KR101696678B1 - Transgenic rice producing high level of flavonoids and manufacturing method thereof - Google Patents
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Abstract
본 발명은 안토시아닌 색소합성에 관여하는 유전자를 센스 및 안티센스 방향으로 포함하는 식물 형질전환용 벡터에 관한 것이다. 본 발명은 또한 상기 벡터로 형질전환된 플라보노이드 생산이 증가된 형질전환 식물체 및 식물 종자에 관한 것이다. 본 발명은 또한 1) 서열번호 1, 서열번호 2 또는 서열번호 3의 염기서열을 갖는 유전자를 센스 및 안티센스 방향으로 삽입한 형질전환용 발현벡터를 제조하는 단계; 2) 상기 제조된 발현벡터를 이용하여 식물 형질전환용 아그로박테리움을 제조하는 단계; 및 3) 상기 형질전환용 아그로박테리움을 이용하여 벼를 형질전환 하는 단계;를 포함하는 것을 특징으로 하는 플라보노이드 생산이 증가된 형질전환 벼의 제조방법에 관한 것이다. The present invention relates to a plant transformation vector comprising a gene involved in anthocyanin pigment synthesis in the sense and antisense direction. The present invention also relates to transgenic plants and plant seeds with increased production of flavonoids transformed with said vectors. The present invention also provides a method for producing a transgenic plant, comprising the steps of: 1) preparing a transgene expression vector in which a gene having the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 is inserted in the sense and antisense direction; 2) preparing Agrobacterium for plant transformation using the expression vector prepared above; And 3) transforming rice with the transforming Agrobacterium. The present invention also relates to a method for producing transformed rice having increased flavonoid production.
Description
본 발명은 플라보노이드 생산이 증가된 형질전환 벼 및 이의 제조방법에 관한 것이다.
The present invention relates to a transgenic rice with increased flavonoid production and a method for producing the same.
식물체는 다양한 기능성(항암, 항염, 항산화 성분)을 지니는 수만종의 파이토케미컬(phytochemical)을 생산하는 것으로 알려져 있다. 이러한 파이토케미컬 중에서도 플라보노이드계 물질은 뛰어난 항산화 기능 외에도 다양한 기능성을 지니는 것으로 알려져 있다. 식물체에서 플라보노이드 물질은 식물의 잎, 종자, 과실, 꽃등에 축적되어 UV광으로부터 식물을 보호하는 역할을 수행하며, 미생병원균, 곤충과 상호작용하는 것으로 알려져 있다. 또한 플라보노이드계 물질들은 항암, 항염, 항산화등의 다양한 기능성이 보고되어 여러 산업분야(건강증진 및 의료분야)에서 이용되고 있다. 쌀은 남아시아, 중동, 라틴아메리카와 서인도를 포함한 많은 인구를 위한 주요한 식량작물이고, 여러 연구를 통해서 유색쌀의 경우에는 일반쌀에 비해서 호분층에 플라보노이드계열, 안토시아닌계열 또는 프로안토시아닌 계열 물질이 축적되어 있음이 보고되고 있다. Plants are known to produce hundreds of thousands of phytochemicals with various functionalities (anti-cancer, anti-inflammatory, antioxidant). Among these phytochemicals, flavonoids are known to possess various functionalities besides excellent antioxidant functions. In plants, flavonoid substances are accumulated in plant leaves, seeds, fruits, flowers, etc., and protect plants from UV light. They are known to interact with microbes and insects. In addition, flavonoids have been reported in various functionalities such as anticancer, anti-inflammation, and antioxidation and are used in various industrial fields (health promotion and medical field). Rice is a major food crop for many populations, including South Asia, the Middle East, Latin America and the West Indies. Studies have shown that flavonoids, anthocyanins or proanthocyanins accumulate in the colony Is being reported.
한편, 식물의 유용한 2차 대사물질의 합성을 증대하거나 특정대사경로로 물질합성을 조절하기 위하여 다양한 대사공학적 방법들이 이용되고 있다. 2차 대사산물을 생산량을 증대하고자 생합성경로의 구조유전자의 발현시키거나 전사인자를 이용하는 방법들이 이용되고 있다. 토마토에서 안토시아닌 생산을 증대시키고자 구조유전자 CHI, FNS, FNS와 CHI유전자를 발현 증대시킨 결과 과피에서 플라보노이드의 함량이 증대되는 결과는 얻었으나, 과실 내에서의 물질생산은 증가되지 않았다(비특허문헌 1). 전사인자를 이용한 연구에서는, 옥수수의 안토시아닌 색소합성 전사인자인 Lc와 C1유전자를 과발현시킨 결과, 플라보놀의 함량은 20배 이상 증가되었으나 과실에서는 안토시아닌의 축적이 관찰되지 않았다. 토마토유래의 Myb 계열 전사인자인 ANT1을 이용한 경우에는 과실의 과피와 전신에서 안토시아닌의 축적이 관찰되었다 (비특허문헌 2). 토마토 과실의 안토시아닌 색소합성 증가는 과실특이프로모터를 이용하여 금어초의 전사인자인 Delia와 Rosea 유전자를 발현시킨 결과, 대조구에 비해 델피리딘의 함량이 적게는 30배 (230ug/g FW)에서 많게는 150배 (2,800ug/g FW)까지 증대된 토마토를 얻을 수 있었다. 기능성 파이토케미컬이 증대된 토마토를 쥐에 먹여 본 결과 평균적으로 대조구에 비해 40일가량 수명이 연장됨을 확인하였다(비특허문헌 3). 대사공학연구를 통해서 기능성 천연색소물질의 생산기작을 이해하고 이를 적용하여 식물유래 기능성 물질 대량생산 연구 및 기능성식품 소재개발 및 건강증진 효과 등을 확인하는 연구가 활발히 진행되고 있다. On the other hand, various metabolic engineering methods have been used to increase the synthesis of useful secondary metabolites in plants or to control the synthesis of substances by specific metabolic pathways. Methods for expressing structural genes of biosynthetic pathway or using transcription factors have been used to increase production of secondary metabolites. As a result of increasing expression of the structural genes CHI, FNS, FNS and CHI genes to increase anthocyanin production in tomato, the content of flavonoid increased in the periplasm, but the production of material in the fruit was not increased One). In the study using transcription factors, the overexpression of Lc and C1 genes, which are transcription factors of anthocyanin pigment in corn, resulted in an increase of flavonol content more than 20 times, but no accumulation of anthocyanin was observed in fruits. When ANT1, a Myb-related transcription factor derived from tomato, was used, accumulation of anthocyanin was observed in fruit pericarp and whole body (Non-Patent Document 2). The expression of Delia and Rosea genes, which are transcription factors of Snapdragon, was increased by 30% (230 ug / g FW) to 150 fold more than that of the control by using a fruit specific promoter (2,800ug / g FW). It was confirmed that mice fed with the functional phytochemical-enhanced tomatoes were on average about 40 days longer than the control (Non-Patent Document 3). Studies on the mass production of functional materials derived from plants and development of functional food materials and health promotion effects have been actively under way by understanding the production mechanism of functional natural coloring matter materials through metabolic engineering research.
플라보노이드계열의 천연물질은 가공식품첨가제, 천연방부제등 다양한 산업분야에 이용되고 있을 뿐 아니라 뛰어난 항산화 효과와 더불어서 다양한 기능성으로 인해서 의료용으로도 주목받고 있다. 벼는 3대 주곡작물로써 전세계의 많은 인류의 식량공급원으로 사용되고 있다. 하지만 다양한 기능성을 지니는 물질들은 종자의 호분층에 존재하고 호분층이 도정되어 먹는 백미의 경우 기능성물질의 섭취가 상대적으로 낮아진다는 한계가 있다. Flavonoid-based natural materials are not only used in various industrial fields such as processed food additives, natural preservatives, but also have excellent antioxidative effects and are attracting attention for medical use due to their various functionalities. Rice has been used as a food source for many humans all over the world as the three most important crops. However, there are limitations in that various functional materials are present in the horny layer of the seed, and the consumption of the functional material is relatively low in the case of the horny layer.
이에 본 발명자들은 벼 배유 내로 기능성 안토시아닌 등의 물질생성을 축적하고자 벼 내재의 안토시아닌 생성 기작의 이해를 바탕으로 플라보노이드 생성과 관련한 유전자를 선발하여 발현운반체를 제작하였다. 또한 이를 이용해 형질전환 식물체를 개발함으로써, 종자 내의 플라보노이드 생성 가능성을 확인하고자 하였다.
Therefore, in order to accumulate the production of functional anthocyanin and the like in rice embryo, the present inventors selected an expression carrier for the flavonoid production based on an understanding of the anthocyanin generation mechanism in the rice embryo. We also developed a transgenic plant using this to determine the possibility of flavonoid production in seeds.
본 발명의 목적은 안토시아닌 색소합성에 관여하는 유전자를 센스 및 안티센스 방향으로 포함하는 식물 형질전환용 벡터를 제공하는 것이다.It is an object of the present invention to provide a plant transformation vector comprising a gene involved in anthocyanin pigment synthesis in the sense and antisense direction.
본 발명의 다른 목적은 상기 식물 형질전환용 벡터로 형질전환된 식물체를 제공하는 것이다.Another object of the present invention is to provide a plant transformed with said plant transformation vector.
본 발명의 또 다른 목적은 식물 형질전환용 벡터로 형질전환된 플라보노이드 생산이 증가된 형질전환 식물 종자를 제공하는 것이다.Another object of the present invention is to provide a transformed plant seed having increased production of flavonoids transformed with a plant transformation vector.
본 발명의 또 다른 목적은 플라보노이드 생산이 증가된 형질전환 벼의 제조방법을 제공하는 것이다.It is another object of the present invention to provide a method for producing transgenic rice with increased flavonoid production.
본 발명의 또 다른 목적은 플라보노이드 생산 증대 방법을 제공하는 것이다.
It is a further object of the present invention to provide a method for increasing flavonoid production.
상기 목적을 달성하기 위하여, 본 발명은 서열번호 1, 서열번호 2 또는 서열번호 3으로 표시되는 안토시아닌 색소합성에 관여하는 유전자를 센스 및 안티센스 방향으로 포함하는 식물 형질전환용 벡터를 제공한다. In order to achieve the above object, the present invention provides a plant transformation vector comprising a gene involved in the synthesis of anthocyanin pigment represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 in the sense and antisense direction.
본 발명에서 용어, "형질전환"은 DNA 염기서열 상의 삽입, 결실 또는 대체 등의 변이를 통해 특정 형질이 발생하거나 사라지거나 조절되는 모든 행위를 의미하며, 특히 본 발명에서는 외부로부터 주어진 DNA에 의하여 생물의 유전적인 성질이 변하는 것을 의미한다. 본 발명에서 용어, "형질전환 식물체"는 형질전환으로 인해 생성된 형질전환 식물을 의미하며, 유전자 재조합 기술을 이용하여 특정 유전자의 변형 또는 활성의 변이가 유발되어 생성된 유전자 재조합체를 포함한다.The term "transformed" in the present invention means all the activities in which a specific trait occurs, disappears or is controlled through mutation such as insertion, deletion or substitution on the DNA base sequence. In particular, in the present invention, Which means that the genetic properties of In the present invention, the term "transgenic plant " refers to a transgenic plant produced by transformation, and includes genetic recombinants produced by mutation of a specific gene or activation of an activity using genetic recombination technology.
본 발명에서 용어, "재조합 벡터"는 특정 서열이 변이, 삽입 또는 제거되는 유전자 재조합 과정을 거친 벡터를 의미하며, 바람직하게는 발현 유도 서열이 삽입된 벡터를 의미하고, 더욱 바람직하게는 해당 발현 유도 서열을 pMJ-PGLUB1-RFA의 발현 조절 부위에 삽입할 수 있는 기능을 하는 재조합 벡터를 의미한다.In the present invention, the term "recombinant vector" means a vector that has undergone a gene recombination process in which a specific sequence is mutated, inserted or deleted, preferably a vector into which an expression inducing sequence is inserted, Means a recombinant vector capable of inserting the sequence into the regulatory region of pMJ-PGLUB1-RFA.
본 발명의 상기 서열번호 1, 서열번호 2 또는 서열번호 3으로 표시되는 안토시아닌 색소합성에 관여하는 유전자는 각각 안토시아닌 색소합성 전체를 조절하기 위한 서열번호 1로 표시되는 MYB 관련 전사인자 1종(OsC1), 서열번호 2로 표시되는 bHLH 관련 전사인자 OsB1 및 서열번호 3으로 표시되는 bHLH 관련 전사인자 OsB2일 수 있다. 상기 유전자는 본 발명의 구체예에 따른 프라이머로 벼 식물체로부터 분리할 수 있다. The gene involved in the synthesis of the anthocyanin pigment represented by SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 of the present invention includes one MYB-related transcription factor (OsC1) shown in SEQ ID NO: 1 for controlling the total synthesis of anthocyanin pigment, , A bHLH-related transcription factor OsB1 represented by SEQ ID NO: 2, and a bHLH-related transcription factor OsB2 represented by SEQ ID NO: 3. The gene can be isolated from rice plants as a primer according to embodiments of the present invention.
본 발명의 벡터는 바람직하게는 벼 종자특이 유전자인 glutelin B1유전자의 프로모터영역과 gateway용 RFA 유전자가 연결된 gateway용 종자발현용 과발현 식물발현운반체 pMJ-PGLUB1-RFA를 이용할 수 있다. 상기 벡터를 이용하는 경우 벼 종자 내로 플라보노이드 계열 물질을 축적할 수 있다. Preferably, the vector of the present invention can utilize an overexpressed plant expression carrier pMJ-PGLUB1-RFA for expression of a seed for a gateway to which a promoter region of glutelin B1 gene, which is a rice seed specific gene, and an RFA gene for a gateway are connected. When the vector is used, the flavonoid-based material can be accumulated in rice seeds.
상기 종자발현용 과발현 식물발현운반체는 서열번호 1, 서열번호 2 또는 서열번호 3으로 표시되는 안토시아닌 색소합성에 관여하는 유전자를 센스 및 안티센스 방향으로 포함하여 안토시아닌 대사증진 운반체로 제작될 수 있다. 상기 플라보노이드는 안토시아닌일 수 있다. 본 발명의 구체예에서, 상기 안토시아닌 대사증진 운반체는 각각 PGluB1-OsC1, PGluB1-OsB1 또는 PGluB1-OsB2일 수 있다. The overexpression plant expression carrier for seed expression can be produced as an anthocyanin metabolic enhancement carrier including a gene involved in the synthesis of an anthocyanin pigment represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 in the sense and antisense direction. The flavonoid may be anthocyanin. In an embodiment of the present invention, the anthocyanin metabolism enhancer may be PGluB1-OsC1, PGluB1-OsB1 or PGluB1-OsB2, respectively.
본 발명은 또한 상기 식물 형질전환용 벡터로 형질전환된 아그로박테리움 투머파시엔스 (Agrobacterium tumefaciens)를 제공한다. 본 발명의 구체예에서, 바람직하게는 아그로박테리움 LBA4404를 이용할 수 있다. 바람직하게는 상기에서 제조한 3종의 안토시아닌 대사 증진 운반체를 상기 아그로박테리움에 트리파렌탈 메이팅(triparental mating)을 통해 형질전환을 수행할 수 있다. The present invention also provides Agrobacterium tumefaciens transformed with said plant transformation vector. In an embodiment of the present invention, Agrobacterium LBA4404 is preferably used. Preferably, the three kinds of anthocyanin metabolism enhancers produced above can be transformed into the Agrobacterium through triparental mating.
본 발명은 또한 상기 식물 형질전환용 벡터 또는 상기 아그로박테리움 투머파시엔스로 형질전환된 것을 특징으로 하는 플라보노이드 생산이 증가된 형질전환 식물체를 제공한다.The present invention also provides a transgenic plant having an increased production of flavonoids, which is transformed with said plant transformation vector or said Agrobacterium tumefaciens.
본 발명에서 용어, " 형질 전환 식물체"는 형질전환의 대상이 될 수 있는 모든 식물을 포함하며, 특히, 종자 내에 플라보노이드의 축적 증진이 필요한 식물은 모두 제한 없이 포함한다. 사용 가능한 형질전환용 식물의 보다 구체적인 예로는 벼, 보리, 밀, 호밀, 기장, 조, 옥수수, 귀리 및 수수 등의 식량작물을 비롯하여 피, 바랭이, 강아지풀, 잔디, 이탈리안 라이그라스, 겨풀, 쇄출, 둑새풀, 조개풀, 넓은 잎개수염, 알방동사니, 방동사니대가리, 강피 및 좀바랭이 등의 화본과 식물을 포함할 수 있으나, 바람직하게는 상기 식물 형질 전환체는 벼(Oryza sp.)일 수 있다. The term "transgenic plant" in the present invention encompasses all plants that can be subjected to transformation, and in particular, all plants that require the promotion of the accumulation of flavonoids in seeds include without limitation. More specific examples of the transgenic plants that can be used include food crops such as rice, barley, wheat, rye, millet, barley, corn, oats and sorghum, as well as food crops such as blood, barnyard grass, grass rolls, grass, Italian rice, The plant transformant may be rice paddy (Oryza sp.). However, the plant transformant may be rice paddy (Oryza sp.).
본 발명의 구체예에서, 상기 형질전환 식물체는 상기 플라보노이드 대사증진 운반체 3종을 이용하여 수행할 수 있다. 벼 형질전환방법으로는 5일 내지 7일간 벼 종자로부터 캘러스를 유도하고, 아그로박테리움을 접종하여 형질전환체를 선발하는 조기형질전환법을 이용하여 수행할 수 있다.
In an embodiment of the present invention, the transgenic plants can be carried out using three kinds of flavonoid metabolism enhancing carriers. Rice transformation can be carried out using an early transformation method in which callus is induced from rice seeds for 5 days to 7 days, and Agrobacterium is inoculated to select a transformant.
본 발명은 또한 상기 식물 형질전환용 벡터 또는 상기 아그로박테리움 투머파시엔스로 형질전환된 것을 특징으로 하는 플라보노이드 생산이 증가된 형질전환 식물 종자를 제공한다. 본 발명의 벡터는 바람직하게는 벼 종자특이 유전자인 glutelin B1유전자의 프로모터영역과 gateway용 RFA 유전자가 연결된 gateway용 종자발현용 과발현 식물발현운반체 pMJ-PGLUB1-RFA를 이용할 수 있는바, 벼 종자 특이적으로 플라보노이드 계열 물질을 축적할 수 있다.
The present invention also provides a transformed plant seed having increased flavonoid production, which is transformed with said plant transformation vector or said Agrobacterium tumefaciens. Preferably, the vector of the present invention can utilize overexpressed plant expression carrier pMJ-PGLUB1-RFA for the expression of a seed for a gateway to which a promoter region of the glutelin B1 gene, which is a rice seed specific gene, and an RFA gene for a gateway are connected, To accumulate the flavonoid-based material.
본 발명은 또한,The present invention also relates to
1) 서열번호 1, 서열번호 2 또는 서열번호 3의 염기서열을 갖는 유전자를 센스 및 안티센스 방향으로 삽입한 형질전환용 발현벡터를 제조하는 단계; 2) 상기 제조된 발현벡터를 이용하여 식물 형질전환용 아그로박테리움을 제조하는 단계; 및 3) 상기 형질전환용 아그로박테리움을 이용하여 벼를 형질전환 하는 단계를 포함하는 것을 특징으로 하는 플라보노이드 생산이 증가된 형질전환 벼의 제조방법을 제공한다. 1) preparing a transgene expression vector in which a gene having the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 is inserted in the sense and antisense direction; 2) preparing Agrobacterium for plant transformation using the expression vector prepared above; And 3) transforming rice with Agrobacterium for transformation. The present invention also provides a method for producing transformed rice having increased flavonoid production.
본 발명의 구체예에서, 상기 1) 단계의 서열번호 1, 서열번호 2 또는 서열번호 3으로 표시되는 안토시아닌 색소합성에 관여하는 유전자는 각각 안토시아닌 색소합성 전체를 조절하기 위한 서열번호 1로 표시되는 MYB 관련 전사인자 1종 OsC1, 서열번호 2로 표시되는 bHLH 관련 전사인자 OsB1 및 서열번호 3으로 표시되는 bHLH 관련 전사인자 OsB2일 수 있다. 상기 유전자는 본 발명의 구체예에 따른 프라이머를 이용하여 벼 식물체로부터 분리할 수 있다. In the embodiment of the present invention, the genes involved in the synthesis of the anthocyanin pigment shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 in the
본 발명의 상기 벡터는 바람직하게는 벼 종자특이 유전자인 glutelin B1유전자의 프로모터영역과 gateway용 RFA 유전자가 연결된 gateway용 종자발현용 과발현 식물발현운반체 pMJ-PGLUB1-RFA를 이용할 수 있다. 상기 벡터를 이용하는 경우 벼 종자 내로 플라보노이드 계열 물질을 축적할 수 있다. Preferably, the vector of the present invention can utilize overexpressed plant expression carrier pMJ-PGLUB1-RFA for expression of a seed for a gateway to which a promoter region of a glutelin B1 gene, which is a rice seed specific gene, and an RFA gene for a gateway are connected. When the vector is used, the flavonoid-based material can be accumulated in rice seeds.
상기 종자발현용 과발현 식물발현운반체는 서열번호 1, 서열번호 2 또는 서열번호 3으로 표시되는 안토시아닌 색소합성에 관여하는 유전자를 센스 및 안티센스 방향으로 포함하여 안토시아닌 대사증진 운반체로 제작될 수 있다. 상기 플라보노이드는 안토시아닌일 수 있다. 본 발명의 구체예에서, 상기 안토시아닌 대사증진 운반체는 각각 PGluB1-OsC1, PGluB1-OsB1 또는 PGluB1-OsB2일 수 있다. The overexpression plant expression carrier for seed expression can be produced as an anthocyanin metabolic enhancement carrier including a gene involved in the synthesis of an anthocyanin pigment represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 in the sense and antisense direction. The flavonoid may be anthocyanin. In an embodiment of the present invention, the anthocyanin metabolism enhancer may be PGluB1-OsC1, PGluB1-OsB1 or PGluB1-OsB2, respectively.
본 발명의 구체예에서, 상기 2) 단계는 바람직하게는 아그로박테리움 LBA4404를 이용할 수 있다. 바람직하게는 상기에서 제조한 3종의 안토시아닌 대사 증진 운반체를 상기 아그로박테리움에 트리파렌탈 메이팅(triparental mating)을 통해 형질전환을 수행할 수 있다. In an embodiment of the present invention, the step 2) may preferably use Agrobacterium LBA4404. Preferably, the three kinds of anthocyanin metabolism enhancers produced above can be transformed into the Agrobacterium through triparental mating.
본 발명의 구체예에서, 상기 3) 단계는 바람직하게는 상기 플라보노이드 대사증진 운반체 3종을 이용하여 벼 형질전환을 수행할 수 있다. 상기 식물 형질 전환체는 벼(Oryza sp.)일 수 있다. In an embodiment of the present invention, step 3) is preferably carried out using three flavonoid metabolic enhancer carriers. The plant transformant may be rice (Oryza sp.).
벼 형질전환 방법으로는 5일 또는 7일간 벼 종자로부터 캘러스를 유도하여 아그로박테리움을 접종하여 형질전환체를 선발하는 조기형질전환법을 이용하여 수행할 수 있다. 가장 바람직하게는 유전자가 들어있는 아그로박테리움과 접종한 후 3일간 공동배양 후, 아그로박테리움을 제거한 후 PPT와 carbenicillin이 포함된 선발배지에 캘러스를 배양할 수 있다. 그 후 형질전환된 캘러스는 신초유기배지와 뿌리유기배지에 배양하여 완전한 식물체를 확보할 수 있다. 그 후, 0.3% 바스타를 처리하여 한번 더 선발과정을 거친 후 온실에 이식하여 종자를 수확하여 형질전환 벼 종자를 획득할 수 있다.
Rice transformation can be carried out by using an early transformation method in which a callus is induced from rice seeds for 5 days or 7 days and Agrobacterium is inoculated to select a transformant. Most preferably, it is inoculated with Agrobacterium containing the gene, co-cultivated for 3 days, Agrobacterium is removed, and callus can be cultured in a selection medium containing PPT and carbenicillin. Then, the transformed callus can be cultured in shoot organism medium and root organic medium to obtain complete plant. After that, 0.3% basta is treated and the seeds are harvested by transplanting into the greenhouse after one more selection process to obtain transgenic rice seeds.
본 발명은 또한 1) 서열번호 1, 서열번호 2 또는 서열번호 3의 염기서열을 갖는 유전자를 포함하는 형질전환용 발현벡터를 제조하는 단계; 2) 상기 제조된 발현벡터를 이용하여 식물 형질전환용 아그로박테리움을 제조하는 단계; 3) 상기 형질전환용 아그로박테리움을 이용하여 벼를 형질전환 하는 단계; 및 4) 상기 형질전환된 식물체의 종자에서 플라보노이드를 추출하는 단계;를 포함하는 플라보노이드 생산 증대 방법을 제공한다.
The present invention also provides a method for producing a transgenic plant, comprising the steps of: 1) preparing a transforming expression vector comprising a gene having the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3; 2) preparing Agrobacterium for plant transformation using the expression vector prepared above; 3) transforming rice with the transforming Agrobacterium; And 4) extracting the flavonoid from the seed of the transformed plant.
본 발명의 구체예에서, 상기 형질전환용 벡터를 이용하여 벼를 형질전환한 결과, 형질전환된 벼에서 안토시아닌의 축적이 관찰되었다. 또한 안토시아닌 생성 전사인자가 도입된 벼 종자를 이용해 종자 내의 플라보노이드 생성유무를 관찰한 결과, 형질전환 벼 종자의 호분층, 배 및 배유 등에서 플라보노이드가 축적된 것을 관찰할 수 있었다. 이상의 결과는 안토시아닌 생성 전사인자들을 종자내에서 발현함으로써, 종자 내의 플라보노이드 생합성 유전자의 발현을 증대시킬 수 있고, 플라보노이드 계열 물질을 생성할 수 있음을 의미한다. 또한 배유 조직에 플라보노이드를 축적하기 위해서 초기단계의 여러 유전자를 다수로 발현시키지 않고, 전사인자만을 발현시키는 것만으로도 충분히 기능성 플라보노이드 물질을 생성할 수 있음을 의미한다.
In an embodiment of the present invention, anthocyanin accumulation was observed in the transformed rice as a result of transformation of rice with the above transformation vector. In addition, the presence of flavonoids in seeds using rice seeds transfected with anthocyanin - producing transcription factors was observed, and it was observed that flavonoids were accumulated in the stratum corneum, the embryo and the endosperm of transgenic rice seeds. These results indicate that expression of anthocyanin-producing transcription factors in seeds can increase expression of flavonoid biosynthesis genes in seeds and produce flavonoid-based materials. In addition, it means that sufficient functional flavonoid materials can be produced by expressing only the transcription factor without expressing many genes at the early stage in order to accumulate flavonoids in the endosperm tissue.
다른 식으로 정의되지 않는 한, 본 명세서에서 사용된 모든 기술적 및 과학적 용어들은 본 발명이 속하는 기술분야에서 숙련된 전문가에 의해서 통상적으로 이해되는 것과 동일한 의미를 갖는다. 일반적으로, 본 명세서에서 사용된 명명법은 본 기술분야에서 잘 알려져 있고 통상적으로 사용되는 것이다.
Unless otherwise defined, 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 is well known and commonly used in the art.
본 발명의 서열번호 1, 서열번호 2 또는 서열번호 3으로 표시되는 안토시아닌 색소합성에 관여하는 유전자를 이용하여 형질전환한 식물체는 식물체 및 종자에서 플라보노이드의 생산이 증가될 수 있다. 이러한 플라보노이드 생산이 증가된 벼 개발을 통해 쌀 이용성 증대에 기여할 수 있다.
Plants transformed with genes involved in the synthesis of anthocyanin pigments of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 of the present invention may increase the production of flavonoids in plants and seeds. The development of rice with increased flavonoid production can contribute to the enhancement of rice availability.
도 1 은 본 발명의 구체예에 따른 종자특이 과발현 운반체의 모식도이다(GluB1: glutelin B1유전자의 프로모터, PinII: 터미네이터, 35S: 항시발현프로모터, Bar: phosphinothricin acetyltransferase, Nos: 터미네이터).
도 2는 본 발명의 구체예에 따른 안토시아닌 대사증진 운반체 3종 (PGluB1-OsB1, PGluB1-OsB2, PGluB1-OsC1)의 모식도이다.
도 3은 본 발명의 구체예에 따른 벼 형질전환과정을 나타낸 모식도이다.
도 4는 본 발명의 구체예에 따른 유색벼(흑진주)와 일반벼(일미)의 형질전환체의 표현형을 나타낸 사진이다.
도 5는 본 발명의 구체예에 따른 PCR 검정을 통해 벼 형질전환체에 OsB1, OsB2, OsC1 유전자의 도입여부를 확인한 결과이다.
도 6은 본 발명의 구체예에 따른 서던 블롯 분석을 통해 도입유전자(OsB1, OsB2, OsC1)의 카피수를 확인한 결과이다.
도 7은 본 발명의 구체예에 따른 Taqman PCR 분석을 통한 동형접합체의 조기선발 시스템을 나타낸다. ( * : 동형접합체인 control과 동일한 copy를 지니는 개체)
도 8은 본 발명의 구체예에 따른 DPBA 염색법을 통한 종자내의 플라보노이드 축적 여부를 확인한 결과이다. (좌: T1세대의 종측, 횡측 절단개체들의 DPBA 염색사진; 우: DPBA염색 대표종자사진).
도 9는 본 발명의 구체예에서 도입유전자(OsB1, OsB2, OsC1)에 따른 현미종자의 총 플라보노이드 함량(Total flavonoid content: TFC)을 나타낸 그래프이다.
도 10은 본 발명의 구체예에서 도입유전자(OsB1, OsB2, OsC1)에 따른 현미종자의 총 플라보노이드 함량(Total flavonoid content: TFC)을 나타낸 그래프이다. FIG. 1 is a schematic diagram of a seed specific overexpression carrier according to an embodiment of the present invention (GluB1: glutelin B1 gene promoter, Pin II: terminator, 35S: always expression promoter, Bar: phosphinothricin acetyltransferase, Nos: terminator).
2 is a schematic diagram of three kinds of anthocyanin metabolism enhancing carriers (PGluB1-OsB1, PGluB1-OsB2, PGluB1-OsC1) according to an embodiment of the present invention.
3 is a schematic diagram showing a rice transformation process according to an embodiment of the present invention.
4 is a photograph showing the phenotype of the transformant of colored rice (black pearl) and general rice (sun light) according to an embodiment of the present invention.
FIG. 5 shows the result of confirming the introduction of the OsB1, OsB2, and OsC1 genes into the rice transformants through a PCR assay according to an embodiment of the present invention.
FIG. 6 is a result of confirming copy number of transgene (OsB1, OsB2, OsC1) through Southern blot analysis according to an embodiment of the present invention.
Figure 7 shows an early selection system of homozygotes with Taqman PCR analysis according to embodiments of the present invention. (*: An object with the same copy as a homozygous control)
FIG. 8 is a result of confirming accumulation of flavonoids in the seeds by the DPBA staining method according to the embodiment of the present invention. (Left: T1 generation, DPBA staining photograph of transverse cut individuals; right: DPBA staining representative seed photograph).
FIG. 9 is a graph showing the total flavonoid content (TFC) of brown rice seeds according to the transgene (OsB1, OsB2, OsC1) in the embodiment of the present invention.
10 is a graph showing the total flavonoid content (TFC) of brown rice seeds according to the transgene (OsB1, OsB2, OsC1) in the embodiment of the present invention.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는 것은 이 기술분야에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.
Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not construed as being limited by these embodiments.
실시예Example 1. 실험 방법 1. Experimental Method
실시예Example 1-1. 기능성 안토시아닌 색소 합성이 증진된 벼 식물체 개발을 위한 운반체 제작 및 형질전환 1-1. Production and Transformation of Carrier for Development of Rice Plants Enhanced Functional Anthocyanin Pigment Synthesis
플라보노이드계열의 천연물질은 가공식품첨가제, 천연방부제등 다양한 산업분야에 이용되고 있을뿐 아니라 뛰어난 항산화효과와 더불어서 다양한 기능성으로 인해서 의료용으로도 주목받고 있다. 벼는 3대 주곡작물로써 전세계의 많은 인류의 식량공급원으로 사용되고 있다. 하지만 다양한 기능성을 지니는 물질들은 종자의 호분층에 존재하고 호분층이 도정되어 먹는 백미의 경우 기능성물질의 섭취가 상대적으로 낮아진다. 본 연구에서는 벼 배유내로 기능성 안토시아닌 물질생성을 축적하고자 벼 내재의 안토시아닌 생성 기작을 이해하고 이를 바탕으로 물질생성과 관련한 유전자를 선발하여 발현운반체를 제작하여 형질전환 식물체를 만들었고, 종자내의 물질생성 가능성을 확인하고자 하였다.
Flavonoid-based natural materials are not only used in various industrial fields such as processed food additives, natural preservatives, but also have excellent antioxidative effects and are attracting attention for medical use due to their various functionalities. Rice has been used as a food source for many humans all over the world as the three most important crops. However, materials with diverse functionalities are present in the horny layer of the seed, and in the case of white rice, the consumption of functional materials is relatively low. In order to accumulate functional anthocyanin production in rice embryo, we studied the anthocyanin production mechanism of rice intrinsic factor, and selected transgenic genes for the production of transgenic plants. Respectively.
실시예Example 1-1-1. 안토시아닌 생성 유전자 탐색 1-1-1. Search for anthocyanin-producing genes
기능성물질생성과 관련한 전사인자를 분리하고자 KOME, NCBI, GRAMENE등에서 안토시아닌 색소합성과 관련한 유전자 정보를 탐색하였다. 안토시아닌 색소합성 전체를 조절하기 위해서 MYB관련 전사인자 1종(OsC1), bHLH관련 전사인자 2종(OsB1과 OsB2)을 PCR을 이용하여 증폭하였다. 본실험에 유전자 클로닝을 위해 사용된 프라이머는 표 1에 정리하여 나타내었다. 증폭한 DNA의 염기서열을 확인하기 위해서 각각을 pGEM-T Easy Vector (Promega)에 클로닝하여 염기서열을 분석하였다. 표 1은 본 실험에 사용된 유전자 및 프로모터 클로닝을 위한 프라이머 염기서열을 나타낸다.
In order to isolate the transcription factors involved in the production of functional substances, gene information related to the synthesis of anthocyanin pigments was searched in KOME, NCBI, and GRAMENE. In order to control the synthesis of anthocyanin pigment, one MYB-related transcription factor (OsC1) and two bHLH-related transcription factors (OsB1 and OsB2) were amplified by PCR. Table 1 summarizes the primers used for gene cloning in this experiment. In order to confirm the nucleotide sequence of the amplified DNA, each was cloned into pGEM-T Easy Vector (Promega) to analyze the nucleotide sequence. Table 1 shows the primer base sequences for the gene and promoter cloning used in this experiment.
실시예Example 1-1-2. 식물발현운반체 제작 1-1-2. Production of plant expression carrier
벼 종자내에 플라보노이드계열 물질을 축적하기 위해서는 배유내의 물질축적을 위한 프로모터가 필요하다. 종자특이 프로모터로 glutelin B1유전자의 프로모터영역을 분리하고 gateway용 운반체 제작을 위해서 RFA 유전자와 연결하여 종자발현용 과발현 식물발현운반체를 제작완료하였다. 플라보노이드 대사증진을 위하여 전사인자 3종의 과발현 운반체를 구축하였고 아그로박테리움 LBA4404에 트리파렌탈 메이팅(triparental mating)을 통해 형질전환을 수행하였다.
In order to accumulate flavonoid-based materials in rice seeds, a promoter for accumulating substances in the feed oil is required. The promoter region of glutelin B1 gene was isolated with a seed - specific promoter and ligated with the RFA gene to construct a carrier for the gateway. In order to promote flavonoid metabolism, three transcription factor overexpression carriers were constructed and transfection was carried out through triparental mating to Agrobacterium LBA4404.
실시예Example 1-2. 안토시아닌 증진 운반체를 이용한 새로운 천연색소 생산 식물 소재 개발 1-2. Development of new natural pigment production plant material using anthocyanin enhancer
실시예Example 1-2-1. 형질전환 1-2-1. Transformation
플라보노이드 대사증진 운반체 3종을 이용하여 벼 형질전환을 수행하였다. 벼 형질전환방법으로는 5일 또는 7일간 벼 종자로부터 캘러스를 유도하여 Agrobacterium을 접종하여 형질전환체를 선발하는 조기형질전환법을 이용하여 수행하였다(Toki et al., 2006). 유전자가 들어있는 Agrobacterium과 접종한 후 3일간 공동배양 후, Agrobacterium을 제거한 후 PPT와 carbenicillin이 포함된 선발배지에 캘러스를 배양한 후 형질전환된 캘러스는 신초유기배지와 뿌리유기배지에 배양하여 완전한 식물체를 확보하고 0.3% 바스타를 처리하여 한번 더 선발과정을 거친 후 온실에 이식하여 종자를 수확하였다.
Rice transformation was carried out using three kinds of flavonoid metabolism enhancer. Rice transformation was carried out using an early transformation method (Toki et al., 2006), in which transgenes were selected by inoculating Agrobacterium with callus induction from rice seeds for 5 days or 7 days. After incubation for 3 days with Agrobacterium containing the gene, the Agrobacterium was removed and the callus was cultured in the selection medium containing PPT and carbenicillin. The transformed callus was cultured in shoot organism medium and root organic medium, And 0.3% basta. After the selection process, seeds were harvested by transplanting in the greenhouse.
실시예Example 1-2-2. 벼 형질전환체의 분자세포생물학적 분석 1-2-2. Molecular Cell Biological Analysis of Rice Transformants
형질전환체의 유전자도입여부를 확인하고자 벼의 잎 조직에서 genomic DNA를 분리하여 attB1과 attB2 프라이머를 이용하여 PCR 검정을 실시하였다. PCR 조건은 95°C에서 3분간 전변성과정을 거친후 95°C에서 15초, 60°C에서 30초, 72°C에서 2분을 1회로 하여 30회를 실시후 72°C에서 10분간 extension을 실시하였다. PCR 산물은 1% agarose gel에 전기영동하여 확인하였다. Genomic DNA was isolated from the leaves of rice plants and attB1 and attB2 primers were used for PCR. The PCR conditions were 95 ° C for 3 minutes, 95 ° C for 15 seconds, 60 ° C for 30 seconds, 72 ° C for 2 minutes, 30 cycles, and 72 ° C for 10 minutes extension. PCR products were confirmed by electrophoresis on 1% agarose gel.
도입유전자의 copy수를 확인하고자 Kim (2002)의 방법을 이용하여 genomic DNA를 분리하였고, 20 μg DNA를 제한효소 EcoRI로 처리하여 0.8% agarose gel에 전기영동한 다음, capillary transfer 방법 (Southern 1975)으로 nylon membrane에 전이시켰다. Membrane은 0.5 M sodium phosphate buffer(pH 7.2), 7% SDS, 1% BSA, 1 mM EDTA, 0.1 mg/ml denatured salmon sperm DNA가 첨가된 용액에서 3시간 (65℃)동안 prehybridization 한 다음, [α-32P]dCTP로 표지된 Mar DNA를 첨가하여 12시간 hybridization하였다. Membrane은 2 X SSC, 0.1% SDS 용액 (65℃)에서 20분, 1 X SSC, 0.1% SDS용액 (65℃)에서 20분 세척한 후 Bio-imaging analyzer (BAS-2000; Fuji Photo Film, Japan)로 분석하였다. Genomic DNA was isolated using the method of Kim (2002) to confirm the copy number of transgene. 20 μg of DNA was treated with restriction enzyme EcoRI and electrophoresed on 0.8% agarose gel. Then, capillary transfer method (Southern 1975) To the nylon membrane. Membrane was prehybridized for 3 hours (65 ° C) in a solution containing 0.5 M sodium phosphate buffer (pH 7.2), 7% SDS, 1% BSA, 1 mM EDTA and 0.1 mg / ml denatured salmon sperm DNA. -32P] dCTP-labeled Mar DNA was added and hybridized for 12 hours. Membrane was washed with 2 × SSC, 0.1% SDS solution (65 ° C.) for 20 minutes, 1 × SSC, 0.1% SDS solution (65 ° C.) for 20 minutes and then transferred to a Bio-imaging analyzer (BAS-2000; Fuji Photo Film, Japan ).
조기에 도입유전자의 동형접합체(homozygote)을 선발하기 위해서 TaqMAN PCR을 실시하였다. 운반체에 포함된 BAR영역의 Nos terminator부분을 probe와 primer로 제작한 것과 식물체내의 tubulin유전자를 reference 유전자로 PCR을 실시하고 분석하였다.TaqMan PCR was performed to select homozygotes homologous to the transgene early. Nos terminator of the BAR region in the carrier was constructed with probes and primers, and tubulin gene in the plant body was analyzed by PCR using the reference gene.
전사인자가 도입된 벼 형질전환체의 종자에서의 플라보노이드 생성여부를 확인하고자 DPBA 염색을 실시하였다 (Murphy et al., 2000). 수정후 20일정도의 벼 종자를 횡축과 종축으로 절단하여 0.25% DPBA와 0.02% Triton X-100을 녹인 용액에 상온에서 15-20분에 염색을 실시한 후 450-490nm에서 관찰하였다. DPBA staining was performed to determine whether flavonoids were produced in seeds of transgenic rice plants transfected with transcription factors (Murphy et al., 2000). After fertilization, 20 seeds of rice were cut into transverse and longitudinal axes and stained in a solution of 0.25% DPBA and 0.02% Triton X-100 at room temperature for 15-20 minutes and observed at 450-490 nm.
전사인자가 도입된 벼 형질전환체의 종자에서의 플라보노이드 생성량을 확인하고자, 왕겨를벗긴 현미종자와 도정한 도정종자를 이용하여 종자내의 총 플라보노이드 함량을 분석하였다. 시료를 마쇄하여 50% 메탄올을 가하여 실온추출한 후 추출물 시료 0.5ml에 diethylene glycol 5ml씩 가하여 혼합하였다. 그후 1N NaOH를 0.5ml을 가하여 잘 혼합한 후 37℃에서 1시간동안 반응시켰다. 1시간후 UV-VIS spectrophotometer로 420nm 흡광도를 측정하였다. 표준물질은 naringin의 표준검량곡선을 작성하여 총 플라보노이드 함량을 % naringin equivalent로 환산하였다.
To determine the amount of flavonoids produced in seeds of rice transgenic plants transfected with transcription factor, the total flavonoid content in the seeds was analyzed using rice husks isolated from rice husks and cultivated seeds. After the sample was ground, 50% methanol was added and the mixture was extracted at room temperature. 5 ml of diethylene glycol was added to 0.5 ml of the extract sample. Then, 0.5 ml of 1N NaOH was added, mixed well, and reacted at 37 ° C for 1 hour. After 1 hour, absorbance at 420 nm was measured by UV-VIS spectrophotometer. The standard calibration curves of naringin were prepared and the total flavonoid content was converted to% naringin equivalent.
실시예Example 2. 실험 결과 2. Experimental results
실시예Example 2-1. 기능성 안토시아닌 색소합성을 벼 식물체개발을 위한 운반체 제작 및 형질전환 2-1. Production and Transformation of Carrier for Development of Rice Plants by Functional Anthocyanin Pigment Synthesis
실시예Example 2-1-1. 안토시아닌 생성 유전자 탐색 2-1-1. Search for anthocyanin-producing genes
안토시아닌 색소합성 증대를 위하여 안토시아닌 색소를 축적하는 벼 식물체를 이용하여 bHLH관련 전사인자 2종(OSB1과 OSB2)와 MYB관련 전사인자 1종(OsC1)을 분리하였다. 표 2는 벼 안토시아닌 대사증진 유전자를 나타낸다.
To increase the synthesis of anthocyanin pigment, two bHLH - related transcription factors (OSB1 and OSB2) and one MYB - related transcription factor (OsC1) were isolated using rice plants that accumulate anthocyanin pigments. Table 2 shows the rice anthocyanin metabolism promoting gene.
실시예Example 2-1-2. 식물발현운반체 제작 2-1-2. Production of plant expression carrier
벼 종자특이 유전자인 glutelin B1유전자의 프로모터영역과 gateway용 RFA 유전자와 연결하여 gateway용 종자발현용 과발현 식물발현운반체 pMJ-PGLUB1-RFA를 제작 완료하였다 (도 1). 종자특이적으로 플라보노이드물질을 증진시키고자 대사증진운반체 3종을 제작 완료하였다 (도 2).
The overexpressed plant expression carrier pMJ-PGLUB1-RFA for the expression of the seed for the gateway was completed by connecting the promoter region of the glutelin B1 gene and the RFA gene for the gateway to the rice seed specific gene (Fig. 1). Three kinds of metabolism promotion carriers were produced to promote flavonoid substances specifically for seeds (Fig. 2).
실시예Example 2-2. 안토시아닌 증진 운반체를 이용한 새로운 천연색소 생산 식물 소재 개발 2-2. Development of new natural pigment production plant material using anthocyanin enhancer
실시예Example 2-2-1. 형질전환 2-2-1. Transformation
기능성 플라보노이드 물질을 증진시키고자 안토시아닌을 생성하는 흑진주벼와 일반벼 일미품종을 이용하여 도 3와 같은 과정을 통하여 벼형질전환을 실시하였다. 흑진주 벼를 이용하여 OsC1유전자를 단독으로 형질전환시킨 개체에서 잎, 화경, 엽설, 엽귀 등의 조직에서 안토시아닌의 축적이 관찰되었으나, 일미벼를 이용하여 형질전환시킨 경우에는 잎, 화경, 엽설, 엽귀 등을 포함한 모든 조직에서 안토시아닌의 축적이 관찰되지 않았다 (도 4). 이러한 결과는 천연색소 안토시아닌을 생산하는 흑진주벼의 경우에는 안토시아닌 대사증진을 위해 OsC1 유전자만이 발현되어도 충분히 물질생성이 이루어짐을 나타내나, 안토시아닌 물질을 생성하지 못하는 일미벼의 경우에는 OsC1 유전자 이외에도 색소합성을 위한 추가적인 유전자가 필요하다고 판단된다.
Rice transgenic transformation was carried out by using a black rice paddy rice and an ordinary paddy rice cultivar which produce anthocyanin to enhance the functional flavonoid material through the process as shown in Fig. Anthocyanin accumulation was observed in tissues such as leaves, flower buds, leaf buds, and lobules in the case of OsC1 gene alone transformed using the black pearl rice. However, when transformed with the rice leaf, leaves, flower buds, But no accumulation of anthocyanin was observed in all tissues (Fig. 4). These results show that in the case of black pepper rice, which produces natural pigment anthocyanin, only the OsC1 gene is expressed sufficiently for the promotion of anthocyanin metabolism. However, in the case of Ilmiri, which does not produce anthocyanin substances, Additional genes are needed.
실시예Example 2-2-2. 벼 형질전환체의 분자세포생물학적 분석 2-2-2. Molecular Cell Biological Analysis of Rice Transformants
형질전환 벼의 잎 조직에서 genomic DNA를 분리하여 PCR 검정을 통하여 유전자의 도입여부를 확인하였다. 도 5에서처럼 선발된 식물체의 대부분은 도입된 유전자가 증폭되어 안정적으로 유전자가 도입된 것으로 확인되었다.
The genomic DNA was isolated from the leaf tissue of transgenic rice and the gene was introduced by PCR. As shown in FIG. 5, it was confirmed that most of the selected plants amplified the introduced gene and stably introduced the gene.
Southern blot 분석을 통하여 유전자 도입된 개체의 copy수를 확인하였다(도 6). 도 6에서 보는 바와 같이 OSB2 유전자가 도입된 형질전환체에서는 도입유전자수가 1개에서 6개까지 다양하게 나타났으며, MAR 영역이 recombintion된 개체들도 확인되었다. OSB1유전자가 단독으로 형질전환된 개체들은 3개의 유전자가 도입되었음을 확인하였고, OSC1유전자로 형질전환된 개체에는 도입유전자의 수가 2개 또는 3개 도입됨과 더불어서 MAR 영역이 recombintion된 개체들도 확인되었다.
Southern blot analysis confirmed the copy number of the transgenic individuals (Fig. 6). As shown in FIG. 6, in the transformants into which the OSB2 gene was introduced, the number of introduced genes varied from one to six, and recombinant MARs were also identified. The OSB1 gene alone transfected with three genes was transfected with the OSC1 gene, and the transgenic cells were transfected with two or three transgenes.
유전자의 기능을 확인하기 위해서는 조기에 도입유전자의 동형접합체 (homozygote)을 확인하는 것이 요구된다. T0 세대에서 1 copy의 유전자가 도입된 OSB2-4 라인의 T1식물체를 분석하여 조기에 도입유전자를 동형접합체로 가지고 있는 개체들을 선발하는 시스템을 구축하였다. 내재유전자는 벼의 tubulin 유전자를 이용하고 T-DNA의 Nos 터미네이터 부분을 이용하여 taqMAN PCR을 실시하였다 (도 7). 도 7 에서처럼 총 21개체의 식물체를 분석한 결과 동형접합체인 control과 동일한 copy를 지니는 개체가 7개체 확인되었고, 나머지 개체들은 이형접합체로 확인되었다. 결과적으로 21개의 분석개체들은 동형접합체:이형접합체=7:14로 분리됨을 확인할 수 있었고, 이런 결과는 예상되는 결과(homo: hetero=1:2)와도 일치하였다. 이런 방법을 통하여 조기에 동종접합체를 선발함으로 유전자의 기능을 좀 더 신속하고 정확하게 확인하는 것이 가능하리라 판단된다.
In order to confirm the function of the gene, it is necessary to confirm the homozygote of the transgene early. In the T0 generation, a system was constructed to analyze the T1 plants of the OSB2-4 lines into which one copy of the gene was introduced and to identify individuals having the transgene as an early transgene. The inherent gene was subjected to taqMAN PCR using the rice tubulin gene and the Nos terminator portion of T-DNA (Fig. 7). As shown in FIG. 7, when a total of 21 plants were analyzed, 7 individuals having the same copy as the homozygous control were identified, and the remaining individuals were identified as heterozygotes. As a result, it was confirmed that 21 analytes were homozygous: heterozygote = 7: 14, and this result was consistent with the expected result (homo: heter = 1: 2). In this way, it is possible to identify the function of genes more quickly and accurately by early selection of homozygotes.
안토시아닌 생성 전사인자가 도입된 일미 형질전환체의 T1 종자를 이용하여 DPBA 염색법을 통하여 종자내의 플라보노이드 생성유무를 관찰하였다(도 8). 벼의 경우에는 보통 호분층과 배에서 플라보노이드가 축적되는 것으로 보고되고 있으며, 본 실험에서도 일미 비형질전환체에서 호분층과 배에서 플라보노이드가 관찰되었다. 안토시아닌 생성 전사인자가 도입된 형질전환체의 종자를 살펴보면 배와 호분층 이외에도 종자내부의 배유조직에서도 플라보노이드 물질의 축적이 확인되었다. 이러한 결과는 종자특이적으로 발현유도를 위해 사용된 GluB1 프로모터가 기존의 보고와 동일하게 종자의 호분층 외에도 종자내의 배유에 강한 발현을 보인다는 것과 일치하는 결과이다(Qu와 Takaiwa, 2004). 이상의 결과는 안토시아닌 생성 전사인자들이 종자내에서 발현함으로 종자내의 플라보노이드 생합성 유전자의 발현을 증대시키고 플라보노이드계열 물질을 생성하였음을 의미하고 있으며 배유조직에 플라보노이드를 축적하기 위해서 초기단계의 여러 유전자를 다수로 발현시키지 않고, 전사인자만을 발현시키는 것만으로도 충분히 기능성 플라보노이드 물질생성을 긍정적인 영향을 미침을 확인하였다.
The presence of flavonoids in the seeds was observed by DPBA staining using T1 seeds of S. aureus transformants transfected with anthocyanin-producing transcription factors (Fig. 8). In the case of rice, it is reported that flavonoid is accumulated in the normal phloem and abdomen. In this experiment, flavonoid was also observed in the phloem and abdomen of the non - transformant. The accumulation of flavonoid substances was confirmed in the endosperm tissue of the seeds as well as in the embryo and hornbone layers when the anthocyanin transcription factor was introduced into the transgenic seeds. These results are consistent with the fact that the GluB1 promoter used for seed-specific induction of expression is strongly expressed in the endosperm of the seed as well as in the horn of the seed (Qu and Takaiwa, 2004). These results indicate that anthocyanin-producing transcription factors are expressed in seeds, thereby increasing expression of flavonoid biosynthesis genes in seeds and producing flavonoid-based materials. In order to accumulate flavonoids in endosperm, It was confirmed that the expression of only the transcription factor had a positive effect on the production of the functional flavonoid substance sufficiently.
벼 종자내의 플라보노이드 생성량을 확인하고자 현미 종자와 도정미 종자를 이용하여 플라보노이드 총함량을 분석하였다. 벼는 일반적으로 현미 상태의 경우, 호분층과 배에서도 플라보노이드가 축적된다. 그러나 도정을 한 백미의 경우에는 유용한 플라보노이드의 성분이 거의 존재하지 않는다. 따라서 백미의 배유 내로 플라보노이드 성분을 축적시키면 효과적으로 기능성 물질을 섭취하는 것이 용이할 수 있다. 이에 따라 본 실험에서는 도정미를 분석함으로써 배유 내의 플라보노이드 축적을 확인하였다. Total amount of flavonoids was analyzed by using brown seeds and rice seeds to determine the amount of flavonoids produced in rice seeds. Rice generally accumulates flavonoids in the hornbanks and stomachs in the case of brown rice. However, there are few components of useful flavonoids in the case of white rice. Therefore, if the flavonoid component is accumulated in the dietary fiber of white rice, it may be easy to effectively ingest the functional substance. In this experiment, the flavonoid accumulation in the endosperm was confirmed by analyzing the rice bran.
먼저 현미종자의 총 플라보노이드 함량을 분석한 결과, bHLH계열 유전자인 OsB2의 경우에는 대조구인 일미에 비해서 총 플라보노이드 함량이 14 - 28% 증가하였고, OsB1의 경우에는 총 플라보노이드 함량이 10 - 13% 증가됨이 확인되었다 (표 3). MYB계열인 OsC1의 경우에는 대조구인 일미(비형질전환체)와 비슷한 수준 또는 10% 정도 총 플라보노이드 함량이 증가되었다. 이러한 결과는 종자내의 전사인자의 발현으로 총 플라보노이드 함량이 증가된 것으로 생각된다. 전사인자별 플라보노이드 증대효과를 살펴보면, OsB2> OsB1> OsC1 의 순으로 유전자로 플라보노이드 함량이 증가되었다 (도 9). As a result of analysis of total flavonoid content of brown rice seeds, the total flavonoid content was increased by 14 - 28% in the bHLH gene gene OsB2 compared to that of the control group, and in the case of OsB1, the total flavonoid content was increased by 10 - 13% (Table 3). In the MYB line OsC1, the total flavonoid content was increased by about 10% or about the same level as that of the control (untransformed). These results suggest that the total flavonoid content is increased by the expression of transcription factors in the seeds. The increase in flavonoid by transcription factor was increased in the order of OsB2> OsB1> OsC1 (Fig. 9).
도정 종자의 총 플라보노이드 함량을 분석한 결과, bHLH계열 유전자인 OsB2의 경우에는 대조구인 일미에 비해서 총 플라보노이드 함량이 비슷한 수준 또는 20% 증가됨을 확인할 수 있었고, OsB1의 경우에는 총 플라보노이드 함량이 비슷한 수준 또는 28% 정도 증가됨이 확인되었다 (표 3). MYB계열인 OsC1의 경우에는 대조구인 일미와 비슷한 수준 또는 5% 정도 총 플라보노이드 함량이 증가되었다. 전사인자별 도정종자내의 플라보노이드 함량 증대효과를 살펴보면, OsB1> OsB2> OsC1의 순으로 증가됨을 확인할 수 있었다. 표 3은 전사인자별 현미종자와 도정종자내의 총 플라보노이드 함량을 나타낸다.
As a result of analysis of total flavonoid content in the seeds, it was confirmed that the total flavonoid content was increased by 20% or more in OsB2 gene of the bHLH gene group compared with that of control group, and in the case of OsB1, the total flavonoid content was similar or (Table 3). In the case of MYB line OsC1, the total flavonoid content was increased by 5% or similar to that of control. The increase of flavonoid contents in transgenic seeds by transcription factors was increased in the order of OsB1>OsB2> OsC1. Table 3 shows the total flavonoid content in the brown seed and the marine seed by transcription factor.
이상의 결과를 살펴보면, 플라보노이드 생합성과 관련된 bHLH 전사인자와 MYB전사인자를 종자내에 발현시킴으로 종자내의 플라보노이드 생합성을 증대하는 효과를 확인할 수 있었다.These results indicate that the expression of bHLH transcription factor and MYB transcription factor related to flavonoid biosynthesis in seeds enhances flavonoid biosynthesis in seeds.
<110> REPUBLIC OF KOREA <120> TRANSGENIC RICE PRODUCING HIGH LEVEL OF FLAVONOIDS AND MANUFACTURING METHOD THEREOF <130> P14R12D0962 <160> 15 <170> KopatentIn 2.0 <210> 1 <211> 819 <212> DNA <213> Artificial Sequence <220> <223> OsC1 nucleotide sequences <400> 1 atggggagga gagcttgctg cgcaaaggaa gggatgaaga gaggggcatg gacgagcaag 60 gaggacgacg tgcttgcctc ctacatcaag tcccatggcg aaggcaagtg gcgcgaggtc 120 ccccaacgag ctggtttgag gcggtgcggc aagagctgca ggctccggtg gctcaactat 180 ctccggccta acatcaagcg cggcaacatc gacgacgacg aggaggagct catcgtcagg 240 ctccacaccc tcctcggcaa caggtggtct ctcattgcag gcaggctgcc gggccgaaca 300 gacaatgaaa tcaagaacta ctggaacagc acgctcagcc gcaagatcgg caccgccgcc 360 accgccgccg ccggcagccg cggtggcagc acgccggaca ccgccagagc gacggacgcg 420 gcgtcgtcca gctccgtcgt gcagccgggc cagcagcagc agccagcctc ccgcgccgac 480 accgacacag caacggcagc ggcggcggcg gcggcgacga cgaccaccgt gtgggcgccc 540 aaggccgtgc ggtgcacgcg cgggttcttc ttccacgacc gtgaaacggc gccgctcgcc 600 gcggcggcgc cggcgccggc aggggaatta ggagacggcg atgacgtcga ctgcgactac 660 tactgcagcg gcagcagctc ggcggcgacg acgacgtcgt cgagctcatt accggcggtc 720 gtcgagccgt gcttctccgc cggcgacgac tggatggacg acgtgagagc cttggcgtcg 780 tttcttgaca ccgacgacgc ctggaacttg tgtgcgtga 819 <210> 2 <211> 1767 <212> DNA <213> Artificial Sequence <220> <223> OsB1 nucleotide sequences <400> 2 atggaagaga cccctctgcc atccgggaag aacttcagga gccagcttgc tgctgcagcg 60 aggagcatca attggacgta tgccatattt tggtccattt caaccagccg cccaggagtt 120 ctgacttgga aggacggctt ctacaacggc gagataaaga cgaggaagat cacgaactcc 180 atgaacctca cggccgacga gctggtcctg cagagaagcg agcagctgag ggagctctac 240 gactctctcc tctccggcga gtgcggccac cgagcgagga ggcccgtcgc tgcactgttg 300 ccggaagatc tcggggacac ggaatggtac tacgtcgtct gcatgaccta cgccttcggc 360 ccccgccaag ggttgccagg caaaagcttc gcaagcaatg aatttgtttg gctgacaaac 420 gctcagtctg cagatagaaa actattccat cgcgcgctta tagcaaagag tgcatctatt 480 aagacaatcg tctgcgtgcc atttatcatg catggtgtcc ttgagctcgg gaccactgat 540 ccgatttcgg aggacccggc tctcgtcgac cgtatcgcgg cgtcgttctg ggatacgccg 600 ccccgcgcgg cgttctcgtc ggaggcggga gacgccgaca tcgtcgtctt cgaagacctc 660 gaccatggca acgccgccgt cgaagcgacg acgacgacgg tcccggggga gccacacgcg 720 gtagccggcg gcgaggtcgc cgagtgcgag cccaactccg acaacgacct cgagcagatc 780 accatggacg acatcggcga gctctacagc ctctgcgagg agctggacgt cgtgcgccct 840 ctcgacgacg acagtagcag ctgggcggtc gcggatccct ggtcgtcctt tcagctagtt 900 ccgacgtctt ctccggcgcc ggatcaggcg ccggcggcgg aagctactga cgtcgacgac 960 gtcgtcgtcg ccgctttaga cagtagctcc attgatggat cttgcaggcc gtcgccgtcg 1020 agttttgtgg cgtggaagag gacggcggac tcggacgagg tgcaggccgt gccgctcatc 1080 agcggagagc cgccacagaa gttgctgaag aaagctgtcg ccggagccgg tgcctggatg 1140 aacaatggtg acagcagcgc ggcggcgatg acgactcaag gaagcagcat caagaaccat 1200 gtcatgtcag agagaaggcg ccgggagaag ctcaacgaga tgttcctgat tctcaaatca 1260 gttgtcccgt ccattcacag ggtggacaaa gcatccattc tcgcagaaac gatagcctac 1320 ctcaaagagc tggagaaaag agtggaagag ctggaatcca gcagccaacc atcgccatgt 1380 ccattggaaa caagaagcag gcgaaagtgc cgtgagatca ctgggaagaa ggtttctgca 1440 ggagcgaaga gaaaggcgcc ggcgccggag gtggccagcg acgacgacac cgacggggag 1500 cggcgccatt gtgtgagcaa cgtgaacgtc accatcatgg acaacaagga ggttctcctc 1560 gagctgcaat gccagtggaa ggaattgctg atgacgagag tgttcgacgc gatcaaggga 1620 gtctccctgg atcctctcgg tgcaggcatc aacatcggat ggtctccttg gactgaagat 1680 acaagccaag tttgcctcat ctgctgccgt cgaacctggg atgattacag aagctctccg 1740 gaaagctata gcaagctagc tagctag 1767 <210> 3 <211> 1356 <212> DNA <213> Artificial Sequence <220> <223> OsB2 nucleotide sequences <400> 3 atggcatctg ctcctccagt tcaggaagaa gccctacagc cagggacgaa ccacttcagg 60 agccggcttg ctgctgctgt gaggagcatc agttggagtt acaccatctt ttggtccact 120 tcaaccagtc tcccgggagt tctgacttgg aacgatggat tctacaacgg cgaggtaaag 180 acgaggaaga tatccaactt agaggacctc accgctgacc agctcgtcct gcggagaagc 240 gagcaactga gcgagctcta ctactctctc ctgtccggcg agtgcgacca ccgggcaagg 300 aagcccgtcg ccgcactgtc gccggaagat atcgcggaca cagaatggta ctacgttgtc 360 tgcatgacct acgcctttcg acccggccaa gggttacccg gcagaagcta tgcaagcaat 420 cgatctgttt ggttgtgcaa tgctcagtct gcagatagca aaacctttct acgtgcgctc 480 ttagcgaaga gcgcgtctat tcagacaatt gtctgcatcc ccttcatgag tggcgtgctt 540 gagctgggaa ccactgatcc ggtttcggag gacccaaact tggtaaaccg aatcgttgca 600 tatttgaagg agcttcagtt tccgatatgc ttagaggtac cgagttctac tccttctcca 660 gacgaaacag aagatgccga caccgtgttc gatggcctca ttgaagagga ccagatggtc 720 atactccagg gagaagacga gctaggcgac gtcgtcgtcg ctgagtgcga gaccaacggc 780 gccaaccccg aaacgatcac catggagacc gacgagttct acagcctctg cgaggagctg 840 gacctggacc tcggttctta tcagctagtc ccgacgtcgg cacgggagac ggtggctgcg 900 gcggcggcgg cggctaacga tgtcgacggc gttgcatact ctcacgcctc gtgtttcgtg 960 tcatggaaaa gagcgaaccc ggcggagaag gtggtggccg tgccgatgac tgcaggcata 1020 gagtcacaga agttgctgaa gaaagctgta ggcggcggca ccgcatggat gagtaatatt 1080 gatgatcgtg gtagcgtggc aataacgacg actccaggaa gtaacatcaa gagccatgtc 1140 atgtcagaga gaaggcgccg agagaagctc aacgagatgt tcctcattct gaagtcacta 1200 ctcccctccg tccgcaaggt tgacaaggca tccatacttg cagaaacgat aacctacctc 1260 aaagtgttag agaaaagagt gaaagagctg gagtccagca gcagggagcc atcgcgttgg 1320 cggccaaccg aaattggaca ggggaaggcg ccgtaa 1356 <210> 4 <211> 2298 <212> DNA <213> Artificial Sequence <220> <223> GluB1 promoter nucleotide sequences <400> 4 acagattctt gctaccaaca acttcacaaa gtagtagtca accaaaacta tgctaaggaa 60 tcacctcact tccgcccatg accgtgagca cgactgttca aacagtttgt taatctctac 120 aaagaaggta cactttacct acacaacgcc actaacctga gttacccagc ccatgcaaaa 180 tagccacgtc ttgtgactta agggatttcg cgacaaggca tttcgaaagc ccacacaagg 240 acaccttatg aaaactggag gggtcccaca gaccaacaac aagttaggtc ccaaaccatg 300 ttgtgccagg aaaaatccaa ggggtcctcc ccaacaccac cccgacaaat ccacttgtcc 360 attggcatca agatttgcct gacctagcta attactcagc caggcatgtc acaattcacc 420 catgtggtca cacatgttat ggttggatga aattctaaag gaatcggtcc atatgagcaa 480 gaacgagaaa accataccac cagtacttct accgaaatac gagtttagta aactcatttg 540 ttttcaaggc accgacccag gtgtgtcggg ttttccaggg attttgtaaa cccaagtttt 600 acccatagtt gatcattcaa attttgagga gggtcattgg tatccgtacc tgagggcacg 660 aatactgaga cctagcattg tagtcgacca aggaggttaa tgcagcaatt gtaggtgggg 720 cctgttggtt atattgcaaa ctgcggccaa catttcatgt gtaatttaga gatgtgcatt 780 ttgagaaatg aaatacttag tttcaaatta tgggctcaaa ataatcaaag gtgacctacc 840 ttgcttgata tcttgagctt cttcctcgta ttccgcgcac taggactctt ctggctccga 900 agctacacgt ggaacgagat aactcaacaa aacgaccaag gaaaagctcg tattagtgag 960 tactaagtgt gccactgaat agatctcgat ttttgaggaa ttttagaagt tgaacagagt 1020 caatcgaaca gacagttgaa gagatatgga ttttctaaga ttaattgatt ctctgtctaa 1080 agaaaaaaag tattattgaa ttaaatggaa aaagaaaaag gaaaaagggg atggcttctg 1140 cctttttggg ctgaaggcgg cgtgtggccc agcgtgctgc gtgggcacag ccgagcgaac 1200 acacgacgga gcagctacga cgaacggggg accgagtgga ccggacgagg atgtggccta 1260 ggacgagtgc acaaggctag tggactcggt cccccgcgcg gtatcccgag tgggtccact 1320 cgtctgcaaa cacgattcac atagagcggg agcacgcggg gagccgtcct aggtgcacgg 1380 gaagcaaatc cgtgcgcctg ggtggatttg agtgacacgg gcccacgtgt agcctcacag 1440 ctctccgtgg tcagatgtgt aaaattatca taatatgtgt ttttcaaata gttaaataat 1500 atatataggc aagttatatg ggtcaataag cagtaaaaag gcttatgaca tggtaaaatt 1560 acttacacca atatgcctta ctgtctgata tattttacat gacaacaaag ttacaagtac 1620 gtcatttaaa aatacaagtt acttatcaat tgtagtgtat caagtaaatg acaacaaacc 1680 tacaaatttg ctattttgaa ggaacactta aaaaaatcaa taggcaagtt atatagtcaa 1740 taaactgcaa gaaggcttat gacatggaaa aattacatac accaatatgc tttattgtcc 1800 ggtatatttt acaagacaac aaagttataa gtatgtcatt taaaaataca agttacttat 1860 caattgtcaa gtaaatgaaa acaaacctac aaatttgtta ttttgaagga acacctaaat 1920 tatcaaatat agcttgctac gcaaaaatga caacatgctt acaagttatt atcatcttaa 1980 agttagactc atcttctcaa gcataagagc tttatggtgc aaaaacaaat ataatgacaa 2040 ggcaaagata catattaaga gtatggatag acatttcttt aacaaactcc atttgtatta 2100 ctccaaaagc accagaagtt tgtcatggct gagtcatgaa atgtatagtt caatcttgca 2160 aagttgcctt tccttttgta ctgttttaac actacaagcc atatattgtc tgtacgtgca 2220 acaaactata tcaccatgta tcccaagatg cgtttttatt gctatataaa ctagcttggt 2280 ctgtctttga actaagct 2298 <210> 5 <211> 272 <212> PRT <213> Artificial Sequence <220> <223> OsC1 amino acid sequences <400> 5 Met Gly Arg Arg Ala Cys Cys Ala Lys Glu Gly Met Lys Arg Gly Ala 1 5 10 15 Trp Thr Ser Lys Glu Asp Asp Val Leu Ala Ser Tyr Ile Lys Ser His 20 25 30 Gly Glu Gly Lys Trp Arg Glu Val Pro Gln Arg Ala Gly Leu Arg Arg 35 40 45 Cys Gly Lys Ser Cys Arg Leu Arg Trp Leu Asn Tyr Leu Arg Pro Asn 50 55 60 Ile Lys Arg Gly Asn Ile Asp Asp Asp Glu Glu Glu Leu Ile Val Arg 65 70 75 80 Leu His Thr Leu Leu Gly Asn Arg Trp Ser Leu Ile Ala Gly Arg Leu 85 90 95 Pro Gly Arg Thr Asp Asn Glu Ile Lys Asn Tyr Trp Asn Ser Thr Leu 100 105 110 Ser Arg Lys Ile Gly Thr Ala Ala Thr Ala Ala Ala Gly Ser Arg Gly 115 120 125 Gly Ser Thr Pro Asp Thr Ala Arg Ala Thr Asp Ala Ala Ser Ser Ser 130 135 140 Ser Val Val Gln Pro Gly Gln Gln Gln Gln Pro Ala Ser Arg Ala Asp 145 150 155 160 Thr Asp Thr Ala Thr Ala Ala Ala Ala Ala Ala Ala Thr Thr Thr Thr 165 170 175 Val Trp Ala Pro Lys Ala Val Arg Cys Thr Arg Gly Phe Phe Phe His 180 185 190 Asp Arg Glu Thr Ala Pro Leu Ala Ala Ala Ala Pro Ala Pro Ala Gly 195 200 205 Glu Leu Gly Asp Gly Asp Asp Val Asp Cys Asp Tyr Tyr Cys Ser Gly 210 215 220 Ser Ser Ser Ala Ala Thr Thr Thr Ser Ser Ser Ser Leu Pro Ala Val 225 230 235 240 Val Glu Pro Cys Phe Ser Ala Gly Asp Asp Trp Met Asp Asp Val Arg 245 250 255 Ala Leu Ala Ser Phe Leu Asp Thr Asp Asp Ala Trp Asn Leu Cys Ala 260 265 270 <210> 6 <211> 588 <212> PRT <213> Artificial Sequence <220> <223> OsB1 amino acid sequences <400> 6 Met Glu Glu Thr Pro Leu Pro Ser Gly Lys Asn Phe Arg Ser Gln Leu 1 5 10 15 Ala Ala Ala Ala Arg Ser Ile Asn Trp Thr Tyr Ala Ile Phe Trp Ser 20 25 30 Ile Ser Thr Ser Arg Pro Gly Val Leu Thr Trp Lys Asp Gly Phe Tyr 35 40 45 Asn Gly Glu Ile Lys Thr Arg Lys Ile Thr Asn Ser Met Asn Leu Thr 50 55 60 Ala Asp Glu Leu Val Leu Gln Arg Ser Glu Gln Leu Arg Glu Leu Tyr 65 70 75 80 Asp Ser Leu Leu Ser Gly Glu Cys Gly His Arg Ala Arg Arg Pro Val 85 90 95 Ala Ala Leu Leu Pro Glu Asp Leu Gly Asp Thr Glu Trp Tyr Tyr Val 100 105 110 Val Cys Met Thr Tyr Ala Phe Gly Pro Arg Gln Gly Leu Pro Gly Lys 115 120 125 Ser Phe Ala Ser Asn Glu Phe Val Trp Leu Thr Asn Ala Gln Ser Ala 130 135 140 Asp Arg Lys Leu Phe His Arg Ala Leu Ile Ala Lys Ser Ala Ser Ile 145 150 155 160 Lys Thr Ile Val Cys Val Pro Phe Ile Met His Gly Val Leu Glu Leu 165 170 175 Gly Thr Thr Asp Pro Ile Ser Glu Asp Pro Ala Leu Val Asp Arg Ile 180 185 190 Ala Ala Ser Phe Trp Asp Thr Pro Pro Arg Ala Ala Phe Ser Ser Glu 195 200 205 Ala Gly Asp Ala Asp Ile Val Val Phe Glu Asp Leu Asp His Gly Asn 210 215 220 Ala Ala Val Glu Ala Thr Thr Thr Thr Val Pro Gly Glu Pro His Ala 225 230 235 240 Val Ala Gly Gly Glu Val Ala Glu Cys Glu Pro Asn Ser Asp Asn Asp 245 250 255 Leu Glu Gln Ile Thr Met Asp Asp Ile Gly Glu Leu Tyr Ser Leu Cys 260 265 270 Glu Glu Leu Asp Val Val Arg Pro Leu Asp Asp Asp Ser Ser Ser Trp 275 280 285 Ala Val Ala Asp Pro Trp Ser Ser Phe Gln Leu Val Pro Thr Ser Ser 290 295 300 Pro Ala Pro Asp Gln Ala Pro Ala Ala Glu Ala Thr Asp Val Asp Asp 305 310 315 320 Val Val Val Ala Ala Leu Asp Ser Ser Ser Ile Asp Gly Ser Cys Arg 325 330 335 Pro Ser Pro Ser Ser Phe Val Ala Trp Lys Arg Thr Ala Asp Ser Asp 340 345 350 Glu Val Gln Ala Val Pro Leu Ile Ser Gly Glu Pro Pro Gln Lys Leu 355 360 365 Leu Lys Lys Ala Val Ala Gly Ala Gly Ala Trp Met Asn Asn Gly Asp 370 375 380 Ser Ser Ala Ala Ala Met Thr Thr Gln Gly Ser Ser Ile Lys Asn His 385 390 395 400 Val Met Ser Glu Arg Arg Arg Arg Glu Lys Leu Asn Glu Met Phe Leu 405 410 415 Ile Leu Lys Ser Val Val Pro Ser Ile His Arg Val Asp Lys Ala Ser 420 425 430 Ile Leu Ala Glu Thr Ile Ala Tyr Leu Lys Glu Leu Glu Lys Arg Val 435 440 445 Glu Glu Leu Glu Ser Ser Ser Gln Pro Ser Pro Cys Pro Leu Glu Thr 450 455 460 Arg Ser Arg Arg Lys Cys Arg Glu Ile Thr Gly Lys Lys Val Ser Ala 465 470 475 480 Gly Ala Lys Arg Lys Ala Pro Ala Pro Glu Val Ala Ser Asp Asp Asp 485 490 495 Thr Asp Gly Glu Arg Arg His Cys Val Ser Asn Val Asn Val Thr Ile 500 505 510 Met Asp Asn Lys Glu Val Leu Leu Glu Leu Gln Cys Gln Trp Lys Glu 515 520 525 Leu Leu Met Thr Arg Val Phe Asp Ala Ile Lys Gly Val Ser Leu Asp 530 535 540 Pro Leu Gly Ala Gly Ile Asn Ile Gly Trp Ser Pro Trp Thr Glu Asp 545 550 555 560 Thr Ser Gln Val Cys Leu Ile Cys Cys Arg Arg Thr Trp Asp Asp Tyr 565 570 575 Arg Ser Ser Pro Glu Ser Tyr Ser Lys Leu Ala Ser 580 585 <210> 7 <211> 451 <212> PRT <213> Artificial Sequence <220> <223> OsB2 amino acid sequences <400> 7 Met Ala Ser Ala Pro Pro Val Gln Glu Glu Ala Leu Gln Pro Gly Thr 1 5 10 15 Asn His Phe Arg Ser Arg Leu Ala Ala Ala Val Arg Ser Ile Ser Trp 20 25 30 Ser Tyr Thr Ile Phe Trp Ser Thr Ser Thr Ser Leu Pro Gly Val Leu 35 40 45 Thr Trp Asn Asp Gly Phe Tyr Asn Gly Glu Val Lys Thr Arg Lys Ile 50 55 60 Ser Asn Leu Glu Asp Leu Thr Ala Asp Gln Leu Val Leu Arg Arg Ser 65 70 75 80 Glu Gln Leu Ser Glu Leu Tyr Tyr Ser Leu Leu Ser Gly Glu Cys Asp 85 90 95 His Arg Ala Arg Lys Pro Val Ala Ala Leu Ser Pro Glu Asp Ile Ala 100 105 110 Asp Thr Glu Trp Tyr Tyr Val Val Cys Met Thr Tyr Ala Phe Arg Pro 115 120 125 Gly Gln Gly Leu Pro Gly Arg Ser Tyr Ala Ser Asn Arg Ser Val Trp 130 135 140 Leu Cys Asn Ala Gln Ser Ala Asp Ser Lys Thr Phe Leu Arg Ala Leu 145 150 155 160 Leu Ala Lys Ser Ala Ser Ile Gln Thr Ile Val Cys Ile Pro Phe Met 165 170 175 Ser Gly Val Leu Glu Leu Gly Thr Thr Asp Pro Val Ser Glu Asp Pro 180 185 190 Asn Leu Val Asn Arg Ile Val Ala Tyr Leu Lys Glu Leu Gln Phe Pro 195 200 205 Ile Cys Leu Glu Val Pro Ser Ser Thr Pro Ser Pro Asp Glu Thr Glu 210 215 220 Asp Ala Asp Thr Val Phe Asp Gly Leu Ile Glu Glu Asp Gln Met Val 225 230 235 240 Ile Leu Gln Gly Glu Asp Glu Leu Gly Asp Val Val Val Ala Glu Cys 245 250 255 Glu Thr Asn Gly Ala Asn Pro Glu Thr Ile Thr Met Glu Thr Asp Glu 260 265 270 Phe Tyr Ser Leu Cys Glu Glu Leu Asp Leu Asp Leu Gly Ser Tyr Gln 275 280 285 Leu Val Pro Thr Ser Ala Arg Glu Thr Val Ala Ala Ala Ala Ala Ala 290 295 300 Ala Asn Asp Val Asp Gly Val Ala Tyr Ser His Ala Ser Cys Phe Val 305 310 315 320 Ser Trp Lys Arg Ala Asn Pro Ala Glu Lys Val Val Ala Val Pro Met 325 330 335 Thr Ala Gly Ile Glu Ser Gln Lys Leu Leu Lys Lys Ala Val Gly Gly 340 345 350 Gly Thr Ala Trp Met Ser Asn Ile Asp Asp Arg Gly Ser Val Ala Ile 355 360 365 Thr Thr Thr Pro Gly Ser Asn Ile Lys Ser His Val Met Ser Glu Arg 370 375 380 Arg Arg Arg Glu Lys Leu Asn Glu Met Phe Leu Ile Leu Lys Ser Leu 385 390 395 400 Leu Pro Ser Val Arg Lys Val Asp Lys Ala Ser Ile Leu Ala Glu Thr 405 410 415 Ile Thr Tyr Leu Lys Val Leu Glu Lys Arg Val Lys Glu Leu Glu Ser 420 425 430 Ser Ser Arg Glu Pro Ser Arg Trp Arg Pro Thr Glu Ile Gly Gln Gly 435 440 445 Lys Ala Pro 450 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PGluB1 forward primer <400> 8 acagattctt gctaccaaca ac 22 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PGluB1 reverse primer <400> 9 agttcaaaga cagaccaagc tagt 24 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> OsC1 forward primer <400> 10 atggggagga gagcttgctg cg 22 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> OsC1 reverse primer <400> 11 tcatcgccgt ctcctaattc 20 <210> 12 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> OsB1 forward primer <400> 12 atggaagaga cccctctgcc atccggg 27 <210> 13 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> OsB1 reverse primer <400> 13 tgaccaagac gatactgatc tcaaccttc 29 <210> 14 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> OsB2 forward primer <400> 14 atggcatctg ctcctccagt tcagga 26 <210> 15 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> OsB2 reverse primer <400> 15 tcatagacct ccttcccggt gatctta 27 <110> REPUBLIC OF KOREA <120> TRANSGENIC RICE PRODUCING HIGH LEVEL OF FLAVONOIDS AND MANUFACTURING METHOD THEREOF <130> P14R12D0962 <160> 15 <170> Kopatentin 2.0 <210> 1 <211> 819 <212> DNA <213> Artificial Sequence <220> <223> OsC1 nucleotide sequences <400> 1 atggggagga gagcttgctg cgcaaaggaa gggatgaaga gaggggcatg gacgagcaag 60 gaggacgacg tgcttgcctc ctacatcaag tcccatggcg aaggcaagtg gcgcgaggtc 120 ccccaacgag ctggtttgag gcggtgcggc aagagctgca ggctccggtg gctcaactat 180 ctccggccta acatcaagcg cggcaacatc gacgacgacg aggaggagct catcgtcagg 240 ctccacaccc tcctcggcaa caggtggtct ctcattgcag gcaggctgcc gggccgaaca 300 gacaatgaaa tcaagaacta ctggaacagc acgctcagcc gcaagatcgg caccgccgcc 360 accgccgccg ccggcagccg cggtggcagc acgccggaca ccgccagagc gacggacgcg 420 gcgtcgtcca gctccgtcgt gcagccgggc cagcagcagc agccagcctc ccgcgccgac 480 accgacacag caacggcagc ggcggcggcg gcggcgacga cgaccaccgt gtgggcgccc 540 aaggccgtgc ggtgcacgcg cgggttcttc ttccacgacc gtgaaacggc gccgctcgcc 600 gcggcggcgc cggcgccggc aggggaatta ggagacggcg atgacgtcga ctgcgactac 660 tactgcagcg gcagcagctc ggcggcgacg acgacgtcgt cgagctcatt accggcggtc 720 gtcgagccgt gcttctccgc cggcgacgac tggatggacg acgtgagagc cttggcgtcg 780 tttcttgaca ccgacgacgc ctggaacttg tgtgcgtga 819 <210> 2 <211> 1767 <212> DNA <213> Artificial Sequence <220> <223> OsB1 nucleotide sequences <400> 2 atggaagaga cccctctgcc atccgggaag aacttcagga gccagcttgc tgctgcagcg 60 aggagcatca attggacgta tgccatattt tggtccattt caaccagccg cccaggagtt 120 ctgacttgga aggacggctt ctacaacggc gagataaaga cgaggaagat cacgaactcc 180 atgaacctca cggccgacga gctggtcctg cagagaagcg agcagctgag ggagctctac 240 gactctctcc tctccggcga gtgcggccac cgagcgagga ggcccgtcgc tgcactgttg 300 ccggaagatc tcggggacac ggaatggtac tacgtcgtct gcatgaccta cgccttcggc 360 ccccgccaag ggttgccagg caaaagcttc gcaagcaatg aatttgtttg gctgacaaac 420 gctcagtctg cagatagaaa actattccat cgcgcgctta tagcaaagag tgcatctatt 480 aagacaatcg tctgcgtgcc atttatcatg catggtgtcc ttgagctcgg gaccactgat 540 ccgatttcgg aggacccggc tctcgtcgac cgtatcgcgg cgtcgttctg ggatacgccg 600 ccccgcgcgg cgttctcgtc ggaggcggga gacgccgaca tcgtcgtctt cgaagacctc 660 gccatggca acgccgccgt cgaagcgacg acgacgacgg tcccggggga gccacacgcg 720 gtagccggcg gcgaggtcgc cgagtgcgag cccaactccg acaacgacct cgagcagatc 780 accatggacg acatcggcga gctctacagc ctctgcgagg agctggacgt cgtgcgccct 840 ctcgacgacg acagtagcag ctgggcggtc gcggatccct ggtcgtcctt tcagctagtt 900 ccgacgtctt ctccggcgcc ggatcaggcg ccggcggcgg aagctactga cgtcgacgac 960 gtcgtcgtcg ccgctttaga cagtagctcc attgatggat cttgcaggcc gtcgccgtcg 1020 agttttgtgg cgtggaagag gacggcggac tcggacgagg tgcaggccgt gccgctcatc 1080 agcggagagc cgccacagaa gttgctgaag aaagctgtcg ccggagccgg tgcctggatg 1140 aacaatggtg acagcagcgc ggcggcgatg acgactcaag gaagcagcat caagaaccat 1200 gtcatgtcag agagaaggcg ccgggagaag ctcaacgaga tgttcctgat tctcaaatca 1260 gttgtcccgt ccattcacag ggtggacaaa gcatccattc tcgcagaaac gatagcctac 1320 ctcaaagagc tggagaaaag agtggaagag ctggaatcca gcagccaacc atcgccatgt 1380 ccattggaaa caagaagcag gcgaaagtgc cgtgagatca ctgggaagaa ggtttctgca 1440 ggagcgaaga gaaaggcgcc ggcgccggag gtggccagcg acgacgacac cgacggggag 1500 cggcgccatt gtgtgagcaa cgtgaacgtc accatcatgg acaacaagga ggttctcctc 1560 gagctgcaat gccagtggaa ggaattgctg atgacgagag tgttcgacgc gatcaaggga 1620 gtctccctgg atcctctcgg tgcaggcatc aacatcggat ggtctccttg gactgaagat 1680 acaagccaag tttgcctcat ctgctgccgt cgaacctggg atgattacag aagctctccg 1740 gaaagctata gcaagctagc tagctag 1767 <210> 3 <211> 1356 <212> DNA <213> Artificial Sequence <220> <223> OsB2 nucleotide sequences <400> 3 atggcatctg ctcctccagt tcaggaagaa gccctacagc cagggacgaa ccacttcagg 60 agccggcttg ctgctgctgt gaggagcatc agttggagtt acaccatctt ttggtccact 120 tcaaccagtc tcccgggagt tctgacttgg aacgatggat tctacaacgg cgaggtaaag 180 acgaggaaga tatccaactt agaggacctc accgctgacc agctcgtcct gcggagaagc 240 gagcaactga gcgagctcta ctactctctc ctgtccggcg agtgcgacca ccgggcaagg 300 aagcccgtcg ccgcactgtc gccggaagat atcgcggaca cagaatggta ctacgttgtc 360 tgcatgacct acgcctttcg acccggccaa gggttacccg gcagaagcta tgcaagcaat 420 cgatctgttt ggttgtgcaa tgctcagtct gcagatagca aaacctttct acgtgcgctc 480 ttagcgaaga gcgcgtctat tcagacaatt gtctgcatcc ccttcatgag tggcgtgctt 540 gagctgggaa ccactgatcc ggtttcggag gacccaaact tggtaaaccg aatcgttgca 600 tatttgaagg agcttcagtt tccgatatgc ttagaggtac cgagttctac tccttctcca 660 gacgaaacag aagatgccga caccgtgttc gatggcctca ttgaagagga ccagatggtc 720 atactccagg gagaagacga gctaggcgac gtcgtcgtcg ctgagtgcga gaccaacggc 780 gccaaccccg aaacgatcac catggagacc gacgagttct acagcctctg cgaggagctg 840 gacctggacc tcggttctta tcagctagtc ccgacgtcgg cacgggagac ggtggctgcg 900 gcggcggcgg cggctaacga tgtcgacggc gttgcatact ctcacgcctc gtgtttcgtg 960 tcatggaaaa gagcgaaccc ggcggagaag gtggtggccg tgccgatgac tgcaggcata 1020 gagtcacaga agttgctgaa gaaagctgta ggcggcggca ccgcatggat gagtaatatt 1080 gatgatcgtg gtagcgtggc aataacgacg actccaggaa gtaacatcaa gagccatgtc 1140 atgtcagaga gaaggcgccg agagaagctc aacgagatgt tcctcattct gaagtcacta 1200 ctcccctccg tccgcaaggt tgacaaggca tccatacttg cagaaacgat aacctacctc 1260 aaagtgttag agaaaagagt gaaagagctg gagtccagca gcagggagcc atcgcgttgg 1320 cggccaaccg aaattggaca ggggaaggcg ccgtaa 1356 <210> 4 <211> 2298 <212> DNA <213> Artificial Sequence <220> <223> GluB1 promoter nucleotide sequences <400> 4 acagattctt gctaccaaca acttcacaaa gtagtagtca accaaaacta tgctaaggaa 60 tcacctcact tccgcccatg accgtgagca cgactgttca aacagtttgt taatctctac 120 aaagaaggta cactttacct acacaacgcc actaacctga gttacccagc ccatgcaaaa 180 tagccacgtc ttgtgactta agggatttcg cgacaaggca tttcgaaagc ccacacaagg 240 acaccttatg aaaactggag gggtcccaca gaccaacaac aagttaggtc ccaaaccatg 300 ttgtgccagg aaaaatccaa ggggtcctcc ccaacaccac cccgacaaat ccacttgtcc 360 attggcatca agatttgcct gacctagcta attactcagc caggcatgtc acaattcacc 420 catgtggtca cacatgttat ggttggatga aattctaaag gaatcggtcc atatgagcaa 480 gaacgagaaa accataccac cagtacttct accgaaatac gagtttagta aactcatttg 540 ttttcaaggc accgacccag gtgtgtcggg ttttccaggg attttgtaaa cccaagtttt 600 acccatagtt gatcattcaa attttgagga gggtcattgg tatccgtacc tgagggcacg 660 aatactgaga cctagcattg tagtcgacca aggaggttaa tgcagcaatt gtaggtgggg 720 cctgttggtt atattgcaaa ctgcggccaa catttcatgt gtaatttaga gatgtgcatt 780 ttgagaaatg aaatacttag tttcaaatta tgggctcaaa ataatcaaag gtgacctacc 840 ttgcttgata tcttgagctt cttcctcgta ttccgcgcac taggactctt ctggctccga 900 agctacacgt ggaacgagat aactcaacaa aacgaccaag gaaaagctcg tattagtgag 960 tactaagtgt gccactgaat agatctcgat ttttgaggaa ttttagaagt tgaacagagt 1020 caatcgaaca gacagttgaa gagatatgga ttttctaaga ttaattgatt ctctgtctaa 1080 agaaaaaaag tattattgaa ttaaatggaa aaagaaaaag gaaaaagggg atggcttctg 1140 cctttttggg ctgaaggcgg cgtgtggccc agcgtgctgc gtgggcacag ccgagcgaac 1200 acacgacgga gcagctacga cgaacggggg accgagtgga ccggacgagg atgtggccta 1260 ggacgagtgc acaaggctag tggactcggt cccccgcgcg gtatcccgag tgggtccact 1320 cgtctgcaaa cacgattcac atagagcggg agcacgcggg gagccgtcct aggtgcacgg 1380 gaagcaaatc cgtgcgcctg ggtggatttg agtgacacgg gcccacgtgt agcctcacag 1440 ctctccgtgg tcagatgtgt aaaattatca taatatgtgt ttttcaaata gttaaataat 1500 atatataggc aagttatatg ggtcaataag cagtaaaaag gcttatgaca tggtaaaatt 1560 acttacacca atatgcctta ctgtctgata tattttacat gacaacaaag ttacaagtac 1620 gtcatttaaa aatacaagtt acttatcaat tgtagtgtat caagtaaatg acaacaaacc 1680 tacaaatttg ctattttgaa ggaacactta aaaaaatcaa taggcaagtt atatagtcaa 1740 taaactgcaa gaaggcttat gacatggaaa aattacatac accaatatgc tttattgtcc 1800 ggtatatttt acaagacaac aaagttataa gtatgtcatt taaaaataca agttacttat 1860 caattgtcaa gtaaatgaaa acaaacctac aaatttgtta ttttgaagga acacctaaat 1920 tatcaaatat agcttgctac gcaaaaatga caacatgctt acaagttatt atcatcttaa 1980 agttagactc atcttctcaa gcataagagc tttatggtgc aaaaacaaat ataatgacaa 2040 ggcaaagata catattaaga gtatggatag acatttcttt aacaaactcc atttgtatta 2100 ctccaaaagc accagaagtt tgtcatggct gagtcatgaa atgtatagtt caatcttgca 2160 aagttgcctt tccttttgta ctgttttaac actacaagcc atatattgtc tgtacgtgca 2220 acaaactata tcaccatgta tcccaagatg cgtttttatt gctatataaa ctagcttggt 2280 ctgtctttga actaagct 2298 <210> 5 <211> 272 <212> PRT <213> Artificial Sequence <220> <223> OsC1 amino acid sequences <400> 5 Met Gly Arg Arg Ala Cys Cys Ala Lys Glu Gly Met Lys Arg Gly Ala 1 5 10 15 Trp Thr Ser Lys Glu Asp Asp Val Leu Ala Ser Tyr Ile Lys Ser His 20 25 30 Gly Glu Gly Lys Trp Arg Glu Val Pro Gln Arg Ala Gly Leu Arg Arg 35 40 45 Cys Gly Lys Ser Cys Arg Leu Arg Trp Leu Asn Tyr Leu Arg Pro Asn 50 55 60 Ile Lys Arg Gly Asn Ile Asp Asp Asp Glu Glu Glu Leu Ile Val Arg 65 70 75 80 Leu His Thr Leu Leu Gly Asn Arg Trp Ser Leu Ile Ala Gly Arg Leu 85 90 95 Pro Gly Arg Thr Asp Asn Glu Ile Lys Asn Tyr Trp Asn Ser Thr Leu 100 105 110 Ser Arg Lys Ile Gly Thr Ala Ala Thr Ala Ala Ala Gly Ser Arg Gly 115 120 125 Gly Ser Thr Pro Asp Thr Ala Arg Ala Thr Asp Ala Ser Ser Ser Ser 130 135 140 Ser Val Val Gln Pro Gly Gln Gln Gln Gln Pro Ala Ser Arg Ala Asp 145 150 155 160 Thr Asp Thr Thr Thr Thr Thr Thr Thr Thr Thr Thr Thr 165 170 175 Val Trp Ala Pro Lys Ala Val Arg Cys Thr Arg Gly Phe Phe Phe His 180 185 190 Asp Arg Glu Thr Ala Pro Leu Ala Ala Ala Ala Pro Ala Pro Ala Gly 195 200 205 Glu Leu Gly Asp Gly Asp Asp Val Asp Cys Asp Tyr Tyr Cys Ser Gly 210 215 220 Ser Ser Ala Ala Thr Thr Ser Ser Ser Ser Leu Pro Ala Val 225 230 235 240 Val Glu Pro Cys Phe Ser Ala Gly Asp Asp Trp Met Asp Asp Val Arg 245 250 255 Ala Leu Ala Ser Phe Leu Asp Thr Asp Asp Ala Trp Asn Leu Cys Ala 260 265 270 <210> 6 <211> 588 <212> PRT <213> Artificial Sequence <220> <223> OsB1 amino acid sequences <400> 6 Met Glu Glu Thr Pro Leu Pro Ser Gly Lys Asn Phe Arg Ser Gln Leu 1 5 10 15 Ala Ala Ala Arg Ser Ile Asn Trp Thr Tyr Ala Ile Phe Trp Ser 20 25 30 Ile Ser Thr Ser Arg Pro Gly Val Leu Thr Trp Lys Asp Gly Phe Tyr 35 40 45 Asn Gly Glu Ile Lys Thr Arg Lys Ile Thr Asn Ser Met Asn Leu Thr 50 55 60 Ala Asp Glu Leu Val Leu Gln Arg Ser Glu Gln Leu Arg Glu Leu Tyr 65 70 75 80 Asp Ser Leu Leu Ser Gly Glu Cys Gly His Arg Ala Arg Arg Pro Val 85 90 95 Ala Ala Leu Leu Pro Glu Asp Leu Gly Asp Thr Glu Trp Tyr Tyr Val 100 105 110 Val Cys Met Thr Tyr Ala Phe Gly Pro Arg Gln Gly Leu Pro Gly Lys 115 120 125 Ser Phe Ala Ser Asn Glu Phe Val Trp Leu Thr Asn Ala Gln Ser Ala 130 135 140 Asp Arg Lys Leu Phe His Arg Ala Leu Ile Ala Lys Ser Ala Ser Ile 145 150 155 160 Lys Thr Ile Val Cys Val Pro Phe Ile Met His Gly Val Leu Glu Leu 165 170 175 Gly Thr Thr Asp Pro Ile Ser Glu Asp Pro Ala Leu Val Asp Arg Ile 180 185 190 Ala Ala Ser Phe Trp Asp Thr Pro Pro Arg Ala Ala Phe Ser Ser Glu 195 200 205 Ala Gly Asp Ala Asp Ile Val Val Phe Glu Asp Leu Asp His Gly Asn 210 215 220 Ala Ala Val Glu Ala Thr Thr Thr Val Pro Gly Glu Pro His Ala 225 230 235 240 Val Ala Gly Gly Glu Val Ala Glu Cys Glu Pro Asn Ser Asp Asn Asp 245 250 255 Leu Glu Gln Ile Thr Met Asp Asp Ile Gly Glu Leu Tyr Ser Leu Cys 260 265 270 Glu Glu Leu Asp Val Val Arg Pro Leu Asp Asp Asp Ser Ser Serp 275 280 285 Ala Val Ala Asp Pro Trp Ser Ser Phe Gln Leu Val Pro Thr Ser Ser 290 295 300 Pro Ala Pro Asp Gln Ala Pro Ala Ala Glu Ala Thr Asp Val Asp Asp 305 310 315 320 Val Val Val Ala Leu Asp Ser Ser Ile Asp Gly Ser Cys Arg 325 330 335 Pro Ser Ser Ser Phe Val Ala Trp Lys Arg Thr Ala Asp Ser Asp 340 345 350 Glu Val Gln Ala Val Pro Leu Ile Ser Gly Glu Pro Pro Gln Lys Leu 355 360 365 Leu Lys Lys Ala Val Ala Gly Ala Gly Ala Trp Met Asn Asn Gly Asp 370 375 380 Ser Ser Ala Ala Ala Met Thr Ser Ser Ile Lys Asn His 385 390 395 400 Val Met Ser Glu Arg Arg Arg Glu Lys Leu Asn Glu Met Phe Leu 405 410 415 Ile Leu Lys Ser Val Val Ser Ile His Arg Val Asp Lys Ala Ser 420 425 430 Ile Leu Ala Glu Thr Ile Ala Tyr Leu Lys Glu Leu Glu Lys Arg Val 435 440 445 Glu Glu Leu Glu Ser Ser Ser Gln Pro Ser Pro Cys Pro Leu Glu Thr 450 455 460 Arg Ser Arg Arg Lys Cys Arg Glu Ile Thr Gly Lys Lys Val Ser Ala 465 470 475 480 Gly Ala Lys Arg Lys Ala Pro Ala Pro Glu Val Ala Ser Asp Asp Asp 485 490 495 Thr Asp Gly Arg Arg His Cys Val Ser Asn Val Asn Val Thr Ile 500 505 510 Met Asp Asn Lys Glu Val Leu Leu Glu Leu Gln Cys Gln Trp Lys Glu 515 520 525 Leu Leu Met Thr Arg Val Phe Asp Ala Ile Lys Gly Val Ser Leu Asp 530 535 540 Pro Leu Gly Ala Gly Ile Asn Ile Gly Trp Ser Pro Trp Thr Glu Asp 545 550 555 560 Thr Ser Gln Val Cys Leu Ile Cys Cys Arg Arg Thr Trp Asp Asp Tyr 565 570 575 Arg Ser Ser Pro Glu Ser Tyr Ser Lys Leu Ala Ser 580 585 <210> 7 <211> 451 <212> PRT <213> Artificial Sequence <220> <223> OsB2 amino acid sequences <400> 7 Met Ala Ser Ala Pro Pro Val Gln Glu Glu Ala Leu Gln Pro Gly Thr 1 5 10 15 Asn His Phe Arg Ser Ser Leu Ala Ala Ala Val Arg Ser Ser Serp 20 25 30 Ser Tyr Thr Ile Phe Trp Ser Thr Ser Ser Ser Pro Pro Gly Val Leu 35 40 45 Thr Trp Asn Asp Gly Phe Tyr Asn Gly Glu Val Lys Thr Arg Lys Ile 50 55 60 Ser Asn Leu Glu Asp Leu Thr Ala Asp Gln Leu Val Leu Arg Arg Ser 65 70 75 80 Glu Gln Leu Ser Glu Leu Tyr Tyr Ser Leu Leu Ser Gly Glu Cys Asp 85 90 95 His Arg Ala Arg Lys Pro Val Ala Ala Leu Ser Pro Glu Asp Ile Ala 100 105 110 Asp Thr Glu Trp Tyr Tyr Val Val Cys Met Thr Tyr Ala Phe Arg Pro 115 120 125 Gly Gln Gly Leu Pro Gly Arg Ser Tyr Ala Ser Asn Arg Ser Val Trp 130 135 140 Leu Cys Asn Ala Gln Ser Ala Asp Ser Lys Thr Phe Leu Arg Ala Leu 145 150 155 160 Leu Ala Lys Ser Ala Ser Ile Gln Thr Ile Val Cys Ile Pro Phe Met 165 170 175 Ser Gly Val Leu Glu Leu Gly Thr Thr Asp Pro Val Ser Glu Asp Pro 180 185 190 Asn Leu Val Asn Arg Ile Val Ala Tyr Leu Lys Glu Leu Gln Phe Pro 195 200 205 Ile Cys Leu Glu Val Ser Ser Thr Ser Ser Pro Asp Glu Thr Glu 210 215 220 Asp Ala Asp Thr Val Phe Asp Gly Leu Ile Glu Glu Asp Gln Met Val 225 230 235 240 Ile Leu Gln Gly Glu Asp Glu Leu Gly Asp Val Val Val Ala Glu Cys 245 250 255 Glu Thr Asn Gly Ala Asn Pro Glu Thr Ile Thr Met Glu Thr Asp Glu 260 265 270 Phe Tyr Ser Leu Cys Glu Glu Leu Asp Leu Asp Leu Gly Ser Tyr Gln 275 280 285 Leu Val Pro Thr Ser Ala Arg Glu Thr Val Ala Ala Ala Ala Ala 290 295 300 Ala Asn Asp Val Asp Gly Val Ala Tyr Ser His Ala Ser Cys Phe Val 305 310 315 320 Ser Trp Lys Arg Ala Asn Pro Ala Glu Lys Val Val Ala Val Pro Met 325 330 335 Thr Ala Gly Ile Glu Ser Gln Lys Leu Leu Lys Lys Ala Val Gly Gly 340 345 350 Gly Thr Ala Trp Met Ser Asn Ile Asp Asp Arg Gly Ser Val Ala Ile 355 360 365 Thr Thr Pro Gly Ser Asn Ile Lys Ser His Val Met Ser Glu Arg 370 375 380 Arg Arg Arg Glu Lys Leu Asn Glu Met Phe Leu Ile Leu Lys Ser Leu 385 390 395 400 Leu Pro Ser Val Arg Lys Val Asp Lys Ala Ser Ile Leu Ala Glu Thr 405 410 415 Ile Thr Tyr Leu Lys Val Leu Glu Lys Arg Val Lys Glu Leu Glu Ser 420 425 430 Ser Ser Arg Glu Pro Ser Arg Trp Arg Pro Thr Glu Ile Gly Gln Gly 435 440 445 Lys Ala Pro 450 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PGluB1 forward primer <400> 8 acagattctt gctaccaaca ac 22 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PGluB1 reverse primer <400> 9 agttcaaaga cagaccaagc tagt 24 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> OsC1 forward primer <400> 10 atggggagga gagcttgctg cg 22 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> OsC1 reverse primer <400> 11 tcatcgccgt ctcctaattc 20 <210> 12 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> OsB1 forward primer <400> 12 atggaagaga cccctctgcc atccggg 27 <210> 13 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> OsB1 reverse primer <400> 13 tgaccaagac gatactgatc tcaaccttc 29 <210> 14 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> OsB2 forward primer <400> 14 atggcatctg ctcctccagt tcagga 26 <210> 15 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> OsB2 reverse primer <400> 15 tcatagacct ccttcccggt gatctta 27
Claims (7)
2) 상기 제조된 발현벡터를 이용하여 식물 형질전환용 아그로박테리움을 제조하는 단계; 및
3) 상기 형질전환용 아그로박테리움을 이용하여 벼를 형질전환 하는 단계
를 포함하는 것을 특징으로 하는 플라보노이드 생산이 증가된 형질전환 벼의 제조방법.1) preparing a transforming expression vector comprising the gene represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 and the promoter represented by SEQ ID NO: 4;
2) preparing Agrobacterium for plant transformation using the expression vector prepared above; And
3) Transformation of rice with the above-mentioned Agrobacterium for transformation
Wherein the flavonoid production is increased.
2) 상기 제조된 발현벡터를 이용하여 식물 형질전환용 아그로박테리움을 제조하는 단계;
3) 상기 형질전환용 아그로박테리움을 이용하여 벼를 형질전환 하는 단계; 및
4) 상기 형질전환된 식물체의 종자에서 플라보노이드를 추출하는 단계;를 포함하는 플라보노이드 생산 증대 방법.
1) preparing a transforming expression vector comprising the gene represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 and the promoter represented by SEQ ID NO: 4;
2) preparing Agrobacterium for plant transformation using the expression vector prepared above;
3) transforming rice with the transforming Agrobacterium; And
4) Extracting the flavonoid from the seed of the transformed plant.
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