title: Construction of a self-cloning system in the unicellular green alga Pseudochoricystis ellipsoidea.
abstract: BACKGROUND
  Microalgae have received considerable interest as a source of biofuel production. The unicellular green alga Pseudochoricystis ellipsoidea (non-validated scientific name) strain Obi appears to be suitable for large-scale cultivation in outdoor open ponds for biodiesel production because it accumulates lipids to more than 30 % of dry cell weight under nitrogen-depleted conditions. It also grows rapidly under acidic conditions at which most protozoan grazers of microalgae may not be tolerant. The lipid productivity of this alga could be improved using genetic engineering techniques; however, genetically modified organisms are the subject of regulation by specific laws. Therefore, the aim of this study was to develop a self-cloning-based positive selection system for the breeding of P. ellipsoidea.
  
  
  RESULTS
  In this study, uracil auxotrophic mutants were isolated after the mutagenesis of P. ellipsoidea using either ultraviolet light or a transcription activator-like effector nuclease (TALEN) system. The cDNA of the uridine monophosphate synthase gene (PeUMPS) of P. ellipsoidea was cloned downstream of the promoter of either a beta-tubulin gene (PeTUBULIN1) or the gene for the small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (PeRBCS) to construct the pUT1 or pUT2 plasmid, respectively. These constructs were introduced into uracil auxotroph strains, and genetically complementary transformants were isolated successfully on minimal agar plates. Use of Noble agar as the solidifying agent was essential to avoid the development of false-positive colonies. It took more than 6 weeks for the formation of colonies of pUT1 transformants, whereas pUT2 transformants formed colonies in 2 weeks. Real-time PCR revealed that there were more PeUMPS transcripts in pUT2 transformants than in pUT1 transformants. Uracil synthesis (Ura(+)) transformants were also obtained using a gene cassette consisting solely of PeUMPS flanked by the PeRBCS promoter and terminator.
  
  
  CONCLUSIONS
  A self-cloning-based positive selection system for the genetic transformation of P. ellipsoidea was developed. Self-cloned P. ellipsoidea strains will require less-stringent containment measures for large-scale outdoor cultivation.
authors.affiliation: Department of Biological Sciences, Faculty of Science and Engineering, Chuo University, Kasuga 1-13-27, Bunkyo-ku, Tokyo, 112-8551 Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Kasai
authors.affiliation: Department of Biological Sciences, Faculty of Science and Engineering, Chuo University, Kasuga 1-13-27, Bunkyo-ku, Tokyo, 112-8551 Japan.
authors.forename: Kohei
authors.initials: K
authors.lastname: Oshima
authors.affiliation: Department of Biological Sciences, Faculty of Science and Engineering, Chuo University, Kasuga 1-13-27, Bunkyo-ku, Tokyo, 112-8551 Japan.
authors.forename: Fukiko
authors.initials: F
authors.lastname: Ikeda
authors.affiliation: Department of Biological Sciences, Faculty of Science and Engineering, Chuo University, Kasuga 1-13-27, Bunkyo-ku, Tokyo, 112-8551 Japan.
authors.forename: Jun
authors.initials: J
authors.lastname: Abe
authors.affiliation: Department of Biological Sciences, Faculty of Science and Engineering, Chuo University, Kasuga 1-13-27, Bunkyo-ku, Tokyo, 112-8551 Japan ; Research Laboratories, Denso Corporation, Nisshin, Aichi 470-0111 Japan.
authors.forename: Yuya
authors.initials: Y
authors.lastname: Yoshimitsu
authors.affiliation: Department of Biological Sciences, Faculty of Science and Engineering, Chuo University, Kasuga 1-13-27, Bunkyo-ku, Tokyo, 112-8551 Japan.
authors.forename: Shigeaki
authors.initials: S
authors.lastname: Harayama
keywords: Genetic transformation; Green algae; Microalgae; Molecular breeding; Pseudochoricystis ellipsoidea; Self-cloning; Uracil auxotroph