Utilizing Large-Scale Sequencing to Select Housekeeping Genes as Comparative Standards for Measuring the Expressions of Nitrogen and Phosphorus Absorption-Related Genes in Marine Diatoms

Transcriptome analysis is a good method to study how genes involved in nutrient uptake are regulated in marine phytoplankton. For a specific target gene, evidence for differential expression usually comes from nutrient enrichment experiments, but constructing transcriptomes for so many samples at every station can be difficult. At a station, when the only sample available is the in situ sample, a set of housekeeping genes may be identified in this transcriptome with their expression levels used as proxies of nutrient-enriched and nutrient-deprived expression levels of the target gene. Thus, the evaluation of the target gene expression in a single transcriptome becomes possible. To test this idea, oceanographic cruises were conducted in the coastal zone of the East China Sea. Phytoplankton samples were collected by a plankton net and subsamples were incubated with various nitrogen and phosphorus treatments. Subsequently, total RNA was extracted from each subsample and corresponding meta- transcriptomes were constructed. In each transcriptome, reads belonging to the diatom genus, Skeletonema, was collected by mapping short sequences. Housekeeping genes were then selected by the geNorm algorithm developed by Vandesompele et al. (2002), which compared ratios between the reads of all combinations of gene pairs across all samples at a station. Several such generated housekeeping genes had expression levels near the nutrient-enriched expression of the target gene. They were designated as the nutrient-enriched proxies. A set of nutrient-deprived proxies were similarly selected. Nutrient-enriched and nutrient-deprived relationships obtained this way were applied to sampling stations at which nutrient enrichment experiment was not performed.