碩士 / 國立臺灣海洋大學 / 生物科技研究所 / 94 / The goal of this thesis was to expolore the arsenic methylating ability of marine and freshwater algae. Experimental analysis by hydride generation - packed coldfinger trap - atomic absorption spectrometric (HG-PCFT-AAS) showed that marine alga Tetraselmis chuii cultrued with 0.133�嵱 arsenate for 7 days generated monomethyl arsenic species (MMA) and dimethyl arsenic species (DMA), but freshwater alga Chlorella pyrenoidosa showed no methylating activity under the same condition. Tetraselmis chuii and Chlorella pyrenoidosa both showed no methylating activity when cultrued with 0.133�嵱 arsenite, a fast decrease in arsenite concentration in medium was detected after culturing Chlorella pyrenoidosa with 0.133�嵱 arsenite suggesting a rapidly binding of arsenite to algal cells. The arsenic concentration in Tetraselmis chuii medium decrease slowly indicating the weak capability of Tetraselmis chuii uptake the arsenite may be the reason why Tetraselmis chuii show no methylating activity or arsenite undergo another metabolism pathway. The addition of reducing agent GSH showed that GSH accelerate the rate of arsenic methylation and the arsenic concentration decrease more rapidly . We suggest that GSH is a important reducing factor in Tetraselmis chuii for arsenic methylation.
For producing an alga species capable of methylating arsenic in freshwater, a 1011-bp cDNA carrying the complete open reading frame of mouse arsenic methyltransferase(cyt 19) was cloned into the expression vector pBIN19 under the control of a Pnos promotor and cyt19 ORF was transformed into Chlorella via infection with Agrobacterium tumefaciens.
A successful transformation was confirmed by PCR,and RT-PCR showed that the expression of cyt19-m-RNA in Chlorella pyrenoidosa Analysis by HG-PCFT-AAS showed that transformed cyt19-transformed Chlorella pyrenoidosa have no arsenic methylation activity. A low but clearly detectable level of arsenic methylation was produced in cyt19-transformed with the presence of GSH and DTT.
2-D gel electrophoresis of crude extract proteins of T. chuii cultured in the presence or absence of 0.133 μM sodium arsenite for 3 days, A 40 kDa polypeptide having a PI about 6 and a 25kDa polypeptide possessing a PI about 5 were found to increase significantly in T. chuii.
| Identifer | oai:union.ndltd.org:TW/094NTOU5111022 |
| Date | January 2006 |
| Creators | Zhen-Hao Liao, 廖振皓 |
| 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 | 65 |
Page generated in 0.0118 seconds