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標題: 利用轉錄體分析探討廣鹽性恆河稻田魚在低溫時誘導的基因表現及訊息途徑變化
Investigation of low-temperature induced gene expressions and pathway alterations in euryhaline medaka, Oryzias dancena, by transcriptomic analyses
作者: Pin-Jhu Liao
關鍵字: 次世代定序
Next generation sequencing
low temperature
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摘要: The purpose of this thesis is to systemically investigate molecular modulations when euryhaline brackish medaka, Oryzias dancena under cold stress. Fresh water (FW)- and seawater (SW)-acclimated brackish medaka were transferred from 28°C to 18°C. After cold exposure to 18°C for a week, brain, gill and liver were sampled. Next generation sequencing was applied for de novo assembly and transcriptome analyses. The assembled transcripts were further used to KEGG annotation. The numbers of genes in KEGG mapping results were mainly categorized into signal transduction group in all three organs under low temperature. In brain, no specific gene groups or signaling pathways were up-regulated under low temperature comparing with other organs. Expression of some specific genes in pathways belong to environmental information processing were activated in the gill. Up-regulations of gene groups in the liver were mainly found among the pathway of metabolic groups especially in carbohydrate metabolism. Nevertheless, gill is the only organ found with largest numbers of up-regulated gene under low temperature. After cold transfer, the following experiments were performed. In the gill of SW-acclimated brackish medaka, the mRNA abundance of anti-apoptosis factor, baculoviral inhibitor of apoptosis repeat-containing 5 (birc5) and protein level of cleaved-caspase-3 increased. The osmoregulatory protein, Na+, K+-ATPase (NKA) protein and activity declined and ATP-sensitive inward rectifier potassium channel 1 (kcnj1) mRNA abundance increased. On the other hand, decreased mRNA abundance of tight junction and related gene crumbs homolog 3 (crb3) mRNA abundance with increased Na+, K+, 2Cl--cotransporter (NKCC) and kcnj1 were found in the gill of FW-acclimated brackish medaka. In this thesis, large numbers of complete coding sequences and pathways classified of mRNA were found to advantage next experimental usage. To combine pathway speculate and physiological change, different adaptation to 18°C of brackish medaka were found: To compare 18°C and 28°C medaka, kcnj1 mRNA abundant were upregulated at both SW18°C and FW18°C, but apoptosis protein were only upregulated at SW18°C. Coupling with mRNA abundant relating to tight junction were downregulated and NKCC mRNA abundant were upregulated at FW18°C. In conclusion, cell permeability of gill and different reaction of ionocyte to cold stress were critical issues in euryhaline fish.
為了系統性地了解廣鹽性恆河稻田魚(Oryzias dancena)面對低溫緊迫時的調節機制,本研究將淡、海水馴養的恆河稻田魚從28°C轉移至18°C一週,分別採樣其腦、鰓以及肝臟,使用次世代定序蒐集序列進行轉錄體分析,並利用生物反應路徑資料庫(KEGG)命名組裝完成的轉錄體。三個器官在低溫時分類於訊息傳遞的基因群上升數目都佔最大部分,其中腦與其他器官相比,並沒有獨特的訊息傳遞路徑基因群在低溫時上升,鰓的基因表現在環境適應類群路徑有特定的活躍路徑,肝臟則是在代謝基因群的尤其是基因屬於醣類代謝的訊息傳遞路徑的數目有所上升,最後可以發現低溫表現上升的基因數量在鰓佔最大部分。在海水馴化的恆河稻田魚鰓中,細胞凋亡抑制因子5 (birc5)基因表現量伴隨著剪切活化的第三型硫胱胺酸蛋白酶(cleaved-Caspase-3)增加,同時離子調節細胞上的鈉鉀幫浦(Na+, K+-ATPase)蛋白表現量及活性下降,內向整流鉀離子通道家族蛋白基因1 (kcnj1) 基因表現量上升;淡水馴化的恆河稻田魚鰓中,細胞緊密連結途徑主要相關蛋白基因下降,伴隨細胞極性調控同源基因3 (crb3)的基因表現量下降,離子調節細胞上鈉鉀氯共轉運蛋白(NKCC)的基因及kcnj1基因表現量則顯著上升。在本論文中,大量完整的編碼序列被定序並且組裝出來,除了利於未來的實驗應用之外,結合訊息途徑的探索及生理的變化,發現了不同鹽度馴化的恆河稻田魚其鰓對於18度的低溫有不同的適應能力:雖然淡、海水馴化的恆河稻田魚鰓上離子調節細胞中的KCNJ1表現量皆上升,海水馴化的恆河稻田魚鰓上卻有較多的細胞凋亡蛋白增加;再加上淡水馴化的恆河稻田魚鰓上細胞緊密連結的下降,離子調節細胞中NKCC基因表現量的上升,綜合以上結果,鰓上細胞間的通透性及離子細胞在低溫時的不同反應,可能是馴養於淡水的稻田魚較能適應低溫的關鍵。
文章公開時間: 2018-08-28
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