CLONING, SEQUENCING AND TRANSCRIPTIONAL ANALYSIS OF UBIQUITIN GENE FROM ALEXANDRIUM CATENELLA UNDER DIFFERENT NUTRIENT CONDITIONS

Ubiquitin is a small conserved protein ubiquitous in all eukaryotic organisms. It plays a critical role in intracellular protein turnover, being the tag that marks proteins for proteolysis. Beside proteolysis, it plays vital role in many cellular processes where protein actively needs to be modified or relocated. Sequence of ubiquitin selected from the differentially expressed gene library of NGS data of Alexandrium catenella was compared with the NCBI database for homologous sequences by blast. Primers were designed for amplification of partial fragment and gene expression analysis using real-time PCR in cells cultured at different nutritional conditions. Dino SL primer reported in the preceding studies was utilized to obtain a complete 5' end. A full-length cDNA of ubiquitin gene was obtained upon the assembly of the amplified products. Conserved domains of ubiquitin including the hydrophobic surface conserved amino acid regions and Ulp1-Smt3 interaction sites were observed in the cloned sequence. Phylogenetic tree constructed from the deduced amino acid sequence revealed two distinct clades representing ubiquitin sequences from dinoflagellates. The amino acid sequence of A. catenella has shown to possess 100% identity at conserved regions with another dinoflagellate, Pfiesteria piscicida. The result of qRTPCR indicated that the gene was significantly upregulated under all tested conditions. We speculate that under high nitrogen (HN) and high nitrogen phosphorous condition (HNP), the up-regulation of genes is due to rapid growth of the algae where proteins are also produced in excess amount. On the other hand, at low nitrogen and phosphorous conditions (LNP), the algae were under the effects of reactive oxygen species and as a result, the activation of ubiquitin-proteasome system is triggered that results in upregulation of ubiquitin family proteins to clear off the damaged proteins. The phenomenon is well supported by previous reports in other organisms. This study provides an important first look into the relationship between nutrient supply and expression analysis of ubiquitin in dinoflagellates.

Automated summary

This study examined the ubiquitin gene in Alexandrium catenella and found significant changes in gene expression under different nutritional conditions. Upregulation of the gene in high nitrogen and nitrogen-phosphorous conditions was associated with rapid algae growth, while under low nitrogen and phosphorous conditions, oxidative stress led to gene upregulation and activation of the ubiquitin-proteasome system.