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研究生:洪翊綾
研究生(外文):Yi-LingHong
論文名稱:柱孢藻在生長及加氯氧化下毒素生成及基因表現變化之研究
論文名稱(外文):Effect of chlorination and cell growth on toxin production and gene expression in Cylindrospermopsis raciborskii
指導教授:林財富林財富引用關係
指導教授(外文):Tsair-Fuh Lin
學位類別:碩士
校院名稱:國立成功大學
系所名稱:環境工程學系
學門:工程學門
學類:環境工程學類
論文種類:學術論文
論文出版年:2020
畢業學年度:108
語文別:中文
論文頁數:103
中文關鍵詞:拉氏柱孢藻柱孢藻毒素加氯氧化基因表現mRNA
外文關鍵詞:Cylindrospermopsiscylindrospermopsinchlorinationgene expressionmRNA
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藍綠菌及其代謝物為淨水處理程序中的一大難題,嚴重影響台灣的飲用水安全,為台灣飲用水中重要議題之一。水庫或淨水廠常以氧化法處理藻類,然而低劑量氧化劑對於藻類細胞的破壞,不像高劑量氧化劑立即及明顯,並存在低劑量氧化劑對藍綠菌細胞形成環境壓力之可能,使得藍綠菌的基因表現量改變,進而導致代謝物濃度變化增加,造成後端處理的負荷。本研究透過瞭解低劑量氧化劑對藍綠菌細胞之影響,評估細胞後續破裂行為以及基因表現與產毒量改變,以提供飲用水水源及水廠控制技術及風險管理基礎與參考。
研究中首先建立並優化藍綠菌產毒基因表現量分析技術,應用反轉錄即時定量聚合酶連鎖反應(Reverse Transcription Real-time Quantitative Polymerase Chain Reaction, RT-qPCR)為基礎,定量樣品的mRNA,其步驟包括萃取mRNA,去除殘餘DNA,並以反轉錄(Reverse Transcription)程序將mRNA轉換為cDNA後,利用即時定量聚合酶連鎖反應進行定量分析。研究中並將優化後mRNA定量方法成功應用於拉氏柱孢藻(Cylindrospermopsis raciborskii)的樣品中的mRNA量測試,樣品中同步測試毒素濃度,建立mRNA基因量與毒素間的相關性,並可搭配mRNA基因當量(Gene quota)回推水體中毒素生成的風險。
mRNA定量分析技術後續並應用在氧化批次實驗中,搭配活菌聚合酶連鎖反應(Viability Polymerase Chain Reaction, vPCR)與PMA染劑評估藻體細胞的完整性,酵素連結免疫吸附分析法(ELISA)分析毒素,以評估氧化劑對藻類產毒基因表現的影響。次氯酸鈉氧化劑量為0.2 mg/L下,不論柱孢藻數的高低,皆能在氧化5分鐘內造成80-95%左右的細胞受損,而當次氯酸鈉氧化劑量為0.1 mg/L下,在氧化5分鐘內則仍有40%的細胞存活,因此皆能觀察到剩餘存活細胞受到次氯酸鈉的刺激,使得氧化後相對產毒基因表現量增加2-2.6倍,進而導致毒素濃度增加的現象。研究結果再次證實,當藻類細胞未被完全破壞時,殘餘之氧化劑量對於藻體造成刺激,進而提高毒素增加之風險。本研究結果顯示氧化劑對藍綠菌產生的環境壓力不容小覷,並可提供後續研究或水庫及水廠使用氧化劑時之參考。
Cylindrospermopsis raciborskii is a filamentous, nitrogen fixing cyanobacterium found in many source water in Taiwan and many other countries, with the capability of producing cyanotoxin, cylindrospermopsin. Recent studies for oxidation of cyanobacteria in w atertreatment systems have been only focused on high-dose of oxidants. However, applications of low-dose oxidants do not quickly lyse cells, and their effect on the cells is xepcetd to be dissimilar to that for high-dose oxidation. The difference might include change of gene expression and change of production rate of toxins. Therefore, it is important to study the interaction between cyanobacteria and oxidants under low dose conditions.
In this study, oxidation experiments of C. raciborskii were conducted and cell integrity, gene expression of mRNA, and cyanotoxin concentration during the experiments were measured. Viability Polymerase Chain Reaction (vPCR) coupled with PMA dye was used to evaluate cell integrity. RNA was extracted and quantified with transcription real-time quantitative polymerase chain reaction (RT-qPCR) for the mRNA of C. raciborskii. Enzyme-linked immunosorbent analysis (ELISA) was used to measure cylindrospermopsin concentration.
The results of oxidation experiments showed a 2-2.6 fold upregulation of toxin-producing genes and an increase of toxin concentration after chlorination at 0.1 mg/L, indicating the response of cyanobacteria to the oxidative stress from chlorine. The results of this study showed that the environmental pressure caused by low-dose oxidants to C. raciborskii cannot be ignored.
摘要 I
Extended Abstract III
目錄 VII
表目錄 X
圖目錄 XI
第 1 章 緒論 1
1.1 研究動機 1
1.2 研究目的 2
第 2 章 文獻回顧 3
2.1 藍綠菌對飲用水安全的危害 3
2.1.1 藍綠菌 3
2.1.2 藍綠菌代謝物 4
2.2 去除水中藍綠菌及藻毒之技術 15
2.2.1 藍綠菌及藻毒去除方法 15
2.2.2 氧化作用對藍綠菌的影響 16
2.3 環境壓力對藍綠菌的影響 18
2.4 分子生物技術 21
2.4.1 即時定量聚合酶連鎖反應(Real-time Quantitative Polymerase Chain Reaction, qPCR) 21
2.4.2 活菌聚合酶連鎖反應(Viability Polymerase Chain Reaction, vPCR) 23
2.4.3 反轉錄即時定量聚合酶連鎖反應(Reverse Transcription Real-time Quantitative Polymerase Chain Reaction, RT-qPCR) 24
第 3 章 實驗設備與方法 26
3.1 研究架構 26
3.2 氧化批次實驗流程 27
3.3 藻類選用與培養 28
3.3.1 藻類來源 28
3.3.2 藻類培養 29
3.3.3 藻類計數方式 31
3.4 DNA萃取 34
3.5 DNA純化 36
3.6 RNA萃取(最佳化流程) 38
3.7 RNA純化(DNase) 40
3.8 反轉錄(Reverse Transcription) 41
3.9 活菌聚合酶連鎖反應(Viability Polymerase Chain Reaction, vPCR) 42
3.10 即時定量聚合酶連鎖反應(Real-time Quantitative Polymerase Chain Reaction, qPCR) 43
3.11 酵素連結免疫吸附分析法(Enzyme Linked Immunosorbent Analysis, ELISA) 45
第 4 章 結果與討論 48
4.1 mRNA定量方法最佳化 48
4.1.1 萃取方法最佳化 48
4.1.2 純化(DNase)效率 53
4.1.3 反轉錄效率 55
4.2 柱孢藻生長曲線 57
4.3 氧化批次實驗 66
4.3.1 以柱孢藻為目標,氧化劑量 0.2 mg/L 66
4.3.2 以柱孢藻為目標,氧氧化劑量 0.1 mg/L 74
4.3.3 以柱孢藻為目標,藻數 10^5 cells/mL 82
4.3.4 比較不同藻種的氧化作用 83
4.4 氧化動力模式模擬 88
第 5 章 結論與建議 93
5.1 結論 93
5.2 建議 96
第 6 章 參考文獻 97
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