Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/23565
標題: 高鹽甲烷太古生物之相容質甜菜鹼自體生合成酵素特性分析並探討其應用於模式生物阿拉伯芥與斑馬魚抗鹽抗旱的可行性
Characterization of de novo betaine synthesizing methyltransferases from Methanohalophilus portucalensis FDF1T and application in salt and drought tolerance of Arabidopsis and Brachydanio
作者: 賴姝蓉
Lai, Shu-Jung
關鍵字: methanoarchaeon;甲烷太古生物;osmolyte betaine;application in salt and drought tolerance;相容質甜菜鹼;抗鹽抗旱應用
出版社: 生命科學系所
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摘要: 
高鹽甲烷太古生物Methanohalophilus portucalensis FDF1T為目前已知可由glycine三次甲基化反應,依序生合成sarcosine、dimethylglycine及甜菜鹼的六種微生物之一。由M. portucalensis FDF1T細胞粗萃取液分別純化出低催化效率的glycine sarcosine dimethylglycine N-methyltransferase (GSDMT)及高催化效率的sarcosine dimethylglycine N-methyltransferase (SDMT)兩套甜菜鹼自體生合成系統,顯示glycine sarcosine N-methyltransferase (GSMT)與SDMT扮演主要甲基化glycine生合成甜菜鹼之角色。本研究成功獲得完整的Mpgsmt-sdmt基因組序列,並以異源表現蛋白並測試其in vitro活性。以北方墨漬法證實gsmt-sdmt受到溫度及鹽逆境誘導其基因相對轉錄量。相較於其他高鹽細菌或藍綠菌之GSMT蛋白,MpGSMT具有受鉀離子及鈉離子濃度正向調控之特性,由MpGSMT蛋白之二級與四級結構分析證實,MpGSMT蛋白會隨著鉀離子濃度增加,逐漸形成二聚體蛋白,且提高其和受質glycine及sarcosine之親合性。不同於MpSDMT及其他高鹽細菌之GSMT/SDMT蛋白,MpGSMT之甲基化活性顯著受到鉀離子及鈉離子之正向調控,且顯著受到終產物甜菜鹼的負向調控,顯示MpGSMT蛋白於高鹽甲烷太古生物適應高鹽環境時,連結salt in與相容質累積兩種滲透壓調控機制,並由累積的高濃度甜菜鹼關閉glycine methyltransferase (GMT)活性以精確的調控胞內能量的有效利用。點突變實驗結果推測GSMT之Arg167為受質glycine或sarcosine結合的位置,SDMT之Gly79、His146及Pro172可能與基質結合位置有關,而Tyr16及Asp142於結構上能穩定甲基轉移過程中SN2機制之過渡時期。由異源共表現MpGSMT及MpSDMT於E. coli中其具有in vivo甜菜鹼累積,並提升大腸桿菌對高鹽逆境的耐受性。本研究進一步藉由農桿菌感染及顯微注射方式分別將Mpgsmt及Mpsdmt基因送入阿拉伯芥與斑馬魚中表現。比較野生型與基因轉殖阿拉伯芥於鹽逆境下生長狀況證實,異源表現MpGSMT及MpSDMT能提升轉殖株於高鹽逆境之存活率。由基因轉殖斑馬魚證實異源表現之MpGSMT及MpSDMT蛋白確實具有蛋白活性,此為目前為止成功於斑馬魚中異源表現高鹽甲烷太古生物之甜菜鹼自體生合成酵素之首例。由以上結果推測異源共表現MpGSMT及MpSDMT蛋白可以提升其它經濟作物或動物於高滲透壓逆境下存活率之潛力。

Halophilic methanoarchaeon Methanohalophilus portucalensis FDF1T is one of the six organisms that can de novo synthesize betaine through three steps of methylation from glycine. There are two betaine de novo synthesizing systems- GSDMT and SDMT- purified from M. portucalensis FDF1T with low and high catalytic efficiency respectively. This implies that GSMT_SDMT system possesses the major role to synthesize betaine. In this study, the complete gene cluster of Mpgsmt-sdmt were obtained and heterologous expressed for in vitro activity assays. The transcription levels of Mpgsmt-sdmt were induced by temperature and salt stresses which were verified by Northern hybridization. In contrast with GSMT from other halophilic bacteria or cyanobacteria, MpGSMT have unique characteristics of dramatic up regulation by potassium and sodium concentrations. The secondary and quaternary structure analyses of MpGSMT showed the dimer formation as increased potassium level that caused the substrate glycine and sarcosine binding affinity improved. The dramatic activating effects of sodium and potassium ions on the glycine and sarcosine methyltransferase activities of MpGSMT, but not MpSDMT and bacterial GSMT/SDMT, and the strong end product betaine inhibitory effect on MpGSMT suggested it is a key player in osmoregulation. Moreover, this osmoregulatory mechanism of halophilic methanogen is couple with the salt-in and osmolyte osmoadaptative strategies. For MpGSMT, Arg167 is the direct substrate binding residue with site-directed mutation evidence. For MpSDMT, Gly79, His146 and Pro172 are related to substrate binding while Tyr16 and Asp142 stabilized the SN2 reaction in methyltransfer process. Heterologous expressed MpGSMT/SDMT in E. coli could accumulate betaine and overcome high salt stress. In this study, the Mpgsmt and Mpsdmt genes were transformed into Arabidopsis and zebrafish via Agrobacterium-mediated transformation and microinjection respectively. Morphological comparison of wild-type and transgenic Arabidopsis seedlings suggested that heterologous expressed MpGSMT and MpSDMT could improve the viability of transgenic Arabidopsis under salt stress. In transgenic zebrafish study, this is the first report that demonstrates heterologous expression of betaine de novo synthesizing enzymes in zebrafish. This study suggested that co-expressed MpGSMT and MpSDMT are feasible of application in crops or animals to improve the viability under osmotic stress.
URI: http://hdl.handle.net/11455/23565
其他識別: U0005-0111201114231300
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