黃鵬鵬臺灣大學：漁業科學研究所廖柏凱Liao, Bo-KaiBo-KaiLiao2007-11-282018-07-062007-11-282018-07-062004http://ntur.lib.ntu.edu.tw//handle/246246/59343淡水魚主要由水體中吸收鈣離子，超過百分之八十的鈣吸收是由鰓上的富含粒線體細胞主動運輸所達成。現今的魚類鈣吸收模式中，鈣離子首先被動地透過一個未知的非膜電位依賴性鈣離子通道進入細胞質中，並在底側膜端由鈉鈣交換蛋白以及細胞膜鈣幫浦主動地打入血液中。但是目前並沒有分子的證據可以支持這個模式。本論文之目的在於研究當魚類的鰓面臨不同環境的鈣離子濃度時，這些運輸蛋白之基因表現與調控。 在第一章中以莫三比克吳郭魚（Oreochromis mossambicus）作為模式動物。經由分子選殖取得吳郭魚上皮細胞鈣離子通道的基因序列。由演化樹分析推測魚類上皮細胞鈣離子通道基因相對於哺乳類只有一型。原位雜合試驗顯示吳郭魚的上皮細胞鈣離子通道、鈉鈣交換蛋白第一型以及細胞膜鈣幫浦第二型均表現在鰓上的富含粒線體細胞中。由定量PCR實驗鑑定在高低鈣環境下馴化的吳郭魚鰓之基因表現，吳郭魚的上皮細胞鈣離子通道在低鈣時表現量顯著地高於高鈣環境時，而鈉鉀幫浦Freshwater fish absorb Ca2+ predominantly from ambient water, and more than 80% of Ca2+ uptake is achieved by active transporting through gill mitochondria-rich cells (MR cells). In the current model for Ca2+ uptake in gill MR cells, Ca2+ may enter passively the cytosol via the apical un-identified voltage-independent Ca2+ channels, and then, is extruded into the plasma through the basolateral Na+/Ca2+ exchanger (NCX) and Ca2+-ATPase (PMCA). However, there was no molecular evidence to support this model. The present study was aimed to examine the regulation of the gene expressions of these transporters in fish gills upon acclimation to different ambient Ca2+ levels. In the Chapter 1, tilapia (Oreochromis mossambicus) were used as model animal. Homology-based cloning strategies were used to clone the tilapia epithelial Ca2+ channel (ECaC). The phylogeny tree of ECaC suggested that tilapia has only one form, which is a homologue of the mammalian CaT1, an isoform of ECaC dominantly found in small intestines. In situ hybridization showed that tECaC, tPMCA2 and tNCX1 were expressed in the MR cells of gills. Quantitative-PCR analysis showed that tilapia acclimated to low-Ca2+ freshwater expressed gill ECaC approximately 4-fold higher than did those acclimated to high-Ca2+ freshwater, however no significant difference was found in the expressions of tNCX1, tPMCA2 and sodium pump alpha-1 subunit between the 2 groups of tilapia. Furthermore, low-Ca2+ acclimated tilapia showed a higher Ca2+ influx rate compared with the counterpart. According to the findings of Chapter 1, the tNCX1 was expressed weakly in gill and different isoform of PMCA compared to mammalian model. In the Chapter 2, zebrafish (Danio rerio), a species with abundant sequence database, was selected to identify all the isoform of PMCA and NCX. A strategy of BLAST from the zebrafish (Danio rerio) genome database was used to clone these genes. Putative full-length open reading frames of zebrafish PMCAs and NCXs were first obtained by homology-based exon/intron prediction, and then were confirmed with the EST database. Subsequently, RT-PCR was used to clone these PMCA and NCX candidates from zebrafish. Five NCX (zNCX1, zNCX2, zNCX3, zNCX4a, and zNCX4b) genes were identified. Phylogenetic analysis showed that two prototypes of NCX (zNCX4a and zNCX4b) were found. All the zNCX genes contain a large exon in the front site, but the zNCX4a and zNCX4b have one and two intron inserts, respectively, in this exon. Six PMCA (zPMCA1a, zPMCA1b, zPMCA2, zPMCA3a, zPMCA3b, and zPMCA4) were identified. The expression patterns analysis showed that all the zPMCAs were abundant expressed in brain, and the zPMCA2 and zPMCA4 were ubiquitously expressed in various tissues. In situ hybridization showed that the zNCX1 was expressed in the MR cells of embryo skin, but the zPMCA1a, zPMCA4 and zNCX4a were not. Ca2+-binding proteins facilitate Ca2+ transport, but large number of homologous related to this function. In Chapter 3, cDNA microarray was used to screen Ca2+-binding protein candidates. Microarray analysis on the gill cDNAs of high- and low-Ca2+ acclimated zebrafish showed that cortisol-related kinase and some cytoskeleton genes were up-regulated in the low-Ca2+ gills, suggesting the involvement of cortisol and epithelial cell morphology changes in the long-term regulation of Ca2+ uptake. Nine Ca2+-binding protein candidates, ictacalcin, zS100 P isoform a, zParvalbumin1b, zParvalbumin1d, zParvalbumin3a, zParvalbumin4a, zParvalbumin4b, zCalretinin 1 and zCalretinin 2, were expressed in gills by reverse microarray analysis or RT-PCR. Based on above findings and the model of Ca2+ uptake, molecular mechanism were proposed for epithelial Ca2+ transport in fish MR cells. Ca2+ may first passively enter the cytosol via the apical ECaC, and then, are bound by Ca2+-binding protein (calretinin and/or parvalbumin3a) for the buffering and decreasing Ca2+ toxicity in the cytosol. Ca2+ is finally extruded into the plasma through the basolateral NCX1 and PMCA2. For environmental Ca2+ challenge, the PMCA2 and NCX1 are maintained in a basal level, while ECaC is a gatekeeper channel in gill MR cells and is the major regulatory target.致謝….……….…………..…………………..…….…………………………1 Abbreviations….……….……………………………..…….…………………………4 中文摘要………….………………………………….…………………………6 Abstract ……………………………………………………………………………..8 Background …………………………………………….………………………………11 Purpose…………………………………….……………….……………………………17 Chapter 1. Involvement of ECaC, NCX and PMCA in Gill Ca2+ Uptake of Tilapia (Oreochromis mossambicus) Introduction………….………………………………….…………………………19 Experimental Designs….…………………………………..………………………21 Materials and Methods….…………………………………..………………………22 Results….…………………………………….…………….….……………………28 Discussion….…………………………………….……………….…………………30 Chapter 2. Identification of PMCA and NCX isofoms from Zebrafish (Danio rerio) Genome Introduction………….………………………………….…………….……………34 Experimental Designs….………………………………….………………………36 Materials and Methods….………………………………….………………………37 Results….…………………………………….…………….………………………41 Discussion….…………………………………….……………………….…………44 Chapter 3. Ca2+-binding Protein Candidates and DNA Microarray Analysis on Gill cDNA of High- and Low-Ca2+ Acclimated Zebrafish Introduction………….………………………………….…………….……………48 Experimental Designs….………………………………….………………………50 Materials and Methods….………………………………….………………………51 Results….…………………………………….…………….………………………54 Discussion….…………………………………….……………………….…………57 Conclusion and Perspective….…………………………………….……………………61 References….…………………………………….……………………………..………63 Tables…….….…..…………………………………..…….………………………76 Figures……….….…………………………………..…….………………………833768024 bytesapplication/pdfen-US富含粒線體細胞鈉鈣交換蛋白上皮細胞鈣離子通道鈣幫浦鈣吸收Calcium uptakePMCAECaCNCXmitochondria-rich cellotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/59343/1/ntu-93-R91243006-1.pdf