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. 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.