQ ID NO: 1. 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 seed of the plant. Preferably, the seed is a seed of Arabidopsis or rice.

The present invention also provides a composition for detecting glucose of a plant comprising the OsHXK5 gene. The OsHXK5 gene acts as a glucose sensor in plants, so it can detect glucose in plants. The OsHXK5 gene may be preferably composed of the nucleotide sequence of SEQ ID NO: 1. The plant is preferably rice. OsHXK5 gene is characterized by having a dual targeting ability for the mitochondria and the nucleus (see FIGS. 1 and 2).

The present invention also comprises the steps of transforming the plant with OsHXK5 gene; And

It provides a method for determining the glucose sensing ability of the OsHXK5 gene according to the plant comprising the step of measuring the growth of the plant in a glucose-containing medium.

Method for determining the glucose sensing ability of the OsHXK5 gene according to the plant of the present invention first comprises transforming the plant with OsHXK5 gene.

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. Calcium / polyethylene glycol method for protoplasts (Krens, FA et al., 1982, Nature 296, 72-74), electroporation of protoplasts (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), particle bombardment methods (DNA or RNA-coated) of various plant elements (Klein TM et. al., 1987, Nature 327, 70), infection with (incomplete) virus in agrobacterium tumerfaciens mediated gene transfer by invasion of plants or transformation of mature pollen or vesicles, and the like. have. 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.

"Plant cell" used for transformation of a plant may be any plant cell. The plant cell may be any of a cultured cell, a cultured tissue, a culture or whole plant, preferably a cultured cell, a cultured tissue or culture medium, and more preferably a cultured cell. Preferably, the plant is rice.

"Plant tissue" refers to the tissues of differentiated or undifferentiated plants, such as, but not limited to, roots, stems, leaves, pollen, seeds, cancer tissues and various types of cells used in culture, ie single cells, protoplasts. (protoplast), shoots and callus tissue. The plant tissue may be in planta or in an organ culture, tissue culture or cell culture.

Method for determining the glucose sensing ability of the OsHXK5 gene according to the plant of the present invention comprises the step of measuring the growth of the plant in a glucose-containing medium. The degree of growth of the plant can be determined, for example, by measuring the length of shoots and the like. By comparing the length of shoots of the transformants with respect to the control, the glucose sensing ability of the OsHXK5 gene according to the plant can be determined. That is, if the length of shoots is large compared to the control of the transgenic plant, the OsHXK5 gene may be judged to have a large glucose detection ability.

The present invention also provides a method for regulating the growth of a plant, comprising the step of transforming the plant with OsHXK5 gene. OsHXK5 gene plays a role as a glucose sensor, it is possible to regulate the growth of the plant according to the glucose treatment. The method according to one embodiment of the present invention provides a method of inhibiting plant growth in a glucose-containing medium comprising transforming and overexpressing an OsHXK5 gene in a plant. Plants transformed with OsHXK5 gene significantly inhibited plant growth in glucose containing medium (see FIG. 7). Growth inhibition of the plant is manifested in phenotypes such as pale yellow leaves and reduced plant length.

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.

Example

Materials and methods

Plant material and growth

ABRC (Ohio State University, Columbus, OH; www.biosci.ohio-state.edu/~plantbio/Facilities/abrc/) wild-type Arabidopsis thaliana (ecotype L er) and gin2-1 plants and transgenic plants are provided by the 22 Raised in soil with photoperiod at 16 ° C./8 h cancer. Wild-type rice ( Orysa sativa L. cv. Dongjin) and transgenic plants were grown in greenhouses at 30 ° C during the day and 20 ° C at night with 14h light / 10h cancer cycle for several generations. For protoplast separation, wild-type corn ( Zea mays L. cv. Yeonnong) and rice plants were grown in dark and growth conditions at 25 ° C. for 7 days and 8-10 days, respectively, and young leaves were used.

Vector building

In order to investigate the cellular location of OsHXK5 and OsHXK6, termination codon, and then the total length of each cDNA fragment containing the 3'-UTR amplification by the addition of Xba I and Xho I seat CaMV35S promoter and sGFP in the pJJ1450 vector (Chiu et al. (1996) Curr Biol 6: 325-330). OsHXK5ΔmTP (amino acids 25-507, GenBank no.DQ116387) and OsHXK6 ΔmTP (amino acids 29-506, GenBank no.DQ116388) were prepared by lacking the mitochondrial targeting signal at its N-terminus and subcloned into the pJJ1450 vector. Primer pairs used for PCR amplification are as follows. OsHXK5 is 5'-GCTCTAGAAGGGAAGGCGGAGCAGCGGTG-3 '(SEQ ID NO: 2) and 5'-CCCTCGAGAGTCGATCTCGGCATACTGGGA-3' (SEQ ID NO: 3), OsHXK6 are 5'-GCTCTAGAGGAAGGAGGAGGAGTAGGACGC-3 '(SEQ ID NO: 4) and 5'-CCCTCGAGACTCGACGCTAGCATACTGGGA-3' (SEQ ID NO: 5), OsHXK5ΔmTP Reverse primers of 5'-GCTCTAGAATGCGGAGGCGGAGGAGGAGGGAC-3 '(SEQ ID NO: 6) and OsHXK5 , OsHXK6ΔmTP were 5'-GCTCTAGAATGCGGAGGAGGAGGAGCAAGCGG-3' (SEQ ID NO: 7) and reverse primers of OsHXK6 .

To overexpress OsHXK5 and OsHXK6 in Arabidopsis and rice, respectively, cDNAs were placed under the control of the CaMV35S promoter using the pPZP2Ha3 (+) vector. Gly (G) conserved at the ATP binding site and Ser (S) at the phosphoryl transfer site to generate mutants free of the enzymatic activity of OsHXK5 and OsHXK6 were PCR-mediated targeted mutagenesis. By Asp (D) and Ala (A), respectively. Primer pairs used for PCR amplification are as follows. OsHXK5-G113D Silver 5'-GGAAGTTGGTGTCTCCAAGATCCAATGCAT-3 '(SEQ ID NO: 8) and 5'-GATCTTGGAGACACCAACTTCCGCGTCCTG-3' (SEQ ID NO: 9), OsHXK5-S186A is 5'-CACTGGGAAGGCAAAGGTGAAGCCCAGCTC-3 '(SEQ ID NO: 10) and 5'-GCTTCACCTTTGCCTTCCCAGTGAGCCAGA-3' (SEQ ID NO: 11), OsHXK6-G112D is 5'-GGAAATTGGTGTCCCCAAGATCGAGAGCAT-3 '(SEQ ID NO: 12) and 5'-CGATCTTGGGGACACCAATTTCCGTGTTAT-3' (SEQ ID NO: 13), OsHXK6-S185A 5'-CACTGGGAAAGCAAAGGTGAAGCCTAACTC-3 '(SEQ ID NO: 14) and 5'-GCTTCACCTTTGCTTTCCCAGTGCACCAAA-3' (SEQ ID NO: 15) were used. The amplified mutant alleles cleaved with Xba I and Xho I were cloned into pPZP2Ha3 (+) vectors and the resulting constructs were named OsHXK5-G113D , OsHXK5-S186A , OsHXK6-G112D and OsHXK6-S185A .

To establish a working vector for transient gene expression analysis, a rice mutant alleles OsHXK5, OsHXK6 and the enzymatic activity of the gene is removed in hexokinase was placed under the control of the CaMV35S promoter of the vector pJJ1549. To construct the reporter vector, the promoter of RAmy3D was amplified by PCR using primers 5'-CGGGATCCGATCTTCAACCACCTGTGCTAGCT-3 '(SEQ ID NO: 16) and 5'-TGCCATGGATCTGTGTAAGCTGAAACCGTGTT-3' (SEQ ID NO: 17). Amplification products digested with Bam HI and Nco I were linked to the firefly LUC gene to produce OsRAmy3D :: LUC . Corn RbcS promoter :: LUC construct (ZmRbcS :: LUC) derived from ZmRbcS :: CAT was used as an additional reporter molecule. Corn ubiquitin derived from pGA1611 binary vector digested with Hin dIII and Bam HI to generate ZmUBQ :: GUS , an internal control reporter construct The promoter was linked to the β - glucuronidase ( GUS ) gene linked to the terminator of the nopaline synthase gene. OsHXK-Myc fusion constructs were prepared by linking the Myc sequence to the C-terminus of OsHXK5 , OsHXK6 and the mutant allele.

Total length cDNAs of OsHXK5 , OsHXK6 and mutant alleles from which the enzymatic activity of these genes were removed were amplified by PCR by addition of Xba I and Xho I sites and subcloned into Spe I and Xho I sites of yeast shuttle vector pDR196 ( Wipf et al. (2003) Genome 46: 177-181).

Intracellular Location of OsHXK-GFP Protein

GFP fusion constructs were delivered to corn and Arabidopsis chloroplasts using polyethylene glycol (PEG) -calcium mediated methods (Hwang and Sheen (2001) Nature 413: 383-389) and incubated for 12-24 hours for transient expression. It was. Mitochondria were observed by staining with MitoTracker Orange CMTMRos (Molecular probes), and nuclei were observed by staining with Cyto staining (Molecular probes).

Chlorophyll autofluorescence was used as chloroplast marker. Expression of this fusion construct was observed using confocal microscopy (LSM 510 META, Carl Zeiss, Jena, Germany).

Yeast Complementarity Analysis

Hexokinase-deficient yeast triple mutant YSH7.4-3C ( hxk1 , hxk2 , glk1 ; De Winde et al. (1996) Eur J Biochem 241: 633-643) was depleted in the enzyme activity of OsHXK5 , OsHXK6 and these genes. Total length of the mutant allele was used for transformation with cDNAs. Yeast complementarity assay methods have been described previously (Cho et al. (2006) Planta 224: 598-611).

Transient Expression Analysis Using Corn and Rice Leaf Protoplasts

Corn leaf protoplasts (1-2 × 10 ⁵ cells / sample) were isolated from the second leaf of the sulfurized plant according to the method of Sheen (2001) (Plant Physiol 127: 1466-1475). mgh.harvard.edu/sheenweb). Rice protoplasts (3-6 × 10 ⁵ cells / sample) were isolated from the yellowed leaves by the modified Chen et al. (2006) method (Mol Plant Pathol 7: 417-427). The isolated protoplasts for transient expression analysis were simultaneously transfected with glucose reactive reporter constructs and working constructs by PEG-calcium mediated methods (Hwang and Sheen (2001) Nature 413: 383-389). ZmUBQ :: GUS was included in each sample as an internal control and was not affected by glucose treatment. Transfected protoplasts were incubated for 6 hours with 0.5 mM or 5 mM glucose and then harvested. Harvested protoplasts were suspended in lysis buffer and used for LUC and GUS analysis. LUC analysis was performed using the LUC analysis system (Promega), and GUS analysis was performed by the method described previously (Jefferson (1987) EMBO J 20: 3901-3907). Fluorescence generated by LUC and GUS activity was measured by the VICTOR2 1420 multilabel counter (PerkinElmer life sciences). In each sample, the measured LUC activity was divided by GUS activity for leveling variance data under experimental conditions, and all transient expression experiments were repeated three times with the same result. Expression of the effector protein in corn protoplasts transfected with OsHXK5, OsHXK6 or mutant alleles from which the enzymatic activity of these genes was removed was confirmed by protein-blot analysis using an anti-Myc antibody (Upstate, Clone A46).

Arabidopsis transformation

To prepare transgenic plants that overexpress OsHXK5, OsHXK6 and mutant alleles from which the enzymatic activity of these genes have been removed, the Agrobacterium tumerfaciens GV3101 strain comprising each vector construct contains 25 mg / L kanamycin. The LB liquid medium was incubated at 28 ° C. at 250 rpm until stationary, and the gin2-1 plants were transformed by the floral dip method described above (Clough and Bent (1998) Plant J 16: 735-743). All transgenic plants were selected in Gamborg B5 medium containing 25 mg / L hygromycin.

Rice Transformation

To prepare transgenic rice that overexpress OsHXK5 and OsHXK6 , Agrobacterium tumerfaciens LBA4404 strains containing the respective vector constructs were incubated for 3 days at 28 ° C. in AB medium containing 25 mg / L kanamycin. Rice transformation was performed with the Agrobacterium-mediated coculture method described above (Jeon et al. (2000) Plant J 22: 561-570). Transformed rice plants were re-differentiated from the transgenic callus in a selection medium comprising 50 mg / L hygromycin and 250 mg / L cytotaxy. Transgenic plants have been grown in greenhouses for generations to produce homozygous transgenic rice plants with OsHXK5 or OsHXK6 .

Glucose inhibition assay

For glucose inhibition assays in Arabidopsis, seedlings were grown for 6 days in 1/2 MS medium containing 6% glucose or mannitol. To examine the growth phenotype, transformants expressing OsHXK5, OsHXK6, or mutant alleles lacking the enzymatic activity of these genes, had low light conditions (70 μmol ⁻² s ⁻¹ ) and high light conditions (240 μmol ⁻² s ^{−). 1} ) in the soil for 18 days.

In rice, the shelled seeds of wild type and transgenic rice plants were sterilized for 10 minutes in 70% EtOH and 30 minutes in 0.8% NaOCl, respectively, and then washed with sterile distilled water. Surface-sterilized seeds were germinated in water agar medium without glucose or with 30 mM glucose and 30 mM sorbitol, respectively. For infiltration of sterile seeds, Petri dishes were placed for 24 hours in dark at 37 ° C. and then placed in the growth chamber at 25 ° C. under constant light for 7-10 days. Water agar medium was used instead of MS medium to rule out interference of sugar signaling reactions by nitrogen sources. To investigate the inhibition of the RbcS gene, RNA was isolated from the second and third leaves of seedlings.

RNA isolation and PCR analysis

Total RNA was isolated from seedlings using Trizol reagent and reverse transcribed using oligo-dT primers and First-Strand cDNA Synthesis Kit (Roche) for RT-PCR. In Arabidopsis plants, PCR was performed by Moore et al. (2003) method (Science 300: 332-336) using the following primers. CAB (At3g27690) is 5'-ATGGCCACTTCAGCAATCCAA-3 '(SEQ ID NO: 18) and 5'-CACAACTTGACACGCCCATAT-3' (SEQ ID NO: 19), SBP (At3g55800) is 5'-ATGGAGACCAGCATCGCGTG-3 '(SEQ ID NO: 20) and 5 '-CTTCCACTGGACCTCCCAT-3' (SEQ ID NO: 21), CAA (At5g14740) are 5'-TGAATACGCTGTCTTGCACC-3 '(SEQ ID NO: 22) and 5'-TGTGATGGTGGTGGTAGCGA-3' (SEQ ID NO: 23), ubiquitin4 ( UBQ , At5g20620) used primers 5'-GTGGTGCTAAGAAGAGGAAGA-3 '(SEQ ID NO: 24) and 5'-TCAAGCTTCAACTTCTTCTTT-3' (SEQ ID NO: 25).

For quantitative real-time PCR, gene specific PCR primers and fluorescent probes for TaqMan analysis were designed with Assays-by-Design Service (Applied Biosystems, Forester City, CA). Gene expression was analyzed according to manufacturer's instructions using TaqMan Universal PCR Master Mix and ABI PRISM 7000 Sequence Detector (Applied Biosystems). Gene specific primers and probes used for quantitative real-time PCR for the analysis of rice plants are as follows. RbcS was RbcS- using forward 5'-AGCAATGGCGGCAGGAT-3 '(SEQ ID NO: 26), RbcS- reverse 5'-GAACTTCTTGATGCCCTCAATCG-3' (SEQ ID NO: 27) and RbcS- probe FAM-CACACCTGCATGCACC-NFQ (SEQ ID NO: 28) , ubiquitin5 ( UBQ5 ) is UBQ5- forward 5'-CCGCCTCCGCAAGGA-3 '(SEQ ID NO: 29), UBQ5- reverse 5'-AAGTGGTTGGCCATGAAGGT-3' (SEQ ID NO: 30) and UBQ5- probe FAM-CCAACGCCGAGTGCG-NFQ (SEQ ID NO: 31 ) Was used. UBQ5 gene expression was used to level real-time PCR results (Jain et al. (2006) Biochem Biophys Res Commun 342: 645-651).

Example 1: Arabidopsis Glucose Sensor AtHXK1 Identification of Rice Hexokinase Equivalent to

The glucose sensor AtHXK1, which is known to be primarily associated with mitochondria, is known to be detectable to some extent in the nucleus and is known to bind glucose and work with their partners as transcriptional inhibitors. To determine the presence of an N-terminal free sequence comprising a mitochondrial targeting peptide to isolate rice homologous hexokinase of AtHXK1, the TargetP program (Emanuelsson et al. (2000) J Mol Biol 300: 1005-1016; http: //www.cbs.dtu.dk/services/TargetP) and predictNLS program (Cokol et al. (2000) EMBO Rep 1: 411-415; http: // cubic. The intracellular location of OsHXKs was estimated using bioc.columbia.edu/services/predictNLS). Of the 10 OsHXKs, OsHXK5 and OsHXK6 were the most reliable with 1MGKGTVVGTAVVVCAAAAAAVG. The two proteins also have 25RRRRR29 (SEQ ID NO: 34) or 28RRRDLELVEGAAAERKRK45 (SEQ ID NO: 35) for OsHXK5, the expected nuclear localization signal within the N-terminal domain, and 29RRRRSKREAEEERRRR44 (SEQ ID NO: 36) for OsHXK6. Indicated. Together with the phylogenetic analysis of rice HXKs earlier, this suggests that OsHXK5 and OsHXK6 are evolutionarily closely related to the Arabidopsis glucose sensor AtHXK1 .

To determine the intracellular location of the two rice homologous proteins for AtHXK1 , GFP fusions of OsHXK5 and OsHXK6 were prepared under CaMV35S promoter control. Intracellular localization experiments were confirmed by co-location experiments with MitoTracker, and the signals of OsHXK5-GFP and OsHXK6-GFP fusion proteins were mainly observed in mitochondria of corn protoplasts (Fig. 1) and mitochondria of data of Arabidopsis protoplasts (data not shown). This means that two hexokinases are associated with mitochondria. To investigate whether two OsHXKs have dual targeting capabilities in mitochondria and nuclei, we removed the expected N-terminal mitochondrial targeting peptide sequence and linked it to GFP . OsHXK mutants OsHXK5ΔmTP and OsHXK6ΔmTP were prepared. Interestingly, the signals of OsHXK5ΔmTP-GFP and OsHXK6ΔmTP-GFP were not observed in mitochondria but in the nucleus and cytoplasm as confirmed by co-location studies by Cyto staining (FIG. 2). The results suggest the possibility that OsHXKs can be targeted to the mitochondria and also targeted to the nucleus, increasing the likelihood that OsHXK5 and OsHXK6 are equivalent to Arabidopsis glucose sensors AtHXK1 .

Example 2: In the Protoplasts of Corn and Rice OsHXK5 , OsHXK6  And expression of mutant alleles of these genes

The sugar recognition and signaling function of AtHXK1 has not been shown to depend on its glucose phosphorylation activity (Moore et al. (2003) Science 300: 332-336). To determine their ability to sugar recognition and signaling from glucose kinase activity, targeted mutagenesis experiments were performed to prepare mutants from which the enzyme activity of OsHXK5 and OsHXK6, which are candidates for the rice glucose sensor, was removed. In mutant alleles, Gsp (D) conserved in the phosphate 1 domain of the ATP-binding site to eliminate ATP binding and Ser (S) conserved in the sugar-binding domain to prevent phosphoryl transfer are Asp (D) and Each to Ala (A) (Kraakman et al. (1999) Biochem J 343 Pt 1: 159-168). These mutant alleles were named OsHXK5-G113D , OsHXK5-S186A , OsHXK6-G112D , and OsHXK6-S185A depending on their mutation site (FIG. 3A). To determine if their enzymatic activity disappeared from the mutant allele, each cDNA clone in YSH7.4-3C ( hxk1 , hxk2 , glk1 ), a yeast triple mutant lacking endogenous hexokinase activity, had a complementary function. Tested. Yeast cells transformed with wild type cDNAs of OsHXK5 and OsHXK6 can grow in selection medium, while yeast cells transformed with OsHXK mutant allele or empty pDR196 vector did not grow in selection medium containing glucose as the only carbon source ( 3B). This indicates that the enzyme activity of the mutant OsHXKs is deficient.

Using glucose inhibition assay in corn and rice leaf protoplasts (Sheen, 2001), we found that two OsHXKs and alleles from which the enzyme activity of the gene was removed have glucose sensing and signaling in monocot plant species. Was tested. In the present invention, a reporter construct was constructed by linking the reporter gene luciferase ( LUC ) after the promoter of the small subunit gene ( ZmRbcS ) of maize ribulose-1,6-bisphosphate carboxylase, well known as a glucose inhibitory gene. . Expression of the rice amy3D ( RAmy3D ) gene encoding α-amylase is known to be rapidly suppressed in response to glucose treatment (Yu et al. (1996) Plant Mol Biol 30: 1277-1289). Thus, the RAmy3D promoter :: LUC fusion was constructed as an additional reporter construct. First, high glucose treatment (5 mM) decreased reporter gene expression linked to ZmRbcS or RAmy3D promoters in maize and rice leaf protoplasts, while not at low glucose treatment (0.5 mM) (FIGS. 4A and B). This supports previous experiments that transient gene expression analysis using lobe protoplasts is effective for the analysis of sugar detection and signaling (Sheen (2001) Plant Physiol 127: 1466-1475). Next, the expression of OsHXK5 or OsHXK6 dramatically reduced LUC expression induced by ZmRbcS or RAmy3D promoters in response to 0.5 mM glucose (FIG. 4, A and B), which was found in the corn and rice leaf protoplasts. It indicates that glucose-dependent inhibition of these genes has increased. Moreover, for OsHXK5 and OsHXK6 Expression of the OsHXK allele with enzyme activity removed suppressed expression of the reporter gene in response to glucose treatment (FIG. 4, A and B). CaMV35S :: OsHXK-Myc protein gel using a fusion construct-blot analysis shows that OsHXK5, OsHXK6 and mutant alleles thereof is expressed in a similar level in the mesophyll protoplasts (Figure 4C). In addition, these Myc fusion constructs are supported by the ZmRbcS or RAmy3D promoters. Glucose-dependent LUC expression has been shown to exhibit approximately the same inhibitory effect (data not shown). These results strongly suggest that OsHXK5 and OsHXK6 function as conserved glucose sensors in corn and rice.

Example 3: Transformation Expressing OsHXK5, OsHXK6, or Mutant Alleles thereof gin2-1 Analysis of plants

In order to investigate the possible roles of the two rice hexokinase isoforms OsHXK5 and OsHXK6 as glucose sensors, the present invention investigated whether these OsHXKs could complement Arabidopsis glucose insensitive2-1 ( gin2-1 ). To express OsHXK5, OsHXK6 and the enzymatic inactive mutant alleles OsHXK5-G113D , OsHXK5-S186A , OsHXK6-G112D and OsHXK6-S185A , cDNAs of genes were placed under the control of the CaMV35S promoter. The resulting constructs were transformed into gin2-1 mutants by the floral-dip method. At least 10 independent transgenic plants for each construct were selected in hygromycin resistant medium. The expression level of the transgene in the transgenic plant was measured by RNA gel-blot analysis (data not shown).

To determine if the OsHXK5, OsHXK6 and its mutant alleles restore glucose-sensitive responses in the gin2-1 background, the progeny of all selected transgenic gin2-1 plants with OsHXKs contain high glucose (6%). Planted in 1/2 MS medium. The growth of transgenic plants with any OsHXKs was shown to be rapidly inhibited in response to 6% glucose with short length of hypocotyl and anthocyanin accumulation (FIG. 5). All transgenic plants tested showed no difference in response to treatment with 6% mannitol. This means that high glucose effects are not attributable to osmotic stress in transgenic gin2-1 plants expressing OsHXK5, OsHXK6 or mutant alleles thereof.

Glucose sensor AtHXK1 is in response to high glucose treatment RbcS, chlorophyll a / b-binding protein 2 (CAB2), three roads heptul Ross-glucose, such as bis-phosphate (sedoheptulose-biphosphatase, SBP) and carbonic anhydrase (carbonic anhydrase, CAA) - It is widely known to inhibit dependent gene expression. In order to investigate whether rice OsHXKs can inhibit expression of target genes in a similar manner, mRNA expression levels of CAB , SBP and CAA genes in transgenic gin2-1 plants were examined (FIG. 5). The results showed that transgenic plants expressing wild type and OsHXK5, OsHXK6 or mutant alleles significantly inhibited photosynthetic gene expression. In contrast, gin2-1 did not inhibit glucose-dependent gene expression. These results indicate that any of these genes restores glucose-dependent gene expression in the gin2-1 background.

AtHXK1 was observed to have a function of growth enhancement by the growth deficient phenotype observed at high light conditions. To investigate whether the overexpression of hexokinase rice can complement the growth deficiency phenotype of gin2-1, OsHXK5, OsHXK6 or low gin2-1 a transgenic plant expressing the mutant allele thereof light conditions (70 μmolm ^-2 s ^-1 ) And under high light conditions (240 μmol ⁻² s ⁻¹ ). Under low light conditions, wild type, gin2-1 and transgenic plants did not show significant differences in their growth (FIG. 6). In contrast, gin2-1 plants exhibited a severe growth deficient phenotype under high light conditions, while transgenic plants expressing OsHXK5, OsHXK6 and their mutant alleles were able to restore plant growth and leaf expansion as wild type plants (FIG. 6). ). This shows that OsHXK5 and OsHXK6 can replace AtHXK1 's function in promoting growth in Arabidopsis.

Example 4: OsHXK5  or OsHXK6 Analysis of transgenic rice plants expressing

For a glucose sensor in rice plants to investigate further the ability of OsHXK5 and OsHXK6, it was produced in the transgenic rice plants expressing a CaMV35S :: OsHXK5 or CaMV35S :: OsHXK6. Two independent transgenic rice plants for each OsHXK gene were selected for further analysis based on high expression of the transgene (data not shown). Individuals from plants of homozygous conjugation of selected plants were germinated in water agar medium containing 30 mM glucose. Growth of transgenic rice seedling plants expressing OsHXK5 and OsHXK6 was more severely inhibited in glucose containing medium than in wild type rice plants (FIG. 7A). Transgenic rice plants have been shown to increase glucose-dependent growth inhibition such as pale yellow leaves and reduced plant length compared to wild type controls (FIGS. 7, A and B). In addition to phenotypic observation, expression of the rice RbcS gene was more sensitively inhibited by transgenic rice plants than wild type in response to glucose treatment (FIG. 7C). Sorbitol was treated as a control to eliminate the general effects caused by osmotic stress. In the results, no significant plant growth inhibition and RbcS gene expression could be observed when sorbitol was treated with rice. This means that the result obtained by the glucose treatment is not due to osmotic stress. Therefore, glucose-inhibitory experiments further support that OsHXK5 and OsHXK6 play the role of glucose sensors in rice plant as well as Arabidopsis gin2-1 mutant backgrounds.

1 is a diagram showing the intracellular location of OsHXK5-GFP and OsHXK6-GFP fusion proteins in transgenic maize protoplasts. A, OsHXK5-GFP. B, OsHXK6-GFP. C, GFP. Chlorophyll autofluorescence and MitoTracker were used as chloroplast and mitochondrial markers, respectively. Blue shows chlorophyll autofluorescence to distinguish chlorophyll from fluorescence of MitoTracker, green shows GFP signal and red shows mitochondrial signal stained with MitoTracker. It also showed light-field images as well as merged images of chlorophyll autofluorescence, GFP and MitoTracker.

2 shows the intracellular location of OsHXK5ΔmTP-GFP and OsHXK6ΔmTP-GFP fusion proteins in maize protoplasts. A, OsHXK5ΔmTP-GFP. B, OsHXK6 mTP-GFP. OsHXK5ΔmTP-GFP and OsHXK6ΔmTP-GFP fusion proteins are present in the cytoplasm as well as the nucleus. Chlorophyll autofluorescence and Cyto staining were used as chloroplast and nuclear markers respectively. Blue light was used as chlorophyll autofluorescence to distinguish chlorophyll from the fluorescence of Cyto staining. The GFP signal is green and the nuclear signal stained with Cyto stain is red. C, OsHXK5ΔmTP-GFP. No interaction between OsHXK5ΔmTP-GFP and mitochondria was detected. Similar results were seen with OsHXK6 mTP-GFP fusion protein (data not shown).

3 shows the results of transformation of the mutant from which the enzyme activity of OsHXK5 and OsHXK6 was removed into the yeast hexokinase mutant. A schematic of A, OsHXK5, OsHXK6 and mutagenesis with the agonist activity of these genes removed. Mitochondrial targeting signals and nuclear localization signals are shown as white (M) and black (N) rectangles. 1, 2 and A represent the adenosine interaction regions within the conserved phosphate 1, 2 and ATP-binding sites, respectively. Region S represents the conserved sugar binding domain. Complementary experiments of hexokinase-deficient yeast triple mutant YSH7.4-3C ( hxk1 , hxk2 , glk1 ) using B, OsHXK5, OsHXK6 and mutant alleles with depleted enzymatic activity of these genes. Transformed colonies were plated in medium containing 2% D-glucose as the only carbon source and incubated at 30 ° C. for 3 days. The YSH7.4-3C mutant strain transformed with pDR196 vector was used as a control.

Figure 4 is a result showing the expression of a response to the glucose processing CaMV35S promoter regulatory under AtHXK1, OsHXK5, OsHXK6 or OsHXK the mutant allele as a transient maize glucose response gene in mesophyll protoplast the Specifications ZmRbcS (A) and Ramy3D (B) . ZmUBQ :: GUS was included in each sample as an internal control and control protoplasts were transfected with empty vectors. Promoter activity of the glucose reactive reporter construct was shown as relative LUC / GUS activity and all transient expression experiments were repeated three times with similar results. C, constant expression of effector protein was detected by protein-blot analysis using anti-Myc antibodies.

5 shows the complementarity results of Arabidopsis gin2-1 mutants by expression of enzymatically inactive OsHXK5 and OsHXK6 mutant alleles. (Top) Seedlings of homozygous of the transgene, gin2-1 and wild type (WT), grown for 6 days in 1/2 MS medium containing 6% glucose or mannitol. (Bottom) Expression levels of CAB , SBP and CAA were analyzed by RT-PCR in transformants, gin2-1 and wild type plants. UBQ was used as a control.

FIG. 6 shows the complementarity results of the growth deficient phenotype of Arabidopsis gin2-1 due to overexpression of alleles in which enzyme activity of OsHXK5 and OsHXK6 was removed in the gin2-1 background. A, Results showing growth phenotypes of wild type (WT), gin2-1 and transgenic plants under low light conditions (70 μmol ⁻² s ⁻¹ ) or high light conditions (240 μmol ⁻² s ⁻¹ ). B, wild type (WT), gin2-1 and transgenic plants grown in low light and high light soils were compared to the phenotype of the sixth leaf of the same age.

7 shows OsHXK5 or OsHXK6 in response to glucose treatment. Gene-expressing Results show the growth phenotype of wild type (WT) and transformed rice seedlings. A, a growth phenotype of seedlings grown in water agar medium containing no glucose (0), 30 mM glucose (G30) and 30 mM sorbitol (S30), respectively. Rod = 1 cm. B, length of shoot of wild-type and transgenic rice plants grown in different media. Each data point represents the mean from three separate experiments (ㅁ SD). C, Relative expression of the rice RbcS gene in the second and third leaves of wild type grown in different media and overexpressed OsHXK5 and OsHXK6 . The expression value of seedlings grown on agar plate without glucose for each plant was arbitrarily set to 1.

<110> University-Industry Cooperation Group of Kyung Hee University <120> Use of OsHXK5 gene as glucose sensor <130> PN08131 <160> 36 <170> KopatentIn 1.71 <210> 1 <211> 2058 <212> DNA <213> Oryza sativa <400> 1 ctctcgtcct cctttctcct acgcggccgg agagaggagg ggggaagaga ggatctcgca 60 gagcgccata gcctcggatc cagaagcgga attcggtgga ggtctcgggc acctggatcg 120 atcggaggaa gggaaggcgg agcagcggtg atggggaagg cggcggcggt ggggacggcg 180 gtggtggtgg ccgcggcggt cggggtggcg gtggtgctgg cgcggaggcg gaggaggagg 240 gacctggagc tggtggaggg agccgcggcg gagaggaaga ggaaggtggc ggcggtgatc 300 gaggacgtgg agcacgcgct gtcgaccccg acggcgctgc tgcggggcat ctcggacgcc 360 atggtcaccg agatggagcg aggcctgcgc ggggacagcc acgccatggt taagatgctc 420 atcacctacg tcgacaacct ccccaccgga aatgaacagg ggttgtttta tgcattggat 480 cttggaggaa ccaacttccg cgtcctgcga gtccaactcg gtggcaagga gaaacgtgtc 540 gtccaacaac agtacgagga agtctcaatt ccaccacatc tgatggttgg aacttccatg 600 gaactgtttg attttattgc ttctgcattg tcaaaatttg ttgatactga aggtgatgat 660 ttccacctcc cagaagggag acagagagag ctgggcttca ccttttcctt cccagtgagc 720 cagacatcaa tatcgtcagg aacgctcatc aagtggacaa agggtttctc catcaatgac 780 gcggttggcg aagatgttgt atctgagttg ggcaaggcca tggagaggca gggattagat 840 atgaaaattg cagcattggt taatgacact gtcggcacat tggctggtgg gaggtatgcg 900 gataacagtg ttgttgctgc tataatattg ggcactggta caaatgcagc atatgttgag 960 aatgctaatg caattcctaa atggaccggt ttactgccta ggtccggaaa tatggtaatc 1020 aacacagaat gggggagctt taaatcagat aagcttcctc tttcagaatt cgataaagca 1080 atggattttg aaagcttgaa tcctggagag cagatatatg aaaagttgat ttctggcatg 1140 tatcttggag agatagtgcg aagaatcttg ctgaagcttg ctcatgatgc agctttgttt 1200 ggggatgttg ttccatctaa gctagagcaa ccgtttgtac taaggacacc ggatatgtca 1260 gccatgcatc atgactcgtc acatgacctt aaaactgttg gagctaagct aaaggatatc 1320 gtcggggtcc cagatacttc cctggaagta agatacatta ccagtcacat ttgtgacata 1380 gttgcagagc gtgctgcacg cttggctgct gctggcatat atggggtcct aaagaagcta 1440 ggtcgggaca agatgccaaa agacggcagt aagatgccta ggactgtcat tgccttggat 1500 ggtgggctct atgaacatta caagaagttc agcagttgct tagaatcaac tctaacagac 1560 cttcttgggg atgatgtctc gtcttcggtg gttaccaagc tggccaacga tggttctggc 1620 attggagctg ctcttctcgc agcctcgcac tcccagtatg ccgagatcga ctagctttaa 1680 ggatgatctt gatgaatgat gaatcaaact ccgtttgtag gttctcattt cccccttcaa 1740 aatccacata atactcctgg ctcccccctt gaaatcttac catctttttt tggctattct 1800 gagggcaaac ataagtgcct ctgcagcggg atatagctag tatagcgcca atgagtttgg 1860 aggttttcta atggcataaa acgttggatg gcagtagcag actaacaggg aaatggaggc 1920 acaggcaatt tccattcctg ttctgtcaga ttcttttccc ccttaattga tgttgagaac 1980 caagattttt ttgctctgta ttttctcttc gtaataaaga aggggacata atctaattgc 2040 tcttgtttga tctcataa 2058 <210> 2 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Forward primer <400> 2 gctctagaag ggaaggcgga gcagcggtg 29 <210> 3 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer <400> 3 ccctcgagag tcgatctcgg catactggga 30 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Forward primer <400> 4 gctctagagg aaggaggagg agtaggacgc 30 <210> 5 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer <400> 5 ccctcgagac tcgacgctag catactggga 30 <210> 6 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Forward primer <400> 6 gctctagaat gcggaggcgg aggaggaggg ac 32 <210> 7 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Forward primer <400> 7 gctctagaat gcggaggagg aggagcaagc gg 32 <210> 8 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Forward primer <400> 8 ggaagttggt gtctccaaga tccaatgcat 30 <210> 9 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer <400> 9 gatcttggag acaccaactt ccgcgtcctg 30 <210> 10 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Forward primer <400> 10 cactgggaag gcaaaggtga agcccagctc 30 <210> 11 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer <400> 11 gcttcacctt tgccttccca gtgagccaga 30 <210> 12 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Forward primer <400> 12 ggaaattggt gtccccaaga tcgagagcat 30 <210> 13 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer <400> 13 cgatcttggg gacaccaatt tccgtgttat 30 <210> 14 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Forward primer <400> 14 cactgggaaa gcaaaggtga agcctaactc 30 <210> 15 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer <400> 15 gcttcacctt tgctttccca gtgcaccaaa 30 <210> 16 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Forward primer <400> 16 cgggatccga tcttcaacca cctgtgctag ct 32 <210> 17 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer <400> 17 tgccatggat ctgtgtaagc tgaaaccgtg tt 32 <210> 18 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Forward primer <400> 18 atggccactt cagcaatcca a 21 <210> 19 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer <400> 19 cacaacttga cacgcccata t 21 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer <400> 20 atggagacca gcatcgcgtg 20 <210> 21 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer <400> 21 cttccactgg acctcccat 19 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer <400> 22 tgaatacgct gtcttgcacc 20 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer <400> 23 tgtgatggtg gtggtagcga 20 <210> 24 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Forward primer <400> 24 gtggtgctaa gaagaggaag a 21 <210> 25 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer <400> 25 tcaagcttca acttcttctt t 21 <210> 26 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Forward primer <400> 26 agcaatggcg gcaggat 17 <210> 27 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer <400> 27 gaacttcttg atgccctcaa tcg 23 <210> 28 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> RbcS probe <400> 28 cacacctgca tgcacc 16 <210> 29 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Forward primer <400> 29 ccgcctccgc aagga 15 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer <400> 30 aagtggttgg ccatgaaggt 20 <210> 31 <211> 15 <212> DNA <213> Artificial Sequence <220> 223 UBQ5 probe <400> 31 ccaacgccga gtgcg 15 <210> 32 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> Mitochondria targeting peptide for OsHXK5 <400> 32 Met Gly Lys Ala Ala Ala Val Gly Thr Ala Val Val Val Ala Ala Ala   1 5 10 15 Val Gly Val Ala Val Val Leu Ala              20 <210> 33 <211> 28 <212> PRT <213> Artificial Sequence <220> <223> Mitochondria targeting peptide for OsHXK6 <400> 33 Met Gly Lys Gly Thr Val Val Gly Thr Ala Val Val Val Cys Ala Ala   1 5 10 15 Ala Ala Ala Ala Val Gly Val Ala Val Val Val Ser              20 25 <210> 34 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Nuclear localization signal for OsHXK5 <400> 34 Arg Arg Arg Arg Arg   1 5 <210> 35 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Nuclear localization signal for OsHXK5 <400> 35 Arg Arg Arg Asp Leu Glu Leu Val Glu Gly Ala Ala Ala Glu Arg Lys   1 5 10 15 Arg lys         <210> 36 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Nuclear localization signal for OsHXK6 <400> 36 Arg Arg Arg Arg Ser Lys Arg Glu Ala Glu Glu Glu Arg Arg Arg Arg   1 5 10 15  

Claims (12)

delete delete delete delete delete delete delete delete delete Transforming the plant with an OsHXK5 gene consisting of the nucleotide sequence of SEQ ID NO: 1; And Method for determining the glucose detection ability of the OsHXK5 gene according to the plant comprising the step of measuring the growth of the plant in a glucose-containing medium. Method of controlling the growth of a plant comprising the step of transforming the plant OsHXK5 gene consisting of the nucleotide sequence of SEQ ID NO: 1. 12. The method of claim 11, wherein the method comprises the step of transforming and overexpressing the OsHXK5 gene into a plant.

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