Generation and Characterization of Transgenic Chlamydomonas ReinhardtII Overexpressing the Novel Gene CIA7

The constant increase in environmental pollution, in particular heavy metal pollution is alarming. Cia7, the gene understudy has conserved cysteine residues. These residues could be involved in metal binding; and the CIA7 protein might be associated to metal homeostasis in Chlamydomonas reinhardtii. The successful overexpression of Cia7 in C. reinhardtii could enhance its metal tolerance. The core hypothesis is that CIA7 plays a role in metal tolerance and in metal binding. The aim of this research is to characterize the Cia7 gene and to assess its potential role in metal homeostasis. The objectives of the research are (1) to overexpress CIA7 in C. reinhardtii, (2) to perform lead tolerance assays in WT overexpressing CIA7, and (3) to compare cell growth, chlorophyll content and bioaccumulation of heavy metals in CC5013 mutant, CC4425 wild-type and the selected transformants under the presence or absence of lead. The gene was overexpressed in wild-type (WT) and mutant (MT) C. reinhardtii cells. Lead tolerant colonies, the wild-type and the mutant strains were subjected to a chlorophyll content, bioaccumulation and cell growth analyses to determine if there is any difference among them. Chlorophyll content and cell growth do support the hypothesis that CIA7 could play a role in heavy metal tolerance. In the metal bioaccumulation analysis, CC4425 (WT) when compared to CC5013 (mutant), CC5013 shows higher lead bioaccumulation than CC4425. Both the parental and transformant cell consistently showed higher lead bioaccumulation, suggesting that absence or presence of the gene did not significantly affect lead bioaccumulation. However, its absence or disruption in the mutant resulted in an increase in lead bioaccumulation. Thus, it could be inferred that Cia7 helps cells detoxify lead. The findings of this research could provide a basis to further investigate the (1) use of Cia7 as a biomarker for metal contamination, or to (2) make the CIA7 protein be overexpressed in C. reinhardtii or in another organism for use in bioremediation