Cloning, expression and potential roles of chloride channel 3 (ClC-3) in gills of euryhaline medaka, Oryzias dancena
| Autor: | Huei-Ming Lin, 林彙茗 |
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| Rok vydání: | 2011 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 99 The ion absorption and secretion mechanisms by diverse membrane transport proteins in gill mitochondrion-rich (MR) cells of various teleostean species have been illustrated. This study used the brackish medaka (Oryzias dancena) to characterize expressions of chloride channel 3 (ClC-3) from the genetic to cellular level in gills. The combination of Japanese medaka (O. latipes) genome database and gene cloning with RACE- PCR were used to identify the gene isoforms of ClC-3, including three isoform sequences of 5’, two isoform sequences of 3’, and one isoform sequence of conserved region from the gills of the brackish medaka. In gills, the results of real-time PCR revealed that the a-isoform of 5’ and short- (S-) isoform of 3’ were dominantly expressed with salinity dependence. In the brackish medaka, encoding sequence of ClC-3 gene was widely expressed in diverse tissues of the medaka. In addition, the genetic locations of ClC-3 were found in MR cells by whole-mount in situ hybridization. The present study further examined the distribution and expression of branchial ClC-3 protein when acclimated to environments of various salinities, including fresh water (FW), brackish water (BW), seawater (SW), and hypersaline seawater (HSW). In response to environmental salinity, the levels of transcript of branchial ClC-3 were similar in all groups. Moreover, the protein abundances of ClC-3 protein in gill membrane fraction were both 1.7-fold higher in FW and HSW fish compared to the BW individuals. To identify whether the expression of branchial ClC-3-like protein specifically responded to lower environmental [Cl-], the brackish medaka were acclimated to artificial waters with normal (the Control group) or lower Cl- concentration (the Low Cl-group). Immunoblotting of membrane fractions of gill ClC-3 protein, however, showed constant expression in these two groups. Branchial ClC-3 protein was not affected in response to acute changes of environmental salinity. On the other hand, branchial ClC-3 immunoreactive (IR) protein was colocalized to the basolateral membrane of Na+/K+-ATPase (NKA) IR cells in the FW fish using whole-mount double immunofluorescent staining. Moreover, the highest intensities of ClC-3-IR signals were found in gill NKA-IR cells of the FW-acclimated medaka. The results of ClC-3-IR combined with cell size and number of NKA-IR cells were used to elucidate the patterns of immunoblotting profile. Taken together, ClC-3-like protein expresses in basolateral membrane of MR cells, the protein amounts are associated with the size and number of MR cells. The existence of ClC-3-like protein may trigger the step of basolateral Cl- absorption to carry out iono- and osmoregulatory functions in gill epithelial cells of the brackish medaka. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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