Diversity of Phytoplankton Nitrate Transporter Sequences from Isolated Single Cells and Mixed Samples from the East China Sea and mRNA Quantification

Abstract

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.

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

“…, Kang et al . , ). The remaining variation can be handled by conducting relative expression assays and calculating the Nrt2 index (eqn. )…”

“…The sequence diversity of the Nrt2 gene has been extensively surveyed in the East China Sea (ECS), enabling the design of species‐specific primers for assessment of the nitrogen status of ecologically important phytoplankton (Kang et al . ). In addition, a relative expression assay has been developed to determine the maximum and minimum Nrt2 transcript levels; these values can be used as standards when examining the transcript levels of untreated samples (Kang et al .…”

Pluses and minuses of ammonium and nitrate uptake and assimilation by phytoplankton and implications for productivity and community composition, with emphasis on nitrogen-enriched conditions

“…GenBank accession numbers for panel A are provided in parentheses and in panel B are provided in the original paper. Reproduced from Kang et al (‐panel A), and from Kang and Chang (‐panel B) with permission of the American Society for Microbiology (A) and the J. Mar. Sci.…”

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

“…Multiple nitrate transporter genes, NRT2 s, have been identified in numerous diatom species (Hildebrand and Dahlin , Song and Ward , Kang et al. ), haptophytes (Kang et al. , Song and Ward , Charrier et al.…”