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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/15371

Title: 東海超微原核浮游植物磷酸鹽指標基因的表現:引子設計與實地測試
The expression of pstS in the East China Sea picoplankton community:
Authors: Shr-Hau Hung
洪仕豪
Contributors: NTOU:Institute of Marine Environmental Chemistry and Ecology
國立臺灣海洋大學:海洋環境化學與生態研究所
Keywords: 藍綠細菌;聚球藻;原核綠藻;及時定量聚合酶連鎖反應;東亞沙塵暴;東海;磷酸鹽
Cyanobacteria;Synechococcus;Prochlorococcus;pstS;real time PCR;the Asia dust storm;the East China Sea;phosphate
Date: 2009
Issue Date: 2011-06-30T08:11:45Z
Abstract: 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酶連鎖反應(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.
URI: http://ethesys.lib.ntou.edu.tw/cdrfb3/record/#G0M96830002
http://ntour.ntou.edu.tw/ir/handle/987654321/15371
Appears in Collections:[海洋環境與生態研究所] 博碩士論文

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