KR101570771B1 - Recombinant vector for polyunsaturated fatty acids biosynthesis and plant transformed by the same - Google Patents
Recombinant vector for polyunsaturated fatty acids biosynthesis and plant transformed by the same Download PDFInfo
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- KR101570771B1 KR101570771B1 KR1020130139794A KR20130139794A KR101570771B1 KR 101570771 B1 KR101570771 B1 KR 101570771B1 KR 1020130139794 A KR1020130139794 A KR 1020130139794A KR 20130139794 A KR20130139794 A KR 20130139794A KR 101570771 B1 KR101570771 B1 KR 101570771B1
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Abstract
본 발명은 고도불포화지방산 생합성에 관한 것으로, 보다 상세하게는 pAO815 벡터를 이용한 세 개의 유전자 McD6DES와 AsELOVL5 및 PtD5DES가 동시에 발현되도록 다중발현 벡터를 만들어 식물체에 형질전환하고, 상기 식물체의 종자에서 오메가-3 및 오메가-6 고도불포화지방산인 GLA, STA, DGLA, ETA, ARA, EPA, DTA 및 DPA가 종자에서 생합성되는 것을 확인하였으므로, 이를 고도불포화지방산 생산 자원으로 유용하게 사용할 수 있다.The present invention relates to a polyunsaturated fatty acid biosynthesis, and more particularly, to a method for producing a polyunsaturated fatty acid by transforming a plant into a multiple expression vector so that three genes McD6DES , AsELOVL5 and PtD5DES are simultaneously expressed using the pAO815 vector, And omega-6 polyunsaturated fatty acids such as GLA, STA, DGLA, ETA, ARA, EPA, DTA and DPA were biosynthesized in seeds.
Description
본 발명은 고도불포화지방산 생합성에 관련된 일련의 효소를 암호화하는 유전자들을 이용하여 식물체에서 고도불포화지방산을 생산하는 것에 대한 것이다.
The present invention is directed to the production of polyunsaturated fatty acids in plants using genes encoding a series of enzymes involved in polyunsaturated fatty acid biosynthesis.
불포화지방산(Unsaturated fatty acid)은 한 분자 속에 하나 이상의 이중결합을 가지는 사슬 모양 화합물로 동식물 속에 널리 분포하며, 일반적으로 탄소수 12 내지 20개 정도의 짝수 개로 구성되어 있다. 지방산 분자가 단 하나의 이중결합을 갖고 있으면 단일불포화지방산이라고 하며, 두 개 이상의 이중결합을 갖고 있으며 다가불포화지방산이라고 부른다. 불포화지방산은 이중결합 바로 다음의 수소원자가 결합된 형태에 따라 시스 형(cis configuration)과 트랜스 형(trans configuration)으로 나뉜다. 시스 형은 인접한 수소원자가 이중결합과 같은 방향에 있는 것을 말하며, 트랜스 형은 다음 2개의 수소원자가 이중결합과 반대의 방향으로 결합한 것을 말한다. 지방산 사슬에서 이중결합이 형성되면 수소원자가 제거되어야 하는데, 이 때문에 포화지방산에서 '포화'라는 용어는 수소원자가 포화되었다는 것을 의미한다. 세포내 대사에서 수소-탄소 결합은 에너지 생산을 위해 결합이 깨지거나 산화된다. 따라서 불포화지방산은 같은 크기의 포화지방산에 비해 에너지를 더 적게 함유한다. 또한, 불포화지방산은 더 낮은 녹는점을 갖고 있어서 세포막의 유동성을 증가시키는 작용도 하는 것으로 알려졌다. Unsaturated fatty acids are chain-like compounds having one or more double bonds in a molecule and are widely distributed in plants and animals, and are generally composed of even numbers of about 12 to 20 carbon atoms. When a fatty acid molecule has only one double bond, it is called a monounsaturated fatty acid and has two or more double bonds and is called a polyunsaturated fatty acid. Unsaturated fatty acids are divided into a cis configuration and a trans configuration depending on the type of double bond followed by the hydrogen atom. The cis type means that the adjacent hydrogen atom is in the same direction as the double bond, and the trans type means that the next two hydrogen atoms are bonded in the opposite direction to the double bond. When a double bond is formed in a fatty acid chain, the hydrogen atom has to be removed, so the term " saturated " in saturated fatty acids means that the hydrogen atom is saturated. In intracellular metabolism, hydrogen-carbon bonds are broken or oxidized for energy production. Thus, unsaturated fatty acids contain less energy than saturated fatty acids of the same size. It is also known that unsaturated fatty acids have a lower melting point, which also increases the fluidity of the cell membrane.
포화지방산의 섭취는 고콜레스테롤혈증(hypercholesterolemia), 동맥경화(atherosclerosis) 등과 같은 심혈관계 질환(cardiovascular disease)의 위험성과 관련이 깊은 반면, 불포화지방산의 섭취는 혈중 콜레스테롤의 감소 및 동맥경화의 발병 위험 감소와 관련이 있는 것으로 잘 알려져 있다. 불포화지방산의 예로는 팔미톨레익산(palmitoleic acid), 올레익산(oleic acid), 미리스톨레익산(myristoleic acid), 리놀레익산(linoleic acid), 아라키도닉산(arachidonic acid), 에이코사펜타에노익산(eicosapentaenoic acid, EPA), 도코사헥사에노익산(docosahexaenoic acid, DHA) 등이 있다. 이러한 불포화지방산을 함유하는 식품으로는 아보카도, 견과류, 카놀라유나 올리브유와 같은 식물성 기름 등이 있다. 비록 불포화지방산이 포화지방산보다는 건강에 유익하지만, 미 식품의약국(FDA)에서는 불포화지방산의 섭취량이 하루 칼로리 섭취량의 30%를 넘지 않도록 권고한다. Saturated fatty acid intake is associated with the risk of cardiovascular diseases such as hypercholesterolemia and atherosclerosis while the intake of unsaturated fatty acids is associated with a decrease in blood cholesterol and a reduced risk of developing atherosclerosis Is known to be associated with. Examples of unsaturated fatty acids include palmitoleic acid, oleic acid, myristoleic acid, linoleic acid, arachidonic acid, eicosapentaenoic acid, Eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and the like. Foods containing such unsaturated fatty acids include avocados, nuts, vegetable oils such as canola oil and olive oil. Although unsaturated fatty acids are healthier than saturated fatty acids, the Food and Drug Administration (FDA) recommends that the intake of unsaturated fatty acids should not exceed 30% of the daily calorie intake.
오메가6(ω6) 및 오메가3(ω3) 라인(line, 계통)의 고도불포화지방산(Polyunsaturated fatty acids: PUFA)인 아라키도닉산(arachidonic acid, ARA, C20:4 ω6)와 에이코사펜타에노익산(eicosapentaenoic acid, EPA, C20:5 ω3), 도코사펜타에노익산(docosahexaenoic acid, DHA, C22:6 ω3)는 동물 세포막의 필수 구성성분으로 포스포파티딜콜린(phosphatidylcholine)과 결합한 형태로 세포막의 인지질 층에 삽입되어 세포막의 유동성을 좋게 하고, 혈중 콜레스테롤을 감소시키며, 암 증식을 억제하는 등의 질병예방 효과가 뛰어나다. 특히 DHA는 뇌 기능 강화와 기억력 증진을 통한 영, 유아의 뇌 기능 성장발달과 학습기능 향상, 통증을 알려주는 프로스타글란딘(prostaglandin)과 같은 호르몬의 전구체로서 호르몬 조절 작용 등의 중요한 기능이 규명되었다. 최근 식품첨가제와 화장품 원료 및 의약품 분야에서 큰 시장을 형성하고 있다. 포유동물은 ω6/Δ12 및 ω3/Δ15 불포화효소(desaturase)가 발현되지 않아 PUFA의 de novo 합성을 할 수 없으며, 정상적인 성장과 발달을 위하여 리놀레익산(linoleic acid, LA, C18:2 ω6)와 알파-리놀레익산(α-linolenic acid, ALA, C18:3 ω3)를 반드시 음식물로 섭취해야만 한다. It has been reported that arachidonic acid (ARA, C20: 4 ω6), polyunsaturated fatty acids (PUFAs) of the omega 6 (ω6) and omega 3 (ω3) lines and eicosapentaenoic acid (docosahexaenoic acid, DHA, C22: 6 ω3) is an essential constituent of animal cell membranes and is a form of binding to phosphatidylcholine, To enhance the fluidity of the cell membrane, to reduce blood cholesterol, and to inhibit cancer proliferation. In particular, DHA has been shown to have important functions such as enhancement of brain function and memory, enhancement of brain function and learning of young children, enhancement of learning function, and hormone regulating action as a precursor of hormones such as prostaglandin. Recently, it has formed a big market in the fields of food additives, cosmetic raw materials and pharmaceuticals. In mammals, linoleic acid (LA, C18: 2 ω6) and ω6 / δ12 and ω3 / Δ15 desaturase were not expressed and thus, de novo synthesis of PUFA was not possible. Alpha-linolenic acid (ALA, C18: 3 ω3) must be ingested as food.
DHA는 주로 참치와 같은 등 푸른 생선으로부터 생산하고 있으나, 다량의 DHA를 식품에 첨가하게 되면 물고기 특유의 비린내 때문에 상품성에 문제가 되어 그 냄새를 제거하기 위한 순수 분리, 정제가 관건이 되고 있다. 해양 조류를 통해서도 DHA 생산이 가능하나, 낮은 성장률로 말미암아 DHA의 대량 생산을 위해서는 고가의 장비를 필요로 한다. 최근 이러한 문제점들을 해결하고 저비용 고효율의 DHA를 생산하고자 식물 종자에서의 발현 시스템을 이용한 PUFA 생산에 대한 관심이 높아지고 있다. PUFA은 전구체인 LA와 ALA로부터 일련의 불포화화(desaturation)와 2탄소 사슬 연장(elongation) 과정을 통해 생합성 된다 (도 4 참고). 식물은 PUFA를 생합성 하지 못하며, LA와 ALA로부터 PUFA를 생합성 하기 위해서는 Δ6 불포화효소(Δ6 desaturase, Δ6DES), Δ6 사슬 연장효소(Δ6 elongase, Δ6ELS), 그리고 Δ5 불포화효소(Δ5 desaturase, Δ5DES)의 도입이 필요하다. DHA is mainly produced from blue fish such as tuna. However, if a large amount of DHA is added to food, it becomes problematic in terms of merchantability due to fish-specific smell of fish, and pure separation and purification for removing the odor are the key. DHA production can also be achieved through marine algae, but due to the low growth rate, high-volume equipment is required for the mass production of DHA. In order to solve these problems and produce low-cost and high-efficiency DHA, interest in the production of PUFAs using expression systems in plant seeds is increasing. PUFAs are biosynthesized through a series of desaturation and 2-carbon chain elongation steps from the precursors LA and ALA (see FIG. 4). Plants do not biosynthesize PUFAs and the introduction of Δ6 desaturase (Δ6 desaturase, Δ6DES), Δ6 chain extension enzyme (Δ6 elongase, Δ6ELS), and Δ5 desaturase (Δ5 DES) to biosynthesize PUFAs from LA and ALA Is required.
현재까지 자연계에는 3 종류의 PUFA 생합성의 경로가 알려져 있다[Qiu X (2003) Biosynthesis of docosahexaenoic acid (DHA, 22:6-4, 7, 10, 13, 16, 19): two distinct pathways. Prostaglandins Leukot Essent Fatty Acids 68, 181-186.]. (1) 물 곰팡이류에 속하는 Saprolegnia는 전구물질인 ALA에 대하여 Δ6 불포화, 연장 및 Δ5 불포화 반응을 통하여서 EPA를 합성하는 경로를 이용하는 반면[Pereira SL 등 (2004a) A novel omega 3-fatty acid desaturase involved in the biosynthesis of eicosapentaenoic acid. Biochem J 378, 665-671.], (2) Euglena 등의 원생생물은 Δ9 연장, Δ8 불포화 및 Δ5 불포화에 의해 EPA를 생합성 한다[Wallis과 Browse, (1999) The Delta8-desaturase of Euglena gracills: an alternate pathway for synthesis of 20-carbon polyunsaturated fatty acids. Arch Biochem Biophys 365, 307-316.]. 위의 두 경로를 통해서 생합성 된 EPA는 Δ5 연장과 Δ4 불포화에 의해 최종적으로 DHA를 생합성 한다[Pereira 등, 2004b]. 포유동물은 EPA로부터 4단계의 더 복잡한 경로 즉, β-산화에 의한 역전환(retro-conversion) 과정을 거쳐 DHA를 생합성 하게 된다[Sprecher 등(1995) Reevaluation of the pathways for the biosynthesis of polyunsaturated fatty acids. J Lipid Res 36, 2471-2477., 1995; Wallis 등, Polyunsaturated fatty acid synthesis: what will they think of next Trends Biochem Sci 27, 467-473.]. (3) 해양 조류(Schizochytrium)와 해양 미생물(Shewanella)은 PKSe(polyketide synthas) 경로를 이용하여 DHA를 생합성 한다[Metz 등, (2001) Production of polyunsaturated fatty acids by polyketide synthases in both prokaryotes and eukaryotes. Science 293, 290-293.]. 이와 같은 다양한 PUFA 생합성 경로에 사용되는 효소들을 재구성하여 유전자원으로 활용할 경우 주로 생선을 통해서만 섭취하던 EPA와 DHA을 식물체에서 손쉽게 생산할 수 있는 유전공학적 기반을 구축하기 위한 연구가 진행되어 왔으나, 이미 알려진 유전자들이라고 해도 서로 다른 유래의 유전자들을 조합하여 식물체의 종자에서 실제로 고도불포화지방산을 생산하는 것이 용이하지 않았다.
To date, three pathways of PUFA biosynthesis have been known in nature [Qiu X (2003) Biosynthesis of docosahexaenoic acid (DHA, 22: 6-4, 7, 10, 13, 16, 19): two distinct pathways. Prostaglandins Leukot Essent Fatty Acids 68, 181-186.]. (1) Saprolegnia belonging to the water mold family uses a pathway for synthesizing EPA through Δ6 unsaturation, prolongation and Δ5 unsaturation on the precursor ALA [Pereira SL et al. (2004a)], while novel omega 3-fatty acid desaturase involved in the biosynthesis of eicosapentaenoic acid. Biochem J 378, 665-671.), (2) protoplasts such as Euglena biosynthes EPA by Δ9 extension, Δ8 unsaturation and Δ5 unsaturation [Wallis and Browse, (1999) The Delta8-desaturase of Euglena gracills: an alternate pathway for synthesis of 20-carbon polyunsaturated fatty acids. Arch Biochem Biophys 365, 307-316.]. EPA biosynthesized through the above two pathways ultimately biosynthesize DHA by Δ5 extension and Δ4 unsaturation [Pereira et al., 2004b]. Mammalian biosynthesizes DHA via a more complex four-step pathway from EPA, that is, retro-conversion by β-oxidation (Sprecher et al., 1995). . J Lipid Res 36, 2471-2477., 1995; Wallis et al., Polyunsaturated fatty acid synthesis: what will they think of next Trends Biochem Sci 27, 467-473.]. (3) Schizochytrium and marine microorganism (Shewanella) biosynthesize DHA using PKSe (polyketide synthase) pathway [Metz et al., (2001) Production of polyunsaturated fatty acids by polyketide synthases in both prokaryotes and eukaryotes. Science 293,290-293.]. When the enzymes used in the various PUFA biosynthetic pathways are reconstructed and used as genetic resources, studies have been carried out to construct a genetic engineering base for easily producing EPA and DHA, which were mainly consumed only by fish, in plants. However, It was not easy to produce polyunsaturated fatty acids from the seeds of plants by combining different derived genes.
이에 본 발명자들은 고등생물 체내에서 혈액순환계, 호르몬 분비계 및 면역계 등 여러 가지 생리활성을 갖는 고도불포화지방산을 합성하는 데 관여하는 갯장어로부터 분리한 Δ6 불포화화 효소(desaturase) 유전자 McD6DES, 조류 유래 Δ5 불포화화 효소(desaturase) 유전자 PtD5DES 및 돔으로부터 분리한 지방산 사슬연장효소(elongase) 유전자 AsELOVL5가 동시에 발현되도록 식물 형질전환용 벡터를 제작하였으며, 이의 애기장대 종자에서의 고도불포화지방산의 생합성 활성을 확인함으로써 그 기능을 확인하고 본 발명을 완성하였다.
Accordingly, the present inventors have found that the Δ6 desaturase gene McD6DES isolated from marsupials, which is involved in the synthesis of polyunsaturated fatty acids having various physiological activities such as blood circulation system, hormone secretion system, and immune system in high organisms, Δ5 unsaturated The desaturase gene PtD5DES and A fatty acid chain separated from the dome extending enzyme (elongase) was making a vector for plant transformation such gene AsELOVL5 is expressed at the same time, completed the present invention and verify its functionality by identifying its Arabidopsis high biosynthetic activity of unsaturated fatty acids in the rod seeds Respectively.
본 발명의 목적은 서열번호 1의 염기서열로 이루어진 유전자, 서열번호 2의 염기서열로 이루어진 유전자 및 서열번호 3의 염기서열로 이루어진 유전자를 포함하는 식물 형질전환용 재조합 벡터를 제공하기 위한 것이다.An object of the present invention is to provide a recombinant vector for plant transformation comprising a gene consisting of a nucleotide sequence of SEQ ID NO: 1, a nucleotide sequence of SEQ ID NO: 2 and a nucleotide sequence of SEQ ID NO: 3.
또한, 본 발명의 다른 목적은 상기 식물 형질전환용 재조합 벡터로 형질전환한 형질전환 식물체를 제공하기 위한 것이다.Another object of the present invention is to provide a transgenic plant transformed with the recombinant vector for plant transformation.
또한, 본 발명의 다른 목적은 고도불포화지방산을 생산하는 식물체를 제조하는 방법을 제공하기 위한 것이다.Another object of the present invention is to provide a method for producing a plant producing a polyunsaturated fatty acid.
아울러, 본 발명의 또 다른 목적은 상기 형질전환 식물체의 종자로부터 고도불포화지방산을 생산하는 방법을 제공하기 위한 것이다.
It is another object of the present invention to provide a method for producing polyunsaturated fatty acids from the seeds of the transgenic plants.
본 발명은 서열번호 1의 염기서열로 이루어진 유전자, 서열번호 2의 염기서열로 이루어진 유전자 및 서열번호 3의 염기서열로 이루어진 유전자를 포함하는 식물 형질전환용 재조합 벡터를 제공한다. The present invention provides a recombinant vector for plant transformation comprising a gene consisting of a nucleotide sequence of SEQ ID NO: 1, a nucleotide sequence of SEQ ID NO: 2 and a nucleotide sequence of SEQ ID NO: 3.
또한, 본 발명은 상기 식물 형질전환용 재조합 벡터로 형질전환한 형질전환 식물체를 제공한다. The present invention also provides a transgenic plant transformed with the recombinant vector for plant transformation.
또한, 본 발명은 고도불포화지방산을 생산하는 식물체를 제조하는 방법을 제공한다.The present invention also provides a method for producing a plant producing a polyunsaturated fatty acid.
아울러, 본 발명은 상기 형질전환 식물체의 종자로부터 고도불포화지방산을 생산하는 방법을 제공한다.
In addition, the present invention provides a method for producing polyunsaturated fatty acids from the seeds of the transgenic plants.
본 발명에 따른 고도불포화지방산 생합성에 관련된 일련의 효소를 암호화하는 delta-6 불포화효소 유전자 (McD6DES)와 지방산 탄소사슬 연장 효소 유전자 (AsELOVL5), delta-5 불포화효소 유전자 (PtD5DES)가 동시에 발현되는 식물 형질전환용 재조합 벡터로 형질전환된 식물체의 종자를 고도불포화지방산 생산에 활용할 수 있다.
A plant in which a delta-6 unsaturated enzyme gene ( McD6DES ) encoding a series of enzymes involved in polyunsaturated fatty acid biosynthesis according to the present invention, a fatty acid carbon chain extension enzyme gene ( AsELOVL5 ) and a delta-5 unsaturated enzyme gene ( PtD5DES ) The seeds of the plant transformed with the recombinant vector for transformation can be utilized for the production of polyunsaturated fatty acids.
도 1은 본 발명의 McD6DES, AsELOVL5 및 PtD5DES 유전자가 각각 도입된 식물 형질전환용 재조합 벡터의 모식도이다.
도 2는 본 발명의 식물 형질전환용 재조합 벡터에 McD6DES, AsELOVL5 및 PtD5DES 유전자 카세트가 도입된 것을 확인한 도이다.
도 3은 본 발명의 식물 형질전환용 재조합 벡터로 형질전환된 애기장대 160 개체에 McD6DES, AsELOVL5 및 PtD5DES 유전자의 도입을 확인한 도이다.
도 4는 형질전환 애기장대 160 개체에서 확보한 T2 종자에 대한 일련번호 및 지질 함량을 나타낸 도이다.
도 5는 본 발명의 형질전환 애기장대 라인(line, 계통)별 T2 종자의 오메가-6 및 오메가-3 지방산 조성을 나타낸 도이다.
도 6은 본 발명의 형질전환 애기장대 라인(line, 계통)별 T2 종자의 오메가-6 및 오메가-3 지방산이 차지하는 함량을 나타낸 도이다.
도 7은 본 발명의 형질전환 애기장대 라인(line, 계통)별 T2 종자에서 오메가-3 지방산인 EPA가 차지하는 함량을 나타낸 도이다.
도 8은 라인(line, 계통)번호 63의 형질전환 애기장대 T2 종자의 고도불포화지방산을 GC로 확인한 도이다.
도 9는 라인(line, 계통)번호 63의 형질전환 애기장대 T2 종자의 고도불포화지방산 함량을 나타낸 도이다.
도 10은 본 발명의 형질전환 애기장대의 종자에서의 오메가-3 및 오메가-6 지방산 생합성 전환율을 나타낸 도이다.BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a graphical representation of the McD6DES , AsELOVL5 And PtD5DES Is a schematic view of a plant transformation vector.
Fig. 2 is a graph showing the expression profiles of McD6DES , AsELOVL5 And the PtD5DES gene cassette were introduced.
Figure 3 shows that 160 Arabidopsis plants transformed with the recombinant vector for plant transformation of the present invention were assigned to McD6DES , AsELOVL5 And the introduction of the PtD5DES gene.
FIG. 4 is a diagram showing serial numbers and lipid contents of T2 seeds obtained from 160 transformed Arabidopsis thaliana.
FIG. 5 is a graph showing omega-6 and omega-3 fatty acid compositions of T2 seeds per line of transgenic Arabidopsis thaliana lines of the present invention.
FIG. 6 is a graph showing the content of omega-6 and omega-3 fatty acids in the T2 seeds of the transformed Arabidopsis thaliana lines of the present invention.
FIG. 7 is a graph showing the contents of EPA, which is an omega-3 fatty acid, in T2 seeds according to the transgenic Arabidopsis thaliana lines of the present invention.
FIG. 8 is a graph showing GC analysis of polyunsaturated fatty acids of transgenic Arabidopsis T2 seeds of
FIG. 9 is a graph showing the polyunsaturated fatty acid content of the transformed Arabidopsis T2 seed of
FIG. 10 is a diagram showing the conversion rates of omega-3 and omega-6 fatty acid biosynthesis in seeds of the transformed Arabidopsis thaliana according to the present invention.
이하, 본 발명을 상세히 설명한다.
Hereinafter, the present invention will be described in detail.
본 발명에서 나타내는 지방산의 표기에서 "16:0"을 예로 들면, 16은 탄소사슬의 개수, 뒤의 번호는 불포화도를 나타낸다. 즉, 0은 포화지방산, 1,2 등은 불포화지방산을 의미한다.
In the description of the fatty acid shown in the present invention, "16: 0" is taken as an example, 16 indicates the number of carbon chains, and the numbers after the letters indicate unsaturation. That is, 0 means saturated fatty acid and 1, 2 means unsaturated fatty acid.
본 발명에서 사용되는 용어 "고도불포화지방산(polyunsaturated fatty acid, PUFA)"은, 지방산 중에 이중결합을 4개 이상 포함하는 것을 말한다. 오메가 6 및 오메가 3 지방산 등이 포함되며, 이 기술분야에 고도불포화지방산의 종류는 널리 알려져 있다. 본 발명에서는 특히, DGLA(dihomo-gamma-linolenic acid), ARA(arachidonic acid), DTA(docosatetraenoic acid), STA(stearidonic acid), ETA(eicosatetraenoic acid), EPA(eicosapentaenoic acid), DPA(docosapetaenoic acid) 및 DHA(docosahexaenoic acid)가 포함된다.
As used herein, the term " polyunsaturated fatty acid (PUFA) " refers to a fatty acid containing four or more double bonds. Omega-6 and omega-3 fatty acids, and the class of polyunsaturated fatty acids in the art is well known. In the present invention, it is particularly preferable to use dihomo-gamma-linolenic acid (DGLA), arachidonic acid (ARA), docosatetraenoic acid (DTA), stearidonic acid (STA), eicosatetraenoic acid (ETA), eicosapentaenoic acid (EPA), docosapetaenoic acid And docosahexaenoic acid (DHA).
본 발명에서 사용되는 용어 "n-3 지방산" 또는 "오메가 3(ω3) 지방산"은 지방산의 메틸기 말단부터 세어서 3번째의 탄소가 이중결합을 갖는 일련의 지방산을 말한다.
As used herein, the term "n-3 fatty acid" or "omega 3 (omega 3) fatty acid" refers to a series of fatty acids in which the third carbon has a double bond, counted from the methyl group end of the fatty acid.
본 발명에서 사용되는 용어 "n-6 지방산" 또는 "오메가 6(ω6) 지방산"은 지방산의 메틸기 말단부터 세어서 6번째의 탄소가 이중결합을 갖는 일련의 지방산을 말한다.
As used herein, the term "n-6 fatty acid" or "omega 6 (omega 6) fatty acid" refers to a series of fatty acids in which the sixth carbon is double bond, counted from the methyl group end of the fatty acid.
본 발명은 서열번호 1의 염기서열로 이루어진 유전자(McD6DES), 서열번호 2의 염기서열로 이루어진 유전자(PtD5DE) 및 서열번호 3의 염기서열로 이루어진 유전자(AsELOVL5)를 포함하는 식물 형질전환용 재조합 벡터를 제공한다. The present invention provides a recombinant vector for plant transformation comprising a gene ( McD6DES ) consisting of the nucleotide sequence of SEQ ID NO: 1, a gene ( PtD5DE ) comprising the nucleotide sequence of SEQ ID NO: 2 and a gene ( AsELOVL5 ) comprising the nucleotide sequence of SEQ ID NO: Lt; / RTI >
상기 유전자 McD6DES는 갯장어 유래의 델타6 불포화화 효소(Δ6 desaturase)를 암호화하는 것이 바람직하나, 이에 한정되지 않는다.The gene McD6DES is preferably, but not limited to, encoding the delta-6 desaturase derived from the mussel.
상기 유전자 PtD5DE는 조류 유래의 델타5 불포화화 효소(Δ5 desaturase)를 암호화하는 것이 바람직하나, 이에 한정되지 않는다.The gene PtD5DE preferably encodes a delta-5 desaturase from algae, but is not limited thereto.
상기 유전자 AsELOVL5는 돔 유래의 지방산 사슬연장효소(elongase)를 암호화하는 것이 바람직하나, 이에 한정되지 않는다.The gene AsELOVL5 preferably encodes a fatty acid chain elongase derived from dome, but is not limited thereto.
본 발명의 서열번호 1, 2 또는 3으로 표시되는 유전자는 단백질을 코딩하는 게놈 DNA와 cDNA를 모두 포함한다. The gene represented by SEQ ID NO: 1, 2 or 3 of the present invention includes both the genomic DNA encoding the protein and the cDNA.
또한, 상기 염기 서열의 변이체가 본 발명의 범위 내에 포함된다. 구체적으로, 상기 유전자는 서열번호 1, 2 또는 3의 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.Variants of the above base sequences are also included within the scope of the present invention. Specifically, the gene has a sequence homology of at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% with the nucleotide sequence of SEQ ID NO: 1, 2 or 3 May contain a base sequence. "% Of sequence homology to polynucleotides" is ascertained by comparing the comparison region with two optimally aligned sequences, and a portion of the polynucleotide sequence in the comparison region is the reference sequence for the optimal alignment of the two sequences (I. E., A gap) relative to the < / RTI >
상기 식물 형질전환용 재조합 벡터는 vicillin 프로모터 및 octopine 터미네이터 사이에 상기 서열번호 1의 염기서열로 이루어진 유전자, 서열번호 2의 염기서열로 이루어진 유전자 및 서열번호 3의 염기서열로 이루어진 유전자가 각각 한개 씩 순차적으로 포함되는 것이 바람직하나, 이에 한정되지 않는다.The recombinant vector for plant transformation comprises a gene consisting of the nucleotide sequence of SEQ ID NO: 1, a gene consisting of the nucleotide sequence of SEQ ID NO: 2 and a nucleotide sequence of SEQ ID NO: 3 in sequence between the vicillin promoter and the octopine terminator But it is not limited thereto.
상기 식물 형질전환용 재조합 벡터는 제초제 저항성 Bar 유전자가 포함된 것이 바람직하나, 이에 한정되지 않는다.Preferably, the recombinant vector for plant transformation comprises a herbicide resistance Bar gene, but is not limited thereto.
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 것으로서 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. The recombinant cell can express a gene or a gene fragment that is not found in the natural form of the cell in one of the sense or antisense form. In addition, the recombinant cell can express a gene found in a cell in its natural state, but the gene has been modified and reintroduced intracellularly by an 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 이용 가능한 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" means a recombinant DNA molecule comprising a desired coding sequence and a suitable nucleic acid sequence necessary for expressing a coding sequence operably linked in a particular host organism. Promoters, enhancers, termination signals and polyadenylation signals available in eukaryotic cells are known.
본 발명의 벡터는 선택표지로서, 통상의 기술분야에서 통상적으로 이용되는 항생제 내성 유전자를 포함할 수 있으며, 예를 들어 암피실린, 겐타마이신, 카베니실린, 클로람페니콜, 스트렙토마이신, 카나마이신, 게네티신, 네오마이신 및 테트라사이클린에 대한 내성 유전자가 있다.The vector of the present invention may be a selection marker and may include an antibiotic resistance gene commonly used in the conventional art. Examples of the vector include ampicillin, gentamycin, carbenicillin, chloramphenicol, streptomycin, kanamycin, There is a resistance gene for neomycin and tetracycline.
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), 글루포시네이트암모늄(glufosinate ammonium) 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 a property that can be selected by a chemical method, and includes all genes capable of distinguishing a transformed cell from a non-transformed cell. Examples include herbicide resistance genes such as glyphosate, glufosinate ammonium or phosphinothricin, kanamycin, G418, Bleomycin, hygromycin, ), Chloramphenicol (chloramphenicol), but are not limited thereto.
본 발명의 재조합 벡터에서, 프로모터는 vicillin, 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 the recombinant vector of the present invention, the promoter may be vicillin, CaMV 35S, actin, ubiquitin, pEMU, MAS or histone promoter, but is not limited thereto. The term "promoter " refers to a region of DNA upstream from a structural gene and refers to a DNA molecule to which an RNA polymerase binds to initiate transcription. A "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, constitutive promoters do not limit selectivity.
본 발명의 재조합 벡터에서, 통상의 터미네이터를 사용할 수 있으며, 그 예로는 노팔린 신타아제(NOS), 벼 α-아밀라아제 RAmy1 A 터미네이터, 파세올린(phaseoline) 터미네이터 및 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 알려져 있다. 그러므로, 터미네이터의 사용은 본 발명의 내용에서 매우 바람직하다.
In the recombinant vector of the present invention, conventional terminators can be used. Examples thereof include nopaline synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator and Agrobacterium tumefaciens (Agrobacterium tumefaciens ) Octopine gene terminator, but the present invention is not limited thereto. Regarding 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.
또한, 본 발명은 본 발명에 따른 식물 형질전환용 재조합 벡터로 형질전환한 형질전환 식물체를 제공한다. 본 발명의 벡터를 안정하고 연속적으로 종자에서 발현시킬 수 있는 형질전환체는 식물체가 바람직하다. The present invention also provides a transgenic plant transformed with a recombinant vector for plant transformation according to the present invention. Plants that are capable of expressing the vector of the present invention stably and continuously in seeds are preferred.
상기 식물체는 서열번호 1의 염기서열로 이루어진 유전자, 서열번호 2의 염기서열로 이루어진 유전자 및 서열번호 3의 염기서열로 이루어진 유전자의 발현에 의해 종자 내의 오메가-3(omega-3) 및 오메가-6 지방산의 함량이 증진된 것이 바람직하나, 이에 한정되지 않는다.The plant is characterized by the expression of the gene consisting of the nucleotide sequence of SEQ ID NO: 1, the nucleotide sequence of SEQ ID NO: 2 and the nucleotide sequence of SEQ ID NO: 3, and the omega-3 and omega- It is preferable that the content of the fatty acid is increased, but not limited thereto.
상기 식물체는 애기장대 또는 유지식물(oil plant)인 것이 바람직하며, 상기 유지식물은 참깨, 유채, 해바라기, 아주까리, 땅콩, 콩, 코코야자, 기름야자 및 호호바로 이루어지는 군으로부터 선택되는 것이 바람직하나, 이에 한정되지 않는다.Preferably, the plant is an Arabidopsis thaliana or an oil plant. The plant is preferably selected from the group consisting of sesame, rapeseed, sunflower, castor bean, peanut, soybean, coconut, oil palm, and jojoba, But is not limited thereto.
또한, 본 발명의 벡터를 진핵 세포에 형질전환시키는 경우에는 숙주세포로서, 효모(Saccharomyce cerevisiae), 곤충세포, 사람세포 (예컨대, CHO 세포주 (Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2, 3T3, RIN 및 MDCK 세포주), 식물세포 및 식물체 등이 이용될 수 있다. 숙주세포는 바람직하게는 식물체이다. When the vector of the present invention is transformed into eukaryotic cells, yeast ( Saccharomyce cerevisiae ), insect cells, human cells (for example, Chinese hamster ovary, W138, BHK, COS-7, 293 , HepG2, 3T3, RIN and MDCK cell lines), plant cells and plants. The host cell is preferably a plant.
본 발명의 벡터를 숙주세포 내로 운반하는 방법은, 숙주세포가 진핵세포인 경우에는, 미세주입법, 칼슘포스페이트 침전법, 전기천공법, 리포좀-매개 형질감염법, 아그로박테리움-매개 형질 감염법, DEAE-덱스트란 처리법, 및 유전자 밤바드먼트 등에 의해 벡터를 숙주세포 내로 주입할 수 있다.When the host cell is a eukaryotic cell, the vector of the present invention may be delivered by a microinjection method, a calcium phosphate precipitation method, an electroporation method, a liposome-mediated transfection method, an Agrobacterium-mediated transfection method, DEAE-dextran treatment, and gene bombardment, etc., into a host cell.
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 the hybrid DNA according to the present invention into suitable progenitor cells. The method is based on the 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) (Shillito RD et al., 1985 Bio / Technol. 3, 1099-1102), microinjection into plant elements (Crossway A. et al., 1986, Mol. Gen. Genet. 202,179-185 (Klein et al., 1987, Nature 327, 70), the infiltration of plants or the transformation of mature pollen or vesicles into Agrobacterium tumefaciens Infection by viruses (non-integrative) in virus-mediated gene transfer (
본 발명에 따른 상기 식물체는 벼, 밀, 보리, 옥수수, 대두, 감자, 밀, 팥, 귀리 및 수수로 이루어진 군에서 선택된 식량 작물류; 애기장대, 배추, 무, 고추, 딸기, 토마토, 수박, 오이, 양배추, 참외, 호박, 파, 양파 및 당근으로 이루어진 군에서 선택된 채소 작물류; 인삼, 담배, 목화, 참깨, 사탕수수, 사탕무, 들깨, 땅콩 및 유채로 이루어진 군에서 선택된 특용 작물류; 사과나무, 배나무, 대추나무, 복숭아, 양다래, 포도, 감귤, 감, 자두, 살구 및 바나나로 이루어진 군에서 선택된 과수류; 장미, 글라디올러스, 거베라, 카네이션, 국화, 백합 및 튤립으로 이루어진 군에서 선택된 화훼류; 및 라이그라스, 레드클로버, 오차드그라스, 알파알파, 톨페스큐 및 페레니얼라이그라스로 이루어진 군에서 선택된 사료 작물류일 수 있다.
The plant according to the present invention is selected from the group consisting of food crops selected from the group consisting of rice, wheat, barley, corn, soybean, potato, wheat, red bean, oats and millet; Vegetable crops selected from the group consisting of Arabidopsis, cabbage, radish, red pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, squash, onions, onions and carrots; Ginseng, tobacco, cotton, sesame, sugar cane, sugar beet, perilla, peanut and rapeseed; Apple trees, pears, jujubes, peaches, sheep grapes, grapes, citrus fruits, persimmons, plums, apricots and banana; Roses, gladiolus, gerberas, carnations, chrysanthemums, lilies and tulips; And feed crops selected from the group consisting of Ryegrass, Red Clover, Orchardgrass, Alpha Alpha, Tall Fescue, and Fereniallaigrus.
또한, 본 발명은 In addition,
1) 제 1항의 식물 형질전환용 벡터를 아그로박테리움에 형질도입하는 단계;1) introducing the vector for plant transformation of
2) 상기 단계 1)의 아그로박테리움을 식물체에 감염시키는 단계;2) infecting the plant with the Agrobacterium of step 1);
3) 제초제를 처리하여 형질전환 식물체를 선별하는 단계; 및3) treating the herbicide to select transgenic plants; And
4) 선별된 식물체의 종자에서 불포화지방산 함량을 확인하는 단계를 포함하는 고도불포화지방산을 생산하는 식물체를 제조하는 방법을 제공한다.
4) determining the content of unsaturated fatty acids in the seeds of the selected plants. The present invention also provides a method for producing a plant producing a polyunsaturated fatty acid.
아울러, 본 발명은 본 발명에 따른 형질전환 식물체의 종자로부터 고도불포화지방산을 생산하는 방법을 제공한다.In addition, the present invention provides a method for producing polyunsaturated fatty acids from seeds of a transgenic plant according to the present invention.
상기 고도불포화지방산은 DGLA(dihomo-gamma-linolenic acid), ARA(arachidonic acid), DTA(docosatetraenoic acid), STA(stearidonic acid), ETA(eicosatetraenoic acid), EPA(eicosapentaenoic acid) 및 DPA(docosapetaenoic acid)로 이루어진 군으로부터 선택되는 하나 이상인 것이 바람직하나, 이에 한정되지 않는다.The polyunsaturated fatty acids may be selected from the group consisting of dihomo-gamma-linolenic acid (DGLA), arachidonic acid, docosatetraenoic acid (DTA), stearidonic acid (STA), eicosatetraenoic acid (ETA), eicosapentaenoic acid (EPA), and docosapetaenoic acid , But the present invention is not limited thereto.
본 발명에서 생산되는 GLA(gamma-linolenic acid), DGLA(dihomo-gamma-linolenic acid), ARA(arachidonic acid) 및 DTA(docosatetraenoic acid)는 형질전환 식물체인 애기장대(arabidopsis thaliana)가 생산하는 Linoleic acid (LA; C18:2n-6)를 기질로 이용하여 생산된 오메가-6 불포화지방산이며, STA(stearidonic acid), ETA(eicosatetraenoic acid), EPA(eicosapentaenoic acid), DPA(docosapetaenoic acid) 및 DHA(docosahexaenoic acid)는 형질전환 식물체인 애기장대(arabidopsis thaliana)가 생산하는 α-linolenic acid (ALA; C18:3n-3)를 기질로 이용하여 생산된 오메가-3 불포화지방산이다.
The gamma-linolenic acid (DGLA), arachidonic acid (ARA) and docosatetraenoic acid (DTA) produced by the present invention are produced by the transformation of linoleic acid (DGLA) produced by the transgenic Arabidopsis thaliana (EA), eicosapentaenoic acid (ETA), eicosapentaenoic acid (EPA), docosapetaenoic acid (DPA), and docosahexaenoic acid (DHA), which are omega-6 unsaturated fatty acids produced by using LA (C18: 2n- acid is an omega-3 unsaturated fatty acid produced by using α-linolenic acid (ALA; C18: 3n-3) produced by the transgenic Arabidopsis thaliana as a substrate.
본 발명 일 실시예에서는 갯장어 유래의 Δ6 불포화화 효소(desaturase) 유전자 McD6DES, 조류 유래의 Δ5 불포화화 효소(desaturase) 유전자 PtD5DES 및 돔 유래의 지방산 사슬연장효소(elongase) 유전자 AsELOVL5를 하나의 벡터에 도입하여 식물 형질전환용 재조합 벡터를 제작하였으며, 이를 LA 및 ALA를 생산하는 애기장대에 형질전환하고, 이로부터 발현된 유전자들이 형질전환 애기장대 T2 종자에서 고도불포화지방산들을 성공적으로 생산함을 GC로 확인하였다. 이를 통해 본 발명의 McD6DES, PtD5DES 및 AsELOVL5를 함께 포함하는 식물 형질전환용 재조합 벡터로 형질전환된 식물체가 오메가-6 경로 및 오메가-3 경로를 통해 포유동물에게 유용한 고도불포화지방산들을 생산하여 제공할 수 있음을 알 수 있었다.
The present invention in one embodiment introducing pike eel derived from the Δ6 desaturation enzymes (desaturase) gene McD6DES, the birds derived Δ5 desaturation enzyme (desaturase) extended fatty acid chain of a gene PtD5DES and dome-derived enzyme (elongase) gene AsELOVL5 in a vector The transgenic recombinant vectors were constructed and transfected into Arabidopsis thaliana producing LA and ALA, and the genes expressed from these transgenic lines were successfully produced by GC in the transgenic Arabidopsis thaliana T2 seeds Respectively. Thus, plants transformed with a recombinant vector for plant transformation containing the McD6DES , PtD5DES and AsELOVL5 of the present invention can produce and provide polyunsaturated fatty acids useful for mammals through the omega-6 pathway and the omega-3 pathway .
이하, 본 발명을 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.
Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.
<< 실시예Example 1> 1> McD6DESMcD6DES , , PtD5DESPtD5DES 및 And AsELOVL5AsELOVL5 유전자 발현 식물 형질전환용 벡터 제작 Generation of vectors for transgenic plant transformation
Δ6 불포화화 효소(desaturase) 유전자 McD6DES, Δ5 불포화화 효소(desaturase) 유전자 PtD5DES 및 지방산 사슬연장효소(elongase) 유전자 AsELOVL5를 동시에 발현하는 벡터를 제작하였다. 구체적으로, 갯장어(Muraenesox cinereus)로부터 분리한 Δ6 불포화화 효소(desaturase) 유전자 McD6DES(서열번호 1), 미세 조류(Phaeodactylum ticornutum KMCC B-128) 유래 Δ5 불포화화 효소(desaturase) 유전자 PtD5DES(서열번호 2) 및 돔(Acanthopagrus schlegeli)으로부터 분리한 지방산 사슬연장효소(elongase) 유전자 AsELOVL5(서열번호 3)를 각각 종자-특이발현 vicillin 프로모터와 octopine 터미네이터 사이에 재조합하여 유전자 카세트를 제작하였다. 제작한 유전자 카세트 및 pCAMBIA3300을 이용하여 3개의 유전자가 동시에 발현되도록 하는 식물체 형질전환용 다중 발현 운반체 pCam-VpAsELOVL5OCS-VpMcD6DOCS-VpPtD5DESOCS를 구축하였다 (도 1). 벡터 제작을 위하여 하기 표 1의 프라이머들을 이용하였다.A vector expressing the Δ6 desaturase gene McD6DES , the Δ5 desaturase gene PtD5DES and the fatty acid chain elongase gene AsELOVL5 was prepared . Specifically, the Δ6 desaturase gene McD6DES (SEQ ID NO: 1) isolated from the marsupial ( Muraenesox cinereus ), the microalgae ( Phaeodactylum the desaturase gene PtD5DES (SEQ ID NO: 2) derived from ticornutum KMCC B-128 and Dome ( Acanthopagrus The gene cassette was prepared by recombining the fatty acid chain elongase gene AsELOVL5 (SEQ ID NO: 3) isolated from Schlegeli with a seed-specific expression vicillin promoter and an octopine terminator, respectively. The constructed gene cassette and pCAMBIA3300 were used to construct pCam-VpAsELOVL5OCS-VpMcD6DOCS-VpPtD5DESOCS (Fig. 1) for expression of three genes simultaneously. The primers of Table 1 below were used for vector production.
그 결과, 상기에서 제작한 다중 발현 운반체에 상기 3개의 유전자 카세트가 정상적으로 도입되었음을 PCR을 통하여 확인하였다 (도 2).
As a result, it was confirmed by PCR that the three gene cassettes were normally introduced into the multi-expression carrier prepared above (Fig. 2).
<< 실시예Example 2> 형질전환 애기장대 제작 2> Production of transgenic Arabidopsis
애기장대에 상기 <실시예 1>에서 제작한 다중 발현 운반체를 형질전환시키기 위해, 아그로박테리움(Agrobacterium) GV3101에 도입하였다. 다중 발현 운반체가 형질도입된 애기장대의 T1 종자를 파종한 뒤, 본잎이 4개 전개되었을 때 0.3%의 바스타를 처리하여 형질전환 애기장대 160 개체를 선별하였다. 그 후, 선별한 160 개체의 애기장대에서 꽃대가 올라오기 전에 각각 잎 1 내지 2개를 채취하여 genomic DNA를 분리하였다. 상기에서 분리한 DNA에서 AsELOVL5 및 PtD5DES를 상기 표 1의 프라이머들을 이용하여 PCR을 통해 확인하였으며, ORF 사이즈 1338 bp가 PtD5DES ORF 사이즈 1410 bp와 겹쳐서 젤 상에 적용되기 때문에 McD6DES 프라이머 (서열번호 10 및 11)로는 625 bp 산물을 내는 ORF내의 염기서열을 사용하였다.
The Arabidopsis thaliana was introduced into Agrobacterium GV3101 for transformation of the multi-expression carrier prepared in Example 1 above. After the seeds of the Arabidopsis thaliana seedlings transduced with the multimodal transporter were inoculated, the transformed Arabidopsis thaliana seedlings were selected by treating with 0.3% of Basta when the four leaves were developed. Thereafter, 1-2 selected leaves were picked from the 160 Arabidopsis thaliana poles, and genomic DNA was isolated. AsELOVL5 and PtD5DES were confirmed by PCR using the primers shown in Table 1 above. Since the ORF size 1338 bp overlapped with the
그 결과, 상기 3개의 유전자가 온전히 모두 형질전환된 애기장대 라인(line, 계통)을 선별할 수 있었다 (도 3).
As a result, it was possible to select Arabidopsis lines in which the above three genes were completely transformed (Fig. 3).
<< 실시예Example 3> 형질전환 애기장대 3> Transgenic Arabidopsis thaliana T2T2 종자 확보 Securing seeds
상기 <실시예 2>에서 제작한 160개체의 형질전환 애기장대 T1을 포트에 심은 뒤 T2 종자를 확보하였다. 상기에서 확보된 종자에 대해 일련번호를 새롭게 부여하였다 (도 4).
160 transgenic Arabidopsis thaliana T1 plants prepared in Example 2 were planted in the pot to obtain T2 seeds. A serial number was newly assigned to the seed thus obtained (Fig. 4).
<< 실시예Example 4> 형질전환 애기장대 4> Transgenic Arabidopsis thaliana T2T2 종자의 지방산 조성 확인 Identification of fatty acid composition of seeds
McD6DES, AsELOVL5 및 PtD5DES 유전자가 삽입되어 delta 6-desaturase, fatty acid elongase 및 delta 5-desaturase를 발현하는 형질전환 애기장대 라인(line, 계통)들(1, 2, 5, 11, 12, 14, 15, 18, 20, 21, 23, 26, 27, 28, 33, 41, 44, 63, 64 및 65)의 T2 종자 (0.1 g)를 수득한 뒤, 각각 분쇄하였다. 잘 분쇄된 0.1g의 종자에 클로로포름 및 메탄올이 2:1로 혼합된 용액 5 ml과 내부표준물질 (pentadecanoic acid in MeOH, 1000 ppm, 1 mg.ml) 1 ml를 넣고 1시간 동안 초음파로 충격을 주어 지질이 유기 용매 쪽으로 추출되도록 하였다. 그 후 0.58% NaCl 5 ml를 넣고 10분 동안 초음파 충격을 가하고 2000 rpm에서 20분 동안 원심분리를 통해 층을 분리하였다. 분리된 층 중 하층의 지질층에서 용매를 질소가스로 제거하고 지질만 남은 샘플에 톨루엔 0.5 ml과 0.5N NaOH 2 ml를 넣고 85℃에서 5분 동안 반응시켰다. 반응 후, 이를 냉각시키고 이에 14% BF3 2.5 ml를 넣은 뒤 85℃에서 5분 동안 반응시켰다. 반응액을 새 튜브로 옮기고 petroleum ether 10 ml 및 ddH2O 15 ml를 넣고 볼텍싱하였다. 그 후 2000 rpm에서 20분 동안 원심분리하여 얻은 상층액에서 용매를 질소가스로 제거하여 지질을 얻었다. 상기 지질을 petroleum ehter 300 ㎕로 녹인 뒤 상업화된 지질 표준물질 (Sigma, St. Louis, MO)를 기초로 하여 가스크로마토그래피(GC) (GC-2010 Plus, SHIMADZU)로 분석하여 물질이 피크로 나온 시간대를 비교하고 이들에 대해 GC-MS를 분석하여 메탄올 농도에 의한 시간대별 지방산 생성물을 총 지방산 생성물에 대한 라인(line, 계통)별 T2 종자 각각의 지방산 함량을 %로 표시하였다.
McD6DES , AsELOVL5 And transgenic lines (1, 2, 5, 11, 12, 14, 15, 18, and 18) transduced with the PtD5DES gene and expressing delta 6-desaturase, fatty acid elongase and delta 5- T2 seeds (0.1 g) of 20, 21, 23, 26, 27, 28, 33, 41, 44, 63, 64 and 65 were obtained and then pulverized. 5 ml of a 2: 1 mixture of chloroform and methanol and 0.1 ml of internal standard (pentadecanoic acid in MeOH, 1000 ppm, 1 mg.ml) were added to 0.1 g of well pulverized seeds and shocked with ultrasonic waves for 1 hour. The lipids were extracted to the organic solvent. Then, 5 ml of 0.58% NaCl was added, ultrasonicated for 10 minutes, and the layers were separated by centrifugation at 2000 rpm for 20 minutes. In the lower layer of the separated layer, the solvent was removed with nitrogen gas, and 0.5 ml of toluene and 2 ml of 0.5 N NaOH were added to the sample containing only lipid, followed by reaction at 85 ° C for 5 minutes. After the reaction, the mixture was cooled, 2.5 ml of 14% BF 3 was added thereto, and the mixture was reacted at 85 ° C for 5 minutes. The reaction solution was transferred to a new tube, 10 ml of petroleum ether and 15 ml of ddH 2 O were added and vortexed. After that, the supernatant was centrifuged at 2000 rpm for 20 minutes and the solvent was removed with nitrogen gas to obtain a lipid. The lipid was dissolved in 300 μl of petroleum ehter and analyzed by gas chromatography (GC) (GC-2010 Plus, SHIMADZU) based on commercialized lipid standard (Sigma, St. Louis, MO) The GC-MS was analyzed by time zone, and the fatty acid content of the T2 seeds by the methanol concentration and the fatty acid contents of the T2 seeds by the line for the total fatty acid products were expressed in%.
그 결과, 본 발명의 벡터에 도입된 세 유전자가 애기장대 자체에서 생산하는 linoleic acid (LA; 18:2n-6) 및 α-linolenic acid (ALA; 18:3n-3)을 기질로 하여 오메가-6 지방산 합성 경로에서는 형질전환 유전자 McD6DES에 의해 발현된 delta 6-desaturase에 의하여 LA 지방산에 이중결합이 형성되어 gamma-linolenic acid (GLA; 18:3n-6)로 전환되었고, 이 GLA는 형질전환 유전자 AsELOVL5에 의해 발현된 fatty acid elongase에 의하여 탄소 사슬이 2개 연장되어 di-homo-linolenic acid (DGLA, 20:3n-6)로 전환되었다. DGLA는 형질전환 유전자 PtD5DES에 의해 발현된 delta 5-desaturase에 의하여 이중결합이 형성되어 arachidonic acid (ARA; 20:4n-6)로 전환되었다. 이 ARA는 위의 AsELOVL5에 의하여 다시 탄소 사슬 2개가 연장되어 docosatetraenoic acid (DTA, 22:4n-6)로 전환되었다. 또한, 상기 세 유전자는 오메가-3 지방산 합성 경로에 따라 ALA를 기질로 하여 차례로 stearidonic acid (STA, 18:4n-3), eicosatetraenoic acid (ETA, 20:4n-3), eicosapentaenoic acid (EPA, 20:5n-3) 및 docosapentaenoic acid (DPA, 222:5n-3)를 합성하였다. 이에 대한 결과를 도 4에 전체적인 각 라인(line, 계통)별 다중불포화지방산 생합성의 함량의 수준에 따라 강/중/약의 색깔로 표시하였으며, 도 5에 새롭게 합성된 오메가-6 및 오메가-3 지방산의 총 함량에 대해 이들이 차지하는 함량을 %로 표시하였다 (도 5 및 도 6). 이에 따르면, 오메가-3 고도불포화지방산 EPA의 함량을 0.20-0.25% 차지하는 형질전환체는 63, 64 및 12이었다. 0.15-0.19% 차지하는 형질전환체는 44, 33, 18, 15 및 11이었으며, 0.07-0.11% 차지하는 형질전환체는 65, 41, 23, 21, 20 및 14이었다. 0.02-0.05% 차지하는 형질전환체는 28, 27, 26, 5, 2 및 1이었다. 도 7에 각 형질전환체 라인(line, 계통)별의 EPA 함량만을 그래프로 나타내었다.
As a result, it was confirmed that the three genes introduced into the vector of the present invention are omega-3 fatty acids using linoleic acid (LA; 18: 2n-6) and α-linolenic acid (ALA; 18: 3n- 6 fatty acid synthesis pathway, a double bond was formed in the LA fatty acid by delta 6-desaturase expressed by the transgene McD6DES and converted to gamma-linolenic acid (GLA; 18: 3n-6) by a fatty acid elongase expression by AsELOVL5 the carbon chain is extended two di-homo-linolenic acid (DGLA , 20: 3n-6) was converted to. DGLA was converted to arachidonic acid (ARA; 20: 4n-6) by the formation of double bonds by delta 5-desaturase expressed by the transgene PtD5DES . This ARA was converted back to docosatetraenoic acid (DTA, 22: 4n-6) by extending the two carbon chains back by AsELOVL5. The three genes were sequestered with stearidonic acid (STA, 18: 4n-3), eicosatetraenoic acid (ETA, 20: 4n-3) and eicosapentaenoic acid (EPA, 20 : 5n-3) and docosapentaenoic acid (DPA, 222: 5n-3) were synthesized. The results are shown in FIG. 4 in terms of the intensity of the polyunsaturated fatty acid biosynthesis in each line, and the results are shown in FIG. 5 as the intensity of the newly synthesized omega-6 and omega-3 The content of fatty acids in terms of their total content is expressed in% (Figs. 5 and 6). According to the results, the transformants containing 0.20-0.25% of the content of omega-3 polyunsaturated fatty acid EPA were 63, 64 and 12, respectively. The transformants that were 0.15-0.19% were 44, 33, 18, 15 and 11, and the transformants which were 0.07-0.11% were 65, 41, 23, 21, 20 and 14, respectively. The transformants that were 0.02-0.05% were 28, 27, 26, 5, 2, and 1, respectively. FIG. 7 is a graph showing only the EPA content of each transformant line.
<< 실시예Example 5> 63번 라인(line, 계통) 형질전환 애기장대 5> line 63 (line, line) Transgenic Arabidopsis thaliana T2T2 종자의 지방산 조성 확인 Identification of fatty acid composition of seeds
상기 <실시예 4>에서 확인한 라인(line, 계통) 중 EPA를 많이 생산하는 형질전환체 63번 라인(line, 계통)의 T2 종자 일정량(0.1g)으로부터 지질을 추출하여 GC 분석을 수행하였다.
GC analysis was performed by extracting lipids from a predetermined amount of T2 seed (0.1 g) of a line (line) of a transformant line (line) that produces a large amount of EPA among the lines identified in Example 4 above.
GC 분석 결과, 본 발명의 벡터에 도입된 세 유전자가 63번 라인(line, 계통)의 애기장대에서 오메가-6 지방산 합성 경로에 따라 LA를 기질로 사용하여 GLA, DGLA, ARA 및 DTA를 합성한 것을 확인하였으며, 또한, 상기 세 유전자가 오메가-3 지방산 합성 경로에 따라 ALA를 기질로 하여 STA, ETA, EPA 및 DPA를 합성한 것을 확인하였다 (도 8). 아울러, 63번 라인(line, 계통) 애기장대의 생산 지방산 조성을 도 9에 나타내었다.
As a result of GC analysis, it was found that the three genes introduced into the vector of the present invention synthesized GLA, DGLA, ARA and DTA using LA as a substrate according to the omega-6 fatty acid synthesis pathway in the Arabidopsis thaliana line (line 63) And that the three genes synthesized STA, ETA, EPA and DPA using ALA as a substrate according to the omega-3 fatty acid synthesis pathway (FIG. 8). In addition, the production fatty acid composition of the
이상 살펴본 바와 같이, 애기장대가 가지고 있는 기질 ALA (오메가-3 경로에 이용)가 LA (오메가-6 경로에 이용)보다 적음에도 불구하고, STA (오메가-3)로의 전환이 GLA (오메가-6)보다 높게 나타났으며, DHA 생성 바로 두 단계 전의 DPA (오메가-3)의 생성이 DTA (오메가-6) 보다 효율이 높았다. 따라서, 형질전환에 이용된 3개 유전자가 고도불포화지방산을 생성하는 경로 중 오메가-3 지방산 생합성에 더 강하게 작용하고 있음을 알 수 있다 (도 10).As described above, the conversion of STA (omega-3) to GLA (omega-6) was not observed even though the substrate ALA (used for the omega-3 pathway) ) And the production of DPA (omega - 3) just before two stages of DHA production was higher than that of DTA (omega - 6). Thus, it can be seen that the three genes used for transformation are more strongly involved in omega-3 fatty acid biosynthesis in the pathway for producing polyunsaturated fatty acids (FIG. 10).
<110> republic of Korea <120> Recombinant vector for polyunsaturated fatty acids biosynthesis and plant transformed by the same <130> p131050 <160> 11 <170> KopatentIn 2.0 <210> 1 <211> 1335 <212> DNA <213> Muraenesox cinereus <400> 1 atggggggcg gaggtcaaca gacggagtcg gaatcgagct gtgggcgagg aggcggcgtt 60 ttcacctggg aagaggtgca gcggcattcc cacaaggggg accagtggtt ggtaatcgac 120 agaaaggtgt gcaacatcac cgactgggtg aaaagacatc caggcggcgc ccgggtcatc 180 agccactatg ccggggagga cgccacggat gcttttgctg cattccatcc agagcctgac 240 tttgtgcgca agtttctgaa gccactgctg attggtgagc ttgcaacctc tgagcccaac 300 caggaccggg acaaaaattc tgtgttgaca caggctttcc gcgaactgcg cgaggaggta 360 gagagggagg gcctcttcag gactcagccg ctgttctttt gcctccacct ggggcatatt 420 cttctcctgg aggccctggc ttacttgctg atttgggtgt atggaacagg ctggctccag 480 accctgctgt gtgcagtaat actggccacc tcacagtctc aggccggatg gctccagcat 540 gacttcggcc acctttccgt cttcaaaaag tcccgctgga accacctgct gcacaagttt 600 gtcatcggac accttaaggg tgcttcggct aactggtgga accatcggca tttccagcac 660 catgcaaaac ccaacatctt cagtaaagac cctgatgtca acatgctcca cacctttgtg 720 ctaggaaaga cccagccagt ggagtatgga ataaagaaga tcaagtacat gccttacaac 780 caccagcacc agtacttctt cctcattgga cctcctatgc ttatcccagt gtatttccac 840 atccagatca tgcaaaccat gttctttcga cgtgattggg tggacctcgt ctggtcaatg 900 agctactacc tgcgctactt cacctgctac acgccctttt acggagtttt tggagctgtg 960 gcgctcatca gcttcgttag gtttctggag agccactggt ttgtgtgggt gacccaaatg 1020 aaccacattc ccatggacat tgaccacgag aagcacgagg attggctgac catgcagctg 1080 aaggccacct gcaacattga gcagtcattc ttcaacgact ggttcagcgg acacctcaac 1140 ttccaaatcg aacaccatct gtttcccaca atgccccggc acaactactg ccgcgtggcc 1200 cctctggtgc gcaaggtgtg tgagaagcat ggcgtcacct accaggagaa gaccctgtgg 1260 agcggcttca gagacgtggt cagctctcta cgggagtctg gagatctgtg gctggatgcg 1320 tatctccata aataa 1335 <210> 2 <211> 1410 <212> DNA <213> Phaeodactylum tricornutum <400> 2 atggctccgg atgcggataa gcttcgacaa cgccagacga ctgcggtagc gaagcacaat 60 gctgctacca tatcgacgca ggaacgcctt tgcagtctgt cttcgctcga cggcgaagaa 120 gtctgcatcg acggaatcat ctatgacctc caatcattcg atcatcccgg gggtgaaacg 180 atcaaaatgt ttggtggcaa cgatgtcact gtacagtaca agatgattca cccgtaccat 240 accgagaagc atttggaaaa gatgaagcgc gtcggcaagg tgacggattt cgtctgcgag 300 tacaagttcg ataccgaatt tgaacgcgaa atcaaacgag aagtcttcaa gattgtgcga 360 cgaggcaagg atttcggtac tttgggatgg ttcttccgtg cgttttgcta cattgccatt 420 ttcttctacc tgcagtacca ttgggtcacc acgggaacct cttggctgct ggctgtggcc 480 tacggagtct cccaagcgat gattggcatg aatgtccagc acgatgccaa ccacggggcc 540 acctccaagc gtccctgggt caacgacatg ctaggcctcg gtgcggattt tattggtgga 600 tccaagtggc tctggcagga acaacactgg acccaccacg cttacaccaa tcacgccgag 660 atggatcccg atagctttgg tgccgaacca atgctcctat tcaacgactg tcccttggat 720 catcccgctc gtacctggct acatcgcttt caagcagtct tttacatgcc cgtcttggct 780 ggatactggt tgtccgctgt cttcaatcca caaattcttg acctccagca acgcggcgca 840 ctttccgtcg gtatccgtct cgacaacgct ttcattcact cgcgacgcaa gtatgcggtt 900 ttctggcggg ccgtgtacat tgcggtgaac gtgattgctc cgttttacac caactccggc 960 ctcgaatggt cctggcgtgt ctttggaaac attatgctca tgggtgtggc ggaatcgctc 1020 gcgctggcgg tcctgttttc gttgtcgcac aatttcgaat ccgccgatcg cgatccgacc 1080 gccccactga aaaagacggg agaaccagtc gactggttca agacacaggt cgaaacttcc 1140 tgcacttacg gtggattcct ttccggttgc ttcacgggag gtctcaactt tcaggttgaa 1200 caccacttgt tcccacgcat gagcagcgcc tggtacccct acattgcccc caaggtccgc 1260 gaaatttgcg ccaaacacgg cgtgcactac gcctactacc cgtggatcca ccaaaacttt 1320 ctctccaccg tccgctacat gcacgcggcc gggaccggtg ccaactggcg ccagatggcc 1380 agagaaaatc ccttgaccgg acgggcgtaa 1410 <210> 3 <211> 885 <212> DNA <213> Acanthopagrus schlegeli <400> 3 atggagacct tcaatcacaa actgaacgtt tacttcgaga catggatggg tccccgagat 60 cagcgggtgc ggggatggct actgctcgac aactacccac caacctttgc actcacagtc 120 atgtaccttc tgatcgtgtg gatggggccc aagtacatga aacaccggca gccgtacccc 180 tgcagaggcc tcctggtgct ctacaatctg ggcctcacac tcctctcctt ctacatgttc 240 tatgagcttg ttactgctgt gtggtatggc ggctacaatt tctactgcca ggacactcac 300 agtgcacagg aagtggataa taagatcata aatgtccttt ggtggtacta cttctccaag 360 ctcattgagt tcatggacac ctttttcttc atactacgga agaataatca ccagatcacc 420 tttcttcaca cctaccacca cgccagcatg ctgaatatct ggtggtttgt tatgaactgg 480 gtaccctgcg gccactcgta cttcggtgcc tccctaaaca gcttcgtcca cgtcgtgatg 540 tattcttact acggcctctc agccatccca gccatgcggc cgtacctttg gtggaagaag 600 tacatcacac agttccagct gatccagttc tttttaacca tgtcccagac aatatttgca 660 gtcatatggc cgtgtggctt ccccgacgga tggctttact tccaaatagg ttacatggtc 720 acgctaattt tcctgttctc aaacttctac attcagacct acaacaagca cagtgcatct 780 ctaaggaagg agcaccagaa cggctctcct ctatcaacaa atggacatgc aaacgggacg 840 ccatcgatgg agcacactgc acacaagaaa ctgagagtgg attga 885 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> McD6D-F primer <400> 4 atggggggcg gaggtcaaca gacg 24 <210> 5 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> McD6D-R primer <400> 5 ttatttatgg agatacgcat ccagccacag atct 34 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PtD5D-F primer <400> 6 atggctccgg atgcggataa gctt 24 <210> 7 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> PtD5D-R primer <400> 7 ttacgcccgt ccggtcaagg gatttt 26 <210> 8 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> AsELOVL5-F primer <400> 8 atggagacct tcaatcacaa actgaacgtt tac 33 <210> 9 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> AsELOVL5-R primer <400> 9 tcaatccact ctcagtttct tgtgtgcagt gt 32 <210> 10 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> McD6D-106-F primer <400> 10 tggttggtaa tcgacagaaa ggtgt 25 <210> 11 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> McD6D-730-R primer <400> 11 tctttcctag cacaaaggtg tggag 25 <110> republic of Korea <120> Recombinant vector for polyunsaturated fatty acids biosynthesis and plant transformed by the same <130> p131050 <160> 11 <170> Kopatentin 2.0 <210> 1 <211> 1335 <212> DNA <213> Muraenesox cinereus <400> 1 atggggggcg gaggtcaaca gacggagtcg gaatcgagct gtgggcgagg aggcggcgtt 60 ttcacctggg aagaggtgca gcggcattcc cacaaggggg accagtggtt ggtaatcgac 120 agaaaggtgt gcaacatcac cgactgggtg aaaagacatc caggcggcgc ccgggtcatc 180 agccactatg ccggggagga cgccacggat gcttttgctg cattccatcc agagcctgac 240 tttgtgcgca agtttctgaa gccactgctg attggtgagc ttgcaacctc tgagcccaac 300 caggaccggg acaaaaattc tgtgttgaca caggctttcc gcgaactgcg cgaggaggta 360 gagagggagg gcctcttcag gactcagccg ctgttctttt gcctccacct ggggcatatt 420 cttctcctgg aggccctggc ttacttgctg atttgggtgt atggaacagg ctggctccag 480 accctgctgt gtgcagtaat actggccacc tcacagtctc aggccggatg gctccagcat 540 gacttcggcc acctttccgt cttcaaaaag tcccgctgga accacctgct gcacaagttt 600 gtcatcggac accttaaggg tgcttcggct aactggtgga accatcggca tttccagcac 660 catgcaaaac ccaacatctt cagtaaagac cctgatgtca acatgctcca cacctttgtg 720 ctaggaaaga cccagccagt ggagtatgga ataaagaaga tcaagtacat gccttacaac 780 caccagcacc agtacttctt cctcattgga cctcctatgc ttatcccagt gtatttccac 840 atccagatca tgcaaaccat gttctttcga cgtgattggg tggacctcgt ctggtcaatg 900 agctactacc tgcgctactt cacctgctac acgccctttt acggagtttt tggagctgtg 960 gcgctcatca gcttcgttag gtttctggag agccactggt ttgtgtgggt gacccaaatg 1020 aaccacattc ccatggacat tgaccacgag aagcacgagg attggctgac catgcagctg 1080 aaggccacct gcaacattga gcagtcattc ttcaacgact ggttcagcgg acacctcaac 1140 ttccaaatcg aacaccatct gtttcccaca atgccccggc acaactactg ccgcgtggcc 1200 cctctggtgc gcaaggtgtg tgagaagcat ggcgtcacct accaggagaa gaccctgtgg 1260 agcggcttca gagacgtggt cagctctcta cgggagtctg gagatctgtg gctggatgcg 1320 tatctccata aataa 1335 <210> 2 <211> 1410 <212> DNA <213> Phaeodactylum tricornutum <400> 2 atggctccgg atgcggataa gcttcgacaa cgccagacga ctgcggtagc gaagcacaat 60 gctgctacca tatcgacgca ggaacgcctt tgcagtctgt cttcgctcga cggcgaagaa 120 gtctgcatcg acggaatcat ctatgacctc caatcattcg atcatcccgg gggtgaaacg 180 atcaaaatgt ttggtggcaa cgatgtcact gtacagtaca agatgattca cccgtaccat 240 accgagaagc atttggaaaa gatgaagcgc gtcggcaagg tgacggattt cgtctgcgag 300 tacaagttcg ataccgaatt tgaacgcgaa atcaaacgag aagtcttcaa gattgtgcga 360 cgaggcaagg atttcggtac tttgggatgg ttcttccgtg cgttttgcta cattgccatt 420 ttcttctacc tgcagtacca ttgggtcacc acgggaacct cttggctgct ggctgtggcc 480 tacggagtct cccaagcgat gattggcatg aatgtccagc acgatgccaa ccacggggcc 540 acctccaagc gtccctgggt caacgacatg ctaggcctcg gtgcggattt tattggtgga 600 tccaagtggc tctggcagga acaacactgg acccaccacg cttacaccaa tcacgccgag 660 atggatcccg atagctttgg tgccgaacca atgctcctat tcaacgactg tcccttggat 720 catcccgctc gtacctggct acatcgcttt caagcagtct tttacatgcc cgtcttggct 780 ggatactggt tgtccgctgt cttcaatcca caaattcttg acctccagca acgcggcgca 840 gt; ttctggcggg ccgtgtacat tgcggtgaac gtgattgctc cgttttacac caactccggc 960 ctcgaatggt cctggcgtgt ctttggaaac attatgctca tgggtgtggc ggaatcgctc 1020 gcgctggcgg tcctgttttc gttgtcgcac aatttcgaat ccgccgatcg cgatccgacc 1080 gccccactga aaaagacggg agaaccagtc gactggttca agacacaggt cgaaacttcc 1140 tgcacttacg gtggattcct ttccggttgc ttcacgggag gtctcaactt tcaggttgaa 1200 caccacttgt tcccacgcat gagcagcgcc tggtacccct acattgcccc caaggtccgc 1260 gaaatttgcg ccaaacacgg cgtgcactac gcctactacc cgtggatcca ccaaaactt 1320 ctctccaccg tccgctacat gcacgcggcc gggaccggtg ccaactggcg ccagatggcc 1380 agagaaaatc ccttgaccgg acgggcgtaa 1410 <210> 3 <211> 885 <212> DNA <213> Acanthopagrus schlegeli <400> 3 atggagacct tcaatcacaa actgaacgtt tacttcgaga catggatggg tccccgagat 60 cagcgggtgc ggggatggct actgctcgac aactacccac caacctttgc actcacagtc 120 atgtaccttc tgatcgtgtg gatggggccc aagtacatga aacaccggca gccgtacccc 180 tgcagaggcc tcctggtgct ctacaatctg ggcctcacac tcctctcctt ctacatgttc 240 tatgagcttg ttactgctgt gtggtatggc ggctacaatt tctactgcca ggacactcac 300 agtgcacagg aagtggataa taagatcata aatgtccttt ggtggtacta cttctccaag 360 ctcattgagt tcatggacac ctttttcttc atactacgga agaataatca ccagatcacc 420 tttcttcaca cctaccacca cgccagcatg ctgaatatct ggtggtttgt tatgaactgg 480 gtaccctgcg gccactcgta cttcggtgcc tccctaaaca gcttcgtcca cgtcgtgatg 540 tattcttact acggcctctc agccatccca gccatgcggc cgtacctttg gtggaagaag 600 tacatcacac agttccagct gatccagttc tttttaacca tgtcccagac aatatttgca 660 gtcatatggc cgtgtggctt ccccgacgga tggctttact tccaaatagg ttacatggtc 720 acgctaattt tcctgttctc aaacttctac attcagacct acaacaagca cagtgcatct 780 ctaaggaagg agcaccagaa cggctctcct ctatcaacaa atggacatgc aaacgggacg 840 ccatcgatgg agcacactgc acacaagaaa ctgagagtgg attga 885 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> McD6D-F primer <400> 4 atggggggcg gaggtcaaca gacg 24 <210> 5 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> McD6D-R primer <400> 5 ttatttatgg agatacgcat ccagccacag atct 34 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> ≪ 223 > PtD5D-F primer <400> 6 atggctccgg atgcggataa gctt 24 <210> 7 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> PtD5D-R primer <400> 7 ttacgcccgt ccggtcaagg gatttt 26 <210> 8 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> AsELOVL5-F primer <400> 8 atggagacct tcaatcacaa actgaacgtt tac 33 <210> 9 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> AsELOVL5-R primer <400> 9 tcaatccact ctcagtttct tgtgtgcagt gt 32 <210> 10 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> McD6D-106-F primer <400> 10 tggttggtaa tcgacagaaa ggtgt 25 <210> 11 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> McD6D-730-R primer <400> 11 tctttcctag cacaaaggtg tggag 25
Claims (12)
A △ 6 desaturase gene isolated from the marsupial ( Muraenesox cinereus ) described in the nucleotide sequence of SEQ ID NO: 1, a △ 6 desaturase gene isolated from the microalgae ( Phaeodactylum ticornutum KMCC B-128) described in the nucleotide sequence of SEQ ID NO: 5 stearidonic acid and DPA (docosapetaenoic acid) in plant seeds, including the desaturase gene of SEQ ID NO: 3 and the fatty acid chain elongase gene isolated from the dome ( Acanthopagrus schlegeli ) acid to increase the production efficiency of the plant.
The recombinant vector for plant transformation according to claim 1, wherein the recombinant vector for plant transformation comprises a gene consisting of the nucleotide sequence of SEQ ID NO: 1 between a vicillin promoter and an Octopine terminator, A gene consisting of a nucleotide sequence and a gene consisting of a nucleotide sequence of SEQ ID NO: 3, respectively.
A transgenic plant transformed with the recombinant vector for plant transformation of claim 1.
The plant according to claim 6, wherein the plant is selected from the group consisting of a △ 6 desaturase gene isolated from the marsupial ( Muraenesox cinereus ) described in the nucleotide sequence of SEQ ID NO: 1, a microorganism ( Phaeodactylum tricornutum Expression of the Δ5 desaturase gene isolated from KMCC B-128 and the expression of a fatty acid chain elongase gene isolated from dome ( Acanthopagrus schlegeli ), which is described in the nucleotide sequence of SEQ ID NO: 3, wherein the production efficiency of stearidonic acid and docosapetaenoic acid (DPA) is enhanced.
The transgenic plant according to claim 6, wherein the plant is a Arabidopsis thaliana plant or an oil plant.
9. The transgenic plant according to claim 8, wherein the preserved plant is selected from the group consisting of sesame, rapeseed, sunflower, castor bean, peanut, soybean, coconut, oil palm and jojoba.
2) 상기 단계 1)의 아그로박테리움을 식물체에 감염시키는 단계;
3) 제초제를 처리하여 형질전환 식물체를 선별하는 단계; 및
4) 선별된 식물체의 종자에서 불포화지방산 함량을 확인하는 단계를 포함하는 식물 종자 내 STA(stearidonic acid) 및 DPA(docosapetaenoic acid)의 생성 효율이 증가된 식물체를 제조하는 방법.
1) introducing the vector for plant transformation of claim 1 into Agrobacterium;
2) infecting the plant with the Agrobacterium of step 1);
3) treating the herbicide to select transgenic plants; And
4) A method for producing a plant having increased production efficiency of stearidonic acid (STA) and docosapetaenoic acid (DPA) in plant seeds, comprising the step of confirming the content of unsaturated fatty acids in the seeds of the selected plants.
A method for producing polyunsaturated fatty acids from seeds of the transgenic plant of claim 6.
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