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標題: | 微囊藻產毒基因檢測技術開發與微囊藻毒素釋放生理研究 Concerns of the application of mcy genes analysis in the toxic Microcystis population screening and observations on the release of microcystins |
作者: | Jian-Zhi Lin 林健智 |
指導教授: | 周宏農 |
關鍵字: | 微囊藻,微囊藻毒,定量PCR,藻毒釋放, Microcystis,Microcystins,Real-Time PCR,Toxin release, |
出版年 : | 2007 |
學位: | 碩士 |
摘要: | 本研究論文分為兩大主題,一為探討利用定量PCR技術,檢測水體中微囊藻族群數的可行性;另一實驗則觀察本實驗室一株由桃園單離培養的微囊藻株,其藻毒合成與釋放的相互關係。
實驗一,藉由設計七組不同產毒基因片段(mcyA ~ E and J及mcy promoter)之引子,並以台灣地區單離培養的微囊藻種(8株產毒,四株不產毒)作為定量PCR最佳檢測目標基因的測試。定量PCR結果顯示:(1)所有產毒微囊藻皆具有mcys,而不產毒微囊藻則含有微量或不含有mcys;(2)PCR產物進行鎔點分析,發現mcyD的序列最為保守,適合作為產毒微囊藻的定量目標基因;(3)微囊藻細胞數與定量PCR的Ct值呈直線相關;(4)定量PCR的分析結果不受野外樣品與實驗室藻混合樣品的影響。定量PCR具備有快速且靈敏的優點,相當適合用於環境微囊藻族群的監測之用。 實驗二,過去的研究相信,微囊藻毒主要存在於細胞中,只有當細胞老化或死亡時,才會將毒素釋出,然本實驗室發現一特殊的微囊藻株M.TY-1,此藻株在指數生長期時具有大量藻毒釋放的現象。藉由McyH蛋白質序列比對,發現其與不釋放藻毒的藻株僅一個胺基酸發生突變,由於McyH被認為有藻毒釋放與合成藻毒的雙重功能,因此M.TY-1將可提供為後續藻毒釋放研究的模式品系。 The suitability of using mcy genes analysis for screening toxic Microcystis populations and the release of microcystins were determined. Seven characteristic segments, mcyA~E, J, and the promoter of the microcystin synthetase gene cluster, designed in a Q-PCR amplification were applied for the quantitative measurement of toxic populations in environmental samples. Observations during the method-development experiments against 8 toxic and 4 non-toxic clones of Microcystis were: (i) the expected specific amplicons were found in all toxic clones but absent or less amount in non-toxic clones; (ii) all the toxic clones showed consistent Tm values of mcyD, implying this partial mcyD segment to be the most conserved one among tested mcys; (iii) a linear correlation was obtained between the microscopically determined cell numbers and the PCR threshold cycles; (iv) cell concentration of the toxic Microcystis from Q-PCR measurement was not affected by field sample and the addendum of non-toxic populations. Q-PCR is sensitivity and provides an efficient technology suitable for toxic Microcystis population screening. Microcystins are considered primary intracellular and microcystins release was generally considered to be linked to a decrease in the integrity of the cells. However, from a strain of M. aeruginosa designated as M.TY-1, it was found that microcystins are released during the log phase of growth and continuously accumulated in the cell-free medium. A single amino acid difference of the sequence McyH was found in the toxin-releasing strain in comparison with other non-toxin-releasing strains. Sequence of McyH was known to be homologous to the bacteria ABC transporter, but also known as a key protein that maintained the integrity of microcystin synthetase clusters. This significant toxin release strain would be a model for the research of the M. aeruginosa toxin release system. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/30759 |
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顯示於系所單位: | 漁業科學研究所 |
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