Identification of the UMP Synthase Gene by Establishment of Uracil Auxotrophic Mutants and the Phenotypic Complementation System in the Marine Diatom Phaeodactylum tricornutum

Abstract

Abstract Uridine-5′-monophosphate synthase (UMPS), the critical step of the de novo pyrimidine biosynthesis pathway, which is a housekeeping plastid process in higher plants, was investigated in a marine diatom, the most crucial primary producer in the marine environment. A mutagenesis using an alkylation agent, N-ethyl-N-nitrosourea, was carried out to the marine diatom Phaeodactylum tricornutum. Cells were treated with 1.0 mg mL−1  N-ethyl-N-nitrosourea and were screened on agar plates containing 100 to 300 mg L−1 5-fluoroorotidic acid (5-FOA). Two clones survived the selection and were designated as Requiring Uracil and Resistant to FOA (RURF) 1 and 2. The 50% effective concentration of 5-FOA on growth of RURF1 was about 5 mm, whereas that in wild-type cells was 30 μm. The ability to grow in the absence of uracil was restored by a P. tricornutum gene that potentially encoded UMPS or the human umps gene, HUMPS. Because the P. tricornutum gene was able to restore growth in the absence of uracil, it was designated as ptumps, encoding a major functional UMPS in P. tricornutum. RNA interference to the ptumps targeting the 5′ region of ptumps resulted in the occurrence of a clear RURF phenotype in P. tricornutum. This RNA interference phenotype was reverted to the wild type by the insertion of HUMPS, confirming that the ptumps encodes UMPS. These results showed direct evidence of the occurrence of novel-type UMPS in a marine diatom and also revealed the potential usage of this gene silencing and complementation system for molecular tools for this organism.