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WO2018084522A1 - Gène issu d'oryza sativa permettant d'améliorer la résistance à la germination avant récolte, et son utilisation - Google Patents

Gène issu d'oryza sativa permettant d'améliorer la résistance à la germination avant récolte, et son utilisation Download PDF

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WO2018084522A1
WO2018084522A1 PCT/KR2017/012132 KR2017012132W WO2018084522A1 WO 2018084522 A1 WO2018084522 A1 WO 2018084522A1 KR 2017012132 W KR2017012132 W KR 2017012132W WO 2018084522 A1 WO2018084522 A1 WO 2018084522A1
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gene
osphs1
plant
rice
seed
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윤인선
김범기
이강섭
허선미
한혜주
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Korea Rural Development Administration
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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
    • 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
    • A01H6/46—Gramineae or Poaceae, e.g. ryegrass, rice, wheat or maize
    • A01H6/4636—Oryza sp. [rice]
    • 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 an Oryza sativa pre-harvest sprouting 1 gene derived from rice ( Oryza sativa ) to enhance the germination resistance, and more particularly, to a plant cell comprising a recombinant vector comprising an OsPHS1 gene derived from rice.
  • a method for enhancing the germination resistance of plants compared to the wild type comprising the step of over-expressing the OsPHS1 gene, and transforming the plant cell with a recombinant vector comprising the rice-derived OsPHS1 gene in the plant cell, overexpressing the OsPHS1 gene
  • Composition for enhancing the germination resistance of a plant containing a recombinant vector comprising a method for producing a transgenic plant with improved germination resistance compared to the wild type, a transgenic plant and seeds thereof, and an OsPHS1 gene derived from rice It is about.
  • Seed dormancy is a physiological trait that inhibits seed germination under appropriate environmental conditions such as moisture, light and temperature (Bewley, Plant Cell , 9: 1055-1066, 1997). In general, the dormancy normally generated during seed development is maintained for a certain period of time in mature seeds, and the dormancy breaks down over time.
  • Seed dormancy is a fundamental trait necessary for plant survival, for example, the dormant seed has the opportunity to spread to more distant areas without germination, and does not germinate under adverse environmental conditions, so severe environmental stress conditions You can wait for a good environment while maintaining your life.
  • PHS pre-harvest sprouting
  • seed dormancy of wild rice is strong, japonica-type rice cultivars developed by breeding for a long time have weakened the dormancy easily for farming, and the degree of seed dormancy varies among developed rice cultivars.
  • embryos are differentiated within about one week after emergence and embryonic maturation is obtained by about 25 days, which is called primary dormancy.
  • Seed primary dormancy is a genetically complex characteristic that is affected by a number of internal and environmental factors (Li and Foley, Trends Plant Sci . , 2: 384-389, 1997). As seeds mature, dormancy is gradually lost or broken down by low temperature or other environmental stimuli.
  • Plant hormone ABA abcisic acid
  • Plant hormone ABA is known as an important regulator involved in the process of acquiring and maintaining dormancy of seeds (Gubler et al ., Curr Op in Plant Biol. , 8: 183-187, 2005; Kermode, J Plant Growth Regulation. , 24: 319-344, 2005).
  • ABA biosynthesis and ABA reactivity are factors that influence seed dormancy of major grains such as rice and corn.
  • the dormancy of rice seeds is influenced not only by the ABA but also by the environmental conditions of the seed maturation period, post-harvest seed storage temperature, moisture content, oxygen, nitrogen and carbon dioxide (Roberts, J Exp Botany , 13: 75-94, 1962).
  • a recombinant vector which enhances the germination resistance of plants compared to wild type, which includes an Oryza sativa pre-harvest sprouting 1 (OsPHS1) gene derived from rice ( Oryza sativa );
  • OsPHS1 Oryza sativa pre-harvest sprouting 1
  • Another object of the present invention is to improve the germination resistance of plants compared to wild type comprising the step of transforming the recombinant vector to plant cells, overexpressing the OsPHS1 gene, using the gene of the transgenic plant with improved germination resistance
  • a transgenic plant with improved sprouting resistance produced by the method and seeds thereof.
  • Still another object of the present invention is to provide a composition for enhancing germination resistance of a plant containing a recombinant vector containing a rice-derived OsPHS1 gene.
  • the present invention provides a recombinant vector for transformation comprising the gene represented by SEQ ID NO: 1 to enhance the germination resistance of plants.
  • pre-harvest sprouting refers to a phenomenon in which the seed that reaches the physiological maturation germinates while the seed is attached to the ear under the wet conditions caused by rainfall, which is an important agricultural trait in grain. It is known to be related to seed dormancy.
  • seed dormancy is a phenomenon that does not germinate for a certain period of time even if a suitable germination condition is given to mature seeds. Previously immature seeds are attached to the ear, causing germination, which causes germination.
  • the gene represented by SEQ ID NO: 1 "OsPHS1 ( Oryza sativa pre-harvest sprouting 1)" is a gene derived from rice ( Oryza sativa ), an Os03g20770 gene whose function is unknown so far located on chromosome 3 to be.
  • the rice-derived OsPHS1 gene which is selected as a spontaneous causative gene of the present invention, is specifically expressed in seed, and that the function of the OsPHS1 gene, which is not known until now, is closely related to the seed. It was found (FIG. 4 and FIG. 5).
  • the seed germination resistance of rice seeds was enhanced through overexpression of OsPHS1 gene (FIG. 9), and the seed dormancy was increased in comparison with wild type Arabidopsis transformed with OsPHS1 overexpressed Arabidopsis transform (FIG. 11).
  • wild type refers to a control of a transgenic plant comprising the rice-derived OsPHS1 gene of the present invention.
  • the plant is rice or Arabidopsis, in the case of rice includes all varieties of rice known in the art. More specifically, the rice plant may be Dongjin rice, but is not limited thereto.
  • plant refers to a body of a type possessed by a plant and may include the whole of the plant, part of the plant, seeds or plant cells.
  • pre-harvest sprouting tolerance is a property that the seeds do not germinate with the ear before harvesting, even if the ear reaching physiological maturity is subjected to wet conditions caused by rainfall, the seed dormancy This increases the resistance of plants to inhibit germination of seeds for a period of time.
  • the term "recombinant” refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a polypeptide 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 the wild-type form of the cell in either the sense or antisense form.
  • Recombinant cells can also express genes found in wild-type cells, but the genes are modified and reintroduced into cells by artificial means.
  • the OsPHS1 gene sequence can be inserted into a recombinant vector.
  • vector of the present invention is used when referring to DNA fragment (s), nucleic acid molecules to be delivered into a cell.
  • Vectors can replicate DNA and be reproduced independently in host cells.
  • recombinant vector is meant bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell viral vector, or other vector.
  • 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 the sequence of each OsPHS1 gene 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.
  • the expression vector 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 promoter may be suitable promoters for transformation, preferably, CaMV 35S promoter, actin promoter, ubiquitin promoter, pEMU promoter, MAS promoter, histone promoter or Clp promoter, but is not limited thereto.
  • promoter refers to a region of DNA over a structural gene and refers to a DNA molecule to which an RNA polymerase binds to initiate transcription.
  • 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, and the use of a constitutive promoter may be desirable in the present invention. Thus, constitutive promoters do not limit the selectability.
  • a conventional terminator can be used, examples of which are the octopine gene of nopalin synthase (NOS), rice ⁇ -amylase RAmy1 A terminator, Agrobacterium tumerfaciens (Agrobacteriumtumefaciens) Terminator, phaseoline (phaseoline) terminator, E. coli rrnB1 / B2 terminator, but is not limited thereto.
  • NOS nopalin synthase
  • rice ⁇ -amylase RAmy1 A terminator Agrobacterium tumerfaciens (Agrobacteriumtumefaciens) Terminator, phaseoline (phaseoline) terminator, E. coli rrnB1 / B2 terminator, but is not limited thereto.
  • phaseoline phaseoline terminator
  • E. coli rrnB1 / B2 terminator but is not limited thereto.
  • the recombinant vector may 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, hygromycin, chloramphenicol, G418, and antibiotics such as bleomycin. Resistance gene, aadA gene, and the like, but are not limited thereto.
  • the present invention provides a transgenic plant with a resistant subalah promote transformed with a recombinant vector containing the OsPHS1 (Oryza sativa pre-harvest sprouting 1) gene of the present invention.
  • the term "transformation” means that the trait of an individual or cell is genetically changed by DNA, which is a genetic material given from the outside, and in the present invention, rice represented by SEQ ID NO: 1 ( Oryza sativa) (Oyza sativa pre-harvest sprouting 1) gene derived from) means that the germination resistance of the transformed plants is enhanced by the introduction of the gene.
  • transformation with the recombinant expression vector of the present invention may be performed by transformation techniques known to those skilled in the art.
  • transformation techniques known to those skilled in the art.
  • microprojectile bombardment, particle gun bombardment, silicon carbide whiskers, sonication, electroporation, PEG-mediated fusion PEG-mediated fusion, microinjection, liposome-mediated method In planta transformation, Vacuum infiltration method, floral meristem dipping method), or Agrobacterium sp. mediated method, and the like, but are not limited thereto.
  • transformer refers to a host cell transformed with a recombinant expression vector containing OsPHS1 ( Oryza sativa pre-harvest sprouting 1) gene derived from rice which enhances the germination resistance of the present invention.
  • OsPHS1 Oryza sativa pre-harvest sprouting 1
  • the transformants, and microorganisms are preferred, but are not limited to, Escherichia coli (Escherichia coli), Bacillus subtilis (Bacillus subtilis), Streptomyces (Streptomyces), Pseudomonas (Pseudomonas), Proteus Mira Billy's (Proteus mirabilis ), Staphylococcus and Agrobacterium tumefaciens , and more preferably Agrobacterium tumefaciens .
  • the present invention is an overexpression of the OsPHS1 gene by transforming a plant cell with a recombinant vector comprising an Oryza sativa pre-harvest sprouting 1 gene derived from rice ( Oryza sativa ) represented by SEQ ID NO: 1. It provides a method for improving the sprouting resistance of plants compared to wild type comprising the step of.
  • the method for transforming the plant cells is as described above.
  • the OsPHS1 gene may be composed of the nucleotide sequence of SEQ ID NO: 1, but is not limited thereto.
  • homologues of the above nucleotide sequences are included within the scope of the present invention.
  • the gene has a base sequence having a sequence homology of at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% with the nucleotide sequence of SEQ ID NO: 1, respectively. It may include.
  • the "% sequence homology" for a polynucleotide is identified by comparing two optimally arranged sequences with a comparison region, wherein part of the polynucleotide sequence in the comparison region is the reference sequence (addition or deletion) for the optimal alignment of the two sequences. It may include the addition or deletion (ie, gap) compared to).
  • the present invention is to improve the germination resistance compared to wild type comprising the step of overexpressing the OsPHS1 gene by transforming plant cells with a recombinant vector comprising a rice-derived OsPHS1 gene represented by SEQ ID NO: 1
  • a recombinant vector comprising a rice-derived OsPHS1 gene represented by SEQ ID NO: 1
  • the method for transforming the plant cells is as described above, the method of the present invention also comprises the step of regenerating the transformed plant from the transformed plant cells.
  • the method of regenerating the transgenic plant may use any method known in the art. Techniques for the regeneration of mature plants from callus or protoplast cultures are well known in the art for numerous different species (Handbook of Plant Cell Culture, Vol. 1-5, 1983-1989 Momillan, N.Y.).
  • the OsPHS1 gene may be composed of the nucleotide sequence of SEQ ID NO: 1, but is not limited thereto.
  • the present invention provides a transgenic plant with enhanced germination resistance and seeds thereof produced by the above method.
  • the plant may be a monocotyledonous plant or a dicotyledonous plant, preferably an Oryza sativa plant or Arabidopsis thaliana ), but is not limited thereto.
  • the present invention provides a composition for enhancing the germination resistance of plants containing a recombinant vector comprising the OsPHS1 gene derived from rice consisting of the nucleotide sequence of SEQ ID NO: 1.
  • the composition is to improve the germination resistance of the plant by transforming the plant with a recombinant vector comprising the OsPHS1 gene consisting of the nucleotide sequence of SEQ ID NO: 1 as an active ingredient.
  • the plants transformed with the rice-derived OsPHS1 gene have improved germination resistance compared to wild-type plants, plant development and crops that can grow strongly even under poor conditions in which the risk of spoiling damage due to abnormal climate is increased. It is effective in increasing the productivity of the plant, and it can be useful for constructing the environment stress resistant crop development platform.
  • FIG. 1 is a diagram showing the results of the germination investigation (A) and seed germination rate (B) of the sporadogenic mutant phs1 .
  • Figure 2 is a schematic diagram showing the position of the Ds transfer factor inserted into the spontaneous mutant phs1 .
  • 3 is a diagram confirming the expression level of OsPHS1, a gene related to germination resistance, in the seed of the spontaneous mutant phs1 .
  • OsPHS1 gene which is a gene related to budding resistance through a public DB search.
  • FIG. 5 is a photograph showing the expression of GUS expression according to seed development in transgenic rice seed into which the OsPHS1 promoter-GUS vector of the present invention is introduced.
  • An is anther, St is stigma, Ov is ovule, Ovt is ovule track, Em is embryo, En is endosperm, and Al is whistle (aleurone layer).
  • Figure 6 is a micrograph observing the fluorescence distribution of OsPHS1-GFP protein transiently expressed in the protoplasts of rice introduced OsPHS1-GFP fusion protein of the present invention.
  • FIG. 7 is a diagram showing a diagram of the OsPHS1 gene transformation vector for the production of transgenic plants to enhance the sprouting resistance of the present invention.
  • TL is the T-DNA left border
  • 35ST is the cauliflower mosaic virus (CaMV) 35S terminator
  • 35SP is the cauliflower mosaic virus (CaMV) 35S promotor
  • NOS is the nopaline synthase
  • OsPHS1 CDS is the Oryza sativa pre-harvest
  • TR is the T-DNA right border.
  • FIG. 8 is a diagram showing the results of analyzing the OsPHS1 gene expression level of the over-expressed rice transformant OsPHS1 gene of the present invention using the RT-PCR method.
  • WT is a wild type Dongjinbyeong, the original species
  • Figure 9 is a diagram showing the result of measuring the germination rate of immature seed of the transgenic rice overexpressing OsPHS1 gene of the present invention.
  • Col-0 is a wild-type Arabidopsis
  • 35SP OsPHS1 (1, 3, 6, 9, 11, 13) is a Arabidopsis transformed so that the OsPHS1 gene is introduced and the gene is overexpressed.
  • FIG. 11 is a view showing the seed germination rate measurement results (A) and seed germination inhibition rate analysis results (B) according to the ABA treatment of the OsPHS1 gene introduction overexpression Arabidopsis transformants of the present invention.
  • Col-0 (Lane 1) is a wild-type Arabidopsis
  • 35SP OsPHS1 (Lane 2, 3, 4, 5) is a Arabidopsis transformed so that the OsPHS1 gene is introduced and the gene is overexpressed.
  • PHS Pre-harvest sprouting
  • mutants with weak germination resistance are selected through mutant population analysis in which Ds transfer factor has been inserted in Dongjin rice. It was.
  • Example 1-1 Ds Transition factor Insert mutant population analysis and Sprouting Mutant ( phs1 ) Selection
  • the phs1 mutant showed a germination phenomenon, which resulted in a weaker germination resistance than the control dongjin rice (FIG. 1A), and also in the seed germination rate, the seed at 25 DAH (day after heading), immature seed stage, in the control of seed germination.
  • phs1 mutants showed more than 40% seed germination at 25 DAH (day after heading).
  • genomic DNA of the spontaneous phs1 mutant was isolated and restriction enzyme RsaI was used. Cut using. Then, FST (Flanking Sequence Tag) gene analysis was performed using the Adaptor-PCR technique.
  • Example 1-2 In the spontaneous phs1 mutant selected in Example 1-2, it was confirmed that the Ds transfer factor was inserted into the second exon (Exon II) of the Os03g20770 gene, that is, the Oryza sativa pre-harvest sprouting 1 gene OsPHS1. It was confirmed by RT-PCR analysis that OsPHS1 gene was not expressed in the seed of the spontaneous phs1 mutant.
  • RNA was isolated from the seeds of the control dongjinbyeong and the spontaneous phs1 mutant, respectively, to synthesize cDNA, and OsPHS1 gene specific primer set (OsPHS1-1F: 5'-ATGGCGGCAAGCCAATACATGG-3 '(SEQ ID NO: 2); OsPHS1 -618R: RT-PCR was performed using 5'-CTAGGCAGAGGAGCTCGATGTTG-3 '(SEQ ID NO: 3)).
  • the rice-derived OsPHS1 gene selected from the Ds rice insertion mutant strain was a germinal causative gene, and thus, the gene could be usefully used for developing a germination resistant crop.
  • OsPHS1 gene ( Os03g20770 through Example 2 above) Gene was identified as a causative causative gene.In order to develop rice crops and seeds of germinal resistance by utilizing this, a public database (Rice Oligonucleotide Array Database) was used to express the expression patterns of OsPHS1 genes by rice tissue. I analyzed it.
  • OsPHS1 gene was specifically expressed in the embryo of rice seed.
  • a vector (OsPHS1 promoter-GUS vector) connected to GUS, a reporter gene, was isolated by separating the top 2 kb region of the start codon of the OsPHS1 gene, and the seed of the transformed rice introduced therein was analyzed by GUS protein staining. It was. At this time, the site where the promoter of OsPHS1 gene is operated by GUS staining was stained blue by GUS staining.
  • OsPHS1 gene which is selected as a spontaneous causative gene, is actually seed-specifically expressed. It can be inferred that the function of OsPHS1 gene, which is not known until now, is closely related to seeds.
  • OsPHS1 gene selected as a congenital causal gene has very low homology with the known protein, it is determined to have novel characteristics, and the expression pattern of OsPHS1 protein encoded by the OsPHS1 gene is determined. I analyzed it.
  • OsPHS1-GFP fusion protein was prepared in which GFP protein was connected to the C-terminal of OsPHS1 protein, and when it was transiently expressed in the protoplasts of rice, As shown, it was confirmed that located in the membrane of the endoplasmic reticulum (ER, golgi body).
  • cDNA was synthesized using RNA isolated from seeds of Dongjin rice as a template, and the OsPHS1 gene specific primer set (OsPHS1-1F: 5'-ATGGCGGCAAGCCAATACATGG-3 '(sequence) OsPHS1-618R: 5'-CTAGGCAGAGGAGCTCGATGTTG-3 '(SEQ ID NO: 3)) was used to isolate the beginning and end of the OsPHS1 gene, or ORF (open reading frame). After confirming the nucleotide sequence for this, as shown in Figure 7, the pCAMBIA1300-OsPHS1 overexpression vector was prepared by inserting the isolated OsPHS1 gene into the pCAMBIA1300 series of rice transformation vectors.
  • an OsPHS1 overexpression vector was constructed so that the OsPHS1 gene was always expressed in plants by connecting OsPHS1 genes to Xba I and BamH I restriction enzymes downstream of the CaMV35S promoter (35SP), wherein the hygromycin (Hygromycin) was used as an antibiotic resistance marker. ) Resistance genes were used.
  • Example 1-1 the mutant phs1 with poor germination resistance was selected from the mutant population line in which the Ds transfer factor was inserted in Dongjin rice . Based on this, caregiver Shing mutants by inserting a pCAMBIA1300-OsPHS1 vector produced in Example 6-1 to prepare a phs1 the rice transformants so that OsPHS1 gene is overexpressed, and wherein the vector is properly inserted is the gene OsPHS1 Overexpression in transgenic rice was analyzed by RT-PCR.
  • phs1 Callus derived from mutant seeds was inoculated with Agrobacterium containing the pCAMBIA1300-OsPHS1 vector, and transformed calli in a medium containing 30 g / ml hygromycin. After selecting loose, the stems and roots were induced to produce transgenic rice.
  • RNA was isolated from wild type (WT) Dongjin rice, which is a control group, and from the leaves of the transformed rice prepared to overexpress the OsPHS1 gene, and cDNA was prepared by using a template, and the OsPHS1 gene was specific.
  • RT-PCR was performed using a primer set (OsPHS1-1F: 5'-ATGGCGGCAAGCCAATACATGG-3 '(SEQ ID NO: 2); OsPHS1-618R: 5'-CTAGGCAGAGGAGCTCGATGTTG-3' (SEQ ID NO: 3)).
  • OsPHS1 gene which is a gene related to germination resistance, was stronger than Dongjin rice.
  • OsPHS1 gene which is a gene related to germination resistance, prepared in Example 6-2 Overexpressed rice transformants were grown in the field, and the ear was harvested at 25 and 40 days after harvesting, respectively. Seeds were separated from the ears and placed in a petri dish, incubated for 5 days under 100% relative humidity, temperature of 30 ° C. and dark conditions, and the seed germination rate was measured. At this time, the control group used a mutant phs1 with weak germination resistance.
  • OsPHS1 gene overexpression was prepared by preparing rice transformants and measuring the germination rate of seeds obtained from the prepared rice transformants. It was confirmed that the rice transformants had enhanced resistance to sprouting germ associated with seed dormancy.
  • OsPHS1 gene overexpressed Arabidopsis was produced in order to confirm whether seed co-plants overexpressing OsPHS1 gene could be enhanced, and germination rate and ABA reactivity of the seeds obtained therefrom were analyzed.
  • OsPHS1 gene overexpressed Arabidopsis transformant was prepared, and whether the OsPHS1 gene was overexpressed in the Arabidopsis transformant RT-PCR. Analysis was performed.
  • the OsPHS1 gene inserted in the above-described OsPHS1 gene insertion pCAMBIA1300-OsPHS1 overexpression vector ( Agrobacterium ) is transformed into a Arabidopsis thaliana Arabidopsis thaliana by floral dipping method (OsPHS1 gene) Arabidopsis transformants were overexpressed. After preparation, RT-PCR was analyzed to see if the vector was properly inserted and OsPHS1 gene was overexpressed in the Arabidopsis transformants.
  • a wild type Arabidopsis pellucida (Col-0) without introducing the OsPHS1 gene was used as a control, and RT-PCR was an OsPHS1 gene-specific primer set (OsPHS1-1F: 5'-ATGGCGGCAAGCCAATACATGG-3 '(SEQ ID NO: 2)).
  • OsPHS1-618R 5'-CTAGGCAGAGGAGCTCGATGTTG-3 '(SEQ ID NO: 3)).
  • OsPHS1 transgenic Arabidopsis transformants were wild-type Arabidopsis (Col). Compared with -0), OsPHS1 gene, which is related to germination resistance, was strongly expressed.
  • Example 8-2 OsPHS1 Seed Germination Rate and ABA Reactivity Analysis of Gene Overexpressed Arabidopsis
  • seed germination rate and seed dormancy were obtained using the seeds obtained from the OsPHS1 gene overexpression Arabidopsis prepared in Example 8-1. Seed germination was analyzed for responsiveness to ABA (abscisic acid), a key regulator involved in the maintenance process. At this time, as a control, a seed of wild type Arabidopsis (Col-0) without introducing OsPHS1 gene was used.
  • ABA abcisic acid
  • OsPHS1 overexpressed Arabidopsis transformant 35SP: OsPHS1
  • 35SP Arabidopsis transformant
  • OsPHS1 gene isolated from rice Overexpression was found to be useful for enhancing seed dormancy of dicotyledonous plants.

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  • Developmental Biology & Embryology (AREA)
  • Environmental Sciences (AREA)
  • Natural Medicines & Medicinal Plants (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne le gène 1 de germination avant récolte, d'Oryza sativa (OsPHS1) issu d'Oryza sativa permettant d'améliorer la résistance à la germination avant récolte, et son utilisation. Selon la présente invention, par comparaison avec un corps végétal de type sauvage, un corps végétal transformé avec un gène OsPHS1 issu d'Oryza sativa présente une résistance améliorée à la germination avant récolte, ce qui est efficace dans l'augmentation de la productivité des cultures et dans le développement de corps végétaux, qui peuvent très bien croître même dans des conditions médiocres dans lesquelles le risque de dommages par germination avant récolte causés par un climat anormal est accru ; et pouvant être utilisé dans la construction d'une plate-forme pour développer des cultures résistantes à un stress environnemental.
PCT/KR2017/012132 2016-11-07 2017-10-31 Gène issu d'oryza sativa permettant d'améliorer la résistance à la germination avant récolte, et son utilisation Ceased WO2018084522A1 (fr)

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KR10-2016-0147333 2016-11-07
KR1020160147333A KR101790010B1 (ko) 2016-11-07 2016-11-07 수발아 저항성을 증진시키는 벼 유래 유전자 및 이의 용도

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Families Citing this family (7)

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KR101790010B1 (ko) * 2016-11-07 2017-10-26 대한민국 수발아 저항성을 증진시키는 벼 유래 유전자 및 이의 용도
KR102080827B1 (ko) * 2018-06-21 2020-02-24 대한민국 수발아 저항성을 증진시키는 벼 유래의 ＯｓＰＨＳ３ 유전자 및 이의 용도
KR102266975B1 (ko) * 2019-10-31 2021-06-21 대한민국 수발아 저항성을 증진시키는 벼 유래 OsPHS5 유전자 및 이의 용도
KR102285720B1 (ko) * 2019-10-31 2021-08-06 대한민국 내염성 또는 수발아 저항성을 증진시키는 벼 유래 dp1405 유전자 및 이의 용도
KR102285723B1 (ko) * 2019-10-31 2021-08-06 대한민국 내염성 또는 수발아 저항성을 증진시키는 벼 유래 dp1405 유전자 및 이의 용도
KR102762745B1 (ko) * 2021-12-13 2025-02-12 대한민국 밀 수발아 내성 증진을 위한 유전자 편집용 조성물 및 이를 이용한 유전자 편집방법
CN116267584A (zh) * 2023-03-29 2023-06-23 江苏丘陵地区镇江农业科学研究所 一种培育耐穗芽粳稻品种的育种方法

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