Cloning of proliferating cell nuclear antigen gene from the dinoflagellate Prorocentrum donghaiense and monitoring its expression profiles by real-time RT-PCR

Abstract

The growth rate of marine phytoplankton at the species level is an important parameter for estimating a phytoplankton population in the field. Therefore, monitoring this rate may have potential to be useful for providing early warnings for red tide blooms. We investigated the proliferating cell nuclear antigen (PCNA) gene of the red tide species Prorocentrumdonghaiense Lu et Goebel to study the relationship between its expression level and the growth rate. The gene was isolated and the full cDNA sequence encoding PCNA was obtained. It consisted of 1,057 base pairs (bp), comprising 81 bp of the 5′ untranslated region (UTR) and 196 bp of the 3′ UTR, and had an open reading frame (ORF) of 780 bp, encoding 259 amino acid residues. The coding region was highly conserved compared with other organisms PCNA gene. Using real-time PCR, we found that the average of PCNA transcript, normalized to per cell basis, decreased obviously from 26.12 ± 3.04 copies in the exponential and transition phase to 5.95 ± 0.79 copies in the stationary phase. However, in the decline phase, the copies of PCNA transcription per cell is below one copy. Furthermore, the expression level of PCNA changed consistently with the growth rate as assessed by the relative quantitation method. These results indicate that PCNA has a potential as a molecular marker of growth phase and rate for P. donghaiense. This may help in forecasting blooms, allowing for effective control measures to be implemented in a timely fashion.