The unicellular green algae Chlamydomonas reinhardtii is a classic model for the study of flagella/cilia, photosynthesis and recently it also has 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.    

Researchers from Institute of Hydrobiology, Chinese Academy of Sciences (IHB), established a one-step procedure to construct an artificial miRNA precursor by annealing eight ~40nt oligonucleotides. In the final construct, the Gussia luciferase (G-luc) gene is positioned between the promotor and the artificial miRNA precursor so that knockdown strains can quickly be screened by visualizing luciferase luminescence with 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 incorporates the promotor of the nitrate reductase gene to allow inducible expression of the artificial miRNA. These vectors will facilitate the application of the artificial miRNA, provide tools for studying the mechanism of epigenetics in Chlamydomonas and can also be adapted for use in other model organisms.   

The study was done by Hu Jinlu, Xuan Deng et al, and the corresponding author is Dr. Kaiyao Huang and Gaohong Wang. The study was funded by National Science Foundation and the National Development and Investment Company (Group). Relevant paper "Rapid construction and screening of artificial miRNA systems in Chlamydomonas reinhardtii" was published online in "The Plant Journal". 

The strategy of construction an artificial microRNA (amiRNA) forChlamydomonasVIPP1gene.  (a). Eight 40-45 oligonucleotides were designed and annealed to make the entire amiRNA precursor in one step. Oligonucleotides1, 2, 5, and 8 are based on the MIR1157 backbone; 3, 4, 6, and 7 are gene specific.EcoRI andEcoRV sites were added for cloning.  (b). The amiRNA knock down vector (pHK226) includes thePsaDpromotor (PPsaD),theGussia luciferasegene and aVIPP1-amiRNA precursor.After transcription and translation inChlamydomonas, theluciferasegene was expressed, and the amiRNA precursor was processed to form a miRNA targeting theVIPP1gene.