Rapid construction and screening of artificial microRNA systems in Chlamydomonas reinhardtii

Abstract

SUMMARYThe unicellular green algae Chlamydomonas reinhardtii is a classic model for the study of flagella/cilia and photosynthesis, and it has recently been exploited for producing biopharmaceuticals and biofuel. Due to the low frequency of homologous recombination, reverse genetic manipulation in Chlamydomonas relies mainly on miRNA-and siRNA-based knockdown methods. However, the difficulty in constructing artificial miRNA vectors, laborious screening of knockdown transformants, and undesired epigenetic silencing of exogenous miRNA constructs limit their application. We have established a one-step procedure to construct an artificial miRNA precursor by annealing eight oligonucleotides of approximately 40 nucleotides. In the final construct, the Gaussia princeps luciferase gene (G-Luc) is positioned between the promoter and the artificial miRNA precursor so that knockdown strains may quickly be screened by visualizing luciferase luminescence using a photon-counting camera. Furthermore, the luciferase activity of transformants correlates with the knockdown level of two test target proteins: the chloroplast protein VIPP1 (vesicle inducing protein in plastids 1) and the flagellar protein CDPK3 (calcium-dependent protein kinase 3). Adding an intron from RBCS2 (ribulose bisphosphate carboxylase/oxygenase small subunit 2) to the miRNA construct enhanced both the luciferase activity and the miRNA knockdown efficiency. A second miRNA vector incorporated the promoter of the nitrate reductase gene to allow inducible expression of the artificial miRNA. These vectors will facilitate application of the artificial miRNA and provide tools for studying the mechanism of epigenetics in Chlamydomonas, and may also be adapted for use in other model organisms.

Intraflagellar transport protein IFT52 recruits IFT46 to the basal body and flagella

“…SDS-PAGE and immunoblotting assays were performed as previously described (Hu et al, 2014). Primary and secondary antibodies used in this study are listed in Table S4.…”

“…The Chlamydomonas cells were grown in liquid Tris-acetate-phosphate (TAP) (Harris et al, 1989) or minimal (MI) (Sager and Granick, 1953) ) in 96-well plates, 250 ml Erlenmeyer flasks or 5 l aerated glass containers (Hu et al, 2014). For long-term storage, the Chlamydomonas cells were maintained on solid TAP medium supplemented with 1.5% agar at 20°C with a cycle of 14 h of light and 10 h of dark.…”

“…Whole-cell soluble proteins were extracted as described previously (Fowkes and Mitchell, 1998;Hu et al, 2014). Nuclear proteins were extracted with CelLytic PN Plant Nuclei Isolation/Extraction Kit (Sigma-Aldrich, #CELLYTPN1) according to the manufacturer's guidelines.…”

“…Electroporation was performed as described previously (Brown et al, 1991;Hu et al, 2014;Shimogawara et al, 1998).…”

“…Nuclear proteins were extracted with CelLytic PN Plant Nuclei Isolation/Extraction Kit (Sigma-Aldrich, #CELLYTPN1) according to the manufacturer's guidelines. The protein concentration was determined by using the Amido black 10B binding method with BSA as a standard (Hu et al, 2014;Schaffner and Weissmann, 1973).…”