Cloning and comparative studies of proliferating cell nuclear antigen (PCNA) genes for nine dinoflagellates

摘要

Proliferating cell nuclear antigen (PCNA), a co-factor of DNA polymerases delta and epsilon, is associated with active cell proliferation, particularly with the S phase of the eukaryotic cell cycle, and the expression of this gene has been therefore proposed as a molecular marker for estimating phytoplankton growth rate and even population dynamics. In this study, the full-length cDNA sequences of PCNA for nine dinoflagellate species (ten strains) were obtained, analyzed, and further characterized with the expression in response to alterations of growth stage and life cycle. All the ten genes have a 777 or 780 bp ORF and encode a protein of 258 or 259 amino acids, similar to PCNAs from vertebrates and plants. The deduced amino acid sequences include conservative motif characteristic to the PCNA gene family, implying conserved functions of PCNA among organisms of different taxa. Alignment analysis in combination with other sequences available in NCBI database exhibited high amino acid similarities (79.2-96.9%) among PCNAs from 17 dinoflagellate species. Phylogenetic tree derived from 37 PCNA sequences of dinoflagellates was consistent with the taxonomic affiliation of these taxa. Then, we used the cosmopolitan, toxic, and resting cyst-producing dinoflagellate Scrippsiella trochoidea as a representative of HAB-forming dinoflagellates to investigate PCNA (StPCNA) expression at different stages of growth and life cycle and the results of real-time quantitative PCR revealed clearly a stage-dependent pattern in the transcription of StPCNA. The transcripts dramatically reduced from an exponential to a stationary growth stage and then further significantly decreased from a stationary stage to dormant stage (resting cysts), a pattern in accordance with that previously reported from the dinoflagellates Alexandrium catenella and Prorocentrum donghaiense. In terms of its minimal implication, this work enhances the previous perception about the function and possible application of PCNA gene, and more importantly, our results indicated that PCNA is not silent during the dormant stage of dinoflagellates.