WO2014025137A1 - Gène uip1 pour augmenter la résistance de plantes au stress de sécheresse et utilisation de celui-ci - Google Patents

Gène uip1 pour augmenter la résistance de plantes au stress de sécheresse et utilisation de celui-ci Download PDF

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Publication number
WO2014025137A1
WO2014025137A1 PCT/KR2013/005589 KR2013005589W WO2014025137A1 WO 2014025137 A1 WO2014025137 A1 WO 2014025137A1 KR 2013005589 W KR2013005589 W KR 2013005589W WO 2014025137 A1 WO2014025137 A1 WO 2014025137A1
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plant
stress
uip1
gene
protein
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PCT/KR2013/005589
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English (en)
Korean (ko)
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김주곤
박수현
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명지대학교 산학협력단
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Publication of WO2014025137A1 publication Critical patent/WO2014025137A1/fr

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    • 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)
    • 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

Definitions

  • the present invention relates to rice-derived 3 'Uranslated region interacting protein (UIP1) and its use for increasing dry stress tolerance.
  • UIP1 rice-derived 3 'Uranslated region interacting protein
  • Plants are immobilized organisms that can not move. Unlike animals, when exposed to stress, there is no way to avoid this. Therefore, it affects more stress than animals. Therefore, plants are able to survive only by reacting more quickly than animals to stress. The life of a plant can be said to be a series of antagonism and antagonism against stress.
  • Biological stresses include diseases, pests, and weeds.
  • Abiotic stresses include silver, moisture, wind, soil, drugs, and air pollutants. Abiotic stress is more in detail due to domestic climate factors.
  • the present invention deals with abiotic stress.
  • RNA molecules There are three experimental methods for determining the protein binding to RNA molecules.
  • SELEX systematic evolution of ligands by exponential enrichment
  • RIP-chip RNA immunoprecipitation-chip.
  • Yeast-three hybrids are proteins and RNAs that are expected to bind in vivo,
  • the RNA that is linked to the reporter gene identifies the protein through three hybridizations.
  • the SELEX method performed in vitro can be performed by using an RNA ligand
  • RJP-chip is a method to find the target RNA of known RNA-binding proteins (RBPs), and it is precipitated with a tagged antibody linked to RBP in a complementary sample between RNA and protein, In silico (experimentation within computer) [ 0 ], a microarray analysis is performed by separating target RNAs through a series of procedures.
  • RBPs RNA-binding proteins
  • the size is lm, and it blooms in August.
  • the temperature, the amount of irradiation and the amount of rainfall directly affect the growth of rice, and the physical and chemical characteristics of the soil are also affected by the production of rice. It has a big influence. In addition, various disease layers, soil microorganisms and weeds, It is very important.
  • the abiotic stress tolerance was investigated using a recombinant vector containing rice-derived UIP1 gene.
  • the identified UIP1 gene was used in the yeast three hybrid method.
  • the present invention relates to a method for improving the dry stress tolerance of a plant by using a rice recombinant vector
  • the purpose of the increase is to increase.
  • the present invention also provides a method for improving the tolerance to abiotic stresses such as high salt and low temperature in addition to the drying stress, Method.
  • the present invention provides a protein for enhancing tolerance to dry stress of a plant, comprising an amino acid sequence of SEQ ID NO: 2
  • Lys lie Asn Gly Thr Lys lie Gly Gly Gly Arg Gly Ser Pro Thr Phe
  • Val lie Gly He Val Thr Arg Ala Asp Val Leu Arg Glu Leu Glu Ala
  • the protein comprises a protein having the amino acid sequence of SEQ ID NO: 2 and 104 amino acids 10 to 203 of the amino acid sequence of SEQ ID NO: 2, ie, the CBS domain of cystathionine - beta-synthase domain)
  • the protein may include a gene encoding a protein comprising the nucleotide sequence of SEQ ID NO: 1 below, preferably a recombinant vector into which the gene is inserted.
  • the invention includes recombinant vector-transformed host cells as described above. More preferably, the host cell is obtained from a plant.
  • the present invention provides a method for producing a recombinant plasmid comprising the steps of: preparing a recombinant plasmid with the recombinant vector; And transforming plant cells using the recombinant vector; And regenerating the transformed plant from the transformed plant cell.
  • the present invention also provides a method for producing a transgenic plant.
  • the present invention features a method for promoting abiotic stress tolerance of a plant by transforming plant cells with a recombinant vector, including overexpressing the gene.
  • the present invention features a method for increasing the grain yield of a plant through the method of enhancing the abiotic stress tolerance.
  • the abiotic stress is characterized by being dry, high salt or low temperature.
  • the plant transformed by the above-described method comprises plants with increased tolerance to abiotic stress and plants with increased grain yield. More preferably, the plant is rice.
  • the composition for promoting stress tolerance of a plant or the composition for increasing grain yield may include the gene encoding the protein through the above-described method as an active ingredient.
  • the transforming method and the above method include a transformed plant.
  • the 3'untranslated region (3'UTR) of Rubisco small subunk (RbcS), which rapidly abolishes mRNA under dry stress, is effective in increasing tolerance of plants susceptible to abiotic stress using a recombinant vector.
  • the present inventors have shown that the resistance to abiotic stress is largely increased by increasing the resistance even under the conditions of high salt and low temperature in addition to the drying stress in the growth period. You can contribute.
  • FIG. 1 shows the identification of the UIPI gene through yeast three-hybrid (Y3H) assay.
  • Y3H yeast three-hybrid
  • FIG. 2 shows changes in composition of UIP1 and 3'RbcS in a histidine-deficient medium containing 3-aminotrizole (3-AT).
  • FIG. 3 relates to selective composition changes between UIP1 and RbcS of the present invention.
  • FIG. 4 shows the expression of the OsCcl: UIP1 agonist and UIP1 transgene, OsCcl promoter-driven UIP1 overexpressing transporter.
  • FIG. 5 is a graph showing changes in the leaf and root tissues of seedlings grown for 4 weeks in the present invention
  • FIG. 6 is a graph showing the results of the dry stress treatment in the present invention.
  • FIG. 7 shows changes in chlorophyll fluorescence (Fv / Fm) of rice plants under (a) drying, (b) high salt and (c) low temperature stress conditions.
  • FIG. 8 shows the amino acid sequence of UIP1 and a deletion mutant region
  • Figure 9 shows the mutual binding between the unilamellar mutants of UIP1 and MS2, 3'RbcS-MS2 and inverted 3'RbcS-MS2 RNA.
  • the present invention provides a method for producing a transgenic plant which increases tolerance to drying stress.
  • Transformation of a plant means any method of transferring DNA to a plant. Such transfection methods do not necessarily have tissue regeneration or incubation periods. Transformation of plant species is now common for plant species including both terminal plants as well as dicotyledons. Any transformation method can be used to introduce the hybrid DNA according to the present invention into suitable progenitor cells.
  • the method can be carried out using a chimney / polyethylene glycol method for protoplasts (Krens, FA et al 1982, nature 296, 72-74; Negrutiu I. et al. June 1987, Plant Mol. Biol. 8, 363-373) (Shillito RD et al. 1985 Bio Technol. 3, 1099-1102), microinjection into plant elements (Crossway A. et al., 1986, Mol. Gen. Genet. 202, 179-185) (DNA or RNA-coded) particle impact method of various plant elements (Klein et al. 1987, Nature 327, 70).
  • Plant cells can be cultured cells, cultured tissues, culture or whole plants, preferably cultured cells, cultured tissues or culture media, and further cultured cells. Preferably, any form of cultured cells is possible.
  • Plant tissue refers to cells of various types used in the culture of silvered or undifferentiated plants, such as, but not limited to, roots, stems, leaves, pollen, seeds, Plant cells may be treated in plants, or may be in an organ culture, a tissue culture, or a cell culture.
  • 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, heterologous peptide or heterologous nucleic acid.
  • a recombinant cell can express a gene found in a cell in its natural state, but the gene (s) or gene fragment (s) can not be found in the natural form of the cell. Are modified and reintroduced into cells by artificial means.
  • vector is used to refer to a DNA fragment, nucleic acid molecule, which is delivered into a cell.
  • a vector replicates DNA and can be independently reproduced in the host cell.
  • Adenylate acid polyadenylation signals are known.
  • a preferred example of an expression vector is a Ti-plasmid vector which, when present in a suitable host, such as Agrobacterium tumefaciensens, is able to transfer, in part, the so-called T-region into plant cells.
  • Smith vector is currently being used to transfer hybrid DNA sequences to plant cells, or to protoplasts where new plants can be produced that appropriately insert hybrid DNA into the genome of the plant.
  • Suitable vectors that may be used to introduce the DNA of the present invention into a plant host include virus vectors such as those derived from double-stranded plant viral vectors (eg, CaMV) and single-stranded viruses, For example, non-complete plant viral vectors.
  • virus vectors such as those derived from double-stranded plant viral vectors (eg, CaMV) and single-stranded viruses, For example, non-complete plant viral vectors.
  • the use of such vectors could be advantageous, especially when it is difficult to transform plant hosts appropriately.
  • the host includes eukaryotic cells such as prokaryotes such as Escherichia coli, bacillus, yeast, stratum corneum, plant cells, animal cells, and the like, preferably an animal other than human.
  • eukaryotic cells such as prokaryotes such as Escherichia coli, bacillus, yeast, stratum corneum, plant cells, animal cells, and the like, preferably an animal other than human.
  • the present invention relates to a method for improving tolerance to biological stress
  • the present invention provides a gene for expressing UIP1 comprising a cystathionine-beta-synthase domain.
  • the present invention also provides a method for screening a biosynthetic stress tolerance gene.
  • a PT-PCR method is used for the measurement
  • the gene selected using the screening method of the present invention can be used in a variety of ways that are related to the abiotic stress of plants, i.e., resistance to abrasion, salt tolerance and cold tolerance. It is available as a candidate gene.
  • the present invention also provides a transgenic plant having increased stress resistance produced by the method of the present invention.
  • plants that can increase stress resistance by the method of the present invention include food crops including pepper, wheat, barley, corn, soybean, potato, peel, oat, and sorghum; Arabidopsis, cabbage, radish, Vegetable crops including tomatoes, watermelons, cucumbers, cabbages, melons, squash, onions, carrots, and special crops including ginseng, tobacco, cotton, sesame seeds, sugarcane, , Grapevines such as rose, gladiolus, gerbera, carnation, chrysanthemum, lilies, lilies, and ragwas, including pears, pear trees, jujube trees, , Red clover, orchard glass, alpha-alpha, top-cue, perennial, etc.
  • food crops including pepper, wheat, barley, corn, soybean, potato, peel, oat, and sorghum
  • Arabidopsis cabbage, radish, Vegetable crops including tomatoes, watermelons, cucumbers, cabbages, melons, squash, onions,
  • Feed crops are included.
  • Plants with increased stress resistance according to the present invention can be produced by conventional
  • the plant of the present invention can be obtained through reproductive processes such as a process of producing seeds through the moisture process of flowers and propagating from the seeds.
  • a plant can be transformed with a recombinant vector containing the UIP1 gene according to the present invention, and then transformed into a callus inducing, rooting and soil purifying process .
  • the cells After culturing on a suitable medium known in the art, the cells are cultured under an appropriate condition to induce callus formation.
  • shoots When shoots are formed on the cultivated plants, they are transferred to a hormone-free medium and cultured. After about two weeks, the shoots are transferred to rooting medium to induce roots. After roots are induced, they are transformed into soil and purified, and plants with increased stress resistance can be obtained.
  • the transgenic plant may include not only whole plants but also tissues, cells or seeds obtainable therefrom.
  • RNA binding protein containing the CBS domain through Y3H screening.
  • the RBP refers to UIP1, which interacts with the 3'UTR of RbcS mRNA involved in stress-induced mRNA degradation under dry, high salt conditions.
  • the deletion of the six base sequences at the N-terminal and C-terminal of the UIP1 gene shows the linkage of the UIP1 gene domain.
  • the various combinations of three hybrid RNA groups and their compositions show that 51 N-terminal amino acids are removed as a result of strand-specific pseudoblock loss in the co-transformed yeast.
  • deletions to regions between amino acids 52 to 212 are shown for 3 ' RbcS, which binds essentially to the CBS domain of UIP1.
  • Overexpression of UIP1 transgenic in rice resulted in increased tolerance to dry stress in the growing season. Under the stress conditions of dry, high salinity and low temperature, the maximum photosynthetic efficiency in the photosystem ⁇ of UIP1 plants was higher than that of plants not transformed.
  • UIP1 is associated with the 3'UTR of RbcSl mRNA and provides a method for increasing the resistance of a transgranular overexpressed to dry stress.
  • RNA-binding proteins play an important role in the regulation of transcriptional pre-transcription of genes.
  • RBPs involve the transcription of a second RNA polymerase from the onset of transcription in the cytoplasm to their degeneration. The first transcription is at the 3 ' end
  • Adenylic acid polymerization reaction These translocations are influenced by mRNA transport, mRNA stabilization and degradation, and translation in the nuclear envelope.
  • RbcS contains a major mRNA region in a common shape sent with strcss-induced mRNA decay under dry and high salt conditions.
  • SMD is in the mRNA of photosynthetic genes that rapidly decrease when plants are exposed to stress.
  • UIP1 To elucidate the major association sites in UIP1, we examined the deletions in the amino acid strands of the protein. In addition, responses to overexpression of UIP1-type transduction were investigated under other types of abiotic stress conditions. More specifically, the expression of RbcS mRNA We propose UIP1 compositions containing the 3'UTR and the expression of UIP1, which increases resistance to a variety of abiotic stresses in transgenic plants at the growing stage.
  • Example 1 Yeast three-hvbrid (Y3U)
  • Y3H screening was performed in order to find a protein binding to 3'UTR of RbcS rapidly abolishing mRNA under dry stress.
  • a plasmid required for the Y3H system was prepared, CDNA library was constructed on plasmid pAD-GAL4 using whole mRNA extracted from seedlings.
  • the DNA oligonucleotide salt was designed to express a variety of RNA strands, and the pIIIA / MS2-l or pIIIA./MS2-2 unique Smal site
  • RNA hybrid 3'RbcS
  • hybrid protein 2 cDNA library
  • the amino acid sequence of UIP1 contains the CBS domain.
  • a deletion mutant strain was constructed as shown in FIG. 8 to find a direct domain for 3 'RbcS and mutual binding.
  • the complete mutations of UIP1 and UIP1 were confirmed by yeast co-transformation with MS2, 3'RbcS-MS2 and inverted 3'RbcS-MS2 RNAs, respectively.
  • the amino acid sequence of UIP1 52 to 212 sites are domains that directly play a role in 3'RbcS and mutual binding.
  • UIP1 The N-terminal end of UIP1 is expressed in AD- ⁇ N1, AD- ⁇ N2, and AD- ⁇ N3.
  • FIG. 8 (FIG. 8)
  • the semi-q-RT-PCR was performed at 95 ° C. for 10 minutes, followed by 30 seconds at 95 ° C., 30 seconds at 55 ° C., 30 seconds at 72 ° C., and 4 ° C. at the last cycle It proceeds with 28 cycles in 10 minutes.
  • RbcSl and Tub genes are used for RNA loading verification.
  • extended PCR extended PCR
  • the resulting composition is repeated to ensure identity.
  • the primer pairs are as follows.
  • transgenic and NT-treated 4-week-old seedlings were not watered for 2 days, and then water was added for 6 days.
  • Transformants and NT rice seeds were grown and grown in half-strength MS solid medium in a growth chamber for 14 days (28 ° C, 16 hours light treatment / 8 hours dark condition treatment).
  • the green portion of 1.0 transformers was cut using scissors for stress pretreatment in vitro All stress treatments were constructed under continuous light conditions at 150 mtnol / m2s
  • All stress treatments were constructed under continuous light conditions at 150 mtnol / m2s
  • High stress was induced by incubation at 400Cm NaCl for over 19 hours at 28 ° C.
  • the plants were treated for 3.5 hours at 28 ° C dry air.
  • the Fv / Fm values were measured using the above-described values, so that the results showed that the expression of UIP1 in transgenic rice increased the tolerance of the plant to dry, high salinity, and low stress conditions during the growing season, Showing a higher tolerance to drying stress.

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Abstract

La présente invention concerne une transformation de plante en utilisant un gène de protéine interagissant avec une région 3' non traduite (UIP1), qui est dérivé d'Oryza sativa, en tant que vecteur recombinant. Plus particulièrement, la présente invention concerne l'augmentation de la résistance d'une plante au stress de sécheresse et à un stress non biologique, tel qu'une salinité élevée et une température basse. Selon la présente invention, étant donné que la résistance d'une plante monocotylédone et d'autres plantes à un stress non biologique peut être augmentée par l'intermédiaire d'une transformation en utilisant UIP1, la présente invention peut grandement contribuer à la production en masse de produits agricoles utiles et à l'amélioration d'espèces végétales. Il est considéré que la présente invention doit être utilisée dans les domaines agricole et alimentaire, de manière à contribuer à des préparations pour des pénuries alimentaires futures. Dans le futur, la présente invention contribuera grandement à assurer la compétitivité nationale dans la guerre des semences due à des pénuries alimentaires et impliquant tous les pays du monde entier.
PCT/KR2013/005589 2012-08-06 2013-06-25 Gène uip1 pour augmenter la résistance de plantes au stress de sécheresse et utilisation de celui-ci WO2014025137A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110218247A (zh) * 2019-05-27 2019-09-10 北京林业大学 PwRBP1和PwNAC1两种蛋白互作协同提高植物耐逆性及其应用

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KR20110121784A (ko) * 2010-05-03 2011-11-09 수원대학교 산학협력단 벼 유래의 ＯｓＡＢＦ1 유전자 및 이의 용도

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110218247A (zh) * 2019-05-27 2019-09-10 北京林业大学 PwRBP1和PwNAC1两种蛋白互作协同提高植物耐逆性及其应用
CN110218247B (zh) * 2019-05-27 2022-04-05 北京林业大学 PwRBP1和PwNAC1两种蛋白互作协同提高植物耐逆性及其应用

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