Journal of Aquaculture (한국양식학회지)

The Korean Society of Fisheries and Aquatic Science (한국양식학회)
권호 표지

Transcriptional Response of Major Antioxidant Enzyme Genes to Heat Stress in Mud Loach (Misgurnus mizolepis)

고온 스트레스에 대한 미꾸라지(Misgurnus mizolepis) 항산화 효소 유전자들의 발현 특징

  • Cho Young-Sun (Department of Aquaculture & Institute of Marine Living Modified Organisms (iMLMO), Pukyong National University(PKNU)) ;
  • Lee Sang-Yoon (Department of Aquaculture & Institute of Marine Living Modified Organisms (iMLMO), Pukyong National University(PKNU)) ;
  • Bang In-Chul (Department of Marine Biotechnology, Soonchunhyang University) ;
  • Kim Dong-Soo (Department of Aquaculture & Institute of Marine Living Modified Organisms (iMLMO), Pukyong National University(PKNU)) ;
  • Nam Yoon-Kwon (Department of Aquaculture & Institute of Marine Living Modified Organisms (iMLMO), Pukyong National University(PKNU))
  • 조영선 (부경대학교 양식학과/해양수산형질전환생물연구소) ;
  • 이상윤 (부경대학교 양식학과/해양수산형질전환생물연구소) ;
  • 방인철 (순천향대학교 해양생명공학과) ;
  • 김동수 (부경대학교 양식학과/해양수산형질전환생물연구소) ;
  • 남윤권 (부경대학교 양식학과/해양수산형질전환생물연구소)
  • Published : 2006.08.01
Download PDF
⟨ Previous Next ⟩

Abstract

Expression of major antioxidant enzyme (AOE) including Cu/Zn superoxide dismutase (Cu/Zn-SOD), catalase (CAT), glutathione-S-transferase (GST) and 3 glutathione peroxidase isotypes (GPXs) at mRNA levels during heat stress was examined in mud loach (Misgurnus mizolepis) liver. Based on the semi-quantitative RT-PCR, real-time RT-PCR and/or northern dot blot hybridization, the antioxidant enzyme genes were generally up-regulated during elevation of water temperature from $23^{\circ}C$ up to $32^{\circ}C$. GPXs and SOD displayed the most significant elevation of mRNA levels (up to 3 and 2 folds, respectively) while CAT showed the steady-state expression irrespective of thermal conditions. GST represented the relatively moderate response (1.3-fold increase) in its transcription to thermal stress. The transcriptional activation of AOE genes was not significant at the treatment temperature lower than $29^{\circ}C$. Increased mRNA levels of GPX (extracellular form) and SOD genes in the fish exposed to $32^{\circ}C$ was readily detectable 1 day after exposure to heat stress.

우리나라 주요 담수 어종인 미꾸라지를 ecotoxicogenomic 연구 모델 어류로 개발하기 위한 연구의 일환으로 본 어종이 고온 스트레스 자극에 노출되었을때 야기되는 산화성 스트레스를 검출하고자 항산화 효소(antioxidant enzyme; AOE) 유전자의 발현 양상을 분석하였다. 주요 항산화 효소인 superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST) 및 glutathione peroxidases (GPXs)의 transcript들을 특이적으로 정량화할 수 있는 semi-quantitative RT-PCR, real-time PCR 또는 northern blot분석을 통해 $23^{\circ}C$에서 $32^{\circ}C$까지 설정된 실험어의 간 조직내 AOE유전자들의 mRNA level을 분석하였다. 고온에 노출되었을 때 본 어종의 AOE들은 일반적으로 증가된 유전자 발현 양상을 나타내었고, 특히 SOD (2배)와 plasma GPX (3배) 유전자가 가장 유의적인 mRNA 증가를 나타내었다. GST의 경우 상대적으로 적은 증가량을 나타내었고 CAT의 경우 고온자극에 반응하지 않았다. 본 어종은 $29^{\circ}C$ 이상에서 AOE 유전자의 발현 증가를 나타내었고 $32^{\circ}C$에 노출되었을 때 1일째부터 SOD와 plasma GPX mRNA의 증가가 관찰되었다.

Keywords

References

  1. Barber, R. D., D. W. Harmer, R. A. Coleman and B. J. Clark, 2005. GAPDH as a housekeeping gene: analysis of GAPDH mRNA expression in a panel of 72 human tissues. Physiol. Genomics, 21, 389-395 https://doi.org/10.1152/physiolgenomics.00025.2005
  2. Bernabucci, U., B. Ronchi, N. Lacetera and A. Nardone, 2002. Markers of oxidative status in plasma and erythrocytes of transition dairy cows during hot season. J. Dairy Sci., 85, 2173- 2179 https://doi.org/10.3168/jds.S0022-0302(02)74296-3
  3. Buda, C., I. Dey, N. Balogh, L. I. Horvath, K. Maderspach, M. Juhasz, Y. K. Yeo and T. Farkas, 1994. Structural order of membranes and composition of phospholipids in fish brain cells during thermal acclimatization. Proc. Natl. Acad. Sci. USA., 91, 8234-8238
  4. Chen, J., D. A. Rider and R. Ruan, 2006. Identification of valid housekeeping genes and antioxidant enzyme gene expression change in the aging rat liver. J. Gerontol. A Biol. Sci. Med. Sci., 61, 20-27
  5. Chen, R., R. Lochmann, A. Goodwin, K. Praveen, K. Dabrowski and K. J. Lee, 2003. Alternative complement activity and resistance to heat stress in golden shiners (Notemigonus crysoleucas) are increased by dietary vitamin C levels in excess of requirements for prevention of deficiency signs. J. Nutr., 133, 2281-2286
  6. Cho, Y. S., B. N. Choi, E.-M. Ha, K. H. Kim, S. K. Kim, D. S. Kim and Y. K. Nam, 2005. Shark, Scyliorhinus torazame metallothionein: cDNA cloning, genomic sequence and expression analysis. Mar. Biotechnol., 7, 350-362 https://doi.org/10.1007/s10126-004-0043-y
  7. Cho, Y. S., B. N. Choi, K.-H. Kim, S. K. Kim, D. S. Kim, I. C. Bang and Y. K. Nam, 2006. Differential expression of Cu/Zn superoxide dismutase mRNA during exposures to heavy metals in rockbream (Oplegnatus fasciatus). Aquaculture, 253, 667-679 https://doi.org/10.1016/j.aquaculture.2005.05.047
  8. Deindl, E. K., Boengler, N. van Royen and W. Schaper, 2002. Differential expression of GAPDH and beta3-actin in growing collateral arteries. Mol. Cell. Biochem., 236, 139-146 https://doi.org/10.1023/A:1016166127465
  9. Dey, I., C. Buda, T. Wiik, J. E. Halver and T. Farkas, 1993. Molecular and structural composition of phospholipid membranes in livers of marine and freshwater fish in relation to temperature. Proc. Natl. Acad. Sci. USA., 90, 7498-7502
  10. Gilsbach, R., M. Kouta, H. Bonisch and M. Bruss, 2006. Comparison of in vitro and in vivo reference genes for internal standardization of real-time PCR data. Biotechniques, 40, 173-177 https://doi.org/10.2144/000112052
  11. Heise, K., S. Puntarulo, M. Nikinmaa, D. Abele and H. O. Portner, 2006. Oxidative stress during stressful heat exposure and recovery in the North Sea eelpout Zoarces viviparus L. J. Exp. Biol., 209, 353-363 https://doi.org/10.1242/jeb.01977
  12. Hwang, D.-F. and T.-K. Lin, 2002. Effect of temperature on dietary vitamin C requirement and lipid in common carp. Comp. Biochem. Physiol., 131, 1-7 https://doi.org/10.1016/S1096-4959(01)00449-3
  13. Imai, H and Y. Nakagawa, 2003. Biological significance of phospholipid hydroperoxide glutathione peroxidase (PHGPx, GPx4) in mammalian cells. Free Radic. Biol. Med., 34, 145-169 https://doi.org/10.1016/S0891-5849(02)01197-8
  14. Jeong.,Y. J., H. W. Choi, H.S. Shin, X. S. Cui, N. H. Kim, G. L. Gerton and J. H. Jun, 2005. Optimization of real time RT-PCR methods for the analysis of gene expression in mouse eggs and preimplantation embryos. Mol. Reprod. Dev., 71, 284-289 https://doi.org/10.1002/mrd.20269
  15. Johnson, P., 2002. Antioxidant enzyme expression in health and disease: effects of exercise and hypertension. Comp. Biochem. Physiol., Part C 133, 493-505 https://doi.org/10.1016/S1096-4959(02)00095-7
  16. Klumpp, D.W., C. Humphrey, H. Huasheng and F. Tao, 2002. Toxic contaminants and their biological effects in coastal waters of Xiamen, China. II. Biomarkers and embryo malformation rates as indicators of pollution stress in fish. Mar. Poll. Bull., 44, 761-769 https://doi.org/10.1016/S0025-326X(02)00054-1
  17. Lopes, P. A., T. Pinheiro, M. C. Santos, M da L. Mathias, M. J. Collares-Pereira and A. M. Viegas-Crespo, 2001. Response of antioxidant enzymes in freshwater fish populations (Leuciscus alburnoides complex) to in organic pollutants exposure. Sci. Total Environ., 280, 153-163 https://doi.org/10.1016/S0048-9697(01)00822-1
  18. Marcinkowska, A., R. Danielewicz R and M. Wolny, 1990. The effect of temperature on catalytic function of glyceraldehyde- 3-phosphate dehydrogenase from muscle of pig and carp Cyprinus carpio. Comp. Biochem. Physiol., Part B 97, 49-54
  19. Nam, Y.K. and D.S. Kim, 2002. Screening of potential stressresponsive and immune-related genes by expressed sequence tags in mud loach (Misgurnus mizolepis). J. Fish Pathol., 15, 83-92
  20. Nam, Y.K., 2006. Tailoring of fish genome and transgenic manipulation as exemplified by mud loach (Misgurnus mizolepis). Fish Genet. Breed. Sci., 35, 113-121
  21. Ozturk, O. and S. Gumuslu, 2004. Age-related changes of antioxidant enzyme activities, glutathione status and lipid peroxidation in rat erythrocytes after heat stress. Life Sci., 75, 1551- 1565 https://doi.org/10.1016/j.lfs.2004.03.020
  22. Pandey, S., S. Parvez, I. Sayeed, R. Haque, B. Bin-Hafeez and S. Raisuddin, 2003. Biomarkers of oxidative stress: a comparative study of river Yamuna fish Wallago attu (Bl. & Schn.). Sci. Total Environ., 309, 105-115 https://doi.org/10.1016/S0048-9697(03)00006-8
  23. Parihar, M. S., A. K. Dubey, T. Faveri and P. Prakash, 1996. Changes in lipid peroxidation, superoxide dismutase activity, ascorbic acid and phospholipids content in liver of freshwater catfish Heteropneustes fossilis exposed to elevated temperature. J. Therm. Biol., 21, 323–330 https://doi.org/10.1016/0306-4565(95)00016-X
  24. Rhee, S. G., K. S. Yang, S. W. Kang, H. A. Woo and T. S. Chang, 2005. Controlled elimination of intracellular H(2)O(2): regulation of peroxiredoxin, catalase, and glutathione peroxidase via post-translational modification. Antioxid. Redox. Signal., 7, 619-626 https://doi.org/10.1089/ars.2005.7.619
  25. Rodriguez-Mulero, S. and E. Montanya, 2005. Selection of a suitable internal control gene for expression studies in pancreatic islet grafts. Transplantation, 80, 650-652 https://doi.org/10.1097/01.tp.0000173790.12227.7b
  26. Whitfield, A.K. and M. Elliott, 2002. Fishes as indicators of environmental and ecological changes within estuaries: a review of progress and some suggestions for the future. J. Fish Biol., 61, 229-250 https://doi.org/10.1111/j.1095-8649.2002.tb01773.x