KR102093591B1 - Novel gene related to plant cold stress tolerance and use thereof - Google Patents

Novel gene related to plant cold stress tolerance and use thereof Download PDF

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KR102093591B1
KR102093591B1 KR1020200009457A KR20200009457A KR102093591B1 KR 102093591 B1 KR102093591 B1 KR 102093591B1 KR 1020200009457 A KR1020200009457 A KR 1020200009457A KR 20200009457 A KR20200009457 A KR 20200009457A KR 102093591 B1 KR102093591 B1 KR 102093591B1
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김우택
민혜조
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연세대학교 산학협력단
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
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    • 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
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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
    • C12N15/67—General methods for enhancing the expression

Abstract

본 발명은 식물의 저온 스트레스 저항성을 증진시키는 신규 유전자 및 이의 용도 에 관한 것으로서, 보다 상세하게 본 발명은 OsCSR1(Oryza sativa Cold Stress Resistant 1) 단백질 또는 OsCSR2(Oryza sativa Cold Stress Resistant 2) 단백질을 코딩하는 유전자 서열을 포함하는, 식물체의 저온 스트레스에 대한 저항성 증진용 조성물, 상기 조성물로 형질전환된 형질전환 식물 세포, 형질전환 식물체 등에 관한 것이다. 본 발명의 식물체 저온 스트레스에 대한 저항성 증진용 조성물은 식물체의 저온 스트레스에 대한 저항성을 증진시키며, 본 발명의 형질전환 식물 세포 및 식물체는 저온 스트레스에 대한 내성이 탁월하여 중요 식량 작물인 벼의 재배 단계에서 발생할 수 있는 이상 저온 기후에 의한 작물 생산량 손실을 줄일 수 있는 신기능 작물로 유용하게 이용될 수 있다.The present invention relates to a novel gene and its use to enhance the low temperature stress resistance of plants, and in more detail the present invention encodes OsCSR1 (Oryza sativa Cold Stress Resistant 1) protein or OsCSR2 (Oryza sativa Cold Stress Resistant 2) protein It relates to a composition for enhancing resistance to low temperature stress of a plant, including a gene sequence, a transgenic plant cell transformed with the composition, a transgenic plant, and the like. The composition for enhancing resistance to low-temperature stress of a plant of the present invention enhances resistance to low-temperature stress of a plant, and the transformed plant cells and plants of the present invention have excellent resistance to low-temperature stress, thereby cultivating rice, an important food crop. It can be useful as a new function crop that can reduce the loss of crop production due to an abnormal low temperature climate.

Description

식물의 저온 스트레스 저항성을 증진시키는 신규 유전자 및 이의 용도{Novel gene related to plant cold stress tolerance and use thereof}BACKGROUND OF THE INVENTION 1. Field of the Invention Novel gene related to plant cold stress tolerance and use thereof

본 발명은 식물의 저온 스트레스 저항성을 증진시키는 신규 유전자 및 이의 용도에 관한 것으로서, 보다 상세하게 본 발명은 OsCSR1(Oryza sativa Cold Stress Resistant 1) 단백질 또는 OsCSR2(Oryza sativa Cold Stress Resistant 2) 단백질을 코딩하는 유전자 서열을 포함하는, 식물체의 저온 스트레스에 대한 저항성 증진용 조성물, 상기 조성물로 형질전환된 형질전환 식물 세포, 형질전환 식물체 등에 관한 것이다.The present invention relates to a novel gene for enhancing cold stress resistance of plants and their use, and in more detail, the present invention encodes OsCSR1 (Oryza sativa Cold Stress Resistant 1) protein or OsCSR2 (Oryza sativa Cold Stress Resistant 2) protein. It relates to a composition for enhancing resistance to low temperature stress of a plant, including a gene sequence, a transformed plant cell transformed with the composition, a transformed plant, and the like.

벼는 가장 중요한 작물 중에 하나이며, 단자엽 식물의 대표적인 모델 식물이다. 주로 아열대 지역이나 열대 지역에서 재배되는 벼는 저온 환경에 노출될 경우 발달이 저해되며, 이는 결과적으로 쌀 생산량의 감소를 초래하게 된다. 특히 지대가 높은 지역이나 아열대지역은 기온 변화의 폭이 크기 때문에 벼의 재배시기 동안 종종 20℃ 이하의 저온에 노출되어 손상을 받을 수 있다. 저온은 생장을 저해하고, 광합성 효율을 낮추며, 분얼의 발달 및 벼의 스파이크(spike) 발달을 방해하여 수확량(Grain yield)을 크게 감소시킨다(Suh J. et al., 2010). 실제로 한국에서는 생식 생장 기간 동안에 저온 기후가 발생하였던 1971년, 1980년, 그리고 1993년에 전체 벼 생산면적의 각각 17, 18, 20%가 손실을 입었으며, 1980년에는 최대 3.9 t/ha에 해당하는 손실을 기록한 바 있다(Zhang et al., 2013). Rice is one of the most important crops, and is a representative model plant of monocotyledons. Rice grown mainly in subtropical or tropical regions is hindered when exposed to a low-temperature environment, resulting in a decrease in rice production. In particular, high-land regions or subtropical regions may be damaged by exposure to low temperatures of 20°C or less during the cultivation period of rice because the range of temperature changes is large. Low temperature inhibits growth, lowers photosynthetic efficiency, and significantly reduces grain yield by interfering with the development of manure and rice spikes (Suh J. et al., 2010). In fact, in Korea, 17, 18, and 20% of the total rice production area was lost in 1971, 1980, and 1993, respectively, when a low-temperature climate occurred during the reproductive growth period, and in 1980 it was a maximum of 3.9 t/ha. It has recorded a loss (Zhang et al., 2013).

환경 스트레스에 대한 식물의 반응 및 적응 기작에 단백질 턴-오버 과정이 중요하게 작용하는 것으로 알려지고 있다. 단백질 턴-오버 과정 중에서도 유비퀴틴화-26S 프로테아좀(ubiquitination-26S proteasome) 시스템은 식물의 호르몬 생합성, 호르몬 시그널링, 형태발생(morphogenesis) 및 환경 스트레스 반응 기작에 관여하는 주요한 과정이다(Vierstra, R. D., 2009). It is known that the protein turn-over process plays an important role in the plant's response to environmental stress and the mechanism of adaptation. Among the protein turn-over processes, the ubiquitination-26S proteasome system is a major process involved in plant hormone biosynthesis, hormone signaling, morphogenesis, and environmental stress response mechanisms (Vierstra, RD, 2009).

특히 식물의 저온 스트레스 대응 과정에 작용하는 유전자들에 대한 연구가 많이 이루어졌으며, 저온 스트레스에 대처하기 위한 식물의 세포학적, 생리학적 반응들도 보고되고 있다(Thomashow MF., 2010). E3 유비키틴 리가아제(E3 ubiquitin ligase)가 식물의 저온 스트레스 반응 기작에 관여하는 주요한 단백질의 턴-오버를 매개한다고 밝혀지면서, 저온 스트레스에 대응하여 기능하는 E3 유비키틴 리가아제에 관한 연구의 중요성이 더욱 부각되고 있다. In particular, many studies have been conducted on genes that act in the process of responding to cold stress in plants, and cytological and physiological responses of plants to cope with cold stress have also been reported (Thomashow MF., 2010). As E3 ubiquitin ligase was found to mediate the turn-over of a major protein involved in the mechanism of cold stress response in plants, the importance of research on E3 ubiquitin ligase that functions in response to cold stress is important. It is becoming more prominent.

식물의 저온 스트레스를 해결하기 위한 기술이 개발되고 있으나, 아직까지 저온 스트레스에 대한 내성이나 민감성에 관여하는 유전자들의 생물학적 기능들에 대한 지식은 여전히 부족한 실정이다. 따라서 저온 스트레스 환경에서 작물의 생산성을 증가시키기 위해 저항성을 갖는 유전자에 대한 연구가 지속적으로 요구되고 있는 실정이다.Technologies for solving low temperature stress in plants are being developed, but knowledge of the biological functions of genes involved in resistance or sensitivity to low temperature stress is still insufficient. Therefore, in order to increase the productivity of crops in a low-temperature stress environment, research on genes having resistance is continuously required.

본 발명자들은 식물의 저온 스트레스에 대한 저항성을 증진시킬 수 있는 유전자를 발굴함으로써 궁극적으로 식물의 생산성을 향상시키는 방법을 개발하기 위해 예의 노력한 결과, 벼의 OsCSR1 또는 OsCSR2 유전자의 발현을 증가시킬 경우 저온 환경에서 저항성이 강화되는 것을 확인함으로써 본 발명을 완성하였다.The present inventors have made diligent efforts to develop a method that ultimately improves the productivity of plants by discovering genes that can increase the resistance to low temperature stress of plants. As a result of increasing the expression of OsCSR1 or OsCSR2 genes in rice, low temperature environment The present invention was completed by confirming that the resistance is strengthened.

본 발명의 목적은 식물체의 저온 스트레스 저항성 증진용 조성물을 제공하는 것이다.It is an object of the present invention to provide a composition for improving the resistance to low temperature stress of a plant.

본 발명의 또 다른 목적은 본 발명의 조성물로 형질전환된 저온 스트레스 저항성이 증진된 식물 세포 및 식물체를 제공하는 것이다.Another object of the present invention is to provide plant cells and plants transformed with the composition of the present invention with improved resistance to low temperature stress.

본 발명의 또 다른 목적은 본 발명의 조성물을 식물 세포에 도입시키는 단계를 포함하는 식물체의 저온 스트레스 저항성 증진 방법을 제공하는 것이다.Another object of the present invention is to provide a method for enhancing low temperature stress resistance of a plant comprising the step of introducing the composition of the present invention into plant cells.

본 발명의 일 양태에 따르면, 본 발명은 OsCSR1(Oryza sativa Cold Stress Resistant 1) 단백질 또는 OsCSR2(Oryza sativa Cold Stress Resistant 2) 단백질을 코딩하는 유전자 서열을 포함하는, 식물체의 저온 스트레스에 대한 저항성 증진용 조성물을 제공한다.According to one aspect of the present invention, the present invention comprises a gene sequence encoding OsCSR1 (Oryza sativa Cold Stress Resistant 1) protein or OsCSR2 (Oryza sativa Cold Stress Resistant 2) protein, for enhancing resistance to cold stress in plants The composition is provided.

본 발명자들은 식물의 저온 스트레스에 대한 저항성을 향상시킬 수 있는 유전자를 발굴함으로써 궁극적으로 식물의 생산성을 향상시키고자 예의 연구 노력하였다. 그 결과, 벼의 OsCSR1 또는 OsCSR2 유전자의 발현을 증가시킬 경우 상술한 저온 스트레스에 대한 강화된 내성을 수득할 수 있음을 확인하였다.The present inventors have made intensive research efforts to ultimately improve the productivity of plants by discovering genes that can improve the resistance of plants to low temperature stress. As a result, it was confirmed that when the expression of the OsCSR1 or OsCSR2 gene of rice is increased, enhanced resistance to the above-described low temperature stress can be obtained.

본 발명의 일 구현예에 따르면, 본 발명의 상기 OsCSR1 단백질은 서열번호 1로 표시되는 아미노산 서열을 포함할 수 있으며, 상기 OsCSR1 단백질을 코딩하는 유전자는 서열목록 2로 표시되는 뉴클레오티드 서열을 포함할 수 있다.According to an embodiment of the present invention, the OsCSR1 protein of the present invention may include an amino acid sequence represented by SEQ ID NO: 1, and the gene encoding the OsCSR1 protein may include a nucleotide sequence represented by SEQ ID NO: 2. have.

또한 본 발명의 상기 OsCSR2 단백질은 서열번호 3으로 표시되는 아미노산 서열을 포함할 수 있으며, 상기 OsCSR2단백질을 코딩하는 유전자는 서열목록 4로 표시되는 뉴클레오티드 서열을 포함할 수 있다.In addition, the OsCSR2 protein of the present invention may include an amino acid sequence represented by SEQ ID NO: 3, and the gene encoding the OsCSR2 protein may include a nucleotide sequence represented by SEQ ID NO: 4.

본 발명에 따르면, 식물체에 다양한 비생물학적 스트레스를 가해준 뒤 유전자 발현 프로파일을 조사한 결과, 건조, 고염 스트레스 및 ABA 호르몬을 가한 경우 본 발명의 OsCSR1 및 OsCSR2 유전자의 발현이 증가하나, 저온 스트레스를 가한 경우 상기 유전자의 발현 증가가 나타나지 않았다. 이러한 발현 패턴에도 불구하고, 하기 실시예에서 입증한 바와 같이 상기 유전자를 과발현시킨 경우 저온 스트레스 환경에서도 그 생존률이 크게 증가함을 확인하였다. According to the present invention, as a result of examining the gene expression profile after applying various abiotic stresses to plants, when dry, high salt stress and ABA hormone are added, the expression of the OsCSR1 and OsCSR2 genes of the present invention increases, but when low temperature stress is applied There was no increase in the expression of the gene. In spite of this expression pattern, it was confirmed that the survival rate of the gene was significantly increased even in a low temperature stress environment when the gene was overexpressed as demonstrated in the following examples.

후술하는 실시예에서 보는 바와 같이, 상기 유전자를 과발현시킨 식물체는 저온 스트레스가 가해지는 환경에서도 그 생존률이 크게 증가함을 확인하였다.As shown in the examples to be described later, it was confirmed that the survival rate of the plant overexpressing the gene significantly increased even in an environment where low temperature stress was applied.

본 발명에서 이용되는 뉴클레오티드 서열은 첨부한 서열목록에 기재된 뉴클레오티드 서열에 한정되지 않는다는 것은 당업자에게 명확하다.It is clear to those skilled in the art that the nucleotide sequence used in the present invention is not limited to the nucleotide sequence described in the attached sequence listing.

뉴클레오티드에서의 변이는 단백질에서 변화를 가져오지 않는 것도 있다. 이러한 핵산은 기능적으로 균등한 코돈 또는 동일한 아미노산을 코딩하는 코돈 (예를 들어, 코돈의 축퇴성에 의해, 아르기닌 또는 세린에 대한 코돈은 여섯 개이다), 또는 생물학적으로 균등한 아미노산을 코딩하는 코돈을 포함하는 핵산분자를 포함한다.Some nucleotide variations do not change the protein. Such nucleic acids include functionally equivalent codons or codons encoding the same amino acid (e.g., by the degeneracy of codons, there are six codons for arginine or serine), or codons encoding biologically equivalent amino acids. It includes a nucleic acid molecule.

본 명세서에서 용어 "핵산 분자(nucleic acids)"는 DNA(gDNA, cDNA 및 CDS) 그리고 RNA 분자를 포괄적으로 포함하는 의미를 가지며, 핵산 분자에서 기본 구성 단위인 뉴클레오티드는 자연의 뉴클레오티드 뿐만 아니라, 당 또는 염기 부위가 변형된 유사체(analogue)도 포함한다(Scheit, Nucleotide Analogs, John Wiley, New York(1980); Uhlman 및 Peyman, Chemical Reviews, 90:543-584(1990)).In the present specification, the term "nucleic acids" is meant to encompass DNA (gDNA, cDNA and CDS) and RNA molecules inclusively, and nucleotides, which are basic structural units in nucleic acid molecules, are not only natural nucleotides, but also sugars or Analogs with modified base sites are also included (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90:543-584 (1990)).

상술한 생물학적 균등 활성을 갖는 변이를 고려한다면, 비생물학적 스트레스-유도성 OsCSR1 단백질(서열목록 1) 또는 OsCSR2 단백질(서열목록 3)을 코딩하는 본 발명에서 이용되는 핵산 분자는 서열목록에 기재된 서열과 실질적인 동일성(substantial identity)을 나타내는 서열도 포함하는 것으로 해석된다. 상기의 실질적인 동일성은, 상기한 본 발명의 서열과 임의의 다른 서열을 최대한 대응되도록 얼라인(align)하고, 당업계에서 통상적으로 이용되는 알고리즘을 이용하여 얼라인된 서열을 분석한 경우에, 최소 60%의 상동성, 일 구현예에 따르면 70%의 상동성, 어떠한 구현예에 따르면 80%의 상동성, 특정 구현예에 따르면 90%의 상동성을 나타내는 서열을 의미한다. 서열비교를 위한 얼라인먼트(alignment) 방법은 당업계에 공지되어 있다. 얼라인먼트에 대한 다양한 방법 및 알고리즘은 Smith and Waterman, Adv. Appl. Math. 2:482(1981); Needleman and Wunsch, J. Mol. Bio. 48:443(1970); Pearson and Lipman, Methods in Mol. Biol. 24: 307-31(1988); Higgins and Sharp, Gene 73:237-44(1988); Higgins and Sharp, CABIOS 5:151-3(1989); Corpet et al., Nuc. Acids Res. 16:10881-90(1988); Huang et al., Comp. Appl. BioSci. 8:155-65(1992) and Pearson et al., Meth. Mol. Biol. 24:307-31(1994)에 개시되어 있다. NCBI Basic Local Alignment Search Tool(BLAST)(Altschul et al., J. Mol. Biol. 215:403-10(1990))은 NBCI(National Center for Biological Information) 등에서 접근 가능하며, 인터넷 상에서 blastp, blasm, blastx, tblastn and tblastx와 같은 서열 분석 프로그램과 연동되어 이용할 수 있다. BLSAT는 http://www.ncbi.nlm.nih.gov/BLAST/에서 접속 가능하다. 이 프로그램을 이용한 서열 상동성 비교 방법은 http://www.ncbi.nlm.nih.gov/BLAST/blast_help.html에서 확인할 수 있다.Considering the above-described mutation having biologically equivalent activity, the nucleic acid molecule used in the present invention encoding the abiological stress-inducing OsCSR1 protein (SEQ ID NO: 1) or OsCSR2 protein (SEQ ID NO: 3) is It is interpreted to include sequences exhibiting substantial identity. The actual identity of the above is the minimum when aligning the sequence of the present invention and any other sequence described above so as to correspond as much as possible, and analyzing the aligned sequence using an algorithm commonly used in the art. It refers to a sequence showing 60% homology, 70% homology according to an embodiment, 80% homology according to an embodiment, and 90% homology according to a specific embodiment. Alignment methods for sequence comparison are known in the art. Various methods and algorithms for alignment are described in Smith and Waterman, Adv. Appl. Math. 2:482 (1981); Needleman and Wunsch, J. Mol. Bio. 48:443 (1970); Pearson and Lipman, Methods in Mol. Biol. 24: 307-31 (1988); Higgins and Sharp, Gene 73:237-44 (1988); Higgins and Sharp, CABIOS 5:151-3 (1989); Corpet et al., Nuc. Acids Res. 16:10881-90 (1988); Huang et al., Comp. Appl. BioSci. 8:155-65 (1992) and Pearson et al., Meth. Mol. Biol. 24:307-31 (1994). NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403-10 (1990)) can be accessed from NBCI (National Center for Biological Information), etc. It can be used in conjunction with sequence analysis programs such as blastx, tblastn and tblastx. The BLSAT is accessible at http://www.ncbi.nlm.nih.gov/BLAST/. A method for comparing sequence homology using this program can be found at http://www.ncbi.nlm.nih.gov/BLAST/blast_help.html.

본 명세서의 용어 "저온 스트레스"는 식물체의 일반적인 생육 환경보다 낮은 온도에 의한 스트레스를 의미한다. 예컨대 벼의 경우 4℃ 이하에서 저온 스트레스를 받을 수 있으나, 품종에 따라 4℃ 보다 높은 온도에서도 스트레스를 줄 수 있다. 본 발명의 저온 스트레스는 4℃에서 3일 또는 4일 동안 배양하는 것으로 실시하지만, 이에 한정되는 것은 아니다.The term "low temperature stress" as used herein refers to a stress caused by a temperature lower than that of a general growth environment of a plant. For example, rice can be subjected to low-temperature stress at 4°C or less, but depending on the variety, stress can be applied at temperatures higher than 4°C. The low temperature stress of the present invention is carried out by culturing for 3 or 4 days at 4°C, but is not limited thereto.

본 발명의 다른 양태에 따르면, 본 발명은 상술한 본 발명의 뉴클레오티드 서열; 상기 뉴클레오티드 서열에 작동가능하게 결합(operatively linked)되어 있고 식물세포에서 RNA 분자를 형성시키는 프로모터; 및 식물세포에서 작용하여 RNA 분자의 3'-말단의 폴리아데닐화를 야기시키는 폴리 A 시그널 서열을 포함하는 식물발현용 재조합 벡터를 포함하는 식물체의 저온 스트레스 저항성 증진용 조성물을 제공한다.According to another aspect of the present invention, the present invention provides the above-described nucleotide sequence of the present invention; A promoter operatively linked to the nucleotide sequence and forming an RNA molecule in a plant cell; And it provides a composition for enhancing low temperature stress resistance of a plant comprising a recombinant vector for plant expression comprising a poly A signal sequence that acts on the plant cell to cause polyadenylation of the 3'-end of the RNA molecule.

본 명세서에서 용어 "작동가능한 결합"은 핵산 발현 조절 서열(예: 프로모터, 시그널 서열, 또는 전사조절인자 결합 위치의 어레이)과 다른 핵산 서열사이의 기능적인 결합을 의미하며, 이에 의해 상기 조절 서열은 상기 다른 핵산 서열의 전사 및/또는 트랜스레이션을 조절하게 된다.As used herein, the term "operable linkage" refers to a functional linkage between a nucleic acid expression control sequence (eg, a promoter, a signal sequence, or an array of transcriptional regulatory factor binding sites) and another nucleic acid sequence, whereby the control sequence is It controls the transcription and/or translation of the other nucleic acid sequence.

본 발명의 벡터 시스템은 당업계에 공지된 다양한 방법을 통해 구축될 수 있으며, 이에 대한 구체적인 방법은 Sambrook et al. Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press(2001)에 개시되어 있으며, 이 문헌은 본 명세서에 참조로서 삽입된다.The vector system of the present invention can be constructed through various methods known in the art, and specific methods for this are described by Sambrook et al. Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001), which is incorporated herein by reference.

본 발명의 벡터는 전형적으로 클로닝을 위한 벡터 또는 발현을 위한 벡터로서 구축될 수 있다. 또한, 본 발명의 벡터는 원핵 세포 또는 진핵 세포를 숙주로 하여 구축될 수 있다.The vector of the present invention can typically be constructed as a vector for cloning or as a vector for expression. In addition, the vector of the present invention can be constructed using a prokaryotic cell or a eukaryotic cell as a host.

본 발명의 벡터를 안정되면서 연속적으로 클로닝 및 발현시킬 수 있는 숙주 세포는 당업계에 공지되어 어떠한 숙주 세포도 이용할 수 있으며, 예컨대, E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, 바실러스 서브틸리스, 바실러스 츄린겐시스와 같은 바실러스 속 균주, 그리고 살모넬라 티피무리움, 세라티아 마르세슨스 및 다양한 슈도모나스 종과 같은 장내균과 균주 등이 있다.Host cells capable of stably and continuously cloning and expressing the vector of the present invention are known in the art, and any host cell can be used, for example, E. coli JM109, E. coli BL21, E. coli RR1, E. strains of the genus Bacillus such as coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus thuringiensis, and Salmonella typhimurium, Serratia marsessons, and various Pseudomonas species. Enterobacteriaceae and strains.

또한, 본 발명의 벡터를 진핵 세포에 형질전환시키는 경우에는 숙주 세포로서, 이스트(Saccharomyce cerevisiae), 곤충 세포, 사람 세포(예컨대, CHO 세포주(Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2, 3T3, RIN 및 MDCK 세포주) 및 식물세포 등이 이용될 수 있다.In addition, when transforming the vector of the present invention into eukaryotic cells, as host cells, yeast ( Saccharomyce cerevisiae ), insect cells, human cells (e.g., CHO cell line (Chinese hamster ovary), W138, BHK, COS-7, 293 , HepG2, 3T3, RIN and MDCK cell lines) and plant cells, and the like can be used.

예를 들어, 본 발명의 벡터가 발현 벡터이고, 원핵 세포를 숙주로 하는 경우에는, 전사를 진행시킬 수 있는 강력한 프로모터(예컨대, pLλ 프로모터, trp 프로모터, lac 프로모터, T7 프로모터, tac 프로모터 등), 해독의 개시를 위한 라이보좀 결합 자리 및 전사/해독 종결 서열을 포함하는 것이 일반적이다. 숙주 세포로서 E. coli가 이용되는 경우, E. coli 트립토판 생합성 경로의 프로모터 및 오퍼레이터 부위(Yanofsky, C., J. Bacteriol., 158:1018-1024(1984)) 그리고 파아지 λ의 좌향 프로모터(pLλ 프로모터, Herskowitz, I. and Hagen, D., Ann. Rev. Genet., 14:399-445(1980))가 조절 부위로서 이용될 수 있다.For example, when the vector of the present invention is an expression vector and a prokaryotic cell is used as a host, a strong promoter capable of promoting transcription (e.g., pL λ promoter, trp promoter, lac promoter, T7 promoter, tac promoter, etc.) , It is common to include a ribosome binding site for initiation of translation and a transcription/translation termination sequence. When E. coli is used as a host cell, the promoter and operator site of the E. coli tryptophan biosynthetic pathway (Yanofsky, C., J. Bacteriol., 158:1018-1024 (1984)) and the left-handed promoter of phage λ (pL) λ promoter, Herskowitz, I. and Hagen, D., Ann. Rev. Genet., 14:399-445 (1980)) can be used as a regulatory site.

숙주 세포 내로 주입된 벡터는 숙주 세포 내에서 발현되며, 이러한 경우에는 다량의 OsCSR1 단백질 또는 OsCSR2 단백질을 얻게 된다.The vector injected into the host cell is expressed in the host cell, in which case a large amount of OsCSR1 protein or OsCSR2 protein is obtained.

한편, 본 발명에 이용될 수 있는 벡터는 당업계에서 종종 사용되는 플라스미드(예: pSK349, pSC101, ColE1, pBR322, pUC8/9, pHC79, pGEX 시리즈, pET 시리즈 및 pUC19 등), 파지(예: λgtㆍλ4B, λ-Charon, λΔz1 및 M13 등) 또는 바이러스(예: SV40 등)를 조작하여 제작될 수 있다.On the other hand, vectors that can be used in the present invention include plasmids often used in the art (e.g., pSK349, pSC101, ColE1, pBR322, pUC8/9, pHC79, pGEX series, pET series and pUC19, etc.), phage (e.g., λgt ㆍCan be produced by manipulating λ4B, λ-Charon, λΔz1 and M13, etc.) or viruses (eg SV40, etc.).

본 발명의 벡터는 그로부터 발현되는 OsCSR1 단백질 또는 OsCSR2 단백질의 정제를 용이하게 하기 위하여, 다른 서열과 융합될 수도 있다. 융합되는 서열은 예컨대, 글루타티온 S-트랜스퍼라제(Pharmacia, USA), 말토스 결합 단백질(NEB, USA), FLAG(IBI, USA) 및 6x His(hexahistidine; Quiagen, USA) 등이 있다. 상기 정제를 위한 추가적인 서열 때문에, 숙주에서 발현된 단백질은 친화성 크로마토그래피를 통하여 신속하고, 용이하게 정제된다.The vector of the present invention may be fused with other sequences to facilitate purification of the OsCSR1 protein or OsCSR2 protein expressed therefrom. Sequences to be fused include, for example, glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA) and 6x His (hexahistidine; Quiagen, USA). Because of the additional sequence for this purification, the protein expressed in the host is rapidly and easily purified through affinity chromatography.

본 발명의 유전자는 식물(예컨대, 벼)에서 분리되었고, 저온 스트레스에 대한 식물체의 내성을 증진하는 작용을 할 수 있으므로, 식물에 대하여 가장 바람직한 유용성을 갖는다. 따라서, 본 발명의 벡터가 식물 세포에 적용되는 경우, 본 발명에 적합한 프로모터는, 식물체의 유전자 도입을 위해 당업계에서 통상적으로 이용되는 어떠한 것도 이용될 수 있으며, 예를 들어, SP6 프로모터, T7 프로모터, T3 프로모터, PM 프로모터, 옥수수의 유비퀴틴 프로모터, 컬리플라워 모자이크 바이러스(CaMV) 35S 프로모터, 노팔린 씬타아제(nos) 프로모터, 피그워트 모자이크 바이러스 35S 프로모터, 수가크레인 바실리폼 바이러스 프로모터, 콤멜리나 엘로우 모틀 바이러스 프로모터, 리불로오스-1,5-비스-포스페이트 카르복실라아제 스몰 서브유티트(ssRUBISCO)의 광유도성 프로모터, 벼 사이토졸 트리오스포스페이트 이소머라아제(TPI) 프로모터, 아라비돕시스의 아데닌 포스포리보실트랜스퍼라아제(APRT) 프로모터 및 옥토파인 신타아제 프로모터를 포함한다. 본 발명의 특정 구현예에 따르면, 본 발명에서 이용되는 프로모터는 옥수수의 유비퀴틴 프로모터(pUbi)이다.The gene of the present invention has been isolated from plants (eg, rice) and can function to enhance the resistance of plants to low temperature stress, and thus has the most desirable utility for plants. Therefore, when the vector of the present invention is applied to a plant cell, any promoter suitable for the present invention may be used any commonly used in the art for gene introduction into a plant, for example, the SP6 promoter, the T7 promoter. , T3 promoter, PM promoter, corn ubiquitin promoter, cauliflower mosaic virus (CaMV) 35S promoter, nopaline sintase (nos) promoter, pigwort mosaic virus 35S promoter, sucrine basiliform virus promoter, commelina yellow mottle virus Promoter, photoinducible promoter of ribulose-1,5-bis-phosphate carboxylase small subunit (ssRUBISCO), rice cytosol triose phosphate isomerase (TPI) promoter, adenine phosphoribosyltransfer of Arabidopsis Raase (APRT) promoter and octopine synthase promoter. According to a specific embodiment of the present invention, the promoter used in the present invention is a corn ubiquitin promoter ( pUbi ).

본 발명의 일 구현예에 따르면, 본 발명에 적합한 3'-말단의 폴리아데닐화를 야기시키는 폴리 A 시그널 서열은 아그로박테리움 투메파시엔스의 노팔린 신타아제 유전자로부터 유래된 것(NOS 3' end) (Bevan et al. Nucleic Acids Research, 11(2):369-385(1983)), 아그로박테리움 튜머페이션스의 옥토파인 신타아제 유전자로부터 유래된 것, 토마토 또는 감자의 프로테아제 억제자 I 또는 Ⅱ 유전자의 3' 말단 부분, CaMV 35S 터미네이터 및 OCS 터미네이터(octopine synthase terminator) 서열을 포함하나, 이에 한정되지 않는다. 본 발명의 특정 구현예에 따르면, 본 발명에 적합한 폴리아데닐화를 야기시키는 3'-말단 폴리 A 시그널 서열은 노팔린합성효소(nopaline synthase) 유전자의 종결서열(Tnos)이다.According to one embodiment of the present invention, the poly A signal sequence causing polyadenylation at the 3'-end suitable for the present invention is derived from the nopaline synthase gene of Agrobacterium tumefaciens (NOS 3'end ) (Bevan et al. Nucleic Acids Research, 11(2):369-385(1983)), derived from the octopine synthase gene of Agrobacterium tumerfaces, protease inhibitor I or II gene of tomato or potato The 3'end portion of the, CaMV 35S terminator and OCS terminator (octopine synthase terminator) sequences, including, but not limited to. According to a specific embodiment of the present invention, the 3'-terminal poly A signal sequence causing polyadenylation suitable for the present invention is a nopaline synthase gene termination sequence (Tnos).

선택적으로, 상기 벡터는 리포터 분자(예: 루시퍼라아제 및 β-글루쿠로니다아제)를 코딩하는 유전자를 추가적으로 운반할 수 있다. 또한, 본 발명의 벡터는 선택 표지로서 항생제(예: 네오마이신, 카베니실린, 카나마이신, 스펙티노마이신, 하이그로마이신 등) 내성 유전자(예: 네오마이신 포스포트랜스퍼라아제(nptⅡ), 하이그로마이신 포스포트랜스퍼라아제(hpt) 등)를 포함할 수 있다.Optionally, the vector may additionally carry a gene encoding a reporter molecule (eg, luciferase and β-glucuronidase). In addition, the vector of the present invention is an antibiotic (eg, neomycin, carbenicillin, kanamycin, spectinomycin, hygromycin, etc.) resistance genes (eg, neomycin phosphotransferase (nptII), hygromycin, etc.) Mycin phosphotransferase (hpt), etc.).

본 발명의 일 구현예에 따르면, 본 발명의 식물발현용 재조합벡터는 아그로박테리움(Agrobacterium) 바이너리 벡터이다.According to one embodiment of the present invention, the recombinant vector for plant expression of the present invention is an Agrobacterium binary vector.

본 명세서에서 용어 "바이너리 벡터(binary vector)"는 Ti(tumor inducible) 플라스미드에서 이동에 필요한 부분인 LB(left border)와 RB(right border)를 가지는 플라스미드와 타겟 뉴클레오티드를 옮기는데 필요한 유전자를 가진 플라스미드를 두 개로 나누어 놓은 벡터를 말한다. 본 발명의 형질전환용 아그로박테리움은 본 발명의 상기 뉴클레오타이드 서열의 발현에 적합한 것이면 어느 것이라도 좋고, 본 발명의 특정 구현예에 따르면, 본 발명에서 식물 형질전환용 아그로박테리움 균주는 아그로박테리움 투메파시엔스(Agrobacterium tumefaciens) LBA4404이다.In the present specification, the term "binary vector" refers to a plasmid having a left border (LB) and a right border (RB), which are parts necessary for migration in a Ti (tumor inducible) plasmid, and a plasmid having a gene required to transfer a target nucleotide. It is a vector divided into two. Agrobacterium for transformation of the present invention may be any one suitable for expression of the nucleotide sequence of the present invention, and according to a specific embodiment of the present invention, the Agrobacterium strain for plant transformation in the present invention is Agrobacterium Tumefaciens (Agrobacterium tumefaciens) LBA4404.

본 발명의 재조합 벡터를 아그로박테리움에 도입하는 방법은 당업자에게 공지된 다양한 방법을 통해 실시될 수 있으며, 예를 들면 입자 충격법(particle bombardment), 전기천공법(electroporation), 형질감염법(transfection), 리튬아세테이트법(lithium acetate method), 열충격법(heat shock) 및 냉동-해빙법(freezethaw method) 등이 있으나, 이에 제한되지 않는다. 본 발명의 특정 구현예에 따르면, 전기천공법(electroporation)을 사용한다.The method of introducing the recombinant vector of the present invention into Agrobacterium can be carried out through various methods known to those skilled in the art, for example, particle bombardment, electroporation, and transfection. ), lithium acetate method, heat shock, and freezethaw method, but are not limited thereto. According to a specific embodiment of the invention, electroporation is used.

본 발명의 또 다른 양태에 따르면, 본 발명은 본 발명의 조성물로 형질전환된 저온 스트레스 저항성이 증진된 식물세포를 제공한다.According to another aspect of the present invention, the present invention provides a plant cell transformed with the composition of the present invention with improved resistance to low temperature stress.

본 발명의 또 다른 양태에 따르면, 본 발명은 본 발명의 조성물로 형질전환된 저온 스트레스 저항성이 증진된 식물체를 제공한다.According to another aspect of the present invention, the present invention provides a plant transformed with the composition of the present invention with improved resistance to low temperature stress.

본 명세서에서, 용어 "식물체(또는 식물)"는 성숙한 식물뿐만 아니라 성숙한 식물로 발육할 수 있는 식물 세포, 식물 조직, 식물 세포 또는 조직으로부터 유래된 캘러스 및 식물의 종자 등을 모두 포함하는 의미로서 이해된다.In the present specification, the term "plant (or plant)" is understood as including both a mature plant as well as a plant cell, plant tissue, callus derived from a plant cell or tissue, and seeds of a plant that can develop into a mature plant. do.

본 발명에서 형질전환의 대상이 되는 식물체는 벼, 밀, 보리, 옥수수, 콩, 감자, 밀, 팥, 귀리 및 수수를 포함하는 식량 작물류; 애기장대, 배추, 무, 고추, 딸기, 토마토, 수박, 오이, 양배추, 참외, 호박, 파, 양파 및 당근을 포함하는 채소 작물류; 인삼, 담배, 목화, 참깨, 사탕수수, 사탕무우, 들깨, 땅콩 및 유채를 포함하는 특용 작물류; 사과나무, 배나무, 대추나무, 복숭아, 양다래, 포도, 감귤, 감, 자두, 살구 및 바나나를 포함하는 과수류; 장미, 글라디올러스, 거베라, 카네이션, 국화, 백합 및 튤립을 포함하는 화훼류; 및 라이그라스, 레드클로버, 오차드그라스, 알파알파, 톨페스큐 및 페레니얼라이그라스를 포함하는 사료작물류를 포함하나, 이에 제한되는 것은 아니다. 본 발명의 일 구현예에 따르면, 본 발명에서 형질전환의 대상이 되는 식물체는 채소 작물류 또는 특용 작물류이다. 본 발명의 어떠한 구현예에 따르면, 본 발명에서 형질전환의 대상이 되는 식물체는 벼(Oryza sativa L.)이다.Plants subject to transformation in the present invention include rice, wheat, barley, corn, beans, potatoes, wheat, red beans, food crops including oats and sorghum; Vegetable crops including Arabidopsis, Chinese cabbage, radish, pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, pumpkin, green onion, onion and carrot; Specialty crops including ginseng, tobacco, cotton, sesame, sugar cane, sugar beet, perilla, peanut and rapeseed; Fruit trees including apple tree, pear tree, jujube tree, peach, parsley, grapes, tangerines, persimmons, plums, apricots and bananas; Flowers including roses, gladiolus, gerberas, carnations, chrysanthemums, lilies and tulips; And feed crops including ryegrass, red clover, orchardgrass, alpha alpha, tolpescue, and perennial ryegrass, but are not limited thereto. According to one embodiment of the present invention, the plant to be transformed in the present invention is vegetable crops or special crops. According to certain embodiments of the present invention, the plant to be transformed in the present invention is rice ( Oryza sativa L.).

특정 뉴클레오티드 또는 이를 포함하는 식물발현용 재조합 벡터를 식물세포에 도입하는 방법은 당업계에 공지된 다양한 방법으로 실시될 수 있다.A method of introducing a specific nucleotide or a recombinant vector for plant expression containing the same into plant cells may be carried out by various methods known in the art.

형질전환된 식물세포의 선별은 형질전환 배양물을 선택제(예: 대사 억제제, 항생제 및 제초제)에 노출시켜 실시될 수 있다. 형질전환되고 선택제 내성을 부여하는 표지 유전자를 안정되게 포함하고 있는 식물세포는 상기한 배양물에서 성장하고 분할한다. 예시적인 표지는, 하이그로마이신 포스포트랜스퍼라아제 유전자, 글리코포스페이트 내성 유전자 및 네오마이신 포스포트랜스퍼라아제(nptII) 시스템을 포함하나, 이에 한정되는 것은 아니다.Selection of transformed plant cells may be carried out by exposing the transformed culture to a selection agent (eg, metabolic inhibitors, antibiotics, and herbicides). Plant cells that have been transformed and stably contain a marker gene that confers selection agent resistance are grown and divided in the above-described culture. Exemplary labels include, but are not limited to, the hygromycin phosphotransferase gene, the glycophosphate resistance gene, and the neomycin phosphotransferase (nptII) system.

본 발명의 형질전환 식물세포 및 형질전환 식물체를 제조하기 위하여 당업 계에 일반적으로 공지된 방법(Methods of Enzymology, Vol. 153, (1987))에 따라 실시될 수 있다. 외래성 폴리뉴클레오티드를 플라스미드나 바이러스 등과 같은 벡터 등의 운반체에 삽입하여 식물을 형질전환시킬 수 있고, 아그로박테리움 박테리아를 매개체로 사용할 수 있으며(Chilton et ai. Cell 11:263:271(1977), 미국 등록특허 제 5,004,863호, 제5,349,124호 및 제5,416,011호), 직접 외래성 폴리뉴클레오티드를 식물 세포 내로 도입시켜 식물을 형질전환시킬 수 있다(Lorz et ai. Mol. Genet. 199:178-182;(1985)). 예를 들어, T-DNA 부위를 포함하지 않는 벡터를 이용하는 경우에는 전기천공법(electroporation, Neumann, E., et al., EMBO J., 1:841(1982)), 입자충격법(microparticle bombardment, Yang, et al., Proc. Natl. Acad. Sci., 87:9568-9572(1990)), 폴리에틸렌 글리콜 침전법(polyethylene glycol-mediated uptake)을 이용할 수 있다. 일반적으로 식물을 형질전환시킴에 있어 많이 사용되는 것이 외래성 폴리뉴클레오티드로 형질전환 된 아그로박테리움 투메페이시언스(Agrobacterium tumefaciens)로 식물 세포나 종자 등을 감염시키는 방법이다(참조: 미합중국 특허 제 5,004,863, 5,349,124 및 5,416,011 호).In order to prepare the transformed plant cell and the transformed plant of the present invention, it may be carried out according to a method generally known in the art (Methods of Enzymology, Vol. 153, (1987)). Plants can be transformed by inserting foreign polynucleotides into carriers such as plasmids or viruses, etc., and Agrobacterium bacteria can be used as mediators (Chilton et ai. Cell 11:263:271 (1977), USA Patent Nos. 5,004,863, 5,349,124, and 5,416,011), it is possible to transform plants by directly introducing exogenous polynucleotides into plant cells (Lorz et ai. Mol. Genet. 199:178-182; (1985)) ). For example, in the case of using a vector that does not contain a T-DNA site, electroporation (electroporation, Neumann, E., et al., EMBO J., 1:841 (1982)), microparticle bombardment method (microparticle bombardment) is used. , Yang, et al., Proc. Natl. Acad. Sci., 87:9568-9572 (1990)), polyethylene glycol-mediated uptake can be used. In general, it is a method of infecting plant cells or seeds with Agrobacterium tumefaciens transformed with foreign polynucleotides that are widely used in transforming plants (see: U.S. Patent No. 5,004,863, 5,349,124 and 5,416,011).

당업자는 공지된 적절한 조건하에서 형질전환된 식물 세포나 종자를 배양 또는 재배하여 식물로 발육시킬 수 있다. 식물 원형질 또는 다양한 익스플랜트(explant)로부터 식물체의 발달 또는 재분화시키는 방법은 당업계에 잘 알려져 있다. 아그로박테리움에 의해 도입된 외래 유전자를 포함하는 식물체의 발달 또는 재분화는 당업계에 공지된 방법에 따라 달성될 수 있다(참조: 미합중국 특허 제 5,004,863, 5,349,124 및 5,416,011 호).Those skilled in the art can develop a plant by culturing or cultivating transformed plant cells or seeds under known appropriate conditions. Methods for the development or regeneration of plants from plant protoplasms or various explants are well known in the art. The development or regeneration of plants containing foreign genes introduced by Agrobacterium can be achieved according to methods known in the art (see U.S. Patent Nos. 5,004,863, 5,349,124 and 5,416,011).

본 발명의 일 구현예에 따르면, 본 발명의 형질전환은 아그로박테리움을 매개체로 이용한 아그로박테리움 시스템을 이용하여 실시되며, 다른 구현예에 따르면 아그로박테리움 튜메파시엔스(Agrobacterium tumefaciens)-바이너리 벡터 시스템을 이용하여 실시된다.According to one embodiment of the present invention, the transformation of the present invention is carried out using an Agrobacterium system using Agrobacterium as a medium, and according to another embodiment, Agrobacterium tumefaciens-binary vector It is implemented using the system.

아그로박테리움 시스템을 이용하는 방법에 있어서, 구체적인 일 실시예는 다음의 단계를 포함한다: In the method of using the Agrobacterium system, a specific embodiment includes the following steps:

(a) 식물세포의 게놈 DNA에 삽입될 수 있고 다음의 서열을 갖는 벡터가 내재되어 있는 아그로박테리움 튜머파시엔스로 식물체의 익스플랜트(예, 캘러스)를 감염시키는 단계: (a) Infecting an extract (eg, callus) of a plant with Agrobacterium tumerfaciens, which can be inserted into the genomic DNA of a plant cell and contains a vector having the following sequence:

(i) 본 발명의 OsCSR1 단백질 또는 또는 OsCSR2 단백질을 코딩하는 뉴클레오티드 서열; (ii) 상기 뉴클레오티드 서열에 작동적으로 결합되며 식물세포에서 작용하여 RNA 분자를 형성시키는 프로모터; (iii) 식물세포에서 작용하여 상기 RNA 분자의 3'-말단의 폴리아데닐화를 야기시키는 3'-비-해독화 부위;(i) the nucleotide sequence encoding the OsCSR1 protein or the OsCSR2 protein of the present invention; (ii) a promoter operably linked to the nucleotide sequence and acting in plant cells to form RNA molecules; (iii) a 3'-non-translational site that acts in plant cells to cause polyadenylation of the 3'-end of the RNA molecule;

(b) 상기 감염된 익스플랜트를 재분화 배지에서 재분화하여 형질전환 식물체를 얻는 단계.(b) obtaining a transgenic plant by re-differentiating the infected extract in a re-differentiation medium.

식물 세포의 형질전환은 Ti 플라스미드를 포함하는 아그로박테리움 튜메파시엔스를 가지고 실시된다(Depicker, A., et al., Plant cell transformation by Agrobacterium plasmids. In Genetic Engineering of Plants, Plenum Press, New York(1983)).Transformation of plant cells is carried out with Agrobacterium tumefaciens containing Ti plasmid (Depicker, A., et al., Plant cell transformation by Agrobacterium plasmids. In Genetic Engineering of Plants, Plenum Press, New York (Depicker, A., et al., Plant cell transformation by Agrobacterium plasmids. 1983)).

본 발명의 어떠한 구현예에 따르면, pBin19, pRD400, pRD320, pGA1611 및 pGA1991과 같은 바이너리 벡터 시스템이 이용된다(An, G., et al., Binary vectors" In Plant Gene Res. Manual, Martinus Nijhoff Publisher, New York(1986); An et al., 1988; 및 Lee et al., 1999). 본 발명에 적합한 바이너리 벡터는 (i) 식물에서 작동하는 프로모터; (ii) 상기 프로모터에 작동적으로 연결된 구조 유전자; 및 (iii) 폴리아데닐화 시그널 서열을 포함한다. 선택적으로, 상기 벡터는 리포터 분자(예: 루시퍼라아제 및 글루쿠로니다아제)를 코딩하는 유전자를 추가적으로 운반한다. 바이너리 벡터에 이용되는 프로모터의 예는 옥수수 유비퀴틴 프로모터, CaMV 35S 프로모터, 1 프로모터, 2 프로모터 및 노팔린 씬타아제(nos) 프로모터를 포함하나, 이에 한정되는 것은 아니다.According to certain embodiments of the present invention, binary vector systems such as pBin19, pRD400, pRD320, pGA1611 and pGA1991 are used (An, G., et al., Binary vectors" In Plant Gene Res. Manual, Martinus Nijhoff Publisher, New York (1986); An et al., 1988; and Lee et al., 1999).A binary vector suitable for the present invention includes (i) a promoter operating in plants; (ii) a structural gene operably linked to the promoter And (iii) a polyadenylation signal sequence Optionally, the vector further carries a gene encoding a reporter molecule (eg luciferase and glucuronidase) Promoter used in binary vectors Examples include, but are not limited to, the corn ubiquitin promoter, CaMV 35S promoter, 1 promoter, 2 promoter, and nopaline sintase (nos) promoter.

아그로박테리움 튜메파시엔스에 의한 캘러스(callus)의 감염은 당업계에 공지된 방법을 포함한다. 본 발명의 일구현예에 따르면, 상기 감염 과정은 아그로박테리움 튜메파시엔스의 배양물에 익스플랜트를 함침시켜 공동배양하는 과정을 포함한다. 이를 통해 아그로박테리움 튜메파시엔스는 식물내로 감염된다.Infection of callus by Agrobacterium tumefaciens includes methods known in the art. According to an embodiment of the present invention, the infection process includes a process of co-culture by impregnating an extract in a culture of Agrobacterium tumefaciens. Through this, Agrobacterium tumefaciens infects the plant.

아그로박테리움 튜메파시엔스에 의해 형질전환된 익스플랜트는 재분화 배지에서 재분화되며, 이는 최종적으로 형질전환 식물체를 형성한다.Explants transformed by Agrobacterium tumefaciens are re-differentiated in re-differentiation medium, which finally form transgenic plants.

본 발명에 따라 형질전환된 식물체는 당업계에 공지된 방법에 의해 형질전환 여부가 확인된다. 예를 들어, 형질전환된 식물체의 조직으로부터 얻은 DNA 시료를 이용하여, PCR을 실시하면 형질전환 식물체의 게놈에 삽입된 외래 유전자가 규명될 수 있다. 택일적으로, 노던 또는 서던 블롯팅을 실시하여 형질전환 여부를 확인할 수 있다(Maniatis, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.(1989)).Plants transformed according to the present invention are confirmed to be transformed by methods known in the art. For example, when PCR is performed using a DNA sample obtained from a tissue of a transformed plant, a foreign gene inserted into the genome of a transformed plant can be identified. Alternatively, Northern or Southern blotting can be performed to confirm whether or not transformation (Maniatis, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989)).

본 발명의 형질전환 식물세포 및 식물체는 상술한 본 발명의 벡터를 이용하여 제조되기 때문에, 이 둘 사이에 공통된 내용은 본 명세서의 과도한 복잡성을 피하기 위하여 그 기재를 생략한다.Since the transformed plant cells and plants of the present invention are prepared using the vector of the present invention described above, descriptions of the contents in common between the two are omitted in order to avoid undue complexity in the present specification.

본 발명의 또 다른 양태에 따르면, 본 발명은 본 발명의 조성물을 식물 세포에 도입시키는 단계를 포함하는 식물체의 저온 스트레스 저항성 증진방법을 제공한다.According to another aspect of the present invention, the present invention provides a method for enhancing low temperature stress resistance of a plant comprising the step of introducing the composition of the present invention into plant cells.

본 발명의 방법은 상술한 조성물과 관련 아미노산 서열, 뉴클레오티드 서열 및 이들을 포함하는 식물발현용 재조합 벡터를 공통으로 하기 때문에, 상기 조성물과의 관계에서 공통된 내용은 본 명세서의 과도한 복잡성을 피하기 위하여 그 기재를 생략한다.Since the method of the present invention shares the above-described composition with the related amino acid sequence, nucleotide sequence, and a recombinant vector for plant expression comprising them, the description in common with the composition is described in order to avoid excessive complexity of the present specification. Omit it.

본 발명의 식물체 냉해 스트레스에 대한 저항성 증진용 조성물은 식물체의 저온 스트레스에 대한 저항성을 증진시키며, 본 발명의 형질전환 식물 세포 및 식물체는 냉해 스트레스에 대한 내성이 탁월하여 중요 식량 작물인 벼의 재배 단계에서 냉해 환경 스트레스에 의해 발생하는 작물 생산량 손실을 줄일 수 있는 신기능 작물로 유용하게 이용될 수 있다.The composition of the present invention for improving resistance to cold stress in plants improves resistance to low temperature stress in plants, and the transgenic plant cells and plants of the present invention have excellent resistance to cold damage stress, so that the cultivation step of rice, an important food crop It can be usefully used as a new functional crop that can reduce the loss of crop production caused by environmental stress in the cold sea.

도 1은 식물의 비생물학적 스트레스인 건조, 고염, ABA, 및 저온 스트레스에 대한 OsCSR1 및 OsCSR2 유전자 발현의 변화를 RT-PCR로 확인한 결과이다.
도 2는 본 발명의 일실시예에 따른 OsCSR1 과다발현 형질전환 식물체와 OsCSR2 과다발현 형질전환 식물체에서 각각의 유전자의 과다발현 여부를 RT-PCR로 확인한 결과이다.
도 3은 본 발명의 일실시예에 따른 OsCSR1 과다발현 형질전환 식물체와 OsCSR2 과다발현 형질전환 식물체에서 OsCSR1 및 OsCSR2 단백질 발현 수준 변화를 SDS-PAGE로 확인한 결과이다.
도 4는 본 발명의 일실시예에 따른 OsCSR1 과다발현 형질전환 식물체(#1, #2)의 저온 스트레스 저항성 증가를 확인한 결과이다.
도 5는 본 발명의 일실시예에 따른 OsCSR2 과다발현 형질전환 식물체(#13)의 저온 스트레스 저항성 증가를 확인한 결과이다.1 is a result of confirming changes in the expression of OsCSR1 and OsCSR2 genes against dry, high salt, ABA, and low temperature stress, which are abiotic stresses of plants, by RT-PCR.
2 is a result of confirming whether each gene is overexpressed in the transgenic plant overexpressing OsCSR1 and the transgenic plant overexpressing OsCSR2 according to an embodiment of the present invention by RT-PCR.
3 is a result of confirming changes in the expression levels of OsCSR1 and OsCSR2 proteins in the OsCSR1 overexpressing transgenic plant and the OsCSR2 overexpressing transgenic plant according to an embodiment of the present invention by SDS-PAGE.
4 is a result of confirming the increase in resistance to low temperature stress of the transgenic plants (#1, #2) overexpressing OsCSR1 according to an embodiment of the present invention.
5 is a result of confirming the increase in resistance to low temperature stress of the transgenic plant (#13) overexpressing OsCSR2 according to an embodiment of the present invention.

이하, 실시예를 통하여 본 발명을 더욱 상세하게 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Below, The present invention will be described in more detail through examples. These examples are for illustrative purposes only, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not construed as being limited by these examples.

실시예 1. 비생물학적 스트레스에 대한 Example 1. Against abiotic stress OsCSR1OsCSR1 및 And OsCSR2OsCSR2 유전자 발현 분석Gene expression analysis

식물의 비생물학적 스트레스인 건조, 고염, 저온 스트레스와 식물의 스트레스 호르몬인 ABA(앱시스산, abscisic acid)에 의해 OsCSR1와 OsCSR2의 유전자 발현이 어떻게 변화하는지 RT-PCR을 통해 확인하였다. RT-PCR confirmed how the gene expression of OsCSR1 and OsCSR2 was changed by the abiotic stress of plants, such as dry, high salt, low temperature stress, and ABA (abscisic acid), the stress hormone of plants.

구체적으로, 공기 중에 노출시켜 물을 공급하지 않는 상태에서 0.5 ~ 2시간 동안 유지시키는 건조 조건, 200 mM NaCl 하에 1.5 ~ 3시간 동안 노출시키는 고염 조건, 100 μM 앱시스산에서 1.5 ~ 3시간 동안 노출시키는 ABA 조건, 및 4℃에서 6 ~ 12시간 동안 노출시키는 저온 조건으로 비생물학적 스트레스를 준 7일령 벼 유모에서 총 RNA를 추출하였다. RT-PCR 분석 시 사용한 프라이머는 하기 표 1에 나타내었다.Specifically, drying conditions that are exposed to air and maintained for 0.5 to 2 hours without supplying water, high salt conditions exposed to 200 mM NaCl for 1.5 to 3 hours, and exposed to 100 μM abcisic acid for 1.5 to 3 hours. Total RNA was extracted from 7-day-old rice hairs subjected to abiological stress under ABA conditions and low temperature conditions exposed at 4° C. for 6 to 12 hours. Primers used in the RT-PCR analysis are shown in Table 1 below.

건조, 고염 스트레스 및 ABA 호르몬에 대한 양성 대조군으로 OsRab16b 유전자를 이용하였고, 저온 스트레스에 대한 양성 대조군으로 OsDREB1A를 이용하였으며, OsUBQ10은 로딩(loading) 대조군로 사용하였다. OsRab16b gene was used as a positive control for dry, high salt stress and ABA hormone, OsDREB1A was used as a positive control for low temperature stress, and OsUBQ10 was used as a loading control.

서열번호Sequence number 프라이머primer 서열(5'→3')Sequence (5'→3') 55 OsUBQ10 RT FW OsUBQ10 RT FW 5’-ATGCAGATCTTTGTGAAGAC-3’5’-ATGCAGATCTTTGTGAAGAC-3’ 66 OsUBQ10 RT RV OsUBQ10 RT RV 5’-TTACTGACCACCACGGAGGC-3’5’-TTACTGACCACCACGGAGGC-3’ 77 OsCSR1 RT FW OsCSR1 RT FW 5’-TAT AGTACACGCGCGAGCTTTGT-3’5’-TAT AGTACACGCGCGAGCTTTGT-3’ 88 OsCSR1 RT RV OsCSR1 RT RV 5’-GCAGAGTTGCAAACCACTTTAGT-3’5’-GCAGAGTTGCAAACCACTTTAGT-3’ 99 OsCSR2 RT FW OsCSR2 RT FW 5’-TCGCACATACTCTGTTCCTCTTT-3’5’-TCGCACATACTCTGTTCCTCTTT-3’ 1010 OsCSR2 RT RV OsCSR2 RT RV 5’-CAAACAGCGTCACAAACAGCAA-3’5’-CAAACAGCGTCACAAACAGCAA-3’ 1111 OsRab16b RT FW OsRab16b RT FW 5’-ACAAGGGCAACAACCACCAG-3’5’-ACAAGGGCAACAACCACCAG-3’ 1212 OsRab16b RT RV OsRab16b RT RV 5’-GCTTGCAATGGCATCACAAG-3’5’-GCTTGCAATGGCATCACAAG-3’ 1313 OsDREB1A RT SE OsDREB1A RT SE 5’-AGGCCGTCGAGGACTTCTT-3’5’-AGGCCGTCGAGGACTTCTT-3’ 1414 OsDREB1A RT AS OsDREB1A RT AS 5’-TAGTAGCTCCAGAGTGGAGT-3’5’-TAGTAGCTCCAGAGTGGAGT-3’

그 결과, 도 1에 나타낸 바와 건조, 고염 스트레스 및 ABA 호르몬 하에서 OsCSR1와 OsCSR2 유전자의 발현량이 증가하는 반면, 저온 스트레스를 가한 경우 발현량이 감소함을 확인하였다. As a result, as shown in FIG. 1, it was confirmed that the expression levels of OsCSR1 and OsCSR2 genes increased under dry, high salt stress, and ABA hormone, while the expression levels decreased when low temperature stress was applied.

실시예 2. OsCSR1 및 OsCSR2 과다발현 형질전환 벼 식물체 제작 Example 2. Production of transgenic rice plants overexpressing OsCSR1 and OsCSR2

OsCSR1 또는 OsCSR2을 과다발현하기 위해 각 유전자의 cDNA를 pENTR SD-TOPO 벡터(Invitrogen)에 삽입한 후, LR 크로나아제 효소(Invitrogen)를 이용하여 pGA2897 운반 벡터에 치환하였다. 이를 벼 식물체에 발현시키기 위해 각각의 플라스미드(pUbi:2xFlag-OsCSR1 및 pUbi:2xFlag-OsCSR2)를 아그로박테리움 튜메파시엔스 LBA4404에 전기천공법으로 넣어 형질 전환하였다. In order to overexpress OsCSR1 or OsCSR2, the cDNA of each gene was inserted into the pENTR SD-TOPO vector (Invitrogen), and then replaced with the pGA2897 transport vector using LR chronase enzyme (Invitrogen). In order to express this in rice plants, each plasmid (pUbi:2xFlag-OsCSR1 and pUbi:2xFlag-OsCSR2) was transformed into Agrobacterium tumefaciens LBA4404 by electroporation.

야생종인 동진벼의 종자를 40% 락스(유한-크로락스)에 30분간 살균 처리한 후 멸균한 물로 5회 이상 세척한 다음, 1일 동안 저온 처리하였다. 처리된 종자를 3% 수크로스, CHU 배지(4 g/L, Duchedfa Biochem), 카사미노산(casamino acid, 300 mg/L), L-프롤린(L-proline, 2.9 g/L), 2,4D(2 mg/L), 0.002% 겔라이트(gelite)로 구성된 N6D 배지에 심고 28℃에서 7일 이상 배양하여 배아로부터 캘러스(callus, 세포 덩어리)를 유도하였다. 이 캘러스의 형질전환은 상기에서 형질 전환된 아그로박테리움 튜메파시엔스 LBA4404와 4일간 배양하여 수세하고, 하이그로마이신 B(hygromycin B) 항생제를 이용하여 새롭게 자라나는 캘러스를 선별하였다. 선별된 캘러스는 옥신과 싸이토키니닌이 포함된 재분화 배지에 배양하여(Sohn et al., Korean J. Breed. 38(3): 173~179 (2006)) 식물체를 획득하였다. 제작한 형질 전환 식물체는 흙으로 옮겨 온실에서 키웠다. The seeds of the wild species Dongjin rice were sterilized in 40% Lax (Yuhan-Clorax) for 30 minutes, washed 5 or more times with sterilized water, and then treated at low temperature for 1 day. Treated seeds were 3% sucrose, CHU medium (4 g/L, Duchedfa Biochem), casamino acid (300 mg/L), L-proline (2.9 g/L), 2,4D (2 mg/L), 0.002% gelite was planted in N6D medium and cultured at 28° C. for 7 days or longer to induce callus from embryos. Transformation of this callus was cultured with Agrobacterium tumefaciens LBA4404 transformed above for 4 days, washed with water, and newly grown callus was selected using hygromycin B antibiotics. The selected callus was cultured in a re-differentiation medium containing auxin and cytokininin (Sohn et al., Korean J. Breed. 38(3): 173~179 (2006)) to obtain plants. The produced transgenic plants were transferred to soil and grown in a greenhouse.

실시예 3. Example 3. OsCSR1 OsCSR1 및 And OsCSR2 OsCSR2 유전자의 발현 확인 Confirmation of gene expression

3-1. 총 RNA 추출 방법3-1. Total RNA extraction method

영양생장기(vegetative stage)의 야생종, OsCSR1 또는 OsCSR2 과다발현 형질전환 벼 식물체의 잎을 액체질소를 이용해 얼린 후 곱게 갈아서 가루로 만들고 2 ml/g 의 RNA 추출 완충액과 섞어주었다. 이어서 200 μl의 클로로포름(chloroform)을 넣어서 다시 섞어준 다음, 4℃에서 13,000 rpm으로 원심분리하여 상층액을 동량의 침전 완충액과 섞어주었다. 다시 4℃에서 13,000 rpm으로 원심분리하여 RNA를 침전하는 방법으로 총 RNA(total RNA)를 추출하였다. The leaves of the transgenic rice plant overexpressing the wild species, OsCSR1 or OsCSR2 overexpressing the vegetative stage were frozen with liquid nitrogen and then finely ground into powder and mixed with 2 ml/g of RNA extraction buffer. Subsequently, 200 μl of chloroform was added and mixed again, followed by centrifugation at 13,000 rpm at 4°C to mix the supernatant with the same amount of precipitation buffer. Total RNA (total RNA) was extracted by centrifuging again at 13,000 rpm at 4° C. to precipitate RNA.

3-2. 유전자 발현 확인: 역전사 중합효소연쇄반응(RT-PCR, Reverse transcription polymerase chain reaction)3-2. Confirmation of gene expression: Reverse transcription polymerase chain reaction (RT-PCR)

상기 3-1에서 획득한 RNA를 정량하고, 각 샘플당 2 μg씩의 동량의 총RNA 로부터 cDNA를 합성하기 위한 역전사 효소반응을 진행하였다. 올리고 dT(oligo dT) 프라이머와 TOPscript역전사효소(enzynomics)를 이용하여 총 RNA로부터 단일가닥 cDNA를 합성하였다. 합성한 cDNA 를 주형으로 하여 OsCSR1와 OsCSR2 유전자를 특이적으로 증폭시키는 PCR을 진행하였다. 10X Taq 폴리머라아제 완충용액(Intron) 2 ㎕, dNTP 혼합액(각 1.25 mM) 2 ㎕, 각 유전자에 상보적인 정방향 프라이머와 역방향 프라이머를 각각 10 pmole 씩, 그리고 1 유닛의 Taq DNA 폴리머라아제(intron)를 넣어 반응의 부피를 20 ㎕ 로 만들었다. BIORAD C-1000 DNA 써멀 사이클러로 PCR을 수행하였다. 95℃에서 3분간 변성시킨 후, 95℃에서 30초, 55℃에서 20초, 72℃에서 40초를 반복하는 것을 수행하였고, 마지막 단계에서 72℃에서 3분간 추가로 중합반응을 수행한 반응물을 전기영동하여 확인하였으며 그 결과를 도 2에 나타내었다. The RNA obtained in 3-1 was quantified, and reverse transcriptase reaction for synthesizing cDNA from the same amount of total RNA of 2 μg for each sample was performed. Single-stranded cDNA was synthesized from total RNA using an oligo dT (oligo dT) primer and TOPscript reverse transcriptase (enzynomics). Using the synthesized cDNA as a template, PCR was performed to specifically amplify the OsCSR1 and OsCSR2 genes. 2 µl of 10X Taq polymerase buffer (Intron), 2 µl of dNTP mixture (1.25 mM each), 10 pmoles each of the forward and reverse primers complementary to each gene, and 1 unit of Taq DNA polymerase (intron). ) Was added to make the volume of the reaction 20 µl. PCR was performed with a BIORAD C-1000 DNA thermal cycler. After denaturing at 95°C for 3 minutes, repeating at 95°C for 30 seconds, at 55°C for 20 seconds, and at 72°C for 40 seconds was performed, and in the last step, the reaction product was subjected to an additional polymerization reaction at 72°C for 3 minutes. It was confirmed by electrophoresis and the results are shown in FIG. 2.

본 실험에 사용된 프라이머는 상기의 표 1과 같으며, 로딩 대조군으로 OsUBQ10을 사용하였다. The primers used in this experiment are shown in Table 1 above, and OsUBQ10 was used as a loading control.

상기 실시예 2에서 제작한 형질전환 식물체에서 RT-PCR을 수행한 결과, mRNA 수준에서 야생종에 비해 OsCSR1 및 OsCSR2의 유전자 발현이 증가되는 것을 확인할 수 있었다. As a result of performing RT-PCR in the transgenic plant prepared in Example 2, it was confirmed that the gene expression of OsCSR1 and OsCSR2 was increased compared to wild species at the mRNA level.

3-3. 단백질 발현 확인: 폴리아크릴아마이드 겔 전기 영동법(SDS-PAGE)3-3. Confirmation of protein expression: polyacrylamide gel electrophoresis (SDS-PAGE)

상기 실시예 2에서 제작한 OsCSR1 과발현 식물체와 OsCSR2 과발현 식물체에서 각각의 유전자가 단백질 수준에서도 발현이 증가하는지 확인하고자 야생종과 각각의 형질전환 식물체에서 단백질을 추출한 후 SDS-PAGE 분석을 수행하였다. 구체적으로, 야생종과 형질전환 식물체에서 추출한 단백질을 폴리아크릴아마이드 겔 상에서 분리시키고 이를 멤브레인에 옮겨서 항-Flag(anti-Flag) 항체로 면역블롯팅(immunoblot)을 수행하였다. In the OsCSR1 overexpressing plant and the OsCSR2 overexpressing plant prepared in Example 2, in order to determine whether the expression of each gene increases even at the protein level, proteins were extracted from wild species and each transgenic plant, and then SDS-PAGE analysis was performed. Specifically, proteins extracted from wild species and transgenic plants were separated on a polyacrylamide gel, transferred to a membrane, and immunoblot was performed with an anti-Flag (anti-Flag) antibody.

그 결과 도 3에 나타낸 바와 같이, OsCSR1 과발현 식물체 #1, #2에서 OsCSR1 단백질이 관찰되었고, OsCSR2 과발현 식물체 #13에서 OsCSR2 단백질이 관찰되었다. 단백질 수준에서의 발현 증가가 확인된 형질전환 식물체를 하기 실시예 4의 저온 스트레스 저항성 표현형 관찰에 사용하였다.As a result, as shown in FIG. 3, OsCSR1 protein was observed in OsCSR1 overexpressing plants #1 and #2, and OsCSR2 protein was observed in OsCSR2 overexpressing plants #13. Transgenic plants with increased expression at the protein level were used to observe the low temperature stress resistance phenotype of Example 4 below.

실시예 4. OsCSR1 또는 OsCSR2 과다발현 형질전환 벼 식물체의 저온 저항성 표현형 분석Example 4. Low temperature resistance phenotype analysis of transgenic rice plants overexpressing OsCSR1 or OsCSR2

4-1. 식물체 준비, 저온 스트레스 처리 및 표현형 분석 방법 4-1. Plant preparation, cold stress treatment and phenotypic analysis method

OsCSR1 및 OsCSR2 유전자가 과발현 되었을 때 저온 스트레스에 대한 저항성이 변화되는지 확인하기 위해 야생종 식물체와 각각의 유전자에 대한 과발현 T3 식물체 종자(#1, #2, #13)를 40% 락스(유한-크로락스)에 30분간 소독한 후, 4℃에 이틀 동안 저온 처리하였다. 처리한 종자를 각각 0.7% 피토아가(phytoagar)가 포함된 ㅍMS 배지(MS medium 2.2 g/L(duchefa), 3% 수크로스)에 심어서 28℃ 챔버에서 수직으로 발아 및 생장시킨다. 발아일로부터 10일이 지나면 하나의 화분에 왼쪽에 야생종을 심고 오른쪽에 각각의 과발현 식물체가 위치하도록 식물을 옮겨주고 28℃ 배양실에서 4주 동안 성장시켰다(16시간 광 조건/8시간 암 조건). 위 화분을 4℃ 챔버에 넣어 저온 환경 스트레스에 3일 또는 4일 동안 노출시킨 후, 다시 28℃ 배양실로 옮겨주었다. 생존율은 생장을 재개하거나 회복 시점으로부터 10일 이상이 지났을 때에도 줄기 부분에 세포 사멸 등이 진행되지 않아 초록색을 띠는 것을 기준으로 분석하였다. In order to check whether the resistance to cold stress changes when OsCSR1 and OsCSR2 genes are overexpressed, 40% of wild species plants and overexpressing T3 plant seeds (#1, #2, #13) for each gene were added to 40% Lax (Limited-Clorax). ) Was sterilized for 30 minutes, and then subjected to low temperature treatment at 4°C for two days. The treated seeds were planted in MS medium (MS medium 2.2 g/L (duchefa), 3% sucrose) each containing 0.7% phytoagar, and germinated and grown vertically in a 28°C chamber. After 10 days from the germination date, wild species were planted on the left in one pot, and the plants were transferred so that each overexpressing plant was located on the right, and grown for 4 weeks in a 28°C incubation room (16 hours light condition / 8 hours dark condition). The above pollen was placed in a 4° C. chamber and exposed to low temperature environmental stress for 3 or 4 days, and then transferred to a 28° C. culture chamber. The survival rate was analyzed based on the fact that the stem part had a green color even when the growth was resumed or when more than 10 days had elapsed from the time of recovery.

그 결과 도 4 및 도 5에 나타낸 바와 같이, OsCSR1 및 OsCSR2 유전자를 과발현시킨 형질전환 벼의 경우 식물체의 저온 스트레스에 대한 저항성이 크게 향상되었으며, 생장이 저해되고 생존하기 어려운 저온 환경에서도 생존 확률이 높아지는 것을 확인할 수 있었다.As a result, as shown in Figs. 4 and 5, in the case of the transgenic rice that overexpresses the OsCSR1 and OsCSR2 genes, the resistance to the low temperature stress of the plant is greatly improved, and the survival probability is increased even in a low-temperature environment where growth is inhibited and it is difficult to survive. I could confirm that.

이처럼 식물체의 OsCSR1 및 OsCSR2 단백질을 코딩하는 뉴클레오티드를 도입한 과다발현 형질전환 벼 식물체는 저온 스트레스에 대한 저항성이 탁월하여 중요 식량 작물인 벼의 재배 단계에서 저온 환경 스트레스에 의해 발생하는 작물 생산량 손실을 줄일 수 있는 신기능 GM 작물로 유용하게 이용될 수 있다. In this way, the overexpressed transgenic rice plant, which introduced the nucleotide encoding the OsCSR1 and OsCSR2 proteins of the plant, has excellent resistance to low temperature stress, reducing the loss of crop production caused by low temperature environmental stress in the cultivation stage of rice, an important food crop. It can be usefully used as a new functional GM crop that can be used.

<110> Industry-Academic Cooperation Foundation, Yonsei University <120> Novel gene related to plant cold stress tolerance and use thereof <130> 1063060 <160> 14 <170> KoPatentIn 3.0 <210> 1 <211> 690 <212> PRT <213> Artificial Sequence <220> <223> OsCSR1_aa <400> 1 Met Thr Pro Pro Pro Pro Pro Pro Arg Arg Arg Met Leu Ala Met Pro 1 5 10 15 Ala Val Cys Pro Cys Glu Asp Ile Ser Pro Gly Thr Leu Leu Ala Ser 20 25 30 Leu Ala Thr Leu Ser Ala Asp Val Ala Asp Gly Cys Asp Val Asp Arg 35 40 45 Leu Pro Ala Leu Arg Gly Gly Val Gly Val Ala Val Arg Val Ala Gly 50 55 60 Leu Leu Arg Glu Phe Leu Glu Glu Val Arg Trp Ala Ala Ala Ala Glu 65 70 75 80 Leu Pro Gly Gly Ser Val Leu Gly Met Ser Glu Leu His Val Ala Leu 85 90 95 Gln Lys Met Arg Phe Leu Leu Glu Asp Cys Gly Arg Lys Gly Ala Arg 100 105 110 Met Trp Val Leu Met Asn Ala Glu Ala Val Ala Ser Glu Leu Arg Val 115 120 125 Val Leu Gly Ser Val Ala Thr Ala Met Asp Val Leu Pro Ala Gly Val 130 135 140 Val Ala Ala Ser Asp Asp Ala Arg Glu Phe Ala Ala Leu Val Ser Gln 145 150 155 160 Gln Ala Trp Arg Ala Ala Val Arg Pro Asp Glu Glu Asp Ser Arg Ala 165 170 175 Ala Arg Ser Val Arg Ser Met Leu Ala Arg Phe Arg Ser Gly Ala Thr 180 185 190 Pro Asp Ala Glu Asp Ala Arg Leu Val Leu Gly Arg Val Gly Val Ala 195 200 205 Ser Trp Trp Asp Cys Ser Gln Glu Val Ser Phe Leu Glu Ala Glu Met 210 215 220 Leu Glu Arg Leu Glu Ala Gly Gly Glu Asn Asp Asn Asp Leu Val Leu 225 230 235 240 Ile Ser Gly Leu Leu Thr Phe Leu Leu Tyr Cys Arg Val Val Leu Phe 245 250 255 Asp Arg Ile Asp Tyr Gly Lys Ala Asp Glu Pro Ala Pro Ala Pro Ala 260 265 270 Pro Arg Ala Ala Ser Tyr Leu Ala Arg Ile Asn Pro Glu Gly Leu Gln 275 280 285 Cys Pro Ile Thr Leu Glu Leu Met Thr Asp Pro Val Thr Leu Ala Thr 290 295 300 Gly Gln Thr Tyr Asp Arg Ala Ser Ile Lys Arg Trp Val Lys Ser Gly 305 310 315 320 Cys Arg Thr Cys Pro Val Thr Gly Glu Lys Leu Arg Ser Ala Asp Val 325 330 335 Val Pro Asn Val Ala Val Arg Gly Ile Val Glu Gln Leu Leu Leu Ser 340 345 350 Ser Gly Val Ser Leu His Glu Pro Ser Ser Lys His Arg Cys Ala Val 355 360 365 Asp Lys Thr Ala Ser Pro Phe Gly Ala Ala Ala Ala Gly Gly Ala Arg 370 375 380 Leu Ala Val Ala Phe Leu Val Ser Lys Leu Cys Arg Gly Thr Pro Glu 385 390 395 400 Glu Gln Lys Lys Ala Thr Tyr Glu Cys Arg Lys Leu Ser Lys Arg Asn 405 410 415 Val Phe His Arg Ala Cys Leu Val Asp Ala Gly Ala Val Pro Trp Leu 420 425 430 Leu His Leu Leu Ser Ser Pro Asp Ala Ser Val Gln Asp Asn Ala Val 435 440 445 Ala Gly Leu Leu Asn Leu Ser Lys His Pro Ala Gly Arg Arg Ala Leu 450 455 460 Val Glu Ala Gly Gly Leu Gly Leu Ile Val Asp Ala Val Ser Val Ala 465 470 475 480 Ala Lys Val Glu Ala Gln Gln Asn Ala Ala Ala Ile Leu Phe Tyr Leu 485 490 495 Ser Ser Asp Ala Gly Tyr Cys Asp Glu Ile Ser Arg Ile Pro Glu Ala 500 505 510 Ile Pro Thr Leu Val Arg Leu Val Arg Glu Gly Ala Tyr Arg Gly Arg 515 520 525 Lys Asn Ala Leu Val Ser Leu Tyr Gly Val Leu Gln Arg Gly Ala Gly 530 535 540 Gly His Gly Arg Ala Val Ser Ala Gly Ala Val Ala Ala Leu Ala Ser 545 550 555 560 Leu Leu Pro Gly Asp Arg Asp Asp Leu Ala Asn Asp Ala Val Ala Leu 565 570 575 Leu Ala Arg Leu Ala Glu Gln Pro Ala Gly Ala Ala Ala Val Leu Ser 580 585 590 Ser Ser Ala Leu Val Thr Arg Leu Val Asp Phe Leu Gly Ala Ser Ala 595 600 605 Ser Arg Ser Ala Lys Asp His Cys Ala Ala Leu Leu Ala Ser Leu Cys 610 615 620 Arg His Gly Gly Asp Ala Val Val Ala Leu Leu Gly Lys Thr Pro Gly 625 630 635 640 Leu Met Pro Ser Leu Tyr Ala Leu Ile Ala Asp Gly Gly Ala Gln Gly 645 650 655 Ser Lys Lys Ala Arg Trp Leu Val Asn Glu Ile His Arg His Tyr Glu 660 665 670 Gln Arg Gln Pro Pro Val Ala Ala Pro Pro Ala Gly Asp Arg Val Ile 675 680 685 Arg Val 690 <210> 2 <211> 2073 <212> DNA <213> Artificial Sequence <220> <223> OsCSR1_nt <400> 2 atgacgccgc cgccgccgcc gccgcgccgc cggatgctgg cgatgccggc ggtgtgccca 60 tgcgaggaca tctcgcccgg gacgctgctg gcgtcgctcg ccacgctttc ggccgacgtc 120 gccgacgggt gcgacgtgga caggctgccg gcgctccggg gcggcgtcgg cgtggccgtg 180 cgcgtcgccg ggttgctccg ggagttcctt gaggaggtcc ggtgggcggc ggcggcggag 240 ctccccggtg gctctgtgct ggggatgtcc gagctgcacg tggcgttgca gaagatgcgg 300 ttcttgctag aggattgcgg gaggaagggg gcgcggatgt gggtgctgat gaacgcggag 360 gctgtggcgt cggagctgcg ggtggtgctc ggctcggtgg cgacggcgat ggacgtgctc 420 cccgccggcg tggtcgcggc gtccgacgac gccagggagt tcgcggcgct ggtgtcgcag 480 caggcgtggc gcgccgcggt gcggccggac gaggaggact cccgcgcggc acggagcgtg 540 cggtcgatgc tggcgcggtt caggagcggc gccacgccgg acgccgagga cgcgaggctg 600 gtgctcggcc gcgtcggcgt cgccagctgg tgggactgct cccaggaggt gtccttcctc 660 gaggccgaga tgctcgagcg gctggaggcc ggcggcgaga acgacaacga cctcgtgctc 720 atcagcggcc tcttgacgtt cctgctctac tgccgcgtcg tcctgttcga ccgcattgat 780 tacggcaagg ctgatgagcc ggcgccggcg ccggcgccga gggcggcgag ctacttggcg 840 cgaatcaatc cggaggggct gcaatgcccg atcacgctgg agctgatgac tgatccggtg 900 acattggcca ccggccagac ctacgaccgc gcgtcgatca agcggtgggt gaagagcggg 960 tgccggacgt gccccgtcac cggcgagaag ctccgtagcg ccgacgtcgt gccgaacgtc 1020 gcggtgcgcg ggatcgtcga gcagctgctg ctcagcagcg gcgtctcgct ccacgagccg 1080 agcagcaagc accggtgcgc ggtcgacaag acggcctcgc cgttcggtgc cgcggcggcc 1140 ggcggcgcgc gcctcgccgt ggcgttcctc gtgtccaagc tgtgccgtgg cacgccggag 1200 gagcagaaga aggcgacgta cgagtgccgg aagctgtcga agcggaacgt gttccaccgg 1260 gcgtgcctcg tcgacgccgg cgccgtgcca tggctgctcc acctcctctc ctccccggac 1320 gcctccgtcc aggacaatgc cgtggctggc ctcctcaacc tctccaagca ccccgccggc 1380 aggagggcgc tcgtcgaggc cggcgggctg ggcctcatcg tcgacgccgt gagcgtggcc 1440 gccaaggtgg aggcgcagca gaacgcggcg gccatcctgt tctacctctc gtcggacgcc 1500 ggctactgcg atgagatcag ccgcatcccg gaggcgatcc cgacgctggt ccggctcgtg 1560 cgggagggcg cgtaccgcgg ccggaagaac gcgctggtga gcctctacgg cgtgctccag 1620 cgcggcgcgg gcggccacgg cagggcggtg tccgccggcg ccgtggcggc gctcgcgtcc 1680 ctgctgcccg gcgaccgcga cgacctcgcc aacgacgccg tcgcgctgct ggcgaggctc 1740 gccgagcagc ccgccggcgc ggcggccgtc ctctccagct cggcgctcgt cacgcggctc 1800 gtcgacttcc tcggcgcgtc ggcgtcgcgg tcggccaagg accactgcgc ggcgctgctg 1860 gcgtcgctgt gccggcacgg cggcgacgcg gtggtcgccc tgctggggaa gacgccgggg 1920 ctgatgccgt ccctgtacgc gctcatcgcc gacggcggcg cgcaggggag caagaaggcg 1980 aggtggctcg tcaacgaaat ccaccggcat tacgagcagc ggcagccgcc ggtggccgcg 2040 ccgccggccg gcgaccgtgt cattcgagta tag 2073 <210> 3 <211> 712 <212> PRT <213> Artificial Sequence <220> <223> OsCSR2_aa <400> 3 Met Ser Ser Pro Pro Pro Pro Pro Pro Ser Lys Pro Lys Arg Arg Arg 1 5 10 15 Leu Leu Ser Leu Pro Ala Val Tyr Pro Cys Glu Asp Ile Ala Pro Ala 20 25 30 Pro Leu Leu Ala Ser Leu Leu Ser Leu Ala Ala Asp Val Ala Ser Arg 35 40 45 Arg Ala Ala Asp Val Asp Ala Phe Pro Val Leu Arg Cys Gly Val Arg 50 55 60 Lys Ala Val Arg Leu Ala Gly Ile Leu Leu Ala Phe Leu Glu Glu Val 65 70 75 80 Gln Asp Ala Ala Ala Ala Ala Ala Leu Pro Ser Ser Ala Val Leu Gly 85 90 95 Leu Thr Glu Leu His Val Ala Met Gln Lys Leu Arg Phe Leu Leu Ala 100 105 110 Asp Cys Ala Arg Arg Gly Ala Arg Leu Trp Val Leu Val Asn Ala Gly 115 120 125 Met Val Ala Ser Glu Leu Arg Leu Val Leu Gly Ser Val Ala Ala Ala 130 135 140 Met Asp Ala Leu Pro Arg Ser Val Ala Glu Ala Ser Val Glu Ala Gly 145 150 155 160 Glu Leu Ala Arg Val Val Ser Glu Gln Ala Trp Arg Ala Ala Val Arg 165 170 175 Pro Asp Gly Ala Asp Glu Arg Ala Ala Arg Ser Val Arg Ser Ile Leu 180 185 190 Asp Gln Phe Lys Asp Gly Val Ala Pro Asp Ala Asp Asp Val Arg Arg 195 200 205 Val Leu Arg Arg Val Arg Val Gly Ser Trp Ser Asp Cys Ser Glu Glu 210 215 220 Ile Ala Phe Leu Glu Ser Glu Ile Cys Ala Arg Leu Asp Ala Gly Asp 225 230 235 240 Glu Asn Ser Asn Asp Val Leu Val Met Asn Ser Leu Met Thr Phe Leu 245 250 255 Val Tyr Cys Arg Val Val Leu Phe Asp His Ile Asp Ala Ser Lys Ser 260 265 270 Gln Pro Ala Ala Ala Ala Ala Pro Ala Pro Ala Arg Cys Pro Glu Trp 275 280 285 Ile Arg Pro Glu Ala Leu Gln Cys Pro Ile Thr Leu Asp Leu Met Thr 290 295 300 Asp Pro Val Thr Val Ser Thr Gly Gln Thr Tyr Asp Arg Ala Ser Ile 305 310 315 320 Thr Arg Trp Met Lys Ala Gly Cys Arg Thr Cys Pro Val Thr Gly Glu 325 330 335 Arg Leu Ser Thr Ala Asp Leu Val Pro Asn Thr Val Leu Arg Gly Ile 340 345 350 Ile Glu Arg Met Leu Leu Ile Asn Gly Val Thr Leu Pro Glu Leu Ser 355 360 365 Ala Ala Gly Gly Gly Gly His Arg His Gly Ala Val Ala Asn Thr Ala 370 375 380 Val Pro Phe Gly Pro Ala Ala Ala Gly Ala Ala Arg Leu Ala Val Ala 385 390 395 400 His Ile Val Ala Gln Leu Ser Arg Gly Ser Thr Glu Glu Arg Arg Lys 405 410 415 Ala Thr Ser Glu Ala Arg Lys Leu Ser Lys His Ser Val Phe Tyr Arg 420 425 430 Ala Cys Leu Val Asp Ala Asn Ala Val Pro Trp Leu Leu Cys Leu Leu 435 440 445 Ser Ser Thr Asp Ala Ala Val Gln Asp Asn Ala Val Ala Ser Leu Leu 450 455 460 Asn Leu Ser Lys His Pro Ala Gly Arg Thr Ala Ile Val Glu Val Gly 465 470 475 480 Gly Val Gly Leu Val Val Asp Val Ile Asn Val Gly Ala Lys Ala Glu 485 490 495 Ala Gln His Asn Ala Ala Ala Val Leu Phe Tyr Leu Ser Ser Asn Ser 500 505 510 Pro Asp Ser Ala Glu Glu Ile Gly Arg Ile Pro Glu Ala Ile Pro Thr 515 520 525 Leu Val Gln Leu Ile Arg Asp Gly Ala Tyr Arg Gly Arg Lys Asn Ala 530 535 540 Met Val Ser Leu Tyr Gly Leu Leu Gln Ser Ala Ala Asn His Gly Arg 545 550 555 560 Ala Ile Ala Ala Gly Ala Val Ser Ala Leu Ala Ala Leu Leu Leu Ser 565 570 575 Ala Asp Arg Asp Asp Leu Ala Gly Asp Ser Val Ala Leu Leu Ala Arg 580 585 590 Ile Ala Glu Gln Pro Ser Gly Ala Ala Ala Val Leu Ser Gln Pro Gly 595 600 605 Leu Val Ala Arg Leu Ala Glu Ala Leu Ala Ala Ser Ser Ala Ser Ser 610 615 620 Ser Arg Ser Ala Arg Asp His Ser Val Ser Leu Leu Ala Ser Leu Cys 625 630 635 640 Arg His Gly Gly Ala Lys Val Val Ala Val Leu Gly Arg Met Pro Gly 645 650 655 Leu Met Ala Ser Leu Tyr Ser Leu Val Ala Asp Gly Gly Ser Pro Gln 660 665 670 Thr Ser Lys Lys Ala Arg Ala Leu Leu Asn Glu Ile His Arg His Tyr 675 680 685 Glu Val Ala Pro Pro Pro Pro Ala Ser Ser Ala Ser Ser Asp Ala Gly 690 695 700 Gly Asp Arg Val Val Arg Val Leu 705 710 <210> 4 <211> 2139 <212> DNA <213> Artificial Sequence <220> <223> OsCSR2_nt <400> 4 atgtcgtcgc cgccgccgcc gccgccgagc aagccgaagc gccggcggct actgtctctt 60 ccggcggtct acccgtgcga ggacatcgcg ccggcgccgc tgctcgcctc gctgctctcc 120 ctcgcggcgg atgtcgccag ccgtcgggcg gccgacgtcg acgccttccc cgttctccgc 180 tgcggcgtgc gcaaggccgt gcggctcgcc gggatcctcc tcgcgttcct cgaggaggtt 240 caggatgcgg cggcggcggc ggcgttgccg tcgtctgcgg tgcttgggct gacggagctg 300 cacgtggcga tgcagaagct gcggttcctg ctcgcggact gcgcgcggcg tggcgcgcgg 360 ctgtgggtgc tcgtgaacgc cgggatggtg gcgtcggagc tcaggttggt gctcgggtcg 420 gtcgcggcgg ccatggacgc gcttccgagg agcgtcgcgg aggcgtcggt ggaggccggg 480 gagctcgcgc gggtggtgtc ggagcaggcg tggcgcgcgg cggtgcggcc cgacggcgcc 540 gacgagcgcg cggcgcggag cgtgcggtcg atcctggatc agttcaagga cggcgtcgcg 600 ccggacgccg acgacgtgag gcgggtgctc cgccgggtca gggtcgggag ctggtcggat 660 tgctccgagg agatcgcgtt cctggagagc gagatttgcg cgcggctgga cgccggcgac 720 gagaacagca atgacgttct cgtcatgaac agcttaatga cgttcctggt gtactgccgc 780 gtcgtgctgt tcgaccacat tgacgcgagc aagtcgcaac cggcggcggc ggcagcgccg 840 gcgccggcga ggtgcccgga atggatcaga ccggaagcgc tgcagtgccc gatcactctt 900 gacctcatga ccgatccggt gaccgtgtcc accggccaga catacgaccg ggcgtcgatc 960 acccggtgga tgaaggccgg ttgccgcacg tgcccggtca ccggcgagag gctgagcacc 1020 gccgacctcg tccccaacac cgtgctccgc gggatcatcg agcggatgct tctcatcaat 1080 ggcgtcaccc tcccggagct gagcgccgcc ggcggcggtg gccaccgcca cggcgccgtc 1140 gcgaacacgg ccgtgccgtt cggcccggcc gccgcgggcg ccgcgcgcct cgccgtcgcg 1200 cacatcgtgg cgcagctctc gagggggtcg acggaggagc ggaggaaggc gacgtcggag 1260 gcacggaagc tgtcgaagca cagcgtgttc taccgggctt gcctcgtcga cgccaacgcc 1320 gtgccgtggt tgctgtgcct cctctcctcc accgacgccg ccgtgcagga caatgccgtg 1380 gcgtcgctgc tcaacctctc gaagcacccc gccggcagga cggccatcgt ggaggtcggc 1440 ggcgtcggcc tcgtcgtcga cgtgatcaac gtcggcgcca aggccgaggc gcagcacaac 1500 gcggcggccg tcctcttcta cctctcgtcg aacagcccgg actccgccga ggagatcggc 1560 cgcatcccgg aggcgatccc gacgctggtc cagctcatcc gggacggcgc gtaccgcggg 1620 cggaagaacg ccatggtcag cctgtacggg ctgctccaga gcgccgccaa ccacggcagg 1680 gccatcgccg cgggcgccgt gtcagcgctc gccgcgctgc tgctctccgc cgaccgcgac 1740 gacctcgccg gggacagcgt cgcgctgctg gcgaggatcg ccgagcagcc gtctggcgcc 1800 gcggccgtcc tctcgcagcc ggggctcgtc gcccgcctcg ccgaggcgct cgccgcgtcg 1860 tcggcgtcgt cgtcccggtc ggcgagagac cacagcgtgt cgctcctcgc ctcgctgtgc 1920 cggcacggcg gcgccaaggt ggtcgccgtg ctggggcgga tgccggggct gatggcgtcg 1980 ctctactcgc tcgtcgccga cggcggcagc ccgcagacga gcaagaaggc gcgggcgctg 2040 ctcaacgaga tccaccggca ttacgaggtg gcgccgccgc cgccggcgtc gtcggcgtcg 2100 tcggacgccg gcggtgaccg tgtcgttcgc gtgctatag 2139 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> OsUBQ10 RT FW <400> 5 atgcagatct ttgtgaagac 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> OsUBQ10 RT RV <400> 6 ttactgacca ccacggaggc 20 <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> OsCSR1 RT FW <400> 7 tatagtacac gcgcgagctt tgt 23 <210> 8 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> OsCSR1 RT RV <400> 8 gcagagttgc aaaccacttt agt 23 <210> 9 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> OsCSR2 RT FW <400> 9 tcgcacatac tctgttcctc ttt 23 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> OsCSR2 RT RV <400> 10 caaacagcgt cacaaacagc aa 22 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> OsRab16b RT FW <400> 11 acaagggcaa caaccaccag 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> OsRab16b RT RV <400> 12 gcttgcaatg gcatcacaag 20 <210> 13 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> OsDREB1A RT SE <400> 13 aggccgtcga ggacttctt 19 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> OsDREB1A RT AS <400> 14 tagtagctcc agagtggagt 20 <110> Industry-Academic Cooperation Foundation, Yonsei University <120> Novel gene related to plant cold stress tolerance and use thereof <130> 1063060 <160> 14 <170> KoPatentIn 3.0 <210> 1 <211> 690 <212> PRT <213> Artificial Sequence <220> <223> OsCSR1_aa <400> 1 Met Thr Pro Pro Pro Pro Pro Pro Arg Arg Arg Met Leu Ala Met Pro 1 5 10 15 Ala Val Cys Pro Cys Glu Asp Ile Ser Pro Gly Thr Leu Leu Ala Ser 20 25 30 Leu Ala Thr Leu Ser Ala Asp Val Ala Asp Gly Cys Asp Val Asp Arg 35 40 45 Leu Pro Ala Leu Arg Gly Gly Val Gly Val Ala Val Arg Val Ala Gly 50 55 60 Leu Leu Arg Glu Phe Leu Glu Glu Val Arg Trp Ala Ala Ala Ala Glu 65 70 75 80 Leu Pro Gly Gly Ser Val Leu Gly Met Ser Glu Leu His Val Ala Leu 85 90 95 Gln Lys Met Arg Phe Leu Leu Glu Asp Cys Gly Arg Lys Gly Ala Arg 100 105 110 Met Trp Val Leu Met Asn Ala Glu Ala Val Ala Ser Glu Leu Arg Val 115 120 125 Val Leu Gly Ser Val Ala Thr Ala Met Asp Val Leu Pro Ala Gly Val 130 135 140 Val Ala Ala Ser Asp Asp Ala Arg Glu Phe Ala Ala Leu Val Ser Gln 145 150 155 160 Gln Ala Trp Arg Ala Ala Val Arg Pro Asp Glu Glu Asp Ser Arg Ala 165 170 175 Ala Arg Ser Val Arg Ser Met Leu Ala Arg Phe Arg Ser Gly Ala Thr 180 185 190 Pro Asp Ala Glu Asp Ala Arg Leu Val Leu Gly Arg Val Gly Val Ala 195 200 205 Ser Trp Trp Asp Cys Ser Gln Glu Val Ser Phe Leu Glu Ala Glu Met 210 215 220 Leu Glu Arg Leu Glu Ala Gly Gly Glu Asn Asp Asn Asp Leu Val Leu 225 230 235 240 Ile Ser Gly Leu Leu Thr Phe Leu Leu Tyr Cys Arg Val Val Leu Phe 245 250 255 Asp Arg Ile Asp Tyr Gly Lys Ala Asp Glu Pro Ala Pro Ala Pro Ala 260 265 270 Pro Arg Ala Ala Ser Tyr Leu Ala Arg Ile Asn Pro Glu Gly Leu Gln 275 280 285 Cys Pro Ile Thr Leu Glu Leu Met Thr Asp Pro Val Thr Leu Ala Thr 290 295 300 Gly Gln Thr Tyr Asp Arg Ala Ser Ile Lys Arg Trp Val Lys Ser Gly 305 310 315 320 Cys Arg Thr Cys Pro Val Thr Gly Glu Lys Leu Arg Ser Ala Asp Val 325 330 335 Val Pro Asn Val Ala Val Arg Gly Ile Val Glu Gln Leu Leu Leu Ser 340 345 350 Ser Gly Val Ser Leu His Glu Pro Ser Ser Lys His Arg Cys Ala Val 355 360 365 Asp Lys Thr Ala Ser Pro Phe Gly Ala Ala Ala Ala Gly Gly Ala Arg 370 375 380 Leu Ala Val Ala Phe Leu Val Ser Lys Leu Cys Arg Gly Thr Pro Glu 385 390 395 400 Glu Gln Lys Lys Ala Thr Tyr Glu Cys Arg Lys Leu Ser Lys Arg Asn 405 410 415 Val Phe His Arg Ala Cys Leu Val Asp Ala Gly Ala Val Pro Trp Leu 420 425 430 Leu His Leu Leu Ser Ser Pro Asp Ala Ser Val Gln Asp Asn Ala Val 435 440 445 Ala Gly Leu Leu Asn Leu Ser Lys His Pro Ala Gly Arg Arg Ala Leu 450 455 460 Val Glu Ala Gly Gly Leu Gly Leu Ile Val Asp Ala Val Ser Val Ala 465 470 475 480 Ala Lys Val Glu Ala Gln Gln Asn Ala Ala Ala Ile Leu Phe Tyr Leu 485 490 495 Ser Ser Asp Ala Gly Tyr Cys Asp Glu Ile Ser Arg Ile Pro Glu Ala 500 505 510 Ile Pro Thr Leu Val Arg Leu Val Arg Glu Gly Ala Tyr Arg Gly Arg 515 520 525 Lys Asn Ala Leu Val Ser Leu Tyr Gly Val Leu Gln Arg Gly Ala Gly 530 535 540 Gly His Gly Arg Ala Val Ser Ala Gly Ala Val Ala Ala Leu Ala Ser 545 550 555 560 Leu Leu Pro Gly Asp Arg Asp Asp Leu Ala Asn Asp Ala Val Ala Leu 565 570 575 Leu Ala Arg Leu Ala Glu Gln Pro Ala Gly Ala Ala Ala Val Leu Ser 580 585 590 Ser Ser Ala Leu Val Thr Arg Leu Val Asp Phe Leu Gly Ala Ser Ala 595 600 605 Ser Arg Ser Ala Lys Asp His Cys Ala Ala Leu Leu Ala Ser Leu Cys 610 615 620 Arg His Gly Gly Asp Ala Val Val Ala Leu Leu Gly Lys Thr Pro Gly 625 630 635 640 Leu Met Pro Ser Leu Tyr Ala Leu Ile Ala Asp Gly Gly Ala Gln Gly 645 650 655 Ser Lys Lys Ala Arg Trp Leu Val Asn Glu Ile His Arg His Tyr Glu 660 665 670 Gln Arg Gln Pro Pro Val Ala Ala Pro Pro Ala Gly Asp Arg Val Ile 675 680 685 Arg Val 690 <210> 2 <211> 2073 <212> DNA <213> Artificial Sequence <220> <223> OsCSR1_nt <400> 2 atgacgccgc cgccgccgcc gccgcgccgc cggatgctgg cgatgccggc ggtgtgccca 60 tgcgaggaca tctcgcccgg gacgctgctg gcgtcgctcg ccacgctttc ggccgacgtc 120 gccgacgggt gcgacgtgga caggctgccg gcgctccggg gcggcgtcgg cgtggccgtg 180 cgcgtcgccg ggttgctccg ggagttcctt gaggaggtcc ggtgggcggc ggcggcggag 240 ctccccggtg gctctgtgct ggggatgtcc gagctgcacg tggcgttgca gaagatgcgg 300 ttcttgctag aggattgcgg gaggaagggg gcgcggatgt gggtgctgat gaacgcggag 360 gctgtggcgt cggagctgcg ggtggtgctc ggctcggtgg cgacggcgat ggacgtgctc 420 cccgccggcg tggtcgcggc gtccgacgac gccagggagt tcgcggcgct ggtgtcgcag 480 caggcgtggc gcgccgcggt gcggccggac gaggaggact cccgcgcggc acggagcgtg 540 cggtcgatgc tggcgcggtt caggagcggc gccacgccgg acgccgagga cgcgaggctg 600 gtgctcggcc gcgtcggcgt cgccagctgg tgggactgct cccaggaggt gtccttcctc 660 gaggccgaga tgctcgagcg gctggaggcc ggcggcgaga acgacaacga cctcgtgctc 720 atcagcggcc tcttgacgtt cctgctctac tgccgcgtcg tcctgttcga ccgcattgat 780 tacggcaagg ctgatgagcc ggcgccggcg ccggcgccga gggcggcgag ctacttggcg 840 cgaatcaatc cggaggggct gcaatgcccg atcacgctgg agctgatgac tgatccggtg 900 acattggcca ccggccagac ctacgaccgc gcgtcgatca agcggtgggt gaagagcggg 960 tgccggacgt gccccgtcac cggcgagaag ctccgtagcg ccgacgtcgt gccgaacgtc 1020 gcggtgcgcg ggatcgtcga gcagctgctg ctcagcagcg gcgtctcgct ccacgagccg 1080 agcagcaagc accggtgcgc ggtcgacaag acggcctcgc cgttcggtgc cgcggcggcc 1140 ggcggcgcgc gcctcgccgt ggcgttcctc gtgtccaagc tgtgccgtgg cacgccggag 1200 gagcagaaga aggcgacgta cgagtgccgg aagctgtcga agcggaacgt gttccaccgg 1260 gcgtgcctcg tcgacgccgg cgccgtgcca tggctgctcc acctcctctc ctccccggac 1320 gcctccgtcc aggacaatgc cgtggctggc ctcctcaacc tctccaagca ccccgccggc 1380 aggagggcgc tcgtcgaggc cggcgggctg ggcctcatcg tcgacgccgt gagcgtggcc 1440 gccaaggtgg aggcgcagca gaacgcggcg gccatcctgt tctacctctc gtcggacgcc 1500 ggctactgcg atgagatcag ccgcatcccg gaggcgatcc cgacgctggt ccggctcgtg 1560 cgggagggcg cgtaccgcgg ccggaagaac gcgctggtga gcctctacgg cgtgctccag 1620 cgcggcgcgg gcggccacgg cagggcggtg tccgccggcg ccgtggcggc gctcgcgtcc 1680 ctgctgcccg gcgaccgcga cgacctcgcc aacgacgccg tcgcgctgct ggcgaggctc 1740 gccgagcagc ccgccggcgc ggcggccgtc ctctccagct cggcgctcgt cacgcggctc 1800 gtcgacttcc tcggcgcgtc ggcgtcgcgg tcggccaagg accactgcgc ggcgctgctg 1860 gcgtcgctgt gccggcacgg cggcgacgcg gtggtcgccc tgctggggaa gacgccgggg 1920 ctgatgccgt ccctgtacgc gctcatcgcc gacggcggcg cgcaggggag caagaaggcg 1980 aggtggctcg tcaacgaaat ccaccggcat tacgagcagc ggcagccgcc ggtggccgcg 2040 ccgccggccg gcgaccgtgt cattcgagta tag 2073 <210> 3 <211> 712 <212> PRT <213> Artificial Sequence <220> <223> OsCSR2_aa <400> 3 Met Ser Ser Pro Pro Pro Pro Pro Pro Ser Lys Pro Lys Arg Arg Arg 1 5 10 15 Leu Leu Ser Leu Pro Ala Val Tyr Pro Cys Glu Asp Ile Ala Pro Ala 20 25 30 Pro Leu Leu Ala Ser Leu Leu Ser Leu Ala Ala Asp Val Ala Ser Arg 35 40 45 Arg Ala Ala Asp Val Asp Ala Phe Pro Val Leu Arg Cys Gly Val Arg 50 55 60 Lys Ala Val Arg Leu Ala Gly Ile Leu Leu Ala Phe Leu Glu Glu Val 65 70 75 80 Gln Asp Ala Ala Ala Ala Ala Ala Leu Pro Ser Ser Ala Val Leu Gly 85 90 95 Leu Thr Glu Leu His Val Ala Met Gln Lys Leu Arg Phe Leu Leu Ala 100 105 110 Asp Cys Ala Arg Arg Gly Ala Arg Leu Trp Val Leu Val Asn Ala Gly 115 120 125 Met Val Ala Ser Glu Leu Arg Leu Val Leu Gly Ser Val Ala Ala Ala 130 135 140 Met Asp Ala Leu Pro Arg Ser Val Ala Glu Ala Ser Val Glu Ala Gly 145 150 155 160 Glu Leu Ala Arg Val Val Ser Glu Gln Ala Trp Arg Ala Ala Val Arg 165 170 175 Pro Asp Gly Ala Asp Glu Arg Ala Ala Arg Ser Val Arg Ser Ile Leu 180 185 190 Asp Gln Phe Lys Asp Gly Val Ala Pro Asp Ala Asp Asp Val Arg Arg 195 200 205 Val Leu Arg Arg Val Arg Val Gly Ser Trp Ser Asp Cys Ser Glu Glu 210 215 220 Ile Ala Phe Leu Glu Ser Glu Ile Cys Ala Arg Leu Asp Ala Gly Asp 225 230 235 240 Glu Asn Ser Asn Asp Val Leu Val Met Asn Ser Leu Met Thr Phe Leu 245 250 255 Val Tyr Cys Arg Val Val Leu Phe Asp His Ile Asp Ala Ser Lys Ser 260 265 270 Gln Pro Ala Ala Ala Ala Ala Pro Ala Pro Ala Arg Cys Pro Glu Trp 275 280 285 Ile Arg Pro Glu Ala Leu Gln Cys Pro Ile Thr Leu Asp Leu Met Thr 290 295 300 Asp Pro Val Thr Val Ser Thr Gly Gln Thr Tyr Asp Arg Ala Ser Ile 305 310 315 320 Thr Arg Trp Met Lys Ala Gly Cys Arg Thr Cys Pro Val Thr Gly Glu 325 330 335 Arg Leu Ser Thr Ala Asp Leu Val Pro Asn Thr Val Leu Arg Gly Ile 340 345 350 Ile Glu Arg Met Leu Leu Ile Asn Gly Val Thr Leu Pro Glu Leu Ser 355 360 365 Ala Ala Gly Gly Gly Gly His Arg His Gly Ala Val Ala Asn Thr Ala 370 375 380 Val Pro Phe Gly Pro Ala Ala Ala Gly Ala Ala Arg Leu Ala Val Ala 385 390 395 400 His Ile Val Ala Gln Leu Ser Arg Gly Ser Thr Glu Glu Arg Arg Lys 405 410 415 Ala Thr Ser Glu Ala Arg Lys Leu Ser Lys His Ser Val Phe Tyr Arg 420 425 430 Ala Cys Leu Val Asp Ala Asn Ala Val Pro Trp Leu Leu Cys Leu Leu 435 440 445 Ser Ser Thr Asp Ala Ala Val Gln Asp Asn Ala Val Ala Ser Leu Leu 450 455 460 Asn Leu Ser Lys His Pro Ala Gly Arg Thr Ala Ile Val Glu Val Gly 465 470 475 480 Gly Val Gly Leu Val Val Asp Val Ile Asn Val Gly Ala Lys Ala Glu 485 490 495 Ala Gln His Asn Ala Ala Ala Val Leu Phe Tyr Leu Ser Ser Asn Ser 500 505 510 Pro Asp Ser Ala Glu Glu Ile Gly Arg Ile Pro Glu Ala Ile Pro Thr 515 520 525 Leu Val Gln Leu Ile Arg Asp Gly Ala Tyr Arg Gly Arg Lys Asn Ala 530 535 540 Met Val Ser Leu Tyr Gly Leu Leu Gln Ser Ala Ala Asn His Gly Arg 545 550 555 560 Ala Ile Ala Ala Gly Ala Val Ser Ala Leu Ala Ala Leu Leu Leu Ser 565 570 575 Ala Asp Arg Asp Asp Leu Ala Gly Asp Ser Val Ala Leu Leu Ala Arg 580 585 590 Ile Ala Glu Gln Pro Ser Gly Ala Ala Ala Val Leu Ser Gln Pro Gly 595 600 605 Leu Val Ala Arg Leu Ala Glu Ala Leu Ala Ala Ser Ser Ala Ser Ser 610 615 620 Ser Arg Ser Ala Arg Asp His Ser Val Ser Leu Leu Ala Ser Leu Cys 625 630 635 640 Arg His Gly Gly Ala Lys Val Val Ala Val Leu Gly Arg Met Pro Gly 645 650 655 Leu Met Ala Ser Leu Tyr Ser Leu Val Ala Asp Gly Gly Ser Pro Gln 660 665 670 Thr Ser Lys Lys Ala Arg Ala Leu Leu Asn Glu Ile His Arg His Tyr 675 680 685 Glu Val Ala Pro Pro Pro Ala Ser Ser Ala Ser Ser Asp Ala Gly 690 695 700 Gly Asp Arg Val Val Arg Val Leu 705 710 <210> 4 <211> 2139 <212> DNA <213> Artificial Sequence <220> <223> OsCSR2_nt <400> 4 atgtcgtcgc cgccgccgcc gccgccgagc aagccgaagc gccggcggct actgtctctt 60 ccggcggtct acccgtgcga ggacatcgcg ccggcgccgc tgctcgcctc gctgctctcc 120 ctcgcggcgg atgtcgccag ccgtcgggcg gccgacgtcg acgccttccc cgttctccgc 180 tgcggcgtgc gcaaggccgt gcggctcgcc gggatcctcc tcgcgttcct cgaggaggtt 240 caggatgcgg cggcggcggc ggcgttgccg tcgtctgcgg tgcttgggct gacggagctg 300 cacgtggcga tgcagaagct gcggttcctg ctcgcggact gcgcgcggcg tggcgcgcgg 360 ctgtgggtgc tcgtgaacgc cgggatggtg gcgtcggagc tcaggttggt gctcgggtcg 420 gtcgcggcgg ccatggacgc gcttccgagg agcgtcgcgg aggcgtcggt ggaggccggg 480 gagctcgcgc gggtggtgtc ggagcaggcg tggcgcgcgg cggtgcggcc cgacggcgcc 540 gacgagcgcg cggcgcggag cgtgcggtcg atcctggatc agttcaagga cggcgtcgcg 600 ccggacgccg acgacgtgag gcgggtgctc cgccgggtca gggtcgggag ctggtcggat 660 tgctccgagg agatcgcgtt cctggagagc gagatttgcg cgcggctgga cgccggcgac 720 gagaacagca atgacgttct cgtcatgaac agcttaatga cgttcctggt gtactgccgc 780 gtcgtgctgt tcgaccacat tgacgcgagc aagtcgcaac cggcggcggc ggcagcgccg 840 gcgccggcga ggtgcccgga atggatcaga ccggaagcgc tgcagtgccc gatcactctt 900 gacctcatga ccgatccggt gaccgtgtcc accggccaga catacgaccg ggcgtcgatc 960 acccggtgga tgaaggccgg ttgccgcacg tgcccggtca ccggcgagag gctgagcacc 1020 gccgacctcg tccccaacac cgtgctccgc gggatcatcg agcggatgct tctcatcaat 1080 ggcgtcaccc tcccggagct gagcgccgcc ggcggcggtg gccaccgcca cggcgccgtc 1140 gcgaacacgg ccgtgccgtt cggcccggcc gccgcgggcg ccgcgcgcct cgccgtcgcg 1200 cacatcgtgg cgcagctctc gagggggtcg acggaggagc ggaggaaggc gacgtcggag 1260 gcacggaagc tgtcgaagca cagcgtgttc taccgggctt gcctcgtcga cgccaacgcc 1320 gtgccgtggt tgctgtgcct cctctcctcc accgacgccg ccgtgcagga caatgccgtg 1380 gcgtcgctgc tcaacctctc gaagcacccc gccggcagga cggccatcgt ggaggtcggc 1440 ggcgtcggcc tcgtcgtcga cgtgatcaac gtcggcgcca aggccgaggc gcagcacaac 1500 gcggcggccg tcctcttcta cctctcgtcg aacagcccgg actccgccga ggagatcggc 1560 cgcatcccgg aggcgatccc gacgctggtc cagctcatcc gggacggcgc gtaccgcggg 1620 cggaagaacg ccatggtcag cctgtacggg ctgctccaga gcgccgccaa ccacggcagg 1680 gccatcgccg cgggcgccgt gtcagcgctc gccgcgctgc tgctctccgc cgaccgcgac 1740 gacctcgccg gggacagcgt cgcgctgctg gcgaggatcg ccgagcagcc gtctggcgcc 1800 gcggccgtcc tctcgcagcc ggggctcgtc gcccgcctcg ccgaggcgct cgccgcgtcg 1860 tcggcgtcgt cgtcccggtc ggcgagagac cacagcgtgt cgctcctcgc ctcgctgtgc 1920 cggcacggcg gcgccaaggt ggtcgccgtg ctggggcgga tgccggggct gatggcgtcg 1980 ctctactcgc tcgtcgccga cggcggcagc ccgcagacga gcaagaaggc gcgggcgctg 2040 ctcaacgaga tccaccggca ttacgaggtg gcgccgccgc cgccggcgtc gtcggcgtcg 2100 tcggacgccg gcggtgaccg tgtcgttcgc gtgctatag 2139 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> OsUBQ10 RT FW <400> 5 atgcagatct ttgtgaagac 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> OsUBQ10 RT RV <400> 6 ttactgacca ccacggaggc 20 <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> OsCSR1 RT FW <400> 7 tatagtacac gcgcgagctt tgt 23 <210> 8 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> OsCSR1 RT RV <400> 8 gcagagttgc aaaccacttt agt 23 <210> 9 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> OsCSR2 RT FW <400> 9 tcgcacatac tctgttcctc ttt 23 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> OsCSR2 RT RV <400> 10 caaacagcgt cacaaacagc aa 22 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> OsRab16b RT FW <400> 11 acaagggcaa caaccaccag 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> OsRab16b RT RV <400> 12 gcttgcaatg gcatcacaag 20 <210> 13 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> OsDREB1A RT SE <400> 13 aggccgtcga ggacttctt 19 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> OsDREB1A RT AS <400> 14 tagtagctcc agagtggagt 20

Claims (8)

식물체의 저온 스트레스에 대한 저항성 증진용 조성물로서,
서열번호 3으로 표시되는 아미노산 서열을 포함하는 OsCSR2(Oryza sativa Cold Stress Resistant 2) 단백질을 코딩하는 유전자 서열을 포함하는, 식물체의 저온 스트레스에 대한 저항성 증진용 조성물.
As a composition for enhancing the resistance of plants to cold stress,
A composition for enhancing resistance to cold stress of a plant, comprising a gene sequence encoding an OsCSR2 (Oryza sativa Cold Stress Resistant 2) protein comprising the amino acid sequence represented by SEQ ID NO: 3.
제1항에 있어서,
상기 OsCSR2 단백질을 코딩하는 유전자 서열은 서열번호 4로 표시되는 뉴클레오티드 서열을 포함하는 것을 특징으로 하는, 식물체의 저온 스트레스에 대한 저항성 증진용 조성물.
According to claim 1,
Gene sequence for encoding the OsCSR2 protein is characterized in that it comprises a nucleotide sequence represented by SEQ ID NO: 4, the composition for promoting resistance to cold stress of plants.
제1항에 있어서,
상기 식물체는 단자엽 식물인 것을 특징으로 하는, 식물체의 저온 스트레스에 대한 저항성 증진용 조성물.
According to claim 1,
The plant is characterized in that the monocotyledonous plant, the composition for enhancing the resistance to low-temperature stress of the plant.
제1항에 있어서,
상기 식물체는 벼인 것을 특징으로 하는, 식물체의 저온 스트레스에 대한 저항성 증진용 조성물.
According to claim 1,
The plant is characterized in that the rice, the composition for promoting resistance to cold stress of the plant.
식물체의 저온 스트레스에 대한 저항성 증진용 조성물로서,
서열번호 3으로 표시되는 아미노산 서열을 포함하는 OsCSR2(Oryza sativa Cold Stress Resistant 2) 단백질을 코딩하는 뉴클레오티드 서열; 상기 뉴클레오티드 서열에 작동 가능하게 결합되어 있고 식물 세포에서 RNA 분자를 형성시키는 프로모터; 및 식물세포에서 작용하여 RNA 분자의 3'-말단의 폴리아데닐화를 야기시키는 폴리 A 시그널 서열을 포함하는 식물발현용 재조합 벡터를 포함하는, 식물체의 저온 스트레스에 대한 저항성 증진용 조성물.
As a composition for enhancing the resistance of plants to cold stress,
A nucleotide sequence encoding an OsCSR2 (Oryza sativa Cold Stress Resistant 2) protein comprising the amino acid sequence represented by SEQ ID NO: 3; A promoter operably linked to the nucleotide sequence and forming an RNA molecule in a plant cell; And a recombinant vector for plant expression comprising a poly A signal sequence that acts on a plant cell and causes 3'-terminal polyadenylation of an RNA molecule, a composition for enhancing resistance to low temperature stress of a plant.
제1항 내지 제5항 중 어느 한 항의 조성물로 형질전환된 저온 스트레스 저항성이 증진된 식물 세포.
A plant cell with improved low temperature stress resistance transformed with the composition of any one of claims 1 to 5.
제1항 내지 제5항 중 어느 한 항의 조성물로 형질전환된 저온 스트레스 저항성이 증진된 식물체.
A plant having improved low temperature stress resistance transformed with the composition of any one of claims 1 to 5.
제1항 내지 제5항 중 어느 한 항의 조성물을 식물 세포에 도입시키는 단계를 포함하는 식물체의 저온 스트레스 저항성 증진 방법.A method for enhancing low temperature stress resistance of a plant, comprising introducing the composition of any one of claims 1 to 5 into plant cells.
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