對於偵測超微原核浮游植物在寡營養鹽海洋環境中的缺磷狀態而言，細胞壁上的磷酸鹽運輸蛋白基因（phosphate-binding transporter gene，pstS）是一個有潛力的指標基因。為了在東海以及時定量聚合?連鎖反應（real-time quantitative reverse transcription polymerase chain reaction，簡稱 Q-RT-PCR）量測 pstS mRNA 的豐富度，首先必須依照本海域超微浮游植物 pstS 基因序列的歧異度製作適合的引子對。從 2006 年三月與 2007年四月及七月的航次中，本研究一共蒐集了 71 條東海中的pstS序列。經由親源分析顯示：在 2006 三月航次於黑潮得到的序列大部分為原核綠藻的，而在 2007 年四月航次於黑潮得到的序列則是聚球藻 Calde II與原核綠藻各半，而同年七月在長江口往黑潮延伸的陸棚則得到屬於 Calde II, III, V 的聚球藻序列；針對以上排序設計了偵測聚球藻 pstS 的引子對 Q-pstS-Syn，以及偵測原核綠藻 pstS 的引子對 Q-pstS-Pro。在使用純種藍綠細菌的 genomic DNA 進行測試後，發現這兩對引子對目標物種有良好的專一性，但是 Q-pstS-Pro因增值效率不佳而被淘汰。之後再以增殖效率較好的聚球藻引子對Q-pstS-Syn在聚球藻（Synechococcus WH7803）缺磷培養中，以Q-RT-PCR 相對定量及絕對定量法探討細胞缺磷的程度和 pstS mRNA 表現量的關係，結果顯示當缺磷組聚球藻的色素合成受到缺磷的影響時，pstS相對表現量會明顯地高於控制組。最後在2006、2007、及 2008三月以及四月東亞沙塵暴期間，運用聚球藻引子對 Q-pstS-Syn 於台灣東北部黑潮測站進行連續日期的實地測量，發現海洋中表水（深度2 m）的聚球藻族群 pstS 表現量變化與磷酸鹽的濃度變化以及 24 小時前大氣懸浮微粒 (PM10) 呈現良好的反比關係，而和混合層深度沒有關係，顯示了沙塵暴的落塵效應比起攪拌強度更能解除聚球藻的缺磷狀態。

The cell wall-associated phosphate-binding transporter gene (pstS) is potentially a diagnostic marker for phosphorus stress in photosynthetic picoplankton. In order to detect the mRNA levels in the East China Sea by Q-RT-PCR (real-time quantitative reverse transcription polymerase chain reaction), suitable primer sets have to be designed according to the gene diversity in the same region. For this purpose, 71 homologous pstS sequences were collected during three cruises conducted in Mar. 2006, Apr. 2007, and Jul. 2007. The phylogenetic analysis indicated that most sequences obtained from Mar. 2006 cruise belonged to Prochlorococcus. In contrast, sequences obtained from Apr. 2007 cruise were equally distributed between Synechococcus Clade II and Prochlorococcus. Moreover, sequences belong to Calde II, III, V were obtained from the continental shelf region between Changjiang river mouth and the Kuroshio current in the summer of 2007. According to sequence alignments, 2 degenerated primer sets were designed for Synechococcus (Q-pstS-Syn) and Prochlorococcus (Q-pstS-Pro), respectively. The specificity of primers was confirmed using genomic DNA extracted from several cultured strains, but the Q-pstS-Pro was excluded from field applications because of its unsatisfactory amplification efficiency. Subsequently, the Q-pstS-Syn primer pair with better performance in amplification efficiency was further tested in monitoring pstS expression pattern in a P-deficient culture of Synechococcus WH7803 using both the relative and the absolute quantification methods. The result showed that when the pigment content in Synechococcus decreased due to P-deficiency, the relative expression level of pstS became significantly higher than that in the control culture. Finally, during the Asian dust storm period in the springs of 2006, 2007, and 2008, the pstS mRNA abundance was measured by the Synechococcus-specific primer set in the Kuroshio Current at a station off the northeast coast of Taiwan. It was found that the pstS expression pattern correlated inversely with the concentration of inorganic phosphate in surface waters and the atmospheric particle concentration (PM10) 24 hours prior to sampling. However, the expression level did not correlate with mixed layer depth. In conclusion, our results suggest that dust storm events may relieve P-deficiency of Synechococcus more effectively through the deposition of particles but less effectively through deepening the mixed layer depth in Kuroshio Current.

摘要 i
Abstract iii
第一章、前言 1
第二章、實驗材料與方法 5
實驗材料 5
實驗方法 5
1. 藻種保存及培養 5
2. 海洋實驗 6
3. pstS基因選殖 7
4. 比對序列與製作親源樹（phylogenetic tree） 15
5. 設計及測試及時定量聚合?連鎖反應 (Q-PCR) 的引子 16
6. 即時定量聚合?連鎖反應 (Q-PCR) 16
7. 絕對定量法標準曲線的製作 17
8. 以絕對定量法定量目標基因的 mRNA 17
9. 以相對基因量定量法定量目標基因的 mRNA 含量 19
第三章、結果 20
東海樣本中的基因選殖和定序 20
設計及測試及時定量聚合?連鎖反應 (Q-PCR) 的引子 20
以純種聚球藻培養測試引子適用性 21
在東海測試引子的適用性 22
第四章、討論 25
蒐集東海超微浮游植物 pstS 序列 25
設計及測試 Q-RT-PCR 引子的專一性 26
退化性引子在定量上的考量 27
相對定量與絕對定量的比較 28
沙塵暴對聚球藻缺磷狀態的影響 29
參考文獻 31
附表 37
附圖 40







表目錄

表一、針對增殖東海 pstS 基因所設計的各式引子 35
表二、得自東海不同測站超微原核浮游植物 pstS 序列分類群統計 36
表三、不同 Q-RT-PCR 引子的專一性測試 37


























圖目錄

圖一、東海測站位置圖 39
圖二、藍綠細菌 pstS 基因序列的多重排序以及各式引子所在位置示意圖。 40
圖三、東海 pstS 基因序列所構成的 neighbor- joining 親源關係樹 42
圖四、Q-RT-PCR 引子測試之電泳圖 44
圖五、各引子對的 Q-RT-PCR 標準曲線 45
圖六、聚球藻 Synechococcus sp. WH7803 培養細胞生理變化圖 46
圖七、聚球藻 Synechococcus sp. WH7803 培養外觀圖 47
圖八、東海南部 2006 年航次黑潮測站 (K14) 各項環境與生物參數日變化圖 48
圖九、東海南部 2006 年航次黑潮測站 (K14) 各項水文參數垂直深度變化圖 49
圖十、東海南部 2007 年航次黑潮測站 (K14) 各項環境與生物參數日變化圖 50
圖十一、東海南部 2007 年航次黑潮測站 (K14) 各項水文參數垂直深度變化圖 51
圖十二、東海南部 2008 年航次黑潮測站 (K14) 各項環境與生物參數日變化圖 52
圖十三、東海南部 2008 年航次黑潮測站 (K14) 各項水文參數垂直深度變化圖 53
圖十四、pstS 基因表現量絕對定量與相對定量之關係圖 54
圖十五、各項環境參數與 pstS 絕對定量及相對定量之關係 55

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