臺灣博碩士論文加值系統

The present study investigated the HSPs expression induced by hypotonic stress in gills of euryhaline milkfish. Since heat shock response is a predominant cellular stress response, two of its major components, heat shock protein 70 (HSP70) and 90 were examined in this study. Four hsp genes were first identified from the transcriptome database, divided into inducible (hsp under stress) and constitutive (hsp unstressed) forms. The alignment and phylogenetic analysis of the sequences revealed the four hsps (mfhsp) groups of milkfish, including mfhsp70, mfhsc70, mfhsp90α, and mfhsp90β. In addition, the mRNA and protein expression were examined through seawater (SW; 35‰) and freshwater (FW) acclimation experiments (> 1 month) and time-course (short term) experiments by direct transfer from SW to FW. In acclimation experiments, although the mRNA abundance of gill hsc70 of the FW-acclimated group was similar to that of the SW-acclimated group, gill hsp70 of the FW group was significantly higher (5.9 folds) than the SW group. Meanwhile, the mRNA abundacne of gill hsp90α of FW milkfish was slightly higher (1.5 folds) compared to the SW group, while the mRNA abundance of hsp90β was not different between the two groups. At the protein level, the abundance of HSP70 and HSP90 of milkfish gills were significantly higher (2.8 and 2.5 folds, respectively) in FW rather than in SW. On the other hand, analysis of gill hsp genes expression in short-term experiments revealed that gills hsc70 mRNA abundance increased within 3 hrs and declined gradually to 168 hrs. However, mRNA amounts of hsp70 and hsp90α rapidly increased at the first 3 hrs post-transfer (16 and 5.8 fold, respectively). Compared to the control group, the hsp90α showed significant increase at 168 hrs post-transfer, whereas hsp90β was not changed. The immunoblots revealed that relative abundance of gill HSP70 was significantly increased at 3 and 24 hrs post-transfer (about 4.3 and 3 folds, respectively). Relatvie protein amounts of the HSP90, however, were not significantly different even though the expression was still higher about 1.7 fold at 3 hrs post-transfer. The interaction between HSP70/90 and Na+, K+-ATPase (NKA), demonstrated by immunoprecipitation, indicated that increasing expression of gill HSPs might protect misfolded or abnormal NKA due to hypotonic stress of the milkfish. Taken together, our findings revealed that four hsps of milkfish displayed different patterns upon hypotonic stress. Higher expression of HSPs demonstrated that inducible HSP70/90 were more efficient in responses to hypotonic stress. The interaction between HSP70/90 and NKA further illustrated the involvement of HSPs in osmoprotective roles for maintaining cellular homeostasis of milkfish gills under hypotonic stress.

ACKNOWLEDGMENT i
Abstract ii
Contents iv
1. Introduction 1
1.1 Environmental stresses to organisms 1
1.2 Fish responses to environmental stresses 1
1.3 Impact of salinity on osmoregulatory organs 3
1.4 Roles of stress proteins in fish 4
1.5 Euryhaline teleosts and milkfish (Chanos chanos) 5
1.6 The aims of the study 6
2. Material and Methods 8
2.1 Experimental fish and environments 8
2.2 RNA extraction and reverse transcription 8
2.3 Molecular cloning of hsps 9
2.4 Phylogenetic tree 9
2.5 Molecular model prediction 11
2.6 Real-time PCR 11
2.7 Antibodies 11
2.8 Gill homogenates 12
2.9 Immunoblotting 13
2.10 Co-immunoprecipitation 14
2.11 Statistical analyses 14
3. Results 15
3.1 Sequence characterization and phylogenetic tree of hsps 15
3.2 Acclimation experiments of milkfish 16
3.3 Time-course experiments of milkfish 16
3.4 Interaction between HSP70/90 and NKA in milkfish gills 18
4. Discussion 19
4.1 Characterization of heat shock proteins in the milkfish 19
4.2 Expression of HSPs in acclimation to environmental stresses 23
4.3 Time-course changes in gill HSPs upon hypotonic challenge 27
4.4 Interaction of HSP70/90 and NKA in milkfish gills 31
5. References 33
6. Tables and Figures 50
7. Appendix 75

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