Stable nuclear transformation of Chlamydomonas reinhardtii by using a C. reinhardtii gene as the selectable marker.
We have developed a stable nuclear transformation system for the unicellular green alga Chlamydomonas reinhardtii. Transformation was accomplished by introducing the cloned C. reinhardtii oxygen-evolving enhancer protein 1 (OEE1) gene into C. reinhardtii cells by bombardment with DNA-coated tungsten particles. The recipient strain was an OEE1-deficient, nonphotosynthetic, acetate-requiring mutant, which recovered photosynthetic competence after transformation, and was therefore able to grow in the absence of acetate. Analysis of several transformants indicates that transformation has proceeded via second-site integration of the cloned gene, leaving the endogenous mutant gene intact. In genetic crosses of transformants with wild type, both mutant and wild-type phenotypes were recovered, showing that the photosynthetic competence of transformants was due not to reversion of the original locus but rather to expression of the introduced gene. We suggest that the success of the present system is largely due to using a homologous C. reinhardtii gene, leading to stable maintenance and expression of the gene. Transformation with heterologous genes may be problematic because of poor expression due to an unusual codon bias in C. reinhardtii.
Mayfield
S P
SP
Department of Molecular Biology, Research Institute of Scripps Clinic, La Jolla, CA 92037.
Kindle
K L
KL
D020418:Algal Proteins; D001483:Base Sequence; D002696:Chlamydomonas / Q000235:genetics*; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics; D003433:Crosses, Genetic; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D005819:Genetic Markers; D008969:Molecular Sequence Data; D009154:Mutation; D010788:Photosynthesis; D010940:Plant Proteins / Q000235:genetics*; D010957:Plasmids; D015183:Restriction Mapping; D014170:Transformation, Genetic
D020418:Algal Proteins; D003062:Codon; D005819:Genetic Markers; D010940:Plant Proteins; C052389:oxygen-evolving enhancer protein 1, plant