| Summary: | 碩士 === 國立臺灣海洋大學 === 海洋環境化學與生態研究所 === 98 === To evaluate the effects of light-dark cycle and nutrient concentration on the physiological activities of a marine diatom, Skeletonema costatum, three different media, including nutrient replete h/2, low ammonium ([NH4+]:24 μM), and low phosphate ([PO43-]:1 μM), were prepared to grow the diatom in either a continuous light or a L:D = 12:12 environment. The method of real-time quantitative reverse transcription polymerase reaction (Q-RT-PCR) was used to detect mRNA expressions of nitrate transporter (Nrt2) and phosphate transporter (PHO) genes. The results showed that the expression levels of both Nrt2 and PHO changed with nitrogen/phosphorus ratios as will as nutrient concentrations. However, when nutrient conditions were fixed, the time courses of gene expressions were very similar between the culture under continuous light and that under light-dark cycle environments. For culture grown under a light-dark cycle, 24-hour intensive sampling was performed on day 2, 3, 5 and 8. The daily geometric mean of Nrt2 expression increased with incubation time. The increase was especially prominent in the low ammonium treatment with a daily mean of 8.24 µmole (mole 18S rRNA)-1 on day 2 and 4680 µmole (mole 18S rRNA)-1 on day 8. Expression levels in other treatment were relatively stable. The time variation in PHO expression was relatively small compared with Nrt2. Expression levels in the low phosphate treatment increased from 188 µmole (mole 18S rRNA)-1 on day 2 to 6606 µmole (mole 18S rRNA)-1 on day 8, and those in h/2 treatment increasd from 82.8 µmole (mole 18S rRNA)-1 on day 2 to 2423 µmole (mole 18S rRNA)-1 on day 8. The variation of PHO expression was small in the low ammonium treatment, which varied from 61.3 ~ 160 µmole (mole 18S rRNA)-1. In addition, a statistical analysis was used to detect the existence of a diel cycle in gene expression. Diel rhythms of Nrt2 expression only appeared on day 3 in the nutrient replete treatment, days 2 and 3 in the low ammonium treatment. In contrast, diel rhythms of PHO only appeared on day 2 in the nutrient replete treatment. The expression levels in other treatments did not show significant diel rhythms. These results indicated the Nrt2 and PHO are acceptable marker genes for nitrogen and phosphorus stresses, respectively. However, since the expression levels of PHO in low phosphate and the h/2 treatments showed no significant difference, further test is needed to confirm its suitability. In oceanographic applications, since the variation due to diel rhythms did not exceed the variation due to nutrient stress, it is not necessary to restrict the sampling of phytoplankton to a special period of a day.
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