Mentor: Dr. W. Kelley Thomas, UNH Hubbard Center for Genome Studies
Molecular Diversity and Functional Analysis of the Opsin Genes Expressed in Daphnia Pulex
Daphnia are branchiopod crustaceans that have been the subject of study by researchers for over 300 years. Daphnia’s ability to detect and use light in the environment may serve important functions in the detection of mates and/or predators and in regulating their migration in the water column. Little is known about the molecular mechanisms of vision in Daphnia. This project is to find and characterize the genes for the light-sensitive opsin proteins in Daphnia.
Part one of this project was to discover the diversity of opsin genes in the Daphnia genome. Hence, DNA sequences of seven known opsin genes from Drosophila melanogaster were compared with the Daphnia genome assembly using TBLASTX. The search yielded a set of approximately 50 genes located on different scaffolds within the genome assembly. Using sequences from their filtered models, 29 putative opsin genes in Daphnia were translated and compared to characterized opsins from other invertebrates using CLUSTALX. Phylogenetic analyses were then implemented in MEGA, producing a neighbor-joining tree which was used for inferring the function of these genes.
The next part of the project consisted of laboratory experiments which were conducted to provide empirical evidence of the predicted opsin genes. Primers were designed from the predicted sequences of the genes for PCR and sequencing. Subsequent analysis confirmed 27 of the 29 genes are represented in the expressed genes of Daphnia. Furthermore, rtPCR experiments were used to confirm the expression of these genes and proposed gene (filter) models for each of the putative opsins.
Recent Daphnia Genome Consortium (DGC) data, introduced the possibility of 51 putative opsin genes, allowing for the expansion of putative genes to include genes previously omitted due to quality issues. Future experiments will generate more sequence data for the Daphnia cDNA libraries in an effort to refine the current gene filter models.