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標題: 浮游植物氮源利用相關基因之表現及其在海洋生態上之應用
Expression of nitrogen-utilization related genes in marine phytoplankton and its application to the study of marine ecology
作者: 康利國
Kang, Lee-Kuo
關鍵字: phytoplankton;浮游植物;nitrate transporter;nitrogen-utilization related genes;nitrogen maker gene;硝酸運輸基因;氮源利用相關基因;氮指標基因
出版社: 生命科學系所
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浮游植物是水域生態系中最主要的初級生產者,而在海洋環境中它們的生產力常受到含氮營養鹽的限制,為了深入暸解各種海洋浮游植物受限制的程度,必須發展分子指標以直接評估浮游植物的生理狀態。在本研究中,首先挑選在氮吸收及同化途徑中重要的基因,包括硝酸運輸蛋白 (Nrt2)、銨運輸蛋白 (Amt),硝酸還原酵素 (Nr)、及glutamine synthetase (glnII) 等做為具有潛力之氮指標基因,其次在純化馴養的藻種中,包括矽藻綱之Skeletonema costatum及Thalassiosira weissflogii、著鞭毛藻綱之Isochrysis galbana、及綠鞭藻綱之Tetraselmis chui進行基因之選殖。之後以I. galbana為實驗室之模式藻種,利用定量反轉錄聚合酶連鎖反應偵測不同氮生長條件下之基因表現情形,結果顯示其中以Nrt2基因的表現對於氮源之種類及濃度最為敏感。在缺氮時呈現飢餓反應並且大量表現,當以硝酸為氮源時則維持中等之表現量,但是在以銨為氮源時則明顯降低表現量,而且在缺氮時的表現量是銨培養下的160倍。顧及此種Nrt2基因表現範圍可能隨物種而變化,於是要發展相對定量法來正確判讀海洋中所測到的Nrt2表現量。在使用I. galbana和Thalassiosira pseudonana為實驗材料的測試中,發現添加銨進行培養可獲得Nrt2基因的最低表現量,而以缺氮培養可獲得Nrt2的最高表現量,藉此定出海洋中浮游植物Nrt2基因之表現範圍做為判斷氮鹽利用情形的依據。另一方面,本研究廣泛搜尋和定序已知矽藻種類之Nrt2序列,以瞭解物種間Nrt2基因在序列上的差異,同時在東海運用退化性引子定序海洋樣本中之Nrt2同源基因,將海洋中獲得的序列與已知序列比對,發現在馬祖海域Nrt2序列有96% 和Skeletonema屬類似;而在湧升流海域中除了有60% 與Skeletonema屬類似外,還有10% 與Chaetoceros屬類似。這些結果有助於針對特定海域設計出具專一性之引子,配合上定量反轉錄聚合酶連鎖反應便可以偵測到海洋樣本中特定種類之Nrt2基因表現。這種使用指標基因表現量的方法,可以方便的以單一技術同時研究不同浮游植物如何受到含氮營養鹽的影響,而且可以擴充到其他的指標基因來探討浮游植物在海洋中的各種生理狀態。

Phytoplankton serve as the primary producer in marine food chain, but their productivities are often limited by the availability of nitrogen in the ocean. For a better understanding of nitrogen limitation among phytoplankton species, it is necessary to develop suitable molecular probes to directly evaluate their physiological states. In this study, 4 genes involved in nitrogen uptake and assimilation, including nitrate transporter genes (Nrt2), ammonium transporter genes (Amt), nitrate reductase genes (Nr) and glutamine synthetase genes (glnII), were selected as candidates for a nitrogen-stress maker. In the first stage, 4 sets of degenerate primers were designed to clone fragments of these genes in cultured marine phytoplankton species, including Skeletonema costatum (Bacillariophyceae), Thalassiosira weissflogii (Bacillariophyceae), Isochrysis galbana (Haptophyceae) and Tetraselmis chui (Prasinophyceae). Next, transcript levels of Nrt2, Amt, and glnII genes in I. galbana grown under various forms and concentrations of nitrogenous nutrients were monitored by quantitative real-time PCR. Our results demonstrated that the mRNA levels of Nrt2 responded sensitively to ambient nitrogen sources with a moderate expression under nitrate-sufficient condition and a high expression under nitrogen-deprived condition. In contrast, the transcript level was severely decreased in the presence of ammonium, which was 160-fold lower than that under nitrogen deprivation. Therefore, Nrt2 genes were chosen to be the first candidate gene for evaluating the nitrogen status of phytoplankton in the ocean. Furthermore, for the proper interpretation of measured Nrt2 transcript abundances in the ocean, a relative expression assay was proposed and tested in I. galbana and Thalassiosira pseudonana. Results from both species indicated that the minimal and the maximal Nrt2 mRNA abundances could be achieved by ammonium addition and nitrogen deprivation, respectively. Considering the range of Nrt2 expression may vary from species to species, the relative expression assay will allow a more precise evaluation of nitrogen deficiency in untested species. On the other hand, to investigate sequence diversity of Nrt2 genes in phytoplankton, data banks were searched and Nrt2 gene fragments of several cultured diatoms were cloned. In addition, Nrt2 fragments were amplified with degenerate primers from samples collected in the East China Sea. Sequence alignments revealed that 96% of Nrt2 sequences at the Matsu station clustered with Skeletonema Nrt2 sequences. In the upwelling area, 60% of sequences clustered with Skeletonema and 10% clustered with Chaetoceros Nrt2. According to the sequences alignments, specific primers targeted to particular phytoplankton Nrt2 were designed. Using these primers with quantitative real-time PCR, our preliminary work demonstrated that the Nrt2 mRNA abundances of specific phytoplankton could be detected in the ocean. By detecting the expression levels of maker genes, not only the nitrogen-utilization status of various phytoplankton can be evaluated using a unified and simple procedure, the same algorithm can also be expanded to study other physiological activities of phytoplankton in the ocean.
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