The actin gene promoter-driven bar as a dominant selectable marker for nuclear transformation of Dunaliella salina.

A 5'-flanking region of an actin gene from the green unicellular alga Dunaliella salina (D. salina) was cloned using a genome-walking method by PCR and its structural features were characterized. Two repetitive sequences found, over 75 bp in length each, were located at position -573 and -424 bp,respectively, relative to the AUG codon. The actin gene promoter region of D. salina displayed a consensus sequence of GCTC (G/C) AAGGC, a CCAAT motif and two TATA-like motifs that did not have a canonical sequence of a TATA box. The 5' flanking region of the actin gene was exploited to direct expression of the bialaphos resistance gene (bar) from Streptomyces hygroscopicus as a dominant marker in the nuclear transformation of D. salina. Direct selection of bar resistant transformants was achieved by allowing a 24 h period of recovery of cells transformed by biolistic procedure, followed by growth of the cells for one week under standard condition prior to harvesting and plating on the solid medium containing 0.5 microg/mL of phosphinothricin (PPT). Five colonies picked from the plate were analyzed, of which the integration of the bar gene was demonstrated in the nuclear genome. Southern blotting revealed that only one of five transformants contained a single copy of the bar gene whereas others contained multiple copies,suggesting that nuclear transformation of D. salina mainly occurred through illegitimate recombination events,resulting in ectopic integration of the introduced DNA. The integration patterns of the foreign DNA in this experiment appeared not to influence the bar gene expression in the transformants containing single or multiple inserts. The bar gene expression in the five transformants was verified by RT-PCR, confirming transcription of the chimeric DNA. These transformants were maintained on agar plates in the absence of PPT for more than seven months and retained resistance to the herbicide at 1 microg/mL. This work demonstrates that the actin gene promoter-driven expression of the bar gene may be used as a dominant selectable marker for nuclear transformation of D. salina.