WO2018164293A1 - Rice-derived roc10 gene for increasing plant resistance to drought stress, and use thereof - Google Patents

Rice-derived roc10 gene for increasing plant resistance to drought stress, and use thereof 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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French (fr)
Korean (ko)
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김주곤
방승운
이동근
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서울대학교산학협력단
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    • C—CHEMISTRY; METALLURGY
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    • 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
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    • 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)

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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

The present invention relates to: a method for controlling plant resistance to environmental stress by transforming plant cells with a recombinant vector comprising a gene coding for an Oryza sativa-derived rice outermost cell-specific gene 10 (Roc10) protein; a method for producing a transgenic plant of which the resistance to environmental stress is controlled by transforming plant cells with a recombinant vector comprising a gene coding for the Roc10 protein; a transgenic plant produced by the method and of which the resistance to environmental stress is controlled, and the seeds thereof; and a composition for controlling plant resistance to environmental stress, comprising, as an active ingredient, a gene coding for the Roc10 protein.

Description

식물의 가뭄 스트레스 내성을 증가시키는 벼 유래의 Ｒｏｃ10 유전자 및 이의 용도Rice-derived ROCc10 gene and its use to increase drought stress tolerance in plants
본 발명은 식물의 가뭄 스트레스 내성을 증가시키는 벼 유래의 Roc10 유전자 및 이의 용도에 관한 것이다.The present invention relates to a Roc10 gene derived from rice and its use to increase drought stress tolerance of plants.
가뭄은 작물의 생산성에 부정적인 영향을 미치는 비생물적 스트레스 중 하나로, 정상 생장 조건에서 생산성의 불이익 없이 가뭄 내성 작물을 개발하는 것은 중요하다. 많은 연구를 통해 가뭄 내성 기작에 관여하는 AP2/ERF, NAC, bZIP, NF-Y, 징크 핑거(Zinc finger) 및 MYB 등과 같은 다양한 전사인자에 관한 보고가 있었으며, 상기 전사인자 활성의 분자적 변형이 가뭄 스트레스에 대한 식물체의 저항성을 향상시키는 것이 보고되었다. 일례로, OsERF71, OsAP37, OsNAC5, OsNAC6 , OsNAC9/OsSNAC1, OsNAC10, OsNF -YA7, OsbZIP23, 또는 OsbZIP45의 과발현은 벼 식물체에 증가된 가뭄 내성을 부여하는 것으로 알려졌다.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. In one example, overexpression of OsERF71 , OsAP37 , OsNAC5 , OsNAC6 , OsNAC9 / OsSNAC1 , OsNAC10 , OsNF- YA7 , OsbZIP23 , or OsbZIP45 is known to confer increased drought tolerance to rice plants.
모든 진핵 생물체는 60개의 보존된 아미노산으로 특징되는 호메오도메인(homeodomain, HD) 전사인자를 가지고 있다. 특히, 식물체 특이적 호메오도메인 루신 지퍼(homeodomain leucine zipper, HD-zip) 전사인자는 호메오도메인과 함께 루신 지퍼 도메인을 포함한다. HD-zip 전사인자는 식물체의 생장 및 발달 뿐만 아니라 스트레스 반응에도 중요하게 작용한다. 최근의 발표에 의하면, HD-zip 클래스 Ⅰ 전사인자의 과발현은 식물체의 가뭄 내성을 증가시키고, 표현형적 변화를 야기하는 것으로 보고되었다. 예를 들면, 해바라기 HaHB4 전사인자를 애기장대에 과발현시킨 경우, 가뭄과 고염에 대한 내성이 증가되고 짧은 절간(internode), 둥근형태의 잎 및 조밀한 화서(inflorescences) 등과 같은 애기장대의 생장 및 발달에도 영향이 미치는 것으로 확인되었다. 해바라기 HaHB1 및 애기장대 AtHB13 전사인자 과발현 형질전환 애기장대 식물체는 세포막의 안정화를 통해 가뭄 및 고염에 대한 내성이 증가되고, 곡식수량이 향상되었다.All eukaryotic organisms have homeodomain (HD) transcription factors that are characterized by 60 conserved amino acids. In particular, plant-specific homeodomain leucine zipper (HD-zip) transcription factors include leucine zipper domains along with homeodomains. HD-zip transcription factors play an important role in stress response as well as plant growth and development. According to a recent announcement, overexpression of HD-zip class I transcription factors has been reported to increase plant drought tolerance and cause phenotypic changes. For example, 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.
벼 HD-zip 클래스 Ⅳ는 게놈 상에 11개의 멤버로 구성되었는데, R ice o utermost c ell-specific gene(Roc)으로 명명되었다(Javelle M et al., 2011, Plant Physiol. 157:790-803). Roc1, 2, 3, 4 및 5 유전자는 영양 분열조직 주변의 잎원기(leaf primordia)의 표피에 주로 발현하고 있어 표피 분화와 관련되어 있음을 암시하였다. 클래스 Ⅳ의 11개 멤버 중, 오직 Roc4 및 Roc5 만이 각각 장일 조건에서 우선적으로 개화시기 및 기동세포(bulliform cell)의 조절을 통한 잎말림과 연관된다는 것이 기능적으로 규명되었다. 가뭄 내성과 연관되어 있는 HD-zip 클래스 I 유전자와는 다르게, HD-zip 클래스 Ⅳ Roc 유전자는 가뭄 내성 기작과 관련되어 알려진 바가 없다.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. Of the 11 members of class IV, only 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. Unlike the HD-zip Class I gene, which is associated with drought tolerance, the HD-zip Class IV Roc gene is unknown with regard to drought tolerance mechanisms.
이에 본 발명자들은 Roc10 유전자가 과발현 또는 녹다운된 벼 형질전환 식물체를 제작하고, 정상 및 가뭄 스트레스 조건에서 비형질전환체 및 각 형질전환체의 생육을 비교한 결과, Roc10 과발현 형질전환 식물체가 비형질전환 식물체 및 녹다운 형질전환 식물체에 비해 증가된 가뭄 내성과 향상된 곡물 생산성을 보여줌을 확인하였다.Therefore, 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.
한편, 한국공개특허 제2012-0111715호에는 '식물체의 한발 스트레스 저항성을 증가시키는 OsbHLH148 유전자 및 이의 용도'가 개시되어 있고, 한국등록특허 제1372114호에는 '벼 징크 핑거 단백질 전사인자 DST 및 가뭄 및 염 내성을 조절하기 위한 이의 용도'가 개시되어 있으나, 본 발명의 식물의 가뭄 스트레스 내성을 증가시키는 벼 유래의 Roc10 유전자 및 이의 용도에 대해서는 기재된 바가 없다.On the other hand, 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.
본 발명은 상기와 같은 요구에 의해 도출된 것으로서, 본 발명자들은 다양한 환경 스트레스 조건 하에서 Roc10 유전자의 발현 수준이 감소됨을 확인하였고, Roc10 유전자를 과발현시킨 형질전환 식물체가 비형질전환체 및 Roc10 녹다운 형질전환 식물체에 비해서, 가뭄 스트레스에 대한 내성이 증가되었으며, 생산량 결정에 중요한 농업형질들이 현저히 증가된 것을 확인함으로써, 본 발명을 완성하였다.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.
상기 과제를 해결하기 위해, 본 발명은 벼(Oryza sativa) 유래 Roc10 (Rice outermost cell-specific gene 10) 단백질을 코딩하는 유전자를 포함하는 재조합 벡터로 식물세포를 형질전환시켜 Roc10 유전자의 발현을 조절하는 단계를 포함하는 식물체의 환경 스트레스 내성을 조절하는 방법을 제공한다.In order to solve the above problems, 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.
또한, 본 발명은 벼 유래 Roc10 단백질을 코딩하는 유전자를 포함하는 재조합 벡터로 식물세포를 형질전환하는 단계; 및 상기 형질전환된 식물세포로부터 식물을 재분화하는 단계를 포함하는 환경 스트레스 내성이 조절된 형질전환 식물체의 제조방법을 제공한다.In addition, 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.
또한, 본 발명은 상기 방법에 의해 제조된 환경 스트레스 내성이 조절된 형질전환 식물체 및 이의 종자를 제공한다.In addition, the present invention provides a transgenic plant and seed thereof having a controlled environmental stress resistance produced by the above method.
또한, 본 발명은 벼 유래 Roc10 단백질을 코딩하는 유전자를 유효성분으로 함유하는 식물체의 환경 스트레스 내성 조절용 조성물을 제공한다.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.
본 발명에 따르면, 벼 유래 Roc10 단백질을 코딩하는 유전자를 이용하면 가뭄 스트레스에 대한 내성이 증가된 식물체를 제조할 수 있으므로, 작물의 생산량 증대 및 식량 수요의 충족에 유용할 것으로 사료된다.According to the present invention, since 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.
도 1은 Roc10 유전자 과발현 또는 녹다운 형질전환 식물체 제작을 위한 벡터맵의 도식이다.1 is a schematic of a vector map for the production of Roc10 gene overexpression or knockdown transgenic plants.
도 2는 야생형(비형질전환) 벼에 가뭄, 앱시스산, 고염 및 저온 스트레스를 처리하고 Roc10 유전자의 발현 경향을 분석한 결과이다.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.
도 3은 비형질전환체, Roc10 과발현 형질전환 식물체 및 Roc10 녹다운 형질전환 식물체에 가뭄 스트레스를 처리하고, 각 식물체의 가뭄 내성을 확인한 결과이다.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.
도 4는 비형질전환체, Roc10 과발현 형질전환 식물체 및 Roc10 녹다운 형질전환 식물체에 12일간 가뭄 처리를 하고, 해당 기간 동안 펄스 증폭 변조(PAM, Pulse-Amplitude Modulation) 분석을 수행한 결과이다. NT, 비형질전환 식물체; Roc10 ox, Roc10 과발현 형질전환 식물체; Roc10 RNAi, Roc10 녹다운 형질전환 식물체.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.
도 5는 비형질전환체, Roc10 과발현 형질전환 식물체 및 Roc10 녹다운 형질전환 식물체에 12일간 가뭄 처리를 하고, 해당 동안 JIP 분석을 수행한 결과이다. NT, 비형질전환 식물체; Roc10 ox, Roc10 과발현 형질전환 식물체; Roc10 RNAi, Roc10 녹다운 형질전환 식물체.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.
본 발명의 목적을 달성하기 위하여, 본 발명은 벼(Oryza sativa) 유래 Roc10 (Rice outermost cell-specific gene 10) 단백질을 코딩하는 유전자를 포함하는 재조합 벡터로 식물세포를 형질전환시켜 Roc10 유전자의 발현을 조절하는 단계를 포함하는 식물체의 환경 스트레스 내성을 조절하는 방법을 제공한다.In order to achieve the object of the present invention, 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.
본 발명에 따른 벼 유래 Roc10 단백질의 범위는 서열번호 2로 표시되는 아미노산 서열을 갖는 단백질 및 상기 단백질의 기능적 동등물을 포함한다. "기능적 동등물"이란 아미노산의 부가, 치환 또는 결실의 결과, 상기 서열번호 2로 표시되는 아미노산 서열과 적어도 70% 이상, 바람직하게는 80% 이상, 더욱 바람직하게는 90% 이상, 더 더욱 바람직하게는 95% 이상의 서열 상동성을 갖는 것으로, 서열번호 2로 표시되는 단백질과 실질적으로 동질의 생리활성을 나타내는 단백질을 말한다. "실질적으로 동질의 생리활성"이란 식물의 환경 스트레스에 대한 내성을 증가시키는 활성을 의미한다.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.
또한, 본 발명은 상기 벼 유래 Roc10 단백질을 코딩하는 유전자를 제공한다. 본 발명의 유전자는 Roc10 단백질을 코딩하는 DNA 또는 RNA일 수 있다. DNA는 cDNA, 게놈 DNA 또는 인위적인 합성 DNA를 포함한다. DNA는 단일 가닥 또는 이중 가닥일 수 있고, 코딩(coding) 가닥 또는 넌코딩(noncoding) 가닥일 수 있다. 바람직하게는, 본 발명의 벼 유래 Roc10 단백질을 코딩하는 유전자는 서열번호 1로 표시되는 염기서열을 포함할 수 있다. 또한, 상기 염기서열의 상동체가 본 발명의 범위 내에 포함된다. 구체적으로, 상기 유전자는 서열번호 1의 염기 서열과 각각 70% 이상, 더욱 바람직하게는 80% 이상, 더 더욱 바람직하게는 90% 이상, 가장 바람직하게는 95% 이상의 서열 상동성을 가지는 염기서열을 포함할 수 있다. 폴리뉴클레오티드에 대한 "서열 상동성의 %"는 두 개의 최적으로 배열된 서열과 비교 영역을 비교함으로써 확인되며, 비교 영역에서의 폴리뉴클레오티드 서열의 일부는 두 서열의 최적 배열에 대한 참고 서열(추가 또는 삭제를 포함하지 않음)에 비해 추가 또는 삭제(즉, 갭)를 포함할 수 있다.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. Preferably, the gene encoding the rice-derived Roc10 protein of the present invention may comprise a nucleotide sequence represented by SEQ ID NO: 1. In addition, homologues of the above nucleotide sequences are included within the scope of the present invention. Specifically, 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).
본 발명의 식물체의 환경 스트레스 내성을 조절하는 방법에 있어서, 상기 "유전자 발현 조절"은 식물체 내의 벼 유래 Roc10 유전자의 발현을 증가시키거나 또는 감소시키는 것을 말한다.In the method for regulating the environmental stress resistance of the plant of the present invention, the "regulation of gene expression" refers to increasing or decreasing the expression of the Roc10 gene derived from rice in the plant.
본 발명의 일 구현 예에 따른 식물체의 환경 스트레스 내성을 조절하는 방법에서, 식물체의 환경 스트레스 내성을 조절할 수 있는 방법으로는, 서열번호 1의 염기서열로 이루어진 유전자를 포함하는 재조합 벡터로 식물세포를 형질전환시켜 Roc10 유전자를 과발현시켜 식물체의 환경 스트레스 내성을 증가시키는 것일 수 있으나, 이에 제한되지 않는다.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.
상기 "유전자 과발현"이란 야생형 식물에서 발현되는 수준 이상으로 상기 유전자가 발현되도록 하는 것을 의미한다. 식물체 내로 상기 유전자를 도입하는 방법으로는 프로모터의 조절을 받는 상기 유전자가 포함된 재조합 발현 벡터를 이용하여 식물체를 형질전환하는 방법이 있다.The "gene overexpression" means that the gene is expressed above the level expressed in wild-type plants. As 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.
용어 "재조합 발현 벡터"는 세균 플라스미드, 파아지, 효모 플라스미드, 식물 세포 바이러스, 포유동물 세포 바이러스, 또는 다른 벡터를 의미한다. 대체로, 임의의 플라스미드 및 벡터는 숙주 내에서 복제 및 안정화할 수 있다면 사용될 수 있다. 상기 발현 벡터의 중요한 특성은 복제 원점, 프로모터, 마커 유전자 및 번역 조절 요소(translation control element)를 가지는 것이다.The term "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.
벼 유래 Roc10 단백질을 코딩하는 DNA 서열 및 적당한 전사/번역 조절 신호를 포함하는 발현 벡터는 당업자에 주지된 방법에 의해 구축될 수 있다. 상기 방법은 시험관내 재조합 DNA 기술, DNA 합성 기술 및 생체내 재조합 기술 등을 포함한다. 상기 DNA 서열은 mRNA 합성을 이끌기 위해 발현 벡터 내의 적당한 프로모터에 효과적으로 연결될 수 있다. 또한 발현 벡터는 번역 개시 부위로서 리보좀 결합 부위 및 전사 터미네이터를 포함할 수 있다.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.
본 발명의 재조합 벡터의 바람직한 예는 아그로박테리움 투머파시엔스(Agrobacterium tumefiaciens)와 같은 적당한 숙주에 존재할 때 그 자체의 일부, 소위 T영역을 식물 세포로 전이시킬 수 있는 Ti플라스미드 벡터이다. 다른 유형의 Ti플라스미드 벡터 (EP 0 116 718 B1호 참조)는 현재 식물 세포, 또는 잡종 DNA를 식물의 게놈 내에 적당하게 삽입시키는 새로운 식물이 생산될 수 있는 원형질체로 잡종 DNA 서열을 전이시키는데 이용되고 있다. Ti플라스미드 벡터의 특히 바람직한 형태는 EP 0 120 516 B1호 및 미국 특허 제4,940,838호에 청구된 바와 같은 소위 바이너리(binary) 벡터이다. 본 발명에 따른 DNA를 식물 숙주에 도입시키는데 이용될 수 있는 다른 적합한 벡터는 이중 가닥 식물 바이러스(예를 들면, CaMV) 및 단일 가닥 바이러스, 게미니 바이러스 등으로부터 유래될 수 있는 것과 같은 바이러스 벡터, 예를 들면 비완전성 식물 바이러스 벡터로부터 선택될 수 있다. 그러한 벡터의 사용은 특히 식물 숙주를 적당하게 형질전환하는 것이 어려울 때 유리할 수 있다.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. Other suitable vectors that can be used to introduce the DNA according to the invention into a plant host are viral vectors, such as those which can be derived from double stranded plant viruses (eg CaMV) and single stranded viruses, gemini viruses, etc. For example, 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.
식물의 형질전환에 이용되는 "식물 세포"는 어떤 식물 세포도 된다. 식물 세포는 배양 세포, 배양 조직, 배양기관 또는 전체 식물이다. "식물 조직"은 분화된 또는 미분화된 식물의 조직, 예를 들면 이에 한정되진 않으나, 뿌리, 줄기, 잎, 꽃가루, 종자, 암 조직 및 배양에 이용되는 다양한 형태의 세포들, 즉 단일 세포, 원형질체(protoplast), 싹 및 캘러스 조직을 포함한다. 식물 조직은 인 플란타(in planta)이거나 기관 배양, 조직배양 또는 세포 배양 상태일 수 있다.The "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.
본 발명의 일 구현 예에 따른 식물체의 환경 스트레스 내성을 조절하는 방법에 있어서, 상기 환경 스트레스는 가뭄, 고염 또는 저온의 스트레스일 수 있고, 바람직하게는 가뭄 스트레스일 수 있으나, 이에 제한되지 않는다.In the method for controlling the environmental stress resistance of the plant according to an embodiment of the present invention, the environmental stress may be drought, high salt or low temperature stress, preferably drought stress, but is not limited thereto.
본 발명은 또한, 벼 유래 Roc10 단백질을 코딩하는 유전자를 포함하는 재조합 벡터로 식물세포를 형질전환하는 단계; 및The present invention also comprises the steps of transforming plant cells with a recombinant vector comprising a gene encoding a rice-derived Roc10 protein; And
상기 형질전환된 식물세포로부터 식물을 재분화하는 단계를 포함하는 환경 스트레스 내성이 조절된 형질전환 식물체의 제조방법을 제공한다.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.
본 발명의 일 구현 예에 따른 형질전환 식물체의 제조방법에 있어서, 상기 벼 유래 Roc10 단백질은 서열번호 2의 아미노산 서열로 이루어진 것일 수 있고, 그 범위는 전술한 바와 같다.In the method for producing a transgenic plant according to an embodiment of the present invention, the rice-derived Roc10 protein may be composed of the amino acid sequence of SEQ ID NO: 2, the range is as described above.
본 발명의 일 구현 예에 따른 형질전환 식물체의 제조방법에서, 식물체의 환경 스트레스 내성을 조절할 수 있는 방법으로는, 상기 벼 유래 Roc10 단백질을 코딩하는 유전자를 포함하는 재조합 벡터로 식물세포를 형질전환시켜 Roc10 유전자를 과발현시켜 식물체의 환경 스트레스 내성을 증가시키는 것일 수 있으나, 이에 제한되지 않는다.In the method for producing a transgenic plant according to an embodiment of the present invention, 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.
본 발명의 방법은 벼 유래 Roc10 단백질을 코딩하는 유전자를 포함하는 재조합 벡터로 식물 세포를 형질전환하는 단계를 포함하는데, 상기 형질전환은 예를 들면, 아그로박테리움 튜머파시엔스(Agrobacterium tumefiaciens)에 의해 매개될 수 있다. 또한, 본 발명의 방법은 상기 형질전환된 식물 세포로부터 형질전환 식물을 재분화하는 단계를 포함한다. 형질전환 식물 세포로부터 형질전환 식물을 재분화하는 방법은 당업계에 공지된 임의의 방법을 이용할 수 있다.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.
형질전환된 식물세포는 전식물로 재분화되어야 한다. 캘러스 또는 원형질체 배양으로부터 성숙한 식물의 재분화를 위한 기술은 수많은 여러 가지 종에 대해서 당업계에 주지되어 있다.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.
본 발명의 일 구현 예에 따른 형질전환 식물체의 제조방법에 있어서, 상기 환경 스트레스는 가뭄, 고염 또는 저온의 스트레스일 수 있고, 바람직하게는 가뭄 스트레스일 수 있으나, 이에 제한되지 않는다.In the method for producing a transformed plant according to an embodiment of the present invention, the environmental stress may be drought, high salt or low temperature stress, preferably drought stress, but is not limited thereto.
또한, 본 발명은 상기 방법에 의해 제조된 환경 스트레스 내성이 조절된 형질전환 식물체 및 이의 종자를 제공한다. 본 발명의 상기 환경 스트레스 내성이 조절된 형질전환 식물체는 바람직하게는 야생형에 비해 환경 스트레스에 대한 내성이 증가된 형질전환 식물체이다.In addition, 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.
본 발명의 상기 식물체는 벼, 보리, 밀, 호밀, 옥수수, 사탕수수, 귀리, 양파 등의 단자엽 식물 또는 애기장대, 감자, 가지, 담배, 고추, 토마토, 우엉, 쑥갓, 상추, 도라지, 시금치, 근대, 고구마, 샐러리, 당근, 미나리, 파슬리, 배추, 양배추, 갓무, 수박, 참외, 오이, 호박, 박, 딸기, 대두, 녹두, 강낭콩, 완두 등의 쌍자엽 식물일 수 있고, 바람직하게는 단자엽 식물일 수 있으며, 더욱 바람직하게는 벼일 수 있으나, 이에 제한되지 않는다.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.
또한, 본 발명은 벼 유래 Roc10 단백질을 코딩하는 유전자를 유효성분으로 함유하는 식물체의 환경 스트레스 내성 조절용 조성물을 제공한다.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.
본 발명의 식물체의 환경 스트레스 내성 조절용 조성물은 유효성분으로 본 발명의 벼 유래 Roc10 단백질을 코딩하는 유전자 또는 상기 유전자를 포함하는 재조합 벡터를 포함하며, 상기 Roc10 유전자 또는 이를 포함하는 재조합 벡터를 식물체에 형질전환시킴으로써 식물체의 환경 스트레스 내성을 조절할 수 있는 것이다. 바람직하게는, 상기 Roc10 유전자를 과발현시켜 식물체의 환경 스트레스 내성을 증가시킬 수 있으나, 이에 제한되지 않는다.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. Preferably, the Roc10 gene may be overexpressed to increase environmental stress tolerance of the plant, but is not limited thereto.
이하, 본 발명을 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.
재료 및 방법Materials and methods
플라스미드 구축 및 아그로박테리움 매개 벼 형질전환Plasmid Construction and Agrobacterium Mediated Rice Transformation
Roc10 유전자 과발현 형질전환 식물체의 제작을 위해, Roc10 전장 cDNA(AK100441; Os08g0292000)를 역전사 중합효소연쇄반응을 통해 증폭하였다. 6x myc 태그 코딩서열이 플랭크된 pE3c 벡터(Dubin MJ et al., 2008, Plant Methods 4:3)로 상기 증폭된 서열을 삽입하였다. 그 후, pE3c-Roc10 벡터로부터 Roc10-6x myc 서열을 게이트웨이 시스템(Invitrogen, 미국)을 이용하여 PGD1 프로모터를 가진 p700 벼 형질전환 벡터로 서브클로닝하였다. 이렇게 제작된 플라스미드를 Roc10 ox로 명명하고, 항시적 Roc10 유전자 과발현체 제작을 위해 사용하였다. 또한, Roc10 유전자 녹다운 형질전환 식물체의 제작을 위해, Roc10 유전자의 코딩 영역으로부터 321bp(ATG로부터 2103~2423 사이)를 p700-GOS2-RNAi 백터(Lee et al., 2016, Plant Physiol. 172:575-588)의 GUS 서열에 의해 분리된 두 개의 자리에 삽입하였고, 상기 플라스미드를 Roc10 RNA1로 명명하였다(도 1). For the production of Roc10 gene overexpressing transgenic plants, 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. In addition, for the production of 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).
제작된 플라스미드는 야생형의 벼(Oryza sativa L. var. Japonica cv. Illmi)에 아그로박테리움 튜머파시엔스(Agrobacterium tumefaciens) LBA4404 균주 매개 공배양법을 사용하여 도입시켰다. 각각의 플라스미드에 대해, 세 개의 독립적인 동형접합성의 형질전환 라인을 선별하고, T4(과발현) 및 T3(녹다운) 세대를 분석을 위해 사용하였다.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.
정량적 실시간 중합효소연쇄반응(quantitative Real Time PCR, qRT-PCR)Quantitative Real Time PCR (qRT-PCR)
유전자 발현 수준을 분석하기 위해서, 제1가닥 cDNA 합성 키트(Fermentas, 미국)와 oligo-dT를 사용하여 cDNA를 합성하였다. qRT-PCR은 2X qRT-PCR Pre-mix, 20X EvaGreenTM(Solgent, 한국)을 사용하여 수행하였다. 증폭반응은 10ng의 cDNA, 0.25μM의 프라이머 및 1㎕의 20X EvaGreenTM을 포함하는 20㎕의 반응액을 이용하여 95℃에서 10분 변성 후, 95℃ 20초, 58℃ 40초 및 72℃ 20초의 과정을 40회 반복하여 수행하였다. qRT-PCR 반응은 Stratagene Mx3000p (Stratagene, 미국)을 이용하여 수행하였다. 벼 ubiquitin 1(AK121590) 유전자를 표준화 대조구로 사용하였으며, 다양한 비생물적 스트레스에 대한 Roc10 유전자의 발현 경향을 분석하기 위해, 비형질전환 식물체를 유리온실 하의 토양에서 2주 동안 16시간 명/8시간 암의 광주기로 28~30℃ 온도 조건에서 재배하였다. 가뭄 처리는 지시된 시간 동안 흙을 제거함으로써 28℃에서 전체 식물체를 공기 건조시켜 처리하였다. 앱시스산, 염 및 저온 처리를 위해, 전체 식물체를 각각 100μM의 앱시스산을 포함하고 있는 물, 400mM의 염화나트륨을 포함하고 있는 물 또는 4℃로 노출된 물에 옮기고 지시된 시간 동안 두었다.To analyze gene expression levels, 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 ™ (Solgent, Korea). The amplification reaction was performed after 10 minutes of denaturation at 95 ° C. using 20 μl of a reaction solution containing 10 ng of cDNA, 0.25 μM primer and 1 μl 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 ℃ 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 μM of abscidic acid, water containing 400 mM sodium chloride or water exposed to 4 ° C. and left for the indicated time.
중합효소연쇄반응을 위한 프라이머 정보Primer Information for Polymerase Chain Reaction
유전자명Gene name 정방향(5'→3')Forward direction (5 '→ 3') 역방향(5'→3')Reverse direction (5 '→ 3')
Roc10Roc10 TGCGAACCTTCTAGTCCTAC (서열번호 3)TGCGAACCTTCTAGTCCTAC (SEQ ID NO: 3) GGTGAGGAGTTGGTATCTTC (서열번호 4)GGTGAGGAGTTGGTATCTTC (SEQ ID NO: 4)
OsUBI1OsUBI1 ATGGAGCTGCTGCTGTTCTA (서열번호 5)ATGGAGCTGCTGCTGTTCTA (SEQ ID NO: 5) TTCTTCCATGCTGCTCTACC (서열번호 6)TTCTTCCATGCTGCTCTACC (SEQ ID NO: 6)
엽록소 형광 측정Chlorophyll Fluorescence Measurement
Fv/Fm 및 PItotal 값을 측정하기 위해서, 엽록소 형광을 최소 1시간의 충분한 암 적응을 위해 암 조건에서 Handy-PEA 형광측정기(Plant Efficiency Analyzer; Hansatech Instruments, 영국)를 사용하여 측정하였다. Roc10 과발현(Roc10 ox), Roc10 녹다운(Roc10 RNAi) 형질전환 식물체 및 비형질전환 식물체를 6주 동안 28~30℃의 온도 조건에서 재배하였다. 각 식물체로부터 10장의 잎에 대해 엽록소 형광을 측정하였으며, Handy PEA 소프트웨어(1.31 버전)와 Biolyzer 4HP 소프트웨어(v4.0.30.03.02)를 사용하여 분석하였다.To determine the F v / F m and PI total values, 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 ) transformed plants and non-transformed plants were cultivated at 28-30 ° C. for 6 weeks. 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).
가뭄 스트레스 처리Drought stress treatment
비형질전환 및 형질전환 벼 식물체의 종자를 생장챔버 내 MS 배지 상에서 28℃ 암 조건에서 3일, 그 후 30℃의 명 조건에서 2일 동안 두어 발아시킨다음, 비형질전환 및 형질전환 식물체의 각 90개의 유묘를 포트(4x4x6cm, 포트 당 3식물체)에 옮겨심고, 16시간 명/8시간 암의 광주기 및 28~30℃의 온도 조건인 유리온실에서 5주 동안 재배하였다. 가뭄 스트레스는 3일 동안 포트에 물공급을 중단하여 처리하였고, 후에 다시 물을 재공급하였다. JIP 분석을 위해, 6주령의 비형질전환 및 형질전환 식물체를 보다 큰 포트(6kg)로 옮겨심고 12일 동안 가뭄 스트레스에 노출시켰다.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. For JIP analysis, 6-week-old untransformed and transformed plants were transferred to larger pots (6 kg) and exposed to drought stress for 12 days.
야외 포장에서 재배한 벼 식물체의 Of rice plants grown in the outdoor pavement 농업형질Agriculture 분석 analysis
정상 농지 조건에서 형질전환 벼 식물체의 농업형질을 분석하기 위해, Roc10 과발현 형질전환체(Roc10 ox) 및 Roc10 녹다운 형질전환체(Roc10 RNAi)의 세 개의 독립적인 T3 라인을 경북대학교 군위(128:34E/36:15N)의 논에 심었다. 무작위 선정에 따라 3곳의 다른 대지에 심어 3반복 수행하였다. 수량 구성 요소는 3곳의 다른 대지로부터 각 계통 당 30개의 식물체를 사용하여 측정하였다. 가뭄 농지 조건에서 형질전환 식물체의 수량 변수들을 측정하기 위해서, 출수 전 및 출수 후 단계 동안 두 번의 가뭄 스트레스를 처리하였다. 비형질전환 식물체가 가뭄 스트레스의 달관 증상(잎 말림)을 보일 때 재관수하였으며, 동일 과정을 반복하였다. 두 번의 가뭄 스트레스 처리 후에, 재관수된 식물체를 수확때까지 재배하였다.To analyze the agricultural traits of transformed rice plants under normal farmland conditions, three independent T 3 lines of Roc10 overexpressing transformants ( Roc10 ox ) and Roc10 knockdown transformants ( Roc10 RNAi ) were used. 34E / 36: 15N). Three repetitions were performed by planting on three different sites according to random selection. Quantity components were measured using 30 plants per line from three different sites. In order to measure the yield parameters of transgenic plants in drought farmland conditions, two drought stresses were treated before and after the withdrawal phase. When the transgenic plants showed drought stress symptomatic symptoms (leaf curls), they were re-watered and the same process was repeated. After two drought stress treatments, the irrigated plants were grown until harvest.
실시예 1. 비생물적 스트레스 조건에서 Example 1 Under Abiotic Stress Conditions Roc10Roc10 유전자의 발현 경향 분석 Analysis of Gene Expression Trend
가뭄 스트레스를 포함한 비생물적 스트레스에 대한 반응으로 Roc10 유전자의 발현 경향이 어떻게 변화하는지 확인하기 위해서 가뭄, 고염, 앱시스산 및 저온 스트레스를 야생형(비형질전환) 벼에 처리하였다.To determine how the expression of the Roc10 gene changes in response to abiotic stress, including drought stress, drought, high salt, abscis acid and cold stress were treated in wild-type (nontransformed) rice.
그 결과, Roc10 유전자의 발현은 벼 식물체의 줄기 부분에서 가뭄, 고염 및 저온 스트레스 시에 급격하게 발현양이 감소되는 것이 확인되었고, 앱시스산 처리에서는 발현 수준이 크게 감소되지 않는 것이 확인되었다(도 2).As a result, it was confirmed that the expression of Roc10 gene is drastically reduced in the stem part of rice plants during drought, high salt, and low temperature stress, and that the expression level is not significantly reduced in the treatment of absic acid (FIG. 2). ).
실시예 2. Example 2. Roc10Roc10 유전자 과발현에 의한 가뭄 저항성 검증 Drought Resistance Verification by Gene Overexpression
전술한 실시예 1을 통해 가뭄 스트레스 조건에서 Roc10 유전자가 강하게 반응함을 확인하였다. 이에 Roc10 유전자의 가뭄 내성에 대한 잠재적인 기능을 확인하기 위해, 비형질전환체, Roc10 과발현 및 녹다운 형질전환 식물체에 가뭄 스트레스를 처리하였다.In Example 1 described above, it was confirmed that the Roc10 gene reacts strongly under drought stress conditions. In order to confirm the potential function of drought resistance of the Roc10 gene, drought stress was applied to non-transformers, Roc10 overexpression and knockdown transgenic plants.
그 결과, 가뭄 스트레스 처리 2일 후, 비형질전환 식물체는 잎의 말림 및 시들음과 같은 가뭄 유도 손상의 약한 증상들이 확인되었고, 가뭄 스트레스 처리 시간의 증가와 함께 증상은 심각해졌으며, 가뭄 처리 3일째에 가뭄 유도에 의한 강한 손상 증상을 나타내었다. 최종적으로, 비형질전환 식물체의 잎은 모두 말라버렸으며, 물을 재공급하여도 가뭄 스트레스 유도에 의한 손상으로부터 식물체는 회복되지 않았다. 또한, Roc10 녹다운 형질전환 식물체도 비형질전환 식물체의 결과와 유사하게 확인되었는데, Roc10 녹다운 형질전환 식물체 역시 가뭄 스트레스 처리 시간이 경과할수록 가뭄 스트레스 유도 손상의 증상이 심하게 나타났으며, 물을 재공급한 후에도 손상으로부터 회복되지 않았다. 반면, Roc10 과발현 형질전환 식물체는 가뭄 스트레스에 대한 강한 저항성을 보여주었는데, Roc10 과발현 형질전환 식물체는 가뭄 스트레스 처리 2일 후에도 가뭄 스트레스 무처리 대조구와 거의 동일한 상태를 나타냈으며, 가뭄 스트레스 3일 후에는 스트레스 유도 손상의 증상을 약하게 보였으나, 가뭄 스트레스 처리 비형질전환 식물체에 비해 증상의 정도가 약하게 확인되었으며, 더욱이 물 공급을 재개하자 가뭄 스트레스 유도 손상으로부터 회복되는 것이 관찰되었다(도 3). 상기의 결과를 통해 Roc10 유전자가 가뭄 스트레스에 대한 저항성을 증진시킬 수 있음을 알 수 있었다.As a result, after 2 days of drought stress treatment, the non-transformed plants were found to have weak symptoms of drought-induced damage such as leaf curling and wilting, and the symptoms became severe with increasing drought stress treatment time. Drought induction showed strong injury symptoms. Finally, the leaves of the non-transformed plants were all dried, and the plants did not recover from damage caused by drought stress induced by water replenishment. In addition, Roc10 knockdown transformed plants were confirmed to be similar to the results of non-transformed plants. Roc10 knockdown transformed plants also exhibited severe symptoms of drought stress induced damage as drought stress treatment time elapsed. There was no recovery from damage afterwards. On the other hand, Roc10 overexpressing transgenic plants showed strong resistance to drought stress. 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.
가뭄 저항성에 있어서 Roc10 유전자의 기능을 확인하기 위해서, Roc10 과발현 형질전환 식물체와 Roc10 녹다운 형질전환 식물체 독립적인 3개 라인을 사용하여 12일간의 가뭄처리를 통한 펄스 증폭 변조(PAM, Pulse-Amplitude Modulation) 시험과 JIP 분석을 수행하였다. In order to confirm the function of the Roc10 gene in drought resistance, pulse-amplitude modulation (PAM) was performed after 12 days of drought treatment using three independent lines of Roc10 overexpressing and Roc10 knockdown transgenic plants. Testing and JIP analysis were performed.
PAM 분석의 Fv/Fm 값과 JIP 분석의 PItotal 값은 식물에 있어서 비생물적 스트레스의 영향을 관찰하는데 사용되는데, 가뭄 스트레스 처리에 의해 비형질전환 식물체와 Roc10 녹다운 형질전환 식물체는 Fv/Fm 값의 강한 감소를 나타내었다. 비형질전환 식물체와 Roc10 녹다운 형질전환 식물체에서 스트레스 무처리 시 Fv/Fm 값은 0.8이었으나, 가뭄 스트레스 처리 6일 이후부터 Fv/Fm 값의 감소를 보이기 시작하였고, 스트레스 처리 시간의 증가와 함께 감소세가 가속화되는 결과를 보여주었다. 반면에, Roc10 과발현 형질전환 식물체는 가뭄 스트레스에 의한 Fv/Fm 값의 변화가 없음을 확인하였다(도 4). PItotal 값은 Roc10 과발현 형질전환 식물체의 경우 서서히 감소되고 비형질전환 식물체와 Roc10 녹다운 형질전환 식물체는 급격히 감소됨을 확인하였다. 또한, Roc10 녹다운 형질전환 식물체의 PItotal 값은 비형질전환 식물체보다 더욱 급격히 감소됨을 확인하였다(도 5). 이상의 결과는 Roc10 유전자의 과발현이 가뭄에 대한 식물체의 저항성을 증진시킴을 뒷받침하였다.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. On the other hand, Roc10 overexpressing transgenic plants were confirmed that there is no change in the F v / F m value due to drought stress (Fig. 4). The PI total value was gradually decreased in Roc10 overexpressing transgenic plants and rapidly decreased in non-transforming plants and Roc10 knockdown transgenic plants. In addition, it was confirmed that the PI total value of the Roc10 knockdown transformed plants was reduced more rapidly than the non-transformed plants (FIG. 5). These results supported that overexpression of the Roc10 gene enhanced plant resistance to drought.
실시예Example 3.  3. Roc10Roc10 유전자 과발현에 의한 가뭄 스트레스 조건하의  Under Drought Stress Conditions by Gene Overexpression 농업형질Agriculture 검증 Verification
전술한 실시예 2를 통해 Roc10 유전자의 과발현이 가뭄에 대한 식물체의 저항성을 증진시킴을 확인하였다. 이에 Roc10 과발현 식물체의 가뭄에 대한 야외 포장 조건에서 농업형질을 확인하기 위해, Roc10 과발현 형질전환 식물체의 농업형질을 조사하였다.In Example 2 described above, it was confirmed that overexpression of the Roc10 gene enhances plant resistance to drought. In order to identify the agricultural traits under outdoor field conditions for drought of Roc10 overexpressing plants, the agricultural traits of Roc10 overexpressing transgenic plants were examined.
그 결과, Roc10 과발현 형질전환 식물체의 포기당 이삭 수(no. of panicles per hill), 총 낟알 무게(total grain weight)와 충진율(filling rate) 등 생산량 결정에 중요한 항목들이 비형질전환 및 Roc10 녹다운 형질전환 식물체에 비해 현저히 증가된 것을 확인하였다(표 2).As a result, items important for production determination such as no. Of panicles per hill, total grain weight and filling rate of Roc10 overexpressing transgenic plants were identified as non-transgenic and Roc10 knockdown traits. It was confirmed that the increase significantly compared to the conversion plants (Table 2).
Figure PCTKR2017002482-appb-T000001
Figure PCTKR2017002482-appb-T000001

Claims (10)

  1. 벼(Oryza sativa) 유래 Roc10 (Rice outermost cell-specific gene 10) 단백질을 코딩하는 유전자를 포함하는 재조합 벡터로 식물세포를 형질전환시켜 Roc10 유전자의 발현을 조절하는 단계를 포함하는 식물체의 환경 스트레스 내성을 조절하는 방법.Rice (Oryza sativa) derived Roc10 (Rice outermost cell-specific gene 10) by transforming the plant cell with a recombinant vector comprising a gene encoding a protein the environmental stress tolerance of a plant comprising the step of regulating the expression of Roc10 gene How to adjust.
  2. 제1항에 있어서, 상기 Roc10 단백질은 서열번호 2의 아미노산 서열로 이루어진 것을 특징으로 하는 식물체의 환경 스트레스 내성을 조절하는 방법.According to claim 1, wherein the Roc10 protein is a method for regulating the environmental stress resistance of the plant, characterized in that consisting of the amino acid sequence of SEQ ID NO: 2.
  3. 제1항에 있어서, 상기 Roc10 유전자를 과발현시켜 식물체의 환경 스트레스 내성을 증가시키는 것을 특징으로 하는 식물체의 환경 스트레스 내성을 조절하는 방법.The method of claim 1, wherein the Roc10 gene is overexpressed to increase the environmental stress resistance of the plant.
  4. 제1항에 있어서, 상기 환경 스트레스는 가뭄, 고염 또는 저온인 것을 특징으로 하는 식물체의 환경 스트레스 내성을 조절하는 방법.The method of claim 1, wherein the environmental stress is drought, high salt, or low temperature.
  5. 벼(Oryza sativa) 유래 Roc10 (Rice outermost cell-specific gene 10) 단백질을 코딩하는 유전자를 포함하는 재조합 벡터로 식물세포를 형질전환하는 단계; 및Transforming 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 ); And
    상기 형질전환된 식물세포로부터 식물을 재분화하는 단계를 포함하는 환경 스트레스 내성이 조절된 형질전환 식물체의 제조방법.A method for producing a transformed plant with controlled environmental stress resistance comprising the step of regenerating the plant from the transformed plant cells.
  6. 제5항에 있어서, 상기 Roc10 단백질은 서열번호 2의 아미노산 서열로 이루어진 것을 특징으로 하는 환경 스트레스 내성이 조절된 형질전환 식물체의 제조방법.The method of claim 5, wherein the Roc10 protein is composed of the amino acid sequence of SEQ ID NO. 2.
  7. 제5항에 있어서, 상기 Roc10 단백질을 코딩하는 유전자를 과발현시켜 식물체의 환경 스트레스 내성을 증가시키는 것을 특징으로 하는 환경 스트레스 내성이 조절된 형질전환 식물체의 제조방법.The method of claim 5, wherein the environmental stress tolerance of the plant is increased by overexpressing the gene encoding the Roc10 protein.
  8. 제5항 내지 제7항 중 어느 한 항의 방법에 의해 제조된 환경 스트레스 내성이 조절된 형질전환 식물체.A transgenic plant with controlled environmental stress tolerance produced by the method of any one of claims 5 to 7.
  9. 제8항의 환경 스트레스 내성이 조절된 식물체의 형질전환된 종자.A transformed seed of a plant in which the environmental stress resistance of claim 8 is regulated.
  10. 벼(Oryza sativa) 유래 Roc10 (Rice outermost cell-specific gene 10) 단백질을 코딩하는 유전자를 유효성분으로 함유하는 식물체의 환경 스트레스 내성 조절용 조성물.A composition for regulating environmental stress resistance of plants containing a gene encoding a Roc10 (Rice outermost cell-specific gene 10) protein derived from rice ( Oryza sativa ) as an active ingredient.
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