KR20180031326A - Transgenic plants transformed with activin A gene and methods of producing activin A protein by using the same - Google Patents

Transgenic plants transformed with activin A gene and methods of producing activin A protein by using the same Download PDF

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KR20180031326A
KR20180031326A KR1020160119795A KR20160119795A KR20180031326A KR 20180031326 A KR20180031326 A KR 20180031326A KR 1020160119795 A KR1020160119795 A KR 1020160119795A KR 20160119795 A KR20160119795 A KR 20160119795A KR 20180031326 A KR20180031326 A KR 20180031326A
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박기영
위수진
권태호
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순천대학교 산학협력단
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Abstract

The present invention relates to a plant transformed by an activin A gene, and a method for producing an activin A protein using the same. According to the invention, in an attempt to design a gene expression composition to produce the activin A protein in plants, a stepwise experiment was conducted. Finally, the produced activin A composition directly induced by a 35S promoter is applied to transformation of a tobacco plant so as to produce transgenic tobacco plant which is stabilized, thereby being useful for producing the activin A protein to be used industrially through molecular agriculture.

Description

액티빈 A 유전자로 형질전환된 식물체 및 및 이를 이용하여 액티빈 A 단백질을 생산하는 방법 {Transgenic plants transformed with activin A gene and methods of producing activin A protein by using the same}[0001] The present invention relates to a plant transformed with an Actin A gene and a method for producing an Actin A protein using the same,

본 발명은 액티빈(activin) A 유전자로 형질전환된 식물체 및 및 이를 이용하여 액티빈 A 단백질을 생산하는 방법에 관한 것으로, 보다 구체적으로는 액티빈 유전자가 도입된 발현벡터, 이를 이용하여 형질전환된 식물체 및 이를 이용하여 액티빈 A 단백질을 생산하는 방법에 관한 것이다. The present invention relates to a plant transformed with an activin A gene and a method for producing an actin A protein using the same, and more particularly, to an expression vector into which an actin gene has been introduced, And a method for producing actin A protein using the same.

액티빈(activin)은 TGF-β superfamily의 하나로서 액티빈에는 3가지 기본적인 액티빈 형태(A, B, AB)가 존재하는데, 이들은 2개의 밀접하게 관련된 β 소단위 (βAβA, βBβB, βAβB)의 동종 또는 이형이합체가 존재한다 (Cronin et al. 1998; Guo and Wang, 2009). TGF-β signaling 중에서 Nodal signaling과 유사한 기전을 갖는 성장인자 단백질로서 척추동물의 경우에는 activin의 고농도에서 내배엽성 유도를 촉진한다. 또한, 내배엽성 유도가 빠르게 일어나도록 촉진하는 역할을 하고 있어 조직이 분화하는데 걸리는 시간을 단축해주기 때문에 줄기세포의 분화에서 활용하면서 경제적인 이점이 큰 물질로 예상되고 있는 이합체 (dimer) 폴리펩티드 성장 인자이다 (Tsuchida et al. 2009). 특히 액티빈은 과립막 세포, 영양막 세포, 성선 성장 세포들을 포함한 다양한 세포들의 성장과 분화에 관여하여 여러 역할을 하는 것으로 알려진 물질들로 알려져 있으며, 난포의 생성, 배아의 성장에도 관여하는 것으로 알려져 있을 뿐만 아니라 인슐린유사성장인자 (insulin-like growth factor, IGF)와 그 수용체, 그리고 인슐린유사성장인자 결합단백질 (insulin-like growth factor binding protein, IGFBP)과도 관련된다는 것이 연구되고 있다(Guo and Wang, 2009; Moustakas and Heldin, 2009; Tsuchida et al. 2009). Activin is one of the TGF-β superfamily, and there are three basic types of actibin (A, B, AB) in actin, which are the homologs of two closely related β subunits (βAβA, βBβB, βAβB) Or heterodimers (Cronin et al., 1998; Guo and Wang, 2009). TGF-β signaling is a growth factor protein with a mechanism similar to nodal signaling and promotes endodomolytic induction at high concentrations of activin in vertebrates. In addition, since it plays a role of promoting the rapid induction of the endodermal induction and shortens the time required for differentiation of the tissue, it is a dimer polypeptide growth factor which is expected to be an economical advantageous material in utilization of stem cell differentiation (Tsuchida et al. 2009). Particularly, actin is known to play various roles in the growth and differentiation of various cells including granulosa cells, trophoblast cells and gonadal growth cells, and is known to be involved in follicle production and embryo development It has also been studied that it is associated with insulin-like growth factor (IGF) and its receptor, and insulin-like growth factor binding protein (IGFBP) (Guo and Wang, 2009 ; Moustakas and Heldin, 2009; Tsuchida et al., 2009).

TGF-β 류에서, 액티빈, 특히 액티빈 A는 난소와 태반 세포에서 호르몬 생산을 촉진하고, 신경 세포 생존을 뒷받침하고, 세포 유형에 따라 세포-주기 진행에 긍정적으로 또는 부정적으로 영향을 주고, 최소한 양서류 배아에서 중배엽 분화(mesodermal differentiation)를 유도할 수 있는 독특한 다기능성 인자이다(DePaolo et al. 1991; Dyson et al. 1997; Woodruff, 1998). 또한, 자극된 인간 단구성 백혈병 세포로부터 분리된 적혈구 분화 인자(erythroid differentiation factor, EDF)가 액티빈 A에 동일한 것으로 밝혀졌다(Murata et al. 1988).In the TGF-β family, actibin, particularly actin A, promotes hormone production in ovarian and placental cells, supports neural cell survival, positively or negatively affects cell-cycle progression, (DePaolo et al., 1991; Dyson et al., 1997; Woodruff, 1998), which is capable of inducing mesodermal differentiation in at least amphibian embryos. In addition, erythroid differentiation factor (EDF) isolated from stimulated human monocytic leukemia cells was found to be identical to actin A (Murata et al. 1988).

액티빈 A는 골수(bone marrow)에서 적혈구 생성(erythropoiesis)의 자연적인 양성 조절인자로서 기능하는 것으로 제안되었으며, 여러 조직에서 액티빈 신호전달은 관련된 이형이합체, 인히빈(inhibin)에 의해 길항작용을 나타낸다. 뇌하수체(pituitary)로부터 난포선-자극 호르몬(follicle-stimulating hormone, FSH)의 방출 동안, 액티빈은 FSH 분비와 합성을 촉진하는 반면, 인히빈은 FSH 분비와 합성을 차단하기도 한다. 액티빈은 생물 활성을 조절하기도 하고 액티빈에 결합하는 다른 단백질에는 폴리스타틴(follistatin, FS), 폴리스타틴-관련된 단백질(follistatin-related protein, FSRP), α2-거대글로불린(macroglobulin), 케르베로스(Cerberus), 엔도글린(endoglin) 등이 알려져 있다 (Niemuller et al. 1995; Schier, 2003).Actin A has been proposed to function as a natural positive regulator of erythropoiesis in bone marrow, and actin signaling in many tissues is antagonized by a related dendritic dimer, inhibin. . During the release of the follicle-stimulating hormone (FSH) from the pituitary, actin promotes FSH secretion and synthesis, while inhynin blocks FSH secretion and synthesis. Actibin also regulates biological activity and other proteins that bind to actin include follistatin (FS), follistatin-related protein (FSRP), a2-macroglobulin, Cerberus ), Endoglin, etc. (Niemuller et al., 1995; Schier, 2003).

액티빈 A는 TGF-β 그룹과 유사하게 생체 내에서 매우 중요한 기능을 수행하고 있는데, 특히 다양한 세포 분화 및 증식을 조절하며, 또한 몇 종류의 인체 암세포주의 생장도 저해하는 것으로 알려지고 있으며(Roberts et al. 1985), 뼈와 관련된 골밀도 증가에도 매우 중요한 역할을 수행한다고 알려져 있다. 액티빈 A는 활성이 높은 단백질이지만, 발현이 매우 어려우며 가격이 비싸고 반감기가 짧아 원활한 사용에 제약이 있다.Actin A, which is similar to the TGF-β group, plays an important role in vivo, and is known to regulate various cell differentiation and proliferation and to inhibit the growth of several types of human cancer cells (Roberts et 1985), it is known to play an important role in increasing bone mineral density. Actin A is a highly active protein, but its expression is very difficult, its cost is high and its half-life is short, which limits its smooth use.

액티빈 A가 임신-비특이적 병인 자간전증(子癎前症)의 생체표지자(biomarker)로 보고되고 있어 이를 이용한 진단키트 개발이 가능하다는 제안이 있다(Yu et al. 2012). 액티빈 A는 신장, 췌장 및 전립선 등 다양한 조직에서 발견되며 여러 세포들에 작용하여 분화 및 발생을 유도하는 역할을 한다는 보고도 있다. 특히 췌장 형성을 유도하고, 췌장 세포 내의 PAX4와 neurogenin 3(Ngn3)의 발현을 조절하여 내분비 세포 분화를 촉진하는 것으로 알려져 있다 (Li et al. 2004). Actin A has been reported to be a biomarker of pregnancy-nonspecific preeclampsia (Yu et al. 2012). Actin A is found in various tissues such as the kidney, pancreas and prostate, and has been reported to act on various cells to induce differentiation and development. In particular, it is known that inducing pancreatic formation and promoting endocrine differentiation by regulating the expression of PAX4 and neurogenin 3 (Ngn3) in pancreatic cells (Li et al. 2004).

최근에는 줄기세포 배양의 인자로서 수요가 늘고 있으나 단백질의 특성상 미생물에서의 재조합 단백질의 생산이 불가능한 상황으로 대부분을 동물세포(Pangas and Woodruff, 2002) 및 곤충세포(Cronin et al, 1998) 배양을 통하여 생산하고 있다. 액티빈 A는 homo dimer의 형태로 이루어지는 관계로 미생물에서의 생산이 되지 않아 현재 동물세포에서 생산하여 사용되는 매우 고가 단백질이다. In recent years, the demand for stem cell culture has been increasing. However, due to the nature of the protein, production of recombinant proteins in microorganisms is impossible. Most of them are cultured in animal cells (Pangas and Woodruff, 2002) and insect cells (Cronin et al, 1998) Production. Actin A is a highly expensive protein that is produced in animal cells because it is in the form of a homo dimer and thus can not be produced in microorganisms.

따라서 식물에서의 유전자재조합 액티빈 A 생산 기술 개발은 상업적 가치가 매우 클 것으로 예상되며 액티빈 A는 당질화가 일어나지 않는 단백질 (non-glycosylation)이므로 식물유래 단백질 생산에서 걸림돌이 되고 있는 문제가 없어서 식물에서 생산된 재조합 단백질은 향후 의료용 사용 가능성이 매우 높으나, 아직까지 그 연구가 미미한 실정이다. Therefore, the development of recombinant actin A production technology in plants is expected to have a very high commercial value. Since actin A is a non-glycosylation protein, there is no problem in producing plant-derived proteins, The recombinant proteins produced are very likely to be used for medical use in the future, but the research is still limited.

따라서, 본 발명에서 해결하고자 하는 기술적 과제는 본 발명에서 해결하고자 하는 기술적 과제는 액티빈 A 유전자가 도입된 식물체 발현용 발현벡터를 제공하기 위한 것이다.Accordingly, an object of the present invention is to provide an expression vector for expression of a plant into which an Actin A gene has been introduced.

또한, 본 발명에서 해결하고자 하는 다른 기술적 과제는 상기 발현벡터로 형질전환된 형질전환 식물체를 제공하기 위한 것이다.Another technical problem to be solved by the present invention is to provide a transgenic plant transformed with the expression vector.

또한, 본 발명에서 해결하고자 하는 다른 기술적 과제는 상기 발현벡터를 이용하여 식물체를 형질전환하는 방법을 제공하기 위한 것이다.Another object of the present invention is to provide a method for transforming a plant using the expression vector.

또한, 본 발명에서 해결하고자 하는 다른 기술적 과제는 상기 형질전환 식물체를 이용하여 재조합 액티빈 A 단백질을 생산하는 방법을 제공하기 위한 것이다.Another object of the present invention is to provide a method for producing recombinant actin A protein using the transgenic plant.

상기한 기술적 과제를 달성하기 위하여, 본 발명에서는 서열번호 1의 염기서열을 가지는 액티빈 A의 유전자를 포함하는 식물체 발현용 발현벡터를 제공한다.In order to accomplish the above object, the present invention provides an expression vector for plant expression comprising the gene of Actin A having the nucleotide sequence of SEQ ID NO: 1.

또한, 본 발명에서는 상기한 기술적 과제를 달성하기 위하여, 상기 발현벡터로 형질전환된 식물체를 제공한다.The present invention also provides a plant transformed with the expression vector to achieve the above technical object.

또한, 본 발명에서는 상기한 다른 기술적 과제를 달성하기 위하여, 상기 발현벡터를 이용하여 식물체를 형질전환하는 방법을 제공한다.According to another aspect of the present invention, there is provided a method for transforming a plant using the expression vector.

또한, 본 발명에서는 상기한 다른 기술적 과제를 달성하기 위하여, 상기 서열번호 1의 염기서열을 가지는 액티빈 A의 유전자를 포함하는 식물체 발현용 발현벡터로 형질전환된 식물체를 이용하여 재조합 액티빈 A 단백질을 생산하는 방법을 제공한다. According to another aspect of the present invention, there is provided a recombinant mutant Actin A protein comprising a plant transformed with an expression vector for plant expression comprising a gene of Actin A having the nucleotide sequence of SEQ ID NO: 1, Of the present invention.

본 발명에 따르면, 액티빈 A의 유전자가 과발현된 식물체에 생산 목적의 재조합 단백질의 유전자를 도입하여 동시 발현시킨다면 추가적인 탈당질화 과정이 생략될 수 있어 이의 활용도를 높일 수 있다. According to the present invention, when a recombinant protein gene for production is introduced into a plant overexpressing the gene of actin A, the simultaneous expression of the recombinant protein can eliminate the additional desalting nitrification process, thereby increasing its utilization.

본 발명에서 액티빈 A의 유전자는 서열번호 7의 염기서열 및 서열번호 8의 아미노산 서열을 가지는 단백질로서, 서열번호 8의 아미노산 서열을 가지는 액티빈 A의 유전자를 코딩하는 단백질과 기능적 동등물일 수 있다. In the present invention, the gene of Actin A is a protein having the nucleotide sequence of SEQ ID NO: 7 and the amino acid sequence of SEQ ID NO: 8 and may be a functional equivalent to a protein coding for the gene of Actin A having the amino acid sequence of SEQ ID NO: .

상기 "기능적 동등물"이란, 아미노산의 부가, 치환 또는 결실의 결과, 서열번호 1로 표시되는 아미노산 서열과 적어도 60%, 바람직하게는 70%, 보다 바람직하게는 80% 이상의 서열 상동성을 갖는 것으로서 본 발명의 액티빈 A를 코딩하는 단백질과과 실질적으로 동질의 활성을 나타내는 단백질을 의미한다. The "functional equivalents" have a sequence homology of at least 60%, preferably 70%, more preferably 80% or more, with the amino acid sequence of SEQ ID NO: 1 as a result of addition, substitution or deletion of amino acids Quot; means a protein that exhibits substantially the same activity as the protein encoding actin A of the present invention.

상기 기능적 동등물에는, 예를 들어, 서열번호 2의 아미노산 서열의 아미노산 중 일부가 치환되거나, 결실 또는 부가된 아미노산 서열 변형체가 포함된다. 아미노산의 치환은 바람직하게는 보존적 치환이다. 천연에 존재하는 아미노산의 보존적 치환의 예는 다음과 같다; 지방족 아미노산(Gly, Ala, Pro), 소수성 아미노산(Ile, Leu, Val), 방향족 아미노산(Phe, Tyr, Trp), 산성 아미노산 (Asp, Glu), 염기성 아미노산(His, Lys, Arg, Gln, Asn) 및 황 함유 아미노산(Cys, Met). 아미노산의 결실은 바람직하게는 본 발명의 액티빈 A의 활성에 직접 관여하지 않는 부분에 위치한다. 또한 상기 기능적 동등물의 범위에는 액티빈 A의 기본 골격 및 이의 생리활성을 유지하면서 단백질의 일부 화학 구조가 변형된 단백질 유도체도 포함된다. 예를 들어, 본 발명의 단백질의 안정성, 저장성, 휘발성 또는 용해도 등을 변경시키기 위한 구조변경 및 생리활성을 유지하면서 GFP(Green Fluorescent Protein)와 같은 다른 단백질과 융합으로 만들어진 융합단백질 등이 이에 포함된다.Such functional equivalents include, for example, amino acid sequence variants in which some of the amino acids of the amino acid sequence of SEQ ID NO: 2 are substituted, deleted or added. Substitution of amino acids is preferably conservative substitution. Examples of conservative substitutions of amino acids present in nature are as follows: (Gly, Ala, Pro), hydrophobic amino acids (Ile, Leu, Val), aromatic amino acids (Phe, Tyr, Trp), acidic amino acids (Asp, Glu), basic amino acids (His, Lys, Arg, Gln, Asn ) And sulfur-containing amino acids (Cys, Met). The deletion of the amino acid is preferably located at a site which is not directly involved in the activity of theactivin A of the present invention. The functional equivalents also include protein derivatives in which the basic skeleton of Actin A and its chemical structure is modified while maintaining its physiological activity. These include, for example, fusion proteins made by fusion with other proteins such as GFP (Green Fluorescent Protein) while retaining the structural modification and physiological activity to change the stability, storage stability, volatility or solubility of the protein of the present invention .

본 발명의 구체적인 실시양태에 따르면, 액티빈 A 유전자를 형질전환용 벡터, pTRAkt vector에 삽입하여 형질전환용 벡터인 pTRAkt-액티빈 A를 제작한 후, 이를 이용하여 담배 형질전환체를 제조하였으며, 형질전환 담배식물체에서 액티빈 A의 발현을 확인하였다.According to a specific embodiment of the present invention, pTRAkt-actibin A, which is a transformation vector, was prepared by inserting the Actin A gene into a transfection vector, pTRAkt vector, Expression of actin A was detected in the transgenic tobacco plants.

상기와 같이 액티빈 A 유전자는 서열번호 1에 기재된 염기서열을 가지는데, 상기 유전자는 첨부한 서열목록에 기재된 서열번호 7의 염기서열에 한정되지 않으며, 기능적으로 균등한 코돈 또는 동일한 아미노산을 코딩하는 코돈, 또는 생물학적으로 균등한 아미노산을 코딩하는 코돈을 포함하는 염기서열을 포함한다. 생물학적으로 균등 활성을 갖는 변이를 고려한다면, 본 발명에서 이용되는 염기서열은 서열목록에 기재된 서열과 실질적인 동일성(substantial identity)을 나타내는 서열도 포함하는 것으로 해석된다. 상기의 실질적인 동일성은, 상기한 본 발명의 서열과 임의의 다른 서열을 최대한 대응되도록 얼라인하고, 당업계에서 통상적으로 이용되는 알고리즘을 이용하여 얼라인된 서열을 분석한 경우에, 최소 60%의 상동성, 보다 바람직하게는 70%의 상동성, 보다 더 바람직하게는 80%의 상동성, 가장 바람직하게는 90%의 상동성을 나타내는 서열을 의미한다. As described above, the Actin A gene has the nucleotide sequence as shown in SEQ ID NO: 1, and the gene is not limited to the nucleotide sequence of SEQ ID NO: 7 described in the attached Sequence Listing and may be a nucleotide sequence encoding a functionally equivalent codon or the same amino acid Or a nucleotide sequence comprising a codon that encodes a biologically equivalent amino acid. Considering a mutation having biologically equivalent activity, the nucleotide sequence used in the present invention is interpreted to include a sequence showing substantial identity with the sequence described in the sequence listing. The above-mentioned substantial identity is determined by aligning the sequence of the present invention with any other sequence as much as possible and analyzing the aligned sequence using an algorithm commonly used in the art. Homology, more preferably 70% homology, even more preferably 80% homology, and most preferably 90% homology.

본 발명의 하나의 구현예에 따르면, 본 발명에서는 서열번호 7의 염기서열을 가지는 액티빈 A의 유전자를 포함하는 식물체 발현용 플라스미드에 도입시킨 발현벡터를 제공한다. 상기 플라스미드로는 식물체 발현용으로, 그 종류에는 제한이 없다. 본 발명에서는 pTRAkt vector를 사용하였다.According to one embodiment of the present invention, the present invention provides an expression vector introduced into a plant expression plasmid containing the gene of Actin A having the nucleotide sequence of SEQ ID NO: 7. The plasmid is used for expression of a plant, and there is no restriction on its kind. In the present invention, a pTRAkt vector was used.

본 발명의 바람직한 실시양태에 따르면, 서열번호 7의 염기서열을 가지는 액티빈 A의 유전자를 포함하는 식물체 발현용 발현벡터는 pTRAkt-액티빈 A이다.According to a preferred embodiment of the present invention, the plant expression vector comprising the gene of Actin A having the nucleotide sequence of SEQ ID NO: 7 is pTRAkt-Actin A.

본 발명에서 "형질전환용 재조합 벡터"란 유전자 삽입물이 발현되도록 작동가능하게 연결된 필수적인 조절 요소를 포함하는 유전자 작제물로, 플라스미드 벡터, 코스미드 벡터, 박테리오파지 벡터 및 바이러스 벡터 등을 포함한 통상의 모든 벡터를 포함한다.In the present invention, a "transgenic recombinant vector" is a gene construct containing an essential regulatory element operatively linked to the expression of a gene insert, and includes all plasmid vectors, cosmid vectors, bacteriophage vectors, .

본 발명의 "형질전환용 재조합 벡터"는 상기 액티빈 A 유전자가 발현될 수 있도록, 발현조절 서열과 기능적으로 연결되어 있다. 예를 들어, 벡터는 프로모터, 오퍼레이터, 개시코돈, 종결코돈, 폴리아데닐화 시그널, 인핸서 같은 발현 조절 요소 외에도 막 표적화 또는 분비를 위한 신호 서열 또는 리더 서열을 포함하며 목적에 따라 다양하게 제조될 수 있다. 또한, 벡터는 선택성 마커를 포함할 수 있으며, 벡터는 자가 복제하거나 숙주 DNA에 통합될 수 있다. 본 발명의 벡터는 당해 기술 분야에서 잘 알려진 유전자 재조합 기술을 이용하여 제조할 수 있으며, 부위-특이적 DNA 절단 및 연결은 당해 기술 분야에서 일반적으로 알려진 효소 등을 사용한다.The "recombinant vector for transformation" of the present invention is operably linked to an expression control sequence so that the Actin A gene can be expressed. For example, the vector may comprise a signal sequence or leader sequence for membrane targeting or secretion in addition to an expression regulatory element such as a promoter, an operator, an initiation codon, a stop codon, a polyadenylation signal, an enhancer, . In addition, the vector may comprise a selectable marker, and the vector may be self-replicating or integrated into the host DNA. The vector of the present invention can be produced using gene recombination techniques well known in the art, and site-specific DNA cleavage and linkage are performed using enzymes generally known in the art.

본 발명의 다른 하나의 양태로서, 본 발명은 상기 재조합 벡터에 의해 형질전환된 식물체를 제공한다. In another aspect of the present invention, the present invention provides a plant transformed with said recombinant vector.

본 명세서에서, 용어 "식물체"는 성숙한 식물체 뿐만 아니라 성숙한 식물로 발육할 수 있는 식물 세포, 식물 조직 및 식물의 종자 등을 모두 포함하는 의미로서 이해된다. 본 발명에서 상기 벡터로 식물체를 형질전환하는 것은 당업자에게 공지된 형질전환기술에 의해 수행될 수 있다. 바람직하게는 아그로박테리움을 이용한 형질전환방법, 미세사출법(microprojectile bombardment), 일렉트로포레이션(electroporation), PEG-매개 융합법(PEG-mediated fusion), 미세주입법(microinjection), 리포좀 매개법(liposome-mediated method), 인-플란타 형질전환법(In planta transformation), 진공 침윤법(Vacuum infiltration method), 화아침지법(floral meristem dipping method), 및 아그로박테리아 분사법(Agrobacteria spraying method)을 이용할 수 있으며, 더 바람직하게는 아그로박테리움을 이용한 형질전환방법을 이용할 수 있다.As used herein, the term "plant" is understood to include not only mature plants but also plant cells, plant tissues and plant seeds capable of developing into mature plants. Transformation of a plant with the vector in the present invention can be carried out by a transformation technique known to a person skilled in the art. The microorganism is preferably transformed with Agrobacterium, microprojectile bombardment, electroporation, PEG-mediated fusion, microinjection, liposome (liposome) method, mediated method, In planta transformation, Vacuum infiltration method, floral meristem dipping method, and Agrobacteria spraying method can be used. And more preferably, a transformation method using Agrobacterium can be used.

본 발명에서 상기 식물체는 벼, 밀, 보리, 옥수수, 콩, 감자, 밀, 팥, 귀리 또는 수수를 포함하는 식량 작물류; 애기장대, 배추, 무, 고추, 딸기, 토마토, 수박, 오이, 양배추, 참외, 호박, 파, 양파 또는 당근을 포함하는 채소 작물류; 인삼, 담배, 목화, 참깨, 사탕수수, 사탕무우, 들깨, 땅콩 또는 유채를 포함하는 특용작물류; 사과나무, 배나무, 대추나무, 복숭아, 양다래, 포도, 감귤, 감, 자두, 살구 또는 바나나를 포함하는 과수류; 장미, 글라디올러스, 거베라, 카네이션, 국화, 백합 또는 튤립을 포함하는 화훼류; 및 라이그라스, 레드클로버, 오차드그라스, 알파알파, 톨페스큐 또는 페레니얼라이그라스를 포함하는 사료작물류로 이루어진 군으로부터 선택된 어느 하나이며, 바람직하게는 인삼, 담배, 목화, 참깨, 사탕수수, 사탕무우, 들깨, 땅콩 또는 유채를 포함하는 특용작물류이며, 더욱 바람직하게는 담배이나, 이에 제한되지 않는다. In the present invention, the plant is a food crop including rice, wheat, barley, corn, soybean, potato, wheat, red bean, oats or millet; Vegetable crops including Arabidopsis, cabbage, radish, pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, squash, onions, onions or carrots; Special crops including ginseng, tobacco, cotton, sesame, sugar cane, beet, perilla, peanut or rapeseed; Fruit trees including apple trees, pears, jujube trees, peaches, sheep grapes, grapes, citrus fruits, persimmons, plums, apricots or bananas; Roses, gladioluses, gerberas, carnations, mums, lilies or tulips; And feed crops including raglass, red clover, orchardgrass, alpha-alpha, tall fescue or perennial rice, preferably ginseng, tobacco, cotton, sesame, sugarcane, beet, Perilla, peanut or rapeseed, more preferably cigarette, but not limited thereto.

본 발명의 구현에에 따르면, 상기 발현벡터를 이용하여 식물체를 형질전환하는 방법을 제공한다.According to an embodiment of the present invention, there is provided a method for transforming a plant using the expression vector.

본 발명에서 용어 "유전자의 발현"은 유전자의 전사를 의미하며, 구체적으로는 mRNA로의 전사를 의미한다. 상기 "전사"는 DNA의 전사 및 생성된 mRNA 산물의 가공을 포함한다. 본 발명에서, 상기 용어 "향상"은 본 발명에서 정의된 대조군 식물과 비교하여 적어도 5% 또는 10%, 바람직하게는 적어도 20% 또는 40%, 더욱 바람직하게는 50%, 60%, 70% 또는 80% 이상의 내건성을 의미하며, 식물이 원래 가지고 있는 내건성에 비해 상기 유전자를 도입하여 내건성이 증가되는 것을 의미한다. The term "expression of a gene" in the present invention means transcription of a gene, and specifically, transcription into mRNA. Said "transcription" involves the transcription of DNA and the processing of the resulting mRNA product. In the present invention, the term " enhancement "is intended to mean at least 5% or 10%, preferably at least 20% or 40%, more preferably 50%, 60%, 70% Means 80% or more resistant to weathering, and means that the resistance to harshness is increased by introducing the gene as compared with the original resistance of the plant.

본 발명의 하나의 구현예에 따르면, 서열번호 1의 염기서열을 가지는 액티빈 A의 유전자를 포함하는 식물체 발현용 발현벡터로 식물체를 형질전환시킨 다음 배양하여 액티빈 A 단백질을 생산하는 방법을 제공한다. 상기 형질전환 및 배양에는 목적하는 재조합 액티빈 A 단백질을 고수율로 효과적으로 발현시킬 수 있기만 하면 유전공학 분야에서 통상적인 어떠한 방법이라도 사용될 수 있다. According to one embodiment of the present invention, there is provided a method for producing an Actin A protein by transforming a plant with an expression vector for plant expression comprising a gene of Actin A having the nucleotide sequence of SEQ ID NO: 1, followed by culturing do. Any method known in the field of genetic engineering can be used for the transformation and culturing as long as the desired recombinant actin A protein can be efficiently expressed at a high yield.

이와 같이, 본 발명에 따라 액티빈 A 단백질을 식물체서 생산하기 위한 유전자 발현 구성물을 설계하기 위하여 단계적 실험을 수행하여 최종적으로 35S 프로모터에 의해 직접 유도되는 액티빈 A 구성물을 제조하여 담배 식물체에 형질전환시켜 안정된 형질전환 식물체를 제조한 하여 형질전환 담배식물체를 제공하여 분자농업을 통해 산업적으로 사용할 액티빈 A 단백질을 생산하는데 사용할 수 있다.Thus, in order to design gene expression constructs for producing Actin A protein in plants according to the present invention, step-by-step experiments were conducted to finally produce actin A constructs directly induced by the 35S promoter, To provide a transgenic tobacco plant, which can be used to produce industrially useful actin A protein via molecular agriculture.

도 1은 액티빈 A 유전자의 염기서열과 이로부터 유도되는 아미노산 서열을 나타낸 결과이다.
도 2는 액티빈 A 유전자를 포함하는 pUC118-Activin A 벡터의 모식도를 나타낸 것이다.
도 3은 고효율로 발현되는 재조합 단백질 발현용 시스템 (pTRAkt vector)에 재조합 액티빈 A 유전자가 35S 프로모터에 의해 발현되도록 디자인한 35S:: Activin A construct의 모식도와 클로닝을 확인한 결과이다.
도 4는 아그로박테리움(Agrobacterium)에 형질전환된 액티빈 A 유전자를 확인한 결과이다 (M, 1kb marker; lane 4~9, 아그로박테리움에서 분리한 재조합 액티빈 A 플라스미드 DNA; lane 10, 재조합 액티빈 A 플라스미드 DNA).1 shows the nucleotide sequence of the Actin A gene and the amino acid sequence derived therefrom.
Fig. 2 is a schematic diagram of a pUC118-Activin A vector containing an Actin A gene.
FIG. 3 shows a schematic and cloning result of a 35S :: Activin A construct designed to express a recombinantactivin A gene by a 35S promoter in a highly efficient expression system for recombinant protein expression (pTRAkt vector).
Figure 4 shows the results of confirming the actin A gene transfected into Agrobacterium (M, 1 kb marker; lane 4 to 9, recombinant Actin A plasmid DNA isolated from Agrobacterium; lane 10, recombinant solution Tin A plasmid DNA).

이하, 실시예 등을 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예 등은 단지 본 발명을 보다 구체적으로 설명하기 위한 것으로서, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예 등에 의해 제한되지 않는다는 것은 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자에게 있어서 자명할 것이다. Hereinafter, the present invention will be described in more detail by way of examples and the like. It is to be understood that the present invention is not limited by these examples in view of the spirit and scope of the present invention, It will be obvious to you.

<< 실시예Example 1>  1> 액티빈Activin A 유전자의 분리 Isolation of A gene

인간 액티빈 A 유전자를 분리하기 위하여 ㈜엔비엠 권태호 대표가 제조한 액티빈 A 유전자를 PCR을 통해 분리하여 사용하였다. NcoI 제한효소 site를 포함한 포워드 방향 프라이머와 XbaI 제한효소 site를 포함한 리버스 방향 프라이머를 주문 제조하였다.In order to isolate the human Actin A gene, actin A gene, which was produced by President Kwon Tae-ho of ENVIM, Inc., was isolated by PCR. A reverse direction primer including a forward direction primer containing an NcoI restriction site and an XbaI restriction site was customized.

Figure pat00001
Figure pat00001

각각의 유전자 특이적인 프라이머를 주문 제조한 후 PCR 반응은 94℃에서 5분간 전변성(pre-denaturation) 시킨 후, 94℃에서 30초간 변성(denaturation), 60℃에서 30초간 어닐링(annealing), 72℃에서 1분간 익스텐션(extension) 과정을 35 cycles로 하였으며 1% (w/v) 아가로오즈 겔 상 에서 증폭된 밴드를 확인한 결과, 예상된 DNA 크기의 1,335bp의 PCR 산물을 확인하고 최종적으로 염기서열분석을 의뢰하여 인간 액티빈 A 유전자를 획득하였다.PCR was carried out by pre-denaturation at 94 ° C for 5 minutes, followed by denaturation at 94 ° C for 30 seconds, annealing at 60 ° C for 30 seconds, The amplification was carried out on a 1% (w / v) agarose gel, and the PCR product of 1,335 bp of the expected DNA size was confirmed. Finally, Sequence analysis was commissioned to obtain human actin A gene.

획득된 인간 액티빈 A 유전자는 NcoI과 XbaI 제한효소 site를 포함하고 있으므로 식물체 발현용 구성물(construct) 제조가 용이하도록 pUC118 cloning vector (TaKaRa)에 먼저 라이게이션(ligation)하였다. 그 결과, 3,162bp의 벡터 단편과 1,335bp의 인설트(액티빈 A) 단편을 획득하여 구성물의 재조합 유무를 확인하였으며 최종적으로 염기서열분석을 통하여 확인하였다. Since the obtained human Actin A gene contains NcoI and XbaI restriction enzyme sites, the pUC118 cloning vector (TaKaRa) was firstly ligated to facilitate plant expression constructs. As a result, a vector fragment of 3,162 bp and an insulat (activin A) fragment of 1,335 bp were obtained, and the recombination of the constructs was confirmed and finally confirmed by sequencing.

도 2는 액티빈 A 유전자를 포함하는 pUC118-Activin A 벡터의 모식도를 나타낸 것이다. Fig. 2 is a schematic diagram of a pUC118-Activin A vector containing an Actin A gene.

<< 실시예Example 2>  2> 액티빈Activin A 유전자가 도입된 식물체 발현용 벡터 제조 A gene expressing plant expression vector

실제 식물에서 액티빈 A 유전자를 생산하기 위해 분자농업을 활용하기 위해서는 식물에서 안정적으로 액티빈 A가 생산되어야 하므로 식물에서 액티빈 A가 과발현 되도록 재조합 유전자 구성물을 제조하였다. 재조합 단백질 발현용 시스템 (pTRAkt vector)에 full-length의 인간 액티빈 A 유전자를 도입하도록 유전자를 디자인하였다 (도 3A). 또한 액티빈 A 단백질의 정제를 용이하도록 만들기 위하여 His6-tag의 염기서열(CACCATCACCATCACCAT)을 첨가하였다. In order to utilize molecular agriculture for production of actin A gene in actual plants, recombinant gene constructs are prepared so that actin A is overexpressed in plants because plants need to produce actin A stably. The gene was designed to introduce a full-length human actin A gene into the recombinant protein expression system (pTRAkt vector) (Fig. 3A). In addition, the His 6-tag base sequence (CACCATCACCATCACCAT) was added to facilitate the purification of actin A protein.

도 3은 고효율로 발현되는 재조합 단백질 발현용 시스템 (pTRAkt vector)에 재조합 액티빈 A 유전자가 35S 프로모터에 의해 발현되도록 디자인한 35S:: Activin A construct의 모식도와 클로닝을 확인한 결과이다. FIG. 3 shows a schematic and cloning result of a 35S :: Activin A construct designed to express a recombinantactivin A gene by a 35S promoter in a highly efficient expression system for recombinant protein expression (pTRAkt vector).

식물에서 고효율로 발현되는 의약용 단백질을 생산하기 위한 식물세포용 프로모터로는 독일 프라운호퍼 연구소 소장인 R. Fisher 박사와의 논의를 거쳐서 사용하도록 제공받은 변형 35S 프로모터를 갖고 있는 식물발현용 pTRAkt vector를 사용하였다. 이 벡터에 인간 액티빈 A의 구성물(construct)을 제한효소 NcoI과 XbaI 자리가 생긴 형질전환용 벡터에 삽입하여 라이게이션(ligation)하여 식물체 발현용 구성물을 제조하였으며 최종적으로 염기서열까지 확인하여 식물 발현 벡터에 클로닝 되었음을 확인하였다 (도 3B).As a plant cell promoter for producing high-yielding medicinal proteins expressed in plants, a plant-expressing pTRAkt vector having a modified 35S promoter provided for use after discussion with Dr. R. Fisher, the director of the Fraunhofer Institute in Germany, is used Respectively. In this vector, constructs of human Actin A were inserted into restriction enzyme NcoI and the transformation vector in which XbaI site was formed and ligation was carried out to prepare constructs for expressing the plants. Finally, the sequence of the plant was expressed Vector (Fig. 3B).

<< 실시예Example 3>  3> 액티빈Activin A 유전자를 이용한 아그로박테리움( Agrobacterium using A gene ( AgrobacteriumAgrobacterium ) 형질전환) Transformation

인간 액티빈 A 유전자가 도입된 식물발현벡터를 Tri-parental mating 방법으로 아그로박테리움(Agrobacterium tumafaciens GV3101)에 형질전환하여 얻은 콜로니를 배양하여 alkai lysis 방법에 근거한 quick-screen 방법으로 플라스미드 DNA를 분리하여 PCR을 통해 인간 액티빈 A 유전자가 도입되었음을 확인하였다.Plasmid DNA was isolated by quick-screen method based on alkai lysis method by culturing colonies obtained by transformation of plant expression vector into which human actin A gene was introduced into Agrobacterium (A grobacterium tumafaciens GV3101) by tri-parental mating method And it was confirmed that the human actin A gene was introduced by PCR.

도 4는 아그로박테리움(Agrobacterium)에 형질전환된 액티빈 A 유전자를 확인한 결과이다 (M, 1kb marker; lane 4~9, 아그로박테리움에서 분리한 재조합 액티빈 A 플라스미드 DNA; lane 10, 재조합 액티빈 A 플라스미드 DNA). Figure 4 shows the results of confirming the actin A gene transfected into Agrobacterium (M, 1 kb marker; lane 4 to 9, recombinant Actin A plasmid DNA isolated from Agrobacterium; lane 10, recombinant solution Tin A plasmid DNA).

<< 실시예Example 4> 재조합  4> Recombination 액티빈Activin A 유전자가 도입된 형질전환 담배 식물체 제조 Transgenic tobacco plant with A gene introduced

인간 액티빈 A 유전자가 도입된 아그로박테리움 배양액(OD600=1.5)에 야생형 담배 식물체의 잎 절편(0.5cm2)을 1분 동안 감염시킨 후 MS shooting 유도배지(0.1 mg/L NAA + 1 mg/L BAP, pH5.8)에 치상하여 2일 동안 암 배양하였다. 공조배양이 끝난 후 담배 잎 절편은 항생제 cefotaxime (250mg/L)과 kanamycin(100 mg/L)이 첨가된 MS shooting 배지에서 2주 간격으로 계대배양하면서 형질전환된 라인을 선별하였다. 항생제 선발로부터 살아남은 형질전환체는 뿌리 유도배지에 옮겨 흙에 순화시켰는데 지속적으로 형질전환 실험을 반복하였다. 항생제 선발로부터 신초가 형성되어 살아남은 형질전환체는 뿌리 유도배지에 옮겨 뿌리 유도를 수행하여 형질전환담배 식물체를 제조하였다. (0.5 cm 2 ) of a wild-type tobacco plant was infected with Agrobacterium broth (OD 600 = 1.5) in which the human Actin A gene was introduced for 1 minute, followed by addition of MS shooting induction medium (0.1 mg / L NAA + 1 mg / L BAP, pH 5.8) and cultured for 2 days. After incubation, tobacco leaves were transfected with MS cultivation medium supplemented with antibiotics cefotaxime (250 mg / L) and kanamycin (100 mg / L) at intervals of 2 weeks. Transformants surviving the selection of antibiotics were transferred to roots induction medium and purified to soil, and the transformation experiment was repeated continuously. Transgenic plants surviving the shoot formation from antibiotic selection were transferred to root induction medium and root induction was carried out to produce transgenic tobacco plants.

이제까지 본 발명에 대하여 그 바람직한 실시 예들을 중심으로 살펴보았다. 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자는 본 발명이 본 발명의 본질적인 특성에서 벗어나지 않는 범위에서 변형된 형태로 구현될 수 있음을 이해할 수 있을 것이다. 그러므로 개시된 실시예들은 한정적인 관점이 아니라 설명적인 관점에서 고려되어야 한다. 본 발명의 범위는 전술한 설명이 아니라 특허청구범위에 나타나 있으며, 그와 동등한 범위 내에 있는 모든 차이점은 본 발명에 포함된 것으로 해석되어야 할 것이다.The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

<110> Industry-academic cooperation foundation of sunchon national university <120> Transgenic plants transformed with activin A gene and methods of producing activin A protein by using the same <130> PA-16-0172 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 1335 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence of activin gene <400> 1 ccatggatta ttacatcaaa acaaaaaatg cctttgcttt ggctgagagg ttttctactt 60 gctagttgct ggattattgt gaggtcgtcc ccaactccag gatctgaggg gcactcggcg 120 gcaccggact gcccgtcctg tgcgctcgcc gcgctcccta aggatgtgcc caactctcaa 180 ccagagatgg tggaggccgt taagaagcac atattaaaca tgctgcattt gaagaagcgc 240 cccgatgtta cacagccagt accaaaggct gcgcttctga acgcgatcag aaagctccat 300 gtgggtaaag tcggggagaa tggctacgtg gagatcgagg atgacattgg aaggagggcc 360 gagatgaatg agcttatgga gcaaacctcg gagatcataa cgtttgccga gtcaggaact 420 gcgcgcaaga cgctccattt cgagatttcc aaggaaggct cggatctgtc agtggtggag 480 cgtgctgagg tttggctctt cctaaaagtc cccaaggcca ataggacaag gaccaaagtc 540 accatccgcc tcttccagca acagaagcac ccacagggaa gcctggacac aggcgaagag 600 gcggaggaag tgggcctcaa gggggaaagg agtgaacttc tgctctctga gaaagtagtt 660 gacgctcgga agagcacctg gcatgtcttc cctgtctcca gtagcatcca acgcttgttg 720 gaccagggca agagttccct ggacgttcgg attgcctgtg agcaatgcca ggagagtggg 780 gccagcctag ttttgctggg caagaagaag aagaaagagg aggaggggga agggaagaag 840 aagggcggag gtgagggtgg agctggagct gatgaggaga aggaacagtc gcacagacct 900 ttcctcatgt tacaggcgcg ccagtctgaa gaccaccctc atgacgatga tgacaagggc 960 ctcgaatgcg acggcaaggt caacatctgc tgcaagaaac agttctttgt cagtttcaag 1020 gacatcggct ggaatgactg gatcattgct ccgtctggct atcatgccaa ctactgcgag 1080 ggtgagtgcc cgagccatat agctggcact tccggatctt cactgtcgtt ccactcaaca 1140 gtcatcaacc actaccggat gcgaggccac agcccctttg ccaatctcaa atcatgctgt 1200 gtgccgacca agctccggcc aatgtctatg ttgtactatg atgatggtca aaatatcatc 1260 aagaaggata ttcaaaacat gatcgtggag gagtgtgggt gctcacacca tcaccatcac 1320 cattgatagt ctaga 1335 <210> 2 <211> 440 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of activin gene <400> 2 Leu Arg Ser Gln Gly Ser Arg Ile Ile Thr Ser Lys Gln Lys Met Pro 1 5 10 15 Leu Leu Trp Leu Arg Gly Phe Leu Leu Ala Ser Cys Trp Ile Ile Val 20 25 30 Arg Ser Ser Pro Thr Pro Gly Ser Glu Gly His Ser Ala Ala Pro Asp 35 40 45 Cys Pro Ser Cys Ala Leu Ala Ala Leu Pro Lys Asp Val Pro Asn Ser 50 55 60 Gln Pro Glu Met Val Glu Ala Val Lys Lys His Ile Leu Asn Met Leu 65 70 75 80 His Leu Lys Lys Arg Pro Asp Val Thr Gln Pro Val Pro Lys Ala Ala 85 90 95 Leu Leu Asn Ala Ile Arg Lys Leu His Val Gly Lys Val Gly Glu Asn 100 105 110 Gly Tyr Val Glu Ile Glu Asp Asp Ile Gly Arg Arg Ala Glu Met Asn 115 120 125 Glu Leu Met Glu Gln Thr Ser Glu Ile Ile Thr Phe Ala Glu Ser Gly 130 135 140 Thr Ala Arg Lys Thr Leu His Phe Glu Ile Ser Lys Glu Gly Ser Asp 145 150 155 160 Leu Ser Val Val Glu Arg Ala Glu Val Trp Leu Phe Leu Lys Val Pro 165 170 175 Lys Ala Asn Arg Thr Arg Thr Lys Val Thr Ile Arg Leu Phe Gln Gln 180 185 190 Gln Lys His Pro Gln Gly Ser Leu Asp Thr Gly Glu Glu Ala Glu Glu 195 200 205 Val Gly Leu Lys Gly Glu Arg Ser Glu Leu Leu Leu Ser Glu Lys Val 210 215 220 Val Asp Ala Arg Lys Ser Thr Trp His Val Phe Pro Val Ser Ser Ser 225 230 235 240 Ile Gln Arg Leu Leu Asp Gln Gly Lys Ser Ser Leu Asp Val Arg Ile 245 250 255 Ala Cys Glu Gln Cys Gln Glu Ser Gly Ala Ser Leu Val Leu Leu Gly 260 265 270 Lys Lys Lys Lys Lys Glu Glu Glu Gly Glu Gly Lys Lys Lys Gly Gly 275 280 285 Gly Glu Gly Gly Ala Gly Ala Asp Glu Glu Lys Glu Gln Ser His Arg 290 295 300 Pro Phe Leu Met Leu Gln Ala Arg Gln Ser Glu Asp His Pro His Asp 305 310 315 320 Asp Asp Asp Lys Gly Leu Glu Cys Asp Gly Lys Val Asn Ile Cys Cys 325 330 335 Lys Lys Gln Phe Phe Val Ser Phe Lys Asp Ile Gly Trp Asn Asp Trp 340 345 350 Ile Ile Ala Pro Ser Gly Tyr His Ala Asn Tyr Cys Glu Gly Glu Cys 355 360 365 Pro Ser His Ile Ala Gly Thr Ser Gly Ser Ser Leu Ser Phe His Ser 370 375 380 Thr Val Ile Asn His Tyr Arg Met Arg Gly His Ser Pro Phe Ala Asn 385 390 395 400 Leu Lys Ser Cys Cys Val Pro Thr Lys Leu Arg Pro Met Ser Met Leu 405 410 415 Tyr Tyr Asp Asp Gly Gln Asn Ile Ile Lys Lys Asp Ile Gln Asn Met 420 425 430 Ile Val Glu Glu Cys Gly Cys Ser 435 440 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Forward primer <400> 3 gggccatgga ttattacatc aaaacaa 27 <210> 4 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer <400> 4 gggtctagac tatcaatggt gatggtg 27 <110> Industry-academic cooperation foundation of sunchon national university <120> Transgenic plants transformed with activin gene and methods of          producing activin protein by using the same <130> PA-16-0172 <160> 4 <170> KoPatentin 3.0 <210> 1 <211> 1335 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence of activin gene <400> 1 ccatggatta ttacatcaaa acaaaaaatg cctttgcttt ggctgagagg ttttctactt 60 gctagttgct ggattattgt gaggtcgtcc ccaactccag gatctgaggg gcactcggcg 120 gcaccggact gcccgtcctg tgcgctcgcc gcgctcccta aggatgtgcc caactctcaa 180 ccagagatgg tggaggccgt taagaagcac atattaaaca tgctgcattt gaagaagcgc 240 cccgatgtta cacagccagt accaaaggct gcgcttctga acgcgatcag aaagctccat 300 gtgggtaaag tcggggagaa tggctacgtg gagatcgagg atgacattgg aaggagggcc 360 gagatgaatg agcttatgga gcaaacctcg gagatcataa cgtttgccga gtcaggaact 420 gcgcgcaaga cgctccattt cgagatttcc aaggaaggct cggatctgtc agtggtggag 480 cgtgctgagg tttggctctt cctaaaagtc cccaaggcca ataggacaag gaccaaagtc 540 accatccgcc tcttccagca acagaagcac ccacagggaa gcctggacac aggcgaagag 600 gcggaggaag tgggcctcaa gggggaaagg agtgaacttc tgctctctga gaaagtagtt 660 gacgctcgga agagcacctg gcatgtcttc cctgtctcca gtagcatcca acgcttgttg 720 gaccagggca agagttccct ggacgttcgg attgcctgtg agcaatgcca ggagagtggg 780 gccagcctag ttttgctggg caagaagaag aagaaagagg aggaggggga agggaagaag 840 aagggcggag gtgagggtgg agctggagct gatgaggaga aggaacagtc gcacagacct 900 ttcctcatgt tacaggcgcg ccagtctgaa gaccaccctc atgacgatga tgacaagggc 960 ctcgaatgcg acggcaaggt caacatctgc tgcaagaaac agttctttgt cagtttcaag 1020 gacatcggct ggaatgactg gatcattgct ccgtctggct atcatgccaa ctactgcgag 1080 ggtgagtgcc cgagccatat agctggcact tccggatctt cactgtcgtt ccactcaaca 1140 gtcatcaacc actaccggat gcgaggccac agcccctttg ccaatctcaa atcatgctgt 1200 gtgccgacca agctccggcc aatgtctatg ttgtactatg atgatggtca aaatatcatc 1260 aagaaggata ttcaaaacat gatcgtggag gagtgtgggt gctcacacca tcaccatcac 1320 cattgatagt ctaga 1335 <210> 2 <211> 440 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of activin gene <400> 2 Leu Arg Ser Gln Gly Ser Arg Ile Ile Thr Ser Lys Gln Lys Met Pro   1 5 10 15 Leu Leu Trp Leu Arg Gly Phe Leu Ala Ser Cys Trp Ile Ile Val              20 25 30 Arg Ser Ser Pro Thr Pro Gly Ser Glu Gly His Ser Ala Ala Pro Asp          35 40 45 Cys Pro Ser Cys Ala Leu Ala Ala Leu Pro Lys Asp Val Pro Asn Ser      50 55 60 Gln Pro Glu Met Val Glu Ala Val Lys Lys His Ile Leu Asn Met Leu  65 70 75 80 His Leu Lys Lys Arg Pro Asp Val Thr Gln Pro Val Pro Lys Ala Ala                  85 90 95 Leu Leu Asn Ala Ile Arg Lys Leu His Val Gly Lys Val Gly Glu Asn             100 105 110 Gly Tyr Val Glu Ile Glu Asp Asp Ile Gly Arg Arg Ala Glu Met Asn         115 120 125 Glu Leu Met Glu Gln Thr Ser Glu Ile Ile Thr Phe Ala Glu Ser Gly     130 135 140 Thr Ala Arg Lys Thr Leu His Phe Glu Ile Ser Lys Glu Gly Ser Asp 145 150 155 160 Leu Ser Val Val Glu Arg Ala Glu Val Trp Leu Phe Leu Lys Val Pro                 165 170 175 Lys Ala Asn Arg Thr Arg Thr Lys Val Thr Ile Arg Leu Phe Gln Gln             180 185 190 Gln Lys His Pro Gln Gly Ser Leu Asp Thr Gly Glu Glu Ala Glu Glu         195 200 205 Val Gly Leu Lys Gly Glu Arg Ser Glu Leu Leu Leu Ser Glu Lys Val     210 215 220 Val Asp Ala Arg Lys Ser Thr Trp His Val Phe Pro Val Ser Ser Ser 225 230 235 240 Ile Gln Arg Leu Leu Asp Gln Gly Lys Ser Ser Leu Asp Val Arg Ile                 245 250 255 Ala Cys Glu Gln Cys Gln Glu Ser Gly Ala Ser Leu Val Leu Leu Gly             260 265 270 Lys Lys Lys Lys Lys Glu Glu Glu Gly Glu Gly Lys Lys Lys Gys Gly         275 280 285 Gly Glu Gly Gly Ala Gly Ala Asp Glu Glu Lys Glu Gln Ser His Arg     290 295 300 Pro Phe Leu Met Leu Gln Ala Arg Gln Ser Glu Asp His Pro His Asp 305 310 315 320 Asp Asp Lys Gly Leu Glu Cys Asp Gly Lys Val Asn Ile Cys Cys                 325 330 335 Lys Lys Gln Phe Phe Val Ser Phe Lys Asp Ile Gly Trp Asn Asp Trp             340 345 350 Ile Ile Ala Pro Ser Gly Tyr His Ala Asn Tyr Cys Glu Gly Glu Cys         355 360 365 Pro Ser His Ile Ala Gly Thr Ser Gly Ser Ser Leu Ser Phe His Ser     370 375 380 Thr Val Ile Asn His Tyr Arg Met Met Gly His Ser Pro Phe Ala Asn 385 390 395 400 Leu Lys Ser Cys Cys Val Pro Thr Lys Leu Arg Pro Met Ser Met Leu                 405 410 415 Tyr Tyr Asp Asp Gly Gln Asn Ile Ile Lys Lys Asp Ile Gln Asn Met             420 425 430 Ile Val Glu Glu Cys Gly Cys Ser         435 440 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Forward primer <400> 3 gggccatgga ttattacatc aaaacaa 27 <210> 4 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer <400> 4 gggtctagac tatcaatggt gatggtg 27

Claims (6)

서열번호 1의 염기서열을 가지는 액티빈 A 유전자를 포함하는 식물체 발현용 발현벡터.An expression vector for expressing a plant comprising an Actin A gene having a nucleotide sequence of SEQ ID NO: 1. 제 1 항에 있어서,
pTRAkt-Activin A 발현벡터.The method according to claim 1,
pTRAkt-Activin A expression vector. 제 1 항에 따른 액티빈 A 유전자를 포함하는 식물체 발현용 발현벡터로 형질전환된 식물체.A plant transformed with an expression vector for plant expression comprising the Actin A gene according to claim 1. 제 4 항에 있어서,
상기 식물체가 담배인 형질전환된 식물체.5. The method of claim 4,
Wherein said plant is tobacco. 제 1 항에 따른 액티빈 A의 유전자를 포함하는 식물체 발현용 발현벡터를 이용하여 식물체를 형질전환하는 방법. A method for transforming a plant using an expression vector for plant expression comprising the gene of Actin A according to claim 1. 제 1 항에 따른 액티빈 A의 유전자를 포함하는 식물체 발현용 발현벡터로 식물체를 형질전환한 다음 배양하여 재조합 액티빈 A 단백질을 생산하는 방법. A method for producing a recombinant actin A protein by transforming a plant with an expression vector for plant expression comprising the gene of Actin A according to claim 1 and culturing the same.
KR1020160119795A 2016-09-20 2016-09-20 Transgenic plants transformed with activin A gene and methods of producing activin A protein by using the same KR20180031326A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109293764A (en) * 2018-10-26 2019-02-01 中国农业科学院特产研究所 Deer subfamily activin A albumen and its preparation and application

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109293764A (en) * 2018-10-26 2019-02-01 中国农业科学院特产研究所 Deer subfamily activin A albumen and its preparation and application
CN109293764B (en) * 2018-10-26 2021-11-16 中国农业科学院特产研究所 Lu's subfamily activin A protein and preparation and application thereof

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