WO2016159560A1 - Gène ibhppd dérivé de la patate douce pour réguler la tolérance des plantes à une contrainte environnementale, et utilisation correspondante - Google Patents

Gène ibhppd dérivé de la patate douce pour réguler la tolérance des plantes à une contrainte environnementale, et utilisation correspondante Download PDF

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WO2016159560A1
WO2016159560A1 PCT/KR2016/002916 KR2016002916W WO2016159560A1 WO 2016159560 A1 WO2016159560 A1 WO 2016159560A1 KR 2016002916 W KR2016002916 W KR 2016002916W WO 2016159560 A1 WO2016159560 A1 WO 2016159560A1
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ibhppd
gene
plant
environmental stress
protein
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Korean (ko)
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곽상수
이행순
정재철
지창윤
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한국생명공학연구원
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    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A01H5/10—Seeds
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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)

Definitions

  • Sweet potato ( Ipomoea batatas ) can be grown on relatively poor lands and is grown in more than 100 countries around the world. It is the seventh major crop in the world after wheat, rice, corn, potatoes, barley and cassava. It is a representative root crop produced. Sweet potato leaves and petioles contain ingredients that are not found in ordinary grains such as vitamins B and C, tocopherol, calcium and iron, and have a wide range of polyphenols, making them highly valuable as vegetables and rich in carbohydrates in sweet potato storage roots. become a source of energy.
  • Tocopherol is one of the hydrophobic low molecular weight antioxidants synthesized in the chloroplast. It protects plants from singlet oxygen and protects photosystems and membrane lipids by preventing lipid peroxidation. In addition, tocopherol prevents lipid peroxidation during seed germination and is resistant to stress such as excessive metal ions and low temperatures.
  • Pectobacterium chrysanthemi is infected through the wound, and black or brown symptoms appear throughout the sweet potato stems, petioles, and storage roots. It is a plant disease that is a problem worldwide with a large loss. However, there are no resistant varieties in sweet potatoes and there is no clear control method.
  • Korean Patent No. 121277 discloses ' IbLEA14 gene derived from sweet potato root and its use'
  • Korean Patent No. 0930593 discloses 'swDREB1 protein derived from sweet potato root and gene encoding the same'
  • the present invention is derived by the request as described above, in the present invention by using the plant with the expression of IbHPPD gene regulation, it was confirmed that IbHPPD gene is involved in environmental stress tolerance of a plant control. In particular, it was confirmed that the IbHPPD gene is strongly expressed in the leaves, especially under environmental stress conditions such as pathogen infection as well as drying and oxidative stress. Accordingly, the present invention was completed by confirming that the plant can produce a plant and its seeds whose environmental stress resistance is controlled using the gene.
  • the present invention provides an amino acid sequence of SEQ ID NO: 2, IbPOPD ( Ipomoea batatas 4-hydroxyphenylpyruvate dioxygenase) protein that regulates environmental stress resistance.
  • IbPOPD Ipomoea batatas 4-hydroxyphenylpyruvate dioxygenase
  • the present invention also provides a gene encoding the protein.
  • the present invention also provides a host cell transformed with the recombinant vector.
  • the present invention also provides a method of controlling the environmental stress resistance of a plant comprising the step of transforming the plant cell with the recombinant vector to control the expression of the IbHPPD gene.
  • the present invention provides a method for producing a transformed plant with controlled environmental stress resistance, comprising the step of transforming plant cells with the recombinant vector.
  • the present invention also provides a composition for controlling environmental stress resistance of plants containing a gene encoding IbHPPD protein derived from sweet potato as an active ingredient.
  • the present invention relates to a potato-derived to control the environmental stress tolerance of a plant IbHPPD gene and use thereof, IbHPPD gene of potato origin according to the invention is especially under environmental stress conditions, such as drying, oxidizing and pathogen infection, strong in leaf Since expression is induced, it may be usefully used to develop transgenic plants and biofuel crops that are resistant to environmental stresses suitable for conditioned adverse areas.
  • environmental stress conditions such as drying, oxidizing and pathogen infection, strong in leaf Since expression is induced, it may be usefully used to develop transgenic plants and biofuel crops that are resistant to environmental stresses suitable for conditioned adverse areas.
  • 1 is a view showing the nucleotide sequence of the sweet potato-derived IbHPPD gene according to the present invention and the amino acid sequence deduced therefrom.
  • Figure 2 is a diagram showing the domain of the deduced protein of sweet potato-derived IbHPPD gene according to the present invention.
  • IbHPPD is a sweet potato-derived gene
  • LsHPPD is a gene derived from lettuce
  • BnHPPD is a rapeseed gene
  • AtHPPD is a gene derived from Arabidopsis
  • MiHPPD is a gene derived from mango
  • ZmHPPD is a gene derived from corn.
  • FIG. 4 is a view showing a flexible relationship between the HPPD gene of the amino acid sequence and various plants (lettuce, rapeseed, Arabidopsis thaliana, mango and corn) of the deduced protein of potato-derived IbHPPD gene according to the invention.
  • IbHPPD is a sweet potato-derived gene
  • LsHPPD is a gene derived from lettuce
  • BnHPPD is a rapeseed gene
  • AtHPPD is a gene derived from Arabidopsis
  • MiHPPD is a gene derived from mango
  • ZmHPPD is a gene derived from corn.
  • Figure 5 is a graph of real-time PCR analysis of the expression pattern of the sweet potato for the IbHPPD gene according to the present invention.
  • L is the leaf
  • S is the stem
  • FR is the fibrous root
  • TR is the thick pigmented root
  • SR is the storage root.
  • Figure 6 is treated with 30% PEG (polyethylene glycol 6000) on the sweet potato leaves after the 0, 2, 4, 6 and 12 hours, IbHPPD gene expression of the graph analyzed by Realtime PCR.
  • PEG polyethylene glycol 6000
  • Figure 9 is a graph (B) showing the expression pattern and tocopherol content of IbHPPD gene in the leaves (A) of tobacco plants inoculated with Agrobacterium mediated IbHPPD gene.
  • FIG. 10 is a graph (B) showing plant photos (A) and survival rates when oxidative stress was applied to Arabidopsis non-transformants (WT) and Arabidopsis transformants overexpressing IbHPPD genes.
  • the present invention provides an Ipomoea batatas 4-hydroxyphenylpyruvate dioxygenase (IbHPPD) protein that regulates environmental stress resistance, consisting of the amino acid sequence of SEQ ID NO: 2.
  • IbHPPD 4-hydroxyphenylpyruvate dioxygenase
  • the range of IbHPPD proteins according to the present invention includes proteins having an amino acid sequence represented by SEQ ID NO: 2 isolated from sweet potatoes and functional equivalents of the proteins.
  • “Functional equivalent” means at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 70% of the amino acid sequence represented by SEQ ID NO: 2 as a result of the addition, substitution, or deletion of the amino acid Is 95% or more of sequence homology, and refers to a protein that exhibits substantially homogeneous physiological activity with the protein represented by SEQ ID NO: 2.
  • substantially homogeneous physiological activity is meant an activity that enhances the plant's resistance to environmental stress.
  • the invention also includes fragments, derivatives and analogues of the IbHPPD protein.
  • fragment refers to a polypeptide that retains the same biological function or activity as the IbHPPD polypeptide of the invention.
  • Fragments, derivatives, and analogs of the present invention comprise (i) polypeptides substituted with one or more conservative or nonconservative amino acid residues (preferably conservative amino acid residues), wherein the substituted amino acid residues are encoded by a genetic code.
  • the present invention also provides a gene encoding the IbHPPD protein.
  • the gene of the present invention may be DNA or RNA encoding IbHPPD protein.
  • DNA includes cDNA, genomic DNA or artificial synthetic DNA.
  • DNA can be single stranded or double stranded.
  • DNA may be a coding strand or a noncoding strand.
  • the gene of the present invention may include the nucleotide sequence represented by SEQ ID NO: 1.
  • polynucleotide encoding a polypeptide refers to a polynucleotide encoding a polypeptide, or a polynucleotide further comprising additional coding and / or noncoding sequences.
  • the invention also relates to variants of said polynucleotides encoding polypeptides comprising the same amino acid sequence as described herein, or fragments, analogs, and derivatives thereof.
  • Polynucleotide variants may be naturally occurring allelic variants or non-naturally occurring variants. Such nucleotide variants include substitutional variants, deletional variants, and insertional variants.
  • allelic variants are alternatives to polynucleotides, which may include one or more substitutions, deletions, or inserted nucleotides, which do not result in a substantial functional change in the polypeptide encoded by the variant. Do not.
  • the present invention provides a poly-hybridized hybridization with a sequence having at least 50%, preferably at least 70%, more preferably at least 80% identity with a nucleotide sequence of SEQ ID NO. It relates to nucleotides.
  • the present invention particularly relates to polynucleotides that hybridize to the polynucleotides described herein under stringent conditions.
  • stringent conditions include (1) hybridization and washing under 0.2 ⁇ SSC, 0.1% SDS, lower ionic strength such as 60 ° C.
  • hybridizable polynucleotide are identical to the biological function and activity of the mature polypeptide represented by SEQ ID NO: 2.
  • the IbHPPD gene is preferably derived from sweet potatoes.
  • embodiments of the invention have high homology (eg, 60% or more, i.e. 70%, 80%, 85%, 90%, 95%, even 98% sequence identity) with the sweet potato IbHPPD gene and other It also includes other genes derived from plants. Sequencing methods and means for determining sequence identity or homology (eg BLAST) are well known in the art.
  • recombinant refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a protein encoded by a peptide, a heterologous peptide, or a heterologous nucleic acid.
  • Recombinant cells can express genes or gene fragments that are not found in their natural form in either the sense or antisense form.
  • Recombinant cells can also express genes found in natural cells, but the genes are modified and reintroduced into cells by artificial means.
  • the polynucleotide sequence encoding the IbHPPD protein can be inserted into a recombinant expression vector.
  • recombinant expression vector means a bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus, or other vector. In principle, any plasmid and vector can be used as long as it can replicate and stabilize in the host.
  • An important feature of the expression vector is that it has an origin of replication, a promoter, a marker gene and a translation control element.
  • Expression vectors comprising IbHPPD protein-encoding DNA sequences and appropriate transcriptional / translational control signals can be constructed by methods well known to those of skill in the art. Such methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence can be effectively linked to a suitable promoter in the expression vector to drive mRNA synthesis. Expression vectors may also include ribosomal binding sites and transcription terminators as translation initiation sites.
  • Preferred examples of recombinant vectors of the present invention are Ti plasmid vectors capable of transferring part of themselves, the so-called T region, to plant cells when present in a suitable host such as Agrobacterium tumerfaciens.
  • Another type of Ti plasmid vector (see EP 0 116 718 B1) is currently used to transfer hybrid DNA sequences to protoplasts from which plant cells or new plants can be produced that properly insert hybrid DNA into the plant's genome.
  • a particularly preferred form of the Ti plasmid vector is the so-called binary vector as claimed in EP 0 120 516 B1 and US Pat. No. 4,940,838.
  • viral vectors such as those which can be derived from double stranded plant viruses (eg CaMV) and single stranded viruses, gemini viruses, etc.
  • it may be selected from an incomplete plant viral vector.
  • the recombinant vector may be, but is not limited to, a pKBS11 vector, a pBI101 vector, and a pCAMBIA vector.
  • the expression vector will preferably comprise one or more selectable markers.
  • the marker is typically a nucleic acid sequence having properties that can be selected by chemical methods, and all genes that can distinguish transformed cells from non-transformed cells. Examples include herbicide resistance genes such as glyphosate or phosphinothricin, kanamycin, G418, bleomycin, hygromycin, and chloramphenicol. Resistance genes include, but are not limited to.
  • the promoter may be, but is not limited to, CaMV 35S, actin, ubiquitin, pEMU, MAS or histone promoter.
  • the term “promoter” refers to a region of DNA upstream from a structural gene and refers to a DNA molecule to which an RNA polymerase binds to initiate transcription.
  • a "plant promoter” is a promoter capable of initiating transcription in plant cells.
  • a “constitutive promoter” is a promoter that is active under most environmental conditions and developmental conditions or cell differentiation. Constitutive promoters may be preferred in the present invention because selection of the transformants may be made by various tissues at various stages. Thus, the constitutive promoter does not limit the selection possibilities.
  • terminators can be used, for example nopalin synthase (NOS), rice ⁇ amylase RAmy1 A terminator, phaseoline terminator, Agrobacterium tumefaciens (Agrobacterium tumefaciens) Terminator of the octopine gene, etc., but is not limited thereto.
  • NOS nopalin synthase
  • rice ⁇ amylase RAmy1 A terminator Phaseoline terminator
  • Agrobacterium tumefaciens Agrobacterium tumefaciens
  • Terminator of the octopine gene etc.
  • the present invention also provides a host cell transformed with the recombinant vector of the present invention.
  • the host cell capable of continuously cloning and expressing the vector of the present invention in a prokaryotic cell while being stable can be used in any host cell known in the art, for example, E. coli JM109, E. coli BL21, E. coli RR1. , Bacillus genus strains, such as E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus thuringiensis, and Salmonella typhimurium, Serratia marcensons, and various Pseudomonas Enterobacteria such as species and strains.
  • yeast Saccharomyce cerevisiae
  • insect cells human cells
  • human cells e.g., CHO cell line (Chinese hamster ovary), W138, BHK, COS7, 293, HepG2) , 3T3, RIN and MDCK cell lines
  • the host cell is preferably a plant cell.
  • the method of carrying the vector of the present invention into a host cell includes a CaCl 2 method, one method (Hanahan, D., J. Mol. Biol., 166: 557580 (1983)) and when the host cell is a prokaryotic cell. It may be carried out by a method of drilling.
  • the vector can be injected into the host cell by microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, DEAEdextran treatment, gene bombardment, or the like. .
  • the invention also, by transforming a plant cell with a recombinant vector containing the gene encoding the IbHPPD protein of potato-derived provides a method for controlling the environmental stress tolerance of a plant comprising the step of regulating the expression of IbHPPD gene.
  • IbHPPD proteins includes proteins having the amino acid sequence represented by SEQ ID NO: 2 and functional equivalents of the proteins.
  • the “gene expression control” of the present invention refers to increasing or decreasing the expression of the IbHPPD gene in the plant.
  • a method of controlling the environmental stress resistance of the plant by transforming the plant cell with a recombinant vector comprising the gene of SEQ ID NO: 1 overexpressing the IbHPPD gene to the environmental stress resistance of the plant May be increased, but is not limited thereto.
  • the environmental stress may be dry, oxidative or pathogen infection stress, preferably pathogen infection stress, most preferably sweet potato bacterial stem root rot pathogen ( Pectobacetrium chrysanthemi ) Infection stress may be, but is not limited to.
  • the "gene overexpression” means that the gene is expressed above the level expressed in wild-type plants.
  • a method of introducing the gene into a plant there is a method of transforming a plant using an expression vector containing the gene under the control of a promoter.
  • Plant cells used for plant transformation may be any plant cells.
  • Plant cells are cultured cells, cultured tissues, culture organs or whole plants.
  • Plant tissue refers to the tissues of differentiated or undifferentiated plants, such as, but not limited to, roots, stems, leaves, pollen, seeds, cancer tissues and various types of cells used in culture, ie single cells, protoplasts. (protoplast), shoots and callus tissue.
  • Plant tissue may be in planta or in organ culture, tissue culture or cell culture.
  • the present invention comprises the steps of transforming plant cells with a recombinant vector comprising a gene encoding a potato-derived IbHPPD protein;
  • It provides a method for producing a transformed plant with controlled environmental stress resistance comprising the step of regenerating the plant from the transformed plant cells.
  • the IbHPPD protein may be composed of the amino acid sequence of SEQ ID NO: 2, but is not limited thereto.
  • the gene encoding the IbHPPD protein may be the nucleotide sequence of SEQ ID NO: 1, but is not limited thereto.
  • the environmental stress may be dry, oxidative or pathogen infection stress, preferably pathogen infection stress, most preferably sweet potato bacterial stem root rot pathogen ( Pectobacetrium chrysanthemi ) Infection stress may be, but is not limited to.
  • the method of the present invention comprises transforming plant cells with a recombinant vector according to the present invention, which transformation can be mediated by, for example, Agrobacterium tumefiaciens.
  • the method also includes the step of regenerating the transgenic plant from said transformed plant cell.
  • the method for regenerating the transformed plant from the transformed plant cell may use any method known in the art.
  • Transformed plant cells should be re-differentiated into whole plants. Techniques for regeneration of mature plants from callus or protoplast cultures are well known in the art for many different species.
  • the present invention provides a transgenic plant and seed thereof having a controlled environmental stress resistance produced by the above method.
  • the plant may be a monocotyledonous plant or a dicotyledonous plant, but is not limited thereto.
  • the present invention also provides a composition for controlling environmental stress resistance of a plant containing a gene encoding the IbHPPD protein as an active ingredient.
  • composition for controlling environmental stress resistance of a plant of the present invention includes a gene encoding the IbHPPD protein of the present invention or a recombinant vector comprising the same as an active ingredient, and the plant environment is transformed by transforming the plant with the gene or the recombinant vector comprising the same. You can control your stress tolerance.
  • the IbHPPD gene may be overexpressed to increase environmental stress tolerance of the plant, but is not limited thereto.
  • Example 1 Sweet Potato IbHPPD Gene cloning, sequencing and softness analysis
  • Sweet potato ( Ipomoea batatas (L.) Lam. Cv. Yulmi) plants were grown in soil for 3 months at 25 ⁇ 3 ° C. under long-term conditions (16 hours and 8 hours cancer treatment).
  • the grown leaf tissue RNAs were isolated by RNase-free DNase-treated TRIzol method (Invitrogen).
  • the RNA was performed cDNA synthesis using the M-MLV reverse transcription system cDNA synthesis kit (Clontech) and primers were designed based on the known gene sequence (GeneBank accession no: KP306528). Sequence information of the HPPD gene was obtained from a transcript library constructed from RNA-seq analysis of sweet potato leaves and storage roots in the laboratory.
  • the total length of the IbHPPD cDNA gene was 1,323 bp, indicating that the coding region was composed of 440 amino acids (FIG. 1).
  • the IbHPPD protein has a SCOP and glyoxalase domain (Fig. 2).
  • the coding region of Ipomoea batatas IbHPPD was examined by BlastP, it showed 74% homology with LsHPPD of Lactuca sativa at the amino acid level and 73% with AtHPPD of Arabidopsis thaliana . The homology of was shown (Fig. 3).
  • Realtime PCR was performed to analyze tissue expression patterns of IbHPPD genes derived from sweet potatoes according to the present invention.
  • cDNA was synthesized by MMLV reverse transcription system cDNA synthesis kit (Clontech) using 2 ⁇ g of RNA.
  • IbHPPD gene specific primers and Accel Taq Premix kit (Genedocs) were used to investigate the expression of IbHPPD gene.
  • primers specific for IbHPPD the primers described in Table 1 were used.
  • the IbHPPD gene was most strongly expressed in the leaf and very weakly expressed in the stem. Storage roots in the root system were relatively strongly expressed compared to fibrous roots and thick pigmented roots. Therefore, the IbHPPD gene isolated from the sweet potato storage root was found to be the gene mainly expressed in the leaves under normal growth conditions.
  • IbHPPD gene responds to various stress treatments, including drying, treatment with various stress conditions, drying (30% PEG, polyethylene glycol), oxidation (400 mM hydrogen peroxide, H 2 O 2 ) and pathogens ( Pectobacterium chrysanthemi ) Afterwards, the expression changes of IbHPPD gene were analyzed using Realtime PCR.
  • IbHPPD In order to analyze the degree of reaction according to the PEG treatment of the gene, after drying the sweet potato leaf tissue according to time zones (0, 2, 4, 6 and 12 hours), RNA was analyzed by the method described in Example 2. After separation, cDNA was synthesized, respectively. IbHPPD used in Example 2 above Realtime PCR was performed with specific primers of the genes. As a result, the IbHPPD gene showed the strongest expression level in the leaf tissue after 4 hours of drying compared to the negative control (No control) (FIG. 6).
  • IbHPPD To determine whether the gene is related to the resistance to Pectobacterium chrysanthemi , the pathogens were treated by time zones (0, 3, 6 and 12 hours) and analyzed for gene expression patterns. Pathogen inoculation was carried out by culturing the pathogen on the leaves and adding 1x10 5 to 10mM MgCl 2 as an infiltration buffer The solution was suspended to a concentration of cfu / ml. As a mock, leaves treated with 10 mM MgCl 2 were used. RNA was extracted, cDNA was synthesized in the same manner as above, and realtime PCR was performed. As a result, 6 hours after treatment with pathogens, IbHPPD compared to Mock It was confirmed that the expression level of the gene is increased rapidly.
  • IbHPPD gene is responsive to various stresses. Therefore, IbHPPD gene is expected to be helpful in the development of environmentally resistant plants (including sweet potatoes) that improve growth even in drying, oxidative stress and pathogen infection.
  • Example 4 From Sweet Potato IbHPPD Analysis of alpha-tocopherol content in the leaves of tobacco plants by transient overexpression of genes
  • Alpha-tocopherol content was analyzed through transient overexpression of the Agrobacterium mediated IbHPPD gene. After inoculating Agrobacterium on the leaves of three-week-old tobacco plants, three days later, the IbHPPD gene expression was confirmed by realtime PCR analysis, and the alpha-tocopherol content was measured by HPLC analysis (FIG. 9A). As a result, not only the IbHPPD gene expression increased significantly compared with the empty vector (control) in the leaves of tobacco plants were inoculated IbHPPD gene, alpha-tocopherol was found that the content is increased to 81% (Fig. 9B). Therefore, IbHPPD gene is involved in the tocopherol biosynthesis pathway, it was confirmed that plays a role in increasing the content of tocopherol, a low-molecular antioxidant.

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Abstract

La présente invention concerne : un gène IbHPPD dérivé de la patate douce permettant de réguler la tolérance des plantes au stress environnemental ; et une utilisation correspondante, le gène pouvant être utile pour la mise au point d'une plante transgénique et d'une plante pour biocarburant, qui ont une tolérance au stress environnemental permettant aux plantes de convenir à des régions moins favorables grâce au gène.
PCT/KR2016/002916 2015-04-02 2016-03-23 Gène ibhppd dérivé de la patate douce pour réguler la tolérance des plantes à une contrainte environnementale, et utilisation correspondante WO2016159560A1 (fr)

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US20050289664A1 (en) * 2004-01-26 2005-12-29 Monsanto Technology, L.L.C. Genes encoding 4-Hydroxyphenylpyruvate dioxygenase (HPPD) enzymes for plant metabolic engineering
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US20040045051A1 (en) * 2002-08-05 2004-03-04 Norris Susan R. Tocopherol biosynthesis related genes and uses thereof
US20050289664A1 (en) * 2004-01-26 2005-12-29 Monsanto Technology, L.L.C. Genes encoding 4-Hydroxyphenylpyruvate dioxygenase (HPPD) enzymes for plant metabolic engineering
KR20090043754A (ko) * 2007-10-30 2009-05-07 한국생명공학연구원 고구마 뿌리 유래의 ＩｂＴＩＰ1 유전자 및 그의 프로모터
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