Establishment of the transposon-mediated insertional mutagenesis system in the green alga Chlamydomonas reinhardtii
Majid Eshaghi and Jianhua Liu
Systems Biology, Genome Institute of Singapore, A*STAR, 60 Biopolis Street, Singapore 138672, Singapore
Microalgae are believed to be a major source for renewable energy. Genetic engineering of microalgae is needed for improving the microalgae bioenergy productivity. The green alga Chlamydomonas reinhardtii has been a model organism for the study of bioenergy production. However, this is hampered by the poor DNA transformation efficiency in C. reinhardtii. Identification of mutations generated by exposure to chemical or physical mutagens requires transformation of DNA in complementation analysis. Though plasmid-based insertional mutations can be determined by thermal asymmetric interlaced (TAIL)-PCR, generation of insertional mutations requires transformation of plasmid DNA. To bypass the low efficient DNA transformation procedure, we have established the transposon-mediated insertional mutagenesis system. In this system, we only need to construct two strains via DNA transformation: the transposon-containing strain of one mating type and the transposase-containing strain of the opposite mating type. Mating of the two strains generates zygospores that are further induced for meiosis and sporulation. The resulting haploid cells containing transposon but not transposase are collected for phenotypic assessment and TAIL-PCR to determine the genomic locus of transposon insertions. We will show our results on the characterization of mutations generated by the transposon-mediated mutagenesis system. This work is supported by the Agency for Science and Technology and Research (A-STAR), Singapore.
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