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Rapid construction and screening of artificial micro RNA systems in C hlamydomonas reinhardtii
Author(s) -
Hu Jinlu,
Deng Xuan,
Shao Ning,
Wang Gaohong,
Huang Kaiyao
Publication year - 2014
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1111/tpj.12606
Subject(s) - chlamydomonas reinhardtii , gene knockdown , luciferase , biology , chlamydomonas , gene , biochemistry , transfection , mutant
Summary The unicellular green algae C hlamydomonas 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 C hlamydomonas relies mainly on mi RNA ‐ and si RNA ‐based knockdown methods. However, the difficulty in constructing artificial mi RNA vectors, laborious screening of knockdown transformants, and undesired epigenetic silencing of exogenous mi RNA constructs limit their application. We have established a one‐step procedure to construct an artificial mi RNA 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 mi RNA 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 VIPP 1 (vesicle inducing protein in plastids 1) and the flagellar protein CDPK 3 (calcium‐dependent protein kinase 3). Adding an intron from RBCS2 (ribulose bisphosphate carboxylase/oxygenase small subunit 2) to the mi RNA construct enhanced both the luciferase activity and the mi RNA knockdown efficiency. A second mi RNA vector incorporated the promoter of the nitrate reductase gene to allow inducible expression of the artificial mi RNA . These vectors will facilitate application of the artificial mi RNA and provide tools for studying the mechanism of epigenetics in C hlamydomonas , and may also be adapted for use in other model organisms.