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WO2012148121A2 - Protéine atpg7 conférant à une plante des caractéristiques d'augmentation du rendement, de retardement du vieillissement et de résistance au stress, gène de ladite protéine et utilisation de celle-ci - Google Patents

Protéine atpg7 conférant à une plante des caractéristiques d'augmentation du rendement, de retardement du vieillissement et de résistance au stress, gène de ladite protéine et utilisation de celle-ci Download PDF

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WO2012148121A2
WO2012148121A2 PCT/KR2012/002966 KR2012002966W WO2012148121A2 WO 2012148121 A2 WO2012148121 A2 WO 2012148121A2 KR 2012002966 W KR2012002966 W KR 2012002966W WO 2012148121 A2 WO2012148121 A2 WO 2012148121A2
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plant
gene
atpg7
aging
seq
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WO2012148121A3 (fr
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이동희
김국진
이인철
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Genomine Inc
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Genomine Inc
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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)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
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    • 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
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    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
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    • 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
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    • 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
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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
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    • 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
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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)
    • C12N15/8241—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 ATPG7 protein, a gene thereof, and a use thereof having a productivity enhancing function, a aging delay function, and a stress resistance function of a plant.
  • Plant aging is the final stage of plant development, an age-dependent process of decay at the cellular, tissue, organ, or organismal level, leading to the lethal stage through the growth and development stages. As aging progresses, the plant gradually loses its ability to synthesize and loses its homeostasis as its intracellular structures and macromolecules break down sequentially (Thomas et al., 1993). The aging of these plants is genetically planned as a series of consecutive biochemical and physiological phenomena that are very sophisticated and active at the level of cells, tissues and organs.
  • the initial phenomenon of aging in cell structure is the degradation of chloroplasts, which are organelles containing more than 70% of leaf protein. In metabolic terms, this means that carbon assimilation in plants is converted to catabolism of chlorophyll and macromolecules such as proteins, membrane lipids, and RNA. Increased catabolic activity through aging induces the conversion of cellular components accumulated in the assimilated leaves during growth into ventilated cellular components supplied for the development of seeds or other storage organs.
  • aging of plants is thought to be a process of cell degeneration as well as a genotype actively acquired to adapt to the environment during evolution (Buchanan-Wollaston et al., 2003; Lim and Nam, 2005; Nam, 1997).
  • cytokinin is a physiologically delayed aging hormone and many aging control techniques have been reported.
  • the Amasino group developed a method for regulating aging-specific cytokinin synthesis by recombining the IPT gene into a senescence-specific SAG12 gene promoter, which showed a 50% increase in productivity in cigarettes that delayed aging.
  • GmSARK in soybean one of the receptor-like kinases, is up-regulated not only in naturally occurring aging but also in the artificial aging process by cancer treatment. Inhibition of this gene is known to cause delay in leaf aging (Li et al. al., 2006).
  • ROS reactive oxygen species
  • An object of the present invention is to provide an ATPG7 protein which has a function of delaying aging of plants and a function of increasing productivity.
  • Another object of the present invention is to provide a gene encoding the protein.
  • Still another object of the present invention is to provide a method for producing a plant having aging delay characteristics.
  • Still another object of the present invention is to provide a method for producing a plant having a yield increasing characteristic.
  • Still another object of the present invention is to provide a method for producing a plant having stress resistance.
  • the present invention relates to an ATPG7 protein which has a function of delaying aging of plants and a function of increasing productivity.
  • the inventor (s) is to isolate the gene of the protein based on the nucleotide sequence of the DNA binding protein-related protein (DNA-binding protein-related, GeneBank accession number NP 194012.1) as confirmed in the following Examples
  • DNA-binding protein-related GeneBank accession number NP 194012.1
  • the productivity increase characteristics such as the increase in the biomass and / or the seed productivity of the individual are also apparent, and in addition to the drought stress or the oxidative stress It was also confirmed that the resistance characteristics were also apparent.
  • the present inventors have named the gene to gene and protein ATPG7 ATPG7 (AT -hook p rotein of G enomine 7), these nucleotide sequences and amino acid sequences are disclosed in SEQ ID NOS: 1 and 2, respectively.
  • the ATPG7 protein of the present invention is one of the polypeptides of (a), (b) and (c) below.
  • protein are used interchangeably with each other in the same sense as a polypeptide
  • gene is used interchangeably with each other in the same sense as a polynucleotide.
  • 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.
  • deletion of the N-terminal or C-terminal moiety as well as other moieties may still have the function of the original polypeptide.
  • one of ordinary skill in the art will be able to ascertain whether such deleted polypeptide still has the function of the original polypeptide within the scope of its usual ability.
  • the present specification discloses the nucleotide sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 2 and further, the polypeptide encoded by the nucleotide sequence of SEQ ID NO: 1 and consisting of the amino acid sequence of SEQ ID NO: 2 increases the delaying function and productivity of the plant
  • the present invention confirms whether a polypeptide having a partial deletion in the amino acid sequence of SEQ ID NO: 2 still retains the function of the polypeptide including the amino acid sequence of SEQ ID NO: 2. It becomes very clear that it can fully confirm within a normal capability range.
  • polypeptide substantially similar to the polypeptides of (a) and (b) includes a function comprising one or more substituted amino acids, but comprising the amino acid sequence of SEQ ID NO: 2, ie aging of a plant. It refers to a polypeptide having a delaying function and a productivity increasing function.
  • the degree of activity or amino acid substitution of the polypeptide is not a problem as long as the polypeptide containing at least one substituted amino acid retains the function of delaying aging and increasing productivity of the plant.
  • 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.
  • the polypeptide having such substituted amino acid (s) may be It will still retain the function of the original polypeptide.
  • a negatively charged amino acid such as glutamic acid
  • another negatively charged amino acid such as aspartic acid
  • 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 comprising one or more substituted amino acids still has this function.
  • the present specification discloses an example in which the base sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 2 are disclosed, and the polypeptide comprising the amino acid sequence of SEQ ID NO: 2 has a function of delaying aging and increasing productivity of plants. It is clear that the "polypeptide substantially similar to the polypeptide of (a) and (b)" of the present invention can be easily implemented by those skilled in the art.
  • 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.
  • the polypeptide has at least 60% sequence homology at the lower end of sequence homology, while at the upper limit of sequence homology, it is preferred that the polypeptide has 100% sequence homology. More specifically, the above 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%, 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% in order of higher.
  • 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, which are 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.
  • the method for producing a delayed aging plant 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) a phenotype of delayed aging. It comprises a step of selecting a plant having a.
  • 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.
  • 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 of the present invention.
  • 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 of the present invention.
  • 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 ATPG7 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 ATPG7 protein consisting of the amino acid sequence of SEQ ID NO: 2, in particular, the gene ATPG7 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 ATPG7 protein consisting of the amino acid sequence of SEQ ID NO: 2, in particular, the gene ATPG7 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. Genomes include altered plants, plant seeds, plant cells.
  • an ATPG7 protein and its gene having a function of delaying aging and increasing productivity of plants. Since the gene has a function of delaying aging and has a function of increasing productivity, when transforming a plant with this gene, the gene may be delayed in aging of the plant and have a function of increasing productivity of the plant.
  • Fig. 1 shows the structure (schematic diagram) of the pCSEN-ATPG7 recombinant vector in which the ATPG7 gene, which has a function of delaying aging of plants and has a function of increasing productivity, is introduced in the sense direction.
  • Figure 2 is a photograph of the Arabidopsis grown 60 days after germination of T 1 plants transformed with the pCSEN-ATPG7 recombinant vector of Figure 1 above.
  • AT7-4 Arabidopsis T 1 plant transformed with pCSEN-ATPG7 recombinant vector
  • Figure 3 shows the results of analyzing the expression of the ATPG7 gene in Arabidopsis thalass cultivated for 20 days after cotyledon generation T 2 line transformed with the pCSEN-ATPG7 recombinant vector of Figure 1 through qRT-PCR will be.
  • ATPG7 ox-4 Arabidopsis T 2 line transformed with pCSEN-ATPG7 recombinant vector
  • ATPG7 ox-5 Arabidopsis T 2 line transformed with pCSEN-ATPG7 recombinant vector
  • ATPG7 ox-6 Arabidopsis T 2 line transformed with pCSEN-ATPG7 recombinant vector
  • Figure 4 shows the results of analyzing the expression of the ATPG7 gene in the various plant organs of Arabidopsis wild type through qRT-PCR.
  • Figure 5 is a photograph of the Arabidopsis grown 50 days and 70 days after germination of the Arabidopsis T 2 line transformed with the pCSEN-ATPG7 recombinant vector of FIG.
  • ATPG7 ox-4 Arabidopsis T 2 line transformed with pCSEN-ATPG7 recombinant vector
  • ATPG7 ox-5 Arabidopsis T 2 line transformed with pCSEN-ATPG7 recombinant vector
  • ATPG7 ox-6 Arabidopsis T 2 line transformed with pCSEN-ATPG7 recombinant vector
  • FIG. 6 is a diagram for increasing the productivity of the Arabidopsis line of FIG.
  • ATPG7 ox-4 Arabidopsis T 2 line transformed with pCSEN-ATPG7 recombinant vector
  • ATPG7 ox-5 Arabidopsis T 2 line transformed with pCSEN-ATPG7 recombinant vector
  • ATPG7 ox-6 Arabidopsis T 2 line transformed with pCSEN-ATPG7 recombinant vector
  • FIG. 7 shows the Arabidopsis wild type (Con), delayed aging-induced variants ATPG7 ox-4, ATPG7 ox-5 , and ATPG7 ox-6 from day 3 and 12, respectively.
  • a picture of the phenotype of a leaf observed up to 40 days per day.
  • FIG. 8 shows every 4-4 leaves of the Arabidopsis wild type (Con), delayed aging-induced variants ATPG7 ox-4, ATPG7 ox-5 , and ATPG7 ox-6 from day 12 after cotyledon production.
  • Figure shows the chlorophyll content of leaves for up to 40 days per day.
  • FIG. 10 shows every 4-4 leaves of the Arabidopsis wild-type (Con), delayed aging-induced variants ATPG7 ox-4, ATPG7 ox-5 , and ATPG7 ox-6 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 ACT 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 of the Arabidopsis wild-type (Con), ATPG7 ox-4, ATPG7 ox-5 , and ATPG7 ox-6 3-4 days after germination, to maintain cancer
  • Figure 12 detaches the left lobe of the Arabidopsis wild-type (Con), delayed-induced mutants ATPG7 ox-4, ATPG7 ox-5 , and ATPG7 ox-6 3-4 days after germination to maintain cancer status. A picture of the chlorophyll content of leaves every 12 days until 12 days.
  • FIG. 13 shows detachment of the left lobe of the Arabidopsis wild-type (Con), delayed-induced mutants ATPG7 ox-4, ATPG7 ox-5 , and ATPG7 ox-6 3-4 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. 14 shows detachment of the left lobe of the Arabidopsis wild-type (Con), ATPG7 ox-4, ATPG7 ox-5 , and ATPG7 ox-6 3-4 days after germination to maintain cancer status.
  • the expression patterns of aging marker genes on the leaves every 12 days were analyzed by qRT-PCR, and ACT was used as a PCR positive control.
  • CAB2, SEN4, and SAG12 are aging marker genes.
  • FIG. 15 shows a 12-day drought treatment of Arabidopsis wild-type or control (Con) and aging-induced variants ATPG7 ox-4, ATPG7 ox-5 , and ATPG7 ox-6 at 30 days after germination , The figure shows the phenotypic change of.
  • FIG. 16 shows a 12-day drought treatment of Arabidopsis wild-type or control (Con) and aging-induced variants ATPG7 ox-4, ATPG7 ox-5 , and ATPG7 ox-6 at 30 days after germination , The figure shows the change in the weight of a leaf.
  • FIG. 17 shows a 6-day H 2 detachment of the Arabidopsis wild-type or control group (Con) and the left lobe 3-4 of the variants ATPG7 ox-4, ATPG7 ox-5 , and ATPG7 ox-6 at 25 days after germination.
  • Figure shows the phenotype change of leaves treated with O 2 .
  • Figure 18 day 25 Arabidopsis thaliana wild-type or control (Con) and aging delays induced mutant ATPG7 ox-4, ATPG7 ox- 5, and to detach the left lobe of three to four times ATPG7 ox-6 6 days after germination H 2 Figure shows changes in chlorophyll content of leaves treated with O 2 .
  • Example 1 It has a function of delaying aging of the plant from the Arabidopsis and increasing productivity ATPG7 Isolation of genes
  • 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.
  • DNA binding protein-related protein DNA binding protein-related protein
  • Arabidopsis the forward primer (PacI / AT4G22810 SOE), which is represented by SEQ ID NO: 3 and contains the sequence of restriction enzyme PacI -F, 5'-TTA ATT AAA TGG ATC CAG TAC AAT CTC ATG G -3 ') and reverse primer (XbaI / AT4G22810 SOE-R, 5'-), represented by SEQ ID NO: 4 and containing the sequence of restriction enzyme XbaI TCT AGA TCA ATA CGG TGG TCG TCC CGT-3 ′) was synthesized.
  • 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.
  • ATPG7 AT -hook p rotein of G enomine 7
  • the isoelectric point of the ATPG7 protein encoded by the gene was found to be 6.8 (hereinafter, the gene is called " ATPG7 " or " ATPG7 gene” using italics, and the protein is called "ATPG7" or "ATPG7 protein”).
  • a transgenic Arabidopsis in which the ATPG7 gene was introduced in the sense direction was prepared to change the expression of the ATPG7 transcript.
  • ATPG7 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-ATPG7 recombinant vector, a sense construct for the ATPG7 gene. It was.
  • the SEN1 promoter has specificity for the gene expressed according to the growth stage of the plant.
  • FIG. 1 is a diagram showing a pCSEN-ATPG7 recombinant vector in which the ATPG7 gene is introduced in the sense direction into the pCSEN vector.
  • BAR indicates a phosphinothricin acetyltransferase gene ( BAR gene) that confers resistance to a Vaster herbicide
  • RB is a right border
  • LB is a left border
  • P35S is a CaMV 35S promoter
  • PSEN is the SEN1 promoter
  • Nos-A refers to the polyA of the nopaline synthase gene.
  • the pCSEN-ATPG7 recombinant vector was introduced into an Agrobacterium tumefaciens using an electroporation method.
  • the transformed Agrobacterium cultures were incubated at 28 ° C. until the OD 600 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 Infiltration Medium (IM; 1X MS SALTS, 1X B5 vitamin, 5% sucrose, 0.005% Silwet L-77, Lehle Seed, USA) medium until the final OD 600 value was 2.0.
  • IM Infiltration Medium
  • 1X MS SALTS 1X B5 vitamin, 5% sucrose, 0.005% Silwet L-77, Lehle Seed, USA
  • the Arabidopsis was placed in a polyethylene bag for 24 hours. Thereafter, the transformed Arabidopsis cultivars continued to grow to harvest seeds (T 1 ).
  • T 1 a non-transformed wild type Arabidopsis or a Arabidopsis transformed with only a vector (pCSEN vector) containing no ATPG7 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, the pollen was treated 5 times with the Basta herbicide during the growth of the transformed Arabidopsis larvae, and the Arabidopsis growth change in each pollen was investigated.
  • T 1 Arabidopsis AT7-4 and AT7-8 transformed with pCSEN-ATPG7 vector were treated with a control group (a vector without the ATPG7 gene (pCSEN vector) transformed with a Arabidopsis or wild-type Arabidopsis), and their germination 60 days after germination.
  • pCSEN vector a vector without the ATPG7 gene transformed with a Arabidopsis or wild-type Arabidopsis
  • the AT7-4 and AT7-8 variants showed distinct aging delay characteristics, and the difference in aging delay characteristics in transgenic individuals was due to the overexpression of genes. (FIG. 2).
  • the phenotypes of these lines were examined by receiving T 2 transgenic seeds from the T 1 transgenic Arabidopsis.
  • T 2 transformed Arabidopsis thawed for 3 days at low temperature (4 °C) T 2 transformed seedlings were grown in pollen, and transgenic Arabidopsis was selected through the treatment of Basta herbicide.
  • the synthesized cDNA was used as a template, and PCR was performed using the primers specific to the following [Table 1] for the ATPG7 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.
  • ATPG7 ox-4, ATPG7 ox-5 and ATPG7 ox-6 variants showed significantly increased expression of ATPG7 genes compared to the Arabidopsis wild - type, and this fact proves that these variants are overexpressions of the ATPG7 gene.
  • cDNA was synthesized by extracting RNA from organs at various stages of development of Arabidopsis wild-type ATPG7 gene and ACT gene used as 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 ATPG7 gene is mainly made in the stem, it can be seen that the expression is also made in the leaves and young leaves during the aging process. On the other hand, gene expression was significantly lower in seedlings, roots and flowers in early development.
  • Phenotyping of selected Arabidopsis T 2 transformation lines was performed 50 days and 70 days after germination (FIG. 5).
  • the ATPG7 ox-4, ATPG7 ox-5 and ATPG7 ox-6 variant lines with pCSEN-ATPG7 constructs showed a marked delay in aging of plants as compared to the T 1 variant.
  • these variants resulted in not only a delayed aging phenotype but also a marked increase in individual size and seed yield during aging delay.
  • 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.
  • ATPG7 ox-5 which has a strong aging trait, does not have a large phenotypic difference in productivity
  • ATPG7 ox-4 which does not have a strong aging trait, has a large phenotypic characteristic compared to a control group in productivity. Appeared. 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.
  • productivity improvement indicators such as seed yield by line of the variants ATPG7 ox-4, ATPG7 ox-5 and ATPG7 ox-6 are provided.
  • the application was compared with the Arabidopsis control.
  • Productivity indicators applied include plant height, silique number (NTS), biomass (Wet-W), dry-W, total seed weight (TSW), total seed number (TNS), And 1,000 seed weights (1,000 SW), and the result is the average of 20 individuals per line.
  • the ATPG7 ox-4, ATPG7 ox-5 and ATPG7 ox-6 variant lines all increased in seed weight and seed count more than 1.5 times compared to Arabidopsis control, and the weight of 1,000 seeds was significantly different from the control. There was no. And in the biomass and biodry weight, the overexpressed mutant was found to increase about 2.8 times more than the control. This fact is believed that the ATPG7 gene causes an increase in crop productivity, such as individual size, seed yield, and the like, with aging delay (FIG. 6). Therefore, the application of other crops of this gene is considered to be very valuable in terms of productivity.
  • Chlorophyll was extracted from each sample leaf using 80% (V / V) acetone to measure chlorophyll content. Chlorophyll content was measured according to the method of Lichtenthaler and Wellburn ( Biochemical Society Transduction 603: 591 ⁇ 592, 1983) using extinction coefficients of 663.2 nm and 664.8 nm. As a result, as shown in Figure 8, the chlorophyll content in the wild species showed a sharp decrease from 20 days after the cotyledon production and the chlorophyll content was almost 0% at 32 days, but ATPG7 ox-4, ATPG7 ox-5 and ATPG7 ox-6 showed more than 80% of chlorophyll content at the beginning of measurement even when 36 days after cotyledon production.
  • Photosynthetic efficiency was measured using Oh et al . ( Plant Mol. Biol. 30: 939, 1996). First, the leaves of each DAE (day after emersion) were treated with cancer for 15 minutes, and then the fluorescence of chlorophyll was measured using a Plant Efficiency Analyzer (Hansatech). Photosynthetic efficiency was expressed by the photochemical efficiency of PSII (photosystem II) using chlorophyll fluorescence properties, which was the maximum variable fluorescence (Fv) versus the maximum value of fluorescence (Fm). It is expressed as the ratio of (Fv / Fm). Higher values indicate better photosynthetic efficiency.
  • Quantitative analysis of the ATPG7 gene and marker genes for aging was confirmed by Quantitative real-time PCR (qRT-PCR) using Applied Bio-systems' 7300 Real Time PCR System. Aging marker gene was used as a SAG12, SEN4 and CAB2 genes, as qRT-PCR positive control was used the ACT gene.
  • the primers used are shown in Table 2 below.
  • the expression patterns of the ATPG7 gene showed that the expression levels of the ATPG7 gene overexpressed were significantly higher than those of the wild type, and gradually decreased, although there was a slight difference in the lines during aging. However, despite this reduction, it was found to maintain a high expression sequence compared to the wild type (Fig. 10). Taken together, the ATPG7 gene delays the onset of aging at the molecular level and subsequently regulates physiological phenomena such as chlorophyll content and photosynthetic efficiency, resulting in phenotypically prolonged leaf life.
  • the overexpression of the ATPG7 gene showed that the greening of the leaves was still progressing during drought treatment, and the weight of the leaves was much higher than that of the wild type. This means that ATPG7 provides maximum resistance to plant moisture retention even under drought stress, providing resistance to drought stress.
  • ATPG7 provides resistance to oxidative stress in plants.
  • the ATPG7 gene is expected to provide many advantages in developing stress-resistant crops by providing resistance to drought and oxidative stress in plants.

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Abstract

L'invention concerne une protéine ATPG7 qui confère à une plante des caractéristiques d'augmentation du rendement, de retardement du vieillissement et de résistance au stress. Elle concerne également un gène de cette protéine et une utilisation de celle-ci. Le corps de la plante, transformé et surexprimé, présente non seulement des caractéristiques d'augmentation du rendement, mais également de retardement du vieillissement et de résistance au stress.
PCT/KR2012/002966 2011-04-26 2012-04-18 Protéine atpg7 conférant à une plante des caractéristiques d'augmentation du rendement, de retardement du vieillissement et de résistance au stress, gène de ladite protéine et utilisation de celle-ci Ceased WO2012148121A2 (fr)

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WO2014209036A1 (fr) * 2013-06-28 2014-12-31 제노마인(주) Protéine atpg10 et son gène, ayant des fonctions d'augmentation du rendement des plantes, de résistance au stress et de retard la sénescence, et son utilisation
WO2014209060A1 (fr) * 2013-06-28 2014-12-31 제노마인(주) Protéine atpg6 et son gène, ayant des fonctions d'augmentation du rendement des plantes, de résistance au stress et de retard la sénescence, et son utilisation

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KR101536894B1 (ko) * 2013-12-24 2015-07-15 명지대학교 산학협력단 벼 유래 OsMDB1 유전자를 이용한 초장이 조절된 형질전환 식물체의 제조방법 및 그에 따른 식물체
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
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

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KR100604195B1 (ko) * 2004-09-02 2006-07-25 고려대학교 산학협력단 식물 노화에 특이적으로 발현되는 유전자 및 그 유전자의프로모터
KR100861524B1 (ko) * 2007-06-01 2008-10-02 고려대학교 산학협력단 식물체에서 내한성 증가를 유도하는 벼 유래의프롤린과다단백질 유전자
KR100907308B1 (ko) * 2007-09-19 2009-07-13 건국대학교 산학협력단 산화적 스트레스에 저항성을 가지는 애기장대 돌연변이체

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Publication number Priority date Publication date Assignee Title
WO2014209036A1 (fr) * 2013-06-28 2014-12-31 제노마인(주) Protéine atpg10 et son gène, ayant des fonctions d'augmentation du rendement des plantes, de résistance au stress et de retard la sénescence, et son utilisation
WO2014209060A1 (fr) * 2013-06-28 2014-12-31 제노마인(주) Protéine atpg6 et son gène, ayant des fonctions d'augmentation du rendement des plantes, de résistance au stress et de retard la sénescence, et son utilisation

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