Isolation and characterization of pigment deficient insertional mutants in the chlorophyte Chlamydomonas reinhardtii

Abstract

In the present work, a large collection of mutants was obtained by insertional mutagenesis from 704 (cw15 Arg7+ Nia1 : Ars mt+) strain of Chlamydomonas reinhardtii, using a linearized fragment of the pSI03 plasmid. About 2100 insertional mutants resistant to the antibiotic paromomycin were isolated and screened to identify Chlamydomonas reinhardtii mutants: i) sensitive to high light, ii) with an altered pigment composition and iii) with an irregular response to high light stress. The insertion site in 15 of the selected mutants was amplified by Restriction enzyme site-directed amplification (RESDA) PCR or inverse PCR. Cloning, sequencing and alignment of the amplified DNA with the Chlamydomonas database allowed the identification of the region adjacent to the insertion in most of the mutants studied. We observed that in 77 % of the transformants analysed, insertion took place in intragenic regions. Almost half of the mutants (46 %) had insertions in genome loci with unknown functions. Among disrupted genes with known functions, we found genes involved in a great diversity of functions, from flagella motion to regulatory or signal transduction processes, suggesting that the sensitivity to high light and the synthesis of pigments are complex and very regulated processes.