WO2018164293A1 - Gène roc10 dérivé du riz pour accroître la résistance des plantes au stress hydrique et utilisation correspondante - Google Patents

Gène roc10 dérivé du riz pour accroître la résistance des plantes au stress hydrique et utilisation correspondante Download PDF

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WO2018164293A1
WO2018164293A1 PCT/KR2017/002482 KR2017002482W WO2018164293A1 WO 2018164293 A1 WO2018164293 A1 WO 2018164293A1 KR 2017002482 W KR2017002482 W KR 2017002482W WO 2018164293 A1 WO2018164293 A1 WO 2018164293A1
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roc10
plant
gene
rice
environmental stress
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PCT/KR2017/002482
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Korean (ko)
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김주곤
방승운
이동근
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서울대학교산학협력단
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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)

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  • the present invention relates to a Roc10 gene derived from rice and its use to increase drought stress tolerance of plants.
  • Drought is one of the abiotic stresses that negatively affects crop productivity, and it is important to develop drought resistant crops without the disadvantages of productivity under normal growth conditions.
  • Many studies have reported on a variety of transcription factors such as AP2 / ERF, NAC, bZIP, NF-Y, zinc fingers and MYB, which are involved in drought tolerance mechanisms. Improved plant resistance to drought stress has been reported.
  • overexpression of OsERF71 , OsAP37 , OsNAC5 , OsNAC6 , OsNAC9 / OsSNAC1 , OsNAC10 , OsNF- YA7 , OsbZIP23 , or OsbZIP45 is known to confer increased drought tolerance to rice plants.
  • HD-zip transcription factors play an important role in stress response as well as plant growth and development.
  • overexpression of HD-zip class I transcription factors has been reported to increase plant drought tolerance and cause phenotypic changes.
  • overexpression of the sunflower HaHB4 transcription factor in Arabidopsis can increase the resistance to drought and high salts , and the growth and development of Arabidopsis such as short internodes, rounded leaves and dense inflorescences. It was confirmed that it also affects.
  • Sunflower HaHB1 And Arabidopsis AtHB13 transcription factor overexpression The transgenic Arabidopsis plants have increased resistance to drought and high salt through stabilization of cell membranes and improved grain yield.
  • Rice HD-zip is class IV consisted of 11 members in the genome, ice o R c utermost was named as ell-specific gene (Roc) ( Javelle M et al, 2011, Plant Physiol 157:.. 790-803) .
  • Roc 1, 2, 3, 4 and 5 genes are mainly expressed in the epidermis of the leaf primordia around the nutrient meristem, suggesting that it is related to epidermal differentiation.
  • Roc4 and Roc5 It has been found functionally that only bays are associated with leaf curling through the regulation of flowering time and bulliform cells under long-term conditions, respectively.
  • the HD-zip Class IV Roc gene is unknown with regard to drought tolerance mechanisms.
  • the present inventors prepared a rice transformed plant overexpressing or knocking down the Roc10 gene, and comparing the non-transformers and the growth of each transformant under normal and drought stress conditions, the Roc10 overexpressing transformed plants were non-transformed. Compared to plants and knockdown transgenic plants, it showed increased drought tolerance and improved grain productivity.
  • Korean Patent Publication No. 2012-0111715 discloses 'OsbHLH148 gene and its use to increase the drought stress resistance of plants
  • Korean Patent No. 1372114 is a rice zinc finger protein transcription factor DST and drought and salt Its use to modulate resistance 'is disclosed, but there is no description of Roc10 gene from rice and its use to increase drought stress resistance of plants of the present invention.
  • the present invention was derived by the above requirements, the present inventors confirmed that the expression level of the Roc10 gene is reduced under various environmental stress conditions, transgenic plants overexpressing the Roc10 gene is non-transformant and Roc10 knockdown transformation Compared to plants, the present invention was completed by confirming that the resistance to drought stress was increased and the agricultural traits important for production determination were significantly increased.
  • the invention is by transforming the plant cell with a recombinant vector containing the gene encoding the derived Roc10 (Rice outermost cell-specific gene 10) protein, rice (Oryza sativa) that regulate the expression of Roc10 gene It provides a method for controlling the environmental stress tolerance of plants comprising the step.
  • the present invention comprises the steps of transforming plant cells with a recombinant vector comprising a gene encoding a rice-derived Roc10 protein; And it provides a method for producing a transformed plant is controlled environmental stress resistance comprising the step of regenerating the plant from the transformed plant cells.
  • the present invention provides a transgenic plant and seed thereof having a controlled environmental stress resistance produced by the above method.
  • the present invention also provides a composition for controlling environmental stress resistance of plants containing a gene encoding a Roc10 protein derived from rice as an active ingredient.
  • a gene encoding a Roc10 protein derived from rice can be used to produce a plant having increased resistance to drought stress, it may be useful for increasing crop yield and meeting food demand.
  • 1 is a schematic of a vector map for the production of Roc10 gene overexpression or knockdown transgenic plants.
  • Figure 2 shows the results of treatment of drought, abscitic acid, high salt and cold stress in wild-type (non-transformed) rice and the expression trend of Roc10 gene.
  • FIG. 3 shows the results of treating drought stress on non-transformers, Roc10 overexpressing transformed plants, and Roc10 knockdown transformed plants, and confirming drought resistance of each plant.
  • FIG. 4 shows the results of a 12-day drought treatment on non-transformers, Roc10 overexpressing transformed plants, and Roc10 knockdown transformed plants, and a pulse-amplitude modulation (PAM) analysis during the corresponding period.
  • NT untransformed plant
  • Roc10 ox Roc10 overexpressing transgenic plant
  • Roc10 RNAi Roc10 knockdown transgenic plants.
  • FIG. 5 shows the results of a 12-day drought treatment on non-transformers, Roc10 overexpressing transformed plants, and Roc10 knockdown transformed plants, during which JIP analysis was performed.
  • NT untransformed plant
  • Roc10 ox Roc10 overexpressing transgenic plant
  • Roc10 RNAi Roc10 knockdown transgenic plants.
  • the present invention transforms plant cells with a recombinant vector comprising a gene encoding a rice outer (Rice outermost cell-specific gene 10) protein derived from rice ( Oryza sativa ) to express the expression of the Roc10 gene. It provides a method of controlling the environmental stress tolerance of plants comprising the step of controlling.
  • the range of rice-derived Roc10 protein according to the present invention includes a protein having the amino acid sequence represented by SEQ ID NO: 2 and a functional equivalent of the protein.
  • “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 increases the plant's resistance to environmental stress.
  • the present invention also provides a gene encoding the rice-derived Roc10 protein.
  • the gene of the present invention may be DNA or RNA encoding Roc10 protein.
  • DNA includes cDNA, genomic DNA or artificial synthetic DNA.
  • DNA may be single stranded or double stranded, and may be a coding strand or a noncoding strand.
  • the gene encoding the rice-derived Roc10 protein of the present invention may comprise a nucleotide sequence represented by SEQ ID NO: 1.
  • 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 "regulation of gene expression” refers to increasing or decreasing the expression of the Roc10 gene derived from rice in the plant.
  • a method for controlling the environmental stress resistance of the plant in the method for controlling the environmental stress resistance of the plant according to an embodiment of the present invention, a method for controlling the environmental stress resistance of the plant, plant cells with a recombinant vector comprising a gene consisting of the nucleotide sequence of SEQ ID NO: 1 It may be to increase the environmental stress resistance of the plant by overexpressing the Roc10 gene by transformation, but is not limited thereto.
  • 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 a recombinant expression vector containing the gene under the control of a promoter.
  • 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 DNA sequences encoding rice derived Roc10 proteins 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.
  • a preferred example of the recombinant vector of the present invention is a Ti plasmid vector capable of transferring part of itself, the so-called T region, to plant cells when present in a suitable host such as Agrobacterium tumefiaciens .
  • 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.
  • 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.
  • 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 environmental stress may be drought, high salt or low temperature stress, preferably drought stress, but is not limited thereto.
  • the present invention also comprises the steps of transforming plant cells with a recombinant vector comprising a gene encoding a rice-derived Roc10 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 rice-derived Roc10 protein may be composed of the amino acid sequence of SEQ ID NO: 2, the range is as described above.
  • the method for producing a transgenic plant as a method for controlling the environmental stress resistance of the plant, by transforming the plant cell with a recombinant vector containing the gene encoding the rice-derived Roc10 protein
  • By overexpressing the Roc10 gene may be to increase the environmental stress resistance of the plant, but is not limited thereto.
  • the method of the present invention comprises transforming plant cells with a recombinant vector comprising a gene encoding a Roc10 protein derived from rice, wherein the transformation is, for example, Agrobacterium tumerfaciens ( 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 environmental stress may be drought, high salt or low temperature stress, preferably drought stress, but is not limited thereto.
  • the present invention provides a transgenic plant and seed thereof having a controlled environmental stress resistance produced by the above method.
  • the transgenic plant with controlled environmental stress resistance of the present invention is preferably a transgenic plant with increased resistance to environmental stress compared to wild type.
  • the plant of the present invention is a monocotyledonous plant such as rice, barley, wheat, rye, corn, sugar cane, oats, onions or Arabidopsis, potato, eggplant, tobacco, pepper, tomato, burdock, garland chrysanthemum, lettuce, bellflower, spinach, Chard, sweet potato, celery, carrot, buttercup, parsley, cabbage, cabbage, mustard, watermelon, melon, cucumber, pumpkin, gourd, strawberry, soybean, green beans, green beans, peas and other dicotyledonous plants, preferably monocotyledonous plants It may be, and more preferably may be rice, but is not limited thereto.
  • the present invention also provides a composition for controlling environmental stress resistance of plants containing a gene encoding a Roc10 protein derived from rice as an active ingredient.
  • the composition for regulating environmental stress resistance of a plant of the present invention comprises a gene encoding a rice-derived Roc10 protein of the present invention or a recombinant vector including the gene, and transforms the plant into a plant with the Roc10 gene or the recombinant vector including the same. By switching, the plant's environmental stress tolerance can be controlled.
  • the Roc10 gene may be overexpressed to increase environmental stress tolerance of the plant, but is not limited thereto.
  • Roc10 Full length cDNA (AK100441; Os08g0292000) was amplified by reverse transcription polymerase chain reaction. The amplified sequence was inserted into a pE3c vector (Dubin MJ et al ., 2008, Plant Methods 4: 3) flanked by a 6x myc tag coding sequence. Then, using the myc sequence Roc10-6x the Gateway system (Invitrogen, USA) from pE3c-Roc10 vector was subcloned into p700 rice plant transformed with a vector PGD1 promoter. The plasmid thus prepared was named Roc10 ox and used for the production of constitutive Roc10 gene overexpression.
  • Roc10 gene knockdown transgenic plants 321 bp (between 2103 and 2423 from ATG) from the coding region of the Roc10 gene was transferred to the p700-GOS2-RNAi vector (Lee et al ., 2016, Plant Physiol. 172: 575-). 588) was inserted into two sites separated by the GUS sequence, and the plasmid was named Roc10 RNA1 (FIG. 1).
  • the prepared plasmid was Agrobacterium tumerfaciens ( Agrobacterium ) in wild type rice ( Oryza sativa L. var. Japonica cv. Illmi). tumefaciens ) was introduced using LBA4404 strain mediated coculture. For each plasmid, three independent homozygous transformation lines were selected and T 4 (overexpression) and T 3 (knockdown) generations were used for analysis.
  • cDNA was synthesized using a first strand cDNA synthesis kit (Fermentas, USA) and oligo-dT.
  • qRT-PCR was performed using 2X qRT-PCR Pre-mix, 20X EvaGreen TM (Solgent, Korea). The amplification reaction was performed after 10 minutes of denaturation at 95 ° C. using 20 ⁇ l of a reaction solution containing 10 ng of cDNA, 0.25 ⁇ M primer and 1 ⁇ l of 20 ⁇ EvaGreen TM , followed by 95 ° C. 20 seconds, 58 ° C. 40 seconds, and 72 ° C. The procedure was repeated 40 times.
  • qRT-PCR reactions were performed using Stratagene Mx3000p (Stratagene, USA).
  • the rice ubiquitin 1 (AK121590) gene was used as a standardized control, and in order to analyze the trend of expression of the Roc10 gene for various abiotic stresses, nontransformed plants were used for 16 hours in 8 weeks / 8 hours in soil under glass greenhouse. It was cultivated at a temperature of 28 ⁇ 30 °C with photoperiod of cancer. Drought treatment was performed by air drying the entire plant at 28 ° C. by removing the soil for the indicated time. For acetic acid, salt and low temperature treatment, the whole plant was transferred to water containing 100 ⁇ M of abscidic acid, water containing 400 mM sodium chloride or water exposed to 4 ° C. and left for the indicated time.
  • chlorophyll fluorescence was measured using a Handy-PEA fluorometer (Plant Efficiency Analyzer; Hansatech Instruments, UK) under dark conditions for sufficient cancer adaptation of at least 1 hour.
  • Roc10 overexpression Roc10 ox
  • Roc10 knockdown Roc10 RNAi
  • Chlorophyll fluorescence was measured on 10 leaves from each plant and analyzed using Handy PEA software (version 1.31) and Biolyzer 4HP software (v4.0.30.03.02).
  • Seeds of non-transformed and transformed rice plants were germinated for 3 days at 28 ° C. dark conditions and 2 days at 30 ° C. light conditions on MS medium in the growth chamber, and then germinated.
  • Ninety seedlings were transferred to pots (4 ⁇ 4 ⁇ 6 cm, 3 plants per pot) and grown for 5 weeks in glass greenhouses with photoperiod of 16 hours light / 8 hours dark and temperature conditions of 28-30 ° C.
  • Drought stress was treated by stopping the water supply to the pot for 3 days, after which water was resupplied.
  • 6-week-old untransformed and transformed plants were transferred to larger pots (6 kg) and exposed to drought stress for 12 days.
  • Example 1 it was confirmed that the Roc10 gene reacts strongly under drought stress conditions.
  • drought stress was applied to non-transformers, Roc10 overexpression and knockdown transgenic plants.
  • Roc10 overexpressing transgenic plants showed almost the same condition as the untreated drought stress control group even after 2 days of drought stress, and 3 days after drought stress. Although the symptoms of induced damage were weaker, the degree of symptoms was weaker than that of drought stress-treated non-transformed plants, and moreover, it was observed that recovery from drought stress-induced damage was resumed when water supply was resumed (FIG. 3). The results suggest that the Roc10 gene may enhance resistance to drought stress.
  • PAM pulse-amplitude modulation
  • F v / F m values and the PI total value of JIP analysis of PAM analysis in the plant are used to observe the influence of abiotic stress, non-transgenic plant by drought stress treatment and Roc10 knockdown transgenic plant is F v It showed a strong decrease in the / F m value.
  • the F v / F m value was 0.8 when unstrained and Roc10 knockdown transformed plants were not treated with stress, but after 6 days of drought stress treatment, the F v / F m value began to decrease and the stress treatment time increased. In addition, the decline was accelerated.
  • Roc10 overexpressing transgenic plants were confirmed that there is no change in the F v / F m value due to drought stress (Fig. 4).
  • Example 2 it was confirmed that overexpression of the Roc10 gene enhances plant resistance to drought.
  • the agricultural traits of Roc10 overexpressing transgenic plants were examined.

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Abstract

La présente invention concerne : un procédé de régulation de la résistance d'une plante à un stress environnemental par transformation de cellules végétales avec un vecteur recombinant comprenant un gène codant pour une protéine de gène 10 (Roc10) spécifique à une cellule la plus externe de riz dérivée d'Oryza sativa ; un procédé de production d'une plante transgénique dont la résistance au stress environnemental est régulée par transformation de cellules végétales avec un vecteur recombinant comprenant un gène codant pour la protéine Roc10 ; une plante transgénique produite par le procédé et dont la résistance au stress environnemental est régulée et les graines correspondantes ; et une composition pour réguler la résistance de plantes à un stress environnemental, comprenant, en tant que principe actif, un gène codant pour la protéine Roc10.
PCT/KR2017/002482 2017-03-08 2017-03-08 Gène roc10 dérivé du riz pour accroître la résistance des plantes au stress hydrique et utilisation correspondante WO2018164293A1 (fr)

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Cited By (1)

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CN113862387A (zh) * 2021-08-27 2021-12-31 上海市农业生物基因中心 水稻耐旱性调控基因OsNAC6的分子标记及其应用

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113862387A (zh) * 2021-08-27 2021-12-31 上海市农业生物基因中心 水稻耐旱性调控基因OsNAC6的分子标记及其应用
CN113862387B (zh) * 2021-08-27 2023-10-24 上海市农业生物基因中心 水稻耐旱性调控基因OsNAC6的分子标记及其应用

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