KR100615803B1 - Detection of DNA Mutations in Porcine ACADM Gene Transcription Regulators and Their Use as DNA Markers for Early Selection of Pigs with High Myocardial Fat - Google Patents
Detection of DNA Mutations in Porcine ACADM Gene Transcription Regulators and Their Use as DNA Markers for Early Selection of Pigs with High Myocardial Fat Download PDFInfo
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Abstract
본 발명은 ACADM(acyl-coA dehydrogenase, medium-chain) 유전자에서 나타나는 DNA 다형성 분석 결과와 이를 활용한 돼지의 근내지방도와의 연관성 분석에 관한 것으로서, 근내지방도가 높은 돼지를 조기선발 할 수 있는 DNA 마커의 개발에 관한 것이다.The present invention relates to a DNA polymorphism analysis result of the ACADM (acyl-coA dehydrogenase, medium-chain) gene and the correlation analysis between pig intramuscular fat using the same, and a DNA marker capable of early selection of pigs with high intramuscular fat. It is about the development of.
본 발명은, 돼지 ACADM 유전자의 조절부위에서 염기변이 부위를 발견하였고, 이 부위를 증폭할 수 있는 primer를 제작하여 증폭한 후 PCR-RFLP 분식을 실시하였다.In the present invention, the base mutation site was found in the regulatory region of the porcine ACADM gene, a primer was prepared and amplified by PCR-RFLP.
근내지방도가 측정된 각각의 개체와 유전자형과의 연관성 분석 결과, 근내지방도와 높은 연관성을 보이는 유전자형을 발견하였으며, 이는 근내지방도가 높을것으로 예상되는 돼지를 조기에 선발할 수 있는 검사 방법으로 활용할 수 있다.As a result of analysis of the association between the individual and the genotypes, the genotypes were found to be highly associated with intramuscular fat, which can be used as an early screening method for pigs that are expected to have high intramuscular fat. .
돼지, 근내지방도, ACADM, 유전자, 다형성, DNA 마커, 조기선발Swine, myopia, ACADM, gene, polymorphism, DNA marker, early selection
Description
도 1은 본 발명에서 밝혀낸 돼지 ACADM 유전자 내 조절부위의 염기서열로서, 시작코돈으로부터 상위부위에 위치하는 41 (R), 77 (K), 100 (S), 450 (Y)번째 부위에서의 점돌연변이와 587~590번의 4 bp의 결실변이를 확인할 수 있다.1 is a nucleotide sequence of a regulatory region in the porcine ACADM gene disclosed in the present invention, which is located at the 41 (R), 77 (K), 100 (S), and 450 (Y) sites located above the start codon. Mutations and deletion mutations of 4 bp 587-590 can be identified.
도 2는 돼지 ACADM 유전자 내 조절부위의 각각의 서열을 나타내는 염기서열 분석결과를 보여주는 것으로, 도 1에 표시된 4개 부위에서 변이가 발생하였음을 확인할 수 있다.Figure 2 shows the sequencing results showing the sequence of each sequence of the regulatory region in the pig ACADM gene, it can be seen that the mutation occurred in the four sites shown in FIG.
도 3은 돼지 ACADM 유전자의 조절부위를 증폭한 결과 (A)와 변이 2를 대상으로 SmaI 제한효소를 이용하여 PCR-RFLP 분석을 수행한 결과 (B)이다.Figure 3 shows the results of amplifying the regulatory region of porcine ACADM gene (A) and mutation 2, PCR-RFLP analysis using Sma I restriction enzyme (B).
본 발명은 돼지의 ACADM(acyl-coA dehydrogenase, medium-chain) 유전자 전사조절부위에서 돌연변이의 탐지와 이를 이용하여 근내지방도와의 연관성 분석을 통한 높은 근내지방도를 나타낼 수 있는 돼지의 조기 선발방법에 관한 것이다. 더욱 상세하게는 돼지 ACADM 유전자를 포함하는 BAC clone을 선발하고, DNA 염기서열분석을 통하여 전사조절부위의 DNA 염기서열을 확보하였다. 국내에서 주로 사육되는 4개 품종 (Landrace, Large White, Duroc, Birkshire)와 한국재래돼지를 이용하여 전사조절부의를 PCR로 증폭하고, DNA 염기서열분석, 다중염기서열비교등을 통하여 4개 지역에서 3개의 점 돌연변이와 1개의 결실돌연변이를 탐지하였다. 이중 시작코돈으로부터 상위방향으로 77번째 존재하는 점 돌연변이 (G 또는 T)를 이용하여 근내지방도가 측정된 F2 세대 486두의 근내지방도와의 연관성 분석을 시행에 하여 유의적인 연관성(P〈0.01)을 파악하였다. 이와 같은 방법으로 ACADM 유전자의 전사조절부위 변이들을 이용하여 향상된 육질을 갖는 개체를 확인할 수 있는 DNA 마커를 개발함으로서, 시간적, 경제적으로 보다 손쉽게 근내지방도가 높은 돼지의 조기검사 및 선발하기 위한 방법에 관한 것이다.The present invention relates to a method for early selection of pigs that can exhibit high intramuscular fat through detection of mutations in the ACADM (acyl-coA dehydrogenase, medium-chain) gene transcription control region of pigs and analysis of the association with intramuscular fat using the same. will be. More specifically, the BAC clone including the pig ACADM gene was selected and DNA sequencing of the transcriptional regulatory region was obtained through DNA sequencing. PCR amplification of the transcriptional regulator using four varieties (Landrace, Large White, Duroc, Birkshire) and Korean native pigs, and DNA sequencing and multiple nucleotide sequences in four regions. Three point mutations and one deletion mutation were detected. To the upper direction from the double start codon 77th present point mutation (G or T) significant correlation (P <0.01) by the performed correlation analysis of intramuscular fat help of the F 2 generation 486 with intramuscular local road is measured both using the I figured out. In this way, by developing DNA markers that can identify individuals with improved meat quality by using transcriptional control region mutations of the ACADM gene, a method for early screening and selection of pigs with high intramuscular fat more easily in time and economics. will be.
돼지는 전 세계적으로 가장 중요한 동물성 단백질 공급원이며, 각각의 시대 상황 및 사람들의 기호에 맞게 사육되어져 왔다. 최근 들어 소비자의 의식수준이 향상됨으로서, 보다 좋은 육질의 돈육을 선호함에 따라 육질형질인 근내지방도, 육색, 연도 및 보수력 등에 관련된 다각적인 연구 및 육종 기술의 개발이 이루어지고 있다. 특히, 근내지방도는 돼지의 육질을 결정하는 가장 중요한 여러 요인들 중 하나로 근내지방도가 높을수록 육질이 부드러워지고 풍미가 좋아지며, 육색이나 보수 력이 향상된다.Pigs are the world's most important source of animal protein and have been reared to suit the needs of each age and situation. Recently, as consumer's level of consciousness has been improved, various researches and development of breeding techniques related to meat quality such as intramuscular fat, meat color, year and water retention have been made according to preference for better quality pork. In particular, intramuscular fat is one of the most important factors that determine the quality of pigs. The higher the intramuscular fat, the smoother the meat, the better the flavor, and the better the color and water retention.
현재 근내지방도의 확인은 돼지를 100 kg 내외까지 사육한 후 도살하고 시료를 채취하여 생화학적 방법으로 측정할 수밖에 없다. 그러나 100 kg 내외까지 사육하는데 필요한 시간적, 경제적 비용부담이 매우 크기 때문에 적지 않은 애로 요인이 되고 있다. 또한 최근에 소에서 초음파를 이용한 마블링지수의 측정을 이용하고 있으나, 아직 돼지를 대상으로는 사용되지 못하고 있다. 특히 돼지에서는 마블링지수와 근내지방도간의 연관성이 높지 않아 초음파검사만으로는 높은 근내지방도에 대한 정확한 확인이 어렵다. 이에 따라 근내지방도가 높은 종돈의 조기 선발을 위한 신속하고 간편한 방법으로써 DNA 마커의 활용이 요구되고 있다.At present, the determination of intramuscular fat can only be measured by biochemical methods by slaughtering pigs, collecting samples, and raising pigs to around 100 kg. However, the time and economic cost of raising up to about 100 kg is very high, which is a cause for many difficulties. In addition, recently, the measurement of the marbling index using ultrasound in cattle, but has not yet been used in pigs. In particular, the correlation between the marbling index and intramuscular fat in pigs is not high, so it is difficult to accurately identify high intramuscular fat by ultrasonography alone. Accordingly, the use of DNA markers is required as a quick and simple method for the early selection of high sows.
유전공학의 발달에 따라 DNA를 이용한 다양한 실험기법들이 개발되었으며, 이에 따라 가축의 유전적 특징이나 이와 연관된 다양한 DNA typing 기술이 개발되어 돼지의 육종에서도 많이 이용되고 있는 실정이다. 특히, 가축의 경제형질과 연관된 양적형질좌인 (quantitative trait loci)를 밝히고 그 위치에 존재하는 유전자들의 DNA 다형성과 경제형질과의 연관성 분석을 위한 많은 노력들이 집중되고 있다. 최근 들어 돼지 6번 염색체 내에 근내지방도를 포함한 육질과 연관된 경제 형질에 영향을 주는 QTL이 존재한다고 보고되어 있다. Gerbens(2000)와 De Koning 등(2000)은 염색체 6번에 근내지방도에 관련된 QTL이 존재한다고 보고하였고, Grandflek 등(2000)은 6번 염색체 초위성체 마커인 SW1823과 S0228사이에 존재한다고 보호하였으며, Ovilo 등(2002)은 6번 연색체 96cM∼108cM에 존재한다고 보고하였다.According to the development of genetic engineering, various experimental techniques using DNA have been developed. Accordingly, genetic characteristics of livestock and various DNA typing techniques related to livestock have been developed, and are widely used in breeding of pigs. In particular, a great deal of effort has been focused on identifying the quantitative trait loci associated with livestock economic traits and analyzing the association between the DNA polymorphism and economic traits of the genes present at that location. Recently, QTL has been reported to affect economic traits related to meat quality, including intramuscular fat, in pig chromosome 6. Gerbens (2000) and De Koning et al. (2000) reported the presence of QTL related to intramuscular adipose on chromosome 6, and Grandflek et al. (2000) protected that they exist between SW1823 and S0228, the chromosome 6 satellite markers. Ovilo et al. (2002) reported that they are present in the chromosome 6 96cM-108cM.
ACADM은 근내지방도와 관련된 QTL이 존재하는 것으로 보고된 6번 염색체 108cM주위에 존재하며 지방대사와 밀접한 연관을 가지고 있다.ACADM is located around 108cM on chromosome 6, which has been reported to have a QTL associated with intramuscular fat and is closely associated with fat metabolism.
따라서 본 발명의 목적은 도축 후 근내지방도 양을 판별하는 기존의 시간적, 경제적으로 비효율적인 방법에서 벗어나, 높은 근내지방도를 갖는 돼지를 조기에 선택적으로 선발 할 수 있는 분자유전학적인 기법을 확립하기 위한 DNA 변이의 탐색과 이를 활용한 DNA 마커의 개발에 있다.Therefore, an object of the present invention is to escape from the existing time- and economically inefficient method for determining the amount of intramuscular fat after slaughter, and to establish a molecular genetic technique that can selectively select pigs with high intramuscular fat early. In the search for mutations and the development of DNA markers using them.
근내지방도와 연관된 돼지 6번 염색체에 위치하는 ACADM 유전자의 조절부위 내에서 발견된 DNA 변이의 탐지와 이를 활용한 한국재래돼지와 Landrace와의 교잡에 의해 생산된 F2 486두와의 근내지방도 유의성 검정 결과 높은 유의성를 보이는 DNA 마커를 개발하여 핵돈군, 증식돈군 및 상업돈군애 대해 우수한 근내지방도를 나타내는 돼지의 조기선발에 이용하고자 한다.Detection of DNA mutations found in the regulatory region of the ACADM gene located on pig chromosome 6 associated with intramuscular fat and the significance of the intramuscular fat with F 2 486 produced by hybridization between Korean native pigs and Landrace. DNA markers showing high significance will be developed and used for early selection of pigs that show excellent intramuscular fat for nucleated pigs, multiplied pigs and commercial pigs.
본 발명은 한국재래돼지와 Landrace 각각 20두와 두 품종의 교잡으로 생산된 F2 486두의 혈액으로부터 DNA를 추출하는 단계; ACADM 유전자의 조절부위를 증폭하기 위한 primer를 제작하여 증폭하는 단계; 두 품종간의 염기서열을 분석하여 품종 간의 변이를 확인하는 단계; F2 486두의 DNA로부터 증폭된 PCR 산물에 SmaI 제한효소를 처리하여 절단하는 단계; F2 486두 각각에서 분석된 근내지방도와 염기변이와의 연관성 분석 단계로 구성된다.The present invention comprises the steps of extracting DNA from the blood of F 2 486 produced by the hybridization of 20 breeds and two breeds of Korean native pigs and Landrace, respectively; Preparing and amplifying a primer for amplifying the regulatory region of the ACADM gene; Analyzing the nucleotide sequence between the two varieties to identify the variation between the varieties; F 2 further comprising: cutting process the Sma I restriction enzyme to a PCR product amplified from the DNA of two 486; The analysis of the association between intramuscular fat and nucleotide variations analyzed in each of F 2 486 heads.
제1단계, 돼지 혈액으로부터 DNA 추출First step, DNA extraction from pig blood
한국재래돼지와 Landrace 각각 20두와 이 두 품종의 교잡으로부터 생산된 F2 486두로부터 혈액을 채취하여 EDTA가 처리된 튜브에 넣어서 응고되지 않도록 잘 섞어준 후 약 300 μl를 이용하여 Genomic DNA 추출 키트 (Promega, USA)로 DNA를 추출하였으며, 4℃에 보관하였다.Blood samples were collected from 20 Korean pigs and Landrace and 2 486 F 2 produced from the hybridization of these two varieties.They were mixed in an EDTA-treated tube and mixed well to prevent coagulation. DNA was extracted (Promega, USA) and stored at 4 ℃.
제2단계. primer 제작 및 PCR 증폭Second step. Primer Preparation and PCR Amplification
돼지 ACADM 유전자 내 조절부위를 증폭하기 위하여 분석된 염기서열을 토대로 forward primer (5'-CAG TAA TAG ACA GCC TAG GG-3') 및 reverse primer (5'-CCT AAA CAT CGC TGC CAT GC -3')를 제작하였다. 상기 준비된 한국재래돼지와 Landrace 각각 20두의 DNA 50ng과 10 μM의 primer 2μl에 10x PCR buffer 5μl 25mM MgCl2 4μl 각각2.5mM dNTPs 4μl 와 2.5unit의 Taq DNA 중합효소(TaKaRa, USA) 및 3차 증류수를 첨가하여 총 50 μl 반응액이 되도록 하였다. 반응은 94℃에서 5분 동안 초기반응을 시키고 94℃에서 1분 (denaturation), 60℃에서 1분(annealing), 72℃에서 1분 (extension)의 순서대로 5주기를 수행하고 94℃에서 1분(denaturation), 58℃에서 1분 (annealing), 72℃에서 1분 (extension)의 순서대로 35주기를 수행한 후 72℃에서 5분간 1회를 수행하여 종료하였으며, 증폭된 PCR 산물은 1% 아가로스 겔 상에서 반응유무를 확인하였다.Forward primer (5'-CAG TAA TAG ACA GCC TAG GG-3 ') and reverse primer (5'-CCT AAA CAT CGC TGC CAT GC -3') based on the sequence analyzed to amplify the regulatory region in porcine ACADM gene ) Was produced. 10 × PCR buffer 5μl 25mM MgCl 2 4μl 4μl and 2.5units of Taq DNA polymerase (TaKaRa, USA) and tertiary distilled water Was added to make a total of 50 μl reaction solution. The reaction was performed at 94 ° C. for 5 minutes, followed by 5 cycles in the order of 1 minute (denaturation) at 94 ° C., 1 minute (annealing) at 60 ° C., and 1 minute (extension) at 72 ° C., followed by 1 at 94 ° C. 35 cycles were performed in order of denaturation, 1 min at 58 ° C., 1 min at 72 ° C., and then 5 min at 72 ° C., and the amplified PCR product was 1 The reaction was confirmed on% agarose gel.
제3단계. 염기서열 분석Third step. Sequencing
상기 두 품종의 PCR 산물의 염기서열을 분석하기 위하여 각각의 최종산물을 QIAEX
제4단계. 제한효소 (restriction enzyme) 처리Fourth step. Restriction enzyme treatment
앞서 돼지 ACADM 유전자 내 조절부위를 증폭하는 과정을 그대로 사용하여 한국 재래돼지와 Landrace의 교잡으로 생산된 F2 486두를 증폭한 후 PCR 산물에 K지역을 인지하는 SmaI(CCC GGG) 제한효소를 첨가하여 PCR-RFLP 분석을 실시하였다. 3 μl의 PCR 산물에 3unit의 SmaI 제한효소, 0.1배의 10x buffer 및 3차 증류수를 첨가하여 총 10 μl 반응액이 되도록 한 후 30℃에서 4시간 동안 반응시켰다. 그 후 4% 아가로즈 겔 상에서 전기영동을 실시한 결과 3개의 형태 (AA, AB, BB)가 확인되었으며, 그 결과를 도 3에 나타내었다.Prior to accept the process for amplifying a pig ACADM gene within the control region for Sma I (CCC GGG) restriction enzyme that recognizes the K area in the PCR product was amplified for the F 2 486 both produced by hybridization of Korea native pig and Landrace PCR-RFLP analysis was performed by addition. To 3 μl of the PCR product, 3 units of Sma I restriction enzyme, 0.1 × of 10 × buffer and tertiary distilled water were added to make a total of 10 μl reaction solution and then reacted at 30 ° C. for 4 hours. After electrophoresis on 4% agarose gel, three forms ( AA, AB, BB ) were identified, and the results are shown in FIG. 3.
제5단계. 염기변이와 근내지방도와의 연관성 분석5th step. Analysis of association between base variation and intramuscular fat map
도 3에서 제시된 3개의 유전자형과 생장형질 및 지방관련 형질들과의 연관성은 SAS 통계 프로그램의 GLM 과정을 통하여 분석하였으며, 연관성 분석을 위해서 아래의 모델식을 사용하였다.The association between the three genotypes and growth traits and fat-related traits shown in FIG. 3 was analyzed through the GLM process of the SAS statistical program, and the following model equation was used for the correlation analysis.
Y = sex + genotype + covariate + eY = sex + genotype + covariate + e
여기서 Y는 표현형 값을, sex는 성의 효과를 그리고 genotype은 유전자형의 효과를 나타내는 것이며, covariate(공변량)은 30주령 체중에 대한 등지방두께와 총지방량의 회귀계수로써 등지방두께와 총지방량에 대해서만 사용하였으며, e는 잔차를 의미한다.Where Y is the phenotype value, sex is the sex effect, genotype is the genotype effect, and covariate is the regression coefficient of back fat thickness and total fat mass for 30-week-old body weight, and only for back fat thickness and total fat mass. E is the residual.
3개의 유전자형 중에서 AA형은 30주령에서 5% 유의차를 보이며 정의 상관관계를 보였으며, 근내지방도와는 1 % 유의차를 보이며 정의 상관관계를 보인 반면 등지방두께는 3개의 유전형과는 어떠한 유의성도 보이지 않았음을 표 1에서 확인할 수 있다. 이런 결과는 AA형과 AB형이 등지방두께와는 독립적으로 높은 근내지방도를 나타내고 있어 높은 근내지방도 개체의 선발을 위한 DNA 마커로써 활용할 수 있다.Among the three genotypes, AA type showed positive correlation with 5% difference at 30 weeks of age, positive correlation with 1% difference with intramuscular fat level, while back fat thickness had no significant correlation with three genotypes. It can be seen in Table 1 that not shown. These results suggest that type AA and AB show high intramuscular fat independently of backfat thickness, so that high intramuscular fat can be used as a DNA marker for the selection of individuals.
이상의 실시 예를 통하여 입증된 바와 같이, 본 발명은 분자유전학적 기법을 이용하여 근내지방도가 높은 집단과 낮은 집단에서차별화 되는 염기서열을 분석하고 그로부터 돼지의 주요형질, 특히 돼지의 근내지방도와 연관된 ACADM 유전자 내 조절부 위의 유전자형을 발견하였다.As demonstrated through the above examples, the present invention analyzes the nucleotide sequences that are differentiated in the high and low intramuscular fat group using molecular genetic techniques, and from there the ACADM associated with the major traits of pigs, especially the intramuscular fat of pigs. A genotype above the regulatory region was found.
ACADM 유전자 내 조절부위의 점 돌연변이는, 도축 후 근내지방도를 판별하는 기존의 방법과는 다르게 근내지방도가 높은 돼지를 조기에 선발할 수 있는 DNA 마커로써 뛰어난 효과가 있으므로, 축산업에 있어서 매우 유용한 발명일 것이다.The point mutation of the regulatory region in the ACADM gene is a very useful invention for livestock industry because it has an excellent effect as a DNA marker for early selection of pigs with high intramuscular fat, unlike conventional methods for determining intramuscular fat after slaughter. will be.
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| KR20040039059A (en) * | 2002-10-30 | 2004-05-10 | 김철욱 | Detection method of DNA marker associated to average daily gain(ADG), backfat thickness(BFT) , and loin muscle area(LMA) in pig. |
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