생명과학회지 (Journal of Life Science)

  • 제23권2호
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  • Pages.190-196
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  • 2013
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  • 1225-9918(pISSN)
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  • 2287-3406(eISSN)
한국생명과학회 (Korean Society of Life Science)
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당 생합성 효소 PGK 유전자 프로모터 변이와 물렁증 저항성 멍게의 선별

Gene Promoter Variation of Phosphoglycerate Kinase, a Glucose Metabolism Enzyme, is a Biomarker for Selection of Disease-resistant Sea Squirt, Halocynthia Roretzi

  • 조현국 (국립수산과학원 생명공학연구소) ;
  • 허영백 (남해수산연구소 양식환경연구센터) ;
  • 정재훈 (부산대학교 분자생물학과)
  • Cho, Hyun Kook (Biotechnology Research Center, National Fisheries Research and Development Institute) ;
  • Hur, Young Baek (Aquaculture Environment Institute) ;
  • Cheong, Jae Hun (Department of Molecular Biology, Pusan National University)
  • 투고 : 2013.01.07
  • 심사 : 2013.02.21
  • 발행 : 2013.02.28
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초록

멍게의 물렁증 발병으로 인해 멍게 양식에 커다란 타격을 받고 있는 가운데 물렁증 발병에 대한 분자적인 접근을 위해 정상 멍게와 물렁증 걸린 멍게에서 DEG 법을 수행하였다. 이번 연구에서 멍게의 당 생합성 효소인 PGK가 물렁증에 걸린 멍게 개체에서 발현이 감소하는 것을 다양한 실험을 통해서 확인하였다. PGK에 대한 유전자 서열을 확보함과 동시에 이 유전자의 발현에 영향을 미치는 부위인 프로모터를 클로닝 하였다. 프로모터 부위의 단일 염기 다형성 분석을 통해서 -106과 -254 위치의 염기에서 다형성이 일어나는 것을 확인하였다. 물렁증에 대한 저항성을 가진 멍게와 다른 개체군의 멍게와의 염기서열을 비교한 결과 많은 차이가 나타남을 확인하였다. 이러한 염기의 차이가 PGK의 발현에 영향을 미치는 지 확인하기 위해서 각각의 멍게 개체군에서 genomic DNA와 total RNA를 분리하여 genotyping과 RT-PCR을 수행하였다. 그 결과 -106과 -254 위치의 염기가 AA와 GT인 개체에서 PGK의 발현량이 상대적으로 많은 것을 확인할 수 있었다. 이러한 결과는 물렁증에 대한 저항성을 가진 멍게를 선발육종하기 위한 분자적인 마커의 개발에 활용됨으로써 물렁증 발병을 줄일 수 있음을 시사하였다.

The sea squirt, Halocynthia roretzi, has experienced mass mortality due to softness syndrome. The identification of disease-induced genes can provide insights into the development of this syndrome. To identify the genes, we performed differentially expressed gene (DEG) analysis. The expression of the phosphoglycerate kinase (HrPGK) gene was significantly decreased in diseased sea squirts compared to normal ones. We confirmed the result of the DEG analysis through RT-PCR and real-time PCR. In addition, we detected single nucleotide polymorphisms at position -106 (A/T) and -254 (G/T) in the HrPGK gene promoter by genotyping analysis. At the -106 site of the HrPGK gene, the frequency of the AA allele in disease-resistant sea squirts was about two-fold higher than that of sensitive ones, and the frequency of the TT allele in the disease-resistant sea squirts was about six-fold lower. At the -254 site of the HrPGK gene, the frequency of the GT and the GG allele was approximately two-fold higher and two-fold lower, respectively, in the disease-resistant sea squirts compared to the disease-sensitive ones. Analysis of the relationship between the genotypic variation at the -106/-254 promoter and the expression of HrPGK mRNA showed that HrPGK mRNA expression was higher in the -106/-254 AA/GT genotype samples than in the -106/254 TT/GG genotype ones. These results show that sea squirts harboring the AA/GT genotype may have more resistance to mortality than the sea squirts with other genotypes.

키워드

참고문헌

  1. Azumi, K., Nakamura, S., Kitamura, S., Jung, S. J., Kanehira, K., Iwata, H., Tanabe, S. and Suzuki, S. 2007. Accumulation of organotin compounds and marine birnavirus detection in Korean ascidians. Fish Sci 73, 263-269.
  2. Breathnach, R. and Chambon, P. 1981. Organization and expression of eucaryotic split genes coding for proteins. Annu Rev Biochem 50, 349-383. https://doi.org/10.1146/annurev.bi.50.070181.002025
  3. Burighel, P. and Cloney, R. A. 1997. Microscopic Anatomy of Invertebrates, Volume 15: Hemichordata, Chaetognatha, and the Invertebrate Chordates, pp. 221-347. Wiley-Liss, New York City, NY, USA.
  4. Cho, H. K., Nam, B. H., Kong, H. J., Han, H. S., Hur, Y. B., Choi, T. J., Choi, Y. H., Kim, W. J. and Cheong, J. 2008. Identification of softness syndrome-associated candidate genes and DNA sequence variation in the sea squirt, Halocynthia roretzi. Mar Biotechnol (NY) 10, 447-456. https://doi.org/10.1007/s10126-008-9084-y
  5. Choi, D. L., Jee, B. Y., Choi, H. J., Hwang, J. Y., Kim, J. W. and Berthe, F. C. J. 2006. First report on histology and ultrastructure of an intrahemocytic paramyxean parasite (IPP) from tunicate Halocynthia roretzi in Korea. Dis Aquat Organ 72, 65-69. https://doi.org/10.3354/dao072065
  6. Cohen, D. M., Wasserman, J. C. and Gullans, S. R. 1991. Immediate early gene and HSP70 expression in hyperosmotic stress in MDCK cells. Am J Physiol 261, C594-C601
  7. Corden, J., Wasylyk, B., Buchwalder, A., Sassone-Corsi, P., Kedinger, C. and Chambon, P. 1980. Promoter sequences of eukaryotic protein-coding genes. Science 209, 1406-1414. https://doi.org/10.1126/science.6251548
  8. Fujii, H., Kanno, H., Hirono, A., Shiomura, T. and Miwa, S. 1992. A single amino acid substitution (157 Gly ${\rightarrow}$ Val) in a phosphoglycerate kinase variant (PGK Shizuoka) associated with chronic hemolysis and myoglobinuria. Blood 79, 1582-1585.
  9. Hultmark, D., Klemenz, R. and Gehring, W. J. 1986. Translational and transcriptional control elements in the untranslated leader of the heat-shock gene hsp22. Cell 44, 429-438. https://doi.org/10.1016/0092-8674(86)90464-2
  10. Kadonaga, J. T. 2002. The DPE, a core promoter element for transcription by RNA polymerase II. Exp Mol Med 34, 259-264. https://doi.org/10.1038/emm.2002.36
  11. Karnsakul, W., Gillespie, S., Skitarelic, K. and Hummel, M. 2010. Obesity and reversed growth retardation in a child with type Ia glycogen storage disease. J Pediatr Endocrinol Metab 23, 507-512.
  12. Lee, Y. C., Chang, C. J., Bali, D., Chen, Y. T. and Yan, Y. T. 2011. Glycogen-branching enzyme deficiency leads to abnormal cardiac development: novel insights into glycogen storage disease IV. Hum Mol Genet 20, 455-465. https://doi.org/10.1093/hmg/ddq492
  13. Njolstad, P. R., Sagen, J. V., Bjørkhaug, L., Odili, S., Shehadeh, N., Bakry, D., Sarici, S. U., Alpay, F., Molnes, J., Molven, A., Søvik, O. and Matschinsky, F. M. 2003. Permanent neonatal diabetes caused by glucokinase deficiency: inborn error of the glucose-insulin signaling pathway. Diabetes 52, 2854-2860. https://doi.org/10.2337/diabetes.52.11.2854
  14. Rosa, R., George, C., Fardeau, M., Calvin, M. C., Rapin, M. and Rosa, J. 1982. A new case of phosphoglycerate kinase deficiency: PGK Creteil associated with rhabdomyolysis and lacking hemolytic anemia. Blood 60, 84-91.
  15. Servidei, S. and DiMauro, S. 1989. Disorders of glycogen metabolism of muscle. Neurol Clin 7, 159-178.
  16. Shaulian, E. and Karin, M. 2001. AP-1 in cell proliferation and survival. Oncogene 20, 2390-2400. https://doi.org/10.1038/sj.onc.1204383
  17. Shaulian, E. and Karin, M. 2002. AP-1 as a regulator of cell life and death. Nat Cell Biol 4, 131-136. https://doi.org/10.1038/ncb0502-e131
  18. Wiese, S., Schliess, F. and Haussinger, D. 1998. Osmotic regulation of MAP-kinase activities and gene expression in H4IIE rat hepatoma cells. Biol Chem 379, 667-671.