Optimization of Electroporation Conditions and Screening of Nitrate Reductase Gene Mutants in Dunaliella viridis

To achieve efficient genetic transformation in Dunaliella viridis, this study optimized key parameters including culture media, antibiotic selection markers, and electroporation conditions. The results demonstrated that adding 0.5% agar to solid media enabled normal algal growth. The D. viridis strain showed insensitivity to glufosinate-ammonium but was susceptible to chloramphenicol and chlorate, with growth significantly inhibited by 400 μg/mL chloramphenicol in liquid media and 300 μg/mL in solid plates. For electroporation, cells were harvested by centrifugation at 1 700g for 3 min to achieve optimal cell collection efficiency. Using the NR gene knockout vector pKS-diaCas9-sgRNA-NRKO delivered via electroporation, nitrate reductase (NR)-deficient mutants were successfully selected on 100 mmol/L (30 g/L) sodium chlorate plate. Sequencing confirmed precise knockout at the second exon of the NR gene. This work establishes a foundational protocol for employing D. viridis as a bioreactor in genetic engineering applications.