博士 / 國立臺灣海洋大學 / 海洋生物研究所 / 103 / Chaetoceros spp. are ecologically important ocean diatoms, and it is hypothesized that their genetic adaptations to phosphorus (P) stress may be different from the adaptations of model species with fully sequenced genomes. To investigate how phosphorus availability affects gene expression at the mRNA level, next-generation sequencing (NGS) was used to construct transcriptomes for C. affinis (CCMP 160) cultures grown under low-phosphate and nutrient-replete conditions. This operation generated 29,285 scaffolds, and subsequent sequence comparisons resulted in the identification of 10,972 expressed genes, of which 503 were up-regulated and 553 were down-regulated by P stress. The differential expression patterns of 41 of these genes were confirmed by performing quantitative reverse-transcription polymerase chain reactions (Q-RT-PCR). These results demonstrated that many genes related to nutrient acquisition are up-regulated in C. affinis under P deficiency. Moreover, C. affinis is unique in that it possesses a “classical” alkaline phosphatase and two type II Na+/Pi cotransporters. Three genes homologous to ones involved in the higher plant regulatory circuit for P uptake were also identified. P deficiency resulted in enhanced intracellular recycling of P in C. affinis via the upregulation of several ribonuclease genes. However, the expression of a gene involved in sulfolipid production was unaffected. Regarding photochemical reactions, the high transcription levels of light-harvesting complex genes, but low maximum quantum efficiency (Fv/Fm), implied that excess energy was dissipated under P deficiency. Furthermore, the NAD-malic enzyme was up-regulated to generate pyruvate via an alternative pathway that is less dependent on P. In low-P cultures, decreased rates of cell proliferation were found during the early stationary phase, consistent with the significant decrease in transcription of genes encoding cell division control protein 45 and ribonucleoside-diphosphate reductase (Shih et al. 2015). Under nutrient stresses, diatoms increase the yield of polysaccharides, which inhibits enzyme activities in Q-RT-PCR. In this study, two commercial reagent kits, and a formula reported in the literatures were compared for efficiencies in extracting diatom RNA. Subsequently, reverse transcriptases from various manufacturers were employed to quantify the transcript levels of elongation factor like gene, EFL, in these RNA templates. The results showed that extraction by TRIzol with high salt solution followed by Epicentre reverse transcriptase performed the best in Q-RT-PCR. Furthermore, the technique of pyrosequencing was used to investigate genetic composition and mRNA expression profiles in the ocean. In an upwelling system off the northern coast of Taiwan, planktons greater than 20 μm in size were collected and both genomic DNA and mRNA were sequenced. Such generated databased were searched for diatom genes related to nitrogen, phosphorus, silicate, and iron utilization. Through the procedures of gene annotation and function identification, the results showed that, in comparison with species related to C. neogracile, Chaetoceros spp., and T. miniscula, the RNA/DNA ratios were higher in the species related to P. fraudulenta, R. Setigera. This observation implied that the latter species might have a higher growth rate. Several reads of flavodoxin gene were observed in species related to P. fraudulenta, which suggested that these species might be under iron stress. A great number of reads belonging to ammonium and nitrate/nitrite transporter genes were observed in species related to C. neogracile, Chaetoceros sp. P. fraudulenta, and R. setigera. It was implied that these diatom were using ammonium as the N source. Only a species of Chaetoceros sp. was evaluated as using nitrate. In genes associated with P utilization, reads of type III Na+/Pi transporter genes were found in species related to S. menzelii and Thalassiosira sp. Similarly, reads of alkaline phosphatase gene were found in species related to Chaetoceros sp.. These observations suggested a status of P stress. By contrast, although the reads of type II Na+/Pi transporter gene occurred in species related to O. sinensis and C. neogracile, the existence of P stress was uncertain. Diatom species related to Chaetoceros sp., C. debilis, C. dichaeta, C. neogracile, E. antarctica, P. delicatissima, and Staurosira complex sp. might be under the pressure of silicate stress because they contained abundant reads of silicate transporter genes.
| Identifer | oai:union.ndltd.org:TW/103NTOU5270011 |
| Date | January 2015 |
| Creators | Shih, Chi-Yu, 時繼宇 |
| Contributors | Chang, Jeng, 張正 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 76 |
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