한우와 Holstein종의 κ -casein 유전자의 발현조절부위의 염기서열 분석

상병찬,류승희,이상훈,Sang, Byung Chan,Ryoo, Seung Heui,Lee, Sang Hoon 충남대학교 농업과학연구소 2000 농업과학연구 Vol.27 No.1

본 연구는 한우와 Holstein종의 $\kappa$ -CN 유전자 발현조절부위, 즉 $\kappa$ -CN gene 5'-flanking region에 대한 특성을 구명하기 위하여 specific primer를 이용하여 PCR(polymerase chain reaction) 방법으로 분석하였다. PCR 증폭 결과, 349bp 크기의 증폭산물을 얻었으며 한우와 Holstein종의 $\kappa$ -CN 유전자의 발현조절부위의 염기서열 분석결과에 있어서는 -82bp에서 -431bp까지 분석되었으며, 종간의 염기서열 비교에서 -386bp에서는 Holstein종에서는 T 염기가 한우에서는 C 염기로 치환되었으며, -241bp와 -192bp에서는 Holstein종에서는 T 및 C 염가가 존재하였으나, 한우에서는 결실되었다. 또한, -183bp에서는 한우에서는 T 염기가 존재하였으나, Holstein종에서는 염기가 결실되어 있음을 알 수 있었다. 또한 한우와 Holstein 종간의 상동성 분석에 있어서는 한우 $\kappa$ -CN 유전자 발현조절부위의 염기서열이 Holstein종과 98.9% 의 상동성을 보였다. This study was carried out to investigate the characterizations of $\kappa$ -CN genes of milk protein. In order to find out the characterizations of $\kappa$ -CN genes, the nucleotide sequences of 5'-flanking regions of $\kappa$ -CN genes were analyzed by PCR(polymerase chain reaction) technique with specific primers in order to investigate the characterizations of these promoters in Korean Native cattle and Holstein. PCR products obtained 349bp fragment and the nucleotide sequences of Korean Native cattle and Holstein of $\kappa$ -CN gene S'-flanking region was analyzed to -82bp from -431bp. On the comparison of each breed, Holstein substituted T$\rightarrow$C at -386bp, and -241bp(T) and -192bp(C) existed, but Korean Native cattle was deleted. Also, Korean Native cattle was existed T at -183bp but Holstein was not. The homology analysis of between Korean Native cattle and Holstein was showed 98.9% homology for $\kappa$ -CN promoter.

Identification of SNPs in TG and EDG1 genes and their relationships with carcass traits in Korean cattle (Hanwoo)

Muhammad Cahyadi(카야디),Dyah Maharani(디아 마하라니),류승희(Seung Heui Ryoo),Seung Hwan Lee(이승환),Jun Heon Lee(이준헌) 충남대학교 농업과학연구소 2012 농업과학연구 Vol.39 No.3

Thyroglobulin (TG) gene was known to be regulated fat cell growth and differentiation and the endothelial differentiation sphingolipid G-protein-coupled receptor 1 (EDG1) gene involves blood vessel formation and known to be affecting carcass traits in beef cattle. The aim of this study was to identify the single nucleotide polymorphisms (SNPs) in both TG and EDG1 genes and to analyze the association with carcass traits in Korean cattle (Hanwoo). The T354C SNP in TG gene located at the 3’ flanking region and c.-312A>G SNP located at 3’-UTR of EDG1 gene were used for genotyping the animals using PCR-RFLP method. Three genotypes were identified in T354C SNP in TG gene and only two AA and AG genotypes were observed for the c.-312A>G SNP in EDG1 gene. The results indicated that T354C SNP in TG gene was not significantly associated with carcass traits. However, the c.-312A>G SNP in EDG1 gene had significant effects on backfat thickness (BF) and yield index (YI). These results may provide valuable information for further candidate gene studies affecting carcass traits in Korean cattle and may use as marker assisted selection for improving the quality of meat in Hanwoo.

소의 체세포핵이식태반과 정상태반간의 차등 발현 유전자 분석

유성란 ( Seong Lan Yu ),정행진 ( Hang Jin Jeong ),상병찬 ( Byung Chan Sang ),류승희 ( Seung Heui Ryoo ),정기철 ( Kie Chul Jung ),윤종택 ( Jong Taek Yoon ),성환후 ( Hwan Hoo Seong ),진동일 ( Dong Il Jin ),이준헌 ( Jun Heon Lee ) 한국동물자원과학회 2008 한국축산학회지 Vol.50 No.5

한우 후보종모우 및 칡소와 흑소에서 MC1R 유전자의 유전자형 분석

진실(Shil Jin),심정미(Jung-Mi Shim),서동원(Dong-Won Seo),정우영(Woo-Young Jung),류승희(Seung-Heui Ryoo),김진호(Jinho Kim),이준헌(Jun-Heon Lee) 충남대학교 농업과학연구소 2011 농업과학연구 Vol.38 No.3

The MC1R (Melanocortin 1 receptor) gene has been known as a causative gene of the coat colors in mammals and responsible for the E (Extension) locus which has three alleles (E<SUP>D</SUP>, E<SUP>+</SUP>, e) that determines coat colors. The dominant allele E<SUP>D</SUP> produces black or brown colors due to the missense mutation and the recessive e allele has frameshift mutation which shows red or yellow coat colors. Whereas the wild type E<SUP>+</SUP> produces variety of colors due to the interaction with A (Agouti) locus. In this study, PCR-RFLP was performed using two restriction enzymes (BsrF I and MspA1 I) in order to obtain MC1R genotypes in Korean brindle cattle and black cattle. The results showed that all of the animals have the E<SUP>+</SUP> alleles, indicating the E<SUP>+</SUP> allele might related with black coat colors. Later on, the experiments expanded to the 260 Korean candidate bulls whether these animals have the same E<SUP>+</SUP> allele. Among 260 samples investigated, 5% (13/260) of the animals had E+e genotypes, indicating the E<SUP>+</SUP> allele is also present in the candidate bulls in a low frequency. Even though we expected that A locus also affect the black coat color in cattle, all the black coat color animals (brindle and black) have E<SUP>+</SUP> alleles in this study. Therefore, the genotyping of the MC1R gene in candidate bulls will recommended be applied for eliminating of black coat colors in Hanwoo population, if the farmers need to have the brown coat colors only.

한국형 홀스타인종 젖소의 BLV env 유전자의 특성분석

정행진(Hang-Jin Jeong),유성란(Seong-Lan Yu),이준헌(Jun-Heon Lee),도창희(Chang-Hee Do),서국현(Guk-Hyun Suh),류승희(Seung-Heui Ryoo),정상일(Sang-Il Chung),상병찬(Byung-Chan Sang) 충남대학교 농업과학연구소 2011 농업과학연구 Vol.38 No.2

This study was performed to investigate the characterization of infectious BLV env gene isolated form Korean Holstein Cattle and to determine its incoming origin. Gp51 region of BLV env gene known as having important role in immunological function was characterized using PCR-RFLP sequencing and phylogenetic analysis. BLV env gene was grouped into PCR-RFLP patterns with three restriction endonucleases including Pvu Ⅱ, BamHI and HaeⅢ, and we identified two new RFLP patterns from nucleotide sequences of each group. Phylogenetic analysis showed that 80% of the Korean Holstein was included in the USA and Japanese group. These results here can provide a valuable information about the character of the BLV env gene and research on infection route of BLV.

홀스타인종 젖소에 있어서 PCR과 ELISA기법을 이용한 BLV 감염진단

정행진(Hang-Jin Jeong),유성란(Seong-Lan Yu),이준헌(Jun-Heon Lee),도창희(Chang-Hee Do),서국현(Guk-Hyun Shu),류승희(Seung-Heui Ryoo),상병찬(Byung-Chan Sang) 충남대학교 농업과학연구소 2011 농업과학연구 Vol.38 No.1

This study was conducted to investigate the farm situation about bovine leukemia virus(BLV) infection that greatly influence productivity in dairy cattle and compare the accuracy of diagnosis for BLV infection between PCR and ELISA techniques. Blood samples of 193 heads from 5 herds in Chungnam and Chungbuk area were used to analyze BLV gene and serum, and the results were obtained as follows. The amplified BLV gene in dairy cattle by PCR technique resulted in 226 bp, 596 bp and 434 bp, respectively, for gag, pol and env, which were well amplified. The infection rates of BLV virus diagnosed by PCR and ELISA techniques ranged from 80.55 to 100% and from 22.22 to 86.95%, respectively, and the infection rates among 5 herds were significantly different in both methods (P<0.05). Further, the average infection rates of 5 herds were 87.05 and 63.21%, respectively, for PCR and ELISA techniques. Kappa statistics for examining consistency of diagnosis by PCR and ELISA techniques showed 0.246, which represents low consistency. Consequently, PCR based BLV technique was considered as a corrective measure for diagnosis of BLV infection in Holstein dairy cattle.

한국 Holstein종 유우집단의 DGAT1 유전자의 특성분석

손지영(Ji-Young Son),정행진(Hang Jin-Jeong),유성란(Seong-Lan Yu),이준헌(Jun-Heon Lee),도창희(Chang-Hee Do),류승희(Seung-Heui Ryoo),상병찬(Byung-Chan Sang) 충남대학교 농업과학연구소 2009 농업과학연구 Vol.36 No.2

본 연구는 한국형 Holstein종 젖소집단의 DGAT1 유전자의 특성을 구명하고, DGAT1의 유전적 다형과 산유형질인 유량 및 유지량 간의 연관성을 구명하여 젖소집단의 유전적 개량을 위한 분자유전학적 접목을 위하여 실험을 실시하였다. Holstein종의 genomic DNA로부터 PCR기법을 이용하여 DGAT1 유전자좌를 specific primers로 증폭한 후, 1.5% agarose gel에 전기영동한 결과 411 bp의 단편이 양호하게 증폭되었음을 확인하였다. DGAT1 유전자의 염기서열을 분석한 결과 DGAT1 Q 대립유전자의 216-218 bp의 염기서열이 AUG(lysine, K)였으나, 동위치의 대립유전자 q의 염기서열은 GCG(alanine, A)로 치환되어 있음을 확인할 수 있었다. 한편 한국 Holstein종과 NCBI에서 보고된 bovine DGAT1 유전자 단편의 염기서열간에는 100% 상동성을 보였다. 한국 Holstein종 유우집단의 DGAT1 유전자형의 분포는 DGAT1 QQ, Qq 및 qq 유전자 분포가 각각 16.43, 36.43 및 47.14%로 qq 유전자형빈도가 다른 유전자형에 비하여 높았으며, 유전자빈도는 DGAT1 Q 및 q 빈도가 각각 0.35 및 0.65로 q의 빈도가 높았다. DGAT1 유전자형과 산유량인 유량 및 유지량간의 연관성에 있어서는 DGAT1 유전자형이 유량 및 유지량에서 유의적인 차이(P<0.05)를 보였으며, DGAT1 Qq 유전자형이 QQ 및 qq 유전자형에 비하여 유량과 유지량에서 유의적인 차이(P<0.05)로 높은 수치를 나타냈다. This study was conducted to characterize the DGAT1 gene in Korean Holstein dairy cattle population and examine the relationship of DGAT1 polymorphisms with milk yield and milk fat yield for the genetic improvement of Korean Holstein dairy cattle. Results indicated that the 411 bp PCR products were successfully amplified by DGAT1 specific primers. Sequence analysis indicated that the DGTA1 Q allele had AUG (Lysine, K) nucleotide sequences in 216-218 bp and q allele had GCG (Alanine, A) sequences in the same position. Nucleotide sequence homology between the DGAT1 sequences generated in this study showed 100% homology with bovine DGAT1 sequences in the NCBI database. The genotype frequencies of DGAT1 QQ, Qq, and qq were 16.43, 36.43, and 47.14%, respectively, in Korean Holstein dairy cattle population. The observed Q and q allele frequencies were 0.35 and 0.65, respectively. Statistically significant (P<0.05) results were identified for milk yield and milk fat yield for the DGAT1 genotypes. The Qq genotype Holsteins have significantly higher milk yield and milk fat yield than those of the QQ and qq genotype Holsteins(P<0.05).

홀스타인종과 한우에 있어서 BoLA-DRB3 유전자의 단일염기다형과 반수체 분석

정행진(Hang-Jin Jeong),유성란(Seong-Lan Yu),라세둘(M. R. Hoque),이준헌(Jun-Heon Lee),도창희(Chang-Hee Do),류승희(Seung-Heui Ryoo),상병찬(Byung-Chan Sang) 충남대학교 농업과학연구소 2011 농업과학연구 Vol.38 No.1

BoLA (bovine leukocyte antigens) have been known as gene complex related with bovine diseases and immunological traits. This study was conducted to find out the characteristics of BoLA-DRB3 gene related to mastitis and BL(bovine leukocyte) from 280 cattle [193 animals of Holstein cattle and 87 animals of Hanwoo]. As a result, five PCR-RFLP types (b, d, e, f and g) using HaeⅢ restriction enzyme, three BstYI restriction patterns (b, d and e) and eight RsaI restriction types(b, d, f, I, j, n, o and w) were identified. Moreover, we identified new d' type (197→175/22), having one more cutting site by BstYI enzyme than d type allele and n' type (180→169/11) having one more cutting site by RsaI enzyme than n allele was additionally identified. Next, we identified 53 SNPs in BoLA-DRB3 exon2 from 280 cattle. SNP frequency and heterozygosity of Holstein and Hanwoo were investigated in all the SNP genotype. These results might be based on research for identifying marker associated with bovine diseases.

한국재래산양 β-lactoglobulin 유전자 5'flanking 영역의 염기서열 분석

류승희,한성욱,서길웅,상병찬 충남대학교 농업과학연구소 2001 농업과학연구 Vol.28 No.2

본 연구는 한국 재래산양의 β-lactoglobulin(β- LG) 유전자 발현조절부위의 특성을 구명하기 위하여 PCR기법으로 specific primer를 이용하여 β-LG 발현조절부위를 증폭한 후 염기서열을 분석하여 Sheep종의 β-LG유전자 발현조절부위의 염기서열상의 차이를 분석하였다. 한국 재래산양의 genomic DNA로부터 PCR기법을 이용하여 β-LG 유전자 발현조절 부위를 증폭한 결과 Sheep종에서 보고된 바와 같이 1,077bp의 단편크기로 증폭되었음을 확인 할 수 있었다, Sheep종에서 보고된 β-LG 발현조절 부위의 염기서열과 한국재래산양에서 분석된 염기서열간의 차이는 총 897개의 염기중 46개의 nucleotide에서 차이를 나타내어 94.9%의 상동성을 보였다. 특히 한국재래산양과 Sheep종간 염기서열상의 차이는 Sheep종의 T염기가 재래산양의 C염기로 치환되었거나 반대로 Sheep종의 C염기가 재래산양에서 T염기로 치환되어 있음을 확인할 수 있었다. 이상의 결과에서 β-LG유전자 발현조절부위의 염기서열은 재래산양과 Sheep 종간에 높은 상동성을 보였으나 이들 종간의 발현조절부위의 염기서열상의 차이에 대한 유전자 발현조절 관계와 전사요소는 앞으로 연구가 더 진행되어야 할 것으로 사료된다. This study was analyzed by PCR technique with specific primer in order to investigate the characteristics of β-lactoglobulin (β-LG) gene 5'flanking region in Korean native goat. This work confirmed amplified product of 1,077 bp fragments obtained from the amplification of β-LG promoter from genomic DNA using PCR in Korean native goat. The nucleotide sequence of β-LG gene 5'flanking region in Korean native goat as compared with that of sheep β-LG were different at 46 base of 897 nucleotides, and showed high homology as about 94.9% each breed. Especially we confirmed that the difference of nucleotide sequences between Korean native goat and sheep were consisted of T→C substitution and C→T substitution reversely. As a consequences, the sequences of β-LG gene 5'flanking region showed a high homology between Korean native goat and sheep. Furthermore we should be studied that relationships between the control of gene expression and nucleotide sequences of transcription factor in Korean native goat.

PCR 기법을 이용한 한국재래산양 β-casein 유전자의 특성

김지애,류승희,유성란,이준헌,서길웅,김선균,상병찬 충남대학교 농업과학연구소 2002 농업과학연구 Vol.29 No.2

본 연구는 한국 재래 산양 112두와 유산양인 Saanen종 7두의 혈액으로부터 genomic DNA를 추출하고, PCR-RFLP 방법에 의해 β-casein 유전자의 특성을 분석하여 한국재래산양의 효율적인 유전자원의 보전 및 개량을 위한 기초 자료로 제공하고자 실시하였다. 한국재래산양의 genomic DNA로부터 PCR기법을 이용하여 β-casein의 유전자좌를 증폭한 결과 각각 481bp 크기의 단편이 양호하게 증폭되었음을 확인하였다. β-casein 유전자좌의 증폭산물에 대한 Bal Ⅰ의 제한효소를 처리한 결과, β-casein AB형은 481bp, 284bp 및 197bp의 단편을, 그리고 BB형은 284BP와 197BP의 단편을 한국재래산양과 유산양인 Saanen 종에서 확인 할 수 있었다, 유전자형 빈도에 있어서는 한국재래산양에서 β-casein AB 및 BB의 빈도는 각각 6.25 및 93.75%이었고, 유산양인 Saanen 종은 각각 57.14 및 42.86%이었다. 유전자빈도에 있어서는 한국재래산양의 β-casein A 및 B의 빈도가 각각 0.031 및 0.969이었고, Saanen 종에서는 각각 0.286 및 0.714의 빈도를 보였다 한국재래산양의 β-casein 유전자의 염기서열과 이미 보고되어 있는 goat의 염기서열(GeneBank accession Number M90556)간에는 총 11개의 염기서열에 차이를 나타내어 97.71%의 상동성을 보였다, 따라서 한국재래산양의 β-casein 유전자의 다형성과 염기서열 분석에 의한 분자유전학적 특성의 규명은 한국재래산양의 유전자원의 보전 및 개량을 위한 기초 및 응용 자료로 이용될 수 있을 것으로 생각된다. This study was performed to provide the basic data for preservation and improvement of genetic resources according to finding genetic construction obtained from analysis of genetic characteristics of β-casein gene in Korean Native goat and Saanen using the PCR-RFLP. This study confirmed the amplified products of 481bp fragments obtained from the amplification of β-casein loci by PCR. The β-casein AB genotype showed 481, 284 and 197bp, and β-casein BB genotype showed 284 and 197bp fragments in Korean Native goat and Saanen. The frequencies of β-casein genotype in Korean Native goat were 6.25 and 93.75% for AA and AB and the frequencies of β-casein genotype in Saanen were 57.14 and 42.86% for AA and AB types. The frequencies of β-casein A and B alleles were 0.031 and 0.969 in Korean Native goat and the frequencies of β-casein A and B alleles are 0.286 and 0.714 in Saanen, respectively. The nucleotide sequence of β-casein gene of Korean Native goat was 97.71% higher homology with 11 nucleotide sequences difference of that of goat reported in GeneBank (M90556). Therefore, this study of molecular genetic characteristics by the analysis of genetic polymorphism and sequencing for β-casein gene should be used as basic and applying data for preservation and improvement of genetic resources in Korean Native goat breeding.