Three DNA fragments belonging to genes involved in nitrogen uptake and assimilation, including a nitrate transporter gene (IgNrt2), an ammonium transporter gene (IgAmt), and a glutamine synthetase gene (IgglnII, were obtained from the marine haptophyte, Isochrysis galbana. Based on alignments of the deduced amino acid sequences, the IgNRT2 fragment shared 47%,, identity with the Cylindrotheca fusiformis (Bacillariophyceae) nitrate transporter, the IgAMT fragment shared 48%, identity with the C. fusiformis ammonium transporter, and the IgGSII fragment shared 61% identity with Skeletonema costatum (Bacillariophyceae) glutamine synthetase. Southern hybridization indicated that both IgNrt2 and IgAmt may have more than one copy in the genome of L galbana, but only a single copy was detected for IgglnII. The transcript abundances of these three genes under various nitrogen-supply conditions were monitored simultaneously by quantitative real-time polymerase chain reaction (PCR). In the presence of ammonium, both IgNrt2 and IgAmt were severely repressed with mean mRNA concentrations of 0.67 and 3.45 mu mol (mol 18S rRNA)(-1). IgNrt2 and IgAmt mRNA transcripts increased 41-fold and 8.3-fold, respectively, in the presence of nitrate, and increased 160-fold and 15-fold, respectively, in the ni trogen-depleted condition. The mRNA level of IgglnII was low in cells grown in the presence of ammonium [3.98 mu mol (mol 18S rRNA)(-1)], but mild increases in the range of 2- to 4-fold were observed in cells grown in both nitrate-containing and nitrogen-depleted cultures. Our results demonstrated that the expression patterns of IgNrt2, IgAmt, and IgglnII shared a common trend with their homologous genes in diatoms and green algae under various nitrogen-supply conditions. This group of genes could be used as indicators in natural environments to estimate the degree of nitrogen deficiency and chemical forms of nitrogenous nutrients in use on a species-specific basis

Nitrate transporter genes (Nrt2) encode high-affinity nitrate transporters in marine phytoplankton, and their transcript levels are potential markers of nitrogen deficiency in eukaryotic phytoplankton. For the proper interpretation of measured Nrt2 transcript abundances, a relative expression assay was proposed and tested in Isochrysis galbana Parke (Prymnesiophyceae) and Thalassiosira pseudonana (Hust.) Hasle et Heimdal (Bacillariophyceae). The minimal transcript levels of Nrt2 genes were achieved by the addition of 100 μM ammonium, which led to a rapid decline in Nrt2 transcripts in 10-30 min. Experiments using a concentration series revealed that the effective dosage of ammonium to create a minimal transcript level of ∼1 μmol · mol(-1) 18S rRNA was ≥25 μM in both species. On the other hand, the addition of l-methionine sulfoximine (MSX), an inhibitor of glutamine synthetase, enhanced the Nrt2 transcript level in I. galbana but did not affect that in T. pseudonana. Nitrogen deprivation was used as an alternative means to create maximal Nrt2 transcript levels. By transferring cells into N-free medium for 24 h, Nrt2 transcript levels increased to ∼90 μmol · mol(-1) 18S rRNA in I. galbana, and to ∼800 μmol · mol(-1) 18S rRNA in T. pseudonana. The degree of nitrogen deficiency thus can be determined by comparing original Nrt2 transcript levels with the minimal and maximal levels.

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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. On the other hand, 291 sequences homologous to Nrt2 were retrieved from mixed-species sequencing using degenerate primers, and these sequences were clustered into 12 major groups according to a phylogenetic analysis. Based on sequence alignments, 11 pairs of group-specific PCR primers were designed to detect Nrt2 mRNA levels in the ECS, and 3 of these primer pairs showed high specificity to target species. In ECS phytoplankton samples, environmental RNA was amplified via antisense RNA amplification followed by cDNA production. Subsequently, Nrt2 transcript levels were readily detected using quantitative PCR. Our results indicated that investigating sequence diversity followed by careful primer design and evaluation is a good strategy to quantify the expression of genes of ecologically important phytoplankton.

cTo 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. Next, the sequence diversity of diatom assemblages was evaluated by obtaining 32 EFL and 29 TBP homologous gene fragments from the East China Sea (ECS). Based on sequence alignments, EFL and TBP primer sets were designed for Chaetoceros and Skeletonema groups in the ECS. An evaluation of primer specificity and PCR efficiency indicated that the EFL primer sets performed better. To demonstrate the applicability of EFL primer sets in the ECS, they were employed to measure mRNA levels of the FcpB (fucoxanthin-chlorophyll protein) gene in diatoms. The results correctly revealed prominent diel variations in FcpB expression and confirmed EFL as a good reference gene.