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

Abstract

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.