Acid-base regulation and stress-resistant of Japanese medaka embryos under ocean acidification

近年由於大氣中二氧化碳(CO2)濃度升高，導致海洋生態系統受到嚴重影響，其中包括海水溫度、含氧量、酸鹼值等變異。 本研究運用日本種稻田魚擁有廣鹽性適應特性、完整基因體資源庫與發生學研究等實驗操作優勢，用以探討當硬骨魚仔稚時期面臨高CO2酸化的海水環境中，個體抗逆境蛋白、表皮離子細胞分化相關基因、以及酸鹼調控基因之表現相。實驗發現海水酸化會造成仔稚魚在2~5 dpf時期面臨發育生長瓶頸，同時酸鹼調控基因之表現有顯著提升現象；另一方面，抗逆境蛋白olhsp27a之表現可能在細胞面臨高碳酸緊迫壓力時扮演著重要角色。利用蛋白免疫染色技術發現olhsp27a表現於離子細胞，進一步運用反義核酸 (morpholino antisense oligonucleotides)抑制olHsp27a蛋白表現後，除了顯著抑制仔魚排酸機制，而酸鹼調節相關基因的表現亦發生回饋補償效應。綜合以上結果我們推論：離子細胞內hsp27a會受細胞外CO2的刺激啟動離子運輸蛋白的轉錄後修飾，進而調控表皮細胞的酸鹼恆定。Contents…………………………………………………………………………………1 中文摘要……………………………………………………………………..………….3 Abstract……………..………………………………………………………………….4 Introduction…………………………………………………………………………...6 Ocean acidification (OA) ……………………………………………………………..6 OA impacts on marine organisms……………………………………………………7 Resistance to ambient stress…………………………………………………………10 Materials and Methods…………………………………………………..………..12 Experimental animals………………………………………………………………12 CO2 perturbation experiments.……………………………………………………12 Preparation of total RNA. …………………………………………………………13 Real-time quantitative PCR (qPCR). ……………………………………………14 Whole-mount in situ hybridization. …………………………………………………15 Whole-mount immunocytochemistry. ………………………………………………16 Western blot analysis………………………………………………………………..17 Translational knockdown with antisense morpholino oligonucleotides (MOs)……17 SIET and measurement of ionic gradients.…………………………………………18 Statistical analysis. …………………………………………………………………20 Results…………………………………………………………………………………21 Responses of acid–base machinery in embryos……………………………………21 Gene expressions in various developmental stages…………………………………21 Expressions of stress-resistant heat shock proteins between control and CO2 conditions…………………………………………………………………………22 olhsp27a mRNA expressions in medaka……………………………………………22 Spatial localization of olhsp27a mRNAs in medaka epithelial ionocytes…………23 Examination of olHSP27a abrogation efficiency by Western blot analysis ………23 Effects of olhsp27a knockdown on physiological responses in medaka embryos…..24 Effects of olhsp27a knockdown on H+ gradients in larvae exposed to CO2 conditions……………………………………………………………………………24 Effects of olhsp27 knockdown on acid-base regulation relative genes……………..25 Discussion……………………………………………………………………………..26 Conclusion…………………………………………………………………………….33 References……………………………………………………………………………...34 Figures and Table…………………………………………………………………..4