臺灣博碩士論文加值系統

微藻之分類繁瑣，隨著培養條件的不同其形態會跟著有所變化，造成分類上的困難，近年來，已有許多研究利用分子生物學的方法進一步鑑定藻種間的親緣關係，其中利用18S rDNA片段序列來作為藻種親緣關係的依據最為普遍。因此本研究目的，在於分離與純化魚塭地區之魚塭微藻，形態特徵及其18S rDNA片段序列來探討其親緣關係。結果顯示：以Marine enrinchment medium f/2淡水培養基在光照70~80 ?慆ole photon m-2s-1，溫度32±0.5℃的培養條件下，已分離純化高雄茄萣魚塭地區的柵藻屬，分別編號為Scenedesmus AELCNU-JDB1 008與Scenedesmus AELCNU-JDB1 009。以光學顯微鏡檢培養於不同pH值或不同鹽濃度的洋菜培養基中的柵藻屬綠藻，顯示形態有所變異。此外，在螢光顯微鏡下觀察經尼羅紅染色處理後的兩株柵藻屬綠藻，無論培養於正常f/2 medium或缺氮培養的結果，皆無油脂反應。兩株以外部形態鑑定為Scenedesmus (柵藻屬)之Scenedesmus linearis var. linearis及Scenedesmus acuminatus var. actiformis之綠藻，經分子生物技術分析其約1700bp之18S rDNA基因序列，以及經NCBI網站比對，顯示目前資料只能確認其為Scenedesmus (柵藻屬)，但無法以其18S rDNA基因序列，比對鑑定至種的分類階層。

The classification of microalgae is complicated and mixed. The morphology of microalgae could vary with different culture conditions, these cause the classification of microalgae more difficult. Recently, many studies used molecular biology method to identify further phylogenetic relationship of algae, 18S rDNA sequencing techniques for identification basis is the most common. Therefore, this study focused on the isolation, identification and phylogenetic relationship with 18S rDNA gene sequence techniques of microalgae Scenedesmus (Chlorophyceae) taken from Kaoshiung Jiading fish farm. The result showed the native fish farm microalgae Scenedesmus: Scenedesmus AELCNU-JDB1 008; Scenedesmus AELCNU-JDB1 009 in Kaoshiung Jiading could be isolated from the by the fish farm Marine enrinchment medium f/2 fresh water medium under conditions of 32±0.5℃, and light intensity 70~80 ?慆ole photon m-2s-1. Therefore, the observation of Scenedesmus in different pH and salinity that show the morphology had changed. By the test of Nile red, these two Scenedesmus don’t have any oil reaction. In the morphology identification, they may be Scenedesmus linearis var. linearis and Scenedesmus acuminatus var. actiformis.
The analysis of molecular biotechnology of the 18S rDNA gene sequence, the sequence is about 1700bp. The identification of NCBI web site, those were Scenedesmus, but couldn’t identify the genus level by 18S rDNA gene sequence.

中文摘要 ………………………………………………………………………………… Ⅰ
英文摘要 …………………………………………………………………………………… Ⅱ
本文目錄 …………………………………………………………………………………… Ⅲ
圖目錄 ……………………………………………………………………………………. Ⅶ
表目錄 ……………………………………………………………………………………. Ⅸ
附圖目錄 ................................................................ X
縮寫表 .................................................................. XI
第一章 緒論 ……………………………………………………………………………… 1
1.1 文獻回顧 ……………………………………………………………………………… 1
1.1.1 國外文獻關於微藻的介紹 …………………………………………………………1
1.1.2 綠藻門柵藻屬之特性 ................................................ 2
1.1.3 以分子生物學方法鑑定藻種之研究 .................................... 4
1.1.4 微藻脂質功能的相關研究 ………………………………………………………… 6
1.1.5 微藻的培養技術 .................................................... 8
1.1.6 微藻在生物科技方面的應用 ……………………………………………………… 9
1.2 研究目的 ………………………………………………………………………………10
第二章 材料與方法 ………………………………………………………………………. 11
2.1 高雄縣茄萣鄉，茄萣魚塭採樣位置圖 …………………………………………….. 11
2.2 採樣的方法 ………………………………………………………………………….. 11
2.2.1 微藻之採集 ………………………………………………………………………… 11
2.3 實驗室的培養 …………………………………………………………………………11
2.3.1 藻種的分離與純化 .................................................. 12
2.3.2 培養基的配製 ...................................................... 12
2.3.3 藻種培養 .......................................................... 13
2.3.4 藻種之斜面保存 ................................................. 13
2.4 顯微鏡之觀察 ................................................... 14
2.4.1 光學顯微鏡觀察 .................................................... 14
2.5 製作藻種的生長曲線 .................................................. 14
2.5.1 OD620吸光值生長記錄 ............................................... 14
2.5.2 細胞數目生長記錄 .................................................. 15
2.6 產油微藻之快速篩檢方法 .............................................. 15
2.6.1 尼羅紅測試 ........................................................ 16
2.6.1.1 尼羅紅的特性 .................................................... 16
2.6.1.2 以尼羅紅測試純化的微藻油脂含量 .................................. 16
2.6.1.2.1染色前處理 ..................................................... 16
2.6.1.2.2染色後的螢光顯微鏡觀察 ......................................... 16
2.7藻種親緣關係分析 ..................................................... 17
2.7.1 微藻DNA的萃取 .................................................... 17
2.7.2 微藻DNA定量 ....................................................... 18
2.7.3 微藻18S rDNA 之PCR放大技術 …………………………………………………. 18
2.7.4 電泳分析 ......................................................... 20
2.7.5 PCR產物定序與比對 ................................................. 20
2.7.6 18S rDNA序列之分析 ................................................ 21
2.8微藻之藻種保存測試 ................................................... 21
第三章 結果 ……………………………………………………………………………... 22
3.1茄萣魚塭微藻的初步調查 ………………………………………………………….. 22
3.2藻種的純化 ……………………………………………………………………………. 22
3.2.1以光學顯微鏡鑑定Scenedesmus（柵藻屬）藻種編號Scenedesmus AELCNU-JDB1 008………………………………………………………………………………………….. 22
3.2.2 以光學顯微鏡鑑定Scenedesmus（柵藻屬）藻種編號Scenedesmus AELCNU-JDB1 009…………………………………………………………………………………………..23
3.3 Scenedesmus（柵藻屬）藻種編號Scenedesmus AELCNU-JDB1 008與Scenedesmus AELCNU-JDB1 009及Chlorella（小球藻屬）藻種編號Chlorella AELCNU-STR 010的18S rDNA基因序列……………………………………………………………………………… 23
3.4 Scenedesmus（柵藻屬）藻種編號Scenedesmus AELCNU-JDB1 008與Scenedesmus AELCNU-JDB1 009及Chlorella（小球藻屬）藻種編號Chlorella AELCNU-STR 010之18S rDNA基因序列與NCBI基因資料庫之比對…………………………………v……………… 28
3.5 純化藻種的耐鹽性分析 ……………………………………………………………… 29
3.6 藻種純化培養的生長記錄 …………………………………………………………… 31
3.7 以Nile Red探討純化藻種的油脂含量 ……………………………………………. 32
3.8 Scenedesmus（柵藻屬）藻種編號Scenedesmus AELCNU-JDB1 008與Scenedesmus AELCNU-JDB1 009在不同低溫環境，保存4天後回復培養結果 …………………………33
第四章 討論 ……………………………………………………………………………... 34
4.1 分離純化微藻的困難 ……………………………………………………………….. 34
4.2 不同鹽濃度與pH值影響藻細胞生長形態 .................................. 34
4.3 綠藻產油潛能 ………………………………..……………………………………… 35
4.4利用DNA序列鑑定綠藻的重要價值 …………………………………………………. 35
4.5 18S rDNA與NCBI基因資料庫之比對....................................... 37
第五章 結論 ……………………………………………………………………………... 40
参考文獻 …………………………………………………………………………………. 42
圖表 ………………………………………………………………………………………. 46
附錄 ………………………………………………………………………………………. 83

圖1. 高雄縣茄萣鄉，茄萣魚塭採樣位置圖 ……………………………………....... 46
圖2. 高雄縣茄萣鄉淡水魚塭微藻的初步調查 ................................. 47
圖3. 高雄縣茄萣鄉淡水魚塭B1樣區所分離純化的藻種編號Scenedesmus AELCNU-JDB1 008………………………………………………………………………………………... 48
圖4. 高雄縣茄萣鄉淡水魚塭B1樣區所分離純化的藻種編號Scenedesmus AELCNU-JDB1
009……………………………………………………………………………………………49
圖5. 藻種編號Scenedesmus AELCNU-JDB1 008在32℃，pH6.5及pH7.5淡水f/2 medium中的生長狀況 ........................................... ………………………50
圖6. 藻種編號Scenedesmus AELCNU-JDB1 009在32℃，pH6.5及pH7.5淡水f/2 medium中的生長狀況 .............................................................. 50
圖7. 藻種編號Scenedesmus AELCNU-JDB1 008在32℃，pH8.0不同鹽濃度的培養條件下之形態特徵 ................................................................. 51
圖8. 藻種編號Scenedesmus AELCNU-JDB1 009在32℃，pH8.0不同鹽濃度的培養條件下之形態特徵 ................................................................. 51
圖9. 以OD值記錄藻種編號Scenedesmus AELCNU-JDB1 008與Scenedesmus AELCNU-JDB1 009的生長曲線 ……………………………………………………………………………… 52
圖10. 以細胞數目記錄藻種編號Scenedesmus AELCNU-JDB1 008與Scenedesmus AELCNU-JDB1 009 ………………………………………………………………………………… 52
圖11. 以光學顯微鏡及螢光顯微鏡比較以細胞數目記錄藻種編號Scenedesmus AELCNU-JDB1 008在淡水f/2 medium生長及缺氮f/2 medium中，經Nile Red染色後的油脂含量 …………………………………………………………………………………………….. 53
圖12. 以光學顯微鏡及螢光顯微鏡比較以細胞數目記錄藻種編號Scenedesmus AELCNU-JDB1 009在淡水f/2 medium生長及缺氮f/2 medium中，經Nile Red染色後的油脂含量 …….………………………………………………………………………………………… 54
圖13. Scenedesmus藻屬的DNA片段設計的primer進行PCR放大技術Scenedesmus AELCNU-JDB1 008 之18S rDNA片段電泳圖……………………………………………………... 55
圖14. Scenedesmus藻屬的DNA片段設計的primer進行PCR放大技術Scenedesmus AELCNU-JDB1 009 之18S rDNA片段電泳圖………………………………………………………..56
圖15. Chlorella藻屬的DNA片段設計的primer進行PCR放大技術Chlorella AELCNU-STR 010 之18S rDNA片段電泳圖…………………………………………………………….. 57
圖16. 藻種編號Scenedesmus AELCNU-JDB1 008（JDB1 008）與Scenedesmus AELCNU-JDB1 009（JDB1 009）以及藻種編號Chlorella AELCNU-STR 010（STR 010）之18S rDNA基因序列之排序比對圖………………………………………………………………………….. 58

表1. 採樣點的經緯度 ..................................................... 64
表2. 培養基Marin enrichment medium f/2 營養鹽成分配方 ................... 64
表3. 高雄茄萣魚塭，各個採樣點的鹽度與pH值 ............................... 65
表4. 藻種編號Scenedesmus AELCNU-JDB1 008在32℃，以pH6.5及pH7.5及不同鹽濃度所配製的洋菜培養基中的耐鹽度分析 ………………………………………………………. 65
表5. 藻種編號Scenedesmus AELCNU-JDB1 009在32℃，以pH6.5及pH7.5及不同鹽濃度所配製的洋菜培養基中的耐鹽度分析 ……………………………………………………….. 66
表6. 藻種編號Scenedesmus AELCNU-JDB1 008在32℃，pH8.0與不同鹽濃度所配製的洋菜培養基中的耐鹽度分析 ………………….………………………………………………… 66
表7. 藻種編號Scenedesmus AELCNU-JDB1 009在32℃，pH8.0與不同鹽濃度所配製的洋菜培養基中的耐鹽度分析 ………….………………………………………………………… 67
表8. 藻種編號Scenedesmus AELCNU-JDB1 008在32℃，pH8.6與不同鹽濃度所配製的洋菜培養基中的耐鹽度分析 ……….……………………………………….…………………… 67
表9. 藻種編號Scenedesmus AELCNU-JDB1 009在32℃，pH8.6與不同鹽濃度所配製的洋菜培養基中的耐鹽度分析 …………….………………………………………………………. 68
表10. 藻種編號Scenedesmus AELCNU-JDB1 008與Scenedesmus AELCNU-JDB1 009在不同低溫環境中，保存4天後的回復培養結果…………………………………………………. 68
表11. 藻種編號Scenedesmus AELCNU-JDB1 008之18S rDNA片段，經NCBI資料庫的比對結果所得的相近藻種…………………………………………………………………………… 69
表12. 藻種編號Scenedesmus AELCNU-JDB1 009之18S rDNA片段，經NCBI資料庫的比對結果所得的相近藻種…………………………………………………………………………… 74
表13. 藻種編號Chlorella AELCNU-STR 010之18S rDNA片段，經NCBI資料庫的比對結果所得的相近藻種……………………………………………………………………………... 79

附圖1. 批次培養藻類生長速率的表現圖 ……………………………………………... 86
附圖2. Scenedesmus obliquus細胞暴露在補食者水蚤之下生長72小時後的細胞數目比例圖….................................................................... 86
附圖3. 核醣體基因位置圖………………………................................ 87
附圖4. 分類階層範圍圖…………………………………………………………………. 88
附圖5. Scenedesnms obliquus在生長期與缺氮培養期間的總脂肪酸構成之相關曲線圖..................................................................... 88
附圖6. 微藻之不同的衍生物在商業上的運用圖................................ 89
附圖7. Scenedesmus柵藻屬藻種編號Secenedesmus AELCNU-JDB1 008之部分18S rDNA 5’端定序圖…………………………………………………………………………………….90
附圖8. Scenedesmus柵藻屬藻種編號Secenedesmus AELCNU-JDB1 008之部分18S rDNA 3’端定序圖………………………………………………………………………………….. 91
附圖9. Scenedesmus柵藻屬藻種編號Secenedesmus AELCNU-JDB1 009之部分18S rDNA 5’端定序圖………………………………………………………………………………………92
附圖10. Scenedesmus柵藻屬藻種編號Secenedesmus AELCNU-JDB1 009之部分18S rDNA 3’端定序圖………………………………………………………………………………………93
附圖11. Chlorella小球藻屬藻種編號Chlorella AELCNU-STR010之部分18S rDNA 5’端定序圖……………………………………………………………………………………………94
附圖12. Chlorella小球藻屬藻種編號Chlorella AELCNU-STR010之部分18S rDNA 3’端定序圖……………………………………………………………………………………………95
附表1. 綠藻及相關種系的總脂肪酸含量比較…………………………………………. 96
附表2. 美國國家生物技術資料中心（NCBI）GenBank 可搜尋比對之Scenedesmus（柵藻屬）綠藻之18S rDNA基因序列索引編碼 ........……………………………………… 97
附表3. 美國國家生物技術資料中心（NCBI）GenBank 可搜尋比對之Chlorella（小球藻屬）綠藻之18S rDNA基因序列索引編碼........……………………………………… 100

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