南海為低氮鹽的海域，具有固氮潛力的微生物可將溶於海水中的氮氣還原成氨提供新氮源，增加海域基礎生產力。本研究主要目標為(1)設計單細胞異營性固氮微生物(unicellular hetrotrophic diazotroph, proteobacteria)的探針引子；(2)以定量聚合酶反應(Quantitative Polymer Chain Reaction, qPCR)偵測南海單細胞異營性固氮微生物之生物量。建立南海單細胞固氮微生物之nifH基因庫(clone libiary)，依據nifH基因之親緣關係得到(1) γ變形菌(γ-proteobacteria) 的兩個subgroups： γ-2477A411, Gamma 4；及(2) α變形菌(α-proteobacteria) 的兩個subgroups： α-Group1, α-Group13。其中γ-2477A411在南海已有文獻報導，Gamma 4在南海為首次發現，但曾在南太平洋被報導過。而α-Group1在南海首次發現，但曾在北大西洋及北太平洋被報導過，卻未設計探針引子或偵測其生物量，α-Group13在南海已有文獻報導，雖有設計探針引子，但對α-Group13的偵測靈敏度低。本研究針對α-Group1及α-Group13設計新的探針引子，而γ-2477A411及Gamma 4之探針引子已有文獻發表，因此延用不再另行設計。
　　利用這些探針引子針對南海北部海域以定量聚合酶鏈鎖反應，偵測其生物量，包括三個不同季節的航次(晚春、秋季及冬季)，在20o-22oN，116o-123oE間採集三個陸棚站、兩個陸坡及六個海盆測站之表水到200 公尺樣本，少數海盆測站有採集深水(400-1500 m)樣本。結果發現γ-proteobacteria由表水累計至200公尺水柱的生物量總和最高為105 nifH gene copies m-2。其中γ-2477A411在各樣本內皆有偵測到生物量(102-104 nifH gene copies L-1)，但Gamma 4的生物量皆小於本研究之偵測極限(42 gene copies L-1)。γ-2477A411的生物量在春秋季高於冬季，主要分佈在200 m以淺的水層，深水樣本中雖也可測得，但其生物量均低於本研究之定量極限(Ct value=40)。γ-2477A411在各季節的生物量與單細胞固氮藍綠藻group-A(UCYN-A)相近，但高於單細胞固氮藍綠藻group-B, -C(UCYN-B, -C)。 γ-2477A411水柱累計0-200公尺的生物量(103-105 nifH gene copies m-2)與表水溫度及UCYN-A, -C水柱累計0-200公尺的生物量呈正相關，與表水硝酸鹽濃度、表水葉綠素a濃度、表水密度及混合層深度呈負相關，推測與其需有機碳及光源以支持其細胞生長，及其可能被高硝酸鹽濃度抑制生長有關。
　　α-proteobacteria水柱累計0-200公尺的生物量總和最高和γ-proteobacteria一樣，為105 nifH gene copies m-2。相較於γ-2477A411，α-proteobacteria並非每一個季節及水層的樣本皆可偵測到，但可分布至較深水層。其中α-Group1生物量只在冬季的樣本偵測到(103-104 nifH gene copies L-1)，可分佈至400公尺深的水層(生物量為103 nifH gene copies L-1)；α-Group13的生物量(102-103 nifH gene copies L-1)在秋冬季高於春季，其分佈比α-Group1更深可至1500公尺(生物量為102 nifH gene copies L-1)。α-proteobacteria水柱累計0-200公尺的生物量(103-105 nifH gene copies m-2)與環境因子間皆未呈現顯著關係。
　　和過去的只偵測單細胞藍綠藻之生物量或是單細胞固氮變型菌之生物量的研究相比，本研究除了針對兩個不同類群的α-proteobacteria設計新的引子探針，並偵測包括單細胞藍綠藻及變型菌的共八種不同的單細胞固氮生物在南海的生物量。

關鍵字：南海、α-, γ-proteobacteria、nifH基因、親源分析樹、探針引子、qPCR

Biological dinitrogen (N2) fixation, the reduction of atmospheric N2 gas to ammonium, is a process carried out by certain nifH gene possessing microorganisms known as diazotrophs. N2 fixation is active in the oligotrophic ocean like South China Sea (SCS). In this study highly specific primers and probes were designed and used to quantitatively detect the nifH gene copies of α- and γ-proteobacteria in the northern SCS. We established the nifH PCR clone library and derived according to the phylogenetic diversity analysis two subgroups of γ-proteobacteria (γ-24774A11 and Gamma 4) and α-proteobacteria (α-Group1 and α-Group13), respectively. γ-24774A11 has been reported in the SCS. Gamma 4 has been reported in the south Pacific but was the first time recovered in the SCS. α-Group1 has been reported in the north Atlantic and north Pacific but was the first time recovered in the SCS. The need to design suitable primers and probes for detecting α-Group1 and α-Group13 became obvious as existing primers and probes are absent (α-Group1) or in low specificity (α-Group13).
In the abundance study, primes and probes of 8 different unicellular diazotrophs were used to detect their biomass in the northern SCS. Water samples were collected generally at depths ranging 0-200 m from 3-shelf, 2-slope and 6-basin stations in 20o-22oN, 116o-123oE. The sampling were carried out during three cruises of different seasons (late spring, fall and winter). Deep sea water samples (400-1500m) were collected in basin stations. Results showed that the maximum integrated (0-200 m) nifH gene copies of γ-24774A11 was 105 nifH gene copies m-2. The nifH gene copies of γ-24774A11 ranged between 102-104 nifH gene copies L-1 was detected in all samples. In contrast, Gamma 4 was not detectable with a limit of 42 gene copies L-1. γ-24774A11 distributed in upper 200 m and its abundance were higher in late spring and fall than in winter. Its nifH gene was detectable in the deep sea water samples but the gene copies was all below the quantitative limitation (Ct value=40). The abundance of γ-24774A11 was similar to those of unicellular cyanobacteria A, UCYN-A but higher than those of UCYN-B and UCYN-C. The abundance of γ-24774A11 was significantly positively correlated with surface temperature and the abundances of UCYN-A and UCYN-C, and was significantly negatively correlated with the concentration of surface nitrate and nitrite, Chl. a, density and mixing layer depth. Suggesting the need for DOC and light to support its growth. On the other hand, high concentrations of nitrate and nitrite might inhibit the growth of γ-24774A11.
Similar to γ-proteobacteria, the maximum integrated nifH gene copies of α-proteobacteria was 105 nifH gene copies m-2. The nifH gene copies of α-proteobacteria was not detectable in all samples but many in deeper water samples. The nifH gene copies of α-Group1 was only detectable in the winter samples (103-104 nifH gene copies L-1) from as deep as 400 m. The abundance of α-Group13 was higher in fall and winter than in late spring. It was detectable at 102 nifH gene copies L-1 as deep as 1500 m. There was no significant correlation between the abundance of α-proteobacteria and environment factors.
Our results revealed that unlike α-proteobacteria, the abundance of γ-proteobacteria in the northern SCS were related with many environmental factors. α-proteobacteria was distributed in deeper water than γ-proteobacteria.


Keywords：South China Sea, α-, γ-proteobacteria, nifH gene, phylogenetic analysis, primer and probe, qPCR

論文審定書............................................................................................................... i
公開授權書.............................................................................................................. ii
誌謝........................................................................................................................... iii
中文摘要................................................................................................................. iv
英文摘要................................................................................................................ vii
目錄......................................................................................................................... viii
圖次........................................................................................................................... x
表次.......................................................................................................................... xii
第ㄧ章 前言............................................................................................................. 1
第二章 文獻回顧..................................................................................................... 3
2.1 固氮生物的....................................................................................... 3
2.2 單細胞固氮藍綠藻........................................................................... 3
2.3 單細胞固氮變型菌........................................................................... 4
2.4 南海固氮生物................................................................................... 6
2.5 單細胞固單生物調查方法分析...................................................... 6
2.6 南海地形及水文環境...................................................................... 7
第三章 材料方法..................................................................................................... 9
3.1 探針引子設計................................................................................... 9
3.2 野外樣本生物量偵測...................................................................... 16
3.3 統計方法分析.................................................................................. 19
3.4 水溫資料因子定義及符號.............................................................. 19
第四章 結果............................................................................................................ 20
4.1 探針引子設計.................................................................................. 20
4.2 野外樣本生物量偵測...................................................................... 25
第五章 討論............................................................................................................ 37
5.1 探針引子設計.................................................................................. 37
5.2 各類單細胞固氮生物之生物量偵測.............................................. 39
參考文獻.................................................................................................................. 43

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