WO2013125835A1 - Protéine atpg3 ayant une capacité à augmenter la productivité, une capacité à retarder la sénescence et une capacité à conférer aux plantes une résistance au stress, gène de la protéine atpg3 et leur utilisation - Google Patents

Protéine atpg3 ayant une capacité à augmenter la productivité, une capacité à retarder la sénescence et une capacité à conférer aux plantes une résistance au stress, gène de la protéine atpg3 et leur utilisation Download PDF

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WO2013125835A1
WO2013125835A1 PCT/KR2013/001311 KR2013001311W WO2013125835A1 WO 2013125835 A1 WO2013125835 A1 WO 2013125835A1 KR 2013001311 W KR2013001311 W KR 2013001311W WO 2013125835 A1 WO2013125835 A1 WO 2013125835A1
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gene
atpg3
plant
seq
aging
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Korean (ko)
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이동희
이인철
김국진
김동수
박경목
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제노마인
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8209—Selection, visualisation of transformants, reporter constructs, e.g. antibiotic resistance markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
    • C12N15/8266—Abscission; Dehiscence; Senescence
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the present invention relates to an ATPG3 protein having its productivity enhancing function, delaying aging function and stress resistance function, a gene thereof and use thereof.
  • Plant senescence is the last step in determining the longevity of a plant's developmental process and is a genetic phenomenon that is a very sophisticated and active life phenomenon at the cellular, tissue, organ and individual level.
  • the chloroplasts which make up 70% of the leaf proteins, are gradually degraded as they degrade, and proteins, membrane lipids, RNA, and other cellular structures or macromolecules are sequentially degraded to lose cell homeostasis. Leads to The products of cell breakdown produced during aging are transferred to new organs or storage organs such as seeds and reused as plant nutrients.
  • the present invention also discloses a gene having a function of delaying aging and the like, which can improve the productivity of crops and the like.
  • the present invention relates to an ATPG3 protein having a productivity enhancing function of a plant, a delaying aging function, and a stress resistance function.
  • the inventor isolates the gene of the protein based on the sequence of AT-HOOK MOTIF NUCLEAR-LOCALIZED PROTEIN (GeneBank accession number NP_566232.1), as confirmed in the following example
  • productivity increase characteristics such as the increase in biomass and / or seed productivity of the individual are apparent
  • the aging delay of the plant is apparent
  • the resistance to drought stress or oxidative stress is also obvious. It was confirmed.
  • the present inventors have named the gene to gene and protein ATPG3 ATPG3 (AT -hook p rotein of G enomine 3), these nucleotide sequences and amino acid sequences are disclosed in SEQ ID NOS: 1 and 2, respectively.
  • the ATPG3 protein of the invention is one of the polypeptides of (a), (b) and (c) below.
  • a "polypeptide comprising a substantial portion of the amino acid sequence set forth in SEQ ID NO: 2" is still sufficient to retain plant aging delay and productivity enhancing functions as compared to the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: And a polypeptide comprising a portion of the amino acid sequence of SEQ ID 2 of.
  • the length of the polypeptide and the degree of activity of such a polypeptide does not matter since it still needs to be sufficient to retain the function of delaying aging and increasing productivity of the plant.
  • polypeptide As such a polypeptide, the polypeptide which deleted the N-terminal part or C-terminal part in the polypeptide containing the amino acid sequence of SEQ ID NO: 2 is mentioned. This is because it is generally known in the art that even if the N- or C-terminal portion is deleted, such a polypeptide has the function of the original polypeptide. Of course, in some cases, the N-terminal or C-terminal moiety is necessary for maintaining the function of the protein, so that a polypeptide deleted with the N-terminal or C-terminal moiety does not exhibit this function. It is within the ordinary skill of one of ordinary skill in the art to distinguish and detect inactive polypeptides from active polypeptides.
  • polypeptide comprising one or more substituted amino acids may be used in plant aging. It is included in the present invention as long as it has a delay function and a productivity increase function. Even if one or more amino acids are substituted, if the amino acid before substitution is chemically equivalent to the substituted amino acid, the polypeptide comprising such substituted amino acid will still retain the function of the original polypeptide.
  • polypeptide having such substituted amino acid (s) will still retain the function of the original polypeptide even if its activity is low. will be.
  • a polypeptide comprising amino acid (s) substituted at the N-terminal or C-terminal portion of the polypeptide will still retain the function of the original polypeptide.
  • Those skilled in the art can produce a polypeptide comprising at least one substituted amino acid as described above, while still retaining the aging delaying function and productivity enhancing function of the polypeptide comprising the amino acid sequence of SEQ ID NO: 2.
  • polypeptide substantially similar to the polypeptide of (a) or (b) above should be understood as meaning including all polypeptides that contain one or more substituted amino acids but still have the ability to delay aging and increase productivity of plants.
  • polypeptide substantially similar to the polypeptide of (a) or (b) is meant to include all polypeptides that contain one or more substituted amino acids but still have the ability to delay aging and increase productivity of plants, but nevertheless active
  • the polypeptide is preferably higher in sequence homology with the amino acid sequence of SEQ ID NO.
  • polypeptides substantially similar to the polypeptides of (a) and (b) above are not only “polypeptides substantially similar to polypeptides comprising the entire amino acid sequence of SEQ ID NO: 2", All descriptions above are intended to include “substantially similar to polypeptides comprising the entire amino acid sequence of SEQ ID NO: 2" as well as “amino acids of SEQ ID NO: 2", since the polypeptide comprising substantially part thereof is included. The same applies to polypeptides that are substantially similar to polypeptides comprising substantial portions of the sequence.
  • polypeptide as described above refers to a polypeptide comprising the entire amino acid sequence of SEQ ID NO: 2, a polypeptide comprising a substantial portion of the amino acid sequence of SEQ ID NO: 2, having a function of delaying aging and increasing productivity of a plant, and In addition to including polypeptides substantially similar to the above polypeptides, it is meant to include all polypeptides of the preferred embodiments as described above.
  • the polynucleotide of the present invention is substantially isolated from the isolated polynucleotide and the polypeptides encoding the polypeptide including all or a substantial part of the amino acid sequence described in SEQ ID NO: 2, while having a function of delaying aging and increasing productivity of plants.
  • Encoding an isolated polypeptide includes an isolated polynucleotide, and furthermore, in a preferred embodiment, an isolation that encodes all polypeptides having the sequence homology in the sequence sequence homology as described above, with the ability to delay aging and increase productivity of plants.
  • Polynucleotides When amino acid sequences are found, those skilled in the art can readily prepare polynucleotides encoding such amino acid sequences based on those amino acid sequences.
  • isolated polynucleotide herein includes both chemically synthesized polynucleotides, polynucleotides isolated from organisms, especially Arabidopsis thaliana , and polynucleotides containing modified nucleotides, and are either single stranded or double stranded. It is defined as including all polymers of stranded RNA or DNA.
  • the present invention relates to a method for producing a plant with delayed aging.
  • aging delay refers to a property of prolonged plant life as compared to wild-type plants, and specifically, the yellowing and / or necrosis of leaves and / or stems is delayed compared to wild-type plants or the chlorophyll content of plants is wild-type. It is more characteristic than plants or photosynthetic efficiency of plants is higher than wild type plants.
  • plant is meant to include mature plants, immature plants (plants), plant seeds, plant cells, plant tissues and the like.
  • plant cells or plant tissues are described in European Patent EP0116718, European Patent EP0270822, International Patent WO 84/02913, Gould et al. 1991, Plant Physiol 95,426-434, etc., can be used to develop and grow into mature plants.
  • plant includes all plants for which delayed aging can give useful results to humans.
  • the delay in aging is directly related to an increase in production, i.e. an increase in seed productivity and / or biomass of an individual, so in the sense of the above plants, productivity is primarily useful for human crops such as rice, wheat, barley, corn, soybeans, potatoes and red beans.
  • gene consisting of a sequence similar to the nucleotide sequence of SEQ ID NO: 1 is a gene encoding the first amino acid of SEQ ID NO: 2 while having a base sequence different from that of the gene of SEQ ID NO: 1 due to codon degeneracy
  • genes consisting of the nucleotide sequence of SEQ ID NO: 1 all of the nucleotide sequence of SEQ ID NO: 1 and other nucleotide sequences due to the evolutionary pathways different according to the type of plant, having the aging delay function of the plant It is meant to include genes.
  • the gene consisting of a sequence similar to the nucleotide sequence of SEQ ID NO: 1 is preferably higher in sequence homology with the nucleotide sequence of SEQ ID NO: 1, and most preferably, having 100% sequence homology.
  • the gene has a sequence homology of 60% or more with the nucleotide sequence of SEQ ID NO: 1.
  • sequence homology is 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73 %, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, Higher in order of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% is preferred.
  • overexpression means expression above the level expressed in wild-type plants.
  • Such “overexpression” can be directly determined by quantifying the gene of SEQ ID NO: 1 or a gene consisting of a sequence similar to the nucleotide sequence of SEQ ID NO: 1, or indirectly by quantifying the protein encoded by the gene. have. It can also be confirmed by the phenotype that appears according to the characteristics of the gene.
  • the step (a) may be performed by a genetic engineering method.
  • Genetic engineering method includes the steps of: (i) inserting a gene having the sequence of SEQ ID NO: 1 or a sequence similar to the sequence of SEQ ID NO: 1 into an expression vector to be operably linked to a regulatory sequence capable of overexpressing it, and ( ii) transforming the expression vector into a plant.
  • operably means that the transcription and / or translation of a gene is linked to be affected. For example, if a promoter influences the transcription of a gene linked to it, the promoter and the gene are operably linked.
  • regulatory sequence is meant to include all sequences whose presence may affect the transcription and / or translation of a gene linked thereto, and such regulatory sequences include a promoter sequence and a polyadenylation signal. ), The replication start point.
  • promoter follows the conventional meaning known in the art, specifically located upstream (5 'side) based on the transcription initiation point of a gene and binding to DNA-dependent RNA polymerase.
  • nucleic acid sequences having the function of controlling transcription of one or more genes including sites, transcriptional initiation sites, transcription factor binding sites, and the like.
  • Such a promoter may be a TATA box upstream of the transcription initiation point (usually at the transcription initiation point (+1) -20 to -30 position), CAAT box (usually approximately -75 position relative to the transcription initiation site if it is of eukaryotes) Present), a 5 'enhancer, a transcription repression factor, and the like.
  • Usable promoters include constitutive promoters (promoters which induce constant expression in all plant tissues), inducible promoters (expression of target genes in response to specific external stimuli) as long as they are promoters capable of overexpressing the gene of SEQ ID NO. 1 linked thereto. Promoters that induce expression or promoters that specifically induce expression in specific developmental periods or specific tissues).
  • constitutive promoters include the promoter of the 35S RNA gene of cauliflower mosaic virus (CaMV), and the ubiquitin family of promoters (Christensen et al., 1992, Plant Mol). Biol. 18, 675-689; EP0342926; Cornejo et al., 1993, Plant Mol. Biol.
  • rice actin promoter Zhang et al. 1991, The Plant Cell 3, 1155-1165
  • rice actin promoter Zhang et al. 1991, The Plant Cell 3, 1155-1165
  • inducible promoters include the yeast metallothionein promoter (Mett et al., Proc. Natl. Acad. Sci., USA, 90: 4567, 1993), which is activated by copper ions, substituted by substituted benzenesulfonamides.
  • In2-1 and In2-2 promoters (Hershey et al., Plant Mol. Biol., 17: 679, 1991), GRE regulatory sequences regulated by glucocorticoids (Schena et al., Proc. Natl. Acad.
  • the transcription termination sequence is a sequence that acts as a poly (A) addition signal (polyadenylation signal) to enhance the integrity and efficiency of transcription.
  • transcription termination sequences include the transcription termination sequence of the nopaline synthase (NOS) gene, the transcription termination sequence of the rice ⁇ -amylase RAmy1 A gene, and the transcription termination of the Octopine gene of Agrobacterium tumefaciens.
  • NOS nopaline synthase
  • the expression vector may include a selection marker gene.
  • marker gene refers to a gene encoding a trait that enables the selection of a plant comprising such a marker gene.
  • the marker gene may be an antibiotic resistance gene or may be a herbicide resistance gene.
  • suitable selectable marker genes include genes of adenosine deaminase, genes of dihydrofolate reductase, genes of hygromycin-B-phosphortransferase, genes of thymidine kinase, genes of xanthine-guanine phosphoribosyltransfer Laze gene, phosphinnothricin acetyltransferase gene, etc. are mentioned.
  • the term "transformation” refers to a modification of the genotype of a host plant by the introduction of a hereditary gene, and regardless of the method used for the transformation, the herb gene is a host plant, more precisely a cell of the host plant. Introduced into and integrated into the genome of a cell.
  • the hereditary genes include homologous and heterologous genes, wherein “homologous genes” refer to endogenous genes of a host organism or the same species, and “heterologous genes” are genes that do not exist in the organism to which they are transformed.
  • Arabidopsis derived genes are homologous to Arabidopsis plants, but heterologous to tomato plants.
  • a method of transforming a plant with an exogenous gene may use a method known in the art, such as a direct gene transfer method using a gene gun, an in planta transformation method using a floral dip, pollen mediation, and the like. Transformation methods, protoplast transformation methods, viral mediated transformation methods, liposome mediated transformation methods, and the like can be used.
  • a transformation method suitable for a specific plant for example, a method for transforming corn is described in US Pat. No.
  • Generally used in transforming plants is a method of infecting seedlings, plant seeds and the like with the transformed Agrobacterium.
  • Such Agrobacterium mediated transformation methods are well known in the art (Chilton et al., 1977, Cell 11: 263: 271; European Patent EP 0116718; US Patent US 4,940,838), and methods suitable for particular plants are also known in the art.
  • the Agrobacterium mediated transformation method uses Ti-plasmid, which will contain left and right border sequences that allow the integration of T-DNA into the genome of plant cells.
  • the (b) screening step is to develop and grow the transformed plant, and to visually select through the progress of the leaf yellowing or the progression of leaf necrosis, or when the selection marker gene is transformed
  • the selection may be performed using a selection marker gene, and further, may be selected through a method of quantifying chlorophyll content, photosynthetic efficiency, and the like, and a method of mixing the above methods.
  • the present invention relates to a method for producing a plant having productivity enhancing properties of the present invention.
  • Method for producing a plant having a productivity enhancing feature of the present invention comprises the steps of (a) overexpressing a gene having a nucleotide sequence of SEQ ID NO: 1 or a gene having a sequence similar to the nucleotide sequence of SEQ ID NO: 1 in the plant and (b) increased productivity And selecting the plant having the characteristic.
  • productivity enhancing properties means that the biomass (size and / or mass) of the whole, stem, root and / or leaves of the plant is increased compared to wild type plants and / or the productivity of the seed of the plant (plant 1). Number and / or mass of seeds per individual) is increased compared to wild type plants.
  • Step (a) may be carried out genetically, as described above with respect to the method for producing a aging delayed plant of the present invention for this genetic engineering method.
  • the step (b) may be selected by comparing the biomass and / or seed productivity of the plant, or when the selection marker gene is transformed together at the time of transformation, may be selected using the selection marker gene, or a method thereof It may be selected by mixing.
  • the present invention relates to a method for producing a stress resistant plant.
  • the method of producing a stress resistant plant of the present invention comprises the steps of: (a) overexpressing a gene having the nucleotide sequence of SEQ ID NO: 1 or a gene having a sequence similar to the nucleotide sequence of SEQ ID NO: 1 in the plant, and (b) having a stress resistant phenotype Comprising plant screening.
  • stress refers to drought stress and / or oxidative stress.
  • Step (a) may be performed genetically, as described above with respect to the method for producing an aging delayed plant of the present invention for this genetic engineering method.
  • the step (b) is selected by comparing the stress resistance of the plant (e.g., the progress of leaf sulfidation, the progression of leaf necrosis, the biomass of the leaves and / or stems, chlorophyll content, photosynthetic efficiency, etc.)
  • the selection marker gene When the selection marker gene is transformed together at the time, the selection marker gene may be used for selection, or a combination thereof may be used for selection.
  • the present invention relates to a method for delaying aging of a plant.
  • the method for delaying aging of a plant of the present invention is to (a) operably link a gene having a nucleotide sequence of SEQ ID NO: 1 or a gene having a sequence similar to that of SEQ ID NO: 1 to a regulatory sequence capable of overexpressing it. Inserting into the expression vector and (b) transforming the expression vector into a plant.
  • the present invention relates to a method for increasing productivity of a plant.
  • the method for increasing the productivity of a plant of the present invention includes (a) an expression vector such that a gene having a nucleotide sequence of SEQ ID NO: 1 or a gene having a sequence similar to that of SEQ ID NO: 1 is operably linked to a control sequence capable of overexpressing it And (b) transforming the expression vector into a plant.
  • the present invention relates to a method for increasing stress resistance of a plant.
  • the method of increasing the stress resistance of a plant of the present invention is (a) operably linking a gene having a nucleotide sequence of SEQ ID NO: 1 or a gene having a sequence similar to that of SEQ ID NO: 1 to a regulatory sequence capable of overexpressing it Inserting into the expression vector preferably and (b) transforming the expression vector into a plant.
  • Steps (a) and (b) in the above methods are the same as those described in connection with the method for producing the delayed aging plant of the present invention.
  • the present invention is the delayed aging of the gene having a nucleotide sequence of SEQ ID NO: 1 or a gene having a sequence similar to the nucleotide sequence of SEQ ID NO: 1 obtained by the method for producing an aging delayed plant of the present invention
  • the present invention relates to a transgenic plant having characteristics.
  • the plant is a transgenic plant having delayed aging characteristics by being introduced into a gene encoding the ATPG3 protein consisting of the amino acid sequence of SEQ ID NO: 2, in particular, a gene having a nucleotide sequence of SEQ ID NO: 1 and overexpressed.
  • the present invention is an overexpression of a gene having a nucleotide sequence of SEQ ID NO: 1 or a gene having a sequence similar to the nucleotide sequence of SEQ ID NO: 1 obtained by the method for producing a plant having a productivity enhancing feature of the present invention
  • the present invention relates to a transgenic plant having improved productivity.
  • the plant is a transgenic plant having productivity enhancing properties by introducing and overexpressing a gene encoding the ATPG3 protein consisting of the amino acid sequence of SEQ ID NO: 2, in particular the gene ATPG3 having the nucleotide sequence of SEQ ID NO: 1.
  • the present invention provides an increase in productivity in which a gene having a nucleotide sequence of SEQ ID NO: 1 or a gene having a sequence similar to the nucleotide sequence of SEQ ID NO: 1 obtained by the method for preparing a stress resistant plant of the present invention is overexpressed.
  • the present invention relates to a transgenic plant having characteristics.
  • the plant is a transgenic plant having stress resistance by introducing and overexpressing a gene encoding the ATPG3 protein consisting of the amino acid sequence of SEQ ID NO: 2, in particular, the gene ATPG3 having the nucleotide sequence of SEQ ID NO: 1.
  • the "transformed plant” refers to a plant cell, a plant tissue, or a plant seed capable of developing and growing as a mature plant, when the gene is introduced and transformed, as well as by mating with the transformed plant.
  • the resulting genome includes modified plants, plant seeds, plant cells.
  • the ATPG3 protein and its gene which have the function of increasing the productivity of the plant and also have the function of delaying aging and stress resistance. Since the gene provides a function of increasing productivity and delaying aging and stress resistance of the plant, when the plant is transformed with the gene, not only can the productivity be increased, but also a plant having the aging delay and stress resistance of the plant can be produced. .
  • Figure 1 shows the structure (schematic) of the pCSEN-ATPG3 recombinant vector in which the ATPG3 gene, which has a function of increasing productivity of plants and has a aging delay and stress resistance function, is introduced in the sense direction.
  • Figure 2 is a photo of the Arabidopsis grown 50 days and 70 days after germination of the Arabidopsis T 2 line transformed with the pCSEN-ATPG3 recombinant vector of FIG.
  • ATPG3 ox-1 Arabidopsis T 2 line transformed with pCSEN-ATPG3 recombinant vector
  • ATPG3 ox-2 Arabidopsis T 2 line transformed with pCSEN-ATPG3 recombinant vector
  • ATPG3 ox-8 Arabidopsis T 2 line transformed with pCSEN-ATPG3 recombinant vector
  • Figure 3 shows the results of analyzing the expression of the ATPG3 gene of Arabidopsis thalass cultivated for 20 days after cotyledon generation of the Arabidopsis T 2 line transformed with the pCSEN-ATPG3 recombinant vector of Figure 1 through qRT-PCR will be.
  • ATPG3 ox-1 Arabidopsis T 2 line transformed with pCSEN-ATPG3 recombinant vector
  • ATPG3 ox-2 Arabidopsis T 2 line transformed with pCSEN-ATPG3 recombinant vector
  • ATPG3 ox-8 Arabidopsis T 2 line transformed with pCSEN-ATPG3 recombinant vector
  • Figure 4 shows the results of analyzing the expression of the ATPG3 gene in various plant organs of Arabidopsis wild type through qRT-PCR.
  • FIG. 5 is a diagram for increasing the productivity of the Arabidopsis line of FIG.
  • ATPG3 ox-1 Arabidopsis T 2 line transformed with pCSEN-ATPG3 recombinant vector
  • ATPG3 ox-2 Arabidopsis T 2 line transformed with pCSEN-ATPG3 recombinant vector
  • ATPG3 ox-8 Arabidopsis T 2 line transformed with pCSEN-ATPG3 recombinant vector
  • Height height
  • NTS long shell and number
  • Dry-W biodry weight
  • TSW total seed weight
  • TNS total seed number
  • 1,000SW 1,000 seed weight
  • FIG. 6 shows every 4-4 leaves of the Arabidopsis wild-type (Con), delayed aging-induced variants ATPG3 ox-1, ATPG3 ox-2 , and ATPG3 ox-8 from day 12 after cotyledon production. A picture of the phenotype of a leaf observed up to 40 days per day.
  • FIG. 7 shows the Arabidopsis wild-type (Con), aging-induced mutants ATPG3 ox-1, ATPG3 ox-2 , and ATPG3 ox-8 from 3-4 days after cotyledon generation.
  • Figure shows the chlorophyll content of leaves for up to 40 days per day.
  • FIG. 8 shows every 4-4 leaves of the Arabidopsis wild-type (Con), delayed senescence induced variants ATPG3 ox-1, ATPG3 ox-2 , and ATPG3 ox-8 from day 12 after cotyledon production.
  • the photosynthetic efficiency of leaves up to 40 days per day was investigated by Fv / Fm.
  • FIG. 9 shows every 4-4 leaves of the Arabidopsis wild-type (Con), delayed senescence-induced variants ATPG3 ox-1, ATPG3 ox-2 , and ATPG3 ox-8 from day 12 after cotyledon production.
  • Expression of senescence marker genes in leaves up to 40 days per day was analyzed by qRT-PCR, and ACT2 was used as a PCR positive control.
  • CAB2 is a chlorophyll a / b binding protein gene
  • SEN4 and SAG12 are aging genes and aging marker genes.
  • FIG. 11 shows detachment of the left lobe 3-4 of the Arabidopsis wild-type (Con), delayed aging induced variants ATPG3 ox-1, ATPG3 ox-2 , and ATPG3 ox-8 at 21 days after germination to maintain cancer status.
  • Figure 12 detaches the left lobe of the Arabidopsis wild-type (Con), delayed aging-induced variants ATPG3 ox-1, ATPG3 ox-2 , and ATPG3 ox-8 3 days after germination to maintain cancer status. This is a picture of Fv / Fm of photosynthetic efficiency of leaves up to 12 days every 2 days.
  • FIG. 13 shows detachment of the left lobe of the Arabidopsis wild-type (Con), ATPG3 ox-1, ATPG3 ox-2 , and ATPG3 ox-8 3 days after germination, to maintain cancer status.
  • the expression patterns of the aging marker genes of the leaves were analyzed by qRT-PCR up to 12 days every 2 days, and ACT2 was used as a PCR positive control.
  • CAB2, SEN4, and SAG12 are aging marker genes.
  • FIG. 14 shows that ATPG3 ox-1, ATPG3 ox-2 , and ATPG3 ox-8 treated with Arabidopsis wild-type or control (Con) and delayed aging at 30 days after germination were treated with drought for 14 days.
  • the figure shows the phenotypic change of.
  • ATPG3 ox-1 Arabidopsis T 2 line transformed with pCSEN-ATPG3 recombinant vector
  • ATPG3 ox-2 Arabidopsis T 2 line transformed with pCSEN-ATPG3 recombinant vector
  • ATPG3 ox-8 Arabidopsis T 2 line transformed with pCSEN-ATPG3 recombinant vector
  • FIG. 15 shows drought treatment of ATPG3 ox-1, ATPG3 ox-2 , and ATPG3 ox-8 for 14 days after drought treatment with Arabidopsis wild-type or control (Con) and delayed aging at 30 days after germination, and 7 and 14 days.
  • the figure shows the weight change of the plant leaves that occurred during the period.
  • Figure 16 is to detach the germinated after 25 days Arabidopsis thaliana wild-type or control (Con) and aging delays induced mutant ATPG3 ox-1, ATPG3 ox- 2, and three to four times the left lobe of ATPG3 ox-8 6 H 2 ilgan Figure shows the phenotype change of leaves treated with O 2 .
  • ATPG3 ox-1 Arabidopsis T 2 line transformed with pCSEN-ATPG3 recombinant vector
  • ATPG3 ox-2 Arabidopsis T 2 line transformed with pCSEN-ATPG3 recombinant vector
  • ATPG3 ox-8 Arabidopsis T 2 line transformed with pCSEN-ATPG3 recombinant vector
  • FIG. 20 shows detachment of the left lobe 3-4 of the ATPG3 ox-1, ATPG3 ox-2 , and ATPG3 ox-8 induced Arabidopsis wild-type or control (Con) and delayed aging at 25 days after germination, and for 2 days H 2 Figure shows changes in chlorophyll content of leaves treated with O 2 .
  • Example 1 Isolation of ATPG3 gene from Arabidopsis to regulate plant productivity, delay aging and provide stress tolerance
  • Arabidopsis cultivars were grown in pots containing soil or Petri dishes containing MS (Murashige and Skoog salts, Sigma, USA) medium containing 2% sucrose (pH 5.7) and 0.8% agar. . When cultivated in a pollen, it was grown in a growth chamber controlled at a light cycle of 16/8 hours at a temperature of 22 ° C.
  • the forward primer (PacI / At4g14465 SOE-F, represented by SEQ ID NO: 3 and containing the sequence of restriction enzyme PacI) 5'- TTA ATT AAA TGG CAA ACC CTT GGT GGA CG -3 ') and a reverse primer (XbaI / At4g14465 SOE-R, 5'-TCT AGA TCA GTA AGG) as shown in SEQ ID NO: 4 and containing the sequence of restriction enzyme XbaI TGG TCT TGC GTG G-3 ').
  • the two primers were used to amplify and isolate full-length cDNA from polymerase chain reaction (PCR) from Arabidopsis cDNA prepared in Example 1-2.
  • the isolated cDNA has a transcriptional translation frame (ORF) of 846 bp encoding 281 amino acids having a molecular weight of about 29.5 kDa, it was confirmed that it consists of one exon, AT- It has a hook domain and named it ATPG3 (AT-hook protein of Genomine 3).
  • the isoelectric point of the ATPG3 protein encoded by the gene was 6.65 (hereinafter, the gene is called " ATPG3 " or " ATPG3 gene” using italics, and the protein is called "ATPG3" or "ATPG3 protein").
  • a transgenic Arabidopsis in which the ATPG3 gene was introduced in the sense direction was prepared to change the expression of the ATPG3 transcript.
  • ATPG3 cDNA was amplified by PCR from Arabidopsis cDNA using a forward primer represented by SEQ ID NO: 3 and a sequence of restriction enzyme PacI and a reverse primer represented by SEQ ID NO: 4 and a sequence of restriction enzyme XbaI .
  • the DNA was digested with restriction enzymes PacI and XbaI and cloned in the sense direction into a pCSEN vector prepared to be controlled by the SEN1 promoter, an inducible promoter, to prepare a pCSEN-ATPG3 recombinant vector, a sense construct for the ATPG3 gene. It was.
  • the SEN1 promoter has specificity for the gene expressed according to the growth stage of the plant.
  • the pCSEN-ATPG3 recombinant vector was introduced into Agrobacterium tumefaciens using an electroporation method.
  • the transformed Agrobacterium culture was incubated at 28 ° C. until the OD600 value was 1.0, and the cells were harvested by centrifugation at 25 ° C. at 5,000 rpm for 10 minutes.
  • Harvested cells were suspended in medium of Infiltration Medium (IM; 1X MS SALTS, 1X B5 vitamin, 5% sucrose, 0.005% Silwet L-77, Lehle Seed, USA) until the final OD600 value was 2.0.
  • IM Infiltration Medium
  • the Arabidopsis was placed in a polyethylene bag for 24 hours. Thereafter, the transformed Arabidopsis cultivars continued to grow to harvest seeds (T1).
  • T1 a non-transformed wild type Arabidopsis or a Arabidopsis transformed with only a vector (pCSEN vector) containing no ATPG3 gene was used.
  • Seeds harvested from the transformed Arabidopsis larvae as in ⁇ Example 2-1> were selected by immersing and incubating for 30 minutes in a 0.1% Bassta herbicide (light, South Korea) solution. Thereafter, during the growth of the transformed Arabidopsis, the pollen was treated 5 times with the Basta herbicide, and the transformed Arabidopsis was selected from each pollen.
  • the variants of T1 Arabidopsis transformed with the pCSEN-ATPG3 vector compared with their control phenotypes (PCSEN vectors or wild-type Arabidopsis transformed with only the pCSEN vector) without the ATPG3 gene, surprisingly, variants were markedly aged. Delayed properties.
  • T2 transgenic seeds were received from the T1 transgenic Arabidopsis and the phenotypes of these lines were examined.
  • T2 transformed seeds which had been cold treated (4 ° C.) for 3 days were grown in pots, and then T2 transgenic Arabidopsis was selected through the treatment of Basta herbicide.
  • Phenotyping of selected Arabidopsis T2 transformation lines was performed 50 days and 70 days after germination (FIG. 2).
  • the ATPG3 ox-1, ATPG3 ox-2 and ATPG3 ox-8 variant lines with pCSEN-ATPG3 constructs showed a pronounced delay in aging of plants compared to Arabidopsis control (Con). These variants had a delayed aging phenotype when grown for 50 days after germination, but were similar or slightly smaller in size than the wild type in terms of individual size.However, when grown for 70 days after germination, not only did the aging phenotypes but also the individual size and seed yield. There was a marked increase in the Arabidopsis wild type. The delay in aging and the increase in productivity were slightly different for each line. This is because the overexpression of genes is slightly different for each line as shown in FIG. 3.
  • PCR was performed using the primers specific to the following [Table 1] for the ATPG3 gene and the ACT gene used as a PCR positive control.
  • PCR denatured template DNA by heating at 94 ° C. for 2 minutes and then 1 minute at 94 ° C .; 1 minute 30 seconds at 55 ° C; And 1 cycle at 72 °C one cycle was performed a total of 30 times, followed by a final reaction for 15 minutes at 72 °C.
  • the PCR product was confirmed by 1% agarose gel electrophoresis, the results are shown in FIG.
  • ATPG3 ox-1 As compared to Arabidopsis thaliana wild-type is ATPG3 ox-1, ATPG3 ox- 2 and ATPG3 expression of ATPG3 gene of ox-8 mutants were able to see that the significant increase in total, this fact has the mutants have demonstrated the overexpression cheim of ATPG3 gene .
  • ATPG3 ox-1, ATPG3 ox-2 and ATPG3 ox-8 variants with high relative expression levels of ATPG3 genes showed higher productivity-enhancing traits such as individual size and seed yield than the control group.
  • the ATPG3 ox-8 variant with low relative expression of genes has a weaker effect on delaying aging than the ATPG3 ox-1 and ATPG3 ox-2 variants. Therefore, the expression level control of the present gene is thought to be able to arbitrarily produce plants with phenotypic characteristics for increased productivity and delay aging.
  • ATPG3 can be used as an excellent gene source for the development of high productivity crops, as it is easy to manufacture plants that have increased harvesting time and the same productivity as the Arabidopsis wild type through the regulation of gene expression.
  • cDNA was synthesized by extracting RNA from organs at various stages of development of Arabidopsis wild-type ATPG3 gene and ACT gene used as a PCR positive control. PCR was performed using the specific primers shown in Table 1 below. As a result, as shown in Figure 4, it was confirmed that the expression of the ATPG3 gene is mainly made in the leaves, it can be seen that the expression is also made in the stem and Inflorescence organ. On the other hand, gene expression was significantly lower in seedlings and roots during early development.
  • Productivity indicators applied include plant height, silique number (NTS), dry-W, total seed weight (TSW), total seed number (TNS), and 1,000 seed weights (1,000). SW), and the result is the average value of 20 objects per line.
  • ATPG3 provides resistance to oxidative stress in plants.

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Abstract

La présente invention concerne une protéine ATPG3 ayant la capacité à augmenter la productivité, une capacité à retarder la sénescence et la capacité à conférer à des plantes une résistance au stress, un gène de la protéine ATPG3, et une utilisation associée. La plante transformée et surexprimée présente les caractéristiques de productivité accrue ainsi que des caractéristiques de retard de sénescence, et des caractéristiques de résistance au stress.
PCT/KR2013/001311 2012-02-20 2013-02-20 Protéine atpg3 ayant une capacité à augmenter la productivité, une capacité à retarder la sénescence et une capacité à conférer aux plantes une résistance au stress, gène de la protéine atpg3 et leur utilisation WO2013125835A1 (fr)

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KR20180038718A (ko) * 2016-10-07 2018-04-17 제노마인(주) 식물의 생산성 증대 기능, 스트레스 내성 기능 및 노화 지연 기능을 갖는 MtATPG2 단백질과 그 유전자 및 이들의 용도
KR20180038717A (ko) * 2016-10-07 2018-04-17 제노마인(주) 식물의 생산성 증대 기능과 노화 지연 기능을 갖는 MtATPG1 단백질과 그 유전자 및 이들의 용도

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WO2005098015A2 (fr) * 2004-04-06 2005-10-20 Metanomics Gmbh Procede de production de produits chimiques fins
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US20020040490A1 (en) * 2000-01-27 2002-04-04 Jorn Gorlach Expressed sequences of arabidopsis thaliana
US20040019925A1 (en) * 2001-04-18 2004-01-29 Heard Jacqueline E. Biochemistry-related polynucleotides and polypeptides in plants
WO2005098015A2 (fr) * 2004-04-06 2005-10-20 Metanomics Gmbh Procede de production de produits chimiques fins
US20090070894A1 (en) * 2005-12-01 2009-03-12 Cropdesign N.V. Plants having improved growth characteristics and methods for making the same

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