Abstract / Bemerkung
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, unselectable reporter gene. The expression of the co-bombarded gluc gene in transformants and the induction of gluc by heat shock were demonstrated through bioluminescence assays. Conclusion Stable nuclear transformation of P. morum using the particle bombardment technique is now feasible. Functional expression of heterologous genes is achieved using heterologous flanking sequences from Volvox carteri and Chlamydomonas reinhardtii. The aphVIII gene of the actinobacterium S. rimosus can be used as a selectable marker for transformation experiments in the green alga P. morum. The gluc gene of the marine copepod G. princeps, expressed under control of heterologous promoter elements, represents a suitable reporter gene for monitoring gene expression or for other applications in P. morum.
Selectable markers; Green algae; Reporter genes; Streptomyces rimosus aphVIII gene; Heterologous expression; Genetic engineering; Gaussia princeps luciferase gene; Volvocine algae; Co-transformation; Volvocaceae
Lerche K, Hallmann A. Stable nuclear transformation of Pandorina morum. BMC Biotechnology. 2014;14(1):65.
Lerche, K., & Hallmann, A. (2014). Stable nuclear transformation of Pandorina morum. BMC Biotechnology, 14(1), 65. doi:10.1186/1472-6750-14-65
Lerche, K., and Hallmann, A. (2014). Stable nuclear transformation of Pandorina morum. BMC Biotechnology 14, 65.
Lerche, K., & Hallmann, A., 2014. Stable nuclear transformation of Pandorina morum. BMC Biotechnology, 14(1), p 65.
K. Lerche and A. Hallmann, “Stable nuclear transformation of Pandorina morum”, BMC Biotechnology, vol. 14, 2014, pp. 65.
Lerche, K., Hallmann, A.: Stable nuclear transformation of Pandorina morum. BMC Biotechnology. 14, 65 (2014).
Lerche, Kai, and Hallmann, Armin. “Stable nuclear transformation of Pandorina morum”. BMC Biotechnology 14.1 (2014): 65.
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