WO2016175351A1 - Gène osiaa6 dérivé du riz permettant d'augmenter la résistance d'une plante au stress de sécheresse et son utilisation - Google Patents

Gène osiaa6 dérivé du riz permettant d'augmenter la résistance d'une plante au stress de sécheresse et son utilisation Download PDF

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WO2016175351A1
WO2016175351A1 PCT/KR2015/004311 KR2015004311W WO2016175351A1 WO 2016175351 A1 WO2016175351 A1 WO 2016175351A1 KR 2015004311 W KR2015004311 W KR 2015004311W WO 2016175351 A1 WO2016175351 A1 WO 2016175351A1
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plant
osiaa6
stress
rice
gene
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Korean (ko)
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김주곤
정하린
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SNU R&DB Foundation
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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
    • C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
    • 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

  • the present invention relates to a rice-derived OsIAA6 gene and its use for increasing drought stress resistance of plants, and more particularly, to a recombinant vector comprising a gene encoding a rice-derived OsIAA6 (Oryza sativa Aux / IAA 6) protein.
  • a method of controlling resistance to environmental stress of a plant comprising the step of transforming a cell, transforming plant cells with a recombinant vector comprising a gene encoding the rice-derived OsIAA6 (Oryza sativa Aux / IAA 6) protein
  • the present invention relates to a method for producing a transgenic plant having a controlled resistance to environmental stress of a plant, a transgenic plant having a controlled resistance to environmental stress produced by the method, and a seed thereof.
  • Drought stress is one of the most serious factors affecting crop production worldwide. Exposure of plants to drought conditions leads to the induction of many stress-related genes, the products of which are thought to function as cellular protective agents against stress-induced damage. The expression of stress-related genes is usually regulated by specific transcription factors, which account for about 6% of the genome total gene number in rice and Arabidopsis.
  • the AUX / IAA (auxin / indoleacetic acid) gene encodes a family of proteins whose expression is tightly regulated by the plant hormone auxin.
  • the plant hormone auxin is involved in a variety of processes such as cell division, cell expansion and differentiation, embryonic patterning, growth control of vasculature or other tissues, root and side roots or stem meristem.
  • AUX / IAA proteins generally have four conserved amino acid sequence motifs (domains I, II, III and IV) and nuclear position signal sequences. Domains I and II play a role in degrading AUX / IAA proteins by recognizing auxin signals as degradation domains.
  • Domains III and IV are protein-protein interaction dimerization domains known to form homodimers with AUX / IAA proteins or heterodimers with ARF proteins.
  • AUX / IAA proteins inhibit ARF activity as negative regulators of auxin response factors (ARFs) that regulate expression of auxin-responsive genes.
  • ARFs auxin response factors
  • the AUX / IAA protein is ubiquitized through interaction with the auxin-SCFTIR1 complex and then degraded by proteasome action.
  • Korean Patent No. 0742194 discloses a method of increasing environmental stress resistance of a plant using environmental stress resistance control genes
  • Korean Patent No. 1427180 discloses rice which increases drought stress resistance of plants.
  • Derived OsCTR1 gene and its use ' is disclosed, but there is no known about the OsIAA6 gene derived from rice and its use to increase drought stress resistance of plants as in the present invention.
  • the present invention has been derived by the above-described requirements, the present inventors transformed the plant with a recombinant vector comprising a rice-derived OsIAA6 protein coding gene and the recombinant vector to produce a rice transgenic plant overexpressing the OsIAA6 gene, By confirming that the OsIAA6 overexpressed transgenic rice plants increased drought stress resistance compared to wild type plants, the present invention was completed.
  • the present invention provides a plant-resistant resistance to environmental stress, comprising the step of transforming plant cells with a recombinant vector comprising a gene encoding a rice-derived OsIAA6 (Oryza sativa Aux / IAA 6) protein It provides a way to adjust.
  • the present invention comprises the steps of transforming plant cells with a recombinant vector comprising a gene encoding a rice-derived OsIAA6 (Oryza sativa Aux / IAA 6) protein;
  • It provides a method for producing a transformed plant is controlled resistance to environmental stress comprising the step of regenerating the plant from the transformed plant cells.
  • the present invention provides a transgenic plant and its seed having a controlled resistance to environmental stress produced by the method.
  • plants overexpressing the genes encoding the rice-derived OsIAA6 (Oryza sativa Aux / IAA 6) protein have increased stress resistance compared to wild type under drought stress conditions. Therefore, the gene encoding the rice-derived OsIAA6 (Oryza sativa Aux / IAA 6) protein of the present invention can be usefully used in the development of transgenic plants with increased resistance to drought stress.
  • the gene encoding the rice-derived OsIAA6 (Oryza sativa Aux / IAA 6) protein of the present invention can be usefully used in the development of transgenic plants with increased resistance to drought stress.
  • Figure 2 is a result of confirming the change in the expression level of OsIAA6 gene when the plant was treated with NAA (naphthalene-1-acetic acid), an analogue of Auxin, a growth hormone of rice.
  • NAA naphthalene-1-acetic acid
  • Auxin an analogue of Auxin
  • OsIAA6 transgenic plants The results confirm the expression level change of the gene in OsIAA6 (PGD1 OsIAA6).
  • OsIAA6 transgenic plants a result of comparing the drought stress tolerance of (PGD1 OsIAA6) and wild type plants,
  • A is a photograph of the drought stress treatment and the plant state of after the water resupply (rewatering),
  • B This is a graph analyzing the change of Fv / Fm value according to drought stress treatment.
  • the present invention is directed to the environmental stress of plants comprising the step of transforming a plant cell with a recombinant vector comprising a gene encoding a rice-derived OsIAA6 (Oryza sativa Aux / IAA 6) protein Provides a way to control tolerance.
  • a rice-derived OsIAA6 Oryza sativa Aux / IAA 6
  • Method according to an embodiment of the present invention is characterized by increasing the plant's resistance to environmental stress by overexpressing a recombinant vector comprising a gene encoding a rice-derived OsIAA6 (Oryza sativa Aux / IAA 6) protein in plant cells
  • a recombinant vector comprising a gene encoding a rice-derived OsIAA6 (Oryza sativa Aux / IAA 6) protein in plant cells
  • a recombinant vector comprising a gene encoding a rice-derived OsIAA6 (Oryza sativa Aux / IAA 6) protein in plant cells
  • a recombinant vector comprising a gene encoding a rice-derived OsIAA6 (Oryza sativa Aux / IAA 6) protein in plant cells
  • it is not limited thereto.
  • environmental stress refers to an external factor that lowers the growth or productivity of a plant and is largely classified into a biological stress and an abiotic stress.
  • Biological stresses typically include pathogens, and abiotic stresses include high concentrations of salt, drought (dry), low temperature, high temperature and oxidative stress.
  • Environmental stress tolerance refers to a trait in which a decrease in growth or a decrease in productivity of a plant caused by environmental stress is suppressed or delayed.
  • the environmental stress may be a drought stress, but is not limited thereto.
  • the range of proteins includes proteins having the amino acid sequence represented by SEQ ID NO: 2 and functional equivalents of such 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 means activity that modulates the environmental stress tolerance of a plant.
  • 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 intracellularly reintroduced by artificial means.
  • the OsIAA6 gene sequence may 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.
  • Expression vectors comprising the OsIAA6 gene sequence of the present invention and appropriate transcriptional / translational control signals can be constructed by methods well known to those skilled 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 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 used to transfer hybrid DNA sequences to protoplasts from which current plant cells or new plants can be produced that properly insert hybrid DNA into the plant's genome. have.
  • a particularly preferred form of the Ti-plasmid vector is 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.
  • CaMV double stranded plant viruses
  • gemini viruses single stranded viruses
  • it may be selected from an incomplete plant viral vector.
  • the use of such vectors can be advantageous especially when it is difficult to properly transform a plant host.
  • the expression vector 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 gene, aadA gene, and the like, but are not limited thereto.
  • the promoter may be, but is not limited to, CaMV 35S, actin, ubiquitin, pEMU, MAS, histone promoter, Clp 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, and the rrnB1 / B2 terminator of Escherichia coli, but are not limited thereto.
  • NOS nopalin synthase
  • rice ⁇ -amylase RAmy1 A terminator phaseoline terminator
  • Agrobacterium tumefaciens Agrobacterium tumefaciens
  • Terminator of the octopine gene and the rrnB1 / B2 terminator of Escherichia coli, but are not limited thereto.
  • terminators With regard to the need for terminators, such regions are generally known to increase the certainty and efficiency of transcription in plant cells. Therefore, the use of terminators is highly desirable in the context of the present invention
  • yeast Saccharomyce cerevisiae
  • insect cells human cells
  • human cells e.g., CHO cell line (Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2) , 3T3, RIN and MDCK cell lines
  • the host cell is preferably a plant cell.
  • the method of transporting the vector of the present invention into the host cell may be performed by injecting the vector into the host cell by microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, DEAE-dextran treatment, gene bombardment, or the like. can do.
  • the present invention comprises the steps of transforming plant cells with a recombinant vector comprising a gene encoding a rice-derived OsIAA6 (Oryza sativa Aux / IAA 6) protein; And it provides a method for producing a transformed plant is controlled resistance to environmental stress comprising the step of regenerating the plant from the transformed plant cells.
  • a recombinant vector comprising a gene encoding a rice-derived OsIAA6 (Oryza sativa Aux / IAA 6) protein
  • Method according to an embodiment of the present invention is characterized by increasing the resistance to environmental stress of the plant by overexpressing a recombinant vector comprising a rice-derived OsIAA6 gene in plant cells, but is not limited thereto.
  • the OsIAA6 protein may be composed of the amino acid sequence of SEQ ID NO: 2.
  • the environmental stress may be a drought stress, but is not limited thereto.
  • the method of the present invention comprises the step of transforming a plant cell with a recombinant vector according to the present invention, the transformation may be mediated by, for example, Agrobacterium tumefiaciens ( 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 its seed having a controlled resistance to environmental stress produced by the method.
  • the transgenic plant and its seeds are transgenic plants and their seeds with increased resistance to environmental stress.
  • the plant is Arabidopsis, potato, eggplant, tobacco, pepper, tomato, burdock, garland chrysanthemum, lettuce, bellflower, spinach, chard, sweet potato, celery, carrot, buttercup, parsley, cabbage, cabbage, gatchi, watermelon, melon, cucumber, Dicotyledonous plants such as pumpkins, gourds, strawberries, soybeans, green beans, kidney beans, peas, or monocotyledonous plants, such as rice, barley, wheat, rye, corn, sugarcane, oats, and onions, preferably monocotyledonous plants, and more.
  • it may be a rice plant, but is not limited thereto.
  • the present invention also provides a composition for regulating environmental stress resistance of plants, comprising a gene encoding the rice-derived OsIAA6 (Oryza sativa Aux / IAA 6) protein consisting of the amino acid sequence of SEQ ID NO: 2 as an active ingredient.
  • the composition contains a gene encoding an OsIAA6 protein consisting of the amino acid sequence of SEQ ID NO: 2 as an active ingredient, the plant is transformed into a plant by transforming the gene or a recombinant vector containing the gene into an environmental stress tolerance, preferably drought. It can increase stress tolerance.
  • the present invention also provides the use of a gene encoding a rice-derived OsIAA6 (Oryza sativa Aux / IAA 6) protein consisting of the amino acid sequence of SEQ ID NO: 2 to regulate the environmental stress resistance of plants.
  • the gene encoding the OsIAA6 protein can preferably be used to increase the drought stress resistance of the plant.
  • OsIAA6 (AK068600) and full-length cDNA are the template of total RNA extracted from rice ('ilmi' cultivar), and the OsIAA6 forward (5'-caccatggaagaagggtccaacaaa-3 '; SEQ ID NO: 3) and the OsIAA6 reverse (5'-ttagaccctagcagtagctcca -3') SEQ ID NO: 4) using a primer.
  • PGD1: OsIAA6 vectors were introduced into plants via the Agrobacterium mediated co-culture method (Jang et al., 1999, Mol. Breeding 5: 453-461).
  • NAA naphthalene-1-acetic acid
  • nontransformed (NT, 'Ilmi' cultivar) rice plants were grown for 4 weeks in soil in a plastic house.
  • Four-week-old transgenic plants were air dried for three hours for drought stress treatment.
  • the whole plant was transferred to 200 mM sodium chloride solution for salt stress for 24 hours, and the whole plant was placed in a 4 ° C. low temperature chamber for 24 hours for low temperature stress treatment.
  • non-transformed (NT, 'Ilmi' varieties) rice plants were grown in a chamber for 6 days, and the plants were transferred to MS medium containing 100 uM NAA for 24 hours.
  • RNA RNA was used for first strand cDNA synthesis using the Superscript II cDNA synthesis kit (Invitrogen, USA).
  • Mx3000p real-time PCR instrument and Platinum SYBR Quantitative PCR was performed using the Green qPCR SuperMix-UDG system (Invitrogen, USA).
  • Rice ubiquitin transcript was used as a standardization index, and OsIAA6 gene specific primers were prepared at 3 'UTR and 3' exon sites. Primers used for quantitative RT-PCR are shown in Table 1 below.
  • PGD1 OsIAA6 transgenic plants were grown with soil for 4 weeks in the soil and then treated with drought stress by removing water from the soil. After 5 days of drought stress treatment, water was replenished and visual symptoms of drought stress in PGD1: OsIAA6 transgenic plants and non-transformed plants were photographed and observed.
  • OsIAA6 transgenic plants were grown in the soil for two weeks with non-transgenic plants, and then leaves were collected from each plant and allowed to acclimate for 10 minutes.
  • drought conditions were treated in a growth chamber in a time-lapse fashion, and at each indicated time, chlorophyll fluorescence emitted from the upper surface of the leaves was extracted using a pulse modulated fluorophotometer (mini-PAM, Walz, Germany).
  • mini-PAM mini-PAM, Walz, Germany
  • the Fv / Fm value was measured by sensing.
  • Fv / Fm values indicate activity of light system II.
  • OsIAA6 gene was increased 6 times in drought stress, 4 times in high salt stress, and 2.3 times in low temperature stress compared to the control group (FIG. 1).
  • Auxins are one of the major hormones in plants that regulate their growth and development.
  • the auxin reaction regulates the expression of ARF genes through the signaling system by accurately recognizing the amount of auxin in the plant.
  • the Aux / IAA protein is a negative regulator of the ARF protein. Fine tune the In order to check whether OsIAA6 gene, which is significantly increased in abiotic stress conditions among 31 Aux / IAA genes in rice, reacted with auxin, NAA, an auxin analogue, was treated with 100 uM for 24 hours in wild type rice grown 6 days. . As a result, the OsIAA6 gene showed about 2.7-fold increase in expression compared to the untreated control (FIG. 2). As a result, it was confirmed that OsIAA6 gene, which is significantly increased under abiotic stress conditions of rice, reacts auxin.
  • Example 1 it was confirmed that OsIAA6 gene was strongly expressed under drought stress conditions.
  • OsIAA6 overexpressed transgenic rice plants were prepared, and the expression pattern of OsIAA6 gene was confirmed in the overexpression (FIG. 3).
  • drought stress was treated to identify drought resistance in overexpression of 5 lines. After 3 days of drought stress treatment, the wild type transgenic plants showed mild symptoms of drought induced damage such as leaf curling and wilting. With the increase of drought stress treatment time, the symptoms of wild type transgenic plants became severe, and on the 5th day of drought treatment, there was a strong symptom of drought induction.
  • OsIAA6 overexpressed transgenic plants showed delayed decrease in Fv / Fm values due to drought stress.
  • Three OsIAA6 overexpressing transgenic plants maintained higher values than wild type transgenic plants until 2 hours of drought stress treatment.

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Abstract

La présente invention concerne : un procédé de régulation de la résistance d'une plante à un stress environnemental, comprenant une étape de transformation des cellules de la plante avec un vecteur recombiné comprenant un gène codant pour une protéine d'Oryza sativa Aux/IAA 6 (OsIAA6) dérivée du riz; un procédé de production d'un corps de plante transgénique dont la résistance d'une plante à un stress environnemental est régulée, comprenant une étape de transformation des cellules d'une plante avec le vecteur recombiné comprenant un gène codant pour la protéine OsIAA6; un corps de plante transgénique produit par le procédé et dont la résistance au stress environnemental est régulée; et des semences correspondantes.
PCT/KR2015/004311 2015-04-29 2015-04-29 Gène osiaa6 dérivé du riz permettant d'augmenter la résistance d'une plante au stress de sécheresse et son utilisation Ceased WO2016175351A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20120034588A (ko) * 2009-04-29 2012-04-12 바스프 플랜트 사이언스 게엠베하 향상된 수확량 관련 형질을 갖는 식물 및 이의 제조 방법
US20120115232A1 (en) * 2009-04-30 2012-05-10 Osaka University Method for inducing degradation of protein in mammalian cell
US20120151629A1 (en) * 2009-08-25 2012-06-14 Basf Plant Science Company Gmbh Nematode-Resistant Transgenic Plants

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20120034588A (ko) * 2009-04-29 2012-04-12 바스프 플랜트 사이언스 게엠베하 향상된 수확량 관련 형질을 갖는 식물 및 이의 제조 방법
US20120115232A1 (en) * 2009-04-30 2012-05-10 Osaka University Method for inducing degradation of protein in mammalian cell
US20120151629A1 (en) * 2009-08-25 2012-06-14 Basf Plant Science Company Gmbh Nematode-Resistant Transgenic Plants

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
SONG, YALING ET AL.: "Comprehensive Expression Profiling Analysis of OsIAA Gene Family in Developmental Processes and in Response to Phytohormone and Stress Treatments", PLANTA, vol. 229, 26 November 2008 (2008-11-26), pages 577 - 591, XP019715457 *
SONG, YALING ET AL.: "Ectopic Overexpression of an AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) Gene OsIAA4 in Rice Induces Morphological Changes and Reduces Responsiveness to Auxin", INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, vol. 14, 28 June 2013 (2013-06-28), pages 13645 - 13656, XP055325996 *

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