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Gene silencing by artificial microRNAs in Chlamydomonas
Tao Zhao 1 , Wei Wang 1,2 , Xue Bai 1,2 and Yijun Qi 1,
1 National Institute of Biological Sciences, No.7 Science Park Road, Zhongguancun Life Science Park, Beijing 102206, China
2 Graduate Program in Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China.
ABSTRACT
Chlamydomonas reinhardtii is a unicellular green alga. It is a model system for studying functions of chloroplast, basal body and flagella. The completion of Chlamydomonas genomic sequence makes it possible to use reverse genetic approaches in this organism. Chlamydomonas contains a set of endogenous microRNAs (miRNAs) that down-regulate their target gene expression through mRNA cleavage. Here we developed an artificial miRNA-based strategy to knock down gene expression in Chlamydomonas. Using an endogenous Chlamydomonas miRNA precursor as the backbone, we constructed two artificial miRNAs (amiRNAs) targeting the MAA7 and RBCS1/2 genes, respectively. When overexpressed, these two amiRNAs could cleave their respective targets precisely at the predicted sites, resulting in greatly decreased accumulation of MAA7 and RBCS1/2 transcripts and expected mutant . We further showed the two amiRNAs could be produced simultaneously from a dimeric amiRNA precursor. We anticipate that the amiRNA technology developed in this study will be very useful in assessing the functions of individual genes and in genome-wide approaches.
