Expression of nitrogen-utilization related genes in marine phytoplankton and its application to the study of marine ecology
| Autor: | Lee-Kuo Kang, 康利國 |
|---|---|
| Rok vydání: | 2008 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 96 Phytoplankton serve as the primary producer in marine food chain, but their productivities are often limited by the availability of nitrogen in the ocean. For a better understanding of nitrogen limitation among phytoplankton species, it is necessary to develop suitable molecular probes to directly evaluate their physiological states. In this study, 4 genes involved in nitrogen uptake and assimilation, including nitrate transporter genes (Nrt2), ammonium transporter genes (Amt), nitrate reductase genes (Nr) and glutamine synthetase genes (glnII), were selected as candidates for a nitrogen-stress maker. In the first stage, 4 sets of degenerate primers were designed to clone fragments of these genes in cultured marine phytoplankton species, including Skeletonema costatum (Bacillariophyceae), Thalassiosira weissflogii (Bacillariophyceae), Isochrysis galbana (Haptophyceae) and Tetraselmis chui (Prasinophyceae). Next, transcript levels of Nrt2, Amt, and glnII genes in I. galbana grown under various forms and concentrations of nitrogenous nutrients were monitored by quantitative real-time PCR. Our results demonstrated that the mRNA levels of Nrt2 responded sensitively to ambient nitrogen sources with a moderate expression under nitrate-sufficient condition and a high expression under nitrogen-deprived condition. In contrast, the transcript level was severely decreased in the presence of ammonium, which was 160-fold lower than that under nitrogen deprivation. Therefore, Nrt2 genes were chosen to be the first candidate gene for evaluating the nitrogen status of phytoplankton in the ocean. Furthermore, for the proper interpretation of measured Nrt2 transcript abundances in the ocean, a relative expression assay was proposed and tested in I. galbana and Thalassiosira pseudonana. Results from both species indicated that the minimal and the maximal Nrt2 mRNA abundances could be achieved by ammonium addition and nitrogen deprivation, respectively. Considering the range of Nrt2 expression may vary from species to species, the relative expression assay will allow a more precise evaluation of nitrogen deficiency in untested species. On the other hand, to investigate sequence diversity of Nrt2 genes in phytoplankton, data banks were searched and Nrt2 gene fragments of several cultured diatoms were cloned. In addition, Nrt2 fragments were amplified with degenerate primers from samples collected in the East China Sea. Sequence alignments revealed that 96% of Nrt2 sequences at the Matsu station clustered with Skeletonema Nrt2 sequences. In the upwelling area, 60% of sequences clustered with Skeletonema and 10% clustered with Chaetoceros Nrt2. According to the sequences alignments, specific primers targeted to particular phytoplankton Nrt2 were designed. Using these primers with quantitative real-time PCR, our preliminary work demonstrated that the Nrt2 mRNA abundances of specific phytoplankton could be detected in the ocean. By detecting the expression levels of maker genes, not only the nitrogen-utilization status of various phytoplankton can be evaluated using a unified and simple procedure, the same algorithm can also be expanded to study other physiological activities of phytoplankton in the ocean. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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