麩胺酸合成酵素透過消耗能量的化學反應將一氨分子與一穀胺酸合成為麩胺酸。此酵素參與在生物體內含氮分子(如核

Glutamine synthetase (GS; EC 6.3.1.2; L-glutamate ammonialigase) catalyzes the ATP-dependent conversion of glutamate and ammonia into glutamine. Due to its key role in nitrogen metabolism, including nucleotide, amino acid and urea biosynthesis, the enzyme has been ascribed an extraordinarily long evolutionary history. Thus, GS has been used as a molecular clock to establish phylogenetic relationship between different species. Through the National Center of Biotechnology Information (NCBI) using Basic Local Alignment Search Tool (BLAST) programs BLASTx (translated nucleotide-protein alignment) and BLASTn (nucleotide-nucleotide alignment) system, we obtained the complete cDNA of GS from tilapia cDNA liberary. Furthermore, the results of the alignment of tilapia GS sequence with that of other species indicated a close relationship between tilapia GS and other fishes. We also found that there is 79% homology between mammal and tilapia within the open read frame (ORF) of GS. However, sequence analysis by computer software revealed the fact that the size (0.5 kb) of GS 3’untranslated region (3’-UTR) of tilapia GS is different from that of mammals. Moreover, there is the complete distinct sequence of the 3’-UTR of tilapia GS from that of mammals. The 3'-UTR of many eukaryotic mRNAs has been implicated in the control of mRNA stability, processing, polyadenylation, and translational regulation. Accordingly, to comprehend the role of 3’-UTR in GS phylogenesis, we examine whether the 3'-UTR of tilapia GS is involved in the regulation of GS expression in mammals. We first generated the construct using pEGFP-N2 carrying the ORF (1.1kb) of tilapia GS gene (ORF-GFP) or the full length (1.6kb) of tilapia GS gene (Full-GFP). Transient or stable transfection of C6 gliomal cells with ORF-GFP indicated that GS mRNA and protein was expressed. When C6 cells were stably transfected with Full-GFP, the expression of GS mRNA, but not its protein, was found. Adenine/uridine-rich sequence elements (AREs) of the 3’-UTR have been known to regulate mRNA stability of certain chemokines. Four AREs are also found in the 3’-UTR of tilapia GS. We further generated the constructs with tilapia ORF-GFP and its 3’-UTR containing 1-4 AREs (A1-GFP, A2-GFP, A3-GFP and A4-GFP). Stable transfection of C6 cells with the different constructs indicated that tilapia GS mRNA is normally transcripted, while there was no expression of GS proteins in stable transfectants. The findings suggest tilapia GS protein expression in mammals by its 3’-UTR and unidentified evolutionary role of the 3’-UTR region of GS.

中文摘要 2
英文摘要 4
目錄 6
圖目錄 8
表目錄 9
壹、 前言 10
一、 吳郭魚的研究 10
二、 穀安酸及麩胺酸在吳郭魚腦部之分佈、發育性分化的角色 10
三、 以吳郭魚做為演化指標 11
四、 麩胺酸合成酵素 12
五、 麩胺酸合成酵素在演化上的角色 13
六、 MRNA非轉譯區在生物體內的調控 14
七、 實驗目的 17
貳、 材料與方法 18
一、 材料 18
1. 細胞培養材料 18
2. 化學藥品 18
3. 試劑組 19
4. 抗體 19
二、 方法 20
1. 大白鼠神經膠質瘤細胞株 (C6 glioma cell line) 20
2. 生物資訊分析 20
3. RNA萃取與北方點墨轉印法（Northern blotting） 21
4. 質體(plasmid)DNA的製備 24
5. 細胞轉染(transfection) 25
6. 反轉錄(reverse transcription)與聚合

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