KR20230062281A - Dwarfism chrysanthemum overexpressing DgGA20 oxidase 1 gene and manufacturing method thereof - Google Patents
Dwarfism chrysanthemum overexpressing DgGA20 oxidase 1 gene and manufacturing method thereof Download PDFInfo
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Abstract
Description
본 발명은 분화용 국화로부터 분리한 DgGA20 oxidase 1 유전자를 과발현시켜 왜성이 유도된 국화 식물체 및 이의 제조방법에 관한 것이다.The present invention relates to a chrysanthemum plant in which dwarfism is induced by overexpressing the
국화(Chrysanthemum morifolium)는 세계 및 국내 3대 절화작물 중 하나이며, 장미에 이어 두 번째로 비중이 큰 작물이다. 현재까지 국화의 품종 육성은 전통적 교배 육종을 통해 주로 이루어져 왔으며, 이를 통하여 다양한 품종들이 개발되고 있다. 그러나 국화는 교배 육종에 있어서 유전자원의 한계, 배수성, 교배 등의 어려움으로 인하여 생명공학기법을 이용한 신품종 개발의 필요성이 증대되고 있다. 최근에는 분자 유전학적 형질전환 방법을 이용하여 유용 유전자가 도입된 형질전환체를 개발하는 방법이 대안으로 제시되고 있다. 그러나 아직 상대적으로 낮은 형질전환율은 국화에서 대단위의 유전자 기능분석을 통한 분자육종법의 개발을 어렵게 하고 있다. Chrysanthemum morifolium is one of the three major cut flower crops in the world and Korea, and is the second largest crop after roses. Until now, breeding of chrysanthemum has been mainly done through traditional cross-breeding, and various varieties have been developed through this. However, due to the limitations of genetic resources, ploidy, and difficulties in crossbreeding, the need for developing new varieties using biotechnology is increasing in chrysanthemum breeding. Recently, a method of developing a transformant into which a useful gene is introduced using a molecular genetic transformation method has been proposed as an alternative. However, the relatively low transformation rate still makes it difficult to develop molecular breeding methods through large-scale gene function analysis in chrysanthemum.
한편, 왜성(矮性; dwarfism)은 생물의 크기가 그 종(種)의 표준 크기에 비하여 작게 자라는 형질, 또는 그런 형질을 가진 품종을 의미한다고 할 수 있다. 이러한 왜성 형질은 농업 및 원예업의 다양한 관점에서 중요하다. 예컨대 식물 높이 또는 줄기 길이를 축소함으로써 새로운 미적 가치를 갖는 관상용 식물을 제조할 수 있고, 채소 또는 과일을 소형화함으로써 먹기에 적당한 크기(bite-sized)를 갖는 등의 새로운 상업적 가치를 갖는 농작물을 제조할 수 있다.On the other hand, dwarfism can be said to mean a trait in which the size of an organism grows smaller than the standard size of the species, or a breed with such a trait. These dwarf traits are important in various aspects of agriculture and horticulture. For example, ornamental plants with new aesthetic value can be produced by reducing plant height or stem length, and crops with new commercial value such as bite-sized vegetables or fruits can be produced by miniaturizing vegetables or fruits. can
이러한 왜성 품종은 단기간에 전통육종으로 쉽게 확보되기 어려운 상황으로 생장조절에 관여하는 유전자를 이용한 GM 화훼 개발이 외국에서는 활발히 이루어지고 있다. 이 중에서 애기장대 gai-1 유전자 이용 왜화 페튜니아 를 개발하거나 Agrobacterium rhizogenes rol 유전자 이용 왜화 칼란코에를 개발하였고 애기장대 유래 SHI 유전 자의 과발현을 통하여 개화시기, 잎모양 등 다른 화훼특징을 보유하면서 크기가 작아진 왜화 칼란코에를 생산하였으며 포플러에서 애기장대 유래 SHI 유전자를 과발현시켜 작아지는 왜성 특성을 유도하였다.Such dwarf varieties are difficult to obtain through traditional breeding in a short period of time, so the development of GM flowers using genes involved in growth control is being actively carried out in foreign countries. Among them, dwarfed petunia was developed using the Arabidopsis gai - 1 gene or dwarfed Kalanchoe was developed using the Agrobacterium rhizogenes rol gene. Gene dwarf kalanchoe was produced, and the Arabidopsis-derived SHI gene was overexpressed in poplar to induce dwarf characteristics.
이외에도 다양한 작물에서 왜성을 유도할 수 있는 크기 관련 유전자들을 발굴하고 조절하여 이용하는 방법에 대한 연구가 진행 중에 있으나, 국화로부터 분리한 지베렐린(GA20) 산화효소 유전자를 포함하는 재조합 벡터로 형질전환되어 왜성이 유도된 국화과 식물체에 대해서는 보고된 내용이 없다.In addition, research on methods for discovering, regulating, and using size-related genes capable of inducing dwarfism in various crops is in progress. There are no reports on induced Asteraceae plants.
본 발명이 해결하고자 하는 과제는 국화 유래 DgGA20 oxidase 1 유전자의 발현이 증가된, 왜성(dwarfism)이 유도된 형질전환 국화 및 이의 제조방법을 제공하는 것이다.An object to be solved by the present invention is to provide a transgenic chrysanthemum in which the expression of the chrysanthemum-derived
본 발명은 서열번호 2로 표시되는 DgGA20 oxidase 1 단백질을 암호화하는 서열번호 1의 유전자의 발현이 증가된, 왜성(dwarfism)이 유도된 형질전환 국화이다.The present invention is a transgenic chrysanthemum in which dwarfism is induced, in which the expression of the gene of SEQ ID NO: 1 encoding the
상기 왜성은 비형질전환 국화에 비해 초장(plant height)이 20-60% 감소된 것일 수 있다.The dwarf may have a 20-60% reduction in plant height compared to non-transformed chrysanthemums.
상기 국화(Chrysanthemum morifolium)는 블랙마블, 보라미, 체리블로섬, 포리스트아로마, 골든페스티벌, 가마, 가든파티, 화월, 신마, 문페스티벌, 무지개, 문라이트, 오렌지메모리, 오렌지마블, 퓨어앤젤, 프리마돈나, 프레디져드아모르, 피크, 핑크프라이드, 핑크팡팡, 핑크바이오렛, 스위트카펫, 설악, 스위트핑크,서니팡팡, 비비드스칼렛, 화이트팡팡, 화이트윙, 에스데이, 엘로우엘레강스, 예스해피, 예스모닝, 예스나우, 예스팡팡, 예스스타, 예로우 팡팡, 예스스타, 에스스완, 예스 투게더, 예스타임, 399, 피스핑크, 피스킹, 피스코퍼, 피스옐로우, 피스퀸, 피스로망스,피스앤젤로 이루어진 군에서 선택된 1종 이상의 품종일 수 있다.The chrysanthemum ( Chrysanthemum morifolium ) is Black Marble, Borami, Cherry Blossom, Forest Aroma, Golden Festival, Gama, Garden Party, Flower Moon, Shinma, Moon Festival, Rainbow, Moonlight, Orange Memory, Orange Marble, Pure Angel, Prima Donna, Freddy Judd Amour, Peak, Pink Pride, Pink Pang Pang, Pink Violet, Sweet Carpet, Seorak, Sweet Pink, Sunny Pang Pang, Vivid Scarlet, White Pang Pang, White Wing, S-Day, Yellow Elegance, Yes Happy, Yes Morning, Yes Now, 1 selected from the group consisting of Yes Pang Pang, Yes Star, Yes Pang Pang, Yes Star, S Swan, Yes Together, Yes Time, 399, Peace Pink, Peace King, Peace Copper, Peace Yellow, Peace Queen, Peace Romance, and Peace Angel There may be more than one variety.
또한, 본 발명은 서열번호 2로 표시되는 DgGA20 oxidase 1 단백질을 암호화하는 서열번호 1의 유전자를 과발현하는 재조합 벡터를 제작하는 단계; 상기 벡터를 아그로박테리움을 이용하여 국화 품종에 형질전환하는 단계; 형질전환 개체를 선발하는 단계를 포함하는, 초장이 20-60% 감소된 형질전환 국화의 제조방법이다.In addition, the present invention comprises the steps of preparing a recombinant vector overexpressing the gene of SEQ ID NO: 1 encoding the
또한, 본 발명은 상기 제조방법에 의해 제조된 형질전환 국화의 형질전환 종자이다.In addition, the present invention is a transgenic seed of a transgenic chrysanthemum prepared by the above production method.
또한, 서열번호 2로 표시되는 DgGA20 oxidase 1 단백질을 암호화하는 서열번호 2의 유전자를 과발현하는 재조합 벡터를 제작하는 단계; 상기 벡터를 아그로박테리움을 이용하여 국화 품종에 형질전환하는 단계; 및 형질전환 개체를 선발하는 단계를 포함하는, 국화의 초장을 20-60% 감소시키는 방법이다.In addition, constructing a recombinant vector overexpressing the gene of SEQ ID NO: 2 encoding the
또한, 본 발명은 서열번호 2로 표시되는 DgGA20 oxidase 1 단백질을 암호화하는 서열번호 1의 유전자의 발현이 증가된, 국화의 초장을 20-60% 감소시키기 위한 조성물이다.In addition, the present invention is a composition for reducing the plant height of chrysanthemum by 20-60%, in which the expression of the gene of SEQ ID NO: 1 encoding the
본 발명에 따른 DgGA20 oxidase 1 단백질 또는 이를 암호화하는 DgGA20 oxidase 1 유전자의 발현을 증가시킴으로써 왜성이 감소된 국화 식물체를 제조할 수 있으며, 이를 이용하여 화훼 및 원예 분야에서 다양한 크기 및 모양을 갖는 기능성 작물 개발 용도로 활용할 수 있다. 또한, 화단 소형 국화를 만드는데 적심이나 왜화제를 사용하지 않아 생장조절제로 인한 발암 및 토양오염을 방지할 수 있다.By increasing the expression of the
도 1은 DgGA20 oxidase 1 유전자가 도입된 과발현용 벡터의 구성을 나타낸 것이다.
도 2는 DgGA20 oxidase 1 유전자가 도입된 과발현용 벡터로 형질전환된 형질전환 국화의 표현형을 나타낸 것이다.
도 3은 DgGA20 oxidase 1 유전자가 과발현된 형질전환 개체 OX-12에서의 GA 유전자 발현량 변화를 나타낸 것이다.
도 4는 지베렐린산(GA) 경로에서의 피드백 조절(feedback regulation) 참고도를 나타낸 것이다.Figure 1 shows the construction of a vector for overexpression into which the
Figure 2 shows the phenotype of transgenic chrysanthemums transformed with the overexpression vector into which the
Figure 3 shows the change in GA gene expression level in the transgenic object OX-12 in which the
Figure 4 shows a reference diagram of feedback regulation in the gibberellic acid (GA) pathway.
달리 정의되지 않는 한, 본 명세서에서 사용된 모든 기술적 및 과학적 용어들은 본 발명이 속하는 기술 분야에서 숙련된 전문가에 의해서 통상적으로 이해되는 것과 동일한 의미를 갖는다. 일반적으로, 본 명세서에서 사용된 명명법 및 이하에 기술하는 실험 방법은 본 기술 분야에서 잘 알려져 있고 통상적으로 사용되는 것이다.Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein and the experimental methods described below are those well known and commonly used in the art.
본 발명에서 개시된 각각의 설명 및 실시형태는 각각의 다른 설명 및 실시 형태에도 적용될 수 있다. 즉, 본 발명에서 개시된 다양한 요소들의 모든 조합이 본 발명의 범주에 속한다. 또한, 하기 기술된 구체적인 서술에 의하여 본 발명의 범주가 제한된다고 볼 수 없다.Each description and embodiment disclosed herein can be applied to each other description and embodiment. That is, all combinations of the various elements disclosed herein fall within the scope of the present invention. In addition, it cannot be seen that the scope of the present invention is limited by the specific descriptions described below.
본 발명은 일 관점에서, 서열번호 2로 표시되는 DgGA20 oxidase 1 단백질을 암호화하는 서열번호 1의 유전자의 발현이 증가된, 왜성(dwarfism)이 유도된 형질전환 국화에 관한 것이다.In one aspect, the present invention relates to a transgenic chrysanthemum in which dwarfism is induced, in which the expression of the gene of SEQ ID NO: 1 encoding the
본 발명에서 DgGA20 oxidase 1 의 구체적인 단백질의 아미노산 서열 또는 염기서열 정보는 NCBI에 공지되어 있다. 구체적으로 DgGA20 oxidase 1 단백질은 서열번호 2의 아미노산 서열로 구성될 수 있으나, 이에 제한되는 것은 아니다. In the present invention, information on the amino acid sequence or base sequence of the specific protein of
또한, 본 발명의 DgGA20 oxidase 1 단백질은 상기 서열번호 2와 적어도 50%, 80%, 90%, 95%, 96%, 97%, 98%, 또는 99% 상동성을 가지는 폴리펩티드를 포함할 수 있으나 이에 제한되지 않는다. 또한, 이러한 상동성을 가지며 상기 단백질에 상응하는 효능을 나타내는 아미노산 서열이라면, 일부 서열이 결실, 변형, 치환 또는 부가된 아미노산 서열을 갖는 보조 단백질도 본 발명에서 사용될 수 있음은 자명하다.In addition, the
또한, 코돈 축퇴성(codon degeneracy)에 의해 상기 서열번호 2로 구성된 아미노산 서열을 가지는 단백질 또는 이와 상동성을 가지는 단백질로 번역될 수 있는 폴리뉴클레오티드 역시 포함될 수 있음은 자명하다. 또는 공지의 유전자 서열로부터 조제될 수 있는 프로브, 예를 들면, 상기 염기 서열의 전체 또는 일부에 대한 상보서열과 엄격한 조건 하에 하이드리드화하여, 서열번호 2로 구성된 아미노산 서열을 포함하는 단백질과 동일 또는 유사한 활성을 가지는 단백질을 암호화하는 서열이라면 제한 없이 포함될 수 있으며, 바람직하게는 서열번호 1로 구성된 염기서열일 수 있다.In addition, it is obvious that a polynucleotide that can be translated into a protein having the amino acid sequence of SEQ ID NO: 2 or a protein having homology thereto by codon degeneracy may also be included. Or a probe that can be prepared from a known gene sequence, for example, a protein that is identical to or Any sequence that encodes a protein having a similar activity may be included without limitation, and preferably may be a nucleotide sequence composed of SEQ ID NO: 1.
상기 "상동성"은 두 개의 폴리뉴클레오티드 또는 폴리펩타이드 모이어티(moiety) 사이의 동일성의 퍼센트를 말한다. 주어진 아미노산 서열 또는 염기 서열과 일치하는 정도를 의미하며 백분율로 표시될 수 있다. 본 명세서 에서, 주어진 아미노산 서열 또는 염기 서열과 동일하거나 유사한 활성을 가지는 그의 상동성 서열이 "% 상동성"으로 표시된다. 하나의 모이어티로부터 다른 하나의 모이어티까지의 서열 간 상동성은 알려진 당해 기술에 의해 결정될 수 있다. 예를 들면, 점수(score), 동일성(identity) 및 유사도(similarity) 등의 매개 변수 (parameter)들을 계산하는 표준 소프트웨어, 구체적으로 BLAST 2.0을 이용하거나, 정의된 엄격한 조건하에서 써던 혼성화 실험에 의해 서열을 비교함으로써 확인할 수 있으며, 정의되는 적절한 혼성화 조건은 해당 기술 범위 내이고, 당업자에게 잘 알려진 방법(예컨대, J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2 nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; F.M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley& Sons, Inc., New York)으로 결정될 수 있다.The "homology" refers to the percent identity between two polynucleotide or polypeptide moieties. It means the degree of matching with a given amino acid sequence or base sequence and can be expressed as a percentage. In this specification, a homologous sequence having the same or similar activity as a given amino acid sequence or nucleotide sequence is expressed as "% homology". Homology between sequences from one moiety to another can be determined by known art techniques. For example, by using standard software, specifically BLAST 2.0, which calculates parameters such as score, identity and similarity, or by Southern hybridization experiments under defined stringent conditions, sequence It can be confirmed by comparing, and appropriate hybridization conditions defined are within the scope of the art and methods well known to those skilled in the art (e.g., J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press , Cold Spring Harbor, New York, 1989; F.M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York).
또한, DgGA20 oxidase 1 유전자는 서열번호 1의 염기서열 또는 서열번호 1과 적어도 50%, 80%, 90%, 95%, 96%, 97%, 98%, 또는 99% 상동성을 가지는 폴리뉴클레오티드를 포함할 수 있으나 이에 제한되지 않는다. 또한, 본 발명의 왜성이 유도된 형질전환 식물은 DgGA20 oxidase 1 단백질의 상동단백질 또는 이를 암호화하는 유전자의 발현이 증가된 것일 수 있다. In addition , the
상기"상동단백질"이란 같은 단백질에서 파생된 여러 단백질들을 의미하는 것으로서, 아미노산 배열의 유사성과 3차원 구조의 유사성을 통해 상동 단백질인지 알 수 있다. 본 발명의 상기 단백질들은 아미노산 배열이 유사하고, 3차원 구조가 유사하며, 식물의 초장을 감소시키는 기능을 갖는 외부 N-말단 모티프의 서열이 유사한 바, 이들은 상동 단백질임이 확인되었다.The "homologous protein" refers to several proteins derived from the same protein, and it can be determined whether the protein is a homologous protein through similarity in amino acid sequence and similarity in three-dimensional structure. The proteins of the present invention have similar amino acid sequences, similar three-dimensional structures, and similar sequences of external N-terminal motifs that have a function of reducing plant plant height, and thus it was confirmed that they are homologous proteins.
본 발명에서는 구체적으로 국화 분화국 피스코퍼로부터 분리된 것일 수 있으나, 이에 제한되는 것은 아니다. In the present invention, it may be specifically separated from chrysanthemum chrysanthemum piscoper, but is not limited thereto.
본 발명의 용어 "발현 증가"란, DgGA20 oxidase 1 단백질 또는 이를 암호화하는 유전자가 도입되어 발현되거나, 식물체가 가진 내재적 발현 또는 변형 전 발현에 비하여 발현이 증가된 것을 의미한다. 상기 단백질의 "도입"은, 식물체가 본래 가지고 있지 않았던 특정 단백질의 활성이 자연적 혹은 인위적으로 나타나게 되는 것을 의미한다. 예를 들어, 상기 발현 증가는 외래의 DgGA20 oxidase 1 단백질 또는 이를 암호화하는 유전자를 도입하여 발현을 증가시키는 것; 또는 내재적 DgGA20 oxidase 1 단백질 또는 이를 암호화하는 유전자의 발현을 증가시키는 것을 모두 포함할 수 있다. The term "increased expression" of the present invention means that the
구체적으로, 1)상기 DgGA20 oxidase 1 단백질을 암호화하는 폴리뉴클레오티드의 카피수 증가, 2)상기 폴리뉴클레오티드의 발현이 증가하도록 발현조절 서열의 변형, 3)상기 단백질의 발현이 증가되도록 염색체 상의 폴리뉴클레오티드 서열의 변형, 또는 4)이의 조합에 의해 발현이 증가되도록 변형하는 방법 등에 의하여 수행될 수 있으나, 이에 제한되지 않는다. Specifically, 1) increase in copy number of the polynucleotide encoding the
상기 1)폴리뉴클레오티드의 카피수 증가는, 특별히 이에 제한되지 않으나, 벡터에 작동 가능하게 연결된 형태로 수행되거나, 대상 식물체의 염색체 내로 삽입됨으로써 수행될 수 있다. 또한 카피수 증가의 한 양태로, 단백질의 활성을 나타낼 수 있는 폴리뉴클레오티드를 대상 식물체 내로 도입하여 수행될 수 있다. 상기 도입은 공지된 형질전환 방법을 당업자가 적절히 선택하여 수행될 수 있으며, 대상 식물체 내에서 상기 도입된 폴리뉴클레오티드가 발현됨으로써 단백질의 발현이 증가될 수 있다. 1) The copy number increase of the polynucleotide is not particularly limited thereto, but may be performed in a form operably linked to a vector or inserted into the chromosome of a target plant. In addition, as one aspect of copy number increase, it may be performed by introducing a polynucleotide capable of exhibiting protein activity into a target plant. The introduction can be performed by appropriately selecting a known transformation method by a person skilled in the art, and expression of the protein can be increased by expressing the introduced polynucleotide in the target plant.
다음으로, 2)폴리뉴클레오티드의 발현이 증가하도록 하는 발현조절 서열의 변형시킬 수 있다. 상기 발현조절 서열은, 특별히 이에 제한되지 않으나 프로모터, 오퍼레이터 서열, 리보좀 결합 부위를 암호화하는 서열, 전사 및 해독의 종결을 조절하는 서열 등을 포함할 수 있다. 구체적으로, 폴리뉴클레오티드 발현 단위의 상부에는 본래의 프로모터 대신 강력한 이종 프로모터가 연결될 수 있다. 본 발명에서는 상기 프로모터로서 35S 프로모터를 사용할 수 있지만, 이에 제한되지 않는다. Next, 2) the expression control sequence to increase the expression of the polynucleotide can be modified. The expression control sequence may include, but is not particularly limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, a sequence that regulates termination of transcription and translation, and the like. Specifically, a strong heterologous promoter may be ligated to the top of the polynucleotide expression unit instead of the original promoter. In the present invention, the 35S promoter may be used as the promoter, but is not limited thereto.
아울러, 3)염색체 상의 폴리뉴클레오티드 서열의 변형은, 특별히 이에 제한되지 않으나, 발현조절 서열상의 변이를 유도하여 수행하거나, 더욱 강한 활성을 갖도록 개량된 폴리뉴클레오티드 서열로 교체함에 의하여 수행될 수 있다. In addition, 3) modification of the polynucleotide sequence on the chromosome is not particularly limited thereto, but may be performed by inducing a mutation on the expression control sequence or by replacing it with an improved polynucleotide sequence to have a stronger activity.
마지막으로, 4)상기 1) 내지 3)의 조합에 의해 발현이 증가되도록 변형하는 방법은, 상기 단백질을 암호화하는 폴리뉴클레오티드의 카피수 증가, 이의 발현이 증가하도록 발현조절 서열의 변형, 염색체 상의 상기 폴리뉴클레 오티드 서열의 변형 중 하나 이상의 방법을 함께 적용하여 수행될 수 있다. 상기 폴리뉴클레오티드는 기능을 할 수 있는 폴리뉴클레오티드 집합체인 경우 유전자로 기재될 수 있다. 본 발명에서 폴리뉴클레오티드와 유전자는 혼용될 수 있으며, 폴리뉴클레오티드 서열과 뉴클레오티드 서열은 혼용될 수 있다. Finally, 4) a method for modifying to increase expression by a combination of 1) to 3) above is to increase the copy number of the polynucleotide encoding the protein, modify the expression control sequence to increase its expression, Modification of the polynucleotide sequence may be performed by applying one or more methods together. The polynucleotide may be described as a gene if it is a set of polynucleotides capable of functioning. In the present invention, polynucleotides and genes may be used interchangeably, and polynucleotide sequences and nucleotide sequences may be used interchangeably.
본 발명은 DgGA20 oxidase 1 유전자를 도입하여 DgGA20 oxidase 1 단백질 또는 이를 암호화하는 유전자의 발현을 증가시킴으로써 왜성이 유도된 형질전환 국화를 제조하였다. 또한, 상기 DgGA20 oxidase 1 유전자는 프로모터에 작동가능하게 연결된 상태로 도입될 수 있지만 이에 제한되는 것은 아니다. In the present invention, dwarfism-induced transgenic chrysanthemums were prepared by introducing the
상기 "프로모터"란, 전사조절인자들이 결합할 수 있는 DNA 염기서열을 의미하는데, 상기 프로모터는 전사조절 인자를 매개로 하여 RNA 중합효소와 결합함으로써, 그의 하류에 위치한 개방해독틀(ORF)의 전사를 유도할 수 있다. 구체적으로 35S 프로모터를 이용할 수 있으나, 이에 제한되는 것은 아니다. The "promoter" refers to a DNA sequence to which transcriptional regulators can bind, and the promoter binds to RNA polymerase via the transcriptional regulator, thereby transcription of the open reading frame (ORF) located downstream thereof can induce Specifically, the 35S promoter may be used, but is not limited thereto.
상기 "작동가능하게 연결(operably linked)"이란, 일반적 기능을 수행하도록 핵산 발현조절 서열과 목적하는 단백질 또는 RNA를 암호화하는 핵산 서열이 기능적으로 연결(functional linkage)되어 있는 상태를 의미다. 예를 들어 프로모터와 단백질 또는 RNA를 암호화하는 핵산 서열이 작동가능하게 연결되어 암호화서열의 발현에 영향을 미칠 수 있다. 발현 벡터와의 작동적 연결은 당해 기술분야에서 잘 알려진 유전자 재조합 기술을 이용하여 제조할 수 있으며, 부위-특이적 DNA 절단 및 연결은 당해 기술 분야에서 일반적으로 알려진 효소 등을 사용할 수 있다.The term "operably linked" means a state in which a nucleic acid expression control sequence and a nucleic acid sequence encoding a protein or RNA of interest are functionally linked to perform a general function. For example, a promoter and a nucleic acid sequence encoding a protein or RNA may be operably linked to affect expression of the coding sequence. Operational linkage with the expression vector can be prepared using genetic recombination techniques well known in the art, and site-specific DNA cleavage and linkage can be performed using enzymes generally known in the art.
상기 "왜성이 유도된"이란, 식물의 생장이 억제되어 초장(plant height)이 감소된 것을 의미한다. 따라서, 초장이 감소된 식물은 식물체의 높이, 줄기의 길이가 감소하고, 이에 의해 잎의 크기 또는 꽃의 크기 등이 감소된 표현형을 나타낼 수 있다. 구체적으로 식물의 길이 생장이 억제된 것일 수 있으나, 이에 제한되는 것은 아니다.The term "dwarfism induced" means that plant growth is inhibited and plant height is reduced. Therefore, a plant with reduced plant height may show a phenotype in which the height of the plant body and the length of the stem are reduced, thereby reducing the size of leaves or flowers. Specifically, the length of the plant may be inhibited, but is not limited thereto.
본 발명의 일실시예에서, DgGA20 oxidase 1 유전자를 과발현시켜 형질전환된 국화를 선발하여 표현형을 검정한 결과, 비형질전환 국화에 비하여 초장이 20-60% 감소된 것을 확인하였다.In one embodiment of the present invention, chrysanthemums transformed by overexpressing the
따라서, 본 발명의 왜성이 유도된 형질전환 국화는 초장이 20-60% 감소된 것일 수 있다.Therefore, the dwarfism-induced transgenic chrysanthemum of the present invention may have a plant height reduced by 20-60%.
상기 국화(Chrysanthemum morifolium)는 블랙마블, 보라미, 체리블로섬, 포리스트아로마, 골든페스티벌, 가마, 가든파티, 화월, 신마, 문페스티벌, 무지개, 문라이트, 오렌지메모리, 오렌지마블, 퓨어앤젤, 프리마돈나, 프레디져드아모르, 피크, 핑크프라이드, 핑크팡팡, 핑크바이오렛, 스위트카펫, 설악, 스위트핑크,서니팡팡, 비비드스칼렛, 화이트팡팡, 화이트윙, 에스데이, 엘로우엘레강스, 예스해피, 예스모닝, 예스나우, 예스팡팡, 예스스타, 예로우 팡팡, 예스스타, 에스스완, 예스 투게더, 예스타임, 399, 피스핑크, 피스킹, 피스코퍼, 피스옐로우, 피스퀸, 피스로망스,피스앤젤로 이루어진 군에서 선택된 1종 이상의 품종일 수 있다.The chrysanthemum ( Chrysanthemum morifolium ) is Black Marble, Borami, Cherry Blossom, Forest Aroma, Golden Festival, Gama, Garden Party, Flower Moon, Shinma, Moon Festival, Rainbow, Moonlight, Orange Memory, Orange Marble, Pure Angel, Prima Donna, Freddy Judd Amour, Peak, Pink Pride, Pink Pang Pang, Pink Violet, Sweet Carpet, Seorak, Sweet Pink, Sunny Pang Pang, Vivid Scarlet, White Pang Pang, White Wing, S-Day, Yellow Elegance, Yes Happy, Yes Morning, Yes Now, 1 selected from the group consisting of Yes Pang Pang, Yes Star, Yes Pang Pang, Yes Star, S Swan, Yes Together, Yes Time, 399, Peace Pink, Peace King, Peace Copper, Peace Yellow, Peace Queen, Peace Romance, and Peace Angel There may be more than one variety.
또한, 본 발명은 서열번호 2로 표시되는 DgGA20 oxidase 1 단백질을 암호화하는 서열번호 1의 유전자를 과발현하는 재조합 벡터를 제작하는 단계; 상기 벡터를 아그로박테리움을 이용하여 국화 품종에 형질전환하는 단계; 형질전환 개체를 선발하는 단계를 포함하는, 초장이 20-60% 감소된 형질전환 국화의 제조방법에 관한 것이다.In addition, the present invention comprises the steps of preparing a recombinant vector overexpressing the gene of SEQ ID NO: 1 encoding the
상기 "재조합"은 세포가 이종의 핵산을 복제하거나, 상기 핵산을 발현하거나 또는 펩티드, 이종의 펩티드 또는 이종의 핵산에 의해 암호된 단백질을 발현하는 세포를 지칭하는 것이다. 재조합 세포는 상기 세포의 천연 형태에서는 발견되지 않는 유전자 또는 유전자 절편을, 과발현(센스) 또는 저발현(안티센스) 형태 중 하나로 발현할 수 있다. 또한 재조합 세포는 천연 상태의 세포에서 발견되는 유전자를 발현할 수 있으며, 상기 유전자는 변형된 것으로서 인위적인 수단에 의해 세포 내 재도입된 것이다. 본 발명에서 DgGA20 oxidase 1 유전자 서열을 재조합 벡터 내로 삽입할 수 있다.The "recombinant" refers to a cell that replicates a heterologous nucleic acid, expresses the nucleic acid or expresses a peptide, a protein encoded by a heterologous peptide or a heterologous nucleic acid. A recombinant cell may express a gene or gene segment not found in the cell's natural form, either in an overexpressed (sense) or underexpressed (antisense) form. In addition, recombinant cells can express genes found in cells in their natural state, and the genes have been reintroduced into cells by artificial means as modified ones. In the present invention, the
"재조합 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."Recombinant vector" means a bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus, or other vector. In general, any plasmid and vector may be used provided that it is capable of replicating and stabilizing in the host.
본 발명의 DgGA20 oxidase 1 유전자 서열 및 적당한 전사/번역 조절 신호를 포함하는 발현 벡터는 당업자에 주지된 방법에 의해 구축될 수 있다. 상기 방법은 시험관 내 재조합 DNA 기술, DNA 합성 기술 및 생체 내 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.An expression vector containing the
본 발명의 발현 벡터는 바람직하게는 하나 이상의 선택성 마커를 포함할 수 있다. 상기 마커는 통상적으로 화학적인 방법으로 선택할 수 있는 특성을 갖는 핵산 서열로, 형질전환된 세포를 비형질전환 세포로부터 구별할 수 있는 모든 유전자가 이에 해당될 수 있다. 그 예로는 글리포세이트(glyphosate) 또는 포스피노트리신(phosphinothricin)과 같은 제초제 저항성 유전자, 카나마이신(kanamycin), G418, 블레오마이신(Bleomycin), 하이그로마이신(hygromycin), 클로람페니콜(chloramphenicol)과 같은 항생제 내성 유전자, aadA 유전자 등이 있으나, 이에 한정되는 것은 아니다.The expression vector of the present invention may preferably contain one or more selectable markers. The marker is a nucleic acid sequence having a characteristic selectable by a conventional chemical method, and may be any gene capable of distinguishing transformed cells from non-transformed cells. Examples include herbicide resistance genes such as glyphosate or phosphinothricin, antibiotics such as kanamycin, G418, bleomycin, hygromycin, and chloramphenicol. Resistance genes, aadA genes, etc., but are not limited thereto.
상기 “형질전환(transformation)”이란 외부로부터 주어진 유전물질인 DNA에 의해 개체 또는 세포의 형질이 유전적으로 변화하는 것을 의미하는 것으로, 본 발명의 재조합 벡터로 형질전환하는 것은 당업자에게 공지된 형질전환기술에 의해 수행될 수 있다. 예를 들어, 미세사출법(microprojectile bombardment), 입자 총 충격법(particle gun bombardment), 실리콘 탄화물 위스커(Silicon carbide whiskers), 초음파 처리(sonication), 일렉트로포레이션(electroporation), PEG-매개 융합법(PEG-mediated fusion), 미세주입법(microinjection), 리포좀 매개법(liposome-mediated method), 인-플란타 형질전환법(In planta transformation), 진공 침윤법(Vacuum infiltration method), 화아침지법(floral meristem dipping method), 또는 아그로박테리움(Agrobacterium sp.) 매개에 의한 방법 등을 사용할 수 있으나, 이에 한정되는 것은 아니다.The term "transformation" means genetic change in the character of an individual or cell by DNA, which is a genetic material given from the outside, and transformation with the recombinant vector of the present invention is a transformation technique known to those skilled in the art. can be performed by For example, microprojectile bombardment, particle gun bombardment, silicon carbide whiskers, sonication, electroporation, PEG-mediated fusion ( PEG-mediated fusion), microinjection, liposome-mediated method, in-planta transformation, vacuum infiltration method, floral meristem dipping method), or a method by Agrobacterium sp. mediation may be used, but is not limited thereto.
또한, 본 발명은 상기 제조방법에 의해 제조된 형질전환 국화의 형질전환 종자에 관한 것이다. In addition, the present invention relates to a transgenic seed of a transgenic chrysanthemum prepared by the above production method.
상기 형질전환 종자는 초장이 감소된 형질전환 국화의 종자로 전술한 바와 동일하다.The transgenic seeds are seeds of transgenic chrysanthemums with reduced plant height, and are the same as described above.
또한, 본 발명은 서열번호 2로 표시되는 DgGA20 oxidase 1 단백질을 암호화하는 서열번호 1의 유전자를 과발현하는 재조합 벡터를 제작하는 단계; 상기 벡터를 아그로박테리움을 이용하여 국화 품종에 형질전환하는 단계; 형질전환 개체를 선발하는 단계를 포함하는, 국화의 초장을 20-60% 감소시키는 방법에 관한 것이다.In addition, the present invention comprises the steps of preparing a recombinant vector overexpressing the gene of SEQ ID NO: 1 encoding the
상기 DgGA20 oxidase 1 단백질, 과발현 재조합 벡터, 초장이 감소된 형질전환 국화 등은 전술한 바와 동일하다.The
또한, 본 발명은 서열번호 2로 표시되는 DgGA20 oxidase 1 단백질을 암호화하는 서열번호 1의 유전자의 발현이 증가된, 국화의 초장을 20-60% 감소시키기 위한 조성물에 관한 것이다.In addition, the present invention relates to a composition for reducing the plant height of chrysanthemum by 20-60%, in which the expression of the gene of SEQ ID NO: 1 encoding the
상기 조성물은 유효성분으로 국화 유래 DgGA20 oxidase 1 단백질을 암호화하는 유전자를 포함하며, 상기 유전자를 과발현시켜 식물체에 형질전환함으로써 초장을 감소시킬 수 있다.The composition contains a gene encoding the chrysanthemum-derived
이하, 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있도록 본 발명의 실시예에 대하여 첨부한 도면을 참고로 하여 상세히 설명한다. 그러나 본 발명은 여러 가지 상이한 형태로 구현될 수 있으며 여기에서 설명하는 실시예에 한정되지 않는다.Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings so that those skilled in the art can easily carry out the present invention. However, the present invention may be embodied in many different forms and is not limited to the embodiments described herein.
<실시예 1> 국화 형질전환용 벡터 제작<Example 1> Construction of vector for chrysanthemum transformation
국화 분화국 피스코퍼 품종에서 DgGA20 oxidase 1 유전자(1445 bp)를 분리하였다. The
하기의 표 1에 DgGA20 oxidase 1 유전자의 염기서열(서열번호 1) 및 아미노산 서열(서열번호 2)을 표기하였다.Table 1 below shows the base sequence (SEQ ID NO: 1) and amino acid sequence (SEQ ID NO: 2) of the
(서열번호1)
(SEQ ID NO: 1)
cccttaaaga ggatcaacga aaatcattcg ttttcgatgc atccgtgcta aaacatgaaa 120
ccaatatacc ccaacagttc atatggcccg atcatgagaa acctaactcc caaaaatcaa 180
aggaacttga ggttcctttg atagacctag gaggctttct ttctggacga tcgagttcaa 240
ctaaggaagc ctccagtctt gtgggcgagg cttgtcaaaa acatggtttt ttcctagtcg 300
tcaaccatgg agttgatgca aacctaatat ccgatgccca acgatacatg gatttgttct 360
ttgagctccc gctttctgta aagcaaagag ctcaaagaaa agttggtgaa agttgtggct 420
atgctagtag tttcactgga cggttttcct ctaagctgcc ttggaaagag acactctctt 480
tccgattttc agctgaggag aacgcatcca acatagttaa ggactacttt gagaacacaa 540
tgggagaaga gtttattcga cttgggaagg tttaccaaga atattgcaat gccatgagta 600
gactctctct tgggatcatg gagttactag ggatgagcct tggtgtgagc cgagcttact 660
acaaagaatt tttcgaagaa cataattcaa taatgaggct taactactat ccaccatgtc 720
aaaaaccaga ccttacctta ggcacgggtc ctcactgtga tccaacatca ttgacaatcc 780
tacatcaaga taacgtaggt ggactagaag tctttgtgga caatgagtgg cgtttaatcg 840
tacccaattc aaatgctttt gttgtaaaca ttggtgacac attcatggca ctttcaaatg 900
gaagatacaa aagttgcttg catagggctg tggtaaacag caaaactact agaaagtcac 960
ttgctttctt cttatgtcca aagaaggata aggtagtgat cccaccaaaa gaattggtgg 1020
acgaaaataa ccctagaata tatccagatt ttacatggtc tacctttctt gagttcactc 1080
agaagcatta cagggctgac aagaataccc ttcaagcttt ctcaacctgg gttcgacaga 1140
aaaccacttg atgtcgaacc aatttgacat gcatgcaaaa agacaattag aactgtaaga 1200
tgaacaccaa agaggatgca tgaagagtta tgcatgcttt gctttcaaag ctttcacctt 1260
tatgtttctt tagggcacac aaatgaagga agaaaacaat cttaaggacc ctagataggc 1320
cagattttaa gtaatcttta tgggcgctaa actagtagga ataaagagaa gcataatata 1380
atgagccagt tttgtaattt gttttggact actatgtaga aagcttatgc agttataatc 1440
actagctagtgattt ttgtgcaatg gctatagatt gcatgatcaa gactacatca aacatgccat 60
cccttaaaga ggatcaacga aaatcattcg ttttcgatgc atccgtgcta aaacatgaaa 120
ccaatatacc ccaacagttc atatggcccg atcatgagaa acctaactcc caaaaatcaa 180
aggaacttga ggttcctttg atagacctag gaggctttct ttctggacga tcgagttcaa 240
ctaaggaagc ctccagtctt gtgggcgagg cttgtcaaaa acatggtttt ttcctagtcg 300
tcaaccatgg agttgatgca aacctaatat ccgatgccca acgatacatg gatttgttct 360
ttgagctccc gctttctgta aagcaaagag ctcaaagaaa agttggtgaa agttgtggct 420
atgctagtag tttcactgga cggttttcct ctaagctgcc ttggaaagag acactctctt 480
tccgattttc agctgaggag aacgcatcca acatagttaa ggactacttt gagaacacaa 540
tgggaagaaga gtttattcga cttgggaagg tttaccaaga atattgcaat gccatgagta 600
gactctctct tgggatcatg gagttactag ggatgagcct tggtgtgagc cgagcttact 660
acaaagaatt tttcgaagaa cataattcaa taatgaggct taactactat ccaccatgtc 720
aaaaaccaga ccttacctta ggcacgggtc ctcactgtga tccaacatca ttgacaatcc 780
tacatcaaga taacgtaggt ggactagaag tctttgtgga caatgagtgg cgtttaatcg 840
tacccaattc aaatgctttt gttgtaaaca ttggtgacac attcatggca ctttcaaatg 900
gaagatacaa aagttgcttg catagggctg tggtaaacag caaaactact agaaagtcac 960
ttgctttctt cttatgtcca aagaaggata aggtagtgat cccaccaaaa gaattggtgg 1020
acgaaaataa ccctagaata tatccagatt ttacatggtc tacctttctt gagttcactc 1080
agaagcatta cagggctgac aagaataccc ttcaagcttt ctcaacctgg gttcgacaga 1140
aaaccacttg atgtcgaacc aatttgacat gcatgcaaaa agacaattag aactgtaaga 1200
tgaacaccaa agaggatgca tgaagagtta tgcatgcttt gctttcaaag ctttcacctt 1260
tatgtttctt tagggcacac aaatgaagga agaaaacaat cttaaggacc ctagataggc 1320
cagattttaa gtaatcttta tgggcgctaa actagtagga ataaagagaa gcataatata 1380
atgagccagt tttgtaattt gttttggact actatgtaga aagcttatgc agttataatc 1440
(서열번호2) DgGA20 oxidase 1 amino acid
(SEQ ID NO: 2)
LIDLGGFLSG RSSSTKEASS LVGEACQKHG FFLVVNHGVD ANLISDAQRY MDLFFELPLS 120
VKQRAQRKVG ESCGYASSFT GRFSSKLPWK ETLSFRFSAE ENASNIVKDY FENTMGEEFI 180
RLGKVYQEYC NAMSRLSLGI MELLGMSLGV SRAYYKEFFE EHNSIMRLNY YPPCQKPDLT 240
LGTGPHCDPT SLTILHQDNV GGLEVFVDNE WRLIVPNSNA FVVNIGDTFM ALSNGRYKSC 300
LHRAVVNSKT TRKSLAFFLC PKKDKVVIPP KELVDENNPR IYPDFTWSTF LEFTQKHYRA 360
DKNTLQAFST WVRQKTTMAIDCMIKTT SNMPSLKEDQ RKSFVFDASV LKHETNIPQQ FIWPDHEKPN SQKSKELEVP 60
LIDLGGFLSG RSSSTKEASS LVGEACQKHG FFLVVNHGVD ANLISDAQRY MDLFFELPLS 120
VKQRAQRKVG ESCGYASSFT GRFSSKLPWK ETLSFRFSAE ENASNIVKDY FENTMGEEFI 180
RLGKVYQEYC NAMSRLSLGI MELLGMSLGV SRAYYKEFFE EHNSIMRLNY YPPCQKPDLT 240
LGTGPHCDPT SLTILHQDNV GGLEVFVDNE WRLIVPNSNA FVVNIGDTFM ALSNGRYKSC 300
LHRAVVNSKT TRKSLAFFLC PKKDKVVIPP KELVDENNPR IYPDFTWSTF LEFTQKHYRA 360
DKNTLQAFST WVRQKTT
상기 DgGA20 oxidase 1 유전자가 도입된 국화 형질전환용 벡터를 하기와 같이 제작하여 도 1에 나타내었다.A vector for chrysanthemum transformation into which the
국화 고유 프로모터(CL-actin-Pro)를 이용하여 Genome Walker의 방법으로 분리한 actin ORF의 업스트림 프로모터 영역을 분리하여 염기서열을 분석하였고, actin 유전자 첫 번째 exon을 포함시킨 1432 bp의 영역을 GUS와 연결하여 형질전환용 과발현 벡터를 제작하였다.Using the chrysanthemum-specific promoter (CL-actin-Pro), the upstream promoter region of the actin ORF isolated by the Genome Walker method was isolated and sequenced. by connecting An overexpression vector for transformation was constructed.
도 1은 DgGA20 oxidase 1 유전자가 도입된 과발현용 벡터의 구성을 나타낸 것이다. 1 is
<실시예 2> <Example 2> DgGA20 oxidaseDgGA20 oxidase 1 One 유전자 도입 형질전환 국화Genetically Transgenic Chrysanthemum
상기 실시예 1에서 제작된 재조합 벡터를 아그로박테리움 튜메파시엔스(Agrobactrium tumefaciens) LBA4404를 사용하여 국화(Chrysanthemum morifolium) 분화국(pot-mum)의 일종인 피스코퍼(peace copper) 품종에 형질전환하였다. The recombinant vector constructed in Example 1 was transformed into a peace copper variety, a kind of pot-mum of Chrysanthemum morifolium, using Agrobacterium tumefaciens LBA4404. .
구체적으로, Hosh 등(A Simple and general method for transferring genes into plants, 1985)의 방법을 변형한 리프 디스크(leaf disk) 방법으로 기내에서 키운 피스코퍼 잎을 약 5 mm 정도 사각형이 되게 자른 후 전배양 (preculture) 배지 암조건에서 2일간 배양하였다. 벡터를 포함한 아그로박테리움을 액체 배지에서 전배양한 다음(2-3일) 약 1.5 mL 정도를 100 mL의 배지에서 다시 접종하여 OD 600=1.0 정도가 되게 키워 수확(harvesting)한다. 원심분리로 가라앉힌 펠렛(pellet)을 공동배양배지에 희석한 후 전배양한 식물체에 약 10분간 침지한 다음 물기를 제거하고 캘러스 유기배지에 이틀간 암배양하였다. 이틀 후 공동배양한 잎 조각을 항생제 배지로 옮겨 7일간 아그로박테리움을 제거하고 슈트(shoot) 선발 배지로 옮겨 형질전환 슈트를 선발하였다. 전 과정에 걸쳐 배지에 사용한 호르몬은 IAA 0.5 mg/L, BA 0.5 mg/L로 사용하였으며, 공동배양 이후 세포탁심 (cefotaxime)은 400 mg/L로 사용하였고 슈트선발 항생제는 카나마이신(kanamycin) 15 mg/L을 사용하였다. 국화 형질전환에서는 슈트 선발 후 뿌리 배지에서 2차 선발을 수행하였는데 이때 사용한 항생제 농도는 20 mg/L로 하였다.Specifically, the leaf disk method, which is a modification of the method of Hosh et al. (A Simple and general method for transferring genes into plants, 1985), cuts the piscoper leaves grown in vitro into squares of about 5 mm and then precultures (preculture) The medium was cultured for 2 days under dark conditions. Agrobacterium containing the vector is pre-cultured in a liquid medium (2-3 days), and then about 1.5 mL is re-inoculated in a 100 mL medium, grown to an OD 600=1.0, and harvested. After diluting the pellet (pellet) settled by centrifugation in the co-culture medium, it was immersed in the pre-cultured plants for about 10 minutes, then the water was removed and cultured in the dark in the organic callus medium for two days. Two days later, the co-cultured leaf pieces were transferred to an antibiotic medium to remove Agrobacterium for 7 days, and transferred to a shoot selection medium to select transgenic shoots. Hormones used in the medium throughout the entire process were used at 0.5 mg/L of IAA and 0.5 mg/L of BA, cefotaxime was used at 400 mg/L after co-culture, and 15 mg of kanamycin was used as a suit selection antibiotic. /L was used. In chrysanthemum transformation, secondary selection was performed in the root medium after shoot selection, and the antibiotic concentration used at this time was 20 mg/L.
뿌리가 발생한 슈트를 우선으로 잎 3-4매를 이용하여 담배와 동일한 방법으로 genomeDNA를 추출하고 PCR을 수행하여 형질전환 개체를 선발하였다.First, 3-4 leaves were used to extract genomic DNA in the same way as tobacco, and PCR was performed to select transgenic individuals.
<실시예 3> 형질전환 국화의 초장 관찰 및 표현형 분석<Example 3> Plant growth observation and phenotype analysis of transgenic chrysanthemum
형질전환체로 확인된 개체들은 기내증식용으로 증식 후 남은 부분(줄기, 마디 뿌리 부분)을 온실에서 순화한 다음 화분에 심어 충분히 생육한 다음 7월에 다시 마디증식하여 발근시킨 후 화분에 정식하여 10월말에 초장 등의 표현형을 확인하였다.Individuals identified as transformants were propagated for in vitro propagation, and the remaining parts (stems, nodes, roots) were purified in a greenhouse, planted in pots, grown sufficiently, and then propagated in nodes again in July, rooted, and planted in pots for 10 days. At the end of the month, phenotypes such as plant height were confirmed.
도 2는 DgGA20 oxidase 1 유전자를 포함하는 재조합 벡터로 형질전환된 형질전환 국화의 표현형을 나타낸 것이다. 도 2를 참고하면, 형질전환 개체로 확인된 오른쪽의 12개 개체는 왼쪽의 대조구인 비형질전환 국화에 비해 20% 이상 초장(plant height)이 감소되었으며, OX-91 라인의 경우 대조구 대비 초장이 57.8% 감소되었다. 따라서, DgGA20 oxidase 1 유전자의 발현 조절을 통해 초장이 조절된 형질전환 국화를 제작할 수 있다.Figure 2 shows the phenotype of transgenic chrysanthemum transformed with a recombinant vector containing the
<실시예 4> 형질전환 국화의 <Example 4> Transgenic chrysanthemum GAGA 유전자 발현 분석 Gene expression analysis
초장이 감소한 국화로부터 total RNA를 추출하여 1st strand기반 cDNA를 합성하였다. 지베렐린산(GA) 관련 유전자(GA2, GA3, GA20) 염기서열에 기반하여 200bp미만의 합성된 프라이머를 표 2에 표기하였다. 상기 프라이머를 이용하여 실시간 PCR을 수행해 GA 유전자 발현량의 변화를 관찰하였다.Total RNA was extracted from chrysanthemums with reduced plant height and 1st strand-based cDNA was synthesized. The synthesized primers of less than 200 bp based on the nucleotide sequences of gibberellic acid (GA)-related genes ( GA2, GA3, GA20 ) are shown in Table 2. Real-time PCR was performed using the primers to observe changes in the expression level of the GA gene.
(서열번호3 / 서열번호4) GA2- forward / GA2- reverse
(SEQ ID NO: 3 / SEQ ID NO: 4)
(서열번호5 / 서열번호6) GA3- forward / GA3- reverse
(SEQ ID NO: 5 / SEQ ID NO: 6)
(서열번호7 / 서열번호8) GA20- forward / GA20- reverse
(SEQ ID NO: 7 / SEQ ID NO: 8)
도 3은 DgGA20 oxidase 1 유전자가 과발현된 형질전환 개체 OX-12에서의 GA 유전자 발현량 변화를 나타낸 것이다. 도 3을 참고하면, 대조구에 비해 GA2, GA3 및 GA20 유전자의 발현량이 증가되었음을 확인할 수 있다. 지베렐린산(GA) 경로에서의 피드백 조절(feedback regulation)을 참고하면(도 4), GA20 oxidase1의 발현 증가가 GA3의 발현을 증가시키고 식물체내 GA가 증가하면 GA 불활성화에 관여하는 GA2 oxidase1의 발현을 촉진시켜, 초장이 감소하는 것으로 판단된다.Figure 3 shows the change in GA gene expression level in the transgenic object OX-12 in which the
<110> REPUBLIC OF KOREA(MANAGEMENT : RURAL DEVELOPMENT ADMINISTRATION) <120> Dwarfism chrysanthemum overexpressing DgGA20 oxidase 1 gene and manufacturing method thereof <130> 2021-0433-10-A_DP20210242 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 1445 <212> DNA <213> Artificial Sequence <220> <223> DgGA20 oxidase 1_gene <400> 1 ctagtgattt ttgtgcaatg gctatagatt gcatgatcaa gactacatca aacatgccat 60 cccttaaaga ggatcaacga aaatcattcg ttttcgatgc atccgtgcta aaacatgaaa 120 ccaatatacc ccaacagttc atatggcccg atcatgagaa acctaactcc caaaaatcaa 180 aggaacttga ggttcctttg atagacctag gaggctttct ttctggacga tcgagttcaa 240 ctaaggaagc ctccagtctt gtgggcgagg cttgtcaaaa acatggtttt ttcctagtcg 300 tcaaccatgg agttgatgca aacctaatat ccgatgccca acgatacatg gatttgttct 360 ttgagctccc gctttctgta aagcaaagag ctcaaagaaa agttggtgaa agttgtggct 420 atgctagtag tttcactgga cggttttcct ctaagctgcc ttggaaagag acactctctt 480 tccgattttc agctgaggag aacgcatcca acatagttaa ggactacttt gagaacacaa 540 tgggagaaga gtttattcga cttgggaagg tttaccaaga atattgcaat gccatgagta 600 gactctctct tgggatcatg gagttactag ggatgagcct tggtgtgagc cgagcttact 660 acaaagaatt tttcgaagaa cataattcaa taatgaggct taactactat ccaccatgtc 720 aaaaaccaga ccttacctta ggcacgggtc ctcactgtga tccaacatca ttgacaatcc 780 tacatcaaga taacgtaggt ggactagaag tctttgtgga caatgagtgg cgtttaatcg 840 tacccaattc aaatgctttt gttgtaaaca ttggtgacac attcatggca ctttcaaatg 900 gaagatacaa aagttgcttg catagggctg tggtaaacag caaaactact agaaagtcac 960 ttgctttctt cttatgtcca aagaaggata aggtagtgat cccaccaaaa gaattggtgg 1020 acgaaaataa ccctagaata tatccagatt ttacatggtc tacctttctt gagttcactc 1080 agaagcatta cagggctgac aagaataccc ttcaagcttt ctcaacctgg gttcgacaga 1140 aaaccacttg atgtcgaacc aatttgacat gcatgcaaaa agacaattag aactgtaaga 1200 tgaacaccaa agaggatgca tgaagagtta tgcatgcttt gctttcaaag ctttcacctt 1260 tatgtttctt tagggcacac aaatgaagga agaaaacaat cttaaggacc ctagataggc 1320 cagattttaa gtaatcttta tgggcgctaa actagtagga ataaagagaa gcataatata 1380 atgagccagt tttgtaattt gttttggact actatgtaga aagcttatgc agttataatc 1440 actag 1445 <210> 2 <211> 377 <212> PRT <213> Artificial Sequence <220> <223> DgGA20 oxidase 1_aa <400> 2 Met Ala Ile Asp Cys Met Ile Lys Thr Thr Ser Asn Met Pro Ser Leu 1 5 10 15 Lys Glu Asp Gln Arg Lys Ser Phe Val Phe Asp Ala Ser Val Leu Lys 20 25 30 His Glu Thr Asn Ile Pro Gln Gln Phe Ile Trp Pro Asp His Glu Lys 35 40 45 Pro Asn Ser Gln Lys Ser Lys Glu Leu Glu Val Pro Leu Ile Asp Leu 50 55 60 Gly Gly Phe Leu Ser Gly Arg Ser Ser Ser Thr Lys Glu Ala Ser Ser 65 70 75 80 Leu Val Gly Glu Ala Cys Gln Lys His Gly Phe Phe Leu Val Val Asn 85 90 95 His Gly Val Asp Ala Asn Leu Ile Ser Asp Ala Gln Arg Tyr Met Asp 100 105 110 Leu Phe Phe Glu Leu Pro Leu Ser Val Lys Gln Arg Ala Gln Arg Lys 115 120 125 Val Gly Glu Ser Cys Gly Tyr Ala Ser Ser Phe Thr Gly Arg Phe Ser 130 135 140 Ser Lys Leu Pro Trp Lys Glu Thr Leu Ser Phe Arg Phe Ser Ala Glu 145 150 155 160 Glu Asn Ala Ser Asn Ile Val Lys Asp Tyr Phe Glu Asn Thr Met Gly 165 170 175 Glu Glu Phe Ile Arg Leu Gly Lys Val Tyr Gln Glu Tyr Cys Asn Ala 180 185 190 Met Ser Arg Leu Ser Leu Gly Ile Met Glu Leu Leu Gly Met Ser Leu 195 200 205 Gly Val Ser Arg Ala Tyr Tyr Lys Glu Phe Phe Glu Glu His Asn Ser 210 215 220 Ile Met Arg Leu Asn Tyr Tyr Pro Pro Cys Gln Lys Pro Asp Leu Thr 225 230 235 240 Leu Gly Thr Gly Pro His Cys Asp Pro Thr Ser Leu Thr Ile Leu His 245 250 255 Gln Asp Asn Val Gly Gly Leu Glu Val Phe Val Asp Asn Glu Trp Arg 260 265 270 Leu Ile Val Pro Asn Ser Asn Ala Phe Val Val Asn Ile Gly Asp Thr 275 280 285 Phe Met Ala Leu Ser Asn Gly Arg Tyr Lys Ser Cys Leu His Arg Ala 290 295 300 Val Val Asn Ser Lys Thr Thr Arg Lys Ser Leu Ala Phe Phe Leu Cys 305 310 315 320 Pro Lys Lys Asp Lys Val Val Ile Pro Pro Lys Glu Leu Val Asp Glu 325 330 335 Asn Asn Pro Arg Ile Tyr Pro Asp Phe Thr Trp Ser Thr Phe Leu Glu 340 345 350 Phe Thr Gln Lys His Tyr Arg Ala Asp Lys Asn Thr Leu Gln Ala Phe 355 360 365 Ser Thr Trp Val Arg Gln Lys Thr Thr 370 375 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GA2-forward <400> 3 gtggtctcgg gtaagggatt 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GA2-reverse <400> 4 ggagtggtta cctggtaccc 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GA3-forward <400> 5 cgcatgcatg gttatcgcta 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GA3-reverse <400> 6 ttttctacca gcagcctcca 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GA20-forward <400> 7 ccaacagttc atatggcccg 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GA20-reverse <400> 8 catgtatcgt tgggcatcgg 20 <110> REPUBLIC OF KOREA(MANAGEMENT : RURAL DEVELOPMENT ADMINISTRATION) <120> Dwarfism chrysanthemum overexpressing DgGA20 oxidase 1 gene and manufacturing method its <130> 2021-0433-10-A_DP20210242 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 1445 <212> DNA <213> artificial sequence <220> <223> DgGA20 oxidase 1_gene <400> 1 ctagtgattt ttgtgcaatg gctatagatt gcatgatcaa gactacatca aacatgccat 60 cccttaaaga ggatcaacga aaatcattcg ttttcgatgc atccgtgcta aaacatgaaa 120 ccaatatacc ccaacagttc atatggcccg atcatgagaa acctaactcc caaaaatcaa 180 aggaacttga ggttcctttg atagacctag gaggctttct ttctggacga tcgagttcaa 240 ctaaggaagc ctccagtctt gtgggcgagg cttgtcaaaa acatggtttt ttcctagtcg 300 tcaaccatgg agttgatgca aacctaatat ccgatgccca acgatacatg gatttgttct 360 ttgagctccc gctttctgta aagcaaagag ctcaaagaaa agttggtgaa agttgtggct 420 atgctagtag tttcactgga cggttttcct ctaagctgcc ttggaaagag acactctctt 480 tccgattttc agctgaggag aacgcatcca acatagttaa ggactacttt gagaacacaa 540 tgggaagaaga gtttattcga cttgggaagg tttaccaaga atattgcaat gccatgagta 600 gactctctct tgggatcatg gagttactag ggatgagcct tggtgtgagc cgagcttact 660 acaaagaatt tttcgaagaa cataattcaa taatgaggct taactactat ccaccatgtc 720 aaaaaccaga ccttacctta ggcacgggtc ctcactgtga tccaacatca ttgacaatcc 780 tacatcaaga taacgtaggt ggactagaag tctttgtgga caatgagtgg cgtttaatcg 840 tacccaattc aaatgctttt gttgtaaaca ttggtgacac attcatggca ctttcaaatg 900 gaagatacaa aagttgcttg catagggctg tggtaaacag caaaactact agaaagtcac 960 ttgctttctt cttatgtcca aagaaggata aggtagtgat cccaccaaaa gaattggtgg 1020 acgaaaataa ccctagaata tatccagatt ttacatggtc tacctttctt gagttcactc 1080 agaagcatta cagggctgac aagaataccc ttcaagcttt ctcaacctgg gttcgacaga 1140 aaaccacttg atgtcgaacc aatttgacat gcatgcaaaa agacaattag aactgtaaga 1200 tgaacaccaa agaggatgca tgaagagtta tgcatgcttt gctttcaaag ctttcacctt 1260 tatgtttctt tagggcacac aaatgaagga agaaaacaat cttaaggacc ctagataggc 1320 cagattttaa gtaatcttta tgggcgctaa actagtagga ataaagagaa gcataatata 1380 atgagccagt tttgtaattt gttttggact actatgtaga aagcttatgc agttataatc 1440 actag 1445 <210> 2 <211> 377 <212> PRT <213> artificial sequence <220> <223> DgGA20 oxidase 1_aa <400> 2 Met Ala Ile Asp Cys Met Ile Lys Thr Thr Ser Asn Met Pro Ser Leu 1 5 10 15 Lys Glu Asp Gln Arg Lys Ser Phe Val Phe Asp Ala Ser Val Leu Lys 20 25 30 His Glu Thr Asn Ile Pro Gln Gln Phe Ile Trp Pro Asp His Glu Lys 35 40 45 Pro Asn Ser Gln Lys Ser Lys Glu Leu Glu Val Pro Leu Ile Asp Leu 50 55 60 Gly Gly Phe Leu Ser Gly Arg Ser Ser Ser Thr Lys Glu Ala Ser Ser 65 70 75 80 Leu Val Gly Glu Ala Cys Gln Lys His Gly Phe Phe Leu Val Val Asn 85 90 95 His Gly Val Asp Ala Asn Leu Ile Ser Asp Ala Gln Arg Tyr Met Asp 100 105 110 Leu Phe Phe Glu Leu Pro Leu Ser Val Lys Gln Arg Ala Gln Arg Lys 115 120 125 Val Gly Glu Ser Cys Gly Tyr Ala Ser Ser Phe Thr Gly Arg Phe Ser 130 135 140 Ser Lys Leu Pro Trp Lys Glu Thr Leu Ser Phe Arg Phe Ser Ala Glu 145 150 155 160 Glu Asn Ala Ser Asn Ile Val Lys Asp Tyr Phe Glu Asn Thr Met Gly 165 170 175 Glu Glu Phe Ile Arg Leu Gly Lys Val Tyr Gln Glu Tyr Cys Asn Ala 180 185 190 Met Ser Arg Leu Ser Leu Gly Ile Met Glu Leu Leu Gly Met Ser Leu 195 200 205 Gly Val Ser Arg Ala Tyr Tyr Lys Glu Phe Phe Glu Glu His Asn Ser 210 215 220 Ile Met Arg Leu Asn Tyr Tyr Pro Pro Cys Gln Lys Pro Asp Leu Thr 225 230 235 240 Leu Gly Thr Gly Pro His Cys Asp Pro Thr Ser Leu Thr Ile Leu His 245 250 255 Gln Asp Asn Val Gly Gly Leu Glu Val Phe Val Asp Asn Glu Trp Arg 260 265 270 Leu Ile Val Pro Asn Ser Asn Ala Phe Val Val Asn Ile Gly Asp Thr 275 280 285 Phe Met Ala Leu Ser Asn Gly Arg Tyr Lys Ser Cys Leu His Arg Ala 290 295 300 Val Val Asn Ser Lys Thr Thr Arg Lys Ser Leu Ala Phe Phe Leu Cys 305 310 315 320 Pro Lys Lys Asp Lys Val Val Ile Pro Pro Lys Glu Leu Val Asp Glu 325 330 335 Asn Asn Pro Arg Ile Tyr Pro Asp Phe Thr Trp Ser Thr Phe Leu Glu 340 345 350 Phe Thr Gln Lys His Tyr Arg Ala Asp Lys Asn Thr Leu Gln Ala Phe 355 360 365 Ser Thr Trp Val Arg Gln Lys Thr Thr 370 375 <210> 3 <211> 20 <212> DNA <213> artificial sequence <220> <223> GA2-forward <400> 3 gtggtctcgg gtaagggatt 20 <210> 4 <211> 20 <212> DNA <213> artificial sequence <220> <223> GA2-reverse <400> 4 ggagtggtta cctggtaccc 20 <210> 5 <211> 20 <212> DNA <213> artificial sequence <220> <223> GA3-forward <400> 5 cgcatgcatg gttatcgcta 20 <210> 6 <211> 20 <212> DNA <213> artificial sequence <220> <223> GA3-reverse <400> 6 ttttctacca gcagcctcca 20 <210> 7 <211> 20 <212> DNA <213> artificial sequence <220> <223> GA20-forward <400> 7 ccaacagttc atatggcccg 20 <210> 8 <211> 20 <212> DNA <213> artificial sequence <220> <223> GA20 reverse <400> 8 catgtatcgt tgggcatcgg 20
Claims (7)
상기 왜성은 비형질전환 국화에 비해 초장(plant height)이 20-60% 감소된 것인, 형질전환 국화.According to claim 1,
Transgenic chrysanthemum, wherein the dwarf is reduced in plant height by 20-60% compared to non-transformed chrysanthemum.
상기 상기 국화(Chrysanthemum morifolium)는 블랙마블, 보라미, 체리블로섬, 포리스트아로마, 골든페스티벌, 가마, 가든파티, 화월, 신마, 문페스티벌, 무지개, 문라이트, 오렌지메모리, 오렌지마블, 퓨어앤젤, 프리마돈나, 프레디져드아모르, 피크, 핑크프라이드, 핑크팡팡, 핑크바이오렛, 스위트카펫, 설악, 스위트핑크,서니팡팡, 비비드스칼렛, 화이트팡팡, 화이트윙, 에스데이, 엘로우엘레강스, 예스해피, 예스모닝, 예스나우, 예스팡팡, 예스스타, 예로우 팡팡, 예스스타, 에스스완, 예스 투게더, 예스타임, 399, 피스핑크, 피스킹, 피스코퍼, 피스옐로우, 피스퀸, 피스로망스,피스앤젤로 이루어진 군에서 선택된 1종 이상의 품종인 것인, 형질전환 국화.According to claim 1,
The chrysanthemum ( Chrysanthemum morifolium ) is Black Marble, Borami, Cherry Blossom, Forest Aroma, Golden Festival, Gama, Garden Party, Flower Month, Shinma, Moon Festival, Rainbow, Moonlight, Orange Memory, Orange Marble, Pure Angel, Prima Donna, Freddie Amor, Peak, Pink Pride, Pink Pang Pang, Pink Violet, Sweet Carpet, Seorak, Sweet Pink, Sunny Pang Pang, Vivid Scarlet, White Pang Pang, White Wing, S-Day, Yellow Elegance, Yes Happy, Yes Morning, Yes Now , Yes Pang Pang, Yes Star, Yes Pang Pang, Yes Star, S Swan, Yes Together, Yes Time, 399, Peace Pink, Peace King, Peace Copper, Peace Yellow, Peace Queen, Peace Romance, Peace Angel A transgenic chrysanthemum that is more than a species.
상기 벡터를 아그로박테리움을 이용하여 국화 품종에 형질전환하는 단계; 및
형질전환 개체를 선발하는 단계를 포함하는, 초장(plant height)이 20-60% 감소된 형질전환 국화의 제조방법.constructing a recombinant vector overexpressing the gene of SEQ ID NO: 1 encoding the DgGA20 oxidase 1 protein represented by SEQ ID NO: 2;
Transforming the vector into chrysanthemum varieties using Agrobacterium; and
A method for producing a transgenic chrysanthemum having a plant height reduced by 20-60%, comprising the step of selecting a transgenic individual.
상기 제조방법에 의해 제조된 형질전환 국화의 형질전환된 종자.According to claim 4,
Transformed seeds of the transgenic chrysanthemum prepared by the above production method.
상기 벡터를 아그로박테리움을 이용하여 국화 품종에 형질전환하는 단계; 및
형질전환 개체를 선발하는 단계를 포함하는, 국화의 초장(plant height)을 20-60% 감소시키는 방법.constructing a recombinant vector overexpressing the gene of SEQ ID NO: 1 encoding the DgGA20 oxidase 1 protein represented by SEQ ID NO: 2;
Transforming the vector into chrysanthemum varieties using Agrobacterium; and
A method for reducing the plant height of chrysanthemum by 20-60%, comprising selecting a transgenic individual.
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| KR101359457B1 (en) | 2011-11-23 | 2014-02-07 | 상명대학교 천안산학협력단 | Method for producing transgenic Compositae flowering plant with regulated flower shape or color and the plant thereof |
| KR102004057B1 (en) | 2017-12-29 | 2019-07-25 | 경희대학교 산학협력단 | A gene involved in plant cell elongation and production of dwarf plants |
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| KR101359457B1 (en) | 2011-11-23 | 2014-02-07 | 상명대학교 천안산학협력단 | Method for producing transgenic Compositae flowering plant with regulated flower shape or color and the plant thereof |
| KR102004057B1 (en) | 2017-12-29 | 2019-07-25 | 경희대학교 산학협력단 | A gene involved in plant cell elongation and production of dwarf plants |
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