Chlamydomonas reinhardtii, a unicellular eukaryotic green alga, serves as a model organism to study the circadian clock in plants and animals. Rhodopsins are blue/green-light photoreceptors also found in C. reinhardtii. Chlamyrhodopsin (COP), the most abundant eyespot protein, was reported to have no role in the phototactic and photophobic responses in C. reinhardtii. Its function is yet unknown. In the present study, we hypothesized that the function of COP is to mediate entrainment of the circadian clock by light. In order to test this hypothesis, a C. reinhardtii selection marker conferring resistance to the antibiotic paromomycin was cloned into a COP RNAi construct obtained from another lab. Firstly, the COP RNAi construct was restriction digested to linearize it. The linearized plasmid was then blunt ended with T4 DNA polymerase and dephosphorylated with phosphatase. The linearized fragment was ligated with the paromomycin resistance marker obtained by restriction digestion of the plasmid containing it and transformed into E.coli. The recombinant clones obtained were confirmed by restriction digests. Fusion regions and the orientation of the insert in the recombinant plasmid were confirmed by sequencing. An attempt was made to transform C. reinhardtii with the construct, but this was not successful. Future studies will be required to optimize the C. reinhardtii transformation method. Transformants with reduced COP amounts can then be tested for defects in resetting the clock after light pulses. This will determine whether chlamyrhodopsin is involved in the circadian input pathway or not. The results of the complete project are expected to contribute to our understanding of the circadian clock of many other organisms including humans.
| Identifer | oai:union.ndltd.org:WKU/oai:digitalcommons.wku.edu:theses-1215 |
| Date | 01 December 2010 |
| Creators | Maddi, Shravya Reddy |
| Publisher | TopSCHOLAR® |
| Source Sets | Western Kentucky University Theses |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Masters Theses & Specialist Projects |
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