KR20100073281A - METHOD FOR PRODUCING TRANSGENIC PLANT PRODUCING THE HUMAN LACTOFERRIN AND BIFIDOBACTERIAL β-GALACTOSIDASE AND PLANT PRODUCED BY THE SAME - Google Patents

METHOD FOR PRODUCING TRANSGENIC PLANT PRODUCING THE HUMAN LACTOFERRIN AND BIFIDOBACTERIAL β-GALACTOSIDASE AND PLANT PRODUCED BY THE SAME Download PDF

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KR20100073281A
KR20100073281A KR1020080131911A KR20080131911A KR20100073281A KR 20100073281 A KR20100073281 A KR 20100073281A KR 1020080131911 A KR1020080131911 A KR 1020080131911A KR 20080131911 A KR20080131911 A KR 20080131911A KR 20100073281 A KR20100073281 A KR 20100073281A
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rice
galactosidase
human lactoferrin
lactoferrin
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임성렬
김일기
이진형
서석철
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한림대학교 산학협력단
서울대학교산학협력단
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Abstract

PURPOSE: A method for producing human lactoferrin and lactobacillus-derived beta-galactosidase is provided to produce seeds with high lactose digestive enzyme and milky opalescence lactoferrin. CONSTITUTION: A transgenic plant is manufactured by transforming with a recombinant plant expression vector containing gene encoding human lactoferrin and lactobacillus-derived beta-galactosidase protein. The gene coding human lactoferrin and lactobacillus-derived beta-galactosidase comprises nucleotides of sequence numbers 1 and 2. The plant is a monocot rice plant. The recombinant plant expression vector is pVE9-TFTβ. The beta-galactosidase is produced from a plant by overexpressing the gene encoding the human lactoferrin and lactobacillus-derived beta-galactosidase.

Description

인간 락토페린 및 유산균 유래의 β-갈락토시다제 단백질을 생산하는 식물체의 제조방법 및 상기 방법에 의해 제조된 식물체{Method for producing transgenic plant producing the human lactoferrin and bifidobacterial β-galactosidase and plant produced by the same}Method for producing transgenic plant producing the human lactoferrin and bifidobacterial β-galactosidase and plant produced by the same}

본 발명은 인간 락토페린 및 유산균 유래의 β-갈락토시다제 단백질을 생산하는 식물체의 제조방법 및 상기 방법에 의해 제조된 식물체에 관한 것으로, 본 발명에 따른 인간 락토페린 및 유산균 유래의 β-갈락토시다제 단백질을 생산하는 형질전환 식물체는 유단백질 락토페린 및 유당 소화효소인 β-갈락토시다제 단백질 함량이 높은 종자를 생산할 수 있는 고부가가치의 식물체로 제공될 수 있다.The present invention relates to a method for producing a plant that produces β-galactosidase protein derived from human lactoferrin and lactic acid bacteria, and to a plant produced by the method, and β-galactosidase derived from human lactoferrin and lactic acid bacteria according to the present invention. The transformed plant producing the first protein may be provided as a high value plant capable of producing a seed having a high content of β-galactosidase protein, a lactoferrin and a lactose digestive enzyme.

인간 락토페린은 포유동물 젖에 고농도로 존재하며 철이 결합된 당단백질로서 분자량은 80kDa로 2개의 로브로 구성되어 있고 각 로브안에 각각 한 개의 철 결합 부위가 존재하고 있다(Metz-Boutigue et al., 1984, Eur. J. Biochem., 145, 659-676). 락토페린은 살균 및 정균작용, 세포증식조절작용, 과산화지질 생성억제작용, 면역계 조절작용, 철 흡수 조절작용, 감염부위 염증발생 제어작용, 항바이러스 작용, 대장균의 장세포 부착방지, 비피더스균 증식작용 등의 다양한 생체방어 작용에 관여하고, 유익한 생리활성을 갖고 있다(Yu, Korean Dairy Techno., 1997, 15, 83-89). 락토페린을 함유한 주요 제품은 유아용 조제분유를 비롯하여 화장품, 식품첨가물, 항설사약, 복막 투석용제, 임상영양제, 여성위생용품, 안약제품, 껌 등이 있다(Nam et al., 1996, Korean J. Dairy Sci., 18, 289-298; Tomita, 1999, Morinaga Milk Industry Co., Ltd).Human lactoferrin is present in mammalian milk at high concentrations and is an iron-bound glycoprotein with a molecular weight of 80 kDa consisting of two lobes, each containing one iron binding site (Metz-Boutigue et al., 1984). , Eur. J. Biochem., 145, 659-676). Lactoferrin is bactericidal and bacteriostatic, cell proliferation control, lipid peroxidation suppression, immune system regulation, iron absorption regulation, control of inflammation site of infection, antiviral effect, intestinal cell adhesion prevention of E. coli, bifidus bacteria growth Is involved in various bioprotective actions and has beneficial physiological activity (Yu, Korean Dairy Techno., 1997, 15, 83-89). The main products containing lactoferrin include infant formula, cosmetics, food additives, antidiarrheal drugs, peritoneal dialysis agents, clinical nutrition, feminine hygiene products, eye drops and gums (Nam et al., 1996, Korean J. Dairy). Sci., 18, 289-298; Tomita, 1999, Morinaga Milk Industry Co., Ltd).

비피도박테리움 속은 유제품 산업에서 다양한 유제품 발효에 가장 널리 이용되는 유형의 박테리아 배양물 중 하나이다. 또한, 비피도박테리움을 함유하는 제품의 섭취는 건강을 촉진하는 효과가 있다. 이러한 효과는 장내 내용물의 pH 감소에 의해서만이 아니라, 항생제 복용 등으로 인해 장내 균총(flora)이 파괴된 개인에서 장내 균총을 복구할 수 있는 비피도박테리움의 능력에 의해서도 달성된다. 또한, 비피도박테리움은 잠재적으로 해로운 장내 미생물들을 배출시키는 (outcompeting) 능력도 보유한다. 효소 β-갈락토시다제는 통상적으로 락토스를 단당류인 D-글루코스 및 D-갈락토스로 가수분해한다. β-갈락토시다제의 정상적인 효소 반응에서, 상기 효소는 락토스를 가수분해하고 갈락토스 단당류를 갈락토스-효소 복합체 중에서 일시적으로 결합하였다가 상기 갈락토스를 물의 히드록실기에 전달함으로써, D-갈락토스 및 D-글루코스를 유리시킨다. 그러나, 락토스가 고농도인 경우, 일부의 β-갈락토시다제는 트랜스갈락토실화라고 지칭되는 과정을 통해 갈락토스를 D-갈락토스 또는 D-글루코스의 히드록실기에 전달하여 갈락토-올리고당류를 생산할 수 있다.The Bifidobacterium genus is one of the most widely used types of bacterial cultures for fermenting various dairy products in the dairy industry. In addition, the intake of a product containing Bifidobacterium has the effect of promoting health. This effect is achieved not only by reducing the pH of the intestinal contents, but also by the Bifidobacterium's ability to recover the intestinal flora in individuals whose intestinal flora has been destroyed, for example, by taking antibiotics. Bifidobacterium also possesses the ability to outcompeting potentially harmful enteric microorganisms. The enzyme β-galactosidase typically hydrolyzes lactose to the monosaccharides D-glucose and D-galactose. In the normal enzymatic reaction of β-galactosidase, the enzyme hydrolyzes lactose and temporarily binds the galactose monosaccharides in the galactose-enzyme complex and then transfers the galactose to the hydroxyl group of water, thereby providing D-galactose and D- Releases glucose. However, at high concentrations of lactose, some β-galactosidase may deliver galactose to the hydroxyl groups of D-galactose or D-glucose through a process called transgalactosylation to produce galacto-oligosaccharides. Can be.

한편, 한국등록특허 제0551713호에서 '식물세포배양에서 인간 락토페린을 대 량 생산하는 방법'과 대한민국공개특허 제2003-0016275호에서 '비피도박테륨에서 단리한 베타-갈락토시다제'를 개시하고 있으나, 본 발명에서와 같이 인간 락토페린 및 유산균 유래의 β-갈락토시다제 단백질을 동시에 생산할 수 있는 식물체에 관한 내용은 밝혀진 바가 전혀 없다.Meanwhile, Korean Patent No. 0551713 discloses a method for mass production of human lactoferrin in plant cell culture, and Korean Patent Publication No. 2003-0016275 discloses beta-galactosidase isolated from Bifidobacterium. However, as described in the present invention, there is no information on a plant capable of simultaneously producing β-galactosidase protein derived from human lactoferrin and lactic acid bacteria.

본 발명은 상기와 같은 요구에 의해 안출된 것으로서, 본 발명은 인간 락토페린 및 유산균 유래의 β-갈락토시다제 단백질 코딩 유전자를 포함하는 재조합 식물 발현 벡터로 형질전환된 식물체의 종자에서 활성이 있는 인간 락토페린 및 β-갈락토시다제의 발현이 증가되었음을 확인함으로써 상기와 같은 형질전환 식물체를 제조하는 방법 및 상기 재조합 단백질을 풍부하게 포함한 쌀 가공식품을 제공하고자 한다.The present invention has been made in accordance with the above requirements, the present invention is a human having activity in the seed of the plant transformed with a recombinant plant expression vector comprising β-galactosidase protein coding gene derived from human lactoferrin and lactic acid bacteria By confirming the increased expression of lactoferrin and β-galactosidase, it is intended to provide a method for producing such a transgenic plant and a rice processed food rich in the recombinant protein.

상기 과제를 해결하기 위해, 본 발명은 인간 락토페린 및 유산균 유래의 β-갈락토시다제 단백질 코딩 유전자를 포함하는 재조합 식물 발현 벡터로 형질전환된 식물체를 제공한다.In order to solve the above problems, the present invention provides a plant transformed with a recombinant plant expression vector comprising a β-galactosidase protein coding gene derived from human lactoferrin and lactic acid bacteria.

본 발명은 또한, 인간 락토페린 및 유산균 유래의 β-갈락토시다제 단백질 코딩 유전자를 포함하는 재조합 식물 발현 벡터를 제공한다.The present invention also provides a recombinant plant expression vector comprising a β-galactosidase protein coding gene derived from human lactoferrin and lactic acid bacteria.

본 발명은 또한, 상기 재조합 식물 발현 벡터를 식물체에 도입하여 인간 락토페린 및 유산균 유래의 β-갈락토시다제 단백질 코딩 유전자를 과발현하는 단계를 포함하는 식물체에서 인간 락토페린 및 유산균 유래의 β-갈락토시다제 단백질 대량생산 방법을 제공한다.The present invention also provides β-galactosid derived from human lactoferrin and lactic acid bacteria in a plant, comprising the step of introducing the recombinant plant expression vector into the plant to overexpress the β-galactosidase protein coding gene derived from human lactoferrin and lactic acid bacteria. First protein mass production method is provided.

본 발명은 또한, 상기 재조합 식물 발현 벡터를 식물체에 형질전환하는 단계를 포함하는 인간 락토페린 및 유산균 유래의 β-갈락토시다제를 생산하는 식물체 의 제조방법을 제공한다.The present invention also provides a method for producing a plant for producing β-galactosidase derived from human lactoferrin and lactic acid bacteria, the method comprising transforming the plant with the recombinant plant expression vector.

본 발명은 또한, 상기 방법에 의해 제조된 벼로부터 얻은 쌀을 함유하는 쌀 가공식품을 제공한다.The present invention also provides a rice processed food containing rice obtained from the rice produced by the method.

본 발명에 따른 인간 락토페린 및 유산균 유래의 β-갈락토시다제 단백질을 생산하는 형질전환 식물체는 유단백질 락토페린 및 유당 소화효소인 β-갈락토시다제 단백질 함량이 높은 종자를 생산할 수 있는 고부가가치의 식물체로서, 상기 식물체의 종자를 유효성분으로 하는 가공식품의 제조에 유용하게 사용될 수 있다.Transgenic plants producing β-galactosidase protein derived from human lactoferrin and lactic acid bacteria according to the present invention are high-value plants capable of producing seeds having a high content of β-galactosidase protein, which is a milk protein lactoferrin and lactose digestive enzyme. As, it can be usefully used for the production of processed foods containing the seed of the plant as an active ingredient.

본 발명의 목적을 달성하기 위하여, 본 발명은 인간 락토페린 및 유산균 유래의 β-갈락토시다제 단백질 코딩 유전자를 포함하는 재조합 식물 발현 벡터로 형질전환된 식물체를 제공한다. In order to achieve the object of the present invention, the present invention provides a plant transformed with a recombinant plant expression vector comprising a β-galactosidase protein coding gene derived from human lactoferrin and lactic acid bacteria.

식물의 형질전환은 DNA를 식물에 전이시키는 임의의 방법을 의미한다. 그러한 형질전환 방법은 반드시 재생 및(또는) 조직 배양 기간을 가질 필요는 없다. 식물 종의 형질전환은 이제는 쌍자엽 식물뿐만 아니라 단자엽 식물 양자를 포함한 식물 종에 대해 일반적이다. 원칙적으로, 임의의 형질전환 방법은 본 발명에 따른 잡종 DNA를 적당한 선조 세포로 도입시키는데 이용될 수 있다. 방법은 원형질체에 대한 칼슘/폴리에틸렌 글리콜 방법(Krens, F.A. et al., 1982, Nature 296, 72-74; Negrutiu I. et al., June 1987, Plant Mol. Biol. 8, 363-373), 원형질체의 전기천공법(Shillito R.D. et al., 1985 Bio/Technol. 3, 1099-1102), 식물 요소로의 현미주사법(Crossway A. et al., 1986, Mol. Gen. Genet. 202, 179-185), 각종 식물 요소의 (DNA 또는 RNA-코팅된) 입자 충격법(Klein T.M. et al., 1987, Nature 327, 70), 식물의 침윤 또는 성숙 화분 또는 소포자의 형질전환에 의한 아그로박테리움 투머파시엔스 매개된 유전자 전이에서 (비완전성) 바이러스에 의한 감염(EP 0 301 316호) 등으로부터 적당하게 선택될 수 있다. 본 발명에 따른 바람직한 방법은 아그로박테리움 매개된 DNA 전달을 포함한다. 특히 바람직한 것은 EP A 120 516호 및 미국 특허 제4,940,838호에 기재된 바와 같은 소위 이원 벡터 기술을 이용하는 것이다.Transformation of a plant means any method of transferring DNA to a plant. Such transformation methods do not necessarily have a regeneration and / or tissue culture period. Transformation of plant species is now common for plant species, including both terminal plants as well as dicotyledonous plants. In principle, any transformation method can be used to introduce hybrid DNA according to the invention into suitable progenitor cells. Method is calcium / polyethylene glycol method for protoplasts (Krens, FA et al., 1982, Nature 296, 72-74; Negrutiu I. et al., June 1987, Plant Mol. Biol. 8, 363-373), protoplasts Electroporation (Shillito RD et al., 1985 Bio / Technol. 3, 1099-1102), microscopic injection into plant elements (Crossway A. et al., 1986, Mol. Gen. Genet. 202, 179-185 ), (DNA or RNA-coated) particle bombardment of various plant elements (Klein TM et al., 1987, Nature 327, 70), Agrobacterium tumulopasis by plant infiltration or transformation of mature pollen or vesicles And infection with (incomplete) virus (EP 0 301 316) in en mediated gene transfer. A preferred method according to the present invention comprises Agrobacterium mediated DNA delivery. Especially preferred is the use of the so-called binary vector technology as described in EP A 120 516 and US Pat. No. 4,940,838.

본 발명의 일 구현예에 따른 상기 인간 락토페린 및 유산균 유래의 β-갈락토시다제 단백질 코딩 유전자는 각각 서열번호 1 및 서열번호 2의 염기서열로 이루어진 것을 특징으로 한다. 또한, 상기 염기 서열의 변이체가 본 발명의 범위 내에 포함된다. 구체적으로, 상기 유전자는 서열번호 1 및 서열번호 2의 염기 서열과 각각 70% 이상, 더욱 바람직하게는 80% 이상, 더 더욱 바람직하게는 90% 이상, 가장 바람직하게는 95% 이상의 서열 상동성을 가지는 염기 서열을 포함할 수 있다. 폴리뉴클레오티드에 대한 "서열 상동성의 %"는 두 개의 최적으로 배열된 서열과 비교 영역을 비교함으로써 확인되며, 비교 영역에서의 폴리뉴클레오티드 서열의 일부는 두 서열의 최적 배열에 대한 참고 서열(추가 또는 삭제를 포함하지 않음)에 비해 추가 또는 삭제(즉, 갭)를 포함할 수 있다.The β-galactosidase protein coding gene derived from human lactoferrin and lactic acid bacteria according to an embodiment of the present invention is characterized by consisting of the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2, respectively. Variants of the above base sequences are also included within the scope of the present invention. Specifically, the gene has at least 70%, more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95% sequence homology with the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2, respectively. Branches can include base sequences. The "% sequence homology" for a polynucleotide is identified by comparing two optimally arranged sequences with a comparison region, wherein part of the polynucleotide sequence in the comparison region is the reference sequence (addition or deletion) for the optimal alignment of the two sequences. It may include the addition or deletion (ie, gap) compared to).

본 발명의 일 구현예에 따른 상기 식물체는 옥수수, 보리, 밀, 벼, 귀리, 호밀 및 사탕수수 중에서 선택되는 것을 특징으로 하는 단자엽 식물일 수 있으나, 바 람직하게는 벼이다.The plant according to one embodiment of the present invention may be a monocotyledonous plant, characterized in that selected from corn, barley, wheat, rice, oats, rye and sugar cane, preferably rice.

본 발명은 또한, 인간 락토페린 및 유산균 유래의 β-갈락토시다제 단백질 코딩 유전자를 포함하는 재조합 식물 발현 벡터를 제공한다.The present invention also provides a recombinant plant expression vector comprising a β-galactosidase protein coding gene derived from human lactoferrin and lactic acid bacteria.

용어 "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, 센스 또는 안티센스 형태 중 하나로 발현할 수 있다. 또한 재조합 세포는 천연 상태의 세포에서 발견되는 유전자를 발현할 수 있으며, 그러나 상기 유전자는 변형된 것으로써 인위적인 수단에 의해 세포 내 재도입된 것이다.The term "recombinant" refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a protein encoded by a peptide, heterologous peptide or heterologous nucleic acid. Recombinant cells can express genes or gene fragments that are not found in their natural form in either the sense or antisense form. Recombinant cells can also express genes found in natural cells, but the genes have been modified and reintroduced into cells by artificial means.

용어 "벡터"는 세포 내로 전달하는 DNA 단편(들), 핵산 분자를 지칭할 때 사용된다. 벡터는 DNA를 복제시키고, 숙주세포에서 독립적으로 재생산될 수 있다. 용어 "전달체"는 흔히 "벡터"와 호환하여 사용된다. 용어 "발현 벡터"는 목적한 코딩 서열과, 특정 숙주 생물에서 작동가능하게 연결된 코딩 서열을 발현하는데 필수적인 적정 핵산 서열을 포함하는 재조합 DNA 분자를 의미한다. 진핵세포에서 이용가능한 프로모터, 인핸서, 종결신호 및 폴리아데닐레이션 신호는 공지되어 있다.The term "vector" is used to refer to a DNA fragment (s), nucleic acid molecule, which is transferred into a cell. The vector replicates the DNA and can be independently regenerated in the host cell. The term "carrier" is often used interchangeably with "vector". The term “expression vector” refers to a recombinant DNA molecule comprising a coding sequence of interest and a suitable nucleic acid sequence necessary to express a coding sequence operably linked in a particular host organism. Promoters, enhancers, termination signals and polyadenylation signals available in eukaryotic cells are known.

식물 발현 벡터의 바람직한 예는 아그로박테리움 투머파시엔스와 같은 적당한 숙주에 존재할 때 그 자체의 일부, 소위 T-영역을 식물 세포로 전이시킬 수 있는 Ti-플라스미드 벡터이다. 다른 유형의 Ti-플라스미드 벡터(EP 0 116 718 B1호 참조)는 현재 식물 세포, 또는 잡종 DNA를 식물의 게놈 내에 적당하게 삽입시키는 새로운 식물이 생산될 수 있는 원형질체로 잡종 DNA 서열을 전이시키는데 이용되고 있다. Ti-플라스미드 벡터의 특히 바람직한 형태는 EP 0 120 516 B1호 및 미국 특허 제4,940,838호에 청구된 바와 같은 소위 바이너리(binary) 벡터이다. 본 발명에 따른 DNA를 식물 숙주에 도입시키는데 이용될 수 있는 다른 적합한 벡터는 이중 가닥 식물 바이러스(예를 들면, CaMV) 및 단일 가닥 바이러스, 게미니 바이러스 등으로부터 유래될 수 있는 것과 같은 바이러스 벡터, 예를 들면 비완전성 식물 바이러스 벡터로부터 선택될 수 있다. 그러한 벡터의 사용은 특히 식물 숙주를 적당하게 형질전환하는 것이 어려울 때 유리할 수 있다.Preferred examples of plant expression vectors are Ti-plasmid vectors which, when present in a suitable host such as Agrobacterium tumerfaciens, can transfer part of themselves, the so-called T-region, into plant cells. Another type of Ti-plasmid vector (see EP 0 116 718 B1) is used to transfer hybrid DNA sequences to protoplasts from which current plant cells or new plants can be produced that properly insert hybrid DNA into the plant's genome. have. A particularly preferred form of the Ti-plasmid vector is a so-called binary vector as claimed in EP 0 120 516 B1 and U.S. Patent No. 4,940,838. Other suitable vectors that can be used to introduce the DNA according to the invention into a plant host are viral vectors, such as those which can be derived from double stranded plant viruses (eg CaMV) and single stranded viruses, gemini viruses, etc. For example, it may be selected from an incomplete plant viral vector. The use of such vectors can be advantageous especially when it is difficult to properly transform a plant host.

발현 벡터는 바람직하게는 하나 이상의 선택성 마커를 포함할 것이다. 상기 마커는 통상적으로 화학적인 방법으로 선택될 수 있는 특성을 갖는 핵산 서열로, 형질전환된 세포를 비형질전환 세포로부터 구별할 수 있는 모든 유전자가 이에 해당된다. 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 유전자가 있으나, 이에 한정되는 것은 아니다.The expression vector will preferably comprise one or more selectable markers. The marker is typically a nucleic acid sequence having properties that can be selected by chemical methods, and all genes that can distinguish transformed cells from non-transformed cells. Examples include herbicide resistance genes such as glyphosate or phosphinothricin, kanamycin, G418, bleomycin, hygromycin, and chloramphenicol. Resistance genes include, but are not limited to.

본 발명의 일 구현예에 따른 재조합 식물 발현 벡터에서, 프로모터는 CaMV 35S, 액틴, 유비퀴틴, pEMU, MAS 또는 히스톤 프로모터일 수 있으나, 이에 제한되지 않는다. "프로모터"란 용어는 구조 유전자로부터의 DNA 업스트림의 영역을 의미하며 전사를 개시하기 위하여 RNA 폴리머라아제가 결합하는 DNA 분자를 말한다. " 식물 프로모터"는 식물 세포에서 전사를 개시할 수 있는 프로모터이다. "구성적(constitutive) 프로모터"는 대부분의 환경 조건 및 발달 상태 또는 세포 분화하에서 활성이 있는 프로모터이다. 형질전환체의 선택이 각종 단계에서 각종 조직에 의해서 이루어질 수 있기 때문에 구성적 프로모터가 본 발명에서 바람직할 수 있다. 따라서, 구성적 프로모터는 선택 가능성을 제한하지 않는다.In a recombinant plant expression vector according to one embodiment of the present invention, the promoter may be, but is not limited to, CaMV 35S, actin, ubiquitin, pEMU, MAS or histone promoter. The term "promoter " refers to the region of DNA upstream from the structural gene and refers to a DNA molecule to which an RNA polymerase binds to initiate transcription. "Plant promoter" is a promoter capable of initiating transcription in plant cells. A "constitutive promoter" is a promoter that is active under most environmental conditions and developmental conditions or cell differentiation. Constructive promoters may be preferred in the present invention because the choice of transformants can be made by various tissues at various stages. Thus, the constitutive promoter does not limit the selection possibilities.

상기 터미네이터는, 통상의 터미네이터를 사용할 수 있으며, 그 예로는 노팔린 신타아제(NOS), 벼 α-아밀라아제 RAmy1 A 터미네이터, 파세올린(phaseoline) 터미네이터, 아그로박테리움 투메파시엔스(agrobacterium tumefaciens)의 옥토파인(Octopine) 유전자의 터미네이터 등이 있으나, 이에 한정되는 것은 아니다. 터미네이터의 필요성에 관하여, 그러한 영역이 식물 세포에서의 전사의 확실성 및 효율을 증가시키는 것으로 일반적으로 알고 있다. 그러므로, 터미네이터의 사용은 본 발명의 내용에서 매우 바람직하다.The terminator may be a conventional terminator, and examples thereof include nopaline synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, agrobacterium tumefaciens (ocrobacterium tumefaciens) Terminator of the Fine (Octopine) gene, etc., but is not limited thereto. With regard to the need for terminators, it is generally known that such regions increase the certainty and efficiency of transcription in plant cells. Therefore, the use of a terminator is highly desirable in the context of the present invention.

본 발명의 일 구현예에 따른 재조합 식물 발현 벡터에서, 상기 인간 락토페린 및 유산균 유래의 β-갈락토시다제 단백질 코딩 유전자는 각각 서열번호 1 및 서열번호 2의 염기서열로 이루어진 것을 특징으로 한다.In the recombinant plant expression vector according to an embodiment of the present invention, the β-galactosidase protein coding gene derived from human lactoferrin and lactic acid bacteria is characterized by consisting of the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

본 발명의 일 구현예에 따른 재조합 식물 발현 벡터에서, 상기 벡터는 도 1의 pVE9-TFTβ이나, 이에 한정되지 않는다.In a recombinant plant expression vector according to an embodiment of the present invention, the vector is pVE9-TFTβ of FIG. 1, but is not limited thereto.

본 발명은 또한, 상기 재조합 식물 발현 벡터를 식물체에 도입하여 인간 락토페린 및 유산균 유래의 β-갈락토시다제 단백질 코딩 유전자를 과발현하는 단계를 포함하는 식물체에서 인간 락토페린 및 유산균 유래의 β-갈락토시다제 단백질 대량생산 방법을 제공한다.The present invention also provides β-galactosid derived from human lactoferrin and lactic acid bacteria in a plant, comprising the step of introducing the recombinant plant expression vector into the plant to overexpress the β-galactosidase protein coding gene derived from human lactoferrin and lactic acid bacteria. First protein mass production method is provided.

본 발명의 일 구현예에 따른 방법에서, 상기 식물체는 바람직하게는 단자엽 식물이며, 더욱 바람직하게는 벼이다.In the method according to one embodiment of the invention, the plant is preferably a monocotyledonous plant, more preferably rice.

본 발명은 또한, 상기 재조합 식물 발현 벡터를 식물체에 형질전환하는 단계를 포함하는 인간 락토페린 및 유산균 유래의 β-갈락토시다제를 생산하는 식물체의 제조방법을 제공한다.The present invention also provides a method for producing a plant for producing β-galactosidase derived from human lactoferrin and lactic acid bacteria, comprising the step of transforming the plant with the recombinant plant expression vector.

본 발명의 일 구현예에 따른 방법에서, 상기 식물체는 바람직하게는 단자엽 식물이며, 더욱 바람직하게는 벼이다.In the method according to one embodiment of the invention, the plant is preferably a monocotyledonous plant, more preferably rice.

본 발명은 또한, 상기 방법에 의해 제조된 벼로부터 얻은 쌀을 함유하는 쌀 가공식품을 제공한다. 상기 가공식품의 종류에는 특별한 제한은 없다. 락토페린은 살균 및 정균작용, 세포증식조절작용, 과산화지질 생성억제작용, 면역계 조절작용, 철 흡수 조절작용, 감염부위 염증발생 제어작용, 항바이러스 작용, 대장균의 장세포 부착방지, 비피더스균 증식작용 등의 여러 활성을 나타내는 유용한 유단백질이고, β-갈락토시다제는 유당 분해효소로서 이 두 단백질이 동시에 생산되는 쌀은 육류, 소세지, 빵, 쵸코렛, 캔디류, 스넥류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알콜 음료 및 비타민 복합제 등에 첨가할 수 있다. 또한, 유아용 조제분유를 비롯하여 화장품, 식품첨가물, 항설사약, 복막 투석용제, 임상영양제, 여성위생용품, 안약제품, 껌 등의 제조에도 유용하게 사용될 수 있다. 특히 소화 흡수가 미숙한 유아 및 어린이의 이유식에 유용하게 사용될 수 있는 것이다.The present invention also provides a rice processed food containing rice obtained from the rice produced by the method. There is no particular limitation on the type of processed food. Lactoferrin is bactericidal and bacteriostatic, cell proliferation control, lipid peroxidation suppression, immune system regulation, iron absorption regulation, control of inflammation site of infection, antiviral effect, intestinal cell adhesion prevention of E. coli, bifidus bacteria growth Β-galactosidase is a lactose-degrading enzyme. Rice produced by these two proteins simultaneously is meat, sausage, bread, chocolate, candy, snacks, confectionary, pizza, ramen, other noodles, It can be added to dairy products including gums and ice creams, various soups, beverages, teas, drinks, alcoholic beverages and vitamin complexes. In addition, it can be usefully used in the manufacture of infant formula, cosmetics, food additives, antidiarrheal drugs, peritoneal dialysis solvents, clinical nutrition, feminine hygiene products, eye drops, chewing gum and the like. In particular, the digestive absorption can be usefully used for baby foods and infants immature.

본 발명에서 락토페린과 β-갈락토시다제는 형질 전환 식물체인 벼의 종자인 쌀에서 동시에 발현됨으로, 벼 잎이나 담배 식물체에서 한 개의 유전자가 발현되는 것과는 다른 복합 유전자 발현이며, 벼 잎이나, 담배 식물체에서 인간 락토페린 및 β-갈락토시다제를 발현시켜 사용하는 용도가 상이하고, 인간 락토페린과 유산균 β-갈락토시다제가 생산되는 쌀은 인체에 흡입시 영양 성분 및 면역 강화 성분등 각종 기능성 성분을 인체에 제공하여 주는 동시에, 유제품의 소화 기능 작용을 향상시켜 인체 건강에 좋은 영향을 제공한다. 특히 모유를 먹는 아기들의 영양성분 및 항 병원성 면역력을 보강시켜 주며, 소화 불량을 예방시켜주는 기능을 가지고 있다. 또한 하나의 쌀 품종에서 두개의 단백질이 생산되므로, 각 단백질을 분리 정제하여 기능성 식의약품 원료나 첨가물, 또는 가축 사료용 첨가물로 상업화 할 경우 하나의 단백질을 분리 발현 시킨 벼 품종보다 경제적 효과가 매우 높아 질 수 있는 장점이 있다. 그리고, 본 발명은 형질전환 벼 식물체를 계통선발 육종 과정을 5세대에 걸쳐 노지에 재배하여, 도입 유전자가 안정화된 형질 전환 품종으로서 다른 특허와 차별성이 있다. In the present invention, since lactoferrin and β- galactosidase are simultaneously expressed in rice, which is a seed of a transgenic plant, rice is a complex gene expression different from that in which one gene is expressed in rice leaves or tobacco plants, Plants expressing human lactoferrin and β- galactosidase have different uses, and rice produced with human lactoferrin and lactic acid bacterium β- galactosidase has various functional ingredients such as nutritional and immune enhancing components when inhaled by the human body. In addition to providing to the human body, while improving the digestive function of dairy products provides a good effect on human health. In particular, it boosts the nutritional and anti-pathogenic immunity of breast-feeding babies and prevents indigestion. In addition, since two proteins are produced from a single rice variety, it is highly economical than rice varieties that express one protein separately when the proteins are separated and purified and commercialized as functional food ingredients, additives, or animal feed additives. There are advantages to it. In addition, the present invention is differentiated from other patents as a transgenic variety in which the transgenic rice plant is cultivated in the open field for five generations through a phylogenetic selection breeding process.

이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하기로 한다. 이들 실시예는 단지 본 발명을 예시하기 위한 것이므로, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는다. Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and thus the scope of the present invention is not construed as being limited by these embodiments.

재료 및 방법Materials and methods

1. 인간 락토페린(Human lactoferrin) cDNA의 변형1. Modification of Human Human Lactoferrin cDNA

인간 락토페린(Human lactoferrin, HLF) cDNA를 미국의 베일러 의과 대학(Baylor College of Medicine)의 폴린 워드(Paulin Ward) 박사로부터 제공받은 플라스미드 pGEM-HLFc로부터, PCR 방법을 통해 염기서열이 변형된 상태로 분리하였다. 변형된 상태로 분리하기 위해, 인터넷 진뱅크(GenBank)의 HLF cDNA 염기서열 (Accession No. AY13740)을 바탕으로 두개의 올리고머 정방향의 cHLF-F와 역방향의 cHLF-R을 합성하였다. 올리고머 cHLF-R은 HLF cDNA의 5‘-말단에 단백질 변환 도메인(protein transduction domain, PTD) tat 부위가 연결되도록 합성하였다. 역방향 주형 DNA (pGEM-HLFc) 100 ng, 200 mM dNTPs, 두개의 올리고머 100 pmoles/ul (cHLF-F(서열번호 3); 5'-AGGTCGACATGAAACTTGTCTTCCTC-3, cHLF-R(서열번호 4); 5'-AGGTCGACCTTCCTGAGGAATTCACA-3', HincII), 그리고 2 유닛의 Pwo DNA 폴리머라제를 넣고, 전체 부피가 50ul가 되도록 멸균 증류수를 첨가하였다. PCR 반응조건은 변성(denaturation) 94℃ 30초, 어닐링(annealing) 48℃ 1분, 중합(polymerization) 72℃ 1분 30초로 하여 30회 반복하였다. 증폭된 PCR 산물은 0.8% 아가로스 젤(agarose gel)에 전기영동한 후, UV 조사를 통해 확인한 다음, QIAGEN사의 젤 추출 키트(Gel extraction kit)로 정제하였다. 정제한 HLF cDNA 절편을 제한효소 HincII로 완전히 절단한 후, pBluescript II KS(+)의 동일한 제한효소 위치에 삽입하였다. 재조합된 플라스미드를 pHLF로 명명하였다. 쌀에서 발현된 락토페린을 인체 세포내로의 침투효율을 높이기 위하여 tat 염기성 도메인(tat basic domain, PTD; 아미노산 47-59, RKKRRQRRR) (Wender et al., 2000, Proc Nat Acad Sci USA 97: 13003-13008)을 HLF cDNA의 양쪽에 결합시키고자 하였다. 이를 위해서 우선 PTD 코딩 염기서열을 합성하여 이중 가닥(double-strand)으로 제작한 다음 pBluescript II KS(+) 삽입하여, 이를 pBks-tat로 명명하였다. 그리고 pHLF를 HincII로 잘라 HLF cDNA 절편을 분리하여, pBks-tat의 EcoRV 부위에 삽입시켰다. 완성된 벡터를 pHLF-PTD이라고 명명하였다. 이 벡터를 이용하여 Tat PTD와 HLF cDNA의 연결을 염료 터미네이터 시퀀싱(dye terminator sequencing) 방법을 이용하여 ABI prism 377 염기서열분석기로 분석 및 확인하였다.Human lactoferrin (HLF) cDNA was isolated from plasmid pGEM-HLFc provided by Dr. Pauline Ward of Baylor College of Medicine in the United States with modified nucleotide sequences by PCR method It was. In order to isolate in a modified state, two oligomers forward cHLF-F and a reverse cHLF-R were synthesized based on the HLF cDNA sequence of GenBank (Accession No. AY13740). The oligomeric cHLF-R was synthesized such that the tat site of the protein transduction domain (PTD) was connected to the 5'-end of the HLF cDNA. Reverse template DNA (pGEM-HLFc) 100 ng, 200 mM dNTPs, two oligomers 100 pmoles / ul (cHLF-F (SEQ ID NO: 3); 5'-AG GTCGAC ATGAAACTTGTCTTCCTC-3, cHLF-R (SEQ ID NO: 4)); 5'-AG GTCGAC CTTCCTGAGGAATTCACA-3 ' , Hinc II), and insert the Pwo DNA polymerase in the second unit, the sterile distilled water was added so that the total volume of 50ul. PCR reaction conditions were repeated 30 times with denaturation at 94 ° C for 30 seconds, annealing at 48 ° C for 1 minute, and polymerization at 72 ° C for 1 minute and 30 seconds. The amplified PCR product was electrophoresed on a 0.8% agarose gel (agarose gel), confirmed by UV irradiation, and purified by gel extraction kit (Gel extraction kit) of QIAGEN. Purified HLF cDNA fragments were completely digested with restriction enzyme Hinc II and inserted into the same restriction enzyme positions of pBluescript II KS (+). Recombinant plasmid was named pHLF. Lactoferrin expressed in rice to improve the efficiency of penetration into human cells tat basic domain (tat basic domain, PTD; amino acids 47-59, RKKRRQRRR) (Wender et al., 2000, Proc Nat Acad Sci USA 97: 13003-13008 ) Was bound to both sides of the HLF cDNA. To this end, PTD coding sequences were first synthesized, double-stranded, and then inserted into pBluescript II KS (+), which was named pBks-tat. And cut pHLF to Hinc II to remove the HLF cDNA fragment was inserted into the Eco RV site of the tat-pBks. The completed vector was named pHLF-PTD. Using this vector, the association of Tat PTD with HLF cDNA was analyzed and confirmed by ABI prism 377 sequencing using dye terminator sequencing.

2. β-갈락토시다제 (β-gal) 유전자의 변형2. Modification of the β-galactosidase (β-gal) Gene

비피도박테리움 아돌레스센티스 Int57(Bifidobacterium adolescentis Int57) 균주의 β-갈락토시다제 (β-gal) (Park et al., 2001, J Microbiol Biotech 11: 106-111) 유전자를 식물형질 전환 벡터에 삽입하기 위하여, PCR 방법을 이용하여 변형하였다. PCR에 사용된 올리고머는 GAL-1(서열번호 5)(AGGTTAACATGACTCAACGTAGAGCCTATCGTTGGCCC, 밑줄부분:HpaI 제한효소 절단부위)과 GAL-2(서열번호 6) (AGCTGCAGTCAAAAGATAGCAAGCCTTTGTTCGGGCGA, 밑줄부분:PstI 제한효소 절단부위)이며, 주형 DNA는 β-갈락토시다제 유전자가 삽입된 pBR-βgal 벡터이다. PCR 반응은 pBR-βgal 1㎍, 200 mM dNTPs, 각각 100 pmoles/ul의 올리고머(GAL-1, GAL-2), 1 유닛의 Pwo DNA 폴리머라제를 넣고, 전체 부피가 50ul가 되도록 멸균 증류수를 첨가하여 실시하였다. PCR 반응조건은 변성(denaturation) 95℃ 30초, 어닐링(annealing) 52℃ 45초, 중합(polymerization) 72℃ 2분으로 하여 30 회 반복하였다. 증폭된 DNA는 0.8% 아가로스 젤에 전기영동을 통해 확인하였다. PCR 증폭에서 얻어진 DNA 절편은 PCR 정제 키트(Qiagen)로 정제하여, DNA 절단효소 HpaI과 PstI으로 절단하여 다음 실험을 위하여 보관하였다.Β-galactosidase (β-gal) gene of Bifidobacterium adolescentis Int57 strain (Park et al., 2001, J Microbiol Biotech 11: 106-111) was transferred to a plant transformation vector. To insert, modifications were made using the PCR method. The oligomers used for PCR were GAL-1 (SEQ ID NO: 5) (AG GTTAAC ATGACTCAACGTAGAGCCTATCGTTGGCCC, underlined: Hpa I restriction enzyme cleavage site) and GAL-2 (SEQ ID NO: 6) (AG CTGCAG TCAAAAGATAGCAAGCCTTTGTTCGGGCGA, underlined: Pst I restriction enzyme Cleavage site), and the template DNA is a pBR-βgal vector into which the β-galactosidase gene is inserted. PCR reaction was performed by adding 1 μg of pBR-βgal, 200 mM dNTPs, 100 pmoles / ul oligomer (GAL-1, GAL-2), 1 unit of Pwo DNA polymerase, and adding sterile distilled water to a total volume of 50ul. It was carried out by. PCR reaction conditions were repeated 30 times with denaturation 95 degreeC 30 second, annealing 52 degreeC 45 second, polymerization 72 degreeC 2 minutes. Amplified DNA was confirmed by electrophoresis on 0.8% agarose gel. DNA fragments obtained from PCR amplification were purified by PCR purification kit (Qiagen), cut with DNA cleavage enzymes Hpa I and Pst I and stored for the next experiment.

3. 벼 형질 전환 벡터의 제작3. Construction of Rice Transformation Vector

위에서 변형시킨 인간 락토페린 유전자와 β-갈락토시다제 유전자를 벼에 도입하기 위하여, 식물 형질전환 바이너리 벡터 pVE9을 이용하였다. 이 형질전환용 바이너리 벡터 pVE9는 CaMV35S 프로모터 및 옥토파인 신타아제 터미네이터(octopin synthase terminator, Ocs))로 이루어진 발현카세트와 옥수수 폴리유비퀴틴 프로모터(maize polyubiquitin promoter) 및 애기장대 루비스코 소단위체 터미네이터(arabidopsis rubisco small subunit terminater, ArbcS)로 이루어진 유전자 발현 카세트 이외에 선발 마커로 제초제 저항성 바(bar) 유전자와 리포터(reporter) 유전자인 smGFP 유전자로 이루어져 있다. 우선 인체 락토페린 변형 유전자를 옥수수 폴리유비퀴틴 프로모터 및 애기장대 루비스코 스몰 서브유닛 터미네이터로 이루어진 유전자 발현 카세트 사이에 삽입시키고, β-갈락토시다제 유전자를 CaMV35S 프로모터 및 옥토파인 신타아제 터미네이터로 이루어진 발현카세트에 삽입시켜 락토페린 유전자와 β-갈락토시다제 유전자가 서로 반대 방향으로 발현이 이루어지게 하였다. 이렇게 제작된 벡터를 pVE9-TFTβ로 명명하였다(도 1). pVE9-TFTβ 벡터를 전기천공법(electroporation)에 의하여 아그로박테리움(Agrobacterium tumefaciens EHA105)에 도입시켜 카나마이신 (50 ug/mL)이 포함된 LB 고체 배지에서 배양한 다 음, pVE9-TFTβ 벡터를 지닌 아그로박테리움(Agrobacterium tumefaciens EHA105) 균주를 선발하였다.In order to introduce the human lactoferrin gene and β-galactosidase gene modified in the above into the rice, the plant transformation binary vector pVE9 was used. This transformation binary vector pVE9 is an expression cassette consisting of a CaMV35S promoter and an octopin synthase terminator (OCS), a maize polyubiquitin promoter and a Arabidopsis rubisco subterminator (arabidopsis rubisco small) In addition to the gene expression cassette consisting of a subunit terminater (ArbcS), the selection marker consists of the herbicide resistant bar gene and the reporter gene, the smGFP gene. First, a human lactoferrin modified gene is inserted between a gene expression cassette consisting of a corn polyubiquitin promoter and a Arabidopsis rubis small subunit terminator, and the β-galactosidase gene is inserted into an expression cassette consisting of a CaMV35S promoter and an octopine synthase terminator. By insertion, lactoferrin gene and β-galactosidase gene were expressed in opposite directions. The vector thus produced was named pVE9-TFTβ (FIG. 1). The pVE9-TFTβ vector was introduced into Agrobacterium tumefaciens EHA105 by electroporation and cultured in LB solid medium containing kanamycin (50 ug / mL), followed by agro with pVE9-TFTβ vector. Bacterium ( Agrobacterium tumefaciens EHA105) strains were selected.

4. 벼의 형질전환4. Transformation of Rice

본 실험에 사용된 벼는 농진청에서 분양 받은 낙동벼(Oryza sativa L. cv. Nakdong)를 이용하였다. 우선 벼 종자의 껍질을 제거하고 75% 에탄올에 2분간 침적한 후 멸균 증류수로 2-3회 세척하였다. 1% 소듐 히포클로라이트(Sodium hypochlorite, NaClO) 용액에 20분간 교반하면서 살균하였다. 살균된 종자를 멸균 증류수로 10회 세척하고 2N6 (N6 염 및 비타민, 2 mg/L 2,4-D(2,4-dichlorophenoxyacetic acid), 30 g/L 글루코스, 0.25% 피타겔(phytagel), pH 5.8) (Chu et al., 1975, Sci Sin 18: 659-668) 캘러스 유도 배지에 종자를 치상하고 27℃에서 3-4주간 암배양하여 유도된 캘러스에서 배발생 캘러스(embryogenic callus)를 육안으로 선별하여 새로운 2N6 배지에 치상하고 3-4일 동안 전 배양하였다. 이 캘러스를 pVE9-TFTβ이 포함된 아그로박테리움(Agrobacterium tumefaciens EHA105) 균주가 OD600이 0.8-1.2 정도로 배양된 아세토시링원(acetosyringone)이 포함된 AA 액체배지 (AA 염 및 아미노산, B5 비타민, 20 g/L 수크로스, 2 mg/L 2,4-D, 0.2 mg/L 키네틴, 100 mM 아세토시링원, pH 5.8) (Toriyama K, Hirata K, 1985, Plant Sci 41: 179-183)에 15분간 침적시켜 혼합배양시켰다. 접종된 캘러스는 멸균된 여과지로 여분의 아그로박테리움 현탁액을 제거한 후, 2N6-AS (2N6, 10 g/L 글루코 스, 100 mM 아세토시링원) 고체배지 1개당 25개의 캘러스를 치상한 다음, 3일간 24℃의 암조건에서 아그로박테리움과 공동배양하였다. 공동배양된 캘러스로부터 아그로박테리움을 제거하기 위하여 250 ug/mL의 카베니실린(carbenicillin)이 포함된 멸균 증류수에 캘러스를 3회 세척하였다. 세척된 캘러스를 멸균된 여과지로 여분의 물기를 제거한 후, 2N6-CP (2N6, 250 mg/L 카베니실린, 3 mg/L 포스피노트리신) 선발배지에 치상하였고 27℃에서 2주 동안 암배양하였다. 이후 왕성하게 분열한 캘러스만 선발하여 다시 2N6-CP 배지에 옮겨준 후 2주 가량의 암배양을 통해 증식된 캘러스만을 선발하였다. 선발된 생육이 양호한 캘러스를 MS 재분화 배지(MS 염 및 비타민, 30 g/L 수크로스, 0.5 mg/L NAA(naphthaleneacetic acid), 2 mg/L 키네틴, 0.4% 피타겔, 3 mg/L 포스피노트리신, 250 mg/L 카베니실린)에 치상하고 28℃에서 12시간 간격으로 명배양/암배양을 통해 재분화를 유도하였다. 재분화 배지로부터 분화된 형질전환 식물체를 생장조절물질이 첨가되지 않은 MS배지에 이식하여 뿌리를 유도하였고 뿌리가 유도된 식물체를 3-4일 동안 기내 순화과정을 거친 후, 화분에 옮겨 온실에서 종자를 수확할 때까지 재배하였다. The rice used in this experiment was Oryza sativa L. cv. Nakdong. First, the rice seed was removed from the shell, soaked in 75% ethanol for 2 minutes, and washed 2-3 times with sterile distilled water. Sterilization was performed in 1% sodium hypochlorite (NaClO) solution with stirring for 20 minutes. The sterilized seeds were washed 10 times with sterile distilled water and washed with 2N6 (N6 salt and vitamin, 2 mg / L 2,4-D (2,4-dichlorophenoxyacetic acid), 30 g / L glucose, 0.25% phytagel, pH 5.8) (Chu et al., 1975, Sci Sin 18: 659-668): Seeds are seeded in callus-inducing medium, and embryogenic callus is visualized in callus induced by cancer culture at 27 ° C. for 3-4 weeks. Were screened and inoculated into fresh 2N6 medium and pre-cultured for 3-4 days. This callus was used as AA liquid medium containing acetosyringone in which Agrobacterium tumefaciens EHA105 strain containing pVE9-TFTβ was cultured at 0.8-1.2 of OD 600 (AA salt and amino acid, B5 vitamin, 20 g / L sucrose, 2 mg / L 2,4-D, 0.2 mg / L kinetin, 100 mM acetocyring source, pH 5.8) (Toriyama K, Hirata K, 1985, Plant Sci 41: 179-183) It was mixed for a minute by immersion. Inoculated callus was sterile filter paper to remove the extra Agrobacterium suspension, then wound 25 callus per 2N6-AS (2N6, 10 g / L glucose, 100 mM acetoshiring source) solid medium, 3 Co-cultured with Agrobacterium at 24 ° C. in a dark condition. Callus was washed three times in sterile distilled water containing 250 ug / mL carbenicillin to remove Agrobacterium from the cocultured callus. The washed callus was drained with sterile filter paper and then wound on a 2N6-CP (2N6, 250 mg / L carbenicillin, 3 mg / L phosphinothricin) selection medium and treated for 2 weeks at 27 ° C. Incubated. Since only the fissely divided callus was selected and transferred to 2N6-CP medium again, only callus propagated through cancer culture for about 2 weeks was selected. Selected well-grown callus was harvested from MS regeneration medium (MS salt and vitamin, 30 g / L sucrose, 0.5 mg / L naphthaleneacetic acid (NAA), 2 mg / L kinetin, 0.4% pitagel, 3 mg / L phosphino) Tricine, 250 mg / L carbenicillin) and induce differentiation via light culture / cancer culture at 28 ° C. at 12 hour intervals. Transgenic plants differentiated from the regeneration medium were transplanted into MS medium without growth regulators to induce roots. After the root-derived plants were subjected to in-flight purification for 3-4 days, they were transferred to pots and seeded in greenhouses. Cultivation until harvest.

5. 벼 형질 전환체에 도입된 유전자 확인(서던 블럿 분석)5. Identification of Genes Introduced in Rice Transformants (Southern Blot Analysis)

형질전환 벼 식물체와 야생형 벼의 종자 및 엽육조직 등 각각 1g씩을 채취하여 액체질소로 분쇄한 후 Dellaporta 등 (1983. A plant DNA minipreparation:version II. Plant Mol Biol Rep. 1: 19-21)의 방법으로 추출하였다. DNA 5 μg을 HindIII 제한효소로 절단하여 1% 아가로스 젤에 분획한 후 모세관 현상을 이용한 이동(capillary transfer) 방법으로 나일론 막(Hybond-N+, Amersham)에 전이시켰다. 전이된 DNA 단편들을 고정시키기 위하여 UV-가교(UV-crosslink, 1200×μJ/cm2)를 실시한 후 막을 혼성화 버퍼(hybridization buffer, 5X SSPE,1.5% SDS, 5X denhardt's solution, 5 mg/mL salmon sperm testicle DNA)로 65℃에서 2시간 동안 예비혼성화(pre-hybridization)하였다. 이후 Amersham Ready-To-GoTM DNA labelling Beads (GE Healthcare)를 이용하여 1.6 kb의 HLF cDNA 단편과 1.0 Kb의 β-갈락토시다제 유전자 단편을 각각 32P로 표지한 프로브로 65℃에서 16-18시간 동안 교잡시켰다. 이 과정이 종료된 후 막을 37℃에서 세정 버퍼 1 (2X SSC, 0.1% SDS)으로 5분간 2회 세척해 준 다음 세정 버퍼 2 (1X SSC, 0.1% SDS)로 65℃에서 15분간 2회 세척한 후, X-ray 필름(Super RX, Fuji)에 감광하였다.1g each of transformed rice plants, wild type rice seeds, and foliar tissues were collected and pulverized with liquid nitrogen, followed by Dellaporta et al. (1983. A plant DNA minipreparation: version II. Plant Mol Biol Rep. 1: 19-21) Extracted with. 5 μg of DNA was digested with Hind III restriction enzyme, fractionated into 1% agarose gel, and transferred to nylon membrane (Hybond-N +, Amersham) by capillary transfer. UV-crosslinking (1200 × μ J / cm 2 ) to fix the transferred DNA fragments was followed by hybridization buffer (5X SSPE, 1.5% SDS, 5X denhardt's solution, 5 mg / mL salmon). sperm testicle DNA) was pre-hybridized at 65 ° C. for 2 hours. Afterwards, Amersham Ready-To-GoTM DNA labeling beads (GE Healthcare) were used to label 16 kb of HLF cDNA fragment and 1.0 Kb of β-galactosidase gene fragment with 32 P, respectively. Hybridized for hours. After this procedure, the membranes were washed twice with 5 minutes of washing buffer 1 (2X SSC, 0.1% SDS) at 37 ° C and then twice with 15 minutes at 65 ° C with washing buffer 2 (1X SSC, 0.1% SDS). Then, it was photosensitive to X-ray film (Super RX, Fuji).

6. 노던 블럿 분석6. Northern blot analysis

형질전환 벼 식물체와 야생형 벼의 종자 및 엽육조직 등 각각 300 mg을 채취하여 액체질소로 분쇄한 후 트리졸 시약(BioRad)을 사용하여 총 RNA를 추출하였다. 정제된 RNA 10 μg을 포름알데히드가 포함된 1% 아가로스 젤에 분리하였다. 이후 검출과정은 서던 블럿 분석법과 동일한 방법으로 진행하였다.300 mg of each of transformed rice plants, wild type rice seeds, and foliar tissues were collected, pulverized with liquid nitrogen, and total RNA was extracted using Trizol reagent (BioRad). 10 μg of purified RNA was isolated on a 1% agarose gel containing formaldehyde. The detection process was then carried out in the same way as Southern blot analysis.

7. 웨스턴 분석7. Western analysis

형질전환 식물체의 엽육조직 (1 g)과 종자 (1 g), 야생형 벼의 엽육조직 (1 g)과 종자 (1 g)을 액체질소로 분쇄한 후 단백질 추출 버퍼 (50 mM Tris-HCl pH 7.5, 0.1% SDS)를 첨가하고 얼음에 30분간 방치하였다. 균질현탁액(Homogenate)을 4℃ 12,000 rpm의 속도로 10분간 원심분리하여 얻은 상등액을 브레드포드 분석법(Bradford MM, 1976, Anal Biochem 72: 248-254)를 통해 상등액의 총 단백질을 정량하였다. 총 단백질을 10% SDS-폴리아크릴아미드 젤에 전기영동하여 분리한 후 PVDF 막 (Immobilon-P, Millipore)으로 전기적 블럿팅(electroblotting)을 수행하였다. 웨스턴 블럿은 WesternBreeze Chemiluminescent Western Blot Immunodetection Kit (Invitrogen)에서 제시한 방법에 따라 수행하였다.Cultivation of leaf tissues (1 g) and seeds (1 g) of transgenic plants, leaf tissues (1 g) and seeds (1 g) of wild-type rice with liquid nitrogen and protein extraction buffer (50 mM Tris-HCl pH 7.5 , 0.1% SDS) was added and left on ice for 30 minutes. The supernatant obtained by centrifuging the homogenate (Homogenate) for 10 minutes at a speed of 12,000 rpm at 4 ℃ was quantified the total protein of the supernatant by the Bradford assay (Bradford MM, 1976, Anal Biochem 72: 248-254). The total protein was separated by electrophoresis on a 10% SDS-polyacrylamide gel, followed by electroblotting with PVDF membrane (Immobilon-P, Millipore). Western blot was performed according to the method suggested by WesternBreeze Chemiluminescent Western Blot Immunodetection Kit (Invitrogen).

8. β-갈락토시다제 분석8. β-galactosidase assay

형질전환 벼 종자와 야생형 벼 종자 각각 1 g을 액체질소로 분쇄하고 50 mM Tris-Cl (pH 7.5)를 가하여 총 단백질을 추출하였다. 각각 총 단백질 2 ug을 FluorAce™ ß-galactosidase Reporter Assay Kit (Bio-rad)를 사용하여 Fluoromark Microplate Fluorometer (Bio-rad)로 360nm와 460nm 파장에서 형광을 측정하여 분석하였다.1 g of each of the transformed rice seeds and wild-type rice seeds was ground with liquid nitrogen, and 50 mM Tris-Cl (pH 7.5) was added to extract total protein. 2 ug of total protein was analyzed by Fluormark Microplate Fluorometer (Bio-rad) using the FluorAce ™ ß-galactosidase Reporter Assay Kit (Bio-rad).

9. ELISA(Enzyme-linked immunosorbance assay)9.Enzyme-linked immunosorbance assay (ELISA)

형질전환 T4 종자에서 HLF 발현양은 BIOXYTECH Lacto-EIA (OxisResearch) 방법을 이용한 ELISA로 정량하였다. 미세역가 플레이트의 웰(well)에 0.5 ug의 총단 백질을 넣고 37℃에서 1시간 동안 정치시킨 후, 세정 버퍼로 5회 세척하고 항-락토트란스페린(anti-lactotransferrin, LTF) 용액 100 ul를 넣고 37℃에서 1시간 동안 정치시켰다. 세정 버퍼로 5회 세척하고 각 웰에 100 ul의 Streptavidin-HRP를 넣고 37℃에서 15분 동안 반응시킨 후, 세정버퍼로 5회 세척하였다. 이후 100 ul의 OPD(o-phenylenediamine) 용액을 첨가하고 37℃에서 5-10분 동안 정치시키면서 색의 변화를 관찰한 후, 각 웰에 50 ul의 1 M H2SO4를 첨가하여 반응을 중지시켰다. Microplate Reader로 450nm에서 흡광도를 측정하였다.The amount of HLF expression in transgenic T4 seed was quantified by ELISA using BIOXYTECH Lacto-EIA ( Oxis Research) method. 0.5 ug of total protein was added to the well of the microtiter plate and allowed to stand at 37 ° C. for 1 hour, followed by washing 5 times with a washing buffer, and 100 ul of anti-lactotransferrin (LTF) solution. It was left to stand at 37 ° C for 1 hour. After washing 5 times with a washing buffer, 100 ul of Streptavidin-HRP was added to each well and reacted at 37 ° C. for 15 minutes, and then washed 5 times with a washing buffer. After 100 ul of OPD (o-phenylenediamine) solution was added and the color change was observed while standing at 37 ° C. for 5-10 minutes, the reaction was stopped by adding 50 ul of 1 MH 2 SO 4 to each well. . Absorbance was measured at 450 nm with a Microplate Reader.

10. 계통선발 및 형질전환 벼 육종10. Lineage Selection and Transgenic Rice Breeding

벼 형질전환체로부터 서던 블럿과 노던 블럿 실험을 수행한 결과, 유전자 도입이 확인된 12 계통의 벼 형질 전환체로부터 T0 종자를 수확하였다. 이중 유전자가 1 카피가 도입되어 있는 벼가 8계통, 2 카피가 도입된 벼가 4계통인 것이 확인되었다. 선발된 12계통 형절전환체를 포스피노트리신(PPT)이 포함되어 있는 배지에 치상하여 제초제 저항성 분리비를 검정하여 본 결과, 각각 분리비가 3:1 이상으로 나타나는 6계통을 선발하였다. 이중에 1 카피 도입 벼가 4계통, 2 카피 도입 벼가 2계통이었다. 이 계통의 벼를 온실의 실험용 포트에 파종하였고, 각각의 형질전환체에 제초제 basta를 처리하여 살아남은 개체들을 농진청 GMO 포장에 이앙하였다. 이 계통으로부터 각 계통마다 15개체의 T1 종자를 수확하였다. 이 중 포장에서 재배 과정 중 관찰한 결과 임성이 양호하고 생육상태가 좋은 3개의 개체를 각 계통마다 선발하였다. 선발된 T1 종자의 각 계통 3개의 개체들을 위와 같은 방법으로 처리하여 바스타 제초제에 저항성이 있는 개체들을 GMO 포장에 이앙하였다. 이로부터 각 계통당 6개체의 T2 종자를 수확하였고, 이 중 상태가 우수한 3개의 개체를 최종 선발하였다. 이 개체들을 위와 같은 방법으로 반복하여 T2 종자를 파종하여, 각 계통당 8개체의 T3 종자를 수확하였다. 수확한 각각의 종자로부터 단백질을 분리하여, 인간 락토페린에 대한 ELISA 실험과, β-갈락토시다제 분석을 수행하여 인간 락토페린과 β-갈락토시다제의 발현이 좋은 3개의 개체를 각 계통당 선발하였다. 선발된 각 계통 3개의 개체를 위와 같은 방법으로 재차 제초제 바스타 저항성 시험을 거쳐, GMO 포장에 이앙하였다. 이양된 벼 중에서 발육상태가 양호하고, 임성이 양호하며, 생육상태가 좋은 T4 세대 종자들을 각 계통별로 8개체씩 수확하였다. Southern blot and northern blot experiments were carried out from rice transformants, and T 0 seeds were harvested from 12 strains of rice transformants which were confirmed to be transgenic. It was confirmed that 8 genes of rice in which one copy of the gene was introduced, and 4 lines of rice in which two copies were introduced. The selected 12 systemic transformants were placed on a medium containing phosphinothricin (PPT) and assayed for herbicide resistance separation ratios. As a result, six lines each having a separation ratio of at least 3: 1 were selected. Among them, one copy introduced rice had four lines and two copy introduced rice had two lines. Rice of this strain was sown in experimental pots in the greenhouse, and surviving individuals were transferred to the RMO GMO package by treating herbicide basta with each transformant. From this strain, 15 T1 seeds were harvested for each strain. Among them, three individuals with good fertility and good growth were selected for each line as observed during the cultivation process in the field. Three individuals from each strain of selected T1 strains were treated in the same manner as above to transfer the individuals resistant to Basta herbicide to GMO packaging. From this, six T2 seeds were harvested for each line, and three of them were finally selected. These individuals were repeated in the same manner as above, sowing T2 seeds, and eight T3 seeds were harvested for each line. Proteins were separated from each of the harvested seeds, and ELISA experiments on human lactoferrin and β-galactosidase assay were performed to select three individuals with good expression of human lactoferrin and β-galactosidase per line. It was. Three individuals of each strain were screened again in the same manner as the herbicide bath resistance test, and transferred to the GMO packaging. Among the transferred rice, eight T4 generation seeds of good growth, good fertility, and good growth were harvested for each strain.

11. 포장 및 포장상태에서 제초제 저항성 검증11. Verification of herbicide resistance in packaging and packaging

각 세대의 형질전환 벼 식물체 계통으로부터 수확한 종자를 30℃의 항온기에서 수분을 흡수 발아시킨 종자를 모판에 파종하여 30일간 유모를 육성하고, 논에 이양하여 육종한 다음, 가을에 추수하였다. 이러한 과정을 거쳐 T4 세대의 종자까지 얻었다. 제초제 저항성 검증은 각 세대별 종자를 증식 후 60일이 지난 시점에 야생형과 형질전환 벼 식물체에 각각 제초제를 처리하였고 1주일 후 변화를 관찰하였다. 제초제로는 시판되고 있는 비선택성 제초제인 바스타 (유효성분 18%, 바이엘크롭 사이언스사)를 3 g/L 농도로 살포하였다.Seeds harvested from the transgenic rice plant lines of each generation were sown on 30 ° C. thermostats and germinated on seedlings to nurture nurses for 30 days, seeded in rice fields, and harvested in autumn. Through this process, seeds of the T4 generation were obtained. The herbicide resistance test was conducted 60 days after the growth of each generation of seed and treated with herbicides in wild type and transgenic rice plants, and observed changes after one week. As a herbicide, a commercially available non-selective herbicide, Vasta (18% active ingredient, Bayer Crop Science, Inc.) was sprayed at a concentration of 3 g / L.

12. 벼 형질전환체로부터 인체 락토페린 단백질의 분리 정제12. Isolation and Purification of Human Lactoferrin Protein from Rice Transformant

형질전환된 벼에서 수확한 종자로부터 도정하여 얻은 쌀을 분쇄기로 곱게 갈아 1:10 (100 g/L)의 비율로 추출버퍼 (20 mM Tris-Cl pH 7.4, 0.5 M NaCl)에 섞은 후 실온에서 밤이 지나도록 교반하였다. 균질현탁액(Homogenate)을 4겹의 무명천으로 여과하고 여과된 용액을 4℃에서 10,000 rpm으로 1시간 동안 원심분리하였다. 상등액을 셀룰로오스 아세테이트(celluose acetate) 재질의 3 um 및 0.45 um의 필터에 순차적으로 여과시켰다. 여과된 상등액을 결합 버퍼 A (20 mM Tris-Cl pH 7.4, 0.5 M NaCl)로 평형화시킨 Con A 세파로스 컬럼 (GE Healthcare)에 4 ml/분의 속도로 통과시키고 결합버퍼 A로 세척하였다. Con A 세파로스에 결합한 단백질은 용출버퍼 (결합 버퍼 A + 0.1 M 만노시드)로 용출하였다. rHLF를 포함하는 용출액을 90% 암모늄 설페이트로 침전시키고 원심분리하여 침전물을 회수하였고 50 kD cut-off 막 (Spectra/Por)으로 50 mM 소듐 포스페이트 버퍼 (pH 7.0)에 투석하였다. 투석한 단백질을 결합 버퍼 B (50 mM 소듐 포스페이트 pH 7.0)로 평형화시킨 SP-세파로스 컬럼에 1 ml/분의 속도로 통과시키고 결합 버퍼 B로 세척한 후, 1 M NaCl을 포함하는 결합 버퍼 B로 용출하였다. 최종적으로 용출된 단백질을 PBS에 투석하고 -80℃에 보관하였다. 정제된 재조합 인체 락토페린은 SDS-PAGE로 분석하였다.The rice obtained from the seed harvested from the transformed rice was finely ground with a grinder, mixed in an extraction buffer (20 mM Tris-Cl pH 7.4, 0.5 M NaCl) at a ratio of 1:10 (100 g / L), and then at room temperature. Stirred overnight. Homogenate was filtered through four layers of cotton cloth and the filtered solution was centrifuged at 10,000 rpm for 1 hour at 4 ° C. The supernatant was sequentially filtered through 3 um and 0.45 um filters made of cellulose acetate. The filtered supernatant was passed through a Con A Sepharose column (GE Healthcare) equilibrated with binding buffer A (20 mM Tris-Cl pH 7.4, 0.5 M NaCl) at a rate of 4 ml / min and washed with binding buffer A. Proteins bound to Con A Sepharose were eluted with an elution buffer (Binding Buffer A + 0.1 M Mannoside). The eluate containing rHLF was precipitated with 90% ammonium sulfate and centrifuged to recover the precipitate and dialyzed in 50 mM sodium phosphate buffer (pH 7.0) with a 50 kD cut-off membrane (Spectra / Por). The dialyzed protein was passed through a SP-Sepharose column equilibrated with binding buffer B (50 mM sodium phosphate pH 7.0) at a rate of 1 ml / min and washed with binding buffer B, followed by binding buffer B with 1 M NaCl. Eluted. Finally eluted protein was dialyzed in PBS and stored at -80 ° C. Purified recombinant human lactoferrin was analyzed by SDS-PAGE.

13. 쥐를 이용한 락토페린의 면역 활성 테스트13. Test of Immune Activity of Lactoferrin in Mice

한림대학교 실험동물센터로부터 6-7주령의 6마리의 수컷 마우스를 분양받았 다. 마우스는 40-60% 습도, 23℃, 12시간 광주기/암주기의 제어된 환경에서 사육하였다. 마우스를 2마리씩 3개의 그룹 - PBS, 비 형질전환체(non-transgenic), 형질전환체(transgenic)-으로 나누어 한 그룹에는 완충용액인 300 ul의 PBS 용액을, 비 형질전환체와 형질전환체는 각각 1 mg의 비 형질전환 벼 총 단백질(non-transgenic rice total protein)과 형질전환 벼 총 단백질(transgenic rice total protein)을 1주일 동안 매일 1회 경구 투여하였다. 투여기간 후, 각 그룹의 마우스를 마취시키고 소장을 적출하여 고유판(lamina propria)에 존재하는 면역글로불린(Immunoglobulin) A를 생산하는(IgA+) 세포에 대한 면역형광테스트를 수행하였다. 면역형광테스트는 실험용 쥐의 소장을 동결하여 미세절편기로 7 um 두께로 잘라 폴리-라이신(poly-lysine)이 코팅된 슬라이드 글라스(slide glass)에 부착시켰다. 항-마우스 (α-chain specific) FITC 컨주게이트 항체가 1/100로 희석된 PBS로 37℃에서 30분 동안 반응시키고 PBS로 2회 세척한 후 형광현미경 (Model BX51T, Olympus)으로 400배의 배율로 10 필드당 IgA+ 세포수를 측정하였다.Six male mice 6-7 weeks old were fed from Hallym University Experimental Animal Center. Mice were bred in a controlled environment of 40-60% humidity, 23 ° C., 12 hours photoperiod / dark cycle. Two mice were divided into three groups, PBS, non-transgenic, and transgenic. One group contained 300 ul of PBS solution as a buffer solution. 1 mg of non-transgenic rice total protein and transgenic rice total protein were administered orally once daily for one week. After the administration period, mice in each group were anesthetized and the small intestine was extracted to perform immunofluorescence tests on cells producing immunoglobulin A (IgA +) present in the lamina propria. In the immunofluorescence test, the small intestine of the rats was frozen and cut into 7 um thickness using a micro slicer and attached to a slide glass coated with poly-lysine. Anti-mouse (α-chain specific) FITC conjugate antibody was reacted for 30 minutes at 37 ° C. with PBS diluted to 1/100, washed twice with PBS, and then magnified 400 times with a fluorescence microscope (Model BX51T, Olympus). IgA + cell number per 10 fields was measured.

<실시예 1. 형질전환 식물체의 선발 및 증식>Example 1 Selection and Propagation of Transgenic Plants

2N6 캘러스 유도배지에 종자를 치상하여 3주 후 유도된 캘러스에 pVE9-TFTβ가 도입된 아그로박테리움(Agrobacterium)을 접종한 후, 카베니실린(carbenicillin)과 PPT가 포함된 2N6 선발배지에 배양하였다. 이후 재분화 배지에서 shoot를 유도한 다음, 재분화 배지로부터 분화된 식물체를 생장조절물질이 첨가되지 않은 MS배지에 이식하여 뿌리를 유도하였다. 뿌리가 유도된 형질전환 식물 체를 순화시킨 후 화분에 옮겨 온실에서 재배 관리하였다(도 2).Seeds were seeded in 2N6 callus-induced medium and inoculated with Agrobacterium introduced pVE9-TFTβ into the callus induced after 3 weeks, and then cultured in 2N6 selection medium containing carbenicillin and PPT. . After the shoot was induced in the regeneration medium, plants differentiated from the regeneration medium were transplanted into MS medium to which no growth regulator was added to induce roots. The root-derived transgenic plant was purified and then transferred to a flowerpot and managed in a greenhouse (FIG. 2).

<실시예 2. 형질전환 벼 육종을 통한 계통 선발><Example 2. Lineage Selection through Transgenic Rice Breeding>

벼 형질전환체로부터 서던 블럿과 노던 블럿 실험을 수행한 결과, 유전자 도입이 확인된 12 계통의 벼 형질전환체로부터 T0 종자를 수확하였다. 이 중 유전자가 1 카피가 도입되어 있는 벼가 8계통(Line 1, 2, 3, 5, 6, 9, 11, 12), 2 카피가 도입된 벼가 4계통(Line 4, 7, 13, 14)인 것이 확인되었다. 선발된 12계통 형절전환체의 종자를 PPT가 포함되어 있는 배지에 치상하여 제초제 저항성 분리비가 3:1 이상으로 나타나는 6계통(1 카피 4계통 식물체 2,5,9,11; 2 카피 2계통 식물체 7, 13)을 선발하였다. 선발된 계통마다 재배 과정 중 생육상태가 양호한 3개체씩을 선발하여 세대별 육종을 실시하였다. 이러한 과정을 T4 종자를 수확할 때까지 5세대에 걸쳐 분자육종을 실시하였다.Southern blot and northern blot experiments were carried out from rice transformants, and T 0 seeds were harvested from 12 strains of rice transformants which were confirmed to be transgenic. Among them, 8 genes of rice (1, 2, 3, 5, 6, 9, 11, 12) with 1 copy of the gene introduced, and 4 genes of rice (Lines 4, 7, 13, 2, with 2 copies introduced) 14) was confirmed. Six strains (1 copy 4 strains 2,5,9,11; 2 copy 2 strains) in which selected seed strains of 12 strain transformants are soaked in a medium containing PPT and the herbicide resistance separation ratio is 3: 1 or higher. 7, 13) were selected. For each of the selected lines, three individuals with good growth conditions were selected during the cultivation process, and breeding was performed by generation. This process was followed by molecular breeding for five generations until T4 seed was harvested.

<실시예 3. 포장상태에서 제초제 저항성 검증><Example 3. Verification of herbicide resistance in packaging state>

T4 세대의 형질전환 벼 식물체와 야생형의 벼 식물체를 GMO 포장의 재배 상태에서 비선택성 제초제인 바스타를 처리하고 1주 후 잎의 변화를 관찰한 결과, 도 3에서 보여주는 바와 같이 야생형은 갈변하여 고사되었지만 형질전환 식물체는 외형적으로 아무런 피해를 입지 않았다. 이와 같은 결과는 벼 게놈에 삽입된 포스피노트리신 아세틸트란스퍼라제(phosphinothricin acetyltransferase) 유전자가 정상 적으로 발현되는 것을 확인할 수 있었다.The transgenic rice plants of the T4 generation and wild type rice plants were treated with the non-selective herbicide, Basta, in the state of cultivation of GMO packaging, and the leaves were changed after 1 week. As shown in FIG. The transgenic plants were not damaged in appearance. These results confirmed that the phosphinothricin acetyltransferase gene inserted into the rice genome is normally expressed.

<실시예 4. 계통 선발된 벼 형질전환체내에서의 도입 유전자 확인><Example 4. Confirmation of the introduced gene in the line selection rice transformants>

형질전환 벼 식물체의 계통 선발 육종을 통해 얻어진 T4 세대의 5계통과 야생형 벼로부터 추출한 게놈 DNA를 HindIII로 처리한 후 HLF cDNA 단편(1.6 kb) 및 β-갈락토시다제 유전자 단편(1.0 kb)을 각각 프로브로 이용하여 서던블럿 분석을 실시하였다. 계통을 유지시킨 형질전환 식물체 모두에서 밴드가 검출되었으며 야생형에서는 어떠한 밴드도 검출되지 않았다 (도 4). 이는 T0 세대에서 T4 세대까지 5세대 동안 형질전환 식물체를 유지하면서 삽입된 유전자가 이탈되지 않고 각 계통에서 안정적으로 유지되고 있다는 것을 보여준다. 특히 계통 식물체 7(10-1-6-1)과 식물체 13(5-2-6-1)은 카피 수가 2개로 관찰되었다(도 4A, 레인 2 및 5). 이는 아그로박테리움을 이용한 형질전환시 T-DNA 부위가 식물체 염색체 상으로 도입될 때 유전자의 재배열로 인해 나온 결과로 사료된다.HLF cDNA fragments (1.6 kb) and β-galactosidase gene fragments (1.0 kb) were treated with Hind III of genomic DNA extracted from five lines of T4 and wild-type rice obtained from lineage breeding of transgenic rice plants. Southern blot analysis was performed using each probe. Bands were detected in all of the transgenic plants maintained in lineage and no bands were detected in wild type (FIG. 4). This shows that the inserted genes remain stable in each line without leaving the transgenic plant for five generations, from generation T0 to generation T4. In particular, lineage plants 7 (10-1-6-1) and plant 13 (5-2-6-1) were observed to have two copies (FIG. 4A, lanes 2 and 5). This may be due to the rearrangement of the gene when the T-DNA site is introduced into the plant chromosome when transformed with Agrobacterium.

<실시예 5. 벼 형질전환체내에서의 도입 유전자의 전사 확인(노던 블럿 분석)><Example 5. Confirmation of transcription of introduced gene in rice transformant (Northern blot analysis)>

형질전환 벼 식물체로부터 서던 블럿 분석을 통해 유전자 도입이 유지되고 있는 것을 확인한 T4 세대의 형질전환 벼 엽육조직과 야생형 벼의 엽육조직으로부터 총 RNA를 분리하여 노던 블럿 분석을 수행하였다. 이 실험으로부터 형질전환된 벼에서는 벼에 도입된 HLF cDNA와 β-갈락토시다제 유전자의 RNA가 발견되었지만 (도 5), 형질전환이 안된 야생형 벼에서는 도입 유전자의 RNA가 검색되지 않았다. 이 실험 결과로 벼 식물체로 도입된 HLF cDNA와 β-갈락토시다제 유전자가 T4 세대에서도 안정적으로 발현되고 있다는 것을 확인할 수 있었다.Northern blot analysis was performed by separating total RNA from T4 generation of transgenic rice leaf tissues and wild type rice leaf tissues, which confirmed that gene introduction was maintained through Southern blot analysis from the transformed rice plants. RNA of HLF cDNA and β-galactosidase gene introduced into rice was found in the transformed rice from this experiment (FIG. 5), but the RNA of the transgene was not detected in the wild type rice without transformation. As a result of this experiment, it was confirmed that HLF cDNA and β-galactosidase genes introduced into rice plants were stably expressed in the T4 generation.

<실시예 6. 형질전환 벼의 쌀에서의 인체 모유 단백질 락토페린 검색(웨스턴 블럿 분석)><Example 6. Human breast milk lactoferrin search in transgenic rice rice (Western blot analysis)>

T4 세대 형질전환 식물체 벼의 쌀에서 인체 모유 단백질 락토페린을 검색하기 위해서 웨스턴 블럿을 수행한 결과, T4 세대까지 유지된 각 계통의 종자에서 인체 모유 단백질 락토페린이 생산되고 있다는 것을 확인할 수 있었다 (도 6, 레인 1-5). 그러나, 형질전환되지 않은 벼 식물체 쌀에서는 락토페린이 검색되지 않았다.Western blot was performed to search for human breast milk protein lactoferrin in rice of T4 generation transgenic plant rice. As a result, it was confirmed that human breast milk protein lactoferrin was produced in each strain maintained until T4 generation (FIG. 6, Lanes 1-5). However, lactoferrin was not detected in untransformed rice plant rice.

<실시예 7. 형질전환 벼의 쌀에서의 유산균 단백질 β-갈락토시다제 검색><Example 7. Lactobacillus protein β-galactosidase search in rice of transformed rice>

T4 세대 형질전환 식물체 벼의 쌀에서 형질전환을 위해 도입된 유산균 단백질 β-갈락토시다제의 발현을 검색하기 위하여, 앞의 실험 방법에서 언급한 것과 같이, X-gal 기질을 이용한 β-갈락토시다제 효소활성 시험을 실시하였다. 이 실험으로부터 T4 세대까지 유지된 각 계통의 종자에서 유산균 단백질 β-갈락토시다제가 생산되고 있다는 것을 확인할 수 있었다 (도 7, 레인 1-4). 그러나, 형질전환되지 않은 벼 식물체 쌀에서는 β-갈락토시다제가 검색되지 않았다.In order to detect the expression of lactic acid protein β-galactosidase introduced for transformation in rice of T4 generation transgenic plant rice, as mentioned in the previous experimental method, β-galacto using X-gal substrate The oxidase enzyme activity test was performed. From this experiment, it was confirmed that lactic acid bacteria protein β-galactosidase was produced in the seeds of each strain maintained up to T4 generation (Fig. 7, lanes 1-4). However, β-galactosidase was not detected in untransformed rice plant rice.

<실시예 8. 유산균 단백질 β-갈락토시다제 활성 및 모유 락토페린의 발현양 분석><Example 8. Lactobacillus protein β-galactosidase activity and expression analysis of breast milk lactoferrin>

각 계통의 T4 세대 벼의 쌀에서 유산균 단백질 β-갈락토시다제 활성과 인체 락토페린의 발현양을 위의 재료 및 방법에서 언급한 방법대로 측정하였다. 각 계통의 T4 세대 벼의 쌀에서 유산균 효소 단백질 β-갈락토시다제 활성과 인체 락토페린의 발현이 검색되었고, 그 중에 형질전환 벼 계통 13(5-2-6-1)에서 β-갈락토시다제 활성과 인체 락토페린의 발현이 가장 강하게 나타나고 있음이 확인되었다(표 1). 특히 인체 모유 락토페린이 쌀의 전체 가용성 단백질의 약 1.5% 정도로 발현되고 있는 것으로 밝혀졌다. 이와 반대로 야생형 종자에서는 β-갈락토시다제 활성이 아주 약하게 나타났으며, 인체 모유 락토페린은 전혀 검출되지 않았다. 야생형 종자에서 β-갈락토시다제 활성이 아주 약하게 나타나는 것은, 벼 자체에 β-갈락토시다제의 활성이 있기 때문인 것으로 추측되어진다.The lactobacillus protein β-galactosidase activity and the amount of human lactoferrin expression in T4 generation rice of each strain were measured as described in the above materials and methods. The lactobacillus enzyme protein β-galactosidase activity and human lactoferrin expression were detected in T4 generation rice of each strain, among which β-galactosidase in transgenic rice strain 13 (5-2-6-1). It was confirmed that the first activity and the expression of human lactoferrin are the strongest (Table 1). In particular, human breast milk lactoferrin was found to be expressed in about 1.5% of the total soluble protein of rice. In contrast, wild-type seeds showed very weak β-galactosidase activity, and no human breast lactoferrin was detected. It is presumed that the reason why the β-galactosidase activity is very weak in wild-type seeds is that the rice itself has β-galactosidase activity.

표 1. T4 형질전환 벼 종자에서 β-갈락토시다제의 활성 및 인간 락토페린의 발현 정도Table 1. Activity of β-galactosidase and expression level of human lactoferrin in T4 transgenic rice seeds

Figure 112008088189398-PAT00001
Figure 112008088189398-PAT00001

<실시예 9. 형질전환 계통선발된 T4 세대 벼의 쌀로부터 인체 모유 락토페린의 정제>Example 9 Purification of Human Breast Milk Lactoferrin from Transgenic Lineage Selected T4 Generation Rice

형질전환 T4 세대의 벼를 각 계통을 모두 혼합하여 얻은 쌀로부터 단백질을 추출하였다. 우선 쌀을 분쇄기로 곱게 마쇄한 다음 추출 용액으로 쌀의 전체 단백질을 추출하였다. 그런 다음 재료 및 방법에 적혀 있는 방식에 따라 친화성 크로마토그라피(Affinity chromatography) 및 Con A 세파로스, 그리고 이온 교환 크로마토그라피(Ion exchange chromatography) 방법과 SP-세파로스를 이용하여 단백질을 정제하였다. 정제되어진 단백질을 SDS-PAGE를 이용하여 분석하였다(도 8). 이 정제 과정을 통하여 순수한 인체 모유 단백질을 정제하여 얻을 수 있었다(도 8, 레인 6 및 7). 실험에 의하여 얻어진 SDS-PAGE를 락토페린 항체를 이용하여 웨스턴 블럿 분석을 실시하여 확인해본 결과 정제된 단백질이 락토페린임이 확인되었다(레인 8 및 9).Proteins were extracted from the rice obtained by mixing all strains of the transgenic T4 generation rice. First, the rice was ground finely with a grinder, and then the whole protein of the rice was extracted with the extraction solution. The proteins were then purified using Affinity chromatography and Con A Sepharose, and ion exchange chromatography and SP-Sepharose according to the methods described in Materials and Methods. Purified protein was analyzed using SDS-PAGE (FIG. 8). Through this purification process, pure human breast milk protein was purified and obtained (FIG. 8, lanes 6 and 7). The SDS-PAGE obtained by the experiment was confirmed by Western blot analysis using a lactoferrin antibody, and it was confirmed that the purified protein was lactoferrin (lanes 8 and 9).

<실시예 10. 쥐를 이용한 형질전환 쌀에서 생산된 인체 모유 락토페린의 생리활성 효과><Example 10. The physiological activity of human breast milk lactoferrin produced from transformed rice using rats>

형질전환 벼 T4 세대의 쌀에서 생산된 인체 락토페린의 생리 활성 효과를 실험용 쥐를 이용하여 검색하였다. 실험용 쥐에게 형질전환 쌀 추출물을 일정 기간 동안 경구 투여하여 해부한 다음, 소장의 고유판(lamina propria)에서 IgA+ 세포의 수치를 측정함으로서 면역 반응 활성을 측정하였다. 형질전환 쌀의 추출물을 경구 투여한 쥐의 소장 고유판에서 IgA+ 세포의 수치가 증가하였으나, 대조군(PBS 및 야 생형 쌀 추출물)에서는 고유판에서 IgA+ 세포의 수치가 증가하지 않았음을 확인할 수 있었다. 이 실험을 통하여 형질전환 쌀에서 생산된 인체 모유 락토페린이 동물 세포에서도 면역 반응 생리 활성 효과가 있음을 알 수 있었다(도 9). The physiological activity of human lactoferrin produced in the rice of the T4 generation of transformed rice was searched using a mouse. Experimental rats were dissected by oral administration of the transformed rice extract for a period of time, and then immune response activity was measured by measuring the levels of IgA + cells in the lamina propria of the small intestine. IgA + cells were increased in the small intestine lamina propria of oral administration of the extract of transgenic rice, but the control (PBS and wild-type rice extract) did not increase the levels of IgA + cells in the lamina propria. Through this experiment, human breast milk lactoferrin produced from transformed rice was found to have an immune response physiological activity in animal cells (FIG. 9).

도 1은 벼 형질전환용 벡터 pVE9-TFTβ의 구조를 나타내는 그림이고(Ubi-P, 옥수수 폴리유비퀴틴 프로모터; Tat, 단백질 변환 도메인; HLF, 인간 락토페린 cDNA; bar, 포스피노트리신 아세틸트란스퍼라제 유전자; ArbcS, 애기장대 루비스코 소단위체; 35S, 콜리플라워 모자이크 바이러스 (CaMV) 35S RNA 프로모터; RB, 오른쪽 보더; LB, 왼쪽 보더), 1 is a diagram showing the structure of the vector pVE9-TFTβ for rice transformation (Ubi-P, corn polyubiquitin promoter; Tat, protein conversion domain; HLF, human lactoferrin cDNA; bar, phosphinothricin acetyltransferase gene ArbcS, Arabidopsis rubisco subunit; 35S, Cauliflower Mosaic Virus (CaMV) 35S RNA promoter; RB, right border; LB, left border),

도 2는 형질전환 벼 식물체를 나타내는 그림이고(A. 배발생 캘러스로부터 shoot의 유도; B. 뿌리 유도 배지에서 뿌리의 재분화; C. 포트에서 자란 형질전환 벼 식물체), 2 is a diagram showing a transformed rice plant (A. induction of shoots from embryogenic callus; B. regeneration of roots in root-induced medium; C. transformed rice plants grown in pots),

도 3은 야생형(왼쪽) 및 T4 형질전환 벼 식물체(오른쪽)에서 바스타 제초제의 영향을 나타내는 그림이고, FIG. 3 is a diagram showing the effects of bastard herbicides in wild-type (left) and T4 transgenic rice plants (right),

도 4는 T4 형질전환 벼 식물체의 서던 블럿 분석을 나타내는 그림이고(A. 1.6kb HLF cDNA 프로브 이용, B. 1.0kb β-갈락토시다제 cDNA 프로브 이용 PC, 2.1 kb HLF cDNA(양성 대조구); NC, 비 형질전환 벼 식물체 DNA(음성 대조구). 레인 1-5, 형질전환 식물체 2(6-2-3-1), 7(10-1-6-1), 9(19-3-6-1), 11(22-1-2-1), 13(5-2-6-1)), 4 is a Southern blot analysis of T4 transgenic rice plants (A. using 1.6 kb HLF cDNA probe, B. PC using 1.0 kb β-galactosidase cDNA probe, 2.1 kb HLF cDNA (positive control); NC, non-transformed rice plant DNA (negative control) Lane 1-5, transgenic plant 2 (6-2-3-1), 7 (10-1-6-1), 9 (19-3-6 -1), 11 (22-1-2-1), 13 (5-2-6-1)),

도 5는 T4 형질전환 벼 식물체에서 HLF cDNA (A) 및 β-갈락토시다제 유전자(B)의 발현을 나타내는 그림이고(NC, 비 형질전환 벼 식물체 DNA(음성 대조구). 레인 1-5, 형질전환 식물체 2(6-2-3-1), 7(10-1-6-1), 9(19-3-6-1), 11(22-1-2-1), 13(5-2-6-1)), Figure 5 is a diagram showing the expression of HLF cDNA (A) and β-galactosidase gene (B) in T4 transgenic rice plants (NC, non-transformed rice plant DNA (negative control). Transformed Plants 2 (6-2-3-1), 7 (10-1-6-1), 9 (19-3-6-1), 11 (22-1-2-1), 13 (5 -2-6-1)),

도 6은 T4 형질전환 쌀 알에서 웨스턴 블럿 분석을 이용하여 인간 락토레핀의 검출을 알아본 그림이고(M, 마커; PC, 상업용 HLF (250 ng, 시그마); NC, 비형질전환체; 레인 1-5, 형질전환 식물체 2(6-2-3-1), 7(10-1-6-1), 9(19-3-6-1), 11(22-1-2-1), 13(5-2-6-1)), FIG. 6 shows the detection of human lactorepin using Western blot analysis in T4 transgenic rice eggs (M, marker; PC, commercial HLF (250 ng, Sigma); NC, nontransformer; lane 1 -5, transgenic plant 2 (6-2-3-1), 7 (10-1-6-1), 9 (19-3-6-1), 11 (22-1-2-1), 13 (5-2-6-1)),

도 7은 T4 형질전환 쌀알에서 박테리아 β-갈락토시다제 활성 테스트를 나타내는 그림이고(N1, 트리스 버퍼 대조구, N2, 비형질전환 쌀알, 레인 1-4, 형질전환 식물체 2(6-2-3-1), 9(19-3-6-1), 11(22-1-2-1), 13(5-2-6-1) (X-gal 농도; 250 mg/L), FIG. 7 shows bacterial β-galactosidase activity test in T4 transgenic rice grains (N1, Tris buffer control, N2, untransformed rice grains, lanes 1-4, transgenic plant 2 (6-2-3) -1), 9 (19-3-6-1), 11 (22-1-2-1), 13 (5-2-6-1) (X-gal concentration; 250 mg / L),

도 8은 SDS-PAGE로 인간 락토페린의 정제 분석을 나타내는 그림이고(M, 분자 마커; 레인 1, 상업용 인간 락토페린 (시그마); 2, Con A 세파로스에 원액을 로딩; 3, Con A 세파로스에 결합되지 않은 분획; 4, Con A 세파로스 컬럼 용출액 (암모늄 설페이트 침전 후); 5, SP-세파로스에 결합되지 않은 분획물; 6 및 7, SP-세파로스 컬럼의 인간 락토페린. 레인 8 및 9; 인간 락토페린 항체에 대한 웨스턴 블럿), FIG. 8 shows purification analysis of human lactoferrin by SDS-PAGE (M, molecular marker; lane 1, commercial human lactoferrin (Sigma); 2, loading stock solution into Con A Sepharose; 3, Con A Sepharose Unbound fraction 4, Con A Sepharose column eluate (after ammonium sulfate precipitation) 5, fraction not bound to SP-Sepharose 6 and 7, human lactoferrin on SP-Sepharose column Lane 8 and 9; Western blot against human lactoferrin antibody),

도 9는 경구 투여에 의한 쥐 고유판(lamina propria)에서 형질전환 쌀알로부터 얻은 인간 락토페린의 면역 생리활성을 조사한 결과이다. 비형질전환 및 형질전환 쌀알로부터 추출한 총 단백질 1mg을 1주일동안 1일에 쥐에 투여하였고, 대조구는 300 ul PBS-버퍼(PBS)를 투여하였다. 9 is a result of examining the immune physiological activity of human lactoferrin obtained from the transformed rice grains in rat lamina propria by oral administration. 1 mg of total protein extracted from untransformed and transformed rice grains was administered to mice on day 1 for one week, and the control group received 300 ul PBS-buffer (PBS).

<110> Industry Academic Cooperation Foundation, Hallym University <120> Method for producing transgenic plant producing the human lactoferrin and bifidobacterial beta-galactosidase and plant produced by the same <130> PN08244 <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 2136 <212> DNA <213> Oryza sativa <400> 1 atgaaacttg tcttcctcgt cctgctgttc ctcggggccc tcggactgtg tctggctggc 60 cgtaggagaa ggagtgttca gtggtgcgcc gtatcccaac ccgaggccac aaaatgcttc 120 caatggcaaa ggaatatgag aaaagtgcgt ggccctcctg tcagctgcat aaagagagac 180 tcccccatcc agtgtatcca ggccattgcg gaaaacaggg ccgatgctgt gacccttgat 240 ggtggtttca tatacgaggc aggcctggcc ccctacaaac tgcgacctgt agcggcggaa 300 gtctacggga ccgaaagaca gccacgaact cactattatg ccgtggctgt ggtgaagaag 360 ggcggcagct ttcagctgaa cgaactgcaa ggtctgaagt cctgccacac aggccttcgc 420 aggaccgctg gatggaatgt ccctacaggg acacttcgtc cattcttgaa ttggacgggt 480 ccacctgagc ccattgaggc agctgtggcc aggttcttct cagccagctg tgttcccggt 540 gcagataaag gacagttccc caacctgtgt cgcctgtgtg cggggacagg ggaaaacaaa 600 tgtgccttct cctcccagga accgtacttc agctactctg gtgccttcaa gtgtctgaga 660 gacggggctg gagacgtggc ttttatcaga gagagcacag tgtttgagga cctgtcagac 720 gaggctgaaa gggacgagta tgagttactc tgcccagaca acactcggaa gccagtggac 780 aagttcaaag actgccatct ggcccgggtc ccttctcatg ccgttgtggc acgaagtgtg 840 aatggcaagg aggatgccat ctggaatctt ctccgccagg cacaggaaaa gtttggaaag 900 gacaagtcac cgaaattcca gctctttggc tcccctagtg ggcagaaaga tctgctgttc 960 aaggactctg ccattgggtt ttcgagggtg cccccgagga tagattctgg gctgtacctt 1020 ggctccggct acttcactgc catccagaac ttgaggaaaa gtgaggagga agtggctgcc 1080 cggcgtgcgc gggtcgtgtg gtgtgcggtg ggcgagcagg agctgcgcaa gtgtaaccag 1140 tggagtggct tgagcgaagg cagcgtgacc tgctcctcgg cctccaccac agaggactgc 1200 atcgccctgg tgctgaaagg agaagctgat gccatgagtt tggatggagg atatgtgtac 1260 actgcatgca aatgtggttt ggtgcctgtc ctggcagaga actacaaatc ccaacaaagc 1320 agtgaccctg atcctaactg tgtggataga cctgtggaag gatatcttgc tgtggcggtg 1380 gttaggagat cagacactag ccttacctgg aactctgtga aaggcaagaa gtcctgccac 1440 accgccgtgg acaggactgc aggctggaat atccccatgg gcctgctctt caaccagacg 1500 ggctcctgca aatttgatga atatttcagt caaagctgtg cccctgggtc tgacccgaga 1560 tctaatctct gtgctctgtg tattggcgac gagcagggtg agaataagtg cgtgcccaac 1620 agcaaggaga gatactacgg ctacactggg gctttccggt gcctggctga gaatgctgga 1680 gacgttgcat ttgtgaaaga tgtcactgtc ttgcagaaca ctgatggaaa taacaatgag 1740 gcatgggcta aggatttgaa gctggcagac tttgcgctgc tgtgcctcga tggcaaacgg 1800 aagcctgtga ctgaggctag aagctgccat cttgccatgg ccccgaatca tgccgtggtg 1860 tctcggatgg ataaggtgga acgcctgaaa caggtgctgc tccaccaaca ggctaaattt 1920 gggagaaatg gatctgactg cccggacaag ttttgcttat tccagtctga aaccaaaaac 1980 cttctgttca atgacaacac tgagtgtctg gccagactcc atggcaaaac aacatatgaa 2040 aaatatttgg gaccacagta tgtcgcaggc attactaatc tgaaaaagtg ctcaacctcc 2100 cccctcctgg aagcctgtga attcctcagg aagtaa 2136 <210> 2 <211> 2349 <212> DNA <213> Oryza sativa <400> 2 atgactcaac gtagagccta tcgttggccc gtcccgttgt ctggacagca ggaacgcatc 60 tggtacggag gtgactacaa cccggaccag tggcccgagg aagtgtggga tgatgacgtc 120 cgtctgatga agaaggcggg cgtgaacctc gtgtccgtcg gcatcttctc atgggcgaag 180 atcgagacga gcgaaggcgt gtacgatttc gactggctcg accgcatcat cgacaagctg 240 ggcgaggccg gcgtcgccgt ggatttggcg tccgccaccg cttcgccgcc gatgtggctc 300 acccaggcgc atcccgaggt gctgtggaag gactaccgcg gcgacgtgtg ccagcctggc 360 gcacgccagc attggaggcc cacgagcccg gtgttccgcg agtacgcgct gaagctgtgc 420 cgtgcgatgg ccgagcatta caagggcaat ccgtacgtgg tcgcgtggca tgtgagcaac 480 gagtacggct gccacaaccg tttcgactat tccgaggacg ccgaacatgc gttccagcag 540 tggtgcgagg aacgctacgg caccatcgac gcggtgaacg acgcgtgggg gaccgcgttc 600 tgggcacagc gcatgaacga cttcagtgag atcgtgcctc cgcgcttcat cggcgacggc 660 aacttcatga acccgggcaa gctgctcgac ttcaagcggt tcagctcgga cgcgttgaag 720 gcgttctaca tcgccgagcg cgacacgctt gcgagatcac gccggatctg ccctgaccac 780 gaacttatgg tgtccgcgtc cggttcggtg ttggactacg acgattgggg cgacgaggtc 840 gatttcgtgt cgaacgacca ttacttcatt ccgggcgagg cccacttgga cgagctcgcg 900 ttctccgcga gcctggtcga cggcatcgca cgcaaggatc cgtggttcct gatggagcat 960 tccaccagcg cggtcaactg gcgtgagatc aactaccgca aggagcccgg ccagctggtg 1020 cgcgactctt tggctcatgt ggccatgggt gcggacgcgg tgtgctactt ccagtggagg 1080 cagtccaagg caggcgcgga gaagttccat tccgcgatgg tgccccacgc gggcgaggat 1140 tccgccgtgt tccgcgacgt gtgcgagctg ggcgccgatc tgaacaaact gtccgacgag 1200 ggcattctgg gatcgggctc gcgaagtccc gtgtggccgt ggtattcgac atacgagtcc 1260 gaatgggcca ccgaacacac cgccacgccg acccagcacg tgcatcacgt ggacgagcca 1320 ttggcgtggt tccgcgcttt ggccgatcag ggtgtgaccg ccgacgtggt gccggtgcgc 1380 ggcgcctggg atgactatga gatggtcgta ttgccgagtg tatacctgct ttccgaggaa 1440 acaccgcagg gtgcgcgatt acgtcgtggg cggcggtcga ctggtcgtga cctactacac 1500 gggcatctcg gacgagaagg accacgtgtg gctcggcggg tatccgggct cgatccggac 1560 gtggtcggcg tgcgcgtgga ggaattcggt gaaggttctc gtaccctcat cgtcggtgag 1620 gttgatcgtc acgtttttgc cgtcgttgga ggtggagaac ggacggcctc cggcaagcca 1680 gatcatcgtg tcgtagaagc tggcgtcacc cgaatcggct gcgatgtaca cgccgatgct 1740 tttcagcttt ttggccgcct cgtaatagtc gtcccacgtg cgaatcttgg aggcatcgat 1800 gtctacctgc tcgaacacgc tgtcgttgta gaagaacgcc attggaccgg aatccatggg 1860 aaggccgtac acgcggccgt tcagctgcac cgaagcccac gtaccgggcg tgtagaaatc 1920 gccgtagcct tccgtacgcc cggtgatcgc caccagctgg ccgcttaccg cgtactggcg 1980 caacgcatag tattccaatt gcaccacgtc gggaatgccg taaccgtctt gaatggcgtt 2040 gttgagattg tcgtaaccgc ttgtgttctt ccaaataacg tggatgtcgg gattttcctt 2100 ttcgaaaccg gcaatgactt gtttcatgct cggctcccac gaccatacgg tgatttcagt 2160 tcgggaatcg acgttggctc cgcatgcagc cgcactggcc gccatgatgg ccgcacagca 2220 gatggcgatg agtcgtttca ccagtttcaa gccgattcct tccgtttgac gttttcgtac 2280 gaaaacgtac gtagacatac gcattcgagt gtacgtcagt cgcccgaaca aaggcttgct 2340 atcttttga 2349 <210> 3 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 aggtcgacat gaaacttgtc ttcctc 26 <210> 4 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 aggtcgacct tcctgaggaa ttcaca 26 <210> 5 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 aggttaacat gactcaacgt agagcctatc gttggccc 38 <210> 6 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 agctgcagtc aaaagatagc aagcctttgt tcgggcga 38 <110> Industry Academic Cooperation Foundation, Hallym University <120> Method for producing transgenic plant producing the human          lactoferrin and bifidobacterial beta-galactosidase and plant          produced by the same <130> PN08244 <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 2136 <212> DNA <213> Oryza sativa <400> 1 atgaaacttg tcttcctcgt cctgctgttc ctcggggccc tcggactgtg tctggctggc 60 cgtaggagaa ggagtgttca gtggtgcgcc gtatcccaac ccgaggccac aaaatgcttc 120 caatggcaaa ggaatatgag aaaagtgcgt ggccctcctg tcagctgcat aaagagagac 180 tcccccatcc agtgtatcca ggccattgcg gaaaacaggg ccgatgctgt gacccttgat 240 ggtggtttca tatacgaggc aggcctggcc ccctacaaac tgcgacctgt agcggcggaa 300 gtctacggga ccgaaagaca gccacgaact cactattatg ccgtggctgt ggtgaagaag 360 ggcggcagct ttcagctgaa cgaactgcaa ggtctgaagt cctgccacac aggccttcgc 420 aggaccgctg gatggaatgt ccctacaggg acacttcgtc cattcttgaa ttggacgggt 480 ccacctgagc ccattgaggc agctgtggcc aggttcttct cagccagctg tgttcccggt 540 gcagataaag gacagttccc caacctgtgt cgcctgtgtg cggggacagg ggaaaacaaa 600 tgtgccttct cctcccagga accgtacttc agctactctg gtgccttcaa gtgtctgaga 660 gacggggctg gagacgtggc ttttatcaga gagagcacag tgtttgagga cctgtcagac 720 gaggctgaaa gggacgagta tgagttactc tgcccagaca acactcggaa gccagtggac 780 aagttcaaag actgccatct ggcccgggtc ccttctcatg ccgttgtggc acgaagtgtg 840 aatggcaagg aggatgccat ctggaatctt ctccgccagg cacaggaaaa gtttggaaag 900 gacaagtcac cgaaattcca gctctttggc tcccctagtg ggcagaaaga tctgctgttc 960 aaggactctg ccattgggtt ttcgagggtg cccccgagga tagattctgg gctgtacctt 1020 ggctccggct acttcactgc catccagaac ttgaggaaaa gtgaggagga agtggctgcc 1080 cggcgtgcgc gggtcgtgtg gtgtgcggtg ggcgagcagg agctgcgcaa gtgtaaccag 1140 tggagtggct tgagcgaagg cagcgtgacc tgctcctcgg cctccaccac agaggactgc 1200 atcgccctgg tgctgaaagg agaagctgat gccatgagtt tggatggagg atatgtgtac 1260 actgcatgca aatgtggttt ggtgcctgtc ctggcagaga actacaaatc ccaacaaagc 1320 agtgaccctg atcctaactg tgtggataga cctgtggaag gatatcttgc tgtggcggtg 1380 gttaggagat cagacactag ccttacctgg aactctgtga aaggcaagaa gtcctgccac 1440 accgccgtgg acaggactgc aggctggaat atccccatgg gcctgctctt caaccagacg 1500 ggctcctgca aatttgatga atatttcagt caaagctgtg cccctgggtc tgacccgaga 1560 tctaatctct gtgctctgtg tattggcgac gagcagggtg agaataagtg cgtgcccaac 1620 agcaaggaga gatactacgg ctacactggg gctttccggt gcctggctga gaatgctgga 1680 gacgttgcat ttgtgaaaga tgtcactgtc ttgcagaaca ctgatggaaa taacaatgag 1740 gcatgggcta aggatttgaa gctggcagac tttgcgctgc tgtgcctcga tggcaaacgg 1800 aagcctgtga ctgaggctag aagctgccat cttgccatgg ccccgaatca tgccgtggtg 1860 tctcggatgg ataaggtgga acgcctgaaa caggtgctgc tccaccaaca ggctaaattt 1920 gggagaaatg gatctgactg cccggacaag ttttgcttat tccagtctga aaccaaaaac 1980 cttctgttca atgacaacac tgagtgtctg gccagactcc atggcaaaac aacatatgaa 2040 aaatatttgg gaccacagta tgtcgcaggc attactaatc tgaaaaagtg ctcaacctcc 2100 cccctcctgg aagcctgtga attcctcagg aagtaa 2136 <210> 2 <211> 2349 <212> DNA <213> Oryza sativa <400> 2 atgactcaac gtagagccta tcgttggccc gtcccgttgt ctggacagca ggaacgcatc 60 tggtacggag gtgactacaa cccggaccag tggcccgagg aagtgtggga tgatgacgtc 120 cgtctgatga agaaggcggg cgtgaacctc gtgtccgtcg gcatcttctc atgggcgaag 180 atcgagacga gcgaaggcgt gtacgatttc gactggctcg accgcatcat cgacaagctg 240 ggcgaggccg gcgtcgccgt ggatttggcg tccgccaccg cttcgccgcc gatgtggctc 300 acccaggcgc atcccgaggt gctgtggaag gactaccgcg gcgacgtgtg ccagcctggc 360 gcacgccagc attggaggcc cacgagcccg gtgttccgcg agtacgcgct gaagctgtgc 420 cgtgcgatgg ccgagcatta caagggcaat ccgtacgtgg tcgcgtggca tgtgagcaac 480 gagtacggct gccacaaccg tttcgactat tccgaggacg ccgaacatgc gttccagcag 540 tggtgcgagg aacgctacgg caccatcgac gcggtgaacg acgcgtgggg gaccgcgttc 600 tgggcacagc gcatgaacga cttcagtgag atcgtgcctc cgcgcttcat cggcgacggc 660 aacttcatga acccgggcaa gctgctcgac ttcaagcggt tcagctcgga cgcgttgaag 720 gcgttctaca tcgccgagcg cgacacgctt gcgagatcac gccggatctg ccctgaccac 780 gaacttatgg tgtccgcgtc cggttcggtg ttggactacg acgattgggg cgacgaggtc 840 gatttcgtgt cgaacgacca ttacttcatt ccgggcgagg cccacttgga cgagctcgcg 900 ttctccgcga gcctggtcga cggcatcgca cgcaaggatc cgtggttcct gatggagcat 960 tccaccagcg cggtcaactg gcgtgagatc aactaccgca aggagcccgg ccagctggtg 1020 cgcgactctt tggctcatgt ggccatgggt gcggacgcgg tgtgctactt ccagtggagg 1080 cagtccaagg caggcgcgga gaagttccat tccgcgatgg tgccccacgc gggcgaggat 1140 tccgccgtgt tccgcgacgt gtgcgagctg ggcgccgatc tgaacaaact gtccgacgag 1200 ggcattctgg gatcgggctc gcgaagtccc gtgtggccgt ggtattcgac atacgagtcc 1260 gaatgggcca ccgaacacac cgccacgccg acccagcacg tgcatcacgt ggacgagcca 1320 ttggcgtggt tccgcgcttt ggccgatcag ggtgtgaccg ccgacgtggt gccggtgcgc 1380 ggcgcctggg atgactatga gatggtcgta ttgccgagtg tatacctgct ttccgaggaa 1440 acaccgcagg gtgcgcgatt acgtcgtggg cggcggtcga ctggtcgtga cctactacac 1500 gggcatctcg gacgagaagg accacgtgtg gctcggcggg tatccgggct cgatccggac 1560 gtggtcggcg tgcgcgtgga ggaattcggt gaaggttctc gtaccctcat cgtcggtgag 1620 gttgatcgtc acgtttttgc cgtcgttgga ggtggagaac ggacggcctc cggcaagcca 1680 gatcatcgtg tcgtagaagc tggcgtcacc cgaatcggct gcgatgtaca cgccgatgct 1740 tttcagcttt ttggccgcct cgtaatagtc gtcccacgtg cgaatcttgg aggcatcgat 1800 gtctacctgc tcgaacacgc tgtcgttgta gaagaacgcc attggaccgg aatccatggg 1860 aaggccgtac acgcggccgt tcagctgcac cgaagcccac gtaccgggcg tgtagaaatc 1920 gccgtagcct tccgtacgcc cggtgatcgc caccagctgg ccgcttaccg cgtactggcg 1980 caacgcatag tattccaatt gcaccacgtc gggaatgccg taaccgtctt gaatggcgtt 2040 gttgagattg tcgtaaccgc ttgtgttctt ccaaataacg tggatgtcgg gattttcctt 2100 ttcgaaaccg gcaatgactt gtttcatgct cggctcccac gaccatacgg tgatttcagt 2160 tcgggaatcg acgttggctc cgcatgcagc cgcactggcc gccatgatgg ccgcacagca 2220 gatggcgatg agtcgtttca ccagtttcaa gccgattcct tccgtttgac gttttcgtac 2280 gaaaacgtac gtagacatac gcattcgagt gtacgtcagt cgcccgaaca aaggcttgct 2340 atcttttga 2349 <210> 3 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 aggtcgacat gaaacttgtc ttcctc 26 <210> 4 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 aggtcgacct tcctgaggaa ttcaca 26 <210> 5 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 aggttaacat gactcaacgt agagcctatc gttggccc 38 <210> 6 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 agctgcagtc aaaagatagc aagcctttgt tcgggcga 38  

Claims (12)

인간 락토페린 및 유산균 유래의 β-갈락토시다제 단백질 코딩 유전자를 포함하는 재조합 식물 발현 벡터로 형질전환된 식물체.A plant transformed with a recombinant plant expression vector comprising a β-galactosidase protein coding gene derived from human lactoferrin and lactic acid bacteria. 제1항에 있어서, 상기 인간 락토페린 및 유산균 유래의 β-갈락토시다제 단백질 코딩 유전자는 각각 서열번호 1 및 서열번호 2의 염기서열로 이루어진 것을 특징으로 하는 식물체.The plant of claim 1, wherein the β-galactosidase protein coding gene derived from human lactoferrin and lactic acid bacteria is composed of the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2, respectively. 제1항에 있어서, 상기 식물체는 단자엽 식물인 것을 특징으로 하는 식물체.The plant of claim 1, wherein the plant is a monocotyledonous plant. 제3항에 있어서, 상기 단자엽 식물은 벼인 것을 특징으로 하는 식물체.The plant according to claim 3, wherein the monocotyledonous plant is rice. 인간 락토페린 및 유산균 유래의 β-갈락토시다제 단백질 코딩 유전자를 포함하는 재조합 식물 발현 벡터.A recombinant plant expression vector comprising β-galactosidase protein coding gene derived from human lactoferrin and lactic acid bacteria. 제5항에 있어서, 상기 인간 락토페린 및 유산균 유래의 β-갈락토시다제 단백질 코딩 유전자는 각각 서열번호 1 및 서열번호 2의 염기서열로 이루어진 것을 특징으로 하는 재조합 식물 발현 벡터.The recombinant plant expression vector according to claim 5, wherein the β-galactosidase protein coding gene derived from human lactoferrin and lactic acid bacteria is composed of the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2, respectively. 제5항에 있어서, 상기 벡터는 도 1의 pVE9-TFTβ인 것을 특징으로 하는 벡터.The vector of claim 5, wherein the vector is pVE9-TFTβ of FIG. 1. 제5항의 재조합 식물 발현 벡터를 식물체에 도입하여 인간 락토페린 및 유산균 유래의 β-갈락토시다제 단백질 코딩 유전자를 과발현하는 단계를 포함하는 식물체에서 인간 락토페린 및 유산균 유래의 β-갈락토시다제 단백질 대량생산 방법.A large amount of β-galactosidase protein derived from human lactoferrin and lactic acid bacteria in a plant, comprising the step of introducing the recombinant plant expression vector of claim 5 into the plant to overexpress the β-galactosidase protein coding gene derived from human lactoferrin and lactic acid bacteria. Production method. 제8항에 있어서, 상기 식물체는 벼인 것을 특징으로 하는 방법.The method of claim 8, wherein the plant is rice. 제5항의 재조합 식물 발현 벡터를 식물체에 형질전환하는 단계를 포함하는 인간 락토페린 및 유산균 유래의 β-갈락토시다제를 생산하는 식물체의 제조방법.A method for producing a plant for producing β-galactosidase derived from human lactoferrin and lactic acid bacteria, the method comprising transforming the plant with the recombinant plant expression vector of claim 5. 제10항에 있어서, 상기 식물체는 벼인 것을 특징으로 하는 방법.The method of claim 10, wherein the plant is rice. 제11항의 방법에 의해 제조된 벼로부터 얻은 쌀을 함유하는 쌀 가공식품.A processed rice product containing rice obtained from the rice produced by the method of claim 11.
KR1020080131911A 2008-12-23 2008-12-23 METHOD FOR PRODUCING TRANSGENIC PLANT PRODUCING THE HUMAN LACTOFERRIN AND BIFIDOBACTERIAL β-GALACTOSIDASE AND PLANT PRODUCED BY THE SAME KR20100073281A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105238858A (en) * 2015-09-25 2016-01-13 浙江大学 Method for specificity identification and flanking sequence detection of transgenic recombinant human lactoferrin rice strain G281

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105238858A (en) * 2015-09-25 2016-01-13 浙江大学 Method for specificity identification and flanking sequence detection of transgenic recombinant human lactoferrin rice strain G281
CN105238858B (en) * 2015-09-25 2019-02-05 浙江大学 It is a kind of for detecting the flanking sequence and its specificity identification method that turn restructuring lactoferrin rice strain G281

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