碩士 / 國立成功大學 / 生物科技研究所碩博士班 / 100 / BL10 is a marine heterotrophic microalga that isolated from the stream outlet of northern Taiwan, it has many unique characteristics, including (1) euryhaline, it able to grow within the salinity range from 2 to 35 ppt; (2) saccharophilic, its growth not inhibited when the cell cultured in medium containing high glucose concentrations (140 gL-1); (3) oleaginous, its oil accumulated more than 80% of the dry biomass; (4) culture easily, the biomass can reach 120 gL-1 at 72 hour that inoculated with the initial cell concentrations of 0.01gL-1; (5) unique lipid metabolism pathway, and (6) special life cycle in BL10 that contains mobile zoospores and irregular-shaped amoeboid cells (the cell with crawling ability), making BL10 a nice candidate to become a new model organism, which can be used to study osmotic regulation, the production and accumulation of lipid, the long chain polyunsaturated fatty acids synthetic pathway of microalgae, and cell migration. However, the available information is insufficient to establish a model organism, including (1) the taxonomy of BL10 (2) the genome size and karyotype of BL10 (3) gene manipulation of BL10. Therefore, the purpose of this study is to resolve the above issues.
First, in order to identify the species of BL10, the 18S rDNA sequence were used to construct the phylogenetic tree. The results of a molecular phylogenetic analysis showed that BL10 belongs to the genus Aurantiochytrium and closely relates to A. limacinum. From the results of morphological observation, we found that BL10 can be easily observed the formation of amoeboid cells, and the amoeboid cells can divide into numerous zoospores after the cell settlement down. The above results showed that BL10 is similar to A. limacinum, while BL10 is unable to form the sporangium that significant different with A. limacinum.However, we consider that these results can not support the taxonomy of BL10.
On the other hand, we found that amoeboid cell with thin cell wall (or without cell wall), and the percentage of amoeboid cells could be enhanced up to 16% of total cells by regulating the medium composition and acquired at specific time. Those results provide information that it's possible to do gene transformation or dsRNA delivery for genomic functional analysis of BL10.
Then we established the genome size by flow cytometry, and used Saccharomyces cerevisiae W303-1A (12 Mbp) as control to calculate the genome size of BL10 (40.5 Mbp) that based on FL3 intensity. And in the karyotype analysis experiment, we used 8-hydroxyquinoline to arrest the cell cycle at the metaphase, and the chromosomes of BL10 were stained with DAPI and observed by fluorescence microscopy. The experimental results showed that BL10 has three chromosomes. Then we proved that the BL10 is a haploid using fluorescence in situ hybridization with a 45S rDNA probe. According to our results, we believe that the work of whole genome sequencing is possible.
| Identifer | oai:union.ndltd.org:TW/100NCKU5111118 |
| Date | January 2012 |
| Creators | Yu-MingSu, 蘇昱銘 |
| Contributors | Yi-Min Chen, 陳逸民 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 89 |
Page generated in 0.0118 seconds