Cloning of nitrate transporter genes in a diatom Ditylum brightwellii and assessing their gene expression under different nitrogen status

• Main Authors: Huang, Ssu-Ying, 黃思穎
• Other Authors: Kang, Lee-Kuo
• Format: Others
• Language: zh-TW
• Published: 2017
• Online Access: http://ndltd.ncl.edu.tw/handle/d2s4hf

碩士 === 國立臺灣海洋大學 === 海洋環境與生態研究所 === 105 === Nitrogen is often considered as a major nutrient limiting phytoplankton growth in the marine environments. Nitrate transporter protein located on the cell membrane is thought to play an important role in nitrate uptake, and its gene, Nrt2, possesses different gene expression in response to different nitrogen environments. In this study, the gene expression of Nrt2 genes was monitored in a diatom, Ditylum brightwellii to compare with that in other diatoms species. In order to confirm the Nrt2 gene family in D. brightwellii, Nrt2 similar sequences were searched in the D. brightwellii transcriptome databases in Marine Microbial Eukaryote Transcriptome Sequencing Project (MMETSP) and confirmed by PCR from genomic DNA and sequencing. In total, four Nrt2 genes were identified, and DbNrt2A and DbNrt2D have not been reported in the litertures. When D. brightwellii was grown with nitrate as the sole nitrogen source, cell density increased exponentially for 5 days with a growth rate of 1.06 day-1. However, its growth was significantly affected by ammonium, especially when the initial ammonium concentration is higher than 100 μM. Monitoring the expression pattern of nitrate transporter genes under different nitrogen conditions, only the transcript level of DbNrt2C was repressed by ammonium, but induced under nitrogen deficiency. This gene expression pattern was consistent with that of T. pseudonana Nrt2.1 and Nrt2.2, but the transcription levels varied in D. brightwellii was only 1/100-fold of that in T. pseudonana. These results suggested that D. brightwellii is less sensitive to regulate the gene expression of Nrt2 gene than T. pseudonana, which might associated with its growth limited by ammonium.