Characterization of de novo betaine synthesizing methyltransferases from Methanohalophilus portucalensis FDF1T and application in salt and drought tolerance of Arabidopsis and Brachydanio

• Main Authors: Shu-Jung Lai, 賴姝蓉
• Other Authors: Mei-Chin Lai
• Format: Others
• Language: zh-TW
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/45907773285325203449

博士 === 國立中興大學 === 生命科學系所 === 100 === 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.