tailieunhanh - Stable nuclear transformation of Pandorina morum

Volvocine green algae like Pandorina morum represent one of the most recent inventions of multicellularity diverged from their unicellular relatives. The 8–16 celled P. morum alga and its close multicellular relatives constitute a model lineage for research into cellular differentiation, morphogenesis and epithelial folding, sexual reproduction and evolution of multicellularity. | Lerche and Hallmann BMC Biotechnology 2014 14 65 http 1472-6750 14 65 BMC Biotechnology METHODOLOGY ARTICLE Open Access Stable nuclear transformation of Pandorina morum Kai Lerche and Armin Hallmann Abstract Background Volvocine green algae like Pandorina morum represent one of the most recent inventions of multicellularity diverged from their unicellular relatives. The 8-16 celled P. morum alga and its close multicellular relatives constitute a model lineage for research into cellular differentiation morphogenesis and epithelial folding sexual reproduction and evolution of multicellularity. Pandorina is the largest and most complex organism in the volvocine lineage that still exhibits isogamous sexual reproduction. So far molecular-biological investigations in P. morum were constricted due to the absence of methods for transformation of this species which is a prerequisite for introduction of reporter genes and modified genes of interest. Results Stable nuclear transformation of P. morum was achieved using chimeric constructs with a selectable marker a reporter gene promoters and upstream and downstream flanking sequences from heterologous sources. DNA was introduced into the cells by particle bombardment with plasmid-coated gold particles. The aminoglycoside 3 -phosphotransferase VIII aphVIII gene of Streptomyces rimosus under control of an artificial heterologous promoter was used as the selectable marker. The artificial promoter contained a tandem arrangement of the promoter of both the heat shock protein 70A hsp70A and the ribulose-1 5-bisphosphat-carboxylase -oxygenase S3 rbcS3 gene of Volvox carteri. Due to the expression of aphVIII transformants gained up to 333-fold higher resistance to paromomycin in comparison to the parent wild-type strain. The heterologous luciferase gluc gene of Gaussia princeps which was previously genetically engineered to match the nuclear codon usage of Chlamydomonas reinhardtii was used as a co-transformed .