碩士 / 國立臺灣海洋大學 / 生物科技研究所 / 95 / The goal of this research was to explore the ability of marine and freshwater algae to detoxify the heavy metal arsenic (As) via biomethylation. The marine alga Tetraselmis chuii cultured with 20.17μg/L arsenate (As5+) and 28.85μg/L arsenite (As3+) for 5 days started to generate monomethyl arsenic species (MMA) and dimethyl arsenic species (DMA) as analyzed by hydride generation - packed coldfinger trap - atomic absorption spectrometry (HG-PCFT-AAS). T. chuii cultured with 201.7μg/L As5+ generated significantly higher level of methylated metabolites. The freshwater alga C. pyrenoidosa showed no methylating activity under either arsenate or arsenite during in vivo or in vitro methylation assay, yet the concentration of inorganic arsenite showed a rapid decrease after 16-h culturing, possibly mediated by extracellular adsorption. Inclusion of 1 mM dithiothreitol (DTT), but not 1 mM glutathione (GSH), in medium culturing T. chuii was shown to enhance the concentration of methylated arsenic metabolites apparently. Hence, DTT might be an important cofactor for arsenic methylation in T. chuii.
For producing C. pyrenoidosa with arsenic methylation ability, a 1011-bp cDNA encoding the open reading frame of mouse arsenic methyltransferase (cyt19) was cloned into the agrobacterium expression vector pBIN19 under the control of a Pnos promoter and cyt19 ORF was transformed into C. pyrenoidosa via infection with Agrobacterium tumefaciens. Five successful integration of cyt19 gene into algal genomic DNA was confirmed by PCR, and cyt19 gene expression was confirmed by RT-PCR. HG-PCFT-AAS analysis showed that transfected Chlorella pyrenoidosa displayed no detectable arsenate methylation ability, yet transfected C. pyrenoidosa cultured with 288.5μg/L As3+ and 201.7μg/L As5+ generated slightly methylated arsenic metabolites. The result of this research showed that marine alga T. chuii and fresh alga C. pyrenoidosa detoxify aquatic As by totally different mechanisms. Both of them can adsorb inorganic As in the environment, and can be used in bioremediation objectives.
Key word: C. pyrenoidosa, T. chuii, arsenic methyl transferase, atomic adsorption, methylated As, agrobacterium mediated transformation
| Identifer | oai:union.ndltd.org:TW/095NTOU5111042 |
| Date | January 2007 |
| Creators | Ming-Shuo Chen, 陳銘碩 |
| Contributors | Todd Hsu, 許濤 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 66 |
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