미호종개 metallothionein 유전자의 구조 및 중금속 노출과 고온 자극에 대한 MT mRNA의 발현 특징 분석

Gene Structure and Altered mRNA Expression of Metallothionein in Response to Metal Exposure and Thermal Stress in Miho Spine Loach Cobitis choii (Cobitidae; Cypriniformes)

  • 이상윤 (부경대학교 해양바이오신소재학과) ;
  • 남윤권 (부경대학교 해양바이오신소재학과)
  • Lee, Sang-Yoon (Department of Marine Bio-Materials & Aquaculture, Pukyong National University) ;
  • Nam, Yoon-Kwon (Department of Marine Bio-Materials & Aquaculture, Pukyong National University)
  • 투고 : 2011.02.11
  • 심사 : 2011.03.28
  • 발행 : 2011.03.31

초록

멸종위기 어류 미호종개(Cobitis choii)로부터 중금속해독 단백질(metallothionein) 유전자를 분리, 클로닝하고 중금속 및 고온 스트레스에 대한 전사 발현 특정을 분석하였다. 미호종개 metallothionein는 gDNA, mRNA 및 아미노산 서열 모두에서 경골 어류 MT들의 구조적 특징을 잘 보전하고 있었으며, 생물정보분석을 통해 미호종개 MT 유전자 5'-upstream 영역은 중금속 조절, 면역 반응 및 온도 반응에 관여하는 다양한 전사 조절인자들의 부착 위치들을 포함하는 것으로 관찰되었다. 카드뮴(Cd), 구리(Cu), 니켈(Ni), 망간(Mn) 및 아연(Zn)을 이용한 침지 노출 실험(0.5 및 $1.0\;{\mu}M$; 24시간)에서 미호종개 MT mRNA 발현은 구리 및 카드뮴 처리군에서 가장 많이 유도되었고($1.0\;{\mu}M$ Cu 처리군에서 최대 10배), 망간 처리군에서는 비교적 적은 양의 MT 발현이 유도된 반면(2배), 아연 및 니켈 노출 군에서는 유의적인 MT 발현의 증감이 관찰되지 않았다. 또한 미호종개 MT 전사 발현은 고온 자극 ($25^{\circ}C$로부터 $31^{\circ}C$까지 증가)에도 민감하게 반응하는 것으로 나타나, $31^{\circ}C$ 도달시점에서 $25^{\circ}C$ 초기 MT mRNA 발현 수준보다 9배 높은 mRNA 발현이 관찰되었다. 본 연구 결과는 MT 기반의 유전자 발현 분석을 이용함으로써, 향후 멸종위기 어류 미호종개의 스트레스 반응 연구에 유용한 기초 자료를 제공할 수 있다고 기대된다.

Gene and promoter structures of metallothionein(MT) from Miho spine loach (Cobitis choii; Cypriniformes) were characterized, and the transcriptional responses to experimental exposures to heavy metals and heat stress were examined. The C. choii metallothionein displayed well-conserved features of teleostean metallothioneins at gDNA, mRNA and amino acid levels. Bioinformatic analysis predicted that the C. choii MT regulatory region potentially possessed various motifs or elements targeted by various transcription factors associated with metal-coordinating regulation (e.g., metal transcription factor-1), immune responses (e.g., nuclear factor kappa B), and thermal modulations (e.g., heat shock factor). Acute heavy-metal exposures to 0.5 or $1.0\;{\mu}M$ of cadmium (Cd), copper (Cu), manganese (Mn), nickel (Ni) or zinc (Zn) showed that MT transcription was significantly stimulated by Cd (9.6-fold relative to non-exposed control) and Cu (10.4-fold), only moderately by Mn (2.4-fold), but hardly by Ni and Zn. Elevation of water temperature from $25^{\circ}C$ to $31^{\circ}C$ caused a rapid modulation of MT mRNAs toward upregulation to 9.5-fold; however, afterward the elevated mRNA level slightly decreased during further incubation at $31^{\circ}C$ for 6 h. Results from this study suggest that MT-based expression assay could be a useful basis for better understanding the metal- and/or heat-caused stresses in this endangered fish species.

키워드

참고문헌

  1. Amiard, J.C., C. Amiard-Triquet, S. Barka, J. Pellerin and P.S. Rainbow. 2006. Metallothioneins in aquatic invertebrates: their role in metal detoxification and their use as biomarkers. Aquat. Toxicol., 76: 160-202. https://doi.org/10.1016/j.aquatox.2005.08.015
  2. Atif, F., M. Kaur, S. Yousuf and S. Raisuddin. 2006. In vitro free radical scavenging activity of hepatic metallothionein induced in an Indian freshwater fish, Channa punctatta Bloch. Chem. Biol. Interact., 162: 172-180. https://doi.org/10.1016/j.cbi.2006.06.006
  3. Bang, I.C., W.J. Kim and I.R. Lee. 2009. Characterization of polymorphic microsatellite loci in the endangered Miho spine loach (Iksookimia choii) and cross-species amplification within the Cobitidae family. Mol. Ecol.Resour., 9: 281-284. https://doi.org/10.1111/j.1755-0998.2008.02452.x
  4. Bi, Y., G.X. Lin, L. Millecchia and Q. Ma. 2006. Superinduction of metallothionein I by inhibition of protein synthesis: role of a labile repressor in MTF-I mediated gene transcriptioin. J. Biochem. Mol. Toxicol., 20: 57-68. https://doi.org/10.1002/jbt.20116
  5. Bourdineaud, J.P., M. Baudrimont, P. Gonzalez and J.L. Moreau. 2006. Challenging the model for induction of metallothionein gene expression. Biochimie, 88: 1787-1792. https://doi.org/10.1016/j.biochi.2006.07.021
  6. Buckley, B.A., A.Y. Gracey and G.N. Somero. 2006. The cellular response to heat stress in the goby Gillichthys mirabilis: a cDNA microarray and protein-level analysis. J. Exp. Bio., 209: 2660-2677. https://doi.org/10.1242/jeb.02292
  7. Chen, W.Y., J.A.C. John, C.-H. Lin, H.-F. Lin, S.-C. Wu, C.-H. Lin and C.-Y. Chang. 2004. Expression of metallothionein gene during embryonic and early larval development in zebrafish. Aquat. Toxicol., 69: 215-227. https://doi.org/10.1016/j.aquatox.2004.05.004
  8. Cheung, A.P.L., V.K.L. Lam and K.M. Chan. 2005. Tilapia metallothionein genes: PCR-cloning and gene expression studies. Biochim. Biophys. Acta., 1731: 191-201. https://doi.org/10.1016/j.bbaexp.2005.09.006
  9. 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
  10. Cho, Y.S., I.C. Bang, I.R. Lee and Y.K. Nam. 2009a. Hepatic expression of Cu/Zn-superoxide dismutase transcripts in response to acute metal exposure and heat stress in Hemibarbus mylodon (Teleostei: Cypriniformes). Fish. Aqua. Sci., 12: 179-184.
  11. Cho, Y.S., S.Y. Lee, C.H. Noh, Y.K. Nam and D.S. Kim. 2006. Survey of genes responsive to long-term heat stress using a cDNA microarray analysis in mud loach (Misgurnus mizolepis) liver. Kor. J. Ichthyol., 18: 65-77.
  12. Cho, Y.S., S.Y. Lee, K.H. Kim, S.K. Kim, D.S. Kim and Y.K. Nam. 2009b. Gene structure and differential modulation of multiple rockbream (Oplegnathus fasciatus) hepcidin isoforms resulting from different biological stimulations. Dev. Comp. Immunol., 33: 46-58. https://doi.org/10.1016/j.dci.2008.07.009
  13. Cho, Y.S., S.Y. Lee, K.-Y. Kim, I.C. Bang, D.S. Kim and Y.K. Nam. 2008. Gene structure and expression of metallothionein during metal exposures in Hemibarbus mylodon, Ecotoxicol. Environ. Saf., 71: 125-137. https://doi.org/10.1016/j.ecoenv.2007.08.005
  14. Cho, Y.S., S.Y. Lee, K.-Y. Kim and Y.K. Nam. 2009c. Two metallothionein genes from mud loach Misgurnus mizolepis (Teleostei; Cypriniformes): Gene structure, genomic organization, and mRNA expression analysis. Comp. Biochem. Physiol. B Biochem. Mol. Biol., 153: 317-326. https://doi.org/10.1016/j.cbpb.2009.04.002
  15. Dragun, Z., M. Podrug and B. Raspor. 2009. Combined use of bioindicators and passive samplers for the assessment of the river water contamination with metals. Arch. Environ. Contam. Toxicol., 57: 211-220. https://doi.org/10.1007/s00244-008-9264-9
  16. Falfushynska, H.I., L.L. Gnatyshyna, C.V. Priydun, O.B. Stoliar and Y.K. Nam. 2010. Variability of responses in the crucian carp Carassius carassius from two Ukrainian ponds determined by multi-marker approach. Ecotoxicol. Environ. Saf., 73: 1896-1906. https://doi.org/10.1016/j.ecoenv.2010.08.029
  17. Ferencz, A. and E. Hermesz. 2009. Identification of a splice variant of the metal-responsive transcription factor MTF-1 in common carp. Comp. Biochem. Physiol. C Toxicol. Pharmacol., 150: 113-117. https://doi.org/10.1016/j.cbpc.2009.03.006
  18. Fu, Z., J. Guo, L. Jing, R. Li, T. Zhang and S. Peng. 2010. Enhanced toxicity and ROS generation by doxorubicin in primary cultures of cardiomyocytes from neonatal metallothionein-I/II null mice. Toxicol. In Vitro, 24: 1584-1591. https://doi.org/10.1016/j.tiv.2010.06.009
  19. Gao, D., G.T. Wang, X.T. Chen and P. Nie. 2009. Metallothionein- 2 gene from the mandarin fish Siniperca chuatsi: cDNA cloning, tissue expression, and immunohistochemical localization. Comp. Biochem. Physiol. C Toxicol. Pharmacol., 149: 18-25. https://doi.org/10.1016/j.cbpc.2008.05.014
  20. Guo, R., H. Ma, F. Gao, L. Zhong and J. Ren. 2009. Metallothionein alleviates oxidative stress-induced endoplasmic reticulum stress and myocardial dysfunction. J. Mol. Cell. Cardiol., 47: 228-237. https://doi.org/10.1016/j.yjmcc.2009.03.018
  21. He, P., M. Xu and H. Ren. 2007. Cloning and functional characterization of 5′-upstream region of metallothionein- I gene from crucian carp (Carassius cuvieri). Int. J. Biochem. Cell Biol., 39: 832-841. https://doi.org/10.1016/j.biocel.2007.01.008
  22. Hermesz, E., M. Abraham and J. Nemcsok. 2001. Tissuespecific expression of two metallothionein genes in common carp during cadmium exposure and temperature shock. Comp. Biochem. Physiol. C Toxicol. Pharmacol., 128: 457-465. https://doi.org/10.1016/S1532-0456(01)00165-X
  23. Kalman, J., I. Riba, T. Angel DelValls and J. Blasco. 2010. Comparative toxicity of cadmium in the commercial fish species Sparus aurata and Solea senegalensis. Ecotoxicol. Environ. Saf., 73: 306-311. https://doi.org/10.1016/j.ecoenv.2009.10.013
  24. Kim, K.-Y., S.Y. Lee, Y.S. Cho, I.C. Bang, D.S. Kim and Y.K. Nam. 2008. Characterization and phylogeny of two beta-cytoskeletal actins from Hemibarbus mylodon (Cyprinidae, Cypriniformes), a threatened fish species in Korea. DNA Seq., 19: 87-97. https://doi.org/10.1080/10425170701445691
  25. Klaassen, C.D., J. Liu and B.A. Diwan. 2009. Metallothionein protection of cadmium toxicity. Toxicol. Appl. Pharmacol., 238: 215-220. https://doi.org/10.1016/j.taap.2009.03.026
  26. Knapen, D., E.S. Redeker, I. Inácio, W. De Coen, E. Verheyen and R. Blust. 2005. New metallothionein mRNAs in Gobio gobio reveal at least three gene duplication events in cyprinid metallothionein evolution. Comp. Biochem. Physiol. C Pharmacol. Toxicol. Endocrinol., 140: 347-355. https://doi.org/10.1016/j.cca.2005.03.002
  27. Laity, J.H. and G.K. Andrews. 2007. Understanding the mechanisms of zinc-sensing by metal-response element binding transcription factor-1 (MTF-1). Arch. Biochem. Biophys., 463: 201-210. https://doi.org/10.1016/j.abb.2007.03.019
  28. Lee, S.Y., O. Stoliar and Y.K. Nam. 2010. Transcriptional alteration of two metallothionein isoforms in mud loach (Misgurnus mizolepis) fry during acute heavy metal exposure. Fish. Aqua. Sci., 13: 112-117.
  29. Lee, Y.A., Y.E. Yun, Y.K. Nam and I.C. Bang. 2008. Genetic diversity of an endangered fish Iksookimia choii (Cypriniformes), from Korea as assessed by amplified fragment length polymorphism. Kor. J. Limnol., 41: 98-103.
  30. Lin, C.H., J.A.C. John, L.W. Ou, J.C. Chen, C.H. Lin and C.Y. Chang. 2004. Cloning and characterization of metallothionein gene in ayu Plecoglossus altivelis. Aquat. Toxicol., 66: 111-124. https://doi.org/10.1016/j.aquatox.2003.06.003
  31. Ren, H., M. Xu, P. He, N. Muto, N. Itoh, K. Tanaka, J. Xing and M. Chu. 2006. Cloning of crucian carp (Carassius cuvieri) metallothionein-II gene and characterization of its gene promoter region. Biochem. Biophys. Res. Commun., 342: 1297-1304. https://doi.org/10.1016/j.bbrc.2006.02.082
  32. Rhee, J.S., S. Raisuddin, D.S. Hwang, K.W. Lee, I.C. Kim and J.S. Lee. 2009. Differential expression of metallothionein (MT) gene by trace metals and endocrinedisrupting chemicals in the hermaphroditic mangrove killifish, Kryptolebias marmoratus. Ecotoxicol. Environ. Saf., 72: 206-212. https://doi.org/10.1016/j.ecoenv.2008.06.001
  33. Ringwood, A.H., M.J. Hameedi, R.F. Lee, M. Brower, E.C. Peters, G.I. Scott, S.N. Luoma and R.T. Di Giulio. 1999. Bivalve biomarker workshop: overview and discussion group summaries. Biomarkers, 4: 391-399. https://doi.org/10.1080/135475099230561
  34. Rodriguez-Ortega, M.J., A. Rodriguez-Ariza, J.L. Gomez- Ariza, A. Muñoz-Serrano and J. Lopez-Barea. 2009. Multivariate discriminant analysis distinguishes metalfrom non metal-related biomarker responses in the clam Chamaelea gallina. Mar. Pollut. Bull., 58: 64-71. https://doi.org/10.1016/j.marpolbul.2008.09.006
  35. Son, Y.-M. and H.-K. Byeon. 2005. The ichthyofauna and dynamics of the fish community in Miho stream, Korea. Kor. J. Ichthyol., 17: 271-278 (in Korean).
  36. Song, H.Y., W.J. Kim, W.O. Lee and I.C. Bang. 2008. Morphological development of egg and larvae of Iksookimia choii (Cobitidae). Korean J. Limnol., 41: 104-110.
  37. Swindell, W.R. 2011. Metallothionein and the biology of aging. Ageing Res. Rev., 10: 132-145. https://doi.org/10.1016/j.arr.2010.09.007
  38. Thirumoorthy, N., K.T.M. Kumar, A.S. Sundar, L. Panayappan and M. Chatterjee. 2007. Metallothionein: an overview, World J. Gastroenterol., 13: 993-996. https://doi.org/10.3748/wjg.v13.i7.993
  39. Wang, L., L. Song, D. Ni, H. Zhang and W. Liu. 2009. Alteration of metallothionein mRNA in bay scallop Argopecten irradians under cadmium exposure and bacteria challenge. Comp. Biochem. Physiol. C Pharmacol. Toxicol. Endocrinol., 149: 50-57. https://doi.org/10.1016/j.cbpc.2008.07.001
  40. Woo, S., S. Yum, J.H. Jung, W.J. Shim, C.H. Lee and T.K. Lee. 2006. Heavy metal-induced differential gene expression of metallothionein in Javanese medaka, Oryzias javanicus. Mar. Biotechnol., 8: 654-662. https://doi.org/10.1007/s10126-006-6046-0
  41. Yan, C.H. and K.M. Chan. 2004. Cloning of zebrafish metallothionein gene and characterization of its gene promoter region in HepG2 cell line. Biochim. Biophys. Acta., 1679: 47-58. https://doi.org/10.1016/j.bbaexp.2004.04.004