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標題: 嗜鹽性甲烷太古生物相容質甜菜鹼自體生合成基因的選殖
Cloning the gene of Osmolyte Betaine Synthesizing Enzyme from Methanohalophilus portucalensis strain FDF1
作者: 蘇旭梅
Su, Hsu-Mei
關鍵字: archaea;太古生物;methanogen;halophilic;osmolyte;compatible solute;osmoprotectants;osmotic stress;glycine betaine;methyltransferase;甲烷菌;嗜鹽性;相容質;滲透壓逆境;甜菜鹼;甲基轉移酵素
出版社: 生命科學系
高鹽甲烷太古生物Methanohalophilus portucalensis strain FDF1為少數會自體生合成相容質甜菜鹼 (glycine betaine, N, N, N-trimethylglycine) 的生物,甜菜鹼為最普遍且滲透壓保護係數最高的相容質。自體生合成甜菜鹼的方式是由甘氨酸 (Glycine) 經過三次甲基化合成,並由腺苷甲硫氨酸 (SAM, S-adenosylmethionine) 提供甲基。本研究針對太古生物M. portucalensis FDF1的甜菜鹼生合成基因進行選殖,主要針對高鹽細菌甜菜鹼生合成酵素的SAM binding motif序列,以及M. portucalensis FDF1甜菜鹼生合成酵素Glycine sarcosine dimethylglycine N-methyltransferase (GSDMT) 已知的N端15個胺基酸設計引子,進行聚合酶連鎖反應 (PCR) 以及反轉錄聚合酶連鎖反應 (RT-PCR)。結果以M. portucalensis FDF1的GSDMT蛋白N端胺基酸參考FDF1慣用密碼子設計的退化引子以及oligo dT引子,經由RT-PCR得到具有GSDMT蛋白N端14個胺基酸NEYFVRMGDGERIS的基因片段RT-2與RT-3,此兩片段僅存在於胞外不含甜菜鹼的M. portucalensis FDF1全細胞RNA中,胞外有甜菜鹼存在的情況下並不存在。M. portucalensis FDF1的GSDMT蛋白經LC-MS/MS分析後經由比對發現其具有與甲烷菌Methanosarcina barkeri dimethylamine methyltransferase MtbB1相似的片段,於RT-2與RT-3的胺基酸序列中則發現有該片段的存在,且RT-2與RT-3亦具有M. portucalensis FDF1的GSDMT蛋白經由Chymotrypsin酵素切割後之片段的N端胺基酸KINEA,因此確認RT-2與RT-3應為M. portucalensis FDF1甜菜鹼生合成基因的部分序列。

Methanohalophilus portucalensis strain FDF1 is one of the few organisms that can de novo synthesize osmolyte glycine betaine from glycine through threefold methylation. And S-adenosylmethionine (SAM) was verified as a methyl group donor. Glycine betaine is the common osmolyte and has the highest osmoprotection efficiency. The strategy for cloning the gene of the glycine betaine synthesizing enzyme was performed by the polymerase chain reaction technology (PCR) and the reverse transcription polymerase chain reaction technology (RT-PCR). Primers were designed from the fifteen amino acid sequences of the N-terminal glycine betaine synthesizing enzyme glycine sarcosine dimethylglycine N-methyltransferase (GSDMT) of M. portucalensis FDF1 and SAM binding motifs of glycine betaine de novo synthesizing enzymes from halophilic bacteria. The fragment RT-2 and RT-3, which was from RT-PCR with the degenerate primer from N-terminal amino acid sequences of GSDMT of M. portucalensis FDF1 and the oligo dT primer, was present only under the condition with no external glycine betaine addition. RT-2 and RT-3 contained fourteen N-terminal amino acid sequences of glycine betaine de novo synthesizing enzyme of M. portucalensis FDF1 and the N-terminal amino acid sequences of KINEA of chymotrypsin digested GNMT. The LC-MS/MS results showed that GSDMT contained the amino acid fragment similar to Methanosarcina barkeri dimethylamine methyltransferase MtbB1. And the amino acid fragment that similar to MtbB1 is also found in RT-2 and RT-3. Therefore the RT-2 and RT-3 amplified by RT-PCR are glycine betaine de novo synthesizing partial gene of M. portucalensis FDF1.
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