| Summary: | 碩士 === 國立高雄海洋科技大學 === 海洋生物技術研究所 === 103 === Heat shock proteins (Hsps) are a group of highly conserved molecule involved in a wide range of biochemical processes in response to external stressors. In this study, the full length cDNA of heat shock protein 60 was cloned from liver of orange-spotted grouper Epinephelus coioides and designated as EcHsp60. Its cDNA was 2341 bp in length and encoded a putative protein of 578 amino acids (Accession no.: KM189816). Based on the result of the multiple sequence alignment and phylogenetic analysis, it revealed that the EcHsp60 gene shared a high similarity with other known Hsp60 sequences, most similar to E. akaara, and contained all three classical Hsp60 family signatures. According to the real-time quantitative PCR analysis, the mRNA of Hsp60, Hsp70 and Hsp90AB was constitutively expressed in all examined tissues of untreated orange-spotted grouper, including muscle, liver, intestine, gill, heart, head kidney, spleen and brain. In order to understand the relation between Hsps and immune responses in orange-spotted grouper, the grouper were injected with poly I:C and lipopolysaccharide, LPS and the mRNA expression was analysed after 24 hours. For the stimulation with poly I:C, mRNA expressions of Hsp60 and Hsp70 were significantly up-regulated in brain but only Hsp60 increased in head kidney. After stimulation with LPS, mRNA expressions of Hsp60 and Hsp70 were significantly up-regulated but Hsp90AB down-regulated in both head kidney and spleen. We also infected groupers with nervous necrosis virus (NNV) and Vibrio harveyi, respectively. Following NNV infection, the mRNA expression of Hsp60 increased at 6, 24 and 72 h in spleen, also at 48 and 72 h in head kidney. Hsp70 transcript levels increased at 6 h in spleen, but decreased at 72 h in head kidney. The Hsp90AB mRNA expression was down-regulated at 6, 24, 48 and 72 h in spleen, but up-regulated at 48 h in head kidney. After V. harveyi challenge, Hsp60 transcript levels increased at 72 h in spleen, also at 6, 48 and 72 h in head kidney. Hsp70 levels increased at 6 h in spleen, but decreased at 24 and 72 h in head kidney. The Hsp90AB expression was down-regulated at 24 h in spleen, also at 6 and 24 in head kidney. These results implicated Hsps in the immune response of orange-spotted grouper, and they represented different expression patterns induced by virus and bacteria. The data presented in this study is the first report describing an effect of both NNV and live V. harveyi on Hsp expression in grouper fish.
|
|---|
