[go: up one dir, main page]

WO2021133049A1 - Peptides de transit de chloroplastes et leur utilisation - Google Patents

Peptides de transit de chloroplastes et leur utilisation Download PDF

Info

Publication number
WO2021133049A1
WO2021133049A1 PCT/KR2020/018969 KR2020018969W WO2021133049A1 WO 2021133049 A1 WO2021133049 A1 WO 2021133049A1 KR 2020018969 W KR2020018969 W KR 2020018969W WO 2021133049 A1 WO2021133049 A1 WO 2021133049A1
Authority
WO
WIPO (PCT)
Prior art keywords
protein
chloroplast
amino acid
polypeptide
seq
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/KR2020/018969
Other languages
English (en)
Korean (ko)
Inventor
박중혁
성순기
김한울
윤준선
한윤정
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
FarmHannong Co Ltd
Original Assignee
FarmHannong Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by FarmHannong Co Ltd filed Critical FarmHannong Co Ltd
Publication of WO2021133049A1 publication Critical patent/WO2021133049A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • 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)
    • 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
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
    • 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
    • C12N15/8274—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 herbicide 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/001—Oxidoreductases (1.) acting on the CH-CH group of donors (1.3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y103/00—Oxidoreductases acting on the CH-CH group of donors (1.3)
    • C12Y103/03—Oxidoreductases acting on the CH-CH group of donors (1.3) with oxygen as acceptor (1.3.3)
    • C12Y103/03004—Protoporphyrinogen oxidase (1.3.3.4)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/08—Fusion polypeptide containing a localisation/targetting motif containing a chloroplast localisation signal

Definitions

  • a polypeptide capable of translocating to the chloroplast of a plant cell, and use of the polypeptide for transport of a protein of interest to the chloroplast are provided.
  • Plant cells contain unique organelles, commonly referred to as “pigments”, that are delimited by distinct membrane structures and perform differentiated intracellular functions.
  • plastids are also responsible for the synthesis and storage of chemical compounds. Plastids have systems for gene expression and protein synthesis independent of the nucleus, but exist within the cell in a semi-autonomous manner that relies on close cooperation with the nuclear-cytoplasmic system.
  • chloroplast A representative plastid is a chloroplast. Although the most essential function of chloroplasts is the performance of photosynthesis, they also perform many other biosynthetic processes important to plant cells. In addition, chloroplasts are one of the important targets in the agrochemical industry. For example, many herbicides are known to act by blocking functions performed within the chloroplast.
  • chloroplast proteins are encoded in the nucleus of plant cells, synthesized as precursor proteins in the cytoplasm, and transported to the chloroplast after translation. Precursor proteins destined for chloroplast localization contain an N-terminal extension known as Chloroplast Transit Peptide (CTP).
  • CTP Chloroplast Transit Peptide
  • the N-terminal transit peptide may be usefully used as a means for targeting a target substance (eg, a target protein) to the chloroplast.
  • a target substance eg, a target protein
  • chloroplast Transit Peptide CTP
  • target substance thereof for targeting the chloroplast a novel chloroplast transit peptide (Chloroplast Transit Peptide, CTP) and a target substance thereof for targeting the chloroplast are provided.
  • One example provides a novel chloroplast transit peptide.
  • a fusion protein comprising the chloroplast transit peptide and a target protein.
  • the target protein may be directly linked to the C-terminus, the N-terminus, or both ends of the chloroplast transit peptide through or without a peptide linker, for example, it may be linked to the C-terminus of the chloroplast transit peptide.
  • Another example provides a polynucleotide encoding the chloroplast transit peptide or the fusion protein.
  • Another example provides a recombinant vector carrying (or comprising) the polynucleotide.
  • Another example provides a recombinant cell comprising the polynucleotide or recombinant vector.
  • Another example is at least one selected from the group consisting of a fusion protein comprising a chloroplast transit peptide and a target protein, a polynucleotide encoding the same, a recombinant vector comprising the polynucleotide, and a recombinant cell comprising the polynucleotide or the recombinant vector It provides a composition for transporting the chloroplast of the target protein, comprising a.
  • Another example provides a chloroplast transport method of the target protein, comprising the step of introducing a fusion protein comprising a chloroplast transit peptide and a target protein, or a polynucleotide encoding the same into a cell.
  • the cell may be selected from all cells including chloroplasts, and may be, for example, cells of plants or algae.
  • Another example provides a polynucleotide encoding a fusion protein comprising a chloroplast transit peptide and a target protein, or a transformant transformed with a recombinant vector comprising the polynucleotide.
  • Another example provides a method for producing a transformant, comprising transforming a host cell with a polynucleotide encoding a fusion protein comprising a chloroplast transit peptide and a target protein or a recombinant vector comprising the polynucleotide.
  • the host cell may be a plant cell.
  • chloroplast Transit Peptide CTP
  • target substance thereof for targeting the chloroplast a novel chloroplast transit peptide (Chloroplast Transit Peptide, CTP) and a target substance thereof for targeting the chloroplast are provided.
  • a polynucleotide which may be used interchangeably with “gene” or a polypeptide (which may be used interchangeably with “protein”) is "comprising a specific nucleic acid sequence or amino acid sequence, consisting of a specific nucleic acid sequence or amino acid sequence, Or it is expressed as a specific nucleic acid sequence or amino acid sequence” is an equivalent meaning and interchangeable expressions, and may mean that the polynucleotide or polypeptide essentially includes the specific nucleic acid sequence or amino acid sequence, and the polynucleotide or polypeptide necessarily includes the specific nucleic acid sequence or amino acid sequence.
  • nucleic acid sequences or amino acid sequences provided herein can be subjected to conventional mutagenesis methods, such as directed evolution and/or site-specification, to the extent that they retain their original or desired functions. It may include those modified by site-directed mutagenesis or the like.
  • reference to a polynucleotide or polypeptide “comprising a specific nucleic acid sequence or amino acid sequence” means that the polynucleotide or polypeptide (i) consists of or consists essentially of the specific nucleic acid sequence or amino acid sequence; or (ii) at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 95% of the specific nucleic acid sequence or amino acid sequence. It may mean that it consists of or consists essentially of a sequence having at least 96%, at least 97%, at least 98%, or at least 99% homology and maintains the original function and/or the desired function. As used herein, the original function may refer to a chloroplast transition (transport, localization, and/or targeting) function.
  • nucleic acid sequence described herein is within a range that does not change the amino acid sequence and / or function of the protein expressed from the coding region, considering the codon preferred in the organism to express the protein due to the degeneracy of the codon.
  • Various modifications can be made to the coding region in .
  • identity or homology refers to the degree of agreement with a given nucleic acid sequence or amino acid sequence, and may be expressed as a percentage (%).
  • algorithm BLAST according to the literature (Karlin and Altschul, Pro. Natl. Acad. Sci. USA, 90, 5873, 1993) or FASTA by Pearson (see Methods Enzymol) ., 183, 63, 1990) can be used.
  • BLASTN BLASTX has been developed (refer to http://www.ncbi.nlm.nih.gov).
  • polypeptide (or protein) and polynucleotide (or gene) comprising an amino acid sequence or nucleic acid sequence specified by SEQ ID NO:' refers to a sequence 100% identical to the sequence of SEQ ID NO: 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96
  • a polypeptide or polynucleotide comprising a sequence having at least %, at least 97%, at least 98%, or at least 99% homology, and retaining the original function and/or desired function of a polypeptide or polynucleotide comprising a sequence that is 100% identical.
  • the protein provided herein is a protein in a form in which methionine (the first amino acid residue from the N-terminus) encoded by the initiation codon is excluded from the defined amino acid sequence (eg, naturally occurring or recombinantly such as chemically synthesized) protein produced by a method other than the method).
  • Chloroplast Transit Peptide (CTP) is provided.
  • the chloroplast transit peptide may refer to a peptide localizable in the chloroplast in a cell including the chloroplast.
  • the chloroplast transit peptide may be a polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 1 to 77, a fragment of the polypeptide, and/or an extension of the polypeptide.
  • the chloroplast transit peptide is SEQ ID NO: 2, 3, 5, 14, 17, 18, 21, 23, 26, 27, 28, 30, 31, 32, 34, 39, 40, 42, 43, an amino acid sequence selected from 47, 48, 52, 53, 54, 57, 59, 60, 62, 63, 65, 69, 70, 71, 73, 75, 76, and 77, such as SEQ ID NOs: 42, 43, and It may be a polypeptide comprising an amino acid sequence selected from 63 or a fragment of the polypeptide.
  • the polypeptide fragment comprises (1) an amino acid sequence selected from SEQ ID NOs: 1 to 77, (2) SEQ ID NOs: 2, 3, 5, 14, 17, 18, 21, 23, 26, 27, 28, 30, 31, 32, 34, 39, 40, 42, 43, 47, 48, 52, 53, 54, 57, 59, 60, 62, 63, 65, 69, 70, 71, 73, 75, 76, and 77 an amino acid sequence, or (3) 20 or more, 25 or more, 30 or more, 35 or more, 40 consecutive from the N-terminus (the first amino acid residue) of the amino acid sequence selected from SEQ ID NOs: 42, 43, and 63 more than 42, more than 45, more than 47, more than 50, more than 52, more than 55, more than 56, more than 57, more than 58, more than 59, more than 60, more than 61 , 62 or more, 63 or more, 64 or more, 65 or more, 66 or more, 67 or more, 68 or more, 69 or more, or 70 or more
  • the polypeptide extension consists of 1 to 50, 1 to 45, 1 to 40, 1 to 35, 1 to 30, or 1 to 25 amino acids in addition to the amino acid sequence selected from SEQ ID NOs: 1 to 77. It may further include an extension sequence, and the extension sequence may be linked to the N-terminus or C-terminus, such as the C-terminus of the amino acid sequence.
  • SEQ ID NO: 319 with 5 amino acids added to the C-terminus of SEQ ID NO: 63, SEQ ID NO: 320 with 10 amino acids added, SEQ ID NO: 321 with 15 amino acids added, etc. Examples may be provided, but the present invention is not limited thereto.
  • Said polypeptide, polypeptide fragment and/or polypeptide elongate may be administered intracellularly, specifically in a chloroplast, such as in a stroma, a peptidase such as a Stromal Processing Peptidase (SPP; e.g. Arabidopsis thaliana).
  • SPP Stromal Processing Peptidase
  • At5g42390 in the case of Chlamydomonas microorganisms, it may include a cleavage site that is cleaved by SPP-1, SPP-2, etc.).
  • the chloroplast transit peptide is an N-terminal region of Arabidopsis Protoporphyrinogen Oxidase 1 (PPO1), such as the N-terminal (first amino acid residue) of the amino acid sequence of SEQ ID NO: 326.
  • PPO1 Arabidopsis Protoporphyrinogen Oxidase 1
  • the N-terminal region is intracellular, specifically in the chloroplast, e.g., in the stroma, a peptidase, e.g., Stromal Processing Peptidase (SPP; e.g., Arabidopsis thaliana)
  • SPP Stromal Processing Peptidase
  • At5g42390 in the case of Chlamydomonas microorganisms, it may include a cleavage site that is cleaved by SPP-1, SPP-2, etc.).
  • the N-terminal region of Arabidopsis protoporphyrinogen oxidase 1 comprises at least the amino acid sequence of SEQ ID NO: 334, 335, 336, 337, 338, or 339 among the amino acid sequence of SEQ ID NO: 326 it could be
  • a fusion protein comprising the chloroplast transit peptide and a target protein.
  • the target protein may be directly linked to the C-terminus, the N-terminus, or both ends of the chloroplast transit peptide through or without a peptide linker, for example, it may be linked to the C-terminus of the chloroplast transit peptide.
  • the target protein may be selected from all proteins to be transported to or expressed in the chloroplast.
  • the fusion protein when the target protein is recombinantly produced and linked to the C-terminus of the chloroplast transit peptide, the N-terminus encoded by the initiation codon in the amino acid sequence of the target protein provided or known herein
  • the remaining sequence excluding the first amino acid residue methionine (Met, M)) may be linked to the C-terminus of the chloroplast transit peptide. That is, when the target protein is recombinantly produced, the fusion protein may be a protein in which the second amino acid residue from the C-terminus of the chloroplast transit peptide and the N-terminus of the target protein are linked.
  • the target protein may be a herbicide resistance protein.
  • the manufacturer-resistant protein is a protein capable of conferring and/or enhancing resistance to PPO activity inhibitory herbicides such as PPO (protoporphyrinogen oxidase) protein (PPO activity inhibitory herbicide resistance protein); Proteins capable of conferring and/or enhancing resistance to glyphosate, such as 5-enolpyruvylshikimate-3-phosphate synthase (EPPSS) protein (glyphosate resistance protein); a protein capable of conferring and/or enhancing resistance to glufosinate, such as phosphinothricin-N-acetyltransferase (PAT) protein (glufosinate resistance protein); DMO (dicamba monooxygenase), such as resistance to dicamba and / or enhanceable protein (dicamba resistance protein); 2,4-D monooxygenase, AAD (aryloxyalkanoate dioxygenase), etc.
  • the herbicide resistance protein may be a protein related to resistance to PPO activity inhibitory herbicides.
  • the protein related to resistance to the PPO activity inhibitory herbicide is a PPO protein derived from prokaryotes (eg, cyanobacteria), and may be a herbicide-resistant PPO protein having resistance to PPO inhibitory herbicides, for example, Oscilatoria nigro-viri. display (Oscillatoria nigro-viridis) (e. g., Oscillatoria nigro-viridis PCC 7112 strain) derived PPO protein, ring via-in (Lyngbya sp.) strain (e.g., Lyngbya sp.
  • PCC 8106 strain derived PPO protein, halo tese in (Halothece sp.) (eg, Halothece sp. PCC 7418 strain) derived PPO protein, Thermosynechococcus elongatus (eg, Thermosynechococcus elongatus BP-1 or PKUAC-SCTE542) strain PPO protein, Synechococcus genus ( Synechococcus sp.) (eg, Synechococcus sp.
  • strain-derived PPO protein Spirulina subsalsa- derived PPO protein, Thermosynechococcus sp.) (eg, ( Thermosynechococcus sp. NK55a) strain-derived PPO protein, Cyanobacteria bacterium (eg Cyanobacteria bacterium J003) strain-derived PPO protein, Thermosynechococcus vulcanus ( Thermosynechococcus vulcanus ) (eg Thermosynechococcus vulcanu NIES-2134)-derived PPO protein, PPO protein from Auxenochlorella protothecoides , PPO group from Myxococcus xanthus One or more selected from the group consisting of white matter, PPO activity and/or PPO activity inhibitory herbicides, etc. may be exemplified, but the present invention is not limited thereto.
  • the herbicide resistance protein is a glyphosate herbicide resistant EPSPS (glyphosate resistant 5-enolpyruvylshikimate-3-phosphate synthase, such as cp4 epsps, mepsps, 2mepsps, CP4EPSPS, etc.), glyphosate oxidase (GOX), glyphosate- N-acetyltransferase) or glyphosate decarboxylase; glufosinate herbicide resistant PAT (phosphinothricin-N-acetyltransferase); dicamba herbicide tolerant DMO (dicamba monooxygenase); 2,4-D herbicide tolerant 2,4-D monooxygenase or aryloxyalkanoate dioxygenase (AAD); ALS inhibitory sulfonylurea herbicide-resistant ALS (acetolactate synthase), AHAS (acetohydroxyacid)
  • the above herbicide tolerance protein is not only wild-type protein, but also 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97 % or more, 98% or more, 99% or more, 99.2% or more, 99.4% or more, 99.6% or more, 99.8% or more, or 99.9% or more sequence homology, conferring and/or enhancing herbicide tolerance to plants and/or algae Variant proteins that retain activity may also be included.
  • the target protein may be at least one selected from the group consisting of biologically active substances and synthesizing enzymes of metabolites.
  • the biologically active material includes all proteins exhibiting useful effects in vivo, and may include, for example, medical proteins, cosmetic raw materials (eg, growth factors, etc.), synthesizing enzymes of metabolites, and the like.
  • the medical protein may be a protein and/or peptide having a desired activity in a living body (eg, prevention, alleviation, and/or therapeutic activity of a specific disease or symptom, or an activity of replacing a substance necessary for a living body), for example, enzymatic activity of proteins (e.g., proteases, kinases, phosphatases, etc.), receptor proteins, transporter proteins, bactericidal and/or endotoxin-binding proteins, structural proteins, immune polypeptides such as antibodies, toxins, antibiotics, hormones, growth factors, vaccines It may be one or more selected from the group consisting of.
  • proteins e.g., proteases, kinases, phosphatases, etc.
  • receptor proteins e.g., transporter proteins, bactericidal and/or endotoxin-binding proteins
  • structural proteins e.g., immune polypeptides such as antibodies, toxins, antibiotics, hormones, growth factors, vaccines It may be one
  • the target protein may be one or more selected from the group consisting of hormones, cytokines, tissue plasminogen activators, immunoglobulins (eg, antibodies or antigen-binding fragments or variants thereof), and the like.
  • the target protein is insulin, hormones such as human growth hormone (hGH), insulin-like growth factor, epidermal growth factor (EGF), various growth factors such as vascular endothelial growth factor (VEGRF), various receptors, tissue plasminogen activator (tPA), erythropoietin (EPO), cytokines (eg, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, Interleukins such as IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18), interferon ( IFN)-alpha, -beta, -gamma,
  • IFN interfer
  • the metabolite may mean, for example, one or more useful metabolites selected from the group consisting of isoprenoids, phenolic compounds, flavonoids, carotenoids, tocopherol, retinol, and the like, and the synthesizing enzyme of the metabolite is It may be selected from among the enzymes involved in the metabolic pathway of the one or more metabolites.
  • the synthetase is 1-deoxy-D-xylulose 5-phosphate synthase (DOXP synthase), 1-deoxy-D-xylulose 5-phosphate reductase (DOXP reductase), 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase (CDPME synthase), 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (CDPME kinase), 2-C-Methyl-D-erythritol-2 ,4-cyclopyrophosphate synthase (MEcPP synthase), (E)-1-hydroxy-2-methyl-but-2-enyl-4-diphosphate synthase (HMBPP synthase), (E)-1-hydroxy-2-methyl -but-2-enyl-4-diphosphate reduct
  • DOXP synthase 1-deoxy
  • Another example provides a polynucleotide encoding the chloroplast transit peptide or the fusion protein.
  • Another example provides a recombinant vector carrying (or comprising) the polynucleotide.
  • Another example provides a recombinant cell comprising the polynucleotide or recombinant vector.
  • Another example is at least one selected from the group consisting of a fusion protein comprising a chloroplast transit peptide and a target protein, a polynucleotide encoding the same, a recombinant vector comprising the polynucleotide, and a recombinant cell comprising the polynucleotide or the recombinant vector It provides a composition for chloroplast transport and / or chloroplast expression of the target protein, comprising a.
  • Another example provides a chloroplast transport method of the target protein, comprising the step of introducing a fusion protein comprising a chloroplast transit peptide and a target protein, or a polynucleotide encoding the same into a cell.
  • the cell may be selected from all cells including chloroplasts, and may be, for example, cells of plants or algae.
  • Another example provides a transformant transformed with a polynucleotide encoding a fusion protein comprising a chloroplast transit peptide and a target protein or a vector comprising the polynucleotide.
  • Another example provides a method for producing a transformant, comprising transforming with a polynucleotide encoding a fusion protein comprising a chloroplast transit peptide and a target protein or a vector comprising the polynucleotide.
  • the transformation may be performed on cells, protoplasts, callus, hypocotyls, seeds, cotyledons, or whole bodies of algae or plants.
  • the transformant may be an alga or plant cell, protoplast, callus, hypocotyl, seed, cotyledon, whole, or clone or progeny thereof.
  • the transformant may have a chloroplast transport ability of the target protein, or have superior chloroplast transport ability of the target protein compared to the same plant or alga before or without transformation.
  • the polynucleotide may be designed to include a codon optimized for a cell to be transduced among codons encoding each amino acid.
  • the optimization codon can be easily recognized by those of ordinary skill in the art (eg, "http://www.genscript.com/codon-opt.html”, “http://sg .idtdna.com/CodonOpt”, etc.).
  • the polypeptide chloroplast transit peptide, target protein, or fusion protein
  • the mutation may be an amino acid substitution that does not entirely alter the activity of the molecule, and such amino acid substitutions are known in the art.
  • the amino acid substitution is amino acid residues Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thr/Phe, substitutions between Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, or Asp/Gly.
  • the chloroplast transit peptide, target protein, or fusion protein is one or more amino acids phosphorylation, sulfation, acylation, glycosylation, methylation, farnesylation (farnesylation) ) may be modified with one or more selected from the group consisting of.
  • the chloroplast transit peptide, the target protein, or the fusion protein may include a variant in which structural stability to heat, pH, etc. of the protein is increased or protein activity is increased by amino acid mutation and/or modification.
  • sequence homology is intended to indicate a degree of similarity to a wild type or a reference amino acid sequence or base sequence, and is 60% or more, 65% or more, 70% or more with the amino acid sequence of the herbicide-resistant PPO protein variant. and/or the chloroplast transit peptide comprising amino acid residues that are at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical. Alternatively, it may be included in the scope of the present invention as long as it has biologically equivalent activity to the target protein. Such protein homologues may contain the same active site as the target protein.
  • the chloroplast transit peptide, target protein, or fusion protein can be obtained by extraction and purification from nature by methods well known in the art. Alternatively, it can be obtained as a recombinant protein using genetic recombination technology.
  • a nucleic acid encoding a chloroplast transit peptide and/or a target protein is inserted into an appropriate expression vector, the vector is transformed into a host cell, the host cell is cultured so that the target protein is expressed, and the host cell is removed from the host cell. It can be obtained by the process of recovering the chloroplast transit peptide and/or the target protein.
  • biochemical separation techniques such as treatment with a protein precipitating agent (salting out method), centrifugation, sonication, ultrafiltration, dialysis, molecular sieve chromatography (Gel filtration), adsorption chromatography, ion exchange chromatography, various chromatography such as affinity chromatography, etc. can be used, and two or more of these can be used in combination to separate high-purity proteins.
  • chloroplast transit peptide and/or the nucleic acid molecule (polynucleotide) encoding the protein of interest may be isolated or prepared using standard molecular biology techniques, for example, chemical synthesis or recombinant methods, or commercially available ones.
  • chloroplast transit peptide, target protein, or fusion protein described herein may be introduced into a plant or alga by various methods known in the art, and preferably, an expression vector for plant or algae transformation may be used.
  • any conventionally used in the art for gene introduction into plants may be used, for example, SP6 promoter, T7 promoter, T3 promoter, PM promoter , Arabidopsis or corn ubiquitin promoter, cauliflower mosaic virus (CaMV) 35S promoter, nopaline synthase (nos) promoter, pigwort mosaic virus 35S promoter, sugacrane basiliform virus promoter, Commelina yellow mottle virus promoter, Lee Photoinducible promoter of fluorose-1,5-bis-phosphate carboxylase small subunit (ssRuBisCO), rice cytosolic triose phosphate isomerase (TPI) promoter, Arabidopsis adenine phosphoribosyltransferase (APRT) promoter, an octopine synthase promoter, and one or more selected from the group consisting of a blue copper binding protein (BCB) promoter, etc. may be used,
  • the vector may include a poly A signal sequence causing polyadenylation at the 3'-end, for example, those derived from the apple actin gene ( Malus domestica actin 3'end), Agrobacterium tume those derived from the nopaline synthase gene of Fasiens (NOS 3' end), the terminator derived from the octopine synthase gene of Agrobacterium tumefaciens, the protease inhibitor I of tomatoes or potatoes, or 3' terminal portion of the II gene, CaMV 35S terminator, rice ⁇ -amylase RAmy1 A terminator, and phaseolin terminator, but are not limited thereto.
  • a poly A signal sequence causing polyadenylation at the 3'-end for example, those derived from the apple actin gene ( Malus domestica actin 3'end), Agrobacterium tume those derived from the nopaline synthase gene of Fasiens (NOS 3' end), the terminator derived from the octop
  • a chloroplast-specific promoter in the case of avian transformation, a chloroplast-specific promoter, a nuclear promoter, a constitutive promoter, or an inducible promoter may be used as the promoter.
  • the chloroplast transit peptide, protein of interest, or fusion protein provided herein can be designed to be operably linked to a 5'UTR or 3'UTR to exert a function in the nucleus of an avian.
  • the vector may further include a transcriptional control sequence suitable for avian transformation. Recombinant genes conferring herbicide tolerance may be integrated into, but not limited to, the genome of the nucleus or the genome of the chloroplast in the host bird.
  • the vector may optionally further include a gene encoding a selection marker as a reporter molecule
  • a selection marker include antibiotics (eg, neomycin, carbenicillin, kanamycin, spectinomycin, hygromycin, bleomycin, ampicillin, chloramphenicol, etc.) or herbicides (glyphosate, glufosinate, phosphinothricin, etc.) resistance genes, and the like, but are not limited thereto.
  • the recombinant vector for plant expression does not include a binary vector, a cointegration vector, or a T-DNA (transfer DNA) region, but a general vector designed to be expressed in plants may be used.
  • Agrobacterium as a transformation strain for introducing the recombinant vector into a plant ( Agrobacterium- mediated transformation), in which case Agrobacterium tumefaci Ens ( Agrobacterium tumefaciens ) or Agrobacterium rhizogenes ( Agrobacterium rhizogenes ) can be used.
  • Agrobacterium tumefaci Ens Agrobacterium tumefaciens
  • Agrobacterium rhizogenes Agrobacterium rhizogenes
  • electroporation, particle bombardment, polyethylene glycol-mediated uptake, etc. are used to introduce the recombinant plasmid into the plant. can be used
  • Transgenic plants into which genes are introduced in the above manner can be redifferentiated into plants through processes such as callus induction, rooting and soil acclimatization using standard techniques known in the art.
  • Plants to be transformed in the present specification are, as well as mature plants, plant cells (including suspension-cultured cells) that can develop into mature plants, protoplasts, callus, hypocotyl, seeds (seed) ), cotyledon, shoot, and the like are understood to include all.
  • the transformant category of the present specification includes the transformant into which the gene is introduced, as well as clones or progeny thereof (T 1 generation, T 2 generation, T 3 generation, T 4 generation, T 5 generation, or more) It includes, for example, asexual or sexual progeny of a plant transformed with a chloroplast transit peptide, a target protein, or a fusion protein, and plants that have inherited herbicide tolerance traits are also included in the scope of the transgenic plants of the present invention. Also included within the scope of the present invention are all mutants and variants exhibiting the characteristics of an initially transformed plant, along with all crosses and fusion products of plants transformed with the genes herein.
  • seeds, flowers, stems, fruits, leaves, roots, tubers, and/or tubers originating from the transformed plants previously transformed by the method of the present invention, or their progeny and consisting of at least a part of transformed cells, and Parts of the same plant are also included in the scope of the present invention.
  • Plants to which the present invention can be applied are not particularly limited, and may be at least one selected from the group consisting of monocotyledonous or dicotyledonous plants. It may also be herbaceous or woody.
  • the monocotyledonous plants are Alismataceae, Hydrocharitaceae, Juncaginaceae, Scheuchzeriaceae, Potamogetonaceae, Najadaceae, Zosteraceae, Liliaceae), Haemodoraceae, Agavaceae, Amaryllidaceae, Dioscoreaceae, Pontederiaceae, Iridaceae, Burmanniaceae, Juncaceae, Chicken coleoptera , Eriocaulaceae, Gramineae, Poaceae, Araceae, Lemnaceae, Sparganiaceae, Typhaceae, Musaceae (Cyperaceae), Musaceae ), Ginger family (Zingiberaceae), Cannace
  • the dicotyledonous plants are family (Diapensiaceae), Alderaceae (Clethraceae), Pyrolaceae, Ericaceae, Myrsinaceae, Primulaceae, Plumbaginaceae, Plumbaginaceae, , Styracaceae, Symplocaceae, Symplocaceae, Oleaceae, Loganiaceae, Gentianaceae, Menyanthaceae, oleaceae , Apocynaceae), Asclepiadaceae, Rubiaceae, Polemoniaceae, Convolvulaceae, Boraginaceae, Verbenaceae, Labiatae, Solanaceae (Scrophulariaceae), Bignoniaceae, Acanthaceae, Pedaliaceae, Orobanchaceae.
  • Gesneriaceae Lentibulariaceae, Phrymaceae, Plantaginaceae, Caprifoliaceae, Adoxaceae, Valerianaceae, Dipsacaceae Campanulaceae), Compositae, Myricaceae, Juglandaceae, Salicaceae, Betulaceae, Fagaceae, Ulmaceae, Moraceae , Urticaceae, Santalaceae, Loranthaceae, Polygonaceae, Phytolaccaceae, Nyctaginaceae, Aizoaceae, Purslane (Portulacaceae), Caryophyllaceae, Chenopodiaceae, Amaranthaceae, Cactaceae, Magnoliaceae, Illiciaceae, Lauraceae, Cercidiphyllaceae, Ranunculaceae, Berberidaceae, Lardizabalaceae, Menispermaceae, Nymphaeaceae, Cera
  • the plant includes food crops including rice, wheat, barley, corn, soybean, potato, red bean, oat and sorghum; vegetable crops including Chinese cabbage, radish, red pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, pumpkin, green onion, onion and carrot; specialty crops including ginseng, tobacco, cotton, forage, grass, sesame, sugar cane, sugar beet, perilla, peanut, rapeseed, grass and castor; fruit trees including apple trees, pear trees, jujube trees, peaches, poplars, grapes, tangerines, persimmons, plums, apricots and bananas; woody trees including pine, palm oil and eucalyptus; flowers including roses, gladiolus, gerberas, carnations, chrysanthemums, lilies and tulips; And ryegrass, red clover, orchard grass, alfalfa, may be at least one selected from the group consist
  • the plant is Arabidopsis thaliana, potato, eggplant, tobacco, red pepper, tomato, burdock, mustard greens, lettuce, bellflower, spinach, chard, sweet potato, celery, carrot, water parsley, parsley, Chinese cabbage, cabbage, radish, watermelon, melon , dicot plants such as cucumber, pumpkin, gourd, strawberry, soybean, mung bean, kidney bean, or pea; and one or more selected from the group consisting of monocotyledonous plants such as rice, wheat, barley, corn, and sorghum, but is not limited thereto.
  • Algae to which the present invention can be applied is not particularly limited, and may be prokaryotic algae or eukaryotic algae.
  • the algae may be cyanobacteria, green algae, red algae, brown algae, macroalgae or microalgae.
  • Cyanobacteria include, but are not limited to, the phylum Chroococcales (e.g., Aphanocapsa, Aphanothece, Chamaesiphon, Chondrocystis, Chroococcus, Chroogloeocystis, Crocosphaera, Cyanobacterium, Cyanobium, Cyanodictyon, Cyanosarcina, Cyanothecesis, Dactylococcia, Radiocystis, Rhabdoderma, Snowella, Synechococcus, Synechocystis, Thermosynechococcus, Woronichinia), phyla Gloeobacteria, phyla Nostocales (e.g., Microchaetaceae, Nostocaceae, Rivulariaceae, Scytonemataceae), phyla Oscillatoriales (e.g., Arthroulinalema, Bcytonemataceae), phyla
  • algae may be exemplified by Chlorophyta, Chlamydomonas, Volvacales, Dunaliella, Scenedesmus, Chlorella, or Hematococcm.
  • algae include Phaeodactylum tricornutum, Amphiprora hyaline, Amphora spp., Chaetoceros muelleri, Navicula saprophila, Nitzschia communis, Scenedesmus dimorphus, Scenedesmus obliquus obliquus, Tetraselmis suecica, Chlamydomonas reinhardtii, Chlorocella vialis, Neococolec , Gloeobacter violaceus, Synechocystis, Thermosynechococcus elongatus, Nannochloropsis oculata, Nannochloropsis salina, Nannochloropsis gaditana, Isochrysis galbana, Botryococcus sudeticus, Euglena gracilis, Neochloris oleoabundans, Nitzschia palea, Pleurochrysis carterae, Tetraselmis chu
  • the chloroplast transit peptide provided herein can effectively target the target protein into the chloroplast so that the target protein can better exert a desired effect, thereby imparting useful properties to plants or further enhancing it.
  • FIG. 1 is a schematic diagram of a plant expression vector according to an embodiment.
  • 2A and 2B are fluorescence images showing the intracellular expression location of the green fluorescent protein linked to the C-terminus of the candidate CTP protein in Arabidopsis protoplasts.
  • 3 is a fluorescence image showing the intracellular expression location of the green fluorescent protein linked to the C-terminus of the candidate CTP protein in corn protoplasts.
  • FIG. 4 is a fluorescence image showing the intracellular expression location of the green fluorescent protein linked to the C-terminus of the candidate CTP protein GmCTP44-67 in Arabidopsis thaliana and corn protoplasts.
  • FIG. 5 is a fluorescence image showing the intracellular expression location of green fluorescent protein linked to the C-terminus of candidate CTP proteins GmCTP45-56, GmCTP45-57, GmCTP45-58, GmCTP45-59, and GmCTP45-60 in Arabidopsis and corn protoplasts. to be.
  • FIG. 6 is a fluorescence image showing the intracellular expression location of green fluorescent protein linked to the C-terminus of candidate CTP proteins GmCTP65-45, GmCTP65-50, and GmCTP65-55 in Arabidopsis thaliana and corn protoplasts.
  • FIG. 7 is a fluorescence image showing the intracellular expression location of the green fluorescent protein linked to the C-terminus of the candidate CTP proteins GmCTP65-70, GmCTP65-75, and GmCTP65-80 in corn protoplasts.
  • FIG. 8 schematically shows an example of a plant transformation vector including a gene encoding a fusion protein in which a target protein is fused to the C-terminus of a CTP candidate protein for use in the construction of a transgenic plant.
  • FIG. 9 is a plant transformation vector comprising a gene encoding a fusion protein in which a target protein (mCyPPO10 SEQ ID NO: 327, or CP4EPSPS SEQ ID NO: 330) is fused to the C-terminus of a CTP candidate protein for use in the construction of a transgenic plant schematically shows an example of
  • FIG. 10 is an image showing resistance to tiafenacil of an Arabidopsis transformant (T 2 generation) into which a gene encoding a fusion protein in which a target protein is fused to the C-terminus of the CTP candidate protein is introduced.
  • 11 is an image showing resistance to glyphosate of an Arabidopsis transformant (T 2 generation) into which a gene encoding a fusion protein in which a target protein is fused to the C-terminus of a CTP candidate protein is introduced.
  • FIG. 12 is an image showing resistance to isoxaflutole of an Arabidopsis transformant (T 2 generation) into which a gene encoding a fusion protein in which a target protein is fused to the C-terminus of the CTP candidate protein is introduced.
  • FIG. 13 is an image showing resistance to glufosinate, tiafenacil, or glyphosate of an Arabidopsis transformant (T 2 generation) into which a gene encoding a fusion protein in which a target protein is fused to the C-terminus of the CTP candidate protein is introduced.
  • FIG. 14 is a schematic diagram of a vector for plant transformation of p35S-AtPPO1TP-CyPPO2-HA according to an embodiment.
  • 15 is a result of western blotting of proteins expressed in cells of Arabidopsis plants transformed using a vector for transformation of p35S-AtPPO1TP-CyPPO2-HA using AtPPO1TP of various lengths.
  • 16 is a schematic diagram of a vector for p35S-AtPPO1TP-CyPPO2-YFP plant transformation according to an embodiment.
  • 17A and 17B are fluorescence images showing intracellular protein expression positions in tobacco transformed using a vector for transformation of p35S-AtPPO1TP-CyPPO2-YFP using AtPPO1TP of various lengths.
  • 19 is a schematic diagram of a vector for transformation of p35S-AtPPO1TP-37-mCyPPO8 soybean according to an embodiment.
  • 21 is a schematic diagram of a vector for transformation of p35S-AtPPO1TP-37-mCyPPO10 soybean according to an embodiment.
  • 22A and 22B are images showing the resistance to various herbicides of AtPPO1TP-37 and mCyPPO10 introduced soybean transformants (T 2 generation).
  • CTP chloroplast transit peptide
  • the DNA sequence (Table 2) of the candidate gene encoding the 77 kinds of CTP putative protein sequences selected in Example 1 was amplified by PCR, and restriction enzyme digestion/ligation or In-Fusion® HD Cloning Kit (Clontech) , A plant expression vector having the structure of FIG. 1 was prepared by inserting it between the CaMV35S promoter (Cauliflower mosaic virus 35S promoter, p35S) and GFP (Green fluorescent protein) of the plant expression vector.
  • CaMV35S promoter Cauliflower mosaic virus 35S promoter, p35S
  • GFP Green fluorescent protein
  • the PCR reaction solution is Template (Maverick bean gDNA, 100 ng/ ⁇ l) 1 ⁇ l, 10X buffer 5 ⁇ l, dNTP mixture (10 mM each) 1 ⁇ l, forward primer (see Table 3; 10 uM) 1 ⁇ l, reverse Primer (see Table 3; 10 uM) 1 ⁇ l, DDW 40 ⁇ l, and 50 ⁇ l of Pfu-X (Solgent, 2.5 unit/ ⁇ l) 1 ⁇ l, etc., were reacted at 94° C. for 4 minutes, followed by 30 cycles ( 30 seconds at 94°C, 30 seconds at 57°C, and 1.5 minutes at 72°C) and amplified by reaction at 72°C for 5 minutes.
  • the primer sequences used for gene amplification as described above are summarized in Table 3 below. :
  • the restriction enzyme treatment was performed under the following conditions:
  • PCR product 30 ⁇ l, BamHI and StuI (New England Biolabs) 0.5 ⁇ l each, 10X buffer 4 ⁇ l, water 5 ⁇ l; Restriction enzyme reaction 37°C, 2 hours
  • the ligation reaction was carried out under the following conditions:
  • PCR product 3 ⁇ l, H 2 O 4 ⁇ l 5X In-Fusion HD Enzyme Premix 2 ⁇ l, restriction enzyme-treated vector 1 ⁇ l (100 ng); 50°C, 10 min.
  • a method of expressing a gene by injecting a high concentration of a plant expression vector into a protoplast from which the cell wall of a plant cell has been removed, and analyzing the intracellular expression location of a protein is well known (Nat. Protoc. (2007) 2, 1565-1572, BMC). Biotechnology (2017) 17:29).
  • GFP expression in Arabidopsis thaliana protoplasts was confirmed by the following procedure: After culturing wild-type Arabidopsis Colombia-0 (Col-0) for 4 weeks, the leaves were chopped and enzyme solution (1% Cellulase R- 10, 0.25% Macerozyme R-10, 0.1% BSA, 0.4 M mannitol, 20 mM KCl, 10 mM CaCl 2 , 20 mM MES, pH 5.7) and incubated for 6 hours at room temperature to separate protoplasts.
  • enzyme solution 1% Cellulase R- 10, 0.25% Macerozyme R-10, 0.1% BSA, 0.4 M mannitol, 20 mM KCl, 10 mM CaCl 2 , 20 mM MES, pH 5.7
  • MMG solution 0.4 M mannitol, 15 mM MgCl 2 , 4 mM MES, pH 5.7
  • PEG solution 0.4 M mannitol, 100 mM CaCl 2 , 40% PEG4000
  • Protoplasts were recovered using a small centrifuge (1,000 rpm, 1 min), and placed in a 12-well plate containing 1 mL of W5 solution and incubated at room temperature for more than 6 hours. Using a fluorescence microscope (Olympus IX70), the intracellular expression location of the GFP protein was confirmed.
  • FIGS. 2A and 2B The obtained results are shown in FIGS. 2A and 2B.
  • GFP As shown in Figures 2a and 2b, in the cells injected with the p35S-GFP vector into which GmCTP is not inserted, GFP is spread throughout the cytoplasm and GFP is not observed in the chloroplast, whereas 77 plant expression vectors were introduced. Among the cells, GFP was observed only in the chloroplast in the cells in which GFP was fused to 37 GmCTP (see FIGS. 2A and 2B ) tested.
  • the 37 kinds of GmCTP in dicotyledonous plants can move GFP fused to the C-terminal to the chloroplast, and the target protein is transferred to the chloroplast by replacing the GFP with the desired protein sequence. It was confirmed that it can be used for Using this method, a dicot transgenic plant, cell line, seed, etc. in which the target protein is transferred to the chloroplast can be prepared.
  • Example 3 the function of moving the target protein to the chloroplast using GmCTP in Arabidopsis protoplasts, a dicot plant, was analyzed using corn protoplasts to confirm whether the same function could be performed in monocot plants.
  • GmCTP44, GmCTP45, and GmCTP65 are selected among GmCTPs whose function has been confirmed in Arabidopsis, and protein length information is added to add GmCTP44-87 (CTP containing from the first to the 87th amino acid of the N-terminal among the amino acid sequence of GmCTP44). ; later named in the same way), GmCTP45-76, and GmCTP65-65.
  • GFP expression in corn protoplasts was confirmed by the following procedure: After culturing wild-type corn (Ilmichalic) for 2 weeks, the leaves were chopped and the enzyme solution (1% Cellulase R-10, 0.25% Macerozyme R-10, 0.1 % BSA, 0.4 M mannitol, 20 mM KCl, 10 mM CaCl 2 , 20 mM MES, pH 5.7) and incubated for 6 hours at room temperature to separate protoplasts.
  • the enzyme solution 1% Cellulase R-10, 0.25% Macerozyme R-10, 0.1 % BSA, 0.4 M mannitol, 20 mM KCl, 10 mM CaCl 2 , 20 mM MES, pH 5.7
  • MMG solution 0.4 M mannitol, 15 mM MgCl 2 , 4 mM MES, pH 5.7
  • PEG solution 0.4 M mannitol, 100 mM CaCl 2 , 40% PEG4000
  • Protoplasts were recovered using a small centrifuge (1,000 rpm, 1 min), and placed in a 12-well plate containing 1 mL of W5 solution and incubated at room temperature for more than 6 hours. Using a fluorescence microscope (Olympus IX70), the intracellular expression location of the GFP protein was confirmed.
  • each p35S-GmCTP-GFP vector was estimated using each p35S-GmCTP-GFP vector GmCTP44-67 with a different number of amino acids based on the cleavage site (CTP including up to the 67th amino acid of the N-terminal in the GmCTP44 protein sequence; named in the same manner hereinafter), GmCTP45-56, GmCTP45-57, GmCTP45-58 , GmCTP45-59, GmCTP45-60, GmCTP65-45, GmCTP65-50, and plant expression vectors containing GmCTP65-55 were constructed.
  • each of the p35S-GmCTP-GFP vectors prepared in Example 2 was used as a template and the primers in Table 4 were used to prepare vectors having different lengths of the GmCTP region by site-directed mutagenesis. .
  • GFP was observed not only in the chloroplast but also in the cytoplasm, and from GmCTP45-58 including up to the 58th amino acid to the chloroplast. It was confirmed that the transport function was normally performed.
  • GmCTP65 including GmCTP65-45 including up to the 45th amino acid presumed to be a cleavage site, and even GmCTP65-55, GFP is not observed in the chloroplast but in the entire cell, including the cytoplasm, and performs a normal function. was found not to be possible.
  • GmCTP65-70, GmCTP65-75 and GmCTP65-80 were cloned into a plant expression vector.
  • the protein sequences and DNA sequences used for cloning are shown in Tables 5 and 6.
  • GmCTP65-70, GmCTP65-75 and GmCTP65-80 performed the chloroplast migration function in Arabidopsis thaliana and corn. Among them, it was confirmed that GmCTP65-80 had the best efficiency.
  • a PPO (Protoporphyrinogen Oxidase) herbicide resistance gene was fused to the C-terminus of three GmCTPs (GmCTP44-67, GmCTP45-58, or GmCTP65-80), which were confirmed to move the target protein to the chloroplast in dicotyledonous and monocot plants.
  • a plant transformation vector (refer to FIGS. 8 to 9) was prepared and transformed into Agrobacteria, and transformants were obtained by inoculating in the fire of Arabidopsis thaliana using a transformation method mediated by Agrobacteria.
  • a mutant of PPO derived from Thermosynechococcus elongatus BP-1 (mCyPPO10: SEQ ID NO: 327), Halothece sp.
  • Genes encoding a variant of PPO derived from PCC 7418 (mCyPPO8: SEQ ID NO: 328), and a variant of PPO derived from the Synechococcus sp. JA-3-3Ab strain (mCyPPO13-2: SEQ ID NO: 329), respectively was used.
  • glyphosate resistance gene CP4EPSPS glyphosate resistant 5-enolpyruvylshikimate-3-phosphate synthase: SEQ ID NO: 330
  • PfHPPD plastid inhibitory herbicide resistance gene
  • Arabidopsis transformants ( ⁇ CTP-mCyPPO10) were prepared using a plant transformation vector in which no CTP was inserted at the N-terminus of mCyPPO10 (SEQ ID NO: 327).
  • Arabidopsis transformation was performed as follows: Each of the vectors prepared above was introduced into Agrobacterium tumefaciens GV3101 competent cells by freeze-thaw method to transform them. Each transformed Agrobacterium was cultured and selected in antibiotic medium (LB agar containing spectinomycin). The selected colonies were liquid-cultured in LB broth. After harvesting Agrobacterium cells from this culture medium, the absorbance (OD 600 ) was suspended in a 5% (w/v) sucrose solution at a concentration of 0.8, and then 0.05% (v/v) Silwet L-77 (Momentive Performance Materials) was added. added. Transformed into wild-type Arabidopsis thaliana (Col-0) by the Floral dipping method, T 1 seeds were harvested.
  • antibiotic medium LB agar containing spectinomycin
  • T2 seeds were harvested from selected plants by spraying BASTA (50 mg/L) or Glyphosate (0.06%) according to the selection marker of each transformant.
  • T2 seeds were sown in each selection medium (25 uM glufosinate or 100 uM glyphosate 1/2 MS medium), and lines showing a separation ratio of about 3:1 (Live:Dead) were selected, transplanted into soil, and then cultured for 3 weeks. .
  • tiafenacil 4.3 g ai/ha
  • glyphosate (1 kg ai/ha
  • isoxaflutole 24 g ai/ha
  • Example 8 A method for transferring a target protein to the chloroplast of a dicotyledon using AtPPO1 CTP
  • the AtPPO1 gene is a PPO1 gene of Arabidopsis, and encodes an AtPPO1 protein that performs an important function in chlorophyll synthesis in chloroplasts.
  • the N-terminal portion of the AtPPO1 protein (SEQ ID NO: 326) has a sequence that functions as a CTP, and when the AtPPO1 protein moves to the chloroplast, it is cleaved at a specific position.
  • a tool for estimating the CTP cleavage site (ChloroP, http://www.cbs.dtu.dk/services/ChloroP/)
  • the 33rd to 35th amino acid positions of the AtPPO1 protein sequence were estimated as the cleavage site.
  • AtPPO1TP-28 CTP including up to the 28th amino acid of the N-terminal in the AtPPO1 amino acid sequence (SEQ ID NO: 326); hereinafter named in the same manner
  • PPO without sequence; SEQ ID NO: 325) was inserted between the coding genes, and a hemagglutinin (HA) tag was linked to the C-terminus of CyPPO2 (see FIG. 14 ).
  • AtPPO1TP-28 amino acid sequences of AtPPO1TP-28, AtPPO1TP-30, AtPPO1TP-31, AtPPO1TP-32 AtPPO1TP-33, AtPPO1TP-35, AtPPO1TP-37, and AtPPO1 TP-52 are shown in Table 8 below:
  • Each plant expression vector was transformed into Arabidopsis by the method of Example 7, transformed into Arabidopsis plants to extract proteins, and western blot was performed using an HA antibody.
  • AtPPO1TP normally moves CyPPO2 into the chloroplast within the cell, it is cleaved at a specific amino acid position and the size of the protein band is similar to the original size of CyPPO2 during western blot. detected.
  • proteins having a large size were identified from AtPPO1TP-28 (28aa) to AtPPO1TP-32 (32aa), whereas proteins having a small size were identified from AtPPO1TP-33 (33aa).
  • These results show that the cleavage site of the N-terminal site having CTP activity of AtPPO1 is after the 33rd amino acid residue (C-terminal side).
  • AtPPO1TP-25 (MELSLLRPTTQSLLPSFSKPNLRLN; SEQ ID NO: 340), AtPPO1TP-26 (MELSLLRPTTQSLLPSFSKPNLRLNV; SEQ ID NO: 341), AtPPO1TP-30 (SEQ ID NO: 333), AtPPO1TP-31 (SEQ ID NO: 334), AtPPO1TP-32 (SEQ ID NO: 335) , AtPPO1TP-33 (SEQ ID NO: 336), AtPPO1TP-35 (SEQ ID NO: 337), AtPPO1TP-37 (SEQ ID NO: 338), and AtPPO1TP-52 (SEQ ID NO: 339) of the coding genes of the plant transformation vector CaMV35S promoter ( p35S) and the CyPPO2 gene, and YFP was inserted at the 3' end of the CyPPO2 gene (see FIG. 16 ).
  • FIGS. 17a and 17b After injecting the prepared plant transformation vector into tobacco ( Nicotiana benthamiana ) by Agro-infiltration transformation, it was cultured for 2 days to be expressed, and the protein expression position was confirmed with a fluorescence microscope, and the results are shown in FIGS. 17a and 17b. .
  • FIGS. 17A and 17B As shown in FIGS. 17A and 17B , when using AtPPO1TP-31, AtPPO1TP-32, AtPPO1TP-33, AtPPO1TP-35, AtPPO1TP-37, chloroplasts were targeted, whereas AtPPO1TP-25, AtPPO1TP-26, and AtPPO1TP-30 were used. When used, expression was also confirmed in the cytoplasm.
  • AtPPO1TP-31, AtPPO1TP-32, AtPPO1TP-33, AtPPO1TP-35, AtPPO1TP-37, etc. in dicotyledonous plants can transfer the target protein and fluorescent protein fused to the C-terminal to the chloroplast.
  • Based on the Western blotting results and fluorescence microscopy analysis it is determined that a sequence of at least AtPPO1TP-37 or higher is required for normal CTP function. Using this method, a dicot transgenic plant, cell line, seed, etc. in which the target protein is transferred to the chloroplast can be prepared.
  • AtPPO1TP-37 (SEQ ID NO: 338) followed by (C-terminal) CyPPO2 (SEQ ID NO: 325)
  • T 1 transformant containing a gene encoding a fusion protein was cultured for 4 weeks, and tiafenacil (2 g ai/ha) was evenly sprayed with 0.05% (v/v) Silwet L-77.
  • tiafenacil (2 g ai/ha) was evenly sprayed with 0.05% (v/v) Silwet L-77.
  • a separate plant transformation vector was prepared by fusion of AtPPO1TP-37 followed by mCyPPO8 (mutated PPO gene derived from Halothece PCC7418, SEQ ID NO: 328) to prepare soybean transformants (FIG. 19).
  • AtPPO1TP-37 and mCyPPO8 were amplified by PCR.
  • Entry vector was prepared by cloning PCR product into pENTR-TOPO vector (Invitrogen) using pENTR Directional TOPO cloning kits (Invitrogen). From the prepared entry vector, a vector for soybean transformation was prepared using the Gateway LR Clonase II Enzyme Mix kit (Invitrogen).
  • Each of the prepared vectors was transformed into Agrobacterium EHA105 by the electro-transformation method (Trans Res. (1993) 2:208-218), and a soybean (Gwangan) transformant was obtained using this.
  • the seeds immersed in sterilized tertiary water were cut vertically up to the hypocotyl, and the seed coat was removed. After cutting the hypocotyl at about 1 cm below the cotyledon, the side where the embryonic axis is attached was cut 8 times.
  • the explants were mixed with the transformed EHA105, sonicated for 20 seconds, and inoculated for 30 minutes. After removing the water from the explants, place the adaxial part facing down on solid CCM (Co-cultivation medium; Gamborg B5 0.32 g/L, MES 4.26 g/L, Sucrose 30 g/L, Agar 0.7%).
  • Each plate was sealed with micropore tape, and then incubated at 25 °C and a photoperiod for 18 hours. After 2 weeks, only the shoot part of the explant was cut, and the adaxial part was facing down, and the puncture was applied to the SIM containing the selective antibiotic PPT 10 mg/L.
  • a separate plant transformation vector was prepared by fusion of AtPPO1TP-37 followed by mCyPPO10 (SEQ ID NO: 327) to prepare a soybean transformant (FIG. 21).
  • soybean transformants were prepared in the same manner as above, and T 2 seeds were obtained from T 1 plants.
  • wild-type soybean Gwangan (Kwangan) was all killed, whereas the transformation event exhibited strong tolerance in all the herbicide combinations analyzed (Fig. 22a (Fig. wild type) and 22b (transformants)).

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Plant Pathology (AREA)
  • Botany (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Lubricants (AREA)

Abstract

L'invention concerne des polypeptides capables de migrer vers les chloroplastes de cellules végétales, et l'utilisation des polypeptides pour transporter une protéine cible vers les chloroplastes.
PCT/KR2020/018969 2019-12-24 2020-12-23 Peptides de transit de chloroplastes et leur utilisation Ceased WO2021133049A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR10-2019-0174547 2019-12-24
KR20190174547 2019-12-24

Publications (1)

Publication Number Publication Date
WO2021133049A1 true WO2021133049A1 (fr) 2021-07-01

Family

ID=76575604

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2020/018969 Ceased WO2021133049A1 (fr) 2019-12-24 2020-12-23 Peptides de transit de chloroplastes et leur utilisation

Country Status (3)

Country Link
KR (1) KR20210082101A (fr)
AR (1) AR120895A1 (fr)
WO (1) WO2021133049A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN120004999A (zh) * 2025-04-16 2025-05-16 北京国锐生科有限公司 叶绿体转运肽

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20010039484A (ko) * 1999-10-11 2001-05-15 백경환 프로토포르피리노겐 옥시다아제 유전자를 이용한 작물의수량 또는 바이오매스의 증대 방법
US20090029861A1 (en) * 2007-02-26 2009-01-29 Monsanto Technology Llc Chloroplast transit peptides for efficient targeting of dmo and uses thereof
US20120042412A1 (en) * 2010-08-13 2012-02-16 Pioneer Hi-Bred International, Inc. Methods And Compositions For Targeting Sequences Of Interest To The Chloroplast
KR20180038048A (ko) * 2015-08-20 2018-04-13 다우 아그로사이언시즈 엘엘씨 엽록체 전이 펩티드

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20010039484A (ko) * 1999-10-11 2001-05-15 백경환 프로토포르피리노겐 옥시다아제 유전자를 이용한 작물의수량 또는 바이오매스의 증대 방법
US20090029861A1 (en) * 2007-02-26 2009-01-29 Monsanto Technology Llc Chloroplast transit peptides for efficient targeting of dmo and uses thereof
US20120042412A1 (en) * 2010-08-13 2012-02-16 Pioneer Hi-Bred International, Inc. Methods And Compositions For Targeting Sequences Of Interest To The Chloroplast
KR20180038048A (ko) * 2015-08-20 2018-04-13 다우 아그로사이언시즈 엘엘씨 엽록체 전이 펩티드

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
HA S B,ET AL: "The plastidic Arabidopsis protoporphyrinogen IX oxidase gene, with or without the transit sequence, confers resistance to the diphenyl ether herbicide in rice.", PLANT CELL AND ENVIRONMENT, vol. 27, no. 1, 1 January 2004 (2004-01-01), GB, pages 79 - 88, XP002344705, ISSN: 0140-7791, DOI: 10.1046/j.0016-8025.2003.01127.x *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN120004999A (zh) * 2025-04-16 2025-05-16 北京国锐生科有限公司 叶绿体转运肽

Also Published As

Publication number Publication date
KR20210082101A (ko) 2021-07-02
AR120895A1 (es) 2022-03-30

Similar Documents

Publication Publication Date Title
KR102305484B1 (ko) 남세균 유래 프로토포르피리노겐 ix 옥시다아제의 변이체를 이용하는 제초제 내성 부여 및/또는 증진을 위한 조성물 및 방법
KR102250017B1 (ko) 프로토포르피리노겐 옥시다아제 또는 이의 변이체를 이용하는 제초제 내성 부여 및/또는 증진을 위한 조성물 및 방법
KR101827545B1 (ko) 프로토포르피리노겐 옥시다아제를 이용한 식물 및/또는 조류의 제초제 저항성 부여 또는 증진 방법
CN109641940B (zh) 原卟啉原氧化酶变体及使用其赋予和/或增强除草剂耐受性的方法和组合物
KR102227354B1 (ko) Ppo의 변이체를 이용하는 제초제 내성 부여 및/또는 증진을 위한 조성물 및 방법
WO2020251313A2 (fr) Procédés et compositions permettant de conférer et/ou améliorer la tolérance aux herbicides à l'aide de protoporphyrinogène ix oxydase de diverses cyanobactéries ou d'un variant associé
WO2021133049A1 (fr) Peptides de transit de chloroplastes et leur utilisation
WO2023191588A1 (fr) Procédés et compositions pour conférer et/ou améliorer la tolérance à la trifludimoxazine à l'aide d'un variant de protoporphyrinogène oxydase

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20906836

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20906836

Country of ref document: EP

Kind code of ref document: A1