Screening for fluorescent and chromoproteins from South African sea anemones
Sea anemones (Order Actinaria) are a diverse order from the Class Anthozoa. They are found in all marine habitats at all depths and their symbiotic relationships play an important role in energy transfers especially in the benthic-pelagic community. The evolutionary background and phylogenetics of the class is poorly understood due to a lack of correspondence between taxonomic and molecular data (Daly et al. 2008). Therefore, a deeper exploration into Cnidarian molecular biology is needed to establish these as an evolutionary model organism. Gene discovery from various marine invertebrates has facilitated the recovery of anti-cancer drugs, antibiotics and reporter genes (Faulkner, 2000; Allen and Jaspars, 2009). The most commercially lucrative products from sea anemones are fluorescent and chromoproteins (FP/CP), which are used as non-invasive real-time reporter genes. The applications for these proteins are extensive and range from monitoring cellular processes such as protein localisation and interactions to imaging (Alieva et al. 2008). Therefore, novel FP and CPs have potential for commercialization. The aims of the project were to analyze basic molecular diversity of the sea anemones Pseudactinia varia, Pseudactina flagellifera and Bunodosoma capensis and evaluate a new screening method to isolate novel FP and CPs. To assess the basic molecular diversity, of the sea anemones and their associated symbionts 16S rRNA and 18S rRNA clone libraries were generated. The sea anemones used in this study clustered together with those of the Family Actiniidae. The bacterial associations observed based on the closest relative BLAST analysis were dominated by Proteobacteria (gamma, alpha and epsilon) as well as Bacteroides. The associate bacterial symbionts possibly produce compounds that range from polyunsaturated fatty acids, polyhydroxyalkanoates to anti-microbial compounds that aid the host in various processes. In order to screen for FPs and CPs from sea anemones three types of cDNA libraries were generated to be screened either by sequence based or activity based approaches. Novel primers were designed which could be applied for the screening of a variety of Anthozoans. A positive control was also designed and synthesised in order to test the capability of the designed primers and optimise the amplification. Although amplicons were generated from gDNA and cDNA libraries from each of the sea anemones they were found to be non-specific products. The detection limit is likely to be the limiting factor. The construction of an activity based library was not achieved due to technical constraints, which highlights the need for new molecular tools in this field or improvements to the existing ones.