碩士 / 國立臺灣海洋大學 / 海洋生物研究所 / 105 / Skeletonema is a dominant diatom genus in the coastal waters near Matsu Islands. Skeletonema contains nitrate transporter genes (Nrt2), the expression level of which reflects the physiological status of this diatom in the utilization of ammonium and nitrate. In order to correctly interpret metatranscriptomes of phytoplankton in Matsu waters and to design highly efficient primers for quantitative polymerase chain reaction (qPCR), single-colony sequencing was employed to identify genes belonging to the same species within Skeletonema. In June and July 2016, plankton samples were collected at several stations near Nangan Island, Matsu. From these samples, 12 Skeletonema colonies were isolated with a micromanipulator. Next, the total amount of DNA in each colony was increased by multiple substitution amplification (MDA). Using the amplified DNA as templates, gene fragments of 18S rRNA, rbcL, Nrt2 and EFL were cloned by PCR and sequenced. Among them, 18S rRNA and rbcL sequences were used to confirm the taxonomical association of individual colonies, and the corresponding Nrt2 and EFL sequences were regarded as genes belonging to the verified species. In the 12 Skeletonema colonies, a total of 48 sequences were obtained for genes mentioned above. According to 18S rRNA and rbcL sequences, there were 7 colonies identified as S. subsalsum and the other 4 colonies were S. tropicum. Each colony was then sequenced to reveal Nrt2 sequences. The results showed that S. subsalsum and S. tropicum each contained two forms of homologous Nrt2 genes. As for the reference gene, only one EFL sequence was cloned from the S. tropicum coloies. In contrast, EFL sequences belonging to S. subsalsum could be categorized into four groups on the phylogenetic tree. Through these sequencing efforts, sequences from individual Skeletonema colonies were used to identify genes of unknown species in metatranscriptomes, and species-specific qPCR primers could be designed with more confidence.
| Identifer | oai:union.ndltd.org:TW/105NTOU5270005 |
| Date | January 2016 |
| Creators | Liao, Pei-Chi, 廖珮綺 |
| 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 | 35 |
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