臺灣博碩士論文加值系統

矽藻中含有硝酸根運輸蛋白 ( nitrate transporter，基因名稱Nrt2 ) 以及鈉磷共運輸蛋白 ( sodium phosphate cotransporter，基因名稱Npt2bl )，分別負責將細胞外的硝酸鹽和磷酸鹽吸收進入細胞內。因為兩者的代謝途徑皆與光合作用有關，於是猜測Nrt2和Npt2bl之mRNA表現量在自然環境中會具有日夜週期變化。本實驗利用2017年6月及7月航次，於馬祖沿海測站進行24小時連續採樣，並於兩個時間點進行營養鹽調控培養。針對矽藻Skeletonema subsalsum以定量聚合酶鎖鏈反應偵測Nrt2、Npt2bl、FcpB ( fucoxanthin-chlorophyll protein 之編碼基因)以及TBP ( TATA Box binding protein之編碼基因) 之表現量，並以餘弦函數進行迴歸分析，將Nrt2與Npt2bl的結果與FcpB和TBP的表現量以及一日內光照變化進行比較，藉以判別Nrt2以及Npt2bl是否具有日週變化。結果顯示，六月航次中FcpB表現量之回歸方程式在中午時段具有最大值，並在凌晨12點時達到最低值。迴歸分析後得到振幅為對數值1.32並且在統計上達到顯著水準 ( p < 0.05 ) ；七月的FcpB的表現量迴歸後振幅並非顯著，但是也在中午時段以及晚間11點左右分別具有明顯的最大值以及最低值。而TBP表現量雖然在迴歸分析的結果中顯示具有日週變化，但是振幅很小導致日週變化不明顯。相較之下，兩個航次的Nrt2及Npt2bl的表現量在一日內會有上下波動，但經迴歸分析後振幅均為不顯著。經由營養鹽調控培養，Nrt2表現量會受到環境中含氮營養鹽濃度的影響，而且改變幅度遠大於一日中現場表現量的變化幅度。同樣的，Npt2bl表現量會受到磷酸鹽濃度的影響，改變幅度也是遠大於一日中現場表現量的變化幅度。若將營養鹽調控培養的結果與現場測值相比，在六月和七月航次中Nrt2現場表現量都非常接近添加銨鹽培養組的測值，顯示馬祖海域中的骨藻並沒有積極製造Nrt2蛋白；在Npt2bl表現量的部分，測量結果顯示在六月航次中骨藻積極製造Npt2bl蛋白，而七月則是以中等的努力製造Npt2bl蛋白。

Diatom cells possess a distinctive nitrate transporter (encoded by Nrt2) and a sodium/phosphate cotransporter (encoded by Npt2bl), to enable the uptake of nitrate and phosphate from the environment. Since the metabolic pathways of nitrogen and phosphorus are both related to photosynthesis, the mRNA levels of Nrt2 and Npt2bl are assumed to have a diel variation in natural environments. In this study, research cruises were conducted in the coastal region rear Matsu Islands in June and July, 2017, respectively. During each cruise, a 24-h sampling program was established with additional nutrient-manipulative experiments performed at 2 time points. In a locally dominant diatom, Skeletonema subsalsum, RT-qPCR was used to measure the mRNA levels of the following genes, Nrt2, Npt2bl, FcpB (encoding fucoxanthin-chlorophyll protein), and TBP (encoding TATA Box binding protein). Next, nonlinear regression was applied to fit a cosine function to measured expression levels. The patterns of FcpB and TBP expressions were used as comparison standards to determine if diel variation existed in the expression patterns of Nrt2 and Npt2bl. Experimental results indicated that gene expression of FcpB reached the maximum at noontime and decreased to the minimum at midnight in June. Regression analysis generated a cosine function with an amplitude of 1.32 (logarithmic scale), which was statistically significant at p < 0.05. In July, the amplitude of FcpB regression equation was not statistically significant, but the maximum and minimum values still appeared at noontime and midnight. The amplitudes of TBP expressions were statistically significant in June and July, but the amplitudes were too small to properly reveal a diel change. In comparison, the gene expressions of Nrt2 and Npt2bl fluctuated in a day, but the amplitudes of regression curves were not statistically significant. In nutrient-manipulative experiments, the gene expressions of Nrt2 were mainly affected by the availability nitrogenous nutrients. Similarly, the gene expressions of Npt2bl were mainly affected by phosphate levels. For both genes, range of gene expression due to nutrient-manipulation was much wider than the range of variation in a day. Comparing the results of nutrient-manipulative experiments to those of in situ samples, the in situ expressions of Nrt2 were similar to ammonium-added treatments in June and July, indicating that S. subsalsum did not actively produce nitrate transporter. By contrast, gene expressions of Npt2bl revealed that S. subsalsum actively produced phosphate transporter in June, but the effort was moderate in July.

目次 IV
表次 VI
圖次 VII
前言 1
材料與方法 4
1. 探測航次與樣本採集 4
2. 船艙內的培養實驗 4
3. 浮游植物細胞計數 5
4. 確認馬祖海域矽藻之基因序列 5
4.1. 全體 RNA 萃取 5
4.2. 建構混合種類全轉錄體 5
4.3. 序列比對與分析 6
5. 測量目標基因之表現量 6
5.1. 製作 cDNA 6
5.2. RT-qPCR引子設計 6
5.3. 定量聚合酶連鎖反應 ( qPCR ) 7
5.4. 定量 PCR的計算與分析 7
6. 使用曲線迴歸偵測日週變化 8
結果 9
1. 水文資料概況 9
2. 浮游植物種類組成 9
3. 淡鹽骨藻各種基因表現量之日週變化 10
3.1 FcpB表現量日週變化 10
3.2 TBP表現量日週變化 10
3.3 Nrt2 表現量日週變化 10
3.4 Npt2bl表現量 11
4. FcpB基因在不同氮磷處理下的表現量 11
5. TBP基因在不同氮磷處理下的表現量 12
6. Nrt2基因在不同氮磷處理下的表現量 12
7. Npt2bl基因在不同氮磷處理下的表現量 13
討論 14
1. FcpB兩航次的日夜週期變化與營養鹽之調控 14
2. TBP日夜週期變化與營養鹽調控之比較 14
3. Nrt2日夜週期變化與營養鹽調控之比較 15
4. Nrt2bl日夜週期變化與營養鹽調控之比較 16
5. 結論 16
參考文獻 18
附錄 36

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