High-frequency nuclear transformation of Chlamydomonas reinhardtii.

TLDR

The authors used a cell‑wall‑deficient Chlamydomonas reinhardtii strain, agitating cells with plasmid DNA, glass beads, and polyethylene glycol, and selected for nitrate‑utilizing transformants by growth on nitrate, achieving ~10³ transformants per µg DNA. The transformation yielded low‑copy genomic integration, with 10–50 % cotransformation of an unselected gene, enabling efficient introduction of any cloned gene and facilitating isolation of genes from genomic libraries, thereby providing a powerful tool for studying chloroplast biogenesis, photosynthesis, and nuclear‑chloroplast genome interactions.

Abstract

By using a method in which cell-wall-deficient Chlamydomonas reinhardtii cells were agitated in the presence of DNA, glass beads, and polyethylene glycol, nuclear transformation rates of approximately 10(3) transformants per micrograms of plasmid DNA were achieved. The nitrate reductase gene from wild-type Chlamydomonas was used to complement a mutation in the corresponding gene of a strain containing nit1-305. Transformants were selected by growth with nitrate as sole source of nitrogen. The transforming DNA integrated into the genome at a low-copy number in nit+ transformants. When cells carrying nit1-305 were agitated in the presence of two plasmids, one with the gene for nitrate reductase and the second with an unselected gene, the unselected gene was present in 10-50% of nit+ transformants. This high frequency of cotransformation will allow any cloned gene to be introduced into Chlamydomonas. Moreover, the overall efficiency of transformation should be high enough to permit isolation of genes from genomic libraries by complementation of stable nuclear mutants. The availability of efficient nuclear and chloroplast transformation in Chlamydomonas provides specific advantages for the study of chloroplast biogenesis, photosynthesis, and nuclear-chloroplast genome interactions.