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Plant Cell. 2016 Feb;28(2):367-87. doi: 10.1105/tpc.15.00465. Epub 2016 Jan 13.

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

Author information

1
Department of Plant Biology, Carnegie Institution for Science, Stanford, California 94305.
2
Department of Plant Biology, University of Minnesota, St. Paul, Minnesota 55108.
3
Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095.
4
Department of Plant Biology, Carnegie Institution for Science, Stanford, California 94305 mjonikas@carnegiescience.edu.

Abstract

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.

PMID:
26764374
PMCID:
PMC4790863
DOI:
10.1105/tpc.15.00465
[Indexed for MEDLINE]
Free PMC Article

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