博士 / 國立海洋大學 / 海洋生物研究所 / 92 / In order to understand the molecular mechanism of growth regulation in phytoplankton, and to develop novel growth-status indicators, subtracted cDNA libraries were constructed by using the mRNA extracted from Skeletonema costatum and Tetraselmis chui in rapid-growth (RG) and death (DS) (early-stationary phase in the case of T. chui) phases, respectively. Subsequently, real-time quantitative reverse transcription- polymerase chain reaction was applied to estimate the mRNA expression levels of these growth-dependent genes under different cultural conditions.
In S. costatom, four RG-related cDNA fragments, RG#14, RG#21, RG#25, RG#42, and one DS-related cDNA fragment, DS#2, were obtained. According to the results of sequence analysis, RG#21 and RG#42 belonged to the vesicle-associated membrane protein family (VAMP family) and minichromosome maintenance factors family (MCM family), respectively. Other fragments including RG#14, RG#25, and DS#2 were novel molecules based on GenBank database. Under continuous illumination, the RG-related mRNA expression levels increased from 10- (RG#21) to 1000-fold (RG#25) with increasing growth rate. In contrast, mRNA expression level of DS#2 increased dramatically when the cell population aged. Except RG#21, all growth-dependent genes showed similar diel-oscillation in expression patterns with high mRNA levels appeared at the light-period. In exponential phase, the maximum and minimum RG#25 mRNA levels during a 24-hour period were 13- to 40-fold higher than those in stationary phase. On the other hand, maximum and minimum DS#2 mRNA levels in stationary phase were 30- to 370-fold higher than those in exponential phase. Between the growth temperatures of 20oC to 30oC, RG#25 maintained high mRNA expression levels at 160 mmole (mole 18S rRNA)-1, which was about 210-fold higher than that at 15oC. As for the influence of light intensity, RG#25 mRNA expression stayed at a high level ranging from 7.40 to 16.02 mmole (mole 18S rRNA)-1 under intense illumination (101.5 to 145 mE m-2 sec-1), which was about 40-fold higher than that under weak illumination (14.5 mE m-2 sec-1). As expected, DS#2 showed high mRNA expression level at 33.01 mmole (mole 18S rRNA)-1 under weak illumination.
In T. chui, a high-affinity phosphate transporter gene, TcPHO, was isolated from the early-stationary subtracted cDNA library. The full-length cDNA of TcPHO was 1993-bp in length and encoded an open reading frame of 610 amino acids. The deduced amino acid sequence of TcPHO shared 51.6% and 49.8% sequence similarity with those of PHO89 from Saccharomyces cerevisiae and PHO4 from Neurospora crassa, respectively. The expression of TcPHO mRNA was dependent on phosphate availability. With low-phosphate treatment, the TcPHO mRNA concentration increased sharply to 2.72 fmole (mg total RNA)-1 from days 1 to 2 and remained at this high level from days 2 to 4. Furthermore, rescue treatment with either phosphate or p-nitrophenyl phosphate addition effectively inhibited TcPHO mRNA expression. In contrast, TcPHO mRNA expression stayed at a low level ranging from 0.25 to 0.28 fmole (mg total RNA)-1 under a low-nitrate condition.
The identification of these novel growth-dependent gene fragments from S. costatum and T. chui provided valuable information concerning mechanisms of growth regulation in phytoplankton. Moreover, these genes may be used as growth-status indicators and may develop into a completely new approach in the study of marine phytoplankton ecology.
| Identifer | oai:union.ndltd.org:TW/092NTOU0270005 |
| Date | January 2004 |
| Creators | Chih-Ching Chung, 鍾至青 |
| Contributors | Jeng Chang, Shang-Ping L. Hwang, 張正, 黃聲蘋 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 66 |
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