Quantification of Diatom Gene Expression in the Sea by Selecting Uniformly Transcribed mRNA as the Basis for Normalization

Abstract

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.

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

“…EFL was used as the reference gene; the specificity and amplification efficiency of the EFL primer pairs were reported by Kang et al . (). To detect of Nrt2 mRNA, several primers were redesigned based on preliminary evaluations (Table ); the specificities and amplification efficiencies of these primers were tested in this study.…”

“…Group‐specific primers for Chaetoceros and Skeletonema (Table ) were designed against the divergent regions of the Nrt2 and EFL genes; such regions were revealed by sequence alignments that have previously been described (Kang et al . , ). EFL was used as the reference gene; the specificity and amplification efficiency of the EFL primer pairs were reported by Kang et al .…”

“…With the selection of the elongation factor‐like gene, EFL , as the basis for normalization (Kang et al . ), a quantitative reverse transcription–PCR (Q‐RT–PCR)‐based platform is ready for evaluating the status of nitrogen deficiency in natural diatom assemblages for which species/genus‐level primers have been designed.…”

Evaluation and selection of reference genes for ecotoxicogenomic study of the green alga Closterium ehrenbergii using quantitative real-time PCR

“…(Cao et al 2012), Chlamydomonas sp. ICE-L (Liu et al 2012), the dinoflagellate Prorocentrum minimum (Guo and Ki 2012a, b), and the diatoms Ditylum brightwellii (Guo et al 2013), Skeletonema costatum, Chaetoceros affinis (Kang et al 2012), Talasiossira pseudonana (Alexander et al 2012), and Pseudo-nitzschia multistriata (Adelfi et al 2014).…”

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

“…EFL was used as the reference gene (Kang et al. ). To separately determine the transcript levels of each NRT2 gene, each primer contained at least four mismatching nucleotides compared to homologous segments belonging to the other two forms of NRT2 and a single mismatch at the 3′ terminal.…”