臺灣博碩士論文加值系統

在海洋環境中氮源經常被認為是限制浮游植物成長的營養源。細胞膜上的硝酸運輸蛋白被認為在硝酸利用上扮演重要的角色，其基因Nrt2在因應不同氮環境的改變有不同的基因表現情形。在本研究中以體型較大矽藻Ditylum brightwellii 為模式物種，藉由監測其 Nrt2 基因表現並與其他矽藻種類進行比較，希望探討不同體型大小矽藻對於氮源利用之間的差異。為了確認D. brightwellii 中的Nrt2 基因家族，利用海洋真核微生物全轉錄體資料庫 (MMETSP) 中進行D. brightwellii Nrt2 相似序列之比對，並利用實驗室培養的D. brightwellii藻種再次定序確認，一共定序出四條Nrt2基因，其中DbNrt2A及DbNrt2D 為過去文獻尚未報導之形式。將D. brightwellii培養在不同氮環境下，發現培養在以硝酸鹽為氮源的情況有較高生長率 (1.06 day-1)，而培養在以銨為氮源下細胞生長明顯受到高濃度銨的抑制，只能在低銨 (100 μM )濃度下生長。比較D. brightwellii 在不同氮環境下硝酸運輸基因的表現模式，發現僅有DbNrt2C會受到銨的抑制，但在缺氮底下會提高表現，此結果與Thalassiosira pseudonana Nrt2.1及Nrt2.2的表現模式相似，但基因表現量變化範圍僅其1/100倍。由此顯示細胞較大的D. brightwellii 在Nrt2基因調節上相對於小細胞T. pseudonana較不敏感，可能與D. brightwellii 生長受銨鹽限制有關。

Nitrogen is often considered as a major nutrient limiting phytoplankton growth in the marine environments. Nitrate transporter protein located on the cell membrane is thought to play an important role in nitrate uptake, and its gene, Nrt2, possesses different gene expression in response to different nitrogen environments. In this study, the gene expression of Nrt2 genes was monitored in a diatom, Ditylum brightwellii to compare with that in other diatoms species. In order to confirm the Nrt2 gene family in D. brightwellii, Nrt2 similar sequences were searched in the D. brightwellii transcriptome databases in Marine Microbial Eukaryote Transcriptome Sequencing Project (MMETSP) and confirmed by PCR from genomic DNA and sequencing. In total, four Nrt2 genes were identified, and DbNrt2A and DbNrt2D have not been reported in the litertures. When D. brightwellii was grown with nitrate as the sole nitrogen source, cell density increased exponentially for 5 days with a growth rate of 1.06 day-1. However, its growth was significantly affected by ammonium, especially when the initial ammonium concentration is higher than 100 μM. Monitoring the expression pattern of nitrate transporter genes under different nitrogen conditions, only the transcript level of DbNrt2C was repressed by ammonium, but induced under nitrogen deficiency. This gene expression pattern was consistent with that of T. pseudonana Nrt2.1 and Nrt2.2, but the transcription levels varied in D. brightwellii was only 1/100-fold of that in T. pseudonana. These results suggested that D. brightwellii is less sensitive to regulate the gene expression of Nrt2 gene than T. pseudonana, which might associated with its growth limited by ammonium.

致謝 I
摘要 II
Abstract III
目次 IV
表次 V
圖次 VI
前言 1
材料與方法 4
一、培養實驗 4
二、矽藻計數 4
三、族群指數生長率 5
四、光合作用最大量子效率 (Fv/Fm) 5
五、基因表現量 5
結果 9
一、Ditylum brightwellii 硝酸運輸基因之選殖 9
二、培養氮源條件之測試 9
三、監測Nrt2 基因在不同氮條件下之表現情形 10
四、Nrt2 基因表現量 11
討論 12
一、引用全轉錄體基因庫搜尋基因家族 12
二、矽藻基因家族成員 12
三、不同氮源下之培養狀況 12
四、參考基因之選擇 13
五、不同形式之表現情形 13
六、細胞大或小是否會影響基因調控 13
參考文獻 15
附錄 33

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