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Cloning of Cia7 Gene and Generation of Transgenic Mutants of Chlamydomonas Reinhardtii

Chlamydomonas reinhardtii is a green haploid microalga that stands as an exemplary model organism in the study of heavy metal tolerance and homeostasis of photosynthetic organisms. Within C. reinhardtii's genome resides Cia7, a novel gene that encodes a protein (CIA7) of 104 amino acids with a conserved sequence of four cysteine residues. Given that thiol moieties are essential to metal‐binding proteins, it has been hypothesized that CIA7 might play a role in heavy metal homeostasis of C. reinhardtii. In the path of elucidating the function of the gene, previous studies have focused on exposing C. reinhardtii cells to heavy metal stress and metal bioaccumulation studies. This research study, on the other hand, aimed to isolate and overexpress the CIA7 protein in Escherichia coliand in C. reinhardtii. Polymerase chain reaction (PCR) was used to amplify Cia7 from C. reinhardtii's cDNA library. The PCR product was purified and ligated to the maltose‐binding protein expression vector pMAL‐c5x. The recombinant DNA with the Cia7 insert was used to transform E. coli. The presence of the insert was verified by restriction enzyme digests. In this study, we report the protein overexpression of CIA7. The overexpressed protein will be used for the in vitro studies of the novel protein, CIA7. In addition, a recombinant DNA generated by using the psL72 vector and the Cia7 insert was transformed into C. reinhardtii cells (wild type, cc4425 and mutant of Cia7, cc5013). The obtained transgenic algae will be used in the future for metal bioaccumulation analysis. Overall, this study will provide the clones and transgenic algae necessary to characterize the function of the gene and to determine whether Cia7 confers heavy metal resistance to C. reinhardtii.