臺灣博碩士論文加值系統

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

吳慧敏 (2005)。東海浮游植物族群組成與分佈暨藍綠細菌屬核醣核酸遺傳組成之研究。碩士論文，生物科技研究所，國立台灣海洋大學，64 頁。
范子霈 (2006)。鐵限制蛋白A（idiA）與氮限制控制基因A（ntcA）在東海超微浮游植物間之歧異度及偵測引子之設計。碩士論文，海洋生物研究所，國立台灣海洋大學，64 頁。
龔國慶 (2006)。The effects of Asian dust storm on biological productivity in the oligotrophic Kuroshio waters of subtropical northwest Pacific Ocean. 國科會 95 學年度海洋學門成果發表會：會議報告。
陳雅玲 (2008)。以葉綠素螢光法偵測藍綠細菌之光合效率：測量之設定與在不同生 理狀態下之反應。碩士論文，海洋生物研究所，國立台灣海洋大學，44 頁。
Benitez-Nelson, C. R. 2000. The biogeochemical cycling of phosphorus in marine systems. Earth-Sci. Rev. 51: 109-135.
Bishop, J. K. B., R. E. Davis, and J. T. Sherman. 2002. Robotic observations of dust storm enhancement of carbon biomass in the North Pacific. Science 298: 817-821.
Chen, Y.-L. L., H.-Y. Chen, W.-H. Lee, C.-C. Hung, G. T. F. Wong, and J. Kanda. 2001. New production in the East China Sea, comparison between well-mixed winter and stratified summer conditions. Contin. Shelf Res. 21: 751-764.
Chiang, K. P., M. C. Kuo, J. Chang, R. H. Wang, and G. C. Gong. 2002. Spatial and temporal variation of the Synechococcus population in the East China Sea and its contribution to phytoplankton biomass. Contin. Shelf Res. 22: 3-13.
Chung, C. C., S. P. L. Hwang, and J. Chang. 2005. Cooccurrence of ScDSP gene expression, cell death, and DNA fragmentation in a marine diatom, Skeletonema costatum. Appl. Environ. Microbiol. 71: 8744-8751.
Falkowski, P. G., R.T. Barber, V. Smetacek. 1998. Biogeochemical controls and feedbacks on ocean primary production. Science. 281: 200-206
Fisher, T. R., E. R. Peele, J. W. Ammerman, and L. W. Harding. 1992. Nutrient Limitation of Phytoplankton in Chesapeake Bay. Mar. Ecol. Prog. Ser. 82: 51-63.
Fuller, N. J., D. Marie, F. Partensky, D. Vaulot, A. F. Post, and D. J. Scanlan. 2003. Clade-specific 16S ribosomal DNA oligonucleotides reveal the predominance of a single marine Synechococcus clade throughout a stratified water column in the Red Sea. Appl. Environ. Microbiol. 69: 2430-2443.
Fuller, N. J. West, N. J., D. Marie, M. Yallop, T. Rivlin, A. F. Post, and D. J. Scanlan 2005. Dynamics of community structure and phosphate status of picocyanobacterial populations in the Gulf of Aqaba, Red Sea. Limnol. Oceanogr. 50: 363-375.
Ganeshram, R. S., T. F. Pedersen, S. E. Calvert, and J. W. Murray. 1995. Large changes in oceanic nutrient inventories from glacial to interglacial periods. Nature 376: 755-758.
Gong, G.-C., Y.-L. Chen and K.-K. Liu. 1996. Chemical hydrography and chlorophyll a distribution in the East China Sea in summer: implications of nutrient dynamics. Conti. Shelf Res. 16: 1561-1590.
Graziano, L. M., R. J. Geider, W. K. W. Li, and M. Olaizola. 1996. Nitrogen limitation of North Atlantic phytoplankton: Analysis of physiological condition in nutrient enrichment experiments. Aquat. Microb. Ecol. 11: 53-64.
Guillard R.R.L. and Ryther J.H. 1962. Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt and Detonula confervacea Cleve. Can. J. Microbiol. 8: 229-239.
Hung, C.-C., G.-C. Gong, W.-C. Chung, W.-T. Kuo, and F.-C. Lin. 2009. Enhancement of particulate organic carbon export flux induced by atmospheric forcing in the subtropical oligotrophic northwest Pacific Ocean. Mar. Chem. 113: 19-24.
Hsu, Shih-Chieh, George T.F. Wong, Gwo-Ching Gong, Fuh-Kwo Shiah, Yi-Tang Huang, Shuh-Ji Kao, Fujung Tsai, Shih-Chun Candice Lung, Fei-Jan Lin, I-I Lin, Chin-Chang Hung, and Chun-Mao Tseng. Sources, solubility, and dry deposition of aerosol trace elements over the East China Sea. Mar. Chem. in press.
John, D., S. Patterson, and J. Paul. 2007. Phytoplankton-Group Specific Quantitative Polymerase Chain Reaction Assays for RuBisCO mRNA Transcripts in Seawater. Mar. Biotech. 9: 747-759.
Karl, D. M., R. Letelier, D .Hebel, L. Tupas, J. Dore, J. Christian, and C. Winn. 1995. Ecosystem changes in the North Pacific subtropical gyre attributed to the 1991-92 El Nino. Nature 373: 230-234.
Kester, D. R., Duedall, I. W., Connors, D. N. and Pytkowicz, R. M. 1967. Preparation of Artificial Seawater. Limnol. Oceanogr. 12: 176-179.
Lalli, C. M. and T. R. Parsons. 1997. Biological Oceanography : An Introduction, 2nd ed. Elsevier.
Li, W. K. W. 1994. Primary production of prochlorophytes, cyanobacteria, and eukaryotic ultraphytoplankton - measurements from flow cytometric sorting. Limnol. Oceanogr. 39: 169-175.
Li, Y. H. 1994. Material exchange between the East China Sea and the Kuroshio Current. Terr. Atmos. Ocean. Sci. 5: 625-631.
Lindell, D., and A. F. Post. 2001. Ecological aspects of ntcA gene expression and its use as an indicator of the nitrogen status of marine Synechococcus spp. Appl. Environ. Microbiol. 67: 3340-3349.
Louis, P., and H. J. Flint. 2007. Development of a semiquantitative degenerate real-time PCR-based assay for estimation of numbers of butyryl-coenzyme A (CoA) CoA transferase genes in complex bacterial samples. Appl. Environ. Microbiol. 73: 2009-2012.
Liu, H., H. A. Nolla, and L. Campbell. 1997. Prochlorococcus growth rate and contribution to primary production in the equatorial and subtropical North Pacific Ocean. Aquat. Microb. Ecol. 12: 39-47.
Mackey, K. R. M., R. G. Labiosa, M. Calhoun, J. H. Street, A. F. Post, and A. Paytan. 2007. Phosphorus availability, phytoplankton community dynamics, and taxon-specific phosphorus status in the Gulf of Aqaba, Red Sea. Limnol. Oceanogr. 52: 873-885.
Martin, J. H., and S. E. Fitzwater. 1988. Iron-deficiency limits phytoplankton growth in the Northeast Pacific Subarctic. Nature 331: 341-343.
Martiny, A. C., M. L. Coleman, and S. W. Chisholm. 2006. Phosphate acquisition genes in Prochlorococcus ecotypes: Evidence for genome-wide adaptation. Proc. Natl. Acad. Sci. 103: 12552-12557.
Migon, C., and V. Sandroni. 1999. Phosphorus in rainwater: partitioning inputs and impact on the surface coastal ocean. Limnol. Oceanogr. 44: 1160-1165.
Moore, L. R., G. Rocap, and S. W. Chisholm. 1998. Physiology and molecular phylogeny of coexisting Prochlorococcus ecotypes. Nature 393: 464-467.
Partensky, F., W. R. Hess, and D. Vaulot. 1999. Prochlorococcus, a Marine Photosynthetic Prokaryote of Global Significance. Microbiol. Mol. Biol. Rev. 63: 106-127.
Rocap, G., D. L. Distel, J. B. Waterbury, and S. W. Chisholm. 2002. Resolution of Prochlorococcus and Synechococcus ecotypes by using 16S-23S ribosomal DNA internal transcribed spacer sequences. Appl. Environ. Microbiol. 68: 1180-1191.
Scanlan, D. J., N. H. Mann, and N. G. Carr. 1993. The Response of the picoplanktonic marine cyanobacterium Synechococcus Species WH7803 to phosphate starvation involves a protein homologous to the periplasmic phosphate-binding protein of Escherichia coli. Mol. Microbiol. 10: 181-191.
Su, Z., V. Olman, and Y. Xu. 2007. Computational prediction of Pho regulons in cyanobacteria. BMC Genomics 8: 156.
Stellwag and Metzenberg. 1984. Ribosomal RNA synthesis during phosphorus starvation in wild type Neuropora and in phosphorus regulatory mutants. Mol. Gen. Genet. 194: 105-110.
Van Cappellen, P., and E. D. Ingall. 1996. Redox stabilization of the atmosphere and oceans by phosphorus-limited marine productivity. Science 271: 493-496.
Veldhuis, M. J. W., G. W. Kraay, J. D. L. Vanbleijswijk, and M. A. Baars. 1997. Seasonal and spatial variability in phytoplankton biomass, productivity and growth in the northwestern Indian Ocean: The southwest and northeast monsoon, 1992-1993. Deep-Sea Res. Part I Oceanogr. Res. Pap. 44: 425-449.
Wanner, B. L.1996. Phosphorus assimilation and control of the phosphate regulon, p. 1357-1381. In F.C. Neidhardt, R. Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger, Escherichia coli and Salmonella: cellular and molecular biology. ASM Press.
Waterbury, J.B., S.W. Watson, F.W. Valois, D.G. Franks. 1986. Biological and ecological characterization of the marine unicellular cyanobacterium Synechococcus. Vol. 214: p.71-120. In Platt, T., W.K.W. Li, Photosynthetic Picoplankton. Canadian Bulletin of Fisheries and Aquatic Sciences.
Wu, J. F., W. Sunda, E. A. Boyle, and D. M. Karl. 2000. Phosphate depletion in the western North Atlantic Ocean. Science 289: 759-762.
Zwirglmaier, K., J. L. Heywood, K. Chamberlain, E. M. Woodward, M. V. Zubkov, and D. J. Scanlan. 2007. Basin-scale distribution patterns of picocyanobacterial lineages in the Atlantic Ocean. Environ. Microbiol. 9: 1278-1290.
Zwirglmaier, K., J. Ludwig, M. Ostrowski, S. Mazard, L. Garczarek, D. Vaulot, F. Not, R. Massana, O. Ulloa, and D. J. Scanlan. 2008. Global phylogeography of marine Synechococcus and Prochlorococcus reveals a distinct partitioning of lineages among oceanic biomes. Environ. Microbiol. 10: 147-161.