An Indexed, Mapped Mutant Library Enables Reverse Genetics Studies of Biological Processes in Chlamydomonas reinhardtii.

TitleAn Indexed, Mapped Mutant Library Enables Reverse Genetics Studies of Biological Processes in Chlamydomonas reinhardtii.
Publication TypeJournal Article
Year of Publication2016
AuthorsLi, X, Zhang, R, Patena, W, Gang, SS, Blum, SR, Ivanova, N, Yue, R, Robertson, JM, Lefebvre, PA, Fitz-Gibbon, ST, Grossman, AR, Jonikas, MC
JournalPlant Cell
Volume28
Issue2
Pagination367-87
Date Published2016 Feb
ISSN1532-298X
Abstract

<p>The green alga Chlamydomonas reinhardtii is a leading unicellular model for dissecting biological processes in photosynthetic eukaryotes. However, its usefulness has been limited by difficulties in obtaining mutants in specific genes of interest. To allow generation of large numbers of mapped mutants, we developed high-throughput methods that (1) enable easy maintenance of tens of thousands of Chlamydomonas strains by propagation on agar media and by cryogenic storage, (2) identify mutagenic insertion sites and physical coordinates in these collections, and (3) validate the insertion sites in pools of mutants by obtaining >500 bp of flanking genomic sequences. We used these approaches to construct a stably maintained library of 1935 mapped mutants, representing disruptions in 1562 genes. We further characterized randomly selected mutants and found that 33 out of 44 insertion sites (75%) could be confirmed by PCR, and 17 out of 23 mutants (74%) contained a single insertion. To demonstrate the power of this library for elucidating biological processes, we analyzed the lipid content of mutants disrupted in genes encoding proteins of the algal lipid droplet proteome. This study revealed a central role of the long-chain acyl-CoA synthetase LCS2 in the production of triacylglycerol from de novo-synthesized fatty acids.</p>

DOI10.1105/tpc.15.00465
Alternate JournalPlant Cell
PubMed ID26764374
PubMed Central IDPMC4790863
Grant ListR24 GM092473 / GM / NIGMS NIH HHS / United States
R24GM092473 / GM / NIGMS NIH HHS / United States