Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/5072
DC FieldValueLanguage
dc.contributor張育傑zh_TW
dc.contributor張怡塘zh_TW
dc.contributor.advisor洪俊雄zh_TW
dc.contributor.author許智強zh_TW
dc.contributor.authorHsu, Chih-Chiangen_US
dc.contributor.other中興大學zh_TW
dc.date2012zh_TW
dc.date.accessioned2014-06-06T06:33:57Z-
dc.date.available2014-06-06T06:33:57Z-
dc.identifierU0005-2607201116503100zh_TW
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dc.identifier.urihttp://hdl.handle.net/11455/5072-
dc.description.abstract藻類為水域生態系中最重要之生產者,其種類眾多數量龐大,廣泛分布於各種環境中,當水中藻類族群數量組成改變時,顯示水體可能受到污染且水質可能受到藻類滋生所影響。因此,監測水體中藻類族群之變化成為相當重要之課題。以往大多使用顯微鏡進行水中藻類之觀察鑑定工作,不僅耗時費力,且因藻體特徵不易辨識或細胞過小而容易導致誤判情況產生。近年來許多學者以分子生物技術鑑定水體中藻類族群結構,發現分子生物方法具有操作容易、靈敏度高且可於短時間內處理大量樣本等優點,適合用於檢測水體中藻類族群。但目前少有文獻針對水體中藻類進行定量工作,因此本研究嘗試以PCR-DGGE與Real-Time PCR針對水中藻類進行定性及定量分析,並將結果與鏡檢分析相互比較,期望建立快速檢測水體中藻類族群結構及數量之分析技術。 本研究以23S plastid rDNA基因序列進行分子生物技術分析,利用PCR-DGGE進行藻類族群鑑定,並以Real-Time PCR進行藻類定量分析,比較分子生物技術與顯微鏡觀察分析結果。同時為瞭解針對水中特定藻類族群進行定性及定量是否可以獲取較準確之結果,本研究嘗試以18S rRNA基因序列針對矽藻族群進行鑑定,比較針對特定藻類族群進行分子生物方法與鏡檢分析結果之差異程度。 研究結果顯示,以顯微鏡觀察水體中藻類族群結構,發現優勢藻類族群以矽藻為主,但利用23S plastid rDNA基因序列進行藻類族群結構分析時,所得結果卻以藍藻門之Synechococcus sp.為最主要之藻類族群,且未偵測到任何矽藻族群存在,此外於鏡檢下也未發現Synechococcus sp.。以Real-Time PCR定量進行藻類定量後發現,定量結果與鏡檢計數結果具有不同之趨勢變化。以18S rRNA基因序列進行矽藻族群在鑑定時,可成功檢測出樣本中主要存在之矽藻族群,進一步以Real-Time PCR針對矽藻族群進行定量並將定量結果與鏡檢下矽藻族群數量進行比較,可發現兩者趨勢變化相似,顯示兩者具有正相關性存在。zh_TW
dc.description.abstractAlgae are the most important primary producers in the aquatic ecosystems and could be found in different habitats with complex phylogenetic diversity. Once the aquatic environment was polluted, indigenous algae community changed correspondently. This effect might not just a reduction in the number of species, but rather a change in the species composition of the community. Therefore monitoring the changes in population of algae become an important issue. Conventional approaches use microscopy to identify the species and numbers of algae. However, microscopic identification is not only time-consuming but also being biased by individual experiences. Furthermore, among different algae species it is not unusual to find very similar shapes, and phenotype-based species often mask genetic diversity as well. Molecular methods is a useful tool for studying algae communities in freshwater. Several recent studies have demonstrated the effectiveness of molecular methods to estimate the diversity of natural prokaryotic communities in aquatic environments. They demonstrated that molecular methods are not only fast and simple but also highly sensitive for the detection and identification of algae. The aims of this study is to compare the community difference between conventional approach (microscopic examination) and molecular methods in attempt to develop a reliable and faster procedure for the detection and quantitative determination of algae in environmental samples. Performance of PCR-DGGE method on exploring the algae diversity was examined in this study. Real-Time PCR method targeting universal 23S plastid rDNA genes was also applied for quantification means. The difference between molecular-based methods (PCR-DGGE, Real-Time PCR) and microscopic analysis were discussed. Furthermore, a set of diatom specific primer targeting 18S rDNA was applied for exploring the communities of diatom. The microscopic examination results showed that the algal communities were dominated by diatoms. However, completely different results were obtained by the 23S plastid rDNA-based PCR-DGGE method in which the algae community were dominated by Synechococcus sp. PCR-DGGE analysis cannot detect any diatoms in all sampling points using this primer set. No Synechococcus sp. could be identified by microscopic examination. We found that there were not in total agreement with the results from Real-Time PCR quantification and cell counting by microscopy. On the other hand, when the diatom specific primer was applied to assess the diatoms community composition, satisfied results were obtained in which the major diatom groups can be identified by PCR-DGGE method. The Real-Time PCR quantification results also presented more correlation with the microscopic analysis on the diatom issus.en_US
dc.description.tableofcontents目錄 中文摘要.................................................I Abstract..................................................II 表目錄...................................................VII 圖目錄..................................................VIII 第一章 前言...............................................1 第一節 研究緣起.........................................1 第二節 研究目的.........................................2 第二章 文獻回顧...........................................3 第一節 藻類及其對水體之影響.............................3 一、 藻類之特性與分類.................................3 二、 藻類對於水體之影響...............................4 (一) 優養化現象.......................................4 (二) 藻毒.............................................6 (三) 臭味問題.........................................7 (四) 消毒副產物.......................................8 三、 藻類對淨水處理程序之影響.........................9 四、 藻類之去除......................................10 第二節 以藻類作為生物指標..............................12 第三節 藻類檢測方法....................................15 一、 顯微鏡觀察......................................15 二、 葉綠素濃度分析..................................16 三、 高效能液相層析(HPLC)............................17 四、 流式細胞儀測定..................................20 五、 分子生物方法....................................21 (一) 聚合酶鏈鎖反應(PCR).............................22 (二) 變性梯度凝膠電泳法(DGGE)........................24 (三) 基因選殖(Gene Cloning)..........................25 (四) 即時定量聚合酶鏈鎖反應(Real-Time PCR)...........27 第四節 分子生物方法應用於藻類族群鑑定..................30 一、 藻類種類鑑定....................................30 (一) 針對特定藻類族群................................30 (二) 針對原核與真核藻類族群..........................32 二、 特定藻類族群定量................................36 第五節 文獻閱讀心得與擬定研究方向......................39 第三章 材料與方法........................................40 第一節 實驗架構........................................40 第二節 實驗設備........................................41 第三節 樣品來源........................................42 一、 鯉魚潭水庫......................................42 二、 中興湖湖水......................................43 第四節 分析方法........................................44 一、 樣本前處理......................................44 二、 DNA萃取.........................................44 三、 聚合酶鏈鎖反應(PCR).............................46 四、 瓊脂膠糖明膠電泳分析(Agarose Gel)...............47 五、 變性梯度明膠電泳(DGGE)..........................48 六、 DNA純化.........................................49 七、 定序............................................49 八、 基因選殖(Gene Cloning)..........................49 (一) 目標DNA序列增殖.................................49 (二) 接合反應(Ligation reaction).....................50 (三) 轉形作用(Transformation)........................50 (四) 藍白菌落篩選(Blue/White Screening)..............50 (五) 聚合酶鏈鎖反應(PCR).............................50 (六) 質體萃取........................................51 九、 即時定量聚合酶反應(Real-time PCR)...............51 十、 葉綠素a測定.....................................52 十一、 顯微鏡觀察......................................53 第四章 結果與討論........................................54 第一節 水庫藻類族群分析................................54 一、 顯微鏡觀察......................................54 二、 葉綠素a濃度測定.................................59 三、 PCR-DGGE分析....................................60 四、 Real-Time PCR定量結果...........................62 (一) Real-Time PCR反應偵測標準曲線...................62 (二) 鯉魚潭水庫樣本藻類定量..........................64 第二節 分析方法改善試驗................................67 一、 中興湖水樣本....................................67 二、 鯉魚潭水庫樣本..................................70 第三節 藻類專一性引子對(p23SrV)純藻定量試驗............78 第四節 不同時期水庫藻類族群分析........................80 第五節 特定藻類族群分析................................85 第六節 綜合討論........................................92 第五章 結論與建議........................................98 第一節 結論............................................98 第二節 建議............................................99 參考文獻.................................................100zh_TW
dc.language.isoen_USzh_TW
dc.publisher環境工程學系所zh_TW
dc.relation.urihttp://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-2607201116503100en_US
dc.subjectalgae communitiesen_US
dc.subject藻類族群結構zh_TW
dc.subjectPCR-DGGEen_US
dc.subjectReal-Time PCRen_US
dc.subjectPCR-DGGEzh_TW
dc.subjectReal-Time PCRzh_TW
dc.title利用分子生物方法檢測水中藻類族群結構之研究zh_TW
dc.titleApplication of molecular method for the detection of freshwater microalgaeen_US
dc.typeThesis and Dissertationzh_TW
item.openairecristypehttp://purl.org/coar/resource_type/c_18cf-
item.openairetypeThesis and Dissertation-
item.cerifentitytypePublications-
item.fulltextno fulltext-
item.languageiso639-1en_US-
item.grantfulltextnone-
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