Identification and Expression Analyses of the Nitrate Transporter Gene (NRT2) Family Among Skeletonema species (Bacillariophyceae).

High-affinity nitrate transporters are considered to be the major transporter system for nitrate uptake in diatoms. In the diatom genus Skeletonema, three forms of genes encoding high-affinity nitrate transporters (NRT2) were newly identified from transcriptomes generated as part of the marine microbial eukaryote transcriptome sequencing project. To examine the expression of each form of NRT2 under different nitrogen environments, laboratory experiments were conducted under nitrate-sufficient, ammonium-sufficient, and nitrate-limited conditions using three ecologically important Skeletonema species: S.

Evaluation of the Relationship Between the 18S rRNA/rDNA Ratio and Population Growth in the Marine Diatom Skeletonema tropicum via the Application of an Exogenous Nucleic Acid Standard.

Ribosomal RNA (rRNA) has been regarded as a proxy for metabolic activity and population growth in microbes, but the limitations and assumptions of this approach should be better defined, particularly in eukaryotic microalgae. In this study, the 18S rRNA/rDNA ratio of a marine diatom, Skeletonema tropicum, was examined in batch and semi-continuous cultures subjected to low nitrogen and phosphorus treatments at a temperature of 20 °C. In the semi-continuous cultures, the measured 18S rRNA/rDNA ratio ranged from 4.

The expression of nitrate transporter genes reveals different nitrogen statuses of dominant diatom groups in the southern East China Sea.

In this study, the mRNA levels of the Nrt2 nitrate transporter gene were used as a molecular indicator of nitrogen status in two dominant diatom groups, Skeletonema and Chaetoceros, which inhabit the southern East China Sea (ECS). To accurately interpret the abundance of Nrt2 transcripts in situ, maximum and minimum expression levels were determined under conditions of nitrogen deprivation and ammonium addition, respectively. In August 2010, Nrt2 transcript levels in Skeletonema at the inner shelf region exhibited a mean of 111 mmole/(mole EFL); at the mid-shelf region, the mean Nrt2 mRNA levels were 298 mmole/(mole EFL), which was very close to the maximum levels observed under nitrogen starvation.

Quantification of diatom gene expression in the sea by selecting uniformly transcribed mRNA as the basis for normalization.

To quantify gene expressions by quantitative reverse transcription-PCR (Q-RT-PCR) in natural diatom assemblages, it is necessary to seek a biomass reference specific to the target species. Two housekeeping genes, TBP (encoding the TATA box-binding protein) and EFL (encoding the translation elongation factor-like protein), were evaluated as candidates for reference genes in Q-RT-PCR assays. Transcript levels of TBP and EFL were relatively stable under various test conditions including growth stages, light-dark cycle phases, and nutrient stresses in Skeletonema costatum and Chaetoceros affinis, and TBP expression was more stable than that of EFL.

Diversity of phytoplankton nitrate transporter sequences from isolated single cells and mixed samples from the East China Sea and mRNA quantification.

The transcript abundances of nitrate transporter genes (Nrt2) were proposed as potential markers for nitrogen deficiency in marine diatoms. To correctly quantify diatom Nrt2 mRNA in the East China Sea (ECS), we utilized both mixed-species sequencing and single-cell PCR to expand the sequence database for this region. Using the single-cell method of PCR, 9 new diatom Nrt2 sequences belonging to 5 genera, the Nrt2 sequences of which have never been reported before, were obtained.