KR20180054373A - A transformant comprising MYH3 gene and use thereof - Google Patents
A transformant comprising MYH3 gene and use thereof Download PDFInfo
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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Abstract
Description
본 발명은 MYH3 유전자를 포함하는 형질전환체 및 이의 이용에 관한 것으로, 구체적으로 서열번호 1의 염기서열로 이루어진 MYH3 유전자를 포함하는 재조합 벡터를 인간을 제외한 개체의 수정란에 형질전환시키는 단계를 포함하는 형질전환체의 육질을 향상시키는 방법에 관한 것이다.The present invention relates to a transformant comprising a MYH3 gene and a use thereof, and more particularly to a method for transforming a fertilized egg of a human, except for a human, comprising a recombinant vector comprising a MYH3 gene consisting of the nucleotide sequence of SEQ ID NO: 1 And a method for improving the meat quality of a transformant.
돼지는 사람이 필요로 하는 단백질을 제공하는 가장 기본적인 가축으로서, 약 9000년 전에 인도의 동부 지방에서 사육된 이래로, 각각의 시대와 상황 및 사람들의 기호에 따라 사육되어 왔다. 돼지의 육질과 육량을 결정하는 요인에는 여러 가지가 있는데, 이 중에서도 근내지방 함량의 수준은 육질, 등심단면적은 육량을 평가하는 가장 중요한 척도이며, 적색육은 소비자가 가장 선호하는 고기 색깔로서 구매 시 우선적으로 눈에 띄는 육질 형질이기도 하다.Pigs are the most basic livestock that provide the protein they need, since they were raised in the eastern part of India about 9,000 years ago, and have been breed in accordance with the times and circumstances and the preferences of the people. There are many factors that determine meat quality and weight of pigs. Among them, meat level is the most important measure of muscle fat content and meat weight is the most important measure of meat weight. Red meat is the favorite meat color of consumers. It is also a predominantly meat quality trait.
이러한 상황에 맞추어 시판용 비육돈을 생산하기 위해 일당증체량, 면역능력, 산자수, 모유량, 육질 등에서 우수한 형질을 갖는 품종을 교배하여 돼지 신품종을 만들어 왔으며, 그 수는 현재 200여 종에 이른다. 그러나, 원하는 형질을 갖는 품종을 얻기에는 육종 과정이 복잡하며, 오랜 시간이 걸린다는 단점이 있었다. 이에 따라, 유전형을 변형시켜 가축의 육질을 향상시키기 위한 기술의 개발이 활발히 이루어지고 있으며, 그 결과로, 돼지의 육질 형질, 등지방 두께 및 흑모색에 관여하는 유전자의 SNP 형질을 이용하여 품질이 향상된 돼지를 제조하는 방법 등이 개발된 바 있다(한국 등록특허공보 제10-1457942호). 그러나, 여전히 돼지의 육질을 간편하고 효과적으로 향상시킬 수 있는 방법에 대해서는 개발되지 않은 상태이며, 또한, MYH3 유전자를 이용하여 돼지의 육질을 향상시키는 방법에 대해서는 개발된 바 없었다.In order to produce commercially available finishing pigs, breeding pigs with excellent traits such as body weight gain, immune ability, number of litter, milk yield, and meat quality have been breeded to produce new kinds of pigs. However, the breeding process is complicated and takes a long time to obtain the breed having the desired traits. Accordingly, techniques for improving the meat quality of livestock have been actively carried out by modifying the genotype. As a result, the quality of the pigs is improved by using the SNP traits of the genes involved in the meat quality, And an improved method of producing pigs have been developed (Korean Patent Registration No. 10-1457942). However, a method for easily and effectively improving the meat quality of pigs has not yet been developed, and a method for improving the meat quality of pigs using the MYH3 gene has not been developed.
이러한 배경하에, 본 발명자들은 유전 형질을 변형하여 가축의 육질을 향상시킬 수 있는 방법을 개발하고자 예의 연구노력한 결과, MYH3 유전자가 돼지의 육질 형질을 결정하는 유전자임을 규명하였으며, 상기 유전자가 도입된 마우스는 야생형에 비하여 육질의 근내지방함량과 적색도가 향상됨을 확인함에 따라, 상기 유전자를 포함하는 재조합 벡터를 이용하면 육질을 향상시킬 수 있음을 확인하고, 본 발명을 완성하였다.Under these circumstances, the present inventors have made extensive efforts to develop a method for improving the meat quality of livestock by modifying the genetic traits, and as a result, they have found that the MYH3 gene is a gene determining the meat quality of pigs, Of the present invention showed that the meat content and redness of the meat were improved compared to the wild type, and that the meat quality could be improved by using a recombinant vector containing the gene. Thus, the present invention has been completed.
본 발명의 하나의 목적은 서열번호 1의 염기서열로 이루어진 MYH3 유전자를 포함하는 재조합 벡터를 인간을 제외한 개체의 수정란에 형질전환시키는 단계를 포함하는, 형질전환체의 육질을 향상시키는 방법을 제공하는 것이다.One object of the present invention is to provide a method for improving the meat quality of a transformant, which comprises transforming a recombinant vector comprising the MYH3 gene consisting of the nucleotide sequence of SEQ ID NO: 1 into embryos of an individual other than a human will be.
상기 목적을 달성하기 위하여, 본 발명의 하나의 양태는 서열번호 1의 염기서열로 이루어진 MYH3 유전자를 포함하는 재조합 벡터를 인간을 제외한 개체의 수정란에 형질전환시키는 단계를 포함하는, 형질전환체의 육질을 향상시키는 방법을 제공한다.In order to achieve the above object, one aspect of the present invention provides a method for transforming a recombinant vector comprising a MYH3 gene consisting of the nucleotide sequence of SEQ ID NO: 1 into embryos of an individual other than a human, Of the present invention.
본 발명에서는, MYH3 유전자에 의해 돼지의 근내지방함량 및 적색도가 결정됨을 확인하였고, 상기 유전자가 도입된 마우스 형질전환체는 야생형에 비하여 육질의 근내지방함량 및 적색도가 향상됨을 확인함에 따라, 상기 유전자의 mRNA 또는 단백질의 발현 수준을 측정하면 육질을 판단할 수 있음을 확인하여 본 발명을 완성하였다.In the present invention, it was confirmed that the intramuscular fat content and redness of pigs were determined by the MYH3 gene, and that the transgenic mice transfected with the gene showed an improvement in the intramuscular fat content and the redness of the meat compared to the wild type, The present inventors have completed the present invention by confirming that the meat quality can be determined by measuring the expression level of mRNA or protein of the present invention.
본 발명의 용어, "MYH3 유전자"는 동물의 근육을 구성하는 마이오신에 포함된 중쇄단백질의 하나인 myosin heavy chain 3을 코딩하는 유전자를 의미하며, 이의 염기서열은 NCBI의 GenBank 등 공지의 데이터베이스에서 얻을 수 있다(예: GenBank Accession No. XM_013981330). 구체적인 예로, 상기 유전자는 돼지로부터 유래한 유전자일 수 있으나, 이에 제한되는 것은 아니다. 본 발명에서, 상기 유전자는 서열번호 1로 표시되는 폴리뉴클레오티드일 수 있다.The term "MYH3 gene" of the present invention refers to a gene encoding myosin
본 발명의 용어, "재조합 벡터"는 적당한 숙주세포에서 목적 단백질 또는 목적 RNA을 발현할 수 있는 벡터로서, 유전자 삽입물이 발현되도록 작동가능하게 연결된 필수적인 조절요소를 포함하는 유전자 작제물을 말한다.The term "recombinant vector" of the present invention refers to a gene construct containing an essential regulatory element operably linked to express a desired gene or target RNA in a suitable host cell, such that the gene insert is expressed.
본 발명의 재조합 벡터는 상기 MYH3 유전자를 포함하는데, 구체적으로 도 6의 개열지도로 표시되는 것일 수 있으며, 더욱 구체적으로 서열번호 2의 염기서열로 이루어진 폴리뉴클레오티드를 포함하는 것일 수 있다.The recombinant vector of the present invention includes the MYH3 gene, specifically, it may be represented by a cleavage map of FIG. 6, and more specifically, may comprise a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2.
또한, 상기 재조합 벡터는 CAGGS 프로모터(CAGGS promoter)를 포함하는 것일 수 있고, 더욱 구체적으로 CAGGS 프로모터(CAGGS promoter)가 MYH3 유전자와 작동 가능하게 연결된 것일 수 있다.In addition, the recombinant vector may include a CAGGS promoter (CAGGS promoter), and more specifically, a CAGGS promoter may be operably linked to a MYH3 gene.
본 발명의 용어, "프로모터"는 일반적으로 전사를 개시하는 지점을 포함하는 암호화 부위의 상류 쪽에 위치하는 유전자 부위로, 흔히 유전자 발현을 조절하는 TATA 박스, CAAT 박스 부위, 외부 자극에 반응하여 유전자 발현에 영향을 주는 부위 및 위치와 방향에 상관없이 거의 모든 유전자의 발현을 촉진하는 인핸서(enhancer) 등으로 이루어져 있다. 또한, 이러한 프로모터 중에는 모든 조직에서 상시적으로 발현을 유도하는 프로모터(constitutive promoter) 및 시간 또는 위치 특이적으로 발현을 유도하는 프로모터(inducible promoter)가 있으며, 구체적으로 본 발명에 따른 프로모터는 CAGGS 프로모터일 수 있다.The term "promoter" of the present invention is a gene site located upstream of an encoding site, which generally includes a point at which transcription is initiated. The promoter is a TATA box that controls gene expression, a CAAT box site, And an enhancer which promotes the expression of almost all genes regardless of the position and direction. In addition, among these promoters, there are a constitutive promoter that constantly induces expression in all tissues and an inducible promoter that induces time or location-specific expression. Specifically, the promoter according to the present invention is a CAGGS promoter .
상기 용어, '작동가능하게 연결된'(operably linked)은 일반적인 기능을 수행하도록 핵산 발현조절 서열과 목적하는 단백질 또는 RNA를 코딩하는 핵산 서열이 기능적으로 연결(functional linkage)되어 있는 것을 말한다. 예를 들어 프로모터와 단백질 또는 RNA를 코딩하는 핵산 서열이 작동가능하게 연결되어 코딩하는 핵산 서열의 발현에 영향을 미칠 수 있다. 재조합 벡터와의 작동적 연결은 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 term 'operably linked' refers to a functional linkage between a nucleic acid expression control sequence and a nucleic acid sequence encoding a desired protein or RNA to perform a general function. For example, a promoter and a nucleic acid sequence encoding a protein or RNA can affect the expression of a nucleic acid sequence that is operably linked. The operative linkage with the recombinant vector can be produced using genetic recombination techniques well known in the art, and site-specific DNA cleavage and linkage are made using enzymes generally known in the art.
본 발명의 용어, "개체"는 마우스, 돼지, 닭, 소 등의 모든 동물을 의미한다. 구체적인 예로, 포유류, 조류 등의 가축일 수 있으나, 근육 및 지방 등으로 구성된 식육을 갖는 한, 이에 제한되는 것은 아니다. 본 발명에서, 상기 개체는 마우스 또는 래트일 수 있으며, 상기 마우스 품종의 구체적인 예로는 C57BL/6n, BCF 하이브리드, FVB/n 등이 있으나, 이에 제한되는 것은 아니다.The term "individual" of the present invention means all animals such as a mouse, a pig, a chicken, a cow, and the like. Specific examples include livestock such as mammals and birds, but the present invention is not limited thereto as long as it has meat and meat composed of muscle and fat. In the present invention, the subject may be a mouse or a rat, and specific examples of the mouse variety include, but are not limited to, C57BL / 6n, BCF hybrid, FVB / n and the like.
본 발명의 용어, "형질전환"은 외부로부터 주어진 DNA에 의하여 생물의 유전적인 성질을 변화시키는 것을 의미한다. 형질전환시키는 방법으로는 종래 알려진 다양한 방법, 구체적인 예로, 미세주입법(microinjection), 전기천공법(electroporation), 입자 분사법(particle bombardment), 정자를 이용하는 방법(sperm-mediated gene transfer), 바이러스 감염법(viral infection), 직접근육주입법(direct muscle injection), 인슐레이터(insulator) 및 트랜스포존(trnasposon)을 이용한 기법 등이 있으며, 당업자라면 발명의 목적에 맞게 적절한 방법을 선택할 수 있을 것이다.The term "transformed" of the present invention means altering the genetic properties of an organism by exogenously given DNA. As a method of transforming, various known methods such as microinjection, electroporation, particle bombardment, sperm-mediated gene transfer, virus infection method, viral infection, direct muscle injection, insulator, and trnasposon. Those skilled in the art will be able to select appropriate methods for the purpose of the invention.
본 발명의 용어, "형질전환체"는 형질전환에 의하여 유전적 성질이 변한 개체를 의미한다. 구체적으로, 마우스, 돼지, 닭, 소 등의 모든 동물, 더더욱 구체적으로 포유류, 조류 등의 가축일 수 있으나, 이에 제한되는 것은 아니다.The term "transformant" of the present invention means an individual whose genetic properties have been changed by transformation. Specifically, it may be any animal such as a mouse, a pig, a chicken, a cow, or more specifically, a mammal, a bird or the like, but is not limited thereto.
본 발명의 용어, "육질"은 뼈를 제외한 도축물의 상태를 나타내는 다양한 표현 형질을 의미하는데, 상기 표현 형질은 특별히 이에 제한되지 않으나, 근내지방함량, 육색, 보수력, 전단력 등이 될 수 있으며, 더욱 구체적으로 근내지방함량 또는 육색이 될 수 있다. 근내지방함량은 근육 내 포함된 지방의 함량, 육색은 육안으로 판단되는 고기의 색, 보수력은 축육이 수분을 유지하는 능력, 전단력은 고기의 찢었을 때 질긴 정도로서, 근내지방함량은 높을수록, 육색은 적색을 나타낼수록, 보수력 및 전단력은 낮을수록 육질의 품질이 높아진다. 이 중에서도, 근내지방함량의 수준은 육질을 평가하는 가장 중요한 척도이며, 육색은 소비자의 신선도 및 선호도 평가에 영향을 주는 주된 요인이다.The term "meat quality" of the present invention means various expression traits indicating the state of the slaughter body except for bones. The expression traits include, but are not limited to, intramuscular fat content, meat color, water holding capacity, shearing force, Specifically, it may be an intramuscular fat content or a meat color. The muscle fat content is the content of fat contained in the muscle, the color is the color of the meat judged by the naked eye, the water holding capacity is the ability of the poultry to maintain moisture, the shear force is tough when the meat is torn, the higher the intramuscular fat content, The lower the water holding capacity and the shearing force, the higher the quality of the meat. Among these, the level of muscle fat content is the most important measure for evaluating meat quality, and meat color is the main factor affecting consumer 's freshness and preference evaluation.
본 발명의 구체적인 일 실시예에서, MYH3 유전자를 포함하는 재조합 벡터를 미세주입법(microinjection)을 이용하여 C57BL/6n 마우스의 수정란에 형질전환시킴으로써 돼지 MYH3 유전자를 발현하는 형질전환 마우스를 제조하였다(도 5 내지 7). 또한, 상기 형질전환 마우스는 야생형 마우스에 비하여 뒷다리 근육에 적색근 섬유 및 지방 조직이 보다 많이 존재하며, 적색근(slow type fibers)에 영향을 미치는 유전자의 mRNA 및 단백질; 및 지방분화 유전자의 mRNA 발현이 월등히 증가함을 확인하였다(도 9 및 10).In a specific embodiment of the present invention, a transgenic mouse expressing the porcine MYH3 gene was prepared by transforming the recombinant vector containing the MYH3 gene into embryos of C57BL / 6n mice using microinjection (Fig. 5 7). In addition, the transgenic mice have more red muscle fibers and adipose tissues in the hind leg muscles than the wild type mice, and mRNAs and proteins of genes that affect the slow type fibers; And mRNA expression of lipogenic genes was significantly increased (Figs. 9 and 10).
이는, MYH3 유전자를 포함하는 재조합 벡터를 이용하여 개체를 형질전환시키면, 형질전환체의 육질을 향상시킬 수 있음을 시사하는 것이다.This suggests that transforming an individual using a recombinant vector containing the MYH3 gene can improve the meat quality of the transformant.
본 발명의 MYH3 유전자를 포함하는 재조합 벡터로 형질전환된 형질전환체는 야생형에 비하여 근내지방함량 및 적색도가 향상되는 효과를 나타내므로, 상기 벡터는 형질전환체의 육질을 향상시키는데 이용될 수 있다.Since the transformant transformed with the recombinant vector containing the MYH3 gene of the present invention has an effect of improving intramuscular fat content and redness as compared with the wild type, the vector can be used to improve the meat quality of the transformant.
도 1은 도입종인 랜드레이스(a)와 재래종인 제주재래돼지(b)의 외모 및 등심사진을 비교한 이미지이다.
도 2는 육질 형질 결정 유전자의 QTL(quantitative trait locus) 영역 확인에 따른 LDLA(linkage-linkage disequilibrium analysis) 결과를 보여주는 그래프이다. a는 제주재래돼지와 랜드레이스 돼지를 교배하여 얻어진 자손(LK축군), b는 제주재래돼지와 듀록 돼지를 교배하여 얻어진 자손(DK축군)에 관한 것이다.
도 3은 육질 형질 결정 유전자의 QTL 영역에 존재하는 유전자를 나타내고 있는 LALD mapping 결과이다.
도 4는 QTL 영역에 존재하는 유전자의 mRNA 발현양을 비교한 그래프이다. a는 등심(최장근, longissimus), b는 후지(대퇴사두근, quadriceps)에 관한 것이다.
도 5는 CAGGS-EGFP-Puro 벡터의 개열지도를 보여준다.
도 6은 MYH3 유전자가 재조합된 CAGGS-MYH3-Flag 발현 벡터의 개열지도 및 상기 벡터 내 유전자의 순서를 보여준다.
도 7은 MYH3 유전자가 재조합된 벡터로 형질전환된 마우스의 MYH3 유전자 활성 및 발현 양상을 보여준다. a는 MYH3 유전자의 전사 활성을 보여주는 이미지이다. P/C는 양성대조군(positive control), 붉은색으로 표시된 숫자(21, 24, 26, 27, 28)는 MYH3 유전자 전사활성을 나타내는 형질전환 마우스, 검정색으로 표시된 숫자(19, 20, 22, 23, 25)는 형질전환은 되었으나, MYH3 유전자의 전사활성이 없는 마우스를 의미한다. b는 MYH3 유전자의 단백질 발현양을 분석한 웨스턴블롯 결과를 나타내는 이미지이다.
도 8은 돼지 MYH3 유전자가 삽입된 형질전환 마우스의 모습이다.
도 9는 야생형 마우스(WT) 및 형질전환 마우스(TG)의 뒷다리 근육 외관(a) 및 근육 조직(b, c)의 모습을 보여주는 이미지이다. b는 뒷다리 근육 조직을 미오신 ATPase로 조직화학염색한 이미지로서, 적색 화살표는 적색근의 일종인 Type 1/oxidative/slow fibers, 청색 화살표는 백색근의 일종인 Type 2a 및 청색 삼각형은 백색근의 일종인 Type 2b의 Type 2/glycolytic/fast fibers를 가리킨다. 스케일바(Scale bar)는 50 ㎛을 의미한다. c는 뒷다리 근육 조직을 Oil red O 염색한 이미지이다. 스케일바는 100 ㎛을 의미하고, 직사각형 내에 존재하는 영역을 확대하여 그 아래에 나타내었다.
도 10은 돼지 MYH3 유전자가 삽입된 형질전환 마우스의 근육 내 유전자 발현 패턴을 확인한 결과를 보여준다. a는 근섬유 type과 관련한 유전자의 mRNA 및 단백질 발현을 qRT-PCR 및 웨스턴블롯을 통해 분석한 결과를 보여준다. 4개월령의 야생형 마우스(n=3)와 형질전환 마우스(n=5)를 이용하였다. b는 뒷다리 근육의 slow-type(좌) 및 fast-type(우) 근육과 관련한 유전자의 mRNA 발현양을 qRT-PCR을 통해 분석한 결과를 보여준다. 4개월령의 야생형 마우스(n=3)와 형질전환 마우스(n=4)를 이용하였다. 결과값은 독립적인 세 번의 실험을 통해 얻었으며, 이를 평균±SEM으로 나타내었다(*P<0.05, **P<0.01). c는 뒷다리 근육을 헤마톡실린 및 에오신으로 염색한 결과를 보여준다. 화살표는 근주막(perimysium), 삼각형은 근내막(endomysium)을 가리킨다. d는 지방분화 유전자의 mRNA 발현양을 qRT-PCR을 통해 분석한 결과를 보여준다.
도 11은 돼지 MYH3 유전자의 구조 및 QTN(quantitative trait nucleotide)을 보여주는 이미지이다.
도 12는 육질에 영향을 미치는 원인 염기변이를 HpyCH4IV 제한효소를 이용하여 절단한 패턴을 보여주는 이미지이다. LANDRACE 품종(q/q)에서 유래한 1/1은 비 절단형, 랜드레이스와 제주재래돼지의 교배 품종(q/Q)에서 유래한 1/2는 절단형, 제주재래돼지 품종(Q/Q)에서 유래한 2/2는 절단형을 보여준다.Fig. 1 is an image comparing appearance and sirloin photographs of the land race (a), which is an introduced species, and Jeju native pig (b), a native species.
FIG. 2 is a graph showing the results of linkage-linkage disequilibrium analysis (LDLA) according to confirmation of a QTL (quantitative trait locus) region of a meat quality trait determining gene. a is a descendant (LK axis group) obtained by crossing Jeju traditional pig and a land race pig, and b is a progeny (DK axis group) obtained by crossing Jeju traditional pig and durok pig.
FIG. 3 shows the result of LALD mapping showing the gene existing in the QTL region of the meat quality trait determining gene.
4 is a graph comparing mRNA expression levels of genes present in the QTL region. a is the sirloin (long root, longissimus), and b is the fuji (quadriceps).
Figure 5 shows a cleavage map of the CAGGS-EGFP-Puro vector.
FIG. 6 shows the cleavage map of the CAGGS-MYH3-Flag expression vector in which the MYH3 gene was recombined and the order of the genes in the vector.
FIG. 7 shows the MYH3 gene activity and expression pattern of a mouse transformed with a recombinant vector of MYH3 gene. a is an image showing the transcriptional activity of the MYH3 gene. P / C is a positive control; numbers marked in red (21, 24, 26, 27, 28) are transgenic mice showing MYH3 gene transcription activity; numbers in black (19, 20, 22, 23 , ≪ / RTI > 25) refers to a mouse that has been transformed but lacks the transcriptional activity of the MYH3 gene. b is an image showing the result of Western blotting analysis of the expression amount of MYH3 gene.
Fig. 8 shows a transformed mouse into which a porcine MYH3 gene is inserted.
Figure 9 is an image showing the appearance of the hind leg muscle (a) and muscle tissue (b, c) of the wild type mouse (WT) and the transgenic mouse (TG). b is a histochemical staining of the hindlimb muscle tissue with myosin ATPase, Type 1 / oxidative / slow fibers, red arrows are Type 1 / oxidative / slow fibers, blue arrows are Type 2a, a type of white muscle, Type 2b /
FIG. 10 shows the results of confirming the intramuscular gene expression pattern of a transgenic mouse into which a porcine MYH3 gene has been inserted. a shows the results of qRT-PCR and Western blot analysis of mRNA and protein expression of genes related to the type of muscle fibers. Four months old wild-type mice (n = 3) and transgenic mice (n = 5) were used. b shows the results of qRT-PCR analysis of mRNA expression levels of genes related to the slow-type (left) and fast-type (right) muscles of the hind limb muscles. Four months old wild-type mice (n = 3) and transgenic mice (n = 4) were used. The results were obtained in three independent experiments, which were expressed as mean ± SEM (* P <0.05, ** P <0.01). c shows the result of staining the hindlimb muscles with hematoxylin and eosin. The arrow indicates the perimysium, and the triangle indicates the endomysium. d shows the results of qRT-PCR analysis of the mRNA expression level of the lipogenic gene.
11 is an image showing the structure and QTN (quantitative trait nucleotide) of the porcine MYH3 gene.
Fig. 12 is an image showing a pattern of cleavage of a causative base mutation affecting meat quality using HpyCH4IV restriction enzyme. 1/1 from the LANDRACE varieties (q / q), 1/2 from the mating varieties (q / Q) of the non-cutting, landrace and Jeju native pigs, ) 2/2 shows the truncated form.
이하 본 발명을 실시예를 통하여 보다 상세하게 설명한다. 그러나 이들 실시예는 본 발명을 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.
실시예 1. 돼지의 육질 형질 육안 비교Example 1. Visual comparison of meat with pig meat
제주재래흑돼지와 랜드레이스 돼지의 육질 형질(근내지방함량 및 적색육)을 비교하기 위하여, 등심의 형태를 분석하였다.In order to compare the meat quality of Korean traditional black pork and land lace pig (intramuscular fat content and red meat), the shape of the sirloin was analyzed.
그 결과, 도 1에서 볼 수 있듯이, 도입종인 랜드레이스의 등심 고기는 백색이면서 창백하나, 제주재래돼지의 등심 고기는 흑모색이면서 적색육이며, 특히 마블링 침착이 우수함을 확인하였다.As a result, as shown in FIG. 1, it was confirmed that the fillet meat of the land race, which was introduced, was whitish and pale, while the fillet meat of Jeju native pig was black and red meat. Especially, marbling deposition was excellent.
상기 결과를 통해, 재주재래돼지는 랜드레이스에 비하여 육질이 우수함을 알 수 있었다.From the above results, it can be seen that the regenerating pigs are superior in meat quality to land races.
실시예Example 2. 돼지의 육질 형질 2. Meat quality of pigs 양적특성자리A quantitative characteristic spot (quantitative trait locus; (quantitative trait locus; QTLQTL ) 분석) analysis
실시예 2-1. 육질 유전자의 QTL 영역 확인Example 2-1. Identification of QTL regions of meat quality genes
돼지의 육질 형질을 결정하는 유전자를 규명하기 위하여, 제주재래돼지와 랜드레이스 돼지를 교배하여 얻어진 자손(LK축군), 및 제주재래돼지와 듀록 돼지를 교배하여 얻어진 자손(DK축군)을 대상으로 돼지의 육질 형질(적색도(a*), 근내지방함량(IMF))에 대해 양적특성자리(quantitative trait locus; QTL) 분석을 수행하였다.(LK axis group) obtained by crossing Jeju traditional pig and land lace pig and a progeny (DK axis group) obtained by crossing Jeju traditional pig and durok pig were examined to identify genes determining the meat quality of pigs. Quantitative trait locus (QTL) analysis was performed on the meat quality (redness (a *), intramuscular fat content (IMF)).
그 결과, 도 2의 a에서 볼 수 있듯이, LK축군에서 수직 점선은 12번 염색체의 661 kb 영역 상에 위치함을 확인하였고, 또한, 도 2의 b에서 볼 수 있듯이, KD축군에서도 수직 점선은 12번 염색체의 661 kb 영역 상에 위치함을 확인하였다.As a result, as shown in FIG. 2 (a), it was confirmed that the vertical dotted line in the LK axis group was located on the 661 kb region of
상기 결과를 통해, 적색육(적색도)과 근내지방함량을 결정하는 유전자는 돼지의 품종에 상관없이 동일한 위치에 존재함을 확인하였고, 이는 12번 염색체의 661kb 영역 내에 존재함을 알 수 있었다.From the above results, it was confirmed that the genes determining the red meat (redness) and intramuscular fat content were present in the same position irrespective of the variety of pigs, and they were found to exist within the 661 kb region of
실시예 2-2. 돼지 육질 형질 유전자 규명Example 2-2. Identification of Pig Flesh Transcript Genes
상기 실시예 2-1를 통해 돼지 육질 형질을 결정하는 유전자는 12번 염색체의 661kb 영역 내에 존재함을 확인함에 따라, 상기 영역 내에 존재하는 유전자를 LALD mapping을 통해 확인하였다.As a result of confirming that the gene for determining porcine meat quality was present within the 661 kb region of
그 결과, 도 3에서 볼 수 있듯이, MYH3, MYH1, MYH2, MYH13, ADPRM, SCO1, TMEM220, ENSSSCG00000029441, ENSSSCG00000018006의 9개 유전자가 상기 육질 형질 유전자 영역 내에 존재함을 확인하였다.As a result, as shown in FIG. 3, it was confirmed that nine genes of MYH3, MYH1, MYH2, MYH13, ADPRM, SCO1, TMEM220, ENSSSCG00000029441 and ENSSSCG00000018006 were present in the above meaty trait gene region.
실시예 2-3. 돼지 육질 형질 관련 유전자 선발Examples 2-3. Selection of genes related to pig meat quality traits
상기 실시예 2-2를 통해 육질 형질을 결정하는 9개 유전자를 확인함에 따라, 상기 유전자 중에서도 육질 형질의 결정에 가장 관련성이 높은 유전자를 선발하고자 하였다.By confirming the nine genes determining the meat quality trait through the above Example 2-2, the genes most relevant to the determination of the meat quality traits among the genes were selected.
랜드레이스(LANDRACE)와 재래돼지(KNP)의 등심(최장근, longissimus) 및 후지(대퇴사두근, quadriceps)에서, 상기 9개 유전자의 상대적인 mRNA 발현양을 분석하였다.Relative mRNA expression levels of the nine genes were analyzed in LANDRACE and KNP loin (longissimus, longissimus) and Fuji (quadriceps).
구체적으로, 재래돼지(LANDRACE)와 랜드레이스로 등심과 후지를 근육조직을 채취하였고, Trizol 시약(Ambion)을 이용하여 RNA를 분리하였다. 각 조직별 RNA농도를 5 ㎍으로 동일하게 맞춘 후 TOPscript cDNA 합성 키트(Enzynomics)를 이용하여 cDNA(complement DNA)를 합성하였다. 이후 각 조직별 cDNA를 이용하여 qRT-PCR 실험을 수행하였다. qRT-PCR 실험은 95℃에서 20초, 60℃에서 20초, 72℃에서 20초를 40회 반복하였으며 qRT-PCR 실험 수행을 위해 QuatiTect SYBR Green PCR 키트(Qiagen)를 사용하였고, Roter-Gene Q thermal cycler(Qiagen) 장비를 이용하여 실시간으로 분석하였다. 총 9개의 유전자 분석을 위해 사용한 프라이머는 하기 표 1에 나열하였다.Specifically, muscle tissue was sampled from the pork loin (Landrace) and the land race, and RNA was isolated using Trizol reagent (Ambion). After adjusting the RNA concentration of each tissue to 5 ㎍, cDNA (complement DNA) was synthesized using TOPscript cDNA synthesis kit (Enzynomics). Then, qRT-PCR experiment was performed using cDNA of each tissue. The qRT-PCR experiment was repeated 40 times at 95 ° C for 20 seconds, at 60 ° C for 20 seconds, and at 72 ° C for 20 seconds. The QRT-PCR experiment was performed using the QuatiTect SYBR Green PCR kit (Qiagen) and analyzed in real time using a thermal cycler (Qiagen) instrument. Primers used for a total of 9 gene analyzes are listed in Table 1 below.
그 결과, 도 4의 a 및 b에서 볼 수 있듯이, 랜드레이스에 비하여, 재래돼지의 등심 및 후지에서 MYH3 유전자의 mRNA 발현이 월등히 높음을 확인하였다. As a result, as shown in Figs. 4A and 4B, mRNA expression of the MYH3 gene was significantly higher in the sirloin and fuji of the native pig than in the landrace.
상기 결과를 통해, MYH3 유전자가 돼지의 육질 형질 중에서도 특히, 적색도와 근내지방함량을 결정하는 주요 유전자임을 알 수 있었다.From the above results, it was found that the MYH3 gene is a major gene that determines the red color and the intramuscular fat content, among the meat quality traits of pigs.
실시예 3. 돼지 MYH3 유전자가 삽입된 형질전환 마우스의 제작Example 3: Construction of transgenic mouse into which porcine MYH3 gene was inserted
상기 실시예 2를 통해 MYH3 유전자가 돼지의 육질 형질 중에서도 특히, 적색도와 근내지방함량을 결정하는 유전자임이 규명됨에 따라, 상기 유전자의 생체 내(in vivo) 활성을 확인하기 위하여, 하기 3-1 내지 3-2에 따른 방법으로 돼지 MYH3 유전자가 삽입된 형질전환 마우스를 제작하여 이를 이용하였다.In order to confirm the in vivo activity of the gene, the MYH3 gene was identified as a gene determining the redness and muscle fat content among the meaty traits of pigs in Example 2, Transgenic mice transfected with the porcine MYH3 gene were prepared by the method according to 3-2.
실시예 3-1. 형질전환 벡터의 제작Example 3-1. Production of Transformation Vector
먼저, 돼지 MYH3 유전자가 삽입된 형질전환 마우스를 제작하고자, MYH3 유전자가 삽입된 재조합 CAGGS-MYH3-Flag 발현벡터를 제조하였다. First, a recombinant CAGGS-MYH3-Flag expression vector into which a MYH3 gene was inserted was prepared in order to construct a transgenic mouse into which a porcine MYH3 gene was inserted.
구체적으로, 돼지 MYH3를 과발현하는 벡터를 제작하기 위해, 돼지 MYH3 유전자의 mRNA 전체 염기서열을 확인한 후, 총 4부분으로 나누어 DNA 단편을 각각 인위적으로 합성하였다. 첫 번째 단편은 1,417bp로 양쪽에 XbaI과 BglII 효소 site를 양끝 단면에 추가하여 인위적 합성하였다. 두 번째 단편은 1,745bp로 BglII와 SalI를 합성하였으며, 세 번째 단편은 1,777bp로 SalI와 SacII를 합성하였고, 마지막 네 번째 944bp로 SacII와 EcoRI으로 합성하였다. 완성된 4개의 DNA 단편을 말단에 인위적으로 결합시킨 효소 site를 이용하여 결합(Ligation)시켜 최종적으로 돼지 MYH3 유전자의 전체 mRNA를 포함하는 유전자 단편을 완성할 수 있었다.Specifically, in order to prepare a vector overexpressing the porcine MYH3, the whole base sequence of the mRNA of the porcine MYH3 gene was confirmed, and the DNA fragments were artificially synthesized by dividing into four portions in total. The first fragment was 1,417 bp, which was artificially synthesized by adding XbaI and BglII enzyme sites on both ends to both ends. BglII and SalI were synthesized at 1,745 bp for the second fragment, SalI and SacII at 1,777 bp for the third fragment, and SacII and EcoRI at the fourth and final 944 bp. The completed four DNA fragments were ligated using an enzyme site artificially bound to the ends to finally complete a gene fragment containing the entire mRNA of the porcine MYH3 gene.
이후 과발현 벡터에 도입하기 위해 도 5에 나타낸 바와 같은 CAGGS-EGFP-Puro 벡터를 XbaI과 EcoRI으로 절단한 후, 앞서 제작한 돼지 MYH3 유전자 mRNA의 전체 단편을 삽입시킴으로써 최종적으로 돼지 MYH3 형질전환벡터를 제작을 완료하였다(도 6).Then, the CAGGS-EGFP-Puro vector as shown in Fig. 5 was digested with XbaI and EcoRI for introduction into the overexpressed vector, and then a whole fragment of the prepared porcine MYH3 gene mRNA was inserted to finally produce a porcine MYH3 transformant vector (Fig. 6).
상기 벡터의 구조는 6에 나타내었으며, 벡터의 염기 서열은 서열번호 2로 표시하였다.The structure of the vector is shown in FIG. 6, and the nucleotide sequence of the vector is shown in SEQ ID NO: 2.
실시예 3-2. 형질전환 마우스의 제작Example 3-2. Construction of transgenic mice
실시예 3-2-1. 형질전환 마우스의 제작 방법Example 3-2-1. How to make transgenic mice
상기 실시예 3-1을 통해 제작한 벡터를 이용하여 형질전환 마우스를 제작하였다. Transgenic mice were constructed using the vectors prepared in Example 3-1.
구체적으로, C57BL/6n 마우스의 수정란을 획득한 후 미세주입기법(microinjection)을 사용하여 상기에 제작된 형질전환 벡터를 수정란의 핵 안으로 도입하였다. Specifically, embryos of C57BL / 6n mice were obtained and the transformed vectors prepared above were introduced into the nucleus of embryos using microinjection.
그 결과, 총 47마리의 F0 founder를 확인하였다.As a result, a total of 47 F0 founders were identified.
실시예 3-2-2. mRNA 발현 분석을 통한 외래 유전자 도입 여부 확인Example 3-2-2. mRNA expression analysis to confirm the introduction of foreign gene
상기 실시예 3-2-1을 통해 제작한 형질전환 마우스에 MYH3 유전자가 도입되었는지 확인하기 위해, 상기 유전자의 mRNA 발현 여부를 분석하였다.In order to confirm whether the MYH3 gene was introduced into the transgenic mouse prepared in Example 3-2-1, mRNA expression of the gene was analyzed.
구체적으로, PCR 실험 조건은 95℃에서 30초, 60℃에서 30초, 72℃에서 30초를 40회 반복하였으며, 사용한 프라이머는 하기 표 2에 나열하였다.Specifically, PCR conditions were repeated 40 times at 95 ° C for 30 seconds, at 60 ° C for 30 seconds, and at 72 ° C for 30 seconds, and the primers used were listed in Table 2 below.
그 결과, 도 7의 a에서 볼 수 있듯이, MYH3 유전자가 삽입되어 mRNA를 발현하는 형질전환 마우스(21, 24, 26, 27, 28번)를 확인할 수 있었다. As a result, as shown in FIG. 7 (a), transgenic mice (21, 24, 26, 27, and 28) in which the MYH3 gene was inserted and expressed mRNA could be identified.
실시예 3-2-3. 단백질 발현 분석을 통한 외래 유전자 도입 여부 확인Example 3-2-3. Confirmation of introduction of foreign gene through analysis of protein expression
상기 실시예 3-2-1을 통해 제작한 형질전환 마우스에 MYH3 유전자가 도입되었는지 확인하기 위해, 상기 유전자의 단백질 발현 여부를 분석하였다.In order to confirm whether the MYH3 gene was introduced into the transgenic mouse prepared in Example 3-2-1, the expression of the gene was analyzed.
구체적으로, 야생형 마우스(WT)와 형질전환 마우스(TG)의 후지 근육조직을 채취한 후, RIPA 버퍼에 단백질 분해 억제제를 첨가하여 초음파 분쇄함으로써 조직을 분해하였다. 이후, 저온원심분리기를 통해 조직과 단백질을 분리하여 상층액에서 단백질을 회수하였다. 회수한 단백질은 BSA 단백질 어세이 시약(bio-rad)을 이용하여 정량한 후, 4x 단백질 로딩 버퍼(1x)로 100℃에서 약 10분간 가열하였다. 준비된 단백질을 이용하여 웨스턴블롯을 수행하기 위해, SDS-PAGE 겔에 약 2시간 동안 상기 단백질을 영동하였다. 이후, 영동된 단백질을 PVDF 멤브레인으로 옮긴 후, 5% skim milk를 이용하여 1시간 동안 블로킹하였고, 실험 목적에 맞는 일차 항체(Anti-Flag M2(F1804, Sigma-Aldrich) 및 b-actin(#4970, Cell signaling))를 첨가하여 4℃에서 밤새 반응시켰다. 다음날 TBST 용액으로 세척한 후 이차 항체를 2시간 동안 반응시킨 뒤, ECL 시약을 처리하였고, LAS-300 luminescent image analyzer system(Fujifilm)을 이용하여 단백질에 결합한 항체의 신호를 확인하였다. 실험에 사용된 이차 항체는 일차 항체에 따라 달리 사용하였다.Specifically, after removing the fuzzy muscle tissue of the wild-type mouse (WT) and the transgenic mouse (TG), the tissue was degraded by adding the protease inhibitor to the RIPA buffer and ultrasonication. Subsequently, the protein and the protein were separated through a low-temperature centrifuge to recover proteins from the supernatant. The recovered protein was quantified using a BSA protein assay reagent (bio-rad), and then heated at 100 ° C. for about 10 minutes with 4 × protein loading buffer (1 ×). To perform Western blotting using the prepared protein, the protein was subjected to SDS-PAGE gel for about 2 hours. Then, the proteins were transferred to a PVDF membrane and blocked with 5% skim milk for 1 hour. The primary antibody (Anti-Flag M2 (F1804, Sigma-Aldrich) and b-actin , Cell signaling) was added and reacted overnight at 4 ° C. The next day, the cells were washed with TBST solution, reacted with the secondary antibody for 2 hours, treated with ECL reagent, and the signal of the antibody bound to the protein was confirmed using a LAS-300 luminescent image analyzer system (Fujifilm). The secondary antibodies used in the experiments were used differently according to the primary antibodies.
그 결과, 도 7의 b에서 볼 수 있듯이, 형질전환 마우스 중에서도 24번에서 MYH3 유전자의 단백질 발현량이 가장 높음을 확인할 수 있었다. 이에 따라, 형질전환 유전자의 단백질의 발현도가 가장 높았던 24번 마우스를 활용하여 교배 후 번식시켰으며, 그 모습은 도 8에 나타내었다.As a result, as shown in FIG. 7 (b), it was confirmed that the expression level of the MYH3 gene was the highest among the transgenic mice at 24 hours. Thus, the 24 transgenic mouse having the highest expression level of the transgene was used for breeding after mating, and its appearance is shown in FIG.
실시예 3-3. 형질전환 마우스의 근육 형태 확인Example 3-3. Identification of muscle type in transgenic mice
육질 형질 결정에 관한 MYH3 유전자의 기능을 확인하기 위해, 상기 실시예 3-2를 통해 제작한 형질전환 마우스의 육질 형질을 조사하였다.In order to confirm the function of the MYH3 gene related to the determination of the meat quality trait, the meat quality of the transgenic mouse prepared in Example 3-2 was examined.
먼저, 도 9의 a에서 볼 수 있듯이, 돼지 MYH3 유전자가 형질전환된 마우스(우; TG)의 뒷다리의 근육은 야생형 마우스(좌; WT)에 비해 더욱 붉은색을 띄는 것을 확인할 수 있었다.First, as can be seen from FIG. 9 (a), it was confirmed that the muscles of the hind legs of the pig MYH3 transgenic mice (W: TG) were redder than the wild type mice (WT).
이후, 근육 형태를 더욱 자세히 확인하기 위하여, ATPase 염색을 수행하였다. 야생형 및 형질전환 마우스의 뒷다리 근육을 회수하여 30% 수크로스(sucrose) 용액에 밤새처리 한 후, OCT compound를 이용하여 -25℃에서 10㎛ 크기로 조직절편(tissue-section)한 뒤, 4% PEA으로 1시간 동안 고정하였다. 이후 흐르는 물에서 10분 동안 세척하였고, 60% 이소프로판올(isopropanol)을 이용하여 세척한 후, ATPase 염색 키트(ATPase stain lyopilized powder for histoenzymatic reaction kit, Bio optica사)를 이용하여 제조사에서 제시한 방법에 따라 실험을 수행하였다.Afterwards, ATPase staining was performed to further confirm the muscle morphology. The hindlimb muscles of wild-type and transgenic mice were harvested, treated overnight in a 30% sucrose solution, tissue-sectioned to 10 쨉 m at -25 째 C using an OCT compound, And fixed with PEA for 1 hour. After washing with running water for 10 minutes and washing with 60% isopropanol, ATPase stain lyopilized powder for histoenzymatic reaction kit (Bio-optica) was used according to the manufacturer's instructions Experiments were performed.
그 결과, 도 9의 b에서 볼 수 있듯이, 야생형 마우스(좌; WT)에 비하여, 상기 유전자가 삽입된 형질전환 마우스(우; TG)는 뒷다리 근육에 적색근이 더 많이 분포함을 확인할 수 있었다.As a result, as shown in FIG. 9 (b), it was confirmed that the transgenic mouse (right; TG) in which the gene was inserted contained more red muscle in the hind limb muscle than the wild type mouse (left; WT) .
추가적으로, 근육조직 내 지방의 분포를 확인하기 위하여 Oil Red O 염색을 수행하였다. 구체적으로, 상기 조직절편을 0.3% Oil Red O 용액에 1시간 동안 반응시켰다.In addition, Oil Red O staining was performed to determine the distribution of fat in muscle tissue. Specifically, the tissue slice was reacted with 0.3% Oil Red O solution for 1 hour.
그 결과, 도 9의 c에서 볼 수 있듯이, 야생형 마우스(좌; WT)에 비하여, 형질전환 마우스(우; TG)에서 염색 정도가 더 강함을 확인함으로써, 형질전환 마우스의 뒷다리 근육에는 지방의 함량이 더 많음을 확인할 수 있었다. As a result, as shown in FIG. 9 (c), it was confirmed that the degree of staining in the transgenic mouse (TG) was stronger than in the wild type mouse (left; WT) Of the total population.
이를 종합하면, MYH3 유전자는 적색근을 축적시켜 적색도를 향상시키며, 근내지방함량을 축적시키는 원인 유전자임을 알 수 있었다.These results suggest that MYH3 gene is a causative gene that accumulates red muscle and improves redness and accumulates muscle fat content.
실시예 3-4. 형질전환 마우스의 근육 내 유전자 발현 패턴 확인Example 3-4. Identification of intramuscular gene expression patterns in transgenic mice
실시예 3-4-1. 백색근/적색근 형성 유전자의 발현 패턴 확인Example 3-4-1. Identification of expression pattern of white muscle / red muscle roughening gene
육질 형질 결정에 관한 MYH3 유전자의 기능을 확인하기 위해, 상기 실시예 3-2를 통해 제작한 형질전환 마우스의 백색근/적색근 형성 유전자의 발현 패턴을 조사하였다.In order to confirm the function of the MYH3 gene related to the meat quality characterization, the expression pattern of the white muscle / red muscle roughening gene of the transgenic mouse prepared in Example 3-2 was examined.
구체적으로, 야생형 마우스(WT)와 형질전환 마우스(TG)의 후지 근육조직을 채취하였고, 상기 실시예 2-3에 따른 방법으로 qRT-PCR을 수행하였다. 이때 사용한 프라이머는 하기 표 3에 나열하였다.Specifically, fuzzy muscle tissue of wild-type mouse (WT) and transgenic mouse (TG) was collected and qRT-PCR was performed by the method according to Example 2-3. The primers used at this time are listed in Table 3 below.
또한, 상기 실시예 3-2-3에 따른 방법으로 웨스턴블롯을 수행하였으며, 이때 사용한 일차항체는 다음과 같다: Anti-Flag M2(F1804, Sigma-Aldrich), MYH7(SC-53089, Santa cruz biotechnology), MYH4(H00004622-B01P, Abnova) 및 b-actin(#4970, Cell signaling).Western blot was performed using the method according to Example 3-2-3, wherein the primary antibody used was Anti-Flag M2 (F1804, Sigma-Aldrich), MYH7 (SC-53089, Santa Cruz Biotechnology ), MYH4 (H00004622-B01P, Abnova) and b-actin (# 4970, Cell signaling).
그 결과, 도 10의 a에서 볼 수 있듯이, 야생형 마우스에 비하여, 형질전환 마우스에서는 적색근의 일종으로 slow type 유전자인 MYH7 유전자의 mRNA 및 단백질의 발현이 급격하게 증가함을 확인하였다.As a result, as shown in FIG. 10 (a), mRNA and protein expression of MYH7 gene, which is a type of red muscle, was dramatically increased in transgenic mice compared with wild type mice.
또한, 도 10의 b에서 볼 수 있듯이, 야생형 마우스에 비하여, 형질전환 마우스에서는 백색근의 일종인 fast type 유전자(Aldoa, Pvalb, Tnnf3, Tnnl2, Tnnc2)는 발현량에서 유의적인 차이가 없으나, 적색근의 일종인 slow type에 영향을 미치는 유전자(Myoglobin, Tnnt1, Tnnl1, Tnnc1)들은 모두 mRNA 발현양이 증가함을 확인하였다. As shown in FIG. 10 (b), in the transgenic mice, the fast type genes (Aldoa, Pvalb, Tnnf3, Tnnl2, and Tnnc2), which are a kind of white muscle, The genes affecting the slow type, myoglobin, Tnnt1, Tnnl1, and Tnnc1, all showed increased mRNA expression.
실시예 3-4-2. 지방 축적 패턴 및 관련 유전자의 발현 패턴 확인Example 3-4-2. Identification of expression patterns of fat accumulation patterns and related genes
아울러, 육질 형질 결정에 관한 MYH3 유전자의 기능을 확인하기 위해, 상기 실시예 3-2를 통해 제작한 형질전환 마우스의 지방 축적 패턴 및 관련 유전자의 발현 패턴을 조사하였다. In addition, in order to confirm the function of the MYH3 gene in the determination of meat quality traits, the fat accumulation pattern of the transgenic mouse prepared in Example 3-2 and the expression pattern of the related gene were examined.
먼저, 지방 축적 패턴을 확인하기 위해 조직상의 형태를 확인하고자 하였고, 이를 위해 H&E(Hematoxylin and eosin) 염색을 수행하였다. 야생형 마우스(WT) 및 형질전환 마우스(TG)의 근육 조직을 이용하여 파라핀 엠베딩(Paraffin embedding)한 후, 4㎛의 두께로 조직을 절편하였다. 이후 자일렌(xylene)을 이용하여 파라핀을 제거하고, 100%, 95%, 75%, 50% 알코올을 순서대로 이용하여 탈수한 후, 흐르는 물에 5분간 세척하였다. 이후, Myyer's 헤마톡실린(hematoxylin) 용액에 1분간 처리한 후 흐르는 물에 20분간 세척하고, 에오신(esoin)에 1분간 처리한 후 탈수 및 클리어링(clearing) 과정을 수행하였고, 이후 현미경 상에서 조직의 염색 상태를 확인하였다.First, to confirm the fat accumulation pattern, we tried to identify the morphology of the tissue, and we performed H & E (Hematoxylin and eosin) staining. Paraffin embedding was carried out using the muscle tissues of wild-type mouse (WT) and transgenic mouse (TG), and the tissue was cut into a thickness of 4 탆. Then, paraffin was removed by using xylene, dehydrated in the order of 100%, 95%, 75% and 50% alcohol, and then washed in running water for 5 minutes. Thereafter, the cells were treated with Myyer's hematoxylin solution for 1 minute, washed with flowing water for 20 minutes, treated with esoin for 1 minute, followed by dehydration and clearing, And the state of staining was confirmed.
또한, 형질전환 마우스의 근육조직에서 지방 축적 유전자의 발현 패턴을 확인하기 위하여, 상기 실시예 2-3에 따른 방법으로 qRT-PCR을 수행하였다. 이때 사용한 프라이머는 하기 표 4에 나열하였다.In order to confirm the expression pattern of the fat accumulation gene in the muscle tissue of the transgenic mouse, qRT-PCR was performed by the method according to Example 2-3. The primers used at this time are listed in Table 4 below.
그 결과, 도 10의 c 및 d에서 볼 수 있듯이, 야생형 마우스에 비하여, 형질전환 마우스에서는 근세포 사이 간격이 크며, 그 사이로 지방이 침착되는데, 지방분화 유전자의 발현 또한 유의적으로 증가함을 확인하였다.As a result, as shown in FIGS. 10C and 10D, it was confirmed that the expression of the lipogenic gene was significantly increased in the transgenic mouse as compared with the wild-type mouse in that the interval between the muscle cells was large and the fat was deposited therebetween .
상기 결과를 통해, MYH3 유전자는 적색근 관련 유전자의 발현 증가에 영향을 미칠 뿐만 아니라 근육 내 지방 형성을 향상시키는 원인 유전자임을 알 수 있었다.These results indicate that the MYH3 gene not only affects the expression of the red muscle-associated gene but also promotes the formation of intramuscular fat.
실시예 4. MYH3 유전자의 유전형 분석Example 4. Genotype analysis of MYH3 gene
MYH3 유전자의 유전형을 분석하기 위하여, 돼지 MYH3 유전자의 전사시작지점(transcription start site; TSS)으로부터 5'-UTR 3kb, 및 종결코돈(stop codon)으로부터 3'-UTR 1kb 까지의 염기서열을 해독하였다.To analyze the genotype of the MYH3 gene, the 5'-UTR 3kb from the transcription start site (TSS) of the porcine MYH3 gene and the 3'-UTR 1kb from the stop codon were decoded .
그 결과, 도 11에서 볼 수 있듯이, 육질에 영향을 미치는 MYH3 유전자에서 QTN(i.e., MYH3-1805_-1810delGGACTG)을 확인하였다(적색점으로 표시). 또한, 랜드레이스(LANDLACE)와 제주재래돼지(KNP) 사이에 염기 변이가 존재하며, 이는 엑손(exon) 3에 있는 개시코돈(ATG)으로부터 5'-UTR에 위치함을 확인하였다. 제주재래돼지의 경우 -1805 bp 내지 -1810 bp의 부위가 결손되었음을 확인하였으며, 이는 근육형성 조절 인자(myogenesis regulatory factor; MRF)의 결합 부위임을 확인하였다.As a result, QTN (i.e., MYH3-1805_-1810delGGACTG) was identified (indicated by a red dot) in the MYH3 gene that affects meat quality, as can be seen from Fig. Also, it was confirmed that a base mutation exists between LANDLACE and KNP, which is located at the 5'-UTR from the initiation codon (ATG) in
실시예 5. MYH3 유전자의 유전형 확인을 통한 돼지의 품종 판단 Example 5. Judgment of Pig Breed through Identification of Genotype of MYH3 Gene
상기 실시예 3을 통해 랜드레이스 및 제주재래돼지의 MYH3 유전자 간에 염기 변이가 존재함을 확인함에 따라, 상기 염기 변이를 이용하여 돼지의 육질을 판단하고자 하였다.As a result of confirming the presence of a base mutation between the MYH3 gene of Landrace and Jeju native pig in Example 3, the base mutation was used to determine the meat quality of the pig.
구체적으로, 프라이머(forward: 5'-TGG TCT TTC CTA ATT GGT GAC AT-3' (서열번호 65), reverse: 5'-AGT TTT GAG CAA GGC TTT TGT T-3' (서열번호 66))를 이용하여 상기 염기 변이를 포함한 부분을 증폭하였다. 각 돼지의 혈액으로부터 분리한 100 ng/㎕의 DNA를 주형으로 하였으며, 10×반응 완충액, 20 mM dNTP, 각각 200 mM의 정방향 및 역방향 프라이머, 1.5 units Taq DNA polymerase(TaKaRa, Japan)에 멸균한 탈이온수를 첨가하여 25 ㎕ 용량으로 PCR 반응을 수행하였다. PCR 증폭은 PTC-200(MJ Research, USA)을 이용하여 총 30 cycle을 수행하였고, 프라이머의 어닐링 온도는 60℃에서 진행하였다. 증폭 산물은 EtBr(ethidium bromide)가 함유된 2% 아가로스 겔 상에서 전기영동한 후, UV 하에서 유전자 증폭 여부를 확인하였다.Specifically, a primer (forward: 5'-TGG TCT TTC CTA ATT GGT GAC AT-3 '(SEQ ID NO: 65), reverse: 5'-AGT TTT GAG CAA GGC TTT TGT T-3' To amplify a portion containing the base mutation. 100 ng / ㎕ of DNA isolated from the blood of each pig was used as a template, and 10 × reaction buffer, 20 mM dNTP, 200 mM forward and reverse primers, and 1.5 units of Taq DNA polymerase (TaKaRa, Japan) The PCR reaction was carried out at 25 용량 by adding ionized water. PCR amplification was performed for 30 cycles using PTC-200 (MJ Research, USA). The annealing temperature of the primer was 60 ° C. The amplified product was electrophoresed on 2% agarose gel containing EtBr (ethidium bromide), and then amplified under UV.
이후, HpyCH4IV 제한효소를 이용하여 상기 원인 염기 변이의 염기서열 중에서 'A▼CGT' 부분을 절단하였다. 상기 증폭한 PCR 산물을 제한효소(HpyCH4Ⅳ)를 이용하여 절단하였고, 제한효소반응 조건은 공급자의 지시에 따라 PCR 증폭 산물 3㎕와 10X buffer 1㎕, 제한효소 0.3㎕, DW 5.7㎕를 혼합하여 37℃에서 밤새 반응하였다. EtBr(ethidium bromide)가 함유된 2% 아가로스 겔 상에서 전기영동하여, 제한효소에 의한 랜드레이스 및 제주재래돼지 MYH3 유전자의 절단 양상을 확인하였다.Then, the 'A ▼ CGT' portion of the base sequence of the causative base mutation was cleaved by using HpyCH4IV restriction enzyme. The amplified PCR product was digested with restriction enzymes (HpyCH4IV). The restriction enzyme reaction conditions were 3 μl of PCR amplification product, 1 μl of 10X buffer, 0.3 μl of restriction enzyme and 5.7 μl of DW, Lt; 0 > C overnight. Electrophoresis was performed on 2% agarose gel containing EtBr (ethidium bromide) to confirm the cleavage pattern of the landrace and Jeju native pig MYH3 gene by restriction enzyme.
그 결과, 도 12에서 볼 수 있듯이, 육질이 좋지 않은 랜드레이스(LANDLACE) 돼지는 유전자가 절단되지 않아 250 bp에서 한 개의 밴드로 나타남을 확인하였고(1/1; 비 절단형), 육질이 우수한 제주재래돼지(KNP)는 유전자가 절단되어 167 및 77 bp에서 두 개의 밴드로 나타냄을 확인하였다(2/2; 절단형). 또한, 랜드레이스와 제주재래돼지의 교배 품종은 250 및 167 bp에서 두 개의 밴드로 나타냄을 확인하였다(1/2; 절단형).As a result, as shown in FIG. 12, it was confirmed that the LANDLACE pig having poor meat quality did not cleave the gene, resulting in one band at 250 bp (1/1; non-cleaved type) The Korean traditional pig (KNP) was confirmed to have two bands at 167 and 77 bp by cleavage of the gene (2/2; truncated form). In addition, it was confirmed that the hybrid cultivars of Landrace and Jeju native pigs were represented by two bands at 250 and 167 bp (1/2, cut type).
상기 결과를 통해, 돼지의 육질 형질을 결정하는 유전자인 MYH3의 변이를 확인함으로써 외래돼지와 토종돼지를 구별할 수 있을 뿐만 아니라, 이를 통해 돼지의 육질을 판단할 수 있음을 확인하였다.Through the above results, it was confirmed that the pigs could be distinguished from the native pigs by confirming the mutation of MYH3, which is a gene that determines the meat quality of pigs, as well as judging the meat quality of the pigs.
이상의 설명으로부터, 본 발명이 속하는 기술분야의 당업자는 본 발명이 그 기술적 사상이나 필수적 특징을 변경하지 않고서 다른 구체적인 형태로 실시될 수 있다는 것을 이해할 수 있을 것이다. 이와 관련하여, 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적인 것이 아닌 것으로서 이해해야만 한다. 본 발명의 범위는 상기 상세한 설명보다는 후술하는 특허 청구범위의 의미 및 범위 그리고 그 등가 개념으로부터 도출되는 모든 변경 또는 변형된 형태가 본 발명의 범위에 포함되는 것으로 해석되어야 한다.From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.
<110> REPUBLIC OF KOREA(MANAGEMENT : RURAL DEVELOPMENT ADMINISTRATION) Jeju National University Industry-Academic Cooperation Foundation <120> A transformant comprising MYH3 gene and use thereof <130> KPA161033-KR <160> 66 <170> KoPatentIn 3.0 <210> 1 <211> 5823 <212> DNA <213> Sus scrofa <400> 1 atgagcagcg acactgaaat ggaagtgttc ggcatagccg ctcccttcct ccgcaagtcg 60 gaaaaggaga ggatcgaggc gcagaaccag cccttcgatg ccaaaaccta ctgcttcgtg 120 gtcgactcga aggaagagta cgccaagggg aaaattaaga gcacccagga tgggaaggtc 180 acggtggaga ccgaagacaa caggaccctg gtggtgaagc cggaggatgt gtatgccatg 240 aaccccccca agtttgaccg gatcgaggac atggccatgc tgacgcacct gaacgagccg 300 gccgtgctgt acaacctcaa ggaccggtac acctcctgga tgatctatac ctactcgggc 360 ctcttctgtg tcaccgtcaa cccctacaag tggctgccgg tgtacaaccc cgaggtggtg 420 gagggctacc gaggcaaaaa gcgccaggag gccccgcccc acatcttctc catctccgac 480 aacgcctatc agttcatgtt gacagatcgt gaaaaccagt ccattctgat caccggagaa 540 tccggggcgg gaaagactgt gaacaccaag agggtcatcc agtactttgc aacaattgct 600 gccactgggg acctcgccaa gaagaaggac tccaagatga aggggactct ggaggaccag 660 atcatcagcg ccaacccgct gctggaggcc ttcggcaacg ccaagaccgt gaggaacgac 720 aactcgtccc gcttcggcaa gttcatccga atccattttg gtaccaccgg gaagctggcc 780 tccgcagaca tcgaaacata tctgctcgaa aaatcgagag tgaccttcca gctgaaggct 840 gagaggagct accacatctt ctaccagatt ctctccaaca agaagccgga gctcatagag 900 ctgctgctca ttacaaccaa ccccttcgac tacccgttca tcagccaggg cgagatcctc 960 gtggccagca ttgatgatgc cgaggagctg ctagccacgg atagtgccat cgacatcctg 1020 ggcttcaccc cagaggagaa atctggactc tacaagctga cgggcgccgt gatgcactac 1080 gggaacatga agttcaagca gaagcaacgg gaggagcagg cagagccgga cggcacggaa 1140 gtggctgaca agacagccta cctcatgggc ctgaactctt cggacctcct gaaggctttg 1200 tgcttcccca gagtcaaagt tgggaacgag tatgttacca agggccagac cgtggatcag 1260 gtgcaccacg cggtgaacgc actctccaaa tccgtctacg agaagctctt cctgtggatg 1320 gtcacccgca tcaaccagca gctggacacc aagctgccca ggcagcactt catcggcgtc 1380 ttggacatcg cgggctttga gatctttgag tataacagcc tggagcagct gtgcatcaac 1440 ttcaccaacg agaaactgca acagtttttc aaccaccaca tgttcgtgct ggagcaggag 1500 gagtacaaga aggaaggcat cgagtggacg ttcatcgact tcgggatgga cctggccgcc 1560 tgcatcgagc tcatcgagaa gcccatgggc atcttctcca tcctggagga ggagtgcatg 1620 ttccccaagg ccacagacac ctccttcaag aacaagctct atgaccagca cctgggcaag 1680 tctgccaact tccagaagcc caaggtgctc aagggcaggg ccgaggccca cttctccctg 1740 atccactacg cgggcaccgt ggactacagt gtctcgggct ggctggagaa gaacaaggac 1800 cccctgaacg agacggtggt cgggctgtac cagaagtcct ccaaccggct cctggcgcac 1860 ctctacgcga ccttcgccac ggccgacgct gacagcggaa agaagaaagt tgccaagaaa 1920 aagggttctt ccttccaaac cgtctctgcc cttttcaggg aaaacctgaa caagctgatg 1980 tcgaatttaa ggacgactca ccctcacttt gtgcgctgta tcattcccaa tgaaaccaaa 2040 accccagggg ccatggaaca tagcctggtc ctgcaccagc tgaggtgtaa cggcgtgctg 2100 gagggcatcc gcatctgcag gaagggcttc cccaatcgga tcctctacgg ggattttaaa 2160 caaagatacc gcgtgctgaa tgccagcgcc atccccgagg gacagttcat cgacagcaag 2220 aaggcgtgtg aaaagctgtt ggcgtccatc gatattgacc acactcagta caaattcgga 2280 cacaccaagg tgttcttcaa ggccggcttg ctgggaaccc tggaggaaat gcgggacgac 2340 cgcctggcca agctcatcac ccggacgcaa gccgtgtgca ggggcttcct catgcgcgtg 2400 gaattccaga agatggtgca gagaagggag tccatcttct gcatccagta caacatccga 2460 gccttcatga acgtcaagca ctggccctgg atgaaactct tcttcaagat caagcctctg 2520 ctgaagagcg cagagaccga gaaggagatg gccaccatga aggaggagtt ccagaaaacc 2580 aaggaggaac tcgccaagtc agaggcaaag aggaaggaac tggaggaaaa gatggtgact 2640 ctggtacaag agaagaacga cctgcagctc caagtacaag ctgaaagtga aaacttgttg 2700 gatgcagagg aaagatgtga ccagctgatc aaagccaagt ttcagctgga ggccaaaatc 2760 aaggaggtga ctgagagagc tgaggatgag gaagagatca atgccgagct gacggccaag 2820 aagaggaaac tggaggatga gtgctcagaa ctgaagaaag acatcgatga ccttgagctg 2880 acactggcca aggttgaaaa ggagaaacat gccacagaga acaaggttaa aaaccttact 2940 gaggaactgg ccggcttgga tgaaaccatt gcgaagttga ccagggagaa gaaggccctc 3000 caggaggccc accagcagac cctggatgac ctccaagctg aagaggacaa agtcaactcc 3060 ttaagcaaaa tcaagagcaa actggaacag caggtggacg atctggaaag ctccctagaa 3120 caagagaaga agctccgggt cgacctggaa aggaataaaa ggaagcttga gggtgacttg 3180 aagcttgccc aggagtccat actggatctg gagaatgaca agcaacagct ggatgaaagg 3240 ctcaagaaga aggattttga gtccagtcag ctgcagagca aagtggaaga tgagcagacc 3300 ctgggcctcc agtttcagaa gaaaattaaa gagctccagg ctcgaatcga ggagctggag 3360 gaagagattg aggccgagag ggccacccgc gccaagacgg agaagcagcg cagcgactac 3420 gcccgggagc tggaggagct gagcgagcgg ctggaggagg cggggggcgt cacatccacg 3480 cagatcgagc tgaacaagaa gcgcgaggcc gagttcctga agctgcgccg ggacctggag 3540 gaggccaccc tgcagcacga ggccacagtg gctgcgctga ggaagaagca cgcggacagc 3600 gtggctgagc tgggggagca gatcgacaac ctgcagaggg tcaagcagaa gctggagaag 3660 gagaagagcg aattcaagct ggagctggac gacctggccg gcaacgtgga gagcgtgtcc 3720 aaggcgaagg ccaacctgga aaaaatctgc cgcaccctgg aggatcagct aagcgaggcc 3780 aggggcaaga acgaggaaat ccagaggagc atgagcgagc tgaccatgca gaagtcccgt 3840 cttcagacgg aggccggtga gctgagtcgt caactcgaag agaaagagag cacagtatca 3900 cagctttcca gaagcaagca agcgtttacc cagcaaatag aagagctcaa gaggcagctg 3960 gaggaggaga gcaaggccaa gaacgccctg gcgcacgccc tgcagtcctc ccgccacgac 4020 tgcgacctgc tgcgggaaca gtatgaggag gagcaggaag ccaaggccga gctgcagagg 4080 gcgctgtcca aggccaacag cgaggttgcc cagtggagga ccaagtacga gacggacgcc 4140 atccagcgca cggaggagct ggaggaggcc aagaaaaagc ttgctcagcg ccttcaggat 4200 tccgaggagc aggtggaggc agtgaacgcc aagtgtgcct ccctggagaa gaccaagcag 4260 aggctgcaag cggaggtgga ggacctgatg gtggacgtgg acagagccaa ctctctggct 4320 gctgcgctgg acaagaagca gagaaacttc gacaaggtgc tcgccgagtg gaaaacaaag 4380 tgtgaggaga gccaggcaga gctggaggca gctctgaagg aatcccgctc cttgagcacc 4440 gagctcttca agctgaaaaa tgcctacgaa gaagccttag atcaacttga aactgtgaag 4500 cgagaaaata agaacttgga gcaggagata gcagatctca cagaacaaat tgctgagaac 4560 ggtaaaacca tccatgaact ggagaaatca agaaagcaga tcgagctgga gaaggctgat 4620 atccagctgg ctcttgagga agcagaggcc gcccttgagc acgaagaggc caagatcctc 4680 cgaatccagc tggaactgac ccaggtgaaa tcggaaatcg acaggaagat ggctgagaaa 4740 gacgaagaga tcgagcagct caagaggaac taccagagaa ccgtggagac gatgcagagc 4800 gccttggacg ccgaggtgcg gagccgcaac gaggccatca ggatcaagaa gaagatggag 4860 ggggacctga acgagatcga gatccagctg agccacgcca accgccaggc cgcggagacc 4920 ctcaaacacc tccggggcgt ccaaggacag ctgaaggaca cccagctcca cctggacgac 4980 gctctccgag gccaggagga cctgaaggag cagctggcca tcgtggagcg cagagccagc 5040 ctgctgcagg ccgaggtgga ggagctgcgg gcctccctgg agcagacgga gagggcccgg 5100 aaactggcag agcaggagct cctggacgcc aacgagaggg tgcagctgct ccacacccag 5160 aacaccagcc tcatccacac caagaagaag ctggagacgg acctgatgca gctccagagc 5220 gaggtcgagg acgccagcag ggatgcgagg aacgccgagg agaaggcgaa gaaggccacc 5280 accgacgcgg ccatgatggc cgaggagctg aagaaggagc aggacaccag cgcccacctg 5340 gagcggatga agaagaacct ggagcagacg gtgaaggacc tgcagcaccg cctggacgag 5400 gcggaacagt tggccctgaa gggcgggaag aagcagatcc agaagctgga ggccagaatc 5460 cgagagctgg agtttgagct tgagggggag cagaagaaga acacggagtc tgtcaagggc 5520 ctcaggaaat acgagcggcg ggtcaaggag ttaacttacc agagtgaaga ggacaggaag 5580 aatgtgctga gattacagga cctggtggac aaacttcaag cgaaggtcaa gtcctacaag 5640 aggcaggcgg aggaggctga tgaacaagcc aatgctcatc tcaccaagtt ccgaaaagct 5700 cagcatgagc tcgaggaggc tgaagaacgg gctgatatcg ccgaatctca ggtcaataag 5760 cttcgcgcaa agacccgaga cttcacctcc agccggatgg tggtccatga gagcgaagag 5820 tga 5823 <210> 2 <211> 12238 <212> DNA <213> Artificial Sequence <220> <223> CAGGS-MYH3-Flag vector <400> 2 gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 60 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 120 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 180 ggactttcca ttgacgtcaa tgggtggact atttacggta aactgcccac ttggcagtac 240 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg 300 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg 360 tattagtcat cgctattacc atgggtcgag gtgagcccca cgttctgctt cactctcccc 420 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca 480 gcgatggggg cggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg 540 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt 600 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc 660 gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccgcc 720 ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc 780 gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag 840 ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg 900 tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct gtgagcgctg 960 cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc ggccgggggc 1020 ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg 1080 tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc 1140 cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtgc ggggcgtggc 1200 gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg 1260 ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gccccggagc gccggcggct 1320 gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg 1380 gacttccttt gtcccaaatc tggcggagcc gaaatctggg aggcgccgcc gcaccccctc 1440 tagcgggcgc gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt 1500 cgtgcgtcgc cgcgccgccg tccccttctc catctccagc ctcggggctg ccgcaggggg 1560 acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt gtgaccggcg 1620 gctctagagc caccatgagc agcgacactg aaatggaagt gttcggcata gccgctccct 1680 tcctccgcaa gtcggaaaag gagaggatcg aggcgcagaa ccagcccttc gatgccaaaa 1740 cctactgctt cgtggtcgac tcgaaggaag agtacgccaa ggggaaaatt aagagcaccc 1800 aggatgggaa ggtcacggtg gagaccgaag acaacaggac cctggtggtg aagccggagg 1860 atgtgtatgc catgaacccc cccaagtttg accggatcga ggacatggcc atgctgacgc 1920 acctgaacga gccggccgtg ctgtacaacc tcaaggaccg gtacacctcc tggatgatct 1980 atacctactc gggcctcttc tgtgtcaccg tcaaccccta caagtggctg ccggtgtaca 2040 accccgaggt ggtggagggc taccgaggca aaaagcgcca ggaggccccg ccccacatct 2100 tctccatctc cgacaacgcc tatcagttca tgttgacaga tcgtgaaaac cagtccattc 2160 tgatcaccgg agaatccggg gcgggaaaga ctgtgaacac caagagggtc atccagtact 2220 ttgcaacaat tgctgccact ggggacctcg ccaagaagaa ggactccaag atgaagggga 2280 ctctggagga ccagatcatc agcgccaacc cgctgctgga ggccttcggc aacgccaaga 2340 ccgtgaggaa cgacaactcg tcccgcttcg gcaagttcat ccgaatccat tttggtacca 2400 ccgggaagct ggcctccgca gacatcgaaa catatctgct cgaaaaatcg agagtgacct 2460 tccagctgaa ggctgagagg agctaccaca tcttctacca gattctctcc aacaagaagc 2520 cggagctcat agagctgctg ctcattacaa ccaacccctt cgactacccg ttcatcagcc 2580 agggcgagat cctcgtggcc agcattgatg atgccgagga gctgctagcc acggatagtg 2640 ccatcgacat cctgggcttc accccagagg agaaatctgg actctacaag ctgacgggcg 2700 ccgtgatgca ctacgggaac atgaagttca agcagaagca acgggaggag caggcagagc 2760 cggacggcac ggaagtggct gacaagacag cctacctcat gggcctgaac tcttcggacc 2820 tcctgaaggc tttgtgcttc cccagagtca aagttgggaa cgagtatgtt accaagggcc 2880 agaccgtgga tcaggtgcac cacgcggtga acgcactctc caaatccgtc tacgagaagc 2940 tcttcctgtg gatggtcacc cgcatcaacc agcagctgga caccaagctg cccaggcagc 3000 acttcatcgg cgtcttggac atcgcgggct ttgagatctt tgagtataac agcctggagc 3060 agctgtgcat caacttcacc aacgagaaac tgcaacagtt tttcaaccac cacatgttcg 3120 tgctggagca ggaggagtac aagaaggaag gcatcgagtg gacgttcatc gacttcggga 3180 tggacctggc cgcctgcatc gagctcatcg agaagcccat gggcatcttc tccatcctgg 3240 aggaggagtg catgttcccc aaggccacag acacctcctt caagaacaag ctctatgacc 3300 agcacctggg caagtctgcc aacttccaga agcccaaggt gctcaagggc agggccgagg 3360 cccacttctc cctgatccac tacgcgggca ccgtggacta cagtgtctcg ggctggctgg 3420 agaagaacaa ggaccccctg aacgagacgg tggtcgggct gtaccagaag tcctccaacc 3480 ggctcctggc gcacctctac gcgaccttcg ccacggccga cgctgacagc ggaaagaaga 3540 aagttgccaa gaaaaagggt tcttccttcc aaaccgtctc tgcccttttc agggaaaacc 3600 tgaacaagct gatgtcgaat ttaaggacga ctcaccctca ctttgtgcgc tgtatcattc 3660 ccaatgaaac caaaacccca ggggccatgg aacatagcct ggtcctgcac cagctgaggt 3720 gtaacggcgt gctggagggc atccgcatct gcaggaaggg cttccccaat cggatcctct 3780 acggggattt taaacaaaga taccgcgtgc tgaatgccag cgccatcccc gagggacagt 3840 tcatcgacag caagaaggcg tgtgaaaagc tgttggcgtc catcgatatt gaccacactc 3900 agtacaaatt cggacacacc aaggtgttct tcaaggccgg cttgctggga accctggagg 3960 aaatgcggga cgaccgcctg gccaagctca tcacccggac gcaagccgtg tgcaggggct 4020 tcctcatgcg cgtggaattc cagaagatgg tgcagagaag ggagtccatc ttctgcatcc 4080 agtacaacat ccgagccttc atgaacgtca agcactggcc ctggatgaaa ctcttcttca 4140 agatcaagcc tctgctgaag agcgcagaga ccgagaagga gatggccacc atgaaggagg 4200 agttccagaa aaccaaggag gaactcgcca agtcagaggc aaagaggaag gaactggagg 4260 aaaagatggt gactctggta caagagaaga acgacctgca gctccaagta caagctgaaa 4320 gtgaaaactt gttggatgca gaggaaagat gtgaccagct gatcaaagcc aagtttcagc 4380 tggaggccaa aatcaaggag gtgactgaga gagctgagga tgaggaagag atcaatgccg 4440 agctgacggc caagaagagg aaactggagg atgagtgctc agaactgaag aaagacatcg 4500 atgaccttga gctgacactg gccaaggttg aaaaggagaa acatgccaca gagaacaagg 4560 ttaaaaacct tactgaggaa ctggccggct tggatgaaac cattgcgaag ttgaccaggg 4620 agaagaaggc cctccaggag gcccaccagc agaccctgga tgacctccaa gctgaagagg 4680 acaaagtcaa ctccttaagc aaaatcaaga gcaaactgga acagcaggtg gacgatctgg 4740 aaagctccct agaacaagag aagaagctcc gggtcgacct ggaaaggaat aaaaggaagc 4800 ttgagggtga cttgaagctt gcccaggagt ccatactgga tctggagaat gacaagcaac 4860 agctggatga aaggctcaag aagaaggatt ttgagtccag tcagctgcag agcaaagtgg 4920 aagatgagca gaccctgggc ctccagtttc agaagaaaat taaagagctc caggctcgaa 4980 tcgaggagct ggaggaagag attgaggccg agagggccac ccgcgccaag acggagaagc 5040 agcgcagcga ctacgcccgg gagctggagg agctgagcga gcggctggag gaggcggggg 5100 gcgtcacatc cacgcagatc gagctgaaca agaagcgcga ggccgagttc ctgaagctgc 5160 gccgggacct ggaggaggcc accctgcagc acgaggccac agtggctgcg ctgaggaaga 5220 agcacgcgga cagcgtggct gagctggggg agcagatcga caacctgcag agggtcaagc 5280 agaagctgga gaaggagaag agcgaattca agctggagct ggacgacctg gccggcaacg 5340 tggagagcgt gtccaaggcg aaggccaacc tggaaaaaat ctgccgcacc ctggaggatc 5400 agctaagcga ggccaggggc aagaacgagg aaatccagag gagcatgagc gagctgacca 5460 tgcagaagtc ccgtcttcag acggaggccg gtgagctgag tcgtcaactc gaagagaaag 5520 agagcacagt atcacagctt tccagaagca agcaagcgtt tacccagcaa atagaagagc 5580 tcaagaggca gctggaggag gagagcaagg ccaagaacgc cctggcgcac gccctgcagt 5640 cctcccgcca cgactgcgac ctgctgcggg aacagtatga ggaggagcag gaagccaagg 5700 ccgagctgca gagggcgctg tccaaggcca acagcgaggt tgcccagtgg aggaccaagt 5760 acgagacgga cgccatccag cgcacggagg agctggagga ggccaagaaa aagcttgctc 5820 agcgccttca ggattccgag gagcaggtgg aggcagtgaa cgccaagtgt gcctccctgg 5880 agaagaccaa gcagaggctg caagcggagg tggaggacct gatggtggac gtggacagag 5940 ccaactctct ggctgctgcg ctggacaaga agcagagaaa cttcgacaag gtgctcgccg 6000 agtggaaaac aaagtgtgag gagagccagg cagagctgga ggcagctctg aaggaatccc 6060 gctccttgag caccgagctc ttcaagctga aaaatgccta cgaagaagcc ttagatcaac 6120 ttgaaactgt gaagcgagaa aataagaact tggagcagga gatagcagat ctcacagaac 6180 aaattgctga gaacggtaaa accatccatg aactggagaa atcaagaaag cagatcgagc 6240 tggagaaggc tgatatccag ctggctcttg aggaagcaga ggccgccctt gagcacgaag 6300 aggccaagat cctccgaatc cagctggaac tgacccaggt gaaatcggaa atcgacagga 6360 agatggctga gaaagacgaa gagatcgagc agctcaagag gaactaccag agaaccgtgg 6420 agacgatgca gagcgccttg gacgccgagg tgcggagccg caacgaggcc atcaggatca 6480 agaagaagat ggagggggac ctgaacgaga tcgagatcca gctgagccac gccaaccgcc 6540 aggccgcgga gaccctcaaa cacctccggg gcgtccaagg acagctgaag gacacccagc 6600 tccacctgga cgacgctctc cgaggccagg aggacctgaa ggagcagctg gccatcgtgg 6660 agcgcagagc cagcctgctg caggccgagg tggaggagct gcgggcctcc ctggagcaga 6720 cggagagggc ccggaaactg gcagagcagg agctcctgga cgccaacgag agggtgcagc 6780 tgctccacac ccagaacacc agcctcatcc acaccaagaa gaagctggag acggacctga 6840 tgcagctcca gagcgaggtc gaggacgcca gcagggatgc gaggaacgcc gaggagaagg 6900 cgaagaaggc caccaccgac gcggccatga tggccgagga gctgaagaag gagcaggaca 6960 ccagcgccca cctggagcgg atgaagaaga acctggagca gacggtgaag gacctgcagc 7020 accgcctgga cgaggcggaa cagttggccc tgaagggcgg gaagaagcag atccagaagc 7080 tggaggccag aatccgagag ctggagtttg agcttgaggg ggagcagaag aagaacacgg 7140 agtctgtcaa gggcctcagg aaatacgagc ggcgggtcaa ggagttaact taccagagtg 7200 aagaggacag gaagaatgtg ctgagattac aggacctggt ggacaaactt caagcgaagg 7260 tcaagtccta caagaggcag gcggaggagg ctgatgaaca agccaatgct catctcacca 7320 agttccgaaa agctcagcat gagctcgagg aggctgaaga acgggctgat atcgccgaat 7380 ctcaggtcaa taagcttcgc gcaaagaccc gagacttcac ctccagccgg atggtggtcc 7440 atgagagcga agaggattac aaggatgacg acgataagtg agaattcact cctcaggtgc 7500 aggctgccta tcagaaggtg gtggctggtg tggccaatgc cctggctcac aaataccact 7560 gagatctttt tccctctgcc aaaaattatg gggacatcat gaagcccctt gagcatctga 7620 cttctggcta ataaaggaaa tttattttca ttgcaatagt gtgttggaat tttttgtgtc 7680 tctcactcgg aaggacatat gggagggcaa atcatttaaa acatcagaat gagtatttgg 7740 tttagagttt ggcaacatat gccatatgct ggctgccatg aacaaaggtg gctataaaga 7800 ggtcatcagt atatgaaaca gccccctgct gtccattcct tattccatag aaaagccttg 7860 acttgaggtt agattttttt tatattttgt tttgtgttat ttttttcttt aacatcccta 7920 aaattttcct tacatgtttt actagccaga tttttcctcc tctcctgact actcccagtc 7980 atagctgtcc ctcttctctt atgaagatcc ctcgacctgc agttggggtt gcgccttttc 8040 caaggcagcc ctgggtttgc gcagggacgc ggctgctctg ggcgtggttc cgggaaacgc 8100 agcggcgccg accctgggtc tcgcacattc ttcacgtccg ttcgcagcgt cacccggatc 8160 ttcgccgcta cccttgtggg ccccccggcg acgcttcctg ctccgcccct aagtcgggaa 8220 ggttccttgc ggttcgcggc gtgccggacg tgacaaacgg aagccgcacg tctcactagt 8280 accctcgcag acggacagcg ccagggagca atggcagcgc gccgaccgcg atgggctgtg 8340 gccaatagcg gctgctcagc agggcgcgcc gagagcagcg gccgggaagg ggcggtgcgg 8400 gaggcggggt gtggggcggt agtgtgggcc ctgttcctgc ccgcgcggtg ttccgcattc 8460 tgcaagcctc cggagcgcac gtcggcagtc ggctccctcg ttgaccgaat caccgacctc 8520 tctccccagg gggatccacc ggagcttacc atgaccgagt acaagcccac ggtgcgcctc 8580 gccacccgcg acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt cgccgactac 8640 cccgccacgc gccacaccgt cgatccggac cgccacatcg agcgggtcac cgagctgcaa 8700 gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc 8760 gccgcggtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag 8820 atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggaa 8880 ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc 8940 tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcg 9000 gccgagcgcg ccggggtgcc cgccttcctg gagacctccg cgccccgcaa cctccccttc 9060 tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc 9120 tggtgcatga cccgcaagcc cggtgcctga cgcccgcccc acgacccgca gcgcccgacc 9180 gaaaggagcg cacgacccca tgcatcggta cctttaagac caatgactta caaggcagct 9240 gtagatctta gccacttttt aaaagaaaag gggggactgg aagggctaat tcactcccaa 9300 cgaagacaag atctgctttt tgcttgtact gggtctctct ggttagacca gatctgagcc 9360 tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga 9420 gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga 9480 cccttttagt cagtgtggaa aatctctagc agtagtagtt catgtcatct tattattcag 9540 tatttataac ttgcaaagaa atgaatatca gagagtgaga ggaacttgtt tattgcagct 9600 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca 9660 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgctgcagc 9720 ccaagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca 9780 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg 9840 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg 9900 tgccagcgga tccgcatctc aattagtcag caaccatagt cccgccccta actccgccca 9960 tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt 10020 ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag 10080 gcttttttgg aggcctaggc ttttgcaaaa agctaacttg tttattgcag cttataatgg 10140 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc 10200 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggatcc gctgcattaa 10260 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg 10320 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag 10380 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa 10440 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc 10500 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca 10560 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg 10620 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct 10680 caatgctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt 10740 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 10800 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc 10860 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac 10920 actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga 10980 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc 11040 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg 11100 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca 11160 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt 11220 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca 11280 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg 11340 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca 11400 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt 11460 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt 11520 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca 11580 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca 11640 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga 11700 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact 11760 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga 11820 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg 11880 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc 11940 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga 12000 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat 12060 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt 12120 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt 12180 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctg 12238 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> pMYH3 F-primer <400> 3 aaaagctcag catgagctcg a 21 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> pMYH3 R-primer <400> 4 agggtcagga accatgaaaa t 21 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> pMYH1 F-primer <400> 5 gttctgaaga gggtggtac 19 <210> 6 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> pMYH1 R-primer <400> 6 agatgcggat gccctcca 18 <210> 7 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> pMYH2 F-primer <400> 7 gggctcaaac tggtgaagc 19 <210> 8 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> pMYH2 R-primer <400> 8 agatgcggat gccctcca 18 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> pMYH13 F-primer <400> 9 cacagggctc tggccgacat 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> pMYH13 R-primer <400> 10 cgtgcgcaca ggggtgtagt 20 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> pADPRM F-primer <400> 11 catcctgaga ccgtgccttc a 21 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> pADPRM R-primer <400> 12 ttccgcattt gggttgtgct 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> pSCO1 F-primer <400> 13 tcctcacgga ctcggggttt 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> pSCO1 R-primer <400> 14 gtggggtctc tgctgccctt 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> pTMEM220 F-primer <400> 15 cccagacgca gaactgtggg 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> pTMEM220 R-primer <400> 16 gttgtatgcc aagccggcag 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> pENSSSCG00000029441 F-primer <400> 17 tcgtgctgga gcaggaggag 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> pENSSSCG00000029441 R-primer <400> 18 aggtgtctgt ggccttgggg 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> pENSSSCG00000018006 F-primer <400> 19 agaaccagcc cttcgatgcc 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> pENSSSCG00000018006 R-primer <400> 20 tggcatacac atcctccggc 20 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> pGAPDH F-primer <400> 21 gggcatgaac catgagaagt 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> pGAPDH R-primer <400> 22 gggcatgaac catgagaagt 20 <210> 23 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> pMYH3 F-primer <400> 23 ccgagagctg gagtttga 18 <210> 24 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> pMYH3 R-primer <400> 24 ctcccatatg tccttccgag t 21 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Myh7 F-primer <400> 25 agtcccaggt caacaagctg 20 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Myh7 R-primer <400> 26 ttccacctaa agggctgttg 20 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Myh2 F-primer <400> 27 agtcccaggt caacaagctg 20 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Myh2 R-primer <400> 28 gcatgaccaa aggtttcaca 20 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Myh1 F-primer <400> 29 agtcccaggt caacaagctg 20 <210> 30 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Myh1 R-primer <400> 30 cacattttgc tcatctcttt g 21 <210> 31 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Myh4 F-primer <400> 31 agtcccaggt caacaagctg 20 <210> 32 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Myh4 R-primer <400> 32 tttctcctgt cacctctcaa ca 22 <210> 33 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Myoglobin F-primer <400> 33 gcaaggccct ggagctcttc 20 <210> 34 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Myoglobin R-primer <400> 34 gcttggtggg ctggacagtg 20 <210> 35 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Tnnt1 F-primer <400> 35 cccccgaaga ttccagaagg 20 <210> 36 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Tnnt1 R-primer <400> 36 tgcggtcttt tagtgcaatg ag 22 <210> 37 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Tnni1 F-primer <400> 37 atgccggaag ttgagaggaa a 21 <210> 38 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Tnni1 R-primer <400> 38 tccgagaggt aacgcacctt 20 <210> 39 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Tnnc1 F-primer <400> 39 gcggtagaac agttgacaga g 21 <210> 40 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Tnnc1 R-primer <400> 40 ccagctcctt ggtgctgat 19 <210> 41 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Aldoa F-primer <400> 41 actctctgct gaccgggctc t 21 <210> 42 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Aldoa R-primer <400> 42 aatgcttccg gtggactcat 20 <210> 43 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Pvalb F-primer <400> 43 atcaagaagg cgataggagc c 21 <210> 44 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Pvalb R-primer <400> 44 ggccagaagc gtctttgtt 19 <210> 45 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Tnnt3 F-primer <400> 45 ggaacgccag aacagattgg 20 <210> 46 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Tnnt3 R-primer <400> 46 tggaggacag agcctttttc tt 22 <210> 47 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Tnni2 F-primer <400> 47 agagtgtgat gctccagata gc 22 <210> 48 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Tnni2 R-primer <400> 48 agcaacgtcg atcttcgca 19 <210> 49 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Tnnc2 F-primer <400> 49 atggcagcgg tactatcgac t 21 <210> 50 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Tnnc2 R-primer <400> 50 ccttcgcatc ctctttcatc tg 22 <210> 51 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> GAPDH F-primer <400> 51 gaagggcatc ttgggctaca c 21 <210> 52 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> GAPDH R-primer <400> 52 gcagcgaact ttattgatgg tatt 24 <210> 53 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> CD36 F-primer <400> 53 aatggcacag acgcagcct 19 <210> 54 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> CD36 R-primer <400> 54 ggttgtctgg attctgga 18 <210> 55 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LPL F-primer <400> 55 gtacctgaag actcgctctc 20 <210> 56 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LPL R-primer <400> 56 agggtgaagg gaatgttctc 20 <210> 57 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Fabp4 F-primer <400> 57 gatgcctttg tgggaacctg 20 <210> 58 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Fabp4 R-primer <400> 58 tcctgtcgtc tgcggtgatt 20 <210> 59 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Fto F-primer <400> 59 gtcagagaga aggccaatga 20 <210> 60 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Fto R-primer <400> 60 tagcagtctc cctggtgaag 20 <210> 61 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Pgc1 alpha F-primer <400> 61 ccctgccatt gttaagacc 19 <210> 62 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Pgc1 alpha R-primer <400> 62 tgctgctgtt cctgttttc 19 <210> 63 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Adiponectin F-primer <400> 63 aatggcacac caggccgtga t 21 <210> 64 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Adiponectin R-primer <400> 64 tctccaggct ctcctttcct g 21 <210> 65 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> MYH3 variation F-primer <400> 65 tggtctttcc taattggtga cat 23 <210> 66 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> MYH3 variation R-primer <400> 66 agttttgagc aaggcttttg tt 22 <110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) Jeju National University Industry-Academic Cooperation Foundation <120> A transformant comprising MYH3 gene and use thereof <130> KPA161033-KR <160> 66 <170> KoPatentin 3.0 <210> 1 <211> 5823 <212> DNA <213> Sus scrofa <400> 1 atgagcagcg acactgaaat ggaagtgttc ggcatagccg ctcccttcct ccgcaagtcg 60 gaaaaggaga ggatcgaggc gcagaaccag cccttcgatg ccaaaaccta ctgcttcgtg 120 gtcgactcga aggaagagta cgccaagggg aaaattaaga gcacccagga tgggaaggtc 180 acggtggaga ccgaagacaa caggaccctg gtggtgaagc cggaggatgt gtatgccatg 240 aaccccccca agtttgaccg gatcgaggac atggccatgc tgacgcacct gaacgagccg 300 gccgtgctgt acaacctcaa ggaccggtac acctcctgga tgatctatac ctactcgggc 360 ctcttctgtg tcaccgtcaa cccctacaag tggctgccgg tgtacaaccc cgaggtggtg 420 gagggctacc gaggcaaaaa gcgccaggag gccccgcccc acatcttctc catctccgac 480 aacgcctatc agttcatgtt gacagatcgt gaaaaccagt ccattctgat caccggagaa 540 tccggggcgg gaaagactgt gaacaccaag agggtcatcc agtactttgc aacaattgct 600 gccactgggg acctcgccaa gaagaaggac tccaagatga aggggactct ggaggaccag 660 atcatcagcg ccaacccgct gctggaggcc ttcggcaacg ccaagaccgt gaggaacgac 720 aactcgtccc gcttcggcaa gttcatccga atccattttg gtaccaccgg gaagctggcc 780 tccgcagaca tcgaaacata tctgctcgaa aaatcgagag tgaccttcca gctgaaggct 840 gagaggagct accacatctt ctaccagatt ctctccaaca agaagccgga gctcatagag 900 ctgctgctca ttacaaccaa ccccttcgac tacccgttca tcagccaggg cgagatcctc 960 gtggccagca ttgatgatgc cgaggagctg ctagccacgg atagtgccat cgacatcctg 1020 ggcttcaccc cagaggagaa atctggactc tacaagctga cgggcgccgt gatgcactac 1080 gggaacatga agttcaagca gaagcaacgg gaggagcagg cagagccgga cggcacggaa 1140 gtggctgaca agacagccta cctcatgggc ctgaactctt cggacctcct gaaggctttg 1200 tgcttcccca gagtcaaagt tgggaacgag tatgttacca agggccagac cgtggatcag 1260 gtgcaccacg cggtgaacgc actctccaaa tccgtctacg agaagctctt cctgtggatg 1320 gtcacccgca tcaaccagca gctggacacc aagctgccca ggcagcactt catcggcgtc 1380 ttggacatcg cgggctttga gatctttgag tataacagcc tggagcagct gtgcatcaac 1440 ttcaccaacg agaaactgca acagtttttc aaccaccaca tgttcgtgct ggagcaggag 1500 gagtacaaga aggaaggcat cgagtggacg ttcatcgact tcgggatgga cctggccgcc 1560 tgcatcgagc tcatcgagaa gcccatgggc atcttctcca tcctggagga ggagtgcatg 1620 ttccccaagg ccacagacac ctccttcaag aacaagctct atgaccagca cctgggcaag 1680 tctgccaact tccagaagcc caaggtgctc aagggcaggg ccgaggccca cttctccctg 1740 atccactacg cgggcaccgt ggactacagt gtctcgggct ggctggagaa gaacaaggac 1800 cccctgaacg agacggtggt cgggctgtac cagaagtcct ccaaccggct cctggcgcac 1860 ctctacgcga ccttcgccac ggccgacgct gacagcggaa agaagaaagt tgccaagaaa 1920 aagggttctt ccttccaaac cgtctctgcc cttttcaggg aaaacctgaa caagctgatg 1980 tcgaatttaa ggacgactca ccctcacttt gtgcgctgta tcattcccaa tgaaaccaaa 2040 accccagggg ccatggaaca tagcctggtc ctgcaccagc tgaggtgtaa cggcgtgctg 2100 gagggcatcc gcatctgcag gaagggcttc cccaatcgga tcctctacgg ggattttaaa 2160 caaagatacc gcgtgctgaa tgccagcgcc atccccgagg gacagttcat cgacagcaag 2220 aaggcgtgtg aaaagctgtt ggcgtccatc gatattgacc acactcagta caaattcgga 2280 cacaccaagg tgttcttcaa ggccggcttg ctgggaaccc tggaggaaat gcgggacgac 2340 cgcctggcca agctcatcac ccggacgcaa gccgtgtgca ggggcttcct catgcgcgtg 2400 gaattccaga agatggtgca gagaagggag tccatcttct gcatccagta caacatccga 2460 gccttcatga acgtcaagca ctggccctgg atgaaactct tcttcaagat caagcctctg 2520 ctgaagagcg cagagaccga gaaggagatg gccaccatga aggaggagtt ccagaaaacc 2580 aaggaggaac tcgccaagtc agaggcaaag aggaaggaac tggaggaaaa gatggtgact 2640 ctggtacaag agaagaacga cctgcagctc caagtacaag ctgaaagtga aaacttgttg 2700 gatgcagagg aaagatgtga ccagctgatc aaagccaagt ttcagctgga ggccaaaatc 2760 aaggaggtga ctgagagagc tgaggatgag gaagagatca atgccgagct gacggccaag 2820 aagaggaaac tggaggatga gtgctcagaa ctgaagaaag acatcgatga ccttgagctg 2880 acactggcca aggttgaaaa ggagaaacat gccacagaga acaaggttaa aaaccttact 2940 gaggaactgg ccggcttgga tgaaaccatt gcgaagttga ccagggagaa gaaggccctc 3000 caggaggccc accagcagac cctggatgac ctccaagctg aagaggacaa agtcaactcc 3060 ttaagcaaaa tcaagagcaa actggaacag caggtggacg atctggaaag ctccctagaa 3120 caagagaaga agctccgggt cgacctggaa aggaataaaa ggaagcttga gggtgacttg 3180 aagcttgccc aggagtccat actggatctg gagaatgaca agcaacagct ggatgaaagg 3240 ctcaagaaga aggattttga gtccagtcag ctgcagagca aagtggaaga tgagcagacc 3300 ctgggcctcc agtttcagaa gaaaattaaa gagctccagg ctcgaatcga ggagctggag 3360 gaagagattg aggccgagag ggccacccgc gccaagacgg agaagcagcg cagcgactac 3420 gcccgggagc tggaggagct gagcgagcgg ctggaggagg cggggggcgt cacatccacg 3480 cagatcgagc tgaacaagaa gcgcgaggcc gagttcctga agctgcgccg ggacctggag 3540 gaggccaccc tgcagcacga ggccacagtg gctgcgctga ggaagaagca cgcggacagc 3600 gtggctgagc tgggggagca gatcgacaac ctgcagaggg tcaagcagaa gctggagaag 3660 gagaagagcg aattcaagct ggagctggac gacctggccg gcaacgtgga gagcgtgtcc 3720 aaggcgaagg ccaacctgga aaaaatctgc cgcaccctgg aggatcagct aagcgaggcc 3780 aggggcaaga acgaggaaat ccagaggagc atgagcgagc tgaccatgca gaagtcccgt 3840 cttcagacgg aggccggtga gctgagtcgt caactcgaag agaaagagag cacagtatca 3900 cagctttcca gagagagag agcgtttacc cagcaaatag aagagctcaa gaggagagg 3960 gaggaggaga gcaaggccaa gaacgccctg gcgcacgccc tgcagtcctc ccgccacgac 4020 tgcgacctgc tgcgggaaca gtatgaggag gagcaggaag ccaaggccga gctgcagagg 4080 gcgctgtcca aggccaacag cgaggttgcc cagtggagga ccaagtacga gacggacgcc 4140 atccagcgca cggaggagct ggaggaggcc aagaaaaagc ttgctcagcg ccttcaggat 4200 tccgaggag aggtggaggc agtgaacgcc aagtgtgcct ccctggagaa gaccaagcag 4260 aggctgcaag cggaggtgga ggacctgatg gtggacgtgg acagagccaa ctctctggct 4320 gctgcgctgg acaagaagca gagaaacttc gacaaggtgc tcgccgagtg gaaaacaaag 4380 tgtgaggaga gccaggcaga gctggaggca gctctgaagg aatcccgctc cttgagcacc 4440 gagctcttca agctgaaaaa tgcctacgaa gaagccttag atcaacttga aactgtgaag 4500 cgagaaaata agaacttgga gcaggagata gcagatctca cagaacaaat tgctgagaac 4560 ggtaaaacca tccatgaact ggagaaatca agaaagcaga tcgagctgga gaaggctgat 4620 atccagctgg ctcttgagga agcagaggcc gcccttgagc acgaagaggc caagatcctc 4680 cgaatccagc tggaactgac ccaggtgaaa tcggaaatcg acaggaagat ggctgagaaa 4740 gacgaagaga tcgagcagct caagaggaac taccagagaa ccgtggagac gatgcagagc 4800 gccttggacg ccgaggtgcg gagccgcaac gaggccatca ggatcaagaa gaagatggag 4860 ggggacctga acgagatcga gatccagctg agccacgcca accgccaggc cgcggagacc 4920 ctcaaacacc tccggggcgt ccaaggacag ctgaaggaca cccagctcca cctggacgac 4980 gctctccgag gccaggagga cctgaaggag cagctggcca tcgtggagcg cagagccagc 5040 ctgctgcagg ccgaggtgga ggagctgcgg gcctccctgg agcagacgga gagggcccgg 5100 aaactggcag agcaggagct cctggacgcc aacgagaggg tgcagctgct ccacacccag 5160 aacaccagcc tcatccacac caagaagaag ctggagacgg acctgatgca gctccagagc 5220 gaggtcgagg acgccagcag ggatgcgagg aacgccgagg agaaggcgaa gaaggccacc 5280 accgacgcgg ccatgatggc cgaggagctg aagaaggagc aggacaccag cgcccacctg 5340 gagcggatga agaagaacct ggagcagacg gtgaaggacc tgcagcaccg cctggacgag 5400 gcggaacagt tggccctgaa gggcgggaag aagcagatcc agaagctgga ggccagaatc 5460 cgagagctgg agtttgagct tgagggggag cagaagaaga acacggagtc tgtcaagggc 5520 ctcaggaaat acgagcggcg ggtcaaggag ttaacttacc agagtgaaga ggacaggaag 5580 aatgtgctga gattacagga cctggtggac aaacttcaag cgaaggtcaa gtcctacaag 5640 aggcaggcgg aggaggctga tgaacaagcc aatgctcatc tcaccaagtt ccgaaaagct 5700 cagcatgagc tcgaggaggc tgaagaacgg gctgatatcg ccgaatctca ggtcaataag 5760 cttcgcgcaa agacccgaga cttcacctcc agccggatgg tggtccatga gagcgaagag 5820 tga 5823 <210> 2 <211> 12238 <212> DNA <213> Artificial Sequence <220> <223> CAGGS-MYH3-Flag vector <400> 2 gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 60 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 120 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 180 ggactttcca ttgacgtcaa tgggtggact atttacggta aactgcccac ttggcagtac 240 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg 300 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg 360 tattagtcat cgctattacc atgggtcgag gtgagcccca cgttctgctt cactctcccc 420 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca 480 gcgatggggg cggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg 540 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt 600 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc 660 gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccgcc 720 ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc 780 gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag 840 ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg 900 tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct gtgagcgctg 960 cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc ggccgggggc 1020 ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg 1080 tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc 1140 cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtgc ggggcgtggc 1200 gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg 1260 ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gccccggagc gccggcggct 1320 gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg 1380 gacttccttt gtcccaaatc tggcggagcc gaaatctggg aggcgccgcc gcaccccctc 1440 tagcgggcgc gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt 1500 cgtgcgtcgc cgcgccgccg tccccttctc catctccagc ctcggggctg ccgcaggggg 1560 acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt gtgaccggcg 1620 gctctagagc caccatgagc agcgacactg aaatggaagt gttcggcata gccgctccct 1680 tcctccgcaa gtcggaaaag gagaggatcg aggcgcagaa ccagcccttc gatgccaaaa 1740 cctactgctt cgtggtcgac tcgaaggaag agtacgccaa ggggaaaatt aagagcaccc 1800 aggatgggaa ggtcacggtg gagaccgaag acaacaggac cctggtggtg aagccggagg 1860 atgtgtatgc catgaacccc cccaagtttg accggatcga ggacatggcc atgctgacgc 1920 acctgaacga gccggccgtg ctgtacaacc tcaaggaccg gtacacctcc tggatgatct 1980 atacctactc gggcctcttc tgtgtcaccg tcaaccccta caagtggctg ccggtgtaca 2040 accccgaggt ggtggagggc taccgaggca aaaagcgcca ggaggccccg ccccacatct 2100 tctccatctc cgacaacgcc tatcagttca tgttgacaga tcgtgaaaac cagtccattc 2160 tgatcaccgg agaatccggg gcgggaaaga ctgtgaacac caagagggtc atccagtact 2220 ttgcaacaat tgctgccact ggggacctcg ccaagaagaa ggactccaag atgaagggga 2280 ctctggagga ccagatcatc agcgccaacc cgctgctgga ggccttcggc aacgccaaga 2340 ccgtgaggaa cgacaactcg tcccgcttcg gcaagttcat ccgaatccat tttggtacca 2400 ccgggaagct ggcctccgca gacatcgaaa catatctgct cgaaaaatcg agagtgacct 2460 tccagctgaa ggctgagagg agctaccaca tcttctacca gattctctcc aacaagaagc 2520 cggagctcat agagctgctg ctcattacaa ccaacccctt cgactacccg ttcatcagcc 2580 agggcgagat cctcgtggcc agcattgatg atgccgagga gctgctagcc acggatagtg 2640 ccatcgacat cctgggcttc accccagagg agaaatctgg actctacaag ctgacgggcg 2700 ccgtgatgca ctacgggaac atgaagttca agcagaagca acgggaggag caggagagc 2760 cggacggcac ggaagtggct gacaagacag cctacctcat gggcctgaac tcttcggacc 2820 tcctgaaggc tttgtgcttc cccagagtca aagttgggaa cgagtatgtt accaagggcc 2880 agaccgtgga tcaggtgcac cacgcggtga acgcactctc caaatccgtc tacgagaagc 2940 tcttcctgtg gatggtcacc cgcatcaacc agcagctgga caccaagctg cccaggcagc 3000 acttcatcgg cgtcttggac atcgcgggct ttgagatctt tgagtataac agcctggagc 3060 agctgtgcat caacttcacc aacgagaaac tgcaacagtt tttcaaccac cacatgttcg 3120 tgctggagca ggaggagtac aagaaggaag gcatcgagtg gacgttcatc gacttcggga 3180 tggacctggc cgcctgcatc gagctcatcg agaagcccat gggcatcttc tccatcctgg 3240 aggaggagtg catgttcccc aaggccacag acacctcctt caagaacaag ctctatgacc 3300 agcacctggg caagtctgcc aacttccaga agcccaaggt gctcaagggc agggccgagg 3360 cccacttctc cctgatccac tacgcgggca ccgtggacta cagtgtctcg ggctggctgg 3420 agaagaacaa ggaccccctg aacgagacgg tggtcgggct gtaccagaag tcctccaacc 3480 ggctcctggc gcacctctac gcgaccttcg ccacggccga cgctgacagc ggaaagaaga 3540 aagttgccaa gaaaaagggt tcttccttcc aaaccgtctc tgcccttttc agggaaaacc 3600 tgaacaagct gatgtcgaat ttaaggacga ctcaccctca ctttgtgcgc tgtatcattc 3660 ccaatgaaac caaaacccca ggggccatgg aacatagcct ggtcctgcac cagctgaggt 3720 gtaacggcgt gctggagggc atccgcatct gcaggaaggg cttccccaat cggatcctct 3780 acggggattt taaacaaaga taccgcgtgc tgaatgccag cgccatcccc gagggacagt 3840 tcatcgacag caagaaggcg tgtgaaaagc tgttggcgtc catcgatatt gaccacactc 3900 agtacaaatt cggacacacc aaggtgttct tcaaggccgg cttgctggga accctggagg 3960 aaatgcggga cgaccgcctg gccaagctca tcacccggac gcaagccgtg tgcaggggct 4020 tcctcatgcg cgtggaattc cagaagatgg tgcagagaag ggagtccatc ttctgcatcc 4080 agtacaacat ccgagccttc atgaacgtca agcactggcc ctggatgaaa ctcttcttca 4140 agatcaagcc tctgctgaag agcgcagaga ccgagaagga gatggccacc atgaaggag 4200 agttccagaa aaccaaggag gaactcgcca agtcagaggc aaagaggaag gaactggagg 4260 aaaagatggt gactctggta caagagaaga acgacctgca gctccaagta caagctgaaa 4320 gtgaaaactt gttggatgca gaggaaagat gtgaccagct gatcaaagcc aagtttcagc 4380 tggaggccaa aatcaaggag gtgactgaga gagctgagga tgaggaagag atcaatgccg 4440 agctgacggc caagaagagg aaactggagg atgagtgctc agaactgaag aaagacatcg 4500 atgaccttga gctgacactg gccaaggttg aaaaggagaa acatgccaca gagaacaagg 4560 ttaaaaacct tactgaggaa ctggccggct tggatgaaac cattgcgaag ttgaccaggg 4620 agaagaaggc cctccaggag gcccaccagc agaccctgga tgacctccaa gctgaagagg 4680 acaaagtcaa ctccttaagc aaaatcaaga gcaaactgga acagcaggtg gacgatctgg 4740 aaagctccct agaacaagag aagaagctcc gggtcgacct ggaaaggaat aaaaggaagc 4800 ttgagggtga cttgaagctt gcccaggagt ccatactgga tctggagaat gacaagcaac 4860 agctggatga aaggctcaag aagaaggatt ttgagtccag tcagctgcag agcaaagtgg 4920 aagatgagca gaccctgggc ctccagtttc agaagaaaat taaagagctc caggctcgaa 4980 tcgaggagct ggaggaagag attgaggccg agagggccac ccgcgccaag acggagaagc 5040 agcgcagcga ctacgcccgg gagctggagg agctgagcga gcggctggag gaggcggggg 5100 gcgtcacatc cacgcagatc gagctgaaca agaagcgcga ggccgagttc ctgaagctgc 5160 gccgggacct ggaggaggcc accctgcagc acgaggccac agtggctgcg ctgaggaaga 5220 agcacgcgga cagcgtggct gagctggggg agcagatcga caacctgcag agggtcaagc 5280 agaagctgga gaaggagaag agcgaattca agctggagct ggacgacctg gccggcaacg 5340 tggagagcgt gtccaaggcg aaggccaacc tggaaaaaat ctgccgcacc ctggaggatc 5400 agctaagcga ggccaggggc aagaacgagg aaatccagag gagcatgagc gagctgacca 5460 tgcagaagtc ccgtcttcag acggaggccg gtgagctgag tcgtcaactc gaagagaaag 5520 agagcacagt atcacagctt tccagaagca agcaagcgtt tacccagcaa atagaagagc 5580 tcaagaggca gctggaggag gagagcaagg ccaagaacgc cctggcgcac gccctgcagt 5640 cctcccgcca cgactgcgac ctgctgcggg aacagtatga ggaggagcag gaagccaagg 5700 ccgagctgca gagggcgctg tccaaggcca acagcgaggt tgcccagtgg aggaccaagt 5760 acgagacgga cgccatccag cgcacggagg agctggagga ggccaagaaa aagcttgctc 5820 agcgccttca ggattccgag gagcaggtgg aggcagtgaa cgccaagtgt gcctccctgg 5880 agaagaccaa gcagaggctg caagcggagg tggaggacct gatggtggac gtggacagag 5940 ccaactctct ggctgctgcg ctggacaaga agcagagaaa cttcgacaag gtgctcgccg 6000 agtggaaaac aaagtgtgag gagagccagg cagagctgga ggcagctctg aaggaatccc 6060 gctccttgag caccgagctc ttcaagctga aaaatgccta cgaagaagcc ttagatcaac 6120 ttgaaactgt gaagcgagaa aataagaact tggagcagga gatagcagat ctcacagaac 6180 aaattgctga gaacggtaaa accatccatg aactggagaa atcaagaaag cagatcgagc 6240 tggagaaggc tgatatccag ctggctcttg aggaagcaga ggccgccctt gagcacgaag 6300 aggccaagat cctccgaatc cagctggaac tgacccaggt gaaatcggaa atcgacagga 6360 agatggctga gaaagacgaa gagatcgagc agctcaagag gaactaccag agaaccgtgg 6420 agacgatgca gagcgccttg gacgccgagg tgcggagccg caacgaggcc atcaggatca 6480 agaagaagat ggagggggac ctgaacgaga tcgagatcca gctgagccac gccaaccgcc 6540 aggccgcgga gaccctcaaa cacctccggg gcgtccaagg acagctgaag gacacccagc 6600 tccacctgga cgacgctctc cgaggccagg aggacctgaa ggagcagctg gccatcgtgg 6660 agcgcagagc cagcctgctg caggccgagg tggaggagct gcgggcctcc ctggagcaga 6720 cggagagggc ccggaaactg gcagagcagg agctcctgga cgccaacgag agggtgcagc 6780 tgctccacac ccagaacacc agcctcatcc acaccaagaa gaagctggag acggacctga 6840 tgcagctcca gagcgaggtc gaggacgcca gcagggatgc gaggaacgcc gaggagaagg 6900 cgaagaaggc caccaccgac gcggccatga tggccgagga gctgaagaag gagcaggaca 6960 ccagcgccca cctggagcgg atgaagaaga acctggagca gacggtgaag gacctgcagc 7020 accgcctgga cgaggcggaa cagttggccc tgaagggcgg gaagaagcag atccagaagc 7080 tggaggccag aatccgagag ctggagtttg agcttgaggg ggagcagaag aagaacacgg 7140 agtctgtcaa gggcctcagg aaatacgagc ggcgggtcaa ggagttaact taccagagtg 7200 aagaggacag gaagaatgtg ctgagattac aggacctggt ggacaaactt caagcgaagg 7260 tcaagtccta caagaggcag gcggaggagg ctgatgaaca agccaatgct catctcacca 7320 agttccgaaa agctcagcat gagctcgagg aggctgaaga acgggctgat atcgccgaat 7380 ctcaggtcaa taagcttcgc gcaaagaccc gagacttcac ctccagccgg atggtggtcc 7440 atgagagcga agaggattac aaggatgacg acgataagtg agaattcact cctcaggtgc 7500 aggctgccta tcagaaggtg gtggctggtg tggccaatgc cctggctcac aaataccact 7560 gagatctttt tccctctgcc aaaaattatg gggacatcat gaagcccctt gagcatctga 7620 cttctggcta ataaaggaaa tttattttca ttgcaatagt gtgttggaat tttttgtgtc 7680 tctcactcgg aaggacatat gggagggcaa atcatttaaa acatcagaat gagtatttgg 7740 tttagagttt ggcaacatat gccatatgct ggctgccatg aacaaaggtg gctataaaga 7800 ggtcatcagt atatgaaaca gccccctgct gtccattcct tattccatag aaaagccttg 7860 acttgaggtt agattttttt tatattttgt tttgtgttat ttttttcttt aacatcccta 7920 aaattttcct tacatgtttt actagccaga tttttcctcc tctcctgact actcccagtc 7980 atagctgtcc ctcttctctt atgaagatcc ctcgacctgc agttggggtt gcgccttttc 8040 caaggcagcc ctgggtttgc gcagggacgc ggctgctctg ggcgtggttc cgggaaacgc 8100 agcggcgccg accctgggtc tcgcacattc ttcacgtccg ttcgcagcgt cacccggatc 8160 ttcgccgcta cccttgtggg ccccccggcg acgcttcctg ctccgcccct aagtcgggaa 8220 gt; accctcgcag acggacagcg ccagggagca atggcagcgc gccgaccgcg atgggctgtg 8340 gccaatagcg gctgctcagc agggcgcgcc gagagcagcg gccgggaagg ggcggtgcgg 8400 gaggcggggt gtggggcggt agtgtgggcc ctgttcctgc ccgcgcggtg ttccgcattc 8460 tgcaagcctc cggagcgcac gtcggcagtc ggctccctcg ttgaccgaat caccgacctc 8520 tctccccagg gggatccacc ggagcttacc atgaccgagt acaagcccac ggtgcgcctc 8580 gccacccgcg acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt cgccgactac 8640 cccgccacgc gccacaccgt cgatccggac cgccacatcg agcgggtcac cgagctgcaa 8700 gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc 8760 gccgcggtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag 8820 atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggaa 8880 ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc 8940 tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcg 9000 gccgagcgcg ccggggtgcc cgccttcctg gagacctccg cgccccgcaa cctccccttc 9060 tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc 9120 tggtgcatga cccgcaagcc cggtgcctga cgcccgcccc acgacccgca gcgcccgacc 9180 gaaaggagcg cacgacccca tgcatcggta cctttaagac caatgactta caaggcagct 9240 gtagatctta gccacttttt aaaagaaaag gggggactgg aagggctaat tcactcccaa 9300 cgaagacaag atctgctttt tgcttgtact gggtctctct ggttagacca gatctgagcc 9360 tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga 9420 gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga 9480 cccttttagt cagtgtggaa aatctctagc agtagtagtt catgtcatct tattattcag 9540 tatttataac ttgcaaagaa atgaatatca gagagtgaga ggaacttgtt tattgcagct 9600 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca 9660 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgctgcagc 9720 ccaagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca 9780 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg 9840 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg 9900 tgccagcgga tccgcatctc aattagtcag caaccatagt cccgccccta actccgccca 9960 tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt 10020 ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag 10080 gcttttttgg aggcctaggc ttttgcaaaa agctaacttg tttattgcag cttataatgg 10140 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc 10200 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggatcc gctgcattaa 10260 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg 10320 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag 10380 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa 10440 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc 10500 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca 10560 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg 10620 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct 10680 caatgctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt 10740 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 10800 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc 10860 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac 10920 actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga 10980 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc 11040 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg 11100 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca 11160 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt 11220 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca 11280 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg 11340 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca 11400 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt 11460 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt 11520 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca 11580 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca 11640 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga 11700 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact 11760 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga 11820 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg 11880 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc 11940 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga 12000 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat 12060 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt 12120 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt 12180 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctg 12238 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> pMYH3 F-primer <400> 3 aaaagctcag catgagctcg a 21 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> pMYH3 R-primer <400> 4 agggtcagga accatgaaaa t 21 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> pMYH1 F-primer <400> 5 gttctgaaga gggtggtac 19 <210> 6 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> pMYH1 R-primer <400> 6 agatgcggat gccctcca 18 <210> 7 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> pMYH2 F-primer <400> 7 gggctcaaac tggtgaagc 19 <210> 8 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> pMYH2 R-primer <400> 8 agatgcggat gccctcca 18 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> pMYH13 F-primer <400> 9 cacagggctc tggccgacat 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> pMYH13 R-primer <400> 10 cgtgcgcaca ggggtgtagt 20 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> pADPRM F-primer <400> 11 catcctgaga ccgtgccttc a 21 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> pADPRM R-primer <400> 12 ttccgcattt gggttgtgct 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> pSCO1 F-primer <400> 13 tcctcacgga ctcggggttt 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> pSCO1 R-primer <400> 14 gtggggtctc tgctgccctt 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> pTMEM220 F-primer <400> 15 cccagacgca gaactgtggg 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> pTMEM220 R-primer <400> 16 gttgtatgcc aagccggcag 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> pENSSSCG00000029441 F-primer <400> 17 tcgtgctgga gcaggaggag 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> ≪ 223 > pENSSSCG00000029441 R-primer <400> 18 aggtgtctgt ggccttgggg 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> pENSSSCG00000018006 F-primer <400> 19 agaaccagcc cttcgatgcc 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> pENSSSCG00000018006 R-primer <400> 20 tggcatacac atcctccggc 20 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> pGAPDH F-primer <400> 21 gggcatgaac catgagaagt 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> pGAPDH R-primer <400> 22 gggcatgaac catgagaagt 20 <210> 23 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> pMYH3 F-primer <400> 23 ccgagagctg gagtttga 18 <210> 24 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> pMYH3 R-primer <400> 24 ctcccatatg tccttccgag t 21 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Myh7 F-primer <400> 25 agtcccaggt caacaagctg 20 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Myh7 R-primer <400> 26 ttccacctaa agggctgttg 20 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Myh2 F-primer <400> 27 agtcccaggt caacaagctg 20 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Myh2 R-primer <400> 28 gcatgaccaa aggtttcaca 20 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Myh1 F-primer <400> 29 agtcccaggt caacaagctg 20 <210> 30 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Myh1 R-primer <400> 30 cacattttgc tcatctcttt g 21 <210> 31 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Myh4 F-primer <400> 31 agtcccaggt caacaagctg 20 <210> 32 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Myh4 R-primer <400> 32 tttctcctgt cacctctcaa ca 22 <210> 33 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Myoglobin F-primer <400> 33 gcaaggccct ggagctcttc 20 <210> 34 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Myoglobin R-primer <400> 34 gcttggtggg ctggacagtg 20 <210> 35 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Tnnt1 F-primer <400> 35 cccccgaaga ttccagaagg 20 <210> 36 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Tnnt1 R-primer <400> 36 tgcggtcttt tagtgcaatg ag 22 <210> 37 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Tnni1 F-primer <400> 37 atgccggaag ttgagaggaa a 21 <210> 38 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Tnni1 R-primer <400> 38 tccgagaggt aacgcacctt 20 <210> 39 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Tnnc1 F-primer <400> 39 gcggtagaac agttgacaga g 21 <210> 40 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Tnnc1 R-primer <400> 40 ccagctcctt ggtgctgat 19 <210> 41 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Aldoa F-primer <400> 41 actctctgct gaccgggctc t 21 <210> 42 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Aldoa R-primer <400> 42 aatgcttccg gtggactcat 20 <210> 43 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Pvalb F-primer <400> 43 atcaagaagg cgataggagc c 21 <210> 44 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Pvalb R-primer <400> 44 ggccagaagc gtctttgtt 19 <210> 45 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Tnnt3 F-primer <400> 45 ggaacgccag aacagattgg 20 <210> 46 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Tnnt3 R-primer <400> 46 tggaggacag agcctttttc tt 22 <210> 47 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Tnni2 F-primer <400> 47 agagtgtgat gctccagata gc 22 <210> 48 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Tnni2 R-primer <400> 48 agcaacgtcg atcttcgca 19 <210> 49 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Tnnc2 F-primer <400> 49 atggcagcgg tactatcgac t 21 <210> 50 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Tnnc2 R-primer <400> 50 ccttcgcatc ctctttcatc tg 22 <210> 51 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> GAPDH F-primer <400> 51 gaagggcatc ttgggctaca c 21 <210> 52 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> GAPDH R-primer <400> 52 gcagcgaact ttattgatgg tatt 24 <210> 53 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> CD36 F-primer <400> 53 aatggcacag acgcagcct 19 <210> 54 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> CD36 R-primer <400> 54 ggttgtctgg attctgga 18 <210> 55 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LPL F-primer <400> 55 gtacctgaag actcgctctc 20 <210> 56 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LPL R-primer <400> 56 agggtgaagg gaatgttctc 20 <210> 57 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Fabp4 F-primer <400> 57 gatgcctttg tgggaacctg 20 <210> 58 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Fabp4 R-primer <400> 58 tcctgtcgtc tgcggtgatt 20 <210> 59 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Fto F-primer <400> 59 gtcagagaga aggccaatga 20 <210> 60 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Fto R-primer <400> 60 tagcagtctc cctggtgaag 20 <210> 61 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Pgc1 alpha F-primer <400> 61 ccctgccatt gttaagacc 19 <210> 62 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Pgc1 alpha R-primer <400> 62 tgctgctgtt cctgttttc 19 <210> 63 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Adiponectin F-primer <400> 63 aatggcacac caggccgtga t 21 <210> 64 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Adiponectin R-primer <400> 64 tctccaggct ctcctttcct g 21 <210> 65 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> MYH3 variation F-primer <400> 65 tggtctttcc taattggtga cat 23 <210> 66 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> MYH3 variation R-primer <400> 66 agttttgagc aaggcttttg tt 22
Claims (4)
1. A method for improving the meat quality of a transformant, comprising the step of transforming a recombinant vector comprising the MYH3 gene consisting of the nucleotide sequence of SEQ ID NO: 1 into embryos of an individual other than a human.
2. The method of claim 1, wherein the subject is a mammal.
3. The method of claim 2, wherein the mammal is a mouse.
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