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2005
LSU-HHMI Summer Undergraduate Research Program |
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Krishen Cunnusamy and James Moroney, Biological Sciences
RNAi Analysis of Carbonic Anhydrases Cah3, Cah6 and Cah7 in Chlamydomonas reinhardtii
Carbonic anhydrases (CAs) are a widely expressed family of zinc-containing metalloenzymes that catalyze the reversible hydration of CO2. First identified in 1933, in red blood cells, CAs have since been found to be abundant in bacteria and all mammalian tissues where they play a key role in the regulation of pH and fluid balance. In plants and algae, CAs are involved in photosynthesis and in the Carbon Concentrating Mechanism. In Chlamydomonas reinhardtii, the eight CAs that have been identified. They facilitate CO2 and HCO3- uptake, thus enhancing photosynthesis by overcoming the slow diffusion of CO2 in water. Our project is aimed at targeting some of those CAs namely CAH3, CAH6 and CAH7 by RNA interference and determining the relative effects of their respective silencing. To accomplish this, we amplified particular fragments of each gene from the cDNA library and genomic DNA and fused them into the pSL72 vector. The resulting constructs were linearized and transformed into Chlamydomonas by electroporation. Successful transformants, grown on paromomycin plates, were then screened on low CO2 to determine whether the reduction of these CA messages affect photosynthesis. Preliminary evidence suggests that the expression of CAH3 and CAH6 has been reduced using these constructs. Additionally, some of the strains with potentially reduced CA expression grow slowly under low CO2 conditions.
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