한국수산과학회지 (Korean Journal of Fisheries and Aquatic Sciences)

  • 제35권5호
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  • Pages.490-496
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  • 2002
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  • 0374-8111(pISSN)
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  • 2287-8815(eISSN)
한국수산과학회 (The Korean Society of Fisheries and Aquatic Science)
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Denaturing gradient gel electrophoresis와 real time PCR 방법을 이용한 연어 유전자들의 DNA 이형 다양성 검색

DNA Heteropolymorphism of Chum Salmon Detected by Denaturing Gradient Gel Electrophoresis and Real Time PCR

  • 함승협 (강릉대학교 해양생명공학부) ;
  • 이석근 (강릉대학교 치의학과) ;
  • 한현섭 (국립수산진흥원 서해수산연구소) ;
  • 진덕희 (강릉대학교 해양생명공학부)
  • Ham Seung Hub (Department of Marine Bioscience and Technology, College of Life Science) ;
  • Lee Suk Keun (Department of Oral Pathology, College of Dentistry, Kangnung National University) ;
  • Han Hyon Sob (West Sea Fisheries Research Institute, National Fisheries Research and Development Institute) ;
  • Jin Deuk Hee (Department of Marine Bioscience and Technology, College of Life Science)
  • 발행 : 2002.09.01
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초록

한국, 미국, 일본지역에 서식하는 연어에서 추출한 genomic DNA를 이용하여 연어의 mtDNA NDI 영역, D-loop 영역, growth hormone, IGF-I, MCH2, histone H3의 염기서열을 분석하여, 최적의 primer를 제작하여 PCR을 실시한 결과, mtDNA NDI 영역은 Ks12, Ks24, As11, As14, Js13, Js15에서 증폭된 DNA를 확인하였으며, D-loop 영역, growth hormone, IGF-I, histone H3, MCH2에서는 모든 시료에서 증폭된 DNA를 확인하였다. DGGE 분석의 결과, mtDNA NDI 영역 (AF133701, 449-880), D-loop 영역 (AF125518, 11-514)과 growth hormone (AFO05927, 181-530)에서는 이형다양성을 확인하였으며, IGF-I (AF063216, 962-1461)과 MCH2 (M27281, 70-593)는 모두 이형다양성이 나타났으나, histone H3 (AF017147, 7-487)는 모두 이형다양성이 관찰되지 않았다. 그리고 real time PCR 관찰 결과는 DGGE의 결과와 유사한 점을 찾을 수 없었지만, real time PCR도 각각의 유전자에 따라 서로 다른 DNA 생성 패턴을 보여 DNA 변이를 쉽게 구별하는데 보조적인 도움이 되었다.

In order to detect the DNA heteropolymorphism of chum salmon, selected essential genes were examined in different regional chum salmons, i.e., Korean, Japanese and American by denaturing gradient gel electrophoresis (DGGE) and real time PCR methods. From the promoter regions and introns of growth hormone, mtDNA NDI region, D-loop region, IGF-I, histone H3 and MCH2 several representative primer pairs were obtained and employed for the DGGE with the PCR products from the genomic DNAs of the different regional chum salmons. mtDNA NDI, D-loop region and IGE-I genes showed marked heteropolymorphism between Korean and American chum salmons. Intron C of growth hormone also showed a heteropolymorphism between Korean and Japanese chum salmons. Whereas heteropolnnorphism of histone liH and MCH2 genes was detected among in Korean, Japanese and Asnerican chum salmons in the examined region. The real time PCR disclosed the characteristic incremental production of target DNAs dependent on the heteropolymorphic conditions of genomic DNAa of chum salmons, thus the different regional chum salmons could be grouped by the variable incremental curies. Although the DGGE and real time PCR did not produce the identical results in this study, we suggest that the DGGE and real time PCR could be used for the primary screening of the DNA heteropolymorphism of different animal genome.

키워드

참고문헌

  1. Cronin, A.A, W.M. Spearman. R.L. Wilmot, J.C. Patton and J.W. Bickham. 1993. Mitochondrial DNA variation in chinook (Oncorhynchus tshawytscha) and chum salmon (0. Ji:eta) detected by restriction enzyme analysis of polymerase chain reaction products. Can. J. Fish. Aquat. Sci., 50, 708-715 https://doi.org/10.1139/f93-081
  2. Verspoor E., E.M. Mccarthy and D. Knox. 1999. The phylogeogaphy of European atlantic salmon (.Salmo salar L.) based on RFLP analysis of the ND1/16sRNA region of the mtDNA. Bio. J. of the L.S., 68, 129-146 https://doi.org/10.1111/j.1095-8312.1999.tb01162.x
  3. Hanley, S., T.J. Smith, F. Muller, N. Maclean, S. Uzbekova, P. Prunet and B. Breton. 1998. Isolation and functional analysis of the histone H3 promoter from atlantic salmon (Salmo salar L.). Mol. Mar. Biol. Biotechnol., 7, 165-172
  4. Kam, L. and T. Edward. 2001. Bacterial community dynamics in liquid swine manure during storage; moleuclar analysis using DGGE/PCR of 16S rDNA. FEMS Microbiol. Ecology., 38, 169-177 https://doi.org/10.1111/j.1574-6941.2001.tb00895.x
  5. Kavan, V.M., A.P. Koval, VA. Grebenjuk, S.J. Chan, D.F. Steiner, C. T. Robert Jr. and D. Leroith. 1993. Structure of the chum salmoninsulin-like growth factor I gene. DNA Cell Biol., 11, 729-737
  6. Koval, A., V. Kulik, S. Duguay, E. Plisetskaya, M.L. Adamo, D.T. Roverts Jr., D. Leroith and V. Kavsan. 1994. Characterization of a salmon insulin-like growth factor I promoter. DNA Cell BioL, 13, 1057-1062 https://doi.org/10.1089/dna.1994.13.1057
  7. Lee, H.J., WJ. Kim, J.H. Lee, K.H. Min, M.A. Yoo, W.H. Lee and J.Y. Park. 1999. Genetic relationships of six salmonids in korean waters: direct sequence analysis of the mitochondnal DNA control region
  8. Maiy, V. John., Iqbal Parwez, V. Mylavarapu, S. Sivaram, Shilpa Mehta, Nidhi Marwah and Sher Ali. 1996. Analysis of VNTRloci in fish genomes using synthetic oligodeoxyhbonucleotide probes. Gene., 172, 191-197 https://doi.org/10.1016/0378-1119(96)00173-4
  9. Mette, H.N. and B.R. Niels. 2002. Denaturing gradient gel elelctrophoresis (DGGE) approaches to study the diversity of ammonia-oxidizing bacteria. J. Micorbiol. methods., 50, 189-203 https://doi.org/10.1016/S0167-7012(02)00026-X
  10. Michael, J., Domanico Ruth and B. Phillips. 1995. Phylogenetic analysis of pacific salmon (Genus Oncorhynchus) based on mitochondiial DNA sequence data. Mol. phylogenetics and evolution, 4, 366-371 https://doi.org/10.1006/mpev.1995.1034
  11. Motoo Kimura. 1980. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol., 16, 111-120 https://doi.org/10.1007/BF01731581
  12. Nelson, R.J., T.D. Beacham and M.P. Small. 1998. Microsatellite analysis of the population structure of a vancouver island sockeye salmon (.Oncorhynchus nerka) stock complex using nondenaturing gel electrophoresis. Mol. Mar. Biol. BiotechnoL, 7, 312-319
  13. Nico, B., D.W. Wim, V. Willy and M.T. Eva. 2002. Evaluation of nested PCR-DGGE (denaturing gradient gel electrophoresis) with group-speciGc 16S rRNA primers for the analysis of bacterial communities from different wastewater treatment plants. FEMS Microbiol. Ecology, 39, 101-112
  14. Nilsson, J, R. Gross, T. Asplund, 0. Dove, H. Jansson, J. Kelloniemi, K. Kohlmann, A. Loytynoja, E.E. Nielsen, T. Paaver, C.R. Primmer, S. Titov, A. Vasemagi, A. Veselov, T. Ost and J. Lumme. 2001. Matrilinear phylogeography of Atlantic salmon(Salmo salar L.) in Europe and postglacial colonization of the Baltic Sea area. Mol. Ecol., 10, 89-102 https://doi.org/10.1046/j.1365-294X.2001.01168.x
  15. Oakley, T.H and R.B. Phillips. 1999. Phylogeny of salmon fishes based on growth hormone introns: atlantic (Salmo) and pacific (Oncorhynchus) salmon are not sister taxa. Mol. Phylogenet. Evol., 11, 381-393 https://doi.org/10.1006/mpev.1998.0599
  16. Pusterla, N., C.M. Leutenegger, B. Sigrist, J.-S. Chae, H. Lutz and J.E. Madigan. 2000. Detection and quantitation of Ehrlichia nsticii genomic DNA in infected horses and snails by real-time PCR. Veterinary Parasitology, 90, 129-135 https://doi.org/10.1016/S0304-4017(00)00227-2
  17. Stephan stubner. 2002. Enumeration of 16S rDNA Desulfotomaculum lineage 1 in rice field soil by real-time PCR with $SybrGreen^{TM}$ detection. J. Microbiol. Methods, 50, 155-164 https://doi.org/10.1016/S0167-7012(02)00024-6
  18. Takayama, Y., C. Wada, H. Kawauchi and M. Ono. 1989. Structures of two genes coding for melanin-concentrating hormone of chum salmon. Gene., 80, 65-73 https://doi.org/10.1016/0378-1119(89)90251-5

피인용 문헌

  1. 넙치의 원산지 판별을 위한 ND-4유전자의 다양성 분석 vol.14, pp.4, 2002, https://doi.org/10.5352/jls.2004.14.4.627