Use of the cytochrome c6 (CYC6) and carbonic anhydrase (CAH1) inducible promoters to drive gene expression and silencing of endogenous genes in Chlamydomonas reinhardtii
Paola Ferrante1, Dennis Diener2, Joel Rosenbaum2, Giovanni Giuliano1
1) ENEA, Casaccia Res. Ctr., Rome, Italy
2) Dept. Mol. Cell. Dev. Biol., Yale Univ., New Haven, CT, USA
Inducible promoters are useful tools for studying gene function and for regulating cellular activities. In this work we use the metal-responsive CYC6 promoter to induce the silencing of a chloroplast sulfur transporter (SulP) and of a radial spoke protein (Rsp3) using artificial microRNA (amiRNA). Real Time RT-PCR and Western blot analyses show that 3 days after nickel addition the mRNA (SulP) and the protein (Rsp3) levels were reduced in the induced transformants relative to uninduced cultures. Inducible SULP silencing is potentially useful in switching cultures to sulfur-starved conditions, thus inducing production of hydrogen. Moreover, inducible RSP3 silencing could be employed to improve harvesting of cells after growth in a photo-bioreactor. We also present data on the use of the CYC6 and carbonic anhydrase promoters to rescue a paralyzed flagella mutant, pf14, lacking a functional copy of the RSP3 gene. In both cases only a small percentage of the cells could swim after induction, despite the fact that under inducing conditions the endogenous genes were strongly induced. Acknowledgments: Supported by grants of the Italian Ministries of Research and Agriculture to GG and NIH GM14642 to JR.
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